Hyperpolarization Of Cell Membrane Research Articles

Epinephrine also acts on beta cells and insulin secretion.

In a recent review examining neurotransmitter modulation of insulin secretion, the significant impact of epinephrine was not addressed. Its primary action involves inhibiting insulin release via alpha-adrenergic receptors, thereby reducing the response to insulin secretion stimulators, through the activation of K+ channels and resulting in membrane hyperpolarization in beta cells.

Physiological role of potassium channels in mammalian germ cell differentiation, maturation, and capacitation

Ion channels are essential for differentiation and maturation of germ cells, and even for fertilization in mammals. Different types of potassium channels have been identified, which are grouped into voltage-gated channels (Kv), ligand-gated channels (Kligand ), inwardly rectifying channels (Kir ), and tandem pore domain channels (K2P ). The present review includes recent findings on the role of potassium channels in sperm physiology of mammals. While most studies conducted thus far have been focused on the physiological role of voltage- (Kv1, Kv3, and Kv7) and calcium-gated channels (SLO1 and SLO3) during sperm capacitation, especially in humans and rodents, little data about the types of potassium channels present in the plasma membrane of differentiating germ cells exist. In spite of this, recent evidence suggests that the content and regulation mechanisms of these channels vary throughout spermatogenesis. Potassium channels are also essential for the regulation of sperm cell volume during epididymal maturation and for preventing premature membrane hyperpolarization. It is important to highlight that the nature, biochemical properties, localization, and regulation mechanisms of potassium channels are species-specific. In effect, while SLO3 is the main potassium channel involved in the K+ current during sperm capacitation in rodents, different potassium channels are implicated in the K+ outflow and, thus, plasma membrane hyperpolarization during sperm capacitation in other mammalian species, such as humans and pigs. Potassium conductance is essential for male fertility, not only during sperm capacitation but throughout the spermiogenesis and epididymal maturation.

Kv7 channel activation reduces brain endothelial cell permeability and prevents kainic acid-induced blood-brain barrier damage.

Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions.NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.

Fisetin-Mediated Perturbations of Membrane Permeability and Intracellular pH in Candida albicans.

The antifungal activity of fisetin against Candida albicans is explored, elucidating a mechanism centered on membrane permeabilization and ensuing disruption of pH homeostasis. The Minimum Inhibitory Concentration (MIC) of fisetin, indicative of its interaction with the fungal membrane, increases in the presence of ergosterol. Hoechst 33342 and propidium-iodide staining reveal substantial propidium-iodide accumulation in fisetin-treated C. albicans cells at their MIC, with crystal violet uptake assays confirming fisetin-induced membrane permeabilization. Leakage analysis demonstrates a significant release of DNA and proteins in fisetin-treated cells compared to controls, underscoring the antifungal effect through membrane disruption. Green fluorescence, evident in both the cytoplasm and vacuoles of fisetin-treated cells under BCECF, AM staining, stands in contrast to controls where only acidic vacuoles exhibit staining. Ratiometric pH measurements using BCECF, AM reveal a noteworthy reduction in intracellular pH in fisetin-treated cells, emphasizing its impact on pH homeostasis. DiBAC4(3) uptake assays demonstrate membrane hyperpolarization in fisetin-treated cells, suggesting potential disruptions in ion flux and cellular homeostasis. These results provide comprehensive insights into the antifungal mechanisms of fisetin, positioning it as a promising therapeutic agent against Candida infections.

The Role of Potassium Channels in the Regulation of the Transport Function of Rats Lymph Nodes during Inflammation

