Bridge-like lipid transfer protein 3A (BLTP3A) is associated with membranes of the late endocytic pathway and is an effector of CASM.

Recent studies have identified a family of rod-shaped proteins which includes VPS13 and ATG2 and are thought to mediate unidirectional lipid transport at intracellular membrane contacts by a bridge-like mechanism. Here, we show that one such protein, BLTP3A/UHRF1BP1, associates with VAMP7-positive vesicles via its C-terminal region and anchors them to lysosomes via the binding of its chorein domain containing N-terminal region to Rab7. Upon damage of lysosomal membranes and resulting mATG8 recruitment to their surface by CASM, BLTP3A first dissociates from lysosomes but then reassociates with them via an interaction of its LIR motif with mATG8. Such interaction is mutually exclusive to the binding of BLTP3A to vesicles and leaves its N-terminal chorein domain, i.e. the proposed entry site of lipids into this family of proteins, available for binding to another membrane, possibly the ER. Our findings reveal that BLTP3A is an effector CASM, potentially as part of a mechanism to help repair or minimize lysosome damage by delivering lipids.

Alkyl Pyridinol Compounds Exhibit Antimicrobial Effects against Gram-Positive Bacteria.

Background/Objectives. The rise of antibiotic-resistant pathogens represents a significant global challenge in infectious disease control, which is amplified by the decline in the discovery of novel antibiotics. Staphylococcus aureus continues to be a highly significant pathogen, causing infections in multiple organs and tissues in both healthcare institutions and community settings. The bacterium has become increasingly resistant to all available antibiotics. Consequently, there is an urgent need for novel small molecules that inhibit the growth or impair the survival of bacterial pathogens. Given their large structural and chemical diversity, as well as often unique mechanisms of action, natural products represent an excellent avenue for the discovery and development of novel antimicrobial treatments. Anaephene A and B are two such naturally occurring compounds with significant antimicrobial activity against Gram-positive bacteria. Here, we report the rapid syntheses and biological characterization of five novel anaephene derivatives, which display low cytotoxicity against mammalian cells but potent antibacterial activity against various S. aureus strains, including methicillin-resistant S. aureus (MRSA) and the multi-drug-resistant community-acquired strain USA300LAC. Methods. A Sonogashira cross-coupling reaction served as the key step for the synthesis of the alkyl pyridinol products. Results/Conclusions. Using the compound JC-01-074, which displays bactericidal activity already at low concentrations (MIC: 16 µg/mL), we provide evidence that alkyl pyridinols target actively growing and biofilm-forming cells and show that these compounds cause disruption and deformation of the staphylococcal membrane, indicating a membrane-associated mechanism of action.

Evaluation of the Antibacterial Potential of Two Short Linear Peptides YI12 and FK13 against Multidrug-Resistant Bacteria.

The accelerating spread of antibiotic resistance has significantly weakened the clinical efficacy of existing antibiotics, posing a severe threat to public health. There is an urgent need to develop novel antimicrobial alternatives that can bypass the mechanisms of antibiotic resistance and effectively kill multidrug-resistant (MDR) pathogens. Antimicrobial peptides (AMPs) are one of the most promising candidates to treat MDR pathogenic infections since they display broad-spectrum antimicrobial activities and are less prone to achieve drug resistance. In this study, we investigated the antibacterial capability and mechanisms of two machine learning-driven linear peptide compounds termed YI12 and FK13. We reveal that YI12 and FK13 exhibit broad-spectrum antibacterial properties against clinically significant bacterial pathogens, inducing no or minimal hemolysis in mammalian red blood cells. We further ascertain that YI12 and FK13 are resilient to heat and acid-base conditions, and exhibit susceptibility to hydrolytic enzymes and divalent cations under physiological conditions. Initial mechanistic investigations reveal that YI12 and FK13 compromise bacterial membrane integrity, leading to membrane potential dissipation and excessive reactive oxygen species (ROS) generation. Collectively, our findings highlight the prospective utility of these two cationic amphiphilic peptides as broad-spectrum antibacterial agents.

Biofilm Disruption from within: Light-Activated Molecular Drill-Functionalized Polymersomes Bridge the Gap between Membrane Damage and Quorum Sensing-Mediated Cell Death.

