Induced Membrane Depolarization Research Articles

Wnt-5a/Ca2+ pathway modulates endogenous current and oocyte structure of Xenopus laevis

Wnt signaling plays an essential role in cellular processes like development, maturation, and function maintenance. Xenopus laevis oocytes are a suitable model to study not only the development but also the function of different receptors expressed in their membranes, like those receptors expressed in the central nervous system (CNS) including Frizzled 7. Here, using frog oocytes and recordings of endogenous membrane currents in a two-electrode path configuration along with morphological observations, we evaluated the role of the non-canonical Wnt-5a ligand in oocytes. We found that acute application of Wnt-5a generated changes in endogenous calcium-dependent currents, entry oscillatory current, the membrane's outward current, and induced membrane depolarization. The incubation of oocytes with Wnt-5a caused a reduction of the membrane potential, potassium outward current, and protected the ATP current in the epithelium/theca removed (ETR) model. The oocytes exposed to Wnt-5a showed increased viability and an increase in the percentage of the germinal vesicle breakdown (GVBD), at a higher level than the control with progesterone. Altogether, our results suggest that Wnt-5a modulates different aspects of oocyte structure and generates calcium-dependent endogenous current alteration and GVDB process with a change in membrane potential at different concentrations and times of the exposition. These results help to understand the cellular effect of Wnt-5a and present the use of Xenopus oocytes to explore the mechanism that could impact the activation of Wnt signaling.

Deciphering the Intracellular Action of the Antimicrobial Peptide A11 via an In-Depth Analysis of Its Effect on the Global Proteome of Acinetobacter baumannii.

The potential antimicrobial activity and low propensity to induce the development of bacterial resistance have rendered antimicrobial peptides (AMPs) as novel and ideal candidate therapeutic agents for the treatment of infections caused by drug-resistant pathogenic bacteria. The targeting of bacterial membranes by AMPs has been typically considered their sole mode of action; however, increasing evidence supports the existence of multiple and complementary functions of AMPs that result in bacterial death. An in-depth characterization of their mechanism of action could facilitate further research and development of AMPs with higher potency. The current study employs biophysics and proteomics approaches to unveil the mechanisms underlying the antibacterial activity of A11, a potential candidate AMP, against Acinetobacter baumannii, a leading cause of hospital-acquired infections (HAIs) and consequently, a serious global threat. A11 peptide was found to induce membrane depolarization to a high extent, as revealed by flow cytometry and electron microscopy analyses. The prompt intracellular penetration of A11 peptide, observed using confocal microscopy, was found to occur concomitantly with a very low degree of membrane lysis, suggesting that its mode of action predominantly involves a nonlytic killing mechanism. Quantitative proteomics analysis employed for obtaining insights into the mechanisms underlying the antimicrobial activity of A11 peptide revealed that it disrupted energy metabolism, interfered with protein homeostasis, and inhibited fatty acid synthesis that is essential for cell membrane integrity; all these impacted the cellular functions of A. baumannii. A11 treatment also impacted signal transduction associated with the regulation of biofilm formation, hindered the stress response, and influenced DNA repair processes; these are all crucial survival mechanisms of A. baumannii. Additionally, robust antibacterial activity was exhibited by A11 peptide against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of A. baumannii; moreover, A11 peptide exhibited synergy with levofloxacin and minocycline as well as low propensity for inducing resistance. Taken together, the findings emphasize the therapeutic potential of A11 peptide as an antibacterial agent against drug-resistant A. baumannii and underscore the need for further investigation.

Zebrafish ANGPT4, member of fibrinogen-related proteins, is an LTA-, LPS- and PGN-binding protein with a bacteriolytic activity

