Welcome to a new year full of new ideas. The first Bioelectricity Buzz of 2021 highlights exciting and diverse advances in detection of signaling events at the subcellular scale, cancer detection, tissue regeneration, tissue engineering, energy from food waste, a novel COVID therapy, and an exploration of the ionic properties of bacterial walls.
Boning Up on Macrophage Function: Enhancing Diabetic Bone Repair with Electroactive Materials
Diabetic individuals are more susceptible to bone fracture, delayed healing, and increased risk of bone graft failure due to macrophage-mediated inflammation. Dai et al. demonstrate that electroactive implant materials can drive bone repair under diabetic (hyperglycemic) conditions by altering macrophage phenotype.
Xiaohan Dai, Boon Chin Heng, Yunyang Bai, Fuping You, Xiaowen Sun, Yiping Li, Zhangui Tang, Mingming Xu, Xuehui Zhang, Xuliang Deng. Restoration of electrical microenvironment enhances bone regeneration under diabetic conditions by modulating macrophage polarization. Bioactive Materials 2021; 6:2029–2038. DOI: 10.1016/j.bioactmat.2020.12.020.
Healing often stalls at the proinflammatory stage in diabetic tissues, with high levels of inflammatory factors, including interleukin (IL)-1β and IL-6. In diabetic bone, grafts can facilitate repair, but these may fail due to increased proinflammatory mediators, inhibition of new blood vessel growth, and impaired function of osteoblasts, which synthesize new bone. Macrophages are innate immune cells that control healing and bone repair by balancing their polarized proinflammatory M1 and prohealing M2 activation states. Polarization phenotype is triggered by the microenvironment, including endogenous electrical cues at the wound site. Therefore, Dai et al. proposed that electroactive biomaterials that mimic endogenous cues could enhance osteogenesis under hyperglycemic conditions to aid diabetic bone repair by promoting M2 macrophage polarization.
To test this idea, Dai et al. prepared a ferroelectric BaTiO3/poly (vinylidene fluoridetrifluoroethylene) [BTO/P(VDF-TrFE)] nanocomposite membrane with a piezoelectric coefficient that approximates that of native bone. Human macrophages cultured on electroactive membranes displayed cytokine production profiles consistent with attenuated M1 polarization and increased M2 polarization. Human bone marrow mesenchymal stem cells (BM-MSCs) grown on the membranes in macrophage-conditioned medium showed osteogenic gene expression and mineral deposition under hyperglycemic conditions.
The authors then tested the ability of the electrically active membrane to improve bone deposition in damaged parietal bone in a diabetic rat model (high-fat diet and streptozotocin). Flow cytometry analysis confirmed that the increased M2 polarization observed in vitro also occurred in vivo. Microcomputed tomography imaging revealed newly regenerated bone filling the defect eight weeks after implantation of an electrically active, polarized membrane, but not the unpolarized membrane, and histology showed mature osteoid tissue in defects treated with the polarized membrane. Microarray analysis revealed that the polarized, electroactive membrane mediates the phenotypic M2 switch in macrophages mechanistically through the AKT-interferon regulatory factor 5 (AKT2-IRF5) signaling pathway.
Collectively, the data suggest a model under hyperglycemic conditions, by which M1 macrophages are induced to the prohealing M2 phenotype by inhibition of AKT2-IRF5 signaling and IL-6. This would create a favorable osteoimmunomodulatory microenvironment near the polarized nanocomposite membranes, triggering endogenous native BM-MSC differentiation to osteoblasts, with consequent bone regeneration.
This work paves the way to new bioimplant design using materials that mimic the electrical microenvironment in situations prone to poor healing. How the electrical environment at the polarized membrane compares to that in natural injuries in diabetic bone remains unknown. Future refinements might also target soft, biodegradable, porous, resorbable piezoelectric materials.
Do It Yourself: At Home Breast Cancer Screening from Metabolites in Urine
The International winner of the 2020 Dyson Award for promising inventors was Judit Giró Benet, a Biomedical engineer from Barcelona who was inspired to develop a new method for cancer diagnosis by learning that almost 40% of women did not attend mammogram screening appointments, decreasing the chance to identify cancer at an early stage. She designed “The Blue Box,” an inexpensive device that can be used at home to analyze urine samples for cancer biomarkers.
https://www.jamesdysonaward.org/2020/project/the-blue-box-1/
The first prototype of the low cost (∼$30 per unit device) demonstrated sensitivity of 75% for 90 test subjects and the second prototype, using an artificial intelligence algorithm, improved this to >95%. The user inserts a plastic box containing a urine sample into the unit where six chemical sensors in contact with the urine sample record the signal. The Blue Box relays the data to the cloud through a dedicated app downloaded to the user's phone. The analysis algorithm is run in the cloud and the results are relayed back to the user's phone. Impressively, The Blue Box has a lower false positive rate (5%) than other technologies (97% from mammograms). This powerful system has the potential to improve cancer screening by increasing compliance and its ease of use, low cost, and portability give it the potential for use in remote regions, provided there is a mobile phone signal.
