Cell culture plays an important role in virology. It provides a platform for the detection and isolation of viruses as well as for the biochemistry and molecular biology based studies of viruses. In the present work, a new system that could permits multiple (different) cell lines to be simultaneously cultured in one dish was developed. In the system, each cell line was cultured in an isolated zone in the same dish or well and the system is therefore called an isolated co-culture system. The usefulness of this novel approach for virus isolation was demonstrated using a model system based on adenovirus.

Objective To investigate the corelation between platelet activating factor acetylhydrolase (PAF-AH) genetic polymorphism and ischemic stroke. Methods The plasma PAF-AH genotype was determined in 205 patients with iachemic stroke and 114 normal subjects by the polymerase chain reaction. The levels of plasma platelet activating factor (PAF), platelet α-granule membrane glycoprotein-140(GMP-140), β-thromboglobulin (β-TG) and the levels of platelet factor 4 (PF4) were analyzed. Results The prevalence of the mutation genotype and plasma PAF, GMP-140, β-TG and PF4 in the patients with isehemic stroke [42.44%,(91.08 ± 39.10) ng/L, (36.46 ± 13.10) μg/L, (41.75 ± 11.18) μg/L, (29.05 ± 9.16) g/L, respectively] were significantly higher than those in the controls[21.05%,(64.30 ± 18.81) ng/L, (18.27 ± 7.68) μg/L, (30.94 ± 8.47) μg/L, (18.75 ± 6.06) μg/L](P< 0.01). The levels of plasma PAF, GMP-140 were significantly higher in mutation genotype patients than those in the normal genotype patients (P < 0.01). Conclusions The activation function of platelet in the acute phase of patients with ischemic stroke increases, and it is associated with genetic polymorphism of PAF-AH. The PAF-AH gene mutation may be a novel genetic marker for high risk of ischemic stroke.

Objective To investigate the effect of electroacupuncture at points Xiaohai and Xiajuxu on the expressions of tumour necrosis factor-α (TNF-α) and choline acetyltransferase (ChAT) in serum and nicotinic acetylcholine receptorα 7 (α 7 nAchR) in duodenal tissues in a rat model of duodenal ulcer (DU) and preliminarily explore the relative specificity of He-Sea point in “treating visceral diseases with He-Sea point”.Methods Forty healthy SD rats were randomized into blank (A), model (B), Xiaohai (C) and Xiajuxu (D) groups, 10 rats each. A rat model of DU was made by subcutaneous injection of 10% cysteamine hydrochloride at the right buttock. After successful model making, group C was given electroacupuncture at point Xiaohai and group D, at point Xiajuxu. Duodenal tissue ulcer was macroscopically observed and scored in every group of rats. Rat serum expression of TNF-α was determined by double antibody sandwich enzyme-linked immunosorbent assay (ELISA); rat serum expression of ChAT, by ultraviolet spectrophotometry & colorimetry; rat duodenal expression ofα7 nAchR, by Western blot.Results After model making, the duodenal ulcer score was significantly higher in groups B, C and D than in group A (allP<0.01) and significantly lower in groups C and D than in group B (bothP<0.01) and in group D than in group C (P<0.01). TNF-α expression was significantly higher in group B than in group A (P<0.01) and significantly lower in groups C and D than in group B (bothP<0.01) and in group D than in group C (P<0.01). ChAT expression tended to increase in group C compared with group B but there was no statistically significant difference (P>0.05) and was significantly higher in group D than in group B (P<0.01) or C (P<0.05).α7 nAchR expression was significantly higher in groups C and D than in group B (bothP<0.01). There was a positive correlation between ChAT andα7 nAchR expressions in every group (r=0.444,P=0.007).Conclusions Electroacupuncture at both points Xiaohai and Xiajuxu can reduce the duodenal ulcer score and serum TNF-α expression and increase serum ChAT and duodenalα7 nAchR expressions in DU rats. The results show that the therapeutic effect of electroacupuncture on duodenal ulcer may be produced by regulating TNF-α. Its mechanism may be activating cholinergic anti-inflammatory pathway to produce an anti-inflammatory effect. The effect being better in group D than in group C suggests that Xiajuxu has the relative specificity.

