Genome-wide re-sequencing of multidrug-resistant Mycobacterium leprae Airaku-3.

Genotyping and molecular characterization of drug resistance mechanisms in Mycobacterium leprae enables disease transmission and drug resistance trends to be monitored. In the present study, we performed genome-wide analysis of Airaku-3, a multidrug-resistant strain with an unknown mechanism of resistance to rifampicin. We identified 12 unique non-synonymous single-nucleotide polymorphisms (SNPs) including two in the transporter-encoding ctpC and ctpI genes. In addition, two SNPs were found that improve the resolution of SNP-based genotyping, particularly for Venezuelan and South East Asian strains of M. leprae.

Identification of hepatitis C virus subtype 2c by sequencing analysis in patients from Córdoba, Argentina

In Argentina, most information on hepatitis C virus (HCV) genotype distribution comes from studies carried out in Buenos Aires (east province). In order to identify HCV subtypes in central Argentina, nucleotide sequencing of core region was performed in samples from 36 patients living in Córdoba, the second most populated province of Argentina. The sequence analysis identified subtype 2c as the most prevalent (50%), followed by subtype 1b (33%) and to a lesser extent by subtypes 1a (11%), 3a (3%) and 4a (3%). This is the first report of circulation of HCV subtype 2c in this region of Argentina and also such high prevalence has never been found before in the genotype distribution of South America.

Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA) and an eucalyptus arboretum (EAA). PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.

Construction of bovine adenovirus type 3 E1 and E3, substitution plasmids and the sequencing analysis of DNA pol and pTP /

The relative ease to concentrate and purify adenoviruses, their well characterized mid-sized genome, and the ability to delete non-essential regions from their genome to accommodate foreign gene, made adenoviruses a suitable candidate for the construction of vectors. The use of adenoviral vectors in gene therapy, vaccination, and as a general vector system for expressing foreign genes have been documented for some time. In this study, the objective was to rescue a BAV3 E1 or E3 recombinant vector carrying the kanamycin resistant gene, a dominant selectable marker with useful applications in studying vectored gene expression in mammalian cells. To accomplish the objective of this study, more information about BAV3 DNA sequences was required in order to make the manipulation of the virus genome accessible. Therefore, sequencing of the BAV3 genome from 1 1 .7% to 30.8% was carried out. Analysis of the determined sequences revealed the primary structure of important viral gene products coded by E2 including BAV3 DNA pol and precursor to terminal protein. Comparative analysis of these proteins with their counterparts from human and non human adenoviruses revealed important insights as to the evolutionary lineage of BAV3. In order to insert the kanamycin resistance gene in either E1 or E3, it was necessary to delete BAV3 sequences to accommodate the foreign gene so as not to exceed the limit of the packaging capacity of the virus. To construct a recombinant BAV3 in which a foreign gene was inserted in the deleted E1 region, an E1 shuttle vector was constructed. This involved the deletion from the viral sequences a region between 1.3% to 9% and inserting the kanamycin resistance gene to replace the deletion. The E1 shuttle vector contained the left (0%- 53.9%) segment of the genome and was expected to generate BAV3 recombinants that can be grown and propagated in cells that can complement the missing E1 functions. To construct a similar shuttle vector for E3 deletion, DNA sequences extending from 78.9% to 82.5% (1281 bp) were deleted from within the E3 region that had been cloned into a plasmid vector. The deleted region corresponds to those that have been shown to be non-essential for viral replication in cell culture. The resulting plasmid was used to construct another recombinant plasmid with BAV3 DNA sequences extending from 37.1% to 100% and with a deletion of E3 sequences that were replaced by kanamycin resistance gene. This shuttle plasmid was used in cotransfections with digested viral DNA in an attempt to rescue a recombinant BAV3 carrying the kanamycin resistance gene to replace the deleted E3. In spite of repeated attempts of transfection, El or E3 recombinant BAV3 were not isolated. It seems that other approaches should be applied to make a final conclusion on BAV3 infectivity.