Fibrinogen-related proteins (FREPs) are a family of glycoproteins that contain a fibrinogen-like (FBG) domain. Many members of FREPs have been shown to play an important role in innate immune response in both vertebrates and invertebrates. Here we reported the immune functional characterization of ANGPT4, member of FREPs, in zebrafish Danio rerio. Quantitative real time PCR showed that the expression of zebrafish ANGPT4 gene is up-regulated by the challenge with lipoteichoic acid (LTA) or lipopolysaccharides (LPS), hinting its involvement in innate immune response. The recombinant ANGPT4 (rANGPT4) could bind to both gram-positive bacteria Staphylococcus aureus and Bacillus subtilis and the gram-negative bacteria Escherichia coli and Aeromonas hydrophila as well as the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LTA, LPS and peptidoglycan (PGN), suggesting it capable of identifying pathogens via LTA, LPS and PGN. In addition, rANGPT4 also displayed strong bacteriolytic activities against both gram-positive and -negative bacteria tested via inducing membrane depolarization and intracellular ROS production. Moreover, the bacterial clearance assay in vivo showed that the rANGPT4 could also accelerate the clearance of bacteria in zebrafish embryos/larvae. Finally, we showed that the eukaryotically expressed recombinant ANGPT4 maintained antibacterial activity and binding activity to bacteria and LTA, LPS and PGN. All these suggested that ANGPT4 could not only capable of recognizing pathogens via LTA, LPS and PGN, but also capable of killing the Gram-positive and Gram-negative bacteria, in innate immune response. This work also provides further information to understand the biological roles of FREPs and the innate immunity in vertebrates.

A membrane targeted multifunctional cationic nanoparticle conjugated fusogenic nanoemulsion (CFusoN): induced membrane depolarization and lipid solubilization to accelerate the killing of Staphylococcus aureus.

Bacterial infections caused by Staphylococcus aureus are one of the growing concerns for human health care management globally. Antibiotic-associated adverse effects and the emergence of bacterial resistant strains necessitate the development of an alternative yet effective approach. Nanoemulsion-based therapy has emerged as a potential therapeutic strategy to combat bacterial infestation. Herein, we designed a cationic metal nanoparticle-conjugated fusogenic nanoemulsion (CFusoN) as a lipid solubilizing nanovesicle for the effective treatment of S. aureus infection with a killing efficiency of 99.999%. The cationic nanoparticle-conjugated nanoemulsion (viz. NECNP) (24.4 ± 2.9 mV) electrostatically bound with the negatively charged bacterial cell membrane (-10.2 ± 3.7 mV) causing alteration of the bacterial surface charge. The fluorometric and flow cytometry studies confirmed the bacterial membrane depolarization and altered cell membrane permeability leading to cell death. The atomic force microscopic studies further demonstrated the damage of the cellular ultrastructure, while the transmission electron microscopic image and membrane lipid solubilization analysis depicted the solubilization of the bacterial membrane lipid bilayer along with the leakage of the intracellular contents. The cell membrane fatty acid analysis revealed that the methyl esters of palmitic acid, stearic acid and octadecadienoic acid isomers were solubilized after the treatment of S. aureus with CFusoN. The bactericidal killing efficiency of CFusoN is proposed to occur through the synergistic efficacy of the targeted attachment of CNP to the bacterial cells along with the lipid solubilization property of NE. Interestingly, NECNP didn't elicit any in vitro hemolytic activity or cytotoxicity against red blood cells (RBCs) and L929 fibroblast cells, respectively, at its bactericidal concentration. Furthermore, a porcine skin wound infection model exhibited the enhanced wound cleansing potency of CFusoN in comparison to the commercially available wound cleansers. The obtained antibacterial activity, biocompatibility and skin wound disinfection efficacy of the NECNP demonstrated the formulation of a cell targeted CFusoN as a promising translatable strategy to combat bacterial infection.

Photodynamic eradication of intratumoral microbiota with bacteria‐targeted micelles overcomes gemcitabine resistance of pancreatic cancer

AbstractIncreasing evidence suggests that intratumoral microbiota plays a pivotal role in tumor progression, immunosurveillance, metastasis, and chemosensitivity. Particularly, in pancreatic ductal adenocarcinoma, tumor‐resident Gammaproteobacteria could transform the chemotherapeutic drug gemcitabine (Gem) into its inactive form, thus rendering chemotherapy ineffective. Herein, a strategy for selectively eradicating intratumoral bacteria was described for overcoming Gem resistance in a pancreatic cancer animal model. An antimicrobial peptide was linked with photosensitizer through a poly (ethylene glycol) chain, which can self‐assemble into micelles with a diameter of ∼20 nm. The micelles could efficiently kill bacteria under light irradiation by inducing membrane depolarization, thereby inhibiting Gem metabolism. In a bacteria‐resident pancreatic cancer animal model, the selective photodynamic eradication of intratumoral bacteria was demonstrated to efficiently reverse Gem resistance. This research highlights antibacterial photodynamic therapy as a promising adjuvant strategy for cancer therapy by modulating intratumoral microbiota.

