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

OBJECTIVES: 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.

Existing M. tuberculosis antigens do achieve a limited sensitivity and negative predictive value to rule out a diagnosis of tuberculosis.M. tuberculosis antigens may help to rule in a diagnosis of active or latent tuberculosis in clinical setting among the high burden tuberculosis countries.This study is the largest retrospective, descriptive, cohort study to evaluate the clinical utilization of existing M. tuberculosis antigens integrating M. tuberculosis immunogens in patients with suspected active tuberculosis in high-burden country.

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.

Immobilization of Phospholipase A1 Using a Protein-Inorganic Hybrid System.

In this study, four kinds of phospholipase A1-metal (Al/Co/Cu/Mn) hybrid nanostructures were prepared for enhancing the stability of the free PLA1. The formed hybrid complexes were characterized by scanning electron microscope (SEM), Fourier infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The stability and substrate specificity of immobilized enzymes were subsequently determined. After immobilization, the temperature tolerance of PLA1-metal hybrid nanostructures was enhanced. The relative activity of PLA1-Al/Co/Cu hybrid nanostructures remained above 60% at 50 °C, while that of free enzyme was below 5%. The thermal transition temperature measured by differential scanning calorimetry (DSC) was found to increase from 65.59 °C (free enzyme) to 173.14 °C, 123.67 °C, 96.31 °C, and 114.79 °C, referring to PLA1-Cu/Co/Al/Mn hybrid nanostructures, respectively. Additionally, after a storage for fourteen days at 4 °C, the immobilized enzymes could exhibit approximately 60% of the initial activity, while the free PLA1 was inactivated after four days of storage. In brief, using Co2+, Cu2+, Al3+, and Mn2+ as the hybridization materials for immobilization could improve the catalytic properties and stability of the free PLA1, suggesting a promising method for a wider application of PLA1 in many fields such as food, cosmetics, and the pharmaceutical industry.

Enhancing Geranylgeraniol Production by Metabolic Engineering and Utilization of Isoprenol as a Substrate in Saccharomyces cerevisiae.

The amount of geranylgeranyl diphosphate (GGPP) is vital for microbial production of geranylgeraniol (GGOH) in Saccharomyces cerevisiae. In this study, a GGPP synthase with stronger catalytic ability was used to increase the supply of GGPP, and an engineered strain producing 374.02 mg/L GGOH at the shake flask level was constructed. Then, by increasing the metabolic flux of the mevalonate (MVA) pathway and the supply of isopentenyl pyrophosphate (IPP), the titer was further increased to 772.98 mg/L at the shake flask level, and we achieved the highest GGOH titer to date of 5.07 g/L in a 5 L bioreactor. This is the first report on the utilization of isoprenol for increasing the amount of IPP and enhancing GGOH production in S. cerevisiae. In the future, these strategies and engineered strains can be used to enhance the production of other terpenoids in S. cerevisiae.

Overproduction of α-Farnesene in Saccharomyces cerevisiae by Farnesene Synthase Screening and Metabolic Engineering.

Maximizing the flux of farnesyl diphosphate (FPP) to farnesene biosynthesis is the main challenge of farnesene overproduction in Saccharomyces cerevisiae. In this study, we screened α-farnesene synthase from soybean (Fsso) with a higher catalytic ability. Combining the overexpression of the mevalonate (MVA) pathway with the expression of Fsso, an engineered yeast strain producing 190.5 mg/L α-farnesene was screened with poor growth. By decreasing the copies of 3-hydroxy-3-methylglutaryl-coenzyme (HMGR) overexpressed, the titer was increased to 417.8 mg/L. Then, the coexpression of Fsso and HMGR under the control of the GAL promoter and inactivation of lipid phosphate phosphatase encoded by DPP1 promoted the titer to 1163.7 mg/L. The titer was further increased to 1477.2 mg/L at the shake flask level with better growth by the construction of a prototrophic strain. Finally, the highest α-farnesene production of 10.4 g/L in S. cerevisiae was obtained by fed-batch fermentation in a 5 L bioreactor.

Affinity adsorption of phospholipase A1 with designed ligand binding to catalytic pocket.

An affinity ligand was designed from 1-aminocyclohexane based on the crystal structure of Streptomyces albidoflavus phospholipase A1 (saPLA1) by using Discovery Studio software. The molecular docking results indicated that the designed ligand could interact with the active pocket of saPLA1. Epichlorohydrin, cyanuric chloride and 1-aminocyclohexane were used to synthesize the affinity ligand, which was composed to Sepharose beads. The density of the ligand on Sepharose beads was 22.5 ± 1.1 µmol/g wet gel. Adsorption analysis of the sorbent indicated the maximum adsorption (Qmax) of the enzyme was 10.7 ± 0.29 mg/g and the desorption constant (Kd) was 426.6 ± 29.7 µg/mL. The sorbent could bind the enzyme in the supernatant of disrupted recombinant Escherichia coli through one step of affinity adsorption. After the optimization of the purification process, a single band was obtained at approximately 30 kDa, which was confirmed as saPLA1 by the matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry and activity assay. The purity of the isolated enzyme was about 96.6% with the purify fold at 7.62, and the activity recovery was 52.5%.