Expression,purification and bioinformatics analysis of PE＿PGRS35 protein of Mycobacterium tuberculosis H37Rv strain / 中国生物制品学杂志

@#Abstract： Objective To clone PE_PGRS35 gene of Mycobacterium tuberculosis（MTB），construct recombinant vector pET28a⁃PE_PGRS35，express and purify the PE_PGRS35 protein of MTB H37Rv heterologously，and explore a new target against MTB after bioinformatics analysis. Methods The PE_PGRS35 coding gene was amplified by PCR and used to construct the expression vector pET28a⁃PE_PGRS35 by recombinant cloning technology，which was transformed to E. coli BL21（DE3）after successful sequencing and induced by using IPTG. The obtained PE_PGRS35 protein was purified by Ni column affinity chromatography and analyzed by bioinformatics. Results The pET28a⁃PE_PGRS35 prokaryotic expression vector was constructed correctly as identified by sequencing. The PE_PGRS35 protein was mainly expressed in the form of inclusion bodies，with a relative molecular mass of about 53 000 and a purity of 90%. Bioinformatics analysis showed that PE_PGRS35 protein was an acid⁃labile protein，with main secondary structure of β⁃sheet and random coil，and no transme⁃ mbrane region，which was presumed to be an extramembrane protein with 39 phosphorylation sites and two conserved domains. Total 10 proteins，including Rv1769，PPE8，PPE64，PPE54，PPE24，PPE16，PPE35，PPE6，PPE28 and PE2， interacted with PE_PGRS35 protein. Conclusion PE_PGRS35 protein with high purity was successfully obtained，which provided a reference for the further development of new targets for drugs against MTB.

Hypoxia-Related Signature Is a Prognostic Biomarker of Pancreatic Cancer.

Background: Hypoxia plays a significant role in the pathogenesis of pancreatic cancer, but the effect of hypoxia-related genes in pancreatic cancer remains to be elucidated. This study aimed to identify hypoxia-related genes related to pancreatic cancer and construct a prognostic signature. Methods: Pancreatic cancer datasets were retrieved from TCGA database. Cox regression analyses were used to identify hypoxia-related genes and construct a prognostic signature. Datasets from International Cancer Genome Consortium and GEO databases were used as validated cohorts. The CIBERSORT method was applied to estimate the fractions of immune cell types. DNA methylation and protein levels of the genes in pancreatic cancer were examined. Results: Three hypoxia-related genes (TES, LDHA, and ANXA2) were identified as associated with patient survival and selected to construct a prognostic signature. Patients were divided into high- and low-risk groups based on the signature. Those in the high-risk group showed worse survival than those in the low-risk group. The signature was shown to be involved in the HIF-1 signaling pathway. The time-dependent ROC analyses of three independent validated cohorts further revealed that this signature had a better prognostic value in the prediction of the survival of pancreatic cancer patients. Immune cells analysis for three datasets demonstrated that high-risk signature was significantly associated with macrophages and T cells. DNA methylation and protein levels of the three genes validated their aberrant expression in pancreatic cancer. Conclusions: Our research provided a novel and reliable prognostic signature that composes of three hypoxia-related genes to estimate the prognosis of pancreatic cancer.

Exosomal HMGA2 protein from EBV-positive NPC cells destroys vascular endothelial barriers and induces endothelial-to-mesenchymal transition to promote metastasis.

Increased vascular permeability facilitates metastasis. Cancer-secreted exosomes are emerging mediators of cancer-host crosstalk. Epstein-Barr virus (EBV), identified as the first human tumor-associated virus, plays a crucial role in metastatic tumors, especially in nasopharyngeal carcinoma (NPC). To date, whether and how exosomes from EBV-infected NPC cells affect vascular permeability remains unclear. Here, we show that exosomes from EBV-positive NPC cells, but not exosomes from EBV-negative NPC cells, destroy endothelial cell tight junction (TJ) proteins, which are natural barriers against metastasis, and promote endothelial-to-mesenchymal transition (EndMT) in endothelial cells. Proteomic analysis revealed that the level of HMGA2 protein was higher in exosomes derived from EBV-positive NPC cells compared with that in exosomes derived from EBV-negative NPC cells. Depletion of HMGA2 in exosomes derived from EBV-positive NPC cells attenuates endothelial cell dysfunction and tumor cell metastasis. In contrast, exosomes from HMGA2 overexpressing EBV-negative NPC cells promoted these processes. Furthermore, we showed that HMGA2 upregulates the expression of Snail, which contributes to TJ proteins reduction and EndMT in endothelial cells. Moreover, the level of HMGA2 in circulating exosomes is significantly higher in NPC patients with metastasis than in those without metastasis and healthy negative controls, and the level of HMGA2 in tumor cells is associated with TJ and EndMT protein expression in endothelial cells. Collectively, our findings suggest exosomal HMGA2 from EBV-positive NPC cells promotes tumor metastasis by targeting multiple endothelial TJ and promoting EndMT, which highlights secreted HMGA2 as a potential therapeutic target and a predictive marker for NPC metastasis.

