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Dihydrofolate reductase (DHFR), a housekeeping enzyme in primary metabolism, has been extensively studied as a model of acid-base catalysis and a clinic drug target. Herein, we investigated the enzymology of a DHFR-like protein SacH in safracin (SAC) biosynthesis, which reductively inactivates hemiaminal pharmacophore-containing biosynthetic intermediates and antibiotics for self-resistance. Furthermore, based on the crystal structure of SacH-NADPH-SAC-A ternary complexes and mutagenesis, we proposed a catalytic mechanism that is distinct from the previously characterized short-chain dehydrogenases/reductases-mediated inactivation of hemiaminal pharmacophore. These findings expand the functions of DHFR family proteins, reveal that the common reaction can be catalyzed by distinct family of enzymes, and imply the possibility for the discovery of novel antibiotics with hemiaminal pharmacophore.
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Over the past few years, hepatitis type E has been increasingly recognized as an underestimated global disease burden. Populations with severe infection-related injuries or deaths include pregnant women, patients with underlying liver disease, and the elderly. Vaccines are the most effective means to prevent hepatitis type E virus (HEV) infection. However, the development of inactivated or attenuated vaccines is not feasible due to the lack of an efficient HEV cell culture system, so researchers have conducted in-depth research on recombinant vaccines. The capsid protein (pORF2), which the virion's open reading frame 2 encodes, contains almost exclusively the HEV neutralization site. Several candidate vaccines based on pORF2 have demonstrated potential for primate protection, with two being well tolerated and highly effective in preventing hepatitis type E in adults. Hecolin® (HEV 239 vaccine), the world's first hepatitis type E vaccine, was approved for marketing in China in 2012.
Subject(s)
Pregnancy , Animals , Humans , Female , Hepatitis E virus , Hepatitis , ChinaABSTRACT
Objective: To determine the optimal manipulation parameters in Liu's back tuina therapy for remission-stage cough variant asthma (CVA) in kids. Methods: A 5-factor 3-level L18(37) orthogonal design was adopted. The experimental factors and levels were selected: A, the number of times of Rou-kneading Feishu (BL 13); B, the frequency of Rou-kneading Feishu (BL 13); C, the number of times of Tui-pushing in a '介'-like pattern; D, the frequency of Tui-pushing in a '介'-like pattern; E, the intensity of red color after Ca-scrubbing Feishu (BL 13) with salt. A1, A2 and A3 stood for 10 times, 20 times and 30 times, respectively; B1, B2 and B3 stood for 200 times/min, 240 times/min and 280 times/min, respectively; C1, C2 and C3 stood for 50 times, 75 times and 100 times, respectively; D1, D2 and D3 stood for 150 times/min, 175 times/min and 200 times/min, respectively; E1, E2 and E3 stood for pinkish, bright red and dark red, respectively. A total of 108 kids with CVA in remission stage were divided into 18 groups using the random number table method, with 6 cases in each group. Changes in the cough score and fractional exhaled nitric oxide (FENO) level were observed after 15-day treatment. Results: The cough score and FENO level changed significantly in every group after treatment (all P<0.01). By the effect in improving cough score, the factors were listed as D, B, A, C and E in descending order; the most optimal manipulation parameter combination was group 17 (A3B2C1D3E1); the second optimal parameter combinations were group 7 (A3B1C2D1E3), group 13 (A2B1C2D3E1) and group 14 (A2B2C3D1E2). Regarding the effect in down-regulating the level of FENO, the factors were listed as C, A, E, B and D in descending order; the most optimal manipulation parameter combination was group 13 (A2B1C2D3E1); the second optimal parameter combinations were group 18 (A3B3C2D1E2), group 16 (A3B1C3D2E3) and group 14 (A2B2C3D1E2).Conclusion: Two most effective manipulation parameter combinations were selected out of 18 different parameter combinations in Liu's back tuina therapy for remission-stage CVA in kids. Doctors can choose an appropriate manipulation based on the symptom features of the disease.
