The Human Serum Metabolome

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca

Xpert MTB/RIF Ultra and Xpert MTB/RIF assays for extrapulmonary tuberculosis and rifampicin resistance in adults.

BACKGROUND: Xpert MTB/RIF Ultra (Xpert Ultra) and Xpert MTB/RIF are World Health Organization (WHO)-recommended rapid nucleic acid amplification tests (NAATs) widely used for simultaneous detection of Mycobacterium tuberculosis complex and rifampicin resistance in sputum. To extend our previous review on extrapulmonary tuberculosis (Kohli 2018), we performed this update to inform updated WHO policy (WHO Consolidated Guidelines (Module 3) 2020). OBJECTIVES: To estimate diagnostic accuracy of Xpert Ultra and Xpert MTB/RIF for extrapulmonary tuberculosis and rifampicin resistance in adults with presumptive extrapulmonary tuberculosis. SEARCH METHODS: Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, Science Citation Index, Web of Science, Latin American Caribbean Health Sciences Literature, Scopus, ClinicalTrials.gov, the WHO International Clinical Trials Registry Platform, the International Standard Randomized Controlled Trial Number Registry, and ProQuest, 2 August 2019 and 28 January 2020 (Xpert Ultra studies), without language restriction. SELECTION CRITERIA: Cross-sectional and cohort studies using non-respiratory specimens. Forms of extrapulmonary tuberculosis: tuberculous meningitis and pleural, lymph node, bone or joint, genitourinary, peritoneal, pericardial, disseminated tuberculosis. Reference standards were culture and a study-defined composite reference standard (tuberculosis detection); phenotypic drug susceptibility testing and line probe assays (rifampicin resistance detection). DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and assessed risk of bias and applicability using QUADAS-2. For tuberculosis detection, we performed separate analyses by specimen type and reference standard using the bivariate model to estimate pooled sensitivity and specificity with 95% credible intervals (CrIs). We applied a latent class meta-analysis model to three forms of extrapulmonary tuberculosis. We assessed certainty of evidence using GRADE. MAIN RESULTS: 69 studies: 67 evaluated Xpert MTB/RIF and 11 evaluated Xpert Ultra, of which nine evaluated both tests. Most studies were conducted in China, India, South Africa, and Uganda. Overall, risk of bias was low for patient selection, index test, and flow and timing domains, and low (49%) or unclear (43%) for the reference standard domain. Applicability for the patient selection domain was unclear for most studies because we were unsure of the clinical settings. Cerebrospinal fluid Xpert Ultra (6 studies) Xpert Ultra pooled sensitivity and specificity (95% CrI) against culture were 89.4% (79.1 to 95.6) (89 participants; low-certainty evidence) and 91.2% (83.2 to 95.7) (386 participants; moderate-certainty evidence). Of 1000 people where 100 have tuberculous meningitis, 168 would be Xpert Ultra-positive: of these, 79 (47%) would not have tuberculosis (false-positives) and 832 would be Xpert Ultra-negative: of these, 11 (1%) would have tuberculosis (false-negatives). Xpert MTB/RIF (30 studies) Xpert MTB/RIF pooled sensitivity and specificity against culture were 71.1% (62.8 to 79.1) (571 participants; moderate-certainty evidence) and 96.9% (95.4 to 98.0) (2824 participants; high-certainty evidence). Of 1000 people where 100 have tuberculous meningitis, 99 would be Xpert MTB/RIF-positive: of these, 28 (28%) would not have tuberculosis; and 901 would be Xpert MTB/RIF-negative: of these, 29 (3%) would have tuberculosis. Pleural fluid Xpert Ultra (4 studies) Xpert Ultra pooled sensitivity and specificity against culture were 75.0% (58.0 to 86.4) (158 participants; very low-certainty evidence) and 87.0% (63.1 to 97.9) (240 participants; very low-certainty evidence). Of 1000 people where 100 have pleural tuberculosis, 192 would be Xpert Ultra-positive: of these, 117 (61%) would not have tuberculosis; and 808 would be Xpert Ultra-negative: of these, 25 (3%) would have tuberculosis. Xpert MTB/RIF (25 studies) Xpert MTB/RIF pooled sensitivity and specificity against culture were 49.5% (39.8 to 59.9) (644 participants; low-certainty evidence) and 98.9% (97.6 to 99.7) (2421 participants; high-certainty evidence). Of 1000 people where 100 have pleural tuberculosis, 60 would be Xpert MTB/RIF-positive: of these, 10 (17%) would not have tuberculosis; and 940 would be Xpert MTB/RIF-negative: of these, 50 (5%) would have tuberculosis. Lymph node aspirate Xpert Ultra (1 study) Xpert Ultra sensitivity and specificity (95% confidence interval) against composite reference standard were 70% (51 to 85) (30 participants; very low-certainty evidence) and 100% (92 to 100) (43 participants; low-certainty evidence). Of 1000 people where 100 have lymph node tuberculosis, 70 would be Xpert Ultra-positive and 0 (0%) would not have tuberculosis; 930 would be Xpert Ultra-negative and 30 (3%) would have tuberculosis. Xpert MTB/RIF (4 studies) Xpert MTB/RIF pooled sensitivity and specificity against composite reference standard were 81.6% (61.9 to 93.3) (377 participants; low-certainty evidence) and 96.4% (91.3 to 98.6) (302 participants; low-certainty evidence). Of 1000 people where 100 have lymph node tuberculosis, 118 would be Xpert MTB/RIF-positive and 37 (31%) would not have tuberculosis; 882 would be Xpert MTB/RIF-negative and 19 (2%) would have tuberculosis. In lymph node aspirate, Xpert MTB/RIF pooled specificity against culture was 86.2% (78.0 to 92.3), lower than that against a composite reference standard. Using the latent class model, Xpert MTB/RIF pooled specificity was 99.5% (99.1 to 99.7), similar to that observed with a composite reference standard. Rifampicin resistance Xpert Ultra (4 studies) Xpert Ultra pooled sensitivity and specificity were 100.0% (95.1 to 100.0), (24 participants; low-certainty evidence) and 100.0% (99.0 to 100.0) (105 participants; moderate-certainty evidence). Of 1000 people where 100 have rifampicin resistance, 100 would be Xpert Ultra-positive (resistant): of these, zero (0%) would not have rifampicin resistance; and 900 would be Xpert Ultra-negative (susceptible): of these, zero (0%) would have rifampicin resistance. Xpert MTB/RIF (19 studies) Xpert MTB/RIF pooled sensitivity and specificity were 96.5% (91.9 to 98.8) (148 participants; high-certainty evidence) and 99.1% (98.0 to 99.7) (822 participants; high-certainty evidence). Of 1000 people where 100 have rifampicin resistance, 105 would be Xpert MTB/RIF-positive (resistant): of these, 8 (8%) would not have rifampicin resistance; and 895 would be Xpert MTB/RIF-negative (susceptible): of these, 3 (0.3%) would have rifampicin resistance. AUTHORS' CONCLUSIONS: Xpert Ultra and Xpert MTB/RIF may be helpful in diagnosing extrapulmonary tuberculosis. Sensitivity varies across different extrapulmonary specimens: while for most specimens specificity is high, the tests rarely yield a positive result for people without tuberculosis. For tuberculous meningitis, Xpert Ultra had higher sensitivity and lower specificity than Xpert MTB/RIF against culture. Xpert Ultra and Xpert MTB/RIF had similar sensitivity and specificity for rifampicin resistance. Future research should acknowledge the concern associated with culture as a reference standard in paucibacillary specimens and consider ways to address this limitation

