OBJECTIVE: To develop a fast, sensitive and cost-effective method based on resonance light scattering (RLS) for characterization of protein solubility to facilitate detection of changes in solubility of mutant proteins. METHODS: We examined the response curve of RLS intensities to the protein concentrations in synchronous scanning mode. The curve intersection points were searched to predict the maximal concentrations of the protein in dispersion state, which defined the solubility of the protein in this given state. Bovine serum albumin (BSA, 0-50 g/L) was used as the model to investigate the influences of pH values (6.5, 7.0, and 7.4) and salt concentrations (0.05, 0.10, 0.15, and 0.20 mol/L) on the determined solubility. The solubility of glutathione S-transferase isoenzymes alpha (GSTA, 0-27.0 g/L) and Mμ (GSTM, 0-20.0 g/L) were estimated for comparison. The RLS-based method was used to determine the solubility of uricase (MGU, 0-0.4 g/L) to provide assistance in improving the solubility of its mutants. RESULTS: We identified two intersection points in the RLS response curves of the tested proteins, among which the lower one represented an approximation of the maximal concentration (or the solubility of the protein) in single molecular dispersion, and the higher one the saturated concentration of the protein in multiple molecular aggregation. In HEPES buffer, the two intersection points of BSA (isoelectric point 4.6) both increased with the increase of pH (6.5-7.4), and their values were ~1.2 g/L and ~33 g/L at pH 7.4, respectively; the latter concentration approached the solubility of commercial BSA in the same buffer at the same pH. The addition of NaCl reduced the values of the two intersection points, and increasing salt ion concentration decreased the values of the lower intersection points. Further characterizations of GSTA and GSTM showed that the low concentration intersection points of the two proteins were ~0.7 g/L and ~0.8 g/L, and their high concentration intersection points were ~10 g/L and ~11 g/L, respectively, both lower than those of BSA, indicating the feasibility of the direct characterization of protein solubility by RLS. The two concentration intersection points of MGU were 0.24 g/L and 0.30 g/L, respectively, and the low concentration intersection point of its selected mutant was increased by 2 times. CONCLUSIONS RLS allows direct characterization of the solubility of macromolecular proteins. This method, which is simple and sensitive and needs only a small amount of proteins, has a unique advantage for rapid comparison of solubility of low-abundance protein mutants.
Hydrogen-Ion Concentration ; Light ; Scattering, Radiation ; Solubility ; Spectrum Analysis

Adult ; Chromatin ; DNA Fragmentation ; Humans ; Hydrogen-Ion Concentration ; Infertility, Male ; Male ; Middle Aged ; Multivariate Analysis ; Principal Component Analysis ; Semen Analysis/methods* ; Sperm Count ; Sperm Motility ; Spermatozoa/metabolism*

We prepared a polymorphic form of valnemulin hydrogen tartrate (Form I) to overcome the instability and irritating odor of valnemulin hydrochloride that affect its use in the production and application of veterinary drugs. The physicochemical properties of Form I were characterized by scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analysis. The results showed the crystal structure and thermal properties of Form I were very different from those of a commercially available form of valnemulin hydrogen tartrate (Form II). Form I and Form II were more stable than valnemulin hydrochloride after storage under irradiation and high humidity conditions, respectively. The solubility of Form I was 2.6 times that of Form II, and Form I was selected for use in pharmaceutical kinetics experiments in vivo. Compared to valnemulin hydrochloride, after oral administration at a dose of 10 mg/kg in pigs, Form I had similar pharmaceutical kinetic behavior but a slightly higher area under the concentration–time curve from time zero to the last measurable concentration. Consequently, Form I should be suitable for the development of simple formulations and be effective in the clinical application of veterinary drugs.
Administration, Oral ; Calorimetry, Differential Scanning ; Humidity ; Hydrogen ; Kinetics ; Microscopy, Electron, Scanning ; Odors ; Pharmacokinetics ; Powder Diffraction ; Solubility ; Spectrum Analysis ; Swine ; Veterinary Drugs

