Objective The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains is exacerbating the global burden of tuberculosis (TB), highlighting the urgent need for new treatment strategies for TB. Methods The recombinant adenovirus vaccine expressing cyclic di-adenosine monophosphate (c-di-AMP) phosphodiesterase B (CnpB) (rAd-CnpB), was administered to normal mice via mucosal immunization, either alone or in combination with drug therapy, to treat Mtb respiratory infections in mice.Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of antibodies in serum and bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was performed to assess the transcription levels of cytokines interferon γ(IFN-γ) and interleukin 10(IL-10) in mouse lungs. Flow cytometry was used to determine the proportions of CD4⁺ and CD8⁺ T cell subsets in the lungs and spleens. ELISA was employed to measure the levels of cytokines IFN-γ, IL-2, IL-10, inflammatory factors IL-6, and tumor necrosis factor α (TNF-α) secreted by spleen cells following antigen stimulation. The bacteria loads in the lungs and spleens of Mtb-infected mice were enumerated by plate counting methods. Resluts Intranasal immunization with rAd-CnpB induced high titers of IgG in mouse serum and the production of IgG and IgA in BALF, along with alterations in T lymphocyte subsets in the lungs and spleens. Administration of rAd-CnpB, either alone or in combination with drugs, to Mtb-infected mice significantly increased serum IgG levels as well as IgA and IgG levels in BALF. rAd-CnpB immunization promoted the secretion of CnpB-specific cytokines and inflammatory factors by splenocytes in Mtb-infected mice. However, rAd-CnpB immunotherapy, either alone or combined with drugs, did not significantly affect the bacterial loads in the lungs and spleens of mice with Mtb respiratory infections. Conclusion Mucosal immunization with rAd-CnpB induced significant mucosal, humoral and cellular immune responses in mice, and significantly enhanced CnpB-specific cellular immune responses in Mtb-infected mice.
Animals ; Adenoviridae/immunology* ; Mycobacterium tuberculosis/genetics* ; Mice ; Female ; Phosphoric Diester Hydrolases/genetics* ; Tuberculosis Vaccines/administration & dosage* ; Tuberculosis/prevention & control* ; Mice, Inbred BALB C ; Cytokines ; Lung/microbiology* ; Immunization ; Bronchoalveolar Lavage Fluid/immunology* ; Immunity, Mucosal

Objective To develop and evaluate the equivalence of the Mandarin test material for tracking of noise tolerance(TNT)test.Methods Six different speech materials were developed(themes including daily life,entertainment,family,festivals,outdoors,and school).Four-minute TNT tests were measured in 21 normal hear-ing subjects using six different test materials.For each session,the tolerable noise level(TNL)and TNT scores were acquired and calculated for 3 time windows(31～240 s,31～120 s,151～240 s).Results Statistic analysis showed significant differences in the TNL(F=43.611,P＜0.05)among the normal hearing listeners.There were statistically significant differences in standardize z-scored TNT scores of the six different materials in the three time windows(P＜0.05).Post-hoc comparisons revealed that all significant differences involved the family and daily life themes.Conclusion Entertainment,festival,outdoors and school themed test materials can serve as the materials of Mandarin tracking of noise tolerance test and can be appied in research and clinical testing.

