Hyperosmotic Environment Research Articles

Salinity significantly affects intestinal microbiota and gene expression in striped catfish juveniles.

In the present study, juvenile striped catfish (Pangasianodon hypophthalmus), a freshwater fish species, have been chronically exposed to a salinity gradient from freshwater to 20 psu (practical salinity unit) and were sampled at the beginning (D20) and the end (D34) of exposure. The results revealed that the intestinal microbial profile of striped catfish reared in freshwater conditions were dominated by the phyla Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Alpha diversity measures (observed OTUs (operational taxonomic units), Shannon and Faith's PD (phylogenetic diversity)) showed a decreasing pattern as the salinities increased, except for the phylogenetic diversity at D34, which was showing an opposite trend. Furthermore, the beta diversity between groups was significantly different. Vibrio and Akkermansia genera were affected differentially with increasing salinity, the former being increased while the latter was decreased. The genus Sulfurospirillium was found predominantly in fish submitted to salinity treatments. Regarding the host response, the fish intestine likely contributed to osmoregulation by modifying the expression of osmoregulatory genes such as nka1a, nka1b, slc12a1, slc12a2, cftr, and aqp1, especially in fish exposed to 15 and 20 psu. The expression of heat shock proteins (hsp) hsp60, hsp70, and hsp90 was significantly increased in fish reared in 15 and 20 psu. On the other hand, the expression of pattern recognition receptors (PRRs) were inhibited in fish exposed to 20 psu at D20. In conclusion, the fish intestinal microbiota was significantly disrupted in salinities higher than 10 psu and these effects were proportional to the exposure time. In addition, the modifications of intestinal gene expression related to ion exchange and stressful responses may help the fish to adapt hyperosmotic environment. KEY POINTS: • It is the first study to provide detailed information on the gut microbiota of fish using the amplicon sequencing method. • Salinity environment significantly modified the intestinal microbiota of striped catfish. • Intestinal responses may help the fish adapt to hyperosmotic environment.

Molecular characterization and structural dynamics of Aquaporin1 from walking catfish in lipid bilayers

Aquaporin's (AQPs) are the major superfamily of small integral membrane proteins that facilitates transportation of water, urea, ammonia, glycerol and ions across biological cell membranes. Despite of recent advancements made in understanding the biology of Aquaporin's, only few isoforms of aquaporin 1 (AQP1) some of the teleost fish species have been characterized at molecular scale. In this study, we made an attempt to elucidate the molecular mechanism of water transportation in AQP1 from walking catfish (Clarias batrachus), a model species capable of breathing in air and inhabits in challenging environments. Using state-of-the-art computational modelling and all-atoms molecular dynamics simulation, we explored the structural dynamics of full-length aquaporin 1 from walking catfish (CbAQP1) in lipid mimetic bilayers. Unlike AQP1 of human and bovine, structural ensembles of CbAQP1 from MD revealed discrete positioning of pore lining residues at the intracellular end. Snapshots from MD simulation displayed differential dynamics of aromatic/arginine (ar/R) filter and extracellular loop C bridging transmembrane (TM) helix H3 and H4. Distinct conformation of large extracellular loops, loop bridging TM2 domain and HB helix along with positioning of selectivity filter lining residues controls the permeability of water across the bilayer. Moreover, the identified unique and conserved lipid binding sites with 100% lipid occupancy signifies lipid mediated structural dynamics of CbAQP1. All-together, this is the first ever report on structural-dynamics of aquaporin 1 in walking catfish which will be useful to understand the molecular basis of transportation of water and other small molecules under varying degree of hyperosmotic environment.

