A broad pH range and processive chitinase from a metagenome library

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

Accuracy of the Timed Up and Go test for predicting sarcopenia in elderly hospitalized patients

OBJECTIVES: The ability of the Timed Up and Go test to predict sarcopenia has not been evaluated previously. The objective of this study was to evaluate the accuracy of the Timed Up and Go test for predicting sarcopenia in elderly hospitalized patients. METHODS: This cross-sectional study analyzed 68 elderly patients (≥60 years of age) in a private hospital in the city of Salvador-BA, Brazil, between the 1st and 5th day of hospitalization. The predictive variable was the Timed Up and Go test score, and the outcome of interest was the presence of sarcopenia (reduced muscle mass associated with a reduction in handgrip strength and/or weak physical performance in a 6-m gait-speed test). After the descriptive data analyses, the sensitivity, specificity and accuracy of a test using the predictive variable to predict the presence of sarcopenia were calculated. RESULTS: In total, 68 elderly individuals, with a mean age 70.4±7.7 years, were evaluated. The subjects had a Charlson Comorbidity Index score of 5.35±1.97. Most (64.7%) of the subjects had a clinical admission profile; the main reasons for hospitalization were cardiovascular disorders (22.1%), pneumonia (19.1%) and abdominal disorders (10.2%). The frequency of sarcopenia in the sample was 22.1%, and the mean length of time spent performing the Timed Up and Go test was 10.02±5.38 s. A time longer than or equal to a cutoff of 10.85 s on the Timed Up and Go test predicted sarcopenia with a sensitivity of 67% and a specificity of 88.7%. The accuracy of this cutoff for the Timed Up and Go test was good (0.80; IC=0.66-0.94; p=0.002). CONCLUSION: The Timed Up and Go test was shown to be a predictor of sarcopenia in elderly hospitalized patients. .

Mitochondrial DNA mutations and male infertility.

Infertility can be defined as difficulty in conceiving a child after 1 year of unprotected intercourse. Infertility can arise either because of the male factor or female factor or both. According to the current estimates, 15% of couples attempting their first pregnancy could not succeed. Infertility is either primary or secondary. Mitochondria have profound effect on all biochemical pathways, including the one that drivessperm motility. Sperm motility is heavily dependent on the ATP generated by oxidative phosphorylation in the mitochondrial sheath. In this review, the very positive role of mitochondrial genome's association with infertility is discussed