The cystine knot (CK) motif comprises an internal ring formed by two disulfide bonds and their connecting backbone segments which is threaded by a third disulfide bond .It is present in peptides and proteins of a variety of species ,in-cluding fungi ,plants ,marine molluscs ,insects and spiders .CK polypeptide is one of the ideal model molecules for drug design and molecular engineering research because of its stable structure and variety of bioactivities .Here we summarized the main structural features of both inhibitor cystine knot (ICK) peptide and cyclic cystine knot (CCK) peptide ,including primary se-quence ,topology ,permutation ,synthesis and folding characteristics ,as well as its applications on drug design and molecular engineering .

Objective To evaluate the effect of diammonium glycyrrhizinate extracted from the Chinese traditional medicine licorice root on the growth of human hair follicles cultured in vitro,and to detect the expression of wnt/β-catenin signaling pathway-related molecules.Methods Isolated hair follicles were cultured with diammonium glycyrrhizinate at different concentrations of 0.1,0.01,0.001 and 0.000 1 μmol/L for 10 days,and the hair follicles cultured in Williams' E medium without diammonium glycyrrhizinate served as a control group.The length of hair follicles was measured under a microscope every day,the morphologic changes of hair follicles were observed,and photos were taken.Immunofluorescence assay was performed to assess the proliferation of hair matrix cells,as well as to determine the expression of β-catenin,glycogen synthase kinase 3β (GSK3β),phosphorylated GSK3β (p-GSK3β) and lymphoid enhancer factor-1 (Lef1) in the Wnt/β-catenin signaling pathway.Statistical analysis was carried out by repeated-measures analysis of variance and one-way analysis of variance.Results As repeated-measures analysis of variance showed,only 0.01 μmol/L diammonium glycyrrhetate showed significantly promotive effect on the growth of hair follicles compared with the medium alone (P ＜ 0.05),and there were no significant differences in the length of hair follicles between the other concentration groups and the control group.Compared with the control group,the transition to the catagen phase of human hair cycle was delayed in the 0.01-μmol/L diammonium glycyrrhetate group,while it did not change in the other diammonium glycyrrhetate groups and control group.Immunofluorescence assay showed that the number of ki67-positive hair matrix cells was obviously increased in the 0.1-,0.01-,0.001-μmol/L diammonium glycyrrhizinate groups compared with the control group,while there was no difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group.One-way analysis of variance revealed that the expression of β-catenin,p-GSK3β and Lef1 significantly differed among all the groups (F =12.604,16.65,15.266 respectively,P ＜ 0.05),while no significant difference in the expression of GSK3β was found among these groups (F =1.472,P ＞ 0.05).Least significant difference (LSD)-t test revealed that the expre-ssion of β-catenin,p-GSK3β and Lef1 in the hair matrix cells was significantly higher in the 0.1-,0.01-,0.001-μmol/L diammonium glycyrrhizinate groups than in the control group (all P ＜ 0.05),but there was no significant difference between the 0.000 1-μmol/L diammonium glycyrrhizinate group and the control group (P ＞ 0.05).Conclusion Diammonium glycyrrhetate at the concentration of 0.01 μmol/L shows markedly promotive effect on the in vitro growth of hair follicles,and can increase the proliferative activity of hair matrix cells and delay the transition to the catagen phase,which may be associated with the activation of Wnt/β-catenin signaling pathway.

Objective To investigate the clinical characteristics and the experience of multi-disciplinary team (MDT) on recurrence of primary hyperoxaluria (PH) type I after renal transplantation. Methods One case presenting with unexplained rapid decline of renal allograft function after allogeneic renal transplantation was discussed by MDT. The role of MDT in diagnosing rare hereditary diseases and improving the long-term survival of renal transplant recipients was summarized. Results After MDT consultation, the patient was diagnosed with recurrence of PH type I. Routine immunosuppressive regimen was initiated after the exclusion of rejection. The patient was instructed to drink a large quantity of water, and given with high-quality protein and low-phosphorus diet, vitamin B6, calcium and other conservative therapies to actively prevent and treat postoperative complications. The deterioration of renal graft function was delayed. Nevertheless, regular hemodialysis was resumed at 5 months after renal transplantation until the submission date of this manuscript. Conclusions Recurrence of PH type I after renal transplantation is relatively rare. The main clinical manifestations are recurrent kidney stones and decreased renal function with multiple complications and poor prognosis. The condition of the patient is consulted by MDT for confirming the diagnosis, determining the optimal treatment scheme, delaying the progression and improving the clinical prognosis.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of chronic liver diseases worldwide and covers a series of pathological manifestations from hepatic steatosis to inflammation, fibrosis, and even liver cirrhosis. NAFLD is associated with a wide range of extrahepatic diseases, including metabolic syndrome, cardiovascular disease, chronic kidney disease, endocrine diseases, obstructive sleep apnea-hypopnea syndrome, psoriasis, and skeleton-muscle diseases. The major causes of death in patients with NAFLD include cardiovascular disease, malignancies, and liver-related complications, suggesting that extrahepatic diseases associated with NAFLD should be taken seriously by clinicians. This article reviews the research advances in extrahepatic diseases associated with NAFLD, so as to provide ideas for clinical assessment and treatment.

