Changes In Membrane Potential Research Articles

KCa 2.2 (KCNN2): A physiologically and therapeutically important potassium channel.

One group of the K+ ion channels, the small-conductance Ca2+ -activated potassium channels (KCa 2.x, also known as SK channels family), is widely expressed in neurons as well as the heart, endothelial cells, etc. They are named small-conductance Ca2+ -activated potassium channels (SK channels) due to their comparatively low single-channel conductance of about ~10 pS. These channels are insensitive to changes in membrane potential and are activated solely by rises in the intracellular Ca2+ . According to the phylogenic research done on the KCa 2.x channels family, there are three channels' subtypes: KCa 2.1, KCa 2.2, and KCa 2.3, which are encoded by KCNN1, KCNN2, and KCNN3 genes, respectively. The KCa 2.x channels regulate neuronal excitability and responsiveness to synaptic input patterns. KCa 2.x channels inhibit excitatory postsynaptic potentials (EPSPs) in neuronal dendrites and contribute to the medium afterhyperpolarization (mAHP) that follows the action potential bursts. Multiple brain regions, including the hippocampus, express the KCa 2.2 channel encoded by the KCNN2 gene on chromosome 5. Of particular interest, rat cerebellar Purkinje cells express KCa 2.2 channels, which are crucial for various cellular processes during development and maturation. Patients with a loss-of-function of KCNN2 mutations typically exhibit extrapyramidal symptoms, cerebellar ataxia, motor and language developmental delays, and intellectual disabilities. Studies have revealed that autosomal dominant neurodevelopmental movement disorders resembling rodent symptoms are caused by heterozygous loss-of-function mutations, which are most likely to induce KCNN2 haploinsufficiency. The KCa 2.2 channel is a promising drug target for spinocerebellar ataxias (SCAs). SCAs exhibit the dysregulation of firing in cerebellar Purkinje cells which is one of the first signs of pathology. Thus, selective KCa 2.2 modulators are promising potential therapeutics for SCAs.

VDAC1 Protein Regulation of Oxidative Damage and Mitochondrial Dysfunction-Mediated Cytotoxicity by Silica Nanoparticles in SH-SY5Y Cells.

Silica nanoparticles (SiNPs) have been widely used in industry, electronics, and pharmaceutical industries. In addition, it is also widely used in medicine, tumor treatment and diagnosis, as well as other biomedical and biotechnology fields. The opportunities for people to contact SiNPs through iatrogenic, occupational, and environmental exposures are gradually increasing. The damage and biological effects of SiNPs on the nervous system have attracted widespread attention in the field of toxicology. Central nerve cells are rich in mitochondria. It is suggested that the effects of SiNPs on mitochondrial damage of nerve cells may involve the maintenance of neuronal membrane potential, the synthesis and operation of neurotransmitters, and the transmission of nerve pulses, and so on. We established an experimental model of SH-SY5Y cells to detect the cell survival rate, apoptosis, changes of reactive oxygen species and mitochondrial membrane potential, and the expression of mitochondrial function-related enzymes and proteins, so as to reveal the possible mechanism of SiNPs on neuronal mitochondrial damage. It was found that SiNPs could cause oxidative damage to cells and mitochondria, destroy some normal functions of mitochondria, and induce apoptosis in SH-SY5Y cells. The voltage-dependent anion channel 1(VDAC1) protein inhibitor DIDS could effectively reduce intracellular oxidative stress, such as the reduction of ROS content, and could also usefully restore some functional proteins of mitochondria to normal levels. The inhibition of VDAC1 protein may play an important role in the oxidative damage and dysfunction of neuronal mitochondria induced by SiNPs.

