Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa.

Volume regulation is essential for cell homeostasis and physiological function. Amongst the sensory molecules that have been associated with volume regulation is the transient receptor potential vanilloid 4 (TRPV4), which is a non-selective cation channel that in conjunction with aquaporins, typically controls regulatory volume decrease (RVD). Here we show that the interaction between orthologous AQP4 (Aqp4a) and TRPV4 (Trpv4) is important for regulatory volume increase (RVI) in post-activated marine fish spermatozoa under high osmotic stress. Based upon electrophysiological, volumetric, and in vivo and ex vivo functional experiments using the pharmacological and immunological inhibition of Aqp4a and Trpv4 our model suggests that upon ejaculation and exposure to the hypertonic seawater, spermatozoon shrinkage is initially mediated by water efflux through Aqp1aa in the flagellar tail. The shrinkage results in an increase in intracellular Ca2+ concentration, and the activation of sperm motility and a Na+/K+/2Cl- (NKCC1) cotransporter. The activity of NKCC1 is required for the initiation of cell swelling, which secondarily activates the Aqp4a-Trpv4 complex to facilitate the influx of water via Aqp4a-M43 and Ca2+ via Trpv4 and L-type channels for the mediation of RVI. The inhibitory experiments show that blocking of each of these events prevents either shrinkage or RVI. Our data thus reveal that post-activated marine fish spermatozoa are capable of initiating RVI under a high hypertonic stress, which is essential for the maintenance of sperm motility.

Natural variation in yeast reveals multiple paths for acquiring higher stress resistance.

BACKGROUND: Organisms frequently experience environmental stresses that occur in predictable patterns and combinations. For wild Saccharomyces cerevisiae yeast growing in natural environments, cells may experience high osmotic stress when they first enter broken fruit, followed by high ethanol levels during fermentation, and then finally high levels of oxidative stress resulting from respiration of ethanol. Yeast have adapted to these patterns by evolving sophisticated "cross protection" mechanisms, where mild 'primary' doses of one stress can enhance tolerance to severe doses of a different 'secondary' stress. For example, in many yeast strains, mild osmotic or mild ethanol stresses cross protect against severe oxidative stress, which likely reflects an anticipatory response important for high fitness in nature. RESULTS: During the course of genetic mapping studies aimed at understanding the mechanisms underlying natural variation in ethanol-induced cross protection against H2O2, we found that a key H2O2 scavenging enzyme, cytosolic catalase T (Ctt1p), was absolutely essential for cross protection in a wild oak strain. This suggested the absence of other compensatory mechanisms for acquiring H2O2 resistance in that strain background under those conditions. In this study, we found surprising heterogeneity across diverse yeast strains in whether CTT1 function was fully necessary for acquired H2O2 resistance. Some strains exhibited partial dispensability of CTT1 when ethanol and/or salt were used as mild stressors, suggesting that compensatory peroxidases may play a role in acquired stress resistance in certain genetic backgrounds. We leveraged global transcriptional responses to ethanol and salt stresses in strains with different levels of CTT1 dispensability, allowing us to identify possible regulators of these alternative peroxidases and acquired stress resistance in general. CONCLUSIONS: Ultimately, this study highlights how superficially similar traits can have different underlying molecular foundations and provides a framework for understanding the diversity and regulation of stress defense mechanisms.

The osmoregulated metabolism of trehalose contributes to production of type 1 fimbriae and bladder colonization by extraintestinal Escherichia coli strain BEN2908.

In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.

Multi-omics analysis of green lineage osmotic stress pathways unveils crucial roles of different cellular compartments.

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages. 76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants, and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.

Nanoscale intracellular ultrastructures affected by osmotic pressure using small-angle X-ray scattering.

