You get what you test for: The killing effect of phage lysins is highly dependent on buffer tonicity and ionic strength.

The phage lysin field has done nothing but grow in the last decades. As a result, many different research groups around the world are contributing to the field, often with certain methodological differences that pose a challenge to the interpretation and comparison of results. In this work, we present the case study of three Acinetobacter baumannii-targeting phage lysins (wild-type endolysin LysMK34 plus engineered lysins eLysMK34 and 1D10) plus one lysin with broad activity against Gram-positive bacteria (PlySs2) to provide exemplary evidence on the risks of generalization when using one of the most common lysin evaluation assays: the killing assay with resting cells. To that end, we performed killing assays with the aforementioned lysins using hypo-, iso- and hypertonic buffers plus human serum either as the reaction or the dilution medium in a systematic manner. Our findings stress the perils of creating hypotonic conditions or a hypotonic shock during a killing assay, suggesting that hypotonic buffers should be avoided as a test environment or as diluents before plating to avoid overestimation of the killing effect in the assayed conditions. As a conclusion, we suggest that the nature of both the incubation and the dilution buffers should be always clearly identified when reporting killing activity data, and that for experimental consistency the same incubation buffer should be used as a diluent for posterior serial dilution and plating unless explicitly required by the experimental design. In addition, the most appropriate buffer mimicking the final application must be chosen to obtain relevant results.

Hydration status in adults with metabolic disorders in relation to socioeconomic, lifestyle and health factors.

INTRODUCTION: Adequate hydration is essential for maintaining the health and functionality of the human body. This study aimed to examine the association between selected socioeconomic, lifestyle, and health factors and the hydration status of adults with metabolic disorders by analyzing their urine osmolality. METHODS: The study involved 290 adults aged 18-70 years with metabolic disorders. Separate multivariate logistic regression models were conducted to evaluate the factors associated with urine osmolality in tertiles for women and men. Odds Ratios (OR) and 95% Confidence Intervals (95% CI) were calculated. RESULTS: In women, the following factors of urine osmolality were identified in 1st tertile: age (OR:1.04), physical activity (moderate/high vs. no/low; OR:0.38), and headaches (no vs. yes; OR:1.55), in 2nd tertile: physical activity (moderate/high vs. no/low; OR:2.46) and fatigue during the day (sometimes vs. never/very rarely; OR:0.45), and in 3rd tertile: age (OR:0.94), professional status ('I work part-time/I study and I work' vs. 'I do not work/I study'; OR:0.27), fatigue during the day (very often vs. never/very rarely; OR:2.55), and headaches (no vs. yes; OR:0.44). In men, the following factors of urine osmolality were identified in 1st tertile: place of residence (city vs. village; OR:2.72) and health assessment (average vs. poor; OR:0.32). CONCLUSION: Different factors affecting urine osmolality have been identified in women and men. These results highlight the need to implement studies to clarify the relationship between socioeconomic, lifestyle and health factors, and hydration status in adults with metabolic disorders.

Effects of ionic strength and bentonite ratio on the migration of Cr(VI) in clayey soil-bentonite engineered barrier.

Soil-bentonite (S-B) barriers have been widely used for heavy metal pollution containment. This study conducted batch adsorption tests and diffusion-through tests to evaluate how ionic strength and bentonite ratio influence the migration of Cr(VI) in natural clay-bentonite mixtures. The test results indicated that the adsorption of Cr(VI) exhibited an obvious anion adsorption effect, the pH of the soil mixture increased with the addition of bentonite, resulting in a decrease in the positive surface charge. This change led to a decrease in Cr(VI) adsorption capacity, from 775.19 mg/kg for pure clay to 378 mg/kg for mixture samples with excessive bentonite. Furthermore, as the ionic strength increases from 0 to 0.1 M, the Cr(VI) adsorption capacity increases slightly due to the weakening of electrostatic repulsion on the clay particle surface, but the effective diffusion coefficient (De) increases by 21.97%. The compression of the diffusion double layer (DDL) under high ionic strength conditions enlarges the diffusion path and enhances the migration of Cr(VI) through the pore flow paths. Moreover, hydrated bentonite effectively fills the interaggregate pores of natural clay, thus creating narrower and more tortuous flow paths. However, excessive bentonite increases the pH value and pore volume, resulting in changes to the soil microstructure and disrupting the continuous skeleton of natural clay, which is unfavorable for Cr(VI) containment. Based on the study of the Cr(VI) contaminated site, a bentonite ratio of 2:10 is recommended for optimal natural performance of the natural clay-bentonite barrier.

