Magnesium Flux Modulates Ribosomes to Increase Bacterial Survival.

Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.

Exploiting protein language models for the precise classification of ion channels and ion transporters.

This study introduces TooT-PLM-ionCT, a comprehensive framework that consolidates three distinct systems, each meticulously tailored for one of the following tasks: distinguishing ion channels (ICs) from membrane proteins (MPs), segregating ion transporters (ITs) from MPs, and differentiating ICs from ITs. Drawing upon the strengths of six Protein Language Models (PLMs)-ProtBERT, ProtBERT-BFD, ESM-1b, ESM-2 (650M parameters), and ESM-2 (15B parameters), TooT-PLM-ionCT employs a combination of traditional classifiers and deep learning models for nuanced protein classification. Originally validated on an existing dataset by previous researchers, our systems demonstrated superior performance in identifying ITs from MPs and distinguishing ICs from ITs, with the IC-MP discrimination achieving state-of-the-art results. In light of recommendations for additional validation, we introduced a new dataset, significantly enhancing the robustness and generalization of our models across bioinformatics challenges. This new evaluation underscored the effectiveness of TooT-PLM-ionCT in adapting to novel data while maintaining high classification accuracy. Furthermore, this study explores critical factors affecting classification accuracy, such as dataset balancing, the impact of using frozen versus fine-tuned PLM representations, and the variance between half and full precision in floating-point computations. To facilitate broader application and accessibility, a web server (https://tootsuite.encs.concordia.ca/service/TooT-PLM-ionCT) has been developed, allowing users to evaluate unknown protein sequences through our specialized systems for IC-MP, IT-MP, and IC-IT classification tasks.

Inroads into saline-alkaline stress response in plants: unravelling morphological, physiological, biochemical, and molecular mechanisms.

MAIN CONCLUSION: This article discusses the complex network of ion transporters, genes, microRNAs, and transcription factors that regulate crop tolerance to saline-alkaline stress. The framework aids scientists produce stress-tolerant crops for smart agriculture. Salinity and alkalinity are frequently coexisting abiotic limitations that have emerged as archetypal mediators of low yield in many semi-arid and arid regions throughout the world. Saline-alkaline stress, which occurs in an environment with high concentrations of salts and a high pH, negatively impacts plant metabolism to a greater extent than either stress alone. Of late, saline stress has been the focus of the majority of investigations, and saline-alkaline mixed studies are largely lacking. Therefore, a thorough understanding and integration of how plants and crops rewire metabolic pathways to repair damage caused by saline-alkaline stress is of particular interest. This review discusses the multitude of resistance mechanisms that plants develop to cope with saline-alkaline stress, including morphological and physiological adaptations as well as molecular regulation. We examine the role of various ion transporters, transcription factors (TFs), differentially expressed genes (DEGs), microRNAs (miRNAs), or quantitative trait loci (QTLs) activated under saline-alkaline stress in achieving opportunistic modes of growth, development, and survival. The review provides a background for understanding the transport of micronutrients, specifically iron (Fe), in conditions of iron deficiency produced by high pH. Additionally, it discusses the role of calcium in enhancing stress tolerance. The review highlights that to encourage biomolecular architects to reconsider molecular responses as auxiliary for developing tolerant crops and raising crop production, it is essential to (a) close the major gaps in our understanding of saline-alkaline resistance genes, (b) identify and take into account crop-specific responses, and (c) target stress-tolerant genes to specific crops.

Mechanisms of cerebrospinal fluid secretion by the choroid plexus epithelium: Application to various intracranial pathologies.

The choroid plexus (CP) is a small yet highly active epithelial tissue located in the ventricles of the brain. It secretes most of the CSF that envelops the brain and spinal cord. The epithelial cells of the CP have a high fluid secretion rate and differ from many other secretory epithelia in the organization of several key ion transporters. One striking difference is the luminal location of, for example, the vital Na+-K+-ATPase. In recent years, there has been a renewed focus on the role of ion transporters in CP secretion. Several studies have indicated that increased membrane transport activity is implicated in disorders such as hydrocephalus, idiopathic intracranial hypertension, and posthemorrhagic sequelae. The importance of the CP membrane transporters in regulating the composition of the CSF has also been a focus in research in recent years, particularly as a regulator of breathing and hemodynamic parameters such as blood pressure. This review focuses on the role of the fundamental ion transporters involved in CSF secretion and its ion composition. It gives a brief overview of the established factors and controversies concerning ion transporters, and finally discusses future perspectives related to the role of these transporters in the CP epithelium.

