Hypo-osmotic Stress Research Articles

Adaptation to space conditions of novel bacterial species isolated from the International Space Station revealed by functional gene annotations and comparative genome analysis

BackgroundThe extreme environment of the International Space Station (ISS) puts selective pressure on microorganisms unintentionally introduced during its 20+ years of service as a low-orbit science platform and human habitat. Such pressure leads to the development of new features not found in the Earth-bound relatives, which enable them to adapt to unfavorable conditions.ResultsIn this study, we generated the functional annotation of the genomes of five newly identified species of Gram-positive bacteria, four of which are non-spore-forming and one spore-forming, all isolated from the ISS. Using a deep-learning based tool—deepFRI—we were able to functionally annotate close to 100% of protein-coding genes in all studied species, overcoming other annotation tools. Our comparative genomic analysis highlights common characteristics across all five species and specific genetic traits that appear unique to these ISS microorganisms. Proteome analysis mirrored these genomic patterns, revealing similar traits. The collective annotations suggest adaptations to life in space, including the management of hypoosmotic stress related to microgravity via mechanosensitive channel proteins, increased DNA repair activity to counteract heightened radiation exposure, and the presence of mobile genetic elements enhancing metabolism. In addition, our findings suggest the evolution of certain genetic traits indicative of potential pathogenic capabilities, such as small molecule and peptide synthesis and ATP-dependent transporters. These traits, exclusive to the ISS microorganisms, further substantiate previous reports explaining why microbes exposed to space conditions demonstrate enhanced antibiotic resistance and pathogenicity.ConclusionOur findings indicate that the microorganisms isolated from ISS we studied have adapted to life in space. Evidence such as mechanosensitive channel proteins, increased DNA repair activity, as well as metallopeptidases and novel S-layer oxidoreductases suggest a convergent adaptation among these diverse microorganisms, potentially complementing one another within the context of the microbiome. The common genes that facilitate adaptation to the ISS environment may enable bioproduction of essential biomolecules need during future space missions, or serve as potential drug targets, if these microorganisms pose health risks.17AcJHQ5oWAqHhb9n7GQHSVideo

Intracellular vacuoles induced by hypo-osmotic stress visualized by coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging

We analyzed the effects of a hypo-osmotic environment on rat Schwann cells, a type of glial cell surrounding neurons, using ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy. After hypo-osmotic treatment, we detected vacuole-like components in the cytoplasm using both bright-field and CARS spectroscopic imaging. An approach integrating both morphological examination and a spectroscopic analysis based on multiple vibrational bands revealed that these structures are predominantly water-filled, and their characteristics closely resembled those of the vacuoles observed in plant cells.

Nutritional value of a novel EMS-induced cell wall-defective strain of Saccharomyces cerevisiae yeast for Pacific oyster (Magallana gigas) juveniles

The difficulties and high costs of live microalgae production have driven the search for alternative diets in bivalve aquaculture. The baker's yeast (Saccharomyces cerevisiae) represents a promising, cost-effective, high-quality protein for aquaculture. However, its application is restricted by poor digestibility due to its thick, rigid cell wall. Previously, we observed that the deletion of mnn9 gene in S. cerevisiae, leading to defective mannan synthesis and increased β-glucan exposure, enhances growth and immune response in Magallana gigas oysters. Nevertheless, using ∆mnn9 in aquaculture is not feasible due to restrictions on genetically modified organisms (GMO) containing antibiotic-resistance genes. This study aimed to create a non-GMO cell-wall defective yeast mutant (JH40) via ethyl methanesulfonate (EMS) mutagenesis and evaluate its nutritional value for M. gigas juveniles. Phenotypical differences between wild-type S. cerevisiae (WT), ∆mnn9, and JH40 were characterized using fluorescein isothiocyanate (FITC)-labeled lectin analysis, microscopy, cell diameter measurements, growth curves, and osmosensitivity tests. The nutritional value of a microalgae-based diet (Chaetoceros muelleri:Tisochrysis lutea, 50:50 based on dry weight) and its 50% substitution with JH40 or WT was assessed over 21 days in M. gigas juveniles. A complementary test also explored higher substitution levels (63%, 75%, and 100%) of the based diet with JH40 and the addition of extra JH40 (+25 %) in the 63% and 75% replacement diets. Results indicated that JH40 forms clumps, grows slower than the WT, has a larger cell diameter, higher binding affinity to WGA and LEL lectins than the WT, and is highly sensitive to hypo-osmotic stress, which are characteristics of cell-wall defective yeasts. Oysters fed a diet with 50% JH40 substitution showed significantly higher growth compared to those fed the WT-containing diet. JH40 could replace 50% of the algal diet without significantly affecting oyster growth and survival. However, higher inclusion levels of JH40 (62 %, 75 %, and 100 %) did not achieve the oyster growth observed with the algae-based diet, even with additional yeast. These findings are attributed to the limited polyunsaturated fatty acid content in yeast cells. Further research on dosing and administration frequencies is needed to evaluate the immunostimulatory potential of JH40.