Urine osmolality assessment through the integration of urea hydrolysis and impedance measurement.

We present the development and validation of an impedance-based urine osmometer for accurate and portable measurement of urine osmolality. The urine osmolality of a urine sample can be estimated by determining the concentrations of the conductive solutes and urea, which make up approximately 94% of the urine composition. Our method utilizes impedance measurements to determine the conductive solutes and urea after hydrolysis with urease enzyme. We built an impedance model using sodium chloride (NaCl) and urea at various known concentrations. In this work, we validated the accuracy of the impedance-based urine osmometer by developing a proof-of-concept first prototype and an integrated urine dipstick second prototype, where both prototypes exhibit an average accuracy of 95.5 ± 2.4% and 89.9 ± 9.1%, respectively in comparison to a clinical freezing point osmometer in the hospital laboratory. While the integrated dipstick design exhibited a slightly lower accuracy than the first prototype, it eliminated the need for pre-mixing or manual pipetting. Impedance calibration curves for conductive and non-conductive solutes consistently yielded results for NaCl but underscored challenges in achieving uniform urease enzyme coating on the dipstick. We also investigated the impact of storing urine at room temperature for 24 hours, demonstrating negligible differences in osmolality values. Overall, our impedance-based urine osmometer presents a promising tool for point-of-care urine osmolality measurements, addressing the demand for a portable, accurate, and user-friendly device with potential applications in clinical and home settings.

Osmolar gap in hyponatraemia: An exploratory study.

Hyponatraemia has indeed been extensively studied from multiple angles, including volume status, tonicity, and aetiology; however, the specific consideration of the osmolar gap (OG) within the context of hyponatraemia and its potential impact on their overall outcomes received limited attention in research. The current study represents an effort to address this gap in our understanding. This prospective exploratory study was conducted on adults aged 14 years and older at the Indus Hospital, Karachi, from 2017 to 2020. The study involved categorising severity of hyponatraemia and volume status. The osmolar gap (OG) was calculated and categorised as either increased (OG>10) or normal (OG<10). Among the 262 patients included in the study, there were 139 females and 123 males. Elevated OG was observed in 141(53.8%) patients. There were 28 (10.7%) recorded fatalities and majority of these individuals had an elevated OG. These findings underscore the importance for clinicians to consider the osmolar gap when managing patients with hyponatraemia.

Mathematical modeling of intracellular osmolarity and cell volume stabilization: The Donnan effect and ion transport.

The presence of impermeant molecules within a cell can lead to an increase in cell volume through the influx of water driven by osmosis. This phenomenon is known as the Donnan (or Gibbs-Donnan) effect. Animal cells actively transport ions to counteract the Donnan effect and regulate their volume, actively pumping Na+ out and K+ into their cytosol using the Na+/K+ ATPase (NKA) pump. The pump-leak equations (PLEs) are a system of algebraic-differential equations to model the membrane potential, ion (Na+, K+, and Cl-), and water flux across the cell membrane, which provide insight into how the combination of passive ions fluxes and active transport contribute to stabilizing cell volume. Our broad objective is to provide analytical insight into the PLEs through three lines of investigation: (1) we show that the provision of impermeant extracellular molecules can stabilize the volume of a passive cell; (2) we demonstrate that the mathematical form of the NKA pump is not as important as the stoichiometry for cell stabilization; and (3) we investigate the interaction between the NKA pump and cation-chloride co-transporters (CCCs) on cell stabilization, showing that NCC can destabilize a cell while NKCC and KCC can stabilize it. We incorporate extracellular impermeant molecules, NKA pump, and CCCs into the PLEs and derive the exact formula for the steady states in terms of all the parameters. This analytical expression enables us to easily explore the effect of each of the system parameters on the existence and stability of the steady states.

