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Risk effects of XRCC1 Arg399Gln and XPD Lys751Gln gene polymorphisms in de novo acute myeloid leukemia – A study from India

Sat, 01 Jul 2017 00:00:00 GMT

Title: Risk effects of XRCC1 Arg399Gln and XPD Lys751Gln gene polymorphisms in de novo acute myeloid leukemia – A study from India Authors: Santhi, S; Sangeetha, Vijay; Sureshkumar, R; Sreeja, L; George, Preethi Sara; Geetha, N; Hariharan, S Abstract: Defects in the DNA repair pathway are connected to many malignancies including leukemia. Variability in the repair rates leading to genotoxic damage have been shown to be associated with polymorphisms of XRCC1 Arg399Gln and XPD Lys 751 Gln, involved in the base excision repair and the nucleotide excision repair pathway. The present study focused on the risk effects of these polymorphisms in acute myeloid leukemia (AML). Genotypes were analyzed in 211 AML patients and control samples using polymerase chain reaction and restriction fragment length polymorphism. Logistic regression analysis was carried out to find the association of various polymorphisms with susceptibility to AML. Kaplan-Meier survival analysis was carried out to find the influence of these polymorphism on patients overall survival. The carriers of XRCC1 399 GA genotype were at increased risk for AML than with carriers of GG genotype. Subjects carrying XPD 751 AC as well as CC genotype were at an increased risk for the onset of the disease. The CC genotype was found to be having the higher risk than other genotypes with an OR of 5. 29 (P < 0. 001). In the combination group XRCC1 399 GG/ XPD 751 AC + CC and XRCC1 399 GA+AA/ XPD 751 AA show an increased risk for AML with P value 0. 010 and 0. 004 respectively. While a 6.8 fold increased risk was shown by the both variant genotype combination. A significant association for the XRCC and XPD genotype with overall survival was not detected in the present study. Our findings suggests that genetic polymorphism in the DNA repair genes may modulate susceptibility to AML. Page(s): 275-283

Cell surface display of rabbit MCP1 on human embryonic kidney 293T cell line

Sat, 01 Jul 2017 00:00:00 GMT

Title: Cell surface display of rabbit MCP1 on human embryonic kidney 293T cell line Authors: Rahimmanesh, Ilnaz; Khanahmad, Hossein; Boshtam, Maryam; Kouhpayeh, Shirin; Hejazi, Zahra Abstract: As atherosclerosis is a prevalent non-communicable disease, and yet, no definitive medical treatment found for it, Trapping MCP1 as a key factor in inflammation could be effective. Therefore, we decided to display rabbit MCP-1 (R-MCP1) on human embryonic kidney 293T cell line surface.Firstly, R-MCP1 plasmid (pR-MCP1) containing kappa chain signal sequence, R-MCP1 sequence and PDGFR intra membrane domain was constructed. The delivered pR-MCP1 was transformed in E.coli TOP10F’, and the resulted clones were assessed by PCR and digestion. After linearizing pR-MCP1 by BglII, HEK cells were transfected by them. MCP1 gene integration and expression was confirmed at RNA and protein levels by real- time PCR and flow cytometry, respectively.PCR product gel electrophoresis on genomic DNA of transfected HEK cells showed a 737 bp band.Based on real- time PCR results, We observed R-MCP1 gene expression significantly increased in transfected cells (272.26±37.32) compare to untransfected HEK 293T cells (2.67±0.12) (p=0.001).The results of flow cytometry showed that about 85% of transfected cells were positive and express R-MCP1. Therefore, cell surface display of R-MCP1 has successfully been performed and the produced cells can be used in future research to prepare diagnostic and therapeutic agents like aptamers. Page(s): 284-288

Expression of Omp16 and L7/L12 Brucella abortus protective antigens as secretory fusion proteins in mammalian cells

Sat, 01 Jul 2017 00:00:00 GMT

Title: Expression of Omp16 and L7/L12 Brucella abortus protective antigens as secretory fusion proteins in mammalian cells Authors: Prusty, Bikash R; Tabassum, Rizwana; Chaudhuri, Pallab; Saini, Mohini; Chaturvedi, V K; Mishra, B P; Gupta, Praveen K Abstract: Brucella abortus is a facultative intracellular bacterial pathogen infecting animals and humans. This preliminary study was designed to express two immunogenic genes of Brucella abortus as recombinant fusion proteins in mammalian cells for being studied as a vaccine candidate in mammalian hosts, especially mice and cattle, in our next study. The complete open reading frame sequences of two immune dominant genes of Brucella abortus namely, Omp16 and L7/L12 were fused with an intermediate spacer along with N-terminal fusion with secretory signal sequence from immunoglobulin M. The complete fusion gene sequence was codon optimized for expression in mammalian cells. For expression analysis, the codon optimized synthetic gene was cloned in pDsRed-Express-N1 mammalian expression vector, with C-terminal fusion with red fluorescent protein sequence. On transfection in MDBK and HEK-293 cells, appearance of red fluorescence in transfected cells indicated expression of Omp16- L7/L12 fusion proteins along with RFP. The Omp16-L7/L12 fusion construct without RFP sequence was also expressed in mammalian cells. The expressed Omp16-L7/L12 fusion proteins were confirmed through both indirect fluorescence antibody test and western blot. This preliminary study suggested that the codon optimized Omp16-L7/L12 fusion construct is ready to be studied in hosts like mice and cattle for its vaccine efficacy. Page(s): 289-295

3D polymerase gene of foot and mouth disease virus is a potential target for siRNA mediated inhibition of viral replication

Sat, 01 Jul 2017 00:00:00 GMT

Title: 3D polymerase gene of foot and mouth disease virus is a potential target for siRNA mediated inhibition of viral replication Authors: Prabhakaran, Arun V; John, Lijo; Nagaraj, Vijay; Vijayapillai, Umapathi; Reddy, Golla Ramalinga; Joyappa, Dechamma Hosur Abstract: Foot and mouth disease (FMD) is a highly contagious viral disease of the cloven hoofed animals and is a constant threat to animal husbandry industry world over. Control of the disease is difficult in India since conventional vaccination is the only control measure available which sometimes leads to post vaccination outbreaks. Hence alternate therapeutics to control the spread of the disease is the need of the hour, to reduce the severity and loss to livestock industry. RNA interference (RNAi) an antiviral strategy is followed in the present study. We designed four pairs of siRNA oligonucleotides targeting FMDV 3D gene and systematically evaluated the effects of siRNA on FMDV polymerase 3D gene expression and FMDV replication in BHK-21 cells. The study showed that out of four siRNA’s, siRNA 2 delivered by pSIREN vector could inhibit FMDV 3D protein expression in BHK-21 cells and FMD Asia I virus replication was inhibited >80% in vitro cell culture. The results suggest that transient expression of FMDV hairpin RNA is competent to trigger an antiviral response to FMDV in BHK-21 cells. Page(s): 296-303