Objective:The human peripheral blood lymphocyte cell line 8E5 is capable of secreting non-infectious HIV-1 viral particles. By targeting the POL region of the HIV-1 proviral gene integrated into the genome of 8E5 cell line and constructing a monoclonal cell line containing a drug resistance mutation site in the POL region using CRISPR/Cas9 point mutation technology, safe and stable HIV-1 genotypic drug resistance test quality control materials can be prepared.Methods:8E5 cells were co-transfected with sgRNA (single guide RNA) and Cas9 coexpression vector and Donor ssODN (donor single-stranded oligonucleotides) carrying the target mutation sites. The positive monoclonal cell lines were obtained through flow microtiter plate sorting, and the editing efficacy of the targeted mutations was validated by Sanger sequencing. Sanger sequencing was performed to verify the editing effect of the targeted mutations on the HIV virus particles secreted into the supernatant of the monoclonal cell lines cultured to the 3rd, 5th and 7th generations.Results:A double sgRNA and Cas9 coexpression vector was successfully constructed and co-transfected with a Donor ssODN carrying the drug-resistant mutation site Q151M to the 8E5 cell line, resulting in the desired outcome. The sequencing result of the target site confirmed the successful mutation at the resistance site and the establishment of a monoclonal homozygous cell line. The Q151M mutation site was detected in non-infectious HIV-1 virus particles secreted by the 8E5 Q151M cell line after transmission. Conclusions:The cell line 8E5 Q151M was successfully constructed using CRISPR/Cas9 point mutation technology to stably carry the Q151M drug resistance mutation site, which provides a new technological platform for the preparation of quality-control materials for testing HIV-1 genotypic drug resistance.

Objective To screen the quality evaluation indicators of Bushen Huoxue Prescription(BHP)in the treatment of diabetic retinopathy(DR)by network pharmacology and molecular docking technology,and to establish content determination of active components in BHP by ultra-high-performance liquid chromatography triple-quadrupole mass spectrometry(UHPLC-QqQ-MS/MS).Methods Network pharmacology was used to screen the disease-related targets and key components,followed by molecular docking to further verify the interaction between them and confirm the active ingredients in BHP as the quality evaluation indicators.A UHPLC-QqQ-MS/MS method for the content determination of the active ingredients in BHP was established.A ZORBAX SB-C18 column(2.1 mm×50 mm,1.8 μm)was used,and methanol(A)-0.1％formic acid solution(B)was used as mobile phase.Gradient elution was performed in multiple reaction monitor mode.The flow rate was 0.3 mL·min-1 and the injection volume was 1 μL.Results Network pharmacology and molecular docking revealed five core targets and 12 BHP-related components(verbascoside,echinacoside,isoacteoside,tanshinone ⅡA,cryptotanshinone,dihydrotanshinone I,ginsenoside Re,Rd and Rb1,puerarin,daidzin,biochanin A)for the treatment of DR.There was a strong binding affinity between them(binding energy≤-5.0 kcal·mol-1).The established quantitative method demonstrated each component presented a good linearity within the specified range(r>0.999 5).The average recovery was in the range of 97.57％～101.48％.The contents of 12 components in eight batches of BHP samples were 0.027 9％～0.050 6％,0.006 4％～0.022 0％,0.017 1％～0.041 5％,0.009 2％～0.015 4％,0.012 6％～0.020 5％,0.004 4％～0.007 6％,0.334％～0.643％,0.238％～0.530％,0.353％～0.693％,3.411％～6.048％,1.023％～1.352％,0.000 8％～0.001 8％,respectively.Conclusion Based on network pharmacology,molecular docking and UHPLC-QqQ-MS/MS,a method for rapid screening and determination of 12 active components of BHP in the prevention and treatment of DR was established.This study provided a reference for comprehensive assessment of the quality and effectiveness of BHP.

Objective To compare and analyze the differences in CD8+naive,effector,memory,exhausted and regulatory T cells in order to investigate the impact of gender on the differentiation fate of CD8+T cells in the context of type 1 diabetes (T1D)based on female and male non-obese diabetic (NOD)mice and healthy Institute for Cancer Research (ICR)mice.Methods The frequencies and phenotypes of CD8+T cell differentiation subsets including naive T cells (TN),central memory T cells (TCM),effector T cells(TEFF),effector precursor T cells (TEP),exhausted T cells (TEX),precursor exhausted T cells (TPEX)and regulatory T cells (Tregs)in the spleen,pancreatic draining lymph nodes (pLN)and pancreas infiltrating lymphocytes (PIL)of male and female NOD mice were detected by flow cytometry.Results The frequencies of IFN-γ+,CD107a+and CCL5+CD8+TEFF in pLN and PIL of female NOD mice were significantly higher than those of male NOD mice.However,the frequencies of CD8+TN,CD8+TCM,CD8+TEX,CD8+TPEX and CD122+CD8+Tregs subsets in the spleen were significantly decreased.While there were no significant differences in the above CD8+T cell subsets except CD8+Tregs between female and male ICR mice. Conclusion Androgen may inhibit the differentiation of memory T cells into effector T cells and promote the exhaustion of effector T cells,leading to the difference in morbidity between the male and female mice.

