Identification of a lncRNA prognostic signature-related to stem cell index and its significance in colorectal cancer.

Background: The relationship between long noncoding RNAs (lncRNAs) and the mRNA stemness index (mRNAsi) in colorectal cancer (CRC) is still unclear. Materials & methods: The mRNAsi, mRNAsi-related lncRNAs and their clinical significance were analyzed by bioinformatic approaches in The Cancer Genome Atlas (TCGA)-COREAD dataset. Results: mRNAsi was negatively related to pathological features but positively related to overall survival and recurrence-free survival in CRC. A five mRNAsi-related lncRNAs prognostic signature was further developed and showed independent prognostic factors related to overall survival in CRC patients, due to the five mRNAsi-related lncRNAs involved in several pathways of the cancer stem cells and malignant cancer cell phenotypes. Conclusion: The present study highlights the potential roles of mRNAsi-related lncRNAs as alternative prognostic markers.

Lay abstract Previous evidence has indicated that the mRNA stem index (mRNAsi) is representative of the stemness of cancer stem cells (CSCs), whereas long noncoding RNAs (lncRNAs) may be crucial regulators in CSC phenotype. Nevertheless, the relationship between lncRNAs and mRNAsi in CRC is still unclear. Our results show that the mRNAsi was negatively related to pathological features and positively related to prognosis in CRC. Five mRNAsi-related lncRNAs were further identified and developed as a prognostic signature that could independently predict survival in CRC patients due to the five mRNAsi-related lncRNAs being involved in several pathways of CSCs and malignant cancer cell phenotypes, indicating the potential roles of mRNAsi-related lncRNAs as alternative prognostic markers.

Structural basis for the inhibition of the SARS-CoV-2 RNA-dependent RNA polymerase by favipiravir-RTP

The RNA polymerase inhibitor, favipiravir, is currently in clinical trials as a treatment for infection with SARS-CoV-2, despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 [A]. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, non-productive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

Integrative Analysis Confirmed the Association between Osteoprotegerin and Osteoporosis / 中国医学科学杂志(英文版)

Objective This study aimed to verify the association between osteoprotegerin gene () and its variants with osteoporosis (OP) by performing integrative analysis.Methods We used the KGG software to perform gene-based association analysis, which integrated all publicly available single-nucleotide polymorphism (SNP)-based values and obtained an overall value for the . The significant SNPs were screened for expression quantitative trait loci (eQTLs). Meta-analysis was used to combine the associations between the variants of and bone mineral density (BMD) reported in the literatures. Then we performed dual-luciferase reporter gene systems for the functional verification of the variants of .Results In the gene-based association analysis, the over all value of was 6.24×10 for BMD at femoral neck (FN) and 7.37×10 for BMD at lumbar spine (LS), indicating the importance of for OP. The publicly available eQTL database identified 5 eQTLs which exert cis-regulation effects on at FN and LS. Literature searching found that rs2073617 (known as T950C) was the hot spot SNP. There were 13 relevant studies on rs2073617 besides the GEFOS-2 study identified from the PubMed. Significant differences among TT, TC and CC genotypes at FN (= 0.047) and LS (= 0.025) were shown by meta-analysis, demonstrating the associations between T950C polymorphism and BMD. Luciferase gene expression was significantly higher at the presence of allele C than allele T in the 293T cells (=-9.47, <0.01). Conclusion The integrative analysis further confirmed the importance of in OP and the correlation of T950C polymorphism with BMD of OP. The strategy can be used as a reference for functional interpretation of other disease-related genes.

Hepatitis B spliced protein enhanced NF-κB activities via interaction with ubiquitously expressed transcript splice variant 1 / 中国人兽共患病学报

We investigated whether hepatitis B spliced protein affect the NF-kappa B activities by interacting with ubiquitously expressed transcript splice variant 1 (UXT-V1).The HBSP-UXT-V1 protein interactions were screened by yeast two hybrid assay,and confirmed by immunoprecipitation,confocal microscopy,mammalian two hybrid assay,and GST-Pulldown assay.The reporter plasmids driven by NF-kappa B promoter were transfected into UXT-knockdown HBSP stably expressed cell lines,and the reporter genes were detected after transfection.Results showed that the interaction between UXT-V1 and HBSP in yeast was demonstrated.Furtherly,HBSP could interact with UXT-V1 in mammalian cells.HBSP could enhance NF-kappa B activities,and this effect was partly achieved by the interaction with UXT-V1.In conclusion,the effect of HBSPUXT-V1 interaction on the NF-kappa B pathway in hepatocytes may have an impact on HBV related liver diseases.

