Comparative genomics of ParaHox clusters of teleost fishes: gene cluster breakup and the retention of gene sets following whole genome duplications.

BACKGROUND: The evolutionary lineage leading to the teleost fish underwent a whole genome duplication termed FSGD or 3R in addition to two prior genome duplications that took place earlier during vertebrate evolution (termed 1R and 2R). Resulting from the FSGD, additional copies of genes are present in fish, compared to tetrapods whose lineage did not experience the 3R genome duplication. Interestingly, we find that ParaHox genes do not differ in number in extant teleost fishes despite their additional genome duplication from the genomic situation in mammals, but they are distributed over twice as many paralogous regions in fish genomes. RESULTS: We determined the DNA sequence of the entire ParaHox C1 paralogon in the East African cichlid fish Astatotilapia burtoni, and compared it to orthologous regions in other vertebrate genomes as well as to the paralogous vertebrate ParaHox D paralogons. Evolutionary relationships among genes from these four chromosomal regions were studied with several phylogenetic algorithms. We provide evidence that the genes of the ParaHox C paralogous cluster are duplicated in teleosts, just as it had been shown previously for the D paralogon genes. Overall, however, synteny and cluster integrity seems to be less conserved in ParaHox gene clusters than in Hox gene clusters. Comparative analyses of non-coding sequences uncovered conserved, possibly co-regulatory elements, which are likely to contain promoter motives of the genes belonging to the ParaHox paralogons. CONCLUSION: There seems to be strong stabilizing selection for gene order as well as gene orientation in the ParaHox C paralogon, since with a few exceptions, only the lengths of the introns and intergenic regions differ between the distantly related species examined. The high degree of evolutionary conservation of this gene cluster's architecture in particular - but possibly clusters of genes more generally - might be linked to the presence of promoter, enhancer or inhibitor motifs that serve to regulate more than just one gene. Therefore, deletions, inversions or relocations of individual genes could destroy the regulation of the clustered genes in this region. The existence of such a regulation network might explain the evolutionary conservation of gene order and orientation over the course of hundreds of millions of years of vertebrate evolution. Another possible explanation for the highly conserved gene order might be the existence of a regulator not located immediately next to its corresponding gene but further away since a relocation or inversion would possibly interrupt this interaction. Different ParaHox clusters were found to have experienced differential gene loss in teleosts. Yet the complete set of these homeobox genes was maintained, albeit distributed over almost twice the number of chromosomes. Selection due to dosage effects and/or stoichiometric disturbance might act more strongly to maintain a modal number of homeobox genes (and possibly transcription factors more generally) per genome, yet permit the accumulation of other (non regulatory) genes associated with these homeobox gene clusters.

Spotted fever group Rickettsia infecting ticks (Acari: Ixodidae) in the state of Santa Catarina, Brazil

During 2006-2008, a total of 260 adult ticks were collected from domestic and wild animals in different regions of the state of Santa Catarina (SC), Brazil, including areas where human cases of Brazilian spotted fever have been reported. Collected ticks belonging to nine species (Amblyomma aureolatum, Amblyomma cajennense, Amblyomma dubitatum, Amblyomma longirostre, Amblyomma ovale, Amblyomma tigrinum, Dermacentor nitens, Rhipicephalus microplus and Rhipicephalus sanguineus) were tested by polymerase chain reaction (PCR) for rickettsial infection. Overall, eight (3.1%) ticks were found to be infected with Rickettsia species. After sequencing the PCR products, we determined that the sequences generated from three A. aureolatum, one A. ovale and one R. sanguineus from the municipality of Blumenau, one A. ovale from the municipality of Águas Mornas and one A. ovale from the municipality of Urussanga were identical to the corresponding partial rickettsial ompA gene sequence of Rickettsia parkeri strain Atlantic rainforest. The sequence generated from one A. longirostre from Blumenau was 100% identical to the corresponding partial rickettsial ompA gene sequence of Rickettsia amblyommii strain AL. Because R. parkeri strain Atlantic rainforest was recently shown to have caused two cases of human spotted fever in other states of Brazil, the role of this rickettsial agent as a possible etiological agent of spotted fever in SC is discussed.

