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Brown adipose tissue (BAT) is a specialized tissue in mammals related to thermogenesis. The Astragalus polysaccharide (APS) is the major natural active component of Astragalus membranaceus, which has been recognized as one of the most popular herbal medicines worldwide. The role and possible mechanisms of APS on brown adipocytes differentiation is not well defined. Here, we explored the effect of APS on the differentiation of brown adipocytes in C3H10T 1/2 cells. The results showed that APS promoted the differentiation of brown adipocytes and improved insulin sensitivity along with significant increases in the expression of brown adipogenic marker proteins (C/EBPα, C/EBPβ, and PPARγ), thermogenesis marker proteins (UCP1, PRDM16, and PGC-1α), and insulin sensitivity marker protein (GLUT4). Meanwhile, the results showed that the amount of the phosphorylation of insulin receptor substrate 1 (p-IRS1) and phospho-AKT (p-AKT) which are critical factors in the insulin signaling pathway was increased without changing the total amount of IRS and AKT. Furthermore, the results of RNA-seq showed that APS altered the expression profiles of various miRNAs, and among which the expression of miR-6911 as a universal regulatory factor was significantly decreased. Importantly, we found that miR-6911 regulated the differentiation of brown adipocytes by targeting PR domain-containing 16 (Prdm16). In addition, after transfection of miR-6911 mimics, compared with the control and inhibitor group, PRDM16 protein expression significantly decreased, which was accompanied by the decrease of PPARγ, UCP1, and PGC-1α. Collectively, our results indicated that APS regulated brown adipocytes differentiation in C3H10T 1/2 cells via miRNA-6911 targeting Prdm16.

Background This retrospective study evaluated the performance of a lipoarabinomannan (LAM)-based immunological method for diagnosing pleural tuberculosis (TB) from pleural effusion samples. Results were compared to those obtained using conventional culture and molecular testing methods. Methods Suspected pleural TB patients who visited Beijing Chest Hospital for medical care between January 2016 and June 2017 were retrospectively analysed in the study. Pleural effusion samples were tested for Mycobacterium tuberculosis (MTB) using the BACTEC MGIT 960 System, GeneXpert, and an anti-LAM antibody assay (LAM assay). Results Pleural effusion samples were collected from a total of 219 retrospectively recruited participants suspected of having pleural TB. Thirteen of 155 confirmed pleural TB cases tested positive for MTB via MGIT culture, for a sensitivity of 8.4% [95% confidence interval (CI): 4.0–12.8%]. In addition, GeneXpert and LAM testing identified 22 and 55 pleural TB cases, for sensitivities of 14.2% (95% CI: 8.7–19.7%) and 35.5% (95% CI: 28.1–43.6%), respectively. The specificities of these two assays were 100.0% (95% CI: 92.9–100.0%) and 96.9% (95% CI: 88.2–99.5%), respectively. Combined application of culture and LAM testing identified 60 positive cases, for a sensitivity of 38.7% (95% CI: 31.0–46.4%) that was significantly higher than that of MGIT culture alone (P

We investigated the epidemiology of extrapulmonary tuberculosis (TB) among patients admitted to Beijing Chest Hospital, Beijing, China, during January 2008-December 2017. Of 19,279 hospitalized TB patients, 33.4% (6,433) had extrapulmonary TB and 66.6% (12,846) had pulmonary TB. The most frequent forms of extrapulmonary TB observed were skeletal TB (41.1%) and pleural TB (26.0%). Younger, female patients from rural areas were more likely to have extrapulmonary TB. However, patients with diabetes mellitus were less likely to have extrapulmonary TB compared with patients without diabetes. A higher proportion of multidrug-resistant (MDR) TB was observed among patients with extrapulmonary TB than among patients with pulmonary TB. We observed a large increase in MDR TB, from 17.3% to 35.7%, for pleural TB cases. The increasing rate of drug resistance among extrapulmonary TB cases highlights the need for drug susceptibility testing and the formulation of more effective regimens for extrapulmonary TB treatment.

Summary Background The management of latent Mycobacterium tuberculosis infection is a new priority action for the WHO End Tuberculosis (TB) Strategy. However, national guidelines on latent tuberculosis infection testing and treatment have not yet been developed in China. Here, we present the results from the 2-year follow-up of a study that aimed to track the development of active disease in individuals with latent tuberculosis infection, identify priority populations for latent infection management, and explore the most suitable latent infection diagnostic approach. Methods A population-based multicentre prospective study was done in four sites in rural China, between 2013 and 2015. The baseline survey in 2013 measured the prevalence of latent tuberculosis infection using QuantiFERON-TB Gold In-Tube (QFT) and tuberculin skin test (TST) in eligible participants. During the follow-up phase between 2014–15, we assessed individuals who had tuberculosis infection at baseline (QFT-positivity or TST tuberculin reaction size [induration] of ≥10 mm) for the development of active disease through active case finding. Eligible participants included in follow-up survey had a birth date before June 1, 2008 (5 years or older in 2013), and continuous residence at the study site for 6 months or longer in the past year. Participants with current active tuberculosis at baseline survey were excluded. Findings Between Sept 1, 2013, and Aug 31, 2015, 7505 eligible participants (aged 5 years or older) were included in tuberculosis infection test positive cohorts (4455 were QFT positive, 6404 had TST induration ≥10 mm, and 3354 were positive for both tests) after baseline examination. During the 2-year follow-up period, 84 incident cases of active tuberculosis were diagnosed. Of participants who developed active tuberculosis, 75 were diagnosed with latent infection by QFT, 62 were diagnosed by TST, and 53 were diagnosed by both tests. An incidence rate of 0·87 (95% CI 0·68–1·07) per 100 person-years was observed for individuals who tested positive with QFT, 0·50 (0·38–0·63) per 100 person-years for those who tested positive with TST (p Interpretation Our results suggest that high-risk populations in communities in rural China, such as individuals at a high risk of disease reactivation from previous tuberculosis, should be targeted for latent infection screening and treatment with an interferon-γ releasing assay rather than a TST. Funding National Science and Technology Major Project of China, Program for Changjiang Scholars and Innovative Research Team in University of China, CAMS Innovation Fund for Medical Sciences, and Sanming Project of Medicine in Shenzhen.

