High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

Complete nucleotide sequence analysis of a Brazilian dengue virus type 2 strain

In the last decade, dengue fever (DF) in Brazil has been recognized as an important public health problem, and an increasing number of dengue haemorrhagic fever (DHF) cases have been reported since the introduction of dengue virus type 2 (DEN-2) into the country in 1990. In order to analyze the complete genome sequence of a DEN-2 Brazilian strain (BR64022/98), we designed primers to amplify contiguous segments of approximately 500 base pairs across the entire sequence of the viral genome. Twenty fragments amplified by reverse transcriptase-PCR were cloned, and the complete nucleotide and the deduced amino acid sequences were determined. This constitutes the first complete genetic characterization of a DEN-2 strain from Brazil. All amino acid changes differentiating strains related to the Asian/American-Asian genotype were observed in BR64022/98, indicating the Asiatic origin of the strain.

Draft genome of the globally widespread and invasive Argentine ant (Linepithema humile).

Ants are some of the most abundant and familiar animals on Earth, and they play vital roles in most terrestrial ecosystems. Although all ants are eusocial, and display a variety of complex and fascinating behaviors, few genomic resources exist for them. Here, we report the draft genome sequence of a particularly widespread and well-studied species, the invasive Argentine ant (Linepithema humile), which was accomplished using a combination of 454 (Roche) and Illumina sequencing and community-based funding rather than federal grant support. Manual annotation of >1,000 genes from a variety of different gene families and functional classes reveals unique features of the Argentine ant's biology, as well as similarities to Apis mellifera and Nasonia vitripennis. Distinctive features of the Argentine ant genome include remarkable expansions of gustatory (116 genes) and odorant receptors (367 genes), an abundance of cytochrome P450 genes (>110), lineage-specific expansions of yellow/major royal jelly proteins and desaturases, and complete CpG DNA methylation and RNAi toolkits. The Argentine ant genome contains fewer immune genes than Drosophila and Tribolium, which may reflect the prominent role played by behavioral and chemical suppression of pathogens. Analysis of the ratio of observed to expected CpG nucleotides for genes in the reproductive development and apoptosis pathways suggests higher levels of methylation than in the genome overall. The resources provided by this genome sequence will offer an abundance of tools for researchers seeking to illuminate the fascinating biology of this emerging model organism.

Ocurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil

A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris) in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR) with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon) evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania) chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L.) chagasi, the natural transmission of T. (T.) evansi occurs in the area under study.

Molecular and morphological characterization of heterorhabditid entomopathogenic nematodes from the tropical rainforest in Brazil

Despite massive losses of primary forest, the Amazonian rainforest remains an extremely rich source of biodiversity. In recent years, entomopathogenic nematodes (EPNs) have been isolated from soil in various parts of the world and used successfully as biological control agents against numerous insect pests. Therefore, a sampling in the rainforest of Monte Negro, Rondônia, Brazil was conducted with the aim of discovering new strains and/or species of EPNs for future development as biological control agents. From 156 soil samples taken at nine collecting sites, 19 isolates were obtained, all of them belonging to the genus Heterorhabditis. Four strains were subjected to detailed morphological and molecular evaluation. Based on morphometrics and internal transcribed spacer (ITS) sequence data, the strains LPP1, LPP2 and LPP4 were identified as Heterorhabditis indica, whereas LPP7 was considered Heterorhabditis baujardi. Comparative analysis of the ITS1 sequence of H. indica and H. baujardi isolates showed a polymorphic site for the restriction enzyme Tth 111 that could be used to distinguish the two species. Consequently, strains LPP1, LPP2, LPP3, LPP4, and LPP9 were identified as H. indica, whereas LPP5, LPP7, LPP8 and LPP10 were identified as H. baujardi.