Dynamic Regulation of Ions and Amino Acids in Adult Asian Hard Clams Meretrix lusoria Upon Hyperosmotic Salinity

The dynamic regulation of ions and amino acids in the gills and mantle of the Asian hard clam, Meretrix lusoria, following the exposure to a hyperosmotic environment was hitherto unclear. The present study revealed that the osmolality as well as the Na+ and Cl– concentrations in the hemolymph were significantly increased 3 h after transferring the clams from an environment with the salinity of their natural habitat (brackish water; BW; 20‰) to one with hyperosmotic salinity (seawater; 35‰). In addition, we found that the specific activities of Na+/K+-ATPase, a key enzyme that plays a significant role in cell osmoregulation, in the gills and mantle of clams were significantly increased at 72 and 12 h post-transfer, respectively, during acclimation to hyperosmotic salinity. Similarly, the contents of free amino acids (FAAs) such as taurine, alanine, and glycine were significantly elevated during hyperosmotic salinity acclimation. Previous research indicates that taurine is the most abundant FAA in the gills and mantles of Asian hard clams and that the taurine transporter (TAUT) plays an important role in taurine accumulation. The present study showed that TAUT mRNA and protein expression were significantly and transiently increased in the mantle of Asian hard clams following exposure to seawater; although the expression of TAUT mRNA in the gills of Asian hard clams was also transiently stimulated by exposure to hyperosmotic salinity, the relative TAUT protein abundance decreased only at later stages. Accordingly, the findings of this study improve our understanding of the dynamic processes of ion and amino acid regulation in the peripheral tissues of bivalves under hyperosmotic stress.

Correlations between environmental salinity levels, blood biochemistry parameters, and steroid hormones in wild juvenile American alligators (Alligator mississippiensis)

American alligators (Alligator mississippiensis) inhabit freshwater wetlands that are vulnerable to salinization caused by anthropogenic alterations to freshwater flow, in addition to storm surges, sea level rise, and droughts. Salinization of coastal freshwater habitats is a growing concern in a changing climate due to increased frequency and intensity of storm surges and drought conditions. This study opportunistically sampled juvenile male and female wild alligators in various salinities each month excluding November, December, and January for one year at Rockefeller Wildlife Refuge in coastal Louisiana. Blood plasma biochemistry parameters including electrolyte levels were subsequently measured. In addition, levels of various renin–angiotensin–aldosterone system hormones, glucocorticoids, androgens, estrogens, and progestogens were analyzed using liquid chromatography and tandem mass spectrometry. Only males were sampled in hyperosmotic environments (> 10‰) during dry conditions in late summer 2018. In juvenile males, plasma Na+, Cl−, and the progestogen 17α,20β-dihydroxypregnenone were significantly and positively correlated with environmental salinity. However, variation in glucocorticoids, androgens, and estrogens were not associated with hypersaline water while sex steroids showed significant seasonal variation. This study demonstrated significant correlation of environmental salinity with electrolyte levels and a sex steroid in wild juvenile alligators, and to our knowledge represents the first measurement of 17α,20β-dihydroxypregnenone in alligators.

Magnesium transport in the aglomerular kidney of the Gulf toadfish (Opsanus beta).

Despite having an aglomerular kidney, Gulf toadfish can survive in water ranging from nearly fresh up to 70 parts per thousand salinity. In hyperosmotic environments, the major renal function is to balance the passive Mg2+ load from the environment with an equal excretion. However, the molecular transporters involved in Mg2+ secretion are poorly understood. We investigated whether environmental MgCl2 alone or in combination with elevated salinity affected transcriptional regulation of genes classically involved in renal Mg2+ secretion (slc41a1, slc41a3, cnnm3) together with three novel genes (trpm6, trpm7, claudin-19) and two isoforms of the Na+/K+-ATPase α-subunit (nka-α1a, nka-α1b). First, toadfish were acclimated to 5, 9, 35, or 60 ppt water (corresponding to ~ 7, 13, 50 and 108mmolL-1 ambient [Mg2+], respectively) and sampled at 24h or 9days. Next, the impact of elevated ambient [Mg2+] was explored by exposing toadfish to control (50mmolL-1 Mg2+), or elevated [Mg2+] (100mmolL-1) at a constant salinity for 7days. Mg2+ levels in this experiment corresponded with levels in control and hypersaline conditions in the first experiment. A salinity increase from 5 to 60 ppt stimulated the level of all investigated transcripts in the kidney. In Mg2+-exposed fish, we observed a 14-fold increase in the volume of intestinal fluids and elevated plasma osmolality and [Mg2+], suggesting osmoregulatory challenges. However, none of the renal gene targets changed expression compared with the control group. We conclude that transcriptional regulation of renal Mg2+ transporters is induced by elevated [Mg2+] in combination with salinity rather than elevated ambient [Mg2+] alone.

