Plasma Membrane Depolarization Research Articles

Visible-light-driven photocatalytic inactivation of S. aureus in aqueous environment by hydrophilic zinc oxide (ZnO) nanoparticles based on the interfacial electron transfer in S. aureus/ZnO composites

Waterborne diseases caused by pathogenic microorganisms pose severe threats to human health. ZnO nanoparticles (NPs) hold great potentials as an effective, economical and eco-friendly method for water disinfection, but the exact antimicrobial mechanism of ZnO NPs under visible-light illumination is still not clear. Herein, we investigate the visible-light-driven photocatalytic inactivation mechanism of amino-functionalized hydrophilic ZnO (AH-ZnO) NPs against Staphylococcus aureus (S. aureus) in aqueous environment from the perspective of electron transfer theory. The results show that the antibacterial effects of AH-ZnO NPs are dependent on the AH-ZnO NPs concentration and treatment time. The bulk ORP value and released Zn2+ concentration in AH-ZnO NPs solutions increase with AH-ZnO NPs concentration. The SEM and intracellular protein leakage results indicate that AH-ZnO NPs can adhere to S. aureus surface without causing obvious cell membrane disruption. The photoluminescence (PL) intensity and fluorescence lifetime of AH-ZnO NPs are remarkedly decreased after adding S. aureus, which confirms the electron transfer from S. aureus to AH-ZnO NPs. Moreover, the ΔPL intensity is closely correlated with the inactivation efficiency, demonstrating that the interfacial electron transfer in S. aureus/AH-ZnO NPs composites contributes to the antibacterial activity, which is speculated to disrupt the normal respiratory electron transfer chain of S. aureus, thereby causing intracellular ROS generation, cell membrane depolarization and eventually apoptosis-like death.

NMDAR in cultured astrocytes: Flux‐independent pH sensor and flux‐dependent regulator of mitochondria and plasma membrane‐mitochondria bridging

Glutamate N-methyl-D-aspartate (NMDA) receptor (NMDAR) is critical for neu-rotransmission as a Ca2+ channel. Nonetheless, flux-independent signaling has also been demonstrated. Astrocytes express NMDAR distinct from its neuronal counter-part, but cultured astrocytes have no electrophysiological response to NMDA. We recently demonstrated that in cultured astrocytes, NMDA at pH6 (NMDA/pH6) act-ing through the NMDAR elicits flux-independent Ca2+ release from the Endoplasmic Reticulum (ER) and depletes mitochondrial membrane potential (mΔΨ). Here we show that Ca2+ release is due to pH6 sensing by NMDAR, whereas mΔΨ deple-tion requires both: pH6 and flux-dependent NMDAR signaling. Plasma membrane (PM) NMDAR guard a non-random distribution relative to the ER and mitochon-dria. Also, NMDA/pH6 induces ER stress, endocytosis, PM electrical capacitance reduction, mitochondria-ER, and -nuclear contacts. Strikingly, it also produces the formation of PM invaginations near mitochondria along with structures referred to here as PM-mitochondrial bridges (PM-m-br). These and earlier data strongly sug-gest PM-mitochondria communication. As proof of the concept of mass transfer, we found that NMDA/pH6 provoked mitochondria labeling by the PM dye FM-4-64FX. NMDA/pH6 caused PM depolarization, cell acidification, and Ca2+ release from most mitochondria. Finally, the MCU and microtubules were not involved in mΔΨ depletion, while actin cytoskeleton was partially involved. These findings demon-strate that NMDAR has concomitant flux-independent and flux-dependent actions in cultured astrocytes. This work has been recently accepted for publication in The FASEB J.

