Osmotic Conditions Research Articles

Exploring the flavor formation mechanism under osmotic conditions during soy sauce fermentation in Aspergillus oryzae by proteomic analysis.

Aspergillus oryzae is a common starter in the soy sauce industry and struggles to grow under complex fermentation conditions. However, little is known about the flavor formation mechanism under osmotic conditions (low-temperature and high-salt) in A. oryzae. This work investigated the flavors and the relative protein expression patterns by gas chromatography-mass spectrometry (GC-MS) and proteomic analysis. Low-temperature and a high-salt content are unfavorable to the secretion of hydrolases and the formation of fragrant aldehydes. The aldehyde contents under osmotic conditions were reduced to 1.4-3.7 times lower than that of the control. Besides, copper amine oxidases which decreased under low-temperature stress and salt stress were shown to be important in catalyzing the oxidative deamination of several amine substrates to fragrant aldehydes. Furthermore, alcohol dehydrogenase and polyketide synthase are beneficial to the formation of alcohols and aromatic flavors under low-temperature stress and salt stress. Particularly, the ethanol content under 16 °C stress was 3.5 times higher than that under 28 °C.

Second-generation ethanol production by Wickerhamomyces anomalus strain adapted to furfural, 5-hydroxymethylfurfural (HMF), and high osmotic pressure

The aims of this work were to improve cell tolerance towards high concentrations of furfural and 5-hydroxymethylfurfural (HMF) of an osmotolerant strain of Wickerhamomyces anomalus by means of evolutionary engineering, and to determine its ethanol production under stress conditions. Cells were grown in the presence of furfural, HMF, either isolated or in combination, and under high osmotic pressure conditions. The most toxic condition for the parental strain was the combination of both furans, under which it was unable to grow and to produce ethanol. However, the tolerant adapted strain achieved a yield of ethanol of 0.43 g g-1glucose in the presence of furfural and HMF, showing an alcohol dehydrogenase activity of 0.68 mU mg protein-1. For this strain, osmotic pressure, did not affect its growth rate. These results suggest that W. anomalus WA-HF5.5strain shows potential to be used in second-generation ethanol production systems.

In vitro seed germination response of corn hybrids to osmotic stress conditions

AbstractDrought is a major environmental stress factor of plants influencing all growth stages. Drought occurring during germination affects both germination percentage and rate. An experiment was conducted to evaluate in vitro seed germination responses of nine corn (Zea mays L.) hybrids to declining osmotic potentials using polyethylene glycol. Time series data for seed germination were generated at different osmotic potentials from 0 to −1.2 MPa at −0.3‐MPa intervals at 25 °C. Maximum seed germination (MSG) and seed germination rate (SGR) were estimated by fitting the data to a three‐parameter sigmoid function. The minimum osmotic potentials at which MSG and SGR rate reached zero (MSGbase and SGRbase) were derived by first fitting linear and quadratic regression models as a function of osmotic potential and then calculating intercepts. Maximum seed germination and SGR decreased as osmotic potential decreased, and differences were identified among hybrids for MSG, SGR, MSGbase, and SGRbase. An osmotic stress response index was developed by summing individual response indices of each parameter for each hybrid and was used to classify the corn hybrids into tolerance groups. AgriGold A6659 and Terral REV 25BHR26 were identified as the most‐ and least‐tolerant corn hybrids during germination, with AgriGold A6659 performing 29% better than REV 25BHR26. Previous studies have used similar methods using polyethylene glycol to create osmotic stress but have not used the parameters used in this study. These parameters establish a new potential screening tool to identify osmotic stress tolerance in corn germplasm.

Manipulation and Assessment of Human Red Blood Cells with Tunable “Tug-of-War” Optical Tweezers

Optical trapping is important in assessing the deformability of red blood cells (RBCs), providing quick, noncontact force measurement in liquid medium with piconewton resolution. Traditional techniques, though, can involve complicated processes or cause photodamage. This study employs ``tug-of-war'' (TOW) optical tweezers with object-adapted optical potentials, to simply and stably trap, stretch, and squeeze a single RBC under different osmotic conditions, thus enabling the assessment of RBC deformability. The large forces of the TOW tweezers can cause larger deformations than traditional dual-trap tweezers at the same power conditions, which can greatly reduce photodamage.

