Cell Membrane Conductance Research Articles

Biophysical phenotyping of single-cell based on impedance and application for individualized precision medicine

Single-cell biophysical characterization based on impedance measurement is an advantageous approach due to its label-free, high-efficiency, cost-effective and real-time capability. Biophysical phenotyping can yield timely and rich information on physiological and pathological state of cells for disease diagnosis, drug screening, precision medicine, etc. However, precise measurement on single-cell impedance is challenging, particularly hard to figure out the detailed biophysical parameters of single cell due to coupling and complexity of impedance model. Here, we propose an analytic determination method to decode single-cell electrophysiological parameters (including cell-substrate interface capacitance, cell membrane capacitance, cell membrane conductivity, and cytoplasm conductivity) from the impedances measured at optimized frequencies by using analytic solution rather than spectrum fitting. With this simple and fast analytic solution method, the physiological parameters of single cell in natural adhesion state can be accurately determined in real time. We validate this cell parameter determination method in monitoring the change of cell adhesion under hydraulic effects and exploring electrophysiological differences among MCF-7, HeLa, Huh7, and MDA-MB-231 cell lines. Particularly, we apply the approach to optimize tumor treating fields (TTFields) therapy, realizing individualized precision medicine. Our work provides an accurate and efficient approach for characterizing single-cell biophysical properties with real-time, in-situ, label-free, and less invasive advantages.

Normal vision and development in mice with low functional expression of Kir7.1 in heterozygosis for a blindness-producing mutation inactivating the channel.

K+ channel Kir7.1 expressed at the apical membrane of the retinal pigment epithelium (RPE) plays an essential role in retinal function. An isoleucine-to-threonine mutation at position 120 of the protein is responsible for blindness-causing vitreo-retinal dystrophy. We have studied the molecular mechanism of action of Kir7.1-I120T in vitro by heterologous expression and in vivo in CRISPR-generated knockin mice. Full-size Kir7.1-I120T reaches the plasma membrane but lacks any activity. Analysis of Kir7.1 and the I120T mutant in mixed transfection experiments, and that of tandem tetrameric constructs made by combining wild type (WT) and mutant protomers, leads us to conclude that they do not form heterotetramers in vitro. Homozygous I120T/I120T mice show cleft palate and tracheomalacia and do not survive beyond P0, whereas heterozygous WT/I120T develop normally. Membrane conductance of RPE cells isolated from WT/WT and heterozygous WT/I120T mice is dominated by Kir7.1 current. Using Rb+ as a charge carrier, we demonstrate that the Kir7.1 current of WT/I120T RPE cells corresponds to approximately 50% of that in cells from WT/WT animals, in direct proportion to WT gene dosage. This suggests a lack of compensatory effects or interference from the mutated allele product, an interpretation consistent with results obtained using WT/- hemizygous mouse. Electroretinography and behavioral tests also show normal vision in WT/I120T animals. The hypomorphic ion channel phenotype of heterozygous Kir7.1-I120T mutants is therefore compatible with normal development and retinal function. The lack of detrimental effect of this degree of functional deficit might explain the recessive nature of Kir7.1 mutations causing human eye disease.NEW & NOTEWORTHY Human retinal pigment epithelium K+ channel Kir7.1 is affected by generally recessive mutations leading to blindness. We investigate one such mutation, isoleucine-to-threonine at position 120, both in vitro and in vivo in knockin mice. The mutated channel is inactive and in heterozygosis gives a hypomorphic phenotype with normal retinal function. Mutant channels do not interfere with wild-type Kir7.1 channels which are expressed concomitantly without hindrance, providing an explanation for the recessive nature of the disease.

Uncoupling of stomatal conductance and photosynthesis at high temperatures: mechanistic insights from online stable isotope techniques.

The strong covariation of temperature and vapour pressure deficit (VPD) in nature limits our understanding of the direct effects of temperature on leaf gas exchange. Stable isotopes in CO2 and H2 O vapour provide mechanistic insight into physiological and biochemical processes during leaf gas exchange. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a leaf temperature range of 5-40°C, while maintaining a constant leaf-to-air VPD (0.8 kPa) without soil water limitation. Above the optimum temperature for photosynthesis (30°C) under the controlled environmental conditions, stomatal conductance (gs ) and net photosynthesis rate (An ) decoupled across all tested species, with gs increasing but An decreasing. During this decoupling, mesophyll conductance (cell wall, plasma membrane and chloroplast membrane conductance) consistently and significantly decreased among species; however, this reduction did not lead to reductions in CO2 concentration at the chloroplast surface and stroma. We question the conventional understanding that diffusional limitations of CO2 contribute to the reduction in photosynthesis at high temperatures. We suggest that stomata and mesophyll membranes could work strategically to facilitate transpiration cooling and CO2 supply, thus alleviating heat stress on leaf photosynthetic function, albeit at the cost of reduced water-use efficiency.

