Lymphocyte subset analyses in healthy adults vaccinated with yellow fever 17DD virus

In this study the kinetics of humoral and cellular immune responses in first-time vaccinees and re-vaccinees with the yellow fever 17DD vaccine virus was analyzed. Flow cytometric analyses were used to determine percentual values of T and B cells in parallel to the yellow fever neutralizing antibody production. All lymphocyte subsets analyzed were augmented around the 30th post vaccination day, both for first-time vaccinees and re-vaccinees. CD3+ T cells increased from 30.8% (SE ± 4%) to 61.15% (SE ± 4.2%), CD4+ T cells from 22.4% (SE ± 3.6%) to 39.17% (SE ± 2%) with 43% of these cells corresponding to CD4+CD45RO+ T cells, CD8+ T cells from 15.2% (SE ± 2.9%) to 27% (SE ± 3%) with 70% corresponding to CD8+CD45RO+ T cells in first-time vaccinees. In re-vaccinees, the CD3+ T cells increased from 50.7% (SE ± 3%) to 80% (SE ± 2.3%), CD4+ T cells from 24.9% (SE ± 1.4%) to 40% (SE ± 3%) presenting a percentage of 95% CD4+CD45RO+ T cells, CD8+ T cells from 19.7% (SE ± 1.8%) to 25% (SE ± 2%). Among CD8+CD38+ T cells there could be observed an increase from 15 to 41.6% in first-time vaccinees and 20.7 to 62.6% in re-vaccinees. Regarding neutralizing antibodies, the re-vaccinees presented high titers even before re-vaccination. The levels of neutralizing antibodies of first-time vaccinees were similar to those presented by re-vaccinees at day 30 after vaccination, indicating the success of primary vaccination. Our data provide a basis for further studies on immunological behavior of the YF 17DD vaccine.

Challenges of phylogenetic analyses of aDNA sequences

One of the crucial steps of authentication of aDNA sequences is phylogenetic consistency. Amplified sequences should fit into the phylogenetic framework of their supposed origin. An inherent property of aDNA sequences however, is their short sequence length. Additionally, genes for aDNA studies are often chosen by their preservation potential rather than by phylogenetically informative content. This poses potential challenges regarding their analyses, and might result in an inaccurate reflection of the supposed phylogenetic history of the sequence or organism under study. In this paper some fundamental problems of phylogenetic analysis and interpretation of aDNA datasets are discussed. Suggestions for character sampling and treatment of missing data are made. The publication is the result of a talk from the 1st PAMINSA Meeting in Rio de Janeiro, July 2005.

Re-mapping the molecular features of the human immunodeficiency virus type 1 and human T-cell lymphotropic virus type 1 Brazilian sequences using a bioinformatics unit established in Salvador, Bahia, Brazil, to give support to the viral epidemiology studies

The analysis of genetic data for human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type 1 (HTLV-1) is essential to improve treatment and public health strategies as well as to select strains for vaccine programs. However, the analysis of large quantities of genetic data requires collaborative efforts in bioinformatics, computer biology, molecular biology, evolution, and medical science. The objective of this study was to review and improve the molecular epidemiology of HIV-1 and HTLV-1 viruses isolated in Brazil using bioinformatic tools available in the Laboratório Avançado de Sáude Pública (Lasp) bioinformatics unit. The analysis of HIV-1 isolates confirmed a heterogeneous distribution of the viral genotypes circulating in the country. The Brazilian HIV-1 epidemic is characterized by the presence of multiple subtypes (B, F1, C) and B/F1 recombinant virus while, on the other hand, most of the HTLV-1 sequences were classified as Transcontinental subgroup of the Cosmopolitan subtype. Despite the high variation among HIV-1 subtypes, protein glycosylation and phosphorylation domains were conserved in the pol, gag, and env genes of the Brazilian HIV-1 strains suggesting constraints in the HIV-1 evolution process. As expected, the functional protein sites were highly conservative in the HTLV-1 env gene sequences. Furthermore, the presence of these functional sites in HIV-1 and HTLV-1 strains could help in the development of vaccines that pre-empt the viral escape process.

Quality assurance in road traffic analyses in Switzerland.

