Intermittent subdiffusion of short nuclear actin rods due to interactions with chromatin.

The interior of cellular nuclei, the nucleoplasm, is a crowded fluid that is pervaded by protein-decorated DNA polymers, the chromatin. Due to the complex architecture of chromatin and a multitude of associated nonequilibrium processes, e.g., DNA repair, the nucleoplasm can be expected to feature nontrivial material properties and hence anomalous transport phenomena. Here, we have used single-particle tracking on nuclear actin rods to probe such transport phenomena. Our analysis reveals that short actin rods in the nucleus show an intermittent, antipersistent subdiffusion with clear signatures of fractional Brownian motion. Moreover, the diffusive motion is heterogeneous with clear signatures of an intermittent switching of trajectories between at least two different mobilities, most likely due to transient associations with chromatin. In line with this interpretation, hyperosmotic stress is seen to stall the motion of nuclear actin rods, whereas hypo-osmotic conditions yield a reptationlike motion. Our data highlights the heterogeneity of transport in the nucleoplasm that needs to be taken into account for an understanding of nucleoplasmic organization and the mechanobiology of nuclei.

Structure and mechanism of the osmoregulated choline transporter BetT.

The choline-glycine betaine pathway plays an important role in bacterial survival in hyperosmotic environments. Osmotic activation of the choline transporter BetT promotes the uptake of external choline for synthesizing the osmoprotective glycine betaine. Here, we report the cryo-electron microscopy structures of Pseudomonas syringae BetT in the apo and choline-bound states. Our structure shows that BetT forms a domain-swapped trimer with the C-terminal domain (CTD) of one protomer interacting with the transmembrane domain (TMD) of a neighboring protomer. The substrate choline is bound within a tryptophan prism at the central part of TMD. Together with functional characterization, our results suggest that in Pseudomonas species, including the plant pathogen P. syringae and the human pathogen Pseudomonas aeruginosa, BetT is locked at a low-activity state through CTD-mediated autoinhibition in the absence of osmotic stress, and its hyperosmotic activation involves the release of this autoinhibition.

Toxic and signaling effects of the anaesthetic lidocaine on rice cultured cells.

Most studies on anesthesia focus on the nervous system of mammals due to their interest in medicine. The fact that any life form can be anaesthetised is often overlooked although anesthesia targets ion channel activities that exist in all living beings. This study examines the impact of lidocaine on rice (Oryza sativa). It reveals that the cellular responses observed in rice are analogous to those documented in animals, encompassing direct effects, the inhibition of cellular responses, and the long-distance transmission of electrical signals. We show that in rice cells, lidocaine has a cytotoxic effect at a concentration of 1%, since it induces programmed reactive oxygen species (ROS) and caspase-like-dependent cell death, as already demonstrated in animal cells. Additionally, lidocaine causes changes in membrane ion conductance and induces a sharp reduction in electrical long-distance signaling following seedlings leaves burning. Finally, lidocaine was shown to inhibit osmotic stress-induced cell death and the regulation of Ca2+ homeostasis. Thus, lidocaine treatment in rice and tobacco (Nicotiana benthamiana) seedlings induces not only cellular but also systemic effects similar to those induced in mammals.

[Metabolic engineering of Klebsiella pneumoniae for 1, 3-propanediol production].

1, 3-propanediol is an important monomer for the production of polytrimethylene terephthalate (PTT). Currently, it is mainly produced by microbial fermentation, which, however, has low production efficiency. To address this problem, this study employed atmospheric room temperature plasma (ARTP) mutagenesis technology and high-throughput screening to obtain a strain with high tolerance to osmotic pressure, which achieved a 1, 3-propanediol titer of 87 g/L. Furthermore, the gene expression elements suitable for Klebsiella pneumoniae were screened, and metabolic engineering was employed to block redundant metabolic pathways (deletion of ldhA, budA, and aldA) and enhance the synthesis pathway (overexpression of dhaB and yqhD). The titer of 1, 3-propanediol produced by the engineered strain increased to 107 g/L. Finally, in a 5 L fermenter, the optimal strain KP-FMME-6 achieved a 1, 3-propanediol titer of 118 g/L, with a glycerol conversion rate of 42% and productivity of 2.46 g/(h·L), after optimization of the fermentation parameters. This study provides a reference for the industrial production of 1, 3-propanediol.

Cell size regulates human endoderm specification through actomyosin-dependent AMOT-YAP signaling.

Cell size is a crucial physical property that significantly impacts cellular physiology and function. However, the influence of cell size on stem cell specification remains largely unknown. Here, we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly, cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore, the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements, we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically, the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation, which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together, our study has established a novel connection between cell size diminution and DE differentiation, which is mediated by AMOT nuclear translocation. Additionally, our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation.