Diversity of cells and signals in the cardiovascular system.

This white paper is the outcome of the seventh UC Davis Cardiovascular Research Symposium on Systems Approach to Understanding Cardiovascular Disease and Arrhythmia. This biannual meeting aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2022 Symposium was 'Cell Diversity in the Cardiovascular System, cell-autonomous and cell-cell signalling'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies, and challenges in examining cell and signal diversity, co-ordination and interrelationships involved in cardiovascular function. This paper originates from the topics of formal presentations and informal discussions from the Symposium, which aimed to develop a holistic view of how the multiple cell types in the cardiovascular system integrate to influence cardiovascular function, disease progression and therapeutic strategies. The first section describes the major cell types (e.g. cardiomyocytes, vascular smooth muscle and endothelial cells, fibroblasts, neurons, immune cells, etc.) and the signals involved in cardiovascular function. The second section emphasizes the complexity at the subcellular, cellular and system levels in the context of cardiovascular development, ageing and disease. Finally, the third section surveys the technological innovations that allow the interrogation of this diversity and advancing our understanding of the integrated cardiovascular function and dysfunction.

Heterologous expression reveals unique properties of Trk K+ importers from nonconventional biotechnologically relevant yeast species together with their potential to support Saccharomyces cerevisiae growth.

In the model yeast Saccharomyces cerevisiae, Trk1 is the main K+ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K+ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins' abilities to support the growth of cells under various cultivation conditions such as low K+ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb+ . Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K+ and high salt and that the overproduction of S. cerevisiae's own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li+ ions.

Physiological and Transcriptional Analyses Provide Insight into Maintaining Ion Homeostasis of Sweet Sorghum under Salt Stress.

Sweet sorghum is an important bioenergy grass and valuable forage with a strong adaptability to saline environments. However, little is known about the mechanisms of sweet sorghum coping with ion toxicity under salt stresses. Here, we first evaluated the salt tolerance of a sweet sorghum cultivar "Lvjuren" and determined its ion accumulation traits under NaCl treatments; then, we explored key genes involved in Na+, Cl-, K+ and NO3- transport using transcriptome profiling and the qRT-PCR method. The results showed that growth and photosynthesis of sweet sorghum were unaffected by 50 and 100 mM NaCl treatments, indicative of a strong salt tolerance of this species. Under NaCl treatments, sweet sorghum could efficiently exclude Na+ from shoots and accumulate Cl- in leaf sheaths to avoid their overaccumulation in leaf blades; meanwhile, it possessed a prominent ability to sustain NO3- homeostasis in leaf blades. Transcriptome profiling identified several differentially expressed genes associated with Na+, Cl-, K+ and NO3- transport in roots, leaf sheaths and leaf blades after 200 mM NaCl treatment for 6 and 48 h. Moreover, transcriptome data and qRT-PCR results indicated that HKT1;5, CLCc and NPF7.3-1 should be key genes involved in Na+ retention in roots, Cl- accumulation in leaf sheaths and maintenance of NO3- homeostasis in leaf blades, respectively. Many TFs were also identified after NaCl treatment, which should play important regulatory roles in salt tolerance of sweet sorghum. In addition, GO analysis identified candidate genes involved in maintaining membrane stability and photosynthetic capacity under salt stresses. This work lays a preliminary foundation for clarifying the molecular basis underlying the adaptation of sweet sorghum to adverse environments.

Morphological and Functional Remodeling of Vascular Endothelium in Cardiovascular Diseases.