Improved tolerance of Escherichia coli to oxidative stress by expressing putative response regulator homologs from Antarctic bacteria.

Response regulator (RR) is known a protein that mediates cell's response to environmental changes. The effect of RR from extremophiles was still under investigation. In this study, response regulator homologs were mined from NGS data of Antarctic bacteria and overexpressed in Escherichia coli. Sixteen amino acid sequences were annotated corresponding to response regulators related to the two-component regulatory systems; of these, 3 amino acid sequences (DRH632, DRH1601 and DRH577) with high homology were selected. These genes were cloned in pRadGro and expressed in E. coli. The transformant strains were subjected to various abiotic stresses including oxidative, osmotic, thermal stress, and acidic stress. There was found that the robustness of E. coli to abiotic stress was increased in the presence of these response regulator homologs. Especially, recombinant E. coli overexpressing drh632 had the highest survival rate in oxidative, hypothermic, osmotic, and acidic conditions. Recombinant E. coli overexpressing drh1601 showed the highest tolerance level to osmotic stress. These results will be applicable for development of recombinant strains with high tolerance to abiotic stress.

Cholesterol Deficiency Causes Impaired Osmotic Stability of Cultured Red Blood Cells.

Ex vivo generation of red blood cells (cRBCs) is an attractive tool in basic research and for replacing blood components donated by volunteers. As a prerequisite for the survival of cRBCs during storage as well as in the circulation, the quality of the membrane is of utmost importance. Besides the cytoskeleton and embedded proteins, the lipid bilayer is critical for membrane integrity. Although cRBCs suffer from increased fragility, studies investigating the lipid content of their membrane are still lacking. We investigated the membrane lipid profile of cRBCs from CD34+ human stem and progenitor cells compared to native red blood cells (nRBCs) and native reticulocytes (nRETs). Ex vivo erythropoiesis was performed in a well-established liquid assay. cRBCs showed a maturation grade between nRETs and nRBCs. High-resolution mass spectrometry analysis for cholesterol and the major phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin and lysophosphatidylcholin, demonstrated severe cholesterol deficiency in cRBCs. Although cRBCs showed normal deformability capacity, they suffered from increased hemolysis due to minimal changes in the osmotic conditions. After additional lipid supplementation, especially cholesterol during culturing, the cholesterol content of cRBCs increased to a subnormal amount. Concurrently, the osmotic resistance recovered completely and became comparable to that of nRETs. Minor differences in the amount of phospholipids in cRBCs compared to native cells could mainly be attributed to the ongoing membrane remodeling process from the reticulocyte to the erythrocyte stage. Obtained results demonstrate severe cholesterol deficiency as a reason for enhanced fragility of cRBCs. Therefore, the supplementation of lipids, especially cholesterol during ex vivo erythropoiesis may overcome this limitation and strengthens the survival of cRBCs ex vivo and in vivo.

A Genome-Wide Identification of Osmotic Stress-Responsive MicroRNAs in Pyropia haitanensis (Bangiales, Rhodophyta)

Pyropia haitanensis, one of the most economically important marine crops worldwide, can recover from up to 80% water loss during emersion in the intertidal zone. However, little is currently known regarding the molecular mechanism involved in the response to osmotic stress at the post-transcriptional level. In this study, we profiled the transcriptional patterns of microRNAs (miRNAs) in P. haitanensis under the conditions of dehydration and succeeding rehydration. Forty-seven miRNAs were sequenced and characterized, which included 2 evolutionarily conserved and 45 novel miRNAs. Among these, 32 miRNAs were differentially expressed under osmotic stress conditions. The target genes were predicted to be related to catalytic activity, binding, transporter activity, transcription factor activity, signal transducer activity, and antioxidant activity. The miRNAPC-3p-99769_194 exhibited constant upregulation during dehydrated stress, whereas its target gene, a carbonic anhydrase, was downregulated as detected via qPCR. Furthermore, we identified genes that encode Dicer and Argonaute (AGO) proteins. The Dicer protein is encoded by two genes, each partially harboring several conserved domains. In this study, we discovered that there was a negative correlation in the miRNAPC-3p-99769_194 transcriptional pattern under osmotic stress, which revealed the essential role of miRNAs in the suppression of carbon fixation to maintain basic living conditions. Additionally, the Pyropia-specific organization of Dicer genes may be indicative of the distinctive structural features of miRNAs. This study provided novel insights into the miRNA-mediated regulatory mechanisms that are essential for desiccation-tolerance in P. haitanensis.