Lymph formed in tissues necessarily passes through lymph nodes (LN), which not only perform an immune function, but also take part in lymph flow through rhythmic high-amplitude contractions. During inflammation, inducible NO synthase (iNOS) is expressed in the lymph nodes, which promotes relaxation of the LN capsule. This study examined the role of KATP- and BKCa-channels in sepsis-induced LN remodeling. Sepsis was induced in rats by cecal ligation-puncture surgery. After 12 and 24 h, mesenteric LN were removed and examined in a myograph. KATP-channels were activated by pinacidil and blocked by glibenclamide. BKCa-channels blocked TEA and activated NS 1619. The strength of tonic contraction of the LN under the action of activators and blockers was assessed. LN of septic rats named low level of tone during standard stretching. Pinacidil led to greater relaxation of LN in septic rats compared to the control group; the effect of glibenclamide was accompanied by an increase in tone. Pinacidil combined with glibenclamide did not lead to significant changes in LN tone. The use of NS 1619 was accompanied by relaxation of the LN; in the LN of septic rats, the effect was more pronounced. TEA (3 mM) led to an increase in LN tone; the LN of septic rats responded to the use of TEA with a greater contraction. We concluded that NO produced by expressed iNOS in animals with sepsis directly or indirectly activates KATP- and BKCa-channels of smooth muscle cells of the capsule in the LN, which leading to hyperpolarization of the smooth muscle cell membrane and their relaxation, which that promotes relaxation of the LN capsule and their hypertrophy of LN. In the future, KATP- and BKCa-channels of smooth muscle cells of the lymph node LN capsule may be a potential target for therapeutic intervention to correct the immune response by slowing down or accelerating the flow of lymph through the LN.

Gold Nanoparticles Capped with a Novel Titanium(IV)-Containing Polyoxomolybdate Cluster: Selective and Enhanced Bactericidal Effect Against Escherichia coli.

Bacterial infections are a public health threat of increasing concern in medical care systems; hence, the search for novel strategies to lower the use of antibiotics and their harmful effects becomes imperative. Herein, the antimicrobial performance of four polyoxometalate (POM)-stabilized gold nanoparticles (Au@POM) against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as Gram-negative and Gram-positive bacteria models, respectively, is studied. The bactericidal studies performed, both in planktonic and sessile forms, evidence the antimicrobial potential of these hybrid nanostructures with selectivity toward Gram-negative species. In particular, the Au@GeMoTi composite with the novel [Ti2 (HGeMo7 O28 )2 ]10- POM capping ligand exhibits outstanding bactericidal efficiency with a minimum inhibitory concentration of just 3.12µm for the E. coli strain, thus outperforming the other three Au@POM counterparts. GeMoTi represents the fourth example of a water-soluble TiIV -containing polyoxomolybdate, and among them, the first sandwich-type structure having heteroatoms in high-oxidation state. The evaluation of the bactericidal mechanisms of action points to the cell membrane hyperpolarization, disruption, and subsequent nucleotide leakage and the low cytotoxicity exerted on five different cell lines at antimicrobial doses demonstrates the antibiotic-like character. These studies highlight the successful design and development of a new POM-based nanomaterial able to eradicate Gram-negative bacteria without damaging mammalian cells.

The osmotic stress of Vallisneria natans (Lour.) Hara leaves originating from the disruption of calcium and potassium homeostasis caused by MC-LR

Aquatic plants are potentially impacted by microcystins (MCs) in lakes experiencing harmful algal blooms. However, how these plants respond, and possibly adapt to osmotic stress caused by MCs is unclear. Vallisneria natans is a pioneer taxon with a global distribution in eutrophic lakes. In this study, we investigated the effect of MC-LR on morphological structure, water retention, osmoregulatory ability, and homeostasis of calcium (Ca2+) and potassium (K+) ions in V. natans leaves. Results showed that the morphological changes caused by MC-LR included increased volumes of epidermal and mesophyll cells, changes in their lignification level, and the degradation of chloroplast structure and dissolution of starch granules. The increased moisture content and water potential with MC-LR concentration were consistent with the occurrence of osmotic stress, and the decreased osmotic potential implied the activation of osmoregulation. Soluble sugar and free amino acid concentrations increased at MC-LR treatments ≥10 μg/L, while inorganic ion K+ content increased in all MC-LR treatments. Although instantaneous K+inflow and Ca2+outflow occurred at 10 μg/L and 100 μg/L MC-LR, respectively, ≥1 μg/L MC-LR resulted in continuous K+ inflow and Ca2+ outflow within 24 h. Moreover, plasma membrane hyperpolarization was caused by MC-LR, especially at 1 and 10 μg/L. We suggest that Ca2+ efflux served as a signal molecule from the cytoplasmic matrix via Ca2+-ATPase, and the uptake of K+ was activated passively through transporters in response to MC-LR-induced plasma membrane hyperpolarization. Therefore, the uptake of K+ was a part of the response but not an adaptation to MC-LR stress, and is considered the cause for the uptake of water in leaves. Ca2+ and K+ homeostasis of V. natans leaves was disrupted by MC-LR concentrations as low as 1 μg/L, suggesting that aquatic plants in most eutrophic lakes may experience negative impacts such as Ca2+ loss, impacts to cell water balance, and alteration in cellular morphology, due to osmotic stress caused by MC-LR.

