20th IUPAB International Congress, 45th SBBf Congress and 50th Annual Meeting of SBBq

AA - Biomimetic systems and membrane biophysics

BA - Membrane Permeation: Channels and Transporters

BB - Membrane-Active Peptides

CA - Applications in Biomedical and Materials Science

CB - Biotechnology and Biomaterials (agricultural, human and animal)

CC - Biotechnology and Biomaterial (Industrial Process)

CD - Drug Discovery and Delivery

DA - Protein Studies and Interactions

DB - Structural Biology

EA - Bioenergetics and Metabolism

EB - Photobiology, Optogenetics and Neural Systems

EC - Redox Processes

FA - Modeling, Big Data and Collective Behaviour

FB - Systems Biology

GA - Arthropods and Mechanobiology

GB - Glycobiology

GC - Microorganisms and Pathogens

GD - Natural Products

GE - Plants and Synthetic Biology

HA - Molecular Mechanisms of Diseases

JA - Signaling, Gene Regulation and Proliferation

JB - Immune System

KA - Computational Biophysics and Biochemistry

NB - Biociências Nucleares

SE - Science Education

08839 - Poster Session

AA.01 - Effects of phase state and charge on membrane fusion using a novel fusogenic system composition

Rafaela Ramos Mororo Cavalcanti ¹, Rafael Bezerra de Lira², Karin do Amaral Riske¹

¹Biophysics, Universidade Federal de Sao Paulo (Sao Paulo, Brazil)

²Molecular Biophysics, Groningen University (Groningen, the Netherlands)

INTRODUCTION

Cell membranes are believed to be heterogeneous and to exhibit lipid domains (or rafts) of coexisting phases, which can govern cellular functions and processes. As previously reported by us, membrane fusion efficiency was modulated by controlling membrane charge and phase state in giant unilamellar vesicles (GUVs, 10-50 μm) used as biomimetic membranes.

OBJECTIVES

In this work, we proposed a new composition of the fusogenic system preferentially in the gel phase at room temperature but fluid in physiological temperatures; additionally, membrane fusion efficiency was also investigated.

MATERIALS AND METHODS

The fusogenic system was based on large unilamellar vesicles (LUVs, 100-150 nm) and composed of an equimolar mixture of neutral and positively charged gel lipids, whereas the GUVs were composed of a mixture of zwitterionic and/or negatively charged fluid lipids. Initially, the fusogenic system was characterized using dynamic light scattering (DLS), zeta potential, anisotropy and differential scanning calorimetry (DSC). Then, fusion efficiency was characterized using microscopy-based fluorescence assays (lipid and content mixing).

DISCUSSION AND RESULTS

The DLS and zeta potential measurements exhibited a size average of ≈ 120 nm and a surface charge of ≈ 42 mV, respectively. The anisotropy and DSC showed that the lipid mixture was in the gel phase at room temperature. The incubation of the fusogenic system with neutral GUVs did not lead to significant lipid mixing and therefore no/low membrane fusion. However, negatively charged GUVs exhibited high lipid and content mixing, as expected in systems with efficient membrane fusion (Lira et al, in preparation). Interestingly, the highly membrane fusion efficiency induced phase-separation in the GUVs.

CONCLUSION

We demonstrated that membrane fusion can be controlled by modulating the membrane charge of the system in spite of the membrane phase state of the fusogenic system. These findings can play an important role in regulating the interaction between cells and liposomes used in drug delivery systems.

Keywords: fusogenic liposomes, biomimetic membranes, phase separation

Supported by: FAPESP

08099 - Poster Session

AA.02 - Reactivity and pH mapping with Catalytic Peptides

Caroline Dutra Lacerda ¹, Maria Aparecida Juliano², Cleber Wanderlei Liria¹, Maria Terêsa Machini¹, Hernan Chaimovich¹, Iolanda Midea Cuccovia¹

¹Bioquímica, Universidade de São Paulo (SÃO PAULO, Brasil)

²Biofísica, Universidade Federal de São Paulo (SÃO PAULO, Brasil)

INTRODUCTION

The sulfhydryl group (-SH) of cysteine (Cys) is essential for the activity of several enzymes, and its apparent p K_a determines the reactivity. Amphiphilic aggregates are useful models for studying chemical reactivity in dimensionally restricted environments, such as enzyme´s active sites. The acid dissociation, and the ensuing reactivity, of weak acids, such as RSH is determined by the local proton/hydroxide (H ⁺ /OH ^- ) distribution.

OBJECTIVES

Characterize H ⁺ /OH ^- radial distribution in vesicles and micelles as a function of the distance from the surface using the following Cys-containing peptides: Hexadecyl-Arg-(Gly)n-Cys-His-NH₂ and Hexadecyl-Lys-(Gly)n-Cys-NH₂, where n is the number of glycine residues.

MATERIALS AND METHODS

The pKap of -SH groups and rate constants (kobs) of the thiolysis of p-nitrophenyl-octanoate (NPO), in micelles of N,N,N-trimethyl-N-hexadecyl ammonium chloride (CTAC), and N,N-dimethyl-N,N-dihexadecylammonium chloride (DHDAC), vesicles were determined spectrophotometrically.

DISCUSSION AND RESULTS

The p Ka of -SH in water was 8.9 for all peptides; in 20 mM CTAC the values were 7.5, 7.8 and 8.3, for n = 3, 6 or 9 Gly, respectively. The p Kap change, as a function of n (1/2/3/4/5/6 or 9) shows that the SH groups are located at different distances from the interface. The kobs of NPO thiolysis by the same peptide series decreased with Gly content. The kobs were 18 times higher in DHDAC and 10 times higher in CTAC at pH 6, for probes with one Gly, compared to the reaction in water at pH 10. The cationic aggregates surfaces have high OH - concentration, a microenvironment in which the sulfhydryl is deprotonated, thus more available for the reaction with NPO. The kobs were not significantly different between probes containing histidine.

CONCLUSION

These results will lead to the first experimental mapping of the H + /OH - distribution on the aggregate's surfaces as a function of distance.

Keywords: micelle, pH probe, catalytic peptides

08051 - Poster Session

AA.03 - SOLUBILIZATION OF BIOMIMETIC MEMBRANES BY DETERGENTS WITH DIFFERENT PHYSICAL-CHEMICAL CHARACTERISTICS

Mariana Silva e Silva de Oliveira ¹, Karin Riske¹

¹Biophysics, Federal University of Sao Paulo (SP, Brazil)

INTRODUCTION

Detergents are used to solubilize biomembranes and extract their components. Previously, our group has shown that, in giant unilamellar vesicles (GUVs) of ternary biomimetic compositions, the detergent Triton X-100 promotes Lo/Ld phase separation followed by solubilization of the Ld phase only, while Lo phase remains unimpaired.

OBJECTIVES

The present work extends the study to detergents with different physical-chemical characteristics: Triton X-165, Dodecyl Maltoside (DDM), C10E5, Octyl Glucopyranoside (OG), Tween 20, CTAB, SDS and Chaps. The membrane compositions studied were Ld phase (pure POPC), Lo phase (SM:cholesterol 7:3), and the ternary mixture POPC:SM:cholesterol 2:1:2. We aim to study different composition vesicles solubilization by different detergents and analyze their interactions.

MATERIALS AND METHODS

The solubilization profile of each detergent was followed by turbidity measurements on large unilamellar vesicles (LUVs). Then, optical microscopy was used to observe GUVs in the presence of the detergents.

DISCUSSION AND RESULTS

Except for CTAB and SDS, all detergents could completely solubilize vesicles in the Ld phase. On the other hand, Lo vesicles were completely or partially insoluble to all detergents. Two different interactions with Ld phase were determined: increase in GUV surface area and turbidity before solubilization (C10E5, TX165, Tween 20 and OG) and vesicle rupturing/bursting (DDM, Chaps, CTAB and SDS) that pairs with no increase on solubilization profile acquired by turbidity measurements.

CONCLUSION

Detergents that promote increase in GUV surface area and turbidity before solubilization apparently can insert on Ld phase and make flip-flop across the membrane. The other detergents are not able to make flip-flop, so their insertion on Ld phase causes tension on the membrane curvature, which is alleviate by bursting. This might be due to polar head size and charge.

Keywords: Biomimetic Membrane, Detergents, Solubilization

Supported by: CNPq

08333 - Poster Session

AA.04 - A biomimetic device combining microfluidics with nanotechnology allows studying the adhesion of erythrocytes to blood vessels.

Nicolás Andrés Saffioti ¹, María Florencia Leal Denis², Vanesa Herlax³, Pablo Schwarzbaum², Diego Pallarola¹

¹Instituto de Nanosistemas, Universidad Nacional de San Martín, (Argentina)

²Instituto de Química y Fisicoquímica Biológicas, CONICET/UBA, (Argentina)

³Instituto de Investigaciones Bioquímicas de La Plata, Universidad Nacional de La Plata, (Argentina)

INTRODUCTION

Erythrocytes under pathological conditions undergo eryptosis, a process characterized by biochemical and morphological changes such as phosphatidylserine (PS) exposure to the plasma membrane external layer. Eryptotic erythrocytes adhere to the endothelial cells (ECs), which may be important in the pathology of bacterial infections and congenital diseases like sickle cell disease. Externalized PS can bind to receptors expressed on ECs under pathological conditions.

OBJECTIVES

To understand the adhesion mechanism, we designed a device combining microfluidics with nanostructured surfaces to mimic the capillary architecture and the expression of adhesive molecules by the activated ECs.

MATERIALS AND METHODS

Microfluidic chips were prepared in PDMS using molds fabricated by photolithography. Nanostructured surfaces were synthesized by block copolymer lithography and consisted of a glass surface covered with 7 nm diameter gold nanoparticles (AuNPs), arranged in a quasi-hexagonal array. The microfluidic chip was adhered to the nanostructured surface by an O₂ plasma treatment. The AuNPs were functionalized with a polyethylene glycol chain (PEG) that binds to the AuNPs by a thiol at one end and has a nitriloacetic group (NTA) at the other end. The NTA binds proteins expressing a His-Tag. The surface not occupied by AuNPs was covered with PLL-g-PEG.

DISCUSSION AND RESULTS

We corroborated the specific AuNPs functionalization by quartz microbalance and fluorescence microscopy using a GFP-His Tag. Then, we studied the erythrocytes adhesion to a device functionalized with Annexin V-His Tag at different flows. Two conditions that promote eryptosis, incubation at 50° C or treatment with ionomycin, significantly increased the adhesion of erythrocytes at flows up to 1.5 dyn/cm ² , in comparison with untreated erythrocytes. However, eryptotic erythrocytes also showed adherence to a device functionalized with a PEG lacking NTA groups. This indicates that erythrocytes adhesion may not be mediated only by PS receptors.

CONCLUSION

Future experiments will test the erythrocytes adhesion elicited by proteins usually expressed by the activated ECs.

Keywords: endothelium, nanostructured, biomimetic

Supported by: FONCYT (PICT-2019 03218), CONICET, The Company of Biologists (travel grant), UBACYT 20820160401330BA.

08928 - Poster Session

AA.05 - From Langmuir monolayers to miniemulsions: an approach to understand the liquid/liquid interfaces

Milagro Mottola ^1,2, María Angélica Perillo^1,2

¹Depto. de Química. Cátedra de Química Biológica, Universidad Nacional de Córdoba, Facultad de Ciencias Exactas, Físicas y Naturales. (Córdoba, Argentina)

²CONICET, Instituto de Investigaciones Biológicas y Tecnológicas (IIBYT) (Córdoba, Argentina)

Lipid miniemulsions (ME) are oil in water (O/W) dispersions stabilized by an interfacial layer of a surfactant and are systems commonly used to encapsulate, maintain, and release molecules of pharmacological interest. In this context, Langmuir monomolecular films at the liquid-liquid interface can be used as experimental models to investigate the dynamic behavior of surfactants at the oil/water (O/W) interface in ME. We have used this technique to characterize the composition and thermal behavior of monomolecular layers of L-α-phosphatidylcholine (EPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at the Vaseline/water interface (VAS/W) by using a homemade Langmuir interfacial trough. Besides, we have studied the interfacial behavior, at the air/water interface (A/W), of VAS/DPPC pseudo-binary mixtures with different VAS/PC molar ratios. This combined analysis was carried out with the aim of theoretically predict and design stable formulations of ME. From our study of adsorption isotherms of DPPC at the VAS/W interface, we obtained the saturation equilibrium pressure of DPPC (πeq, sat ∼ 27.3 ± 0.1 mN / m), which could be interpolated in the π-MMA isotherm at the VAS/W interface to calculate the minimum area of DPPC (MMAmin) corresponding to the equilibrium pressure of DPPC at this interface. With the aid of geometric calculations, the MMAmin could be used as a starting point to estimate a suitable DPPC:VAS ratio to formulate stable miniemulsions particles according to the desired size and concentration. Controlling the VAS:DPPC molar ratio, the theoretical predictions made from the Langmuir film model were satisfactory for the synthesis of particles with average diameters in the range of 100 nm to 400 nm and with low DPPC concentrations (0.01mM) in the global system. The ME obtained were characterized by DLS and were stable at least for one month in suspension.

Keywords: Langmuir films, nanoemulsions, liquid/liquid interface

Supported by: CONICET, FONCYT, SeCyT-UNC.

08788 - Poster Session

AA.06 - Impact of macromolecular crowding on the mesomorphic behaviour of lipid self-assemblies

Agustin Mangiarotti¹, Luis Alberto Bagatolli¹

¹Biofísica, Instituto de Investigación Médica Mercedes y Martín Ferreyra - INIMEC (CONICET)- Universidad Nacional de Córdoba, Córdoba, Argentina (, Argentina)

INTRODUCTION

We established that intracellular water dynamics in yeast displaying oscillatory glycolysis, is coupled to, and oscillates synchronously with, the concentration of ATP. These cytosolic water oscillations propagate to the membrane interface. According to the Association Induction Hypothesis (AIH), this phenomenon involves changes in the structure of intracellular proteins. We hypothesize that metabolic changes may regulate mesomorphic changes in lipid self-assemblies via changes in the activity of intracellular water.

OBJECTIVES

We attempt to test in vitro, whether structural features of polymers may influence the structure of lipid self-assemblies via changes in water activity.

MATERIALS AND METHODS

We used LAURDAN fluorescence and Raman measurements. In our experiments the polymers are not in direct molecular contact with the membranes (using a dialysis bag).

DISCUSSION AND RESULTS

Water dynamics seeing by LAURDAN in DOPC bilayers is differentially regulated by the presence of crowded suspensions of different proteins (HSA, IgG, Gelatin) and PEG. Specifically, we found that the extent of water dynamics correlates with an increased fraction of randomly oriented configurations in the polymers, as Gelatin>PEG>IgG>HSA. Also we found that structural transitions from globular to extended conformations in proteins induced lamellar to non-lamellar phase transitions in mixtures of DOPC and monoolein. In addition, Raman experiments show that high proportions of randomly oriented conformations display increased fractions of tetracoordinated water, a configuration that is dominant in ice. This indicates a greater capacity of this extended polymers for polarizing water and consequently reducing its chemical activity.

CONCLUSION

This effect is in line with the tenets of the AIH, which predicts a long-range dynamic structuring of water molecules via their interactions with proteins showing extended conformations. Our results suggest a crucial role of water in promoting couplings between structural changes in macromolecules and supramolecular arrangements of lipids. This mechanism may be of relevance to cell structure/function when the crowded nature of the intracellular milieu is considered.

Keywords: lipid polymorphism, macromolecular crowding, membrane hydration

08096 - Poster Session

AA.07 - Micellar catalysis of the reaction of 4-nitro-naphthalimides with thiols. Quantitative analysis and analytical applications in biology

Iolanda Midea Cuccovia ¹, Vanessa dos Santos Martins¹, Eduardo triboni Triboni², João Baptista Sargi Bonilha³, Larissa Martins Gonçalves¹, Laura Mortara¹, Larissa Anastácio da Costa Carvalho¹, Caroline Lacerda Dutra¹, Flavia Carla Meotti¹, Mario Jose Politi¹, Hernan Chaimovich¹

¹Biochemistry, Chemistry Institute, USP (São Paulo, Brasil)

²Chemistry Engineering, Escola de Engenharia de Lorena-USP (São Paulo, Brasil)

³Chemistry, Faculdade de Filosofia, Ciencias e Letras, Ribeirão Preto, USP (São Paulo, Brasil)

INTRODUCTION

Cysteine (Cys), a reactive amino acid, is essential in proteases, phosphatases, in peptides such as glutathione (GSH) and may also maintain protein structures through disulfide bonds. Thiols react with 4-Nitro-Naphthalimides displacing the nitro group and producing fluorescent compounds. Micelles can accelerate different reactions and are a convenient alternative to organic solvents for the solubilization of hydrophobic compounds.

OBJECTIVES

Here we describe the aromatic nucleophilic substitution reaction of the -NO2 group of 4-nitro-N-ethylene-N, N´-dimethyl, N´´-hexadecyl ammonium-1,8-naphthalimide bromide, 4-nitro-NEHN, and 4-nitro-n-butyl-naphthalimide, 4-nitro-NBN, by Cys, GSH, hexadecyl-cysteinamide (HCys), and the enzyme peroxiredoxin 2, PRDX2, in the presence of N, N, N-trimethyl-N-hexadecyl ammonium chloride, CTAC, micelles and analyze the effect of micelles on the reaction rates (kψ) and the dissociation constant of the thiols (pKa).

MATERIALS AND METHODS

Reaction rates, quantification of SH groups and pKas were determined by UV and fluorescence.

DISCUSSION AND RESULTS

The pKa of all thiols decreased as a function of [CTAC] to a minimum and increased at higher detergent concentration. CTAC micelles accelerated all reactions to a maximum in kψ. Micellar rate enhancement reached ca. 2.5 x 10 ⁷ for the reaction of HCys with 4-nitro-NEHN at pH 5.5 and 2.5 x 10 ⁶ with 4-NBN at the same pH. The main factors leading to the observed effects were pKas changes, substrates concentrations, and changes in the intrinsic micellar rate constant (km). Product fluorescence allowed SH´s quantification. The thiol detection limit was 1 x 10 ^-6 M at pH 7.4. The addition of H2O2 did not change the fluorescence intensity.

CONCLUSION

Micelles of CTAC strongly catalyzed the reaction of thiols and 4-nitro-naphthalimides. 4-nitro-NBN and 4-nitro-NEHN are excellent probes for thiol quantification, and the products are stable in oxidant conditions.

Keywords: 4-Nitro-naphthalimides, catalysis, micelles

Supported by: FAPESP, CNPq, INCT-FCx, NAP-FCx

08888 - Poster Session

AA.08 - Topographic Analysis of Annexin A6-Proteoliposomes by Atomic Force Microscopy

Ekeveliny Amabile Veschi ¹, Maytê Bolean¹, Ana Paula Ramos¹, Pietro Ciancaglini¹, Slawomir Pikula², Thierry Granjon³

¹Chemistry, University of São Paulo (, Brazil)

²Biochemistry, Nencki Institute of Experimental Biology (, Poland)

³Chemistry and Biochemistry, University of Lyon, Lyon (, France)

INTRODUCTION

The liposomes are the most used membrane mimetic model. They have been developed to facilitate the study of the physicochemical properties of biological membranes and the interaction between lipids and proteins. Annexin A6 (AnxA6) is a protein found in three distinct regions of the matrix vesicle (MVs) membrane. These vesicles provide a suitable microenvironment for mediation of bone mineralization.

OBJECTIVES

The present study aimed to evaluate whether AnxA6 interacts with liposomes composed by lipids present in MVs´ membrane- (dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS) and cholesterol (Chol)), using atomic force microscopy (AFM).

MATERIALS AND METHODS

For this, liposomes (10 mg. mL ^-1 ) and proteoliposomes harboring AnxA6 (1:100 protein:lipid molar ratio) composed of pure DPPC and 5:4:1 (molar ratios) DPPC:Chol:DPPS were prepared. Liposomes and proteoliposomes were fixed using glutaraldehyde, dropped onto the surface of freshly cleaved mica and dried at room temperature. Measurements were performed a Shimadzu SPM-9600 Scanning Probe Microscope operating in tapping phase mode.

DISCUSSION AND RESULTS

Liposomes composed of DPPC and 5:4:1 DPPC:Chol:DPPS were identified as spherical particles with uniform size distribution and a smooth and homogeneous surface without any evidence phase segregation. Conversely, proteoliposomes harboring AnxA6 showed surface irregularities formed by protrusions that appear to agglomerate at various sites. The AFM topographic cross sections revealed that these protrusions were 21.37±4.83 nm wide and 1.15±0.41 nm height (N = 20) for DPPC proteoliposomes, whereas they were 26.97±7.62 nm wide and 2.01±0.89 nm tall (N=22) for DPPC:Chol:DPPS (5:4:1) proteoliposomes. This finding is consistent with the size of AnxA6 dimers or trimers assigned to the V-shaped AnxA6 with two flexible 6 nm-in-length loops.

CONCLUSION

AFM images showed protrusions that suggest dimeric or trimeric AnxA6 domains on the surface of proteoliposomes, revealing that AnxA6 may adopt different conformations upon interaction with the lipid membrane.

Keywords: Annexin A6, proteoliposome, atomic force microscopy

Supported by: CAPES

08212 - Poster Session

AA.09 - Matrix vesicle biomimetics carrying Annexin A5 and Alkaline Phosphatase bind to native collagen produced by human smooth muscle cell transdifferentiated in osteo/chondrocyte cells.

Maytê Bolean ¹, Benedetta Izzi², Soetkin van Kerckhoven³, Massimo Bottini⁴, Ana Paula Ramos¹, José Luiz Millàn⁵, Marc Hoylaerts⁶, Pietro Ciancaglini¹

¹Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - Universidade de São Paulo (São Paulo, Brasil)

²Department of Epidemiology and Prevention, IRCCS NEUROMED (Pozzilli (IS), Italy)

³Functiemetingen Pneumologie, UZ Leuven Gasthuisberg (Leuven, Belgium)

⁴Department of Experimental Medicine, University of Rome Tor Vergata (Rome, Italy)

⁵Sanford Burnham Prebys Medical Discovery Institute, Sanford Burnham Prebys Medical Discovery Institute (CA, EUA)

⁶Department of Cardiovascular Sciences,, University of Leuven (Leuven, Belgium)

INTRODUCTION

Vascular smooth muscle cells (VSMCs) transdifferentiated ectopically trigger vascular calcifications, contributing to clinical cardiovascular disease in the aging population. AnxA5 and TNAP play a crucial role in (patho)physiological mineralization.

OBJECTIVES

The goal is study the affinity of proteoliposomes harboring AnxA5 and/or TNAP and different types of collagen matrix to simulate the MVs function during the ectopic calcification conditions.

MATERIALS AND METHODS

We performed affinity studies between DPPC and 9:1 DPPC:DPPS-proteoliposomes carrying AnxA5 and/or TNAP and different types of collagen matrix: type I, II, I+III and native collagenous extracellular matrix (ECM) produced from VSMCs with or without differentiation.

DISCUSSION AND RESULTS

AnxA5-proteoliposomes had the highest affinity for collagens, specially for type II. TNAP-proteoliposomes bound poorly and the simultaneous presence of TNAP in the AnxA5-proteoliposomes disturbed interactions between AnxA5 and collagen. DPPC proteoliposomes-AnxA5 affinities for ECM from transdifferentiating cells went up 2-fold compared to that from native VSMCs. The affinities of DPPC:DPPS-proteoliposomes were high for ECM from VSMCs with or without differentiation, underscoring a synergistic effect between AnxA5 and DPPS. Co-localization studies uncovered binding of proteoliposomes harboring AnxA5 or TNAP+AnxA5 to various regions of the ECM, not limited to type II collagen. AnxA5-proteoliposomes showed highest affinities for type II collagen, deposited during chondrocyte mineralization in joint cartilage. In all, TNAP in the lipid/protein microenvironment disturbs interactions between AnxA5 and collagen.

CONCLUSION

These findings arise the hypothesis that TNAP would be cleaved from the MVs membrane just before ECM binding, that such will facilitate MVs anchoring to ECM via AnxA5 interaction. Proteoliposomes as MVs biomimetics are useful in the understanding of mechanisms that regulate the process and essential for the development of novel therapeutic strategies to prevent or inhibit ectopic mineralization. Acknowledgments: FAPESP, CNPq and CAPES.

Keywords: proteoliposomes, collagen, matrix vesicles

Supported by: Capes-PNPD

08944 - Poster Session

AA.10 - A plug-and-play microfluidic device for efficient generation of monodisperse giant unilamellar vesicles

Hiroaki Suzuki ¹, R., Ushiyama¹, K., Koiwai²

¹Dept. Precision Mechanics, Tokyo University of Marine Science and Technology (Tokyo, Japan)

²Laboratory of Genome Science, Tokyo University of Marine Science and Technology (Tokyo, Japan)

Giant unilamellar vesicles, or GUVs, are cell-sized compartments widely used in biological and chemical applications. The monodispersity of GUVs is essential in terms of the quantitative analysis and the experimental reproducibility as biomimetic microreactors. Here we established a highly reproducible plug-and-play microfluidic-based method to generate the monodisperse GUVs without surface treatment[1] of the channel or precise flow-rate adjustment[2]. In this technique, the water-in-oil (W/O) droplets were generated in the microfluidic channel by hydrodynamic flow focusing and transferred across the W-O interface to form thin-shelled water-in-oil-in-water (W/O/W) droplets as a precursor of GUVs. The success rate of the W/O droplets transferring reached nearly 100% by stabilizing the co-flow of oil and water as well as adjusting the curvature of the interface. Owing to interfacial energy minimization, the thin oil layer of the W/O/W droplets was accumulated and detached to form GUVs. We performed a membrane protein insertion assay to confirm the unilamellarity of the GUVs and shrinkage of the GUVs caused by osmotic pressure. The present device can be operated right after bonding the monolithically replicated PDMS channel and plugging tubes. The stable state of the W/O/W droplet generation can be obtained within a short time (1–2 min) so that the amount of inner solution can be reduced down to ~20 μl. GUVs generated by the dewetting of the excess oil can be used as a unilamllar biological compartment as a cell mimic, and the present technology is especially useful to conduct rare or expensive reactions. We believe that this method will become one of the major platforms for artificial cell studies and quantitative biochemical studies which involve the lipid membrane. References [1] Deshpande, S. et. al., Nat. Commun. 2016, 7, 1–9. [2] Deng, N. N. Et al., J. Am. Chem. Soc. 2016, 138 (24), 7584–7591.

Keywords: microfluidics, monodisperse GUVs, bioreactor

Supported by: JSPS grant

08729 - Poster Session

AA.11 - Effects of insecticide acephate on lipid monolayers and bilayers

Luis Fernando do Carmo Morato ¹, Gilia Cristine Marques Ruiz¹, Wallance Moreira Pazin¹, Carlos José Leopoldo Constantino¹

¹Departamento de Física, Universidade Estadual Paulista – FCT/UNESP (São Paulo, Brasil)

INTRODUCTION

Due the large production of agricultural products in Brazil, the use of pesticides, such as herbicides, fungicides and insecticides, is highly widespread in the country. The irregular use of these chemicals (above the maximum limit or used in crops other than the allowed) is a concern, once they can be harmful to the environment and human health. Acephate is an insecticide of the organophosphorus family and it is the most irregularly used in Brazil.

OBJECTIVES

Once the main effect of this pesticide on insects and mammals is the inhibition of the enzyme acetylcholinesterase, it was chosen in order to assess its effects on lipid monolayers and bilayers as mimetic systems of the cell membrane.

MATERIALS AND METHODS

Three types of mimetic system were chosen: the Langmuir films (monolayers, analyzed by π-A isotherms), formed by DPPC (zwitterionic lipid), DODAB or DPTAP (cationic lipids); the giant unilamellar vesicles (GUVs - bilayers, analyzed by phase contrast microscopy), formed by hydration of DPPC films and hydration followed by vortex of DODAB or DPTAP films; and the large unilamellar vesicles (LUVs - bilayers, characterized by dynamic light scattering (DLS) and zeta potential), formed by the extrusion of the DPPC, DODAB and DPTAP multilamellar vesicles previously provided.

DISCUSSION AND RESULTS

The Langmuir films show that the acephate affects the cationic monolayers (DODAB and DPTAP) in concentration equal to 10 ^-4 M, while the GUVs did not show any morphological effects in the presence of acephate at concentration of 0.8x10 ^-4 M. On the other hand, the cationic LUVs in the presence of acephate at concentrations of 0, 0.1, 10, 25, 50, 75 and 100 μM show an increasing on their diameter and a diminishing of the superficial charge, mainly at the higher concentrations.

CONCLUSION

Although the pesticide did not change the GUVs morphology, the results indicate that the acephate affects preferably the cationic monolayers and bilayers (LUVs).

Keywords: acephate, vesicles, lipids

Supported by: FAPESP (2018/01984-8), CNPq, CAPES

08277 - Poster Session

AA.12 - Comparative study of the native and mutant TNAP in in vitro biomineralization

Bruno Zoccaratto Favarin ^1,2, Ana Paula Ramos², Antonio Costa FIlho¹, Pietro Ciancaglini²

¹Physics, University of Sao Paulo (São Paulo, Brasil)

²Chemistry, University of Sao Paulo (São Paulo, Brasil)

³Human genetics program, Sanford Burnham Prebys Medical Discovery Institute (USA, USA)

INTRODUCTION

Osteoblasts are cells responsible for the bone biomineralization in a process mediated by the release of matrix vesicles (MVs). The MV´s membrane are enriched in Tissue Non-specific Alkaline Phosphatase (TNAP) when compared to the plasma membrane. TNAP is a phosphomonohydrolase enzyme able to produce inorganic phosphate (Pi) through PPi or ATP hydrolysis, thus initiating biomineralization.

OBJECTIVES

We also measured the ability of native and mutant TNAP either in solution or anchored to DMPC-liposomes, in propagating mineralization in vitro.

MATERIALS AND METHODS

In the present study, specific amino acids at positions Pro-244, Pro-307 or Ala-420 in native TNAP were replaced by cysteines for spin labeling and subsequent ESR analyses. Since TNAP is inserted in the membrane through a GPI anchor, we evaluated the influence of lipid microenvironments, which could cause conformational and functional changes in the properties of TNAP

DISCUSSION AND RESULTS

The ability of the proteoliposomes to induce biomineralization in vitro was assessed by turbidimetry changes at 340nm/μg TNAP. Circular dichroism was used to confirm the structure of the mutants while kinetic studies were carried out to characterize phosphate production. We observed that in vitro mineral propagation for the TNAP-244 mutant and for the native TNAP was higher both in solution and into proteoliposomes. This finding agreed with the increase in the values of kcat/K0.5 (higher for TNAP in solution) previously obtained. FTIR results revealed changes in the intensity of the PO4-3/C=0 bands similar to apatite crystals for proteoliposomes containing TNAP-244 and TNAP-420. Unfortunately, ESR signals were of low intensity hindering any further analysis

CONCLUSION

However, with the aid of other techniques, we proved that the mutants with cysteines farther from the catalytic site and from the GPI anchor yielded higher mineralization propagation and higher catalytic efficiency for ATP hydrolysis. Finally, the lipid bilayer present in the proteolipossome were able to enhance in vitro mineral propagation.

Keywords: TNAP, Proteoliposomes, Matrix vesicles

Supported by: FAPESP (2016/21236-0, 2018/12092-0, 2019/08568-2), CNPq and CAPES.

08210 - Poster Session

AA.13 - Localization of Annexin A6 in Matrix Vesicles During Physiological Mineralization

Ekeveliny A. Veschi¹, Mayte Bolean¹, Agnieszka Strzelecka‐Kiliszek², Joanna Bandorowicz-Pikula², Slawomir Pikula², Thierry Granjon³, Mebarek Saida³, David Magne³, Ana Paula Ramos¹, José Luis Millán⁴, Rene Buchet³, Massimo Bottini⁵, Pietro Ciancaglini ¹

¹Dep. de Química, Universidade de São Paulo (São Paulo, Brasil)

², Nencki Institute of Experimental Biology (Warsaw, Poland)

³Chimie Biochimie, Universite Lyon 1 (, France)

⁴, Sanford Burnham Prebys Medical Discovery Institute (CA, USA)

⁵Dep. of Experimental Medicine, University of Rome Tor Vergata (, Italy)

INTRODUCTION

Annexin A6 (AnxA6, ~68 kDa) is the largest member of the annexin family of proteins present in matrix vesicles (MVs). MVs serve as nucleation sites for crystal deposition during physiological mineralization. Biochemical analyses revealed that AnxA6 is present in three distinct regions of the MV membrane. The first, corresponds to Ca2+-bound AnxA6 interacting with the inner leaflet of MV membrane, the second, is AnxA6 localized on the surface of the outer leaflet of MV membranes, and the third, is AnxA6 inserted in the membrane’s hydrophobic bilayer and co-localized with cholesterol.

OBJECTIVES

Here, we assess the localization of AnxA6 in the MV membrane using native MVs and MVs biomimetics.

MATERIALS AND METHODS

Using monolayers and proteoliposomes composed of either dipalmitoylphosphatidylcholine (DPPC) to mimic the outer leaflet of the MV bilayer or a 9:1 DPPC:dipalmitoylphosphatidylserine (DPPS) mixture to mimic the inner leaflet, we confirmed that AnxA6 interacts differently with MV membranes in agreement with the biochemical data.

DISCUSSION AND RESULTS

Thermodynamic analysis based on the measurement of the surface pressure exclusion, enthalpy and phase transition cooperativity (Δt1/2) showed that AnxA6 interacts with both the lipid models and that this interaction increases in the presence of cholesterol. The selective recruitment of AnxA6 by cholesterol molecules was observed in MVs as probed by the addition of methyl-β-cyclodextrin (MβCD). AnxA6-lipid interaction was Ca2+-dependent as evidenced by the greater increase in surface pressure in negatively charged 9:1 DPPC:DPPS monolayers and a larger decrease in enthalpy in 9:1 DPPC:DPPS proteoliposomes caused by the addition of AnxA6 in presence of Ca2+ compared to zwitterionic bilayers composed of DPPC.

CONCLUSION

We conclude that the different localizations and ways of interaction of AnxA6 with the lipid membrane suggest distinct functions in MV during biomineralization.

Keywords: Annexin A6, Proteoliposomes, Biomineralization

Supported by: FAPESP, CNPq and CAPES

08857 - Poster Session

AA.14 - Modulating membrane shape and mechanics by light

Mina Aleksanyan ¹, A, Grafmueller¹, F. Crea ², V. Georgiev¹, J. Heberle¹

¹Science Park, Max Planck Institute of Colloids and Interfaces (Potsdam, Germany)

²Department of Physics, Freie Universitaet Berlin (Berlin, Germany)

Over the years, light has been widely used as an effective trigger in biotechnology to interrogate biological systems and provide a conditional control over the complex cellular processes. Of particular advantage are the reversibility, physiological compatibility and high spatiotemporal precision of photo-induced processes. Understanding the fundamental biophysics of light-triggered changes in bio-systems is crucial for cell viability and optimization of clinical applications of light-induced processes in optogenetics and photopharmacology. Here, we employ cell-sized giant unilamellar vesicles (GUVs) to investigate light-triggered changes on the material properties of membranes doped with azobenzene-phosphatidylcholine (azo-PC ) as a photoswitch. In particular, we examined light-triggered vesicle area change, reversibility and kinetics of photoswitching and membrane bending rigidity. We show that light can be used to manipulate the shape and mechanics of these synthetic cells. Light-induced changes in vesicle area were quantified by employing vesicle electro-deformation. Our studies demonstrated membrane area increase due to cis photoisomerization of azo-PC which is consistent with results from molecular dynamics simulations. At high azo-PC fractions (>50 mol%), large and complex shape transformations were observed. Photoswitching was reversible but showed faster kinetics from cis to trans isomerization. Trans-to-cis isomerization of azo-PC rendered the membrane softer. With increasing fractions of trans azo-PC the GUV membrane stiffens, while increasing cis-azo-PC fraction softens the membrane. Our results demonstrate that membrane mechanics could be easily controlled for azo-PC containing membranes through light.

Keywords: biomembranes, biophysics, photoswitchable lipids

08734 - Poster Session

AA.15 - Self-organization of protocell accompanied by micro phase-segregation in a crowding solution

Fumika Fujita ¹, Hiroki Sakuta¹, Kanta Tsumoto², Koichiro Sadakane¹, Takahiro Kenmotsu¹, Kenichi Yoshikawa¹

¹Faculty of Life and Medical Sciences, Doshisha University (, Japan)

²Faculty of Engineering, Mie University (, Japan)

INTRODUCTION

Living cells on the Earth maintain their lives through the self-organization of their structure under the crowding conditions of biopolymers on the order of 30 - 40 weight%. However, the underlying physico-chemical mechanism why and how lives utilize the crowding condition still remains as a matter of unsolved problem.

OBJECTIVES

In this study, we aimed to unveil the fundamental problem how intracellular order, stability and functions are controlled by creating protocells under crowding environment. We report self-generation of a cell-like structure for water/water microdroplets in a crowding polymer solution.

MATERIALS AND METHODS

When the aqueous solution of poly(ethylene glycol) (PEG) and dextran (DEX) is mixed in the two-phase region near the binodal line, phase separation occurs and microdroplets of several 10 - 100 μm are formed.

DISCUSSION AND RESULTS

We found that long DNA (λ-DNA 49kbp) are entrapped spontaneously inside the droplets. In addition, we report the generation of a cell-sized droplet covered with phospholipid membrane, containing DNA molecules in a self-organized manner. We evaluated the stability of the droplets by experimenting with the time variation of the droplets. Compared to the control droplets, the droplets with DNA and lipid membranes were found to be more stable. Such experimental observations indicate that the droplets covered by lipid are rather stable over several hours, suppressing the fusion between the droplets.

CONCLUSION

Based on these experimental observations on the spontaneous formation of stable cell-like structure, we propose a novel working hypothesis on the generation of primitive cell on the Earth.

Keywords: aqueous two phase separation, cell model, self-organization

08961 - Poster Session

AA.16 - Lipid hydroperoxide impacts in the lipid bilayer structure and decreases the bending modulus

Gustavo Scanavachi Moreira Campos ¹, A. Coutinho^2,3,4, A. Fedorov^2,4, Manuel Prieto^2,4, A. Melo^2,4, Rosangela Itri¹

¹Institute of Physics, University of São Paulo (São Paulo, 05508-090, Brazil)

²iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa (Av. Rovisco Pais, Lisboa, Portugal)

³Dep. Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa (Campo Grande, Lisboa, Portugal)

⁴Associate Laboratory i4HB—Institute for Health and Bioeconomy , Instituto Superior Técnico, Universidade de Lisboa (Av. Rovisco Pais, Lisboa, Portugal)

It is well known that oxidized lipids play an important role in diseases and cell death. Here, we studied how the presence of a lipid hydroperoxide derived from POPC, the lipid POPC-OOH, impacts in the lipid bilayer by coupling advanced fluorescence techniques and small-angle X-ray scattering (SAXS). SAXS data analysis pointed out the decrease of lipid bilayer bending modulus, which increased the swelling between different stacked bilayers. Furthermore, we used the fluorescent probes TMA-DPH and Laurdan to investigate the impact of the POPC-OOH in the apolar/polar interface. The TMA-DPH time resolved fluorescence analysis showed a decrease in the mean fluorescence lifetime in function of the hydroperoxide concentration, revealing a higher content of water molecules at the membrane interface. Also, there was an increase of the microviscosity in the TMA-DPH vicinity. Laurdan relaxation process in pure POPC-OOH membranes indicated a higher viscosity and hydration near the -OOH group. In conclusion, our results reveal that POPC-OOH alters the membrane order, increase hydration at the membrane interface, increase the microviscosity and decrease the bending rigidity.

Keywords: biophysics, oxidized lipid, x-ray scattering

Supported by: CNPq, CAPES, Ciências sem Fronteiras - CAPES, FAPESP, FCT

08861 - Poster Session

AA.17 - Can inhibitors of ATPase/Alkaline Phosphatase modulate the activity of Matrix Vesicles released by Chondrocytes?

Heitor Gobbi Sebinelli ¹, Bruno Z Favarin¹, Marcos Antonio Cruz¹, Massimo Bottini², José Luis Millán³, Saida Mebarek Mebarek⁴, Ana Paula Ramos¹, René Buchet⁴, Pietro Ciancaglini¹

¹Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - USP (São Paulo, Brasil)

²Department of Experimental Medicine, University of Rome Tor Vergata (Rome, Italy)

³Human Genetics Program, Sanford Burnham Prebys Medical Discovery Institute (CA, United States of America)

⁴Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Université Lyon 1 (Lyon, France)

INTRODUCTION

Na,K-ATPase(NKA) is physiologic essential for many cells and found in appreciable concentration in the matrix vesicles(MV) membrane[1]. We hypothesized that the NKA-membrane orientation may be used to understand the process of MVs biogenesis. Therefore, rightside/inside-out orientations could reveal whether the vesicles were protruded from the apical cell membrane or if first invaginated and later released and if NKA can be a backup mineralization pathway.

OBJECTIVES

Determine MV and NKA-proteoliposomes activity and ability to mineralize in vitro in the presence of ATPase inhibitors.

MATERIALS AND METHODS

MV released from growth-plate femurs chondrocytes of chicken embryos by collagenase were isolated[2]. Turbidity changes of synthetic cartilage lymph(SCL), 31P-NMR and colorimetric techniques along with inhibitors were used to estimate MV activity and mineralization. Precipitates analyzed by FTIR. DPPC and DPPC:DPPE(1:1w/w) proteoliposomes prepared by co-solubilization[3].

DISCUSSION AND RESULTS

MV of 212nm diameter were used[2] for ³¹ P-NMR and colorimetric techniques estimated the MV activity in the presence of ouabain is ±95%, Levamisole and SBI-450 were more effective because they inhibit TNAP, which is the main phosphatase in MV. The proteoliposomes lipidic compositions are known to distribute different positions of NKA into the membranes. AFM images revealed the formation of protrusions related to the NKA insertion into the liposomes, associated with the height dimensions of the (αβ)-unit orientation[3]. The α-subunit domain, containing the active site of NKA, has a 4nm height, while the β-subunit domain is 8nm high. The height of the protrusions found in DPPC-NKA and DPPC:DPPE-NKA proteoliposomes were 2.1 and 0.5nm, respectively, revealing the exposure of ATP binding site outwards for DPPC:DPPE-NKA. The DPPC-NKA-proteoliposomes preserved activity was 61%, DPPC:DPPE-NKA 91%.

CONCLUSION

Proteoliposomes with reconstituted NKA achieved mineral propagation by ATP hydrolysis and may be helpful to probe the mechanism of MVs mineral propagation. [1]Thouverey et al.(2011) J. Proteomics,74:1123. [2]Buchet et al.(2013) Methods Mol. Biol.1053:115. [3]Sebinelli et al.(2019) Soft matter,15:2737. Funding: FAPESP, CNPq and CAPES

Keywords: Na,K-ATPase, Matrix vesicles, Inhibitors

08176 - Poster Session

AA.18 - OBTAINING, CHARACTERIZING AND RECONSTITUTING THE SALMONELLA ENTERICA TYPHIMURIUM PGTE PROTEASE IN LIPOSOMES: A STUDY OF THE MECHANISM OF RESISTANCE TO ANTIMICROBIAL PEPTIDES

Pamela Jacque de Souza Nascimento da Silva ¹, Vitor Marcelo Siqueira Bueno Brandão de Oliveira¹, Marcelo Ferreira Marcondes Machado¹, Katia Regina Perez ¹

¹Departamento de Biofísica, Escola Paulista de Medicina - Universidade Federal de São Paulo (São Paulo, Brasil)

INTRODUCTION

Multi-resistant bacteria represent one of the most prominent public health problems. Bacteria use to produce proteases to inactivate the host's defense proteins, including antimicrobial peptides. PgtE is an aspartyl proteases present in the outer membrane of Salmonella spp. enterica tiphymurium that cleaves antimicrobial peptides rich in arginine.

OBJECTIVES

Obtaining of the recombinant protein PgtE in its soluble and active form to reincorporate it in liposomes to study the role of the lipid composition in PgtE insertion into lipid bilayer.

MATERIALS AND METHODS

The recombinant PgtE was obtained by cloning its gene from the DNA of the S. tiphy 5535 strain using the vector pET 28a (+) in E. coli BL21 (DE3). To obtain soluble and active form of the protein, the fractions of bacterial membrane were incubated with different detergents and later ultracentrifuged. The solubilization yield was evaluated by SDS-PAGE, protein dosage and the proteolytic activity in the supernatant.

DISCUSSION AND RESULTS

Our results indicate a good level of PgtE expression and a better recovery of the enzyme in its active form using the CHAPS detergent. The solubilized and active PgtE was reincorporated into DPPC or DPPC: DPPG 1: 1 liposomes using the co-solubilization method. The incorporation of the protein in the membrane was evaluated by differential scanning calorimetry (DSC), protein and lipid dosage in the liposomes and assays of proteolytic activity using the FRET method. The DSC tests showed a significant change in the profile of the membrane lipids phase transition thermogram including relevant changes in the ∆H and in the cooperativity of the lipids of proteoliposomes of DPPC: DPPG. In addition, the kinetic assays of PgtE in DPPC:DPPG membranes showed higher proteolytic activity.

CONCLUSION

The presence of the negative charge on the membrane is an important factor in modulating PgtE insertion in the lipid bilayer.

Keywords: antimicrobial resistance, proteases, proteoliposome

08388 - Poster Session

AA.19 - Effect of Annexin A5 in the interaction between DPPS-enriched membranes and Ca2+

Claudio dos Reis Ferreira¹, Marcos Antônio Eufrásio Cruz¹, Ana Paula Ramos¹, Maytê Bolean¹, Pietro Ciancaglini¹

¹Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (São Paulo, Brazil)

INTRODUCTION

The mineralization of bone is a process driven by matrix vesicles (MVs), which produce and release the first mineral particles into the collagenous extracellular matrix. Annexin A5 (AnxA5) is believed to play a crucial role in the mineral nucleation inside MVs influenced by both calcium and DPPS.

OBJECTIVES

We aimed to evaluate the stability of membranes composed of DPPC and DPPS in a medium containing Ca ²⁺ ions, as well as the role of AnxA5 in this phenomenon and its implication for the biomineralization process.

MATERIALS AND METHODS

We assessed the effect of Ca ²⁺ ions on the organization of membranes constituted by pure DPPC, 10%DPPS, 20%DPPS and pure DPPS (numbers depict molar percentage) formed both as bilayers (liposomes, characterized by differential scanning calometry (DSC) and dynamic light scattering (DLS) and monolayers (Langmuir monolayers, characterized by π-A isotherms and fluorescence microscopy)).

DISCUSSION AND RESULTS

Our data revealed that calcium ions in the concentration range of 0.5-2.0 mM induced the formation of DPPS-enriched domains that make liposomes prone to vesicle fusion/aggregation, as observed by DSC and DLS measurements. However, in the presence of AnxA5, these domains are stabilized in such a way that the calcium fusogenic effect is retarded. In fact, fusion was observed only in concentrations higher than 1.0 mM Ca ²⁺ while in the absence of protein it started at 0.5 mM Ca ²⁺ . Microscopy images revealed the formation of small condensed lipid domains for pure DPPC, that became larger when DPPS was present and even larger when AnxA5 was present.

CONCLUSION

We believe that this ternary behavior is central to the mineral nucleation inside the MVs that requires a significant amount of calcium and phosphate ions concentrated on a small region of the membrane, since the negatively charged DPPS-membrane associated to AnxA5 facilitates the nucleation, growth and release of mineral.

Keywords: DPPS, AnxA5, Calcium

Supported by: FAPESP, CNPq, CAPES

08234 - Poster Session

AA.21 - Effect of senescence on the expression of tyrosine hydroxylase and S100B in the Substantia nigra pars compacta of wistar rats (rattus norvegicus

Gabriel Sousa da Rocha ¹, Karina Maia Paiva^1,2, Rodrigo Oliveira Freire^1,2, Marco Aurélio de Moura Freire¹, Débora Lopes Souza ^1,2, Paulo Leonardo Araújo de Góis Morais¹, José Rodolfo Lopes de Paiva Cavalcanti^1,2

¹Departamento de Ciências Biomédicas, Universidade do Estado do Rio Grande do Norte (Rio Grande do Norte, Brazil)

², Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular (PMBqBM-UERN) (, Brazil)

Senescence is the natural aging process in which the living being undergoes a series of changes that make him more fragile and susceptible to pathological states. The brain is responsible for several cognitive, motor and metabolic functions, and over the years the functions are compromised due to the degeneration of neuronal circuits and structures that are coordinated. The senescence causes considerable changes in the substantia nigra pars compacta. This nuclei of dopaminergic neurons is part of a movement modulation circuit, which once damaged, causes a decline in the subject's motricity pattern. S100B is a calcium-binding protein, expressed mainly in astrocytes, which plays several intracellular and extracellular roles, and its release is increased in cases of trauma to the nervous tissue, and for this reason it is very useful as a marker of injury to the nervous system central. The aim of this study was to analyze the effect of senescence on neuronal and glial cells of substantia nigra pars compacta. In this study, 12 male wistar rats were used, 6 young (3 months) and 6 elderly (23 months), and in both groups the brains were removed, sectioned and submitted to immunohistochemistry for S100B and Tyrosine-hydroxylase. Later, the slides were analyzed in an optical microscope. The results show a large degeneration of S100B+ astrocytes in Substantia nigra pars compacta, however there were no statistically significant changes in the density of TH+ neurons in the same region. On the other hand, there was considerable degeneration of the projection fibers, as well as the immunoreactivity of TH+ neurons. We conclude that senescence is related to the anatomical and neurochemical changes observed in our animal group. Therefore, it is important that further studies are carried out with different experimental techniques, analyzing other cell markers, to see if these results can be supported or not.

Keywords: Aging, dopamine, astrocytes

Supported by: CNPq and CAPES.

08665 - Poster Session

AA.22 - Clotrimazole fluidizes phospholipid membranes and localizes at the hydrophobic part near the polar part of the membrane

JUAN CARMELO GÓMEZ FERNANDEZ ¹, Alessio Ausili¹, Ilya Yakimenko¹, José A. Teruel¹

¹Bioquímica y Biología Molecular A, Universidad de Murcia (, España)

INTRODUCTION

Clotrimazole (1-[(2-chlorophenyl)-diphenylmethyl]-imidazole) is an azole antifungal drug belonging to the imidazole subclass that is being widely used in pharmacology and that it may incorporate in membranes.

OBJECTIVES

The objective to understand its interaction with model membranes.

MATERIALS AND METHODS

We have used DMPC and POPC as phospholipids and DSC, and 1H NOESY MAS-NMR and Molecular Dynamic Simulations as studying techniques.

DISCUSSION AND RESULTS

We have studied its interaction with DMPC phospholipid vesicles by using differential scanning calorimetry showing that clotrimazole decreases the temperature of the phase transition and at high concentrations it forms areas of high proportions of clotrimazole. 1H-NMR and 1H NOESY MAS-NMR was employed to investigate the location of clotrimazole in POPC phospholipid membranes. In the presence of clotrimazole all the resonances originating from POPC were shifted upfield but mainly those corresponding to C2 and C3 of the fatty acyl chains suggesting that the clotrimazole aromatic rings preferentially locate near these carbons. In the same way 2D-NOESY measurements showed that the highest cross-relaxation rates between protons of clotrimazole and POPC were with those bound to the C2 and C3 carbons of the fatty acyl chains. After molecular dynamics simulations it was seen that indicating that clotrimazole is located near the top of the hydrocarbon chains phase, the nitrogen atoms of the imidazole ring of clotrimazole being closest to the polar group of the carbonyl moiety. These results are in close agreement with the NMR ones and the conclusion is that clotrimazole is located near the water-lipid interface and located in the upper part of the hydrophobic bilayer.

CONCLUSION

Clotrimazole fluidizes phospholipid membranes and localizes at the hydrophobic part near the polar part of the membrane.

Keywords: 1H NOESY MAS-NMR, clotrimazole, DSC

08080 - Poster Session

AA.23 - A complete membrane fusion system: determining fusion intermediates, kinetics and efficiency

Rafael Bezerra de Lira ^1,2, Rafaela M. Cavalcanti^1,3, Karin do Amaral Riske³, Wouter H. Roos¹

¹Moleculaire Biofysica, Zernike Instituut, Rijksuniversiteit Groningen (Groningen, Netherlands)

²Max Planck Institute of Colloids and Interfaces, (MPICI) (Potsdam, Germany)

³Departament of Biophysics, Universidade Federal de Sao Paulo (Sao Paulo, Brazil)

Membrane fusion is ubiquitous for cells to carry out seemingly unrelated processes such as fertilization and neurotransmission. Fusion occurs through specific intermediates; (i) docking, when the membranes bind, (ii) hemifusion, when lipids in the outer leaflet mix, and (iii) full-fusion (content mixing), when lipids on both leaflets as well as their aqueous contents mix. As a result, the end product is a larger compartment whose dimensions are the sum of the fusing membrane areas. Due to the high complexity, fusion is often studied using spectroscopic methods and reconstituted lipid vesicles, but these systems carry a number of limitations - the inability to detect intermediates, the increase in vesicle area, and the inherent complicated kinetics. Recently, we have developed a reconstituted system based on the fusion of charged large and giant unilamellar vesicles (LUVs and GUVs) that is able to circumvent these limitations (Lira et al., Bioph. J. 2019). It enables the detection of fusion intermediates, efficiency and kinetics in real-time using microfluidic devices. Here, we extended its capabilities by using a combination of independent and quantitative imaging and micromanipulation techniques, allowing us to quantify fusion efficiency, to estimate membrane’s composition upon fusion in real-time and measure membrane mechanics on the level of a single vesicle. The quantification of area increase upon fusion is reported for the first time. The system is extremely efficient, and tens of thousands LUVs fused to a single GUV. Using a double optical tweezer setup coupled to a confocal microscope, we simultaneously detect fusion intermediates and measure the forces associated with the interaction of the fusing membranes, which is in the picoNewton force range. We anticipate that the insights here will help to unravel how regulatory factors mediate fusion in cells as well as it will support the development of highly efficient drug delivery systems.

Keywords: Membrane fusion, fusion intermediates, reconstituted systems

Supported by: University of Groningen, MaxSynBio, Fapesp

08506 - Poster Session

AA.24 - The role of NPP1 in the biomineralization process: In vitro propagation of calcium phosphate minerals.

Luiz Henrique da Silva Andrilli ¹, Bruno Zocaratto Favarin², Ana Paula Ramos¹, Pietro Ciancaglini¹

¹Chemistry Department, University of São Paulo (São Paulo, Brazil)

²Physics Department, University of São Paulo (São Paulo, Brazil)

INTRODUCTION

The process of bone mineralization is mediated by matrix vesicles (MVs) that act as starting sites of hydroxyapatite formation. These vesicles are originated by the membrane budding of mineralization-competent cells with specific composition. Ecto-pyrophosphatase/phosphodiesterase (NPP’s) consists of a large family of enzymes expressed in several mammalian tissue.

OBJECTIVES

Evaluate the behavior of NPP1 when incorporated into liposomes. Evaluate its influence on biophysical parameters in incorporated liposomes. Estimate whether NPP1 has the ability to propagate minerals in vitro.

MATERIALS AND METHODS

The expression of NPP1 was performed as described by Simão et al. 2010. Liposomes consisting of 1.5 mg/mL dipalmitoylphosphatidyl choline (PC) were prepared as described by Bolean et al. 2020. The incorporation of NPP1 into PC-liposomes was performed as described by Favarin, et al. 2019. For in vitro assays, NPP1:PC-proteoliposomes were incubated in SCL buffer, 74.0 mM Tris-HCl buffer (pH 7.4) and 2.0 mM of ATP, for 24h, at 37 ºC.The mineral formation was followed by changes in the turbidity at 340 nm. Infrared spectroscopy was used to investigate the chemical composition of the minerals precipitated.

DISCUSSION AND RESULTS

The mean diameter of the NPP1:PC-proteoliposomes was 115.8 with a polydispersivity index PI of 0.22, while 101.2 nm and PI 0.08 were obtained for the liposomes, indicating heterogeneity in the enzyme incorporation. Calorimetric analysis revealed a ΔHtrans of 4.3 and 5.8 kcal/mol for the NPP1:PC-proteoliposomes and PC-liposomes, respectively. Tc close to 39.8 ºC and t1/2 of 4.1 were obtained for the proteoliposomes.NPP1:DPPC-proteoliposomes induced mineral propagation only after incubation with either PS-CPLX or in the absence of nucleators, as indicated by increase in the turbidity close to 53 and 45%, respectively.

CONCLUSION

We conclude that NPP1 is present in the studied liposomes, as well as affecting the thermodynamic properties of these liposomes. The in vitro mineralization data suggests that NPP1:DPPC-proteoliposome supports the propagation of phosphate minerals in a biomineralization model.

Keywords: Biomineralization, Liposomes, Proteoliposomes

Supported by: CNPq

08588 - Poster Session

AA.25 - Interaction of metallic nanoparticles with DOPC vesicles by dynamic light scattering

Cibely da Silva Martin Sonvesso ¹, Carlos José Leopoldo Constantino¹

¹Física, Universidade Estadual Paulista (SP, Brasil)

INTRODUCTION

Nanomaterials of all sorts have been widely explored in scientific studies, and new properties and applications are constantly being discovered. In biotechnology, including biomedical applications, the nanomaterials stand out to integrate a new generation of cell probes or carriers for drug delivery, especially because of their small size and tunable surface properties. Thus, is important to evaluate the interaction of nanoparticles with the membranes component to understand the interaction mechanism.

OBJECTIVES

Thus, in this work, we evaluated the interaction of silver nanoparticles (AgNp), silica-coated silver nanoparticles (AgNp@SiO2), and gold nanoparticles (AuNp) with the DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), a phospholipid present in the cell membrane.

MATERIALS AND METHODS

Thus DOPC large unilamellar vesicles (LUVs) with ~130 nm (biomimetic system) were exposed to 200 μL of AgNp, AgNp@SiO2, or AuNp colloid for 180 minutes and evaluated by dynamic light scattering (DLS).

DISCUSSION AND RESULTS

The variation of DOPC LUVs sizes was performed in function of intensity, volume, and number percentage. A significant decrease in the sizes, as well as the number of vesicles in the system, was observed only for the AgNp up to 90 minutes. A small variation was observed to AgNp@SiO2 up to 90 minutes, which can be scribed to AgNp residues present in the AgNp@SiO2 colloid, once the AgNp was applied as a precursor in the nanoparticle synthesis. No changes were observed for DOPC LUVs in presence of AuNp. The results suggesting a high surface reactivity for AgNp onto DOPC lipid, which promotes a break in DOPC bilayer. The latter can be related to the action mechanism as an antimicrobial agent. On the other hand, the SiO2 coat decreases the surface reactivity, and as well as AuNp can be classified as biocompatible nanoparticles

CONCLUSION

The DLS measurements showed to be an important tool for analysis of the nanoparticle interaction at the biomimetic system

Keywords: pesticides, surface properties, vesicles

Supported by: FAPESP

08926 - Poster Session

AA.26 - Characterization of lipid membranes fluidity in Bacillus subtilis adaptive response to cold shock.

Yenisleidy de las Mercedes Zulueta Díaz ¹, Daniela Albanesi¹, Diego de Mendoza¹

¹Institute of Molecular and Cell Biology of Rosario (IBR), Department of Microbial Physiology, Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario (Rosario, Argentina)

Homoviscous adaptation in Bacillus cells upon a decrease in the ambient growth temperature occurs by an increment in the proportion of low-melting-point fatty acids in the membrane lipids. The short- term adaptation involves desaturating the acyl chains of their membrane phospholipids. The temperature dependence of unsaturated fatty acids (UFAs) biosynthesis relies on the transcriptional control of the des gene, which codes for the sole desaturase of this microorganism, Δ5-Des, and is upregulated upon a cold shock. The increased expression of des in response to low temperatures is strictly controlled by a canonical two-component system constituted by the histidine kinase DesK and its cognate response regulator DesR. This regulatory pathway has been dissected at the genetic, biochemical and structural levels. However, the membrane biophysical changes regulating this pathway remain poorly studied. Therefore, the main objective of this work is to characterize and determine parameters associated with lipid membrane fluidity changes in Bs, as well as with model membranes with a similar composition, upon temperature variations. In order to obtain membranes with different lipid composition we resorted to a set of Bs strains. A wild type strain exhibits 2 % UFAs at 37ºC and 8 %UFAs at 25ºC in the acyl chains of membrane phospholipids. Overexpression of the des gene (Des+ strain) leads to membranes containing up to 12% and 20% UFAs at 37ºC and 25ºC, respectively. In contrast, a mutant strain lacking the des gene (Des-) is unable to produce UFAs. Lipid membranes of these strains were extracted and purified. We have determined quantitative parameters associated with membrane fluidity by fluorescence spectroscopy and microscopy techniques. These studies provide the first step towards the biophysical characterization of B. subtilis plasma membrane adaptation upon a temperature decrease. The information obtained here could be extended to other bacteria and signaling pathways.

Keywords: Bacillus subtilis, lipid membranes fluidity, Fluorescence Spectroscopy

Supported by: This work was supported by the Consejo Nacional de Investigaciones Científcas y Técnicas (CONICET). Y.M.Z.D. is a CONICET fellow.

08185 -

AA.28 - The Protective Effect of Artepillin C in Healthy Model Membrane Structure Against Reactive Oxygen Species

Wallance Moreira Pazin ¹, Marcelo José dos Santos Oliveira¹, Pedro Henrique Benites Aoki², Carlos José Leopoldo Constantino¹

¹Department of Physics, São Paulo State University, School of Technology and Applied Sciences (São Paulo, Brasil)

²Department of Biotechnology, São Paulo State University, School of Sciences, Humanities and Languages (São Paulo, Brasil)

INTRODUCTION

Artepillin C is the majority compound found in Brazilian green propolis, a well-known product used for disease and antioxidant prophylaxis. Independent of its protonation state, artepillin C interacts with model membranes, either by disrupting the lipid organization when in neutral state (acidic pH) or only by interacting preferentially in the surface when in deprotonated state (physiological pH), without affecting structurally the membrane. The latter enforces the advantage of using Artepillin C against lipid peroxidation caused by free radicals, which is a first step before degenerative diseases.

OBJECTIVES

The protective effect of artepillin C against lipid peroxidation was evaluated by the analysis of the surface pressure (SP) stability of Langmuir monolayers, after inducing reactive oxygen species (ROS) generation by photoactivation of erythrosin in the presence of model membranes.

MATERIALS AND METHODS

Lipid films were formed of dioleoylphophatidylcholine (DOPC). The pH of the system was controlled by using a Hepes buffer solution at 10 mM + 150 mM NaCl. Langmuir monolayers were formed after spreading 0.5 mg/mL of DOPC onto the subphase containing erythrosin and/or artepillin C (10 μM), and the SP stability analyses were performed when the lipid film reaches 30 mN/m, keeping the trough area constant over data acquirement. Erythrosin was photoactivated by using a LED source at 530 nm.

DISCUSSION AND RESULTS

Without photoactivation, the SP of the lipid film has decreased near 10% for all system, independent of the presence or the absence of erythrosin and artepillin C. When erythrosin was photoactivated, the ROS generation lead the lipids to a lysis after peroxidation, increasing the area per lipid in the film and, consequently, leading the SP for even lower values.

CONCLUSION

The antioxidant potential of artepillin C and its interaction with Langmuir monolayer preclude the lipid peroxidation caused by the ROS.

Keywords: Artepillin C, Model Membranes, Lipid Peroxidation

Supported by: CAPES; CNPq; FAPESP

08396 -

AA.30 - Triglyceride lenses at the air-water interface as a model system for studying the initiating stage in the biogenesis of lipid droplets

Benjamín Caruso ¹, Natalia Wilke², María Angélica Perillo¹

¹CONICET-UNC, Instituto de Investigaciones Biológicas y Tecnológicas (Córdoba, Argentina)

²CONICET-UNC, Centro de Investigaciones en Química Biológica de Córdoba (Córdoba, Argentina)

INTRODUCTION

Lipid Droplets (LD) are intracellular structures consisting of an apolar lipid core, composed mainly of triglycerides (TG) and steryl esters, coated by a lipid-protein mixed monolayer. The mechanisms underlying LD biogenesis at the endoplasmic reticulum membrane are a matter of many current investigations. Although models explaining the budding-off of protuberances of phase-segregated TG inside bilayers have been proposed recently, the assumption of such initial blisters needs further empirical support.

OBJECTIVES

To evaluate the dispersability and wettability of TG-phase (lenses) excluded from a PC monolayer. To describe the oil-water interfacial tension at the lens in relation to the compression of the laterally surroundng PC monolayer.

MATERIALS AND METHODS

Langmuir films of PC/TG where compressed up to lateral packing where TG molecules are excluded from the monolayer into Collapse Structures (CS). Surface Spectral Fluorescence Microscopy (SSFM) was used characterize the solvatochromism of Nile Red (NR) both in monolayers and inside CS. Brewster Angle Microscopy (BAM) was used to characterize the topography of films and thickness and lateral size of individual CS.

DISCUSSION AND RESULTS

Upon TG exclusion from Langmuir TG and PC/TG films, CS exhibited highly reproducible lateral size (∼1 micron lateral radius) not varying with lateral packing changes, and being highly stable at surface pressures beyond collapse. NR solvatochromism both in monolayers and inside CS indicated that CS corresponded to a phase of liquid TG. These lenses were dramatically flattened when PC was present (6-12 nm compared to 30-50 nm for lenses on PC/TG and TG films, respectively). The oil-water interfacial tension acting at each individual microscopic lens varied with compression states of the laterally surrounding monolayer at the air-water interface

CONCLUSION

Lenses formed on air-water Langmuir films can serve to assess variables of relevance to the initial step of LD biogenesis -such as the degree of dispersion of excluded-TG phase and shape, spatial distribution and oil-water interfacial tension of TG lenses-.

Keywords: Lipid Droplets Biogenesis, Langmuir monolayers, Neutral Lipids

08050 -

AA.31 - Stability of membranes containing different types of anionic lipids

Fernanda dos Santos Costa Leomil ^1,2, Rumiana Dimova², Karin do Amaral Riske¹

¹Biophysics Department, Universidade Federal de São Paulo (São Paulo, Brasil)

²Department of Theory & Bio-systems, Max Planck Institute of Colloids and Interfaces (Potsdam, Germany)

INTRODUCTION

Membrane stability is fundamental to sustaining life. When subjected to strong stimuli, such as electric pulses, pores open in the membrane. Usually these pores reseal after that and membrane integrity is restored, as observed in neutral PC (phosphatidylcholine) giant unilamellar vesicles (GUVs). However, GUVs containing charged lipids display a different response to strong DC electric pulses: some micron-sized pores open indefinitely leading to vesicle burst within less than a second after the pulse while GUVs that apparently restore their integrity after macropore closure exhibit a long-lasting high permeability revealing the persistence of sub-microscopic pores minutes after the end of the pulse. These phenomena are supposed to correlate with the fraction of the anionic lipid used.

OBJECTIVES

Here, the stability of GUVs composed of PC and increasing fractions of other physiologically relevant anionic lipids, such as cardiolipin (CL), phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2) is investigated with phase contrast optical microscopy.

MATERIALS AND METHODS

First, the occurrence frequency of disturbing events is quantified in a large population of GUVs. Then, the dynamics of vesicle contrast loss due to long-lasting permeability is assessed on individual GUVs. Finally, the edge tension, which reflects the energy penalty per unit length to arrange lipids in pore rims, is measured from the dynamics of macropore closure.

DISCUSSION AND RESULTS

A significant membrane destabilization occurs already at 10 mol% CL, whereas for PI and PIP2-containing membranes substantial vesicle perturbation was induced only at 50 mol% PI or PIP2. The edge tension for membranes composed of 50% of anionic lipid were significatively reduced when compared to pure PC membranes.

CONCLUSION

Membranes containing higher fractions of charged lipids are more unstable as a result of a reduced pore edge tension. The results obtained are important to understanding the response of cells to electroporation, a widely used protocol that renders biomembranes transiently permeable for several medical applications.

Keywords: electroporation, GUVs, anionic lipids

Supported by: CAPES and FAPESP 2016/13368-4

08295 - Poster Session

BA.01 - (Na+,K+)-ATPase activity in different tissues.

Romário da Silva Portilho ¹, Romario da Silva Portilho¹, Mila Marluce Lima Fernandes¹, Henrique Ranieri Covali Pontes¹, Igor Leal Brito¹, Jeandre Augusto dos Santos Jaques¹, Malson Neilson de Lucena¹

¹Instituto de Biociências, Universidade Federal do Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

The (Na ⁺ , K ⁺ )-ATPase, found in the plasma membranes of all animal cells, underpins many homeostatic processes and is directly responsible for the asymmetrical, electrogenic counter-transport of Na ⁺ and K ⁺ that results in strong ionic gradients across their membranes. The (Na ⁺, K ⁺)-ATPase also hydrolyzes other phosphate-donating substrates (K+-phosphatase activity), such as p-nitrophenylphosphate, O-methylfluorescein phosphate and acetyl phosphate.

OBJECTIVES

The activities of (Na ⁺ , K ⁺ )-ATPase from three different rat tissues homogenates (kidney, brain and heart) were characterized using the synthetic substrate p-nitrophenylphosphate (PNPP

MATERIALS AND METHODS

The tissues were homogenized and centrifuged at 10,000 × g for 35 min at 4 °C, the supernatant was rapidly frozen in liquid nitrogen and stored at –20 °C. PNPP hydrolysis (PNPPase activity) by the gill microsomal fraction was assayed at 37 °C, monitoring the release of the p-nitrophenolate ion. The difference in measured PNPPase activity in the absence and presence of ouabain was considered to represent the K ⁺ -phosphatase activity.

DISCUSSION AND RESULTS

K ⁺ -phosphatase activity in homogenate from kidney is 5- and 12-fold higher than in homogenate from brain and heart, respectively. Differences are also observed in sensitivity to ouabain

CONCLUSION

The results suggest that different isoforms of (Na ⁺ , K ⁺ )-ATPase are expressed in these tissues and the (Na ⁺ , K ⁺ )-ATPase can be regulated to the different cellular needs.

Keywords: peptides, proteins, scorpion

Supported by: CNPq, CAPES and UFMS

08664 - Poster Session

BA.02 - Mechanosensitivity and electric field modulates the water flow through plant and animal aquaporins

Marcelo Ozu ¹, Juán José Alvear-Arias^3,4, Miguel Angel Fernández Zuñiga^3,4, Agustín Francisco Caviglia^1,2, José Antonio Gárate^3,4, Gabriela Amodeo^1,2, Carlos González^3,4

¹CONICET-UBA, Instituto de Biodiversidad y Biología Animal y Aplicada (, Argentina)

²Facultad de Ciencias Exactas y Naturales, UBA, Departamento de Biodiversidad y Biología Experimental (, Argentina)

³Universidad de Valparaíso, Centro Interdisciplinario de Neurociencia de Valparaíso (, Chile)

⁴Universidad de Valparaíso, Facultad de Ciencias, (Chile)

INTRODUCTION

Aquaporins (AQPs) are part of a large but conserved family of transmembrane tetrameric proteins that include water channels as well as water-solute and/or gas channels. Each subunit has its own permeable pathway. Experiments in oocytes show that the water-transport rate of plant and animal aquaporins decreases by increasing the osmotic gradient and that this effect correlates with membrane tension increments. By other side, molecular dynamic (MD) simulations predict how the water molecules move through the permeable pathway as well as how they might respond to electric fields.

OBJECTIVES

Using the heterologous xenopus oocytes system, we studied the mechanosensitivity of homo and heterotetramers of the plant FaPIP2;1. In addition, to test the effects of electric fields on the water transport rate we performed molecular dynamic simulations on homotetramers of FaPIP2;1 and AQP4.

MATERIALS AND METHODS

Functional parameters were obtained from the kinetics of cell volume changes with different osmotic gradients. Simulations were performed with NAMD v.2.7 and the CHARMM27 force field, using a homology model of FaPIP2;1 developed with the crystal of SoPIP2;1 (PDB 2B5F) and the structural data of human AQP4 (PDB 3GD8).

DISCUSSION AND RESULTS

Our experimental results show that FaPIP2;1 behaves as a mechanosensitive aquaporin. In analogy with the study of ion channels, the transport capacity of AQPs can be evidenced in a plot of water flux versus osmotic gradient (Jw-∆osm). For mechanosensitive AQPs the Jw-∆osm plots show deviations from linearity with high gradients. On the other hand, molecular dynamic simulations reveal that FaPIP2;1 has higher water load capacity than other aquaporins, such as SoPIP2; 1 and AQP4. In addition, MD simulations predict that the water transport rate can change with the applied electric field in both AQP4 and FaPIP2;1.

CONCLUSION

Our results suggest that changes of membrane tension or electric field perturbates the water flow through aquaporin channels.

Keywords: mechanosensitivity, gating, electric field

Supported by: CONICYT, Millennium Scientific Initiative of the Chilean Ministry of Economy, Development, and Tourism (Chile); CONICET, ANPCYT, UBA (Argentina).

08140 - Poster Session

BA.03 - Transitions During the Voltage Sensor Activation in the Voltage-gated Proton Channel (Hv1) Changed According to the Absolute pH

Emerson Carmona¹, Osvaldo Alvarez^1,2, Alan Neely¹, Carlos Gonzalez¹

¹Instituto de Neurociencias, Centro Interdisciplinario de Neurociencia de Valparaíso, Universidad de Valparaíso (, Chile)

²Departamento de Biología, Facultad de Ciencias, Universidad de Chile, (Chile)

INTRODUCTION

The voltage-gated proton channel (Hv1) is a dimeric membrane protein able to dissipate acute acid loads in cells. This is achieved because the aperture of the channel is regulated by both the voltage and ΔpH across the membrane. Structurally, its function relays in the voltage sensor domain, as deletion of the N- and C-terminal domain produced monomers that maintains the biophysical properties of the dimer.

OBJECTIVES

We wanted to study the coupling between the voltage and pH sensing in the monomeric Hv1.

MATERIALS AND METHODS

We measured Hv1 currents to study the pH dependence of the monomeric channel in excised membrane patches of Xenopus laevis oocytes.

DISCUSSION AND RESULTS

As in the dimeric channel, the monomeric channel G-V curves were shifted according to the ΔpH established across the membrane, but this was not the case for the kinetics of activation, which were different for the same ΔpH. Then, we measured the effect of pH in gating currents of the monomeric Hv1 using a non-conducting mutant channel. In this case, the Q-V curves were changed according to the ΔpH established across the membrane, but like the ionic currents, the kinetics of decay of the ON-gating currents were not similar at the same ΔpH. Using different voltage protocols, we seek to find the transitions effected at different pHs during the movement of the voltage sensor. Finally, we fit our data using a Markov chain Monte Carlo method to try to quantify the modulation by pH.

CONCLUSION

Our results showed that for the case of the monomeric channel, ΔpH determines the initial and final states during activation of the channel, but the kinetic mechanism to reach the states does not simply depend on the ΔpH, but it changed according to the absolute internal and external pH.

Keywords: voltage-gated proton channel, pH-dependence, gating currents

Supported by: Supported by CONICYT-PFCHA/Doctorado Nacional/2017-21170395 to E.C., Fondecyt 1180464 to C.G., and 1161672 to A.N. Millennium Scientific Initiative of the Chilean Ministry of Economy, Development, and Tourism (P029-022-F).

08714 - Poster Session

BA.04 - Automatized and optimized analysis of video-registered volume time courses of aquaporin-expressing Xenopus oocytes

Marcos Oliver Gibson ^1,2, Florencia Guastaferri^1,2, Agustin Caviglia^1,2, Amodeo Gabriela^1,2, Marcelo Ozu^1,2, Luciano Galizia^1,2

¹Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, UBA (Ciudad Autónoma de Buenos Aires, Argentina)

²UBA, CONICET, Instituto de Biodiversidad y Biología Experimental y Aplicada (Ciudad Autónoma de Buenos Aires, Argentina)

INTRODUCTION

Biophysical characterization of aquaporins expressed in oocytes is based on monitoring cell volume changes. This model allows the recording of sequential images or videos with low-cost videomicroscopy equipment. However, one bottleneck in the experimental workflow is image processing. Timelapses recorded at high temporal resolution may contain thousands of images from where oocyte volume information must be extracted. The first step is oocyte segmentation, a user-dependent process that requires training and experimental criteria and is prone to bias included by the operator. Afterwards, oocyte area in each image is computed and converted to a relative volume curve (V_t/V₀), assuming spherical shape of oocytes. Then, the osmotic permeability coefficient (P_f) is calculated using the slope of the V_t/V₀ curve. To achieve an accurate P_f estimation, detection of the initial frame is mandatory, and not trivial to determine. This initial frame corresponds to an in-focus and stable oocyte image. Moreover, this frame must be temporarily located as near as possible to the instant the oocyte is subjected to the osmotic gradient.

OBJECTIVES

The challenge of this work is to present a new Phyton-based tool which allows us to process images, extract required information, and calculate the initial frame, V_t/V₀ curves and P_f of each oocyte.

MATERIALS AND METHODS

We developed this new script to produce an automatic, faster, and operator-independent end-to-end solution to perform P_f analysis. To develop this tool we used various image and data processing techniques such as shape recognition algorithms and in-depth classification of image focus using cluster analysis.

DISCUSSION AND RESULTS

Comparison of results with the traditional approach show equal P_f values but with better determination of the initial frame and significantly lower time of analysis.

CONCLUSION

With this tool it is possible to greatly increase the number of oocytes per experiment, gaining statistical power and substantially reducing the efforts spent in manual analysis.

Keywords: Cell volume, Aquaporins, Image processing

Supported by: UBA (UBACyT 2018-2020 number 20020170200049BA and UBACyT 2020-2021 number 20020190200141BA awarded to M.O) and ANPCyT (PICT 2017-0368 awarded to M.O. and PICT2017-2338 to G.A.)

08735 - Poster Session

BA.05 - Hydrolysis of nucleotide and non-nucleotide substrates by the Spf1p P5A-ATPase.

Luciana Romina Mazzitelll ¹, Julia Adriana Arpi Barrera¹, Gerardo Raul Corradi¹, Hugo Pedro Adamo¹

¹Facultad de Farmacia y Bioquimica, Instituto de Química y Fisicoquímica Biológicas (Buenos Aires, Argentina)

INTRODUCTION

P-ATPases are a large and ubiquitous family of transporters that use the energy of ATP hydrolysis to keep the concentrations of different compounds on both sides of biological membranes out of thermodynamic equilibrium. The P5-ATPases have atracted much interest after the reports of mutations in the human P5-ATPase ATP13A2 that cause a juvenile form of Parkinson's disease. Spf1p from Saccharomyces cerevisiae is the best characterized P5A-ATPase. Recent studies showed that Spf1p has a peptidyl translocase activity and directly interacts with the transmembrane segment of certain proteins, allowing their translocation when they are incorrectly inserted into the ER membrane and place P5-ATPAses within the cellular proteostasis control network.

OBJECTIVES

The present work aimed to explore the hydrolytic activity of Spf1p against different phosphorylated compounds using a purified preparation of the recombinant protein in C12E10 micelles.

MATERIALS AND METHODS

The reaction media contained Tris-HCl 50mM (pH de 7,2 a 28°C), MgCl2 2mM free, Na3N5mM, mM EGTA 0,5mM, the indicated amount of ATP o NTP and 1 μg of Spf1p supplemented with 40 μg of PC andC1 2E10. Pi was estimated with Baginski method after 20 minutes.

DISCUSSION AND RESULTS

We found that in addition to ATP, Spf1p was capable of hydrolyzing other nucleotidic substrates as ITP, GTP and, to a lesser extent, UTP. Interstingly, Spf1p did also hydrolyze non-nucleotide substrates, as pNPP with a maximum velocity approximately 4 times higher than that of ATP. As with ATP, the activity increased with the concentration of pNPP along a double hyperbolic curve with two components of low and high affinity. Based in these results, it might be possible that under conditions of low cellular energy Spf1p function is preserved by using alternative substrates

CONCLUSION

Knowledge of the functioning of P5A-ATPases can contribute to the design of strategies to avoid the loss of cellular proteostasis that underlies different types of human diseases.

Keywords: NTPs, P5-ATPASE, SPF1

08910 - Poster Session

BA.06 - Mechanistic insight into pH gating in PIP aquaporins: role of specific loopD amino acids

Agustina Canessa Fortuna ^1,2, Gerardo Zerbetto De Palma^1,2,3, Victoria Vitali^1,2, Jonathan Chevriau², Ari Zeida⁵, Dario Estrin⁶, Karina Alleva^1,2

¹Bioquímica, Departamento de Fisicomatemática, Cátedra de Física, Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, (Buenos Aires, Argentina)

²Instituto de Química y Fisicoquímica Biológicas “Prof. Alejandro C. Paladini”, Universidad de Buenos Aires-CONICET (Buenos Aires, Argentina)

³Instituto de Biotecnología, Universidad Nacional de Hurlingham (Villa Tesei, Argentina)

⁴Laboratorio de Fisiología de Proteínas, IQUIBICEN, Universidad de Buenos Aires-CONICET, Facultad de Ciencias Exactas y Naturales (Buenos Aires, Argentina)

⁵Departamento de Bioquímica, Facultad de Medicina and Centro de Investigaciones Biomédicas (Ceinbio), Universidad de la Republica (Montevideo, Uruguay)

⁶Departamento de Química Inorgánica, Analítica y Química Física (INQUIMAE), Universidad de Buenos Aires-CONICET, Facultad de Ciencias Exactas y Naturales (Bueno Aires, Argentina)

Aquaporins are membrane channels that transport water and other solutes. Their transport activity is regulated by different stimuli such as pH, binding of cations, phosphorylation/dephosphorylation or interaction with other proteins. All aquaporins share a general fold and tetrameric quaternary structure. Each protomer present two conserved regions that regulate the specificity of transport: the Asn-Pro-Ala (NPA) motives and the aromatic/arginine (Ar/R) selectivity filter. The plant PIP subfamily of aquaporins distinguish from other members of the family due to a longer intracellular loopD which is involved in its gating mechanism. The open/closed conformational transition in PIP channels is triggered by intracellular acidification, and different pH0,5 are found for homotetramers and heterotertamers form by PIP1 and PIP2 paralogues. Our goal is to elucidate the role of specific LoopD’ s amino acids in the modulation of PIP gating mechanism by intracellular pH. Our in vitro and in silico experiments, for wild type and mutant PIP show that: i- a conserved Leu residue in the cytoplasmic constriction is the structural element that determines pore blockage and, ii- a Pro residue present in PIP2 but not in PIP1 channels is involved in differential pH0.5 of homo and heterotetramers dose-response curves. So, two loopD residues, Leu 206 and Pro 194, works in combination with the pH sensor His 202 to control effective pore closing.

Keywords: aquaporin, gating, water transport

Supported by: Universidad de Buenos Aires (UBACYT 2018), Agencia Nacional de Promoción Científica y Tecnológica (PICT-2017-0244, PICT-2019-0387), and Universidad Nacional de Hurlingham (PIUNHAUR-5 2018).

08369 - Poster Session

BA.07 - EXPLORING THE CX26 HEMICHANNEL using MD simulations

Juan M. R. Albano^1,2, Jara Gabriel³, Julio FACELLI⁴, MARTA FERRARO^1,2, Monica Pickholz^1,2

¹Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina (Ciudad Autónoma de Buenos Aires, Argentina)

²Física, CONICET- Universidad de Buenos Aires, IFIBA (Ciudad Autónoma de Buenos Aires, Argentina)

³Institute of Chemistry, University of Campinas, , (Campinas, SP, 13083-861, Brazil)

⁴Biomedical Informatics, University of Utah (UTAH, United States)

INTRODUCTION

Gap junctions provide a communication pathway between adjacent cells. They are formed by two connexons that correspond to different cells. In this work, we investigated the dynamic behavior of a Cx26 connexon in a POPC lipid bi-layer using Molecular Dynamics simulations.

OBJECTIVES

Specific amino acid interactions with calcium and their stability were found. Few of these sites such as, GLU42, GLU47, GLY45 and ASP50, were already suggested in the literature. Besides, we identified novel calcium biding sites: ASP2, ASP117, ASP159, GLU114, GLU119, GLU120 and VAL226. .

MATERIALS AND METHODS

Molecular Dynamics simulations

DISCUSSION AND RESULTS

Furthermore, we focus our attention on the membrane protein interactions and the ion flux though the connexon pore. We analyzed extensive atomistic simulations with and without calcium ions. We found that lipid-protein interactions were mainly mediated by hydrogen bonds. Specific amino acids were identified forming hydrogen bonds with the POPC lipids (ARG98, ARG127, ARG165, ARG216, LYS22, LYS221, LYS223, LYS224, SER19, SER131, SER162, SER219, SER222, THR18 and TYR97, TYR155, TYR212, and TYR217). In the presence of calcium ions, we found subtle differences on the HB lifetimes.

CONCLUSION

Finally, these MD simulations are able to identify and explain differential chlorine flux through the pore depending on the amount of the calcium ions and its distribution within the pore

Keywords: CONNEXIN, Molecular Dynamics, POPC

Supported by: Universidad de Buenos Aires, CONICET and ANPCyT

08484 - Poster Session

BA.09 - A solution NMR study of a prokaryotic Na+/Ca2+ exchanger incorporated in detergent micelles

Luis Andrés Casavilca Ramírez ¹, Phelipe Augusto Mariano Vitale¹, Gustavo Penteado Battesini Carretero¹, Iolanda Midea Cuccovia¹, Roberto Kopke Salinas¹

¹Departamento de Bioquímica, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Na+/Ca2+ exchangers (NCXs) are essential for the maintenance of Ca2+ homeostasis in different cell types and are therefore considered important pharmacological targets. However, high-resolution structures for eukaryotic NCXs have not been obtained to date. NCX-Mj, an orthologue from the thermophilic archaea M. jannashii, has emerged as a structural model of the NCX transmembrane domain. While NCX-Mj kinetic properties have been extensively studied, only its outward-facing state structure has been solved and, hence, a complete model of the ion translocation mechanism is missing.

OBJECTIVES

This project aims to study the NCX-Mj intrinsic dynamics in DDM micelles by solution NMR spectroscopy.

MATERIALS AND METHODS

A synthetic gene coding for NCX-Mj was cloned in pET24 to express a protein in fusion with a 9xHis tag at the C-terminal end. E. coli C43 harbouring the pET24a-NCX-Mj plasmids were cultured in minimal media with 15NH4Cl as nitrogen source. Harvested cells were sonicated and membrane proteins were solubilised with 2% sarkosyl. After centrifugation, NCX-Mj was purified from the supernatant by batch Ni2+-affinity chromatography, at which point sarkosyl was exchanged for 0.3% DDM. NCX-Mj was further separated by size-exclusion chromatography, yielding 2 mg NCX-Mj per culture litre.

DISCUSSION AND RESULTS

SEC-MALS analyses revealed monodispersity of the protein detergent complex (PDC) fractions, as well as different elution times for empty micelles and PDCs, albeit with surprisingly longer retention times for the latter. These analyses suggested that monodispersity could be compromised by changes in the protein:detergent ratio.

CONCLUSION

The observation of well-dispersed peaks along the 1H dimension in preliminary 1H-15N TROSY spectra is consistent with a folded protein. Analyses of NCX-Mj mutants that stabilise a given state (outward- or inward-facing) are underway. By comparing 1H-15N TROSY spectra of wild-type and mutated NCX-Mj we expect to identify sets of peaks for each state.

Keywords: Na+/Ca2+ exchanger, Solution NMR spectroscopy, Structural biology

Supported by: FAPESP

08867 - Poster Session

BA.10 - BaCopA, a Cu(I) ATPase from the Antarctic bacterium Bizionia argentinensis

Noelia Inés Burgardt¹, Noelia Agostina Melian¹, Francisco Luis González Flecha¹

¹Departamento de Química Biológica, Instituto de Química y Fisicoquímica Biológicas (, Argentina)

INTRODUCTION

Copper ions are cofactor for several enzymes and participate in some cellular redox reactions. Intracellular excess of copper ions generates reactive radicals that cause damage to DNA, proteins and lipids. For this reason, intracellular levels must be regulated to avoid toxic concentrations. A subfamily of P-ATPases (denoted as PIB-1) are present in prokaryotic and eukaryotic organisms, and constitute one of the main transporters responsible for the elimination of excess copper ions from the cytosol.

OBJECTIVES

In this work we characterize a putative PIB-type ATPase belonging to ⟨i⟩Bizionia argentinensis⟨/i⟩(BaCopA), a gram-negative bacterium isolated from the superficial seawater of Potter Cove, Antarctica.

MATERIALS AND METHODS

BaCopA was cloned and expressed in ⟨i⟩Saccharomyces cerevisiae⟨/i⟩ as a GFP-fusion His-tagged protein for its subsequent purification and detection.

DISCUSSION AND RESULTS

Activity assays indicate that purified BaCopA is able to catalyze ATP hydrolysis at 5°C. ATPase activity of BaCopA increases when Cu (I) and ATP are added to the reaction medium.However, an inhibitory effect of ATPase activity occurs with the addition of vanadate, a specific inhibitor of P-ATPase-type enzymes.A structural model was built by homology modeling using the resolved structure of ⟨i⟩L. pneumophila⟨/i⟩CopA as template (PDB: 4BBJ). The structural alignment shows a high degree of similarity, with the typical topological pattern of PIB-1 ATPases.

CONCLUSION

Comparison with its mesophilic and hyperthermophilic counterparts led to the identification of key residues conserved in functional domains and differences in non-covalent interactions and surface charges. The detailed analysis of this interaction network suggests greater structural flexibility in BaCopA and, therefore, a better adaptation to low temperatures.

Keywords: ion transport ATPases, psychrophilic enzymes, bioinformatics

08659 -

BA.11 - The VRAC blocker DCPIB opens Ca2+-activated BK channels and increases intracellular Ca2+ in melanoma and pancreatic duct carcinoma cell lines

Paolo Zuccolini¹, Loretta Ferrera¹, Alessia Remigante¹, Cristiana Picco¹, Raffaella Barbieri¹, Sara Bertelli¹, Oscar Moran¹, Paola Gavazzo¹, Michael Pusch¹

¹IBF, Istituto di Biofisica, CNR (Liguria, Italy)

INTRODUCTION

The Volume Regulated Anion Channel (VRAC) is known to be involved in cancer cell behavior and response to therapies. Since ion channels play an increasingly recognized role in cancer we investigated the effect of DCPIB, a presumably specific VRAC blocker, in pancreatic duct adenocarcinoma (PDAC) and melanoma.

OBJECTIVES

We sought to define the mechanisms of DCPIB in two PDAC lines (Panc-1, MiaPaCa-2), as well as on a primary (IGR39) and a metastatic (IGR37) melanoma line.

MATERIALS AND METHODS

We performed whole-cell patch clamp electrophysiology, gene expression analysis and calcium measurements.

DISCUSSION AND RESULTS

DCPIB induced a dramatic increase of currents in Panc1, MiaPaca2 and IGR39, but not in IGR37 cells. Currents were sensitive to tetraethylammonium and thus not mediated by K2P channels, known to be activated by DCPIB. Rather, currents were mostly mediated by the Ca2+-dependent BK channel. DCPIB activation of BK as verified in transfected HEK293 cells. Further experiments showed that in IGR39, and to a smaller degree also in Panc-1 cells, DCPIB induces a rapid Ca2+ influx. This, in turn, indirectly potentiates not only BK but, in IGR39 cells, additionally activates other Ca2+-dependent channels. However, Ca2+ influx is not required for BK activation by DCPIB. The direct activation of BK by DCPIB involves the extracellular part of the protein, as no effect was detectable when DCPIB was delivered inside the cell via the patch pipette.

CONCLUSION

We conclude that the BK channel is a new target of DCPIB, and that the compound can acutely increase intracellular Ca2+, elongating the list of DCPIB side-effects that need to be taken into consideration for future development of DCPIB-based activators/inhibitors of ion channels and other membrane proteins.

Keywords: BK channel, PDAC cancer, Melanoma

Supported by: AIRC, MIUR

08451 - Poster Session

BB.01 - Experimental and simulation study: Interaction of CARC peptides from HlyA with lipid membranes

Lucia Cané ¹, Fanny Guzmán², Galo Ezequiel Balatti^3,4, Maria Antonieta Daza Millone⁵, María Elena Vela⁵, María Florencia Martini^4,6, Sabina Maté¹, Vanesa Herlax¹

¹La Plata, Instituto de Investigaciones Bioquímicas de La Plata, CCT- La Plata, CONICET. Facultad de Ciencias Médicas. Universidad Nacional de La Plata. (Buenos Aires, Argentina)

²Valparaíso, Núcleo Biotecnológico de Curauma, Pontificia Universidad Católica de Valparaíso. (Valparaíso, Chile)

³Quilmes, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes. (Buenos Aires, Argentina)

⁴CABA, Instituto de Química y Metabolismo del Fármaco, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). (Buenos Aires, Argentina)

⁵La Plata, Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas, CCT- La Plata, CONICET. Universidad Nacional de La Plata. (Buenos Aires, Argentina)

⁶CABA, Cátedra de Química Medicinal, Facultad de Farmacia y Bioquímica, Junín 956, (1113). (Buenos Aires, Argentina)

INTRODUCTION

Escherichia coli alpha hemolysin (HlyA) is a pore-forming protein which belongs to the 'Repeat in toxins' (RTX) family. Although HlyA does not need cholesterol as a receptor to be active, the presence of this lipid in target cells enhances its activity. Several CRAC (Cholesterol Recognition/interaction Aminoacid Consensus sequence) and CARC (similar to CRAC but with the opposite orientation) were found in HlyA sequence. Only one CARC is present in the membrane insertion domain of the toxin.

OBJECTIVES

The aim of this work was to study the role of the CARC domain present in the N-terminal portion of the toxin in the interaction with membranes.

MATERIALS AND METHODS

Two peptides derived from HlyA were synthesized: PEPY: corresponds to a CARC sequence in the transmembrane domain of HlyA and PEPA: similar to PEPY but with residue Y ³⁴⁷ substituted by A. Peptides were synthesized by the solid phase peptide synthesis method (Fmoc strategy) and purified by HPLC; peptide molecular mass and structure were determined by mass spectrometry and circular dichroism. Langmuir monolayer assays and Surface Plasmon Resonance (SPR) were performed to study insertion and association of the peptides into different POPC:Cho lipid mixtures (1:0, 4:1; 2:1). In addition, Molecular Dynamics (MD) simulations of the peptides with bilayers of those same compositions were carried out by using the united-atoms force field GROMOS.

DISCUSSION AND RESULTS

MD simulations and SPR assays show that PEPY has more affinity for POPC:Cho (4:1) membranes and also that PEPA presents less affinity for lipid bilayers in general. On Langmuir monolayer experiments, the same results were observed regarding insertion into monolayers.

CONCLUSION

These results indicate that the CARC peptide derived from the N-terminal region of HlyA has a strong affinity for POPC:Cho (4:1) membranes, and that peptide-lipid interaction strongly depends on the presence of residue Y ³⁴⁷.

Keywords: alpha hemolysin, biophysics, cholesterol

Supported by: CONICET, UNLP.

08677 - Poster Session

BB.02 - PROJECT - In vitro evaluation of the antitumor potential of the Pep 5 peptide and its effects on Na,K-ATPase activity and analysis of the lipid composition of the plasma membrane

Priscilla Oliveira Gil ¹, Vanessa Faria Cortes¹, Debora de Oliveira Lopes¹

¹Bioquímica e Biologia Molecular, Universidade Federal de São João del-Rei (Minas Gerais, Brasil)

INTRODUCTION

Cancer is one of the most studied diseases today, as it has a high mortality rate and affects people of all ages. The development of this disease is due to the disordered growth of cells that invade tissues and organs and tend to be very aggressive and uncontrollable. The Na,K-ATPase (NKA) pump is an integral enzyme of the plasma membrane that depends on ATP for the active transport of the Na+ and K+ ions and is responsible for maintaining a low intracellular Na+ / K+ ratio. The enzyme has three subunits: The alfa subunit, responsible for catalytic activity and ionic transport; The beta subunit, responsible for the normal activity of NKA and contains several glycosylation sites; And the gama (or FXYD2) subunit that has a modulating function and influences the kinetic properties of the enzyme. This pump plays a fundamental role in the survival of cells, and is closely related to the amount of phospholipids and cholesterol in the membrane, which are necessary for its correct operation. Generally, the NKA pump has its expression increased in tumor cell lines compared to non-tumor cells, which makes this enzyme an excellent target for anticancer therapy. Bacteriocins are peptides or proteins secreted by bacteria that have antimicrobial and anticancer properties.

OBJECTIVES

Therefore, the present study aims to evaluate the interaction of the bacteriocin-like peptide Pep 5, secreted by Staphylococcus epidermidis 5, with the NKA pump and the plasma membrane in tumor cells.

MATERIALS AND METHODS

Cytotoxicity, cell migration, enzymatic activity and expression of its subunits will be performed, in addition to determining the dosage of phospholipids in the membrane of lung carcinoma cells, in order to propose new treatments against cancer, more effective and with lower side effects.

DISCUSSION AND RESULTS

CONCLUSION

Keywords: Cancer, NKA, Bacteriocins

Supported by: Universidade Federal de São João del-Rei - UFSJ

08357 - Poster Session

BB.03 - CaDef2.1G27-K44, an antimicrobial peptide bioinspired on defensin from C. annuum fruits: anti-Candida and antimycobacterial activities, mechanisms of action on yeasts and cytotoxicity on mammalian cells

Gabriel Bonan Taveira ¹, Érica de Oliveira Mello¹, Thatiana Lopes Biá Ventura^2,3, Milena Belei Cherene¹, André de Oliveira Carvalho¹, Michelle Frazão Muzitano², Elena Lassounskaia³, Olney Vieira da Motta⁴, Valdirene Moreira Gomes¹

¹Centro de Biociências e Biotecnologia/LFBM, Universidade Estadual do Norte Fluminense Darcy Ribeiro (RJ, Brasil)

²Pólo Novo Cavaleiro-IMMT/LPBio, Universidade Federal do Rio de Janeiro (RJ, Brasil)

³Centro de Biociências e Biotecnologia/LBR, Universidade Estadual do Norte Fluminense Darcy Ribeiro (RJ, Brasil)

⁴Centro de Ciências Tecnologias e Agropecuárias/LSA, Universidade Estadual do Norte Fluminense Darcy Ribeiro (RJ, Brasil)

INTRODUCTION

The growing resistance of microorganisms to multiple drugs associated with the high host toxicity of drugs in the current scenario is a major concern in Public Health. Therefore, new therapeutic strategies are necessary to cope with these problems. Antimicrobial peptides, natural or synthetic, appear as promising molecules for antimicrobial therapy because of their both broad antimicrobial activity and mechanism of action.

OBJECTIVES

To determine the anti-Candida and antimycobacterial activities, mechanism of action on yeasts, cytotoxicity activity on mammalian cells in the presence of CaDef2.1_G27-K44 peptide (sequence under patent review).

MATERIALS AND METHODS

The CaDef2.1_G27-K44 peptide was designed meeting the following criteria: positive net charge; low molecular weight (

DISCUSSION AND RESULTS

CaDef2.1_G27-K44 inhibited the growth of C. albicans, C. buinensis, C. parapsilosis and C. tropicalis with MIC100 values ranging from 12.5 to 25μM. For C. albicans, C. buinensis and C. tropicalis the MIC100 value corresponds to the lethal dose that kill 100% of the treated cell. CaDef2.1_G27-K44 showed inhibitory activity against laboratory Mycobacterium tuberculosis strain H37Rv and highly virulent strain of M. tuberculosis with MIC50 of 33.2 and 55.4μM, respectively. Additionally, we demonstrate that CaDef2.1_G27-K44 is active against yeasts even at different salt concentrations, induced morphological alterations such as, smaller cell size and cytoplasm condensation, presented cell membrane permeabilization and increased ROS levels, causes loss of mitochondrial functionality and activation of caspases. CaDef2.1_G27-K44 has low cytotoxicity against mammalian cells.

CONCLUSION

CaDef2.1_G27-K44 has great potential as an antimicrobial agent with low toxicity to host cells.

Keywords: Antimicrobial peptide, Candida sp., Mycobacterium tuberculosis

Supported by: FAPERJ, UENF, CNPq, CAPES

08786 - Poster Session

BB.04 - Design, synthesis, purification and characterization of the wild type and four analogues of the SARS-CoV-2 fusion peptide (FP2) to evaluate the specific interaction with calcium ions

Matheus Marchetti Melo¹, Luís Guilherme Mansor Basso², Eduardo Festozo Vicente¹

¹Departamento de Engenharia de Biossistemas, Universidade Estadual Paulista (Unesp), Faculdade de Ciências e Engenharia, Campus de Tupã (São Paulo, Brasil)

²Centro de Ciência e Tecnologia, Laboratório de Ciências Físicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro (Rio de Janeiro, Brasil)

INTRODUCTION

The SARS-CoV-2 fusion process occurs through interaction with membranes of the so-called fusion peptide, a relatively hydrophobic and highly conserved amino acid fragment of the Spike protein within the Coronaviridae family, which is essential for the virus's fusogenic activity. It has been hypothesized that coordination of calcium ions at peptide sites is essential for the peptide binding to the target membranes.

OBJECTIVES

Thus, this study evaluates the importance of calcium ions in the modulation of the fusion peptide conformation.

MATERIALS AND METHODS

For this, solid phase peptide synthesis of the wild type fusion peptide (FP2) was performed, designed with a polyglycyl-lysine tail at C-terminus to increase peptides’ solubility. In addition, three peptide analogues were synthesized containing strategic substitutions at positions 4 (Glu), 5 and 15 (Asp) by Gln and Asn, respectively, to determine which residues modulate calcium binding. Also, another peptide analogue containing a Asn replacement at position 9 by Asp was synthesized, aiming to hypothetically increase fusogenicity, by potentially creating a second calcium binding site. Further, purification, characterization of the synthesized peptides by HPLC and Mass Spectrometry were successfully evaluated and Circular Dichroism was performed to analyze secondary structure formation with and without calcium ions in solution and in lysophosphatidylglycerol micelles.

DISCUSSION AND RESULTS

Interestingly, the results evidenced that the secondary structures of FP2, Asn5 and Ans15 analogues are modulated by calcium binding, indicating that both Asp5 and Asp15 residues of the FP2 may not represent the calcium coordination sites. In contrast, the secondary structures of both Asp9 and Gln4 analogues are no longer affected by calcium binding, even in the micellar environment. This result suggests that the FP2 Glu4 residue is a putative calcium binding site.

CONCLUSION

In conclusion, our results showed that a Ca2+-dependent secondary structure formation of FP2 and its analogues, identifying the putative Ca2+-coordination site, which most likely affects the peptide fusogenicity.

Keywords: Biophysics, Peptides, SARS-CoV-2

Supported by: FAPESP

08668 - Poster Session

BB.05 - Effect of membrane potential on entry of lactoferricin B-derived antimicrobial peptide into single bacterial cells and lipid vesicles

Farzana Hossain¹, Hideo Dohra², Masahito Yamazaki^1,3

¹Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University (Shizuoka, Japan)

²Research Institute of Green Science and Technology, Shizuoka University (Shizuoka, Japan)

³Research Institute of Electronics, Shizuoka University (Shizuoka, Japan)

INTRODUCTION

Recently, we reported that antimicrobial peptide (AMP)-induced damage of membrane, increased with increasing negative membrane potential (∆φ) (JBC. 2019). AMP LfcinB(4-9) (sequence-RRWQWR) derived from lactoferricinB, can enter E. coli cells without damaging cell membranes. Thus, LfcinB(4-9) is a cell-penetrating peptide (CPP)-type AMP.

OBJECTIVES

Effect of ∆φ on the action of CPP-type AMPs is not well known. In this study, we investigated the effect of ∆φ, on interactions of Rh-LfcinB(4-9), with single E. coli cells, spheroplasts and giant unilamellar vesicles (GUVs) to reveal the mechanism of its antimicrobial activity (J. Bacteriol, 2021).

MATERIALS AND METHODS

First, we investigated interaction of Rh-LfcinB(4-9) with single E. coli cells and spheroplasts containing calcein by adding peptide to their vicinity and observed their fluorescence intensity change using CLSM. Next, we examined effect of ∆φ on interaction of Rh-LfcinB(4-9) with E. coli lipid-GUVs containing AF647 and small GUVs using single GUV method.

DISCUSSION AND RESULTS

At low peptide concentrations, Rh-LfcinB(4-9) entered cytosol of E. coli and spheroplasts without damaging membranes and the protonophore carbonyl cyanide m-chloro-phenylhydrazone (CCCP) suppressed its entry. Studies of time-kill method indicate that these low concentrations of peptides exhibit antimicrobial activity, but CCCP inhibits this activity. We also found that, Rh-LfcinB(4-9) entered the GUV lumen without pore formation in the presence of ∆φ and the rate of peptide entry into GUV lumen increased with increasing ∆φ.

CONCLUSION

All the results show that ∆φ increases the rate of Rh-LfcinB(4-9) entry into the cytosol of E. coli cells and spheroplasts, and the lumen of GUVs without pore formation. To the best of our knowledge, this is the first report demonstrating the role of ∆φ in the antimicrobial activity of CPP-type AMPs and in their entry into the cytoplasm, which is a key process to determine their antimicrobial activity. These results indicate that antimicrobial activity of Rh-LfcinB(4-9) and hence LfcinB(4-9) increases with increasing negative ∆φ.

Keywords: antimicrobial activity, membrane potential, entry

Supported by: JSPS

08652 - Poster Session

BB.06 - Translocation of the Nonlabeled Antimicrobial Peptide PGLa across Lipid Bilayers and its Entry into Vesicle Lumens without Pore Formation

Ali M.H¹, Shuma M.S¹, Dohra H², Yamazaki, M.^1,3

¹Integrated Bioscience Section, Shizuoka University (Shizuoka, Japan)

²Research Institute of Green Science and Technology, Shizuoka University (Shizuoka, Japan)

³Research Institute of Electronics, Shizuoka University (Shizuoka, Japan)

INTRODUCTION

Fluorescent probe-labeled peptides are used to investigate the interaction of antimicrobial peptides (AMPs) with cells and lipid vesicles to elucidate the mechanism of their activity. However, it is reported that labeling can significantly change the interactions of AMPs with plasma and lipid membranes.

OBJECTIVES

To eliminate the effect of fluorescent probe labeling, we investigated the entry of nonlabeled AMP PGLa into the lumen of single giant unilamellar vesicles (GUVs) using the new method devloped in our lab (Biochemistry, 2020).

MATERIALS AND METHODS

First, we investigated using confocal laser scanning microscopy the interaction of nonlabeled PGLa with single DOPG/DOPC (4/6)-GUVs containing fluorescent probe AF647 and DOPG/DOPC (8/2)-large unilamellar vesicles with self-quenched calcein in their lumen. Second, we simultaneously measured the PGLa-induced increase in fluorescence intensity of the GUV lumen due to calcein (I_calcein) and the PGLa-induced fractional area change of the GUV membrane (δ), as well as the lumen intensity due to AF647.

DISCUSSION AND RESULTS

In the interaction of nonlabeled PGLa with single GUVs, (I_calcein) increased with time without leakage of AF647 after a lag time from starting the interaction. This result clearly shows that PGLa can enter into the GUV lumen without damaging the GUV membrane, i.e., without pore formation. With increasing PGLa concentration, the fraction of PGLa entry increased. The results of simultaneous measurement show that PGLa entry occurs during the second increase in (δ), indicating that PGLa enters the lumen only during the translocation of PGLa from the outer to the inner leaflet of GUV membrane. Membrane tension due to an external force elevated the rate of PGLa entry.

CONCLUSION

This is the first report to demonstrate experimentally that nonlabeled AMPs enter the vesicle lumens without pore formation (BBA-Biomembrane, 2021). Based on these results, we discuss the mechanism of nonlabeled PGLa entry into lipid vesicle.

Keywords: PGLa, entry, giant unilamellar vesicle

Supported by: JSPS

08462 - Poster Session

BB.07 - Semi-quantitative RT-PCR analysis of an antimicrobial peptide expression in hemolymph and hepatopancreas of shrimp Macrobrachium rosenbergii (De Man, 1879) challenged with Aeromonas hydrophila

Flávia Bordignon Hendges ¹, Victor Donatti Machado², Gabrielle Caroline Peiter¹, Hércules Tancredo Moreira¹, Andressa Piacentini Betti², Flavio Augusto Vicente Seixas³, Adriana Fiorini Rosado¹, Eduardo Luis Cupertino Ballester⁴, Fabio Rogério Rosado¹

¹Programa de Pós-Graduação em Bioquímica e Biologia Molecular, Universidade Federal do Paraná – Setor Palotina (Paraná, Brazil)

²Departamento de Biociências, Universidade Federal do Paraná – Setor Palotina (Paraná, Brazil)

³Departamento de Tecnologia, Universidade Estadual de Maringá – Campus Umuarama (Paraná, Brazil)

⁴Departamento de Zootecnia, Universidade Federal do Paraná – Setor Palotina (Paraná, Brazil)

INTRODUCTION

Macrobrachium rosenbergii, known as freshwater giant shrimp, stands out for being one of the most economically important crustaceans with a high market value, being cultivated on a large scale in the world. The species was introduced in Brazil in 1970 and is currently the most favored for cultivation in the country. In the production environment, shrimp are exposed to several microorganisms that can burden the rearing systems, causing the crop to be threatened. Thus, crustaceans need an efficient and active immune system, responding through quick reactions to stimuli caused by the environment. Small chains of amino acids called antimicrobial peptides (AMPs) are secreted and actively react to fight infections. AMPs are considered essential molecules of the innate immune system, being present in several species.

OBJECTIVES

This work aimed to evaluate the production of a specific type of AMP expressed in M. rosenbergii, named Pellino, in response to septic injury caused by Aeromonas hydrophila after 12 hours of injection.

MATERIALS AND METHODS

The transcriptional profile of genes involved in the production of Pellino was analyzed by semi-quantitative RT-PCR analysis, with the β-Actin gene as expression normalizer. Different primers were used for the cDNA reaction.

DISCUSSION AND RESULTS

Results indicated that it was possible to express Pellino AMP under the conditions tested.

CONCLUSION

It was observed Pellino gene expression in hemolymph of animals challenged with A. hydrophila and no expression was observed in the control sample, indicating a possible immune defense activation.

Keywords: freshwater shrimp, hepatopancreas, immune system

Supported by: Fundação Araucária and CAPES

08915 - Poster Session

BB.08 - Introducing the multispectral phasors: a tool for the analysis of fluorescence excitation-emission matrix and spectral signal unmixing

Luis Benito Pérez Socas ¹, Ernesto E. Ambroggio¹

¹Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica-Ranwel Caputto (Haya de la Torre y Medina Allende s/n. Córdoba (X5000HUA), Argentina)

The use of phasors to analyze fluorescence data was first introduced for time-resolved studies in order to reduce the mathematical complexity of the models used. Recently, this approach was extrapolated to steady-state experiments, in what is called spectral phasors (SP), which are obtained from the Fourier transform of the fluorescence emission spectrum. In this work, we revise key mathematical aspects that lead us to re-interpret SP as the characteristic function of a probability distribution. This allows us to introduce a new tool, called multispectral phasor (MSP), that seize not only the information from the emission spectrum, but from the full excitation-emission matrix (EEM). Additionally, here we also developed an open-source Java software in order to facilitate the data processing. Using simulated spectra, we prove that there is a tight relationship between the MSP polar coordinates and the characteristics of the EEM, similar to what is known for SP. Due to our mathematical re-interpretation, we also show that MSP can be a powerful tool to tackle spectral signal unmixing problems in a more general case than SP. In addition, we proposed two main biophysical applications of MSP: protein conformational studies and Föster Resonance Energy Transfer (FRET). As a prove of concept, we experimentally measure the EEM changes upon denaturation of the human serum albumin (HSA) and during a HSA-ANS FRET. In this sense, we were able to use MSP to interpret the results in a simpler and finer way and in complete agreement with what is known in the literature. Understanding a protein’s EEM as a molecular conformation fingerprint will open a door for the use of MSP as a tool to analyze and comprehend protein conformational changes.

Keywords: Spectral phasor, fluorescence excitation-emission matrix, signal unmixing

Supported by: CONICET (PIP2013-2015), FonCyT (PICT2012-1377, PICT2015-2575). L.B.P.S. holds a PhD fellowship from CONICET and E.E.A is a Career Member of CONICET. We are also grateful to all the members of the biophysical area at CIQUIBIC for helpful discussions

08473 -

BB.09 - Biophysical and structural studies of an antimicrobial membrane-modifying peptide of Piscidin family

Kelton Rodrigues de Souza^1,2, Lucio Otávio Nunes¹, Evgeniy Salnikov², Talita Lopes Santos¹, Victor Hugo de Oliveira Munhoz¹, Jarbas Magalhães Resende³, Christopher Aisenbrey², Rodrigo Moreira Verly¹, Burkhard Bechinger^2,4

¹Chemistry, Federal University of Jequitinhonha and Mucuri Valleys (Minas Gerais, Brasil)

²Chemistry, University of Strasbourg (Grand Est, France)

³Chemistry, Federal University of Minas Gerais (Minas Gerais, Brasil)

⁴Chemistry, Institut Universitaire de France (, France)

INTRODUCTION

Antimicrobial peptides are potential templates for the design of novel antibacterial drugs due to their low propensity for resistance development and their ability to kill multi-drug-resistant bacteria. The ecPis-4s peptide isolated from the fish species Epinephelus coioides has been described as a potent antimicrobial agent against several bacterial and fungal strains1. It is composed of 22 residues (FFRHIKSFWKGAKAIFRGARQG-NH2) and carries a natural C-terminal carboxamide. The mode of interaction of ecPis-4s with membrane mimetic media is studied employing a set of biophysical techniques.

OBJECTIVES

The mode of interaction of ecPis-4s with membrane mimetic media is studied employing a set of biophysical techniques.

MATERIALS AND METHODS

Firstly, the ecPis-4s was chemically synthetized by SPPS using the Fmoc strategy, purified by HPLC, and characterized by mass spectrometry.

DISCUSSION AND RESULTS

Circular dichroism (CD) spectroscopy shows a high content of peptide helical structure when it is associated with POPC:POPG vesicles, and a low helix structuration in the absence of membranes. Solution NMR experiments have been performed in the presence of negatively charged SDS micelles to determine the three-dimensional structure of the peptide. The structure confirmed the helical conformation throughout the linear chain of ecPis-4s. Proton-decoupled 15N and 2H solid-state NMR of macroscopically oriented lipid bilayers have been used to determine the peptide topology in membranes made of POPC:POPG or E. coli lipid extract. The chemical shift around 70 ppm indicates that the peptide adopts a surface orientation with a tilt angle close to 90°. Carboxyfluorescein (CF) release experiments demonstrate a lytic activity of this peptide in the presence negatively charged vesicles made of POPC:POPG.

CONCLUSION

In conclusion, the ecPis-4s peptide presents a high affinity to anionic membranes where it adopts an amphipathic helical conformation that aligns parallel to the phospholipid bilayer surface. This mode of interaction suggests a membrane-lytic activity once the local concentration or peptide reaches a threshold.

Keywords: antimicrobial peptide, peptide-membrane interaction, peptide topology

08460 - Poster Session

CA.01 - Luminescence of complex cis-[Ru(phen)2(3,4Apy)2]2+ in model membranes

Maria Laura da Cruz Garcia ¹, Rose Maria Carlos¹

¹Departamento de Química, Universidade Federal de São Carlos (São Paulo, Brazil)

INTRODUCTION

The study of the interaction between the transition metal complex cis-[Ru(phen)₂(3,4Apy)₂] ²⁺ (RuApy) and cell model membranes was motivated by previous studies from our laboratory that demonstrate that RuApy has spectroscopic properties (in physiological environment pH 7,4: λ_abs=480nm, ε_480nm=9500 mol ^-1 Lcm ^-1 , λ_em=655 nm, τ_em=120ns) that allow its use as a luminescent probe for the β-amyloid peptide(Aβ).

OBJECTIVES

These properties are important as the toxicity of Aβ can be influenced by those in neuronal cell membranes. In this context, this work investigates the interaction between RuApy and cell model membranes such as large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs) prepared with the negatively charged lipid DOPG

MATERIALS AND METHODS

The luminescence auto suppression studies of RuApy in aqueous solution pH 7.4 (phosphate buffer) indicated a limit for the steady state luminescence studies

DISCUSSION AND RESULTS

at 60 μM. The complex was a sensitive probe in the presence of the DOPG(LUVs) vesicle, and this can be observed by the continuous suppression of RuApy emission at 655 nm. Studies carried out to determine the sensitivity of the RuApy complex against the LUV vesicle of DOPG indicated that 10 μM of complex is sensitive to a concentration of 5μM of vesicle.

CONCLUSION

The results obtained so far indicate an electrostatic interaction between RuApy and DOPG(LUVs).

Keywords: fluorescence, model membranes, ruthenium complex

Supported by: CNPq, FAPESP and Capes

08780 - Poster Session

CA.02 - Improvement of the Methodological Strategies to Product Functionalizes Antibodies using Small Angle Neutron Scattering (SANS)

Beatriz Tremarin ¹, Fabiano Yokaichiya¹, Guinther Kellermann¹, Margareth Kazuyo Kobayashi Dias Franco ³, Joachim Storsberg²

¹Departamento de Física, Universidade Federal do Paraná (Paraná, Brazil)

²Healthcare, Biomaterials and Cosmeceuticals, Fraunhofer-Institute for Applied Polymer Research (Potsdam, Germany)

³Rejeitos Radioativos - GRR, Instituto de Pesquisas Energéticas e Nucleares (São Paulo , Brazil)

INTRODUCTION

Antibodies are used by jawed vertebrates for defense against invading pathogens. Usage of those versatile tools in a plethora of settings in clinics and biomedical sciences hinges on functionalization strategies that retain native antibody reactivity. To this date, antibody functionalization is performed by trial and error.

OBJECTIVES

We aim to reduce costs by providing general principles to allow the full spectrum of antibody functionalization by correlating functionalized antibody reactivity to cognate antigen by small angle neutron scattering, SANS, measurements and mathematical modeling of antibody and antibody-antigen super-complexes, obtained by titration experiments.

MATERIALS AND METHODS

For this research we have used for as antibody pure goat anti rabbit immunoglobulin, and for the antigen, pure Horseradish Peroxidase Preliminary results show that the systems (antibody and antibody-antigen complexes) do not change in the range of a temperature related to storage temperature (25º C), body temperature (37º C) and 40º C.

DISCUSSION AND RESULTS

These results will give us the pair distribution function of these systems and the results will be viewed in light of published precedence to highlight areas where future effort is needed to refine such versatile tools and improve their production. However, between the antibody and the complexes structure, different conformations were observed. The antibody has a globular structure with a radius of gyration around 33 Å, and the complexes display an elongated cylindrical shape with radius of gyration around 63 Å.

CONCLUSION

This study shows how the scattering techniques (SANS) can provide useful information about the conformation of the antibody and antibody-antigen formation and help to shed light in the understanding the physical, chemical, and structural changes on the organization of these important antibody functionalization for the immunological system.

Keywords: Antibody-Antigen, Biophysics, SANS

Supported by: CNPq

08343 - Poster Session

CA.03 - Evaluation of Photoinduced Membrane Damage by Substituted Magnesium Porphyrazines

Otávio Augusto de Oliveira Reis ¹, Camila Fontes Neves Silva¹, Thiago Teixeira Tasso², Helena Couto Junqueira¹, Maurício da Silva Baptista¹

¹Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo (São Paulo, Brasil)

²Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais (Minas Gerais, Brasil)

INTRODUCTION

Photodynamic Therapy (PDT) is an innovative and efficient treatment modality for a wide array of diseases, including infectious and many types of cancer. PDT is usually based on a photosensitizer (PS), its excitation by light and the subsequent energy or electron transfer to molecular oxygen (³ O₂), generating reactive oxygen species (ROSs), specially singlet oxygen ( ¹ O₂). However, new experimental evidences suggest that contact-dependent reactions between the PS and biomolecules in the cell membrane – mainly lipids – are essential to cause membrane leakage, therefore unbalancing the chemical gradients that keeps the cell functioning and generating cell death. In order to advance in the development of this treatment, research on PSs that act by the mechanism of contact-dependent reactions with the cell membranes is essential.

OBJECTIVES

We have prepared a series of magnesium porphyrazines octa-substituted with fluor and trifluoromethyl groups (FMgPz and CF₃MgPz, respectively) and have investigated their properties in interaction with membrane models, more specifically SUVs and GUVs (small and giant unilamellar vesicles, respectively), which are made of unsaturated lipids.

MATERIALS AND METHODS

The membrane damage was investigated in the presence of the porphyrazines after red light irradiation (λ=630nm), which is the wavelength of maximum absorption by the porphyrazines (ε = ~ 6x10 ⁴ L.mol ^-1 .cm ^-1 ). Both FMgPz and CF₃MgPz have equal ¹ O₂ quantum yields (Φ_Δ=0,34).

DISCUSSION AND RESULTS

Our results showed a greater photoinduced membrane leakage in the presence of CF₃MgPz when compared to FMgPz. We attributed the higher efficiency of CF₃MgPz to the contact-dependent reactions between the CF₃MgPz and the vesicles, which also causes PS photobleaching. Interestingly, the photobleaching rate of CF₃MgPz was also far greater than that of FMgPz, indicating that the greater membrane damage is parallel with the higher photobleaching rate.

CONCLUSION

Therefore, in order to develop more efficient PS, we need to consider strategies to have the bleached photosensitizer replenished during PDT.

Keywords: Redox Biochemistry, Photochemistry, Membrane

Supported by: FAPESP

08566 - Poster Session

CA.04 - Effects of Amburana cearensis dichloromethane extract in cerebral ischemia models focusing on glial cells

Rafael Short Ferreira ^1,2, Juliana Helena Castro e Silva ¹, Juliana Bender Hoppe³, Monique Marylin Alves de Almeida^1,2, Francesca Pieropan², Erica Patricia Lima Pereira¹, Andrea Rivera², Beatriz Correia de Lima Ferreira¹, Gustavo Borges da Silva Andrade¹, Paulo Roberto Ribeiro⁴, Luzimar Gonzaga Fernandez⁵, Christianne Gazzana Salbego³, Jose Claudio Fonseca Moreira³, Silvia Lima Costa¹, Arthur Morgan Butt², Victor Diogenes Amaral da Silva^1,2

¹Laboratory of Neurochemistry and Cell Biology, Institute of Life Sciences, Federal University of Bahia (Bahia, Brazil), ²Institute of Biomedical and Biomolecular Sciences, University of Portsmouth (, England), ³Department of Biochemistry, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul (, Brazil), ⁴Department of Organic Chemistry, Institute of Chemistry, Federal University of Bahia (Bahia, Brazil), ⁵Biochemistry, Biotechnology and Bioproducts Laboratory, Federal University of Bahia (Bahia, Brazil)

INTRODUCTION

Glutamatergic excitotoxicity is a pathophysiological mechanism present in chronic neurodegenerative diseases (Alzheimer's Disease) and acute (Brain Ischemia), and it especially affects the hippocampus, as it has a high density of glutamatergic neurons. Under these conditions, neurons and oligodendrocytes can be severely affected. However, astrocytes can attenuate or prevent cell death through this mechanism by re-uptake excess glutamate. It is known that secondary metabolites of Amburana cearensis may be related to neuroprotection mechanisms against excitotoxic damage.

OBJECTIVES

The aim of this work was to investigate the neuroprotective effects associated with treatment with A. cearensis Dichloromethane Extract (EDAC) in models of cerebral ischemia.

MATERIALS AND METHODS

Hippocampal slices from wild-type Wistar rats (P6-8) or transgenic SOX10-EGFP and GFAP-EGFP reporter mice (P10-12) were used to identify oligodendrocytes and astrocytes, respectively. These slices were submitted in two models treated with EDAC: 1) oxygen and glucose supply (OGN) or deprivation (OGD); and 2) in organotypic culture (OHSC) submitted to glutamate excitotoxicity. Protein expression, cell morphology, cell viability and genetic transcription tests were performed.

DISCUSSION AND RESULTS

Under OGD conditions, our results showed that EDAC prevented the reduction of cellular processes of SOX10 expression, without an increase in the expression of astrocytic proteins between the OGN and OGD control groups. However, EDAC increased GFAP expression under OGD conditions. In OHSC, we observed that excess glutamate induced an increase in cell death and that this was inhibited by treatment with EDAC. However, under these conditions, EDAC does not protect neurons. On the other hand, GFAP, GLT1, GLAST and GS were overexpressed in cultures treated with EDAC under glutamatergic excitotoxicity. We also observed, by RT-qPCR, a slight increase in transcription in GDNF, GLT1, GS, NGF and OLIG2 in EDAC-treated hippocampal slices.

CONCLUSION

Our results demonstrate that EDAC has a potential pharmacological effect in brain ischemia models.

Keywords: Amburana cearensis, Neuroprotection, Glia

Supported by: FAPESB, CAPES and CNPq

08405 - Poster Session

CA.05 - PROJECT: Multiplatform Metabolomic Prospection of Biomarkers and Biochemical Aspects of Sickle Cell Disease and Osteonecrosis Secondary to Sickle Cell Disease.

Tayla Da Cruz Santos Pereira ¹, Paulo Roberto Ribeiro de Jesus¹

¹Departamento De Química Orgânica, Universidade Federal Da Bahia, (Brasil), ²Departamento De Química Orgânica, Universidade Federal Da Bahia, (Brasil)

INTRODUCTION

Sickle cell disease is the most frequent hemoglobinopathy in Brazil and is a worldwide public health problem, with a great impact on the morbidity and mortality of the affected population. In patients with this pathology, the predominant clinical manifestation of the joint is osteonecrosis, which commonly progresses to a terminal disease. Metabolite profiling approaches in complex systems have become a powerful tool to investigate metabolic processes, identify potential biomarkers and unravel metabolic reprogramming in various diseases. Thus, metabolomic studies can reveal new ways to study this pathology as well as a better understanding of the sickling process of red blood cells, dysregulated pathways, unrecognized biomarkers, and new therapeutic possibilities.

OBJECTIVES

In this context, this work aims at identifying biomarkers related to osteonecrosis secondary to sickle cell disease, using nuclear magnetic resonance and high-performance liquid chromatography in a multiplatform metabolomics approach.

MATERIALS AND METHODS

Blood plasma from control individuals and from sickle cell disease patients with and without osteonecrosis will be used. Based on the clinical stage of osteonecrosis, graded with the modified Ficat and Arlet method, the samples will be classified as early, intermediate and advanced stage of osteonecrosis. After processing the data, the identification of metabolites will be carried out using the Chenomx NMR Suite 8.4 software and the search in a database (Metlin, HMDB). The metabolic pathways will be associated through the KEGG metabolic pathway bank. The statistical treatments of the data will be performed in MetaboAnalyst 3.0 software.

DISCUSSION AND RESULTS

Preliminary results identified differences between the metabolic profile of the study groups in this work, as well as a map of metabolic alterations associated with sickle cell disease.

CONCLUSION

The present study may provide support for the validation of biomarkers for osteonecrosis secondary to sickle cell disease.

Keywords: metabolomics, osteonecrosis, sickle cell disease

08519 - Poster Session

CA.06 - Interleukin‐10 gene promoter region polymorphism is associated with worse sudden cardiac death risk and echocardiographic parameters in chronic Chagas disease

João Paulo da Silva Liberalino ^1,2, Dayane Carla Costa Paiva Dantas¹, Valeria Duarte Almeida¹, Micássio Fernandes de Andrade^1,2, Cleber de Mesquita Andrade¹, Wogelsanger Oliveira Pereira^1,2, Christiane Medeiros Bezerra³, Thales Allyrio Araújo de Medeiros Fernandes^1,2

¹Departamento de Ciências Biomédicas, Universidade do Estado do Rio Grande do Norte (Rio Grande do Norte, Brazil), ²PMBqBM-UERN, Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular (Rio Grande do Norte, Brazil), ³Departamento de Microbiologia e Parasitologia, Universidade Federal do Rio Grande do Norte (Rio Grande do Norte, Brazil)

INTRODUCTION

Functional genetic polymorphisms involved in the immune response may modulate the clinical variability in Chagas disease (CD). Several studies have proposed a protective role for IL-10 in CD pathophysiology. One study found an association between the G allele in rs1800896 single nucleotide polymorphism (SNP) and the cardiac form of CD, while others were not able to find the same results.

OBJECTIVES

This study aimed to evaluate the association between rs1800896 SNP and clinical and echocardiographic manifestations in CD patients.

MATERIALS AND METHODS

We conducted a cross-sectional study with 185 chronic CD patients, of which 95 were male and 90 females, with a mean age of 47,9 ± 11,6 years. They were genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. The clinical forms, risk of sudden death (Rassi score), the score of cardioembolic ischemic stroke scores, and echocardiographic parameters were obtained by accessing the patients' medical records. P-value < 0.05 was considered significant.

DISCUSSION AND RESULTS

We observed that 72 patients presented the AA genotype, 96 AG and 17 GG. The expected and observed genotype frequencies obeyed the Hardy-Weinberg equilibrium. The statistical analyses conducted showed no significant associations between the allelic and genotypic frequencies and the clinical forms of chronic CD (cardiac, digestive, or cardiodigestive) nor the score of cardioembolic ischemic stroke. On the other hand, the G allele was associated with higher sudden cardiac death risk (Rassi score) (p=0,025), systolic (p=0,004) and diastolic (p=0,035) left ventricular diameters above echocardiographic reference values and lower left ventricular ejection function (p=0,026).

CONCLUSION

This study found an association between the G allele of rs1800896 and worse sudden death risk and echocardiographic parameters compatible with CD dilated cardiomyopathy pathophysiology. Therefore, rs1800896 polymorphism seems to be a promising biomarker for CD, although other studies may be necessary to reinforce those findings.

Keywords: Chagas Disease, Interleukin-10, Single Nucleotide Polymorphism

Supported by: CNPq and CAPES

08678 - Poster Session

CA.07 - Molecular cloning, expression and purification of Fbxl17 fragments for antibodies produce

Camila Rolemberg Santana Travaglini Berti de Correia ¹, Patricia Passos¹, Valentine Spagnol¹, Felipe Roberti Teixeira¹

¹Departamento de Genética e Evolução, Universidade Federal de São Carlos (SP, Brasil)

INTRODUCTION

Fbxl17 is one of the 69 human F-box proteins, which interacts with SKP, Cullin and RBX to form the SCF-type E3 ubiquitin ligases. In addition to the F-box domain of interaction with SKP1, Fbxl17 contains the LRR domain (Leucine Rich Repeat) which interacts with its substrates. Analysis of 1992 patient samples from the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium), showed that FBXL17 was mutated in 135 samples in the region that encodes its LRR domain. Truncating Fbxl17 LRRs impaired its association with other SCF components and decreased its ubiquitination activity. We are investigating the consequences of FBXL17 truncations in cellular level, therefore, we are interested in producing antibodies for use in western blotting and immunofluorescence.

OBJECTIVES

Clonning, expression and purification of the N-terminus, F-box and C-terminus fragments of Fbxl17 from bacterial expression vectors. To obtain anti-Fbxl17 antibodies from these fragments that interact with Fbxl17 in mammalian cells by western blotting.

MATERIALS AND METHODS

Synthetic FBXL17 codon usage to bacteria was used as template to cloning Fbxl17 fragments into pET28a vector. Induction of heterologous expression was performed with IPTG 0,2 μM at 37 ºC for 4 hours. Insoluble Fbxl17 N-terminus were purified by affinity chromatography and submitted to SDS-PAGE. The Coomassie stained band of Fbxl17 N-terminus was excised and used to immunization of rabbits. The specificity of total immunoglobulins obtained were tested in western blotting.

DISCUSSION AND RESULTS

Cloning of the Fbx117 fragments into pET28a was confirmed by sequencing. The N-terminus fragment was expressed in insoluble fraction and purified. The anti-Fbxl17 N-terminus was specific and selective for the purified fragment, however did not probe Fbxl17 from cell extracts.

CONCLUSION

The anti-Fbxl17 N-terminus obtained was unable to identify Fbxl17 in cell extracts, but it will be used in protein-protein interaction assays when this fragment was used as a bait.

Keywords: Fbxl17, breast cancer, E3 ubiquitin ligase

Supported by: FAPESP

08993 - Poster Session

CA.08 - Development of an electrochemical device for the fast and label free detection of the biomarker ADAM-33 related to the triple negative breast cancer

Calaça G. N.¹, Lima, D.², Santos, C. S.³, Manica, G.C.M⁴, Klassen, G⁴, Pessôa, C.A.², Wohnrath, K.², Juliana Inaba ¹

¹Bioquimica, Instituto Federal do Paraná (Paraná, Brasil), ²Departamento de Química, Universidade Estadual de Ponta Grossa (Paraná, Brasil), ³Bioquimica, Instituto Federal Farroupilha (Rio Grande do Sul, Brasil), ⁴Departamento de Patologia Básica, Universidade Federal do Paraná (Paraná, Brasil)

The classification of breast cancer is defined by clinicopathologic parameters and immunohistochemical markers. Recently, the lack of expression of ADAM-33 was related to the triple negative breast cancer, basal-like markers and correlated with shorter overall survival and metastasis-free survival. Usually, the detection of ADAM-33 is performed by immuhistochemistry, which is laborious and time consuming. In this context, electrochemical immunosensors are a good alternative for protein detection as they may provide fast, straightforward, and label-free determinations. We developed an electroanalytical immunosensing platform consisting of a gold electrode modified with self-assembled monolayers of cystamine with anti-ADAM-33 IgG monoclonal antibodies covalently attached. Cyclic voltammetry and electrochemical impedance spectroscopy (EIS) experiments were employed to characterize the immunosensor. The ADAM-33 detection was based on the variation of the charge transfer resistance (∆Rct), obtained through EIS using [Fe(CN)6]3-/4- as probe, after incubating the immunosensor in the presence of the biomarker. ∆Rct increased linearly with ADAM-33 concentration in the range of 0.5-1.5 μg mL-1, with a detection limit of 0.31 μg mL-1. The immunosensor was able to successfully detect ADAM-33 in cell extracts from human breast cancer lines, without suffering significant influence from other coexisting biomolecules. The results suggest that the developed device is a promising, feasible, and sensitive tool to detect ADAM-33 in breast cancer isolated material. Thus, it could consist in an alternative to support fast breast cancer diagnosis and prognosis.

Keywords: breast cancer, ADAM-33, electrochemical immunosensor

Supported by: UEPG, Propesp, C-LABMU, Fundação Araucária, CNPq and CAPEs

08819 -

CA.09 - Direct visualization of virus removal process in hollow fiber membrane using an optical microscope

Takayuki Nishizaka ¹

¹Dept. Phys., Gakushuin University (Tokyo, Japan)

INTRODUCTION

Virus removal filters developed for the decontamination of small viruses from biotherapeutic products are widely used in basic research and critical step for drug production due to their long-established quality and robust performance. A variety of imaging techniques have been employed to elucidate the mechanism(s) by which viruses are effectively captured by filter membranes, but they are limited to ‘static’ imaging. Here, we propose a novel method for detailed monitoring of ‘dynamic process’ of virus capture; specifically, direct examination of biomolecules during filtration under an ultra-stable optical microscope. Samples were fluorescently labeled and infused into a single hollow fiber membrane comprising cuprammonium regenerated-cellulose. While proteins were able to pass through the membrane, virus-like particles (VLP) accumulated stably in a defined region of the membrane. After injecting the small amount of sample into the fiber membrane, the real-time process of trapping VLP in the membrane was quantified beyond the diffraction limit. The method presented here serves as a preliminary basis for determining optimum filtration conditions, and provides new insights into the structure of novel fiber membranes.

OBJECTIVES

For details, please refer the following publication. https://www.nature.com/articles/s41598-020-78637-z

MATERIALS AND METHODS

For details, please refer the following publication. https://www.nature.com/articles/s41598-020-78637-z

DISCUSSION AND RESULTS

For details, please refer the following publication. https://www.nature.com/articles/s41598-020-78637-z

CONCLUSION

For details, please refer the following publication. https://www.nature.com/articles/s41598-020-78637-z

Keywords: molecular imaging, dynamics of virus filtration, VLP visualization

08552 -

CA.10 - Imidazolium-based ionic liquids as additives to preserve green fluorescent protein activity at room-temperature and under stress

Nathalia Vieira Veríssimo ^1,2,3, Carolina Falaschi Saponi^1,2, Timothy M. Ryan⁴, Ricardo Pinheiro de Souza Oliveira³, Tamar L. Greaves², Jorge Fernando Brandão Pereira^5,1

¹Department of Engineering of Bioprocesses and Biotechnology, São Paulo State University (São Paulo, Brazil), ²School os Science, Royal Melbourne Institute of Technology (VIC, Australia), ³School of Pharmaceutical Sciences, São Paulo University (São Paulo, Brazil), ⁴SAXS, Australian Synchrotron (VIC, Australia), ⁵Department of Chemical Engineering, University of Coimbra (, Portugal)

INTRODUCTION

Advances in biotechnology have allowed the development of fluorescent proteins (FP) for several industrial applications. However, there are still difficulties in their use at large scales and in novel fields due to the low stability of FP, which limit their application, distribution, and storage. The discovery of additives capable of preserving the activity of FP at room temperature and under stress conditions is needed to help expand and facilitate their commercial use.

OBJECTIVES

Hence, we aimed to evaluate the use of ionic liquids (ILs) as additives capable of preserving the activity of the Enhanced Green Fluorescent Protein (EGFP), an important biomarker and biosensor, at different storage times and under unfavorable conditions.

MATERIALS AND METHODS

We evaluated the effect of 1-alkyl-3-methylimidazolium chloride-based ILs ([Cnmim]Cl) aqueous solutions on EGFP fluorescence at short (48 h) and long-term studies (3 months), and then their ability to protect the EGFP in the presence of denaturing agents.

DISCUSSION AND RESULTS

All [Cnmim]Cl ILs (at 0.100 M) were able to preserve EGFP fluorescence for longer than the phosphate-saline buffer (PBS) and NaCl solutions, increasing from 1 to 3 months. ILs solutions with shorter to medium cation alkyl chain length were the most effective for maintaining EGFP fluorescence, as well as protectors of EGFP activity in the presence of the surfactant SDS, an acid of guanidine hydrochloride, and for H2O2. [Cnmim]Cl solutions can be added to aqueous solutions to preserve EGFP fluorescence activity at room temperature for long-storage times and to reduce the negative impact of denaturing agents on EGFP.

CONCLUSION

Therefore, there is a massive potential for the application of ILs as additives to preserve FP in the long-term without refrigeration and under unfavorable conditions, which is fundamental to expanding their industrial and commercial uses.

Keywords: protein stability, ionic liquids, preservatives

Supported by: FAPESP (projects 2018/50009-8, 2014/19793-3, 2014/16424-7, 2018/25511-1, 2016/07529-5, 2018/06576-5, 2020/14144-8, 2018/01858-2, 2018/20833-0, CAPES (001), CNPq, ATN, FCT (projects UIDB/EQU/00102/2020 and UIDP/EQU/00102/2020).

08053 - Poster Session

CB.01 - A new approach for purification of the catalytic site of the Angiotensin Conversion Enzyme, N domain, mediated by the ELP-Inten system

Carolina Machado dos Santos¹, Suelen de Barros Sampaio², Fagner Santana ¹, Rodrigo Costa Leite¹, Beatriz Angelo Prata¹, Regina Affonso ¹

¹Centro de Biotecnologia, Instituto de Pesquisas Energéticas e Nucleares (São Paulo, Brasil), ²Bioquímica e Biologia Molecular, Instituto de Química, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Angiotensin-converting enzyme I, ACE, is a key part of the renin-angiotensin system whose main function is to regulate blood pressure and balance of salts in the body. ACE1 has two isoforms, somatic, sACE, and testicular, tACE. sACE possesses two domains, N- C-, with catalytic sites which exhibit 60% sequence identity. These domains differ in terms of chloride-ion activation profiles, rates of peptide hydrolysis and sensitivities to various inhibitors. N-domain has specific action in the hydrolyze of Alzheimer’s diseases beta amyloid bodies and angiotensin 1-7, which active the MAS receptor and triggering anti-thrombotic and anti-inflammatory actions.

OBJECTIVES

The objective this work was to obtain catalytic site Ala361 to Gli468 of the N-domain region, csACEN, isolation without chromatographic and denaturant chemical process.

MATERIALS AND METHODS

For that, a new methodology was used in the expression of the csACEN peptide, in which the peptide was linked to the elastin-like polypeptide, ELP, and Intein, and expressed at 37C. The characterization of catalytic site was made by SDS-PAGE and dot blotting.

DISCUSSION AND RESULTS

The culture temperature at 37C significantly increased the expression of the ELP/Intein/csACEN fusion protein. This culture was lysed at a low temperature allowing the fusion protein to become soluble. The precipitation of ELP at high concentrations of ammonium sulfate were obtained in 0.57 M and 0.8 M. Intein autocleavage occurs at acidic pH and it is important to pay attention to: pI 6.65 for csACEN and pI 6.87 for ELPcsACEN, which are very low. The best autocleavage efficiency was with MES and TriHCl buffers, pH 6.3 and 6.8, respectively, in which pure csACEn peptide was obtained.

CONCLUSION

The strategy used to obtain the Ala361 to Gli468 catalytic site in soluble and pure form was obtained with success and the protocol for obtaining similar peptides was established.

Keywords: N Catalytic site of ACE1, elastin-like polypeptide/Intein, high temperature of cultivation

08227 - Poster Session

CB.02 - ENTREPRENEURIAL UNIVERSITY: THE SEARCH FOR TRANSFER OF TECHNOLOGY FROM A SCHISTOSOMIASIS DIAGNOSTIC KIT.

Michelli Dos Santos ¹, Laís Moreira Nogueira¹, Jonatas Oliveira Da Silva¹, Mariana Teixeira Faria¹, Juliana Martins Machado¹, André Vinícius Fernandes Ferreira¹, Ana Amélia Maia Silva¹, Mariana Campos Da Paz Lopes Galdino², Alexsandro Sobreira Galdino¹

¹Laboratório de Biotecnologia de Microrganismos, Universidade Federal de São João Del-Rei, Campus Centro Oeste (Minas Gerais, Brazil), ²Laboratório de NanoBiotecnologia & Bioativos, Universidade Federal de São João Del-Rei, Campus Centro Oeste (Minas Gerais, Brazil)

INTRODUCTION

Schistosomiasis is a neglected tropical disease caused by the helminth infection from species of the genus Schistosoma. Globally, it is estimated that the disease affects over 250 million people in 78 countries of the world and is responsible for some 280,000 deaths each year. In the Americas, the only pathogenic human schistosome species is Schistosoma mansoni (S. mansoni). Available methods for the diagnosis of schistosomiasis comprise microscopic, molecular and serological approaches, with the latter detecting antigens or antibodies associated with S. mansoni. Throughout the last decades, efforts are aimed at developing news strategy for diagnostic to be used especially for low intensity infections in lowly endemic areas.

OBJECTIVES

With a view to innovation and technology transfer from the university to the private company, the objective of this work is to evaluate the optimization parameters of an ELISA diagnostic kit using rationally designed chimeric proteins.

MATERIALS AND METHODS

Thus, one of the steps evaluated was coating buffer using PBS pH7.2, Tris-HCl pH8.5 and coating buffer provided by the company were used. The conditions established for the ELISA were: 96-well polystyrene microplates, concentration of protein 140ng/well, dilution of serum 1:20, the conjugate was an anti-IgG antibody peroxidase-conjugated dilution of 1:5.000 (v/v), and measure the absorbance at 405 nm. The ratio of positive to negative was used to determine which of them is the best to discriminates the samples.

DISCUSSION AND RESULTS

The Tris-HCl pH 8.5 coating buffer showed the best discrimination between positive and negative samples when used SM1 protein; however, the company's coating buffer was better to discriminate the same samples groups when used SM2 protein. In this way, SM2 protein is more advantageous than SM1 protein and it had better results with the company’s coating buffer standardized.

CONCLUSION

In conclusion, the protein SM2 is a potential tool to be investigated for schistosomiasis diagnosis on an industrial scale.

Keywords: S. mansoni, innovation, diagnosis

Supported by: UFSJ, CNPq, and Capes

08131 - Poster Session

CB.03 - The Presence of Solvents can Induce Cubic-to-Inverted Micelar and the Cubic-to-Hexagonal-to-Inverted Micellar phase transitions on phytantriol-based cubosomes

Mayra Lotierzo¹, Bruna Renata Casadei¹, Barbara Malheiros¹, Leandro Ramos Souza Barbosa ^2,1

¹Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Universidade de São Paulo (SP, Brazil), ²General Physics, Institute of Physics, Universidade de São Paulo (SP, Brazil)

INTRODUCTION

Cubosomes are nanoparticles composed of a specific combination of amphiphilic lipids, like phytantriol (PHY), and a nonionic polymer. They have a high hydrophobic volume, as compared to regular liposomes and are good candidates for drug delivery applications. Due to their unique structure, these nanoparticles possess the ability to incorporate highly hydrophobic drugs. A challenge for the encapsulation of hydrophobic molecules is the use of organic solvents in the sample preparation process.

OBJECTIVES

In this study, we investigated the structural influence of different solvents (acetone, ethanol, chloroform, octane, DMSO, and methanol) on the inner structure of cubosomes.

MATERIALS AND METHODS

Small Angle X-ray Scattering (SAXS) technique was used to get the structural information and the phase behavior of cubosomes.

DISCUSSION AND RESULTS

The presence of high amount of acetone and ethanol (around 16% v/v) are not able to change the cubic symmetry (Pn3m), despite the increase in the unit cell parameter, observed in the case of ethanol. In both cases, a cubic-to-inverted micellar phase transition was observed. Chloroform and Octane, on the other hand, have different effects over the cubosomes as compared to acetone and ethanol, both of them induced a cubic-to-reversed hexagonal phase transition at ~0.25 and 2.5 v/v% for chloroform and octane respectively. those are attributed to the insertion of the solvent in the hydrophobic region of the cubosomes, increasing its volume and inducing such a transition. Before this phase transition, no change in the unit cell parameter was observed at smaller solvent concentrations. Moreover, a second phase transition from reversed hexagonal-to-inverted micellar was observed 1.0 and 17 v/v% for chloroform and octane, respectively. Our data also suggest that after 24 hours of solvent/cubosome incubation, some structural features of cubosomes changes.

CONCLUSION

We believe that this study could shed light on researchers working on drug delivery systems using cubosomes.

Keywords: Cubosome, nanoparticles, solvents, SAXS, phase diagram, lyotropic liquid crystal, LLC,

Supported by: FAPESP, CNPq, CAPES

08218 - Poster Session

CB.04 - RUBELLA DIAGNOSTIC ASSAY USING RP1, A NOVEL RATIONALLY DESIGNED ANTIGEN

Jonatas Oliveira Da Silva¹, Michelli dos Santos¹, André Vinícius Fernandes Ferreira¹, Juliana Martins Machado¹, Ana Amélia Maia Silva¹, Lais Moreira Nogueira¹, Mariana Campos da Paz¹, Alexsandro Sobreira Galdino¹

¹Microbial Biotechnology Laboratory (LABIOM), Universidade Federal São João Del Rei (Minas Gerais, Brasil)

INTRODUCTION

Rubella is a disease of epidemiological importance, especially when pregnant women are infected, causing congenital rubella syndrome. Thus, the specific diagnosis is essential since the clinical manifestations are similar to other diseases. In this context, the development of local antigens with high sensitivity and specificity, at low cost, can be a promising alternative compared to imported antigens.

OBJECTIVES

This study aimed to the development of an in-house IgG Rubella-specific ELISA using a novel antigen.

MATERIALS AND METHODS

The new antigen was designed and predicted by bioinformatic tools. After synthesis, two in-house ELISA assays were performed: The first one, for titration of the peptide and the other one to evaluate the reactivity of the peptide against different human sera.

DISCUSSION AND RESULTS

Two potential epitopes were predicted as the potential antigenic target was selected, originating the RP1. The results of the in-house ELISA showed an optimal concentration of 200 ng of peptide/well. This new peptide was able to discriminate between positive and negative human samples.

CONCLUSION

Our results demonstrate the potential of the peptide for the detection of rubella-specific IgG antibodies, being use full for the development of national kits in the future.

Keywords: rubella virus, german measles, diagnosis rubella

Supported by: CNPq

08407 - Poster Session

CB.06 - Evaluation of the immunomodulatory effect of the rhodium chloride/polycyclodextrin complex

Juliane de Carvalho Albuquerque¹, Paloma Esteves de Carvalho¹, Caio César de Souza Alves², Alessandra de Paula Carli², Sandra Bertelli Ribeiro de Castro¹, Jeferson Gomes da Silva¹, Ângelo Márcio Leite Denadai¹

¹Institute of Life Science, Universidade Federal de Juiz de Fora (Minas Gerais, Brasil), ²Faculty of Medicine, Universidade Federal dos Vales do Jequitinhonha e Mucuri (Minas Gerais, Brasil)

INTRODUCTION

The Rhodium chloride (RhCl3) shows several biological activities, such as anti-inflammatory, and its complexation with polycyclodextrin (RhCl3/pCD) could enhance its biological activities.

OBJECTIVES

Objectives: The aim of this study was to synthesize and characterize the RhCl3/pCD and to evaluate its immunomodulatory effect in macrophage response and in carrageenan-induced paw edema model.

MATERIALS AND METHODS

Materials and methods: Physical-chemistry characterizations of RhCl3/pCD were performed by FTIR, H-NMR and ITC. RAW264.7 was cultured in 96-well plates at 2×10 5 cells·mL -1 in supplemented RPMI-1640. Cells were maintained for 3h in the presence or absence of RhCl3/pCD. NF-κB expression was measured in RAW264.7 cells stimulated with LPS (1 μg·mL -1 ) and IFN-γ (0.9 ng·mL -1 ). The paw edema model was induced by carrageenan (2.5%) injection (20 μL) into the left footpad, and 20 μL of PBS into the right footpad, of all groups. The left and the right paws were measured at 1, 2, 3 and 4h after the injection of carrageenan and the difference were calculated. 30 minutes before the paw edema induction, the RhCl3/pCD (5 mg/Kg) were administered intraperitoneally (100 μL).