Adaptation reactions of common wheat (Triticum aestivum L.) and emmer (T. dicoccum Schrank ex Schübl.) seedlings under osmotic stress and treatment with metal nanoparticles

Aim. To study the adaptive reactions of common wheat (Triticum aestivum L.) and emmer (T. dicoccum Schrank ex Schübl.) seedlings using the parameters of oxidative homeostasis under polyethylene glycol (PEG)-induced osmotic stress and metal (Fe, Cu, Mn, Zn) mixture nanoparticles treatment. Methods. Biochemical assays: spectrophotometric measurements of the activity of antioxidant enzymes (SOD, CAT), the content of proline and products of lipid peroxidation; mathematical statistics. Results. The state of oxidative homeostasis of common wheat (cv. Favorytka, cv. Trypilska) and emmer (cv. Holikovska) seedlings under PEG-induced osmotic stress conditions was investigated and analyzed. The development of lipid peroxidation processes under stress conditions was observed only in the cv. Trypilska seedlings. It was established that proline content in common wheat seedlings of both cultivars under stress had signifi cant (10 : 1) predominance in roots, while in roots and shoots of emmer seedlings proline content increased equally. SOD activity in the leaves of the studied cultivars under stress conditions was not changed. At the same time, an increase of SOD activity by 30 % under osmotic stress was shown in the roots of common wheat of cv. Favorytka, while it decreased by 25 % in the roots of cv. Trypilska. It was established that CAT activity in the roots of both cultivars of common wheat decreased by 25 and 38 %, respectively, whereas in emmer of cv. Holikovska this parameter increased by 35 % under osmotic stress. Presowing treatment using a colloidal solution of a mixture of biogenic metal nanoparticles contributed to the induction of SOD activity in the roots of cv. Favorytka, CAT activity in the leaves of cv. Trypilska, and CAT activity in the roots and leaves of cv. Holikovska compared to non-treated plants. It should be noted that the treatment of plants with metal nanoparticles promoted the restoration of CAT activity in the roots under osmotic stress conditions to the level of the control plants in both wheat varieties. Conclusions. It was established that osmotic stress provoked the development of oxidative processes and inhibition of the activities of antioxidant enzymes, in particular, SOD and CAT, in the seedling roots of common wheat cultivars. It was shown that emmer wheat seedlings of cv. Holikovska can maintain redox homeostasis and avoid oxidative damage under osmotic stress conditions. It was found that the seedlings of common wheat and emmer demonstrated different strategies of osmotic regulation under osmotic stress, which is confi rmed, in particular, at the level of proline accumulation. It was shown that the application of colloidal solutions of metal nanoparticles induced an antioxidant protection system and reduced the oxidative processes, which are inevitable effects of drought. The obtained results indicate that common wheat cultivars are more susceptible to drought compared to emmer wheat of cv. Holikovska.

Root Growth Adaptation is Mediated by PYLs ABA Receptor-PP2A Protein Phosphatase Complex.

