Plasma Membrane Depolarization Research Articles

Magnetic manipulation of Fe3O4@BaTiO3 nanochains to regulate extracellular topographical and electrical cues.

Magnetic fields play an essential role in material science and biomedical engineering. Magnetic-responsive materials can be arranged orderly in matrix to realize the construction of an aligned scaffold under magnetic induction. However, a single topological cue is insufficient to activate neural tissue regeneration, demanding more cues to promote regeneration synergistically, such as electrical stimulation and a biomimetic matrix. Herein, we propose one-dimensional (1D) magnetoelectric Fe3O4@BaTiO3 nanochains with controllable lengths under the regulation of a magnetic field. These nanochains can be oriented in the biomimetic hydrogel under magnetic guidance and induce the hydrogel microfiber to align along the direction of the nanochains, which is beneficial for cell-oriented outgrowth. This aligned hydrogel enabled wireless electrical stimulation mediated by magnetoelectric nanochains under magnetic stimulation, thereby activating the voltage-gated ion channel. Consequently, topological and electrical cues in this multifunctional biomimetic hydrogel synergistically enhanced the expression of neural functional proteins, facilitating synapse remodeling and neural regeneration. Predictably, the construction of multifunctional hydrogels based on low-cost and facile synthesis of magnetoelectric nanochains is an emerging patient-friendly and effective therapeutic strategy for neural or other tissue regeneration. STATEMENT OF SIGNIFICANCE: A facile and controllable magnetic strategy is established to manipulate 1D nanomaterial growth, matrix topography, and wireless electrical stimulation of cells. First, the magnetic-assisted interface co-assembly was used to control the length of Fe3O4@BaTiO3 nanochains with enhanced magnetoelectric effect. Then, the motion of the magnetic-induced nanochains guided the orientation of nanofibers in a 3D biomimetic hydrogel matrix. Finally, wireless electrical signals and topological cues in the biomimetic matrix synergistically promoted orderly aligned cell outgrowth and membrane depolarization by Ca2+ influx, thus enhancing nerve cell synaptic plasticity and functional expression. Consequently, this work provides a conceptual strategy from material design to extracellular matrix signal manipulation and synergistic induction of tissue regeneration.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

The enhancement in toxic potency of oxidized functionalized polyethylene-microplastics in mice gut and Caco-2 cells

Microplastics (MPs) are inevitably oxidized in the environment, however, to date, no studies have discussed the biological toxicity of oxidized polyethylene (Ox-PE) MPs. In this study, oxidized low-density polyethylene (Ox-LDPE), a representative Ox-PE, was prepared using a selective oxidation method. The difference in toxicity between LDPE-MPs and Ox-LDPE-MPs were evaluated in C57BL/6 mice and Caco-2 cells. The proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared (FTIR) spectroscopy analyses revealed that some hydrocarbon-containing groups were transformed into carboxyl and ketone groups during selective oxidation. In vivo experiment results showed that LDPE-MPs and Ox-LDPE-MPs exists in the intestinal (duodenum and colon) of mice, and Ox-LDPE-MPs caused more severe intestinal histological changes, oxidative stress, and inflammatory response. The gut microbiota data showed that the relative abundance of Lactobacillus decreased significantly in the LDPE-MP- and Ox-LDPE-MP-exposed groups (P < 0.05). The predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway suggested that exposure to LDPE-MPs or Ox-LDPE-MPs inhibited glycan biosynthesis and metabolism in the flora (P < 0.05). In vitro experiment results showed that selective oxidation to LDPE promoted its uptake by cells and aggravated adverse effects on cells, including reduced cell viability, damaged cell membrane, oxidative stress, and mitochondrial depolarization. The major mechanism of the increased toxicity of Ox-LDPE-MPs may be its easier accumulation and the ionic effect of oxygen-containing functional groups. Overall, these findings provide insights on the differences in toxicity between LDPE-MPs and Ox-LDPE-MPs. They also provide new perspectives for understanding the biohazards of MPs, which are necessary to accurately assess the potential environmental and health risks of these plastic pollutants.

