Objective: To analyse the genomic characteristics of Novirus(NoV) GII.4 genotype JN010 strain isolated form Jinan in 2017. Methods: Seven pairs of primers were designed and used to amplify the JN010 genome. Sequence analyses, alignment and phylogenetic trees of ORF1 and ORF2 genes were performed using the software Lasergene7.1 and MEGA5.2. At the same time the major protein VP1 amino acid mutations were analyzed. Results: The 7 516 bp complete genome sequence of JN010 strain was obtained, the most mutation sites were located in P2 subdomain of VP1. Two substitutions I293N and H373N of VP1 were locate neighboring epitope A, and R297H mutation happened within epitope A and the site A that binding with histo-blood group antigens(HBGAs). The JN010 strain was GII.Pe/GII.4 genotype and genetically closest to the strains found in Osaka of Japan(GenBank accession number LC066046) and the strains in Zhongshan city of Guangdong (GenBank accession number KY407064) respectively according to ORF1 and ORF2 gene homologous and phylogenetic analysis. Conclusions The NoV GII.4 variant strain JN010 has occurred mutations in the key site of the epitope A and site A that bind with HBGAs, and maybe affect its antigenicity and interaction with HBGAs.

Objective:To evaluate the role of hsa_circ_0081596 in oxygen-glucose deprivation and restoration (OGD/R) injury to human neurons. Methods:SK-N-SH cells were cultured and the cells within 5 generations were divided into 4 groups ( n=20 each) using a random number table method: control group (group C), OGD/R group (group O), OGD/R+ siRNA group (group S) and OGD/R+ siRNA negative control group (group I). The cells in C group were cultured under normal conditions of 37 ℃ and 5% CO 2.The cells in group O were placed in 6- or 96-well plates until they were completely attached to the wall, and then subjected to oxygen-glucose deprivation for 4 h, followed by restoration of oxygen-glucose for 24 h. In group S and group I, the cells were transfected with hsa_circ_0081596 siRNA and its negative control, respectively, and 72 h later OGD/R model was established.The expression of hsa_circ_0081596 and mitochondrial fission protein 1 (Fis1) mRNA was detected using quantitative real-time polymerase chain reaction.The expression of Fis1 was determined by Western blot, the cell survival rate was determined by CCK-8 assay and the apoptosis rate was determined by flow cytometry. Results:Compared with group C, the expression of hsa_circ_0081596, Fis1 and its mRNA was significantly up-regulated, the cell survival rate was decreased, and the apoptosis rate was increased in group O ( P<0.05). Compared with group O, the expression of hsa_circ_0081596 and Fis1 was significantly down-regulated, the cell survival rate was increased and the apoptosis rate was decreased in group S, and the expression of hsa_circ_0081596 and Fis1 was significantly up-regulated, the cell survival rate was decreased and the apoptosis rate was increased in group I ( P>0.05). Conclusion:hsa_circ_0081596 is involved in the pathophysiological mechanism of OGD/R through up-regulating the expression of Fis1 in human neurons.