A Bright Idea: Harnessing Solar Energy from Crop Waste
Keeping with the Dyson Award theme, the Sustainability winner for 2020 was Carvey Ehren Maigue, who developed an ingenious transparent film created from crop waste that attaches to windows and walls to sequester photons from the sun's UV light to provide clean energy.
https://www.jamesdysonaward.org/2020/project/aureus-aurora-renewable-energy-uv-sequestration/
The device is called AuREUS (Borealis Solar Window and Astralis Solar Wall) because it mimics the phenomenology of the Northern and Southern Lights; high-energy particles are absorbed by luminescent particles embedded in a thin sheet and they are reemitted as visible light. Luminescent particles derived from certain fruits and vegetables are embedded in a resin material. When UV light hits the particles, they absorb it and visible light is emitted at the edges of the material due to internal reflection. The captured light is then converted to useful direct current electricity.
Unlike typical photovoltaic cells, it does not need to be mounted facing the sun directly and even works when mounted vertically on any side of a building, even in shaded, narrow streets. Given the huge number of skyscrapers in crowded urban areas, this device has enormous potential to provide energy if the technology were to be fitted to windows and walls. In addition, it provides a use for crop waste, benefitting farmers. Everybody wins, especially the planet.
Calming the Cytokine Storm: A Role for the Vagus Nerve in Severe COVID-19?
The viral pandemic remains a global health concern. For some people, the clinical symptoms of COVID-19 are especially severe, but predicting who is most susceptible to severe illness and how best to treat those symptoms remain significant challenges. Bonaz et al. propose that vagal nerve tone could be a predictive marker of severe COVID-19 and that electrical stimulation of the vagus nerve could be used to treat it.
Bruno Bonaz, Valérie Sinniger, Sonia Pellissier. Targeting the cholinergic anti-inflammatory pathway with vagus nerve stimulation in patients with Covid-19? Bioelectronic Medicine 2020; 6:15. DOI: 10.1186/s42234-020-00051-7.
Despite the rapid development of several promising vaccines, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) SARS-CoV-2 virus responsible for COVID-19 is likely to persist in global populations. Although most people infected with SARS-CoV-2 remain asymptomatic or have relatively manageable symptoms, others develop an immune reaction called a cytokine storm, which impacts the integrity and function of many vital organ systems severely, including the lungs, brain, and gut. The basis of this potentially fatal storm is the pathogen-stimulated release of high levels of proinflammatory cytokines such as IL-1β, IL-6, tumor necrosis factor (TNF), and the release of chemokines by epithelial cells in the airways, dendritic cells, and macrophages. Excessive activation of the immune system causes inflammation of the airways, gastrointestinal symptoms, breakdown of the blood–brain barrier, and acute lung damage, which may progress to acute respiratory distress syndrome.
One response to septic shock is elevation of TNF levels and activation of the cholinergic anti-inflammatory pathway (CAP), which links stimulation of the vagus nerve and subsequent release of the neurotransmitter acetylcholine with dampening of TNF levels. Electrical stimulation of the vagus nerve is also known to reduce levels of proinflammatory molecules involved in the cytokine storm such as TNF, IL-1β, and IL-6, but not IL-10, which acts as an anti-inflammatory agent. Because the vagus nerve innervates organs that are adversely affected in COVID-19 (e.g., lungs and the gastrointestinal tract), Bonaz et al. propose that vagus nerve stimulation could dampen or prevent the cytokine storm by targeting the CAP pathway.
The optimal stimulation parameters necessary to resolve cytokine storm activity via the CAP mechanism are unknown, but Bonaz et al. propose that high frequency stimulation (20–30 Hz) such as that used for drug-resistant epilepsy would target vagal afferent nerve fibers with few predicted side effects. Alternatively, low-frequency stimulation (5–10 Hz) could target both vagal efferent and afferent fibers. In practical terms, it would be undesirable to use invasive electrode placement on the vagus nerve, but transcutaneous stimulation may be feasible.
