Immunophenotypic and Ultrastructural Analysis of Mast Cells in Hermansky-Pudlak Syndrome Type-1: A Possible Connection to Pulmonary Fibrosis.

Hermansky-Pudlak Syndrome type-1 (HPS-1) is an autosomal recessive disorder caused by mutations in HPS1 which result in reduced expression of the HPS-1 protein, defective lysosome-related organelle (LRO) transport and absence of platelet delta granules. Patients with HPS-1 exhibit oculocutaneous albinism, colitis, bleeding and pulmonary fibrosis postulated to result from a dysregulated immune response. The effect of the HPS1 mutation on human mast cells (HuMCs) is unknown. Since HuMC granules classify as LROs along with platelet granules and melanosomes, we set out to determine if HPS-1 cutaneous and CD34+ culture-derived HuMCs have distinct granular and cellular characteristics. Cutaneous and cultured CD34+-derived HuMCs from HPS-1 patients were compared with normal cutaneous and control HuMCs, respectively, for any morphological and functional differences. One cytokine-independent HPS-1 culture was expanded, cloned, designated the HP proMastocyte (HPM) cell line and characterized. HPS-1 and idiopathic pulmonary fibrosis (IPF) alveolar interstitium showed numerous HuMCs; HPS-1 dermal mast cells exhibited abnormal granules when compared to healthy controls. HPS-1 HuMCs showed increased CD63, CD203c and reduced mediator release following FcɛRI aggregation when compared with normal HuMCs. HPM cells also had the duplication defect, expressed FcɛRI and intracytoplasmic proteases and exhibited less mediator release following FcɛRI aggregation. HPM cells constitutively released IL-6, which was elevated in patients' serum, in addition to IL-8, fibronectin-1 (FN-1) and galectin-3 (LGALS3). Transduction with HPS1 rescued the abnormal HPM morphology, cytokine and matrix secretion. Microarray analysis of HPS-1 HuMCs and non-transduced HPM cells confirmed upregulation of differentially expressed genes involved in fibrogenesis and degranulation. Cultured HPS-1 HuMCs appear activated as evidenced by surface activation marker expression, a decrease in mediator content and impaired releasibility. The near-normalization of constitutive cytokine and matrix release following rescue by HPS1 transduction of HPM cells suggests that HPS-1 HuMCs may contribute to pulmonary fibrosis and constitute a target for therapeutic intervention.

UTR introns, antisense RNA and differentially spliced transcripts between Plasmodium yoelii subspecies.

BACKGROUND: The rodent malaria parasite Plasmodium yoelii is an important animal model for studying host-parasite interaction and molecular basis of malaria pathogenesis. Although a draft genome of P. yoelii yoelii YM is available, and RNA sequencing (RNA-seq) data for several rodent malaria species (RMP) were reported recently, variations in coding regions and structure of mRNA transcript are likely present between different parasite strains or subspecies. Sequencing of cDNA libraries from additional parasite strains/subspecies will help improve the gene models and genome annotation. METHODS: Here two directional cDNA libraries from mixed blood stages of a subspecies of P. yoelii (P. y. nigeriensis NSM) with or without mefloquine (MQ) treatment were sequenced, and the sequence reads were compared to the genome and cDNA sequences of P. y. yoelii YM in public databases to investigate single nucleotide polymorphisms (SNPs) in coding regions, variations in intron-exon structure and differential splicing between P. yoelii subspecies, and variations in gene expression under MQ pressure. RESULTS: Approximately 56 million of 100 bp paired-end reads were obtained, providing an average of ~225-fold coverage for the coding regions. Comparison of the sequence reads to the YM genome revealed introns in 5' and 3' untranslated regions (UTRs), altered intron/exon boundaries, alternative splicing, overlapping sense-antisense reads, and potentially new transcripts. Interestingly, comparison of the NSM RNA-seq reads obtained here with those of YM discovered differentially spliced introns; e.g., spliced introns in one subspecies but not the other. Alignment of the NSM cDNA sequences to the YM genome sequence also identified ~84,000 SNPs between the two parasites. CONCLUSION: The discoveries of UTR introns and differentially spliced introns between P. yoelii subspecies raise interesting questions on the potential role of these introns in regulating gene expression and evolution of malaria parasites.