Antioxidant and Antimutagenic Activities of Different Fractions from the Leaves of Rhododendron arboreum Sm. and Their GC-MS Profiling

In this era of urbanization and environmental pollution, antioxidants and antimutagens derived from plants are promising safeguards for human health. In the current investigation, we analyzed the antioxidant and antimutagenic effects of the hexane, chloroform, and ethyl acetate fractions of Rhododendron arboreum Sm. leaves and determined their chemical composition. The different fractions inhibited lipid peroxidation, repressed the production of nitric oxide radicals, and prevented deoxyribose degradation. The antimutagenic activity of the leaf fractions was analyzed against 4-nitro-O-phenylenediamine, sodium azide and 2-aminofluorene mutagens in two test strains (TA-98 and TA-100) of Salmonella typhimurium. The experiment was conducted using pre- and co-incubation modes. The best results were obtained in the pre-incubation mode, and against indirect acting mutagen. The presence of a number of bioactive constituents was confirmed in the different fractions by GC-MS analysis. The study reveals the strong antioxidant and antimutagenic activity of R. arboreum leaves. We propose that those activities of R. arboreum might correspond to the combined effect of the phytochemicals identified by GC-MS analysis. To the best of our knowledge, this is the first report on the antimutagenic activity of R. arboreum leaves

Ursolic acid Modulates cellular ROS levels.

