Objective To establish a duplex fluorescence PCR for detection of HIV proviral DNA and to evaluate its application for early diagnosis of HIV infection in infants .Methods A duplex fluores-cence PCR system was set up based on TaqMan technology for detection of human ribonuclease P ( RNase P) gene and long terminal repeat ( LTR) region of HIV.A recombinant plasmid containing the targeted gene fragment , pTG19-T, was constructed by TA cloning technique and used as the template for evaluation of sen -sitivity of the assay .Blood samples from 11 healthy individuals and 98 HIV-infected patients were collected and detected to validate the assay specificity .The assay of duplex fluorescence PCR was then carried out to detect 96 infant blood samples collected from several maternal and child health hospitals in Zhejiang province from January 2011 to September 2012 for early diagnosis of HIV infection .The results were compared with those by using the Roche HIV DNA qualitative detection kit .Results The established duplex fluorescence PCR could specifically detect HIV proviral DNA with a specificity of 100%and a detection sensitivity of 100 cps per reaction .The coincidence rate between the established assay and the Roche HIV DNA qualitative de -tection kit was 100%in the detection of 96 blood samples .Conclusion The duplex fluorescence PCR as-say showed advantages of cost-effectiveness , convenience , good specificity and accuracy with high sensitivi-ty.It could be used for early diagnosis of HIV infection in infants and also as a general technical platform for the detection of HIV proviral DNA .

Helicobacter pylori (Hp) is a microaerophilic Gram-negative bacterium that colonizes the gastric mucosa. After colonization, many proteins expressed by Hp, such as Helicobacter pylori urease (HPU), neutrophil-activating protein (NAP), outer membrane protein (OMP), cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA), may serve as antigens to mediate neutrophil chemotaxis. Thereafter, neutrophils infiltrate in gastric mucosa and play roles in mucosal immunity via secreting cytokines and releasing reactive oxygen species (ROS) and myeloperoxidase (MPO). This article reviewed the advances in study on the role of neutrophil infiltration in Hp infection-related diseases.

Objective: To understand the molecular subtypes and recombinant structure of HIV-1 infection in Zhejiang Province. Methods: According to the method of stratified random sampling method, a total of 302 newly HIV infected people (excluding AIDS patients) were selected as the subjects of the investigation from April to June, 2015, who were all newly HIV positive patients reported in Zhejiang Province and were diagnosed as HIV positive and had not received antiretroviral treatment. Questionnaires were used to collect information such as gender, residency, marital status, route of transmission and case reporting area. At least 8 ml of venous blood was collected from HIV infected people, reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR were used to amplify the pol gene sequence of HIV-1 strains and circulating recombination forms (CRF) and unique recombinant form (URF) strains were screened using phylogenetic tree, and molecular subtype analysis and recombinant analysis were performed. Results: Of the 302 HIV infected people, 18 cases were infected with new CRFs or URFs, among which 15 were male (83.3%), 3 were heterosexual transmission (17.7%); 8 cases (44.4%) were infected with new CRFs, which included CRF55_01B 5(27.8%), CRF67_01B 2 (11.1%), CRF69_01B 1 (5.6%); 10 cases (55.6%) were infected with URFs, in which 6 cases were infected with two groups of recombinant strains composed of CRF01_AE subtype and CRF07_BC subtypes in pol region, 3 cases were infected with three groups of recombinant strains of the two subtypes in pol region, and 1 case could not be accurately determined of the recombinant structure. The results of recombinant source analysis showed that the newly recombinant strains found from Zhejiang province were highly homologous to the strains from Guangdong, Anhui, Hebei, Yunnan, Liaoning province, and the sequence similarity was 94.4%-98.0%. Conclusion The new recombinant strains of Zhejiang province included CRFs strains and URFs strains with CRF01_AE and CRF07_BC as the main recombinant structure mode, and all of them were introduced from other places and formed a trans-regional chain of HIV transmission.

