Real-time quantitative PCR assay for measurement of bird telomeres

We present the application of a real-time quantitative PCR assay, previously developed to measure relative telomere length in humans and mice, to two bird species, the zebra finch Taeniopygia guttata and the Alpine swift Apus melba. This technique is based on the PCR amplification of telomeric (TTAGGG)(n) sequences using specific oligonucleotide primers. Relative telomere length is expressed as the ratio (T/S) of telomere repeat copy number (T) to control single gene copy number (S). This method is particularly useful for comparisons of individuals within species, or where the same individuals are followed longitudinally. We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a single control gene. In both species, we validated our PCR measurements of relative telomere length against absolute measurements of telomere length determined by the conventional method of quantifying telomere terminal restriction fragment (TRF) lengths using both the traditional Southern blot analysis (Alpine swifts) and in gel hybridization (zebra finches). As found in humans and mice, telomere lengths in the same sample measured by TRF and PCR were well correlated in both the Alpine swift and the zebra finch.. Hence, this PCR assay for measurement of bird telomeres, which is fast and requires only small amounts of genomic DNA, should open new avenues in the study of environmental factors influencing variation in telomere length, and how this variation translates into variation in cellular and whole organism senescence.

Identificação por PCR dos alelos-S associados à compatibilidade gametofítica em ameixeira japonesa

O objetivo deste trabalho foi identificar e caracterizar molecularmente os alelos-S de cultivares de ameixeira japonesa e verificar a compatibilidade gametofítica entre estes. Foram utilizados dois pares de iniciadores específicos na amplificação dos alelos via PCR, em 18 cultivares: Pluma 7, Gulf Rubi, Blood Plum, Wickson, América, Santa Rosa, Rosa Mineira, Estrela Púrpura, Amarelinha, The First, Harry Pieckstone, Santa Rita, Seleção 16, Seleção A26 (Burbank), Seleção 21, Seleção 19, Methley e Simka. As condições da PCR e as combinações de oligonucleotídeos iniciadores utilizadas permitiram a identificação de alelos-S nas cultivares, bem como a indicação dos polinizadores mais compatíveis. As cultivares América e Santa Rosa, bem como Blood Plum, Wickson, Rosa Mineira, Estrela Púrpura e Seleção 21 apresentam incompatibilidade total entre si, por compartilharem os mesmos alelos.

eggNOG v4.0: nested orthology inference across 3686 organisms

With the increasing availability of various 'omics data, high-quality orthology assignment is crucial for evolutionary and functional genomics studies. We here present the fourth version of the eggNOG database (available at http://eggnog.embl.de) that derives nonsupervised orthologous groups (NOGs) from complete genomes, and then applies a comprehensive characterization and analysis pipeline to the resulting gene families. Compared with the previous version, we have more than tripled the underlying species set to cover 3686 organisms, keeping track with genome project completions while prioritizing the inclusion of high-quality genomes to minimize error propagation from incomplete proteome sets. Major technological advances include (i) a robust and scalable procedure for the identification and inclusion of high-quality genomes, (ii) provision of orthologous groups for 107 different taxonomic levels compared with 41 in eggNOGv3, (iii) identification and annotation of particularly closely related orthologous groups, facilitating analysis of related gene families, (iv) improvements of the clustering and functional annotation approach, (v) adoption of a revised tree building procedure based on the multiple alignments generated during the process and (vi) implementation of quality control procedures throughout the entire pipeline. As in previous versions, eggNOGv4 provides multiple sequence alignments and maximum-likelihood trees, as well as broad functional annotation. Users can access the complete database of orthologous groups via a web interface, as well as through bulk download.

Premenopausal serum androgens and breast cancer risk: a nested case-control study.