Antimicrobial photodynamic inactivation with curcumin against Staphylococcus saprophyticus, in vitro and on fresh dough sheet

Photodynamic inactivation (PDI), as a new non-thermal decontamination and preservation technology, exhibits the advantages of low cost, free of residue and non-microbial resistance. Curcumin (CUR) as a natural photosensitizer could effectively inactivate food spoilage bacteria at an absorbance wavelength (420–470 nm) light illumination. In this study, the in vitro influence of PDI process parameters on the inactivation of Staphylococcus saprophyticus and the antimicrobial mechanism was analyzed. Approximate 5 log CFU/ml reduction was obtained when treated with 25 μM CUR and 4.32 J/cm2 of blue LED. Obvious cell wrinkles, cell membrane disruption, intracellular contents leakage and increased membrane permeability of S. saprophyticus cell were observed after CUR-PDI treatment. Further, generation of intracellular reactive oxygen species, along with decreased intracellular enzyme activities and increased degree of cell membrane depolarization verified cell damage. The in vivo photo-antibacterial effect (1.04 log CFU/g and 0.81 log CFU/g) was validated on fresh millet dough sheet at two initial bacterial loads (5 log CFU/g and 3 log CFU/g, respectively). These findings illustrated that CUR mediated PDI could be applied to inhibit S. saprophyticus growth and possibly prevent spoilage of fresh dough products.

Ethnic-Related Sodium Voltage-Gated Channel α Subunit 5 Polymorphisms Shape the In Vitro Pharmacological Action of Amiodarone upon Nav1.5.

Nav1.5-derived Na+ current (INa) exerts a pivotal role in the depolarization phase of cardiomyocytes' action potential, and, therefore, changes in INa can contribute to fatal arrhythmias. Nav1.5 displays naturally occurring ethnicity-related polymorphisms, which might alter the functioning and pharmacology of the channel. Some studies have shown how single-nucleotide polymorphism can change the response to antiarrhythmic drugs. Investigations on the role of Nav1.5 in arrhythmogenesis associated with its functional polymorphisms are currently growing as well as the possible variability in the antiarrhythmic pharmacotherapy among ethnic groups. The influence of the ethnicity-related polymorphisms (S524Y, S1103Y, R1193Q, V1951L) on the responsiveness, selectivity, and pharmacological efficacy of the clinically used antiarrhythmic amiodarone (AMIO) is not completely known. Our objectives were to analyze biophysical and pharmacological aspects of four ethnicity-related polymorphisms before and after exposure to AMIO. Polymorphisms caused reduced AMIO potency compared with wild type (WT), which can vary by up to 4× between them. AMIO shifted the voltage dependency for current inactivation without significant effect in voltage-dependent activation to a similar extent in WT and polymorphisms. The recovery from inactivation was altered between the polymorphisms when compared with WT. Finally, the use dependency of AMIO differed between studied groups, especially at a more depolarized cell membrane. Thus, our work may guide future studies focusing on the efficiency of AMIO in treating different arrhythmias and establish more individualized guidelines for its use depending on the Nav1.5 polymorphism after validating our findings using in vivo studies. SIGNIFICANCE STATEMENT: Sodium voltage-gated channel α subunit 5 (SCN5A) gene encodes the α subunit of Nav1.5, the main cardiac voltage-gated Na+ channel. Interestingly, ethnicity-related polymorphisms are found in SCN5A. Amiodarone is used in clinical practice, and some of its effects are attributed to interaction with Nav1.5. Important, amiodarone efficacy is variable among patients. Here we show that ethnicity-related SCN5A polymorphisms lead to altered Nav1.5-amiodarone interaction, which may be the cause for the variable efficacy observed in clinical usage of amiodarone.

