MicroRNAs (miRNAs) play an important role in infection and replication of virus in host cells. To identify cellular miRNAs involved in the host response to enterovirus 71 (EV71) infection, we examined miRNAs effects on the replication of EV71 in rhabdomyosarcoma cells. We constructed target gene of miRNAs screening system. 3'untranslated region (UTR) dual luciferase reporter analysis was used to identify putative miRNA targets in the EV71 virus genome. First, 13 segments of EV71 virus genomes were inserted to the pMIR vector and the luciferase expression were assayed to identify the target gene of putative miRNA. The reporter gene expression of the cells transfected with the vector containing 5'-UTR was significantly downregulated. Then we screened the miRNAs that may target to 5'-UTR using online analysis programs. Furthermore, Western blotting and real-time PCR test were performed to investigate the effect of miRNAs on viral replication. The study showed that miR373 and miR542-5p could suppress the replication of EV71 virus through binding to the 5'-UTR gene. Cellular miRNAs could regulate the replication of EV71 virus in host cells, and our paper should report the role of miR373 and miR542-5p in this regulation for the first time. Our findings supported the notion that the cellular miRNAs might be essential in the host-pathogen interactions.
3' Untranslated Regions ; 5' Untranslated Regions ; Down-Regulation ; Enterovirus A, Human ; physiology ; Enterovirus Infections ; virology ; Gene Expression ; Host-Pathogen Interactions ; Humans ; MicroRNAs ; genetics ; Rhabdomyosarcoma ; virology ; Tumor Cells, Cultured ; Virus Replication

The artificial 5-helix can inhibit the formation of trimer-of-hairpins structure during the course of HIV-directed membrane fusion and then inhibit human immunodeficiency virus (HIV) infecting target cells. But 5-helix was apt to form inclusion body when expressed directly in prokaryotic cell and was difficult to renature, which causes inconvenience to future study. We found a proper expression vector by simulating protein structure. We simulated its proper conformation in two vectors pGEX-6P-1 and pET44b by homology modeling. The contrast of conformations showed that the energy of salvation of its fusion protein with NusA in vector pET44b was higher than its fusion protein with glutathione-S-transferase (GST) in pGEX-6P-1 and its restriction site lay on the surface of its fusion protein in vector pET44b. 5-helix gene was amplified from pGEX-6P-1-5H by PCR, and was ligated to pET44b to construct recombinant vector pET44b-PSP-5Helix after tested correctly by enzymes digestion. The recombinant vector was transformed into Escherichia coli BL21 (DE3) to express 5-helix protein at different temperatures. Aim protein was purified with Ni column and GST column, and was determined by SDS-PAGE. Then the purified 5 -Helix was used to test the inhibitive activity of pseudo HIV virus infecting GHOST-CXCR4. Results show that its fusion protein with NusA can be effectively soluble expressed and easier to be cleaved, and that the purified 5-helix can efficiently inhibit pseudo HIV virus infecting GHOST-CXCR4 and its IC50 value is (22.77 +/- 5.64) nmol/L, which lay the foundation to further discuss the application in HIV-1 infection.
Carrier Proteins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; HIV Envelope Protein gp41 ; metabolism ; HIV-1 ; genetics ; Peptides ; genetics ; Repetitive Sequences, Nucleic Acid ; genetics ; Viral Fusion Proteins ; genetics ; Virus Internalization ; drug effects

The M and NP genes of H5N1 avian influenza virus (A/chicken/Hubei/489/2004) were amplified by RT-PCR from viral RNA,and cloned into pMD 18-T vector respectively.The expression plasmid containing the M gene (pHM6-m) or the NP gene (pHM6-np) was then constructed by inserting the M or NP gene into the pHM6 eukaryote expression vector; the constructed plasmid was then sequenced.32 BALB/c mice (6-week-old) were divided into four groups at random.Three groups of BALB/c mice were inoculated one time the intramuscular route with either 30 μg of plasmid pHM6-m,30 μg of plasmid pHM6-np or the mixture of plasmid pHM6-m (15 μg ) and pHM6-np(15 μg) respectively.A additional group of mice were injected with 100 μ1 PBS as controls.Two weeks later,all mice were challenged with homologous H5N1 avian influenza virus,and observed in the following 12 days.The survival rates of mice in the pHM6-m group,the pHM6-np group and mixed plasmids group were 62.5% ,25.0% and 50.0%,respectively.Results showed that effective protection could be provided by either pHM6-m or pHM6-np,but pHM6-m provided a better protective effect than pHM6-np.