Uncovering the risk factors of pulmonary hypertension and its mechanisms is crucial for the prevention and treatment of the disease.In the current study,we showed that experimental periodontitis,which was established by ligation of molars followed by orally smearing subgingival plaques from patients with periodontitis,exacerbated hypoxia-induced pulmonary hypertension in mice.Mechanistically,periodontitis dysregulated the pulmonary microbiota by promoting ectopic colonization and enrichment of oral bacteria in the lungs,contributing to pulmonary infiltration of interferon gamma positive(IFNγ+)T cells and aggravating the progression of pulmonary hypertension.In addition,we identified Prevotella zoogleoformans as the critical periodontitis-associated bacterium driving the exacerbation of pulmonary hypertension by periodontitis,and the exacerbation was potently ameliorated by both cervical lymph node excision and IFNγ neutralizing antibodies.Our study suggests a proof of concept that the combined prevention and treatment of periodontitis and pulmonary hypertension are necessary.

AIM:To explore the effect of polyphenolic compound 3,5-dihydroxy-4-methoxybenzyl alcohol(DHMBA)on hypoxia/reoxygenation(H/R)injury of human umbilical vein endothelial cells(EA.hy926 cells)and its po-tential mechanisms.METHODS:To construct an H/R model,the EA.hy926 cells were cultured in an acidic hypoxia buffer while in an anaerobic workstation.The cells were divided into control,H/R,H/R+different doses of DHMBA,H/R+edaravone(antioxidant)and H/R+reactive oxygen species(ROS)inducer oligomycin A+DHMBA groups.Cell viability was measured by CCK-8 assay,and tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)levels in cells were mea-sured by ELISA.Phosphorylation of endothelial nitric oxide synthase(eNOS)and nuclear factor-κB(NF-κB)p65 were measured by Western blot.Intracellular NO levels were determined by laser confocal microscopy.Glutathione(GSH)/glu-tathione disulfide(GSSG)oxidation balance was determined by the dinitrobenzoic acid chromogenic method.Intracellular ROS levels were measured by flow cytometry.Lactate dehydrogenase(LDH)leakage was determined using nitro blue tet-razolium staining.Scratch assays were performed to assess cell migration.RESULTS:DHMBA exhibited no significant cytotoxicity between 125 and 1 000 μmol/L.In H/R-injured human umbilical vein endothelial cells,DHMBA improved cell survival,inhibited phosphorylation of NF-κB p65,reduced the content of TNF-α and IL-6,and increased phosphory-lation of eNOS and NO levels.DHMBA also suppressed ROS overload and restored the ratio between GSH and oxidized GSH,decreased in LDH leakage and increased cell migration in H/R-injured human umbilical vein endothelial cells.CONCLUSION:DHMBA can alleviate H/R-induced oxidative stress,inflammation,cellular damage,and dysfunction,which are associated with the ability of DHMBA to inhibit ROS production in human umbilical vein endothelial cells.

OBJECTIVE: To explore the clinical and genetic characteristics of four patients with medium-chain acyl-CoA dehydrogenase deficiency (MCADD). METHODS: Four children who had presented at the Children's Hospital Affiliated to Zhengzhou University between August 2019 and August 2021 were selected as the study subjects. Clinical data of the children were collected. The children were subjected to whole exome sequencing (WES). RESULTS: All of the four children were diagnosed with MCADD. Blood amino acid and ester acyl carnitine spectrum test showed that the concentration of octanoyl carnitine (C8) was significantly increased. The main clinical manifestations included poor mental response (3 cases), intermittent diarrhea with abdominal pain (1 case), vomiting (1 case), increased transaminase (3 cases), and metabolic acidosis (2 cases). Five variants were identified by genetic testing, among which c.341A>G (p.Y114C) was unreported previously. Three were missense variants, one was frameshift variant and one was splicing variant. CONCLUSION The clinical heterogeneity of MCADD is obvious, and the severity of the disease may vary. WES can assist with the diagnosis. Delineation of the clinical symptoms and genetic characteristics of the disease can facilitate early diagnosis and treatment of the disease.
Child ; Humans ; Acyl-CoA Dehydrogenase/genetics* ; Carnitine ; Genetic Testing ; Lipid Metabolism, Inborn Errors/genetics* ; Neonatal Screening

