Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium

Tools for the genetic manipulation of Trypanosoma cruzi are largely unavailable, although several vectors for transfection of epimastigotes and expression of foreign or recombinant genes have been developed. We have previously constructed several plasmid vectors in which recombinant genes are expressed in T. cruzi using the rRNA promoter. In this report, we demonstrate that one of these vectors can simultaneously mediate expression of neomycin phosphotransferase and green fluorescent protein when used to stably transfect cultured epimastigotes. These stably transfected epimastigotes can be selected and cloned as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for relevant genes. Thus, the methodologies outlined herein provide important new tools for the genetic dissection of this important parasite.

A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.

Hepatitis C prevalence and risk factors in hemodialysis patients in Central Brazil: a survey by polymerase chain reaction and serological methods

An hemodialysis population in Central Brazil was screened by polymerase chain reaction (PCR) and serological methods to assess the prevalence of hepatitis C virus (HCV) infection and to investigate associated risk factors. All hemodialysis patients (n=428) were interviewed in eight dialysis units in Goiânia city. Blood samples were collected and serum samples screened for anti-HCV antibodies by an enzyme-linked immunosorbent assay (ELISA). Positive samples were retested for confirmation with a line immunoassay (LIA). All samples were also tested for HCV RNA by the PCR. An overall prevalence of 46.7% (CI 95%: 42-51.5) was found, ranging from 20.7% (CI 95%: 8.8-38.1) to 90.4% (CI 95%: 79.9-96.4) depending on the dialysis unit. Of the 428 patients, 185 were found to be seropositive by ELISA, and 167 were confirmed positive by LIA, resulting in an anti-HCV prevalence of 39%. A total of 131 patients were HCV RNA-positive. HCV viremia was present in 63.5% of the anti-HCV-positive patients and in 10.3% of the anti-HCV-negative patients. Univariate analysis of risk factors showed that the number of previous blood transfusions, transfusion of blood before mandatory screening for anti-HCV, length of time on hemodialysis, and treatment in multiple units were associated with HCV positivity. However, multivariate analysis revealed that blood transfusion before screening for anti-HCV and length of time on hemodialysis were significantly associated with HCV infection in this population. These data suggest that nosocomial transmission may play a role in the spread of HCV in the dialysis units studied. In addition to anti-HCV screening, HCV RNA detection is necessary for the diagnosis of HCV infection in hemodialysis patients.

An outbreak of gastroenteritis associated with astrovirus serotype 1 in a day care center, in Rio de Janeiro, Brazil

Between June 4th and June 20th1996 rotavirus, adenovirus, and astrovirus (HAstrV) were investigated in fecal samples from 27 children under three years old with acute diarrhea, attending the Bertha Lutz day care center, in Rio de Janeiro. All fecal samples were analyzed by polyacrylamide gel electrophoresis (PAGE), reverse transcriptase polymerase chain reaction (RT-PCR), enzyme immunoassays (EIA), and electron microscopy (EM). Nine of them (33%) showed positive results for HAstrV by at least one of the employed methodologies. Eight were positive by RT-PCR and EIA, and six by EM. All positive samples were inoculated onto HT-29 (human colon adenocarcinoma) cultured cells for HAstrV isolation and seven were positive after three passages. The sequencing analysis of eight RT-PCR products (449 bp) from gene that codifies VP2 protein, showed a total nucleotide identity among them and 98% with HAstrV-1 (strain Oxford type 1). This is the first report of a gastroenteritis outbreak associated with HAstrv-1 in a day care center in Rio de Janeiro and it reinforces the importance of this virus in association with infantile acute gastroenteritis.

A Heminested Polymerase Chain Reaction for the Detection of Brazilian Rabies Isolates from Vampire Bats and Herbivores

A heminested-PCR (hn-PCR) using primers to the nucleoprotein-coding gene in a nested set was evaluated in the detection of Brazilian strains of rabies virus (RV). A representative number of RV nucleoprotein sequences belonging to genotype 1 were aligned. Based on such alignment, primers were directed to highly conserved regions. All 42 clinical samples positive by both fluorescent antibody and mouse inoculation tests were also positive by the hn-PCR. Brain tissue that had been left to decompose, obtained from an experimentally inoculated mouse was tested by hn-PCR and yielded positive results. In conclusion, primers designed here were capable of amplifying Brazilian RV isolates obtained from a rural epidemiological cycle.