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Objective To investigate the epidemiological characteristics and main risk factors of chronic kidney disease in the elderly in Tianmen, Hubei Province. Methods A descriptive method was adopted in this study. Using stratified sampling, a total of 720 local residents over 65 years old in Tianmen of Hubei Province were selected from January 2017 to January 2020 as the research subjects. The general information of patients with chronic kidney disease was compared, and the difference in the characteristics of patients with different disease progression was compared. The risk factors for chronic kidney disease and disease progression were analyzed. Results Through the diagnosis of chronic kidney disease, a total of 82 patients with chronic kidney disease were found, including 33 patients in stage I, 26 patients in stage II and 23 patients in stage III. As the disease progressed, the patient's age, serum uric acid, triglyceride, total cholesterol, fasting blood glucose, serum creatinine, and the incidence of chronic diseases all increased significantly. The results of correlation analysis showed that the incidence of chronic kidney disease and the progression of the disease were positively correlated with age, body mass index, serum uric acid, triglyceride, total cholesterol, fasting blood glucose, serum creatinine level, diabetes, hypertension, and dyslipidemia, which were independent risk factors for chronic kidney disease. Conclusion The prevalence of chronic kidney disease in Tianmen was 11.39%. Higher age, serum uric acid, blood lipid, blood pressure and blood glucose levels are all important factors for the progression of chronic kidney disease.
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Accumulating evidence suggested that long noncoding RNAs (lncRNAs) possess a potential role in prostate cancer (PCa) diagnosis and prognosis. Rapid biochemical recurrence (BCR) is considered as a sign for clinical recurrence metastasis and PCa-specific mortality. Hence, the aim of the present study was to identify a lncRNA signature that can predict BCR of PCa accurately. Bioinformatics analysis, Kaplan-Meier analyses, Cox regression analyses, and Gene Set Enrichment Analysis (GSEA) were performed in a publicly available database with 499 PCa tissues and 52 matched normal tissues. A signature was identified. All these lncRNAs were differentially expressed between tumor and normal tissues and differentially expressed between high Gleason score and low Gleason score tissues. Furthermore, we developed a seven lncRNAs signature that can predict PCa BCR. Patients classified into low-risk group showed better BCR survival significantly than the patients in the high-risk group (hazard ratio = 0.32, 95% CI: 0.20-0.52, concordance index = 0.63). The area under the curve was 0.68 for BCR. The signature also had good discrimination for BCR in men with Gleason 7 PCa. In conclusion, our results suggest that the seven lncRNAs signature is a new biomarker of BCR and high risk in PCa. In addition, the individual lncRNA warrants further study to uncover the associated mechanisms of PCa progression and the signature could be used to design direct clinical trials for adjuvant therapy.
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Objective:To establish a method for rapidly screening human antibodies recognizing HEV capsids proteins from pe-ripheral blood.The antibodies recognizing HEV capsids proteins were screened from the peripheral blood of vaccinator and the properties of the antibodies were analyzed.Methods:The HEV capsids proteins specific memory B cells in peripheral blood were obtained by flow cytometry sorting.Then antibody variable genes were acquired through single-cell RT-PCR and recombined to express in eukaryocyte.Finally,the properties analysis of recombinant expressed human monoclonal antibodies were carried out.Results:Six hu-manized monoclonal antibodies recognizing HEV capsids proteins were successfully obtained,and most of them had binding activity and neutralizing activity.Conclusion:The sequence of human monoclonal antibodies recognizing HEV capsid proteins is successfully screened and successfully expressed in the eukaryocyte.The properties of the antibodies are identified,which lay the foundation for studying antibody evolution in the human body after vaccination.
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Xiangxi (the western part of Hunan province) Liu's infantile tuina,as one main school of current infantile tuina in China,highlights the compatibility of the specific points of Wujing in children,the idea of treatment by syndrome differentiation,and produces unique efficacy in the treatment of common diseases in children.Exogenous fever in children can be treated with this method with excellent efficacy.Based on the clinical experience and effective cases treated by this tuina school,the authors elaborated the clinical thought and experience from the perspective view of tuina,for the promotion of Xiangxi Liu's infantile tuina in clinic.