Suitability of DNA extracted from archival specimens of fruit-eating bats of the genus Artibeus (Chiroptera, Phyllostomidae) for polymerase chain reaction and sequencing analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Whole genome sequencing analysis of Plasmodium vivax using whole genome capture

Background: Malaria caused by Plasmodium vivax is an experimentally neglected severe disease with a substantial burden on human health. Because of technical limitations, little is known about the biology of this important human pathogen. Whole genome analysis methods on patient-derived material are thus likely to have a substantial impact on our understanding of P. vivax pathogenesis and epidemiology. For example, it will allow study of the evolution and population biology of the parasite, allow parasite transmission patterns to be characterized, and may facilitate the identification of new drug resistance genes. Because parasitemias are typically low and the parasite cannot be readily cultured, on-site leukocyte depletion of blood samples is typically needed to remove human DNA that may be 1000X more abundant than parasite DNA. These features have precluded the analysis of archived blood samples and require the presence of laboratories in close proximity to the collection of field samples for optimal pre-cryopreservation sample preparation. Results: Here we show that in-solution hybridization capture can be used to extract P. vivax DNA from human contaminating DNA in the laboratory without the need for on-site leukocyte filtration. Using a whole genome capture method, we were able to enrich P. vivax DNA from bulk genomic DNA from less than 0.5% to a median of 55% (range 20%-80%). This level of enrichment allows for efficient analysis of the samples by whole genome sequencing and does not introduce any gross biases into the data. With this method, we obtained greater than 5X coverage across 93% of the P. vivax genome for four P. vivax strains from Iquitos, Peru, which is similar to our results using leukocyte filtration (greater than 5X coverage across 96% of the genome). Conclusion: The whole genome capture technique will enable more efficient whole genome analysis of P. vivax from a larger geographic region and from valuable archived sample collections.

Comparative genomic sequencing analysis of a region in human chromosome 11p15.3, mouse chromosome 7 and analysis of the novel STK33, Stk33 gene

The comparative genomic sequence analysis of a region in human chromosome 11p15.3 and its homologous segment in mouse chromosome 7 between ST5 and LMO1 genes has been performed. 158,201 bases were sequenced in the mouse and compared with the syntenic region in human, partially available in the public databases. The analysed region exhibits the typical eukaryotic genomic structure and compared with the close neighbouring regions, strikingly reflexes the mosaic pattern distribution of (G+C) and repeats content despites its relative short size. Within this region the novel gene STK33 was discovered (Stk33 in the mouse), that codes for a serine/threonine kinase. The finding of this gene constitutes an excellent example of the strength of the comparative sequencing approach. Poor gene-predictions in the mouse genomic sequence were corrected and improved by the comparison with the unordered data from the human genomic sequence publicly available. Phylogenetical analysis suggests that STK33 belongs to the calcium/calmodulin-dependent protein kinases group and seems to be a novelty in the chordate lineage. The gene, as a whole, seems to evolve under purifying selection whereas some regions appear to be under strong positive selection. Both human and mouse versions of serine/threonine kinase 33, consists of seventeen exons highly conserved in the coding regions, particularly in those coding for the core protein kinase domain. Also the exon/intron structure in the coding regions of the gene is conserved between human and mouse. The existence and functionality of the gene is supported by the presence of entries in the EST databases and was in vivo fully confirmed by isolating specific transcripts from human uterus total RNA and from several mouse tissues. Strong evidence for alternative splicing was found, which may result in tissue-specific starting points of transcription and in some extent, different protein N-termini. RT-PCR and hybridisation experiments suggest that STK33/Stk33 is differentially expressed in a few tissues and in relative low levels. STK33 has been shown to be reproducibly down-regulated in tumor tissues, particularly in ovarian tumors. RNA in-situ hybridisation experiments using mouse Stk33-specific probes showed expression in dividing cells from lung and germinal epithelium and possibly also in macrophages from kidney and lungs. Preliminary experimentation with antibodies designed in this work, performed in parallel to the preparation of this manuscript, seems to confirm this expression pattern. The fact that the chromosomal region 11p15 in which STK33 is located may be associated with several human diseases including tumor development, suggest further investigation is necessary to establish the role of STK33 in human health.

