Hepatotoxicity and ALAD Activity Profile for Prediction of NOAEL of Metal Welding Fumes in Albino Rats.

Metal fume pollutants of urban Kano, a city of over 10 million people, and widespread metal works have increased exposure with related health effects. Few data on metal fume toxicity and atmospheric levels have been documented in Nigeria and Kano in particular. Hence, the work was aimed at evaluating the metal fume toxicity to laboratory rat species for setting the permissible limit of exposure in urban Kano. The investigation involved the collection of metal welding fumes and subsequent laboratory analysis. Experimental animals were then exposed intratracheally to varying doses of the fumes which were equivalent to normal metal workers' daily routine of 2, 4, and 8 h for 3, 5, 10, and 20 years. Following euthanization, whole blood samples were collected and functions of liver and delta-aminolevunilic acid dehydratase were evaluated in the serum. Exposure to the fumes has caused significant mortality that was observed to be dose-dependent and statistically different (p < 0.05); moreover, the fumes had synergistically affected the functions of liver. In addition, the fumes had increased (statistically) the activity delta-aminolevinilic acid dehydratase. This has indicated that exposure to metal welding fumes being multi-elemental is toxic and had produced mortality at exposure to higher doses of metal welding fumes. It was therefore established from the study that no-observed-adverse-effect level (NOAEL) for metal welding fumes is 25.73 mg with LD50 of 270 mg which corresponds to the metal worker's 4-h shifts daily for 5 years under existing working conditions. It was recommended that regular monitoring should be put in place to limit exposure and extent of engagement in metal works beyond NOAEL levels.

Laboratory Findings Associated With Severe Illness and Mortality Among Hospitalized Individuals With Coronavirus Disease 2019 in Eastern Massachusetts.

Importance: The coronavirus disease 2019 (COVID-19) pandemic has placed unprecedented stress on health systems across the world, and reliable estimates of risk for adverse hospital outcomes are needed. Objective: To quantify admission laboratory and comorbidity features associated with critical illness and mortality risk across 6 Eastern Massachusetts hospitals. Design, Setting, and Participants: Retrospective cohort study of all individuals admitted to the hospital who tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by polymerase chain reaction across these 6 hospitals through June 5, 2020, using hospital course, prior diagnoses, and laboratory values in emergency department and inpatient settings from 2 academic medical centers and 4 community hospitals. The data were extracted on June 11, 2020, and the analysis was conducted from June to July 2020. Exposures: SARS-CoV-2. Main Outcomes and Measures: Severe illness defined by admission to intensive care unit, mechanical ventilation, or death. Results: Of 2511 hospitalized individuals who tested positive for SARS-CoV-2 (of whom 50.9% were male, 53.9% White, and 27.0% Hispanic, with a mean [SD ]age of 62.6 [19.0] years), 215 (8.6%) were admitted to the intensive care unit, 164 (6.5%) required mechanical ventilation, and 292 (11.6%) died. L1-regression models developed in 3 of these hospitals yielded an area under the receiver operating characteristic curve of 0.807 for severe illness and 0.847 for mortality in the 3 held-out hospitals. In total, 212 of 292 deaths (72.6%) occurred in the highest-risk mortality quintile. Conclusions and Relevance: In this cohort, specific admission laboratory studies in concert with sociodemographic features and prior diagnosis facilitated risk stratification among individuals hospitalized for COVID-19.

A colorimetric assay method for measuring d-glutamate cyclase activity.

In mammals, metabolism of free d-glutamate is regulated by d-glutamate cyclase (DGLUCY), which reversibly converts d-glutamate to 5-oxo-d-proline and H2O. Metabolism of these d-amino acids by DGLUCY is thought to regulate cardiac function. In this study, we established a simple, accurate, and sensitive colorimetric assay method for measuring DGLUCY activity. To this end, we optimized experimental procedures for derivatizing 5-oxo-d-proline with 2-nitrophenylhydrazine hydrochloride. 5-Oxo-d-proline was derivatized with 2-nitrophenylhydrazine hydrochloride in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a catalyst to generate the acid hydrazides, whose levels were then determined using a colorimetric method. Under optimized conditions, we examined the sensitivity and accuracy of the colorimetric method and compared our technique with other methods by high-performance liquid chromatography with ultraviolet-visible or fluorescence detection. Moreover, we assessed the suitability of this colorimetric method for measuring DGLUCY activity in biological samples. Our colorimetric method could determine DGLUCY activity with adequate validity and reliability. This method will help to elucidate the relationship among DGLUCY activity, the physiological and pathological roles of d-glutamate and 5-oxo-d-proline, and cardiac function.