OBJECTIVE: To explore the clinical features and genetic basis of a child with Galactosemia. METHODS: A child who had presented at the Children's Hospital Affiliated to Zhengzhou University on November 20, 2019 was selected as the study subject. Clinical data of the child was collected. Whole exome sequencing was carried out for the child. Candidate variants were validated by Sanger sequencing. RESULTS: Clinical manifestations of the child have included anemia, feeding difficulty, jaundice, hypomyotonia, abnormal liver function and coagulation abnormality. Tandem mass spectrometry showed increased citrulline, methionine, ornithine and tyrosine. Urine organic acid analysis showed increased phenyllactic acid, 4-hydroxyphenylacetic acid, 4-hydroxyphenyllactic acid, 4-hydroxyphenylpyruvate and N-acetyltyrosine. Genetic testing revealed that the child has harbored compound heterozygous variants of the GALT gene, namely c.627T>A (p.Y209*) and c.370G>C (p.G124R), which were respectively inherited from her healthy parents. Among these, c.627T>A (p.Y209*) was known as a likely pathogenic variant, while c.370G>C (p. G124R) was unreported previously and also predicted as a likely pathogenic variant(PM1+PM2_Supporting+PP3_Moderate+PPR). CONCLUSION Above discovery has expanded the spectrum of the GALT gene variants underlying Galactosemia. Patients with thrombocytopenia, feeding difficulties, jaundice, abnormal liver function and coagulation abnormality without obvious causes should be analyzed by screening of metabolic diseases in combination with genetic testing.
Child ; Female ; Humans ; Galactosemias/genetics* ; Genetic Testing ; Health Status ; Methionine ; Muscle Hypotonia ; Mutation

Objective:To investigate the clinical characteristics, genetic characteristics and diagnosis of spinocerebellar ataxia type 2 (SCA2) patients with childhood onset.Methods:The clinical data of a SCA2 pedigree who diagnosed at Neurogenetic Metabolic Disease Clinic of Children′s Hospital Affiliated to Zhengzhou University in July 2019 were collected, and the reported cases of childhood-onset SCA2 were reviewed. The CAG repeat of ATXN2 gene was detected by polymerase chain reaction, capillary gel electrophoresis and Sanger sequencing techniques.Results:A total of 9 people in 4 generations of the family were affected, showing an autosomal dominant inheritance. The proband was a 3 years and 4 months old boy, who showed abnormal symptoms at 9 months which manifested as developmental retardation. At 1 year old, he developed progressive regression which represented neither to be amused, recognize others, stand and walk alone, nor had language development. Meanwhile, he had difficulty swallowing, long-term constipation, and a history of convulsions. His sister and mother were not yet sick. His grandmother could not walk, had slurred speech accompanied by nystagmus, and magnetic resonance imaging showed cerebellar atrophy. His granduncles and grandaunts had unstable walking and dysarthria. His great-grandfather required wheelchair to walk. This pedigree showed an autosomal dominant inheritance. One of the ATXN2 gene alleles of the proband, his sister, mother and grandmother all showed abnormal amplification with 99, 55, 44, and 43 times respectively and no inserting CAA sequence. A total of 14 literatures reported 20 cases of childhood-onset SCA2 patients who were genetically diagnosed. The majorities had onset in infancy, and few can develop into school age. The main clinical manifestations were developmental delay, dystonia or insufficiency, myoclonus or infantile spasms, motor retardation, abnormal eye movement, retinitis pigmentosa and dysphagia, while the classic cerebellar syndrome was only partially present. Abnormal rhythm was found on electroencephalogram, cerebellar atrophy on magnetic resonance imaging or CT of the head.Conclusions:This case is the youngest genetically-confirmed SCA2 patient reported in China. Reported patients usually have onset in infancy with excessive repeat sequence expansion. Their clinical characteristics are different from the classic patients and could only be diagnosed by dynamic mutation detection.