The vascular endothelium plays a vital role during embryogenesis and aging and is a cell monolayer that lines the blood vessels. The immune system recognizes the endothelium as its own. Therefore, an abnormality of the endothelium exposes the tissues to the immune system and provokes inflammation and vascular diseases such as atherosclerosis. Its secretory role allows it to release vasoconstrictors and vasorelaxants as well as cardio-modulatory factors that maintain the proper functioning of the circulatory system. The sealing of the monolayer provided by adhesion molecules plays an important role in cardiovascular physiology and pathology.

Downregulation of NHE-3 (SLC9A3) expression by MicroRNAs in intestinal epithelial cells.

Na+/H+ exchanger-3 (NHE-3) is the major apical membrane transporter involved in vectorial Na+ absorption in the intestine. Dysregulation of NHE-3 expression and/or function has been implicated in pathophysiology of diarrhea associated with gut inflammation and infections. Therefore, it is critical to understand the mechanisms involved in the regulation of NHE-3 expression. MicroRNAs (miRNAs) are highly conserved small RNAs that can regulate gene expression at the posttranscriptional level. To date, however, very little is known about the regulation of NHE-3 expression by microRNAs. Therefore, current studies were undertaken to examine the potential miRNA candidates that can regulate the expression of NHE-3 in intestinal epithelial cells. In silico analysis, using different algorithms, predicted several miRNAs that target NHE-3. MicroRNAs with highest context and target score, miR-326, miR-744-5p, and miR-330-5p, were selected for the current study. Human NHE-3 gene 3' untranslated region [3'UTR; 160 base pair (bp)] was cloned into pmirGLO vector upstream of luciferase reporter and transiently transfected with mimics of miR-326, miR-744-5p, and miR-330-5p into Caco-2, HT-29, and SK-CO15 cells. Cotransfection of NHE-3 3' UTR with miR-326 and -miR-330-5p mimics resulted in a significant decrease in relative luciferase activity. Transfection of miR-326 and -330-5p mimics into SK-CO15 cells significantly decreased the NHE-3 protein expression, with no change in NHE-3 messenger ribonucleic acid (mRNA) levels. Our findings demonstrate a novel mechanism for posttranscriptional regulation of NHE-3 by miR-326 and -330-5p by translational repression. We speculate that miR-326 and -330-5p dependent pathways may be involved in modulating NHE-3 expression under physiological and pathophysiological conditions.

Lubiprostone is non-selective activator of cAMP-gated ion channels and Clc-2 has a minor role in its prosecretory effect in intestinal epithelial cells.

Loss of prosecretory Cl- channel CFTR activity is considered as the key cause of gastrointestinal disorders in cystic fibrosis including constipation and meconium ileus. Clc-2 is proposed as an alternative Cl- channel in intestinal epithelia that can compensate for CFTR loss-of-function. Lubiprostone is an FDA-approved drug with Clc-2 activation as its presumed mechanism of action. However, relative contribution of Clc-2 in intestinal Cl- secretion and the mechanism of action of lubiprostone remain controversial due to lack of selective Clc-2 inhibitors. Using recently identified selective Clc-2 inhibitor AK-42, we characterized the roles of Clc-2 in Cl- secretion in human intestinal epithelial T84 cells. Clc-2 inhibitor AK-42 had minimal (15%) inhibitory effect on secretory short-circuit current (Isc) induced by cAMP agonists, where subsequently applied CFTR inhibitor (CFTRinh-172) caused 2-3 fold greater inhibition. Similarly, AK-42 inhibited lubiprostone-induced secretory Isc by 20%, whereas CFTRinh-172 caused 2-3 fold greater inhibition. In addition to increasing CFTR and Clc-2-mediated apical Cl- conductance, lubiprostone increased basolateral membrane K+ conductance, which was completely reversed by cAMP-activated K+ channel inhibitor BaCl2 All components of lubiprostone-induced secretion (Clc-2, CFTR and K+ channels) were inhibited by ~65% with the extracellular Ca2+-sensing receptor (CaSR) activator cinacalcet that stimulates cAMP hydrolysis. Lastly, EP4 prostaglandin receptor inhibitor GW627368 pretreatment inhibited lubiprostone-induced secretion by 40% without any effect on forskolin response. Our findings suggest that Clc-2 has minor role in cAMP-induced intestinal Cl- secretion; and lubiprostone is not a selective Clc-2 activator, but general activator of cAMP-gated ion channels in human intestinal epithelial cells. Significance Statement Cl- channel Clc-2 activation is the proposed mechanism of action of the FDA-approved constipation drug lubiprostone. Using first-in-class selective Clc-2 inhibitor AK-42, we showed that Clc-2 has minor contribution in intestinal Cl- secretion induced by lubiprostone and cAMP agonists. We also found that lubiprostone is a general activator of cAMP-gated ion channels in human intestinal epithelial cells (via EP4 receptors). Our findings clarify the roles of Clc-2 in intestinal Cl- secretion and elucidate the mechanism of action of approved-drug lubiprostone.