Regulation of localized corrosion of 316L stainless steel on osteogenic differentiation of bone morrow derived mesenchymal stem cells

Localized corrosion has become a concerning issue in orthopedic implants as it is associated with peri-implant adverse tissue reactions and implant failure. Here, the pitting corrosion of 316 L stainless steels (316 L SSs) was initiated by electrochemical polarization to simulate the in vivo localized corrosion of orthopedic implants. The effect of localized corrosion on osteogenic differentiation of bone marrow derived mesenchymal stem cells (BMSCs) was systematically studied. The results suggest that pitting corrosion of 316 L SS reduced the viability, adhesion, proliferation, and osteogenic differentiation abilities of BMSCs, especially for the cells around the corrosion pits. The relatively high concentrations of metallic ions such as Cr3+ and Ni2+ released by pitting corrosion could cause cytotoxicity to the BMSCs. The inhomogeneous electrochemical environment resulted from localized corrosion could promote reactive oxygen species (ROS) generation around the corrosion pits and cause oxidative stress of BMSCs. In addition, localized corrosion could also electrochemically interact with the cells and lead to cell membrane depolarization. The depolarized cell membranes and relatively high levels of ROS mediated the degradation of the osteogenic capacity of BMSCs. This work provides new insights into corrosion-mediated cell function degeneration as well as the material-cell interactions.

The effects of daptomycin on cell wall biosynthesis in Enterococcal faecalis

Daptomycin is a cyclic lipodepsipeptide antibiotic reserved for the treatment of serious infections by multidrug-resistant Gram-positive pathogens. Its mode of action is considered to be multifaceted, encompassing the targeting and depolarization of bacterial cell membranes, alongside the inhibition of cell wall biosynthesis. To characterize the daptomycin mode of action, 15N cross-polarization at magic-angle spinning NMR measurements were performed on intact whole cells of Staphylococcus aureus grown in the presence of a sub-inhibitory concentration of daptomycin in a chemically defined media containing l-[ϵ-15N]Lys. Daptomycin-treated cells showed a reduction in the lysyl-ε-amide intensity that was consistent with cell wall thinning. However, the reduced lysyl-ε-amine intensity at 10 ppm indicated that the daptomycin-treated cells did not accumulate in Park’s nucleotide, the cytoplasmic peptidoglycan (PG) precursor. Consequently, daptomycin did not inhibit the transglycosylation step of PG biosynthesis. To further elucidate the daptomycin mode of action, the PG composition of daptomycin-susceptible Enterococcus faecalis grown in the presence of daptomycin was analyzed using liquid chromatography-mass spectrometry. Sixty-nine muropeptide ions correspond to PG with varying degrees of modifications including crosslinking, acetylation, alanylation, and 1,6-anhydrous ring formation at MurNAc were quantified. Analysis showed that the cell walls of daptomycin-treated E. faecalis had a significant reduction in PG crosslinking which was accompanied by an increase in lytic transglycosylase activities and a decrease in PG-stem modifications by the carboxypeptidases. The changes in PG composition suggest that daptomycin inhibits cell wall biosynthesis by impeding the incorporation of nascent PG into the cell walls by transpeptidases and maturation by carboxypeptidases. As a result, the newly formed cell walls become highly susceptible to degradation by the autolysins, resulting in thinning of the cell wall.

