Introduction of an automated system for the diagnosis and quantification of hepatitis B and hepatitis C viruses.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections pose major public health problems because of their prevalence worldwide. Consequently, screening for these infections is an important part of routine laboratory activity. Serological and molecular markers are key elements in diagnosis, prognosis and treatment monitoring for HBV and HCV infections. Today, automated chemiluminescence immunoassay (CLIA) analyzers are widely used for virological diagnosis, particularly in high-volume clinical laboratories. Molecular biology techniques are routinely used to detect and quantify viral genomes as well as to analyze their sequence; in order to determine their genotype and detect resistance to antiviral drugs. Real-time PCR, which provides high sensitivity and a broad dynamic range, has gradually replaced other signal and target amplification technologies for the quantification and detection of nucleic acid. The next-generation DNA sequencing techniques are still restricted to research laboratories.The serological and molecular marker methods available for HBV and HCV are discussed in this article, along with their utility and limitations for use in Chronic Hepatitis B (CHB) diagnosis and monitoring.

Exploration of alpine orographic precipitation patterns with radar image processing and clustering techniques

A methodology of exploratory data analysis investigating the phenomenon of orographic precipitation enhancement is proposed. The precipitation observations obtained from three Swiss Doppler weather radars are analysed for the major precipitation event of August 2005 in the Alps. Image processing techniques are used to detect significant precipitation cells/pixels from radar images while filtering out spurious effects due to ground clutter. The contribution of topography to precipitation patterns is described by an extensive set of topographical descriptors computed from the digital elevation model at multiple spatial scales. Additionally, the motion vector field is derived from subsequent radar images and integrated into a set of topographic features to highlight the slopes exposed to main flows. Following the exploratory data analysis with a recent algorithm of spectral clustering, it is shown that orographic precipitation cells are generated under specific flow and topographic conditions. Repeatability of precipitation patterns in particular spatial locations is found to be linked to specific local terrain shapes, e.g. at the top of hills and on the upwind side of the mountains. This methodology and our empirical findings for the Alpine region provide a basis for building computational data-driven models of orographic enhancement and triggering of precipitation. Copyright (C) 2011 Royal Meteorological Society .

Natural abundance of stable isotopes (15N and 13C) and isotopic fractionation of sea bream muscle as markers of food and culture conditions and its relationship with metabolic and proteomic analyses

Els isòtops estables com a traçadors de la cadena alimentària, s'han utilitzat per caracteritzar la relació entre els consumidors i els seus aliments, ja que el fraccionament isotòpic implica una discriminació en contra de certs isòtops. Però les anàlisis d'isòtops estables (SIA), també es poden dur a terme en peixos cultivats amb dietes artificials, com la orada (Sparus aurata), la especie más cultivada en el Mediterráneo. Canvis en l'abundància natural d'isòtops estables (13C i 15N) en els teixits i les seves reserves poden reflectir els canvis en l'ús i reciclatge dels nutrients ja que els enzims catabòlics implicats en els processos de descarboxilació i desaminació mostren una preferència pels isòtops més lleugers. Per tant, aquestes anàlisis ens poden proporcionar informació útil sobre l'estat nutricional i metabòlic dels peixos. L'objectiu d'aquest projecte va ser determinar la capacitat dels isòtops estables per ser utilitzats com a marcadors potencials de la capacitat de creixement i condicions de cria de l'orada. En aquest sentit, les anàlisis d'isòtops estables s'han combinat amb altres metabòlics (activitats citocrom-c-oxidasa, COX, i citrat sintasa, CS) i els paràmetres de creixement (ARN/ADN). El conjunt de resultats obtinguts en els diferents estudis realitzats en aquest projecte demostra que el SIA, en combinació amb altres paràmetres metabòlics, pot servir com una eina eficaç per discriminar els peixos amb millor potencial de creixement, així com a marcador sensible de l'estat nutricional i d'engreix. D'altra banda, la combinació de l'anàlisi d'isòtops estables amb les eines emergents, com ara tècniques de proteòmica (2D-PAGE), ens proporciona nous coneixements sobre els canvis metabòlics que ocorren en els músculs dels peixos durant l‟increment del creixement muscular induït per l'exercici.

