Hyperosmotic Stress Research Articles

Study on the osmotic response and function of myo-inositol oxygenase in euryhaline fish nile tilapia (Oreochromis niloticus).

To understand the role of myo-inositol oxygenase (miox) in the osmotic regulation of Nile tilapia, its expression was analyzed in various tissues. The results showed that the expression of miox gene was highest in the kidney, followed by the liver, and was significantly upregulated in the kidney and liver under 1 h hyperosmotic stress. The relative luminescence efficiency of the miox gene transcription starting site (-4,617 to +312 bp) under hyperosmotic stress was measured. Two fragments (-1,640/-1,619 and -620/-599) could induce the luminescence activity. Moreover, the -1,640/-1,619 and -620/-599 responded to hyperosmotic stress and high-glucose stimulation by base mutation, suggesting that osmotic and carbohydrate response elements may exist in this region. Finally, the salinity tolerance of Nile tilapia was significantly reduced after the knocking down of miox gene. The accumulation of myo-inositol was affected, and the expression of enzymes in glucose metabolism was significantly reduced after the miox gene was knocked down. Furthermore, hyperosmotic stress can cause oxidative stress, and MIOX may help maintain the cell redox balance under hyperosmotic stress. In summary, MIOX is essential in osmotic regulation to enhance the salinity tolerance of Nile tilapia by affecting myo-inositol accumulation, glucose metabolism, and antioxidant performance.NEW & NOTEWORTHY Myo-inositol oxygenase (MIOX) is the rate-limiting enzyme that catalyzes the first step of MI metabolism and determines MI content in aquatic animals. To understand the role of miox in the osmotic regulation of Nile tilapia, we analyzed its expression in different tissues and its function under hyperosmotic stress. This study showed that miox is essential in osmotic regulation to enhance the salinity tolerance of Nile tilapia by affecting myo-inositol accumulation, glucose metabolism, and antioxidant performance.

Quantitative proteomic and metabolomic profiling reveals different osmoregulation mechanisms of tilapia cells coping with different hyperosmotic stress

This study aimed to investigate the different regulatory mechanisms of euryhaline fish under regular hyperosmotic and extreme hyperosmotic stress. The OmB (Oreochromis mossambicus brain) cells were exposed to three treatments: control, regular hyperosmotic stress and extreme hyperosmotic stress. After 12 h exposure, proteomics, metabolomics analyses and integrative analyses were explored.Both kinds of stress lead to lowering cell growth and morphology changes, while under regular hyperosmotic stress, the up-regulated processes related with compatible organic osmolytes synthesis are crucial strategy for the euryhaline fish cell line to survive; On the other hand, under extreme hyperosmotic stress, the processes related with cell apoptosis and cell cycle arrest are dominant. Furthermore, down-regulated pyrimidine metabolism and several ribosomal proteins partially participated in the lowered cell metabolism and increased cell death under both kinds of hyperosmotic stress. The PI3K-Akt and p53 signaling pathways were involved in the stagnant stage of cell cycles and induction of cell apoptosis under both kinds of hyperosmotic stress. However, HIF-1, FoxO, JAK-STAT and Hippo signaling pathways mainly contribute to disrupting the cell cycle, metabolism and induction of cell apoptosis under extreme hyperosmotic stress. SignificanceIn the past, the research on fish osmoregulation mainly focused on the transcription factors and ion transporters of osmoregulation, the processes between osmotic sensing and signal transduction, and the associations between signaling pathways and regulation processes have been poorly understood.Investigating fish cell osmoregulation and potential signal transduction pathways is necessary. With the advancements in omics research, it is now feasible to investigate the relationship between environmental stress and molecular responses.In this study, we aimed to explore the signaling pathways and substance metabolism mode during hyper-osmoregulation in OmB cell line, to reveal the key factors that are critical to cell osmoregulation.

Hyperosmotic stress alters enzyme stoichiometry of glucosomes

Hyperosmotic stress alters enzyme stoichiometry of glucosomes

Exogenous Pregnane X Receptor Does Not Undergo Liquid-Liquid Phase Separation in Nucleus under Cell-Based In Vitro Conditions.