Heat shock protein gp96 is a glycoprotein which was found several years ago. Besides its function as a molecular chaperone, it is also reported to play important roles both in innate immunity and adaptive immunity. Gp96 can stimulate the maturation of antigen presenting cells (especially dendritic cells) and the secretion of cytokines. Gp96 and its associated peptides could stimulate peptide specific cytotoxic T lymphocyte reaction (CTL), which was very promising in the designing of anti-virus and anti-tumor vaccines. However the expression level of whole length gp96 was relatively low in E. coli and the purity of gp96 are not very suitable for further study. We successfully cloned the carboxy terminal fragment (560aa-751aa) of murine gp96 into the pGEX-6p-1 vector and expressed in BL21 strain. This fragment contains the peptide binding domain and the dimerization domain. After purification, the recombinant fusion protein was cleaved with the PreScission Protease and analyzed by Gelfiltration. The results show that this fragment may be related to the dimerization of gp96 and make an foundation for further investigations of the protein.
Animals ; Antigens, Neoplasm ; genetics ; Blotting, Western ; Chromatography, Gel ; Cloning, Molecular ; Mice ; Peptide Fragments ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification

Two Heptad repeat motifs (HR1 and HR2) from paramyxoviruses F protein could form thermostable heterodimers containing high alpha-helix while virus infected host cell. Following that the viral membrane and the host cell membrane were juxtaposed, which leads to membrane fusion. Mumps virus (MuV) is a member of the genus Rubulavirus in the family of Paramyxoviridae. MuV could use similar infection mechanism as well as other paramyxoviruses. In this study the HR1 and HR2 regions of MuV F protein were predicted by a computer program and expressed in E. coli with the GST fusion expression system. The GST fusion or GST-removed proteins were purified with Gluthathion Sepharose 4B Column. GST pull-down experiment suggested the interaction of HR1 and HR2 peptides, and analysis of gel filtration showed two peptides could form multimer, which indicates that the HR regions of MuV F protein may play an important role in virus fusion.
Membrane Fusion ; genetics ; Mumps virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Repetitive Sequences, Amino Acid ; Viral Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

Objective: Genetic high-risk assessment combines hereditary breast, ovarian and pancreatic cancer into one syndrome. However, there is a lack of data for comparing the germline mutational spectrum of the cancer predisposing genes between these three cancers. Methods: Patients who met the criteria of the hereditary breast, ovarian and pancreatic cancer were enrolled and received multi-gene sequencing. Results: We enrolled 730 probands: 418 developed breast cancer, 185 had ovarian cancer, and 145 had pancreatic cancer. Out of the 18 patients who had two types of cancer, 16 had breast and ovarian cancer and 2 had breast and pancreatic cancer. A total of 167 (22.9%) patients had 170 mutations. Mutation frequency in breast, ovarian and pancreatic cancer was 22.3%, 33.5% and 17.2%, respectively. The mutation rate was significantly higher in patients with double cancers than those with a single cancer (p<0.001). BRCA1 and BRCA2 were the most dominant genes associated with hereditary breast and ovarian cancer, whereas ATM was the most prevalent gene related to hereditary pancreatic cancer. Genes of hereditary colon cancer such as lynch syndrome were presented in a part of patients with pancreatic or ovarian cancer but seldom in those with breast cancer. Families with a history of both ovarian and breast cancer were associated with a higher mutation rate than those with other histories. Conclusion The mutation spectrum varies across the three cancer types and family histories. Our analysis provides guidance for physicians, counsellors, and counselees on the offer and uptake of genetic counseling.