Selection of references for quantitative real-time PCR analysis of microRNAs in Nile tilapia (Oreochromis niloticus) under osmotic stress.

MicroRNAs play crucial regulatory roles in various aspects of development and physiology, including environmental adaptation and stress responses in teleosts. RT-qPCR is the most commonly used method for studying microRNA expression, with the accuracy and reliability of results depending on the use of an appropriate reference gene for normalization. This study aimed to evaluate seven miRNAs (U6, Let-7a, miR-23a, miR-25-3, miR-103, miR-99-5, and miR-455) expression stability in different tissues of Nile tilapia subjected to osmotic stress. Fish were divided into two groups: a control and an experimental group, raised in 0 and 12 ppt salinity water respectively. After 21 days, brain, gills, liver, and posterior intestine were collected for analysis. Different mathematical algorithms (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) were employed to identify the most suitable reference miRNAs. The results indicate that the miR-455/miR-23a combination is a robust reference for normalizing miRNA expression levels in studies of osmotic stress responses in Nile tilapia. The stability of miRNA expression can vary depending on specific stress conditions and biological processes, underscoring the necessity of selecting appropriate normalizing miRNAs for each experimental context. This study identifies reliable reference genes for future RT-qPCR analyses of miRNA expression, thereby enhancing our understanding of molecular responses in fish to environmental challenges. These insights are fundamental to the development of new technologies for the improved management and sustainability of aquaculture practices.

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.

Cartilage extracellular matrix polymers: hierarchical structure, osmotic properties, and function.

Proteoglycans are hierarchically organized structures that play an important role in the hydration and the compression resistance of cartilage matrix. In this study, the static and dynamic properties relevant to the biomechanical function of cartilage are determined at different levels of the hierarchical structure, using complementary osmotic pressure, neutron scattering (SANS) and light scattering (DLS) measurements. In cartilage proteoglycans (PGs), two levels of bottlebrush structures can be distinguished: the aggrecan monomer, which consists of a core protein to which are tethered charged glycosaminoglycan (GAG) chains, and complexes formed of the aggrecan monomers attached around a linear hyaluronic acid backbone. The principal component of GAG, chondroitin sulfate (CS), is used as a baseline in this comparison. The osmotic modulus, measured as a function of the proteoglycan concentration, follows the order CS < aggrecan < aggrecan-HA complex. This order underlines the benefit of the increasing complexity at each level of the molecular architecture. The hierarchical bottlebrush configuration, which prevents interpenetration among the bristles of the aggrecan monomers, enhances both the mechanical properties and the osmotic resistance. The osmotic pressure of the collagen solution is notably smaller than in the proteoglycan systems. This is consistent with its known primary role to provide tensile strength to the cartilage and to confine the aggrecan-HA complexes, as opposed to load bearing. The collective diffusion coefficient D governs the rate of recovery of biological tissue after compressive load. In CS solutions the diffusion process is fast, D ≈ 3 × 10-6 cm2 s-1 at concentrations comparable with that of the GAG chains inside the aggrecan molecule. In CS solutions D is a weakly decreasing function of calcium ion concentration, while in aggrecan and its complexes with HA, the relaxation rate is insensitive to the presence of calcium.

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.

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 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.

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.

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.

Deficiency in Opu Systems Imparts Salt-Sensitivity to Weizmannia coagulans.

Weizmannia coagulans can be used as a starter strain in fermented foods or as a probiotic. However, it is salt-sensitive. Here, W. coagulans genomes were compared with the genomes of strains of Bacillus species (B. licheniformis, B. siamensis, B. subtilis, and B. velezensis) that were isolated from fermented foods and show salt tolerance, to identify the basis for the salt-sensitivity of W. coagulans. Osmoprotectant uptake (Opu) systems transport compatible solutes into cells to help them tolerate osmotic stress. B. siamensis, B. subtilis, and B. velezensis each possess five Opu systems (OpuA, OpuB, OpuC, OpuD, and OpuE); B. licheniformis has all except OpuB. However, W. coagulans only has the OpuC system. Based on these findings, the opuA and opuB operons, and the opuD and opuE genes, were amplified from B. velezensis. Expression of each of these systems, respectively, in W. coagulans increased salt-tolerance. W. coagulans expressing B. velezensis opuA, opuD, or opuE grew in 10.5% NaCl (w/v), whereas wild-type W. coagulans could not grow in 3.5% NaCl. The salt resistance of B. subtilis was also increased by overexpression of B. velezensis opuA, opuB, opuD, or opuE. These results indicate that the salt-susceptibility of W. coagulans arises because it is deficient in Opu systems.