In addition to treating COVID-19 symptoms with vagal stimulation, Bonaz et al. propose that monitoring the level of vagal resting tone can also be a predictor of vulnerability to virus infection, inflammation, and severe illness. This is because people with very low vagal tone may be at higher risk of dysregulated (excessive) proinflammatory responses. Since vagal tone can be monitored noninvasively from heart rate variability, it is possible to determine whether low tone is a predictor of the course of COVID-19 disease severity. Patients with low vagus tone who develop severe symptoms may therefore benefit from vagus nerve stimulation to calm or prevent the cytokine storm, improving outcome.
It's All Happening: Imaging Simultaneous Intracellular Signaling Networks
Imagine you enter a room of friends with everyone talking at once about where to go for dinner. Although it is chaotic, somehow a collective decision emerges as each idea is considered and integrated. That's what it's like inside a cell all the time, where multiple overlapping, sometimes chaotic signaling cascades are activated, yet, important collective decisions are made continuously about differentiation fate, mitosis, migration, and countless other actions. Technology that allowed you to listen selectively to your friend's ideas, and then assimilate them to make a decision would be useful. Linghu et al. describe a new technology that permits scientists to listen selectively to several intracellular signaling networks simultaneously, integrating the information afterward, allowing new understanding of how complex networks make collective decisions.
Changyang Linghu, Shannon L Johnson, Pablo A Valdes, Or A Shemesh, Won Min Park, Demian Park, Kiryl D Piatkevich, Asmamaw T Wassie, Yixi Liu, Bobae An, Stephanie A Barnes, Orhan T Celiker, Chun-Chen Yao, Chih-Chieh Jay Yu, Ru Wang, Katarzyna P Adamala, Mark F Bear, Amy E Keating, Edward S Boyden. Spatial multiplexing of fluorescent reporters for imaging signaling network dynamics. Cell 2020; 183:1682–1698. DOI: 10.1016/j.cell.2020.10.035.
Tagging proteins and other signaling molecules to decode complex intracellular signaling pathways can involve modifying cells genetically, sometimes using distinct fluorophores to tag specific signaling molecules in one cell. However, technology limits the number of possible simultaneous fluorophore combinations and the tags may interfere with normal function of the molecules being monitored, or even the health of the cell.
Linghhu et al. have designed a modular reagent that reveals relationships between the signals that convert cell stimulus input into cell behavioral output. Using fluorescent reporters coupled to pairs of self-assembling peptides they showed that distinct fluorescent clusters formed intracellularly, which they called signaling reporter islands (SiRIs). Existing fluorescent indicators can be fused to pairs of self-assembling peptides to form clusters stochastically at distinct points inside the cell with an intensity 100–1000-fold higher than background. Each “island” represents a signal from one signaling species, with the punctae remaining stable and stationary in live cells for at least an hour, permitting subsequent fixation for post hoc immunolabeling to identity each cluster.
To test the system SiRIs for Ca2+, cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA) were used in cultured hippocampal neurons and slices of mouse hippocampus to simultaneously to map the relationship between the signaling molecules before and after stimulation with forskolin. Neurons that had shorter-latency cAMP and Ca2+ responses to forskolin exhibited stronger PKA activation than neurons with longer latency cAMP and Ca2+ responses. These data from the same cells suggest that an output signal like PKA is dictated by the properties of the upstream signals, an interpretation that would not be possible using standard fluorescent signaling imaging from data averaged from multiple separate cells.
Although the system is readily adaptable by modifying the peptide subunits to target specific signaling molecules, the punctae form about a micron apart, which limit spatial resolution but permit imaging of many different signals simultaneously within a living cell, even if they all have the same fluorescence spectrum. Future work will likely address the spatial resolution to extend its utility. The ability to use such a signal to resolve responses to electrical stimuli will surely benefit the field of bioelectricity.
Thinking Small with Voltair: Not a Tiny French Philosopher, but a Nanoscale Voltmeter for Organelle Membranes
The electrical potentials of cell and organelle membranes drive diverse cell functions. Saminathan et al. describe a new molecular tool that is administered noninvasively to monitor organelle membrane potential in vitro with impressive selectivity and spatial resolution.
Anand Saminathan, John Devany, Aneesh Tazhe Veetil, Bhavyashree Suresh, Kavya Smitha Pillai, Michael Schwake, Yamuna Krishnan. A DNA-based voltmeter for organelles. Nature Nanotechnology 2020; 16:96–103. DOI: 10.1038/s41565-020-00784-1.
