Objective To observe the transfection efficiency and anti-fibrotic effect of miR-29b transfected by anti-TGF-β Ⅱ R ScFv/Ck/tP fusion protein (new vector) in hepatic stellate cell (HSC),and to provide a new vector in gene therapy for liver fibrosis.Methods The liposome vector,new vector,and lentiviral vector were used as transfection reagents to transfect miR-29b into HSC.Transfection efficiency was observed under fluorescence microscope and flow cytometry.Collagen α1 (Ⅰ) mRNA and protein expression in different groups were analyzed by real-time RT-PCR and Western Blot,respectively.Results Compared to the control,transfection efficiencies in lentiviral vector,new vector,and liposome vector groups were about 70％,58％,and 29％,respectively.Collagen α1 (Ⅰ) mRNA expression in lentiviral vector,new vector,and liposome vector groups was decreased by about 70％,50％,and 38％,respectively ((t =6.316,P ＜0.01 ; t =4.082,P ＜0.01 ; t =3.014,P ＜0.05).Collagen α1(Ⅰ) protein expression in lentiviral vector,new vector,and liposome vector groups was decreased by about 59％,41％,and 27％,respectively (t =4.209,P ＜0.01; t =4.033,P ＜0.01; t =2.842,P ＜0.05).Conclusions The new vector constructed by us has a high transfection efficiency.MiR-29b transfected by the new vector has a good anti-liver fibrosis effect.

Objective: To learn the genotypic drug resistance of men who have sex with men ( MSM ) with HIV who failed in antiviral therapy in Yunnan Province, in order to provide basis for improving the effect of antiviral therapy. Methods: The patients who were infected with HIV-1, homosexual transmitted and failed in antiviral therapy in Yunnan Province from 2014 to 2019 were recruited. Their plasma samples were tested by reverse transcription nested polymerase chain reaction ( RT-nPCR ) , the fragments were spliced using ContigExpress, and the resistance to 8 protease inhibitors ( PIs ) , 7 nucleoside reverse transcriptase inhibitors ( NRTIs ) and 5 non-nucleoside reverse transcriptase inhibitors ( NNRTIs ) were obtained from the HIV drug resistance data website of Stanford University. Results: A total of 205 HIV/AIDS cases were included, 169 positive plasma samples were amplified, 112 cases were drug resistant, and the rate of drug resistance was 66.27%. The patients who were aged 30-49 years ( 76.09% ) , had genotype of CRF01_AE ( 76.34% ) or treated by AZT+3TC+NVP ( 77.08% ) had higher resistance rate. The resistance rates of NNRTIs, NRTIs and PIs were 62.72%, 49.70% and 2.96%, respectively; the resistance rates of NVP and EFV in NNRTIs were 62.72% and 61.54%. The main mutation site associated with NNRTIs was K103, accounting for 21.89% ( 37 cases ) ; the main mutation site associated with NRTIs was M184, accounting for 39.64% ( 67 cases ) ; the main mutation sites associated with PIs were M46L/K, accounting for 2.96% ( 5 cases ) , resulting in high resistance to NFV. Conclusions The drug resistance rate of HIV-infected MSM with failure of antiviral therapy in Yunnan Province is relatively high, with CRF01_AE as the main gene subtype of drug resistance. The drug resistance rate of NNRTIs is relatively high, especially NVP and EFV.

Objective To investigate the subtypes distribution of human immunodeficiency virus (HIV)-1 epidemic strains and the characteristics of amino acid variation in different areas of Yunnan Province.Methods Totally 800 HIV/AIDS plasma specimens and epidemiological information were collected between 2012 and 2015 from 14 areas of Yunnan.Viral RNA was extracted and amplified using RT polymerase chain reaction (PCR).4.5 kb 5'halves fragments were obtained and directly sequenced.Subtypes of strains were identified by Genotyping,MEGA 6.06 and BLAST.Grouping was analyzed by location and subtype.Entropy software was used to analyze the difference of amino acid sequences between different groups according to the sampling location and subtypes to analyze the regional distribution and genetic variation of HIV-1 subtypes in Yunnan Province.Results Of the 800 plasma specimens,a total of 446 genomic sequences from 12 areas were successfully amplified and sequenced.After genotypes were identified,the subtypes of HIV-1 strains prevalent in Yunnan were CRF08_BC (58.3％),CRF01_AE (19.3％),CRF07_BC (11.6％),unknown recombinant forms (7.1％),B(B') (1.7％) and C (1.3％).The geographical distribution in Yunnan was analyzed.The CRF01_AE predominated in Dehong,Xishuangbarma and Wenshan.The CRF08_BC predominated in Lincang,Honghe and Puer (more than 70.0％) and CRF08_BC was prevalent in the other areas.But CRF07_BC in Kunming,Yuxi and Dali accounted for more than 20 ％.The constitutions of amino acid of three majors CRF08_BC,CRF01 _AE and CRF07_BC were different on 17,14 and 18 amino acid sites with statistical differences in the eastern and western regions of Yunnan Province (均P ＜ 0.05).Conclusions HIV-1 strains transmit and vary genetically in the province widely.The amino acid mutation sites of eastern and western strains are different.This difference represents that the same subtype strains in different geographical distribution vary on different genetic background and are selected by immune responses.The epidemic trends need to be closely monitored.