The role of protein kinase R in placental inflammation, mtUPR and apoptosis

IntroductionPlacental inflammation is implicated in the pathophysiology of many pregnancy complications, including fetal growth restriction, preeclampsia, gestational diabetes, and choriocarcinoma. Mitochondrial dysfunction, one of the outcomes of placental inflammation, is characterized by loss of membrane potential, accumulation of oxygen radicals, mitochondrial protein folding defects, and disturbances in mitochondrial dynamics. Protein kinase R (PKR) is stimulated by double-stranded RNA and bacterial endotoxins in the presence of pathogens and is a critical immune response enzyme. PKR is also correlated with the cell death response during endoplasmic reticulum stress. In this study, we aim to investigate the effects of PKR activity stimulated by lipopolysaccharide (LPS), and double-stranded RNA analog (Poly I:C) on mitochondrial unfolded protein response (mtUPR), mitochondrial membrane potential, apoptosis, and oxidative stress in placental trophoblasts. MethodsWe applied LPS and Poly I:C to BeWo cells to induce PKR activation. In addition, cells were treated with 2-aminopurine (2-AP) to inhibit the kinase activity of PKR. Protein levels of ATP-dependent Clp protease proteolytic subunit (CLPP) and heat shock protein 60 (HSP60) were determined after treatments. Apoptotic markers were detected by real-time PCR and flow cytometry. PKR-induced reactive oxygen radicals (ROS) accumulation and mitochondrial membrane potential change were assessed by flow cytometry. ResultsIt was determined that PKR activation-induced apoptosis in BeWo cells by reducing the levels of mtUPR proteins (CLPP and HSP60) and caused a decrease in mitochondrial membrane potential. PKR inhibition was sufficient for decreases in apoptotic markers and caused a reduction in the ratio of depolarized and ROS (+) cells. DiscussionOur results showed that LPS and Poly I:C administration stimulated PKR in BeWo cells in vitro. Furthermore, PKR activation is correlated with the levels of proteins involved in mitochondrial homeostasis and apoptosis. Our findings will contribute to understanding the role of PKR activation in placental inflammation and related diseases.

Triterpene acids from Rosa roxburghii Tratt fruits exert anti-hepatocellular carcinoma activity via ROS/JNK signaling pathway-mediated cell cycle arrest and mitochondrial apoptosis

BackgroundRosa roxburghii Tratt (RRT) is a famous healthy and medicinal edible fruit in southwest China and has been shown to have some hepatoprotective properties. However, whether the active components, such as the triterpene acids from Rosa roxburghii Tratt fruits (TAR), have anti-hepatocellular carcinoma (HCC) effects and the potential molecular mechanisms are still unclear. PurposeThis study aimed to investigate the anti-HCC effects and potential action mechanisms of triterpene components in RRT fruits. MethodsThe triterpene acids in TAR were analyzed by using UPLC-Q-Exactive Orbitrap/MS, and the main components were virtual screening for targets based on pharmacophore and then performed enrichment analysis. HepG2 cells were used for in vitro experiments, including MTT assay, wound healing assay, and flow cytometry to detect cell cycle, reactive oxygen species (ROS) level, caspase-3 activity, and mitochondrial membrane potential (MMP) changes. Moreover, the western blot was used to detect mitochondrial apoptosis and ROS/ c-Jun N-terminal kinase (JNK) signaling pathway-related proteins. ResultsThe main components in TAR are pentacyclic triterpene acids (mainly euscaphic acid and roxburic acid). TAR could inhibit cell viability, cell migration ability and suppress the proliferation of HepG2 cells through G2/M cell cycle arrest. On the other hand, TAR could induce HepG2 cells apoptosis, which was achieved by causing the accumulation of ROS and activation of the JNK signaling pathway, and our research showed that this apoptosis was mediated through the mitochondrial pathway. In addition, the free radical scavenger N-acetyl cysteine (NAC) could attenuate TAR-induced ROS accumulation and JNK signaling pathway activation, which ultimately reversed mitochondrial apoptosis. ConclusionTAR could activate the ROS/JNK signaling pathway, which could inhibit the proliferation through G2/M cell cycle arrest and promote apoptosis through the mitochondrial pathway in HCC cells. This supports the anti-tumor potential in RRT fruits.

Vanillic acid exhibits potent antiproliferative and free radical scavenging effects under in vitro conditions