TRPV4-β-catenin axis is a novel therapeutic target for dry skin-induced chronic itch

Dry skin induced chronic pruritus is an increasingly common and debilitating problem, especially in the elderly. Although keratinocytes play important roles in innate and adaptive immunity and keratinocyte proliferation is a key feature of dry skin induced chronic pruritus, the exact contribution of keratinocytes to the pathogenesis of dry skin induced chronic pruritus is poorly understood. In this study, we generated the acetone-ether-water induced dry skin model in mice and found that epidermal hyperplasia induced by this model is partly dependent on the β-catenin signaling pathway. XAV939, an antagonist of β-catenin signaling pathway, inhibited epidermal hyperplasia in dry skin model mice. Importantly, dry skin induced chronic pruritus also dramatically reduced in XAV939 treated mice. Moreover, acetone-ether-water treatment-induced epidermal hyperplasia and chronic itch were decreased in Trpv4−/− mice. In vitro, XAV939 inhibited hypo-osmotic stress induced proliferation of HaCaT cells, and hypo-osmotic stress induced proliferation of in HaCaT cells and primary cultured keratinocytes were also significantly reduced by blocking TRPV4 function. Finally, thymic stromal lymphopoietin release was examined both in vivo and in vitro, which was significantly inhibited by XAV939 treatment and Trpv4 deficiency, and anti-TSLP antibody treatment significantly decreased AEW-induced scratching behavior. Overall, our study revealed a unique ability of TRPV4 expressing keratinocytes in the skin, which critically mediated dry skin induced epidermal hyperplasia and chronic pruritus, thus provided novel insights into the development of therapies for chronic pruritus in the elderly.

The effects of ACE inhibitor Enalapril on Mytilus galloprovincialis: Insights into morphological and functional responses

In the last decades, pharmaceuticals have emerged as a new class of environmental contaminants.Antihypertensives, including angiotensin-converting enzyme (ACE) inhibitors, are of special concern due to their increased consumption over the past years. However, the available data on their putative effects on the health of aquatic animals, as well as the possible interaction with biological systems are still poorly understood.This study analysed whether and to which extent the exposure to Enalapril, an ACE inhibitor commonly used for treating hypertension and heart failure, may induce morpho-functional alterations in the mussel Mytilus galloprovincialis, a sentinel organism of water pollution. By mainly focusing on the digestive gland (DG), a target tissue used for analysing the effects of xenobiotics in mussels, the effects of 10-days exposure to 0.6 ng/L (E1) and 600 ng/L (E2) of Enalapril were investigated in terms of cell viability and volume regulation, morphology, oxidative stress, and stress protein expression and localization. Results indicated that exposure to Enalapril compromised the capacity of DG cells from the E2 group to regulate volume by limiting the ability to return to the original volume after hypoosmotic stress. This occurred without significant effects on DG cell viability. Enalapril unaffected also haemocytes viability, although an increased infiltration of haemocytes was histologically observed in DG from both groups, suggestive of an immune response. No changes were observed in the two experimental groups on expression and tissue localization of heat shock proteins 70 (HSPs70) and HSP90, and on the levels of oxidative biomarkers.Our results showed that, in M. galloprovincialis the exposure to Enalapril did not influence the oxidative status, as well as the expression and localization of stress-related proteins, while it activated an immune response and compromised the cell ability to face osmotic changes, with potential consequences on animal performance.