Objective To investigate the influence factors of mortality among human immunodeficiency virus (HIV)-infected children under highly active antiretroviral therapy (HAART).Methods Retrospective cohort study of 652 children initiated HAART from 2005 to 2014 was conducted,and enrolled patients were followed-up until December,2015.Survival data was analyzed using Kaplan-Meier method and Cox regression model was used to identify independent predictors of mortality among these children on HARRT.Chi-square test and Fisher's exact test were used for comparison between groups.Results Overall,26 of the children died over a follow-up period of 3 116.24 child-years,with a mortality rate of 0.83 per 100 child-years.Twelve (46％)of deaths occurred during the first six months after starting HAART.Cox regression analysis of variables showed that the World Health Organization (WHO) clinical stages Ⅲ/Ⅳ (hazard rate [HR] =10.717,95％confidence interal [95％ CI]:4.189-4.749,P =0.000),baseline hemoglobin ＜ 80 g/L (HR =14.768,95 ％ CI:5.721-38.125,P =0.000),tuberculosis co-infection (HR =4.794,95％ CI:2.105-10.918,P =0.000),baseline CD4+T lymphocyte ＜ 50 cells/μL (HR =4.219,95％ CI:1.524-11.680,P =0.006),weight-for-age z-score ＜-2 (HR =2.983,95 ％ CI:1.094-8.135,P =0.033) were independently associated with death,whereas the age ＜ 7 year-old at HAART initiation was protectire (HR =0.293,95％ CI:0.126-0.684,P =0.005).Conclusions The mortality of children receiving HAART is strongly associated with WHO stages Ⅲ/Ⅳ,hemoglobin ＜ 80 g/L,weight-for-age z-score ＜-2,tuberculosis co-infection and older age at treatment.

Objective To detect the molecular regulatory mechanism of co-treatment with LA and PCA on P38 MAPK signaling Pathway in the Neurons of Wilson's Disease Model-TX mice.Methods The neurons of TX suckling mice were isolated and cultured by primary method,and were divided into control group,model group,ALA group,PCA group and combined group.Flow cytometry was used to analyze the expression of ROS and JC-1.Western blot was used to detect the expression of P38 MAPK,Cyt C,Caspase 9 and Caspase 3.Results Flow cytometry results showed that MFI of ROS was 59.29±1.22,53.19±1.34 and 52.46±1.23 in ALA,PCA and co-treatment.ALA,PCA and co-treatment could significantly reduce the release of ROS and enhance the fluorescence intensity of JC-1 (P＜0.01).Compared with ALA group and PCA group,combined group could reduce the release of ROS and significantly enhance the fluorescence intensity of JC-1.Western blot indicated that the expression levels of P38 MAPK,Cyt C,Caspase 9,Caspase 3 in the neurons of model group had a remarkable increase compared with control group.Compared with the model group,the three treatment groups could decrease the expression levels of P38 MAPK,Cyt C,Caspase 9 and Caspase 3 in the neurons of TX suckling mice (P＜0.01).Meanwhile,the protein levels of P38 MAPK,Cyt C,Caspase 9 and Caspase 3 had a significant decrease compared with ALA group and PCA group.Conclusion he present findings suggest that co-treatment with LA and PCA can increase the copper excretion,reduce copper-induced mitochondria damage and attenuate the neurotoxicity,which in turn decrease neuronal apoptosis and improve neurological impairment of WD.