Cells endure oxidative stress as a result of an imbalance between the status of body’s reactive oxygen species (ROS) and antioxidants. Higher production of ROS or weak antioxidant defense mechanism in the cell could lead to various pathological disorders, including carcinogenesis. The present study investigated the in vitro free radical scavenging activity and antiproliferative efficacy of vanillic acid using a spectrum of radical scavenging assays and cytotoxic assays, respectively. Vanillic acid’s antioxidant ability was investigated using in vitro antioxidant assays such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydroxyl, and superoxide radical scavenging assays. The current study observed an excellent free radical scavenging capacity of vanillic acid, which was comparable to that of ascorbic acid (reference drug). The antiproliferative effect of vanillic acid was assessed in mammary cancer cells (Michigan Cancer Foundation-7 [MCF-7]) by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, ROS generation potential, changes in mitochondrial membrane potential, and by evaluating its apoptotic induction potential. The cell viability of breast cancer cells was drastically decreased by vanillic acid, and its antiproliferative potential in MCF-7 cells could be due to its ability to induce excessive generation of ROS and its apoptotic induction potential. The present investigation thus explores antioxidant and antiproliferative properties of vanillic acid under in vitro conditions.

Anti-glioma mechanism of pterostilbene by regulating apoptosis and GSDME-mediated pyroptosis pathways: a study based on network pharmacology and experimental research

This study aimed to explore the anti-glioma effect of natural compound pterostilbene(PTE) through regulating pyroptosis and apoptosis pathways, and to analyze the possible anti-glioma pathways and targets of PTE by network pharmacology and molecular docking. In this study, the action targets of PTE and the glioma targets were obtained by network pharmacology to construct a target network and a protein-protein interaction(PPI) network to predict the possible action targets of PTE against glioma. Molecular docking was performed on the core targets by AutoDock and the action pathways of PTE against glioma were predicted by enrichment analysis. In addition, the effect of PTE on the viability of U87MG and GL261 glioma cells was detected by CCK-8 assay. Clone formation assay and cell scratching assay were used to explore the effect of different concentrations of PTE on the proliferation and migration, respectively of glioma cells. Hoechst staining was used to observe PTE-induced apoptosis in glioma cells. The changes in mitochondrial membrane potential were detected by JC-1 staining. The pyroptosis-inducing effect of PTE on glioma cells was observed by inverted microscopy and lactate dehydrogenase(LDH) assay. Hoechst 33342/PI dual staining assay was performed to detect the integrity of glioma cell membranes. The expressions of pyroptosis and apoptosis-related proteins in glioma cells after PTE induction were determined by Western blot. In this study, 37 anti-glioma targets of PTE were obtained, and enrichment analysis suggested that PTE exerted anti-glioma effects through various signaling pathways including cancer pathway, proteoglycan in cancer, PI3K/AKT pathway, and apoptosis regulatory pathway. Molecular docking revealed that PTE had good binding activity with the main targets. Compared with the control group, PTE significantly reduced the viability as well as the proliferation, migration and adhesion abilities of U87MG and GL261 cells; it induced the apoptosis of the two glioma cells and the decrease of mitochondrial membrane potential in U87MG cells, and the effects increased with the increase of drug concentration. Compared with the conditions in the control group, glioma cells in the PTE group had increased pyroptosis-specific appearance and gradually increased LDH release; the number of PI positive cells was significantly elevated with the increase of PTE concentration as revealed by Hoechst 33342/PI staining; the expression levels of apoptosis-related factors cleaved PARP1 and B-cell lymphoma-2(Bcl-2) associated X(BAX) in the PTE group were markedly up-regulated, while the expression level of Bcl-2 was markedly down-regulated; the activation levels of pyroptosis-related proteins cleaved caspase-3 and gasdermin E-N(GSDME-N) had a remarkable rise in the PTE group, while no significant changes were found in the activation levels of gasdermin D-N(GSDMD-N) and cleaved caspase-1. In summary, PTE plays an anti-glioma role by inhibiting cell viability, proliferation, and migration and activating the caspase-3/GSDME-mediated pyroptosis pathway and mitochondrial apoptosis pathway.