The key role of the cell membrane potential in driving processes as diverse as nerve action potentials, the electromechanics of the cochlea, cell proliferation, cancer progression, cell volume control, and secretion is well established. This understanding emerged mostly from standard electrophysiological methods that report the relative voltage difference between the tip of a sensing electrode inserted inside the cell and a reference electrode on the outside of the intact cell membrane. Conversely, the role of the organelle membrane potential in regulating organelle biology is relatively unexplored. This is due to the combined challenges of measuring the potentials of very small dynamic vesicles in living cells and because existing fluorescent tools have limitations due to their sensitivity to pH, which is codependent with lumenal pH. In addition, apart from mitochondria, it has been impossible to target voltage-sensitive dyes to specific organelles.
Saminathan et al. have addressed these challenges by designing Voltair, a ratiometric, fluorescent DNA-based construct that reports the absolute membrane potential in organelle membranes. Structurally, Voltair is a 38-base pair DNA duplex composed of three elements: (1) a 38-mer single-stranded DNA conjugated at its 3′ end to a fluorescent voltage-sensing dye. (2) A reference probe that corrects for inhomogeneous probe distribution and consequent signal intensity. (3) A targeting moiety that is distinct for each target organelle and can be modified to suit the application.
When Voltair is added to the culture medium, the fluorescent reporter probe becomes anchored to the cell surface in a defined orientation, which is retained due to the charge properties of the molecular complex. It then labels intracellular organelles by exploiting the ability of the specific duplex DNA to engage with endocytic scavenger receptors to target specific organelles. Once in place in the organelle, Voltair compares the fluorescence of the reporter probe in the lumenal side of the membrane to that of a reference probe located outside the membrane at a different wavelength. The reference fluorophore serves dual roles for ratiometry, and as an endocytic tracer, permitting dynamic organelles to be monitored within living cells as they move, mature, or are recycled.
When Saminathan et al. used Voltair to measure the membrane potential of endocytic organelles in HEK 293T cells, they reported in situ membrane potential values for the first time; +153 mV for early endosomes and +46 mV for late endosomes. When modified to target recycling endosomes and the trans-Golgi network, they reported organelle membrane potential values of +65 and +121 mV, respectively (both lumen positive), the first reported measurements for these organelles.
Voltair is likely to find its place in bioelectricity research, especially in the context of understanding how electrical stimulation at the cell and tissue levels impact organelle function and the implications of those findings for new clinical electrotherapies. There is scope for further development, however. For example, it would be desirable to use such a system to monitor neuron synaptic vesicles, but their small size provides further challenges with respect to imaging. In addition, the fluorescent reporter probe used by Saminathan et al. is not amenable to two photon imaging, so its utility is currently restricted to thin transparent tissues or surface cell imaging, but these limitations can be overcome with further work.
An Attractive Idea for Tissue Repair: Magneto-Patterned 3D Hydrogels for Complex Tissue Engineering
The goal of repairing tissue damage with bioengineered materials has been hindered by the collective challenges of mimicking the complex natural properties of cellular architecture, tissue mechanics, and biochemistry in three dimensions (3D). Zlotnick et al. describe an experimental system that cleverly leverages a nontraditional magnetic approach to bioengineer an artificial 3D cartilage construct.
Hannah M. Zlotnick, Andy T. Clark, Sarah E. Gullbrand, James L. Carey, Xuemei M. Cheng, Robert L. Mauck. Magneto-driven gradients of diamagnetic objects for engineering complex tissues. Advanced Materials 2020; 32:2005030. DOI: 10.1002/adma.202005030.
Magnetic forces can be used to coax cells, molecules, or small particle cargos into defined positions within 3D bioengineered constructs. Typically, this requires changing the magnetic properties of the target particles or cells by adding magnetic tags, which can interfere with their biological properties. Zlotnick et al. used a different approach by enhancing the magnetic properties of the hydrogel precursor solution surrounding the cells or particles, leaving the magnetic properties of the suspended cells or particles unaltered. Although such an approach has been used before to manipulate cells, the authors state that this is the first report of magneto-patterning of unaltered cells in a 3D stabilized (photo-crosslinked) hydrogel that subsequently formed heterogeneous tissues after several weeks in culture.
