Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls.

Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen­deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups. This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis. Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD. The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.

Groundwater well optimization to minimize contaminant movement from a surficial shallow aquifer to a lower water supply aquifer using stochastic simulation-optimization modeling techniques: Strategy formulation.

The interaction between surficial shallow aquifers of poorer quality and semi-confined water-supply aquifers poses a potential risk for degradation of the water supply. Groundwater engineers and hydrogeologists use groundwater models to synthesize field data, conceptualize hydrological processes, and improve understanding of the groundwater system to support informed decision-making. Models for decision-making, called management models, aid in the efficient planning and sustainable management of groundwater systems. Management models search for the best or least-cost management strategy satisfying hydrologic and environmental regulations. In management models, a simulation model is linked or coupled with an optimization formulation. Widely used optimization formulations are linear, non-linear, quadratic, dynamic, and global search models. Management models are applied but are not limited to maximizing withdrawals, minimizing drawdown, pumping costs, and saltwater intrusion, and determining the best locations for production wells. This paper theoretically presents the development of groundwater wellfield management strategies and the corresponding modeling framework for each strategy's evaluation. Depending on the strategy, the modeling effort applies deterministic (simulation) and stochastic (simulation-optimization) techniques. The goals of the optimization strategies are to protect wells from potential contaminant sources, identify optimal future well installation sites, mitigate risks, and extend the life of wells that may face water contamination issues.•Several management strategies are formulated addressing well depth, seasonal pumping operation, and mapping no-drilling or red zones for new well installation.•Modeling methodologies are laid down that apply thousands of numerical simulations for each strategy to simulate and evaluate recurring patterns of contaminant movement.•The simulation model integrates MODFLOW and MODPATH to simulate 3D groundwater flow and advective contaminant movement, respectively and is transferred via FloPy to couple with the optimization/decision model using a custom Python script.

Impaired intestinal function is associated with lower muscle and cognitive health and well-being in patients with congestive heart failure.

BACKGROUND: Small- and large-intestinal perturbations have been described as prevalent extracardiac systemic manifestations in congestive heart failure (CHF), but alterations in protein digestion and absorption and plasma short-chain fatty acid (SCFA) concentrations and the potential link with other systemic effects (muscle and cognitive health) have not been investigated in CHF. METHODS: We analyzed protein digestion and absorption with dual stable tracer method in 14 clinically stable, noncachectic CHF outpatients (mean left ventricular ejection fraction: 35.5% [95% CI, 30.9%-40.1%]) and 15 controls. Small-intestinal non-carrier-mediated permeability and active carrier-mediated glucose transport were quantified by sugar permeability test. Plasma SCFA (acetate, propionate, butyrate, isovalerate, valerate) concentrations were measured as intestinal microbial metabolites. Muscle function was assessed by isokinetic dynamometry, cognition by a battery of tests, and well-being by questionnaire. RESULTS: Protein digestion and absorption were impaired by 29.2% (P = .001) and active glucose transport by 38.4% (P = .010) in CHF. Non-carrier-mediated permeability was not altered. Whereas plasma propionate, butyrate, and isovalerate concentrations were lower in CHF (P < .05), acetate and valerate concentrations did not differ. Overall, intestinal dysfunction was associated with impaired leg muscle quality, emotional distress, and cognitive dysfunction (P < .05). CONCLUSIONS: We identified impaired protein digestion and absorption and altered SCFA concentrations as additional intestinal dysfunctions in CHF that are linked to reduced muscle and cognitive health and well-being. More research is needed to implement strategies to improve intestinal function in CHF and to investigate the mechanisms underlying its link with other systemic manifestations.

Glycogen supercompensation is due to increased number, not size, of glycogen particles in human skeletal muscle.