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<p><b>OBJECTIVE</b>To investigate the clinical characteristics and surgical treatment of penile Paget's disease.</p><p><b>METHODS</b>We retrospectively analyzed the treatment and follow-up data of 10 cases of penile Paget's disease surgically treated in Jiangsu Provincial Government Hospital and Jiangsu Provincial People's Hospital from 2008 to 2012.</p><p><b>RESULTS</b>All the 10 patients received expanded local resection of the lesion with reconstruction of the defects with scrotal skin flaps or free skin flaps from the thigh. All surgeries were successful and the postoperative course was uneventful with complete graft survival and no lymph node metastasis. IIEF scores obtained before and 1 -2 months after surgery showed no statistically significant differences in the penile erectile function (P = 0.229), sexual orgasm (P = 0.761), and sexual satisfaction (P = 0.801) of the patients.</p><p><b>CONCLUSION</b>When penile skin lesions suggest the possibility of Paget's disease, biopsy should be performed and surgery should follow as soon as possible. The ideal surgical option is expanded local resection of the lesion with reconstruction of the defects with scrotal skin flaps or free flaps according to the patient's specific conditions.</p>
Subject(s)
Adult , Aged , Humans , Male , Middle Aged , Follow-Up Studies , Paget Disease, Extramammary , General Surgery , Penile Neoplasms , General Surgery , Retrospective Studies , Treatment OutcomeABSTRACT
<p><b>OBJECTIVE</b>To explore the long-term survival and prognosis of prostate cancer patients after treated by androgen deprivation therapy.</p><p><b>METHODS</b>We conducted a follow-up study of 124 patients with prostate cancer treated by androgen deprivation therapy, and compared the survival times of the patients with different pathological grades and clinical characteristics using Kaplan-Meiers survival curves.</p><p><b>RESULTS</b>The mean survival time of the 124 patients after androgen deprivation therapy was 5. 912 years, with the median survival time of 7.81 years. The patients with bone metastases showed a shorter survival time than those with non-bone metastasis (P = 0.04). Pathological grades and PSA levels were not prognostic factors. No significant differences were found in the mean survival time between those died of prostate cancer (n = 35) and those from other factors (n = 23) (P = 0.50).</p><p><b>CONCLUSION</b>Bone metastasis is an important prognostic factor in advanced prostate cancer following androgen deprivation therapy, which is more significantly correlated with the survival time of the patients than tumor grades and clinical classification.</p>
Subject(s)
Adult , Aged , Aged, 80 and over , Humans , Male , Middle Aged , Androgen Antagonists , Therapeutic Uses , Bone Neoplasms , Disease-Free Survival , Follow-Up Studies , Prognosis , Prostatic Neoplasms , Mortality , TherapeuticsABSTRACT
<p><b>OBJECTIVE</b>To determine the antigen characteristics of different fragments of SARS-CoV N protein expressed in E. Coli and their application in the serological diagnosis.</p><p><b>METHODS</b>Based on preliminary analysis of 39 different segments of the N protein, We choosed six purified N protein for further antigenicity characterization in this study, including that PN360 (1 -360aa), PN301 (1-301aa), PN199 (30-228aa), PN185 (30-214aa), PN155b (60-214aa), and PN125 (90-214aa). We developed Western-Bolt and ELISA to detect antibody reactivity between truncated N fragments with sera from SARS-CoV-negative normal adults or SARS-CoV patient convalescent sera.</p><p><b>RESULTS</b>Western-Bolt results show that all the six fragments have reacted with the SARS patient convalescent sera, but the PN360 and PN301 showed obvious cross-reaction with sera from SARS-CoV-negative normal adults; sensitivity analysis using an ELISA coating with PN199, PN185, PN155b, PN125 as antigen showed that the PN185 and PN155b are better than PN125.</p><p><b>CONCLUSION</b>Truncated N protein PN185 and PN155b expressed in E. Coli are better antigen candidates used for detection of SARS-CoV specific antibody.</p>
Subject(s)
Humans , Antibodies, Viral , Blood , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Genetics , Nucleocapsid Proteins , Allergy and Immunology , Peptide Fragments , Allergy and Immunology , Recombinant Proteins , Allergy and Immunology , Serologic Tests , Severe Acute Respiratory Syndrome , DiagnosisABSTRACT
To investigate the expression and localization of various functional domains of ORF1 polyprotein and ORF3 protein of hepatitis E virus in host cells, the coding sequences of the various functional domains (RdRp, HEL, MET, PLP, X) of ORF1 were separately cloned into pcDNA3. 1-GFP vectors for constructing the recombinant plasmids which were verified by enzyme digestion and sequencing. The exact expression of the fusion proteins were detected by Western Blot, and the distribution and localization were observed by the laser scanning confocal microscope(LSCM). In huh7 cells, GFP-RdRp proteins were found mainly in the nuclei, GFP-HEL proteins were distributed vesicularly around the nucleus, GFP-MET proteins were distributed granularly both in the nuclei and the cytoplasm, GFP-PLP proteins had polar distribution around the nucleus, and unknown GFP-X proteins were distributed uniformly both in the nuclei and the cytoplasm. Different localization of these proteins verified the previous data obtained from in vitro studies, providing a support for further research on the biological functions of various proteins coded by HEV genome.
