The Use Of Rap-PCR In Studying Mycobacterium tuberculosis Intracellular Gene During Macrophage Infection

Mycobacterium tuberculosis is the second leading cause of death from infectious agent. This study sought to detect M. tuberculosis genes, which were specifically expressed, or upregulated during intracellular infection ofJ774 murine macrophages; as such genes may be potential targets for novel drug action. J774 murine macrophage cell line was infected with M. tuberculosis (H37Rv strain) at 10:1 multiplicity of infection (MOI). RNA wasdifferentially extracted from M. tuberculosis infecting J774 macrophage cell line. The control in this case was RNA from extracellular broth grown bacteria. Approximately 50 ng of RNA from intracellular derived bacteria and extracellular derived bacteria (control) were subjected to random arbitrarily primed PCR (RAP-PCR) using 50 primer combinations. Eleven differential RAP-PCR products were observed. All RAP-PCR products were cloned into pCR®2.1 and sequenced in order to determine the identity of the products. Four of the eleven products were derived from macrophage genes and another 4 products were derived from the M. tuberculosis rRNA genes (three 23S and one 16S rRNA). The 3 remaining RAP-PCR products were found to be mycobacterial genes other than ribosomal genes. The three products were genes encoding enzyme involving in a shikimate pathway, a putative carboxyphosphonoenolpyruvate phosphonomutase and a serine protease with homology to HtrA. Of the 3 mycobacterial genes other than ribosomal genes detected, none were specifically expressed during intracellular infection but competitive RT-PCR showed that aroF gene was upregulated two-fold in intracellular derived bacilli

Development and evaluation of a patient centered cardiovascular health education program for insured patients in rural Nigeria (QUICK - II)

<p>Abstract</p> <p>Background</p> <p>In Sub Saharan Africa, the incidence of hypertension and other modifiable cardiovascular risk factors is growing rapidly. Poor adherence to prescribed prevention and treatment regimens by patients can compromise treatment outcomes. Patient-centered cardiovascular health education is likely to improve shortcomings in adherence. This paper describes a study that aims to develop a cardiovascular health education program for patients participating in a subsidized insurance plan in Nigeria and to evaluate the applicability and effectiveness in patients at increased risk for cardiovascular disease.</p> <p>Methods/Design</p> <p><it>Design: </it>The study has two parts. Part <it>1 </it>will develop a cardiovascular health education program, using qualitative interviews with stakeholders. Part <it>2 </it>will evaluate the effectiveness of the program in patients, using a prospective (pre-post) observational design.</p> <p><it>Setting: </it>A rural primary health center in Kwara State, Nigeria.</p> <p><it>Population: </it>For part 1: 40 patients, 10 healthcare professionals, and 5 insurance managers. For part 2: 150 patients with uncontrolled hypertension or other cardiovascular risk factors after one year of treatment.</p> <p><it>Intervention: </it>Part <it>2</it>: patient-centered cardiovascular health education program.</p> <p><it>Measurements: </it>Part 1: Semi-structured interviews to identify stakeholder perspectives. Part 2: Pre- and post-intervention assessments including patients' demographic and socioeconomic data, blood pressure, body mass index and self-reporting measures on medication adherence and perception of care. Feasibility of the intervention will be measured using process data.</p> <p><it>Outcomes: </it>For program development (part 1): overview of healthcare professionals' perceptions on barriers and facilitators to care, protocol for patient education, and protocol implementation plan.</p> <p>For program evaluation (part 2): changes in patients' scores on adherence to medication and life style changes, blood pressure, and other physiological and self-reporting measures at six months past baseline.</p> <p><it>Analysis: </it>Part 1: content analytic technique utilizing MAXQDA software. Part 2: univariate and multilevel analysis to assess outcomes of intervention.</p> <p>Discussion</p> <p>Diligent implementation of patient-centered education should enhance adherence to cardiovascular disease prevention and management programs in low income countries.</p> <p>Trial Registration</p> <p><a href="http://www.controlled-trials.com/ISRCTN47894401">ISRCTN47894401</a></p

A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines

Introduction: Inhibiting the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of breast carcinoma cells, and this is linked to human epidermal growth factor receptor 2 (HER2) signaling pathways in models of simultaneous expression of FASN and HER2. Methods: In a xenograft model of breast carcinoma cells that are FASN+ and HER2+, we have characterised the anticancer activity and the toxicity profile of G28UCM, the lead compound of a novel family of synthetic FASN inhibitors. In vitro, we analysed the cellular and molecular interactions of combining G28UCM with anti-HER drugs. Finally, we tested the cytotoxic ability of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib, that we developed in our laboratory. Results: In vivo, G28UCM reduced the size of 5 out of 14 established xenografts. In the responding tumours, we observed inhibition of FASN activity, cleavage of poly-ADPribose polymerase (PARP) and a decrease of p-HER2, p- protein kinase B (AKT) and p-ERK1/2, which were not observed in the nonresponding tumours. In the G28UCM-treated animals, no significant toxicities occurred, and weight loss was not observed. In vitro, G28UCM showed marked synergistic interactions with trastuzumab, lapatinib, erlotinib or gefitinib (but not with cetuximab), which correlated with increases in apoptosis and with decreases in the activation of HER2, extracellular signal-regulated kinase (ERK)1/2 and AKT. In trastuzumab-resistant and in lapatinib-resistant breast cancer cells, in which trastuzumab and lapatinib were not effective, G28UCM retained the anticancer activity observed in the parental cells. Conclusions: G28UCM inhibits fatty acid synthase (FASN) activity and the growth of breast carcinoma xenografts in vivo, and is active in cells with acquired resistance to anti-HER2 drugs, which make it a candidate for further pre-clinical development

