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Published in final edited form as: J Cell Physiol. 2019 Sep 5;235(3):2590–2598. doi: 10.1002/jcp.29163

Dysbiotic 1-carbon metabolism in cardiac muscle remodeling

Akash K George 1,*, Mahavir Singh 1,*,†, S Pushpakumar 1, Rubens P Homme 1, Shanna J Hardin 1, Suresh C Tyagi 1
PMCID: PMC6899188  NIHMSID: NIHMS1048826  PMID: 31489638

Abstract

Unless there is a genetic defect/mutation/deletion in a gene, the causation of a given disease is chronic dysregulation of gut metabolism. Most of the time, if not always, starts within the gut; i.e. what we eat. Recent research shows that the imbalance between good versus bad microbial population, especially in the gut, causes systemic diseases. Thus, an appropriate balance of the gut microbiota (eubiosis over dysbiosis) needs to be maintained for normal health (Veeranki and Tyagi 2017). However; during various diseases such as metabolic syndrome, inflammatory bowel disease, diabetes, obesity and hypertension the dysbiotic gut environment tends to prevail. Our research focuses on homocysteine (Hcy) metabolism that occupies a center-stage in many biochemically relevant epigenetic mechanisms. For example, dysbiotic bacteria methylate promoters to inhibit gene activities. Interestingly, the product of the 1-carbon metabolism is Hcy, unequivocally. Emerging studies show that host resistance to various antibiotics occurs due to inverton promoter inhibition, presumably because of promoter methylation. This results from modification of host promoters by bacterial products leading to loss of host’s ability to drug compatibility and system sensitivity. In this study, we focus on the role of high methionine diet (HMD), an ingredient rich in red meat and measure the effects of a probiotic on cardiac muscle remodeling and its functions. We employed wild type (WT) and cystathionine beta synthase heterozygote knock-out (CBS+/−) mice with and without HMD and with and without a probiotic; PB (Lactobacillus) in drinking water for 16 weeks. Results indicate that matrix metalloproteinase-2 (MMP-2) activity was robust in CBS+/− fed with HMD and that it was successfully attenuated by the PB treatment. Cardiomyocyte contractility and ECHO data revealed mitigation of the cardiac dysfunction in CBS+/−+HMD mice treated with PB. In conclusion, our data suggest that probiotics can potentially reverse the Hcy-meditated cardiac dysfunction.

Keywords: Betaine, Carnitine, Epigenetics, Eubiosis, Microbiome

1. INTRODUCTION

Biochemically, 1-carbon metabolism is the hallmark of epigenetics and DNA/RNA/protein methylation that controls gene expression activity via chromatin/promoter modification (Selhub 2002). Interestingly, the product of this methylation is the homocysteine (Hcy). Elevated levels of Hcy, i.e. hyperhomocysteinemia (HHcy) participates in numerous cardiovascular diseases (Tyagi S. C. 1999) (Veeranki and Tyagi 2017). Diet that is rich in methionine (Met) such as red meat causes HHcy, presumably due to dysbiosis. During eubiosis phosphatidylcholine (PC), carnitine, trimethylamine (TMA), Hcy, methionine (Met) and dimethylglycine (DMG) are regulated by phosphatidylcholine phosphatase (PCP) and betaine-Hcy S-methyltransferase (BHMT) in the gut (Fig.1A). However; during dysbiosis the elevated levels of flavin monooxygenase (FMO) create trimethylamine N-oxide (TMAO; a most potent factor that is responsible for cardiovascular stiffness) (Jiang et al. 2019), instigating remodeling and decreasing regenerative capacity of the heart (Brzezinski and Zundel 1993). More recently, studies on host resistance to antibiotics was shown to be the result of inverton promoter inhibition via methylation as induced by dysbiotic bacterial antigens in turn forcing the host to lose its competitive edge on pathogenic bacteria (Bjorndal et al. 2015, Jiang et al. 2019). During eubiosis, the epigenetically controlled reversible DNA methylation by DNA methyl transferase (DNMT) and phosphatidylethanolamine methyltransferase (PEMT) allows normal gene regulation but during dysbiosis the irreversible DNA methylation causes HHcy which is responsible for cardiovascular complications most likely as an onslaught of gut dysbiotic environment leading to the metabolic syndrome (Chaturvedi et al. 2014) (Fig. 1B). Cystathione beta synthase (CBS) metabolizes Hcy and clears Hcy by the transsulfuration pathway. Methylation of CBS promoter has been shown (Behera et al. 2019), leading to reduced CBS expression and this may also contribute to inverton and Hcy-mediated antibiotic resistance. Therefore, the hypothesis of this study is that dysbiotic 1-carbon metabolism causes cardiovascular diseases and treatment with probiotics mitigates dysbiosis and cardiovascular diseases.

