EspR promotes mycobacteria survival in macrophages by inhibiting MyD88 mediated inflammation and apoptosis.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb), leading to about a million deaths each year. EspR is a DNA binding protein of Mtb which regulates expression of multiple genes and the activity of ESX-1 secretion system of the bacteria, with itself being secreted out as a substrate of ESX-1. We explored the function of secreted EspR in host cells by overexpressing the protein in murine macrophage cell line RAW264.7, infecting the cells with BCG which does not secrete EspR, and evaluating the antimicrobial responses of the cells. We found that EspR resulted in an increased intracellular bacteria load in macrophages. This is due to its inhibition on BCG induced expression of inflammatory cytokines and inducible nitric oxide synthase (iNOS), as well as host cell apoptosis. Mechanism study showed that EspR directly interacted with adaptor protein myeloid differentiation factor 88 (MyD88), suppressed MyD88 dependent Toll-like receptor (TLR) and IL-1R signal activation, thus reduced inflammatory responses and apoptosis in macrophages and promoted mycobacteria survival.

LpqT improves mycobacteria survival in macrophages by inhibiting TLR2 mediated inflammatory cytokine expression and cell apoptosis.

Tuberculosis is a severe infectious disease caused by Mycobacterium tuberculosis (Mtb). LpqT is a lipoprotein of Mtb identified as a candidate virulence factor by a high-throughput screen searching for genes important for mycobacteria intracellular survival. To investigate its function, we constructed M. smegmatis strains deficient of LpqT or overexpressing LpqT. Wildtype or LpqT modified M. smegmatis strains were used to infect macrophages and mice, and intracellular survival of mycobacteria was measured. We found that LpqT can improve M. smegmatis survival in macrophage cell line, bone marrow derived macrophages (BMDMs), and murine lungs. This survival promoting effect is dependent on TLR2 and Myd88. Western blot analysis of M. smegmatis infected macrophages showed that LpqT suppressed M. smegmatis induced NF-κB and MAPK phosphorylation, indicating that LpqT hampered TLR2 signal activation. In consistent with this, LpqT inhibited M. smegmatis induced inflammatory cytokine expression and cell apoptosis in macrophages, thus supported mycobacteria intracellular survival.

MptpB Promotes Mycobacteria Survival by Inhibiting the Expression of Inflammatory Mediators and Cell Apoptosis in Macrophages.

Tuberculosis is a severe contagious disease caused by Mycobacterium tuberculosis (Mtb). To develop new vaccines and medicine against TB, there is an urgent need to provide insights into the mechanisms by which Mtb induces tuberculosis. In this study, we found that secreted Mtb virulence factor MptpB significantly enhanced the survival of H37Rv in macrophages. MptpB suppressed the production of iNOS, the expression of inflammatory factors IL-1ß and IL-6, as well as the apoptosis of the macrophage in Mtb infected RAW264.7 cells. Mechanism investigation showed that MptpB simultaneously hampered the NF-κB and MAPK signal pathways, evidenced by its blocking of p65, IKKα, Erk1/2, and p38 phosphorylation induced by Mtb infection. MptpB also inhibited host cell p53 expression. The results demonstrated that MptpB contributed to the survival of H37Rv by inhibiting host inflammatory responses and apoptosis through impeding the NF-κB and MAPK signal pathways and p53 expression in the macrophage.

[Determination of 14 heterocyclic aromatic amines in wine by liquid chromatography-ion trap-time of flight tandem mass spectrometry].

A rapid qualitative and quantitative analytical method was developed for the simultaneous determination of 14 heterocyclic aromatic amines (HAAs) in wine by liquid chromatography-ion trap-time of flight tandem mass spectrometry (LC-IT-TOF MS). HAAs were extracted from the samples by ethyl acetate under alkaline condition. The quantitation was carried out using internal standard method. The separation of HAAs was carried out based on Phenomenex Kinetex C18 100A column (100 mm x 2.1 mm, 2.6 microm), with a gradient elution of acetonitrile and 30 mmol/L ammonium formate at a flow rate of 0.4 mL/min. The analytes were detected under positive-ion electrospray ionization mode. The results showed that the linear ranges of the 14 HAAs were 1-500 microg/L with limits of detection (signal/noise = 3) of 0.33-1.77 microg/L. The average recoveries of all the compounds spiked in wine samples at three levels of 10, 50, 100 microg/L were in the ranges of 71.6%-96.4%, 72.9%-101.9%, 74.5%-103.3%, with the corresponding relative standard deviations (RSDs, n = 6) of 2.9%-7.9%, 1.7%-5.3%, 1.8%-4.8%, respectively. The established method is simple, rapid, accurate, and has wide linear range and high sensitivity. It can be applied to the simultaneous analysis of the HAAs in wine.

[Determination of sulfonamides, quinolones, benzimidazoles and the metabolites of benzimidazoles in chicken livers by dispersive solid-phase extraction and ultra performance liquid chromatography-tandem mass spectrometry].