Subject(s)
Humans , Blotting, Western , Cells, Cultured , Hepatitis E virus , Genetics , Open Reading Frames , Viral Proteins , Genetics , PhysiologyABSTRACT
In our previous study, a panel of 52 broadly cross-reactive H5-specific monoclonal antibodies (MAbs) were generated and characterized. The 13D4, one of these MAbs, has been demonstrated to protect mice against lethal challenge by 4 strains of H5N1 avian influenza virus representing the currently prevailing genetic populations, clades 1, 2.1, 2.2, and 2.3. Here, we further cloned the gene of the 13D4 MAb and constructed a single-chain variable fragment. Then, the 13D4 single-chain antibody (scFv) was expressed in secretory maner in Pichia pastoris. The supernatant of the culture was concentrated and subjected to ammonium sulfate precipitation. The purity of the 13D4 scFv was around 90% in SDS-PAGE following ion-exchange chromatography. We further investigated its binding property using hemagglutination inhibition (HI) test and blocking ELISA. The results indicated that the 13D4 scFv shared the same binding sites and comparable HI titer with the prototype murine 13D4 Mab. In conclusion, an anti-H5 single-chain wide-spectrum neutralizing antibody is prepared successfully in yeast system.
Subject(s)
Antibodies, Viral , Genetics , Hemagglutination Inhibition Tests , Immunoglobulin Fragments , Genetics , Allergy and Immunology , Influenza A Virus, H5N1 Subtype , Allergy and Immunology , Pichia , Genetics , Single-Chain Antibodies , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the correlation between the polymorphism of the tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and the genetic susceptibility to prostate cancer (PCa) in the Chinese Han population in Nanjing.</p><p><b>METHODS</b>We performed a case control study on 187 cases of PCa and 237 cancer-free healthy controls. Peripheral blood genome DNA was extracted from the subjects for analysis of the polymorphism of the TRAIL-716 locus by polymerase chain reaction-ligase detection reaction (PCR-LDR). The correlations between the susceptibility to PCa and different genotypes were compared.</p><p><b>RESULTS</b>An SNP (-716A/G) was found in the promoter of the TRAIL gene. AA, AG and GG genotypes were identified. Logistic regression analysis suggested that AG, GG and AG + GG genotypes had no significant correlation with the risk of PCa (OR = 0.89, 95% CI = 0.54 -1.47; OR = 0.94, 95% CI = 0.69 -1.27; OR = 0.87, 95% CI = 0.54 - 1.41).</p><p><b>CONCLUSION</b>The TRAIL-716 polymorphism is not directly related with the genetic susceptibility to PCa in the Chinese Han population of Nanjing.</p>
Subject(s)
Aged , Aged, 80 and over , Humans , Male , Middle Aged , Asian People , Genetics , Case-Control Studies , China , Genetic Predisposition to Disease , Genotype , Polymorphism, Single Nucleotide , Prostatic Neoplasms , Genetics , TNF-Related Apoptosis-Inducing Ligand , GeneticsABSTRACT
Objective To study changes and correlations in ultrasound acoustic parameters, bone density and microstructure of the cancellous bone at different stages of decalcification. Methods Fifteen defatted porcine cancellous bone specimens were decalcified at different decalcification stages, and the bone density, microstructure and acoustic parameters were measured by Micro CT and ultrasound system, respectively, before and after the decalcification. Correlations between acoustic parameters, bone density and microstructure were investigated. Results With the loss of calcium in bone specimens, BMD (bone mass density), BS/TV and BV/TV decreased continuously. Microstructure parameters SMI and BS/BV increased, while Tb.Th and Tb.N decreased with Tb. Sp increasing. Degree of anisotropy (DA) increased. Acoustic parameter SOS increased at first, and then decreased, with nBUA slightly decreasing. High correlation was found between acoustic parameters, BMD and bone microstructure parameters. Conclusions Ultrasound acoustic parameters are correlated with BMD and bone microstructure. This study may provide some reference information for the early diagnosis of osteoporosis based on ultrasound.
