Objective To clone and express immunodominant fragment of glycoprotein G of HSV-2 (FgG-2). Methods The target gene was amplified by polymerase chain reaction (PCR). The PCR products were ligated into directional TOPO expression vector. After identification, the recombinant expression vector was transferred into BL21 StarTM cell for expression. Finally, recombinant protein of FgG-2 (rFgG-2) was detected by Western Blot (WB). Results A 616 bp DNA fragment was obtained with PCR and then confirmed in recombinant vector by PCR and sequencing, bearing 99.5% consistent sequence with target gene. Highest recombinant protein production was obtained at the time point of 3 hours. Expression of target protein was confirmed by WB with anti-gG monoclonal antibody. Conclusions The immunodominant fragment of gG-2 has been successfully cloned and expressed in E.coli, which might be used for the development of serum diagnostics assay kits for HSV-2 infection.

Objective To study LI gene sequence of HPV cp6108 from 5 cases of condyloma acuminata. Methods T-A cloning and direct sequencing of PCR product were used. Results The LI gene sequences of HPV cp6108 from 5 specimens were presented with the homology of 99% to reference sequence in GenBank. A total of 3 gene mutations were found, including a nonsense mutation of G70A, a missense mutation of D77N, and a missense mutation of Tl16P. Conclusions In comparison with the sequence in GenBank, at least 3 gene mutations of HPV CP6108, i.e. one nonsense mutation of G70A and missense mutations of D77N and Tl 16P, are found in the present study.

Objective:To analyze the variation and evolution characteristics of hemagglutinin (HA) and neuraminidase (NA) genes of influenza B virus circulating in Jining from 2017 to 2020.Methods:Throat swab specimens were collected from patients with influenza-like symptoms in sentinel hospitals and influenza outbreaks in Jining from 2017 to 2020 and tested for influenza B virus nucleic acid. After virus isolation, 20 representative strains of influenza B virus were selected to sequence the full length of HA and NA genes. Phylogenetic trees were constructed and the molecular characteristics were analyzed using bioinformatics software. Results:A total of 4 575 specimens were collected and 842 of them were positive for influenza virus, including 398 (8.7%, 398/4 575) influenza B virus-positive specimens. The positive rate of influenza B virus was 47.27% (398/842). The isolated influenza B virus strains of Victoria (BV) and Yamagata (BY) lineages from 2017 to 2020 shared 98.7%-98.8% and 98.5%-99.1% homology in HA gene with vaccine strains, respectively. The BV lineage strains isolated from 2018 to 2020 belonged to Victoria clade 1A branch and the BY lineage strains isolated from 2017 to 2018 belonged to Yamagata clade 3 branch. Mutations were detected in several antigenic sites, but not in the sites related to NA inhibitor resistance. Conclusions:Mutations in several antigenic sites caused antigenic changes in influenza B virus of BV and BY lineages, which might be related to the outbreaks of influenza B virus infection in Jining during 2017 to 2020.

Objective:To investigate the effect of melatonin (MEL) influence on lipopolysaccharide (LPS)-induced long-term anxiety-like behavior and activation of astrocytes in septic neonatal rats.Methods:Sprague-Dawley rats were randomly(random number) assigned to the control group, LPS group and LPS+MEL group. Sepsis model was intraperitoneally injected with LPS (1 mg/kg), and neonatal rats in the MEL group were administered with MEL (10 mg/kg) 30 min after LPS injection. At different time points after injection, rats in each group were divided into three subgroups: 3 d, 7 d and 28 d. The expression of GFAP and TNF-α in the corpus callosum was detected by immunofluorescence staining and Western blot. Open-field test was applied to observe anxiety-like behaviors. In vitro, cultured neonatal SD rat astrocytes were divided into the control group, LPS group, LPS+MEL group, and LPS+MEL+luzindole group. Immunofluorescence staining was used to observe the expression of GFAP and TNF-α. Expression of GFAP, TNF-α, p-NF-κBp65, NF-κBp65 protein in astrocytes were assessed by Western blot. RT-qPCR was used to investigate the mRNA expression of GDNF and BDNF. One-way ANOVA and two-way ANOVA were used for comparison of multiple groups of variables. A P<0.05 was considered statistically significant. Results:LPS reduced the duration of movement in the central area and distance in the central area/total distance in open-field test, while melatonin evidently reversed the LPS-induced anxiety-like behavior. Compared with the LPS group, the expressions of GFAP and TNF-α were significantly decreased in the corpus callosum at 3 d and 7 d in the MEL group ( P< 0.05). Compared with the LPS group, MEL could significantly decrease the expression of GFAP, TNF-α and p-NF-κBp65 in astrocytes ( P< 0.05), which could be blocked by Luzindole. In addition, compared with the LPS group, MEL pretreatment could reverse the down regulation of GDNF and BDNF induced by LPS ( P<0.05). Conclusions:MEL can relieve LPS-induced long-term anxiety-like behavior in septic neonatal rats. The mechanism may be related to the inhibition of astrocyte activation and inflammatory reaction through NF - κ B pathway.