Species-specific metabolome changes during salinity downshift in sub-Arctic populations of Mytilus edulis and M. trossulus

The blue mussels Mytilus edulis and Mytilus trossulus are ecologically and economically important species distributed widely across the Northern Hemisphere. Understanding their behavioral and physiological disparities is crucial for assessing their ecological success and aquacultural value. The recent finding of non-native M. trossulus in the White Sea raises concerns regarding its potential competition with native M. edulis and its prospective spread in light of climate change and surface water freshening. We investigated the responses of M. edulis and M. trossulus to salinity variations by examining shell closure thresholds and tissue levels of 35 metabolic intermediates in mussels acclimated to different salinities (25, 16, and 10). The salinity threshold for valve closure was similar in both studied species, but M. trossulus consistently opened at lower salinities (by 0.2–0.7 practical salinity units) compared to M. edulis. Salinity-induced changes in metabolite levels were similar between the two species. Taurine emerged as the dominant osmolyte, comprising over 50% of the total free amino acid pool, with aspartate and glycine contributing 15–30%. Concentrations of taurine, glycine, and total free amino acids declined with decreasing salinity. Taurine to glycine ratios were higher in M. edulis and increased in both species with declining salinity. Acclimation salinity significantly influenced urea cycle intermediates and methionine sulfoxide content, a cellular biomarker of amino acid oxidation. Species-specific differences were observed in purine metabolism, with higher levels of GMP and AMP found in M. edulis. Likewise, aromatic amino acids and histidine levels were higher in M. edulis compared to M. trossulus. However, no evidence suggests superior adaptation of M. trossulus metabolism to hypoosmotic stress compared to M. edulis. Further research is necessary to elucidate the functional implications of subtle metabolic differences between these Mytilus congeners and their ecological consequences in changing marine environments.

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.

The deletion of AQP4 and TRPV4 affects astrocyte swelling/volume recovery in response to ischemia-mimicking pathologies.

Astrocytic Transient receptor potential vanilloid 4 (TRPV4) channels, together with Aquaporin 4 (AQP4), are suspected to be the key players in cellular volume regulation, and therefore may affect the development and severity of cerebral edema during ischemia. In this study, we examined astrocytic swelling/volume recovery in mice with TRPV4 and/or AQP4 deletion in response to in vitro ischemic conditions, to determine how the deletion of these channels can affect the development of cerebral edema. We used three models of ischemia-related pathological conditions: hypoosmotic stress, hyperkalemia, and oxygenglucose deprivation (OGD), and observed their effect on astrocyte volume changes in acute brain slices of Aqp4-/-, Trpv4-/- and double knockouts. In addition, we employed single-cell RT-qPCR to assess the effect of TRPV4 and AQP4 deletion on the expression of other ion channels and transporters involved in the homeostatic functioning of astrocytes. Quantification of astrocyte volume changes during OGD revealed that the deletion of AQP4 reduces astrocyte swelling, while simultaneous deletion of both AQP4 and TRPV4 leads to a disruption of astrocyte volume recovery during the subsequent washout. Of note, astrocyte exposure to hypoosmotic stress or hyperkalemia revealed no differences in astrocyte swelling in the absence of AQP4, TRPV4, or both channels. Moreover, under ischemia-mimicking conditions, we identified two distinct subpopulations of astrocytes with low and high volumetric responses (LRA and HRA), and their analyses revealed that mainly HRA are affected by the deletion of AQP4, TRPV4, or both channels. Furthermore, gene expression analysis revealed reduced expression of the ion transporters KCC1 and ClC2 as well as the receptors GABAB and NMDA in Trpv4-/- mice. The deletion of AQP4 instead caused reduced expression of the serine/cysteine peptidase inhibitor Serpina3n. Thus, we showed that in AQP4 or TRPV4 knockouts, not only the specific function of these channels is affected, but also the expression of other proteins, which may modulate the ischemic cascade and thus influence the final impact of ischemia.