Figure 1.

Figure 1

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Mechanism of methionine-homocysteine-betaine-cycle during eubiosis versus dysbiosis (A) PC, phosphatidylcholine; PCP, phosphatidylcholine phosphatase; Hcy, homocysteine; Met, methionine; TMA, trimethyl amine; FMO, Flavin (FAD)-monooxygenase; TMAO, trimethyl amine-oxide; BHMT, betaine-homocysteine S-methyl transferase; DMG, dimethyl glycine. Epigenetic mechanisms favor reversible and irreversible DNA methylation patters during eubiosis versus dysbiosis that is responsible for causation of hyperhomocysteinemia (HHcy) (B). PE, phosphatidylethanolamine; PC, phosphatidylcholine; PEMT, phosphatidylethanolamine methyltransferase; SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine; SAHH, S-adenosyl homocysteine hydrolase; DNMT, DNA methyltransferase.

2. MATERIALS AND METHODS

2.1. Animal protocols

Male and female 10–12 weeks old mice, WT (C57BL/6J) and CBS+/− (B6.129P2-Cbstm1Unc/J 002853) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The animal procedures were reviewed and subsequently approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Louisville School of Medicine, Louisville, Kentucky, USA. Further, the animal care and guidelines of the National Institutes of Health (NIH, USA) were also adhered to. To create HHcy condition, the mice were fed with a high methionine diet HMD (Harlan Laboratories) and with or without oral probiotic; PB (Lactobacillus rhamnosus GG @ 2.5×105 CFU for a period of 16 weeks in drinking water. Control mice groups were fed the standard chow diet. All mice were allowed water ad libitum.

2.2. Genotyping of CBS+/− mice and probiotic (PB) administration

The mice were weaned and genotyped according to the Jackson Laboratory’s recommendations. Tail samples were collected, and genotypic analyses were performed using PCR, as shown in Fig 2. Heterozygotes CBS+/− mice produced two bands (450 and 308 bp) while CBS+/+ mice presented only one band (308 bp). Western blot of the CBS levels in the heterozygote mice in comparison to the WT has previously been performed by our group and others (George et al. 2019, Robert et al. 2003). Unless otherwise mentioned, three to five mice were used in each group for all the experiments. Mice were treated with PB for 16 weeks @ 2.5×105 CFU while the control mice (without PB) were given normal water. At the end of the experiment, animals were euthanized by using 2 X tribromoethanol (TBE). Mice were grouped as follows: (i) WT: Control group, (ii) WT+PB: Treatment group, (iii) CBS+/−+HMD: Control group and (iv) CBS+/−+HMD+PB: Treatment group. Body weight (BW) in grams before and after the PB treatment were measured. To ensure probiotic treatment, the fecal levels of acetates were also measured (Baxter et al. 2019).

Figure 2.

Figure 2

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Genotyping of the mice strains with PCR for identifying the CBS+/− (heterozygote knockout; KO) from the wild type (WT, CBS+/+) (A). The amplified reaction products were loaded using the agarose gel electrophoresis procedure to identify the DNA bands of interest. Lane 1, DNA marker; lane 2, CBS+/−, lanes 3 and 4, WT (CBS+/+); lanes 5 and 6, CBS+/−. Acetate levels in the fecal samples of mice treated with or without high methionine diet (HMD, an ingredient high in red meat causing further dysbiosis) (B). The Box-plot showing the abundance and modulation of the acetic acid concentrations in experimental mice groups as obtained by pairwise Wilcoxon test (p < 0.05). The WT mice were also fed HMD with and without PB. The results suggested that PB mitigated decrease in acetate levels in WT mice fed with HMD.