Objective To systematically investigate the molecular epidemiological profiles of human papillomavirus (HPV) in patients with condyloma acuminata(CA). Methods Two hundred and one samples of HPV DNA isolated from CA were PCR amplified by the PGMY09/11 primer system. The PCR products were simultaneously hybridized to 37 specific HPV probes immobilized on a nylon strip and then genotyped. All DNA templates were further PCR amplified using HPV 6 and 11 type specific primers for verification. Results All samples were HPV DNA positive consisting of totally 31 genotypes, the types of which were type 11(53.7%, 108/201), 6(43.8%, 88/201), 16(6.5%, 13/201), 52(6.0%, 12/201), 33(5.5%, 11/201), cp6108 (5.5%, 11/201) and 42 (5.0%, 10/201). The samples infected with a single and mixed types of HPV accounted for 60.2% (121/201) and 39.8% (80/201) respectively. Consistent results were found with the detection of HPV6 and 11 between hybridization assay and type-specific PCR. Conclusions At least 31 HPV genotypes are associated with CA. HPV 11 predominates while 68, 40, 54, 67, 73, 82, 35, 64 and 83 are rare in CA. Type cp6108 is detected in CA for the first time with a high prevalence. HPV26, 69, 70, 71,72 and IS39 might be not associated with CA. CA infected with a single and mixed HPV types accounts for 60.2% and 39.8%, respectively.

Objective To discuss the diagnosis and treatment of normotensive pheochromocyto-ma. Methods The clinical data of 22 patients with normotensive pheochromocytoma were reviewed. Inclusion criteria for normotensive pheochromocytoma were no previous history of hypertension and episode of symptoms suggesting high blood pressure. The blood pressure on admission was 90-130/ 60-90 mm Hg with an average of 113/72 mm Hg. Seven patients were found adrenal mass by routine ultrasonic examination. Twelve patients presented with superior abdominal or flank pain. Four pa-tients were present with fatigue, and 2 patients had fever. Headache and palpitation were found in 1 patient. Most of patients were present with large and round mass with low density area in the center of the tumor by uhrosonography and CT. Four patients had elevated level of plasma epinephrine and nor-epinephrine. 24 hours urine CA and VMA were elevated in 5 and 4 patients respectively. Seven pa-tients were prepared with infusion preoperatively to expand intravascular volume, and 2 patients were given prazosin 1.5 mg/d for 5 to 7 days. Results During the operation, seventeen patients had ele-vated blood pressure and 5 patients had no changed. One of seven patients with preoperative prepara-tion had obvious hypertension during operation, and 11 of 15 patients without preoperative preparation had obvious hypertension. The tumors were removed successfully in 21 patients. All the patients were diagnosed pheochromocytoma pathologically. Twenty-one patients had normal blood pressure with no recurrence during the follow-up from 1 month to 7 years. Conclusions The patients with normotensive pheochromocytomas may have lower catecholamine in their plasma and urine. The application of α-blockers and the expanding intravascular volume before operation could be important for the patients safe.