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Effective and specific RNA interference (RNAi) elements are essential for the RNAi-based anti-HIV-1 research which has achieved extensive application. vif37 targeted to HIV-1 vi f is a highly effective and conserved RNAi target obtained from the previous study on screening. In this study, we explored the construction of artificial miRNAs to induce RNAi targeted to vif37, which had advantages on inhibition efficiency and flexibility of promoter selection. Three artificial miRNA targeted to vif37 were constructed by walking method using native miR-155 as a backbone and expressed by RNA polymerase II promoter. Then, expression vectors of artificial miRNA were co-transfected with HIV-1 infectious clone pNL4-3 to score its inhibition ability and showed that only miR-vif37 had the significant inhibition efficiency similar to shRNA-vif37. Subsequently, co-transfections with luciferase reporter plasmids into which different target sequences were inserted proved the specificity of miR-vif37 H. The replication of HIV-1 was inhibited in MT-4-miR37 H cells which could express miR-vif37 H stably and were cloned from MT-4 cells transducted with recombinant lentiviral vectors containing the miR-vif37 H expression element. Real-time RT-PCR revealed that miR-vif37 H had much lower expression level than shRNA-vif37. Results also showed that intracellular miR-181 and miR-16 expression levels and stat1 mRNA levels were not effected by the expression of miR-vif37 H in MT-4-miR37 H cells. We conclude miR-vif37 is a specific and highly effective artificial miRNA which will promote the further application of vif37 target.
Subject(s)
Humans , Cell Line , Gene Targeting , Methods , Genetic Vectors , Genetics , Metabolism , HIV Infections , Virology , HIV-1 , Genetics , Physiology , Lentivirus , Genetics , Metabolism , MicroRNAs , Genetics , Metabolism , Nucleic Acid Conformation , RNA Interference , vif Gene Products, Human Immunodeficiency Virus , Chemistry , Genetics , MetabolismABSTRACT
E2 is a recombinant hepatitis E virus capsid protein including its main antigenic determinants but lacking of the particle assembling domain. P239 was the C-terminal extending protein of E2 and could self-assemble to form virus like particles, which might serve as mimicry of virions both structurally and antigenically. We previously used yeast two-hybrid system to screen proteins interacting with E2 based on a human hepatocyte cDNA library. One candidate was identified as the segment (aa388-437) of cytochrome P450 2A6 protein, which is predominantly expressed in liver and important for metabolization. Some studies have demonstrated that hepatitis virus infection may altered cell metabolic clearance of coumrarin which were rapidly matebolised by CYP2A6. In this research, we demonstrated that the protein interaction between HEV capsid proteins and CYP2A6 by pull-down and co-immunoprecipitation. It was also found that their interaction could decrease the CYP2A6 catalytic activity when p239 was incubated within the CYP2A6-transfected Huh7 cells. These results suggested that CYP2A6 might be related to the pathological process when HEV invaded host cells.