Objective:To elucidate the changes of cytokine expression profiles in hand, foot and mouth disease (HFMD) patients infected with non-EV-A71 enteroviruses in Jinan city, and explore the characteristics of cytokines expression.Methods:The serum samples of acute and convalescent phases were collected from non-EV-A71 enterovirus-infected HFMD patients in Jinan from 2014 to 2017. The serum samples of healthy subjects were collected as control group. The Bio-plex liquid chip platform was used for high-throughput detection of 27 cytokines. GraphPad Prism and SPSS 22.0 were used for description and statistical analysis.Results:Twenty-two serum cytokines significantly changed in non-EV-A71 infected patients, including 11 kinds of interleukin (IL), 5 kinds of chemokines, 2 kinds of growth factors, granulocyte colony stimulating factor (G-CSF), interferon-γ, tumor necrosis factor-α (TNF-α) and granulocyte-macrophage colony stimulating factor (GM-CSF). There were 21 kinds (mean ranks 17.06-19.00 pg/ml vs 5.50-8.80 pg/ml, P < 0.05) and 20 kinds (mean ranks 16.41-19.00 pg/ml vs 5.50-9.90 pg/ml, P < 0.05) of cytokines expression in acute stage and convalescent stage respectively were higher than those in healthy control group for coxsackievirus A16 (CV-A16) infected patients, and GM-CSF expression (mean ranks 9.65 pg/ml vs 21.40 pg/ml, 9.59 pg/ml vs 21.50 pg/ml, P < 0.05) were both lower than those in healthy control group. For HFMD patients infected CV-A6, there were 19 kinds (mean ranks 11.92-13.50 pg/ml vs 5.50-6.45 pg/ml, P < 0.05) and 21 kinds (mean ranks 12.00-13.50 pg/ml vs 5.50-6.40pg/ml, P < 0.05) of cytokines expression with acute and convalescent stage respectively were higher than those in healthy control group. GM-CSF expression decreased only in acute phase (mean ranks 5.00 pg/ml vs 10.60 pg/ml, P < 0.05) compared with healthy control group. Double serum analysis showed that interleukin 6 (22.79pg/ml vs 35.88 pg/ml) and interferon-induced protein 10 (IFNγ -induced protein 10) (793.56 pg/ml vs 2 157.32 pg/ml) expression in patients with CV-A16 infection during convalescent stage were lower than that in acute stage; IL-7 (3.13 pg/ml vs 1.165 pg/ml), IL-15 (27.84 pg/ml vs 16.005 pg/ml) and regulated upon activation normal T cell expressed and secreted (RANTES) (22 605.96 pg/ml vs 7 040.90 pg/ml) expression in patients with CV-A6 infection during convalescent stage increased compared with the acute stage. Conclusions:There are extensive changes in cytokine expression profile in HFMD patients with non-EV-A71 enterovirus infection. Different pathogens infection and different clinical course of HFMD have different characteristics of cytokine expression. These findings could provide scientific data for finding indicators that are meaningful for disease progression, clinical diagnosis and immunotherapy.

Objective:To evaluate the role of has_circ_0008039 and miR-484 in oxygen-glucose deprivation/reoxygenation (OGD/R) injury in SK-N-SH cells and the relationship with Fis1.Methods:SK-N-SH cells were cultured in vitro to logarithmic growth stage and divided into 5 groups ( n=25 each) according to the random number table method: control group (group C), OGD/R group, has_circ_0008039 siRNA group (group S), hsa_circ_0008039 over-expression group (group E) and has_circ_0008039 siRNA plus miR-484 inhibitor group (group S+ I). Cells were cultured in normal condition in group C. In S, E and S+ I groups, after the cells were transfected with hsa_circ_0008039 siRNA, has_circ_0008039 over-expression vector, hsa_circ_0008039 siRNA and miR-484 inhibitor, the cells were subjected to oxygen-glucose deprivation for 12 h followed by 24 h restoration of O 2-glucose supply to develop the OGD/R model.At 24 h of restoration of O 2-glucose supply, the cell viability and amount of lactic dehydrogenase (LDH) released were measured using CCK-8 assay, the expression of hsa_circ_0008039, miR-484 and Fis1 mRNA was detected using real-time polymerase chain reaction, and the expression of Fis1 was detected by Western blot.A dual-fluorescein experimental report was used to verify the targeting relationship between hsa_circ_0008039 and miR-484. Results:Compared with group C, the cell viability was significantly decreased, and the amount of LDH released was increased in the other 4 groups, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in OGD/R and E groups, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.05). Compared with group OGD/R, the cell viability was significantly decreased, and the amount of LDH released was increased in E and S+ I groups, the cell viability was significantly increased, and the amount of LDH released was decreased in group S, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in group E, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and the expression of miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.05). Compared with group S, the cell viability was significantly decreased, the amount of LDH released was increased, the expression of miR-484 was down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.01). The dual-fluorescein experimental report verified that miR-484 was the target of hsa_circ_0008039 which binded to miR-484 specifically. Conclusions:has_circ_0008039 is involved in OGD/R injury in SK-N-SH cells by targetedly binding to miR-484, which is associated with up-regulation of Fis1 expression.