Transformation in band energetics of CuO nanoparticles as a function of solubility and its impact on cellular response

Nanoparticles under a reactive microenvironment have the propensity to undergo morphological and compositional changes, which can translate into band edge widening. Although cell membrane depolarization has been linked with the electronic band structure of nanomaterials in their native state, the change in band structure as a consequence of a soluble nanoparticle system is less studied. Therefore we studied the consequence of dissolution of CuO nanoparticles on the band structure and correlated it with its ability to induce intracellular oxidative stress. The temporal variation in bandgap, fermi energy level and valence band maxima were evaluated on the remnant CuO nanoparticles post dissolution. CuO nanoparticles showed a very high dissolution in simulated body fluid (51%) and cell culture media (75%). This dissolution resulted in an in situ physico-chemical transformation of CuO nanoparticles. A temporal increase in the bandgap energy as a result of media interaction was up to 107%. Temporal variation in the flat band potentials with the generation of intracellular ROS, cell viability, late and early apoptosis in addition to necrosis on RAW 264.7 cells was established due to biological redox potential overlap. The mRNA expression for TNF-α, IL-6, IL-1β and IL-10 in response to the particle treatment was also evalulated for 6 h. Through this study, we establish that the toxicological potential of CuO nanoparticles is a temporal function of band energies (its overlap with the intracellular redox potential) followed by release of ionic species in the cytotoxic regime.

A pattern-triggered immunity-related phenolic, acetosyringone, boosts rapid inhibition of a diverse set of plant pathogenic bacteria

BackgroundAcetosyringone (3,5-dimethoxy-4-hydroxyacetophenone, AS) is a syringyl-type phenolic compound rarely found in plants in free form. It has been shown earlier to inhibit the growth of Pseudomonas bacteria in the presence of hydrogen peroxide and peroxidase (AS mix).ResultsWe detected elevated levels of free AS in Nicotiana tabacum and N. benthamiana plants after inducing pattern-triggered immunity (PTI) by injecting bacterial elicitor flg22, or pathogenicity-mutant Pseudomonas syringae pv. syringae 61 hrcC- bacteria; but not after inoculations with compatible or incompatible pathogens at the time of PTI onset. In this study, we demonstrate that the antibacterial effect of the AS mix is general, as growth of several Gram-negative and -positive phytopathogenic bacteria was characteristically inhibited. The inhibition of bacterial metabolism by the AS mix was rapid, shown by the immediate drop of luminescence intensity of P. syringae pv. tomato DC3000 lx strain after addition of AS mix. The mechanism of the bacteriostatic effect was investigated using fluorescent reporter dye assays. SYTOX Green experiments supported others’ previous findings that the AS mix does not result in membrane permeabilization. Moreover, we observed that the mode of action could be depolarization of the bacterial cell membrane, as shown by assays carried out with the voltage sensitive dye DIBAC4(3).ConclusionsLevel of free acetosyringone is elevated during plant PTI responses in tobacco leaves (N. tabacum and N. benthamiana). When combined with hydrogen peroxide and peroxidase (AS mix), components of the mix act synergistically to inhibit bacterial metabolism and proliferation rapidly in a wide range of plant pathogens. This effect is related to depolarization rather than to permeabilization of the bacterial cell membrane. Similar AS mixture to the in vivo model might form locally at sites of invading bacterial attachment to the plant cells and the presence of acetosyringone might have an important role in the inhibition of bacterial proliferation during PTI.

Bactericidal Properties of Rod-, Peanut-, and Star-Shaped Gold Nanoparticles Coated with Ceragenin CSA-131 against Multidrug-Resistant Bacterial Strains.

