Objective To identify the pathogens that cause hand, foot and mouth disease (HFMD) in adults and analyze the nucleotide sequences characteristics of enterovirus 71 (EV71). Methods The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the enterovirus from the samples of four adult HFMD patients. The 227 bp amplified segments of EVT1 were then sequenced and compared with the sequences of previously isolated EVT1 strains available from GenBank by homogeneity and phylogenetic tree analyses. Results All the results of RT-PCR with enterovirus universal primers and EVT1 specific primers were positive. The EV71 sequences analysis showed that the four new sequences (named as GZ19610, GZ99310, GZ99355 and GZ46477) shared 96.0% to 99.1% nucleotide identify themselves and shared 96.9% to 100.0% homology with the strain Fuyang/17.08/3 isolated in 2008 from Fuyang, Anhui Province. Phylogenetic tree analysis showed that the genotype of the four new sequences was all subtype C4, they were the same sub-genotype as those strains isolated from Chinese mainland and Chinese Taiwan in 2004, and the genetic distance between them was most closely. Conclusions EV71 can cause adult HFMD. Compared with the nucleotide sequences of EV71 strains that isolated now and formerly in China, there is no large variation of the EV71 sequences isolated from four adult HFMD patients in Guangzhou this time. The adult HFMD patients should be isolated for treatment to avoid them transmitting the virus and causing disease spreading.

detection of HIV-1 quasispecies in HIV-1 infected populations with low level viral load.

Objective To investigate the characteristics of V3 loop amino acid sequences of human immunodeficiency virus type 1 (HIV-1) quasi-species in long-term non-progressors (LTNP)infected with HIV. Methods End-point limiting dilution polymerase chain reaction (EPLD PCR) was used to amplify the env gene c2-v3-c3 region of single HIV-1 provirus from five LTNPs at sequential time points. The PCR products were then sequenced and the amino acid sequences of V3 loop were analyzed by sequence confirm analysis technology. Results The results showed that there were one to ten kinds of polymorphisms in the V3 region of HIV-1 quasi-species which were found from the serial samples of the five LTNP. However, the sequences of the predominant strains were either completely consistent or at most changed at one or two residues in the serial samples of individual patient. The tetramer compositions of the tip of V3 loop were consistent in each patient. It was GPGR in four patients and GPGK in one patient. It was speculated the co-receptor of HIV-1 was CC chemokine receptor (CCR)-5 based on the amino acids at the residue 11 and residue 25 of V3 loop and the net charge of V3 loop. Conclusions There are various polymorphisms at the HIV V3 loop in LTNP. However, the tetramer composition of the tip part of V3 loop is stable. The LTNP are very likely infected with non-syncytium inducing (NSI) strain.

Objective To identify the sequence of hepatitis B virus S gene"a"determinant in patients with positive HBsAg and anti-HBs.Methods Nested-PCR Was used to amplify the HBV S gene in 4 patients with positive HBsAg and anti-HBs,and the PCR products were sequenced directly or after cloning.The sequences of"a" determinants were then analyzed by sequence alignment.Results Direct sequencing of PCR products showed that there was one amino acid (aa)residue in"a"determinant less conserved region emerging polymorphism in all 4 patients.Clone sequencing showed that aa residue at 126 of "a"determinant in patient 1 miSht be Thr,Ile and Set,at 134 might be Phe and Set;the aa at 126 in patient 2 misht be Ala and Thr.and in patient 3 might be Ile and Asn;aa polymorphism was not found in patient 4.Conclusion The polymorphism of"a"determinant in HBV S gene might be associated with positivity of both HBsAg and anti-HBs in hepatitis B patients.