Objective To investigate the effect of cone beam CT with DynaCT on liver cancer patients undergoing transcatheter arterial chemoembolization (TACE) and its influence on the prognosis of patients. Methods A total of 73 patients with primary liver cancer who attended The Second Affiliated Hospital of North Sichuan Medical College from May 2017 to May 2019 were enrolled and randomly divided into observation group with 38 patients and control group with 35 patients. The patients in the control group underwent TACE under 2D-DSA angiography, and those in the observation group underwent DynaCT angiography after 2D-DSA angiography. The two groups were compared in terms of time of operation, X-ray exposure, amount of contrast agent used, intrahepatic tumor lesions detected and blood supplying arteries displayed by 2D-DSA angiography versus DynaCT angiography, and lipiodol deposition in tumor lesions evaluated by postoperative two-dimensional X-ray fluoroscopy versus plain DynaCT scan. The two-independent-samples t test was used for comparison between two groups, and the chi-square test was used for comparison of categorical data between two groups; the Kaplan-Meier survival curve was plotted for survival analysis, and the log-rank test was used for comparison between two groups. Results There were no significant differences between the two groups in time of operation, X-ray exposure, and amount of contrast agent used (all P >0.05). For the observation group, a total of 93 tumor lesions were detected, among which 79 (84.95%) were positive for blood supplying arteries, while in the control group, a total of 61 tumor lesions were detected, among which 34 (55.74%) were positive for blood supplying arteries, suggesting that the proportion of lesions positive for blood supplying arteries in the observation group was significantly higher than that in the control group ( χ 2 =16.088, P < 0.05). After surgery, 113 lesions of the two groups were analyzed for lipiodol deposition; two-dimensional X-ray fluoroscopy showed that lipiodol was evenly deposited in 89 lesions and was partially or completely missing in 24 lesions, while plain DynaCT scan showed that lipiodol was evenly deposited in 78 lesions and was partially or completely missing in 35 lesions. The observation group had significantly better overall survival than the control group ( χ 2 =4.347, P =0.037). Conclusion DynaCT can increase the detection rate of ischemic lesions and overlapping lesions in the liver without increasing the amount of intraoperative X-ray exposure and contrast agent used, thereby improving the accuracy of intubation and reducing the patient's vascular injury, and at the same time, it can be used to evaluate the deposition of lipiodol after embolization. It has an important application value in TACE for liver cancer and can help to improve the survival of patients after surgery.

Objective:To evaluate the relationship between hippocampal miR-3065-5p and insulin-like growth factor-1/phosphatidylinositol 3-kinase/protein kinase B(IGF-1/PI3K/Akt)signaling pathway in a mouse model of perioperative neurocognitive disorder (PND).Methods:Eighty clean-grade healthy male C75BL/6 mice, aged 12-14 weeks, weighing 20-30 g, were divided them into 4 groups ( n=20 each) using the random number table method: control group (C group), PND group, miR-3065-5p agonist group (Ag group) and miR-3065-5p agonist negative control group (Ag-NC group). PND model was prepared by internal fixation of tibial fracture under anesthesia with 1.5% isoflurane. Two days before developing the model, miR-3065-5p agomir 2 μl was injected into the lateral ventricle in Ag group, miR-3065-5p agomir negative control 2 μl was injected into the lateral ventricle in Ag-NC group. Morris water maze test and open field test were performed at 7 days after surgery. The mice were sacrificed after the end of test, and hippocampal tissues were obtained for determination of the expression of miR-3065-5p, IGF-1 mRNA and Bcl-2 mRNA (by quantitative real-time polymerase chain reaction) and expression of IGF-1, phosphorylated Akt (p-Akt), phosphorylated glycogen synthase kinase-3β (p-GSK3β) and Bcl-2 (by Western blot). Results:There was no significant difference in each parameter in the open field test among the four groups ( P>0.05). Compared with group C, the postoperative escape latency was significantly prolonged, the percentage of time of stay at the target quadrant was decreased, the number of crossing the original platform was reduced, the expression of miR-3065-5p was up-regulated, and the expression of IGF-1 mRNA, Bcl-2 mRNA, IGF-1, p-Akt, p-GSK3β and Bcl-2 was down-regulated in the other three groups ( P<0.05). Compared with PND group and Ag-NC group, the postoperative escape latency was significantly prolonged, the percentage of time of stay at the target quadrant was decreased, the number of crossing the original platform was reduced, the expression of miR-3065-5p was up-regulated, and the expression of IGF-1 mRNA, Bcl-2 mRNA, IGF-1, p-Akt, p-GSK3β and Bcl-2 was down-regulated in Ag group ( P<0.05). Conclusions:Up-regulation of miR-3065-5p can inhibit the activation of IGF-1/PI3K/Akt signaling pathway, which might be one of the mechanisms of PND developed in mice.

