Pattern recognition of Inductively Coupled Plasma Atomic Emission Spectroscopy of human scalp hair for discriminating between healthy and Hepatitis C patients

Inductively Coupled Plasma Atomic Emission Spectroscopy measurements of six trace elements were performed on the scalp hair of 155 donors, 73 of which have been diagnosed with Hepatitis C and 82 Controls. Principal Components Analysis (PCA) was employed to visualise the separation between groups and show the relationship between the elements and the diseased state. Pattern recognition methods for classification involving Quadratic Discriminant Analysis and Partial Least Squares Discriminant Analysis (PLS-DA) were applied to the data. The number of significant components for both PCA and PLS were determined using the bootstrap. The stability of training set models were determined by repeatedly splitting the data into training and test sets and employing visualisation for two components models: the percent classification ability (CC), predictive ability (PA) and model stability (MS) were computed for test and training sets.     

Fluorescence study of the dynamic interaction between E1(145–162) sequence of hepatitis GB virus C and liposomes

The physicochemical characterization of the peptide sequence E1(145–162) corresponding to the structural protein E1 of the hepatitis G virus was done by studying its interaction with model membranes. Small unilamellar vesicles (SUVs) of dimyristoylphosphatidylglycerol or dimyristoylphosphatidylcholine were chosen as mimetic membranes. Peptide incorporation and location in the phospholipid bilayer was investigated by fluorescence anisotropy with SUVs labeled with diphenylhexatriene (DPH) or trimethylammonium–DPH. The addition of the peptide E1(145–162) showed significant changes in the anisotropy values of the probe located at the air/water interface. These results indicate that the peptide E1(145–162) preferably interacts with the lipid surface without penetrating inside the bilayer. A series of fluorescence experiments based on tryptophan peptide fluorescence were modeled by means of multivariate curve resolution-alternating least squares (MCR-ALS) algorithm to further study the peptide interaction with bilayers at different temperatures. The preliminary results obtained with MCR-ALS showed how the peptide concentration decay is directly linked to the appearance of a new specie, which corresponds to the lipid-peptide binding. These results provide useful information for the design of synthetic immunopeptides that can be incorporated into a liposomal system with potential to promote a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, thus increasing the immuno response from the host. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression and Characterization of a Recombinant Truncated Capsid Protein of Hepatitis E Virus in Pichiapastoris

Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus(HEV).HEV is a nonenveloped virus that has been classified in the family of Caliciviridae.The virus appears to be a polyadenylated,positive-stranded RNA virus with three major open reading frames(ORFs).The capsid protein of HEV is encoded by the open reading frame 2(ORF2).We attempted to produce a truncated capsid protein,designed p293,in Pichia pastoris.The p293 gene encoding amino acids(aa) 382-674 of HEV ORF2 was designed based on the full length of HEV ORF2,cloned into the yeast vector pPIC9K,and expressed in P.pastoris strain GS115.SDS-PAGE and Western blotting demonstrated that the recombinant protein p293 could well be expressed in P.pastoris.Under optimized conditions (culture medium pH,6.0―6.5;methanol concentration added daily,3.0%;inoculum density,OD600=60;induction time point,72―96h),the yield of soluble p293 was approximately 80 mg/L.We also observed p293 secretory expressed in P.pastoris to be 30 nm viral like particles by using electron microscopy.These results show that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV,and serve as a useful antigen for both diagnostic and vaccine purposes.     

基于双层纳米金修饰的高灵敏电位型乙肝表面抗原免疫传感器研究

基于电沉积和层层自组装技术，提出了一种新的生物分子固定化方法，研制成一种高灵敏电位型乙肝表面抗原免疫传感器。利用L-半胱胺酸（LCys）的双官能团结合双层纳米金，从而通过比表面积大，生物相容性好的纳米金胶吸附大量抗体，同时用聚乙烯醇缩丁醛（PVB）薄膜的笼效应把乙肝表面抗体（HBsAb）和纳米金固定在玻碳电极上，从而制得了高灵敏度、高稳定性的电位型免疫传感器。采用循环伏安法（CV）对电极的层层自组装过程进行了考察，并对该免疫传感器的性能进行了详细的研究。该免疫传感器线性范围是8．5～256．0ng／mL，线性相关系数为0．9978，灵敏度为89．0，检出限为3．1ng／mL。已用于病人的血清样品分析。     

HBVDNA与抗HBcIgM在临床检测中的价值

采用地高辛标记探针以分子杂交的方法对８５１例血清进行ＨＢＶＤＮＡ的检测,采用ＥＬＩＳＡ的方法对相同标本检测了抗－ＨＢｃＩｇＭ、ＨＢＶ二对半、抗－ＨＡＶＩｇＭ、抗－ＨＣＶ及抗－ＨＥＶＩｇＭ,发现ＨＢＶＤＮＡ阳性检出率与ＨＢｅＡｇ出现相关,且ＨＢｅＡｇ阴性的标本中仍有１０．２５％（４９／４７８）ＨＢＶＤＮＡ阳性,说明仅以ＨＢｅＡｇ出现与否判定是否存在ＨＢＶ的复制是不可靠的。同时,还发现ＨＢＶＤＮＡ的出现与肝炎类型无关。抗－ＨＢｃＩｇＭ阳性率则与ＨＢｅＡｇ的出现无关,而与肝炎类型相关。     