[Protective effect of astrocyte exosomes on hypoxic-ischemic neurons].

OBJECTIVE: To study the effect of astrocyte exosomes on hypoxic-ischemic neurons. METHODS: Rat astrocytes were cultured in vitro, and differential centrifugation was used to obtain the exosomes from the cell supernatant. Transmission electron microscopy, Nanosight, and Western blot were used for the identification of exosomes. BCA method was used to measure the concentration of exosomes. Rat neurons were cultured in vitro and then divided into control group, exosome group, oxygen glucose deprivation (OGD) group, and OGD+exosome group (n=3 each). The OGD and OGD+exosome groups were cultured in glucose-free medium under the hypoxic condition. The exosome and OGD+exosome groups were treated with exosomes at a final concentration of 22 μg/mL. The control and OGD groups were given an equal volume of phosphate-buffered saline. ELISA was used to measure the level of lactate dehydrogenase (LDH) in neurons. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to measure the apoptotic index of neurons. RESULTS: The identification of exosomes showed that the exosomes extracted by differential centrifugation had the features of exosomes. Compared with the control and exosome groups, the OGD group had significant increases in LDH level and apoptotic index (P<0.05). Compared with the OGD group, the OGD+exosome group had significant reductions in LDH level and apoptotic index (P<0.05). CONCLUSIONS: The exosomes from astrocytes have a protective effect on neurons with hypoxic-ischemic injury.

Physiology underlies the assembly of ecological communities.

Trait-based community ecology promises an understanding of the factors that determine species abundances and distributions across habitats. However, ecologists are often faced with large suites of potentially important traits, making generalizations across ecosystems and species difficult or even impossible. Here, we hypothesize that key traits structuring ecological communities may be causally dependent on common physiological mechanisms and that elucidating these mechanisms can help us understand the distributions of traits and species across habitats. We test this hypothesis by investigating putatively causal relationships between physiological and behavioral traits at the species and community levels in larvae of 17 species of dragonfly that co-occur at the landscape scale but segregate among lakes. We use tools borrowed from phenotypic selection analyses to show that physiological traits underlie activity rate, which has opposing effects on foraging and predator avoidance behaviors. The effect of activity on these behaviors ultimately shapes species distributions and community composition in habitats with either large-bodied fish or invertebrates as top predators. Remarkably, despite the inherent complexity of ecological communities, the expression of just two biomolecules accounts for a high proportion of the variation in behavioral traits and hence, dragonfly community composition between habitats. We suggest that causal relationships among traits can drive species distributions and community assembly.

A continuous assay for l-talarate/galactarate dehydratase using circular dichroism.

l-Talarate/galactarate dehydratase (TGD) is a member of the enolase superfamily of enzymes and catalyzes the dehydration of either meso-galactarate or l-talarate to form 5-keto-4-deoxy-d-glucarate (5-KDG). To facilitate study of this enzyme and other galactarate dehydratases, a continuous circular dichroism-based assay has been developed. Using recombinant enzyme from Salmonella typhimurium (StTGD), the rates of StTGD-catalyzed conversion of m-galactarate to 5-KDG were determined by following the change in ellipticity at 323 nm. The apparent molar ellipticity ([θ]323) for the 5-KDG formed was determined to be 202 ±â€¯2 deg cm2 dmol-1, which was used to convert observed rates (Δθ/Δt) into concentration-dependent rates (Δc/Δt). The kinetic parameters Km, kcat, and kcat/Km were 0.38 ±â€¯0.05 mM, 4.8 ±â€¯0.1 s-1, and 1.3 (±0.2) × 104 M-1s-1, respectively. These values are in excellent agreement with those published previously [Yew, W.S. et al. (2007) Biochemistry46, 9564-9577] using a coupled assay system. To demonstrate the utility of the assay, the inhibition constant (Ki = 10.7 ±â€¯0.4 mM) was determined for the competitive inhibitor tartronate. The continuous CD-based assay offers a practical and efficient alternative method to the coupled assay that requires access to 5-KDG aldolase, and to the labor-intensive, fixed-time assays.