Supplemental Digital Content is available in the text, Vitamin D is associated with the susceptibility of tuberculosis and might have an adjunctive effect on anti-tuberculosis treatment. This study aims to investigate the association of vitamin D deficiency and vitamin D receptor (VDR) gene polymorphisms with susceptibility and severity to multidrug-resistant tuberculosis (MDR-TB) in comparison with drug-sensitive tuberculosis (DS-TB) and health controls in China. A total of 180 patients with pulmonary TB (128 DS-TB, 52 MDR-TB) and 59 healthy controls were enrolled into 3 groups. Vitamin D levels and VDR genotypes at FokI, BsmI, ApaI, and TaqI sites of all the participants and clinical characteristics of patients with TB were measured and collected. Statistical analysis revealed that vitamin D levels were lower in both TB groups. Patients with TB with bilateral lesions and patients with MDR-TB with extrapulmonary TB had lower vitamin D levels. The frequencies of ff genotype and f allele were higher in both TB groups. Patients with Ff genotype and ff genotype had lower proportion of extrapulmonary TB. Patients with ff genotype had higher proportion of retreatment. Male patients with ff genotype had higher proportion of cavity formation. Patients with DS-TB with AA genotype had higher proportion of cavity formation. Our findings demonstrate that vitamin D deficiency and ff genotype may be the risk factors of TB in Chinese population. In addition, patients with TB with lower level of vitamin D may have a greater risk of bilateral lesions and extrapulmonary TB. VDR genotypes may be associated with TB clinical characteristics.

Allergic bronchopulmonary aspergillosis (ABPA) is a disease of the lungs resulting from a hypersensitivity reaction to spores of Aspergillus fumigatus. Here we report the case of a 13-year-old girl with ABPA who presented with productive cough, bronchiectasis and decline in lung function, and review the clinical features and treatment for pediatric ABPA.

Rapid and accurate detection of multidrug resistance (MDR) in Mycobacterium tuberculosis is essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) of rpoB , using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 stored M. tuberculosis clinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78 M. tuberculosis culture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistant M. tuberculosis in clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDR M. tuberculosis , particularly in high prevalence settings with limited resources.

Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively ( P < 0.01). All four of these tests showed good specificities: 88.9% for the adenosine deaminase assay and 100% for the remaining three assays. The T-SPOT.TB assay with pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS 6110 per ml of pleural effusion and showed good accordance of the results between repeated tests ( r = 0.978, P = 2.84 × 10 −10 ). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology.

A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis by targeting resistance-associated mutations in the katG , mabA-inhA promoter, rpoB , and gyrA genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical M. tuberculosis isolates was selected for development and evaluation of HRMA. PCR amplicons from the katG , mabA-inhA promoter, rpoB , and gyrA genes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified katG and/or mabA-inhA promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates, rpoB mutations in 51 of 54 (94.4%) rifampin-resistant isolates, and gyrA mutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in M. tuberculosis which could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.

16S rDNA sequence analysis is the most accurate method for definitive species identification of nocardiae. However, conflicting results can be found due to sequence errors in gene databases. This study tested the feasibility of species identification of Nocardia by partial (5’-end 606-bp) 16S rDNA sequencing, based on sequence comparison with “reference” sequences of well-annotated strains. This new approach was evaluated using 96 American Type Culture Collection (n=6), and clinical (n=90) Nocardia isolates. Nucleotide sequence-based polymorphisms within species were indicative of “sequence types” for that species. Sequences were compared with those in the GenBank, Bioinformatics Bacteria Identification and Ribosomal Database Project databases. Compared with the reference sequence set, all 96 isolates were correctly identified using the criterion of ≥99% sequence similarity. Seventy-eight (81.3%) were speciated by database comparison; alignment with reference sequences resolved the identity of 14 (15%) isolates whose sequences yielded 100% similarity to sequences in GenBank under >1 species designation. Of 90 clinical isolates, the commonest species was Nocardia nova (33.3%) followed by Nocardia cyriacigeorgica (26.7%). Recently-described or uncommon species included Nocardia veterana (4.4%), Nocarida bejingensis (2.2%) and, Nocardia abscessus and Nocardia arthriditis (each n=1). Nocardia asteroides sensu stricto was rare (n=1). There were nine sequence types of N. nova, three of Nocardia brasiliensis with two each of N. cyriacigeorgica and Nocardia farcinica. Thirteen novel sequences were identified. Alignment of sequences with reference sequences facilitated species identification of Nocardia and allowed delineation of sequence types within species, suggesting that such a barcoding approach can be clinically useful for identification of bacteria.

