Objective:To explore the genetic basis of eighteen patients with tetrahydrobiopterin deficiency (BH4D) from Gansu Province.Methods:Eighteen patients diagnosed with BH4D at Gansu Provincial Maternal and Child Health Care Hospital from January 2018 to December 2021 were selected as the study subjects. Whole exome sequencing was carried out, and candidate variants were verified by Sanger sequencing.Results:All of the thirty-six alleles of the eighteen patients were successfully determined by molecular genetic testing. Sixteen patients were found to harbor variants of the PTS gene, and two had harbored variants of the QDPR gene. Ten variants were detected in the PTS gene, with the most common ones being c. 259C>T (34.38%) and c. 286G>A (15.63%). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c. 259C>T was classified as a pathogenic variant, whilst the c. 286G>A, c. 166G>A, c. 200C>T, c. 272A>G, c. 402A>C, c. 421G>T, c. 84-291A>G and c. 317C>T were classified as likely pathogenic variants. A novel c. 289_290insCTT variant was classified as likely pathogenic (PM1+ PM2_Supporting+ PM3+ PP3+ PP4). The two variants (c.478C>T and c. 665C>T) detected in the QDPR gene were both classified as variants of uncertain significance (PM1+ PM2_Supporting+ PP3+ PP4). Conclusion:Genetic testing has clarified the pathogenic variants in these BH4D patients, which has enabled timely and accurate clinical intervention and treatment, and provided a reference for genetic counseling and reproductive guidance for their families.

OBJECTIVE: To investigate the potential mechanisms that mediate the inhibitory effect of porcine recombinant NKlysin (prNK-lysin) against liver cancer cell metastasis. METHODS: HPLC-tandem mass spectrometry was used to identify the differentially expressed proteins in prNK-lysin-treated hepatocellular carcinoma SMMOL/LC-7721 cells in comparison with the control and PBS-treated cells. GO functional annotation and KEGG pathway analysis of the differentially expressed proteins were performed using GO and KEGG databases. RT-qPCR was used to determine the mRNA expression levels of polypeptide-N-acetylgalactosaminotransferase 13 (GALNT13), transmembrane protein 51 (TMEM51) and FKBP prolyl isomerase 3 (FKBP3) in the cells, and the protein expression of FKBP3 was verified using Western blotting. RESULTS: Proteomic analysis identified 1989 differentially expressed proteins in prNK-lysin-treated cells compared with the control cells, and 2753 compared with PBS-treated cells. Fifteen proteins were differentially expressed between PBS-treated and the control cells, and 1909 were differentially expressed in prNK- lysin group compared with both PBS and control groups. These differentially expressed proteins were involved mainly in the viral process, translational initiation and RNA binding and were enriched mainly in ribosome, protein process in endoplasmic reticulum, and RNA transport pathways. RT-qPCR showed that compared with the control group, prNK-lysin treatment significantly increased the mRNA expressions of GALNT13 (P < 0.05) and TMEM51 (P < 0.01) and lowered FKBP3 mRNA expression (P < 0.05). Western blotting also showed a significantly decreased expression of FKBP3 protein in prNK-lysin-treated cells (P < 0.001). CONCLUSION Treatment with prNK-lysin causes significant changes in protein expression profile of SMMOL/LC-7721 cells and inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 protein and affecting the cellular oxidative phosphorylation and glycolysis pathways.
Animals ; Swine ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Oxidative Phosphorylation ; Proteomics ; Glycolysis ; RNA, Messenger