Insulin secretory actions of ethanolic extract of Acacia arabica bark in high fat-fed diet-induced obese Type 2 diabetic rats.

Acacia arabica commonly known as 'babul' has been widely used for the treatment of numerous diseases, including diabetes due to their potential pharmacological actions. The aim of the present study was to investigate the insulinotropic and antidiabetic properties of ethanol extract of Acacia arabica (EEAA) bark through in vitro and in vivo studies in high fat-fed (HFF) rats. EEAA at 40-5000 µg/ml significantly increased (P<0.05-0.001) insulin secretion with 5.6 and 16.7 mM glucose, respectively, from clonal pancreatic BRIN BD11 β-cells. Similarly, EEAA at 10-40 µg/ml demonstrated a substantial (P<0.05-0.001) insulin secretory effect with 16.7 mM glucose from isolated mouse islets, with a magnitude comparable to 1 µM glucagon-like peptide-1 (GLP-1). Diazoxide, verapamil, and calcium-free conditions decreased insulin secretion by 25-26%. The insulin secretory effect was further potentiated (P<0.05-0.01) with 200 µM isobutylmethylxanthine (IBMX; 1.5-fold), 200 µM tolbutamide (1.4-fold), and 30 mM KCl (1.4-fold). EEAA at 40 µg/ml, induced membrane depolarization and elevated intracellular Ca2+ as well as increased (P<0.05-0.001) glucose uptake in 3T3L1 cells and inhibited starch digestion, glucose diffusion, dipeptidyl peptidase-IV (DPP-IV) enzyme activity, and protein glycation by 15-38%, 11-29%, 15-64%, and 21-38% (P<0.05, 0.001), respectively. In HFF rats, EEAA (250 mg/5 ml/kg) improved glucose tolerance, plasma insulin, and GLP-1 levels, and lowered DPP-IV enzyme activity. Phytochemical screening of EEAA revealed the presence of flavonoids, tannins and anthraquinone. These naturally occurring phytoconstituents may contribute to the potential antidiabetic actions of EEAA. Thus, our finding suggests that EEAA, as a good source of antidiabetic constituents, would be beneficial for Type 2 diabetes patients.

Kinetically regulated one-pot synthesis of cationic gold nanoparticles and their size-dependent antibacterial mechanism

Cationic gold nanoparticles (cAuNPs) have been regarded as promising candidates for antibacterial applications due to their high surface charge density, favorable biocompatibility, and controllable surface chemistry. Nevertheless, the complicated fabrication process and unclear antibacterial mechanism have greatly hindered the further biomedical application of cAuNPs. Herein, we have developed a simple and controllable strategy for synthesizing cAuNPs with tailored size and antibacterial behavior by kinetically modulating the reaction process. Specifically, a functional ligand, (11-mercaptoundecyl)-N, N, N-trimethylammonium bromide (MUTAB), was chosen to chemically manipulate the positive surface charge of cAuNPs via a one-step strategy. The size of cAuNPs could be flexibly adjusted from 1.1 to 14.8 nm by simply elevating the stirring speed of the reaction from 0 to 1500 rpm. Further studies revealed that the antibacterial effect of cAuNPs was strongly correlated with the particle size. MUTAB-protected ultrasmall gold nanoclusters (MUTAB-AuNCs) were able to eradicate E. coli at a concentration as low as 1.25 μg mL–1, while the minimum inhibitory concentration of MUTAB-AuNPs with a large size for E. coli was 5 μg mL–1. Mechanistic investigation revealed that MUTAB-AuNPs were able to damage the bacterial membrane and stimulate the production of reactive oxygen species more effectively than MUTAB-AuNCs. Conversely, MUTAB-AuNCs were more active in inducing membrane depolarization in contrast to MUTAB-AuNPs, suggesting the unique size-dependent antibacterial manner of cAuNPs. This study presents a new strategy for the controlled preparation of cAuNPs with distinct sizes and antibacterial behavior, laying a valuable foundation for developing efficient cationic NP-based bactericidal agents.