Plant root architecture dynamically adapts to various environmental conditions, such as salt‐containing soil. The phytohormone abscisic acid (ABA) is involved among others also in these developmental adaptations, but the underlying molecular mechanism remains elusive. Here, a novel branch of the ABA signaling pathway in Arabidopsis involving PYR/PYL/RCAR (abbreviated as PYLs) receptor‐protein phosphatase 2A (PP2A) complex that acts in parallel to the canonical PYLs‐protein phosphatase 2C (PP2C) mechanism is identified. The PYLs‐PP2A signaling modulates root gravitropism and lateral root formation through regulating phytohormone auxin transport. In optimal conditions, PYLs ABA receptor interacts with the catalytic subunits of PP2A, increasing their phosphatase activity and thus counteracting PINOID (PID) kinase‐mediated phosphorylation of PIN‐FORMED (PIN) auxin transporters. By contrast, in salt and osmotic stress conditions, ABA binds to PYLs, inhibiting the PP2A activity, which leads to increased PIN phosphorylation and consequently modulated directional auxin transport leading to adapted root architecture. This work reveals an adaptive mechanism that may flexibly adjust plant root growth to withstand saline and osmotic stresses. It occurs via the cross‐talk between the stress hormone ABA and the versatile developmental regulator auxin.

Biosynthesis of the Stress-Protectant and Chemical Chaperon Ectoine: Biochemistry of the Transaminase EctB.

Bacteria frequently adapt to high osmolarity surroundings through the accumulation of compatible solutes. Ectoine is a prominent member of these types of stress protectants and is produced via an evolutionarily conserved biosynthetic pathway beginning with the L-2,4-diaminobutyrate (DAB) transaminase (TA) EctB. Here, we studied EctB from the thermo-tolerant Gram-positive bacterium Paenibacillus lautus (Pl) and show that this tetrameric enzyme is highly tolerant to salt, pH, and temperature. During ectoine biosynthesis, EctB converts L-glutamate and L-aspartate-beta-semialdehyde into 2-oxoglutarate and DAB, but it also catalyzes the reverse reaction. Our analysis unravels that EctB enzymes are mechanistically identical to the PLP-dependent gamma-aminobutyrate TAs (GABA-TAs) and only differ with respect to substrate binding. Inspection of the genomic context of the ectB gene in P. lautus identifies an unusual arrangement of juxtapositioned genes for ectoine biosynthesis and import via an Ehu-type binding-protein-dependent ABC transporter. This operon-like structure suggests the operation of a highly coordinated system for ectoine synthesis and import to maintain physiologically adequate cellular ectoine pools under osmotic stress conditions in a resource-efficient manner. Taken together, our study provides an in-depth mechanistic and physiological description of EctB, the first enzyme of the ectoine biosynthetic pathway.

Selection of appropriate reference genes for quantitative real-time reverse transcription PCR in Betula platyphylla under salt and osmotic stress conditions.

Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative real-time reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.

Chaperone activity of serine protease HtrA of Helicobacter pylori as a crucial survival factor under stress conditions

BackgroundSerine protease HtrA exhibits both proteolytic and chaperone activities, which are involved in cellular protein quality control. Moreover, HtrA is an important virulence factor in many pathogens including Helicobacter pylori, for which the crucial stage of infection is the cleavage of E-cadherin and other cell-to-cell junction proteins.MethodsThe in vitro study of H. pylori HtrA (HtrAHp) chaperone activity was carried out using light scattering assays and investigation of lysozyme protein aggregates. We produced H. pylori ∆htrA deletion and HtrAHp point mutants without proteolytic activity in strain N6 and investigated the survival of the bacteria under thermal, osmotic, acidic and general stress conditions as well as the presence of puromycin or metronidazole using serial dilution tests and disk diffusion method. The levels of cellular and secreted proteins were examined using biochemical fraction and Western blotting. We also studied the proteolytic activity of secreted HtrAHp using zymography and the enzymatic digestion of β-casein. Finally, the consequences of E-cadherin cleavage were determined by immunofluorescence microscopy.ResultsWe demonstrate that HtrAHp displays chaperone activity that inhibits the aggregation of lysozyme and is stable under various pH and temperature conditions. Next, we could show that N6 expressing only HtrA chaperone activity grow well under thermal, pH and osmotic stress conditions, and in the presence of puromycin or metronidazole. In contrast, in the absence of the entire htrA gene the bacterium was more sensitive to a number of stresses. Analysing the level of cellular and secreted proteins, we noted that H. pylori lacking the proteolytic activity of HtrA display reduced levels of secreted HtrA. Moreover, we compared the amounts of secreted HtrA from several clinical H. pylori strains and digestion of β-casein. We also demonstrated a significant effect of the HtrAHp variants during infection of human epithelial cells and for E-cadherin cleavage.ConclusionHere we identified the chaperone activity of the HtrAHp protein and have proven that this activity is important and sufficient for the survival of H. pylori under multiple stress conditions. We also pinpointed the importance of HtrAHp chaperone activity for E- cadherin degradation and therefore for the virulence of this eminent pathogen.