丙型肝炎病毒包膜蛋白E1在小鼠和家兔中免疫应答研究

探讨丙型肝炎病毒包膜蛋白E1作为丙肝候选疫苗的可行性．用大肠杆菌表达的非糖基化HCV(丙型肝炎病毒)E1包涵体蛋白免疫小鼠和家兔，分析该包涵体蛋白在小鼠和家兔中所引起的免疫应答及其安全性．该E1蛋白具有良好的免疫原性，能诱导小鼠和家兔产生针对E1的特异性体液免疫应答．小鼠CD8^ T细胞数量在免疫后有明显升高．免疫小鼠未见明显的毒副作用．推测HCV E1这种包涵体结构可能有利于E1抗原的呈递．而HCV E1蛋白的糖基化并不是其免疫原性所必须的．     

Computational prediction of hybridization patterns between hepatitis C viral genome and human microRNAs

Hepatitis C virus (HCV) infection causes acute and chronic hepatitis leading to cirrhosis and hepatocellular carcinoma which are the major health problems around the world including Thai population. Many recent studies were shown an important role of microRNA (miRNA) to inhibit or promote viral replication. This study aimed to investigate human miRNAs and analyze hybridization patterns between cellular microRNA (miRNA) and whole genome of hepatitis C virus (HCV) especially genotypes 1a, 3a and 6. Computational prediction was performed by using miRBase and RNAhybrid. Candidate human miRNAs were analyzed based on minimum free energy (MFE) and hybridization patterns between HCV viral target genes and the miRNAs. An individual genotype of HCV was targeted by different miRNAs due to highly genetic variation among different genotypes of HCV. The genome of HCV genotypes 1a, 3a and 6 was served as a target for hybridization with 26, 34 and 32 human miRNAs, respectively. There were only 3 miRNAs including hsa-miR-24-3p (for genotypes 3a & 6), hsa-miR-624 (for genotypes 1a & 3a) and hsa-miR-1915-5p (genotypes 1a & 3a) target with multiple genotypes of HCV. The results revealed several candidate miRNAs that should be further confirmed by experimental analysis to ensure the effect of each candidate miRNAs. Nevertheless, the predicted miRNAs targeting HCV might be useful and have a potential role for inhibition of hepatitis C viral replication in the future.     

Ultrasensitive Detection of Hepatitis C Virus DNA Subtypes Based on Cucurbituril and Graphene Oxide Nano-composite

Infectious of hepatitis C viruses(HCVs)lead to hepatic fibrosis,cirrhosis even hepatoma.Developing rapid and sensitive diagnostic method for HCV is of great importance.Based on the host-and-guest interaction between cucurbit[7]uril(CB[7])and methylene blue(MB),a CB[7]-graphene nano-composite(CB[7]-N3-GO)is raised for the electrochemical detection of HCV DNA.The method is able to linearly detect the HCV nucleic acid in the range of 0.2—10 nmol/L with detection limit as low as 160.4 pmol/L.The proposed detection strategy is able to discriminate the lb and 6k subtypes of HCV and has a prospective potential in the blood screen for HCV in clinical diagnosis.     

基于毛细管液滴技术的血液乙型肝炎病毒DNA提取

基于毛细管液滴技术,建立了提取血液中乙型肝炎病毒(HBV)DNA的方法.方法的基本原理是向聚四氟乙烯毛细管中连续引人油相和水相溶液时,由于表面张力作用,可以形成稳定的油包水型液滴.依次引人含有不同试样的液滴,在毛细管中完成进样、DNA结合、洗涤以及洗脱等过程.实验表明,采用蛋白酶K裂解和提高洗脱温度有利于提高DNA回收...     

Sandwich Immunoassay for Hepatitis C Virus Non-Structural 5A Protein Using a Glassy Carbon Electrode Modified with an Au-MoO3/Chitosan Nanocomposite

A novel electrochemical immunosensor was prepared for the detection of the hepatitis C virus non-structural 5A protein. A glassy carbon electrode was modified with an Au-MoO₃/Chitosan nanocomposite that warranted good conductivity and biocompatibility. Mesoporous silica with a large specific surface served as a nanocarrier for horseradish peroxidase and the polyclonal antibody as the reporter probe. The immunosensor was characterized by scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. Following the sandwich-type immunoreaction, horseradish peroxidase was efficiently captured on the surface of the electrode to catalyze the decomposition of hydrogen peroxide. The analytical signal was obtained as an amperometric i-t curve (chronoamperometry). The assay reported here had a wide detection range (1 ng mL^?1 ?50 µg mL^?1) and detection limit as low as 1 ng mL^?1 of hepatitis C virus non-structural 5A protein. The electrochemical biosensor experiments showed excellent reproducibility, high selectivity, and outstanding stability for the determination of hepatitis C virus non-structural 5A protein, and it was successfully applied to the detection of the analyte in real serum samples.