Association of Knowledge and Health Habits with Physiological Hydration Status.

The association of hydration knowledge and health habits with hydration status and fluid intake is rarely examined. We sought to determine whether knowledge or physical health behaviors predict physiological hydration status and fluid intake. Ninety-six participants (59 female; 27 ± 10 year) completed the previously validated hydration survey. Participants then recorded total fluids consumed (TFC), collected urine, and tracked void frequency for 24 h. Hydration status was assessed via 24 h urine specific gravity (USG) and osmolality (Uosm). Health behaviors included self-reported physical activity, BMI, smoking, alcoholic drinking, and sleep status. TFC was significantly correlated with 24 h USG (r = -0.390; p < 0.001), Uosm (r = -0.486; p < 0.001), total urine volume (r = 0.675; p < 0.001), and void frequency (r = 0.518; p < 0.001). Hydration knowledge was not correlated with 24 h USG (r = 0.085; p = 0.420), Uosm (r = 0.087; p = 0.419), urine total volume (r = 0.019; p = 0.857), void frequency (r = 0.030; p = 0.771), or TFC (r = 0.027; p = 0.813). Hydration knowledge did not predict 24 h USG (LR+ = 1.10; LR- = 0.90), Uosm (LR+ = 0.81; LR- = 1.35), or TFC (LR+ = 1.00; LR- = 1.00). Health habits did not predict 24 h USG, Uosm, or TFC. In conclusion, self-reported 24 h diet and fluid log recording is comparable to hydration status verification via 24 h urine collection. Hydration knowledge and health habits are not related to, or predictive of, hydration status.

The MATH test. A three-phase assay?

This study aimed to investigating the possible interference caused by glass test tubes on the quantification of bacterial adhesion to hydrocarbons by the MATH test. The adhesion of four bacteria to hexadecane and to glass test tubes was evaluated employing different suspending polar phases. The role of the ionic strength of the polar phase regarding adhesion to glassware was investigated. Within the conditions studied, Gram-positive bacteria adhered to both the test tube and the hydrocarbon regardless of the polar phase employed; meanwhile, Escherichia coli ATCC 25922 did not attach to either one. The capacity of the studied microorganisms to adhere to glassware was associated with their electron-donor properties. The ionic strength of the suspending media altered the patterns of adhesion to glass in a strain-specific manner by defining the magnitude of electrostatic repulsion observed between bacteria and the glass surface. This research demonstrated that glass test tubes may interact with suspended bacterial cells during the MATH test under specific conditions, which may lead to overestimating the percentage of adhesion to hydrocarbons and, thus, to erroneous values of cell surface hydrophobicity.

Urea transporter UT-A1 as a novel drug target for hyponatremia.

Hyponatremia is the most common disorder of electrolyte imbalances. It is necessary to develop new type of diuretics to treat hyponatremia without losing electrolytes. Urea transporters (UT) play an important role in the urine concentrating process and have been proved as a novel diuretic target. In this study, rat and mouse syndromes of inappropriate antidiuretic hormone secretion (SIADH) models were constructed and analyzed to determine if UTs are a promising drug target for treating hyponatremia. Experimental results showed that 100 mg/kg UT inhibitor 25a significantly increased serum osmolality (from 249.83 ± 5.95 to 294.33 ± 3.90 mOsm/kg) and serum sodium (from 114 ± 2.07 to 136.67 ± 3.82 mmol/L) respectively in hyponatremia rats by diuresis. Serum chemical examination showed that 25a neither caused another electrolyte imbalance nor influenced the lipid metabolism. Using UT-A1 and UT-B knockout mouse SIADH model, it was found that serum osmolality and serum sodium were lowered much less in UT-A1 knockout mice than in UT-B knockout mice, which suggest UT-A1 is a better therapeutic target than UT-B to treat hyponatremia. This study provides a proof of concept that UT-A1 is a diuretic target for SIADH-induced hyponatremia and UT-A1 inhibitors might be developed into new diuretics to treat hyponatremia.

