Establishing an Intravesical Doublet Chemotherapy Clinic for Nonmuscle-Invasive Bladder Cancer Patients.

Introduction: Intravesical sequential doublet chemotherapy (SDC) is being used increasingly as a rescue treatment for nonmuscle-invasive bladder cancer failing bacillus Calmette-Guérin (BCG), as single-agent chemotherapies are less effective, especially for carcinoma in situ. Considering the current BCG shortage, intravesical SDC also provides an efficacious alternative to BCG. Our aim is to detail the implementation to assist with establishing an efficient and practical intravesical SDC clinic for urologic practice.

Expression of fluorescent proteins within the repeat long region of the Marek's disease virus genome allows direct identification of infected cells while retaining full pathogenicity.

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus and causes Marek's disease (MD) in chickens. RLORF4 is an MDV-specific gene located in the repeat long (RL) regions of the genome and is directly involved in attenuation. In this report, we generated recombinant (r)MDVs in which eGFP or mRFP was inserted in-frame of the 3' end of the RLORF4 gene.

Interaction network of proteins associated with human cytomegalovirus IE2-p86 protein during infection: a proteomic analysis.

Human cytomegalovirus protein IE2-p86 exerts its functions through interaction with other viral and cellular proteins. To further delineate its protein interaction network, we generated a recombinant virus expressing SG-tagged IE2-p86 and used tandem affinity purification coupled with mass spectrometry. A total of 9 viral proteins and 75 cellular proteins were found to associate with IE2-p86 protein during the first 48 hours of infection.

Alternative splicing of the human cytomegalovirus major immediate-early genes affects infectious-virus replication and control of cellular cyclin-dependent kinase.

The major immediate-early (MIE) gene locus of human cytomegalovirus (HCMV) is the master switch that determines the outcomes of both lytic and latent infections. Here, we provide evidence that alteration in the splicing of HCMV (Towne strain) MIE genes affects infectious-virus replication, movement through the cell cycle, and cyclin-dependent kinase activity. Mutation of a conserved 24-nucleotide region in MIE exon 4 increased the abundance of IE1-p38 mRNA and decreased the abundance of IE1-p72 and IE2-p86 mRNAs.

[Construction of phage antibody library with predetermined CDR3 gene and screening of humanized Fab of anti-human integrin alphanubeta3 monoclonal antibody].

Aim: To construct phage antibody library with predetermined CDR3 and to screen humanized Fab of anti-human integrin alphanubeta(3) monoclonal antibody (mAb) by epitope guided selection.

Methods: LCDR3 gene of mAb E10 was inserted into human light chain variable region gene library. Hybrid phage antibody library was constructed by cloning E10 chimeric Fd gene and human light chain variable region gene into pComb3.

[Construction and expression of anti-human integrin alphavbeta3 scFv].

Aim: To construct single chain antibody (scFv) gene of mAb E10 against human integrin alphavbeta3.

Methods: The VH and VL genes were amplified from hybridoma cells secreting mAb E10 by RT-PCR and connected with the use of linker (Gly4Ser)3 to assemble scFv gene. The scFv gene was cloned into prokaryotic expression vector pTIG-TRX and expressed in E.

[Construction of eucaryotic expression plasmid carrying the BMP7 gene and expression in mesenchymal stem cells].

Objectives: To construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs.

Methods: The BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1.

In vitro assay for HCV serine proteinase expressed in insect cells.

Aim: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells.

Methods: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells.

[Study on the methods for virus epidemiology in epitope level].

A method for studying virus epidemiology in epitope level was established via phage random peptide library and thioredoxin surface display technique and the method was proved by test with core protein of HCV.

[Selection and characterization of peptides that specifically binding to hepatitis C virus serine protease].

The HCV NS3 serine protease that plays important role in the processing of polyprotein and the replication of virus is a prime target for antiviral drugs and therapy research. Based on the crystallographic structure of HCV sreine protease, a single-chain protease was contstructed in which the central sequence of NS4A was fused to the N-terminus of NS3 serine protease domain via a flexible linker and it was expressed at high level in soluble form in E. coli.

Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single-chain protease.

Aim: To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors.

Methods: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing.

Identification of B cell epitopes of hepatitis C virus RNA dependent RNA polymerase.

The aim of this study was to identify the B cell epitopes of hepatitis C virus (HCV) NS5B RNA dependent RNA polymerase (RdRp). The truncated HCV NS5B protein NS5B-dc21 was expressed in Escherichia coli and its antigenicity was confirmed by Enzyme-Linked Immunosorbent Assay (ELISA) using 130 HCV-positive human sera and 15 negative sera. Antibodies specific to NS5B-dc21 protein were purified by affinity chromatography using sepharose-4B coupled with the recombinant protein.