Membrane Permeability Dominates over Electrostatic Interactions in Dictating Drug Transport in Osmotically Shocked Escherichia coli.

To combat surging multidrug-resistant Gram-negative bacterial infections, better strategies to improve the efficacy of existing drugs are critical. Because the dual membrane cell envelope is the first line of defense for these bacteria, it is crucial to understand the permeation properties of the drugs through it. Our recent study shows that isosmotic conditions prevent drug permeation inside Gram-negative bacteria, Escherichia coli, while hypoosmotic stress enhances the process. Here, we unravel the reason behind such differential drug penetration. Specifically, we dissect the roles of electrostatic screening and low membrane permeability in the penetration failure of drugs under osmotically balanced conditions. We compare the transport of a quaternary ammonium compound malachite green in the presence of an electrolyte (NaCl) and a wide variety of commonly used organic osmolytes, e.g., sucrose, proline, glycerol, sorbitol, and urea. These osmolytes of different membrane permeability (i.e., nonpermeable sucrose and NaCl, freely permeable urea and glycerol, and partially permeable proline and sorbitol) clarify the role of osmotic stress in cell envelope permeability. The results showcase that under balanced osmotic conditions, drug molecules fail to penetrate inside E. coli cells because of low membrane permeabilities and not because of electrostatic screening imposed by the osmolytes. Contribution of the electrostatic interactions, however, cannot be completely overruled as at osmotically imbalanced conditions, drug transport across the bacterial subcellular compartments is found to be dependent on the osmolytes used.

The Impacts of Osmotic Stress During the Critical Window of Development on Protein and Histone PTM Alterations in California grunion

The California Grunion, Leuresthes tenuis, can experience a broad range of salinities during their early life stages, with their nursery grounds measuring salinities of 21 to 42ppt under normal conditions. Due to the unique subterrestrial incubation of their eggs within the sand of California beaches, it is not unlikely for this marine fish to hatch in non-seawater conditions, whereafter the larvae may transition to relatively diluted estuaries or concentrated harbors to develop as juveniles for several months. Consequently, they are an ideal system to study the impacts of ecologically relevant salinity stress on the alteration of proteins and histone post-translational modifications (PTMs) during early development. The study of the relationship between the critical window of development (CWD), in which sexual bipotential is lost, and the longevity of protein landscape and histone PTM changes has not been widely explored. The CWD is of interest as this is when the gametes of this species become set. Therefore, we hypothesize that histone PTM changes that occur during this time may thus be a form of heritable epigenetics. Thus, hypo-and-hyperosmotic exposures of L. tenuis hatchlings during this window, and past it, attempt to identify if the CWD is relevant to the presence and persistence of protein and histone PTM changes under salinity stress. We hypothesize that exposure to osmotic stress during the CWD will result in induced compensatory mechanisms in the protein landscape of the California grunion larvae, as well as persistent alterations in the histone PTMs in the larvae. To elucidate the relationship between these factors, California grunion larvae were exposed in replicate to one of three chronic salinity treatments (16ppt, 32ppt, or 40ppt) for the duration of their CWD (68dph), with 16ppt and 40ppt inducing hypoosmotic and hyperosmotic stress respectively. Chronic post-CWD exposures and recovery times until 98dph were used to determine whether the CWD or prolonged salinity exposures were key to alterations in the histone PTM and protein landscape of the larvae. After each timepoint, L. tenuis were culled and processed for histone PTM enrichment and proteomic analysis. At the time of culling, there were no significant differences in survivorship between replicates and experimental groups, with statistically significant different deaths between groups occurring only shortly after hatching. Skyline and MSstats were used in the analysis of the statistical significance of differential regulation of proteins and histone PTMs under stress conditions versus the controls. Of special interest is the osmoregulatory mechanisms involved in the myo-inositol biosynthesis pathway, with analysis focusing on the abundance of histone acetylation, amidation, ubiquitylation, and 4-hydroxynonelation in histone PTMs, as the accumulations of these have been found to be correlated with osmotic stress in some fish tissues. Funding provided by NSF IOS-2209383 and NSF GRFP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genome-wide identification of salinity stress-associated lncRNAs in Nile tilapia