The level of ascorbate peroxidase is enhanced in benznidazole-resistant populations of Trypanosoma cruzi and its expression is modulated by stress generated by hydrogen peroxide

Ascorbate peroxidases (APX) are class I heme-containing enzymes that convert hydrogen peroxide into water molecules. The gene encoding APX has been characterized in 11 strains of Trypanosoma cruzi that are sensitive or resistant to benznidazole (BZ). Bioinformatic analysis revealed the presence of two complete copies of the T. cruzi APX (TcAPX) gene in the genome of the parasite, while karyotype analysis showed that the gene was present in the 2.000-kb chromosome of all of the strains analyzed. The sequence of TcAPX exhibited greater levels of similarity to those of orthologous enzymes from Leishmania spp than to APXs from the higher plant Arabidopsis thaliana. Northern blot and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no significant differences in TcAPX mRNA levels between the T. cruzi strains analyzed. On the other hand, Western blots showed that the expression levels of TcAPX protein were, respectively, two and three-fold higher in T. cruzi populations with in vitro induced (17 LER) and in vivo selected (BZR) resistance to BZ, in comparison with their corresponding susceptible counterparts. Moreover, the two BZ-resistant populations exhibited higher tolerances to exogenous hydrogen peroxide than their susceptible counterparts and showed TcAPX levels that increased in a dose-dependent manner following exposure to 100 and 200 µM hydrogen peroxide.

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.

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.

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.

Characterisation of pks15/1 in clinical isolates of Mycobacterium tuberculosis from Mexico

Tuberculosis (TB) is an infectocontagious respiratory disease caused by members of the Mycobacterium tuberculosis complex. A 7 base pair (bp) deletion in the locus polyketide synthase (pks)15/1 is described as polymorphic among members of the M. tuberculosis complex, enabling the identification of Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to characterise this locus in TB isolates from Mexico. One hundred twenty clinical isolates were recovered from the states of Veracruz and Estado de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the locus pks15/1, while genotypic characterisation was performed by spoligotyping. One hundred and fifty isolates contained the 7 bp deletion, while five had the wild type locus. Lineages X (22%), LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates were identified as Beijing and two (1%) EAI-Manila. The wild type pks15/1 locus was observed in all Asian lineage isolates tested. Our results confirm the utility of locus pks15/1 as a molecular marker for identifying Asian lineages of the M. tuberculosis complex. This marker could be of great value in the epidemiological surveillance of TB, especially in countries like Mexico, where the prevalence of such lineages is unknown.

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

Genetic basis of triatomine behavior: lessons from available insect genomes

Triatomines have been important model organisms for behavioural research. Diverse reports about triatomine host search, pheromone communication in the sexual, shelter and alarm contexts, daily cycles of activity, refuge choice and behavioural plasticity have been published in the last two decades. In recent times, a variety of molecular genetics techniques has allowed researchers to investigate elaborate and complex questions about the genetic bases of the physiology of insects. This, together with the current characterisation of the genome sequence of Rhodnius prolixus allows the resurgence of this excellent insect physiology model in the omics era. In the present revision, we suggest that studying the molecular basis of behaviour and sensory ecology in triatomines will promote a deeper understanding of fundamental aspects of insect and, particularly, vector biology. This will allow uncovering unknown features of essential insect physiology questions for a hemimetabolous model organism, promoting more robust comparative studies of insect sensory function and cognition.

Next-generation sequencing of experimental mouse strains.

Since the turn of the century the complete genome sequence of just one mouse strain, C57BL/6J, has been available. Knowing the sequence of this strain has enabled large-scale forward genetic screens to be performed, the creation of an almost complete set of embryonic stem (ES) cell lines with targeted alleles for protein-coding genes, and the generation of a rich catalog of mouse genomic variation. However, many experiments that use other common laboratory mouse strains have been hindered by a lack of whole-genome sequence data for these strains. The last 5 years has witnessed a revolution in DNA sequencing technologies. Recently, these technologies have been used to expand the repertoire of fully sequenced mouse genomes. In this article we review the main findings of these studies and discuss how the sequence of mouse genomes is helping pave the way from sequence to phenotype. Finally, we discuss the prospects for using de novo assembly techniques to obtain high-quality assembled genome sequences of these laboratory mouse strains, and what advances in sequencing technologies may be required to achieve this goal.

