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.

Nanobubble transport in porous media: Towards agro- and environmental applications.

Nanobubbles have been increasingly used in various applications involving porous media, such as groundwater remediation and irrigation. However, the fundamental scientific knowledge regarding the interactions between nanobubbles and the media is still limited. The interactions can be repulsive, attractive, or inert, and can involve reversible or irreversible attachment as well as destructive mechanisms. Specifically, the stability and mobility of nanobubbles in porous media is expected to be dependent on the dynamic conditions and the physicochemical properties of the porous media, solutions, and nanobubbles themselves. In this study, we investigated how changes in solution chemistry (pH, ionic strength, and valence) and media characteristics (size and wettability) affect the size and concentration of nanobubbles under dynamic conditions using column experiments. Quartz crystal microbalance with dissipation monitoring provided a deeper understanding of irreversible and elastic nanobubbles' interactions with silica-coated surfaces. Our findings suggest that nanobubbles are less mobile in solutions of higher ionic strength and valence, acidic pH and smaller porous media sizes, while the wettability of porous media has a negligible influence on the retention of nanobubbles. Overall, our findings provide insights into the underlying mechanisms of nanobubble interactions and suggest potential strategies to optimize their delivery in various applications.

Angle between DNA linker and nucleosome core particle regulates array compaction revealed by individual-particle cryo-electron tomography.

The conformational dynamics of nucleosome arrays generate a diverse spectrum of microscopic states, posing challenges to their structural determination. Leveraging cryogenic electron tomography (cryo-ET), we determine the three-dimensional (3D) structures of individual mononucleosomes and arrays comprising di-, tri-, and tetranucleosomes. By slowing the rate of condensation through a reduction in ionic strength, we probe the intra-array structural transitions that precede inter-array interactions and liquid droplet formation. Under these conditions, the arrays exhibite irregular zig-zag conformations with loose packing. Increasing the ionic strength promoted intra-array compaction, yet we do not observe the previously reported regular 30-nanometer fibers. Interestingly, the presence of H1 do not induce array compaction; instead, one-third of the arrays display nucleosomes invaded by foreign DNA, suggesting an alternative role for H1 in chromatin network construction. We also find that the crucial parameter determining the structure adopted by chromatin arrays is the angle between the entry and exit of the DNA and the corresponding tangents to the nucleosomal disc. Our results provide insights into the initial stages of intra-array compaction, a critical precursor to condensation in the regulation of chromatin organization.

Osmolality in oral supplements drives ileostomy output: Defining the Goldilocks zone.

BACKGROUND: Patients with an ileostomy often have impaired quality of life, sodium depletion, secondary hyperaldosteronism, and other organ-specific pathologies. The osmolality of oral supplements influences ileostomy output and increases sodium loss. We hypothesized the existence of an osmolality range in which fluid absorption and secondary natriuresis are optimal. METHODS: This was a single-center, quasi-randomized crossover intervention study, including patients with an ileostomy and no home parenteral support. After an 8-h fasting period, each patient ingested 500 mL of 3-18 different oral supplements and a standardized meal during the various intervention periods, followed by a 6-h collection of ileostomy and urine outputs. The primary outcome was 6-h ileostomy output. RESULTS: A total of 14 ileostomy patients with a median age of 65 years (interquartile range 38-70 years) were included. The association between osmolalities (range 5-1352 mOsm/kg) and ileostomy output forecasted an S-curve. A linear association between osmolality of oral supplements (range 290-600 mOsm/kg) and ileostomy output was identified and assessed with a mixed-effects model. Ileostomy output increased by 57 g/6 h (95% confidence interval (CI) 21-94) when the oral supplement osmolality increased by 100 mOsm/kg (p = 0.005). CONCLUSION: Osmolality in oral supplements correlated with ileostomy output. Our results indicate that patients with an ileostomy may benefit from increased ingestion of oral supplements with osmolalities between 100 and 290 mOsm/kg. We define this range as the Goldilocks zone, equivalent to optimal fluid and electrolyte absorption.

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.

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.

Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

Differences in urine creatinine and osmolality between black and white Americans after accounting for age, moisture intake, urine volume, and socioeconomic status.