DISCUSSION AND RESULTS

Results and Discussion: FTIR and 1 H-NMR confirmed the formation of the complex in the solid state and in solution. Moreover, ITC suggest a 1:3 RhCl3/pCD stoichiometry, per monomer of pCD. The RhCl3/pCD was more potent to reduce the NF-κB expression in RAW 264.7 and J774 A.1, than RhCl3, at all tested concentrations. Moreover, both RhCl3 and RhCl3/pCD were able to reduce paw edema.

CONCLUSION

Conclusions: Both RhCl3 and RhCl3/pCD were able to reduce the paw edema. However, the RhCl3/pCD showed better ability to reduce the NF-κB expression than RhCl3, suggesting a better imunomudulator effect. Further studies are necessary to determine the molecular mechanisms of the immunomodulation.

Keywords: RhCl3, Cyclodextrin, immunomodulation

Supported by: FAPEMIG, CNPq, CAPES and UFJF

08341 - Poster Session

CB.07 - Project - Efficacy and safety of magnetic-fluorescent nanoparticles for tracking mesenchymal stem cells

Willian Pinheiro Becker¹, Gabriela Salvador Valle², Juliana Barbosa Torreão Dáu², Rosana Bizon Vieira Carias², Jasmin Jasmin¹

¹NUMPEX-BIO, Universidade Federal do Rio de Janeiro (RJ, Brasil), ²Centro de Medicina Regenerativa, Faculdade de Medicina de Petrópolis (RJ, Brasil)

INTRODUCTION

Stem cells are characterized by their capacity for self-renewal and differentiation into several specialized cell types. Interest in these cells has increased in recent years due to their plasticity and regenerative potential. For the implementation of advanced therapies, such as therapies with mesenchymal stem cells, pre-clinical and clinical studies that demonstrate the absence of adverse effects and the presence of desired therapeutic effects are essential. Stem cell tracking allows obtaining information on the lifespan and better route of cell administration, enabling a greater understanding of the effects of these new therapies.

OBJECTIVES

The superparamagnetic iron oxide nanoparticles have been explored with the aim of tracking stem cells, as they have applicability in clinical studies in the construction of magnetic resonance images, allowing the comparison of functional data with tracking data simultaneously, in addition to being biocompatible. This project proposes evaluate the efficacy and safety of magnetic-fluorescent nanoparticles to label mesenchymal stem cells for posterior tracking.

MATERIALS AND METHODS

The nanoparticles used in this project will be the Molday ION Rhodamine-B (MIRB), efficacy and safety evaluations will be carried out in mesenchymal stem cells extracted from human adipose tissue. To assess viability, the MTT colorimetric test will be performed; proliferation and cell death will be evaluated by immunocytochemistry using antibodies to anti-ki67 and anti-caspase 3, respectively; and the cells will be induced to differentiate into adipocytes, osteocytes and chondrocytes.

DISCUSSION AND RESULTS

CONCLUSION

With this project we hope to determine if MIRB is efficient and safe for tracking mesenchymal stem cells for long-term use in preclinical models.

Keywords: mesenchymal stem cells, cell tracking, superparamagnetic iron oxide nanoparticl

Supported by: FAPERJ and CNPq

08520 - Poster Session

CB.08 - PROJECT: In Silico molecular characterization of the prenyltransferase gene family and its contribution to the genetic improvement of Ricinus communis

Nara Emília Santos Benedicto¹, Paulo Roberto Ribeiro¹

¹Instituto de Química, Universidade Federal da Bahia (Bahia, Brasil)

INTRODUCTION

Ricinus communis L. is an oilseed species with great potential for pharmaceutical and other industrial applications. Thus, it is important to research mechanisms to guarantee its production despite the abiotic stress conditions that most crops are subjected to, since the largest planting in Brazil is in the semi-arid area of the northeast region. The prenyltransferase (PT) gene family is responsible for the formation of enzymes that catalyze the formation and structural modification of isoprenoids that give rise to an extensive group of natural products present in plants and involved in primary and secondary metabolisms and responses to environmental stresses. These products can have cis and trans stereochemistry and in other species, such as A. thaliana, they have highly conserved domains.

OBJECTIVES

In this project, bioinformatics will be applied to characterize this important gene family, whereas in silico gene expression patterns of PT genes will be analyzed by microarray.

MATERIALS AND METHODS

R. communis PT genes will be submitted to several bioinformatics tools such as phylogenetic analysis, identification of conserved domains, prediction of cell sublocation, physical-chemical characteristics as well as analysis of the promoter upstream regions.

DISCUSSION AND RESULTS

The presence of conserved domains PF00432, PF01239, PF01255, PF01040 and PF00348 referring to prenyltransferases identified based in PFAM domain database will assist the characterization of the genes

CONCLUSION

The results of this project aim to promote the understanding of the genes of the PT family and establish a basis for the study of this family in R. communis, as well as the possibility of developing strains more resistant to abiotic stresses.

Keywords: bioinformatics, prenyltransferases, R. communis

08431 - Poster Session

CB.09 - Development of Sars-CoV2 Spike protein binding peptides for covid diagnosis

Anderson Albino Gomes¹, Leonardo Antônio Fernandes¹, Lina Maria Salazar Echeverri¹, Ketriane Mota de Souza¹, Bruna Andersen Pereira de Jesus¹, Gustavo Felippe da Silva¹, Maria de Lourdes Borba Magalhães¹

¹DEPARTAMENTO DE PRODUCAO ANIMAL E ALIMENTOS CAV, Universidade do Estado de Santa Catarina - Centro de Ciências Agroveterinária (Santa Catatina, Brasil)

INTRODUCTION

COVID-19 is an infectious disease caused by the new coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus is highly contagious and can be transmitted through direct and indirect contact with infected people through respiratory droplets when sneezing, coughing, or even speaking. Real-time polymerase chain reaction (RT-PCR) testing is the standard reference for confirming Sars-CoV2 infection, and with the rapid increase in the number of infected people, most countries are finding a shortage of diagnostic kits.

OBJECTIVES

This project intends to develop a new molecule capable of binding to the virus that causes COVID-19 to develop new diagnostic kits.

MATERIALS AND METHODS

Through protein engineering techniques and prior knowledge about the interaction between the virus and its receptors in the human host cell, the project generated a chimeric peptide with binding capabilities to SARS-CoV-2.

DISCUSSION AND RESULTS

Computational analyses were performed to create a peptide with the binding domain of the human ACE-2 coupled to a rigid peptide that improves stability. The synthetic gene for this peptide was acquired, followed by protein expression and purification. ELISA assays using the Sars-CoV2 Spike protein as an antigen were successful.

CONCLUSION

Further studies are necessary to validate new diagnostics tests using the protein developed in this study.

Keywords: diagnosis, Immunoassays, SARS-CoV-2

Supported by: CAPES

08198 - Poster Session

CB.10 - Initial Characterization of Thin Films Containing Giant Hemoglobins

Claudemir Oliveira Souza¹, Valquíria da Cruz Rodrigues³, Lierge Ramos^1,2, Isis Sebastião^1,2, Patrícia Soares Santiago²

¹Bioquímica, Universidade Estadual Paulista "Júlio de Mesquita Filho" (São Paulo, Brazil), ²Agronomia, Universidade Estadual Paulista "Júlio de Mesquita Filho" (São Paulo, Brazil), ³Polímeros, Universidade de São Paulo (São Paulo, Brazil)

INTRODUCTION

Giant hemoglobins are currently widely studied for blood replacement systems due to their important characteristics such as size, molecular mass, number of heme and the ability to carry O₂. Recent studies have also tested their ability to sense heavy metals.

OBJECTIVES

Initially characterization of films grown with alternating layers of polyethyleneimine (PEI) and Amynthas gracilis hemoglobin (HbAg) through the action of heavy metals.

MATERIALS AND METHODS

The films are formed by 10 bilayers of PEI/HbAg grown on a printed carbon sensor for use in voltammetric measurements and analyze the growth of the film by quartz crystal microbalance (QCM) measurements, performed on the gold surface were used. The metals used are cadmium (Cd), copper (Cu) and zinc (Zn) in increasing concentrations from 0 to 20 μmol.L ^-1 for each metal analyzed.

DISCUSSION AND RESULTS

For cyclic voltammetry, the cadmium, copper and zinc metals had similar saturation points, close to 10 μmol.L ^-1 of each metal. The bilayer brought into contact with copper provided us a linearity coefficient of 0.985. For QCM, the growth of the analyzed films was satisfactory, with wide variation in frequency for each bilayer deposited.

CONCLUSION

The deposition of the material on the electrode was efficient in the analysis by QCM. The metals were sensible by the PEI/HbAg bilayer on the carbon electrode.

Keywords: Annelid, ciclic voltammetry, quartz crystal microbalance

Supported by: CAPES

08213 - Poster Session

CB.11 - Identification of beta-casein A1 and A2 in cow kinds of milk using a chicken IgY based ELISA

Bruna Andersen Pereira de Jesus¹, Lina María Salazar Echeverri¹, Anderson Albino Gomes¹, Leonardo Antônio Fernandes¹, Maria de Lourdes Borba Magalhães¹, Anelize Felício Ramos¹, Gustavo Felippe da Silva¹

¹Engenharia Florestal, Centro de Ciências Agroveterinárias (Santa Catarina, Brasil)

INTRODUCTION

Beta-caseins A2 and A1 are the most abundantly proteins found in bovine milk. Each animal, depending on its genetics, can produce proteins A1 and A2 simultaneously or independently protein A1 or A2. The difference between protein A2 (ancestral origin), is a mutation suffered in the codon responsible for position 67, with a substitution of a proline for a histidine. A bioactive peptide known as beta-casomorphine-7 (BCM7), which is derived mainly from partial proteolysis of version A1, has been linked, by several clinical studies, as the trigger of several serious disorders of the human gastrointestinal system.

OBJECTIVES

The objective of the project was to develop an immunodiagnostic method based on antibodies capable of recognizing, in a discriminatory way, beta-caseins A1 and A2 in milk samples from producing cows. With this detection using our method, due to the low cost of the methodology when compared to genetic sequencing methods, we will be able to identify A2A2 milk producing matrices of the order of thousands of animals in a relatively short time, expand the milk market A2 and ascertain the quality of batches produced in the dairy industry.

MATERIALS AND METHODS

In the immunodiagnostic test type ELISA, IgYs were used as primary antibodies from international production and another from national production. For better test reliability, the 25 animal samples were previously genotyped.

DISCUSSION AND RESULTS

The sensitivity and specificity were evaluated, obtaining sensitivity and specificity of 100% when using the commercial IgY anti-A1 antibody and 95.2% sensitivity and 100% specificity when using the national anti-A1 antibody.

CONCLUSION

Thus, the technique developed with both primary antibodies could differentiate the A2A2 milk from A1A1 and A1A2 types.

Keywords: Dairy industry, ELISA, Milk A2,

08826 - Poster Session

CB.12 - Project: Use of anti-pgl 1 serology as an active search tool for sources of infection by Mycobacterium leprae in hyperendemic municipalities – Mossoró/RN

Lais Fernanda De Pontes Santos¹, Caio Augusto Martins Aires¹

¹Departamento de Ciências Biomédicas , Universidade do Estado do Rio Grande do Norte (Rio Grande do Norte, Brasil)

INTRODUCTION

Leprosy is an infectious and contagious disease, caused by the bacillus Mycobacterium leprae, which can cause physical disabilities, becoming a worldwide public health problem. In 2019, Brazil ranked second in the detection of new cases, with Rio Grande do Norte being the state with the lowest detection coefficient, however, from 2001 to 2013, about 20% of its university students had very high or hyperendemic coefficients. Among them is the municipality of Mossoró, in the western region.

OBJECTIVES

Therefore, the present study intends to investigate the usefulness of anti PGL-1 serology through the rapid test to detect cases of multibacillary leprosy in residents of the hyperendemic area of ​​the city of Mossoró / RN, contributing to blocking the transmission of M. leprae to the community, through an innovative strategy.

MATERIALS AND METHODS

The collection of 500 people belonging to the male sex has been carried out since June 2021 at the Laboratory of the Clinical Center Vingt-un Rosado, in Mossoró / RN. The greetings are identified by the laboratory team, after identification, they are transported in a thermos box to the Laboratory of Biochemistry and Molecular Biology - BIOMOL of the State University of Rio Grande do Norte - (UERN). Serology for anti-PGL-1 analysis is performed by depositing 5 μl of serum into the lateral flow test receptacle (ML Flow) together with carrier solution. Results are interpreted after 10 minutes, and the test positivity is scaled up according to the color intensity of the test line.

DISCUSSION AND RESULTS

According to the serological analyzes of 101 that were carried out from June to July 2021, with approximately 17 analyzed weekly, a total of 8 desired tested positive and 7 were considered doubtful, even with the preliminary results, this is a project that aims to screen patients and colonize through clinical diagnoses.

CONCLUSION

Keywords: Leprosy, Epidemiology, Serology.

08361 - Poster Session

CB.13 - TOXICITY EVALUATION OF Bauhinia monandra EXTRACT CONTAINING PHYTOCHEMICALS AND LEAF LECTIN (BmoLL) ON EMBRYOS OF Biomphalaria glabrata

Luana Cassandra Breitenbach Barroso Coelho ¹, Silvio Assis de Oliveira Ferreira², Kaio Henrique de Freitas¹, Letícia da Silva Santos¹, Magda Rhayanny Assunção Ferreira³, Luiz Alberto Lira Soares³, Ana Maria Mendonça de Albuquerque Melo⁴, Mônica Camelo Pessoa de Azevedo Albuquerque⁵, Hallysson Douglas Andrade de Araújo¹, André de Lima Aires⁵, Thierry Wesley Albuquerque Aguiar¹

¹Departamento de Bioquímica, Universidade Federal de Pernambuco, (Brasil), ²Departamento de Bioquímica, Universidade Federal de Pernambuco, (Brasil), ³Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, (Brasil), ⁴Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco, (Brasil), ⁵Departamento de Medicina Tropical, Universidade Federal de Pernambuco, (Brasil)

INTRODUCTION

Biomphalaria glabrata mollusks are the main vector of schistosomiasis in Brazil and the search for natural molluscicides interrupting the parasite evolutionary cycle is necessary. Plants of the Bauhinia genus are widely found in endemic continents for the disease and leaves of Bauhinia monandra contain a BmoLL lectin with biocidal action.

OBJECTIVES

To analyze the extract (E) from leaves of B. monandra and the embryotoxic effect on B. glabrata.

MATERIALS AND METHODS

E was obtained with powder from B. monandra leaves in 10 mM citrate-phosphate buffer, pH 6.5, containing 0.15 M NaCl. Secondary metabolites were investigated by thin-layer chromatography (TLC); quantitative protein, hemagglutinating activity (HA) and specific HA (SHA) were evaluated. B. glabrata embryos at the blastula, gastrula, trocophore, veliger and hippo stages (300 of each) were exposed to E for 24 h at different concentrations (12.5-600 μg/mL). Subsequently, the embryos were washed and transferred to plates with filtered and dechlorinated water, analyzed with a magnifying glass for their viability (normal) and unviable forms (malformations and death).

DISCUSSION AND RESULTS

TLC detected cinnamic derivatives, flavonoids and saponins; protein concentration (21.2 mg/mL) and SHA (96.6) confirmed the lectin presence. In the concentration of 100 μg/mL, E provided 65.3% and 88.7% of unviable blastula and gastrula, respectively, while at 200 μg/mL 100% of unviable embryos were observed for both stages. In trocophore 400 and 600 μg/mL of E made 90.3% and 100% of non-viable embryos. At the same E concentrations, the velliger and hippo stages showed lower unviability at 400 μg/mL of 59.3% and 51.6%, respectively, and 100% of malformations and/or deaths at 600 μg/mL.

CONCLUSION

E showed teratogenic/toxic effects in all evolutionary stages of B. glabrata, proving to be an efficient molluscicide.

Keywords: Lectin, Embryotoxicity, Schistosomiasis

08193 - Poster Session

CB.14 - Bactericide activities of purified tripsin inhibidor from seeds of Enterolobium timbouva

Paula Bêlit Mazacote Zenteno¹, Caio Fernando Ramalho de Oliveira¹, Janaina de Cassia Orlandi Sardi¹, Maria Lígia Rodrigues Macedo¹

¹Instituto de Biociências – INBIO, Universidade Federal de Mato Grosso do Sul (Mato Grosso Do Sul, Brasil)

INTRODUCTION

Among the biggest problems facing the world, microorganism resistance to antibiotics could, by 2050, kill more than 10 million people. Within the biomolecules studied for new antibiotics, protease inhibitors from plant organs have shown high antimicrobial and antibiotic potential.

OBJECTIVES

In this study we aimed to verify the bactericidal, antibiofilm and synergistic activities of a trypsin inhibitor from the seeds of Enterolobium Timbouva (EtTI).

MATERIALS AND METHODS

Using five Gram-positive and five Gram-negative bacterial species, the minimum inhibitory concentration was defined as the lowest concentration of EtTI capable of inhibiting visible microbial growth by the Müller Hinton broth microdilution technique. The minimum bactericidal concentration was the lowest concentration of EtTI that did not allow bacterial growth on solid medium. The effects of EtTI on bacterial biofilm inhibition and on mature biofilms were done in 96-well microplates and after the incubation period, seeded onto BHI agar plates for counting. Mechanism of action: SYTOX Green was used as indicator of membrane damage. To evaluate the synergistic activity, the test, also called "chessboard", was based on the CLSI protocol.

DISCUSSION AND RESULTS

EtTI had a MIC/MBC for S. haemolyticus, S. saprophyticus and S. epidermidis and only MIC for E. coli. EtTI showed the best result for the S. saprophyticus strain, with MIC and MBC of 4.5 μM. This strain was the chosen for antibiofilm treatments with MIC and 10 X MIC that caused reduction 61,5% in biofilm formation and 50% in mature biofilm. EtTI had a linear damage kinetics pattern, with 48% death after 2 hours. In addition, EtTI was shown to be synergistic with the standard drug vancomycin (ΣFIC: 0,28μg/mL), reducing the MIC value for EtTI by 32 times and the MIC of vancomycin by 4 times.

CONCLUSION

In conclusion, the biological activity of EtTI shows the potential as antimicrobial and, as further steps are taken, possible drug.

Keywords: Biofilm, Synergism , SYTOX Green

Supported by: FUNDECT

08954 - Poster Session

CB.15 - Metal-Organic Frameworks Formed by Nucleotides, Transition Metals and Quantum Dots

Renata Lara Mori¹, Lucas Rodrigues de Mello¹, Emerson Rodrigo da Silva¹

¹Departamento de Biofísica, Universidade Federal de São Paulo (SP, Brazil)

INTRODUCTION: Nucleotides are excellent metal ligands able to stablish coordination interactions with metal ions, specially with transition metals. The co-assembly of nucleotides and metal ions through coordination interactions at specific sites give raise to nanoscopic scaffolds called metal-organic frameworks (MOFs). These materials display a range of promising applications in biomedicine including drug loading, molecular delivery and bioimaging. In the current work, we perform the synthesis and structural characterization of MOFs based on coordination polymers formed between nucleotides, metal ions and quantum dots. OBJECTIVE: our purpose is to develop a synthesis protocol to produce complexes involving purine-based nucleotides, metal ions and ZnO quantum dots. We aim to provide detailed characterization on the structure of these nanostructured materials and test their in vitro behavior toward tumor cell lines. MATERIALS AND METHODS: the synthesis protocol consists in the solubilization of guanosine or adenosine monophosphates, which have versatile binding sites, in the presence of metal ions such as Ca, Mg, Mn, Fe, Co, Cu, Ni and Ag. ZnO quantum dots (QDs) are prepared through a hydrolisis reaction of zinc acetate in the presence of sodium hydroxide and methanol. This procedure leads to the formation of QDs that emit greener light upon UV irradiation. The QDs are co-solubilized with nucleotides and metal ions leading to the spontaneous formation of nanoparticles over a course of hours or days. To perform structural and morphological characterization of the complexes, we have used fluorescence microscopy, atomic force microscopy (AFM), fluorimetric assays and circular dichroism (CD). RESULTS AND DISCUSSION: the characterization techniques allowed to select complexes that are most viable for the research. Interestingly, metal ions that participate in biological roles (e.g., Ca and Mg) have led to better results in comparison to those that do not have a biological function. The formation of complexes has been observed for atoms with neighbors regarding their valence layers and having relatively similar atomic masses. Importantly, the incorporation of ZnO has been observed and complexes have been found to form well ordered, crystal-like, nanoscopic assemblies. CONCLUSION: a viable protocol to produce ordered nanoparticles with QDs embedded in the crystal structure has been developed. From the results obtained so far, the nanostructured materials are promising for tests with cell membranes.

Keywords: coordination polymers, quantum dots, metal-organic frameworks

Supported by: CNPq

08480 - Poster Session

CB.16 - PROJECT Characterization of the antifungal activity of metabolites from native brazilian plants against the strains of the genus Cryptococcus and Candida

Mônica Maria de Almeida¹, Karen Luise Lang¹, Gabriella Freitas Ferreira¹

¹Multicêntrico em Bioquímica e Biologia Molecular (PMBqBM), Universidade Federal de Juiz de Fora campus Governador Valadares (Minas Gerais, BRASIL)

INTRODUCTION

The frequency of invasive fungal infections caused by opportunistic pathogens has increased worldwide. Cryptococcosis and candidiasis are systemic fungal infections of great medical relevance worldwide. Therapeutic options for the treatment of these infections are restricted, both because of the increased resistance of Cryptococcus and Candida strains and because of the drugs used and the high toxicity of antifungal agents for the human body, all of which have made drug treatment difficult. Thus, the search for prototypes for the development of new antifungal agents is an important public health issue and requires investment in research so that new drug candidates can be discovered.

OBJECTIVES

Thus, this work aims to characterize the antifungal activity of native plants in Brazil against strains of the genus Cryptococcus and Candida.

MATERIALS AND METHODS

The extracts and fractions and/or subfractions with the best anticryptococcal and anticandida activity will be subjected to chemical characterization (high performance liquid chromatography and nuclear magnetic resonance) and cytotoxicity analysis. Regarding the antifungal activity, the Minimum Inhibitory Concentration (MIC) and the fungicidal/fungistatic concentration against Cryptococcus and Candida strains will be determined. After this initial screening, tests will be carried out to understand the effect of extracts on the oxidative stress of fungal cells (dosing of antioxidant enzymes, reactive oxygen and nitrogen species, and analysis of mitochondrial and lysosomal stability by flow cytometry) in the cell membrane (ergosterol dosage), cell wall (sorbitol test), capsule (morphometry and zeta potential) and cell volume (morphometry).

DISCUSSION AND RESULTS

CONCLUSION

Thus, it is expected that this project can contribute to the management of candidiasis and cryptococcosis, since native plants are an excellent option for these diseases that are often neglected.

Keywords: Cryptococcus, Candida, treatment

Supported by: FAPEMIG

08766 - Poster Session

CB.17 - Detection of SARS-Cov-2 Protein Using an ACE2-Like Protein

Leonardo Antonio Fernandes¹, Anderson Albino Gomes¹, Lina Maria Salazar Echeverri¹, Bruna Andersen Pereira de Jesus¹, Ketriane Mota de Souza ¹, Maria de Lourdes Borba Magalhães¹, Gustavo Felippe da Silva¹

¹Departamento de Engenharia Florestal, UNIVERSIDADE DO ESTADO DE SANTA CATARINA (Santa Catarina, BRAZIL)

INTRODUCTION

COVID19 is a respiratory disease caused by the SARS-CoV-2 virus and is transmitted by aerosols. The penetration of SARS-CoV-2 into respiratory tract cells is mediated by the Spike protein using the ACE-2 (angiotensin-converting enzyme 2) receptor as a gateway. The structure of ACE-2 responsible for the interaction with the viral Spike protein is constituted by an alpha-helix. Based on this, by protein engineering technique, an ACE-2 peptidomimetic denominated K2ECA2 was developed to bind SARS-CoV-2 spike protein.

OBJECTIVES

The aim of this study is to develop new technologies for SARS-CoV-2 detection.

MATERIALS AND METHODS

The protein was successfully expressed in E. coli and purified to homogeneity. The protein gene was cloned into pET32a and transformed into E. coli BL21 PLysS bacteria for expression. ELISA assays were performed by sensitizing a 96-well plate with 75 ng ECD (Spike Extracellular Domain) protein and detected with 5 μg/mL biotinylated K2ECA2 protein. A Dot Blot was performed by immobilizing 100 ng of ECD on a nitrocellulose membrane and detected with increasing amounts of K2ECA2 protein.

DISCUSSION AND RESULTS

Protein was expressed during 6 hours of induction. In both ELISA and Dot Blot, the recombinant protein was able to detect the immobilized ECD protein. The K2ECA2 protein was able to detect the ECD portion of the Spike protein of SARS-CoV-2.

CONCLUSION

More complex trials using samples obtained from infected patients will be performed.

Keywords: COVID19, Diagnosis , Immunoassays

Supported by: CAPES

08536 - Poster Session

CB.18 - Construction of a recombinant chimeric protein using epitopes of T. evansi ISGs75

Gabriella Bassi das Neves¹, Amanda Martins Ungri ¹, Júlia Marques ¹, Lina Maria Salazar Echeverri¹, Sandra Regina de Mello¹, Luiz Claudio Miletti¹

¹Departamento de Produção Animal e Alimentos, Universidade do Estado de Santa Catarina (Santa Catarina, Brasil)

INTRODUCTION

Trypanosoma evansi, the causative agent of Surra, main strategy of evading the host's immune systemis antigenic variation, where VSGs undergo remodeling instead of ISGs that do not present this variation. Previous bioinformatics analyzes demonstrate a variety of unique epitopes in T. evansi ISGs 75. This fact, combined with the potential use of chimeric proteins, can enhance their use for differential diagnosis. In which, these tests using recombinant antigens generally have greater specificities when compared to raw antigens.

OBJECTIVES

The aim of this work was the expression of a chimeric protein consisting of sequential peptides epitopes of the T. evansi ISGs75.

MATERIALS AND METHODS

The synthesis of peT31 with the peptide chimera was made by the company FastBio. The expression was made according to the peT31 manual. To determine the best expression time, the total protein profile was analyzed before the addition of IPTG, T0 (time zero) and after (0.1 mM IPTG) until T6 (six hours). Pellets were sonicated (0°C) and centrifuged to obtain total proteins.

DISCUSSION AND RESULTS

The analysis and visualization of the protein profile (E. coli, pLysS) and determination of the best expression time was performed by SDS-PAGE. And confirmation of the expression was made by marking the His-Tag by Dotblotting. In the analysis of the electrophoretic profile of the cell lysate, bands of proteins with different molecular masses were found. The recombinant protein is located at the 21KDa position, between 20 and 29 kDa markers, and the best expression time was 6 hours. For confirmation, the soluble and insoluble T6 fractions were subjected to Dotbloting and anti-His-Tag antibodies were detected.

CONCLUSION

Successful construction and expression of the recombinant chimera was achieved. It was possible to confirm the presence of the recombinant protein and from that, new studies will be developed in order to use the chimera as a target for diagnosis or vaccine production.

Keywords: Glycoprotein, Recombinant protein, Surra

08746 - Poster Session

CB.19 - PROJECT: Biochemical and physiological effects of plant growth promoting bacteria (PGPB) as nutrients solubilizer in the soil during the growth and vegetative development of Zea mays

Higor Oliveira dos Santos Borges ¹, Rafael Da Conceição Simões ¹

¹Centro das Ciências Biológicas e da Saúde - CCBS, Universidade Federal do Oeste da Bahia (Bahia, Brasil), ²Centro das Ciências Biológicas e da Saúde - CCBS, Universidade Federal do Oeste da Bahia (Bahia, Brasil)

INTRODUCTION

Corn (Zea mays L.), belongs to Poaceae family, originates from a tropical American region. The adaptive qualities are a result of the work done by Native Americans. However, the domestication of primitive corn became extremely dependent on man. Submitted to suitable field conditions, the seed develops from the absorption of water, starting its plant development. The corn crop presents particularities in the absorption of chemical compounds during its cultural cycle. Fertilization using chemical compounds as a means of nutritional gain for the soil is one of the pillars of modern agriculture. However, the exaggerated use of mineral fertilizers in agriculture has become a problem. Plant growth promoting bacteria (PGPB) are microorganisms known in the literature due to their great potential for agricultural production. Plant macronutrients such as phosphorus are often found in large amounts in Brazilian soils, but the vast majority are not available for absorption due to mobility, insolubility or their structural shape. In this context, the PGPB present a sustainable alternative for soil management, acting as biofertilizers, which can help in cultural growth and development, providing micro and macronutrients for plant absorption

OBJECTIVES

The aim of this master's degree project is to evaluate the physiological and biochemical changes of corn seedlings germinated in different PGPB preparations.

MATERIALS AND METHODS

Physiological parameters such as germinability, emergence speed, fresh mass, radicle and shoot growth and biochemical parameters such as Sugar Composition, Production of Non-enzymatic Antioxidant Compounds, Activity Determination of Catalase and Superoxide Desmutase Antioxidant Enzymes, Identification of Isoform of SOD in addition to evaluation of the micronutrient and macronutrient content of the soil before planting and after seedling establishment

DISCUSSION AND RESULTS

With the results obtained, it is expected to indicate a PGPB lineage capable of improving corn production.

CONCLUSION

Keywords: Corn, Antioxidant Activity, Plant Biochemistry

08426 - Poster Session

CB.20 - Histopathological evaluation of ZEUS® toxicity on the gills of Oreochromis niloticus

Alaide Cristina de Bem Matos¹, Caroline Kuhnen Lima², Mariele Abadia Elias³, Indianara Fernanda Barcarolli⁴, Carla Ivane Ganz Vogel⁵, Karim Hahn Luchmann⁶

¹PPGBQBM, Universidade do Estado de Santa Catarina (Santa Catarina, Brasil), ²Medicina Veterináia, Universidade do Estado de Santa Catarina (Santa Catarina, Brasil), ³Biotecnologia, Instituto Federal de Santa Catarina (Santa Catarina, Brasil), ⁴Engenharia Ambiental e Sanitária, Universidade do Estado de Santa Catarina (Santa Catarina, Brasil), ⁵Produção Animal, Universidade do Estado de Santa Catarina (Santa Catarina, Brasil), ⁶Educação Científica e Tecnológica, Universidade do Estado de Santa Catarina (Santa Catarina, Brasil)

INTRODUCTION

Corn is one of the most cultivated crop worldwide, but is commonly affected by stink bugs. To avoid crop losses due to pests, insecticides have been used. However, insects acquire resistance, requiring the use of new compounds. One of them is the insecticide ZEUS® (Ihara), a combination of dinotefuran and lambda cyhalothrin, produced in order to reduce their environmental impacts.

OBJECTIVES

Therefore, our objective was to analyse the histopathological effects of sublethal concentrations of ZEUS® on the gills of Nile tilapia (O. niloticus).

MATERIALS AND METHODS

Juveniles tilapia were purchased from a commercial fish farm in Santa Catarina state, Brazil, acclimatized for 81 days, and then exposed for 96 hours to 0.01 mg.L-1 (T1), 0.05 mg.L-1 (T2) and 0.1 mg.L-1 (T3) ZEUS® concentrations, in addition to the control group (CT). After the exposure period, fish were sacrificed and their gills were fixed in buffered formaldehyde, followed by dehydration, diaphonization and inclusion in paraffin. The hematoxylin and eosin stained slides were analysed under light microscope at 1,000 x.

DISCUSSION AND RESULTS

The histopathological alterations in the gills were hyperplasia, lamellar fusion, cell necrosis, parasitosis, and aneurysm. For all ZEUS® concentrations, the most frequent damage was the aneurysm, which can lead to the disruption of pillar cells and structural disarrangement of the branchial lamellae, possibly affecting the gas exchange and osmotic regulation. In general, fish of CT, T1 and T2 groups were classified as containing soft damages, while the T3 group presented moderate damage. Although damage was apparently different among treatments, there was no statistical differences between exposed and control group.

CONCLUSION

Therefore, we can suggest that 0.1 mg.L-1 of ZEUS® can cause concerning histopathological changes on O. niloticus gills.

Keywords: Histology, Insecticide, Toxicity

08437 - Poster Session

CB.21 - Selection of binding peptides against the Spike protein of SARS-CoV2 using Phage display

Lina Maria Salazar Echeverri ¹, BRUNA ANDERSEN PEREIRA DE JESUS¹, LEONARDO ANTONIO FERNANDES¹, ANDERSON ALBINO GOMES¹, MARIA DE LOURDES BORBA MAGALHAES¹, GUSTAVO FELIPPE DA SILVA¹

¹Departamento de engenharia Florestal, Universidade do estado de Santa Catarina centro de ciências agroveterinarias (Santa Catarina, Brasil)

INTRODUCTION

The worldwide outbreak caused by the pathogen called SARS-CoV-2 originated from Wuhan in China has spread to 6 continents and claimed to date over 4 million human lives. Therefore, the development of fast diagnostic strategies, therapy, and vaccines is urgent. On the other hand, the Spike protein of SARS-CoV-2 and its proteins derivatives such as the ECD (ExtraCellular Domain) or even the tiny receptor-binding domains (RBD) has become an essential target to many researchers since these proteins are needed to recognize the ACE2 human cell receptors and subsequent invasion. Therefore, specific ligands identified against RBD or ECD may be required tools for developing new diagnostic or even drug candidates. The phage display technology allows the display of peptides or proteins on the surface of bacteriophages, a technique used to identify specific ligands to virtually any biological target moderately fast.

OBJECTIVES

Our goal was to test a synthetic Kunitz phage-displayed library to discover specific ligands to the ECD SARS-CoV-2.

MATERIALS AND METHODS

After five rounds of library selection against ECD protein immobilized on ELISA microwells, phages binders were collected and re propagated in E. coli XL1 Blue bacteria. Input and output Phage populations were quantified on each selection round to assess the evolution of sub-libraries. In the last round, some colonies were selected, amplified individually, and tested as monoclonal phage in ELISA.

DISCUSSION AND RESULTS

We obtained two clones that showed strong binding to the ECD protein compared to the control. Thus, our synthetic Kunitz phage display technique was able to identify specific ligands against SARS CoV-2 ECD protein.

CONCLUSION

The phage display technique proved to be efficient for detecting ligands and possible inhibitors

Keywords: Peptide library, ELISA, Kunkel mutagenes

Supported by: UDESC

08674 - Poster Session

CB.22 - Ultrastructural and viability analysis of biofilm formed by oral streptococci under the action of N-acetylcysteine

Gisele da Silva Sarkis¹, Ana Angélica Macedo², Fernando Mendes³, Daniel Saito¹, Cristiane Pereira Borges Saito¹

¹Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular, Universidade do Estado do Amazonas (Amazonas, Brasil), ²Laboratório de Pesquisa, Instituto Federal do Maranhão (campus Imperatriz) (Maranhão, Brasil), ³Instituto Politécnico de Coimbra, Escola Superior de Tecnologia da Saúde de Coimbra (Coimbra, Portugal)

INTRODUCTION

Biofilm is the name given to the aggregated microbial life form, possible through the adhesion of these microorganisms to a matrix or extracellular polymeric substance (EPS), which provides stability, protection and resistance to disinfecting agents. N-acetylcysteine (NAC) has been proposed as an alternative for the control of bacterial biofilm in several human infections, as it disturbs the stability of the biofilm matrix and exposes microorganisms to antimicrobials.

OBJECTIVES

The aim of this study is to evaluate the ultrastructure of oral biofilms, under the action of NAC, formed by oral streptococci from the groups: S. gordonii, S. mitis, S. mutans, S. oralis, S. salivarius, S. sanguinis, and the structure of the multispecies biofilm, under scanning electron microscopy (SEM) and confocal microscopy (MC), in hydroxyapatite (HA) discs.

MATERIALS AND METHODS

The bacterial inoculum was standardized by means of spectrophotometry due to optical density (630 nm). Final bacterial concentrations of 1.5x10 ⁸ CFU/mL were obtained, which corresponds to tube No. 0.5 on the MacFarland scale. Biofilm susceptibility to NAC at different concentrations (0.78 to 25 mg/mL) was evaluated by colorimetric assay with crystal violet.

DISCUSSION AND RESULTS

NAC at a concentration of 12.5 mg/mL was shown to inhibit biofilm formation for all species, while, concentrations of 0.78 to 25 mg/mL inhibited biofilm formation by S.oralis. On the next step, HA discs will be soaked in saliva and then distributed in 24-well microplates along with bacterial inocula to obtain mono and multispecies biofilms. Biofilm degradation activity will be evaluated under different concentrations of NAC: 6,25 and 12,5 mg/mL. The ultrastructure of biofilms after treatment will be analyzed under SEM and CM, using SYTO 9 (excitation wavelength of 488 nm and emission wavelength of 500-550 nm) to analyze the biofilm in x–y planes. The parameters of the coefficient of thickness and roughness of biofilms will be measured using the MATLAB software.

CONCLUSION

Keywords: oral biofilm, orals streptococos, scanning electron andconfocal microscopy

Supported by: Fundação de Âmparo à Pesquisa do Amazonas

08522 - Poster Session

CB.23 - PROJECT: Ecotoxicological effects of microplastic leachates in Ruditapes decussatus

Juliano Marcelo Vilke^1,2, Maria Bebianno², Karim Lüchmann¹

¹Departamento de Educação Científica e Tecnológica, Universidade do Estado de Santa Catarina (Santa Catarina, Brasil), ²Centro de Investigação Marinha e Ambiental, Universidade do Algarve (Faro, Portugal)

INTRODUCTION

Plastics are the main category of waste reported in the oceans; they are ubiquitous in all ecosystems, especially marine environments. These compounds became notably important because they are made from raw materials like natural gas or refined crude oil; most are not biodegradable. Plastics can be produced in different size scales, from macro-, meso-, micro- and nanoplastics. When in the marine environment they can suffer from the action of waves, photodegradation, and weathering, leading to their fragmentation into smaller particles. These processes can also leach of their structural chemical components, which in turn might cause negative effects on marine biota, especially on filter-feeding organisms.

OBJECTIVES

Therefore, this project aims to evaluate the response of biochemical biomarkers in different tissues of the clam Ruditapes decussatus exposed to microplastic leachates under laboratory conditions.

MATERIALS AND METHODS

Clams will be exposed for 30 days to two environmentally relevant concentrations of plastic leachates (1 mg/L and 100 mg/L), made from plastics collected at different beaches of the Algarve coast, south Portugal. The biochemical biomarkers include those involved in the antioxidant defence system: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), xenobiotic biotransformation system: glutathione S-transferase (GST), oxidative damage: levels of lipid peroxidation (LPO) and protein carbonylation (PC), neurotoxicity: acetylcholinesterase (AChE), genotoxicity (comet assay) and filtration rate (FR). Chemical analyses will be performed to identify the chemical compounds present in the microplastics leachate. Finally, bioaccumulation analysis of contaminants will also be evaluated in the clam tissues.

DISCUSSION AND RESULTS

Overall, we expect to provide valuable information on the toxicity of microplastics leachate in order to guide future decisions on the use and destination of plastics worldwide

CONCLUSION

Keywords: biomarkers, environmental pollution, chemical analysis

Supported by: RESPONSE - JPI OCEANS

08747 - Poster Session

CB.24 - Bothrops jararacussu ontogenetic venom variability: from birth to adulthood

Weslei da Silva Aguiar^1,2, Nathália da Costa Galízio^1,2, Kathleen Fernandes Grego¹, Giovanni Perez Machado da Silveira¹, Karen de Morais-Zani¹, Anita Mitico Tanaka-Azevedo¹

¹Laboratório de Herpetologia, Instituto Butantan (São Paulo, Brasil ), ²Instituto de Ciências Biomédicas, Universidade de São Paulo (São Paulo, Brasil )

INTRODUCTION

Variability in activity and composition of venoms is a fact previously described and occurs at various levels, such as ontogenetic, geographic, sexual, diet, inter and intra-specific. Ontogenetic changes are observed in snake species from different genus. Bothrops jararacussu snake belongs to Viperidae family. These individuals show sexual dimorphism in both size and color. Their diet undergoes ontogenetic change, young individuals feeding on ectothermic animals and adults predominantly preying on small mammals. Individuals of this species have large amounts of myotoxic PLA₂ in their venom composition.

OBJECTIVES

This present study aims to perform an ontogenetic analysis of venom from B. jararacussu, following the development of newborn individuals in Instituto Butantan until adulthood.

MATERIALS AND METHODS

Venom samples were obtained from B. jararacussu snakes born from the same litter at the Laboratory of Herpetology of Instituto Butantan. Venom samples were subjected to SDS-PAGE, followed by HPLC and L-amino acid oxidase enzymatic activity.

DISCUSSION AND RESULTS

A protein band within the range of 15-10 kDa appears on male individuals around 9 months of age, while in females this band appears around 15 months. HPLC profile shows a peak eluted around 45-minute that also increases over time, while the peaks eluted at 80-90 region decrease in same proportion. This peak/band, possibly a PLA₂ with myotoxic activity, increases in intensity with each subsequent extraction. LAAO activity was higher in female individuals up to 15 months of age, when the difference with male individuals started to became less noticeable.

CONCLUSION

We observed that transition of protein profile from young to adult occurs around 15 months in female and 9 months in male individuals. We observed that this change happens gradually. A trend of increased LAAO activity in these individuals was observed. The greatest advantage of our work was following the growth of venom from same individuals, from birth to adulthood.

Keywords: variability, ontogeny, biotechnology

Supported by: CAPES, CNPq, FAPESP (2018/25786-0; 2017/16908-2; 2018/20651-0)

08376 - Poster Session

CB.25 - Structural and mechanical characterization of self-assembled guanosine-hydrogels loaded with methylene blue

Juliana Sakamoto Yoneda ^1,4, Daniele Ribeiro de Araujo², Francesco Spinozzi³, Paolo Mariani³, Rosangela Itri¹

¹Física Aplicada, Instituto de Física - Universidade de São Paulo (São Paulo, Brasil), ²Centro de Ciências Naturais e Humanas, Universidade Federal do ABC (São Paulo, Brasil), ³Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche (, Italy), ⁴Brazilian Synchrotron Light Laboratory , Brazilian Center for Research in Energy and Materials (São Paulo, Brasil)

INTRODUCTION

Hydrogels are attractive biomaterials finding many applications, including the biomedical field. Guanosine is a low molecular weight gelator, being a building block to produce supramolecular hydrogels. Its self-assembling in aqueous medium can produce flexible and long fibers (G-quadruplex), which can entangle into a 3D network. Being a natural compound, the guanosine-derived hydrogel exhibits biocompatibility and biodegradability, which promote its bio-application.

OBJECTIVES

The aim was characterizing the guanosine-based hydrogels loaded with methylene blue (MB) comparing to the drug-free formulation.

MATERIALS AND METHODS

Stable hydrogels were formed by mixing: Guanosine (G)-neutral and Guanosine 5’-monophosphate (GMP)-negatively charged (G:GMP molar ratio = 1:6; 1:2; 1:1). AFM, rheology and SAXS were applied to study the structural and mechanical features of the G-hydrogels.

DISCUSSION AND RESULTS

AFM revealed the G-wires in the sample containing MB like those in the absence. The Tgel-sol obtained by rheology was 62.3°C, 58.2°C, and 47.2°C for MB-free and 62.2°C, 57.4°C and 45.5°C for MB-loaded to 1:1, 1:2, and 1:6 samples, respectively. The MB-loaded hydrogels were less viscous compared to the MB-free, mainly for the 1:6 sample, due to the higher GMP proportion, enhancing the electrostatic interaction with MB (positively charged). The volume-fraction distribution of the different chemical groups from SAXS showed the MB position overlaps with the ribose and phosphate groups, with slight differences related to the G:GMP and MB concentration. It was shown that the Gibb’s free energy for MB binding to GMP is lower (-30 kJ/mol) than to G, (-20 kJ/mol) demonstrating a preference of MB to bind to GMP. Other interesting properties of guanosine-hydrogels such as a pH-dependent swelling behavior and drug release, besides the self-healing property were demonstrated.

CONCLUSION

Concluding, the encapsulation of a drug model did not significantly perturb the structure of guanosine-based hydrogel. The results qualify the G:GMP hydrogel as an excellent biomaterial that can entrap and deliver bioactive molecules.

Keywords: biomaterial, drug encapsulation, supramolecular hydrogel

Supported by: FAPESP - grant number: 2018/07194-9, CNPq and CAPES

08817 - Poster Session

CB.26 - PROJECT: COMPARATIVE STUDY ON YIELD, CHEMICAL COMPOSITION AND ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS FROM Croton blanchetianus Baill FROM DIFFERENT POPULATIONS IN SERRA DO LIMA, PATU-RN

Carlos Walber Batista Henrique ¹, Cynthia Cavalcante de Albuquerque¹

¹CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE DO ESTADO DO RIO GRANDE DO NORTE (RIO GRANDE DO NORTE, BRASIL)

INTRODUCTION

Croton blanchetianus Baill (Quince), belonging to the Euphorbiaceae family, is a widespread species in the Caatinga biome of northeastern Brazil. Several studies report the importance of this species' essential oil for folk medicine and pharmaceuticals.

OBJECTIVES

Therefore, the objective of this study will be to analyze the yield, chemical composition and antifungal activity of essential oils from different populations of C. blanchtianus from Serra do Lima in Patu, Rio Grande do Norte

MATERIALS AND METHODS

For the study, branches and leaves will be collected over a year, both in the dry season and in the rainy season, at 6, 12 and 17 h. The collected plant material will be packed in nylon bags and taken to the Plant Physiology and Biochemistry Laboratory (LFBP) for the extraction of essential oils, which after being extracted will be dehydrated and sent for analysis of chemical compounds by means of gas chromatography. The oils will also be evaluated for their antifungal activity against Fusarium oxysporium, an important pathogen that causes disease in bananas. For this step, tests will be carried out on spore germination and mycelial antifungal activity, using a 96-well plate, each well containing 100 μL of different concentrations of essential oils at different concentrations and 100 μL of potato-dextrose broth containing spores . To inhibit the mycelia, a thin layer of PDA medium will be deposited in a Petri dish, where they will be deposited on a solid surface, the plates will be incubated at 35°C for 72h and the diameters of the inhibition zones will be observed every 24h

DISCUSSION AND RESULTS

It is expected to identify in which period of the year (dry or rainy) there is greater production of essential oils, and if there will be any change in their chemical composition. It is expected to prove antifungal activity of the oil against the deposited fungus.

CONCLUSION

Keywords: Antifungal activity, C. blanchetianus, Essential oil

08386 - Poster Session

CB.27 - PROJECT - ANALYSIS OF THE EFFECT OF EXPOSURE TO PESTICITIES IN PREGNANTS AND NEWBORNS AND ITS RELATIONSHIP WITH CHANGES IN OXIDATIVE METABOLISM AND DNA REPAIR

Graziela Rodrigues Ródio ¹, Ana Paula Carneiro Brandalize¹, Isac George Rosset¹

¹Programa Multicêntrico de Pós-graduação em Bioquímica e Biologia Molecular, Universidade Federal do Paraná (Paraná, Brasil), ²Programa de Pós-Graduação em Biotecnologia, Universidade Federal do Paraná (Paraná, Brasil), ³Faculdade de Medicina, Universidade Federal do Paraná (Paraná, Brasil)

INTRODUCTION

In today's world human beings are exposed to pesticides throughout their entire lives, including during pregnancy due to the passage of these products via the placenta. Exposure to these compounds has been associated with changes in oxidative metabolism and DNA, with consequences on the development and health of the newborn. Despite the evidence pointing to a relationship between fetal exposure to pesticides and the development of diseases, knowledge about the genotoxic effects of exposure to pesticides on the fetus is negligible, with no conclusive studies.

OBJECTIVES

Therefore, the objective of this work is to evaluate the association between exposure to pesticides, occurrence of DNA damage and changes in DNA repair pathways in mothers and newborns.

MATERIALS AND METHODS

The present study will be carried out from maternal peripheral blood and umbilical cord blood samples from newborns treated at a city hospital in Palotina/PR. The samples will be submitted to tests to detect the presence of organophosphates, carbamates, organochlorines and glyphosate, as well as to verify possible damage to the genetic material through micronucleus tests, comet assay and expression tests of genes encoding repair proteins. In addition, a possible state of oxidative stress will be investigated by checking the activity of the enzyme Glutathione Reductase, the products of lipid peroxidation and the levels of 8-oxodg.

DISCUSSION AND RESULTS

It is expected with this to verify the existence of transplacental passage of pesticides, and consequent exposure of the fetus still in the gestational period. Through this, we will be able to understand how exposure to these products can damage the genetic material and interfere with maternal-fetal health.

CONCLUSION

Keywords: DNA damage, Pesticides, Pregnancy

08697 - Poster Session

CB.28 - Project: Development oh the Genome Surveillance Methodology of SARS-CoV-2 Independent of Genetic Sequencing

Mayanna Moreira Costa Fogaça ¹, Jaime Henrique Amorim Santos¹

¹Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular, Universidade Federal Do Oeste Da Bahia (Ba, Brasil)

INTRODUCTION

COVID-19 is a disease caused by the Sars-CoV-2 virus that belongs to the Coronaviridae family and the Betacoronavirus genus, which comprises a group of large RNA positive viruses simple tape. This disease, which currently affects more than 205 million confirmed cases of COVID-19 in the world, and more than 4 million deaths, due to the high transmissibility and the seriousness of the cases. The COVID-19 pandemic has generated a worrying epidemiological situation that highlights the importance of epidemiological surveillance in ensuring effective actions for rapid diagnosis, tracking of the disease for early control, in order to minimize transmission and prevent future outbreaks. Among the laboratory techniques used, the test considered standard for diagnosis is the RT-PCR, and for genomic analysis and alterations it is the genetic sequencing method. However, one of the biggest concerns is the emergence of viral variants, with genetic mutations that confer greater transmissibility to the virus, which emphasizes the need for studies of methodologies that help genomic surveillance more quickly.

OBJECTIVES

This study is an applied research that will be carried out at the Laboratory of Infectious Agents and Vectors (LAIVE) of the Federal University of Western Bahia - UFOB, which aims to develop an alternative method of genomic surveillance, in a less expensive and simpler way.

MATERIALS AND METHODS

An analysis of the genomic sequences of SARS-CoV-2 will be performed to find conserved regions that will be targeted for amplification and subsequent enzymatic restriction. Through the analysis of the restriction fragmanides polymorphism (RFLP) it is expected to find a specific pattern that identifies the different viral variants.

DISCUSSION AND RESULTS

CONCLUSION

With this research, it is expected that an alternative, simpler and cheaper genomic surveillance methodology capable of differentiating the SARS-CoV-2 variants will be developed. And with that, knowledge is generated that will help in preventive measures to control the disease.

Keywords: COVID-19, Coronavírus, Mutation

Supported by: FAPESB

08597 - Poster Session

CB.29 - Biochemical characterization of plant growth-promoting fungus Penicillium chrysogenum 34-P to enhance the salinity tolerance of non-halophytic crops

Rodrigo Mattos Silva Galeano ¹, Samanta Monção Silva¹, Murilo Kioshi Aquino Yonekawa¹, Nelciele Cavalieri de Alencar Guimarães¹, Douglas Chodi Masui¹, Giovana Cristina Giannesi¹, Marivaine Silva Brasil², Fabiana Fonseca Zanoelo¹

¹Laboratório de Bioquímica Geral e de Microrganismos, Instituto de Biociências, Universidade Federal de Mato Grosso do Sul (MS, Brazil), ²Laboratório de Genética e Microbiologia, Universidade Federal de Mato Grosso do Sul - Campus do Pantanal (MS, Brazil)

INTRODUCTION

Soil salinity is one of the major causes of abiotic stress in plants, decreasing food productivity in many regions of the world. Plant growth-promoting fungi can help plants to tolerate stressful conditions and confer an improved growth on saline soils.

OBJECTIVES

In the present study, we evaluated the potential of Penicillium chrysogenum 34-P to promote plant growth under salinity stress.

MATERIALS AND METHODS

The tolerance of isolate to saline conditions was investigate in Potato-Dextrose-Agar (PDA) with different concentrations of NaCl (0-20% w/v). The fungal was grown in PD broth (0 and 5% NaCl) for 7 days at 30ºC (110 rpm) to measure dry weight. The strain has been investigated for indole-3-acetic acid (IAA), ammonia and siderophores production, and phosphate solubilization in presence of NaCl at 5% (w/v). Amylase, CMCase, pectinase, protease and xylanase production was investigated under saline conditions (5% w/v) in TLE medium for 168 hours.

DISCUSSION AND RESULTS

P. chrysogenum 34-P showed tolerance to different concentrations of NaCl (3, 5, 10 and 15% w/v). The fungus growth in PD broth with NaCl showed significant decrease (27.9%) of dry weight when compared with the control, from 257 to 201 mg, respectively. The presence of NaCl reduced in 71% the synthesis of IAA (17.67 μg mL-1). On the other hand, phosphate solubilization was increased by an average of 24% in relation to control (92.54 mg L-1). The isolate exhibited ammonia and siderophores production under NaCl presence. The enzymatic activity of amylase (11.3 U mL-1 in 72 h) and xylanase (5.82 U mL-1 in 120 h) were decreased in presence of salt, however, activities of CMCase (0.84 U mL-1 in 48 h), pectinase (0.81 U mL-1 in 120 h) and protease (10.02 U mL-1 in 120 h) were increased.

CONCLUSION

Our results indicate that the P. chrysogenum 34-P displays plant growth-promoting abilities showing potential for applications in saline environments.

Keywords: salt stress, plant growth-promoting fungi, enzymes

Supported by: CNPq and CAPES

08496 - Poster Session

CB.30 - Project: Study of the interaction and modulating effect of lectin from seeds of Dioclea violacea and the antibiotic neomicin applied to skin wounds using carboximethylcellulose and alginate biological membrane.

LUIZ NELDECILIO ALVES VITOR ¹, CLAUDENER SOUZA TEIXEIRA¹

¹BIOQUÍMICA E BIOLOGIA MOLECULAR, UNIVERSIDADE FEDERAL DO CARIRI (CE, BRASIL)

INTRODUCTION

Skin lesions can increase the risk of local and systemic infections by pathogens. To speed up the healing process, bacterial contamination levels need to be reduced and dressings associated with traditional antibiotics have been an option in this treatment as they release the drugs in a controlled manner, reducing the risk of toxic effects in patients. Lately, studies of the action of lectins have converged in this purpose of fighting infections, especially when associated with aminoglycoside antibiotics, since lectins have DRC (Carbohydrate Recognition Domain).

OBJECTIVES

This work will aim to analyze the modulating effect of Dioclea violacea (DVL) seed lectin on the antibiotic activity of Neomycin in multiresistant bacterial strains present in mouse skin lesions.

MATERIALS AND METHODS

The DVL lectin will be extracted and purified from the seeds of D. violacea which, by means of a hemagglutinating assay, will characterize its interaction with neomycin. Inoculums of multiresistant bacterial strains will be added to wounds induced in Swiss mice that will be divided into 06 groups, 01 uninfected group, with aseptic wounds treated with saline (50 μL); and 05 infected groups treated with: saline (50 μL); the formulation (membrane); formulation containing the D. violacea lectin; formulation containing the neomycin and formulation containing the lectin and neomycin, respectively. In each group with its defined treatment, clinical analysis of the lesions, count of viable bacteria and histopathological analysis will be performed

DISCUSSION AND RESULTS

Results will be expressed as the mean ± standard error of the mean. Inhibition percentages will be calculated as the mean of the inhibitions obtained for each individual experiment. The statistical evaluation of the results will be performed using analysis of variance (ANOVA), followed by the Boferroni and Kruskal-Wallis test, for parametric and non-parametric data, respectively. The significance level equal to 0.05 will be adopted.

CONCLUSION

Keywords: lectins, membrane, neomycin

08754 - Poster Session

CB.31 - Ontogenetic and sexual comparison of Bothrops erythromelas (jararaca-da-seca) snake venom

Daniela Miki Hatakeyama ^1,2, Giovanni Perez Machado da Silveira¹, Sávio Stefanini Sant'Anna¹, Kathleen Fernandes Grego¹, Karen de Morais Zani^1,2, Anita Mitico Tanaka Azevedo^1,2

¹Laboratório de Herpetologia, Insitituto Butantan (São Paulo, Brazil), ²Biotecnologia, Interunidades em Biotecnologia, Instituto de Ciências Biomédicas – Instituto Butantan – Instituto de Pesquisas Tecnológicas (São Paulo, Brazil)

INTRODUCTION

The genus Bothrops is responsible for approximately 85% of the more than 27,000 snakebite envenomations reported yearly in Brazil. In the northeast region, Bothrops erythromelas is one of the most medically important species. The snakes from this species shift their diet from predominantly amphibians and lizards to mainly mammals as they develop from juveniles to adults.

OBJECTIVES

As ontogenetic changes in snake venoms were observed in other species, we aimed to analyze the shift in the venom of B. erythromelas snakes from newborns to adults.

MATERIALS AND METHODS

Thirteen B. erythromelas snakes born and maintained in captivity in the Laboratory of Herpetology at Butantan Institute had their venoms milked and pooled every six months after they were born, separated in males and females (at 24 months-old the venoms were stored individually). Additionally, thirteen adult snake venoms were milked and stored individually. All venoms were submitted to protein quantification by Bradford method, phospholipase A₂ (PLA₂; 4-nitro-3-(octanoyloxy)benzoic acid substrate), L-amino acid oxidase (L-methionine substrate), and caseinolytic (azocasein substrate) activities; the latter was also performed with previous incubation with antibothropic serum to measure this activity’s inhibition.

DISCUSSION AND RESULTS

Venoms from newborns presented less protein quantification, PLA₂, and caseinolytic activities, which increased as the snakes aged. Male venoms had slightly higher PLA₂ and proteolytic activities in general. L-amino acid activity, however, was not detected in any of the pools and was low in 6 individuals. Individual variation was observed among the venoms in all assays, including inhibition of the caseinolytic by the antivenom, which inhibited male venom slightly better (average of 10.9% and 9.9% inhibition of male and female venoms, respectively).

CONCLUSION

In conclusion, the B. erythromelas venoms studied in this work showed that ontogeny might influence them more than gender, individual variation is also remarkable in this species, and antivenom is not able to efficiently neutralize proteolytic activity over casein.

Keywords: Bothrops erythromelas, Ontogeny, Individual variation

Supported by: CNPq (140872/2019-1) and FAPESP (2018/25786-0)

08546 - Poster Session

CB.32 - Bactericidal and antioxidant potential of microalgae Skeletonema costatum and Diacronema lutheri, by the influence of temperature and light incidence variation.

Fabrine Souza de Andrade ¹, Suzana Telles da Cunha Lima¹

¹Departamento de Biologia Geral, Laboratório de Bioprospecção e Biotecnologia - Instituto de Biologia/Universidade Federal da Bahia (Bahia, Brazil)

INTRODUCTION

Microalgae have been a growing object of scientific interest due to their relevance in several areas. With their photosynthesizing characteristics and amplitude of occurrence in the planet, they are an efficient alternative producer of bioactive compounds with application in several areas and with high biological and biotechnological value. Modifications in abiotic conditions can promote changes in the production of metabolites and, consequently, in antibacterial and antioxidant properties of microalgae.

OBJECTIVES

The project proposes to evaluate the increase in the production of metabolites with antibacterial and antioxidant properties of the microalgae Skeletonema costatum and Diacronema lutheri, through responses induced by temperature variation and light incidence, in their production of algal biomass.

MATERIALS AND METHODS

For temperature conditions, the project will use the regular cultivation one (22ºC) and 30ºC. For light incidence, microalgae will be subject to 70 and 100 μmol of photons m ^-2 s ^-1 . Extracts will be prepared by maceration in 99.5% ethanol. To evaluate the antibacterial activity, the method of disk-diffusion in agar and microdilution in broth will be performed for six strains of bacteria. The antioxidant activity (AAO) will be evaluated by the radical scavenging activity method using 2.2-diphenyl-1-picrilhidrazil radical (DPPH). With the results obtained from the previous steps, the profile of the microalgae metabolites with the best potential in the activities tested will be analyzed using the Ultra-Efficiency Liquid Chromatography (CLUE) method coupled to a Mass Spectrometer (MS).

DISCUSSION AND RESULTS

As an expected result, the project aims to obtain the profile of metabolites to understand how abiotic changes in the cultivation of these species can alter the production of their bioactive compounds.

CONCLUSION

In addition, to identify the antibiotic and antioxidant potential of the studied microalgae, contributing to the isolation or synthesis of new pharmacological targets.

Keywords: Bioactivity, Cultivation condition, Microalgae

Supported by: CAPES, FINEP

08804 - Poster Session

CB.33 - In vitro antitumor effects of sodium selenite in chronic myeloid leukemia cell lineages.

Matheus Alves De Moura ¹, Luisa Andrea Ketzer¹

¹ Campus Duque de Caxias, Universidade Federal do Rio de Janeiro, Rio de Janeiro,, Núcleo Multidisciplinar de Pesquisa UFRJ-Xerém em Biologia (Rio de Janeiro, Brasil)

INTRODUCTION

Selenium compounds have been studied as an antitumor agent due their oxidant role and the negative interaction with glycolysis pathway, leading cancer cells to death by starvation.

OBJECTIVES

In this scenario, the objective of this work is to evaluate an antitumor activity of sodium selenite (Na2SeO3) in chronic myeloid leukemia cell lineages K562 and LUCENA.

MATERIALS AND METHODS

LUCENA cells is a resistant lineage obtained through treatment with low doses of vincristine, an antitumor agent used in the clinic treatment. Cell viability was measured by MTT assay and the mitochondrial membrane potential (MMP) was performed by JC-1 probe.

DISCUSSION AND RESULTS

The cells were previously treat with 10 μM, 100 μM or 1000 μM of Na2SeO3 for 48 hours, where a proliferation of 75% of control K562 cells was observed, while a significant reduction was observed for the concentrations of 100 μM and 1000 μM of Na2SeO3. For the LUCENA lineage, the proliferation rate was 63% for the control, whereas a 66% reduction in cell number occurred for the 1000 μM concentration of Na2SeO3. On these conditions, the cell viability was significant reduced only in 100 μM or 1000 μM of Na2SeO3 for both cell types. To MMP, the treatment was performed for 24 hours, and the result obtained was a significant reduction for all concentration for K562 cells, while for the LUCENA lineage only at concentrations of 100 μM and 1000 μM of Na2SeO3 was performed a significant reduction.

CONCLUSION

The preliminary results of this work show that K562 and LUCENA cells are sensitive to Na2SeO3 supplementation and that the treatment affects mitochondrial physiology.

Keywords: metabolism, cancer, selenium

Supported by: FAPERJ

08328 - Poster Session

CB.34 - The use of metformin as adjuvant to restore allantoin-reduced cisplatin cytotoxicity in vitro

Grazielle Silva Paz ¹, Janaina Fernandes¹, Gisele Amorim¹

¹NUMPEX-BIO, UNIVERSIDADE FEDERAL DO RIO DE JANEIRO (RIO DE JANEIRO, BRAZIL)

INTRODUCTION

The tumor lysis syndrome (TLS) is a metabolic disorder frequently associated to hyperuricemia. As a tool for the treatment of TLS, rasburicase has been employed, producing allantoin. In recent studies by our group, was observed that high levels of allantoin promote the reduction of the cytotoxic effect of cisplatin both in H460 and K562 cells lines. Therefore, in view of the maintenance of the concomitant use of cisplatin and rasburicase, search for adjuvant agents, like metformin, that can maintain their efficience it is of great interest. Recently, our group demonstrated the capacity of metformin in restores cisplatin cytotoxicity in the presence of allantoin in K562 cell line.

OBJECTIVES

Through this observation, this study sought to understood how metformin promoted the recuperation of cisplatin cytotoxicity.

MATERIALS AND METHODS

To realize this study, the K562 cell line was maintained in RPMI 1640 media supplemented with 10% of fetal bovine serum and 0.5% of penicilin-streptomicin at 37ºC and 5% of CO2. For the assays, the cells were incubated in RPMI 1640 low glucose media (0.5mM) for 2h and treated with metformin (0.5 to 3mM), cisplatin (15 to 33μM), allantoin (100 and 200μg/ml), rotenone (0.125 to 2μM) and their combination for 48h. Cell viability, cell cycle, morphology analysis and NMR assays were performed.

DISCUSSION AND RESULTS

Our results showed: (1) the synergism between metformin and cisplatin, (2) the cytotoxicity recuperation of cisplatin by metformin at morphology level, (3) the alterations of cisplatin effect in cell cycle after combination with allantoin, (4) the absence of metformin-cisplatin interaction, (5) the interaction between metformin and allantoin, (6) the increase of cisplatin cytotoxicity after its combination with rotenone and (7) the similarity between metformin and rotenone effects in restores cisplatin cytotoxicity in the presence of allantoin.

CONCLUSION

This study demonstrated that metformin restores allantoin-reduced cisplatin cytotoxicity independently and dependently of the mitochondria.

Keywords: Metformin, Cisplatin, Allantoin

Supported by: FAPERJ

08694 - Poster Session

CB.35 - PROJECT: FTIR FOR DIAGNOSIS AND PROGNOSIS OF COVID-19

JESSICA PIRES FARIAS ¹, JAIME SANTOS¹

¹Programa Multicêntrico de Pós-graduação em Bioquímica e Biologia Molecular, UNIVERSIDADE FEDERAL DO OESTE DA BAHIA (BA, Brasil)

INTRODUCTION

The Sars-Cov-2 virus belongs to the Coronaviridae Family, is the causative agent of the Coronavirus Disease of 2019. The gold standard for COVID-19 diagnosis is Reverse Transcription Polymerase Chain Reaction (RT-PCR) and uses the respiratory tract specimen for the analysis. Among the main COVID-19 biomarkers included acute phase proteins like C Reactive Protein (CRP), IL-6 cytokine and Lactate Dehydrogenase (LDH). However, the collection of respiratory tract specimen brings a series of limitations that range from patient discomfort and high risk of transmission to health operators. Vibrational spectroscopic techniques are applied for the human disease’s diagnosis and are already well knowledge in the literature for biological fluids analysis such as urine, blood and saliva. The advantages of its use are reproducibility, small amount of sample and specificity in the biochemical arrangement recognizing of the different biomolecule’s classes.

OBJECTIVES

Therefore, the aim of this project is evaluating the use of Fourier Transform Infrared absorption spectroscopy (FTIR), with multivariate analysis, as a new diagnosis tool, besides detect and quantify salivary biomarkers associated with clinical forms of COVID-19 for determining patient’s prognosis.

MATERIALS AND METHODS

This is an epidemiological study, prospective cohort, to be carried out at the Laboratory of Infectious Agents and Vectors -LAIVE of the Federal University of Western Bahia- UFOB. The sample will consist of 200 patients attended in the LAIVE services. The method to be used involves the following steps: (i) saliva and nasopharyngeal swab collection; (ii) samples processing; (iii) PCR analysis; (iv) implementation and validation of the FTIR diagnostic method; (v) implementation and validation of the quantification of salivary biomarkers by FTIR.

DISCUSSION AND RESULTS

CONCLUSION

Thus, a new, faster and less expensive diagnostic and prognostic method of COVID-19 it is expected, and that will be able to prioritize the analysis of non-invasive biological samples, ensuring patient comfort, safety of the health operator and good results.

Keywords: Sars-CoV-2, Spectroscopy, Saliva

08492 - Poster Session

CB.36 - PROJECT: PHARMACOLOGICAL ACTIVITY OF CARVACROL CIGARETTE IN A SMOKE-INDUCED ACUTE LUNG INJURY

Carlos Demócedes Luís de França A. Moreira ¹, A.K.H PEREIRA³, C.S Borges³, C.C.A Santos³, G.M Silva³, Crystianne Calado Lima⁴, P Oliveira-Melo⁵, Emanuel Kennedy Feitosa¹

¹Bioquímica e Biologia Molecular, Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular (RN, Brazil), ²Bioquímica e Biologia Molecular, Universidade do Estado do Rio Grande do Norte (RN, Brazil), ³Departamento de Ciências da Saúde, Universidade Federal Rural do Semi-Árido, Mossoró-RN, Brazil (RN, Brazil), ⁴Instituto Superior de Ciências Biomédicas, Universidade do Estado do Ceará (CE, Brazil), ⁵Instituto do Coração, Universidade de São Paulo (SP, Brazil)

INTRODUCTION

Acute lung injury is a condition characterized by tissue damage and increase of oxidative stress and inflammation, increasing inflammatory marker such as inflammatory cells and cytokines. Cigarette smoking is one of the risk factors for reactive oxygen species (ROS) generation, which induces oxidative stress and lung inflammation. Carvacrol (CAR) is a promissor terpene that have been used against pulmonary inflammation and antioxidative activity.

OBJECTIVES

This study aims to evaluate of anti-inflammatory and antioxidant activity of carvacrol in Cigarette smoke-induced acute lung injury.

MATERIALS AND METHODS

Mice C57BL/6, male, (25-28g) will be divided into two groups: control and cigarette smoke (CS). The CS group will be exposed to 12 cigarettes per day for 5 days. The control group will be exposed to sham smoking (without cigarrete smoke). The CS group will be treated with CAR (1, 3 or 10 mg/mL) or vehicle by inhalation (15 min/daily) for 5 days. After 5 days bronchoalveolar lavage (BAL) to analyses of cytokines release (TNF-α, IL-6, KC and IL-10) by ELISA. The trachea will be used to airway hiperresponsivines analyse by dose-response curve to carbachol (CCh; 0,01 – 100 μM) and potassium chloride (KCl; 10 – 80 mM). Right lung will be collected to morphological analysis such as lung inflammation score, bronchoconstriction index and count of inflammatory cells (macrophages and neutrophil) and left lung will be used to redox marks analyses (superoxide dismutase and catalase activity and, malondialdehyde levels).Statistical analysis were performed by Analysis Variance (ANOVA) followed by Tukey´s post hoc test.

DISCUSSION AND RESULTS

Significant difference when p < 0.05. The literature suggests that terpenes, such as carvacrol, has anti-inflammatory and antioxidant activity.

CONCLUSION

Therefore, we expect that carvacrol will act reducing inflammation and oxidative damage, consequently being a promissory substance to treat the acute lung injury induced by cigarette smoke.

Keywords: Carvacrol, Antioxidant, Antiinflamatory

Supported by: UFERSA

08465 - Poster Session

CB.37 - PROJECT: Toxicological and antitumor evaluation of fabaceae family lectins

ADRIELLE RODRIGUES COSTA ¹, CLAUDENER SOUZA TEIXEIRA^1,1

¹Faculdade de Medicina, Universidade Federal do Cariri (Ceará, Brazil)

INTRODUCTION

Cancer is one of the major public health problems worldwide, accounting for about 10 million deaths per year. In Brazil, as malignant neoplasms are considered as the second cause of death among non-communicable diseases, which usually arise through a slow process, and it may take several years before they are diagnosed, however, anticancer drugs are often ineffective, present a number of side effects and drug resistance. In this context, more efficient scientific research in the search for drugs has grown considerably, and products of plant origin are a viable alternative.

OBJECTIVES

In view of the above, the objective of this work is to evaluate an antitumor and toxicological activity of vegetable lectins from Vatairea macrocarpa (VmL) and Dioclea violacea (DvL).

MATERIALS AND METHODS

The purification of the lectins will be carried out by affinity chromatography techniques. In vitro and in vivo techniques will be developed to evaluate possible mechanisms of cytotoxic actions, genotoxicity and oxidative stress markers. In vitro tests will be evaluated for cytotoxicity with cancerous and healthy cells, mechanisms of apoptosis, mitochondrial transmembrane potential, cell cycle, DNA fragmentation and levels of gene expression of proteases (caspases 2, 3, 6, 8, 9 and Apaf-1), in addition to evaluating damage caused to the cell's DNA, by the comet assay. The in vivo experiments will be carried out with Drosophila melanogaster, a promising invertebrate in tests to evaluate toxicological parameters, such as: cell viability, total thiols, non-protein thiols, lipid peroxidation, determination of free Fe2+ levels and nitrite levels.

DISCUSSION AND RESULTS

CONCLUSION

This research aims to contribute to the advancement of the knowledge of the therapeutic properties of lectins, that once these properties of the species under study have been proven, it will be possible to favor the development of a new anticancer substance.

Keywords: Cancer, Oxidative stress, Lectins

08749 - Poster Session

CB.38 - Cloning and expression of γBjPLI, a natural inhibitor of phospholipase A2 from Bothrops jararaca snake blood

Caroline Serino Silva ^1,2, Verônica de Morais Manzato³, Karen de Morais-Zani^2,1, Kathleen Fernandes Grego², Aparecida Sadae Tanaka³, Anita Mitico Tanaka-Azevedo ²

¹Interunidades em Biotecnologia, USP, Ibut and IPT, Universidade de São Paulo (SP, Brasil), ²Laboratório de Herpetologia, Instituto Butantan (SP, Brasil), ³Departamento de Bioquímica, Universidade Federal de São Paulo (SP, Brasil)

INTRODUCTION

γBjPLI is a phospholipase A₂ inhibitor (PLI) present in low concentrations the Bothrops jararaca snake blood serum. This protein is classified as a gamma type based on its characteristic structural domains. γBjPLI has approximately 22 kDa and an inhibitory activity of enzymatic, edematogenic and myonecrotic activities, suggesting a role of this inhibitor for protection of these snakes against self-envenomation. Phospholipase A₂ acts in the cell membrane phospholipids resulting in fatty acids, lysophospholipids and deconstructing the cell membrane. This protein is commonly responsible for the local effect of snake envenomation, causing tissue inflammation in the affected area.

OBJECTIVES

To improve the yield in obtaining this inhibitor, this work aims to clone and express γBjPLI in its recombinant form.

MATERIALS AND METHODS

First, by PCR, a 543 bp fragment was amplified from a cDNA library produced from the liver of B. jararaca snake. The γBjPLI fragment was inserted into the pGEM-T easy vector, cloned using the DH5αstrain. The fragment was purified and introduced into a pET28a expression vector and cloned again into the DH5α strain. Plasmid pET28a+γBjPLI was isolated and sequenced to confirm the composition of the nucleotide sequence. Finally, by heat shock, pET28a+γBjPL was inserted into the expression strain Shuffle of Escherichia coli, the expression was induced with different concentrations of IPTG for 16 h at 37 °C, and analyzed by 15% SDS-PAGE.

DISCUSSION AND RESULTS

In the first step, 2 were positive for the pGEM-T+γBjPLI vector. In the next step, 4 were positive for the presence of pET28a+γBjPLI. The nucleotide sequence was confirmed, and the 4 colonies were able to express a 22 kDa band with IPTG 0,1 mM and 0,4 mM.

CONCLUSION

In this work, the cloning and expression of γBjPLI in its recombinant form was carried out for the first time, using the pET28a vector and the SHuffle expression strain of the E. coli bacterial species.

Keywords: Snake plasma, Phospholipase A2 Inhibitor, Bothrops jararaca

Supported by: FAPESP

08833 - Poster Session

CC.01 - Investigation of the use of xanthan gum as a controlled release system for doxycycline

Kátia Maria De Oliveira Almeida ¹, Ângelo Danadai¹, GABRIELLA FERREIRA¹

¹Departamento de Farmácia, Universidade Federal de Juiz de Fora Campus Governador Valadares (Minas Gerais, Brasil)

INTRODUCTION

Xantham gums or Xanthans are biopolymers obtained by fermentation of bacteria from the Xanthomonas genus. They are biodegradable and biocompatible, being widely used by the pharmaceutical industry, especially as controlled drug release systems.

OBJECTIVES

Concerning their anionic feature, the present work aimed to investigate the formation of the complex spontaneously formed with the cationic antibiotic doxycycline (DOX), as well as its biological activity.

MATERIALS AND METHODS

The samples were characterized in solid state by FTIR and thermal analysis (TGA and DTA). The interactions between doxycycline and xanthan were characterized in solution by ITC, zeta potential (ZP) and rheological titrations. The antimicrobial action of the doxycycline/xanthan complex and its precursors was evaluated against Staphylococcus aureus 323886023, by determining the median lethal dose (LD50) and death curve.

DISCUSSION AND RESULTS

FTIR and TGA/DTA data confirmed the solid state interaction. ZP titrations showed the gradual neutralization of xanthans by DOX. The ITC data showed two distinct sites of interaction (one exothermic and the other endothermic). The rheological titration experiments showed a strong reduction in viscosity, suggesting that the DOX/xanthan interaction causes a collapse in the polymer structure, breaking the entanglements. The results obtained showed that the complexation led to an increase in antimicrobial activity, by reducing the LD50 and inhibition time,

CONCLUSION

suggesting that the complex is promising for the development of new formulations for a antimicrobial drug controlled release.

Keywords: xanthan gum, controlled drug release, doxycycline

Supported by: FAPEMIG

08375 - Poster Session

CC.02 - Combination of biocatalysis and sonochemistry in the ethyl oleate production in a co-solvent free system

Isac George Rosset ¹, Natália Almeida¹

¹Engenharias e Exatas, Universidade Federal do Paraná (Paraná, Brazil)

INTRODUCTION

Fatty acid alkyl esters (FAAEs) are a family of natural neutral lipids and can be produced cleanly and sustainably by esterification of free fatty acids (FFAs) with short chain alcohols using enzymatic catalysts. Recently alkyl esters have attracted much attention for their application in food, beverage, and chemical industries. Fatty acid methyl or ethyl esters are also widely used as biofuel in diesel engines. Due to inherent merits of enzymatic reactions, e.g., milder conditions, less pollution, etc., enzymatic production of fatty acid alkyl esters such as ethyl esters, have received much attention.

OBJECTIVES

In this study, it was evaluated the use of lipases in enzymatic esterifications of oleic acid with ethanol using the combination of biocatalysis and sonochemistry (ultrasound) in the absence of co-solvents.

MATERIALS AND METHODS

Reaction parameters, such as type of lipase, amount of enzyme, reaction time, alcohol hydration level and enzyme turnover were evaluated. Reactions were carried out in 5 mL Eppendorf® tubes containing 200 mg of oleic acid, 600 μL of ethanol, 20 mg (10%) of lipase and 20 mg of molecular sieves. The mixtures were incubated in ultrasound bath at 35ºC for 10 hours. For quantification, synthetic ethyl oleate (acid catalysis) was prepared and used as a standard.

DISCUSSION AND RESULTS

Eleven enzymes were initially tested and just C. antarctica lipase provided yields above 95% in less than 10 h with 10% (m/m) of catalyst. The use of hydrous ethanol (5% of water) showed a slight drop in yield but remained above 90% of ethyl oleate production and C. antarctica lipase showed no loss of efficiency even after 10 reaction cycles.

CONCLUSION

The combination of biocatalysis and ultrasound radiation greatly increased the reaction yield, showing that this combination may be a good choice for ethyl oleate enzymatic synthesis.

Keywords: lipase, sonochemistry, ethyl oleate

Supported by: CNPq and CAPES

08232 - Poster Session

CC.03 - Production and application of prebiotic xylo-oligosaccharides obtained from agro-industrial residues using Thermomyces lanuginosus endoxylanase.

Andreza Gambelli Lucas Costa Nascimento ¹, Érica Sabrina Zuppa¹, Maiara Zonin¹, Alexandre Maller¹, José Luis da Conceição Silva¹, Rita de Cássia Garcia Simão¹, Marina Kimiko Kadowaki¹

¹Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná - Cascavel (Paraná, Brasil)

INTRODUCTION

Xylooligosaccharides (XOS) are short chain oligomers containing 𝛽 e_x1D6FD;-1,4-linked xylose residues that have prebiotic action and human health benefits due to stimulation of the growth of bacteria such as Lactobacilli and Bifidobacteria.

OBJECTIVES

The aim of this study was to evaluate the production of XOS from agro-industrial residues using Thermomyces lanuginosus endoxylanase, and to test these XOS in the growth of the probiotic Lactobacillus acidophilus.

MATERIALS AND METHODS

Hemicelluloses were extracted from corn straw and brewery bagasse after alkaline pretreatment with 6% hydrogen peroxide, and subsequently hydrolyzed with T. lanuginosus pure endoxylanase at 65ºC for 1h. Subsequently, the probiotic bacterium was grown in microplate containing basal MRS medium supplemented with XOS and incubated, at 37°C under anaerobic conditions for 24h. To monitor the growth of bacteria was measured by optical density at a wavelength of 600 nm at cultivation times 0, 4, 8, 12 and 24 h.

DISCUSSION AND RESULTS

The XOS released after the hydrolysis of each of the hemicelluloses by endoxylanase were mainly composed of xylobiose, xylotriose, xylotetraose and xylopentaose (X2-X5), and the yield of the XOS mixture was 4.06 mg / mL and 2.86 mg / mL for corn straw and brewery bagasse, respectively. The stimulus on the growth of the probiotic microorganism in the presence of XOS was verified after 12 h of incubation. Therefore, these XOS obtained from agro-industrial residues used as carbon source to probiotic bacterium culture were effective in promoting the growth of L. acidophilus, comparable to the control culture with glucose.

CONCLUSION

Thus, these XOS obtained from agro-industrial residues show prebiotic and promising potential for biotechnological exploitation.

Keywords: agro-industrial residues, probiotics, xylooligossaccharides

Supported by: CAPES and Fundação Araucária

08690 - Poster Session

CC.04 - Production of pectinases by Aspergillus japonicus and Aspergillus sp. (M1) for juice clarification using agro-industrial waste as a carbon source

Nathalia Nunes Glienke ¹, Ana Lorena de Oliveira Simas ¹, Nelciele Cavalieri de Alencar Guimarães ¹, Rodrigo Mattos Silva Galeano¹, Douglas Chodi Masui ¹, Fabiana Fonseca Zanoelo ¹, Giovana Cristina Giannesi ¹

¹Laboratório de Bioquímica Geral e de Microrganismos, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brazil)

INTRODUCTION

Pectinases are a group of enzymes capable of degrading polysaccharides of the plant cell wall, facilitating the obtaining of carbon source by microorganisms as a result of cell fragmentation. Filamentous fungi are the most used for enzymatic production on an industrial scale, with application in the fruit juice clarification, among these are fungi of the genus Aspergillus.

OBJECTIVES

The goal was to evaluate the production and characterize pectinases produced by Aspergillus japonicus and Aspergillus sp. M1 from agro-industrial waste and apply these enzymes in the fruit juice clarification.

MATERIALS AND METHODS

Pectinase production was evaluated using different carbon sources in solid medium (5 grams) and the fungi were inoculated using 10 ml of a Salt Solution with spores. Optimal growth time was evaluated using Tereré Herb residue as carbon source in solid medium for 192h under stationary condition at 35°C. Enzyme activity was measured at temperatures between 30 and 70°C and McIlvaine buffer pH 3 to 7 using the DNS method. Juice clarification by pectinases was evaluated measuring transmittance according to Rosmine et al. (2017).

DISCUSSION AND RESULTS

Aspergillus sp. M1 had optimal production in Tereré Herb residue after 72h of growth and maximum activity at pH 4 (2.54 U/ml) and 60°C (4.05 U/ml). A. japonicus had optimal production in Tereré Herb residue after 48h and maximum activity with McIlvaine pH 4 buffer (1.09 U/ml) and at 60°C (2.16 U/ml). Pectinase from A. japonicus obtained maximum results in the clarification of guava juice at 50°C, with 49.3% ±1.8 clarification.

CONCLUSION

The results showed that pectinases from both fungi have viable features for application in fruit juice clarification, whose tests with A. japonicus proved their potential for industrial application.

Keywords: pectinases, fruit juice clarification, Aspergillus sp.

Supported by: CNPq

08047 - Poster Session

CC.05 - BIOETHANOL PRODUCTION FROM PRETREATED SUGARCANE BAGASSE UNDER OPTIMISED CONDITIONS USING SELECTED FUNGI

Adebare Johnson Adeleke ^1,2, Hayatu Mohammed Raji³, Dimitris G. Hatzinikolaou⁴, Sunday A. Odunfa²

¹Department of Microbiology , Modibbo Adama University of Technology (Yola, Nigeria , Nigeria), ²Department of Microbiology , University of Ibadan (Ibadan Nigeria , Nigeria), ³Department of Natural and Environmental Science , American University of Nigeria (Yola, Nigeria , Nigeria), ⁴Enzyme and Microbial Biotechnology Unit, Department of Biology, National and Kapodistrian University of Athens (Athens, Greece, Greece)

INTRODUCTION

Sugarcane Bagasse (SB), a waste of the sugar industry contains cellulose and hemicellulose which can be converted to bioethanol. However, its recalcitrant nature demands optimal pretreatment method to make sugar components available for enzymatic depolymerisation.

OBJECTIVES

This study was designed to optimally pretreat SB and to identify appropriate fungi for enhanced bioethanol yield.

MATERIALS AND METHODS

Yeasts isolated from SB dumpsite were screened based on their ability to convert pentose and hexose sugars to bioethanol using different nitrogen sources. Optimisation of pretreatment of SB at different concentrations of potassium hydroxide (KOH), temperature and treatment time was determined using Response Surface Methodology (RSM). Pretreated SB was hydrolysed using A. niger XY and a commercial hemicellulase mixture. Fermentation of pretreated SB hydrolysate with selected yeasts using Simultaneous Hydrolysis and Fermentation (SHF) as well as Simultaneous Saccharification and Fermentation (SSF) of pretreated SB were carried out. Bioethanol yield was determined by gas chromatography.

DISCUSSION AND RESULTS

Eleven yeasts grew on both glucose and xylose and were identified as Pichia kudriavzevii (7), Saccharomyces cerevisiae (1), and Candida tropicalis (3). All yeasts converted glucose to ethanol but only C. tropicalis Y5 converted xylose to ethanol (4.83g/L) with urea as the best nitrogen source. Optimum pretreatment conditions were: 150mg/g bagasse (KOH), 86oC and 120 minutes. Hydrolysis with hemicellulase yielded reducing sugars of 600 mg/g bagasse within 20 hours while hydrolysis with A. niger XY took a longer time (12 days) and yielded 18.8 mg/g bagasse. Bioethanol yield using SHF and SSF were 19 g/L and 30 g/L, respectively

CONCLUSION

Alkaline pretreatment followed by enzymatic hydrolysis gave a higher yield of total reducing sugars. Candida tropicalis Y5 converted both pentose and hexose to bioethanol and showed good prospect for its use in commercial fermentation of sugarcane bagasse.

Keywords: Bioethanol, Candida tropicalis Y5, KOH

08443 - Poster Session

CC.06 - Project: Production, purification, biochemical characterization, and biotechnological application of amylase from the thermophilic fungus Humicola brevis var. thermoidea

Camila Langer Marciano ^1,2, Aline Pereira de Almeida^1,2, Aline Reginaldo dos Santos¹, Fabiana Fonseca Zanoelo^1,2, Giovana Cristina Giannesi^1,2, Roberto Ruller^1,2, Douglas Chodi Masui^1,2

¹Laboratório de Bioquímica Geral e de Microrganismos-LBq , Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil), ²Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

Starch is a polysaccharide commonly found in plants and, therefore, is a significant source in industrial applications. Amylases are enzymes capable of degrading starch and represent 25% of the world market for enzymes, of which several markets use them, such as in ethanol production and bakery. This enzyme belongs mainly to the class of hydrolases and cleaves starch in different regions depending on its classification of α;-amylase, β;-amylase, and γ;-amylase. Microorganisms can produce amylases, such as bacteria and fungi, therefore, distinct means of production studied them for characterization of amylase to promote starch bioprocesses. In this context, previous studies have shown that the thermophilic fungus Humicola brevis var. thermoidea in solid culture medium is a good amylase producer

OBJECTIVES

Hence, this work aims to produce, purify, and biochemically characterize the amylase obtained from H. brevis and its biotechnological application in the depolymerization of the different starch sources

MATERIALS AND METHODS

Particularly, the native amylase will be purified from the crude extract obtained by solid-state fermentation (SSF). The maximal production will be performed using the one-factor-at-time method (OFAT) to determine the best source for overexpression of amylase activity. The amylolytic activity will be assayed using 1% starch, in optimal pH and temperature conditions, using the dinitrosalicylic acid method to reducing sugars to available starch hydrolysis. The homogeneity of purified enzyme will be available using electrophoresis in denaturing conditions SDS-PAGE. The biochemical characterization of crude extract and purified amylolytic activity will be realized by evaluating the optimal pH and temperature, stabilities, the effect of the presence of different ions, organic compounds, and detergent

DISCUSSION AND RESULTS

Thus, it is expected to obtain the standardization of the expression of amylase, as well as the biochemical characterization and purification of the enzyme in the crude extract, so that can become an alternative for the starch liquefaction process.

CONCLUSION

Keywords: Amylase, Humicola brevis var. thermoidea, Starch

08727 - Poster Session

CC.07 - Immobilization of fungi in polyurethane for production of phosphatases

Ester Vieira Gonçalves ¹, Marcos Henrique Dias Martins¹, Solange Xavier dos Santos¹, Samantha Salomão Caramori¹

¹Câmpus Central, Universidade Estadual de Goiás (Goiás, Brasil)

INTRODUCTION

Phosphatase, an enzyme that catalyzes organic phosphorus, is involved in biotechnological processes, including bioremediation of domestic and industrial effluents, heavy metal removal, and plant fertilization. Enzyme catalysis can be optimized with simple methods of immobilizing cells in different matrices. The polyurethane foam matrix was applied in this work because it offers high porosity, good mechanical resistance and low cost.

OBJECTIVES

This work proposed the production of phosphatase by fungal immobilized cells Aspergillus niger, Aspergillus flavus and Trichoderma sp. entrapped in polyurethane foams.

MATERIALS AND METHODS

The isolated fungi was incubated in a minimum culture media containing potassium phosphate varied from 1g L ^-1 to 4g ^L-1 to induce phosphatase production. The commercial macroporous polyurethane foam (0.71 mm) was cut into 1.0 cm x 1.0 cm x 0.7 cm parallelepiped. Then, the biomass of A. niger, A. flavus, Thichoderma sp.A and Trichoderma sp.B were inoculated in minimal medium containing 0.5 g of previously autoclaved foam cubes. The microorganisms grew for 12 days and during this time the fungal biomass inside the foams was monitored for the appearance of the characteristic color of each isolate. The acid phosphatase activity with p-nitrophenyl phosphate was measured.

DISCUSSION AND RESULTS

Induction of phosphatase production occurred with KH2PO4 at 4 gL ^-1 for Aspergillus and at 1 gL ^-1 for Trichoderma sp. All fungi produced phosphatase, ie. A. niger for 12 days, followed by Trichoderma sp. B, 11 days, and 7 days for A. flavus and Trichoderma sp. The A. niger and A. flavus isolates had higher phosphatase activity (229 μmol/h), after 192 hours and 96 hours, respectively. The two Trichoderma sp. showed better enzyme activity in 72 hours, with 78 μmol/h for Trichoderma sp. B and 36.7 μmol/h for Trichoderma sp. A. Reusability of Trichoderma sp. A was 4 cycles and A. flavus in 2 cycles.

CONCLUSION

Those isolated fungi are good candidates for phosphatase bioremediation.

Keywords: entrapment, biocatalysis, biorremediation

Supported by: CAPES; UEG

08971 - Poster Session

CC.08 - Expression and characterization of a thermostable endo-arabinanase

Vandierly Sampaio de Melo ¹, Brisa Moreira Gomes¹, Felipe Santiago Chambergo Alcalde¹

¹Escola de Artes, Ciências e Humanidades, Universidade de São Paulo (São Paulo, Brazil)

INTRODUCTION

Arabinan is an important constituent of plant biomass, this monosaccharide has several applications in the biofuel, food and pharmaceutical industries. Its hydrolysis by the enzymes endo-1,5-α-L-arabinanases and α-L-arabinofuranosidases results in L-arabinose releasing. Based on the evidences that no arabinase from Geobacillus sp. JS12 has been described, we characterized the recombinant enzyme endo-1,5-α-L-arabinanase.

OBJECTIVES

Produce and characterize the recombinant enzyme endo-1,5-α-L-arabinanase from Geobacillus sp. JS12.

MATERIALS AND METHODS

To develop this work, we first synthesized and cloned the putative enzyme gene, obtained from the CAZY database, in E. coli BL21. The recombinant enzyme was purified by affinity chromatography on a nickel-sepharose column. The amino acid sequence analyzes were performed in silico, the enzymatic activity analysis were performed in different synthetic and natural substrates. The influence of pH, temperature and additives, on the enzymatic activity, was verified by DNS method and the kinetic parameters were determined by Michaelis-Menten equation.

DISCUSSION AND RESULTS

The results showed that endo-1,5-α-L-arabinanase had greater activity at pH 7.0, at 70°C, on the specific substrate arabinan debranched. In tests with additives, the enzyme exhibited almost the same activity as previously, in the presence of EDTA and, its activity, was slightly improved in cobalt presence. It was found that the enzyme has good thermal stability at 70°C, especially in cobalt ion presence, after 48h. Also, the enzyme showed specific activity and kinetic parameters on debranched arabinan, what was similar to other endo-arabinanase already characterized.

CONCLUSION

In this sense, the endo-1,5-α-L-arabinanase maximum activity, at high temperatures, and its thermostability turns the enzyme a potential candidate for obtaining and applying L-arabinose in industrial process.

Keywords: arabinan, glycosyl hydrolases, endo-α-1,5-L-arabinanase

Supported by: CAPES

08395 - Poster Session

CC.09 - Purification, characterization, and evaluation of the biotechnological potential of endo-xylanase from the thermophilic fungus Humicola brevis var. thermoidea.

Aline Pereira de Almeida ^1,2, Aline Reginaldo dos Santos¹, Camila Langer Marciano^1,2, Fabiana Fonseca Zanoelo^1,2, Giovana Cristina Giannesi^1,2, Roberto Ruller^1,2, Douglas Chodi Masui^1,2

¹Laboratório de Bioquímica Geral e de Microrganismos, Instituto de Biociências, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil), ²Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

The biodegradation of lignocellulosic biomass, especially hemicellulose, depends on a group of enzymes (hemicellulolytic complex) produced mainly by fungi and bacteria. Among these hemicellulases, endo-xylanases have been widely used to enhance hemicellulose in bioprocess applications.

OBJECTIVES

In this context, this work aimed to purify, characterize and evaluate the biotechnological potential of endo-xylanase from Humicola brevis var. thermoidea in the saccharification of lignocellulosic biomass.

MATERIALS AND METHODS

For this purpose, the native endo-xylanase (XylHb) was purified from the crude extract obtained by solid-state fermentation.

DISCUSSION AND RESULTS

The biophysical characterization of secondary structures by circular dichroism indicated the predominance of the β-sheet, a typical characteristic of the GH11 family endo-xylanases, and the intrinsic fluorescence of the tryptophan analysis indicated that the protein has its tertiary folding with Trp residues immersed in a hydrophobic environment. The results of the biochemical and kinetic characterizations of XylHb indicated that the enzyme under study had a greater tolerance in a wide range of pH, temperature, NaCl, and ethanol, having a better performance in the hydrolysis of birch xylan at temperatures of 50 and 70 ºC in comparison with commercial xylanase (Cellic ^® HTec2). The hydrolysis assays of hydrothermally pretreated sugarcane bagasse demonstrated that the supplementation of Cellic ^® CTec2 with XylHb showed synergistic interactions and significantly increased the release of glucose and total reducing sugars compared to the individual Cellic ^® Ctec2 commercial standard.

CONCLUSION

Thus, a purified XylHb from the enzymatic extract of H. brevis var. thermoidea, due to its physical-active properties, performance, and synergism with commercial cellulase, which are important characteristics for application in an industrial environment, can contribute as a promising biotechnological alternative, application, and valorization of hemicellulose in the production of bioproducts.

Keywords: Endo-xylanase, Halotolerant enzyme, Sugarcane bagasse hydrolysis

Supported by: CAPES and CNPq

08703 - Poster Session

CC.10 - PROJECT: Obtainment and application of antimicrobial coating based on green banana starch for post-harvest fruit conservation

BÁRBARA REGES FEITOSA ¹, VOLNEI SOUZA¹

¹Centro das Ciências Biológicas e da Saúde, Universidade Federal do Oeste da Bahia (BA, Brasil)

INTRODUCTION

Post-harvest losses of vegetables have been associated with the stages of transport to storage, highlighting the importance of developing appropriate packaging with the development of post-harvest technologies, with a view to optimizing transport costs, reducing losses and maintenance of fruit quality. In addition, the high perishability of post-harvest vegetables makes reference to the production of ethylene in climacteric fruits, providing rapid senescence of the vegetables, reducing their useful life even before reaching the consumer, generating damage to the small producer and the community. With this in mind, we have studied the production of biodegradable and low-cost coatings, obtained through natural polymers, with the insertion of antimicrobial systems as a way to increase the shelf life of post-harvest fruits, using active packaging technology.

OBJECTIVES

In view of this, this research project aims to produce an antimicrobial coating based on green banana starch and apply it to the conservation of postharvest papaya.

MATERIALS AND METHODS

The methodology applied will be to obtain and characterize the starch, evaluating its yield, composition, water solubility, gelatinization capacity and syneresis, in addition to producing the coating, applying to the fruits and analyzing their shelf life, through the evaluation of total soluble solids, titratable acidity, color, weight loss and mold and yeast count. The data will be evaluated by analysis of variance (ANOVA) with Tukey's means comparison test (p < 0.05 of significance). The statistical package SAS (Statistic Analysis System) version 9 will be used to carry out the analyzes.

DISCUSSION AND RESULTS

Expected results: In this work is expected to obtain a coating capable of increasing the shelf life of fruits compared to traditional methods of conservation.

CONCLUSION

Keywords: active packaging, biopolymers, food conservation

08972 - Poster Session

CC.11 - Characterization of lignocellulosic enzymes for hemicellulose degradation

Brisa Moreira Gomes ¹, Vandierly Sampaio de Melo^1,1, Felipe Santiago Chambergo Alcade¹

¹Department of Biotechnology, University of Sao Paulo (São Paulo, Brazil)

INTRODUCTION

Environmental problems caused by the release of greenhouse gases (GHG) in the burning of fossil fuels have motivated the search for ecological and sustainable consumerism. One strategy is to exploit renewable and sustainable biomass resources. Lignocellulosic materials are renewable, low cost and abundantly available on the planet, and their product is widely used in various industrial sectors. One of the ways of converting this material is through the biochemical route, based on hydrolysis and fermentation processes. This process is carried out by several lignocellulolytic enzymes responsible for the degradation of lignocellulosic biomass in fermentable sugars. Some of this enzymes are α-L-arabinofuranosidases, β-xylosidases and xylanases, responsible for the degradation of hemicellulose.

OBJECTIVES

Therefore, this work seeks to characterize an α-L-arabinofuranosidase/β-xylosidase (TerARA) from termite intestine metagenoma and an endo-1,4-β-xylanase (PaeXYL2) from Paenibacillus sp < br>

MATERIALS AND METHODS

The putative enzyme genes were selected through the CAZy database, synthesized and cloned, and the recombinant proteins were expressed in E. coli BL21. Until this moment, the characterization of the TerARA enzyme is nearing completion and the PaeXYL2 enzyme has already been cloned, with initial production and purification tests underway. TerARA was purified by affinity chromatography on a nickel-sepharose column. In silico analysis of the amino acid sequence, analysis of enzyme activity in different synthetic and natural substrates, and the influence of pH, temperature and ions on the enzyme activity were carried out.

DISCUSSION AND RESULTS

The results show the bifunctionality of the enzyme to pNP-X and pNP-A, greater activity at pH 7.0 and temperature at 40 ºC. In tests with additives, the enzyme exhibited expressive inhibition effects in presence of Cu2+ and Fe2+ and EDTA. A slightly positive effects was exhibited in Ca2+ Mg2+ and Mn2+. The enzyme didn’t show efficient thermal stability on the pNP-A substrate.

CONCLUSION

The already results obtained show that TerARA has interesting characteristics for application in the deconstruction of plant biomass.

Keywords: hemicellulases, glycosyl hydrolases, bifunctional enzymes

08381 - Poster Session

CC.12 - High ethanol yield in mead fermentation after yeast immobilization and enzyme treatment of must

João Vitor Rios Mayrinck ⁵, Marcel Menezes Lyra da Cunha^5,5

¹Núcleo Multidisciplinar de Pesquisa UFRJ – Xerém em Biologia, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Mead is an alcoholic beverage made from fermented honey. Publications about meads are growing in number since 2016 and several articles focus on fermentation problems due to lack of important nutrients, e.g. free amino nitrogen (FAN), in honey musts. Our work addressed a systematic review of mead literature and fermentation issues common to mead production, such as FAN and sugar availability

OBJECTIVES

. We hypothesised that sugarcane could be used both as juice for source of sugars and nutrients but also, its bagasse, as structural support to immobilize yeast for fermentation. In addition, commercial enzymes common to beer production were tested to improve nutritional characteristics of the honey must.

MATERIALS AND METHODS

A systematic search was done to understand all published scientific knowledge. Search in Google Scholar, PubMed and Web Of Science databases, choosing keywords: NMR (Nuclear magnetic resonance), HPLC (High performance liquid chromatography), Fermentation, Yeast, Saccharomyces cerevisiae, QA23, Honey, Mead, Beer, Wine, Cider. The results were extracted and submitted on organization software on-line Rayyan QCRI. 147 articles were found that describe the state of the art on the subject.

DISCUSSION AND RESULTS

FAN on musts treated or not with amylases, proteases and pectinases was measured by ninhydrin method. After protease only, FAN was increased by 55,5% and, after fermentation, final ethanol concentration was 49% higher.

CONCLUSION

Morphology of sugarcane bagasse and yeast adhesion were analyzed by scanning electron microscopy. Bagasse pretreated with NaOH and heat resulted in greater cell adhesion, better cell dispersion per bagasse and higher fermentation yield. Our results shed light on the use of enzymes and support for yeast on the production of mead and provide strategies for this application on larger scale experiments. Despite the increase in publications, the literature is still incomplete and with many gaps and much still needs to be discussed.

Keywords: Mead, Fermentation, Free amino nitrogen

Supported by: faperj

08430 - Poster Session

CC.13 - Detection of Wheat stripe mosaic virus (WhSMV) using loop-mediated isothermal amplification.

ANDERSON VARELA DE ANDRADE¹, FERNANDO SARTORI PEREIRA¹, FABIO NASCIMENTO DA SILVA¹, GUTAVO FELIPPE DA SILVA¹, MARIA DE LOURDES BORBA MAGALHÃES¹

¹DEPARTAMENTO DE PRODUÇÃO ANIMAL E ALIMENTOS, UNIVERSIDADE DO ESTADO DE SANTA CATARINA - cENTRO AGROVETERINÁRIO (SANTA CATARINA, BRASIL)

INTRODUCTION

Wheat (Triticum spp.) is one of the most cultivated cereals worldwide. According to the national supply company (CONAB), Brazil is the sixteenth country in wheat production, yielding 5.2 tons per year according to the United States Department of Agriculture (USDA). In 2020, the southern Brazilian region was responsible for producing 90% of wheat, according to the Technical Office for Economic Studies of the Northeast (ETENE). The Wheat stripe mosaic virus (WhSMV) has been recently identified, whose infection leads to 50% wheat production loss.

OBJECTIVES

Our goal is to develop a point of care diagnostic test for the WhSMV virus using the Loop-Mediated Isothermal Amplification (LAMP) technique.

MATERIALS AND METHODS

The use of point of care strategies, such as the LAMP technique, allows the rapid identification of the pathogens in the field, improving decision-making, and expediting early treatment to reduce production losses.LAMP technique is a new DNA amplification method for pathogen detection that shows high speed, specificity, and efficiency, under isothermal conditions.

DISCUSSION AND RESULTS

Furthermore, the process is simple, low cost, and less sensitive to the presence of inhibitors. Our goal is to develop a new molecular test for WhSMV detection using LAMP.

CONCLUSION

The rapid identification and early treatment of such a pathogen may improve grain production, thus making wheat and its derivatives more economically profitable for producers and millions of consumers worldwide.

Keywords: Wheat, WhSMV, LAMP

Supported by: PROMOP

08981 - Poster Session

CC.14 - Development and Stability study of Microemulsionate peelin of Papain

MAHALA CORREIA CLAUDIO ¹, J.G. Amaral², J.S. De Freitas¹

¹BIOQUIMICA E BIOLOGIA MOLECULAR, UNIVERSIDADE ESTADUAL DA BAHIA (BAHIA, Brasil), ²BIOCIENCIAS, UNIVERSIDADE FEDERAL DA BAHIA (Bahia, Brasil)

INTRODUCTION

The word “cosmetic” refers to products that amplify the skin´s appearance, instensify cleanliness and promote beauty.The dissemination of the increasing appreciation of appearance by the media, the cosmetic industry is expanding and little shaken by the economic crisis.Therefore, investiments in new product development have increased, and along with it, the appearance of new vehicles and used raw materials. Among them are the use of enzymocosmetics, an expanging area with promising results. To convey such products, several are used to improve their performance, and on of them is the conversion of regular materials into nanometric formulations.

OBJECTIVES

The objective of this work was develop and study the stability of a papain nanoemulsified enzymatic peeling.

MATERIALS AND METHODS

At first, a pseudoternary phase diagram was developed. The microemulsified systems were selected for delivery of 2% papain, followed by its characterization (size, zeta potential, PDI, conductivity, pH, preliminary stability, thermal stress.In the phase diagram, a wide region of transparent nanodisperse systems was identified, with PDI values below 0.2, and characteristic droplet sizes for this type of system.

DISCUSSION AND RESULTS

Formulation presented the average droplet size of 35.28. The values of PDI were 0.28. Macroscopic aspect of the formulation analyzed without separation of phases. In thermal stress, temperature generated large oscillations in the Zeta Potential values, but at values far from zero, which configures as main feature of ensuring system stability. PDI values have undergoing changes, as well as that of size, but still, if kept stable during the heating ramp.

CONCLUSION

After conducting preliminar tests, the formulation becomes stable to deliver papain.Protein dosage tests, accelerated stability, and safety, should be performed to perform papaya delivery in this namoemulsified system

Keywords: Cosmetic, Nanotechnology, Papain

Supported by: FAPESB

08315 - Poster Session

CC.15 - Evaluation of Trim21 as a molecular tool for antibody purification

Anelize Ramos¹, Leonardo Fernandes¹, Gustavo Felippe da Silva¹, Maria de Lourdes Borba Magalhaes¹

¹BIOQUIMICA E BIOLOGIA MOLECULAR, CAV- UNIVERSIDADE ESTADUAL DE SANTA CATARINA (Santa Catarina, Brasil)

INTRODUCTION

The use of antibodies in therapeutics and diagnostic is an expanding segment of the pharmaceutical and biotechnology industries. Antibodies can be purified using chromatographic resins based on bacterial proteins A and G. However, these resins use acidic elution conditions, which may cause antibody denaturation. Therefore, improved methods of IgG purification are a need of the biotechnology industries. Trim21 is an intracellular protein that mediates intracellular antibody-mediated proteolysis and binds the Fc domain of IgGs and IgAs. The presence of increasing ionic strength weakens IgG binding. Therefore, Trim21 might be a good candidate for the development of chromatographic resins for IgG purification since elution conditions can occur at neutral pH conditions in the presence of salt. Previous studies from our group developed a chimeric protein consisting of the Fc binding domain of TRIM21 (PRYSPRY domain) linked to a streptavidin moiety to facilitate TRIM21 immobilization biotinylated matrices (PCT/BR2018/050310, 2018)

OBJECTIVES

Test the performance of the chimeric protein Trim21-streptavidin immobilized in biotinylated resins for the purification of G-type immunoglobulins (IgG).

MATERIALS AND METHODS

Trim21-streptavidin (Trim21-SA) was expressed in E. coli (BL21 (DE3) pLysS in the presence of 0.1mM isopropyl-β-D-thiogalactopyranoside (IPTG). The protein's inclusion bodies were denatured using 8M urea and refolded, followed by purification using Ni-NTA resin.Trim21-SA was immobilized in biotinylated resin, and the resultant resin was tested for IgG purification from human serum.Trim21-SA was efficiently immobilized in biotinylated resins, suggesting that the streptavidin moiety of the chimeric protein was efficiently folded.

DISCUSSION AND RESULTS

The resulting matrix was used to purify IgG from human serum. Neutral pH elution conditions are currently being tested. TRIM21-SA appears to be a promising tool in the purification of antibodies.

CONCLUSION

This tool can expand the purification capacity of antibodies used in the manufacture of medicines, diagnostic, and research reagents, updating the biotechnological processes.

Keywords: Affinity chromatography, Biotin resin, Immunoglobulin G

08293 - Poster Session

CC.16 - EXPRESSION OF THE Cryptococcus flavus AMYLASE IN Pichia pastoris (Komagataella phaffii) FOR INDUSTRIAL APPLICATIONS

ANA AMELIA MAIA SILVA¹, THAÍS PAIVA PORTO DE SOUZA¹, JONATAS OLIVEIRA DA SILVA¹, MICHELLI DOS SANTOS¹, RENATO RAMOS GODOI², DANIEL BONOTO GONÇALVES¹, WILLIAM JAMES NOGUEIRA LIMA³, MARINA QUÁDRIO RAPOSO BRANCO RODRIGUES⁴, RONALDO ALVES PINTO NAGEM², ALEXSANDRO SOBREIRA GALDINO¹

¹Divinópolis, Federal University of São João del-Rei (Minas Gerais, Brasil), ²Belo Horizonte, Federal University of Minas Gerais (Minas Gerais, Brasil), ³Montes Claros, Federal University of Minas Gerais (Minas Gerais, Brasil), ⁴Alfenas, Federal University of Alfenas (Minas Gerais, Brasil)

INTRODUCTION

The expression of recombinant proteins through heterologous systems is a promisiing alternative for the production of enzymes. The available models have adequate machinery to increase the production levels of a certain protein with the yield of expressed protein being higher than in native systems.

OBJECTIVES

The main goal of this study was to develop a recombinant Pichia pastoris (Komagataella phaffii) strain for the production of Cryptococcus flavus α-amylase (Amy1), using methanol as a sole carbon source.

MATERIALS AND METHODS

The nucleotide sequence encoding Amy1 was optimized with codon usage biased for expression in K. phaffii. The resulting recombinant plasmid was used to transform yeasts cells. Potential recombinant clones were then evaluated for their ability to degrade starch using FUWA method protocol qualitatively and quantitatively.

DISCUSSION AND RESULTS

The starch-degrading plate assay showed that Amy1 was successfully expressed in several clones and extracellular amylolytic activity increased during growth in fermentation, reaching a maximal value (5.45/5.64 U/mL) at 46h of incubation. These values were higher than those found in the literature.

CONCLUSION

Preliminary results showed that the gene was successfully cloned and expressed in K. phaffii cells. However, additional studies on expression conditions need to be conducted. Therefore, this study was able to develop a recombinant K. phaffii strain with potential application in industrial starch degradation.

Keywords: Enzymes, Biochemistry, Recombinant

Supported by: CAPES, CNPq and FAPEMIG.

08440 - Poster Session

CD.01 - Details of the cooperative binding of piperlongumine with rat serum albumin obtained by spectroscopic and computational analyses

ANA PAULA RIBEIRO POVINELLI ¹, Gabriel Zazeri¹, Marcelo de Freitas Lima², Marinonio Lopes Cornélio¹

¹Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas (, Brasil), ²Departamento de Química, Instituto de Biociências, Letras e Ciências Exatas (, Brasil)

INTRODUCTION

Piperlongumine (PPL) has presented a variety of important pharmacological activities. Although PPL is present in the bloodstream, no information is found on the interaction between PPL and rat serum albumin (RSA). In this sense, the present study elucidated the mechanism of interaction between PPL and RSA, using in conjunction spectroscopic and computational techniques.

OBJECTIVES

The goal of present investigation is to elucidate the molecular biophysical mechanisms of the interaction between RSA and PPL. Two approach lines of the molecular biophysical field were applied: one experimental, based on spectroscopy; another computational, based on molecular docking and dynamics.

MATERIALS AND METHODS

Emission fluorescence was measured in an ISS PC1 spectrofluorometer with temperature controlled at 288, 298, 308 K.The sample was excited at 295 nm, and the emission spectrum was obtained in the region of 305-500 nm. Inner filter correction was applied for the excitation and for all the emission wavelengths. In the synchronous fluorescence experiments, excitation wavelength varied from 240 to 350 nm with the excitation and emission wavelengths interval (Δλ) set to 60 nm. The molecular docking was calculated with Autodock 4.2 and the molecular dynamics of the complex were executed by GROMACS/5.1.4, with GROMOS96/53a force field.

DISCUSSION AND RESULTS

Our work shows the importance of applying inner filter correction over the entire fluorescence spectrum prior to any conclusion regarding changes in the polarity of the fluorophore microenvironment. Thermodynamic parameters revealed that PPL binds to RSA spontaneously (ΔG < 0) and the process is entropically driven. Interaction density function method (IDF) indicated that PPL accessed two cooperative sites in RSA, with moderate binding constants. The molecular docking described the microenvironment of the interaction sites, rich in apolar residues. The stability of the RSA-PPL complex was checked by molecular dynamics.

CONCLUSION

The RSA-PPL complex was characterized by a molecular biophysical approach with the fluorescence technique and computational methods.

Keywords: Spectrosocopy, Molecular dynamics, Protein-ligand interaction

Supported by: CAPES

08757 - Poster Session

CD.02 - Inhibitory effect of icetexane diterpenoids on Leishmania braziliensis old yellow enzyme

Silvia Helena Libardi¹, Thais Carvalho de Moura¹, Anees Ahmad ², Antonio Carlos Bender Burtoloso¹, Francis Barbosa Ferreira³, Ronaldo Junio Oliveira⁴, Ícaro Putinhon Caruso⁵, Fernando Alves Melo⁵, Júlio Cesar Borges¹

¹Instituto de Química de São Carlos, Universidade de São Paulo (São Paulo, Brazil), ²Instituto de Química, Universidade Estadual de Campinas (São Paulo, Brazil), ³FAZU, Faculdades Associadas de Uberaba (Minas Gerais, Brazil), ⁴Instituto de Ciências Exatas, Naturais e Educação, Universidade Federal do Triângulo Mineiro (Minas Gerais, Brazil), ⁵Instituto de Biociências, Letras e Ciências Exatas , Universidade Estadual Paulista (São Paulo, Brazil)

INTRODUCTION

Old Yellow enzymes (OYEs) are NAD(P)H flavin-dependent redox enzymes that promote the asymmetric reduction of activated α, β-unsaturated alkenes (e.g. as ketones, aldehydes, carboxylic acids). OYE are present in protozoan parasites such as Leishmania braziliensis (LbOYE) and could be a potential target for treatments of parasitic pathologies. Icetexane diterpenoid is a class of compounds from plants that have been reported with antiparasitic activity.

OBJECTIVES

In this study, we aimed to evaluate the inhibition of LbOYE activity by synthetic diterpene brussonol, a natural product first isolated from Salvia brussonetti, and two analogs AN-08 and AN-35.

MATERIALS AND METHODS

Recombinant LbOYE (rLbOYE) was produced in Escherichia coli cells and purified until homogeneity had its activity measured using N-ethylmaleimide (NEM) as substrate and NADPH as reductant. The kinetic experiments were performed following NADPH concentration decay at 340 nm in the presence of brussonol and analogs.

DISCUSSION AND RESULTS

The compound brussonol did not show significant inhibition while the analogs AN-08 and AN-35 did. Furthermore, the interaction of compounds with rLbOYE was carried out by fluorescence quenching with Kd on the order of μM. STD-NMR also demonstrates the interaction of rLbOYE and all ligands. The differences in the inhibition were better understood by molecular docking that showed differences of brussonol position in relation to the FMN group comparing to the analogs.

CONCLUSION

These results demonstrate that icetexane diterpenoids modifications improved the inhibitory effects on the LbOYE activity.

Keywords: old yellow enzymes, Leishmania braziliensis, flavoenzyme

Supported by: FAPESP (2018/05576-1)

08879 - Poster Session

CD.03 - Antimicrobial activity of nanostructured lipid carriers prepared with natural oils and loaded with ciprofloxacin

Talita Cesarim Mendonça ¹, Simone Ramos Castro^1,1, Lígia Ribeiro^2,2, Ludmilla Moura¹, Gabriela Geronimo¹, Gustavo Rodrigues¹, Fabíola Carvalho¹, Eneida De Paula¹

¹Department of Biochemistry and Tissue Biology, University of Campinas (São Paulo, Brazil), ²Institute of Biotechnology, Federal University of Uberlândia (Minas Gerais, Brazil)

INTRODUCTION

The fluoroquinolone ciprofloxacin (CIP) is a broad-spectrum antibiotic with activity against gram-positive and gram-negative bacteria of clinical interest such as Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus and Enterobacter aerogenes. Despite its low solubility, CIP inhibits the bacterial DNA gyrase, favoring degradation and blocking DNA replication, transcription, and DNA repair. Lipid-based drug delivery systems (DDS) such as nanostructured lipid carriers (NLC) have high upload capacity for lipophilic drugs such as CIP, and can be used to improve their bioavailability, chemical stability, and therapeutic action. Natural products can bring additional therapeutic properties if used as excipients of DDS.

OBJECTIVES

This study aimed to develop NLC composed of natural lipids with encapsulated CIP and evaluate its antimicrobial activity in vitro.

MATERIALS AND METHODS

The formulation was prepared using the emulsification-ultrasonication method, consisting of beeswax (7%), andiroba oil (3%), Pluronic F-68 (2.5%) and 3% encapsulated CIP.

DISCUSSION AND RESULTS

The optimized formulation presented 5.1 ± 0.2 x 1013 particles/mL, average diameters between 259-316 nm, low size polydispersity (< 0.20), negative zeta potentials (|28,6 - 35,3| mV), high encapsulation efficiency (94.6%) and shelf stability for 12 months of storage at 25 oC. In the antimicrobial susceptibility testing the diameters of the inhibitory halo of 10 bacteria strains (S. aureus - S.A., BEC9393, and RIB 1- S. Epidermidis, P. aeruginosa - 31NM, PA 26, PA-ATCC 25619 and 76JF - E. coli ATCC 129214 and K. pneumoniae KP230) treated with increasing concentrations of NLCCIP were compared to those of free CIP.

CONCLUSION

The results showed that NLCCIP was effective against the 10 strains, and that encapsulation improved by 30-45% the antimicrobial activity of CIP. These results point out the potential use of NLC prepared with natural compounds – to join the intrinsic therapeutic properties of its lipid excipients to that of the fluoroquinolone antibiotics – in the treatment of bacterial resistant strains.

Keywords: Ciprofloxacin, nanostructured lipid carriers, Drug delivery

Supported by: FAPESP ; CNPq/PIBITI; CAPES

08127 - Poster Session

CD.04 - Peptide-conjugated silver nanostars as anticancer agent

Ítala Cristine Silva ¹, Miguel Peixoto de Almeida², Eulália Pereira², Octavio Luiz Franco^3,4,5, Nuno Correia Santos¹, Sónia Gonçalves Abreu¹

¹Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa (Lisboa, Portugal), ²LAQV/REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto (Porto, Portugal), ³Centro de Análises Proteômicas e Bioquímicas, Pós-graduação Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília (Brasília, Brazil), ⁴Programa de Pós-Graduação em Patologia Molecular, Universidade de Brasília (Brasília, DF, Brazil), ⁵S-Inova Biotech, Pós-graduação em Biotecnologia, Universidade Católica Dom Bosco (Campo grande, MS, Brazil)

INTRODUCTION

The challenge of bacterial resistance and the problems caused by the decreased efficacy of conventional antibiotics have led to the search new therapeutic strategies. Antimicrobial peptides (AMPs) have been identified as promising alternatives to the conventional molecules used nowadays against infection. Some of them have been shown to have dual activity, both as antimicrobial and anticancer peptides. Nanoparticles have been used as improved drug delivery systems and as diagnostic tools for the imaging of cancer cells. Silver nanoparticles (AgNPs) have high antibacterial activity, being a promising solution to treat intracellular infections. In addition to the use of AgNPs as therapeutic agent, their use as carrier, conjugating with molecules of biomedical importance, has been increasing in the last years.

OBJECTIVES

In the present work we conjugated the AMP PaMAP 1.9 with star-shaped silver nanoparticles (AgNSs) to improve the peptide efficacy against cancer cells.

MATERIALS AND METHODS

The peptide, AgNSs and conjugates were evaluated in terms of stability by the following methodologies: Nanoparticle Tracking Analysis, Transmission electron microscopy, Ultraviolet–visible spectroscopy, and Dynamic light scattering. Their anticancer activity was assayed through a XTT viability test.

DISCUSSION AND RESULTS

The biophysical characterization of the peptide-conjugated nanoparticles showed consistent size and stability. AgNSs induced cell death in MCF-7 cancer cells, at low concentrations, and the peptide itself exhibit cytotoxic activity on these cells. However, when combined with AgNSs, there was an enhancement of the percentage of cell death, showing that the anticancer activity of the peptide is amplified. The peptide and the nanoparticles were screened for cytotoxic effects in healthy cells as well. PaMAP1.9 has cytotoxic effects on MCF10-A cells above the IC50 of cancer cells. Furthermore, the peptide-AgNSs conjugate displays lower cytotoxicity to healthy cells in comparison with the peptide itself.

CONCLUSION

Results indicate that PaMAP1.9 conjugated with AgNSs, provide lower toxicity and improve their anticancer effect.

Keywords: antimicrobial peptides, silver nanoparticles, drug delivery

Supported by: Portuguese national funding agency for science, research and technology (FCT)

08321 - Poster Session

CD.06 - Articaine-loaded NLC functionalized with copaiba oil: Improvement of anesthetic effect in zebrafish and in murine inflammatory pain model

Gustavo Henrique Rodrigues da Silva ¹, Gabriela Geronimo¹, Julia B. P. Lemes², Ludmilla David de Moura¹, Fabíola V. Carvalho¹, Talita Cesarim Mendonça¹, Juan Pablo García-López³, Carmen Gloria Feijóo³, Carlos Amilcar Parada², Eneida de Paula¹

¹Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (São Paulo, Brazil), ²Department of Structural and Functional Biology, Institute of Biology, University of Campinas (São Paulo, Brazil), ³Department of Biological Sciences, Faculty of Life Sciences, Andres Bello University (, Chile)

INTRODUCTION

Articaine (ATC) is the second most used amino-amide local anesthetic in dentistry. Due a peculiarity in its structure (an additional ester group), ATC can be used at higher doses, with lower systemic adverse effects that other anesthetics. However, like other local anesthetics agents, ATC is not effective on inflamed tissues.

OBJECTIVES

Articaine was encapsulated in nanostructured lipid carriers (NLC) prepared with copaiba oil (CO) as a functional excipient, to enhance its anesthetic and anti-inflammatory properties over inflamed tissues.

MATERIALS AND METHODS

DISCUSSION AND RESULTS

After development and characterization, the NLC-CO-ATC formulation (particles size = 217.7 ± 0.8 nm, PDI = 0.174 ± 0.004, zeta potential = -40.2 ± 1.1 mV and ATC loading efficiency = 78.4 ± 0.1%) was tested regarding its anesthetic effectiveness using two in vivo models (Zebrafish larvae and murine). In Zebrafish larvae (5 days post fertilization), the NLC-CO-ATC formulation administered in the larvae medium was orally absorbed and promoted effects on the CVS (60% > bradycardia) and CNS (100% higher response to touch) than free articaine. In rats, nociception was measured by the electronic Von Frey test, using the carrageenan-induced inflammatory pain model and NLC-OC-ATC formulation increased by 30% the anesthetic efficacy compared to free ATC. Furthermore, NLC-CO-ATC prolonged the anesthetic effect until the end of the experiment (3 hours) while free ATC caused anesthesia for no more than 1 hour.

CONCLUSION

Thus, the results found show that the encapsulation of ATC in NLC-OC is a promising strategy for the treatment of inflamed tissues, where the lower pH curbs the effectiveness of anesthetic agents. In addition, the formulation was found efficient to control postoperative pain (carrageenan-induced inflammatory pain model), probably due to ATC sustained release but also to the functionalization with CO, which is another advantage of this system.

Keywords: Articaine, NLC, inflammatory pain

Supported by: FAPESP (#17/15174-5, G.H.R.S. Ph.D scholarship and internship at Andres Bello University—Chile; E.P. grant #14/14457-5)

08570 - Poster Session

CD.07 - PROJECT: Study of neuroprotective potential of Amburana cearensis compounds in aminochrome in vitro model of Parkinson's disease

Gabriel de Jesus Ferrolho ¹, Victor Diógenes Amaral da Silva^1,1,1,1, Paulo Roberto Ribeiro de Jesus^1,1,1,1, Silvia Lima Costa^1,1,1,1

¹Departamento de Bioquímica e Biofísica, Universidade Federal da Bahia (BA, Brasil)

INTRODUCTION

Parkinson's Disease (PD) is a slowly progressing neurodegenerative disease with alarming epidemiological data, but without a cure. Some neuroprotective compounds derived from plants from the flora of the Brazilian caatinga have been prospected in order to inhibit dysfunctions and cell death in the pathogenesis of PD. Amburana Cearenses seed extracts have shown promising results in in vitro studies, as a potent inhibitor of neuronal death induced by glutamatergic excitotoxicity.

OBJECTIVES

In this sense, this work aims to study the potentially therapeutic effects of A. cearenses seed extract obtained by extraction with dichloromethane (EDAC), and its isolated coumarin.

MATERIALS AND METHODS

PD in vitro model will be induced by aminochrome. It is a molecule derived from the oxidation of dopamine and capable of inducing molecular alterations characteristic of PD, such as lysosomal and mitochondrial dysfunction, therefore, it has been used as an inducer in an experimental model of PD. Therefore, cell cultures of the PC12 lineage differentiated with nerve growth factor (NGF) treated with aminochrome and/or EDAC or coumarin will be used. In this model, cell viability will be evaluated by MTT, lysosomal acidification by staining with acridine orange and Lysosensor, as well as the evaluation of mitochondrial membrane potential by staining with 5.5', 6.6' tetrachlorine iodide 1 ,1,3,3' tetraethylbenzimidazolylcarbocyanine (JC-1) to investigate the association of aminochrome with lysosomal dysfunction and altered mitochondrial function.

DISCUSSION AND RESULTS

CONCLUSION

Thus, this work may contribute to the characterization of the neuroprotective effect of compounds from A. cearenses and will reveal potential pharmacological application of the plant and its constituents in the treatment of the disease.

Keywords: Amburana cearenses, neurodegeneration, neuroprotection

08942 - Poster Session

CD.08 - PROJECT: Evaluation of the antiproliferative and cytotoxic actions of Viridicatumtoxin A combined with antitumor drugs in 2D and 3D cell culture models.

Emylli Areco Pereira ¹, Renata Trentin Perdomo¹

¹Departamento de Biologia Molecular e Cultura Celular, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul , Brasil)

INTRODUCTION

The viridicatumtoxins belong to a rare class of polyketide antibiotics of the tetracycline group produced by endophytic fungi of various species within the Penicillium, Aspergillus and Paecilomyces family. These toxins have positive functions in the pharmaceutical industry, acting as antibiotics. Furthermore, studies show their cytotoxicity in certain cancer cells, demonstrating that their use as a chemotherapeutic against cancer is a promising perspective.

OBJECTIVES

The objective of this proposal is to test the viridicatumtoxin A obtained from Penicillium, in human cancer cell lines (prostate,PC-03; kidney,786-0; colon,HT-29; MCF-7 breast and multidrug-resistant breast, MDA-MB-231) and a non-neoplastic one (fibroblast,HFF1) to obtain the selectivity index, in culture model 2D and 3D, since the three-dimensional model mimics the in vivo, where cells occupy the three dimensions of space, generating multicellular spheroid structures that exhibit aspects very similar to those observed in tumors in living organisms.

MATERIALS AND METHODS

In the 2D model, the antiproliferative activity in these strains will be evaluated, by staining with Sulforhodamine B, and then the strain with the highest antiproliferative activity will be selected by GI50 concentration for evaluations in the 3D model (spheroids) obtained by molds from a cell density of 1x106. These spheroids will be treated with viridicatumtoxin A and evaluated for apoptotic effect using fluorescent dyes and by flow cytometry for annexin V concentration, activation of caspase 3 and cell cycle arrest by 7AA-D. To estimate the oxidative stress action of the treatment, enzymatic and non-enzymatic markers will be evaluated.

DISCUSSION AND RESULTS

At the end of the study it is expected that it will be possible to determine the cytotoxic potential of the substance, as well as the selectivity index, cytostatic and cytocidal effect, and the possible pathways of antiproliferative activity from apoptosis and oxidative stress generated.

CONCLUSION

Keywords: cytotoxicity, viridicatumtoxin A, 3D cell culture

08303 - Poster Session

CD.09 - Combining Elmann’s Colorimetric Assay and (STD)1H-NMR to Unravel Inhibitory Activity of a Ru(II) Polypyridyl Complex Towards human Acetylcholinesterase.

Marlon Augusto Profeta de Almeida ¹, Flávio Kock¹, Tiago Venâncio¹, Rose Carlos¹

¹Chemistry, Federal University of Sao Carlos (, Brazil)

INTRODUCTION

Cholinergic hypothesis for Alzheimer’s disease (A.D.) implicates neurotransmitter acetylcholine (ACh) cleavage by acetylcholinesterase (AChE) enzyme as the responsible event related to A.D symptoms. Anticholinergic drugs are the only available FDA approved treatment for A.D, although with several side effects and poor memory enhancement results. Ergo the search for acetylcholinesterase inhibitors remains important. Here, we report the inhibitory activity of cis-[Ru(bpy)2(EtPy)2].2PF6 (bpy = 2,2’ bipyridine, EtPy = 4,2-ethylamino pyridine) against hAChE probed by classic Elmann assay and (STD)1H-NMR technique, that gives quantitative information about molecular orientation and surface interaction based on magnetic saturation transfer by NOE effect from enzyme to complex, enabling the fine tunning of the structure to enhance the results.

OBJECTIVES

Evaluate the cis-[Ru(bpy)2(EtPy)2].2PF6 inhibitory activity against hAChE by Elmann’s assay and which ligand plays the key role in inhibition by (STD)1H-NMR.

MATERIALS AND METHODS

Reagents, solvents and hAChE were purchased from Sigma-Aldrich and used without purification. Elmann’s assay was conducted in an Agilent 8453 Uv-Vis spectrophotometer using a Helma two window quartz cuvette (3 mL 1 cm length path) NMR experiments were conducted in a Bruker Avance 600 MHz.

DISCUSSION AND RESULTS

Michaelis-Mentem evaluation of Elmann’s assay indicates that the complex strongly inhibits hAChE activity (inhibition constant Ki = 11,7 μM and IC50 = 39 μM) presenting a competitive mechanism. (STD)1H-NMR indicates that the aminopyridyl ligand interacts strongly with the enzyme surface (100% STD intensity) than bpy ligand (73%), probably due to structural resemblance between ethyl amino group and choline molecule.

CONCLUSION

The complex presents strong inhibitory activity against hAChE as demonstrated by Elmann’s assay. (STD)1H-NMR shows that the amino pyridine ligand is in close interaction to the surface of the enzyme, being most relevant to the result, which implies that structural changes in the other ligands may enhance the inhibitory activity of this complex.

Keywords: Acethylcholinesterase, Enzyme inhibition, Ru(II) complex

Supported by: CAPES (proc number 88882.332778/2019-01) and FAPESP (proc numbers 2018/16040-5, 2018/09145-5 and 2019/21143-0)

08722 - Poster Session

CD.10 - Role of residual Sb(III) in the therapeutic efficacy of topical meglumine antimoniate against cutaneous leishmaniasis

Ana Carolina Borges de Oliveira ¹, Frédéric Jean Georges Frézard¹, Virgínia Mendes Russo Vallejos¹, Juliane Sousa Lanza¹, Cynthia Demicheli ², Henrique José Fabrino²

¹Fisiologia e Biofísica, UNIVERSIDADE FEDERAL DE MINAS GERAIS (MG, Brasil), ²Departamento de Química, UNIVERSIDADE FEDERAL DE MINAS GERAIS (MG, Brasil)

INTRODUCTION

Leishmaniasis is a global health problem and, in Brazil, the pentavalent antimonial meglumine antimoniate (MA) has been the drug of choice for the treatment of cutaneous leishmaniasis (CL) since 1943. This drug is used parenterally and is believed to act as prodrug, being reduced from Sb(V) into Sb(III) active form. Unfortunately, MA is highly toxic and the WHO strongly recommends the development of new therapeutic strategy.

OBJECTIVES

In this context, we investigate a topical formulation of MA for CL and the possibility of optimizing its efficacy through manipulation of the degree of polymerization of MA and the residual Sb(III) content.

MATERIALS AND METHODS

Three different forms of MA were used: synthetic MAs prepared from either SbCl5 (MA-SbCl5) or KSb(OH)6 (MA-KSb(OH)6); and commercial MA (Glucantime®). As demonstrated previously by our group, Glucantime® is more polymerized than the synthetic MAs and MA-SbCl5 exhibits the highest level of Sb(III) residue. These compounds were formulated as propylene glycol-containing hydrogels at 12%(w/v) Sb concentration. These formulations were applied daily for 30 days at the tail base of BALB/c mice after infection with Leishmania amazonensis. Control groups were treated topically with drug-free gel or remained non-treated. The efficacy of topical treatments was compared to that of intraperitoneal treatment with Glucantime® (200 mg Sb/kg/day).

DISCUSSION AND RESULTS

Treatment with the topical formulation of MA-SbCl5 promoted significant reductions in the lesion size and parasite load in comparison to controls, to a similar level as the parenteral treatment. On the other hand, Glucantime® or MA-KSb(OH)6 did not show significant efficacy.

CONCLUSION

This study strongly supports the role of residual Sb(III) in the therapeutic efficacy of topical MA against CL. MA-SbCl5 also emerges as a promising chemical form of MA for the topical treatment of CL.

Keywords: antimony, topical formulation, speciation

Supported by: CNPq

08203 - Poster Session

CD.11 - Hot spots identification and molecular fragment screening as innovative approaches to development Leishmania major diidroorotate dehydrogenase inhibitors

Thamires Quadros Froes ¹, Marcelo Castilho^2,2, Flávio Emery^1,1,1, Maria Cristina Nonato^1,1,1,1

¹Biomolecules Science, School of Pharmaceutical Sciences of Ribeirão Preto (São Paulo, Brazil), ²LABIMM/LACRIMA, School of Pharmacy (Bahia, Brazil)

INTRODUCTION

Although more than 1 billion people are at risk by neglected tropical diseases (NTD) and 35,000 deaths/day are due to such ailments worldwide, the R&D investment from both private and public sectors to fight NTD is disproportionally small. Among NTDs, cutaneous leishmaniasis (CL) is spread over 85 countries and causes up to 119,600 deaths, per year, in Brazil. The available drugs display high toxicity and low efficacy, highlighting the need for novel therapeutic alternatives to fight CL.

OBJECTIVES

In order to accomplish this goal, dihydroorotate dehydrogenase (DHODH), a key enzyme from the de novo pyrimidine biosynthetic pathway, has been exploited.

MATERIALS AND METHODS

In contrast to previous studies that target DHODH active site (orotate-BS), this work focus on a putative druggable pockets, identified with FTmap server (https://ftmap.bu.edu/). Aiming at identifying compounds that bind outside orotate-BS, the following screening strategy was designed: Fragments that shift DHODH Tm (melting temperature) in Thermofluor assays (DTm ≥ 1.0 ºC), but not in ThermoFMN were considered as promising compounds, since FMN binding site is close to orotate-BS.

DISCUSSION AND RESULTS

Thermal stabilization in ThermoFMN assays is highly correlated to competitive inhibitors, which bind to orotate-BS, whereas Thermofluor assay accounts for interactions to any hydrophobic surface that is exposed upon protein denaturation. Accordingly, compounds that are inactive in ThermoFMN, but active in Thermofluor might behave as non-competitive LmDHODH. Accordingly, a series 76 compounds had its effect over DHODH thermal stability investigated at a single concentration (0.5 mM). This strategy led to the identification of 15 hits.

CONCLUSION

Currently, the concentration-response behavior of these compounds is being investigated by Microscale Thermophoresis to exclude false-positives and proceed with kinetic assays that support the non-competitive mechanism of action for those compounds.

Keywords: Dihydroorotate dehydrogenase, Fragment screening, Leishmania major

Supported by: FAPESP and CNPq

08917 - Poster Session

CD.12 - An N-capping asparagine-lysine-proline (NKP) motif contributes to a hybrid flexible/stable multifunctional peptide scaffold

Marlon Henrique e Silva Cardoso ^1,2, Lai Yue Chan³, Elizabete de Souza Cândido^1,2, Marcelo D.T. Torres⁴, Karen G.N. Oshiro^1,5, Ítala C. Silva⁶, S. Gonçalves⁶, Danieli Fernanda Buccini¹, Timothy K. Lu⁷, Nuno C. Santos⁶, Cesar de la Fuente-Nunez⁴, David J. Craik³, Octávio L. Franco^1,2,5

¹S-inova Biotech, Programa de PG em Biotecnologia, Universidade Católica Dom Bosco (Campo Grande, MS, Brazil), ²Centro de Análises Proteômicas e Bioquímicas, PG em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, (Brasília, DF, Brazil), ³Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland (Brisbane, Australia), ⁴Machine Biology Group, Dep of Psychiatry and Microbiology, Institute for Biomedical Informatics, Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, and Department of Bioengineering, University of Pennsylvania (Philadelphia, PA, USA), ⁵PG em Patologia Molecular, Faculdade de Medicina, Universidade de Brasília (Distrito Federal, Brazil), ⁶Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa (Lisbon, Portugal), ⁷Synthetic Biology Group, MIT Synthetic Biology Center, The Center for Microbiome Informatics and Therapeutics; Research Lab of Electronics, Dep of Biological Engineering, and Dep of Electrical Engineering and Computer Science, MIT (Cambridge, MA, USA)

Structural diversity is intrinsically related to biological activities in peptide drug candidates. Therefore, numerous peptide scaffolds have been reported to date and, in some cases, correlated with a given biological activity. Here, we describe an unusual N-capping asparagine-lysine-proline (NKP) motif that confers a hybrid flexible/stable multifunctional scaffold to a computationally designed peptide (PaDBS1R7). PaDBS1R7 has a shorter α-helix segment than two other computationally designed peptides of similar sequence but with key residue substitutions. Nevertheless, although the NKP motif acts as an α-helix breaker in PaDBS1R7, the N-terminus asparagine presents exclusive N-capping effects, resulting in a highly amphipathic and stable α-helix from Pro7-Ile17. The solution nuclear magnetic resonance structures, along with temperature coefficient spectra and computational peptide mutants reinforced the role of the NKP motif for a coil/N-cap/α-helix scaffold. Biological studies revealed that all PaDBS1 peptides presented antibacterial activities, without interfering with bacterial surfaces at their minimal inhibitory concentration. However, only PaDBS1R7 displayed anticancer properties, completely eradicated Pseudomonas aeruginosa biofilms, decreased bacterial counts from 100-1,000 times in an abscess mouse model and reduced LPS-induced macrophages stress, without modulating the expression of TNF-α and IL-1β. This multifunctionality was also investigated in terms of peptide/mimetic vesicle interactions, revealing that in all conditions tested PaDBS1R7 preserved its lower α-helical content, but with higher ability to disrupt bacterial-like and cancer cells-like vesicles. Based on these results, we are confident that this study extends our understanding of an N-capping NKP motif to developing structurally hybrid peptide drug candidates with multiple biological activities.

Keywords: N-capping, peptide scaffold, multifunctionality

Supported by: FAP-DF, CNPq, FUNDECT and CAPES

08807 - Poster Session

CD.13 - Production of metallic nanoparticles-polymeric hydrogels hybrid systems for tooth whitening applications

Vinicius Ikezu Saito ¹

¹CCNH - Human and Natural Sciences Center, Federal University of ABC (SP, Brazil), ²Fundamental Chemistry, Universitty of São Paulo (SP, Brazil), ³Biotechnology, University of Sorocaba (SP, Brazil)

INTRODUCTION

The search for white teeth in aesthetic character by the world population pushed scientists to search for new technologies targeting at better whitening-process performances, allied to minimizing possible side effects such as teeth sensitivity and enamel wear. In this sense, new technologies have been developed associating different types of systems such as combining metallic nanoparticles and micellar hydrogels.

OBJECTIVES

This study aimed at the preparation and physico-chemical characterization of hybrid systems composed of metallic oxide nanoparticles and their further dispersion in thermosensitive hydrogels for tooth whitening purposes.

MATERIALS AND METHODS

Titanium nanoparticles (titanium oxide II, IV and isopropoxide, 5 mM) were prepared and dispersed into poloxamer 407-P407/10 or 90 kDa hydropxypropyl methylcellulose-HPMC (20/0.5 % m/v).

DISCUSSION AND RESULTS

Sol-gel transition temperatures (Tsol-gel) were determined by rheological analysis, with values from ~18.8 to 24.9 °C, according to titanium isopropoxide structures incorporation into 10 kDa HPMC-P407 hydrogels. Elastic (G’) and viscous (G”) modulii relationships were from 30 to 10-times for all systems. Ti-isopropoxide incorporation showed an influence on hydrogels structural organization considering the presence of P407 isolated or in association with 10 or 90 KDa HPMC. Scanning Electron Microscopy revealed an amorphous character with rough and porous surfaces for all formulations. FTIR analysis showed asymmetric and symmetric CH2 stretching (2922 and 2860 cm-1, respectively) and C=O stretching vibrations (1100 cm-1) suggesting an interaction between Ti nanoparticles and P407.

CONCLUSION

The results revealed that all Ti nanoparticles were incorporated into PL-HPMC hydrogels and their differential influence on systems structural organization according to HPMC molecular weight, maintaining the hydrogels bioadhesive properties looking forward tooth whitening applications.

Keywords: Hydrogels, Metallic nanoparticles, Tooth whitening

08302 - Poster Session

CD.14 - A new arylsulfanyl-benzo-2,1,3- thiadiazoles derivative has anti-amnesic effect on behavioral in mice

Karline da Costa Rodrigues ¹, Karline da Costa Rodrigues¹

¹Centro de Ciências Químicas, Farmacêuticas e de alimentos, Universidade Federal de Pelotas (Rio Grande do Sul, Brasil), ²Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas (rio grande do sul, brasil), ³Catálise Orgânica e Biocatálise, Universidade Federal de Grande Dourados (MS, brasil)

INTRODUCTION

Dementia is one the major complications involved memory loss, characterized by cognitive decline and it is commonly associated with behavioral disturbance in Alzheimer`s disease. Available treatments are based on symptom reduction, but they not stop the progression of cognitive disorder. This way, the search for new treatments is of great value. Faced with this, our research group showed recently the ability to inhibit in vitro acetylcholinesterase (AChE) activity in the cerebral cortices of mice by an arylsulfanyl-benzo-2,1,3-thiadiazoles derivative, 5-((4-methoxyphenyl)thio)benzo[c][1,2,5]thiadiazole (MTDZ).

OBJECTIVES

In this context, the aim of the present study was to investigate the binding affinity of MTDZ with AChE and effect anti-amnesic of compound in a scopolamine (SCO)-induced model in mice.

MATERIALS AND METHODS

The binding affinity of MTDZ with AChE was investigated by molecular docking analyses. For experimental model, male Swiss mice were treated with MTDZ (10mg/kg, intragastrically (i.g.)) or canola oil (10ml/kg, i.g.) and induced, thirty minutes after, with injection of SCO (0.4 mg/kg, intraperitoneally (i.p.)) or saline (0.9%, 5 ml/kg, i.p.). On days 6 and 7, mice were submitted to object recognition and location, and on day 8 were submitted to Y-maze task.

DISCUSSION AND RESULTS

We verified that MTDZ interacts with residues of the AChE active site, similar to donepezil. SCO caused amnesia in mice by reducing the exploratory preference for new object and for new location of object, as well as, by reducing the spontaneous alternation behavior. MTDZ treatment attenuated the behavioral changes caused by SCO, with actions similar to donepezil.

CONCLUSION

In conclusion, MTDZ presented dual inhibition activity of AChE, presenting anticholinesterasic action and it ameliorated the SCO-induced behavioral changes in learning- and memory-impaired mice.

Keywords: dementia, amnesia, thiadiazoles

Supported by: CNPq, FAPERGS, CAPES, UFPel

08574 - Poster Session

CD.15 - Outcomes of Se-DMC therapy for inflammatory response, nociception and oxidative stress in a mono-arthritis model in mice

Caren Aline Ramson da Fonseca ¹, Paola dos Santos Soares¹, Angélica Schiavom dos Reis¹, Ketlyn Pereira da Motta¹, Briana Barros Lemos¹, William Borges Domingues², Eduardo Bierhals Blodorn², Daniela Rodrigues Araujo³, Angelita Manke Barcellos³, Gelson Perin³, Mauro Pereira Soares⁴, Vinicius Farias Campos², Cristiane Luchese¹, Ethel Antunes Wilhelm¹

¹Programa de Pós-Graduação em Bioquímica e Bioprospecção, Universidade Federal de Pelotas (Rio Grande do Sul, Brazil), ²Programa de Pós-Graduação em Biotecnologia, Universidade Federal de Pelotas (Rio Grande do Sul, Brazil), ³Programa de Pós-Graduação em Química, Universidade Federal de Pelotas (Rio Grande do Sul, Brazil), ⁴Laboratório Regional de Diagnóstico, Faculdade de Veterinária, Universidade Federal de Pelotas (Rio Grande do Sul, Brazil)

INTRODUCTION

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation of the synovial membrane, causing damage and destruction of the cartilage and bone tissue that form the joints. It is estimated that 1% of the world population is affected by RA. Pain is the most prevalent and debilitating symptom reported by RA patients and it is associated with inflammation of the peripheral joints. There is currently no cure for RA and its treatment aims to reduce pain and inflammation and improve the patients' quality of life. Despite major advances, not all patients achieve disease remission, and there is still an unmet need for new therapeutic approaches.

OBJECTIVES

Thus, the objective of the present study was to investigate the therapeutic potential Se-[(2,2-dimethyl-1,3-dioxolan-4-yl) methyl] 4-chlorobenzoselenolate (Se-DMC) on mono-arthritis model induced by complete Freund’s adjuvant (CFA).

MATERIALS AND METHODS

The effect of Se-DMC and Meloxicam (5 mg/kg, oral route), both administered to animals daily for 14 days, on mechanical sensitivity (Von Frey test), inflammation (expression of tumor necrosis factor-alpha (TNF-α) and nuclear factor-κB (NF-κB) and paw histology) and oxidative stress (reactive species (RS) levels, non-protein thiol (NPSH) content and superoxide dismutase (SOD) activity) was evaluated in male Swiss mice exposed to intraplantar injection CFA (0.1 ml, 10 mg/ml).

DISCUSSION AND RESULTS

Se-DMC reduced the paw withdrawal threshold and mRNA relative expression levels of TNF-α and NF-κB induced by CFA. Paw histopathological results revealed the antiedematogenic potential of the compound, evidenced by the smaller amount of dilated lymphatic vessels when compared with the CFA group. In addition, Se-DMC reduced the RS levels and restored the SOD activity in paw of CFA mice. No alteration in the NPSH content was observed after treatments.

CONCLUSION

Our results demonstrated that Se-DMC reduced inflammatory and nociceptive responses and exerts antioxidant actions, representing a new therapeutic approach for the treatment of the RA.

Keywords: arthritis, hyperalgesia, inflammation

Supported by: UFPel, CNPq, CAPES, FAPERGS and L’ORÉAL-UNESCO-ABC for Women in Science

08938 - Poster Session

CD.16 - Potential Biomarkers with Quercetin and Lanthanides

Diane Correia de Araújo Lima ¹, Janaína dos Anjos², Severino Alves-Jr², Thereza Soares^2,3

¹Departamento de química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto - USP (, Brazil), ²Departamento de química fundamental, Universidade Federal de Pernambuco (, Brazil), ³Departamento de Física, Universidade de São Paulo (, Brazil)

INTRODUCTION

Flavonoids are ubiquitous plant compounds with great capacity as metal chelators and scavengers of free radicals. Some flavonoids such as myricetin have been shown to express antimicrobial activity by curbing the development of biofilms without killing bacteria. However, the molecular basis of the antimicrobial activity of this class of compounds is poorly known. The development of molecular probes to study the antimicrobial mechanism of flavonoids can provide useful insights into the action of these compounds.

OBJECTIVES

In this study, we have investigated the potential of quercetin and lanthanides ions ( Eu ³⁺ , Nd ³⁺ ) to coordinate to produce luminescent complexes. The flavonoid quercetin is a more affordable chemical variant of myricetin, and for this reason, has been chosen as a preliminary model.

MATERIALS AND METHODS

The synthesis of quercetin was obtained via hydrolysis of rutin in the presence of HCl. Subsequently, we have deprotonated the hydroxyl groups using NaOH, which was subsequently complexed with the Eu ³⁺ and Nd ³⁺ ions.

DISCUSSION AND RESULTS

The preliminary characterization of these complexes showed, via fluorescence spectrometry, luminescence intensification capacity of these complexes upon variations of the chemical environment.

CONCLUSION

This observation points to the potential use of these complexes as biological markers that can respond to changes in the chemical environment in the cell, such as seen in the penetration in the cell membrane.

Keywords: Lanthanides, Markers, Quercetin

Supported by: FACEPE, CNPq and CAPES.

08297 - Poster Session

CD.17 - Aging aggravates acute oxaliplatin-induced peripheral neuropathy in mice through the increase of the oxidative stress and modulation of Na+, K+ - ATPase: Beneficial effects of 7-chloro-4-(phenylselanyl) quinoline

Julia da Silva Chaves ¹, Angélica Schiavon dos Reis², Carolina Cristóvão Martins², Henrique Peres da Mota³, Manoela do Sacramento³, Diego da Silva Alves³, Cristiane Luchese², Ethel Antunes Wilhelm²

¹Curso de Farmácia (CCQFA), Universidade Federal de Pelotas (Rio Grande do Sul, Brasil), ²Programa de Pós-graduação em Bioquímica e Bioprospecção (CCQFA), Universidade Federal de Pelotas (Rio Grande do Sul, Brasil), ³Programa de Pós-graduação em Química (CCQFA), Universidade Federal de Pelotas (Rio Grande do Sul, Brasil)

INTRODUCTION

Cancer is considered a major public health problem and its incidence increases with age. Chemotherapy remains one of the primary treatment approaches for patients with cancer. Despite the advancement in cancer treatment, several chemotherapeutic drugs, as oxaliplatin (OXA), promote peripheral neuropathy. Almost 90% of patients develop acute neuropathic pain immediately after OXA treatment. 7-Chloro-4-(phenylselanyl) quinoline (4-PSQ) is an organoselenium compound with potential antioxidant, anti-inflammatory, and neuroprotective actions.

