Experimental and computational applications of microarray technology for malaria eradication in Africa

Various mutation assisted drug resistance evolved in Plasmodium falciparum strains and insecticide resistance to female Anopheles mosquito account for major biomedical catastrophes standing against all efforts to eradicate malaria in Sub-Saharan Africa. Malaria is endemic in more than 100 countries and by far the most costly disease in terms of human health causing major losses among many African nations including Nigeria. The fight against malaria is failing and DNA microarray analysis need to keep up the pace in order to unravel the evolving parasite’s gene expression profile which is a pointer to monitoring the genes involved in malaria’s infective metabolic pathway. Huge data is generated and biologists have the challenge of extracting useful information from volumes of microarray data. Expression levels for tens of thousands of genes can be simultaneously measured in a single hybridization experiment and are collectively called a “gene expression profile”. Gene expression profiles can also be used in studying various state of malaria development in which expression profiles of different disease states at different time points are collected and compared to each other to establish a classifying scheme for purposes such as diagnosis and treatments with adequate drugs. This paper examines microarray technology and its application as supported by appropriate software tools from experimental set-up to the level of data analysis. An assessment of the level of microarray technology in Africa, its availability and techniques required for malaria eradication and effective healthcare in Nigeria and Africa in general were also underscored

Transcription analysis of apple fruit development using cDNA microarrays

The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and maturation thus potentially involved in fruit quality traits. The expression profile of 1,536 transcripts was analysed by microarray hybridisation. A total of 177 genes resulted to be differentially expressed in at least one of the developmental stages considered. Gene ontology annotation was employed to univocally describe gene function, while cluster analysis allowed grouping genes according to their expression profile. An overview of the transcriptional changes and of the metabolic pathways involved in fruit development was obtained. As expected, August and September are the two months where the largest number of differentially expressed genes was observed. In particular, 85 genes resulted to be up-regulated in September. Even though most of the differentially expressed genes are involved in primary metabolism, several other interesting functions were detected and will be presented

Comparability of Microarray Data between Amplified and Non Amplified RNA in Colorectal Carcinoma

Microarray analysis reaches increasing popularity during the investigation of prognostic gene clusters in oncology. The standardisation of technical procedures will be essential to compare various datasets produced by different research groups. In several projects the amount of available tissue is limited. In such cases the preamplification of RNA might be necessary prior to microarray hybridisation. To evaluate the comparability of microarray results generated either by amplified or non amplified RNA we isolated RNA from colorectal cancer samples (stage UICC IV) following tumour tissue enrichment by macroscopic manual dissection (CMD). One part of the RNA was directly labelled and hybridised to GeneChips (HG-U133A, Affymetrix), the other part of the RNA was amplified according to the ?Eberwine? protocol and was then hybridised to the microarrays. During unsupervised hierarchical clustering the samples were divided in groups regarding the RNA pre-treatment and 5.726 differentially expressed genes were identified. Using independent microarray data of 31 amplified vs. 24 non amplified RNA samples from colon carcinomas (stage UICC III) in a set of 50 predictive genes we validated the amplification bias. In conclusion microarray data resulting from different pre-processing regarding RNA pre-amplification can not be compared within one analysis

Transcriptional and Proteomic Analysis of a Ferric Uptake Regulator (Fur) Mutant of Shewanella oneidensis: Possible Involvement of Fur in Energy Metabolism, Transcriptional Regulation, and Oxidative Stress

The iron-directed, coordinate regulation of genes depends on the fur (ferric uptake regulator) gene product, which acts as an iron-responsive, transcriptional repressor protein. To investigate the biological function of a fur homolog in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1, a fur knockout strain (FUR1) was generated by suicide plasmid integration into this gene and characterized using phenotype assays, DNA microarrays containing 691 arrayed genes, and two-dimensional polyacrylamide gel electrophoresis. Physiological studies indicated that FUR1 was similar to the wild-type strain when they were compared for anaerobic growth and reduction of various electron acceptors. Transcription profiling, however, revealed that genes with predicted functions in electron transport, energy metabolism, transcriptional regulation, and oxidative stress protection were either repressed (ccoNQ, etrA, cytochrome b and c maturation-encoding genes, qor, yiaY, sodB, rpoH, phoB, and chvI) or induced (yggW, pdhC, prpC, aceE, fdhD, and ppc) in the fur mutant. Disruption of fur also resulted in derepression of genes (hxuC, alcC, fhuA, hemR, irgA, and ompW) putatively involved in iron uptake. This agreed with the finding that the fur mutant produced threefold-higher levels of siderophore than the wild-type strain under conditions of sufficient iron. Analysis of a subset of the FUR1 proteome (i.e., primarily soluble cytoplasmic and periplasmic proteins) indicated that 11 major protein species reproducibly showed significant (P < 0.05) differences in abundance relative to the wild type. Protein identification using mass spectrometry indicated that the expression of two of these proteins (SodB and AlcC) correlated with the microarray data. These results suggest a possible regulatory role of S. oneidensis MR-1 Fur in energy metabolism that extends the traditional model of Fur as a negative regulator of iron acquisition systems