Objective To understand the characteristics of distribution on HIV-1 subtypes and the transmission clusters in Yiwu in Zhejiang province.Methods A cross-sectional study of molecular epidemiology was carried out on newly reported H1V/AIDS cases in Yiwu.RNA was extracted from 168 plasma samples,followed by RT-PCR and nest-PCR for pol gene amplification,sequencing,phylogenetic tree construction used for analyzing the subtypes and transmission clusters.Mutations on drug resistance was analyzed by CPR 6.0 online tool.Results Subjects were mainly males (86.3％,145/168),with average age as (39.1 ± 13.4) years old and most of them were migrants (66.7％,112/168).The major routes of transmission included homosexual (51.2％,86/168) and heterosexual (48.8％,82/168) contacts.The rate of success for sequence acquisition was 89.9％ (151/168).The dominant subtypes showed as CRF01_AE (74,49.0％) and CRF07_BC (64,42.4％),followed by CRF08_BC (5,3.3％),CRF55_01B (3,2.0％),each case of subtype B,CRF45_cpx,CRF59_01B,CRF85_BC and URF (B/C).CRF45_cpx and CRF85_BC were discovered the first time in Zhejiang province.Twenty-six transmission clusters involving 65 cases were found,with the total clustered rate as 43.0％ (65/151),in which the CRF01_AE clustered rate appeared as 54.1％ (40/74),higher than that of CRF07_BC (21/64,32.8％).The average size of cluster was 2.5 cases/cluster,with average size of cluster in CRF01_AE patients infected through heterosexual transmission as the largest (3.5 cases/cluster).The prevalence of transmitted drug resistance was 4.6％ (7/151).Seven cases with surveillance drug resistant mutations (SDRM) were found,including 5 cases of M46L (3.3％),and one case of F77L or Y181C.Conclusion HIV genetic diversity and a variety of transmission clusters had been noticed in this study area (Yiwu).Programs on monitoring the subtypes and transmission clusters should be continued and strengthened.

Objective:To investigate the factors associated with delay in anticoagulant therapy in patients with cerebral venous sinus thrombosis (CVST) and its effect on outcome.Methods:Patients with CVST admitted to Changhai Hospital, Naval Medical University from January 2010 to August 2021 were retrospectively enrolled. Patients were divided into early anticoagulation group and late anticoagulation group by the median time interval from first symptom to initiation of anticoagulation. The modified Rankin Scale was used for outcome assessment at 90 d after onset. 0-2 scores were defined as good outcome and 3-6 were defined as poor outcome. Demographic and clinical data were compared for the early versus late anticoagulation group and for the good versus poor outcome groups. Multivariable logistic regression was used to identify independent influencing factors of delay in anticoagulation and the correlation of delay in anticoagulation with poor outcome. Results:A total of 131 patients were included, their age was 40.07±15.11 years old, and 68 (51.91%) were male. Of these, 65 patients (49.62%) were in the early anticoagulation group and 14 (10.69%) were in the poor outcome group. Compared with the late anticoagulation group, the early anticoagulation group had a significantly higher proportion of patients with seizures and brain parenchymal damage as well as higher D-dimer levels on admission, while the proportion of patients with visual impairment/papilloedema was significantly lower (all P<0.05). Compared with the good outcome group, the poor outcome group had significantly higher proportions of patients with seizures, dyskinesia, impaired consciousness, low Glasgow Coma Scale score, and brain parenchymal damage as well as higher D-dimer, total cholesterol and low density lipoprotein cholesterol levels, sites of thrombus involvement were more common in the superior sagittal and straight sinuses, and significantly lower proportions of patients with headache and lower albumin levels on admission (all P<0.05). Multivariate logistic regression analysis showed that visual impairment/papilloedema (odds ratio [ OR] 0.119, 95% confidence interval [ CI] 0.030-0.473; P=0.002) and brain parenchymal damage ( OR 1.341, 95% CI 1.042-1.727; P=0.023) were independently associated with a delay in anticoagulation treatment, and a delay in anticoagulation treatment ( OR 6.102, 95% CI 1.185-30.504; P=0.030) and D-dimer level on admission ( OR 1.299, 95% CI 1.141-1.480; P<0.001) were the independent predictors of poor outcome in patients with CVST. Conclusions:Visual impairment/papilloedema and absence of brain parenchymal damage on cranial imaging are the independent risk factors for delay in anticoagulation in patients with CVST. The delay in anticoagulation is strongly associated with the poor outcome in patients with CVST.