Urine osmolality is used throughout research to determine hydration levels. Prior studies have found black individuals to have elevated urine creatinine and osmolality, but it remains unclear which factors explain these findings. This cross-sectional, observational study sought to understand the relationship of self-reported race to urine creatinine and urine osmolality after accounting for age, socioeconomic status, and fluid intake. Data from 1,386 participants of the 2009-2012 National Health and Nutrition Examination Survey were utilized. Age, poverty-to-income ratio (PIR), urine flow rate (UFR), fluid intake, estimated lean body mass (LBM), urine creatinine, and urine osmolality were measured. In a sex-specific manner, black and white participants were matched on age, dietary moisture, UFR, and PIR. Urine creatinine was greater in black men (171 mg/dL) than white men (150 mg/dL) and greater in black women (147 mg/dL) than white women (108 mg/dL) (p < .001). Similarly, urine osmolality was greater in black women than white women (723 vs. 656 mOsm/kg, p = .001), but no difference was observed between white and black men (737 vs. 731 mOsm/kg, p = .417). Estimated LBM was greater in black men (61.8 kg) and women (45.5 kg) than in white men (58.9 kg) and women (42.2 kg) (p≤.001). The strongest correlate of urine osmolality in all race-sex groups was urine creatinine (Spearman ρ = .68-.75). These results affirm that individuals identifying as black produce higher urine creatinine concentrations and, in women, higher urine osmolality after matching for age, fluid intake, and socioeconomic status. The findings suggest caution when comparing urine hydration markers between racial groups.

Drug Crystal Precipitation in Biorelevant Bicarbonate Buffer: A Well-Controlled Comparative Study with Phosphate Buffer.

The purpose of the present study was to clarify whether the precipitation profile of a drug in bicarbonate buffer (BCB) may differ from that in phosphate buffer (PPB) by a well-controlled comparative study. The precipitation profiles of structurally diverse poorly soluble drugs in BCB and PPB were evaluated by a pH-shift precipitation test or a solvent-shift precipitation test (seven weak acid drugs (pKa: 4.2 to 7.5), six weak base drugs (pKa: 4.8 to 8.4), one unionizable drug, and one zwitterionic drug). To focus on crystal precipitation processes, each ionizable drug was first completely dissolved in an HCl (pH 3.0) or NaOH (pH 11.0) aqueous solution (450 mL, 50 rpm, 37 °C). A 10-fold concentrated buffer solution (50 mL) was then added to shift the pH value to 6.5 to initiate precipitation (final volume: 500 mL, buffer capacity (ß): 4.4 mM/ΔpH (BCB: 10 mM or PPB: 8 mM), ionic strength (I): 0.14 M (adjusted by NaCl)). The pH, ß, and I values were set to be relevant to the physiology of the small intestine. For an unionizable drug, a solvent-shift method was used (1/100 dilution). To maintain the pH value of BCB, a floating lid was used to avoid the loss of CO2. The floating lid was applied also to PPB to precisely align the experimental conditions between BCB and PPB. The solid form of the precipitants was identified by powder X-ray diffraction and differential scanning microscopy. The precipitation of weak acids (pKa ≤ 5.1) and weak bases (pKa ≥ 7.3) was found to be slower in BCB than in PPB. In contrast, the precipitation profiles in BCB and PPB were similar for less ionizable or nonionizable drugs at pH 6.5. The final pH values of the bulk phase were pH 6.5 ± 0.1 after the precipitation tests in all cases. All precipitates were in their respective free forms. The precipitation of ionizable weak acids and bases was slower in BCB than in PPB. The surface pH of precipitating particles may have differed between BCB and PPB due to the slow hydration process of CO2 specific to BCB. Since BCB is a physiological buffer in the small intestine, it should be considered as an option for precipitation studies of ionizable weak acids and bases.

Short communication: Storage time and temperature affect plasma osmolality values in field-collected blood samples.

As climate change alters the hydric regime of many habitats, understanding the hydric physiology of animals becomes increasingly important. Plasma osmolality is a popular metric to assess an organism's hydration, but samples often need to be stored before being analyzed, under varying conditions and for different lengths of time. Previous studies on plasma storage conditions, and how they impact sample integrity, are minimal and have focused more on clinical applications than field studies. We studied the stability of osmolality values from wild rattlesnake plasma samples stored in commonly used plastic snap-cap tubes under different time (0, 2, 3, 7, 29 days) and temperature (refrigerated at 2 °C and frozen at -18 °C) treatments. We hypothesized that frozen samples would remain more stable (e.g., retain osmolality values more similar to baseline values) than refrigerated samples because freezing the plasma would reduce evaporation. We found that osmolality of samples increased over time at both temperatures, becoming significantly higher than baseline after 7 days. Contrary to our prediction, osmolality increased more in frozen samples than in refrigerated samples. We discuss possible reasons for our results, along with their implications. To obtain the most accurate plasma osmolality values, we recommend refrigerating plasma samples for as short a time as possible, 3 days or fewer, before analyzing them on an osmometer.