P-096 Prediction of fertilization success by voltage-dependent intracellular Ca2+ dynamics in non-normozoospermic human semen samples undergoing in vitro fertilization: preliminary results

Abstract Study question Does the change in intracellular calcium levels evoked by artificial membrane depolarizations predict fertilization success in a non-normozoospermic semen sample undergoing assisted reproductive techniques? Summary answer The increase in the intracellular calcium after artificial plasma membrane depolarization correlates negatively with fertilization rates in conventional IVF but not with ICSI What is known already Sperm capacitation-related features, like the regulation of the intracellular calcium concentration ([Ca2+]i) and the hyperpolarization of the plasma membrane potential (Vm), have been proposed as predictors of success during artificial reproduction techniques (ART). Intriguingly, upon Vm depolarization, sperm exhibit a rapid and transitory increase in the [Ca2+]i controlled, likely, by the activation of the sperm-specific calcium channel CatSper. The regulation of CatSper activity is essential for sperm function and is crucial for fertilization. Therefore we wondered if the Vm depolarization-dependent rise in the [Ca2+]i can be used to predict fertilization success rates in non-normozoospermic semen samples of men undergoing ART. Study design, size, duration This prospective study employed unidentified semen samples from 29 non-normozoospermic men (37.8 ± 5.6 years old) undergoing fertility treatment from March 2022 to the present at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and procedures with less than 3 oocytes were excluded. Ten normozoospermic semen samples were analyzed as a control. The depolarization-dependent [Ca2+]i changes were evaluated by flow cytometry under four different Vm conditions: resting (variable among samples), –64, –46, and –32 mV. Participants/materials, setting, methods Semen samples were obtained on the same day as oocyte retrieval. Sperm cells were washed and incubated in capacitating HTF media for further use in conventional IVF or ICSI procedures. When available, the surplus cells were stained with vitality, Ca2+, and Vm-sensitive fluorescent dyes. Fluorescence changes were evaluated by AccuriC6 flow cytometer. The kinetical analysis of the transitory [Ca2+]i increase as well as the statistical comparisons were performed using custom-made R programming language code. Main results and the role of chance Analysis of resting Vm showed that normozoospermic semen samples presented more depolarized Vm than non-normozoospermic controls (–75.7 ± 6.9 vs –67.3 ± 10.5 mV, respectively, p = 0.02). The former group of semen samples was either used for conventional IVF (n = 11) or ICSI (n = 14). The semen samples in these last categories showed a tendency to present a more depolarized Vm than non-normozoospermic samples (–69.9 ± 8.8 and –66.6 ± 11.3 mV, respectively). No differences were seen in the resting Ca2+ levels. We then induced a Vm hyperpolarization by adding the K+ ionophore, valinomycin followed by increasing concentrations of extracellular K+. Each depolarizing stimulus evoked a fast and transitory increase in the [Ca2+]i. We then calculated seven different kinetic parameters of such responses: basal Ca2+ level, increase and decrease velocity, maximum Ca2+ reached, response durability, and area under the curve (AUC). The basal Ca2+ level reached after depolarizing stimuli correlated negatively (R = –0.64, p = 0.03) with the conventional IVF fertilization ratio (number of zygotes/total number of mature oocytes) when the Vm was clamped either to –64 and –46 mV. A negative and significant correlation (R = –0.76, p = 0.006) was seen for the AUC analysis at –64 mV. No relevant correlations were found in the ICSI samples. Limitations, reasons for caution This is an in vitro study, and caution must be taken when extrapolating these results in vivo. The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples (in progress). Wider implications of the findings Further research into the molecular regulation of the CatSper channel could offer promising alternatives for clinicians to appropriately choose between IVF and ICSI for each couple. This protocol is fast and simple to perform in a laboratory equipped with a flow cytometer or any fluorescence-based reader. Trial registration number NA