Mummichog gill and operculum exhibit functionally consistent claudin-10 paralog profiles and Claudin-10c hypersaline response.

ABSTRACTClaudin (Cldn)-10 tight junction (TJ) proteins are hypothesized to form the paracellular Na+ secretion pathway of hyposmoregulating mummichog (Fundulus heteroclitus) branchial epithelia. Organ-specific expression profiles showed that only branchial organs [the gill and opercular epithelium (OE)] exhibited abundant cldn-10 paralog transcripts, which typically increased following seawater (SW) to hypersaline (2SW) challenge. Post-translational properties, protein abundance, and ionocyte localization of Cldn-10c, were then examined in gill and OE. Western blot analysis revealed two Cldn-10c immunoreactive bands in the mummichog gill and OE at ∼29 kDa and ∼40 kDa. The heavier protein could be eliminated by glycosidase treatment, demonstrating the novel presence of a glycosylated Cldn-10c. Protein abundance of Cldn-10c increased in gill and OE of 2SW-exposed fish. Cldn-10c localized to the sides of gill and OE ionocyte apical crypts and partially colocalized with cystic fibrosis transmembrane conductance regulator and F-actin, consistent with TJ complex localization. Cldn-10c immunofluorescent intensity increased but localization was unaltered by 2SW conditions. In support of our hypothesis, cldn-10/Cldn-10 TJ protein dynamics in gill and OE of mummichogs and TJ localization are functionally consistent with the creation and maintenance of salinity-responsive, cation-selective pores that facilitate Na+ secretion in hyperosmotic environments.

Age-Dependent Decline in Salinity Tolerance in a Euryhaline Fish.

Euryhaline teleost fish are characterized by their ability to tolerate a wide range of environmental salinities by modifying the function of osmoregulatory cells and tissues. In this study, we experimentally addressed the age-related decline in the sensitivity of osmoregulatory transcripts associated with a transfer from fresh water (FW) to seawater (SW) in the euryhaline teleost, Mozambique tilapia, Oreochromis mossambicus. The survival rates of tilapia transferred from FW to SW were inversely related with age, indicating that older fish require a longer acclimation period during a salinity challenge. The relative expression of Na+/K+/2Cl− cotransporter 1a (nkcc1a), which plays an important role in hyposmoregulation, was significantly upregulated in younger fish after SW transfer, indicating a clear effect of age in the sensitivity of branchial ionocytes. Prolactin (Prl), a hyperosmoregulatory hormone in O. mossambicus, is released in direct response to a fall in extracellular osmolality. Prl cells of 4-month-old tilapia were sensitive to hyposmotic stimuli, while those of >24-month-old fish did not respond. Moreover, the responsiveness of branchial ionocytes to Prl was more robust in younger fish. Taken together, multiple aspects of osmotic homeostasis, from osmoreception to hormonal and environmental control of osmoregulation, declined in older fish. This decline appears to undermine the ability of older fish to survive transfer to hyperosmotic environments.

Environmental Salinity Modifies Mucus Exudation and Energy Use in European Sea Bass Juveniles.

