Exosomal lncRNA LINC01356 Derived From Brain Metastatic Nonsmall-Cell Lung Cancer Cells Remodels the Blood-Brain Barrier.

Brain metastasis is a severe complication that affects the survival of lung cancer patients. However, the mechanism of brain metastasis in lung cancer remains unclear. In this study, we constructed an in vitro BBB model and found that cells from the high-metastatic nonsmall cell lung cancer (NSCLC) cell line H1299 showed a higher capacity to pass through the blood-brain barrier (BBB), as verified by Transwell assays, than cells from the low-metastatic NSCLC cell line A549. Brain microvascular endothelial cells (BMECs) could internalize H1299-derived exosomes, which remarkably promoted A549 cells across the BBB. The BBB-associated exosomal long noncoding RNA (lncRNA) was selected from the RNA-Seq dataset (GSE126548) and verified by real-time PCR and Transwell assays. LncRNA LINC01356 was significantly upregulated in H1299 cells and exosomes derived from these cells compared to that of A549 cells. Moreover, LINC01356 was also upregulated in serum exosomes of patients with NSCLC with brain metastasis compared with those without metastasis. In addition, BMECs treated with LINC01356-deprived exosomes expressed higher junction proteins than those treated with the control exosomes, and silencing LINC01356 in exosomes derived from H1299 cells could inhibit A549 cells from crossing the BBB. These data might indicate that the exosomal lncRNA LINC01356 derived from brain metastatic NSCLC cells plays a key role in remodeling the BBB system, thereby participating in brain metastasis in lung cancer.

Classification of Pituitary Adenomas Invading the Cavernous Sinus Assisted by Three-Dimensional Multimodal Imaging and Its Clinical Application.

Introduction To help diagnose and evaluate the prognosis of pituitary adenoma with cavernous sinus (CS) invasion and guide endonasal endoscopic surgery (EES) assisted by intraoperative navigation (ION) with three-dimensional multimodal imaging (3D-MMI). We propose a classification of CS invasion based on 3D-MMI. Methods We picked some appropriate cases and reconstructed the 3D-MMI and then classified them into 3 grades according to the stereo relationship among ICA, tumor and CS in 3D-MMI. Then, we applied different strategies according to their grade to remove pituitary adenomas that invaded the CS. Results All 38 patients were divided into 3 grades. Tumors compressing the ICA and CS without CS invasion were divided into grade 1. Tumors encasing the ICA and invading the superior-posterior compartment and/or anterior-inferior compartment but without distinct separation of the ICA and CS lateral wall were deemed as grade 2. Tumors encasing the ICA and filling the lateral compartment of the CS that dissociated the lateral wall from the ICA were deemed as grade 3. The 3D-MMI enabled adequate spatial visualization of the ICA, CS and tumors. All patients were operated on under the guidance of ION with 3D-MMI. Conclusion Classification based on 3D-MMI can better demonstrate the relationships among tumor, ICA and CS in a stereo and multi-angle view, which will have significance in guiding the surgical strategy.

LncRNA PITPNA-AS1 stimulates cell proliferation and suppresses cell apoptosis in glioblastoma via targeting miR-223-3p/EGFR axis and activating PI3K/AKT signaling pathway.

Glioblastoma (GBM) is a kind of malignant primary brain tumor, which is difficult to cure. Continuous researches have underlined that long non-coding RNAs (lncRNAs) get widely involved in the occurrence and progression of tumors, and glioblastoma is included. In this paper, we identified lncRNA PITPNA antisense RNA 1 (PITPNA-AS1) and explored its in-depth regulatory mechanism in glioblastoma cells. Firstly, RT-qPCR examined that PITPNA-AS1 was highly expressed in glioblastoma. Then, PITPNA-AS1 role in glioblastoma was assessed via functional assays. The results demonstrated that depletion of PITPNA-AS1 inhibited the proliferation and promoted the apoptosis of glioblastoma cells. After confirming that PITPNA-AS1 mainly existed in cell cytoplasm, we conducted mechanism assays which disclosed that PITPNA-AS1 sequestered microRNA-223-3p (miR-223-3p) and modulated epidermal growth factor receptor (EGFR) expression, thereby participating in the activation of PI3K/AKT signaling pathway. Eventually, rescue assays validated PITPNA-AS1 sponged miR-223-3p to promote EGFR expression, thus activating PI3K/AKT signaling pathway to accelerate proliferation and inhibit apoptosis of GBM cells. Overall, PITPNA-AS1 played an oncogenic role in glioblastoma which might be developed as a potential biomarker for glioblastoma diagnosis and treatment in the future.[Figure: see text].

Expression, purification and identification of an immunogenic fragment in the ectodomain of prostate-specific membrane antigen.