Cytotoxic effects of endophyte origin nanosilver and its computational studies

The present study aimed in the investigation of anticancer potential of silver nanoparticles (AgNPs) from endophytic bacterium Cupriavidus metallidurans (Cm-AgNPs) and Pantoea anthophila (Pa-AgNPs) of W. indica reported in our earlier studies, against human oral epidermoid carcinoma (KB 3–1) and COLON 26 cell lines. The cytotoxicity response was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). Measurement of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ɅΨm) and apoptotic morphological changes were studied in KB 3–1 cell line. Both the endophytes were found to produce periplasmic nitrate reductase (PNR) that converts the ionic silver into stable nano silver. Hence the structure and function of PNR was determined by computationl tools and evolutionary relationships were constructed using Clustal Omega. The inhibitive properties of AgNP against selected cancer targets were analyzed by molecular docking tool Autodock 4.0. KB 3–1 cell line showed good anticancer response than COLON 26 cell lines. On comparison of AgNPs, Cm-AgNPs exhibited higher cytotoxicity with increased intracellular ROS levels, altered ɅΨm and apoptotic cell death than Pa-AgNPs. The three-dimensional structure of the PNR was modeled by Swiss model and validated using PROCHECK and PROVES. Docking study showed that the AgNP bound exactly in the inhibition site or in their close proximity that may enable the modulation of proteins. Thus the study provides an insight on the anticancer activity of AgNPs fabricated from endophytic extract.

Effects of photodynamic therapy using Red LED-light combined with hypocrellin B on apoptotic signaling in cutaneous squamous cell carcinoma A431 cells

BackgroundThe incidence of cutaneous squamous cell carcinoma (cSCC) has been demonstrating yearly increases. cSCC is a malignant cancer and exerts a major impact on patients' health and quality of life. Thus, the development and use of novel therapies in the treatment of cSCC are needed. It has been reported that LED photodynamic therapy (LED PDT) mediated by Hypocrellin B and its derivatives, a second-generation photosensitizer, can induce apoptosis in a variety of tumor cells, However, its potential pro-apoptotic effects on cSCC have yet to be investigated. ObjectiveThis study aims to investigate the pro-apoptotic effects and molecular mechanisms of HB-LED PDT in cutaneous squamous cell carcinoma A431 cells (Subsequent abbreviation A431 cells). Such information can provide an important theoretical foundation for the clinical translation of HB-LED PDT in the treatment of cSCC. Methods1. Effects of HB on A431 cells were determined using a Cell Counting Kit-8 assay, which method can indirectly reflect the number of living cells. In this way, this assay can then provide a means to identify the optimal concentrations of HB required for the induction of apoptosis in A431 cells. 2. The effects of HB-LED PDT on the morphology of A431 cells and changes in the nuclei after Hoechst33342 staining as determined using inverted fluorescent microscopy. 3. Use of the Annexin V-FITC test kit to detect levels of apoptosis in A431 cells in response to treatment with HB. Changes in reactive oxygen species and mitochondrial membrane potential following HB-LED PDT treatment in A431 cells were determined using fluorescence activated cell sorting (FACS). 4. Real-time quantitative PCR and Western Blot were applied to assess changes in several key factors involved in apoptosis including Bax, Bcl-2, and Caspase-3, at both transcription and translation levels. With these assays, it was possible to investigate the apoptotic signaling pathway in A431 cells in response to HB-LED PDT. ResultsHB-LED PDT inhibited proliferation activity and promoted nuclear fragmentation within these A431 cells. HB-LED PDT inhibited mitochondrial activity, increased reactive oxygen species production, and promoted apoptosis of A431 cells. In addition, several key factors in the apoptotic signaling pathway were increased at both the transcriptional and translational levels in A431 cells in response to the HB-LED PDT, indicating that the apoptotic signaling pathway was activated by HB-LED PDT. ConclusionHB-LED PDT induces apoptosis in A431 cells through a mitochondria-mediated apoptotic pathway. Such findings serve as an important foundation for the development of new approaches in the treatment of cSCC.

The appearance of cytoplasmic cytochrome C precedes apoptosis during Drosophila salivary gland degradation.

Apoptosis is an important process for organism development that functions to eliminate cell damage, maintain homeostasis, and remove obsolete tissues during morphogenesis. In mammals, apoptosis is accompanied by the release of cytochrome C (Cyt-c) from mitochondria to the cytoplasm. However, whether this process is conserved in the fruit fly, Drosophila melanogaster, remains controversial. In this study, we discovered that during the degradation of Drosophila salivary gland, the transcription of mitochondria apoptosis factors (MAPFs), Cyt-c, and death-associated APAF1-related killer (Dark) encoding genes are all upregulated antecedent to initiator and effector caspases encoding genes. The proteins Cyt-c and the active caspase 3 appear gradually in the cytoplasm during salivary gland degradation. Meanwhile, the Cyt-c protein colocates with mito-GFP, the marker indicating cytoplasmic mitochondria, and the change in mitochondrial membrane potential coincides with the appearance of Cyt-c in the cytoplasm. Moreover, impeding or promoting 20E-induced transcription factor E93 suppresses or enhances the staining of Cyt-c and the active caspase 3 in the cytoplasm of salivary gland, and accordingly decreases or increases the mitochondrial membrane potential, respectively. Our research provides evidence that cytoplasmic Cyt-c appears before apoptosis during Drosophila salivary gland degradation, shedding light on partial conserved mechanism in apoptosis between insects and mammals.