ABSTRACT: INTRODUCTION: Prospective epidemiologic studies have consistently shown that levels of circulating androgens in postmenopausal women are positively associated with breast cancer risk. However, data in premenopausal women are limited. METHODS: A case-control study nested within the New York University Women's Health Study was conducted. A total of 356 cases (276 invasive and 80 in situ) and 683 individually-matched controls were included. Matching variables included age and date, phase, and day of menstrual cycle at blood donation. Testosterone, androstenedione, dehydroandrosterone sulfate (DHEAS) and sex hormone-binding globulin (SHBG) were measured using direct immunoassays. Free testosterone was calculated. RESULTS: Premenopausal serum testosterone and free testosterone concentrations were positively associated with breast cancer risk. In models adjusted for known risk factors of breast cancer, the odds ratios for increasing quintiles of testosterone were 1.0 (reference), 1.5 (95% confidence interval (CI), 0.9 to 2.3), 1.2 (95% CI, 0.7 to 1.9), 1.4 (95% CI, 0.9 to 2.3) and 1.8 (95% CI, 1.1 to 2.9; Ptrend = 0.04), and for free testosterone were 1.0 (reference), 1.2 (95% CI, 0.7 to 1.8), 1.5 (95% CI, 0.9 to 2.3), 1.5 (95% CI, 0.9 to 2.3), and 1.8 (95% CI, 1.1 to 2.8, Ptrend = 0.01). A marginally significant positive association was observed with androstenedione (P = 0.07), but no association with DHEAS or SHBG. Results were consistent in analyses stratified by tumor type (invasive, in situ), estrogen receptor status, age at blood donation, and menopausal status at diagnosis. Intra-class correlation coefficients for samples collected from 0.8 to 5.3 years apart (median 2 years) in 138 cases and 268 controls were greater than 0.7 for all biomarkers except for androstenedione (0.57 in controls). CONCLUSIONS: Premenopausal concentrations of testosterone and free testosterone are associated with breast cancer risk. Testosterone and free testosterone measurements are also highly reliable (that is, a single measurement is reflective of a woman's average level over time). Results from other prospective studies are consistent with our results. The impact of including testosterone or free testosterone in breast cancer risk prediction models for women between the ages of 40 and 50 years should be assessed. Improving risk prediction models for this age group could help decision making regarding both screening and chemoprevention of breast cancer.

Identification of parasitoid species using PCR amplification and restriction enzyme digestion

Spodoptera frugiperda is a pest of great economic importance in the Americas. It is attacked by several species of parasitoids, which act as biological control agents. Parasitoids are morphologically identifiable as adults, but not as larvae. Laboratory rearing conditions are not always optimal to rear out parasitic wasps from S. frugiperda larvae collected from wild populations, and it frequently happens that parasitoids do not complete their life cycle and stop developing at the larval stage. Therefore, we explored ways to identify parasitoid larvae using molecular techniques. Sequencing is one possible technique, yet it is expensive. Here we present an alternate, cheaper way of identifying seven species of parasitoids (Cotesia marginiventris, Campoletis sonorensis, Pristomerus spinator, Chelonus insularis, Chelonus cautus, Eiphosoma vitticolle and Meteorus laphygmae) using PCR amplification of COI gene followed by a digestion with a combination of four restriction endonucleases. Each species was found to exhibit a specific pattern when the amplification product was run on an agarose gel. Identifying larvae revealed that conclusions on species composition of a population of parasitic wasps can be biased if only the emerging adults are taken into account.

New diagnostic real-time PCR for specific detection of Parachlamydia acanthamoebae DNA in clinical samples

Given the low sensitivity of amoebal coculture, we developed a specific real-time PCR for the detection of Parachlamydia. The analytical sensitivity was high, and the inter- and intrarun variabilities were low. When the PCR was applied to nasopharyngeal aspirates, it was positive for six patients with bronchiolitis. Future studies should assess the role of Parachlamydia in bronchiolitis.