Identification of a novel splice-site mutation in the CYP1A2 gene.

AIMS: To identify the molecular basis for a low CYP1A2 metabolic status, as determined by a caffeine phenotyping test, in a 71-year-old, nonsmoking, Caucasian woman who presented with very high clozapine concentrations despite being administered a standard dose of the drug. METHODS: The nucleotide sequence of the 7 exons, exon-intron boundaries and 5'-flanking region of the CYP1A2 gene was analysed by direct sequencing. RESULTS: Only one heterozygous point mutation was identified in the donor splice site of intron 6 (3534G > A) of CYP1A2. This mutation could cause abnormal RNA splicing and therefore lead to a truncated nonfunctional enzyme. No other carrier of this mutation was identified in a population of 100 unrelated healthy Caucasians. CONCLUSIONS: This is the first report of a splice-site mutation affecting the CYP1A2 gene. This polymorphism is a likely explanation for the low CYP1A2 activity associated with high clozapine concentrations in this patient.

A novel ABCG-like transporter of Trypanosoma cruziis involved in natural resistance to benznidazole

Benznidazole (BZ) is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzistrains. ATP-binding cassette (ABC) transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison ofTcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1genes of BZ-susceptible and resistant strains were investigated by computational tools.

Effect of immune pressure on hepatitis C virus evolution: insights from a single-source outbreak.

The host's immune response to hepatitis C virus (HCV) can result in the selection of characteristic mutations (adaptations) that enable the virus to escape this response. The ability of the virus to mutate at these sites is dependent on the incoming virus, the fitness cost incurred by the mutation, and the benefit to the virus in escaping the response. Studies examining viral adaptation in chronic HCV infection have shown that these characteristic immune escape mutations can be observed at the population level as human leukocyte antigen (HLA)-specific viral polymorphisms. We examined 63 individuals with chronic HCV infection who were infected from a single HCV genotype 1b source. Our aim was to determine the extent to which the host's immune pressure affects HCV diversity and the ways in which the sequence of the incoming virus, including preexisting escape mutations, can influence subsequent mutations in recipients and infection outcomes. Conclusion: HCV sequences from these individuals revealed 29 significant associations between specific HLA types within the new hosts and variations within their viruses, which likely represent new viral adaptations. These associations did not overlap with previously reported adaptations for genotypes 1a and 3a and possibly reflected a combination of constraint due to the incoming virus and genetic distance between the strains. However, these sites accounted for only a portion of the sites in which viral diversity was observed in the new hosts. Furthermore, preexisting viral adaptations in the incoming (source) virus likely influenced the outcomes in the new hosts.

The evolution of universities' relations with the business sector in Brazil: What national publications between 1980 and 2012 reveal

ABSTRACT This paper addresses the changes in university-industry relations in Brazil regarding innovation activities. It is based on a survey of articles published in major national journals or presented at the most relevant Brazilian and regional conferences, between 1980 and 2012. The year 1980 was chosen due to the creation of the Technological Innovation Offices (NITs), which was the first government initiative to encourage knowledge transfer from universities to companies; the second was the Innovation Act of 2004. Our assumption was that after the Act the number of academic papers on this subject would increase, bringing new ideas and propositions of models to enhance this relationship. The methodology employed a qualitative, exploratory approach, using bibliographical research and a bibliometric analysis of 247 papers. Literature review of international studies shows the discussion of problems and suggestions for improvements, while in Brazil there is still a debate on whether this collaboration should occur, and if this is a legitimate role for the university. Despite the numerical growth, the content analysis showed few papers on new configurations and procedures for partnership management. We conclude that university-industry relations are not a regular and totally accepted process in Brazilian public universities, which reflect an ideological bias against cooperation with firms.