Swiss laboratories performing toxicological road traffic analyses have been authorized for many years by the Swiss Federal Roads Office (FEDRO). In 2003 FEDRO signed a contract with the Swiss Society of Legal Medicine (SSLM) to organize the complete quality management concerning road traffic analyses. For this purpose a multidisciplinary working group was established under the name of "road traffic commission (RTC)". RTC has to organize external quality control, to interpret the results of these controls, to perform audits in the laboratories and to report all results to FEDRO. Furthermore the working group can be mandated for special tasks by FEDRO. As an independent organization the Swiss Center for Quality Control (CSCQ) in Geneva manages the external quality controls in the laboratory over the past years. All tested drugs and psychoactive substances are listed in a federal instruction. The so-called 'zero tolerance substances' (THC, morphine, cocaine, amphetamine, methamphetamine, MDMA and MDEA) and their metabolites have to be tested once a year, all other substances (benzodiazepines, zolpidem, phenobarbital, etc.) periodically. Results over the last years show that all laboratories are generally within the confidence interval of +/-30% of the mean value. In cases of non-conformities measures have to be taken immediately and reported to the working group. External audits are performed triennially but accredited laboratories can combine this audit with the approval of the Swiss Accreditation Service (SAS). During the audits a special checklist filled in by the laboratory director is assessed. Non-conformities have to be corrected. During the process of establishing a new legislation, RTC had an opportunity of advising FEDRO. In collaboration with FEDRO, RTC and hence SSLM can work actively on improving of quality assurance in road traffic toxicological analyses, and has an opportunity to bring its professional requests to the federal authorities.

Characterization of the rice PHO1 gene family reveals a key role for OsPHO1;2 in phosphate homeostasis and the evolution of a distinct clade in dicotyledons.

Phosphate homeostasis was studied in a monocotyledonous model plant through the characterization of the PHO1 gene family in rice (Oryza sativa). Bioinformatics and phylogenetic analysis showed that the rice genome has three PHO1 homologs, which cluster with the Arabidopsis (Arabidopsis thaliana) AtPHO1 and AtPHO1;H1, the only two genes known to be involved in root-to-shoot transfer of phosphate. In contrast to the Arabidopsis PHO1 gene family, all three rice PHO1 genes have a cis-natural antisense transcript located at the 5 ' end of the genes. Strand-specific quantitative reverse transcription-PCR analyses revealed distinct patterns of expression for sense and antisense transcripts for all three genes, both at the level of tissue expression and in response to nutrient stress. The most abundantly expressed gene was OsPHO1;2 in the roots, for both sense and antisense transcripts. However, while the OsPHO1;2 sense transcript was relatively stable under various nutrient deficiencies, the antisense transcript was highly induced by inorganic phosphate (Pi) deficiency. Characterization of Ospho1;1 and Ospho1;2 insertion mutants revealed that only Ospho1;2 mutants had defects in Pi homeostasis, namely strong reduction in Pi transfer from root to shoot, which was accompanied by low-shoot and high-root Pi. Our data identify OsPHO1;2 as playing a key role in the transfer of Pi from roots to shoots in rice, and indicate that this gene could be regulated by its cis-natural antisense transcripts. Furthermore, phylogenetic analysis of PHO1 homologs in monocotyledons and dicotyledons revealed the emergence of a distinct clade of PHO1 genes in dicotyledons, which include members having roles other than long-distance Pi transport.

Integrative analyses of speciation and divergence in Psammodromus hispanicus (Squamata: Lacertidae).

ABSTRACT: BACKGROUND: Genetic, phenotypic and ecological divergence within a lineage is the result of past and ongoing evolutionary processes, which lead ultimately to diversification and speciation. Integrative analyses allow linking diversification to geological, climatic, and ecological events, and thus disentangling the relative importance of different evolutionary drivers in generating and maintaining current species richness. RESULTS: Here, we use phylogenetic, phenotypic, geographic, and environmental data to investigate diversification in the Spanish sand racer (Psammodromus hispanicus). Phylogenetic, molecular clock dating, and phenotypic analyses show that P. hispanicus consists of three lineages. One lineage from Western Spain diverged 8.3 (2.9-14.7) Mya from the ancestor of Psammodromus hispanicus edwardsianus and P. hispanicus hispanicus Central lineage. The latter diverged 4.8 (1.5-8.7) Mya. Molecular clock dating, together with population genetic analyses, indicate that the three lineages experienced northward range expansions from southern Iberian refugia during Pleistocene glacial periods. Ecological niche modelling shows that suitable habitat of the Western lineage and P. h. edwardsianus overlap over vast areas, but that a barrier may hinder dispersal and genetic mixing of populations of both lineages. P. h. hispanicus Central lineage inhabits an ecological niche that overlaps marginally with the other two lineages. CONCLUSIONS: Our results provide evidence for divergence in allopatry and niche conservatism between the Western lineage and the ancestor of P. h. edwardsianus and P. h. hispanicus Central lineage, whereas they suggest that niche divergence is involved in the origin of the latter two lineages. Both processes were temporally separated and may be responsible for the here documented genetic and phenotypic diversity of P. hispanicus. The temporal pattern is in line with those proposed for other animal lineages. It suggests that geographic isolation and vicariance played an important role in the early diversification of the group, and that lineage diversification was further amplified through ecological divergence.