Long noncoding RNAs (lncRNAs) are increasingly recognized as key players in various biological processes, yet their role in fish's response to salinity changes remains largely unexplored. This study undertakes a genome-wide analysis of lncRNAs in Nile tilapia (Oreochromis niloticus), examining their response to salinity stress across a substantial dataset of 98 RNA-seq samples from nine different tissues. We identified 28,819 high-confidence lncRNA candidates, which are characterized by a shorter average length of 788 nucleotides and fewer exons (average 1.84) compared to mRNAs. Specifically, in the gills exposed to salinity 27 for three and a half months, we found 846 differentially expressed lncRNAs (DElncRNAs), including 342 upregulated and 504 downregulated transcripts. The study also highlighted 220 significant DElncRNA-mRNA pairs, indicating potential regulatory interactions. Notably, in response to acute salinity stress, we identified 989 DElncRNAs, with 414 upregulated and 575 downregulated in gills. An analysis of these DElncRNAs revealed 348 high-confidence pairs, with Gene Ontology (GO) and KEGG pathway analyses indicating a prominent role in immune-inflammatory responses for most target genes, such as trim7, trim35, trim47, cd225. Crucially, two DElncRNAs (ENSONIT00000076723 and MSTRG.18195.2) were found to co-localize with QTL intervals associated with salinity tolerance, situated near three significant SNPs. Additionally, a notable cis-acting pair, consisting of mRNA ENSONIT00000087370 (apold1a) and lncRNA ENSONIT00000076723, demonstrated a strong correlation under both acute and chronic hypo-osmotic stress. Our findings significantly broaden the understanding of lncRNAs in salinity stress adaptation and suggest their novel roles as key regulators in the osmoregulatory mechanisms of Nile tilapia.

ROS formation, mitochondrial potential and osmotic stability of the lamprey red blood cells: effect of adrenergic stimulation and hypoosmotic stress.

Semi-anadromous animals experience salinity fluctuations during their life-span period. Alterations of environmental conditions induce stress response where catecholamines (CA) play a central role. Physiological stress and changes in external and internal osmolarity are frequently associated with increased production of reactive oxygen species (ROS). In this work, we studied the involvement of the cAMP/PKA pathway in mediating catecholamine-dependent effects on osmoregulatory responses, intracellular production of ROS, and mitochondrial membrane potential of the river lamprey (Lampetra fluviatilis, Linnaeus, 1758) red blood cells (RBCs). We also investigated the role of hypoosmotic shock in the process of ROS production and mitochondrial respiration of RBCs. For this, osmotic stability and the dynamics of the regulatory volume decrease (RVD) following hypoosmotic swelling, intracellular ROS levels, and changes in mitochondrial membrane potential were assessed in RBCs treated with epinephrine (Epi, 25 μM) and forskolin (Forsk, 20 μM). Epi and Forsk markedly reduced the osmotic stability of the lamprey RBCs whereas did not affect the dynamics of the RVD response in a hypoosmotic environment. Activation of PKA with Epi and Forsk increased ROS levels and decreased mitochondrial membrane potential of the lamprey RBCs. In contrast, upon hypoosmotic shock enhanced ROS production in RBCs was accompanied by increased mitochondrial membrane potential. Overall, a decrease in RBC osmotic stability and the enhancement of ROS formation induced by β-adrenergic stimulation raises concerns about stress-associated changes in RBC functions in agnathans. Increased ROS production in RBCs under hypoosmotic shock indicates that a decrease in blood osmolarity may be associated with oxidative damage of RBCs during lamprey migration.

