Alan Mitchell Durham

DNA Microarray-Based Genome Comparison of a Pathogenic and a Nonpathogenic Strain of Xylella fastidiosa Delineates Genes Important for Bacterial Virulence

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease.

EGene: a configurable pipeline generation system for automated sequence analysis

UNLABELLED EGene is a generic, flexible and modular pipeline generation system that makes pipeline construction a modular job. EGene allows for third-party programs to be used and integrated according to the needs of distinct projects and without any previous programming or formal language experience being required. EGene comes with CoEd, a visual tool to facilitate pipeline construction and documentation. A series of components to build pipelines for sequence processing is provided. AVAILABILITY http://www.lbm.fmvz.usp.br/egene/ CONTACT alan@ime.usp.br; argruber@usp.br SUPPLEMENTARY INFORMATION http://www.lbm.fmvz.usp.br/egene/

TRAP: automated classification, quantification and annotation of tandemly repeated sequences

TRAP, the Tandem Repeats Analysis Program, is a Perl program that provides a unified set of analyses for the selection, classification, quantification and automated annotation of tandemly repeated sequences. TRAP uses the results of the Tandem Repeats Finder program to perform a global analysis of the satellite content of DNA sequences, permitting researchers to easily assess the tandem repeat content for both individual sequences and whole genomes. The results can be generated in convenient formats such as HTML and comma-separated values. TRAP can also be used to automatically generate annotation data in the format of feature table and GFF files.

Non-coding transcription characterization and annotation A guide and web resource for non-coding RNA databases

Large-scale transcriptome projects have shown that the number of RNA transcripts not coding for proteins (non-coding RNAs) is much larger than previously recognized. High-throughput technologies, coupled with bioinformatics approaches, have produced increasing amounts of data, highlighting the role of non-coding RNAs (ncRNAs) in biological processes. Data generated by these studies include diverse non-coding RNA classes from organisms of different kingdoms, which were obtained using different experimental and computational assays. This has led to a rapid increase of specialized RNA databases. The fast growth in the number of available databases makes integration of stored information a difficult task. We present here NRDR, a Non-coding RNA Databases Resource for information retrieval on ncRNA databases (www.ncrnadatabases.org). We performed a survey of 102 public databases on ncRNAs and we have introduced four categorizations to classify these databases and to help researchers quickly search and find the information they need: RNA family, information source, information content and available search mechanisms. NRDR is a useful databases searching tool that will facilitate research on ncRNAs.

A comparative transcriptome analysis reveals expression profiles conserved across three Eimeria spp. of domestic fowl and associated with multiple developmental stages

Coccidiosis of the domestic fowl is a worldwide disease caused by seven species of protozoan parasites of the genus Eimeria. The genome of the model species, Eimeria tenella, presents a complexity of 55-60MB distributed in 14 chromosomes. Relatively few studies have been undertaken to unravel the complexity of the transcriptome of Eimeria parasites. We report here the generation of more than 45,000 open reading frame expressed sequence tag (ORESTES) cDNA reads of E. tenella, Eimeria maxima and Eimeria acervulina, covering several developmental stages: unsporulated oocysts, sporoblastic oocysts, sporulated oocysts, sporozoites and second generation merozoites. All reads were assembled to constitute gene indices and submitted to a comprehensive functional annotation pipeline. In the case of E. tenella, we also incorporated publicly available ESTs to generate an integrated body of information. Orthology analyses have identified genes conserved across different apicomplexan parasites, as well as genes restricted to the genus Eimeria. Digital expression profiles obtained from ORESTES/EST countings, submitted to clustering analyses, revealed a high conservation pattern across the three Eimeria spp. Distance trees showed that unsporulated and sporoblastic oocysts constitute a distinct clade in all species, with sporulated oocysts forming a more external branch. This latter stage also shows a close relationship with sporozoites, whereas first and second generation merozoites are more closely related to each other than to sporozoites. The profiles were unambiguously associated with the distinct developmental stages and strongly correlated with the order of the stages in the parasite life cycle. Finally, we present The Eimeria Transcript Database (http://www.coccidia.icb.usp.br/eimeriatdb), a website that provides open access to all sequencing data, annotation and comparative analysis. We expect this repository to represent a useful resource to the Eimeria scientific community, helping to define potential candidates for the development of new strategies to control coccidiosis of the domestic fowl.

A transcriptome analysis of the Aedes aegypti vitellogenic fat body

Abstract Aedes (Stegomyia) aegypti is an important dengue vector in tropical and subtropical zones throughout the world. A transcriptome of Ae. aegypti vitellogenic fat bodies is described here. The fat body is a dynamic tissue that participates in multiple biochemical functions of intermediate metabolism. A total of 589 randomly selected cDNAs were assembled into 262 clusters based on their primary sequence similarities. The putative translated proteins were classified into categories based on their function in accordance with significant similarity using the BlastX at NCBI FTP site and Pfam (Bateman et al. 2000) and SMART (Schultz et al. 2000) databases. The characterization of transcripts expressed in the fat body of Ae. aegypti at 24 hours post blood meal provides a basic tool for understanding the processes occurring in this organ and could identify putative new genes whose promoters can be used to specifically express transgenes in the fat bodies of Ae. aegypti.

A framework for run-time systems and its visual programming language

Frameworks and domain-specific visual languages are two different reuse techniques, the first targeted at expert programmers, the second at domain experts. In fact, these techniques are closely related. This paper shows how to develop a domain-specific visual language by first developing a white-box framework for the domain, then turning it into a black-box framework, and finally building a graphical front end for it. We used this technique in a compiler to specify run-time systems.

Small RNAs in metastatic and non-metastatic oral squamous cell carcinoma

BackgroundSmall non-coding regulatory RNAs control cellular functions at the transcriptional and post-transcriptional levels. Oral squamous cell carcinoma is among the leading cancers in the world and the presence of cervical lymph node metastases is currently its strongest prognostic factor. In this work we aimed at finding small RNAs expressed in oral squamous cell carcinoma that could be associated with the presence of lymph node metastasis.MethodsSmall RNA libraries from metastatic and non-metastatic oral squamous cell carcinomas were sequenced for the identification and quantification of known small RNAs. Selected markers were validated in plasma samples. Additionally, we used in silico analysis to investigate possible new molecules, not previously described, involved in the metastatic process.ResultsGlobal expression patterns were not associated with cervical metastases. MiR-21, miR-203 and miR-205 were highly expressed throughout samples, in agreement with their role in epithelial cell biology, but disagreeing with studies correlating these molecules with cancer invasion. Eighteen microRNAs, but no other small RNA class, varied consistently between metastatic and non-metastatic samples. Nine of these microRNAs had been previously detected in human plasma, eight of which presented consistent results between tissue and plasma samples. MiR-31 and miR-130b, known to inhibit several steps in the metastatic process, were over-expressed in non-metastatic samples and the expression of miR-130b was confirmed in plasma of patients showing no metastasis. MiR-181 and miR-296 were detected in metastatic tumors and the expression of miR-296 was confirmed in plasma of patients presenting metastasis. A novel microRNA-like molecule was also associated with non-metastatic samples, potentially targeting cell-signaling mechanisms.ConclusionsWe corroborate literature data on the role of small RNAs in cancer metastasis and suggest the detection of microRNAs as a tool that may assist in the evaluation of oral squamous cell carcinoma metastatic potential.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

BackgroundThe implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery.ResultsTen miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer.ConclusionsCritical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients.

BackgroundPlasmodium vivax is the most widely distributed human malaria, responsible for 70–80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected.MethodsA pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10-30 was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminologyResultsA total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them.ConclusionThese are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.