Exploring albumin-sulfonephthalein dyes interactions by a chemometric-assisted and multi-technique equilibria analysis in solution.

Albumin is undoubtedly the most studied protein thanks to its widespread diffusion and biochemistry; despite its binding ability towards different dyes, provoking dye's colour change, has been exploited for decades for quantification purposes, the joint effect of working pH, ionic strength, and dye's pKa still remains only sporadically discussed. In the present study, the interaction of Bovine Serum Albumin (BSA) with five dyes belonging to the sulfonephthalein group, Bromophenol Blue (BPB, pKa = 3.75), Bromocresol Green (BCG, pKa = 4.42), Chlorophenol Red (CPR, pKa = 5.74), Bromocresol Purple (BCP, pKa = 6.05) and Bromothymol Blue (BTB, pKa = 6.72), is investigated at four working pH values (3.5, 6.0, 7.5 and 9.0) and two ionic strength conditions by UV-Vis spectroscopy. Principal Component Analysis is then applied to rationalize dye behavior upon BSA addition at each pH value and to summarize the protein effect on dyes' spectral features, identifying three general behaviors. The most relevant systems are then submitted to further characterization involving a solution equilibria study aimed at determining conditional binding constants for the selected DSA-dye adducts and fluorescence, CD, and 1H NMR spectroscopy to evaluate the binding effect on the species involved.

Assessing neophyte response to daily disposable silicone hydrogel contact lenses: A randomised clinical trial investigation over one month.

OBJECTIVE: This randomised clinical trial assessed the impact on symptoms, tear film dynamics and ocular surface integrity of daily disposable silicone-hydrogel contact lenses (CLs) over a month, paying special attention to lid wiper epitheliopathy (LWE) and its implications for CL discomfort. METHODS: Neophyte CL wearers (n = 44, 21.09 ± 5.00 years old) were randomly assigned to either the experimental (n = 24) or control group (n = 20). Participants assigned to the experimental group were required to wear daily disposable CLs for 1 month for at least 8 h/day and 6 days/week. All participants were healthy subjects (no history of ocular surgery or active ocular disease) with spherical refractive errors between -8.00 and +5.00 D and cylindrical power <0.75 D. At the baseline and 1-month sessions, the Dry Eye Questionnaire 5 (DEQ-5) was completed, together with the measurement of tear film osmolarity with the TearLab osmometer, tear meniscus height (TMH) and lipid layer pattern (LLP) using a slit-lamp with Tearscope Plus attached, fluorescein break-up time (FBUT), maximum blink interval (MBI), corneal staining with fluorescein under cobalt blue light and LWE with lissamine green under slit lamp and halogen white light. RESULTS: At the baseline session, LWE showed a negative correlation with DEQ-5 (r = -0.37, p = 0.02). Significant differences in FBUT and LWE (p = 0.04) and a positive correlation between LWE and DEQ-5 (r = 0.49, p = 0.007) were observed at 1 month. Intrasession analysis at 1 month showed significant differences between the experimental and control groups in DEQ-5, FBUT and LWE (all p ≤ 0.02). Intersession analysis in the experimental group showed variations in DEQ-5, FBUT and LWE (all p ≤ 0.02) but no significant variation in the control group (all p ≥ 0.11). CONCLUSION: The presence of LWE was significantly correlated with higher symptom values in the DEQ-5. Also, participants in the experimental group presented higher values of LWE after 1 month of CL wear, in comparison with the control group.

Inhibition of silica scaling with functional polymers: Role of ionic strength, divalent ions, and temperature.

