Abstract：Objective To improve the replication level of varicella⁃zoster virus（VZV）in human diploid cell line MRC⁃5 and increase the yield of VZV vaccine by reducing the expression of interferon（IFN）related genes via optimizing the cell line MRC⁃5. Methods Interferon receptor 1（IFNAR1）silenced MRC⁃5 cell line（MRC⁃5IFNAR1⁃）was constructed by CRISPR／Cas9 gene editing technology，which was determined for the relative expression of IFNAR1 mRNA，and for those of mRNA of IFN related genes IFNβ and OAS1 after VZV infection by qRT⁃PCR to evaluate the effect of gene silencing. Gene mutation sequences were further identified by sequencing of the silenced sites. The replication of VZV in MRC⁃5 and MRC⁃5IFNAR1⁃ cell lines was compared 168 h after VZV infection by using qRT⁃PCR and plaque formation unit（PFU）assay， to evaluate the effect of MRC⁃5IFNAR1⁃cell line on VZV replication. Results The growth status of MRC⁃5IFNAR1⁃ cell line wasconsistent with that of MRC ⁃ 5 cells，and the relative expression of IFNAR1 mRNA decreased by 73%；The relative expressions of IFNβ and OAS1 mRNA in MRC⁃5IFNAR1⁃ cell line were 61% and 90% lower than those in MRC⁃ 5 cells respectively after VZV infection；In addition，168 h after VZV infection，the level of DNA replication and the titer of VZV increased by 5. 7 folds and 4 folds respectively. Conclusion The successful establishment of MRC⁃5IFNAR1⁃ cell line may be a potential scheme to increase the yield of vaccines based on human diploid cells，and provided a reference for expanding production of VZV vaccine.

OBJECTIVES: To investigate the risk factors for neonatal asphyxia in Hubei Enshi Tujia and Miao Autonomous Prefecture and establish a nomogram model for predicting the risk of neonatal asphyxia. METHODS: A retrospective study was conducted with 613 cases of neonatal asphyxia treated in 20 cooperative hospitals in Enshi Tujia and Miao Autonomous Prefecture from January to December 2019 as the asphyxia group, and 988 randomly selected non-asphyxia neonates born and admitted to the neonatology department of these hospitals during the same period as the control group. Univariate and multivariate analyses were used to identify risk factors for neonatal asphyxia. R software (4.2.2) was used to establish a nomogram model. Receiver operator characteristic curve, calibration curve, and decision curve analysis were used to assess the discrimination, calibration, and clinical usefulness of the model for predicting the risk of neonatal asphyxia, respectively. RESULTS: Multivariate logistic regression analysis showed that minority (Tujia), male sex, premature birth, congenital malformations, abnormal fetal position, intrauterine distress, maternal occupation as a farmer, education level below high school, fewer than 9 prenatal check-ups, threatened abortion, abnormal umbilical cord, abnormal amniotic fluid, placenta previa, abruptio placentae, emergency caesarean section, and assisted delivery were independent risk factors for neonatal asphyxia (P<0.05). The area under the curve of the model for predicting the risk of neonatal asphyxia based on these risk factors was 0.748 (95%CI: 0.723-0.772). The calibration curve indicated high accuracy of the model for predicting the risk of neonatal asphyxia. The decision curve analysis showed that the model could provide a higher net benefit for neonates at risk of asphyxia. CONCLUSIONS The risk factors for neonatal asphyxia in Hubei Enshi Tujia and Miao Autonomous Prefecture are multifactorial, and the nomogram model based on these factors has good value in predicting the risk of neonatal asphyxia, which can help clinicians identify neonates at high risk of asphyxia early, and reduce the incidence of neonatal asphyxia.
Infant, Newborn ; Humans ; Male ; Pregnancy ; Female ; Nomograms ; Retrospective Studies ; Cesarean Section ; Risk Factors ; Asphyxia Neonatorum/etiology*