Reactive oxygen species-specific characteristics of transient receptor potential ankyrin 1 receptor and its pain modulation

Transient receptor potential ankyrin 1 (TRPA1) receptors are major polymodal nociceptors that generate primary pain responses in the peripheral nerve endings of the dorsal root ganglion neurons. Recently, we reported that the activation of TRPA1 receptors by reactive oxygen species (ROS) signaling, which is triggered by Ca&lt;sup&gt;2+&lt;/sup&gt; influx through T-type Ca&lt;sup&gt;2+&lt;/sup&gt; channels, contributes to prolonged pain responses induced by jellyfish toxin. In this review, we focus on the characteristics of the TRPA1 receptor involved in intracellular signaling as a secondary pain modulator. Unlike other transient receptor potential receptors, TRPA1 receptors can induce membrane depolarization by ROS without exogenous stimuli in peripheral and central sensory neurons. Therefore, it is important to identify the functional characteristics of TRPA1 receptors to understand pain modulation under several pathogenic conditions such as neuropathic pain syndromes and autoimmune diseases, which are mediated by oxidative signaling to cause chronic pain in the sensory system.

Purkinje cell dopaminergic inputs to astrocytes regulate cerebellar-dependent behavior

Dopamine has a significant role in motor and cognitive function. The dopaminergic pathways originating from the midbrain have received the most attention; however, the relevance of the cerebellar dopaminergic system is largely undiscovered. Here, we show that the major cerebellar astrocyte type Bergmann glial cells express D1 receptors. Dopamine can be synthesized in Purkinje cells by cytochrome P450 and released in an activity-dependent fashion. We demonstrate that activation of D1 receptors induces membrane depolarization and Ca2+ release from the internal store. These astrocytic activities in turn modify Purkinje cell output by altering its excitatory and inhibitory synaptic input. Lastly, we show that conditional knockout of D1 receptors in Bergmann glial cells results in decreased locomotor activity and impaired social activity. These results contribute to the understanding of the molecular, cellular, and circuit mechanisms underlying dopamine function in the cerebellum, revealing a critical role for the cerebellar dopaminergic system in motor and social behavior.

Identification and functional characterization of zebrafish ELAVL1b as a new member of antimicrobial protein

Previous studies have shown that ELAVL1 play multiple roles and may be associated with immune response. However, it remains largely unknown about the direct roles of ELAVL1 during a bacterial infection. After reporting the zebrafish ELAVL1a is a maternal immune factor that can protect zebrafish embryos from bacterial infection, here we studied the immune function of zebrafish ELAVL1b. In this study, we showed that zebrafish elavl1b was markedly up-regulated by LTA and LPS treatment, suggesting it may be involved in anti-infectious responses. We also showed that zebrafish recombinant ELAVL1b (rELAVL1b) could bind to both the Gram-positive and negative bacteria M. luteus and S. aureus, E. coli and A. hydrophila as well as their signature molecules LTA and LPS, hinting it may act as a pattern recognition receptor, capable of identifying pathogens. In addition, rELAVL1b could directly kill the Gram-positive and negative bacteria tested via inducing membrane depolarization and intracellular ROS production. Collectively, our results indicate that zebrafish ELAVL1b plays an immune-relevant role as a newly-characterized antimicrobial protein. This work also provides further information to understand the biological roles of ELAVL family and the innate immunity in vertebrates.

Photothermal-Enhanced Modulation of Cellular Membrane Potential Using Long-Wavelength-Activated Gold Nanoflowers.