OBJECTIVES

Thus, this study aimed to investigate the impact of aging on acute OXA-induced peripheral neuropathy in Swiss mice and the effect of 4-PSQ as a pharmacological strategy.

MATERIALS AND METHODS

Mice were divided into six groups: young; young+OXA; young+OXA+4-PSQ; old; old+OXA; old+OXA+4-PSQ. Male aged (20 months) and young (2 months) mice were intraperitoneally treated with OXA (10 mg kg ^-1 ) or vehicle on days 0 and 2 of the experimental protocol. On day 2, half an hour after OXA administration, mice received 4-PSQ (1 mg kg ^-1 ) or vehicle, by the intragastric route. The acetone drop test and the Von Frey test were performed 0.5 h after the 4-PSQ-treatment to access the OXA-induced thermal and mechanical sensitivities. Posteriorly, animals were euthanized and the sciatic nerve, spinal cord, and cerebral structures were collected to further assays. The reactive species (RS) and nitrate and nitrite (NOx) levels and their influence on Na ⁺ , K ⁺ -ATPase activity were also analyzed.

DISCUSSION AND RESULTS

It was observed that 4-PSQ reversed hypersensitivity induced by OXA and aging. 4-PSQ reduced the RS and NOx levels increased by OXA exposure in both ages. Na ⁺ , K ⁺ -ATPase activity was inhibited by aging and OXA. However, 4-PSQ reversed the inhibition of Na ⁺ , K ⁺ -ATPase activity OXA-induced, but not caused by aging.

CONCLUSION

4-PSQ might be a good prototype for the development of a more effective drug for the treatment of OXA-induced acute peripheral neuropathy.

Keywords: aging, Na+, K+ - ATPase, oxaliplatin

Supported by: FAPERGS, CNPq, CAPES, L’ORÉAL-UNESCO-ABC for Women in Science

08612 - Poster Session

CD.18 - Literature review of the therapeutic utility of Campomanesia ssp species focusing on the study of Campomanesia adamantium

Isabela Borges da Mota Silveira ^1,2,1, Ana Tereza Gomes Guerreiro^3,2,1, Renata Trentin Perdomo^1,2,3

¹Bioquimica e Biotecnologia, Universidade Federal de Mato Grosso do Sul (BR|MS, Brasil), ²Pesquisa clínica, Fundação Oswaldo Cruz (Rio de Janeiro, Brasil), ³Bioquimica e Biotecnologia, Universidade Federal de Mato Grosso do Sul (BR|MS, Brasil)

INTRODUCTION

The Campomanesia adamantinum, popularly known as guavira, guabiroba or gabiroba-do-mato, is recognized as the symbol fruit of the state of Mato Grosso do Sul because it is a native plant with wide distribution of popular use as food and treatment, and its activities are attributed mainly by its phenolic components, flavonoids, chalcones, carotenoids and vitamins

OBJECTIVES

The present work aims to perform a bibliographic survey in databases about biological activities related to species of the genus Campomanesia.

MATERIALS AND METHODS

The searches were made in the MEDLINE (PubMed) platform, with no lower period limit and no language restriction. The descriptors used in the data collection were Campomanesia; Myrtaceae; pharmacology. The search strategy used was Campomanesia and Myrtaceae and pharmacology. Forty-five articles were found, and 33 articles related to the theme were selected.

DISCUSSION AND RESULTS

The species C. xanthocarpa and C. adamantium were the most studied, with 17 and 7 studies. Other species cited were C. guazumifolia, C. reitziana, C. velutina, C. pubescens and C. phaea. Regarding the part of the plant used, 20 (60.6%) used leaves, 11 (33.3%) used fruits, used roots and used seeds. The species C. xanthocarpa and C. adamantium showed antioxidant, antilipemic and anti-inflammatory effects. C. xanthocarpa also showed trypanocidal, antiglycemic, antiplatelet and hepatoprotective activity and C. adamantium showed immunomodulatory and antidepressant activity. The other species showed anti-inflammatory, antioxidant and antinociceptive activities. And the leaf extracts of Campomanesia species presented pharmacological potential to be studied.

CONCLUSION

Thus, it is concluded that the Campomanesia species have pharmacological potential to be studied.

Keywords: Campomanesia, Campomanesia adamantium, pharmacology

08389 - Poster Session

CD.19 - Acute toxicity assays of nanoencapsulated and free Colchicine in Drosophila melanogaster: Proposal for the repositioning of drugs for the treatment of Covid-19

Franciéle Romero Machado Balok ¹, Stífani Machado Araujo¹, Vandreza Cardoso Bortolotto¹, Mustafa Munir Mustafa Dahleh¹, Eliana Jardim Fernandes¹, Franciane Cabral Pinheiro¹, Ana Cláudia Funguetto Ribeiro², Sandra Elisa Haas², Gustavo Petri Guerra¹, Marina Prigol¹, Silvana Peterini Boeira¹

¹Lab de Avaliações farmacológicas e toxicológicas aplicadas às moléculasbioativas, Universidade Federal do Pampa (, Brazil), ²Laboratório de Farmacologia, Universidade Federal do Pampa (, Brazil)

INTRODUCTION

Much research has been conducted to develop possible treatments for Covid-19 and the repositioning of existing drugs is an opportune option. Colchicine is a promising drug to minimize the inflammatory conditions so characteristic of this disease, its administration through nanoencapsulated formulations may be able to optimize therapy and reduce its adverse effects.

OBJECTIVES

Therefore, the aim of our work is to evaluate the acute toxicity of nanoencapsulated (NCOL) and free (COL) colchicine in an alternative animal model such as Drosophila melanogaster.

MATERIALS AND METHODS

The flies were divided into 5 groups of different concentrations: Control, 0.001 mg/mL, 0.0025 mg/mL, 0.005 mg/mL and 0.010 mg/mL of NCOL and COL exposed for 48 hours. Afterwards, the survival rate, locomotor performance (open field test and negative geotaxis) and biochemical analyses (reactive species (RS) and thiobarbituric acid reactive substances (TBARS)) were analyzed.

DISCUSSION AND RESULTS

We obtained as results that both the NCOL and COL formulations did not affect the fly survival rate compared to the control group. Locomotor performance in the open field did not differ statistically for NCOL, but concentrations of 0.0025 and 0.010 mg/mL (COL) had reduced crossings compared to 0.001 mg/mL (COL). In the negative geotaxis test, there were no significant differences. COL showed an increase in RS levels at concentrations of 0.0025 and 0.005 mg/mL and TBARS levels were elevated in the 0.0025 mg/mL and 0.005 mg/mL groups. In NCOL, no statistical differences were found in RS, however TBARS were elevated by 0.001 mg/mL in relation to the control.

CONCLUSION

It is concluded that acute exposure to COL induced oxidative damage, however the tested concentrations of NCOL proved to be safe and with low toxicity, so further analysis is needed to target it for therapeutic purposes against Covid-19.

Keywords: Nanoencapsulation, Repositioning, Toxicity

Supported by: CAPES

08326 - Poster Session

CD.20 - In silico studies on the interaction between bioactive ligands and DPP-IV: insights on potential candidates for the treatment of type 2 diabetes mellitus

Michelle Cristiane Melo Reis Martins ¹

¹CCNH, Universidade Federal do ABC (São Paulo, Brasil), ²EACH, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Type II diabetes mellitus is a metabolic disorder that involves a failure of insulin secretion by the pancreas and/or of action of insulin in target tissues. In 2019, the number of people with diabetes in the world reached the mark of 463 million and it is estimated that in 2045 we will have 700 million cases. It is known that a metabolic pathway involved in the control of diabetes is the inhibition of dipeptidyl peptidase-IV (DPP-IV), which regulates the degradation of GLP-1 (glucagon-like peptide-1) and GIP (gastric inhibitory peptide), which are responsible for maintaining the insulin levels and reducing the blood glucose levels.

OBJECTIVES

In this study, we employed molecular modeling strategies in a set of inhibitor compounds to understand the main molecular characteristics related to the biological activity of these ligands at the DPP-IV enzyme.

MATERIALS AND METHODS

For this study, a set containing 45 compounds was selected for all analyses. CoMFA (Comparative Molecular Field Analysis) method was used to understand the relationships between molecular features (electrostatic and steric) of the compounds and their biological data. The possible interactions that the set of DPP-IV inhibitors can perform at the active site of the DPP-IV enzyme were analyzed using the molecular docking method, implemented in the GOLD program.

DISCUSSION AND RESULTS

The models obtained from CoMFA presented significant values of internal (r2 = 0.988 e q2 LOO = 0.768) and external (r2pred = 0.986 and r2 m= 0.970) validations. Important interactions between the studied compounds and active site´s residues, such as Glu205, Tyr666, Arg125, Ser630, Phe357 and Tyr662, were also identified.

CONCLUSION

Therefore, from the results obtained in this study, it is possible to propose molecular modifications in the structure of the DPP-IV inhibitors to improve their potential to treat type 2 diabetes.

Keywords: Diabetes, DPP-IV, molecular modeling

Supported by: FAPESP, CNPQ e CAPES

08250 - Poster Session

CD.21 - VIRTUAL SCREENING AND IN VITRO ASSAYS OF LIGANDS WITH ACTIVITY AGAINST DIPEPTIDYL PEPTIDASE-4 (DPP-4) ENZYME

Simone Queiroz Pantaleão ¹, Eric Allison Philot², Heberth de Paula³, Mirela Inês de Sairre¹, Angelica Nakagawa Lima⁴, Raquel Alves dos Santos⁵, Ana Ligia Scott², Kathia Maria Honório^1,6

¹Center for Sciences Natural and Human, Federal University of ABC (São Paulo, Brazil), ²Center for Mathematics, Computing and Cognition, Federal University of ABC (São Paulo, Brazil), ³Center for Exact, Natural and Health Sciences, Federal University of Espírito Santo (Espírito Santo, Brazil), ⁴Center for Engineering, Modeling and Applied Social Sciences, Federal University of ABC (São Paulo, Brazil), ⁵Laboratory of Genetics and Molecular Biology, University of Franca (São Paulo, Brazil), ⁶School of Arts, Sciences and Humanities, University of São Paulo (São Paulo, Brazil)

INTRODUCTION

Diabetes is a metabolic disorder with hyperglycemia and vascular complications due to non-production of insulin or inappropriate use of insulin by the body. One of the treatment strategies is the inhibition of dipeptidyl peptidase-4 (DPP-4).

OBJECTIVES

Conduct virtual screening studies to search new bioactive inhibitors for the DPP-4 enzyme.

MATERIALS AND METHODS

This study involves a virtual screening with the AutoDock Vina software, in the crystallographic structure of DPP-4 (PDB 4A5S), using the "Diversity Set II" database available in the ZINC database. The applied filters were affinity energy, ligand efficiency, positioning at the three DPP-4 binding sites, molecular interactions and pharmacokinetic properties (ADME-Tox). The conformations of the ligands obtained from AutoDock Vina were analyzed using a consensus obtained by using two other algorithms (AutoDock and GOLD). For the biological assay, the "DPPIV-Glo™ protease assay" was used. The cytotoxicity test was performed using the following cells: MCF-10A, MCF-7, HeLa, A549 and GM07492A.

DISCUSSION AND RESULTS

The ZINC 1572309 molecule established π-stacking, π-π, T-stacking and cation-π interactions with important residues, such as Tyr547, Tyr662 and Tyr666; salt bridge with Arg125 and His740 and hydrogen bonding with Ser630. The results of the ADME-Tox prediction for the ZINC 1572309 molecule were compared with the drug sitagliptin. The ZINC 1572309 molecule presented TPSA outside the classification, representing limitations regarding the parameters of lipophilicity and polarity. The assessment of oral bioavailability confirmed the need for molecular modifications. The toxicity prediction of the mutagenic effects (AMES test) showed that both molecules are not toxic. Although the ZINC 1572309 molecule presents an available and accessible synthesis, the results of the ADME-Tox showed the need for structural changes.

CONCLUSION

From in silico and in vitro studies, a molecule was promising as DPP-4 inhibitor that can structurally be optimized to achieve the suitable pharmacodynamic and pharmacokinetic profiles.

Keywords: Diabetes, DPP-4, Drug discovery

08438 - Poster Session

CD.22 - PROJECT - ANALYSIS OF THE EFFECT OF FLAVONOID RUTIN ON MODULATION OF GLIAL RESPONSE AND NEUROPROTECTION IN AMIOTROPHIC LATERAL SCLEROSIS STUDY MODELS

Lucas Matheus Gonçalves de Oliveira ¹, Rodrigo Barreto Carreira¹, Flavia Santos Sanches¹, Irlã Santos Lima¹, Ravena Pereira do Nascimento¹, Silva Lima Costa¹, Victor Diógenes Amaral da Silva¹

¹Laboratório de Neuroquímica e Biologia Celular, Universidade Federal da Bahia (Bahia, Brasil)

INTRODUCTION

Amyotrophic Lateral Sclerosis (ALS) is classified as a disease characterized by the degeneration of motor neurons and consequent loss of motor function. Among molecular and cellular alteration in ALS, mutation in SOD1 and gliosis seems to play a key role in the pathogenesis. It is shown that neuroinflammation associated with microglia and astrocytic reaction to SOD1 aggregates contribute to the motor neuron loss. In this bias, bioprospection of compounds with potential to modulate glial cells response is a strategy for drug discovery for ALS. Studies developed by the Laboratory of Neurochemistry and Cell Biology (LabNq) research group have already demonstrated that rutin has a regulatory effect on the activation of microglia and astrocytes, regulating tumor necrosis factors (TNF) and nitric oxide (NO) in cortical primary glial culture or microglia culture from rats. Therefore, our objective in this project is to evaluate if rutin have the potential to modulate glial cell function involved in ALS pathogenesis, preventing neuronal degeneration.

OBJECTIVES

The aim of this project is to evaluate the protective effect of rutin and modulation of glial response in ALS study model.

MATERIALS AND METHODS

In vitro studies will be performed in cortical primary culture of microglia from mutant SOD1G93A rats. To access the effects of rutin on glial activation and neuroinflammation, immunocytochemistry and westernblotting will be performed for GFAP, GS, IBA-1 and OX42, and qPCR will be performed for IL1b, IL6, TNF, IL10, TGFB1, ARG1. Additionally, in in vivo studies, neurodegeneration and glial activity will be evaluate using immunohistochemistry for GFAP, S100b, IBA-1, ChAT, CD206 and CD68 in spinal cord and nucleus hypoglassus of mutant SOD1G93A rats. This project is a collaborative work with Dr. Luis Barbeito from Pasteur Institut in Montevideo.

DISCUSSION AND RESULTS

We hope that the results obtained will contribute to the development of new therapeutic interventions for ALS.

CONCLUSION

Keywords: Flavonoids, Aminotrophic Lateral Sclerosis, neuroinflammation

Supported by: FAPESB and CAPES

08931 - Poster Session

CD.23 - Characterization of sub products from forced degradation of the antibiotic Cephalexin

LOUISE ELOA ARAUJO SOUZA ¹

¹BIOFISICA , UNIVERSIDADE FEDERAL DE SÃO PAULO (São Paulo, Brasil)

INTRODUCTION

Cephalexin is a first generation & β-lactam antibiotic, largely used in the treatment of diseases caused by gram-positive bacteria such as those appearing in urinary and respiratory infections, being classified as an essencial medicine by the World Health Organisation (WHO). Guidelines for degradation studies of medicines have been stablished by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) to verify how changes on environmental physicochemical conditions influence the quality of pharmaceutical compounds. Through degradation analyses, it is possible to verify the intrinsic stability of the molecule under stressing conditions, and simulate conditions of incorrect storage

OBJECTIVES

to identify byproducts from degradation of cephalexin under stressing conditions, and to investigate the antibiotic behavior of these byproducts against bacterial strains.

MATERIALS AND METHODS

Cephalexin solutions at 2 mg/ml were submitted to heating at 60 °C for 7 days or to UV irradiation to doses of 1.2 million lux-hours. The degraded solutions were studied by liquid chromatography, mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopies to identify sub products. In vitro assays using E. coli, E. cloacae and B. subtilis as bacteria models have been performed to assess how minimum inhibitory concentrations and growth rates of colonies are affected by degradation of the antibiotics.

DISCUSSION AND RESULTS

our data show that Cephalexin is highly sucesptible to degradation under oxidizing and photolytic stress (> 1.2 million lux hours, UV and visible range). Acid and basic conditions appear to promote degradation to a lower extent, whereas very little formation of subproducts is observed in samples submitted to thermal stress (up to 60°C for 7 seven days) and exposure to metal ions.

CONCLUSION

The main sub products generated from cephalexin degradation were cephalosoporanic acid, 7-ACA, and dimers. These species do not retain the antibiotic capacity of cephalexin.

Keywords: antibiotics, degradation, analytical chemistry

08342 - Poster Session

CD.24 - Expression and characterization of scFv antibodies against human tissue kallikrein 7

Rafael Cerioni Tognato¹, Luciano Puzer¹

¹Centro de Ciências Naturais e Humanas, Universidade Federal do ABC (São Paulo, Brasil)

INTRODUCTION

Human tissue kallikrein 7 (hKLK7) is a serine protease associated with skin desquamation. The alteration in its activity is associated with several skin pathologies such as Netherton Syndrome, where high activity of hKLK7 leads to a severe desquamation process. Through phage display, three antibodies were selected against hKLK7 and aimed to be expressed as a single-chain fragment (scFv) in E. coli.

OBJECTIVES

Although numerous challenges of expressing scFv antibodies in E. coli lineages are known, such as aggregation in inclusion bodies, misfolding and existence of rare codons in prokaryotic organisms, this research purposes a study of the three scFv selected sequences, leading to a comparative expression experiments of them in different E. coli strains (BL21(DE3), Origami 2 and Roseta (DE3)), aiming to optimize recombinant scFv expression with different expression conditions (culture volume, concentration of IPTG and temperature).

MATERIALS AND METHODS

Bacteria were transformed and verified by PCR. Bacteria were inoculated in 10 ml flasks filled with LB medium. When OD₆₀₀ reached 0.5, each flask was induced with different concentrations of IPTG (0.05 μM, 0.1 μM, 0.2 μM, 0.5 μM, 0.75 μM, 1 mM) at 30°C. After 2, 4 and 24 hours of induction, 1 ml of each culture was removed and harvested and pellets were loaded on 12% SDS-PAGE and analyzed by Western blotting. Positive results were then repeated in 100 ml of culture for antibody purification.

DISCUSSION AND RESULTS

There were none expressed scFv in BL21(DE3) and Origami 2 cultures. Low IPTG concentration (50 μM) at 30ºC in Rosetta 2(DE3) leaded to a great amount of recombinant protein. When repeated in 100 ml, most of the expressed scFv were in inclusion bodies, needing to be extracted with urea and refolded. Refolding leaded to a partial loss of scFv activity.

CONCLUSION

The partial loss of scFv activity due to refolding is yet a challenge to be studied in this research.

Keywords: kallikrein, phage display, scFv

Supported by: FAPESP (2019/11045-1)

08479 - Poster Session

CD.25 - Lidocaine- and synthetic curcuminoid-activated organogels towards dual drug-carrier formulations for topical applications

Aryane Alves Vigato ¹, Ian Pompermayer Machado², Mirela Inês de Sairre¹, Daniele Ribeiro de Araujo¹

¹Center for Natural and Human Sciences, Federal University of ABC (São Paulo, Brasil), ²Institute of Chemistry, University of São Paulo (São Paulo, Brasil)

INTRODUCTION

Organogels (ORGs) are semi-solid systems, widely investigated for pharmaceutical and cosmetic applications.

OBJECTIVES

This work proposes the development of a dual drug-release system, considering the application of synthetic curcuminoid derivatives (CURs) and lidocaine (LDC) to treat topical inflammatory lesions.

MATERIALS AND METHODS

ORGs were prepared associating an aqueous phase (AP) of Pluronic F127-(30% w/v) and LDC hydrochloride (25 mg/mL) with 2 mL isopropyl myristate (IPM) with lecithin (LEC) 2% as organic phase (OP) , adding the CURs (1 mg/mL). The OP:AP ratio was 1:4 v/v. Physico-chemical characterization was performed by rheology, differential scanning calorimetry, and scanning electron microscopy. In addition, in vitro permeation assays were also performed by using vertical Franz-type diffusion cells and artificial skin-model (Strat-M®).

DISCUSSION AND RESULTS

Results revealed that the LEC-LDC-CURs incorporation into ORGs increased viscosity and elastic/viscous moduli (G’/G”) ratio, favoring the ORGs structural organization. In vitro permeation assays through skin-model showed that LDC permeation flux was enhanced by the LEC/CURs (19,42 ± 1,47 μg·cm−2·h−1) when compared to formulations without LEC/CURs (14,64 ± 1,07 μg·cm−2·h−1). The LDC permeation coefficient was also observed to enhance, from 1,22 ± 0,02 (whitout additives) to 1,62 ± 0,03 cm·h−1 (with additives) after 48h. Moreover, latency time was also increased, i.e., LDC-release starts after longer times in formulations containing the LEC/CURs additives. Porcine ear skin epidermis (EP) were analyzed by Infrared Spectroscopy (FTIR), where the C-H symmetric and asymmetric stretching bands and the splitting of the CH2 scissors band were studied in detail. The lipids from matrix into the stratum corneum were observed to have an orthorhombic structure, which tends to disorganize after 4 and 24 hours after treatment with formulations.

CONCLUSION

Perspectives include Optical Coherence Tomography studies to concept the interaction between the ORGs and the stratum corneum lipidic matrix. In summary, results pointed ORGs as promising formulations for skin-delivery with potential pharmacological application in topical lesions.

Keywords: Organogels, drug-release, skin

Supported by: FAPESP (2019/14773-8, 2019/20303-4), CNPq, CAPES

08154 - Poster Session

CD.26 - Structure-based virtual screening identifies Novobiocin, Montelukast and Cinnarizine as TRPV1 modulators with anticonvulsant activity in-vivo.

Manuel A. Llanos¹, Nicolás Enrique², María Laura Sbaraglini¹, Federico Garofalo¹, Alan Talevi¹, Luciana Gavernet¹, Pedro Martín ²

¹Department of Biological Sciences, Faculty of Exact Sciences, UNLP, Laboratory of Bioactive Research and Development (Buenos Aires, Argentina), ²CONICET— UNLP, Instituto de Estudios Inmunológicos y Fisiopatológicos (Buenos Aires, Argentina)

INTRODUCTION

Transient Receptor Potential Vanilloid 1 (TRPV1) is a nonselective cation channel modulated by endogenous and exogenous ligands, pH, temperature, and voltage. In the last few years, it has been proposed as a promising target to develop novel anticonvulsant compounds. However, thermoregulatory effects associated with channel inhibition have hindered the way towards TRPV1 antagonists becoming marketed drugs.

OBJECTIVES

We conducted a structure-based virtual screening (VS) campaign to repurpose TRPV1 inhibitors among approved drugs, which are known to be safe and thermally-neutral.

MATERIALS AND METHODS

Initially, three homology models of the hTRPV1 were constructed and refined with Rosetta, representing biologically relevant states of the channel: unliganded, open agonist-bound, and closed antagonist-bound. Then, several docking conditions were evaluated to find the best docking model, in terms of pose and score accuracy, able to identify compounds interacting with the capsaicin binding site. Top scoring hits were evaluated in vitro using the patch-clamp technique and, in vivo on the maximal electroshock seizure (MES), the 6 Hz psychomotor (6 Hz) and pentylenetetrazole (PTZ) mice tests.

DISCUSSION AND RESULTS

Among top scoring hits from the VS campaign, Novobiocin, Montelukast, and Cinnarizine were selected for biological testing. The interaction between the selected compounds and the TRPV1 channel heterologously expressed in HEK293 cells was evaluated by their ability to reduce currents induced by 250 nM capsaicin using the patch-clamp technique. All tested compounds showed inhibitory effects on capsaicin-induced TRPV1 currents measured at -100 mV (% inhibition: 60±7 (n=10), 62±8 (n=8) and 40±6 (n=6) for 0,1 μM Novobiocin, 0,1 μM Montelukast and 1 μM Cinnarizine, respectively). Finally, their in vivo anticonvulsant profile was completed, showing promising anticonvulsant activity mainly against maximal electroshock seizure test.

CONCLUSION

Our results further support the modulation of TRPV1 channels as a promising strategy to develop novel antiepileptic drugs.

Keywords: docking, epilepsy, TRPV1

Supported by: PICT 2016-0165 (ANPCyT), Argentina

08767 - Poster Session

CD.27 - Development of nanoparticle-based DNA vaccine against SARS-Cov-2.

Lays Cordeiro Guimarães ¹, Pedro Augusto Carvalho Costa², Heloisa Athaydes Seabra Ferreira¹, Anderson kenedy santos¹, Pedro Henrique Dias Moura Prazeres², Walison Nunes da Silva², Virgínia Mendes Russo Vallejos ¹, Alexander Birbrair², Silvia Guatimosim¹, Frederic Jean Georges Frezard¹, Pedro Pires Goulart Guimaraes¹

¹Department of Physiology and Biophysics, Universidade Federal de Minas Gerais (Minas Gerais, Brasil), ²Department of General Pathology, Universidade Federal de Minas Gerais (Minas Gerais, Brasil)

INTRODUCTION

A safe and effective vaccine with long-term protection against SARS-CoV-2 and variants is an urgent global health priority. Nucleic acid vaccines have emerged as a promising alternative to conventional vaccine approaches. Here, we developed a platform of nanoparticle-based DNA to provide safe and effective delivery of DNA to directly induce antigen presenting cells (APCs) to express antigens implicated in immune recognition of SARS-CoV-2.

OBJECTIVES

Therefore, our objective is to develop a nanoparticle-based DNA vaccine for potently neutralizing responses against SARS-CoV-2.

MATERIALS AND METHODS

Lipid nanoparticles (LNPs) were formulated via rapid microfluidic mixing and characterized with regard to size, zeta potential, pKa and encapsulation efficiency. To assess the immunogenicity and protection against SARS-CoV-2, BALB/c mice were immunized intramuscularly with LNPs containing DNA coding for GFP (Control Group), spike protein (S), nucleocapsid protein (N), or spike protein plus protein nucleocapsid (S+N). Two vaccine schedules were carried out, one in a single dose, while the other received a second dose 3 weeks after initial immunization (prime+boost). To assess cell-mediated immune response, splenocytes from vaccinated mice were incubated in culture with SARS-CoV-2 proteins and evaluated by flow cytometry. To assess neutralization titer, plaque reduction neutralization test was performed.

DISCUSSION AND RESULTS

Single dose and prime+boost schedule were able to induce lung resident memory. In two doses schedule, an antigen-specific CD8+ T cells significantly responded to SARS-CoV-2 proteins (S and N) in animals immunized with S+N-LNPs. In addition, immunizations with S+N LNPs in two doses was able to neutralize significantly Wuhan and P1 variants infections.

CONCLUSION

We developed a LNP platform capable to provide effective delivery of DNA to APCs to express antigens implicated in immune recognition of SARS-CoV-2 and variants. Clinically, S+N-LNP, which induced robust systemic humoral and cell-mediated immune responses, hold promise as a vaccine candidate for preventing SARS-CoV-2 and variants infection.

Keywords: nanoparticle-based DNA, vaccine, COVID-19

Supported by: CNPq, PRPq-UFMG, CAPES and FAPEMIG

08878 - Poster Session

CD.28 - Nanostructured lipid carriers prepared with olive oil shows anti-inflammatory activity, as seen in zebrafish acute inflammatory model

Gabriela Geronimo¹, Gustavo Henrique Rodrigues da Silva¹, Ludmilla David de Moura¹, Fabíola Vieira de Carvalho¹, Talita Cesarim Mendonça¹, Juan Pablo García-López², Carmen Glória Feijoó², Eneida de Paula¹

¹Department Biochemistry and Tissue Biology, University of Campinas (SP, Brazil), ²Department of Biological Sciences, Andres Bello University (Santiago, Chile)

INTRODUCTION

Oleocanthal, a component of olive oil, has a poor bioavailability but anti-inflammatory properties comparable to those of ibuprofen. Nanostructured lipid carriers (NLC) are drug delivery systems composed of a blend of solid and liquid lipid stabilized by surfactants; they are able to encapsulate insoluble drugs, increasing their bioavailability. In here, olive oil was used as the liquid lipid of NLC stabilized with Pluronic F68 (NLC-OO-P) or Tween 80 (NLC-OO-T). The characterization of these particles revealed for NLC-OO-P: size= 236.4 ± 0.5 nm, polydispersity index (PDI)= 0.163 ± 0.03, zeta potential (ZP) = -31.5 ± 0.7mV and 6.08±0.70 x 10∧13 particles/mL. For NLC-OO-T: size = 225.7 ± 0.6 nm, PDI = 0.205 ± 0.02, ZP= -22; 26.5 ± 0.1mV and 4.90 ± 0.21 x 10∧13 particles/mL.

OBJECTIVES

The aim of this work is to verify the anti-inflammatory activity of these nanoformulations.

MATERIALS AND METHODS

Zebrafish larvae (5 days after fertilization, dpf) were used to determine drug absorption through the intestinal mucosa, with both formulations. In the two cases, the lethal dose was >1012 particles/mL. In a well stablished acute inflammation model, which involved caudal fin transection of the 5 dpf Tg (BACmpx:GFP)i114 larvae, the animals were incubated in suspensions with the different NLC formulations for 1 h prior to the caudal fin transection. Neutrophils recruitment to the affected area was analyzed at 3 hours post-damage.

DISCUSSION AND RESULTS

The results obtained for the control group showed an average of 15 neutrophils infiltrated in the damaged zone. The number of neutrophils decreased to 8 and 6, for NLC-OO-P and NLC-OOT, respectively. These results demonstrate that NLC prepared with olive oil have a significant anti-inflammatory effect. In addition, the different surfactant composition did not interfere with the anti-inflammatory activity of the oil.

CONCLUSION

Thus, the use of olive oil as a functional excipient is an interesting approach to achieve anti-inflammatory activity in pharmaceutical formulations.

Keywords: drug delivery, nanostructured lipid carrier, anti-inflammatory effect

Supported by: FAPESP, CNPq/Brazil

08561 - Poster Session

CD.29 - Evaluation of structural changes of benzocaine-loaded, optimized nanostructured lipid carriers using SANS and Raman imaging approaches

Hery Mitsutake ¹, Alessandro Delfino de Souza², Fabiano Yokaichiya³, Margareth Kazuyo Kobayashi Dias Franco⁴, Daniel Clemens³, Ben Kent³, Marcia Cristina Breitkreitz¹, Lígia Nunes de Morais Ribeiro⁵, Eneida de Paula², Heloisa Nunes Bordallo⁶

¹Department of Analytical Chemistry, Institute of Chemistry, University of Campinas (São Paulo, Brazil), ²Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (São Paulo, Brazil), ³Helmholtz-Zentrum Berlin, Helmholtz-Zentrum Berlin (, Germany), ⁴Nuclear and Energy Research Institute, Nuclear and Energy Research Institute (São Paulo, Brazil), ⁵Institute of Biotechnology, Federal University of Uberlândia (Minas Gerais, Brazil), ⁶Niels Bohr Institute, University of Copenhagen (, Denmark)

INTRODUCTION

Local anesthetics are substances that reversibly block the nerve-impulse conduction, alleviating pain without loss of consciousness. Benzocaine, a poorly soluble local anesthetic, is an ester of para-aminobenzoic acid. Several strategies of formulations can be used to improve bioavailability and decrease adverse effects of benzocaine. In this study nanostructured lipid carriers (NLC) were employed. These lipid-based drug delivery carriers have a lipid core composed of a blend of solid and liquid lipids, and a shelf of non-ionic surfactant.

OBJECTIVES

The main aim of this work was to optimize benzocaine-loaded NLC and to investigate structural changes in these nanoparticles, under different temperatures.

MATERIALS AND METHODS

The ratio of excipients (cetyl palmitate, Capmul® PG-8 NF and Pluronic®F68) and benzocaine in the NLC was optimized using a 2 ³ factorial design with respect to the following parameters: particle size, polydispersity index (PDI) and zeta potentials.

DISCUSSION AND RESULTS

The interactions between the factors were found relevant to determine particle size and PDI. Using desirability function, the best formulation conditions were found. Structural changes in optimized NLC were observed with Small-Angle Neutron Scattering (SANS) and Raman imaging, in samples at 27, 37 and 40º C. SANS pointed the formation of lamellar structures inside the NLC, which interlamellar distances increase at higher temperature. Raman imaging showed that the incorporation of P68 and benzocaine in-between the lipids increased at higher temperatures, explaining the changes in Q values (SANS).

CONCLUSION

This work shows how different scattering techniques can provide complementary information and be used together to characterize and understand the physical, chemical, and structural changes on the organization of pharmaceutical carriers in drug delivery system.

Keywords: factorial design, local anesthetic, pharmaceutical formulations

Supported by: FAPESP, CNPq, CAPES, Niels Bohr Fond and Carlsberg Foundation

08901 - Poster Session

CD.30 - Docetaxel-loaded lipid nanoparticles prevent the growth of murine melanoma

Ludmilla David de Moura ¹, Lígia Ribeiro², Fabíola Carvalho¹, Gustavo Silva¹, Hery Mitsutake¹, Fabiano Yokaichiya³, Sergio Brunetto⁴, Eneida de Paula¹

¹Department of Biochemistry and Tissue Biology, University of Campinas (SP, Brazil), ² Institute of Biotechnology, Federal University of Uberlândia (MG, Brazil), ³ Materialien und Energie, Helmholtz-Zentrum (, Berlin), ⁴Nuclear Medicine Division, University of Campinas (, Brazil)

INTRODUCTION

Melanoma is the most aggressive type of skin carcinoma. Considering its low response and resistance to chemotherapy, nanotechnology brings new therapeutic options for melanoma treatment. Nanostructured lipid carriers (NLC) are ideal drug-delivery systems to encapsulate hydrophobic drugs such as docetaxel (DTX).

OBJECTIVES

The aim of this work was to develop an innovative treatment for melanoma. For this, we developed an optimized NLC formulation containing myristyl myristate (65% w/v), Mygliol 812® (25% w/v), Pluronic F68® (3% w/v) and docetaxel (1% w/v).

MATERIALS AND METHODS

The formulations were developed by factorial design (2^4) using the ultrasonication method. Subsequently, the formulations were characterized by: Dynamic light scattering(DLS), Nanoparticle Tracking Analysis(NTA), Cryo-electron microscopy(Cryo-EM), Small-Angle Neutron Scattering(SANS), Raman imaging, and encapsulation efficiency (%EE) by HPLC. The orthotopic model of murine melanoma was used for preclinical trials. Tumor regression was assessed by tumor volume quantification and microPET/CT image analysis. Possible adverse effects were considered.

DISCUSSION AND RESULTS

The physicochemical characterization revealed particles with average sizes of 214.0 ± 10.9 nm, low polydispersity (0.09 ± 0.01), negative zeta potential (-24.2 ± 0.30 mV) and spherical morphology (Cryo-EM) at pH 6.12 ± 0.24, with high DTX-encapsulation (%EE= 97.29 ± 2.64) and stable at long-term storage (12 months). SANS revealed that incorporation of DTX turned the surface of NLC rough and induced the formation of hydrophobic clusters. Miscibility tests by Raman imaging indicated that DTX favored the interaction between excipients (solid/liquid lipids and the surfactant). In vivo tests, indicated that the intratumoral treatment with NLC-DTX was effective, inhibiting the tumor volume rate by 97.9%, in comparison to the commercial formulation (94.9%).

CONCLUSION

The general therapeutic evaluation of NLC-DTX was better than that of the commercial formulation, with lower adverse effects and 100% survival rate. These results endorse such NLCDTX as a promising formulation for the treatment of melanoma.

Keywords: docetaxel, melanoma, nanostructured lipid carriers

Supported by: FAPESP (# 2019-17784-0), CAPES (L.D.M. fellowship)

08344 - Poster Session

CD.31 - Using a fragment based strategy to identify compounds that intectact with Mycobacterium tuberculosis’ MurE ligase.

Nicolas de Oliveira Rossini ¹, Catharina dos Santos Silva¹, Márcio Vinicius Bertacine Dias¹

¹Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

New therapy strategies against tuberculosis are necessary and the enzymes from the cellular wall biosynthesis pathway are interesting targets since they are involved in many pathogenic resistance mechanisms. Accordingly, this work targets MurE, an essential enzyme for peptidoglycan biosynthesis in M. tuberculosis (Mtb).

OBJECTIVES

In this work, we intended to accomplish the identification of fragment-like compounds that interact with Mtb MurE using a fragment library composed of about 600 compounds through biophysical techniques . We also intend to observe ligand interaction using protein crystallography and molecular docking strategies.

MATERIALS AND METHODS

MurE has been cloned in pET28a, expressed in LB media and purified using affinity and gel filtration chromatographies. Thermal shift assays (DSF) were performed and docking experiments simulations were effected using GOLD and Autodock vina softwares.

DISCUSSION AND RESULTS

At the moment, MtbMurE was successfully expressed, purified in a soluble fraction, and used in two Thermal Shift screening strategies, leading to promising fragments. These molecules still need further confirmation through other biophysical techniques such as RMN, ITC, protein co-crystallization. Molecular docking was performed with the most promising compounds in order to predict the interaction with MtbMurE. Since MtbMurE was not prone to crystallization without its substrate, UAG, we were not able yet to confirm the binding mode of the fragments and we intend to use M. thermoresistibile MurE as a surrogate model.

CONCLUSION

The integration of several biophysical techniques will provide key information about the identification of new lead molecules against this target and help in the discovery of new Mtb inhibitors.

Keywords: Mycobacterium tuberculosis, Fragment-Based Drug Discovery, ATP-dependent ligase

Supported by: FAPESP

08605 - Poster Session

CD.32 - MOLECULAR INTERACTION OF NEOCUPROINE AND ITS COPPER(II) COMPLEX WITH MODEL MEMBRANES

Carlos Yáñez Fernández ¹, Luis Felipe Mendes², Bruno Favarin², Analu Rocha³, Alzir Azevedo Batista³, Antonio J. Costa-Filho², Gianella Facchin¹

¹Montevideo, Facultad de Química, Universidad de la República (, Uruguay), ²Ribeirão Preto, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (, Brasil), ³Sao Carlos, Departamento de Química, Universidade Federal de São Carlos (, Brasil)

INTRODUCTION

Metal complexes are a tool in the search of new drugs to improve current cancer treatments and have been used since the discovery of Cisplatin. The aim of this research is the development of new copper complexes with antitumor activity. To reach that goal a series of ternary complexes were synthetized and characterized

OBJECTIVES

The ternary complexes [Cu(phenolic acid)(diimine)] were synthetized using 1,10-phenanthroline (phen) and neocuproine (neo) because they are intercalating agents to DNA. As a second ligands we selected phenylacetate acid and phenylpropionic acid because they stabilize ternary copper complexes by the presence of the phenolic ring.

MATERIALS AND METHODS

Affinity to DNA was studied by UV, determining the Kb for each complex and the binding to DNA was characterized by circular dichroism and variation of viscosity. Although many studies have been focused on the interaction of diimines and their metal complexes with DNA as a biomolecular target molecule for cytotoxic action, little attention has been paid to the interaction with lipidic membranes. The interaction of the complexes with membranes models has been studied by differential scanning calorimetry (DSC) and EPR.

DISCUSSION AND RESULTS

The DNA Kb values ranged from 3x10e3 to 7x10e3, characterization studies have shown that these complexes could act as partial intercalator or binding by the grooves. For complex [CuCl(fenilpropanoato)(neo)] in the thermogram obtained by DSC, a new peak can be observed, a behavior that had not been previously recorded, and in the EPR it was observed that the copper remains completely rigid within the membrane at room temperature, both studies suggests a strong interaction of the complex with the DPPC/DPPS membrane.

CONCLUSION

The complexes are potent cytotoxic agents on cancer cell lines (IC50 in the low micromolar range). Their cytotoxicity was modulated by the diimine: phen complexes < neo complexes.

Keywords: copper, cytotoxity , membranes

08918 - Poster Session

CD.33 - Applications of computational biochemistry techniques in the discovery of drug candidates: in silico, in vitro, and in vivo studies

Paulo Sérgio Alves Bueno ¹, Flávio Augusto Vicente Seixas²

¹Department of Biochemistry, Universidade Estadual de Maringá (Maringá, PR, Brazil), ²Department of Technology, Universidade Estadual de Maringá (Umuarama, PR, Brazil)

This work used computer simulations to prospect new antifungal, hypoglycemic and antiviral drug candidates and investigate their interaction with the target proteins. Firstly, the target protein structures were modeled and subjected to energy minimization procedures. Then, molecular docking protocols were validated for large-scale virtual screening (VS) and molecular dynamics simulations (MD) were performed to calculate the ΔG_binding by MM-PBSA method, aiming to verify the system behavior involved in the stabilization of protein-ligand complexes. The theoretical results were later confirmed by in vitro and in vivo assays. The antifungals were discovered using two targets: homoserine dehydrogenase and chorismate synthase. Four drug candidates were selected by VS and then evaluated against strains of P. brasiliensis, where MICs/MFCs ranged between 8-128 μg.mLμ1 and low cytotoxicity in mammalian cells were found. One of those molecules (CaCS02) demonstrated a strong synergistic effect in combination with amphotericin-B. The in vitro studies using recombinant P. brasiliensis CS showed an IC50 of 29 μM for CaCS02. Regarding the discovery of hypoglycemic compounds, the crystallographic structure of human pancreatic α-amylase was used as target. The hydrolyzed (HTN) and Condensed (CTN) tannins were selected by VS from a library containing different tannins and also the reference ligands acarbose (ACA) and amylose substrate. Theoretical ΔG_binding calculations were performed and indicated that HTN, ACA, and CTN have a high affinity for α-amylase, where their relative affinities are in agreement with the experimental evaluation. Finally, an antiviral against BmNPV, a virus that infects the silkworm targeting the viral cathepsin was discovered. ΔG_binding calculations indicated that Bm5 compound has a high affinity for the enzyme. The in vivo assays using B. mori caterpillars infected with BmNPV and treated with Bm5 increased silkworm survival by 84%. The techniques used in this work selected candidate compounds for applications in human health and agricultural technology.

Keywords: computer simulations, virtual screening, drug discovery

Supported by: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação Araucária (FA)

08419 - Poster Session

CD.34 - Development of new copper coordination compounds as possible drugs to fight cancer

Gianella Facchin ¹, Carlos Yañez¹, Emiliano Garcia¹, Bruno Rosa¹, Celisnolia Leite², Adriana Napoleone¹, Natalia Alvarez¹, Priscilla Freddi³, Luis Felipe Mendes³, Javier A. Ellena⁴, Antonio J. Costa-Filho³, Alzir A. Batista²

¹Estrella Campos, Facultad de Química, Universidad de la República ( Montevideo, Uruguay), ²Departamento de Química, Universidade Federal de São Carlos (São Paulo, Brazil), ³Departamento de Física, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (São Paulo, Brazil), ⁴Instituto de Física de São Carlos, Instituto de Física de São Carlos, Universidade de São Paulo (São Paulo, Brazil)

INTRODUCTION

Metal containing coordination compounds emerged as anticancer drugs since the discovery of Cisplatin´s antitumor activity, whose use in clinic started in 1978 bringing cure to some malignant tumors. Consequently, an intense research aimed at expanding the pharmacological arsenal to fight cancer with coordination compounds started. Our goal is to develop new copper complexes with cytotoxic activity. To that end several series of compounds were synthesized and characterized.

OBJECTIVES

We started using [Cu-dipeptide] complexes, which are very stable systems where a second ligand can be introduced. DNA intercalating agent such as a planar diimines were used as ligands, making [Cu(L-dipeptide)(diimine)] complexes series. The diimine used were: phenanthroline (phen), 5-NO2-phenanthroline (5-NO2-phen), neocuproine (neo), 3,4,7,8-tetramethyl-phenanthroline, (tmp) and bathophenanthroline (batho). Their cytotoxicity was evaluated on different cancer cell lines.

MATERIALS AND METHODS

Affinity to DNA was assessed as Kb (determined by UV) and the binding to the DNA was characterized by circular dichroism and paramagnetic electronic resonance (EPR). Recently, the cellular membrane has emerged a possible target or site of accumulation for metallic complexes. Therefore, we explored the interaction of the complexes with membrane models by calorimetry and Spin-label EPR. We found that the compounds were highly potent cytotoxic agents on cancer cell lines (IC50 in the low micromolar range).

DISCUSSION AND RESULTS

Their activity was modulated mainly by the diimine attached, following the general tendency: 5-NO2-phen complexes ≤ phen complexes ∠tmp complexes ∠ neo complexes≅ batho complexes. Their DNA Kb value ranged from 1x103 to 1x106, structurally the binding occurs through partial and/or groove binding. No direct relation towards the observed cytotoxicity could be determined. The first insights into the interaction of [Cu(L-dipeptide)(phen)] with membranes suggest that it depends both on the dipeptide and phenanthroline ligands, being more marked in DPPG than in DPPC membranes.

CONCLUSION

The complexes present an adequate activity to be tested in vivo as antitumor drugs.

Keywords: copper, cytotoxicity, membranes

Supported by: CSIC, PEDECIBA and ANII (Uruguay), FAPESP and CAPES (Brazil)

08581 - Poster Session

CD.35 - APE1 ENDONUCLEASE INHIBITION STUDY BY MODELING AND MOLECULAR DYNAMICS METHODS

Nathalia Dumas de Paula ¹, Pedro Geraldo Pascutti¹, Claudia de Alencar Santos Janot Pacheco¹

¹Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein responsible for removing lesions in the DNA molecule and to perform about 95% of the incision activity of the Base Excision Repair pathway. The overexpression of APE1 and other DNA repair proteins in cancer cells is associated with greater resistance to treatment using drugs that induce damage to genetic material. This makes these proteins target of studies for pharmacological inhibition and the increase of the efficiency of chemotherapy treatments.

OBJECTIVES

In this work, modeling and molecular dynamics methods were used to characterize the APE1 protein and to optimize ligands proposed in the literature as inhibitors of DNA repair activity.

MATERIALS AND METHODS

For this, the mapping of the protein surface and characterization of the cavities, description of pharmacophore properties, simulations of Molecular Dynamics (MD), clustering of conformations, analysis of the hydrogen bonds of the complexes, and the free energy of binding were performed.

DISCUSSION AND RESULTS

For the 13 cavities identified on the surface of APE1, the active site showed the higher druggability score, thus it was chosen as the site for performing molecular docking of the mc43 and mc47 ligands. Subsequently MD simulations of complexes were performed. The inhibitor mc47 showed a higher prevalence of hydrogen bonds over time and its pharmacophore properties were obtained to optimize the interaction with the enzyme. Through simulations with mixed solvent (ethanol/water), decomposition of the free energy of binding by residues, and computational mapping by probes, the hotspot residues Asn 174, Asn 212, Tyr 171, His 309, Arg 156 and Asp 210, were selected.

CONCLUSION

Ligands modification suggestions were made based on the information obtained from this study.

Keywords: APE1, cancer therapy, molecular dynamics

Supported by: CAPES

08877 - Poster Session

CD.36 - Nanostructured Lipid Carriers loaded with docetaxel for the treatment of breast cancer

Fabíola Vieira de Carvalho¹, Lígia Nunes de Morais Ribeiro^1,2, Ludmilla David de Moura¹, Gustavo Henrique Rodrigues da Silva¹, Gabriela Geronimo¹, Talita Cesarim Mendonça¹, Márcia Cristina Breitkreitz¹, Eneida de Paula¹

¹Biochemistry and Tissue Biology, University of Campinas (São Paulo, Brazil), ²Biotechnology, Federal University of Uberlândia (MG, Brazil)

INTRODUCTION

Breast cancer is the cancer with the highest incidence in women worldwide. Docetaxel (DTX) is a taxoid antineoplastic agent which mechanism of action involves inhibition of microtubule formation and angiogenesis and induction of apoptosis, among others. Technological strategies can improve the effectiveness of chemotherapy by promoting sustained release or directing the drug to tumor cells, thus reducing its systemic toxicity. Nanostructured lipid carriers (NLC) are capable of encapsulating hydrophobic drugs in their blend-of-lipids matrix, which imperfections prevent early expulsion of the drug and increase the colloidal stability of the nanoparticles.

OBJECTIVES

The aim this work is to developed and characterize NLC encapsulated DTX, by factorial design and physicochemical analysis.

MATERIALS AND METHODS

In this work, a formulation of NLC consisting of beeswax (solid lipid), copaiba oil and Miglyol® (liquid lipids), Pluronic F-68 (surfactant) and containing 0.5%DTX was developed. The stability of the formulation (NLCDTX) and its control without DTX (NLCCTL) was evaluated for 3 months regarding size, polydispersity index (PDI), zeta potential (ZP), and particle concentration, besides pH and DTX encapsulation efficiency (%EE). The sustained release of docetaxel encapsulated in the NLC was compared to that of the reference drug using Franz-type cells.

DISCUSSION AND RESULTS

The optimized formulation had particles with relatively homogenous distribution (PDI< 0.200) of sizes (170-240nm) negative ZP (|25–35|mV), ca. 5.1013particles/mL, acidic pH(~5.5), and %EE of almost 100%. The kinetic experiments revealed that the encapsulated DTX was sustained released compared to the reference, the equilibrium being reached after 34 and 10h, respectively.

CONCLUSION

In the search for a less toxic and more effective treatment for breast cancer, the optimized formulation was able to promote sustained release (diminishing the serum levels/systemic toxicity) and its natural excipients (beeswax, copaiba oil) have intrinsic anticancer and analgesic properties, which are valuable in oncology. These pharmacological properties are now being tested over normal and breast cancer cell lines.

Keywords: Breast cancer, Docetaxel, Drug delivery

Supported by: CAPES, FAPESP

08510 - Poster Session

CD.37 - Drug repositioning in treatment of cryptococcosis

Emanuel Almeida Moreira de Oliveira ¹, Gabriella Freitas Ferreira^1,2, Karen Luise Lang^1,2

¹Multicentric Program in Biochemistry and Molecular Biology, Federal University of Juiz de Fora campus Governador Valadares (Minas Gerais, Brazil), ²Department of Pharmacy, Federal University of Juiz de Fora, Governador Valadares (Minas Gerais , Brazil)

INTRODUCTION

Cryptococcosis is a systemic mycosis caused by pathogenic yeasts of the Cryptococcus genus. The cryptococcal meningitis causes 180.000 deaths per year, being the second biggest cause of mortality among individuals with HIV/AIDS. Currently, there are few treatments available, which presents high toxicity and antifungal resistance. Drug repositioning is a strategy for identifying new uses for approved or investigational drugs that are outside the scope of the original medical indication. This strategy is promising in the development of antifungals drugs and can reduce costs, risks and research time.

OBJECTIVES

Evaluate the antifungal activity of 28 drugs with privileged chemical structures, outside its original indication, alone and in combination with fluconazole (FLZ) against C. neoformans (ATCC H99).

MATERIALS AND METHODS

Values of minimum inhibitory concentrations (MICs) were determined using the methods proposed by CLSI M-27-A3. The combinatory effects between drugs and FLZ was measured by checkerboard assay with the fractional inhibitory index (FIC) and by synergism scores in SynergyFinder web application through models Bliss, Loewe, HSA and ZIP.

DISCUSSION AND RESULTS

Albendazole, fenbendazole, flubendazole and mebendazole showed promising anticryptococcal activity with a MIC between 0.047μM and 3.125μM. None of the drugs evaluated had an ICF ≤ 0.5, therefore no synergism between the drugs and FLZ was observed. However, additive effect was observed between FLZ + finasteride (FIN) and FLZ + hydroxyzine (HID) with FIC > 0.5 and ≤ 1.0, respectively. The combination FLZ/HID has mean scores by model Bliss (12.05), HSA (11.35) and ZIP (12.14) and FLZ/FIN combination with average scores by the Bliss (1.06), HSA (4.81) and ZIP (2.29), both suggestive of synergistic or additive effects.

CONCLUSION

Benzimidazoles antiparasitic drugs are promising candidates for repositioning treatment of cryptococcosis, as well as the FLZ/FIN and FLZ/HID combinations. In the future, the phenotypic impacts of these combinations on C. neoformans will be evaluated.

Keywords: Drug repositioning, cryptococcosis, drug combination

Supported by: PROPP/UFJF

08840 - Poster Session

CD.38 - Peptide and antibody phage display: a platform for antibody engineering

Caio Cesar Nogueira Cambui ¹, Ricardo Jose Giordano¹

¹Bioquímica, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Antibodies are important biopharmaceuticals due to their specificity and high affinity for targets. Nevertheless, one of the challenges to generate antibodies with clinical potential is the immune system itself, which avoids targeting highly conserved domains in proteins such as binding sites. Combinatorial techniques (i.e., antibody phage display) may help mitigating these difficulties but it still rely on the immune system to generate the repertoire of complementarity-determining regions (CDR) in order to produce high diversity antibody libraries. Our goal is to by-pass this limitation by combining peptide and antibody phage display technologies.

OBJECTIVES

Design and optimize a platform using peptide, antibody and yeast display to graft bioactive peptides into the CDR of human globulins to facilitate the discovery of high affinity antibodies with clinical potential.

MATERIALS AND METHODS

We have engineered plasmid vectors to build peptide libraries within the CDR of a human IgG1 to select scFv antibody fragments with specificity toward selected targets. To validate our platform, we have engineered an anti-Tie1 antibody. To increase affinity toward its target, we are now combining peptide grafting with yeast display to optimize the remaining CDRs.

DISCUSSION AND RESULTS

We have engineered the variable region of human IgG1 gene to carry restriction sites flanking the heavy and light chain CDR3.Using oligonucleotides encoding the sequence of a Tie1 binding peptide, we have built an artificial antibody phage display library and selected scFv that bind specifically to Tie1. Currently, a new scFv human library is being used to optimize the remaining CDRs to increase affinity towards Tie1.

CONCLUSION

By combining combinatorial methodologies (phage and yeast display), one might overcome an important limitation in biopharmaceutical production, our own immune system. If successful, this work might generate a new anti-angiogenesis antibody and pave the way for a new platform for rational antibody discovery.

Keywords: Angiogenesis, Antibody Engineering, Phage Display

Supported by: FAPESP, CNPq

08872 - Poster Session

CD.39 - Characterization of nonionic cubosomes in thepresence of model proteins: a structural approach

Heidie Da Silva Torres ¹, Leandro Ramos de Souza Barbosa²

¹Dep. de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmaceuticas, USP (SP, Brazil), ²Dep de Física Geral, Instituto de Física da USP (SP, Brazil)

INTRODUCTION

One area of research that has gained much attention in recent years is nanomedicine, with particular attention to drug delivery systems. Among the various nanoparticles used for this purpose, we highlight the systems formed by lipids and polymers, such as liposomes and cubosomes.

OBJECTIVES

The main objective of this research project is to build nanostructured systems capable of acting as antimicrobial systems.

MATERIALS AND METHODS

These systems will be composed of cubosomes in the absence and presence of the enzyme and will be analyzed using several biophysical techniques such as: small angle X-ray scattering (SAXS) and dynamic light scattering (DLS), potential -ζ besides essays in vivo. This cubosomes will be obtein in a equipment, developed by our research group so that we could reproducibly obtain cubosomes. This equipment uses Arduino type electronics, made in a 3D printer, and our objective will be to characterize, in terms of size and polydispersion, the cubosomes formed using different injection speeds.

DISCUSSION AND RESULTS

The results obtained in this research project will be presented at national scientific events (such as the meetings of the Brazilian Biophysical Society) and published in international scientific journals with arbitration.

CONCLUSION

Due to the progress of the research, there are no conclusions about what was initially proposed.

Keywords: lysozyme, cubosomes, nanostructured systems

Supported by: FAPESP, CNPq and CAPES

08578 - Poster Session

CD.40 - Investigation of the trypanocidal activity of naphthoquinone-derived analogues as possible therapeutic agents for leishmaniasis.

Alberto Nogueira Neto ¹, Michelle Chain¹, Ana Bombaça², Rubem Barreto², Raphael Silva¹, Luiz De Melo¹

¹Genética, Instituto Federal de Educação, Ciência e Tecnologia (Rio de Janeiro, Brazil), ²Laboratório de Biologia Celular (IOC), Fundação Oswaldo Cruz (Rio de Janeiro, Brazil.)

INTRODUCTION

Leishmaniasis is caused by protozoa of the genus Leishmania spp. and it affects millions of people, with an increasing number of new cases each year, as well as being neglected by health agencies around the world. The drugs used in leishmaniasis are Glucantine and Pentostam, with Amphotericin B and Pentamidine as a secondary approach in some cases. Such procedures cause adverse effects, which lead to treatment withdrawal, and severe liver damage. Thus, the search for new therapeutic agents is essential to ensure a better quality of life and a higher cure rate. In this context, the project proposes the investigation of promising selective compounds: naphthoquinone analogues.

OBJECTIVES

Determine the IC50 of 2TIONQ for L. amazonensis with viability assay using the MTT method; Investigate the cellular stress caused in proliferative forms of L. amazonensis treated with 2TIO-NQ, quantifying changes in cellular oxygen consumption and production of reactive oxygen species; Investigate the cellular stress caused in proliferative forms of L. amazonensis treated with 2TIO-NQ, quantifying alterations in the mitochondrial membrane potential; Investigate the drug potencial against infection on animal cell lines.

MATERIALS AND METHODS

The current proposal was to deepen the biochemical investigation of the action of 2TIO-NQ compounds in Leishmania amazonensis, which suggest similar results in regard of ROS measurement, mitochondrial depolarization and cell cycle in vitro assays under treatment. Thus, positioning the 2TIO-NQ as a pharmacological perspective in combined treatments in Chagas disease and leishmaniasis.

DISCUSSION AND RESULTS

It is expected similar results as seen in the T. cruzi model, with higher detection of ROS and depolaryzation of mitochindrial membrane upon drug treatment. Cell cycle assay suggets to be arrested on low quantities of drug.

CONCLUSION

The work is still in progress, but show promising end towards the drug being used in further tests. We expect trying other methods to know more about its mecanisms and then proceed to in vivo tests.

Keywords: Leishmania, leishmaniasis, naphtoquinones

Supported by: FAPERJ; IFRJ

08854 - Poster Session

CD.41 - Interaction of chaperone proteins with biomimetic systems of membrane: a structural and spectroscopic approach

Mayra Cristina Gomes Lotierzo ¹, Leandro Ramos de Souza Barbosa²

¹Department of Biochemical and Pharmaceutical Technology, University of São Paulo (São Paulo, SP, Brazil), ²Department of General Physics, University of São Paulo, Physics Institute (São Paulo, SP, Brazil)

Chaperone proteins, also known as heat shock proteins (HSPs), are present in several cell locations, being responsible for maintaining the cell proteostasis. Studies of chaperone proteins have been growing in recent years, mainly due to the great potential to unveil cell protection mechanisms. However, there is still a lot to be analyzed regarding interaction and mechanisms of functioning of these proteins. In particular, it is known that chaperone proteins from HSP70 family can interact with plasma and mitochondrial membranes. In this sense, this research project has as character the study of the interaction of chaperone proteins (HSPA9 and BIP) with biomembranes, investigating how this interaction occurs, which domains interact, in addition to verifying how the protein is internalized in the membrane. To do this, it will be performed a structural characterization study of liposomes before and after the interaction with chaperone proteins. Techniques such as Dynamic Light Scattering will be used (DLS), Isothermal Titration Calorimetry (ITC), Cryo Electron Microscopy of Transmission (Crio-TEM) and Small Angle X-ray Scattering (SAXS), emphasizing for the new possibility of SAXS resolved in time, which will be made available in the Sirius laboratory soon. *Corresponding author E-mail:lbarbosa@if.usp.br

Keywords: chaperone, proteins, biophysics, membrane

Supported by: FAPESP, CNPq and CAPES

08406 -

CD.42 - Identification of novel class of VEGF inhibitors using virtual screening

Erika Piccirillo¹, Lilian C. Alecrim¹, Antonia Tavares do Amaral², Ricardo Jóse Giordano¹

¹Bioquímica, Instituto de Química, Universidade de São Paulo (Sao Paulo, Brasil), ²Química Fundamental, Instituto de Química, Universidade de São Paulo (Sao Paulo, Brasil)

INTRODUCTION

Inhibition of angiogenesis, the formation of new blood vessels from pre-existing ones, is a reality and an important therapeutic option for patients suffering from oncological and ocular diseases. Most angiogenesis inhibitors target the VEGF pathway, the main factor responsible for initiating and maintaining the neovascularization process. Although effective, there are challenges to anti-VEGF therapy, such as side effects and drug resistance. Our group has shown that small molecules targeting the VEGF receptors might be an important alternative for a novel class of VEGF inhibitors (Michaloski, et al., Sci Adv, 2016).

OBJECTIVES

Here, we show that small organic compound mimetic of this peptide identified by virtual screening (VS) inhibit angiogenesis and might be an important drug lead for the development of novel angiogenesis inhibitors.

MATERIALS AND METHODS

Crystal structure of VEGFR-1 complex with VEGF (PDB 1FLT) was used to dock a pre-filtered subset of the ZINC database (7.8x10 ⁶ molecules) with FRED (v. 3.3.03, OpenEye Scientific). Docking poses with good fit were further minimizaded and visually inspected using VIDA (v. 4.1.1, OpenEye Scientific). Most promising compounds were purchased, and their anti-VEGF activity evaluated using in vitro and in vivo angiogenesis assays (VEGF induced-cell proliferation/migration; aorta-ring and oxygen-induced retinopathy — OIR).

DISCUSSION AND RESULTS

The VS campaign suggested 29 possible VEGF inhibitors. Three of them were purchased and tested as anti-VEGF inhibitors. One compound (V2) selectively inhibits the VEGF-induced proliferation of endothelial cells over epithelial ones. V2 also inhibits two tumor cells responsive to VEGF (CAKI-1 and U87). We also analyzed V2 using two angiogenesis models: aorta-ring and OIR. V2 inhibits the neovascularization in both models. Finally, V2 showed low acute toxicity in mice.

CONCLUSION

Altogether, these results suggested that V2 might be an important drug lead for the development of novel anti-VEGF inhibitors.

Keywords: Angiogenesis, Drug Discovery, Virtual Screening

Supported by: FAPESP (2018/24678-0), CNPq, CAPES, CEPID-REDOXOMA

08720 -

CD.43 - Optimization of Lipid Nanoparticle Formulations for DNA delivery in cardiomyocytes

Sérgio Scalzo¹, Anderson K. Santos¹, Heloísa Athaydes¹, Pedro A. Costa², Pedro H. D. M. Prazeres², Lays C. Guimaraes¹, Mário de Morais e Silva¹, Marco T. R. Alves¹, Celso T. R. Viana¹, Alice Pereira Rodrigues¹, Frederic Frezard¹, Silvia Guatimosim¹, Pedro Guimaraes¹

¹Department of Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais (Minas Gerais, Brasil), ²Department of General Pathology, Institute of Biological Sciences, Universidade Federal de Minas Gerais (Minas Gerais, Brasil)

INTRODUCTION

Gene therapy is a promising approach to be applied in cardiac regeneration after myocardial infarction and gene correction for inherited cardiomyopathies. One of the greatest challenges faced is the gene delivery vector. Here, we developed a library of lipid nanoparticles (LNPs) containing plasmid DNA (pDNA) for enhanced transfection efficiency in cardiomyocytes.

OBJECTIVES

Identify an optimized LNP formulation to enhance gene expression in cardiomyocytes in vitro and in vivo.

MATERIALS AND METHODS

pDNA encoding GFP was encapsulated in LNPs consisting of varying lipid molar ratios via rapid microfluidic mixing. LNPs were characterized using DLS, zeta potential and cryo-TEM. pKa of LNPs was assessed via fluorescent reagent 6-(ptoluidinyl) naphthalene-2-sulfonic acid. pDNA concentration was determined using a NanoDrop and encapsulation efficiency was obtained through Qubit dsDNA HS Assay Kit. Primary culture of cardiomyocytes was treated with LNPs at pDNA dosages of 0.00625-0.8μg in vitro. C57/BL6 mice were treated via tail vein injection with LNPs at a dose of 10 μg total of pDNA to determine in vivo GFP expression. Cell viability was assessed by resazurin reduction method. GFP fluorescence was carried out in confocal and Cytation 5.

DISCUSSION AND RESULTS

90-120nm LNPs containing pDNA were formulated via microfluidic mixing. Encapsulation efficiency varied from 71% to 94%. Lead LNP induced higher than 60% transfection efficiency after 24h and 80% after 48h, which remained until day 8. LNPs formulated with higher DOPE and lower cholesterol molar ratio exhibited enhanced GFP expression in cardiomyocytes. In addition, LNP with pKa closer to endosomal pH has shown higher GFP expression and cellular uptake, without cell toxicity. In vivo, lead LNP was able to induce significant gene expression in heart tissue 7 days after intravenous injection, with negligible toxicity.

CONCLUSION

Collectively, the use of our LNPs holds promise to improve pDNA delivery and transfection efficiency for the treatment of cardiovascular diseases.

Keywords: Lipid nanoparticle, DNA delivery, Heart

Supported by: CNPq, CAPES and FAPEMIG

08770 -

CD.44 - PEGylated liposomal formulation of amphotericin B for improved treatment of cutaneous lesihmaniasis

Guilherme Santos Ramos¹, Virgínia Mendes Carregal ¹, Gabriel Silva Marques Borges¹, Ricardo Toshio Fujiwara¹, Lucas Antônio Miranda Ferreira¹, Pedro Pires Goulart Guimaraes¹, Frédéric Jean Georges Frézard¹

¹Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais (MG, Brasil), ²Departamento de Parasitologia, Universidade Federal de Minas Gerais (MG, Brasil), ³Faculdade de Farmácia, Universidade Federal de Minas Gerais (MG, Brasil)

INTRODUCTION

Cutaneous leishmaniasis (CL) is caused by the parasite Leishmania and found in the tropics, subtropics, and southern Europe, and can generate social and psychological stigmas due to the permanent scars generated by injuries. The few drugs available exhibit toxicities, decreased patient compliance and increased risk of resistance. Liposomal amphotericin B (AmB) or Ambisome® is the most effective and safer therapeutic agent currently available against visceral leishmaniasis (VL), but its clinical efficacy is limited in CL and HIV/VL co-infections.

OBJECTIVES

The aim of this work was to develop a formulation of AmB in long-circulating PEGylated liposomes and compare it to Ambisome® regarding physicochemical characteristics and efficacy in a murine model of CL.

MATERIALS AND METHODS

Formulations of AmB in conventional and PEGylated liposomes were developed and characterized for particle size and morphology by DLS and cryomicroscopy, drug encapsulation efficiency by HPLC and AmB aggregation state by circular dichroism (CD). Those were evaluated and compared to Ambisome® in Leishmania amazonensis-infected BALB/c mice for their effects on the lesion size growth and parasite load as determined by qPCR.

DISCUSSION AND RESULTS

The conventional and PEGylated formulations showed hydrodynamic diameter of 125 and 158 nm, with polydispersity indexes of 0.1 and 0.25, respectively. The drug encapsulation efficiencies were higher than 95%. CD spectra demonstrated the interaction of AmB with the membrane mainly under the monomeric form, and differed from that of Ambisome®. Following IV administration in the murine model of CL, only PEGylated liposomal formulation significantly reduced the lesion size growth and the parasite load in comparison to the control group receiving empty liposomes.

CONCLUSION

This work demonstrates for the first time that PEGylation improves the efficacy of a liposomal formulation of AmB in a murine model of CL, presumably through prolonged blood persistence and passive targeting to the skin lesion.

Keywords: Amphotericin B, Liposome, Leishmaniasis

Supported by: CNPq, CAPES

08202 -

CD.45 - Dihydroorotate dehydrogenase: A potential target against schistosomiasis.

Maria Cristina Nonato ¹

¹Ciências Biomoleculares, 1Laboratório de Cristalografia de Proteínas, FCFRP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil (SP, Brazil)

INTRODUCTION

Trematode worms of the genus Schistosoma are the causing agents of schistosomiasis, a parasitic disease responsible for a considerable economic and healthy burden worldwide

OBJECTIVES

It is our interest to exploit the enzyme dihydroorotate dehydrogenase (DHODH) as a target for the development of new therapies against schistosomiasis.

MATERIALS AND METHODS

Combination of structural, biophysical and biochemical studies of dihydroorotate dehydrogenase from Schistosoma mansoni (SmDHODH) demonstrated that SmDHODH is a member of class 2 DHODHs and catalyzes the oxidation of dihydroorotate into orotate using quinone as an electron acceptor by employing a ping-pong mechanism of catalysis.

DISCUSSION AND RESULTS

SmDHODH crystal structure showed the presence of all structural features reported for class 2 DHODH enzymes and reveal the presence of an additional protuberant domain that folds as a flexible loop and is absent in the other known class 2 DHODHs. Molecular dynamics simulations showed that the ligand-free forms of SmDHODH and HsDHODH undergo different rearrangements in solution. Well-known class 2 DHODH inhibitors were tested against SmDHODH and HsDHODH and the results showed that the variable nature of the quinone-binding tunnel between human and parasite enzymes, as well as the differences in structural plasticity involving rearrangements of the N-terminal α-helical domain can be exploited for the design of SmDHODH selective inhibitors. I

CONCLUSION

n our experiments, the antimalarial drug and class 2 DHODH inhibitor atovaquone was found to selectively inhibit SmDHODH in the nanomolar range. Similarly, different series of antimalarial compounds, and their fragments designed based on the selective inhibition of plasmodium spp DHODH were tested and allowed the identification of potent and selective inhibitors. The best inhibitors were tested against against all schistosome stages (cercariae, schistosomule and adult) and revealed potent antischistosomal activity, in particular to the juvenile stage.

Keywords: Dihydroorotate dehydrogenase, schistosomiasis, drug gtarget

08941 -

CD.46 - Drug delivery with lipid nanoparticles and cosmetic applications

Freddy Ignacio Rojas Rodríguez ¹

¹Pharmaceutical and Biochemical Technology , University of São Paulo (São Paulo, Brasil)

INTRODUCTION: Lipid nanoparticles (LNP) are inspired by their natural homologous, the "virus", microscopic agents with a great capacity to deliver RNA or proteins in cells of different species. Knowing the current state of the drugs that use LNPs in the market by reviewing specialized literature provides valuable information for future developments. Objectives: Analyze scientific articles in the area of administration of drugs made with lipid nanoparticles to offer an overview of the forms of application in the human body and the therapeutic objective for which it is currently being used, showing the versatility of technology as a releasing agent of drugs. Materials and methods: Scientific articles from the Scopus and CAS Content Collection databases were investigated and filtered between the years 2000 to 2021.Results: LNPs supplies DNA, RNA, proteins and other agents for pharmacological are used in the treatment of various health problems. The ability of the LNPs to deliver various therapeutic agents is the key to the localized and spatialized delivery of drugs that remain protected and stabilized within the limits of the LNPs to release their contents at a desired time. The versatility of LNPs in their design and assembly has provided simple liposomes that have evolved into next-generation LNPs, such as solid lipid nanoparticles, nanostructured lipid carriers, cubosomes, and cationic lipid-nucleic acid complexes. Databases such as Scopus an CAS Content Collection show the growing interest in publications as evidence that different branches of science seek a better understanding of new generations of LNPs to be applied. Currently about 29 LNPs drugs are registered as approved in the world market. These drugs are distributed in 45% dedicated to cancer treatments, 2.1% in delivery of amino acids for vaccines, 1.3% to antifungal treatment, 1% in analgesics and later they are distributed in hormonal therapies, immunosuppressants and respiratory therapies, etc. Recent FDA approvals for cancer therapies and the well-known Covid-19 vaccine are a product of the versatility of encapsulation and open the opportunity to better understand the importance of technology, exhibiting more complex architectures and stabilities. LNPs have shown versatility in the application routes, where the parental path stands out with 43 %, oral 20 %, topical/transdermal 17 %, nasal 5 %, rectal 4 %, ocular 3 %, pulmonary 3 %, vaginal 2 % and 3 % other routes. In the cosmetic area, applications of LNPs are reported in applications of: sunscreen 22%, antiaging & antiwrinkle 17%, skin moisturizer 12%, shampoos 11%, etc. Finally, the prospects for finding better and more efficient LNPs preparation/application systems are growing the more these modern drug delivery systems are studied. Conclusions: It highlights that the applications of drugs that use lipid nanoparticle technology are applied parentally and are directed in genetic treatments against cancer. Vaccine, hormonal and cosmetic applications are also mentioned which shows the potential of the technique. The interest of scientists and companies is growing in the study and development of new and better lipid nanoparticle formulations.

Keywords: Lipid nanoparticles, Drug delivery, LNPs application routes

08190 -

CD.47 - Hybrid Nanoemulsions for Migraine Treatment

Lígia Nunes de Morais Ribeiro ¹, Verônica Muniz Couto¹, Simone Ramos de Castro¹, Gustavo Henrique Rodrigues da Silva¹, Márcia Cristina Breitkreitz², Carolina S. Martinez³, Daniela E. Igartúa³, Maria Jimena Prieto³, Eneida de Paula¹

¹Bioquímica e Biologia Tecidual, Universidade Estadual de Campinas (SP, Brasil), ²Química Analítica, Universidade Estadual de Campinas (SP, Brasil), ³Department of Science and Technology, National University of Quilmes (, Argentina)

INTRODUCTION

Migraine is a painful, disabling and often chronic disease, with no descriptions of cure so far. Intranasal sumatriptan succinate (SMT) is the gold standard drug in the treatment of migraine symptoms. Conversely, its efficacy by intranasal route is restrained by poor mucoadhesion.

OBJECTIVES

The preparation of hybrid nanoemulsions (NE) with copaiba oil nanoparticles and xanthan, pectin or alginate biopolymers, for the intranasal sustained release of SMT (2%; w/v).

MATERIALS AND METHODS

The blend of SMT and biopolymer matrices were encapsulated in copaiba oil nanoparticles, providing different hybrid NE. Structural characterization was performed by FTIR-ATR, DSC and TEM and the physicochemical stability was followed up a year (25 °C). The efficiency of encapsulation and SMT in vitro release profile were carried out. The nanotoxicity of the alginate-based NE (ALG-NE/SMT) was evaluated in vivo by measuring the mortality, spontaneous movement, morphological changes and cardiotoxicity of Zebrafish larvae.

DISCUSSION AND RESULTS

The hybrid NE exhibited physicochemical stability over a year of storage at 25 °C, with size in the range of 120 nm, low polydispersity (~ 0.2), zeta potential around -25 mV, and concentration of ca. 2.1014 part/mL. The hybrid systems showed excellent SMT encapsulation (41-69 %), prolonging the in vitro SMT release for ca. 24 h, at 37 o C. The structural analyses confirmed the morphology of the particles and details on the molecular arrangements of the hybrid systems. The most suitable formulation (ALG-NE/SMT) was tested in Zebrafish larvae, showing no nanotoxicity in any of the evaluated parameters, after 48 h of treatment.

CONCLUSION

These assays point out the ALG-NE/SMT system as a promising candidate for the treatment of migraine.

Keywords: hybrid nanoemulsion, vegetable oils, biopolymers

08195 -

DA. - Biophysical Studies of Extracellular Hemocyanin of Macrobrachium acanthurus (HcMac) in the Presence of Cationic CTAC

Lierge Ramos ¹, Karyna Satomi Izumi², Claudemir Oliveira Souza^1,2, Giovana Bertini², Patrícia Soares Santiago²

¹Instituto de Química de Araraquara, Universidade Estadual Paulista Júlio de Mesquita Filho (São Paulo, Brasil), ²Laboratório de Biomoléculas, Campus de Registro, UNESP, SP, Brasil (São Paulo, Brasil)

INTRODUCTION

Hemocyanins are considered giant proteins because they have a molecular weight in the order of 0.45 to 3.0 MDa. Hemocyanins function as an oxygen carrier in the immune system of crustaceans.

OBJECTIVES

The aim of this work was to evaluate the effect of the Cetyltrimethylammonium chloride (CTAC) surfactant on the oligomeric structure of the HcMac.

MATERIALS AND METHODS

The HcMac was purified by ultra-centrifugation (250,000 x g) and by size exclusion chromatography (SEC) in Superdex 200 PG column at pH 7.0. The samples were prepared according to the function of surfactants concentration (0 – 10 mmol.L ^-1 ) at pH 7.0 (25ºC), using acetate-phosphate-borate buffer 30 mmol.L ^-1 for 3 h and centrifuged before the spectroscopic measurements.

DISCUSSION AND RESULTS

The absorption spectrum of HcMac showed band with maximum at 280 nm, typical of proteins containing aromatic amino acids, and band at 340 nm due to the active site copper(II)–peroxide complex. The predominance of anionic groups on the protein surface above the isoelectric point (pI) promotes an effective interaction with CTAC. Until 0.1 mmol.L ^-1 , the surfactant promotes the exposition of di-copper center that increasing the absorbance at 340 nm. The surfactant-protein aggregation is intense (0.5 mmol.L ^-1 ), generating an absorbance low at 280 nm, 340 nm and LSI after centrifuged, suggesting a significant electrostatic contribution to the protein-surfactant interaction. Fluorescence data showed that in concentration above 0.4 mmol.L ^-1 , the CTAC promoted the shifts and increase intensity fluorescence changing the maximum fluorescence emission wavelength from 327 to 336 nm. These results can be ascribed to conformational changes in the local environment of the copper active sites.

CONCLUSION

The CTAC, at pH 7.0, induced the unfolding process followed by aggregation with HcMac and pH values seems to be a determinant of the type interaction between protein-surfactant.

Keywords: hemocyanin, surfactant, freshwater shrimp

08456 - Poster Session

DA.01 - The cold shock domain of the glycine-rich protein AtGRP2 shows sequence selectivity and folds upon binding its cognate DNA

Karina de Carvalho Pougy ¹, Clara Malizia Leal Ferreira da Motta¹, Luis Mauricio Trambaioli da Rocha e Lima², Gilberto Sachetto Martins³, Anderson de Sá Pinheiro¹

¹Departamento de Bioquímica, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ²Departamento de Fármacos e Medicamento, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ³Departamento de Genética, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

AtGRP2 (Arabidopsis thaliana glycine rich protein 2) is a glycine-rich, RNA-binding protein that plays key roles in abiotic stress response and flowering time regulation in Arabidopsis thaliana. AtGRP2 consists of an N-terminal cold shock domain (CSD) and two C-terminal retroviral CCHC-type zinc knuckles interspersed with glycine-rich regions. Despite the wealth of information on AtGRP2 function, the molecular mechanisms underlying its biological role is largely unknown.

OBJECTIVES

Thus, the objective of this work was to investigate the structure and systematically evaluate the binding selectivity of AtGRP2-CSD.

MATERIALS AND METHODS

A combination of biophysical technics was employed, including NMR. A set of 25 different 7-mer DNA oligonucleotide sequences was used for the binding specificity analysis through fluorescence spectroscopy.

DISCUSSION AND RESULTS

1D ¹ H NMR spectra exhibited features of a folded protein, including a large amide chemical shift dispersion. In contrast, 2D [ ¹ H, ¹⁵ N] HSQC spectra revealed the presence of an unfolded state in equilibrium with the native, folded protein with exchange kinetics in the slow time regime. Using multidimensional, triple resonance NMR, we unambiguously assigned 95% of the folded state and 68% of the unfolded state resonances. On the fluorescence experiments, it was observed that increasing concentrations of oligonucleotides led to suppression of fluorescence spectra, suggesting that Trp37 is part of the binding site. DNA binding occurred with affinities ranging from low nM to μM. Remarkably, AtGRP2-CSD bound to cognate DNA with a Hill “n” coefficient of 0.4, suggesting negative cooperativity arising from folding upon binding events. NMR titration results identified aromatic residues at strands β2 and β3, as well as loop β3β4 as critical for DNA binding. In addition, we show that full-length AtGRP2, but not AtGRP2-CSD, phase separates into a gel-like condensate.

CONCLUSION

Therefore, these results suggest that AtGRP2 selectively interacts with DNA oligonucleotides with, preferably, T-rich sequences. The phase-separation of full-length AtGRP2 has potential functional implications.

Keywords: AtGRP2, Structure, NMR

Supported by: FAPERJ, CNPq and CAPES

08502 - Poster Session

DA.02 - The gill (Na+, K+)-ATPase from the swamp ghost crab Ucides cordatus is regulated by protein kinases and the FXYD2 peptide

Francisco de Assis Leone ¹, John C. McNamara¹, Malson N. Lucena³, Carlos F.L. Fontes⁴, Rogerio O. Faleiros⁵, Daniela P. Garçon⁶

¹Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, (SP, Brasil), ²Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, (SP, Brasil), ³Instituto de Biociências, Universidade Federal do Mato Grosso do Sul (MS, Brasil), ⁴Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro (RJ, Brasil), ⁵Ciências Agrárias e Biológicas, Universidade Federal do Espírito Santo (ES, Brasil), ⁶Quimica, Universidade Federal do Triângulo Mineiro (MG, Brasil)

INTRODUCTION

Ucides cordatus is a mangrove crab known as the swamp ghost crab. While its osmoregulatory ability is well characterized, little is known of the biochemical processes underlying gill ion transport.

OBJECTIVES

.After 10-days acclimation to different salinities we investigated the crab’s osmotic and ionic regulatory ability, kinetic behavior of the posterior gill (Na ⁺ , K ⁺ )-ATPase and its regulation by protein kinases A and C, the calcium/calmodulin-dependent kinase and the FXYD2 peptide.

MATERIALS AND METHODS

For enzyme activity estimation see Leone et al. (2020) Comp. Biochem. Physiol. 250B: 110507.

DISCUSSION AND RESULTS

Salinity acclimation had no effect on hemolymph osmolality, which was very strongly hyper-/hypo-regulated; [Cl ^- ] was well hyper/hypo-regulated although [Na ⁺ ] was less so, becoming iso-natriuremic at high salinity. An unusual, notable decrease in (Na ⁺ , K ⁺ )-ATPase activity on acclimation to lower salinities suggests that osmolytes other than Na ⁺ and Cl ^- sustain hemolymph osmolality in dilute media. Crabs acclimated to low salinities exhibit a single family of ATP binding sites that obey Michaelis-Menten behavior. At salinities above 18 ‰S, high-affinity ATP binding sites appear and correspond to 10-20% of total (Na ⁺ , K ⁺ )-ATPase activity; both the low- and high-affinity sites exhibit allosteric behavior. Salinity acclimation affected the enzyme’s specificity constant (kcat/K_M), inducing a direct increase in catalytic efficiency under saturation conditions. The endogenous protein kinases A and C inhibited (Na ⁺ , K ⁺ )-ATPase activity while the FXYD2 peptide stimulated activity in a salinity dependent manner.

CONCLUSION

The endogenous calcium/calmodulin-dependent kinase exhibited regulatory phosphorylation of the gill ATPase and represents a novel finding in the regulation of the crustacean (Na+, K+)-ATPase. These data reveal that U. cordatus exhibits a suite of osmoregulatory and enzymatic adjustments to salinity acclimation that sustain its osmotic and ionic homeostasis, an ability particularly important given its challenging, variable salinity environment.

Keywords: Gill (Na+, K+)-ATPase, Protein kinases, FXYD peptide

Supported by: FAPESP, CNPq, FAPEAM, INCT ADAPTA II.

08814 - Poster Session

DA.03 - BoophilinD1 Phage Display Library: Selection for Zika virus NS2b-NS3 protease

Verônica de Moraes Manzato ¹, Ricardo José Soares Torquato¹, Camila de Morais Coelho², Camila Cardoso Di Santo¹, Fernando Allan Abreu Silva ¹, Martin Rodrigo Alejandro Wurtele², Aparecida Sadae Tanaka¹

¹Biochemistry, Universidade Federal de São Paulo (SP, Brazil), ²Instituto de ciência e tecnologia, Universidade Federal de São Paulo (SP, Brazil)

INTRODUCTION

Arboviruses are a worldwide concern due to increased human mortality and incidence. Outbreaks of different arboviruses have been stimulated the search for drugs and vaccines for viral control. Zika virus translates a single polyprotein, containing structural and non-structural proteins. Our interest is the only serine protease (NS3), a chymotrypsin-like, present in the genome. The complex NS2b (cofactor)-NS3 is responsible for the polyprotein processing, together with host enzymes therefore, being essential to viral cycle.

OBJECTIVES

Our objectives were the construction of a phage display library using Boophilin domain1 (D1), thrombin inhibitor of Kunitz family, as a template, and selection of Boophilin D1 mutants for the Zika virus NS2b-NS3 protease.

MATERIALS AND METHODS

DISCUSSION AND RESULTS

Therefore, we produce a BoophilinD1 library containing random mutations at positions P1-P4’ with a title of 2.9x10 ⁶ (cfu). The library was selected for purified Zika NS2b-NS3 protease by three selection cycles. 17 clones were sequenced, and the results showed: 47% of RALHA (P1-P4’) and 11.8% for RASWA, SMRPT or KALIP (wt). The wild type, mutant 12 and 14 containing the sequence KALIP, RALHA and RASWA (P1-P4’), respectively were expressed and purified. They presented Ki values for Zika protease of 0.046, 0.409 and 0.06 nM, respectively. The inhibitors were also able to inhibit DENV2 protease with Ki values in nM range as well. Docking data indicate two possible interactions between enzyme and inhibitor for mutant 12.

CONCLUSION

The protease has an open and shallow pocket, in addition to an allosteric site, which makes stable interaction and selection of compatible inhibitors challenging. Boophilin D1 mutant 14 selected for Zika protease showed low Ki in the same order of Boophilin D1 wt. Our results suggested that those inhibitors are the strongest Zika inhibitors present in the Boophilin D1 phage display library and this library can be used to select inhibitors for other flavivirus proteases.

Keywords: phage display, inhibitor, NS2b-NS3 Zika

Supported by: CNPq

08921 - Poster Session

DA.04 - Production and Characterization of the Recombinant Human Proteins TRAP-1 and CyP-D and in vitro Detection of their Interaction

Eduardo Feliciano de Lima Santos ¹, Marina Del Giudice¹, Silvia Helena Libardi², Carlos Henrique Inacio Ramos³, Júlio César Borges², Lisandra Marques Gava¹

¹Departamento de Genética e Evolução, Universidade Federal de São Carlos (São Paulo, Brazil), ²Departamento de Química e Física Molecular, Universidade de São Paulo (São Paulo, Brazil), ³Departamento de Química Orgânica, Universidade Estadual de Campinas (São Paulo, Brazil)

INTRODUCTION

Proteins are molecules responsible for several biological functions, including maintaining homeostasis within the cell. Molecular chaperones such as the mitochondrial HSP90, TRAP-1, are fundamental to this process due to their ability to ensure correct protein folding and protect the cell from apoptosis. TRAP-1 is a crucial protein in this process and is responsible for maintaining mitochondrial integrity and playing a protective role against reactive oxygen species. Another important protein in this context is Cyclophilin-D (CyP-D), which acts as a co-chaperone, regulating the activities of other proteins. Both proteins are heavily studied as targets for therapies, as imbalances in their functionality are related to diseases, such as cancer.

OBJECTIVES

The main objective of this project was to produce and characterize the two recombinant proteins, as well as detect and assess the in vitro interaction between them.

MATERIALS AND METHODS

Both proteins were produced in E. coli BL21(DE3), and protein expression was induced by isopropyl-β-D-thiogalactoside (IPTG). Purification was achieved through immobilized metal affinity (IMAC) and size-exclusion (gel filtration) chromatographies. The efficiency of the methods was verified by polyacrylamide gel electrophoresis (SDS-PAGE) and protein concentration was determined by spectrophotometric measurements. A pulldown assay was performed to detect the interaction, and both proteins were submitted to analytical gel filtration for characterization.

DISCUSSION AND RESULTS

Both proteins were successfully expressed and purified, with a high degree of purity and homogeneity. They were both characterized by the analytical gel filtration and the pulldown assay was able to detect their interaction.

CONCLUSION

The results show that both proteins can be successfully produced in vitro. The characterization showed that TRAP-1 was an asymmetric dimer in solution, while CyP-D was a globular monomer. The pulldown assay confirmed the physical interaction between both proteins under all tested conditions, which justifies further and more detailed investigation of this interaction.

Keywords: Molecular Chaperone, TRAP-1, CyP-D

Supported by: CNPq e FAPESP

08148 - Poster Session

DA.05 - MACROPHAGE MIGRATION INHIBITORY FACTOR OF LEISHMANIA MAJOR (LmMIF) AND C-TERMINAL TRUNCATED MUTANTS: STRUCTURAL STABILITY AND MIGRATION INHIBITORY ACTIVITY

Elise Marques Freire Cunha^1,2, Dham Khlisman Velozo da Silva¹, Rafael Ricci de Azevedo³, Marcelo Rodrigues Pinto⁴, Bruno Tavares Biagi¹, Andrew Leech⁵, Jeremy C Mottram⁶, Arthur Henrique Cavalcante de Oliveira ¹

¹Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto-USP (São Paulo, Brasil), ²Departamento de Ciências, Instituto Federal de Educação, Ciência e Tecnologia de Rondônia (Rondônia, Brasil), ³Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos,, Faculdade de Medicina de Ribeirão Preto-USP (São Paulo, Brasil), ⁴Departamento de Biopatologia e Biologia Molecular, Universidade de Uberaba (São Paulo, Brasil), ⁵Technology Facility, York Biomedical Research Institute, University of York (, United Kingdom), ⁶Department of Biology, York Biomedical Research Institute, University of York (, United Kingdom)

INTRODUCTION

The macrophage migration inhibitory factor (MIF) is an important proinflammatory and immunoregulatory protein and participates in both innate and adaptive immune responses. MIF orthologues were identified in several parasites, including Leishmania, and have been suggested a possible modulating function of the host immune response to benefit the development of parasites.

OBJECTIVES

In this work, the structural characterization and the migration inhibitory activity of L. major MIF2 (LmMIF2), C-terminal truncated Delta5 (W108FDelta5) and Delta10 (WtDelta10) mutants were performed in solution.

MATERIALS AND METHODS

The recombinant LmMIF2, Delta10 and Delta5-C-terminal mutants were expressed in E. coli and purified by affinity chromatography. The solution stability of the LmMIF2 and mutants were evaluated by circular dichroism, Intrinsic Fluorescence Tryptophan Emission and SEC-MALS analysis. The inhibition of the macrophage migration was evaluated by wound healing assay.

DISCUSSION AND RESULTS

Far UV CD analyzes showed secondary structure is stable between pHs 4 and 10 and that the C-terminal region is important only extreme pHs. The decreased fluorescence emission at acidic pH indicated a tertiary structure adjustment that was most effective in the mutants at pH < 4. ANS binding revealed a molten globule state for LmMIF2 and mutants and chemical denaturation confirmed a decreased stability of WtDelta10 mutant, indicating the importance of the C-terminal portion for the structure. The SEC-MALS experiments revealed a trimeric structure for LmMIF2 and mutants at pH 7 and pH4 indicating that C-terminal portion is not essential in the maintenance of the trimeric quaternary structure. The wound healing assay showed that the macrophage migration inhibitory activity of the C-terminal mutants was preserved.

CONCLUSION

The stability of the solution structure of LmMIF2, resulting from a robust secondary structure associated with tertiary structure adjustments of a trimeric state, allows its migration inhibitory activity independent of the C-terminal region.

Keywords: Protein , Stability, Leishmania

Supported by: FAPESP, CNPq, Capes, PRP-USP

08487 - Poster Session

DA.06 - The tyrosine-rich repeat in chlamydial TarP is intrinsically disordered

Tharin M. A. Blumenschein¹, James Tolchard², Andrew J. Miles³, Bonnie A. Wallace³

¹School of Chemistry, University of East Anglia (NR4 7TJ, United Kingdom), ²PDBe, PDBe, EMBL-EBI (CB10 1SD, United Kingdom), ³Institute of Structural and Molecular Biology, Birkbeck College, University of London (WC1E 7HX, United Kingdom)

INTRODUCTION

TarP (Translocated actin-recruiting phosphoprotein) is a chlamydial effector protein essential for host cell invasion. It is injected into the host cell in the beginning of the infection process via type III secretion. Once inside the host cell, multiple regions in TarP interact with host proteins in different ways to reorganise the actin cytoskeleton and bring chlamydia into the host cell. Some of these regions are common to all chlamydia species, such as the WH2-like region that is capable of directly binding actin monomers and nucleating the formation of new actin filaments, while other regions are absent in some species. Chlamydia trachomatis is the species responsible both for the most commonly diagnosed sexually transmitted disease in the UK and the most common cause of preventable blindness in the world. Unlike some other chlamydia species, its TarP contains a variable number of tyrosine-rich repeats, which get phosphorylated upon injection into the host cell. Once phosphorylated, they interact with SH2 domains in signalling proteins, disrupting cytoskeleton signalling.

OBJECTIVES

To determine the folding status of the Tyr-rich repeats in TarP,

MATERIALS AND METHODS

we have expressed and purified a single Tyr-rich repeat from C. trachomatis TarP, and characterized it using NMR and Synchrotron Radiation Circular Dichroism spectroscopy.

DISCUSSION AND RESULTS

The NMR chemical shifts and the SRCD spectrum showed mainly characteristics of a random coil.

CONCLUSION

The resulting spectra are characteristic of a mostly intrinsically disordered protein, with some residual helical content.

Keywords: chlamydia, intrinsically disordered proteins, NMR

08633 - Poster Session

DA.07 - Biochemical and Biophysical studies of the Neurospora crassa AAA+ RVB-1/RVB-2 protein complex and its novel function in stress response

Jonatas Erick Maimoni Campanella ¹, Vanessa T. R. Kiraly², Paula A. Barbugli³, Antoniel Augusto Severo Gomes⁴, Marcos Roberto de Mattos Fontes⁴, Julio Cesar Borges², Maria Célia Bertolini¹

¹Departamento de Bioquímica e Química Orgânica, Instituto de Química de Araraquara, Universidade Estadual Paulista (São Paulo, Brazil), ²Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo (São Paulo, Brazil), ³Departamento de Materiais Dentários e Prótese, Faculdade de Odontologia de Araraquara, Universidade Estadual Paulista, (São Paulo, Brazil), ⁴Departamento de Física e Biofísica, Instituto de Biociências de Botucatu, Universidade Estadual Paulista (São Paulo, Brazil)

INTRODUCTION

The RVB protein complex, composed of two paralogs (RVB-1 and RVB-2 in Neurospora crassa), belong to the AAA+ (ATPases Associated with various cellular Activities) superfamily present in archaea and eukaryotes. They are highly conserved proteins and can interact with many proteins forming multimolecular complexes involved in many biological processes, such as gene expression, cellular differentiation, chromatin remodeling and tumorigeneses.

OBJECTIVES

The objective of the present work is to characterize the N. crassa RVB-1/RVB-2 complex in the context of stress response and structural properties.

MATERIALS AND METHODS

To characterize the protein complex we used functional assays, and biochemical and biophysical approaches.

DISCUSSION AND RESULTS

Growth of the heterokaryotic mutant cells is severely impaired in the presence of stressors and both proteins are overexpressed under stress-induced by salt and temperature. In addition, both are light-regulated proteins exhibiting opposite expression modulation, while RVB-1 is activated, RVB-2 is repressed by light. Both proteins show a perinuclear location, and their nuclear expression are increased under stress. RVB-1 co-immunoprecipitations assays of cell extracts exposed to osmotic and heat stresses led us to identify components of several chromatin remodelers and the shugoshin-like protein, a protein described as involved in stress response in mouse cells. Biochemical and biophysical studies were also performed with the RVB-1/RVB-2 recombinant proteins. SEC-MALS analysis revealed that the complex predominantly exists as a dimer in solution and that nucleotides (ATP/ADP) influence the oligomerization state, while ATP favors hexamers assembly, ADP favors the formation of multimeric states, likely dodecamers. Molecular Dynamics simulations showed that nucleotides promote a structural rearrangement of the complex, more likely influencing the cooperativity between the monomers. The protein complex binds to dsDNA fragments and exhibits ATPase activity, which is strongly enhanced in the presence of DNA.

CONCLUSION

Our findings show that some properties of this protein complex are fungus specific.

Keywords: SEC-MALS, co-immunoprecipitation, stress response

Supported by: FAPESP, CAPES e CNPq

08587 - Poster Session

DA.08 - Myristoylation and its effects on the Golgi Reassembly and Stacking Protein 55

Emanuel Kava ¹, Antonio José Costa Filho¹

¹Física, Universidade de São Paulo (São Paulo, Brasil), ²Física, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

GRASP55 is a myristoylated protein localized in the medial/trans-Golgi faces and involved in the Golgi structure maintenance and the regulation of unconventional secretion pathways. It is believed that GRASP55 achieves its main functionalities in the Golgi organization by acting as a tethering factor and, when bound to the lipid bilayer, its orientation relative to the membrane surface is restricted to determine its proper trans-oligomerization. Despite the paramount role of myristoylation in GRASP function, the impact of such protein modification on the membrane- anchoring properties and the structural organization of GRASP remains elusive. Here, an optimized protocol for the myristoylation in E. coli of the membrane-anchoring domain of GRASP55 is presented.

OBJECTIVES

Optimization of a myristoylation protocol in GRASP55 in E. coli and analysis of its biophysical properties and interaction with model membranes.

MATERIALS AND METHODS

Protein expression and purification in E.coli, size exclusion chromatography (SEC), liposome electrophoretic mobility assay (LEMSA), near and far-UV CD, fluorescence, MD simulations.

DISCUSSION AND RESULTS

The biophysical properties of the myristoylated/non-myristoylated GRASP55 GRASP domain were characterized in a membrane-mimicking micellar environment. Although myristoylation did not cause any impact on the protein’s secondary structure, according to our circular dichroism data, it had a significant impact on the protein's thermal stability and solubility. Electrophoresis of negatively charged liposomes incubated with the two GRASP55 constructions showed different electrophoretic mobility for the myristoylated anchored protein only, thus demonstrating that myristoylation is essential for the biological membrane anchoring. Molecular dynamics simulations were used to further explore the anchoring process in determining the restricted orientation of GRASPs in the membrane.

CONCLUSION

Less soluble and more stable protein, differences in electrophoretic mobility of liposomes incubated with myristoylated protein, a successful anchoring in model membranes.

Keywords: GRASP, Membrane interaction, Myristoylation

Supported by: CAPES

05032 - Poster Session

DA.09 - Vibrio cholerae YaeO is a structural homologue of RNA chaperone Hfq that inhibits Rhodependent transcription termination by dissociating its hexameric state

Kamalendu Pal ¹, Malti Yadav¹, Sriyans Jain², Biplab Ghosh³, Ranjan Sen², Udayaditya Sen¹

¹Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, HBNI (1/AF Bidhan Nagar, Kolkata 700064, India), ²Laboratory of Transcription, Center for DNA Fingerprinting and Diagnostics (Tuljaguda Complex, 4-1-714 Mouzamjahi Road, Nampally, Hyderabad 500 001, India), ³High Pressure & Synchrotron Radiation Physics Division, Bhabha Atomic Research Centre (Trombay, Mumbai 400085, India)

Efficient and accurate transcription termination is required for the correct regulation of bacterial gene expression. In bacteria, two main mechanisms of transcription termination have been described, Rho-independent and Rho-dependent termination. In Rho-dependent termination, Rho binds to the rho utilizing (rut) sites on the nascent RNA and translocates in the 5' to 3' direction using the ATP hydrolysis energy eventually dislodging the RNA-polymerase from elongation complex [1]. The Psu and YaeO are two established inhibitors of Rho-dependent termination [2,3]. Crystal structure of Psu, solved by our lab, demonstrates novel fold and uniquely knotted dimer [2]. Structure of Escherichia coli YaeO (EcYaeO), solved by NMR, exhibits similarity with Hfq protein [4]. Detailed sequence and phylogenetic analysis demonstrate that V. cholerae YaeO (VcYaeO) is significantly distinct from its E. coli counterpart. VcYaeO causes significant growth defect upon in vivo expression and inhibits in vitro functions of the Rho. Structure of VcYaeO solved at 1.75 Å resolution, the first crystal structure of a YaeO protein, demonstrates a beta-sandwich fold distinct from the NMR-structure of the EcYaeO. Through various biophysical techniques like SEC, DLS and chemical crosslinking we have demonstrated that VcYaeO disrupts the oligomeric state of the VcRho. Interestingly, VcYaeO has structurally more resembles to the Hfq protein and like the latter it exhibits ssDNA/RNA binding properties whereas EcYaeO is unable to do so. Through docking studies and ssDNA/RNA binding properties we propose that VcYaeO inhibits the function of the Rho protein via disruption of the latter’s hexameric assembly and also likely by sequestering the RNA from the Rho primary binding sites. References: [1] T. Platt, Mol. Microbiol. 11 (1994) 983–990. [2] R. Banerjee. et al. J. Biol. Chem. 287 (2012) 44667–44675. [3] S. Pichoff. et al. Mol. Microbiol. 29 (1998) 859–869. [4] P. Gutiérrez. et al. J. Biol. Chem. 282 (2007) 23348–23353. Presenting author e-mail address: kamalendu.pal@saha.ac.in & kpal.biochem@gmail.com

Keywords: X-ray crystallography, anti-termination, disruption of Rho hexamer

08267 - Poster Session

DA.10 - STRUCTURE AND ANTIBACTERIAL PROPERTIES FROM BMOOLAAO-II, A L-AMINO ACID OXIDASE ISOLATED FROM BOTHROPS MOOJENI.

Thales Alves de Melo Fernandes ¹, Tassia Rafaella Costa¹, Lorena Polloni¹, Carlos Henrique Gomes Martins², Ralciane de Paula Menezes², Meliza Arantes de Souza Bessa², Nilson Nicolau Junior³, Veridiana de Melo Rodrigues Ávila¹

¹Laboratório de Bioquímica e Toxinas Animais, Universidade Federal de Uberlândia (MG, Brasil), ²Laboratório de Ensaios Antibacterianos, Universidade Federal de Uberlândia (MG, Brasil), ³Laboratório de Modelagem Molecular, Universidade Federal de Uberlândia (MG, Brasil)

INTRODUCTION

The snake venom is composed of a mixture of macromolecules with a wide variety of biological effects. Due to their pharmacological properties, many components call attention to the treatment of various human diseases, such as L-amino acid oxidases from snake venoms (SV-LAAOs).

OBJECTIVES

The present study aimed to (i) isolate an L-amino acid oxidase (LAAO) isoform from Bothrops moojeni called BmooLAAO-II; (ii) evaluate the bactericidal effect of the toxin against Gram-positive and Gram-negative strains acquired from the American Type Culture Collection and multidrug-resistant clinical isolates; and (iii) investigate the molecular structure to clarify the biological function.

MATERIALS AND METHODS

The purification of the toxin was performed using three chromatographic steps including molecular exclusion, hydrophobic interaction, and affinity. The antibacterial activity was determined by the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (CBM). The structure analysis was performed by molecular modeling, molecular docking, and molecular dynamics.

DISCUSSION AND RESULTS

Purified BmooLAAO-II showed to be a homodimeric acidic glycoprotein with molecular weight around 60 kDa under reducing conditions in SDS-PAGE. The enzyme exhibited high specificity catalysis on amino acid L-Leucine, 4,136,683 U/mg/min. After incubation, the toxin demonstrated a strong bactericidal effect against Staphylococcus aureus and Escherichia coli, additionally the toxins show bacteriostatic action against Enterococcus faecium. Sequence analysis indicates a high sequence identity among SV-LAAOs. Structures studies show highly conserved structural pattern of SV-LAAOs composed by FAD-binding domain, substrate-binding domain, and helical domain.

CONCLUSION

In this sense, BmooLAAO-II demonstrated promising pharmacological properties. These results are important to direct our research for the development of new and more effective antibacterial agents, including the control of resistant bacterial infections. Furthermore, the structure-functional relationship contributes to clarify the mechanism of action and assist the research of potential molecules with biotechnological applications.

Keywords: Bothrops moojeni, L-amino acid oxidase, Pharmacological properties

Supported by: CNPq

08364 - Poster Session

DA.11 - Fibrillation of alpha synuclein probed by surface methylation

Maria Rocio Rial Hawila ¹, Gabriela Elena Gómez¹, Andrés Binolfi², José María Delfino¹

¹Departamento de Química Biológica e IQUIFIB (UBA-CONICET) , Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires (, Argentina), ²IBR-Universidad Nacional de Rosario, Rosario, Santa Fe (, Argentina)

INTRODUCTION

The nature and size of the accessible surface area (SASA) of the polypeptide chain plays a key role in protein folding and complex formation. SASA can be probed with diazirine (DZN), a tiny precursor of the extremely reactive methylene carbene (:CH2). Sparse methylated sites left on the polypeptide provide revealing signs on the conformation. The metric extent of methylation (EM) derived from mass spectra discriminates between native and alternate states. Human alpha synuclein (AS) aggregates into oligomers and amyloid fibrils, constituents of Lewy bodies, a cytosolic hallmark of Parkinson disease.

OBJECTIVES

DZN labeling proves particularly fit to analyzing the conformational plasticity inherent to this intrinsically disordered protein.

MATERIALS AND METHODS

DISCUSSION AND RESULTS

Unlike folded proteins where EM differs markedly between native and unfolded states, AS in buffer or in 6 M GdmCl displays a similarly enhanced value, revealing high solvent accessibility under normal physiological conditions. Strikingly, AS fibrils display an enhanced EM value compared to the monomer, as a consequence of the organization of a hydrophobic interface. In addition, location of methylated sites by multidimensional NMR (1H15N-HSQC) opens a vast panorama. The extent of :CH2 reaction across the surface of AS defines sequential intensity profiles. Differential labeling signs point to the involvement of the N-terminal domain in fibril formation.

CONCLUSION

We anticipate that the combined picture derived from MS and NMR data will serve to elucidate the contribution to fibrillation of the different structural moieties of AS, allowing the appraisal of species belonging to the monomeric ensemble, the lesser-known oligomers, and the fibrillar aggregates.

Keywords: Synuclein, Methylation, Diazirine

Supported by: UBACyT, CONICET and ANPCyT

08894 - Poster Session

DA.13 - Investigation of New Therapeutic Targets in Trypanosoma cruzi Genome Using Different Computational Approaches

Lorrana Faria Fonseca ¹, Manuela Leal da Silva¹

¹Bioinformatic, 1 Institute of Biodiversity and Sustainability, Federal University of Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Trypanosoma cruzi is a protozoan that belongs to the Trypanosomatidae family and an etiologic agent of Chagas disease. It’s transmission to insect vector Rhodnius prolixus. About 6 million to 7 million people worldwide are estimated to be infected with T. cruzi, the parasite that causes Chagas disease. The Benzonidazole is used to treat infection in Brazil, which has no guarantee of the treatment's total effectiveness yet.

OBJECTIVES

The objective of this work is to analyze the hypothetical proteins predicted in the genome of the Trypanosoma cruzi, unveiling their functionalities and interactions using in silico tools aiming at the search for new therapeutic targets for the treatment of Chagas disease.

MATERIALS AND METHODS

We used data of the Trypanosoma cruzi [PRJNA15540] with 19,607 proteins. We separate hypothetical proteins and make a structural prediction through comparative 3D modeling using the MHOLline platform. The 3D models were selected by analyzing the identity-coverage between the alignments, RMSD between model-template and Ramachandran's plot generated by the PROCHECK software. We search for structural similarity using Kpax, CATH and ScopE. Molecular Function was predicted using Gene Ontology.

DISCUSSION AND RESULTS

It was possible to build 808 three-dimensional models from the 19,607 proteins predicted in the T. cruzi genome. After analyzing the favorable-prohibited regions through the Ramachandran’s plot, of these 360 proteins to predict function and resulting in 27 target proteins for the next step.

CONCLUSION

It was possible to build 3D models for 27 proteins from interesting groups previously predicted like hypothetical proteins. The next steps will be to analyze the metabolic pathways of these proteins in KEGG (Kyoto Encyclopedia of Genes and Genomes) to direct these proteins like possible therapeutic targets.

Keywords: Trypanosoma cruzi, Chagas disease, predict function

Supported by: CNPq and CAPES

08337 - Poster Session

DA.14 - Effects of the endocannabinoids AEA and 2-AG on single channel gramicidin A conductance and membrane capacitance of lipid bilayer

Djalma Medeiros ¹, Manoel Arcisio-Miranda²

¹Filosofia, Faculdade de São Bento (São Paulo, Brazil), ²Biofísica, Universidade Federal de São Paulo (São Paulo, Brazil)

INTRODUCTION

AEA and 2-AG are endogenous cannabinoids molecules lipid messengers with amphiphilic character that exercise regulatory functions in nervous system by binding to cell membrane receptors CB1 and CB2. They also exert direct actions, without receptor mediation, regulating the function of ion channels. The regulation model by endocannabinoid specific binding to its target does not promptly explicates its activity modulation of different classes of ion channels.

OBJECTIVES

Our research propound an alternative endocannabinoid mode of regulation of protein ion channel based on a nonspecific and receptor-independent mechanism involving the adjustment of the membrane/protein hydrophobic coupling, which arises from drug-induced local changes in bilayer elastic properties that alter the energetic coupling between the protein and its host bilayer.

MATERIALS AND METHODS

The regulatory action of both AEA and 2-AG on gA ion channel in planar lipid bilayers composed of DOPC and DPhPC was determined using single channel conductance and membrane capacitance measurements together with techniques of continuous theories of elastic deformation of bilayers.

DISCUSSION AND RESULTS

The endocannabinoids do not change the membrane capacitance or the amplitude of single channel current, but produce increase in the channel’s appearance frequency and its open state average lifetime. The observed effects are more intense in bilayer of DOPC then of DPhPC due to higher hydrophobic mismatch of DOPC. The insertion of a protein into the hydrophobic environment of the lipid bilayer has an energy cost related to the adjustments of the bilayer to the membrane protein hydrophobic portion.

CONCLUSION

The correlation between channel kinetics and energetics shows that the action of the endocannabinoids on gA channel has a linear rate-equilibrium relation that provides strong support for the hypothesis that their effects are mediated by a more general and nonspecific modulation mechanism associated with locally adjusting the bilayer hydrophobic thickness to match the channel length.

Keywords: bilayers, channels, endocannabinoids

Supported by: FAPESP and CNPq

08604 - Poster Session

DA.15 - Preliminary over view of the interaction features between the recombinant disintegrin, Jararacin, and platelets: NMR studies

Jorge Eduardo Chang Estrada ¹, Jorge Eduardo Chang Estrada¹, Ariana Azevedo Vasconcelos¹, Victor da Concencão David¹, Leticia dos Reis Machado¹, Russolina Benedeta Zingali¹, Fabio Ceneviva Lacerda de Almeida¹

¹Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro (Rio de Janeiro (RJ), Brazil)

INTRODUCTION

Desintegrins are small cysteine-rich peptides found in snake venom. They have modulating activity over a broad range of Integrins, which are heterodimeric receptors that have a key role in mediating physiological and pathological processes, such as cell adhesion, migration, apoptosis, and platelet aggregation. Integrins, thus, have become a therapeutically target and disintegrins can provide new information about physiopathology activities, such as thrombosis and angiogenesis.

OBJECTIVES

The objective of this study was to express and analyze the activity of a recombinant disintegrin, jararacin (rJarc), over platelet aggregation.

MATERIALS AND METHODS

To complete our objective, we expressed the disintegrin on a Pichia pastoris system. The yeast was transformed with the vector pPIC9 containing the synthetic gene of rJarc. The disintegrin was expressed and secreted in the cultured media and then purify by molecular exclusion chromatography. Also, 15N-rJarc was expressed and purified as described. We confirmed the molecular mass and internal sequence by mass spectrometry and its correct folding by 1H nuclear magnetic resonance spectra and circular dichroism. Then, we performed biological assays to evaluate the activity of rJarc over platelet aggregation. rJarc yield was 30 mg/L approximately.

DISCUSSION AND RESULTS

Also, rJarc was capable of inhibiting platelet aggregation induced by ADP, collagen, and thrombin. Also, presented an inhibitory activity over the adhesion of platelets to collagen (by 500nM) and fibrinogen (by 150 nM) under continuous flow. In addition, preliminary NMR studies, in the presence or absence of platelets, revealed changes in rJarc structure and the residues involved in the platelet-desintegrin interaction.

CONCLUSION

In conclusion, rJarc was expressed in a correct folding and inhibit platelet aggregation in a similar way to the native protein. NMR data shows that rJarc is a useful tool to understand the interaction of disintegrins with platelets and integrins.

Keywords: Disintegrin, Integrin, Platelet aggregation

Supported by: FAPERJ, CNPq and CAPES

08610 - Poster Session

DA.16 - The phosphoprotein of respiratory syncytial virus, a protein of low structural complexity and still an essential player of the viral polymerase

Christina SIZUN ¹, CArdone Christophe¹, Caseau Claire-MArie¹

¹Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique (, France)

INTRODUCTION

The phosphoprotein (P) is an essential co-factor of the polymerase of Mononegavirales order viruses. We focused on the phosphoprotein of respiratory syncytial virus (RSV), which is a major pathogen responsible for pneumonia in children and the archetype of the Orthopneumovirus genus, Pneumoviridae family. The RSV P phosphoprotein is an essential co-factor of the viral RNA polymerase complex. Its primary function is to tether the catalytic subunit of the polymerase (L) to the ribonucleoprotein genome by specific interactions with the viral nucleoprotein (N), by binding to the L protein on the one hand and to the N protein on the other hand. More generally, the RSV P protein acts as a hub protein for the viral RNA polymerase complex by assembling its different components. For example it recruits additional factors like the RSV transcription factor M2-1 and the cellular phosphatase PP1. We used a combination of biochemical, byophysical and NMR approaches to gain structural and dynamic insight into the RSV P protein. We showed that it contains large N- and C-terminal intrinsically disordered regions (IDRs), the only domain with well-defined structure being a small tetramerization domain. We analyzed the full protein and fragments. By NMR we showed that the IDRs display transiently structured regions, some with high helical propensity. The combination of intrinsic disorder and tetrameric organization allows RSV P to bind to multiple partners and endows it with adaptor protein-like properties. We identified several binding regions that correspond to different partners and complexes AND/or that drive compaction of this protein. Finally, we showed that the RSV N-P protein-protein interaction could be targeted by small compound inhibitors to design new antiviral therapeutics.

OBJECTIVES

MATERIALS AND METHODS

DISCUSSION AND RESULTS

CONCLUSION

Keywords: nuclear magnetic resonance, intrinsically disordered protein, viral polymerase complex

Supported by: ANR, Labex LERMIT

08930 - Poster Session

DA.17 - Human TMED 1 GOLD domain: Thermodynamic and structural analysis

Danielly Christine Adriani Maia Mota ¹, Luis Felipe Santos Mendes¹, Iara Aimê Cardoso¹, Renan Minin de Mori¹, Mariana Raquel Bunoro Batista¹, Luís Guilherme Mansor Basso², Maria Cristina Nonato¹, Antônio José Costa-Filho¹

¹Department Physics, University of São Paulo (SP, Brasil), ²Science and Technology Center, Universidade Estadual do Norte Fluminense Darcy Ribeiro (RJ, Brazil)

INTRODUCTION

TMEDs are eukaryotic transmembrane proteins located in all subcompartments of the initial secretory pathway. Although essential during bidirectional transport between the endoplasmic reticulum and the Golgi, information about their structure, oligomeric state and how they anchor the transport cargo is still lacking.

OBJECTIVES

To fulfil this gap, we performed a detailed biophysical characterization of the GOLD domain of human TMED1.

MATERIALS AND METHODS

The recombinant TMED1 GOLD protein was purified in two steps and analysed through: Circular Dichroism, Size Exclusion Chromatography with Multi-Angle Light Scattering, Size Exclusion Chromatography, Differential Scanning Calorimetry, X-ray crystallography, Phylogenetic analyses, Molecular dynamics simulations and MM/PBSA free energy calculation.

DISCUSSION AND RESULTS

The circular dichroism data showed that, after purification, the protein was folded and its secondary structure content was predominantly formed by β-sheets (~44%). The high-resolution structure was determined by protein crystallography and showed a structural organization in a β-sandwich composed of two four-strand antiparallel β-sheets and a conserved disulphide bond. The protein formed a dimer structure in the crystal organization, further confirmed experimentally in solution and showed to be salt-dependent.

CONCLUSION

The proposed dimeric structure was further analysed using molecular dynamics simulation and the residues important for oligomerization were mapped. A model for the TMED docking to the membrane was proposed.

Keywords: TMED, P24 family, Early Secretory Pathway

Supported by: CNPq, CAPES and FAPESP

08483 - Poster Session

DA.19 - Quantitative evaluation of the binding of the scFv antibody NUsc1 to Alzheimer’s Disease-Associated Aβ oligomers using Surface plasmon Resonance

Mariane Fávero Carraro ¹, Ana Paula Masson¹, Raquel Maria de Campos¹, Vitor Marcel Faça¹, Adriano Silva Sebollela¹

¹Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto (SP, Brasil)

INTRODUCTION

Alzheimer disease (AD), the leading cause of dementia worldwide, is characterized by the brain accumulation of protein aggregates, including those formed by the β-amyloid peptide (Aβ). Although the molecular mechanisms underlying the pathogenesis of AD are still not completely understood, mounting evidence implicates soluble Aβ oligomers (AβOs) as key players in disease onset and progression. Here we have used surface plasmon resonance (SPR) to evaluate the binding of a scFv antibody fragment previously selected by our group, named NUsc1, which has been shown to neutralize the neurotoxicity of AβOs, to in vitro-prepared AβOs.

OBJECTIVES

Evaluate the binding of the scFv antibody NUsc1 to soluble Aβ oligomers.

MATERIALS AND METHODS

A SPR CM-5 chip was functionalized with the anti-c-myc IgG 9e10. Next, purified c-myc tagged-NUsc1 was immobilized this chip via interaction with c-myc tag. The interaction of synthetic AβOs with this chip was analyzed by SPR.

DISCUSSION AND RESULTS

The immobilization of NUsc1 to the chip via myc tag, and the binding of AβOs to immobilized NUsc1 were clearly observed. The binding of AβOs at different concentrations revealed that AβOs binds to NUsc1 with high affinity, in the low nanoM range. We also observed the selectivity of NUsc1 binding comparing its binding to AβOs to the response obtained with lysozyme oligomers, which was significantly lower.

CONCLUSION

We have established an efficient strategy to quantitatively evaluate the binding of the scFv antibody NUsc1 to Aβ oligomers. Using this assay, we have determined the high affinity of NUsc1 to AβOs, which is in the low nanoM range. This SPR assay may be used in the future to quantify the affinity of other high-affinity conformation-sensitive antibodies to AβOs, with implications to diagnostic and therapeutic applications in AD.

Keywords: AβOs, Alzheimer, SPR

Supported by: FAPESP, CNPq, CAPES, FAEPA

08599 - Poster Session

DA.20 - Biophysical characterization of the RNA-dependent RNA polimerase (nsP4) protein of Chikungunya virus

Marjorie Caroline Liberato Cavalcanti Freire ¹, Luis Guilherme Mansor Basso², Nathalya C M R Mesquita¹, Glaucius Oliva¹

¹Instituto de Física de São Carlos , Universidade de São Paulo (SP, Brazil), ²Campos dos Goytacazes, Universidade Estadual do Norte Fluminense Darcy Ribeiro (RJ, Brazil)

INTRODUCTION

The alphavirus Chikungunya virus (CHIKV) is the causative agent of Chikungunya fever, an arboviral disease transmitted to humans by the bite of mosquitoes from the Aedes genus. The symptoms include rash, fever, edema, and joint and musculoskeletal pain. The lack of an effective treatment, associated with the debilitating period caused by joint pain, creates the need to intensify studies that may lead to new drugs or vaccines against this virus. The CHIKV belongs to Togaviridae family and is a positive-sense single-stranded RNA virus , whose genetic material encodes two polyproteins (structural and non-structural), which after cleavage give rise to nine mature proteins. Among them, nsP4 is the RNA-dependent RNA polymerase (RdRp) and plays a crucial role in the viral RNA replication. Due to this notorious importance, the nsP4 emerges as a promising target for the search for drugs against CHIKV

OBJECTIVES

So far, its three-dimensional structure has not been elucidated, then biophysical methods can be useful for obtain structural and dynamic informations about this protein.

MATERIALS AND METHODS

Here, the CHIKV nsP4 was cloned, expressed in heterologous system and purified. Differential Scanning Fluorimetry, Circular Dichorism and Differential Scanning Calorimetry were performed to assess stability, secondary structure content, thermal/chemical denaturation profiles and thermodynamic parameters of the transition process.

DISCUSSION AND RESULTS

As results, the CHIKV nsP4 showed a predominance of helical content, which is a common feature of viral polymerases. The chemical denaturation showed a non-cooperative process and the thermal denaturation occurs cooperatively, presenting well defined state transition and this process is kinetically controlled.

CONCLUSION

Based on these findings, studies of ligand interactions will be carried out to search for compounds able of interact with nsP4, aiming the development of novel antiviral drug candidates to treat CHIKV infection.

Keywords: Chikungunya virus, nsP4, thermal denaturation

Supported by: FAPESP

08868 - Poster Session

DA.21 - Structural, Functional and Evolutionary aspects of Calpain/Cactus complex action in Toll pathway modulation

Alison Henrique Ferreira Julio ^1,2, Paloma Dias e Vasconcellos¹, Priscila da Silva Figueiredo Celestino Gomes², Pedro Geraldo Pascutti², Paulo Bisch², Helena Araujo¹

¹ICB , Instituto de Ciências Biomédicas, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil), ²Biofisica, Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Calpain A is a cysteine protease regulated by Ca++ able to cleave Cactus (IKB) resulting in modulation of Toll signaling. However, structural and biophysical requirements involved in the interaction of Calpain A and Cactus remains unknown.

OBJECTIVES

Here, we report the molecular structures and conformational dynamics of Calpain A-Cactus complex and functional analysis of Calpain in D. melanogaster and Rhodnius prolixus.

MATERIALS AND METHODS

All-atom molecular dynamics of Calpain A-Cactus complex were carried out with AMBER14 using AMBER (ff14SB) force field followed by 350 ns simulations.

DISCUSSION AND RESULTS

Molecular dynamic simulations revealed that the N-terminal region of Cactus undergoes wide conformational rearrangement indicating that acts as a modulator in the cleavage process, by approximating the Cactus cleavage site to the catalytic site of Calpain A. Our results also indicate that the complex depends on electrostatic interactions, involving negatively charged regions in Cactus ankyrin repeats and positively charged regions in Calpain A domains PC1 and PEF. Furthermore, the Calpain A-Cactus complex is sustained by salt-bridges formed along the Cactus alpha helix anti-parallel ankyrin repeats and Calpain A EF hands 2, 3, 5 and CBSW domain of Calpain A. Next, we interrogated the conservation of Calpain in modulate Toll signaling in a phylogenetically basal insect R. prolixus. We found amplification of Calpain system in Rhodnius genome. Calpains in R. prolixus revealed differential architecture, with differential EF-Hand domain and containing key mutations at the catalytic residues of CysPc domain of Calpain A/B like, yielding catalytic dead-Calpains. Functional assays by pRNAi revealed phenotypes at oviposition for CalpA1, CalpA4 and CalpC. Interesting, in situ hybridization after catalytic-dead-CalpA2 knockdown indicated disruption of Twist and SoxN territories resulting at fail of germ band elongation.

CONCLUSION

These evidences reveal the structural requirements involved in Calpain modulation of Toll signaling, as well as a different domain-architecture of Calpains including catalytic-dead that influences critical early stages of Rhodnius development.

Keywords: CalpainA-Cactus complex, NFkB, protein-protein interactions

Supported by: CAPES, CNPq, FAPERJ, INCT-EM

08925 - Poster Session

DA.22 - Exploring the Energy Landscape of Chromosomes

Vinícius de Godoi Contessoto ¹, Antonio B. Oliveira Jr¹, José N. Onuchic¹

¹Center for Theoretical Biological Physics (CTBP), Rice University (Houston, TX, USA)

Significant efforts have been made to obtain the genome's three-dimensional structure and understand how chromosomal organization may affect gene regulation and expression. Chromosome conformational capture techniques such as Hi-C have been essential in uncovering the quantitative information needed to determine chromatin organization. Complementing these experiments, co-polymers theoretical methods are necessary to determine the ensemble of three-dimensional structures associated with Hi-C maps. In addition to the structural information, these theoretical advances also shed light on the underlying mechanisms governing genome assembly and function. We perform chromatin dynamics simulations using a maximum entropy approach by applying the MiChroM (Minimal Chromatin Model) energy function to generate the 3D ensemble of chromosome structures. The energy function is transferable between chromosomes, cell lines, cell cycle phases, and different organisms. MiChroM energy function is now available in a fast and scalable software version, which can perform chromosome simulations using GPUs via OpenMM Python API, called Open-MiChroM. Open-MiChroM and 3D ensemble of chromosomal structures are available at the Nucleome Data Bank - https://ndb.rice.edu.

Keywords: Genome Organization, Chromosome Modeling, Chromatin Dynamics

08235 - Poster Session

DA.23 - Structural and biochemical characterization of LsfA, a 1-Cys Prx involved in Pseudomonas aeruginosa virulence

Rogério Luis Aleixo Silva ¹, Renato Mateus Domingos¹, Madia Trujillo², Arnaldo Oliveira Filho³, Cristiano Luis Pinto de Oliveira³, Luis Eduardo Soares Netto¹

¹Genética e Biologia Evolutiva, Universidade de São Paulo (SP, Brasil), ²Center for Free Radical and Biomedical Research, Universidad de la Republica (, Uruguai), ³Instituto de Física, Universidade de São Paulo (SP, Brazil)

INTRODUCTION

Pseudomonas aeruginosa is a ubiquitous, gammaproteobacteria that is the main cause of nosocomial infections and some its strains present multiple antibiotic resistance. Macrophages and neutrophils release oxidants in response to pathogen infection. In turn, P. aeruginosa contains antioxidants such as LsfA, a Cys-based peroxidase of the peroxiredoxin family of enzymes. LsfA belongs to the Prdx6 sub-family and as most of its members displays the 1-Cys Prx mechanism. Prdx6 enzymes are still poorly characterized. Additionally, LsfA is involved in P. aeruginosa virulence.

OBJECTIVES

Therefore, the aim of the present work is the structural and biochemical characterization of LsfA.

MATERIALS AND METHODS

We investigated the kinetics of LsfA by analyzing redox dependent changes in its intrinsic fluorescence and also by a competitive assay, employing heme-dependent peroxidases.

DISCUSSION AND RESULTS

Thereby the second order rate constants for the reactions of LsfA with: H2O2 (107 M-1.s-1), tert-butylhydroperoxide (106 M-1.s-1) and peroxynitrite (107 M-1.s-1) were determined. Furthermore, rate constants of LsfA hyperoxidation by H2O2 (230 ± 2.31 M-1.s-1) and by tert-butylhydroperoxide (286.9 ± 11.27 M-1.s-1) were also determined by the fluorimetric approach and confirmed by western blotting. LsfA is rapidly oxidized by several peroxides, but its hyperoxidation rate constants are lower than for other peroxiredoxin enzymes. Notably, this is the first description of hyperoxidation rate constants for enzymes of the Prdx6 sub-family. Furthermore, two crystallographic structures of LsfA were elucidated in distinct oxidative states (reduced and sulfonic acid), both in the dimeric state; at 2.6 and 2.0 Ǻ resolution, respectively. The native quaternary structure (dimer) was confirmed by SAXS analysis. LsfA presents distinct topology and substrate accessibility in the active site in comparison to human Prx6.

CONCLUSION

Thus, relevant structural and biochemical insights were obtained on an antioxidant enzyme involved in P. aeruginosa virulence that might enable the identification of a specific inhibitor.

Keywords: Peroxiredoxin, antioxidant defence , kinect characterization

Supported by: FAPESP/CNPq

08558 - Poster Session

DA.24 - Effect of hydrogen sulfide on protein tyrosine phosphatase A from Mycobacterium tuberculosis

Karla Oliveira Ortiz ¹, Tomás Bohn Pessatti¹, Angela Camila Orbem Menegatti², Hernán Francisco Terenzi¹

¹Bioquímica , Universidade Federal de Santa Catarina (SC, Brasil), ²Bioquímica , Universidade Federal da Paraíba (Paraíba , Brasil)

INTRODUCTION

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis, in 2019, this disease was the world's 13th leading cause of death. To survive within the host macrophage, M. tuberculosis secretes the protein tyrosine phosphatase A (PtpA), which contributes to the inhibition of phagosome-lysosome fusion. PtpA activity is modulated by nitric oxide (NO), the S-nitrosylation of cysteine 53 decreasing 50% of its activity. Hydrogen sulfide (H₂S) is also involved in the posttranslational modification of cysteine residues.

OBJECTIVES

Thus, we aimed to investigate M. tuberculosis PtpA susceptibility to persulfidation.

MATERIALS AND METHODS

PtpA and its heterologous cysteine mutants (C16A, C53A and C16/53A) were expressed and purified. To perform the analyses, the proteins were incubated in the presence or absence of 1 mM NaSH (H₂S donor) for 30 min at 25 ^o C.

DISCUSSION AND RESULTS

PtpA WT and C53A specific activities, using p < /i>-nitrophenyl phosphate as substrate (p < /i>NPP), were significantly reduced by persulfidation, 2 and 3.5 times, respectively (one-way ANOVA, p < 0.001), while for the Cys16 mutants no difference were observed with the treatments. Using circular dichroism (CD), we observed that the overall far-UV spectra of the proteins were similar, indicating that posttranslational modification of PtpA and mutants do not affect the secondary structure profile. Also, no difference in the thermal denaturation profile of PtpA and mutants were detected in the presence of NaSH.

CONCLUSION

Altogether, our preliminary results indicate that PtpA persulfidation may occur at Cys16 residue and that the posttranslational modification inhibits PtpA activity but does not decrease protein thermal stability.

Keywords: Bacillus of Koch, posttranslational modification, S-sulfhydration

Supported by: CAPES

08673 - Poster Session

DA.25 - Identification of novel substrates for the E3 ubiquitin-ligase SCF(Fbxl17) complex by protein microarrays.

Patrícia Maria Siqueira dos Passos ¹, Camila Correia¹, Tie Koide², Valentine Spagnol², Felipe Teixeira¹

¹Department of Genetics and Evolution, Federal University of São Carlos (São Paulo, Brazil), ²Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo (São Paulo, Brazil)

INTRODUCTION

The SCF(Fbxl17) complex is a human E3 ubiquitin-ligase responsible for substrate recognition and ubiquitination leading them to either proteasomal degradation or modulating its function. Fbxl17 protein has a LRR domain (Leucine Rich Repeat) for substrate interaction and a F-box domain to interact with SKP1 and assemble the complex. Previously, a CGH-array (Comparative Genomic Hybridization) was performed in 746 cancer cell lines, and it was found breaks in FBXL17 in 3 lines of breast cancer (BT-474, HCC38 and HCC1395) and in 1 line of esophageal/gastric cardia adenocarcinoma (OE-19). These breaks led to the generation of Fbxl17 containing the LRR domain truncated. Surprisingly, these truncations impaired Fbxl17 association with the other components of the SCF complex decreasing its activity. The consequences of LRRs truncation regarding substrate binding and ubiquitination were not investigated yet. Moreover, substrates identification for SCF(Fbxl17) remain poorly described.

OBJECTIVES

Identification of potential substrates and the effects of those truncations in substrate interaction and ubiquitination.

MATERIALS AND METHODS

We performed a large-scale in vitro ubiquitination assay using SCF(Fbxl17), SCF(Fbxl17-∆3LRR) complexes or Fbxl17-∆F-box purified from HEK293T cells and protoarrays as substrate source. Targets differentially ubiquitinated by the wild-type complex were selected through statistical analysis and submitted to validation assays.

DISCUSSION AND RESULTS

A total of 194 candidates were ubiquitinated by SCF(Fbxl17) and SCF(Fbxl17-∆3LRR), among them, 92 targets were positive only for the wild-type complex. Functional analysis identified that most targets were involved in RNA metabolism and cell cycle modulation. Several possible substrates were submitted to validation assays, and we obtained promising results with DDB1, a protein that is involved in DNA damage repair and protein ubiquitylation. According to our results, DDB1 interacts with Fbxl17 and is ubiquitinated by SCF(Fbxl17).

CONCLUSION

Mutation in the LRRs domain affect SCF(Fbxl17) activity. DDB1 interacts with Fbxl17 and is ubiquitinated by SCF(Fbxl17).

Keywords: DDB1, SCF(Fbxl17), Ubiquitination

Supported by: CAPES, FAPESP and CNPq

08583 - Poster Session

DA.26 - Biochemical characterization and structure elucidation of a new cytochrome P450 decarboxylase

Letícia Leandro Rade ¹, Amanda Silva de Sousa¹, Suman Das², Wesley Cardoso Generoso¹, Mayara Chagas de Ávila ¹, Plinio Vieira Salmazo¹, Gabriela Felix Persinoti¹, Antonio Bonomi¹, Mario Tyago Murakami¹, Thomas Michael Makris², Leticia Maria Zanphorlin¹

¹LNBR/CNPEM, Brazilian Center for Research in Energy and Materials (CNPEM) (SP, Brasil), ²Molecular and Structural Biochemistry,, North Carolina State University (NORTH CAROLINA, EUA)

INTRODUCTION

Alkenes have an economic appeal, especially in the biofuels field, since they are precursors for drop-in biofuels production, which have similar chemical and physical properties to the conventional fossil fuels, with no oxygen in their composition. After the discovery of the first P450 CYP152 OleT_JE in 2011, reported with its unique property of decarboxylating fatty acids (FA), by using hydrogen peroxide as a cofactor and producing 1-alkenes as the main product, the scientific and technological interest in this family of enzymes vastly increased.

OBJECTIVES

In this context, the present work presents a new decarboxylase (OleT_RN) with low similarity with OleT_JE (32%), its biochemical characterization, and the structure elucidation.

MATERIALS AND METHODS

DISCUSSION AND RESULTS

As main results, OleT_RN presented a high yield of expression and purity, optimum reaction conditions at 35 °C and pH from 6.5 to 8.0, and higher specificity for oleic acid. Besides that, structure-guided mutations were performed, and, according to the functional characterizations, it was observed that some mutations presented different specificity and chemoselectivity by varying the chain-length of FA substrates from 12 to 20 carbons. These results are extremely interesting from a biotechnological perspective as those characteristics could diversify the applications and contribute to designing better cytochrome P450 decarboxylases.

CONCLUSION

Considering that peroxygenases have the potential activity of decarboxylating and hydroxylating fatty acids and that the elucidation of the intriguing mechanistic involved in the decarboxylation preferential from OleT_JE is still a challenge, the elucidation of OleT_RN structure and the functional characterizations of OleT_RN and its mutants contribute with new information about CYP152. Besides that, the work also contributed to the discovery of a new decarboxylase with a different selectivity profile from OleT_JE, which allows a wide range of applications.

Keywords: P450, decarboxylases, alkenes

Supported by: FAPESP, SBBF

08374 - Poster Session

DA.27 - The crystal structure of the exonucleolytic domain of the ribonuclease RRP44 of Trypanosoma brucei reveals details of the catalytic mechanism

Giovanna Cesaro ^2,1, Pierre Legrand³, Ahmed Haouz⁴, Nilson Ivo Tonin Zanchin¹, Beatriz Gomes Guimaraes^1,2

¹ICC, Instituto Carlos Chagas (Paraná, Brasil), ²Programa de pós-graduação ciências-bioquímica, Universidade Federal do Paraná (Paraná, Brasil), ³SOLEIL, Synchrotron SOLEIL (, França), ⁴Instituto Pasteur, Instituto Pasteur (, França)

INTRODUCTION

Parasitic trypanosomatids, such as Trypanosoma brucei, T. cruzi and Leishmania sp. present unique features compared with other eukaryotes regarding RNA processing and maturation. For instance, T. brucei ribosomes contain specific rRNA expansions and the 60S subunit is composed of eight rRNAs molecules instead of the three rRNAs found in most eukaryotes. The role of specific endo- and exonucleases in the maturation of the unusual rRNA precursor of trypanosomatids remains largely unknown. One of the nucleases involved in rRNA processing is Rrp44, a member of RNase II/RNB exonuclease family and an exosome associated ribonuclease in yeast, which is involved in several metabolic RNA pathways.

OBJECTIVES

Here we investigate the structure of T. brucei RRP44 and its exonucleolytic domain.

MATERIALS AND METHODS

Recombinant full-length with mutations at the catalytic sites and a truncated form of TbRRP44 containing the RNB domain (TbRRP44_CSD1-S1) were overexpressed in Escherichia coli and purified by chromatography methods before co-crystallization assays with RNA oligonucleotides.

DISCUSSION AND RESULTS

The structures of TbRRP44_CSD1-S1 in the apo and holo forms were determined at 3.1 Å and 2.6 resolution respectively. The overall folding of the RNase II/RNB family is conserved in TbRRP44, however comparative analyses indicate a distinct path for the substrate entrance, comparing to the human orthologue. The crystallization of TbRRP44 in its active form revealed an intermediate state of the catalytic cycle, between substrate cleavage and product exit, pointing out the role of two conserved arginine residues in catalysis.

CONCLUSION

Our structural analysis of this essential T. brucei protein provides detailed information on the enzyme mechanism and reveals specific features that could be exploited for structure-based drug design.

Keywords: Trypanosoma brucei ribonuclease RRP44 , crystallographic structure, catalytic mechanism

Supported by: CNPq, CAPES, COFECUB, FIOCRUZ, Synchrotron SOLEIL

08513 - Poster Session

DA.28 - Synergistic Effects of Xylanase-Arabinofuranosidase Chimers Against Minimally Pre-treated Lignocellulosic Biomass

Matheus Quintana Barreto ¹, Lara Aparecida Buffoni de Campos Carneiro¹, Carolina Victal Garbelotti², Richard John Ward^1,2

¹Departamento de Bioquímica e Imunologia - FMRP, Universidade de São Paulo (São Paulo, Brasil), ²Departamento de Química - FFCLRP, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Lignocellulosic biomass is an abundant and renewable resource for the production of biofuels, which as an agroindustrial by-product does not compete against food crops. Arabinoxylan is a major constituint of the hemicellulose of cereals and grasses and is composed of a xylopyranoside backbone (β-1,4) substituted by α-L-arabinofuranosyl residues. The release of reducing sugars from arabinoxylan is performed by a series of glycosyl hydrolases, including the endo-β-1,4-xilanase (EC 3.2.1.8), which cleaves internal bonds in the xylan backbone, and arabinofuranosidase (EC 3.2.1.55), responsable for the release of the arabinose moiety.

OBJECTIVES

In this work insertional chimerogenesis between the B. subtilis GH11 endo-β-1,4-xilanase A (XynA, BSU18840) and the GH43 arabinoxylan arabinofuranohydrolase (AXH-m2,3 XynD, BSU18160) was used to create a bifunctional enzyme (AraXyl) for the degradation of arabinoxylan.

MATERIALS AND METHODS

The coding sequences of both enzymes and the chimera were cloned in pET28a, expressed in E. coli BL21 (DE3) Arctic Express and purified by metal affinity chromatrography. Biochemical characterization of both Araxyl and individual enzymes was assayed using wheat arabinoxylan and synthetic substrates.

DISCUSSION AND RESULTS

Biochemical characterization against synthetic substrates revealed that the xylanase and arabinofuranosidase KM values of the paretnal enzymes were generally lower than AraXyl. However a synergistic effect of equimolar mixtures of XynA and XynD and AraXyl against wheat arabinoxylan as compared to individual XynA and XynD was observed. Analysis of arabinose release measured by HPAEC-PAD, however, show that the chimeric enzyme possessed a 6-fold higher activity against sugar cane bagasse alchohol insoluble residue when compared to individual enzymes and XynA/XynD equimolar mixtures.

CONCLUSION

These results indicate that the arabinoxylan conformation in the context of the plant cell influences its susceptability to enzyme hydrolysis, and that protein engineering may be used to generate enzymes that are suitable for releasing sugars from minimally pre-treated lignocellulosic biomass.

Keywords: biomass, biofuels, enzymes

Supported by: CAPES, FAPESP, CNPq

08287 - Poster Session

DA.29 - Criteria for the Development of Structurally Functional Humanized Antibody Molecules

Isabella Gizzi Jiacomini ^1,2, Martina Beltramino¹, João Carlos Minozzo⁴, Juliana Ferreira de Moura¹, Philippe Billiald³, Nicolas Aubrey², Larissa Magalhães Alvarenga¹

¹Patologia Básica, Universidade Federal do Paraná (Paraná, Brazil), ²Sciences Pharmaceutiques, Université de Tours (, France), ³Sciences Pharmaceutiques, Université Paris-Saclay (, France), ⁴CPPI, Centro de Pesquisa de Imunobiológicos (Paraná, Brazil)

INTRODUCTION

Envenomation can be broadly defined as the result of the exposure to a venom or toxin caused by the bite or sting of a venomous animal. In this context, Loxosceles spider envenomation remains an important health concern worldwide, and of medical relevance, especially in the Americas. To this day, there is no established treatment for Loxoscelism, albeit horse polyclonal antivenoms are currently employed. Amongst all Loxosceles venoms components, the Phospholipases D are widely known as the main toxic constituents, reproducing most of the cytotoxic effects observed in Loxoscelism.

OBJECTIVES

Considering this, molecules able to bind these venoms components would be interesting suitors in the proposition of alternative, effective, specific, and safer treatments. In this context, we introduce LimAb7, a monoclonal antibody capable of binding and neutralizing Loxosceles intermedia PLDs and its derived recombinant antibody fragment counterparts. We have previously tried to humanize this antibody and produce it in the scFv and diabody formats, however our results indicated loss in fragment affinity, stability and neutralization leading us to infer that molecule size and format might be of importance to the maintenance of parental antibody features.

MATERIALS AND METHODS

Aiming to develop evolved, more structurally stable antibody fragments, we report for the first time the design of humanized antibody light and heavy V-domains produced and purified as Fab fragments (Fab’) and whole IgG1 molecules. 16 humanized Fab variants of LimAb7 were produced and screened as to their biophysical features.

DISCUSSION AND RESULTS

The best construct was considered our lead molecule, and thus produced in the IgG1 format. Improvements in the constructs were observed in humanized Fabs and IgG1 with respect to thermal and pH stability, production yield, as well as target binding affinity.

CONCLUSION

Overall, our findings shed light on the criteria to be considered when developing functional humanized antibody molecules.

Keywords: antibody humanization, recombinant antibodies, venoms

Supported by: CAPES-PRINT

08903 - Poster Session

DA.30 - Biophysical characterization of human 71 kDa heat shock cognate protein (HSPA8/hHsc70)

Noeli Soares Melo da Silva¹, Luiz Fernando de Camargo Rodrigues ¹, Paulo Roberto Dores-Silva³, Carlos Henrique Inacio Ramos ⁴, Leandro Ramos de Souza Barbosa^2,5, Júlio César Borges¹

¹Molecular Chemistry and Physics Department, Universidade de São Paulo (São Paulo, Brasil), ²General Physics Department, Universidade de São Paulo (São Paulo, Brasil), ³Department of Surgery School of Medicine, University of California San Diego (Califórnia, Estados Unidos), ⁴Organic Chemistry Department, Universidade Estadual de Campinas (São Paulo, Brasil), ⁵Brazilian Synchrotron Light Laboratory, Centro Nacional de Pesquisa em Energia e Materiais (São Paulo, Brasil)

Molecular chaperones are critical for cell proteostasis, with functions related to protein folding and refolding, trafficking and directing poorly folded proteins for degradation. 70 kDa heat shock proteins (Hsp70s) are a family of structurally conserved chaperones with different expression levels and subcellular locations. These proteins are composed of two domains connected by a hydrophobic linker: a nucleotide binding domain (NBD, responsible for ATP binding and hydrolysis) and a protein binding domain (PBD, which binds to client proteins), which can be in an open or closed conformation if bound to ATP or not. Human 71 kDa heat shock cognate protein (HSPA8, or hHsc70) is an essential cytosolic Hsp70 that, unlike other cytosolic Hsp70s, is not induced by heat shock and is present in other cellular functions, such as cell signalling and antigen processing. These features draw attention to this chaperone as a possible therapeutic target for pathologies such as neurodegenerative diseases and cancer. To better understand this chaperone’s structure, we’ve characterized recombinant HSPA8 by means of techniques such as circular dichroism (CD), instrinsic tryptophan fluorescence, small angle x-ray scattering (SAXS), isothermal titration calorimetry (ITC) and chaperone activity assays, which allowed probing its overall biophysical and biochemical features. Our studies have shown that HSPA8 was purified with high α-helical and β-sheet content, and having at least one Trp residue buried within its hydrophobic core. Chaperone activity and kinetics assays highlighted its specificities in ATP hydrolysis rate and aggregation prevention, while ITC has shown some differences regarding its interaction with nucleotides in comparison with other human Hsp70s. SAXS has allowed low resolution envelope calculation and flexibility studies, showing that HSPA8 is a slightly elongated monomer which seems to remain mostly in a compact state. We hope these results help in a better understanding and guiding future studies on the Hsp70 chaperone family. This work was supported by Conselho de Aperfeiçoamento de Pessoal de Nível Superior (PROEX – 0487, grant #88887.508861/2020-00), by CNPq (grants #471415/2013-8, #303129/2015-8, 420567/2016-0, #141986/2017-4 and #303262/2018-4) and by FAPESP (grants #2011/23110-0, #2012/50161-8, #2014/07206-6, #2014/16646-0, #2015/15822-1, #2016/22447-1, #2017/07335-9 and #2017/26131-5).

Keywords: Protein folding, Chaperones, Heat shock proteins

08356 - Poster Session

DA.31 - The double-edged influence of guanidinium chloride on protein aggregation

Lucrecia María Curto ¹, Martin Ezequiel Ballatore¹, José María Delfino¹

¹Dpto. de Química Biológica, FFYB, UBA, Instituto de Química y Fisicoquímica Biológicas (Buenos Aires, Argentina)

INTRODUCTION

Due to its ability to form micelle-like clusters, trifluoroethanol (TFE) promotes conformational changes that can trigger amyloid formation. Unlike classical denaturants, TFE favors protein aggregation mainly at moderate concentrations (~30% v/v). Accordingly, our protein model IFABP (intestinal fatty acid binding protein) shows amyloid-like aggregation above 15% v/v.

OBJECTIVES

As a straightforward correlation has been generally postulated between stability and amyloid propensity, our aim was to address the influence of sub-aggregating TFE concentrations on IFABP stability.

MATERIALS AND METHODS

Urea or guanidinium hydrochloride (GdmCl)-induced unfolding transitions were monitored by fluorescence and circular dichroism (CD) spectroscopy. Insights on the process arise from the comparison of the shape and intensity of the full set of CD spectra obtained from thermal ramps performed with a Chirascan V-100 (Applied Photophysics).

DISCUSSION AND RESULTS

With urea, IFABP can be assimilated to a 2-stage system and its stability remains unchanged. At variance, with GdmCl, the appearance of amyloid-like aggregation becomes more evident as TFE concentration increases. Temperature-induced denaturation profiles show that both additives diminish stability. Whereas a concomitant increase of amorphous aggregation occurs upon heating in the presence of TFE, no aggregation takes place with GdmCl. Conversely, when both additives are present, amyloid-like aggregation initiates immediately.

CONCLUSION

In summary, two aggregation pathways might happen: amorphous or amyloid-like. Upon heating, low TFE concentrations promote the first. If GdmCl is also present the second route takes over. The explanation must reconcile the effects of additives on both the protein and solvent structures. Briefly, TFE desolvates the protein, a process further reinforced by heat. Although GdmCl might prevent thermal aggregation by solubilizing non-native states, this same phenomenon could favor amyloid aggregation. On top of that, the electrolytic-induced segregation of TFE might contribute to the development/stabilization of TFE clusters that might reach a large enough size to act as nucleation-inducing interfaces, thus leading to the observed amyloid aggregation.

Keywords: aggregation, guanidinium, trifluoroethanol

Supported by: UBACYT, CONICET and ANPCYT

08620 - Poster Session

DA.32 - SARS-CoV-2 Orf9b-host protein-protein interaction: molecular and biophysical characterization

Kehinde S. Ayinde¹, Glaucia M.S. Pinheiro¹, Carlos H.I Ramos¹

¹INSTITUTE OF CHEMISTRY, UNIVERSITY OF CAMPINAS (São Paulo, Brazil)

The emergence of Covid-19 pandemic, caused by Severe Acute Respiratory Syndrome (Sars-Cov-2) remains a great threat to the global health despite the early development of vaccines. However, researchers have continued to intensify studies into understanding the molecular characteristics of the viral proteins during infection, replication and host immune evasion. Orf9b an important accessory protein of the Sars-Cov-2 virus is identified to play a critical role in the viral host interaction, targeting a member of the mitochondrial translocase of outer membrane complex, Tom70. This orf9b and Tom70 assembly is implicated in disrupting the mitochondrial antiviral signaling, leading to immune evasion. Here, we described the recombinant expression, purification, and characterization of orf9b using molecular and biophysical techniques. We expressed Orf9b singly and co-expressed the two proteins (Tom70-Orf9b) in an E. coli expression system, followed by purification by affinity and size exclusion chromatography. Structural characterization and protein-protein interaction studies was further accessed. The 97 amino acid protein purified as a homodimer with an approximate molecular weight of 22 kDa as determined by SEC-MALS. Folding and conformational studies using circular dichroism showed that the protein exhibit a random conformation and stable upon 90C. This conformation is believed to be altered when forming a complex with Tom70. Therefore, a series of pull down experiments are underway to characterize the Tom70-Orf9b complex. Our results may contribute to understand the role that Sars-Cov-2 orf9b protein has on virus infection.

Keywords: SAR-CoV-2, Orf9b, Tom70

Supported by: FAPESP, CAPES

08813 - Poster Session

DA.33 - Resolving the fine structure in the energy landscapes of repeat proteins

Vitor Barbanti Pereira Leite ¹, Rodrigo G. Parra², Rafael Viegas¹, Antonio B. Oliveira Jr³, Peter G. Wolynes³, Diego U Ferreiro³

¹IBILCE, Universidade Estadual Paulista (São Paulo , Brasil), ²Instituto de pesquisa científica, EMBL Heidelberg (Heidelberg, Germany), ³Center for Theoretical Biological Physics, Rice University (Houston, United States of America), ⁴Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (Buenos Aires, Argentina)

The Ankyrin (ANK) repeat is a recurrent tandem sequence motif that mediates protein-protein interactions in a diversity of biological functions. Ankyrin repeat-containing proteins have an elongated non-globular shape and a complex folding mechanism that has not been fully elucidated. In this work, we investigated the energy landscape of proteins consisting of 3, 4, and 6 ANK repeats using the Energy Landscape Visualization Method (ELViM).1 For that, we have used biased and unbiased coarse-grained AWSEM simulations to sample conformations along the folding pathway. Combining the ELViM structure-based phase space with energy information from simulations, it is possible to reconstruct a 3-dimensional energy surface, which is funneled towards the native structure. On this surface, it is possible to indicate the existence of on-pathway and off-pathway intermediate states, suggesting the most favorable folding routes. Our results show that folding of all proteins initiates forming internal repeats, whereas a terminal repeat undergoes an unfolding/refolding process along the pathway. Overall, the results are consistent with a cooperative folding, and its pathway may be inferred from the concise ELViM representation.v[1] A.B. Oliveira Jr. et al, J. Chem. Theory Comput., 15, 6482-6490 (2019).