Epstein-Barr Virus Induces Lymphangiogenesis and Lympth Node Metastasis via Upregulation of VEGF-C in Nasopharyngeal Carcinoma.

Lymphatic metastasis is a common clinical symptom in nasopharyngeal carcinoma (NPC), the most common Epstein-Barr virus (EBV)-associated head and neck malignancy. However, the effect of EBV on NPC lymph node (LN) metastasis is still unclear. In this study, we demonstrated that EBV infection is strongly associated with advanced clinical N stage and lymphangiogenesis of NPC. We found that NPC cells infected with EBV promote LN metastasis by inducing cancer-associated lymphangiogenesis, whereas these changes were abolished upon clearance of EBV genomes. Mechanistically, EBV-induced VEGF-C contributed to lymphangiogenesis and LN metastasis, and PHLPP1, a target of miR-BART15, partially contributed to AKT/HIF1a hyperactivity and subsequent VEGF-C transcriptional activation. In addition, administration of anti-VEGF-C antibody or HIF1α inhibitors attenuated the lymphangiogenesis and LN metastasis induced by EBV. Finally, we verified the clinical significance of this prometastatic EBV/VEGF-C axis by determining the expression of PHLPP1, AKT, HIF1a, and VEGF-C in NPC specimens with and without EBV. These results uncover a reasonable mechanism for the EBV-modulated LN metastasis microenvironment in NPC, indicating that EBV is a potential therapeutic target for NPC with lymphatic metastasis. IMPLICATIONS: This research demonstrates that EBV induces lymphangiogenesis in NPC by regulating PHLPP1/p-AKT/HIF1a/VEGF-C, providing a new therapeutic target for NPC with lymphatic metastasis.

A Multicenter, Randomized, Controlled Trial of Electroacupuncture for Perimenopause Women with Mild-Moderate Depression.

OBJECTIVE: Up to 62% of perimenopausal women have depression symptoms. However, there is no efficacy treatment. The aim of this study is to compare the clinical efficacy and safety of EA therapy and escitalopram on perimenopause women with mild-moderate depressive symptom. METHOD: A multicenter, randomized, positive-controlled clinical trial was conducted at 6 hospitals in China. 242 perimenopause women with mild-moderate depressive symptom were recruited and randomly assigned to receive 36 sessions of EA treatment or escitalopram treatment. The primary outcome measure was the 17-item Hamilton Depression Rating Scale (HAMD-17). The secondary outcome measures include menopause-specific quality of life (MENQOL) and serum sexual hormones which include estrogen, follicle-stimulating hormone, and luteinizing hormone. RESULTS: 221 (91.3%) completed the study, including 116 in the EA group and 105 in the escitalopram group. The baseline levels of demographic and outcome measurements were similar in the two groups. In the intervention period, there was no difference between two groups. However, in the follow-up, both HAMD-17 and MENQOL were significantly decreased, and at week 24 the mean differences were -2.23 and -8.97, respectively. There were no significant differences in the change of serum sexual hormones between the two groups. No serious adverse events occurred. CONCLUSION: EA treatment is effective and safe in relieving depression symptom and improving the quality of life in the perimenopausal depression. Further research is needed to understand long-term efficacy and explore the mechanism of this intervention. This study is registered with ClinicalTrials.gov NCT02423694.

Civic/sanctuary orientation and HIV involvement among Chinese immigrant religious institutions in New York City.

Using data from a study of Chinese immigrant religious institutions in New York City (primarily Christian and Buddhist), this paper explores why some religious institutions are more inclined than others to be involved in HIV-related work. Although numerous factors are likely to play a role, we focus on organisations' differing views on social engagement as an explanatory factor. We hypothesise that religious institutions that value social engagement ('civic') will be more inclined towards HIV/AIDS involvement than those that are more inward focused ('sanctuary'). Given that many religious institutions are fundamentally defined by their stance on the appropriateness of social engagement, better understanding of this key characteristic may help to inform community and government organisations aiming to increase religious institutions' involvement in HIV/AIDS-related work. Our analysis suggests that some organisations may be less interested in taking on the challenges of working in HIV/AIDS because of their general view that churches or temples should not be socially engaged. On the other hand, religious institutions that have concerns about social acceptability, fear of infection or lack of capacity--but generally embrace social engagement--may be more open to partnering on HIV/AIDS-related work because of their overriding community service orientation.

[Effect of NAD+ against radiation injury and its dose-effect relationship].