Dodonaea viscosa Jacq. induces cytotoxicity, antiproliferative activity, and cell death in colorectal cancer cells via regulation of caspase 3 and p53.

Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide and is the second leading cause of cancer-related death due to an insufficiency prognosis and is generally diagnosed in the last step of development. The Peruvian flora has a wide variety of medicinal plants with therapeutic potential in several diseases. Dodonaea viscosa Jacq. is a plant used to treat inflammatory process as well as gastrointestinal diseases. The aim of this study was to examine the cytotoxic, antiproliferative, and cell death-inducing effects of D. viscosa on colorectal cancer cells (SW480 and SW620). The hydroethanolic extract was obtained by maceration at 70% ethanol, the phytochemical constituents were identified by LC-ESI-MS. D. viscosa revealed 57 compounds some of them are: isorhamnetin, kaempferol, quercetin, methyl dodovisate B, hardwickiic acid, viscosol, and dodonic acid. Regarding the antitumoral activity, D. viscosa induced cytotoxic and antiproliferative activity in both SW480 and SW620 cancer cells, accompanied with, important changes in mitochondrial membrane potential, formation of the Sub G0/G1 population and increasing levels of apoptotic biomarkers (caspase 3 and the tumor suppressor protein p53) in the metastatic derivative cell line (SW620), suggesting an intrinsic apoptotic process after the treatment with the hydroethanolic extract of D. viscosa.

1776-P: Glucolipotoxic Conditions Affect Membrane Potential Wave Velocity Determined in Intact Islets Using Microelectrode Arrays

Pancreatic β-cells form highly connected networks within pancreatic islets. Each cell shows upon stimulation an oscillatory depolarization of the membrane potential and subsequent changes in [Ca2+]c. These signals propagate through the islet. The extent to which this coordinated islet cell activity is determined by a subset of β-cells is currently under discussion. In order to determine factors that influence this propagation, we have chosen a new method to identify wave propagation. We used high-resolution microelectrode arrays (CMOS-MEA) to measure voltage changes in the membrane potential of islet cells within the intact mouse islet. For network analysis, the individual electrode traces were compared with each other to gain insight into connectivity. The node degree of an islet (treated with 10 mM glucose) appears to be higher in the middle, but on average it was 8±2 degrees. Furthermore, algorithms were established to track the wave propagation from electrode to electrode, i.e. approximately from cell to cell. Treatment of isolated islets from C57BL6 mice with increasing glucose concentrations revealed that 15 mM glucose significantly reduced the wave duration compared to 6, 8 and 10 mM glucose (6 mM glucose: 10±4 s, n=32, vs. 15 mM glucose 7±2 s, n=26, p=0.0007). Additionally, the wave velocity was increased with rising glucose concentrations (6 mM glucose: 72±33 µm/s, n=20, vs. 8 mM glucose 118±69 µm/s, n=25, p=0.009). However, when the islets were pre-incubated with glucolipotoxic medium (25 mM glucose, 100 µM palmitate) for 72 h to mimic pre-diabetic conditions the wave velocity (waves induced by 8 mM glucose) was reduced (control: 87±40 µm/s, n=19, vs. GLT 64±32 µm/s, n=25, p=0.04). The results show that propagating waves of membrane depolarization are likely an important aspect for a functioning islet. The established technique proves to be suitable for tracking signal propagation and opens a wide field for further studies on islet network signaling. Disclosure A.K.Gresch: None. T.Deickert: None. J.D.Hüwel: None. J.Briggs: None. C.Beecks: None. R.K.Benninger: None. M.Düfer: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK102950R01, DK106412)

Simultaneous measurement and ODE-modeling of ion- and water permeability through ion exchange membranes