NEW FINDINGS: What is the central question of this study? Glycogen supercompensation after glycogen-depleting exercise can be achieved by consuming a carbohydrate-enriched diet, but the associated effects on the size, number and localization of intramuscular glycogen particles are unknown. What is the main finding and its importance? Using transmission electron microscopy to inspect individual glycogen particles visually, we show that glycogen supercompensation is achieved by increasing the number of particles while keeping them at submaximal sizes. This might be a strategy to ensure that glycogen particles can be used fast, because particles that are too large might impair utilization rate. ABSTRACT: Glycogen supercompensation after glycogen-depleting exercise can be achieved by consuming a carbohydrate-enriched diet, but the associated effects on the size, number and localization of intramuscular glycogen particles are unknown. We investigated how a glycogen-loading protocol affects fibre type-specific glycogen volume density, particle diameter and numerical density in three subcellular pools: between (intermyofibrillar) or within (intramyofibrillar) the myofibrils or beneath the sarcolemma (subsarcolemmal). Resting muscle biopsies from 11 physically active men were analysed using transmission electron microscopy after mixed (MIX), LOW or HIGH carbohydrate consumption separated by glycogen-lowering cycling at 75% of maximal oxygen consumption until exhaustion. After HIGH, the total volumetric glycogen content was 40% [95% confidence interval 16, 68] higher than after MIX in type I fibres (P < 0.001), with little to no difference in type II fibres (9% [95% confidence interval -9, 27]). Median particle diameter was 22.5 (interquartile range 20.8-24.7) nm across glycogen pools and fibre types, and the numerical density was 61% [25, 107] and 40% [9, 80] higher in the subsarcolemmal (P < 0.001) and intermyofibrillar (P < 0.01) pools of type I fibres, respectively, with little to no difference in the intramyofibrillar pool (3% [-20, 32]). In LOW, total glycogen was in the range of 21-23% lower, relative to MIX, in both fibre types, reflected in a 21-46% lower numerical density across pools. In comparison to MIX, particle diameter was unaffected by other diets ([-1.4, 1.3] nm). In conclusion, glycogen supercompensation after prolonged cycling is exclusive to type I fibres, predominantly in the subsarcolemmal pool, and involves an increase in the numerical density rather than the size of existing glycogen particles.

Low-level soluble chloride extraction in soil.

Three methods of extraction of low-level soluble chloride contents from vadose-zone soil were evaluated in this study. Three methods were employed on a silty sand soil using a 2:1 fluid:soil ratio: 1) Method A utilized three successive rinses with deionized water; 2) Method B applied three successive rinses of 0.0001 M and 0.001 M Na2SO4 solution; and 3) Method C passed deionized water through the soil with a pressurized filtration system three times. Method A had lower standard deviation and yielded more consistent soluble chloride contents per rinse than method C; Method B was ruled out because of concerns that the Na2SO4 reagent contained trace amounts of chloride. Method A was applied with a 1:1 fluid:soil ratio in duplicate to 50 samples from a 34-m thick vadose-zone borehole, yielding a mean difference in duplicates of 13.9% and percent total extracted soluble chloride of 62.4 ± 9.9%, 25.2 ± 7.4%, and 12.4 ± 6.6% in each of the three successive rinses.•Three successive rinses of soil with deionized water achieved consistent extraction results.•Multiple rinses are necessary to extract soluble chloride if chloride contents are low.•This method is amenable to analysis of soil in vadose-zone borehole samples.

Heterogeneity in subcellular muscle glycogen utilisation during exercise impacts endurance capacity in men.