Frequency of Infection of Lutzomyia Phlebotomines with Leishmania braziliensis in a Brazilian Endemic Area as Assessed by Pinpoint Capture and Polymerase Chain Reaction

Leishmania infected of Lutzomyia spp. are rare in endemic areas. We tested the hypothesis that there is clustering of infected vectors by combining pinpoint capture with sensitive L. braziliensis kDNA minicircle specific PCR/dot blot in an endemic area in the State of Bahia. Thirty out of 335 samples (10 to 20 sand flies/sample; total of 4,027 female sand flies) were positive by PCR analysis and dot blot leading to a underestimated overall rate of 0.4% positive phlebotomines. However, 83.3% of the positive samples were contributed by a single sector out of four sectors of the whole studied area. This resulted in a rate of 1.5% Leishmania positive phlebotomines for this sector, far above rates of other sectors. Incidence of American cutaneous leishmaniasis cases for this sector was about twice that for other sectors. Our results show that there is a non-homogeneous distribution of Leishmania-infected vectors. Such a clustering may have implications in control strategies against leishmaniasis, and reinforces the necessity of understanding the ecological and geographical factors involved in leishmanial transmission.

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

Involvement of nuclear factor-kappaB in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin- 1 beta in ectopic endometrial cells.

OBJECTIVE: To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. DESIGN: Prospective study. SETTING: Human reproduction research laboratory. PATIENT(S): Nine women with endometriotic lesions. INTERVENTION(S): Endometriotic lesions were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. RESULT(S): This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. CONCLUSION(S): This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.

Minority quasispecies of drug-resistant HIV-1 that lead to early therapy failure in treatment-naive and -adherent patients.

BACKGROUND: Early virological failure of antiretroviral therapy associated with the selection of drug-resistant human immunodeficiency virus type 1 in treatment-naive patients is very critical, because virological failure significantly increases the risk of subsequent failures. Therefore, we evaluated the possible role of minority quasispecies of drug-resistant human immunodeficiency virus type 1, which are undetectable at baseline by population sequencing, with regard to early virological failure. METHODS: We studied 4 patients who experienced early virological failure of a first-line regimen of lamivudine, tenofovir, and either efavirenz or nevirapine and 18 control patients undergoing similar treatment without virological failure. The key mutations K65R, K103N, Y181C, M184V, and M184I in the reverse transcriptase were quantified by allele-specific real-time polymerase chain reaction performed on plasma samples before and during early virological treatment failure. RESULTS: Before treatment, none of the viruses showed any evidence of drug resistance in the standard genotype analysis. Minority quasispecies with either the M184V mutation or the M184I mutation were detected in 3 of 18 control patients. In contrast, all 4 patients whose treatment was failing had harbored drug-resistant viruses at low frequencies before treatment, with a frequency range of 0.07%-2.0%. A range of 1-4 mutations was detected in viruses from each patient. Most of the minority quasispecies were rapidly selected and represented the major virus population within weeks after the patients started antiretroviral therapy. All 4 patients showed good adherence to treatment. Nonnucleoside reverse-transcriptase inhibitor plasma concentrations were in normal ranges for all 4 patients at 2 separate assessment times. CONCLUSIONS: Minority quasispecies of drug-resistant viruses, detected at baseline, can rapidly outgrow and become the major virus population and subsequently lead to early therapy failure in treatment-naive patients who receive antiretroviral therapy regimens with a low genetic resistance barrier.

Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids.

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid beta-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0. 06 mg g(-1) dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward beta-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via beta-oxidation and that a considerable flow toward beta-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids.

Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata

Simple sequence repeat anchored polymerase chain reaction amplification (SSR-PCR) is a genetic typing technique based on primers anchored at the 5' or 3' ends of microsatellites, at high primer annealing temperatures. This technique has already been used in studies of genetic variability of several organisms, using different primer designs. In order to conduct a detailed study of the SSR-PCR genomic targets, we cloned and sequenced 20 unique amplification products of two commonly used primers, CAA(CT)6 and (CA)8RY, using Biomphalaria glabrata genomic DNA as template. The sequences obtained were novel B. glabrata genomic sequences. It was observed that 15 clones contained microsatellites between priming sites. Out of 40 clones, seven contained complex sequence repetitions. One of the repeats that appeared in six of the amplified fragments generated a single band in Southern analysis, indicating that the sequence was not widespread in the genome. Most of the annealing sites for the CAA(CT)6 primer contained only the six repeats found within the primer sequence. In conclusion, SSR-PCR is a useful genotyping technique. However, the premise of the SSR-PCR technique, verified with the CAA(CT)6 primer, could not be supported since the amplification products did not result necessarily from microsatellite loci amplification.

Polymerase chain reaction and restriction fragment length polymorphism of cytocrome oxidase subunit I used for differentiation of Brazilian Biomphalaria species intermediate host of Schistosoma mansoni

The intermediate hosts of Schistosoma mansoni, in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea, were identified by restriction fragment length polymorphism analysis of the mitochondrial gene cytochrome oxidase I (COI). We performed digestions with two enzymes (AluI and RsaI), previously selected, based on sequences available in Genbank. The profiles obtained with RsaI showed to be the most informative once they were polymorphic patterns, corroborating with much morphological data. In addition, we performed COI digestion of B. straminea snails from Uruguay and Argentina.

Supplementary Report of the HIA to the Minister for Health, in accordance with Section 7E(1)(b) of the Health Insurance Acts, 1994-2009 (Redacted Version)

The purpose of this Supplementary Report is to advise on how the budgetary measures impact on the conclusions in relation to tax credits and stamp duty included in the Authority’s November 2011 Report. in doing so, we will assess the direct impacts and we will discuss some scenarios. However, the Authority’s advice in this area relies on projections of the health insurance market and, in light of the above, there is considerable uncertainty surrounding any projections of how claims inflation or the market size may develop, even in the short and medium terms. Supplementary Report of the HIA to the Minister for Health, in accordance with Section 7E(1)(b) of the Health Insurance Acts, 1994-2009 (Redacted Version) Click here to download PDF 3.2mb

Novel relationship between Vegf expression and retinal pigmented epithelium permeability following light damage in the mouse retina

Purpose:In the retina, the balance between pro- and anti-angiogenic factors is critical for angiogenesis control but is also involved in cell survival and maintenance. For instance, the anti-angiogenic factor PEDF is neuroprotective for photoreceptors (PRs) in models of retinal degeneration. We previously reported upregulation of VEGF (24h to 48h post lesion) in the light-damage (LD) model. Furthermore, systemic delivery of PEDF, as well as lentiviral gene transfer of an anti-VEGF antibody rescue PRs from cell death. Studies in vitro show that VEGF induces retinal endothelial cells apoptosis via the alteration of the Akt1/p38 MAPK signalling pathway under hypoxic conditions. Thus, in this study, we investigate the effect of high levels of VEGF on retinal pigmented epithelium (RPE) permeability and molecular targets expression after light-induced PR degeneration. Methods:To characterize the action of VEGF in the retina during the course of LD, we exposed adult Balb/c mice to 5'000 lux for 1h, and we collected neural retinas and eye-cups (containing RPE) at different time points after the LD. We analysed protein expression by Elisa and Western blotting. In order to study RPE cell permeability after the LD we stained β-catenin on flat mounted RPE. Results:In the neural retina, preliminary results indicate that high levels of VEGF induce a significant upregulation of VEGF receptor 2, whereas VEGF receptor 1 expression is decreased. Concomitantly with VEGF upregulation, LD increases the Src phosphorylation between 24h to 48h. Furthermore, we observe that β-catenin translocates to the cytoplasm of RPE cells between 24h to 36h after the lesion, indicating an increase on the RPE permeability, which could contribute indirectly to the deleterious effect of VEGF observed during light-induced PR apoptosis. Conclusions:This study further involves VEGF in LD and highlights the prime importance of angiogenic factor balance for PR survival. Our results suggest that PR apoptosis is augmented by RPE cell permeability, which may induce high level of VEGF and could be deleterious. The specific action of RPE permeability on PR survival and the role of Src in the retina are under investigation.