Simple SummaryChanges in skin mucus production and composition offer a new means to study how fish cope with changes in the environment. We explored the utility of skin mucus as an indicator of physiological responses and energy use in a reference fish species, the European sea bass. We evaluated the exudation volume of skin mucus and the main stress- and osmoregulation-related biomarkers in both mucus and plasma. We demonstrate the viability to study the exuded volume of skin mucus composition and its parameters as an informative tool of the fish energy waste at different environmental salinities. This study is of great interest for both aquaculture and ecological studies.The European sea bass (Dicentrarchus labrax) is a euryhaline marine teleost that can often be found in brackish and freshwater or even in hypersaline environments. Here, we exposed sea bass juveniles to sustained salinity challenges for 15 days, simulating one hypoosmotic (3‰), one isosmotic (12‰) and one hyperosmotic (50‰) environment, in addition to control (35‰). We analyzed parameters of skin mucus exudation and mucus biomarkers, as a minimally invasive tool, and plasma biomarkers. Additionally, Na+/K+-ATPase activity was measured, as well as the gill mucous cell distribution, type and shape. The volume of exuded mucus increased significantly under all the salinity challenges, increasing by 130% at 50‰ condition. Significantly greater amounts of soluble protein (3.9 ± 0.6 mg at 50‰ vs. 1.1 ± 0.2 mg at 35‰, p < 0.05) and lactate (4.0 ± 1.0 µg at 50‰ vs. 1.2 ± 0.3 µg at 35‰, p < 0.05) were released, with clear energy expenditure. Gill ATPase activity was significantly higher at the extreme salinities, and the gill mucous cell distribution was rearranged, with more acid and neutral mucin mucous cells at 50‰. Skin mucus osmolality suggested an osmoregulatory function as an ion-trap layer in hypoosmotic conditions, retaining osmosis-related ions. Overall, when sea bass cope with different salinities, the hyperosmotic condition (50‰) demanded more energy than the extreme hypoosmotic condition.

Phenotypic and genomic characterization of a Vibrio parahaemolyticus strain causing disease in Penaeus vannamei provides insights into its niche adaptation and pathogenic mechanism

The virulence of Vibrio parahaemolyticus is variable depending on its virulence determinants. A V. parahaemolyticus strain, in which the virulence is governed by the pirA and pirB genes, can cause acute hepatopancreatic necrosis disease (AHPND) in shrimps. Some V. parahaemolyticus that are non-AHPND strains also cause shrimp diseases and result in huge economic losses, while their pathogenicity and pathogenesis remain unclear. In this study, a non-AHPND V. parahaemolyticus , TJA114, was isolated from diseased Penaeus vannamei associated with a high mortality. To understand its virulence and adaptation to the external environment, whole-genome sequencing of this isolate was conducted, and its phenotypic profiles including pathogenicity, growth characteristics and nutritional requirements were investigated. Shrimps following artificial infection with this isolate presented similar clinical symptoms to the naturally diseased ones and generated obvious pathological lesions. The growth characteristics indicated that the isolate TJA114 could grow well under different salinity (10–55 p.p.t.), temperature (23–37 °C) and pH (6–10) conditions. Phenotype MicroArray results showed that this isolate could utilize a variety of carbon sources, amino acids and a range of substrates to help itself adapt to the high hyperosmotic and alkaline environments. Antimicrobial-susceptibility test showed that it was a multidrug-resistant bacterium. The whole-genomic analysis showed that this V. parahaemolyticus possessed many important functional genes associated with multidrug resistance, stress response, adhesions, haemolysis, putative secreted proteases, dedicated protein secretion systems and a variety of nutritional metabolic mechanisms. These annotated functional genes were confirmed by the phenotypic profiles. The results in this study indicated that this V. parahaemolyticus isolate possesses a high pathogenicity and strong environmental adaptability.

NFAT5-Mediated Signalling Pathways in Viral Infection and Cardiovascular Dysfunction.

The nuclear factor of activated T cells 5 (NFAT5) is well known for its sensitivity to cellular osmolarity changes, such as in the kidney medulla. Accumulated evidence indicates that NFAT5 is also a sensitive factor to stress signals caused by non-hypertonic stimuli such as heat shock, biomechanical stretch stress, ischaemia, infection, etc. These osmolality-related and -unrelated stimuli can induce NFAT5 upregulation, activation and nuclear accumulation, leading to its protective role against various detrimental effects. However, dysregulation of NFAT5 expression may cause pathological conditions in different tissues, leading to a variety of diseases. These protective or pathogenic effects of NFAT5 are dictated by the regulation of its target gene expression and activation of its signalling pathways. Recent studies have found a number of kinases that participate in the phosphorylation/activation of NFAT5 and related signal proteins. Thus, this review will focus on the NFAT5-mediated signal transduction pathways. As for the stimuli that upregulate NFAT5, in addition to the stresses caused by hyperosmotic and non-hyperosmotic environments, other factors such as miRNA, long non-coding RNA, epigenetic modification and viral infection also play an important role in regulating NFAT5 expression; thus, the discussion in this regard is another focus of this review. As the heart, unlike the kidneys, is not normally exposed to hypertonic environments, studies on NFAT5-mediated cardiovascular diseases are just emerging and rapidly progressing. Therefore, we have also added a review on the progress made in this field of research.