Although intracellular ultrastructures have typically been studied using microscopic techniques, it is difficult to observe ultrastructures at the submicron scale of living cells due to spatial resolution (fluorescence microscopy) or high vacuum environment (electron microscopy). We investigate the nanometer scale intracellular ultrastructures of living CHO cells in various osmolality using small-angle X-ray scattering (SAXS), and especially the structures of ribosomes, DNA double helix, and plasma membranes in-cell environment are observed. Ribosomes expand and contract in response to osmotic pressure, and the inter-ribosomal correlation occurs under isotonic and hyperosmolality. The DNA double helix is not dependent on the osmotic pressure. Under high osmotic pressure, the plasma membrane folds into form a multilamellar structure with a periodic length of about 6 nm. We also study the ultrastructural changes caused by formaldehyde fixation, freezing and heating.

Hyperosmotic sisomicin infusion: a mouse model for hearing loss.

Losing either type of cochlear sensory hair cells leads to hearing impairment. Inner hair cells act as primary mechanoelectrical transducers, while outer hair cells enhance sound-induced vibrations within the organ of Corti. Established inner ear damage models, such as systemic administration of ototoxic aminoglycosides, yield inconsistent and variable hair cell death in mice. Overcoming this limitation, we developed a method involving surgical delivery of a hyperosmotic sisomicin solution into the posterior semicircular canal of adult mice. This procedure induced rapid and synchronous apoptotic demise of outer hair cells within 14 h, leading to irreversible hearing loss. The combination of sisomicin and hyperosmotic stress caused consistent and synergistic ototoxic damage. Inner hair cells remained until three days post-treatment, after which deterioration in structure and number was observed, culminating in a complete hair cell loss by day seven. This robust animal model provides a valuable tool for otoregenerative research, facilitating single-cell and omics-based studies toward exploring preclinical therapeutic strategies.

Dynamics of the osmotic lysis of mineral protocells and its avoidance at the origins of life.

The osmotic rupture of a cell, its osmotic lysis or cytolysis, is a phenomenon that active biological cell volume regulation mechanisms have evolved in the cell membrane to avoid. How then, at the origin of life, did the first protocells survive prior to such active processes? The pores of alkaline hydrothermal vents in the oceans form natural nanoreactors in which osmosis across a mineral membrane plays a fundamental role. Here, we discuss the dynamics of lysis and its avoidance in an abiotic system without any active mechanisms, reliant upon self-organized behaviour, similar to the first self-organized mineral membranes within which complex chemistry may have begun to evolve into metabolism. We show that such mineral nanoreactors could function as protocells without exploding because their self-organized dynamics have a large regime in parameter space where osmotic lysis does not occur and homeostasis is possible. The beginnings of Darwinian evolution in proto-biochemistry must have involved the survival of protocells that remained within such a safe regime.

The transcriptome of acute dehydration in myeloid leukemic cells.

Human myeloid leukemia cells (HL-60/S4) exposed to hyperosmotic stress with sucrose undergo dehydration and cell shrinkage. Interphase chromatin and mitotic chromosomes congeal, exhibiting altered phase separation (demixing) of chromatin proteins. To investigate changes in the transcriptome, we exposed HL-60/S4 cells to hyperosmotic sucrose stress (~600 milliOsmolar) for 30 and 60 minutes. We employed RNA-Seq of polyA mRNA to identify genes with increased or decreased transcript levels relative to untreated control cells (i.e., differential gene expression). These genes were examined for over-representation of Gene Ontology (GO) terms.  In stressed cells, multiple GO terms associated with transcription, translation, mitochondrial function and proteosome activity, as well as "replication-dependent histones", were over-represented among genes with increased transcript levels; whereas, genes with decreased transcript levels were over-represented with transcription repressors. The transcriptome profiles of hyperosmotically-stressed cells suggest acquisition of cellular rebuilding, a futile homeostatic response, as these cells are ultimately doomed to a dehydrated death.

Long Noncoding RNAs in Response to Hyperosmolarity Stress, but Not Salt Stress, Were Mainly Enriched in the Rice Roots.