Volume 52, no. 5, p. [1540–1548][1], 2014. Page 1547: The addendum shown below should be added. Notice of redundant publication. A paper published in Chinese in the Chinese Journal of Antibiotics in November 2014 (W. Yang, B. Wang, L. Hao, X. Chen, and Y. Lu, Chin J Antibiot 2014:849–855)

Sequence analysis of the Nocardia essential secretory protein SecA1 gene ( secA1 ) for species identification of 120 American Type Culture Collection (ATCC) and clinical isolates of Nocardia (16 species) was studied in comparison with 5′-end 606-bp 16S rRNA gene sequencing. Species determination by both methods was concordant for all 10 ATCC strains. secA1 gene sequencing provided the same species identification as 16S rRNA gene analysis for 94/110 (85.5%) clinical isolates. However, 40 (42.6%) isolates had sequences with secA1 sequences for the species, including 29 Nocardia cyriacigeorgica (96.6 to 98.9% similarity) and 4 Nocardia veterana (91.5 to 98.9% similarity) strains. Discrepant species identification was obtained for 16 (14.5%) clinical isolates, including 13/23 Nocardia nova strains (identified as various Nocardia species by secA1 sequencing) and 1 isolate each of Nocardia abscessus (identified as Nocardia asiatica ), Nocardia elegans ( Nocardia africana ), and Nocardia transvalensis ( Nocardia blacklockiae ); both secA1 gene sequence analysis and deduced amino acid sequence analysis determined the species to be different from those assigned by 16S rRNA gene sequencing. The secA1 locus showed high sequence diversity (66 sequence or genetic types versus 40 16S rRNA gene sequence types), which was highest for N. nova (14 secA1 sequence types), followed by Nocardia farcinica and N. veterana ( n = 7 each); there was only a single sequence type among eight Nocardia paucivorans strains. The secA1 locus has potential for species identification as an adjunct to 16S rRNA gene sequencing but requires additional deduced amino acid sequence analysis. It may be a suitable marker for phylogenetic/subtyping studies.

DNA sequencing analyses have demonstrated relatively limited polymorphisms within the fungal internal transcribed spacer (ITS) regions among Trichophyton spp. We sequenced the ITS region (ITS1, 5.8S, and ITS2) for 42 dermatophytes belonging to seven species ( Trichophyton rubrum , T. mentagrophytes , T. soudanense , T. tonsurans , Epidermophyton floccosum , Microsporum canis , and M. gypseum ) and developed a novel padlock probe and rolling-circle amplification (RCA)-based method for identification of single nucleotide polymorphisms (SNPs) that could be exploited to differentiate between Trichophyton spp. Sequencing results demonstrated intraspecies genetic variation for T. tonsurans , T. mentagrophytes , and T. soudanense but not T. rubrum . Signature sets of SNPs between T. rubrum and T. soudanense (4-bp difference) and T. violaceum and T. soudanense (3-bp difference) were identified. The RCA assay correctly identified five Trichophyton species. Although the use of two “group-specific” probes targeting both the ITS1 and the ITS2 regions were required to identify T. soudanense , the other species were identified by single ITS1- or ITS2-targeted species-specific probes. There was good agreement between ITS sequencing and the RCA assay. Despite limited genetic variation between Trichophyton spp., the sensitive, specific RCA-based SNP detection assay showed potential as a simple, reproducible method for the rapid (2-h) identification of Trichophyton spp.

Small heat shock proteins (sHsps) were found to exhibit efficient chaperone-like activities under stress conditions although their native structures are severely disturbed. Here, using an alternative approach (site-directed mutagenesis), we obtained two structurally and functionally distinct Mycobacterium tuberculosis Hsp16.3 single-site mutant proteins. The G59W mutant protein (with Gly59 substituted by Trp) is capable of exhibiting efficient chaperone-like activity even under non-stress conditions although its secondary, tertiary, and quaternary structures are very different from that of the wild type protein. By contrast, the G59A mutant protein (with Gly59 substituted by Ala) resembles with the wild type protein in structure and function. These observations suggest that the Gly59 of the Hsp16.3 protein is critical for its folding and assembly. In particular, we propose that the exhibition of chaperone-like activity for Hsp16.3 does not require its intact (native) structures but requires the disturbance of its native structures (i.e., the native structure-disturbed Hsp16.3 retains its chaperone-like activity or even becomes more active). In addition, the behavior of such an active mutant protein (G59W) also strongly supports our previous suggestion that Hsp16.3 exhibits chaperone-like activity via oligomeric dissociation.