Pregnane X receptor (PXR) belongs to the nuclear receptor superfamily that plays a crucial role in hepatic physiologic and pathologic conditions. Phase separation is a process in which biomacromolecules aggregate and condense into a dense phase as liquid condensates and coexist with a dilute phase, contributing to various cellular and biologic functions. Until now, whether PXR could undergo phase separation remains unclear. This study aimed to investigate whether PXR undergoes phase separation. Analysis of the intrinsically disordered regions (IDRs) using algorithm tools indicated a low propensity of PXR to undergo phase separation. Experimental assays such as hyperosmotic stress, agonist treatment, and optoDroplets assay demonstrated the absence of phase separation for PXR. OptoDroplets assay revealed the inability of the fusion protein of Cry2 with PXR to form condensates upon blue light stimulation. Moreover, phase separation of PXR did not occur even though the mRNA and protein expression levels of PXR target, cytochrome P450 3A4, changed after sorbitol treatment. In conclusion, for the first time, these findings suggested that exogenous PXR does not undergo phase separation following activation or under hyperosmotic stress in nucleus of cells. SIGNIFICANCE STATEMENT: PXR plays a critical role in hepatic physiological and pathological processes. The present study clearly demonstrated that exogenous PXR does not undergo phase separation after activation by agonist or under hyperosmotic stress in nucleus. These findings may help understand PXR biology.

Molecular mechanisms underlying phenotypic degeneration in Cordyceps militaris: insights from transcriptome reanalysis and osmotic stress studies

Phenotypic degeneration in Cordyceps militaris poses a significant concern for producers, yet the mechanisms underlying this phenomenon remain elusive. To address this concern, we isolated two strains that differ in their abilities to form fruiting bodies. Our observations revealed that the degenerated strain lost the capacity to develop fruiting bodies, exhibited limited radial expansion, increased spore density, and elevated intracellular glycerol levels. Transcriptome reanalysis uncovered dysregulation of genes involved in the MAPK signaling pathway in the degenerate strain. Our RT-qPCR results demonstrated reduced expression of sexual development genes, along with upregulation of genes involved in asexual sporulation, glycerol synthesis, and MAPK regulation, when compared to the wild-type strain. Additionally, we discovered that osmotic stress reduced radial growth but increased conidia sporulation and glycerol accumulation in all strains. Furthermore, hyperosmotic stress inhibited fruiting body formation in all neutralized strains. These findings indicate dysregulation of the MAPK signaling pathway, the possibility of the activation of the high-osmolarity glycerol and spore formation modules, as well as the downregulation of the pheromone response and filamentous growth cascades in the degenerate strain. Overall, our study sheds light on the mechanisms underlying Cordyceps militaris degeneration and identifies potential targets for improving cultivation practices.

Novel high throughput screen reports that benzo(a)pyrene overrides mouse trophoblast stem cell multipotency, inducing SAPK activity, HAND1 and differentiated trophoblast giant cells