Natural Root Cellular Variation in Responses to Osmotic Stress in Arabidopsis thaliana Accessions.

Arabidopsis naturally occurring populations have allowed for the identification of considerable genetic variation remodeled by adaptation to different environments and stress conditions. Water is a key resource that limits plant growth, and its availability is initially sensed by root tissues. The root’s ability to adjust its physiology and morphology under water deficit makes this organ a useful model to understand how plants respond to water stress. Here, we used hyperosmotic shock stress treatments in different Arabidopsis accessions to analyze the root cell morphological responses. We found that osmotic stress conditions reduced root growth and root apical meristem (RAM) size, promoting premature cell differentiation without affecting the stem cell niche morphology. This phenotype was accompanied by a cluster of small epidermal and cortex cells with radial expansion and root hairs at the transition to the elongation zone. We also found this radial expansion with root hairs when plants are grown under hypoosmotic conditions. Finally, root growth was less affected by osmotic stress in the Sg-2 accession followed by Ws, Cvi-0, and Col-0; however, after a strong osmotic stress, Sg-2 and Cvi-0 were the most resilience accessions. The sensitivity differences among these accessions were not explained by stress-related gene expression. This work provides new cellular insights on the Arabidopsis root phenotypic variability and plasticity to osmotic stress.

Effect of salinity on the synthesis and concentration of glycine betaine in osmoregulatory tissues from juvenile shrimps Litopenaeus vannamei

In marine animals, glycine betaine is one of the main osmolytes accumulated under osmotic stress conditions; nevertheless, in penaeids, shrimps little is known about the pathways involved in glycine betaine biosynthesis. In animal cells, glycine betaine is synthesized by the enzyme betaine aldehyde dehydrogenase (BADH). We herein investigated the salinity effect on the synthesis and concentration of glycine betaine on white shrimp Litopenaeus vannamei. Shrimps were subjected to 10, 20, 35, 40, 50, and 60 ppt salinity conditions for seven days. BADH activity increased in hepatopancreas and gills of shrimps subjected to salinities above 35 ppt salinity. In muscle, the BADH activity decreased at 35 ppt salinity. In hepatopancreas from shrimps subjected to 50 and 60 ppt salinities, BADH activity increased 1.1 and 1.7-fold. At 60 ppt salinity, BADH activity increased 1.5-fold respect to 35 ppt in gills. Glycine betaine concentration increased in hepatopancreas, gills, muscle, and hemolymph in shrimps subjected to salinities above 35 ppt. Glycine betaine concentration also increased at 20 ppt salinity, while at 10 ppt, not detected significant differences. The catch of glycine betaine from hemolymph by the cell likely is carried out to avoid protein denaturalization. Ammonia concentration in the aquarium's water only increased at salinities of 20 ppt and 10 ppt (1.1-fold relative to 35 ppt). Our data demonstrated that in L. vannamei, salinity regulates BADH activity and glycine betaine content in a tissue-specific manner.

Knockdown of Gh_A05G1554 (GhDHN_03) and Gh_D05G1729 (GhDHN_04) Dehydrin genes, Reveals their potential role in enhancing osmotic and salt tolerance in cotton

