Identification of miR-93 as a suitable miR for normalizing miRNA in plasma of tuberculosis patients.

Tuberculosis (TB) remains a major public health issue. New tests to aid diagnoses and monitor the response to therapy are urgently required. There is growing interest in the use of microRNA (miRNA) profiles as diagnostic, prognostic or predictive markers in a range of clinical and infectious diseases, including Mycobacterium tuberculosis infection, however, challenges exist to accurately normalise miRNA levels in cohorts. This study examined the appropriateness of 12 miRs and RNU6B to normalise circulating plasma miRNA levels in individuals with active TB from 2 different geographical and ethnic regions. Twelve miRs (let-7, miR-16, miR-22, miR-26, miR-93, miR-103, miR-191, miR-192, miR-221, miR-423, miR-425 and miR-451) and RNU6B were selected based on their reported production by lung cells, expression in blood and previous use as a reference miRNA. Expression levels were analysed in the plasma of newly diagnosed TB patients from Australia and China compared with individuals with latent TB infection and healthy volunteers. Analysis with both geNorm and NormFinder software identified miR-93 as the most suitable reference miR in both cohorts, either when analysed separately or collectively. Interestingly, there were large variations in the expression levels of some miRs, in particular miR-192 and let-7, between the two cohorts, independent of disease status. These data identify miR-93 is a suitable reference miR for normalizing miRNA levels in TB patients, and highlight how environmental, and possibly ethnic, factors influence miRNA expression levels, demonstrating the necessity of assessing the suitability of reference miRs within the study population.

Walnut ointment promotes full-thickness burning wound healing: role of linoleic acid.

PURPOSE: To investigate the active ingredients of walnut ointment (WO) and its mechanism in repairing wounds. METHODS: The ingredients of WO were detected by gas chromatography-mass spectrometry. The effect of linoleic acid (LA) was tested by in vitro Alamar Blue (AB) reagent. Image J software, histological and immunohistochemical analysis were used to confirm the healing effect of LA in the porcine skin model. The animals were euthanized after the experiment by injection of pentobarbital sodium. RESULTS: LA, 24% in WO, promotes keratinocytes and fibroblasts proliferation, which were 50.09% and 15.07% respectively higher than control (p < 0.05). The healing rate of the LA group (96.02% ± 2%, 98.58% ± 0.78%) was higher than the saline group (82.11% ± 3.37%, 88.72% ± 1.73%) at week 3 and week 4 (p < 0.05). The epidermal thickness of the LA was 0.16 ± 0.04 mm greater and the expression of the P63 and CK10 proteins was stronger in the LA group than the control (p < 0.05). CONCLUSIONS: LA, which is the main components in WO can promote full-thickness burning wounds (FBWs) by stimulating cell proliferation and differentiation.

High sensitivity and specificity of a 5-analyte protein and microRNA biosignature for identification of active tuberculosis.

OBJECTIVES: Non-sputum-based tests to accurately identify active tuberculosis (TB) disease and monitor response to therapy are urgently needed. This study examined the biomarker capacity of a panel of plasma proteins alone, and in conjunction with a previously identified miRNA signature, to identify active TB disease. METHODS: The expression of nine proteins (IP-10, MCP-1, sTNFR1, RANTES, VEGF, IL-6, IL-10, TNF and Eotaxin) was measured in the plasma of 100 control subjects and 100 TB patients, at diagnosis (treatment naïve) and over the course of treatment (1-, 2- and 6-month intervals). The diagnostic performance of the nine proteins alone, and with the miRNA, was assessed. RESULTS: Six proteins were significantly up-regulated in the plasma of TB patients at diagnosis compared to controls. Receiver operator characteristic curve analysis demonstrated that IP-10 with an AUC = 0.874, sensitivity of 75% and specificity of 87% was the best single biomarker candidate to distinguish TB patients from controls. IP-10 and IL-6 levels fell significantly within one month of commencing treatment and may have potential as indicators of a positive response to therapy. The combined protein and miRNA panel gave an AUC of 1.00. A smaller panel of only five analytes (IP-10, miR-29a, miR-146a, miR-99b and miR-221) showed an AUC = 0.995, sensitivity of 96% and specificity of 97%. CONCLUSIONS: A novel combination of miRNA and proteins significantly improves the sensitivity and specificity as a biosignature over single biomarker candidates and may be useful for the development of a non-sputum test to aid the diagnosis of active TB disease.