Synergistic effects of benzyl isothiocyanate and resveratrol against Listeria monocytogenes and their application in chicken meat preservation.

This study aimed to investigate the synergistic effects of benzyl isothiocyanate (BITC) and resveratrol (RS) on Listeria monocytogenes and their application in chicken meat preservation. BITC combined with RS (BR) significantly enhanced the antimicrobial activity and inhibited the growth of Listeria monocytogenes within 24h compared to individual treatment, as well as suppressing bacterial swimming and swarming motility, reducing biofilm formation by 56.4%, increasing cell membrane disruption, and inducing intracellular ROS surges. Synergistic effects were associated with the inhibition of biofilm formation, cell membrane destruction, and ROS production. Biofilm removal facilitated the direct antimicrobial action of BR. RS disrupted cell membrane permeability, allowing more BITC into the cells, resulting in increased intracellular antibacterial levels, cell membrane hyperpolarization, and rapid ROS accumulation. Furthermore, BR visibly slowed the microbial growth in chicken flesh stored at 25°C and 4°C. Therefore, BR is expected to be a new strategy for food preservation.

Plasma membrane–chloroplast interactions activated by the hyperpolarizing response in characean cells

Signaling pathways in plant cells often comprise electrical phenomena developing at the plasma membrane. The action potentials in excitable plants like characean algae have a marked influence on photosynthetic electron transport and CO2 assimilation. The internodal cells of Characeae can also generate active electrical signals of a different type. The so called hyperpolarizing response develops under the passage of electric current whose strength is comparable to physiological currents circulating between nonuniform cell regions. The plasma membrane hyperpolarization is involved in multiple physiological events in aquatic and terrestrial plants. The hyperpolarizing response may represent an unexplored tool for studying the plasma membrane–chloroplast interactions in vivo. This study shows that the hyperpolarizing response of Chara australis internodes whose plasmalemma was preliminary converted into the K+-conductive state induces transient changes in maximal (Fm′) and actual (F′) fluorescence yields of chloroplasts in vivo. These fluorescence transients were light dependent, suggesting their relation to photosynthetic electron and H+ transport. The cell hyperpolarization promoted H+ influx that was inactivated after a single electric stimulus. The results indicate that the plasma membrane hyperpolarization drives transmembrane ion fluxes and modifies the ionic composition of cytoplasm, which indirectly (via envelope transporters) affects the pH of chloroplast stroma and chlorophyll fluorescence. Remarkably, the functioning of envelope ion transporters can be revealed in short-term experiments in vivo, without growing plants on solutions with various mineral compositions.

Piezoelectric Bioactive Glasses Composite Promotes Angiogenesis by the Synergistic Effect of Wireless Electrical Stimulation and Active Ions.

Insufficient angiogenesis frequently occurs after the implantation of orthopedic materials, which greatly increases the risk of bone defect reconstruction failure. Therefore, the development of bone implant with improved angiogenic properties is of great importance. Mimicking the extracellular matrix clues provides a more direct and effective strategy to modulate angiogenesis. Herein, inspired by the bioelectrical characteristics of the bone microenvironment, a piezoelectric bioactive glasses composite (P-KNN/BG) based on the incorporation of polarized potassium sodium niobate is constructed, which could effectively promote angiogenesis. It is found that P-KNN/BG has exceptional wireless electrical stimulation performance and sustained active ions release. In vitro cell experiments reveal that P-KNN/BG enhances endothelial cell adhesion, migration, and differentiation via activating the eNOS/NO signaling pathway, which might be contributed to cell membrane hyperpolarization induced by wireless electrical stimulation increase the influx of active ions into the cells. In vivo chick chorioallantoic membrane experiment demonstrates that P-KNN/BG shows excellent pro-angiogenic capacity and biocompatibility. This work broadens the current understanding of bioactive materials with bionic electrical properties, which brings new insights into the clinical treatment of bone defect repair.