1772-P: Alpha-Cell Heterogeneity Contributes to Phasic Glucagon Release

Glucagon, produced by α-cells of pancreatic islets, is one of the body’s primary tools for elevating blood glucose during hypoglycemia. The exact mechanism linking hypoglycemia to glucagon release by islet α-cells is, however, unclear; we therefore aimed to investigate it using optogenetics. We generated mice harboring the light-activated ion channel channelrhodopsin-2 (ChR2) specifically in α-cells and imaged the effect of optically induced depolarization of plasma membrane (10-ms 470-nm light pulses at 20Hz) at the level of membrane potential, cytosolic Ca2+ ([Ca2+]i) and ATP/ADP ratio. At 1 mmol/L glucose, optostimulation depolarized the electrically active α-cells from -76mV to -8mV, induced a transient surge in [Ca2+]i and elevated glucagon secretion by 200%. In contrast, the glucagonostatic effect of high glucose (&amp;gt;6 mmol/L) was mitigated by optostimulation of α-cells. At 1 mmol/L glucose, the stimulatory effects were abolished by the KATP channel activator diazoxide (100μmol/L) and L-type Ca2+ channel blocker isradipine (10μmol/L). Notably, KATP channel inhibitor tolbutamide (100μmol/L) induced electrical activity in the fraction of α-cells insensitive to low glucose (35%). As stimulation of electrical activity persisted significantly beyond optoactivation, we probed the interplay between electrical activity and energy metabolism. The depolarizing stimulus induced by a cocktail of amino acids ((AA) 2 mmol/L of alanine + arginine +glutamine) or a moderate increase of extracellular K+ (from 4 to 15 mmol/L) reduced ATP/ADP in α-cells exhibiting spontaneous Ca2+ oscillations at low glucose. Surprisingly, in the population of inactive α-cells, the stimulation increased ATP/ADP ratio. These data indicate that α-cells are functionally heterogeneous due to metabolic differences. The observed secretory responses reflect the combination of stimulatory and inhibitory effects in electrically active and silent cells, which may contribute to the phasic nature of AA-induced glucagon secretion. Disclosure C.A.Miranda: None. H.Dou: None. J.Tolö: None. A.I.Tarasov: None. P.Rorsman: None. Funding Svenska Sällskapet för Medicinsk Forskning (PD20-0056); Swedish Vetenskap Rådet

Beetroot (Beta vulgaris) extract exerts an antibacterial effect by inducing apoptosis-like death in Bacillus cereus

In this study, we assessed the phytochemicals of beetroot (Beta vulgaris) extract (BE), and examined the antibacterial activity and possible mechanism of BE against Bacillus cereus. The phytochemicals in BE were analyzed by UPLC-ESI-MS-based widely targeted metabolomics analysis, and identified a total of 745 phytochemicals. Nine polyphenols and 19 flavonoids with growth-inhibiting activity against B. cereus were identified. The minimum inhibitory concentration (MIC) of BE against B. cereus was 15 mg/mL. The intracellular adenosine 5′-triphosphate (ATP) levels were decreased and the B. cereus exhibited the features of apoptosis-like death (ALD) [cell membrane depolarization, caspase-like protein activation, phosphatidylserine (PS) translocation, and DNA fragmentation] by depleting reactive oxygen species (ROS) after BE treatment. These results suggest BE is rich in bioactive substances and induces ALD in B. cereus. Thus, the phytochemicals diversity makes BE as a source of bioactive ingredients, especially as a promising potential natural preservative to reduce B. cereus contamination.

Antimicrobial and Antibiofilm Efficacy and Mechanism of Oregano Essential Oil Against Shigella flexneri.

The aim of this study was to assess the antimicrobial activity of oregano essential oil (OEO) against Shigella flexneri and eradication efficacy of OEO on biofilm. The results showed that the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of OEO against S. flexneri were 0.02% (v/v) and 0.04% (v/v), respectively. OEO effectively killed S. flexneri in Luria-Bertani (LB) broth and contaminated minced pork (the initial population of S. flexneri was about 7.0 log CFU/mL or 7.2 log CFU/g), and after treatment with OEO at 2 MIC in LB broth or at 15 MIC in minced pork, the population of S. flexneri decreased to an undetectable level after 2 or 9 h, respectively. OEO increased intracellular reactive oxygen species concentration, destroyed cell membrane, changed cell morphology, decreased intracellular ATP concentration, caused cell membrane depolarization, and destroyed proteins or inhibited proteins synthesis of S. flexneri. In addition, OEO effectively eradicated the biofilm of S. flexneri by effectively inactivating S. flexneri in mature biofilm, destroying the three-dimensional structure, and reducing exopolysaccharide biomass of S. flexneri. In conclusion, OEO exerts its antimicrobial action effectively and also has a valid scavenging effect on the biofilm of S. flexneri. These findings suggest that OEO has the potential to be used as a natural antibacterial and antibiofilm material in the control of S. flexneri in meat product supply chain, thereby preventing meat-associated infections.