The determination of diffusion coefficients in solid materials as a measure of particle mobility is of great scientific interest. This applies both to desired diffusion, such as in fuel cell- and dialysis membranes, and to undesired diffusion in insulating materials. In any case, diffusion is a measure of performance. Especially for solvated ions, the permeability of the solvent must also be considered.In this work, a measurement method for the simultaneous determination of coupled ion diffusion and water permeability in polymer membranes is presented. For this purpose, measurement methods for the determination of concentration-, volume- and membrane-potential changes are integrated into a single, miniaturized concentration cell.A system of ordinary differential equations is developed for the general description of the coupled transport processes. This makes it possible to model the interdependent solute concentration and solvent volume changes. The coefficients of interest are defined by fitting the model parameters to the measured data.To evaluate the new combined measurement setup, a freestanding ion exchange membrane with largely known properties is used. The membrane of choice is a Nafion® NR211 membrane, since Nafion® is one of the most investigated PEM in literature and allows a comparison of computational and experimental results with existing data.

Caffeic Acid Markedly Induced Apoptosis of Human Multiple Myeloma Cells through the Caspase-dependent Pathway

Background Caffeic acid (CA) or 3,4-dihydroxycinnamic acid is a polyphenolic compound primarily found in coffee, herbs, berries, and other fruits. Its antioxidant, anti-inflammatory, and immunomodulatory effects on multiple health conditions have been evaluated and reported. CA’s anti-tumor effect has been reported in solid tumors, but evidence regarding liquid tumors such as multiple myeloma (MM) is limited. The increasing incidence of MM globally provides a justified rationale to explore the potential of CA on human MM cells through caspase-dependent induced apoptosis. Objectives The study explores CA’s therapeutic effect and mechanism on multiple myeloma. Materials and Methods We performed flow cytometry at different concentrations and time points after treating human MM cell lines (MM.1R, RPMI8226, and U266) with CA to identify apoptosis and changes in mitochondrial membrane potential. A Western blot was used to assess the expression of an apoptosis-related protein in MM cell lines. Statistical Analysis Used Student’s t-test was used to evaluate the mean difference between the experimental group and the control group. Results CA markedly induced the apoptosis of MM cells in a dose- and time-dependent manner. After co-incubation with CA, JC-1 (a cationic lipid fluorescent dye was used as an indicator of mitochondrial transmembrane potential), flow cytometry results showed that the mitochondrial membrane potential of MM cells significantly decreased, and the Western blot showed activation and cleavage of caspase-3, which is the classical marker of the mitochondrial apoptosis pathway. The experimental group was statistically significant compared with the control group ( p < 0.01). Conclusion Our research demonstrated that CA induced MM cell apoptosis through disturbing mitochondrial membrane integrity, followed by caspase-3 splitting, and suggested that CA might have tremendous therapeutic potential for MM treatment.

Toxicity and Biotransformation of Carbon-Based Nanomaterials in Marine Microalgae Heterosigma akashiwo.

This work is related to the environmental toxicology risk assessment and evaluation of the possible transformation of carbon-based nanomaterials (CNMs) after contact with marine microalgae. The materials used in the study represent common and widely applied multi-walled carbon nanotubes (CNTs), fullerene (C60), graphene (Gr), and graphene oxide (GrO). The toxicity was evaluated as growth rate inhibition, esterase activity, membrane potential, and reactive oxygen species generation changes. The measurement was performed with flow cytometry after 3, 24, 96 h, and 7 days. The biotransformation of nanomaterials was evaluated after 7 days of microalgae cultivation with CNMs by FTIR and Raman spectroscopy. The calculated toxic level (EC50 in mg/L, 96 h) of used CNMs reduced in the following order: CNTs (18.98) > GrO (76.77) > Gr (159.40) > C60 (414.0). Oxidative stress and membrane depolarization were the main toxic action of CNTs and GrO. At the same time, Gr and C60 decreased the toxic action with time and had no negative impact on microalgae after 7 days of exposure even at the concentration of 125 mg/L. Moreover, C60 and Gr after 7 days of contact with microalgae cells obtained structural deformations.

Super-Resolution Imaging of Voltages in the Interior of Individual, Vital Mitochondria.