Due to their immobility and possession of underground parts, plants have evolved various mechanisms to endure and adapt to abiotic stresses such as extreme temperatures, drought, and salinity. However, the contribution of long noncoding RNAs (lncRNAs) to different abiotic stresses and distinct rice seedling parts remains largely uncharacterized beyond the protein-coding gene (PCG) layer. Using transcriptomics and bioinformatics methods, we systematically identified lncRNAs and characterized their expression patterns in the roots and shoots of wild type (WT) and ososca1.1 (reduced hyperosmolality-induced [Ca2+]i increase in rice) seedlings under hyperosmolarity and salt stresses. Here, 2937 candidate lncRNAs were identified in rice seedlings, with intergenic lncRNAs representing the largest category. Although the detectable sequence conservation of lncRNAs was low, we observed that lncRNAs had more orthologs within the Oryza. By comparing WT and ososca1.1, the transcription level of OsOSCA1.1-related lncRNAs in roots was greatly enhanced in the face of hyperosmolality stress. Regarding regulation mode, the co-expression network revealed connections between trans-regulated lncRNAs and their target PCGs related to OsOSCA1.1 and its mediation of hyperosmolality stress sensing. Interestingly, compared to PCGs, the expression of lncRNAs in roots was more sensitive to hyperosmolarity stress than to salt stress. Furthermore, OsOSCA1.1-related hyperosmolarity stress-responsive lncRNAs were enriched in roots, and their potential cis-regulated genes were associated with transcriptional regulation and signaling transduction. Not to be ignored, we identified a motif-conserved and hyperosmolarity stress-activated lncRNA gene (OSlncRNA), speculating on its origin and evolutionary history in Oryza. In summary, we provide a global perspective and a lncRNA resource to understand hyperosmolality stress sensing in rice roots, which helps to decode the complex molecular networks involved in plant sensing and adaptation to stressful environments.

Material Composition Characteristics of Aspergillus cristatus under High Salt Stress through LC-MS Metabolomics.

Aspergillus cristatus is a crucial edible fungus used in tea fermentation. In the industrial fermentation process, the fungus experiences a low to high osmotic pressure environment. To explore the law of material metabolism changes during osmotic pressure changes, NaCl was used here to construct different osmotic pressure environments. Liquid chromatography-mass spectrometry (LC-MS) combined with multivariate analysis was performed to analyze the distribution and composition of A. cristatus under different salt concentrations. At the same time, the in vitro antioxidant activity was evaluated. The LC-MS metabolomics analysis revealed significant differences between three A. cristatus mycelium samples grown on media with and without NaCl concentrations of 8% and 18%. The contents of gibberellin A3, A124, and prostaglandin A2 related to mycelial growth and those of arabitol and fructose-1,6-diphosphate related to osmotic pressure regulation were significantly reduced at high NaCl concentrations. The biosynthesis of energy-related pantothenol and pantothenic acid and antagonism-related fluvastatin, aflatoxin, and alternariol significantly increased at high NaCl concentrations. Several antioxidant capacities of A. cristatus mycelia were directly related to osmotic pressure and exhibited a significant downward trend with an increase in environmental osmotic pressure. The aforementioned results indicate that A. cristatus adapts to changes in salt concentration by adjusting their metabolite synthesis. At the same time, a unique set of strategies was developed to cope with high salt stress, including growth restriction, osmotic pressure balance, oxidative stress response, antioxidant defense, and survival competition.

Analysis of Hyperosmotic Tolerance Mechanisms in Gracilariopsis lemaneiformis Based on Weighted Co-Expression Network Analysis.