Objective: To explore the safety and efficacy of excimer laser coronary angioplasty (ELCA) for the treatment of degenerated great saphenous vein graft (SVG). Methods: This is a single-center, prospective, single-arm study. Patients, who were admitted to the Geriatric Cardiovascular Center of Beijing Anzhen Hospital from January 2022 to June 2022, were consecutively enrolled. Inclusion criteria were recurrent chest pain after coronary artery bypass surgery (CABG), and coronary angiography confirmed that the SVG stenosis was more than 70% but not completely occluded, and interventional treatment for SVG lesions was planned. Before balloon dilation and stent placement, ELCA was used to pretreat the lesions. Optical coherence tomography (OCT) examination was performed and postoperative index of microcirculation resistance (IMR) were assessed after stent implantation. The technique success rate and operation success rate were calculated. The technique success was defined as the successful passage of the ELCA system through the lesion. Operation success was defined as the successful placement of a stent at the lesion. The primary evaluation index of the study was IMR immediately after PCI. Secondary evaluation indexes included thrombolysis in myocardial infarction (TIMI) flow grade, corrected TIMI frame count (cTFC), minimal stent area and stent expansion measured by OCT after PCI, and procedural complications (Ⅳa myocardial infarction, no reflow, perforation). Results: A total of 19 patients aged (66.0±5.6) years were enrolled, including 18 males (94.7%). The age of SVG was 8 (6, 11) years. The length of the lesions was greater than 20 mm, and they were all SVG body lesions. The median stenosis degree was 95% (80%, 99%), and the length of the implanted stent was (41.7±16.3)mm. The operation time was 119 (101, 166) minutes, and the cumulative dose was 2 089 (1 378, 3 011)mGy. The diameter of the laser catheter was 1.4 mm, the maximum energy was 60 mJ, and the maximum frequency was 40 Hz. The technique success and the operation success rate were both 100% (19/19). The IMR after stent implantation was 29.22±5.95. The TIMI flow grade of patients after ELCA and stent implantation was significantly improved (all P>0.05), and the TIMI flow grade of all patients after stent implantation was Grade Ⅲ. The cTFC decreased significantly after ELCA (33.2±7.8) and after stent placement (22.8±7.1) than preoperative level (49.7±13.0) (both P<0.001). The minimum stent area was (5.53±1.36)mm², and the stent expansion rate was (90.0±4.3)%. Perforation, no reflow, type Ⅳa myocardial infarction and other complications were not observed. However, postoperative high-sensitivity troponin level was significantly increased ((67.937±33.839)ng/L vs. (5.316±3.105)ng/L, P<0.001). Conclusion: ELCA is safe and effective in the treatment of SVG lesions and could improve microcirculation and ensure full expansion of stent.
Male ; Humans ; Aged ; Prospective Studies ; Percutaneous Coronary Intervention ; Lasers, Excimer/therapeutic use* ; Saphenous Vein/transplantation* ; Constriction, Pathologic ; Atherectomy, Coronary/methods* ; Myocardial Infarction ; Coronary Angiography ; Stents ; Treatment Outcome

Objective: To analyze the course of disease and epidemiological parameters of COVID-19 and provide evidence for making prevention and control strategies. Methods: To display the distribution of course of disease of the infectors who had close contacts with COVID-19 cases from January 1 to March 15, 2020 in Guangdong Provincial, the models of Lognormal, Weibull and gamma distribution were applied. A descriptive analysis was conducted on the basic characteristics and epidemiological parameters of course of disease. Results: In total, 515 of 11 580 close contacts were infected, with an attack rate about 4.4%, including 449 confirmed cases and 66 asymptomatic cases. Lognormal distribution was fitting best for latent period, incubation period, pre-symptomatic infection period of confirmed cases and infection period of asymptomatic cases; Gamma distribution was fitting best for infectious period and clinical symptom period of confirmed cases; Weibull distribution was fitting best for latent period of asymptomatic cases. The latent period, incubation period, pre-symptomatic infection period, infectious period and clinical symptoms period of confirmed cases were 4.50 (95%CI:3.86-5.13) days, 5.12 (95%CI:4.63-5.62) days, 0.87 (95%CI:0.67-1.07) days, 11.89 (95%CI:9.81-13.98) days and 22.00 (95%CI:21.24-22.77) days, respectively. The latent period and infectious period of asymptomatic cases were 8.88 (95%CI:6.89-10.86) days and 6.18 (95%CI:1.89-10.47) days, respectively. Conclusion: The estimated course of COVID-19 and related epidemiological parameters are similar to the existing data.
COVID-19 ; Cohort Studies ; Contact Tracing ; Humans ; Incidence ; Prospective Studies