Salinity induced alterations in photosynthetic and oxidative regulation are ameliorated as a function of salt secretion.

Ion secretion facilitates recretohalophytes to tolerate saline and drought conditions but its relative contribution to the survival of many species remains poorly understood. Tamarix chinensis has high potential for restoration of saline deteriorated lands. The water management and high salt tolerance of the plant have highlighted the need to determine the strategies that govern these mechanisms. Here we report the selectivity of this halophyte to transport, utilize, and secrete different cations and anions under various NaCl (0, 100, 200 and 400mM) concentrations. Plant growth, photosynthesis and antioxidant defense responses were also determined to relate them with the function of ion secretion. Results reflected two different sets of strategies adopted by plants to survive low and high salinities. Exposure to highly saline conditions caused reduction in photosynthesis due to stomatal and biochemical limitations. The decreased content of photosynthetic pigments exposed plants to excessive light energy that accelerated production of ROS (i.e., hydrogen peroxide H2O2) and caused damage to cellular membranes. The increased activities of anti-oxidative enzymes (superoxide-dismutase, catalase, ascorbate-peroxidase, and glutathione-reductase) were insufficient to detoxify H2O2. In contrast, plants treated with low salinity did not face stomatal limitations while the photosynthetic pigments increased. As no damage to membranes was detected, the increased content of H2O2 was postulated for its messenger role. The assimilation of essential nutrients was affected due to increased content of toxic ions (Na+ and Cl-) in the growing medium and within the plants. However, the ability to regulate K+ facilitated plants to improve water use efficiency under hyper-osmotic environment. The removal of toxic ions from the photosynthesizing tissues demands high energy, which was evident in the compromised growth of plants. This study offers a window to physiological mechanisms, e.g., potassium retention that ensure salt secretion as a beneficial strategy for prolonged survival of T. chinensis.

TonEBP regulates the hyperosmotic expression of aquaporin 1 and 5 in the intervertebral disc

The central region of the intervertebral disc (IVD) is rich in proteoglycans, leading to a hyperosmotic environment, which fluctuates with daily loading. The cells of the nucleus pulposus (NP cells) have adapted to this environment via the function of tonicity enhancer binding protein (TonEBP), and NP cells have been shown to express several water channels known as aquaporins (AQP). We have previously shown that AQP1 and 5 decrease during IVD degeneration. Here, the regulation of AQP1 and 5 by hyperosmotic conditions and the role of TonEBP in this regulation was investigated. AQP1 and 5 gene expression was upregulated by hyperosmotic conditions mimicking the osmolality of the healthy IVD, which was abrogated by TonEBP knockdown. Furthermore, AQP1 and 5 immunopositivity was significantly reduced in TonEBPΔ/Δ E17.5 mice when compared with wildtype controls, indicating in vivo expression of AQP1 and 5 is controlled at least in part by TonEBP. This hyperosmotic regulation of AQP1 and 5 could help to explain the decreased AQP1 and 5 expression during degeneration, when the osmolality of the NP decreases. Together this data suggests that TonEBP-regulated osmo-adaptation may be disrupted during IVD degeneration when the expression of both AQPs is reduced.