Objective To investigate the effect of interleukin-1β (IL-1β) on the expression of synaptic protein SNAP-25 in the hippocampus in septic neonatal rat induced by systemic lipopolysaceharide (LPS) injection.Methods Sprague-Dawley (SD) rats were randomly divided into two groups:control group and sepsis group.The rat model of sepsis was produced by intraperitoneal injection of 1 mg/kg LPS,and rats in the control group were injected with an equal volume of 0.01 mol/L phosphate buffered saline (PBS).The expression levels of IL-1β and IL-1R1 in the hippocampus at 1,2 and 3 d,and synaptosomal-associated protein 25 (SNAP-25) at 7,14 and 24 d after LPS intraperitoneal injection were detected by Western blot.After cultured for 24 h,primary hippocampal neurons were divided into four groups including the control group,IL-1β (40 ng/mL) treatment group,IL-1β (40 ng/mL) + IL-1Ra (40 ng/mL) treatment group,and IL-1Ra (40 ng/mL) treatment group.The effect of IL-1β on SNAP-25 expression in primary hippocampal neuron was determined by Western blot and real-time PCR.The purity of hippocampal neurons were identified by NeuN immunofluorescence staining and the activity of neurons were detected by CCK-8 assay.All data were analyzed by SPSS version 22.0.The data were analyzed by student-t test and Dunnett-t test.The interaction effects were analyzed by factorial ANOVA.Differences were considered to be statistically significant if P＜ 0.05.Results Compared with the control group,the expressions of IL-1β and IL-1R1 were significantly increased in the hippocampus at 1,2 and 3 d after intraperitoneal injection of LPS (P＜0.05).The expression of SNAP-25 protein was decreased at 7,14,and 28 d after intraperitoneal injection of LPS (P＜0.05).The purity of primary neurons was about up to 92％.The activity of primary neurons was not relatively changed after treated with IL-1β at a dose less than 40 ng/mL.The level of SNAP-25 protein was obviously decreased in primary neurons at 24 h after IL-1β treatment (P＜0.05).IL-1Ra treatment might reverse the effect of IL-1β on primary neurons (P＜0.05).While,the expression of SNAP-25 mRNA was not statistically different in each group (P＞0.05).Conclusions IL-1β may possibly inhibit the expression level of SNAP-25 protein in the hippocampus in the septic rats through its receptor IL-1R1,which would contribute to cognitive dysfunction of septic neonatal rats in later life.

Objective:To reevaluate the upper limit of normal (ULN) of serum alanine aminotransferase (ALT) by retrospectively analyzing the ALT levels in healthy people in Ningbo area.Methods:A total of 56 140 people who underwent health examination and detection of liver biochemical indexes in the Affiliated Hospital of Medical School of Ningbo University and Yinzhou Huamao Hospital of Ningbo from 2018 to 2020 were enrolled. After excluding relevant factors that may lead to liver injury, 11 411 people were included to compare the difference of serum ALT levels among different genders and age groups (20 to 29 years, 30 to 39 years, 40 to 49 years and 50 to 59 years) to determine the ALT ULN in different gender groups. Statistical methods were performed using two independent samples t test and analysis of variance. Results:The serum ALT of males was (19.20±7.90) U/L, which was higher than that of females ((13.75±6.17) U/L), with statistical significance ( t=41.16, P<0.001). The serum ALT ULN in males and in females were 35 U/L and 26 U/L, respectively. The serum ALT levels of 20 to 29, 30 to 39, 40 to 49 and 50 to 59 years old groups were (15.48±7.61) U/L, (16.21±7.40) U/L, (17.36±7.52) U/L and (18.77±7.57) U/L, respectively.The difference was statistically significant ( F=71.51, P<0.001). Serum ALT level in 50 to 59 years old group was higher than that in 20 to 29 years old group, and the difference was statistically significant ( t=13.11, P<0.01). In males, the ALT ULN of 20 to 29 years old was the lowest of 34.43 U/L, and highest of 35.29 U/L in 40 to 49 years old. In females, the ALT ULN in the 20 to 29 years old group was the lowest of 23.01 U/L, and the ALT ULN in the 50 to 59 years old group was the highest of 30.79 U/L. ALT ULN increased with age in females. The serum ALT of males was higher than that of females in all age groups ( t=29.55, 26.91, 13.43 and 4.62, respectively, all P<0.05). Conclusions:The serum ALT level is significantly correlated to gender and age. The serum ALT ULNs of healthy adult are 35 U/L in males and 26 U/L in females in Ningbo area.