Houttuynia cordata Thunb. crude extract inactivates Cronobacter sakazakii: Antibacterial components, antibacterial mechanism, and application as a natural disinfectant

The purpose of this study was to reveal the antibacterial components of Houttuynia cordata Thunb. crude extract (HCCE), analyze the antibacterial activity and mechanism of HCCE against Cronobacter sakazakii, and evaluate the bactericidal effect of HCCE as a natural disinfectant. Using an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-QE-MS) non-target metabolomics technology, 15 kinds oforganic acids (18.65%), 14 kinds of phenols (16.17%), 10 kinds of flavonoids (12.32%), 5 kinds of terpenoids (5.87%), 3 kinds of alkaloids (1.62%), and 3 kinds of aldehydes (1.28%) were detected from HCCE. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of HCCE against C. sakazakii were 7.5 mg/mL and 15 mg/mL, respectively, and when the concentration was 2 MIC, C. sakazakii stagnated in sluggish phase. C. sakazakii exposed to HCCE showed hyperpolarization of cell membrane, increased intracellular ROS content, decreased protein and DNA content, impaired cell membrane integrity, improved cell membrane permeability, cell fluid leakage, and partial cell fragmentation. After treatments with 1 MIC and 2 MIC of HCCE for 5 min, C. sakazakii strains on 8 contact surfaces including stainless steel, tinplate, glass, polystyrene, bamboo, plank, ceramic, and tin foil paper were significantly inhibited (P < 0.05), and the reduction amounts ranged from 3.00 to 3.68 log CFU/100 cm2 and 3.97 to 4.53 log CFU/100 cm2, respectively. These findings demonstrate the potential of HCCE as a natural disinfectant and provide a new idea to reduce the C. sakazakii contamination caused by surface contact.

Genetically-encoded BRET probes enlight ligand bias-induced variable ion selectivity in TRPV1 and P2X5/7.

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels remains, however, challenging even with patch-clamp electrophysiology. In this study, we have developed highly-sensitive Bioluminescence Resonance Energy Transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of TRPV1 and P2X channels pore in real-time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the CAPS-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by Bz-ATP. These new custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

Abstract 11032: Peptide Lv Promotes the Trafficking and Membrane Insertion of Endothelial K Ca 31 Through the MEK1/ERK Pathway

Background: Peptide Lv is an endogenous secretory peptide that is upregulated in the eyes of patients with diabetic retinopathy, an ocular disease with pathological angiogenesis. Peptide Lv promotes vascular endothelial cell proliferation, migration, and sprouting, and blocking peptide Lv with a specific antibody, anti-Lv, dampens neovascularization, so peptide Lv is an angiogenic factor. However, the molecular mechanism of how peptide Lv promotes angiogenesis is not clear. We found that peptide Lv causes endothelial cell membrane hyperpolarization, a step that is essential in endothelium-dependent angiogenesis. Peptide Lv augments the protein expression and current densities of K Ca 3.1, an ion channel underlying the endothelial hyperpolarization. As ion channels require trafficking and insertion to the plasma membrane to be functional after they are expressed, we aimed to determine the downstream signaling of peptide Lv that is involved in the trafficking of K Ca 3.1 in endothelial cells. Hypothesis: Peptide Lv activates the MEK1/ERK signaling pathway to promote the trafficking and membrane insertion of K Ca 3.1 in endothelial cells. Blocking MEK1/ERK will prevent augmentation of K Ca 3.1 by peptide Lv. Methods: Cultured human umbilical vein endothelial cells (HUVECs) were treated with peptide Lv (500 ng/ml) or PBS (vehicle control) in the presence/absence of FR180402 (10 μM; an ERK inhibitor) or PD98059 (10 μM, a MEK1 inhibitor) followed by the patch-clamp electrophysiological recordings and biotinylation assays. Results: Blocking ERK activation attenuated the peptide Lv-meditated membrane hyperpolarization and increase of K Ca 3.1 currents in endothelial cells. Blocking ERK activation did not affect peptide Lv-elicited increases of K Ca 3.1 protein expression, but it reduced the level of membrane-bound K Ca 3.1 Conclusions: The MEK1/ERK signaling pathway mediates peptide Lv-elicited trafficking and membrane insertion of K Ca 3.1 but does not affect the increase of K Ca 3.1 expression by peptide Lv.