Objective:To investigate the effects of mild hypothermia on microglia polarization and janus kinase 2/signal transduction and transcriptional activation factor 3 (JAK2/STAT3) signaling pathway during cerebral ischemia-reperfusion (I/R) in rats.Methods:Forty-five clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 260-280 g, were divided into 3 groups ( n=15 each) by the random number table method: sham operation group (S group), cerebral I/R group (I/R) and mild hypothermia group (H group). In I/R group and H group, cerebral I/R was induced by middle cerebral artery occlusion using a nylon thread in anesthetized animals, the nylon thread was removed to restore the perfusion after 2 h of occlusion, and the rectal temperature was maintained at 36-37 ℃ during the period. Group H was wiped with 75% alcohol for 3 h starting from the time point immediately after reperfusion, and the rectal temperature was maintained at 32-33℃. Modified neurological severity score (mNSS) was evaluated at 24 h of reperfusion. Animals were then sacrificed for determination of the cerebral infarct size (using TTC staining), expression of M1 marker inducible nitric oxide synthase (iNOS), M2 marker arginase 1(Arg-1), phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)(by Western blot), expression of iNOS mRNA and Arg-1 mRNA (by quantitative polymerase chain reaction), and contents of interleukin-6 (IL-6) and IL-10 (by enzyme-linked immunosorbent assay). Results:Compared with group S, mNSS and cerebral infarct size were significantly increased, the expression of iNOS, Arg-1 protein and mRNA in cerebral ischemic penumbral zone was up-regulated, and the p-JAK2/JAK2 ratio, p-STAT3/STAT3 ratio, and contents of IL-6 and IL-10 were increased in the other two groups ( P<0.05). Compared with I/R group, mNSS and cerebral infarct size were significantly decreased, the expression of iNOS protein and mRNA in cerebral ischemic penumbral zone was down-regulated, the expression of Arg-1 and mRNA was up-regulated, and the p-JAK2/JAK2 ratio, p-STAT3/STAT3 ratio and IL-6 content were decreased, and the IL-10 content was increased in group H ( P<0.05). Conclusions:Mild hypothermia can promote the polarization shift of microglia from M1 to M2 phenotype during cerebral I/R and inhibit the central inflammatory responses, and the mechanism may be related to inhibition of JAK2/STAT3 signaling pathway in rats.

Objective:To evaluate the role of miR-124-3p in reduction of oxygen-glucose deprivation and restoration (OGD/R) injury by electrostimulation preconditioning in microglia and its relationship with microglial polarization.Methods:The well-growing BV2 cells were divided into 4 groups ( n=30 each) by the random number table method: control group (group C), OGD/R group, electrostimulation preconditioning group (group E) and miR-124-3p inhibitor group (group I). Group C was cultured under normal conditions, and group OGD/R was deprived of oxygen and glucose for 2 h followed by restoration of oxygen and glucose supply for 24 h to develop the OGD/R injury model. In group E, cells were stimulated with 100 mV/mm direct current for 4 h before oxygen-glucose deprivation, and the other treatments were similar to those previously described in group OGD/R. Group I was transfected with micrOFF? mmu-miR-124-3p inhibitor at 48 h before oxygen-glucose deprivation, and the other treatments were similar to those previously described in group E. The cell survival rate was determined by CCK-8 assay, the concentrations of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and IL-10 in the cell supernatant were measured by enzyme-linked immunosorbent assay. The expression of a surface marker of M1 microglia inducible nitric oxide synthase (iNOS) and a surface marker of M2 microglia arginase 1 (Arg-1) was detected by immunofluorescence and Western blot, respectively. The expression of iNOS and Arg-1 mRNA and miR-124-3p was detected by quantitative polymerase chain reaction. Results:Compared with group C, the cell survival rate was significantly decreased, the concentrations of TNF-α, IL-1β and IL-10 in the supernatant were increased, and the expression of iNOS and Arg-1 protein and mRNA and miR-124-3p was up-regulated in the remaining three groups ( P<0.05). Compared with group OGD/R, the cell survival rate was significantly increased, the concentrations of TNF-α and IL-1β in the supernatant were decreased, the IL-10 concentration was increased, the expression of iNOS protein and mRNA was down-regulated, and the expression of Arg-1 protein and mRNA and miR-124-3p was up-regulated in E and I groups ( P<0.05). Compared with group E, the cell survival rate was significantly decreased, the concentrations of TNF-α and IL-1β in the supernatant were increased, the IL-10 concentration was decreased, the expression of iNOS protein and mRNA was up-regulated, and the expression of Arg-1 protein and mRNA and miR-124-3p was down-regulated in group I ( P<0.05). Conclusions:The mechanism by which electrostimulation preconditioning reduces OGD/R injury in microglia is related to up-regulation of the expression of miR-124-3p, promotion of M2 microglia polarization, inhibition of M1 microglia polarization, and thus inhibiting the inflammatory responses.