Heteroaggregation and deposition behaviors of carboxylated nanoplastics with different types of clay minerals in aquatic environments: Important role of calcium(II) ion-assisted bridging.

The widespread utilization of plastic products ineluctably leads to the ubiquity of nanoplastics (NPs), causing potential risks for aquatic environments. Interactions of NPs with mineral surfaces may affect NPs transport, fate and ecotoxicity. This study aims to investigate systematically the deposition and aggregation behaviors of carboxylated polystyrene nanoplastics (COOH-PSNPs) by four types of clay minerals (illite, kaolinite, Na-montmorillonite, and Ca-montmorillonite) under various solution chemistry conditions (pH, temperature, ionic strength and type). Results demonstrate that the deposition process was dominated by electrostatic interactions. Divalent cations (i.e., Ca2+, Mg2+, Cd2+, or Pb2+) were more efficient for screening surface negative charges and compressing the electrical double layer (EDL). Hence, there were significant increases in deposition rates of COOH-PSNPs with clay minerals in suspension containing divalent cations, whereas only slight increases in deposition rates of COOH-PSNPs were observed in monovalent cations (Na+, K+). Negligible deposition occurred in the presence of anions (F-, Cl-, NO3-, CO32-, SO42-, or PO43-). Divalent Ca2+ could incrementally facilitate the deposition of COOH-PSNPs through Ca2+-assisted bridging with increasing CaCl2 concentrations (0-100 mM). The weakened deposition of COOH-PSNPs with increasing pH (2.0-10.0) was primarily attributed to the reduce in positive charge density at the edges of clay minerals. In suspensions containing 2 mM CaCl2, increased Na+ ionic strength (0-100 mM) and temperature (15-55 ◦C) also favored the deposition of COOH-PSNPs. The ability of COOH-PSNPs deposited by four types of clay minerals followed the sequence of kaolinite > Na-montmorillonite > Ca-montmorillonite > illite, which was related to their structural and surface charge properties. This study revealed the deposition behaviors and mechanisms between NPs and clay minerals under environmentally representative conditions, which provided novel insights into the transport and fate of NPs in natural aquatic environments.

Aqueous size exclusion chromatography applied to polymer analysis: experimental conditions and molecular weight calibration curves.

Aqueous mode size exclusion chromatography (SEC) was employed for the analysis and construction of molecular weight (MW) calibration curves of three water-soluble polymers, namely, polyethylene glycol, polyethylene oxide, and polyacrylic acid sodium salt. Several calibration curves were obtained, varying chromatographic conditions such as columns arrangement, ionic strength, temperature and pH, in addition trends in polymeric chromatographic behavior were examined. The variation in SEC distribution coefficients at different temperatures was found to be below 10 %, indicating that the studied polymers follow an ideal SEC mechanism under the tested conditions. Thus, differences in chromatographic behavior were ascribed to changes in polymer configuration induced by media and/or temperature. These variations in morphology were consistent with the observed SEC behavior. Regarding MW calibration, polynomial regression models ranging from first to fifth order were applied, and the most adequate ones were selected based on their fit and prediction capabilities. Third order polynomials were the preferred models for polyethylene glycol and polyacrylic acid sodium salt, independently of chromatographic conditions. Meanwhile for polyethylene oxide, either third or fifth-order polynomial models were optimal depending on the chromatographic conditions. All the selected regression models presented coefficients of multiple determination (R2) above 0.990, while achieving relative errors of prediction (REP%) in MW ranging from 0.3 to 4 % for cross-validation.