Objective:To evaluate the effect of necrostatin-1 (Nec-1)pre-treatment on postoperative cognitive function in aged rats with chronic pain due to knee arthritis.Methods:One hundred and twenty healthy male Sprague-Dawley rats, aged 22 months, weighing 550-600 g, were divided into 4 groups ( n=30 each) using a random number table method: chronic pain due to knee arthritis group(group P), chronic pain due to knee arthritis + operation group (group PS), dimethyl sulfoxide (DMSO) + chronic pain due to knee arthritis + operation group (DMSO+ PS group), and necrostatin-1 + chronic pain due to knee arthritis + operation group (Nec-1+ PS group). The inflammation-induced knee arthritis model was developed by injecting monosodium iodoacetate (MIA) into the left joint cavity.The exploratory laparotomy under sevoflurane anesthesia was performed at 12 weeks after intra-articular MIA injection. In Nec-1+ PS group and DMSO+ PS group, necrosstatin-1 6.25 mg/kg and the equal dose of DMSO were intraperitoneally injected at 1 h before surgery, respectively. At 7 days after surgery, the Morris water maze test was used to evaluate the cognitive function, the activation of microglial cells in the dentate gyrus of hippocampus was observed by immunofluorescent staining, and the activation rate of microglia cells was calculated, the necrosis rate of neurons was determined by flow cytometry, the expression of receptor-interacting protein kinase 1 (RIPK1)and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) was determined by Western blot, and the contents of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 were determined by enzyme-linked immunosorbent assay. Results:Compared with P group, the escape latency was significantly prolonged, the time of staying at the original platform quadrant was shortened, and the number of crossing the original platform was reduced, the activation rate of microglia cells in the hippocampal dentate gyrus and necrosis rate of hippocampal neurons were increased, the expression of RIPK1 and p-MLKL was up-regulated, and the contents of pro-inflammatory factors TNF-α, IL-1β and IL-6 in hippocampus were increased in PS, DMSO+ PS and Nec-1+ PS groups ( P<0.05). Compared with PS group and DMSO+ PS group, the escape latency was significantly shortened, the time of staying at the original platform quadrant was prolonged, and the number of crossing the original platform was increased, the activation rate of microglia cells in the hippocampal dentate gyrus and necrosis rate of hippocampal neurons were decreased, the expression of RIPK1 and p-MLKL was down-regulated, and the contents of pro-inflammatory factors TNF-α, IL-1β and IL-6 in hippocampus were decreased in Nec-1+ PS group ( P<0.05). Conclusions:Necrostatin-1 pre-treatment can improve postoperative cognitive function in aged rats with chronic pain due to knee arthritis, and the mechanism may be related to inhibition of necrosis in hippocampal neurons and reduction of neuroinflammation.

Purpose/Significance Based on big data,a cardiovascular and cerebrovascular electronic medical record(EMR)analy-sis platform is developed.By utilizing imaging data analysis techniques and clinical document analysis techniques,the platform provides patients with precise diagnosis,treatment plans,scientific administration,prognosis prediction,smart health education prescriptions and other precise services.Method/Process The medical ontology,knowledge rules and knowledge graph for cardiovascular and cerebrovas-cular diseases are developed and constructed by using Protégé.On the basis of constructing a knowledge graph,a knowledge base for clinical diagnosis,treatment,pathological analysis and prognosis judgment of cardiovascular and cerebrovascular diseases is formed.A EMR analysis platform for cardiovascular and cerebrovascular diseases is designed based on the knowledge base.Result/Conclusion The designed cardiovascular and cerebrovascular EMR analysis platform is conducive to providing personalized diagnosis and treatment plans for different populations,and providing patients with various precise diagnosis and treatment services.

Objective:To investigate the application value of totally laparoscopic trans-abdominal-hiatal approach in the radical resection of Siewert type Ⅱ adenocarcinoma of esophago-gastric junction (AEG).Methods:The retrospective and descriptive study was conducted. The clinicopathological data of 45 patients with Siewert type Ⅱ AEG who were admitted to the Affiliated Hangzhou First People′s Hospital, Zhejiang University School of Medicine from May 2017 to December 2020 were collected. There were 28 males and 17 females, aged from 35 to 85 years, with a median age of 64 years. All patients underwent radical resection of AEG by totally laparoscopic trans-abdominal-hiatal approach with gastrointestinal anastomosis using proximal gastrectomy with double-tract anastomosis or total gastrectomy with esophagojejunointestinal anastomosis and digestive reconstruction using transdiaphragmatic-hiatal superior overlap esophagojejunostomy. Observation indicators: (1) surgical and postoperative situations; (2) postoperative histopathological examination; (3) follow-up and survival situations. Follow-up using outpatient examination and telephone interview was performed to detect survival of patients and tumor recurrence and metastasis up to March 2021. Measurement data with normal distribution were presented as Mean± SD and measure-ment data with skewed distribution were presented as M(range). Results:(1) Surgical and post-operative situations: all 45 patients underwent radical resection of AEG by totally laparoscopic transabdominal-hiatal approach successfully, including 35 cases undergoing total gastrectomy with esophagojejunointestinal anastomosis and 10 cases undergoing proximal gastrectomy with double-tract anastomosis. The total operation time, time of lower mediastinal lymph node dissection, time of superior overlap esophagojejunostomy, volume of intraoperative blood loss, time for initial out-of-bed activities, time to first flatus, time to initial liquid diet intake, time to drainage tube removal of the 45 patients were (235±32)minutes, (25±8)minutes, (45±10)minutes, (70±13)mL, (20±8)hours, (2.3±0.2)days, (2.6±0.8)days and (6.2±1.1)days, respectively. Eleven of 45 patients under-went postoperative complications and none of patient died during perioperative period. The post-operative duration of hospital stay of 45 patients was (10±3)days. (2) Postoperative histopatho-logical examination: all 45 patients had negative upper surgical margin. The length of proximal margin, tumor diameter, total number of lymph lodes harvested and number of lower mediastinal lymph nodes harvested were (2.5±0.5)cm, (2.9±0.8)cm, 35.0±4.0 and 2.4±0.8, respectively. Patholo-gical examination showed adenocarcinoma in all 45 patients with pTNM staging as 5 cases of stage ⅠB, 8 cases of stage ⅡA, 21 cases of stage ⅡB and 11 cases of stage ⅢA. (3) Follow-up and survival situations: 45 patients were followed up for 3 to 46 months, with a median follow-up time of 26 months. During follow-up, 8 of 45 patients died. Of the 37 patients survived, 3 cases underwent liver metastasis and 3 cases underwent bone metastasis, lung metastasis or peritoneal metastasis respec-tively.Conclusion:Total laparoscopic transabdominal-hiatal approach is safe and feasible in the treatment of Siewert type II AEG with a satisfactory clinical efficacy.