Investigation of the functional impact of CHED- and FECD4-associated SLC4A11 mutations in human corneal endothelial cells.

Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 (SLC4A11WT) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11MU). SLC4A11WT and SLC4A11MU hCEnC lines were generated to express either SLC4A11 variant 2 (V2WT and V2MU) or variant 3 (V3WT and V3MU), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and NH3-induced membrane conductance. We demonstrate SLC4A11-/- and SLC4A11MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11WT hCEnC. Additionally, SLC4A11-/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11WT hCEnC. Induction with 10mM NH4Cl led SLC4A11WT hCEnC to depolarize; conversely, SLC4A11-/- hCEnC hyperpolarized and the majority of SLC4A11MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH4Cl induction. Immunostaining of primary hCEnC and SLC4A11WT hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.

Screening of Lactiplantibacillus plantarum NML21 and Its Maintenance on Postharvest Quality of Agaricus bisporus through Anti-Browning and Mitigation of Oxidative Damage.

Browning and other undesirable effects on Agaricus bisporus (A. bisporus) during storage seriously affect its commercial value. In this study, a strain, Lactiplantibacillus plantarum NML21, that resists browning and delays the deterioration of A. bisporus was screened among 72 strains of lactic acid bacteria (LAB), and its preservative effect was analyzed. The results demonstrated that gallic acid, catechin, and protocatechuic acid promoted the growth of NML21, and the strain conversion rates of gallic acid and protocatechuic acid reached 97.16% and 95.85%, respectively. During a 15 d storage of the samples, the NML21 treatment displayed a reduction in the browning index (58.4), weight loss (2.64%), respiration rate (325.45 mg kg-1 h-1), and firmness (0.65 N). The treatment further inhibited Pseudomonas spp. growth and polyphenol oxidase activity, improved the antioxidant capacity, reduced the accumulation of reactive oxygen species, and reduced the malonaldehyde content and cell membrane conductivity. Taken together, the optimized concentrations of NML21 may extend the shelf life of A. bisporus for 3-6 d and could be a useful technique for preserving fresh produce.

Diode Character of Local Conductivity of Human Buccal Epithelial Cell Membranes

AFM methods revealed unilateral (diode) local electrical conductivity of the membrane of human buccal epithelium cells at the nanoscale and its correlation with the topology of micromechanical properties.

Influence of Joule heating during single-cell electroporation simulation under IRE and H-FIRE pulses

Conventional irreversible electroporation (IRE) and high-frequency irreversible electroporation (H-FIRE) pulses have been widely employed in many biomedical and food applications. To investigate the effect of Joule heating generated by pulsed electric fields on cell electroporation, single-cell electroporation models without (EP only) and with the consideration of Joule heating (EP+T) were subjected to the equivalent energy IRE and H-FIRE pulses. The simulation results showed that with the consideration of Joule heating, the transmembrane voltage of the anodic point (TMPA) reached the electroporation threshold (∼1 V) faster, and a significant increase in the plasma membrane conductivity (σA) and pore number (kp) was found. We further evaluated the effect of the pulse burst repetition frequency (PBRF) of H-FIRE pulses on cell electroporation based on the EP+T model, and discovered kp increased with frequency when PBRF lower than 2 kHz, whereas it decreased with frequency for PBRF higher than 2 kHz. The accumulation of the ratio of the perforation area to the cell membrane area (sum of Ap/A) during the 400th pulse was also reversed when PBRF was 2 kHz. Our results indicated that Joule heating generated by repetitive pulse delivery would facilitate electroporation, and the effect of PBRF on cell electroporation was non-monotonic.