(+)-CatechinAttenuatesMultipleAtherosclerosis-Associated Processes In Vitro, Modulates Disease-Associated Risk Factors in C57BL/6J Mice and Reduces Atherogenesis in LDL Receptor Deficient Mice by Inhibiting Inflammation and Increasing Markers of Plaque Stability.

A prospective study of 34492 participants shows an inverse association between (+)-catechin intake and coronary heart disease. The effects of (+)-catechin on atherosclerosis and associated risk factors are poorly understood and are investigated. (+)-Catechin attenuates reactive oxygen species production in human macrophages, endothelial cells and vascular smooth muscle cells, chemokine-driven monocytic migration, and proliferation of human macrophages and their expression of several pro-atherogenic genes. (+)-Catechin also improves oxidized LDL-mediated mitochondrial membrane depolarization in endothelial cells and attenuates growth factor-induced smooth muscle cell migration. In C57BL/6J mice fed high fat diet (HFD) for 3 weeks, (+)-catechin attenuates plasma levels of triacylglycerol and interleukin (IL)-1β and IL-2, produces anti-atherogenic changes in liver gene expression, and reduces levels of white blood cells, myeloid-derived suppressor cells, Lin- Sca+ c-Kit+ cells, and common lymphoid progenitor cells within the bone marrow. In LDL receptor deficient mice fed HFD for 12 weeks, (+)-catechin attenuates atherosclerotic plaque burden and inflammation with reduced macrophage content and increased markers of plaque stability; smooth muscle cell and collagen content. This study provides novel, detailed insights into the cardio-protective actions of (+)-catechin together with underlying molecular mechanisms and supports further assessments of its beneficial effects in human trials.

Structural Characterization of the Staphylococcus aureus Targeting Lectin Peptides from Garlic (Allium sativum L) by Liquid Nitrogen Grinding Coupled with the Proteomic and Antimicrobial Mechanism Analysis.

Garlic has long been used as an antimicrobial spice and herbal remedy. The aim of this study was to isolate the antimicrobial agent in garlic water extract against Staphylococcus aureus (S. aureus) and investigate its antimicrobial mechanism. By an activity-guided separation, garlic lectin-derived peptides (GLDPs) with main molecular weight of around 12kDa were extracted by liquid nitrogen grinding and identified with high bactericidal activity toward S. aureus, and the MIC was determined as 24.38μg/mL. In-gel digestion-based proteomic analysis indicated that the peptide sequences were highly identical to the B strain of garlic protein lectin II. Structure analysis suggested that the secondary structure was strongly affected by lyophilization and thus resulted in the inactivation of GLDPs (P < 0.05). Mechanism study revealed that treatment of GLDPs resulted in cell membrane depolarization in a dose-dependent manner, and the disruptions of the cell wall and membrane integrities were observed under electric microscopies. GLDPs could successfully dock with cell wall component lipoteichoic acid (LTA) via van der Waals and conventional bonds in molecular docking analysis. These results suggested that GLDPs were responsible for the S. aureus targeting activity and might be promising candidates for antibiotic development against bacterial infection.

Remodeled connexin 43 hemichannels alter cardiac excitability and promote arrhythmias.