The osmoregulatory mechanism in response to hypoosmotic stress and the key role of ABCC1 in osmoregulation in the mud crab, Scylla paramamosain

Scylla paramamosain is an important mariculture animal. In the present study, we examined the physiological and biochemical characteristics of juvenile S. paramamosain under hypoosmotic stress and further verified the function of ABCC1, a key transport protein in gill tissue. The results showed that Na+/K+-ATPase, Ca2+-ATPase, and Na+/K+/2Cl- cotransporter in gill tissues increased first and then decreased. In addition, SOD and CAT activities also began to gradually increase, protecting gill tissues from oxidative damage. The hemolymph osmolarity decreased rapidly from 792 mOsm/kg to 560 mOsm/kg within 6 h, and then rose to 626.25 mOsm/kg at 48 h, and then remained stable. At the same time, Na+, Cl-, K+, and Ca2+ concentrations were decreased to varying degrees. There were significant changes in free amino acids such as aspartic acid, glutamic acid, and asparagine in hemolymph in response to acute salt depletion. By injection of ABCC1 inhibitor/activator, it was found that while ABCC1 transporter activity in the MK-571 group showed a decreasing trend at 9 h, Na+/K+ -ATPase activity began to increase and reached a peak at 48 h. Compared with the control group, the levels of aspartic acid, glutamic acid, asparagine, glycine, arginine, alanine, lysine, and proline in the MK-571 group showed significant differences at 6 h; Significant differences in glutamate, asparagine, arginine, alanine, lysine, and proline appeared at 12 h in the Thiethylperazine group. These results suggested that the dynamic changes of gill ion transport enzymes, antioxidant enzymes, inorganic ions, and free amino acids in hemolymph occurred in S. paramamosain under hypoosmotic stress, and the ABCC1 transporter played an important role in this regulatory process.

Effects of hypo-osmotic stress on osmoregulation, antioxidant response, and energy metabolism in sea cucumber Holothuria moebii under desalination environment

With global climate changing, hypo-salinity events are increasing in frequency and duration because of continuous rainfall and freshwater inflow, which causes reduced cytosolic osmolarity and cellular stress responses in aquatic animals. Sea cucumbers are considered stenohaline because they lack osmoregulatory organs and are vulnerable to salinity fluctuations. In this study, we performed multiple biochemical assays, de novo transcriptomics, and widely targeted metabolomics to comprehensively explore the osmoregulatory mechanisms and physiological responses of sea cucumber Holothuria moebii to hypo-osmotic stress, which is a representative specie that is frequently exposed to hypo-saline intertidal zones. Our results found that H. moebii contracted their ambulacral feet and oral tentacles, and the coelomic fluid ion concentrations were reduced to be consistent with the environment. The microvilli of intestines and respiratory trees underwent degeneration, and the cytoplasm exhibited swelling and vacuolation. Moreover, the Na+, K+, and Cl− concentrations and Na+/K+-ATPase activity were significantly reduced under hypo-osmotic stress. The decrease in protein kinase A activity and increase in 5′-AMP level indicated a significant inhibition of the cAMP signaling pathway to regulate ion concentrations. And small intracellular organic molecules (amino acids, nucleotides and their derivatives) also play crucial roles in osmoregulation through oxidative deamination of glutamate, nucleotide catabolism, and nucleic acid synthesis. Moreover, lysosomes and peroxisomes removed oxidative damage, whereas antioxidant metabolites, such as N-acetyl amino acids and glutathione, were increased to resist oxidative stress. With prolonged hypo-osmotic stress, glycerophospholipid metabolism was enhanced to maintain membrane stability. Furthermore, acyl-CoA-binding protein activity was significantly inhibited, and only a small amount of acylcarnitine was significantly accumulated, which indicated a disruption in energy metabolism. PPAR signaling pathway and choline content were up-regulated to promote fatty acid metabolism under hypo-osmotic stress. Overall, our results provide new insights into the osmoregulatory mechanisms and physiological responses of sea cucumbers to hypo-osmotic stress.