Bacterial biofilms represent an escalating global health concern with the proliferation of drug resistance and hospital-acquired infections annually. Numerous strategies are under exploration to combat biofilms and preempt the development of antibacterial resistance. Among these, mechanical disruption of biofilms and enclosed bacteria presents a promising avenue, aiming to induce membrane permeabilization and consequent lethal damage. Herein, we introduce a hemithioindigo (HTI) motor activated by visible light, capable of disrupting sessile bacteria when integrated into a polymeric vesicle carrier. Under visible light, bacteria exhibited a notable outer membrane permeability, reduced membrane fluidity, and diminished viability following mechanical drilling. Moreover, various genetic responses pertaining to the cell envelope were examined via qRT-PCR, alongside the activation of a self-lysis mechanism associated with phage stress, which was coupled with increases in quorum sensing, demonstrating a potential self-lysis cascade from within. The multifaceted mechanisms of action, coupled with the energy efficiency of mechanical damage, underscore the potential of this system in addressing the challenges posed by pathogenic biofilms.

Relationship between oligoarginine-induced membrane damage of single Escherichia coli cells and entry of the peptide into the cytoplasm.

Cell-penetrating peptides (CPPs) can enter the cytosol of eukaryotic cells without killing them whereas some CPPs exhibit antimicrobial activity against bacterial cells. Here, to elucidate the mode of interaction of the CPP nona-arginine (R9) with bacterial cells, we investigated the interactions of lissamine rhodamine B red-labeled peptide (Rh-R9) with single Escherichia coli cells encapsulating calcein using confocal laser scanning microscopy. After Rh-R9 induced the leakage of a large amount of calcein, the fluorescence intensity of the cytosol due to Rh-R9 greatly increased, indicating that Rh-R9 induces cell membrane damage, thus allowing entry of a significant amount of Rh-R9 into the cytosol. To determine if the lipid bilayer region of the membrane is the main target of Rh-R9, we then investigated the interaction of Rh-R9 with single giant unilamellar vesicles (GUVs) comprising an E. coli polar lipid extract containing small GUVs and AlexaFluor 647 hydrazide (AF647) in the lumen. Rh-R9 entered the GUV lumen without inducing AF647 leakage, but leakage eventually did occur, indicating that GUV membrane damage was induced after the entry of Rh-R9 into the GUV lumen. The Rh-R9 peptide concentration dependence of the fraction of entry of Rh-R9 after a specific interaction time was similar to that of the fraction of leaking GUVs. These results indicate that Rh-R9 can damage the lipid bilayer region of a cell membrane, which may be related to its antimicrobial activity.

Guidance on the assessment of biocompatibility of biomaterials: Fundamentals and testing considerations.

BACKGROUND: Assessing the biocompatibility of materials is crucial for ensuring the safety and well-being of patients by preventing undesirable, toxic, immune, or allergic reactions, and ensuring that materials remain functional over time without triggering adverse reactions. To ensure a comprehensive assessment, planning tests that carefully consider the intended application and potential exposure scenarios for selecting relevant assays, cell types, and testing parameters is essential. Moreover, characterizing the composition and properties of biomaterials allows for a more accurate understanding of test outcomes and the identification of factors contributing to cytotoxicity. Precise reporting of methodology and results facilitates research reproducibility and understanding of the findings by the scientific community, regulatory agencies, healthcare providers, and the general public. AIMS: This article aims to provide an overview of the key concepts associated with evaluating the biocompatibility of biomaterials while also offering practical guidance on cellular principles, testing methodologies, and biological assays that can support in the planning, execution, and reporting of biocompatibility testing.

Molecular insights reveal how the glycolipids in cell membrane mitigates nanomaterial's invasion.

Nanomaterial-cellular membrane interaction is crucial for the cytotoxicity of such materials in theoretical investigations. However, previous research often used cellular membrane models with one or few lipid types, which deviates significantly from realistic membrane compositions. Here, employing molecular dynamics (MD) simulations, we investigate the impact of a typical nanomaterial, boron nitride (BN), on a cellular membrane model based on the realistic small intestinal epithelial cell (SIEC) membrane. This membrane contains a complex composition, including abundant glycolipids. Our MD simulations reveal that BN nanosheet can partially insert into the SIEC membrane, maintaining a stable binding conformation without causing obvious structural changes. Dynamic analyses suggest that van der Waals (vdW) interactions drive the binding process between BN and the SIEC membrane. Further simulation of the interaction between BN nanosheet and deglycosylated SIEC membrane confirms that BN nanosheet cause significant structural damage to deglycosylated SIEC membranes, completely inserting into the membrane, extracting lipids, and burying some lipid hydrophilic heads within the membrane interior. Quantitative analyses of mean squared displacements (MSD) of membranes, membrane thicknesses, area per lipid, and order parameters indicate that BN nanosheet causes more substantial damage to deglycosylated SIEC membrane than to intact SIEC membrane. This comparison suggests the molecular mechanism involved in mitigating BN invasion by SIEC membrane that the polysaccharide heads of glycolipids in the SIEC membrane form a significant steric hindrance on membrane surface, not only hindering the insertion of BN, but also resisting the lipid extraction by BN. Free energy calculations further support this conclusion. Overall, our MD simulations not only shed new light into the reduced impact of BN nanosheet on the realistic SIEC membrane but also highlight the importance of glycolipids in protecting cell membranes from nanomaterial invasion, contributing to a deeper understanding of nanomaterial-realistic cell membrane interactions.