In this investigation, whole-genome identification and functional characterization of the cotton dehydrin genes was carried out. A total of 16, 7, and 7 dehydrin proteins were identified in G. hirsutum, G. arboreum and G. raimondii, respectively. Through RNA sequence data and RT-qPCR validation, Gh_A05G1554 (GhDHN_03) and Gh_D05G1729 (GhDHN_04) were highly upregulated, and knockdown of the two genes, significantly reduced the ability of the plants to tolerate the effects of osmotic and salt stress. The VIGS-plants recorded significantly higher concentration levels of oxidants, hydrogen peroxide (H2O2) and malondialdehyde (MDA), furthermore, the four stress responsive genes GhLEA2, Gh_D12G2017 (CDKF4), Gh_A07G0747 (GPCR) and a transcription factor, trihelix, Gh_A05G2067, were significantly downregulated in VIGS-plants, but upregulated in wild types under osmotic and salt stress condition. The result indicated that dehydrin proteins are vital for plants and can be exploited in developing a more osmotic and salt stress-resilient germplasm to boost and improve cotton production.

Mitotic Chromosomes in Live Cells Characterized Using High-Speed and Label-Free Optical Diffraction Tomography.

The cell nucleus is a three-dimensional, dynamic organelle organized into subnuclear compartments such as chromatin and nucleoli. The structure and function of these compartments are maintained by diffusion and interactions between related factors as well as by dynamic and structural changes. Recent studies using fluorescent microscopic techniques suggest that protein factors can access and are freely mobile in heterochromatin and in mitotic chromosomes, despite their densely packed structure. However, the physicochemical properties of the chromosome during cell division are not fully understood. In the present study, characteristic properties such as the refractive index (RI), volume of the mitotic chromosomes, and diffusion coefficient (D) of fluorescent probes inside the chromosome were quantified using an approach combining label-free optical diffraction tomography with complementary confocal laser-scanning microscopy and fluorescence correlation spectroscopy. Variations in these parameters correlated with osmotic conditions, suggesting that changes in RI are consistent with those of the diffusion coefficient for mitotic chromosomes and cytosol. Serial RI tomography images of chromosomes in live cells during mitosis were compared with three-dimensional confocal micrographs to demonstrate that compaction and decompaction of chromosomes induced by osmotic change were characterized by linked changes in chromosome RI, volume, and the mobilities of fluorescent proteins.

Nuclear Mechanics in the Fission Yeast.

In eukaryotic cells, the organization of the genome within the nucleus requires the nuclear envelope (NE) and its associated proteins. The nucleus is subjected to mechanical forces produced by the cytoskeleton. The physical properties of the NE and the linkage of chromatin in compacted conformation at sites of cytoskeleton contacts seem to be key for withstanding nuclear mechanical stress. Mechanical perturbations of the nucleus normally occur during nuclear positioning and migration. In addition, cell contraction or expansion occurring for instance during cell migration or upon changes in osmotic conditions also result innuclear mechanical stress. Recent studies in Schizosaccharomyces pombe (fission yeast) have revealed unexpected functions of cytoplasmic microtubules in nuclear architecture and chromosome behavior, and have pointed to NE-chromatin tethers as protective elements during nuclear mechanics. Here, we review and discuss how fission yeast cells can be used to understand principles underlying the dynamic interplay between genome organization and function and the effect of forces applied to the nucleus by the microtubule cytoskeleton.

Interlamellar-induced time-dependent response of intervertebral disc annulus: A microstructure-based chemo-viscoelastic model