Bioinformatic analyses identifies novel protein-coding pharmacogenomic markers associated with paclitaxel sensitivity in NCI60 cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Paclitaxel is a microtubule-stabilizing drug that has been commonly used in treating cancer. Due to genetic heterogeneity within patient populations, therapeutic response rates often vary. Here we used the NCI60 panel to identify SNPs associated with paclitaxel sensitivity. Using the panel's GI50 response data available from Developmental Therapeutics Program, cell lines were categorized as either sensitive or resistant. PLINK software was used to perform a genome-wide association analysis of the cellular response to paclitaxel with the panel's SNP-genotype data on the Affymetrix 125 k SNP array. FastSNP software helped predict each SNP's potential impact on their gene product. mRNA expression differences between sensitive and resistant cell lines was examined using data from BioGPS. Using Haploview software, we investigated for haplotypes that were more strongly associated with the cellular response to paclitaxel. Ingenuity Pathway Analysis software helped us understand how our identified genes may alter the cellular response to paclitaxel.</p> <p>Results</p> <p>43 SNPs were found significantly associated (FDR < 0.005) with paclitaxel response, with 10 belonging to protein-coding genes (<it>CFTR</it>, <it>ROBO1</it>, <it>PTPRD</it>, <it>BTBD12</it>, <it>DCT</it>, <it>SNTG1</it>, <it>SGCD</it>, <it>LPHN2</it>, <it>GRIK1</it>, <it>ZNF607</it>). SNPs in <it>GRIK1</it>, <it>DCT</it>, <it>SGCD </it>and <it>CFTR </it>were predicted to be intronic enhancers, altering gene expression, while SNPs in <it>ZNF607 </it>and <it>BTBD12 </it>cause conservative missense mutations. mRNA expression analysis supported these findings as <it>GRIK1</it>, <it>DCT</it>, <it>SNTG1</it>, <it>SGCD </it>and <it>CFTR </it>showed significantly (p < 0.05) increased expression among sensitive cell lines. Haplotypes found in <it>GRIK1, SGCD, ROBO1, LPHN2</it>, and <it>PTPRD </it>were more strongly associated with response than their individual SNPs.</p> <p>Conclusions</p> <p>Our study has taken advantage of available genotypic data and its integration with drug response data obtained from the NCI60 panel. We identified 10 SNPs located within protein-coding genes that were not previously shown to be associated with paclitaxel response. As only five genes showed differential mRNA expression, the remainder would not have been detected solely based on expression data. The identified haplotypes highlight the role of utilizing SNP combinations within genomic loci of interest to improve the risk determination associated with drug response. These genetic variants represent promising biomarkers for predicting paclitaxel response and may play a significant role in the cellular response to paclitaxel.</p

Proteomic Analysis of Growth Phase-Dependent Expression of Legionella pneumophila Proteins Which Involves Regulation of Bacterial Virulence Traits

Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE) combined with Matrix-Assisted Laser Desorption/Ionization–Mass Spectrometry (MALDI-TOF-MS). Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB) biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins) were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs). Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase

Molecular basis of USP7 inhibition by selective small-molecule inhibitors

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice

Optimization Of Rna Extraction In Mycobacterium Tuberculosis For Studying Intracellular Gene Expression

Mycobacterium tuberculosis is the leading cause of death due to infectious disease after Human immunodeficiency virus. There has been an upsurge in the incidence of tuberculosis since 1980s. In order to reverse this trend, there is need to understand the biology of the organism. This can be brought about by studying gene expression at transcriptional level. The success of this hinges on RNA of good quality. In this paper, five methods (hot phenol, sonication with guanidinium thiocyanate (GTC) solution, beadbeating method with Trizol, FastPrep machine with Divolab as detergent and GTC solution, and FastPrep machine with Trizol) of extracting RNA from bacteria were compared to find which of the method would be suitable for mycobacteria. The study found that physical method of lysing bacteria was necessary for extraction of RNA from mycobacteria. FastPrep machine gave the highest yield and also provided the speed necessary for optimum RNA extraction. FastPrep and Trizol as reagent for extraction of RNA was applied to macrophage infected with M. tuberculosis (H37Rv) after removing the macrophage RNA. We were able to demonstrate the expression of dnaK gene in both intracellular and broth grown bacilli. The expression of dnaK gene was found to be downregulated in macrophage compared to broth. African Journal of Clinical and Experimental Microbiology Vol. 10 (2) 2009: pp. 64-7

Evaluation Of The Effect Of Exogenous Enzymes In Cattle Diet

In a study to evaluate the effect of a crude enzyme preparation on Gamba hay (Andropogon gayanus), 12 yearling Fulani white x Bunaji heifer crosses were randomly allotted to three dietary treatment groups comprising untreated Gamba hay (A), low level enzyme treated Gamba hay (B) and high level enzyme treated Gamba hay (C) in addition to a concentrate supplement for 8 weeks. There was no significant difference (P>0.05) between the feed intake in the different diet groups. At the end of the experiment, animals in group C had the highest (