We conducted transcriptome sequencing on salt-tolerant mutants X5 and X3, and a control (Ctr) strain of Gracilariopsis lemaneiformis after treatment with artificial seawater at varying salinities (30‱, 45‱, and 60‱) for 3 weeks. Differentially expressed genes were identified and a weighted co-expression network analysis was conducted. The blue, red, and tan modules were most closely associated with salinity, while the black, cyan, light cyan, and yellow modules showed a close correlation with strain attributes. KEGG enrichment of genes from the aforementioned modules revealed that the key enrichment pathways for salinity attributes included the proteasome and carbon fixation in photosynthesis, whereas the key pathways for strain attributes consisted of lipid metabolism, oxidative phosphorylation, soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) interactions in vesicular transport, and porphyrin and chlorophyll metabolism. Gene expression for the proteasome and carbon fixation in photosynthesis was higher in all strains at 60‱. In addition, gene expression in the proteasome pathway was higher in the X5-60 than Ctr-60 and X3-60. Based on the above data and relevant literature, we speculated that mutant X5 likely copes with high salt stress by upregulating genes related to lysosome and carbon fixation in photosynthesis. The proteasome may be reset to adjust the organism's proteome composition to adapt to high-salt environments, while carbon fixation may aid in maintaining material and energy metabolism for normal life activities by enhancing carbon dioxide uptake via photosynthesis. The differences between the X5-30 and Ctr-30 expression of genes involved in the synthesis of secondary metabolites, oxidative phosphorylation, and SNARE interactions in vesicular transport suggested that the X5-30 may differ from Ctr-30 in lipid metabolism, energy metabolism, and vesicular transport. Finally, among the key pathways with good correlation with salinity and strain traits, the key genes with significant correlation with salinity and strain traits were identified by correlation analysis.

The N-terminus of the Aspergillus fumigatus group III hybrid histidine kinase TcsC is essential for its physiological activity and targets the protein to the nucleus.

Group III hybrid histidine kinases are fungal-specific proteins and part of the multistep phosphorelay, representing the initial part of the high osmolarity glycerol (HOG) pathway. TcsC, the corresponding kinase in Aspergillus fumigatus, was expected to be a cytosolic protein but is targeted to the nucleus. Activation of TcsC by the antifungal fludioxonil has lethal consequences for the fungus. The agent triggers a fast and TcsC-dependent activation of SakA and later on a redistribution of TcsC to the cytoplasm. High osmolarity also activates TcsC, which then exits the nucleus or concentrates in spot-like, intra-nuclear structures. The sequence corresponding to the N-terminal 208 amino acids of TcsC lacks detectable domains. Its loss renders TcsC cytosolic and non-responsive to hyperosmotic stress, but it has no impact on the antifungal activity of fludioxonil. A point mutation in one of the three putative nuclear localization sequences, which are present in the N-terminus, prevents the nuclear localization of TcsC, but not its ability to respond to hyperosmotic stress. Hence, this striking intracellular localization is no prerequisite for the role of TcsC in the adaptive response to hyperosmotic stress, instead, TcsC proteins that are present in the nuclei seem to modulate the cell wall composition of hyphae, which takes place in the absence of stress. The results of the present study underline that the spatiotemporal dynamics of the individual components of the multistep phosphorelay is a crucial feature of this unique signaling hub. IMPORTANCE: Signaling pathways enable pathogens, such as Aspergillus fumigatus, to respond to a changing environment. The TcsC protein is the major sensor of the high osmolarity glycerol (HOG) pathway of A. fumigatus and it is also the target of certain antifungals. Insights in its function are therefore relevant for the pathogenicity and new therapeutic treatment options. TcsC was expected to be cytoplasmic, but we detected it in the nucleus and showed that it translocates to the cytoplasm upon activation. We have identified the motif that guides TcsC to the nucleus. An exchange of a single amino acid in this motif prevents a nuclear localization, but this nuclear targeting is no prerequisite for the TcsC-mediated stress response. Loss of the N-terminal 208 amino acids prevents the nuclear localization and renders TcsC unable to respond to hyperosmotic stress demonstrating that this part of the protein is of crucial importance.

Osmotic stress response of the coral and oyster pathogen Vibrio coralliilyticus: acquisition of catabolism gene clusters for the compatible solute and signaling molecule myo-inositol.

Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known as osmolytes. The osmotic stress response and compatible solutes used by the coral and oyster pathogen Vibrio coralliilyticus were unknown. In this study, we showed that to alleviate osmotic stress V. coralliilyticus biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not myo-inositol. Myo-inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified myo-inositol (iol) catabolism clusters in V. coralliilyticus and other Vibrio, Photobacterium, Grimontia, and Enterovibrio species. Growth pattern analysis demonstrated that V. coralliilyticus utilized myo-inositol as a sole carbon source, with a short lag time of 3 h. An iolG deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on myo-inositol. Within the iol clusters were an MFS-type (iolT1) and an ABC-type (iolXYZ) transporter and analyses showed that both transported myo-inositol. IolG and IolA phylogeny among Vibrionaceae species showed different evolutionary histories indicating multiple acquisition events. Outside of Vibrionaceae, IolG was most closely related to IolG from a small group of Aeromonas fish and human pathogens and Providencia species. However, IolG from hypervirulent A. hydrophila strains clustered with IolG from Enterobacter, and divergently from Pectobacterium, Brenneria, and Dickeya plant pathogens. The iol cluster was also present within Aliiroseovarius, Burkholderia, Endozoicomonas, Halomonas, Labrenzia, Marinomonas, Marinobacterium, Cobetia, Pantoea, and Pseudomonas, of which many species were associated with marine flora and fauna.IMPORTANCEHost associated bacteria such as Vibrio coralliilyticus encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that myo-inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of myo-inositol could contribute to a breakdown of the coral-algae symbiosis during thermal stress or disrupt the coral microbiome. Phylogenetic analyses showed that the evolutionary history of myo-inositol metabolism is complex, acquired multiple times in Vibrio, but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved iol cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.

Enhanced catabolism of glycine betaine and derivatives provides improved osmotic stress protection in Methylorubrum extorquens PA1.

Integration of metabolites into the overall metabolic network of a cell requires careful coordination dependent upon the ultimate usage of the metabolite. Different stoichiometric needs, and thus pathway fluxes, must exist for compounds destined for diverse uses, such as carbon sources, nitrogen sources, or stress-protective agents. Herein, we expand upon our previous work that highlighted the nature of glycine betaine (GB) metabolism in Methylobacteria to examine the utilization of GB-derivative compounds dimethylglycine (DMG) and sarcosine into Methylorubrum extorquens in different metabolic capacities, including as sole nitrogen and/or carbon sources. We isolated gain-of-function mutations that allowed M. extorquens PA1 to utilize dimethylglycine as a carbon source and dimethylglycine and sarcosine as nitrogen source. Characterization of mutants demonstrated selection for variants of the AraC-like regulator Mext_3735 that confer constitutive expression of the GB metabolic gene cluster, allowing direct utilization of the downstream GB derivatives. Finally, among the distinct isolates examined, we found that catabolism of the osmoprotectant used for selection (GB or dimethylglycine) enhanced osmotic stress resistance provided in the presence of that particular osmolyte. Thus, access to the carbon and nitrogen and osmoprotective effects of GB and DMG are made readily accessible through adaptive mutations. In M. extorquens PA1, the limitations to exploiting this group of compounds appear to exist predominantly at the levels of gene regulation and functional activity, rather than being constrained by transport or toxicity.IMPORTANCEOsmotic stress is a common challenge for bacteria colonizing the phyllosphere, where glycine betaine (GB) can be found as a prevalent osmoprotectant. Though Methylorubrum extorquens PA1 cannot use GB or its demethylation products, dimethylglycine (DMG) and sarcosine, as a sole carbon source, utilization is highly selectable via single nucleotide changes for both GB and DMG growth. The innate inability to use these compounds is due to limited flux through steps in the pathway and regulatory constraints. Herein, the characterization of the transcriptional regulator, Mext_3735 (GbdR), expands our understanding of the various roles in which GB derivatives can be used in M. extorquens PA1. Interestingly, increased catabolism of GB and derivatives does not interfere with, but rather improves, the ability of cells to thrive under increased salt stress conditions, suggesting that metabolic flux improves stress tolerance rather than providing a distinct tension between uses.

Functional analysis of OmpA and its contribution to pathogenesis of Edwardsiella tarda.