The annulus fibrosus of the intervertebral disc exhibits an unusual transversal behavior for which a constitutive representation that considers as well regional effect, chemical sensitivity and time-dependency has not yet been developed, and it is hence the aim of the present contribution. A physically-based model is proposed by introducing a free energy function that takes into account the actual disc annulus structure in relation with the surrounding biochemical environment. The response is assumed to be dominated by the viscoelastic contribution of the extracellular matrix, the elastic contribution of the oriented collagen fibers and the osmo-induced volumetric contribution of the internal fluid content variation. The regional dependence of the disc annulus response due to variation in fibers content/orientation allows a micromechanical treatment of the soft tissue. A finite element model of the annulus specimen is designed while taking into consideration the ‘interlamellar’ ground substance zone between lamellae of the layered soft tissue. The kinetics is designed using full-field strain measurements performed on specimens extracted from two disc annulus regions and tested under different osmotic conditions. The time-dependency of the tissue response is reported on stress-free volumetric changes, on hysteretic stress and transversal strains during quasi-static stretching at different strain-rates and on their temporal changes during an interrupted stretching. Considering the effective contributions of the internal fluid transfer and the extracellular matrix viscosity, the microstructure-based chemo-mechanical model is found able to successfully reproduce the significant features of the macro-response and the unusual transversal behavior including the strong regional dependency from inner to outer parts of the disc: Poisson's ratio lesser than 0 (auxetic) in lamellae plane, higher than 0.5 in fibers plane, and their temporal changes towards usual values (between 0 and 0.5) at chemo-mechanical equilibrium. The underlying time-dependent mechanisms occurring in the tissue are analyzed via the local numerical fields and important insights about the effective role of the interlamellar zone are revealed for the different disc localizations. Statement of significanceThe structural complexity of the annulus fibrosus has only been appreciated through recent experimental contributions and a constitutive representation that considers as well regional effect, chemical sensitivity and time-dependency of the unusual transversal behavior has not yet been developed. Here, a microstructure-based chemo-viscoelastic model is developed to highlight the interlamellar-induced time-dependent response by means of a two-scale strategy. The model provides important insights about the origin of the time-dependent phenomena in disc annulus along with regional dependency, essential for understanding disc functionality.

Untargeted metabolomics of strawberry (Fragaria x ananassa 'Camarosa') fruit from plants grown under osmotic stress conditions.

Plants activate defense mechanisms to cope with adverse environmental conditions, leading to the accumulation and / or depletion of general and specialized metabolites. In this study, a multiplatform untargeted metabolomics strategy was employed to evaluate metabolic changes in strawberry fruit of cv. Camarosa grown under osmotic stress conditions. Liquid chromatography-mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) data from strawberries grown under two water-deficit conditions, irrigated at 95% crop evapotranspiration (ETc) and 85% ETc, and one excess salt condition with a 80 mmol L-1 NaCl solution, were analyzed to determine treatment effects on fruit metabolism. Multivariate principal component analysis, orthogonal projections to latent structures-discriminant analysis (OPLS-DA), and univariate statistical analyses were applied to the data set. While multivariate analyses showed group separation by treatment, T-tests and fold change revealed 12 metabolites differentially accumulated in strawberries from different treatments-among them phenolic compounds, glycerophospholipids, phytosterols, carbohydrates, and an aromatic amino acid. Untargeted metabolomic analysis allowed for the annotation of compounds differentially accumulated in strawberry fruit from plants grown under osmotic stress and non-stressed plants. The metabolic disturbance in plants under stress involved metabolites associated with the inhibition of reactive oxygen species and cell-wall and membrane lipid biosynthesis, which might serve as osmotic stress biomarkers. © 2019 Society of Chemical Industry.

Claudin 19 Is Regulated by Extracellular Osmolality in Rat Kidney Inner Medullary Collecting Duct Cells.

The inner medullary collecting duct (IMCD) is subject to severe changes in ambient osmolality and must either allow water transport or be able to seal the lumen against a very high osmotic pressure. We postulate that the tight junction protein claudin-19 is expressed in IMCD and that it takes part in epithelial adaptation to changing osmolality at different functional states. Presence of claudin-19 in rat IMCD was investigated by Western blotting and immunofluorescence. Primary cell culture of rat IMCD cells on permeable filter supports was performed under different osmotic culture conditions and after stimulation by antidiuretic hormone (AVP). Electrogenic transepithelial transport properties were measured in Ussing chambers. IMCD cells cultivated at 300 mosm/kg showed high transepithelial resistance, a cation selective paracellular pathway and claudin-19 was mainly located in the tight junction. Treatment by AVP increased cation selectivity but did not alter transepithelial resistance or claudin-19 subcellular localization. In contrast, IMCD cells cultivated at 900 mosm/kg had low transepithelial resistance, anion selectivity, and claudin-19 was relocated from the tight junctions to intracellular vesicles. The data shows osmolality-dependent transformation of IMCD epithelium from tight and sodium-transporting to leaky, with claudin-19 expression in the tight junction associated to tightness and cation selectivity under low osmolality.