Objective To investigate the epidemiological, clinical and pathogenic characteristics and prognosis of the first imported case of Chikungunya fever in China in 2008. Methods Epidemiological and clinical data of this mate adult patient were analyzed retrospectively. Chikungunya virus (CHIKV)-IgM was detected using enzyme linked immunosorbent assay (ELISA) and colloidal gold immunoassay. CHIKV-RNA was detected using real-time fluorescent polymerase chain reaction (RT-PCR). Results This patient had onset of fever on March 2 in 2008 and lasted for 5 days. In addition, he felt joint and muscle pains, headache and found generalized engorged maculopapule. The laboratory tests showed leukopenia and thrombocytopenia. CHIKV-IgM was detected positive at day 9 after the onset and CHIKV-RNA was all positive at day 3, 5, 7, 9 after the onset. A 575 bp fragment of RT-PCR product was sequenced and detected the nucleotide homology was 99% compared with CHIKV sequences in GenBank. The patient recovered with symptomatic supportive treatment.Conclusion This imported case of Chikungunya virus infection is reported for the first time in China.It shows similar clinical manifestations with dengue fever.

Objective To detect and analyze the haemagglutinin (HA) gene of the first influenza A-H1N1 viral strain isolated in Guangdong Province during an influenza A pandemic in 2009.Methods A-H1N1 virus strain was isolated from the throat swab of the first patient diagnosed with A-H1N1 virus infection in Guangdong Province in 2009. Viral nucleonic acid was extracted from supernatant of cell culture and amplified using reverse transcriptase-polymerase chain reaction (RT-PCR) with HA gene-specific primers. The product was cloned, sequenced, and the homology was analyzed. Results A 1710 bp HA gene of the first influenza A-H1N1 viral strain in Guangdong Province in 2009 was acquired, which was named as A/GuangzhouSB/01/2009 (H1N1) HA with GenBank access No. GQ268003. The homology of the studied HA gene and the 277 influenza A (H1N1) isolates reported in the epidemic areas was 99.0%-99.8%, and as high as 99.8% when compared with the isolates reported in the United States where the patient had traveled. When the studied HA gene was compared with 25 isolates of Chinese seasonal A-H1N1 virus, the homology was 72.3%-85.6%. Conclusions The homology of the first isolated A-H1N1 viral strain in Guangdong Province in 2009 and epidemic influenza A-H1N1 virus is high, while it is low compared with Chinese seasonal A-H1N1 virus.

Objective: To explore the association between preeclampsia/eclampsia and maternal and fetal angiotensinogen SNPs. Methods: From January 2008 to October 2015, a case-parents/mother-control designed study was conducted among 347 preeclampsia/eclampsia cases and 700 controls to collect related information on their demographic characteristics and to detect the related angiotensinogen SNPs’ genotypes. Both log-linear and unconditional logistic regression methods were employed to investigate the genetic effects of maternal/fetal angiotensinogen SNPs on preeclampsia/eclampsia. Multivariate binary unconditional logistic regression model and covariance were used to analyze the relationship between BMI before pregnancy, weight gain during pregnancy and overweight and obesity in preschool children. Results: Both fetal angiotensinogen rs3789679 GA and AA genotype were associated with the reduced risks of preeclampsia/eclampsia, with ORs as 0.73 (95%CI: 0.55-0.96) and 0.62 (95%CI: 0.39-0.98), respectively. For fetal angiotensinogen rs2493132, individuals that carrying the TT genotype, presented a positive association with the risk of preeclampsia/eclampsia, with OR as 1.60 (95%CI: 1.08-2.37). However, these associations were not statistically significant after the correction of the false discovery rate. It was observed that fetal rs3789679 could reduce the risk of preeclampsia/eclampsia (OR=0.73, 95%CI: 0.55-0.96) under the dominant model (GA+AA/GG) while fetal rs2493132 increased the risk of preeclampsia/eclampsia (OR=1.66, 95%CI: 1.13-2.44) under the recessive model (TT/CC+CT). Maternal rs5051 presented an association with preeclampsia/eclampsia (OR=1.33, 95%CI: 1.01-1.76) under the dominant model (TC+CC/TT). Conclusions Results from the dominant model showed that both fetal rs3789679 GA and AA genotype reduced the risk of preeclampsia/eclampsia and maternal rs5051 TC while CC genotype increased the risk of preeclampsia/eclampsia. Fetal rs2493132 TT genotype seemed to be associated with the risk of preeclampsia/eclampsia under the recessive model.