Outer membrane protein A (OmpA), a major component of outer membrane proteins in gram-negative bacteria, is considered to be an important virulence factor in various pathogenic bacteria, but its underlying mechanisms involved in pathogenic process of Edwardsiella tarda has not yet been fully elucidated. E. tarda is an important facultative intracellular pathogen with a broad host range. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. To address the functions of OmpA and its potential roles in the pathogenesis of E. tarda, ΔompA mutant strain and ΔompA-C complementary strain were constructed by the allelic exchange method in this study. Here, we demonstrate that the abilities of motility, biofilm formation and adherence to RAW264.7 cells of ΔompA were significantly impaired, although there was no difference in growth between wild-type (WT) strain and ΔompA. Moreover, inactivation of ompA rendered E. tarda more sensitive to oxidative, heat shock and osmotic stress, which simulate the in vivo conditions that E. tarda encounters within the intramacrophage environment. Consist with this observation, ΔompA was also found to be markedly attenuated for growth within macrophages. In addition, compared with the WT strain, ΔompA activated macrophages to release more inflammatory mediators, including tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS) and nitric oxide (NO). However, flow cytometry analysis revealed that ΔompA induced less apoptosis of RAW264.7 cells as compared with WT strain, characterized by decreased Annexin V binding and the activation of caspase-3. Overall, our findings suggest an importance of OmpA to E. tarda and provide the first comprehensive insight into its functions and potential roles in the pathogenesis of E. tarda, including its effect on interaction with macrophages.

Nimodipine inhibits spreading depolarization, ischemic injury, and neuroinflammation in mouse live brain slice preparations.

Nimodipine is used to prevent delayed ischemic deficit in patients with aneurysmal subarachnoid hemorrhage (aSAH). Spreading depolarization (SD) is recognized as a factor in the pathomechanism of aSAH and other acute brain injuries. Although nimodipine is primarily known as a cerebral vasodilator, it may have a more complex mechanism of action due to the expression of its target, the L-type voltage-gated calcium channels (LVGCCs) in various cells in neural tissue. This study was designed to investigate the direct effect of nimodipine on SD, ischemic tissue injury, and neuroinflammation. SD in control or nimodipine-treated live mouse brain slices was induced under physiological conditions using electrical stimulation, or by subjecting the slices to hypo-osmotic stress or mild oxygen-glucose deprivation (mOGD). SD was recorded applying local field potential recording or intrinsic optical signal imaging. Histological analysis was used to estimate tissue injury, the number of reactive astrocytes, and the degree of microglia activation. Nimodipine did not prevent SD occurrence in mOGD, but it did reduce the rate of SD propagation and the cortical area affected by SD. In contrast, nimodipine blocked SD occurrence in hypo-osmotic stress, but had no effect on SD propagation. Furthermore, nimodipine prevented ischemic injury associated with SD in mOGD. Nimodipine also exhibited anti-inflammatory effects in mOGD by reducing reactive astrogliosis and microglial activation. The results demonstrate that nimodipine directly inhibits SD, independent of nimodipine's vascular effects. Therefore, the use of nimodipine may be extended to treat acute brain injuries where SD plays a central role in injury progression.

Ratiometric Fluorescence Probes for In Situ Imaging of Membrane Tension in Live Cells.

Membrane tension is an important physical parameter of describing cellular homeostasis, and it is widely used in the study of cellular processes involving membrane deformation and reorganization, such as cell migration, cell spreading, and cell division. Despite the importance of membrane tension, direct measurement remains difficult. In this work, we developed a ratiometric fluorescent probe sensitive to membrane tension by adjusting the carbon chain structure based on polarity-sensitive fluorophores. The probe is sensitive to changes in membrane tension after cells were subjected to physical or chemical stimuli, such as osmotic shock, lipid peroxidation, and mechanical stress. When the polarity of the plasma membrane increases (the green/red ratio decreases) and the membrane tension increases, the relative magnitude of the membrane tension can be quantitatively calculated by fluorescence ratio imaging. Thus, the probe proved to be an efficient and sensitive membrane tension probe.

Germination responses of Lens Culiunaris L. seeds to osmotic potentials at cardinal temperatures using hydrothermal time model.