Mushroom poisoning from Inocybe serotina: A case report from Ningxia, northwest China with exact species identification and muscarine detection.

Mushroom poisoning is a serious food safety issue in China. However, there is insufficient information on many poisoning incidents, including mushroom species and their clinical manifestations, diagnosis, treatments and toxins. Detailed epidemiological investigation was conducted after the occurrence of a mushroom poisoning incident resulting in typical muscarinic syndrome in Ningxia, China. The suspected mushroom species was identified based on morphological and phylogenetic analyses. Muscarine was detected using ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). On September 2, 2019, two patients exhibited typical muscarinic syndrome after consuming wild mushrooms. The clinical manifestations included chills, sweating, salivation and diarrhoea; the incubation period was approximately 2 h. Treatments, including anti-inflammatory, detoxification and nutritional support, were remedial. Full recovery ensued within 24 h. The specimen was identified as Inocybe serotina, and its muscarine content was 324.0 ± 62.4 mg/kg (k = 2, p = 95%). Two patients were poisoned via stimulation of their parasympathetic nervous system due to mistaken consumption of muscarine-containing I. serotina. They fully recovered with supportive treatments. To our knowledge, this is the first case report of I. serotina poisoning worldwide and is the first record of this species in China. Further, a method for muscarine detection was established using UPLC-MS/MS.

Identification of a plasma microRNA profile in untreated pulmonary tuberculosis patients that is modulated by anti-mycobacterial therapy.

OBJECTIVE: microRNA expression profiles are of interest as a biomarker of tuberculosis (TB). How anti-TB therapy effects miRNA profiles is unknown and was examined. METHODS: We identified 87 plasma miRNAs that were significantly modified in an exploratory group of 19 Chinese pulmonary TB (PTB) patients compared to 14 healthy controls. We selected 10 of these miRNAs for analysis in a cohort of 100 PTB patients prior to, and at one, two and six months during treatment. RESULTS: Five miRNAs were differentially expressed in PTB patients compared to controls at diagnosis; miRs -29a and -99b were up-regulated, whilst miRs -21, -146a and -652 were down-regulated. A combination of 5 miRNA distinguished TB from healthy controls with a sensitivity of 94%, a specificity of 88%, and an AUC of 0.976. Within one month of treatment, significant changes in miRs -29a, -99b, -26a and 146a levels occurred in successfully treated patients, although not all miRNAs returned to baseline by treatment completion. CONCLUSION: A 5-miRNA signature shows potential for development as a novel biomarker for TB disease with potential to predict response to treatment. The failure of all miRNA to return to baseline levels may reflect ongoing remodelling in the lung parenchyma that continues after completion of anti-TB therapy.

Dynamic Characteristics and HIV Infection of Men who have Sex with Men from 2011 to 2017 in Yinchuan, Ningxia, China.

OBJECTIVES: Ningxia Hui Autonomous Region, an important area for ethnic Hui settlement in Northwest China, is a low HIV prevalence region. However, HIV infection rates among men who have sex with men (MSM) in Ningxia have increased to an alarming level, despite scale-up of control measures in recent years. This study aimed to understand the demographical and sexual behavior dynamics of MSM and to explore the factors associated with HIV infection. METHODS: Annual cross-sectional surveys were carried out among MSM during 2011~2017 in Yinchuan, the capital city of Ningxia. Information regarding social demographics, sexual behavior and HIV prevention knowledge was collected. Blood samples were taken for HIV, HCV serological and genetic analysis, and syphilis serological analysis. The dynamic trend was analyzed with trend χ2 test and factors associated with HIV infection were identified by multivariate logistic regression analysis. RESULTS: The study found a decreasing trend for mean age of the MSM population over the study period. MSMs with a college education or higher increased significantly, while the proportions that were in a marriage significantly decreased over the study period. The rate of HIV positive among MSM increased during the study period (p<0.05), however, the rate of recently diagnosed infections decreased from 2012 (p<0.05). Overall, a very high proportion (98%) of MSM had basic knowledge of HIV prevention, however, only approximately 40% of them used condoms consistently during anal sex with male partners. Unprotected anal sex was identified as a risk factor associated with HIV infection, as was syphilis infection. Local residency status and MSM who received intervention and detection services were the factors that decreased HIV infection risk. Sequence analysis identified the HIV-1 CRF55_01B subtype from MSM for the first time in Yinchuan. CONCLUSION: The reduction of recent HIV diagnoses is an encouraging sign of successful HIV control measures in MSM in Ningxia. The finding that a high proportion of MSM had knowledge of HIV prevention but still conducted unprotected sex highlights the need for further control measures to change unsafe sexual practices among MSM.