Mechanosensitive channel MscL gating transitions coupling with constriction point shift.

The mechanosensitive channel of large conductance (MscL) acts as an "emergency release valve" that protects bacterial cells from acute hypoosmotic stress, and it serves as a paradigm for studying the mechanism underlying the transduction of mechanical forces. MscL gating is proposed to initiate with an expansion without opening, followed by subsequent pore opening via a number of intermediate substates, and ends in a full opening. However, the details of gating process are still largely unknown. Using in vivo viability assay, single channel patch clamp recording, cysteine cross-linking, and tryptophan fluorescence quenching approach, we identified and characterized MscL mutants with different occupancies of constriction region in the pore domain. The results demonstrated the shifts of constriction point along the gating pathway towards cytoplasic side from residue G26, though G22, to L19 upon gating, indicating the closed-expanded transitions coupling of the expansion of tightly packed hydrophobic constriction region to conduct the initial ion permeation in response to the membrane tension. Furthermore, these transitions were regulated by the hydrophobic and lipidic interaction with the constricting "hot spots". Our data reveal a new resolution of the transitions from the closed to the opening substate of MscL, providing insights into the gating mechanisms of MscL.

Osmotic stress induces the formation of migrasome-like vesicles.

Migrasomes are extracellular vesicles that form on the retraction fibers of migrating cells. In this study, we report the formation of migrasome-like vesicles enriched in tetraspanin 4 and containing cytoplasmic components in response to hypoosmotic stress. When migrating cells were subjected to hypoosmotic stress, vesicles with a size distribution of 0.5 to 2 μm formed on the retraction fibers, and vanished in a few minutes. The vesicles are rich in cholesterol, and their number was reduced when cells were pretreated with lipoprotein-deficient serum. The formation of migrasome-like vesicles upon hypoosmotic stress may provide biophysical cues regarding the cellular response to this external stimulus in cells and tissues.

Short communication: ROS production and mitochondrial membrane potential in hemocytes of marine bivalves, Mytilus galloprovincialis and Magallana gigas, under hypoosmotic stress.

Bivalve mollusks that inhabit low-depth coastal and estuarine areas frequently experience osmotic stress that may be also associated with alterations of antioxidant enzyme activities and markers of oxidative stress. Mitochondria are a major source of reactive oxygen species (ROS) in eucaryotic cells. Overpoduction of ROS induces oxidative stress leading to a damage of intracellular compartments and cell death. In euryhaline bivalves, information concerning cellular ROS production upon osmotic stress and changes in mitochondrial membrane potential is scarce. The present study investigates osmotic stability and hemocytes` regulatory volume decrease (RVD) of Mediterranean mussel (Mytilus galloprovincialis) and the Pacific oyster (Magallana gigas). We also studied dynamic changes in intracellular ROS levels and mitochondrial membrane potential in hemocytes undergoing the RVD response following hypoosmotic swelling. Our data revealed that osmotic stability of mussel and oyster hemocytes did not significantly differ. Loss of environmental osmolarity from 460.0±2.0mOsml-1 to 216.0±4.0mOsml-1 resulted in an increase of hemocyte volume by 60% of the initial cellular volume in mussels and by 28% in oysters. After rapid hypoosmotic swelling hemocytes of both species demonstrated the RVD response. At the end of 60min exposure to hypoosmotic environment, hemocyte volume significantly decreased in both species by 10-12% compared to the maximal hemocyte volume. Hypoosmotic shock induced an increase of mitochondrial membrane potential in hemocytes of mussels and oysters. In mussels, increased mitochondrial membrane potential was accompanied with decreased ROS levels in hemocytes, whereas oyster hemocytes showed enhanced ROS production.