<p>(A) UA increased ROS levels in lymphocytes. Lymphocytes were stained with DCF-DA (20 µM, 30 min at 37°C), treated with UA (0.5–5 µM) for 1 h and fluorescence emission was measured at 535 nm. *p<0.01, as compared to DCF treated cells (B) Effect of UA on intracellular GSH levels. Lymphocytes were treated with UA for 4 h at 37°C and stained with MCB (final concentration 40 µM) for 30 min. Fluorescence emission was measured at 490 nm following excitation at 394 nm. *p<0.01, as compared to MCB treated cells (C) Immunosuppressive effects of UA were independent of cellular redox status. Lymphocytes were incubated with different antioxidants (GSH or NAC or DTT or trolox) for 2 h. These cells were then stimulated with Con A in presence or absence of UA for 24 h at 37°C .The concentration of IL-2 and IFN-γ in the culture supernatant was estimated by ELISA. (D&E) Lymphocytes were stained with CFSE and were incubated with different antioxidants (GSH 10 mM or NAC 10 mM or DTT 100 µM or trolox 100 µM) for 2 h. The cells were then stimulated with Con A in presence or absence of UA for 72 h at 37°C. Cell proliferation was measured from CFSE dye dilution using a flowcytometer. Representative flowcytometric histograms and corresponding bar diagram are shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031318#pone-0031318-g005" target="_blank">Fig. 5D&E</a>). Each bar shows mean±S.E.M from three replicates and two such independent experiments were carried out. *p<0.01, as compared to vehicle treated cells and #p<0.01, as compared to Con A stimulated cells.</p

List of primer sequences used for real time quantitative PCR.

<p>List of primer sequences used for real time quantitative PCR.</p

Ursolic acid suppresses inducible expression of T cell and B cell activation markers and co-stimulatory molecules.

<p>Lymphocytes were treated with UA (5 µM, 4 h) and then stimulated with Con A (10 µg/ml) or LPS (50 µg/ml) for 24 h (A–B) or 48 h(C–H) at 37°C. In each group, 1×10⁶ cells were stained with PE conjugated anti-CD69 (A) or anti-CD25 (B) or anti-CD134 (C) or anti-CD28 (D) or anti-CD19 (E) or anti-CD80 (F) or anti-CD86 (G) or anti-I-A (H) mAbs. Representative flowcytometric histogram and corresponding bar diagram are shown. Data points represent mean±S.E.M. from three replicates and two such independent experiments were carried out. *p<0.01, as compared to vehicle treated cells and #p<0.01, as compared to Con A or LPS stimulated cells.</p

Ursolic acid inhibits lymphocyte proliferation in vitro.

<p>(A) For cell proliferation analysis CFSE labelled lymphocytes were treated with UA (0.25–5 µM, 4 h) and stimulated with the Con A (10 µg/ml) at 37°C for 72 h. Twenty thousand cells were acquired in a flowcytometer. Vehicle treated cells served as control. Percent daughter cells were calculated from decrease in mean fluorescence intensity. (B) Each bar represents percentage of daughter cells in each treatment group. (C & D) UA inhibits anti-CD3 induced T cell proliferation. CFSE labelled lymphocytes were treated with UA (1 & 5 µM, 4 h) and stimulated with coated anti-CD3mAb (1 µg/ml) and soluble anti CD28mAb (1 µg/ml) at 37°C for 72 h. Percent daughter cells were estimated by CFSE dye dilution. Representative histogram and corresponding bar diagram are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031318#pone-0031318-g001" target="_blank">Fig. 1C and D</a> respectively. (E) UA inhibits Con A induced cell cycle progression: Lymphocytes were treated with UA (0.25–5 µM, 4 h) and stimulated with the Con A (5 µg/ml) at 37°C for 72 h. The cells were stained with propidium iodide and twenty thousand cells were acquired in a flowcytometer. The hollow bars represent percentage of cells containing less than 2n DNA (sub-G1/apoptotic cells), light gray bars show cells containing 2n DNA (in G1 phase) and the dark gray bars indicate the cells containing more than 2n DNA (in S+G2/M phase). (F) Evaluation of the potential cytotoxicity of UA to lymphocytes. Lymphocytes were cultured with UA (5 µM) for 24 h and cytotoxicity was measured by PI staining. Vehicle treated cells served as control. Percentage apoptosis (pre-G1 peak) in lymphocytes was estimated and is shown in the histograms. (G) Each bar represents percentage of apoptotic in each treatment group. Data points represent mean±S.E.M. from three replicates and three such experiments were carried out. *p<0.01, as compared to vehicle treated cells and #p<0.01, as compared to Con A or antiCD3/CD28 stimulated cells.</p