Objective:To explore the genotype and clinical phenotype characteristics of patients with pancreatic agenesis caused by GATA6 gene mutations and to improve the clinical understanding of pancreatic agenesis.Methods:The clinical data of a newborn with pancreatic agenesis admitted to the Second Hospital of Shandong University were retrospectively analyzed. Relevant literature published until October 31, 2022, were retrieved from China National Knowledge Infrastructure, Wanfang Database, VIP Database, Chinese Medical Journal Full Text Database, PubMed, Embase and SCI Database with the terms of "pancreatic agenesis", "GATA6", "pancreatic agenesis/hypoplasia" and "GATA6 Translation Factor". The characteristics of gene variants and clinical manifestations of patients diagnosed with pancreatic agenesis caused by GATA6 gene mutation were retrieved and summarized.Results:This case was a full-term male infant who developed insulin dependent hyperglycemia and fatty diarrhea 2 d after birth, accompanied by intrauterine growth restriction, congenital heart disease, and cryptorchidism. Genetic testing showed a novel heterozygous mutation of GATA6 (c.1366C>T) which was consistent with the autosomal dominant inheritance pattern. The phenotype and genotype between the proband and his parents were consistent with the cosegregation. The ACMG mutation was rated as pathogenic variant. Intravenous infusion of insulin, subcutaneous injection of insulin, or long-acting insulin were not effective. After continuous subcutaneous pumping of aspartic insulin combined with oral pancreatic enzyme replacement therapy, the infant's condition was improved and discharged. Follow up to age of 15 months, the patient still relied on continuously subcutaneous pump to control blood glucose, pancreatic exocrine function was back to normal, and the development was generally normal. A total of 59 cases were reported in 22 articles, with the case from our hospital, there were 60 patients in total. Among them, 47 were probands and 13 were family members, about 61.7% (29/47) of which were de novo mutations. There were 39 variants, of which 28.2% (11/39) were missense mutations and 71.8% (28/39) were functional deletion variations. Mutations of GATA6 gene had a broad phenotype spectrum. The phenotypes mainly included neonatal diabetes mellitus ( n=39) and pancreatic exocrine insufficiency ( n=39). Other extra-pancreatic features included different types of congenital heart disease ( n=54), congenital biliary abnormalities ( n=23), intestinal developmental disorders ( n=16), neurocognitive disorders ( n=18) and endocrine abnormalities ( n=15). Conclusions:The heterozygous variations of GATA6 gene lead to pancreatic hypoplasia and a broad phenotype spectrum. The pancreatic phenotypes mainly include neonatal diabetes mellitus and pancreatic exocrine insufficiency, and extra-pancreatic phenotypes include congenital heart disease and other developmental abnormalities.

Objective:To investigate the value of pulse oxygen saturation (SpO 2) monitoring in predicting children with moderate-to-severe obstructive sleep apnea (OSA). Methods:It was a retrospective study involving 341 children with snoring during nighttime sleep who had visited the Children′s Hospital of Soochow University from June 2017 to November 2020 and monitored for polysomnography (PSG) and SpO 2.The SpO 2 parameters mainly included oxygen desaturation index (ODI), oxygen desaturation index ≥3% (ODI3), oxygen desaturation index ≥4% (ODI4), mean pulse blood oxygen saturation (MSpO 2), lowest pulse blood oxygen saturation (LSpO 2), cumulative time spent with blood oxygen saturation below 95%, 92% and 90%(T95, T92 and T90). According to obstructive sleep apnea hypopnea index (OAHI), patients were divided into the snoring and mild OSA group (OAHI≤5 times/h) and moderate-to-severe OSA group (OAHI>5 times/h). Differences in SpO 2 parameters were compared between groups using the Chi- square test and Mann- Whitney U test. Spearman correlation analysis was used to analyze the correlation between SpO 2 parameters and OAHI in all children.The SpO 2 parameters were included in the Logistic regression model.Receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficiency of SpO 2 parameters on moderate-to-severe OSA. Results:A total of 341 patients were recruited, including 206 male and 135 female patients with the mean age, body mass index (BMI) and OAHI of 6.0 (4.0, 7.5) years, 16.2 (15.1, 18.0) kg/m 2 and 0.6 (0.1, 3.0) times /h, respectively.There were 283(83.0%) and 58 (17.0%) patients in the snoring and mild OSA group and moderate-to-severe OSA group.The ODI3[0.7 (0.3, 1.4) times/h vs.7.7 (4.4, 12.8) times/h], ODI4[0.4 (0.1, 0.8) times/h vs.5.3 (2.7, 9.1) times/h], T95[1.4 (0.3, 5.3) min vs.13.7 (7.0, 33.5) min], T92[0.1 (0, 0.5) min vs.1.8 (0.9, 6.0) min] and T90[0 (0, 0.1) min vs.0.6 (0.2, 2.2) min] were significantly lower in the snoring and mild OSA group than those of moderate-to-severe group, while LSpO 2[91.0 (89.0, 93.0)% vs.86.5 (82.0, 88.0)%] and MSpO 2[ 97.0 (97.0, 98.0)% vs.96.0 (96.0, 97.0)%] were significantly higher(all P<0.001). All SpO 2 parameters were significantly correlated with OAHI (all P<0.001), and the correlation coefficient between ODI3 and OAHI was 0.660.ODI3 was an independent predictor of moderate-to-severe OSA ( OR=3.117, 95% CI: 1.635-5.945, P=0.001). The area under the ROC curve of ODI3 in predicting the moderate-to-severe OSA was 0.957, and the cut-off value of 3.45 times/h and specificity of 95.4%.MSpO 2 was an independent predictor of moderate-to-severe OSA ( OR=2.917, 95% CI: 1.589-5.354, P=0.001). Conclusions:ODI3 can be used to predict the moderate-to-severe OSA in children.