BACKGROUND: The left internal thoracic artery (LITA) has been used as the first conduit of choice in coronary artery bypass grafting (CABG) because of excellent long-term patency and outcomes. However, no studies have examined substances other than nitric oxide that could be beneficial for the bypass conduit, native coronary artery or ischemic myocardium. This study was conducted to evaluate differences in metabolic profiles between the LITA and ascending aorta using gas chromatography-time of flight-mass spectrometry (GC-TOF-MS). METHODS: Twenty patients who underwent CABG using the LITA were prospectively enrolled. Plasma samples were collected simultaneously from the LITA and ascending aorta. GC-TOF-MS based untargeted metabolomic analyses were performed and a 2-step volcano plot analysis was used to identify distinguishable markers from two plasma metabolome profiles. Semi-quantitative and quantitative analyses were performed using GC-TOF-MS and enzyme-linked immunosorbent assay, respectively, after selecting target metabolites based on the metabolite set enrichment analysis. RESULTS: Initial volcano plot analysis demonstrated 5 possible markers among 851 peaks detected. The final analysis demonstrated that the L-cysteine peak was significantly higher in the LITA than in the ascending aorta (fold change = 1.86). The concentrations of intermediate metabolites such as L-cysteine, L-methionine and L-cystine in the ‘cysteine and methionine metabolism pathway' were significantly higher in the LITA than in the ascending aorta (2.0-, 1.4- and 1.2-fold, respectively). Quantitative analysis showed that the concentration of hydrogen sulfide (H2S) was significantly higher in the LITA. CONCLUSION: The plasma metabolome profiles of the LITA and ascending aorta were different, particularly higher plasma concentrations of L-cysteine and H2S in the LITA.
Aorta ; Chromatography, Gas ; Coronary Artery Bypass ; Coronary Vessels ; Cysteine ; Cystine ; Enzyme-Linked Immunosorbent Assay ; Humans ; Hydrogen Sulfide ; Mammary Arteries ; Mass Spectrometry ; Metabolism ; Metabolome ; Metabolomics ; Methionine ; Myocardium ; Nitric Oxide ; Plasma ; Prospective Studies ; Spectrum Analysis

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.
Animals ; Aspergillus niger ; Calibration ; Cellulase/analysis* ; Chemistry, Clinical/methods* ; Dextranase/analysis* ; Enterocolitis, Necrotizing/diagnosis* ; Equipment Design ; Flavonoids/analysis* ; Glucose/analysis* ; Glucuronic Acid/analysis* ; Glucuronidase/analysis* ; Glycoside Hydrolases/analysis* ; Hydrogen-Ion Concentration ; Linear Models ; Multienzyme Complexes/analysis* ; Plants, Medicinal ; Polygalacturonase/analysis* ; Rats ; Reproducibility of Results ; beta-Galactosidase/analysis* ; beta-Glucosidase/analysis*

A magnetic metal organic framework (MMOF) was synthesized and used to separate Sr2+ in aqueous solution. The shape and structure of prepared Fe3O4@UiO-66-NH2 were characterized, and the absorbed concentration of strontium was determined through inductively coupled plasma mass spectrometry. The results indicated that Fe3O4 and UiO-66-NH2 combined through chemical bonding. The experimental adsorption results for separation of Sr2+ in aqueous solution indicated that the adsorption of Sr2+ to Fe3O4@UiO-66-NH2 increased drastically from pH 11 to pH 13. The adsorption isotherm model indicated that the adsorption of Sr2+ conformed to the Freundlich isotherm model (R2 = 0.9919). The MMOF thus inherited the superior qualities of magnetic composites and metal organic frameworks, and can easily be separated under an external magnetic field. This MMOF thus has potential applications as a magnetic adsorbent for low level radionuclide 90Sr.
Adsorption ; Ferrosoferric Oxide ; chemistry ; Hydrogen-Ion Concentration ; Metal-Organic Frameworks ; chemical synthesis ; chemistry ; Models, Theoretical ; Nanoparticles ; chemistry ; Strontium ; analysis ; Surface Properties ; Water Pollutants, Radioactive ; analysis ; Water Purification ; methods

Acanthamoeba spp. are free-living protozoa that are opportunistic pathogens for humans. Cysteine proteases of Acanthamoeba have been partially characterized, but their biochemical and functional properties are not clearly understood yet. In this study, we isolated a gene encoding cysteine protease of A. castellanii (AcCP) and its biochemical and functional properties were analyzed. Sequence analysis of AcCP suggests that this enzyme is a typical cathepsin L family cysteine protease, which shares similar structural characteristics with other cathepsin L-like enzymes. The recombinant AcCP showed enzymatic activity in acidic conditions with an optimum at pH 4.0. The recombinant enzyme effectively hydrolyzed human proteins including hemoglobin, albumin, immunoglobuins A and G, and fibronectin at acidic pH. AcCP mainly localized in lysosomal compartment and its expression was observed in both trophozoites and cysts. AcCP was also identified in cultured medium of A. castellanii. Considering to lysosomal localization, secretion or release by trophozoites and continuous expression in trophozoites and cysts, the enzyme could be a multifunctional enzyme that plays important biological functions for nutrition, development and pathogenicity of A. castellanii. These results also imply that AcCP can be a promising target for development of chemotherapeutic drug for Acanthamoeba infections.
Acanthamoeba castellanii ; Acanthamoeba ; Cathepsin L ; Cathepsins ; Cysteine Proteases ; Cysteine ; Fibronectins ; Genes, vif ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; Sequence Analysis ; Trophozoites ; Virulence