The impact of heat treatment on E. coli cell physiology in rich and minimal media considering oxidative secondary stress.

AIMS: This study investigates the cell physiology of thermally injured bacterial cells, with a specific focus on oxidative stress and the repair mechanisms associated with oxidative secondary stress. METHODS AND RESULTS: We explored the effect of heat treatment on the activity of two protective enzymes, levels of intracellular reactive oxygen species, and redox potential. The findings reveal that enzyme activity slightly increased after heat treatment, gradually returning to baseline levels during subculture. The response of Escherichia coli cells to heat treatment, as assessed by the level of superoxide radicals generated and redox potential, varied based on growth conditions, namely minimal and rich media. Notably, the viability of injured cells improved when antioxidants were added to agar media, even in the presence of metabolic inhibitors. CONCLUSIONS: These results suggest a complex system involved in repairing damage in heat-treated cells, particularly in rich media. While repairing membrane damage is crucial for cell regrowth and the electron transport system plays a critical role in the recovery process of injured cells under both tested conditions.

Inter-subspecies diversity of maize to drought stress with physio-biochemical, enzymatic and molecular responses.

Background: Drought is the most significant factor limiting maize production, given that maize is a crop with a high water demand. Therefore, studies investigating the mechanisms underlying the drought tolerance of maize are of great importance. There are no studies comparing drought tolerance among economically important subspecies of maize. This study aimed to reveal the differences between the physio-biochemical, enzymatic, and molecular mechanisms of drought tolerance in dent (Zea mays indentata), popcorn (Zea mays everta), and sugar (Zea mays saccharata) maize under control (no-stress), moderate, and severe drought stress. Methods: Three distinct irrigation regimes were employed to assess the impact of varying levels of drought stress on maize plants at the V14 growth stage. These included normal irrigation (80% field capacity), moderate drought (50% field capacity), and severe drought (30% field capacity). All plants were grown under controlled conditions. The following parameters were analyzed: leaf relative water content (RWC), loss of turgidity (LOT), proline (PRO) and soluble protein (SPR) contents, membrane durability index (MDI), malondialdehyde (MDA), and hydrogen peroxide (H2O2) content, the antioxidant enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT). Additionally, the expression of heat shock proteins (HSPs) was examined at the transcriptional and translational levels. Results: The effects of severe drought were more pronounced in sugar maize, which had a relatively high loss of RWC and turgor, membrane damage, enzyme activities, and HSP90 gene expression. Dent maize, which is capable of maintaining its RWC and turgor in both moderate and severe droughts, and employs its defense mechanism effectively by maintaining antioxidant enzyme activities at a certain level despite less MDA and H2O2 accumulation, exhibited relatively high drought tolerance. Despite the high levels of MDA and H2O2 in popcorn maize, the up-regulation of antioxidant enzyme activities and HSP70 gene and protein expression indicated that the drought coping mechanism is activated. In particular, the positive correlation of HSP70 with PRO and HSP90 with enzyme activities is a significant result for studies examining the relationships between HSPs and other stress response systems. The discrepancies between the transcriptional and translational findings provide an opportunity for more comprehensive investigations into the role of HSPs in stress conditions.

Membrane Damage and Metabolic Disruption as the Mechanisms of Linalool against Pseudomonas fragi: An Amino Acid Metabolomics Study.