RE-AIM analysis of Internet-based psychotherapy

We live in an era where the Internet is expected to be available at the home, the workplace, schools, libraries, and even the corner café. Everyday, more and more of the world's population is connected to this growing minefield of information, many of whom use it to seek out services they need. Health services are amongst the many purchasable products currently available online. The Internet, thus, is a viable method of contacting populations that a provider would not traditionally be able to reach. A growing service in this area is the practice of Internet-based psychotherapy. This goes by many other names as well, such as e-counseling, telecounseling, web therapy, computer mediated counseling, Interapy, and many other variations. ^ This paper reviews the current available literature on the efficacy and associated concerns of Internet-based psychotherapy through the RE-AIM lens. After an evaluation of the selected studies, Internet-based psychotherapy may be able to reach a wider audience than with traditional means and also produces similar efficacy results to in-person therapy. However, providers are still reluctant to adopt Internet-based psychotherapy due to legal concerns, and long-term maintenance of these practices may be an issue. Further research into the effectiveness, cost, and legal issues surrounding Internet-based psychotherapy is recommended. ^

Role of the Protein Tyrosine Kinase 7 gene in human neural tube defects

Les anomalies du tube neural (ATN) sont des anomalies développementales où le tube neural reste ouvert (1-2/1000 naissances). Afin de prévenir cette maladie, une connaissance accrue des processus moléculaires est nécessaire. L’étiologie des ATN est complexe et implique des facteurs génétiques et environnementaux. La supplémentation en acide folique est reconnue pour diminuer les risques de développer une ATN de 50-70% et cette diminution varie en fonction du début de la supplémentation et de l’origine démographique. Les gènes impliqués dans les ATN sont largement inconnus. Les études génétiques sur les ATN chez l’humain se sont concentrées sur les gènes de la voie métabolique des folates du à leur rôle protecteur dans les ATN et les gènes candidats inférés des souris modèles. Ces derniers ont montré une forte association entre la voie non-canonique Wnt/polarité cellulaire planaire (PCP) et les ATN. Le gène Protein Tyrosine Kinase 7 est un membre de cette voie qui cause l’ATN sévère de la craniorachischisis chez les souris mutantes. Ptk7 interagit génétiquement avec Vangl2 (un autre gène de la voie PCP), où les doubles hétérozygotes montrent une spina bifida. Ces données font de PTK7 comme un excellent candidat pour les ATN chez l’humain. Nous avons re-séquencé la région codante et les jonctions intron-exon de ce gène dans une cohorte de 473 patients atteints de plusieurs types d’ATN. Nous avons identifié 6 mutations rares (fréquence allélique <1%) faux-sens présentes chez 1.1% de notre cohorte, dont 3 sont absentes dans les bases de données publiques. Une variante, p.Gly348Ser, a agi comme un allèle hypermorphique lorsqu'elle est surexprimée dans le modèle de poisson zèbre. Nos résultats impliquent la mutation de PTK7 comme un facteur de risque pour les ATN et supporte l'idée d'un rôle pathogène de la signalisation PCP dans ces malformations.

Genome-wide analysis in German shepherd dogs reveals association of a locus on CFA 27 with atopic dermatitis

Humans and dogs are both affected by the allergic skin disease atopic dermatitis (AD), caused by an interaction between genetic and environmental factors. The German shepherd dog (GSD) is a high-risk breed for canine AD (CAD). In this study, we used a Swedish cohort of GSDs as a model for human AD. Serum IgA levels are known to be lower in GSDs compared to other breeds. We detected significantly lower IgA levels in the CAD cases compared to controls (p = 1.1 × 10(-5)) in our study population. We also detected a separation within the GSD cohort, where dogs could be grouped into two different subpopulations. Disease prevalence differed significantly between the subpopulations contributing to population stratification (λ = 1.3), which was successfully corrected for using a mixed model approach. A genome-wide association analysis of CAD was performed (n cases = 91, n controls = 88). IgA levels were included in the model, due to the high correlation between CAD and low IgA levels. In addition, we detected a correlation between IgA levels and the age at the time of sampling (corr = 0.42, p = 3.0 × 10(-9)), thus age was included in the model. A genome-wide significant association was detected on chromosome 27 (praw = 3.1 × 10(-7), pgenome = 0.03). The total associated region was defined as a ~1.5-Mb-long haplotype including eight genes. Through targeted re-sequencing and additional genotyping of a subset of identified SNPs, we defined 11 smaller haplotype blocks within the associated region. Two blocks showed the strongest association to CAD. The ~209-kb region, defined by the two blocks, harbors only the PKP2 gene, encoding Plakophilin 2 expressed in the desmosomes and important for skin structure. Our results may yield further insight into the genetics behind both canine and human AD.