A Mathematical Model of Salivary Gland Duct Cells.

Saliva is produced in two stages in the salivary glands: the secretion of primary saliva by the acinus and the modification of saliva composition to final saliva by the intercalated and striated ducts. In order to understand the saliva modification process, we develop a mathematical model for the salivary gland duct. The model utilises the realistic 3D structure of the duct reconstructed from an image stack of gland tissue. Immunostaining results show that TMEM16A and aquaporin are expressed in the intercalated duct cells and that ENaC is not. Based on this, the model predicts that the intercalated duct does not absorb Na[Formula: see text] and Cl[Formula: see text] like the striated duct but secretes a small amount of water instead. The input to the duct model is the time-dependent primary saliva generated by an acinar cell model. Our duct model produces final saliva output that agrees with the experimental measurements at various stimulation levels. It also shows realistic biological features such as duct cell volume, cellular concentrations and membrane potentials. Simplification of the model by omission of all detailed 3D structures of the duct makes a negligible difference to the final saliva output. This shows that saliva production is not sensitive to structural variation of the duct.

Skeletal muscle phenotype and game performance in elite women football players.

We combined game activity analyses with skeletal muscle phenotypes and comprehensive physiological testing to elucidate factors of importance for physical performance in elite women's football. GPS-data from an experimental game, sprint and endurance testing, and muscle tissue analysis of metabolic enzyme activity, protein expression and fiber type composition were completed for international top-level women players (n = 20; age; 23 ± 4 yrs, height; 166 ± 10 cm, weight; 60 ± 8 kg; VO2max ; 51 ± 6 ml/min/kg). Muscle monocarboxylate transporter 4 (MCT4) protein expression explained 46% of the variance in total game distance, while the ability to maintain high-intensity running (HIR) during the final 15 min of the game correlated to myosin heavy chain 1 (MHCI) and Na+ -K+ ATPase ß1, FXYD1 (phospholemman) and superoxide dismutase 2 (SOD2) protein expression (range: r = 0.51-0.71; all p < 0.05). Total HIR distance correlated with (MHCIIa) protein expression (r = 0.51; p < 0.05), while muscle Na+ /H+ exchanger 1 (NHE1) protein explained 36% of the variance in game sprint distance (p < 0.05). Total game accelerations (actions >4 m/s2 ) correlated with platelet endothelial cell adhesion molecule (PECAM-1) protein expression (r = 0.51; p < 0.05), while concentric knee flexor strength explained 42-62% of the variance in intense decelerations (>4 m/s2 ). In conclusion, for elite women players' game endurance performance and resistance to end-game fatigue were affected by monocarboxylate transporter expression and myosin heavy chain profile. HIR was also correlated to ion transporter expression and muscle antioxidative capacity. Finally, the importance of functional strength and measures of muscle vascularization in relation to total game decelerations and accelerations, respectively, illustrates the complex physiological demands in elite women's football.

Role of Monovalent Ions in the NKCC1 Inhibition Mechanism Revealed through Molecular Simulations.