Objective:To explore the relationship between drug resistance occurrence and the distribution pattern of polymorphic loci in individuals with human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) treated with highly active anti-retroviral therapy (HAART).Methods:HAART-failed HIV/AIDS patients who successfully amplified the gene sequences of the pol region between June 2015 and December 2021 from 16 prefecture-level administrative regions in Yunnan Province were included.The resistant sequences were classified using the human immunodeficiency virus (HIV) basic local alignment search tool (BLAST) and validated through MEGA 6.0, and the obtained sequences were submitted to the Stanford University HIV Drug Resistance Database to identify drug resistance loci. The distribution of polymorphic loci was analyzed across patients exhibiting varying degrees of drug resistance, different treatment regimens and distinct HIV-1 subtypes.Changes of the frequencies of polymorphic loci in patients with different degrees of drug resistance were analyzed using trend chi-square test. Statistical comparisons and further paired comparisons were performed using chi-square test.Results:Gene sequences were amplified from 1 453 patients, and the resistance testing results showed 954 sensitive, 224 potentially or low resistant, 189 moderately resistant, and 86 highly resistant patients. The frequencies of mutations I15V, L19I, D60E in the HIV-1 protease region (PR region) and E36A, T39D, S48T mutations in the HIV-1 reverse transcriptase region (RT region) showed a decreasing trend as the degree of HIV-1 resistance escalated ( χ2trend=19.86, 9.16, 13.66, 37.64, 18.44 and 40.86, respectively, all P<0.01). Conversely, the mutations V77I in the PR region and K122E in the RT region showed an ascending trend ( χ2trend=12.19 and 10.03, respectively, both P<0.01). Distinct treatment groups, namely zidovudine (AZT)+ lamivudine (3TC)+ lopinavir/ritonavir (LPV/r), AZT+ 3TC+ efavirenz (EFV), AZT+ 3TC+ nevirapine (NVP), and tenofovir (TDF)+ 3TC+ EFV, were examined. Statistically significant differences in the frequencies of mutations E35D, M36I, and D60E in the PR region, as well as S48T, K122E, and R211K in the RT region, were observed among these treatment groups ( χ2=22.46, 9.32, 14.46, 26.85, 18.92 and 24.26, respectively, all P<0.05). In paired comparisons, AZT+ 3TC+ LPV/r group displayed higher frequencies of E35D, M36I, and D60E mutations, the AZT+ 3TC+ EFV group showed a higher frequency of S48T mutation, the AZT+ 3TC+ NVP group showed a higher frequency of K122E mutation, and the TDF+ 3TC+ EFV group exhibited a higher frequency of R211K mutation, all with statistically significant differences (all P<0.008). The differences in the frequencies of T12S, I15V, L19I, M36I, V77I, L89M in the PR region and E53D, I135V, S162C, R211K, K277R in the RT region among circulating recombinant form (CRF)08_BC, CRF07_BC and CRF01_AE subtype group were statistically significant ( χ2=693.60, 712.51, 798.11, 434.85, 386.91, 657.78, 932.58, 409.21, 344.39, 469.44 and 260.48, respectively, all P<0.001). In paired comparisons, the frequencies of T12S, I15V, L19I, E53D, I135V, S162C and R211K in CRF08_BC subtype, the frequencies of V77I and K277R in CRF07_BC subtype, and the frequencies of M36I and L89M in CRF01_AE subtype were higher than those in the other two groups, and the differences were all statistically significant (all P<0.017). Conclusions:The polymorphic loci resulting from HIV-1 HAART failure show different distribution patterns across various degrees of drug resistance, treatment regimens and HIV-1 subtypes.These loci demonstrate both specific and shared characteristics. It is necessary to enhance the surveillance of select polymorphic loci.