Objective:To explore the role and the intervention effect of emodin in intestinal neuropathy in rats with severe acute pancreatitis (SAP) through the nucleotide binding oligomerization domain like receptor protein 3/cysteine containing aspartic acid protease-1 (NLRP3/Caspase-1) pathway.Methods:Forty male healthy SD rats aged 6-8 weeks with a weight of approximately 200g were randomly divided into control group, SAP model group, emodin treatment (EMO) group, and NLRP3 knockdown group. SAP were induced by retrograde injection of sodium deoxycholate into the pancreatic duct of rats and serum amylase of which were detected. The effective NLRP3 knockdown sequence was screened for NLRP3 knockdown animal experiments. Fluorescence quantitative polymerase chain reaction was used to detect the expression of NLRP3, Caspase-1, gasdermin-D (GSDMD), interleukin (IL)-1β, IL-18 and tumor necrosis factor-α(TNF-α) in the small intestine of each group. Immunofluorescence staining was used to detect the expression of glial fibrillary acidic protein (GFAP) in the small intestine of each group.Results:The amylase levels of the control group, SAP group, EMO group, and NLRP3 knockdown group were (277.73±24.92) U/L, (1018.57±282.89) U/L, (625.43±134.40) U/L, and (391.01±27.63) U/L, respectively. The SAP and EMO groups were significantly higher than the control group ( P<0.001), while the EMO and NLRP3 knockdown groups were significantly lower than the SAP group (all P<0.001). Compared with control group, the expression levels of NLRP3, Caspase-1, IL-1β, IL-18, TNF-α and GSDMD in SAP group were increased, with statistical significance (all P<0.001). Compared with SAP group, the NLRP3 knockdown group showed the expressionlevels of the above 6 genes were all decreased, and EMO group showed decreased gene expressing levels of NLRP3, IL-1β, IL-18 and TNF-α, with statistical significance (all P<0.05). The relative expression of GFAP in small intestine of control group, SAP group, EMO group and NLRP3 knockdown group were (1.00±0), (1.66±0.11), (1.13±0.02) and (1.13±0.02), respectively. Among them, the expression of GFAP in SAP group was increased compared with the control group; The expression of GFAP in EMO group and NLRP3 knockdown group was lower than that in model group, and the differences were statistically significant (all P<0.05). Conclusions:Emodin and knocking down NLRP3 can both promote the repair of SAP small intestine injury through the NLRP3/Caspase-1 signaling pathway, and thus play a protective role in the intestine.

Rv2742 is a novel gene identified from Mycobacterium tuberculosis H37Rv by the proteogenomics strategy. The aim of this study was to establish a system of soluble expression and purification of the missing protein Rv2742 in M. tuberculosis H37Rv, to provide reference for further research on the biological function of Rv2742. The soluble protein was not successfully induced by prokaryotic expression vectors pGEX-4T-2-Rv2742, pET-32a-Rv2742, pET-28a-Rv2742 and pMAL-c2X-Rv2742. After the codon of novel gene Rv2742 was optimized according to E. coli codon usage frequency, only the recombinant strain containing plasmid pMAL-c2X-Rv2742 could produce soluble products of Rv2742 encoding gene. In addition, the expression effects of the desired fusion protein were also analyzed under different conditions including hosts, culture temperatures and IPTG concentrations. The optimum expression conditions were as follows: Rosetta (DE3) host, 16 °C culture temperature and 0.5 mmol/L IPTG. After being purified by affinity chromatography with amylose resin, the fusion protein sequence was confirmed by LC-MS/MS. These results indicated that the novel gene Rv2742 product could be successfully induced and expressed in a soluble form by the expression system pMAL-c2X with MBP tag. Our findings provide reference for studies on potential interaction and immunogenicity.
Chromatography, Liquid ; Cloning, Molecular ; Escherichia coli ; Mycobacterium tuberculosis ; genetics ; Recombinant Fusion Proteins ; Tandem Mass Spectrometry

Background Thyroid associated ophthalmopathy (TAO) is an autoimmune disease.Current research on the pathogenesis focuses on common autoantigen.Insulin-like growth factor-1 receptor (IGF-1R) is necessary for the function of IGF-1,also IGF-1 plays an important role in signaling pathway of thyroid stimulating hormone receptor (TSHR).Objective This study was to investigate the effects of IGF-1 on the proliferation,expression of IGF-1R and TSHR on cultured orbital fibroblasts (OFs) derived from TAO.Methods Human orbital tissue was obtained from 17 TAO patients who received orbital adipectomy and 4 normal controls who received cosmetic surgery in West China Hospital from March 2016 to June 2016.OFs were cultured by explant culture with DMEM/F12 containing 5％ fetal bovine serum and identified by immunochemistry.The OFs were treated with different concentrations of IGF-1.IGF-1 at different concentrations (0,50,100,125 μg/L) was added into the medium,respectively,and the proliferation of the cells (absorbancy) was detected by MTS.The percentages of IGF-1R and TSHR expressions in the cells were assayed by flow cytometry.Results Cultured cells appeared to be spindle-like in shape and grew well with abundant cytoplasm.The characteristics of the cells derived from TAO patients were consistant with normal ones.The cells showed the positive response for vimentin and absent respose for desmin,S-100,myoglobin and cytokeratin.The proliferative values of OFs were gradually elevated with the increase of IGF-I dose in both TAO group and normal group (Fgroup =219.639,P＜0.001;F ion =17.752,P＜0.001) with the optimal effects in 100 μg/L IGF-1.The expression levels of IGF-1R in the OFs were (0.009 1 ±0.008 7)％,(0.095 3±0.023 3) ％,(0.083 7±0.022 7) ％ and (0.070 9 ± 0.024 1) ％ in the TAO group,and those in the normal group were (0.0023± 0.0006)％,(0.0093±0.0012)％,(0.0073±0.0015)％ and (0.0083±0.0012)％ after treatment of 50,100,125 μg/L IGF-1.The expression levels of IGF-1 R were significantly higher after treatment of 50,100 and 125 μtg/L IGF-1 than those treatment of 0 μg/L IGF-1 in both TAO group and normal group,and the expression levels of IGF-1R in the OFs were significantly increased in the TAO group compared with the normal group (all at P＜0.05).No statistical difference was seen in the TSHR expression between the TAO group and normal group after treatment of 0,50,100 and 125 μg/L IGF-1 (Fgroup =0.133,P ＞ 0.05;F ion =0.004,P ＞ 0.05).Conclusions IGF-1 can promote the proliferation of OFs and up-regulate the expression of IGF-1R in OFs.However,IGF-1 dose not play a regulating effect on the expression of TSHR in OFs.

OBJECTIVE To describe the clinical features of the parapharyngeal space tumors and assess the postoperative complications and outcomes in our hospital.METHODS The clinical data of 135 cases with parapharyngeal space tumor treated from Jan.1995 to Dec.2005 in our hospital were retrospectively studied.RESULTS It included 24 heterogeneous histologies in this group.Neurogenic tumors(72.6 %) were the most common tumors,next were salivary gland tumors(15.6 %),and others 11.8 % tumors were miscellaneous tumors.There were 121(89.6 %) patients with benign lesions and 14(10.4 %) with malignant tumors.Transcervical approach was the most commonly applied route.Only 4 cases recurred in 113 operated benign patients.At end of the follow-up,of 14 patients with malignant tumors,4(28.6 %) were alive with no evidence of disease,5(35.7 %) were alive with disease,5(35.7 %) died of the diseases.CONCLUSION Primary parapharyngeal space neoplasms are rare and the majority of these tumors are benign.Surgery is the mainstay of treatment for parapharyngeal space tumors.Most benign cases with a low rate of complication and recurrence after operation,but malignant neoplasms have a poor prognosis.

AIM: To observe whether arachidonic acid (AA) could induce apoptosis in mouse fibroblast cell line L929 and the potential mechanism involved. METHODS: The viability and damaged degree of L929 was monitored by MTT and the release of lactate dehydrogenase (LDH). Lipid peroxidation in L929 was measured as malondialdehyde (MDA) content by colorimetric assay. Hoechst 33258 staining was used to observe AA-induced morphological changes. Agarose gel electrophoresis was used to detect DNA fragmentation. RESULTS: Treatment of L929 cell with AA for 24 h, in the range of 40-160 ?mol/L, caused a great decrease in cell survival and increased MDA contents and the release of LDH simultaneously( P