Sorption of ciprofloxacin and enrofloxacin on alkaline cropland soil in semiarid regions: Roles of pH, ionic strength, and ion type.

Animals manure and chemical fertilizers are widely applied to agricultural soils to mitigate soil fertility decline resulting from intensive farming practices. However, the use of antibiotics such as ciprofloxacin (CIP) and enrofloxacin (ENR) in these manures introduces certain environmental risks. The sorption of CIP and ENR in soil is influenced by various factors. Soil cations (i.e., Na+, K+, Mg2+, and Ca2+) and artificially introduced ions (NH4+) can affect the sorption behavior of CIP and ENR in alkaline agricultural soils through mechanisms such as ion exchange and competitive sorption. To investigate the effects of ionic strength and ion type on the sorption of antibiotics in alkaline agricultural soil, batch equilibrium experiments were conducted in this study. The results showed that the affinity of alkaline farmland soil to CIP and ENR was poor, and Kd was only 159 L/kg and 89 L/kg, respectively. Increases in temperature and pH inhibited CIP and ENR sorption on soil. Mineral elements in the soil strongly inhibited CIP and ENR sorption. Conversely, NH4+ promoted the Kd values of CIP and ENR by 46% and 221%, respectively. Additionally, under different influencing factors, both the sorption affinity (Kd) and sorption amount of ENR were lower than those of CIP. These findings indicate that ENR has a greater migration potential and poses a greater environmental risk in agricultural soils. Alkaline soil and mineral elements increase the migration potential of CIP, ENR, but the introduction of NH4+ in agricultural production can weaken the migration potential of them.

High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility.

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.

The N-terminus of the Aspergillus fumigatus group III hybrid histidine kinase TcsC is essential for its physiological activity and targets the protein to the nucleus.

Group III hybrid histidine kinases are fungal-specific proteins and part of the multistep phosphorelay, representing the initial part of the high osmolarity glycerol (HOG) pathway. TcsC, the corresponding kinase in Aspergillus fumigatus, was expected to be a cytosolic protein but is targeted to the nucleus. Activation of TcsC by the antifungal fludioxonil has lethal consequences for the fungus. The agent triggers a fast and TcsC-dependent activation of SakA and later on a redistribution of TcsC to the cytoplasm. High osmolarity also activates TcsC, which then exits the nucleus or concentrates in spot-like, intra-nuclear structures. The sequence corresponding to the N-terminal 208 amino acids of TcsC lacks detectable domains. Its loss renders TcsC cytosolic and non-responsive to hyperosmotic stress, but it has no impact on the antifungal activity of fludioxonil. A point mutation in one of the three putative nuclear localization sequences, which are present in the N-terminus, prevents the nuclear localization of TcsC, but not its ability to respond to hyperosmotic stress. Hence, this striking intracellular localization is no prerequisite for the role of TcsC in the adaptive response to hyperosmotic stress, instead, TcsC proteins that are present in the nuclei seem to modulate the cell wall composition of hyphae, which takes place in the absence of stress. The results of the present study underline that the spatiotemporal dynamics of the individual components of the multistep phosphorelay is a crucial feature of this unique signaling hub. IMPORTANCE: Signaling pathways enable pathogens, such as Aspergillus fumigatus, to respond to a changing environment. The TcsC protein is the major sensor of the high osmolarity glycerol (HOG) pathway of A. fumigatus and it is also the target of certain antifungals. Insights in its function are therefore relevant for the pathogenicity and new therapeutic treatment options. TcsC was expected to be cytoplasmic, but we detected it in the nucleus and showed that it translocates to the cytoplasm upon activation. We have identified the motif that guides TcsC to the nucleus. An exchange of a single amino acid in this motif prevents a nuclear localization, but this nuclear targeting is no prerequisite for the TcsC-mediated stress response. Loss of the N-terminal 208 amino acids prevents the nuclear localization and renders TcsC unable to respond to hyperosmotic stress demonstrating that this part of the protein is of crucial importance.