Connexin-43 (Cx43) is the most abundant protein forming gap junction channels (GJCs) in cardiac ventricles. In multiple cardiac pathologies, including hypertrophy and heart failure, Cx43 is found remodeled at the lateral side of the intercalated discs of ventricular cardiomyocytes. Remodeling of Cx43 has been long linked to spontaneous ventricular arrhythmia, yet the mechanisms by which arrhythmias develop are still debated. Using a model of dystrophic cardiomyopathy, we previously showed that remodeled Cx43 function as aberrant hemichannels (non-forming GJCs) that alter cardiomyocyte excitability and, consequently, promote arrhythmias. Here, we aim to evaluate if opening of remodeled Cx43 can serve as a general mechanism to alter cardiac excitability independent of cellular dysfunction associated with a particular cardiomyopathy. To address this issue, we used a genetically modified Cx43 knock-in mouse (S3A) that promotes cardiac remodeling of Cx43 protein without apparent cardiac dysfunction. Importantly, when S3A mice were subjected to cardiac stress using the β-adrenergic agonist isoproterenol (Iso), they displayed acute and severe arrhythmias, which were not observed in WT mice. Pretreatment of S3A mice with the Cx43 hemichannel blocker, Gap19, prevented Iso-induced abnormal electrocardiographic behavior. At the cellular level, when compared with WT, Iso-treated S3A cardiomyocytes showed increased membrane permeability, greater plasma membrane depolarization, and Ca2+ overload, which likely caused prolonged action potentials, delayed after depolarizations, and triggered activity. All these cellular dysfunctions were also prevented by Cx43 hemichannel blockers. Our results support the notion that opening of remodeled Cx43 hemichannels, regardless of the type of cardiomyopathy, is sufficient to mediate cardiac-stress-induced arrhythmogenicity.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

Antibacterial Pattern of Rosa roxburghii Tratt Pomace Crude Extract Against Staphylococcus aureus and Its Application in Preservation of Cooked Beef

Staphylococcus aureus is a common foodborne pathogen and spoilage bacterium in meat products. To develop a natural preservative for meat products, this study revealed the antibacterial activity and mechanism of Rosa roxburghii Tratt pomace crude extract (RRPCE) against S. aureus, and applied RRPCE to the preservation of cooked beef. The diameter of inhibition zone, minimum inhibitory concentration (MIC), and minimum bactericide concentration of RRPCE against S. aureus were 15.85 ± 0.35 to 16.21 ± 0.29 mm, 1.5 mg/mL, and 3 mg/mL, respectively. The growth curve of S. aureus was completely stalled by treatment with RRPCE at 2 MIC. RRPCE results in the decrease of intracellular adenosine 5'-triphosphate (ATP) content, depolarization of cell membrane, leakage of cell fluid including nucleic acid and protein, and destruction of cell membrane integrity and cell morphology. During storage, RRPCE significantly reduced S. aureus viable counts, pH, and total volatile basic nitrogen of cooked beef compared with untreated samples (p < 0.05). In addition, RRPCE could significantly increase the redness (a*) value, decrease lightness (L*) and yellowness (b*) values, and slow down the color change of cooked beef (p < 0.05). These findings suggest that RRPCE can effectively inhibit S. aureus, and has the potential as a natural preservative for the preservation of cooked beef.

In vitro Study of Antioxidant, Antidiabetic and Antimicrobial Activities of &lt;i&gt;Solanum Torvum&lt;/i&gt; Root