OBJECTIVE: To explore the effect of NAD+ against radiation injury and its dose-effect relationship. METHODS: L02 liver cells cultured in RPMI 1640 medium containing 10% fetal calf serum were exposed to X-ray irradiation followed by immediate application of NAD+. The cellular viability was analyzed by MTT assay and the apoptotic cells were detected by TUNEL methods to observe the damages of L02 liver cells induced by X-ray exposure and analyze the dose-effect relationship of NAD+. RESULTS: The viability of L02 liver cells was decreased with increasing dose of X-ray irradiation. The most obvious growth inhibition of L02 cells occurred 24 h after the irradiation. NAD+ significantly increased the cell survival rate after irradiation, and this effect was gradually increased within the concentration range of 100-1000 microg/ml; at higher concentrations, the survival rate of the irradiated L02 cells showed no significant increase. CONCLUSION: NAD+ provides partial protection of the liver cells against radiation injury, and the effect is positively correlated to NAD+ concentration within a certain range.

[Therapeutic effect of para-toluenesulfonamide on transplanted hepatocarcinoma in nude mice].

OBJECTIVE: To study the effect of of percutaneous para-toluenesulfonamide (PTS) injection on transplanted hepatocarcinoma in nude mice. METHODS: Sixty nude mice with subcutaneous transplanted hepatocarcinoma were randomized into 6 groups, namely PTS, chemotherapy, radiotherapy, PTS+chemotherapy, PTS+radiotherapy and control groups. PTS were injected into the tumor in the nude mouse models as indicated, and the tumor growth rate and survival time of the mice were recorded. RESULTS: All the treatments resulted in effective arrest of the tumor growth, but the effects of PTS+chemotherapy and PTS+radiotherapy were more obvious. No significant difference in the survival time of the mice were noted between the groups. CONCLUSION: PTS+chemotherapy and PTS+radiotherapy are safe and reliable, and produces better effects than either radiotherapy or chemotherapy alone.

[Effects of cryoablation on the bronchi of normal pigs].

OBJECTIVE: To investigate the effects of cryoablation on different bronchi of normal pigs and provide experimental bases for the potential clinical application of this technique. METHODS: Six normal pigs were divided into two groups and subjected to percutaneous cryoablation of the lung tissues. Three pigs were sacrificed on day 3 (group A) and another 3 on day 28 (group B) after the ablation, and the morphology and volume of the ablated areas and the pathological changes in different bronchi. RESULTS: In group A, examination of the biopsy samples taken 3 days after the ablation revealed significantly greater maximal longitudinal (t=9.789, P=0.000) and transverse (t=3.253, P=0.023) diameters of the area of freezing damage than those observed immediately after the cryoablation. The diameters of the freezing damage area in group B were significantly smaller than those in group A (t=7.227, P=0.000; t=6.006, P=0.001). The freezing damages to the bronchi worsened with the reduction of the bronchial lumen; the damages to the major bronchi and the second-order bronchi were relatively slight, which also showed better recovery 28 days after the ablation. CONCLUSION: CT-guided percutaneous cryoablation does not produce serious effects on the major bronchi and the second-order bronchus, and can be a minimally invasive therapy for lung tumors with good tolerance and safety.

[Dose-effect relationship of para-toluenesulfonamide for treatment of hepatocellular carcinoma in rats].

OBJECTIVE: To investigate the dose-effect relationship of para-toluenesulfonamide (PTS) for treatment of hepatocellular carcinoma in rats. METHODS: Forty-two SD rats bearing subcutaneous transplanted hepatocellular carcinoma were randomly divided into 6 groups (n=7), in which 0.02, 0.04, 0.06, 0.08, and 0.10 ml PTS and 0.10 ml normal saline were injected into the tumor, respectively. All of the rats were executed 24 h after the injection to observe the pathological changes in the tumor. RESULTS: In rats with saline injection, the tumor tissues exhibited no obvious changes and the tumor cells retained the active proliferation. PTS, in contrast, caused coagulation necrosis of the tumor tissue, and the necrotic area expanded with the increase of the injected doses. The necrotic volume of the tumor was in roughly linear correlation with the dose of PTS injected, with the linear regression equation of V (cm(3))=-0.018+2.595Y (where V represents tumor necrosis volume, and Y the injected dose of PTS). CONCLUSION: The dose-effect relationship of PTS is roughly linear, and the PTS dose for injection can be estimated according to the diameter of the tumor.

[Treatment of chronic idiopathic thrombocytopenic purpura with small dose heparin: a report of 12 cases].

In order to determine the curative effect of small dose heparin for treatment of chronic idiopathic thrombocytopenic purpura (CITP), a total of 12 CITP patients, who were failed with prednisone and immunosuppressants over 6 months, had been treated with subcutaneous injection of small dose heparin. The curative effects were seen in 8 patients and there were no exacerbation of hemorrhage during the therapy. The results showed that it is effective and safe to use this treatment for CITP.