Some aspects of the life of SARS-CoV-2 ORF3a protein in mammalian cells

The accessory protein ORF3a, from SARS-CoV-2, plays a critical role in viral infection and pathogenesis. Here, we characterized ORF3a assembly, ion channel activity, subcellular localization, and interactome. At the plasma membrane, ORF3a exists mostly as monomers and dimers, which do not alter the native cell membrane conductance, suggesting that ORF3a does not function as a viroporin at the cell surface. As a membrane protein, ORF3a is synthesized at the ER and sorted via a canonical route. ORF3a overexpression induced an approximately 25% increase in cell death. By developing an APEX2-based proximity labeling assay, we uncovered proteins proximal to ORF3a, suggesting that ORF3a recruits some host proteins to weaken the cell. In addition, it exposed a set of mitochondria related proteins that triggered mitochondrial fission. Overall, this work can be an important instrument in understanding the role of ORF3a in the virus pathogenicity and searching for potential therapeutic treatments for COVID-19.

Activation of Transient Receptor Potential Vanilloid 4 (TRPV4) channels opens Connexin Hemichannels in Lens Epithelial Cells

ATP release from the lens via connexin hemichannels has been hypothesized as a response to TRPV4 activation when the lens is subjected to osmotic swelling. To explore the apparent linkage between TRPV4 activation and hemichannel opening we performed patch-clamp recordings on cultured mouse lens epithelial cells exposed to the TRPV4 agonist GSK1016790A (GSK) in the presence or absence of the TRPV4 antagonist HC067047 (HC). GSK was found to cause a fast, variable and generally large non-selective increase of whole cell membrane conductance evident as a larger membrane current (Im) over a wide voltage range. This response was prevented by HC. The GSK-induced Im increase was proportionally larger at negative voltages and coincided with fast depolarization and the simultaneous disappearance of an outward current, likely a K + current. The initial presence of this outward current in control conditions appeared to be a reliable predictor of a cell’s response to GSK treatment. In some studies, recordings from single cells combining cell-attached and whole-cell patch clamp configurations, were obtained. This novel approach revealed events with a channel conductance 180-270 pS following GSK application through the patch pipette on the cell-attached side. The findings are consistent with TRPV4-dependent opening of connexin hemichannels. Ongoing studies are aimed at confirming the hemichannels identity. NIH: 5R01EY029171-04 and 2R01EY009532-28A1. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The surface conductance of red blood cells and platelets is modulated by the cell membrane potential.

Dielectrophoretic analysis of cell electrical properties via the Clausius-Mossotti model has been widely used to estimate values of the membrane conductance, membrane capacitance and cytoplasm conductivity of cells. However, although the latter two values produced by this method compare well to those acquired by other electrophysiological methods, the membrane conductance is often substantially larger than that acquired by methods such as patch clamp. In this paper, the electrical properties of red blood cells (RBC) are analysed at two conductivities and following membrane-altering treatments, to develop a mathematical model of membrane conductance. Results suggest that the RBC "membrane conductance" term is primarily dominated by surface conduction, comprising an element related to medium conductivity augmented by conduction in the electrical double layer, which is in turn altered by the cell membrane potential. Validation of the relationship between membrane potential and membrane conductance was performed using platelets, where a similar relationship was observed. This sheds new light on the origin and significance of the membrane conductance term and explains for the first time phenomena of alterations in the parameter counter to changes in membrane potential or cytoplasm conductivity.

Patch clamp studies on TRPV4-dependent hemichannel activation in lens epithelium.

ATP release from the lens via hemichannels has been explained as a response to TRPV4 activation when the lens is subjected to osmotic swelling. To explore the apparent linkage between TRPV4 activation and connexin hemichannel opening we performed patch-clamp recordings on cultured mouse lens epithelial cells exposed to the TRPV4 agonist GSK1016790A (GSK) in the presence or absence of the TRPV4 antagonist HC067047 (HC). GSK was found to cause a fast, variable and generally large non-selective increase of whole cell membrane conductance evident as a larger membrane current (Im) over a wide voltage range. The response was prevented by HC. The GSK-induced Im increase was proportionally larger at negative voltages and coincided with fast depolarization and the simultaneous disappearance of an outward current, likely a K+ current. The presence of this outward current in control conditions appeared to be a reliable predictor of a cell's response to GSK treatment. In some studies, recordings were obtained from single cells by combining cell-attached and whole-cell patch clamp configurations. This approach revealed events with a channel conductance 180-270pS following GSK application through the patch pipette on the cell-attached side. The findings are consistent with TRPV4-dependent opening of Cx43 hemichannels.

Simulated Analysis of Influence of Changes in H+-ATPase Activity and Membrane CO2 Conductance on Parameters of Photosynthetic Assimilation in Leaves.