Fine-mapping and mutation analysis of TRPM1: a candidate gene for leopard complex (LP) spotting and congenital stationary night blindness in horses.

Leopard Complex spotting occurs in several breeds of horses and is caused by an incompletely dominant allele (LP). Homozygosity for LP is also associated with congenital stationary night blindness (CSNB) in Appaloosa horses. Previously, LP was mapped to a 6 cm region on ECA1 containing the candidate gene TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) and decreased expression of this gene, measured by qRT-PCR, was identified as the likely cause of both spotting and ocular phenotypes. This study describes investigations for a mutation causing or associated with the Leopard Complex and CSNB phenotype in horses. Re-sequencing of the gene and associated splice sites within the 105 624 bp genomic region of TRPM1 led to the discovery of 18 SNPs. Most of the SNPs did not have a predictive value for the presence of LP. However, one SNP (ECA1:108,249,293 C>T) found within intron 11 had a strong (P < 0.0005), but not complete, association with LP and CSNB and thus is a good marker but unlikely to be causative. To further localize the association, 70 SNPs spanning over two Mb including the TRPM1 gene were genotyped in 192 horses from three different breeds segregating for LP. A single 173 kb haplotype associated with LP and CSNB (ECA1: 108,197,355- 108,370,150) was identified. Illumina sequencing of 300 kb surrounding this haplotype revealed 57 SNP variants. Based on their localization within expressed sequences or regions of high sequence conservation across mammals, six of these SNPs were considered to be the most likely candidate mutations. While the precise function of TRPM1 remains to be elucidated, this work solidifies its functional role in both pigmentation and night vision. Further, this work has identified several potential regulatory elements of the TRPM1 gene that should be investigated further in this and other species.

Comparison of two statistical methods for rare variant association analysis

Studies have shown that rare genetic variants have stronger effects in predisposing common diseases, and several statistical methods have been developed for association studies involving rare variants. In order to better understand how these statistical methods perform, we seek to compare two recently developed rare variant statistical methods (VT and C-alpha) on 10,000 simulated re-sequencing data sets with disease status and the corresponding 10,000 simulated null data sets. The SLC1A1 gene has been suggested to be associated with diastolic blood pressure (DBP) in previous studies. In the current study, we applied VT and C-alpha methods to the empirical re-sequencing data for the SLC1A1 gene from 300 whites and 200 blacks. We found that VT method obtains higher power and performs better than C-alpha method with the simulated data we used. The type I errors were well-controlled for both methods. In addition, both VT and C-alpha methods suggested no statistical evidence for the association between the SLC1A1 gene and DBP. Overall, our findings provided an important comparison of the two statistical methods for future reference and provided preliminary and pioneer findings on the association between the SLC1A1 gene and blood pressure.^

Floquet stability analysis of the flow around an oscillating cylinder

This investigative work is concerned with the flow around a circular cylinder submitted to forced transverse oscillations. The goal is to investigate how the transition to turbulence is initiated in the wake for cases with different Reynolds numbers (Re) and displacement amplitudes (A). For each Re the motion frequency is kept constant, close to the Strouhal number of the flow around a fixed cylinder at the same Re. Stability analysis of two-dimensional periodic flows around a forced-oscillating cylinder is carried out with respect to three-dimensional infinitesimal perturbations. The procedure consists of performing a Floquet type analysis of time-periodic base flows, computed using the spectral/hp element method. With the results of the Floquet calculations, considerations regarding the stability of the system are drawn, and the form of the instability at its onset is obtained. The critical Reynolds number is observed to change with the amplitude of oscillation. With respect to instabilities, unstable modes with the same symmetry as mode A of a fixed cylinder are observed; however, they present different wavelengths. Also, the instabilities observed for the oscillating cylinder are distinctively stronger in the braid shear layers. Other unstable modes similar to mode B are found. Quasi-periodic modes are observed in the 2S wake, and subharmonic mode occurrences are reported in P + S wakes. (C) 2009 Elsevier Ltd. All rights reserved.