In mammalian cells, plasma membrane potential plays vital roles in both physiology and pathology and it is controlled by a network of membrane-resident ion channels. There is considerable interest in the use of nanoparticles (NPs) to control biological functions, including the modulation of membrane potential. The photoexcitation of gold NPs (AuNPs) tethered close to the plasma membrane has been shown to induce membrane depolarization via localized heating of the AuNP surface coupled with the opening of voltage-gated sodium channels. Previous work has employed spherical AuNPs (AuNS) with absorption in the 500-600 nm range for this purpose. However, AuNP materials with absorption at longer wavelengths [e.g., near-infrared (NIR)] would enable greater tissue penetration depth in vivo. We show here the use of new anisotropic-shaped AuNPs [gold nanoflowers (AuNFs)] with broad absorption spanning into the NIR part of the spectrum (∼650-1000 nm). The AuNFs are directly synthesized with bidentate thiolate ligands, which preserves the AuNF's shape and colloidal stability, while facilitating conjugation to biomolecules. We describe the characterization of the AuNF particles and demonstrate that they adhere to the plasma membrane when bioconjugated to PEGylated cholesterol (PEG-Chol) moieties. The AuNF-PEG-Chol mediated the depolarization of rat adrenal medulla pheochromocytoma (PC-12) neuron-like cells more effectively than AuNS-PEG-Chol and unconjugated AuNS and AuNF when photoexcited at ∼561 or ∼640 nm. Importantly, AuNF induction of depolarization had no impact on cellular viability. This work demonstrates anisotropic AuNFs as an enabling nanomaterial for use in cellular depolarization and the spatiotemporal control of cellular activity.

The systemic disruption of P2rx7 alleviates tau pathology in P301S tau mice via inhibition of extracellular vesicle release

AbstractBackgroundP2X purinoceptor 7 (P2RX7), an ATP‐gated cation channel present abundantly in the microglia, induces membrane depolarization and extracellular vesicle (EV) secretion. Previous study demonstrated that administration of GSK1482160, a P2RX7 selective inhibitor, suppressed EVs secretion from murine microglia resulting in accumulation of Tsg101+ intraluminal vesicles (ILVs) in the hippocampus, and restored partial behavioral deficits in P301S tau mice. However, the specificity of GSK1482160 to P2RX7 has never been investigated in P301S tau mice. It is crucial to validate whether P2RX7 is a potential target for alleviating the tauopathy phenotype in P301S tau mice. The purpose of this study is to determine if deletion of P2RX7 is sufficient to recapiturate the therapeutic effect of GSK1482160 in tauopathy mouse model.MethodFours and nine‐months‐old P2RX7‐/‐:P301S and P301S mice were generated for the evaluation of ILV accumulation and tau pathology. Primary microglia and astrocytes from WT (C57BL/6) and P2RX7‐/‐ mice, treated with GSK1482160 and ATP for EV secretion. EVs were isolated from conditioned media via the sequential centrifugation and ultracentrifugation. Finally, EVs were quantified by nanoparticle tracking analysis and exosome specific marker CD9 ELISA. Effects of GSK1482160 on EVs secretion from astrocytes/microglia were also assessed using Western blot with EV specific markers Tsg101 and CD9.ResultExosome specific marker Tsg101, CD9, and tau expression levels were reduced in the hippocampus of nine‐months‐old P2RX7‐/‐:P301S mice compared to P301S mice. No significant difference between the two groups at 4 months of age was observed. Strikingly, primary cultured microglia and astrocytes showed reduction in EV secretion in both ATP stimulated or unstimulated condition from P2RX7‐/‐ group compared to WT group. Additionally, GSK1482160 pretreatment significantly reduced EV secretion from WT astrocytes and microglia, although it had no effects in P2RX7‐/‐ cells. These findings were validated by ELISA of EV marker CD9 and immunoblotting of Tsg101 and flotillin‐1 using isolated EVs.ConclusionResults demonstrate that P2RX7 is primarily responsible for ATP‐induced EV secretion from astrocytes and microglia, and systemic disruption of P2RX7 mimics the effect of GSK1482160 in P301S tau mice, including accumulation of ILVs in microglia and suppression of tau accumulation in the hippocampal regions.

Protective Effects of Choline against Inflammatory Cytokines and Characterization of Transport in Motor Neuron-like Cell Lines (NSC-34).