Membrane damage mechanism of protocatechualdehyde against Micrococcus luteus and its effect on pork quality characteristics

This study investigated the mechanism of membrane damage by protocatechualdehyde (PCA) against Micrococcus luteus and assessed effects of PCA on the sensory and physicochemical properties of pork. The mechanism of PCA inhibition on M. luteus was studied by determining the minimum inhibitory concentration (MIC) based on membrane potential, intracellular ATP concentration, intracellular pH, confocal laser scanning microscopy (CLSM), and field emission gun scanning electron microscopy (FEG-SEM). The results showed that the MIC of PCA against M. luteus was 1.25 mg/mL. Hyperpolarization of the bacterial cell membrane, a decrease in the intracellular ATP concentration, and intracellular pH indicated that PCA damaged the cell membrane of M. luteus. FEG-SEM observation revealed that PCA could cause surface collapse, cell membrane rupture, and content outflow of M. luteus. Additionally, PCA was found to inhibit increases in the total number of colonies, the thiobarbituric acid reactive substances (TBARS) value growth rate, and moisture mobility in raw pork. Additionally, it improved the color and texture of raw pork, all of which effectively prolonged its shelf life. This study will encourage the application of PCA as a natural antibacterial agent in the food industry.

Genetically-encoded BRET probes shed light on ligand bias–induced variable ion selectivity in TRPV1 and P2X5/7

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

Antibacterial Mechanism of Eugenol Against Shigella sonnei and Its Antibacterial Application in Lettuce Juice.

Shigella sonnei is a species of Shigella, and the infection rate of S. sonnei is increasing year by year. Eugenol is an active ingredient in clove essential oil and is a generally recognized as safe (GRAS)-certified food ingredient. The mechanism of inhibition of S. sonnei by eugenol has been investigated in this study. The minimum inhibitory concentration of eugenol against both S. sonnei ATCC 25931 and S. sonnei CMCC 51592 was 0.5 mg/mL and minimum bactericidal concentration (MBC) for both strains was 0.8 mg/mL. The inhibition effect of eugenol against S. sonnei was due to increased levels of reactive oxygen species in cells, changed cell membrane permeability, and induced cell membrane dysfunction, for instance, cell membrane hyperpolarization and intracellular ATP concentration drops. The results of confocal laser scanning microscope and field emission scanning electron microscopy showed that eugenol leads to decreased cell membrane integrity, resulting in changed cell morphology. Moreover, eugenol inactivated S. sonnei in Luria-Bertani (LB) broth and lettuce juice. These results indicated that eugenol could inactivate S. sonnei and has the potential to control S. sonnei in the food industry.

Evaluation of the antibacterial mechanism and biofilm removal effect of eugenol on Vibrio vulnificus and its application in fresh oysters

Eugenol is a natural active substance with high antibacterial activity, but its antibacterial activity against Vibrio vulnificus has not been extensively studied. The purpose of this study was to investigate the antibacterial and biofilm-clearing abilities and potential mechanisms of eugenol against V. vulnificus, and to provide knowledge for the use of eugenol to prevent oyster contamination. It was found that eugenol had an encouraging antibacterial effect on V. vulnificus with a minimum inhibitory concentration (MIC) of 0.2 mg/mL. The accumulation of reactive oxygen species (ROS) and the increase of malondialdehyde (MDA) content suggest that oxidative stress is involved in the bactericidal mechanism. Moreover, cell membrane hyperpolarization, changes in cell membrane integrity and morphology suggest that eugenol can reduce the permeability and integrity of cell membranes in V. vulnificus. In addition, eugenol produced a significant biofilm clearance effect on V. vulnificus, as evidenced by the reduced amount of biofilm and the reduction of polysaccharides and viable cells in the biofilm. Finally, eugenol was able to effectively inhibit the activity of V. vulnificus in artificially contaminated oyster at 4 °C and 25 °C. But sensory analysis showed that 0.10% eugenol was most acceptable to trained panelists. All of these highlight the great promise of eugenol as a natural bacteriostatic agent for the food industry.

Erg6p is essential for antifungal drug resistance, plasma membrane properties and cell wall integrity in Candida glabrata.