Although radiation therapy is a cornerstone of modern management of malignancies, various side effects are inevitably linked to abdominal and pelvic cancer after radiotherapy. Radiation-mediated gastrointestinal (GI) toxicity impairs the life quality of cancer survivors and even shortens their lifespan. Hydrogen has been shown to protect against tissue injuries caused by oxidative stress and excessive inflammation, but its effect on radiation-induced intestinal injury was previously unknown. In the present study, we found that oral gavage with hydrogen-water increased the survival rate and body weight of mice exposed to total abdominal irradiation (TAI); oral gavage with hydrogen-water was also associated with an improvement in GI tract function and the epithelial integrity of the small intestine. Mechanistically, microarray analysis revealed that hydrogen-water administration upregulated miR-1968-5p levels, thus resulting in parallel downregulation of MyD88 expression in the small intestine after TAI exposure. Additionally, high-throughput sequencing showed that hydrogen-water oral gavage resulted in retention of the TAI-shifted intestinal bacterial composition in mice. Collectively, our findings suggested that hydrogen-water might be used as a potential therapeutic to alleviate intestinal injury induced by radiotherapy for abdominal and pelvic cancer in preclinical settings.
Animals ; Body Weight ; Down-Regulation ; Gastrointestinal Microbiome ; Gastrointestinal Tract ; Humans ; Hydrogen ; Inflammation ; Intestine, Small ; Mice ; Microarray Analysis ; Oxidative Stress ; Pelvic Neoplasms ; Quality of Life ; Radiotherapy ; Survival Rate ; Survivors

Severe eating disorders characterized by repetitive episodes of purging and vomiting can occasionally trigger acute kidney injury. However, interstitial nephritis induced by episodes of repeated vomiting has rarely been reported, and the pathophysiology of this entity remains unknown. A 26-year-old man was admitted to our hospital because of known hypokalemia. His serum electrolyte profile showed: sodium 133 mEq/L, potassium 2.6 mEq/L, chloride 72 mEq/L, total carbon dioxide 50 mEq/L, blood urea nitrogen/creatinine ratio (BUN/Cr) 21.9/1.98 mg/dL, and magnesium 2.0 mg/dL. Arterial blood gas analysis showed: pH 7.557, partial pressure of carbon dioxide 65.8 mmHg, and bicarbonate 58.5 mEq/L. His urinary potassium concentration was 73.2 mEq/L, and Cr was 111 mg/dL. Renal biopsy revealed acute tubular necrosis and tubulointerstitial nephritis with a few shrunken glomeruli. Repeated psychogenic vomiting may precipitate acute kidney injury and interstitial nephritis secondary to volume depletion and hypokalemia. Serum electrolyte levels and renal function should be carefully monitored in patients diagnosed with eating disorders to prevent tubular ischemia and interstitial nephritis.
Acute Kidney Injury ; Adult ; Anorexia Nervosa* ; Anorexia* ; Biopsy ; Blood Gas Analysis ; Carbon Dioxide ; Eating ; Humans ; Hydrogen-Ion Concentration ; Hypokalemia ; Ischemia ; Magnesium ; Male* ; Necrosis ; Nephritis, Interstitial* ; Partial Pressure ; Potassium ; Sodium ; Urea ; Vomiting

BACKGROUND: This study was conducted to investigate the heat stress and semen quality among male workers in a steel industry in Iran and investigate the relationship between heat stress indices and semen parameters. METHODS: The study was conducted on workers exposed (n = 30) and unexposed (n = 14) to heat in a steel industry. After obtaining a brief biography of the selected employees, scrotal temperature, oral temperature, and environmental parameters were measured, and their semen samples were analyzed according to the procedure recommended by the World Health Organization. The heat stress indices, including wet-bulb globe temperature (WBGT) and predicted heat strain (PHS), in their workplace were calculated according to environmental parameters (ISO 7243:1989 and 7933:2004, respectively). RESULTS: Time-weighted averages of WBGT and PHS (35.76°C and 491.56 w/m2 w m 2 , respectively) for the exposed group were higher than threshold limit values. The mean difference of environmental, physiological, and semen parameters (exception: pH of semen), and also WBGT and PHS indices were statistically significant (p < 0.05) between the two groups. Mean semen parameters were in the normozoospermic range. WBGT and PHS indices showed significantly “negative” correlation with physiological parameters (scrotal and oral temperature) and most semen parameters (semen volume, sperm morphology, sperm motility, sperm count; p < 0.05); moreover, the correlation of WBGT with these parameters was stronger than PHS. CONCLUSION: Semen parameters of the studied workers exposed to heat were in the borderline level of normozoospermic range, and their semen parameters were significantly lower than controls. For better assessment of occupational environment concerning physiological and semen parameters in steel industries, WBGT can be a more useful index.
Cross-Sectional Studies* ; Hot Temperature* ; Humans ; Hydrogen-Ion Concentration ; Iran ; Male ; Semen Analysis* ; Semen* ; Sperm Count ; Sperm Motility ; Spermatozoa ; Steel* ; Threshold Limit Values ; World Health Organization