Photosynthesis is an important process in plants which influences their development and productivity. Many factors can control the efficiency of photosynthesis, including CO2 conductance of leaf mesophyll, which affects the CO2 availability for Rubisco. It is known that electrical stress signals can decrease this conductance, and the response is probably caused by inactivation of H+-ATPase in the plasma membrane. In the current work, we analyzed the influence of both CO2 conductance in the plasma membrane, and chloroplast envelopes and H+-ATPase activity on photosynthetic CO2 assimilation, using a two-dimensional mathematical model of photosynthesis in leaves. The model included a description of assimilation on the basis of the Farquhar-von Caemmerer-Berry model, ion transport through the plasma membrane, diffusion of CO2 in the apoplast, and transport of CO2 through the plasma membrane and chloroplast envelope. The model showed that the photosynthetic CO2 assimilation rate was mainly dependent on the plasma membrane and chloroplast envelope conductance; direct influence of the H+-ATPase activity (through changes in pH and CO2/HCO3- concentration ratio) on this rate was weak. In contrast, both changes in CO2 conductance of the plasma membrane and chloroplast envelopes and changes in the H+-ATPase activity influenced spatial heterogeneity of the CO2 assimilation on the leaf surface in the simulated two-dimensional system. These effects were also observed under simultaneous changes in the CO2 conductance of the plasma membrane and H+-ATPase activity. Qualitatively similar influence of changes in the CO2 conductance of the plasma membrane and chloroplast envelopes, and changes in the H+-ATPase activity on photosynthesis were shown for two different densities of stomata in the simulated leaf; however, lowering the density of stomata decreased the assimilation rate and increased the heterogeneity of assimilation. The results of the model analysis clarify the potential influence of H+-ATPase inactivation on photosynthesis, and can be the basis for development of new methods for remote sensing of the influence of electrical signals.

Antibacterial characteristics and mechanisms of some herbal extracts and ϵ-polylysine against two spoilage bacterial

This study aimed to evaluate the antibacterial effects of ε-polylysine (ε-PL) and plant extracts of Thymus vulgaris, Zataria multiflora, and Salvia verticillata against the spoilage bacterium Pseudomonas aeruginosa and Pectobacterium carotovorum. Chemical compounds contained in the plants extracts were detected using HPLC and H NMR assays. To determine the antimicrobial effects, the minimum inhibitory concentration (MIC) was calculated. Scanning electron microscopy (SEM) was used to evaluate the mode of action of plant extracts and ε-polylysine on bacteria. Bacterial membrane electrical conductivity was also measured to investigate the effect of treatments on electrolyte leakage. Based on HPLC results, rosmarinic acid, caffeic acid, gallic acid, and chlorogenic acid were identified as major chemical ingredients of the extracts. Based on HPLC results, chlorogenic acid in the T. vulgaris, Z. multiflora, and S. verticillata extracts with the values of 3.209, 3.163, and 1.455 mg/L, respectively, was the dominant compound. Gallic acid, carvacrol, and p-cymene were detected in the H NMR assay as predominant secondary metabolites. The results of SEM revealed that plant extracts and ε-polylysine caused deformity of bacterial membranes. Minimum inhibitory concentration values for T. vulgaris, ε-polylysine, Z. multiflora, and S. verticillata were determined to be 0.78, 1.56, 6.25, and 25 mg/L; respectively. The electrical conductivity of bacterial cell membranes followed a dose-dependent trend. The highest electrical conductivity was observed in T. vulgaris treatment, followed by ε-polylysine and Z. multiflora.

An Antifungal Role of Hydrogen Sulfide on Botryosphaeria Dothidea and Amino Acid Metabolism Involved in Disease Resistance Induced in Postharvest Kiwifruit.

Botryosphaeria dothidea is a major pathogen responsible for postharvest kiwifruit soft rot. This study aimed to determine the influence of hydrogen sulfide (H2S) on postharvest resistance to kiwifruit soft rot and the antifungal role of H2S against B. dothidea. The results indicated that H2S (20 μl L−1) restricted the lesion area following inoculation with B. dothidea. H2S enhanced the production of shikimic acid, tyrosine, tryptophan, and phenylalanine while also increasing the total phenols, flavonoids, and lignin. H2S upregulated the expression of AcDHQS, AcSDH, AcSK, AcPAL, AcCAD, and AcCHS. Additionally, sodium hydrosulfide (NaHS)-released H2S inhibited mycelial growth. NaHS concentrations of 20 and 40 mmol L−1 significantly decreased the mycelial weight and malondialdehyde content (MDA) content while increasing cell membrane conductivity and membrane leakage. The results indicate that H2S induces resistance in kiwifruit via a microbicidal role and amino acid metabolism involved in postharvest kiwifruit disease resistance.