2.3. Blood pressure

Non-invasive tail cuff method was used to measure blood pressure in conscious mice (CODA, Kent Scientific, Torrington CT). Animals were allowed sufficient time to acclimate in the chambers and warming pad were used prior to data recordings. Blood pressure (BP) was recorded under standard conditions.

2.4. Western blot analysis

Antibodies for CSE, BHMT were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), REDD, DAAM1, DAAM 2 from Abcam (Cambridge, MA, USA), GAPDH was from Boster Biological Technology (Pleasanton, CA, USA) and occludin was purchased from Novus Biologicals (Littleton, CO, USA). PB was purchased from i-Health, Inc. CT, USA. Cardiac tissue protein was isolated using protein extraction buffer (RIPA lysis buffer, protease inhibitor cocktail and PMSF). Lysates were spun in extraction buffer for 12 h and then centrifuged at 12,000 × g for 15 min. Supernatant was transferred to new tubes and protein concentrations were analyzed via Bradford protein estimation assay. Samples were run on a 10/12% sodium dodecyl sulfate (SDS)-polyacrylamide gel with Tris–glycine SDS buffer. The gel was transferred electrophoretically overnight onto a PVDF membrane at 4 °C. The membrane was blocked with a 5% milk solution for 1 h. Primary antibodies were diluted at a concentration 1:1000 in TBST and incubated on the membrane overnight. All membranes were washed in TBST solution four times and then incubated with secondary HRP conjugated antibody solution for 1 h at room temperature. Four TBST washing steps followed before membranes were developed using a chemiluminescent substrate in a BioRad Chemidoc (Hercules, Calif.). Band intensity was determined using densitometry analysis. Beta-actin was used to normalize protein loading. Equal amounts of total protein (50 μg) were resolved on SDS-PAGE and transferred to polyvinylidene membranes. The membranes were probed overnight at 4ºC with primary antibodies followed by 2h incubation in secondary antibodies. The signal capturing was done with the Bio-Rad ChemiDoc XRS+ system and Image Lab software (Bio-Rad, Hercules, CA). The relative optical density of protein bands was analyzed using gel software Image Lab 3.0. The membranes were stripped and re-probed with GAPDH as a loading control.

2.5. MMPs activity

Zymography, using 1% gelatin in the gels were performed on LV tissue homogenates as described (Kundu et al. 2009, Tyagi N. et al. 2010, Tyagi S. C. et al. 1993).

2.6. Echocardiography

Ultrasound through Vevo 2100 imaging system, cardiac and aortic data were collected. The transducer probe was placed on the left hemithorax of the mice in the partial left decubitus position. Two-dimensionally targeted M-mode echocardiograms were obtained from a short-axis view of the left ventricle at or just below the tip of the mitral-valve leaflet and were recorded. LV size and the thickness of LV wall were measured. Only M-mode ECHO with well-defined continuous interfaces of the septum and posterior wall were collected (Kunkel et al. 2019). Experimental animals were placed supine on a warm platform (37 °C), under isoflurane anesthesia and fixed. Using a MS550D (22–25 mHz) transducer, the thoracic cavity was imaged. Aortic arch velocity and cardiographic function were assessed in pulse wave and color Doppler modes.

2.7. Analyses of the single myocyte contraction, calcium transit, and cardiomyocyte contractility measurements

The intact hearts were dissected out quickly from the anaesthetized mice and were perfused with freshly made perfusion buffer with liberase TH as described before (Givvimani et al. 2015). The yield was 80–85% and did not vary between the groups. The isolated myocytes were used immediately for the contractility measurements, calcium transits, flow cytometry analysis, mitochondrial fractionation and oxygen consumption rates (OCR) measurements. The systolic and diastolic on isolated myocytes were recorded by Ion-Optics (Boston). The decay of calcium transient was also recorded as previously described (Veeranki et al. 2016). A sub set of cardiomyocytes were incubated with Fura-2-AM (1.0 μmol/l) for 30 min, and fluorescence measurements are recorded with a dual-excitation fluorescence photomultiplier system (IonOptix) as described before after 1 Hz field stimulation (Givvimani et al. 2015). Myocytes were field stimulated (at a frequency of 1.0 Hz, pulse duration of 4 ms and amplitude of 10 volts) using IonOptix myopacer and the contractions were recorded through SoftEdge™ Acquisition Software as described before (Givvimani et al. 2015). A batch of 5 randomly-selected myocytes were recorded for the contraction parameters at a time from an unstimulated pool and a total of 20 myocyte recordings per heart were collected for further analysis.

