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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

Diurnal rhythm of urinary aquaporin-2 in children with primary monosymptomatic nocturnal enuresis.

Background/aim: Nocturnal enuresis can be frustrating for children and their families as the child ages. Our aim is to evaluate urine aquaporin 2 (AQP-2) as a noninvasive biomarker of water balance in children with primary monosymptomatic nocturnal enuresis (PMNE). Material and methods: The study included 90 children; sixty-eight children suffering from PMNE aged (9.57 ± 2.16) years and 22 healthy children with good toilet control, matched sex and age. All enuretic children were subjected to complete history taking, clinical evaluation, and bed wetting diary. Serum arginine vasopressin (AVP) and urine AQP-2 were tested in the morning (at 9-11 am) and evening (at 9-11 pm). Blood urea, creatinine, Na, glucose, urine osmolality, Ca/Cr, Alb/Cr and specific gravity were tested simultaneously. Results: Serum AVP, urine AQP-2, and urine osmolality were statistically lower in patients than controls. Patients had a significantly lower level of night serum AVP concentrations, urine AQP-2, and urine osmolality than the corresponding morning level. Urine AQP-2 was significantly correlated with urine osmolality (p < 0.05). AQP-2 had a sensitivity of 90% and a specificity of 70%. However, no statistically significant correlation was found between serum AVP and urine AQP-2. Conclusion: Primary monosymptomatic nocturnal enuresis in children could be associated with reduction of urine excretion of AQP-2 at night. Urine AQP-2 is significantly correlated with urine osmolality. Therefore, it may be a noninvasive biomarker of hydration status in children with PMNE, with good sensitivity and specificity.

Kidney collecting duct-derived vasopressin is not essential for appropriate concentration or dilution of urine.

We have previously shown that kidney collecting ducts make vasopressin. However, the physiological role of collecting duct-derived vasopressin is uncertain. We hypothesized that collecting duct-derived vasopressin is required for the appropriate concentration of urine. We developed a vasopressin conditional knockout (KO) mouse model wherein Cre recombinase expression induces deletion of arginine vasopressin (Avp) exon 1 in the distal nephron. We then used age-matched 8- to 12-wk-old Avp fl/fl;Ksp-Cre(-) [wild type (WT)] and Avp fl/fl;Ksp-Cre(+) mice for all experiments. We collected urine, serum, and kidney lysates at baseline. We then challenged both WT and knockout (KO) mice with 24-h water restriction, water loading, and administration of the vasopressin type 2 receptor agonist desmopressin (1 µg/kg ip) followed by the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). We performed immunofluorescence and immunoblot analysis at baseline and confirmed vasopressin KO in the collecting duct. We found that urinary osmolality (UOsm), plasma Na+, K+, Cl-, blood urea nitrogen, and copeptin were similar in WT vs. KO mice at baseline. Immunoblots of the vasopressin-regulated proteins Na+-K+-2Cl- cotransporter, NaCl cotransporter, and water channel aquaporin-2 showed no difference in expression or phosphorylation at baseline. Following 24-h water restriction, WT and KO mice had no differences in UOsm, plasma Na+, K+, Cl-, blood urea nitrogen, or copeptin. In addition, there were no differences in the rate of urinary concentration or dilution as in WT and KO mice UOsm was nearly identical after desmopressin and OPC-31260 administration. We conclude that collecting duct-derived vasopressin is not essential to appropriately concentrate or dilute urine.NEW & NOTEWORTHY Hypothalamic vasopressin is required for appropriate urinary concentration. However, whether collecting duct-derived vasopressin is involved remains unknown. We developed a novel transgenic mouse model to induce tissue-specific deletion of vasopressin and showed that collecting duct-derived vasopressin is not required to concentrate or dilute urine.

Engineering of chimeric enzymes with expanded tolerance to ionic strength.

Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpACD) protein from Enterococcus faecalis. Unlike other enzymes within the M23 family, EnpACD demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpACD fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.IMPORTANCEThese studies demonstrate that the addition of the SH3b-binding domain to the EnpACD results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins' engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.