Background: The ever-growing number of infections caused by multidrug-resistant (MDR) bacterial strains requires an increased effort to develop new antibiotics. Herein, we demonstrate that a new class of gold nanoparticles (Au NPs), defined by shape and conjugated with ceragenin CSA-131 (cationic steroid antimicrobial), display strong bactericidal activity against intractable superbugs. Methods: For the purpose of research, we developed nanosystems with rod- (AuR NPs@CSA-131), peanut-(AuP NPs@CSA-131) and star-shaped (AuS NPs@CSA-131) metal cores. Those nanosystems were evaluated against bacterial strains representing various groups of MDR (multidrug-resistant) Gram-positive (MRSA, MRSE, and MLSb) and Gram-negative (ESBL, AmpC, and CR) pathogens. Assessment of MICs (minimum inhibitory concentrations)/MBCs (minimum bactericidal concentrations) and killing assays were performed as a measure of their antibacterial activity. In addition to a comprehensive analysis of bacterial responses involving the generation of ROS (reactive oxygen species), plasma membrane permeabilization and depolarization, as well as the release of protein content, were performed to investigate the molecular mechanisms of action of the nanosystems. Finally, their hemocompatibility was assessed by a hemolysis assay. Results: All of the tested nanosystems exerted potent bactericidal activity in a manner resulting in the generation of ROS, followed by damage of the bacterial membranes and the leakage of intracellular content. Notably, the killing action occurred with all of the bacterial strains evaluated, including those known to be drug resistant, and at concentrations that did not impact the growth of host cells. Conclusions: Conjugation of CSA-131 with Au NPs by covalent bond between the COOH group from MHDA and NH3 from CSA-131 potentiates the antimicrobial activity of this ceragenin if compared to its action alone. Results validate the development of AuR NPs@CSA-131, AuP NPs@CSA-131, and AuS NPs@CSA-131 as potential novel nanoantibiotics that might effectively eradicate MDR bacteria.

Antibacterial Activity of Chrysanthemum buds Crude Extract Against Cronobacter sakazakii and Its Application as a Natural Disinfectant.

Cronobacter sakazakii is an opportunistic food-borne pathogen that endangers the health of neonates and infants. This study aims to elucidate the antibacterial activity and mechanism of Chrysanthemum buds crude extract (CBCE) against C. sakazakii and its application as a natural disinfectant. The antibacterial activity was evaluated by the determination of the diameter of inhibition zone (DIZ), minimum inhibitory concentration (MIC), and minimum bactericide concentration (MBC). The antibacterial mechanism was explored based on the changes of growth curve assay, intracellular ATP concentration, membrane potential, intracellular pH (pHin), content of soluble protein and nucleic acid, and cell morphology. Finally, the inactivation effects of CBCE against C. sakazakii in biofilm on stainless steel tube, tinplate, glass, and polystyrene were evaluated. The results showed that the DIZ, MIC, and MBC of CBCE against C. sakazakii were 14.55 ± 0.44–14.84 ± 0.38 mm, 10 mg/mL, and 20 mg/mL, respectively. In the process of CBCE acting on C. sakazakii, the logarithmic growth phase of the tested bacteria disappeared, and the concentrations of intracellular ATP, pHin, bacterial protein, and nucleic acid were reduced. Meanwhile, CBCE caused the cell membrane depolarization and leakage of cytoplasm of C. sakazakii. In addition, about 6.5 log CFU/mL of viable C. sakazakii in biofilm on stainless steel tube, tinplate, glass, and polystyrene could be inactivated after treatment with 1 MIC of CBCE for 30 min at 25°C. These findings reveal the antibacterial activity and mechanism of CBCE against C. sakazakii and provide a possibility of using a natural disinfectant to kill C. sakazakii in the production environment, packaging materials, and utensils.

Structural membrane changes induced by pulsed blue light on methicillin-resistant Staphylococcus aureus (MRSA)

BackgroundIn a recent study we showed that blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA) by perturbing, depolarizing, and disrupting its cell membrane. PurposeThe current study presents visual evidence that the observed biochemical changes also result in cell metabolic changes and structural alteration of the cell membrane. MethodsCultures of MRSA were treated with 450 nm pulsed blue light (PBL) at 3 mW/cm2 irradiance, using a sub lethal dose of 2.7 J/cm2 radiant exposure three times at 30-min intervals. Following 24 h incubation at 37 °C, irradiated colonies and control non-irradiated colonies were processed for light and transmission electron microscopy. ResultsThe images obtained revealed three major effects of PBL; (1) disruption of MRSA cell membrane, (2) alteration of membrane structure, and (3) disruption of cell replication. ConclusionThese signs of bacterial inactivation at a dose deliberately selected to be sub-lethal supports our previous finding that rapid depolarization of bacterial cell membrane and disruption of cellular function comprise another mechanism underlying photo-inactivation of bacteria. Further, it affirms the potency of PBL.