Deletion Of XIAP reduces the severity of acute pancreatitis via regulation of cell death and nuclear factor-κB activity.

Severe acute pancreatitis (SAP) still remains a clinical challenge, not only for its high mortality but the uncontrolled inflammatory progression from acute pancreatitis (AP) to SAP. Cell death, including apoptosis and necrosis are critical pathology of AP, since the severity of pancreatitis correlates directly with necrosis and inversely with apoptosis Therefore, regulation of cell death from necrosis to apoptosis may have practicably therapeutic value. X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the inhibitor of apoptosis proteins (IAP) family, but its function in AP remains unclear. In the present study, we investigated the potential role of XIAP in regulation of cell death and inflammation during acute pancreatitis. The in vivo pancreatitis model was induced by the administration of cerulein with or without lipopolysaccharide (LPS) or by the administration of l-arginine in wild-type or XIAP-deficient mice, and ex vivo model was induced by the administration of cerulein+LPS in AR42J cell line following XIAP inhibition. The severity of acute pancreatitis was determined by serum amylase activity and histological grading. XIAP deletion on cell apoptosis, necrosis and inflammatory response were examined. Caspases activities, nuclear factor-κB (NF-κB) activation and receptor-interacting protein kinase1 (RIP1) degradation were assessed by western blot. Deletion of XIAP resulted in the reduction of amylase activity, decrease of NF-κB activation and less release of TNF-α and IL-6, together with increased caspases activities and RIP1 degradation, leading to enhanced apoptosis and reduced necrosis in pancreatic acinar cells and ameliorated the severity of acute pancreatitis. Our results indicate that deletion of XIAP switches cell death away from necrosis to apoptosis and decreases the inflammatory response, effectively attenuating the severity of AP/SAP. The critical role of XIAP in cell death and inflammation suggests that inhibition of XIAP represents a potential therapeutic strategy for the treatment of acute pancreatitis.

Effects of Tocilizumab on Experimental Severe Acute Pancreatitis and Associated Acute Lung Injury.

OBJECTIVE: To examine the therapeutic effects of tocilizumab, an antibody against interleukin-6 receptor, on experimental severe acute pancreatitis and associated acute lung injury. The optimal dose of tocilizumab and the activation of interleukin-6 inflammatory signaling were also investigated. DESIGN: Randomized experiment. SETTING: Research laboratory at a university hospital. SUBJECT: Experimental severe acute pancreatitis in rats. INTERVENTIONS: Severe acute pancreatitis was induced by retrograde injection of sodium taurocholate (50 mg/kg) into the biliopancreatic duct. In dose-study, rats were administered with different doses of tocilizumab (1, 2, 4, 8, and 16 mg/kg) through the tail vein after severe acute pancreatitis induction. In safety-study, rats without severe acute pancreatitis induction were treated with high doses of tocilizumab (8, 16, 32, and 64 mg/kg). Serum and tissue samples of rats in time-study were collected for biomolecular and histologic evaluations at different time points (2, 6, 12, 18, and 24 hr). MEASUREMENTS AND MAIN RESULTS: 1) Under the administration of tocilizumab, histopathological scores of pancreas and lung were decreased, and severity parameters related to severe acute pancreatitis and associated lung injury, including serum amylase, C-reactive protein, lung surfactant protein level, and myeloperoxidase activity, were all significant alleviated in rat models. 2) Dose-study demonstrated that 2 mg/kg tocilizumab was the optimal treatment dose. 3) Basing on multi-organ pathologic evaluation, physiological and biochemical data, no adverse effect and toxicity of tocilizumab were observed in safety-study. 4) Pancreatic nuclear factor-κB and signal transducer and activator of transcription 3 were deactivated, and the serum chemokine (C-X-C motif) ligand 1 was down-regulated after tocilizumab administration. CONCLUSIONS: Our study demonstrated tocilizumab, as a marketed drug commonly used for immune-mediated diseases, was safe and effective for the treatment of experimental severe acute pancreatitis and associated acute lung injury. Our findings provide experimental evidences for potential clinical application of tocilizumab in severe acute pancreatitis and associated complications.

