Hotspots and Mechanisms of Action of the Thermostable Framework of a Microbial Thermolipase

The lipase TrLipB from Thermomicrobium roseum is highly thermostable. However, its thermostable skeleton and mechanism of action should be investigated for industrial applications. Toward this, TrLipB was crystallized using the hanging-drop vapor diffusion method and subjected to X-ray diffraction at 2.0 Å resolution in this study. The rigid sites, such as the prolines on the relatively flexible loops on the enzyme surface, were scanned. Soft substitutions of these sites were designed using both molecular dynamics (MD) simulation and site-directed mutagenesis. The thermostability of several substitutions decreased markedly, while the catalytic efficiencies of the P9G, P127G, P194G, and P300G mutants reduced substantially; additionally, the thermostable framework of the double mutant, P194G/P300G, was considerably perturbed. However, the substitutions on the lid of the enzyme, including P49G and P48G, promoted the catalytic efficiency to approximately 150% and slightly enhanced the thermostability below 80 °C. In MD simulations, the P100G, P194G, P100G/P194G, P194G/P300G, and P100G/P194G/P300G mutants showed high B-factors and RMSD values, whereas the secondary structures, radius of gyration, H-bonds, and solvent accessible surface areas of these mutants were markedly affected. Our observations will assist in understanding the natural framework of a stable lipase, which might contribute to its industrial applications

Loss of FMRP Impaired Hippocampal Long-Term Plasticity and Spatial Learning in Rats

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene that inactivate expression of the gene product, the fragile X mental retardation 1 protein (FMRP). In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate Fmr1 knockout (KO) rats by disruption of the fourth exon of the Fmr1 gene. Western blotting analysis confirmed that the FMRP was absent from the brains of the Fmr1 KO rats (Fmr1(exon4-KO)). Electrophysiological analysis revealed that the theta-burst stimulation (TBS)-induced long-term potentiation (LTP) and the low-frequency stimulus (LFS)-induced long-term depression (LTD) were decreased in the hippocampal Schaffer collateral pathway of the Fmr1(exon4-KO) rats. Short-term plasticity, measured as the paired-pulse ratio, remained normal in the KO rats. The synaptic strength mediated by the a -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) was also impaired. Consistent with previous reports, the Fmr1(exon4-KO) rats demonstrated an enhanced 3,5-dihydroxyphenylglycine (DHPG)-induced LTD in the present study, and this enhancement is insensitive to protein translation. In addition, the Fmr1(exon4-KO) rats showed deficits in the probe trial in the Morris water maze test. These results demonstrate that deletion of the Fmr1 gene in rats specifically impairs long-term synaptic plasticity and hippocampus-dependent learning in a manner resembling the key symptoms of FXS. Furthermore, the Fmr1(exon4-KO) rats displayed impaired social interaction and macroorchidism, the results consistent with those observed in patients with FXS. Thus, Fmr1exon4 KO rats constitute a novel rat model of FXS that complements existing mouse models.</p

Clinical utilization of multiple antibodies of Mycobacterium tuberculosis for serodiagnosis evaluation of tuberculosis: a retrospective observational cohort study

AbstractObjectives We aimed to investigate clinical uncertainties by characterizing the accuracy and utility of commercially available antibodies of Mycobacterium tuberculosis in the diagnostic assessment of suspected tuberculosis in high-burden countries.Methods We conducted a retrospective, descriptive, cohort study among participants aged ≥ 18 years with suspected tuberculosis in Nanning, Guangxi, and China. Participants were tested for M. tuberculosis infection using commercially available antibodies against Mycobacterum tuberculosis. Specificity, sensitivity, negative and positive predictive values, and negative and positive likelihood ratios of the tests were determined. Sputum specimens and bronchoalveolar lavage fluid were sent for mycobacterial culture, Xpert MTB/RIF assay, and cell-free M. tuberculosis DNA or RNA assay. Blood samples were used for IGRAs, T-cell counts (CD3 + CD4+ and CD3 + CD8+), and antibodies to tuberculosis test.Results Of the 1857 participants enrolled in this study, 1772 were included in the analyses, among which, 1311 were diagnosed with active tuberculosis. The specificity of antibody against 16kD for active tuberculosis was 92.7% (95% confidence interval [CI]: 89.3–95.4) with a positive likelihood ratio for active tuberculosis cases of 3.1 (95% CI: 2.1–4.7), which was higher than that of antibody to Rv1636 (90.5% [95% CI: 86.6–93.5]), antibody to 38kD (89.5% [95% CI: 85.5–92.7]), antibody against CFP-10 (82.6% [95% CI: 77.9–86.7]), and antibody against LAM (79.3% [95% CI: 74.3–83.7]). Sensitivity ranged from 15.8% (95% CI: 13.9–17.9) for antibody against Rv1636 to 32.9% (95% CI: 30.4–35.6) for antibody to LAM.Conclusions Commercially available antibodies against to Mycobacterium tuberculosis do not have sufficient sensitivity for the diagnostic evaluation of active tuberculosis. However, antibody against Rv1636 and 16kD may have sufficiently high specificities, high positive likelihood ratios, and correspondingly high positive predictive values to facilitate the rule-in of active tuberculosis