Objective:To summarize the clinical characteristics of central nerve system (CNS) infection and grasp the necessity and possibility of early diagnosis and precise intervention of CNS infection after renal transplantation.Methods:This retrospective study enrolled consecutive recipients of renal transplantation with CNS infection after transplant between January 2000 and December 2020. Correlative factors for CNS infection after renal transplant were determined by comparing the clinical data between recipients with and without CNS infection. After screening 3, 199 consecutive renal transplant recipients, 12 patients with CNS infection post-transplant were identified and recruited. The median age-of-onset was 48.5 (23-65) years. And the median time to disease onset after transplant was 50.5(1-204) months. The most common symptoms of CNS infection after renal transplant included fever (75.00%), consciousness disorder (58.33%), headache (58.33%) and neck rigidity (41.67%).Results:Hepatitis B virus carrier and pulmonary infection were correlated with CNS infection after transplantation ( P<0.05). Nine patients failed to identify the pathogen and only received empirical anti-infective regimen. The outcomes were curing ( n=3) and death ( n=6). Metagenomic sequencing was performed for identifying the pathogen in three recipients and actively adjusting the anti-infective regimen. As a result, 2 were cured and 1 died. The overall mortality was 58.33%. The median time to death or curing from disease onset were 20(2-19) and 25(16-35) days respectively in surviving and non-surviving recipients. Conclusions:The progress of CNS infection after transplantation is rapid with a high mortality. HBV carrier and pulmonary infection are possible risk factors of CNS infection after renal transplantation. Early pathogenic identification and precise etiological intervention are vital for better clinical outcomes.