IntroductionCultured mouse trophoblast stem cells (mTSC) maintain proliferation/normal stemness (NS) under FGF4, which when removed, causes normal differentiation (ND). Hypoxic, or hyperosmotic stress forces trophoblast giant cells (TGC) differentiate. Hypoxic, hyperosmotic, and genotoxic benzo(a)pyrene (BaP), which is found in tobacco smoke, force down-regulation of inhibitor of differentiation (Id)2, enabling TGC differentiation. Hypoxic and hyperosmotic stress induce TGC by SAPK-dependent HAND1 increase. Here we test whether BaP forces mTSC-to-TGC while inducing SAPK and HAND1. MethodsHand1 and SAPK activity were assayed by immunoblot, mTSC-to-TGC growth and differentiation were assayed at Tfinal after 72hr exposure of BaP, NS, ND, Retinoic acid (RA), or sorbitol. Nuclear-stained cells were micrographed automatically by a live imager, and assayed by ImageJ/FIJI, Biotek Gen 5, AIVIA proprietary artificial intelligence (AI) software or open source, CellPose artificial intelligence/AI software. ResultsBaP (0.05-1μM) activated SAPK and HAND1 without diminishing growth. TSC-to-TGC differentiation was assayed with increasingly accuracy for 2–4 N cycling nuclei and >4 N differentiating TGC nuclei, using ImageJ/FIJI, Gen 5, AIVIA, or CellPose AI software. The AIVIA and Cellpose AI software matches human accuracy. The lowest BaP effects on SAPK activation/HAND1 increase are >10-fold more sensitive than similar effects for mESC. RA induces 44–47% 1st lineage TGC differentiation, but the same RA dose induces only 1% 1st lineage mESC differentiation. DiscussionFirst, these pilot data suggest that mTSC can be used in high throughput screens (HTS) to predict toxicant exposures that force TGC differentiation. Second, mTSC differentiated more cells than mESC for similar stress exposures, Third, open source AI can replace human micrograph quantitation and enable a miscarriage-predicting HTS.

Estimation of Structural Sensitivity of Intrinsically Disordered Regions in Response to Hyperosmotic Stress in Living Cells Using FRET.

Intrinsically disordered regions (IDRs) are protein domains that participate in crucial cellular processes. During stress conditions, the physicochemical properties of the cellular environment change, directly impacting the conformational ensemble of IDRs. IDRs are inherently sensitive to environmental perturbations. Studying how the physicochemical properties of the cell regulate the conformational ensemble of IDRs is essential for understanding the environmental control of their function. Here, we describe a step-by-step method for measuring the structural sensitivity of IDRs in living Saccharomyces cerevisiae cells in response to hyperosmotic stress conditions. We present the use of ensemble fluorescence resonance energy transfer (FRET) to estimate how the global dimensions of IDRs change during a progressive increase of hyperosmotic stress imposed on cells with any osmolyte. In addition, we provide a script for processing fluorescence measurements and comparing structural sensitivity for different IDRs. By following this method, researchers can obtain valuable insights into the conformational changes that IDRs undergo in the complex intracellular milieu upon changing environments.

Involvement of CCL2 in Salivary Gland Response to Hyperosmolar Stress Related to Sjögren's Syndrome.

In primary Sjögren's syndrome (pSS) patients, salivary gland (SG) epithelial cells (SGECs) could be exposed to chronic hyperosmotic stress (HOS), consecutive to their destruction and deregulation, that exacerbates an inflammatory response. The aims of this study were to assess the mechanism accounting for C-C motif chemokine ligand 2 (CCL2) expression in an immortalized human salivary gland epithelial acinar cell line (NS-SV-AC) subjected to HOS, as well as the involvement of CCL2 in pSS. CCL2 mRNA and protein levels were determined via RT-qPCR and ELISA. Reporter plasmids and a promoter pull-down assay were used to identify transcription factors associated with CCL2 mRNA increase. Our data showed that HOS-induced CCL2 mRNA increase was independent of the nuclear factor of activated T-cells 5 (NFAT5) and nuclear factor-kappa B (NFkB) but involved Kruppel-like factor 5 (KLF5). CCL2 protein levels, quantified by enzyme-linked immunosorbent assay (ELISA) in sera samples from pSS patients, correlated with the European Alliance of Associations for Rheumatology's Sjogren's syndrome disease activity index (ESSDAI) score for systemic activity. In addition, CCL2 protein levels were higher in patients with biological activity, cutaneous manifestations, and ESSDAI score superior or equal to five. Our data suggest that chronic HOS could exacerbate pSS disease by contributing to the inflammatory process induced by the expression and secretion of CCL2.

Transmembrane Protein CMTM6 Alleviates Ocular Inflammatory Response and Improves Corneal Epithelial Barrier Function in Experimental Dry Eye.