The Pancreatic β-Cell: The Perfect Redox System.

Pancreatic β-cell insulin secretion, which responds to various secretagogues and hormonal regulations, is reviewed here, emphasizing the fundamental redox signaling by NADPH oxidase 4- (NOX4-) mediated H2O2 production for glucose-stimulated insulin secretion (GSIS). There is a logical summation that integrates both metabolic plus redox homeostasis because the ATP-sensitive K+ channel (KATP) can only be closed when both ATP and H2O2 are elevated. Otherwise ATP would block KATP, while H2O2 would activate any of the redox-sensitive nonspecific calcium channels (NSCCs), such as TRPM2. Notably, a 100%-closed KATP ensemble is insufficient to reach the −50 mV threshold plasma membrane depolarization required for the activation of voltage-dependent Ca2+ channels. Open synergic NSCCs or Cl− channels have to act simultaneously to reach this threshold. The resulting intermittent cytosolic Ca2+-increases lead to the pulsatile exocytosis of insulin granule vesicles (IGVs). The incretin (e.g., GLP-1) amplification of GSIS stems from receptor signaling leading to activating the phosphorylation of TRPM channels and effects on other channels to intensify integral Ca2+-influx (fortified by endoplasmic reticulum Ca2+). ATP plus H2O2 are also required for branched-chain ketoacids (BCKAs); and partly for fatty acids (FAs) to secrete insulin, while BCKA or FA β-oxidation provide redox signaling from mitochondria, which proceeds by H2O2 diffusion or hypothetical SH relay via peroxiredoxin “redox kiss” to target proteins.

Physicochemical and transcriptomic responses of Lactobacillus brevis JLD715 to sodium selenite.

Elemental selenium, as a new type of selenium supplement, can be prepared by microorganisms reducing inorganic selenium. In this study, Lactobacillus brevis JLD715 was incubated in broth containing different concentrations of sodium selenite (Na2 SeO3 ). The results showed that the bacterial biomass of L. brevis JLD715 decreased due to the inhibition of Na2 SeO3 . The cell membrane of L. brevis JLD715 treated with Na2 SeO3 was damaged, as evidenced by the reduction of intracellular ATP concentration, depolarization of cell membrane, reduction of intracellular pH and impairment of membrane integrity. In addition, we investigated the metabolism mechanism of Na2 SeO3 by L. brevis JLD715 based on transcriptome sequencing. A total of 461 genes were significantly differentially expressed under Na2 SeO3 treatment, of which 231 genes were up-regulated and 230 genes were down-regulated. These genes were involved in pathways such as pyruvate metabolism, fatty acid biosynthesis, selenocompound metabolism and nucleotide-binding oligomerization domain-like (NOD-like) receptor signaling. Meanwhile, the genes related to sulfhydryl oxidoreductase, electron carrier proteins and transmembrane transport proteins synthesis were significantly up-regulated. To sum up, the findings of this research will contribute to providing support for the application of L. brevis JLD715 in selenium-enriched functional foods. © 2021 Society of Chemical Industry.