Pigments and plasters discovered in the House of Diana (Cosa, Grosseto, Italy): An integrated study between art history, archaeology and scientific analyses

The pigments and the plasters of the Roman frescoes discovered at the House of Diana (Cosa, Grosseto, Italy) were analysed using non-destructive and destructive mineralogical and chemical techniques. The characterization of both pigments and plasters was performed through optical microscopy, scanning electron microscopy and electron microprobe analysis. The pigments were identified by Raman spectroscopy and submitted to stable isotope analysis. The results were integrated with the archaeological data in order to determine and reconstruct the provenance, trade patterns and the employment of the raw materials used for the elaboration of the frescoes.

Genome-wide linkage and association analyses to identify genes influencing adiponectin levels: the GEMS Study.

Adiponectin has a variety of metabolic effects on obesity, insulin sensitivity, and atherosclerosis. To identify genes influencing variation in plasma adiponectin levels, we performed genome-wide linkage and association scans of adiponectin in two cohorts of subjects recruited in the Genetic Epidemiology of Metabolic Syndrome Study. The genome-wide linkage scan was conducted in families of Turkish and southern European (TSE, n = 789) and Northern and Western European (NWE, N = 2,280) origin. A whole genome association (WGA) analysis (500K Affymetrix platform) was carried out in a set of unrelated NWE subjects consisting of approximately 1,000 subjects with dyslipidemia and 1,000 overweight subjects with normal lipids. Peak evidence for linkage occurred at chromosome 8p23 in NWE subjects (lod = 3.10) and at chromosome 3q28 near ADIPOQ, the adiponectin structural gene, in TSE subjects (lod = 1.70). In the WGA analysis, the single-nucleotide polymorphisms (SNPs) most strongly associated with adiponectin were rs3774261 and rs6773957 (P < 10(-7)). These two SNPs were in high linkage disequilibrium (r(2) = 0.98) and located within ADIPOQ. Interestingly, our fourth strongest region of association (P < 2 x 10(-5)) was to an SNP within CDH13, whose protein product is a newly identified receptor for high-molecular-weight species of adiponectin. Through WGA analysis, we confirmed previous studies showing SNPs within ADIPOQ to be strongly associated with variation in adiponectin levels and further observed these to have the strongest effects on adiponectin levels throughout the genome. We additionally identified a second gene (CDH13) possibly influencing variation in adiponectin levels. The impact of these SNPs on health and disease has yet to be determined.

Complete longitudinal analyses of the randomized, placebo-controlled, phase III trial of sunitinib in patients with gastrointestinal stromal tumor following imatinib failure.

PURPOSE: To analyze final long-term survival and clinical outcomes from the randomized phase III study of sunitinib in gastrointestinal stromal tumor patients after imatinib failure; to assess correlative angiogenesis biomarkers with patient outcomes. EXPERIMENTAL DESIGN: Blinded sunitinib or placebo was given daily on a 4-week-on/2-week-off treatment schedule. Placebo-assigned patients could cross over to sunitinib at disease progression/study unblinding. Overall survival (OS) was analyzed using conventional statistical methods and the rank-preserving structural failure time (RPSFT) method to explore cross-over impact. Circulating levels of angiogenesis biomarkers were analyzed. RESULTS: In total, 243 patients were randomized to receive sunitinib and 118 to placebo, 103 of whom crossed over to open-label sunitinib. Conventional statistical analysis showed that OS converged in the sunitinib and placebo arms (median 72.7 vs. 64.9 weeks; HR, 0.876; P = 0.306) as expected, given the cross-over design. RPSFT analysis estimated median OS for placebo of 39.0 weeks (HR, 0.505, 95% CI, 0.262-1.134; P = 0.306). No new safety concerns emerged with extended sunitinib treatment. No consistent associations were found between the pharmacodynamics of angiogenesis-related plasma proteins during sunitinib treatment and clinical outcome. CONCLUSIONS: The cross-over design provided evidence of sunitinib clinical benefit based on prolonged time to tumor progression during the double-blind phase of this trial. As expected, following cross-over, there was no statistical difference in OS. RPSFT analysis modeled the absence of cross-over, estimating a substantial sunitinib OS benefit relative to placebo. Long-term sunitinib treatment was tolerated without new adverse events.