Emergence and characterisation of vanB vancomycin-resistant Enterococcus faecalis in Rio de Janeiro, Brazil

Here we describe the detection and characterisation of three isolates of vancomycin-resistant VanB-type Enterococcus faecalis. Sequence analysis suggested that these isolates harboured the vanB1 gene. The isolates were susceptible to the majority of antimicrobial agents tested, with the exception of chloramphenicol, erythromycin and vancomycin, and showed distinct profiles of high-level resistance to aminoglycosides. Analysis of the clonal relatedness of the vanB E. faecalis isolates showed similar pulsed-field gel electrophoresis profiles. To our knowledge, this is the first report of the occurrence of enterococcal strains carrying vanB genes in Brazil.

Microarray profiling of mononuclear peripheral blood cells identifies novel candidate genes related to chemoradiation response in rectal cancer.

Preoperative chemoradiation significantly improves oncological outcome in locally advanced rectal cancer. However there is no effective method of predicting tumor response to chemoradiation in these patients. Peripheral blood mononuclear cells have emerged recently as pathology markers of cancer and other diseases, making possible their use as therapy predictors. Furthermore, the importance of the immune response in radiosensivity of solid organs led us to hypothesized that microarray gene expression profiling of peripheral blood mononuclear cells could identify patients with response to chemoradiation in rectal cancer. Thirty five 35 patients with locally advanced rectal cancer were recruited initially to perform the study. Peripheral blood samples were obtained before neaodjuvant treatment. RNA was extracted and purified to obtain cDNA and cRNA for hybridization of microarrays included in Human WG CodeLink bioarrays. Quantitative real time PCR was used to validate microarray experiment data. Results were correlated with pathological response, according to Mandard´s criteria and final UICC Stage (patients with tumor regression grade 1-2 and downstaging being defined as responders and patients with grade 3-5 and no downstaging as non-responders). Twenty seven out of 35 patients were finally included in the study. We performed a multiple t-test using Significance Analysis of Microarrays, to find those genes differing significantly in expression, between responders (n = 11) and non-responders (n = 16) to CRT. The differently expressed genes were: BC 035656.1, CIR, PRDM2, CAPG, FALZ, HLA-DPB2, NUPL2, and ZFP36. The measurement of FALZ (p = 0.029) gene expression level determined by qRT-PCR, showed statistically significant differences between the two groups. Gene expression profiling reveals novel genes in peripheral blood samples of mononuclear cells that could predict responders and non-responders to chemoradiation in patients with locally advanced rectal cancer. Moreover, our investigation added further evidence to the importance of mononuclear cells' mediated response in the neoadjuvant treatment of rectal cancer.

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

Emergence of young human genes after a burst of retroposition in primates.

The origin of new genes through gene duplication is fundamental to the evolution of lineage- or species-specific phenotypic traits. In this report, we estimate the number of functional retrogenes on the lineage leading to humans generated by the high rate of retroposition (retroduplication) in primates. Extensive comparative sequencing and expression studies coupled with evolutionary analyses and simulations suggest that a significant proportion of recent retrocopies represent bona fide human genes. We estimate that at least one new retrogene per million years emerged on the human lineage during the past approximately 63 million years of primate evolution. Detailed analysis of a subset of the data shows that the majority of retrogenes are specifically expressed in testis, whereas their parental genes show broad expression patterns. Consistently, most retrogenes evolved functional roles in spermatogenesis. Proteins encoded by X chromosome-derived retrogenes were strongly preserved by purifying selection following the duplication event, supporting the view that they may act as functional autosomal substitutes during X-inactivation of late spermatogenesis genes. Also, some retrogenes acquired a new or more adapted function driven by positive selection. We conclude that retroduplication significantly contributed to the formation of recent human genes and that most new retrogenes were progressively recruited during primate evolution by natural and/or sexual selection to enhance male germline function.