Pseudomonas fragi (P. fragi) is usually detected in low-temperature meat products, and seriously threatens food safety and human health. Therefore, the study investigated the antibacterial mechanism of linalool against P. fragi from membrane damage and metabolic disruption. Results from field-emission transmission electron microscopy (FETEM) and atomic force microscopy (AFM) showed that linalool damage membrane integrity increases surface shrinkage and roughness. According to Fourier transform infrared (FTIR) spectra results, the components in the membrane underwent significant changes, including nucleic acid leakage, carbohydrate production, protein denaturation and modification, and fatty acid content reduction. The data obtained from amino acid metabolomics indicated that linalool caused excessive synthesis and metabolism of specific amino acids, particularly tryptophan metabolism and arginine biosynthesis. The reduced activities of glucose 6-phosphate dehydrogenase (G6PDH), malate dehydrogenase (MDH), and phosphofructokinase (PFK) suggested that linalool impair the respiratory chain and energy metabolism. Meanwhile, genes encoding the above enzymes were differentially expressed, with pfkB overexpression and zwf and mqo downregulation. Furthermore, molecular docking revealed that linalool can interact with the amino acid residues of G6DPH, MDH and PFK through hydrogen bonds. Therefore, it is hypothesized that the mechanism of linalool against P. fragi may involve cell membrane damage (structure and morphology), disturbance of energy metabolism (TCA cycle, EMP and HMP pathway) and amino acid metabolism (cysteine, glutamic acid and citrulline). These findings contribute to the development of linalool as a promising antibacterial agent in response to the food security challenge.

Pore Formation by Amyloid-like Peptides: Effects of the Nonpolar-Polar Sequence Pattern.

One of the mechanisms accounting for the toxicity of amyloid peptides in diseases like Alzheimer's and Parkinson's is the formation of pores on the plasma membrane of neurons. Here, we perform unbiased all-atom simulations of the full membrane damaging pathway, which includes adsorption, aggregation, and perforation of the lipid bilayer accounting for pore-like structures. Simulations are performed using four peptides made with the same amino acids. Differences in the nonpolar-polar sequence pattern of these peptides prompt them to adsorb into the membrane with the extended conformations oriented either parallel [peptide labeled F1, Ac-(FKFE)2-NH2], perpendicular (F4, Ac-FFFFKKEE-NH2), or with an intermediate orientation (F2, Ac-FFKKFFEE-NH2, and F3, Ac-FFFKFEKE-NH2) in regard to the membrane surface. At the water-lipid interface, only F1 fully self-assembles into ß-sheets, and F2 peptides partially fold into an α-helical structure. The ß-sheets of F1 emerge as electrostatic interactions attract neighboring peptides to intermediate distances where nonpolar side chains can interact within the dry core of the bilayer. This complex interplay between electrostatic and nonpolar interactions is not observed for the other peptides. Although ß-sheets of F1 peptides are mostly parallel to the membrane, some of their edges penetrate deep inside the bilayer, dragging water molecules with them. This precedes pore formation, which starts with the flow of two water layers through the membrane that expand into a stable cylindrical pore delimited by polar faces of ß-sheets spanning both leaflets of the bilayer.

Release of ions enhanced the antibacterial performance of laser-generated, uncoated Ag nanoparticles.

Identifying the antibacterial mechanisms of elemental silver at the nanoscale remains a significant challenge due to the intertwining behaviors between the particles and their released ions. The open question is which of the above factor dominate the antibacterial behaviors when silver nanoparticles (Ag NPs) with different sizes. Considering the high reactivity of Ag NPs, prior research has primarily concentrated on coated particles, which inevitably hinder the release of Ag+ ions due to additional chemical agents. In this study, we synthesized various Ag NPs, both coated and uncoated, using the laser ablation in liquids (LAL) technique. By analyzing both the changes in particle size and Ag+ ions release, the impacts of various Ag NPs on the cellular activity and morphological changes of gram-negative (E. coil) and gram-positive (S. aureus) bacteria were evaluated. Our findings revealed that for uncoated Ag NPs, smaller particles exhibited greater ions release efficiency and enhanced antibacterial efficacy. Specifically, particles approximately 1.5 nm in size released up to 55 % of their Ag+ ions within 4 h, significantly inhibiting bacterial growth. Additionally, larger particles tended to aggregate on the bacterial cell membrane surface, whereas smaller particles were more likely to be internalized by the bacteria. Notably, treatment with smaller Ag NPs led to more pronounced bacterial morphological changes and elevated levels of intracellular reactive oxygen species (ROS). We proposed that the bactericidal activity of Ag NPs stems from the synergistic effect between particle-cell interaction and the ionic silver, which is dependent on the crucial parameter of particle size.