Subject(s)
Humans , Aryl Hydrocarbon Hydroxylases , Genetics , Metabolism , Capsid Proteins , Genetics , Metabolism , Cell Line, Tumor , Coumarins , Metabolism , Cytochrome P-450 CYP2A6 , Hepatitis E virus , Metabolism , Imidazoles , Metabolism , Immunoprecipitation , Protein Binding , Recombinant Proteins , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Occult hepatitis B virus (HBV) infection status of blood donors in a southern city in China was investigated by immunological assays and nucleic acid testing. Overall, 17 (0.19%, 95% CI: 0.11%-0.30%) of the 9023 HBsAg negative samples were found to be positive for the presence of HBV DNA. "A" epitope sequences were obtained from 14 among them. Mutation(s) in aa124-aa147 existed in 6 (42.9%, 6/14) samples and 4 (66.7%, 4/6)were G145R mutation. Ratio of genotype C in occult donors (10/17) was statistically higher than HBs-positive donors (0/15, P<0.01), which implied that HBV genotype C leaded to occult infection more easily.
Subject(s)
Adolescent , Adult , Female , Humans , Male , Young Adult , Blood Donors , China , Epidemiology , DNA, Viral , Genetics , Genotype , Hepatitis B , Epidemiology , Allergy and Immunology , Virology , Hepatitis B Surface Antigens , Genetics , Allergy and Immunology , Hepatitis B virus , Classification , Genetics , Allergy and Immunology , Physiology , Immunologic Tests , Mutation , Sequence Alignment , Sequence Analysis, DNAABSTRACT
HPV16 L1 gene was amplified from HPV16 positive vaginal secretion sample by PCR, and inserted into pTO-T7 to obtain the recombinant expression vector pTO-T7-HPV16-L1. Then, the pTO-T7-HPV16-L1 was transformed into E. coil strain ER2566 and the recombinant protein HPV16 L1 was expressed in soluble form. After purification by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interaction chromatography, the recombinant protein HPV16 L1 had a purity of more than 98%. By removing DTT, purified HPV16 L1 proteins self-assembled in vitro into VLPs with the diameter of 50 nm. The vaccination experiments on experimental animals showed the VLPs could elicit high titer of neutralizing antibodies against HPV 16. HPV16 VLPs with high immunogenicity and high purity can be produced easily and effectively from an E. coli expression system in the study, and thus can be used in structure investigation and HPV16 vaccine development.
Subject(s)
Animals , Humans , Male , Rabbits , Antibodies, Viral , Allergy and Immunology , Capsid Proteins , Genetics , Allergy and Immunology , Goats , Human papillomavirus 16 , Genetics , Allergy and Immunology , Oncogene Proteins, Viral , Genetics , Allergy and Immunology , Papillomavirus Infections , Allergy and Immunology , Virology , Recombinant Proteins , Genetics , Allergy and Immunology , Vaccination , Virion , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To study the feasibility of using tetracysteine (TC) reporter in gene therapy.</p><p><b>METHODS</b>Effects of TC reporter and conventional reporter genes encoding green fluorescence protein (GFP) and luciferase (Luc) on expression and function of the therapeutic gene MGMT(P140K) were compared. Cytotoxicity and drug resistance were studied by Western blot. TC reporter used in therapy was analyzed by flow cytometry (FCM).</p><p><b>RESULTS</b>The TC reporter had no toxicity to cells and neither affected the expression or activity of therapeutic gene as compared to GFP and Luc. TC could be used in blood sample detection.</p><p><b>CONCLUSION</b>TC is a new kind of reporter gene for lentiviral vector in future gene therapy.</p>
Subject(s)
Animals , Cricetinae , Humans , CHO Cells , Cricetulus , Cysteine , Genetics , Metabolism , Gene Expression Regulation , Genes, Reporter , Genetic Therapy , Lentivirus , Genetics , Lymphocytes , MetabolismABSTRACT
Western blot, capture-PCR, blocking ELISA and synthetic polypeptides were used to systematically study the recognition epitopes on HEV ORF2 of 23 anti-HEV monoclonal antibodies(McAbs) which were previously generated in our laboratory directed against HEV ORF2. Results showed that seven McAbs recognized linear epitopes that located at aa408-458 of HEV ORF2 and 16 conformation-dependent McAbs, most of which recognized the surface epitopes of native HEV, located at aa459-606 of HEV ORF2. The systematical study of the recognition epitopes of anti-HEV McAbs on HEV ORF2 provides important information for the investigation of virus receptor and HEV infection mechanism, as well as its vaccine and diagnostics development.