Choline, a component of the neurotransmitter acetylcholine, is essential for nervous system functions, brain development, and gene expression. In our study, we investigated the protective effect and transport characteristics of choline in amyotrophic lateral sclerosis (ALS) model cell lines. We used the wild-type (WT) motor neuron-like hybrid cell line (NSC-34/hSOD1WT) as a control and the mutant-type (MT; NSC-34/hSOD1G93A) as a disease model. The uptake of [3H]choline was time-, pH-, and concentration-dependent. [3H]Choline transport was sodium-dependent, and, upon pretreatment with valinomycin, induced membrane depolarization. Gene knockdown of Slc44a1 revealed that choline-like transporter 1 (CTL1) mediates the transport of choline. In NSC-34 cell lines, the specific choline transporter inhibitor, hemicholinium-3 demonstrated significant inhibition. Donepezil and nifedipine caused dose-dependent inhibition of [3H]choline uptake by the MT cell line with minimal half inhibitory concentration (IC50) values of 0.14 mM and 3.06 mM, respectively. Four-day pretreatment with nerve growth factor (NGF) resulted in an inhibitory effect on [3H]choline uptake. Choline exerted protective and compensatory effects against cytokines mediators. Hence, the choline transport system CLT1 may act as a potential target for the delivery of novel pharmacological drugs, and the combination of drugs with choline can help treat symptoms related to ALS.

Asenapine, an atypical antipsychotic, blocks voltage-gated potassium channels in rabbit coronary artery smooth muscle cells

We investigated the effect of asenapine, a commonly used atypical antipsychotic, on voltage-dependent K+ (Kv) channels in rabbit coronary artery smooth muscle cells. Asenapine inhibited the Kv current in a concentration-dependent manner, with an half-inhibitory concentration (IC50) value of 8.59 ± 2.25 μM and Hill coefficient of 0.64 ± 0.06. Although asenapine did not affect the steady-state activation curve of Kv channels, it shifted the voltage dependence of the steady-state inactivation curve toward a more negative potential. Asenapine increased the recovery time constant of channel inactivation and produced use (state)-dependent inhibition of Kv channels at a stimulation frequency of 1 or 2 Hz. Pretreatment with the Kv1.5 subtype inhibitor DPO-1 reduced the Kv current; however, additional application of asenapine did not further inhibit the Kv current. Pretreatment with the Kv2.1 subtype inhibitor guangxitoxin and Kv7 inhibitor linopirdine also reduced the Kv current. However, additional application of asenapine further reduced the Kv current, similar to the application of asenapine alone. Asenapine induced membrane depolarization and vasoconstriction. Based on these results, we conclude that asenapine inhibits the Kv current in concentration- and use (state)-dependent manners by shifting the inactivation curve. The major target of asenapine is the Kv1.5 subtype channel.

Mitochondrial dysfunction due to in vitro exposure to atrazine and its metabolite in striatum.

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) has beendescribed as a potential toxic for dopaminergic metabolism both in vivo and in vitro. Its main metabolite diamino-chloro triazine (DACT) has been shown to achieve higher levels in brain tissue than atrazine. The aim of this study was to evaluate the in vitro effects of atrazine and DACT on striatal mitochondrial function, active oxygen species generation, and nitric oxide (NO) content. Incubation of mitochondria with atrazine (10 µM) was not able to modify oxygen consumption. However, a 50% increase in malate-glutamate state 4 respiratory rates was observed after DACT treatment (100 µM) without changes in respiratory state 3. Atrazine was able to inhibit complex I-III activity by 30% and DACT induced a tendency to decrease by 17% in the striatum. Regardingreactive oxygen species(ROS), DACT increased H2 O2 production by 43%. Also, superoxide anion levels were higher (14%) after atrazine exposure than in control mitochondria. Incubation of striatal mitochondria with atrazine and DACT induced membrane depolarization by 15% and 19%, respectively. Also, atrazine increased NO content by 10% but no significant changes were observed after exposure of mitochondria to DACT. Glutathione peroxidase activity was inhibited (56%) by DACT and atrazine inhibited superoxide dismutase activity by 60%. Also, cardiolipin oxidation (15%) was observed after atrazine treatment. Summing up, the obtained results suggest that in vitro atrazine and DACT induce ROS production affecting striatal mitochondrial function. The atrazine effects would be attributed to a direct effect on the mitochondrialrespiratory chain and superoxide dismutase activity while DACT appears to disturb glutathione-related enzyme system.

The Membrane Activity of the Amphibian Temporin B Peptide Analog TB_KKG6K Sheds Light on the Mechanism That Kills Candida albicans.