The present study aimed to identify, express and purify an immunogenic fragment in the ectodomain of prostate-specific membrane antigen (PSMA) within a fusion protein. The PSMA amino acid sequence published in National Center for Biotechnology Information GenBank was used to determine sequence homology and immunogenic index analyses, additionally using BLASTN, Protean and ExPASy software to predict the polypeptide sequences of immunogenic epitopes. The gene sequence encoding the ectodomain of the polypeptide immunogenic fragments, containing the identified immunogenic epitopes, was generated using whole-gene synthesis. Prokaryotic expression vector pET-32a-r-ectodomain-PSMA was constructed and the recombinant plasmids were transformed into competent BL21 (DE3) Escherichia coli, which was followed by induction of recombinant protein expression using isopropyl-ß-D-thiogalactopyranoside. Fusion proteins were isolated and purified using affinity chromatography and their immune activity was subsequently investigated using western blot analysis. Purified protein was used to immunize BALB/c mice in order to generate polyclonal antibodies, and the binding of polyclonal antibodies to prostate cancer cell lines in vitro was evaluated using flow cytometry. A total of 3 polypeptide fragments with high specificity were identified following analysis using numerous software packages, and the gene sequences encoding regions containing the 2 most immunogenic fragments were synthesized and successfully inserted into the prokaryotic expression vector pET-32a-r-ectodomain-PSMA. The recombinant PSMA protein fragment had a molecular weight of ~50 kDa and 95% purity. Western blot analysis revealed that the r-ectodomain-PSMA fusion protein specifically bound to the anti-PSMA ectodomain monoclonal antibody. Flow cytometry demonstrated that polyclonal antibodies raised against these recombinant proteins could specifically bind to PSMA-positive LNCaP cells, but not to PSMA-negative PC-3 cells. An immunogenic fragment in the ectodomain of PSMA was successfully expressed and purified. The present study, therefore, provides a basis for the preparation of an anti-PSMA small humanized monoclonal antibody.

Comparison of tumor markers using different detection devices.

BACKGROUND: With the development of proteomics, tumor markers have attracted increasing attention for the early diagnosis and treatment of lung cancer. As biochip technology and nanotechnology continues to grow, rapid and highly sensitive joint detection of multi-tumor markers has become possible. METHODS: Eighty-six patients with lung cancer and 42 healthy controls were recruited for this study. Based on analysis of the detection results, we plotted four standard tumor marker graphs, and compared the results of the highly sensitive nanogold probe and protein chip detection with the results of electrochemiluminescence immunoassay and Dickkopf-1 (DKK1) detection used in the clinic. We then analyzed the relationship between the detection results and our clinical data. RESULTS: Four plotted standard protein graphs all had stages with sound linear relationships. It was found in a correlation analysis of the detection results that overall the two methods showed consistency. CONCLUSION: We developed a detection method for ultra-trace protein that can detect four tumor markers, namely carcinoembryonic antigen, cytokeratin-19 fragments, neuron-specific enolase, and DKK1 in a highly sensitive way within 1.5 hours by magnifying the signal of nanogold deposition based on protein chips and nanogold probes. By comparing the results from the different detection devices, we have developed an experimental basis for detection of tumor markers in the clinic.

Genotyping and clinical characteristics of multidrug and extensively drug-resistant tuberculosis in a tertiary care tuberculosis hospital in China.

BACKGROUND: There is a lack of information on the clinical characteristics of multidrug-resistant (MDR) tuberculosis (TB) and extensively drug-resistant (XDR) TB in the Jiangxi Province of China; furthermore, data have not been reported on the utility of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analyses in genotyping Mycobacterium tuberculosis strains isolated from this region. The aim of this study was to analyse the clinical features of patients with MDR and XDR TB from Jiangxi Province and to evaluate the discriminatory power of the 15-loci MIRU-VNTR method. METHODS: A retrospective study was conducted on patients diagnosed with MDR and XDR TB at the Jiangxi Chest Hospital from July 2010 to June 2011. The RD105 deletion-targeted multiplex PCR (DTM-PCR) and the 15-loci MIRU-VNTR method were used to determine the genetic background of the identified MDR and XDR M. tuberculosis clinical isolates. RESULTS: Of 804 M. tuberculosis clinical isolates, 159 (159/804, 19.8%) of the isolates were identified as MDR with first-line drug susceptibility testing. Of the 123 available MDR isolates, 13 (13/123, 10.6%) were XDR. The RD105 deletion-targeted multiplex PCR method identified 85 (85/110, 77.3%) MDR and 12 (12/13, 92.3%) XDR isolates as the Beijing genotype. MIRU-VNTR cluster analysis demonstrated that 101 MDR and 13 XDR strains had unique genotype patterns; the remaining 9 MDR strains were in 4 clusters, namely 1 cluster with 3 strains and 3 clusters with 2 strains, resulting in a low clustering rate (4.06%). The Hunter-Gaston discriminatory index (HGDI) of the 15-loci MIRU-VNTR method was as high as 0.992. In addition, clinical surveys showed that 87 (87/110, 79.1%) MDR TB patients and 10 (10/13, 76.9%) XDR TB patients had been previously treated. Diabetes mellitus was the most common comorbidity in both MDR TB (16/110, 14.5%) and XDR TB (2/13, 15.4%) patients. CONCLUSIONS: Based on our preliminary data, the MDR and XDR M. tuberculosis clinical isolates identified at the Jiangxi Chest Hospital were genetically diverse and clustered at a low frequency. The 15-loci MIRU-VNTR method showed high discriminatory power and may be used as a first-line genotyping tool in investigating the molecular epidemiology of M. tuberculosis in Jiangxi, China. Decisive measures are urgently needed to effectively prevent and manage MDR and XDR tuberculosis in this province.