Variations in fluid chemical potential induce fibroblast mechano-response in 3D hydrogels.

Mechanical deformation of skin creates variations in fluid chemical potential, leading to local changes in hydrostatic and osmotic pressure, whose effects on mechanobiology remain poorly understood. To study these effects, we investigate the specific influences of hydrostatic and osmotic pressure on primary human dermal fibroblasts in three-dimensional hydrogel culture models. Cyclic hydrostatic pressure and hyperosmotic stress enhanced the percentage of cells expressing the proliferation marker Ki67 in both collagen and PEG-based hydrogels. Osmotic pressure also activated the p38 MAPK stress response pathway and increased the expression of the osmoresponsive genes PRSS35 and NFAT5. When cells were cultured in two-dimension (2D), no change in proliferation was observed with either hydrostatic or osmotic pressure. Furthermore, basal, and osmotic pressure-induced expression of osmoresponsive genes differed in 2D culture versus 3D hydrogels, highlighting the role of dimensionality in skin cell mechanotransduction and stressing the importance of 3D tissue-like models that better replicate in vivo conditions. Overall, these results indicate that fluid chemical potential changes affect dermal fibroblast mechanobiology, which has implications for skin function and for tissue regeneration strategies.

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.

Transcriptional upregulation of the myo-inositol biosynthesis pathway is enhanced by NFAT5 in hyperosmotically stressed tilapia cells.

Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia (Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species, the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyperosmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO-challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (P < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (P < 0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO-challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO-induced activation of the MIB pathway.NEW & NOTEWORTHY In our study, we use a multi-pronged synthetic biology approach to demonstrate that the fish homolog of the predominant mammalian osmotic stress transcription factor nuclear factor of activated T-cells (NFAT5) also contributes to the activation of hyperosmolality inducible genes in cells of extremely euryhaline fish. However, in addition to NFAT5 the presence of other strong osmotically inducible signaling mechanisms is required for full activation of osmoregulated tilapia genes.

The TSC22D, WNK, and NRBP gene families exhibit functional buffering and evolved with Metazoa for cell volume regulation.

The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity.

Quantitative assessment of the nanoanatomy of the contractile vacuole complex in Trypanosoma cruzi.

Trypanosoma cruzi uses various mechanisms to cope with osmotic fluctuations during infection, including the remodeling of organelles such as the contractile vacuole complex (CVC). Little is known about the morphological changes of the CVC during pulsation cycles occurring upon osmotic stress. Here, we investigated the structure-function relationship between the CVC and the flagellar pocket domain where fluid discharge takes place-the adhesion plaque-during the CVC pulsation cycle. Using TcrPDEC2 and TcVps34 overexpressing mutants, known to have low and high efficiency for osmotic responses, we described a structural phenotype for the CVC that matches their corresponding physiological responses. Quantitative tomography provided data on the volume of the CVC and spongiome connections. Changes in the adhesion plaque during the pulsation cycle were also quantified and a dense filamentous network was observed. Together, the results suggest that the adhesion plaque mediates fluid discharge from the central vacuole, revealing new aspects of the osmoregulatory system in T. cruzi.

Two faces of one coin: Beneficial and deleterious effects of reactive oxygen species during short-term acclimation to hypo-osmotic stress in a decapod crab.

Exposure to environmental changes often results in the production of reactive oxygen species (ROS), which, if uncontrolled, leads to loss of cellular homeostasis and oxidative distress. However, at physiological levels these same ROS are known to be key players in cellular signaling and the regulation of key biological activities (oxidative eustress). While ROS are known to mediate salinity tolerance in plants, little is known for the animal kingdom. In this study, we use the Mediterranean crab Carcinus aestuarii, highly tolerant to salinity changes in its environment, as a model to test the healthy or pathological role of ROS due to exposure to diluted seawater (dSW). Crabs were injected either with an antioxidant [N-acetylcysteine (NAC), 150 mg·kg-1] or phosphate buffered saline (PBS). One hour after the first injection, animals were either maintained in seawater (SW) or transferred to dSW and injections were carried out at 12-h intervals. After ≈48 h of salinity change, all animals were sacrificed and gills dissected for analysis. NAC injections successfully inhibited ROS formation occurring due to dSW transfer. However, this induced 55% crab mortality, as well as an inhibition of the enhanced catalase defenses and mitochondrial biogenesis that occur with decreased salinity. Crab osmoregulatory capacity under dSW condition was not affected by NAC, although it induced in anterior (non-osmoregulatory) gills a 146-fold increase in Na+/K+/2Cl- expression levels, reaching values typically observed in osmoregulatory tissues. We discuss how ROS influences the physiology of anterior and posterior gills, which have two different physiological functions and strategies during hyper-osmoregulation in dSW.