Biochemical mechanism of chlorine dioxide fumigation in inhibiting Ceratocystis fimbriata and black rot in postharvest sweetpotato.

The inhibitory properties and underlying mechanism of chlorine dioxide (ClO2) fumigation on the pathogen Ceratocystis fimbriata (C. fimbriata) and resultant sweetpotato black rot were investigated in vitro and in vivo. Results revealed that the ClO2 fumigation effectively inhibited fungal growth and induced obvious morphological variation of C. fimbriata mycelia. Furthermore, the mycelial membrane suffered damage, as evidenced by a significant increase in malondialdehyde content and the leakage of protein and nucleic acid from mycelia cells, accompanied by a marked decrease in ergosterol content. Additionally, ClO2 fumigation caused spores cell membrane damage, a notable decrease in spore viability, and induced cell apoptosis as indicated by reductions in spore germination rate, two fluorescence staining observations, and flow cytometry analysis. Moreover, the decay diameter of sweetpotato black rot lesions decreased significantly after ClO2 fumigation, and the growth of C. fimbriata was also inhibited. These findings present a novel and effective technology for inhibiting the progression of sweetpotato black rot.

Biogenic synthesis of selenium nanoparticles from Nyctanthes arbor-tristis L. and evaluation of their antimicrobial, antioxidant and photocatalytic efficacy.

Biogenic synthesis of nanoparticles has been established as an environmentally benign and sustainable approach. This study emphasizes biosynthesis of selenium nanoparticles (SeNPs) utilizing leaf extract of Nyctanthes arbor-tritis L., well known for its abundant bioactive compounds. Various analytical techniques were employed for characterization of synthesized SeNPs. X-ray diffraction (XRD) spectroscopy confirmed the crystalline structure and revealed the average crystalline size of SeNPs to be 44.57 nm. Additionally, UV-Vis spectroscopy confirmed successful synthesis of SeNPs by validating the surface plasmon resonance (SPR) properties of SeNPs. FTIR analysis data revealed different bonds and their corresponding functional groups responsible for the synthesis and stability of synthesized SeNPs. DLS and zeta analysis revealed that 116.5 nm sized SeNPs were stable in nature. Furthermore, field emission scanning electron microscopy (FE-SEM) validated the spherical morphology of SeNPs with a size range of 60-80 nm. Inductively coupled plasma-optical emission spectroscopy (ICP-OES) determined the concentration of SeNPs in the obtained colloidal solution. Antioxidant activity of synthesized SeNPs was evaluated employing DPPH and H2O2 assay, revealed that the synthesized SeNPs were effective antioxidant agent. Additionally, antimicrobial potential was evaluated against a panel of Gram-positive and Gram-negative bacteria and found to be effective at higher concentration of SeNPs. SeNPs also exhibited strong anti-biofilm activity while evaluated against various biofilm producing bacteria like Escherichia coli , Staphylococcus epidermidis and Klebsiella pneumonia. The cytotoxicity of the bio-synthesized SeNPs was evaluated against HEK 293 cell line, exhibited minimal toxicity even at concentration 100 µg/mL with 65% viable cells. SeNPs has also been evaluated for dye degradation which has indicated excellent photocatalytic activity of synthesized SeNPs. The experimental data obtained altogether demonstrated that synthesized SeNPs exhibited significant antimicrobial and anti-biofilm activity against various pathogens, and also showed significant antioxidant and photocatalytic efficiency.

Far-Ultraviolet Light at 222 nm Affects Membrane Integrity in Monolayered DLD1 Colon Cancer Cells.

222 nm far-ultraviolet (F-UV) light has a bactericidal effect similar to deep-ultraviolet (D-UV) light of about a 260 nm wavelength. The cytotoxic effect of 222 nm F-UV has not been fully investigated. DLD-1 cells were cultured in a monolayer and irradiated with 222 nm F-UV or 254 nm D-UV. The cytotoxicity of the two different wavelengths of UV light was compared. Changes in cell morphology after F-UV irradiation were observed by time-lapse imaging. Differences in the staining images of DNA-binding agents Syto9 and propidium iodide (PI) and the amount of cyclobutane pyrimidine dimer (CPD) were examined after UV irradiation. F-UV was cytotoxic to the monolayer culture of DLD-1 cells in a radiant energy-dependent manner. When radiant energy was set to 30 mJ/cm2, F-UV and D-UV showed comparable cytotoxicity. DLD-1 cells began to expand immediately after 222 nm F-UV light irradiation, and many cells incorporated PI; in contrast, PI uptake was at a low level after D-UV irradiation. The amount of CPD, an indicator of DNA damage, was higher in cells irradiated with D-UV than in cells irradiated with F-UV. This study proved that D-UV induced apoptosis from DNA damage, whereas F-UV affected membrane integrity in monolayer cells.