ABSTRACTTemporin B (TB) is a 13-amino-acid-long, cationic peptide secreted by the granular glands of the European frog Rana temporaria. We recently showed that the modified TB peptide analog TB_KKG6K rapidly killed planktonic and sessile Candida albicans at low micromolar concentrations and was neither hemolytic nor cytotoxic to mammalian cells in vitro. The present study aimed to shed light into its mechanism of action, with a focus on its fungal cell membrane activity. We utilized different fluorescent dyes to prove that it rapidly induces membrane depolarization and permeabilization. Studies on model membrane systems revealed that the TB analog undergoes hydrophobic and electrostatic membrane interactions, showing a preference for anionic lipids, and identified phosphatidylinositol and cardiolipin as possible peptide targets. Fluorescence microscopy using fluorescein isothiocyanate-labeled TB_KKG6K in the presence of the lipophilic dye FM4-64 indicated that the peptide compromises membrane integrity and rapidly enters C. albicans cells in an energy-independent manner. Peptide-treated cells analyzed by cryo-based electron microscopy exhibited no signs of cell lysis; however, subcellular structures had disintegrated, suggesting that intracellular activity may form part of the killing mechanism of the peptide. Taken together, this study proved that TB_KKG6K compromises C. albicans membrane function, which explains the previously observed rapid, fungicidal mode of action and supports its great potential as a future anti-Candida therapeutic.IMPORTANCE Fungal infections with the opportunistic human pathogen C. albicans are associated with high mortality rates in immunocompromised patients. This is partly due to the yeast's ability to rapidly develop resistance toward currently available antifungals. Small, cationic, membrane-active peptides are promising compounds to fight against resistance development, as many of them effectuate rapid fungal cell death. This fast killing is believed to hamper the development of resistance, as the fungi do not have sufficient time to adapt to the antifungal compound. We previously reported that the synthetic variant of the amphibian TB peptide, TB_KKG6K, rapidly kills C. albicans. In the current study, the mechanism of action of the TB analog was investigated. We show that this TB analog is membrane-active and impairs cell membrane function, highlighting its potential to be developed as an attractive alternative anti-C. albicans therapeutic that may hinder the development of resistance.

Taste Receptor Cells Generate Oscillating Receptor Potentials by Activating G Protein-Coupled Taste Receptors.

The receptor potentials of taste receptor cells remain unclear. Here, we demonstrate that taste receptor cells generate oscillating depolarization (n = 7) with action potentials in response to sweet, bitter, umami, and salty taste substances. At a lower concentration of taste substances, taste receptor cells exhibited oscillations in membrane potentials with a low frequency and small magnitude of depolarization. Although the respective waves contained no or 1–2 action potentials, the taste receptor cells generated action potentials continuously in the presence of taste stimuli. Both the frequency and magnitude of oscillations increased when the concentration was increased, to 0.67–1.43 Hz (n = 3) and Δ39–53 mV (n = 3) in magnitude from −64.7 ± 4.2 to −18.7 ± 5.9 mV, which may activate the ATP-permeable ion channels. In contrast, a sour tastant (10-mM HCl) induced membrane depolarization (Δ19.4 ± 9.5 mV, n = 4) with action potentials in type III taste receptor cells. Interestingly, NaCl (1 M) taste stimuli induced oscillation (n = 2) or depolarization (Δ10.5 ± 5.7 mV at the tonic component, n = 9). Our results indicate that the frequency and magnitude of oscillations increased with increasing taste substance concentrations. These parameters may contribute to the expression of taste “thickness.”

Double-Edged Nanobiotic Platform with Protean Functionality: Leveraging the Synergistic Antibacterial Activity of a Food-Grade Peptide to Mitigate Multidrug-Resistant Bacterial Pathogens.