The diamagnetic object of choice was suspended in a viscous mixture comprising hydrogel precursor solution (methacrylated hyaluronic acid), a contrast agent (Gadodiamide, Gd) and a photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate). The resulting mixture was injected into a mould positioned 3.8 mm above a permanent magnet (13,200 Gauss; 1.32 T) for 10 min and then the solution was crosslinked with ultraviolet light for 9 min, locking the suspended cells or particles in position. The Gd was subsequently eluted out of the hydrogel by repeated washing to improve cell viability.
After magnetic field exposure (10 min), gradients were established (lowest particle density nearest the magnet) for suspensions of polystyrene beads (5 μm), drug delivery microcapsules (20 μm), and living bovine MSCs, but the distribution of smaller particles (≤1 μm) was not affected by the magnetic field. The ability to manipulate the position of such cargos in the 3D gel and to fix them in position suggests that this system could be used to manufacture implant materials where graded distribution of particulate drug delivery systems or bioactive factors is desired. Indeed, complex patterns of cells and particles could be generated by altering the size and buoyancy of the diamagnetic objects, the magnetic field geometry, and exposure time.
Zlotnick et al. ultimately aimed to prove that the technology could be used in a tissue engineering capacity. They used articular cartilage as a model because in situ it has a natural cellular gradient from the superficial (high cellularity) to the deep zone (low cellularity) of the tissue. The 3D constructs of MSCs were exposed to magnetic fields for 2 or 5 min, fixed in place, washed to remove Gd and grown in chondrogenic medium for 6 weeks. The cells remained viable as a gradient of cells, and after 3 weeks, those exposed to fields for 5 min (but not 2 min) expressed molecular extracellular matrix components in a gradient consistent with cartilage. This relatively straightforward technique has potential applications beyond cartilage. The ability to form complex and stable 3D biomolecular and cellular gradients is an exciting prospect for tissue engineering.
Thinking Positively: Modeling the Ionic and Electrical Properties of Gram-Positive and Gram-Negative Bacterial Walls
Although viruses have been grabbing the headlines lately, understanding the properties of the bacterial cell wall is key to developing new antibiotic and antibacterial reagents and to understanding biofilm formation. Cremin et al. have mapped the properties of gram-negative and gram-positive bacteria, revealing new details of their heterogeneous electrical properties and ionic microenvironments.
Kelsey Cremin, Bryn A. Jones, James Teahan, Gabriel N. Meloni, David Perry, Christian Zerfass, Munehiro Asally, Orkun S. Soyer, Patrick R. Unwin. Scanning ion conductance microscopy reveals differences in the ionic environments of gram-positive and -negative bacteria. Analytical Chemistry 2020; 92:16024–16032. DOI: 10.1021/acs.analchem.0c03653.
Gram staining is typically used to categorize bacteria because the thick peptidoglycan layer of the cell wall retains crystal violet stain in gram-positive bacteria, but the thinner layer in gram-negative bacteria permits it to be subsequently washed away with ethanol. The properties of the two bacterial classes can be very different and Cremin et al. have used scanning ion conductance microscopy (SICM) to map spatially the nanoscale ionic properties and charge environments of living gram-negative Escherichia coli and gram-positive Bacillus subtilis.
SICM can be used to map simultaneously the nanotopgraphy and surface charge of individual live bacteria bathed in electrolyte using a nanopipette filled with electrolyte solution and two quasi-reference counter electrodes. SICM measurements identified significant differences between gram-positive and gram-negative bacteria. Although both exhibited negative surface properties, B. subtilis was more negative compared to E. coli. When fitted to a simple finite element model (FEM), the data indicated that the surface charge on B. subtilis was between −350 and −450 mC m−2, but it was between −80 and −140 mC m−2 for E. coli, which also had a lower cell wall conductivity than B. subtilis. A refined FEM was designed to account for more realistic physical properties of the cell wall, including the peptidoglycan layer variations observed in B. subtilis. The refined FEM analysis suggested that it could be used in future studies to further understand complex ion transport activities at the gram-positive cell wall. This understanding could be exploited to develop better methods to control biofilm development and to inform antibiotic design.
I hope you enjoyed this Bioelectricity Buzz collection and look forward to what the rest of 2021 will bring.
Ann M. Rajnicek, BS, PhD, FRSB
Institute of Medical Sciences
School of Medicine, Medical Sciences and Nutrition
University of Aberdeen
AB25 2ZD Aberdeen
Scotland, UK
Media Editor
Bioelectricity
E-mail: a.m.rajnicek@abdn.ac.uk