From Multistep Enzyme Monitoring to Whole-Cell Biotransformations: Development of Real-Time Ultraviolet Resonance Raman Spectroscopy.

Process analytical technologies (PAT) are used within industry to give real-time measurements of critical quality parameters, ultimately improving the quality by design (QbD) of the final product and reducing manufacturing costs. Spectroscopic and spectrophotometric methods are readily employed within PAT due to their ease of use, compatibility toward a range of sample types, robustness, and multiplexing capabilities. We have developed a UV resonance Raman (UVRR) spectroscopy approach to quantify industrially relevant biotransformations accurately, focusing on nitrile metabolizing enzymes: nitrile hydratase (NHase) and amidase versus nitrilase activity. Sensitive detection of the amide intermediate by UVRR spectroscopy enabled discrimination between the two nitrile-hydrolyzing pathways. Development of a flow-cell apparatus further exemplifies its suitability toward PAT measurements, incorporating in situ analysis within a closed system. Multivariate curve resolution-alternating least-squares (MCR-ALS) was applied to the UVRR spectra, as well as off-line HPLC measurements, to enable absolute quantification of substrate, intermediate, and product. Further application of hard modeling to MCR-ALS deconvolved concentration profiles enabled accurate kinetic determinations, thus removing the requirement for comparative off-line HPLC. Finally, successful quantitative measurements of in vivo activity using whole-cell biotransformations, where two Escherichia coli strains expressing either NHase (transforming benzonitrile to benzamide) or amidase (further conversion of benzamide to benzoic acid), illustrate the power, practicality, and sensitivity of this novel approach of multistep and, with further refinement, we believe, multiple micro-organism biotransformations.

Helicobacter pylori Antigens Inducing Early Immune Response in Infants.

To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4-11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease ß subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.

Spectrophotometric assay for sensitive detection of glycerol dehydratase activity using aldehyde dehydrogenase.

Glycerol dehydratase (GDHt) is a pivotal enzyme for fermentative utilization of glycerol by catalyzing radical-mediated conversion of glycerol into 3-hydroxypropionaldehyde (3-HPA). Precise and sensitive monitoring of cellular GDHt activity during the fermentation process is a prerequisite for reliable metabolic analysis to afford efficient cellular engineering and process optimization. Here we report a new spectrophotometric assay for the sensitive measurement of the GDHt activity with a sub-nanomolar limit of detection (LOD). The assay method employs aldehyde dehydrogenase (ALDH) as a reporter enzyme, so the readout of the GDHt activity is recorded at 340 nm as an increase in UV absorbance which results from NADH generation accompanied by oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP). The GDHt assay was performed under the reaction conditions where the ALDH activity overwhelms the GDHt activity (i.e., 50-fold higher activity of ALDH relative to GDHt activity), affording sensitive detection of GDHt with 360 pM LOD. The ALDH-coupled assay was used to determine kinetic parameters of GDHt for glycerol, leading to KM = 0.73 ± 0.09 mM and kcat = 400 ± 20 s-1 which are in reasonable agreements with the previous reports. Our assay method allowed measurement of even a 104-fold decrease in the cellular GDHt activity during fermentative production of 3-HP, which demonstrates the detection sensitivity much higher than the previous methods.

Machine perfusion versus cold storage of livers: a meta-analysis.

Different organ preservation methods are key factors influencing the results of liver transplantation. In this study, the outcomes of experimental models receiving donation after cardiac death (DCD) livers preserved through machine perfusion (MP) or static cold storage (CS) were compared by conducting a meta-analysis. Standardized mean difference (SMD) and 95% confidence interval (CI) were calculated to compare pooled data from two animal species. Twenty-four studies involving MP preservation were included in the meta-analysis. Compared with CS preservation, MP can reduce the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and hyaluronic acid (HA) and the changes in liver weight. By contrast, MP can enhance bile production and portal vein flow (PVF). Alkaline phosphatase (ALP) levels and histological changes significantly differed between the two preservation methods. In conclusion, MP of DCD livers is superior to CS in experimental animals.