A method for the determination of 12 sulfonamides, 19 quinolones and 8 benzimidazoles and the metabolites of benzimidazoles in chicken livers by quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed. The samples were extracted with 1% acetic acid-acetonitrile solution, cleaned up with amine (NH2) sorbent and defatted with n-hexane. The identification and quantification were achieved by using electrospray ionization in positive ion mode (ESI+) with multiple reaction monitoring (MRM). The matrix-matched internal standard calibration curves were used for quantitative determination. The linear range was from 5 to 100 microg/kg. The average recoveries and relative standard deviations were 72% - 121% and 1.5% -23.4% respectively in the spiked range of 10 -50 microg/kg. The limits of detection were 5 microg/kg and the limits of quantification were 10 microg/kg for the 39 drugs. The method is simple, rapid, sensitive and accurate. It is suitable for the quantitative determination and confirmation of 12 sulfonamides, 19 quinolones, 8 benzimidazoles and the metabolites of benzimidazoles.

[Rapid determination of 6 pesticide residues in tomato paste by ultra performance liquid chromatography-tandem mass spectrometry].

An ultra performance liquid chromatography-tandem mass spectrometric method (UPLC-MS/MS) was established for the simultaneous determination of imidacloprid, carbendazim, thiophanate-methyl, propamocarb, methomyl and dimethomorph residues in tomato paste. The samples were extracted by methanol-water (1: 1, v/v) containing 0.1% (v/v) acetic acid. The separation was performed on a Waters Acquity UPLC system with a BEH C18 column with the gradient elution of methanol and water (containing 10 mmol/L ammonium acetate). The six pesticides were determined in the modes of electrospray positive ionization (ESI+) and multiple reaction monitoring (MRM). The analytes were quantified by matrix-matched standard solution, and the calibration curves showed good linearity within the concentrations of 0.005 to 0.2 mg/L and the correlation coefficients (r) were more than 0.995. The average recoveries of the six pesticides ranged from 66.8% to 102.9% in the three spiked levels of 0.02, 0.05 and 0.2 mg/kg. The relative standard deviations (RSDs) were all less than 15%. The limits of quantification (LOQ, S/N > 10) were 0.02 mg/kg for the all analytes. The results indicate that the method is easier, faster, more sensitive, and suitable for the qualitative and quantitative confirmation of the six pesticide residues from tomato paste.

[Rapid determination of patulin in apple juice by tert-butyldimethylsilyl derivatization-gas chromatography-mass spectrometry].

A gas chromatography-mass spectrometry (GC-MS) method for the quantitative determination and confirmation of patulin in apple juice by tert-butyldimethylsilyl (TBDMS) derivatization was established. The sample was extracted with ethyl acetate-hexane and an aliquot of the supernatant was cleaned up using mixed-mode solid phase extraction (SPE) cartridge containing C18 and graphitized carbon black (GCB), evaporated to dryness under nitrogen gas and the residue was converted to tert-butyldimethylsilyl derivative which was determined with GC-MS in selected ion monitoring (SIM) mode and external standard method was used for quantitative determination. The linear range was from 0.01 to 1 mg/L. The average recoveries were 88% - 98% and relative standard deviations were 5.3% - 13.6% in the spiked range of 2 - 50 microg/kg. The limit of detection was 0.5 microg/kg and the limit of quantification and confirmation was 2 microg/kg. The method is rapid, highly sensitive, accurate, specific, rugged and suitable for the quantitative determination and confirmation of patulin in apple juice.

[Determination and confirmation of cyanuric acid in infant formula using mixed-mode solid-phase extraction cartridge clean up-gas chromatography-mass spectrometry].

A method is presented for the quantitative determination and confirmation of cyanuric acid in infant formula by mixed-mode solid-phase extraction cartridge clean up-gas chromatography-mass spectrometry (GC-MS). Cyanuric acid was extracted from infant formula with an acetic acid solution at 84 degrees C. An aliquot of the supernatant was cleaned up using mixed-mode solid-phase extraction cartridge containing C18 and graphitized carbon black (GCB), and evaporated to dryness under nitrogen. The residues were converted to trimethylsilyl derivatives, then determined by GC-MS in selected ion monitoring (SIM) mode. The linear range was from 0.01 -2 mg/L. The recoveries were 80% - 103% and the relative standard deviations were 7.7% - 14.5% in the spiked range of 0.25 -2.5 mg/kg. The limit of detection was 0.10 mg/kg and the limit of quantification was 0.25 mg/kg. The method is rapid, sensitive, accurate, specific, and rugged. It is suitable for the quantitative determination and confirmation of cyanuric acid in infant formula.

[Determination of melamine and cyanuric acid in milk by gas chromatography-mass spectrometry].

A gas chromatography-mass spectrometry method was developed for the quantitative determination and confirmation of melamine and cyanuric acid in milk. The milk sample was extracted with diethylamine-acetonitrile-water solution. The extract was evaporated to dryness and derivatized with N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) and chlorotrimethylsilane (TMCS), then analyzed using gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. The external standards were used for the quantitative determination. The linear range was from 0.025 to 2 mg/kg. The average recoveries were 84%-87% for melamine and 75%-102% for cyanuric acid, and the relative standard deviations were 5.7%-11.7% for melamine and 4.9%-7.8% for cyanuric acid in the spiked levels at 0.5, 1.0 and 2.5 mg/kg. The limits of detection of melamine and cyanuric acid were 0.05 mg/kg and 0.10 mg/kg, respectively. The method is suitable for the quantitative determination and confirmation of melainine and cyanuric acid residues in milk.