Keywords: Protein folding, Protein folding, Protein folding

Supported by: FAPESP and CNPq

08955 - Poster Session

DA.34 - The stressed life of a lipid in the Zika Virus membrane

Martín Soñora ¹, Exequiel E. Barrera^1,2, Sergio Pantano¹

¹Biomolecular Simulations Group, Institut Pasteur de Montevideo (Mataojo 2020, CP 11400 Montevideo, Uruguay), ²Instituto de Histología y Embriología (IHEM), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Cuyo (CC56, Mendoza, Argentina)

Protein-lipid interactions modulate a plethora of physiopathologic processes and have been the subject of countless studies. However, this kind of interactions in the context of viral envelopes have remained relatively unexplored, partially because the intrinsically small dimensions of the molecular systems escape to the current resolution of experimental techniques. However, coarse-grained and multiscale simulations may fill that niche, providing nearly atomistic resolution at an affordable computational price. Here we used multiscale simulations to characterize the lipid-protein interactions in the envelope of the Zika Virus, a prominent member of the Flavivirus genus. Comparisons between the viral envelope and simpler molecular systems, indicate that the viral membrane is under extreme pressures and asymmetric forces. Furthermore, the dense net of protein-protein contacts established by the envelope proteins creates poorly solvated regions that destabilize the external leaflet leading to a decoupled dynamics between leaflets. These findings lead to the idea that the flaviviral membrane may store elastic energy, playing an active role in the membrane fusion process. On the other hand, we would like to show the developed an optimized pipeline for building and simulating enveloped virus-like particles (VLP). Detailed structural analysis of the protein envelope also shows very good agreement in root-mean-square deviations and B-factors with the experimental data. The level of details attained shows for the first time a possible role for anionic phospholipids in stabilizing the envelope. Combining an efficient and reliable setup procedure with an accurate coarse-grained force field provides a valuable pipeline for simulating arbitrary viral systems or subcellular compartments, paving the way toward whole-cell simulations.

Keywords: Zika Virus VLP Flavivirus, SIRAH force field, Coarse-grained mol dynamics simulation

08196 - Poster Session

DA.35 - Effect of C-terminal and N-terminal dimerization and alanine scan on antibacterial activity of the analogs of the peptide p-BthTX-I

Norival Alves Santos Filho ¹, Gabriela Marinho Righetto³, Julia Pinto Piccoli¹, Larissa Mathias Teizen Almeida¹, Thainá Cristina Leal¹, Ilana Lopes Baratella da Cunha Camargo³, Eduardo Maffud Cilli¹

¹Departamento de Bioquímica e Tecnologia Química, UNESP - Instituto de Química (Sao Paulo, Brasil), ²Física e Ciência Interdisciplinar, Instituto de Física de São Carlos, USP (São Paulo, Brasil)

INTRODUCTION

The study of relationship between peptide structure and antimicrobial activity has been mandatory on the search for novel antimicrobial agents. On previous study, peptide (pBthTX-I)2 and the analogue des-Lys12/Lys13-(p-BthTX-I)2 showed activity against bacteria, indicating a potential specificity uppon prokaryotic cells.

OBJECTIVES

The present study amis to analyze the antimicrobial and hemolytic activities of different dimeric analogs of p-BthTX-I. In addition, we evaluated the importance of each cationic and aromatic amino acid on the antimicrobial and hemolytic activities of des-Cys11,Lys12,Lys13-(p-BthTX-I)2K, the most active analog

MATERIALS AND METHODS

Solid phase peptide synthesis (SPPS) was performed manually using the Fmoc protocol. Minimal Inhibitory Concentration (MIC) was determined by the technique described in the Clinical & Laboratory Standards Institute: CLSI Guidelines.

DISCUSSION AND RESULTS

In this work, the peptide des-Cys11,Lys12,Lys13-(pBthTX-I)2K [(KKYRYHLKPF)2K] was synthesized and dimerization was performed with a Lys instead of Cys residue, beginning the synthesis with a Fmoc-Lys(Fmoc)-OH. This change was important to avoid Cys oxidation, decreasing one step on original peptide synthesis and obtaining a smaller peptide. This peptide showed antimicrobial activity similar or superior to (pBthTX-I)2. Additionally, in order to evaluate the impact of linker position on peptide dimerization, the peptide E(pBthTX-I)2 [E(KKYRYHLKPFCKK)2] was synthesized, using a Fmoc-Glu-OH in the end of the synthesis. The antibacterial activity of this N-terminal dimeric peptide was worse than the original peptide, showing that free N-terminal is important to antimicrobial activity of the (pBthTX-I)2. Additionally, the alanine scan was also performed. Alanine scanning is used in order to identify specific amino acid residues responsible for a peptide’s activity.Interestingly, removal of any single amino acid resulted in lower antimicrobial activity.

CONCLUSION

In this work, we demonstrated that the des-Cys11,Lys12,Lys13-(p-BthTX-I)2K analog, which is shorter and synthesized by an easier process leading to a more stable peptide, is the most antibacterial active peptide against multidrug-resistant bacteria.

Keywords: p-BthTX-I, Antimicrobial peptides, (pBthTX-I)2

08540 - Poster Session

DA.36 - Single Molecule Investigation of Conformation and Dynamics of Macromolecules

Nastaran Hadizadeh¹, Emanuel N. Lissek¹

¹N/A, LUMICKS (Massachusetts, United States)

INTRODUCTION

Studying how macromolecules fold correctly and undergo conformational changes is crucial to understand the underlying biological mechanism of how diseases arise. Single-molecule force spectroscopy represents an ideal tool to study these molecular phenomena because of its unique capability to isolate individual biomolecules and observe conformational transitions as they happen in real-time.

OBJECTIVES

Here, we introduce the high-resolution C-Trap® optical tweezers technology and its application in directly observing conformational changes of individual proteins by measuring their length with sub-nm precision.

MATERIALS AND METHODS

A mutant of AdK was designed as previously described, where cysteine residues were inserted and used for attaching DNA handles to the enzyme. DNA handles were biotin and digoxigenin modified at their respective ends, and coupled to beads surface functionalized with streptavidin and anti-digoxigenin to form a dumbbell configuration for the optical tweezers assay. The setup used was a high-resolution dual-beam optical trap to detect conformational dynamics of the enzyme at the nanoscale level and to determine and quantify the mechanism of action of small molecule inhibitors.

DISCUSSION AND RESULTS

To investigate the equilibrium dynamics of the conformational transitions of Adk, we monitored bead displacement under constant force. We observed that in the absence of kinase inhibitors, conformational transitions of AdK were undetectable as the force load applied stretches and locks AdK in an extended (open) state. However, in the presence of nanomolar concentrations of the AdK inhibitor, AP5A, the two extended and contracted states of AdK were observed over time in a distance range of 1.5-1.7 nm. This agrees with previously described crystal structures and data published.

CONCLUSION

Our data show high-resolution measurements of protein conformational dynamics at the nanoscale level using the C-Trap. Importantly, this feature can be used to determine the mechanism of action of candidate drugs on enzymatic activity relating to conformational changes of the enzymes.

Keywords: Dynamic Single Molecule, Protein/Enzyme Dynamics, Optical Tweezers Technology

08271 - Poster Session

DA.37 - Molecular docking studies of the interaction of the Rose Bengal dye with human serum albumin

Mauricio Ikeda Yoguim ¹, Ignez Caracelli², Valdecir Farias Ximenes¹, Aguinaldo Robinson de Souza¹

¹Química, Universidade Estadual Paulista Júlio de Mesquita Filho (São Paulo, Brasil), ²Física, Universidade Federal de São Carlos (São Paulo, Brasil)

INTRODUCTION

The Human Serum Albumin (HSA) protein is a transport protein. Thus there are several possible binding sites for complex formation. The interaction between biological macromolecules (proteins and DNA) with drugs and/or other ligands is one of the research areas of great importance in the field of life sciences.

OBJECTIVES

These studies allow us to understand the formation of complexes such as: the proposition of interaction and reaction mechanisms, and molecular level recognition studies of binding sites. In the study carried out using molecular docking to form complexes between the (HSA) and the rose bengal dye (RB), crystallographic structures of HSA-ligands complexes, FA free, obtained from the PDB (Protein Data Bank) were considered.

MATERIALS AND METHODS

The selected complexes in the PDB were pdb code 2BXE (HSA-diflunisal) and 2BXF (HSA-azapropazone). With the two crystallographic structures, it was possible to investigate whether RB can accommodate the DS1 and DS2 (Sudlow's nomenclature), FA1 and FA6 sites. Docking was performed with the Gold 2020.20 Program, using the crystallographic ligand as the center of the calculations, adding a radius of 6 Å and the GoldScore adjustment function. As a result, poses (orientation, conformation and position) of the RB were obtained at the sites indicated and selected by the output pattern.

DISCUSSION AND RESULTS

The results showed that most HSA sites studied can be occupied by RB. DS1 Site: Lys199:HZ1-RB:O3 2.21 Å, Glu292:OE2–RB:H1 1.86 Å; His242–RB:Cl33 2.79 Å, Ala292:HB–RB 2.28 Å. FA1 site: Arg117:HH12–RB:O4 2.11 Å; Tyr138:HH–RB:I4 2.48 Å, Arg145:HD1–RB:Cl33 2.50 Å, Arg186:O–RB:Cl36 3.15 Å, Ser192:HB2–RB:I3 3.01 Å. FA6 site: Arg209:HH11–RB:I2 2.40 Å, Lys351:HZ1–RB:O1 1.58 Å, Asp324:OD2–RB:H49 1.97 Å. More possibilities are still under investigation.

CONCLUSION

At least 3 HSA sites as described above. More possibilities are still under investigation.

Keywords: docking molecular, human serum albumin protein, rose bengal

08893 - Poster Session

DA.38 - Structural prediction of proteins related to lesions in the cerebral cortex

Aline Maia Alves ¹, Viviane Gomes da Silva², Arthur Giraldi-Guimarães², Manuela Leal da Silva¹

¹Bioinformatic, Institute of Biodiversity and Sustainability, Federal University of Rio de Janeiro (RJ, Brasil), ²Biotechnology Laboratory, Center for Biosciences and Biotechnology, North Fluminense State University Darcy Ribeiro (RJ, Brasil)

INTRODUCTION

Cellular and molecular alterations resulting from focal lesions cause distinct effects on behavior, tissue response and neuroplasticity.

OBJECTIVES

The objective of this work is to analyze proteins from the perilesional cortical region seven days post-ischemia in the primary sensorimotor cortex, to search for new molecular targets in ischemic lesions.

MATERIALS AND METHODS

The methodology included the creation of a multifast file for proteins with increased expression (UP) and for decreased expression (DOWN) compared to the control group of proteins - with normal expression level - containing 184 proteins in the UP group and 191 proteins in the DOWN group. Both groups were analyzed using Uniprot and Gene Ontology (cellular component, molecular function and biological process). The MHOLline program was used to build three-dimensional protein models, with identity and coverage validation; RMSD between template and model and Ramachandran plot. Behavioral, tissue and neuroplasticity groups were selected: antioxidant activity, cellular anatomical entity, immune system process, behavior and response to stimuli.

DISCUSSION AND RESULTS

Of the 184 UP proteins analyzed, 172 had defined cellular functions and components and 163 biological processes were identified. 7 proteins were classified with antioxidant activity with 3 constructed structures, 167 proteins related to the cellular anatomical entity and 37 structures, 28 proteins in the immune system process (2 structures), 9 proteins in behavior (1 structure ) and in response to stimuli 69 proteins and 27 structures. In DOWN the results showed that of the 191 proteins analyzed, 166 had defined molecular functions, 171 cell components and 170 had identified biological processes. It was possible to generate 153 models for UP and 152 models for DOWN, with 69 models excluded in UP and 74 in DOWN according to the validation criteria.

CONCLUSION

It was possible to build 3D models for 37 UP proteins and 24 DOWN proteins in selected groups. Further analysis needs to be performed to relate these results to ischemia.

Keywords: structural prediction, cerebral cortex, ischemia

08045 - Poster Session

DA.39 - Comparative study between different techniques to obtain affinity between biomolecules

Hamine Cristina de Oliveira ¹, Thiago Revers Dreyer¹, Marcos Roberto de Mattos Fontes¹

¹Departamento de Biofísica e Farmacologia, Instituto de Biociências de Botucatu, Universidade Estadual Paulista (São Paulo, Brasil)

INTRODUCTION

Biophysical methods have been increasingly used as complementary approaches to traditional ones to increase the knowledge of molecular mechanisms. Techniques applied to the study of protein/peptide or protein/protein interaction provide important information to understand the binding process. There are several techniques that theoretically or experimentally predict the affinity between two or more molecules.

OBJECTIVES

The comparison between affinities values obtained by different physical principles need to be used with criterion because these values are not numerically equal, and the objective is to understand the specificities and similarities of one technique in relation to the other.

MATERIALS AND METHODS

In this work, isothermal titration calorimetry (ITC), fluorescence spectroscopy, molecular docking and molecular dynamics simulation were used to study the affinity of nuclear transport protein importin-alpha and nuclear localization sequences (NLS) from cargo proteins.

DISCUSSION AND RESULTS

The experimental techniques studied allowed to calculate similar values in order of magnitude, which indicated the accuracy of the proposed methodology for fluorescence spectroscopy. The computational techniques, on the other hand, differed considerably from the ITC, used as an experimental reference value, as docking calculates affinity from the best orientation of the peptide in the protein and molecular dynamics considers the movement of the peptide in the complex, these dissociation constant values are a little overrated.

CONCLUSION

In conclusion, despite the difficulty of comparing results obtained by different techniques, it is possible, knowing each calculation principle, to relate these results and choose the most adequate for your study, as experimental techniques have their limitations, the use of computational models is very welcome in the understanding of protein/peptide binding.

Keywords: affinity assays, isothermal titration calorimetry, fluorescence spectroscopy

Supported by: FAPESP, CNPq and CAPES

08585 - Poster Session

DA.40 - Three-Finger Toxins from Brazilian Coral Snakes: From Molecular Framework to Insights in Biological Function

Jessica Matos Kleiz Ferreira ¹, Nuria Cirauqui², Edson Araujo Trajano¹, Marcius da Silva Almeida¹, Russolina Benedeta Zingali¹

¹Instituto de Bioquímica Medica Leopoldo de Meis (IBqM), Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil), ²Faculdade de Farmácia, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Brazilian Micrurus venoms are mostly predominant in Three-finger toxins (3FTx), which belong to a family of small non-enzymatic proteins constituted by 58 to 90 amino acid residues. In all members of the family, the protein fold is based on three loops of beta strands that reassemble “fingers" extending to a globular and hydrophobic core, stabilized by four conserved disulfide bonds. Studies on 3FTxs around the world are showing the amazing diversity in these proteins both in structure and function. In Brazil, we have not realized the broad variety of their amino acid sequences and probable diversified structures and targets.

OBJECTIVES

In this context, this work aims to conduct an in silico systematic study on available 3FTxs found in Micrurus species from Brazil.

MATERIALS AND METHODS

We elaborated a specific guideline for this toxin family. First, we grouped them according to their structural homologue predicted by HHPred server and further curated manually. For each group, we selected one sequence and constructed a representative structural model. By looking at conserved features and comparing with the information available in the literature for this toxin family, we managed to point to potential biological functions. In parallel, the phylogenetic relationship was estimated for our database by maximum likelihood analyses and a phylogenetic tree was constructed including the homologous 3FTx previously characterized.

DISCUSSION AND RESULTS

Our results highlighted an astonishing diversity inside this family of toxins, showing some groups with expected functional similarities to known 3FTxs, and pointing out others with potential novel roles and perhaps structures.

CONCLUSION

Moreover, this classification guideline may be useful to aid future studies on these abundant toxins.

Keywords: Three-finger toxins, Sequence variability, Structure-function classification

Supported by: Faperj, Capes, CNPq, Inbeb

08718 - Poster Session

DA.41 - DNA translocase repositions a nucleosome by the lane-switch mechanism

Fritz Nagae ¹, Giovanni Brandani¹, Shoji Takada¹, Tsuyoshi Terakawa¹

¹Biophysics, Kyoto University (, Japan)

INTRODUCTION

Nucleosome is the basic unit of chromatin and contributes to package eukaryotic genome DNA into a nucleus. During DNA transactions such as DNA replication, transcription, and DNA repair, various translocases (e.g., DNA/RNA polymerases, replicative helicases, and exonucleases) unidirectionally move along the genomic DNA. Therefore, these translocases inevitably collide with the nucleosomes. In previous studies, single-molecule experiments have shown that a translocase causes downstream nucleosome repositioning after the collision. However, the molecular mechanism of this repositioning has not been identified.

OBJECTIVES

This study proposes the molecular mechanism of nucleosome repositioning by a DNA translocase.

MATERIALS AND METHODS

In this study, we performed molecular dynamics simulations to observe the molecular details of the collision between a translocase and a nucleosome. We also verified the lane-switch mechanism by restriction enzyme digestion assays and deep sequencing assays.

DISCUSSION AND RESULTS

From these simulation results, we proposed the lane-switch mechanism in which a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding lane to vacant one, and the downstream DNA rewraps, completing the downstream repositioning. The restriction enzyme assays show that T7 RNAP causes downstream repositioning of a nucleosome after unwrapping nucleosomal DNA up to the site proximal to the dyad. The deep sequencing assays show that the repositioning distance is ~100 base-pairs, which is consistent with the estimation from the simulations.

CONCLUSION

The molecular mechanism shown here may improve the understanding of the DNA transactions on nucleosomal DNA.

Keywords: Nucleosome, Molecular dynamics simulation, Biochemical experiment

08698 - Poster Session

DA.42 - Plasma Membrane Calcium ATPase (PMCA) activity is modulated by the biophysical properties of the surrounding bilayer

Marilina de Sautu ¹, Gustavo Scanavachi², Mariela Soledad Ferreira Gomes¹, Juan Pablo Rossi¹, Rosangela Itri², Irene Cecilia Mangialavori¹

¹Departamento de Química Biológica, Cátedra de Fisicoquímica Biológica, Universidad de Buenos Aires, Instituto de Química y Fisicoquímica Biológicas (Buenos Aires, Argentina), ²Departamento de Física Aplicada, Laboratorio de Cristalografia, Instituto de Física, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

PMCA is a P-ATPase involved in the regulation of the cell calcium homeostasis transporting Ca ²⁺ from cytoplasm towards the extracellular medium. PMCA like other integral membrane proteins operates surrounded by a complex and dynamic lipid bilayer, and its activity largely depends on the lipids. Aluminium (Al ³⁺ and other soluble species) is environmentally ubiquitous, providing human exposure and neurotoxic effects in humans and animals. The mechanisms proposed to explain aluminium toxicity are linked to changes in the cellular calcium homeostasis. In previous works, we demonstrated that aluminium inhibits PMCA activity preventing the dephosphorylation of the pump.

OBJECTIVES

The aim of this work is to understand the effect of the surrounding bilayer on the PMCA activity and on the effect of the aluminium. Aluminium would have distinct effect depending on the lipid composition of the cell membrane where the PMCA is located.

MATERIALS AND METHODS

To characterize this effect, mixed micelles of phospholipids and detergent (C₁₂E₁₀) were formed at different molar fractions, and we measured how PMCA activity varied. To evaluate other biophysical changes in the lipid bilayers we performed small Angle X-ray scattering (SAXS) experiments for studying how the lipidic environment was changing alongside different molar fractions and also in the presence of aluminium. In turn, we evaluate changes in the membrane phase properties using two fluorescent probes, laurdan and merocyanine 540.

DISCUSSION AND RESULTS

The results show a biphasic effect of activation and inhibition of the pump that depended on the molar fraction of phospholipids. SAXS measurements indicate that biophysical changes of the bilayer at different molar fractions could explain the difference in enzymatic activity. Moreover, we demonstrated how aluminium interacts with the micelles and influences the biophysical properties that in turn, affected the PMCA activity.

CONCLUSION

PMCA activity largely depends on the biophysical properties of the surrounding bilayer that affects the activity and also the aluminium effect on the pump.

Keywords: ATPase, bilayer, SAXS

Supported by: ANPCYT PICT 2014 0065, CONICET PIP 0250 and Universidad de Buenos Aires: 20020130100254B, LNLS, CAPES, CNPq, FAPESP.

08057 - Poster Session

DA.43 - EfTenA: a protein involved in thiamine biosynthesis in Enterococcus faecalis

Raissa Ferreira Gutierrez ¹, Carsten Wrenger², Alessandro Silva Nascimento¹

¹Sao Carlos Institute of Physics, University of Sao Paulo (São Paulo, Brazil), ²Biomedical Sciences Institute, University of Sao Paulo (Sao Paulo, Brazil)

INTRODUCTION

Bacterial resistance to antibiotics in therapeutic usage is a worldwide concern. The World Health Organization highlights, among other efforts, the urgency of finding new compounds to tackle multidrug resistance. Thiamine biosynthetic pathway (TBS) has been proposed as a source of drug targets for human pathogens, as this could be a pathogen-specific pathway and is absent in humans. TenA is a bifunctional enzyme with aminohydrolase and thiaminase II activity involved in the thiamine salvage pathway; however, the physiological function of this enzyme is not entirely clear.

OBJECTIVES

Here, we aim to structurally characterize and investigate the function of the TenA enzyme from Enterococcus faecalis (EfTenA). E. faecalis is related to healthcare-associated infections, and it is a relevant human pathogen within the context of antibiotic resistance.

MATERIALS AND METHODS

The recombinant protein was overexpressed in E. coli, purified by affinity and size-exclusion chromatography steps, and submitted to biophysical, biochemical, and structural experiments.

DISCUSSION AND RESULTS

Differential scanning fluorimetry assays showed a binding affinity of EfTenA to thiamine > thiamine monophosphate (TMP) > and thiamine pyrophosphate (TPP). The thiaminase II activity, measured by the thiocrome colorimetric method, revealed that EfTenA can hydrolyze thiamine and TMP but is inactive on TPP. After crystallization screening, crystals were submitted to an X-ray diffraction experiment. Two crystallographic structures were solved by molecular replacement. The first one shows a molecule of imidazole bound to the active site of ETenA, while the second structure shows an HMP ring (4-amino-5-hydroxymethyl-pyrimidine) bound to the active site. The HMP ring is the catalysis product of a TMP molecule by EfTenA crystals.

CONCLUSION

Taken together, these results show that the tolerance of the active site decreases with the addition of phosphate groups to the thiamine molecule. As it is accepted that TMP is not a substrate for TenA, our findings can help to understand the TenA role in the TBS pathway.

Keywords: Bacterial resistance, Thiamine biosynthetic pathway, Enterococcus faecalis

Supported by: CAPES and FAPESP

08490 - Poster Session

DA.44 - Trypanosoma evansi interactome with mouse hippocampal neuronal cell (HT22)

Sandra Regina de Mello ¹, Gabriella Bassi das Neves¹, Julia Marques¹, Marcelo Farina², Glorister Alves Alte², Luiz Claudio Miletti¹

¹Produção Animal e Alimentos, Universidade do Estado de Santa Catarina (SANTA CATARINA, Brasil), ²Bioquímica, Universidade Federal de Santa Catarina (SANTA CATARINA, Brasil)

INTRODUCTION

Trypanosoma evansi is the agent of "surra," a trypanosomosis endemic in many areas worldwide being responsible for a major economic problem in rural regions due to the loss of animals.T. evansi has the ability to migrate to other tissues, especially the Central Nervous System (CNS). Few studies have been carried about the interactions between the parasite and the CNS, despite the knowledge of some alterations resulting from this possible contact.

OBJECTIVES

This work consists of identifying which proteins secreted by T. evansimediate the interaction with mouse hippocampal neurons cells (HT22) and may be related to the invasion of the parasite in the CNS.

MATERIALS AND METHODS

T.evansi soluble protein extract at concentrations of 100μg/mL and 200 μg/ml of were incubated with cells of HT22 strains in DMEM 10% FBS culture medium, 100 U/ml penicillin and 100 mg/ml streptomycin, and kept in a 5% CO₂ incubator at 37 ^° C for one, three and five hours. Then, protein extraction was performed from co-culture cells subjected to ten cycles of freezing and thawing at -80 ^° C and 37 °C, and sonicated in an ice bath. Soluble protein supernatant was subjected to SDS-PAGE. Western blot will be used to analyze the soluble T. evansi, proteins that bind to HT22 cells.

DISCUSSION AND RESULTS

CONCLUSION

The present work will be able to elucidate possible interaction mechanisms between the parasites and the neuronal cells.

Keywords: Trypanosoma evansi, cell interaction, CNS

08820 - Poster Session

DA.45 - Search for new therapeutic targets for the treatment of tegumentary leishmaniasis through in silico functional prediction of Leishmania braziliensis proteins

Areza Caputo ^1,2

¹Biologia computacional , Fundação Oswaldo Cruz (Rio de Janeiro, Brasil), ²Biologia computacional , Instituto de Biodiversidade e Sustentabilidade (Rio de Janeiro, Brasil)

INTRODUCTION

Tegumentary leishmaniasis (TL) is a neglected parasitic disease whose main agent is Leishmania braziliensis, it is estimated that 1 million people are diagnosed with the disease annually. Currently, the pharmacological guideline for the treatment of TL is highly toxic and with reports of acquired resistance (monotherapy with meglumine antimoniate).

OBJECTIVES

Through the analyzes of the L. braziliensis genome available at the NCBI (Genbank: GCA_900537975.1) we will suggest new targets for drug repositioning through functional prediction.

MATERIALS AND METHODS

Using the 3,373 proteins with unknown function present in the genome of L. braziliensis and the databases, Uniprot, Psipred, SignalP, Psort, Pfam, CDD, SMART and STRING, a functional prediction based on sequence was made. Using the BATS and FILTERS filters of the MHOLline software, 3D models for these proteins were generated and selected. The selection of 3D models was based on identity (>25%) and coverage (>70%) values ​​and using FastSCOP, SCOPe and CATH databases reached a consensus on the prediction of function of these proteins.

DISCUSSION AND RESULTS

In the classification obtained by BATS, 8 proteins were selected: SYZ66351, SYZ69098, SYZ64647, SYZ69127, SYZ63617, SYZ66198, SYZ66915 and SYZ67025. After the analysis of metabolic pathways, we followed with the protein SYZ64647 with predicted function of UDP-Galactopyranose that is present in the parasite, but not in humans. Using the programs AutoDock Vina, AutoDock Tools and the 3D model UDP-Galactopyranose (PDBid template: 4DSG) the GRID was parameterized based on the amino acids that interact with the ligand (center x: 63.988 y: -9.335 z: -43.684 size x/y/z: 30) and exhaustive tests (8, 16, 64, 100). With the parameters validated, the virtual screening was carried out with the World database, reaching 10 promising substances.

CONCLUSION

With the studies carried out with the SYZ64647 protein, we saw this as a promising target for the repositioning of drugs for the treatment of tegumentary leishmaniasis.

Keywords: Comparative Modeling, Leishmaniasis, Leishmania braziliensis

Supported by: CNPq, CAPES and IOC

08852 - Poster Session

DA.46 - Characterization of Photo-induced Oxidative Modifications of Parkinson´s Disease Related Protein α-Synuclein

Ezequiel Giménez ¹, G.F. Cavazzutti¹, A.M. Toscani¹, Thomas M. Jovin², Henning Urlaub³, Lisandro Jorge FALOMIR LOCKHART¹

¹Laboratorio de Neuroquímica y Biofísica de Enfermedades Neurodegenerativas, INIBIOLP-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de La Plata (La Plata, Argentina), ²Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry (Göttingen, Germany), ³Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry (Göttingen, Germany)

INTRODUCTION and Objectives: A gain of toxic function of the protein alpha-Synuclein (aSyn) has been largely linked to the development and progress of Parkinson’s Disease (PD) and related Synucleinopathies. However, the identity of aSyn toxic species remains elusive. In this way, several post-translational modifications (PTMs) that derived from oxidative stress could be possible modulators of the physiopathology of aSyn in neurons. In this work we investigated the susceptibility of aSyn to be modified when this protein adopts different conformations, allowing us to identify potential pathological modifications. Materials and Methods: Recombinantly expressed aSyn was modified employing optimized photochemical methods that uses Rutheniun (II) Tris(bipyridine) as photosensitizer to produce either Tyrosine (Tyr) crosslinking or, in the presence of NaNO2, nitration. The reactions were characterized and quantified by Fluorescence emission, UV-Vis absorption and Mass Spectrometry. Results and Conclusions: Three conditions were analyzed: disordered aSyn free in solution, bound to membranes (helix-rich structure) and fibril conformation (cross β-sheets). Tyrosine crosslinking of disordered aSyn mainly involved Tyr39 and C-term residues Tyr133 or Tyr136. On the other hand, Tyrosine nitration were identified in all Tyr residues, being Tyr125 the most nitrated during photo-reaction. When bound to negatively charged SUVs, modification of Tyr39 is highly restricted. Similar outcome was found in the fibrillary form. Nitration of C-term residues is only reduced when aSyn interacts with membranes with a negative net charged, not in the unstructured C-term in fibrils. In conclusion, particular modifications, such Tyr39 nitration or Tyr39-Tyr133/136 crosslinking, could only emerge in pathological conditions and their identification in complex biological samples could be employed for the development of innovative tools for the early diagnosis of Synucleopathies.

Keywords: alpha-Synclein, Oxidative Stress, Post-translational Modifications

Supported by: CONICET (Argentina), ANPCyT (Argentina), UNLP (Argentina), Bunge & Born, Max Planck Society and Williams Foundations (Argentina-Germany), DFG (Germany)

08192 - Poster Session

DA.47 - Identification and prediction of HbAg d-chain sequence.

Isis Sebastião ¹, Emerson de Souza Santos³, Leandro de Mattos Pereira⁴, Patrícia Gleydes Morgante¹, Patrícia Soares Santiago^1,2

¹Campus de Registro, UNESP, Campus de Registro (São Paulo, Brasil), ²IQ, Instituto de Química, UNESP - Araraquara (São Paulo, Brasil), ³Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas da USP de Ribeirão Preto (São Paulo, Brasil), ⁴Laboratório de Ecologia Microbiana e Molecular, Instituto de Microbiologia Paulo de Góez, UFRJ (Rio de Janeiro, Brasil)

INTRODUCTION

The giant hemoglobins from annelids have particular biotechnological characteristics. Sequence data from Amynthas gracilis hemoglobin are scarce and the improvement of the knowledge about what sequences are associated to the protein structure is important to biotechnological studies.

OBJECTIVES

The aim of this work was the obtaining of the transcriptome of the earthworm species A. gracilis to identify the coding sequence of one of the Hb subunits, the d-chain.

MATERIALS AND METHODS

A cDNA library was constructed from mRNAs extracted from adult individuals of A. gracilis. The sequencing was carried out on an Illumina MiSeq device, generating 2x300bp paired-end reads. The assembly of the transcriptome was performed using the Trinity 2.0.6 software and the prediction of the open reading frame (ORF) was made with the TransDecoder. The identification of the HbAg d-chain was performed using BLAST against the sequences deposited in the UniprotKB database considering a minimum e-value of 1.0e-20.

DISCUSSION AND RESULTS

: Around 68% of the obtained data showed a phred quality score ≥30, with 25,644,392 reads. The assembly of the transcriptome generated a N50 of 883 bp, the lengh average of the contigs was 626.24 bp and the total of bases assembled was 681845025. After BLAST analysis, six sequences homologous to other annelid species were found, more specifically, sequences homologous to the d-chains of Lumbricus terrestris and Glossoscolex paulistus.

CONCLUSION

A potential ORF corresponding to the d-chain of HbAg was found from the transcriptome obtained.

Keywords: transcriptome, giant proteins, earthworms

Supported by: Fapesp, CNPq

08076 - Poster Session

DA.48 - Architecture and dynamics of the yeast aquaglyceroporin Fps1 in response to osmotic and oxidative stress

Sviatlana Shashkova ^1,2,3, Mikael Andersson⁴, Stefan Hohmann⁵, Mark Leake^1,2

¹Department of Physics, University of York (, United Kingdom), ²Department of Biology, University of York (, United Kingdom), ³Department of Microbiology and Immunology, University of Gothenburg (, Sweden), ⁴Department of Chemistry and Molecular Biology, University of Gothenburg (, Sweden), ⁵Department of Biology and Biological Engineering, Chalmers University of Technology (, Sweden)

INTRODUCTION

Membrane proteins play key roles at the interface between the cell and its environment by mediating selective import and export of molecules via plasma membrane channels. The Fps1 protein is the principal membrane aquaglyceroporin of the yeast Saccharomyces cerevisiae with its primary purpose to regulate intracellular pressure via facilitating glycerol efflux. Fps1 belongs to the Major Intrinsic Proteins superfamily, which is present in the vast majority of organisms from bacteria to plants and humans, fulfilling life essential tasks such as water content regulation and cellular signalling.

OBJECTIVES

Despite a multitude of studies on these transmembrane channels, understanding of their dynamics directly within living systems is limited. To address this, we correlated molecular scale information from living cells with real time changes to their microenvironment.

MATERIALS AND METHODS

We combined traditional molecular biology approaches with the Slimfield microscopy, a powerful new technology which enables detection of biomolecules directly in living cells with millisecond sampling, to track labelled molecules of interest in real time. We use the Fps1 aquaglyceroporin as an exemplar membrane protein to dissect and correlate its stoichiometry and molecular turnover kinetics with various extracellular conditions.

DISCUSSION AND RESULTS

We show that Fps1 resides in multi-tetrameric clusters and is also present as an intracellular pool, while hyperosmotic conditions cause Fps1 reorganization. We also demonstrate that rapid exposure to oxidative stress causes Fps1 assembly into larger foci which are further subjected to hydrogen peroxide-induced degradation and may thus be a regulatory step.

CONCLUSION

Hence, we provide novel insights into understanding of cellular adaptation to the microenvironment through characterisation of the plasma membrane channels.

Keywords: single-molecule, plasma membrane channels, cell stress

Supported by: Adlerbertska forskningsstiftelsen, the Biological Physical Science institute (BPSI), the Royal Society Newton International Fellowship, EPSRC, BBSRC, Marie Curie EU FP7 ITN, and the Leverhulme Trust.

08318 - Poster Session

DA.49 - PROJECT: Membrane interaction of the S. cerevisiae Golgi Reassembly and Stacking Protein

Carolina Gimenes Oliveira ¹, Mariana L. S. Gil¹, N. A. Fontana¹, Antônio J. Costa – Filho¹

¹Physics Department, University of São Paulo (São Paulo, Brazil)

INTRODUCTION

The Golgi reassembly and stacking proteins (GRASPs) were initially identified by in vitro experiments as one of the main factors necessary for stacking the Golgi cisternae in mammalian cells. Over the years, data about GRASPs have reported their participation in many other cellular processes and the additional involvement of members of the GRASP family in unconventional protein secretion pathways. Although we already have some knowledge on their structure and function, such as their presence in membrane of organelles and vesicles that participate in the early and late secretory pathway, no data on how this interaction occurs have been reported this far.

OBJECTIVES

This was the main objective of this project. To understand more about how this protein can anchor in the membrane of organelles, the yeast Saccharomyces cerevisiae, a model organism, was used in this work. This budding yeast has a single homolog of GRASP65, which is called Grh1, and has already been reported in the early secretory pathway. Grh1 interaction with membranes are thought to involve the acetylation of its N-terminal.

MATERIALS AND METHODS

We used the heterologous expression of Grh1 to produce samples destined for fluorescence and circular dichroism experiments so as to unravel the molecular determinants of Grh1-model membrane interactions.

DISCUSSION AND RESULTS

CONCLUSION

Keywords: GRASP65/55, Membrane, protein

Supported by: CAPES

08891 - Poster Session

DA.50 - Exploring novel environments for effective searching of unique xylose isomerases to lignocellulosic materials fermentation

Renan Yuji Miyamoto ^1,2, Ricardo Rodrigues de Melo¹, Douglas Antonio Alvaredo Paixao¹, Gabriela Felix Persinoti¹, Roberto Ruller³, Leticia Maria Zanphorlin¹

¹Brazilian Biorenewables National Laboratory, Brazilian Center for Research in Energy and Materials (Sao Paulo, Brazil), ²Faculty of Pharmaceutical Sciences, State University of Campinas (Sao Paulo, Brazil), ³Institute of Bioscience, Federal University of Mato Grosso do Sul (MS, Brazil)

INTRODUCTION

Xylose is regarded as the second-most abundant sugar in nature. Thus its usage is crucial for the development of a sustainable biobased economy. Xylose isomerases (XI) catalyze the first reaction of xylose metabolism, enabling downstream metabolic steps to convert xylose into a value-added bioproduct. However XIs suffer from low activity at fermentation conditions since most XIs present optimum thermophilic and alkaliphilic range conditions.

OBJECTIVES

Therefore in this work, we explored a metagenomics approach to identify novel XIs from the two unique environments, the mangrove region and Antarctica soil, searching for higher activity at milder conditions.

MATERIALS AND METHODS

To set our targets, we performed a sequence-similarity network (SSN) which indicated three potential XIs (ORF1, ORF3 from mangrove, and AraXI from Antarctica soil). We performed an in-depth biochemical and molecular characterization, from cloning to detailed biochemical characterization.

DISCUSSION AND RESULTS

Our results show a thermophilic behavior even at XI prospected from Antarctica. Although the targets in this study showed higher susceptibility to inhibitors, the ORF3 presented better catalytic properties at fermentation conditions than the control XI. Because XIs are metalloproteins, we studied a range of different conditions biochemically and biophysically, accessing the thermal stabilization through Differential Scanning Fluorimetry. Interestingly, the ion which maximized the activity (Mg ²⁺ ) generated the lowest thermal stabilization. To understand the molecular aspects underpinning these properties, we investigated their high- and low- resolution structures. Small Angle X-ray Scattering experiments revealed XIs are found as homotetramers in solution, and the crystallographic structures are in the refinement process.

CONCLUSION

Our findings suggest that metagenomics approaches exploring different environments gave useful information about the thermostability of XIs; their activities and stabilities are fine-tuned by several reaction conditions; and structural information could give important insights for protein engineering.

Keywords: Biorefinery, Lignocellulose, Xylose

Supported by: FAPESP

08968 - Poster Session

DA.51 - Production, purification, characterization and biotechnological application of endoglucanase isolated from Pycnosporus sanguineus

Dávida Maria Ribeiro Cardoso dos Santos ¹, F. Albuquerque², N.A. Ferreira², S.S. Machado², J.M. Rodrigues², T.P. Silva³, Hugo Juarez Vieira Pereira²

¹Instituto de Ciências Farmacêutica, Universidade Federal de Alagoas (Alagoas, Brasil), ²Instituto de Química e Biotecnologia, Universidade Federal de Alagoas (Alagoas, Brasil), ³Colegiado do Curso de Farmácia, Faculdade Independente do Nordeste-FAINOR (Vitória da Conquista, Bahia)

The increase in the production of agro-industrial residues has required alternatives to minimize the global problem. Clean alternatives have been developed, including the use of agro-industrial residues as a source for fungal growth and production of enzymes of biotechnological interest. The objective of this work was the production, isolation, characterization and biotechnologically application of the endoglucanase produced from the filamentous fungus Pycnosporus sanguineus. Five agro-industrial residues, from industries in Maceió-AL, were used (wheat bran, sugarcane bagasse, sawdust, coconut fiber and paper). It was isolated using ethanol fractionation and ion exchange and chromatography. It is characterized in terms of temperature, pH, halotolerance, enzymatic kinetics and biotechnological application. Wheat bran was the residue where the fungus showed the highest enzymatic production. The ethanolic fractionation fractionated the enzymatic extract into 5 fractions, the 80-100% fraction concentrated greater activity and was subjected to ion exchange chromatography (DEAE-Sepharose) in AKTA pure M1GE, the isolated enzyme was obtained in a single fraction, observed through of the SDS-PAGE. The biochemical characterization of the enzyme determined the optimum temperature of 50°C and thermal stability between 30 and 60°C with up to 50% of the enzymatic activity. The optimum pH was 5.0, with stability at pH between pH 4.0 and 8.0. The isolated enzyme maintain more than 100% of the activity in up to 5 M NaCl with Km of 3.18 ± 100 mg/mL and the Kcat of 4.53 S-1. The isolated enzyme produced 273.096 mg/mL reducing sugars in 24 hours in rice husks. The present work concluded that the fungus Pycnosporus sanguineus is a producer of an important endoglucanase, with biochemical characteristics compatible with various industrial processes, which can be produced, isolated and applied biotechnologically quickly and with low cost.

Keywords: Agro-industrial residues, saccharification, halotolerant

Supported by: CAPES

08354 - Poster Session

DA.52 - Evaluation of the interaction between p53 tumor suppressor protein and heparin

Natália Neves de Souza ^1,1, Gabriela Assis de Lemos¹, Caroline Augusto Barros^2,1, Giulia Diniz da Silva Ferretti¹, Gileno dos Santos de Sousa¹, Jerson Lima da Silva¹, Tuane Cristine Ramos Gonçalves Vieira¹

¹Institute of Medical Biochemistry Leopoldo de Meis, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil (Rio de Janeiro, Brazil), ²Department of Biochemistry and Molecular Biology, Federal Institute of Rio de Janeiro (Rio de Janeiro , Brazil )

INTRODUCTION

The p53 tumor suppressor protein is known as the guardian of the genome due to its functions of maintenance and conservation of cell stability, responsible for DNA repair or blocking of gene alteration. Mutations in the central domain of the protein are present in approximately 50% of human cancers, causing loss of function of p53 functions, promoting rapid cell proliferation, associated with the formation of intracellular protein aggregates. The mechanism of propagation of these structural alterations still needs to be elucidated, and interaction with cofactors may influence this process. Among these molecules, glycosaminoglycans, negatively charged carbohydrates, such as heparan sulfate and chondroitin sulfate are found in amyloid deposits of tumors, and this presence is related to an increase in malignancy.

OBJECTIVES

Our goal is to observe the effects of heparin on p53 aggregation (WT and R280K mutant) and analyze the structural alterations after interaction in vitro and evaluate the effect in cancer cell models.

MATERIALS AND METHODS

We used spectroscopic techniques such as fluorescence, light scattering and circular dichroism to evaluate in vitro interaction, and used p53-expressing tumor cell lines (MDA-MB-231 and MCF-7) to investigate the cytotoxic effects of heparin.

DISCUSSION AND RESULTS

The interaction between p53 and heparin was analyzed through polarization, showing higher affinity for R280K p53. This interaction did not change the secondary and tertiary structures of the protein at 25°C. However, at 37°C, it induced an increase in protein aggregation, time-dependently, leading to the formation of amyloid structures. Regarding cell viability, high concentrations of heparin showed significant cell death.

CONCLUSION

The data emphasize the importance of heparin in p53 aggregation, suggesting that this ligand may remodel p53 structure during its aggregation process, acting as a chaperone. If this remodeling leads to the formation of more or less toxic species it still has to be further evaluated.

Keywords: amyloid, cancer, glycosaminoglycans

Supported by: CAPES, CNPq e FAPERJ

08511 - Poster Session

DA.53 - Kinect characterization of V(H+)-ATPase activity from gill microsomal fractions of two hololimnetic populations from shrimp Macrobrachium amazonicum

Leonardo Milani Fabri ¹, Cintya Mendes Moraes¹, Daniela Pereira Garçon², Francisco Assis Leone³

¹Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (São Paulo, Brasil), ²Campus Universitário de Iturama, Universidade Federal do Triângulo Mineiro (Minas Gerais, Brasil), ³Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto da Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Macrobrachium amazonicum is endemic to South America and it can be separated into hololimnetic and coastal. In freshwater, this shrimp hyper-osmoregulates their hemolymph energized by the gill (Na ⁺ , K ⁺ )- and V(H ⁺ )-ATPase.

OBJECTIVES

This study aimed to characterize the kinetic properties of gill V(H ⁺ )-ATPase activity of M. amazonicum from Grande and Tietê Rivers’ populations.

MATERIALS AND METHODS

The gill microsomal fractions were prepared by differential centrifugation. The ATPase activity was carried out at 25 °C using PK/LDH coupling system and monitored spectrophotometrically at 340 nm (ε_{340nm, pH 7,5} = 6200 M ^-1 cm ^-1 ). The V(H ⁺ )-ATPase activity represents the difference in activity measured in the presence of orthovanadate and orthovanadate plus bafilomycin.

DISCUSSION AND RESULTS

The V(H ⁺ )-ATPase maximum activity was 16.7 ± 1.7 nmol Pi min ^-1 mg ^-1 protein for the fresh-caught shrimps from Grande River and 27.2 ± 1.1 nmol Pi min ^-1 mg ^-1 protein for the Tietê River population. The apparent affinity by ATP and Mg ²⁺ of the gill M. amazonicum V(H ⁺ )-ATPase from the Grande River population was 0.16 ± 0.03 mmol L ^-1 and 0.14 ± 0.02 mmol L ^-1 , respectively. On the other hand, from Tietê river the apparent affinity was 0.27 ± 0.04 mmol L ^-1 and 0.28 ± 0.05 mmol L ^-1 for ATP and Mg ²⁺ , respectively. The gill V(H ⁺ )-ATPase showed a constant enzyme inhibitor for bafilomycin, 35.1 ± 1.1 nmol L ^-1 and 21.2 ± 1.3 nmol L ^-1 for Tietê and Grande rivers populations, respectively.

CONCLUSION

The V(H ⁺ )-ATPase activity shown by those M. amazonicum populations was close to another population inhabiting a São Paulo state reservoir, although it is twice smaller when compared to the cultivated shrimp from broodstock, which was originally collected in the coastal Amazon basin region.

Keywords: V(H+)-ATPase, Macrobrachium amazonicum, osmoregulation

Supported by: Capes, FAPESP, CNPq, FAPEAM, INCT-ADAPTA

08711 - Poster Session

DA.54 - Strategies to release protein material from alpha-lactalbumin protein microparticles: exploiting their pH-responsiveness

Dirk Fennema Galparsoro ¹, Valeria Vetri¹, Vito Foderà²

¹Department of Physics and Chemistry, University of Palermo (Palermo, Italy), ²Department of Pharmacy, University of Copenhagen (Copenhagen, Denmark)

INTRODUCTION

In destabilizing conditions, proteins may create intermolecular interactions and self-assemble into protein aggregates with different size and morphology. A great interest is focused on a peculiar characteristic group of protein aggregates with a common structure (the cross beta-sheet) stabilized by H-bonds named Amyloids. Their assembly and disassembly processes are of special interest for the design of biomaterials. Among amyloid structures, micron-sized spherical aggregates formed at pH near protein`s isoelectric point called protein particulates, have gained relevance for their ideal size, low citotoxicity, the ability to be formed from most of globular proteins and being unrelated to neurodegenerative diseases.

OBJECTIVES

Combining spectroscopic and quantitative fluorescence microscopy methods, we study the assembly and disassembly of protein particulates made of alpha-lactalbumin in different conditions.

MATERIALS AND METHODS

I have formed protein microparticles of alpha-lactalbumin and I have characterized them using advance fluorescence microscopy and spectroscopic techniques.

DISCUSSION AND RESULTS

We show that, structure, size and compactness of protein particulates can be controlled depending on the initial protein concentration and the time of incubation. These properties have a direct effect on the stability of these protein microspheres. We prove that disassembly of protein particulates occur differently varying the pH from hard acidic (pH2) to neutral/basic pH (pH 7.4) and the addition of salt. At hard acidic conditions the disassembly is instantaneous resulting in small oligomeric and/or large fragments depending on the maturation stage of the aggregate. At neutral/basic pH in the presence of salt disassembly is characterised by a slower releasing non-toxic monomeric alpha-lactalbumin.

CONCLUSION

As a conclusion, protein particulates can act as reservoir of protein material presenting different strategies for the release that control not only the speed of the process, but also the final product that is delivered.

Keywords: Protein Microparticles, Disassembly, Amyloids

Supported by: Villum Fonden, University of Copenhagen, University of Palermo

08882 - Poster Session

DA.55 - Exploration of decarboxylase profile of putative P450 peroxygenases

Isabelle Taira Simões ¹, Wesley Cardoso Generoso¹, Leticia Maria Zanphorlin¹

¹LNBR, Centro Nacional de Pesquisa em Energia e Materiais (São Paulo (SP), Brasil)

INTRODUCTION

Cytochrome P450 proteins (CYP) are a superfamily of heme-containing monooxygenase enzymes. CYPs catalyze a wide variety of chemical reactions, making them of great interest for biotechnological applications. In recent years, the P450 peroxygenase OleT (CYP152) has attracted attention due to its ability to catalyze the oxygen removal from medium to long-chain fatty acids, with hydrogen peroxide as co-substrate, and thereby producing terminal alkenes. This characteristic is of great scientific and industrial concern as it can be explored to produce drop-in biofuels. Drop-in biofuels are composed of renewable hydrocarbons, chemically identical to petroleum fuels, and fully compatible with existing petroleum infrastructure. This similarity would allow the development of fuels with less environmental impact without the need for structural changes in vehicles. However, little is known despite the importance of the peroxygenases capable of fatty acid decarboxylation.

OBJECTIVES

This study aimed to discover new putative P450 enzymes previously selected by amino acid sequence analysis based on a unique decarboxylase described in the literature to expand the knowledge in the field.

MATERIALS AND METHODS

The genes were synthesized, fused to a histidine tag, and transformed into BL21 E. coli cells. The purification was performed using nickel-immobilized resin columns.

DISCUSSION AND RESULTS

Out of the six chosen candidates, three of them were possible to be purified and SDS-PAGE analysis together to spectrophotometric assays (Soret-peak ~ 420nm) indicated a high likelihood for functional CYPs. Activity test using C14 was carried out demonstrating a possible activity to this fatty acid.

CONCLUSION

Further experiments will be performed to evaluate peroxygenase activity and the degree of activity against other fatty acids.

Keywords: Cytochrome P450 proteins, Drop-in biofuels., Terminal alkenes

08457 - Poster Session

DA.56 - Structural and DNA binding characterization of the glycine-rich protein AtGRP7 RRM domain

Igor Guimarães Pascoal¹, Gustavo DallOlio Cardoso ¹, Anderson de Sá Pinheiro¹

¹Departamento de Bioquímica, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

AtGRP7 (Arabidopsis thaliana glycine rich protein 7) is a glycine-rich, RNA-binding protein that plays a central role in plant growth, development, and abiotic stress response. AtGRP7 consists of an N-terminal RNA recognition motif (RRM) followed by an intrinsically disordered region enriched in glycines. Despite the role played by AtGRP7 in cold adaptation and flowering time regulation in Arabidopsis thaliana, the biochemical mechanisms underlying its function are largely unknown.

OBJECTIVES

Here, we used a plethora of biophysical techniques to characterize the structure, stability, and DNA binding affinity of AtGRP7-RRM.

MATERIALS AND METHODS

The gene sequence enconding the RRM domain of AtGRP7 (residues 1-90) was cloned into RP1B and expressed as a His6-tag fusion protein. AtGRP7-RRM showed partially soluble expression in Escherichia coli BL21 DE3 at 18οC and 0.5 mM IPTG. Recombinant AtGRP7-RRM was purified by a combination of nickel-affinity and size exclusion chromatography.

DISCUSSION AND RESULTS

Circular dichroism revealed features of a typical RRM fold, containing a mixture of α-helices and β-sheets. AtGRP7-RRM displayed a melting temperature of 38 οC, suggesting a lowly stable protein. AtGRP7-RRM interaction with a 7-mer DNA oligonucleotide consisting of a previously identified binding site was investigated by fluorescence spectroscopy. The fluorescence emission spectrum showed a maximum at ~349 nm, indicating that the sole tryptophan residue is solvent exposed. Increasing concentrations of oligonucleotide led to fluorescence quenching and AtGRP7-RRM interaction with DNA occured with an apparent K_D of 17.9 ± 4.2 μM. Using multidimensional, triple resonance NMR, we unambiguously assigned 90% of AtGRP7-RRM resonances.

CONCLUSION

This is an important first step toward the sctructural determination of AtGRP7-RRM, which will shed light into its mechanism of action.

Keywords: AtGRP7, Arabidopsis, structure

08512 - Poster Session

DA.57 - Partial purification of the posterior gills (Na⁺,K⁺)-ATPase from the swimming crab Callinectes danae by Molecular Sieve Chromatography

Cintya Mendes Moraes¹, Leonardo Milani Fabri¹, Daniela Pereira Garçon², Francisco de Assis Leone³

¹Departamento de Bioquímica, Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo (São Paulo, Brazil), ²Departamento de Química, Campus Universitário de Iturama -Universidade Federal do Triângulo Mineiro (Minas Gerais, Brazil), ³Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto - Universidade de São Paulo (São Paulo, Brazil)

INTRODUCTION

Improving enzymatic activity while keeping the conformational structure of membrane proteins after purification has been a challenge over the past 70 years. The (Na ⁺ ,K ⁺ )-ATPase is a transmembrane protein responsible for transporting 2 K ⁺ in and 3 Na ⁺ out of cells. Although this enzyme has been the main focus of several purification procedures in mammal tissues, it has been slightly neglected for crustaceans.

OBJECTIVES

Therefore, the main goal of this study was to purify the gill (Na ⁺ ,K ⁺ )-ATPase from the swimming crab Callinectes danae using Molecular Sieve Chromatography.

MATERIALS AND METHODS

Aliquots (3.0 mL) of concentrated (Na+,K+)-ATPase microsomal gill fraction was purified in a Sepharose 4B column (2.5 cm X 32.5 cm) equilibrated and eluted with 50 mM HEPES buffer, pH 6.8, containing 250 mM sucrose using the AKTA Pharmacia Protein Purifier. The Electrophoresis and Western Blot analysis, as well as the (Na ⁺ ,K ⁺ )-ATPase activity of each eluted fraction (1.0 mL) were estimated according to Moraes et al. (2020).

DISCUSSION AND RESULTS

two well resolved protein peaks were eluted from the column and only Peak I showed (Na ⁺ ,K ⁺ ) - ATPase activity (154.9 nmol Pi min ^-1 mg ^-1 ) representing a 14-fold purification compared with that of concentrated microsomal fraction. SDS-Page of Peak I revealed a clearer protein profile compared with that of microsomal fraction prior purification and the Western Blotting analysis showed a single immunoreactive band for (Na ⁺ ,K ⁺ )-ATPase α subunit (≈110 kDa). Peak II revealed a more complex protein profile (molecular weight lower than ≈70 kDa), but no (Na ⁺ ,K ⁺ )-ATPase activity was observed.

CONCLUSION

These primary results allow great advances in the area.

Keywords: (Na⁺,K⁺)-ATPase, Purification, Callinectes danae

Supported by: FAPESP, CAPES, CNPq, FAPEAM, INCT-ADAPTA II

08782 - Poster Session

DA.58 - Expression, Purification and Refolding of the Receptor Binding Domain (RBD) of the Spike protein from SARS-COV-2.

Fábio Antonio Vaz Busoli ¹, Gabriel Cerqueira Alves Costa¹, Verônica de Moraes Manzato¹, Fernando Allan Abreu Silva¹, Ricardo José Soares Torquato¹, Aparecida Sadae Tanaka¹

¹Department of Biochemistry, Escola Paulista de Medicina, Universidade Federal de São Paulo (São Paulo, Brasil)

INTRODUCTION

In 2019, a new outbreak of a respiratory disease was identified in Wuhan, China, caused by the new coronavirus SARS-CoV-2, which is a positive sense, single stranded RNA virus. The SARS-CoV-2 spread all over the world very fast leading to a pandemic state declared by the WHO in March 2020, responsible for more than 4 million deaths. The SARS-CoV-2 interacts with Angiotensin 2 Converting Enzyme (ACE-2) on the host cells by spike protein subunit S1 (containing RBD) to enter and initiate the virus replication. The long period of SARS-CoV-2 pandemic state has favored the emergence of mutations. A variant of concerning has been the variant P1 (gamma) identified in Manaus. Despite the development of different vaccines, none of them promoted a complete immunization to control the virus spreading, making it important to continue searching for new inhibitors.

OBJECTIVES

In this scene, the objectives of this work are the expression, purification, and refolding of recombinant RBD and RBD-P1.

MATERIALS AND METHODS

The plasmid containing RBD-wt sequence was kindly given by Dr. Lima from School of Life Sciences, Keele University, UK. The plasmid containing RBD-wt was used as template for the construction of RBD-P1 by side-direct mutagenesis using PCR, which was confirmed by sequencing. Both constructions were used to transform E. coli Bl21(DE3)plyS and protein expression was performed in auto induction media at 37°C for 4 h. Protein expressions were also performed in LB medium and 1 mM IPTG at 30°C for 16 h. The proteins were purified and refolded on an affinity chromatography column.

DISCUSSION AND RESULTS

Both proteins presented a 34 kDa in the SDS-PAGE.

CONCLUSION

We successfully obtained purified recombinant RBD and RBD-P1. The perspectives will be the selection of peptide phage display library for both purified proteins, in attempt to find specific peptide for each protein.

Keywords: SARS-CoV-2, Receptor Binding Domain, Recombinant Protein

08920 - Poster Session

DA.59 - Heterologous Expression, Purification and Characterization of TRAP1 and PINK1

Marina Del Giudice ¹, Eduardo Feliciano de Lima Santos¹, Silvia Helena Libardi², Carlos Sabino de Oliveira², Júlio César Borges², Carlos Henrique Inacio Ramos³, Lisandra Marques Gava Borges ¹

¹Departamento de Genética e Evolução (DGE), Universidade Federal de São Carlos (São Paulo, Brasil), ²Departamento de Química e Física Molecular (DQFM), Universidade de São Paulo (São Paulo, Brasil), ³ Departamento de Química Orgânica (DQO), Universidade Estadual de Campinas (São Paulo, Brasil)

INTRODUCTION

Inside eukaryotic cells are located the mitochondria, organelle responsible for a lot of physiological processes, such as energy production, synthesis of biomolecules and it also has an important role in some mechanisms of cellular apoptosis. Imbalances in its operation can disturb cell homeostasis, resulting in the emergence of many diseases (e.g cancer). Molecular chaperones, including Hsp90 and its mitochondrial homologue TRAP-1, are involved in the good performance of mitochondrial functions. TRAP-1 ensures the correct protein folding and it’s essential for the mitochondrial integrity, protecting cells against apoptosis, besides avoiding toxic effects of oxidants and anti-cancer drugs. To perform its functions, it interacts with other proteins, such as PINK-1, a mitochondrial serine-threonine kinase: a fundamental regulator of the cytoprotective activity of TRAP-1 through the chaperone phosphorylation.

OBJECTIVES

Recombinant proteins production as well as their structural characterization.

MATERIALS AND METHODS

The proteins were produced in E. coli BL21(DE3) strains and protein expression were induced with isopropyl-β-D-thiogalactoside (IPTG). The pellets were lysed and purified using immobilized metal affinity (IMAC) and size-exclusion (gel filtration) chromatographies. The efficiency of the methods was verified by polyacrylamide gel electrophoresis (SDS-PAGE) and protein concentration determination by spectrophotometric measurements. To characterize TRAP-1 and PINK-1 circular dichroism and intrinsic tryptophan fluorescence assays were performed. A preliminary pull-down test was also performed to detect the interaction between the recombinant proteins.

DISCUSSION AND RESULTS

Both proteins were successfully expressed and purified, with a high degree of purity and homogeneity. Both proteins are well folded as characterized by circular dichroism spectrum and intrinsic tryptophan fluorescence assays. The interaction was detected in the pull-down assay, but control optimizations are still needed.

CONCLUSION

Proteins were obtained with a high degree of purity and well folded. As future perspectives, we intend to carry out other tests focusing on the protein interaction, such as the ITC experiments.

Keywords: structural characterization, TRAP1, PINK1

Supported by: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

08144 - Poster Session

DA.60 - Conformational changes of freshwater shrimp hemocyanin induced by urea

Patricia Soares Santiago ¹, Lierge Ramos^2,1, Karina Satomi Izumi¹, Claudemir Oliveira Souza^2,1, Isis Sebastião^2,1, Giovana Bertini¹

¹Campus de Registro, Universidade Estadual Paulista "Júlio de Mesquita Filho" (SP, Brazil), ²Instituto de Química, Araraquara, Universidade Estadual Paulista "Júlio de Mesquita Filho" (SP, Brasil)

INTRODUCTION

Hemocyanins are giant extracellular proteins with molecular weight between 0.45 to 3.0 MDa and have several biotechnological applications associated with their immunogenic properties, which motivates the performance of biophysical studies on these proteins.

OBJECTIVES

In this work the objective was to study the effect of urea on the hemocyanin structure of freshwater shrimp Macrobrachium acanthurus (HcMac) by different spectroscopic techniques.

MATERIALS AND METHODS

The hemolymph was centrifuged at 3,000 x g by 15 min, dialyzed for 12 h in 100 mmol.L-1 Tris-HCl + 20 mmol.L-1 CaCl2 buffer at pH 7.0. Followed of ultracentrifugation at 250.000 x g by 5 h. Spectroscopies analysis of the concentration of HcMac was 1.6 mg.mL-1, prepared in 30 mmol.L-1 acetate-phosphate-borate buffer at pH 5 and 7, equilibrated for 2 h, depending on the urea concentration (0 to 8 M).

DISCUSSION AND RESULTS

Dissociation of the quaternary structure and drastic conformational modifications occur while the concentration of the denaturant increase. Optical absorption spectrum shows changes in the maximum absorption intensity at 340 nm (di-copper center) due to the increase in urea concentration. Fluorescence data show a decrease in fluorescence emission intensity between 0.5 - 4.0 mol.L-1 of urea. Above 4.0 mol.L-1 there is an increase in the emission intensity and an evident shift in the maximum emission wavelength from 330 nm to 347 nm. A reduction in LSI occurs after the addition of urea until it reaches a concentration of 4 mol.L-1, associated with protein dissociation.

CONCLUSION

Concentrations below 4.0 mol.L-1 urea promoted a greater exposure of the di-copper center to the solvent, showing changes in the quaternary and tertiary structure of HcMac. The results suggest that the HcMac denaturation process starts at concentrations greater than 4.0 mol.L-1 of urea, that is, after the protein dissociates.

Keywords: hemoprotein, biophysical characterization, urea

08532 - Poster Session

DA.61 - Phosphatidic acid vesicles induce the formation of prion protein PK-sensitive fibrils with stable secondary structure

Cyntia Alves Conceição ^1,2, Jerson Silva^1,2, Tuane Vieira^1,2

¹Biochemistry, National Institute of Science and Technology for Structural Biology and Bioimaging (Rio de Janeiro, Brazil), ²Structural Biology, Institute of Medical Biochemistry Leopoldo De Meis (Rio de Janeiro, Brazil)

INTRODUCTION

The mechanism of cellular prion protein (PrP ^C ) conversion into prion scrapie (PrP ^Sc ) is considered the most important factor in the development of prion diseases. PrP ^C is anchored to the outer cell membrane into lipid rafts through a GPI anchor. PrP ^C

OBJECTIVES

Our aim was to elucidate PA-induced rPrP conversion into amyloid fibrils, characterizing the aggregate structure, stability, and resistance.

MATERIALS AND METHODS

We used light scattering, circular dichroism, fluorescence spectroscopy, proteinase K (PK) digestion and electron microscopy over different temperatures to obtain structural and stability information.

DISCUSSION AND RESULTS

PA vesicles induced rPrP aggregation and fibrillization in a temperature-dependent manner, the lower the temperature, the greater the effect. Fibrils formed at 25 °C kept their secondary structure unchanged when submitted to high temperatures, but lost thioflavin T binding. Return to 25 °C restored fibril signal. Using acrylamide as fluorescent quencher agent, we observed that tryptophan accessibility to solvent was decreased in lower temperatures, and these residues were more hidden in the fiber core. The fibrils formed were fully digested by PK. Dynamic light scattering showed no significant changes in PA vesicle diameter submitted to high temperatures, but we observed a decrease in laurdan generalized polarization.

CONCLUSION

Our results suggest that rPrP:PA interaction leads to formation of PK-sensitive fibrils. PrP dynamics and flexibility changes are important to follow a fibrillization pathway induced by PA. Vesicle bilayer rigidity is also important for rPrP interaction and conversion. The quaternary structure of these fibrils seems to be sensible to high temperatures, although its secondary structure is stable, forming an intermediate capable of reorganizing into fibers.

Keywords: Amyloid fibrils, Prion, Phosphatidic acid

Supported by: CNPq, FAPERJ, INBEB, CAPES

08550 - Poster Session

DA.62 - Effects of chronic toxicity to ammonia from M. amazonicum

Daniela Pereira Garçon ¹, Leonardo Fabri⁴, Cintya Moraes⁴, Maria Izabel Costa², John McNamara³, Francisco Leone²

¹Campus Iturama, Universidade Federal do Triângulo Mineiro (MG, Brazil), ²Departamento de Química, FFCLRP, Universidade de São Paulo (SP, Brazil), ³Departamento de Biologia, FFCLRP, Universidade de São Paulo (SP, Brazil), ⁴Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo (SP, Brazil)

INTRODUCTION

The Amazon River shrimp Macrobrachium amazonicum is widely distributed throughout South America and shows elevated potential for aquaculture investment. The successful aquaculture activity depends, among other parameters, the water quality, particularly to the ammonia concentration, which increases considerably during cultivation.

OBJECTIVES

We investigate the in a homolimnetic population of the Macrobrachium amazonicum from Paraná/Paraguay River acclimated to “safe” ammonia concentration (0.1 x LC ₅₀ -96h) for 10 days.

MATERIALS AND METHODS

Adult shrimps were collected from the Rio Grande River, state of Minas Gerais, Brazil, near the Água Vermelha Dam.

DISCUSSION AND RESULTS

Western blotting of gill microsomal homogenates from fresh caught shrimps showed a single immunoreactive band of ≅120 kDa, corresponding to the (Na ⁺ , K ⁺ )-ATPase α-subunit. The ammonia-exposed shrimps also exhibited a weak diffuse band of ≅130 kDa. The ammonia-exposure of M. amazonicum shrimps expressed a secund α subunit isoform that is 2.5-fold increased affinity (Na ⁺ , K ⁺ )-ATPase for NH₄ ⁺ . Exposure of M. amazonicum to 4.9 mg L ^-1 total ammonia (1.8 mg L ^-1 un-ionized ammonia) increase gill (Na ⁺ , K ⁺ )-ATPase (1.3-fold), V(H ⁺ )-ATPase (2.5-fold) and K ⁺ -ATPase (2.5-fold) activities; these are key enzymes in the active ammonia excretion process. These changes suggest that the mechanism, still unknown for freshwater shrimp, is not very different from that described for marine species, where NH₄ ⁺ would enter the gill ionocytes via substitution for K ⁺ on the hemolymph-facing basal (Na ⁺ , K ⁺ )-ATPase K ⁺ binding sites, and K ⁺ binding sites on the presumably basal K ⁺ -ATPase, then being excreted into the subcuticular space via the apical Na ⁺ /H ⁺ (NH₄ ⁺ ) exchanger.

CONCLUSION

Although ammonia-exposure does not cause mortality, lethargy or any indication of sulfuring for 10 days, further studies are needed to improve the determination the maximum acceptable toxic concentration in this population shrimps.

Keywords: ammonia toxicology, gill (Na+, K+)-ATPase, freshwater shrimp aquaculture

Supported by: FAPEMIG, CNPQ, FAPESP

08876 - Poster Session

DA.63 - Structural-Functional Study of domains NBD (Nucleotide Binding Domain) and PBD (Peptide Binding Domain) of human HspA1A protein

Carlos Sabino de Oliveira ¹, Noeli Soares Melo da Silva¹, Júlio César Borges¹

¹Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Among the class of molecular chaperones, there is a superfamily called Heat Shock Proteins (Hsps) that are highly expressed in cells under thermal stress conditions and act, in general, on cellular proteostasis. The Hsp70 family, 70 kDa heat shock proteins, have about 40-60% identity, ubiquitous and ATP-dependent monomeric proteins. Among the different members belonging to the human Hsp70 family, the cytosolic HspA1A isoform is an important constituent of the cellular network of molecular chaperones and providers of folding within cells. Structurally, Hsp70 are proteins composed of two domains, the nucleotide-binding domain (NBD), responsible for the weak ATPase activity, and the peptide-binding domain (PBD), responsible for the interaction with sequences of hydrophobic amino acid residues in the client protein. The investigation of the function, dynamics, structure and stability of the individualized PBD and NBD domains of HspA1A, can help in elucidating its molecular mechanism

OBJECTIVES

Thus, the present work consists of evaluating some structural and functional properties of the NBD and PBD domains of the HspA1A protein,

MATERIALS AND METHODS

such as circular dichroism, intrinsic fluorescence of tryptophan and analytical size exclusion chromatography, showing its structural characteristics, hydrodynamic and stability.

DISCUSSION AND RESULTS

The obtained results showed that the recombinant proteins referring to the constructs HspA1A_PBD and HspA1A_NBD were produced in their folded form, with secondary structure content, rich in β-sheets and α-helices, respectively, and behave, respectively, as a mixture of oligomers and monomer in solution. The proteins showed different thermal stability, the PBD construction being partially reversible and the NBD irreversible. Chemical stability, for both, showed two transitions, so that for the PDB, was characteristic of cooperative unfolding and, for NBD, partially cooperative

CONCLUSION

In summary, the results obtained enrich the information about the individual characteristics of the domains and their influence on the canonical HspA1A.

Keywords: Molecular chaperones, HspA1A, domains

08865 - Poster Session

DA.64 - The Cryo-EM-elucidated structure of an early oligomer of alpha Synuclein and its relation to Parkinson’s disease toxicity

Ritobrita Chakraborty ¹, Sandip Dey¹, Pallabi Sil², Simanta Sarani Paul³, Dipita Bhattacharya⁴, Anirban Bhunia⁴, Jayati Sengupta¹, Krishnananda Chattopadhyay¹

¹Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology (4, Raja SC Mullick Road, Kolkata, India), ²Department of Physics, University of Alberta (Edmonton, Canada), ³Department of Medicine, Centre for Prion and Protein Folding Disease, University of Alberta (Edmonton, AB, Canada), ⁴Department of Biophysics, Bose Institute (Centenary Campus, P-1/12C.I.T. Scheme VII-M, Kolkata, India)

The neurodegenerative Parkinson’s disease (PD) is characterized by the aggregation of the intrinsically disordered protein alpha-Synuclein (α-Syn) into fibrils with a cross-β amyloid structure, in the Lewy body plaques within dopaminergic neurons of the mid-brain. The fibrillation pathway of α-Syn encompasses a multitude of transient oligomeric forms differing in size, structure, toxicity and prion-like seeding activity. According to a recent solid state NMR study, the core residues within a fibril of α-Syn are arranged into in-register parallel β sheets with a unique C-terminal Greek key topology (PDB 2N0A). Here, we report the formation of stable, non-toxic (to both liposomal membranes and neuroblastoma cells) ‘mace’-shaped oligomers, when the physiologically-available small molecule heme (hemin chloride) is added at sub-stoichiometric ratios, to either monomeric or aggregated α-Syn. Using cryo-electron microscopy, we found that a tetrameric form of the Greek key model could be fit into the density of these mace oligomers, albeit with a distortion in the head-neck junction of the mace structure. The ‘Greek key oligomer’ fits well as a segment of the previously-described annular oligomers and appears to be its structural predecessor in the hierarchical pathway of fibril formation. We propose that the distortion in the heme-treated oligomer prevents further appending of the twisted units into annular oligomers. Furthermore, heme binds to a crucial histidine (His50) residue located in the inter-protofilament pre-non amyloid β component interface, thus interfering with a salt bridge formation with a Glu57 residue located in the opposite protofilament, thereby weakening the inter-protofilament steric zipper integrity. Overall, we describe a mechanism of inhibition of fibrillation and associated of α-Syn using a physiologically-available small molecule.

Keywords: oligomer, neurodegeneration, Greek key fibril

08353 - Poster Session

DA.65 - Isolation and characterization of Canavalia ensiformis serine proteases: enzymes with pharmacological potential

Thayane Aparecida Alves de Araujo ^1,2, Raquel Elisa da Silva-López¹, Flávia Almada do Carmo²

¹Departamento de Produtos Naturais, Oswaldo Cruz Foundation (Rio de Janeiro, Brazil), ²Faculdade de Farmácia, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Canavalia ensiformis is a tropical legume with high content of active proteins, that have important biological functions, and their pharmacological activities have been extensively investigated. Previous studies from our group demonstrated important serine protease activity from C. ensiformis leaf aqueous extract (CE-A), suggesting a possible pharmaceutical application.

OBJECTIVES

The aims of the present work was to isolate and characterize serine proteases from CE-A.

MATERIALS AND METHODS

Fresh C. ensiformis leaves collected in Plataforma Agroecológica de Fitomedicamentos (PAF), of FIOCRUZ were processed with N₂ and proteins extracted with distilled water. Protein content was determined by Bradford method and protein profile evaluated by SDS-PAGE. Serine proteases were isolated by affinity chromatography using ρ-aminobenzamidine–agarose column.

DISCUSSION AND RESULTS

Serine proteases rich fraction (CE-ApBza) was isolated from CE-A, 3.00 times with total yield of 79.8%. SDS-PAGE showed major proteins molecular weight about 52, 37 and 25 kDa. The enzymatic activity was studied using L-N–α–p–tosyl–L–arginyl–methyl ester (_L-TAME) and N-Benzoyl-L-tyrosine ethyl ester (BTEE) as substrates. The maximum protease activity was observed in typical serine protease pH range (9.0), besides, was expressively inhibited only by serine protease inhibitor, such as benzamidine which inhibited 100% of CE-AρBza using BTEE as substrate and 77% when using _L-TAME. This fraction preserved 88% of activity in 24h at 70ºC using BTEE as substrate and 46% when using _L-TAME. CE-AρBza has similar affinity to BTEE (K_M =11,65 μM) and TAME (14,97μM).

CONCLUSION

These results indicated that CE-AρBza proteases had expressive and thermostable activity, and the methodology for isolation was efficient. Thus, these proteases have potential biotechnological and pharmacological employment.

Keywords: biochemical characterization, Canavalia ensiformis, serine proteases

08526 - Poster Session

DA.66 - AUXIN AS MITIGATING AGENT OF SALT STRESS IN TWO SPECIES OF FORAGE GRASSES

Gilmara Matias de Sousa ¹, Juan Carlos Alvarez-Pizarro²

¹CCAB, Universidade Federal do Cariri (Ceará, Brasil), ²CCAB, Universidade Federal do Cariri (Ceará, Brasil)

INTRODUCTION

PROJECT - The grasses Urochloa brizantha and Megathyrsus maximus have little tolerance to salinity. And Plant hormones are fundamental to the adaptation to salinity by mediating several adaptive responses.

OBJECTIVES

The aim of the proposed study is to evaluate the effect of auxin (indole-3-acetic acid) on salt stress adaptation methods in Urochloa brizantha and Megathyrsus maximus submitted to salinity.

MATERIALS AND METHODS

The seeds will be germinated in substrate moistened with auxin with three repetitions. Beginning on the 30th day, salinity will be applied with a combination of ions (Na+, Mg+2 and Ca+2). On the day of collection, one group of plants will be decapitated at the collum level to collect fluid from the xylem, estimate the concentration of ions Na+ and K+, and estimate the flow rate of xylem sap. In parallel, another group of plants will be divided into leaves, colla and roots to estimate the content of ions Na+ and K+. The quantification of ions in these samples will be performed using flame photometry. The Western blot method and polymerase chain reaction (qPCR) will be used to analyze the expression of transporters involved in the control of the transport of Na+, such as plasma membrane (SOS1) and vacuolar (NHX) antiporters in roots.

DISCUSSION AND RESULTS

CONCLUSION

Treatment with indole-3-acetic acid is expected to stimulate the Na+ homeostasis regulating mechanism. Thus, Urochloa brizantha and Megathyrsus maximus could have perform better under conditions of salinity.

Keywords: Auxin, Forage grasses, Tolerance to salinity

08693 - Poster Session

DA.67 - Inositol monophosphatase 2 (IMPase 2) and its inhibition with lithium(l) and hesperetin

Rafaella Trevisan Scandiuzzi ¹, Guilherme Crispim de Faria Cruz ¹, Ljubica Tasic¹

¹Departamento de química orgânica , Universidade Estadual de Campinas (, Brasil)

INTRODUCTION

Individuals with bipolar disorder (BD) have a characteristic and sudden change in mood from euphoria to depression. In Brazil, there are around 6 million people diagnosed with BD. The most used drug to alleviate BD symptoms is lithium along with other drugs and therapies, but Li(I) medicinal dose is close to toxic. Besides, the benefits of lithium are observed just in 60% of patients with BD that use this drug. In addition, the use of lithium therapy provokes a drop in myo-inositol, which is a precursor of phosphatidylinositol that plays an important role in intracellular signal transduction through the production of second messengers. Thus, an important connection between bipolar disorder and the altered performance of enzymes from the family of inositol monophosphatase (IMPase), especially IMPase 2, was hypothesized.

OBJECTIVES

This project aimed to propose a flavone, hesperetin, as a possible phytotherapeutic agent for BD and an inhibitor of IMPase 2.

MATERIALS AND METHODS

To perform the in silico assays and preliminary experiments conducted in vitro, it was first necessary to obtain the protein using the recombinant DNA technique. IMPase 2 was purified using affinity chromatography and characterized using biophysical and biochemical techniques as a folded and active enzyme. Then, the interactions among hesperetin-IMPase 2, lithium-IMPase 2, and hesperetin+lithium-IMPase 2 were studied.

DISCUSSION AND RESULTS

The results obtained in circular dichroism, fluorescence, and enzymatic activity experiments pointed to an interaction between the flavone of interest and IMPase 2, and that this interaction enabled to inhibit the enzyme's action. Finally, when associated with lithium, hesperetin inhibited the IMPase 2 hydrolytic properties, especially against inositol-1-monophosphate.

CONCLUSION

There are still to determine the IMPase 2 hesperetin binding site, and propose the alteration in the IMPase 2 mechanism of action in the presence of the flavone and lithium.

Keywords: Inhibitors , Enzymes , Flavone

Supported by: FAPESP

08397 - Poster Session

DA.68 - Mechanism elucidation of not conventional peroxygenases applied on production of drop in biofuels

Mayara Chagas de Ávila ^1,3, AMANDA SOUSA¹, LETICIA RADE¹, WESLEY GENEROSO¹, PLINIO SALMAZO VIEIRA¹, THOMAS MAKRIS², Gabriela Felix Persinoti¹, Carlos Henrique Inacio Ramos³, Renan Yuji Miyamoto ¹, Letícia Maria Zanphorlin Murakami¹

¹National Laboratory of Biorenewables , Brazillian Center for Research in Energy and Materials (Sao Paulo, Brazil), ²Department of Molecular and Structural Biochemistry, North Carolina State University (North Carolina , United States), ³Instituto de Química, Universidade de Campinas (São Paulo, Brasil)

INTRODUCTION

Alkenes or olefins are precursors for drop-in biofuels production, such as biokerosene and green diesel. OleTJE is the only studied enzyme from the P450 family that can promote decarboxylation activity on free fatty acids, the reaction that produces alkenes as a final product.

OBJECTIVES

Our research intends to find new enzymes with decarboxylation activity to understand what drives this type of reaction and propose a biotechnology route to produce drop-in biofuels.

MATERIALS AND METHODS

Herein, we discovered a novel decarboxylase P450 (named RN) using bioinformatics tools that cluster enzymes by their activity and amino acid sequences. RN enzyme was expressed in a host cell (E.coli) and purified by chromatography methods. The purified enzyme was submitted to SEC-MALS, SAXS, crystallization, and X-ray diffraction experiments to determine its molecular properties. Moreover, the spectroscopic characterization was done by methods as UV-Visible to calculate dissociation constant (Kd) with different chain size substrates (C12, C14, C16, C18, and C20) and circular dichroism (CD) to analyze the secondary structure besides to melting temperature (Tm).

DISCUSSION AND RESULTS

Hydrodynamic studies revealed RN as a dimer, conformation never reported in this class of enzymes, and conserved amino acids in its active site, written in OleTJE as crucial to the decarboxylation route. The native enzyme presented higher affinity by myristic acid (C14, Kd: 0.59 μM) and Tm of 52.23 °C. To better understand the role of dimer conformation on RN, mutants S26F and E405F were investigated. The mutants were confirmed as monomers, presenting a similar CD profile and Tm (53.3 and 49.5°C). Still, there is a lower affinity for myristic acid (Kd: 0.91 and 2.07 μM) and alkene production compared to the wild-type enzyme.

CONCLUSION

The results presented here expand the knowledge on the molecular bases for decarboxylation activity of an enigmatic hydrocarbon-producing P450 before based on a unique structure available in the literature.

Keywords: alkenes, decarboxilation, drop in biofuels

Supported by: FAPESP

08656 -

DA.69 - Antimicrobial resistant bacteria as treasure troves of protein evolution

Rodrigo Machado¹, Fernanda Stephens-Hermes¹, Ana Paula Assef¹, Paulo Redner¹, Vincent Delorme³, L. Caetano Antunes¹, Teca Calcagno Galvão ¹

¹Instituto Oswaldo Cruz, Fiocruz (, Brazil), ²CRPHF-ENSP, Fiocruz, (Brazil), ³Tuberculosis Research Laboratory, Institute Pasteur Seoul, (Brazil)

INTRODUCTION

The intensive use of antibiotics in hospital settings and in the management of animals for human consumption, coupled with limited success in the development of new classes of antimicrobials, has created the possibility of an imminent public health crisis: the number of infections caused by antimicrobial resistant bacteria is on the rise and there are estimates that by 2050 more people will die of infections by resistant bacteria than due to cancer.

OBJECTIVES

Our aim is to evaluate how substitutions resulting from antimicrobial selective pressure lead to resistance, by looking at their effect on protein function and structure. Specifically, we look at the transcriptional repressor EthR of Mycobacterium tuberculosis and the two component systems PhoPQ and PmrAB of Klebsiella pneumoniae.

MATERIALS AND METHODS

We employ bacterial genetics and protein biochemistry to assay how substitutions change DNA binding, protein-protein interaction, and or enzymatic properties.

DISCUSSION AND RESULTS

We find that certain substitutions in EthR and PmrAB and PhoPQ occur in key functional regions. In the case of EthR we show that two substitutions enhance transcriptional repression, likely by disrupting allosteric changes predicted to happen upon ligand binding. In the case of PmrAB and PhoPQ we show that mutations selected by polymyxin treatment fall in discrete functional regions, hinting at the mechanisms of resistance.

CONCLUSION

Albeit a major health problem, antibiotic selective pressure on bacteria works as an experiment on the evolution of protein function and structure.

Keywords: AMR, bacterial two component systems, M. tuberculosis

Supported by: Fiocruz; Institute Pasteur International Network

08223 -

DA.71 - A biophysical and in vitro characterization of an amyloidogenic and highly tolerated cell penetrating peptide

Lucas Rodrigues de Mello ¹, Li Porosk², Thiago Lourenço¹, Bianca Garcia¹, Carlos Costa³, Sang Han¹, Juliana Souza⁴, Ulo Langel^5,2, Emerson Silva¹

¹Biofísica, Universidade Federal de São Paulo - Escola Paulista de Medicina (São Paulo, Brasil), ²Institute of Technology, University of Tartu (, Estonia), ³Laboratório Nacional de Nanotecnologia, Centro Nacional de Pesquisa em Energia e Materiais (, Brasil), ⁴Centro de Ciências Naturais e Humanas, Universidade Federal do ABC (São Paulo, Brasil), ⁵Biochemistry and Biophysics, Stockholm University (, Suécia)

INTRODUCTION

Due to the increasing number of new therapies based in the delivery of nucleic acids or even drugs with great therapeutic appeal that are poorly absorbed by the tissue/cells, we face an increasing demand for biocompatible carriers. In the last 30 years, nearly 2000 cell penetrating peptides (CPPs) have been reported in the literature, CPPs are small amino acid sequences with the capacity of translocating through cell membranes, and since the presence of cationic residues such as arginine and lysine are reported as potential enhancers for the translocation of these sequences, most CPPs are highly hydrophilic and cationic. However, this increase in charge is generally followed by an increase in cytotoxicity, which is a major problem for the use of CPPs in living organisms.

OBJECTIVES

In this work we propose the formation of non-covalent complexes between the sequence PFVYLI, a hydrophobic and non-charged CPP, conjugated with nucleic acids

MATERIALS AND METHODS

The self-assembly of PFVYLI was characterized by classical biophysical techniques such as small angle scattering X-ray (SAXS), Circular dichroism (CD) and Atomic force microscopy-based infrared spectroscopy (AFM-IR). the delivery of non-covalent aggregates between PFVYLI and a model fragmented DNA into HeLa cells was observed by fluorescence confocal microscopy. Cytoxicity was determined by MTT assays.

DISCUSSION AND RESULTS

It was possible to observe the self-assemble of PFVYLI into rod-like supersctructures with lengths ranging from a few nanometers to micrometers, and the the AFM-IR, fluoresnce in the presence of Thioflavin and CD assays indicates an Amyloid-rich secondary structure. We also succesfully formulated non-covalent aggregates between this peptide and nucleic acids, which were well tolerated and internalized by HeLa cells, possibly via an endocytic pathway.

CONCLUSION

The peptide PFVYLI self-assembled into rod-like super structures with an intrinsic β-amyloid secondary structure, we also successfully produced conjugates between this peptide and DNA that were well tolerated and internalized for HeLa cells.

Keywords: biophysics, molecular biology, cell penetrating peptides

Supported by: FAPESP, CAPES

08134 -

DA.72 - An anterior transcriptional repression mechanism in the segmentation cascade of Drosophila

Luiz Paulo Moura Andrioli ¹

¹EACH, Escola de Artes Ciências e Humanidades USP (São Paulo, Brasil)

INTRODUCTION

This cascade is formed by sequential transcription factors that set the polarity of the egg and establish the division of the body in segments. The cascade comprises three hierarchical levels formed by gap, pair-rule and segment polarity genes.

OBJECTIVES

Our goal is to understand transcription regulation during development. To that end, we investigate the segmentation cascade that specifies the antero-posterior axis in Drosophila during its syncytial blastoderm. In the laboratory we study the segmentation cascade in the anterior region of the embryo.

MATERIALS AND METHODS

Our approach is to combine genetics, cellular, biochemical and bioinformatics to understand molecular mechanisms underlying transcription at DNA regulatory regions.

DISCUSSION AND RESULTS

According to our results, a mechanism comprised by the combined activity of gap repressors prevents the formation of pair-rule stripes in the anterior blastoderm.

CONCLUSION

So far, we identified Sloppy-paired, Tailless and Huckebein as repressors that operate in additive manner to set the borders of anterior stripes of pair-rule genes.

Keywords: Drosophila, repression, transcription

08635 -

DA.73 - The conformational dynamics of the flanking polyQ regions in the membrane-bound state of huntingtin exon 1

Tânia Sousa^1,2, Nuno Bernardes^1,2, Ana Coutinho^1,2,3, Manuel Prieto^1,2, Ana M. Melo ^1,2

¹iBB—Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa (Lisbon, Portugal), ²Associate Laboratory i4HB—Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa (Lisbon, Portugal), ³Dep. Química e Bioquímica, Faculdade de Ciências (Lisbon, Portugal)

INTRODUCTION

The pathological expansion of the polyglutamine (polyQ) repeat within the first exon of huntingtin (HTTex1) protein is a hallmark of Huntington’s disease (HD). Multiple evidences support that the membrane interaction of huntingtin is critical for its misfolding and aggregation in HD.

OBJECTIVES

Here, we focus on obtaining a detailed understanding of the initial steps of HTTex1-lipid interaction and the conformational dynamics of the flanking polyQ regions.

MATERIALS AND METHODS

DISCUSSION AND RESULTS

We initially employed single-molecule approaches based mainly on fluorescence correlation spectroscopy (FCS) to monitor the binding of HTTex1 to synthetic lipid vesicles. The dependence on membrane curvature and lipid composition was studied, considering as models anionic lipids, raft-mimicking mixtures, and total brain lipid extract. The FCS results show a preferential binding of HTTex1 towards POPS (anionic lipid)-containing vesicles. Moreover, time-resolved fluorescence intensity and anisotropy measurements of HTTex1 site-specifically single labeled at the adjacent polyQ regions reveal distinct conformational dynamics of these flanking regions in the HTTex1 membrane-bound state. Notably, the proline-rich domain remains highly flexible and solvent exposed even upon membrane binding. Instead, the N17 segment converts into a less dynamic state.

CONCLUSION

Our findings provide unique insight into how membrane composition and flanking polyQ sequences modulate the early stages of HTTex1 aggregation at the membrane surface.

Keywords: Intrinsically disordered proteins, lipid-protein interaction, Neurodegeneration

Supported by: Work supported by FCT-Portugal (PTDC/BIA-BFS/30959/2017 grant and CEECIND/00884/2017 contract to AMM, and UIDB/04565/2020 funding to iBB-IST).

08805 -

DA.75 - P53 protein and Liquid Liquid Phase Separation (LLPS): Aggregation studies in anticancer therapy

Elaine da Conceição Petronilho ¹, Murilo Martins Pedrote¹, Mayra de Amorim Marques¹, Yulli Moraes Passos¹, Michelle Ferreira Mota¹, Benjamin Jakobus³, Gileno dos Santos de Sousa¹, Filipe Pereira da Costa¹, Adriani de Lima Felix ¹, Giulia Diniz da Silva Ferreti¹, Fernando Pereira de Almeida¹, Yraima Cordeiro¹, Tuane Cristine Ramos Gonçalves Vieira¹, Guilherme Augusto Piedade de Oliveira¹, Jerson Lima Silva¹

¹Institute of Medical Biochemistry Leopoldo de Meis, Federal University of Rio de Janeiro (Rio de Janeiro, Brasil), ²Faculty of Pharmacy, Federal University of Rio de Janeiro (Rio de Janeiro, Brasil), ³Modal Informática Ltda, Modal Informática Ltda (Rio de Janeiro, Brasil), ⁴National Center for Structural Biology and Bioimaging (CENABIO), Federal University of Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

The p53 tumor suppressor protein is a cellular sensor for DNA damage that induces cell cycle abrogation to avoid misbehavior on cellular phenotype. In this sense, mutants of the TP53 gene have been found in more than 50% of cancer cells. The hotspot mutations are placed on the DNA-binding domain of the p53 protein and they are related to loss of function that results in tumor development. On the other hand, the mutations can also induce gain of oncogenic function by the formation of amyloid aggregates that impair DNA damage recognition.

OBJECTIVES

The goal of this work is to investigate whether the p53 aggregation route involves liquid-liquid phase separation (LLPS).

MATERIALS AND METHODS

The first step was the preparation of soluble p53, p53 WT-EGFP and p53 mutants, by heterologous expression. After, we tested whether p53 LLPS could occur, varying some conditions as temperature and molecular crowding, using Differential Interference Contrast (DIC) and fluorescence recovery after photo bleaching (FRAP).

DISCUSSION AND RESULTS

The results showed that p53 phase separates in the presence of the molecular crowding agent ficoll under low temperatures. Longer incubation times or increased temperature led to the transition of p53 to aggregates. Polyanions, such as heparin and RNA, were able to modulate the phase separation and phase transition in vitro. Heparin led the p53 condensates in a gel-like state, whereas RNA resulted in the conversion into a solid-like state of the protein, similar to that found in proteins involved in neurodegenerative diseases.

CONCLUSION

The possibility to probe different conformational states on the misfolding pathway at an atomic level has highlighted microscopy tests as the state of the art in the structural biology field to study invisible intermediate states.

Keywords: Liquid Liquid Phase Separation, protein, p53

Supported by: FAPERJ, FINEP, CNPq and CAPES

08953 - Poster Session

DB.01 - Structure of a Short Peptide Derived from the Transform Growth Factor beta1

Thiago da Costa Lourenço ¹, Lucas Rodrigues de Mello¹, Bruna Barbosa da Silva¹, Emerson Rodrigo da Silva¹

¹Departamento de Biofísica, Universidade Federal de São Paulo (SP, Brazil)

INTRODUCTION: Peptide nanostructures have been studied as alternative therapeutics due to their potential selectivity for certain molecular targets. One of the promising uses is related to autoimmune diseases, which in many cases are correlated to either central or peripheral tolerance processes. The peripheral process is mediated by the transform growth factor beta 1 (TGF-β1), which is secreted by T-regulator cells, and plays anti-inflammatory function in autoimmune diseases. In this context, the structural characterization of peptide fragments derived from TGF-β1 may assist the elucidation of mechanisms implied in the action and open possibilities for developing biomimetic nanomaterials based on this growth factor [1]. OBJECTIVE: We aimed to provide detailed characterization on both self-assembly and structure of a heptamer with amino acid sequence Ac-ESPLKRQ, derived from TGF- β1, and correlate structure and anti-inflammatory action. MATERIALS AND METHODS: The peptide has been synthesized through standard solid-phase approaches. Characterization has been performed through a range of biophysical techniques including fluorescence, circular dichroism and infrared spectroscopy, small-angle X-ray scattering, and electron and atomic force microscopy. RESULTS AND DISCUSSIONS: Fluorescence assays have shown the formation of stable aggregates above a critical concentration ~ 3 mg/mL, with secondary structure dominated by disordered conformations; however, at higher concentrations, these supramolecular assemblies present signature of α-helix conformations, the same secondary structure exhibited by the active in the native TGF-β1 protein, which may be the conformation responsible for docking to cell receptors. The aggregates present nanometer lengths, composed by subunits with globular shape and sizes ~60 nm, which may assist interaction with cell membranes. CONCLUSION: The peptide sequence mimetizing TGF-β1 investigated here is able to form nanoscopic aggregates with secondary structure that reproduces conformations of active sites found in the native protein. These characteristics make this fragment a promising candidate to produce bioactive nanoparticles able to exert similar anti-inflammatory responses to those observed for TGF-β1.

Keywords: peptide self-assembly, TGF-beta1, biomaterials

Supported by: CAPES, CNPq, FAPESP

08346 - Poster Session

DB.02 - Structural and functional characterization of the N-terminal domain of the human coronavirus hCoV-HKU1 nucleocapsid protein

Aline de Luna Marques ^1,2, Ícaro Putinhon Caruso^3,2, Peter Reis Bezerra², Fábio Ceneviva Lacerda de Almeida², Gisele Cardoso de Amorim^1,2

¹Multidisciplinary Center for Research in Biology (Numpex-Bio), Federal University of Rio de Janeiro, Campus Duque de Caxias (Rio de Janeiro, Brazil), ²National Center for Nuclear Magnetic Resonance (CNRMN), Federal University of Rio de Janeiro (Rio de Janeiro, Brazil), ³Multiuser Center for Biomolecular Innovation (CMIB), São Paulo State University (São Paulo, Brazil)

INTRODUCTION

Since 2002, three beta coronaviruses (bCoVs) have gained notoriety: SARS-CoV, MERS-CoV, and SARS-CoV-2, which is causing the Covid-19 pandemic. Besides these more aggressive CoVs, there are endemic human viruses, such as hCoV-HKU1, associated with mild respiratory tract disease, that can progress to acute respiratory failure. During infection, the transcription of the viral genome generates full-length and subgenomic RNAs, through a discontinuous process regulated by the transcriptional regulatory sequences (TRSs). The nucleocapsid (N) protein of CoVs acts as an RNA chaperone and its N-terminal domain (N-NTD) binds to TRS, forming a high-affinity complex. Comparing the structure, dynamics, and TRS interaction of HKU1-N-NTD with those of more aggressive CoVs will enable the understanding of the molecular factors leading to severe infections and the development of treatment strategies.

OBJECTIVES

Determining the structure of HKU1-N-NTD, mapping its interaction with different DNA/RNA fragments, including TRS, and probing its dsDNA/RNA melting activity.

MATERIALS AND METHODS

NMR assignments of the HKU1-N-NTD were determined using a set of classical 2D and 3D experiments. Structure calculation was performed using ARIA and CS-Rosetta softwares. HKU1-N-NTD interaction with TRS was evaluated by protein NMR titration with DNA/RNA. HKU1-N-NTD melting activity towards double-stranded DNA/RNA was assessed by fluorescence.

DISCUSSION AND RESULTS

The calculated structural model presents 4 β-strands and 4 flexible regions, including an extended loop known as the finger. In the presence of TRS, chemical shift perturbation (CSP) was observed for several residues, including Asp57, Gln59, and Val105, which presented high CSP for all evaluated protein-fragment interactions. N-NTD melting activity was greater for dsRNA.

CONCLUSION

HKU1-N-NTD interacts with DNA and RNA fragments and promotes double-strand melting. CSP data indicate that the main binding region is located between the finger and the central β-sheet. These results may help better understand the role of N protein in the discontinuous transcription, enabling new approaches to fight CoVs.

Keywords: hCoV-HKU1, nucleocapsid protein, TRS

08919 - Poster Session

DB.03 - Determination of the three-dimensional Structure of Schistosoma mansoni Universal Stress G4LZI3 Protein by X-ray Crystallography

Priscilla MASAMBA ¹, Geraldene Munsamy¹, Brandon Weber², Bryan Trevor Sewell², Abidemi Paul Kappo¹

¹Molecular Biophysics and Structural Biology (MBSB) Group, Department of Biochemistry and Microbiology, University of Zululand (KwaDlangezwa 3886, South Africa), ²Aaron Klug Centre for Imaging and Analysis, University of Cape Town (Rondebosch 7701, Cape Town, South Africa)

Schistosomiasis is the most potent water-based disease in tropical and subtropical regions of the world from a global health perspective, infecting more than 252million people with 90% resident in SSA. Despite its existence from antiquity, efforts to eradicate and control the disease have failed and unfortunately current treatment now displays drug resistance and reduced efficacy. Schistosomes have over time developed mechanisms to cope with the myriad of stresses they encounter throughout their developmental cycle through the up-regulation of Universal Stress Proteins (Usp). The G4LZI3 USP has since been identified as a potential ‘lead’ molecule in schistosomal treatment hence, the aim of this study was to determine the 3D structure of this protein and its biological function. The G4LZI3 protein was over-expressed in M15 cells and purified using Ni-NTA affinity chromatography and gel filtration. Characterization of the protein was done by Differential Scanning Calorimetry, Fluorescence Spectroscopy, Fourier-transform Infrared Spectroscopy, Mass Spectrometry and 1D Nuclear Magnetic Resonance. These showed the protein was folded, consisted of various secondary structure elements and is thermodynamically-stable. Pooled purified fractions were concentrated to yield samples for crystallization trials. Various conditions yielded small crystals that were seeded and thereafter used to generate considerably-sized 3-dimensionally-shaped crystals. Diffraction data was remotely collected at Diamond Light Source (United Kingdom) and processed at the University of Cape Town using MR Rosetta Software tools. Coot was thereafter used for model building and refinement of the structure. Lastly, bioinformatics tools were used to identify small molecule inhibitors which were docked onto the protein. Those with the best scores were used to determine parameters such as molecular dynamic simulations, post dynamic analysis and binding free energy calculations. These results provide basis for further investigation of the G4LZI3 protein towards the design of new anti-schistosomals.

Keywords: Characterization, Crystallography, G4LZI3

Supported by: Diamond Light Source (UK), National Research Foundation (NRF).

08184 - Poster Session

DB.04 - SAXS e DLS: a theoretical review

Amanda Santos Palma ¹, Leandro Ramos de Souza Barbosa¹

¹Departament of General Physics, Institute of Physics, University of São Paulo (São Paulo, Brazil.)

INTRODUCTION

In order to determine nanoparticles structure and size, SAXS and DLS techniques are the best to be used. DLS is a simple technique and can return parameters as particle size and polidispersity, which mean how is the size distribution of the sample. SAXS technique can determine particle structure and supply information about lattice parameter and water channel disposition. This technique can also be used to determine protein and other molecules structures.

OBJECTIVES

The objective of this study is to go through an overview around parameters and the theory behind DLS and SAXS techniques. It is focused on its use to analyse cubosomes, an important lipid nanoparticle with cubic phase.

MATERIALS AND METHODS

As an theoretical study about the techniques mentioned above, this work followed theory placed in articles and books.

DISCUSSION AND RESULTS

The SAXS and DLS techniques are reliable to determine the parameters discussed above. In one hand, DLS is a straightfoward technique and provides the results right after the experiment is performed. On the other hand, SAXS needs some analyzing, in which the positions of peaks are used to determine particle structure. Through this analysis, it can be seen coexistence of cubic phases and phase transitions.

CONCLUSION

Therefore, these techniques are the finest to use to determine particle structure and particle size. The analysis made is rather simple and the results obtained are accurate. In addition, SAXS and DLS techniques are often used by researches to characterize nanoparticles samples.

Keywords: characterization, DLS, SAXS

Supported by: FAPESP

08719 - Poster Session

DB.06 - Exfoliative protein C (ExhC) of Staphylococcus sciuri: inactivation and structural determination of necrotic activity

Carolina Gismene ¹, Angela Rocio Nino Santisteban¹, Ricardo Barros Mariutti¹, Raghuvir Krishnaswamy Arni¹

¹Centro Multiusuário de Inovação Biomolecular, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista "Júlio de Mesquita Filho" (SP, Brasil)

INTRODUCTION

Staphylococcus sciuri is pathogenic bacterium of significant clinical and veterinary relevance. Recently, a strain of S. sciuri has been described as the etiologic agent of exudative epidermitis in pigs in China and the main virulence factor involved in this clinical manifestation was the Exfoliative protein C (ExhC). ExhC of S. sciuri, in addition to causing epidermal exfoliation in pigs and neonatal rats, it was able to induce cell necrosis in vitro, specifically in the cell line of renal fibroblasts of neonatal hamsters (BHK-21) a property hitherto fore unobserved in exfoliative toxins (ETs). The production of ExhC recombinant fragments allowed us to conclude that the domain containing residues 79-128 is essential for the observed necrotic activity.

OBJECTIVES

The aim of this research was to verify whether the mutations of specific amino acid residues inactivate necrotic activity and to determine the structural parameters of this region and correlate it with necrosis.

MATERIALS AND METHODS

The amino acids of the necrotic domain were aligned with the corresponding regions of other ETs which indicated the presence of conserved amino acid residues or with similar biochemical properties in most of the ETs except in ExhC of S. sciuri. This in silico evaluation formed the basis for the design, expression and purification of ExhC protein with mutation in four amino acid residues and wild type ExhC for in vitro tests with the cell line BHK-21.

DISCUSSION AND RESULTS

The mutant ExhC loss of necrotic activity was observed in the in vitro tests with the cell line BHK-21, different results from those observed for wild type ExhC which causes a significant decrease in cellular viability.

CONCLUSION

The crystal structure of mutant ExhC was determined with 1,57 Å and the stereochemical structural parameters have been correlated with the results of the in vitro tests and provide details of the steric requirements for the observed activity.

Keywords: exfoliative protein C, Staphylococcus sciuri, necrotic activity

Supported by: FAPESP

08889 - Poster Session

DB.07 - Human fumarase cocrystallization: revealing metabolite-fumarase interactions

Iara Aimê Cardoso ¹, Mariana Araújo Ajalla Aleixo^1,2, Kevin G. Hicks³, Jared Rutter³, Maria Cristina Nonato¹

¹Department of Biomolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (SP, Brazil), ²Brazilian Nanotechnology National Laboratory, Brazilian Nanotechnology National Laboratory, Brazilian Center for Research in Energy and Materials (SP, Brazil), ³Department of Biochemistry, University of Utah School of Medicine (Utah, United States of America)

INTRODUCTION

Fumarases are enzymes that catalyze the estereospecific and reversible hydratation of fumarate to L-malate. Human fumarase (HsFH) belongs to class II and in mitochondria participates in the tricarboxylic acid (TCA) cycle, and in the cytosol can act in the metabolism of amino acids and also play a key role in DNA damage response to DNA double strand breaks. HsFH gene mutations have been mainly associated with heritable diseases: fumarate hydratase deficiency (FHD), multiple cutaneous and uterine leiomyomatosis (MCUL) and hereditary leiomyomatosis and renal cell cancer (HLRCC). There are few inhibitors and activators reported for fumarases, and its regulation mechanisms remains unclear.

OBJECTIVES

This present work aimed the evaluation of different metabolites interactions in the human fumarase structure.

MATERIALS AND METHODS

A mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) was performed to evaluate protein-metabolite interactions. The cocrystallization experiments were performed with the recombinant HsFH and the metabolites, by sitting drop method and using PEG 10 K as precipitant agent.

DISCUSSION AND RESULTS

The preliminary analysis of MIDAS allowed the identification of maleic acid and D-2-amino-3-phosphono-propionic acid as potential ligands. Cocrystallization experiments were carried out in order to obtain the complex structure, and datasets (at 1.8 Å and 2.15 Å resolution for maleic acid and D-2-amino-3-phosphono-propionic acid, respectively) were collected. The solved structures revealed the presence of maleic acid in a pocket formed by two different chains, far away from the active site, while the D-2-amino-3-phosphono-propionic acid was found bound at the active site.

CONCLUSION

MIDAS was proved to be a reliable method to identify enzyme-metabolite interactions and the results obtained so far are promising and revealed a new site of interaction in the HsFH structure.

Keywords: fumarase, enzyme-metabolite interaction, cocrystallization

Supported by: FAPESP and CNPq

08630 - Poster Session

DB.08 - Structural studies of a J-domain co-chaperone by NMR

Glaucia Pinheiro de Castro ¹, Carolina O Matos¹, Fabio CL Almeida², Carlos HI Ramos¹

¹Organic Chemistry, University of Campinas (São Paulo, Brasil), ²Institute of Medical Biochemistry and Nucleus for Structural Biology and Bioim, Federal University of Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

HSP40 co-chaperones cooperate with HSP70 in aiding proteins to fold in the cell. HSP40 binds a partially folded protein and delivers it to be folded by HSP70. Several lines of investigation have been used to understand the interaction between the J-domain and HSP70, however, a detailed mechanism of interaction is still missing.

OBJECTIVES

This work presents a high-resolution structure of the J-domain of Sis1, an HSP40 from Saccharomyces cerevisiae, solved by NMR. Chemical shift protection approaches were used to identify the residues that change conformation in the full-length protein and are affected by the presence of HSP70.

MATERIALS AND METHODS

All NMR samples were prepared in 25 mM Tris-HCl pH 7.5, 200 mM NaCl and 10% D2O. Sis1 was at 200 uM, Sis1 J-domain at 100 to 250 uM, and HSP70 peptide at 10 mM. NMR spectra were acquired at 30°C on Bruker 900, 800 MHz, or 600 MHz spectrometers at CNRMN-UFRJ.

DISCUSSION AND RESULTS

Six residues were the most affected in both cases: V2, D9, R27, T39, F52, and R73 in the full-length protein and A29, H34, T39, F45, F52, and Y67 in the presence of DnaK/HSP70. Since two of the residues, T39 and F52, as well as two regions 27-39 and 67-73, appear to be equally affected, this work suggests that the J-domain in the full-length HSP40 may be in a conformation favorable to bind HSP70, facilitating the interaction between the two proteins.

CONCLUSION

The structure of the isolated J-domain and J-domain in Sis1 are similar, but there are remarkable small differences, which may account for the Sis1 function.

Keywords: NMR, co-chaperone, J-domain

Supported by: Fapesp

08603 - Poster Session

DB.09 - Potential Targets For Leishmaniasis Disease: New Found Binding Sites And Biophysical Characterizations

Natanael Endrew Souto Maior Torres Bonfim^1,2, Simone Queiroz Pantaleão², Leonardo de Azevedo Calderon¹, Ana Lígia Barbour Scott²

¹Departamento de Medicina, Centro de Estudos de Biomoléculas Aplicadas à Saúde, Fundação Oswaldo Cruz, Fiocruz Rondônia e Universidade Federal de Rondônia (Porto Velho-RO, Brazil), ²CMCC, Laboratory of Computational Biophysical and Biology , Universidade Federal do ABC (Santo André-SP, Brazil)

INTRODUCTION

Peptides can participate of in several physiological processes, from biological responses as well as modulation of biochemical responses. Their biological activity is determined by the sequences of specific amino acids which enables them to design short synthetic peptides with high specificity, stability and to ease of synthesis. In silico experiments are widely used in pharmacokinetic experiments reducing the time it takes for a drug to reach the market. However, the selection of targets and definition of the region where the drug will interact is charge for the design of peptide to act as a inhibitor.

OBJECTIVES

We propose a pipeline to identify sites that can be good target for these peptides inhibitors.

MATERIALS AND METHODS

Until now, we tried this protocol with 6 potential target proteins for Leishmaniasis described in the literature. Binding sites were detected and characterized with PeptiMap < /i> and FTMap < /i> during a first step. In a second moment, we investigated the volume and area of these sites with CHIMERA, analyzing several aspects as: i) electrostatic map for the target (using the server BLUUES), ii) protein surface accessibility and electrostatic contribution of polar residues (using the server TKSA-MC) were considered. Finally, the collective motions and structural dynamics was analysed using l elastic network modes models with the server DYNOMICS.

DISCUSSION AND RESULTS

We found 6 new potential binding sites for each target protein, considering peptides as inhibitors . We present the results for each protein, descrining their characteristics studied.

CONCLUSION

The initial results showed good results for these targets. This pipeline will be improved and tried with another systems in order to be used in the drug design process with the purpose to obtain a better specificity of these compounds.

Keywords: Biophysical Characterization, Rational Drug Design, Computational Biology

Supported by: FAPERO, FAPESP, CNPq and CAPES

08428 - Poster Session

DB.10 - Evaluation of bovine lactoferrin as an antiprion drug

Caroline Augusto Barros ¹, Natalia Ferreira³, Jerson Silva^2,4, Tuane Vieira^1,2,4

¹Biotechnology, Federal Institute of Rio de Janeiro (Rio de Janeiro, Brazil), ²UFRJ, National Institute of Science and Technology for Structural Biology and Bioimaging (Rio de Janeiro (RJ), Brazil), ³NIH, National Institutes of Health (Montana, USA), ⁴UFRJ, Institute of Medical Biochemistry Leopoldo de Meis (Rio de Janeiro, Brazil)

INTRODUCTION

The cellular prion protein (PrPC) is found in various tissues but abundantly in the central nervous system. A structural modification can occur on its endogenous rich α-helix form to a pathogenic isoform, PrP scrapie (PrPSc), turning into a β-sheet rich structure. This conversion triggers protein aggregation, which accumulates in the nervous tissue and progressively causes the loss of neuronal cells. Bovine lactoferrin (bLf) is known by its multiple functions, such as antiviral, antimicrobial and others. Lf is found in brain cells that were damaged by various neurodegenerative diseases, such as Parkinson’s and Alzheimer's disease. It is important to investigate the possible antiprion activity of bLf because little is known about the role of this protein in prion disease.

OBJECTIVES

Our goal was to evaluate the interaction between recombinant PrP and bLf, characterizing the molecular details involved in this interaction.

MATERIALS AND METHODS

The interaction of the complex PrP-bLf was monitored by techniques such as polarization, dinamic and static light scattering, circular dichroism and isotermal titration calorimetry. The RT-QuIC assay was performed to induce the in vitro formation of fibrillar aggregates. The dot-blot assay was used to assess whether apo and holo-bLf were able to decrease the presence of proteinase k resistant PrP in ScN2a cells.

DISCUSSION AND RESULTS

We showed that apo and holo bLf were able to interact with similar affinity, but holo bLf induces the formation of an oligomeric complex. PrP N-terminal domain modulated this interaction. We observed that bLf was able to decrease the presence cellular proteinase K resistant PrPSc. Both apo and holo-bLf were able to totally inhibit in vitro fiber formation even at very low concentrations. This effect was observed using infected brain homogenates, liquor from CJD patients or in vitro produced fibrils as seeds.

CONCLUSION

These studies are important to understand the possible application of bLf as an antiprion agent.

Keywords: bovine lactoferrin, prion protein, interaction

Supported by: FAPERJ, CNPq, INBEB and CAPES

08768 - Poster Session

DB.11 - Structural insights into SARS-CoV-2 Main Protease maturation process

Gabriela Dias Noske¹, Aline Minali Nakamura¹, Victor Oliveira Gawriljuk¹, Rafaela Sachetto Fernandes ¹, Andre Schutzer Godoy¹, Glaucius Oliva¹

¹Instituto de Física de São Carlos, Universidade de São Paulo (, Brazil)

INTRODUCTION

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the global pandemic. The viral genome contains the ORF1ab, that codifies two polyproteins containing the non-structural proteins, essential for viral replication. The Main Protease (Mpro) is responsible for cleavage of the viral polyproteins in 11 sites, including its own N and C-terminus. Due to its importance in the viral cycle, it represents an important target for antiviral development. Yet, the details of its self-maturation process remain still poorly understood.

OBJECTIVES

In here, we used the MANACA beamline at Sirius to characterize the mechanism of self-maturation of the SARS-CoV-2 Mpro.

MATERIALS AND METHODS

For that, three different constructs were obtained: the immature form (IMT Mpro), the native form (Mpro) and an inactive mutant (C145S Mpro). All constructs were biochemically and structurally characterized

DISCUSSION AND RESULTS

The IMT Mpro crystal structure revealed several structural changes compared with native protein, including differences in the substrate binding pocket. Also, this form of the enzyme exhibited reduced activity when compared with mature form, as well as to form monomers in solution. The crystal structure of C145S Mpro revealed a complex with the endogenous N and C-terminal peptides substrates during the formation of the tetrameric complex. Further biochemical characterization revealed how oligomeric state shifts overtime in this sample. Based on all evidences, we propose a mechanism for Mpro maturation process that start with the formation of an intermediate immature dimer for N-terminal cleavage, followed by the association of two dimers and C-terminal cleavage resulting in the mature form of the enzyme.

CONCLUSION

The elucidation of this process sheds light in our understanding of viral cycle, and can be used to propose specific inhibitors targeting intermediate states of the SARS-CoV-2 Mpro.

Keywords: Main-Protease, maturation, SARS-CoV-2

08336 - Poster Session

DB.12 - Structural characterization of the type VI secretion system of Klebsiella pneumoniae: the inner tube Hcp protein

Marcos Caique Santana Silva ¹, Nadine Tonelli Cavalari¹, Fábio Ceneviva Lacerda de Almeida¹, Marcel Menezes Lyra da Cunha¹, Gisele Cardoso de Amorim¹

¹Campus Duque de Caxias, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil), ²Centro Nacional de Ressonância Magnética Nuclear, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil), ³Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Klebsiella pneumoniae is the causative agent of acute infections of the respiratory and urinary tracts. These infections represent a major challenge to public health, as strains resistant to multiple antibiotics are circulating throughout the world. The comprehension of the mechanisms associated with virulence is important for understanding and fighting these infections. Comparative analysis of the genomes of K. pneumoniae revealed that all strains analyzed contain genes encoding type VI secretion system (T6SS) proteins. T6SS is a needle-like complex made up of about 13 families of proteins. It's used by the bacteria to deliver toxins into target eukaryotic and prokaryotic cells. However, the molecular mechanisms involved in the secretion of effector proteins have not yet been fully elucidated.

OBJECTIVES

In this work, we intend to characterize the structure of the Hemolysin Co-regulated Protein (Hcp), responsible for the formation of the inner tube of the T6SS and for the transport of effector proteins to the interior of a target cell.

MATERIALS AND METHODS

Recombinant expression of Hcp2; Nickel affinity purification; Negative staining transmission microscopy; Thermal Shift.

DISCUSSION AND RESULTS

We successfully overexpressed and purified the K. pneumoniae Hcp2 in Escherichia coli strains, but it has low stability in vitro, forming aggregates and precipitating. Aggregates were processed for negative staining and small structures of different sizes similar to hexameric Hcp were observed by transmission electron microscopy. Also, using Thermal Shift assay, we were able to optimize the ideal pH for the protein buffer, increasing its stability. Aiming to optimize in vitro stability and stabilize a single conformational state of the protein, site-directed mutations were performed. We are currently overexpressing the mutants and doing in vitro stability tests.

CONCLUSION

Our data will contribute to the better understanding of the internal tube assembly and transport of effector proteins through the T6SS of K. pneumoniae, promoting the basis for the developing of new drugs.

Keywords: Klebsiella pneumoniae, HCP, T6SS

Supported by: FAPERJ

08377 - Poster Session

DB.13 - Structure and biophysical characterization of the exfoliative E toxin mutant of Staphylococcus aureus

Angela Rocio Nino Santisteban ¹, Carolina Gismene¹, Ricardo Barros Mariutti¹, Raghuvir Krishnaswamy Arni¹

¹Multiuser Center for Biomolecular Innovation, Department of Physics, Universidade Estadual Paulista "Júlio de Mesquita Filho" (SP, Brazil)

INTRODUCTION

Staphylococcus aureus is a commensal and opportunistic bacterium that can infect a variety of hosts, including humans and different species of cattle, represents a global public health problem, and causes enormous damage to the livestock industry. Exfoliative toxin E (ETE) is a virulence factor that facilitates the spread of the pathogen by inducing the generalized detachment of the granular layer of the epidermis in sheep. This serine protease is inactive in its native state. Occasional secondary interactions, which are still unknown in this system, and uncommon between serine proteinases induce specific hydrolysis of the peptide bond between Glu381-Gly382 of desmoglein 1 (Dsg1).

OBJECTIVES

A better understanding of the mechanism of action of ETs is necessary to develop strategies to inhibit the pathogen. This work seeks to elucidate the mechanisms and modes of action at the atomic level, in interactions with the substrate Dsg1.

MATERIALS AND METHODS

For this, the native and mutant ETE protein (Ser219Ala) were expressed in E. coli BL21 (DE3) -T1R. Subsequently, they were purified by gel filtration, on a Superdex® 75 10/300 GL column in 20 mM MES buffer pH 7.03, and pH 5.5. We analyzed its secondary structure by circular dichroism, showing that both proteins have a predominance of random coil, in dynamic light scattering the particle size for the two proteins was 2.093 ± 0.3 (r.nm).

DISCUSSION AND RESULTS

Thermal unfolding experiments using circular dichroism and differential scanning calorimetry allowed us to establish the values of the melting temperature (49.0 ± 1.4), the calorimetric enthalpy (220 ± 50), and the Van't Hoff enthalpy (270 ± 70). Crystals obtained from the proteins were diffracted in the MANACÁ line of SIRIUS from Campinas-SP, with an initial resolution of 2.4 Å.

CONCLUSION

Data collected from ETEmut diffraction are in the process of refinement and will be used to analyze the functioning of the protein.

Keywords: Staphylococcus aureus, exfoliative toxin E (ETE), X-ray crystallography

Supported by: CAPES

08226 -

EA. - Mechanical properties of mitochondria depend on cytoskeletal integrity

Agustina Belén Fernández Casafuz ¹, María Cecilia De Rossi², Luciana Bruno¹

¹Instituto de Cálculo - CONICET, Universidad de Buenos Aires (, Argentina), ²Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (, Argentina)

INTRODUCTION

Mitochondria are organelles involved in many cellular processes, such as ATP generation and the maintenance of cell homeostasis. The functional requirements of a cell correlate with changes in mitochondrial dynamics, form and positioning. Recent research demonstrated that the cytoskeleton and associated proteins play a crucial role in regulating mitochondrial dynamics, organisation and function. For instance, actin filaments facilitate mitochondrial fission, while microtubules act as tracks for mitochondrial transport mediated by molecular motors. Although the interactions between mitochondria and the cytoskeleton have been found to alter mitochondrial function, the mechanisms underlying this phenomenon are largely unknown.

OBJECTIVES

In this work we explore how the integrity of the cytoskeleton and the forces exerted by molecular motors associated to these networks affect the morphology and the mechanical properties of mitochondria.

MATERIALS AND METHODS

We use an experimental approach in which we analyze confocal microscopy images of mitochondria labeled with mitoTracker Deep Red in Xenopus laevis melanophore cells, both in control conditions or treated with latrunculin, a drug that depolymerizes the actin filaments. Using a filament tracking routine, we recovered the longitudinal coordinate of individual mitochondria from the confocal images and computed the tangent angle along the organelle shape.

DISCUSSION AND RESULTS

We calculated the ensemble average of the tangent angle persistence and found an exponential behaviour. We estimated the persistence length as the characteristic length of an exponential fitting of the data, this parameter is associated with the organelle’s flexural rigidity and its capacity to deform.

CONCLUSION

Our results show that mitochondria are less deformed in the absence of filamentous actin suggesting that contacts with this network are a key factor in the regulation of mitochondrial morphology and probably affect the organelle dynamics.

Keywords: cytoskeleton, mechanical properties, mitochondria

Supported by: Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (Argentina)

08755 - Poster Session

EA.01 - Effects of the exposure of different cell lines to the steroidal hormone gestrinone

Francisco Mota Tostes ¹, Fabiana Carneiro¹, Gisele Amorim¹

¹Núcleo Multidisciplinar de Pesquisa Xerém em Biologia (NUMPEX), Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ²Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Gestrinone is a steroidal hormone with anti-estrogen and anti-gonadotropic actions, used for the treatment of endometriosis, contraception, and other estrogen related conditions. However, there are no studies analyzing the systemic effects of this hormone, what would be important to prove its efficacy and safety.

OBJECTIVES

Thus, we proposed an analysis of its effects on different cell lines: mammary, uterine, endothelial, and hepatic, at different concentrations and exposure times. Cell viability in the presence of the hormone was assessed by MTT.

MATERIALS AND METHODS

So far, we analyzed HuH-7 (hepatocyte) and HUVEC cells (from umbilical cord endothelium), in the absence and presence of different concentrations of gestrinone: 1, 5, 10, 50 and 100 μmol/L.

DISCUSSION AND RESULTS

Both cell lines showed a concentration-dependent reduction in viability when compared to the control after 24h. After 48h, cell viability decrease was observed at the lowest tested hormone concentration (1 μmol/L),

CONCLUSION

suggesting that, in addition to the concentration, the exposure time has also an important impact on cell viability. Gestrinone will be tested in the other mentioned cell lines and the metabolites secreted by these cells will be identified using nuclear magnetic resonance, followed by metabolomic analysis, which may provide us a broad profile of the cellular pathways affected by gestrinone. Keywords: gestrinone, implant, endometriosis Acknowledgements: FAPERJ, Formédica Rio

Keywords: gestrinone, endometriosis, implant

08355 - Poster Session

EA.02 - Artichoke extract reduces DNA damage in the blood of mice submitted to animal model of obesity

Igor da Silva de Souza ¹, Mariella Reinol da Silva¹, Jéssica S. Abel¹, Catarina B. C. Bressan¹, Alexandre Piccinini¹, Isabel B. Becker ¹, Mariana P. Oliveira¹, Gabriela S. Bett¹, Larissa E. Silva ¹, Daniele H. Salla ¹, Talita F. Mendes¹, Adriani P. Damiani ², Lígia S. Dagostin², Vanessa M. Andrade ², Thais C. Vilela ¹, Gislaine T. Rezin¹

¹Laboratório de Neurobiologia de Processos Inflamatórios e Metabólicos, Universidade do Sul de Santa Catarina (Santa Catarina, Brasil), ²Laboratório de Biomedicina Translacional, Universidade do Extremo Sul Catarinense (Santa Catarina, Brasil)

INTRODUCTION

Obesity results from excess fat stored in adipose tissue, developing a low-grade chronic inflammatory disease. Some plants admit substances that help to treat the effects of obesity, artichoke, for example, has beneficial properties to health.

OBJECTIVES

Evaluate the effect of artichoke on the levels of damage to deoxyribonucleic acid (DNA) in mice submitted to an animal model of obesity.

MATERIALS AND METHODS

24 male Swiss mice [Mus musculus] with 30 days of age, weighing from 25 to 35 grams, were used. The mice were divided into two groups, one classified as obese and the other non-obese. 12 mice belonging to the obese group were fed with a high-fat diet and the other 12 were fed with a normolipid diet. Water and feed were provided and maintained in light/dark cycles of 12 hours each and a temperature of 23°C. After six weeks of obesity induction, orally and gavage, the animals received daily treatment with artichoke extract at a concentration of 370 g/ml for four weeks. The mice were killed by decapitation, followed by blood collection and cerebral cortex. Afterwards, the structures were sent to perform the comet assay and check for DNA damage.

DISCUSSION AND RESULTS

Greater damage was observed in the blood and cerebral cortex of animals in the obese group compared to the control group. A significant reduction in DNA damage in the blood was seen with the artichoke-based treatment, but in the cerebral cortex the reduction was not significant. The active ingredient of the artichoke leaf extract was carried out by chromatography. The chromatographs showed peaks corresponding to chlorogenic acid (4.2 per minute), on the other hand, caffeic acid was not expressive.

CONCLUSION

Although the results were positive for blood, further biochemical investigations are important to elucidate the different mechanisms of action of artichok/e extracts.

Keywords: Obesity, artichoke, DNA damage

08403 - Poster Session

EA.03 - Sugarcane arabinoxylan composition profiling using combinations of hemicellulolytic enzymes

Carolina Victal Garbelotti ¹, Richard John Ward¹

¹Química, Faculdade de FIlosofia Ciências e Letras de Ribeirão Preto - Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Lignocellulosic biomass is an abundant source for biofuels and food supplements production, but its recalcitrant and heterogeneous structure is a major factor limiting industrial use. The main plant cell walls components are cellulose, a homopolymer of β-1,4 linked glucoses organized in microfibrils, and hemicellulose, an heterogenous polymer containing pentoses, hexoses and sugar acids. Xylans, the most common hemicellulose, consist of a heteropolysaccharide with a backbone chain of β-1,4 linked xyloses that can be branched with arabinose, glucuronic or 4-O-methyl glucuronic, ferulic and p-coumaric acids and acetyl groups. Use of diverse hemicellulolytic enzymes is an alternative for xylan deconstruction since enzymes break specific linkages, producing less subproducts.

OBJECTIVES

In this work we propose to use combinations of 5 hemicellulolytic enzymes: Arabinofuranosidase (GH43), Endoxylanases (GH10 and GH11), Acetyl Xylan Esterase (CE4) and α-Glucuronidase (GH67) to obtain a composition profile and a better enzyme combination for a most complete degradation of sugarcane extracted arabinoxylan.

MATERIALS AND METHODS

For this, enzymes with sequences cloned in pET-28 vectors were expressed in Escherichia coli STAR strain, purified with nickel affinity chromatography, and reacted against sugarcane DMSO extracted arabinoxylan powder in a 2% suspension in buffer, in different combinations, to access the effect of the enzymes both alone and in groups. Reaction products were analysed by hydrophobic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS) and reducing sugar quantification.

DISCUSSION AND RESULTS

Mass spectrometry results analysis indicated the arabinoxylan has acetyl (Ac) and methyl glucuronic (MG) modifications in the main xylose backbone and arabinofuranosidase activity indicated the presence of arabinoses. A recalcitrant structure, that no enzyme combination was able to hydrolyse, was also observed and the best combination observed for hydrolysis was GH10, CE4 and GH67.

CONCLUSION

This method can be extended to more complex polymers to access its structure and look for a better enzymatic treatment for complete hydrolysis.

Keywords: Hemicellulolytic Enzymes, Arabinoxylan, Mass Spectroscopy

Supported by: FAPESP

08236 - Poster Session

EA.05 - Ahp1 is an important peroxiredoxin under stress by organic peroxide under conditions of peroxisome biogenesis in yeast

CAROLINE GONCALVES DE GOES ¹, Victor Faria¹, Fernando Gomes¹, Luis Eduardo Netto¹

¹Department of Genetics and Evolutionary BiologyInstitute of Biosciences, Universidade de Sao Paulo (, Brazil)

INTRODUCTION

The β-oxidation of fatty acids occurs in peroxisomes, generating H2O2, that may cause oxidative insults. Catalase protect cells against H2O2 and is located in peroxisomes. However, deletion of the catalase gene does not provoke drastic phenotypes, suggesting that other enzymes may also participate in the protection of cells against oxidants derived from the peroxisomes. Peroxiredoxin (Prx) enzymes are Cys-based peroxidases that are highly abundant and efficient in hydroperoxide reduction. Ahp1 (alkyl hydroperoxide reductase 1) is a Prx from Saccharomyces cerevisiae with the ability to efficiently reduce alkylhydroperoxides and other peroxides, such as H2O2. The amino acid sequence (AHL) of this protein presents elements that suggest its localization in peroxisome.

OBJECTIVES

We analyzed the functions of Ahp1 under distinct physiological conditions, employing a ΔAhp1 (cells with the Ahp1 gene deleted) strain.

MATERIALS AND METHODS

Cells treated with 1mM tert-butyl hydroperoxyde (tBHP) underwent a growth arrest, in medium containing glucose or glycerol. By the ferrous oxidation-xylenol orange (FOX) assay, we also observed that the duration of the growth arrest correlated with the amounts of tBHP remaining in culture medium.

DISCUSSION AND RESULTS

In cells treated with 1mM tBHP, the lag time to recover cell growth was longer for ΔAhp1 (about 72 hours) than for wild type cells (about 48 hours) in Synthetic Defined medium (SD medium). By the ferrous oxidation-xylenol orange (FOX) assay, we also observed that the duration of the growth arrest correlated with the amounts of tBHP remaining in culture medium. When tBHP levels dropped, yeast growth was restored in both WT and ΔAhp1 strains. Cell fractionation-western blot assays are ongoing in an attempt to determine the cellular localization of Ahp1 under distinct physiological conditions.

CONCLUSION

Ahp1 protects yeast from exogenously added organic hydroperoxides.

Keywords: Metabolism, Oxidants, Alkylhydroperoxide reductase 1

08911 - Poster Session

EA.06 - The p-chloro-diphenyl diselenide (p-ClPhSe)2, an organoselenium compounds, modulates the glycolytic pathway through an insulin-mimetic action in different experimental models

Caroline Brandão Quines ¹, Flávia Suelen de Oliveira Pereira¹, Alisson G.R. Santos¹, Cristina W. Nogueira², Gilson R. Zeni², Daiana Silva Ávila¹

¹Laboratory of Biochemistry and Toxicology in Caenorhabditis elegans, Federal University of Pampa (Uruguaiana, Rio Grande do Sul, Brasil), ²Federal University of Santa Maria, Laboratory of Synthesis, Reactivity, Pharmacological and Toxicological Evaluation of Organochalcogen Compounds (Rio Grande do Sul, Brasil)

Organoselenium compounds have been described by modulating rodents metabolism by stimulating glucose uptake in their liver and muscles. Notably, p-chloro-diphenyl diselenide (p < /i>-ClPhSe)₂ restores the metabolic defects induced by obesity by regulating the activities of enzymes involved in glucose metabolism, thus suggesting an insulin- like effect. However, to verify this putative mechanism, a genetically tractable animal model was required. We investigated the effect of (p < /i>-ClPhSe)₂ in modulating the insulin-like pathway in Caenorhabditis elegans (C. elegans). Knockout mutants of insulin/IGF-1 like signaling were used in this study, TJ1052 (age-1(hx546) II), VC204 (akt-2(ok393) X), DCR3791 (pfk-1.1(ola72) X) and CF1038 [daf-16 (mu86)], besides Bristol wild-type strain (WT-N2). First, we have confirmed that glucose and triacylglycerides basal levels were reduced by (p-ClPhSe)₂ 30 minutes treatment (from 1μM). Furthermore, we established that (p-ClPhSe)₂ reduced the glucose levels in C. elegans in an age-1, akt-1 and 2 and daf-16 dependent-manner. Considering that DAF- 16 can be activated by other pathways and to confirm that (p-ClPhSe)₂ was acting by this pathway, we submitted age-1 mutants to RNAi feeding to knockdown daf-16 gene. Our results demonstrate that the reduction in glucose and triacylglycerides levels caused by (p-ClPhSe)₂ was lost in age-1/daf-16 worms. In conclusion, (p < /i>-ClPhSe)₂ can reduce glucose and triglycerides levels by modulating insulin/IGF-1 like signaling in an AGE-1/DAF-16 dependent-manner in C. elegans.

Keywords: Organoselenium compounds, metabolism, C.elegans

08493 - Poster Session

EA.07 - Glucocorticoids decrease the thermogenic capacity and increase the triacylglycerol synthesis by glycerokinase activation in brown adipose tissue of rats

Ana Paula de Assis ¹, Karine Emanuelle da Silva¹, Natalia Ennes da Silva Lautherbach¹, Maria Antonieta Rissato Garófalo², Neusa Maria Zanon², Luiz Carlos Carvalho Navegantes², Valéria Ernestânia Chaves³, Isis do Carmo Kettelhut¹

¹Department of Biochemistry and Immunology, Ribeirão Preto Medical School - University of São Paulo (, Brazil), ²Department of Physiology, Ribeirão Preto Medical School - University of São Paulo, (Brazil), ³Laboratory of Physiology, Federal University of São João del-Rei, (Brazil)

INTRODUCTION

The maintenance of adequate triacylglycerol (TAG) stores is essential for normal brown adipose tissue (BAT) functioning and requires a continuous supply of glycerol-3-phosphate (G3P).

OBJECTIVES

This study aimed to investigate the effects of glucocorticoids on thermogenic capacity and in G3P generation pathways for TAG synthesis in interscapular brown adipose tissue (IBAT) of rats.

MATERIALS AND METHODS

Male Hannover rats received a single daily injection of dexamethasone (DEXA) (1 mg/Kg) or saline 0,9% during 7 days (CEUA protocol 195/2018). The mitochondrial proteins content, the temperature after noradrenaline stimulation, and noradrenaline content were measured in IBAT. The generation of G3P was evaluated by glycolysis, glyceroneogenesis, and direct phosphorylation of glycerol, respectively, by 2-deoxyglucose uptake, phosphoenolpyruvate carboxykinase (PEPCK) activity and pyruvate incorporation into TAG-glycerol, and glycerokinase (Gyk) activity and glycerol incorporation into TAG in IBAT.

DISCUSSION AND RESULTS

DEXA treatment increases the IBAT mass and lipid content probably by increasing the de novo fatty acid (FA) synthesis, evaluated by increased glucose-6-phosphate dehydrogenase and ATP citrate lyase activities (79% and 48% respectively), compared to control. DEXA increases the content (~55%) and activity (~41%) of Gyk, without affecting the glucose uptake and glyceroneogenesis. DEXA reduces the glycerol incorporation into TAG (~54%), the AQP7 content (~50%), and the rate of basal glycerol release (~54%) in IBAT. In addition, DEXA decreases the thermogenic capacity of IBAT, evidenced by a reduction in the content of mitochondrial proteins, including UCP-1, and the respiratory complexes, reduction in the noradrenaline content (53%), and the capacity of IBAT to increase the temperature after noradrenaline stimulation.

CONCLUSION

Our data suggest that direct phosphorylation of glycerol by Gyk may be responsible for maintaining the supply of G3P for the increased esterification of FA and TAG synthesis in IBAT from DEXA-treated rats. The reduction of IBAT thermogenic capacity in these animals could be probably due to reduced sympathetic stimulation of IBAT.

Keywords: Brown adipose tissue, Glucocorticoids, Glycerokinase

08681 - Poster Session

EA.08 - High-density lipoprotein remodeling associates with COVID-19 severity: a quantitative proteomic study

Douglas Ricardo de Souza Junior ¹, Amanda Ribeiro Martins da Silva¹, Lívia Rosa-Fernandes², Giuseppe Palmisano², Graziella Eliza Ronsein¹

¹Departamento de Bioquímica, Universidade de São Paulo (São Paulo, Brasil), ²Departamento de Parasitologia, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Coronavirus disease 2019 (COVID-19) has caused more than 4 million deaths worldwide. Alterations in lipid profile, including lower levels of high-density lipoprotein (HDL)-cholesterol and high levels of triglycerides have been linked to disease severity. Although important for lipid metabolism, HDL may also play a role in immune response in infectious diseases. Due to its very complex protein composition, HDL proteome is altered in several diseases, including metabolic, inflammatory and infectious diseases.

OBJECTIVES

We used quantitative proteomics to test whether alterations in HDL proteome associate with COVID-19 severity.

MATERIALS AND METHODS

COVID-19 patients (n=41) were divided into two groups according to disease severity (hospitalized and non-hospitalized subjects). Levels of 29 HDL proteins were quantified by high resolution mass spectrometry.

DISCUSSION AND RESULTS

We showed levels of five proteins were increased by more than 50% in hospitalized patients when compared to non-hospitalized ones. Those proteins were serum amyloid A 1 and 2 (SAA1 and SAA2), pulmonary surfactant-associated protein B (SFTPB), apolipoprotein F (APOF) and inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4). On the other hand, phospholipid transfer protein (PLTP) and apolipoproteins A2 (APOA2) and L1 (APOL1) were reduced by more than 30% in those same hospitalized patients. Apolipoprotein M (APOM) levels within HDL negatively associated with odds of death due to COVID-19. Furthermore, HDL proteins were able to classify COVID-19 subjects into those two groups (error rate of 5%).

CONCLUSION

Our results indicate an inflammatory remodeling of HDL proteome which reflects the severity of COVID-19 infection and contribute to the putative role of HDL in infectious diseases.

Keywords: high-density lipoprotein, proteomics, COVID-19

Supported by: FAPESP

08913 - Poster Session

EA.09 - Metabolic characterization of whole, parotid and submandibular/sublingual saliva: a valuable tool for diagnostics

Eleonora Quartieri ^1,2, Margherita Pezzi³, Rita Antonelli³, Maria Vittoria Viani³, Benedetta Ghezzi³, Giacomo Setti³, Tecla Ciociola¹, Elena Ferrari¹, Emanuela Casali¹, Mariana Gallo¹, Marco,M. Meleti³, Thelma A. Pertinhez^1,2

¹Dept. Medicine and Surgery, University of Parma (Parma, Italy), ²Azienda USL-IRCCS di Reggio Emilia, (, Italy), ³Centro Universitario di Odontoiatria, University of Parma (Parma, Italy)

Whole saliva is a mixture of different fluids secreted by various glands such as parotids and submandibular/sublingual. Since saliva is collectible by non-invasive procedures, a growing number of studies have identified specific salivary biomarkers associated with pathological and physiological alterations, promoting the development of non-invasive tools for diagnostics. (1,2) We collected three different types of saliva (whole; parotid and submandibular – sublingual), from 20 healthy volunteers and mapped every gland-specific metabolic composition. Saliva metabolites profiles have been obtained by 1H-NMR spectroscopy, adding a freeze-drying step in sample preparation. Metabolites quantification was performed using Chenomx and MestReNova software. Statistical analysis revealed a distinct profile for the whole saliva and an overlap for the parotid and the submandibular/sublingual saliva. We identified metabolites originated both from endogenous metabolism and from oral bacterial flora, whose concentration was higher in the whole saliva. By correlating the profile of the whole saliva metabolome with the periodontal health status scores, we identified a panel of 5 metabolites that can distinguish the initial phase of bleeding disease in the healthy population. The identified metabolites are related to oral bacterial metabolism, which could potentially induce a significant host response, including oral inflammation and gingival bleeding. (3) This tool could be used for the development of an early diagnostic tool in oral inflammation. References: [1]García-Villaescusa G., et al., Plos One, 1-12 (2018). [2]Lohavanichbutr P., et al., Plos One, 1-18 (2018). [3]Liebsch C, et al., J Dent Res, 642-651 (2019)

Keywords: Saliva, 1H-NMR, diagnostics

08242 - Poster Session

EA.10 - Systemic investigation of TOR kinase: a strategic regulatory hub for biomass accumulation in sugarcane

João Pedro de Jesus Pereira ^1,2,3, Marina Câmara Mattos Martins Soldi^2,3, Lauana Pereira de Oliveira^2,3, Bruno Viana Navarro^2,3, Marcos Silveira Buckeridge^2,3

¹Departamento de Bioquímica, Instituto de Química (São Paulo, Brazil), ²Departamento de Botânica, Instituto de Biosciências (São Paulo, Brazil), ³Bioetanol, Instituto Nacional de Ciência e Tecnologia (São Paulo, Brazil)

INTRODUCTION

Sugarcane (Saccharum spp) is an important crop in the Brazilian sucroenergetic scenario. In the past two decades, there was an expressively advance in the knowledge behind the physiology and molecular biology of sugarcane motivated by the challenge of exploring the potential of cellulosic ethanol. Thus, since some molecular targets function as regulators of biomass accumulation, one can achieve efficient crop yield improvements through a systemic integration of biological sugar signaling and sensing machinery. A strategic regulatory hub in plants is the Target of Rapamycin (TOR) kinase that synchronizes growth according to the nutritional/energetic status and environmental inputs, triggering important changes in carbon flux through the central and energetic cellular metabolism.

OBJECTIVES

This work aimed at investigating the role of TOR in sugarcane by using an ATP-competitive inhibitor to track how does TOR inhibition alters the primary metabolism.

MATERIALS AND METHODS

There were performed a TOR inhibition experiment with in vitro seedlings obtained through axillary meristem of sugarcane SP80-33280 variety, which were individualized and treated with the drug AZD-8055 (25, 50, and 100 μM). Another strategy adopted to investigate this signaling pathway was through bioinformatic analysis to search and filter high-quality orthologous sequences in seven publicly available genomic/transcriptome sugarcane databases.

DISCUSSION AND RESULTS

TOR suppression in sugarcane did not impact the starch content and only a few amino acids had their levels altered after 12h of treatment, diverging from classical responses of model species. These retrieved 208 sequences from the TOR kinase complex, of which 69 showed their respective predicted protein domains.

CONCLUSION

This project encompasses the guidelines of the National Institute of Science and Technology (INCT) of Bioethanol, which proposes to unveil the theoretical bases for carbon participation in the metabolic status of sugarcane, contributing to set new strategies allowing higher biomass and/or sucrose content.

Keywords: Biomass accumulation, Sugarcane, Target of Rapamycin

Supported by: CNPq (115313/2019-2; 142090/2018-2), CAPES PNPD (20131621-33002010156P0), and FAPESP (2014/50884-5; 2018/03764-5).

08543 - Poster Session

EA.11 - Project: Induction of the enzymatic and non-enzymatic antioxidant system by Trichoderma sp. in Urochloa brizantha cv. Piatã under salt stress

Juan Carlos Alvarez Pizarro¹, Roberta Dávila Pereira de Lima¹

¹Faculdade de Medicina (Famed), Universidade Federal do Cariri (Ceará, Brasil)

INTRODUCTION

Urochloa brizantha is a tropical grass species widely used as pasture for animals. Despite its adaptability, cultivation in soils with high levels of salts reduces its growth and productivity. Salinity contributes to oxidative stress, causing physiological changes and toxicity. The use of symbiotic microorganisms proved to be promising to mitigate effects generated by abiotic stresses, highlighting fungi of the Trichoderma genus. There is evidence that these fungi are producers of auxins, hormones that help tissues development, root and stem growth.

OBJECTIVES

In view of this, the work aims to evaluate the induction potential of the antioxidant system, germination and development of U. brizantha inoculated with Trichoderma sp. under conditions of salt stress.

MATERIALS AND METHODS

Seeds of U. brizantha will be sown and the inoculation of Trichoderma sp. will be simultaneously performed. The seedlings will be subjected to concentrations of 0 (control) and 75 mmol/L of NaCl and collected for analysis, 1, 7 and 14 days after the onset of salt treatment. The fungal root colonization will be determined by microscopy and plant growth will be estimated. Membrane damage will be evaluated by lipid peroxidation and percentage of electrolyte leakage. To evaluate the antioxidant system, the enzymes catalase, guaiacol peroxidase, superoxide dismutase and ascorbate peroxidase will be quantified by spectrophotometry, as well as non-enzymatic compounds ascorbic acid, tocopherols, glutathione. The content of auxins, proline, amino acids and soluble proteins will also be obtained by spectrophotometry. The activity of antioxidant enzymes will be analyzed by zymography with electrophoresis following specific staining for the gels of each enzyme.

DISCUSSION AND RESULTS

With the completion of the project, it is expected to evaluate the action of colonization of Trichoderma sp. in U. brizantha seedlings under conditions of salt stress on antioxidant responses, as well as investigating the production of auxins and determining their influence on the activity of antioxidant enzymes.

CONCLUSION

Keywords: Urochloa brizantha, Trichoderma, oxidative stress

Supported by: Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico

08822 - Poster Session

EA.12 - Fatty acid based in vitro model of Metabolism-Associated Fatty Liver Disease and its effect on oxidative stress biomarkers

Vinícius Marques Arruda ¹, Bruno Quintanilha Faria², Joyce Ferreira da Costa Guerra¹

¹Instituto de Biotecnologia, Universidade Federal de Uberlândia (Minas Gerais, Brasil), ²Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná (Paraná, Brasil)

INTRODUCTION

Metabolism-Associated Fatty Liver Disease (MAFLD) refers to a multisystem metabolic disorder, whose first stage is the accumulation of triacylglycerols within hepatocytes, known as hepatic steatosis. The progression of the disease is strictly associated with oxidative stress, which is defined by the imbalance between reactive species and the neutralizing capacity of defense systems. Among the antioxidant mechanisms, the one involving glutathione system is the main endogenous response and therefore a potential target for MAFLD.

OBJECTIVES

Thus, this study aimed to evaluate the efficiency and oxidative stress biomarkers of an in vitro model of MAFLD.

MATERIALS AND METHODS

To induce lipid accumulation, HepG2 cells were maintained in DMEM medium in absence of fetal bovine serum at 37ºC with 5% CO₂ for 24 hours, and then incubated with a solution of palmitic acid (0.7mM); or a mixture of oleic and palmitic acids (1.0 and 2.0 mM) in a 2:1 ratio, respectively, in the presence of albumin, for 24 hours. Cells were fixed with 4% paraformaldehyde, stained with Oil Red O and lipid content determined spectrophotometrically. The cytotoxicity was evaluated through the lactate dehydrogenase release method and the oxidative stress through the quantification of lipid peroxidation biomarkers by thiobarbituric acid reactive substances assay (TBARS) and total glutathione levels.

DISCUSSION AND RESULTS

All tested concentrations shown difference (p < 0.001) in intracellular lipid content compared to control, not resulting in cytotoxic effect. Also, no increase in lipid peroxidation was detected. However, reduced glutathione levels were observed in cells exposed to 1.0 (p < 0.05) and 2.0mM (p < 0.01) concentrations. The results indicate that the treatment with fatty acids is efficient in inducing steatosis in vitro, especially at 1.0 and 2.0mM.

CONCLUSION

Despite not observing changes in TBARS levels, reduced GSH levels, indicate a depletion of the intracellular antioxidant system thus, this model is a valuable tool for preclinical study of MAFLD and treatment targets related to oxidative stress.

Keywords: MAFLD, glutathione, hepatic steatosis

Supported by: FAPEMIG, CAPES, UFU

08367 - Poster Session

EA.13 - Project Analyzes of MC4R variants in patients undergoing bariatric surgery

Ricardo Batista de Oliveira ¹, Dhébora Mozena Dall'Igna¹, Ketriane Mota de Souza¹, Carla Ivane Ganz Vogel¹

¹Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular, Centro de Ciências Agroveterinárias da Universidade do Estado de Santa Catarina (Santa Catarina, Brazil)

INTRODUCTION

Obesity is a public health problem that becomes a risk factor for numerous metabolic diseases, greatly impacting healthcare costs worldwide. It is influenced by diet, exercise, and a susceptible genotype in 50-75% of early-onset severe cases. Among the most prevalent monogenic forms of obesity are variants of the MC4R gene (Melanocortin-4 receptor), responsible for generating the transmembrane receptor, acting in the central pathway of appetite regulation.

OBJECTIVES

The present study aims to evaluate the coding region of the MC4R gene of 56 patients who underwent bariatric surgery at Hospital Tereza Ramos in the city of Lages-SC, from 2016 to 2018, investigating the genotype-phenotype relationship of carriers of gene variants.

MATERIALS AND METHODS

Amplifications of the genomic region of the MC4R gene from 56 individuals were carried out using PCR and gel electrophoresis, followed by Sanger sequencing.

DISCUSSION AND RESULTS

So far, 18% of the material has been sequenced. A male patient, who had hypertension and altered blood glucose, has the missense alteration V103I.

CONCLUSION

Although bariatric surgery is the most efficient treatment of choice, there are studies in individuals with MC4R variants highlighting post-surgical weight loss failure. There are few data related to these variants in the Brazilian population, making it necessary to provide additional clinical follow-up to these patients, as well as to reduce the costs of ineffective treatments.

Keywords: MC4R, Obesity, Variant

Supported by: FAPESC, PAP 2020

08450 - Poster Session

EA.14 - Project: Biochemical and physiological characterization of the pequizeiro (Caryocar coriaceum Wittm.) submitted to saline stress.

Antonio Viana Lopes Neto¹, Juan Carlos Alvarez Pizarro¹

¹Centro de Ciências Agrárias e Biodiversidade, Universidade Federal do Cariri (Ceará, Brasil)

INTRODUCTION

The pequi tree (Caryocar coriaceum) is an important plant in the Brazilian Northeast. Soil salinity is responsible for losses in agriculture and wild environments.

OBJECTIVES

Check if the pequi tree is resistant to salinity.

MATERIALS AND METHODS

The seedlings in this work will be produced by cuttings with different concentrations of auxin (50, 150, 250, 350, 450 μg/mL). The substrate for the cutting will be sand: earth: organic compost (1:2:1). Rooted, the plants will be cultivated for two months and submitted to three levels of salinity: 0 mM (control); 50 mM (NaCl) and 100 mM salt (NaCl). For responses to salt stress, gas exchanges are verified with the Li-Cor 6400 Equipment; chlorophyll fluorescence with the Li-Cor Equipment 6400-40 (Li-Cor Inc., Lincoln, NE, USA); the chlorophyll content in acetone (80%) will be measured in DMSO extracts; potassium and sodium ions will be measured by flame photometry; chlorine will be measured by titration with silver nitrate. Proline will be quantified by a protocol using 3% aqueous sulfosalicylic acid and absorption measured by a chromophore containing toluene; the soluble sugars in ethanol extracts will be determined by the reaction with phenol/H2SO4. The leaf anatomy will be by light microscopy with the help of software. For proteomic analysis and enzyme zymogram, proteins will be extracted in phosphate buffer (pH 7.0). The mass spectrometry will be with the Gel-free/label-free protocol that analyzes the total proteins without using a 2D gel and the zymogram will use the leaf zymography technique. The activities of catalase enzymes are through the destruction of hydrogen peroxide; of superoxide dismutase by the photoreduction of nitro blue tetrazolium; aspartate peroxidase will be measured by ascorbate consumption; the guaiacol peroxidase will be determined through the oxidation of guaiacol.