Objective:To evaluate the role of glutathione S-transferase μ1 (GSTM1) expression in mild hypothermia-induced mitigation of cerebral ischemia-reperfusion (I/R) injury and the relationship with microglial polarization.Methods:Eighty clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 260-280 g, were divided into 4 groups ( n=20 each) using a random number table method: sham operation group (S group), cerebral I/R group (I/R group), mild hypothermia group (H group), and GSTM1 inhibitor + mild hypothermia group (IH group). The rat model of cerebral I/R injury was prepared using the filament occlusion method. The filament was removed to restore blood flow after the left middle cerebral artery was blocked for 2 h, and the rats′ brain and rectal temperature were maintained at 36-37 ℃ during the period. The vessels were only isolated and ligated without occlusion in S group. In H group, the entire body was wiped with 75% ethanol immediately after removing the filament, and the brain and rectal temperatures were maintained at 32-33 ℃ for 3 h, and the other procedures were the same as those previously described in I/R group. In IH group, GSTM1 inhibitor itaconic acid 8.6 mg/kg was intraperitoneally injected at 24 and 1 h before developing the model, and the other procedures were the same as those previously described in H group. Neurological deficits were evaluated using a modified neurological severity score (mNSS) at 24 h of reperfusion, and then the animals were sacrificed and the brains were removed for observation of cerebral infarction (by TTC staining) and for determination of the expression of GSTM1, M1-type microglial marker inducible nitric oxide synthase (iNOS), and M2-type microglial marker arginase-1 (Arg-1) (by Western blot), expression of GSTM1, iNOS and Arg-1 mRNA (quantitative real-time polymerase chain reaction) and contents of interleukin-6 (IL-6), IL-10, tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) (by enzyme-linked immunosorbent assay). Results:Compared with S group, the mNSS and percentage of cerebral infarct size were significantly increased, and the expression of iNOS and Arg-1 protein and mRNA was up-regulated, the expression of GSTM1 and mRNA was down-regulated, and the contents of IL-6, TNF-α, IL-10 and TGF-β were increased in the other three groups ( P<0.05). Compared with I/R group and IH group, the mNSS and percentage of cerebral infarct size were significantly decreased, and the expression of iNOS protein and mRNA was down-regulated, the expression of Arg-1 protein and mRNA and GSTM1 was up-regulated, the contents of TNF-α and IL-6 were decreased, and the contents of TGF-β and IL-10 were increased in H group ( P<0.05). Conclusions:Up-regulated expression of GSTM1 is involved in mild hypothermia-induced mitigation of cerebral I/R injury, which is associated with inhibition of microglial polarization toward the M1 phenotype and promotion of polarization toward the M2 phenotype.

Objective: To analyze the VP1 gene characteristics of Coxsackievirus A16 (CV-A16) isolated in(hand, foot and mouth disease, HFMD) patients of Jinan 2017. Methods: The samples collected from patients with HFMD in Jinan city in 2017 were analyzed and some of the CV-A16 nucleic acid positive samples were choosen with simple random sampling method and pretreated to inoculate RD cells. The whole VP1 gene sequences of CV-A16 stains which had obvious cytopathic effect in RD cells were amplified and sequenced. MEGA5.2 software was used to constructed phylogenetic tree and Lasergene software was used to analysis the homology consistency of nucleotide and amino acid of VP1 gene. Results: All the 10 CV-A16 strains isolated from Jinan were located 3 clusters belonged to B1b genotypes, with a nucleotide similarity of 94.9%~100% and deduced amino acid similarity of 99.3%~100%. The Jinan CV-A16 strains were nearest related with Shanghai strains KX871333(nucleotide similarity: 99.7%), Henan strains KM260134(99.0%), Jiangsu strains KP751580、KR138327(98.7%) and Guangdong strains MH004016(98.5%). Compared with some reference strains from our country, amino acid mutation were identified at positions 14, 59, and 145 in VP1 proteins of Jinan CV-A16 strains. Conclusions CV-A16 strains B1b genotypes were the strains prevailed in Jinan city, and 3 amino acid substitutions in VP1 proteins were happened in CV-A16 strains isolated from Jinan.