The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function.

The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the gamma peptide which co-purifies with Na,K-ATPase. Immuno-radiolabeling of epitope-tagged gamma subunits in intact oocytes and protease protection assays show that the gamma peptide is a type I membrane protein lacking a signal sequence and exposing the N-terminus to the extracytoplasmic side. Co-expression of the rat or Xenopus gamma subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K-ATPase. The gamma peptide does not influence the formation and cell surface expression of functional Na,K-ATPase alpha-beta complexes. On the other hand, the gamma peptide itself needs association with Na,K-ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. Gamma subunits do not associate with individual alpha or beta subunits but only interact with assembled, transport-competent alpha-beta complexes. Finally, electrophysiological measurements indicate that the gamma peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the gamma subunit of Na,K-ATPase.

Novel Chlamydiales strains isolated from a water treatment plant.

Chlamydiae are obligate intracellular bacteria infecting free-living amoebae, vertebrates and some invertebrates. Novel members are regularly discovered, and there is accumulating evidence supporting a very important diversity of chlamydiae in the environment. In this study, we investigated the presence of chlamydiae in a drinking water treatment plant. Samples were used to inoculate Acanthamoeba monolayers (Acanthamoeba co-culture), and to recover autochthonous amoebae onto non-nutritive agar. Chlamydiae were searched for by a pan-chlamydia 16S rRNA gene PCR from both Acanthamoeba co-cultures and autochthonous amoebae, and phylotypes determined by 16S rRNA gene sequencing. Autochthonous amoebae also were identified by 18S rRNA gene amplification and sequencing. From a total of 79 samples, we recovered eight chlamydial strains by Acanthamoeba co-culture, but only one of 28 amoebae harboured a chlamydia. Sequencing results and phylogenetic analysis showed our strains belonging to four distinct chlamydial lineages. Four strains, including the strain recovered within its natural host, belonged to the Parachlamydiaceae; two closely related strains belonged to the Criblamydiaceae; two distinct strains clustered with Rhabdochlamydia spp.; one strain clustered only with uncultured environmental clones. Our results confirmed the usefulness of amoeba co-culture to recover novel chlamydial strains from complex samples and demonstrated the huge diversity of chlamydiae in the environment, by identifying several new species including one representing the first strain of a new family.

Genome-wide mRNA expression correlates of viral control in CD4+ T-cells from HIV-1-infected individuals.

There is great interindividual variability in HIV-1 viral setpoint after seroconversion, some of which is known to be due to genetic differences among infected individuals. Here, our focus is on determining, genome-wide, the contribution of variable gene expression to viral control, and to relate it to genomic DNA polymorphism. RNA was extracted from purified CD4+ T-cells from 137 HIV-1 seroconverters, 16 elite controllers, and 3 healthy blood donors. Expression levels of more than 48,000 mRNA transcripts were assessed by the Human-6 v3 Expression BeadChips (Illumina). Genome-wide SNP data was generated from genomic DNA using the HumanHap550 Genotyping BeadChip (Illumina). We observed two distinct profiles with 260 genes differentially expressed depending on HIV-1 viral load. There was significant upregulation of expression of interferon stimulated genes with increasing viral load, including genes of the intrinsic antiretroviral defense. Upon successful antiretroviral treatment, the transcriptome profile of previously viremic individuals reverted to a pattern comparable to that of elite controllers and of uninfected individuals. Genome-wide evaluation of cis-acting SNPs identified genetic variants modulating expression of 190 genes. Those were compared to the genes whose expression was found associated with viral load: expression of one interferon stimulated gene, OAS1, was found to be regulated by a SNP (rs3177979, p = 4.9E-12); however, we could not detect an independent association of the SNP with viral setpoint. Thus, this study represents an attempt to integrate genome-wide SNP signals with genome-wide expression profiles in the search for biological correlates of HIV-1 control. It underscores the paradox of the association between increasing levels of viral load and greater expression of antiviral defense pathways. It also shows that elite controllers do not have a fully distinctive mRNA expression pattern in CD4+ T cells. Overall, changes in global RNA expression reflect responses to viral replication rather than a mechanism that might explain viral control.