DISCUSSION AND RESULTS

CONCLUSION

The perspective is to create an orchard and check if the pequi tree is resistant to salinity.

Keywords: pequi, salinity, enzymes

08803 - Drug design and delivery

EA.15 - PROJECT: Environmental contaminants with disruptor endocrine action - effects of neonatal phthalate exposure on metabolism

Thayná Martins Macario ¹, Ana Paula Santos da Silva de Oliveira^1,2

¹Núcleo Multidisciplinar de Pesquisa em Biologia UFRJ - Xerém, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil ), ²Laboratório de Endocrinologia Experimental - LEEx- ICB, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil )

INTRODUCTION

According to the World Health Organization, the number of obese people has tripled since 1975 (WHO, 2020). Exposure to different endocrine disruptors during critical periods of development, such as pregnancy, lactation and adolescence, can influence this condition. There is a class of endocrine disruptors known as obesogenic, due to its ability to modulate the metabolism, contributing to the development not only of obesity, but also other metabolic disorders such as cardiovascular disease, thyroid dysfunction, diabetes and metabolic syndrome. Phthalates, derivatives of phthalic acid and alcohols, are present in polyvinyl chloride (PVC) film, hospital materials, cosmetics and consumer products. Phthalates is classified as obesogenic endocrine disruptors and its exposure is associated with hormonal, hepatic, renal and pulmonary alterations, as well as with the appearance of some tumors. Exposure to endocrine disruptors has been well described in adult animals and humans as harmful however in critical stages with great plasticity and susceptibility to external factors as lactation and phthalate exposure and its consequences is not elucidated.

OBJECTIVES

Objective: to research the phthalates exposure (bis-(2-ethylhexyl) phthalate) during lactation by breast milk and its endocrine-metabolic consequences at short-term (weaning) and long-term (adulthood).

MATERIALS AND METHODS

Methods: Nutritional assessment, glycemic homeostasis, central adiposity, serum adipokines and hormone levels, hypothalamic regulation of energy homeostasis and lipid profile

DISCUSSION AND RESULTS

Expected results: We hope to find an association between exposure during the critical phase of lactation and a possible initially altered metabolic profile, which would lead to obese profile of the adult life

CONCLUSION

Conclusion: We suggest that phthalate is a potential agent that would be associated with the onset of late obesity.

Keywords: phthalates, metabolism, obesity

Supported by: Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro - FAPERJ

08360 - Poster Session

EA.16 - Quantitative Metabolic Analysis of Alcoholic Fermentation in Brewing Process by Nuclear Magnetic Resonance

Werner Florentino Brandão ¹, Marcel Menezes Lyra da Cunha¹, Gisele Cardoso de Amorim¹, Celso Barbosa de Sant’Anna Filho²

¹Campus Duque de Caxias, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil), ²Metrologia Aplicada a Ciências da Vida, Instituto Nacional de Metrologia Qualidade e Tecnologia (Rio de Janeiro, Brazil)

INTRODUCTION

Wort fermentation by yeast is the initial step at beer production and has a high impact on its flavor. Elucidate how the fermentation can be influenced by the source and the sugar concentration is interesting for the academy and industry. A complete map yeasts’ synthesis and consumption can help in the fine adjustment of fermentation and be a tool to improve beer production yield.

OBJECTIVES

This project aims the detailed analysis of alcoholic fermentation of high sugar content to accurately identify the metabolites and analyze the correlations between synthesis and consumption of compounds with sensory interest in beer.

MATERIALS AND METHODS

Saccharomyces cerevisiae strains were inoculated in malt extract medium (15% sugar) at 20 ° C. The 1D ¹H NOESY and ¹H ¹³C HSQC spectra were obtained on a Bruker Avance 500 MHz spectrometer for 3 days with intervals of approximately 2 hours between the series, and at intervals of 1, 7 and 14 days with variation of 3 initial inoculation conditions. The spectra were processed using the Topspin 3.5 software and the compounds were identified on the Colmar NMR platform and in the literature. The 1H NOESY and 13C 1H HSQC spectra were used using online metabolomic databases, the Chenomx program and previous work.

DISCUSSION AND RESULTS

We quantify the consumption of glucose and maltose as a function of time and relate it to the production of ethanol. Other molecules related to the fermentation processes were mapped and, in the case of acetate esters, their production was increased in the condition of greater yeast inoculum.

CONCLUSION

The results indicate that NMR can be a strong allied for the efficient identification of substances in beer. Further investigations are underway to expand the identification and the relationship between the compounds produced during fermentation and the common variables of modern beer production, such as sugars, adjuvants and different yeast strains.

Keywords: Alcoholic Fermentation, Yeast Metabolomic, Nuclear Magnetic Resonance

Supported by: Faperj

08531 - Poster Session

EA.17 - Modulations of respiratory parameters in glucose metabolism of C2C12 myoblasts and myotubes induced by capsaicin

Julia Mello Barros ^1,2, Sara Eloy de Oliveira¹, Lorena de Oliveira Fernandes Siqueira², Luisa Andrea Ketzer^1,2

¹Núcleo Multidisciplinar de Pesquisa UFRJ (NUMPEX-Bio), Universidade Federal do Rio de Janeiro - Campus Duque de Caxias (Rio de Janeiro, Brasil), ²Laboratório de Bioquímica de Vírus - Instituto de Bioquímica Médica (IBqM), Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Capsaicin (CAP) is a selective agonist for the transient receptor potential vanilloid subtype 1 and gives pungent characteristics in the genus Capsicum plants. Previous studies show that CAP can modulate muscle energy metabolism. However, the molecular mechanisms of action of the CAP are not elucidated.

OBJECTIVES

Thus, the objective of this study is to investigate the effects of CAP in glucose metabolism of skeletal muscle.

MATERIALS AND METHODS

C2C12 myoblasts and myotubes were grown in DMEM-high glucose supplemented with fetal bovine serum and antibiotics. 24 hours after growth, the culture medium was changed for DMEM- high (HG, 25 mM), medium (MG, 15 mM), or low glucose (LG, 5.5 mM) and treated with CAP concentrations for 24 h or 48 h for the experiments. Cell viability was determined by MTT assay. Oxygen consumption rate was measured by high-resolution respirometry.

DISCUSSION AND RESULTS

After 24 h of treatment with CAP (25-400 μM), compared with a control (DMSO), only the 400 μM concentration was able to induce cytotoxicity in myoblasts, while in myotubes the reduction was already observed in 150 μM in HG. The treatment with CAP 25 μM for 24 h in myoblasts stimulated in the HG condition a significant increase in basal (65,70 ± 9,91 nmol O2/ml HG X 29,24 ± 8,54 nmol O2/ml LG), maximum (94.74 ± 15.16 nmol O2/ml HG X 43.74 ± 11.33 nmol O2/ml LG) and non-mitochondrial respiration (33.80 ± 9.93 nmol O2/ml HG X 7.95 ± 2.37 nmol O2/ml LG) of the cells compared with LG. In myotubes, different concentrations of glucose and treatment with CAP seem to alter only the maximum respiration, but now, this increase was observed in LG compared with HG treatment.

CONCLUSION

Our results show that glucose has a key role in muscle energy metabolism and treatment with capsaicin can modulate cell respiration and may improve glucose signaling.

Keywords: capsaicin, skeletal muscle, glucose metabolism

Supported by: FAPERJ, CNPq

08390 -

EA.18 - Overexpression of the argonaute ALG-1 induces miRNA biogenesis and promotes oxidative stress resistance in C. elegans

Carlos Alberto Vergani Junior ¹, Raissa de Paula Moro¹, Thiago Leite Knittel¹, Silas Pinto da Silva¹, Evandro Araújo de Souza¹, Katlin Brauer Massirer¹, Marcelo Alves da Silva Mori¹

¹Departamento de Bioquímica e Biologia Tecidual, Universidade Estadual de Campinas (SP, Brasil)

INTRODUCTION

Down-regulation of components of the miRNA biogenesis pathway has been associated with chronic noncommunicable diseases and aging. Argonautes are key proteins involved in miRNA synthesis/function and have been shown to play a role in metabolism and longevity. ALG-1 is one of the argonautes required for miRNA maturation and function in C. elegans.

OBJECTIVES

We sought to investigate the mechanisms controlling the expression and function of ALG-1 in C. elegans and test whether ALG-1 overexpression is sufficient to promote miRNA biogenesis, increase stress resistance and extend lifespan.

MATERIALS AND METHODS

Worms overexpressing ALG-1 (ALG-1/OE) were subjected to lifespan and oxidative stress resistance assays (i.e., paraquat or arsenite). To identify how alg-1 is transcriptionally regulated, we used modENCODE data to search for proteins that bind to alg-1 promoter. We also performed quantitative proteomic analysis and miRNA sequencing in ALG-1/OE worms. Moreover, using knock-out mutants of miRNAs overexpressed by ALG-1/OE, we evaluated the influence of those miRNAs in the oxidative stress response of C. elegans.

DISCUSSION AND RESULTS

ALG-1/OE worms showed more resistance to oxidative stress, despite a normal lifespan under unstressed conditions. Among the transcription regulators that bind to alg-1 promoter, we experimentally confirmed that GEI-2, NHR-28, NHR-77, R02D3.7 and SKN-1 suppress ALG-1 expression. Interestingly, silencing of nhr-28 and R02D3.7 promotes stress resistance in an ALG-1 dependent manner. Global miRNA expression is increased upon ALG-1 O/E, and among the upregulated miRNAs, 8 were also upregulated in long-lived, stress resistant glp-1 mutants, where ALG-1 is also increased. The proteomic analysis revealed differences in proteins related to nucleotide binding, reproduction and DNA mismatch repair – all processes commonly related to aging.

CONCLUSION

Together, these results support the notion that ALG-1 expression can be dynamically modified to confer protection against oxidative stress, contributing to the general healthspan of C. elegans.

Keywords: miRNAs, Oxidative Stress, Metabolism

Supported by: FAPESP

08589 - Poster Session

EB.01 - Melanoma cell migration in response to red and near-infrared low-level light

Carolina Gouvêa de Souza Contatori ¹, Mayara Santana Pinto¹, Martha Simões Ribeiro¹

¹CELAP, Nuclear and Energy Research Institute (São Paulo, Brazil)

INTRODUCTION

Cell migration plays an important role in tissue formation and cancer progression. In vitro scratch assay has been used for many years to study cell migration to mimic the migration of in vivo cells, and, thus, to evaluate cancer growth. Low-level red and near-infrared light (LLL) can increase normal cell migration. However, the impact of LLL on tumor cells remains unclear.

OBJECTIVES

In this work, we aimed to evaluate the effects of a single LLL dose on melanoma cell migration.

MATERIALS AND METHODS

B16F10 (murine melanoma) cells were cultivated in RPMI medium with 10% of fetal bovine serum until they reached 80% confluency. The cell line was seeded in a 6-well plate at a density of 2x10 ⁵ cells/well in triplicate at two different moments. A wound scratch was performed to disrupt the confluent cell monolayer with a 10 μL pipette tip. Immediately after the injury, the cells were submitted to the LLL at two distinct wavelengths (660 and 780 nm) provided by a LED and a laser, respectively, delivering 3 different energies (1.3, 3.6, and 6 J) at an irradiance of 4.2 mW/cm ² . The control group was not irradiated. Cells were photographed immediately and at 3, 12, 24, and 36 h after the scratch. The wound closure was measured using ImageJ software. To evaluate the overall migration, we calculated the areas under the curve for each group.

DISCUSSION AND RESULTS

Cells exposed to the red laser at 6 J migrated slower than control. In contrast, LLL at 780 nm promoted faster cell migration when irradiated with 3.6 J.

CONCLUSION

These results suggest that low-level LEDs at 660 nm could prevent melanoma progression in higher energies. However, 780 nm should be avoided at middle energies.

Keywords: Melanoma, Photobiomodulation therapy, Scratch-wound assay

Supported by: CNPq and CAPES

08700 - Poster Session

EB.02 - Mechanisms of membrane protection by deuterated PUFA

Márcia Silvana Freire Franco ¹

¹Departamento de Bioquímica, Universidade de São Paulo - Instituto de Química (São Paulo, Brasil), ²Departamento de Física Aplicada, Universidade de São Paulo - Instituto de Física (São Paulo, Brasil), ³Drug Discovery, Retrotope (Califórnia , United States)

INTRODUCTION

Polyunsaturated fatty acids (PUFAs) constitute one of the most abundant and important components found in membrane bilayers. PUFAs stabilize protein complexes and modulate membrane properties, ensuring homeostasis of organelles and cells. However, PUFAs are highly susceptible to oxidative damage through lipid peroxidation (LPO) chain reaction which triggers atherosclerosis, cancer and neurodegenerative diseases. Selective hydrogen replacement of bis-allylic PUFA hydrogens by deuterium offers protection against LPO, but the protection mechanism is not fully understood.

OBJECTIVES

To understand the protection mechanism by deuterium substitution, Giant Unilamellar Vesicles (GUV) were prepared with H-Lin-PC, in the presence of small amounts of D-PUFAs.

MATERIALS AND METHODS

We analyzed photo-induced oxidation in the presence of 1μM of Al(III) Phthalocyanine tetrasulfonic acid chloride. The initial steps of the membrane oxidation, which consists of lipid hydroperoxidation by singlet oxygen, are characterized by fluctuations and area expansion of the GUVs. Membrane permeabilization results from further oxidation steps, forming lipid truncates aldehyds.

DISCUSSION AND RESULTS

We show that the presence of 20% of D-PUFA in the 80%of H-Lin-PC matrix of vesicles, prevents substantially the fluctuation/area increase, and the loss of contrast. The presence of tocopherol, following the same proportion of D2-PUFA-PC in H-Lin-PC, is effective in preventing the formation of pores/membrane permeabilization, however it does not inhibit the formation of hydroperoxides, resulting in area fluctuation and increase.

CONCLUSION

These findings demonstrate that a small proportion of D-PUFAs is sufficient for the protection of both contact-dependent and contact-independent oxidation processes. Deuterium reinforced lipids offer membrane protection and the relief of the oxidative stress, mitigating several diseases.

Keywords: Lipid peroxidation, Giant Unilamellar vesicles, Deuterium reinforced lipids

Supported by: FUSP - RETROTOPE

08905 - Poster Session

EB.03 - Effects of photodynamic inactivation mediated by Zn(II) porphyrin on promastigote and amastigote forms of Leishmania amazonensis

Tiago Henrique dos Santos Souza ^1,5, Camila Andrade¹, Fernanda Cabral², José Sarmento-Neto³, Júlio Rebouças ³, Beate Santos⁴, Martha Ribeiro², Regina Figueiredo⁵, Adriana Fontes¹

¹Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco (Pernambuco, Brazil), ²Centro de Lasers e Aplicações, Instituto de Pesquisas Energéticas e Nucleares (São Paulo, Brazil), ³Departamento de Química, Universidade Federal da Paraíba (Paraíba, Brazil), ⁴Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco (Pernambuco, Brazil), ⁵Instituto Aggeu Magalhães, Fundação Oswaldo Cruz (Pernambuco, Brazil)

INTRODUCTION

Photodynamic inactivation (PDI) has been attracting attention as an innovative technology to treat topical diseases, such as cutaneous leishmaniasis (CL) and infections caused by multidrug-resistant microorganisms. Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (ZnTnHex-2-PyP ⁴⁺ ) is a lipophilic water-soluble Zn(II) porphyrin with improved photophysical properties, high chemical stability, and cationic/amphiphilic character that can enhance its interaction with cells.

OBJECTIVES

Thus, this study aimed to investigate the PDI effects mediated by ZnTnHex-2-PyP ⁴⁺ on Leishmania amazonensis.

MATERIALS AND METHODS

Confocal fluorescence microscopy was explored to study the interaction of ZnTnHex-2-PyP ⁴⁺ with promastigotes. The PDI action was analyzed by cell membrane integrity, mitochondrial membrane potential (ΔΨm), and cell morphology. Promastigotes were incubated with ZnTnHex-2-PyP ⁴⁺ for 5 min at 0.62 and 1.25 μM and irradiated by a LED (410 nm) for 1 or 3 min (2.3 and 3.4 J/cm ² , respectively). PDI on amastigotes and the cytotoxicity on macrophages were also analyzed (3.4 J/cm ² ).

DISCUSSION AND RESULTS

Fluorescence microscopy revealed that parasites efficiently uptake ZnTnHex-2-PyP ⁴⁺ and displayed a punctate labeling pattern along with the cytoplasm. An intense ΔΨm depolarization was also observed, which in association with microscopy results, suggests that ZnTnHex-2-PyP ⁴⁺ may accumulate in the mitochondrion, or other well-defined structures close to it. Moreover, ZnTnHex-2-PyP ⁴⁺ at concentration as low as 0.62 μM led to the immediate inactivation of >95% of promastigotes, regardless of the light dose used. Loss of the fusiform shape and plasma membrane wrinkling were also observed. After a single treatment session in amastigotes, PDI led to a reduction of 70% in the infection index. No considerable toxicity was observed on mammalian cells.

CONCLUSION

Thus, PDI of Leishmania parasites showed in vitro efficiency at a submicromolar concentration of ZnTnHex-2-PyP ⁴⁺ , with short pre-incubation and irradiation times. The results encourage further studies in CL pre-clinical assays and PDI of other microorganisms.

Keywords: Cutaneous leishmaniasis, photodynamic therapy, ZnTnHex-2-PyP4+

Supported by: CAPES, CNPq, FACEPE, FAPESP, IAM/FIOCRUZ, FINEP and INCT-INFo.

08549 - Poster Session

EB.04 - Blue light supports the aging of the skin of Swiss mice

Isabella Favarato Benedito¹, Nayara Zanetti Comério¹, Lucas G. Rabello², Clairton Marcolongo-Pereira¹, Maurício Lawrence Freitas¹, Maurício S. Baptista³, Orlando Chiarelli-Neto ¹

¹Faculdade de Medicina, Centro Universitário do Espírito Santo (Espirtio Santo, Brasil), ²Faculdade de Medicina, Escola Superior de Ciências da Santa Casa de Misericórdia (Espirtio Santo, Brasil), ³Departamento de Bioquímica , Instituto de Química, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Solar irradiation causes skin aging by generating biomolecular damage and oxidized structure accumulations. Although the effects of ultraviolet (UV) light are well characterized, little is known about the effects of visible light. In vitro studies showed that UV-A and blue light stimulate redox processes with the generation of reactive oxygen species (ROS) and nitrogen (RNS) damaging skin cells. However, there are no in vivo studies that show the consequences of blue light on the skin and its association with UV-A. In addition there is no evidence of factors that block or even minimize damage.

OBJECTIVES

The objective here was to verify the effects of blue light (465nm) and in synergy with UV-A (365nm) in the skin of Swiss mice.

MATERIALS AND METHODS

Animal experimentation was approved by the Animal Use Ethics Committee (CEUA – 613296), with seven groups (G) (8 animals/group) divided into G0 (control group), G1 (UV-A), G2 (blue light) and G3 (UV-A+blue light). Groups G4, G5 and G6 followed the same order, but were added sunscreen (PP Photo Ultra ISDIN active unify 99) before each irradiation cycle in BlackBox Smart equipment (BioLambda – SP) with UV-A and blue doses, 20J/cm² and 100J/cm² respectively. All animals underwent dorsal trichotomy before irradiation, with subsequent photographic recording and biopsy in 3 times: zero, fifth and tenth irradiation. Statistics was performed by Origin 8.0.

DISCUSSION AND RESULTS

Through macro and microscopic analysis (HE technique at 40x magnification) it was noted that groups irradiated without PP, showed greater hyperpigmentation of the skin, indicative of melanogenesis. G3 stands out, in which 50% of the animals showed hyperkeratosis and 12% elastosis. Furthermore, accumulations were observed in G2 (blue light), suggestive of lipofuscin or hemosiderin.

CONCLUSION

Therefore, blue light was more harmful and accelerated the aging process, especially when associated with UV-A.

Keywords: Blue light, photoaging, Swiss mice

Supported by: Fundação de Amparo a Pesquisa e Inovação do Espírito Santo - FAPES

08669 - Poster Session

EB.05 - Sun-induced modifications on collagen molecular structure explored by fluorescence spectroscopy and advanced microscopy techniques

Giuseppe De Luca¹, Giuseppe Sancataldo², Valeria Vetri²

¹Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo (Exterior, Italy), ²Department of Physics and Chemistry - Emilio Segrè, University of Palermo (Exterior, Italy)

INTRODUCTION

UV light is known to induce dangerous modifications on various biological molecule, which are linked to onset of pathological conditions. Since collagen is a structural protein ubiquitous in all human body, exploring the modifications induced by sun radiation and particularly focusing on UV range, is of crucial interest.

OBJECTIVES

We here present an experimental study based on steady state fluorescence spectroscopy and time resolved non-linear optical imaging aimed at characterizing collagen modifications at molecular level.

MATERIALS AND METHODS

Collagen was exposed to single wavelength UV lamp radiation (270 nm) and to solar simulator which emits a radiation with similar characteristic to the one Sun produces in term of spectrum and power.

DISCUSSION AND RESULTS

The results were a reduction of auto-fluorescence signal, attributed to the aromatic residues and to cross-links, and a decrease of the turbidity of samples, this highlighting an overall loss of supramolecular structure, associated to a solubility increase. CD spectra also reveal structural modifications which we investigate more in depth using 8-Anilino-1-naphthalenesulfonic acid (ANS). ANS is a fluorescent dye known to bind to collagen fibers and whose fluorescence is affected by its molecular environment, and in particular its hydrophobicity. We carried out fluorescence lifetime imaging (FLIM) measurements of ANS-stained samples, analyzed by phasor approach to observe alterations in the molecular structure of collagen triple helix. As already hypothesized from ANS steady state fluorescence measurement, FLIM analysis confirm a reduction of binding sites affinity, showing a high grade of lifetime distributions heterogeneity and an overall reduction of the ANS fluorescence lifetime.

CONCLUSION

Moreover, since we observe that irradiated collagen isn’t able to self-organize in fibers, we conclude that solar radiation degrades the hydrophobic terminals of collagen triple helix, vital for its fibrillogenesis, and that ANS provide a useful and simple tool to investigate them.

Keywords: Collagen, UV-light, FLIM

08904 - Poster Session

EB.06 - Evaluating Re(I) complexes as anticancer agent

Tayná Saraiva de Lavor ¹, Tayná Saraiva^1,1,1,1, Tayná Saraiva de Lavor¹, Tayná Saraiva¹

¹Instituto de Química, Universidade Federal de Uberlândia (, Brasil)

INTRODUCTION

In recent decades, many organometallic compounds (ie, compounds containing at least one metal-carbon bond) have been extremely promising as candidates for anticancer drugs. Rhenium compounds have several intrinsic advantageous properties for the development of new anticancer drug candidates. Considering that some Re complexes have shown to be promising as candidates for anticancer agents, but have been little explored. We selected these 3 chemical structures of Re complexes, whose ligands are based on phenanthroline and lipophilicity increases as a ring is added to the ligand, to study their therapeutic potential and evaluate the interaction of these compounds with biomolecules. In addition, we also propose the use of nanocarriers to improve the therapeutic effect of the compounds.

OBJECTIVES

Study the properties of Rhenium (I) and its ability to interact with biomolecules, as well as evaluate its toxicity before and after encapsulated in polymeric nanospheres.

MATERIALS AND METHODS

Fluorescence spectroscopy was used to investigate the interaction of the Rhenium(I) complex with the biomolecules. For encapsulation of compounds, the polymeric system chosen was carboxymethylcellulose and apple pectin. The technique used consists in coacervation, which occurs the separation of phases in a colloidal solution under certain conditions, i.e. temperature, pH and solubility of the dissolution medium.

DISCUSSION AND RESULTS

The interaction studies between Rhenium complexes and BSA analyzed by Stern-Volmer treatment, indicated that as more hydrophobic is the compound stronger is the interaction with protein. Also, nanocarriers composed of carboxymethylcellulose or apple pectin has been successfully prepared by coacervation proposed method, but some characterizations are still ongoing.

CONCLUSION

Our tested compounds can be useful as anticancer agents, since preliminar data suggest stronger interaction with biomolecule. However, further studies are still ongoing for solid conclusion.

Keywords: Rhenium (I) complexes, biomolecule interaction, nanocarrier systems

08495 - Poster Session

EB.07 - Effect of membrane deformation on electrical firing in rat cortical neurons during electrophysiological measurements

Bogdana Cepkenovic ^1,2, Vanessa Maybeck¹, Andreas Offenhäusser¹

¹IBI-3 Institute of Bioelectronics, Forschungszentrum Jülich (, Germany), ²Faculty 1, RWTH Aachen (, Germany)

INTRODUCTION

From patch-clamp to 3D nanoelectrodes, tight mechanical coupling with the neuronal membrane is essential to secure the high amplitude electrical recording. As a byproduct, both approaches induce membrane deformation. In the former, the membrane is acutely deformed by the glass pipette, while in the latter, the membrane spontaneously engulfs the 3D vertical nanostructure.

OBJECTIVES

In line with the discoveries pointing to the existence of mechanosensitive ion channels in neurons, we combined electrophysiology with functional imaging to test the effect of acute and chronic membrane deformation on rat cortical neurons’ electrical properties and firing dynamics.

MATERIALS AND METHODS

To estimate the effect of patch-clamp induced acute deformation, we combined semi-blind patch-clamp with calcium-imaging. Additionally, we utilized patch-clamp to investigate whether the long-term exposure to vertical topology on 3D nanoelectrodes influences the neurons’ electrical properties. All measurements were performed on rat cortical neurons starting from 2 weeks in culture.

DISCUSSION AND RESULTS

Calcium-imaging measurements during the formation of giga-seal have demonstrated that patch-clamp targeted neurons respond to the mechanical perturbation with plateau-shaped calcium signals (N = 29). Moreover, up to 100% of neurons in 0.185 mm ² area responded in a similar trend. This finding suggests that acute deformation affects not only the targeted neuron, but also the immediate network. Furthermore, the comparison of neurons on flat surface and neurons on 3D nanoelectrodes showed no statistically significant difference in excitability and action potential firing.

CONCLUSION

Overall, these results recognize the effects of acute, patch-clamp mediated mechanical perturbation on the targeted neuron, as well as the immediate network. On the other hand, no changes were present with chronic membrane deformations during the spontaneous engulfment of vertical nanostructures.

Keywords: electrophysiology, membrane, neurons

08423 - Poster Session

EB.08 - Modulators of dopaminergic neurotransmission: effects on the behavior and expression of receptors in an experimental model of Epilepsy

Gabriela Machado Parreira ¹, Ivan Lobo de Sousa Marques¹, Márcio Flávio Dutra Moraes², Leonardo de Oliveira Guarnieri^2,3, Rafael Pinto Vieira¹

¹Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (Minas Gerais, Brazil), ²Departamento de Fisiologia e Biofísica,Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (Minas Gerais, Brazil), ³Departamento de Engenharia Eletrônica, Escola de Engenharia, Universidade Federal de Minas Gerais (Minas Gerais, Brazil)

INTRODUCTION

Epilepsies are Central Nervous System disorders characterized by irregular and recurrent electrical discharges and, frequently, epileptic patients are also affected by neuropsychiatric comorbidities, such as depression and anxiety. Both Epilepsy and comorbidities may be related to changes in dopaminergic neurotransmission.

OBJECTIVES

In the context of drug repositioning, we investigated behavioral and biochemical alterations induced by modulators of dopaminergic pathways in an experimental model of Epilepsy – Wistar Audiogenic Rats (WARs).

MATERIALS AND METHODS

Male WAR and Wistar rats were divided in four groups: Levodopa/Carbidopa (LC, 30 and 15 mg/kg), Levodopa/Benserazide (LB, 30 and 2 mg/kg), Haloperidol (H, 0.3 mg/kg) and control group (S, 0.9% saline). Doses were administered for 14 days, once a day, according to the protocol approved by Ethics Committee – 233/2019. Rotarod and open field tests were used to evaluate motor and exploratory behaviors.

DISCUSSION AND RESULTS

A significant decrease in the first fall latency in rotarod was observed in S/WAR group when compared to the S/Wistar group. Treatments with LC, LB and H in WARs attenuated the first fall latency when compared to S/WAR group. In the open field test, a significant decrease in the mobility rate, locomotion speed and total distance was observed in S/WAR group when compared to the S/Wistar group. No changes were observed in these parameters in all WAR treatment group when compared to the S/WAR groups. Interestingly, a significant increase in the type 1 dopamine receptor (DR1) expression was observed in S/WAR group when compared to the S/Wistar group. In addition, a decrease in DR1 expression was also observed in all WAR groups treated with the modulators when compared to the S/WAR groups.

CONCLUSION

In summary, our treatments were able to change behavioral aspects in WAR animals when compared to Wistar animals, also modifying the expression of receptors related to dopaminergic neurotransmission in the WAR model.

Keywords: dopamine, behavior, drug repositioning

Supported by: FAPEMIG, CNPq and CAPES

08892 - Poster Session

EB.09 - Characterization of the effects of Photodynamic therapy on membrane mimetics composed by 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt

Pedro Nunes de Oliveira Júnior ¹, David Mioto¹, Tayana Tsubone², Rosangela Itri¹

¹DFAP, University of São Paulo (São Paulo, Brazil), ²IQUFU, Federal University of Uberlândia (Minas Gerais, Brazil)

INTRODUCTION

Photodynamic therapy (PDT) is an important technique that uses chemical compounds called photosensitizers to oxidize unsaturated bonds in organic molecules in the presence of light.To better understand the response of bacteria membranes to induced photodamage, we used GUVs (Giant Unilamellar Vesicles) as a simple model of its membranes, where DOPG (1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt) is one of the main lipids on bacteria membrane.

OBJECTIVES

In order to understand the effects of oxidative stress induced in the membrane of bacterias, GUVs composed by DOPG are made and subjected to light irradiation with wavelength of the order of 655 nm in the presence of two different isomer photosensitizers (PS): Methylene Blue (MB) and Dimethyl Methylene Blue (DMMB). The objective is to compare the effect of these two photosensitizers and analyze qualitatively its effects on the liposome.

MATERIALS AND METHODS

Phase-contrast microscopy was the principal technique used in this work. It guarantees time resolution images about de GUVs. With the images, we extract information about the contrast between internal and external parts, being able to observe fluctuations of the liposome area, loss of contrast, poration, etc.

DISCUSSION AND RESULTS

Irradiation of DOPG GUVs in the presence of DMMB resulted in the contrast loss and changes in liposome area, getting smaller than before. In general, this effect was associated with a bud emission. MB had a similar effect, but the time demanded for this was greater than for its isomer.

CONCLUSION

To conclude, we consider that a better understanding of the action mechanisms of the different PS can improve the use of PDT to treat a variety of diseases. DMMB seems to be more efficient than MB in allowing the lipid bilayer to be permeated and then broken down, on the other hand, there are still many other factors to be evaluated when talking about effectiveness in implementing this for the treatment of diseases.

Keywords: DOPG, membrane mimetics, Photodynamic therapy

Supported by: conselho nacional de desenvolvimento científico e tecnológico (CNPq)

08449 - Poster Session

EB.10 - Highly fluorescent nanoprobe based on quantum dots and Enterolobium contortisiliquum trypsin inhibitor (EcTI)

Natália Regina Melo Santos ¹, Weslley Felix de Oliveira¹, Mariana Paola Cabrera³, Clovis Macêdo Bezerra Filho⁴, Maria Luiza Vilela Oliva⁴, Paulo Euzébio Cabral Filho², Adriana Fontes², Maria Tereza dos Santos Correia¹

¹Departamento de Bioquímica , Universidade Federal de Pernambuco (Pernambuco, Brasil), ²Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco (Pernambuco, Brasil), ³Departamento de Química Fundamental, Universidade Federal de Pernambuco (Pernambuco, Brasil), ⁴Departamento de Bioquímica , Universidade Federal de São Paulo (São Paulo, Brasil)

INTRODUCTION

Serine proteases play crucial roles in life maintenance, and their activities are controlled through their inhibitors. The dysregulation in this activation/deactivation mechanism can trigger diseases. EcTI is a serine protease inhibitor purified from Enterolobium contortisiliquum seeds. EcTI has high biotechnological potential, showing anti-cancer, anti-inflammatory, larvicide, and insecticide activities. Currently, fluorescent nanoprobes, able to providing information at molecular levels, are being increasingly requested. In this regard, the quantum dots (QDs) stand out due to their unique optical properties. Thus, a nanotool consisted of between QDs and EcTI can provide valuable information on the location and interaction of specific serine proteases in biological systems by fluorescence techniques.

OBJECTIVES

To develop a highly fluorescent nanoprobe based on the conjugation of QDs and EcTI for studies of the interaction with serine proteases in biological processes.

MATERIALS AND METHODS

Carboxyl QDs were synthesized and optically characterized, then covalently conjugated to EcTI. Conjugates were characterized by absorption and emission spectroscopy. The conjugation efficiency was evaluated through fluorescence correlation spectroscopy (FCS) and fluorescent microplate assay (FMA).

DISCUSSION AND RESULTS

The QDs-EcTI conjugate presented high fluorescence, and its absorption spectra did not present noteworthy changes compared to bare QDs. Using FCS, a shorter diffusion time (135.87 ± 13.77 μs) was obtained for bare QDs compared with the QDs-EcTI conjugate (529.14 ± 46.90 μs). From diffusion times, hydrodynamic sizes were estimated as 3.4 ± 0.3 nm and 13.1 ± 1.2 nm for bare QDs and QD-EcTI conjugates, respectively. Moreover, from FMA a relative fluorescence of 3,814.8% ± 533.8% was obtained compared to controls (only QDs or EcTI).

CONCLUSION

EcTI was effectively conjugated to QDs and the proposed conjugate can be a potential nanotool for studies involving serine proteases in biological systems through fluorescence-based techniques.

Keywords: quantum dots, protease inhibitor, fluorescence

Supported by: CAPES, CNPq, FACEPE, INCT-INFo, and LARNANO-UFPE

08684 - Poster Session

EB.11 - Molecular dynamics simulations and in vitro assay of POPC membrane models in the presence of phenothiazinium photosensitizers

Camila Fontes Neves da Silva ¹, Drielli Gomes Vital-Fujii¹, Helena Couto Junqueira^1,1, Maria Cecilia Oliveira^2,2, Ronei Miotto², Mark Wainwright³, Mauricio Silva Baptista¹

¹Departamento de Bioquímica, Universidade de São Paulo (Sao Paulo, Brazil), ²Centro de Ciências Naturais e Humanas, Universidade Federal do ABC (Sao Paulo, Brazil), ³School of Pharmacy & Biomolecular Sciences, Liverpool John Moores University (Merseyside, United Kingdom)

INTRODUCTION

Photodynamic therapy (PDT) treatment have been widely investigated in order to understand the photophysical mechanisms. PDT has been used for skin diseases, inactivation of virus and cancer treatment. In general PDT involves a photosensitizer (PS) molecule that it can be activate after light irradiation and generate radical species that it causes several effects in the target. In this way cell membranes composed by proteins and lipids are the major targets for photoinduced cell inactivation. After light irradiation, the free radicals and contact dependent reactions by PSs can produces oxidized lipids through lipid peroxidation process. The formation of these species can lead to membrane damage and causes cell death.

OBJECTIVES

To perform in vitro and theorical studies to analyzed PSs photochemical properties and membrane damage effects.

MATERIALS AND METHODS

Preparation of SUVs and GUVs vesicles with POPC lipid and molecular dynamics (MD) simulation using Gromacs software.

DISCUSSION AND RESULTS

Experiments performed in small unillamelar vesicles (SUVs) and giant unilamellar vesicles (GUVs), with a POPC (unsaturated lipid), showed that MB, DO15, DO16 and DO37 cause membrane damage in a small concentration, (1 μM), after light irradiation (630 nm). To better understand the results found in the experimental tests, we performed the molecular dynamics (MD) simulation of the PSs described above in a POPC membrane model. MD studies reinforce the importance of PS interaction with the cell membrane in PDT efficiency. As with the experimental results, the simulation suggests that DO15 is more efficient in generating damage to the cell membrane. Due to its proximity to the double bond of lipids, by a contact-dependent mechanism, followed by DO16, DO37, and finally MB.

CONCLUSION

All compounds entered the membrane, and those that came closer to the double bond, internalized perpendicularly to the membrane with the piperidine ring coming close to the lipid double bond, suggesting that the site of action is in this region.

Keywords: Photodynamic therapy, Photosensitizers, Membrane damage

Supported by: Fapesp

08890 - Poster Session

EB.12 - Reconstitution of Leishmania plasma membrane to understand the photodynamic effect

Maressa Donato Ferreira de Souza ^1,2, Rosangela Itri², Martha Simões Ribeiro¹

¹Center for Laser and Applications , Nuclear and Energy Research Institute (São Paulo, Brasil), ²Applied Physics, Institute of Physics (São Paulo, Brasil)

INTRODUCTION

Leishmaniasis is an important neglected disease. Photodynamic therapy (PDT) has been used to fight cutaneous leishmaniasis showing good results. However, PDT mechanisms in Leishmania parasites are not yet completely clarified.

OBJECTIVES

In this work, our objective was to develop a protocol to produce giant plasma membrane vesicles (GPMVs) from Leishmania amazonensis promastigotes to understand the mechanisms of action of methylene blue (MB)-mediated PDT on the cell membrane of parasites.

MATERIALS AND METHODS

For membrane extraction, several techniques were tested. The osmotic shock was the technique that presented the best yield and effectiveness. Phosphate and protein measurements were performed to confirm membrane extraction. For the growth of GPMVs, the best technique was electroforming using different frequencies and voltages in 4 cycles. Reconstituted GPMVs were observed by phase-contrast light microscopy. Subsequently, PDT was applied to GPMVs dispersed in an aqueous solution containing 50 μM MB and we verified the changes in permeability before and after exposure to light. The same process was applied to giant unilamellar vesicles (GUVs) with lipid compositions similar to the parasite membrane.

DISCUSSION AND RESULTS

The electroforming technique with the protocol developed in this work made it possible to obtain GPMVs from a promastigote membrane isolate of L. amazonensis. The membrane isolation technique was effective to extract the parasite's membrane while preserving lipids and proteins. In GUVs we observe an increase in the area during PDT in different compositions and loss of contrast. The GPMVs showed a loss of contrast as well as the GUVs but did not show an increase in area.

CONCLUSION

This factor could be explained by the high degree of complexity of the membrane, which contains membrane proteins in addition to containing lipids.

Keywords: Leishmania amazonensis, GPMVs (Giant Plasma Membrane Vesicles), PDT (Photodynamic Therapy)

08278 - Poster Session

EB.13 - Enhanced action of nanoencapsulated herbicide on photosynthesis and antioxidant activity in spinach leaves: toward a sustained weed control?

Montcharles da Silva Pontes ¹, Débora Ribeiro Antunes², Mariana Monteiro Lima Forini², Jaqueline Silva Santos¹, Gilberto José Arruda¹, Anderson Rodrigues Lima Caires³, Etenaldo Felipe Santiago¹, Renato Grillo²

¹Centro de Estudos em Recursos Naturais, Universidade Estadual de Mato Grosso do Sul (MS, Brazil), ²Departamento de Física e Química, Universidade Estadual Paulista (SP, Brazil), ³Instituto de Física, Universidade Federal de Mato Grosso do Sul (MS, Brazil)

INTRODUCTION

Despite a wide range of possible applications of nano-enabled pesticides, the mechanisms involved in their enhanced action remain largely unknown. Understanding the interaction between nanopesticides and plants is crucial for evaluating their potential safety application.

OBJECTIVES

Using an experimental and theoretical approach, this study aimed to investigate the target effect of paraquat-loaded chitosan/tripolyphosphate nanoparticles on the photosystem I (PSI).

MATERIALS AND METHODS

Chitosan/tripolyphosphate nanoparticles carrying paraquat was prepared by ionic gelation method. Nanoformulation was characterized, and the amount of lipid peroxidation, photooxidizable P700 reaction center content, NADPH/NADP+ ratio levels, and antioxidant enzymes were evaluated in spinach leaf tissue exposed to the nanoherbicide compared to the non-encapsulated herbicide.

DISCUSSION AND RESULTS

Biochemical traits of PSI were significantly decreased in spinach leaf tissue exposed to the nanoherbicide. Our data also revealed that nanoformulation might act promoting oxidative stress by changes observed on antioxidant enzymes. Also, the molecular docking results showed a preferential disposition of the herbicide paraquat and paraquat-tripolyphosphate complex (TPP:PQ) into the ligand domain close to FAD and Glu312. Due to the inhibitor's strategic position into the catalytic pocket, a model of electron-capture is proposed, where the herbicide disturbs the redox process NADP+ ⇌ NADPH by capturing electrons to reduce itself.

CONCLUSION

Our findings provide important insights into changes induced on targeted action mechanisms may play a key role in its increased herbicidal efficiency. Thus, our findings contribute to a better understanding of the mode of action of herbicides encapsulated in polymeric nanoparticles.

Keywords: Nano-enabled agrochemicals, Enzymes, Photosynthetic electron transport

Supported by: CAPES

08593 - Poster Session

EB.14 - Red LED irradiation impacts the cytotoxic response of murine breast cancer cells to ionizing radiation

Mayara Santana Pinto ¹, Camila Ramos Silva^1,1, Camila de Almeida Salvego^1,1, Martha Ribeiro Simões^1,1

¹CELAP, Instituto de Pesquisas Energéticas e Nucleares (São Paulo, Brasil)

INTRODUCTION

Breast cancer is a disease of worldwide importance since it is considered the 5th leading cause of cancer deaths. Triple-negative breast cancer (TNBC) is a molecular subtype that presents resistance to conventional radiotherapy, demanding high doses of ionizing radiation (IR) for a prolonged period of treatment. On the other hand, low-level light irradiation (LLLI) has been studied to sensitize cells before IR exposure. However, the literature is poor regarding the association of both techniques in TNBC cells.

OBJECTIVES

Thus, we aimed to assess the effect of LLLI before IR exposure on two TNBC cell lineages.

MATERIALS AND METHODS

MDA-MB-231 (human TNBC) and 4T1 (murine TNBC) were cultivated, seeded at a density of 2.5 x 10 ⁵ cells/cm², and maintained in an incubator (37ºC, 5% of CO₂) overnight. LLLI was performed with a red LED (λ= 660 ± 11 nm, 38.2 mW/cm²) delivering energies of 1.2 J and 6.0 J. One-h after LLLI, the cells were submitted to both 2.5 and 5.0 Gy doses from a ⁶⁰ CO source. After 24-h, mitochondrial activity (MA) was quantified by MTT assay with n= 9/group.

DISCUSSION AND RESULTS

Our data showed that 4T1 cells exposed to LLLI at 1.2 J exhibited higher MA than cells exposed to IR2.5. In contrast, cells exposed to 6 J of LLLI showed lower MA than IR5. Concerning MDA-MB231 cells, no statistically significant differences were noticed among groups regardless of IR and LLLI doses.

CONCLUSION

These findings indicate that LLLI before IR could sensitize only murine breast cancer. Besides, an appropriate combination of IR and LLLI doses seems to play a role to kill TNBC cells.

Keywords: Photobiology, radiotherapy, radiomodifier

Supported by: CAPES, CNPq, CNEN

08855 - Poster Session

EB.15 - Fetuin detection by a promising optical-magnetic multimodal nanoprobe functionalized with Cramoll lectin

Weslley Felix de Oliveira^1,2, Mariana Paola Cabrera³, João Victor Araújo de Lima ², Luana Cassandra Breitenbach Barroso Coelho¹, Giovannia Araújo de Lima Pereira³, Beate Saegesser Santos⁴, Paulo Euzébio Cabral Filho², Maria Tereza dos Santos Correia¹, Adriana Fontes²

¹Departamento de Bioquímica, Universidade Federal de Pernambuco (PE, Brazil), ²Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco (PE, Brazil), ³Departamento de Química Fundamental, Universidade Federal de Pernambuco (PE, Brazil), ⁴Departamento de Farmácia, Universidade Federal de Pernambuco (PE, Brazil)

INTRODUCTION

Many researchers are seeking to develop smart nanomaterials due to the diversity of potential applications. Bimodal nanoprobes (BNPs) have gained attention, especially those consisted of quantum dots (QDs) and iron oxide nanoparticles (SPIONs), due to the possibility of combining the advantageous superparamagnetic response of SPIONs and the singular optical properties of QDs. Furthermore, BNPs can be conjugated with biomolecules, such as Cramoll lectin, a glucose/mannose-binding protein purified from Cratylia mollis seeds, to become site-specific.

OBJECTIVES

To obtain a multimodal system (BNPs-Cramoll) to detect the fetuin glycoprotein whose levels may be altered in pathologies.

MATERIALS AND METHODS

Carboxyl-coated QDs were covalently combined to aminated SPIONs, and then BNPs were conjugated with Cramoll. The optical properties and the zeta potential of nanosystems were determined. Candida albicans yeasts were incubated with BNPs-Cramoll and analyzed through fluorescence microscopy and flow cytometry to evaluate the specificity/efficiency of the nanoprobe. Fetuin detection was performed through fluorimetry.

DISCUSSION AND RESULTS

The QD absorption band was absent in the supernatant of BNPs, indicating effective conjugation with SPIONs. There was a redshift in the maximum emission of BNPs compared to bare QDs; lectin conjugation did not cause a spectral shift. BNPs-Cramoll had a less negative surface charge than BNPs. Approximately 90% of yeast cells were homogeneously labeled by BNPs-Cramoll and after inhibition with methyl-α-D-mannopyranoside, a labeling reduction of ca. 3x was observed. When incubated with different concentrations of fetuin (0.675-10.8 mg/mL), a linear decay in the BNPs-Cramoll fluorescence was identified. Incubation with bovine serum albumin (control) did not significantly decrease the fluorescence.

CONCLUSION

BNPs-Cramoll showed to be a specific fluorescent-magnetic nanoprobe able to detect fetuin, promising for the biosensing of this glycoprotein.

Keywords: quantum dot, magnetic nanoparticle, lectin

Supported by: CAPES, CNPq, FACEPE, INCT-INFo, and LARNANO-UFPE.

05089 - Poster Session

EB.16 - Interaction of ruthenium complex with biomolecules and its outcomes of photodynamic efficiency

Patrícia Alves de Matos¹, Patrícia Alves de Matos ¹

¹Instituto de Química, Universidade Federal de Uberlândia (Minas Gerais, Brazil)

INTRODUCTION

Photodynamic therapy (PDT) is related to two non-toxic actors, which are combined to induce cellular and tissue effects in an oxygen-dependent manner. The main actors are the photosensitizer (PS), light absorption and oxygen, which together generate reactive oxygen species (ROS) to inactivate undesirable cells. The PSs approved by FDA involve compounds from the classes of porphyrin, chlorins and phthalocyanines. Recently, metal complexes and especially polypyridine Ru(II) complexes have received increasing attention due to their intriguing photophysical and biological properties. TLD-1433 Ru(II) complex is the first inorganic complex (not belonging to the classes of porphyrins, chlorins and phthalocyanines) to enter phase II clinical trials as an PS for PDT against bladder cancer.

OBJECTIVES

Study properties of Rubpy and Rubpe and their ability to bind and photooxidize biomolecules in order to correlate the PS-biomolecule interaction and photodynamic efficiency.

MATERIALS AND METHODS

UV-Vis spectrophotometer was used to study properties of PS such as absorption spectra in organic solvent and aqueous solution as well as determination of pka values. Fluorescence spectroscopy was used to evaluate interaction of Rubpe and Rubpy with lipids (liposomes made of DMPC) and proteins (BSA).

DISCUSSION AND RESULTS

Rubpe exhibited binding constant (Kb) value 2.5 times more than Rubpy in liposomes made of DMPC. Also, the interaction of Rubpe with BSA (protein) were larger compared to Rubpy. Among the 2 PS studied, one of them presents an ethylene group separating two pyridine rings, providing more hydrophobicity to the compound which probably increase PS interaction to biomolecules. Studies of photosensitization eukariotic cells with both compounds are ongoing in order to correlate the stronger interaction of Rubpe and the phototoxicity.

CONCLUSION

The ethylene group added between the pyridine rings increases the ability of PS to interact and photo-oxidize biomolecules such as lipid and protein. It may improve photodynamic effects.

Keywords: Photodynamic Therapy, Ruthenium complex, biomolecule interaction

08951 - Poster Session

EB.17 - How to make protoporphyrin IX more efficient?

Debora Cristina Kawasaki Codognato^1,2, Iouri Bosissevitch^2,3, Ana Paula Ramos¹

¹Physical-Chemistry of Colloids and Surfaces, FFCLRP, University of São Paulo (Ribeirão Preto, SP, Brazil), ²Department of Physics, FFCLRP, University of São Paulo (Ribeirão Preto, SP, Brazil), ³Institute of Physics, Federal University of Goiania, (Goiania,GO, Brazil)

INTRODUCTION: Protoporphyrin IX (PpIX) is a derivative of ALA (5-aminolevulic acid) an intrinsic photosensitizer (PS) of the human body. PpIX is highly fluorescent, however it photobleaches rapidly under production of singlet oxygen ^a which makes PpIX an efficient FS used in photochemotherapy. The affinity of PpIX for diseased tissues can be improved by the use of carriers ^b . OBJECTIVES: To study the interaction between PpIX and Bovine Serum Albumin (BSA) to be used as a carrier justified by its similarity with Human Serum Albumin (HSA) that has affinity to cancer cells ^b . METHODOLOGY: Optical absorption and fluorescence emission spectroscopy, particle size and zeta potential were performed to characterize PpIX and its complexes with BSA. Langmuir monolayers containing Dipalmitoylphosphatidylcholine (DPPC) were used as a model to study the interaction between both PpIX and PpIX-BSA complex with biological membranes. All experiments were carried out in acidic (4.5) and physiological (7.3) pH buffer solutions. RESULTS: Changes in the profile of the optical absorption spectra and fluorescence emission show that BSA binds to PpIX. These photophysical changes do not impair PpIX photochemical efficiency. Particle size measures showed the formation PpIX-BSA complexes depending on the BSA concentration with sizes ranging from 5-1000 nm at pH 4.5 and from 3-400 nm at pH 7.3. The formation of the complex was favored at pH 4.5, close to the BSA isoelectric point. Surface pressure (π–A) curves measured through the Langmuir monolayers show that both porphyrin and its albumin complex interact with the DPPC monolayer. CONCLUSION: PpIX binds to BSA with enhanced photophysical properties. Both PpIX and PpIX-BSA interact with the cell membrane model suggesting enhanced performance of PpIX as PS. a LEE, H.-S.; LEE, J.-B.; YUN, S. J.; et al. Spectrofluorometric Determination of Protoporphyrin IX in Cells Using Acridine as Internal Standard. Bull. Korean Chem. Soc., v. 27, n. 7, p. 1067–1070, 2007. b ELSADEK, B.; KRATZ, F. Impact of albumin on drug delivery — New applications on the horizon. Journal of Controlled Release, v. 157, n. 1, p. 4–28, jan. 2012.

Keywords: Protoporphyrin XI, BSA, Langmuir Monolayer

Supported by: FAPESP, CNPq and CAPES

08594 -

EB.18 - Photodynamic therapy associated with ionizing radiation in the treatment of triple-negative breast cancer cells

Camila Ramos Silva ¹, Mayara Santana Pinto¹, Martha Simões Ribeiro¹

¹Centro de Lasers e Aplicações, Instituto de Pesquisas Energéticas e Nucleares (São Paulo, Brasil)

INTRODUCTION

Breast cancer is the most common cancer for women worldwide. According to the World Health Organization, it is considered the 5 th leading cause of death from cancer. Triple-negative breast cancer (TNBC) is a subtype of this disease that represents around 20% of all invasive breast cancer, whose main characteristics are resistance to conventional treatments, such as exposure to ionizing radiation (IR). On the order hand, the photodynamic therapy (PDT) using porphyrins and their derivatives has been described in the literature as a potential therapy against cancer.

OBJECTIVES

Thus, our goal in this work was to associate PDT and IR in the treatment of TNBC.

MATERIALS AND METHODS

MDA-MB-231 cells at a concentration of 2x104 cells were submitted to PDT using TMPyP porphyrin (30 μM) and a red light (660 ±11 nm) with fluences of the 23 and 57.5 J/cm 2 (57.3 mW/cm 2). Immediately post-PDT, cells were divided into groups: non-treated (control), only IR and PDT associated with IR (PDT57+IR and PDT23+IR) and then, exposed to IR with a dose of 2.5 Gy. Past 24-h of the PDT-session, the cell viability, clonogenicity and total glutathione were verified.

DISCUSSION AND RESULTS

Cells exposed to IR not presented statistically significance difference compared to the control group. However, treated groups showed around 38% lower cell viability in relation to the control and IR groups. For the clonogenic assay a reduction of the approximately 65% was observed between IR and treated groups. Regarding to the total glutathione, all groups showed an increase when compared to control group. Nonetheless, no were identified differences between IR and treated groups.

CONCLUSION

Taken together, our results indicate that PDT associate with IR may be an ally in TNBC treatment.

Keywords: radiotherapy, combined therapy, cancer

Supported by: CNPQ

08125 -

EB.20 - Combination Therapy of Antimicrobial Photodynamic Inactivation: Potential of Nanoparticles and Plant Based Compounds

Khatereh Khorsandi ¹, Reza Hosseinzadeh²

¹Department of Photodynamic, Medical Laser Research Center, Yara Institute, ACECR, Tehran, Iran (, IRAN), ²Department of Medical Laser, Medical Laser Research Center, Yara Institute, ACECR, Tehran, Iran (, IRAN)

INTRODUCTION

The multidrug resistance of pathogenic bacteria has become a serious problem to public health, finding novel approaches to combat multidrug resistant bacteria have therefore become increasingly important. Microbes in biofilm form can tolerate higher levels of antibiotics than their planktonic form. An “ideal” anti-biofilm agent should be able to penetrate the matrix and/or to inhibit/interfere with its accumulation; combined with the ability to recruit immune cells and/or modulate the host immune response would be an added value. One promising approach is antimicrobial photodynamic therapy (APDT) which involves the use of photosensitizer (nontoxic dyes) that are excited by visible light and produce oxygen free radicals in the presence of oxygen. APDT can be combined with other agents or drugs, improving the overall result while reducing individual concentrations and avoiding host tissue damaging. Among these options nano formulations of photosensitizer or using nano vehicle for drug delivery got enormous interest and advance in the recent. Also polyphenol compounds from plants showed antibacterial activity against different pathogen which could be consider as an adjuvant in APDT.

MATERIALS AND METHODS

In our work, after performing MTT assay (to evaluate photosensitizer toxicity on human fibroblast cells), the effect of APDT on bacteria in the planktonic and biofilm forms were investigated. We combined APDT based methylene blue as photosensitizer with polyphenols or used curcumin nano particle as photosensitizer.

DISCUSSION AND RESULTS

Our result showed that there was a reduction in the number of bacteria in planktonic condition, bacterial biofilm production and also enhance in destruction of the biofilm in combination mode compared to single mode.

CONCLUSION

Therefore, combination therapy with APDT could be suggested as novel approach in the treatment of multi drug resistant bacteria in chronic ulcer condition.

Keywords: Antimicrobial photodynamic inactivation, Combination therapy, Nano particles

08875 - Poster Session

EC.01 - Plasmalogens pro-oxidant action by generation of excited singlet molecular O ₂ (¹Δg) in the dark

Rodrigo Lucas de Faria ¹, Sayuri Miyamoto¹, Adriano Britto Chaves Filho¹

¹Departamento de Bioquímica,, Instituto de Química da Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Plasmalogens are glycerophospholipid with a vinyl–ether linkage at the sn-1 position of the glycerol backbone. Even though they are found in all human tissues, being especially abundant in the brain and heart, the biological role of plasmalogens remains unclear. It has been suggested that plasmalogens are antioxidants because of the high reactivity of their vinyl ether groups with reactive oxygen species (ROS).

OBJECTIVES

However, plasmalogen reaction with singlet molecular oxygen (O2 (1∆g) can produce two primary unstable oxidation products, a hydroperoxide and a short-lived dioxetane intermediate whose decomposition can produce O2 (1∆g) by transferring energy to triplet molecular oxygen.

MATERIALS AND METHODS

Herein, we describe evidences of the generation of O2 (1∆g) by chemical trapping and monomol IR light emission at 1,270 nm. We have also characterized the main O2 (1∆g)-oxidation products of phosphatidylethanolamine plasmalogen (pPE) by high resolution tandem mass spectrometry (ESI-MS/MS).

DISCUSSION AND RESULTS

The results from mass spectrometry confirms the formation of pPE hydroperoxides and unstable dioxetanes, which were degraded to formyl phosphoethanolamine (formyl-PE), lyso-PE and fatty aldehydes with 15 and 17 carbons.

CONCLUSION

These findings demonstrate that although plasmalogens are considered antioxidants, they can act as a pro-oxidant by promoting singlet molecular oxygen generation in the dark.

Keywords: photochemistry in the dark, Plamalogen, singlet molecular oxygen

Supported by: FAPESP

08306 - Poster Session

EC.02 - The contrast agent 2,3,5-triiodobenzoic acid (TIBA) induces cell death in tumor cells through the generation of reactive oxygen species

Jéssica Sodré Silva de Abreu ¹, Janaína Fernandes¹

¹Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

TIBA is a iodine contrast agents used for diagnosis of tissue structures in X-ray techniques. However, its ionic form increases blood osmolarity, generating physiological complications such as contrast-induced nephropathies (CIN). The CIN leads to an increase in the generation of reactive oxygen species (ROS) in tubules and renal epithelium, causing the death of these cells. An antitumor activity of TIBA has been described in the non-small cell lung cancer (H460). In this model, TIBA induced cell death for mitochondrial intrinsic pathway, in considerably lower concentrations than iodinated contrast agents used in the clinic. But the subcellular mechanisms involved in TIBA-induced cell death are still unknown.

OBJECTIVES

Thus, the objective of this work was to evaluate whether the anti-tumor activity of TIBA involves ROS increase, in cell lines of non-small cell lung cancer (H460), chronic myeloid leukemia (K562), and its cytotoxicity in normal renal epithelial (VERO).

MATERIALS AND METHODS

The MTT assay was used for evaluation of cell viability, the fluorescent probe H2DCFDA to evaluate ROS induction, cell cycle analysis using flow cytometry to measure cell death, and immunofluorescence with annexin/7-AAD, to assess the association of cell death with the ROS generation.

DISCUSSION AND RESULTS

TIBA decreases cell viability in a dose-dependent manner for the H460 and K562. However, VERO cells showed less response to the drug, with 70% viable cells after 72 hours of treatment in the highest concentration of the drug. And the tumor cells with only 20% viable cells. Thus, tumor cells exhibited higher DNA fragmentation, compared to the renal line (VERO with 5% of fragmented DNA, H460 with 26%, and 56% in K562). Finally, TIBA-induced ROS and apoptosis in all lines, which is significantly decreased after treatment with the antioxidant N-acetyl-cysteine (NAC).

CONCLUSION

These data demonstrate the relationship between the increased cellular oxidative stress and the anti-tumor action of the TIBA.

Keywords: TIBA, Cancer, Contrast Agent

Supported by: FAPERJ

08795 - Poster Session

EC.03 - Antioxidant capacity of Petroselinum crispum (parsley) in yeast cells

Danniel Cosme Neves Grillo ¹, Karine Santos¹, Neide Mara de Menezes Epifânio², Douglas Siqueira de Almeida Chaves², Cristiano Jorge Riger¹

¹Departamento de Bioquímica, Universidade Federal Rural do Rio de Janeiro (Rio de Janeiro, Brasil), ²Departamento de Ciências Farmacêuticas, Universidade Federal Rural do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Oxidative stress is caused by an imbalance that occurs between oxidizing species such as reactive oxygen species (ROS), and the insufficiency of intracellular defense mechanisms. These species cause damage to cells and tissues and can be precursors of a series of pathologies. For this reason, several studies investigate the prevention of the generation of this oxidative stress through substances that have an antioxidant action capable of maintaining intracellular redox homeostasis. In our previous studies was demonstrated the antioxidant activity of apiin, a glycosylated flavonoid present in 6% in the aqueous extract of Petroselinum crispum (parsley) in Saccharomyces cerevisiae cells

OBJECTIVES

The aim of this study was to verify whether non-cytotoxic concentrations of the aqueous extract of parsley would present in S. cerevisiae cells a protective antioxidant effect upon oxidative stress induced by hydrogen peroxide.

MATERIALS AND METHODS

Toxicity and cell viability assays by colony forming units as well as damage to membrane lipids (TBARS) were performed in yeast cells.

DISCUSSION AND RESULTS

Both concentrations of 0.113 and 0.226g.L-1 of the aqueous extract did not show toxicity to the cells, obtaining survival percentages of 81.67 ± 4.16% and 105.33 ± 6.81% respectively. In relation to the extract, both concentrations showed a similar protection to oxidative stress. Analysis of cells under stress revealed 26.50 ± 8.88% cell viability while pre-incubation with extracts it increased to 54.25 ± 20.67% and 46.33 ± 5.51% respectively. Pretreatment with the extract also showed a reduction in lipid peroxidation, so with significant results when evaluated at the concentration of 0.565g.L-1 of parsley extract.

CONCLUSION

In conclusion, it is likely that this results with previous antioxidant analyzes using the flavonoids apiin and apigenin are intrinsically related to the antioxidant potential of P. crispum extract, mainly to the glycosylated flavonoid apiin.

Keywords: apiin, Petroselinum crispum, Saccharomyces cerevisiae

Supported by: FAPERJ, CNPq and CAPES

08791 - Poster Session

EC.04 - Oxidative and membrane parameters in Wistar rat hippocampus with intrauterine development in the absence of maternal melatonin or in the presence of valproic acid

Eduardo Henrique de Paula Ribeiro ¹

¹Bioquímica e Biologia Molecular , Universidade Federal de São João del Rei (Minas Gerais, Brasil)

INTRODUCTION

O desenvolvimento de Desordens do Espectro autista (DEA) pode vir não apenas por componentes genéticos, mas também por fatores ambientais, como a exposição a toxinas, fármacos e infecções que levam a uma possível indução em alterações epigenéticas. A exposição pré-natal ao ácido valproico (VPA) é um fator importante para o desenvolvimento de DEA e, por isso, é um modelo robusto utilizado para o estudo dessas desordens do sistema neurológico. Portadores de DEA podem sofrer com alterações no sono e isso pode ser consequência dos níveis insuficientes de melatonina, um hormônio produzido pela glândula pineal que tem um papel fundamental na regulação do ciclo circadiano, além de apresentar funções antioxidantes. Elevações nos parâmetros de estresse oxidativo também são relatadas em indivíduos com DEA. No intuito de estudar as relações entre estresse oxidativo, DEA e melatonina, o presente trabalho avaliará as possíveis alterações nos parâmetros oxidativos e membranares nos hipocampos de ratos induzidos a DEA por injeção pré-natal de VPA, comparando a proles de ratas Wistar pinealectomizadas, ou seja, com desenvolvimento pré-natal na ausência da glândula pineal.

OBJECTIVES

Avaliar as possíveis alterações nos parâmetros oxidativos e membranares no hipocampo de prole induzida a DEA por injeção de VPA, comparando à prole de ratas Wistar pinealectomizadas.

MATERIALS AND METHODS

As amostras de hipocampo serão processadas para a obtenção de um homogenato e uma fração membranar. A determinação da concentração de proteínas totais será feita pelo método de Bradford (1976). Na determinação de parâmetros oxidativos, serão avaliados: atividade da Catalase, Glutationa peroxidase, Superoxidodesmutase e teores de Glutationa reduzida, proteína carbonilada, peroxidação lipídica e formação de peróxido de hidrogênio. Será feita também uma extração lipídica e determinação de fosfolipídeos totais e colesterol total. Todos os experimentos serão realizados em triplicata.

DISCUSSION AND RESULTS

CONCLUSION

Keywords: Autism, Oxidative Stress, Melatonin

Supported by: FAPEMIG

08300 - Poster Session

EC.05 - Piperine in association with N-acetylcysteine protects against acetaminophen-induced hepatotoxicity

Aline Meireles Coelho ¹, Isabela Ferreira Queiroz¹, Wanderson Geraldo de Lima¹, André Talvani¹, Melina Oliveira de Souza², Daniela Caldeira Costa¹

¹Departamento de Ciências Biológicas , Universidade Federal de Ouro Preto (Minas Gerais, Brasil), ²Departamento de Alimentos , Universidade Federal de Ouro Preto (Minas Gerais, Brasil)

INTRODUCTION

Acetaminophen (APAP) is widely used in the world for presenting analgesic properties and antipyretic however, its use in high doses can result irreversible liver damage. Preliminary studies suggest that piperine acts by inhibiting cytochrome P450, whom is involved in the metabolism of different xenobiotics, including paracetamol. With that our hypothesis that piperine could be a possible therapeutic target to minimize APAP-induced hepatotoxicity.

OBJECTIVES

The aim of this study was to evaluate the hepatoprotective effect of piperine in association or not with N-acetylcysteine (NAC) in a model of paracetamol-induced hepatotoxicity in C57BL/6 mice.

MATERIALS AND METHODS

The groups (n=7) were distributed into: control, APAP, APAP+P20, APAP+P40, APAP+NAC, APAP+P20+NAC, APAP+P40+NAC. Paracetamol was administered (500mg/Kg) and after 2 hours the treatments were performed with piperine (20 and 40 mg/Kg) in association or not with NAC (300 mg/kg). All treatments were performed orally through gavage. The animals were euthanized 12 hours after APAP administration. We evaluate hepatic and renal function, in addition to histological analysis and redox status in the liver. All procedures were approved the Ethics Committee on Animal Use (CEUA) from the Federal University of Ouro Preto, Brazil.

DISCUSSION AND RESULTS

The results showed that treatment with piperine 20 mg/kg associated with NAC reduced the activity of the ALT, reduced MMP-9 and increased the sulfhydryl group (-SH) compared to the APAP group. AST, urea and TBARS decreased in NAC, NAC+P20 and NAC+P40 groups when compared to APAP. The area of necrosis, TNF and carbonyl protein decreased in groups P40, NAC, NAC+P20 and NAC+P40 compared to APAP. The cytokine IL-6 reduced in all treated groups compared to APAP.

CONCLUSION

Based on these results, piperine has been shown to be a possible ally with NAC in the treatment of hepatotoxicity.

Keywords: Acetaminophen, Piperine, hepatotoxicity

Supported by: UFOP, FAPEMIG e CAPES

08554 - Poster Session

EC.06 - Bioactive compounds and in vivo and in vitro antioxidant capacity of biquinho pepper (Capsicum chinense)

Tamires de Almeida Cruz ¹, Jéssica Sodré Mendes¹, Vanessa Sales de Oliveira², Bárbara Jardim Mariano², Tatiana Saldanha², Cristiano Jorge Riger¹

¹Bioquímica, Universidade Federal Rural do Rio de Janeiro (Rio de Janeiro, Brasil), ²Tecnologia de Alimentos, Universidade Federal Rural do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Biquinho pepper (Capsicum chinense) is one of the most produced and consumed domesticated peppers in Brazil. Besides its commercial and economic value, this pepper has a significant health-promoting potential, since it contains bioactive compounds, which are responsible for nutritional and antioxidant potential.

OBJECTIVES

The objective of this work was to identify bioactive compounds present in C. chinense, and to evaluate the antioxidant capacity by in vivo and in vitro assays.

MATERIALS AND METHODS

Bioactive compounds were identified by UHPLC-MS analysis. For the antioxidant capacity were used DPPH, ORAC and β-carotene/linoleic acid assays and extracts toxicity and cell viability assays using Saccharomyces cerevisiae cells.

DISCUSSION AND RESULTS

Thirteen compounds were detected, such as carotenoids (β-carotene (m/z 537), lutein (m/z 551), zeaxanthin (m/z 569), antheraxanthin (m/z 585), cryptoxanthin (m/z 735), capsanthin (m/z 767), and capsorubin (m/z 811)), capsaicionoids (nordihydrocapsaicin (m/z 294), capsaicin (m/z 306), dihydrocapsaicin (m/z 308) and homocapsaicin-I (m/z 320)), and capsinoids (nornorcapsiate (m/z 301) and nordihydrocapsiate (m/z 317)). Capsaicionoids and capsinoids are recognized due to their anti-inflammatory, antioxidant, and antitumoral properties. Carotenoids have a series of conjugated C=C bonds that are responsible for their ability of quenching molecular oxygen and scavenging free radicals. C. chinense showed an antioxidant potential in DPPH radical scavenging activity (33.17±0.08%). The oxygen radical absorbance capacity (ORAC) was measured by fluorescein method and it was 32.35±0.02μM TE/g and result of β-carotene/linoleic acid assay was 55.80±1.30%. In vivo antioxidant analyses demonstrated that the pepper extract (170μg/mL) decreased the damage promoted by H₂O₂ in Saccharomyces cerevisiae cells.

CONCLUSION

The antioxidant potential was demonstrated by both in vivo and in vitro assays. Additionally, C. chinense extract showed low toxicity to Saccharomyces cerevisiae cells under the evaluated conditions. Therefore, the biquinho pepper becomes a potential target for more advanced studies, both in the health and food areas, which may be related to disease prevention and potential food preservatives.

Keywords: antioxidant, Capsicum chinense, Saccharomyces cerevisiae

08811 - Poster Session

EC.07 - Modified protocol for redox proteomics in melanoma cells

Elizabeth Sousa da Cunha ¹, Ester Mazepa¹, Michel Batista², Fabricio Klerynton Marchini², Glaucia Regina Martinez¹

¹Departamento de Biologia e Bioquímica Molecular, Universidade Federal do Paraná (Paraná, Brasil), ²Instituto Carlos Chagas, Fundação Oswaldo Cruz/PR (Paraná, Brasil)

INTRODUCTION

In the development and progression of cancer, chemical modification of proteins is as important as the amount of protein synthesized. Although the involvement of reactive oxygen species (ROS) in signaling processes is well established, sensors and targets of these events are still not well defined. In this context, it becomes necessary studies that can clarify the modifications generated in the proteins, that act in the malignancy or even in the resistance of these tumors to the treatment. These modifications are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics emerge as a fundamental tool to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after the enrichment steps may occur

OBJECTIVES

Based on this, our objective was to determined a protocol to analyse redox proteomics in melanoma.

MATERIALS AND METHODS

Protocols for redox proteomics were revised and tested.

DISCUSSION AND RESULTS

Considering the biological importance of thiol’s oxidation in melanoma and based on previous protocols mainly described by Zaccarin e colleagues (2014), and our own insights, we adapted a protocol of Biotin switch assay technique with Biotin-HPDP and NEM for a cell line

CONCLUSION

This adaption surpassed limitations on the traditional method, improving a protocol of redox proteomics focused on thiol-protein studies in melanoma cells, whose biological importance of study concerning thiol´s oxidation were extensively reviewed.

Keywords: redox, proteomics, melanoma

Supported by: CNPQ and CAPES

08209 - Poster Session

EC.08 - Effects of Potential Antioxidants on Insulin-Producing Cells

Ingrid Batista Borges Rodrigues ¹, Kléber Luiz Araújo Souza¹

¹NUMPEX-BIO, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Oxidative stress may be a common link in pancreatic beta cell dysfunction. The use of chemical compounds mimicking the situations encountered in diabetes, such as sodium nitroprusside (SNP), menadione and hydrogen peroxide, has made it possible to clarify the various deleterious or protective molecular mechanisms that act in the beta cell. In contrast, bioactive compounds such as glycyrrhizin and rutin, which may act to prevent or mitigate the consequences of this process, are of great interest.

OBJECTIVES

To evaluate the toxicity of oxidizing agents and nitric oxide donors and to test the possible cytoprotective effects of natural compounds on RINm5F insulin-producing cells

MATERIALS AND METHODS

RINm5F insulin producing cells were grown in an appropriate medium and exposed to cytotoxic compounds (hydrogen peroxide, menadione, and sodium nitroprusside) and antioxidants of interest (glycyrrhizin and rutin). The MTT spectrophotometric reduction method used to cell viability assays. The status of redox (nitroxy) cells, anionic production of intracellular mitochondrial superoxide and detection of phosphatidylserine in the cytoplasmic membrane were determined by the fluorogenic probes in the cytoplasmic membrane were determined by the fluorogenic probes DCFH-DA, MitoSOX and Annexin V - FITC, respectively, by fluorescence microscopy.

DISCUSSION AND RESULTS

There was a significant decrease in cell viability and an increase in the production of intracellular reactive oxygen species against oxidative challenge. However, co-incubation of RINm5F cells with glycyrrhizin had three different types of effects: partial protection against the use of hydrogen peroxide; other deleterious exposure to menadione exposure and no effect on donor sodium nitroprusside (SNP) use.

CONCLUSION

It is possible that cell type and sub-localization of reactive oxygen species production will determine the ultimate effect of glycyrrhizin and cell fate. Although rutin had no protection against oxidative damage induced by hydrogen peroxide, menadione and SNP, no cytotoxic effect on RINm5F cells was observed under the conditions tested.

Keywords: antioxidants, beta-cells, reactive species

Supported by: FAPERJ

08808 - Poster Session

EC.09 - Antioxidant effect of phenolic compounds fermented by probiotics on Saccharomyces cerevisiae strains deficient in antioxidant systems

Nathalia Soares Camargo ¹, Edlene Ribeiro Prudêncio Souza¹, Marcos Dias Pereira², Cristiano Jorge Riger¹

¹Departamento de Bioquímica, Universidade Federal Rural do Rio de Janeiro (Rio de Janeiro, Brasil), ²Departamento de Bioquímica, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Studies show that a diet rich in foods containing phenolic substances helps in the body's redox balance acting against free radicals, which in high concentrations are associated with a series of diseases, such as degenerative and cardiovascular diseases, tumors, diabetes and others. Most bioactive compounds are metabolized by microorganisms present in the intestinal lumen before being absorbed, which directly impacts their biological activity.

OBJECTIVES

The objective these study was to evaluate the influence of microbial biotransformations on the antioxidant activity of the phenolic substances phenethyl ester of caffeic acid (CAPE) and mangiferin in Saccharomyces cerevisiae cells .

MATERIALS AND METHODS

Cells were treated with CAPE and mangifetin (0.1mM) isolated and after fermentation of these phenolics with a probiotic blend, followed by exposure to hydrogen peroxide (2.0 mM) for 1h. Antioxidant analysis was determined by cell viability, membrane lipid peroxidation (TBARS) and redox environment by dichlorofluorescein.

DISCUSSION AND RESULTS

Our results revealed that pure CAPE and mangiferin (0.1 mM) were able to decrease the oxidative damage induced by hydrogen peroxide in the control strain (BY4741) and in the mutant strains Δsod1, Δgsh1 and Δctt1, deficient in superoxide dismutase, glutathione and catalase, respectively. After fermentation, the phenolics maintained their antioxidant capacity, but this activity related only to mangiferin was not relevant in the intracellular viability and oxidation tests.

CONCLUSION

These studies suggest that the antioxidant activity was maintained in the presence of microbial biotransformations, showing that these compounds can be potential antioxidants even after their fermentation by microorganisms present in the intestinal flora using a eukaryotic study model.

Keywords: Saccharomyces cerevisiae, probiotics, phenolics substances

08964 - Poster Session

EC.10 - A novel diselenide attenuates the carrageenan-induced inflammation in mice by inhibiting neutrophil chemotaxis

TÁSSIA LESSA¹, THIAGO MACÊDO CORREIA², TALITA COSTA SANTOS², RAILMARA PEREIRA DA SILVA³, BEATRIZ SILVA³, ALBERT SOUZA PEIXOTO⁴, WILLIAM TADEU FESTUCCIA⁴, REGIANE YATSUDA^2,5, AMÉLIA CRISTINA GUSMÃO^5,2, ALCINDO APARECIDO DOS SANTOS³, FLÁVIA CARLA MEOTTI³, RAPHAEL FERREIRA QUEIROZ⁶

¹Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular, Universidade Estadual do Sudoeste da Bahia (BA, Brasil), ²Ciências Fisiológicas, Universidade Federal da Bahia (BA, Brasil), ³Instituto de Química, Universidade de São Paulo (SP, Brasil), ⁴instituto de Ciências Biomédicas, Universidade de São Paulo (SP, Brasil), ⁵Instituto multidisciplinar de Saúde , Universidade Federal da Bahia (BA, Brasil), ⁶Ciências naturais, Universidade Estadual do Sudoeste da Bahia (BA, Brasil)

INTRODUCTION

Myeloperoxidase (MPO) is a neutrophil haem peroxidase that catalyzes the formation of free radicals and HOCl. Despite their role in immune defense, these species may oxidize host biomolecules and lead to human inflammatory diseases. Therefore, there is interest in the development of MPO inhibitors for clinical application.

OBJECTIVES

Here we investigated the in vitro and in vivo anti-inflammatory effect of a new selenium compound, dibenzyl[diselanediylbis(propane-3,1-diyl)]dicarbamate.

MATERIALS AND METHODS

We first evaluated the reaction of diselenide and HOCl by the taurine-chloramine method. The diselenide effect on oxidative burst of PMA-stimulated d HL-60 was also investigated. Carrageenan-induced peritoneal and paw inflammation models were employed to monitor its anti-inflammatory activity by measuring some redox and inflammation biomarkers. The toxicity was assessed by single dose and brine shrimp assays.

DISCUSSION AND RESULTS

In vitro, the compound rapidly reacted with HOCl (k=9.2×10 ⁷ M ⁻¹ s ⁻¹ ) similarly to glutathione (k=1.2×10 ⁸ M ⁻¹ s ⁻¹ ). It also reduced the HOCl formation by human neutrophils, but tyrosine did not restore it. Interestingly, the diselenide inhibited the oxidative burst of dHL-60 cells (O₂ consumption, O₂ ^●- and H₂O₂ production) in a concentration-dependent manner with no cell toxicity (≤25 μM). In vivo, carrageenan increased leukocyte migration into mouse peritoneal cavity, and the compound (25, 50 and 75 mg/kg, i.p.) reduced the total leukocyte and neutrophil numbers, MPO activity, lipid peroxidation, albumin exudation, nitrite, TNF-α and IL-1β levels in peritoneal fluid. Likewise, the diselenide (50 mg/kg, i.p.) decreased the paw edema after carrageenan-subplantar injection in mice over 4 hours. Histological and immunohistological analysis from the inflamed paws showed a reduction in neutrophil counting, edema area, and marking for MPO and protein carbonyl. No toxicity was observed by the single dose toxicity study (50 mg/kg, i.p.) over 15 days and the brine shrimp bioassay

CONCLUSION

Collectively, these data indicate that the new diselenide attenuates carrageenan-induced inflammation mainly by suppressing neutrophil migration and the resulting oxidative burst-mediated damage.

Keywords: selenium, inflammation, oxidative burst

Supported by: FAPESB

08499 - Poster Session

EC.11 - Air pollution in Recife (PE): evaluation of cell death, inflammatory responses and oxidative stress in A549 cells

Cleonilde Maria do Nascimento¹, Sheilla Andrade de Oliveira ¹, Helotonio Carvalho ²

¹Immunology, Instituto Aggeu Magalhães (Fiocruz-PE) (Pernambuco, Brazil), ²Biophysics and Radiobiology, Universidade Federal de Pernambuco (Pernambuco, Brazil)

INTRODUCTION

The World Health Organization (WHO) estimates that air pollution is responsible for seven million deaths annually, triggering or aggravating cardiorespiratory disorders and some types of cancer. Recife, with more than 1.6 million inhabitants and a fleet of almost 700.000 vehicles, showed a considerable increase in the number of hospitalizations and deaths from diseases associated with air pollution in the last decade, which may be related to the increase in the fleet and greater formation of particulate matter (PM). PM generates reactive oxygen species (ROS) and causes inflammation when inhaled.

OBJECTIVES

This study determined the concentration of PM2.5 in Recife atmosphere (PM-Recife) and evaluated its effects on human alveolar type II cells (A549), comparing with the diesel-derived PM (PM-Diesel).

MATERIALS AND METHODS

We analysed cytotoxicity, cell death, levels of inflammatory cytokines, ROS production, and transcriptional activation of antioxidant enzymes.

DISCUSSION AND RESULTS

Our results show that the concentration of PM2.5 in Recife is according to the current Brazil standard but it is close to WHO standards. PM-Diesel reduced cell viability after 48 h (p < 0.0002) and 72 h (p < 0.0001) of exposure, affecting more than 60% of the cells while PM-Recife reduced cell viability by almost 50% after 72 h (p < 0.03). PM-Diesel increased the levels of IL-6 after 48 h (< 0.03) and 72 h (p < 0.005) of exposure and both PMs increased IL-8 after 72 h (p < 0.02). Both PMs induced apoptosis and intracellular oxidative stress in A549 cells after 72 h (p < 0.02), in addition to transcriptionally activating the enzymes superoxide dismutase, catalase, glutathione peroxidase, thioredoxin and ferredoxin reductase (p < 0.02).

CONCLUSION

We conclude that PM-Recife, like PM-Diesel, is toxic to lung cells, capable of inducing apoptosis, inflammatory responses, oxidative stress and altering the cellular system of antioxidants. Such findings point to the need for further studies to understand the mechanisms involved in the pathogenesis of PM-Recife.

Keywords: Air pollution, Apoptosis, Oxidative stress

Supported by: CNPq, CAPES and FIOCRUZ

08221 - Poster Session

EC.12 - Influence of ferulic and p-cumaric acids on the catalase enzyme in oxidative stress condition

Rodrigo de Paulo Osorio ¹, Carlos Mauricio Sant’Anna¹, Cristiano Riger¹

¹Departamento de Bioquímica, Universidade Federal Rural do Rio de Janeiro (RJ, Brasil), ²Departamento de Química Fundamental, Universidade Federal Rural do Rio de Janeiro (RJ, Brasil)

INTRODUCTION

Oxidative stress is a condition generated by the inability of the antioxidant defense system to control the action of oxidants in cells, which can react with lipids, proteins and DNA, damaging the properties of cells structures. Oxidative stress has been associated with several pathologies, such as neurodegenerative diseases, diabetes and cancer. Ferulic and p < /i>-coumaric acids are exogenous antioxidants related to positive effects against neurodegenerative diseases.

OBJECTIVES

Study the influence of ferulic and p-coumaric acids on catalase in Saccharomyces cerevisiae cells.

MATERIALS AND METHODS

Cells viability, catalase activity, antioxidant activity and molecular modelling experiments were carried out.

DISCUSSION AND RESULTS

Both acids did not show toxicity at the concentration of 10 μg.m/L for cells. The effect of these acids on catalase activity under different concentrations of hydrogen peroxide (0.5mM, and 2.5mM) revealed 77.76% and 53.63%; 127.52% and 104.39%; and 83.67% and 70.61% for ferulic and p < /i>-coumaric acids in relation to the negative control, respectively. Regarding the antioxidant potential, pre-treatment with acids showed an increase in cell viability after oxidative stress with hydrogen peroxide (2.0mM). These results were observed in the control BY4741 (increase greater than 20%) and mutant strain Δctt1 (increase greater than 25%) in catalase. Analyzing by molecular docking (GOLD) and in silico analysis it was found that both acids can establish possible interactions with the enzyme, such as hydrogen bonding with the amino acid Gln163, in addition to hydrophobic interactions with Val111, Pro124, Phe148, Phe149 and Phe159 in catalase, amino acids outside the enzyme catalytic site.

CONCLUSION

In conclusion, ferulic acid and p < /i>-coumaric acid provide cellular protection against oxidative stress and this protection is apparently unrelated to their effect on catalase.

Keywords: hydroxycinnamic acids, catalase, Saccharomyces cerevisiae

Supported by: FAPERJ, CNPq and CAPES

08980 - Poster Session

EC.13 - Time-course of redox status, redox activity-related and mitochondrial dynamics-related gene expression after acute bout of different physical exercise protocols

Ramon Alves Pires¹, Thiago Macedo Lopes Correia², Amanda Alves Almeida², Raildo da Silva Coqueiro², Marco Machado^3,4, Mauro Fernandes Teles⁶, Raphael Ferreira Queiroz^1,5, Rafael Pereira^1,2,6

¹Programa Multicêntrico de Pós-graduação em Bioquímica e Biologia Molecular, Universidade Estadual do Sudoeste da Bahia (Bahia, Brazil), ²Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, Universidade Federal da Bahia (Bahia, Brazil), ³Ciências Auxiliares, Fundação Universitária de Itaperuna (Rio de Janeiro, Brazil), ⁴Laboratório de Fisiologia e Biocinética, Faculdade de Ciências Biológicas e Saúde (Rio de Janeiro, Brazil), ⁵Programa de Pós-graduação em Biociências, Universidade Federal da Bahia (Bahia, Brazil), ⁶Programa de Pós-graduação em Enfermagem e Saúde, Universidade Estadual do Sudoeste da Bahia (Bahia, Brazil)

INTRODUCTION

The magnitude of exercise-induced adaptations depends on exercise parameters, such as intensity, duration and execution mode (continuous or with intervals).

OBJECTIVES

In this context, the present study aimed to investigate the expression of redox activity-related and mitochondrial dynamics-related genes in mice skeletal muscle along 24 hours after an exercise session carried out with different protocols.

MATERIALS AND METHODS

Sixty-five male Swiss mice were allocated into a control group (5 animals) and others 3 groups (20 animals/group), which were submitted to a forced swimming bout with the follow protocols: low-intensity continuous (LIC), high-intensity continuous (HIC) and high-intensity interval (HII). Five animals from each group were euthanized immediately (0h) and at 6h, 12h and 24h after the exercise session. Gastrocnemius muscle was removed for analysis of expression of mitochondrial dynamics-related genes: Ppargc1a (mitochondrial biogenesis), Mfn2 (fusion), Dnm1L (fission), and Park2 (mitophagy); and redox activity-related genes: Nos2 Nfe2l2 and GPx1. Within-group and between-group comparisons were performed with ANOVA, with significance level set as p ≤ 0.05.

DISCUSSION AND RESULTS

Despite opposites in exercise intensity and duration, similar temporal behavior was observed in the expression of the Ppargc1a in LIC and HII, with greater expression at 6 and 24h, while HIC showed significant increases only at 0h and 6h after exercise. Mfn2 was significantly higher at 0h only in HIC and HII, remaining high at 6h only in HII. Only HII exhibited repression of Dnm1L and Park2 genes along 24h after exercise, while HIC was the unique with significant increase. Nos2 was significantly higher only in HIC (0h) and HII (6h). Nfe2l2 increased along the 24h in LIC and HII. GPx1 was significantly higher only in HIC (0h) and HII (24h).

CONCLUSION

The use of intervals during high-intensity exercise could suppress the expression of fission and mitophagy-related genes, and enhances the molecular profile related to mitochondrial biogenesis and fusion, as well as antioxidant defense.

Keywords: activity redox, exercise, mitochondrial dynamics

Supported by: FAPESB

08392 - Poster Session

EC.14 - Neutrophil granules isolation: a new miniaturized method

Gabrielly Alexandria de Moura Freitas ¹, Graziella Eliza Ronsein¹

¹Bioquímica, Universidade de São Paulo (SP, Brazil)

INTRODUCTION

Neutrophils are the most abundant leukocytes in the bloodstream and play a key role in the immune system. The activation and functionality of neutrophils depends on the exocytosis of storage particles, that are divided in 2 groups: granules and secretory vesicles. The granules are subdivided in 3 subtypes: azurophil, specific and gelatinase. These particles are differentiated mainly through their protein content; therefore, abundant specific proteins can be used as markers for each storage particle. The markers are myeloperoxidase (MPO) for azurophil, lipocalin-2 (NGAL) and lactoferrin for specific, gelatinase for gelatinase granules and latent alkaline phosphatase (latent AP) for secretory vesicles. Characterization of granule’s content is essential to comprehend the different functions of these cells, hence the importance of the granule’s isolation. Currently, the protocols of isolation are performed in large density gradients, leading to the necessity of major cell quantities, about 3x108, that makes unfeasible to compare stimuli and biological replicates in the same experiment.

OBJECTIVES

Thus, the main goal of our work was to create a miniaturized isolation method based on a discontinuous percoll density gradient.

MATERIALS AND METHODS

In order to achieve this goal, 9 x 106 neutrophils were isolated, lysed, put on top of a 3-layer percoll density gradient and centrifuged. The resulting gradient (~940μL) was collected from the bottom to the top of the tube in 19 fractions. The protein markers were then analyzed for each fraction using western blot (MPO, lactoferrin and NGAL), gelatin zymography (gelatinase) and enzyme assay (latent AP).

DISCUSSION AND RESULTS

Our results showed we successfully isolated the granules and secretory vesicles using a gradient with less than 1 mL of total volume.

CONCLUSION

The miniaturized method allows new experiments to be conducted, including a comparative study of neutrophill’s response to diverse stimuli.

Keywords: Fractionation, Granules, Neutrophil

Supported by: FAPESP, CAPES e CNPq

08794 - Poster Session

EC.15 - Effects of redox modulation on quiescin/sulfhydryl oxidase activity of melanoma cells with stimulated melanogenesis

Ester Mazepa¹, Ana Luiza Dorigan de Matos Furlanetto¹, Hulyana Brum¹, Lia Sumie Nakao², Elizabeth Sousa da Cunha¹, Glaucia Regina Martinez ¹

¹Bioquímica e Biologia Molecular, Universidade Federal do Paraná (PR, Brasil), ²Patologia Básica, Universidade Federal do Paraná (PR, Brasil)

INTRODUCTION

Secreted quiescin/sulfhydryl oxidase (QSOX) is overexpressed in many tumor cell lines, including melanoma and it is usually associated with a pro-invasive phenotype. Glutathione (GSH) is one of the main responsible to control redox homeostasis in cells. Our previous work described that B16-F10 cells enter in a quiescent state as a protective mechanism against ROS-damage generated by melanogenesis stimulation.

OBJECTIVES

This work aimed to investigate how modulation of GSH levels in B16-F10 murine melanoma cell line under condition of stimulation of melanogenesis affects cell surviving and QSOX activity.

MATERIALS AND METHODS

Melanogenesis was stimulated by culturing B16-F10 cells with RPMI 1640 medium in the presence of L-tyrosine and NH4Cl during 48 h. After 48 h, cells were treated with GSH or BSO for 24 h. Cell viability was evaluated by MTT assay and Crystal violet dye, which stains nucleic acids of adhered and fixed cells. QSOX activity in supernatant was measured using TBR 4100/1025 Free Radical Analyzer. Quantification of total glutathione, GSSG and GSH was performed by enzymatic/colorimetric assay.

DISCUSSION AND RESULTS

The redox homeostasis was impaired by treating cells with GSH in excess or depleting its intracellular levels through BSO treatment. Cells under melanogenesis stimulation showed lower GSH/GSSG ratio (8:1) in comparison with control (non-stimulated) cells (20:1), indicating a pro-oxidative state after stimulation. This was accompanied by a decrease in cell viability after GSH-depletion, and no alterations in QSOX activity.

CONCLUSION

We suggest that melanogenesis stimulation together with redox impairment caused by GSH-depletion enhanced the oxidative stress in these cells, leading to the effects we observed and changing the phenotype of proliferation to quiescence. Another important observation that remains to be elucidated is that GSH-depleted control cells kept high levels of viability, suggesting a possible adaptative mechanism of survival even under low GSH levels.

Keywords: QSOX, melanogenesis, glutathione

Supported by: CAPES and CNPq

08966 - Poster Session

EC.16 - The antioxidant peroxiredoxin AhpC1 is a key protein in Pseudomonas aeruginosa virulence and in protection against oxidative response

Leonardo Silva Rocha¹, Beatriz Pereira Silva², Thiago Macêdo Lopes Correia⁴, Railmara Pereira da Silva², Diogo de Abreu Meireles⁴, Rafael Pereira^1,5, Luis Eduardo Soares Netto⁴, Flavia Carla Meotti², Raphael Ferreira Queiroz ^1,6

¹Programa Multicêntrico de Pós-graduação em Bioquímica e Biologia Molecular, Universidade Estadual do Sudoeste da Bahia (Bahia, Brazil), ²Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo (São Paulo, Brazil), ³Programa Multicêntrico de Pós-graduação Multicêntrico em Ciências Fisiológicas, Universidade Federal da Bahia (Bahia, Brazil), ⁴Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo (Bahia, Brazil), ⁵Departamento de Ciências Biológicas, Universidade Estadual do Sudoeste da Bahia (Bahia, Brazil), ⁶Departamento de Ciências Naturais, Universidade Estadual do Sudoeste da Bahia (Bahia, Brazil)

Pseudomonas aeruginosa is an opportunistic pathogen with a plethora of virulence factors and antioxidant enzymes that help to subvert the immune system. Here we investigated the role of a 2-Cys peroxiredoxin, alkyl-hydroperoxide reductase C1 (AhpC1), in P. aeruginosa (PA14) survival and virulence. In vitro, wild-type and ΔahpC1 PA14 were incubated with HOCl, H2O2, urate hydroperoxide, and human neutrophils before CFU counting. The rate constant for the reaction of urate hydroperoxide and AhpC was determined by stopped flow. Mice were intranasally instilled with wild-type and ΔahpC1 PA14 strains 24-hours before CFU counting in lung, liver, and spleen, and evaluating some lung inflammatory biomarkers. Mice were monitored for survival after intranasal instillation with wild-type, ΔahpC1 and ΔahpC1 complemented with ahpC1 gene in normouricemic and hyperuricemic animals. Deletion of AhpC1 led to a higher sensitivity to all oxidants. ΔahpC1 was more sensitive to the killing by neutrophils, and less virulent in a mice model of infection. All mice instilled with ΔahpC1 survived for 15 days, whereas 100% died within 3 days with wild-type and complemented strain. A significantly lower number of colonies was detected in lung and spleen of ΔahpC1-infected mice. Total leukocytes, neutrophils, myeloperoxidase activity, pro-inflammatory cytokines, nitrite and lipid peroxidation were much lower in lungs or bronchoalveolar liquid of ΔahpC1-infected mice. Purified AhpC reacted with urate hydroperoxide at 2.3±0.1×106 M−1s−1, and only the ΔahpC1 was sensitive to this oxidant. Uric acid, the urate hydroperoxide precursor, impaired the killing of wild-type by neutrophils but improved the killing of ΔahpC1. Hyperuricemic mice presented higher levels of serum cytokines and succumbed much faster to PA14 infection when compared to normouricemic mice. Collectively, ΔahpC1 PA14 presented a lower virulence due to a poorer ability to neutralize the oxidants generated by inflammatory oxidative burst, especially urate hydroperoxide, leading to a more efficient killing by the host. Keywords: inflammation, oxidants, uric acid Funding: FAPESB, FAPESP, UESB, CNPq and CAPES

Keywords: inflammation, oxidants, uric acid

Supported by: FAPESB, FAPESP, UESB, CNPq and CAPES

08498 - Poster Session

FA.01 - Genomic characterization of HIV-1 BC recombinant viruses

Rodrigo Cunha Oliveira¹, Joana Paixão Monteiro-Cunha^1,1

¹Departamento de Bioquímica e Biofísica, Instituto de Ciências da Saúde, Universidade Federal da Bahia (Bahia, Brasil)

INTRODUCTION

The HIV-1 genetic classification presents 4 groups, subdivided into 10 subtypes which may present subsubtypes and circulating or unique recombinant forms. Currently, 11 CRF BC (07, 08, 31, 57, 60, 61, 62, 64, 85, 86 and 88) are described in the Los Alamos database and the CRF108, CFR110 and CRF118 were identified in Spain and China (2020 and 2021). According to the authors, the recombinant forms represent 22.9% of infections worldwide and may represent a challenge in the development of prophylactic agents.

OBJECTIVES

The aim of this study is the characterize the genetic diversity of HIV-1 BC recombinants, mapping the preferential points of recombination (R), exploring the genetic and biological aspects of their evolution.

MATERIALS AND METHODS

We collected 223 BC sequences in Los Alamos database and all sequences were evaluated in the jpHMM tool. To characterize the sequences, where there is recombination, the generated mosaics were used to estimate the frequency between the B and C subtypes along the genes. To estimate the phylogenetic relationship the alignment was performed using MAFFT online and manually edited using BioEdit software and submitted in IQtree.

DISCUSSION AND RESULTS

The dataset contains BC sequences collected between 1992 to 2018 from 15 countries and 220 sequences were used, 134 (60.9%) of them correspond to 14 CRFs. The mosaics revealed a low frequency of B with the maintence of C. Concerning the structural genes, the R occurred more frequently in pol (69%) and env (63%) whereas gag presented only 34% of intrasubtype recombination. This result was not observed in the accessory and regulatory genes of the virus, only the exception of nef gene, which is shown a high rate of recombination (53%).

CONCLUSION

Based on this preliminary results, posteriorly the genetic and biological importance of recombination events will be investigated, in addition to the phylogenetic relationship between the samples and the worldwide distribution.

Keywords: HIV-1, Recombination, BC subtype

Supported by: Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB)

08365 - Poster Session

FA.02 - Quantitative analysis and simulation of a liquid crystal organizational model in human liver tissue

María Subía Potosí ¹, Hernán Morales-Navarrete²

¹Physics, Escuela Politécnica Nacional (, Ecuador), ²Friedrich Miescher Laboratory, Max Planck Society (, Germany)

INTRODUCTION

Cell polarity coordination is a key phenomenon underlying epithelial tissue structure and function. For complex three-dimensional tissues, such as liver tissue, a proper mathematical characterization of cell polarity has proven to be complicated to develop. The first structural model describing the organization of hepatic tissue was developed by Hans Elias in 1949, but could not describe the complex tridimensional organization of the liver. Nowadays, the current development of imaging techniques and digital reconstruction methods have allowed scientists to revisit the principles of liver organization with unprecedented accuracy. Previous studies have quantified the spatial patterns of apico-basal hepatic cell polarity in mouse liver tissue. Through a conceptual and algorithmic framework, it was found that the structure of liver tissue is in an intermediate state between an amorphous structure and a perfect crystal, best described as a liquid crystal.

OBJECTIVES

In this study, we quantify the nematic cell polarity in three-dimensional tissues to the case of human liver tissue under two physiologically relevant conditions: healthy tissue and tissue with the pathology of Non-Alcoholic Fatty Liver Disease (NAFLD). Then, we elucidate the mechanisms underlying the liquid-crystalline order in human liver tissue.

MATERIALS AND METHODS

We apply the methodology developed for the quantification of nematic cell polarity in three-dimensional tissues for the mentioned study cases. Then, we use Monte-Carlo simulations to characterize the mechanisms (i.e. apico-basal repulsion, local cell-cell interactions, external fields interactions, boundary conditions) underlying liquid-crystalline order in human liver tissue.

DISCUSSION AND RESULTS

Our preliminary results show that human liver tissue follows a liquid-crystalline type of organization as the one found in the mouse liver.

CONCLUSION

The use of quantitative tools combined with simulations has helped to identify the physical properties of complex biological systems such as the liver, and it will continue to be useful in understanding the mechanisms underlying the organization and function of tissues.

Keywords: active matter, liquid crystals, tissue biophysics

08866 - Poster Session

FA.03 - Calcium transport by the Plasma Membrane Ca²⁺ pump (PMCA)

Maximiliano Angel Vigil ¹, Nicolas Saffioti², Débora Rinaldi¹, Irene Mangialavori¹, Rolando Rossi¹, Juan Pablo Rossi¹, Mariela Ferreira-Gomes¹

¹Departamento de Química Biológica, Facultad de Farmacia y Bioquímica. UBA, Instituto de Química y Fisicoquímica Biológicas. “Prof. Paladini” (, Argentina), ²-, Instituto de Nanosistemas, Universidad de San Martin (, Argentina)

INTRODUCTION

Calcium (Ca ²⁺ ) is an important second messenger that participates in many cellular activities. Cells display different mechanisms to maintain low levels of cytoplasmic Ca ²⁺ concentration ([Ca ²⁺ ]_cyt) (100-200 nM) needed to regulate its targets with optimal effectiveness. These mechanisms include transport systems at the plasma membrane level (e.g. Ca ²⁺ -ATPases -PMCAs-, Na ⁺ /Ca ²⁺ exchanger) and at certain intracellular organelle membranes (e.g. the sarcoplasmic reticulum Ca ²⁺ ATPase -SERCA-).

OBJECTIVES

The aim of this study was to investigate the Ca ²⁺ transport by the plasma membrane Ca ²⁺ pump (PMCA) in living cells.

MATERIALS AND METHODS

HEK-293T cell line was transfected transiently with PMCA4 and [Ca ²⁺ ]_cyt was measured in real-time by loading cells with the fluorescent probe Fluo-4. [Ca ²⁺ ]_cyt kinetics were examined by studying the alterations in [Ca ²⁺ ]_cyt generated by Ca ²⁺ released from the sarcoplasmic reticulum (ER), and by extracellular Ca ²⁺ entry through store-operated Ca ²⁺ channels (SOCs). Finally, the results were interpreted in terms of a mathematical model of [Ca ²⁺ ]_cyt kinetics and the parameters were obtained.

DISCUSSION AND RESULTS

The typical experiment of [Ca ²⁺ ]_cyt kinetics showed two phases: (a) transient elevation in [Ca ²⁺ ]_cyt generated by the addition of 1 μM thapsigargin, a SERCA inhibitor that causes Ca ²⁺ released from ER and (b) [Ca ²⁺ ]_cyt increase generated by the addition of 4 mM Ca ²⁺ to the external medium which that induced the SOC activity. In both phases, [Ca ²⁺ ]_cyt reached a maximum and then its stationary level was reestablished. Overexpression of hPMCA4 led to a significant decrease in the global [Ca ²⁺ ]_cyt at all times after the stimulus. On the other hand, the results were interpreted in terms of a mathematical model of [Ca ²⁺ ]_cyt kinetics suggesting that Ca ²⁺ transport by PMCA must increase slowly after [Ca ²⁺ ]_cyt increase to explain the [Ca ²⁺ ]_cyt kinetics.

CONCLUSION

In conclusion, the model built in this study is a useful tool to analyze the PMCA Ca ²⁺ transport activity in living cells.

Keywords: Ca2+-signaling, Ca2+ homeostasis, Mathematical model, PMCA, SERCA, SOCS

08843 - Poster Session

FA.04 - Digital holographic microscopy of diatomite embedded in transparent resins

Yuki Ide¹, Yuji Matsukawa¹, Shigeki Mayama², Kazuo Umemura¹

¹Department of Physics, Tokyo University of Science (Tokyo, Japan), ²Advanced Support Center for Science Teachers, Tokyo Gakugei University (Tokyo, Japan)

INTRODUCTION

According to the improvement of digital holographic microscopy (DHM), numbers of academic papers that reported various micron size objects are increasing in the past several years. On the other hand, sample preparation techniques for DHM is still in the primitive stage. In many papers, micron size objects including living cells were just deposited on a glass surface, and then, observed in air or in liquids.

OBJECTIVES

In this work, we propose a new procedure of sample preparation using several types of resins for digital holographic microscopy (DHM).

MATERIALS AND METHODS

Diatomaceous earth (61790-53-2, MP Biomedicals, LLC) was used as a test sample. Diatomite is fossils of diatoms that are photosynthetic planktons. 0.5 mg of diatomite powder was deposited in a Tomodish (Tomocube Inc.), and then, embedded the diatomite in SYLGARD 184 (761036, Sigma-Aldrich), mount media (139-06682, FUJIFILM Wako Pure Chemical Corp.), or 1.5% agar (016-15812, FUJIFILM Wako Pure Chemical Corp.). Finally, the Tomodish was sealed with a coverslip. The prepared samples were observed by a commercially available DHM (HT-2, Tomocube Inc.). For comparison, the diatomite was also observed in water. Although the diatomite includes aggregates of diatom fossils, isolated individual diatomite (a fossil of one diatom cell) was observed.

DISCUSSION AND RESULTS

As a result, the embedding method was effective to stabilize DHM imaging. In the control sample (water), sometimes position of individual diatomite fluctuated. Furthermore, when diatomite was embedded in SYLGARD 184, higher resolution was obtained. With SYLGARD 184, mesoporous structures of diatomite were well resolved in all the 10 independent individuals. With mount media and 1.5% agar, the mesoporous structures were resolved in 1 and 1 sample among 10 samples, respectively. In water, the structures were observed in 4 samples among 10 samples.

CONCLUSION

Our results suggested that the embedding method drastically affects quality of DHM observation.

Keywords: Diatomaceous earth, Digital holographic microscopy, Embedding resin

08716 - Poster Session

FA.05 - Acceleration of Evolutionary Processes by Learning and Extended Fisher's Fundamental Theorem

So Nakashima ¹, Tetsuya J. Kobayashi^1,2,3

¹graduate school of Information Science and Technology, the University of Tokyo (Tokyo, Japan), ²Institution of Industrial Science, the University of Tokyo (, Japan), ³Universal Biology Institute, the University of Tokyo (, Japan)

INTRODUCTION

Natural selection is a universal and powerful concept not only to explain biological evolution but also to design engineering algorithms like genetic algorithms. Although conventional natural selection is passive in that the mutation is directionless and random, there is an increasing interest in the systems where natural selection and learning interplay. Precisely, we are interested in the natural selection of the population of agents that can learn and replicate. In biological systems, we have increasing pieces of evidence that an organism can transmit information to its descendants via epigenetic states and cultures. By learning from such information, an agent might increase the population fitness without relying on natural selection and thereby accelerate the evolutionary process. In engineering systems, the interplay between natural selection and learning is important to extend and to improve the genetic algorithms.

OBJECTIVES

Although these systems are individually discussed in many fields, we lack theoretical foundations. We do not have a proof that learning can actually accelerate natural selection other than numerical simulations. We also do not know what information or communication is sufficient to optimize the population fitness, which is a trait of the population not of individual agents. In addition, we do not have a methodology to quantify the acceleration. In this work, we give a unified theoretical framework and solve these problems.

MATERIALS AND METHODS

To solve the problem, we propose a learning rule called an ancestral learning. Also, we extend the Fisher’s fundamental theorem of natural selection to quantify the acceleration.

DISCUSSION AND RESULTS

We showed that the ancestral learning can accelerate the evolutionary process. Additionally, we successfully extended the Fisher’s fundamental theorem of natural selection.

CONCLUSION

Since ancestral learning uses the information transmitted from ancestors, such ancestral information is sufficient to optimize the population fitness. The extended theorem relates the acceleration to the variance of the individual fitness of the agents.

Keywords: evolution, evolutionary algorithms, learning

Supported by: JSPS and JST

08559 - Poster Session

FB.01 - Different oxidative response between sexes in Drosophila melanogaster exposed to Biphenol S

Elize Aparecida Santos Musachio ¹, Dieniffer Espinosa Janner¹, Marcia Rosula Poetini Silva¹, Luana Barreto Meichtry¹, Eliana Jardim Fernandes¹, Ketnne Hanna Poleto Pinto¹, Marina Prigol¹

¹Laboratory of Pharmacological and Toxicological Assessments Applied to Bioactiv, Universidade Federal do Pampa (Rio Grande do Sul, Brasil)

INTRODUCTION

The intensification of regulation and prohibition of the use of Bisphenol A (BPA) in some countries, led the industry to use other types of bisphenols in the production of utensils and plastic packaging. Even without in-depth studies, Bisphenol S (BPS) is one of the most used substitutes in the production of products called “BPA-free”. Therefore, evaluating the toxicological action of BPS, and establishing whether there is a difference in the action of this chemical between the sexes, is pertinent information to be considered by scientists, legislators and society

OBJECTIVES

Thus, our aim was to evaluate the effect of BPS on the oxidative stress biomarkers of Drosophila melanogaster, male and female, separately.

MATERIALS AND METHODS

Flies were separated by sex, divided into groups: Control (standard diet only), BPS 0.25 mM, BPS 0.5 mM and BPS 1mM (BPS mixed with standard diet). After 7 days of exposure, analyzes were performed on whole body samples to quantify reactive species (RS), lipid peroxidation (LPO), mitochondrial and cell viability.

DISCUSSION AND RESULTS

Female flies exposed to BPS 0.25, 0.5 and 1mM obtained increased levels of RS. In addition, they showed increased lipid peroxidation, decreased mitochondrial and cell viability at concentrations of BPS 0.5 and 1 mM, when compared to the control group. Men exposed to BPS (0.25, 0.5 and 1mM) also showed increased RS levels and decreased mitochondrial viability at BPS 0.5 and 1mM, when compared to the control group. However, male flies did not show changes in lipid peroxidation and cell viability. Changes in RS levels and decreased mitochondrial viability did not alter the cell viability of male flies.

CONCLUSION

Thus, we conclude that BPS triggered different changes between genders, showing greater damage to female flies, and encouraging us to carry out future studies.

Keywords: Bisphenol S, Bisphenol A, Drosophila melanogaster

Supported by: CNPq and CAPES

08715 - Poster Session

FB.02 - Inference and analysis of a gene regulatory network of angiogenesis in an oxygen-induced retinopathy mouse model

Jhonatas Sirino Monteiro ¹, Débora Guerra Peixe¹, Ricardo José Giordano¹, João Carlos Setubal¹

¹Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Angiogenesis, the formation of new blood vessels from pre-existing ones, is an important therapeutic target. Nevertheless, not all patients benefit from current drugs available for angiogenesis-dependent diseases (i.e. cancer and retinopathies). To address this problem, we sought to achieve a better understanding of gene regulation in angiogenesis, which may help the identification of new targets for drug development.

OBJECTIVES

Previous work by our group has identified by RNA-seq several differentially expressed (DE) protein-coding genes in a mouse model of angiogenesis (OIR, oxygen-induced retinopathy). Here, we expanded on these studies to analyze the mRNA isoforms and regulatory RNAs. These data will then be integrated to generate a comprehensive gene network of the OIR model.

MATERIALS AND METHODS

Seven-day-old mice were placed in a hyperoxic environment (75% O₂) for five days and then returned to room air. This causes a sudden hypoxic condition leading to VEGFA overexpression, resulting in abnormal vascular growth and pathological angiogenesis. Retinas were dissected for RNA extraction and sequencing right after the return to room air, but also 3 and 5 days later. The RNA-Seq data were analyzed using bioinformatics pipelines.

DISCUSSION AND RESULTS

We identified 60 DE mRNA isoforms, of which 26 were inferred to be homologous to human isoforms. We also identified 99 DE miRNAs, of which 5 are new candidates. Most of the predicted targets for these miRNAs are related to angiogenesis. Among lncRNAs, we identified 218 DE, of which 57 are already described and 161 are new candidates. We predicted 5421 circRNAs, half of which are novel. The next step is to build the co-expression network using WGCNA to identify the interactions between mRNAs and these regulatory RNAs.

CONCLUSION

Most of the RNAs that were identified as DE are promising candidates for future studies and validation as potential targets for drug therapies, or as diagnostics/prognosis biomarkers for angiogenesis-dependent diseases.

Keywords: angiogenesis, network, RNA

Supported by: FAPESP and CAPES

08414 - Poster Session

FB.03 - Trans fatty acid during the developmental period induces cognitive deficit in Drosophila melanogaster

Luana Barreto Meichtry ¹, Guilherme Silva da Silva¹, Larissa Londero¹, Mustafa Munir Mustafa Dahleh¹, Vandreza Cardoso Bortolotto¹, Stífani Machado Araujo¹, Márcia Rósula Poetini¹, Elize Aparecida Santos Musachio¹, Gustavo Petri Guerra¹, Marina Prigol¹

¹Laftambio, Universidade Federal do Pampa (Rio Grande do Sul, Brasil)

INTRODUCTION

Trans fatty acid (FA) is highly consumed by the population, this FA can be incorporated into neural membranes, causing changes in its structure, fluidity, and permeability, and consequently, impairing cognitive functions. These changes occur more intensely during the development, so the type of FA consumed during this period is important. In this work, we use the model of Drosophila melanogaster, which has demonstrated great homology with human systems.

OBJECTIVES

The objective of the work was to evaluate parameters related to the cognition of Drosophila melanogaster exposed to hydrogenated vegetable fat (HVF) during the developmental period and associated with the incorporation of these fatty acids in the nervous tissue of the fly.

MATERIALS AND METHODS

Progenitors were divided into 4 groups, containing 50 flies of both sexes each: (1) RD (corn flour, sugar, wheat germ, salt, powdered milk, and agar), (2) SHVF (RD fat values were replaced by HVF in the same proportion), (3) HVF 10% (RD with 10% HVF) and (4) HVF 20% (RD with 20% HVF). After 7 days of exposure, the progenitors were removed from the medium, leaving the larvae and eggs. Flies born on this medium (1-3 days) were used to assess short and long-term memory and FA composition in the head.

DISCUSSION AND RESULTS

There was a reduction in memory at 6h and 24h in flies exposed to HVF in all concentrations compared to the RD. An increase in the presence of trans FA was observed in the heads of flies exposed to HVF in all concentrations, compared to the RD. In addition, there was a reduction in monounsaturated FA and an increase in polyunsaturated FA in the group exposed to SHVF when compared to the HVF 10% and HVF 20% groups.

CONCLUSION

It was concluded that trans FA can be incorporated in the flies' heads and thus causing changes related to cognition deficit.

Keywords: memory, membranes, diet

Supported by: CAPES and CNPq

08679 - Poster Session

FB.04 - Biochemical implementation of optimal control for run-and-tumble chemotaxis

Kento Nakamura ¹, Tetsuya J. Kobayashi^1,2,3

¹Graduate school of Information Science and Technology, University of Tokyo (Tokyo, Japan), ²Institute of Industrial Science, University of Tokyo (Tokyo, Japan), ³Universal Biology Institute, University of Tokyo (Tokyo, Japan)

INTRODUCTION

Various organisms search for food, good environments, and other targets with remarkable efficiency. One representative search strategy is run-and-tumble chemotaxis. Organisms can climb a spatial gradient of ligand concentration by controlling the frequency of random directional changes (tumble) based on sensed temporal changes of the concentration during ballistic swimming (run). It has been a fundamental question how efficient the run-and-tumble chemotaxis can be in principle and whether organisms have a biochemical mechanism that achieves that efficiency limit or not. This question has been addressed by deriving optimal control of run-and-tumble motions theoretically and comparing it with measured motor responses of organisms.

OBJECTIVES

Due to theoretical difficulties, the previous derivations of the optimal control ignore sensory noise and nonlinearity of response, hindering the detailed relation between the optimal control and biochemical systems. Here, we overcome such difficulty by deriving the nonlinear optimal control of run-and-tumble motion under sensory noise.

MATERIALS AND METHODS

We combine the nonlinear filtering theory and Kullback-Leibler control of partially observed Markov decision process (POMDP).

DISCUSSION AND RESULTS

The derived optimal control consists of two parts: the filtering dynamics, which extracts necessary information from the noisy signal, and the optimal control function, which converts the information to the motor control. Furthermore, the optimal control reproduces a standard biochemical model of the sensory system of Escherichia coli and a nonlinear response relation observed experimentally, indicating that the E. coli's biochemical pathway can implement the optimal structure for achieving efficient run-and-tumble chemotaxis.

CONCLUSION

We derived the nonlinear optimal control of run-and-tumble chemotaxis under sensory noise and show its implementation in the biochemical pathway of E. coli. Our derivation can work as a theoretical basis for investigating the efficiency of more complex sensory-motor integrations in run-and-tumble chemotaxis.

Keywords: Chemotaxis, Optimal control, Nonlinear filtering

Supported by: JSPS, JST

08521 - Poster Session

FB.05 - PROJECT INVESTIGATION OF ABORTION CASES AT ALMEIDA CASTRO MATERNITY IN MOSSORÓ/RN

TAYSSA BARBOSA¹, DIOGO CAVALCANTI²

¹CIÊNCIAS BIOMÉDICAS, UNIVERSIDADE DO ESTADO DO RIO GRANDE DO NORTE (Rio Grande do Norte, Brasil), ²CIENCIAS DA SAUDE, UNIVERSIDADE FEDERAL DO SEMI ARIDO (Rio Grande do Norte, Brasil)

INTRODUCTION

Abortion is defined as termination of pregnancy in which the fetus is unable to survive outside the uterus. Statistics show that, of diagnosed pregnancies, the incidence of this complication occurs in 10 to 15% of cases and 80% of cases occur in early pregnancy.

OBJECTIVES

Given the above and associated with the scarcity of information on the etiology of these losses, the objective of the research is to analyze the possible causes and effects of abortions that occur in the Almeida Castro maternity hospital, as well as to identify the predisposing factors and factors of pregnancy loss, seeking to characterize the profile of parents and fetus; investigate possible teratological changes, describing the possible macroscopic and microscopic changes associated with each case of abortion.

MATERIALS AND METHODS

This is a quantitative experimental research, carried out in collaboration with the Maternity Almeida Castro in the Municipality of Mossoró-RN. The study population will be composed according to the cases of abortion documented by the Hospital Almeida Castro of the Municipality of Mossoró-RN and the samples will be based on the casuistry. Data collection will be done following a pre-established script. First, the mother's history will be investigated, based on the data described in her medical record. Once selected, in the second moment, the macroscopic analysis of the fetus will be carried out in the maternity ward, if any abnormality or suspicion of alteration is found, in the third subsequent moment, samples of at most 1 cm² from specific regions will be collected. and these punctual biopsies will be forwarded for microscopic analysis at UFERSA's Histopathological Processing Laboratory.

DISCUSSION AND RESULTS

CONCLUSION

It is expected, therefore, to be able to understand the causes and risk factors for abortion cases in this population, as well as to arrive at a diagnosis to understand its causes, which is a much neglected area of research.

Keywords: Abortion, Birth Defects, Teratology

08832 - Poster Session

FB.06 - Blood serum lipidomics of patinete with severe respiratory syndrome, positive and negative for SARS-CoV-2 by NMR techniques

Erik Sobrinho Braga ¹, Lucas Gelain Martins¹, Danijela Stanisic¹, Milka Jadranin², Ljubica Tasic¹

¹Organic Chemistry, Institute of Chemistry, University of Campinas (São Paulo , Brazil), ²Chemistry, Institute of Chemistry (Belgrade, Serbia)

INTRODUCTION

COVID-19 disease, which affects patients infected with the SARS-CoV-2 virus, has a different fatality and behavior from respiratory infections caused by other viruses. In this context, understanding metabolic variations is fundamental to understand how the disease may affect us. Important components of blood serum, lipids are related to several metabolic functions and their variations in quantity and variety related to the disease can guide the understanding of the pathogen's action or the search for therapeutic targets.

OBJECTIVES

Therefore, this study aims to identify the lipids responsible for distinguishing between two classes of patients with severe acute respiratory syndrome (one group of individuals with a positive RT-qPCR test for SARS-CoV-2), and a group of age-matched healthy volunteers.

MATERIALS AND METHODS

Serum samples were obtained from patients and volunteers at the University of Campinas Hospital and stored at -80 °C until the moment of analysis. Lipid extractions were performed by diluting 150 μL of blood serum in 350 μL of MiliQ water, mixed with 500 μL of methanol and chloroform. The samples were shaken for 2 min, then centrifuged at 2200 rpm for 20 min at 25 °C and transferred to a freezer at -20 °C for 30 min. Then the polar and non-polar phases were separated, and both were analyzed by NMR. High resolution 1H-NMR spectra were obtained on the Bruker AVANCE III 600 MHz instrument at 25 °C.

DISCUSSION AND RESULTS

Sera from positive to SARS-CoV-2 patients showed lower levels of cholesterol, polar lipids, and lysophosphocolines. But, higher VLDL and phosphocholine levels were measured in the same cohort. On the other side, serum samples from negative to SARS-CoV-2 patients showed similar trends in levels of lipids, although in lesser extent.

CONCLUSION

The data show that NMR spectroscopy can be applied to patient identification, in addition to demonstrating differences in patient lipid levels and types in blood serum samples.

Keywords: COVID-19 , Respiratory infections , 1H-NMR

Supported by: CAPES

08637 - Poster Session

FB.07 - Peripheral blood as a tool to determine gene expression patterns in patients with psychiatric and neurological disorders: a systematic review and meta-analysis

Alana Castro Panzenhagen ¹, Alexsander Alves-Teixeira¹, Lucas Santos¹, Maikel Varal¹, Giovani Pereira de Andrade¹, Marlene Campos¹, Carolina Saibro Girardi¹, Alexandre Kleber Silveira¹, Daniel Pens Gelain¹, José Cláudio Fonseca Moreira¹

¹Biochemistry, Universidade Federal do Rio Grande do Sul (RS, Brazil)

INTRODUCTION

Psychiatric disorders have been investigated on several biological levels. One that reflects the interaction of genes, proteins, and transcription factors is the transcriptome.

OBJECTIVES

Thus, we aim at summarizing the evidence regarding gene expression levels of peripheral blood samples between subjects with psychiatric and neurological disorders to healthy controls and later comparing those results to genome-wide association studies (GWAS) through systems biology.

MATERIALS AND METHODS

The Gene Expression Omnibus and the ArrayExpress databases were searched with specific strategies to find studies that included blood samples and investigated the following disorders: major depressive disorder (MDD), schizophrenia (SCZ), autism spectrum disorder (ASD), Parkinson’s disease (PD), or Alzheimer’s disease (AD). The duplicates were removed and the records were screened independently by two reviewers. Studies were meta-analyzed using the R environment package metaMA. The genes found were also compared to the GWAS mapped genes. Moreover, analyses concerning protein-protein interaction (PPI) networks were conducted.

DISCUSSION AND RESULTS

Meta-analysis procedures revealed that the expression seems to be consistently altered for 5 genes in the MDD group, 1105 genes in SCZ, 8 genes in ASD, 22 genes in PD, and 717 genes in AD. From the genes found in SCZ, seven are also mapped genes in Schizophrenia GWAS studies. Additionally, from the ones found in ALZ, 10 genes are also mapped genes in Alzheimer’s disease GWAS studies. Furthermore, when PPI networks are investigated based on the differential expressed genes found, they show that their proteins are connected. Enrichment of PPI networks found an association with biological processes related to the synapse, cellular signaling, circadian rhythm and inflammatory response.

CONCLUSION

It is certainly interesting to show that some genes already associated in GWAS were found with a consistently differentially expressed pattern across transcriptome studies. This approach can be an ally to transcriptome-wide association studies in the investigation of genomic variants and their impact on gene expression.

Keywords: biochemistry, neuropsychiatry, transcriptomics

Supported by: CNPq, CAPES, and FAPERGS/PPSUS

08775 - Poster Session

FB.08 - Lutein Nanoparticles Protect Anxious-Like Behavioral Changes and Oxidative Damage in Rat Brain in a Prenatal Model of Autism

Cristini Escobar Viana ¹, Vandreza Bortolotto¹, Stífani Araujo¹, Mustafa Dahleh¹, Franciéle Machado¹, Adson Pereira², Odinei Gonçalves³, Marina Prigol¹, Gustavo Guerra¹

¹Department of Biochemistry - LaftamBio Pampa, Federal University of Pampa (Rio Grande do Sul, Brasil), ²Department of Biochemistry - GPBioFEx, Federal University of Pampa (Rio Grande do Sul, Brasil), ³Department of Biochemistry - PPGTA, Federal Technological University of Paraná (Rio Grande do Sul, Brasil)

INTRODUCTION

Autism Spectrum Disorder (ASD) has increased in prevalence in recent years, but there are still many diagnostic criteria and therapeutic resources to be established. Bioactive compounds such as lutein, an important carotenoid, have antioxidant and neuroprotective potential, as it crosses the blood-brain barrier, demonstrating a neuroprotective effect in diseases such as ASD.

OBJECTIVES

Thus, this study aims to evaluate the action of lutein-loaded nanoparticles on anxiety-like behavior and oxidative stress in the experimental model of valproic acid-induced ASD (VPA) in rats.

MATERIALS AND METHODS

Fifteen adult Wistar rats (90 - 120 days old) were used, constituting the F0. On the 12.5th day, the pregnant females were separated and received an intraperitoneal injection of VPA (600 mg/kg) or saline (0.9% NaCl, 1 mL/kg). On the 21st day, the female offspring (F1) were subdivided into two groups and received nanoparticles loaded with lutein in the form of nanoparticles (5 mg/kg) or saline (1 mL/kg), orally for 14 days. On the 15th day, the open field behavioral test (OFT) was performed and after euthanization, the brain structures were used to quantify reactive species (RS).

DISCUSSION AND RESULTS

To measure the animals' anxiety, the length of stay in the center of the OFT was determined, a characteristic associated with reduced cognitive control in the ASD. Concomitantly, changes in RS cause oxidative stress and neurological dysfunction supporting the pathogenesis and/or severity of ASD. Our results showed that lutein was able to protect changes in the anxious and apathetic-like behavior characteristic of ASD, as well as VPA-induced cerebral oxidative damage in this animal model of autism.

CONCLUSION

These findings suggest that lutein may be a natural alternative to protect the anxiety phenotype and oxidative alterations associated with autism.

Keywords: neurodevelopmental disorder, carotenoid, valproic acid

Supported by: CNPq; CAPES; Unipampa - PPG biochemistry

08573 - Poster Session

FB.09 - Bisphenol A alters the development of Drosophila melanogaster exposed during the embryonic period

Dieniffer Espinosa Janner ¹, Elize Aparecida Santos Musachio¹, Márcia Rósula Poetini¹, Eliana Jardim Fernandes¹, Marina Prigol¹, Gustavo Petri Guerra¹

¹Programa de Pós graduação em Bioquimica, Universidade Federal do Pampa (RS, Brasil)

INTRODUCTION

Bisphenol A (BPA), is a ubiquitous chemical substance used in the synthesis of polycarbonate plastic and epoxy resins, inserted in a variety of everyday products, such as plastic bags and containers, thermal papers, cans for food and beverages, and several other materials. BPA is identified as an endocrine disruptor, in which several studies propose a relationship between exposure to BPA and the emergence of adverse health effects, such as cancer, infertility, diabetes and obesity, among others. Increasing evidence indicates that exposure to BPA is related to a variety of disorders in the reproductive system, which are being studied.

OBJECTIVES

The present study aimed to evaluate the effect of embryo exposure to BPA on the development of Drosophila melanogaster.

MATERIALS AND METHODS

For this study, virgin males and females were divided into 3 groups: 1) Control (standard diet only); 2) BPA 0.5 mM and 3) BPA 1 mM (respective concentrations of BPA mixed with the standard diet). After copulation, the progenitors were removed and the treatment medium containing the embryos was preserved for the following evaluations: a) average of viable eggs, b) hatching percentage in relation to the average number of eggs laid, c) eclosion rate of pupae.

DISCUSSION AND RESULTS

Flies exposed to different concentrations of BPA did not show a statistical difference in the percentage of viable eggs compared to the average number of eggs laid. Flies exposed to BPA 1 mM showed a decrease in the eclosion rate of the pupae when compared to the control group.

CONCLUSION

Our data suggest that exposure to BPA does not interfere with embryo fertility, but exposure during larval development influenced the complete development of the fly, as seen by the decrease in pupal and eclosion rates

Keywords: Drosophila melanogaster, Eclosion, Bisphenol A

Supported by: Unipampa, CAPES and CNPq

08750 - Poster Session

FB.10 - Comparative transcriptomics among archaea to search for circular RNAs

Beatriz Picinato ¹, Vinícius Santos¹, Ricardo Vêncio², Lívia Zaramela¹, Tie Koide¹

¹Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (, Brazil), ²Departamento de Computação e Matemática, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (, Brazil)

INTRODUCTION

Circular RNAs (circRNA), i.e. RNA molecules with no 3' nor 5' loose ends, are pervasive in eukaryotes and progressively being understood not as mere splicing by-products but rather probably functional molecules. In bacteria, on the other hand, circRNAs were not consistently reported. Therefore, archaea, interesting prokaryotes withstanding features from both eukaryotes and bacteria, are well placed organisms to provide insights on evolutionary paths that established circRNAs. A specialized RNA-seq protocol, introducing an RNase R treatment sample preparation step before deep sequencing, is commonly used to search for circRNAs in a high-throughput fashion (Circ-seq). Among archaea, public Circ-seq data is available for Saccharolobus solfataricus, Sulfolobus acidocaldarius and Pyrococcus abyssi, covering only the Crenarchaeota and Thermococci clades.

OBJECTIVES

We performed Circ-seq experiments in Halobacterium salinarum, a prominent extremophile model organism representing the Stenosarchaea group.

MATERIALS AND METHODS

We implemented a bioinformatics pipeline, named Monark-seq, that explores high-quality non-aligned reads from regular RNA-seq pipeline genome alignments searching for circRNAs signatures which also survived RNase R digestion.

DISCUSSION AND RESULTS

Preliminary data yielded 763 putative redundant circRNAs signatures coming from ~80 unique genes in H. salinarum NRC-1 strain, some of which were observed in S. solfataricus and P. abyssi: tRNA ^{Trp < /sup > and both 16S and 23S rRNAs.}

CONCLUSION

Further analysis will probably reveal conserved sequences that may shed light on functional and biogenesis aspects of circRNAs.

Keywords: Archaea, Circular RNAs, RNase R

Supported by: FAPESP, FAEPA and CAPES

08101 - Poster Session

GA.01 - Effect of mealworm meal on immunological parameters, digestive enzymes and midgut microbiota of the shrimp Litopenaeus vannamei

Carlos Peres Silva ¹, Cristina Rios¹

¹Bioquímica, Universidade Federal de Santa Catarina (Santa Catarina, Brasil)

INTRODUCTION

Shrimp farming is one of the most profitable sectors of aquaculture, and the shrimp Litopenaeus vannamei is one of the most cultivated species worldwide. The nutrition of species is a great hindrance of shrimp farming, due to its high cost, in particular due to the use of fishmeal as one of the components in the feeds. Many studies have been carried out to strength the use of insect meal in shrimp feed without financial losses.

OBJECTIVES

The main goal of the present work was to analyze if the substitution of fish meal (TM-0) by mealworm meal (TM-25, TM-50, TM-75 and TM-100) can influence immunological parameters, digestive enzymes and midgut microbiota of L. vannamei.

MATERIALS AND METHODS

Five diets consisting of 0%, 25%, 50%, 75% and 100% of fishmeal replacement by MLM (denominated TM-0, TM-25, TM-50, TM-75 and TM-100) were performed during six weeks of culture. All diets were formulated on a digestive basis to provide 300 g kg-1 protein digestible and 3000 cal kg-1 energy digestible. The total count of hemocyte, intestinal microbiota, total protein concentration, phenoloxidase activity in haemolymph and the activities of trypsin, chymotrypsin, lipase and α-amylase were quantified.

DISCUSSION AND RESULTS

The total count of hemocytes, protein concentration, phenoloxidase activity in haemolymph were not significantly altered when TM-0 was substituted by the insect meals (p > 0.05). However, the agglutinating activity of L. vannamei serum against dog erythrocytes was higher in the shrimp group fed with TM-0. The results of the absolute and specific activities of trypsin, chymotrypsin, lipase and α-amylase demonstrated no significant differences between treatments. The shrimp midgut microbiota profile was similar in bacteria of the genus: Pseudoalteromonas, Rubritalea, Ruegeria, Tenacibaculum and Vibrio.

CONCLUSION

These results support that the mealworm meal can be utilized as protein source for L. vannamei without any loss of digestive capability.

Keywords: Digestion, Aquaculture, Entomophagy

Supported by: Capes

08579 - Poster Session

GA.02 - iTRAQ-based quantitative proteomic analysis of the midgut from the soybean caterpillar Anticarsia gemmatalis in the presence of Bacillus thuringiensis Cry1Ac toxin

Luis Felipe Costa Ramos ¹, Yara Martins Silva¹, Gilberto Domont¹, Cristiane Dinis AnoBom¹, Fábio César Souza Nogueira¹, Magno Junqueira¹, Danielle Maria Perpétua Oliveira¹

¹Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Anticarsia gemmatalis is considered one of the main defoliating pest of soybeans in Brazil, being an important target of pest control. Bacillus thuringiensis is a Gram-positive bacterium, with wide global dispersion, characterized by the production of Cry toxins during the stationary phase of its growth (sporulation), to which an entomopathogenic effect is attributed. That is why this microorganism is used as a biopesticide to control several agricultural pests, including A. gemmatalis. However, insects have developed resistance to this control.

OBJECTIVES

This work aims to obtain the quantitative proteome of the midgut epithelium of A. gemmatalis after challenged or not with the toxin Cry1Ac of B. thuringiensis.

MATERIALS AND METHODS

A bioassay was performed to calculate the lethal concentration (LC50) of B. thuringiensis, using a total of 35 caterpillars in seven different concentrations of the sporulated bacteria, monitoring the survival rate for 96 hours. Midguts were dissected and protein extraction from epithelial tissue was performed using S-Trap column (Protifi®) protocol. Trypsin (Promega®) was used for the digestion step. After an iTRAQ labeling method, a fractionation step by reverse phase chromatography was applied and samples were analyzed in Q-exactive Plus (ThermoScientific®) mass spectrometer. Protein identification was performed using Proteome Discoverer 2.4 (ThermoScientific®).

DISCUSSION AND RESULTS

Bioassay results showed a LC50 of 0.073 mg/mL using B. thuringiensis sporulated bacteria against A. gemmatalis. A total of 2,990 proteins from 24h and 48h exposed caterpillars were identified by the bottom-up proteomics approach. A deeper proteome analysis will be performed since there is no description for this insect midgut. To overcome this difficulty, we plan to identify proteins by de novo sequencing and homology search protocol, used to describe proteomes of organisms with unsequenced genomes, comparing up and down-regulated proteins after B. thuringiensis infection.

CONCLUSION

Proteomics approach can elucidate the mechanisms involved in the response of A. gemmatalis during B. thuringiensis infection.

Keywords: Anticarsia gemmatalis, Bacillus thuringiensis infection, Proteomics

Supported by: CAPES and FAPERJ

08272 - Poster Session

GA.03 - The cytotoxic effect of Amblyomma sculptum tick saliva over cancer cell lines is associated with ΔΨm loss and cleavage of caspases-3 and caspase-9.

Sheron Campos Cogo ^1,2, Nilton de França Junior^1,2, Geison Cambri^1,2, Olivier Micheau², Simone Simons³, Selene Elifio Esposito¹

¹Programa de Pós-graduação em Ciências da Saúde, Pontifícia Universidade Católica do Paraná (Paraná, Brasil), ²UFR Science de Santé, Université de Bourgogne Franche-Comté (Franche-Comté, France), ³Departamento de Parasitologia, Instituto Butantan (São Paulo, Brasil)

INTRODUCTION

Animal secretions are promising sources of bioactive molecules. The crude saliva of Amblyomma sculptum (Fabricius, 1787) has demonstrated cytotoxic effects on different tumor cell lines, with no cytotoxicity in non-tumoral cells. However, little is known about the mechanisms involved in those activities.

OBJECTIVES

Here, we elucidate the effect of A. sculptum crude saliva on activation of programmed cell death pathways in tumoral cell lines.

MATERIALS AND METHODS

The crude saliva (SL) was collected by the Butantan Institute using microcapillary tubes and dopamine stimulation. Breast cancer (MDA-MB-231), colorectal adenocarcinoma (HCT-116), and neuroblastoma (SH-SY5Y, SK-N-SH and Be(2)M17) lineages were treated with 10% of the SL for 72 hours and analyzed to detect apoptosis, loss of mitochondrial potential and caspase cleavage. 5-Aza-2-deoxycytidine (AZA) demethylating agent known to increase caspase-8 expression and the pan-caspase-inhibitor Q-VD-Oph were tested in combination with SL. Protein expression was analyzed by western blotting. To evaluate the role of antiapoptotic proteins and mitochondrial potential control, cells overexpressing Bcl-XL and Mcl-1 recombinant proteins were generated by lentiviral infections. Statistical analyses were performed using GraphPad-Prism6.0.

DISCUSSION AND RESULTS

Apoptosis induction was observed after SL treatment on tested cell lines. Protein expression profiles showed that neuroblastoma lineages are deficient in initiating caspases and death receptors, suggesting SL activity to be independent of extrinsic apoptotic triggers. Which was supported by the fact that caspase-8 recovered expression with AZA did not affect cell death. QVD, however, was able to entirely protect cells against SL. Overexpression of Mcl-1 and Bcl-XL was able to prevent apoptosis and mitochondrial potential loss. Caspase-3 cleavage was no longer present in the generated cell lines, as well as laminin A/C and caspase-9 cleavage.

CONCLUSION

Therefore, we provide evidence that A. sculptum crude saliva induces intrinsic apoptosis in tumor cell lines by mitochondrial potential loss and consequent cleavage of caspase-9 and caspase-3.

Keywords: Tick saliva, Antitumoral, Intrinsic apoptosis

Supported by: CAPES/COFECUB

08362 - Poster Session

GA.04 - Protein profile of Tityus paraguayensis venom and hemolymph

Mila Marluce Lima Fernandes ¹, Henrique Ranieri Covali Pontes¹, Telma Rodrigues Silva Benetti¹, Jeandre Augusto dos Santos Jaques¹, Malson Neilson Lucena¹

¹Setor de Bioquímica, Instituto de Biociências, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

Tityus paraguayensis scorpion is endemic in Mato Grosso do Sul. The chemical composition of the scorpion venom can vary between species and individuals, and season. Another potential source of molecules in scorpions is the hemolymph. Several antimicrobial peptides (AMPs) like defensins and open-end cyclic peptides have been described in scorpion hemolymph.

OBJECTIVES

This study aimed to determine the protein profile of hemolymph and venom from T. paraguayensis scorpion.

MATERIALS AND METHODS

Protein concentration was determined by the Bradford method. In order to analyze the protein profile, the SDS-PAGE electrophoresis was realized under reducing conditions. The concentration of venom protein measured was 3.54 mg/mL.

DISCUSSION AND RESULTS

The electrophoretic profile of venom revealed the presence of seven proteins with different sizes, ranging from 150 to smaller than 10 kDa. The most abundant proteins in the venom have 75 kDa, 70 kDa and 10 kDa. The protein of 10 kDa suggests the presence of low molecular weight proteins or peptides, which are described in scorpion venoms as responsible for the toxicity of the venoms. On the other hand, 70 kDa and 75 kDa proteins correspond to hemocyanins, which are abundant protein complexes in arthropod hemolymph, being involved in gas exchange. To hemolymph, the concentration of plasma fraction measured was 4.23 mg/mL, while hemocytes fraction showed the lowest concentration (1.12 mg/mL). Both hemolymph fractions analyzed revealed a predominant band around 75 kDa. These results demonstrate a high presence of hemocyanin in hemolymph and venom.

CONCLUSION

This study demonstrated that venom and hemolymph of T. paraguayensis not only contains hemocyanin but other proteins, which can serve as a starting point for future research facilitating the identification of the venom and hemolymph composition of scorpions.

Keywords: proteins, peptides, scorpion

Supported by: CNPq, FUNDECT and UFMS

08914 - Poster Session

GA.05 - Acoustofluidic Interferometric Device (AID) for cells' mechanical characterization

Julián Mejía Morales ^1,2,5, Gian-Luca Lippi², Peter Glynne-Jones³, Massimo Vassalli⁴

¹Università di Genova, (Genova, Italy), ²Université Côte d’Azur, (Nice, France), ³University of Glasgow, (Glasgow, UK), ⁴University of Southampton, (Southampton, UK), ⁵Ghent University, (Gent, Belgium)

Here we present a high throughput single cell physical characterization device intended to address the growing need for marker-free tools for biomedical applications. Intended for real time cells Optical Thickness (OT) assessment We designed and implemented a microfluidic chip integrating a Fabry-Perot resonator which allows to measure the OT profile of cells flowing through it with high throughput. To ensure the reliability of the measurement, cell trajectories should be constrained as much as possible on the same plane. A piezoelectric transducer was coupled to the chip, tuned to induce an acoustic standing wave in the chip, thus providing vertical alignment of floating cells. The same experimental setup has been exploited towards cellular mechanotyping. In fact, the size distribution collected with the system depends on the power applied to the acoustic trap, hence, it can be related to the deformability of the cell. Comparing the distribution of elasticity collected at very different values, is possible assess the overall mechanical properties with high statistical relevance aimed at measuring drugs effect or physiological and pathological condition response on this parameter. The analysis of the perturbation enables the assessment of the cell’s optomechanical properties. Measurement of Algae and Yeast cells’ deformability has been carried out to test the instrument’s performance and compared to the equivalent perturbation introduced by Microgel beads and Polystyrene spheres as controls. The results show a change in the optomechanical properties of the Algae, Yeast, and Microgel. Notoriously, the Polystyrene sample remains virtually unchanged, as expected since Polystyrene is much stiffer than a cell and cannot be deformed by the instrument’s pressure field. These results show that the acoustofluidic technique presented here is useful to detect and measure different optomechanical properties which, potentially, can be used as label-free biomarkers in clinical diagnosis. Corresponding author: julian.mejia_morales@ugent.be

Keywords: Microfluidics Cytometry, Cells Mechanics, Acoustofluidic Interferometric Device

08393 - Poster Session

GA.06 - Project: Biochemical and biological characterization of venom and hemolymph from scorpions Tityus mattogrossensis e Tityus confluens

Laís Corrêa de Lima ¹, Malson Neilson de Lucena^1,1

¹Instituto de Biociências (INBIO), Universidade Federal do Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

Scorpions are an order of animals that cause poisoning due to their venom and have been considered a public health problem in several countries as Brazil. Both scorpion venom and hemolymph are important for biochemical, physiological and pathological studies, as well as promising for the development of new biotechnological and pharmacotherapeutic product.

OBJECTIVES

The present project aims to evaluate the biochemical and biological characteristics of the venom and hemolymph of from scorpions Tityus mattogrossensis and Tityus confluens.

MATERIALS AND METHODS

In order to understand and characterize the effects of venom and hemolymph of scorpions, their effects on the activity of enzymes (Na+, K+)-ATPase and Ca2+-ATPase will be evaluated. The proteolytic enzymes, phospholipase A2 and fibrinogenolytics activities will also be investigated. Furthermore, the cytotoxicity of venom and hemolymph against normal and tumor cells, and T. cruzi epimastigote will be assayed.

DISCUSSION AND RESULTS

The dates will be validated by statistical analysis and it is expected to improve the knowledge about these scorpions present in Mato Grosso do Sul, as well as to identify compounds with pharmacological or biotechnological potential. In addition, to contribute for the understanding of the role of these compounds in the clinical overview, promoting a more appropriate treatment of scorpionic accidents.

CONCLUSION

Keywords: Enzymes, scorpion, Tityus sp.

08783 - Poster Session

GA.07 - Construction of a Rhipicephalus microplus salivary peptide library on the surface of filamentous phages

Gabriel Cerqueira Alves Costa ¹, Aparecida Sadae Tanaka¹

¹Bioquímica, Universidade Federal de São Paulo (São Paulo, Brasil)

INTRODUCTION

Rhipicephalus microplus is a tick species present in Brazil and well known to parasite cattle. High levels of parasitism can seriously affect the animals, resulting in reduced milk and meat production and causing substantial economic losses to the livestock. The control of ticks in animals is carried out mainly by using chemical acaricides from different families, but the indiscriminate use of these agents can be responsible for the emergence of resistant tick populations and the environmental contamination. Thus, it is necessary to search for alternative control methods that could be employed in herds and contribute to the reduction of parasitism levels by ticks.

OBJECTIVES

The main objective of the present study is to identify immunogenic targets that could be useful in control of R. microplus through the phage display technique.

MATERIALS AND METHODS

Firstly, salivary glands from partially engorged tick females were dissected and the total RNA was extracted. A cDNA library was synthetized, amplified by PCR and fragmented by a restriction enzymes cocktail. Then, purified DNA fragments were cloned into the pHORF3 phagemid.

DISCUSSION AND RESULTS

A pool of salivary glands from five female ticks (mean weight = 55mg) was used in the cDNA library construction. The cDNA fragments library was used to transform E. coli TOP10F’ competent cells. Transformation titer of 2,6x10 ⁴ and insert rate of 63% were observed, while most of the cloned inserts ranged from 100 to 300 bp. cDNA fragments library will be packaged in M13 phages, that will express and present the salivary peptides on its surfaces. Then, by the phage display technique, a screening of the most reactive M13 phages against IgG from tick-resistant cattle will be performed.

CONCLUSION

It is expected that the selected immunogenic peptides could be useful for the development of an effective vaccine against the R. microplus parasitism.

Keywords: Phage display, R. microplus, salivary glands

Supported by: FAPESP

08305 - Poster Session

GA.08 - Tityus paraguayensis venom increases the K⁺-phosphatase activity of the enzyme (Na⁺/K⁺)-ATPase from rat kidney

Henrique Ranieri Covali Pontes ¹, Mila Marluce Lima Fernandes¹, Igor Leal Brito¹, Jeandre Augusto dos Santos Jaques¹, Malson Neilson de Lucena¹

¹Instituto de Biociências, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

Scorpion venoms have a great chemical diversity, and several biological activities have been described. However, there are a few studies about some species, such as the endemic scorpion Tityus paraguayensis. (Na ⁺ /K ⁺ )-ATPase is important to maintain the osmotic balance for the transport of ions through the membrane and one of the proteins that was shown to interact with (Na ⁺ /K ⁺ )-ATPase in the presence of ouabain is the so called melittin like protein from bee venom.

OBJECTIVES

Thus, this study aims to evaluate the effect of the venom from scorpion Tityus paraguayensis on the (Na ⁺ /K ⁺ )-ATPase enzyme.

MATERIALS AND METHODS

The venom was obtained by an electrical stimulus applied to the scorpion's telson. After this, the venom was diluted in distilled water and centrifuged, followed by lyophilization of the supernatant. Bradford assay was used for protein quantification. To evaluate the K ⁺ -phosphatase activity of the enzyme (Na ⁺ /K ⁺ )-ATPase, reactional medium was prepared with the substrate pNPP, in the presence or the absence of ouabain. The specific activity of the enzyme was obtained by the difference between in absence and presence of ouabain. The reactional medium was incubated for 50 minutes at 37 ^° C with 50 μg of rat kidney homogenate with or without scorpion venom.

DISCUSSION AND RESULTS

An increase of 9% and 20% in K ⁺ -phosphatase activity was observed in the presence of 1 and 10 ug of venom, respectively, when compared to the negative control.

CONCLUSION

These results indicate the presence of components in the venom of Tityus paraguayensis that can modulate the (Na ⁺ /K ⁺ )-ATPase activity, requiring further studies for isolation and characterization of these compounds.

Keywords: ATPase, pNPP, scorpion

Supported by: CNPq, CAPES, FUNDECT and UFMS

08741 - Poster Session

GA.09 - Deformation-dependent relaxation induced by local viscoelasticity in DNA solution

Akinori Miyamoto ¹, Yoshihiro Murayama¹

¹Department of Applied Physics, Tokyo University of Agriculture and Technology, (Tokyo, Japan)

INTRODUCTION

Micro-rheology has been widely used to measure viscoelasticity of a cell and cytoplasm. To understand the highly complexed viscoelasticity, fundamental viscoelastic properties of biopolymer solutions or gel are needed. The viscoelastic properties of DNA solution have been investigated to clarify concentration, length, and topological effects on its viscoelasticity. However, there is little knowledge to explain nonlinear relaxations such as double-exponential or power-law relaxation observed in dense DNA solution. Since there is no crosslinking between DNA molecules, the viscoelastic properties are sensitive to the degree of deformation.

OBJECTIVES

In this study, we focused on the effect of the degree of deformation on microstructure in dense DNA solution.

MATERIALS AND METHODS

We used 0.6 mg/ml Klenow-fragment-treated λ-phage DNA. We observed the relaxation process of a bead after a movement at a certain distance xm at a constant speed (1.0 um/s) by optical tweezers.xm corresponds to the degree of deformation, and we changed the xm from 0.4 to 6.5 um.

DISCUSSION AND RESULTS

We observed that the relaxation changed from a double exponential to a power law with an exponent of -0.5, as the degree of deformation increased. We found that these two nonlinear relaxation can be explained by a simple viscoelastic model with considering fluctuation of viscosity. In our model, viscous force acting on the bead was distinguished from that acting on DNA mesh structure, which enabled us appropriate evaluation of viscoelasticity.

CONCLUSION

The elasticity was independent of xm, and it was equivalent to that of mesh structure. On the other hand, the viscosity increased as increasing xm, resulting from compression of the mesh structure. Micro-rheology focusing on relaxation process could give novel insights to evaluate the viscoelasticity of dense polymer solution and a cell.

Keywords: DNA solution, microrheology, viscoelasticity

08745 - Poster Session

GA.11 - Correlation of cellular traction forces and dissociation kinetics of adhesive protein zyxin revealed by multi-parametric live cell microscopy

Lorena Sigaut¹, Micaela Bianchi², Catalina von Bilderling^3,4, Lía Isabel Pietrasanta^1,2

¹Departamento de Física - Instituto de Física de Buenos Aires (IFIBA, CONICET-UBA, Universidad de Buenos Aires (Buenos Aires, Argentina), ²Centro de Microscopías Avanzadas, Universidad de Buenos Aires (Buenos Aires, Argentina), ³Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Universidad Nacional de La Plata (La Plata, Argentina), ⁴Departamento de Física, Universidad de Buenos Aires (Buenos Aires, Argentina)

INTRODUCTION

Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix.

OBJECTIVES

In this work, we present an approach based on a combination of several microscopies and quantitative data analysis that allowed us to explore the correlation between the generation of traction forces and zyxin dynamics at focal adhesions.

MATERIALS AND METHODS

To this end, we combine techniques such as traction force microscopy (TFM), fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS), in addition to the fabrication of adjustable stiffness polyacrylamide hydrogels and the characterization of their elasticity by force spectroscopy using an atomic force microscope (AFM).

DISCUSSION AND RESULTS

By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by TFM, and characterize the molecular dynamics of the focal adhesion protein zyxin by FCS and FRAP. As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. This correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin.

CONCLUSION

A key advantage in the approach presented here lies in the possibility of an integral and multiparametric single cell analysis. Our results provide further evidence reinforcing the mechanosensitive properties of zyxin, pointing it out as a key protein for cellular traction forces.

Keywords: imaging, traction forces, zyxin

Supported by: UBA, ANPCyT, CONICET

08738 -

GA.12 - Non-invasive force measurement for axonal transport by kinesin and dynein

Kumiko Hayashi ¹

¹Applied Physics, Tohoku University (, Japan)

INTRODUCTION

Neuronal morphology necessitates particularly fast cargo vesicle transport for efficient communication between the cell body and distal processes. Kinesin superfamily proteins and cytoplasmic dynein haul cargo vesicles, such as synaptic cargos and mitochondria, anterogradely toward the terminal and retrogradely toward the cell body.