KEY POINTS: When muscle biopsies first began to be used routinely in research on exercise physiology five decades ago, it soon become clear that the muscle content of glycogen is an important determinant of exercise performance. Glycogen particles are stored in distinct pools within the muscles, but the role of each pool during exercise and how this is affected by diet is unknown. Here, the effects of diet and exercise on these pools, as well as their relation to endurance during prolonged cycling were examined. We demonstrate here that an improved endurance capacity with high carbohydrate loading is associated with a temporal shift in the utilisation of the distinct stores of glycogen pools and is closely linked to the content of the glycogen pool closest to actin and myosin (intramyofibrillar glycogen). These findings highlight the functional importance of distinguishing between different subcellular microcompartments of glycogen in individual muscle fibres. ABSTRACT: In muscle cells, glycogen is stored in three distinct subcellular pools: between or within myofibrils (inter- and intramyofibrillar glycogen, respectively) or beneath the sarcolemma (subsarcolemmal glycogen) and these pools may well have different functions. Here, we investigated the effect of diet and exercise on the content of these distinct pools and their relation to endurance capacity in type 1 and 2 muscle fibres. Following consumption of three different diets (normal, mixed diet = MIX, high in carbohydrate = HIGH, or low in carbohydrate = LOW) for 72 h, 11 men cycled at 75% of V̇O2 max until exhaustion. The volumetric content of the glycogen pools in muscle biopsies obtained before, during, and after exercise were quantified by transmission electron micrographs. The mean (SD) time to exhaustion was 150 (30), 112 (22), and 69 (18) minutes in the HIGH, MIX and LOW trials, respectively (P < 0.001). As shown by multiple regression analyses, the intramyofibrillar glycogen content in type 1 fibres, particularly after 60 min of exercise, correlated most strongly with time to exhaustion. In the HIGH trial, intramyofibrillar glycogen was spared during the initial 60 min of exercise, which was associated with levels and utilisation of subsarcolemmal glycogen above normal. In all trials, utilisation of subsarcolemmal and intramyofibrillar glycogen was more pronounced than that of intermyofibrillar glycogen in relative terms. In conclusion, the muscle pool of intramyofibrillar glycogen appears to be the most important for endurance capacity in humans. In addition, a local abundance of subsarcolemmal glycogen reduces the utilisation of intramyofibrillar glycogen during exercise.

Barriers to receipt of novel oral oncolytics: A single-institution quality improvement investigation.

INTRODUCTION: Novel oral oncolytic agents have become the standard of care and first-line therapies for many malignancies. However, issues impacting access to these drugs are not well explored. As part of a quality improvement project in a large tertiary academic institution, we aim to identify potential barriers that delay treatment for patients who are prescribed novel oral oncolytics. METHODS: This was a retrospective review of adults who were newly prescribed a novel oral oncolytic for Food and Drug Administration-approved indications at a single tertiary care center. Patients were identified via electronic prescription data (e-Scribe). Demographics, insurance information, and prescription dates were extracted from the electronic medical record and pharmacy claims data. Statistical analyses were performed to determine whether time-to-receipt was associated with insurance category, pharmacy transfers, cost assistance, and drug prescribed. RESULTS: Of the 270 successfully filled prescriptions, the mean time-to-receipt was 7.3 ± 10.3 days (range: 0-109 days). Patients with Medicare experienced longer time-to-receipt (9.1 ± 13.1 days) compared to patients with commercial insurance (4.4 ± 3.3). Uninsured patients experienced the longest time-to-receipt (15.7 ± 7.8 days) overall. Pharmacy transfers and cost assistance programs were also significantly associated with longer time-to-receipt. Ten prescriptions remained unfilled 90 days after the study period and were considered abandoned. CONCLUSION: Insurance has a significant effect on the time-to-receipt of newly prescribed novel oral oncolytics. Pharmacy transfers and applying for cost assistance are also associated with longer wait times for patients. Our retrospective analysis identifies areas of improvement for future interventions to reduce wait times for patients receiving novel oral oncolytics.

Citrullinome of Porphyromonas gingivalis Outer Membrane Vesicles: Confident Identification of Citrullinated Peptides.