Objective:To analyze the variation characteristics of gag gene sequence of human immunodeficiency virus type 1 (HIV-1) epidemic strains in four major acquired immunodeficiency syndrome (AIDS) endemic areas in Yunnan Province. Methods:A total of 480 human immunodeficiency virus (HIV)/AIDS patients from designated antiviral treatment institutions in Kunming City, Dehong Dai and Jingpo Autonomous Prefecture, Hani-Yi Autonomous Prefecture of Honghe and Lincang City in Yunnan Province from January 2019 to December 2020 were randomly selected. The epidemiological information of the patients was collected. The plasma samples were collected and sent to the Yunnan Provincial Infectious Diseases Hospital for analysis. HIV-1 gag gene was amplified by nested polymerase chain reaction for genotyping.The phylogenetic tree was constructed by MEGA 6.06, and the characteristic amino acids were analyzed by VESPA online analysis tool. The gene distances of gag and matrix protein p17, capsid protein p24, nucleocapsid protein p7 and p6 protein of gag protein were calculated by Distance program. The ratio of synonymous mutation frequnency and non-synonymous mutation frequnency (Ks/Ka) was analyzed by SNAP program. Statistical comparison among multiple groups was performed using analysis of variance. Results:The gag gene sequences were successfully obtained from 404 patients.Most of these patients were men (250 cases, 61.9%), 59.7%(241/404) of patients were aged 40 to 59 years, and the main transmission route was heterosexual transmission (61.4%, 248/404). The main epidemic subtypes of HIV-1 were circulating recombinant form(CRF)08_BC (38.4%, 155/404), CRF01_AE (18.3%, 74/404), unique recombinant form (URF) (12.9%, 52/404), CRF07_BC(9.7%, 39/404), C subtype (8.4%, 34/404), other subtypes (6.9%, 28/404) and B subtype (5.4%, 22/404). Two main spreading clusters were formed by CRF08_BC in the phylogenetic tree, and there were significant differences in the distribution of characteristic amino acids of eight loci between the two spreading clusters, including No.79, 93, 121, 122, 151, 363, 395 and 396. The gag gene distances of CRF01_ AE, CRF07_ BC and CRF08_ BC were 0.090±0.004, 0.088±0.004 and 0.078±0.002, respectively, and the difference was statistically significant ( F=118.33, P<0.001). The Ks/Ka values of gag of the three subtypes were 4.003±1.309, 4.141±0.860 and 4.514±1.215, respectively, and the difference was statistically significant ( F=1.35, P<0.001). The Ks/Ka values of p17 were 2.590±0.186, 2.831±0.496 and 2.936±0.475, respectively, and the difference was statistically significant ( F=1.59, P<0.001). The Ks/Ka values of p24 were 12.579±1.116, 10.185±0.494 and 8.522±0.595, respectively, and the difference was statistically significant ( F=1.61, P<0.001). The Ks/Ka values of p7 were 10.850±0.711, 9.717±0.932 and 8.522±0.026, respectively, and the difference was statistically significant ( F=4.24, P<0.001). The Ks/Ka values of p6 were 3.122±0.134, 3.040±1.498 and 4.841±0.353, respectively, and the difference was statistically significant ( F=10.68, P<0.001). Conclusions:CRF08_ BC is the main subtype in these four areas in Yunnan Province.The different clusters of CRF08_BC result in a different pattern of amino acid composition.The variation degree of different subtype gag gene is different in each section. Surveillance of HIV variant strains should be strengthened to control the epidemics of HIV.

Objective To study the effect of high copper on Wnt /β-catenin signaling pathway and oxidative stress. Methods BRL-3A cells were incubated with different concentrations of CuSO4.The cell growth and proliferation wre assessed using MRR method.The production of reactive oxygen species(ROS)and mitochondrial membrane potential (JC-1) were examined usingf flow cytometry. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were detected by microplate reader. The expression of protein was detected by Western Blot. Results①The results of MTT showed that CuSO4inhibited the growth and proliferation of BRL-3A cells in a time- and concentration-dependent manner(P<0.01).②Flow cytometry results showed that CuSO4induced a large amount of ROS and significantly decreased the fluorescence intensity of JC-1 in BRL-3A cells (P<0.01). ③ Microplate reader results showed that CuSO4increased the content of MDA and decreased the activity of SOD (P<0.05).④Western blotting assay showed that CuSO4significantly decreased the total expression levels of β-catenin and p-Ser 9-GSK-3β protein as well as nuclear levels of p-(S33+S37)-β-catenin and c-Myc (P<0.01) and increased expression levels of GSK-3β、DKK1、Dishevelled3 protein in BRL-3A cells. Conclusion High copper can induce oxidative stress and induce Wnt /β-catenin signaling pathway to deactivate liver cells,leading to hepatocellular injury.