Resveratrol depolarizes the membrane potential in human granulosa cells and promotes mitochondrial biogenesis

To study the biological effects of resveratrol on the growth, electrophysiology, and mitochondrial function of human granulosa cells (h-GCs). Preclinical study. Electrophysiology laboratory and invitro fertilization unit. This study included h-GCs from seven infertile women undergoing assisted reproductive techniques. Human ovarian Granulosa Cell Tumor (GCT) cell line COV434 and h-GCs obtained after oocyte retrieval were cultured in the absence or presence of resveratrol. Granulosa cells were evaluated for cell viability and mitochondrial activity. Electrophysiological recordings and evaluation of potassium current (IKur) and Ca2+ concentration were also performed. Resveratrol induced mitochondrial activity in a bell-shaped, dose-effect-dependent manner. Specifically, resveratrol treatment (3 μM, 48 hours) increased ATP production and cell viability and promoted the induction of cellular differentiation. These biological changes were associated with mitochondrial biogenesis. Electrophysiological recordings showed that resveratrol reduced the functional expression of an ultra rapid activating, slow inactivating, delayed rectifier potassium current (IKur) that is associated with a plasma membrane depolarization and that promotes an increase in intracellular Ca2+. CONCLUSION(S): The effects of resveratrol on potassium current and mitochondrial biogenesis in h-GCs could explain the beneficial effects of this polyphenol on the physiology of the female reproductive system. These findings suggest there are therapeutic implications of resveratrol in a clinical setting.

Aquatic toxicity of particulate matter emitted by five electroplating processes in two marine microalgae species.

Electroplating is a widely used group of industrial processes that make a metal coating on a solid substrate. Our previous research studied the concentrations, characteristics, and chemical composition of nano- and microparticles emitted during different electroplating processes. The objective of this study was to evaluate the environmental toxicity of particulate matter obtained from five different electrochemical processes. We collected airborne particle samples formed during aluminum cleaning, aluminum etching, chemical degreasing, nonferrous metals etching, and nickel plating. The toxicity of the particles was evaluated by the standard microalgae growth rate inhibition test. Additionally, we evaluated membrane potential and cell size changes in the microalgae H. akashiwo and P. purpureum exposed to the obtained suspensions of electroplating particles. The findings of this research demonstrate that the aquatic toxicity of electroplating emissions significantly varies between different industrial processes and mostly depends on particle chemical composition and solubility rather than the number of insoluble particles. The sample from an aluminum cleaning workshop was significantly more toxic for both microalgae species compared to the other samples and demonstrated dose and time-dependent toxicity. The samples obtained during chemical degreasing and nonferrous metals etching processes induced depolarization of microalgal cell membranes, demonstrated the potential of chronic toxicity, and stimulated the growth rate of microalgae after 72 h of exposure. Moreover, the sample from a nonferrous metals etching workshop revealed hormetic dose-response toxicity in H. akashiwo, which can lead to harmful algal blooms in the environment.

Punicalagin Damages the Membrane of Salmonella Typhimurium

Salmonella, a bacterial foodborne pathogen, can contaminate meat, milk, and vegetables. While appropriate measures are available to control Salmonella, the inhibitory phytochemicals from plants are gaining increased attention. Punicalagin, a natural antimicrobial, is one of the main active tannins isolated from Punica granatum L. To obtain a broader understanding of the effect of punicalagin on the cell membranes of Salmonella Typhimurium, the growth curves, extracellular potassium concentration, release of cell constituents, intracellular pH, membrane potential, and morphological features were characterized to elucidate the mechanisms of action. Treatment with punicalagin induced an increase in the extracellular concentrations of potassium and a release of cell constituents. A higher pH gradient, an increase in the intracellular pH, and cell membrane depolarization were observed after punicalagin treatment. Electron microscopy observations showed that the cell membrane structures of Salmonella Typhimurium were damaged by punicalagin. It is concluded that punicalagin inhibits the proliferation of Salmonella Typhimurium and destroys the integrity of the cell membrane, leading to a loss of cell homeostasis. These findings indicate that punicalagin has the potential to be developed as a future alternative to control Salmonella Typhimurium contamination in foods and reduce the risk of salmonellosis.

Dominant or recessive mutations in the RYR1 gene causing central core myopathy in Brazilian patients.