2.8. Cardiac passive function

The length tension relationship of cardiac stiffness was measured by cardiac ring preparation as described (Tyagi S. C. et al. 1999). In brief ex vivo endothelial-myocyte coupling was measured (Camp et al. 2004). The “deli” shaped LV rings were mounted between two wires in a tissue myobath as described (Tyagi S. C. et al. 1999). The rings were free of any mechanical or hypoxic injuries under these conditions (Tyagi S. C. et al. 1999).

2.9. Histology

Cryosectioning of cardiac tissue was preserved in plastic tissue embedding molds (polysciences) containing tissue freezing media (Triangle Biosciences, Durham, NC). Tissues were stored at − 80 °C. Sections of 8 μm were created using a cryocut (leica CM 1850). Cryosections were placed on superfrost plus (lysine coated) microscope slides and stored at − 80 °C for further use in immuno-histochemistry. Quantitative analysis was performed using Image Pro program (IMAGE-PRO Media Cybernetics, Silver Spring, MD, USA) and data was expressed in marker area (pixels2) averaged form five randomly selected fields per slide.

2.10. Statistical Analysis

Data analyses and graphical presentations were performed with the help of GraphPad InStat 3 and GraphPad Prism, version 6.07 (GraphPad Software, Inc., La Jolla, CA). Data are represented as mean values ± standard error (SE) from 5 independent experiments in all cases. The experimental groups were compared by one-way analyses of variance (ANOVA) if the values were sampled from Gaussian distributions. For a set of data, if ANOVA indicated a significant difference (p < 0.05); Tukey-Kramer multiple comparison tests were used to compare group means. Post-test was only performed if p<0.05. If the value of Tukey-Kramer ‘q’ is less than 4.046, then the p-value is less than 0.05 and considered statistically significant.

3. Results

To determine the genotype of CBS+/− mice, the PCR on the DNAs isolated from tail clips were performed. The results suggested WT mice with two normal copy of same allele molecular weight. The CBS revealed two, normal and disrupted DNA copy in CBS+/− mice (Fig. 2A). To determine whether the treatment of the mice with probiotic (PB) was effective, we measured total fecal acetate levels (a marker of probiotics). The results revealed dysbiosis in CBS+/− with HMD. Interestingly, the treatment with PB normalized the acetate levels and consequently the levels of probiotic (Fig. 2B). The treatment of the mice with PB decreased the body weights in CBS+/− mice as compared to WT mice (Fig. 3 A, B). Although it is known that systemic arterial stiffness causes systemic hypertension, the CBS+/− mice do display arteriosclerotic as well as the high blood pressure phenotypes. Interestingly, treatment with PB was able to decrease the systemic arterial blood pressure in these CBS+/− mice strain (Fig. 3C). The remodeling by its very nature involves the matrix metalloproteinases (MMPs), therefore we decided to measure the cardiac LV MMPs activities employing the zymography procedure, and the results revealed that PB mitigated the MMP-2 activity in all groups. These results suggested for the first time that PB decreases MMPs activities in the hearts of the dysbiotic CBS+/− mice (Fig. 4A). Further, to determine whether the regenerative capacity of the hearts is also improved by PB treatment in the CBS+/− mice hearts, we measured the DAAM1, DAAM2, REDD1, BHMT and the CSE target proteins expression profiles in the lysates from the mice hearts (Fig. 4B). The results suggested improvements in the levels of these key target regenerative molecules in the hearts post PB treatment as demonstrated by others in the past (Nolan et al. 2017).

Figure 3.

Figure 3

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Body weights (BW) of WT and CBS+/− mice before (A) the start of the experiment and after16 weeks of probiotic treatment (B). Values are averages ± standard error, n= 3–5 per group. Bar graph representing the average mean arterial pressure (MAP, mmHg) of WT and CBS+/− mice treated with HMD with and without the probiotic (PB) (C). Values are averages ± standard error, and with a significance within groups, p<0.05. Asterisks indicate significant difference (p < 0.05) from the WT.

Figure 4.