While persistent efforts are being made to develop a novel arsenal against bacterial pathogens, the development of such materials remains a formidable challenge. One such strategy is to develop a multimodel antibacterial agent which will synergistically combat bacterial pathogens, including multidrug-resistant bacteria. Herein, we used pediocin, a class IIa bacteriocin, to decorate Ag° and developed a double-edged nanoplatform (Pd-SNPs) that inherits intrinsic properties of both antibacterial moieties, which engenders strikingly high antibacterial potency against a broad spectrum of bacterial pathogens including the ESKAPE category without displaying adverse cytotoxicity. The enhanced antimicrobial activity of Pd-SNPs is due to their higher affinity with the bacterial cell wall, which allows Pd-SNPs to penetrate the outer membrane, inducing membrane depolarization and the disruption of membrane integrity. Bioreporter assays revealed the upregulation of cpxP, degP, and sosX genes, triggering the burst of reactive oxygen species which eventually cause bacterial cell death. Pd-SNPs prevented biofilm formation, eradicated established biofilms, and inhibited persister cells. Pd-SNPs display unprecedented advantages because they are heat-resistant, retain antibacterial activity in human serum, and alleviate vancomycin intermediate Staphylococcus aureus (VISA) infection in the mouse model. In addition, Pd-SNPs wrapped in biodegradable nanofibers mitigated Listeria monocytogenes in cheese samples. Collectively, Pd-SNPs exhibited excellent biocompatibility and in vivo therapeutic potency without allowing foreseeable resistance acquisition by pathogens. These findings underscore new avenues for using a potent biocompatible nanobiotic platform to combat a wide range of bacterial pathogens.

In vitro and in vivo antihyperglycemic activity of the ethanol extract of Heritiera fomes bark and characterization of pharmacologically active phytomolecules.

This study aimed to demonstrate the mechanistic basis of Heritiera fomes, which has traditionally been used to treat diabetes. Clonal pancreatic β-cells and primary islets were used to measure insulin release. 3T3-L1 cells were used to analyse insulin action, and in vitro systems were used to measure further glucose-lowering activity. In vivo assessment was performed on streptozotocin (STZ)-induced type-2 diabetic rats and reversed-phase-HPLC followed by liquid chromatography mass spectrometry (LC-MS) to detect bioactive molecules. Ethanol extract of Heritiera fomes (EEHF) significantly increased insulin release with stimulatory effects comparable to 1 µM glucagon-like peptide 1, which were somewhat reduced by diazoxide, verapamil and calcium-free conditions. Insulin release was stimulated by tolbutamide, isobutyl methylxanthine and KCl. EEHF induced membrane depolarization and increased intracellular Ca2+ levels. EEHF enhanced glucose uptake in 3T3L1 cells and decreased protein glycation. EEHF significantly inhibited postprandial hyperglycaemia following sucrose loading and inversely elevated unabsorbed sucrose concentration in the gut. It suppressed glucose absorption during in situ gut perfusion. Furthermore, EEHF improved glucose tolerance, plasma insulin and gut motility, and decreased plasma dipeptidyl peptidase IV activity. Procyanidins, epicatechin and proanthocyanidins were some of the identified bioactive constituents that may involve in β-cell actions. This study provides some evidence to support the use of H. fomes as an antidiabetic traditional remedy.

Improving effect of cordycepin on insulin synthesis and secretion in normal and oxidative-damaged INS-1 cells

Diabetes mellitus (DM) has recently become one of the major diseases that have received attention. Cordycepin (molecular formula: C10H13N5O3), is one of the major bioactive components of Cordyceps militaris, decreases blood glucose levels. In this study, the effect and mechanism of cordycepin in normal and oxidative-damaged INS-1 cells were explored by using cell and molecular biology methods. Results showed that cordycepin could enhance insulin synthesis and secretion. The mechanism is possibly related to the elevated ATP content induced membrane depolarisation and increased Ca2+ concentration. At the genetic level, cordycepin upregulated the mRNA level of insulin, pancreatic duodenal homeobox factor-1 (PDX-1) and glucose transporter 1 (GLUT1). At the protein level, cordycepin promoted the expression of PDX-1, GLUT1, serine threonine kinase (Akt) and phosphorylated Akt (P-Akt). These effects may also contribute to the enhancement of insulin synthesis and secretion. Further analysis revealed that cordycepin protected against H2O2-induced damage on INS-1 cells and improved their viability and insulin synthesis/secretion. This effect should be attributed to the reduced intracellular reactive oxygen species (ROS), enhanced mitochondrial membrane potential (MMP), increased activity of superoxide dismutase (SOD) and upregulated genetic and protein expression of catalase (CAT), PDX-1, GLUT1 and P-Akt. In conclusion, cordycepin promotes insulin synthesis and secretion in normal islet β cells and improves this function in oxidative-damaged islet β cells. Given that islet β cells are vulnerable to oxidative stress, the improving effect of cordycepin on the antioxidant capacity and insulin synthesis/secretion of INS-1 cells may be an important mechanism for its hypoglycaemic effect.