OBJECTIVES

The in vivo driving force produced by motor proteins acting on cargo vesicles was investigated through non-invasive force measurements based on non-equilibrium statistical mechanics. Using the method, by inferring the driving force from the fluctuating motion of transported cargo, the number of motor proteins carrying a single cargo was estimated. In this research, the number of motors was estimated for many kinds of cargo vesicles in the cases of healthy neurons and disease neurons.

MATERIALS AND METHODS

Primary culture of neurons was performed. After culture for 4–7 days, the neurons were transfected with the plasmid vector to label cargo vesicles. Cargo movement was observed with a fluorescence microscope. Images were obtained with an sCMOS camera at 100 frames/s. The center position of each cargo vesicle was determined from the recorded images using custom software. The force index χ was defined using the idea of the fluctuation theorem as χ=ln[P(ΔX)/P(-ΔX)]/ΔX.

DISCUSSION AND RESULTS

Using the force index χ, the number of motors carrying a single cargo was estimated as 1–4 anterograde and 1–3 retrograde motors for endosome transport in mice dorsal ganglion neuron, 1-6 anterograde and 1-6 retrograde motors for synaptic cargos in mice hippocampal neurons, 1–3 anterograde FPUs for synaptic cargos in motor neurons of Caenorhabditis elegans. We found that the number of motors was changed for disease neurons.

CONCLUSION

Because multi-motor cooperativity contributes to the stable and long-distance transport of materials along axons to maintain neuronal activity, the number of motors hauling a cargo is considered to be a reasonable indicator of healthy neuronal activity.

Keywords: molecular motor, axonal transport, non-equilibrium physics

Supported by: Japan Science and Technology Agency

08059 - Poster Session

08088 - Poster Session

GB.02 - Anticoagulant activity of a partially hydrolyzed and chemically sulfated xylan from açaí (Euterpe oleracea)

Genilza da Silva Mello ¹, Franciê Assis Melo Faria¹, Carina Boaron¹, Thaisa Moro Cantu-Jungles², Lucimara Mach Cortes Cordeiro¹, Iacomini Marcelo¹, Thales Ricardo Cipriani¹

¹Bioquímica e Biologia Molecular, Universidade Federal do Paraná (PR, Brasil), ²Department of Food Science, Purdue University (Indiana, Estados Unidos)

INTRODUCTION

Thromboembolic events are developed due to changes in the mechanisms of hemostasis and as therapeutic alternatives, heparins stand out, which are naturally sulfated polysaccharides. But, despite effective, these drugs have limitations of use.

OBJECTIVES

In this work, the homoxylan from açaí pulp (Euterpe oleracea Mart.), with uronic acid content of 2.8%, was used in order to obtain a chemically sulfated derivative with linear structure and low molecular weight, as an alternative to low molecular weight heparins (LMWH).

MATERIALS AND METHODS

Native xylan was partially hydrolyzed at three different times (30, 60 and 90 min), at 100 °C, using 0.5 M HCl, to obtain low molecular weight xylans (LMWX). LMWX were chemically sulfated for 2 h, at 50 °C, at a molar ratio of 9:1 (SO3.Pyr:OH on the polysaccharide), and the DS and the ability of the samples to alter the normal aPTT of citrated sheep plasma were evaluated. HSQC and HPSEC analyses were performed for structural elucidation and molecular weight evaluation.

DISCUSSION AND RESULTS

HSQC analyses obtained from LMWX showed 13C/1H correlations characteristic of β-D-xylan (1→4)-linked. The partially hydrolyzed sulfated xylans (LMWXS) showed DS between 0.35 and 0.39. Dose-response curves showed that for each increase of 1 μg/mL of LMWXS, aPTT increased around 14.72 to 18.30 s, an anticoagulant effect greater than that observed for enoxaparin, a LMWH. LMWXS had homogeneous elution profiles in HPSEC, with molecular weights higher than 4.786 x 104 g/mol. However, previous studies have shown that sulfated polysaccharides with a molecular weight around 1 x 104 g/mol are suitable for use as subcutaneously administered anticoagulants, as occurs with LMWH.

CONCLUSION

Despite the remarkable anticoagulant activity of LMWXS, it is still necessary to reduce the molecular weight of LMWX in order to obtain a compound suitable for subcutaneous use, such as LMWH.

Keywords: aPTT, chemical sulfation, polysaccharides

Supported by: CAPES and CNPq

08434 - Poster Session

GB.04 - Effect of oxidative stress in syndecan-1 of endothelial cells.

Beatriz Marin Rocha Campos ¹, Maria Aparecida da Silva Pinhal^1,2, Carina Mucciolo Melo^1,2

¹Biochemistry, Centro Universitário FMABC (SP, Brasil), ²Biochemistry, Universidade de São Paulo (SP, Brasil)

INTRODUCTION

Oxidative stress is defined as a state of overproduction of reactive oxygen species (ROS) that results in pathophysiological changes. Numerous diseases are associated with oxidative stress, such as cardiovascular events that are the first cause of death globally. In vascular diseases, oxidative stress seems to affect glycocalyx. This endothelial dysfunction is one of the first changes that give rise to atherosclerosis. Syndecans are a family of heparan sulfate proteoglycans and are present in the glycocalyx of various tissues. The decrease of syndecan-1 in vessels leads to cell stiffness, alteration in microvascular tone, leucocyte adhesion, and thrombosis.

OBJECTIVES

Analyze syndecan-1 expression during oxidative stress.

MATERIALS AND METHODS

ECV-340, a human endothelial cell line, was treated with hydrogen peroxide (exogenous ROS) in different doses and incubation times. Gene expression analysis was performed using qPCR. In addition, ECV-340 was treated with an inhibitor of glutathione synthesis that raises endogenous ROS, L-buthionine sulphoximine (BSO), in different doses. The increase of ROS was confirmed by 2',7'–dichlorofluorescein diacetate. Besides, the syndecan-1 was analyzed by flow cytometry.

DISCUSSION AND RESULTS

There was a dose-dependent decrease of syndecan-1 mRNA levels with 3 and 18 hours of hydrogen peroxide incubation. The syndecan-1 also decreased with BSO treatment. The flow cytometry confirmed the decrease of syndecan-1 in the cell surface. It is well known that ROS raises sheddases levels; however, our results demonstrated that ROS also may alter syndecan-1 gene expression. This study shows some perspective about the alteration of syndecan-1 gene expression by ROS.

CONCLUSION

Oxidative stress seems to alter gene expression of syndecan-1 in the ECV-340 cell line.

Keywords: Proteoglycans, Reactive oxygen species, Vascular endothelium

Supported by: CNPq, FAPESP, CAPES.

08411 - Poster Session

GB.05 - Analysis of genes that co-express with versican in invasive breast carcinoma

Pedro José Correia Ferraz ¹, Giselle Zenker Justo², Maria Aparecida da Silva Pinhal^1,2, Carina Mucciolo Melo^1,2

¹Departamento de Bioquímica, Faculdade de Medicina do ABC (São Paulo, Brasil), ²Departamento de Bioquímica, Universidade Federal de São Paulo (São Paulo, Brasil)

INTRODUCTION

Breast cancer is one of the major public health problems worldwide, as it not only has a high mortality rate, but is also the most incident in the world among women. Understanding the mechanisms of carcinogenesis and tumor progression is essential to mitigate this disease. Versican (VCAN), a proteoglycan present in the extracellular matrix (ECM), is not expressed in normal breast tissue, but is largely produced by the stroma in the tumor environment, showing the importance of VCAN in breast tumor carcinogenesis. It can be cleaved by proteases, such as matrix metalloproteinase-2 (MMP2), forming two active fragments: the G1 fragment that can modulate the cell cycle, and the G3 fragment that binds to growth factors and other ECM molecules. Although VCAN is important in carcinogenesis, its gene expression regulation has not yet been fully elucidated.

OBJECTIVES

Investigate the genes that co-express with the VCAN gene to give some insight into VCAN regulation.

MATERIALS AND METHODS

The study was conducted using Microarray and RNA-Seq data from two distinct invasive breast carcinoma databases, The Cancer Genomic Atlas (TCGA), Nature 2012 (n=460) and Metabric, Nature 2012/2016 (n=1904). The data was analyzed using the cBioPortal software.

DISCUSSION AND RESULTS

Regardless of breast tumor subtype or staging, linear regression analyses showed a statistically significant correlation between MMP2 and VCAN gene expression in invasive breast carcinoma tissues, both in the TCGA database [p < 0.001; q< 0.001; R=0.846], and in the Metabric database [p < 0.001; q< 0.001; R=0.701]. The mechanism of substrate and enzyme being mutually regulated is very common in various cells and tissues. Confirming bioinformatics analysis, qPCR with different breast cancer cell lines was performed (MCF-7, SKBR3, MDA-MB-231).

CONCLUSION

These results give a better understanding for elucidation of VCAN modulation, since MMP2 and VCAN genes show statistically significant coexpression in breast neoplasm.

Keywords: bioinformatics, breast cancer, proteoglycans

Supported by: CNPq, CAPES, FAPESP

08312 - Poster Session

GB.06 - Dermatan sulfate: antithrombotic effects and stimuli of heparan sulfate proteoglycan synthesis from endothelial cells

João Victor Gerheim-Silva¹, Carlos Magno da Costa Maranduba², Arthur Girardi Carpanez¹, MARTHA DE OLIVEIRA GUERRA³, Vera Maria Peters³, Valquiria Pereira de Medeiros¹, Jair Adriano Kopke De Aguiar ^1,3

¹Biochemistry, Laboratório de Análise de Glicoconjugados, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Minas Gerais, Brazil (Minas Gerais, Brazil), ²Biology, Laboratório de Genética Humana e Terapia Celular, Instituto de Ciências Biológicas, Universidade Federal de Juiz de Fora, Minas Gerais, Brazil; (Minas Gerais, Brasil), ³Biology, Centro de Biologia da Reprodução, Universidade Federal de Juiz de Fora, Minas Gerais, Brazil. (Minas Gerais, Brasil)

INTRODUCTION

Dermatan sulfate (DS) is an anionic, linear, and structurally complex polysaccharide belonging to the glycosaminoglycans (GAG) class, found in a wide variety of tissues in many species attached to a core protein as proteoglycans (PG). DSPGs have been implicated in various cellular processes and cell-matrix interactions. DS chains may also influence the coagulation process, binding to serum proteins and inhibiting thrombin (interacting with HCII).

OBJECTIVES

This study aimed to investigate DS effects upon coagulation hemostasis

MATERIALS AND METHODS

DS from porcine skin (PS) and bovine cornea (BC) extracted by proteolysis and purified by anion exchange chromatography were characterized by agarose gel electrophoresis, molecular mass, chemically (hexosamine, uronic acid, and sulfate contents), and spectroscopically (IR, RAMAN, and RMN). The purified polysaccharides were evaluated in experimental animal models of thrombosis and the stimulation of heparan sulfate proteoglycan synthesis by endothelial cells in culture.

DISCUSSION AND RESULTS

The purified polysaccharides have a distinct polymeric structure. BC-DS showed the highest molecular mass and lower sulfate: hexosamine ratio (DS-BC=0,802, DS-PS=1,189), also it seems to have the highest content of IdoA when compared with PS-DS (GlcAC/HEX: DS-BC=0,619, DS-PS=1,109). Although the spectroscopical analysis of the purified carbohydrates showed similar spectra, it is possible to find some subtle differences that might be influenced by the chemical environment on each of their polymeric structure. No other contaminants were identified on the samples. DS-BC and DS-PS showed similar effects on coagulation, with BC-DS ceasing thrombi formation with a lower dose (50μg/g) compared to DS-PS (75μg/g). Both GAGs were capable of stimulating the synthesis of endothelial HSPG secreted to the medium.

CONCLUSION

We highlight the findings on antithrombotic properties of DS from PS and BC, and presented a new result regarding its properties on the endothelial cell wall, stimulating the synthesis of HS, which have been found to promote angiogenic responses of endothelial cells beyond its antithrombotic properties.

Keywords: Dermatan sulfate, Thrombosis, Coagulation

08641 - Poster Session

GB.07 - Alteration on glycated human erythrocyte by in vitro action of anesthetics: propofol, remifentanil, and vecuronium

Marcus Vinícius Batista da Silva ¹, Analía Ines Alet¹, Horácio Castellini², Nicolás Alet³, Bibiana Doris Riquelme^1,4

¹Área Física, Facultad de Ciencias Bioquímicas y Farmacéuticas - Universidad Nacional de Rosario (Rosario - Santa Fe, Argentina), ²Física, Facultad de Ciencias Exactas, Ingeniería y Agrimensura - Universidad Nacional de Rosario (Rosario - Santa Fe, Argentina), ³Anestesiología, Facultad de Ciencias Médicas - Universidad Nacional de Rosario (Rosario - Santa Fe, Argentina), ⁴Física, Grupo de Física Biomédica - Instituto de Física de Rosario (Rosario - Santa Fe, Argentina)

INTRODUCTION

Glycation refers to slow, non-enzymatic, and spontaneous reactions between the amino groups of proteins and the carbonyl group of a reduction sugar, like glucose. In pathological conditions, such as diabetes, the glucose level in the blood is high. Due to this hyperglycemia, glycation occurs on the red blood cells (RBC), resulting in possible alterations of erythrocyte viscoelasticity. Several authors have demonstrated that anesthetic drugs commonly used in surgery (propofol, remifentanil, and vecuronium) may affect the stationary viscoelastic parameters of red blood cells from healthy donors.

OBJECTIVES

This work aimed to evaluate the hemorheological effect of anesthesia in hyperglycemia.

MATERIALS AND METHODS

Therefore, RBCs from healthy donors were incubated with glucose solutions 0,5% v/v for 5 hours at 37°C under constant stirring. After the glycation procedure, the RBCs were incubated separately and combined with propofol (P, 4 μg/ml whole blood), remifentanil (R, 10 ng/ml plasma), and vecuronium (V, 0.15 μg/ml plasma) for 30 minutes at 37°C under constant stirring. The Reómetro Eritrocitario was used to determine the viscoelastic parameters, obtaining the erythrocyte elastic modulus (μ), surface viscosity (η), and deformability index (DI).

DISCUSSION AND RESULTS

Statistical analysis shows a significant decrease of μ for RBC incubated with R and RV anesthetics (p < 0.05). Nevertheless, μ significant increase for RBC treated with P and PR (p < 0.05). The η values increase in RBC treated with PR and PV (p < 0.05), and V (p < 0.001). The DI was increased for RBC, glycated and no glycated, when treated with R and RV (p < 0.01). However, DI decreases for RBC glycated and incubated with P (p < 0.05).

CONCLUSION

These results suggest an interaction of these anesthetics with the RBC, which could alter the viscoelastic parameters of the erythrocyte membrane at different levels. Consequently, these results would be a contribution to the prevention of complications in surgical procedures.

Keywords: anesthetic drugs, glycated erythrocyte , viscoelastic parameters

Supported by: BIO604 Universidad Nacional de Rosario - UNR

08308 - Poster Session

GB.08 - New bovine intestinal heparin derivated molecule with low anticoagulant activity and its anti-tumoral activity

Roberto Pereira Santos^1,2, Marcos Roberto de Oliveira ¹, Nina Valéria Machado Capillé¹, Ana Maria Freire Tovar¹, Paulo A. S. Mourão¹

¹Department of Bioquemistry, Leopoldo Meis Bioquemistry Institute - Federal University of Rio de Janeiro (Rio de Janeiro, Brazil), ²Department of Neurology, Clementino Fraga Flilho University Hospital - Federal University of Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

During the last few years, heparin's biological effects have been incorporated, which go beyond its anticoagulant activity, such as anti-inflammatory, antiviral, and anti-tumor effects. However, its use for these purposes is limited due to its potential hemorrhagic effect. In this context, heparinoid molecules have been researched in order to obtain molecules with low anticoagulant activity and other therapeutic effects. With this in mind, in our laboratory we were able to purify a fraction of intestinal bovine heparin with low anticoagulant activity (LABH).

OBJECTIVES

In the present work, our objective is to test this molecule for an antitumor activity.

MATERIALS AND METHODS

link the ectopic tumor growth model with Lewis lung carcinoma developed on the back of C57BL / 6 mice treated with daily injections at a dose of 8mg / kg subcutaneously for 4 weeks.

DISCUSSION AND RESULTS

Treatment with LABH reduced: a) cachexia, b) tumor complication, c) death and delayed tumor growth.

CONCLUSION

LABH showed anti-tumor action in this animal model. Further studies are necessary in other models and at other doses in order to investigate the full potential benefit of this molecule.

Keywords: heparin, cancer, coagulation

Supported by: FAPERJ and SENAI

08675 - Poster Session

GB.09 - Extraction, isolation, characterization and biological activity of sulfated polysaccharides present in ascidian viscera Microcosmus exasperatus

Ananda de Araujo Bento ¹, Simone Coutinho Cardoso², Mauro Sergio Gonçalves Pavão³, Mariana Paranhos Stelling¹

¹Bioquímica, Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (Rio de Janeiro, Brasil), ²Instituto de Física, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ³Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Ascidians are marine invertebrate tunicates that synthesize sulfated glycosaminoglycans (GAGs) within their viscera. Ascidian GAGs are considered analogues of mammalian GAGs and possess great potential as bioactive compounds, presenting antitumor and anticoagulant activity. Due to their clinical potential, it is important to understand how ascidian GAGs are produced, their function within the ascidian organism and how they keep their bioactivity after extraction and purification processes. Mammalian heparin is one of the main compounds used to treat thrombosis and related diseases due to their anticoagulant activity. However, heparin presents adverse effects, in this context, ascidian GAGs are proposed as good alternatives for mammalian heparin and, therefore, should be carefully studied.

OBJECTIVES

Our main objectives are to study the ascidian Microcosmus exasperatus regarding GAGs composition, structure, distribution within the viscera and also its biological activity.

MATERIALS AND METHODS

Ascidians were collected by free diving. GAGs were extracted by proteolytic digestion and purified by ion-exchange liquid chromatography and characterized by agarose gel electrophoresis and enzymatic treatments. Anticoagulant activity was evaluated by aPTT assays. Cell cytotoxicity was evaluated by MTT assays using two tumor cell lines (LLC and MC-38). Tumor cell migration activity was evaluated in vitro by wound healing assays.

DISCUSSION AND RESULTS

Our results show that M. exasperatus presents three distinct polysaccharides. These polysaccharides were fractionated into two fractions named PS 1 and PS 2. M. exasperatus produces a low anticoagulant dermatan sulfate (PS 2) and a heparin-like (PS 1) compound, which, interestingly, is not susceptible to heparinases treatment and does not present significant anticoagulant activity. PS 2 shows mild cytotoxicity in LLC tumor cells when in combination with manganese, however it has no effect on the invasive potential of LLC and MC-38 tumor cells.

CONCLUSION

As a conclusion, we hope to establish M. exasperatus GAGs as suitable compounds for future preclinical studies in cancer and vascular disease areas.

Keywords: Microcosmus exasperatus, sulfated glycosaminoglycans, ascidians

Supported by: CNPq, FAPERJ, CAPES, Fundação do Câncer, UFRJ and IFRJ

08319 -

GB.10 - Optimization of anticoagulant activity of chemically sulfated citrus pectin

Carina Boaron ¹, Franciê Assis Melo Faria¹, Genilza da Silva Mello¹, Thales Ricardo Cipriani¹

¹Biochemistry and Molecular Biology, Federal University of Paraná (Paraná, Brazil)

INTRODUCTION

Sulfation and other chemical modifications of polysaccharides can lead to better anticoagulant activity, which is important in searching alternatives to heparin. Citrus pectin (CP) has great potential for this purpose.

OBJECTIVES

Therefore, this research aims to optimize chemical sulfation and carry out other modifications in the CP structure in order to understand and further improve its performance.

MATERIALS AND METHODS

Commercial CP was subjected to dialysis and partial acid hydrolysis, obtaining high molecular weight pectin (CPHW – 298,8 kg/mol) and low molecular weight pectin (CPLW – 7,53 kg/mol). Both fractions were subjected to optimization of chemical sulfation, modifying the molar ratio of sulfation agent to hydroxyl group on the polysaccharide (ηSO3-Pyridine/OH), the total reaction volume to weight of sample (Vt/w) and the reaction time. Anticoagulant activity was evaluated by the ability of derivatives to increase aPTT.

DISCUSSION AND RESULTS

The high and low molecular weight sulfated derivatives with the best anticoagulant activities, CPHWS1 and CPLWS1, were obtained with ηSO3-pyridine/OH=9, Vt/w=100 mL/g, and 8 h and 4 h of reaction, respectively. Subsequently, CPHWS1 and CPLWS1 were carboxyl-reduced (CR; 1 and 4 cycles each), resulting in lower anticoagulant activities, reinforcing the role of carboxyl groups in bioactivity. The sulfated and carboxyl-reduced samples were resulfated (S2), using optimized parameters. The resulting derivatives showed better anticoagulant performances, indicating that replacement of carboxyl by sulfate groups favors anticoagulant activity. The derivatives that showed the best results were CPHWS2CR4 (aPTT at 40 μg/mL=226,2 s; DS=1,27) and CPLWS2CR1 (aPTT at 50 μg/mL=187,1 s; DS=1,30). Interestingly, they were able to inhibit FIIa and FXa without antithrombin, with the effect being increased in the presence of antithrombin, especially on FIIa.

CONCLUSION

New approaches for the development of citrus pectin-based anticoagulants were presented. The optimization of chemical sulfation and the other changes made in CPHW and CPLW have shown to influence their anticoagulant activities.

Keywords: Anticoagulant activity, Citrus pectin, Polysaccharides

Supported by: CNPq

08339 -

GB.11 - Anticoagulant activity of partially hydrolyzed, oxidized and chemically sulfated guar gum

Franciê Assis Melo Faria ¹, Carina Boaron¹, Genilza da Silva Mello¹, Diogo Ricardo Bazan Ducatti¹, Thales Ricardo Cipriani¹

¹Biochemistry and Molecular Biology, Federal University of Paraná (Paraná, Brazil)

INTRODUCTION

Heparin is a naturally sulfated polysaccharide widely used as anticoagulant. Despite its importance, it has some disadvantages such as its high cost, side effects, and risk of contamination. Therefore, researchers seek development of alternative anticoagulants. In the case of polysaccharides, the sulfation process is important to promote anticoagulant activity, but little is known about the importance of uronic acids, which are also present in heparin structure. Guar gum, when partially hydrolyzed and chemically sulfated, showed promising anticoagulant effect in previous studies.

OBJECTIVES

Therefore, this paper aimed to evaluate whether the presence of uronic acids in partially hydrolyzed and chemically sulfated guar gum would contribute to the anticoagulant action.

MATERIALS AND METHODS

Thus, native guar gum (GG) was submitted to partial acid hydrolysis, giving rise to GGH (Mw = 1.47 x 10 ⁴ g/mol). Methylation and HSQC analyses confirmed the galactomannan structure. GGH was oxidized in a pH 10 buffered system using the oxidant 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and co-oxidant chloroisocyanuric acid (TCCA), giving fractions with low (GGHOP) and high (GGHOT) degree of oxidation according to the amount of co-oxidant used. All fractions were chemically sulfated and the derivatives evaluated for anticoagulant activity using aPTT, PT, TT assays.

DISCUSSION AND RESULTS

The anticoagulant effect was inversely proportional to the degree of oxidation, with the sulfated but not oxidized derivative GGHS1 showing considerably better results. It is noteworthy that GGHS1 showed better anticoagulant activity than the low molecular weight heparin enoxaparin. All fractions had a similar mechanism of action, inhibiting mainly FIIa, especially in the presence of antithrombin. In addition, they were able to respond to the protamine antidote.

CONCLUSION

These results indicated that the presence of uronic acids (after the oxidation process) in partially hydrolyzed and chemically sulfated guar gum decreases its anticoagulant activity, reinforcing the anticoagulant potential of GGHS1.

Keywords: Anticoagulant activity, Selective oxidation, Guar gum

Supported by: CNPq

08284 -

GB.12 - New bovine intestinal heparin derivated molecule with low anticoagulant activity and its anti-metastatic activity

Roberto Pereira Santos^1,2, Marcos Roberto de Oliveira¹, Nina Valeria Machado Capille¹, Ana Maria Freire Tovar¹, Paulo A. S. Mourão¹

¹Leopoldo de Meis Biochemistry Institute, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil), ²Department of Neurology, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Evidence has emerged that the biological effects of heparin go beyond the anticoagulant activity. Studies have demonstrated anti-inflammatory, anti-viral, anti-tumor effects. However, it is not possible to use it for these therapeutic purposes due to its potential hemorrhagic effect. In view of this, new molecules have been researched in order to bypass this obstacle. Our laboratory we were able to purify a low anticoagulant activity fraction of bovine intestinal heparin (LABH).

OBJECTIVES

In this work, our goal is to test this molecule for anti-metastatic activity.

MATERIALS AND METHODS

Lung metastasis model induced by intravenous injection of Melanoma cells in C57BL/6 mice previously treated with a single dose of LABH (2, 4, 8 or 20 mg / kg) or saline, subcutaneous administered one hour before the melanoma cells injection. After 4 weeks of follow-up, the lungs were weighed and the metastatic foci counted. Results: Treatment with LABH reduced: a) cachexia, b) mortality and c) amount of lung metastasis.

DISCUSSION AND RESULTS

Treatment with LABH reduced: a) cachexia, b) mortality and c) amount of lung metastasis.

CONCLUSION

LABH showed anti-metastatic action in this experimental model. Further studies in other models and other doses are necessary in order to investigate the full potentiality of the benefit of this molecule.

Keywords: heparin, cancer, coagulation

Supported by: FAPERJ and SENAI

08323 -

GB.13 - Polysaccharides isolated from Piper regnelli (Pariparoba) leaves: structural characterization and antinociceptive activity

Giuliana Cozzella Campo Grande ¹, Bruna Barbosa da Luz², Maria Fernanda de Paula Werner², Daniele Maria-Ferreira², Marcello Iacomini¹, Thales Ricardo Cipriani¹

¹Bioquímica e Biologia Molecular, Universidade Federal do Paraná (Paraná, Brasil), ²Farmacologia, Universidade Federal do Paraná (Paraná, Brasil)

INTRODUCTION

Piper regnellii is a medicinal plant popularly known as pariparoba and is widely used to treat pain, inflammation, ulcer among others. Amongst the bioactive molecules isolated from plants are the polysaccharides, biopolymers with biological potential to treat many conditions and diseases, as described in scientific literature. Little is known about P. regnellii’ polysaccharides.

OBJECTIVES

This study aims to extract, purify and characterize the polysaccharides of the plant leaves and evaluate its antinociceptive activity.

MATERIALS AND METHODS

From the aqueous extract of P. regnellii leaves (decoction; 15 minutes under boiling), followed by a number of purification processes, such as ethanolic precipitation, freeze thawing and ultrafiltration, was obtained a purified polysaccharide fraction, named PR30R (fraction retained in membrane with 30 kDa cut-off).

DISCUSSION AND RESULTS

The elution profile in HPSEC of PR30R was found to be homogeneous, with a calculated molar mass of 164.000 g/mol. The monosaccharide composition analysis by GC-MS revealed that PR30R was mainly constituted by galactose (51.5%), arabinose (24.9%) and galacturonic acid (11.3%). NMR and methylation analysis showed that PR30R is constituted by a type II arabinogalactan (AGII), with a backbone of β-D-Galp-(1→3) units, O-6 substituted by β-D-Galp-(1→6) side chains, which are O-3 substituted by non-reducing ends of α-L-Araf, and by a type I rhamnogalacturonan (RGI), composed of a backbone of α-D-GalpA-(1→4) units intercalated by α-L-Rhap-(1→2). By integrating the NMR spectra signals of methyl and non-methyl esterified galacturonic acid units, we were able to calculate the degree of esterification of the RGI as 70.5%. Finally, PR30R was evaluated for its antinociceptive activity, using the acetic acid-induced writhing model in mice.

CONCLUSION

We found that the fraction, at a dose of 0.1096 mg/kg, was able to reduce the number of abdominal writhes by 56% when compared to vehicle group, confirming the contribution of P. regnellii polysaccharides to the popular use of the plant leaves tea to treat pain.

Keywords: Antinociceptive Activity, Piper regnellii, Polysaccharides

Supported by: CNPq

08799 - Poster Session

GC.01 - HOST IMMUNE RESPONSE PARTICIPATES IN MALARIA ACUTE KIDNEY INJURY: ROLE OF CD8+ T CELLS

Douglas Esteves Teixeira¹, Alessandro de Sá Pinheiro¹, Leandro de Souza Silva¹, Rodrigo Pacheco da Silva-Aguiar¹, Diogo B Peruchetti¹, Tatiana A Pádua⁴, Mariana Conceição de Souza⁴, Celso Caruso-Neves^1,2,3, Ana Acacia de Sá Pinheiro^1,3

¹Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil), ²Rio de Janeiro Innovation Network in Nanosystems for Health-NanoSAÚDE, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (Rio de Janeiro, Brazil), ³3National Institute of Science and Technology for Regenerative Medicine, Conselho Nacional de Desenvolvimento Científico e Tecnológico (Rio de Janeiro, Brazil), ⁴Bio-Manguinhos, Fundação Oswaldo Cruz (Rio de Janeiro, Brazil)

INTRODUCTION

Severe malaria is attributed to, Plasmodium falciparum infection and entails different pathologies caused directly by parasite infection and host immune response. Malaria acute kidney injury (MAKI) is characterized by glomerular and tubular damage. This process is attributed to oxidative stress and obstruction of renal microvasculature by aggregates of parasitized erythrocytes. Overactivation of the host immune response is also postulated, however its precise role in MAKI is still unknown.

OBJECTIVES

The objective of the work was to evaluate the participation of T cells in MAKI pathogenesis.

MATERIALS AND METHODS

For this, we performed adoptive transfer of splenocytes-derived T cells from C57Bl6 mice infected with P. berghei ANKA to healthy acceptor animals. Renal function as well as homing and immune response were assessed.

DISCUSSION AND RESULTS

Adoptively transfered T cells induced proteinuria (2-fold) and increased UPCr (protein and creatinine ratio; 2.3-fold). Markers of glomerular injury, creatinine clearance, plasma creatinine and plasma urea, did not change. However, we observed an increase in γGT activation in urine (1.6-fold), a marker of renal tubular damage. These results indicate that malaria-responsive T cells induce renal tubular damage without glomerular involvement. Accordingly, there was a remarkable increase in T cell homing to the kidneys, as well as spleen and brain. Moreover, we observed increase in renal proinflammatory cytokines INFγ (2-fold), IL-17 (1.3-fold) and IL-6 (1.5-fold). FACS analysis revealed an increase in the frequency of CD8+ T cells in the kidney, which was accompanied by increased expression of perforin in the renal cortex (1.7-fold). These results indicate that CD8+ T cells are activated during malaria infection and can migrate to the kidney besides brain and spleen. In the kidney, perforin production has a role in inducing renal tubular damage.

CONCLUSION

This work adds new insights into the pathogenesis of MAKI describing it as a consequence of host exacerbated immune response.

Keywords: malaria, kidney injury, immune response

Supported by: Faperj, CNPq e Capes

08334 - Poster Session

GC.02 - Effects Of Natural Compounds On The Peroxidase Activity Of AhpCs From Pathogenic Bacteria

Vitória Isabela Montanhero Cabrera ¹, Melina Cardoso Santos¹, Deborah Pelossi Sessa³, João Henrique Ghilardi Lago³, Luis Eduardo Soares Netto², Marcos Antonio Oliveira¹

¹LABIMES, Universidade Estadual Paulista, Instituto de Biociências, Campus Litoral Paulista (São Paulo, Brasil), ²Departamento de Bioquímica, Universidade De São Paulo, Instituto de Química (, Brazil), ³CCNH, Universidade Federal do ABC, Centro de Ciências Naturais e Humanas (, Brazil)

INTRODUCTION

The resistance of pathogenic bacteria to various antibiotics has increased worldwide in recent years, requiring new antibacterial compounds. Recently, some studies have shown that different antibiotics have the convergent capacity to produce reactive oxygen species (ROS), which and together with the host's oxidative defenses contributes to pathogen annihilation. Bacterial pathogenic cells have several antioxidant enzymes can decompose the ROS and minimize the host defenses and antibiotic oxidant properties. The thiol peroxidase AhpC is considered the major hydroperoxide scavenger in some bacteria species and may represent inhibiting targets to combat bacterial infections. The high reactivity of AhpC relies on a very reactive cysteine (C_P) together with two polar residues (Thr/Ser and Arg) makes up the catalytic triad

OBJECTIVES

The objective of this work is to identify natural compounds can interfer with the AhpC from Pseudomonas aeruginosa (PaAhpC).

MATERIALS AND METHODS

PaAhpC was recombinantly expressed and purified by IMAC. The effects of four compound on PaAhpC activity were evaluated by the NADPH oxidation.

DISCUSSION AND RESULTS

The results revealed that a compound, named here as CN-ABP1, acted as an PaAhpC inhibitor since decreased significatively the initial decomposition rates (v0PaAhpC = 0.22 ± 0.01 μs ^-1 and v0PaAhpC+CNABP1 = 0.09 ± 0.02 μs ^-1 ) and total NADPH consumption after 300sec (PaAhpC = 93μM and CN-ABP1 = 24.30μM, approximately). To better understand this result, we performed molecular docking analysis and our results indicate that CN-ABP1 can be stabilized in the microenvironment of the PaAhpC active site (ΔG = -6.3 to -7.4kcal/mol) by several hydrophobic and polar interactions, including the amino acids from the catalytic triad.

CONCLUSION

Our results also suggest that the inhibition occurs by a Michael addition between an α, β unsaturated carbonyl system of the natural compound and the sulfur of the CP AhpC. Approaches for determining IC₅₀ and evaluating the effects of CN-ABP1 on bacterial cells (MIC₅₀) are underway.

Keywords: Inhibitor , Natural compounds, Peroxiredoxins

Supported by: FAPESP

08590 - Poster Session

GC.03 - CHARACTERIZATION OF THE ORAL VIROME IN HEALTH AND PERIODONTITIS

Pâmela Natasha Mesquita Bentes ¹, Daniel Saito¹, Cristiane Pereira Borges Saito ¹, Fabiana de Souza Cannavan², Siu Mui Tsai²

¹Escola Superior de Ciências da Saúde , Universidade Estadual do Amazonas (Amazonas , Brasil), ²Instituto CENA, Univeraidade de São Paulo (São Paulo , Brasil)

INTRODUCTION

The oral cavity encompasses a highly complex microbiome composed by a wide variety of microorganisms (bacteria, fungi, protozoans and viruses). The interactions between the different components of the oral microbiota can trigger inflammation in the periodontium, leading to the development of periodontitis, a disease that causes significant morbidity at national and global levels.

OBJECTIVES

In this sense, this project aims to study the viral genetic material (viroma) present in the oral cavity of healthy individuals and patients harboring periodontitis.

MATERIALS AND METHODS

For this purpose, 27 saliva samples from healthy (n = 13) and periodontitis individuals (n = 14) were collected and their genetic material was extracted and quantified. The metagenome was submitted to next generation DNA sequencing on the Ilumina Hiseq2500 platform. After nucleotide sequence quality control, reads were paired into larger contiguous sequences (contigs) using the PEAR program, and later annotated with the Kaiju program coupled with the NCBI Refseq virus reference database. Taxa quantification and tabulation was performed using R program packages. The two hundred most abundant taxa were submitted to Linear Discriminant Analysis (LDA) through the LefSe software, on the Galaxy/Hutlab platform.

DISCUSSION AND RESULTS

Overall, the majority of viruses corresponded to bacteriophage species belonging to the Myoviridae and Siphoviridae families. Especifically, Streptococcus spp., Enterococcus spp. and Eggertella spp. phages were most prevalent in the healthy group (LDA ≥ 3.0), while Escherichia spp. and Bacillus spp. phages along with microalgae and protozoan viruses (such as Chrysochromulina ericina, Only Syngen Nebraska, Cafeteria roenbergensis, and Acanthamoeba polyphaga viruses) were most abundant in the periodontitis group (LDA ≤ -3.0). The results helped us gather a better understanding of the nature of the oral virome, revealing previously unreported viruses associated with disease onset and homeostasis.

CONCLUSION

All in all, we believe that the taxonomic units denoted here may represent important biomarkers for future research on oral microbial ecology.

Keywords: Oral, Virome, Virus

Supported by: Fapeam

08314 - Poster Session

GC.04 - Combination effect of isoobtusilactone A and benznidazole on Trypanosoma cruzi

Bruna Castro de Barros¹, Júlio Menta de Almeida¹, Felipe Oliveira Nunes², Letícia Alves da Cruz¹, Inês Aparecida Tozetti¹, Fernanda Rodrigues Garcez², Alda Maria Teixeira Ferreira¹

¹Laboratório de Imunologia, Biologia Molecular e Bioensaios, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil), ²Laboratório de Pesquisa de Produtos Naturais Bioativos, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

Isoobtusilactone A is a butanolide obtained from the active extract of the xylopodium of Aiouea trinervis Meisn. (Lauraceae), which grows in the Cerrado biome of Mato Grosso do Sul, Brazil. This compound has already shown lethal effect and a fast action mechanism on Trypanosoma cruzi, the etiological agent of Chagas Disease. The current treatment of Chagas Disease in Brazil is done with benznidazole, a drug known by its several side effects and its barely activity on the chronic stage of infection. For being the main approved drug for use in Brazil, benznidazole still being target of many studies, which seek its effects under combinations with other drugs.

OBJECTIVES

The aim of this work was to avaliate the combination effect of isoobtusilactone A and benznidazole on epimastigotes forms of T. cruzi.

MATERIALS AND METHODS

A combination assay was done with different proportions (1:3; 1:1; 3:1) and concentrations (2 μM – 12 μM and 5 μM – 60 μM) of isoobtusilactone A and benznidazole, respectively, in 96-well plates during 24h at 28ºC. Viability after was determinated through MTS assay.

DISCUSSION AND RESULTS

The results showed that proportions belonging to IC₉₀ had synergistic effect (CI < 1), and all others concentrations belonging to IC_25-75 had almost additive effect (CI = 1) or antagonist effect (CI > 1). To achieve a synergistic effect, the two drugs needed to be administered in higher concentrations. Despite that, isoobtusilactone A was able to reduce up to 5 times (DRI) the amount of benznidazole needed to reach IC₉₀, which can influence the reduction of side effects attributed to benznidazole.

CONCLUSION

The current work is unprecedented in evaluating the effect of combining a butanolide with benzonidazole and has reinforced our previous data that isoobtusilactone A holds promise in the search for new trypanocidal agents.

Keywords: Benznidazole, Combination assay, Trypanosoma cruzi

08556 - Poster Session

GC.05 - PROJECT: The in vitro role of Trypanosoma evansi Rad51 in hydrogen peroxide resistance

Ketriane Mota de Souza ¹, Carlos Renato Machado², Carla Ivane Ganz Vogel¹

¹Departamento de Produção Animal e Alimentos, Centro de Ciências Agroveterinárias, Universidade do Estado de Santa Catarina (Santa Catarina, Brazil), ²Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (Minas Gerais, Brazil)

INTRODUCTION

Trypanosoma evansi causes the “Surra” disease in animals like equines, cattle, sheep, goats and pigs. This sickness present non-specific clinical signs such as weight loss, anorexia, anemia, drop in milk production and reproductive disorders. Homologous recombination (HR) is able to repair DNA double-strand breaks and damaged replication forks. In HR repair, in which RAD51 protein plays important roles, a homologous DNA sequence serves as a model for restoration of lost sequence information at the break site. Regarding RAD51 in trypanosomatids, one study showed that overexpression of RAD51 in Trypanosoma cruzi promoted increased resistance against hydrogen peroxide treatment (a potent cytotoxic agent). Thus, this work aims to explore TevRad51 (Rad51 protein from T. evansi) in DNA repair and cell survival in T. evansi.

OBJECTIVES

(1) Clone and express the TevRad51 gene; (2) Develop antibodies against the TevRad51 protein and perform immunofluorescence assay; (3) Establish in vitro culture of T. evansi; (4) Perform in vitro cytotoxicity assay with T. evansi and evaluate the TevRad51 expression using the developed antibodies.

MATERIALS AND METHODS

TevRad51 gene was inserted in pGEM-T Easy and will later be inserted in pET-28 a (+) vector for protein expression in Escherichia coli. Rats will be immunized with the TevRad51 protein for production of antibodies. TevRad51 expression in T. evansi during cytotoxicity assay will be evaluated using the developed antibodies. Temporary T. evansi culture was standardized with Ham’s F12 and DMEM culture mediums.

DISCUSSION AND RESULTS

The T. evansi survive for up to 8 days in temporary in vitro culture established. Colony PCR showed positive colony for TevRad51 after ligation gene in pGEM-T Easy vector and bacterial transformation by electroporation.

CONCLUSION

The cytotoxicity assays and TevRad51 expression evaluation will contribute for understanding TevRad51 role in T. evansi survival.

Keywords: DNA repair, TevRad51 , Trypanosoma evansi

Supported by: FAPESC (Edital nº 27/2020), CNPq.

08168 - Poster Session

GC.06 - A Family of T6SS Antibacterial Effectors related to L,D-transpeptidases Targets the Peptidoglycan

Julia Takuno Hespanhol ¹, Stephanie Sibinelli-Souza¹, Gianlucca Gonçalves Nicastro¹, Bruno Yasui Matsuyama², Stephane Mesnage³, Ankur Patel³, Robson Francisco de Souza¹, Cristiane Rodrigues Guzzo¹, Chuck Farah², Ethel Bayer-Santos¹

¹Departmento de Microbiologia, Universidade de São Paulo-Instituto de Ciências Biomédias (São Paulo, Brasil), ²Departamento de Bioquímica, Universidade de São Paulo-Instituto de Química (São Paulo, Brasil), ³Department of Molecular Biology and Biotechnology, University of Sheffield (Sheffield, United Kingdom)

INTRODUCTION

Type VI secretion systems (T6SSs) are contractile nanomachines widely used by bacteria to intoxicate competitors. Salmonella Typhimurium encodes a T6SS within the Salmonella pathogenicity island 6 (SPI-6) that is used during competition against species of the gut microbiota.

OBJECTIVES

Characterize a new SPI-6 T6SS antibacterial effector containing DUF2778 (STM14_0336) and its cognate immunity protein containing DUF2195 (STM14_0335).

MATERIALS AND METHODS

Bioinformatic analyzes of the SPI-6 T6SS cluster revealed a putative effector and immunity protein pair. Effector was cloned with or without N-terminal PelB periplasmic localization sequence and its toxicity was analyzed by microscopy. Searches for DUF2778 homologs at NCBI nr database established its evolutionary relationship. Effector was expressed as recombinant protein for enzymatic assays using muropeptides as substrates and analyzed by RP-HPLC coupled to MS.

DISCUSSION AND RESULTS

STM14_0336, renamed Tlde1 (T6SS L,D-transpeptidase effector 1), was toxic in target-cell periplasm. Its toxicity was neutralized by co-expression with immunity protein Tldi1 (T6SS L,D-transpeptidase immunity 1) (STM14_0335). Time-lapse microscopy revealed that intoxicated cells displayed altered cell division, swelling and lysis, indicating cell wall damage. Bioinformatics analysis showed that DUF2778-containing proteins comprise a superfamily evolutionarily related to L,D-transpeptidases that further divided into three families (Tlde1a, Tlde1b, Tlde1c). Point mutations on conserved His121 and Cys131 residues eliminated toxicity. Co-incubation of purified Tlde1 and peptidoglycan tetrapeptides showed that Tlde1 displays L,D-carboxypeptidase activity, cleaving GM-tetrapeptides between mDAP ³ and D-Ala ⁴ . Results suggest that Tlde1 promotes depletion of acceptor GM-tetrapeptides, thus preventing formation of new crosslinks and weakening the peptidoglycan mesh structure.

CONCLUSION

Tlde1 comprise a new family of antibacterial effectors with L,D-carboxypeptidase activity evolutionarily related to L,D-transpeptidases. DUF2778 superfamily is widespread in Proteobacteria.

Keywords: peptidoglycan, toxin, T6SS

Supported by: FAPESP

08453 - Poster Session

GC.07 - A New Synthetic Antimicrobial Peptide Bioinspired from a Plant Protein is Active against Staphylococcus saprophyticus biofilms

Luís Henrique De Oliveira Almeida ¹, Suellen Rodrigues Ramalho², Janaína de Cássia Orlandi Sard³, Caio Fernando Ramalho de Oliveira², Maria Lígia Rodrigues Macedo³

¹Instituto de Biociências (INBIO), Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brazil), ²Faculdade de Medicina (FAMED), Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brazil), ³Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição (FACFAN), Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, BRAZIL)

INTRODUCTION

Bacterial resistance is the cause of overwhelming number of deaths, a public health problem. One of the causes of bacterial infections, mainly in the urinary tract, is Staphylococcus saprophyticus. Furthermore, S. saprophyticus biofilms are naturally resistant against the available antimicrobial therapies. Antimicrobial peptides (AMP) are short-chain amino acid molecules with a broad spectrum of activity.

OBJECTIVES

Considering the urgent need for discovery of compounds with antibiofilm activity, we investigated antibiofilm properties of a new AMP, designed from the Inga laurina trypsin inhibitor (ILTI) amino acid sequence, named KWI-19

MATERIALS AND METHODS

The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of KWI-19 against S. saprophyticus ATCC 49453 was evaluated according to CLSI Protocol, using a KWI-19 solution prepared in a sterile 0.9% NaCl solution and serially diluted (from 10 to 0.02 μM). The positive control was carried out with vancomycin (from 87 to 0.17 μM), the negative control was prepared with MH broth and bacterial suspension. The effects of KWI-19 on the inhibition of S. saprophyticus ATCC 49453 biofilm formation and the eradication of mature biofilm were also evaluated. The biofilm viability was quantified as a percentage of the total number of viable colony-forming units (CFU).

DISCUSSION AND RESULTS

KWI-19 presented MIC and MBC of 1.25 and 2.5 μM, respectively, while vancomycin presented MIC and MBC of 0.68 μM and 1.36 μM, respectively. The peptide inhibited 40.6% and 46.6% of the biofilm formation at 1.25 and 12.5 μM, respectively. KWI-19 eradicated 48% and 57.9% of 24-h mature biofilms at 1.25 and 12.5 μM, respectively. KWI-19 inhibited the biofilm formation better than vancomycin at MIC, suggesting the potential use of AMP to control biofilm infections.

CONCLUSION

Therefore, KWI-19 showed antibiofilm potential to control S. saprophyticus. The potential of KWI-19 to control infections through in vivo models can be investigated in the future.

Keywords: peptide, antibacterial, antibiofilm

Supported by: FUNDECT, FINEP, CNPq and CAPES

08467 - Poster Session

GC.08 - Antioxidant activity and antimicrobial effect of lyophilized extract of Macrocybe titans giant mushroom

Gabrielle Caroline Peiter ¹, Evandro de Araújo Sitó Junior², Gabrieli Maria Huff³, Fernanda Cristina Buraslan Neves Pereira¹, Vivian Emanuelle Mamede de Santana Justo Justo³, Ednilson Clayton Rogério³, Hércules Moreira¹, Flávia Bordignon Hendges¹, André Rabelo Peixoto Bertocco¹, Adriana Fiorini Rosado³, Thiago Cintra Maniglia⁴, Fábio Rogerio Rosado³

¹Bioquímica e Biologia Molecular , Universidade Federal do Paraná (Paraná, Brasil), ²Biotecnologia, Universidade Federal do Paraná (Paraná, Brasil), ³Biociências, Universidade Federal do Paraná (Paraná, Brasil), ⁴Engenharia de Bioprocessos e Biotecnologia , Universidade Tecnológica Federal do Paraná (Paraná, Brasil)

INTRODUCTION

Over the past few decades, the rise and spread of bacterial and fungal resistance to most clinically available antimicrobial agents, has reached alarming rates. In Brazil, the macrofungus Macrocybe titans was recently reported and there are no records of its antimicrobial potential.

OBJECTIVES

Based on that, this work aimed to evaluate the antimicrobial and antioxidant activity of the lyophilized aqueous extract of M. titans on ATCC strains of pathogenic bacteria and fungi, Bacillus cereus 10876, Salmonella Typhimurium 14028, S. enterididis 13076, Listeria monocytogenes 19111, Escherichia coli 25922, E. coli O157:H7 43888, Pseudomonas aeruginosa 27853, Staphylococcus aureus 6538, B. subtilis subspecie Spizizinni 6633, Klebsiella pneumoniae 13883, Candida albicans (INCQS 40006), C. krusei (INCQS 40147), C. parapsilosis (INCQS 40038), C. glabrata (INCQS 40136) C. tropicalis (750) and one clinical isolate of Trichosporon spp.

MATERIALS AND METHODS

Aqueous extracts of the mushroom were prepared from dried fruting bodies, grounded and diluted in water and then lyophilized. The antimicrobial and antifungal effects were evaluated by the disk-diffusion method, based on CLSI M2-A12 Vol. 35, No 1 and CLSCI, M44-A2 protocols. Antioxidant potential was determined by scavenging free radicals (DPPH), metal reducing power (FRAP), phenolics and flavonoids.

DISCUSSION AND RESULTS

The results obtained with the use of the aqueous extract of M. titans demonstrated a great antimicrobial potential, significantly inhibiting ten bacterial as well as the six yeast tested strains. It was also verified that the extract presented a high rate of phenolic compounds, flavonoids and antioxidant activity.

CONCLUSION

Thus, the results allowed us to state that the aqueous extract of M. titans presented high potential as an antimicrobial agent, requiring the continuation of the study of purification and structural elucidation of its chemical components.

Keywords: Macrocybe titans, disk-diffusion, antimicrobial activity

08764 - Poster Session

GC.09 - PROJECT The Effect of Diaryl Disulfides on the Replicative and Infective Forms of Trypanosoma cruzi.

Anny Beatriz da Silva ¹, Alda Maria Teixeira Ferreira¹, Bruna Migliorini Melo Silva¹, Edson dos Anjos dos Santos²

¹Instituto de Biociências, Universidade Federal de Mato Grosso do Sul (, Brasil), ²Instituto de Biociências, Universidade Federal de Mato Grosso do Sul (, Brasil)

INTRODUCTION

Diaryl disulfides represent a class of synthetic compounds, which have shown an inhibitory action on tubulin protein synthesis in several human neoplastic cell lines, including the MCF-7 lineage. Our Research Group has been studying these compounds and recently it was found that they also have inhibitory activity on replicative forms (epimastigotes) of Trypanosoma cruzi, the etiologic agent of Chagas Disease (CD), and reduced cytotoxicity on Vero cells. Currently, the only drug available in Brazil for the treatment of CD is Benznidazole, but although this drug has been used for approximately five decades, it does not have a fully elucidated mechanism of action, it has several side effects and reduced efficacy in the treatment of the chronic phase of the disease. Due to the high toxicity that this drug has, combined with the lack of therapeutic alternatives, it is necessary to search for new bioactive compounds to combat this neglected disease.

OBJECTIVES

Therefore the objective of this work will be to evaluate the effect of diaryl disulfides on the replicative and infective forms of T. cruzi.

MATERIALS AND METHODS

For that, five compounds of the diaryl disulfides class will be tested in order to evaluate their trypanocidal or trypanostatic effect, through the recovery assay. Epimastigote forms will be treated during 4h of incubation with high concentrations of compounds. After removing the drugs, the growth of the parasites will be monitored through daily counts, in a Neubauer chamber, up to 168 h. The effect of these compounds against the trypomastigote form will also be tested. Analyzes will be performed with the aid of Graph Pad Prism 7 and Microsoft Excel 2013 software. It is hoped that this work can contribute more information about the effects of diaryl disulfides on replicative and infective forms of T. cruzi, in addition to suggesting possible mechanism of action.

DISCUSSION AND RESULTS

CONCLUSION

Keywords: Diaryl disulfides, Trypanosoma cruzi, trypomastigotes

08812 - Poster Session

GC.10 - Can a Proteus mirabilis exotoxin cause neuroinflammation? Evidence of a new role for the urease of P. mirabilis.

Matheus Vinicius Coste Grahl ¹, Evelin Furtado Meirelles², Augusto Frantz Uberti^1,2, Célia Regina Ribeiro da Silva Carlini^1,2

¹Graduate Program in Medicine and Health Sciences, , Pontifícia Universidade Católica do Rio Grande do Sul (, Brazil), ²Brain Institute - InsCer, Pontifícia Universidade Católica do Rio Grande do Sul (, Brazil)

INTRODUCTION

Proteus mirabilis is a gastrointestinal bacillus and an opportunistic uropathogen. The infection for P. mirabilis typically leads to formation of stones in the urinary tract and catheter-associated urinary infections. The urease of P. Mirabilis (PMU) produces ammonia that induces alkalinization of the urine leading to precipitation of urinary salts and formation of stones, which protect the entrapped bacteria. Proteus spp. have been described also as the causative agent of several diseases such as several types of meningitis and is currently associated with Parkinson’s disease.

OBJECTIVES

Better understanding of the contributions of PMU not only to urinary infections, but also to the extra-urinary pathologies associated with this bacterium.

MATERIALS AND METHODS

All assays were performed with three cell lineages: human neuroblastoma SH-SY5Y, murine microglial BV-2 and human embryonic kidney HEK293 and three doses of PMU were used: 63, 126 and 252 nM with incubation of 6 or 24 h. We evaluated the metabolic activity with MTT assay. The amount of intracellular calcium ([Ca2+]i) and the release of reactive oxygen species (ROS) were analyzed by Fluo4 and CM-DFFDA fluorescent probes, respectively. PMU was labeled with Texas Red to study the interaction of the protein with cells. Nuclear localization sequence analyzes were performed using cNLS mapper.

DISCUSSION AND RESULTS

Our results showed that PMU did not alter cell viability in all cell lineages tested. In SH-SY5Y cells PMU induced the increase of [Ca2+]i in both times tested while BV-2 showed a decrease in [Ca2+]i while HEK293 cells were not altered. PMU induced an increase of ROS production in SH-SY5Y and HEK293 cells while no effect was seen for BV-2. Texas Red-labeled PMU was present inside the cytoplasm of all cell lineages, possibly interacting with the nucleus as well. In silico analyzes show the presence of a nuclear localization sequence in PMU.

CONCLUSION

PMU may induce neuroinflammation and contribute to extra-urinary pathologies.

Keywords: Neurotoxin, Parkinson’s Disease, Proteus mirabilis

Supported by: Fapergs, CNPq and CAPES

08580 - Poster Session

GC.11 - Investigation of pro and anti-apoptotic modulators during infection by pathogenic protozoa such as Trypanosoma cruzi and Leishmania amazonensis.

Michelle de Oliveira Chain ¹, Amanda Nogueira¹, Alberto Neto¹, Igor Maciel¹, Thayane Fagundes¹, Ulisses Lopes³, Rosane Silva³, Rafael Mesquita⁴, Luciana Carneiro², Gabriela de Moraes², Luiz de Melo¹

¹Biotecnology, Federal Institute of Education, Science and Technology of Rio de Janeiro (, Brazil), ² Molecular Hemato-Oncology, National Cancer Institute (RJ, Brasil), ³ Biophysics, Federal University of Rio de Janeiro (, Brazil), ⁴Biochemistry, Federal University of Rio de Janeiro (RJ, Brasil)

INTRODUCTION

Trypanosoma cruzi is the etiologic agent of Chagas disease. Leishmania sp. is the etiological agent of leishmaniasis, being Leishmania amazonensis causing the cutaneous manifestation. We believe that both parasites appropriate anti-apoptotic proteins such as Galectin-3 (Gal-3) to subvert the cell death.

OBJECTIVES

The project aims to investigate the cell signaling pathways responsible for modulating apoptosis in cells infected by L. amazonensis or T. cruzi with the participation of the Gal-3 protein.

MATERIALS AND METHODS

Using HeLa lineage silenced for Gal-3 infected with T. cruzi and mouse peritoneal macrophages from C57/Black C6 Wild type(WT) and KO Galectin-3 (KOGal-3) infected with L . amazonensis assays with TMRE, PI and DHE labeling were performed. The protein level of proteins that regulate apoptosis such as Survivin, XIAP, cIAP-1, Bax and Bcl-2 were analyzed by western blotting during the infections. Another assay performed was RNAseq with mRNA analysis of 100 differents genes selected using the Iseq.

DISCUSSION AND RESULTS

In the TMRE labeling assays there was a reduction and in PI labeling there was an increase in cells which presents reduction gal- 3 levels for both infections, in the DHE assay there was no change in during infection with T. cruzi, however there was greater staining with DHE in the peritoneal macrophages of KOGal-3 mice. In the western blotting assay, there was an alteration in the protein levels of IAPs throughout both infections, especially when compared to cells with a reduced level of gal-3, whereas for Bax and Bcl-2 there was no change during T. cruzi infection, but there was change during infection by L. amazonensis. During the RNAseq assay it showed particular patterns during each of the infections, demonstrating the importance of Gal-3.

CONCLUSION

We believe that parasite appropriates of the signaling pathways of the host cell in favor of positively regulating the functions of Gal-3 related to survival and apoptosis inhibition.

Keywords: Apoptosis, Trypanosoma cruzi, Leishmania amazonensis

Supported by: FAPERJ e CNPq

08611 - Poster Session

GC.12 - Gene expression analysis of putative Pho-regulon genes in Vibrio cholerae.

Lucas Batista Rocha ¹, Matheus Lucchetta da Fonseca¹, Paulo Mascarello Bisch¹, Wanda Maria Almeida von Krüger¹

¹Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Cholera is a life-threatening waterborne gastroenteritis caused by the Gram-negative bacterium Vibrio cholerae, naturally occurring in freshwater, estuarine and marine ecosystems worldwide. It can be found free-living or in association with different types of aquatic fauna, flora and certain cyanobacteria species. Natural aquatic environments are generally poor in nutrients such as phosphorus, an essential element for all forms of life. Thus, V. cholerae survival in such media depends on its ability to cope with phosphorus starvation. Most organisms use inorganic phosphate (Pi) as phosphorus main source and respond to Pi deficiency by expressing genes collectively known as the Pho regulon. These genes are under transcriptional control of the PhoR/PhoB, a two-component regulatory system. Under low Pi levels, PhoR gets phosphorylated and then transfers the phosphate group to PhoB. PhoB~B binds to Pho-boxes, in the regulatory of the Pho regulon genes, to regulate their expression. In a previous research, using bioinformatic tools, our group found putative PhoB binding sites in the regulatory regions of many V. cholerae genes, raising the hypothesis they could be members of the Pho regulon.

OBJECTIVES

In this work, we selected five of those genes for further study to evaluate which can be a Pho regulon gene.

MATERIALS AND METHODS

The selection was based on their putative function, position of their Pho-box sequences and similarity to the consensus Pho-box sequence. Using quantitative PCR, we analyzed the expression of those genes in V. cholerae cells, grown under low and high Pi level.

DISCUSSION AND RESULTS

Two genes, vc1592, involved in the metabolism of the second messenger cyclic di-GMP (c-di-GMP) that controls a wide range of cellular processes in bacteria and vc2488, a putative lipid phosphatase of the PAP2 family, were differentially expressed under low Pi levels.

CONCLUSION

This findings suggest that these genes might be new members of the Pho regulon of V. cholerae.

Keywords: Phosphate, Vibrio, Pho regulon

Supported by: CAPES

08732 - Poster Session

GC.13 - Comparative genomics of seven V. parahaemolyticus strains isolated in the Brazilian territory

Leandro de Oliveira Santos ¹, Cristóvão de Lanna¹, Paulo Bisch¹, Wanda von Krüger¹

¹CCS - UFRJ, Instituto de Biofísica Carlos Chagas Filho (Rio de Janeiro, Brasil)

INTRODUCTION

Vibrio parahaemolyticus is a halophilic bacterium found in marine and estuarine environments, free living or associated with biotic or abiotic surfaces. Considered one of the main pathogenic species to humans among, it can cause acute gastroenteritis, through ingestion of contaminated raw fish and/or seafood, and systemic infection. Many virulence factors have been described for this species, including the hemolysins TDH and TRH, type III (T3SS) and type VI (T6SS) secretion systems.

OBJECTIVES

Investigate other factors that might be involved in the pathogenicity of this species. With the increasing number of reports of V. parahaemolyticus in the environment and in clinical specimens in Brazil, we also want to investigate their genomic potential.

MATERIALS AND METHODS

In this work, seven Brazilian isolates of V. parahaemolyticus were analyzed. Three were clinical isolates, and four were from environmental sources. The isolates were cultured, their chromosomal DNAs were isolated and libraries were prepared for sequencing on the MiSeq platform (Illumina). FastQC and Trimmomatic were used to evaluate sequence quality, for genomic assembly Spades v.3.8.1 and the Contiguator were used, and the RAST server was used for annotation.

DISCUSSION AND RESULTS

More than 65 million reads were obtained for the seven genomes, resulting in two circular chromosomes per strain, with average sizes of 3.1 Mbp and 1.8 Mbp. An average of 2833 and 1609 CDS were annotated, respectively, in the chromosomes I and II. All genomes carried the tlh gene, but none had the trh gene. Gene clusters for T3SSI and T6SSII were found in all strains, but only the clinical isolates carried the gene tdh, and gene clusters for T3SSII and T6SSI.

CONCLUSION

In summary, these results emphasize the genomic diversity of V. parahaemolyticus in the Brazilian territory. However, further comparative genomic analysis could provide a better understanding of their distinct pathogenic potential, environmental adaptation, and evolution.

Keywords: V. parahaemolyticus, Comparative genomics, Sequencing

Supported by: CAPES, CNPq, FAPERJ

08412 - Poster Session

GC.14 - Drug repurposing study based on enzymes from energy and lipid metabolism of Trypanosoma cruzi

Caroline Silva Garcia ¹, Milena Pereira Batista¹, Cacilda Tezelli Junqueira Padovani¹, Alda Maria Teixeira Ferreira¹

¹Laboratório de Imunologia, Biologia Molecular e Bioensaios-INBIO, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brazil)

INTRODUCTION

Trypanosoma cruzi is the etiological agent of Chagas disease, one of the main public health diseases of concern in Latin America. The available drugs for the treatment of Chagas disease are Benznidazole and Nifurtimox. Both drugs are more effective in the acute phase and severe side effects are reported. In this context, drug repurposing has become an important strategy, because it is a faster and less costly tool than the development of new drugs.

OBJECTIVES

The aim of this study was to select in silico approved drugs with potential activity against T. cruzi strain Dm28.

MATERIALS AND METHODS

On-line and free databases were used to perform the search and selection of drugs with potential action over homologous enzymes from energy and lipid metabolism of T. cruzi, based on the identification of drug targets with high similarity to pathogen genes.

DISCUSSION AND RESULTS

On TDR Targets database were selected 139 and 151 genes encoding enzymes from energy and lipid metabolism of T. cruzi, respectively. Subsequently, the amino acid sequence of the cited enzymes was obtained on TriTrypDB database. The sequences were inserted into DrugBank and Therapeutic Targets databases to identify homologous drug targets. Among the protein sequences with positive drug matches, only those with E-value T. cruzi strain Dm28 was performed using PUBMED, LILACS and Academic Google databases, resulting in 19 drugs for energy metabolism and 25 for lipid.

CONCLUSION

The in silico analysis suggested that the selected drugs have activity against T. cruzi, with the advantage of being approved drugs for use in humans. In order to demonstrate the biological activity, in vitro tests are being carried out.

Keywords: Drug Repurposing, In Silico, Trypanosoma cruzi

Supported by: FUNDECT, CNPq and UFMS

08896 - Poster Session

GC.15 - Annotation of two novel Saccharibacteria (TM7) genomes assembled from the oral metagenome

Ana Tana Rosas Nascimento Ferreira¹, Cristiane Pereira Borges Saito¹, Anderson Nogueira Barbosa², Leandro Nascimento Lemos³, Tsai Siu Mui³, Daniel Saito¹

¹Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas (Amazonas, Brasil), ²Laboratório de Virologia, Instituto Nacional de Pesquisas da Amazônia (Amazonas, Brasil), ³Centro de Energia Nuclear na Agricultura, Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Saccharibacteria (formerly TM7) is a phylum composed by small cell-size epibiontic bacteria that can inhabit various environments, including soil, water, plants, and animals. This phylum has also been associated with oral diseases in humans, including halitosis and periodontitis. Metagenome-assembled-genomes (MAGs) represent a novel bioinformatics approach with prominent contributions to microbial taxonomy and ecology.

OBJECTIVES

Given the scarcity of investigations targeting the Saccharibacteria phylum, this study aimed to retrieve Saccharibacteria MAGs from the oral cavity, in effort to shed additional light into their role in the oral cavity.

MATERIALS AND METHODS

To this end, saliva samples were collected from 13 healthy individuals and 14 periodontitis patients. Total DNA was extracted and subjected to Illumina Hiseq2500 sequencing. The recovered reads were assembled and the, GC content, integrity, coverage levels and genome size were assessed. Six distinct Saccharibacteria clones were identified, of which only 2 were further selected, based on 97% minimum completeness and 5% maximum contamination values. The assembled datasets were annotated via the IMG-JGI pipeline.

DISCUSSION AND RESULTS

In all, 874 and 866 genes were identified in clones RVB-004 and BGC-009, respectively. Of this total, annotation revealed 816 and 817 general protein encoding genes, 56 and 47 RNA genes, 2 regulatory and miscellaneous system genes, and 563 and 573 functional genes. Overall, the sequenced clones were proved to be genetically distinct, with BGC-009 being phylogenetically closest to Candidatus Saccharibacteria bacterium YM_S32_TM7_50_20 and RVB-004 to Candidatus Saccharibacteria bacterium oral taxon 955 strain FS17P. Both showed important virulence factors such as antibiotic resistance genes (van, pen, mur, mra and mrc), pleomorphism determinants (glm), immune response modulators (imm), efflux pumps (ABC transporters) and motility components (pil, fli and fla).

CONCLUSION

In summary, the present study unveiled novel Saccharibacteria genomes in health and disease conditions, validating the importance of MAGs retrieval for characterization of previously unreported metabolic pathways in the oral cavity.

Keywords: Saccharibacteria, metagenome, periodontitis

Supported by: FAPEAM and PMBqBM

08441 - Poster Session

GC.16 - Antimicrobial properties of a new ultrashort peptide active against multi-resistant bacteria

Maria Caroline de Moura Cavalheiro ¹, Caio Fernando Ramalho de Oliveira¹, Maria Lígia Rodrigues Macedo¹

¹Laboratório de Purificação de Proteínas e suas Funções Biológicas, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

Antimicrobial peptides shared a differential mechanism of action when compared with traditional antibiotics. An expressive amount of research has been looked for ultra-short peptides, molecules with around 10 amino acid residues, able to control multi-resistant bacteria, without presenting side effects against eukaryotic cells.

OBJECTIVES

Thus, we describe the antimicrobial properties of USP_1, our first ultra-short peptide containing 8 amino acid residues.

MATERIALS AND METHODS

The peptide was chemically synthesized and assayed against multi-resistant Gram-negative bacteria, E. coli KPC+ and Acinetobacter baumannii; and Gram-positive Staphylococcus aureus methicillin resistant (MRSA) strains. The synergic effect of USP_1 and ciprofloxacin was assayed against E. coli KPC+. The ability to eradicate A. baumannii multi-resistant mature biofilms was also assayed. USP_1 did not display cytotoxic effects against murine macrophages RAW 264.7 or human erythrocytes up to 100 μM. The peptide showed a minimum inhibitory concentration (MIC) for MRSA ATCC33591 and MRSA ATCC43300 of 8 and 16 μM, respectively. For E. coli KPC+ CI001812446 and A. baumannii CI003321216, USP_1 showed a MIC of 16 and 64 μM, respectively. The peptide and ciprofloxacin showed a Bliss Synergy Score of 3.99 against E. coli KPC+, keeping the antimicrobial activity in concentration of ciprofloxacin reduced by 32-fold and 8-fold the concentration of USP_1. At MIC, USP_1 eradicated 38% of 24-h A. baumannii mature biofilm, impairing both biofilm structure and bacteria viability, results observed through fluorescence microscopy. When administered alone at the MIC, ciprofloxacin increased the A. baumannii biofilm by 12%. CD analyses showed that RK8 assumes a non-ordered structure in water, presenting ~1,28% of α-helix content. Otherwise, in presence of SDS micelles, the peptide assumed an α-helix structure.

DISCUSSION AND RESULTS

Further assays are investigating the resistance of USP_1 against peptidases, to evaluate it potential in preclinical assays.

CONCLUSION

We conclude that USP_1 represents a promising strategy to development of a new antimicrobial agents against multi-resistant bacteria.

Keywords: Antimicrobial peptide, biomimetic, rational design

Supported by: FUNDECT, FINEP, CNPq and CAPES

08733 - Poster Session

GC.17 - Genotypic diversity and pathogenic potential of clinical and environmental Vibrio parahaemolyticus isolates from Brazil

Cristóvão Antunes de Lanna ¹, Leandro Santos¹, Anna Arcanjo¹, Paulo Bisch¹, Wanda von Krüger¹

¹Laboratório de Física-Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Vibrio parahaemolyticus is a leading worldwide agent of acute gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. They also cause wound infections that can lead to septicemia and death. Many virulence factors in different combinations have been identified in clinical and environmental isolates, including the hemolysins TDH and TRH, the adhesive factor VpadF, and pandemic strain-associated markers toxRSnew and orf8, suggesting that V. parahaemolyticus pathogenesis has not been fully elucidated. An increasing number of isolates have been reported in Brazil. However, few studies have characterized their pathogenic potential.

OBJECTIVES

Therefore, we analyzed the genotypic diversity of V. parahaemolyticus isolates from clinical and environmental sources in Brazil, focusing on virulence, pandemic markers, and pathogenic potential.

MATERIALS AND METHODS

V. parahaemolyticus strains recovered from human diarrheal stools (3, one in 1975 and two in 2001) and environmental sources (4, between 2008-2010) were investigated for the presence of virulence genes (trh, tdh, and vpadF), pandemic markers (orf8, toxRSnew), and with respect to their pathogenic potential in mice and Galleria mellonella larvae systemic infection models.

DISCUSSION AND RESULTS

Based on the presence of the genetic markers, all environmental strains were classified as non-pathogenic, while one clinical strain was pathogenic/non-pandemic and the other two were pathogenic/pandemic. All strains, except for the clinical pathogenic/non-pandemic, produced lethal infection in both infection models, regardless of source, serotype, and genotype. Based on mice and larval mortality rates, which were remarkably similar, the pathogenic/non-pandemic strain was classified as avirulent, while the others were considered of high or intermediate virulence.

CONCLUSION

These findings demonstrate that G. mellonella larvae can be used as an alternative model to study the pathogenicity of V. parahaemolyticus. Moreover, they raise doubts about the use of traditional virulence markers to predict pathogenesis of the species and show that reliable models are indispensable to determine the pathogenic potential of environmental isolates considered non-pathogenic.

Keywords: Infection models, Vibrio parahaemolyticus, Virulence and pandemic markers

Supported by: CAPES, CNPq, FAPERJ

08408 - Poster Session

GC.18 - Pan-genome analysis of the difficult-to-identify and multi-drug resistant emerging pathogen Corynebacterium amycolatum

Hendor Neves Ribeiro deJesus ¹, Danilo Jobim Passos Gil da Rocha¹, Jesus Navas Méndez², Vasco Ariston de Carvalho Azevedo³, Eric Roberto Guimarães Rocha Aguiar⁴, Luis Gustavo Carvalho Pacheco¹

¹Departamento de Biotecnologia, Universidade Federal da Bahia (Bahia, Brazil), ²Faculty of Medicine, University of Cantabria (, Spain), ³Departamento de Biologia Geral, Universidade Federal de Minas Gerais (Minas Gerais, Brazil), ⁴Departmento de Ciências Biológicas, Universidade Estadual de Santa Cruz (Bahia, Brazil)

INTRODUCTION

Corynebacterium amycolatum, which is an emerging multidrug-resistant (MDR) opportunistic pathogen, is often misidentified at the clinical microbiology laboratory when using traditional bacterial identification methods. This has hampered the development of studies aimed at understanding the molecular mechanisms involved in the transition from colonization to the invasive MDR phenotype in clinical isolates.

OBJECTIVES

To perform a comprehensive pan-genomic analysis of MDR clinical isolates of C. amycolatum, to obtain information on the genetic factors contributing to infectivity and multidrug resistance in this species.

MATERIALS AND METHODS

We generated 08 preliminary genome sequences from clinical isolates of C. amycolatum MDR from Spain and Tunisia. In addition, 18 complete or draft genomes of C. amycolatum were retrieved from the NCBI. The Bacterial Pan Genome Analysis (BPGA) v.1.3 tool was used to perform pan-genomic analysis. Automated antimicrobial resistance (AMR) gene predictions were performed in the Comprehensive Antibiotic Resistance Database (CARD) and the National Database of Antibiotic-Resistant Organisms (NDARO). Furthermore, virulence-related genes were searched with VFanalyzer, and genomic islands were annotated with IslandViewer4.

DISCUSSION AND RESULTS

The species C. amycolatum presented an open pan-genome (α=0.854905) containing 3,280 gene families distributed among the central (n= 1,690), accessory (n= 1,121), and unique (n= 469) genomes. Four out of nine identified antimicrobial resistance (AMR) genes are associated with resistance to aminoglycosides. In addition, with exception of the housekeeping gene rpsL, all other AMR genes are present in genomic islands, indicating extensive horizontal gene transfer (HGT). We identified 47 putative virulence factors, including 17 associated with iron acquisition and the SpaD-type pili.

CONCLUSION

Species with an open pan-genome have an extended ability to acquire new genes through HGT. Consistently, we found AMR genes predominantly in genomic islands in C. amycolatum isolates. Our results also demonstrate that C. amycolatum has a metabolism particularly adapted to invasive infections.

Keywords: Genomic Island, Pathogenomics, Virulence factor

Supported by: FAPESB

08555 - Poster Session

GC.19 - Analysis of outer membrane vesicles (OMVs) produced by Vibrio cholerae under limitation and abundance of inorganic phosphate and its relationship to the pathogenicity

Matheus Luchetta da Fonseca ¹

¹IBCCF, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Vibrio cholerae, like other bacteria, produces outer membrane vesicles (OMVs), which are released from their surface during growth. OMVs contain phospholipids, lipopolysaccharides, proteins of the cytoplasm, periplasm and of the outer membrane, as well as, DNA and RNA. V. cholerae inhabits phosphorous-poor aquatic media and colonizes the host intestinal tract, environments where it expresses various genes in response inorganic phosphate (Pi) limitation (Pho regulon).

OBJECTIVES

This information led us to verify if V. cholerae OMVs produced in vitro under Pi limitation, could carry factors essential to the pathogenicity of the bacterium.

MATERIALS AND METHODS

For this, V. cholerae was cultivated under Pi abundance and limitation and the OMVs were purified and quantified. About 2 times higher protein content was found in the OMV suspension from MGLP (MOPS, glucose, low [Pi]) than from MGHP (MG, high [Pi]), suggesting greater OMV production at low Pi concentration.

DISCUSSION AND RESULTS

Proteomic analysis of OMVs by SDS-PAGE and mass spectrometry showed that the electrophoretic profile and protein composition of V. cholerae OMVs are dependent on the Pi concentration in the culture medium. Similar results were observed with the bacterial cells grown in MGHP and MGLP. However, V. cholerae proteins in OMVs released in MGHP and MGLP fall into the same functional groups. Among the proteins specific to OMVs released in MGLP, several are products of regulon Pho genes and some are important for the pathogenicity of the bacterium.

CONCLUSION

The lipid composition of OMVs was analyzed by mass spectrometry. OMVs released by V. cholerae in MGHP and MGLP have similar lipid profiles with high levels of ornithine lipids (OLs) that are distinct from those of the original cells. The pathogenic potential of these OMVs in the Galleria mellonella infection model showed different mortality rates, in agreement with our findings that the V. cholerae OMVs generated under Pi limitation carry virulence-related factors.

Keywords: outer membrane vesicles, proteomics, lipidomics

Supported by: cnpq

08442 - Poster Session

GC.20 - Genomic analysis of antimicrobial resistance in OXA-23-producing Acinetobacter baumannii strains in Brazil

Fernanda Jales de Souza ¹, Caio Augusto Martins Aires²

¹Programa Multicêntrico de Pós-graduação em Bioquímica e Biologia Molecular, Universidade do Estado do Rio Grande do Norte (Rio Grande do Norte, Brazil), ²Departamento de Ciências da Saúde, Universidade Federal Rural do Semi-Árido (Rio Grande do Norte, Brazil)

INTRODUCTION

Resistance to carbapenems in Acinetobacter baumannii isolates has become a major public health problem. Among the mechanisms responsible for this phenotype, OXA-type carbapenemases are the main ones, especially OXA-23 in Brazil. Therefore, WHO has designated carbapenem-resistant A. baumannii a threat in human health, with critical priority and need new antibiotics to come.

OBJECTIVES

To identify the main mechanisms of antimicrobial resistance in OXA-23-producing A. baumannii genomes isolates in Brazil

MATERIALS AND METHODS

Data corresponding to bacterial genomes were collected from the genome bank of the National Center for Biotechnology Information using the Pathogen Detection tool. The genome search was done in May 7th, 2021, using as a filter to select the genomes: genotype, bla_OXA-23; organism group, A. baumannii; location, Brazil; type of isolate, clinical; host, Homo sapiens. Data were interpreted through descriptive statistical analysis with simple frequency distribution using the Microsoft Office Excel® software.

DISCUSSION AND RESULTS

The resistance genes of 153 A. baumannii strains were found and analyzed. Isolates were collected between 2008 and 2020. Most of the isolates (98%) presented information about the type of clinical sample which was obtained, being mostly blood samples (33.3%), followed by respiratory tract samples (31.4%). Regarding the location of the insolates, 95% had this specification. The states of São Paulo (68%) and Minas Gerais (7.2%) stands out. A total of 1986 genes were found, with an average of 12.98 genes per isolate. Among the genes found, most were associated with aminoglycoside resistance (33%), followed by b-Lactam antibiotics (27%). Further the bla_OXA-23, the bla_ADC, ant(3'')-IIa and amvA genes were present in all strains.

CONCLUSION

In conclusion, OXA-23-producing A. baumannii strains in Brazil have the ability to carry several resistance genes associated with multiple drugs, thus genomic analysis is an important tool to observe the spread of gene resistance and diversity.

Keywords: Acinetobacter baumannii, Drug Resistance, Carbapenems

Supported by: CAPES

08040 -

GC.21 - Study of SARS-CoV-2 morphogenesis and interaction with the cell by transmission and high resolution scanning electron microscopy

Fabiana Avila Carneiro ^1,2, Lucio Ayres Caldas^1,2, Luiza Mendonça Higa³, Fábio Luis Monteiro³, Gustavo Peixoto da Silva⁴, Luciana Costa⁴, Ingrid Augusto^1,5, Kildare Miranda^1,5, Amilcar Tanuri³, Wanderley de Souza^1,5

¹Instituto de Biofísica Carlos Chagas Filho, LUCHM, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ²Núcleo Multidisciplinar de Pesquisa em Biologia (NUMPEX), Campus Duque de Caxias, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ³Departamento de Genética, Instituto de Biologia , Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ⁴Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ⁵Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

SARS-CoV-2 is a single strand RNA virus, belonging to the betacoronavirus genus, within the Coronaviridae family. This pathogen, transmitted mainly through droplets and aerosol, is responsible for the COVID-19. As occur to many of the positive sensed RNA viruses, its cellular cycle involves a robust membranar rearrangement in the cytosol of the infected cells. This structure delimits and protects the locus of replication and morphogenesis of SARS-CoV-2.

OBJECTIVES

In the present study, we approached the main steps of SARS-CoV-2 morphogenesis and interaction with the cell by transmission and high resolution scanning electron microscopy (HR-SEM).

MATERIALS AND METHODS

The sites of viral replication, and assembly were documented in Vero cells at 24, 48 and 72 hours post-infection by using both electron microscopy modes. In addition, the interactions of this virus with the cell surface, as well as the viral factory and the details of its main components, were also investigated with the aim of HR-SEM, after the removal of infected cells plasma membrane.

DISCUSSION AND RESULTS

This allowed the visualization of unprecedented features of the interactions between this virus and the cell, such as the so-called “virus surfing”, which enables a relatively safe cell-to-cell viral propagation in the tissue. On the other hand, the electron-tomography of these samples showed, for the first time, the presence of SARS-CoV-2 particles in the space between the inner and the outer nuclear envelope.

CONCLUSION

The data obtained in this work contribute to the knowledge of the route of SARS-CoV-2 within the infected cell and the cell biology of their interactions.

Keywords: SARS-CoV-2, Electron Microscopy, Morphogenesis

Supported by: FAPERJ and FINEP

08545 - Poster Session

GD.01 - Evaluation of the antioxidant activity of mandacaru (Cereus jamacaru D.C) from Caatinga of Bahia

Leila Magda Rodrigues Almeida ¹, Brenda Antunes de Andrade Santos¹, Patrícia Campos Santos^1,2, Gabriel Teixeira Medrado¹, Lourdes Cardoso de Souza Neta³, Renato Delmondez de Castro¹, Luzimar Gonzaga Fernandez^1,2

¹Departamento de Bioquímica e Biofísica, Universidade Federal da Bahia (Bahia, Brasil), ²Programa de Doutorado em Biotecnologia – Rede Nordeste de Biotecnologia, Universidade Federal da Bahia (BA, Brasil), ³Departamento de Ciências Exatas e da Terra I, Universidade do Estado da Bahia (BA, Brasil)

INTRODUCTION

Cereus jamacaru (mandacaru) has great potential as a source of medicinal substances. However, despite the great use of this species by the Caatinga population, studies on the antioxidant potential of mandacaru are still scarce.

OBJECTIVES

Thus, the aim of this study is to evaluate the antioxidant activity and determine the content of total phenolic compounds in the cladode of Cereus jamacaru.

MATERIALS AND METHODS

The cladodes were placed to dry at room temperature, being later pulverized in knife mills. The pulverized material was submitted to extraction by maceration with different solvents: ethanol, ethyl acetate, hexane and hydro-ethanolic. The crude extracts were concentrated in a rotary evaporator, under reduced pressure, at temperatures of 40-45ºC. The solvent residue of each extract was removed by evaporation in an exhaust hood. The antioxidant activity was evaluated by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical and the 2,2-azinobis-3-ethyl-benzothiazolin-6-sulfonic acid (ABTS) radical scavenging method and the content of phenolic compounds was determined by the Folin-Ciocalteau method.

DISCUSSION AND RESULTS

In the DPPH assay, the extracts in ethyl acetate (21.07 μgmL-1) and hexane (22.65 μgmL-1) exhibited higherantioxidant activity than the others extracts, without significant differences regarding the IC50 value. The hydro-ethanolic and ethanolic extracts showed higher antioxidant activity by the ABTS method (378.61 μMTrolox.g-1 and 214.24 μMTrolox.g-1, respectively), differing statistically (p > 0.05) from the other extracts. The content of phenolic compounds showed a strong negative correlation with the DPPH antioxidant capacity for the extracts in ethyl acetate (97.23 mgEGA.g-1 / r = 0.95) and hexane (100.36 mgEGA.g-1 / r = 0.95), while the hydro-ethanolic (126.73 mgEGA.g-1 / r = 0.95) and ethanolic (104.23 mgEGA.g-1 / r = 0.99) extracts showed a strong positive correlation with ABTS.

CONCLUSION

Mandacaru cladode extracts have shown potential to be used in future studies showing the bioactive properties related to traditional use.

Keywords: ethnopharmacobotany, cactace, medicinal plants

08266 - Poster Session

GD.02 - Phytochemical analysis and in vitro antioxidant and toxic activities of ethanolic extracts from Amazonian plants

Sthéfanny Caroline Mendes Azevedo ^1,2, Lethícia do Nascimento Marinho², Marcela Martins Vieira², Keyla Maciel Carvalho², Paula Hariana Dantas³, Valdely Ferreira Kinupp⁴, Bruno Sampaio Amorim⁵, Cecília Verônica Nunez⁶, Patrícia Melchionna Albuquerque^1,5

¹Programa Multicêntrico de Pós-graduação em Bioquímica e Biologia Molecular, Universidade do Estado do Amazonas (Amazonas, Brasil), ²Departamento de Ensino e Pesquisa, Fundação Centro de Controle de Oncologia do Estado do Amazonas (Amazonas, Brasil), ³Escola Normal Superior, Universidade do Estado do Amazonas (Amazonas, Brasil), ⁴Departamento de Agroecologia, Instituto Federal do Amazonas (Amazonas, Brasil), ⁵Programa de Pós-graduação em Biotecnologia e Recursos Naturais da Amazônia, Universidade do Estado do Amazonas (Amazonas, Brasil), ⁶Faculdade de Ciências Farmacêuticas, Universidade Federal do Amazonas (Amazonas, Brasil)

INTRODUCTION

The Amazon has plant species with potential in the formulation of new drugs with antioxidant action.

OBJECTIVES

We sought to evaluate the chemical profile and antioxidant activity of ethanol extracts from leaves of Garcinia macrophylla Mart., Tovomita macrophylla Walp. (Clusiaceae); Vismia japurensis Reichardt (Hypericaceae); Cnidoscolus chayamansa McVaugh (Euphorbiaceae); Trema micrantha (L.) Blume (Cannabaceae); and Cecropia concolor Willd (Urticaceae).

MATERIALS AND METHODS

The extracts were obtained by cold maceration. The evaluation of chemical classes was performed using thin layer chromatography with an elution system containing ethyl acetate, hexane, and dichloromethane (1:4:5 ratio). UV light at 254 and 365 nm, as well as aluminum chloride, ceric sulfate, ferric chloride and Dragendorff’s reagent were used as developers. The toxicity of the extracts was evaluated through the bioassay with Artemia salina Leach, and the total phenolic content was determined by the Folin-Ciocalteu method using gallic acid as standard, while the antioxidant activity was measured via the DPPH• radical scavenging technique, using ascorbic acid and quercetin as standards.

DISCUSSION AND RESULTS

The presence of phenolic compounds, flavonoids, terpenes and alkaloids was observed in all the extracts; however, these which were not sufficiently toxic to the microcrustacean A. salina at 1.0, 0.5, 0.25 and 0.125 mg/mL. Three species showed higher phenolic content and noteworthy antioxidant potential: C. concolor, V. japurensis and G. macrophylla, with EC₅₀ values lower than those found for ascorbic acid and quercetin. The extract of T. macrophylla also showed promising results for antioxidant activity.

CONCLUSION

The presence of phenolic compounds and flavonoids may explain the promising antioxidant activity in the extracts, since these substances are known for their ability to react with free radicals. Of the Amazonian species evaluated here, C. concolor showed outstanding antioxidant activity, despite this species having not previously been described in the literature as a source of antioxidant metabolites.

Keywords: phytochemical, antioxidant, C. concolor

Supported by: CAPES

08682 - Poster Session

GD.03 - Effects of Lectin-rich Fraction from Moringa oleifera Seeds on Survival, Biological Cycle Progress and Midgut Development of Aedes aegypti

Livia Lais de Santana Silva¹, Kenner Morais Fernandes², Franciane Rosa Miranda², Stella Cristina Cabral Silva¹, Luana Cassandra Breitenbach Barroso Coelho¹, Daniela Maria do Amaral Ferraz Navarro³, Thiago Henrique Napoleão¹, Gustavo Ferreira Martins², Patrícia Maria Guedes Paiva ¹

¹Departamento de Bioquímica, Universidade Federal de Pernambuco (Pernambuco, Brasil), ²Departamento de Biologia Geral, Universidade Federal de Viçosa (Minas Gerais, Brasil), ³Departamento de Química Fundamental, Universidade Federal de Pernambuco (Pernambuco, Brasil)

INTRODUCTION

The Aedes aegypti is a vector mosquito of the etiologic agents of different arboviruses such as chikungunya, dengue, yellow fever, and zika. The control of this insect is crucial to mitigate the spreading if these diseases. Seeds of Moringa oleifera contain a water-soluble lectin (WSMoL) with larvicidal and ovicidal activities against this insect.

OBJECTIVES

The present work evaluated the effects of a lectin-rich fraction containing WSMoL on the survival and development of A. aegypti individuals.

MATERIALS AND METHODS

The insects were exposed to the fraction (0.05–0.6 mg/mL of protein) at the third larval instar for 24 h and development were followed for 9 days post-exposure. In addition, alterations in the midgut organization of treated larvae, pupae, and adults were investigated.

DISCUSSION AND RESULTS

The fraction induced the death of A. aegypti larvae along the post-exposure period. The mean survival time was reduced to 5.000±0.096 days in the treatment at 0.6 mg/mL, while in control there was no death along the 9 days. The fraction also delayed the developmental cycle, since 90% of the individuals reached the adult stage in control while no adults were present in the treatment with the fraction at 0.1 mg/mL during the 9-day period. The midguts of treated larvae and pupae showed disorganization and epithelial vacuolization, while in treated adults, the epithelium was underdeveloped compared to control. Unlike in control mosquitos, proliferating cells were not detected in treated larvae, and appeared in lower numbers in treated pupae than in control pupae.

CONCLUSION

In conclusion, females that developed from larvae treated with lectin-rich fraction showed damage to midgut organization, which can be linked to the impairment of development and survival.

Keywords: dengue mosquito, insecticidal proteins, development

Supported by: FACEPE, CNPq and CAPES

08304 - Poster Session

GD.04 - Evaluation of the antimicrobial activity of protein extract from Inga laurina seeds (Fabaceae)

Bruna Sanae Moroto ¹, Janaina de Cássia Orlandi Sardi¹, Maria Lígia Rodrigues Macedo¹

¹Instituto de Biociências, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil), ²Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

Inga laurina (Fabaceae) is a tree species native to the Cerrado, widely distributed in Brazil. In its seed extract, ILTI (Inga laurina trypsin inhibitor) has been isolated, a peptidase inhibitor that has antifungal activity.

OBJECTIVES

Thus, this study aimed to evaluate the inhibitory activity of the protein extract from Inga laurina seeds, as well, as its antimicrobial potential.

MATERIALS AND METHODS

Seeds of I. laurina were dried and ground. The extraction of soluble proteins from the flour was performed with 0.1 M phosphate buffer + 0.15 M NaCl, pH 7.6, at 25°C, for 2 h, under magnetic stirring. After centrifugation, the supernatant was denominated crude extract (EB). Protein quantification was performed according to Bradford (1976). For the trypsin inhibition assay, the methodology of Erlanger et al. (1961) and polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmili (1970). Antimicrobial activity was determined by the broth microdilution method according to CSLI standards.

DISCUSSION AND RESULTS

The EB yield was 1.72% in relation to the initial dry mass of seeds. Protein quantification indicated a content of 89.69% of total proteins. The EB inhibited the enzymatic activity of trypsin, and the band of 20 kDa, approximately, in electrophoresis gel confirmed the presence of ILTI in the EB. For most of the yeast tested, EB was active at a concentration of 4 mg/mL, showing better activity against Cryptococcus gattii, at the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 31.25 μg/mL, in addition to show good activity against Candida albicans, Candida guillermondii, Candida krusei and Candida nivariensis, with MIC and MFC of 250 μg/mL for these yeast. EB at a concentration of up to 4 mg/mL did not show activity for the bacterial strains tested.

CONCLUSION

EB showed good antifungal activity, which would allow it to be used to develop a pharmaceutical form for this purpose.

Keywords: antifungal, ILTI, ingá

Supported by: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

08469 - Poster Session

GD.05 - Investigation of Insecticide Properties Of Inga cylindrica Trypsin Inhibitor Against Anagasta kuehniella larvae

Ana Paula Ramos Pereira ¹, Caio Fernando Ramalho de Oliveira¹, Maria Lígia Rodrigues Macedo¹

¹FACFAN, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

Several molecules obtained from plant seeds display insecticidal properties, including trypsin inhibitors.

OBJECTIVES

The aim of the study was to investigate the insecticidal potential of a trypsin inhibitor purified from ⟨i⟩Inga⟨/i⟩ ⟨i⟩cylindrica⟨/i⟩ seeds (IcTI) against the Mediterranean moth, the insect pest ⟨i⟩ Anagasta⟨/i⟩ ⟨i⟩kuehniella⟨/i⟩ (Lepidoptera: Pyralidae).

MATERIALS AND METHODS

IcTI was purified from ⟨i⟩I.⟨/i⟩ ⟨i⟩cylindrica⟨/i⟩ seeds through gel filtration and ion-exchange chromatography. The inhibitory activity against trypsin was assayed using the colorimetric substrate, benzoyl-arginine-p-nitroanilide (BAPNA) and bovine trypsin. For in vivo assays with ⟨i⟩A.⟨/i⟩ ⟨i⟩kuehniella⟨/i⟩, neonate larvae were fed with diet (250 mg) containing different concentrations of IcTI (0; 0.5; 1; 1.5; and 2% w/w). Two independent assays were carried out in 10 replicates (n= 30). The larvae were kept in standard conditions (25± 2 ºC, 12 h photoperiod) for 30 days. Then, the larvae were weighted, the survival rate was determined, and the midguts were removed for enzymatic assays.

DISCUSSION AND RESULTS

The diet containing IcTI prompted a reduction in larval weight by 72%, 84%, 86%, and 88%, in comparison with control-fed larvae. The survival rate was reduced 33% among the treatments. Enzymatic assays are being carried out to investigate the physiological effects triggered by IcTI ingestion.

CONCLUSION

Since peptidase inhibitors can be expressed in transgenic plants, the knowledge of effects of ingestion these molecules by insect pests could be applied as biotechnological alternative for pest control.

Keywords: Biotechnological, Pest Control, Trypsin Inhibitor

Supported by: FAPESP, CNPq and CAPES

08544 - Poster Session

GD.06 - Antifungal potential of Macrocybe titans aqueous extract on Candida albicans

Fernanda Cristina Buraslan Neves Pereira¹, Vivian Emanoelle Mamede de Santana Justo², Gabrielle Caroline Peiter¹, Gabrieli Maria Huff², Fabio Rogério Rosado², Patrícia de Souza Bonfim-Mendonça³, Adriana Fiorini Rosado²

¹Programa de Pós-Graduação em Bioquímica e Biologia Molecular, Universidade Federal do Paraná – Setor Palotina (Paraná, Brasil), ²Departamento de Biociências, Universidade Federal do Paraná – Setor Palotina (Paraná, Brasil), ³Departamento de Análises Clínicas e Biomedicina, Universidade Estadual de Maringá (Paraná, Brasil)

INTRODUCTION

With the rapid emergence of antibiotics resistant microorganisms, the search for new effective therapeutic agents has become a favorable alternative in the fight against diseases caused by fungi, mainly Candida species.

OBJECTIVES

Thus, the present study aimed to verify the antifungal potential of the mushroom Macrocybe titans extracts, recently reported in Brazil and with few reports in the literature about its antimicrobial potential, against Candida albicans (ATCC 90028), as well as the effects on the morphological aspects of this yeast.

MATERIALS AND METHODS

The extracts used were obtained from M. titans, as three aqueous fractions (concentrated, filtered EfraMat-45 and EfraMat-22) and methanolic/ethyl acetate extracts (cold method and Soxhlet). The antifungal susceptibility was evaluated by broth microdilution, according to CLSI′s M27-A3 protocol. Micromorphological aspects were analyzed by microculture on cornmeal agar supplemented with 1% Tween 80, Fluorescence Microscopy using Calcofluor White and propidium iodide, and Scanning Electron Microscopy (SEM).

DISCUSSION AND RESULTS

It was verified that EfraMat-45 showed the best results for Minimum Inhibitory Concentration – MIC (31.25 μg/μl) among extracts. Microculture analysis showed complete inhibition of yeast growth on the culture slides referring to MIC, in all aqueous extracts. Morphological changes were observed on the slides of sub-MIC, with an increased formation of elongated cells and chlamydospores absence, compared to control yeasts. Through SEM, it was possible to observe morphological changes and slight damages on the surface of cells treated with EfraMat-45 MIC, as well as the presence of elongated cells viewed by Fluorescence Microscopy using Calcofluor White. Propidium iodide staining revealed cell death on yeasts treated with the extract. EfraMat-45 showed absence of in vitro cytotoxicity.

CONCLUSION

The results suggest that the aqueous extract of M. titans showed a very promising pattern as regarded to antifungal action, with cellular changes certifying cell death.

Keywords: Candida albicans, Macrocybe titans, natural products

08601 - Poster Session

GD.08 - Development and Characterization of Lipid Nanocapsules with Mauritia flexuosa Pulp Oil as a natural anti-inflammatory

Fernando Freitas de Lima ¹, Priscila Cordeiro Lima Fernandes², Ludmilla David Moura³, Gabriela Geronimo³, Talita Cesarim Mendonça³, Fabiola Vieira Carvalho³, Eneida de Paula³, Adriana Torres Silva e Alves¹, Leila Maria Spadoti¹

¹Centro de Tecnologia de Laticínios, Instituto de Tecnologia de Alimentos (São Paulo, Brazil), ²Faculdade de Ciências Farmacêuticas, Universidade Estadual de Campinas (São Paulo, Brazil), ³Departamento de Bioquímica e Biologia Tecidual, Universidade Estadual de Campinas (São Paulo, Brazil)

INTRODUCTION

The Mauritia flexuosa palm is found in Brazil mainly in the Cerrado region. The fruit of M. flexuosa , locally known as 'buriti', has a pulp layer with a color ranging from orange and red. Among the bioactive compounds, buriti pulp oil (BUPO) has a high content of carotenoids (CA) and fatty acids (FA). Recent studies with BUPO demonstrated an anti-inflammatory effect attributed to the high CA and FA content. However, these compounds are easily degraded and have low bioavailability. To prevent the degradation of these compounds and improve their bioavailability, technologies were developed, example: encapsulation in lipid nanocapsules (LNCs). LNC is a promising and ‘’simple” technique used in the Drug Delivery System (DDS). The development of LNCs replacing synthetic oils with a natural oil with a potential natural anti-inflammatory agent is interesting and promising.

OBJECTIVES

Therefore, the objective of the work was to develop, characterize (size, polydispersion, zeta potential and particle concentration) and evaluate the anti-inflammatory effect of BUPO nanocapsules (NCBU).

MATERIALS AND METHODS

The NCBU were developed using the phase inversion method (liquid lipid, BUPO, nonionic surfactant, hydrogenated soy lecithin and NaCl in Milli-Q water). The anti-inflammatory effect was evaluated by the Carrageenan-induced paw edema method.

DISCUSSION AND RESULTS

The results showed NCBU with average sizes of 55.68 ± 0.37 nm, PDI of 0.162 ± 0.028, zeta potential of -25.50 ± 1.37 and particle concentration of 1.42 ± 0.03 x10 ¹⁵ .mL ^-1 . The NCBU had an anti-inflammatory effect for 4h and edema inhibition of up to 89.69%.

CONCLUSION

The NCBU are viable for more characterization studies and another’s in vivo biological evaluation.

Keywords: natural products, nanocapsules, carotenoids

Supported by: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2021/01237-8); CNPq and CAPES

08427 - Poster Session

GD.09 - Exposure of male Drosophila melanogaster to chronic unpredictable mild stress causes oxidative damage: Role of the antioxidant γ-oryzanol

Stífani Machado Araujo ¹, Vandreza Cardoso Bortolotto¹, Mustafa Munir Mustafa Dahleh¹, Márcia Rósula Poetini¹, Luana Barreto Meichtry¹, Franciane Cabral Pinheiro¹, Elize Aparecida Santos Musachio¹, Franciéle Romero Machado¹, Marina Prigol¹

¹Departamento de Bioquímica, Laboratório de Avaliações Farmacológicas e Toxicoló, Universidade Federal do Pampa (RS, Brazil)

INTRODUCTION

Exposure to chronic unpredictable mild stress (CUMS) leads to the development of a depressive-like state in experimental animals such as Drosophila melanogaster. Evidence suggests that cellular oxidative damage may be related to the development of depressive-like behaviors caused by mitochondrial dysfunction. Therefore, the use of compounds such as γ-oryzanol (ORY) that have a portion of ferulic acid that gives it antioxidant potential is of great value.

OBJECTIVES

Our aim was to evaluate the effect of ORY on oxidative stress caused by CUMS in male Drosophila melanogaster.

MATERIALS AND METHODS

Flies were divided into 4 groups: (1) Control, (2) CUMS, (3) ORY (25 μM), (4) ORY (25 μM) + CUMS. For CUMS the flies were subjected to stress by cold, heat, starvation, sleep deprivation for 10 days. At the end, the survival rate was evaluated, and measurements were made of the levels of reactive species (RS) in the head and body of the flies and the preliminary evaluation of the oxygen flux by high-resolution respirometry (HRR).

DISCUSSION AND RESULTS

As a result, we can observe an increase in RS levels only in the heads of flies exposed to CUMS compared to the control group. There was also an increase in mitochondrial oxygen flux in the HRR, mainly between complexes I and II in the ORY groups alone and treated group when compared to the CUMS group, where there was a decrease in the respiratory rate and a possible mitochondrial compromise. The observed oxidative stress presumably is a contributing factor to the reduced survival rate in the CUMS group.

CONCLUSION

It is concluded that ORY had a beneficial effect on the oxidative stress caused by CUMS, and has the potential to prevent a possible breakdown of cellular energy, thus avoiding the triggering of depressive-like behavior in Drosophila melanogaster.

Keywords: Antioxidant, CUMS, Drosophila

Supported by: FAPERGS, CNPq and CAPES

08798 - Poster Session

GD.10 - Antinociceptive Effect of Schinus terebinthifolia Raddi. (Anacardiaceae) Leaf Lectin in Mice

Amanda de Oliveira Marinho¹, Leydianne Leite de Siqueira Patriota¹, Jéssica de Santana Brito¹, Abdênego Rodrigues da Silva¹, Suéllen Pedrosa da Silva¹, Alisson Macário de Oliveira¹, Patrícia Maria Guedes Paiva¹, Thiago Henrique Napoleão ¹

¹Departamento de Bioquímica, Universidade Federal de Pernambuco (Pernambuco, Brasil)

INTRODUCTION

Pain control remains a clinical challenge and there is a need to seek new therapeutic options for its relief. In this context, natural products stand out, including plant-derived lectins, which are proteins which have shown antinociceptive potential.

OBJECTIVES

This study aimed to evaluate the antinociceptive activity of the Schinus terebinthifolia leaf lectin (SteLL) through acute experimental models of peripheral and central nociception in mice.

MATERIALS AND METHODS

SteLL was isolated by chromatography of leaf extract (in 0.15 M NaCl) on chitin column. The animals were intraperitoneally treated with PBS (control), indomethacin (20 mg/kg), morphine (10 mg/kg) or SteLL (1, 5 and 10 mg/kg). To verify the peripheral antinociceptive effect, the writhing number was determined in the acetic acid-induced abdominal writhing test. In the formalin test, the time spent by each animal licking its paw was recorded during the first 5 minutes (first phase: neurogenic pain) and at the interval of 15 to 30 minutes (second phase: inflammatory pain), after the formalin administration. The involvement of lectin carbohydrate recognition domain (CRD) and the participation of opioid receptors in the antinociceptive effect were verified. Tail immersion test was performed to assess central antinociception. The latency period for tail removal was determined at 30, 60, 90 and 120 min after treatments.

DISCUSSION AND RESULTS

SteLL reduced acetic acid-induced writhing by 84%-100%. In the first phase of the formalin test, SteLL reduced paw licking time by 49%-51%. In the second phase, SteLL reduced paw licking time by 81%-83%. This antinociceptive effect was reversed by ovalbumin (indicating the possible involvement of CRD) and by naloxone (suggesting a modulation of opioid receptors). In the tail immersion test, SteLL reduced the thermal stimulus perception sensitivity even after two hours.

CONCLUSION

SteLL possess both peripheral and central analgesic actions. The peripheral effect involves the lectin CRD and is probably mediated by opioid receptors.

Keywords: Pain, Antinociception, Brazilian pepper tree

Supported by: FACEPE, CNPq and CAPES

08538 - Poster Session

GD.11 - Trypsin inhibitor from Crotalaria spectabilis leaf with anti-Leishmania activity

Patrícia Fernandes Ferreira¹, Érika Maria Gomes Ferreira Teixeira¹, Raquel Elisa Silva-López¹

¹Produtos Naturais, Fundação Oswaldo Cruz (Rio de Janeiro, Brasil)

INTRODUCTION

Crotalaria is a genus of Fabaceae family that includes about 500 species of herbaceous plants and shrubs found in tropics and subtropics. Crotalaria spectabilis, flowering plant, is native to Indian, China, and Southeast Asia. It is tolerant to drought and diseases, and are used as green manure, forages, preventing soil nematodes and in folk medicine.

OBJECTIVES

Thus, the purposes of this work was to isolate and characterize trypsin-like inhibitors from aqueous phosphate extract from fresh leaves of C. spectabilis (CS-P).

MATERIALS AND METHODS

Protein amount was determined by Bradford method and protein profile was evaluated by SDS-PAGE. Isolate and characterize inhibitors by affinity chromatography using trypsin-Sepharose and LC-MS.

DISCUSSION AND RESULTS

The purification was about 6.05-fold yielded 61% of trypsin-like inhibitors obtained from 3,2 mg of protein from CS-P, and this inhibitor was named as CSPI. SDS-PAGE analysis identified three proteins with approximately 32, 37 and 44 kDa. CSPI inhibited the activity of both trypsin and LSPIII, an extracellular serine protease from Leishmania amazonensis (LSPIII), about 67 and 75%, respectively, using Nα-p-Tosyl-L-arginine methyl ester as substrate. Trypsin inhibition by CSPI was the highest at 65°C, but after 24 and 48 h of incubation at 65 ºC, the inhibitor activity was completely abolished. CSPI maintained the activity until 55 days, when stored in freezer (-20°C), however, the extract CS-P preserved the protease inhibitory activity about 2 years, stored in similar conditions, possibly because the high content of flavonids. The main flavonoids identified were: quercetin 3-O-neohesperidoside (756.21130), tricin 7-diglucuronoside (682.53998), quercetin 3,4'-diglucoside (626.14832), and quercetin-3-O-alpha-L-rhamnopyranoside (756.21130). Other serine protease inhibitors were obtained from C. paulina and pallida seeds, and they had different biochemical characteristics of CSPI, isolated from leaves.

CONCLUSION

Furthermore, CSPI was the first plant polypeptide protease inhibitor that inhibited LSPIII, and studies from our group demonstrated that LSPIII inhibition induced the death of L. amazonensis promastigotes and amastigotes in vitro.

Keywords: trypsin inhibitor, , C. spectabilis, , L.amazonenis

Supported by: CNPq e FIOCRUZ

08413 - Poster Session

GD.12 - The extraction method affects the concentration of secundary metabolites present in Aloysia citriodora Palau extracts

Felipe Kreuz Machado ¹, Carla Maria Garlet de Pelegrin^1,1, Marlei Veiga dos Santos ^1,1, Nessana Dartora^1,1

¹Biochemistry, Universidade Federal da Fronteira Sul (Rio Grande do Sul, Brazil)

INTRODUCTION

A native plant Aloysia citriodora is considered a promising species for the development of new drugs, being very widespread in South American folk medicine.

OBJECTIVES

In this work, the leaves of A. citriodora were subjected to extraction with distilled water, simulating the popularly consumed tea, and hydrocolic, in order to structurally isolate and identify the main chemical constituents present in its leaves in different forms of extraction.

MATERIALS AND METHODS

With the high molecular mass separated from the low mass, the crude extracts were precipitated with refrigerated ethyl alcohol, resulting in ethanol-soluble fractions, containing the secondary metabolites, named AC-SBH for the hydroalcoholic extract and AC-SBA for the aqueous extract. AC-SBH and AC-SBA were then analyzed by the techniques of high performance liquid chromatography (HPLC) and mass spectrometry (ESI-MS).

DISCUSSION AND RESULTS

A total of 38 compounds were found in AC-SBH and 37 in AC-SBA, based on retention time (Rt), standards of mass fragmentation and ultraviolet spectrum, with data search in the current literature. The aqueous and hydroalcoholic extracts differed qualitatively and quantitatively, where it was observed that for AC-SBA the major component of chrysoeriol-7-diglucuronide, while for AC-SBH, the major components were verbascoside and chrysoeriol-7- diglucuronide. It could also be noted that the relationship between the peaks of the other components for both extractions is different, that is, the relative abundance of the compounds is different from each other, demonstrating that the extraction method affects the concentration of metabolites. It was found that most of the secondary metabolites of A. citriodora were extracted in greater quantity in the hydroalcoholic extraction (AC-SBH).

CONCLUSION

The data found here are in line with those found in previous research, but also legitimize new research aimed at different extraction methodologies for isolating compounds of interest in the species.

Keywords: biochemistry, bioactive compounds, structural characterization

08373 - Poster Session

GD.13 - Effect of Capsicum annuum var. annuum leaf extract on the development of Callosobruchus maculatus

Milena Bellei Chereme ¹, Sarah Rodrigues Ferreira², Layrana de Azevedo dos Santos ¹, Antônia Elenir Amâncio Oliveira², Rosana Rodrigues³, André Oliveira Carvalho¹, Valdirene Moreira Gomes¹

¹Laboratório de Fisiologia e Bioquímica de Microrganismos, Universidade Estadual do Norte Fluminense Darcy Ribeiro (Rio de Janeiro, Brazil), ²Laboratório de Química e Função de Proteínas e Peptídeos, Universidade Estadual do Norte Fluminense Darcy Ribeiro (Rio de Janeiro, Brazil), ³Laboratório de Melhoramento Genético Vegetal, Universidade Estadual do Norte Fluminense Darcy Ribeiro (Rio de Janeiro, Brazil)

INTRODUCTION

The increase in demand for the safe food productivity for humans and the environment has driven the search for new substances with the objective of controlling and preventing agricultural pests.

OBJECTIVES

The present work aims to evaluate the effect of fractions rich in peptides isolated from Capsicum annuum on the larval development of Callosobruchus maculatus.

MATERIALS AND METHODS

The leaves were extracted using a solution containing 60% methanol and dichloromethane in a 1:1 ratio. The hydromethanolic extract was used in biological tests and purification of peptides in reverse phase chromatography using C18 column and elution with linear gradient of acetonitrile. To test the effect on the development of C. maculatus larvae, artificial seeds of Vigna unguiculata cotyledon flour containing the C. annuum extract were used. Glucose, cholesterol, triglycerides, proteins, lipase and amylase were measured in 20-day-old larvae.

DISCUSSION AND RESULTS

The chromatographic profile of the extract showed 3 fractions eluting in 6%, 24.5% and 27% acetonitrile. The extract and the fractions presented only a single band, with a molecular mass around 5 kDa. The addition of the extract in the concentration of 0.5%, 1.0% and 2.0% in artificial seeds reduced the weight of the larvae by up to 65% and reduced the survival rate of the larvae in seeds, but had no effect of repellency for the laying of eggs. Biochemical parameters are indicative of a delay in development when compared to control larvae. The extract and one of the fractions isolated on HPLC showed inhibitory activity of Tenebrio molitor α-amylase, which may explain the toxicity of this compound to the insect's larvae. The extract and the fractions did not exhibit porcine trypsin and chymotrypsin inhibitory activity.

CONCLUSION

Other experiments will be carried out for biochemical characterization and elucidation of the mechanism of action of the obtained fractions.

Keywords: Capsicum annuum, insecticidal activity, peptides

Supported by: UENF; FAPERJ; CNPq

08644 - Poster Session

GD.14 - (PROJECT) Profile of Medicinal Plants with Antineoplastic Potential Activity in Santa Catarina Plateau.

Jaime Antônio Machado Farias ¹, Victoria Liandra Biava¹, Amanda Leite Bastos Pereira¹

¹Bioquímica e Biologia Molecular, Universidade do Estado de Santa Catarina (, Brazil)

INTRODUCTION

Medicinal plants are popularly known and used, mainly in traditional communities. This knowledge contributes to scientific community, because facilitates identification and classification of species, seeking evidence of their biological and pharmacological activities, for further use in prevention and treatment of diseases, including cancer. Malignant neoplasms are treated by a set of conventional therapies. However, they may be not capable or sufficient, or may lead to undesirable effects. The search for alternative or complementary methods has become increasingly present in oncological treatments, and this, together with popular knowledge about medicinal plants, led to the development of the present study.

OBJECTIVES

It aims to identify, first, through questionnaires, which medicinal plants with antineoplastic purposes are used by residents of Santa Catarina Plateau region.

MATERIALS AND METHODS

From the samples collected in the homes of the interviewees, the botanical and phytochemical composition of the classified plants will be identified. For that, three extracts will be prepared, following the following solvents: distilled water, ethanol and hydroethanolic solution, and through them will be performed phytochemistry analysis, mainly by high performance liquid chromatography (HPLC) assays. The search for a possible antitumor activity will be predicted by in silico tools such as the online PASS (Prediction of Activity Spectra for Substances) or Molsoft Drug-Likeness programs. Afterwards, the activity may be determined by in vitro and/or in vivo methods, using for example Ehrlich´s tumor cells.

DISCUSSION AND RESULTS

CONCLUSION

Therefore, medicinal regional plants, effective against cancer, will be identified, thus proving the benefits of their use, besides to provide news opportunities to regional further investigations.

Keywords: in silico, Medicinal plants, Acca sellowiana

08489 - Poster Session

GD.15 - Metabolomic Profile and Biological Properties of Extracts of Miconia Albicans from the North Coast of Bahia

Gabriel Teixeira Medrado ¹, Patricia Campos Santos², Elis dos Reis Requião², Renato Delmondez de Castro², Lourdes Cardoso de Souza Neta³, Paulo R. Ribeiro de Jesus^1,2,4, Luzimar Gonzaga Fernandez^1,2,4

¹Universidade Federal da Bahia, Programa de Pós-graduação Multicêntrico em Bioquímica e Biologia Molecular (Bahia, Brasil), ²Departamento de Bioquímica e Biofísica, UFBA, Laboratório de Bioquímica, Biotecnologia e Bioprodutos, Instituto de Ciências da Saúde (Bahia, Brasil), ³Departamento de Ciências Exatas e da Terra, Departamento de Ciências Exatas e da Terra (Bahia, Brasil), ⁴Instituto de Química, UFBA, Metabolomics Research Group, Instituto de Química (Bahia, Brasil)

INTRODUCTION

Brazil has one of the biggest biodiversity in the world, with a large collection of medicinal and economic plants that present pharmacological properties and it’s used for the treatment of diseases. This use is based on traditional knowledge, such as Miconia albicans (Canela-de-velho) are widely used by the North Coast of Bahia communities. Thus, it’s necessary to identify the pharmacological properties and their active principles for better therapeutic applications.

OBJECTIVES

In the present study, was to determine the metabolite profiling and evaluate the medicinal potential of Miconia albicans extract for antioxidant and antimicrobial activity.

MATERIALS AND METHODS

Leaves and stem of Canela-de-velho were collected in the Mata de São João-Bahia. Extracts were prepared by maceration in different organic solvents. The cellular metabolome was evaluated using 1H nuclear magnetic resonance (NMR). The determination of the antioxidant activity was 2,2-azinobis-3-ethyl-benzothiazolin-6-sulfonic acid radical scavenging method and total phenols concentration by the Folin-Ciocalteu. The antimicrobial activity was performed against Gram-positive and Gram-negative bacteria, likewise non-filamentous fungi through broth microdilution susceptibility test.

DISCUSSION AND RESULTS

Concerning the antioxidant activity and total phenols the extracts showed potential promising. The total phenols concentration the leaf ethanol extracts had a higher concentration (81.1 mgEGA.g-1) compared to the stem extract (73.7 mgEGA.g-1). The ethanol extracts of M. albicans antimicrobial activity weren’t efficient up to the maximum concentration tested against fungi, gram-positive and gram-negative bacteria. As for the metabolites present in the extracts, there is a strong indication of the presence of compounds such as myricitrin, mearnsetin, ursolic acid and oleanoic acid. But the NMR analysis results still in process.

CONCLUSION

M. albicans extracts present good levels of total phenols. However, their antimicrobial activity was not efficient and new concentrations should be tested. The results of the NMR are still being processed for further identification of biocompounds and correlation with the pharmacological properties and traditional use.

Keywords: Antimicrobial activity, Antioxidant activity, Ethnopharmacology

08568 - Poster Session

GD.16 - Metabolite profiling, phenolic compounds and antioxidante activity of ethanol extracts from Conocarpus erectus L. Var.

BRENDA ANTUNES DE ANDRADE SANTOS¹, RENATO DELONDEZ DE CASTRO^1,2, PATRÍCIA CAMPOS SANTOS^2,1, DANIELE TAKAHASHI BERNAL¹, MARTA BRUNO LOUREIRO¹, LUZIMAR GONZAGA FERNANDEZ^1,2, PAULO ROBERTO RIBEIRO³, LOURDES CARDOSO NETA⁴

¹Departamento de Bioquímica e Biofísica, Laboratório de bioquímica, biotecnologia e bioprodutos (Bahia, Brasil), ²Departamento de Ciências Exatas e da Terra, Rede Nordeste de Biotecnologia (Bahia, Brasil), ³Departamento de Química Orgânica, Instituto de Química (Bahia, Brasil), ⁴Departamento de Ciências Exatas e da Terra, Universidade Estadual da Bahia (Bahia, Brasil)

INTRODUCTION

Phenolic compounds have been related to the antioxidant capacity of vegetable due to different actions in the oxidative and anti-inflammatory processes. Conocarpus erectus L. Var. belongs to the combretaceae family, has a large amount of phenolic compounds and it is used by riverside communities to treat skin infections, anemia and headaches. Due to medicinal use, further studies are needed to identify the plant's bioactive compounds and phytotherapeutic properties.

OBJECTIVES

In the current study, the objective was to determine phenolic compounds, antioxidant activity and evaluate the metabolomic profile of ethanol extracts from C. erectus.

MATERIALS AND METHODS

Samples of the stem and leaf collected in the Pojuca River Mangrove, North Coast of Bahia, were dried at room temperature and ground before preparing the extracts by maceration in etanol. The extracts were submitted to rotaevaporation to acquire the crude extracts. Total phenols were quantified by the Folin-Ciocalteau method and antioxidant activity by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Metabolomic analyzes were performed using liquid chromatography coupled to a mass spectrometer.

DISCUSSION AND RESULTS

The leaf extract showed total phenol content of 66,55 mgEGA.g-1 and high antioxidant activity with EC50 of 18,74 μgmL-1 when compared to stem extract. By metabolomics, secondary metabolites were identified, such as catechin and rutin, which are phenolic compounds and antioxidants

CONCLUSION

Based on the results, it appears that the analyzed extracts have metabolites with possible phytotherapeutic activity. So, C. erectus is a promising species, however further studies are needed to identify the metabolites responsible for the properties that justify its use in traditional medicine.

Keywords: Antioxidant activity, Button mangrove, Metabolomics

Supported by: PIBIC

08324 - Poster Session

GD.17 - Effects of γ-oryzanol on the attenuation of oxidative stress markers in a chronic unpredictable mild stress model of Drosophila melanogaster

Mustafa Munir Mustafa Dahleh ¹, Stífani Machado Araujo¹, Vandreza Cardoso Bortolotto¹, Márcia Rósula Poetini¹, Luana Barreto Meichtry¹, Franciane Cabral Pinheiro¹, Elize Aparecida Santos Musachio¹, Franciéle Romero Machado¹, Marina Prigol¹

¹Department of Biochemistry, LaftamBio, Federal University of Pampa (Rio Grande do Sul, Brazil)

INTRODUCTION

Chronic unpredictable mild stress (CUMS) acts as one of the main factors related to increased oxidative stress, causing changes such as depression, among other neurobiological disorders. In this sense, the investigation of changes resulting from exposure to CUMS becomes important, and for this, the Drosophila melanogaster model stands out, which has high reproducibility, low cost, and good management, in addition to an good acceptance of antioxidant compounds capable of reducing the effects from oxidative stress, such as γ-oryzanol (ORY).

OBJECTIVES

The objective of this work was to evaluate the protective effect of ORY on oxidative stress markers in Drosophila melanogaster submitted to CUMS.

MATERIALS AND METHODS

The flies were divided into 4 groups: Control, CUMS, ORY (25 μM), and ORY (25 μM) + CUMS. For the CUMS model, flies were exposed to heat, cold, starvation and sleep deprivation, all for 10 days. To analyze the effects of oxidative stress, the head and body of the flies were separated, and the formation of reactive species (RS), lipid peroxidation via malondialdehyde (MDA), cell viability by reducing resazurin, and the activity of the enzyme superoxide dismutase (SOD) were evaluated.

DISCUSSION AND RESULTS

It was verified an increase of RS levels in the heads of flies exposed to CUMS, and ORY treatment reduced these levels. There was a significant increase in MDA levels in the heads of flies exposed to CUMS alone compared to the control. Cell viability decreased in the body of flies exposed to CUMS, while ORY treatment prevented this damage. It was also observed a reduction in SOD activity in the heads of flies from the CUMS group, while treatment with ORY prevented this reduction.

CONCLUSION

It is concluded that ORY has antioxidant potential, reducing oxidative markers, mainly in the flies' heads, reducing possible neurobiological disorders derived from CUMS.

Keywords: CUMS, depression, Drosophila melanogaster

Supported by: FAPERGS, CNPq and CAPES

08642 - Poster Session

GD.18 - Flavonoid agathisflavone reprograms microglia towards a neuroprotective inflammatory profile

Balbino Lino dos Santos ^1,2, Cleonice Creusa dos Santos¹, Karina Costa da Silva¹, Victor Diogenes Amaral da Silva^1,3, Maria de Fátima Dias Costa¹, Arthur Butt⁵, Jorge Mauricio David⁶, Silvia Lima Costa^1,3,4

¹Laboratory of Neurochemistry and Cellular Biology, Institute of Health Sciences, (Bahia, Brazil), ²College of Nursing, Federal University of Vale do São Francisco (Pernambuco, Brasil), ³INCT-EN, INCT for Excitotoxicity and Neuroprotection (Rio Grande do Sul, Brasil), ⁴INCT-TN, Instituto Nacional de Ciência e Tecnologia em Excitotoxicidade e Neuroproteção (Rio Grande do Sul, Brasil), ⁵School of Pharmacy and Biomedical Science, University of Portsmouth, United Kingdom (Portsmouth, United Kingdom), ⁶Departament of general and Inorganic Chemistry, Universidade Federal da Bahia (Bahia, Brasil)

INTRODUCTION

In the central nervous system, microglia orchestrate the inflammatory response to diverse insults, including neuroinflammation associated to neurodegenerative diseases (NDD).Microglia recognize damaged cells acquiring a pro-inflammatory cytotoxic profile that can exacerbate brain damage. However, considering microglia plasticity modulation of their inflammatory response to injury may also promote resolution stages of inflammation and tissue regeneration. Agathisflavone, a biflavonoid purified from Poincianella pyramidalis (Tul.) has demonstrated anti-inflammatory and neuroprotective properties in in vitro models of NDD.

OBJECTIVES

Here, we investigated the effects of agathisflavona directly in microglial cells submitted to inflammatory damage in view to elucidate mechanisms of neuroprotection associated to modulation of inflammatory response.

MATERIALS AND METHODS

Microglia were isolated from cortical primary cultures of newborn Wistar rats and were exposed to Escherichia choli lipopolysaccharide (LPS,10 ng/mL) and treated or not with agathisflavone (1-10 μM), for 24h. To investigate possible neuroprotective effects of agathisflavona treatment, differentiated PC12 neuronal cells were exposed to the microglia secretoma (MS) derived from cultures in each experimental condition.

DISCUSSION AND RESULTS

We observed that the inflammatory stimulus with LPS induced the microglia to assume an activated cellular state with pro-inflammatory profile characteristic (increased CD68), confirmed by phenotypic changes with more rounded or amoeboid cells. However, when treated with agathisflavone, microglia up-regulated expression of CD206 (anti-inflammatory) and down-regulated CD68 expression, as well presented mainly more branched-like phenotype, in addition to a reduction in the expression of inflammatory mediators IL-6, IL1-b, TNF, NRLP3 and chemokines CCL5 and CCL2, characterizing change to an anti-inflammatory state.Moreover, we observe the preservation of neurites and regulation in the expression of β-tubulin III and Caspase-3 in PC12 cells exposed to MS derived from agathisflavona and agathisflavona plus LPS treated cultures.

CONCLUSION

Together, these data reinforce the capacity of the flavonoid in reprograming microglia to neuroprotective anti-inflammatory profile standing out as a promising molecule for the treatment or prevention of neurodegenerative diseases.

Keywords: Flavonoids, Anti-inflammatory, Neuroprotection

Supported by: Capes, CNPq e FAPESB

08401 - Poster Session

GD.19 - PROJECT EVALUATION OF THE BIOLOGICAL ACTIVITY OF COMPOUNDS ISOLATED FROM Lepidium meyenii AND ITS SYNTHETIC DERIVATIVES

Fernanda Vidal Carvalho ¹, Paulo Ribeiro¹

¹Metabolomics Research Group, Universidade Federal da Bahia, Instituto de Química (, Brasil)

INTRODUCTION

Lepidium meyenii is a plant traditionally found in the Andean region of Peru that has several biological activities, such as immunomodulatory, antioxidant, and antitumor. Several metabolites have already been identified in L. meyenii roots and leaves, such as alkaloids, phenolic compounds, glucosinolates, macaenes, and macamides. However, to this date few studies have performed large-scale metabolite profiling of L. meyenii extracts and correlating it with its biological activities.

OBJECTIVES

To isolate bioactive compounds from the root of L. meyenii and test their antioxidant, antibacterial and cytotoxic activities.

MATERIALS AND METHODS

The compounds will be isolated from ethyl acetate extracts obtained from the plant root using high performance liquid chromatography (HPLC) and identified through nuclear magnetic resonance (NMR) analysis. The antioxidant activity will be determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method. The antimicrobial activity will be evaluated against Gram-positive and Gram-negative bacteria, as well as non-filamentous fungi through the broth microdilution method and the cytotoxicity will be evaluated against the rat glioma cell line (C6) and astrocytes. Also, macamides will be synthesized and their biological activities will be evaluated.

DISCUSSION AND RESULTS

To isolate and identify new compounds from L. meyenii and evaluate their antioxidant, antibacterial and cytotoxic potential. Therefore, the study could provide empirical support for the medicinal use of this plant, which directly implies benefits for society in the treatment and prevention of pathologies, also contribute to expanding scientific knowledge about the pharmacological potential of L. meyenii.

CONCLUSION

Keywords: Peruvian Maca, Metabolomics, Natural Products

08455 - Poster Session

GD.20 - Bioactivity of natural products from Rhizophora mangle L.: metabolomic profile, phenolic compounds, and antioxidant capacity.

PATRÍCIA CAMPOS SANTOS ^1,2,3, BRENDA ANTUNES DE ANDRADE SANTOS¹, RENATO DELONDEZ DE CASTRO^1,2, MARTA BRUNO LOUREIRO¹, PAULO ROBERTO RIBEIRO^3,1, LUZIMAR GONZAGA FERNANDEZ^1,2,3

¹Bioquímica e Biofísica, Laboratório de Bioquímica, Biotecnologia e Bioprodutos, Instituto de Ciências da Saúde, Universidade Federal da Bahia (Bahia, Brasil), ²Bioquímica e Biofísica, Programa de Doutorado em Biotecnologia – Rede Nordeste de Biotecnologia (RENORBIO), Universidade Federal da Bahia (Bahia, Brasil), ³Química orgânica, Metabolomics Research Group, Instituto de Química, Universidade Federal da Bahia (Bahia, Brasil)

INTRODUCTION

Phenolic compounds are secondary metabolites present in plant species and have important antioxidant properties that neutralize free radicals, favoring the cure of several diseases, such as cardiovascular diseases and infections. Rhizophora mangle, popularly known as red mangrove, of the family Rhizophoraceae, is an obligatory species of mangroves, widely used by traditional and riverside communities as herbal medicines for the treatment of skin infections, but its chemical composition and pharmacological properties is still little known.

OBJECTIVES

This study aimed was to investigate the metabolomic profile of ethanol extracts (CEERm) from the leaf, stem and root of R. mangle, as well as their phenolic compounds and in vitro antioxidant activities.

MATERIALS AND METHODS

Samples of R. mangle were collected in the Pojuca river mangrove, Mata de São João-Bahia, Brazil. CEERm were obtained by maceration in ethanol at room temperature. After maceration was performed, the ethanol was removed under reduced pressure at 40 °C. Liquid chromatography–mass spectrometry was applied for metabolomic analysis, and data processing and compounds identification were performed by Metlin and MetaboAnalyst (V5.0) web-platforms. Total phenolic compounds were quantified by the Folin-Ciocalteau method and antioxidant capacity was assessed by 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay.

DISCUSSION AND RESULTS

The root extract had the highest concentration of phenolic compounds (71.16 mgEGA.g ^-1 ) while the leaf extract had the lowest concentration (46.35 mgEGA.g ^-1 ). The IC50 of the CEERm antioxidant activity ranged from 9.05 (stem) to 31.39 μgmL ^-1 (leaf). The innovative approach used allowed the identification of likely candidate bioactive compounds in the extracts, such as tannins, flavonoids and gallic acid that showed a positive correlation with the antioxidant capacity in different botanical parts of R. mangle.

CONCLUSION

These results also indicate that the R. mangle have antioxidant potential and source of new natural products, probably due to the presence of the phenolic compounds identified in this study.

Keywords: Metabolomics, Secondary metabolites, Rhizophoraceae

Supported by: FAPESB, CNPq, Capes, Prefeitura de Mata de São João, UFBA.

08340 - Poster Session

GD.21 - Trypsin Kunitz-type inhibitor from Cajanus cajan leaves

Erika Maria Gomes Ferreira Teixeira ¹, Erika Teixeira¹, Dario Kalume², Raquel Elisa Silva-López¹

¹Natural Products, Institut of Pharmaceuticals Technology (Rio de Janeiro, Brazil), ²Interdisciplinary Laboratory of Medical Research, Oswaldo Cruz Institute (Rio de Janeiro, Brasil)

INTRODUCTION

Cajanus cajan is a legume widely consumed in Asia, Africa and Americas due to the high protein content in their seeds. It is employed a medicinal plant for the treatment of different pathologies.

OBJECTIVES

Therefore, the aims of this work were isolate and characterizate biochemically and structurally inhibitors of trypsin-like serine proteases obtained from aqueous phosphate extract of C. cajan fresh leaves (CC-P).

MATERIALS AND METHODS

The trypsin-type protease inhibitor was isolated from C. cajan leaf extract CC-P by affinity chromatography with immobilized trypsin in agarose matrix. Protein content was determined by Bradford method and protein profile evaluated by SDS-PAGE.

DISCUSSION AND RESULTS

This inhibitor was isolated with purification of 3.64-fold yielded 54% of trypsin-like inhibitors and was denominated TIC, that had a molecular weight about 15 kDa by SDS-PAGE analysis. TIC was assayed against trypsin, papain, pepsin and collagenase, and inhibited only the trypsin activity. TIC has higher affinity for trypsin (Ki = 1.617 μM) than chymotrypsin (Ki = 6.46013 μM) and was a competitive inhibitor. The primary structure of TIC was studied by mass spectrometry and showed homology with a kunitz type trypsin inhibitor when used NCBI database. The inhibitory activities of TIC was maintained even after the following experimental situations: 24 h treatment at 70 °C, 1h treatments with buffers of different pH values and increasing concentrations of β-mercaptoethanol. However, the activity of TIC was affected at different levels in the presence of oxidizing agents, such as hydrogen peroxide and dimethylsulfoxide.

CONCLUSION

These results highlight that C. cajan leaves are sources of protease inhibitor with important structural stability.

Keywords: C. cajan, trypsin inhibitor, Kunitz-type inhibitor

Supported by: Capes and FIOTEC

08705 - Poster Session

GD.22 - Potentially cytoprotective compounds in the establishment of diabetes-related pancreatic beta cell functions

Diana Baense de Abreu Araújo ¹⁵

¹Biologia celular, Universidade Federal do Rio de Janeiro, Campus Duque de Caxias (, Duque de Caxias)

INTRODUCTION

Chronic hyperglycemia is a characteristic of diabetes. It is known that beta cells play a very important role in glucose homeostasis. Protecting and restoring beta cell function can help minimize the damage from diabetes mellitus. The use of chemical compounds that mimic situations found in the pathogenesis of diabetes, such as glyoxal and methylglyoxal, can help to clarify the various deleterious molecular mechanisms that operate in the cell. Methylglyoxal (MG) is a reactive carbonyl species found at high levels in uncontrolled diabetic patients and may influence the production of reactive oxygen species (ROS). Thus, the use of potentially cytoprotective compounds can have a beneficial effect in situations of high MG levels. Glycyrrhiza glabra, popularly known as licorice, has as one of its main components glycyrrhizin, which belongs to the class of saponins, and, among other properties, it has shown an antioxidant and protective effect in different contexts of cytotoxicity. Rutin is a flavonoid with antioxidant and anti-inflammatory properties, found in different fruits and vegetables, and which has the ability to scavenge reactive species such as hydroxyl, superoxide and peroxyl radicals, thus being a potent attenuator of the harmful effects of MG

OBJECTIVES

the aim of this work was to evaluate in vitro the cytotoxicity of methylglyoxal in RINm5F insulin-producing cells, and to test the cytoprotective effect of potentially antioxidant compounds

MATERIALS AND METHODS

MTT reduction method (cell viability assessment) and intracellular ROS detection by H2-DCFH-DA. Our results indicate that glycyrrhizin partially inhibits MG-induced cytotoxicity and rutin showed a cytoprotective effect, causing an increase in cell viability in cells exposed to MG

DISCUSSION AND RESULTS

The results obtained through the DCFH-DA intracellular ROS detection test showed that MG increases ROS production. Treatments with glycyrrhizin or rutin decrease ROS production when cells were co-incubated with MG.

CONCLUSION

Keywords: diabetes, ROS, methylglyoxal

Supported by: FAPERJ

08494 - Poster Session

GD.23 - Genistein attenuates amyloid-beta-induced cognitive impairment in rats by modulation of hippocampal synaptotoxicity and hyperphosphorylation of Tau

Fernanda dos Santos Petry ¹, Juliana Bender Hoppe¹, Caroline Peres Klein¹, Bernardo Gindri dos Santos¹, Régis Mateus Hözer¹, Felippo Bifi¹, Cristiane Matté^1,2, Christianne Gazzana Salbego^1,2, Vera Maria Treis Trindade^1,2

¹Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Universidade Federal do Rio Grande do Sul (Rio Grande do Sul, Brasil), ²Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul (Rio Grande do Sul, Brasil)

INTRODUCTION

Alzheimer’s disease is a progressive neurodegenerative disorder characterized by extracellular accumulation of amyloid-beta (Aβ) peptide, which induces synaptic dysfunction, alteration of intracellular signaling pathways, hyperphosphorylation of the Tau protein, and cognitive impairment. Genistein, one of the major isoflavones present in soy and soy products, has been shown to modulate some of the pathogenic events associated with the neurodegeneration process. However, its underlying mechanisms remain to be clarified.

OBJECTIVES

The objectives of the present study were to evaluate the ability of genistein to protect against Aβ1-42-induced cognitive impairment in rats and to elucidate some of the possible mechanisms involved in its neuroprotective effects in the hippocampus.

MATERIALS AND METHODS

Adult male Wistar rats (aged 90 days) received bilateral intracerebroventricular infusions of Aβ1-42 (2 nmol) and genistein 10 mg/kg orally for 10 days. Behavioral analyses were initiated 24h after completion of drug treatment and performed sequentially on days 11-15. On the day following completion of the behavioral tasks (day 16), the hippocampi were collected for neurochemical analyses.

DISCUSSION AND RESULTS

The Aβ-infused animals showed significant impairment of memory, which was accompanied by the following neurochemical alterations in the hippocampus: decreased levels of the synaptic proteins synaptophysin and postsynaptic density protein 95 (PSD-95), hyperphosphorylation of Tau with increased activation of glycogen synthase kinase-3β (GSK-3β) and c-Jun N-terminal kinase (JNK), and inactivation of extracellular signal-regulated kinase 1/2 (ERK). Treatment with genistein improved Aβ-induced cognitive impairment by attenuation of synaptotoxicity, hyperphosphorylation of Tau, and inactivation of ERK.

CONCLUSION

These findings provide further evidence of the neuroprotective effect of genistein in an in vivo model of Aβ toxicity and, importantly, extend the current knowledge concerning the mechanisms associated with the neuroprotective effects of this compound in the hippocampus.

Keywords: Alzheimer’s disease, genistein, neuroprotection

Supported by: CNPq and INCT (EN 465671/2014-4)/CNPq

08372 - Poster Session

GE.01 - The gene families of Glutathione peroxidase and Glutathione-S-transferase from Ricinus communis L.: phylogenetic analysis and in silico characterization

Neide da Hora Conceição ^1,2, Amanda Átila A. S. Reis^1,2, Renato Delmondez de Castro¹, Paulo Roberto Ribeiro ³, Luzimar Gonzaga Fernandez^1,2,3

¹Bioquímica e Biofísica, Laboratório de Bioquímica, Biotecnologia e Bioprodutos - UFBA (Bahia, Brasil), ²Bioquímica e Biofísica, Programa Multicêntrico de Pós-graduação em Bioquímica e Biologia Molecular (Bahia, Brasil), ³Química orgânica, Metabolomics Research Group (Instituto de Química/UFBA) (Bahia, Brasil)

INTRODUCTION

Ricinus communis L. (Castor) uses several defense mechanisms against different types of abiotic stresses. Antioxidant enzymes such as glutathione peroxidase (GPX) and glutathione-S-transferase (GST) are responsible for maintaining normal levels of reactive oxygen species (ROS), reducing and/or preventing the cascade of damage caused by oxidative stress in plants. The gene families of these enzymes have several members that are present in various parts of the cell.

OBJECTIVES

The objective of this study was to characterize the glutathione peroxidase (RcGPX) and glutathione-S-transferase (RcGST) gene family of R. communis in comparison with other seven species (Arabidopsis thaliana, Oryza sativa, Zea mays, Populus trichocarpa, Solanum lycopersicum, Glycine max and Sorghum bicolor).

MATERIALS AND METHODS

The sequences were retrieved in the Plant Comparative Genomics of the Joint Genome Institute-Phytozome, after searching the conserved domains in Simple Modular Architecture Research Tool (SMART). Alignment was performed using the MUltiple Sequence Comparison by Log-Expectation (MUSCLE) and phylogenetic analysis using the Molecular Evolutionary Genetics Analysis Software (MEGAX). Finally, cell location prediction was done using subCELLular LOcalization predictor (CELLO) and the verification of conserved motifs in Multiple Em for Motif Elicitation (MEME).

DISCUSSION AND RESULTS

It was found that GPX and GST have several members in their gene families, varied amino acid sequences, which are very conserved in GPX, but not in GST. RcGPX and RcGST share common ancestors with Glycine max and Arabidopsis thaliana, respectively. RcGPX and RcGST consist of multiple isoenzymes with distinct subcellular locations that exhibit different tissue-specific expression patterns, with greater predominance in the cytoplasm. RcGPX has several conserved motifs shared with enzymes from other species, the same not occurring with RcGST, even considering the existing phylogenetic relationships.

CONCLUSION

It was concluded that these enzymes are important in regulating the response to different types of environmental stresses in R. communis and other plant species.

Keywords: Antioxidant enzymes, Castor, Gene analysis.

Supported by: CNPQ; FAPESB; PMBqBM/UFBA and UFBA.

08279 - Poster Session

GE.02 - Responses of different wheat genotypes to Herbaspirillum seropedicae inoculation by proteomic analysis

Adeline Neiverth-Chagas^1,2, Souza , S. G. H.³, Zingali , R.B.⁴, Rezende H. K.², Fernandes, B. V.², Klein, M.K.², Klein, N. K.², Vendruscolo, E. C. G.², Marise Fonseca dos Santos ¹

¹PMBqBM, Universidade Federal do Paraná (Parana, Brasil), ²Departamento de Biociências, Universidade Federal do Paraná (Parana, Brasil), ³Programa de Pós-Graduação em Biotecnologia Aplicada à Agricultura, Universidade Paranaense (Parana, Brasil), ⁴Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

Inoculation with plant growth-promoting bacteria (PGPB) has proven to be a promising strategy in supplying the plant's nitrogen and other important nutrients. Herbaspirillum seropedicae SmR1, a PGPB, promoted different responses in Brazilian wheat (Triticum aestivum var. Lini) cultivars (CD104 and CD120) under in vitro and greenhouse conditions. The aim of this work was to investigate the protein profile in wheat leaves (CD104 and CD120) when inoculated with H. seropedicae to understand the plant physiology and photosynthetic system. ZADOK 2.1 wheat plants were obtained by growth in MS medium without (SN) and with (CN) nitrogen source, without sucrose. After 24h, some tubes of SN medium received bacterial inoculum (SNI). The tubes were kept in a culture room at 24 °C and 14h light /dark for 20 days. Leaves were harvested to obtain protein extract for proteomics based on protein fractionation by SDS-PAGE followed by LC-MS/MS - Q-TOF type. Intact plants were used to obtain nitrogen index (NBI) and chlorophyll (CHL), flavonoids and anthocyanins, chlorophyll fluorescence. As results, CD120 in SNI medium promoted increase in NBI and CHL, reduced anthocyanins, chlorophyll fluorescence in photochemical and non-photochemical extinction. Only one protein was uniquely found in SNI condition, being related to protection of photosystem II. CD 104 in SNI condition presented 5 photosynthesis proteins and others related to hypersensitivity and diseases resistance. Both cultivars in SN presented antioxidant proteins, although chlorophyll fluorescence was not severely affected. The plant-bacteria interaction is genotype dependent and H. seropedicae was able to partially meet the N requirement for the CD120 genotype and promote defense and tolerance responses for the CD104 genotype.

Keywords: Wheat, proteomic analysis, photosynthetic system

Supported by: FAPPR and CAPES

08503 - Poster Session

GE.03 - PROJECT - Secondary metabolites from Zea mays L. corn silk extracts

Natan Rodrigues Santana Da Hora ¹, Paulo Roberto Ribeiro de Jesus¹

¹DEPARTAMENTO DE QUÍMICA ORGÂNICA, UNIVERSIDADE FEDERAL DA BAHIA (BAHIA, BRASIL)

INTRODUCTION

Corn silk is a part of the female flower of Zea mays L., which is well-known in the traditional Chinese medicine for its properties. Several reports have described its pharmacological activities, but few studies have performed a global chemical profiling of the extracts, especially correlating it with the activities. Thus, corn silk extracts may be of great interest as a potential source of bioactive compounds.

OBJECTIVES

The objective of this project is to isolate bioactive compounds from the corn silk ethyl acetate extract.

MATERIALS AND METHODS

The antioxidant activity will be performed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assat, whereas antimicrobial activity will be evaluated by the broth microdilution method against Gram-positive and Gram-negative bacteria, and against non-filamentous fungi. The antitumor potential of the extracts will be evaluated against the murine glioma (C6) cell line. Extracts fractionation will be performed using several chromatographic techniques, such as column chromatography, preparative thin layer chromatography and High-efficiency liquid chromatography (HPLC-UV).

DISCUSSION AND RESULTS

The isolation of new bioactive metabolites will provide an important leads into the understanding of the corn silk medicinal properties in terms of its secondary metabolites. In addition, it will be possible to correlate the metabolites of extracts with antioxidant, cytotoxic and antimicrobial activities.

CONCLUSION

Therefore, providing important clues for the discovery of new active compounds and possibly for the development of new pharmaceutical products. In addition, it will contribute to the sustainable use of Brazilian biodiversity, since the use of medicinal plants with well-established protocols and quality control procedures can expand the range of therapeutic options and thus contribute to health improvement.

Keywords: Corn silk, Metabolomics, Murine glioma C6

Supported by: FAPESB

08584 - Poster Session

GE.04 - Differential proteomic analysis of wheat root (Triticum aestivum var. lini) cv. CD104 in the absence of nitrogen and the presence of the bacteria Herbaspirillum seropedicae Smr1.

Alessandro Augusto Gonzalez ¹, Chagas, A. N.¹, Fernandes, B. V.², Rezende, H. K², Klein, M. K.², Klein, N. K.², Vendruscolo, E. C. G.², Santos, M. F.²

¹PMBqBM, Universidade Federal do Paraná (Parana, Brasil), ²Departamento de Biociência, Universidade Federal do Paraná (Parana, Brasil)

Herbaspirillum seropedicae is a diazotrophic, endophytic bacterium that has shown good results in the interaction with wheat plants (Triticum aestivum L.). This study aimed to investigate proteins present in the roots of wheat cultivar CD104 when inoculated with the bacterium in the absence of nitrogen and carbon source to understand the plant-bacteria interaction. Wherefore, wheat seeds were sterilized, pre-germinated, and transferred to glass test tubes containing liquid MS culture media without sucrose to obtain in vitro cultivation under treatments: with nitrogen source (CN), without nitrogen (SN), and without nitrogen with the bacterium inoculum (SNI) added to SN tubes after 24 hours of seed transfer to tubes of SN and CN All tubes were kept in a culture room at 25 °C and a photoperiod of 14 h of light for 20 days. The roots were taken to obtain protein extracts for shotgun proteomic analysis. Proteins, 1069, were identified and other information on metabolic pathways was obtained from identified proteins in each treatment. The RUBISCO protein and others as beta-1,3-glucanase and chitinases observed in SNI suggest the activity of carbon fixation, hypersensitivity responses, and resistance to diseases in plants promoted by the bacteria. A total of 211 exclusive proteins of H. seropedicae were identified in SNI treatment. Proteins were related to synthesis activity, energy and carbohydrate metabolism, and biological nitrogen fixation activity. Proteins related to antioxidant metabolism, as glutathione S-transferase, were verified in the roots of the SN treatment. The results suggest that wheat roots in the absence of nitrogen were responded to this condition and the presence, decreased responses to stress in the absence of nitrogen, and some pieces of evidence of biological nitrogen fixation in the root.

Keywords: Shotgun proteomics, wheat roots, plant growth promoting bacteria

Supported by: FAPPR

08645 - Poster Session

GE.05 - Investigation of antiglioma activity from Aloysia virgata (Ruíz & Pavón) Juss

Manoela Fernanda Schuster ¹, Nessana Dartora¹, Lauren Lúcia Zamin¹

¹Programa de Pós Graduação em Ambiente e Tecnologias Sustentáveis, Universidade Federal da Fronteira Sul (Rio Grande do Sul, Brasil)

INTRODUCTION

Aloysia virgata is a medicinal plant belonging to the Verbenaceae family, widespread in South America, and its tea is used to treat symptoms of diseases related to the digestive system.

OBJECTIVES

MATERIALS AND METHODS

In this work, the leaves of A. virgata were submitted to two types of extraction, aqueous (AQ), simulating the popularly consumed tea, and hydroalcoholic (ET), which generally extracts the maximum amount of compounds a plant. Both extracts obtained were fractionated by liquid-liquid partition, with the following solvents in increasing order of polarity, chloroform, ethyl acetate and butanol for ET; and ethyl acetate and butanol for AQ. The partition process then yielded 4 fractions for ET, named CE (chloroform ethanolic fraction), ACE (ethyl acetate ethanolic fraction), BE (butanolic ethanolic fraction) and AE (aqueous ethanolic fraction); and 3 fractions for AQ, ACA (aqueous ethyl acetate fraction), BA (aqueous butanolic fraction) and AA (aqueous aqueous fraction). The extracts and fractions were then used in the treatment of glioblastoma cells, initially at three concentrations: 0.01, 0.05 and 0.1%. All treatments were performed in triplicate, for 24, 48 and 72 hours in a 96-well plate, with 5,000 cells per well, using two glioma strains, C6 and U251. Cell viability was measured using the MTT test.

DISCUSSION AND RESULTS

The results obtained so far demonstrate that both strains showed sensitivity to treatments with CE, ACA and ACE, at concentrations of 0.05 and 0.1%, in 48 and 72 hours, reducing cell viability by at least 50%, compared to the control. The other fractions, as well as AQ and ET, did not show a satisfactory decrease. Although these are preliminary results, they are unprecedented when using the plant in glioma cells. Thus, more studies are being conducted to prove the effectiveness of A. virgata compounds in reducing the growth of this type of cells.

CONCLUSION

Keywords: Anti-tumor activity, bioactive compounds, glioblastoma

08924 - Poster Session

GE.06 - Integrated production of high-value aromatic alcohols directly from lignocellulosic biomass

Robson Tramontina ¹, James L. Galman², Timothy D.H. Bugg³, Nicholas J. Turner², Rosana Goldbeck¹, Neil Dixon², Fabio Squina⁴

¹Departamento de Engenharia de Alimentos, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas (Campinas, SP, Brasil), ²Manchester Institute of Biotechnology (MIB), University of Manchester (, UK), ³Department of Chemistry, University of Warwick (Coventry CV4 7AL, United Kingdom), ⁴Program of Technological and Environmental Processes, Universidade de Sorocaba (aposo Tavares Road, km 92.5 - Vila Artura, SP, Brasil)

INTRODUCTION

Sustainable production of fine chemicals from renewable plant biomass offers an excellent alternative to the continued use of finite geological oil reserves for fine chemistry purposes. However, in order to compete with current petrochemical refinery processes, alternative biorefinery processes must overcome significant costs and productivity barriers. The production of high-value aromatic alcohols directly from lignocellulosic biomass is an attractive alternative to add value in biorefineries worldwide.

OBJECTIVES

Herein, we demonstrate the biocatalytic production of the versatile chemical building block, coniferol, directly from lignocellulosic biomass.

MATERIALS AND METHODS

Following the biocatalytic treatment of lignocellulose to release and convert ferulic acid with feruloyl esterase (XynZ), carboxylic acid reductase (CAR) and aldo- keto reductase (AKR).

DISCUSSION AND RESULTS

This whole-cell catalytic cascade not only achieved the equivalent release of ferulic acid from lignocellulose compared to alkaline hydrolysis but also displayed efficient conversion of ferulic acid to coniferol.

CONCLUSION

This system represents a consolidated biodegradation-biotransformation strategy for the production of high-value fine chemicals from waste plant biomass, offering the potential to minimize environmental waste and add value to agro-industrial residues.