Molecular characterization of multidrug- and extensively drug-resistant Mycobacterium tuberculosis strains in Jiangxi, China.

In this study, a total of 77 multidrug-resistant and extensively drug-resistant (MDR and XDR, respectively) isolates of Mycobacterium tuberculosis were characterized among samples from patients living in Jiangxi province, China. The following two approaches were used: (i) genotyping all drug-resistant isolates by the 15-locus MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) method and identifying the Beijing family genotype using the RD105 deletion targeted multiplex PCR and (ii) determining the mutation profiles associated with the resistance to the first-line antituberculous drugs rifampin (RIF) and isoniazid (INH) and the second-line drugs ofloxacin (OFX), kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) with DNA sequencing. Six loci were examined: rpoB (for resistance to RIF), katG and mabA-inhA (INH), gyrA and gyrB (OFX), and rrs (KAN, AMK, and CAP). It is shown that the Beijing genotype was predominant (80.5%) among these strains and that the selected drug-resistant strains were genetically diverse, suggesting that they probably had independently acquired drug resistance. In comparison to the phenotypic data, the sensitivities for the detection of RIF, INH, OFX, and KAN/AMK/CAP resistance by DNA sequencing were 94.8, 80.5, 84.6, and 78.9%, respectively. The most prevalent mutations involved in RIF, INH, OFX, and KAN/AMK/CAP resistance were Ser531Leu in rpoB (44.2%), Ser315Thr in katG (55.8%) and C-15T in mabA-inhA (11.7%), Asp94Gly in gyrA (48.7%), and A1401G in rrs (73.7%), respectively. Five novel katG mutants (Trp191Stop, Thr271Pro, Trp328Phe, Leu546Pro, and Asp695Gly) and six new alleles (Ile569Val, Ile572Met, Phe584Ser, Val615Met, Asp626Glu, and Lys972Thr) in the rpoB gene were identified.

[Immunotherapeutic efficacy of BCG vaccine in pulmonary tuberculosis and its preventive effect on multidrug-resistant tuberculosis].

OBJECTIVE: To evaluate the effect and safety of BCG vaccine on initially treated pulmonary tuberculosis and its controlling effect on multidrug-resistant tuberculosis. METHODS: All 360 volunteers with initially treated pulmonary tuberculosis of positive smear and culture were divided into immunotherapy group (180 cases, also BCG group) and control group (180 cases) at random pair. The patients in BCG group were treated with chemotherapy of a regimen of 2HRZ/2HR and immunotherapy with BCG for 4 months,and the first BCG vaccine was given a month after chemotherapy. Meanwhile, the patients in the control group were treated with chemotherapy of 2HRZ/4HR only. RESULTS: (1) The negative conversion rate of sputum smear in BCG group was 98.3% (177/180), and it was 97.2% (175/180) in control group. There was no significant difference between the two groups both at the ends of 4 and 6 months after treatment (chi2 = 0.1278, P > 0.05). (2) The positive conversion rate of sputum smear in BCG group was 2.3% (4/177), and it was 6.9% (12/175) in control group followed up for 5 years. The successful rate was 96.1% (173/180) in BCG group, and it was significantly higher than that of 90.6% (163/180) in control group (chi2 = 4.4643, P < 0.05). (3) In the 5-year follow up, bacteriologic result was similar to that of X-ray. (4) The occurrence rate of multidrug-resistant tuberculosis was 2.3% (4/177) in BCG group,significantly lower than that of 7.3% (13/178) in the control group (chi2 = 4.9513, P < 0.05). CONCLUSION: As an adjunct chemotherapy,immunotherapy with BCG vaccine should be helpful for patients with initially treated pulmonary tuberculosis. It would further strengthen the effects of chemotherapy and reduce the occurrence rate of multidrug-resistant tuberculosis.