EXTRAÇÃO DE DNA GENÔMICO DE Passiflora spp. PARA ANÁLISES PCR-RAPD

A identificação e caracterização da diversidade genética de plantas por meio de técnicas moleculares envolvem a avaliação de vários indivíduos, necessitando-se, portanto, de métodos rápidos e precisos de extração do DNA. O co-isolamento de polissacarídeos, fenóis e compostos secundários é o principal problema encontrado no isolamento e purificação de DNA vegetal. Folhas das diversas espécies de Passiflora possuem níveis variados desses compostos que podem comprometer este procedimento. O presente estudo foi realizado com o objetivo de avaliar a qualidade e quantidade de DNA de folhas de variedades de Passiflora spp., utilizando-se de três métodos de extração. Os três métodos forneceram DNA em qualidade e quantidade suficientes para a realização da técnica PCR-RAPD.

Discovery of a 29-gene panel in peripheral blood mononuclear cells for the detection of colorectal cancer and adenomas using high throughput real-time PCR.

Colorectal cancer (CRC) is the second leading cause of cancer-related death in developed countries. Early detection of CRC leads to decreased CRC mortality. A blood-based CRC screening test is highly desirable due to limited invasiveness and high acceptance rate among patients compared to currently used fecal occult blood testing and colonoscopy. Here we describe the discovery and validation of a 29-gene panel in peripheral blood mononuclear cells (PBMC) for the detection of CRC and adenomatous polyps (AP). Blood samples were prospectively collected from a multicenter, case-control clinical study. First, we profiled 93 samples with 667 candidate and 3 reference genes by high throughput real-time PCR (OpenArray system). After analysis, 160 genes were retained and tested again on 51 additional samples. Low expressed and unstable genes were discarded resulting in a final dataset of 144 samples profiled with 140 genes. To define which genes, alone or in combinations had the highest potential to discriminate AP and/or CRC from controls, data were analyzed by a combination of univariate and multivariate methods. A list of 29 potentially discriminant genes was compiled and evaluated for its predictive accuracy by penalized logistic regression and bootstrap. This method discriminated AP >1cm and CRC from controls with a sensitivity of 59% and 75%, respectively, with 91% specificity. The behavior of the 29-gene panel was validated with a LightCycler 480 real-time PCR platform, commonly adopted by clinical laboratories. In this work we identified a 29-gene panel expressed in PBMC that can be used for developing a novel minimally-invasive test for accurate detection of AP and CRC using a standard real-time PCR platform.

Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy

Reproductive traits play a key role in pig production in order to reduce costs and increase economic returns. Among others, gene expression analyses represent a useful approach to study genetic mechanisms underlying reproductive traits in pigs. The application of reverse-transcription quantitative PCR requires the selection of appropriate reference genes, whose expression levels should not be affected by the experimental conditions, especially when comparing gene expression across different physiological stages.

Newly described human polyomaviruses Merkel Cell, KI and WU are present in urban sewage and may represent potential environmental contaminants

Recently, three new polyomaviruses (KI, WU and Merkel cell polyomavirus) have been reported to infect humans. It has also been suggested that lymphotropic polyomavirus, a virus of simian origin, infects humans. KI and WU polyomaviruses have been detected mainly in specimens from the respiratory tract while Merkel cell polyomavirus has been described in a very high percentage of Merkel cell carcinomas. The distribution, excretion level and transmission routes of these viruses remain unknown. Here we analyzed the presence and characteristics of newly described human polyomaviruses in urban sewage and river water in order to assess the excretion level and the potential role of water as a route of transmission of these viruses. Nested-PCR assays were designed for the sensitive detection of the viruses studied and the amplicons obtained were confirmed by sequencing analysis. The viruses were concentrated following a methodology previously developed for the detection of JC and BK human polyomaviruses in environmental samples. JC polyomavirus and human adenoviruses were used as markers of human contamination in the samples. Merkel cell polyomavirus was detected in 7/8 urban sewage samples collected and in 2/7 river water samples. Also one urine sample from a pregnant woman, out of 4 samples analyzed, was positive for this virus. KI and WU polyomaviruses were identified in 1/8 and 2/8 sewage samples respectively. The viral strains detected were highly homologous with other strains reported from several other geographical areas. Lymphotropic polyomavirus was not detected in any of the 13 sewage neither in 9 biosolid/sludge samples analyzed. This is the first description of a virus isolated from sewage and river water with a strong association with cancer. Our data indicate that the Merkel cell polyomavirus is prevalent in the population and that it may be disseminated through the fecal/urine contamination of water. The procedure developed may constitute a useful tool for studying the excreted strains, prevalence and transmission of these recently described polyomaviruses.