Objective:To investigate the prognosis of robotic pancreatoduodenectomy after the learning curve and open pancreatoduodenectomy for pancreatic cancer.Methods:The propensity score matching and retrospective cohort study was conducted. The clinicopathological data of 396 patients who underwent curative pancreatoduodenectomy for pancreatic duct adenocar-cinoma in Ruijin Hospital of Shanghai Jiaotong University School of Medicine from January 2017 to December 2018 were collected. There were 244 males and 152 females, aged 64(range, 36?92)years. Of 396 patients, 86 cases undergoing robotic pancreatoduodenectomy were divided into robotic group, 310 cases undergoing open pancreatoduodenectomy were divided into open group. Observa-tion indicators: (1) propensity score matching and comparison of general data between the two groups after matching; (2) follow-up and survival analysis. Follow-up was conducted by telephone interview or outpatient examinations including tumor markers and abdominal imaging examina-tions to detect survival of patients up to March 2022. Overall survival was defined as the time from the surgery date to death or the last follow-up. Disease-free survival was defined as the time from the surgery date to tumor recurrence or the last follow-up. The propensity score matching was conducted by 1∶1 matching using the nearest neighbor method. Normality of measurement data was examined using the Shapiro-Wilk test. Measurement data with skewed distribution were described as M(range), and comparison between groups was analyzed using the Mann-Whitney rank-sum test. Count data were represented as absolute numbers, and comparison between groups was analyzed using the chi-square test. Kaplan-Meier method was used to calculate survival rates and draw survival curves, and Log-Rank test was used for survival analysis. An intent-to-treat analysis was performed in this study, patients who were converted to laparotomy from robotic surgery were still divided into the robotic group. Results:(1) Propensity score matching and comparison of general data between the two groups after matching: 164 of 396 patients had successful matching, including 82 cases in robotic group and open group, respectively. Before propensity score matching, the body mass index, cases in stage T1, T2, T3, T4, cases in N0, N1, N2 were 23.4(range, 21.4?25.3)kg/m 2,24, 41, 10, 11, 52, 27, 7 for the robotic group, versus 22.4(range,20.3?23.9)kg/m 2,57, 144, 22, 87, 131, 132, 47 for the open group, showing significant differences in the above indicators between the two groups ( Z=3.01, 2.63, 3.03, P<0.05). After propensity score matching, cases of males, age, body mass index, cases with American Society of Anesthesiologists (ASA) score as 1, 2, 3, CA19-9, cases with preoperative biliary drainage, cases with portal vein resection, cases with pancreatic resection margin <1 mm, cases in stage T1, T2, T3, T4, cases in stage N0, N1, N2, cases with nerve invasion, cases with tumor differentiation as high-medium differentiation, medium-low differentiation, low differentiation, cases with adjuvant chemotherapy were 51, 65(range, 59?69)years, 23.0(range, 21.0?25.2)kg/m 2, 32, 41, 9, 160.4(range, 46.7?377.2)U/mL, 21, 9, 8, 21, 40, 10, 11, 48, 27, 7, 76, 26, 47, 9, 53 for the robotic group, versus 58, 65(range, 58?69)years, 23.3(range, 21.4?25.3)kg/m 2, 35, 39, 8, 172.0(range, 69.7?402.9)U/mL, 26, 9, 10, 24, 40, 7, 11, 49, 28, 5, 76, 22, 49, 11, 57 for the open group, showing no significant difference in the above indicators between the two groups ( χ2=1.34, Z=0.18, 0.34, 0.49, 0.51, χ2=0.75, 0.00,0.25, Z=0.59, 0.27, χ2=0.00, Z=0.76, χ2=0.44, P>0.05). (2) Follow-up and survival analysis: after propensity score matching, 164 patients were followed up for 54(range, 1?67)months. The follow-up time of patients was 55(range, 51?59)months for the robotic group, versus 54(range, 50?58)months for the open group, respectively, showing no significant difference between the two groups ( Z=0.48, P>0.05). During the follow-up, the 1-year overall survival rate, 3-year overall survival rate, the median survival time, 1-year disease-free survival rate, 3-year disease-free survival rate, the median disease-free survival time, tumor recurrence rate, cases with recurrence pattern as local recurrence, liver recurrence, other distant recurrence, local and distant recurrence were 81.7%, 39.0%, 27 months(95% confidence interval as 19?33 months), 61.0%, 34.2%, 15 months(95% confidence interval as 12?18 months), 54.9%(45/82), 12, 16, 9, 8 for the robotic group. The above indicators were 79.3%, 36.0%, 24 months(95% confidence interval as 19?31 months), 59.8%, 27.5%, 15 months(95% confidence interval as 10?20 months), 58.5% (48/82), 10, 22, 6, 10 for the open group. There was no significant difference in overall survival or disease-free survival between the two groups ( χ2=0.39, 0.47, P>0.05). There was no significant difference in tumor recurrence rate or tumor recurrence site between the two groups either ( χ2=0.22, 1.86, P>0.05). Conclusion:After the learning curve, robotic pancreato-duodenectomy has non-inferior prognosis compared with open pancreatoduodenectomy.

Objective To explore the mechanism of motherwort in the treatment of nerve injury. Methods The active components of motherwort were obtained by searching TCMSP and BATMAN-TCM databases. The action targets of candidate compounds were collected and predicted from TCMSP and SwissTargetPrediction (STP) databases. The target genes corresponding to the active components of motherwort were obtained by using the standardized database of disease targets (Uniprot). The potential targets of motherwort in the treatment of nerve injury were obtained by mapping the disease genes of nerve injury with the three databases of Genecards, DisGenet and OMIM. The network topology analysis software Cytoscape 3.6.0 was used to construct the action target network of motherwort active components. The protein interaction platform database (STRING) was used to construct the interaction relationship between action targets. The target protein interaction (PPI) network was constructed by introducing Cytoscape 3.6.0 software. Through STRING database, GO enrichment analysis and KEGG pathway enrichment analysis were carried out to analyze the target points of motherwort in the treatment of nerve injury. Results 19 active components were screened from motherwort, involving 654 action targets, including 426 action targets related to nerve injury and 6 key targets. These target genes were mainly involved in biological regulation, oxidative stress response and cell communication and other biological processes. Molecular functions were mainly related to protein binding, ion binding and catalytic reduction. They were enriched outside the cell membrane. Its mechanism was related to signal pathways such as MAPK, Toll-like receptor, PI3K-Akt, TNF, IL-17, and apoptosis. Conclusion The active components of motherwort may play a protective role on nerve injury through anti-inflammation, anti-apoptosis and promoting cell growth.