ERG6 gene encodes C-24 methyltransferase, one of the specific enzymes that differ in mammalian and yeast sterol biosynthesis. To explore the function of CgErg6p in the yeast pathogen Candida glabrata, we have constructed the Cgerg6Δ deletion mutant. We found that C. glabrata cells lacking CgErg6p exhibit reduced susceptibility to both antifungal azoles and polyenes. The reduced content of ergosterol in the Cgerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthetic pathway. The absence of CgErg6p leads to plasma membrane hyperpolarization and decrease in its fluidity compared to the parental C. glabrata strain. The absence of sterols containing C-24 alkyls influenced the susceptibility of Cgerg6Δ mutant cells to alkali metal cations and several other metabolic inhibitors. Our results thus show that sterols lacking C-24 alkyls are not sufficient substitutes for maintaining yeast plasma membrane function. The absence of CgErg6p influences also the cell wall integrity and calcineurin signaling in C. glabrata.

The Antimicrobial and Antibiofilm Activity of Oregano Essential Oil against Enterococcus faecalis and Its Application in Chicken Breast.

Oregano essential oil (OEO) possesses anti-inflammatory, antioxidant, and cancer-suppressive properties. Enterococcus faecalis is a foodborne opportunistic pathogen that can be found in nature and the food processing industry. The goal of this investigation was to explore the antimicrobial action and mechanism of OEO against E. faecalis, inactivation action of OEO on E. faecalis in mature biofilms, and its application in chicken breast. The minimum inhibitory concentration (MIC) of OEO against E. faecalis strains (ATCC 29212 and nine isolates) ranged from 0.25 to 0.50 μL/mL. OEO therapy reduced intracellular adenosine triphosphate (ATP) levels, caused cell membrane hyperpolarization, increased the intracellular reactive oxygen species (ROS), and elevated extracellular malondialdehyde (MDA) concentrations. Furthermore, OEO treatment diminished cell membrane integrity and caused morphological alterations in the cells. In biofilms on stainless-steel, OEO showed effective inactivation activity against E. faecalis. OEO reduced the number of viable cells, cell viability and exopolysaccharides in the biofilm, as well as destroying its structure. Application of OEO on chicken breast results in a considerable reduction in E. faecalis counts and pH values, in comparison to control samples. These findings suggest that OEO could be utilized as a natural antibacterial preservative and could effectively control E. faecalis in food manufacturing.

Deciphering the antibacterial activity and mechanism of p-coumaric acid against Alicyclobacillus acidoterrestris and its application in apple juice

Alicyclobacillus acidoterrestris has been regarded as the main hazardous factor causing the spoilage of commercially pasteurized fruit juice due to its unique thermo-acidophilic properties. p-Coumaric acid, primarily isolated from plants and having high biological activity, is supposed to be a viable food additive. Herein, the antibacterial activity and mechanism of p-coumaric acid against A. acidoterrestris and its application in apple juice were investigated. The results showed that p-coumaric acid had an active inhibition against A. acidoterrestris vegetative cells, and both minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were 0.2 mg/mL. MIC of p-coumaric acid against spores was also 0.2 mg/mL, while MBC was >1.6 mg/mL. Treatment of p-coumaric acid caused significant damage of cell integrity, decrease of intracellular ATP, hyperpolarization of cell membrane, degradation of whole cell protein, and malformation of cell morphology. Agarose gel retardation and fluorescence spectroscopy assays revealed that p-coumaric acid could interact with DNA and intercalate into the groove of DNA double helix to disturb normal cellular functions. Furthermore, the addition of p-coumaric acid at MIC caused 2.07 log drop in A. acidoterrestris vegetative cells and effectively inhibited the germination of spores in apple juice storage at 20 °C for 7 days. The low-temperature storage (4 °C) could inactivate A. acidoterrestris cells in apple juice, but the addition of p-coumaric acid further accelerated degradation of vegetative cells and the inactivation time was shortened from 5 days to 3 days compared with control. Importantly, the addition of p-coumaric acid could increase total phenolic content and ABTS radical scavenging activity and had no remarkable influence on pH, total soluble solids, color and sensory qualities of apple juice, regardless of storage temperature. These results revealed that p-coumaric acid could be expected to serve as an alternative or supplement agent for inhibiting the development of A. acidoterrestris in fruit juice.