Effect of pinocembrin on thymocyte proliferation and death

Introduction: Cell volume regulation is critical for cellular proliferation and death. Pinocembrin effectively suppresses the volume regulation in thymocytes under hypoosmotic stress by blocking the volume-sensitive anion channel. This study aims to evaluate the effects of this flavonoid on thymocyte proliferation and death. Methods: Thymocytes were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, and the cell number was determined by cloud-based automated cell counting (Corning). Necrotic and apoptotic cell death were evaluated by propidium iodide- and annexin V-staining, respectively. Results: Pinocembrin at 10–50 μM caused suppression of primary cultured thymocyte proliferation with a half-maximal effect of 28.4 ± 0.2 μM. The cell counts did not fall below the control level at the doses of 100–150 μM. The fraction of spontaneously necrotic cells was ~26% of the total population and increased to ~51% in the presence of dexamethasone. The fraction of spontaneously apoptotic cells increased by this glucocorticoid from 3.6% to 16.7%. Pinocembrin protected thymocytes from necrosis both in spontaneous and dexamethasone-induced death, reducing the fraction of necrotic cells by ~40–50% at 150 μM. Pinocembrin attenuated dexamethasone-induced apoptotic death, reducing the fraction of annexin-positive cells to the control (spontaneous) level. Conclusion: Our results suggest that pinocembrin arrests thymocyte proliferation without essential killing. Under conditions of massive death (e.g., during inflammation, when the level of glucocorticoids increases sharply both physiologically and as a result of pharmacotherapy), pinocembrin protects immuno-competent cells from necrotic and apoptotic death.

Hypo-osmotic Stress Increases Permeability of Individual Barriers in Escherichia coli Cell Envelope, Enabling Rapid Drug Transport.

Survival of foodborne Gram-negative bacteria during osmotic stress often leads to multidrug resistance development. However, despite the concern, how osmoadaptation alters drug penetration across the Gram-negative bacterial cell envelope has remained inconclusive for years. Here, we have investigated drug permeation and accumulation inside hypo-osmotically shocked Escherichia coli. Three different quaternary ammonium compounds (QACs) are used as cationic amine-containing drug representatives; they also serve as envelope permeability indicators in different assays. Propidium iodide fluorescence reveals cytoplasmic accumulation and overall envelope permeability, while crystal violet sorption and second harmonic generation (SHG) spectroscopy reveal periplasmic accumulation and outer membrane permeability. Malachite green sorption and SHG results reveal transport across both the outer and inner membranes and accumulation in the periplasm as well as cytoplasm. The findings are found to be complementary to one another, collectively revealing enhanced permeabilities of both membranes and the periplasmic space in response to hypo-osmotic stress in E. coli. Enhanced permeability leads to faster QACs transport and higher accumulation in subcellular compartments, whereas transport and accumulation both are negligible under isosmotic conditions. The QACs' transport rates are found to be highly influenced by the osmolytes used, where phosphate ion emerges as a key facilitator of transport across the periplasm into the cytoplasm. E. coli is found viable, with morphology unchanged under extreme hypo-osmotic stress; i.e., it adapts to the situation. The outcome shows that the hypo-osmotic shock to E. coli, specifically using phosphate as an osmolyte, can be beneficial for drug delivery.

Improved high-quality reference genome of red drum facilitates the processes of resistance-related gene exploration

Sciaenops ocellatus is among the most important artificially introduced farmed fish across 11 countries and regions. However, the frequent occurrence of extreme weather events and breeding escapes have placed great pressure on local marine biodiversity and ecosystems. We reported the de novo assembly and annotation with a contig N50 of 28.30 Mb using PacBio HiFi sequencing and Hi-C technologies, which resulted in a 283-fold increase in contig N50 length and improvement in continuity and quality in complex repetitive region for S. ocellatus compared to the previous version. In total, 257.36 Mb of repetitive sequences accounted for 35.48% of the genome, and 22,845 protein-coding genes associated with a BUSCO value of 98.32%, were identified by genome annotation. Moreover, 54 hub genes rapidly responding to hypoosmotic stress were identified by WGCNA. The high-quality chromosome-scale S. ocellatus genome and candidate resistance-related gene sets will not only provide a genomic basis for genetic improvement via molecular breeding, but will also lay an important foundation for investigating the molecular regulation of rapid responses to stress.