Objective To reveal the pharmacodynamic material basis of Kaixinsan by using high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)and the integrated analysis of"chemical component spectrum-plasma exposure component spectrum-mitochondrial function".Methods Through a review of literature,databases,and previous studies,the chemical components of ginseng,polygala,poria,and acorus were systematically cataloged.A qualitative analysis method for the chemical constituents in the aqueous extract of Kaixinsan was developed,allowing for the identification of its chemical components.A qualitative analysis for rat plasma based on HPLC-MS/MS was established,which was applied to analyze the plasma exposure component spectrum following oral administration of Kaixinsan aqueous extract in rats.Aerobic respiration was evaluated using a seahorse cell energy metabolism analyzer,and the effect of key components of Kaixinsan on mitochondrial aerobic respiration was assessed.Results Four main types of components were identified in the Kaixinsan aqueous extract,including saponins,oligosaccharide esters,xanthones,and triterpenes,comprising a total of 231 identified compounds.Analysis of rat plasma 30 minutes after gavage with Kaixinsan identified 55 compounds.The analysis revealed that ginsenoside Rg1,3,6'-disinapoylsucrose,polygalaxanthone Ⅲ and poricoic acid B could significantly enhance mitochondrial respiratory capacity using in vitro cellular assays to detect aerobic respiration of four main components entered blood.Conclusions Saponins,oligosaccharide esters,xanthones,and triterpenes may be the material basis for the pharmacological effect of Kaixinsan by improving mitochondrial function.The integrated analysis of"chemical component spectrum-plasma exposure component spectrum-mitochondrial function"provides a new approach for in-depth exploration of the material basis underlying the efficacy of traditional Chinese medicine.