Resolvin D1 protects against inflammation in experimental acute pancreatitis and associated lung injury.

Acute pancreatitis is an inflammatory condition that may lead to multisystemic organ failure with considerable mortality. Recently, resolvin D1 (RvD1) as an endogenous anti-inflammatory lipid mediator has been confirmed to protect against many inflammatory diseases. This study was designed to investigate the effects of RvD1 in acute pancreatitis and associated lung injury. Acute pancreatitis varying from mild to severe was induced by cerulein or cerulein combined with LPS, respectively. Mice were pretreated with RvD1 at a dose of 300 ng/mouse 30 min before the first injection of cerulein. Severity of AP was assessed by biochemical markers and histology. Serum cytokines and myeloperoxidase (MPO) levels in pancreas and lung were determined for assessing the extent of inflammatory response. NF-κB activation was determined by Western blotting. The injection of cerulein or cerulein combined with LPS resulted in local injury in the pancreas and corresponding systemic inflammatory changes with pronounced severity in the cerulein and LPS group. Pretreated RvD1 significantly reduced the degree of amylase, lipase, TNF-α, and IL-6 serum levels; the MPO activities in the pancreas and the lungs; the pancreatic NF-κB activation; and the severity of pancreatic injury and associated lung injury, especially in the severe acute pancreatitis model. These results suggest that RvD1 is capable of improving injury of pancreas and lung and exerting anti-inflammatory effects through the inhibition of NF-κB activation in experimental acute pancreatitis, with more notable protective effect in severe acute pancreatitis. These findings indicate that RvD1 may constitute a novel therapeutic strategy in the management of severe acute pancreatitis.

Knockdown of interleukin-2 by shRNA-mediated RNA interference prolongs liver allograft survival.

Interleukin-2 (IL-2) plays a central role in T-cell activation, expansion, and homeostasis. The failure of IL-2 biosynthesis may play a critical role in tolerance induction. We tested the effect of IL-2 blockade by short hairpin RNA (shRNA) on regulating acute rejection in rat liver transplantation. To this end, we successfully designed and selected an effective interference plasmid, pIL-2B. The IL-2 mRNA expression level in the pIL-2B group was one-fifth of that in the no transfection group. Lewis to BN orthotopic liver transplant model was used to explore the effect of knockdown IL-2 by shRNA in vivo. Recipients treated with pIL-2-shRNA survived longer (median survival time of 16 d range 7-21 d) than those with empty vector (11; range 5-13) or saline (9; range 5-13) (P<0.05), and was inferior to those with CsA (24; range 13-36, P<0.05). The IL-2-shRNA attenuated acute rejection with decreased apoptosis of hepatocytes and reduced cytokine production of IL-2, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in the graft. Our results suggest that IL-2 targeting using RNA interference approach may be of potential interest in organ transplantation.

Pleiotropic effect of tatC mutation on metabolism of pathogen Yersinia enterocolitica / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To analyze the impact of depletion of the twin arginine translocation (TAT) system on virulence and physiology of Yersinia enterocolitica for a better understanding of its pathogenicity.</p><p><b>METHODS</b>We constructed a DeltatatC::SpR mutant of Yersinia enterocolitica by P1 phage mediated transduction using Escherichia coli K-12 DeltatatC::SpR strain as a donor.</p><p><b>RESULTS</b>A P1-mediated genetic material transfer was found between the two species of enterobacteria, indicating a great potential of acquisition of antibiotic resistance in emergency of a new threatening pathogen by genetic material exchanges. Periplasmic trimethylamine N-oxidase reductase activity was detected in the wild type Y. enterocolitica strain and translocation of this enzyme was completely abolished by the DeltatatC::SpR mutation. In addition, the DeltatatC::SpR mutation showed a pleiotropic effect on the metabolism of Y. enterocolitica. However, the tat mutation did not seem to affect the mobility and virulence of Y. enterocolitica under the conditions used.</p><p><b>CONCLUSION</b>Unlike other pathogenic bacteria studied, the TAT system of Y. enterocolitica might play an important role in the pathogenic process, which is distinct from other pathogens, such as Pseudomonas aeruginosa and enterohemorrhagic E. coli O157:H7.</p>