Lung function in idiopathic pulmonary fibrosis--extended analyses of the IFIGENIA trial.

BACKGROUND The randomized placebo-controlled IFIGENIA-trial demonstrated that therapy with high-dose N-acetylcysteine (NAC) given for one year, added to prednisone and azathioprine, significantly ameliorates (i.e. slows down) disease progression in terms of vital capacity (VC) (+9%) and diffusing capacity (DLco) (+24%) in idiopathic pulmonary fibrosis (IPF). To better understand the clinical implications of these findings we performed additional, explorative analyses of the IFGENIA data set. METHODS We analysed effects of NAC on VC, DLco, a composite physiologic index (CPI), and mortality in the 155 study-patients. RESULTS In trial completers the functional indices did not change significantly with NAC, whereas most indices deteriorated with placebo; in non-completers the majority of indices worsened but decline was generally less pronounced in most indices with NAC than with placebo. Most categorical analyses of VC, DLco and CPI also showed favourable changes with NAC. The effects of NAC on VC, DLco and CPI were significantly better if the baseline CPI was 50 points or lower. CONCLUSION This descriptive analysis confirms and extends the favourable effects of NAC on lung function in IPF and emphasizes the usefulness of VC, DLco, and the CPI for the evaluation of a therapeutic effect. Most importantly, less progressed disease as indicated by a CPI of 50 points or lower at baseline was more responsive to therapy in this study.

Homology modeling and metabolism prediction of human carboxylesterase-2 using docking analyses by GriDock: a parallelized tool based on AutoDock 4.0.

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted to developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas less has been done to predict the activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES2. The study involved first a homology modeling of the hCES2 protein based on the model of hCES1 since the two proteins share a high degree of homology (congruent with 73%). A set of 40 known substrates of hCES2 was taken from the literature; the ligands were docked in both their neutral and ionized forms using GriDock, a parallel tool based on the AutoDock4.0 engine which can perform efficient and easy virtual screening analyses of large molecular databases exploiting multi-core architectures. Useful statistical models (e.g., r (2) = 0.91 for substrates in their unprotonated state) were calculated by correlating experimental pK(m) values with distance between the carbon atom of the substrate's ester group and the hydroxy function of Ser228. Additional parameters in the equations accounted for hydrophobic and electrostatic interactions between substrates and contributing residues. The negatively charged residues in the hCES2 cavity explained the preference of the enzyme for neutral substrates and, more generally, suggested that ligands which interact too strongly by ionic bonds (e.g., ACE inhibitors) cannot be good CES2 substrates because they are trapped in the cavity in unproductive modes and behave as inhibitors. The effects of protonation on substrate recognition and the contrasting behavior of substrates and products were finally investigated by MD simulations of some CES2 complexes.

MAPI: towards the integrated exploitation of bioinformatics Web Services.

BACKGROUND. Bioinformatics is commonly featured as a well assorted list of available web resources. Although diversity of services is positive in general, the proliferation of tools, their dispersion and heterogeneity complicate the integrated exploitation of such data processing capacity. RESULTS. To facilitate the construction of software clients and make integrated use of this variety of tools, we present a modular programmatic application interface (MAPI) that provides the necessary functionality for uniform representation of Web Services metadata descriptors including their management and invocation protocols of the services which they represent. This document describes the main functionality of the framework and how it can be used to facilitate the deployment of new software under a unified structure of bioinformatics Web Services. A notable feature of MAPI is the modular organization of the functionality into different modules associated with specific tasks. This means that only the modules needed for the client have to be installed, and that the module functionality can be extended without the need for re-writing the software client. CONCLUSIONS. The potential utility and versatility of the software library has been demonstrated by the implementation of several currently available clients that cover different aspects of integrated data processing, ranging from service discovery to service invocation with advanced features such as workflows composition and asynchronous services calls to multiple types of Web Services including those registered in repositories (e.g. GRID-based, SOAP, BioMOBY, R-bioconductor, and others).