FEATURES OF THE PARAMETERS OF RED BLOOD CELLS AND HEMOSTASIS IN PATIENTS WITH STROKES ASSOCIATED WITH CORONAVIRUS INFECTION

Objective: The purpose is to identify the peculiarities of the parameters of red blood cells (RBC) and hemostasis in patients with strokes associated with coronavirus infection. Design and method: A total of 124 patients (48.5 + 1.9 years) with impairments of cerebral circulation due to COVID-19 (confirmed by positive PCR test) had been examined. Among them, 74 patients had ischemic stroke, 25– transient ischaemic attack, 17– intracerebral hemorrhage, 8– subarachnoid hemorrhage. The parameters of hemostasis were measured by standard methods, electrical, viscoelastic parameters of RBC - by dielectrophoresis. Results: 71 patients (the 1st group) showed signs of intravascular coagulation and thrombosis: accelerated platelet-leukocyte aggregation, increased levels of coagulation products, reduced fibrinolysis activity (p = 0.001–0.04). The levels of D-dimer, fibrinogen, ESR, platelet count were higher in this group compared to the second one (p < 0.01). A moderate increase of RBC summarized rigidity, viscosity was noted. The level of RBC hemolysis was associated with platelet count (r = 0.735,p = 0.03), D-dimer (r = 0.482, p < 0.05), fibrinogen level (r = 0.374, p = 0.04). In 2nd group (53 persons), the markers of thrombosis had moderate deviations. Sharply reduced RBC deformability with increased summarized rigidity, viscosity was dominant coupled with the background of high electrical conductivity of cell membranes compared to the indicators in the 1st group (p < 0.01). There was a decrease of membrane capacity, surface charge, cell dipole moment, polarizability than those in the 1st group (p = 0.0001–0.05). A sharp decrease of RBC deformability creates obstacles to overcoming small-diameter capillaries, leading to violations of microcirculatory blood flow. RBC deformability was associated with levels of ferritin (r = 0.451, p = 0.02), HbA1c (r = 0.480, p = 0.03), uric acid (r = -0.371, p < 0.05), LDL cholesterol (r = 0.461, p = 0.02). Incubation of blood samples in vitro for 10 min with riboflavin, nicotinamide, inosine, which ensures RBC energy metabolism, restored the reduced RBC deformability (p < 0.01), altered cell morphology (p = 0.04), decreased RBC aggregation (p < 0.001). Conclusions: The revealed features of parameters of RBC hemostasis in stroke patients with coronavirus infection are associated with two independent pathogenetic mechanisms: thrombotic and hemorheologic. The thrombotic variant is due to procoagulant state and an activity of inflammation. The hemorheologic variant is caused by decrease of RBC energy metabolism, activity of enzymes.

Eph-ephrin Signaling Affects Eye Lens Fiber Cell Intracellular Voltage and Membrane Conductance.

The avascular eye lens generates its own microcirculation that is required for maintaining lifelong lens transparency. The microcirculation relies on sodium ion flux, an extensive network of gap junction (GJ) plaques between lens fiber cells and transmembrane water channels. Disruption of connexin proteins, the building blocks of GJs, or aquaporins, which make up water and adhesion channels, lead to lens opacification or cataracts. Recent studies have revealed that disruption of Eph-ephrin signaling, in particular the receptor EphA2 and the ligand ephrin-A5, in humans and mice lead to congenital and age-related cataracts. We investigated whether changes in lens transparency in EphA2 or ephrin-A5 knockout (–/–) mice is related to changes in GJ coupling and lens fluid and ion homeostasis. Immunostaining revealed changes in connexin 50 (Cx50) subcellular localization in EphA2–/– peripheral lens fibers and alteration in aquaporin 0 (Aqp0) staining patterns in ephrin-A5–/– and EphA2–/– inner mature fiber cells. Surprisingly, there was no obvious change in GJ coupling in knockout lenses. However, there were changes in fiber cell membrane conductance and intracellular voltage in knockout lenses from 3-month-old mice. These knockout lenses displayed decreased conductance of mature fiber membranes and were hyperpolarized compared to control lenses. This is the first demonstration that the membrane conductance of lens fibers can be regulated. Together these data suggest that EphA2 may be needed for normal Cx50 localization to the cell membrane and that conductance of lens fiber cells requires normal Eph-ephrin signaling and water channel localization.