The secondary active Na-K-Cl cotransporter 1 (NKCC1) promotes electroneutral uptake of two chloride ions, one sodium ion and one potassium ion. NKCC1 regulates Cl- homeostasis, thus being implicated in transepithelial water transport and in neuronal excitability. Aberrant NKCC1 transport is linked to a variety of human diseases. The loop diuretic drugs bumetanide, furosemide, azosemide and ethacrynic acid target NKCC1, but are characterized by poor selectivity leading to severe side effects. Despite its therapeutic importance, the molecular details of the NKCC1 inhibition mechanism remain unclear. Using all-atom simulations, we predict a putative binding mode of these drugs to the zebrafish (z) and human (h) NKCC1 orthologs. Although differing in their specific interactions with NKCC1 and/or monovalent ions, all drugs can fit within the same cavity and engage in hydrophobic interactions with M304/M382 in z/hNKCC1, a proposed ion gating residue demonstrated to be key for bumetanide binding. Consistent with experimental evidence, all drugs take advantage of the K+/Na+ ions, which plastically respond to their binding. This study not only provides atomic-level insights useful for drug discovery campaigns of more selective/potent NKCC1 inhibitors aimed to tackle diseases related to deregulated Cl- homeostasis, but it also supplies a paradigmatic example of the key importance of dynamical effects when drug binding is mediated by monovalent ions.

Physiological and Pathological Significance of Esophageal TRP Channels: Special Focus on TRPV4 in Esophageal Epithelial Cells.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel that is broadly expressed in different human tissues, including the digestive system, where it acts as a molecular sensor and a transducer that regulates a variety of functional activities. Despite the extensive research to determine the role of this channel in the physiology and pathophysiology of different organs, the unique morphological and functional features of TRPV4 in the esophagus remain largely unknown. Ten years ago, TRPV4 was shown to be highly expressed in esophageal epithelial cells where its activation induces Ca2+-dependent ATP release, which, in turn, mediates several functions, ranging from mechanosensation to wound healing. This review summarizes the research progress on TRPV4, and focuses on the functional expression of TRPV4 in esophageal epithelium and its possible role in different esophageal diseases that would support TRPV4 as a candidate target for future therapeutic approaches to treat patients with these conditions.

Genetic and Biochemical Characterization of the Na+/H+ Antiporters of Pseudomonas aeruginosa.

Pseudomonas aeruginosa has four Na+/H+ antiporters that interconvert and balance Na+ and H+ gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we constructed four strains, each of which has only one antiporter, i.e., NhaB, NhaP, NhaP2, and Mrp. We also constructed a quadruple deletion mutant that has no Na+/H+ antiporters. Although the antiporters of P. aeruginosa have been studied previously, the strains constructed here present the opportunity to characterize their kinetic properties in their native membranes and their roles in the physiology of P. aeruginosa. The strains expressing only NhaB or Mrp, the two electrogenic antiporters, were able to grow essentially like the wild-type strain across a range of Na+ concentrations and pH values. Strains with only NhaP or NhaP2, which are electroneutral, grew more poorly at increasing Na+ concentrations, especially at high pH values, with the strain expressing NhaP being more sensitive. The strain with no Na+/H+ antiporters was extremely sensitive to the Na+ concentration and showed essentially no Na+(Li+)/H+ antiporter activity, but it retained most K+/H+ antiporter activity of the wild-type strain at pH 7.5 and approximately one-half at pH 8.5. We also used the four strains that each express one of the four antiporters to characterize the kinetic properties of each transporter. Transcriptome sequencing analysis of the quadruple deletion strain showed widespread changes, including changes in pyocyanin synthesis, biofilm formation, and nitrate and glycerol metabolism. Thus, the strains constructed for this study will open a new door to understanding the physiological roles of these proteins and their activities in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa has four Na+/H+ antiporters that connect and interconvert its Na+ and H+ gradients. We have constructed four deletion mutants, each of which has only one of the four Na+/H+ antiporters. These strains made it possible to study the properties and physiological roles of each antiporter independently in its native membrane. Mrp and NhaB are each able to sustain growth over a wide range of pH values and Na+ concentrations, whereas the two electroneutral antiporters, NhaP and NhaP2, are most effective at low pH values. We also constructed a quadruple mutant lacking all four antiporters, in which the H+ and Na+ gradients are disconnected. This will make it possible to study the role of the two gradients independently.