In this study, the molecular mechanism of astragaloside Ⅳ(AS-Ⅳ) in the treatment of Parkinson's disease(PD) was explored based on network pharmacology, and the potential value of AS-Ⅳ in alleviating neuronal injury in PD by activating the PI3 K/AKT signaling pathway was verified through molecular docking and in vitro experiments. Such databases as SwissTargetPrediction, BTMAN-TAM, and GeneCards were used to predict the targets of AS-Ⅳ for the treatment of PD. The Search Tool for the Retrieval of Interacting Genes/Proteins(STRING) was employed to analyze protein-protein interaction(PPI) and construct a PPI network, and the Database for Annotation, Visualization and Integrated Discovery(DAVID) was used for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Based on the results of GO enrichment analysis and KEGG pathway analysis, the PI3 K/AKT signaling pathway was selected for further molecular docking and in vitro experiments in this study. The in vitro cell model of PD was established by MPP~+. The cell viability was measured by MTT assay and effect of AS-Ⅳ on the expression of the PI3 K/AKT signaling pathway-related genes and proteins by real-time polymerase chain reaction(RT-PCR) and Western blot. Network pharmacology revealed totally 122 targets of AS-Ⅳ for the treatment of PD, and GO enrichment analysis yielded 504 GO terms, most of which were biological processes and molecular functions. Totally 20 related signaling pathways were screened out by KEGG pathway analysis, including neuroactive ligand-receptor interaction, PI3 K/AKT signaling pathway, GABAergic synapse, and calcium signaling pathway. Molecular docking demonstrated high affinity of AS-Ⅳ to serine/threonine-protein kinases(AKT1, AKT2), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma(PIK3 CG), and phosphoinositide-3-kinase, catalytic, alpha polypeptide(PIK3 CA) on the PI3 K/AKT signaling pathway. In vitro experiments showed that AS-Ⅳ could effectively inhibit the decrease of the viability of PC12 induced by MPP~+ and up-regulate the mRNA expression levels of AKT1 and PI3 K as well as the phosphorylation levels of AKT and PI3 K. As an active component of Astragali Radix, AS-Ⅳ acts on PD through multiple targets and pathways. Furthermore, it inhibits neuronal apoptosis and protects neurons by activating the PI3 K/AKT signaling pathway, thereby providing reliable theoretical and experimental supports for the treatment of PD with AS-Ⅳ.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Molecular Docking Simulation ; Network Pharmacology ; PC12 Cells ; Phosphatidylinositol 3-Kinases/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Rats ; Saponins ; Signal Transduction ; Triterpenes

Objective: To screen and analyze the mutations of MITF gene in two children of type Ⅱ Waardenburg syndrome (WS2) from different families in Yunnan,China,and to explore the possible molecular pathogenesis. Methods: With informed consent, medical history collection, physical examinations, audiological evaluation, and high resolution computer tomography (HRCT) scan of temporal bone were performed on the two WS2 probands and their family members. Genomic DNA was extracted from peripheral blood of all individuals. The coding regions including all exons, part of introns and promoters of MITF, PAX3, SOX10, SNAI2, END3, ENDRB, and KITLG genes were sequenced by high-throughput sequencing. According to the results of high-throughput sequencing, pathogenic mutations detected in the probands and their parents were verified by Sanger sequencing. Results: The proband 1 carried c.641_643delGAA mutation in the 7th exon of MITF gene, which was a frame-shift mutation resulting in an amino acid change of p.214delR. It was a de novo mutation as the parents of proband 1 showed no variation on this site. The proband 2 carried heterozygous loss of the large fragment ranging from exon 1 to exon 9 of MITF gene, which defected the function of MITF protein. Conclusion: Genetic examinations provide important evidence for diagnosis of Waardenburg syndrome. Heterozygous mutation c.641_643delGAA and heterozygous loss of the large fragment ranging from exon 1 to exon 9 of MITF gene might be the molecular pathogenesis of the two WS2 probands in this study.
Asians/genetics* ; Child ; China ; Humans ; Mutation ; Pedigree ; SOXE Transcription Factors/genetics* ; Waardenburg Syndrome/genetics*