Molecular mechanisms of hypoxic responses via unique roles of Ras1, Cdc24 and Ptp3 in a human fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans encounters a low oxygen environment when it enters the human host. Here, we show that the conserved Ras1 (a small GTPase) and Cdc24 (the guanine nucleotide exchange factor for Cdc42) play an essential role in cryptococcal growth in hypoxia. Suppressor studies indicate that PTP3 functions epistatically downstream of both RAS1 and CDC24 in regulating hypoxic growth. Ptp3 shares sequence similarity to the family of phosphotyrosine-specific protein phosphatases and the ptp3Δ strain failed to grow in 1% O2. We demonstrate that RAS1, CDC24 and PTP3 function in parallel to regulate thermal tolerance but RAS1 and CDC24 function linearly in regulating hypoxic growth while CDC24 and PTP3 reside in compensatory pathways. The ras1Δ and cdc24Δ strains ceased to grow at 1% O2 and became enlarged but viable single cells. Actin polarization in these cells, however, was normal for up to eight hours after transferring to hypoxic conditions. Double deletions of the genes encoding Rho GTPase Cdc42 and Cdc420, but not of the genes encoding Rac1 and Rac2, caused a slight growth retardation in hypoxia. Furthermore, growth in hypoxia was not affected by the deletion of several central genes functioning in the pathways of cAMP, Hog1, or the two-component like phosphorylation system that are critical in the cryptococcal response to osmotic and genotoxic stresses. Interestingly, although deletion of HOG1 rescued the hypoxic growth defect of ras1Δ, cdc24Δ, and ptp3Δ, Hog1 was not hyperphosphorylated in these three mutants in hypoxic conditions. RNA sequencing analysis indicated that RAS1, CDC24 and PTP3 acted upon the expression of genes involved in ergosterol biosynthesis, chromosome organization, RNA processing and protein translation. Moreover, growth of the wild-type strain under low oxygen conditions was affected by sub-inhibitory concentrations of the compounds that inhibit these biological processes, demonstrating the importance of these biological processes in the cryptococcal hypoxia response.

Unlocking the Power of Big Data at the National Institutes of Health.

The era of "big data" presents immense opportunities for scientific discovery and technological progress, with the potential to have enormous impact on research and development in the public sector. In order to capitalize on these benefits, there are significant challenges to overcome in data analytics. The National Institute of Allergy and Infectious Diseases held a symposium entitled "Data Science: Unlocking the Power of Big Data" to create a forum for big data experts to present and share some of the creative and innovative methods to gleaning valuable knowledge from an overwhelming flood of biological data. A significant investment in infrastructure and tool development, along with more and better-trained data scientists, may facilitate methods for assimilation of data and machine learning, to overcome obstacles such as data security, data cleaning, and data integration.

GOFAST: an integrated approach for efficient and comprehensive membrane proteome analysis.

Membrane proteomics, the large-scale analysis of membrane proteins, is often constrained by the difficulties of achieving fully resolvable separation and resistance to proteolysis, both of which could lead to low recovery and low identification rates of membrane proteins. Here, we introduce a novel integrated approach, GELFrEE Optimized FASP Technology (GOFAST) for large-scale and comprehensive membrane proteins analysis. Using an array of sample preparation techniques including gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), filter-aided sample preparation (FASP), and microwave-assisted on-filter enzymatic digestion, we identified 2 090 proteins from the membrane fraction of a leukemia cell line (K562). Of these, 37% are annotated as membrane proteins according to gene ontology analysis, resulting in the largest membrane proteome of leukemia cells reported to date. Our approach combines the advantages of GELFrEE high-loading capacity, gel-free separation, efficient depletion of detergents, and microwave-assisted on-filter digestion, minimizing sample losses and maximizing MS-detectable sequence coverage of individual proteins. In addition, this approach also shows great potential for the identification of alternative splicing products.

Definição in sílico do transcriptoma de diferentes tipos de tumor / In silico definition of the transcriptome of different types of tumor