Formation of left-handed helices by C2'-fluorinated nucleic acids under physiological salt conditions.

Recent findings in cell biology have rekindled interest in Z-DNA, the left-handed helical form of DNA. We report here that two minimally modified nucleosides, 2'F-araC and 2'F-riboG, induce the formation of the Z-form under low ionic strength. We show that oligomers entirely made of these two nucleosides exclusively produce left-handed duplexes that bind to the Zα domain of ADAR1. The effect of the two nucleotides is so dramatic that Z-form duplexes are the only species observed in 10 mM sodium phosphate buffer and neutral pH, and no B-form is observed at any temperature. Hence, in contrast to other studies reporting formation of Z/B-form equilibria by a preference for purine glycosidic angles in syn, our NMR and computational work revealed that sequential 2'F…H2N and intramolecular 3'H…N3' interactions stabilize the left-handed helix. The equilibrium between B- and Z- forms is slow in the 19F NMR time scale (≥ms), and each conformation exhibited unprecedented chemical shift differences in the 19F signals. This observation led to a reliable estimation of the relative population of B and Z species and enabled us to monitor B-Z transitions under different conditions. The unique features of 2'F-modified DNA should thus be a valuable addition to existing techniques for specific detection of new Z-binding proteins and ligands.

Internalization of PEI-based complexes in transient transfection of HEK293 cells is triggered by coalescence of membrane heparan sulfate proteoglycans like Glypican-4.

Polymer-cationic mediated gene delivery is a well-stablished strategy of transient gene expression (TGE) in mammalian cell cultures. Nonetheless, its industrial implementation is hindered by the phenomenon known as cell density effect (CDE) that limits the cell density at which cultures can be efficiently transfected. The rise in personalized medicine and multiple cell and gene therapy approaches based on TGE, make more relevant to understand how to circumvent the CDE. A rational study upon DNA/PEI complex formation, stability and delivery during transfection of HEK293 cell cultures has been conducted, providing insights on the mechanisms for polyplexes uptake at low cell density and disruption at high cell density. DNA/PEI polyplexes were physiochemically characterized by coupling X-ray spectroscopy, confocal microscopy, cryo-transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR). Our results showed that the ionic strength of polyplexes significantly increased upon their addition to exhausted media. This was reverted by depleting extracellular vesicles (EVs) from the media. The increase in ionic strength led to polyplex aggregation and prevented efficient cell transfection which could be counterbalanced by implementing a simple media replacement (MR) step before transfection. Inhibiting and labeling specific cell-surface proteoglycans (PGs) species revealed different roles of PGs in polyplexes uptake. Importantly, the polyplexes uptake process seemed to be triggered by a coalescence phenomenon of HSPG like glypican-4 around polyplex entry points. Ultimately, this study provides new insights into PEI-based cell transfection methodologies, enabling to enhance transient transfection and mitigate the cell density effect (CDE).

Assessment of Hydration Status Using Conventional Method and Salivary Osmolarity as a Point-of-care Tool.

Dehydration is a well-known problem worldwide, and its assessment can be challenging due to confusing physical signs. The most effective way to assess hydration status is through the costly stable isotope methodology, but this approach has practical limitations. More commonly accepted and utilized indicators of hydration status are hematological and urinary parameters. However, hematological markers require invasive methods, and urinary markers have varying degrees of success in tracking hydration changes. While alterations in body weight can serve as a means of promptly evaluating hydration status, various factors such as food consumption, fluid intake, fecal losses, and urine production can impact these changes. Researchers have turned their attention to saliva as a potential marker and point-of-care (POC) testing to address the limitations of existing biomarkers. Saliva is appealing due to its easy collection process and similarities to extracellular fluid in terms of water and ion concentrations. Recent studies have shown that saliva flow rate, osmolarity/osmolality, and total protein concentration can effectively monitor changes in body mass during acute dehydration. Misdiagnosing dehydration can have severe clinical consequences, leading to morbidity and even mortality. This narrative review focuses on recognizing the significance of hydration assessment, monitoring, and the potential of salivary osmolarity (SOSM) as an assessment tool. Healthcare professionals can improve their practices and interventions to optimize hydration and promote overall wellness using such tools.