Polymerase chain reaction genotyping of Epstein-Barr virus in scraping samples of the tongue lateral border in HIV-1 seropositive patients

The Epstein-Barr virus (EBV) is the etiological agent of oral hairy leukoplakia (OHL), an oral lesion with important diagnostic and prognostic value in acquired immunodeficiency disease syndrome. The two EBV genotypes, EBV-1 and EBV-2, can be distinguished by divergent gene sequences encoding the EBNA-2, 3A, 3B, and 3C proteins. The purpose of this study was to identify the EBV genotype prevalent in 53 samples of scrapings from the lateral border of the tongue of HIV-1 seropositive patients, with and without OHL, and to correlate the genotypes with presence of clinical or subclinical OHL with the clinic data collected. EBV-1 and EBV-2 were identified through PCR and Nested-PCR based on sequence differences of the EBNA-2 gene. EBV-1 was identified in the 31 samples (15 without OHL, 7 with clinical OHL and 9 with subclinical OHL), EBV-2 in 12 samples (10 without OHL, 1 with clinical and 1 subclinical OHL), and a mixed infection in 10 samples (2 without OHL, 3 with clinical and 5 with subclinical OHL). The presence of EBV-1 was higher in women, but a significant statistical result relating one the EBV genotypes to the development of OHL was not found. We conclude that the oral epithelium in HIV-1 seropositive patients can be infected by EBV-1, EBV-2 or by a mixed viral population.

Phylogenetic analysis of Biomphalaria tenagophila (Orbigny, 1835) (Mollusca: Gastropoda)

Mitochondrial DNA of Biomphalaria tenagophila, a mollusc intermediate host of Schistosoma mansoni in Brazil, was sequenced and characterised. The genome size found for B. tenagophila was 13,722 bp and contained 13 messenger RNAs, 22 transfer RNAs (tRNA) and two ribosomal RNAs (rRNA). In addition to sequencing, the mitochondrial DNA (mtDNA) genome organization of B. tenagophila was analysed based on its content and localization of both coding and non-coding regions, regions of gene overlap and tRNA nucleotide sequences. Sequences of protein, rRNA 12S and rRNA 16S nucleotides as well as gene organization were compared between B. tenagophila and Biomphalaria glabrata, as the latter is the most important S. mansoni intermediate host in Brazil. Differences between such species were observed regarding rRNA composition. The complete sequence of the B. tenagophila mitochondrial genome was deposited in GenBank (accession EF433576). Furthermore, phylogenetic relationships were estimated among 28 mollusc species, which had their complete mitochondrial genome deposited in GenBank, using the neighbour-joining method, maximum parsimony and maximum likelihood bootstrap. B. tenagophila was positioned at a branch close to B. glabrata and Pulmonata molluscs, collectively comprising a paraphyletic group, contrary to Opistobranchia, which was positioned at a single branch and constituted a monophyletic group.

Morphological and molecular characterisation of Heliconema hainanensis sp. nov. (Spirurina: Physalopteridae) from congers in the South China Sea, with a key to the species of Heliconema

Heliconema hainanensis sp. nov. collected from Uroconger lepturus (Richardson) (Anguilliformes: Congridae), Muraenesox cinereus (Forsskål) and Congresox talabonoides (Bleeker) (Anguilliformes: Muraenesocidae) in the South China Sea was described using light and scanning electron microscopy. The new species differs from its congeners by the following morphology: pseudolabia, the number and arrangement of caudal papillae (4 pairs of pedunculate precloacal papillae arranged in 2 groups of 2 and 2 pairs and 6 pairs of pedunculate postcloacal papillae arranged in 4 groups of 1, 2, 1 and 2 pairs), the length of spicules [left spicule 0.51-0.69 mm, right spicule 0.20-0.27 mm, spicule (right:left) ratio 1:2.20-2.69] and the morphology of the female tail tip. In addition, specimens of the new species collected from the three different hosts and specimens of an unidentified species of Heliconema collected from U. lepturus were characterised using molecular methods by sequencing the internal transcribed spacer (ITS) of ribosomal DNA. Analyses and comparison of the ITS sequence of H. hainanensis sp. nov. with Heliconema sp. support the validity of the new species based on morphological observations. An identification key to the species of Heliconema is also provided.

Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.

Identification of new MHC class I-restricted human tumor antigens for immunotherapy

Summary Cancer is a leading cause of morbidity and mortality in Western countries (as an example, colorectal cancer accounts for about 300'000 new cases and 200'000 deaths each year in Europe and in the USA). Despite that many patients with cancer have complete macroscopic clearance of their disease after resection, radiotherapy and/or chemotherapy, many of these patients develop fatal recurrence. Vaccination with immunogenic peptide tumor antigens has shown encouraging progresses in the last decade; immunotherapy might therefore constitute a fourth therapeutic option in the future. We dissect here and critically evaluate the numerous steps of reverse immunology, a forecast procedure to identify antigenic peptides from the sequence of a gene of interest. Bioinformatic algorithms were applied to mine sequence databases for tumor-specific transcripts. A quality assessment of publicly available sequence databanks allowed defining strengths and weaknesses of bioinformatics-based prediction of colon cancer-specific alternative splicing: new splice variants could be identified, however cancer-restricted expression could not be significantly predicted. Other sources of target transcripts were quantitatively investigated by polymerase chain reactions, as cancer-testis genes or reported overexpressed transcripts. Based on the relative expression of a defined set of housekeeping genes in colon cancer tissues, we characterized a precise procedure for accurate normalization and determined a threshold for the definition of significant overexpression of genes in cancers versus normal tissues. Further steps of reverse immunology were applied on a splice variant of the Melan¬A gene. Since it is known that the C-termini of antigenic peptides are directly produced by the proteasome, longer precursor and overlapping peptides encoded by the target sequence were synthesized chemically and digested in vitro with purified proteasome. The resulting fragments were identified by mass spectroscopy to detect cleavage sites. Using this information and based on the available anchor motifs for defined HLA class I molecules, putative antigenic peptides could be predicted. Their relative affinity for HLA molecules was confirmed experimentally with functional competitive binding assays and they were used to search patients' peripheral blood lymphocytes for the presence of specific cytolytic T lymphocytes (CTL). CTL clones specific for a splice variant of Melan-A could be isolated; although they recognized peptide-pulsed cells, they failed to lyse melanoma cells in functional assays of antigen recognition. In the conclusion, we discuss advantages and bottlenecks of reverse immunology and compare the technical aspects of this approach with the more classical procedure of direct immunology, a technique introduced by Boon and colleagues more than 10 years ago to successfully clone tumor antigens.

A genomewide association study of smoking relapse in four European population-based samples.

OBJECTIVES: Genomewide association studies (GWAS) have identified clear evidence of genetic markers for nicotine dependence. Other smoking phenotypes have been tested, but the results are less consistent. The tendency to relapse versus the ability to maintain long-term abstinence has received little attention in genetic studies; thus, our aim was to provide a better biological understanding of this phenotype through the identification of genetic loci associated with smoking relapse. METHODS: We carried out a GWAS on data from two European population-based collections, including a total of 835 cases (relapsers) and 990 controls (abstainers). Top-ranked findings from the discovery phase were tested for replication in two additional independent European population-based cohorts. RESULTS: Of the seven top markers from the discovery phase, none were consistently associated with smoking relapse across all samples and none reached genomewide significance. A single-nucleotide polymorphism rs1008509, within the Xylosyltransferase II (XYLT2) gene, was suggestively associated with smoking relapse in the discovery phase (β=-0.504; P=5.6E-06) and in the first replication sample (ALSPAC) (β=-0.27; P=0.004; n=1932), but not in the second sample (KORA) (β=0.19; P=0.138; n=912). We failed to identify an association between loci implicated previously in other smoking phenotypes and smoking relapse. CONCLUSION: Although no genomewide significant findings emerged from this study, we found that loci implicated in other smoking phenotypes were not associated with smoking relapse, which suggests that the neurobiology of smoking relapse and long-term abstinence may be distinct from biological mechanisms implicated in the development of nicotine dependence.