Antibacterial mechanism of the methanol extract of Thamnolia subuliformis (Ehrh.) W. Culb against Staphylococcus aureus.

Thamnolia subuliformis (Ehrh.) W. Culb is a species of lichen with edible and medicinal applications in China. Our previous studies demonstrated that the methanol extract of Thamnolia subuliformis (METS) exhibits broad antibacterial activity and stability against foodborne pathogens. This study aimed to investigate the antibacterial mechanism of METS against Staphylococcus aureus using nontargeted metabolomics, focusing on cell wall and membrane damage. The results revealed that the minimum inhibitory concentration (MIC) was 0.625 mg ml-1 and that METS had good biosafety at this concentration. METS caused significant damage to the cell wall and membrane integrity, based on both morphological observation by electron microscopy and the leakage of alkaline phosphatase, protein, and nucleic acid in the cell cultures. Treatment with METS at the MIC disrupted the lipid metabolism of S. aureus, causing a decrease in the metabolism of various phospholipids and sphingolipids in the cell membrane and an increase in the ratio of saturated fatty acids to unsaturated fatty acids. Moreover, it influenced intracellular amino acid and energy metabolism. These results shed light on the antibacterial mechanism of METS against S. aureus while also serving as a reference for the further development of natural antibacterial compounds derived from Thamnolia subuliformis.

Autologous serum protein stabilized silver quantum clusters as host-specific antibacterial agents.

Aim: To synthesize host-specific serum protein stabilized silver quantum clusters and assess their preclinical safety as potential antibacterial agents.Materials & methods: Ag-QC-NanoSera (Ag-QCNS) were synthesized using bovine, human and murine sera. Antibacterial efficacy was evaluated against E. coli (including antibiotic-resistant strain), S. aureus and P. aeruginosa. Biocompatibility, hemocompatibility and antibacterial mechanism were also investigated. Preclinical safety and biodistribution of autologous Ag-QCNS were assessed in BALB/c mice over 28 days.Results: Ag-QCNS showed high biocompatibility, hemocompatibility and high antibacterial activity at ∼12.72 µg/ml Ag equivalent. Intracellular ROS and bacterial membrane damage were confirmed as antibacterial mechanism. Ag-QCNS were established as preclinically safe.Conclusion: Ag-QCNS demonstrate potential as next-generation host-specific nanotheranostic antibacterial agents, enhancing the safety and efficacy while combating antibiotic resistance.

[Box: see text].

Excitation of Reactive Oxygen Species and Damage to the Cell Membrane, Protein, and DNA are Important Inhibition Mechanisms of CO2 on Shewanella putrefaciens at 4 °C.

To explore whether oxidative stress caused by 100% CO2 is an inhibitory mechanism against Shewanella putrefaciens, the oxidative stress reaction, antioxidant activity, and damage to the cell membrane, protein, and DNA of CO2-incubated S. putrefaciens at 4 °C were evaluated. Research demonstrated that CO2 caused more severe reactive oxygen species (ROS) accumulation. Simultaneously, weaker •OH/H2O2/O2•--scavenging activity and decreased T-VOC and GSH content were also observed. The activities of antioxidant enzymes (SOD, POD, CAT, and GPX) continuously declined, which might be attributed to the CO2-mediated decrease in the pH value. Correspondingly, the cell membrane was damaged with hyperpolarization, increased permeability, and more severe lipid peroxidation. The expression of total and membrane protein decreased, and the synthesis and activity of extracellular protease were inhibited. DNA was also subjected to oxidative damage and expressed at a lower level. All results collaboratively confirmed that ROS excitation and inhibition of antioxidant activity were important inhibition mechanisms of CO2 on S. putrefaciens.

Shoot hydraulic impairments induced by root waterlogging: parallels and contrasts with drought.