NFAT5 promotes oral squamous cell carcinoma progression in a hyperosmotic environment

AbstractEpidermal growth factor receptor (EGFR) is highly expressed in several types of cancer cells including oral squamous cell carcinoma (OSCC). EGF/EGFR signaling is recognized as an important molecular target in cancer therapy. However, cancer cells often become tolerant to EGF/EGFR signaling-targeted therapies. In the tumor microenvironment, the tumor incites inflammation and the inflammation-derived cytokines make a considerable impact on cancer development. In addition, hyperosmolarity is also induced, but the role of osmotic stress in cancer development has not been fully understood. This study demonstrates molecular insights into hyperosmolarity effect on OSCC development and shows that NFAT5 transcription factor plays an important functional role in enhancing the oral cancer cell proliferation by inducing the EGFR translocation from the endoplasmic reticulum to the plasma membrane through increase the expression of DPAGT1, an essential enzyme for catalyzing the first committed step of N-linked protein glycosylation. These results suggest that hyperosmolarity-induced intra-nuclear translocation of NFAT5 essential for DPAGT1 activation and EGFR subcellular translocation responsible for OSCC tumor progression.This study demonstrates that NFAT5 promotes oral squamous cell carcinoma progression in the hyperosmotic environment through increased expression of DPAGT1, an essential enzyme for protein glycosylation, and altered EGFR subcellular localization from the cytoplasm to the plasma membrane in tumor cells.

Recovery from Hypersaline-Stress-Induced Immunity Damage and Intestinal-Microbiota Changes through Dietary β-glucan Supplementation in Nile tilapia (Oreochromis niloticus)

Simple SummaryLong-term hypersaline stress can induce coagulation disorders and splenomegaly and down-regulate the complement pathway in tilapia, which can increase risk in healthy breeding. As a prebiotic, β-glucan dietary supplementation can significantly reduce enlarged spleen resulting from hypersaline stress. The hematological aspects of the red blood cell count, hematocrit, red cell distribution width, platelet count, and plateletcrit were also decreased by supplementation with dietary β-glucan. In the spleen and intestine, β-glucan intake significantly decreased the high expression of immune-related genes due to hypersaline stress resulting from β-glucan intake in tilapia. β-glucan supplementation also significantly increased the abundance of beneficial microbiota such as Lactobacillus, Phycicoccus, and Rikenellaceae in the intestine. In summary, β-glucan intake can relieve tissue damage and optimize the intestinal microbiota of tilapia in brackish water and improve fish health.Long-term exposure to hyperosmotic environments can induce severe immune damage and increase risk in tilapia breeding. As an effective immunoregulator, β-glucan has attracted extensive attention in nutritional research and given rise to high expectations of improving health status and alleviating organismal damage in tilapia, Oreochromis niloticus, in brackish water. In this study, an 8-week cultivation experiment was conducted on tilapia fed a basal diet or diets with β-glucan supplementation in freshwater (control) and brackish water. Growth performance, hematological aspects, immune cytokine expression, and the intestinal microbiota of tilapia were analyzed. The results indicated that supplementation with β-glucan significantly reduced the enlarged spleen of tilapia resulting from hypersaline stress. Tilapia fed β-glucan showed significantly-greater decreases in the red blood cell count, hematocrit, red cell distribution width, platelet count, and plateletcrit than those fed the basal diet. β-glucan significantly decreased the high expression of immune-related genes in the spleen induced by hyperosmotic stress. In the intestine, the high migration inhibitory factor-2 (MIF-2) and IL-1β gene expression induced by hypersaline stress was significantly reduced. β-glucan supplementation also significantly increased the abundance of beneficial microbiota such as Lactobacillus, Phycicoccus, and Rikenellaceae. Therefore, dietary β-glucan supplementation can significantly reduce spleen enlargement and improve immune function in tilapia in brackish water. β-glucan intake can also optimize the intestinal microbiota of tilapia in brackish water and improve fish health.

Contribution of the mitogen-activated protein kinase Hog1 to the halotolerance of the marine yeast Debaryomyces hansenii

Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.