Malaria real-time PCR: correlation with clinical presentation.

Among 112 patients infected only by Plasmodium falciparum, WHO criteria of severity were compared with parasite load assessed by microscopy and quantitative PCR. Clinical severity was significantly correlated with higher parasite load as determined by microscopy (p < 0.001) and by PCR (p < 0.001). Hence, quantitative PCR might be useful to predict outcome.

Schistosoma mansoni and HIV acquisition in fishing communities of Lake Victoria, Uganda: a nested case-control study.

OBJECTIVE: It has been suggested that Schistosoma mansoni, which is endemic in African fishing communities, might increase susceptibility to human immunodeficiency virus (HIV) acquisition. If confirmed, this would be of great public health importance in these high HIV-risk communities. This study was undertaken to determine whether S. mansoni infection is a risk factor for HIV infection among the fishing communities of Lake Victoria, Uganda. We conducted a matched case-control study, nested within a prospective HIV incidence cohort, including 50 HIV seroconverters (cases) and 150 controls during 2009-2011. METHODS: S. mansoni infection prior to HIV seroconversion was determined by measuring serum circulating anodic antigen (CAA) in stored serum. HIV testing was carried out using the Determine rapid test and infection confirmed by enzyme-linked immunosorbent assays. RESULTS: About 49% of cases and 52% of controls had S. mansoni infection prior to HIV seroconversion (or at the time of a similar study visit, for controls): odds ratio, adjusting for ethnicity, religion, marital status, education, occupation, frequency of alcohol consumption in previous 3 months, number of sexual partners while drunk, duration of stay in the community, and history of schistosomiasis treatment in the past 2 years was 1.23 (95% CI 0.3-5.7) P = 0.79. S. mansoni infections were chronic (with little change in status between enrolment and HIV seroconversion), and there was no difference in median CAA concentration between cases and controls. CONCLUSIONS: These results do not support the hypothesis that S. mansoni infection promotes HIV acquisition.

Immunodeficiency and the risk of cervical intraepithelial neoplasia 2/3 and cervical cancer: A nested case-control study in the Swiss HIV cohort study.

HIV-infected women are at increased risk of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC), but it has been difficult to disentangle the influences of heavy exposure to HPV infection, inadequate screening and immunodeficiency. A case-control study including 364 CIN2/3 and 20 ICC cases matched to 1,147 controls was nested in the Swiss HIV Cohort Study (1985-2013). CIN2/3 risk was significantly associated with low CD4+ cell counts, whether measured as nadir [odds ratio (OR) per 100-cell/μL decrease = 1.15, 95% CI: 1.08, 1.22], or at CIN2/3 diagnosis (1.10, 95% CI: 1.04, 1.16). An association was evident even for nadir CD4+ 200-349 versus ≥350 cells/μL (OR = 1.57, 95% CI: 1.09, 2.25). After adjustment for nadir CD4+, a protective effect of >2-year cART use was seen against CIN2/3 (OR versus never cART use = 0.64, 95% CI: 0.42, 0.98). Despite low study power, similar associations were seen for ICC, notably with nadir CD4+ (OR for 50 vs. >350 cells/μL= 11.10, 95% CI: 1.24, 100). HPV16-L1 antibodies were significantly associated with CIN2/3, but HPV16-E6 antibodies were nearly exclusively detected in ICC. In conclusion, worsening immunodeficiency, even at only moderately decreased CD4+ cell counts, is a significant risk factor for CIN2/3 and cervical cancer.