To investigate the impact of transmembrane protein CMTM6 on the pathogenesis of dry eye disease (DED) and elucidate its potential mechanisms. CMTM6 expression was confirmed by database analysis, real-time polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry. Tear secretion was measured using the phenol red thread test. Immune cell infiltration was assessed through flow cytometry. Barrier function was evaluated by fluorescein sodium staining, immunofluorescence staining of zonula occludens 1 (ZO-1), and electric cell-substrate impedance sensing (ECIS) assessment. For silencing CMTM6 expression, siRNA and shRNA were employed, along with lentiviral vector-mediated overexpression of CMTM6. Proinflammatory cytokine levels were analyzed by RT-PCR and cytometric bead array (CBA) analysis. CMTM6 showed high expression in healthy human and mouse corneal and conjunctival epithelium but was notably reduced in DED. Notably, this downregulation was correlated with disease severity. Cmtm6-/- dry eye (DE) mice displayed reduced tear secretion, severe corneal epithelial defects, decreased conjunctival goblet cell density, and upregulated inflammatory response. Additionally, Cmtm6-/- DE mice and CMTM6 knockdown human corneal epithelial cell-transformed (HCE-T) cells showed more severe barrier disruption and reduced expression of ZO-1. Knockdown of CMTM6 in HCE-T cells increased inflammatory responses induced by hyperosmotic stress, which was significantly mitigated by CMTM6 overexpression. Moreover, the level of phospho-p65 in hyperosmolarity-stimulated HCE-T cells increased after silencing CMTM6. Nuclear factor kappa B (NF-κB) p65 inhibition (JSH-23) reversed the excessive inflammatory responses caused by hyperosmolarity in CMTM6 knockdown HCE-T cells. The reduction in CMTM6 expression on the ocular surface contributes to the pathogenesis of DED. The CMTM6-NF-κB p65 signaling pathway may serve as a promising therapeutic target for DED.

The cAMP-PKA signalling crosstalks with CWI and HOG-MAPK pathways in yeast cell response to osmotic and thermal stress.

The yeast Saccharomyces cerevisiae is widely used in food and non-food industries. During industrial fermentation yeast strains are exposed to fluctuations in oxygen concentration, osmotic pressure, pH, ethanol concentration, nutrient availability and temperature. Fermentation performance depends on the ability of the yeast strains to adapt to these changes. Suboptimal conditions trigger responses to the external stimuli to allow homeostasis to be maintained. Stress-specific signalling pathways are activated to coordinate changes in transcription, translation, protein function, and metabolic fluxes while a transient arrest of growth and cell cycle progression occur. cAMP-PKA, HOG-MAPK and CWI signalling pathways are turned on during stress response. Comprehension of the mechanisms involved in the responses and in the adaptation to these stresses during fermentation is key to improving this industrial process. The scope of this review is to outline the advancement of knowledge about the cAMP-PKA signalling and the crosstalk of this pathway with the CWI and HOG-MAPK cascades in response to the environmental challenges heat and hyperosmotic stress.

Pathologic changes in neuronal intranuclear inclusion disease are linked to aberrant FUS interaction under hyperosmotic stress

CGG repeat expansion in NOTCH2NLC is the genetic cause of neuronal intranuclear inclusion disease (NIID). Previous studies indicated that the CGG repeats can be translated into polyglycine protein (N2CpolyG) which was toxic to neurons by forming intranuclear inclusions (IIs). However, little is known about the factors governing polyG IIs formation as well as its molecular pathogenesis. Considering that neurogenetic disorders usually involve interactions between genetic and environmental stresses, we investigated the effect of stress on the formation of IIs. Our results revealed that under hyperosmotic stress, N2CpolyG translocated from the cytoplasm to the nucleus and formed IIs in SH-SY5Y cells, recapitulating the pathological hallmark of NIID patients. Furthermore, N2CpolyG interacted/ co-localized with an RNA-binding protein FUS in the IIs of cellular model and NIID patient tissues, thereby disrupting stress granule formation in cytoplasm under hyperosmotic stress. Consequently, dysregulated expression of microRNAs was found both in NIID patients and cellular model, which could be restored by FUS overexpression in cultured cells. Overall, our findings indicate a mechanism of stress-induced pathological changes as well as neuronal damage, and a potential strategy for the treatment of NIID.

Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties.

A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life.

SCAB1 coordinates sequential Ca2+ and ABA signals during osmotic stress induced stomatal closure in Arabidopsis.

Hyperosmotic stress caused by drought is a detrimental threat to plant growth and agricultural productivity due to limited water availability. Stomata are gateways of transpiration and gas exchange, the swift adjustment of stomatal aperture has a strong influence on plant drought resistance. Despite intensive investigations of stomatal closure during drought stress in past decades, little is known about how sequential signals are integrated during complete processes. Here, we discovered that the rapid Ca2+ signaling and subsequent abscisic acid (ABA) signaling contribute to the kinetics of both F-actin reorganizations and stomatal closure in Arabidopsis thaliana, while STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1) is the molecular switch for this entire process. During the early stage of osmotic shock responses, swift elevated calcium signaling promotes SCAB1 phosphorylation through calcium sensors CALCIUM DEPENDENT PROTEIN KINASE3 (CPK3) and CPK6. The phosphorylation restrained the microfilament binding affinity of SCAB1, which bring about the F-actin disassembly and stomatal closure initiation. As the osmotic stress signal continued, both the kinase activity of CPK3 and the phosphorylation level of SCAB1 attenuated significantly. We further found that ABA signaling is indispensable for these attenuations, which presumably contributed to the actin filament reassembly process as well as completion of stomatal closure. Notably, the dynamic changes of SCAB1 phosphorylation status are crucial for the kinetics of stomatal closure. Taken together, our results support a model in which SCAB1 works as a molecular switch, and directs the microfilament rearrangement through integrating the sequentially generated Ca2+ and ABA signals during osmotic stress induced stomatal closure.

The ankyrin repeat-containing protein PlANK1 mediates mycelial growth, oospore development, and virulence in Peronophythora litchii

Litchi downy blight, caused by Peronophythora litchii, is one of the most serious diseases in major litchi-producing regions worldwide. The ankyrin (ANK) repeat is one of the most common protein-protein interaction motifs found in all kingdoms of life proteins. ANK-containing proteins have been demonstrated to regulate various biological processes in animals, plants, and fungi. However, their functions in phytopathogenic oomycetes remain unknown. Here, we identified 284 non-redundant genes that encode ANK-containing proteins in P. litchii and classified them into 11 subfamilies. Among them, PlANK1 was found to be highly up-regulated in oospores and from zoospores to the infection process. Loss of PlANK1 in P. litchii resulted in impaired mycelial growth and cyst germination, accelerated zoospore encystment, and increased sensitivity to hyperosmotic stresses and Congo red. Furthermore, PlANK1 deletion mutants were defective in oospore formation and development. Inoculation assays showed that the absence of PlANK1 severely diminished the pathogen’s virulence on litchi leaf and fruit. Through transcriptome analysis and nitrogen source utilization assays, we demonstrated that PlANK1 modulates the pathogen’s nitrogen metabolism. Altogether, our findings indicate that PlANK1 is a key regulator of sexual and asexual development, and virulence in P. litchii.

Puerarin alleviates hyperosmotic stress-induced oxidative stress, inflammation, apoptosis and barrier damage of human corneal epithelial cells by targeting SIRT1/NLRP3 signaling