Objective:To explore high-frequency color Doppler ultrasound characteristics of extramammary Paget′s disease (EMPD) .Methods:From January 2015 to October 2019, 32 patients with pathologically confirmed EMPD were collected from the Affiliated Hospital of Jining Medical University, and characteristics of their high-frequency color Doppler ultrasound images were retrospectively analyzed.Results:Of the 32 lesions, 25 were located on the external genitalia (21 on the scrotum, 4 on the perineum) , 4 on the medial thigh, 1 on the lower abdominal wall, 1 in the perianal region, and 1 on the neck. Ipsilateral inguinal lymph node metastasis occurred in 3 patients, and bilateral inguinal lymph node metastasis in 1 patient. High-frequency ultrasonography showed no obvious abnormal ultrasound manifestations in 6 cases, and thickened skin lesions in 26 cases. According to the morphological and high-frequency ultrasound manifestations, 26 skin lesions were classified into 2 types: 21 diffuse-type lesions with unclear borders and irregular shapes, and 5 mass-type lesions with clear borders and irregular shapes. Solid and homogeneously hypoechoic areas were observed in 18 cases, and solid and heterogeneously hypoechoic areas in 8. According to the Adler blood flow classification, the 26 lesions were classified into 4 grades: 2 of grade 0, 5 of grade 1, 8 of grade 2, and 11 of grade 3.Conclusion:EMPD shows characteristic high-frequency color Doppler ultrasound manifestations, which can facilitate its clinical diagnosis.

Objective:A highly sensitive and specific real-time quantitative TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for rapid and accurate detection of Tibet orbivirus (TIBOV).Methods:The TIBOV genomic sequences from GenBank were analyzed by Clustal X 2.1 and the specific primers and probe were designed in the conserved segment of VP4 gene. RNA standard was obtained from in vitro transcription and a TaqMan RT-PCR assay for TIBOV was established. The sensitivity, specificity and repeatability of this method were evaluated. Results:The assay showed a good amplification curve within the range of 1.0×10 2~8 copies/reaction template, the detection limit was 1.0×10 2 copies/reaction, the coefficients of variation of Ct values in repeat detections were all less than 1.5%. No cross-reaction was found in this assay. Variable mosquito samples were screened by this assay and the result showed TIBOV negative. The prepared TIBOV simulated positive samples were 100% detected. Conclusions:The assay developed in this study is specific and sensitive for detection of TIBOV and can be used for laboratory detection and routine surveillance.