Hyperosmotic Adaptation of Pseudomonas protegens SN15-2 Helps Cells to Survive at Lethal Temperatures

Pseudomonas protegens is a gram-negative bacterium with an excellent biological control effect. Compared to standard cells growing in NaCl-free media, the ability of the hyperosmotic cells (450 mM NaCl) to resist high temperatures and freezing was significantly improved. It is of great significance to apply the P. protegens to elaborate on the hyperosmotic adaptation mechanism. RNA-seq was used to sequence P. protegens cultured with 0 mM and 450 mM NaCl. Comparative transcriptomic analyses of the different treatments were performed using gene ontology and the Kyoto encyclopedia of genes and genome. The results revealed that hyperosmotic stress had prominent impacts on the genes involving in multiple cellular functions. The hyperosmotic environment significantly affected carbohydrate, energy, and amino acid metabolism, as well as membrane system and cell motility. Our findings indicated that P. protegens adopted a series of approaches, including the high cytoplasmic concentrations of potassium ions and the uptake or synthesis of osmoprotectants, for surviving hyperosmotic stress. Among these, trehalose and proline synthesis appeared to be an important method to withstand prolonged hyperosmotic stress in P. protegens. These data provide crucial resource that may determine specific responses to the hyperosmotic environment in P. protegens.

Inactivation kinetics modeling of Escherichia coli in concentrated urine for implementing predictive environmental microbiology in sanitation safety planning

Urine concentration (condensation) leads to the inactivation of pathogens in urine owing to a hyperosmotic environment. This study proposed an inactivation kinetic model of Escherichia coli (E. coli), a surrogate of human bacterial pathogens, in concentrated synthetic urine. The model parameters were obtained under an assumption that the inactivation rate of E. coli followed a binomial distribution, which made it possible to accurately simulate the time-course decay of E. coli in synthetic urine. The inactivation rate constant values obtained in concentrated urine samples, ammonium buffer solutions and carbonate buffer solutions indicated that the osmotic pressure was a relatively predominant cause for the inactivation of E. coli. The appropriate storage time was estimated using the approach of quantitative microbial risk assessment, which indicated that the 5-fold concentrated urine could be safely collected after 1-day storage when urea was hydrolyzed, whereas 91-hour storage was required for non-concentrated urine. The occupational risk was not negligible even with 6-month storage at 20 °C when urea was not hydrolyzed, which suggested that the urine storage styles should be clarified more minutely. The present study highlights the importance of “predictive environmental microbiology,” which deals with inactivation kinetic models of microorganisms under varied environmental conditions to fully implement the hazard analysis and critical control point (HACCP) approach for the safe use of human excreta in agriculture.

Effect of salinity acclimation on osmoregulation, oxidative stress, and metabolic enzymes in the invasive Xenopus laevis.

Aquatic animals often display physiological adjustments to improve their biological performance and hydrosaline balance in saline environments. In addition to energetic costs associated with osmoregulation, oxidative stress, and the activation of the antioxidant system are common cellular responses to salt stress in many species, but the knowledge of osmoregulation-linked oxidative homeostasis in amphibians is scarce. Here we studied the biochemical responses and oxidative responses of Xenopus laevis females exposed for 40 days to two contrasting salinities: hypo-osmotic (150 mOsm·kg-1 ·H2 O NaCl, HYPO group) and hyper-osmotic environments (340 mOsm·kg-1 ·H2 O NaCl, HYPER group). We found an increase of plasma osmolality and plasma urea concentration in the animals incubated in the HYPER treatment. Increases in electrolyte concentration were paralleled with an increase of both citrate synthase and cytochrome c oxidase activities in liver and heart. Interestingly, HYPO group had higher catabolic activity of the skin and liver total antioxidant capacity (TAC), compared with animals from the HYPER group. Moreover, there was an inverse relationship between liver TAC and plasma osmolality; and with the metabolic enzymes from liver. These findings suggest that salinity induces changes in urea metabolism and specific activity of metabolic enzymes, which appears to be tissue-dependent in X. laevis. Contrary to our expectations, we also found a moderate change in the oxidative status as revealed by the increase in TAC activity in the animals acclimated to low salinity medium, but constancy in the lipid peroxidation of membranes.