Application of colon capsule endoscopy (CCE) to evaluate the whole gastrointestinal tract: a comparative study of single-camera and dual-camera analysis.

BACKGROUND AND STUDY AIMS Colon capsule endoscopy (CCE) was developed for the evaluation of colorectal pathology. In this study, our aim was to assess if a dual-camera analysis using CCE allows better evaluation of the whole gastrointestinal (GI) tract compared to a single-camera analysis. PATIENTS AND METHODS We included 21 patients (12 males, mean age 56.20 years) submitted for a CCE examination. After standard colon preparation, the colon capsule endoscope (PillCam Colon™) was swallowed after reinitiation from its "sleep" mode. Four physicians performed the analysis: two reviewed both video streams at the same time (dual-camera analysis); one analyzed images from one side of the device ("camera 1"); and the other reviewed the opposite side ("camera 2"). We compared numbers of findings from different parts of the entire GI tract and level of agreement among reviewers. RESULTS A complete evaluation of the GI tract was possible in all patients. Dual-camera analysis provided 16% and 5% more findings compared to camera 1 and camera 2 analysis, respectively. Overall agreement was 62.7% (kappa = 0.44, 95% CI: 0.373-0.510). Esophageal (kappa = 0.611) and colorectal (kappa = 0.595) findings had a good level of agreement, while small bowel (kappa = 0.405) showed moderate agreement. CONCLUSION The use of dual-camera analysis with CCE for the evaluation of the GI tract is feasible and detects more abnormalities when compared with single-camera analysis.

Effectiveness of two interventions based on improving patient-practitioner communication on diabetes self-management in patients with low educational level: study protocol of a clustered randomized trial in primary care.

BACKGROUND In the last decades the presence of social inequalities in diabetes care has been observed in multiple countries, including Spain. These inequalities have been at least partially attributed to differences in diabetes self-management behaviours. Communication problems during medical consultations occur more frequently to patients with a lower educational level. The purpose of this cluster randomized trial is to determine whether an intervention implemented in a General Surgery, based in improving patient-provider communication, results in a better diabetes self-management in patients with lower educational level. A secondary objective is to assess whether telephone reinforcement enhances the effect of such intervention. We report the design and implementation of this on-going study. METHODS/DESIGN The study is being conducted in a General Practice located in a deprived neighbourhood of Granada, Spain. Diabetic patients 18 years old or older with a low educational level and inadequate glycaemic control (HbA1c > 7%) were recruited. General Practitioners (GPs) were randomised to three groups: intervention A, intervention B and control group. GPs allocated to intervention groups A and B received training in communication skills and are providing graphic feedback about glycosylated haemoglobin levels. Patients whose GPs were allocated to group B are additionally receiving telephone reinforcement whereas patients from the control group are receiving usual care. The described interventions are being conducted during 7 consecutive medical visits which are scheduled every three months. The main outcome measure will be HbA1c; blood pressure, lipidemia, body mass index and waist circumference will be considered as secondary outcome measures. Statistical analysis to evaluate the effectiveness of the interventions will include multilevel regression analysis with three hierarchical levels: medical visit level, patient level and GP level. DISCUSSION The results of this study will provide new knowledge about possible strategies to promote a better diabetes self-management in a particularly vulnerable group. If effective, this low cost intervention will have the potential to be easily incorporated into routine clinical practice, contributing to decrease health inequalities in diabetic patients. TRIAL REGISTRATION Clinical Trials U.S. National Institutes of Health, NCT01849731.

False phylogenies on wood mice due to cryptic cytochrome-b pseudogene.