We present super-resolution microscopy of isolated functional mitochondria, enabling real-time studies of structure and function (voltages) in response to pharmacological manipulation. Changes in mitochondrial membrane potential as a function of time and position can be imaged in different metabolic states (not possible in whole cells), created by the addition of substrates and inhibitors of the electron transport chain, enabled by the isolation of vital mitochondria. By careful analysis of structure dyes and voltage dyes (lipophilic cations), we demonstrate that most of the fluorescent signal seen from voltage dyes is due to membrane bound dyes, and develop a model for the membrane potential dependence of the fluorescence contrast for the case of super-resolution imaging, and how it relates to membrane potential. This permits direct analysis of mitochondrial structure and function (voltage) of isolated, individual mitochondria as well as submitochondrial structures in the functional, intact state, a major advance in super-resolution studies of living organelles.

Unraveling the Connection of Epstein-Barr Virus and Its Glycoprotein M146-157 Peptide with Neurological Ailments.

Epstein-Barr virus (EBV) is known to be associated with several cancers along with neurological modalities like Alzheimer's disease (AD) and multiple sclerosis (MS). Previous study from our group revealed that a 12 amino acid peptide fragment (146SYKHVFLSAFVY157) of EBV glycoprotein M (gM) exhibits amyloid-like self-aggregative properties. In the current study, we have investigated its effect on Aβ42 aggregation along with its effect on neural cell immunology and disease markers. EBV virion was also considered for the above-mentioned investigation. An increase in the aggregation of Aβ42 peptide was observed upon incubation with gM146-157. Further, the exposure of EBV and gM146-157 onto neuronal cells indicated the upregulation of inflammatory molecules like IL-1β, IL-6, TNF-α, and TGF-β that suggested neuroinflammation. Besides, host cell factors like mitochondrial potential and calcium ion signaling play a crucial role in cellular homeostasis and alterations in these factors aid in neurodegeneration. Changes in mitochondrial membrane potential manifested a decrease while elevation in the level of total Ca2+ ions was observed. Amelioration of Ca2+ ions triggers excitotoxicity in neurons. Subsequently, neurological disease-associated genes APP, ApoE4, and MBP were found to be increased at the protein level. Additionally, demyelination of neurons is a hallmark of MS and the myelin sheath consists of ∼70% of lipid/cholesterol-associated moieties. Hereby, genes associated with cholesterol metabolism indicated changes at the mRNA level. Enhanced expression of neurotropic factors like NGF and BDNF was discerned postexposure to EBV and gM146-157. Altogether, this study delineates a direct connection of EBV and its peptide gM146-157 with neurological illnesses.

Influence of Growth Medium Composition on Physiological Responses of Escherichia coli to the Action of Chloramphenicol and Ciprofloxacin.

The ability of hydrogen sulfide (H2S) to protect bacteria from bactericidal antibiotics has previously been described. The main source of H2S is the desulfurization of cysteine, which is either synthesized by cells from sulfate or transported from the medium, depending on its composition. Applying electrochemical sensors and a complex of biochemical and microbiological methods, changes in growth, respiration, membrane potential, SOS response, H2S production and bacterial survival under the action of bactericidal ciprofloxacin and bacteriostatic chloramphenicol in commonly used media were studied. Chloramphenicol caused a sharp inhibition of metabolism in all studied media. The physiological response of bacteria to ciprofloxacin strongly depended on its dose. In rich LB medium, cells retained metabolic activity at higher concentrations of ciprofloxacin than in minimal M9 medium. This decreased number of surviving cells (CFU) by 2-3 orders of magnitude in LB compared to M9 medium, and shifted optimal bactericidal concentration (OBC) from 0.3 µg/mL in M9 to 3 µg/mL in LB. Both drugs induced transient production of H2S in M9 medium. In media containing cystine, H2S was produced independently of antibiotics. Thus, medium composition significantly modifies physiological response of E. coli to bactericidal antibiotic, which should be taken into account when interpreting data and developing drugs.