Vacuolar H+-ATPase and Na+/K+-ATPase energize Na+ uptake mechanisms in the nuchal organ of the hyperregulating freshwater crustacean Daphnia magna.

The nuchal organ of the embryos and neonates of the cladoceran, Daphnia magna, has been shown to be a site of Na+ influx and H+, NH4+ and Cl- efflux. This study combines the scanning ion-selective electrode technique with application of inhibitors of specific transporters to assess the mechanisms of Na+ transport across the nuchal organ. Na+ influx across the nuchal organ was inhibited both by inhibitors of the Na+/K+-ATPase (ouabain, bufalin) and by inhibitors of the vacuolar H+-ATPase (bafilomycin, N-ethylmaleimde, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, KM91104, S-nitrosoglutathione). Na+ influx was unaffected by the epithelial Na+ channel blocker benzamil, but was sensitive to ethylisopropyl amiloride and elevated external ammonium concentrations, consistent with roles for Na+/H+ and Na+/NH4+ exchangers in the apical membrane but not Na+ channels. Transport across the basolateral membrane into the haemolymph is proposed to involve the Na+/K+-ATPase and a thiazide-sensitive Na+/Cl- cotransporter.

Physiological responses of freshwater insects to salinity: molecular-, cellular- and organ-level studies.

Salinization of freshwater is occurring throughout the world, affecting freshwater biota that inhabit rivers, streams, ponds, marshes and lakes. There are many freshwater insects, and these animals are important for ecosystem health. These insects have evolved physiological mechanisms to maintain their internal salt and water balance based on a freshwater environment that has comparatively little salt. In these habitats, insects must counter the loss of salts and dilution of their internal body fluids by sequestering salts and excreting water. Most of these insects can tolerate salinization of their habitats to a certain level; however, when exposed to salinization they often exhibit markers of stress and impaired development. An understanding of the physiological mechanisms for controlling salt and water balance in freshwater insects, and how these are affected by salinization, is needed to predict the consequences of salinization for freshwater ecosystems. Recent research in this area has addressed the whole-organism response, but the purpose of this Review is to summarize the effects of salinization on the osmoregulatory physiology of freshwater insects at the molecular to organ level. Research of this type is limited, and pursuing such lines of inquiry will improve our understanding of the effects of salinization on freshwater insects and the ecosystems they inhabit.

Sodium sequestration confers salinity tolerance in an ancestral wild rice.

Wild rice Oryza rufipogon, a progenitor of cultivated rice Oryza sativa L., possesses superior salinity tolerance and is a potential donor for breeding salinity tolerance traits in rice. However, a mechanistic basis of salinity tolerance in this donor species has not been established. Here, we examined salinity tolerance from the early vegetative stage to maturity in O. rufipogon in comparison with a salt-susceptible (Koshihikari) and a salt-tolerant (Reiziq) variety of O. sativa. We assessed their phylogeny and agronomical traits, photosynthetic performance, ion contents, as well as gene expression in response to salinity stress. Salt-tolerant O. rufipogon exhibited efficient leaf photosynthesis and less damage to leaf tissues during the course of salinity treatment. In addition, O. rufipogon showed a significantly higher tissue Na+ accumulation that is achieved by vacuolar sequestration compared to the salt tolerant O. sativa indica subspecies. These findings are further supported by the upregulation of genes involved with ion transport and sequestration (e.g. high affinity K+ transporter 1;4 [HKT1;4], Na+ /H+ exchanger 1 [NHX1] and vacuolar H+ -ATPase c [VHA-c]) in salt-tolerant O. rufipogon as well as by the close phylogenetic relationship of key salt-responsive genes in O. rufipogon to these in salt-tolerant wild rice species such as O. coarctata. Thus, the high accumulation of Na+ in the leaves of O. rufipogon acts as a cheap osmoticum to minimize the high energy cost of osmolyte biosynthesis and excessive reactive oxygen species production. These mechanisms demonstrated that O. rufipogon has important traits that can be used for improving salinity tolerance in cultivated rice.