Diabetes mellitus is a metabolic disorder with characteristics of multiple aetiology of chronic hyperglycaemia. These effects include dysfunction and long-term degeneration of various organs. Characteristics of diabetes mellitus include polyuria, blurred vision, thirst, and weight loss. Many types of insulin are available, for example porcine insulin, beef insulin, and human insulin. Weight gain and hypoglycaemia are the most adverse effects of using insulin. Insulin binds to sulfonylurea receptors on β-cell plasma membranes. In this study, ethanolic extract of Solanum torvum root was used to study ATP-sensitive potassium channels, which lead to depolarized cell membranes. The ethanolic extract was also analysed for antioxidant activities by free radical 1,1-diphenyl-2-picryl hydrazyl (DPPH) scavenging method. The different concentration of extracts exhibited varying result of radical scavenging activity using DPPH as substrate. The root of Solanum torvum exhibited significant α-amylase (79.56%) and α-glucosidase (80.58%) inhibitory activities at the concentration 100 μg/ml respectively when compared with standard acarbose drug. It also showed a good antioxidant activity with 65.47% when compared with standard ascorbic acid. The ethanolic extract of Solanum torvum root has a potential antimicrobial activity against Staphylococcus aureus and Candida tropicalis. Thus, Solanum torvum is a better source for natural antioxidant, anti-diabetic and anti-microbial activities due to presence of natural compounds in it.

PPP3R1 Promotes MSCs Senescence by Inducing Plasma Membrane Depolarization and Increasing Ca2+ Influx.

Aging of mesenchymal stem cells(MSCs) has been widely reported to be strongly associated with aging-related diseases, including osteoporosis (OP). In particular, the beneficial functions of mesenchymal stem cells decline with age, limiting their therapeutic efficacy in age-related bone loss diseases. Therefore, how to improve mesenchymal stem cell aging to treat age-related bone loss is the current research focus. However, the underlying mechanism remains unclear. In this study, protein phosphatase 3, regulatory subunit B, alpha isoform, calcineurin B, type I (PPP3R1) was found to accelerate the senescence of mesenchymal stem cells, resulting in reduced osteogenic differentiation and enhanced adipogenic differentiation in vitro. Mechanistically, PPP3R1 induces changes in membrane potential to promote cellular senescence by polarizing to depolarizing, increasing Ca2+ influx and activating downstream NFAT/ATF3/p53 signaling. In conclusion, the results identify a novel pathway of mesenchymal stem cell aging that may lead to novel therapeutic approaches for age-related bone loss.

NKCC transport mediates the insulinotropic effects of taurine and other small neutral amino acids

AimsDespite its high concentration in pancreatic islets of Langerhans and broad range of antihyperglycemic effects, the route facilitating the import of dietary taurine into pancreatic β-cell and mechanisms underlying its insulinotropic activity are unclear. We therefore studied the impact of taurine on beta-cell function, alongside that of other small neutral amino acids, L-alanine and L-proline. Main methodsPharmacological profiling of insulin secretion was conducted using clonal BRIN BD11 β-cells, the impact of taurine on the metabolic fate of glucose carbons was assessed using NMR and the findings were verified by real-time imaging of Ca2+ dynamics in the cytosol of primary mouse and human islet beta-cells. Key findingsIn our hands, taurine, alanine and proline induced secretory responses that were dependent on the plasma membrane depolarisation, import of Ca2+, homeostasis of K+ and Na+ as well as on cell glycolytic and oxidative metabolism. Taurine shifted the balance between the oxidation and anaplerosis towards the latter, in BRIN BD11 beta-cells. Furthermore, the amino acid signalling was significantly attenuated by inhibition of Na+-K+-Cl− symporter (NKCC). SignificanceThese data suggest that taurine, like L-alanine and L-proline, acutely induces glucose-dependent insulin-secretory responses by modulating electrogenic Na+ transport, with potential role of intracellular K+ and Cl− in the signal transduction. The acute action delineated would be consistent with antidiabetic potential of dietary taurine supplementation.

Relative Membrane Potential Measurements Using DISBAC2(3) Fluorescence in Arabidopsis thaliana Primary Roots.

In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC2(3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC2(3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors. Key features Non-invasive method to relatively quantify membrane potential in plant roots. Method suitable for imaging seedlings root in agar or liquid medium. Straightforward quantification.