A high-throughput screening assay for distinguishing nitrile hydratases from nitrilases.

A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.

A high-throughput screening assay for distinguishing nitrile hydratases from nitrilases

A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.

Dyskerin, tRNA genes, and condensin tether pericentric chromatin to the spindle axis in mitosis.

Condensin is enriched in the pericentromere of budding yeast chromosomes where it is constrained to the spindle axis in metaphase. Pericentric condensin contributes to chromatin compaction, resistance to microtubule-based spindle forces, and spindle length and variance regulation. Condensin is clustered along the spindle axis in a heterogeneous fashion. We demonstrate that pericentric enrichment of condensin is mediated by interactions with transfer ribonucleic acid (tRNA) genes and their regulatory factors. This recruitment is important for generating axial tension on the pericentromere and coordinating movement between pericentromeres from different chromosomes. The interaction between condensin and tRNA genes in the pericentromere reveals a feature of yeast centromeres that has profound implications for the function and evolution of mitotic segregation mechanisms.

Improving the predictive accuracy of identifying exudative effusions.

BACKGROUND: Application of Light's criteria results in misclassification of some transudative effusions as exudative, particularly because of congestive heart failure (CHF). We sought to determine if the serum to pleural fluid albumin (SF-A) and serum to pleural fluid protein (SF-P) gradients increased the predictive accuracy to correctly identify exudative effusions. METHODS: We retrospectively analyzed 1,153 consecutive patients who underwent a diagnostic thoracentesis at the Medical University South Carolina. Univariable logistic regression analyses were used to determine the statistical significance of pleural fluid tests that correctly identified exudative effusions. Tests with significant diagnostic accuracy were combined in multivariable logistic regression models, with calculation of areas under the curve (AUCs) to determine their predictive accuracy. The predictive capability of the best model was compared with Light's criteria and other test combinations. RESULTS: Pleural fluid lactate dehydrogenase (LDH), SF-A gradient, and SF-P gradient had a significant effect on the probability of identifying exudative pleural effusions. When combined together in a multivariable logistic regression, LDH (OR, 14.09 [95% CI, 2.25-85.50]), SF-A gradient (OR, 7.16 [95% CI, 1.24-41.43]), and SF-P gradient (OR, 6.83 [95% CI, 1.56-27.88]) had an AUC of 0.92 (95% CI, 0.85-0.98). CONCLUSIONS: Application of Light's criteria, not uncommonly, misclassifies CHF transudative effusions as exudates. In cases where no cause for an exudative effusion can be identified or CHF is suspected, the sequential application of the fluid LDH, followed by the SF-P and then the SF-A gradients, may assist in reclassifying pleural effusions as transudates.

Pretreatment of therapeutic cells with poly(ADP-ribose) polymerase inhibitor enhances their efficacy in an in vitro model of cell-based therapy in myocardial infarct.