Nesta tese são apresentados dois estudos in silico de conjuntos de dados de expressão. Ambos os estudos originaram-se de conjuntos de dados de expressão feitas de "Open Reading Frame ESTs" ou ORESTES. Estes ORESTES foram gerados no projeto genoma humano de câncer da FAPESP/LICR. A ênfase da primeira parte da tese está no estudo de expressão diferencial de genes em tumor de mama, enquanto a segunda parte da tese está no estudo da expressão de novos genes em câncer colorretal. A combinação do uso de ORESTES e a informação disponível dos bancos de dados de UniGene e SAGE caracterizaram o transcriptoma de células normais e tumorais de mama. Neste estudo, identificamos 154 genes como candidatos de genes que são super-expressos em células tumorais de mama... Neste trabalho demonstramos que a metodologia ORESTES pode contribuir para a descoberta de novos genes. Em câncer colorretal observa-se um tipo específico de instabilidade genômica, caracterizado por alterações no tamanho das unidades simples de seqüência repetitiva ou microsatélites. Muitos dos transcritos de baixa abundância podem ser essenciais para determinar fenótipos celulares normais e patológicos e podem ser responsáveis pelas diferenças fundamentais poucos compreendidas entre os diferentes fenótipos de câncer colorretal... As análises aqui apresentadas poderão contribuir para o entendimento das diferenças fundamentais em características clínicas, patológicas e moleculares dos cânceres coloretais com estabilidade (MSS) e instabilidade (MSI) de microsatélites. Com a abordagem computacional apresentada, observamos que a metodologia ORESTES pode ser complementar a outras tecnologias de larga escala de expressão gênica (SAGE, bibliotecas normalizadas de ESTs) na identificação de genes novos com importantes papéis em tumorigênese...

In this thesis, two in silico studies of expression datasets are presented. Both studies start with datasets of ORESTES or "Open Reading Frame Expressed Sequence Tags". These ORESTES were produced within the human cancer genome project of the FAPESP I LICR. The emphasis of the first part of the thesis is on the differential expression of genes in breast tumors. The second part of the thesis emphasizes the study o f novel genes in colorectal cancer. The combination o f the use of ORESTES and the publicly available information in the databases o f Uni Gene and SAGE lead to the characterization o f the transcriptome o f normal and tumour breast cells. In this study, we identified 154 genes as candidate up-regulated genes in breast tumour cells. Among these, 28 genes have been shown by others to be overexpressed in breast or other tumours. Using RT-PCR, we tested 11 candidate genes and found that 9 were indeed overexpressed in breast tumour cells. Furthermore, 99 genes were validated in silico by SAGE data. Of the 55 genes that were not confirmed by SAGE, 42 have their corresponding cluster composed solely by ESTs. These 42 clusters have no functional annotation and the function of these genes is unknown. The transcripts of the genes that are represented by these 42 clusters are likely to be expressed at low leveis in breast tissue. These results led to a more profound study of low abundance genes among which probably most of the novel genes can be found. As the majority of novel genes are expressed at low leveis, difficulties are encountered in identifying them with gene expression techniques like SAGE (Serial Analysis of Gene Expression) or EST (Expressed Sequence Tag) libraries. In this work we show the contribution of the ORESTES methodology in identifying novel genes. In colorectal cancer, a specific type of genetic instability characterized by length alterations within simple repeated sequences, termed microsatellite instability (MSI) is seen in the majority of hereditary nonpolyposis colorectal cancers (HNPCCs) and in a subset o f sporadic cancers. Many o f the low abundance transcripts could distinguish between different phases of tumorigenesis. The analyses presented in this work could contribute to the understanding of fundamental clinicai, pathological and molecular differences between colorectal cancers with stability in microsatellites and colorectal cancers with instability in microsatellites. With the described computational approach, we observed that the ORESTES methodology could be complementary to other large scale gene expression technologies (SAGE, normalized EST libraries) in the identification of novel genes with important roles in tumorigenesis (AU)

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags.

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximately 23,500 genes, of which only approximately 1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.

ORESTES are enriched in rare exon usage variants affecting the encoded proteins.

A significant fraction of the variability found in the human transcriptome is due to alternative splicing, including alternative exon usage (AEU), intron retention and use of cryptic splice sites. We present a comparison of a large-scale analysis of AEU in the human transcriptome through genome mapping of Open Reading Frame ESTs (ORESTES) and conventional ESTs. It is shown here that ORESTES probe low abundant messages more efficiently. In addition, most of the variants detected by ORESTES affect the structure of the corresponding proteins.

In silico comparison of the transcriptome derived from purified normal breast cells and breast tumor cell lines reveals candidate upregulated genes in breast tumor cells.

Genes that are differentially expressed in tumor tissues are potential diagnostic markers and drug targets. The DNA sequence information available in the public databases can be used to identify transcripts differentially expressed in cancer. We report here the combined use of the ORESTES sequences generated in the FAPESP/LICR Human Cancer Genome Project and information available in the UniGene and SAGE databases to characterize the transcriptome of normal and breast tumor cells. We have identified 154 genes as candidates for overexpression in breast tumor cells. Among these, 28 genes have been shown by others to be overexpressed in breast or other tumors. Using RT-PCR, we tested 11 candidate genes and found that 9 were indeed overexpressed in breast tumor cells.