Coping strategies in response to different levels of elevated water hardness in channel catfish (Ictalurus punctatus): Insight into ion-regulatory and histopathological modulations.

Water hardness above the optimal level can incite toxic effects in fish, which are often species specific. Hence, we aimed at obtaining insights on the potential effects of elevated water hardness as well as coping strategies in channel catfish (Ictalurus punctatus). First, a toxicity assay was performed where the 96 h-LC50 was calculated as 4939 mg/L CaCO3. Thereafter, to gain knowledge on the underlying adaptive strategies to high water hardness, fish were exposed to seven hardness levels (150, 600, 1000, 1500, 2000, 3000 and 4000 mg/L CaCO3 at pH 8.15) for 15 days. Results showed that branchial activities of Ca2+-ATPase and Na+/K+-ATPase, which facilitate Ca2+ uptake, reduced starting respectively from 1000 mg/L and 1500 mg/L CaCO3. Nevertheless, Ca2+ burden in plasma and tissue (gills, liver and intestine) remained elevated. Hardness exposure also disturbed cations (Na+, K+, Mg2+) and minerals (iron and phosphorus) homeostasis in a tissue-specific and dose-dependent manner. Both hemoglobin content and hematocrit dropped significantly at 3000-4000 mg/L CaCO3, with a parallel decline in iron content in plasma and gills. Muscle water content rose dramatically at 4000 mg/L CaCO3, indicating an osmo-regulation disruption. Higher hardness of 3000-4000 mg/L CaCO3 also incited a series of histopathological modifications in gills, liver and intestine; most likely due to excess Ca2+ accumulation. Overall, these data suggest that channel catfish can adapt to a wide range of elevated hardness by modulating Ca2+ regulatory pathways and histomorphological alterations, however, 1500 mg/L CaCO3 and above can impair the performance of this species.

Salt transport by the gill Na+-K+-2Cl- symporter in palaemonid shrimps: exploring physiological, molecular and evolutionary landscapes.

Palaemonid shrimps inhabit osmotic niches from marine to continental waters. They hyper-regulate hemolymph osmolality and ionic concentrations in dilute media, hypo-regulating in concentrated media. Their gill epithelia express ion transporters like the Na+-K+-2Cl- symporter (NKCC) thought to play a role in salt secretion. To examine Cl- hypo-regulatory capability and phylogenetic correlations between gill NKCC mRNA levels and protein expression, we used palaemonids ranging from marine tide pools through estuaries (Palaemon) to coastal and continental fresh waters (Macrobrachium). We established the species' upper critical salinity limits (UL50) and short- (24 h) and long-term (120h) hypo-regulatory abilities at salinities of 80% of their UL50's (80%UL50). The Palaemon species exhibited the highest UL50's and greatest hypo-regulatory capabilities; among the Macrobrachium species, UL50's were higher in the diadromous than in the hololimnetic species. While basal transcript levels of gill NKCC mRNA were highest in P. pandaliformis, levels were unaffected by salinity or exposure time in all species. However, gill NKCC protein abundance increased after 120-h exposure at the 80%UL50 in all Macrobrachium species, except M. potiuna. Unexpectedly, hemolymph hyper-osmoregulatory capability in acclimatization media correlated with gill NKCC protein synthesis, while gill NKCC mRNA expression correlated with hemolymph hyper-Cl- regulation in Macrobrachium. These findings, together with the evolutionary history of osmoregulation in this shrimp clade, suggest a role for the gill NKCC symporter in both salt uptake and secretion. The evolution of NKCC protein expression responsiveness, unlike hemolymph hypo-regulation and NKCC mRNA expression, may have been driven by environmental salinity during niche radiation. SUMMARY STATEMENT: While mRNA expression of the gill Na+-K+-2Cl- symporter is unchanged during acclimation of palaemonid shrimps to saline media, protein expression is up regulated, revealing a role in chloride secretion.