Comparative study of gene expression by cDNA microarray in human colorectal cancer tissues and normal mucosa

The causative molecular pathways underlying the pathogenesis of colorectal cancer (CRC) need to be better characterized. The purpose of our study was to better understand the genetic mechanism of oncogenesis for human colorectal cancer and to identify new potential tumor markers of use in clinical practice. We used cDNA microarrays to compare gene expression profiles of colorectal biopsies from 25 CRC patients and 13 normal mucosa from adjacent non-cancerous tissues. Findings were validated by real-time PCR; in addition, western blotting and immunochemistry analysis were carried out as further confirmation of differential expression at a protein level. Comparing cancerous tissues with normal colonic mucosa we identified 584 known genes differentially expressed to a significant degree (p<0.001). Many of the transcripts that were more abundant in tumors than in non-neoplastic tissues appear to reflect important events for colon carcinogenesis. For example, a significant number of these genes serve as apoptotic inhibitors (e.g. BFAR, BIRC1, BIRC6). Furthermore, we observed the simultaneous up-regulation of HLA-E and the down-regulation of beta2-microglobulin; these genes strongly support a potential tumor escape strategy from immune surveillance in colon cancer tissues. Our study provides new gene candidates in the pathogenesis of human CRC disease. From our results we hypothesize that CRC cells escape immune surveillance through a specific gene expression alteration; moreover, over-expression of several survival genes seems to confer a more anti-apoptotic phenotype. These genes are involved in pathways not previously implicated in CRC pathogenesis and they may provide new targets for therapy.Fil: Bianchini, Michele. Fundación P/la Invest.y Prevención del Cancer. Centro de Investigaciones Oncologicas; ArgentinaFil: Levy, Estrella Mariel. Fundación P/la Invest.y Prevención del Cancer. Centro de Investigaciones Oncologicas; Argentina. Universidad de Buenos Aires; ArgentinaFil: Zucchini, Cinzia. Università di Bologna; ItaliaFil: Pinski, Victor. Universidad de Buenos Aires; ArgentinaFil: Macagno, Carlo. Universidad de Buenos Aires; ArgentinaFil: De Sanctis, Paola. Università di Bologna; ItaliaFil: Valvassori, Luisa. Università di Bologna; ItaliaFil: Carinci, Paolo. Università di Bologna; ItaliaFil: Mordoh, Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Fundación P/la Invest.y Prevención del Cancer. Centro de Investigaciones Oncologicas; Argentin

Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: Insights into the phagosomal environment

Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2–deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon � – and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and �-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of �E-dependent, sodium dodecyl sulfate–regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment

A graph-based representation of Gene Expression profiles in DNA microarrays

This paper proposes a new and very flexible data model, called gene expression graph (GEG), for genes expression analysis and classification. Three features differentiate GEGs from other available microarray data representation structures: (i) the memory occupation of a GEG is independent of the number of samples used to built it; (ii) a GEG more clearly expresses relationships among expressed and non expressed genes in both healthy and diseased tissues experiments; (iii) GEGs allow to easily implement very efficient classifiers. The paper also presents a simple classifier for sample-based classification to show the flexibility and user-friendliness of the proposed data structur

Expression of Regulatory Platelet MicroRNAs in Patients with Sickle Cell Disease

Background: Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile. Methods and Findings: We analyzed the expression of miRNAs isolated from platelets from a primary cohort (SCD = 19, controls = 10) and a validation cohort (SCD = 7, controls = 7) by hybridizing to the Agilent miRNA microarrays. A dramatic difference in miRNA expression profiles between patients and controls was noted in both cohorts separately. A total of 40 differentially expressed platelet miRNAs were identified as common in both cohorts (p-value 0.05, fold change>2) with 24 miRNAs downregulated. Interestingly, 14 of the 24 downregulated miRNAs were members of three families - miR-329, miR-376 and miR-154 - which localized to the epigenetically regulated, maternally imprinted chromosome 14q32 region. We validated the downregulated miRNAs, miR-376a and miR-409-3p, and an upregulated miR-1225-3p using qRT-PCR. Over-expression of the miR-1225-3p in the Meg01 cells was followed by mRNA expression profiling to identify mRNA targets. This resulted in significant transcriptional repression of 1605 transcripts. A combinatorial approach using Meg01 mRNA expression profiles following miR-1225-3p overexpression, a computational prediction analysis of miRNA target sequences and a previously published set of differentially expressed platelet transcripts from SCD patients, identified three novel platelet mRNA targets: PBXIP1, PLAGL2 and PHF20L1. Conclusions: We have identified significant differences in functionally active platelet miRNAs in patients with SCD as compared to controls. These data provide an important inventory of differentially expressed miRNAs in SCD patients and an experimental framework for future studies of miRNAs as regulators of biological pathways in platelets. © 2013 Jain et al