RanBP3L is a NFAT5 target gene and its deficiency in principal cells promotes epithelial mesenchymal transition through MAPK signaling

Inner medullary kidney cells are exposed to an increasing hyperosmolality altering several intracellular processes. As a key regulator involved in adaption to hyperosmotic environment the transcription factor nuclear factor of activated T cells‐5 (NFAT5) was identified. Its nuclear translocation results in activation of different genes to restore homeostasis. By using primarily cultured rat renal inner medullary collecting duct cells (IMCD) and microarray analysis we identified hundreds of genes that showed differences in expression level in a time‐ and osmolality‐dependent manner. The kidney specific Ran binding protein 3 like (RanBP3L) showed the highest changes in expression level and is located in IMCDs. Within the collecting duct its expression is specific to the principal cells. Since studies describing the renal function are missing, we further characterized the cellular and physiological role of RanBP3L in a renal mouse cell line.We used the murine kidney collecting duct cell line mpKCCD creating a RanBP3L deficient cell clone by using CRISPR/Cas9. To investigate its physiological role the gene expression profiles were analyzed by Next Generation Sequencing. We further analyzed the migration and colony forming capability of the cells and used immunofluorescence to study differences in cell morphology and specific signaling pathways. These experiments were performed under isoosmolar (300 mosmol/kg) and hyperosmolar (600 mosmol/kg) conditions. Since RanBP3L expression is induced by hyperosmolality, the NFAT5 dependent expression of RanBP3L was examined through CRISPR/Cas9 mediated NFAT5 knock out.Loss of RanBP3L under 300 mosmol/kg showed no significant changes in the morphology while under 600 mosmol/kg massive changes were induced resulting in a loss of epithelial structures. This correlates with our functional analysis showing a higher migration and invasiveness capacity. Furthermore RanBP3L deficiency is associated with a massive change in gene expression, indicating a dysregulation of stress associated pathways. Immunofluorescence showed that TGF‐β stimulation in wild type induced a similar phenotype to RanBP3L knock out cells. Downstream analysis of MAPK pathway showed that the key proteins JNK, c‐JUN and Smad2 are deregulated in RanBP3L deficient cells resulting in an epithelial mesenchymal transition (EMT) like phenotype, which is a hallmark in cancer development. This matches with the data from the cancer genome atlas from patients with renal clear cell carcinoma (RCC) where low RanBP3L expression level is associated with shorter overall survival. It seems as if the expression level of RanBP3L in kidney cells is crucial for cell protection. Here we could show that RanBP3L expression is reduced in NFAT5 deficient cells, indicating that RanBP3L is a NFAT5 target gene.Taken together we have shown that RanBP3L is expressed NFAT5 dependent and prevents a MAPK associated EMT in renal cells and should be used as a prognostic marker for patients with RCC. Further experiments, like in‐vivo studies of a conditional RanBP3L knock out mouse will give us more insights to resolve the underlying pathway.Support or Funding Informationgerman research foundation

Recent progress on bio-based production of dicarboxylic acids in yeast.

Dicarboxylic acids are widely used in fine chemical and food industries as well as the monomer for polymerisation of high molecular material. Given the problems of environmental contamination and sustainable development faced by traditional production of dicarboxylic acids based on petrol, new approaches such as bio-based production of dicarboxylic acids drew more attentions. The yeast, Saccharomyces cerevisiae, was regarded as an ideal organism for bio-based production of dicarboxylic acids with high tolerance to acidic and hyperosmotic environments, robust growth using a broad range of substrates, great convenience for genetic manipulation, stable inheritance via sub-cultivation, and food compatibility. In this review, the production of major dicarboxylates via S. cerevisiae was concluded and the challenges and opportunities facing were discussed.Key Points• Summary of current production of major dicarboxylic acids by Saccharomyces cerevisiae.• Discussion of influence factors on four-carbon dicarboxylic acids production by Saccharomyces cerevisiae.• Outlook of potential production of five- and six-carbon dicarboxylic acids by Saccharomyces cerevisiae.