Keywords: Lignocellulose, biocatalysis, Coniferol

Supported by: FAPESP (2016/07926-4)

08576 - Poster Session

GE.07 - Identification and quantification of flavonoids in CD150 wheat inoculated with Azospirillum brasilense

Hércules Tancredo Moreira ¹, Flavia Bordignon Hendges¹, Gabrielle Caroline Peiter¹, Adriana Fiorini Rosado¹, Isac George Rosset¹, Fabio Rogério Rosado¹

¹Programa de Pós-Graduação em Bioquímica e Biologia Molecular , Universidade Federal do Paraná - Setor Palotina (Paraná, Brazil), ²Departamento de Engenharia e Exatas, Universidade Federal do Paraná - Setor Palotina (Paraná, Brazil), ³Departamento de Biociências, Universidade Federal do Paraná (Paraná, Brazil)

INTRODUCTION

Several plant organs produce flavonoids that are secondary water-soluble phenolic compounds, whose presence is related to external stimuli, with antioxidant and regulatory functions for plant processes. A bias in the study of flavonoids refers to the role of biocommunication between bacteria and plants such as wheat.

OBJECTIVES

The objective of this work was to determine the flavonoids produced in wheat roots inoculated with native rhizobacteria of biotechnological potential, to identify flavonoids that could increases nitrogen fixation through plant-bacteria communication.

MATERIALS AND METHODS

The in vitro experiment consisted of four treatments and five replications of CD 150 wheat plant, inoculated with Azospirillum brasilense, fed with solutions with or without nitrogen addition. After twenty-one days, the weight and length of roots and leaves were measured. The roots and stems were evaluated, by scanning electron microscopy, to verify the conducting vessels of wheat inoculated and not inoculated with the Azospirillum brasilense bacterium. The extracts were subjected to flavonoid identification by high performance liquid chromatography.

DISCUSSION AND RESULTS

Preliminary data demonstrated a higher concentration of total flavonoids in treatments performed with nitrogen in the presence of Azospirillum brasilense. The extracts showed the presence of three main types of flavonoids (isoflavone, quercetin and kaempferol).

CONCLUSION

The role of these flavonoids in rhizosphere communication, strengthening the idea of chemotaxis between plant and microorganism and their biological nitrogen fixation ability.

Keywords: Bacteria, Flavonoids, Wheat

08985 - Poster Session

GE.08 - Biomass and essential oil content of basil (Ocimum basilicum L.) cultivated in semiarid region.

Bárbara Almeida Dutra ¹, Messulan Rodrigues Meira¹, Maria Caroline Rizério Costa¹, Rute Caires Fonseca¹, Gilberto das Neves Nascimento¹, Mateus Sousa Porto¹, Marcondes dos Santos Nascimento¹, Esaul Lucas Oliveira¹, Crislene Viana Silva¹, Simone Andrade Gualberto¹, Janaína Silva Freitas¹

¹Department of Exact and Natural Sciences, State University of Southwest Bahia (Bahia, Brasil)

INTRODUCTION

Basil is an herbaceous species of Asian origin, used as a condiment and medicinal plant. The production of this species has been the subject of much research around the world given the production of essential oil that arouses commercial interest and has pharmacological properties. The production of the species to obtain essential oils in semiarid regions is challenging for family farming.

OBJECTIVES

Therefore, the objective was to study the production of essential oil of Ocimum basilicum L. under protected cultivation and in full sun.

MATERIALS AND METHODS

The plants were cultivated at UESB, in the city of Itapetinga-BA, in 15cm high containers in a greenhouse and in the field for 75 days, individually watered. After harvest, they were weighed on an analytical balance to determine fresh matter, stored in kraft paper, and conducted for hydrodistillation for two hours. The essential oil was collected and weighed on an analytical balance. The material resulting from the extraction was carried to an oven at 60°C to determine the dry matter. Essential oil content was calculated based on dry matter and data expressed as an average.

DISCUSSION AND RESULTS

The two cultivation systems showed differences in biomass. Plants grown in a greenhouse recorded 129 g of fresh mass and 29 g of dry matter. Plants grown in full sun obtained 107g of fresh biomass and 23g of dry matter, respectively. Consequently, the essential oil content was also higher in protected cultivation, with 0.6% in the greenhouse and 0.4% in the field.

CONCLUSION

The greater production of biomass and essential oil in protected cultivation can be explained by the protection of individuals against herbivory as well as better absorption of water and nutrients by reducing evapotranspiration. However, to obtain biomass and essential oils from basil in semiarid regions, cultivation under protection from photosynthetically active irradiation at 50% is suggested.

Keywords: basil, biomass, essential oil

Supported by: FAPESP, CNPq and CAPES

08424 - Poster Session

GE.09 - In silico characterization of catalase gene family from Ricinus communis L.

Maria Cecília Oliveira dos Santos Silva ^1,2, Neide Hora Conceição ^1,2, Renato Delmondez de Castro¹, Luzimar Gonzaga Fernandez ^1,2

¹Departamento de Bioquímica e Biofísica, Laboratório de Bioquímica Biotecnologia e Bioprodutos Instituto Ciências Saúde, Universidade Federal Bahia (BA, Brasil), ²Departamento de Bioquímica e Biofísica, Programa Multicêntrico de Pós Graduação em Bioquímica e Biologia Molecular, Instituto Ciências Saúde, Universidade Federal Bahia (BA, Brasil)

INTRODUCTION

Ricinus communis (Castor) is an oilseed with industrial and social importance in Brazil and in different parts of the world. Environmental factores can lead to an increase in reatives oxygen species (ROS) and affects metabolic pathways that determined the germination process of it seeds, promoting the oxidative stress. The catalase reaction involves the decomposition of two molecules of hydrogen peroxide (H₂O₂) to water (H₂O) and oxygen (O₂).

OBJECTIVES

The objective of this study was to characterize the catalase genes from Ricinus communis L. (RcCAT) compared to genes of 10 other plant species.

MATERIALS AND METHODS

CAT sequences were retrieved from the Plant Comparative Genomics do Joint Genome Institute (Phytozome) after searching for conserved domains in Simple Modular Architecture Research Tool (SMART). The alignment was performed by MUltiple Sequence Comparison by Log-Expectation (MUSCLE) and the phylogenetic analysis were made in Software Molecular Evolutionary Genetics Analysis (MEGAX). Lastly, cell sublocalization prediction was done using web server for protein Subcellular Localization Predictive System (CELLO).

DISCUSSION AND RESULTS

The CAT protein was show to have well conserved residues (LA(E)F) in comparison with the other species analyzed. All RcCAT contained a catalase core domain as well as an immune response catalase domain. According to phylogenetic analyses, these CAT genes were grouped in clades that suggest the integration of the CAT enzyme subfamilies. Phylogenetic relationships showed that there is a common ancestor between RcCAT and MeCAT (CAT of Manihot esculenta).The prediction of subcellular localization indicated that the candidate sequences they mostly owned peroxisomal localization.

CONCLUSION

It was concluded that the CAT is a multigenic enzyme and has a variation in its number of genes according to each species. They are isoenzymes involved in various functions such as growth, development and stress tolerance with a fundamental role in the ROS response in plants.

Keywords: Antioxidant enzymes, Castor bean, Phylogeny.

Supported by: CNPq; FAPESB and UFBA.

08092 - Poster Session

GE.10 - A minimal pathway for the regeneration of redox cofactors

Michele Partipilo¹, Eleanor Jane Ewins ¹, Jacopo Frallicciardi¹, Tom Robinson², Bert Poolman¹, Dirk Jan Slotboom¹

¹Department of Biochemistry, Groningen Institute of Biomolecular Sciences & Biotechnology, University of Groningen (Nijenborgh 4, 9747 AG Groningen, The Netherlands), ²Department of Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces (Am Mühlenberg 1, 14476 Potsdam, Germany)

Metabolism is an intricate network of biochemical pathways that make a cell out-of-equilibrium, but only a limited group of fundamental molecules have been conserved under evolutionary pressure to carry out specific functions.1The nicotinamide adenine dinucleotides NAD(H) and NADP(H) belong to this category of hub metabolites,2involved mostly in the transfer of reducing equivalents between reactants. An ambitious challenge of building a synthetic cell via bottom-up methodologies requires a metabolic component, thus ensuring the constant availability of nicotinamide cofactors.Here, we constructed a synthetic pathway in which both the redox cofactors involved in cell catabolism and anabolism, respectively NAD+ and NADP+, are continuously reduced and oxidized. This is mediated by the catalysis of the purified enzymes formate dehydrogenase and soluble transhydrogenase. Regulation between the two oxidative states is given by the concentrations of enzymes and substrates required by the specific reactions. The action of formate dehydrogenase leads to NADH accumulation. This allows the reduction of NADP+ over time by soluble transhydrogenase, which simultaneously restores the required NAD+ for the first reaction. Consequently, the formed NADPH becomes available for further redox enzymes that could drive NADP+ regeneration. The coupled reactions are triggered by a membrane permeable redox donor, formic acid. This makes the cofactor regeneration feasible confined in vitro in phospholipid vesicle compartments, as well as in solution. We also investigated the behaviour of our coupled redox system across different compartment scales, using LUVs and GUVs. Developing such coupled redox reactions will provide the necessary energy in terms of reducing equivalents in order to increase the achievable metabolic complexity inside the synthetic cell-like systems. [1] Schmidt, S., et al., Trends Biochem. Sci. 28, 336–341 (2003). [2] Pfeiffer, T., et al., PLoS Biol. 3, 1269–1275 (2005). Acknowledgments: We thank Yi Yang for the provision of the template vector pRDNA3.1-hygro-cyto-iNap1 and Marco Fraaije for assistance and discussion on SthA properties. M.P. and D.J.S. are supported by NWO Gravitation program (Building a Synthetic Cell) grant 024.003.019. B.P. is supported by ERC Advanced Grant (ABCvolume) grant #670578. E.J.E and D.J.S. are supported by Dutch Ministry for Education, Culture and Science (OCW), Bonus Incentive Scheme.

Keywords: Redox cofactors, metabolic pathways, bottom-up approach

08533 - Poster Session

HA.01 - PROJECT: EVALUATION OF THE FUNCTIONAL ROLE OF miRNA IN SIGNALING PATHWAY MODULATION ASSOCIATED WITH THE DEVELOPMENT AND DEGREE OF SEVERITY OF DIABETES MELLITUS TYPE II: IN VIVO AND IN SILICO STUDY.

Victor de Barro Serrano Neves ¹, Simone Garcia Macambira^1,2, Natália Tavares Machado³, Sara Nunes Araújo³

¹Biofísica e bioquímica, Universidade Federal da Bahia (BA, Brasil), ²LETI, Fundação Gonçalo Muniz (BA, Brasil), ³LAIPHE, Fundação Gonçalo Muniz (BA, Brasil)

INTRODUCTION

Diabetes mellitus (DM) is a chronic disease resulting from defects in insulin production, secretion, or signaling. DM affects about 400 million individuals worldwide. It is estimated that the diagnosis is given around 7 years after the onset of the disease and usually with associated comorbidities. In a previous study, miRNA Let-7a, Let-7b, 106b, 93, and 17 had greater sensitivity and specificity for prediabetes and were differentially modulated in the analyzed transcriptome. Hypothesis: The miRNAs Let-7a, Let-7b, 106b, 93, and 17 interfere with glucose metabolism, being involved in several intracellular pathways related to insulin resistance (IR) and the destruction of pancreatic β-cells

OBJECTIVES

Objective: To investigate the functional role of Let-7a, Let-7b, 106b, 93, and 17 miRNAs in glucose metabolism by validating the expression pattern of miRNAs in a cohort of healthy, pre-diabetic, and diabetic individuals.

MATERIALS AND METHODS

Methodology: A prospective cohort study will be carried out with healthy, pre-diabetic, and diabetic individuals, followed by CEDEBA between 2021 to 2025. The patients' blood will be collected for extraction of total RNA and miRNA microarray and Elisa for cytokines. The validation of miRNA targets will proceed in vitro with HSkMC cells transfected with miRNA targets and Western Blotting will be executed for evaluation of the transcription of genes involved in the insulin signaling pathway.

DISCUSSION AND RESULTS

It is expected to detect the miR and target genes responsible for DM.

CONCLUSION

Keywords: Predictive Analysis, Transcriptome, Insulin Resistance

Supported by: FAPESB

08709 - Poster Session

HA.02 - Search for new therapeutic targets for Chagas disease: functional prediction and analysis of metabolic pathways

Raissa Lima ¹, Manuela da Silva^1,2

¹Programa de Pós Graduação em Biologia Computacional e Sistemas, Instituto Oswaldo Cruz (Rio de Janeiro, Brazil), ²Instituto de Biodiversidade e Sustentabilidade (NUPEM), Universidade Federal Do Rio de Janeiro (Macaé, Brazil)

INTRODUCTION

Chagas disease is a social and economic burden even for specialized clinical centers and in 2019, 9,490 deaths were reported worldwide. The treatment of the acute phase is recommended benznidazole (Bnz) and nifurtimox, both highly toxic, and treatment interruption is common. It is necessary to find new targets for the formulation of new drugs in order to improve therapeutic adherence.

OBJECTIVES

The objective is, through the use of different programs, to perform the functional prediction of proteins with unknown function described in the Trypanosoma cruzi proteome (CL Brener strain), in order to find new protein targets.

MATERIALS AND METHODS

For functional prediction, the following programs and databases were used: Pfam, String, Psort, Smart, CDD, Prosite, TritryDB, HMMER, Uniprot, BlastP (NR) and MHOLline (comparative modeling with Modeller vs 10.0). From the data obtained in the MHOLline program, we used the SCOP and SCOPe databases. For the analysis of metabolic pathways we consulted the MetaCyc, Trypanocyc, KEGG and Reactome databases.

DISCUSSION AND RESULTS

From the proteome deposited at the NCBI (GCA_000209065.1) proteins with unknown function were separated, totaling 11,171. These sequences were submitted to MHOLline, which built a 3D model for 1,421 proteins, from which 43 models were selected according to the identity and coverage value (>35% and >70% respectively). Based on the use of the aforementioned programs for functional prediction, the function was predicted for 17 proteins, among the functions are: Adenylate Kinase, Ankyrin repeat, Uridyltransferase, Putative RNA-binding, AcylCoa binding, Ubiqutin-fold, O-acetyl-ADP -ribose, Tetracopeptide, Transcription factor IIB C-Terminal, Mitochondrial membrane anchored protein and RAS-related protein.

CONCLUSION

Among the analyzed metabolic pathways, we saw that the protein Adenylate Kinase, if inhibited, could lead to impairment of the adenosine ribonucleotide pathways of new biosynthesis and purine and pyrimidine metabolism, with direct impacts on the production of the ADP molecule, used as an energy currency within of the cells.

Keywords: Trypanosoma cruzi, treatment, functional prediction

Supported by: Capes and CNPq

08952 - Poster Session

HA.03 - Modification of PPARy-coregulator’s recruitment by Ser273 phosphorylation

Marieli Mariano Gonçalves Dias ^1,2, Fernanda Aparecida Heleno Batista¹, Thais Helena Tittanegro¹, Ana Carolina Migliorini Figueira^1,2

¹Brazilian Center for Research in Energy and Materials (CNPEM), Brazilian Biosciences National Laboratory (LNBio) (Campinas, SP, Brazil), ²Graduate Program in Functional and Molecular Biology, Institute of Biology, State University of Campinas (Campinas, SP, Brazil )

The nuclear receptor PPARg is the master regulator of adipogenesis, lipid, and glucose metabolism. The phosphorylation of adipose tissue subtype of PPARy at Ser273 has been linked to the dysregulation of a subset of specific genes that induce insulin resistance. However, the molecular mechanisms that drive this change in gene expression are unclear. In this work, we aim to investigate how this phosphorylation may disturb the interaction between PPARg and some coregulator proteins as a new mechanism that leads to insulin re-sistance. Through cellular and in vitro assays, we show that PPARg phosphor-ylation inhibition increased the activation of the receptor. Thus, this blocking increases the recruitment of PGC1-a and TIF2 coactivators, whilst decreases the interaction with SMRT and NCoR corepressors. Moreover, we investigate whether the CDK5, responsible for Ser273 phosphorylation could also disturb the PPARg-corregulator’s balance. Finding that CDK5 presence decreases the PPARg interaction with PGC1-a, TIF2, and NCoR, while increases the cou-pling of SMRT. Together these results indicate that the insulin resistance as-sociated with PPARg phosphorylation is linked to a differential coregulators recruitment, which may promote dysregulation in gene expression.

Keywords: PPARg, protein-protein interaction, phosphorylation

Supported by: This work was supported by the CAPES: 8887.373113/2019-00; FAPESP: 19/10274-7; and CNPEM

08461 - Poster Session

HA.04 - Peptides reduce cell viability of glioblastoma cells

Mariany Lopes da Costa Folly ¹, João Eustáquio Antunes^1,1, Raquel Tognon Ribeiro^1,1,1, Michelle Bueno Moura Pereira Antunes¹

¹Programa Multicêntrico Bioquímica e Biologia Molecular, Universidade Federal de Juiz de Fora (Minas Gerais, Brasil), ²Departamento de Farmácia, Universidade Federal de Juiz de Fora (Minas Gerais, Brasil)

INTRODUCTION

Glioblastoma multiforme (GBM) is a spontaneously occurring central nervous system tumor that is highly aggressive and invasive. Two proteins seem to be important for the development of glioblastomas, the Focal Adhesion Kinase (FAK) and the αB-crystallin (CryAB). Previous work has shown that the interaction between FAK/CryAB in cardiomyocytes cells is essential for cell survival and the reduction of this interaction promotes apoptosis. Through a series of biochemical approaches, the peptides involved in this interaction were identified.

OBJECTIVES

propose that the association of FAK with CryAB contributes to the maintenance of cell viability in glioblastoma cells.

MATERIALS AND METHODS

This study was performed in glioblastoma cells (U87-MG). We performed western blotting assays to confirm the expression of both proteins in these tumor cells. We realized co-immunoprecipitation assays to verify the interaction of the proteins FAK/CryAB. We performed MTT assays to verify the viability of the cells after treatment with the peptides previously identified. These peptides were synthesized by company AminoTech Research and Development

DISCUSSION AND RESULTS

We validated the expression of both proteins in the glioblastoma cells and we showed that these proteins also interact in these tumor cells. The treatment of the cells with the peptide FP01 (25μM), derived from the α-Crystalline domain (CryAB85-94) reduced in around 35% of cell viability and the treatment with the peptide FP02 (25μM), derived from the FAT domain (FAK921-930) reduced in around 30% of cell viabilty. Futhermore, we performed immunoprecipitation assays after the treatment of cells with the peptides, in order to verify the decrease in the interaction among the proteins. We also extract proteins from the cell lysate to perform western blotting assays and verify proteins important to the apoptosis process. Both experiments are in progress.

CONCLUSION

These previous results propose that the association of FAK with CryAB contributes to the maintenance of cell viability in glioblastoma cells.

Keywords: FAK, αB-Crystalline, glioblastoma cells

Supported by: CNPq

08586 - Poster Session

HA.05 - Investigation of anti-apoptotic modulators in the course of infection of macrophages by L. (L.) amazonensis.

Thayane Motta Fagundes ¹, Luiz Dione Barbosa de Melo¹, Michelle de Oliveira Chain¹

¹Bioquímica, Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (RJ, Brazil)

INTRODUCTION

Tropical infections such as leishmaniasis are caused by intracellular protozoa of the genus Leishmania, and American Tegumentary Leishmaniasis (ATL) has L. (L) amazonenis as its main etiological agent in the New World. In a pathological state, programmed cell death (apoptosis) pathways should be activated in infected cells, either autocrine or mediated by cytotoxic immune cells, playing a key role in resolving pathogen-induced infections. However, previous results from the group and the literature demonstrate that trypanosomatids can activate pathways promoting a pro-survival state by also inhibiting apoptosis of infected cells. Leishmania spp. and T. cruzi are able to subvert apoptosis, keeping cells alive long enough for infection and intracellular proliferation of new parasites, in a safe niche.

OBJECTIVES

The objective of this project is to investigate in cells infected by L. (L) amazonensis the expression/activation of anti-apoptotic genes related to proliferation and cell cycle progression, mainly IAPs (Inhibitors of Apoptosis Proteins) such as XIAP and Survivin.

MATERIALS AND METHODS

As a tool for the functional assays of host-parasite interaction in infection, the Raw 264.7 strain of murine macrophages and human macrophages differentiated from the monocyte lineage THP-1 will be used. Infected strains will be investigated by flow cytometry to determine changes in mitochondrial membrane potential and an evaluation of protein complex formation by coimmunoprecipitation and mass spectrometry will occur.

DISCUSSION AND RESULTS

Previous studies by the group through RNAseq and other functional approaches revealed an active participation of these proteins in apoptosis resistance events during infection.

CONCLUSION

It is predicted that a greater knowledge about the survival strategies that Leishmania (L) amazonensis uses to protect itself and circumvent the attempts of elimination made by the host should contribute to a better understanding of the mechanisms of subversion of cell death in the pathophysiology of leishmaniasis.

Keywords: Leishmania, apoptosis, parasite-host interaction

Supported by: FAPERJ

08707 - Poster Session

HA.06 - Molecular aspects of the SARS-CoV-2 variants and their virulence

Carolina Corrêa Giron^1,2, Fernando Luís Barroso da Silva¹, Aatto Laaksonen^3,4,5,6

¹Departamento de Ciências Biomoleculares, Faculdade de Ciências Farmacêuticas de Ribeirão Preto (SP, Brasil), ²Hospital de Clínicas, Universidade Federal do Triângulo Mineiro (MG, Brasil), ³Department of Materials and Environmental Chemistry, Stockholm University (, Sweden), ⁴State Key Laboratory of Materials-Oriented and Chemical Engineering, Nanjing Tech University (, PR China), ⁵Centre of Advanced Research in Bionanoconjugates and Biopolymers, Petru Poni Institute of Macromolecular Chemistry, Aleea Grigore Ghica-Voda (, Romania), ⁶Department of Engineering Sciences and Mathematics, Luleå University of Technology (, Sweden)

INTRODUCTION

The SARS-CoV-2 betacoronavirus has emerged as a new threat to global health, demanding its molecular mechanisms to be fully elucidated. The natural evolutive surge of new variants is imposing additional challenges. A molecular understanding is fundamental to the design of efficient diagnostic tools, scientifically verified therapeutical and prevention options.

OBJECTIVES

To quantify the stability of spike trimeric structures and their binding affinities with the human cell receptor for different viral strains at several pH regimes.

MATERIALS AND METHODS

By means of biophysical computational methods (Constant-pH Monte Carlo simulations), the stability of several spike (S) trimeric structures at different conformational states and pH conditions was investigated, as well as the free energy of interactions between the receptor binding domain (RBD) and the Angiotensin Converting Enzyme 2 (ACE2) for the most common variants of concern (Alfa, Beta, Gamma, Kappa, Epsilon, and Iota). Furthermore, the electrostatic epitopes were mapped by the PROCEEDpKa method.

DISCUSSION AND RESULTS

Analyses of the results allowed the observation of the conformational state's influence on the stability of the S protein. The stability is directly dependent on the viral S protein sequence for each strain. For instance, SARS-CoV-1 S protein has a probability of ca. 67% for the open state. Conversely, the wildtype SARS-CoV-2 S protein has a smaller probability, which correlates with a lower infection rate. In contrast, the Beta variant has virtually 100% of probability for the S protein to be at the open state. This indicates a higher possibility of infection. The binding affinities of some variants, including Gamma, show an increased attraction to human cells.

CONCLUSION

SARS-CoV-2 has developed a mechanism that favors the open state, combining more stability with more affinity for ACE2. These results contribute to a better understanding of molecular physiopathology and viral evolution, which consequently helps in the fight against the disease.

Keywords: binding affinities, conformational states, mutations

Supported by: FAPESP; CNPq

08415 - Poster Session

HA.07 - Project association of long non-coding RNA MALAT1 polymorphism and clinical course of patients with Chagas disease

Gilson Aquino Cavalcante ², João Paulo da Silva Liberalino², Ana Paula Andrade Meireles², Valéria Duarte de Almeida², Thales Allyrio Araujo de Medeiros Fernandes²

²Faculdade de Ciências da Saúde - FACS/UERN, Universidade do Estado do Rio Grande do Norte (Rio Grande do Norte, Brasil)

INTRODUCTION

Chagas Disease (CD) is a disease caused by Trypanosoma cruzi and presents variable clinical manifestations. Most patients will not develop the clinically significant disease, but about 30-40% of those infected may develop the defined clinical forms (cardiac, digestive, or cardiodigestive), usually 10-30 years after the initial infection. Several factors are associated with this variability, and among these are the genetic characteristics of the host. Some studies have shown that lncRNAs are involved in the development of cardiovascular disease, MALAT1 stands out. However, to date, no study has evaluated the role of polymorphisms in the gene of this long non-coding RNA with the clinical evolution of patients with CD.

OBJECTIVES

Therefore, this project aims to analyze the association between lncRNA MALAT1 polymorphisms with clinical forms, death and stroke risk scores, and other clinical, electrophysiological, and echographic features of patients with chronic CD.

MATERIALS AND METHODS

A cross-sectional study will be conducted in which peripheral blood samples will be collected from patients with CD to extract DNA, then genotyping of MALAT1 polymorphism by restriction enzyme digestion technique will be performed. Then, the clinical forms of the patients will be determined, and imaging, echocardiogram, and electrocardiographic data will be evaluated by consulting and analyzing the patients' medical records. Finally, the data will be tabulated and evaluated by appropriate statistical analysis.

DISCUSSION AND RESULTS

We expect that the inheritance of certain alleles of the MALAT1 gene is associated with a higher frequency of the clinical cardiac form and higher risk of death and stroke, as well as with echographic and electrophysiological changes in CD patients.

CONCLUSION

This project aims to identify new genetic biomarkers for the prognosis of patients by better understanding the pathophysiological mechanisms that act in the clinical evolution of patients with DC.

Keywords: Chagas Disease, Polymorphism, Long non-coding RNA

Supported by: CAPES

08299 - Poster Session

HA.08 - The rs2910164 polymorphism in miR-146A is not associated with clinical and echocardiographic manifestations and scores in chronic Chagas disease

João Paulo da Silva Liberalino¹, Dayane Carla Costa Paiva Dantas^1,2, Gilson Aquino Cavalcante^1,2, Valéria Duarte Almeida¹, Micássio Fernandes Andrade^1,2, Cleber Mesquita Andrade¹, Nickson Melo Morais¹, Wogelsanger Oliveira Pereira^1,2, Thales Allyrio A. de M. Fernandes ^1,2

¹Ciências Biomédicas, Universidade do Estado do Rio Grande do Norte (Rio Grande do Norte, Brasil), ²SBBq, Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular (Rio Grande do Norte, Brasil)

INTRODUCTION

Functional genetic polymorphisms involved in the immune response may modulate the clinical variability in Chagas disease (CD). Recent studies showed that miR-146A is regulated differentially in multiple conditions, including CD, in which it appears upregulated. The C allele in rs2910164 single nucleotide polymorphism (SNP) in miR-146A gene seems to reduce its production and efficiency and is associated with clinical outcomes in multiple patient populations. However, this association has not been studied in CD patients yet.

OBJECTIVES

This study aimed to evaluate the association between rs2910164 SNP and clinical and echocardiographic manifestations and scores in CD patients.

MATERIALS AND METHODS

We conducted a cross-sectional study with 186 chronic CD patients, of which 93 were male and 93 females, with a mean age of 47,78 ± 11,57 years. They were genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. The clinical forms, the risk of sudden death (Rassi score), the score of cardioembolic ischemic stroke, and echocardiographic parameters were obtained by accessing the patients' medical records.

DISCUSSION AND RESULTS

We observed that 104 patients presented GG genotype, 69 GC, and 13 CC. The expected and observed genotype frequencies obeyed Hardy-Weinberg equilibrium. The statistical analyses conducted showed no significant associations between the allelic and genotypic frequencies and the clinical forms of chronic CD (cardiac, digestive, or cardiodigestive), the risk of sudden death, the score of cardioembolic ischemic stroke nor echocardiographic parameters (such as left atrial and diastolic, systolic left ventricular diameters; and left ventricular ejection function).

CONCLUSION

We conclude that there is no association between rs2910164 polymorphism in miR-146A and clinical and echocardiographic manifestations and scores in CD patients. Therefore, it seems to be more valuable to investigate the influence of other polymorphisms in miR-146A or other molecules in regards to CD, although these results could be reinforced by studies with bigger population samples and more robust methodologies.

Keywords: Chagas Disease, MicroRNAs, Single Nucleotide Polymorphism

Supported by: CAPES; CNPq

08802 - Poster Session

HA.09 - PROJECT: Nanoparticle activity on modulation of in vitro aggregation and neurotoxicity of alpha-synuclein protein

Marcos Eduardo Braga Pacheco ¹, Carolina Alvares da Cunha de Azeredo Braga¹

¹Núcleo Multidisciplinar de Pesquisa em Biologia UFRJ - Xerém, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Parkinson's disease is the second most common neurodegenerative disorder in people over 60 years of age. Among its main symptoms are: bradykinesia, tremor, rigidity and postural instability, in addition to non-motor clinical events such as depression, anxiety, memory loss and olfactory defects. Its main pathological characteristics are the death of dopaminergic neurons in the substantia nigra and the presence of protein inclusions called Lewy bodies, whose main constituent is the alpha-synuclein protein. By appearing in this region, aggregates of the protein in question, and certain species of this aggregation are toxic to neuronal cells, Parkinson's disease is considered an amyloidosis. In this way, there is a need to find molecules and compounds that are capable of acting as neuroprotectors, inhibiting the toxicity of such species, and thus preventing cell death and maintenance of its morphology and viability.

OBJECTIVES

In this work, the effect of iron oxide and cobalt ferrite nanoparticles isolated and coated with organic matter will be evaluated, and their roles as neuroprotectors in human dopaminergic neuroblastoma cells in the presence of alpha-synuclein protein.

MATERIALS AND METHODS

biophysical and biochemical analyses, cytotoxicity assays, flow cytometry and evaluation of cell morphology. In addition to analyzing the role of these nanoparticles in modulating the aggregation kinetics of the alpha-synuclein protein, and its ability to break up previously formed aggregates.

DISCUSSION AND RESULTS

Define the minimum nanoparticle concentration necessary to inhibit aggregation, evaluate the role of the nanoparticle produced by this protocol in inhibiting the toxicity of oligomers and amyloid fibers formed by alpha synuclein, observe whether there is a nuroprotective role of nanoparticles when pre-treating the neuroblastoma cells.

CONCLUSION

Keywords: Alpha synuclein, Nanoparticle, Parkinson

08482 - Poster Session

HA.10 - Genetic diversity of the structural region of hepatitis C virus genotypes 1 and 3 in patients with chronic hepatitis C and its clinical and laboratory implications

Maurício Tavares de Melo ¹, Juliene Antonio Ramos¹, Bianca Catarina Azeredo Cabral², Cristiane Alves Villela-Nogueira³, Rosane Silva², Luísa Hoffmann¹

¹Laboratório de Genética Molecular, Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (Rio de Janeiro, Brasil), ²Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ³Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Around 71 million people are infected with hepatitis C virus (HCV) worldwide and chronic infection is associated with complications such as cirrhosis and hepatocellular carcinoma. HCV has a single-stranded RNA genome of approximately 9.6 kb, which is translated into structural and non-structural proteins. So far, 8 HCV genotypes and 90 subtypes have been described, with genotypes 1 and 3 being the most prevalent in Brazil. In addition, each infected individual has a unique viral set (quasispecies). Due to high genetic diversity, there is still no vaccine for HCV. Therefore, the structural region has been studied as a target in the development of vaccines considering its importance in the viral infectious cycle.

OBJECTIVES

The aim of this work is to comparatively analyze the genetic diversity of the structural region (core, E1 and E2) of HCV genotypes 1a, 1b and 3a in patients with chronic hepatitis C with different clinical and laboratory characteristics.

MATERIALS AND METHODS

From a cohort of twenty patients with chronic hepatitis C, HCV genome was sequenced using a customized panel (Ampliseq On-Demand) (ThermoFisher). After preparing libraries using barcodes, massively parallel sequencing was performed to generate reads on the Ion Proton platform (ThermoFisher). Reads were imported and analyzed using CLC Genomics Workbench v.8.5 (Qiagen) software. Reads were mapped with their respective HCV genome sequence references and the differences were evaluated.

DISCUSSION AND RESULTS

For genotype 3a we observed that the E2 region had the highest percentage of amino acid changes (18.73%) and when there was a change of amino acids in the E2-HVR1 region, there was no change in E2-HVR2, and vice versa. Same analysis are being performed with HCV-1a and -1b.

CONCLUSION

Studying the structural region of HCV is essential to better comprehension of genotypes differences and to assist in the development of effective vaccines.

Keywords: bioinformatics, biotechnology, hepatitis C

Supported by: IFRJ, CNPq, FAPERJ, CAPES

08701 - Poster Session

HA.11 - PROJECT - Evaluation of the wound healing activity of a combination of Artemisia vulgaris and Persea cordata, using the moxatherapy technique,in mice

ROBERTA DICH SIQUEIRA ²

²1Multicenter Program in Postgraduate in Biochemistry and Molecular Biology, Santa Catarina State University (SANTA CATARINA, BRAZIL)

INTRODUCTION

Moxatherapy is a Chinese traditional medicine technique where Artemisia vulgaris burning is used to relieve pain and assist healing process. Data using animal models report that this technique may increase cell proliferation rate, speeding healing, stimulating growth of collagen fibers, possibly mediated by the induction of TGF-β, an important molecule during tissue repair. Medicinal plants may be used during wound healing. One example is Persea cordata, which has been commonly used by the population of Santa Catarina Plateau in the treatment of wounds. This species has its significant wound healing effect possibly due the presence of molecules such as N-Hexane, dichloromethane, ethyl acetate and butanol.

OBJECTIVES

This study aims to associate Persea cordata during moxibustion, to evaluate its healing activity, applied into experimental wounds in mice.

MATERIALS AND METHODS

The experimental protocol will be submitted to local CEUA. Animals will be acquired and kept in standard conditions and are going to be submitted to a cutaneous excisions method. Mice will be divided in experimental groups: 1) No treatment, 2) Moxa (Artemisia) 3) Moxa (Artemisia plus Persea); being treated during fourteen days. Important parameters of tissue repair investigation will be investigated, such as histological analysis, tissues level of hydroxyproline and leucocyte migration (myeloperoxidase and N-acetyl-glucosaminidase enzymatic activities).

DISCUSSION AND RESULTS

The moxibustion therapy using Artemisia vulgaris burning plus Persea cordata, may be effective to increase the rate of wound healing of cutaneous wound in mice, being an excellent new therapeutic option.

CONCLUSION

These results, thus may provide development and innovation using regional medicinal plants.

Keywords: Moxatherapy, wound healing, Persea cordata

08205 - Poster Session

HA.12 - Effect of Uncaria tomentosa aqueous extract on the response to lipotoxicity- induced oxidative stress in cultured skeletal muscle cells

Bruna Letícia de Freitas ¹, Jeniffer Farias dos Santos¹, Myrian Thiago Pruschinski Fernandes¹, Carla Roberta de Oliveira Carvalho², Viviane Abreu Nunes¹

¹Department of Biotechnology, School of Arts, Sciences and Humanities, University of Sao Paulo (SP, Brazil), ²Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo (SP, Brazil)

INTRODUCTION

Type 2 diabetes mellitus (T2DM) is a chronic non-communicable disease with an increasing number of cases in recent years worldwide. It is characterized by chronic hyperglycemia, associated with dyslipidemia, which corresponds to the increase in the concentrations of triglycerides and fatty acids, in tissues such as skeletal muscle. The accumulation of intramuscular fatty acids is related to cell death, redox imbalance and oxidative stress. The use of herbal medicines such as Uncaria tomentosa (Ut) has been proposed as an auxiliary treatment for patients with T2DM, considering the possibility of overlapping conventional therapy

OBJECTIVES

Based on this, the aim of this work was to evaluate the effect of the Ut aqueous extract on these events, induced by the free fatty acid (FFA) palmitate or palmitic acid (PA), in skeletal myoblasts of C2C12 lineage

MATERIALS AND METHODS

Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), at 37 ^o C humidified atmosphere and 5% CO₂. The treatments were performed by the incubation of cells with PA, in different concentrations, in the presence or absence of 250 μg/ml Ut aqueous extract, for 2, 6 or 24 h.

DISCUSSION AND RESULTS

Which resulted in an increase of, at least, 50% in cell viability compared to control. After these periods, oxidative stress was evaluated by fluorescence spectroscopy, using the fluorescent marker DCFDA. The treatment of cells with Ut aqueous extract, for 6 h, followed by exposure to 500 μM PA, resulted in decrease of 38% in the ROS formation, in relation to those incubated with PA only.

CONCLUSION

In summary, the Ut aqueous extract promoted a raise in cell viability, reduced cell death and attenuated ROS formation in cultures incubated with 500 μM PA.

Keywords: Uncaria tomentosa, palmitic acid, oxidative stress

Supported by: FAPESP

08331 - Poster Session

HA.13 - Chrysin restores memory deficit in hypothyroidism mice through the neurotrophinergic system

Vandreza Cardoso Bortolotto ¹, Stífani Machado Araujo¹, Franciane Cabral Pinheiro¹, Márcia Rósula Poetini¹, Luana Barreto Meichtry¹, Mariana G. Fronza², Silvana Peterini Boeira¹, Lucielli Savegnago², Marina Prigol¹

¹Departamento de Bioquímica, Universidade Federal do Pampa (RS, Brasil), ²Departamento de Neurobiotecnologia, Universidade Federal de Pelotas (RS, Brasil)

INTRODUCTION

Hypothyroidism is associated with neuropsychiatric disorders, causing a memory deficit, this cognitive decline is closely related to Alzheimer's disease. The present study evaluated the effects of flavonoid chrysin, a natural compound that demonstrates several beneficial neuronal effects.

OBJECTIVES

In this context, this study investigated the protective effect of chrysin in cognitive impairment in hypothyroid female mice by exploring neuroplasticity.

MATERIALS AND METHODS

Hypothyroidism was induced by continuous exposure to 0.1% methimazole (MTZ) in drinking water for 31 days. Exposure to MTZ was associated with low plasma levels of thyroid hormones T3 and T4 compared with the control group. Subsequently, euthyroid and MTZ-induced hypothyroid mice were intragastrically administered vehicle or chrysin (20 mg/kg) once a day for 28 consecutive days. After treatments, the following behavioral assessments were performed: open-field test (OFT) and morris water maze (MWM). Then, the levels of neurotrophins (BDNF and NGF) in the hippocampus and prefrontal cortex were measured and also tested the affinity of chrysin with neurotrophinergic receptors through molecular docking.

DISCUSSION AND RESULTS

Hypothyroid mice showed a deficit of spatial memory and chrysin treatment reversed this deficit. It also reduced the levels of neurotrophins in both cerebral structures in the hypothyroid mice, meanwhile, the chrysin treatment was able to increased the levels of BDNF in hipocamppus and NGF in both structures. Additionally, molecular docking analysis showed that chrysin potentially binds to the active site of the TrkA, TrkB, and p75NTR receptors.

CONCLUSION

Together, these findings provide a comprehensive effect of chrysin that was able to reverse the behavioral and neurochemical changes associated with memory deficit induced by hypothyroidism, by modulating synaptic plasticity in the neurotrophinergic system.

Keywords: Flavonoid, Memory loss, Neurotrophins

Supported by: FAPERGS, CNPq and CAPES

08564 - Poster Session

HA.14 - Can aging increase the peripheral neuropathy and comorbidities associated with paclitaxel treatment?

Jaini Janke Paltian¹, Angélica Schiavom dos Reis¹, Cristiane Luchese¹, Ethel Antunes Wilhelm¹

¹Programa de Pós-graduação em Bioquímica e Bioprospecção, Universidade Federal de Pelotas (Rio Grande do Sul, Brazil)

INTRODUCTION

The pace of population aging around the world is increasing dramatically. Due to cell aging and lack of hormonal protection characteristic of old age, this group is more susceptible to developing cancer. Chemotherapy drugs are widely used anti-cancer treatments. However, chemotherapeutic agents such as paclitaxel (PTX) can cause peripheral neuropathy and promote emotional and cognitive impairment in cancer survivors. A longer life brings with it opportunities, but the extent of these opportunities and contributions depends heavily on one factor: health.

OBJECTIVES

Thus, elucidating the role of aging in cancer pain and associated comorbidities becomes increasingly relevant. The present study aimed to investigate the impact of aging on PTX-induced peripheral neuropathy and associated comorbidities in BALB/c mice.

MATERIALS AND METHODS

Male mice were divided into four groups: YOUNG (2 months); YOUNG+PTX; OLD (20 months); OLD+PTX. PTX was administered intraperitoneally (i.p.), at a dose of 2 mg/kg, once a day, for 3 consecutive days, or 0.9% saline solution (10 mL/kg, i.p.). Nociceptive response was evaluated on days 4, 8, 11, and 14 of the experimental protocol. To investigate the effects of PTX/aging on comorbidities associated with peripheral neuropathy, the cognitive impairment (on days 6-7, and 12-13), and the anxiety-like behavior (on days 5 and 15) were evaluated.

DISCUSSION AND RESULTS

Our results demonstrated that PTX treatment caused mechanical and thermal hypersensitivity in both young and old mice. Furthermore, both young and old mice that received PTX also demonstrated emotional and cognitive impairments. Importantly, we revealed that older animals had greater mechanical and thermal hypersensitivity and greater cognitive impairment than animals of the YOUNG+PTX group.

CONCLUSION

Based on this evidence, it can be inferred that aging contributes to the exacerbation of neuropathic pain and comorbidities related to PTX treatment. These results demonstrate the importance of searching for new therapeutic approaches that consider the specificities of these patients.

Keywords: paclitaxel, peripheral neuropathy, aging

Supported by: CNPq, CAPES and L’ORÉAL-UNESCO-ABC for Women in Science

08759 - Poster Session

HA.15 - Recognition of Trypanosoma cruzi epitopes by IgG of benznidazole treated Chagas disease patients

Luis Antonio Rodriguez Carnero ¹, Jhonatas Sirino Monteiro¹, João Carlos Setubal¹, Ester Cerdeira Sabino², Edécio Cunha Neto^3,4,5, Ricardo José Giordano^1,5

¹Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP 05508, Brazil (Sao Paulo, Brazil), ²USP, Institute of Tropical Medicine, University of São Paulo School of Medicine, São Paulo, SP, Brazil (Sao Paulo, Brazil), ³Faculty of Medicine, Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, SP, Brazil (São Paulo, Brazil), ⁴Faculty of Medicine, Division of Clinical Immunology and Allergy, University of São Paulo School of Medicine, São Paulo, SP, Brazil (São Paulo, Brazil), ⁵INCT, Institute for Investigation in Immunology (iii), INCT, São Paulo, SP, Brazil (São Paulo, Brazil)

INTRODUCTION

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects millions of people worldwide. Treatment is challenging due to lack of effective drugs and assays to monitor parasite persistence. Here, we use our recent develop gPhage platform to analyze and compare antibody response in Chagas patients, before and after benznidazole treatment, to identify epitopes that could be used as molecular marker for disease status.

OBJECTIVES

Then, we searched for antigens/epitopes recognized preferentially by patients treated with benznidazole that are responders (PCR-negative for T. cruzi) or non-responders (that remain PCR-positive).

MATERIALS AND METHODS

We used IgG purified from sera of patients (N=20) collected before and after treated with benznidazole. Patients were then classified on responders (N=10) and non-responders (N=10) based on their PCR-status. In order to enrich for antigens recognized by IgG from patients before and after treatment, we devised a two-tier biopanning procedure. One of the IgG samples of the same patient (before or after treatment) was first used to pre-clear the T. cruzi genomic phage library (gPhage) before performing the positive selection on the remaining IgG samples. After 4 rounds of selection, phage bound to IgGs were characterized by Next Generation Sequencing. Sequences were assembled and epitopes identified by alignment with T. cruzi genome and clustering.

DISCUSSION AND RESULTS

We observed a significant and inverse correlation between the number of phage display T. cruzi-antigens recovered by IgG from patients and their PCR status. There was a significant enrichment in T. cruzi-specific antigens bound to IgG from non-responders compared to responders. The surface antigen 2 (B13) was the most prevalent antigen recovered, although we also identified other molecular marker of interest.

CONCLUSION

Our work corroborates previous studies indicating the T. cruzi antibody response is a potential marker for cure. Several antigens, including the well-known B13, are potential molecular markers of disease status.

Keywords: Chagas disease, benznidazole, epitopes

Supported by: CNPq, FAPESP, CAPES

08986 - Poster Session

HA.16 - Study of the effects of palmitic acid and pregnancy steroids on the survival of insulin-producing cells

Rafael Teixeira do Nascimento ¹

¹Bioquímica e Biologia Molecular, Programa de Bioquímica e Biologia Molecular da Escola de Artes, Ciências e Humanidades da Universidade de São Paulo (São Paulo, Brasil)

INTRODUCTION

Gestational diabetes (GD) attacks 3-17% of pregnant women, four-times prevalent between obese women, correlating adiposity and GD. Adiposity interferes in adipokines amount, reflecting adipose tissue alterations what could contribute to metabolic diseases.

OBJECTIVES

Study pregnancy steroids and fatty acids effects on viability and DNA fragmentation of insulin-producing-cell line.

MATERIALS AND METHODS

INS-1E were cultivated in RPMI with 5% fbs, bicarbonate, sodium pyruvate, 2-mercaptoethanol and 5% CO2 in humidified atmosphere. Cells were treated with 50, 100, 200 and 300μM palmitic acid (PA) , for 24h or 48h, alone or combined with mixes of progesterone (P4) and estradiol (E2): mix1 (M1: concentrations of P4 and E2 in healthy pregnant women); mix-2 (M2: P4 and E2 in GD pregnant women). Was performed the growth curve for INS-1E (n = 2) and toxicity of PA (n=4) and steroid hormones (n=5) curves. After incubations, membrane integrity and DNA-fragmentation were evaluated by cytometry.

DISCUSSION AND RESULTS

A 3-days folding time for INS-1E was consistent with literature. Treatment with PA caused a slight loss of membrane integrity after 24h (200μM, 8% and 300μM, 10%, regarding to control) and 48h (200μM, 12% and 300μM, 16%, regarding to control). Percentage of DNA-fragmented cells was pronounced: 24h (200μM, 73% and 300μM, 90%, regarding to control) and 48h (200μM, 77% and 300μM, 91%, regarding to control). The toxic effect of PA was dose-dependent without any significant influence of incubation time. Membrane integrity, after treatment with 25μM PA and hormones did not show significant difference in all groups regarding control. Was observed DNA fragmentation in all groups compared to the control (PA: 44%, PA+M1: 44%, PA+M2: 50%, M1: 67% and M2: 68%).

CONCLUSION

Growth curve allowed to establish parameters for experiments with the strain; PA toxicity was dose-dependent, but not time-dependent; only pregnant hormones combinations were more toxic to cells than those with fatty acid.

Keywords: Gestational Diabetes, sex hormones, INS-1E

Supported by: FAOESO

08035 - Poster Session

HA.17 - A conformation sensitive fragment antibody-based assay allows isolation of a neurotoxic oligomeric assembly of the β-amyloid peptide under non-denaturing condition

Raquel Maria de Campos ¹, André Luiz Brandão Bitencourt¹, Erika N. Cline², Mariane Fávero Carraro¹, William L. Klein², Adriano S. Sebollela¹

¹Departament of Biochemistry and Immunology, Ribeirao Preto Medical School, University of Sao Paulo (SP, Brasil), ²Department of Neurobiology, Northwestern University (Illinois, USA)

INTRODUCTION

Soluble oligomers formed by β-amyloid peptide (AβOs) are currently implicated in the pathogenesis cascade of Alzheimer’s disease (AD). The biochemical identity of those oligomers is yet debated, including their molecular mass and conformation. Importantly, the neurotoxicity of AβOs has been shown to be neutralized by the scFv fragment antibody NUsc1, which preferentially targets a subpopulation of AβOs > 50 kDa. NUsc1 antibody is a promising candidate to assist in the isolation and subsequent biochemical characterization of AD- relevant toxic AβOs.

OBJECTIVES

In this work, we describe a method for the isolation of toxic AβOs under non-denaturing conditions based on the use of the conformation-sensitive single-chain antibody fragment NUsc1 as capturing antibody.

MATERIALS AND METHODS

Cobalt-coated magnetic beads were functionalized with His-tagged NUsc1, and used in the isolation of either synthetic or AD-mice brain-derived AβOs. After pull down, the complex NUsc1-AβO was successfully released from the beads using imidazole, as followed by Western Blot (WB). NUsc1-targeted AβO species were stabilized by cross-linking (XL) and molecular mass was analyzed by WB. Alternately, NUsc1-AβOs complex was passed through 50 kDa cutoff filters and retained fraction was also crosslinked and analyzed by WB.

DISCUSSION AND RESULTS

WB analysis of eluates from magnetic beads confirmed that the complex NUsc1-AβO was successfully isolated under non-denaturing conditions, both from synthetic and AD-mice brain-derived AβOs. Interestingly, the majority of cross-linked AβO species targeted by NUsc1 were larger than 100 kDa, in line with the results obtained from NUsc1-AβO cross-linked after complex isolation.

CONCLUSION

Taken together, our findings indicate that NUsc1 preferentially targets AβO species larger than 100 kDa either prepared in vitro or extracted from tissue. The determination of the molecular mass of NUsc1-reactive AβOs by size-exclusion chromatography (SEC) is ongoing, as well as investigations on the molecular shape of these species.

Keywords: AβOs, Alzheimer’s Disease, scFv

08509 - Poster Session

HA.18 - Hsp70 inhibition effects in an ubiquitinated GFP transfected cell line

Milene Nóbrega de Oliveira Moritz ¹, Maria Júlia Mattos Gomes¹, Felipe R. Teixeira², Andrei Leitão¹, Heloisa S Selistre-de-Araujo², Júlio C. Borges¹

¹Departamento de Química e Física Molecular, Universidade de São Paulo (SP, Brazil), ²Departamento de Genética, Universidade Federal de São Carlos (SP, Brazil), ³Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos (SP, Brazil)

INTRODUCTION

Chaperones are specialized in protein folding and cooperate with the ubiquitin/proteasome system during protein quality control in eukaryotic cells. Human heat shock proteins 70 (Hsp70 or HSPA) comprise a highly conserved chaperone family that feature a pivotal role in proteasome system. Studies have been shown that HSPAs inhibitors increase proteotoxicity and suppress proliferation, indicating that these chaperones directly influence cell survival probably through activation of a proteostasis network such as ubiquitin-proteasome degradation system.

OBJECTIVES

Based on that, we hypothesized that the HSPA inhibitor MKT-077 causes proteasome breakdown. To test this hypothesis we used transfected HEK293T cells capable of expressing ubiquitin fused GFP (uGFP) that was directed to proteasome system right after expressed. Once HSPA is inhibited, u-GFP delivering to proteasome is harmed and it leads to GFP accumulation that can be measured by fluorescence.

MATERIALS AND METHODS

Therefore, HEK-uGFP cells were treated with 0.5 to 24 μM of MKT-077 (HSPA ligand) during 5 h and then GFP fluorescence signal was evaluated by flow cytometry and/or plate reader. Besides, cytotoxicity was also evaluated by MTT assay using MKT-077 at 10 to 800 μM during 5 h.

DISCUSSION AND RESULTS

Results demonstrated that the IC50 for MKT-077 was around 200 μM after 5 h of treatment in HEK-uGFP cells and the uGFP accumulation raised with the increased MKT-077 concentrations. These results confirm that HSPA inhibition causes uGFP accumulation since its delivery to proteasome is harmed.

CONCLUSION

We believe that this report model can help to identify interfering features in the intracellular balance between protein folding and protein degradation. It can be a potential strategy to treat diseases involving chaperonepaties such as cancer and neudegenerative diseases. Funding: FAPESP, CNPq and CAPES

Keywords: HSPA, proteasome, co-chaperones

Supported by: FAPESP

08793 - Poster Session

HA.19 - ALBUMIN OVERLOAD IMPAIRS ALBUMIN ENDOCYTOSIS IN PROXIMAL TUBULE CELLS THROUGH INCREASED O-GLCNACYLATION

Rodrigo Pacheco da Silva de Aguiar ¹, Diogo de Barros Peruchetti¹, Maria Paz-Marzolo², Ana Acacia de Sá Pinheiro^1,3, Wagner Barbosa Dias¹, Celso Caruso Neves^1,3,4

¹IBCCF, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil (Rio de Janeiro, Brazil), ²Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile (Santiago, Chile), ³Rio de Janeiro Innovation Network in Nanosystems for Health-NanoSAÚDE/FAPERJ, Fundação de Amparo a Pesquisa do Rio de Janeiro (Rio de Janeiro, Brazil), ⁴National Institute of Science and Technology for Regenerative Medicine, Conselho Nacional de Desenvolvimento Científico e Tecnológico (Rio de Janeiro, Brazil)

INTRODUCTION

Renal disease is strictly associated with urinary protein loss, proteinuria, a condition that reflects protein overload at the proximal tubule epithelial cells, PTECs. It is well known that albumin overload promotes renal disease progression. Identifying the molecular mechanisms mediating this process is essential for the development of new treatments. Our group has previously demonstrated an association among essential hypertension and tubular proteinuria with development of tubule-interstitial injury. The molecular mechanism involved hyper-O-GlcNAcylation in PTECs, but the trigger is unknown.

OBJECTIVES

We aimed to study the possible role of PT albumin overload as a trigger for dysregulated O-GlcNAcylation and its impact in PT albumin endocytosis.

MATERIALS AND METHODS

LLC-PK1 cells, a well characterized PTECs, were incubated overnight with 20 mg/mL albumin mimicking PT albumin overload. LLC-PK1 cells were transfected with minimegalin constructs (mMeg-HA) to study the traffic and expression of megalin, a receptor involved in PT albumin endocytosis. Albumin endocytosis was assessed by albumin-FITC. Surface megalin expression was determined by confocal microscopy. O-GlcNAcylation was also evaluated in 2 different animal models: 1) subclinical acute kidney injury, subAKI; 2) Adriamycin-induced nephropathy (CEUA-045/17).

DISCUSSION AND RESULTS

We observed that the incubation of the cells with albumin induced: 1) an increase in O-GlcNAcylation; 2) a reduction in albumin binding and endocytosis; 3) a reduction of surface mMeg-HA expression. Thiamet G (5 μM), an O-GlcNAcylation enhancer, mimicked these effects. On the other hand, OSM-1, an inhibitor of O-GlcNAcylation, blocked the albumin effect on O-GlcNAcylation, albumin endocytosis and mMeg-HA expression. Importantly, subAKI and adriamycin-induced nephropathy mice models showed increased renal cortex O-GlcNAcylation correlated with decreased proximal tubule albumin endocytosis and reduced megalin expression.

CONCLUSION

Our results indicate that albumin overload impairs PT protein reabsorption by a mechanism involving an increase in O-GlcNAcylation, which decreased megalin surface expression. We propose that this mechanism promotes the development of progressive renal dysfunction in proteinuric conditions.

Keywords: Albumin overload, Megalin, O-GlcNAc

Supported by: FAPERJ, CAPES and CNPq

08525 - Poster Session

HA.20 - INTERACTION OF PROTEINS FROM PLASMA KALLIKREIN-KININ AND PLAMINOGEN ACTIVATOR SYSTEMS WITH BREAST CANCER CELLS.

Katia R. B. Melo¹, Fabio D. Nascimento¹, Marcela M.M.P. Alvarez¹, Renan P. Cavalheiro¹, Carlos M.V. Palomino¹, Samara M.M. Shimon¹, Maria Aparecida S. Pinhal¹, Helena B. Nader¹, Ivarne L.S. Tersariol¹, Guacyara Da Motta ¹

¹Biochemistry/EPM, Universidade Federal de São Paulo (, Brazil)

INTRODUCTION

The proteolytic plasminogen activator system (PAS) plays important role in cancer invasion and metastasis. PAS includes urokinase plasminogen activator (uPA), its membrane-linked receptor (uPAR), plasminogen/plasmin and inhibitors (PAI). The plasma kallikrein-kinin system (KKS) links inflammation and fibrinolysis because plasma kallikrein (PKa) releases bradykinin (BK) from high molecular weight kininogen (HK) and is an activator of pro-uPA.

OBJECTIVES

The aim of this work is to study in breast cancer the interaction of KKS and PAS through HK assembly, BK endocytosis and interaction of PKa/uPA or uPA/syndecans.

MATERIALS AND METHODS

The cell lineages studied were MCF-10A (non-metastatic), MCF-7 (less-metastatic) and MDA-MB-231 (highly-metastatic). The techniques employed were confocal microscopy and RT-PCR. The antibodies used were anti-BK (MBK3 clone), anti-PKa (U691.10), anti-uPA (TC31014, TC21063), anti-syndecan-1 (sc390791) and anti-syndecan-4 (CS12236S).

DISCUSSION AND RESULTS

HK-Dylight-650 bound to cell surface in MCF-10A (1,947.0 ± 0.003 pixels/cell), MCF-7 (648.0 ± 0.002 pixels/cell) and MDA-MB-231 (1,251.00 ± 0.002 pixels/cell). In endocytic vesicles, HK-Dylight-650 and LT-green colocalized in MCF-10A (974.0 ± 0.002 pixels/cell), MCF-7 (61.0 ± 0.001 pixels/cell) and MDA-MB-231 (385.0 ± 0.001 pixels/cell). In comparison the HK binding and endocytosis was MCF10A > MDAMB-231 > MCF-7. BK was detected in endocytic vesicles colocalized with LT-red in MCF-10A (19.7%), MCF-7 (40.9%) and MDA-MB-231 (53%). BK in endocytic vesicles of MDA-MB-231 allows its proliferative effect in metastatic cells. In MDA-MB-231 plasma prekallikrein (PK) mRNA was determined and PKa was present in both non-permeabilized and permeabilized cells. In highly-metastatic cells PKa and uPA colocalized in both conditions non-permeabilized (58.9%) and permeabilized (65.5%) without difference (p=0.1705); syn-4 and uPA colocalization in non-permeabilized (55.3%) and permeabilized (66.8%) was different (p=0.0265); syn-1 and uPA colocalized in non-permeabilized (33.7%) and permeabilized (41.9%) without difference (p=0.2208).

CONCLUSION

Our data suggest that in highly-metastatic breast cancer cells PKa function as pericellular and endogenous protease on HK and pro-uPA that may assemble to membrane by syndecans.

Keywords: cell biology, enzymes, proteoglycans

Supported by: FAPESP, CAPES and CNPq

08667 - Poster Session

HA.21 - Comparison of tumor progression model secretome to molecular subtypes of high-grade serous ovarian cancer

Guilherme Pauperio Lanfredi ¹, John Oluwafemi Teibo¹, Mariana Lopes Grassi¹, Ana Paula Masson¹, Vitor Marcel Faça¹

¹Dept. Biochemistry and Immunology, Ribeirão Preto Medical School (São Paulo, Brazil)

INTRODUCTION

High-grade serous ovarian cancer is the most common and lethal among ovarian cancers, representing around 70% of cases, it has a high mortality rate, mainly due to late diagnosis of severe forms of the disease. Diagnosis, correct classification and treatment can represent improvement of survival rate in 95% of cases, thus generating motivation for understanding the molecular characteristics of each type and discovering biomarkers.

OBJECTIVES

To compare our metastasis model of ovarian cancer cells with analyzes of large groups of patient samples performed by Clinical Proteomic Tumor Analysis Consortium (CPTAC).

MATERIALS AND METHODS

We used the induction of the Epithelial to Mesenchymal Transition (EMT) process in Caov-3 cell line of ovarian cancer by treatment with the epidermal growth factor (EGF) and obtained the culture supernatant on which an analysis was performed with the shotgun proteomic approach and then we compared these results with analysis done by the CPTAC using only the most abundantly expressed proteins of each molecular subtype.

DISCUSSION AND RESULTS

The treatment of Caov-3 cells with EGF showed characteristic markers. The analysis of differentially expressed proteins in the supernatant returned the result of 99 targets with the arbitrary cutoff. While comparing our results with the comprehensive analysis disclosed by the CPTAC study, characteristic profiles of each subtype was provided, highlighting the mesenchymal and proliferative subtype with greater coincidence and structural proteins linked to the location and transport process.

CONCLUSION

The treatment of Caov-3 cells with EGF enabled the occurrence of EMT according to the analysis of characteristic markers of this process. The Caov-3 cell secretome, despite having identified proteins of all subtypes, resembled the mesenchymal subtype, confirming the characteristics required in our model and providing important molecular entities for the study and monitoring of metastasis processes.

Keywords: Ovarian cancer, Epithelial to Mesenchymal transition, Proteomics

Supported by: FAPESP, CNPq and CAPES

08902 - Poster Session

HA.22 - Project - Investigation of neuroinflammatory parameters related to Parkinson's disease

Luciana dos Santos Viana¹, Allyson Guimarães da Costa², Cleiton Fantin Rezende³

¹Programa Multicêntrico de Pós-graduação em Bioquímica e Biologia Molecular, Universidade do Estado do Amazonas (, Brasil), ²Escola de Enfermagem de Manaus, Universidade Federal do Amazonas (, Brasil), ³Departamento de Biologia, Escola Normal Superior, Universidade do Estado do Amazonas (, Brasil)

INTRODUCTION

Parkinson's Disease (PD) is considered an age-related multifactorial disease, comprising environmental and genetic factors. Genetic forms include mutations at 23 loci, and several of these genes are associated with mechanisms linked to immunity. The alpha-synuclein protein is referred to as the center of the immune response that occurs in PD, and its aggregates trigger microglia activation, initiating inflammatory responses in the brain.

OBJECTIVES

The present study aims to evaluate the contribution of different molecular factors related to PD neuroinflammation, by estimating the expression of SNCA, PRKN, DJ-1 and LRRK2 genes and dosage of their products, as well as the quantification of cytokines, chemokines and circulating growth factors.

MATERIALS AND METHODS

Patients will be divided into groups, which will contain 20 patients, according to the severity of the disease. 5 ml of blood will be collected, which will be separated into cells and plasma. The cells will be subjected to total RNA extraction, which will be used to estimate gene expression by RT-qPCR. While plasma will be used for measuring the products of these genes through ELISA, as well as for quantification of cytokines, chemokines and growth factors through cytometry. Immunophenotyping will be evaluated in FlowJo software (v.10) and descriptive and statistical analyzes in GraphPad Prism software (v.8.0.2).

DISCUSSION AND RESULTS

It is expected that the expression of genes and plasma concentrations of the evaluated proteins increase with the severity of the disease, due to the intensification of the neuroinflammatory response.

CONCLUSION

Knowing these factors underlying the neuroinflammatory process associated with PD may contribute to a better understanding of the pathophysiology of the disease and also to the search for therapeutic strategies in order to improve the patient's quality of life. Furthermore, the inference of these correlations may provide support for the definition of predictor biomarkers of disease worsening.

Keywords: Parkinson's disease, neuroinflammation, gene expression

Supported by: FAPEAM

08565 - Poster Session

HA.23 - PROJECT- Functional analysis of HBZ and Tax in the expression of antioxidant response components in ATLL and LTR in the suppression of HTLV-1 infection in T cell lines

Jéssica Sousa ¹, Ricardo Khouri², Leonardo Farias³, Luiz Gustavo Oliveira³, Aline Miranda¹

¹Departamento de Bioquímica e Biofísica, Instituto de Ciências da Saúde, Universidade Federal da Bahia (Bahia, Brazil), ²Laboratório de Enfermidades Infecciosas Transmitidas por Vetores, Instituto Gonçalo Moniz, Fiocruz (Bahia, Brazil), ³Laboratório de Inflamação e Biomarcadores, Instituto Gonçalo Moniz, Fiocruz (Bahia, Brazil)

INTRODUCTION

ATLL is a lymphoproliferative disease triggered by the HTLV-1 virus. There is no cure for ATLL. The development and maintenance of ATLL require contributions from HBZ and Tax viral proteins involved in the modulation of antioxidant response components. HBZ is the only protein that maintains its expression in all ATLL cases. HBZ activates the HMOX-1 expression, an enzyme that acts in the detoxification of free heme and has been described in cell lines transformed by HTLV-1. Tax stimulates viral genes expression, through its interaction with cellular factors and with the non-coding ends, LTRs, of the proviral genome. Expression of Tax has been reported to induce reactive oxygen species, with DNA damage, in primary human CD4+ T lymphocytes. Emerging evidence from several studies indicates that the antioxidant enzyme SOD1, which catalyzes superoxide radical dismutation, is overexpressed in cancers. Thus, its activity may be essential to cells fail to undergo apoptosis. Preliminary studies from our research group demonstrated that the NFE2L2 factor, related to the antioxidant response, is a positive regulator of the most expressed genes in ATLL.

OBJECTIVES

Therefore, our aim is to evaluate the importance of viral proteins HBZ and Tax, and LTRs, for cell proliferation and viral persistence in HTLV-1 infected cell lines.

MATERIALS AND METHODS

The CRISPR/Cas9 gene-editing system will be used to knockout the pX viral region, that encodes HBZ and Tax proteins, and for the cleavage of the LTRs regions.

DISCUSSION AND RESULTS

As a result, we expect to understand the redox balance in HTLV-1 infected cell lines, as well as its importance for the maintenance of leukemogenesis, and promote the removal of the proviral genome to suppress the HTLV-1 infection.

CONCLUSION

Genome editing technology could be a promising therapeutic approach for ATLL.

Keywords: HTLV-1, ATLL, CRISPR/Cas9

08753 - Poster Session

HA.24 - Heparanase and syndecan-1 expression in different breast cancer subtypes

Thérèse Rachell Theodoro ¹, Lara Rodrigues Jeronymo¹, Guilherme Freire Roberto¹, Ariana Carolina Ferreira², Mariane de Barros Ribeiro², Maria Aparecida Pinhal^1,2

¹BIOQUÍMICA/BIOLOGIA MOLECULAR, CENTRO UNIVERSITÁRIO FMABC (SÃO PAULO, BRAZIL), ²BIOQUÍMICA/BIOLOGIA MOLECULAR, UNIVERSIDADE FEDERAL DE SÃO PAULO (SÃO PAULO, BRAZIL)

INTRODUCTION

The heparanase-1 (HPSE) and the heparan sulfate proteoglycan syndecan-1 (SDC1) participate in molecular mechanisms involved in microenvironment tumor communication, carcinogenesis, and tumor metastasis. Targeted therapies and precision medicine demonstrate the importance of identifying specific molecule profiles for each breast cancer subtype besides the current classification defined in luminal tumors A and B, HER2 positive, and triple-negative to obtain a better diagnosis, prognosis, and responses to different therapies.

OBJECTIVES

We decided to investigate HPSE and SDC1 in circulating lymphocytes from patients with different breast cancer subtypes and cell lines representing each molecular subtype.

MATERIALS AND METHODS

Blood samples were obtained from 50 patients with breast cancer and 50 women not affected by cancer (control group). Lymphocytes were obtained following a protocol using the Ficoll Hypaque® method. We also evaluated HPSE and SDC1 in different cell lines and the non-tumor lineage (MCF-10A). Total RNA extraction was performed using the TRIzol® method. Reverse transcriptase SuperScriptIII™ was used to obtain cDNA. The qPCR technique was applied to analyze the relative expression of HPSE and SDC1 using the PowerSybrGreen®. The results were represented by the geometric mean based on endogenous reference genes RPL13a and GAPDH. ethics committee approval protocol number 2.753.436 (CEP/FMABC).

DISCUSSION AND RESULTS

The results showed that lymphocytes from patients with different breast cancer subtypes and different tumor lineages showed higher expression of HPSE and SDC1 compared to control groups. ROC curve analyzes showed a positive predictive value for the luminal B and triple-negative breast cancer subtypes (AUC greater than 0.7).

CONCLUSION

We conclude that HPSE and SDC1 may represent additional molecular markers for breast cancer. Triple-negative tumors are tumors with a worse prognosis and less responsive to conventional therapies. Therefore, HPSE and SDC1 may represent potential target molecules as new treatment alternatives and represent an additional diagnostic factor given the great molecular diversity of triple-negative breast tumors.

Keywords: Heparanase, Syndecan-1, Breast Cancer Subtypes

Supported by: FAPESP, CNPq, CAPES and NEPAS

08370 - Poster Session

HA.25 - Peptide inhibitor for FAK and SRC protein Interaction decreases JAK2 V617F positive cells viability.

Ana Carolina Menezes Mendonça Valente¹, Ana Cristina Ribeiro Bernardo², Gustavo Henrique Lima de Faria², Michelle Bueno de Moura Pereira³, Raquel Tognon Ribeiro ⁴

¹Programa Multicêntrico de Pós-Graduação em Bioquímica e Biologia Molecular, Universidade Federal de Juiz de Fora- Campus Governador Valadares (Minas Gerais, Brasil), ²Graduandos em Farmácia, Universidade Federal de Juiz de Fora- Campus Governador Valadares (Minas Gerais, Brasil), ³Departamento de Ciências Básicas da Vida, Instituto Ciências da Vida, Universidade Federal de Juiz de Fora- Campus Governador Valadares (Minas Gerais, Brasil), ⁴Departamento de Farmácia, Instituto Ciências da Vida, Universidade Federal de Juiz de Fora- Campus Governador Valadares (Minas Gerais, Brasil)

INTRODUCTION

Peptides for protein-protein interaction inhibition has been investigated due to their higher specificity. The tyrosine kinase FAK participate in several cellular process as adhesion, survival, migration and apoptosis and has been described upregulated in many neoplasias. When FAK is activated, it interacts with other proteins, including the tyrosine kinase SRC. The BCR-ABL negative Myeloproliferative Neoplasms (MPN) are hematological neoplasms characterized by excess of proliferation and resistance to apoptosis. The description of genetic alterations as JAK2 V617F mutation contributed to the MPN pathophysiology knowledge, but there is still no curative drug treatment, justifying further studies.

OBJECTIVES

The present work had investigated the effect of a peptide to disrupt FAK and SRC interaction in the viability and apoptosis of HEL 92.1.7, a JAK2 V617F positive cell line.

MATERIALS AND METHODS

For this purpose, it was performed FAK immunoprecipitation (IP) and SRC detection by Western Blot (WB), the peptide sequence was selected using literature data and online proteins databank and the peptide ETDDpYAEIIDEED was synthetized linked to TAT translocator (-YGRKKRRQRRR) by a commercial supplier. HEL cells were treated with the peptide at 1uM, 5uM and 10uM for 48h; 10uM and 25uM for 12h and 25uM for 24h. TAT peptide was used as negative control and doxorubicin was used as positive control. After treating, MTT assay was performed to determine the cell viability and cleaved PARP detected by WB to investigate apoptosis.

DISCUSSION AND RESULTS

It was possible to demonstrated FAK/SRC interaction in HEL 92.1.7 cells and after testing the different concentrations and time of treatment, the lowest cell viability was verified when cells were treated with the peptide at 25uM for 24h (73% of viability compared to control). However, it was not detected cleaved PARP after this treatment.

CONCLUSION

Therefore, the peptide reduced the cell viability, but more studies are necessary to investigate the mechanisms involved.

Keywords: FAK, Peptides, Myeloproliferative Disorders

Supported by: FAPEMIG (APQ02185/16). CT_INFRA2013/FINEP. ACMMV receives master's scholarship from FAPEMIG. ACRB had received PROBIC/FAPEMIG/PROPP/UFJF scholarship and GHLF had received BIC/PROPP/UFJF scholarship.

08301 - Poster Session

HA.26 - Clinical, epidemiological, and laboratory profile of individuals with diagnostic suspicion of leprosy treated at the CREDEN-PES.

Marcos Daniel Silva Pinheiro ¹, Vladmir Machado Rios¹, Amanda Juliane Finardi³, Ida Maria Foschiani Dias-Baptista³, Jessica K Fairley⁴, Lucia Alves de Oliveira Fraga¹

¹Departamento de Ciências Básicas da Vida - DCBV, Universidade Federal de Juiz de Fora - Campus Governador Valadares (MINAS GERAIS, BRASIL), ²DEPARTAMENTO DE MEDICINA, Universidade Federal de Juiz de Fora - Campus Governador Valadares (MINAS GERAIS, BRASIL), ³CIDADE BAURU, INSTITUTO LAURO DE SOUZA LIMA (SAO PAULO, BRASIL), ⁴NÃO INFORMADO, Rollins School of Public Health, Emory University (, ESTADOS UNIDOS DA AMÉRICA)

INTRODUCTION

OBJECTIVES

Leprosy caused by Mycobacterium leprae persists as a public health problem in Brazil. It is an infectious disease of slow and progressive evolution that, if left untreated, can cause the development of permanent physical disabilities. In this context, the fight against leprosy is a priority for the Ministry of Health. The main action strategies are the early detection of cases and examining contacts to promote the breaking of the transmission chain. This study aims to characterize the clinical-epidemiological and laboratory profile of individuals with diagnostic suspicion of leprosy treated at the CREDEN-PES-SMS/GV Center for Endemic Diseases and Special Programs from 2017 to 2021.

MATERIALS AND METHODS

This is a cross-sectional epidemiological study that is being carried out from the survey of secondary data obtained through CREDEN-PES medical records and the application of questionnaires. After signing the consent form, volunteers are submitted to collecting auricular dermal scraping samples for bacilloscopic examination and investigation of strains resistant to Multi-Drug-Therapy (MDT) by the qPCR method.

DISCUSSION AND RESULTS

Samples were collected from 260 individuals, 46.53% female and 53.47% male. About 22.7% of the participants were diagnosed with leprosy, 38 being multibacillary and 21 paucibacillary. Of the cases with leprosy, 64.4% had a bacilloscopic index (BI) ranging from 0.25 to 5, with a mean of 2.26. The age ranged from 9 to 88 years, and 02 cases were registered in children under 18 years of age with positive bacilloscopic index (BI). The vast majority of participants declared skin color brown. Tests to assess resistance to MDT are ongoing.

CONCLUSION

The data presented reinforce the importance of actively searching for new cases and evaluating their contacts due to the active transmission of the disease, especially among children under 18 years of age.

Keywords: leprosy, multi-drug resistance, diagnosis

Supported by: FAPEMIG, NIH/CNPq (Emory University), ILSL, UFJF/GV

08497 - Poster Session

HA.27 - Unraveling the neurotropic potential of the emergent viruses Oropouche and SARS-CoV-2 using adult human brain slice cultures

Glaucia Maria Almeida ^1,2, Niele Dias Mendes³, Giovanna Oslovski Nogueira¹, Juliano Paula Souza², Marjorie Cornejo Pontelli², Guilherme Podolsky-Gondim⁴, Marcelo Volpon Santos⁴, Thiago Mattar Cunha⁵, Luciano Neder³, Eurico Arruda², Adriano Sebollela¹

¹Dept. Biochemistry and Immunology, Ribeirao Preto Medical School, University of Sao Paulo (SP, Brazil), ²Center for Virus Research, Ribeirao Preto Medical School, University of Sao Paulo (SP, Brazil), ³Dept. of Pathology and Forensic Medicine, Ribeirao Preto Medical School, University of Sao Paulo (SP, Brazil), ⁴Dept. of Surgery and Anatomy, Ribeirao Preto Clinics Hospital, Ribeirao Preto Medical School, University of Sao Paulo (SP, Brazil), ⁵Dept. of Pharmacology, Ribeirao Preto Medical School, University of Sao Paulo (SP, Brazil)

INTRODUCTION

Neurotropic viruses can cause central nervous system (CNS) diseases. Indeed, about 30% of confirmed encephalitis cases are attributed to virus infections. Cellular and molecular mechanisms on CNS viral infections have been obtained using rodent models. Despite these advances, significant biochemical and functional differences between rodent and human brains limit their application as disease models in translational neuroscience.

OBJECTIVES

Here we have used slice cultures from adult human brains to investigate whether Oropouche (OROV) and SARS-CoV-2 viruses, reported to cause neurological symptoms in some infected individuals, are capable of infecting human neural cells in a context of preserved brain cytoarchitecture and neural connections.

MATERIALS AND METHODS

Brain tissue was obtained from patients undergoing temporal lobectomy for the treatment of refractory epilepsy (Ethics Committee approval HCRP 17578/15). Cortical fragments were collected at the surgical room and immediately transported to the laboratory, where the tissue was carefully sliced using a vibratome and cultured in 24-well plates. At days in vitro 1-2, brain slices were infected by OROV or SARS-CoV-2 for 2h. Infected slices were cultivated for 24-48 h post-infection.

DISCUSSION AND RESULTS

Our results indicate that both OROV and SARS-CoV-2 infect human neural cells and that these cells support virus replication. Interestingly, while OROV infects mainly microglia, SARS CoV-2 was seen to preferentially infect astrocytes. Both viruses also infected neurons to a lesser extent. OROV infection led to tissue damage and the release of the pro-inflammatory cytokine TNF-α. SARS-CoV-2 infection increased the RNA-expression of pro-inflammatory cytokines such as CCL2, IL-8, and IL-6 by brain slices. We are currently driving efforts to unravel the ultrastructural consequences of OROV and SARS-CoV-2 infections in adult human brain slices using transmission electron microscopy.

CONCLUSION

Given the uncertainties on both acute and long-lasting neurological consequences of neural infection by OROV and SARS-CoV-2, our present work helps to raise awareness about the potential impact of these viruses on the human brain.

Keywords: Human Brain Slice, Oropouche, SARS-CoV-2

08785 - Poster Session

HA.28 - RAGE-associated serum markers along with motor and cognitive clinical parameters as predictors of Parkinson's Disease

Nauana Somensi ¹, Juciano Gasparotto¹, Carolina Saibro Girardi¹, Nayron Medeiros Soares ^2,3,4, Alana Castro Panzenhagen¹, Camila Tiefensee Ribeiro¹, Ana Carolina Leonardi Dutra^2,3, Nathalie Ribeiro Artigas ^2,3, José Cláudio Fonseca Moreira¹, Rosa Maria Martins de Almeida², Arlete Hilbig ^5,4, Carlos Roberto de Mello Rieder^2,4, Daniel Pens Gelain¹

¹Bioquímica, Universidade Federal do Rio Grande do Sul (Rio Grande do Sul, Brasil), ²Faculdade de Medicina, Universidade Federal do Rio Grande do Sul (Rio Grande do Sul, Brasil), ³Clínica Médica, Universidade Federal de Ciências da Saúde de Porto Alegre (Rio Grande do Sul, Brasil), ⁴Neurologia, Hospital de Clínicas de Porto Alegre (Rio Grande do Sul, Brasil), ⁵ Centro de Doença de Parkinson e Distúrbios do Movimento, Santa Casa de Misericórdia de Porto Alegre (Rio Grande do Sul, Brasil)

INTRODUCTION

Parkinson’s disease (PD) affects nearly 10 million people globally. The course of disease is highly variable and there are no established biomarkers with diagnostic value or predictive models. The receptor for advanced glycation end products (RAGE) is crucial in the propagation of inflammatory events, exerting a major role in neuroinflammation and dopaminergic denervation.

OBJECTIVES

We asked if inflammatory parameters and circulating RAGE agonists in serum of patients with Parkinson's disease correlate to clinical assessments. Also, we question how these parameters behave over time and /if they correlate. If so, what are the potential predictors of Parkinson's disease

MATERIALS AND METHODS

Clinical interview, cognitive and motor tests and blood samples were collected from PD patients and controls ("analysis"). Serum parameters were measured by Multiplex and ELISA. We evaluated the correlation of inflammatory cytokines with RAGE agonists in serum, in parallel with cognitive (MoCA) and motor/non-motor (UPDRS) clinical parameters o'f PD. The patients were re-evaluated for the same parameters at a later period of approximately 1 year (reanalysis").

DISCUSSION AND RESULTS

In analysis, increase in HMGB1 in PD is correlated with other RAGE agonists, such as nitrotirosine, 4-HNE, CML along with S100B, but when analyzed together they do not predict the outcome. Although α-synuclein does not differ between control and PD, it is positively correlated to TNF-α; in PD, and together they are factors that predict the disease. The reanalysis regarding the side dish of patients are being processed and correlated to analysis

CONCLUSION

Our preliminary results suggest that more parameters should be developed in order to discover RAGE-associated potential molecular markers that may aid in clinical diagnostic. The reanalysis should inform us how are inflammation and RAGE status and how they correlate with disease progression.

Keywords: Parkinson's Disease, RAGE, biomarkers

Supported by: CNPq, CAPES

08515 - Poster Session

HA.29 - Effects of NT157 on tyrosine kinase signaling pathways in BCR-ABL1 T315I cells

Virgínia Campos Silvestrini ^1,2,3, Alessandra Pinto Vargas^1,2, Ana Paula Masson^1,2, Carolina Hassibe Thomé², Natasha Peixoto Fonseca^3,2, Bruna Alves Fenerich^3,2, Antônio Bruno Alves Silva^3,2, Fabiola Traina^3,2, Vitor Marcel Faça^1,2,3

¹Dept. Biochemistry and Immunology, Ribeirão Preto Medical School (São Paulo, Brasil), ²Cell-Based Therapy Center, Ribeirão Preto Blood Center, Ribeirão Preto Medical School (São Paulo, Brasil), ³Department of Medical Images, Hematology, and Clinical Oncology, Ribeirão Preto Medical School (São Paulo, Brasil)

INTRODUCTION

Chronic myeloid leukemia (CML) is characterized by the presence of oncoprotein BCR-ABL1, which constitutively activates the tyrosine kinase activity triggering hematopoietic stem cells neoplastic transformation. Although, some reports using tyrosine kinase inhibitors (TKI) have represented an advance in CML treatment with up to 20% of patients being resistant to those inhibitors. Studies aiming proteins that bind indirectly to the BCR-ALB1 have identified insulin receptor substrates (IRS) as potential targets, representing new therapeutic strategies for CML.

OBJECTIVES

Evaluation of proteomic alterations in primary samples from patients carrying T315I mutation undergoing treatment with an IGF1R-IRS1/2 inhibitor, NT157.

MATERIALS AND METHODS

Peripheral blood mononuclear cells (PBMCs) from a CML patient with a BCR-ABL1 T315I phenotype were treated with 6.4 μM NT157 for 48 hours and submitted to analysis of cell viability and apoptotic molecular markers. To determine the changes in protein abundances of the cells, we performed a global proteomics analysis. The proteomic data was acquired using a Q-Exactive-HF LC-MS/MS system and processed using Label-Free-Quantification (LFQ) approach with MaxQuant software.

DISCUSSION AND RESULTS

The 6.4 μM NT157 treatment for 48 hours significantly decreased cell viability and increased apoptosis in PBMCs. All 3244 proteins were confidently identified with FDR < 1%. LFQ analysis highlighted a list of 116 upregulated and 85 downregulated proteins differentially detected among cells not treated and cells treated with NT157. In particular, BCR protein, which is directly involved in CML pathogenesis is decreased by the NT157 treatment. Among the processes found altered we can highlight several metabolic pathways involved in TCA cycle, angiogenesis and immune response.

CONCLUSION

NT157 showed antineoplastic effects in primary cells from patients CML BCR-ABL1 T315I including reduced cell viability and increased apoptosis. Inhibition of IGF1R/IRS signaling has revealed a potential therapeutic approach and maybe an alternative to TKI in the context of patient’s inhibitor resistance.

Keywords: Proteomics, NT157, Chronic myeloid leukemia

Supported by: Cnpq

08655 - Poster Session

HA.30 - PROJECT: Molecular and cellular evaluation of the expression of heme oxygenase-1 and peroxisome proliferator-activated receptors and their differential contributions on pathophysiology and clinical complications of sickle cell anemia

João Paulo Moreira Rigueira^1,2, Cibele Velloso Rodrigues^2,3

¹Departamento de Medicina, Universidade Federal de Juiz de Fora - Campus Governador Valadares (MG, Brasil), ²Programa Multicêntrico de Bioquímica e Biologia Molecular, Universidade Federal de Juiz de Fora - Campus Governador Valadares (MG, Brasil), ³Departamento de Ciências Básicas da Vida, Universidade Federal de Juiz de Fora - Campus Governador Valadares (MG, Brasil)

INTRODUCTION

Sickle cell anemia (SCA), the most severe form of sickle cell disease (SCD), is a hereditary hemoglobinopathy with vast distribution worldwide. In SCA, the initial pathological phenomenon consists of the polymerization of S hemoglobin chains, triggering other pathological phenomena such as intravascular hemolysis, production of molecules related to oxidative stress and inflammation. Heme oxygenase-1 (HO-1), acts, in this process, decreasing the availability of free heme in the blood which, in turn, results in cytoprotective, anti-inflammatory and antioxidant effects. The expression of HO-1 is regulated by nuclear factor erythroid 2-related factor 2 (Nrf2), the latter being regulated by receptors activated by proliferation of peroxisomes (PPARs). PPARs are associated with the reduction of inflammation and the control of oxidative stress, and are involved in the regulation of a series of genes associated with those mechanisms.

OBJECTIVES

The objective is investigate differential cellular expression of HO-1 and PPARs in innate immune cells and variants of these genes correlate with intravascular hemolysis, oxidative and inflammatory stress in SCA.

MATERIALS AND METHODS

Therefore, it is intended to evaluate a sample of participants with SCA between six and fifty years old enrolled at the Regional Blood Center of Governador Valadares in a retrospective cohort. Blood samples will be used in flow cytometry and qPCR analysis in order to assess the relation of clinical and laboratory manifestations of severity and hemolysis with the differential expression of HO-1 and PPARs.

DISCUSSION AND RESULTS

As results of these analysis it is expected to find possible mollecular explanations for the different phenotypes of SCA, demostrating the role of HO-1 and PPARs.

CONCLUSION

The clarification of those mechanisms may open field to new prognostic tools, constituting possible predictive markers of severity, and therapeutical approachs, aiming at reducing the clinical complications of the disease.

Keywords: sickle cell anemia, HO-1, PPAR

Supported by: CNPq, CAPES, FAPEMIG, UFJF

08851 - Poster Session

HA.31 - Featuring ACE2 binding SARS-CoV and SARS-CoV-2 through a conserved evolutionary pattern of amino acid residues

Patrícia Pereira Duzi Carvalho ¹, Nelson Augusto Alves¹

¹PHISICS APPLIED TO MEDICINE AND BIOLOGY, UNIVERSIDADE DE SÃO PAULO (SP, BRAZIL)

INTRODUCTION

The importance of the RBM is further explored here in relation to its structural topology. Thus, instead of only analysing specific residues that make contacts with ACE2 after binding, we go a step further and track the molecular origin that drives the viral attachment to this cell receptor. This investigation has revealed a highly conserved amino acid residue sequence Tyr-Gly-Phe in coronavirus variants that employ this receptor. Consequently, we hypothesize that the short sequence-YGF is vital for RBD-ACE2 interaction because of the formation of a hydrophobic pocket proper to the receptor specificity.

OBJECTIVES

Here, we investigate the occurrence and importance of the specific amino acid residue sequence YGF for SARS-CoV and SARS-CoV-2 strains able to use ACE2 proteins as receptors.

MATERIALS AND METHODS

Mutations may affect the spike receptor-binding complexed with hACE2 either leading to higher, lower or even neutral binding affinity. Thus, we apply the fast and accurate MutaBind2 method to estimate the binding free-energy change upon mutation to predict its functional effects.

DISCUSSION AND RESULTS

We investigate the relevance of the hydrophobic pocket driven by the YGFY and YGFQ sequences in promoting the stability of SARS-CoV and SARS-CoV-2 S RBD complexed with hACE2, respectively.

CONCLUSION

YGF-based mechanism can act as a protein signature to distinguish CoVs able to use ACE2 as a cell entry receptor whenever this residue sequence is located at the CoV RBM region. It must be accentuated that the occurrence of other XGF sequences, mainly with X being a hydrophobic residue, in the RBM, or even in the RBD region, can disrupt the proposed topological mechanism for ACE2 binding.

Keywords: SARS-CoV, SARS-CoV-2, ACE2

08542 - Poster Session

HA.32 - Genetic markers in the diagnosis of cholestatic liver diseases in a group of Brazilian patients

Julio Cesar de Jesus Barbosa ¹, Juliene Antonio Ramos¹, Luísa Hoffmann¹

¹Laboratório de Genética Molecular, Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Cholestatic liver diseases are pathophysiological changes characterized by reduced or absent bile flow in the duodenum. Cholestasis can be extra or intrahepatic or due to a functional change in the hepatocyte. It presents clinically by the triad: jaundice, dark urine and pale stool. This group of diseases can have different origins and several subdivisions, three of them well-known: Primary biliary cholangitis (PBC), Primary sclerosing cholangitis (PEC) and Hereditary cholestasis (HC). Because of these different forms, the diagnosis is complicated. Nowadays, diagnosis is mainly made by the set of liver enzymes, imaging tests and histopathological exams. However, a large number of cases do not have a conclusive diagnosis. Thus, it’s very important finding biomarkers to characterize these diseases.

OBJECTIVES

The aim of this project is to evaluate the molecular variants of the ABCB4, ABCB11 and ATP8B1 genes from patients with different forms of cholestatic liver diseases in the search for biomarkers.

MATERIALS AND METHODS

For materials and methods, we will use genotyping by sanger sequencing or real-time PCR TaqMan assay, with subsequent association between genotype and clinical aspects and laboratory tests. We are recruiting patients with different forms of cholestatic liver diseases in the Ambulatório de Doenças Auto-imunes belonging to the Hospital Universitário Clementino Fraga Filho (HUCFF) of the Universidade Federal do Rio de Janeiro (UFRJ).

DISCUSSION AND RESULTS

Clinical and laboratorial characteristics are being evaluated. With this association, we intend to improve the patient’s diagnosis and their life quality, reducing their comorbidities and mortality rate.

CONCLUSION

In addition to cholestatic liver diseases this study can intervene in the clinical improvement of patients in other organ systems potentially damaged by this group of diseases.

Keywords: Cholestasis, Diagnosis, Biomarker

Supported by: FAPERJ, IFRJ

08075 - Poster Session

HA.33 - Metabolic alterations in COVID-19 observed by 1H-NMR

Ljubica Tasic ¹, Ljubica Tasic¹

¹Department of Organic Chemistry, University of Campinas (São Paulo, Brazil)

INTRODUCTION

Blood serum metabolites reflect the body's physiological state and change with the age, disease, or response of the body to any external effect, thus may add-in to a better understanding of viral infections. Our knowledge about the metabolic changes specifically occurring upon SARS-CoV-2 infection is still limited and expected to point to nucleotide, carbohydrate, lipid, and amino acid metabolism alterations.

OBJECTIVES

This work aimed to define serum metabolites that may differentiate patients that tested positive vs. negative for SARS-CoV-2 among two cohorts of patients - in patients with grave symptoms and ones with moderate symptoms by NMR-metabolomics.

MATERIALS AND METHODS

The informed consent was obtained from all participants (n = 350), and the present study was approved by the Research Ethics Committees. The sera were obtained from 5 mL of peripheral blood collected in a dry tube after peripheral venipuncture, realized for the routine hematological procedure. Just 200 μL of samples after dilution with deuterium oxide (250 μL) were used to record the high-resolution 1H-NMR NOESY1D 1D (noesy1dgppr1d), CPMG (cpmgpr1d), and edited by diffusion (stebpgp1s191d) spectra. The spectra were acquired on the Bruker AVANCE III 600 MHz spectrometer using the inverse triple-core probe (TBI) at 25 °C, processed, and analyzed through the MetaboAnalyst platform.

DISCUSSION AND RESULTS

At least fifty serum metabolites were found as strongly affected by the severity of the symptoms, and 60% of those were increased. High concentrations of glucose and lipids were seen, but, cholesterol, lysophosphocholines, and proteins were decreased. Serum metabolite variations in patients with moderate symptoms were also seen in serum lipids, although VLDL and LDL were somewhat less altered when compared to those measured in intensive care patients.

CONCLUSION

So, there must be a connection between glucose excess, high VLDL, and low serum proteins with the disease severity, and NMR could be successfully used to map those.

Keywords: metabolites, infection, 1H-NMR

Supported by: CNPq, and FAEPEX-PRP.

08970 - Poster Session

HA.34 - Project: Analysis of the neuroprotective effects of linseed oil and/or alpha-lipoic acid supplementation of rats with haloperidol-induced orofacial dyskinesia

Rodrigo Freire Oliveira ^1,2, José Rodolfo Lopes de Paiva Cavalcanti^1,2

¹Ciências Biomédicas, Universidade Estadual do Rio Grande do Norte (Rio Grande do Norte, Brasil), ²Ciências Biomédicas, 2Programa de Pós-Graduação Multicêntrico na área de Bioquímica e Biologia Molecular (Rio Grande do Norte, Brasil)

INTRODUCTION

Tardive dyskinesia (TD) is manifested by atypical involuntary movements that are associated by the long-term use of antipsychotic medications, such as haloperidol, that cause the pharmacological blockade of D₂ dopaminergic receptors. Although the origin mechanism of this disorder is not yet fully understood it is pointed a role for oxidative stress, inflammation, and neurotrophic factors levels, which are potential therapeutic intervention tools. Previous studies showed that the flaxseed oil (Linum usitatissimum L.), rich in the polyunsaturated essential fatty acid α-linolenic acid, and the α-lipoic acid are both antioxidants, have anti-inflammatory properties and can influence Brain Derived Neurotrophic Factor (BDNF) levels.

OBJECTIVES

This work aims to evaluate the influence of linseed oil and/or α-lipoic acid supplementation on neuroprotection against the development of orofacial dyskinesia induced by sub-chronic haloperidol administration in rats.

MATERIALS AND METHODS

108 males Wistar Rattus norvegicus will be used, arranged in 6 study groups: I) control: no supplemental diet or haloperidol; II) haloperidol administration; III) haloperidol with lipoic acid administration; IV) haloperidol with linseed oil administration; V) haloperidol with lipoic acid and flaxseed oil administration; VI) control: clozapine administration. After the previous interventions the animals will go under validation tests for the tardive dyskinesia experimental model, euthanasia, antioxidant assessment (TBARS test, SOD activity and GSH levels), molecular (Western Blotting) and immunohistochemical evaluations for TH, BDNF, GFAP, DRD3 markers on the prefrontal cortex, midbrain, and striatum regions.

DISCUSSION AND RESULTS

It is possible to say that both substances may reduce the extrapyramidal effects produced by subchronic use of haloperidol, improving motor function, relieving oxidative stress and restoring BDNF levels.

CONCLUSION

This could advance neuroprotective methods for patients on haloperidol and for other drugs with neurological side effects.

Keywords: haloperidol, linseed oil, thioctic acid

Supported by: CAPES

08322 - Poster Session

HA.35 - Challenges of stable silencing of β3 integrin subunit of triple negative breast adenocarcinoma cell lines (MDA-MB-231) in vitro.

Ana Carolina Caetano Nunes ¹, Wanessa Fernanda Altei^1,2, Heloisa Sobreiro Selistre de Araujo¹

¹Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos (São Paulo, Brasil), ²Departamento de Radioterapia, Hospital de Amor (São Paulo, Brasil)

INTRODUCTION

Breast cancer is the major disease that affects women, especially the triple negative phenotype, which has a metastatic potential leading to a poor prognosis. Metastasis is the main cause of death of cancer patients; however, a better understanding on its underlying mechanisms is essential in the search for more efficient therapies. During metastasis development, the tumor microenvironment and the extracellular matrix (ECM) play important roles, providing the tumor cells a surrounding environment that supports its development and spreading. In addition, the expression profile of integrins changes, including the β3 integrin subunit, which is often overexpressed in tumors.

OBJECTIVES

The objective of this study was to analyze the effects of β3 subunit silencing in tumor cells of triple negative breast adenocarcinoma cell lines (MDA-MB-231) in vitro.

MATERIALS AND METHODS

For that, we produced lentivirus shRNA ITGB3 using third generation system (Plasmids: VSV-G envelope; pLP1 and pLP2 packaging; ITGB3 3’-UTR-shRNA and ITGB3 CDS-shRNA transfection) in human kidney embryonic cell lines (293FT). Viral particles were used for transduction of target cells. The success of silencing was confirmed by qPCR and western blotting.

DISCUSSION AND RESULTS

Although we have confirmed 84% silencing of β3 subunit by RT-qPCR, immunofluorescence assays of cells plated in fibronectin coating suggested recovering of integrin expression but in a different distribution pattern, apparently in the cell border. This intriguing result lead us to develop the hypothesis that the presence of specific ECM components may be able to overcome the silencing mechanism.

CONCLUSION

Considering the complexity of microenvironment within the breast and all the changes that occur during tumor progression, this finding could be a clue for anti-integrin therapies failure. However, new experiments will be done to understand the consequences of silencing β3 subunit in different ECM proteins and tumor progression.

Keywords: β3 integrin, breast cancer, silencing

Supported by: FAPESP, CNPq, CAPES

08478 - Poster Session

HA.36 - Microbial metabolites reduce α-synuclein aggregation in a Saccharomyces cerevisiae model

Edlene Ribeiro Prudêncio de Souza ¹, Rosane Nora Castro², Marcos Dias Pereira³, Cristiano Jorge Riger¹

¹Department of Biochemistry, Federal Rural University of Rio de Janeiro (Rio de Janeiro, Brazil), ²Department of Organic Chemistry, Federal Rural University of Rio de Janeiro (Rio de Janeiro, Brazil), ³Department of Biochemistry, Federal University of Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Parkinson's disease (PD) is a progressive neurodegenerative disease associated mainly with aging. The current understanding of the pathophysiology of PD suggests a central role in the accumulation of the protein α-synuclein and several evidences have been directing that the initial site of this process would be the enteric nervous system. It is known that the intake of phenolic substances contributes to the redox balance of the organism, however its bioactivities are highly impacted by microbial biotransformation that occur in the intestinal lumen.

OBJECTIVES

The objective of this work was to evaluate the influence of phenolic compounds and probiotic microorganisms on the aggregation of α-synuclein protein expressed in the yeast Saccharomyces cerevisiae.

MATERIALS AND METHODS

A pre-inoculum was prepared in SC-GLU at 160rpm/30°C, and after 24 h of growth, cells were transferred to SC-GAL medium supplemented with caffeic acid phenethyl ester (CAPE) or mangiferin; CAPE and mangiferin fermented by probiotic blend; medium fermented by probiotic blend; and control with only SC-GAL. CAPE and mangiferin concentrations were 0.1 mM and cell suspensions were incubated at 160rpm/30°C for 35h. Yeast growth was kinetically monitored by measuring the OD600 and also submitted to fluorescence microscopy, spot plating and detection of metabolites by HPLC-Q-TOF-MS.

DISCUSSION AND RESULTS

Results showed that CAPE and mangiferin without fermentation did not inhibit protein aggregation, but fermentation was able to reduce this aggregation by about 50%. Inhibition of α-synuclein aggregation was correlated with the presence of fermented metabolites. The detection of 3-hydroxyphenylpropionic acid (3-HPPA), a microbial metabolite associated with the reduction of α-sin toxicity converges with recent theories that the microbiota influences the etiology of PD.

CONCLUSION

Therefore, our studies suggest that interactions between the microbiome and certain dietary factors may support new therapeutic strategies to modulate the onset and/or progression of synucleinopathies.

Keywords: Parkinson's disease, phenolic compounds, probiotics

Supported by: FAPERJ, CNPq and CAPES

08713 - Poster Session

HA.37 - An integrated study of transcriptome, lipidome and proteome in search of new therapeutic targets for pathological angiogenesis

Lilian Cristina Costa Alecrim de Oliveira ¹, Alex Inague¹, Jhonatas Sirino Monteiro¹, Marcos Yoshinaga¹, João Carlos Setubal¹, Sayuri Miyamoto¹, Ricardo José Giordano¹

¹Bioquímica, Instituto de Química - Universidade de São Paulo (SP, Brasil)

INTRODUCTION

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential in physiology and pathology. Cancer and retinopathy are examples of diseases for which anti-angiogenic drugs are already available. Despite its success, we need to better understand the molecular mechanisms driving angiogenesis in order to develop a new generation of anti-angiogenesis drugs for these patients. Recently, using RNA-seq from an angiogenesis in vivo animal model (OIR, oxygen-induced retinopathy), our group has shown that differentially expressed genes in the retina of these animals could be used as a prognostic tool for a human angiogenesis dependent disease.

OBJECTIVES

We will expand these studies by characterizing the lipidome and proteome of mice retinas under pathological angiogenesis to integrate with the transcriptome data.

MATERIALS AND METHODS

Samples were collected at different postnatal days (P12, P12.5, P15, and P17) from mouse pups under pathological (OIR model from day 7 to day 12) and physiological development. Total lipid extracts were analyzed through non-targeted lipidomics by HPLC coupled to high-resolution mass spectrometry. For proteome, samples were collected in the same conditions and are being prepared for analysis.

DISCUSSION AND RESULTS

We identified and quantified 301 lipid species. PCA analysis revealed alterations in retinal lipidome mainly according to time, but also to the condition, physiological or pathological angiogenesis. The most significantly altered lipids in pathological angiogenesis correspond to storage lipids (CE and TAG) and membrane lipids (phospholipids). A preliminary integration of these results with transcriptome shows a cholesterol metabolism enzyme as a possible new marker for pathological angiogenesis.

CONCLUSION

Lipidomic analysis suggests that pathological angiogenesis leads to intense remodeling of membrane and storage lipids. Proteomic analysis is ongoing and will provide, along with transcriptome and lipidome data, a better understanding of the different pathways associated with pathological angiogenesis.

Keywords: Angiogenesis, Retinopathy, Oxygen-Induced Retinopathy

Supported by: CAPES, FAPESP and CNPq

08504 - Poster Session

HA.38 - Evaluation of the effect of superparamagnetic iron oxide nanoparticles functionalized with Wedelolactona in modulating the aggregation of alpha-synuclein protein and neurotoxicity of aggregates formed.

Gabriela Ferraz Ribeiro ¹, Luiz de Oliveira¹, Carolina Braga¹

¹NUMPEX-Bio, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Parkinson's disease (PD) has as main pathological characteristics the death of dopaminergic neurons in the substantia nigra and the presence of Lewy Bodies, who have as main constituent the aggregates of alpha-synuclein protein (α-syn), which classifies DP as an amyloidosis.There is thus, a need to find compounds that are of acting as modulators of amyloid aggregation inhibiting the formation of toxic species and thus preventing death and maintaining morphology and cell viability. Previous projects of our group identified two possible compounds with the ability to inhibit α-syn aggregation in vitro and to undo mature fibers, namely, Wedelolactone (WED) and superparamagnetic iron oxide nanoparticles (SPIONs), respectively. WED, studied during my master's degree, has shown to reduce in approximately 90% thioflavin-T-binding, which is a marker for amyloid formation. While SPION,was able to “break” pre-formed aggregates through treatment with hyperthermia, in addition to modulate aggregation demonstrated.

OBJECTIVES

In this way, we will functionalize the SPIONs with Wedelolactone compound, forming the SPIONs@wed and evaluate the role of these in the formation of alpha-synuclein protein aggregates, and in the neurotoxicity of the species formed in their presence.

MATERIALS AND METHODS

The green synthesis of SPIONs was carried out by the sol-gel method, using coconut water as a framework for the reaction. We will start now, the functionalization phase of these with WED, initially only by joining the two molecules in an attempt to make hydrogen bonds between them and in this way we obtain the functionalization.

DISCUSSION AND RESULTS

After confirming the SPIONs@WED formation through characterization by several physicochemical methods, experiments will be carried out in order to evaluate their effect on the aggregation of α-syn protein.

CONCLUSION

We aim with functionalization to increase the effectiveness of aggregation modulation and design a compound with the possibility of being targeted and tracked in the dopaminergic cell.

Keywords: alpha synuclein, amyloidosis, Parkinson's disease

Supported by: FAPERJ

08491 - Poster Session

HA.39 - Project: The hsa-miR-1 expression profile in the evolution of Chagas disease by oral transmission

Eliane de Freitas Oliveira ¹

¹Departamento de Ciências Biomédicas, Universidade do Estado do Rio Grande do Norte (Rio Grande do Norte, Brasil)

INTRODUCTION

Chagas disease is an emerging infectious disease considered a public health problem, characterized by a short acute phase, followed by a long asymptomatic chronic phase known as the chronic indeterminate form, which can last a lifetime or progress to cardiac and /or digestive form. Oral transmission has epidemiological importance, related to a greater manifestation of symptoms, as well as an increase in mortality, mainly associated with myocarditis. In the course of infection, association pathways were predicted between target molecules and miRNAs, so that in the acute phase the gene for cyclin D1 was shown to be potentially affected by the expression of some miRNAs and, in the chronic cardiac phase, this same gene was specifically regulated by hsa-miR-1, in which decreased expression of this miRNA results in a marked translation of cyclin D1 in cardiac tissue.

OBJECTIVES

This research project aims to evaluate the expression profile of hsa-miR-1 in the evolution of patients with Chagas disease after acute infection by oral transmission.

MATERIALS AND METHODS

The study group will consist of eight patients, corresponding to the Chagas disease outbreak diagnosed after ingestion of sugarcane juice in the town of Marcelino Vieira in October 2015. This trial will be conducted in five stages (1, 2, 3, 4 and 5), referring to the acute phase of the infection in October 2015 (time 1) and the chronic phase after one and a half year (time 2), two and a half years (time 3), three and a half years (time 4) and six years (time 5) after acute infection, by collecting peripheral blood for quantitative analysis of hsa-miR-1 using real-time PCR.

DISCUSSION AND RESULTS

As a result, a correlation between hsa-miR-1 expression levels and the evolution of Chagas disease is expected.

CONCLUSION

Keywords: Chagas Disease, hsa-miR-1, Oral Transmission

08777 - Poster Session

HA.40 - The role of histone 3 trimethylations in the pathophysiology of major depression in rats subjected to chronic unpredictable mild stress model

Lucas dos Santos da Silva ¹, Luiza Behrens¹, Camila Barbosa¹, Daniel Peixoto¹, Camila Ribeiro², Helen Silva¹, Pedro Brum³, Daniel Gelain¹, Roberto Almeida⁴, José Cláudio Moreira¹

¹Bioquímica, Universidade Federal do Rio Grande do Sul (, Brazil), ²Montreal Neurological Institute Hospital, Mcgill University (, Canada), ³Center for Molecular Biology, University of Vienna (, Austria), ⁴Departamento de Ciências Biológicas, Universidade Federal de Ouro Preto (, Brazil)

INTRODUCTION

The pathophysiology of Major Depressive Disorder (MDD) is not fully understood, but it is known to be a multifactorial disorder. For preclinical studies, the chronic unpredictable mild stress model (CUMS) is considered a model with high translational potential for the study of TDM. Furthermore, studies show that environmental factors such as stress can cause epigenetic changes. In this context, considering that histones are subject to complex epigenetic modifications, such as methylation, this phenomenon can modify the nucleus-histone interaction. Although many studies have been performed, no study reports methylation of lysine residues (K) 4, 9, 27, 36, and 79 of histone 3 (H3) with the heritability of depression.

OBJECTIVES

However, first, the objective was to investigate possible DNA damage and alterations in the methylation pattern of H3 residues in Wistar rats submitted to CUMS.

MATERIALS AND METHODS

For this, 29 rats were used (8 controls and 21 submitted to CUMS for 42 days). During CUMS, sucrose consumption was performed, followed by open field and object recognition. Finally, blood and bone marrow samples from the femur were collected to perform repair kinetics and micronucleus, while the hypothalamus and hippocampus were used to verify possible epigenetic modifications.

DISCUSSION AND RESULTS

Regarding DNA damage, we found that repair of the damage occurred in a shorter time in control and resilient animals than in depressive-type animals. Finally, evaluating the trimethylations pattern of H3, we observed H3K27 hypermethylation while H3K9 was hypomethylated in the hypothalamus of animals with a depressive-type phenotype. In the hippocampus, we observed hypermethylation of H3K4 and H3K36 in resilient animals and hypermethylation of H3K9 in animals submitted to CUMS.

CONCLUSION

From our results, we elucidate that there are different patterns of trimethylation between depressed and resilient animals. These patterns may be related to the development of the phenotype, as the offspring inherited behaviors similar to those of their parents.

Keywords: Depression, Epigenetics, Histone methylation

08311 - Poster Session

HA.41 - The effect of hypoxia in the motility of MDA-MB-231 breast tumor cells and endothelial cells in vitro

BRUNA CARLA CASALI¹, WANESSA FERNANDA ALTEI^1,2, LARISSA THABATA GOZZER^1,1, HELOISA SOBREIRO SELITRE-DE-ARAUJO^1,1

¹Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos (São Paulo, Brasil), ²Departamento de Radioterapia, Hospital de Amor (São Paulo, Brasil)

INTRODUCTION

Solid tumors, such as breast cancer, may present hypoxic areas that induces angiogenesis and cell migration contributing to metastasis. Integrins are cell surface receptors that play a key role in cell motility. Inhibition of cell migration by blocking such receptors might impair metastasis. Desintegrins are integrin inhibitors, such as DisBa-01, a RGD disintegrin from Bothrops altenatus with high affinity to α_Vβ₃ integrin. DisBa-01 was demonstrated to inhibit cell migration and angiogenesis in different in vitro models in normoxia. This inhibitory effect, however, has never been investigated in low oxygen conditions as usually found in solid tumors.

OBJECTIVES

The goal of this work was to understand the migratory behavior of cells under α_Vβ₃ integrin blocking in hypoxia using two migration models, the Boyden chamber and the wound healing assays.

MATERIALS AND METHODS

MDA-MB-231 cells and HUVEC were treated with DisBa-01 for 30 minutes in room temperature, transferred to 24-well inserts for transwell migration assays in hypoxia (1% O₂, 5% CO₂, and 94% N₂) and normoxia. For wound healing assay, cells were treated with DisBa-01 for 24 hours in the two conditions. For cell morphology analysis, cells were exposed to DisBa-01 for 4 hours in normoxia and hypoxia, and stained with phalloidin.

DISCUSSION AND RESULTS

MDA-MB-231 cell migration was inhibited by DisBa-01 with IC₅₀ values were 9.8 nM and 16.62 nM in normoxia and in hypoxia, respectively, after 16h. DisBa-01 inhibited HUVEC migration in hypoxia after for 24h, without effect in normoxia. In the wound healing assays in hypoxia, DisBa-01 was effective only at its highest concentration. DisBa-01 changed the morphology of MDA-MB-231 cells to a more circular form similarly in both oxygen conditions.

CONCLUSION

We concluded that larger DisBa-01 concentrations are needed to inhibit α_Vβ₃ integrin under hypoxic conditions and therefore to inhibit cell motility.

Keywords: Cell migration, integrin αVβ3, hypoxia

Supported by: FAPESP, CNPq and CAPES

08470 - Poster Session

HA.42 - Exosomes from MDA-MB-231 cell line induce apoptosis gene expression, and phenotype and functional changes in Dendritic Cells: Possible role of Caspase-9 gene and pro-apoptotic miRNAs

Brenda Raissa De Oliveira ¹, Mariana Sousa Vieira¹, Graziela Gorete Romagnoli Castilho², Anderson Kenedy Santos¹, Viviane Aline Oliveira Silva³, Rui Manuel Vieira Rei³, Helder Magno Silva Valadares⁴, José Alexandre Marzagão Barbuto⁵, Fernando Luiz de Lucca⁶, Rodrigo Ribeiro Resende ¹, Helton da Costa Santiago¹, Nayara Delgado André Bortoleto⁴

¹Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, , Universidade Federal de Minas Gerais (Minas Gerais, Brasil), ²Bioquimica, Universidade Estadual Paulista Botucatu (São Paulo, Brasil), ³Fundação Pio XXII, Hospital do Câncer de Barretos (São Paulo, Brasil), ⁴Bioquimica, Universidade Federal de São João Del Rei (São Paulo, Brasil), ⁵Instituto de Ciências Biomédicas IV, Universidade de São Paulo (São Paulo, Brasil), ⁶Bioquimica, Faculdade de Medicina de Ribeirão Preto (São Paulo, Brasil)

INTRODUCTION

Over the recent years, it has been demonstrated that dendritic cells (DCs) from cancer patients have phenotype altered becoming cells with reduced ability to activate immune system. Tumor derived exosomes (TEX) are critical components of intercellular information network between tumor and host, emerging, in recent decades, as important modulators immune response in the cancer development and establishment context.

OBJECTIVES

To evaluate if TEXs isolated from MDA-231 tumor cell line culture supernatant can induce phenotypic alterations functional changes and evaluate caspase 9 and blc-2 gene expression in monocyte-derived DC (Mo-DC) in vitro differentiation. To quantify pro-apoptotic miRNAs (29b, 34a, 155 and 146) TEX expression.

MATERIALS AND METHODS

Exosomes from MDA-231 culture were obtained through ExoQuick-TCTM kit. Structural and morphological exosomes characterization were performed by Western blot, Nanosight and Transmission Electron Microscopy. Mo-DCs were obtained from healthy donors and maintained in culture in presence or absence of TEXs (30μg/mL). Mo-DC phenotypic characterization, cell viability, apoptosis and co-culture T cell proliferation were analyzed by cytometry flow. mRNAs apoptosis genes and miRs expression were identified for qPCR.

DISCUSSION AND RESULTS

Exosomes was successfully isolated and characterized. It was observed TEXs from MDA-231 induced phenotypic Mo-DCs alterations, exhibited by CD80 downregulation (p < 0,05) which corroborated with functional tests that demonstrated reduced ability to stimulate proliferation of CD4 and CD8 cells and induction of tolerogenic T cells differentiation (p < 0,05). Mo-DCs treated with TEX showed 42% death, 37.% in apoptosis process, caspase-9 gene overexpression (p < 0,05) and bcl-2 gene downexpression (p < 0,05). Finally, all pro-apoptotic miRs evaluated presented overexpression in TEX.

CONCLUSION

These results suggest TEX action on the DC functional immunostaining in order to induce a tolerogenic state that suppress an adaptive antitumor immune response favoring tumor evasion. Since casp9, bcl-2 and proapoptotic, miRNAs are altered, it is possible that their overexpression is involved in the apoptosis of Mo-DC.

Keywords: breast cancer, exosome, Dendritic Cells

Supported by: FAPEMIG, CAPES, CNPq

08973 - Poster Session

HA.43 - PROJECT: Analysis of the neuroprotective effects of supplementation with flaxseed oil and/or α-lipoic acid in the hippocampus of rats in haloperidol-induced orofacial dyskinesia model

Karina Maia Paiva ¹, JOSÉ RODOLFO LOPES DE PAIVA CAVALCANTI¹

¹CIÊNCIAS DA SAÚDE, Universidade do Estado do Rio Grande do Norte (, Brasil)

INTRODUCTION

Typical antipsychotics are a class of medications associated with motor disorders when used chronically, such as haloperidol. Tardive dyskinesia (TD) is a motor syndrome that has as its main pharmacological cause the blockade of dopaminergic D2 receptors. Its exact pathophysiology is not defined and theories point to the involvement of inflammatory causes, oxidative stress, poorly adaptive plasticity and neurochemical imbalance. In this context, research on substances with antioxidant action has intensified, such as the use of flaxseed oil (Linum usitatissimum L.) and α-lipoic acid, highlighted by neuroprotective action, in important neuroplastic regions associated with memories, such as the hippocampus.

OBJECTIVES

This study seeks to evaluate the influence of supplementation with flaxseed oil and/or α-lipoic acid on neuroprotection on the development of orofacial dyskinesia induced by sub-chronic administration of haloperidol in the hippocampus of rats.

MATERIALS AND METHODS

Ninety-six Wistar, males, three months, weighing 250-320 g, arranged in 6 groups (n=16) will be divided between the saline control groups; haloperidol administration; submitted to haloperidol administration with supplemental lipoic acid diet; administration of haloperidol with supplemental flaxseed oil diet, haloperidol administration with supplemental diet of lipoic acid and flaxseed oil and submitted to clozapine administration. Evaluations of the behavioral profile of animals will be performed under aspects of motor activity, induction of orofacial dyskinesia and acquisition of memory, anxiety and learning, evaluation of dopamine receptor D3 expression levels, BDNF (brain-derived neurotrophic factor) and GFAP (glial fibrillar acid protein) by quantification by Western Blott, comparison of morphological and morphometric parameters by immunohistochemistry and also evaluation by biochemical assays to measure the activity of antioxidant enzymes from lipoperoxidation assays, evaluation of catalase concentration, enzymatic activity of superoxide dismutase and reduced glutathione.

DISCUSSION AND RESULTS

With this, we aim to promote neuroprotective, anti-inflammatory, neuroplastic, behavioral and antioxidant actions, the neurological damage associated with the administration of neuroleptics in animal models.

CONCLUSION

Keywords: Oxidative Stress, Antioxidant, Tardive Dyskinesia

Supported by: CAPES

08347 - Poster Session

HA.44 - The flavonoid Agathisflavone improve tissue repair in acute spinal cord injury in the rat

Ravena Pereira do Pereira do Nascimento ^1,2, Livia Bacelar de Jesus¹, Markley S. Oliveira-Junior¹, Aurea Maria Almeida¹, Eduardo Luiz Trindade. Moreira¹, Bruno Diaz Paredes³, Jorge Mauricio David¹, Juceni Pereira David¹, Maria de Fátima Dias Costa¹, Arthur Morgan Butt⁴, Victor Diógenes Amaral da Silva¹, Silvia Lima Costa¹

¹Bioquímica e Biofísica, Universidade Federal da Bahia (Bahia, Brasil), ²Biotecnologia, Universidade Estadual de Feira de Santana (Bahia, Brasil), ³D'Or Institute for Research and Education, Hospital São Raphael - Centro de Biotecnologia e Terapia Celular (Bahia, Brasil), ⁴School of Pharmacy and Biomedical Science, University of Portsmouth (UK, Inglaterra)

INTRODUCTION

Spinal cord injury (SCI) is a serious disease that lacks effective therapy. Among therapeutic alternatives, flavonoids as well as mesenchymal cells have been widely studied in the literature. Agathisflavone is a flavonoid capable of inducing neurogenesis and presenting anti-inflammatory properties.

OBJECTIVES

We hypothesize that, the treatment with agathisflavone compared to untreated rMSCs, agathisflavone exposed rMSCs infused after acute spinal cord injury (SCI) increase production of neurotrophic factors and modulate inflammatory damage favoring tissue regeneration.

MATERIALS AND METHODS

rMSCs were obtained from adult Wistar rats and cultured in supplemented DMEM. Cells were exposed to agathisflavone (1μM) and toxicity analyzed by MTT test after 24h and 72h; For in vivo experiments adult male Wistar rats (n=6/group) underwent acute SCI with an F2 fogarty catheter and after 4h were treated with a single application (via caudal vein) of 1x106 control rMSCs or pretreated with agathisflavone (1μM, every 2 days, for 21 days in vitro). Alternatively, animals (n=6/group) were treated with a single dose of methylprednisolone (MP, 60 mg/kg ip), or treated daily with agathisflavone (10 mg/kg ip). After 1, 3 and 6 days treatment BBB scale was used to evaluate the motor functions of the animals; after 7 days of the treatment the SCI area was analyzed after H&E staining, and RT-qPCR was performed to analyze expression neurotrophins and arginase.

DISCUSSION AND RESULTS

Treatment of animals with of 21days agathisflavone-treated rMSCs was able to protect the injured spinal cord tissue, associated with the increase in expression of NGF,GDNF and arginase, and reduction on the macrophage infiltrate, as well as with the improve of the motor functions (with the highest BBB score). Moreover, treatment of animals with agathisflavone alone was also able to protect injured spinal cord tissue, to increase expression of neurotrophins and modulate the inflammatory response

CONCLUSION

Agathisflavone modulates rMSCs metabolic profile and neuroinflammatory response during the spinal cord injury.

Keywords: Agathisflavone, Acute Spinal Cord Injury, Flavonoid

Supported by: FAPESB, CAPES, CNPq

08516 - Poster Session

HA.45 - Project: Effect of rutin on neurogenesis and neuroprotection: study of the pharmacological action of flavonoids in glial biology and neuronal population at different stages of life and in a study model for Parkinson's disease.

Jéssica Souza ^1,2, Fillipe de Araújo¹, Juciele de Oliveira¹, Lívia de Jesus¹, Maria de Fátima Costa¹, Silvia Costa¹, Victor Diogenes da Silva^1,2

¹Departamento de Bioquímica e Biofísica, Universidade Federal da Bahia, Laboratório de Neuroquímica e Biologia Celular (Bahia, Brasil), ²Departamento de Bioquímica e Biofísica, Universidade Federal da Bahia, Programa de Pós-graduação Multicêntrico em Bioquímica e Biologia Molecular (Bahia, Brasil)

INTRODUCTION

Parkinson's Disease (PD) is characterized by a complex pathogenesis that mainly involves mitochondrial damage, oxidative stress, α-synuclein accumulation and neuroinflammation. Such mechanisms responsible for the loss of dopaminergic neurons in the disease are still discussed, however aging is described as the main risk factor for the development of PD. Currently, it is known that the cellular changes inherent to the disease cover several brain regions, including the nigrostriatal system and the cerebellum. The current therapeutic application is only aimed at reducing symptoms and the search for a treatment that reduces the rate of neurodegeneration or that interrupts the development of the disease is continuous. In this sense, flavonoids represent a large class of compounds with neuroprotective, antioxidant and anti-inflammatory potential. More specifically, the flavonoid rutin has the ability to regulate the microglial response and to protect neurons against damage induced by glutamate, as evidenced in previous studies developed by the Laboratório de Neuroquímica e Biologia Celular research group.

OBJECTIVES

Thus, this project aims to investigate the effect of rutin on glial biology and neuronal population at different stages of life and its implications for PD.

MATERIALS AND METHODS

For this, young (3 months) and old (24 months) wistar rats will be treated orally with the flavonoid rutin (10 mg/kg) for 21 days, and on the 14th day the animals will be subjected to behavioural tests. Immunohistochemistry and qPCR will be adopted to assess molecular markers of glial response and neuronal population under conditions of exposure or not to the neurotoxic agent aminochrome a 6 nmol.

DISCUSSION AND RESULTS

It is expected with the development of this project to characterize more broadly the therapeutic potential of rutin for PD, showing its neuroprotective effects against aminochrome toxicity in different regions of the brain, besides to preventing possible changes associated with aging.

CONCLUSION

Keywords: Neuroprotection, flavonoids, aging

Supported by: FAPESP, CNPQ

08387 - Poster Session

HA.46 - The conformational scFv antibody NUsc1 protects differentiated human neuroblastoma cells against Alzheimer’s-associated Aß oligomers toxicity

Nathalia Reges Pinheiro ¹, Giulia Scarcella Cancelliero¹, Izabela Silva Santos¹, Silvana Chedraoui Silva¹, Adriano Silva Sebollela¹

¹Department of Biochemistry and Immunology, University of Sao Paulo (São Paulo, Brazil)

INTRODUCTION

Soluble oligomers of the Aβ peptide (AβOs) are neurotoxins linked to synaptotoxicity and neurodegeneration in Alzheimer's disease (AD). AβOs are heterogeneous in conformation and toxicity and the extent to which different Aβ species contribute to the pathophysiology of AD remains uncertain. In previous work, we selected a scFv fragment antibody, named NUsc1, that distinguishes AβOs from both monomeric and fibrillar Aβ. Different from other conformational anti-Aβ antibodies, NUsc1 preferentially targets a subset of AβOs larger than 50kDa present in AD brain tissue.

OBJECTIVES

Here we aimed to investigate whether NUsc1 is capable of preventing the dysfunction and degeneration induced by AβOs in a human neuronal-like cell model.

MATERIALS AND METHODS

SH-SY5Y neuroblastoma cells were differentiated into mature neurons and challenged with AβOs, either alone or in combination with NUsc1, for 24h. AβO binding to SH-SY5Y cells was assessed by immunofluorescence. Viability was evaluated by both MTT and Live/ Dead assays.

DISCUSSION AND RESULTS

AβO binding to differentiated SH-SY5Y cells revealed a punctate binding pattern. Treatment with AβOs induced a decrease in cell viability as followed with the MTT assay, although not leading to cell death. Importantly, MTT reduction capacity was not affected when the cells were co-treated with AβOs and NUsc1. Additionally, we have compared the neuroprotection conferred by NUsc1 with the protection by the pan anti-Aβ 6E10 antibody, which recognizes multiple forms of Aβ and is known to reduce Aβ-induced toxicity in AD models.

CONCLUSION

We have demonstrated that the conformation-sensitive scFv antibody NUsc1 binds to and neutralizes the neurotoxicity of AβOs on differentiated SH-SY5Y cells. These results support the use of this cell lineage as an in vitro neuronal-like model to study early molecular and cellular events underlying the neurotoxicity triggered by AβOs, and the potential of NUsc1 as a target-selective therapeutic candidate for the treatment of AD.

Keywords: Alzheimer’s Disease, ß-amyloid oligomers, SH-SY5Y neuroblastoma cells

Supported by: FAPESP, CNPq, CAPES, FAEPA

08409 - Poster Session

HA.47 - PROJECT ASSESSMENT OF THE FAK/SRC PATHWAY ROLE IN THE PROLIFERATION AND APOPTOSIS OF JAK2 V617F POSITIVE CELL LINE.

Ana Carolina Menezes Mendonça Valente ¹, Raquel Tognon Ribeiro¹

¹Farmácia, Instituto Ciências da Vida, Universidade Federal de Juiz de Fora campus Governador Valadares (MG, Brazil)

INTRODUCTION

The Chronic Myeloproliferative Neoplasms (MPN) BCR-ABL negative are hematopoietic disease that affect the myeloid lineage cells. These include Polycythemia vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (MF). The identification of the acquired mutation JAK2V617F in patients with PV, TE and MF has led to a better understanding of the pathogenesis of these diseases since the constitutive activation of the JAK2 plays an important role in cell proliferation increases and apoptosis resistance. However, there is still no pharmacological treatment that leads all patients to molecular remission or cure and so, the study of new molecular targets is justified. In turn, the Focal Adhesion Kinase has an important role in cell proliferation, migration and survival and, as it is overexpressed in several neoplasms, it becomes a promising target in the development of cancer drugs.

OBJECTIVES

Thus, the present study proposes to investigate the role of the FAK/SRC signaling pathway in the proliferation and apoptosis of a JAK2V617F positive cell line.

MATERIALS AND METHODS

For this purpose, the following assays will be performed: (1) immunoprecipitation, in order to confirm FAK and SRC interaction in the cell line; (2) FAK inhibition and JAK inhibition assays, alone and in combination, followed by cell viability determination, the detection of the FAK/SRC and JAK/STAT pathways inhibition and apoptosis proteins by Western Blotting.

DISCUSSION AND RESULTS

Considering the role of FAK protein in other neoplasms, it is expected that FAK inhibition will decrease cell viability/proliferation and will induce apoptosis, which will be evidenced by the presence of cleaved PARP and Caspase 3. Moreover, it will be possible infer whether the FAK inhibition interferes with the activation of JAK/STAT pathway.

CONCLUSION

The identification of new molecular targets may contribute to better comprehension of pathophysiology mechanisms and, in the future, to the development of new therapies.

Keywords: Myeloproliferative Neoplasms, FAK, Apoptosis

Supported by: FAPEMIG

08613 - Poster Session

HA.48 - Association of serum levels of IGF-1 with vaso-occlusive crisis in children with sickle cell anemia

Domício Antônio da Costa Júnior ¹, Thaisa Netto Souza Valente³, Ana Paula Pinho Santos ⁵, Andre Barreto Amaral⁵, Célia Maria Silva⁵, Gisele Queiroz Carvalho⁴, Cibele Velloso Rodrigues²

¹Medicine, Federal University of Juiz de Fora - Governador Valadares Campus (Minas Gerais, Brasil), ²Department of Basic Life Sciences, Federal University of Juiz de Fora - Governador Valadares Campus (MINAS GERAIS, Brasil), ³Laboratory of Cell Biology and Molecular Genetics, Federal University of Juiz de Fora - Governador Valadares Campus (MINAS GERAIS, Brasil), ⁴Nutrition, Federal University of Juiz de Fora - Governador Valadares Campus (MINAS GERAIS, Brasil), ⁵Regional Blood Center, Hemominas Foundation (MINAS GERAIS, Brasil)

INTRODUCTION

Sickle cell anemia (SCA) progresses with dysfunction of multiple organs and episodes of acute pain that are caused by vaso-occlusive crises (VOC). Previous studies suggest that insulin-like growth factor I (IGF-1) can act on afferent neurons increasing pain sensitivity.

OBJECTIVES

Assess whether serum IGF-1 levels may be associated with episodes of VOC.

MATERIALS AND METHODS

A cross-sectional population-based study evaluated 39 individuals with SCA, 51,3% (20/39) male, mean age 8.2±2.2 years (range = 3.7-11.9 years). The population was grouped according to the VOC report in the last year. Clinical data and serum levels of IGF-1 were evaluated in each group.

DISCUSSION AND RESULTS

At least one VOC was reported in 59% (23/39) of children. Both groups had similar age (p = 0.66), sex (p = 0.20), body mass index (BMI) SDS (p = 0.52) and height SDS (p = 0.53). The IGF-1 SDS was correlated with the BMI SDS (p = 0.03, r = 0.34), but it did not show any correlation with other anthropometric data evaluated in the research. IGF-1 is produced primarily in the liver under the stimulation of the pituitary growth hormone and is important for height growth, especially for intrauterine growth. Although our study did not show a correlation between the serum level of IGF-1 and birth length (BL), both variables had the same direction in the groups evaluated in the research. Therefore, comparing the groups involved in the study, BL (46.5 cm vs 49.8 cm) and IGF-1 SDS (-1.25 vs -0.62) were significantly (p = 0.02 and 0.03, respectively) higher in the group with episodes of VOC than in the group that did not report any event in the last year.

CONCLUSION

These results suggest that serum level of IGF-1, known to be associated with intrauterine growth, may have an impact on the VOC of children with SCA.

Keywords: IGF-1, vaso-occlusive crisis, sickle cell anemia

Supported by: FAPEMIG/PPSUS MCT, Finep/CT-Infra and PROQUALI-UFJF and CAPES

08790 - Poster Session

HA.49 - Guanosine increases mitochondrial activity via glutamate transport coupling and prevents hippocampal slices damage following oxygen/glucose deprivation

Daniel T. Thomaz¹, Gianni Mancini ¹, Alexandra Latini¹, Carla Ines Tasca ¹

¹Bioquímica, Universidade Federal de Santa Catarina (Santa Catarina, Brasil)

INTRODUCTION

Stroke is a major cause of death and incapacity worldwide and ischemic stroke represents 70% of all strokes globally. The rupture of brain energy metabolism due to reduced oxygen and glucose supply, disruption in ATP synthesis and ROS production, besides glutamate excitotoxicity are hallmarks in the pathogenesis of brain ischemia. Oxygen and glucose deprivation (OGD) in brain tissue preparations reproduces pathological features induced by stroke providing a valuable ex vivo protocol for studying the mechanism of action of neuroprotective agents. We previously showed that the nucleoside guanosine promotes the recovery of ATP, lactate levels and cellular viability in hippocampal slices subjected to 15 min of OGD and followed by 3 hours of reoxygenation (OGD/R).

OBJECTIVES

We here investigated the underlying mechanisms triggered by guanosine on cellular energetic metabolism recovery in an in vitro ischemic model.

MATERIALS AND METHODS

We assessed cellular viability by tackling lactate and glutamate transport and the involvement of mitochondrial oxidative phosphorylation (OXPHOS), through high-resolution respirometry.

DISCUSSION AND RESULTS

OGD/R caused a severe mitochondrial dysfunction through reduction of basal, phosphorylating state and maximal oxygen consumption rates (OCR). Guanosine (100 μM) addition in the reoxygenation period recovered mitochondrial dysfunction, reduction in ATP levels and glutamate uptake into hippocampal slices evoked by ischemia. To understand the neuroprotective mechanism of guanosine, we used α-cyano-4-hydroxycinnamate (4-CIN, 500 μM) a monocarboxylate transporters (MCTs) inhibitor, and DL-threo-β-benzyloxyaspartic acid (TBOA, 10 μM) a non-substrate glutamate transporters inhibitor. 4-CIN prevented guanosine effects on ATP levels and glutamate and lactate transport, but it did not abolished guanosine effects on mitochondrial OCR. On the contrary, TBOA abolished all guanosine effects, indicating a dependence on glutamate transport on the mitochondrial OXPHOS activity.

CONCLUSION

Guanosine treatment prevents mitochondrial dysfunction after OGD/R, pointing to a promising effect of improving brain mitochondria bioenergetics. Therefore, guanosine could act as an adjuvant in battling the sequelae after ischemia, preventing negative outcomes.

Keywords: guanosine, brain ischemia, mitochondria

Supported by: CNPq, INCT-EN, CAPES

08517 -

JA. - On integrative biophysics - Importance of cell receptors for idiopathic and renal hypertension: 50 years research (2021-1970)

Eva Neu ¹, Michael Michailov¹

¹Pharmaco-Physiology, Institute Umweltmedizin c/o ICSD/IAS e.V. POB 340316, 80100 Muenchen, Germany (Int. Council Sci. Develop./Int. Acad. Sci. Berlin-Bratislava-Innsbruck-Muenchen-NewDelhi-Paris-Sofia-Vienna) (, Germany)

INTRODUCTION

During 19.IUPAB-Congress 2017-Edinburgh-[1] and this congress (IUPAB-2021: Michailov/Neu/Martin et-al.) are considered essential approaches to cell-receptors conc. arterial-hypertension&viral-infections. Recently is given information reflecting 50years research as example for future biophysical-education&research in physics-chemistry-biology-medicine-psychology,etc. (post-graduate).

OBJECTIVES

Ref. Michailov-Neu-Martin et-al. [1]-BIOPHYSICS: [a]-IUPAB-2021-Brazil-Virtual (in press), IUPAB-2017-Edinburgh, Eur. Biophysics J. 46/S1:S333(2x), S378,S400. 2014-Brisbane, Proc. p.17,31,73-75. 2011-Beijing, Protein&Cell, S19-03, p.113-4,185-6,581-2. 2008-Long-Beach, Biophys.J., Jan.:125a-126a, 2005-Montpellier, Eur.Biophys.J., 34/6:765&819, 2002-Buenos Aires, Abstract-Book=AB,p.109, 1999-New-Delhi, J.Biosci., 24/S1:3&142, 1996-Amsterdam, Progr. Biophys.&Mol. Biol., 65/S1:170&205, 1993-Budapest, AB-118(2x), 1987-Jerusalem, AB-153(2x), 1984-Bristol, AB-282&283, 1981-Mexico-City, AB-277(2x), 1978-Kyoto, AB-129&157(3x)&231&388, 1975-Copenhagen, AB-146(2x). [b]-EBSA-2011-Budapest, Eur.Biophys.J.(2x) S171,S219, 2007-London, Eur.Biophys.J. 36/Sl:S151&S150, 2005-Montpellier, Eur.Biophys.J. 34/6:765&819, 1997-Orleans, Eur.Biophys.J. 26/1:71. [2]-RADIATION RESEARCH IARR-2019-Manchester (RadRes) Abstract-Book Po.no 116,117,124,192. 2015-Kyoto (part I), p.201,255,825. 2011-Warsaw, p.131-3: Presentations to all congr. 2007-San-Francisco,p.76/2003Brisbane/1999Dublin/1995Würzburg/1991Toronto/1987Edinburgh/1983Amsterdam/1979Tokyo/1974Seattle p.42(2x)/1970Evian p.145. [3]-CHEMISTRY IUPAC-2017-São-Paulo Progr.-Book, p. 121, Po.-No. 2181. 2013-Istanbul. Abstract-Book (ed. Turkish Chem. Soc.&IUPAC), p. 224,611,613,1534. [4]-PHYSIOLOGY. Proc.IUPS 2017-Rio-de-Janeiro, 24, Abs.-ID:977,999,1001,1003. Kyoto-2009/XXII:JPhysiolSci 59/S1:168&214&447-8. Presentations to all congr. New-Delhi-1974/XI+XII:273&378&1133. [5]-PATHOLOGY ESP-IAP(Int.Acad.Pathol.)-2019-Nice, VirchowsArch. Vol.475/Suppl.1, E-PS-25-023/032/033/066. ESP-2018-Bilbao, Eur.J.Pathol. 473/Suppl.1:S180. ESP-2017-Amsterdam, Eur.J.Pathol. 471/Suppl.1:S245/S284/S307. IAP/ESP-2016-Köln, Eur.J.Pathol. 469/Suppl.1:S245. [6]-RAD.ENVIR.BIOPHYS. [a]-20:245-254,1982, [b]-11:289-294,1975, [c]-7:276-282,1971. Experientia 25:621-2/1969

MATERIALS AND METHODS

METHOD: Electro-& pharmacophysiological technique: Electrical&motor activities in-vitro et in-vivo (ref.).

DISCUSSION AND RESULTS

RESULTS: Nicotine&MEG (2-mercaptoethylguanidine, 30mg/100g) changes depressor-reaction=dR to acetylcholine=ACH into biphasic-dR/pR=pressor-one by stimulation of nicotinic-cholinergic receptors=nACHRs, also dR to central-vagal-electrostimulation (55Hz,2ms,5s,5V) (normal&spinal-rats, blood-pressure). MEG potentiates vasopressin=VP pR (0.05-5mU/100g) & dR-bradykinine, also induces inversion of 5-HT dR (0.1-6μg/100g) into pR.

CONCLUSION

Clarification of interaction nicotine-MEG with nACHRs, also alpha-42-beta23, neuronal=VPR1b & vascular=VPR1a and 5-HT1a-f, 5-HT2a-c, 5-HT7 receptors, could open new therapy-approaches of arterial-hypertension, cardiac-arrhythmia, tobacco-pathology, also for molecular-physiological reactions of single-cells incl. corona-virus infections (Michailov/Neu et-al. this congress). This could support UNO-Agenda21 for better health-education-ecology-economy on global level. DEDICATION: Moral&scientific support by Nobel-Laureates: AUSTRIA: K.Lorenz, BELGIUM: I.Prigogine, CHINA-TAIWAN: Y.T.Lee, FRANCE: J-M.Lehn, GB: A.Hewish, B.Josephson, Lord A.Todd, GERMANY: J.Deisenhofer, K.v.Klitzing, H.Michel, E.Neher, ITALY: C.Rubbia, NIGERIA: W.Soyinka, SWEDEN: B.Samuelson, SOUTH AFRICA: Bishop Tutu, USA: P.Anderson, E.Corey, H.Hauptmann, D.Hubel, S.Ting, S.Tonegawa, R.Wilson

Keywords: arterial hypertension, cell receptors, UNO Agenda 21

08459 - Poster Session

JA.01 - EVALUATION OF THE ROLE OF ALLANTOIN IN DRUG RESISTANCE IN LEUKEMIA

RAFAELA RAMOS DE OLIVEIRA DOS SANTOS ¹, JANAINA FERNANDES¹

¹UFRJ - Campus Duque de Caxias, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Leukemia is a hematological cancer characterized by the exacerbated proliferation of cells of hematopoietic tissue. In the beginning of chemotherapy treatment, there may be the development of Tumor Lysis Syndrome (TLS) due to the large amount of intracellular content from lysis of tumor cells. For this, the treatment of TLS, carried out in parallel to the chemotherapy treatment, uses a recombinant urate oxidase enzyme. The enzyme is responsible for converting high serum uric acid levels into allantoin, which is more easily eliminated in the urine. However, there are no clinical studies that show the action of allantoin during chemotherapy treatment until its complete elimination by urine.

OBJECTIVES

Our objective was to investigate if allantoin interferes with the action of cisplatin, in vitro, in sensitive chronic myeloid leukemia cells.

MATERIALS AND METHODS

Chronic myeloid leukemia cells (K562) were cultured in RPMI 1640 culture medium supplemented with 10% fetal bovine serum, 1% antibiotic, and maintained in a stove at 37°C and 5% CO2. The cells were treated with cisplatin 10 μg/ml and allantoin in concentrations of 25 μg/ml, 50 μg/ml, 100 μg/ml and 200 μg/ml. The cell viability assay was performed using the MTT assay, flow cytometry was used to analyze the induction of DNA fragmentation and the loss of mitochondrial membrane potential was observed by fluorescence microscopy.

DISCUSSION AND RESULTS

Our results show that allantoin does not induce cell death and cisplatin leads to decreased viability, in K562 cells. However, we observed that in the presence of allantoin there is a reduction in death caused by cisplatin, allowing us to deduce that allantoin enhanced the survival of K562 cells, preventing the efficient action of cisplatin.

CONCLUSION

Since our results show an alteration in the viability of a sensitive leukemia cell, continuing this study, we will simultaneously investigate the effects of allantoin in sensitive and resistant leukemia cells.

Keywords: allantoin, cisplatin, leukemia

Supported by: FAPERJ

08704 - Poster Session

JA.02 - BIOCHEMICAL ANALYSIS OF THE ACTIVATION OF APOPTOTIC PATHAYS IN THE HIPPOCAMPUS OF RATS FED CAFETERIA DIET AND TREATED WITH ATORVASTATIN

Ítalo Leonardo Diogo ¹, Eduarda Crecêncio Leal¹, Valéria Ernestânia Chaves¹, Leandro Augusto de Oliveira Barbosa¹, Vanessa Faria Cortes¹, Luciana Estefani Drumond de Carvalho¹

¹Campus Centro-Oeste, Universidade Federa de São João del-Rei (Minas Gerais, Brazil)

INTRODUCTION

The accumulation of adipose tissue causes a series of metabolic disturbances. This accumulation can occur due to excessive consumption of food and nutrients and can cause increase in circulating cholesterol levels and in reactive oxygen species, leading to cell death by apoptosis, which can be mediated by Caspase 3. One drug option used to control hypercholesterolemia is atorvastatin.

OBJECTIVES

The aim of this study was to evaluate serum cholesterol levels, adipocyte accumulation, and to assess caspase 3 mediated apoptotic pathway signaling in the hippocampus in rats fed a hyperlipidic, hyperglycemic cafeteria-type diet and treated with atorvastatin.

MATERIALS AND METHODS

Male Wistar rats were divided into four groups: commercial diet - saline (CoSal), commercial diet - atorvastatin (CoArt), cafeteria diet - saline (CafSal), and cafeteria diet - atorvastatin (CafArt). Atorvastatin was administered at doses of 10 mg per rat kg per day and saline at a dose of 0.9%, both by gavage, and the cafeteria groups received water with an addition of 20% of sucrose. Rats were submitted to daily weighing and on the 25th day they were euthanized. Blood was collected for cholesterol quantification and retroperitoneal and epididymal fats were removed for weighing. In addition, hippocampi were collected to evaluate the expression of caspase 3.

DISCUSSION AND RESULTS

All groups gained equivalent weight during the assessed time. However, for the CafSal group, the percentage of fats and serum cholesterol were higher, and atorvastatin (CafArt) was able to return to values equivalent to the control group. In addition, the CoArt group presented values equivalent to the control in all analyzed parameters and the expression of hippocampal caspase 3 did not show differences between the studied groups.

CONCLUSION

Obesity did not alter the total weight but the percentage of fat and circulating cholesterol and atorvastatin reversed this effect. None of the variables affected the expression of caspase 3.

Keywords: Obesity, Neuroprotection, Apoptosis

Supported by: FAPEMIG e UFSJ

08358 - Poster Session

JA.03 - UDESC-CAV/LACEN: Validation of a laboratory for COVID-19 diagnostic support network in Serra Catarinense

Dhébora Mozena Dall'Igna ¹, Ricardo Batista Oliveira¹, Ketriane Mota de Souza¹, Carla Ivane Ganz Vogel¹

¹Programa Multicêntrico de Bioquímica e Biologia Molecular (PMBqBM), Centro de Ciências Agroveterinárias, Universidade do Estado de Santa Catarina (Santa Catarina, Brazil)

INTRODUCTION

Due to the fight against the COVID-19 pandemic, caused by SARS-CoV-2 infection, there is an urgent need to increase the number of exams performed for the diagnosis of this disease. Despite several efforts in creating large-scale diagnostic methods, real-time quantitative reverse transcription polymerase chain reaction PCR (RT-qPCR) is still the gold standard for establishing a COVID‐19 diagnosis. We performed a SARS-CoV-2 RNA RT-qPCR assay methodology using multiple detection temperature (MuDT™) technology, validated by Centers for Disease Control and Prevention (CDC, USA) developed by Charité Virology Institute, from Berlin University, Germany.

OBJECTIVES

Using all the biosafety level 2 (BSL-2) guidelines for research and clinical diagnosis, a commercial SARS-CoV-2 RNA multiple RT-qPCR kit for the E, N and RdRP genes was employed on two Real-Time PCR Detection Systems.

MATERIALS AND METHODS

Molecular SARS-CoV-2 detection involved the steps of RNA viral extraction from oropharyngeal swab (OPS), SARS-CoV-2 reaction mix preparation and RT-qPCR performance. In both RT-PCR detection systems it was possible to validate the results of positive and negative virus detection by Laboratório Central de Saúde Pública of Santa Catarina State (LACEN/SC).

DISCUSSION AND RESULTS

The MuDT™ technology can perform a simultaneous detection and identification of 3 target genes specific for COVID-19, in a one-step reaction workflow. The assay is designed to detect E gene for all Sarbecovirus including SARS-CoV-2 and N and RdRP genes specific for SARS-CoV-2, enabling a performance with high sensitivity and specificity. The kit Internal Control (IC) verify the whole process validation from extraction to PCR whole process control.

CONCLUSION

The validation of a laboratory inside the university with skilled labor using a convenient and reliable methodology can contribute to facing COVID-19 pandemic in Serra Catarinense. This approach allows performing a greater number of exams in a faster way, reinforcing the importance of research work from universities in strategic selection of patients’ management with SARS-CoV-2 infection.

Keywords: COVID-19 pandemic, RT-qPCR, SARS-CoV-2

Supported by: FAPESC, Edital 06/2020.

08740 - Poster Session

JA.04 - Effect of insulator sequence on twist deformation of a single DNA molecule

Shiho ISHII ¹, Kyoko KASHIWAZAKI¹, Haruka NAKAGAWA², Shota KUMAYAMA¹, Naoaki SAKAMOTO², Akinori AWAZU², Yoshihiro MURAYAMA¹

¹Department of Applied Physics, Tokyo University of Agriculture and Technology (Tokyo, Japan), ²Department of Mathematical and Life Sciences, Hiroshima University (Hiroshima, Japan)

INTRODUCTION

Insulator is a DNA element that regulate gene expressions. Insulators have been identified in several organisms, showing enhancer-blocking activity or barrier activity that protect transgenes. Ars Insulator (ArsIns) identified in sea urchin has AT-rich region which is sensitive to nucleotide base modification, suggesting a non-B-DNA structure. This AT-rich region alone exhibits the insulator activity, but specific binding proteins have not been detected, suggesting that local DNA sequence could affect the large deformation necessary for regulation of gene expressions.

OBJECTIVES

In this study, we twisted single DNA molecules with or without ArsIns sequence using opto-magnetic tweezers, and examined the effect of the local sequence on the large deformation of DNA.

MATERIALS AND METHODS

DNA with ArsIns (Ins-DNA, 7058 bp) contained ArsIns (578 bp) approximately in the center of the DNA, and DNA without ArsIns (No-Ins-DNA, 7058 bp) contained a part of lambda-phage DNA instead of ArsIns at the same position. One end of the DNA was attached to glass surface and the other end was attached to a magnetic bead. The DNA was stretched by radiation force of a focused laser to the bead, and twisted by rotating magnetic field created by two pairs of Helmholtz type coil. We obtained relationship between the bead position in height and number of the twist, which is referred as twist curve. The decrease of the bead position during twisting means the formation of plectoneme (super-helical structure with DNA crossing).

DISCUSSION AND RESULTS

Ins- and No-Ins- DNA showed clearly different shapes in the twist curves. Moreover, while only one type of twist curve appeared for No-Ins-DNA, two types of twist curve appeared for Ins-DNA; one of the curve was equivalent to that observed for No-Ins-DNA.

CONCLUSION

These results suggest that large deformation of DNA is sensitive to local DNA sequence, and ArsIns can stochastically switch the large structure of DNA.

Keywords: DNA, Insulator, twist deformation

08034 - Poster Session

JA.05 - Influence of supplementation with polyunsaturated fats of different sources on cell proliferation

Amanda Caroline Rossi de Oliveira¹, Amanda Plaça Bialli², Fabíola Iagher², Marcia Helena Appel ³

¹Departamento de Medicina, Universidade Ferderal do Parana (Parana, Brazil), ²Departamento de Fisiologia, Universidade Federal do Paraná (Parana, Brazil), ³Departamento de Biologia Estrutura, Molecular e Genética, Univesidade Estadual de Ponta Grossa (Parana, Brazil)

INTRODUCTION

Nutraceutics are nutritional supplements from different sources and are claimed to improve health with no or little side effects. Therefore, omega-3 polyunsaturated fats consumption has been associated to longevity. As many cellular events depends on cell membrane lipids characteristics and polyunsaturated fats supplementation is able to modify cell membrane composition. It is important to understand cell behavior in the presence of nutraceutics, such as polyunsaturated fats.

OBJECTIVES

Here in, it is presented the influence of polyunsaturated fats supplementation upon cell proliferation.

MATERIALS AND METHODS

Cell medium was supplemented with fish oil (FO), or shark liver oil (SLO), or oro inca oil (OIO). Each oil was complexed with BSA 1% overnight and They were diluted in cell medium (1:100, 1:200, 1:400). Cell lines with different proliferation ratio and origins were chosen. N2A, HeLa and 3T3 proliferation ratio was tested using Alamar blue die. 3T3 proliferation ratio was tested up to 100h. N2A and HeLa was test up to 180h.

DISCUSSION AND RESULTS

OIO (1:100) was able to decrease N2A, HeLa and 3T3 proliferation. OIO (1:200 and 1:400) decreased 3T3 and increased N2A and HeLa cell proliferation. FO (1:100 and 1:200) had no influence in 3T3 and N2A and increase HeLa proliferation. FO (1:400) decreased 3T3 and increased N2A and HeLa proliferation. SLO (1:100) decreased 3T3 and increased N2A and HeLa proliferation. SLO (1:200 and 1:400) lead to no augment in 3T3 proliferation and increased N2A and HeLa proliferation. The fats tested had different sources omega-3 and composition. OIO is a plant derivative oil rich in ALA which can be converted in EPA and DHA in small amounts. DHA and EPA are present in OF. And SLO has alkylglycerols in composition.

CONCLUSION

Despite the modification in cell lines proliferation influenced by the different oils, cell lines characteristics seems to be determinate to influence proliferation in the presence of polyunsaturated oils.