Blood Loss, Surgical ; prevention & control ; Epilepsy ; chemically induced ; Humans ; Orthopedic Procedures ; Spine ; surgery ; Tranexamic Acid ; adverse effects

AIM:To investigate the role of hydrogen molecule on apoptosis-related proteins in glomerular me-sangial cells cultured with high glucose and to explore its possible mechanism.METHODS:Mouse glomerular mesangial cells cultured in vitro were divided into 4 groups:normal control group(C group,5.5 mmol/L glucose),mannitol group(G group,5.5 mmol/L glucose+19.5 mmol/L mannitol),high glucose group(H group,25 mmol/L glucose),high glu-cose+hydrogen-rich water group(HH group,25 mmol/L glucose+hydrogen-rich water),and cultured for 48 h.The pro-tein levels of Bax,Bcl-2,cleaved caspase-3,nuclear factor E2-related factor-2(Nrf2),heme oxygenase-1(HO-1)and NAD(P)H:quinone oxidoreductase-1(NQO-1)were determined by Western blot ,and the mRNA expression of HO-1 and NQO-1 was determined by RT-PCR.The level of intracellular reactive oxygen species(ROS)was detected by dihydro-ethidium method,and the activity of superoxide dismutase(SOD)was measured by WST-8 assay.RESULTS:Compared with C group,the protein levels of Bax and cleaved caspase-3 were up-regulated,and Bcl-2 was down-regulated in H group(P <0.05).No significantly difference of the protein levels mentioned above between C and HH group was observed. Compared with H group,the protein levels of Bax and cleaved caspase-3 were down-regulated,and Bcl-2 was up-regulated in HH group(P <0.05).The level of intracellular ROS was higher and the activity of SOD was lower in H group than those in C group(P<0.05).However,there was no difference of the SOD activity between C group and HH group.The level of intracellular ROS decreased and the activity of SOD increased in HH group as compared with H group(P<0.05). Compared with C group,clearly reduced protein expression of Nrf2,HO-1 and NQO-1,and decreased mRNA expression of HO-1 and NQO-1 in H group were observed(P<0.05).Compared with H group,the protein levels of Nrf2,HO-1 and NQO-1 as well as the mRNA levels of HO-1 and NQO-1 were obviously increased in HH group(P<0.05 ).CONCLU-SION:Hydrogen molecule inhibits the expression of pro-apoptotic proteins and induces the expression of anti-apoptotic pro-teins in glomerular mesangial cells cultured with high glucose.The mechanism may be related to activation of Nrf 2 signaling pathway.

Ambroxol ; therapeutic use ; Anti-Bacterial Agents ; therapeutic use ; Humans ; Infant ; Male ; Penicillanic Acid ; analogs & derivatives ; therapeutic use ; Pneumonia ; drug therapy ; etiology ; virology ; Poliomyelitis ; immunology ; Vaccines ; adverse effects ; immunology

BACKGROUNDScreening on multidrug-resistant tuberculosis (MDR-TB) has been limited to the serious TB subpopulations excluding the new TB patients. This study aimed to examine MDR-TB burden among the new TB patients.

METHODSWe conducted a study in Zhejiang Province during 2009-2013 to screen for MDR-TB patients among the low MDR-TB risk patients and five subpopulations of high MDR-TB risk patients. The number, prevalence, and trend of MDR-TB were compared while the logistic regression model was used to examine risk factors related to MDR-TB.

RESULTSA total of 200 and 791 MDR-TB cases were, respectively, identified from the 9830 new TB cases and 2372 high-risk suspects who took MDR-TB screening from 2009 to 2013. The MDR-TB rates went down in both of the new TB patients and five MDR-TB high-risk groups over the study time, but the percentage of MDR-TB patients identified from the new TB patients in all diagnosed MDR-TB cases kept stable from 28.3% in 2011 to 27.0% in 2012 to 26.0% in 2013.

CONCLUSIONSThe study indicated that MDR-TB burden among new TB patients was high, thus screening for MDR-TB among the new TB patients should be recommended in China as well as in the similar situation worldwide.