A Note of Caution: Gramicidin Affects Signaling Pathways Independently of Its Effects on Plasma Membrane Conductance.

Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization.

Stroke mechanisms associated with coronavirus disease (COVID-19)

The purpose is to identify the pathophysiological mechanisms of stroke development in patients with COVID-19.MethodsA total of 92 patients (46.72+1.72 years) with impairments of cerebral circulation due to COVID-19 (confirmed by positive PCR test results) had been examined. Among them, 67 patients had an ischemic stroke, 14–a transient ischaemic attack, 11–an intracerebral hemorrhage, 3– a subarachnoid hemorrhage.The parameters of hemostasis were measured by standard methods, electrical and viscoelastic parameters of red blood cells (RBC) by the dielectrophoresis method was carried out.ResultsMost of the surveyed persons (60 patients) showed signs of an intravascular coagulation and thrombosis: accelerated platelet-leukocyte aggregation, increased levels of coagulation products, reduced fibrinolysis activity (p=0.002–0.04). The levels of D-dimer, fibrinogen, ESR, platelet count were significantly higher in this group compared to patients in the second one (p<0.01). A moderate increase in summarized rigidity, viscosity of RBC was noted. The level of RBC hemolysis was associated with platelet count (r=0.727, p<0.05), D-dimer level (r=0.422, p=0.04), and fibrinogen level (r=0.318, p<0.05).In the second group of patients (32 persons with a predominance of men - 25 male subjects), the markers of thrombosis had moderate deviations. A sharply reduced RBC deformability with increased summarized rigidity and viscosity was dominant coupled with the background of high electrical conductivity of cell membranes compared to the indicators in the first group (p<0.01). There was a decrease in membrane capacity, surface charge, electrical dipole moment of the cells and polarizability at all frequencies of the electric field than those in the first group (p=0.0001–0.05). A sharp decrease in RBC deformability creates obstacles to overcoming small-diameter capillaries, leading to violations of microcirculatory blood flow.The RBC deformability was associated with levels of ferritin (r=0.407, p=0.024), HbA1c (r=0.419, p=0.033), uric acid (r=−0.303, p<0.05), and LDL cholesterol (r=0.426, p=0.029).Incubation of blood samples in vitro for 10 min with riboflavin, nicotinamide, inosine, which ensures RBC energy metabolism (anaerobic glycolysis and pentose phosphate pathway) restored the reduced the RBC deformability (p<0.01), and decreased the RBC aggregation (p<0.001). Similar changes were obtained when coenzymes were administered to patients in vivo.ConclusionTwo independent mechanisms of cerebral circulatory disturbances have been identified in COVID-19 stroke patients: both thrombotic and hemorheologic. The thrombotic variant is associated with a procoagulant state and with an activity of inflammation. The hemorheologic variant is caused by a decrease in the activity of the RBC energy metabolism enzymes; it is associated with the presence of metabolic disorders and is to be eliminated by an administration of coenzymes and metabolites.FUNDunding AcknowledgementType of funding sources: Other. Main funding source(s): This research was done within the framework of the topic “Epidemiological monitoring of the population state of health and studies on molecular genetic and molecular biological mechanisms of the development of common internal diseases in Siberia for improvement of the relevant diagnostic, preventive, and therapeutic methods” in State Assignment No. 0324-2018-0001, registration No. AAAA-A17-117112850280-2

Multiphysics modelling of electroporation under uni- or bipolar nanosecond pulse sequences

A nonlinear dispersive multiphysics model of single-cell electroporation was proposed in this paper. The time-domain Debye model was utilised to describe the membrane dispersion while the dynamic pore radius function was deployed to modify the plasma membrane conductivity. The dynamic spatial distributions of the ion concentration were dominated by the Nernst-Planck function. First, a single nanosecond pulsed electric field was applied to verify our model and to explore the effects of dispersion and dynamic pore radius on the redistribution of the electric field. The dispersive membrane was found to increase the transmembrane potential, expedite the electroporation process, and weaken the membrane permeability; however, adding the dynamic pore radius function had the opposite effect on transmembrane potential and membrane permeability. The responses of the cells exposed to unipolar and bipolar nanosecond pulse sequences were subsequently simulated. During the application of unipolar pulse sequences, the pore radius and perforation area showed a step-like accumulation, and significant increases in the perforation area and intracellular ion concentration were observed with higher frequency pulse sequences and wider subpulse intervals. The bipolar cancellation effect was also observed in terms of membrane permeability and pore radius.

Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia.

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.