Objective:To investigate the molecular network of HIV-1 CRF01_AE in Yunnan Province and the factors influencing it.Methods:Demographic data and plasma samples of HIV/AIDS patients in Yunnan drug resistance monitoring database from 2018 to 2021 were collected. HIV-1 pol gene fragments (protease and reverse transcriptase region) were amplified using RT-PCR and then sequenced. The optimal gene distance was selected and a molecular network was constructed based on the sequences of CRF01_AE genotype. Results:In this study, a total of 967 sequences of CRF01_AE genotype were obtained by sequencing. At the optimal gene distance threshold of 1.75%, a total of 320 sequences were involved in the network with a rate of 33.1%, and 84 clusters were identified. In the regional distribution, one cluster dominated by multiple regions, one cluster dominated by Zhaotong, one cluster dominated by Honghe and five clusters dominated by Wenshan were formed in the network. In the network, 75.8% of heterosexual men were connected with other heterosexual men and 54.1% were connected with heterosexual women. There was potential transmission among 66.7% of men who have sex with men (MSM). HIV/AIDS patients in Chuxiong, Dali, Dehong, Honghe, Lincang, Pu′er, Wenshan, Yuxi and Zhaotong were more likely to be involved in the network that those in Kunming. People who were 50 years old and above were more likely to be involved in the network than those less than 25 years old. Factors influencing HIV/AIDS patients with HIV-1 CRF01_AE infection to become high-risk transmitters in Yunnan were not found and further study on this subject was needed.Conclusions:HIV-1 CRF01_AE strains had spread actively in different regions of Yunnan Province and the transmission network was complex. Dynamic monitoring of CRF01_AE strains should be strengthened and a precise intervention for high-risk transmitters should be performed to reduce new infections.

Objective:To investigate the urodynamic characteristics in Parkinson's disease(PD)versus multiple system atrophy(MSA)patients with lower urinary tract symptoms(LUTS).Methods:We performed a retrospective study in PD and MSA patients admitted to the First Affiliated Hospital of Zhengzhou University and undergone urodynamic examinations from January 2016 to June 2019.A total of 178 patients, mean age(59.2±9.7)years were enrolled, with 64 PD patients, 74 MSA patients and 40 normal controls.Urodynamic parameters included maximum flow rate(Qmax), post-voided residual urine volume(PVR), bladder compliance(BC), overactive bladder(OAB), maximum cystometric capacity(MCC)and detrusor pressure at maximum flow rate(PdetQmax). Bladder function was assessed.Results:Frequent urination(68.8%)was the most common LUTS in PD patients, as opposed to urinary retention(91.9%)in MSA patients.The Qmax, PdetQmax and incidence of OAB were higher and the PVR were lower in PD patients than in MSA patients [free-flow(FF)-Qmax: (13.5±7.1)ml/s vs.(10.1±5.2)ml/s, U=26.98, P<0.01]; pressure-flow study(PFS)-Qmax: [(13.6±5.7)ml/s vs.(10.5±3.3)ml/s, U=34.90, P<0.01]; PFS-PdetQmax: [(23.9±11.3)cm H 2O vs.(16.3±8.6)cmH 2O, U=35.04, P<0.01]; OAB: (46.9% vs.27.0%, χ2=5.85, P<0.01); FF-PVR: [(30.4±20.0)ml vs.(161.7±79.8)ml, U=-71.81, P<0.01]; PFS-PVR: [(65.9±30.7)ml vs.(212.6±83.0)ml, U=-65.29, P<0.01]. Compared with the control group, the incidences of OAB and PFS-PVR were increased and the MCC and PdetQmax were decreased in the PD group(OAB: 46.9% vs.7.5%, χ2=6.15, P<0.018); PFS-PVR: [(65.9±30.7)ml vs.(22.2±10.4)ml, U=47.25, P<0.01]; MCC: [(305.1±79.7)ml vs.(389.6±65.2)ml, U=-52.13, P<0.01]; PdetQmax: [(23.9±11.3)cmH 2O vs.(37.3±10.3)cmH 2O, U=-49.88, P<0.01]. Compared also with the control group, the MSA group had a lower Qmax, PdetQmax and MCC, FF-Qmax: [(10.1±5.2)ml/s vs.(16.3±4.7)ml/s, U=-50.11, P<0.01]; PFS-Qmax: [(10.5±3.3)ml/s vs.(13.1±5.0)ml/s, U=-27.54, P<0.05]; PdetQmax: [(16.3±8.6)cmH 2O vs.(37.3±10.3)cmH 2O, U=-84.92, P<0.01]; MCC: [(284.3±71.8)ml vs.(389.6±65.2)ml, U=-39.31, P<0.01], a higher PVR, lower bladder compliance(BC)and a higher incidence of OAB(FF-PVR: [(161.7±79.8)ml vs.(22.0±13.0)ml, U=84.82, P<0.01]; PFS-PVR: [(212.6±83.0)ml vs.(22.2±10.4)ml, U=112.54, P<0.01]; BC: (28.4% vs.7.5%, χ2=6.81, P<0.01); OAB: (27.0% vs.7.5%, χ2=17.62, P<0.01). Conclusions:PD and MSA patients with LUTS have bladder dysfunction.MSA patients have more serious bladder dysfunction than PD patients.