A natural product noraucupatin against multidrug-resistant Enterococcus faecium: an inhibition mechanism study

Background: This work elucidates the antimicrobial activity and mechanism of action of the natural product noraucupatin against MDR Enterococcus faecium. E. faecium has become a major opportunistic pathogen with the worldwide spread of multidrug-resistant (MDR) isolates, especially vancomycin-resistant enterococci (VRE), belongs to “ESKAPE” organisms causing significant problems widely. Hence, there is a pressing need to discover new promising drugs or alternative therapies. Fortunately, we found a natural product noraucupatin (C13H12O3, a biphenyl compound) with “extremely encouraging” anti-clinical drug-resistant bacterial activity isolated from yeast-induced Rowan suspension cells. A comprehensive and in-depth exploration of antimicrobial mechanisms will bring fresh insights for researchers to develop novel antimicrobial strategies against MDR bacteria.Methods: The antibacterial effect of noraucupatin against MDR E. faecium is investigated from a microbial metabolism perspective using microcalorimetry. The antibacterial effect is determined based on the thermodynamic parameters. Based on spectroscopic techniques, microscopy techniques and confocal scanning laser microscopy with membrane probes, the antibacterial mechanism is elucidated definitely.Results: Comparing with the IC50 of noraucupatin against MDR Enterococcus faecalis, MRSA, CRPA, the IC50 of noraucupatin against MDR E. faecium was just 67.54 μM. The growth rate of MDR E. faecium decreases with the increase of concentration of noraucupatin. The bacterial intracellular structure entirely collapses and the slurries flow out under the influence of high levels of noraucupatin by TEM. The changes of membrane potential, permeability and evidences of nucleic acid leakage was obtained by CSLM and UV, the mechanism of noraucupatin against MDR E. faecium we explored.Conclusion: The present study highlights the excellent antibacterial activity of noraucupatin against MDR E. faecium by altering the permeability of the membrane and disrupting the membrane potential leading to electrolyte permeation. In addition, noraucupatin has excellent biocompatibility through its haemolytic activity in rabbit erythrocyte. These findings suggest that noraucupatin could be used in infectious diseases caused by MDR E. faecium.

The Comparative Toxic Impact Assessment of Carbon Nanotubes, Fullerene, Graphene, and Graphene Oxide on Marine Microalgae Porphyridium purpureum.

The growing production and application of carbon-based nanomaterials (CNMs) represent possible risks for aquatic systems. However, the variety of CNMs with different physical and chemical properties and different morphology complicate the understanding of their potential toxicity. This paper aims to evaluate and compare the toxic impact of the four most common CNMs, namely multiwalled carbon nanotubes (CNTs), fullerene (C60), graphene (Gr), and graphene oxide (GrO) on the marine microalgae Porphyridium purpureum. The microalgae cells were exposed to the CNMs for 96 h and measured by flow cytometry. Based on the obtained results, we determined no observed effect level (NOEL), and calculated EC10 and EC50 concentrations for growth rate inhibition, esterase activity, membrane potential, and reactive oxygen species (ROS) generation changes for each tested CNM. According to the sensitivity (growth rate inhibition) of P. purpureum, the used CNMs can be listed in the following order (EC50 in mg/L, 96 h): CNTs (2.08) > GrO (23.37) > Gr (94.88) > C60 (>131.0). The toxicity of CNTs was significantly higher than the toxic effect of the other used CNMs, and only this sample caused an increase in ROS generation in microalgae cells. This effect was apparently caused by the high affinity between particles and microalgae associated with the presence of exopolysaccharide coverage on P. purpureum cells.

Mitochondrial Toxic Effects of Antiepileptic Drug Valproic Acid on Mouse Kidney Stem Cells.

Valproic acid (VPA) is a histone deacetylase inhibitor that is used mainly as an antiepileptic and anticonvulsant drug. The side effects of VPA usually appears as hepatic injury and various metabolic disorders. On the other hand, it is rarely reported to cause kidney injury. Despite the many studies on the influence of VPA exposure on the kidneys, the specific mechanism remains unclear. This study examined the changes after VPA treatment to the mouse kidney stem cells (mKSCs). VPA triggers an increase in mitochondrial ROS, but there was no change in either mitochondrial membrane potential or the mitochondrial DNA copy number in mKSCs. The VPA treatment increased the mitochondrial complex III but decreased complex V significantly compared to the DMSO treatment as a control. The inflammatory marker (IL-6) and the expression of the apoptosis markers (Caspase 3) and were increased by VPA. In particular, the expression of the podocyte injury markers (CD2AP) was increased significantly. In conclusion, VPA exposure has adverse effects on mouse kidney stem cells.