The potential of cell-based therapies in diseases involving ischemia-reperfusion is greatly hampered by the excessive loss of administered cells in the harsh and oxidative environment where these cells are supposed to act. Therefore, we investigated if inhibition of poly(ADP-ribose) polymerase (PARP) in the therapeutically added cells would lead to their increased viability and, subsequently, to an enhanced effect in an in vitro simulated ischemia-reperfusion (I-R) setting. Ischemic conditions were simulated by oxygen and glucose deprivation for 160 min using H9c2 rat cardiomyoblast cells. After 30 min of reperfusion, these cells received 4 types of treatments: no added cells (I-R model), fluorescently labeled (Vybrant DiD) therapeutic H9c2 cells with vehicle (H9c2) or PARP inhibitor (10 µM or 100 µM PJ34) pretreatment. We assessed viability (live, apoptotic and necrotic) of both 'postischemic' and therapeutic cells with flow cytometric analysis using calcein-AM/ethidium homodimer-2 fluorescent staining after 24 h of co-culture. Further measurements on necrosis and metabolic activity were performed using lactate dehydrogenase (LDH) release and resazurin based assays. The percentage of surviving therapeutic cells increased significantly with PARP inhibition (untreated, 52.02±5.01%; 10 µM PJ34, 63.38±4.50%; 100 µM PJ34, 64.99±3.47%). The percentage of necrotic cells decreased in a similar manner (untreated, 37.23±4.40%; 10 µM PJ34, 26.83±3.49%; 100 µM PJ34, 24.96±2.43%). Notably, the survival of the cells that suffered I-R injury was also significantly higher when treated with PARP-inhibited therapeutic cells (I-R model, 36.44±5.05%; H9c2, 42.81±5.11%; 10 µM PJ34, 52.07±5.80%; 100 µM PJ34, 54.95±5.55%), while necrosis was inhibited (I-R model, 43.64±4.00%; H9c2, 37.29±4.55%; 10 µM PJ34, 30.18±4.60%; 100 µM PJ34, 25.52±3.47%). In subsequent experiments, PARP inhibition decreased LDH-release of the observed combined cell population and enhanced the metabolic activity. Thus, our results suggest that pretreating the therapeutically added cells with a PARP inhibitor could be beneficial in the setting of cell-based therapies.

On the variability of experimental data in macromolecular crystallography.

Experimental errors as determined by data-processing algorithms in macromolecular crystallography are compared with the direct error estimates obtained by a multiple crystal data-collection protocol. It is found that several-fold error inflation is necessary to account for crystal-to-crystal variation. It is shown that similar error inflation is observed for data collected from multiple sections of the same crystal, indicating non-uniform crystal growth as one of the likely sources of additional data variation. Other potential sources of error inflation include differential X-ray absorption for different reflections and variation of unit-cell parameters. The underestimation of the experimental errors is more severe in lower resolution shells and for reflections characterized by a higher signal-to-noise ratio. These observations partially account for the gap between the expected and the observed R values in macromolecular crystallography.

[Dependence of protective effect of alpha-tocopherol on its concentration and time of action on PC12 cell under conditions of oxidative stress].

At the short-term incubation (0.5 and 1.5 h) of cells of the PC12 neuronal line with alpha-tocopherol, its protective effect against the cytotoxic hydrogen peroxide action was increased with rise of its concentration in samples; the protection was practically absent at action of nanomolar antioxidant concentrations, but was well expressed at its micromolar concentrations. These data agree with the concept that alpha-tocopherol increases the cell living activity by reacting directly with free radicals, which leads to formation of the less reactive compounds deprived of non-paired electron. The evidence is obtained that at the long-term action on PC12 cells, alpha-tocopherol not only in micro-, but also in nanomolar concentrations increases statistically significantly the cell living activity under conditions of oxidative stress. As follows from the obtained data, an important role in realization of the alpha-tocopherol protective effect at the long-term incubation with it seems to be played by modulation by this antioxidant of activity of protein kinase activated by extracellular signaling, phosphatidylinosite 3-kinase, and protein kinase C.

Establishment and characterization of a fibroblast cell line derived from Mongolian sheep.

The Mongolian sheep ear marginal tissue fibroblast cell line (MSF32) from 32 samples was successfully established by using primary explants technique and cell cryoconservation technology. MSF32 cells were adherent, with a population doubling time of 28.2 h. Chromosome analysis showed that >90.2% of cells were diploid (2n=54) prior to cell passage 4. Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that the MSF22 cells had no cross-contamination with other species. Tests for cell line contamination with bacteria, fungi, viruses and mycoplasmas were also negative. Plasmids encoding the fluorescent proteins pEGFP-N3, pEGFP-C1, pECFP-N1, pECFP-mito, pDsRed1-N1 and pEYFP-N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 12.3% and 63.3%. Every index of the MSF32 cell line meets all the standard quality controls of American Type Culture Collection (ATCC). Not only has the genetic resources of the Mongolian sheep been preserved at the cell level, but also valuable materials had been provided for genome, postgenome and somacloning research.

Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase.

Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase.