Extraction and bioinformatics analysis of Chlamydia trachomatis LpxA.

OBJECTIVE: The aim of this study is to clone the LpxA gene of Chlamydia trachomatis and analyze its biological characteristics. METHODS: Specific primers were designed according to the sequence of Ct LpxA gene. LpxA gene was amplified by PCR and connected to pMD18-T vectors. Positive clones were selected for PCR and DNA sequencing. Finally, bioinformatics software was used to analyze the biological properties of LpxA protein. RESULTS: The total length of LpxA gene was 840 bp, encoding 280 amino acids. LpxA protein has no signal peptide and was located in bacterial cytoplasm. The prediction of secondary structure showed that the α-helix, extended strand, ß-turn and random coil accounted for 19.6%, 32.8%, 11.4% and 36%, respectively. According to the prediction of tertiary structure, three identical LpxA molecules constituted homologous trimers. It was predicted that there were 11 B cell epitopes in LpxA. CONCLUSION: Ct Lpxa gene was cloned, and LpxA protein structure and function were predicted.

Curcumin suppressed proliferation and migration of human retinoblastoma cells through modulating NF-κB pathway.

PURPOSE: To study the effect of curcumin on proliferation and invasion of the human retinoblastoma cells and its potential mechanism. METHODS: A cell line of retinoblastoma (WERI-Rb-1) was treated with various concentrations of curcumin (0-40 µM). Cell number was counted with CCK8 kit, and cell migration was assessed using the Transwell assay. Immunoblotting was performed to detect the proteins of metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF) as well as nuclear translocation of nuclear factor-κB (NF-κB, p65). RESULTS: Proliferation and migration of WERI-Rb-1 cells were significantly inhibited by curcumin in a concentration-dependent manner (0-40 µM). Protein expressions of MMP-2, MMP-9 and VEGF in the WERI-Rb-1 cells were also significantly inhibited by curcumin in a concentration-dependent manner (0-40 µM). Furthermore, nuclear translocation of NF-κB (p65) was significantly inhibited by curcumin in time-dependent manner (6-24 h). CONCLUSION: Curcumin inhibited proliferation and migration of WERI-Rb-1 cells, a cell line of human retinoblastoma, which might be through modulating NF-κB and its downstream proteins including VEGF, MMP-2, and MMP-9.

The expression and purification of LpxA of Chlamydia trachomatis and preparation of its polyclonal antibody.

The purpose of this study is to purify the LpxA protein of Chlamydia trachomatis (Ct) and prepare the polyclonal antibody against LpxA protein, so as to lay a foundation for studying the function of LpxA protein. The LpxA gene was amplified by PCR. The expression plasmid pET28a-LpxA was constructed by using pET28a as the vector. The fusion protein containing 6 histidine tag was induced by IPTG and purified by Ni2+ chromatography gel. The purified His-LpxA protein was used as an immunogen to immunize New Zealand rabbits subcutaneously through the back to prepare polyclonal antibody. Immunoblotting was used to detect the reaction between the antibody and His-LpxA. The determination of polyclonal antibody titer was detected by ELISA. The relative molecular weight of His-LpxA was 32.8 kDa, and it could be expressed in Escherichia coli. The purity of the purified protein was about 95%. After immunizing New Zealand rabbits, the antiserum was able to recognize the recombinant His-LpxA protein with a titer greater than 1:10240. In this study, LpxA protein was successfully purified and antiserum was prepared, which provided an experimental basis for studying the function of LpxA protein.

Foot-and-mouth disease virus induces lysosomal degradation of NME1 to impair p53-regulated interferon-inducible antiviral genes expression.

Nucleoside diphosphate kinase 1 (NME1) is well-known as a tumor suppressor that regulates p53 function to prevent cancer metastasis and progression. However, the role of NME1 in virus-infected cells remains unknown. Here, we showed that NME1 suppresses viral replication in foot-and-mouth disease virus (FMDV)-infected cells. NME1-enhanced p53-mediated transcriptional activity and induction of interferon-inducible antiviral genes expression. FMDV infection decreased NME1 protein expression. The 2B and VP4 proteins were identified as the viral factors that induced reduction of NME1. FMDV 2B protein has a suppressive effect on host protein expression. We measured, for the first time, VP4-induced lysosomal degradation of host protein; VP4-induced degradation of NME1 through the macroautophagy pathway, and impaired p53-mediated signaling. p53 plays significant roles in antiviral innate immunity by inducing several interferon-inducible antiviral genes expression, such as, ISG20, IRF9, RIG-I, and ISG15. VP4 promoted interaction of p53 with murine double minute 2 (MDM2) through downregulation of NME1 resulting in destabilization of p53. Therefore, 5-flurouracil-induced upregulation of ISG20, IRF9, RIG-I, and ISG15 were suppressed by VP4. VP4-induced reduction of NME1 was not related to the well-characterized blocking effect of FMDV on cellular translation, and no direct interaction was detected between NME1 and VP4. The 15-30 and 75-85 regions of VP4 were determined to be crucial for VP4-induced reduction of NME1. Deletion of these VP4 regions also inhibited the suppressive effect of VP4 on NME1-enhanced p53 signaling. In conclusion, these data suggest an antiviral role of NME1 by regulation of p53-mediated antiviral innate immunity in virus-infected cells, and reveal an antagonistic mechanism of FMDV that is mediated by VP4 to block host innate immune antiviral response.

An overview of neurocritical care in China: a nationwide survey.

BACKGROUND: Little quantitative evidence was available regarding the development of NICUs in China. The purpose of this survey was to evaluate the current situation of neurointensive care units (NICUs) across China. METHODS: The directors of NICUs from 100 tertiary care hospitals across China were contacted and asked to complete a closed response questionnaire regarding their NICUs. Basic information, equipment, and technology information available in the units, as well as staffing information were investigated. RESULTS: Seventy-six questionnaires were returned (a 68% response rate). Of 76 NICUs, 43 units constituted the majority. The number of each NICU bed varied from 4 to 45, occupying 2%-30% of the total department beds. Over 70% of NICUs were equipped with many emergency treatment equipments as well as physiological and biochemical monitoring equipments, while 34%-70% of NICUs still lacked some kinds of equipments such as defibrillators. Some specialist equipments were still partially lacking in 62%-95% of NICUs. A vast majority of the NICUs were equipped with neurocritical care directors, full-time attending physicians, and head nurses, but full-time NICU residents and neurocritical care nurses were still lacking in nearly half (53%) and one-third (33%-37%) of NICUs, respectively. In 76 NICUs, full-time neurointensivists and nurses added up to 359 and 852, respectively. In addition, 78%-97% of all the surveyed NICUs were severely short of non-neurological professional staffs. CONCLUSION: In China, neurocritical care has developed rapidly, but there is still a shortage of well-equipped and well-staffed NICUs across the nation currently.

Evaluation of neuro-intensive care unit performance in China: predicting outcomes of Simplified Acute Physiology Score II or Glasgow Coma Scale.

BACKGROUND: Severity scoring systems are useful tools for measuring the severity of the disease and its outcome. This pilot study was to verify and compare the prognostic performance of the Simplified Acute Physiology Score II (SAPS II) and Glasgow Coma Scale (GCS) in neuro-intensive care unit (N-ICU) patients. METHODS: A total of 1684 patients consecutively admitted to the N-ICU at Xuanwu Hospital between January 1, 2005 and December 31, 2011 were enrolled in this study. The data-base included admission data, at 24-, 48-, and 72-hour SAPS II and GCS. Repeated measure data analysis of variance, Logistic regression analysis, the Hosmer-Lemeshow goodness-of-fit statistic, and the area under the receiver operating characteristic were used to evaluate the performance. RESULTS: There was a significant difference between the SAPS II or GCS score at four time points (F = 16.110, P = 0.000 or F = 8.108, P = 0.000). The SAPS II scores or GCS score at four time points interacted with the outcomes with significant difference (F = 116.771, P = 0.000 or F = 65.316, P = 0.000). Calibration of the SAPS II or GCS score at each time point on all patients was good. The percentage of a risk estimate prediction corresponding to observed mortality was also good. The 72-hour score have the greatest consistency. Discriminations of the SAPS II or GCS score at each time were all satisfactory. The 72-hour score had the greatest discriminative power. The cut-off value was 33 (sensitivity of 85.2% and specificity of 74.3%) and 6 (sensitivity of 70.6% and specificity of 65.0%). The SAPS II at each time point on all patients showed better calibration, consistency and discrimination than GCS. The binary Logistic regression analysis identified physiological variables, GCS, age, and disease category as significant independent risk factors of death. After the two variables including underlying disease and type of admission were excluded, we built the simplified SAPS II model. A correlation was suggested between the simplified SAPS II score at each time point and outcome, regardless of the diagnosis. CONCLUSIONS: The GCS scoring system tends to be a little weaker in the predictive power than the SAPS II scoring system in this Chinese cohort of N-ICU patients. The advantage of SAPS II scoring system still exists that it dose not need to take into account the diagnosis or diseases categories, even in the special N-ICU. The simplified SAPS II scoring system is considered a new idea for the estimation of effectiveness.