Advantages of extended bottom-up proteomics using sap9 for analysis of monoclonal antibodies.

Despite the recent advances in structural analysis of monoclonal antibodies with bottom-up, middle-down, and top-down mass spectrometry (MS), further improvements in analysis accuracy, depth, and speed are needed. The remaining challenges include quantitatively accurate assignment of post-translational modifications, reduction of artifacts introduced during sample preparation, increased sequence coverage per liquid chromatography (LC) MS experiment, and ability to extend the detailed characterization to simple antibody cocktails and more complex antibody mixtures. Here, we evaluate the recently introduced extended bottom-up proteomics (eBUP) approach based on proteolysis with secreted aspartic protease 9, Sap9, for analysis of monoclonal antibodies. Key findings of the Sap9-based proteomics analysis of a single antibody include: (i) extensive antibody sequence coverage with up to 100% for the light chain and up to 99-100% for the heavy chain in a single LC-MS run; (ii) connectivity of complementarity-determining regions (CDRs) via Sap9-produced large proteolytic peptides (3.4 kDa on average) containing up to two CDRs per peptide; (iii) reduced artifact introduction (e. g., deamidation) during proteolysis with Sap9 compared to conventional bottom-up proteomics workflows. The analysis of a mixture of six antibodies via Sap9-based eBUP produced comparable results. Due to the reasons specified above, Sap9-produced proteolytic peptides improve the identification confidence of antibodies from the mixtures compared to conventional bottom-up proteomics dealing with shorter proteolytic peptides.

Islands of euchromatin-like sequence and expressed polymorphic sequences within the short arm of human chromosome 21

The goals of the human genome project did not include sequencing of the heterochromatic regions. We describe here an initial sequence of 1.1 Mb of the short arm of human chromosome 21 (HSA21p), estimated to be 10% of 21p. This region contains extensive euchromatic-like sequence and includes on average one transcript every 100 kb. These transcripts show multiple inter- and intrachromosomal copies, and extensive copy number and sequence variability. The sequencing of the "heterochromatic" regions of the human genome is likely to reveal many additional functional elements and provide important evolutionary information.

Applause identification and its relevance to archival of Carnatic music

A Carnatic music concert is made up of a sequence of pieces, where each piece corresponds to a particular genre and ra¯aga (melody). Unlike a western music concert, the artist may be applauded intra-performance inter-performance. Most Carnatic music that is archived today correspond to a single audio recordings of entire concerts.The purpose of this paper is to segment single audio recordings into a sequence of pieces using thecharacteristic features of applause and music. Spectral flux, spectral entropy change quite significantly from music to applause and vice-versa. The characteristics of these features for a subset of concerts was studied. A threshold based approach was used to segment the pieces into music fragments and applauses. Preliminary resultson recordings 19 concerts from matched microphones show that the EER is about 17% for a resolution of 0.25 seconds. Further, a parameter called CUSUM is estimatedfor the applause regions. The CUSUM values determine the strength of the applause. The CUSUM is used to characterise the highlights of a concert.

Social rationality and scales of action: inter-ethnic relations in cockfighting and game-fishing, Raiatea, French Polynesia

This article challenges the notion of economic rationality as a criterion for explaining ethnic boundary maintenance. It offers an ethnographic analysis of inter-ethnic relations in the context of games (cockfights and game-fishing contests) in the island of Raiatea (French Polynesia). Although all players engage in the same basic gambling practices, money is differentially scaled and mobilized by the Tahitian and Chinese participants. Building on recent pragmatic approaches to rationality, it is shown that the players' rationalities differ not from the point of view of economic maximization, but only in so far as they participate in social relations at different scales.