Objective: We aimed to investigate whether 2-[ 18F]fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (2-[ 18F]FDG PET/CT) can aid in evaluating the risk of malignancy in ampullary tumors detected by endoscopy. Materials and Methods: This single-center retrospective cohort study analyzed 155 patients (79 male, 76 female; mean age, 65.7 ± 12.7 years) receiving 2-[ 18F]FDG PET/CT for endoscopy-detected ampullary tumors 5–87 days (median, 7 days) after the diagnostic endoscopy between June 2007 and December 2020. The final diagnosis was made based on histopathological findings. The PET imaging parameters were compared with clinical data and endoscopic features. A model to predict the risk of malignancy, based on PET, endoscopy, and clinical findings, was generated and validated using multivariable logistic regression analysis and an additional bootstrapping method. The final model was compared with standard endoscopy for the diagnosis of ampullary cancer using the DeLong test. Results: The mean tumor size was 17.1 ± 7.7 mm. Sixty-four (41.3%) tumors were benign, and 91 (58.7%) were malignant. Univariable analysis found that ampullary neoplasms with a blood-pool corrected peak standardized uptake value in earlyphase scan (SUVe) ≥ 1.7 were more likely to be malignant (odds ratio [OR], 16.06; 95% confidence interval [CI], 7.13–36.18;P < 0.001). Multivariable analysis identified the presence of jaundice (adjusted OR [aOR], 4.89; 95% CI, 1.80–13.33; P = 0.002), malignant traits in endoscopy (aOR, 6.80; 95% CI, 2.41–19.20; P < 0.001), SUVe ≥ 1.7 in PET (aOR, 5.43; 95% CI, 2.00–14.72; P < 0.001), and PET-detected nodal disease (aOR, 5.03; 95% CI, 1.16–21.86; P = 0.041) as independent predictors of malignancy. The model combining these four factors predicted ampullary cancers better than endoscopic diagnosis alone (area under the curve [AUC] and 95% CI: 0.925 [0.874–0.956] vs. 0.815 [0.732–0.873], P < 0.001). The model demonstrated an AUC of 0.921 (95% CI, 0.816–0.967) in candidates for endoscopic papillectomy. Conclusion Adding 2-[ 18F]FDG PET/CT to endoscopy can improve the diagnosis of ampullary cancer and may help refine therapeutic decision-making, particularly when contemplating endoscopic papillectomy.

Animals ; Humans ; Immunity, Innate ; immunology ; Influenza, Human ; immunology ; Interferons ; immunology ; Orthomyxoviridae Infections ; immunology ; virology

OBJECTIVE: To investigate the efficacy of frankincense and myrrha in the treatment of acute interstitial cystitis/painful bladder syndrome (IC/PBS). METHODS: The effects of frankincense and myrrha on the proliferation and migration of primary human urothelial cells (HUCs) were assessed in vitro. In the animal study, 48 virgin female rats were randomized into 4 groups (12 in each group): (1) control group (saline-injected control); (2) cyclophosphamide (CYP) group (intraperitoneal injected 150 mg/kg CYP); (3) CYP + pentosan polysulfate sodium group (orally received 50 mg/kg pentosan polysulfate sodium); and (4) CYP + frankincense and myrrha group [orally received frankincense (200 mg/kg) and myrrha (200 mg/kg)]. Rats orally received pentosan polysulfate sodium or frankincense and myrrha on day 1, 2, and 3. The experiments were performed on day 4. Pain and cystometry assessment behavior test were performed. Voiding interval values were assessed in rats under anesthesia. Finally, immunohistochemistry and Western blot were used to confirm the location and level, respectively, of cell junction-associated protein zonula occludens-2 (ZO-2) expression. RESULTS: Low dose frankincense and myrrha increased cell proliferation and migration in HUCs compared with control (P<0.05). Rats with acute IC/PBS rats exhibited lower voiding interval values, pain tolerance, and ZO-2 expression (P<0.05). Voiding interval values and pain tolerance were higher in the frankincense and myrrha group than CYP group (P<0.05). ZO-2 expression in the bladder was increased in the CYP + pentosan polysulfate and frankincense + myrrha groups compared with the CYP-induced acute IC/PBS group (P<0.05). CONCLUSION frankincense and myrrha modulate urothelial wound healing, which ameliorates typical features of acute IC/PBS in rats.