High concentrations of dissolved silica in saline industrial wastewaters and brines cause silica scale formation, significantly hampering the efficacy of diverse engineered systems. Applying functional polymers as scale inhibitors in process feedwater is a common strategy to mitigate silica scaling. However, feedwater characteristics often vary widely, depending on the specific processes, making the inhibition of silica scaling challenging and complex. In this study, we systematically investigate the role of ionic composition, specifically ionic strength and divalent ions, and solution temperature, in inhibiting silica scaling using molecularly designed amine/amide polymers. The inhibitor demonstrates effective stabilization of silicic acid, with inhibition efficiency of 74 and 55 % in the absence and presence of 20,000 ppm NaCl, respectively. However, further increasing the ionic strength of oversaturated silicic acid solutions significantly diminishes inhibition performance, rendering it ineffective at 180,000 ppm NaCl. Divalent inorganic cations exhibit a stronger impact on reducing inhibition efficiency compared to sodium ions. Molecular dynamics simulations reveal a competition mechanism between anionic silicic acid reactants (i.e., H3SiO4-) and chlorides for binding to ammonium groups within the polymeric inhibitor. Additionally, cations form clusters with H3SiO4- ions, hindering their stabilization with polymeric inhibitor. Notably, at elevated temperatures, the inhibitor achieves near-perfect inhibition for 500 ppm silicic acid solutions. This comprehensive assessment provides important insights into the effectiveness of silica scaling inhibitors under solution conditions relevant to real-world applications, addressing the challenges posed by varying solution parameters in diverse industrial processes.

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.

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.

Characterization of Freezing Processes in Drug Substance Bottles by Ice Core Sampling.

Freezing of biological drug substance (DS) is a critical unit operation that may impact product quality, potentially leading to protein aggregation and sub-visible particle formation. Cryo-concentration has been identified as a critical parameter to impact protein stability during freezing and should therefore be minimized. The macroscopic cryo-concentration, in the following only referred to as cryo-concentration, is majorly influenced by the freezing rate, which is in turn impacted by product independent process parameters such as the DS container, its size and fill level, and the freezing equipment. (At-scale) process characterization studies are crucial to understand and optimize freezing processes. However, evaluating cryo-concentration requires sampling of the frozen bulk, which is typically performed by cutting the ice block into pieces for subsequent analysis. Also, the large amount of product requirement for these studies is a major limitation. In this study, we report the development of a simple methodology for experimental characterization of frozen DS in bottles at relevant scale using a surrogate solution. The novel ice core sampling technique identifies the axial ice core in the center to be indicative for cryo-concentration, which was measured by osmolality, and concentrations of histidine and polysorbate 80 (PS80), whereas osmolality revealed to be a sensitive read-out. Finally, we exemplify the suitability of the method to study cryo-concentration in DS bottles by comparing cryo-concentrations from different freezing protocols (-80°C vs -40°C). Prolonged stress times during freezing correlated to a higher extent of cryo-concentration quantified by osmolality in the axial center of a 2 L DS bottle.

On the covalent nature of lysine polyphosphorylation.

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell.

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.

NAGase sensing in 3% milk: FET-based specific and label-free sensing in ultra-small samples of high ionic strength and high concentration of non-specific proteins.

Biosensing with biological field-effect transistors (bioFETs) is a promising technology toward specific, label-free, and multiplexed sensing in ultra-small samples. The current study employs the field-effect meta-nano-channel biosensor (MNC biosensor) for the detection of the enzyme N-acetyl-beta-D-glucosaminidase (NAGase), a biomarker for milk cow infections. The measurements are performed in a 0.5 µL drops of 3% commercial milk spiked with NAGase concentrations in the range of 30.3 aM-3.03 µM (Note that there is no background NAGase concentration in commercial milk). Specific and label-free sensing of NAGase is demonstrated with a limit-of-detection of 30.3 aM, a dynamic range of 11 orders of magnitude and with excellent linearity and sensitivity. Additional two important research outcomes are reported. First, the ionic strength of the examined milk is ∼120 mM which implies a bulk Debye screening length <1 nm. Conventionally, a 1 nm Debye length excludes the possibility of sensing with a recognition layer composed of surface bound anti-NAGase antibodies with a size of ∼10 nm. This apparent contradiction is removed considering the ample literature reporting antibody adsorption in a predominantly surface tilted configuration (side-on, flat-on, etc.). Secondly, milk contains a non-specific background protein concentration of 33 mg/ml, in addition to considerable amounts of micron-size heterogeneous fat structures. The reported sensing was performed without the customarily exercised surface blocking and without washing of the non-specific signal. This suggests that the role of non-specific adsorption to the BioFET sensing signal needs to be further evaluated. Control measurements are reported.