Central Core Disease (CCD) is an inherited neuromuscular disorder characterized by the presence of cores in muscle biopsy. CCD is caused by mutations in the RYR1 gene. This gene encodes the ryanodine receptor 1, which is an intracellular calcium release channel from the sarcoplasmic reticulum to the cytosol in response to depolarization of the plasma membrane. Mutations in this gene are also associated with susceptibility to Malignant Hyperthermia (MHS).In this study, we evaluated 20 families with clinical and histological characteristics of CCD to identify primary mutations in patients, for diagnosis and genetic counseling of the families.We identified variants in the RYR1 gene in 19/20 families. The molecular pathogenicity was confirmed in 16 of them. Most of these variants (22/23) are missense and unique in the families. Two variants were recurrent in two different families. We identified six families with biallelic mutations, five compound heterozygotes with no consanguinity, and one homozygous, with consanguineous parents, resulting in 30% of cases with possible autosomal recessive inheritance. We identified seven novel variants, four of them classified as pathogenic. In one family, we identified two mutations in exon 102, segregating in cis, suggesting an additive effect of two mutations in the same allele.This work highlights the importance of using Next-Generation Sequencing technology for the molecular diagnosis of genetic diseases when a very large gene is involved, associated to a broad distribution of the mutations along it. These data also influence the prevention through adequate genetic counseling for the families and cautions against malignant hyperthermia susceptibility.

Biological Properties of New Chiral 2-Methyl-5,6,7,8-tetrahydroquinolin-8-amine-based Compounds.

The synthesis of a small library of 8-substituted 2-methyl-5,6,7,8-tetrahydroquinoline derivatives is presented. All the compounds were tested for their antiproliferative activity in non-cancer human dermal microvascular endothelial cells (HMEC-1) and cancer cells: human T-lymphocyte cells (CEM), human cervix carcinoma cells (HeLa), human dermal microvascular endothelial cells (HMEC-1), colorectal adenocarcinoma (HT-29), ovarian carcinoma (A2780), and biphasic mesothelioma (MSTO-211H). Compounds 3a, 5a, and 2b, showing significant IC50 values against the whole panel of the selected cells, were further synthesized and tested as pure enantiomers in order to shed light on how their stereochemistry might impact on the related biological effect. The most active compound (R)-5a was able to affect cell cycle phases and to induce mitochondrial membrane depolarization and cellular ROS production in A2780 cells.

Red and near-infrared light evokes Ca2+ influx, endoplasmic reticulum release and membrane depolarization in neurons and cancer cells

Low level light therapy uses light of specific wavelengths in red and near-infrared spectral range to treat various pathological conditions. This light is able to modulate biochemical cascade reactions in cells that can have important health implications. In this study, the effect of low intensity light at 650, 808 and 1064 nm on neurons and two types of cancer cells (neuroblastoma and HeLa) is reported, with focus on the photoinduced change of intracellular level of Ca2+ ions and corresponding signaling pathways. The obtained results show that 650 and 808 nm light promotes intracellular Ca2+ elevation regardless of cell type, but with different dynamics due to the specificities of Ca2+ regulation in neurons and cancer cells. Two origins responsible for Ca2+ elevation are determined to be: influx of exogenous Ca2+ ions into cells and Ca2+ release from endoplasmic reticulum. Our investigation of the related cellular processes shows that light-induced membrane depolarization is distinctly involved in the mechanism of Ca2+ influx. Ca2+ release from endoplasmic reticulum activated by reactive oxygen species generation is considered as a possible light-dependent signaling pathway. In contrast to the irradiation with 650 and 808 nm light, no effects are observed under 1064 nm irradiation. We believe that the obtained insights are of high significance and can be useful for the development of drug-free phototherapy.