PS-B02-14: DISCOVERY OF A NEW DRUG FOR PRIMARY ALDOSTERONISM TARGETING THE CYP11B2 AND KCNJ5 MUTANTS

Primary aldosteronism (PA) is a common cause of secondary hypertension. PA induces damage to cardiovascular and renal blood vessels. Therefore, PA is regarded as a problem because it increases the risk of stroke, cardiomyopathy, kidney disease, etc. Aldosterone-producing adenomas (APA) are one of the major causes of PA, and gene mutations are often identified in tumor cells. KCNJ5 mutation is well known as a gene mutation that causes the loss of ion selectivity in APA. KCNJ5 is an inwardly rectifying potassium channel. KCNJ5 induces sodium ion influx due to mutations in APA. The sodium ion influx induces depolarization, and which overexpresses CYP11B2. CYP11B2 is the rate-determining enzyme for aldosterone synthesis, overexpression of CYP11B2 induces aldosterone overproduction. Examples of therapeutic drugs for PA and APA include calcium channel blockers, aldosterone antagonist and β;-blockers. However, therapeutic drugs for PA and APA targeting cell membrane depolarization have not been developed yet. We aim to find compounds that suppress CYP11B2 expression by membrane depolarization to develop novel therapeutic drugs for PA and APA. By developing a therapeutic drug that targets depolarization, we hope to establish an effective therapeutic method for APA caused by gene mutations such as KCNJ5 mutation. We performed high-throughput screening (HTS) using Tohoku University Compound Library (6080 compounds). First, we generated H295R-CYP11B2 N-Luc cells in which the luciferase gene is inserted downstream of the CYP11B2 locus. The cells H295R-CYP11B2 N-Luc were stimulated with KCl to cause depolarization, after which candidate compounds were added and HTS was performed by Luc assay. As a result, 101 compounds were selected. Furthermore, 25 of 101 compounds were selected by examining the cytotoxicity and dose response. Also, 11 of the 25 compounds suppressed CYP11B2 mRNA expression in KCl stimulated H295R cells. Secondary, we generated KCNJ5 mutant cells that expressed the KCNJ5 mutant (L168R) in a Doxycycline-inducible manner. 3 of 11 compounds that suppressed the expression of CYP11B2 mRNA were selected by examination using KCNJ5 mutant cells. After that, 2 of 3 compounds decreased the aldosterone production. These compounds have a different structure from existing therapeutic drugs such as calcium channel blockers. Since these can be novel therapeutic drugs for PA and APA caused by depolarization such as KCNJ5, we are examing the detailed mechanism of action of the compounds. In addition, we would like to further experiment the effects of the compounds in vivo using hypertensive model mice, Task1 /Task3 double KO mice.

Development of neuroprotection approaches for long-term space missions

The study aimed to develop a strategy and methodology for neuroprotection during long-term space missions, which is based on a comprehensive study of the impact of therapeutic hypothermia combined with the action of neuroactive drugs on the key characteristics of synaptic transmission in brain nerve terminals, which change under the influence of planetary dust and conditions of altered gravity. Development of neurotoxicity under conditions of altered gravity may result from excess extracellular glutamate caused by the reverse functioning of glutamate transporters. Under conditions of moderate and deep hypothermia, a gradual decrease in the transporter-mediated release of L-[14C]glutamate from nerve terminals was demonstrated, which is stimulated by plasma membrane depolarization with KCl and dissipation of the proton gradient of synaptic vesicles by the protonophore FCCP. This fact indicates a neuroprotective effect, which increases when hypothermia changes from moderate to deep. The possible risks of using hypothermia in space medicine have been determined. Hypothermia is not able to reduce the extracellular level of L-[14C]glutamate and [3H]GABA, which increases under the conditions of exposure to carbon-containing planetary dust. Hypothermia can lead to a further decrease in the rate of accumulation of neurotransmitters in the presence of carbon-containing planetary dust and to contribute to the development of neurotoxicity, which is a possible risk of using hypothermia in space medicine. In this context, it is important to choose the optimal individual temperature regime for each astronaut.