Porphyromonas gingivalis is a key pathogen in chronic periodontitis and has recently been mechanistically linked to the development of rheumatoid arthritis via the activity of peptidyl arginine deiminase generating citrullinated epitopes in the periodontium. In this project the outer membrane vesicles (OMV) from P. gingivalis W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional HFBA-based separation system combined with LC-MS. For optimal and positive identification and validation of citrullinated peptides and proteins, high resolution mass spectrometers and strict MS search criteria were utilized. This may have compromised the total number of identified citrullinations but increased the confidence of the validation. A new two-dimensional separation system proved to increase the strength of validation, and along with the use of an in-house build program, Citrullia, we establish a fast and easy semi-automatic (manual) validation of citrullinated peptides. For the WT OMV we identified 78 citrullinated proteins having a total of 161 citrullination sites. Notably, in keeping with the mechanism of OMV formation, the majority (51 out of 78) of citrullinated proteins were predicted to be exported via the inner membrane and to reside in the periplasm or being translocated to the bacterial surface. Citrullinated surface proteins may contribute to the pathogenesis of rheumatoid arthritis. For the C351A-OMV a single citrullination site was found and no citrullinations were identified for the ΔPPAD-OMV, thus validating the unbiased character of our method of citrullinated peptide identification.

Mechanistic Investigation of Molybdate-Catalysed Transfer Hydrodeoxygenation.

The molybdate-catalysed transfer hydrodeoxygenation (HDO) of benzyl alcohol to toluene driven by oxidation of the solvent isopropyl alcohol to acetone has been investigated by using a combination of experimental and computational methods. A Hammett study that compared the relative rates for the transfer HDO of five para-substituted benzylic alcohols was carried out. Density-functional theory (DFT) calculations suggest a transition state with significant loss of aromaticity contributes to the lack of linearity observed in the Hammett study. The transfer HDO could also be carried out in neat PhCH2 OH at 175 °C. Under these conditions, PhCH2 OH underwent disproportionation to yield benzaldehyde, toluene, and significant amounts of bibenzyl. Isotopic-labelling experiments (using PhCH2 OD and PhCD2 OH) showed that incorporation of deuterium into the resultant toluene originated from the α position of benzyl alcohol, which is in line with the mechanism suggested by the DFT study.

Performance of pilot-scale constructed wetlands for secondary treatment of chromium-bearing tannery wastewaters.

Tannery operations consist of converting raw animal skins into leather through a series of complex water- and chemically-intensive batch processes. Even when conventional primary treatment is supplemented with chemicals, the wastewater requires some form of biological treatment to enable the safe disposal to the natural environment. Thus, there is a need for the adoption of low cost, reliable, and easy-to-operate alternative secondary treatment processes. This paper reports the findings of two pilot-scale wetlands for the secondary treatment of primary effluents from a full tannery operation in terms of resilience (i.e., ability to produce consistent effluent quality in spite of variable influent loads) and reliability (i.e., ability to cope with sporadic shock loads) when treating this hazardous effluent. Areal mass removal rates of 77.1 g COD/m2/d, 11 g TSS/m2/d, and 53 mg Cr/m2/d were achieved with a simple gravity-flow horizontal subsurface flow unit operating at hydraulic loading rates of as much as 10 cm/d. Based on the findings, a full-scale wetland was sized to treat all the effluent from the tannery requiring 68% more land than would have been assumed based on literature values. Constructed wetlands can offer treatment plant resilience for minimum operational input and reliable effluent quality when biologically treating primary effluents from tannery operations.

Chromium fate in constructed wetlands treating tannery wastewaters.

Nine experimental wetlands were built to determine chromium partitioning inside systems treating tannery wastewaters. Results showed 5-day biochemical oxygen demand and chromium removals of 95 to 99% and 90 to 99%, respectively. The majority of chromium was found in association with media (96 to 98%), followed by effluents (2.9 to 3.9%), and the least was found in plant parts (0.1%). Chemical speciation modeling of solutions and scanning electron microscope analysis suggest two potential chromium removal mechanisms--sorption/coprecipitation with iron hydroxides or oxyhydroxides and biomass sorption. The release of the majority of chromium in the iron- and organic-bound phases during sequential extractions supports the proposed dominant removal mechanisms. The use of a mixture of peat and gravel resulted in lower removal efficiencies and stronger partitioning in organic phases during sequential extractions. Chromium was efficiently removed by wetlands, retained through chemical and biological processes. Future research will focus on further exploring removal mechanisms and proposing management strategies for the chromium-containing wetland media.