Keywords: fish oil, oro inca oil, shark liver oil,

Supported by: Fundação Araucária, CNPq

08628 - Poster Session

JA.06 - Biochemical characterization of the interaction between Fbxo7 and androgen (AR) and estrogen (ER) receptors

Valentine Spagnol ¹, Felipe Teixeira^1,2, Patrícia Passos², Karoline Santos², Camila Correia²

¹Department of Biochemistry and Immunology, University of Sao Paulo (Sao Paulo, Brazil), ²Department of Genetics and Evolution, Federal University of Sao Carlos (Sao Paulo, Brazil)

INTRODUCTION

Ubiquitously eXpressed Transcript isoform 2 (UXT-V2) also known as Androgen Receptor Trapped clone-27 (ART-27) is a prefoldin-like protein involved in NF-kB signaling, apoptosis, androgen and estrogen response. UXT-V2 regulates the androgen and estrogen signaling pathways in prostate and breast cancer, acting as a transcriptional coactivator that binds to the N terminus of the androgen/estrogen receptors (AR/ER). Fbxo7 is a component of E3 ubiquitin ligase that interacts and mediates the proteasomal degradation of UXT-V2.

OBJECTIVES

The aim of this study was to evaluate the interaction of Fbxo7 with AR and ER receptors. We also evaluate if the receptors are substrates of SCF(Fbxo7).

MATERIALS AND METHODS

The interaction of Fbxo7 and AR/ER was evaluated by co-immunoprecipitation in HEK293T cells transfected with Fbxo7 and the receptors plasmids in the presence or not of UXT-V2 plasmid. The in vivo ubiquitination assays were also carried out in HEK293T cells transfected with Fbxo7, ubiquitin-myc and the receptors followed by immunoprecipitation.

DISCUSSION AND RESULTS

Our results suggest that Fbxo7 interacts with AR and that the UXT-V2 protein has a negative effect on this interaction. Furthermore, we also verified a potential interaction between Fbxo7 and the ER receptor.

CONCLUSION

These results suggest a possible interaction of AR and ER receptors with Fbxo7 and the functional consequences of this interaction in the androgen/estrogen signaling pathways are being investigated.

Keywords: Fbxo7, AR/ER receptors, UXT-V2

Supported by: CNPq, CAPES and FAPESP

08425 - Poster Session

JA.07 - Identification of miRNAs in mice and their correlation with intracellular pathways associated with the healing process of sickle cell ulcers.

Rafael Souza de Almeida ¹, Simone Garcia Macambira¹, Vitor Antônio Fortuna¹, Victor de Barros Serrano Neves¹

¹Departamento de Bioquímica e Biofísica, Universidade Federal da Bahia (Bahia, Brazil)

INTRODUCTION

Sickle cell ulcers (UF) are among the most serious complications of sickle cell anemia. Treatment is limited to removal of necrotic tissue and infection control. Understanding the mechanisms involved in the pathogenesis of UF is relevant for the development of therapies and the prognosis of patients. In this context, microRNAs represent a promising tool, as they can influence the modulation of genes and growth factors essential for UF healing.

OBJECTIVES

To study the expression profile of miRNA and its correlation with the intracellular pathways associated with the development of UF.

MATERIALS AND METHODS

Public transcriptome data [access code GEO - GSE121996] were analyzed. Samples were examined using bioinformatics tools on the Galaxy platform. A filtering was performed based on the expression level ratio (fold-change) and the adjusted p value < 0.05, to identify microRNAs differentially expressed in different periods after the induction of the lesion.

DISCUSSION AND RESULTS

Differently expressed miRNAs were identified in comparisons of time 0h vs 6h, 0h vs 24h and 0h vs 5 days. Mir7-2 and Mir511 were underexpressed in 0h vs 24hrs and 0h vs 5 days comparisons. Mirlet7c-2 was underexpressed in 0h vs 6h, 0h vs 24h and 0h vs 5 days comparisons. Mir26a-1 was overexpressed in the 0h vs 24hr and 0h vs 5 day comparisons.

CONCLUSION

Preliminary results show an increase in differentially expressed microRNAs throughout healing. Underexpressed microRNAs are related to cell differentiation and proliferation while Mir26a-1 is involved in synaptic plasticity. The future perspective is to establish a correlation between these microRNAs and their target genes during wound healing from sickle cell ulcers, in addition to validating them in in vivo models.

Keywords: sickle cell ulcers, transcriptome, bioinformatics

Supported by: FAPESB

08987 - Poster Session

JA.08 - PROJECT ANALYSIS OF NEURODEVELOPMENTAL ALTERATIONS IN WISTAR RATS SUBJECTED TO ANTIDEPRESSANT TREATMENT DURING THE PRENATAL PERIOD

ANTONIO CARLOS QUEIROZ DE AQUINO ¹, SARAH SOPHIA GUEDES LINHARES², JOSÉ RODOLFO LOPES DE PAIVA CAVALCANTI¹

¹PROGRAMA MULTICÊNTRICO DE PÓS-GRADUAÇÃO EM BIOQUÍMICA E BIOLOGIA MOLECULAR, UNIVERSIDADE DO ESTADO DO RIO GRANDE DO NORTE (RIO GRANDE DO NORTE, BRASIL), ²PROGRAMA DE PÓS-GRADUAÇÃO EM PSICOBIOLOGIA, UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE (RIO GRANDE DO NORTE, BRASIL)

INTRODUCTION

During pregnancy, women are subject to the development of depression. Selective Serotonin Reuptake Inhibitors (SSRIs), like fluoxetine, are usually the first-line treatment for this disease. However, fluoxetine is able to overcome the transplacental barrier and affect the fetus, causing changes in serotonin levels early in life and in the long term can cause damage to brain circuits that control cognitive and emotional behavior as a result of early exposure fluoxetine during neurodevelopment.

OBJECTIVES

In this study, we will address the question of how exposure to fluoxetine (25mg / kg / day) from the thirteenth day (GD13) to the twenty-first day of pregnancy (GD21) can lead to behavioral changes in men and women offspring at 90 days (PN90).

MATERIALS AND METHODS

For this, we will analyze the performance of individuals in behavioral tests: splash test, open field, discriminative avoidance of the maze in cross and context-based fear, of which they are responsible for assessing behaviors similar to anxiety and depression and learning / memory processing. In addition, we will perform neurochemical experiments to understand the dynamics of serotonergic circuits in neurodevelopment, using neuropeptides as specific markers.

DISCUSSION AND RESULTS

Our study seeks to find out whether there are sexual differences modulated by serotonin during neonatal development in offspring subjected to perinatal exposure to fluoxetine (GD13-GD21) and whether it affects behavioral patterns of anxiety, depression and memory.

CONCLUSION

Keywords: NEURODEVELOPMENT, DEPRESSION, FLUOXETINE

08422 - Poster Session

JB.01 - Association of immune biomarkers and oral biofilm in the induction of dental papilla growth by suction technique.

Daniel Guimarães Xavier ¹, Marcos Daniel Silva Pineiro¹, Dirce Ribeiro de Oliveira¹, Andréa Teixeira Carvalho², Lucia Alves de Oliveira Fraga¹

¹Departamento de Ciências Básicas da Vida - DCBV, Universidade Federal de Juiz de Fora - Campus Governador Valadares (Minas Gerais, Brasil), ²CPqRR, Centro de Pesquisas Rene Rachou (Minas Gerais, Brasil)

INTRODUCTION

Gingival recession is highly prevalent among the low-income population and is one of the main causes of pain, tooth loss, and esthetic deficiency. There are still no reports of non-surgical techniques to repair this damage. Due to this gingival area's small vascularization, alternative treatments have presented low success rates. From observations made in several denture wearers, it was confirmed important growth of palate epithelial tissue into the suction chambers in patients with this prosthetic device. Studies show that this epithelium has no capacity for malignant transformation.

OBJECTIVES

We propose an alternative and innovative approach of low-cost and non-invasive treatment, using the suction plate technique, for gingival neoformation and resolution of recession situations.

MATERIALS AND METHODS

To evaluate 30 individuals separated into group 01 (control) and group 02 (patients with gingival retraction). The patients will be molded with alginate, and then suction plates will be made, which should be used for 28 days. Crevicular fluid samples will be collected on days 0, 1, 7, 14, 21, 28 and stored at -80°C until used. Cytokines, chemokines, growth factors, gene expression of antioxidant enzymes, and the local microbiota will be analyzed at each collection time. A pilot study has already been conducted.

DISCUSSION AND RESULTS

Case follow-up of a 38-year-old patient, leucoderma, no history of morbidities, with the gingival recession in the upper incisor region, where he was wearing dental implants, complaining of functional and aesthetic problems was submitted to the use of suction plate for 28 days. During the entire papilla growth induction process by suction, we observed reddish areas, edema, and sometimes bleeding. This confirms the viability of the technique

CONCLUSION

Preliminary results provide positive subsidies for study designs that will underpin public health policies, aiming to establish more effective non-invasive measures for the control and clinical management of patients with gingival recession.

Keywords: gingival recession, biomarkers, suction technique

Supported by: FAPEMIG, CPqRR/FIOCRUZ-MINAS

08269 - Poster Session

JB.02 - Effect of gene therapy with the factor derived from pigmented epithelium in a murine silicosis model.

Igor Correa da Costa Braga ¹

¹Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

Silicosis is an occupational disease related to the exposure of workers to silica particles. Inhalation of silica leads to chronic inflammation and fibrosis of the lung parenchyma. So far, there is no efficient treatment for this disease. In this context, gene therapy emerges as a favourable treatment for inserting genes with therapeutic potentials for silicosis. The associated adenovirus 8 (AAV8) has emerged as a promising vector since it presents a natural tropism for airway epithelial cells and is poorly immunogenic. The factor derived from pigmented epithelium (PEDF) has anti-inflammatory, antifibrotic, and antioxidant activity.

OBJECTIVES

Thus, the present study investigated the hypothesis that the gene encoding PEDF, delivered by the vector AAV8, reduces inflammation and pulmonary remodeling in a murine silicosis model.

MATERIALS AND METHODS

For this purpose, female C57BL/6 mice were randomly divided into two groups: control (C) and silica (SIL). All experimental groups were subdivided into two subgroups: saline subgroups (C-SAL and SIL-SAL) and PEDF subgroups (C-PEDF and SIL-PEDF). We performed three experimental models: treatment 15 days after sílica instillation and treatment 1 and 28 days before sílica instillation. The relative amount of PEDF mRNA, respiratory mechanics, and the fraction of granuloma area were investigated.

DISCUSSION AND RESULTS

Instillation of silica caused histological changes in the lung parenchyma, such as the presence of granulomas, in addition to causing a high mortality rate. In all models, there were no improvements in pulmonary function and histology, in addition to the low expression of the PEDF gene in the SIL-PEDF subgroup. The treatment was ineffective in improving lung function and granuloma fraction in any of the adopted therapeutic strategies. Besides, a decrease in the efficiency of PEDF expression could be observed in the treated sick animals.

CONCLUSION

Further analysis is necessary for a better understanding of the anti-fibrogenic and anti-inflammatory action of AAV8-PEDF in silicosis.

Keywords: Silicosis, Gene therapy, PEDF

Supported by: FAPERJ, CNPQ e CAPES

08402 - Poster Session

JB.03 - Ursolic acid derivatives reduces carrageenan-induced paw edema

Elaine Carlos Scherrer Ramos ¹, Caio César de Souza Alves², Fernando de Sá Silva¹, Paloma Esteves de Carvalho ¹, Gabriela de Paula Carli ², Alessandra de Paula Carli², Jeferson Gomes da Silva¹, Ydia Mariele Valadares¹, Sandra Bertelli Ribeiro de Castro¹

¹instituto de ciências da vida, Universidade Federal de Juiz de Fora campus Governador Valadares (Minas Gerais, Brasil), ²Faculdade de Medicina do Mucuri da Universidade Federal dos Vales do Jequitinhon, Universidade Federal dos Vales do Jequitinhonha e Mucuri (Minas Gerais, Brasil)

INTRODUCTION

The ursolic acid (UA) already is the target of studies that investigate its anti-inflammatory potential and, therefore, structural modifications can enhance its biological activities.

OBJECTIVES

The aim of this study was to evaluate the immunomodulatory effect of the ursolic acid derivatives (UAD) in macrophage response and in carrageenan-induced paw edema model.

MATERIALS AND METHODS

RAW264.7 was cultured in 96-well plates at 2×105 cells·mL-1 in supplemented RPMI-1640. Cells were maintained for 3h and 48h in the presence or absence of UA or UAD (1-19). Nitric oxide (NO) and NF-κB were measured in RAW264.7 cells stimulated with LPS (1 μg·mL-1) and IFN-γ (0.9 ng·m-1). Cellular viability was measured in non-stimulated cell cultures. The paw edema model was induced by carrageenan (2.5%) injection (20 μL) into the left footpad, and 20 μL of PBS into the right footpad, of all groups. The left and the right paws were measured at 1, 2, 3 and 4 h after the injection of carrageenan and the difference were calculated. Dexamethasone was used as control treatment. 30 minutes before the paw edema induction, the UA, UAD 1 and UAD2 (200 mg/Kg) were administered intraperitoneally (100 μL).

DISCUSSION AND RESULTS

The derivatives UAD2, UAD3, UAD4, UAD7, UAD9, UAD10, UAD11 and UAD14 were able to reduce the NO production in relation to the control (p < 0.05; IC50 > 90 μM). The NF-κB expression was evaluated after 3h of culture. The compounds UA, UAD1, UAD2, UAD3, UAD4, UAD5, UAD6, UAD7, UAD8, UAD9, UAD11, UAD12, UAD13, UAD14, UAD15 and UAD18 showed reduction of the NF-κB expression. The UA, UAD1 and UAD2 were able to reduce edema, as well as dexamethasone.

CONCLUSION

The UA and derivatives were able to reduce inflammatory mediators and paw edema. Further studies are necessary to determine the molecular mechanisms of action of these derivatives.

Keywords: inflammation, RAW264.7, ursolic acid

Supported by: FAPEMIG, CNPq and CAPES

08571 - Poster Session

JB.04 - High levels of circulating immune complexes are observed in VL patients with acute kidney injury

Gabriela Corrêa e Castro ^1,2, Maria Luciana Silva de Freitas², Renata Caetano Kuschnir², Ludmila de Paula³, Maria Rita Teixeira Dutra³, Leonardo Soares Pereira³, Glaucia Fernandes Cota⁴, Alda Maria Da Cruz², Adriano Gomes da Silva⁵, Joanna Reis Santos de Oliveira^1,2

¹Núcleo de Ciências Biomédicas Aplicadas, Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (Rio de Janeiro, Brazil), ²Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (Rio de Janeiro, Brazil), ³Hospital Eduardo de Menezes, Fundação Hospitalar do Estado de Minas Gerais (Minas Gerais, Brazil), ⁴Centro de Pesquisas René Rachou, FIOCRUZ (Minas Gerais, Brazil), ⁵Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz (Rio de Janeiro, RJ)

INTRODUCTION

In Brazil, visceral leishmaniasis (VL) is caused by Leishmania infantum. Active phase of VL can vary from asymptomatic form to severe disease and death. Clinically is observed fever, splenomegaly, hepatomegaly, asthenia, anorexia and acute kidney injury (AKI). Depletion of CD4 ⁺ T lymphocytes, polyclonal activation of T and B cells, microbial translocation, cytokine storm and high levels of anti-Leishmania Igs are also involved in the VL immunopathogenesis. Considering that AKI is an important feature of VL, we hypothesized that this clinical condition could be related to high levels of Igs and mediated by immune complex (IC) deposition.

OBJECTIVES

Our aim was to evaluate the circulating IC (CIC) levels in VL patients and whether there is a relationship with markers of severity and renal failure.

MATERIALS AND METHODS

Fourteen VL patients recruited from Hospital Eduardo de Menezes (BH-MG) were evaluated since the active phase until 12 months post-treatment (mpt). Ten healthy controls (HC) were included. CIC and TNF levels were measured by ELISA and correlated with anti-Leishmania Igs levels and AKI markers.

DISCUSSION AND RESULTS

Twelve VL patients met the criteria for AKI according KDIGO2012 guidelines. Interestingly, the anti-Leishmania IgG1 levels were correlated with serum creatinine in post-treatment (r=0.673;p=0.014) and with the variation of glomerular filtration rate (∆GFR) between the active and post-treatment phases (r=-0.900;p < 0.001). These data support the hypothesis of Igs involvement in renal damage. Indeed, not only IgG1-containing CIC but also IgA-, IgM-, IgG- and IgG3-CIC were higher in VL patients than HC until 6mpt (p < 0,05). Interestingly, the ∆GFR was negatively correlated with IgG1-containing CIC (r=-0.682;p=0.012). Concomitantly, the post-treatment levels of TNF-α, cytokine involved in IC-mediated renal injury, was positively correlated with anti-Leishmania IgG1 levels (r=0.671;p=0.020) and negatively correlated with ∆GFR (r=-0.665;p=0.028).

CONCLUSION

Our results suggest that the AKI in active phase of VL was associated with high levels of IgG1, probably driven by IC deposition and TNF-α expression.

Keywords: Visceral leishmaniasis, renal failure, immune complexes

Supported by: FAPERJ, CNPq, IOC/FIOCRUZ, IFRJ

08371 - Poster Session

JB.05 - CYTOKINE PROFILE IN HPV POSITIVE PATIENTS WITH CERVICAL LESIONS

MARIANA MAYUMI TADOKORO ¹, Inês Aparecida Tozetti ¹, Marco Antonio Moreira Puga², Alda Maria Teixeira Ferreira ¹, Cacilda Tezelli Junqueira Padovani³, Camila Mareti Bonin Jacob ⁴, Adriane Cristina Bovo⁵

¹Bioquímica e Biologia Molecular , Universidade Federal do Mato Grosso do Sul (Mato Grosso do Sul , Brasil ), ²Medicina , Universidade Federal do Mato Grosso do Sul (Mato Grosso do Sul , Brasil ), ³Saúde da Família , Universidade Federal do Mato Grosso do Sul (Mato Grosso do Sul , Brasil ), ⁴Imunologia , Universidade Federal do Mato Grosso do Sul (Mato Grosso do Sul , Brasil ), ⁵Oncologia , Hospital do Amor (Mato Grosso do Sul , Brasil )

INTRODUCTION

Persistent Human papillomavirus (HPV) infection is the main cause of the development of neoplastic lesions and cervical cancer in women. This condition is influenced by several factors, including the immune response profile and HR-HPV (high-risk oncogenic HPV) infection.

OBJECTIVES

The aim of this study was to determine the cytokine profile in patients positive for HR-HPV and with cytological alterations such as cervicitis, cervical lesions, and carcinoma.

MATERIALS AND METHODS

The research carried out was descriptive, observational, cross-sectional involving 85 patients attended at Hospital de Amor, Campo Grande - MS unit, during 2019 and 2020. Patients positive for HR-HPV were selected for the study, using the Cobas® method HPV and underwent cervical biopsy, with 40% of the patients diagnosed with High Grade Intraepithelial Lesions (HSIL), 40% with cervicitis, 14% with Low Grade Intraepithelial Lesions (LSIL) and other less significant changes and 6% with carcinoma. Cytokine dosage was performed by flow cytometry, using the CBA Th1/Th2/Th17 BD™ detection system. This project was approved by CEP UFMS (No. 2,685.400 of 05/30/2018).

DISCUSSION AND RESULTS

The average patient’s age was 41.66, and 41% had completed high school. The beginning of sexual life after 16 years was reported by 67% of women. As for the use of condoms, 71% of patients said they did not use condoms. The dosage of Il-17 was a higher in young adults (from 25 to 35 years old), being more frequent also in patients with cervicitis and HSIL. Higher frequency of IL-6 was observed in patients with carcinoma. IL-17 together with IL-6 were more frequent in patients positive for HPV 16 only or associated with other HR-HPV. Statistical analyzes will be developed to verify the meaning of such findings.

CONCLUSION

Higher levels of IL-17 and IL-6 may be associated with an important participation of these mediators in the inflammatory response and in the progression of lesions.

Keywords: immune response, cytokines, HSIL

Supported by: Fundect Called FUNDECT No. 06/2017 - UNIVERSAL-MS and UFMS

08927 - Poster Session

JB.06 - Saturated cardiolipins and cationic lipids activate TLRs: mechanistic basis for immune response in diseases, gene therapy and vaccine formulation

Malvina Pizzuto ^1,2,3,4, Caroline Lonez^1,3, Alberto Baroja‑Mazo⁴, Helios Martinez‑Banaclocha⁴, Panagiotis Tourlomousis³, Pascal Bigey^5,6, Anne-Marie Lachagès^5,6, Virginie Escriou^5,6, Pablo Pelegrin⁴, Jean-Marie Ruysschaert¹, Monique Gangloff², Clare E. Bryant³, Nicholas J. Gay³

¹SFMB, Université Libre de Bruxelles (Brussels, Belgium ), ²Department of Biochemistry, University of Cambridge (Cambridge, United Kingdom), ³Department of Veterinary Medicine, University of Cambridge (Cambridge, United Kingdom), ⁴Biomedical Research Institute of Murcia (IMIB-Arrixaca), University of Murcia (Murcia, Spain), ⁵Université Paris Descartes, Sorbonne-Paris-Cité University (UTCBS, F-75006 Paris, France), ⁶Chimie ParisTech, PSL Research University (UTCBS, F-75005 Paris, France)

Toll-like Receptors (TLRs) are the main protagonists of the innate immune system. Among them, TLR2 and TLR4/MD2 recognize lipids located in bacterial membrane and induce pro-inflammatory reactions. We found that TLR recognition is not limited to bacterial molecules. TLR4 and TLR2 are able to recognize also synthetic cationic lipids, used as nucleic acid nanocarriers, and the mitochondrial lipid cardiolipin. In silico analysis coupled with in vitro and in vivo experiments brought us to understand the molecular parameters for which a lipid would be inert, an agonist or an antagonist of TLRs: short saturated di-acyl cationic lipids lipopolyamines activate TLR2 and 4, longer(≥C18) saturated cationic lipids activate only TLR2, and ≥C18 unsaturated lipopolyamines do not induce TLR activation(Lonez et al., 2015; Pizzuto et al., 2016, 2018); moreover, saturated tetra-acyl lipids like cardiolipins(CLs), activate TLR4, whereas unsaturated CLs are TLR4 antagonists(Pizzuto et al., 2019). Molecular docking show that all CLs fit into the hydrophobic cavity of MD2, however, our analysis failed to predict the pharmacology of CLs, revealing the limitations of such an approach with this family of molecules. By contrast, docking of lipopolyamines in TLR2 suggested potential TLR2 binding modes reminiscent of natural TLR2 agonist and was able to predict their activity. According to our in silico analysis and in vitro data, lipopolyamines with both unsaturated chains do not fit into the hydrophobic pocket and do not activate TLR2.(Pizzuto et al., 2017 J Control Release). We therefore recommend the use of unsaturated C18 chains for the synthesis of inert transfection agents or TLR4 antagonists to be used as therapeutics for TLR4-related diseases such as sepsis, while we recommend saturated lipopolyamines in vaccine formulation as their immunostimulatory activity coupled to their carrier properties conferred good adjuvant properties in vivo(Pizzuto et al., 2018 J Control Release). The ability of saturated CL to activate TLR4 may boost the development of vaccine adjuvants, the understanding of the immune response to bacteria rich in saturated CLs as well as of the chronic inflammation in disease characterized by the presence of saturated CLs. Corresponding author: malvina.pizzuto@gmail.com

Keywords: Toll-like receptor, Cardiolipin, Cationic lipid

08529 - Poster Session

JB.07 - Project morphological and quantitative analysis of peripheral blood neutrophils from patients with Chagas disease

Amanda Estevam Carvalho ¹, Micássio Fernandes de Andrade^1,1

¹Faculdade de Ciências da Saúde, Universidade Estadual do Rio Grande do Norte (Rio Grande do Norte, Mossoró)

INTRODUCTION

Chagas disease, caused by the protozoan Trypanosoma cruzi affects about 7 million individuals worldwide, it has two phases of infection, acute ( high parasitemia, symptomatic or asymptomatic) and chronic (indeterminate, cardiac, digestive or cardio-digestive). Neutrophils are one of the immune system cells type that act in innate defense, but also modulate the response to chronic inflammation. Characterized by being polymorphonuclear cells, they can also present distinct morphology, with ring-shaped nucleus, in patients affected by diverse pathologies, but there are few reports of this event in individuals with trypanosomiasis. The Neutrophil/Lymphocyte Ratio (NLR) can indicate inflammation, being used as a clinical biomarker and aiding in the prognosis and determination of the severity of several clinical conditions. However, there are no reports in the literature about NLR and trypanosome infections

OBJECTIVES

Evaluate the morphology of the neutrophil nucleus and the neutrophil/lymphocyte ratio in the peripheral blood of patients with the different clinical forms of Chagas disease.

MATERIALS AND METHODS

Patients with Chagas disease, who previously accept to participate in the research and signed an informed consent form, will have blood collected through venipuncture. The material will undergo an automatic total leukocyte count. Then, hematological smears, stained with Leishman dye, will pass through total and differential leukocytes count by optical microscopy. Polymorphonuclear cell's nucleus will be counted and evaluated. Resulting data will be correlated to medical records parameters of different clinical forms of trypanosomiasis using the Graphpad Prism software.

DISCUSSION AND RESULTS

CONCLUSION

This study aims to investigate the presence of ring-shaped cells in peripheral blood of patients with Chagas disease, as well as to evaluate and correlate the NLR in these patients with the different chronic forms of the disease and to understand the participation and presence of these cells during the development of this pathology.

Keywords: Neutrophils, Trypanosoma, Neutrophil lymphocyte ratio

Supported by: Capes

08932 - Poster Session

JB.08 - In silico methods applied to identification of potential biomarkers for the development of diagnostic kits for arboviruses

ALESSANDRA SBANO DA SILVA ¹, MANUELA LEAL DA SILVA²

¹DIMAV, Instituto Nacional de Metrologia, Qualidade e Tecnologia (RJ, Brasil), ²PMPGCF, Universidade Federal do Rio de Janeiro (RJ, Brasil)

INTRODUCTION

The Brazilian population is exposed to infections caused by arboviruses widely distributed on the national territory and associated with humans. Cross-epidemics of different arboviruses are frequently and diagnostic methods for patients suspected by Dengue, Zika and Chikungunya virus are limited in many ways. The development of accessible tools becomes essential, because molecular methods are inaccessible for public health. The epitope mapping is widely used in biotechnological applications and constitutes a fundamental portion of the immune system. Experimental methods for epitope mapping are expensive and time consuming. Advances in epitope mapping by computational prediction have molecular insights into the antigen-antibody complex.

OBJECTIVES

The objective was the b-cell epitope prediction of homologous targets NS3 and NSP2, found respectively in the Flaviviridae and Togaviridae families. The identification of differential epitopes in each virus is the first step in developing diagnostic methods based on epitopes.

MATERIALS AND METHODS

The BepiPred-2.0 is used linear in b-cell epitope prediction. Residues above the 0.5 limit were considered epitopes. The statistical cutoff point is defined based on a training set of the server, which used physicochemical parameters of epitope sequences, elucidated experimentally. The ABCpred server was selected for results validation, because is the first server developed based on a recurrent neural network for b-cell epitope prediction.

DISCUSSION AND RESULTS

The statistical cut-off was the standard of 0.5 in length with 16 residues.To filter the results, the location in the secondary structure and also Hydrophobicity (Eisenberg) are observed.The peptides have different characteristics in the NS3 and NSP2 targets for each viral type.

CONCLUSION

The results were satisfactory and compared to other viruses of the family. It is intended to use the results in molecular docking simulations, and make the t-cell epitope prediction. The lead epitopes will be validated experimentally using ELISA.

Keywords: epitope prediction, diagnostic methods, arboviruses

Supported by: PRONAMETRO (INMETRO) and CAPES Biocomputacional

08444 - Poster Session

JB.09 - E-NTPDase-1 and Ecto5’NTase activities in lymphocytes and platelets

TELMA RODRIGUES DA SILVA BENETTI¹, HENRIQUE RANIERE COVALI PONTES¹, MILA MARLUCE LIMA FERNANDES¹, ROMÁRIO DA SILVA PORTILHO¹, IGOR LEAL BRITO¹, JEANDRE AUGUSTO DOS SANTOS JAQUES¹, MALSON NEILSON DE LUCENA¹

¹SETOR DE BIOQUÍMICA/INSTITUTO DE BIOCIÊNCIAS, UNIVERSIDADE FEDERAL DO MATO GROSSO DO SUL (MS, BRASIL)

INTRODUCTION

The purinergic system is a signalling system, where the purine nucleotides, ATP (Adenosine 5′-triphosphate) and ADP (Adenosine diphosphate), and the nucleoside, adenosine, act as extracellular messengers. CD39 (ecto-nucleoside triphosphate diphosphohydrolase 1, E-NTPDase1) converts ATP or ADP into AMP, and then CD73 (ecto-5'-nucleotidase, Ecto5'NTase) dephosphorylates AMP into adenosine. CD39 and CD73 regulate the function of several immune cell types, including lymphocytes. Furthermore, there is evidence that changes in CD39 expression and activity affects the potential thrombogenic of a tissue.

OBJECTIVES

We evaluated comparatively CD39 and CD73 activities in men and women platelet and lymphocytes.

MATERIALS AND METHODS

It have been realized the collection of peripheral blood mononuclear cells (PBMC) rich in lymphocytes and platelet-rich plasma. ATPase, ADPase and AMPase activities were determined by measuring the concentrations of inorganic phosphate (Pi) with the Malachite Green assay.

DISCUSSION AND RESULTS

Platelet from woman showed increased ATPase and ADPase activity compared to enzymatic activity on platelets from man; however the AMPase activity was equal to women and men. Lymphocyte from both women and men showed the same ADPase activity but the ATPase activity was higher to man.

CONCLUSION

Our results suggest that the activity of CD39 and CD73 is different between men and women.

Keywords: CD39, CD73, purinergic system

08821 - Poster Session

JB.10 - Mesenchymal stem cell therapy in Ulcerative colitis, a neuroinflammatory perspective

Alexandre Kleber Silveira ¹, Henrique Gomes¹, Eduardo Klein¹, Luciana Adam¹, Alexia Sant'Ana¹, Dienifer Sirena², José Cláudio Moreira¹

¹Bioquímica, Universiade Federal do Rio Grande do Sul (Rio Grande do Sul, Brazil), ²Centro de Terapia Gênica, Hospital de Clinicas de Porto Alegre (, Brazil)

INTRODUCTION

Ulcerative colitis (UC), trigger a chronic inflammatory process associated with an increase in the permeability of intestinal mucosa, which may lead to the leaky gut phenomenon. This phenomenon causes entrance of molecules from intestinal lumen in the bloodstream, such as proinflammatory mediators that can promote inflammation in central nervous system.

OBJECTIVES

However, little is known about the neuroinflammatory process in ulcerative colitis. Mesenchymal stem cell (MSC) therapy is a promising treatment for UC. Induced colitis animal models treated with MSCs show a reduction in inflammatory cell infiltrates in the intestine, in addition to histological and clinical improvement. MSCs membrane particles (MPs) alone also present anti-inflammatory effects in UC model, without the thromboembolic problems observed in MSCs therapy.

MATERIALS AND METHODS

We induced UC with Dextran Sodium Sulfate (2%) for 7 days in the drinking water of adult wistar rats. Animals were divided in two groups, receiving tap water or 2% DSS and further divided in 6 groups, treated with Saline MSCs or MPs intravenously at treatment day 5. Animals were euthanized and perfunded for immunohistochemical assays.

DISCUSSION AND RESULTS

There was no statistical difference between groups in weight gain. However, we can observe that the only group that presented weight loss at the end of the treatment was DSS alone group. Two proinflammatory cytokines were quantified in the serum. MSCs and MPs reversed the proinflammatory state caused by DSS, returning to the control level in rats’ serum. MSCs play a central role in tissue regeneration, coordinating the repair mechanism through the secretion of healing factors and promoting an anti-inflammatory action in damaged areas

CONCLUSION

Enteric glia is known to play a key role in the intestinal immune response, similar to neural glia. Immunohistochemical analyzes of proinflammatory markers are currently being carried out. We expect that DSS caused neuroinflammation which may be attenuated by MSCs or MPs treatment

Keywords: Ulcerative Colitis, Neuroinflammation, Mesenchymal Stem Cells

Supported by: CNPq

08907 -

JB.11 - Brillouin and Raman microspectroscopy: a new tool for the chemo-mechanical investigation of human bone and cartilage tissue diseases

Martina Alunni Cardinali ¹, M. Govoni², D. Dallari², L. Vivarelli², C. Stagni², M. Rocchi², M. Tschon³, S. Brogini³, M. Fini³, M. Mattarelli⁴, S. Caponi⁵, D. Fioretto⁴, A. Morresi¹

¹Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 (Perugia, Italy), ²Reconstructive Orthopaedic Surgery and Innovative Techniques, Musculoskeletal Tissue Bank, IRCCS Istituto Ortopedico Rizzoli (Bologna, Italia), ³Complex Structure of Surgical Sciences and Technologies, IRCCS Istituto Ortopedico Rizzoli (Bologna, Italia), ⁴Department of Physics and Geology, University of Perugia (Perugia, Italia), ⁵Istituto Officina Dei Materiali, National Research Council (IOM-CNR), Unit of Perugia, c/o Department of Physics and Geology, University of Perugia (Via A. Pascoli, 06123 Perugia, Italy)

Human bone and cartilage are biological tissues characterized by a sophisticated architecture with a strict relationship between their structure, chemical composition, and mechanical performance. As a consequence, an impairment in even one of the sub-constituents at the micrometre level can lead to loss of function of the entire organ, causing the eruption of severe orthopaedic diseases. Osteoarthritis (OA) is a degenerative disease characterized by the progressive erosion of articular cartilage, which covers the surfaces of the long bones in the joints. It is caused by the establishment of inflammatory processes that affects all the joint constituents and subchondral bone, causing in the patient acute pain. Brillouin and Raman micro-spectroscopy is a correlative technique contact-less and not destructive, that allows the simultaneous investigation of both the mechanical and the chemical properties of samples, thanks to endogenous mechanisms, namely the propagation of thermally activated acoustic waves and the macromolecular vibrations. In recent years, this technique has approached applications in the biomedical field, successfully analysing the properties of single cells, complex biomaterials, and tissues affected by oncological and neurodegenerative diseases. Here, we present the results of mapping and imaging performed on both the cortical and trabecular tissue obtained from resections of a human femoral head and diaphysis. The results of the analysis of tissues in healthy conditions will be used to demonstrate the ability of the technique to recognize the major manifestations of osteoarthritic pathology on the cartilage surface and subchondral bone. This proof-of-concept not only constitutes a first step towards the application of the technique in the diagnosis of osteoarthritis but provides an approach that can be extended to other important issues in biomedical research on bone, such as bone infections and cancer.

Keywords: Brillouin spectroscopy, Raman spectroscopy, bone disease

08702 - Poster Session

KA.01 - Conservation and evolution of splice sites

Renan dos Reis ¹, Ricardo De Marco¹

¹Departamento de Física e Ciência Interdisciplinar, Instituto de Física de São Carlos, Universidade de São Paulo (São Paulo, Brazil)

INTRODUCTION

Introns are sequences that interrupt coding regions of eukaryotic genes. They are removed from the pre-mRNA in a process termed splicing, by the action of the spliceosome. For the correct splicing of introns, the spliceosome must recognize two sites present on the intron’s boundaries: the 5' (5'ss) and 3' (3'ss) splice sites. However, those recognitions are not simple tasks for the spliceosome, which must differentiate pseudo sites from the real ones, that are degenerated. Much of what is known about these sites was gathered from analysis of introns in plants, fungi, and animals. However, there are still open questions about their evolution and fundamental features, which require studies of broader phylogenetic branches of eukaryotes.

OBJECTIVES

In this work, the 5′ss and 3′ss sites of the introns of 29 species of metazoan, fungal, and protozoan clades were studied via the analysis of information content of biological signals and frequency of consensus motifs.

MATERIALS AND METHODS

DISCUSSION AND RESULTS

The results show a strong linear negative correlation between the information content of the 5'ss site and the percentage of pre-mRNAs with introns in the genome. In addition, it is observed high conservation among species of both the consensus sequences of the sites and the most frequent motifs found in them, showing that the conservation of recognition mechanisms reaches not simply the global and total pattern of recognition of splice junctions, but also the precise and local patterns of recognition. Finally, it was also possible to identify how the splice sites adapt to structural factors of the introns in which they reside, such as length and GC content.

CONCLUSION

These results, together with the literature, point to a very malleable evolution of splicing sites throughout the evolutionary history of eukaryotes, driven, among other factors, by the number of introns in organisms, the characteristics of these introns, and mutations in the spliceosome.

Keywords: molecular evolution, introns, splice sites

08900 - Poster Session

KA.02 - Flavonoids from Siparuna cristata inhibitors against SARS-Cov-2 3Clpro? An in silico investigation

Maria Eduarda Alves Esteves ¹, Carla Monteiro Leal^2,3, Suzana Guimarães Leitão⁴, Gilda Guimarães Leitão³, Manuela Leal da Silva^1,5

¹Programa de Pós-graduação em Biologia Computacional e Sistemas, Instituto Oswaldo Cruz (Rio de Janeiro, Brasil), ²Programa de Pós graduação em Biotecnologia Vegetal e Bioprocessos, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ³Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ⁴Faculdade de Farmácia, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ⁵Instituto de Biodiversidade e Sustentabilidade NUPEM, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

The covid−19 is a clinical condition of acute respiratory syndrome caused by SARS−CoV−2, which has claimed millions of victims worldwide from 2019 to the present day.

OBJECTIVES

Considering that there is still no specific treatment for this disease, our work proposes the analysis by computational methodology of Flavonoids from Siparuna cristata, substances belonging to Brazilian biodiversity, as inhibitors of the SARS−CoV−2 protease 3CL_pro.

MATERIALS AND METHODS

We evaluated the interaction of 3CL_pro with the following substances: quercetin (1), retusin (2), and kumatakenin (3) and controls: lopinavir (4), ritonavir (5) and chloroquine (6). The prediction of pKa values of the receptor protein was performed using the PDB2PQR at pH 7.4; the probable protonation states of the ionizable residues were adjusted using the GROMACS computational module. From the ligand Narlaprevir, crystallized with 3CL_pro (PDBid: 6XQT), residues at 5 Å were selected, as well as the catalytic dyad His41/Cys145 and Glu166. The grid box was constructed to accommodate the entire highlighted area, with the center dimensions being determined: x=−11, y=1, z=45, and size x=32, y=35, z=33. The Gasteiger charges were added to the protein with AutoDock tools and the docking simulation was performed with the AutoDock Vina program with exhaustiveness equal to 8. The ligands were prepared using OpenBabel.

DISCUSSION AND RESULTS

Substance 2 has obtained the best result making hydrogen bonding with His41 equals to 2.60Å whereas Cys145 was 2.68Å and the interaction energy of -6.3 Kcal/mol with the 3CL_pro. 1 presented -6.5 Kcal/mol and 2.77Å/Cys145; 3 3.01Å/Cys145, 2.19Å/Glu166 and -7.2 Kcal/mol. Moreover 4 demonstrated, 2.92Å/His41 and -5.1 Kcal/mol; 5: 2.41Å /His41, 2.68Å/Cys145, 2.42Å/Glu166 and -8.1 Kcal/mol; 6: 2.11Å/Glu166 and -6.4 Kcal/mol.

CONCLUSION

Through the analyzes carried out, we intend to suggest these natural substances as possible inhibitors of 3CL_pro due previous interaction results, aiming the treatment for covid-19.

Keywords: 3CLpro, flavonoids, in silico

Supported by: CAPES

08071 - Poster Session

KA.03 - Study of molecular mechanisms involved in neurological disorders caused by NS1 protein of Zika virus

Marcos Freitas Parra¹, Eric Allison Philot¹, Pedro Tulio de Resende Lara^1,2, Ana Ligia Scott^1,2, Angelica Nakagawa Lima ³

¹Laboratório de Biologia Computacional e Bioinformática, Universidade Federal do ABC (SP, Brasil), ²Laboratory of Biology and Applied Pharmacology, École normale supérieure Paris-Saclay, France (, França), ³Centro de Engenharia, Modelagem e Ciências Sociais Aplicadas, Universidade Federal do ABC (SP, Brasil)

INTRODUCTION

The nonstructural protein 1 (NS1) has applications such as diagnostic biomarker, viral antigen and antibody recognition. Therefore, NS1 is a potential candidate for compose a vaccine against Zika virus. This protein is a highly conserved protein among flaviviruses with function is related to virial replication.

OBJECTIVES

The main goal is to understand the function of the NS1 protein, classify the different sequences in circulation in the world and suggest new protein sequences with epitopes optimized for diagnosis and composition of an effective vaccine against the Zika virus.

MATERIALS AND METHODS

In this study, the Normal Modes Analysis (NMA) was applied to investigate the flexibility of NS1 protein and mutants. NMA is an efficient methodology to predict flexibility and global movements. For the calculations, we used Charmm program.

DISCUSSION AND RESULTS

The results showed that the NS1 wild types structures availably in Protein Data Bank (PDBs ID 5GS6, 5K6K and 5IY3) had minor changes in the quaternary structure. However, mutations such as the one at position 146 that changes a charged amino acid negatively (glutamic acid – E) by a positively charged one (lysine – K) can alter regions important in the protein. With NMA, we calculated the movements of NS1 wild type and mutants of proteins from Brazil and other countries.

CONCLUSION

In this study, it is possible to observe structural differences in the three-dimensional structures available in the PDB. These changes may be indicative of the occurrence of microcephaly for the structure detected in Brazil. Regarding mutations, it is possible to notice that the 5K6K structure is more susceptible to mutations than the 5GS6 structure, with exception of the Y122H mutation that changes the flexibility of both structures.

Keywords: molecular simulations, NS1, ZIKA

08624 - Poster Session

KA.04 - Drug design and repurposing with DockThor-VS web server: virtual screening focusing on SARS-CoV-2 therapeutic targets and their non-synonym variants

Isabella Alvim Guedes ¹, Leon Sulfierry Corrêa Costa¹, Karina Baptista dos Santos¹, Ana Luiza Martins Karl¹, Gregório Kappaun Rocha², Iuri Malinoski Teixeira¹, Marcelo Monteiro Galheigo¹, Vívian Medeiros¹, Eduardo Krempser da Silva³, Fábio Lima Custódio¹, Helio José Corrêa Barbosa¹, Marisa Fabiana Nicolás¹, Laurent Emmanuel Dardenne¹

¹Mecânica Computacional, Laboratório Nacional de Computação Científica (Rio de Janeiro, Brasil), ²IFF, Instituto Federal Fluminense (Rio de Janeiro, Brasil), ³Fiocruz, Fundação Oswaldo Cruz (Rio de Janeiro, Brasil)

INTRODUCTION

The COVID-19 caused by the SARS-CoV-2 virus was declared as a pandemic disease in March 2020 by the World Health Organization (WHO). Docking methodologies have been widely used for both new drug development and drug repurposing to find effective treatments against this disease.

OBJECTIVES

In this work, we present the developments implemented in the DockThor-VS web server to provide a virtual screening (VS) platform with curated structures of potential therapeutic targets from SARS-CoV-2 incorporating genetic information regarding relevant non-synonymous variations.

MATERIALS AND METHODS

The web server facilitates repurposing VS experiments providing curated libraries of currently available drugs on the market. The DockThor docking program was specially developed to deal with highly flexible ligands, being recently validated to dock highly flexible peptides. The program uses a phenotypic crowding-based multiple solution steady-state genetic algorithm as the search method and a scoring function based on the MMFF94S force field to score the docked poses. The affinity prediction and ranking of distinct ligands are performed with the empirical linear model (trained using 2959 protein-ligand complexes), DockTScore scoring function, recently developed by our research group.

DISCUSSION AND RESULTS

Currently, DockThor-VS provides ready-for-docking 3D structures for wild type and selected mutations for Nsp3, Nsp5, Nsp12, Nsp15, N protein and Spike. We performed VS experiments of FDA-approved drugs considering the therapeutic targets available at the web server to assess the impact of considering different structures and mutations in the identification of possible new treatments of SARS-CoV-2 infection.

CONCLUSION

The DockThor-VS is freely available at www.dockthor.lncc.br. Guest users are allowed to submit VS experiments with up to 200 compounds, whereas registered users with approved projects can submit up to 5,000 compounds per job. The web server utilises the computational facilities of the Brazilian high-performance platform (SINAPAD, https://www.lncc.br/sinapad/) and the supercomputer SDumont (https://sdumont.lncc.br/).

Keywords: virtual screening, molecular docking, COVID-19

Supported by: CNPq, CAPES, Faperj

08345 - Poster Session

KA.05 - Artificial intelligence applied to the structural study of a protein complex related to the nuclear import of the porcine circovirus type 2

Lucas Hecker Vasques ¹, Ramon Hernany Martins Gomes¹, Igor Rodrigues Araújo de Oliveira¹, Rafael Plana Simões², Angelo José Magro^1,3

¹Departamento de Bioprocessos e Biotecnologia, Universidade Estadual Paulista ‘Júlio de Mesquita Filho’ (São Paulo, Brasil), ²Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista ‘Júlio de Mesquita Filho’ (São Paulo, Brasil), ³Instituto de Biotecnologia (IBTEC), Universidade Estadual Paulista ‘Júlio de Mesquita Filho’ (São Paulo, Brasil)

INTRODUCTION

In recent years, Brazil has achieved a prominent position in pork production. Although intensive breeding methods provide high productivity, there are many challenges in porcine farming, including infectious diseases, such as those caused by the viral pathogen Porcine Circovirus Type 2 (PCV2). The nuclear import of this virus occurs by the classic pathway, with the formation of a complex between the protein Importin-α (Impα) from the host and the Nuclear Localization Sequence (NLS) of the viral capsid protein.

OBJECTIVES

This study aims to characterize this complex (Impα/NLS) using computational methods based on artificial intelligence and deep learning in order to identify structural patterns related to the interaction between the PCV2 NLS and Impα.

MATERIALS AND METHODS

To carry out the deep learning calculation, a training dataset was constituted using homologous complexes. These structures were submitted to molecular dynamics (MD) simulations and the calculated structures were used to generate RGB two-dimensional images (200 x 200 pixels) able to codify information as atom pair distances and electric and Lennard-Jones potential.

DISCUSSION AND RESULTS

Based on these data, we expect to classify the most likely Impα/NLS complexes and identifying their essential interface contacts. Therefore, our results could contribute to a better comprehension of the PCV2 biology and help the future development of antiviral drugs and/or other products of biotechnological interest.

CONCLUSION

Additionally, the artificial intelligence method proposed in this work is potentially disruptive and could be used to clarify the general molecular mechanisms related to the classical pathway of nuclear transport in eukaryotes.

Keywords: Importin-alpha, Nuclear Import, Deep Learning

Supported by: FAPESP

08164 - Poster Session

KA.06 - The Effect of Long-Range Electrostatic Treatment on the Sampling of Molecular Simulations of pDMAEMA Polymer Brushes

Vinicius Firmino dos Santos ¹, Denys Ewerton da Silva Santos¹, Thereza Amélia Soares da Silva^1,2

¹Departamento de Química Fundamental, Universidade Federal de Pernambuco (Pernambuco, Brazil), ²Instituto de Física, Universidade de São Paulo (São Paulo, Brazil)

INTRODUCTION

Polymer brushes are a class of polymers bonded to a surface at one end of the polymer chain. The pDMAEMA, poly(dimethylaminoethyl methacrylate) brush can be used for the rational design of gene delivery vectors. Classic computational models of pDMAEMA and other polymer brushes were created and validated in our research group (Langmuir. 2019, 35: 5037-5049).

OBJECTIVES

Recently, we have found that the use of distinct long-range electrostatic (LRE) treatments can interfere with the conformational behavior of the brush.

MATERIALS AND METHODS

We have used the GROMOS-derived atomic parameters to perform molecular dynamics simulations of 90-mers long pDMAEMA brushes with 4, 9 and 16 chains in different protonation states. To investigate the pDMAEMA behavior without the high density of the brush, we simulated systems containing a single fully protonated polymer chain with 30 or 192 monomers and a system containing 40 protonated free monomers. The systems were solvated with SPC water and chloride ions were added to neutralize the charged moieties of the polymer. Two LRE treatments were tested: the Particle Mesh Ewald (PME) and the Reaction Field (RF).

DISCUSSION AND RESULTS

We found that brushes simulated with PME have a greater thickness and greater radius of gyration (Rg) of the polymer chains than those simulated with RF. This effect is accentuated in fully protonated brushes (~16 nm for PME and ~7 nm for RF). Free chains simulated with the PME have also shown a larger Rg compared to simulations with RF. The system containing only monomers in solution showed differences in the peak intensities of its RDFs for different electrostatic treatments. These differences are accentuated in the brushes.

CONCLUSION

The present results show that the choice of LRE treatment influences the conformational sampling and non-bonded interactions in simulations of pDMAEMA polymer brushes, polymer chains and monomers. This should be carefully considered in atomistic simulations of long brushes.

Keywords: Molecular dynamics simulations, Long-range artefacts, Atomic parameters

Supported by: CNPq, CAPES, FACEPE and FNDE

08608 - Poster Session

KA.07 - Fractal dimension applied in the analysis of 17-β-Estradiol chronic effects on the female rat's brain activity

Wibson Silva¹, Raldney Silva¹, Leandro Aguiar², Jeine Silva¹

¹Departament of Animal Morphology and Physiology, Federal Rural University of Pernambuco (Pernambuco, Brazil), ²Center for Higher Studies of Pinheiro, State University of Maranhão (Maranhão, Brazil)

INTRODUCTION

The 17-β-estradiol acts as a modulator in brain function through its receptors located in various regions of the brain. This steroid hormone is related to behavior and cognition functions and is associated with disorders in the central nervous system.

OBJECTIVES

The present work aims to evaluate whether chronic exposure to 17-β-estradiol promotes changes in brain electrical activity.

MATERIALS AND METHODS

Eighteen female Wistar rats (60-day-old) were distributed into three experimental groups: I - estradiol benzoate (20 μg/kg; 0.1 ml sc; sid); II - peanut oil (0.1 ml sc; sid); III - 0.9% sodium chloride (0.1 ml sc; sid). The respective substances were administered subcutaneously for 90 consecutive days (License CEUA-UFRPE nº 075/2019). The record of electrocorticograms (ECoG) was obtained two weeks later, during free moving. Data were processed and analyzed using the box-counting fractal dimension (FD).

DISCUSSION AND RESULTS

In all experimental groups, the FD for ECoG was lower (I= 0.968 ± 0.049; II= 0.986 ± 0.124; III= 0.941 ± 0.053) than those observed for each brain wave. Additionally, the fractality observed in the different brain waves present in ECoG is characterized by higher FD values associated with faster waves, as beta (I= 2.943 ± 0.006; II= 2.942 ± 0.005; III= 2.942 ± 0.008) and alpha (I= 2.695 ± 0.025; II= 2.697 ± 0.024; III= 2.699 ± 0.014), followed by slower theta (I= 2.280 ± 0.030; II= 2.283 ± 0.032; III= 2.280 ± 0.018) and delta (I= 1.472 ± 0.024; II= 1.452 ± 0.049; III= 1.463 ± 0.031) rhythms. There was no significant difference between the experimental groups (p > 0.05).

CONCLUSION

The administration of 17-β-estradiol during 90 days in young rats did not alter the animals' electrocorticographic patterns. We can consider the absence of chronic effects on the animals' brain activity is because of its rapid metabolism and negative feedback on the biological system.

Keywords: ECoG, Electrophysiology, Mathematical Methods

08089 - Poster Session

KA.08 - In silico analysis of natural products from Brazilian biodiversity in COVID-19 treatment: NuBBEDB against SARSCoV2 PLpro

Caio Felipe de Araujo Ribas Cheohen ^1,2, Diego Allonso², Manuela Leal da Silva¹

¹Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ²Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil)

INTRODUCTION

SARS-CoV-2 emerged as a new human pathogen with rapid spread that may cause COVID-19 (WHO,2021). SARS-CoV-2 PL ^pro is a potential target for coronavirus inhibitors, being essential on virus replication. Natural Products Database (NUBBE_DB) was created as the first natural product library from Brazilian biodiversity being a valuable resource for studies. Residues Asn267, Gln269 and Tyr268 are related to protein flexibility, being defined as the main inhibition residues in the search for PL ^pro inhibitors (Russo et al.2020).

OBJECTIVES

This work aims to find a PL ^pro inhibitor on NUBBE_DB, using in silico strategies.

MATERIALS AND METHODS

PL ^pro crystal structure (PDBid:7JRN) was obtained from Protein Data Bank and processed by PDB2PQR server to assess the pKa prediction at pH7.4. GRL0617 redocking and molecular docking were performed with AutoDock Vina (Vina) and GOLD software's. Parameters were defined based on GRL0617 inhibitor center, found on crystal structure. Grid centers x=13, y=−9, z=30, sizes x=30, y=30, z=30; were used in all cases; exhaustiveness=100, number-of-modes=20 were applied in Vina.

DISCUSSION AND RESULTS

Vina redocking presented bind energy of −9.6 kcal/mol and RMSD 0.45356Å between the best pose and the crystal original bind. GOLD redocking are presented through the score for the GOLD/CHEMPLP and fitness for the GOLD/Gold score. Redocking with GOLD/CHEMPLP scored 90.41, RMSD 0.537Å, and GOLD/Goldscore, fitness 59.35, RMSD 0.651Å, both compared to GRL0617. Molecular docking was performed against NUBBE_DB and a penalty score, based on molecular docking molecule’s classification, was developed aiming to compare the molecular docking outputs for each score function. The better the molecule ranks in the docking result; the less penalty will be suffered. Molecules with ≤0.3 penalty score will be selected to proceed in the study.

CONCLUSION

The results will be reclassified based on the distances in Å from Tyr268, described as the main inhibitor residue. Further analyses, such as interaction profile, ADMET and molecular dynamics will be performed.

Keywords: PLpro, SARS-CoV-2, Natural Products

Supported by: CAPES

08021 - Poster Session

KA.09 - Modeling the Reactivity of Iron-Sulfur Proteins

Felipe Curtolo¹, Murilo Hoias Teixeira¹, Guilherme Menegon Arantes ¹

¹Biochemistry, Instituto de Química da Universidade de São Paulo (Sao Paulo, Brazil)

Iron-sulfur (FeS) clusters are essential metal cofactors, comprising the largest class of metalloenzymes. They are involved in a wide variety of catalytic functions such as natural photosynthesis and cellular respiration. From structural and electronic points of view, FeS clusters sit between transition metal atoms and solids. Their electronic structures show many low-lying and near-degenerate states that may cross, leading to multiple-state reactivity. In polynuclear FeS clusters, strong electron correlation and long-range spin coupling effects complicate enormously the theoretical description. Here we describe the reactivity of FeS clusters including environmental effects with quantum mechanical/molecular mechanical (QM/MM) potentials [1] to model Fe-S bare dissociation and substitution reactions in aqueous solution and in protein environments. We show that sextet and quartet spin states cross during bare dissociation in a protein desolvated microenvironment with an homolytic Fe-S bond cleavage mechanism[3]. For water substitution at neutral and acid media, however, no spin-crossings are observed and bond cleavage is heterolytic due to stabilization of the sextet ground state by solvation effects and leaving-group protonation[2,4]. These results help to understand the catalytic mechanisms, stability and biogenesis of iron-sulfur proteins. [1] Ferric-thiolate bond dissociation studied with electronic structure calculations. Arantes GM and Field MJ. J. Phys. Chem. A, 119, 10084-10090, 2015; [2] Modelling the hydrolysis of iron-sulfur clusters. Teixeira MH, Curtolo F, Camilo SG, Field MJ, Zheng P, Li H and Arantes GM. J. Chem. Inf. Model., 60, 653-660, 2020; [3] Homolytic cleavage of Fe-S bonds in rubredoxin under mechanical stress. Arantes GM, Bhattacharjee A, Field MJ. Angew. Chem. Int. Ed., 52, 8144-8146, 2013; [4] Force-induced chemical reactions on the metal centre in a single metalloprotein molecule. Zheng P, Arantes GM, Field MJ e Li H. Nat. Commun., 6:7569, 2015;

Keywords: iron-sulfur clusters, computer simulation, bioinorganic chemistry

Supported by: FAPESP and CNPq

08268 - Poster Session

KA.10 - Stability of the Delta variant of SARS-CoV-2 and prediction of mutations that maximize antibody-antigen interaction

Micael Davi Lima de Oliveira ¹, Jonathas Nunes da Silva¹, Clarice de Souza Santos², João Alfredo Holanda Bessa Neto², Rosiane de Freitas², Kelson Mota Teixeira de Oliveira¹

¹Laboratory of Theoretical and Computational Chemistry, Federal University of Amazonas (Amazonas, Brazil), ²Institute of Computing, Federal University of Amazonas (Amazonas, Brazil)

INTRODUCTION

The COVID-19 pandemic is of unprecedented impact since the 1918 Spanish flu. The most recent strain of concern of SARS-CoV-2 is Delta (B.1.617.2), responsible for an increase in infections in India and reported with a large increase in viral transmissibility.

OBJECTIVES

We studied the stability of the Delta variant mutations. Finally, was performed a computational screening of mutations that maximize the antibody-antigen interaction.

MATERIALS AND METHODS

We initially performed the stability prediction of the L452R and T478K mutations using the "Residue Scanning" module in the Schrödinger Maestro 2021-2 software. Then, we use the "Affinity Maturation" functionality, whereby the Monte Carlo optimization method we find the mutations that maximize the stability of ACE2-RBD (PDB ID: 6M0J) and antibody-antigen (PDB ID: 7BWJ) binding. Throughout the interpretation of the results, it was considered that the negative sign denotes an increase in stabilization and affinity.

DISCUSSION AND RESULTS

We found that the Delta variant L452R mutation achieved stability at -8.161 kcal/mol and affinity of -0.182 kcal/mol for ACE2-RBD. Although the T478K mutation showed a destabilization at +19.490 kcal/mol, it induced an increase in affinity at -5.046 kcal/mol. In this way, we can see an evolution in search of greater structural stability. These values are relatively far from what the virus could actually achieve in terms of stability, where we obtained the maximized value at -96.252 kcal/mol. This, therefore, indicates that SARS-CoV-2 has been seeking marginal stability as a form of adaptation. Regarding the maximization of the antibody-antigen interaction, we found that in terms of stability T333H, A363M, V510I with -33.543 kcal/mol. In terms of affinity, we obtained T333H, V445Q, H519R with -10.501 kcal/mol.

CONCLUSION

Finally, we are currently running molecular dynamics simulations to confirm these conclusions. Therefore, we hope with these results to maximize the effectiveness of the next generation of vaccines so that they are protected against emerging variants.

Keywords: Delta variant, Monte Carlo, SARS-CoV-2

Supported by: CNPq

08471 - Poster Session

KA.11 - In silico identification of a novel open reading frame in the pseudogene MAP_RS22950 from Mycobacterium avium paratuberculosis

João Paulo da Cruz Farias¹, Ângela Camila Orbem Menegatti²

¹Ciências Biológicas, Campus Professora Cinobelinas Elvas, Universidade Federal do Piauí (Piauí, Brazil), ²Departamento de Biologia Molecular, Centro de Ciências Exatas e da Natureza, Universidade Federal da Paraíba (Paraíba, Brazil)

INTRODUCTION

Mycobacterium avium paratuberculosis (MAP) is the causative agent of Johne's disease in ruminants, a chronic granulomatous inflammatory bowel disease, and strongly associated with Crohn's disease in humans. Similar to the closely related Mycobacterium tuberculosis (Mtb), MAP encodes a functional protein tyrosine phosphatase A, PtpA, but no protein tyrosine phosphatase B, PtpB sequence is annotated as a pseudogene. PtpA and PtpB from Mtb are two secreted virulence factors essential for its pathogenicity, and hence they become interesting targets for anti-tuberculosis drug development.

OBJECTIVES

Based on novel open reading frames (nORFs) discovery, we aimed to further investigate the sequence of the pseudogene MAP_RS22950 for the presence of possible ORFs.

MATERIALS AND METHODS

Using the ORF finder (NCBI), we identified 11 potential ORFs for the pseudogene. To search for encoding hypothetical proteins each translated ORF was analyzed by BLASTp, we found 2 ORFs encoding proteins with similarities (< 80%) to tyrosine phosphatases.

DISCUSSION AND RESULTS

Multiple sequence alignments using Clustal Omega showed that ORF4 (193 amino acids) has the catalytic site of PTPs, the P-loop, but does not have the FPD-loop. Neighbor-joining phylogenetic tree generated with MEGA X showed that the closest related proteins of ORF4 were PTPs from Mycobacterium avium, Mycobacterium lepraemurium and Mycobacterium timonense. The sequence alignment (Clustal Omega) of ORF4 against a PTP from Mycobacterium intracellulare revealed that the FPD-loop would be replaced by the amino acid residues Ala8, Pro9 and Thr10. The theoretical 3D model of ORF4 predicted by SWISS-MODEL was related to the Mtb PtpB (PDB: 2oz5). The temple covered 85% of ORF4 sequence (range 30 to 193) and showed GMQE and QMEAN score of 0.77 and −0.03, respectively.

CONCLUSION

Further experimental validation of the existence and functional roles of these ORFs need to be performed to validate the in silico data, and thus contribute to a better understanding of MAP pseudogene.

Keywords: Mycobacterium, Paratuberculosis, ptpB

Supported by: UFPI

08378 - Poster Session

KA.12 - Experimental and computational studies of essential oils insecticidal action on Calliphoridae flies and identification of their possible molecular targets

Eduardo Jose Azevedo Correa ¹, Frederico Chaves Carvalho², Raquel Cardoso de Melo-Minardi², Leonardo Henrique França de Lima¹

¹Bioquímica e Biologia Molecular, Universidade Federal de São João Del Rey (Minas Gerais, Brazil), ²Departamento de Ciência da Computação, Universidade Federal de Minas Gerais (Minas Gerais, Brazil), ³Departamento de Ciência da Computação, Universidade Federal de Minas Gerais (Minas Gerais, Brazil), ⁴Departamento de Ciências Exatas e Biológicas, Universidade Federal de São João Del Rey (Minas Gerais, Brazil)

INTRODUCTION

Flies in livestock are a huge problem and identification of targets and new compounds to control are crucial in the development of insecticides.

OBJECTIVES

This work aims to identify, through bioinformatic and bioassays, new plant compounds that could be used as fly killers.

MATERIALS AND METHODS

Essential oils were obtained with Clevenger apparatus/gas chromatography and from literature performing 170 database structures/ligands. A laboratory bioassay on blowfly using essential oil of Baccharis dracunculifolia shows, until now, that its essential oil has an LC50 of 3,33 μL/cm2. It is observed a pupae size reduction and adult abnormalities. We have performed in silico experiments to identify the compound and target which affects fly metabolism. Seven fly metabolism targets were modeled. Molecular docking and two score functions have been used to rank compounds with high affinity to receptors. A chemical-physical computational database of high-affinity compounds was made and the euclidean distance of stereochemical restrictions for target and distance of plant essential oils studied in our bio essays was calculated.

DISCUSSION AND RESULTS

Comparing receptors, Octopamine and Agonist Ultraspiracle are most typical presenting the unique stereochemical character as a potential target for selective insecticide. The lowest euclidean distance from other targets was obtained with the Ecdysone receptor suggesting that compounds acting on it could act on the other targets too; it is known that terpenoids act on a myriad of targets. Considering plant species and euclidean distance, B. dracunculifolia’s oil is predicted to interact with different targets, in agreement with insecticide and anti-metamorphosis effects documented. Computational tools could explain bioassay data by the high affinity of oil compounds by Acetylcholinesterase and Antagonist Octopamine affecting the metamorphic cycle binding to Juvenile Hormone and Ecdysone receptors.

CONCLUSION

In general, B. dracunculifolia’s oil is the most promising source for new insecticides, corroborating with bioassays and the literature signaling anti-metamorphic and neurotoxic effects.

Keywords: Computational biology, essential oils, blowfly

Supported by: EPAMIG/UFSJ

08547 - Poster Session

KA.13 - Unveiling Mutation Effects on the Structural Dynamics of the Main Protease from SARS-CoV-2 with Hybrid Simulation Methods

Patricia Gasparini ¹, Jean Carlos Mattos¹, Natanael Endrew Souto Maior Torres-Bonfim¹, Roberto Carlos Navarro Queiroz¹, David Perahia², Angelo José Magro³, Eric Allison Philot¹, Ana Ligia Scott¹

¹Laboratório de Biofísica e Biologia Computacional, Universidade Federal do ABC (São Paulo, Brazil), ²Laboratory of Biology and Applied Pharmacology , École Normale Supérieure Paris-Saclay (Saclay, France), ³Departamento de Bioprocessos e Biotecnologia, Universidade Estadual Paulista (São Paulo, Brazil)

INTRODUCTION

The 2019 coronavirus disease (COVID-19) pandemic, caused by the human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain, is the most health crisis in the past 100 years. Macromolecules adopt several favored conformations in solution depending on their structure and shape, determining their dynamics and function. Integrated methods combining the lowest-frequency movements obtained by Normal Mode Analysis, faster movements from Molecular Dynamics are necessary to establish the correlation between complex structural dynamics of macromolecules and their function.

OBJECTIVES

The aim of this study was to characterize the structural dynamics and global motions of Mpro Protease of SARS-CoV-2 wild-type (WT) and of its 48 mutants, including several mutations that appear in five importants variants of concern.

MATERIALS AND METHODS

PyMOL software was used to generate 48 mutants with single point mutations reported by Amamuddy and colleagues (2020). These mutants were minimized and calculated the first collective modes with an all-atom approach. Twenty seven mutants with a significant difference of RMSF compared to WT were selected and energetically relaxed conformations were generated along the lowest frequency normal modes with the Hybrid method VMOD. After, these structures were analyzed considering several aspects: flexibility, global movements, dyad catalytic and the accessible area for the catalytic site. We used several tools: analysis of variance (ANOVA) test, scripts developed in Python and R-studio, PDBePISA (Proteins, Interfaces, Structures and Assemblies) and TKSA-MC.

DISCUSSION AND RESULTS

The results suggested that a single mutation cause a significant change in the flexibility and collective motions of Mpro, reflecting in importants structural characteristics: energy, electrostatic contribution for free energy, energetically accessible conformations, SASA of important regions as the interface of the dimer and the distance of dyad catalytic. Four mutants (K90R, N151D, P108S and P99L) showed significant energy and SASA reduction and could be more stable.

CONCLUSION

Hence, the mutants present low conservation of these motions compared with WT.

Keywords: Variants of concern, Normal Modes, Molecular Dynamics

Supported by: CNPq, CAPES, Fapesp and UFABC

08897 - Poster Session

KA.14 - Computational Study of Synthetic Aromatic Aldehydes as Potential Inhibitors of GABA-AT

Maíra Rocha¹, Rhaiza Domingues¹, Erich Tahara¹, Lucas Bleicher¹, Rafael Vieira¹

¹Departament of Biochemistry and Immunology, Universidade Federal de Minas Gerais (Minas Gerais, Brazil)

INTRODUCTION

Synthetic aromatic aldehydes are consumed worldwide. They are structurally similar to pyridoxal 5'-phosphate (PLP), a B6 vitamer and essential cofactor of neurological disorders-related enzymes. We investigated 17 aromatic aldehydes as potential cofactors of gamma-aminobutyric acid aminotransferase (GABA-AT), a PLP-enzyme, using a computational model.

OBJECTIVES

We aimed to obtain and validate a computational model of GABA-AT from Saccharomyces cerevisiae. The model was subsequently used in docking procedures to evaluate covalent and non-covalent interactions between the aldehydes and the model.

MATERIALS AND METHODS

A tridimensional structure for P17649 GABAT_YEAST was obtained by homology modeling and compared to the template P80147 GABAT_PIG (PDB 1ohv). Target and template sequences were aligned by Clustal Omega web server. Modeller 9.24 was used to obtain 100 model structures for P17649 GABAT_YEAST. All models were submitted to the composite scoring function QMEAN and Ramachandran plots were analyzed with RAMPAGE to estimate quality. A web-based computational prediction of protonation states at pH value 8.3 in the best model and in the template was performed by H++ server. Covalent docking (CD) by GOLD plataform was performed with aldehydes and PLP, using the best model and the template.

DISCUSSION AND RESULTS

M95 was selected as best model according to its QMEAN value (-2,64) and Ramachandran plot statistics revealed 93.9% of residues in the preferred region. CD was simultaneously performed by two investigators, to compare and validate the classifications. The PLP score values for both M95 and the template varied between 110.6 and 113.3. All the aldehydes presented score values between 99.0 and 118.4, significantly equivalent to PLP results.

CONCLUSION

Methoxybenzaldehyde derivatives displayed values in the 115.6-118.4 range, the higher scores in comparison to PLP. The results stimulate subsequent in vitro assays aiming yeast lifespan modulation, paving the way for aldehyde-based development of human GABA-AT inhibitors as drug candidates for neurological disorders.

Keywords: covalent docking, neurological disorders, synthetic aromatic aldehydes

Supported by: FAPEMIG, CNPq and CAPES

08769 - Poster Session

KA.15 - Niferidil drug for the treatment of short QT syndrome: experiment in silico

Matheus Leonardo Alves de Camargo ¹, Daniel Gustavo Goroso^1,2, Robson Rodrigues da Silva^1,3

¹Núcleo de Pesquisas Tecnológicas, Univesidade de Mogi das Cruzes (SP, Brasil), ²Human Motor Skill Analysis Laboratory, National University of Tucumán (, Argentina), ³Center for Biomedical Engineering, University of Campinas (, Brasil)

INTRODUCTION

Computational tools based on mathematical models have collaborated in the discovery and repositioning of antiarrhythmic drugs, helping in decision-making and speeding up the obtaining of results.

OBJECTIVES

In this context, we developed the computational tool PharmaLab, which simulates the pharmacological action in the mouse ventricular myocyte.

MATERIALS AND METHODS

The new proposed model was based in Mullins & Bondarenko (2013) and pharmacological dynamical based on Hill's equation. The short QT syndrome (SQTS) was simulated as an application of the computational tool. According to Campuzano et al. (2018), this disease alters the cardiac Action Potential (AP) and generates a faster repolarization. The conductance of the fast potassium current (G_Kr = 0.078mS/μF) was multiplied by a factor of 1000 to simulate this condition. To correct the SQTS, it was simulated the application of the drug Niferidil, which acts on the main potassium currents (I_Ktof, I_Kur and I_Kss). The concentrations applied of Niferidil were 25μM, and 50μM.

DISCUSSION AND RESULTS

The experiment in silico showed that SQTS mainly changes the time of 90% of repolarization of AP (APD90) reducing the value from 10.04ms (control) to 8.29ms (test). It was found that the drug Niferidil corrects the APD90 for the concentrations applied, 9.92ms and 11.04ms, respectively. However, it was observated an increase in the time of 50% of repolarization of AP (APD50), this increase being 8.28% and 16.56% compared to control for the concentrations of 25μM and 50μM, respectively.

CONCLUSION

Although new tests are needed to confirm the effects of this drug on AP. The results obtained with the simulations are in accordance with the data reported by Abramochkin et al. (2015).

Keywords: in silico experiment, drug, cardiovascular disease

Supported by: CAPES

08432 - Poster Session

KA.16 - Gene family characterization of superoxide dismutase responsible for tolerance to oxidative stress in Jatropha curcas L.

Luiza Carolina Monteiro Souza ¹, Neide da Hora Conceição^1,2, Renato Delmondez de Castro¹, Luzimar Gonzaga Fernandez^1,2

¹Laboratório de Bioquímica, Biotecnologia, Bioprodutos (LBBB/ICS/UFBA), Universidade Federal da Bahia (Bahia, Brasil), ²Programa Multicêntrico de Pós Graduação em Bioquímica e Biologia Molecular, Universidade Federal da Bahia (Bahia, Brasil)

INTRODUCTION

Jatropha curcas L. (physic nut) is an easily propagated, rapid growth oilseed adapted to semi-arid regions that are low in nutrients. The plant has defense mechanisms which provides resistance to abiotic stress caused by temperature, salinity, drought, heavy metals, among others. The tolerance level is correlated with the expression of antioxidant enzymes such as superoxide dismutase (SOD), which catalyzes the decomposition of O₂ ^- to O₂ and H₂O₂.

OBJECTIVES

The aim of this study was to characterize the superoxide dismutase gene family in J. curcas (JcSOD) and to verify phylogenetic relationship with other species.

MATERIALS AND METHODS

Sequences of JcSOD and four other species (Ricinus communis, Glycine max, Helianthus annuus and Gossypium hirsutum) were retrieved from the NCBI database, followed by confirmation of the presence of JcSOD domains by Simple Modular Architecture Research Tool (SMART). Alignment was performed with the MUltiple Sequence Comparison by Log-Expectation (MUSCLE) and the verification of the phylogenetic relationships by the Software Molecular Evolutionary Genetics Analysis (MEGAX). The subcellular locations were predicted by the protein subCELlular LOcalization prediction (CELLO) and the conserved motifs in the sequences by the Multiple Em for Motif Elicitation (MEME).

DISCUSSION AND RESULTS

The JcSOD genes were characterized based on the phylogenetic analysis, subcellular locations and distribution of conserved protein motifs in relation to the SOD genes of the other analyzed species. The five species contain conserved domains, several SOD isoforms that differ in their metallic ion at the active site, however all species had Cu/ZnSOD and FeSOD in common.

CONCLUSION

It is concluded that SOD is an important enzyme in plant stress tolerance, and the results provide basis for further functional research on the SOD gene family, which is the first line of defense and one of the most efficient antioxidant enzymes against reactive oxygen species in J. curcas.

Keywords: Antioxidant enzymes, Phylogeny, Physic nut

Supported by: CNPq, FAPESB, UFBA

08824 - Poster Session

KA.17 - Computational Modeling of ZIKV MTase NS5-SAH Complex

Lilian Mendonça Alves de Oliveira ⁵²

⁵²Institute of Biophysics Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (RJ, Brasil)

INTRODUCTION

The Zika virus (ZIKV) has emerged as an international public health concern due to its serious symptoms, notably the evidence that has accumulated to conclude that infection during pregnancy is a major cause of microcephaly and other severe fetal brain defects. Parallel to the development of an efficacious vaccine, research on chemotherapy focusing on specific proteins are excellent alternatives for the inhibition of viral replication. The NS5 protein of ZIKV is one of the most important and conserved Flaviviridae enzyme, which contains two domains: a N-terminal methyltransferase (MTase) and a C-terminal RNA polymerase. The function of the MTase domain depends on an S-Adenosyl methionine (SAM) cofactor that acts as a methyl group donor, and is thus converted to S-Adenosyl Homocysteine (SAH). The MTase domain methylates the viral RNA and prevents it from being recognized by the host’s immune system. Therefore, NS5 MTase can be considered a promising target for drug design.

OBJECTIVES

To investigate the NS5 MTase domain in complex with SAH (PDB ID: 5NJU) through Molecular Dynamics (MD) simulations to design an inhibitor for this enzyme.

MATERIALS AND METHODS

MD simulations were performed using AMBER18 package in three different conditions: (I) protein without SAH solvated with 70% water and 30% ethanol. This mixed solvation set-up was carried out using the Packmol software, (II) protein without SAH in simple aqueous system and (III) protein-SAH complex in water.

DISCUSSION AND RESULTS

The mixed solvent induced a quick structural stabilization of NS5 MTase even without SAH binding. Conversely, the apo protein remains more flexible in water solvent. The map of mixed solvent binding microsites on the protein surface showed important regions to explore with pharmacophoric groups for the design of an inhibitor.

CONCLUSION

We sugested a conformational change around the active site on the unliganded NS5 in water, highlighting that a cavity is open and can be explored as a target for allosteric inhibitors.

Keywords: Molecular Dynamics, NS5, ZikV

Supported by: FAPESP, CNPq and CAPES

08298 - Poster Session

KA.18 - Molecular docking experiments from different isoforms of the human importin alpha with nuclear localization signals

Ivan Rossi Moraes ¹, Hamine Cristina de Oliveira¹, Tainá Dorte da Silva¹, Guilherme Henrique Marchi Salvador¹, Cíntia Akemi Fukuda¹, Marcos Roberto de Mattos Fontes¹

¹Departamento de Biofísica e Farmacologia, Universidade Estadual Paulista (São Paulo, Brazil)

INTRODUCTION

The Nuclear Import Classical Pathway, which import proteins that contain the classical nuclear localization sequences (NLSs) are transported into the nucleus by the importin-α/β heterodimer. Importin-α (Impα) contains the NLS recognition site and, importin-β (Impβ) mediates transport through the nuclear membrane pore. Several proteins related to DNA repair are transported to the cell nucleus by the classical NLS-import pathway and among them, there is a heterodimer formed by the proteins MLH1 and PMS2. These proteins belong to the mismatch repair pathway (MMR) which is responsible for repairing error in DNA replication. Previous studies from the heterodimer interacting with Mus musculus Impα (MmImpα) have shown that this complex binds with high affinity. To better understand the importance of the NLS residues, four mutated MLH1 NLSs were chosen to perform many structural assays, including the molecular docking.

OBJECTIVES

The aim is to compare if the human Impα (HsImpα1 and 3) interacts to the MLH1 mutated NLSs in the same way as the Mus musculus Impα.

MATERIALS AND METHODS

Molecular docking using the MLH1 and mutated MHL1 peptides and crystal structures of the HsImpα1 and HsImpα3 was performed by the Autodock Vina tool, present in the software PyRx. The best complexes obtained were chosen by two criterias, the antiparallel conformation of NLS related to the Impα models and Gibbs free energy expressed in kcal/mol.

DISCUSSION AND RESULTS

The HsImpα1 interacts with the MLH1 peptides in a very similar way as the MmImpα, as expected, since they share a very high sequence identity. In contrast, the HsImpα3 only bind to these peptides in the major site, due to natural mutations of some key residues in the minor site.

CONCLUSION

We were able through comparisons to reveal the specificity of the Impα interactions in the Classical Nuclear Import Pathway, and the HsImpα1 and HsImpα3 bind in a different way to the NLSs.

Keywords: Importin, Molecular Docking, Nuclear Localization Signal

Supported by: FAPESP, CNPq, CAPES

08847 - Poster Session

KA.19 - Computational Study of Synthetic Aromatic Aldehydes as Potential Inhibitors of GABA-AT

Maíra Rocha¹, Rhaiza Domingues¹, Lucas Bleicher¹, Erich Tahara¹, Rafael Vieira¹

¹Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais (Minas Gerais, Brazil)

INTRODUCTION

Synthetic aromatic aldehydes are consumed worldwide. They are structurally similar to pyridoxal 5'-phosphate (PLP), a B6 vitamer and essential cofactor of neurological disorders-related enzymes. We investigated 17 aromatic aldehydes as potential cofactors of gamma-aminobutyric acid aminotransferase (GABA-AT), a PLP-enzyme, using a computational model.

OBJECTIVES

We aimed to obtain and validate a computational model of GABA- AT from Saccharomyces cerevisiae. The model was subsequently used in docking procedures to evaluate covalent and non-covalent interactions between the aldehydes and the model.

MATERIALS AND METHODS

A tridimensional structure for P17649 GABAT_YEAST was obtained by homology modeling and compared to the template P80147 GABAT_PIG (PDB 1ohv). Target and template sequences were aligned by Clustal Omega web server. Modeller 9.24 was used to obtain 100 model structures for P17649 GABAT_YEAST. All models were submitted to the composite scoring function QMEAN and Ramachandran plots were analyzed with RAMPAGE to estimate quality. A web-based computational prediction of protonation states at pH value 8.3 in the best model and in the template was performed by H++ server. Covalent docking (CD) by GOLD plataform was performed with aldehydes and PLP, using the best model and the template.

DISCUSSION AND RESULTS

M95 was selected as best model according to its QMEAN value (-2,64) and Ramachandran plot statistics revealed 93.9% of residues in the preferred region. CD was simultaneously performed by two investigators, to compare and validate the classifications. The PLP score values for both M95 and the template varied between 110.6 and 113.3. All the aldehydes presented score values between 99.0 and 118.4, significantly equivalent to PLP results.

CONCLUSION

Methoxybenzaldehyde derivatives displayed values in the 115.6-118.4 range, the higher scores in comparison to PLP. The results stimulate subsequent in vitro assays aiming yeast lifespan modulation, paving the way for aldehyde-based development of human GABA-AT inhibitors as drug candidates for neurological disorders.

Keywords: Covalent docking, Neurological disorders, Synthetic aromatic aldehydes

Supported by: FAPEMIG, CNPq and CAPES

08736 - Poster Session

KA.20 - Development of Coarse-grained model for protein complex

Kazutomo Kawaguchi ¹, Hidemi Nagao¹

¹Institute of Science and Engineering, Kanazawa University (, Japan)

INTRODUCTION

Molecular simulations are useful to investigate dynamics and statics of biomolecules. Coarse-grained (CG) models can reduce computational cost of large-scale and long-time simulation for biomolecules. CG models for single protein have been developed to understand protein folding. CG models for protein complex should be developed to understand protein complex formation, which takes much computational cost than protein folding.

OBJECTIVES

In this work, we develop a CG model for protein complex system and apply our CG model to formation of protein complex.

MATERIALS AND METHODS

In our CG model, intra-molecular interaction was represented by the Go-like model, in which each amino acid residue was described by a single CG particle. Inter-molecular interaction was described by effective interaction between two CG particles, which was calculated as free energy profile as a function of the distance between two amino acid side chain analogs in an explicit water solvent by using all-atom molecular dynamics simulations with the Thermodynamic Integration method. Potential function and parameters were determined from the free energy calculation and applied to CG simulations for protein complex system.

DISCUSSION AND RESULTS

We performed the Langevin dynamics simulations for formation of protein complex systems, such as GCN4-pLI tetramer, cyclin D3 and CDK4 complex, and cytochrome f and plastocyanin complex systems. We showed that protein structure complex obtained from our CG simulation was consistent with the structure obtained from experiments. Complex formation pathway was investigated for GCN4-pLI tetramer. Unfolded intermediate of CDK4 was suggested during the functional cycle. Estimated rate constant of electron transfer from cytochrome f and plastocyanin was consistent with an experimental result.

CONCLUSION

We conclude that our CG model is useful to understand protein complex formation.

Keywords: molecular simulation, coarse-grained model, Langevin dynamics

08923 - Poster Session

KA.21 - Halogen bonding in membrane-ligand recognition: Insights from computational biophysics

Rafael de Santana Nunes ^1,2, D. Vila-Viçosa^1,3, P.J. Costa¹

¹BioISI - Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa (1749-016 Lisboa, Portugal), ²Centro de Química Estrutural, Faculdade de Ciências, Universidade de Lisboa, (1749-016 Lisboa, Portugal), ³Kinetikos, (Coimbra, Portugal)

Halogen bonds (XBs) are noncovalent interactions where halogenated species interact with electronegative acceptors through a region of positive electrostatic potential, named σ–hole [1]. These interactions are known to mediate molecular recognition phenomena in biology, namely protein-ligand complexes or nucleic acids, and hence have found increasing application in drug discovery [2]. In this context, the ability to establish molecular interactions such as hydrogen bonds is a known factor determining the permeability of druglike molecules across biological membranes and may ultimately modulate their pharmacological properties. In contrast, the role of other interactions such as XBs has been largely overlooked, despite halogenated compounds being prevalent in drug discovery and development. Therefore, the eventual role of halogen bonding targeting phospholipid oxygen acceptors merits further investigation. In this work, we used molecular dynamics simulations, explicitly accounting for the σ–hole in halogenated ligands [3], to probe the existence of halogen-phospholipid interactions in a bilayer environment. The results [4] provide direct evidence for the formation of favorable XB interactions, as well as insights into the role of halogen bonding in the partitioning of halogenated molecules across biomembranes. [1] P. J. Costa, Phys. Sci. Rev. 2017, 2, 20170136. [2] P. J. Costa, R. Nunes, D. Vila-Viçosa, Expert Opin. Drug Discov. 2019, 14, 805. [3] R. Nunes, D. Vila-Viçosa, M. Machuqueiro, P. J. Costa, J. Chem. Theory Comput. 2018, 5383. [4] R. S. Nunes, D. Vila-Viçosa, P. J. Costa, J. Am. Chem. Soc. 2021, 143, 4253. Acknowledgements: Fundação para a Ciência e a Tecnologia (FCT), Portugal is acknowledged for funding through doctoral grant SFRH/BD/116614/2016 and projects UIDB/04046/2020, UIDP/04046/2020. This work was also supported by FCT, Programa Operacional Regional de Lisboa (Lisboa 2020), Portugal 2020, FEDER/FN, and the European Union under projects LISBOA-01-0145-FEDER-028455, PTDC/QUI-QFI/28455/2017.

Keywords: halogen bonding, membrane-ligand interactions, molecular dynamics simulations

08282 - Poster Session

KA.22 - Parkinson’s Disease – In Silico Analysis and Molecular Dynamics of Mutations of a-Syn Protein

Aloma Nogueira Rebello da Silva ¹, Gabriel Rodrigues Coutinho Pereira¹, Tiago Fleming Outeiro^2,3,4, Joelma Freire de Mesquita¹

¹Department of Genetics and Molecular Biology, Federal University of the State of Rio de Janeiro (, Brazil), ²Department of Experimental Neurodegeneration, University Medical Center Göttingen (, Germany), ³Translational and Clinical Research Institute, Newcastle University (, United Kingdom), ⁴Experimental Neurodegeneration, Max Planck Institute for Experimental Medicine (, Germany)

INTRODUCTION

Parkinson's disease (PD) is among the most prevalent neurodegenerative diseases that still have no cure. PD is characterized by typical motor symptoms that include resting tremor, postural instability, bradykinesia. 5-10% of the cases of PD have a familial origin, with mutations in the SNCA gene, which encodes the a-Syn.

OBJECTIVES

In this work, we aim to evaluate the effects on the a-Syn protein caused by mutations associated with PD development.

MATERIALS AND METHODS

Effects of mutations on protein function were predicted using the algorithms SIFT, PolyPhen-2, PhD-SNP, PANTHER, PMUT, PROVEAN, and MutPred. The structural evolutionary conservation analysis of a-Syn was performed with the ConSurf algorithm. The in silico mutagenesis was performed using VMD 1.9.3 mutator plugin. The molecular dynamics (MD) simulations of the WT a-Syn and its variants A30P, A53T, and G51D were performed in triplicates using the GROMACS 2018.6 package with an AMBER99SB-ILDN force field and a TIP3P water triclinic box. The molecular systems were neutralized by adding Na+ and Cl− ions and minimized for 5000 steps. After system minimization, NVT and NPT ensembles were performed at 1 atm and 300K for 100 ps.

DISCUSSION AND RESULTS

The A30P mutation was predicted to be deleterious by all algorithms. Consurf results revealed that mutations A30P, E46K, H50Q, G51D, A53E, A53T occur in a highly-conserved sites. MD analysis pointed to flexibility decrease at the N-terminal region of the analyzed variants and flexibility increase in their C-terminal regions compared to the WT. The secondary structure analysis suggested alterations in the G51D variant, particularly by increasing the number of β-sheets formed. Mutation G51D is known to increase a-Syn’s propensity to form b-pleated sheets, which increases its tendency to form toxic protein aggregates.

CONCLUSION

This work suggests that mutation of a-Syn affects the structure and function of the protein, which could be related to the development of PD.

Keywords: Alpha-synuclein, In Silico, Parkinson's Disease

Supported by: CAPES, DAAD, FINEP, NVIDIA, CNPq, FAPERJ and UNIRIO

08781 - Poster Session

KA.23 - Computational studies on inhibition of vivapains of Plasmodium vivax

Mariana Simões Ferreira ¹, Diego Enry Barreto Gomes², Pedro Geraldo Pascutti¹

¹Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brasil), ²Physics, University of Auburn (, USA)

INTRODUCTION

Malaria is one of the most worldwide spreaded diseases, killing 400 thousand people among the 200 million infected per year. Two parasite species cause together 95% of all its cases, which are Plasmodium falciparum and P. vivax. Increasing resistance to treatment against P. vivax infection difficults its combat around the world. New biological targets and pharmacological molecules must be explored to overcome that. In P. vivax, the proteins responsible for breaking hemoglobin, providing amino acids for the replication of the parasite inside lysosomes are the vivapains (VPs).

OBJECTIVES

Here, we study the anti-malarial targets enzymes VPs, specially the vivapain-4, aiming to explore its catalytic and allosteric sites for guiding virtual screening assays to identify potential new inhibitors.

MATERIALS AND METHODS

Since their structures have not yet been solved experimentally, comparative modelling of their sequences has been made with Modeller v9.21 based on the structure of their homologous enzyme, falcipain-3 from P. falciparum, found through the BLASTp server. To map protein pockets and important residues, different systems were run in replicates for 1 μs (water or mixture of water/ethanol solvents), which were analysed in Amber18. Such mapping was also accessed by the FTMAP server (for docking of fragments on the surface of the enzyme). LASSBio drug repository was the source for the virtual screening process, using AutoDock Vina, and further evaluation with RFScore and MM/GBSA.

DISCUSSION AND RESULTS

Mapping of pockets through conformational clusters, as well as from the MD with mixed solvents allowed us to identify important residues for ligand binding interactions in both catalytic and allosteric sites. The Tyr159 and Trp208 are examples, that combined with VS evaluation from MM/GBSA, led us to identify the most interesting ligands, further analysed by MD simulations.

CONCLUSION

The results among the different strategies allowed the identification of potential lead molecules for binding sites of VP4 and perspectives for optimisation.

Keywords: malaria, drug design, molecular dynamics simulations

Supported by: CAPES

08191 - Poster Session

KA.24 - Unravelling how phosphorylation patterns affect the collective motions and the transition states of hDat.

Roberto Carlos Navarro-Quiroz¹, Eric Allison Philot¹, Ana Ligia Scott ¹

¹LBBC, Federal University of ABC (São Paulo, Brasil)

INTRODUCTION

The human dopamine transporter (hDAT) is a member of the subfamily of monoamine transporters which show association with different biological functions such as memory, state of well-being and motor activity that result from the regulation of Dopamine in the sympathetic cleft,this function is highly regulated by the phosphorylations that occur in it.

OBJECTIVES

This study aims to evaluate the effects and molecular mechanism involved with hDAT and dopamine transport (absorption and efflux) under various phosphorylation conditions described in the experimentally reported by several papers. We show how the phosphorylation regulates the m collective motions and the transitions of hDat states as: open , closed and intermediate (outward-facing open, inward-facing open, holo - occluded).

MATERIALS AND METHODS

In the first moment, we built and validated a hDat model using homology and threading modeling for different parts of the protein. In the first moment, we investigate four different phosphorylation conditions using Normal Modes and Hybrid methods ( a CHARMM facility VMOD and MDeNM-Molecular Dynamics with Normal Modes Excited) to verify the collective motions and conformational space for them and define which motions lead to the states. Finally, we used the Markov Chain model to calculate the transition between the states and the free energy barrier.

DISCUSSION AND RESULTS

We observed that some motions promote preferentially one of the three states. When we do not phosphorylate the SER12, the Open states (outward-facing open and IF inward-facing open) are promoted. On the other hand, with SER12 phosphorylated and SER333 not, the intermediate (holo - occluded) states previled.

CONCLUSION

A single phosphorylation alters the collective motions and, consequently, the states populated by the proteins and the free energy barrier between them. In this way, we correlate how different phosphorylations affect the speed of Dopamine uptake and efflux in the nerve cell from the standpoint of collective motions.

Keywords: Nomral Modes, Free Energy, Human Dopamine Transport

08845 - Poster Session

KA.25 - Allosteric inhibition study of p38 MAPK enzyme using computational biology techniques

Thamires Rocco Machado ¹, Rosemberg de Oliveira Soares¹, Mariângela Dametto², Pedro Geraldo Pascutti¹

¹Instituto de Biofísica Carlos Chagas Filho (IBCCF), Universidade Federal do Rio de Janeiro (RJ, Brasil), ²Renato Archer Research Center-CenPRA, Renato Archer Information Technology Center (SP, Brasil)

INTRODUCTION

Recently, more and more research has demonstrated the important roles of p38 MAPK in the development of chronic inflammation and cancer. Allosteric inhibitors, such as BIRB796, have been developed, leading to conformational changes that avoid the ATP binding, preventing the catalytic activity of p38 MAPK.

OBJECTIVES

The present work aims to study the allosteric inhibition of p38α MAPK through molecular dynamics (MD), analysis of correlated movements, and solvent mapping techniques.

MATERIALS AND METHODS

Structures, dynamics and energies of protein-ligand interaction were characterized by simulations of MD of the p38α MAPK in complex with 5 compounds candidates as allosteric inhibitors, using the compound BIRB796 as reference. Discontinuities were investigated at the interface between the protein and the most promising ligand in mixed solvent (water 70% / ethanol 30%) and molecular probing with small ligands. The characterization of new allosteric cavities in the protein was also carried out through MD simulations in mixed solvent (water 70% / ethanol 30%), analysis of correlated movements and molecular probing with small ligands.

DISCUSSION AND RESULTS

The compound LASSBio-1494 showed the best results of free energy of binding between the compounds of LASSBio. Through simulations in ethanol/water and analysis by molecular probes was possible to map solvent molecules stabilized in microsites around the LASSBio-1494 ligand, which will be useful for the proposal of chemical groups to be aggregated in the ligand improving the free energy of interaction with the protein. Through analysis of cavities on the protein surface, potential allosteric sites were identified, with the most promising being characterized, which demonstrated residues with a high prevalence of hydrogen bonds with water and/or ethanol in the simulations of MD and with fragments in the analysis by molecular probes.

CONCLUSION

LASSBio-1494 obtained the most promising results and It was possible to identify potential allosteric sites. The data from this study will be used to propose new p38α inhibitors.

Keywords: drug design, Molecular Dynamics, p38 MAPK inhibition

Supported by: CAPES

08685 - Poster Session

KA.26 - Exploring the conformational landscape of the 33-mer peptide: a computational approach

María Julia Amundarain ¹, Agustín Vietri¹, Verónica Isabel Dodero³, Marcelo Daniel Costabel¹

¹Departamento de Física, Instituto de Física del Sur, Universidad Nacional del Sur,CONICET (Bahía Blanca, Argentina), ²Departamento de Física, Universidad Nacional del Sur (Bahía Blanca, Argentina), ³Fakultät für Chemie, Universität Bielefeld (Bielefeld, Germany)

INTRODUCTION

The 33-mer peptide is a digestion product of the gluten α2-gliadin protein. This proline- and glutamine-rich peptide is a putative trigger of the immune response in coeliac disease patients after gluten ingestion. In addition, in the small intestine, 33-mer is deamidated in three glutamines, which allows its recognition by the immune system, initiating the inflammation process. There is experimental evidence that both peptides (the WT and the deamidated peptide) form aggregates in physiologically relevant conditions, and it has been hypothesized that their oligomerization might be involved in the development of the disease. However, an accurate and complete structural description of the peptide and the initial oligomers has remained elusive.

OBJECTIVES

This work aims to explore computationally the conformational space of both the 33-mer peptide and its deamidated form as a first step to understand their aggregation properties and their influence on gluten-related diseases.

MATERIALS AND METHODS

The methods and force fields to assess Intrinsically Disordered Peptides’ (IDPs) structures have evolved fast-paced in the last years. We performed classical molecular dynamics (MD) simulations with GROMACS2020 using the atomistic AMBER03WS force field (1.5 μs each peptide) and the coarse-grained SIRAH force field (30 μs per peptide), which have been successfully employed to study other IDPs. The starting structures for both simulations were elongated peptides obtained from an experimentally based modelling scheme.

DISCUSSION AND RESULTS

From atomistic unbiased MD simulations, we obtained mainly a wide range of elongated structures. However, through the coarse-grained approach, we were able to assess possible folded and unfolded states. In both representations, the WT peptide visited more compact states than the deamidated one. The representative structures of both types of simulations showed good agreement with the experimental data available.

CONCLUSION

Both the 33-mer peptide and its deamidated form presented several stable structural arrangements, which should be considered to assess the complete aggregation process.

Keywords: Coeliac Disease, IDPs, Molecular Dynamics

Supported by: CONICET, CIC, UNS, de.NBI for computational resources.

08912 - Poster Session

KA.27 - The Hitchhiker’s Guide to the Periplasm: Unexpected Molecular Interactions of Antibiotics in E. coli

Conrado Pedebos ¹, Iain Smith², Alister Boags², Syma Khalid¹

¹Department of Biochemistry, University of Oxford (Oxford OX1 3QU, United Kingdom), ²School of Chemistry, University of Southampton (Southampton, SO17 1BJ, United Kingdom)

Gram-negative bacterial cell envelopes are host to a variety of biomolecules, being delimited by two lipid membranes, namely the inner membrane (IM) and the outer membrane (OM), along with the cell wall, a peptidoglycan layer that divides the periplasmic space. In recent years, complex models have emerged with the development of atomistic models for the OM and the cell wall components, which is allowing us to access novel conformational dynamics that were previously unreachable. In this regard, studies involving an accurate depiction of the bacterial envelope, as well as describing antibiotic mechanisms of action at the molecular level, can provide invaluable insights for experimental efforts to tackle the current antibiotic resistance crisis.Here, we present our work on large scale atomistic-level molecular dynamics simulations of the E. coli cell envelope, with a particular focus on the development of complex cell envelope models and the path taken by polymyxin B1 (PMB1) through various models of the periplasm, aiming to understand how it negotiates the periplasmic space to arrive at the inner membrane. Systems sizes oscillate between 600,000 to 700,000 atoms, due to the presence of OM and cell wall, plus 6 to 9 proteins, water, ions, and antibiotic molecules. Our simulations reveal that PMB1 forms transient interactions with proteins that are free in solution as well as lipoproteins anchored to the outer membrane. Furthermore, the lipid moiety of PMB1 enter the hydrophobic cavities of some lipoprotein carriers. Data obtained also shows that PMB1 binds strongly to the cell wall, diffusing on its surface and altering local morphology, while also allowing us to evaluate possible cell wall crossing mechanisms. Together the results show that PMB1 has a winding, obstacle-ridden path to the inner membrane. It is likely that this observation is true for other antibiotics that rely on diffusion rather than transporters.

Keywords: molecular dynamics, cell envelope, antibiotics

08208 - Poster Session

KA.28 - In Silico Analysis of the A4V and D90A Variants of Human SOD1 related to Amyotrophic Lateral Sclerosis

Gabriel Rodrigues Coutinho Pereira ¹, Joelma Freire De Mesquita¹

¹Bioinformatics and Computational Biology Group, Federal University of the State of Rio de Janeiro (Rio de Janeiro, Brazil)

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neurodegenerative disorder in adults. Missense mutations in superoxide dismutase 1 (SOD1), a major cytoplasmic antioxidant enzyme, are associated with the development of ALS. The A4V and D90A variants account for approximately half of all ALS-SOD1 cases in the United States and Europe.

OBJECTIVES

This work aims to characterize in silico the structural and functional effects of A4V and D90A variants on human SOD1 protein.

MATERIALS AND METHODS

Three-dimensional structures of A4V and D90A protein variants were computationally modeled in the VMD-1.9.1 package using the experimentally determined structure of wild-type SOD1 (PDB ID: 2C9V) as the template. Molecular dynamics (MD) simulations of the wild-type SOD1 protein and its variants A4V and D90A were performed in triplicates using the GROMACS-2018.8 package and AMBER99SB-ILDN force-field. TIP3P water molecules were added to a dodecahedral box system, which was neutralized by the addition of Na+ Cl- ions and then minimized. The system also had its temperature and pressure equilibrated at 1atm and 300K before the start of the simulations, which lasted 300ns. The MD trajectories were concatenated, and the following parameters were analyzed using GROMACS distribution programs: root-mean-square deviation, root-mean-square fluctuation, B-factor, radius of gyration, solvent accessible surface area, secondary structure, and essential dynamics.

DISCUSSION AND RESULTS

The MD analyses pointed to changes in flexibility and essential dynamics in the regions corresponding to the electrostatic and metal-binding loops of the variants, which could impact substrate guidance towards the active site and their enzymatic activity.

CONCLUSION

Our findings pointed to structural alterations in A4V and D90A variants that could have functional implications for SOD1 and explain their association with the development of ALS.

Keywords: Amyotrophic Lateral Sclerosis, Superoxide Dismutase 1, In Silico

Supported by: FAPERJ, CAPES, DAAD, FINEP, NVIDIA, CNPq and UNIRIO

08779 - Poster Session

KA.29 - Evaluation of chemical elements distribution and their inter-elemental correlations in tumor progression

Samella Pontes Salles¹, Simone Coutinho Cardoso², Mauro Sérgio Gonçalves Pavão², Mariana Paranhos Stelling¹

¹Núcleo de Ciências Biomédicas Aplicadas, Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (, Brazil), ²Instituto de Bioquímica Médica Leopoldo De Meis, Universidade Federal do Rio de Janeiro (, Brazil)

INTRODUCTION

Cancer is considered one of the most complex and fatal diseases worldwide. New approaches to study tumor progression and growth are relevant subjects of research. In this context, the particular role of chemical elements in cancer progression is a subject still not fully explored that presents opportunities for investigation.

OBJECTIVES

The main goal of our study is to assess the distribution of chemical elements in cancer progression as well as to discover correlations between elements, observing both the primary tumor and the distant tissues that the tumor cells may affect.

MATERIALS AND METHODS

For simulating tumor progression in vivo, murine Lewis lung carcinoma cells were injected in C57BL/6 mice and data indicating the presence, concentration, and location of different elements in distinct tissues, in both control and experimental groups, were obtained in a time frame of 5 weeks of tumor progression. The data were collected via Synchrotron Radiation X-Ray Fluorescence in the Brazilian Synchrotron Light Laboratory (LNLS). In order to extract relevant information inherent to the voluminous available data, we adopt statistical analysis.

DISCUSSION AND RESULTS

With this work, it was possible to observe the elements’ relevance for biological processes of normal, as well as tumor cells, during its tumor progression. Thus, it was possible to notice indications of tumor influence on distant tissues as well as highlight the importance of elements and their correlations for the tissues, including for processes of tumor progression, such as growth and cellular migration, angiogenesis, among others.

CONCLUSION

This work also confirmed information found in the literature and featured results apparently not yet observed. Moreover, elements and correlations of relevance for more investigation, regarding their role in the processes described, were highlighted to bring to light explanations for such observations not yet noted.

Keywords: elemental distribution, tumor progression, X-Ray fluorescence

08524 - Poster Session

KA.30 - POLYana: a new software for rheological study of polymeric colloidal materials

Anderson Ferreira Sepulveda ¹, Margareth Franco², Fabiano Yokaichiya³, Daniele de Araujo¹

¹Center for Natural and Human Sciences, Federal University of ABC (São Paulo, Brasil), ²RMB, Nuclear and Energy Research Institute (São Paulo, Brazil), ³Physics Department, Federal University of Parana (Parana, Brazil)

INTRODUCTION

POLYAna is a new executable software developed by SISLIBIO group for rheological analysis of hydrogel and organogel systems and other colloidal materials (nanoparticles and micelles). The software development aims to facilitate the analysis of rheology data associated to both temperature- and frequency-dependent analysis, viscosity and curve flow profiles.

OBJECTIVES

The software development aims to facilitate the analysis of rheology data associated to both temperature- and frequency-dependent analysis, viscosity and curve flow profiles.

MATERIALS AND METHODS

From raw data, several models are applied like power-law model for frequency response and curve flow, Boltzmann law to calculate gelation temperature and viscosity response under temperature, Maxwell model to study interchain relationships in addition to other models such as Bingham model, Cross model, and Herschel-Bulkley are also available. POLYana outputs calculates rheological parameters like consistency, adhesion, hysteresis, flow index, G’/G” ratio.

DISCUSSION AND RESULTS

To validate results obtained from POLYana, same data were analyzed by applying other programs and same mathematical models. In this sense, rheological analysis of Poloxamer 407 in water solution (15 %) were performed: from temperature-dependent G’ and G” analysis were obtained gelation temperature of 45.46 ± 0.02 °C, η_0 = 0.08 ± 0.03 mPa*s, η_max = (32.44 ± 0.17) mPa*s and dη/dT = (1.27 ± 0.02) mPa*s/°C by fitting Boltzmann law (R2 = 0.998), which are similar to results obtained by others softwares and found in literature. From temperature-dependent G’ and G” analysis, it gets adhesion value of (1647.15 ± 18.01) mPa*sn calculated from power-law model (R2 = 0.869), also similar to PRISM results.

CONCLUSION

Also, other Poloxamer concentrations and hydrogels types have been evaluated, showing close numbers to that previously reported. In order to stablish structural relationships, one of POLYana tools is also to analyze small-angle neutron scattering (SANS) and develop Monte Carlo simulation for SANS and rheological analysis, simultaneously.

Keywords: Colloidal materials , Rheology, Software

Supported by: CAPES (grant #001), CNPq (307718/2019-0)

08871 - Poster Session

KA.31 - Strategies of Ranking methods for Virtual Screening in SARS-CoV-2 PLpro

Bruce Veiga Andriolo ^1,2, Caio Felipe de Araujo Ribas Cheohen^1,2, Diego Henrique Silvestre¹, Manuela Leal da Silva^1,2

¹Instituto Nupem, Universidade Federal do Rio de Janeiro (Rio de Janeiro , Brazil), ²Diretoria de Metrologia Aplicada as Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia (Rio de Janeiro , Brazil)

INTRODUCTION

In 2019 strange cases of unidentified pneumonia led to the discovery of a new species of coronavirus, the SARS-CoV-2 responsible for COVID-19 pandemic. One of its most important proteins is the PLpro for its ubiquitin and deISGylating capabilities. Considering the costs of developing new drugs, repositioning already approved drugs is an excellent strategy in fighting COVID-19. We used the drugs approved by the Brazilian Health Regulatory Agency ANVISA using in silico techniques to try to predict interaction between these compounds and the PLpro. The importance of this work is based on the huge amount of money and time needed to develop a novel drug, while repositioning is cheaper and faster.

OBJECTIVES

Perform Virtual Screening and use different strategies for ranking the results to determine possible candidates to inhibit SARS-CoV-2 PLpro.

MATERIALS AND METHODS

Virtual Screening was performed by AutoDock Vina using parameters defined in redock with the exception of generation of 20 poses. After VS the candidates whose energy was lower than -8.0 kcal/mol were selected. For ADMETox prediction, SwissADME, admeSAR and pkCSM were used with PAINS and AMES being considered as limiting factors. The distance between the molecules and Y268 from PLpro was measured using PyMOL and molecular weight was used like a filter. The compounds were ranked based on these criteria.

DISCUSSION AND RESULTS

From the original 273 compounds, 44 had the correct energy range. From those, 36 passed the AMES and PAINS test who were ranked and organized in accordance with their usage. Vitamins, unpromising or problematic drugs were excluded.

CONCLUSION

Using ADMETox prediction and information about distance, pocket volume and literature we organize possible drugs to be tested in vitro against PLpro of SARS-CoV-2 and HEK-293 lineage cells.

Keywords: SARS-CoV-2, PLpro, Virtual Screening

Supported by: CNPq and CAPES

08572 -

KA.32 - Immunogenic characterization of the antigen group (gag) gene of strains of the human immunodeficiency virus type 1 (HIV-1) circulating in Brazil,

STEVE BIKO MENEZES HORA ALVES RIBEIRO ¹

¹Biofunção, Universidade Federal da Bahia (BA, Brasil)

INTRODUCTION

A large genetic variability of HIV represents a major obstacle to the control of infection by the host immune system and to the development of effective drugs and vaccines. CTLs recognizing epitopes within the HIV antigen (gag) group gene have been associated with the initial control of infection and these epitopes, uses in current approaches to developing an HIV vaccine.

OBJECTIVES

This study aims to investigate a variability of the non-gag HLA class I ligand binding epitopes of circulating HIV-1 strains in Brazil, available in a publicly accessible database.

MATERIALS AND METHODS

For this purpose, an allelic variability and predominance of HLA class I in the Brazilian population was determined in order to identify the epitopes of the gag gene of circulating HIV-1 strains in Brazil. One was evaluated by target cell (CTL / CD8) and one screened by an assessed target cell (CTL /CD8). , the already defined gene (gag) and the most prevalent HLA alleles in the Brazilian population. The Allele Frequency Net Database (AFND) was used, as gag regions were removed from through the electronic address https://www.hiv.lanl.gov, with the defined genes (gag) and the alleles most prevalent in the Brazilian population. The Immune Epitope Database (IEDB) was used to determine which are the best Brazilian HLA-linked epitopes.

DISCUSSION AND RESULTS

From this research a table with a description of the non-Brazilian circulating epitopes was generated for each HLA class I allele to the HIV-1 subtype and position of the epitope in relation to the gag gene.

CONCLUSION

Thus, it is concluded that the variability of HLA class I ligand epitopes in the gag gene found, generated a sum of importance data to characterize and describe the non-circulating epitopes in Brazil and to show the studies involved with these epitopes not the world, for future future vaccine referrals in Brazilian territory.

Keywords: HIV, Epitopes, Brazil

08518 - Poster Session

NB.01 - Synthesis of a iodine-131-labeled derivative of laminin-111, [131I]I-YIKVAV, and its assessment as a potential radiopharmaceutical for breast cancer diagnosis

Fernanda Ferreira Mendonça ¹, Danielle Vieira Sobral¹, Ana Claudia Ranucci Durante², Ana Claudia Camargo Miranda², Jorge Mejia², Wagner Ricardo Montor¹, Marycel Figols de Barboza², Leonardo Lima Fuscaldi¹, Luciana Malavolta¹

¹Departamento de Ciências Fisiológicas, Faculdade de Ciências Médicas da Santa Casa de São Paulo (Sao Paulo, Brasil), ²Instituto Israelita de Ensino e Pesquisa, Hospital Israelita Albert Einstein (Sao Paulo, Brasil)

INTRODUCTION

Peptide sequences derived from laminin-111 regulate gene expression in breast tumor cells, including the bioactive peptide IKVAV.

OBJECTIVES

Here, we evaluated the potential of the YIKVAV modified fragment radiolabeled with iodine-131 by in vitro interaction with human breast cancer cells and ex vivo biodistribution in mice.

MATERIALS AND METHODS

The YIKVAV modified fragment (25 μg) was radiolabeled with [ ¹³¹ I]NaI (11.1–14.8 MBq) using the chloramine T method (reaction time=90 s). The radiochemical yield was evaluated by ascending chromatography using TLC-SG strips and acetonitrile/water (95:5), as eluent. The radiopeptide, [ ¹³¹ I]I-YIGSR, was incubated with 2x10 ⁶ MDA-MB-231 and MCF-7 cells at 37°C under agitation (500 rpm). In vitro binding and internalization were assessed at 1, 4, and 24 h post-incubation. To evaluate the biodistribution profile, the [ ¹³¹ I]I-YIKVAV was intravenously injected into normal nude female Balb/c mice. Ex vivo biodistribution was performed at 0.5 and 2 h after injection.

DISCUSSION AND RESULTS

The data revealed radiochemical yield >90% (n=10). The In vitro data showed high affinity of the radiopeptide to both human breast cancer cells. The binding percentages were 5.65±0.68, 7.15±0.64, and 7.34±1.17, and the internalization percentages were 68.22±3.70, 73.37±3.73, and 52.00±6.10, at 1, 4, and 24 h, respectively, for MDA-MB-231 cells (n=5). The assay with MCF-7 cells showed binding percentages of 15.21±1.54, 18.10±1.63, and 13.09±2.23, and internalization percentages of 78.42±2.95, 79.23±3.62, and 59.71±5.57, at 1, 4, and 24 h, respectively (n=5). The biodistribution data showed rapid blood clearance and low accumulation of the radiopeptide in the evaluated organs (%ID ¹³¹ I]I-YIKVAV (n=3). Further biodistribution in breast tumor-bearing mice will be performed in order to elucidate in vivo tumor uptake.

CONCLUSION

Therefore, [ ¹³¹ I]I-YIKVAV showed high affinity to breast cancer cells and the biodistribution profile revealed that the radiopeptide do not accumulate in any organ compatible with breast cancer primary tumor or its metastasis.

Keywords: [131I]I-YIKVAV, Breast cancer, Derivative of laminin-111

Supported by: FAPESP, CAPES, and FAP-FCMSCSP

08990 - Poster Session

NB.02 - Effective methodology for maintaining Toxoplasma gondii in vitro using paramagnetic iron nanoparticles to support three-dimensional cell culture

Ana Cristina Gomes Nascimento ¹, Andres Jimenez Galisteo Junior², Giovana Dias da Silva¹, Leonardo Wilans Pereira de Souza Rocha¹, Daniel Perez Vieira¹

¹Laboratory of Radiobiology – Biotechnology Center , Nuclear and Energy Research Institute - IPEN/CNEN, USP, (SP, Brazil), ²Laboratory of Protozoology, Institute of Tropical Medicine of São Paulo - IMTSP, USP (SP, Brazil)

INTRODUCTION

Toxoplasma gondii is a protozoan parasite that infects approximately one billion people worldwide. Upon infection, the host may die due to latent infection or presence with chronic cysts in brain, retina or muscle tissue. Humans can become infected consuming water or foods contaminated with oocysts or eating undercooked meat. Its virulent form is difficult to replicate in vitro, requiring additional steps using experimental animals. The use of nanotechnology can contribute to this in vitro production, through the three-dimensional cultivation of mouse fibroblast cells (NIH / 3T3 ATCC ® CRL-1658™) and nanoparticles synthesized with radiation.

OBJECTIVES

The objective of this work was to demonstrate the three-dimensional culture of fibroblast cells aggregated to nanoparticles for inoculation the T. gondii.

MATERIALS AND METHODS

This methodology was created to facilitate parasite management and replication. For the production of nanoparticles, the work used concentrations of iron sulfate II heptahydrate (Fe2SO4.7H2O, CAS 7782-63-0) and glycine (NH2CH2COOH, CAS 56-40-6) diluted in ultrapure water free of O2 at pH 12. This solution was irradiated by electron beam of the IPEN / CNEN-SP Radiation Technology Center in doses of at least 15 and at most 30kGy. Paramagnetic iron oxide nanoparticles (PION’s) were then adsorbed on cell membranes, and cells were kept together by a magnetic field. Structured spheroids (4 day of culture) were infected with 106 parasites (RH strain) and the infection was evaluated by transmission electron microscopy.

DISCUSSION AND RESULTS

Tachyzoites were found inside 3T3 cells, assuring that the spheroid can be a suitable culture substrate to T. gondii in vitro propagation.

CONCLUSION

A three-dimensional methodology for in vitro cultivation of the parasite is perhaps the key for applications in the study of toxoplasmosis, as it has a fast, cheap, efficient production (yield and reduction of contamination).

Keywords: Toxoplasma gondii, Three-dimensional cell culture, Nanoparticles

Supported by: FAPESP (2017/50332-0) & IPEN/CNEN-SP

08481 - Poster Session

NB.03 - Beneficial effects of fructo-oligosaccharides (FOS) and arginine on the intestinal mucositis, induced by 5- fluorouracil (5-FU)

Maria Emília Rabelo Andrade ¹, Luísa Martins Trindade², Simone Odília Antunes Fernandes¹, Vasco Ariston de Carvalho Azevedo³, Geovanni Dantas Cassali⁴, Simone de Vasconcelos Generoso², Valbert Nascimento Cardoso¹

¹Departamento de Análises Clínicas e Toxicológicas, Universidade Federal de Minas Gerais (MG, Brasil), ²Escola de Enfermagem, Universidade Federal de Minas Gerais (MG, Brasil), ³Departamento de Biologia Geral, Universidade Federal de Minas Gerais (MG, Brasil), ⁴Departamento de Patologia Geral, Universidade Federal de Minas Gerais (MG, Brasil)

INTRODUCTION

Mucositis is one of the most common complications in patients undergoing chemotherapy or radiotherapy. The use of compounds with action on the immune system and intestinal microbiota may be a beneficial alternative for the prevention and/or treatment of mucositis.

OBJECTIVES

So, the aim of this study was to evaluate the effects of FOS and arginine on intestinal damage in experimental mucositis.

MATERIALS AND METHODS

Balb/c mice were randomized into 4 groups: CTL (without mucositis + saline), MUC (mucositis + saline), FOS (mucositis + supplementation with FOS – 1st until 10th day) and ARG (mucositis + supplementation with arginine – 1st until 10th day). On the 7th day, mucositis was induced with an intraperitoneal injection of 300 mg/kg 5-FU. After 72 h, weight variation, intestine length, intestinal permeability (IP), morphometry and histopathology analysis were evaluated by ANOVA two way test. Significance level was set at p < 0.05.

DISCUSSION AND RESULTS

The MUC group showed lost weight, reduced intestine length and increased IP (p < 0.05). Results showed presence of tissue damage, inflammatory cells and ulcerations in the ileum of animals of MUC group. FOS and arginine supplementation reduced lost weight, intestinal permeability and maintained the intestine length at physiologic levels (p < 0.05). However, arginine was more effective in reducing tissue damage and maintaining villus height in the ileum compared with FOS group.

CONCLUSION

In conclusion, the present results show that FOS and arginine restored intestinal barrier, decreased lost weight and the inflammation induced by mucositis. These immunomodulators could be important adjuvants in the prevention and treatment of mucositis.

Keywords: arginine, fructo-oligosaccharides , mucositis

Supported by: CNPq, FAPEMIG and CAPES

05069 - Poster Session

NB.04 - Tucumã extract: phytochemical characterization, acute and subacute oral toxicity studies in Wistar rats

Camille Gaube Guex ¹, Gabriela Buzzati Cassanego², Rafaela Castro Dornelles¹, Rosana Casoti³, Ana Martiele Engelmann⁵, Tatiana Emanuelli⁵, Cristiane Cademartori Danesi⁶, Euler Esteves Ribeiro⁷, Liliane Freitas Bauermann^1,2

¹Pós-graduação em Farmacologia, Universidade Federal de Santa Maria (TS, Brasil), ²Pós-graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria (RS, Brasil), ³Pós-graduação em Química, Universidade Federal do Espírito Santo (ES, Brasil), ⁴Pós-graduação em Medicina Veterinária, Universidade Federal de Santa Maria (RS, Brasil), ⁵Pós-Graduação em Ciência e Tecnologia dos Alimentos, Universidade Federal de Santa Maria (RS, Brasil), ⁶Departamento de Patologia, Universidade Federal de Santa Maria (RS, Brasil), ⁷Gerontologia, Universidade Aberta da Terceira Idade (AM, Brasil)

INTRODUCTION

Tucumã (Arecaceae) is a native palm tree from Brazil, Its fruits are widely consumed by the local population as food and traditionally used to treat the respiratory system, infections, infestations and digestive system disorders. Even though they are natural, its misuse can lead to nocive effects on people. In this scenario, toxicity studies are essential to ensure the safety use of these substances.

OBJECTIVES

Therefore, the aim of this study was to perform the phytochemical characterization of tucumã fruits extract (TFE) and to evaluate its toxicity through acute and repeated-doses toxicity studies in female and male rats.

MATERIALS AND METHODS

TFE was analyzed by HPLC and GC/MS. Acute toxicity consisted in the administration of TFE at a single dose of 2000 mg/kg in female rats. The repeated-doses study was performed in male and female rats, which received TFE for 28 days at doses of 200, 400 and 600 mg/kg. After euthanasia, blood was collected by cardiac puncture for the hematological and biochemical analyses. Liver and kidney were removed to analyze the histopathology and oxidative damage markers/enzymatic activity.

DISCUSSION AND RESULTS

The phytochemical analyses evidenced the presence of carotenoids, flavonoids, unsaturated and saturated fatty acids, and triterpenes. The single dose administration of TFE did not induce mortality nor any sign of toxicity. Thus, TFE was classified as safe in acute toxicity. Regarding the repeated-doses study, the 28 days treatment with TFE at the highest dose showed renal toxicity evidenced by histopathological analysis in male rats, all the other parameters were not altered. In female rats, no signs of toxicity were observed.

CONCLUSION

Therefore, the results suggest that TFE did not induce toxicity after exposure to a single or repeated doses in female rats. However, in males it may be considered safe when given repeatedly in low doses.

Keywords: Carotenoids, Arecaceae, Safety

Supported by: CAPES [#001]

08446 - Poster Session

NB.05 - Estudo sobre a eficiência do radiofármaco PSMA-1007-18F na detecção do câncer de próstata em um estudo pré-clínico in vivo

Carolina Silva Ferreira dos Santos ¹, Maria Helena Bellini², Sofia Nascimentos dos Santos¹, Elaine Bortoleti de Araújo¹

¹Centro de Radiofarmácia, Instituto de Pesquisas Energéticas e Nucleares (São Paulo, Brasil), ²Centro de Biotecnologia, Instituto de Pesquisas Energéticas e Nucleares (São Paulo, Brasil)

INTRODUCTION

O PSMA-1007-18F é caracterizado por uma biodistribuição única em comparação com outros agentes de PSMA, pois é eliminado através do fígado, além de ter captação elevada dentro das células do câncer de próstata.

OBJECTIVES

O objetivo deste trabalho é analisar a especificidade do radiofármaco PSMA-1007-18F em camundongos com modelo tumoral PSMA+ através de um estudo de bloqueio.

MATERIALS AND METHODS

Animais SCID com células tumorais LNCaP foram preparados e divididos em quatro grupos (n=5) de acordo com os tempos de biodistribuição de 30 minutos, uma hora, duas horas e uma hora com agente bloqueador PSMA I&T (100μg/μL), sendo injetado 30 minutos antes do radiofármaco. Foram injetados 5,55 MBq (0,056 MBq/μL) via caudal nos animais e após os tempos pré-determinados foram sacrificados, com os órgãos de interesse coletados, pesados e sua atividade contabilizada. As imagens PET/CT foram realizados para ilustrar a captação do PSMA-1007-18F pelo tumor e por órgãos PSMA+ com e sem bloqueio.

DISCUSSION AND RESULTS

O grupo de 30 minutos apresentou maiores captações, caracterizando o estágio de distribuição. Rins e baço apresentaram alta captação pelo PSMA-1007-18F por serem PSMA+, onde sua especificidade pode ser constata pela drástica diminuição de captação nos animais com agente bloqueador. A captação pelas células tumorais de próstata se mostrou constante durante o período avaliado e foi efetivamente bloqueada pelo excesso de PSMA I&T (imagem), que confirma a ligação do radiofármaco aos receptores de PSMA. Apesar de apresentar uma baixa hidrofilicidade, a razão entre tumor/sangue e tumor/músculo foi de 14,18 ± 3,19 e 4,78 ± 1,52 em 1h, respectivamente.

CONCLUSION

O radiofármaco PSMA-1007-18F apresenta propriedades de ligação que fornecem captação tumoral específica. Sua viabilidade em imagens para câncer de próstata foi demonstrada pela alta especificidade pelas células PSMA+ e pela via de excreção hepatobiliar. Os resultados deste estudo serão úteis para subsidiar o registro e comercialização deste radiofármaco.

Keywords: flúor-18, PSMA-1007, radiofármaco

Supported by: FAPESP

08992 - Poster Session

NB.07 - Molecular docking study of copaiba oil interacting with the spike protein of SARS-CoV-2

Willian Oliveira Santos ¹

¹Instituto de Física, Universidade Federal de Alagoas (Sergipe, Brasil)

INTRODUCTION

COVID-19 triggered by SARS-CoV-2 has caused hundreds of thousands of deaths worldwide. Organic and inorganic compounds have been tested as potential inhibitors of this lethal virus. For these tests, several techniques are used to design molecules of biological interest for drug composition, in which molecular coupling plays an important role.

OBJECTIVES

In the present work, the compounds kaurenoic, copalic and beta-caryophyllene acids that form the copaiba oil were studied as anti-inflammatory and as SARS-CoV-2 inhibitor

MATERIALS AND METHODS

The universal force field (UFF) was selected to perform the calculations. After obtaining the best conformation of the geometries, the structures were submitted to a new optimization at the DFT level using the DMOL3 Code, where the generalized gradient approximation (GGA) considers all the electrons of the molecules.

DISCUSSION AND RESULTS

Molecular docking showed alkyl, pi-alkyl, conventional H-bond, unfavorable bump, and Van der Waals interactions. The calculated electrostatic potential maps showed the nucleophilic and electrophilic regions. The negative binding energies obtained for the three acids suggest the stability of the complexes. The minimum energy states for -caryophyllene are lower than the other compounds analyzed, and it can be predicted that this is the most stable.

CONCLUSION

From the results obtained, it can be inferred that the acids that form the copaiba oil can be used as an inhibitor of COVID-19.

Keywords: Copaiba oil, COVID-19, DFT, Molecular docking

Supported by: FAPESPA, CNPq and CAPES

08382 - Poster Session

NB.08 - Influence of image reconstruction protocol on PET image with 11C

João Vitor do Carmo Barbosa^1,2, Andrea Vidal Ferreira¹, Guilherme Cavalcante de Albuquerque Souza^1,2, Rodrigo Modesto Gadelha Gontijo², Bruno Melo Mendes¹, Marcelo Henrique Mamede Lewer²

¹Serviço de Radiofármacos (SERFA), Centro de Desenvolvimento da Tecnologia Nuclear (Minas Gerais, Brazil), ²Departamento de Anatomia e Imagem (IMA), Universidade Federal de Minas Gerais (Minas Gerais, Brazil)

INTRODUCTION

The small animals positron emission tomography (PET) scanner from Molecular Imaging Laboratory (LIM/CDTN) is dedicated to pre-clinical studies on new ¹⁸ F and ¹¹ C-based radiopharmaceuticals and novel applications for well-known radiopharmaceuticals. According NEMA NU 4-2008 publication, several tests must be performed to ensure the performance of small animal PET scanners.

OBJECTIVES

The aim of this work was to evaluate the influence of image reconstruction protocols on the image quality, accuracy of attenuation and scatter corrections parameters for ¹¹ C PET images.

MATERIALS AND METHODS

A PET image of the Image Quality Phantom filled with ¹¹ C-PK-11195 was acquired and reconstructed using forty-nine different protocols. The reconstruction variables evaluated were the algorithms (FBP, MLEM-3D, OSEM-2D), the resolution mode (high/standard) and the number of iterations (10 to 150). Uniformity, spill-over ratio (SOR) and recovery coefficients (CR) tests were adapted from NEMA NU 4-2008 and performed for each reconstructed image. PMOD ^® software was used for image analysis.

DISCUSSION AND RESULTS

FBP based protocol generated noisier images compared to iterative algorithms (MLEM-3D or OSEM-2D) based protocols. The increase in the number of iterations resulted in higher standard deviation of the analyzed parameters for all reconstructed images. MLEM-3D and OSEM-2D based protocols generated similar results when number of iterations and resolution mode were identical. SOR and RC mean values remained practically stable when the number of iterations ranged from 40 to 150.

CONCLUSION

This study allowed the evaluation of different image reconstruction protocols on important parameters of ¹¹ C PET image quality. Additionally, a standard image reconstruction protocol (MLEM-3D algorithm, 40 iterations, standard resolution mode) for ¹¹ C images reconstruction was defined to be adopted in LIM/CDTN laboratorial routine.

Keywords: PET, Image quality control, Carbon-11

08575 - Poster Session

NB.09 - Application of 99mTc-DTPA for monitoring the intestinal barrier in the pharmacological repositioning of atorvastatin in the treatment of enteric mucositis

Kátia Duarte Vital ¹

¹Departamento de Análises Clínicas e toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte (MG, Brasil), ²Departamento de Microbiologia, Universidade Federal de Minas Gerais (MG, Brasil)

INTRODUCTION

Pharmacological repositioning seeks to find new uses for existing drugs. Nuclear medicine is a dynamic and versatile specialty, allowing a functional and molecular assessment of organic systems. Mucositis is a side effect of chemotherapy characterized by intense inflammation in the gastrointestinal tract, making treatment a challenge. Statins that are inhibitors of cholesterol biosynthesis and that have anti-inflammatory effects that can be used to control enteric mucositis. The assessment of intestinal permeability using 99mTc-DTPA is a precise and simple method that allows the identification of changes in the gastrointestinal barrier caused by mucositis.

OBJECTIVES

The objective of this work is to evaluate the potential of atorvastatin in protecting the gastrointestinal barrier in a model of enteric mucositis.

MATERIALS AND METHODS

Male BALB/c mice weighing 20-25g (n=6) received intraperitoneal injections of 5-FU (30 mg/kg/day) for 5 days, and were treated with atorvastatin (10 mg/kg) given via oral gavage for 7 days and the mucositis group received 0.9% NaCl. Control group mice received intraperitoneal injection and oral gavage of 0.9% NaCl. On the 7th day of the protocol, the animals were anesthetized and euthanized, the ileum was collected for the analysis of MPO, EPO and histology, and the blood collected for the quantification of 99mTc-DTPA.

DISCUSSION AND RESULTS

Animals with mucositis showed a significant increase in intestinal permeability, MPO and EPO (p < 0.05) and the treatment with atorvastatin promoted a reduction in intestinal permeability, also associated with an improvement in small intestine mucosal architecture, suppression of enzyme levels EPO and MPO.

CONCLUSION

Our results indicate that atorvastatin was able to prevent adverse mucositis-induced damage to the intestinal mucosa, proving to be a therapeutic strategy to aid in the treatment of intestinal mucositis.

Keywords: Gastrointestinal mucositis, Atorvastatin, Pharmacological repositioning

Supported by: CAPES

08991 - Poster Session

NB.10 - Radiomodifying effect of black grape juice on hematologic alterations induced by whole brain irradiation in rats

Robson Borba de Freitas ¹, Liliane de Freitas Bauermann^1,1,7,8, Robson Borba de Freitas^1,1,7,8, Edson Ramos de Andrade^1,4,7,8, Juan Pablo Barrio^1,1,7,9

¹Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria (Santa Maria, RS, Brasil), ⁴Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria (Rio Grande do Sul, Brasil), ⁶Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria (Rio Grande do Sul, Brasil), ⁷ Departamento de Sistemática e Ecologia, Universidade Federal da Paraiba (Paraíba, Brasil), ⁸Fisiologia, Universidade de León (Castilha, Espanha)

INTRODUCTION

Whole brain irradiation (WBI) causes a variety of secondary side-effects including anorexia, immunosuppression and osteoradionecrosis. Some natural compounds produced by plants are able to reduce the secondary side effect of irradiation because they react with free radicals (FRs) and reactive oxygen species (ROSs)

OBJECTIVES

In the present study we evaluated the radiomodifying effect of black grape juice (BGJ) in animal model of fractionated whole brain irradiation

MATERIALS AND METHODS

Forty male rats (200–250 g) were exposed to eight sessions of cranial X-ray irradiation. The total dose absorbed was 32 Gy delivered over 2 weeks. Four groups were defined: (a) NG: non-irradiated, glucose and fructose solution-supplemented (GFS); (b) NJ: non-irradiated, BGJ-supplemented; (c) RG: irradiated, GFS-supplemented; and (d) RJ: irradiated, BGJ-supplemented. Rats received daily BGJ or GFS dosing by gavage starting 4 days before, continuing during, and ending 4 days after WBI. Two months after the last dose of WBI, the rats were killed by exsanguination under deep anesthesia induced by i.p. injecting 200 mg/kg body weight pentobarbital. The carotid was cannulated and blood samples were collected in tubes containing EDTA. The hematological parameters analyzed were: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV), and platelet distribution width (PDW). Data were analyzed using ANOVA.

DISCUSSION AND RESULTS

BGJ increased WBC by 118% when compared to that in the irradiated RG. The red blood cell count (RBC) also decreased after irradiation. BGJ increased RBC by 8.3% when compared to that in the RG. HCT increased in BGJ group.

CONCLUSION

The BGJ was able to reduce the hematological syndrome in rats expose to WBI. The BGJ is rich in phenolic compounds which’ reduce oxidative stress induce irradiation.

Keywords: black grape juice, whole brain irradiation, hematological parameters

Supported by: CAPES, Uni. León, UFSM

08330 - Poster Session

NB.11 - Comparative analysis of image quality parameters in three PET systems in Brazil

Rodrigo Modesto Gadelha Gontijo ¹, Andréa Vidal Ferreira², Daniele de Paula Faria³, Carlos Alberto Buchpiguel ³, Samuel Greggio⁴, Gianina Teribele Venturin⁴, Jaderson Costa da Costa⁴, Juliana Batista da Silva², Marcelo Mamede¹

¹Anatomia e Imagem (IMA) , Universidade Federal de Minas Gerais (Minas Gerais, Brasil), ²Serviço de Radiofármacos - SERFA, Centro de Desenvolvimento da Tecnologia Nuclear (Minas Gerais, Brasil), ³Hospital das Clínicas da Faculdade de Medicina, Universidade de São Paulo (São Paulo, Brasil), ⁴Instituto do Cérebro - InsCer, Pontifícia Universidade Católica do Rio Grande do Sul (Rio Grande do Sul , Brasil)

INTRODUCTION

Positron emission tomography (PET) is widely used in preclinical studies, generating images applied to biochemical, metabolic and functional study of organs and tissues of small animals. A comprehensive evaluation of the PET scanner intrinsic parameters is important to optimize the acquired images, providing more reliable qualitative and quantitative analyses.

OBJECTIVES

To evaluate Image Quality (IQ) parameters of Triumph® LabPET-4 systems installed in three different Brazilian preclinical molecular imaging centers.

MATERIALS AND METHODS

Experiments were carried out at the centers: C1 - Molecular Imaging Laboratory (LIM) of the CDTN/CNEN; C2 - Laboratory of Nuclear Medicine (LIM-43) of HCFMUSP; C3 - Preclinical Research Center of the Brain Institute (InsCer) at PUC-RS. IQ phantom PET images were acquired as recommended in NEMA NU 4-2008 standard ( ¹⁸ F-FDG, 3.7 MBq, 20 minutes). Image reconstructions were performed in the system where they were acquired using the same reconstruction protocol (MLEM-3D algorithm, 20 iterations and no high-resolution mode). Data was processed using PMOD® software. The IQ parameters: (i) uniformity, (ii) spill-over ratio (SOR) and (iii) recovery coefficients (RC) were evaluated and compared.

DISCUSSION AND RESULTS

For Uniformity test, the percentage standard deviations of mean activity concentration were 7.8%; 7.3% and 6.4% for Centers 1, 2 and 3 respectively. Cold chambers RSO values in the systems 1, 2 and 3 were respectively 0.16, 0.19 and 0.21 for water; 0.26; 0.28 and 0.30 for air. The RC’s for rod diameters from 1 to 5 mm ranged from 0.08 to 0.91 for the three centers.

CONCLUSION

Results revealed that the three PET systems have appropriate quality parameters for pre-clinical studies, presenting values compatible with international standards for this type of image. This study was able to reveals the performance of preclinical PET system of different Brazilian imaging centers and may support the standardization of a National Quality Control Program.

Keywords: image quality, PET, NEMA

08577 - Poster Session

NB.12 - Photobiomodulation modulates gene expression of pro-inflammatory markers in an experimental model of acute arthritis.

Bruna Silva Gomes ¹, Amanda Bezerra Gonçalves¹, Marcelo Augusto Marretto Esquisatto¹, Gaspar de Jesus Lopes Filho², Fernando Russo Costa do Bomfim^1,2

¹Laboratório de Biologia Molecular, Centro Universitário da Fundação Hermínio Ometto (São Paulo, Brasil), ²Programa de Pós-graduação em Ciência Cirúrgica Interdisciplinar, Universidade Federal de São Paulo (São Paulo, Brasil)

INTRODUCTION

Introduction: Rheumatoid arthritis is an autoimmune, inflammatory disease that causes pain and joint destruction, and its treatment is performed with anti-inflammatory drugs and resources such as photobiomodulation (PBM) with low-level laser.

OBJECTIVES

Objectives: The aim of this study was to evaluate the effects of photobiomodulation on iNOS and C3 gene expression in an experimental model of acute arthritis.

MATERIALS AND METHODS

Materials and Methods: This study was aprproved by CEUA-FHO 077/2017. Eighteen female Wistar rats (200±10g), were kept in light-dark cycles of 12 hours with food and water ad libitum. Animals (n=18) were assigned into three groups (n=6), Control (no induction), Sham (induced arthritis) and PBM (arthritis and low-level laser). Arthritis induction was performed with 200μg of Zymosan injected in the right knee of the animals and twenty-four hours after induction, photobiomodulation was performed with low-level laser by single dose therapy with the following parameters, λ=808nm, 25mW nominal power, fluency of 20J/cm2, beam area of 0.02mm2, time of 33s and total energy of 0.825J. Animals were euthanized after 7 days of arthritis induction by anesthetic overdose and cardiac exsanguination. Synovia samples were submitted to RNA extraction, cDNA synthesis and gene expression was evaluated by real-time PCR (2-ΔΔCt) for β-actin (constitutive), iNOS and C3 genes.

DISCUSSION AND RESULTS

Results: The gene expression, median; min;max 2-ΔΔCt values, of iNOS was higher with PBM therapy, Control 0.8749 (0.8154; 0.9362), Sham 0.8836 (0.8311; 0.9363), PBM 1.095 (0.9476; 1.184) with significant differences between PBMxSham p=0.0022. No statistical differences were observed with PBM in C3 gene expression, Control 0.8770 (0.8220; 0.8960), Sham 0.8815 (0.8720; 0.9630), PBM 0.9329 (0.9184; 1.060).

CONCLUSION

Conclusion: Photobiomodulation up regulated iNOS gene expression, which refers to secondary mitochondrial stimulation that can promote an increase of antioxidant enzyme balance, while C3 levels remained constant and it is suggested that there is no activation of the complement system in the period studied.

Keywords: Arthritis rheumatoid, Low-level laser therapy, Gene expression

Supported by: Propesq-FHO

08989 - Poster Session

NB.13 - A simple and quick method to generate in vitro tridimensional tumor bodies from a human breast adenocarcinoma (MCF7) using magnetic aggregation technique

Mayelle Maria Paz Lima ¹, Pamela Ferreira do Nascimento¹, Ana Cristina Gomes Nascimento¹, Daniel Perez Vieira¹

¹Laboratory of Radiobiology, Center of Biotechnology (CEBIO), Nuclear and Energetic Research Institute (IPEN/CNEN-SP) (SP, Brasil)

INTRODUCTION

Tumor physiology studies have to rely on efficient and representative models, as animal-based or in vitro tridimensional cell constructs. The work used magnetite (Fe3O4) nanoparticles produced by electron-beam induced chemical reduction to give cells the ability to form aggregates when submitted to a magnetic field, and thus to produce micro tumors in vitro.

OBJECTIVES

The work aimed to produce human breast adenocarcinoma mini tumors (BAMT's) in vitro.

MATERIALS AND METHODS

Paramagnetic iron oxide nanoparticles (PION’s) were synthesized through electron-beam induced Fe3+ reduction and subsequent coprecipitation. Due to its poly-L-lysine coating, PION’s were adsorbed on cell membranes of MCF7 (human breast adenocarcinoma). Cells were seeded in 24-well cell culture plates pre-treated overnight with Pluronic® F-127 to prevent cell adhesion and kept in culture conditions under magnetic fields for at least 6 days. BAMT's were differentially stained with Hoescht 33342 and ethidium bromide and imaged by wide-field fluorescence microscopy.

DISCUSSION AND RESULTS

BAMT's appeared as integer and well-defined cellular aggregates, with sparse dead cells stained by ethidium bromide. These structures can be further used for in vitro tumor studies, as BAMT’s are supposed to be more reliable models than monolayer cultures. Treatment of wells with poloxamer caused a mild to moderated cell-repellent effect, similar to those found in commercially available products, only by a fraction of the cost.

CONCLUSION

The experiments succesfully produced mini tumors prone to be used in in vitro studies.

Keywords: breast cancer, 3d culture, magnetic

Supported by: FAPESP (2017/50332-0) & IPEN/CNEN-SP

08291 - Poster Session

NB.14 - Development of a Female Mouse Computational Model Based on CT Images for Dosimetric Assays

Christiana da Silva Leite ¹, Ana Carolina Araújo Bispo¹, Marcelo Mamede³, Andrea Vidal Ferreira¹, Juliana Batista Silva¹, Bruno Melo Mendes²

¹SERFI, Centro de Desenvolvimento da Tecnologia Nuclear (MG, Brasil), ²SECDOS, Centro de Desenvolvimento da Tecnologia Nuclear (MG, Brasil), ³Faculdade de Medicina , Universidade Federal de Minas Gerais (MG, Brasil)

INTRODUCTION

Small animals, such as mice, are used in biodistribution studies and innumerous preclinical investigations involving ionizing radiation. Longitudinal preclinical studies with five or more image procedures (MicroCT and/or PET/SPECT) are not uncommon. However, the cumulated absorbed doses in mice organs and their influence in experimental results is often neglected. Accurate calculation of absorbed doses in mice organs are needed to evaluate potential radiobiological effects that may interfere with in vivo experiments. Based on a previous study of a male mouse computational model known as DM_BRA, this paper is focused on the development of FM_BRA, a female mouse computational model.

OBJECTIVES

Develop and implement for the MCNP code a female computational mouse model for mice radiopharmaceutical dosimetry.

MATERIALS AND METHODS

A set of Micro-CT images of a female mouse kindly available at (https://www.youtube.com/watch?v=-Xg921NVFSs) was selected for the segmentation process. Forty-seven coronal slices were manually segmented using AdobePhotoshop®. In these images each color corresponds to a numerical code that identifies each organ. After the segmentation process, the images were converted into a ".raw" 3D file format. An in house C++ program was used to convert the 3D image into the computational model in the MCNP format.

DISCUSSION AND RESULTS

The new FM_BRA was segmented with 20 tissues/organs. The model matrix has (156 x 366 x 105) voxels and the voxels dimensions are (0.25 x 0.25 x 0.25) mm3. Elemental composition and density of human organs were used in MCNP setup of the model. The total mass of the model is 26.3 g. The masses of segmented organs were compatible with the values ​​found in the literature.

CONCLUSION

A new female mice model was successfully developed and implemented for MCNP. A set of S-values for dosimetry of positron emitting radioisotopes will be available soon.

Keywords: female mouse model, mice dosimetry, Monte Carlo

Supported by: CNPq

05065 -

NB.15 - Radiochemical and biological properties of peptides designed to interact with EGF receptor: Relevance for glioblastoma

Danielle Vieira Sobral¹, Leonardo Lima Fuscaldi¹, Fernanda Ferreira Mendonça¹, Ana Cláudia Camargo Miranda², Jorge Mejia², Marycel Barboza², Luciana Malavolta¹

¹Ciências Fisiológicas, Faculdade de Ciências Médicas da Santa Casa de São Paulo (São Paulo, Brasil), ²Hospital Israelita Albert Einstein, Instituto Israelita de Ensino e Pesquisa (São Paulo, Brazil)

INTRODUCTION

Radiolabeled peptides with high specificity for receptors expressed on tumor cells hold great promise as diagnostic and therapeutic biomarkers.

OBJECTIVES

The main objective of this study was to evaluate the radiochemical and biological properties of two [131I]I-peptides, as well as their interaction with the epidermal growth factor receptor (EGFR), which is overexpressed in a wide variety of tumors, including glioblastoma.

MATERIALS AND METHODS

The peptide EEEEYFELV and its analogue DEDEYFELV, both designed to interact with EGFR, were chemically synthesized, purified and radiolabeled with iodine-131 ([131I]-NaI). Radioiodination was evaluated and optimized using the chloramine-T methodology. Stability, serum protein binding and partition coefficient were evaluated for both radioconjugates. In addition, the binding and uptake fraction of radiopeptides synthesized with rat glioblastoma cells (C6) and with rat brain homogenates from a glioblastoma-induced model were evaluated and ex vivo biodistribution studies were performed.

DISCUSSION AND RESULTS

Under optimized radiolabeling conditions, the peptides showed an average radiochemical yield of 90-95%. Stability studies showed that both peptides remained stable for up to 24 h in reaction medium, saline solution, and human serum. [131I]I-peptides have hydrophilic characteristics and have a binding percentage to serum proteins around 50%, which is highly compatible with clinical applications; showed binding and internalization capacity both in tumor cells (C6) and in rat brain tissues after tumor induction. Biodistribution studies corroborated cell culture studies and confirmed the different binding characteristics derived from a simple two-amino acid change (Glu-->Asp1,3) in their sequences. The results obtained are consistent enough to motivate further studies.

CONCLUSION

The peptides were efficiently synthesized and the tested radiolabeling strategies showed successful results. Moreover, all the peptides demonstrated affinity for the tumor cells evaluated. These results obtained in this study are consistent to adapt in the clinical application.

Keywords: Glioblastoma, Peptides, Radioiodination

Supported by: FAPESP, CAPES AND FAP (FCMSCSP)

08996 -

NB.16 - Metabolic and Structural Signatures in Corticobasal Syndrome: A Multimodal PET/MRI Study

Carneiro, G. C.¹, Parmera, J. B.², Almeida, I. J.³, Oliveira, M. C. B^2,4, Silagi, M. L.⁵, Studart-Neto, A.², Ono, C. R¹, Barbosa, E. R.², Nitrini, R.², Buchpiguel, C. A.¹, Brucki, S. M. D.², Coutinho, A. M.¹

¹Laboratory of Nuclear Medicine, Nuclear Medicine Center and Division, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; ²Department of Neurology, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; ³Department of Physical Therapy, Speech, and Occupational Therapy, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; ⁴Neurology Unit, Instituto do Câncer do Estado de São Paulo, São Paulo, Brazil; ⁵Department of Speech, Language and Hearing Sciences, Universidade Federal de São Paulo, São Paulo, Brazil.

Corticobasal syndrome (CBS) is a progressive neurological disorder related to multiple pathologies, including four-repeat tauopathies, such as corticobasal degeneration and progressive supranuclear palsy, and Alzheimer’s disease (AD). Speech and language are commonly impaired, encompassing a broad spectrum of deficits. To investigate CBS speech and language impairment patterns considering a multimodal imaging approach. They underwent positron emission tomography with [¹⁸F-]fluorodeoxyglucose (FDG-PET) and [¹¹C]Pittsburgh Compound-B (PIB-PET) on a hybrid PET-MRI machine to assess their amyloid status. PIB-PET images were classified based on visual and semi-quantitative analyses. Quantitative group analyses were performed on atrophy patterns on MRI were investigated using voxel-based morphometry (VBM) and FDG-PET data. Thirty healthy participants were recruited as imaging controls. PIB-PET was classified as negative (CBS-Amyloid – group) in n=18/31 and positive (CBS-Amyloid+ group) in n=13/31 patients. The frequency of dysarthria was significantly higher in the CBS-A– group than in the CBS-A+ group (55.6 vs. 7.7%, p = 0.008). They showed brain atrophy mainly at the putamen and opercular frontal gyrus. Relative to the phonemic verbal fluency, we found a positive correlation at the left frontal opercular gyrus (p = 0.0003, R² = 0.3685), the inferior (p = 0.0004, R² = 0.3537), and the middle temporal gyri (p = 0.0001, R² = 0.3993). There was a positive correlation between [¹⁸F]FDG uptake and semantic verbal fluency at the left inferior (p = 0.006, R² = 0.2326), middle (0.0054, R² = 0.2376), and superior temporal gyri (p = 0.0066, R² = 0.2276). Metabolic and structural signatures depicted from this feature provide further insights into the motor speech production network and are also helpful to differentiate CBS variants. In the spectrum of language impairment profile, dysarthria might be helpful to distinguish CBS patients not related to AD.

Keywords: Corticobasal syndrome, amyloid-PET, fluorodeoxyglucose

Funding: FAPESP

Keywords: Corticobasal syndrome, amyloid-PET, fluorodeoxyglucose

Supported by: FAPESP

08534 - Poster Session

SE.01 - Didactic games in Biochemistry: alternative approaches to content absorption

Daniel de Carvalho Santos ¹, Giselle Zenker Justo², Karin Argenti Simon², Nidia Alice Pinheiro²

¹Química, Universidade de São Paulo (, Brasil), ²Ciências Farmacêuticas, Universidade Federal de São Paulo (, Brasil)

INTRODUCTION

Biochemistry is considered by many students as a subject with a high degree of difficulty in view of the massive amount of content, which stimulates the development of new teaching methodologies, generating new approaches to the learning process. Among these methodologies are educational games, which allows the student to interact with content in an alternative way, in addition to stimulating skills such as creativity, reasoning and group work.

OBJECTIVES

Thus, the objective of this work was the development of three educational games as an alternative approach to teaching the main contents of the discipline.

MATERIALS AND METHODS

The games “Quem sou eu, bioquímico?”, “Aprendendo bioquímica por bem ou por mau-mau” and “Lipoprotein Game” were applied to students of Biological Sciences, Pharmacy and Chemistry graduation courses at the Federal University of São Paulo (UNIFESP) - Campus Diadema.

DISCUSSION AND RESULTS

During the proposed activities, students were subjected to different assessment methods such as forms of satisfaction and progress within the course in relation to each point explored, accompanied by teachers and monitors.

CONCLUSION

Subsequent assessment of students’ perceptions with questionnaires revealed enhanced interest in the subject and a better perfomance in the tests was noticed, suggesting that the use of diversified approaches can be effective in the learning of Biochemistry.

Keywords: Didactic games, Biochemistry, teaching methodologies

08363 - Poster Session

SE.02 - Metagame: a proposal for teaching biochemistry

Amanda Borges Colman¹, Malson Neilson de Lucena ¹

¹Instituto de Biociências, Universidade Federal de Mato Grosso do Sul (Mato Grosso do Sul, Brasil)

INTRODUCTION

Biochemistry is an essential subject in most of the undergraduate courses related to health and biologics fields. However, many students find it difficult to understand because of the fragmented way in which this subject is taught. In this context, many educational innovations have been developed, especially games. Games are always in evidence for their ludic and dynamic way of teaching contents and also for stimulating student’s critical thinking.

OBJECTIVES

The objective of this work was to create and develop a card game that would allow the integration of metabolism content in a way that would allow undergraduate students from biomedical and biological areas to understand the connection between the topics covered in the biochemistry subject and their applications on a daily basis

MATERIALS AND METHODS

The game consists of a total of 35 cards, among which five correspond to the “tissue” cards. The other 30 cards correspond to the “problem situation” cards.

DISCUSSION AND RESULTS

The created game was called Metagame, which proposes to work biochemistry from mammalian metabolism contents in a dynamic, integrated and contextualized way, which can be used as an evaluative or fixation activity. Some suggestions for applying and a real example of using the game in a nutrition class are presented to show the practical use of Metagame as a paradidactic instrument to construct biochemistry knowledge.

CONCLUSION

Metagame facilitates the understanding of biochemical concepts considered complex and that are important throughout the curriculum structure of undergraduate courses in the areas of health and biological sciences.

Keywords: didactic model , ludic, metabolism