Walnut ointment promotes full-thickness burning wound healing: role of linoleic acid

Purpose: To investigate the active ingredients of walnut ointment (WO) and its mechanism in repairing wounds. Methods: The ingredients of WO were detected by gas chromatography­mass spectrometry. The effect of linoleic acid (LA) was tested by in vitro Alamar Blue (AB) reagent. Image J software, histological and immunohistochemical analysis were used to confirm the healing effect of LA in the porcine skin model. The animals were euthanized after the experiment by injection of pentobarbital sodium. Results: LA, 24% in WO, promotes keratinocytes and fibroblasts proliferation, which were 50.09% and 15.07% respectively higher than control (p < 0.05). The healing rate of the LA group (96.02% ± 2%, 98.58% ± 0.78%) was higher than the saline group (82.11% ± 3.37%, 88.72% ± 1.73%) at week 3 and week 4 (p < 0.05). The epidermal thickness of the LA was 0.16 ± 0.04 mm greater and the expression of the P63 and CK10 proteins was stronger in the LA group than the control (p < 0.05). Conclusions: LA, which is the main components in WO can promote full-thickness burning wounds (FBWs) by stimulating cell proliferation and differentiation.

[Typing and identification of non-polio enterovirus from acute flaccid paralysis cases in Ningxia, 1997-2011].

OBJECTIVE: To identify the serotype of 73 non-polio enterovirus (NPEV) strains from acute flaccid paralysis (AFP) cases in Ningxia province, during 1997 - 2011. METHODS: Partial sequencing of the VP1 region was amplified by RT-PCR with degenerate primers and sequenced while sequences were compared with the database of GenBank by the BLAST algorithm. Evolution was analyzed by constructing phylogenetic tree using Mega 5.1. RESULTS: In this study, a total of 73 NPEVs were analyzed, including 4 strains un-typed, 69 strains typed by RT-PCR. A total of 27 serotypes were identified, including 8 serotypes of human enterovirus (HEV)-A, 19 serotypes of HEV-B. The HEV-B group (46/69, 66.7%) constituted the largest proportion of isolates, followed by HEV-A (23/69, 33.3%), but no strains were found that belonged to HEV-C or HEV-D group. In the 69 strains, enterovirus 71 was the most frequently seen isolates, followed by coxsackie-virus A4, 16, 9 and echovirus 24, 6. CONCLUSION: HEV-B was the most predominant (46/69, 66.7%) serotype of NPEV in Ningxia during the AFP surveillance, in 1997 - 2011.

[Genetic characteristics of entervirus 71 isolated in Ningxia Hui Autonomous region in 2009].

OBJECTIVE: To analyze the genetic characteristics of EV71 strains isolated from HFMD cases in Ningxia Hui Autonomous Region in 2009. METHODS: In 2009, totally 385 specimens from 344 HFMD cases were collected from Ningxia. Enterovirus isolation was performed in RD cell line from all the specimens. EV71 isolates were identified by specific RT-PCR from the positive cultures, and sequences of complete EV71 VP1 encoding region were determined for farther analyses. RESULTS: Totally from 126 EV strains isolated in this study, 58 EV71 strains (46%) were identified. And complete VP1 sequences of 46 EV71 strains were determined, and genetic analyses were performed. It was showed that the nucleotide identity of 46 Ningxia strains with the representatives of A and B genotypes were 81.7%-82.8% and 83.1%-85.2%, and the amino acid identity were 93.9%-95.9% and 96. 2%-97.9% respectively. The nucleotide identity of NingXia EV71 isolates with representatives of subgenotype C1, C2, C3, C4a, C4b, and C5 were 88.3%-90.6% (97.9%-99.6%), 88.3%-90.1% (97.9%-99.3%), 87.8%-89.0% (97.6%-98.9%), 94.2%-98.9% (97.9%-100%), 91.8%-94.1% (98.6%-99.6%), and 86.7%-89.1% (97.9%-98.9%). Phylogenetic tree analysis revealed that 46 stains were clustered with reference stains of subgenotype C4 and the Ningxia EV71 isolates were belonged to subgenotype C4a. CONCLUSION: EV71 of subgenotype C4a had spread widely in Ningxia in 2009, which was absolutely predominant type in Ningxia in 2009 and also as the predominant type in China mainland since 2005.