Figure 4

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One dimensional (1D) gelatin gel zymographic analysis of MMP-2 (72 kDa) in the hearts of WT and CBS+/− mice treated with HMD and with and without the probiotic. (A) The zymograph is the representative data from 3 independent experiments. Expression analysis of key proteins that are involved in the regenerative response (i.e. DAAM1 & 2, REDD1, BHMT, CSE) in the hearts of different experimental mice groups (B).

To determine single cardiomyocyte contractility, we measured contractility of the single isolated cardiomyocytes, and the results revealed depression in the contraction of CBS+/− mice. Interestingly, the treatment with PB mitigated this depression as revealed by the cardiac contractility (Fig. 5A, B). The rate of relaxation (-dl/dt) and the contraction (dl/dt) were also attenuated in the CBS+/− dysbiotic mice that were fed with HMD, however; the PB treatment mitigated this contractile attenuation rates (Fig. 5 C, D). To determine the nature of remodeling, if any, in the CBS+/− mice, the cardiac fibrosis was measured employing collagen staining with trichrome. The results demonstrated robust fibrotic changes in both LV and RV parenchyma of these CBS+/− mice that were fed with the dysbiotic HMD diet. Interestingly, the treatment with PB mitigated the cardiac fibrosis (Fig.6A).

Figure 5.

Figure 5

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Recording of the single myocyte contraction from WT and CBS+/− mice (A and B) treated with and without the probiotic (PB). Rate of systolic (C) and diastolic (D) cardiomyocyte relaxation in mice treated with and without the probiotic (PB). All experiments were performed at least in triplicates. There was no significant change in the magnitudes, but there was a shift in lengthening peak.

Figure 6.

Figure 6

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Cardiac fibrosis and its mitigation (A). Masson’s trichrome staining was employed for the demonstration of fibrotic changes in the CBS+/− +HMD mice group compared to the WT mice and its alleviation by the prolonged treatment with the probiotic (PB) in CBS+/ -+HMD+PB mice hearts. Relationship between the cardiac ring left ventricle (LV) length-tension (B). Cardiac response to passive stretching of the LV rings in myobath prepared from mice that were treated with and without probiotic (PB). All experiments were performed in 4–6 separate mice from each group. Tension data were significantly decreased in the CBS+/− strain. Interestingly, the PB diet mitigated this decrease in CBS+/− +HMD as well as in WT mice.

To determine whether the above findings regarding the fibrotic changes had any cardiac stiffness effect in the CBS+/− dysbiotic mice, we measured the cardiac passive length-tension relationships in cardiac rings that were prepared from individual mice hearts. The results suggested the blunted length-tension relation in CBS+/− dysbiotic mice as compared to the control normal mice hearts. The intervention with PB mitigated this bluntness and the effects of the cardiac stiffness (Fig. 6B). Additionally, we performed the Echo M-mode and Doppler blood flow measurements in all the groups of the mice under study. The findings revealed the attenuation of the cardiac systolic and diastolic functions as well as the aortic blood flow in the dysbiotic CBS+/− mice. Again, the treatment with PB mitigated these functional parameters in these dysbiotic CBS+/− mice hearts (Fig. 7 A, B).

Figure 7.

Figure 7

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Echocardiography of the mice hearts treated with and without HMD and the PB (A). The ultrasound echocardiography displaying blood flow through aorta as accomplished via Vevo 2100 ultrasound pulse wave system followed by the color Doppler analysis. The M-mode images correspond to the specified experimental group. Arrows indicate diastolic (longer)/systolic (shorter) chamber lengths in respective group. Acquisition of images using the ultrasound Doppler echocardiography (B). Analysis showing the blood flow through aorta as acquired via Vevo 2100 ultrasound pulse wave system followed by the color Doppler analysis. The M-mode images correspond to the specified experimental group. The rate of blood flow is altered in the CBS+/− mice and that was mitigated by the PB treatment. All experiments were performed in 4–6 separate mice from each group. The data showed that CBS+/− +HMD+PB group revealed mitigation of cardiac dysfunction.

