Whole-genome sequencing as a tool for studying the microevolution of drug-resistant serial Mycobacterium tuberculosis isolates.

Treatment of drug-resistant tuberculosis requires extended use of more toxic and less effective drugs and may result in retreatment cases due to failure, abandonment or disease recurrence. It is therefore important to understand the evolutionary process of drug resistance in Mycobacterium tuberculosis. We here in describe the microevolution of drug resistance in serial isolates from six previously treated patients. Drug resistance was initially investigated through phenotypic methods, followed by genotypic approaches. The use of whole-genome sequencing allowed the identification of mutations in the katG, rpsL and rpoB genes associated with drug resistance, including the detection of rare mutations in katG and mixed populations of strains. Molecular docking simulation studies of the impact of observed mutations on isoniazid binding were also performed. Whole-genome sequencing detected 266 single nucleotide polymorphisms between two isolates obtained from one patient, suggesting a case of exogenous reinfection. In conclusion, sequencing technologies can detect rare mutations related to drug resistance, identify subpopulations of resistant strains, and identify diverse populations of strains due to exogenous reinfection, thus improving tuberculosis control by guiding early implementation of appropriate clinical and therapeutic interventions.

A quantitative method for the detection and validation of catalase activity at physiological concentration in human serum, plasma and erythrocytes.

A novel method has been proposed to develop a simple, rapid, sensitive and affordable chromogenic attempt for the quantification of catalase (CAT) activity in blood samples. The method is based on the oxidation of pyrocatechol (PC) to give quinone form which by oxidative coupling with aminyl radical of 4-aminoantipyrine (4-AAP) resulting from H2O2/CAT to produce a pink colored quinone-imine product with λmax = 530 nm in a 100 mmol/L of tris buffer of pH 9.8 at room temperature (30 °C). The linearity of CAT assay was between 0.316 and 10 U/mL. The accuracy ranges for CAT having concentrations of 1.25, 5 and 7.5 µmol/L were 89-105.52, 90-107%, and 91-104.58% respectively. Within-run and between-run precision studies showed CV's of 1.98-3.02% (n = 7) and 2.97-4.40% (n = 7), respectively. The detection and quantification limits of CAT were 0.12 and 0.225 µmol/L, respectively. The Michaelis-Menten constant and maximum velocity of the reaction was Km = 1.052 mM and Vmax = 0.168 µmol/min, respectively. The present method provides a convenient means for investigating the usefulness of CAT measurements in biological sample assessing the potential for free radical-induced pathology.

Enhanced alkaline catalase production by Serratia marcescens FZSF01: enzyme purification, characterization, and recombinant expression

Background: Catalase (CAT) is an important enzyme that degrades H2O2 into H2O and O2. To obtain an efficient catalase, in this study, a new strain of high catalase-producing Serratia marcescens, named FZSF01, was screened and its catalase was purified and characterized. Results: After optimization of fermentation conditions, the yield of catalase produced by this strain was as high as 51,468 U/ml. This catalase was further purified using two steps: DEAE-fast flow and Sephedex-G150. The purified catalase showed a specific activity of 197,575 U/mg with a molecular mass of 58 kDa. This catalase exhibited high activity at 20­70°C and pH 5.0­11.0. Km of the catalase was approximately 68 mM, and Vmax was 1886.8 mol/min mg. This catalase was further identified by LC­MS/MS, and the encoding gene was cloned and expressed in Escherichia coli BL21 (DE3) with a production of 17,267 ± 2037 U/ml. Conclusions: To our knowledge, these results represent one of the highest fermentation levels reported among current catalase-producing strains. This FZSF01 catalase may be suitable for several industrial applications that comprise exposure to alkaline conditions and under a wide range of temperatures.

Polysorbate-80-coated, polymeric curcumin nanoparticles for in vivo anti-depressant activity across BBB and envisaged biomolecular mechanism of action through a proposed pharmacophore model.

Depression is a modern world epidemic. Its main causative factor is oxidative stress, as reported in study subjects. Natural products are yet to show significant therapeutic effects in comparison with synthetic drugs. Current study deals with the preparation of brain-targeted polysorbate-80-coated curcumin PLGA nanoparticles (PS-80-CUR-NP), and their characterisation via Spectral and optical methods. PS-80-CUR-NP were evaluated against the oxidative stress-mediated depressant (OSMD) activity via Force despair, Tail suspension tests and stress biomarker assay (SOD and catalase activity). A significant reduction in immobility (p < 0.01) in force despair and tail suspension test and a significant increase (p < 0.001 and p < 0.01) in SOD and catalase activity was found and compared with stress control, which confirmed the OSMD activity of PS-80-CUR-NP at 5 mg equivalent dose. Further, AUC(0.5-15 h) curve of brain homogenates estimated the curcumin concentration of 1.73 ng/g C max at T max 3 h via HPLC technique.