ObjectiveTo investigate the mechanism of neferine（Nef） in promoting vascular regeneration against cerebral ischemia through modulation of mitochondrial calcium uniporter（MCU） ion channel. MethodTaking the area of subintestinal vessels in microvascular deficiency zebrafish as an index， the vascular regenerative efficacy of Nef was evaluated， and the median effective concentration（EC₅₀） was calculated. Rats were randomly divided into a sham operation group， a model group， a positive drug group（butylphthalide， 6 mg·kg^-1）， and Nef low， medium， and high dose groups（0.125， 0.625， 3.125 μg·kg^-1）. Except for the sham operation group， the middle cerebral artery occlusion（MCAO） model was established in other groups. After modeling， the groups were administered the corresponding dose of drugs by gavage， while the sham operation and model groups received equal volumes of saline， once a day for 7 consecutive days. Neurobehavioral scores were assessed for each group of rats， and the infarct rate of ischemic brain tissue was calculated by 2，3，5-triphenyltetrazolium chloride（TTC） staining. The regional cerebral blood flow（rCBF） of each group was measured using a speckle contrast imaging. Immunofluorescence and Western blot were conducted to detect the expression of vascular endothelial growth factor（VEGF）， platelet endothelial cell adhesion molecule-1（CD31）， and hypoxia-inducible factor-1α（HIF-1α） proteins in each group. Human umbilical vein endothelial cells（HUVECs） were divided into the normal group， model group， positive drug group（astragaloside Ⅳ， 10 μmol·L^-1）， and Nef group （32 nmol·L^-1）. In the verification of mitochondrial protection of Nef and its mechanism in promoting vascular regeneration， the spermine（MCU agonist） and Nef+spermine group were added. HUVECs model of oxygen-glucose deprivation（OGD） was established in all groups except the normal group， the cell viability was assessed using 3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide（MTT） assay， and cell migration ability was evaluated through scratch and tube formation assays. Fluorescent probes（Rhod-2 AM， Fluo-3 AM， JC-1， Calcein AM） and a cellular energy metabolism analyzer were used to analyze the mitochondrial protective effects of Nef. Molecular docking was performed to predict the binding ability of Nef with MCU and HIF-1α， and Western blot was used to detect the effects of Nef on the protein expressions of MCU， B-cell lymphoma-2 associated X protein（Bax）， Caspase-3 and HIF-1α in the OGD model HUVECs. ResultThe results of vascular regeneration in microvascular deficiency zebrafish showed that compared to the normal group， the area of subintestinal vessels in the model group significantly decreased（P<0.01）. Compared to the model group， different concentrations of Nef could significantly increase the area of subintestinal vessels（P<0.01）， with the maximum tolerated concentration of 10.24 μmol·L^-1 and the EC₅₀ of 0.23 μmol·L^-1. Anti-cerebral ischemia results on MCAO rats showed that compared to the sham operation group， the model group had a significant decrease in rCBF and a significant increase in infarct rate， while CD31 expression significantly decreased（P<0.01）， and VEGF and HIF-1α protein expressions significantly increased（P<0.05）. Compared to the model group， the treated groups showed significant increases in rCBF， significant reductions in infarct volume， and significant increases in CD31， VEGF， and HIF-1α protein expression（P<0.01）. Cell experiment results showed that compared to the normal group， the model group had decreased cell viability and migration ability， increased intracellular Ca²⁺ and mitochondrial Ca²⁺ levels， reduced mitochondrial permeability transition pore（MPTP） opening， and decreased mitochondrial energy metabolism capability， with increased expressions of MCU， Bax， Caspase-3 and HIF-1α proteins（P<0.05， P<0.01）. Compared to the model group， the Nef group showed increased cell viability and migration ability， decreased intracellular Ca²⁺ and mitochondrial Ca²⁺ levels， increased MPTP opening， enhanced mitochondrial energy metabolism capability， decreased expressions of MCU， Bax and Caspase-3 proteins， and increased HIF-1α protein expression（P<0.05， P<0.01）. ConclusionNef can stabilize mitochondrial membrane potential and inhibit mitochondrial apoptosis. By down-regulating the expression of MCU， it suppresses the activation of intracellular Bax and Caspase-3 while activating the HIF-1α signaling pathway， enhancing the expression of VEGF and CD31， thereby promoting vascular regeneration to treat ischemic brain injury.

【Objective】 To retrospectively analyze the transfusion records of COVID-19 patients from Feb to Mar 2020 in the Optical Valley Branch of Tongji Hospital in Wuhan, and summarize the clinical features and blood use of those patients. 【Methods】 1) The utilization of blood components in 81 blood recipients were collected and retrospectively analyzed; 2) Propensity score matching (PSM, by the clinical classification of COVID-19) was used to match the transfused and non-transfused patients according to the ratio of 1∶2, and the clinical features of the two groups were compared. 【Results】 The total transfusion rate in our hospital was 5.5%(81/1 463), among which 88.9%(72/81)transfused red blood cell (RBC). Ten patients received RBC transfusion > 20 U, consumed 48.3%(330/680)RBC, 57.5%(53 500/93 100)plasma, 36.2%(42/116)platelets, and 62.3%(114.25/183.25)cryoprecipitates due to ECMO or gastrointestinal bleeding. Compared to non-transfused patients, transfused patients showed worse lab-indexes related to inflammation, infection, and coagulation at admission, higher incidences of acute liver, kidney and cardiac injury, admission to the ICU and mortality(P<0.01). 【Conclusion】 The related functional indexes and prognosis of transfused COVID-19 patients were significantly worse than non-transfused ones. RBC transfusions were dominant, and massive blood transfusions were seldom.