The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact.

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

Kinetic studies on optimized extracellular laccase from Trichoderma harzianum PP389612 and its capabilities for azo dye removal.

BACKGROUND: Azo dyes represent a common textile dye preferred for its high stability on fabrics in various harsh conditions. Although these dyes pose high-risk levels for all biological forms, fungal laccase is known as a green catalyst for its ability to oxidize numerous dyes. METHODS: Trichoderma isolates were identified and tested for laccase production. Laccase production was optimized using Plackett-Burman Design. Laccase molecular weight and the kinetic properties of the enzyme, including Km and Vmax, pH, temperature, and ionic strength, were detected. Azo dye removal efficiency by laccase enzyme was detected for Congo red, methylene blue, and methyl orange. RESULTS: Eight out of nine Trichoderma isolates were laccase producers. Laccase production efficiency was optimized by the superior strain T. harzianum PP389612, increasing production from 1.6 to 2.89 U/ml. In SDS-PAGE, purified laccases appear as a single protein band with a molecular weight of 41.00 kDa. Km and Vmax values were 146.12 µmol guaiacol and 3.82 µmol guaiacol/min. Its activity was stable in the pH range of 5-7, with an optimum temperature range of 40 to 50 °C, optimum ionic strength of 50 mM NaCl, and thermostability properties up to 90 °C. The decolorization efficiency of laccase was increased by increasing the time and reached its maximum after 72 h. The highest efficiency was achieved in Congo red decolorization, which reached 99% after 72 h, followed by methylene blue at 72%, while methyl orange decolorization efficiency was 68.5%. CONCLUSION: Trichoderma laccase can be used as an effective natural bio-agent for dye removal because it is stable and removes colors very well.

Metabolomic pattern associated with physical sequelae in patients presenting with respiratory symptoms validates the aestivation concept in dehydrated patients.

Hypertonic dehydration is associated with muscle wasting and synthesis of organic osmolytes. We recently showed a metabolic shift to amino acid production and urea cycle activation in coronavirus-2019 (COVID-19), consistent with the aestivation response. The aim of the present investigation was to validate the metabolic shift and development of long-term physical outcomes in the non-COVID cohort of the Biobanque Québécoise de la COVID-19 (BQC19). We included 824 patients from BQC19, where 571 patients had data of dehydration in the form of estimated osmolality (eOSM = 2Na + 2K + glucose + urea), and 284 patients had metabolome data and long-term follow-up. We correlated the degree of dehydration to mortality, invasive mechanical ventilation, acute kidney injury, and long-term symptoms. As found in the COVID cohort, higher eOSM correlated with a higher proportion of urea and glucose of total eOSM, and an enrichment of amino acids compared with other metabolites. Sex-stratified analysis indicated that women may show a weaker aestivation response. More severe dehydration was associated with mortality, invasive mechanical ventilation, and acute kidney injury during the acute illness. Importantly, more severe dehydration was associated with physical long-term symptoms but not mental long-term symptoms after adjustment for age, sex, and disease severity. Patients with water deficit in the form of increased eOSM tend to have more severe disease and experience more physical symptoms after an acute episode of care. This is associated with amino acid and urea production, indicating dehydration-induced muscle wasting.NEW & NOTEWORTHY We have previously shown that humans exhibit an aestivation-like response where dehydration leads to a metabolic shift to urea synthesis, which is associated with long-term weakness indicating muscle wasting. In the present study, we validate this response in a new cohort and present a deeper metabolomic analysis and pathway analysis. Finally, we present a sex-stratified analysis suggesting weaker aestivation in women. However, women show less dehydration, so the association warrants further study.