OBJECTIVETo understand the HIV-1 subtype diversity and transmission characteristics in men having sex with men (MSM) in Zhejiang province.

METHODSA total of 233 newly diagnosed as HIV-1 positive patients in 2011 were screened out by BED capture enzyme immunoassay (BED-CEIA). Among them, 107 eligible subjects were enrolled for further molecular epidemiological study. Viral RNA was extracted from plasma samples and followed by reverse transcription PCR and nested PCR for amplification of pol gene fragments, sequencing, and bioinformatics analysis.

RESULTSThere were no statistically significant differences regarding the social demographic distribution between the subjects under study and those recently infected MSM population. The rate of success for sequence acquisition was 94.4% (101/107). The highest proportion of subtype was CRF01_AE (62.4%), followed by CRF07_BC (31.7%) and with three cases of subtype B, one case of CRF55_01B and two cases of unique recombinant form (CRF01_AE/B and CRF01_AE/CRF07_BC). The phylogenetic trees were mainly divided into CRF01_AE cluster 1, cluster 2 and CRF07_BC cluster 3. The strains located in Hangzhou were diffused in the branches of phylogenetic tree. 10 transmission clusters were found, in which 80% involved two or more regions and 90% was associated with patients residing in Hangzhou. Three surveillance drug resistance mutations (M46I, T215S and G190A) were found in three samples (each sample harbored only one resistance mutation). The overall rate of transmitted drug resistance (TDR) was 2.97%.

CONCLUSIONThe increasing complexity of HIV was noticed in MSM in Zhejiang province. However, the prevalence of TDR was low. Cross-regional HIV transmission in MSM was common, which inferred from the study. Hangzhou might play a central regional role in the intra-provincial spread of HIV, to form an interwoven complex network in the MSM population.

China ; epidemiology ; Demography ; Drug Resistance, Viral ; Genes, pol ; HIV Infections ; genetics ; transmission ; HIV-1 ; classification ; Homosexuality ; Humans ; Immunoenzyme Techniques ; Male ; Phylogeny ; Prevalence ; RNA, Viral

Effective expression of pIFN-α in recombinant Pichia pastoris was conducted in a 5 L fermentor. Ethanol accumulation during the late glycerol feeding period inhibited heterologous protein expression. Comparative transcriptome analysis was thus performed to compare the gene transcription profiles of Pichia pastoris KM71H in high and low ethanol concentration environments. The results showed that during the glycerol cultivation stage, 545 genes (265 up-regulated and 280 down-regulated) were differentially expressed with ethanol stress. These genes were mainly involved in protein synthesis, energy metabolism, cell cycle and peroxisome metabolism. During the methanol induction stage, 294 genes (171 up-regulated and 123 down-regulated) were differentially expressed, which were mainly related to methanol metabolism, amino acid metabolism and protein synthesis. Ethanol stress increased protein misfolding and reduced structural integrity of ribosome and mitochondria during cultivation stage, and led to the failure of endoplasmic reticulum stress removal and damaged amino acid metabolism during induction stage in Pichia pastoris.
Amino Acids ; metabolism ; Bioreactors ; Endoplasmic Reticulum Stress ; Energy Metabolism ; Ethanol ; chemistry ; Gene Expression Profiling ; Gene Expression Regulation, Fungal ; Glycerol ; Methanol ; Pichia ; metabolism ; Protein Biosynthesis ; drug effects ; Protein Folding ; Recombinant Proteins ; biosynthesis ; Transcriptome