The increase of tear osmolarity caused by excessive evaporation of tear phase is the main pathological mechanism of dry eye disease (DED). Puerarin, the major bioactive ingredient isolated from the root of the Pueraria lobata (Willd.) Ohwi, has been reported to improve ophthalmic diseases in clinic. However, the effect and the potential regulatory mechanism related to silent information regulator sirtuin 1 (SIRT1)/NOD-like receptor family pyrin domain containing 3 (NLRP3) signaling of puerarin in DED has not been evaluated. In this study, we aimed to explore the effect and mechanism of hyperosmotic stress (Hyp)-induced human corneal epithelial cell line (HCE-2). The viability of HCE-2 cells induced by Hyp with or without puerarin treatment was assessed by a CCK-8 assay. Results indicated that puerarin treatment enhanced cell viability, reduced reactive oxygen species (ROS) content, increased CAT and SOD activities, and elevated the ratio of GSH/GSSG in HCE-2 cells exposed to Hyp. Besides, TNF-α, IL-1β and IL-6 contents were decreased by puerarin. Additionally, puerarin inhibited Hyp-induced apoptosis and barrier disruption of HCE-2 cells. Moreover, molecular docking method suggested that puerarin bound to SIRT1, and upregulated SIRT1 and downregulated NLRP3 inflammasome proteins after puerarin treatment was observed. Furthermore, SIRT1 silencing alleviated the protective effects of puerarin on Hyp-induced HCE-2 cell damage. Collectively, puerarin attenuates Hyp-induced injury of HCE-2 cells by targeting regulating SIRT1/NLRP3 signaling.

Development of chemically defined media for Lactococcus lactis subsp. lactis YF11 to eliminate the influence of hyperosmotic stress.

The online version contains supplementary material available at 10.1007/s13205-023-03788-5.

Osmotic Stress Responses, Cell Wall Integrity, and Conidiation Are Regulated by a Histidine Kinase Sensor in Trichoderma atroviride.

Trichoderma atroviride responds to various environmental stressors through the mitogen-activated protein kinase (MAPK) Tmk3 and MAPK-kinase Pbs2 signaling pathways. In fungi, orthologues to Tmk3 are regulated by a histidine kinase (HK) sensor. However, the role of T. atroviride HKs remains unknown. In this regard, the function of the T. atroviride HK Nik1 was analyzed in response to stressors regulated by Tmk3. The growth of the Δnik1 mutant strains was compromised under hyperosmotic stress; mycelia were less resistant to lysing enzymes than the WT strain, while conidia of Δnik1 were more sensitive to Congo red; however, ∆pbs2 and ∆tmk3 strains showed a more drastic defect in cell wall stability. Light-regulated blu1 and grg2 gene expression was induced upon an osmotic shock through Pbs2-Tmk3 but was independent of Nik1. The encoding chitin synthases chs1 and chs2 genes were downregulated after an osmotic shock in the WT, but chs1 and chs3 expression were enhanced in ∆nik1, ∆pbs2, and ∆tmk3. The vegetative growth and conidiation by light decreased in ∆nik1, although Nik1 was unrequired to activate the light-responsive genes by Tmk3. Altogether, Nik1 regulates responses related to the Pbs2-Tmk3 pathway and suggests the participation of additional HKs to respond to stress.

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO–K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

A genetic selection for Mycobacterium smegmatis mutants tolerant to killing by sodium citrate defines a combined role for cation homeostasis and osmotic stress in cell death.

Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine Mycobacterium smegmatis survival in the presence of the chelator sodium citrate. We observed that instead of restricting M. smegmatis growth, concentrated sodium citrate killed M. smegmatis. RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill M. smegmatis under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, mgtE, treZ, and fadD6, we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen M. tuberculosis. Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When Mycobacterium smegmatis is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the mgtE magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis.

Serine protease 35 regulates the fibroblast matrisome in response to hyperosmotic stress.

Hyperosmotic stress occurs in several diseases, but its long-term effects are largely unknown. We used sorbitol-treated human fibroblasts in 3D culture to study the consequences of hyperosmotic stress in the skin. Sorbitol regulated many genes, which help cells cope with the stress condition. The most robustly regulated gene encodes serine protease 35 (PRSS35). Its regulation by hyperosmotic stress was dependent on the kinases p38 and JNK and the transcription factors NFAT5 and ATF2. We identified different collagens and collagen-associated proteins as putative PRSS35 binding partners. This is functionally important because PRSS35 affected the extracellular matrix proteome, which limited cell proliferation. The in vivo relevance of these findings is reflected by the coexpression of PRSS35 and its binding partners in human skin wounds, where hyperosmotic stress occurs as a consequence of excessive water loss. These results identify PRSS35 as a key regulator of the matrisome under hyperosmotic stress conditions.