The phylogeny and phylogeography of the Old World wood mice (subgenus Sylvaemus, genus Apodemus, Muridae) are well-documented. Nevertheless, the distributions of species, such as A. fulvipectus and A. ponticus remain dubious, as well as their phylogenetic relationships with A. sylvaticus. We analysed samples of Apodemus spp. across Europe using the mitochondrial cytochrome-b gene (cyt-b) and compared the DNA and amino-acid compositions of previously published sequences. The main result stemming from this study is the presence of a well-differentiated lineage of Sylvaemus including samples of various species (A. sylvaticus, A. fulvipectus, A. ponticus) from distant locations, which were revealed to be nuclear copies of the mitochondrial cyt-b. The presence of this cryptic pseudogene in published sequences is supported by different pathways. This has led to important errors in previous molecular trees and hence to partial misinterpretations in the phylogeny of Apodemus.

Seasonal fluctuations of bacterial community diversity in agricultural soil and experimental validation by laboratory disturbance experiments.

Natural fluctuations in soil microbial communities are poorly documented because of the inherent difficulty to perform a simultaneous analysis of the relative abundances of multiple populations over a long time period. Yet, it is important to understand the magnitudes of community composition variability as a function of natural influences (e.g., temperature, plant growth, or rainfall) because this forms the reference or baseline against which external disturbances (e.g., anthropogenic emissions) can be judged. Second, definition of baseline fluctuations in complex microbial communities may help to understand at which point the systems become unbalanced and cannot return to their original composition. In this paper, we examined the seasonal fluctuations in the bacterial community of an agricultural soil used for regular plant crop production by using terminal restriction fragment length polymorphism profiling (T-RFLP) of the amplified 16S ribosomal ribonucleic acid (rRNA) gene diversity. Cluster and statistical analysis of T-RFLP data showed that soil bacterial communities fluctuated very little during the seasons (similarity indices between 0.835 and 0.997) with insignificant variations in 16S rRNA gene richness and diversity indices. Despite overall insignificant fluctuations, between 8 and 30% of all terminal restriction fragments changed their relative intensity in a significant manner among consecutive time samples. To determine the magnitude of community variations induced by external factors, soil samples were subjected to either inoculation with a pure bacterial culture, addition of the herbicide mecoprop, or addition of nutrients. All treatments resulted in statistically measurable changes of T-RFLP profiles of the communities. Addition of nutrients or bacteria plus mecoprop resulted in bacteria composition, which did not return to the original profile within 14 days. We propose that at less than 70% similarity in T-RFLP, the bacterial communities risk to drift apart to inherently different states.

Imprinting of tumor-suppressor genes in human placenta.

Transcriptional deregulation in cancer has been shown to be associated with epigenetic alterations, in particular to tumor-suppressor- gene (TSG) promoters. In contrast, DNA methylation of TSGs is not considered to be present in normal differentiated cells. Nevertheless, we previously showed that the promoter of the tumor-suppressor gene APC is methylated, for one allele only, in normal gastric cells. Recently, RASSF1A has been shown to be imprinted in normal human placenta. To clarify putative TSG methylation in the placenta, 23 normal placental tissues from the first trimester, both decidua and villi, and four normal non-gestational endometrium were screened for DNA methylation by methylation-sensitive single-strand conformation analysis (MS-SSCA) and sequencing after bisulfite modification, on a panel of 12 genes known to be implicated in carcinogenesis. In all placental villi, four TSG promoters-APC, SFRP2, RASSF1A and WIF1-were hypermethylated, whereas all decidua and normal endometrium did not show any methylation. Allele-specific methylation analysis revealed that this methylation was monoallelic. Furthermore, comparison with maternal DNA indicated that APC and WIF1 were methylated on the maternal allele, whereas SFRP2 was methylated on the paternal allele. Sequence analysis of WIF1 mRNA revealed that only the unmethylated paternal allele was transcribed. The imprinting status of these TSGs is conserved during pregnancy. These results indicate that TSG imprinting is pre-existent in normal human placenta and should not be confused with carcinogenesis or pathology-induced methylation.