Soil waterlogging and drought correspond to contrasting water extremes resulting in plant dehydration. Dehydration in response to waterlogging occurs due to impairments to root water transport, but no previous study has addressed whether limitations to water transport occur beyond this organ or whether dehydration alone can explain shoot impairments. Using common bean (Phaseolus vulgaris) as a model species, we report that waterlogging also impairs water transport in leaves and stems. During the very first hours of waterlogging, leaves transiently dehydrated to water potentials close to the turgor loss point, possibly driving rapid stomatal closure and partially explaining the decline in leaf hydraulic conductance. The initial decline in leaf hydraulic conductance (occurring within 24 h), however, surpassed the levels predicted to occur based solely on dehydration. Constraints to leaf water transport resulted in a hydraulic disconnection between leaves and stems, furthering leaf dehydration during waterlogging and after soil drainage. As leaves dehydrated later during waterlogging, leaf embolism initiated and extensive embolism levels amplified leaf damage. The hydraulic disconnection between leaves and stems prevented stem water potentials from declining below the threshold for critical embolism levels in response to waterlogging. This allowed plants to survive waterlogging and soil drainage. In summary, leaf and stem dehydration are central in defining plant impairments in response to waterlogging, thus creating similarities between waterlogging and drought. Yet, our findings point to the existence of additional players (likely chemicals) partially controlling the early declines in leaf hydraulic conductance and contributing to leaf damage during waterlogging.

Acorus calamus L. rhizome extract and its bioactive fraction exhibits antibacterial effect by modulating membrane permeability and fatty acid composition.

ETHNOPHARMACOLOGICAL RELEVANCE: India's ancient texts, the Charak Samhita and Sushruta Samhita, make reference to the traditional medicinal usage of Acorus calamus L. In India and China, it has long been used to cure stomach aches, cuts, diarrhea, and skin conditions. This ability of the rhizome is attributed to its antimicrobial properties. Research studies to date have shown its antimicrobial properties. However, scientific evidence on its mode of action is still lacking. AIM OF THE STUDY: Acorus calamus L. rhizome extract and its bioactive fraction exhibits antibacterial effect by modulating membrane permeability and fatty acid composition. MATERIAL AND METHOD: The secondary metabolites in the rhizome of A. calamus L. were extracted in hexane using Soxhlet apparatus. The ability of the extract to inhibit multidrug resistant bacterial isolates, namely Bacillus cereus, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa were evaluated using checkerboard assay. Further, the extract was purified using thin layer chromatography, gravity column chromatography, and combiflash chromatography. Structure elucidation of the active compound was done using GC-MS, FT-IR, and UV-Vis spectral scan. The mode of action of the bioactive fraction was determined. Bacterial membrane damage was analyzed using SEM, membrane permeability was determined using SYBR green I and PI dye, leakage of cytoplasmic contents were analyzed using Bradford assay and Fehling's reagent. The ability to inhibit efflux pump of A. baumannii was determined using EtBr accumulation assay and ß-lactamase inhibition was analyzed using nitrocefin as substrate. Also, the biofilm inhibition of B. cereus was determined using crystal violet dye. Moreover, the effect of the bioactive fraction on the fatty acid profile of the bacterial membrane was determined by GC-FAME analysis using 37 component FAME mix as standard. RESULTS: Acorus calamus L. rhizome hexane extract (AC-R-H) demonstrated broad-spectrum antibacterial activity against all the isolates tested. AC-R-H extract also significantly reduced the MIC of ampicillin against all tested bacteria, indicating its bacterial resistance modulating properties. The assay guided purification determined Asarone as the major compound present in the bioactive fraction (S-III-BAF). S-III-BAF was found to reduce the MIC of ampicillin against Escherichia coli (100-25 mg/mL), Pseudomonas aeruginosa (15-3.25 mg/mL), Acinetobacter baumannii (12.5-1.56 mg/ml), and Bacillus cereus (10-1.25 mg/mL). Further, it recorded synergistic activity with ampicillin against B. cereus (FICI = 0.365), P. aeruginosa (FICI = 0.456), and A. baumannii (FICI = 0.245). The mode of action of S-III-BAF can be attributed to its ability to disturb the membrane integrity, enhance membrane permeability, reduce biofilm formation, and possibly alter the fatty acid composition of the bacterial cell membranes. CONCLUSION: The bioactive fraction of AC-R-H extract containing Asarone as the active compound showed antibacterial activity and synergistic interactions with ampicillin against the tested bacterial isolates. Such activity can be attributed to the modulation of fatty acids present in bacterial membranes, which enhances membrane permeability and causes membrane damage.