BACKGROUND: Lentil is a significant legume that are consumed as a staple food and have a significant economic impact around the world. The purpose of the present research on lentil was to assess the hydrothermal time model's capacity to explain the dynamics of Lens culinaris L. var. Markaz-09 seed germination, as well as to ascertain the germination responses at various sub-optimal temperatures (T) and water potentials (Ψ). In order to study lentil seed germination (SG) behavior at variable water potentials (Ψs) and temperatures (Ts). A lab experiment employing the hydrothermal time model was created. Seeds were germinated at six distinct temperatures: 15 0С, 20 0С, 25 0С, 30 0С, 35 0С, and 40 0С, with five Ψs of 0, -0.3, -0.6, -0.9, and - 1.2 MPa in a PEG-6000 (Polyethylene glycol 6000) solution. RESULTS: The results indicated that the agronomic parameters like Germination index (GI), Germination energy (GE), Timson germination index (TGI), were maximum in 25 0C at (-0.9 MPa) and lowest at 40 0C in 0 MPa. On other hand, mean germination time (MGT) value was highest at 15 0C in -1.2 MPa and minimum at 40 0C in (-0.6 MPa) while Mean germination rate (MGR) was maximum at 40 0C in (0 MPa) and minimum at 15 0C in (-0.6 MPa). CONCLUSIONS: The HTT model eventually defined the germination response of Lens culinaris L. var. Markaz-09 (Lentil) for all Ts and Ψs, allowing it to be employed as a predictive tool in Lens culinaris L. var. Markaz-09 (Lentil) seed germination simulation models.

Common Bean (Phaseolus vulgaris L.) NAC Transcriptional Factor PvNAC52 Enhances Transgenic Arabidopsis Resistance to Salt, Alkali, Osmotic, and ABA Stress by Upregulating Stress-Responsive Genes.

The NAC family of transcription factors includes no apical meristem (NAM), Arabidopsis thaliana transcription activator 1/2 (ATAF1/2), and cup-shaped cotyledon (CUC2) proteins, which are unique to plants, contributing significantly to their adaptation to environmental challenges. In the present study, we observed that the PvNAC52 protein is predominantly expressed in the cell membrane, cytoplasm, and nucleus. Overexpression of PvNAC52 in Arabidopsis strengthened plant resilience to salt, alkali, osmotic, and ABA stresses. PvNAC52 significantly (p < 0.05) reduced the degree of oxidative damage to cell membranes, proline content, and plant water loss by increasing the expression of MSD1, FSD1, CSD1, POD, PRX69, CAT, and P5CS2. Moreover, the expression of genes associated with abiotic stress responses, such as SOS1, P5S1, RD29A, NCED3, ABIs, LEAs, and DREBs, was enhanced by PvNAC52 overexpression. A yeast one-hybrid assay showed that PvNAC52 specifically binds to the cis-acting elements ABRE (abscisic acid-responsive elements, ACGTG) within the promoter. This further suggests that PvNAC52 is responsible for the transcriptional modulation of abiotic stress response genes by identifying the core sequence, ACGTG. These findings provide a theoretical foundation for the further analysis of the targeted cis-acting elements and genes downstream of PvNAC52 in the common bean.

Exploration of genes associated with induction of the viable but non-culturable state of Campylobacter jejuni.

Campylobacter jejuni is known to enter a viable but non-culturable (VBNC) state when exposed to environmental stresses. Microarray and quantitative real-time polymerase chain reaction (qPCR) analyses were performed to elucidate the genes related to the induction of the VBNC state. The C. jejuni NCTC11168 strain was cultured under low-temperature or high-osmotic stress conditions to induce the VBNC state. mRNA expression in the VBNC state was investigated using microarray analysis, and the gene encoding peptidoglycan-associated lipoprotein, Pal, was selected as the internal control gene using qPCR analysis and software. The three genes showing particularly large increases in mRNA expression, cj1500, cj1254, and cj1040, were involved in respiration, DNA repair, and transporters, respectively. However, formate dehydrogenase encoded by cj1500 showed decreased activity in the VBNC state. Taken together, C. jejuni actively changed its mRNA expression during induction of the VBNC state, and protein activities did not always match the mRNA expression levels.