Antibacterial mechanism of thymol against Enterobacter sakazakii

Thymol has a broad-spectrum antibacterial effect, but few studies have elucidated the antibacterial mechanism of thymol on Enterobacter sakazakii to date. This study aimed to uncover its antimicrobial mechanism against E. sakazakii. The minimum inhibitory concentration (MIC) of thymol was determined using the broth microdilution. Membrane potential, intracellular ATP concentrations, and intracellular pH (pHi) were measured, and the membrane damage was observed using a confocal laser scanning microscopy (CLSM) and a field emission gun scanning electron microscope (FEGSEM) to discuss the antibacterial effect of thymol. The MIC of thymol against E. sakazakii BNCC 186088 was 1.25 mg/mL. Cell membrane depolarization, decreased intracellular ATP concentrations, and lower pHi were observed after treatment with thymol, which indicates broken cell membranes and disrupted intracellular homeostasis. These results suggest that thymol has the potential to prevent bacterial contamination by E. sakazakii in the food industry.

Phosphatidylserine Synthase from Salicornia europaea Is Involved in Plant Salt Tolerance by Regulating Plasma Membrane Stability.

Salinity-induced lipid alterations have been reported in many plant species; however, how lipid biosynthesis and metabolism are regulated and how lipids work in plant salt tolerance are much less studied. Here, a constitutively much higher phosphatidylserine (PS) content in the plasma membrane (PM) was found in the euhalophyte Salicornia europaea than in Arabidopsis. A gene encoding PS synthase (PSS) was subsequently isolated from S. europaea, named SePSS, which was induced by salinity. Multiple alignments and phylogenetic analysis suggested that SePSS belongs to a base exchange-type PSS, which localises to the endoplasmic reticulum. Knockdown of SePSS in S. europaea suspension cells resulted in reduced PS content, decreased cell survival rate, and increased PM depolarization and K+ efflux under 400 or 800 mM NaCl. By contrast, the upregulation of SePSS leads to increased PS and phosphatidylethanolamine levels and enhanced salt tolerance in Arabidopsis, along with a lower accumulation of reactive oxygen species, less membrane injury, less PM depolarization and higher K+/Na+ in the transgenic lines than in wild-type (WT). These results suggest a positive correlation between PS levels and plant salt tolerance, and that SePSS participates in plant salt tolerance by regulating PS levels, hence PM potential and permeability, which help maintain ion homeostasis. Our work provides a potential strategy for improving plant growth under multiple stresses.

NMDAR in cultured astrocytes: Flux-independent pH sensor and flux-dependent regulator of mitochondria and plasma membrane-mitochondria bridging.

Glutamate N-methyl-D-aspartate (NMDA) receptor (NMDAR) is critical for neurotransmission as a Ca2+ channel. Nonetheless, flux-independent signaling has also been demonstrated. Astrocytes express NMDAR distinct from its neuronal counterpart, but cultured astrocytes have no electrophysiological response to NMDA. We recently demonstrated that in cultured astrocytes, NMDA at pH6 (NMDA/pH6) acting through the NMDAR elicits flux-independent Ca2+ release from the Endoplasmic Reticulum (ER) and depletes mitochondrial membrane potential (mΔΨ). Here we show that Ca2+ release is due to pH6 sensing by NMDAR, whereas mΔΨ depletion requires both: pH6 and flux-dependent NMDAR signaling. Plasma membrane (PM) NMDAR guard a non-random distribution relative to the ER and mitochondria. Also, NMDA/pH6 induces ER stress, endocytosis, PM electrical capacitance reduction, mitochondria-ER, and -nuclear contacts. Strikingly, it also produces the formation of PM invaginations near mitochondria along with structures referred to here as PM-mitochondrial bridges (PM-m-br). These and earlier data strongly suggest PM-mitochondria communication. As proof of the concept of mass transfer, we found that NMDA/pH6 provoked mitochondria labeling by the PM dye FM-4-64FX. NMDA/pH6 caused PM depolarization, cell acidification, and Ca2+ release from most mitochondria. Finally, the MCU and microtubules were not involved in mΔΨ depletion, while actin cytoskeleton was partially involved. These findings demonstrate that NMDAR has concomitant flux-independent and flux-dependent actions in cultured astrocytes.