4. DISCUSSION

Short chain fatty acids (SCFA) are the products of fermentation of insoluble fiber from diet (e.g. cellulose, resistant starch) by the bacteria that are present in the gut. These fatty acids have been shown to play an important role in regulating the metabolism in the gut and are closely associated with gastrointestinal diseases such as irritable bowel syndrome and other conditions such as obesity. SCFA have been shown to modify systemic blood pressure as well (Pluznick 2014). By quantifying SCFA in the fecal samples, one can monitor the gut health and the level of inflammation. Recent studies reported that acetate changes the gut microbiota and that can prevent the development of hypertension and heart failure (Marques et al. 2017) and other organs dysfunction (Bluemel et al. 2016). we report that acetate levels are low in hypertensive CBS, interestingly, the supplementation with the probiotic improved the levels of acetate in the gut of CBS. Although CBS+/− heterozygote KO mice are moderately hypertensive, treatment with the probiotic mitigated the hypertension. Because probiotics and fiber rich diet mimic and thus may allow the animals to lose their weights, we too observed decrease in their body weights after probiotic treatment in the CBS+/− heterozygote KO mice, clearly suggesting the role of homocysteine in the epigenetic memory (Reizel et al. 2018, Reizel et al. 2015).

We show for the first time that MMP-2 was reduced in the CBS+/− heterozygote KO mice treated with the probiotic, suggesting the reversal of the remodeling by the prolonged treatment with the probiotic in HHcy-induced heart failure model. The levels of DAAM1 and DAAM2 were also decreased in the HHcy mice, however the treatment with the probiotic mitigated this decrease in DAAM molecules. Further, the levels of REDD1, CSE, and BHMT were shown to be mitigated as well (Ajima et al. 2015, Bluemel et al. 2016, Hayasaka et al. 2014, Kumari et al. 2011).

These results indicate that the cardiac regeneration was severely blunted in CBS+/− heterozygote KO mice with dysbiosis; however, treatment with the probiotic was able to mitigate this bluntness. The cardiac functions were improved post probiotic treatment too as well as cardiac fibrosis in CBS+/− heterozygote KO mice was found to be reduced leading to weakness in the cardiac muscle strength. Interestingly, the muscle weakness was successfully mitigated by the treatment with the probiotic, suggesting that a likely underlying mechanism for this positive effect might be, in some way, connected to a switch in the energy source from fibrous dietary components for the generation of the short-chain fatty acids such as abundance of acetic acid as demonstrated in our study (Fig. 8). The overall findings from our study suggest that a dysbiotic hearts in the CBS+/− mice cause cardiac dysfunction in part as seen by an increase in the remodeling and a concomitant decrease in the regenerative capacity of the cardiac musculature. The study holds potential that despite severe metabolic dysfunction because of dysregulation of the 1-carbon metabolism as further potentiated with dysbiotic gut environment a prolonged treatment with the PB could reverse the remodeling of the cardiac muscle and thus save from the heart failure as depicted via putative mechanism(s) in the schematic (Fig. 8).

Figure 8.

Figure 8

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A schematic illustrating the putative mechanism(s) underlying the protection of myocardium as afforded by the prolonged treatment with probiotic in the dysbiotic hearts during chronic metabolic dysregulation of the 1-carbon metabolic cycle in mice. The treatment could successfully reverse the remodeling of the cardiac muscle and thus save it from heart failure.

LIMITATIONS

The MMP2 activity was quantified in triplicates. Although to determine if the effect is on activity or expression, RT-PCR and Western blotting should also be performed. Also, the regenerative proteins increasing with probiotics at the mRNA level. It would be informative to investigate whether transcription/epigenetic changes are involved in the change in protein levels. These experiments will be performed in future studies.

ACKNOWLEDGMENT

This work was supported by NIH grants: HL74185, HL139047, and AR-71789. Other members of the laboratory are duly recognized for their help and support.

A part of this study was supported by NIH grants: HL74185, HL139047, and AR-71789 and presented at the Experimental Biology Annual Meeting 2019, Orlando, Florida.

Abbreviations

CBS

cystathionine β synthase

CSE

cystathionine ϒ lyase

DAAM1

disheveled associated activator of morphogenesis 1

DNMT

DNA methyltransferase

REDD1

Regulated in development and DNA damage response 1

PEMT

Phosphatidylethanolamine N-Methyltransferase

TMAO

trimethylamine-N-oxide

Footnotes

Data Availability Statement

As desired, we will share the research data including, but not limited to: raw data, processed data, software, algorithms, protocols, methods, materials, etc.

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