Objective To investigate the safety and feasibility of totally laparoscopic transabdominalhiatal approach in the treatment of Siewert type Ⅱ adenocarcinoma of esophagogastric junction (AEG).Methods The retrospective and descriptive study was conducted.The clinicopathological data of 11 patients with Siewert type Ⅱ AEG who were admitted to Affiliated Hangzhou First People's Hospital of Zhejiang University School of Medicine from May 2017 to July 2018 were collected.There were 8 males and 3 females,aged 56-72 years,with an average age of 63 years.Patients underwent radical resection of AEG by totally laparoscopic transabdominalhiatal approach.Observation indicators:(1) surgical situations and postoperative recovery;(2) postoperative pathological examination;(3) follow-up and survival situations.Follow-up using outpatient examination and telephone interview was performed to detect postoperative adjuvant chemotherapy,complications,food intake,anastomosis patency,tumor recurrence and metastasis,and survival up to December 2018.Measurement data with normal distribution were presented as Mean±SD,measurement data with skewed distribution were presented as M (range),and count data were represented as absolute number or percentage.Results (1) Surgical situations and postoperative recovery:all the patients underwent totally laparoscopic radical resection of Siewert type Ⅱ AEG by transabdominal-hiatal approach,without conversion to open surgery or perioperative death.Of the 11 patients,8 underwent total gastrectony including 3 combined with splenic hilar lymph node dissection and 3 underwent proximal gastrectomy with double-tract reconstruction.Operation time,time of superior overlap esophagojejunostomy,volume of intraoperative blood loss,time for initial out-of-bed activities,time to first flatus,time to initial liquid diet intake,time of drainage tube removal were respectively (245± 39)minutes,(60± 12) minutes,(75±23) mL,(24± 8) hours,(2.4± 0.5) days,(3.5 ± 0.8) days,(8.2 ± 1.3) days respectively.There was no serious complication including postoperative hemorrhage,anatomotic fistula or death.Three patients had left pleural effusion,and were cured after thoracic drainage.The duration of postoperative hospital stay was (11.0±3.0) days.(2) Postoperative pathological examination:all the 11 patients had negative upper surgical margin.The length of proximal margin,tumor diameter,total number of lymph lodes harvested,and number of lower mediastinal lymph lodes harvested were (2.1 ±0.2) cm,(2.6±0.9) cm,(36.0±4.0)/case and (2.3± 0.8)/case.Pathological examination showed adenocarcinoma in all the 11 patients.pTNM staging:2 cases were in stage Ⅰ B,4 cases in stage Ⅱ A,3 cases in stage Ⅱ B and 2 cases in stage Ⅲ A.(3) Follow-up and survival situations:11 patients were follow-up for 6-19 months,with a median time of 9 months.Chemotherapy regimeus were formulated according to the pathological examination.Nine patients received postoperative adjuvant chemotherapy,and 2 in stage Ⅱ B received no postoperative adjuvant chemotherapy.During the follow-up,11 patients had no obvious reflux symptom or choking feeling,and the anastomosis was patent as evaluated by oral contrast agent and gastroscopy.There was no tumor recurrence and metastasis or death in the 11 patients.Conclusion Totally laparoscopic transabdominal-hiatal approach applied in the radical resection is safe and feasible for the treatment of Siewert type Ⅱ AEG,with good short-term outcomes.

Objective: To evaluate modified overlap esophagojejunostomy in digestive tract reconstruction of totally laparoscopie total gastrectomy(TLTG)in patients of gastric carcinoma. Methods: The clinicalpathological data of 68 patients admitted to Hangzhou First People′s Hospital from Jun 2015 to Aug 2018 Undergoing totally laparoscopie total gastrectomy and modified overlap esophagojejunostomy were analyzed retrospectively. Results: Procedures were successful in all 68 patients without conversion to open surgery and no perioperative death.The operation time, esophagojejunostomy time, volume of intraoperative blood loss and incision length were respectively (210±30)min, (25±12)min, (50±20)ml and(3.5±1.1)cm. Time for initial out of bed activity, time of initial anal exsufflation, time for postoperative fluid diet intake and duration of postoperative hospital stay were (24±8)h, (2.4±0.5)d, (3.1±0.8)d, and (8±3.6)d. There was anastomotic fistula in one and recovered by conservative treatment, total number of harvested LNs in 68 patients were (38.9±2.3). By pTNM staging: 6 cases in stage ⅠB, 10 cases in stage ⅡA, 20 cases in stage ⅡB , 15 cases in stage ⅢA, 12 cases in stage ⅢB, 5 cases in stage ⅢC. 59 patients were followed up from 3 to 42 months. One with liver metastasis after 22 months, others were all tumor-free. Conclusion The modified overlap esophagojejunostomy method is safe and feasible in digestive tract reconstruction of totally laparoscopie total gastrectomy for gastric carcinoma.

Objective: To understand the status and genotype of norovirus in Wuhu, and provide the basic molecular epidemiological data for norovirus infection control and prevention, 2017. Methods: Anal swab and vomit specimens were collected during outbreaks from patients, primarily using fluorescence quantitative PCR to preliminarily identify the genotype of norovirus; according to preliminary result , through their respective specific primer, RT-PCR was applied, and sequencing was done to identify virus genotypes. Sequence alignment and phylogenetic analysis of norovirus was performed by using biological software. Results: Seventeen norovirus epidemics were observed in Wuhu area, a total of 137 specimens were collected. Real-time fluorescent quantitative PCR result showed that 77 specimens were norovirus nucleic acid testing positive, and all were GⅡ genotype. Through specific primers amplification, sequencing analysis showed that the 2017 outbreaks in Wuhu were caused only by two genotypes, GⅡ.3 and GⅡ. 2, including one case of GⅡ. 3, Six cases of GⅡ.2, and 10 epidemic aggregations, 8 cases of GⅡ. 2, and there were two cases of G Ⅱ. 4 Sydney 2012. Conclusions Norovirus GⅡ.2 genotype was dominant in norovirus outbreaks and the aggregation epidemics, other genotypes were occasionally seen in Wuhu, 2017.