Objective:To investigate the prevalence and the risk factors of Helicobacter pylori( H. pylori) infection in Qinghai Province with a multi-center cross-sectional study. Methods:From May to December in 2021, stratified sampling was conducted in Xining City, Haidong agricultural district, Hainan Tibetan Autonomous Prefecture, Haibei Tibetan Autonomous Prefecture and Yushu Tibetan Autonomous Prefecture by 20 centers, including the Affiliated Hospital of Qinghai University, Qinghai Red Cross Hospital and Qinghai Renji Hospital, etc. A questionnaire survey was carried out among the individuals undergoing routine health checkups. The questionnaire included general information, lifestyle and family history, etc. 13C-urea breath test was performed to diagnose H. pylori infection. Chi-square test, trend Chi-square test, segmentation method and multivariate logistic regression analysis were performed for statistical analysis. Results:The infection rate of H. pylori in Qinghai area was 53.6% (2 531/4 724). The age distribution of infection was statistically significant ( χ2=15.95, P=0.007), the infection rate in individuals aged 15 to<30 was higher than that of individuals aged 60 to< 75 (57.6%, 626/1 086 vs.49.5%, 231/467), and the difference was statistically significant ( χ2=8.83, P=0.003). With the increase of age, the infection rate decreased in female over 30 years old ( χ2trend=5.89, P=0.015). There were statistically significant differences in H. pylori infection rate among different ethnic groups ( χ2=28.13, P<0.001); the infection rate of Tibetan population was higher than that of Han population (62.9%, 313/498 vs. 51.5%, 1 804/3 503), and the difference was statistically significant ( χ2=22.56, P<0.001). The H. pylori infection rate in people living at an altitude >3 500 m was higher than that of people living at an altitude between 1 500 m and 3 500 m (61.8%, 327/529 vs. 52.5%, 2 204/4 195), and the difference was statistically significant ( χ2=16.25, P<0.001). Compared with those without corresponding habits, the infection rate of H. pylori was higher in smokers (62.1%, 1 081/1 740 vs. 48.6%, 1 450/2 984), in people who had the habit of eating hand grabbed mutton (55.4%, 967/1 744 vs. 52.5%, 1 564/2 980), raw garlic (55.5%, 968/1 744 vs. 52.4%, 1 563/2 980), spicy food (55.6%, 1 471/2 647 vs. 51.0%, 1 060/2 077), sweet food (55.2%, 1 187/2 149 vs. 52.2%, 1 344/2 575), the infection rate of tea drinkers was lower (50.5%, 1 135/2 247 vs. 56.4%, 1 396/2 477), and the differences were statistically significant ( χ2=80.94, 3.89, 9.63, 4.36, 4.13 and 16.19, all P<0.05). The results of logistic regression analysis indicated that Tibetan ( OR=1.379, 95% confidence interval (95% CI) 1.120 to 1.699), the Hui people ( OR=1.362, 95% CI 1.117 to 1.662), living at an altitude over 3 500 m ( OR=1.355, 95% CI 1.107 to 1.657), smoking ( OR=1.847, 95% CI 1.629 to 2.094), and spicy food ( OR=1.224, 95% CI 1.084 to 1.382) were independent risk factors of H. pylori infection (all P<0.05). Conclusions:The infection rate of H. pylori in Qinghai Province is higher than the average level in inland China. The infection rate of people with different ages, nationalities, altitudes of residence, occupations, living and eating habits were different. The infection rate of female over 30 years old is decreasing year by year. The infection risk is high in Hui people and Tibetan, spicy food lovers, smokers and people living at an altitude >3 500 m.

Objective:To evaluate the relationship between declined preoperative left ventricular diastolic function and postoperative increased extravascular lung water (EVLW) in the patients undergoing transurethral resection of the prostate (TURP).Methods:A total of 116 patients, aged 55-90 yr, of American Society of Anesthesiologists physical status Ⅰ-Ⅲ, with body mass index of ≤30 kg/m 2, undergoing elective TURP under general anesthesia, without increased EVLW before surgery, were included in the study.Lung ultrasound examination was performed and lung ultrasound scores were assessed before leaving PACU.Increased EVLW was defined as lung ultrasound score ≥20.The occurrence of increased EVLW after operation was recorded, and patients were divided into increased EVLW group and non-increased EVLW group according to whether increased EVLW occurred.Multivariate logistic regression analysis was used to identify the risk factors for postoperative increased EVLW. Results:The results of multivariate logistic regression analysis showed that declined preoperative left ventricular diastolic function was an independent risk factor for postoperative increased EVLW ( P<0.05). Conclusions:Declined preoperative left ventricular diastolic function is an independent risk factor for postoperative increased EVLW in the patients undergoing TURP.