Trypanosoma cruzi XRNA granules colocalise with distinct mRNP granules at the nuclear periphery

BACKGROUND Eukaryotic ribonucleoprotein (RNP) granules are important for the regulation of RNA fate. RNP granules exist in trypanosomatids; however, their roles in controlling gene expression are still not understood. XRNA is a component of granules in Trypanosoma brucei but has not been investigated in Trypanosoma cruzi. OBJECTIVES This study aimed to investigate the TcXRNA dynamic assembly and its interaction with RNP components under conditions that affect the mRNA availability. METHODS We used in vitro metacyclogenesis of T. cruzi to observe changes in RNP granules during the differentiation process. TcXRNA expression was analysed by Western blot and immunofluorescence. Colocalisation assays were performed to investigate the interaction of TcXRNA with other RNP components. FINDINGS TcXRNA is constantly present during metacyclogenesis and is localised in cytoplasmic granules. TcXRNA does not colocalise with TcDHH1 and TcCAF1 granules in the cytoplasm. However, TcXRNA granules colocalise with mRNP granules at the nuclear periphery when mRNA processing is inhibited. MAIN CONCLUSIONS TcXRNA plays a role in mRNA metabolism as a component of mRNP granules whose assembly is dependent on mRNA availability. TcXRNA granules colocalise with distinct RNP granules at the nuclear periphery, suggesting that the perinuclear region is a regulatory compartment in T. cruzi mRNA metabolism.

Amplification of human telomerase reverse transcriptase gene as a diagnostic marker in lung cancer

Analysis of circulating DNA or RNA in plasma can provide a useful marker for earlier lung cancer detection. This study was designed to assess the sensitivity and specificity of a quantitative molecular assay of circulating RNA to identify patients with lung cancer with different grades. The amount of plasma RNA was determined through the use of real-time quantitative polymerase chain reaction [PCR] amplification of the human telomerase reverse transcriptase gene [hTERT] in 19 non-small-cell lung cancer patients and 10 age and sex matched controls. Performance of the assay was calculated through the receiver operating characteristic [ROC] curve. The hTERT mRNA ratio in cancer lung patients showed a mean of 196.34 +/- 307.23 which was higher compared to that of the controls 1.24 +/- 0.80; this difference was statistically highly significant where P<0.01. The median concentration of circulating plasma RNA in patients was higher than the value detected in controls [71.70 v 1.149 ratio]. Plasma RNA was a strong risk factor for lung cancer; concentrations in the patients were associated with a 62-fold higher risk than were those in the controls. The point of the best cut-off value was at 2.24 where sensitivity was 73.7% and specificity was 90%. The area under the ROC curve was 0.704. This study shows that higher levels of free circulating RNA can be detected in patients with lung cancer compared with disease-free heavy smokers by a PCR assay, and suggests a new, noninvasive approach for early detection of lung cancer. Levels of plasma RNA is recommended to be measured as it could also identify higher-risk individuals for lung cancer screening and chemoprevention trials

Rapid detection of dengue viral RNA in mosquitoes by nucleic acid-sequence based amplification (NASBA).

RNA amplification by nucleic acid sequence-based amplification (NASBA) was used to detect serotype specific dengue viruses in artificially-infected female Aedes mosquitoes, in comparison with RT-PCR technique. NASBA could detect dengue virus serotype 2 and 4 below 0.1 PFU, which was more sensitive than RT-PCR, but this technique was as sensitive as RT-PCR when detecting dengue virus serotype 1 and 3. Dengue viruses could be detected at the thorax of mosquitoes at 0, 7, and 14 days after inoculation with dengue virus serotype 2. This method should be useful for virological surveillance of dengue infected Aedes mosquitoes, as an early warning system to predict outbreaks of dengue viruses.

Influence of sublethal ammonia toxicity on some physiological parameters of Labeo rohita (Hamilton-Buchanan) fingerlings.

Fingerlings of Labeo rohita subjected to sublethal unionized ammonia (0.132mg/l) for 30 days exhibited significant changes. Increase in haemoglobin, haematocrit, plasma cortisol, plasma glucose, plasma cholesterol and plasma lactic acid levels whereas, decrease in plasma chloride, liver and muscle glycogen, hepatosomatic index and DNA/RNA ratio of muscles with stable plasma protein was observed. Metabolic recovery was not observed within 30 days of exposure.

Detection of 1583 bp gene transcript in lymphocytes of muscular dystrophy patients.

DMD and BMD are X-lined recessive disorders. RAP-PCR was utilized to investigate differentially expressed gene transcripts in lymphocytes from DMD, BMD and normal individuals as possible diagnostic parameter. A 1583 bp transcript was found to be expressed in both DMD and BMD patients which was unrelated to the known dystrophin gene. This may prove helpful in determining the carrier status of DMD/BMD.

Studies of cocktail therapy with multiple cytokines for neoplasia or infectious disease of the dog I. cDNA cloning of canine IL-3 and IL-6

This paper describes the cloning and sequence analysis of the cDNAs encoding the canine homologues of interleukin-3 (IL-3) and interleukin-6 (IL-6). The coding sequences for canine IL-3 and IL-6 were obtained by using the reverse transcription polymerase chain reaction (RT-PCR) with RNA harvested from canine peripheral blood mononuclear cells (PBMCs). Canine IL-3 cDNA includes a single open reading frame of 432 nucleotides, which encodes a 143 amino acid polypeptide and has 44.7, 42.4, 37 and 23.7% homology with the cow, sheep, human and rat IL-3 sequences, respectively. Canine IL-6 cDNA (GenBank accession number; AF275796) encodes a putative 20-amino acid signal peptide followed by a 187-amino acid mature protein. The predicted amino acid sequence of canine IL-6 shares 60.4, 77.2, 71.0, 55.8 and 42.0% sequence identity with those of human, feline, porcine, sheep and rat IL-6, respectively.

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott(r)), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95 percent) sera from patients in the study group and negative in 38 of the 39 (97 percent) sera from those in the control group, showing an accuracy of 96 percent. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

Secuenciacion directa de un producto amplificado de una muestra de suero / Direct sequence of an amplified product from a serum sample

Se reporta la secuencia nucleotida y aminoacidica de una zona de gran variabilidad en el genoma del virus dengue 2 a partir del ARN del virus original sin pase en ningun sistema de aislamiento y se compara con la primera cepa de dengue 2 aislada durante la epidemia de 1981 con 4 pases en raton lactante. Los resultados demuestran que la secuencia nucleotidica del suero y de la cepa A15 son iguales

Determinación del RNA genómico y replicativo del virus de la hepatitis C en pacientes tratados con interferón / Determination of genomic RNA and replicative hepatitis C viruses in patients treated with interferon

En este ensayo se trató de correlacionar la evolución clínica e histológica de un grupo de pacientes tratados con IFN, con la presencia del RNA genómico y replicativo del HCV en plasma y tejido hepático. Se estudiaron 11 pacientes con hepatitis crónica "C" diagnosticada por elevación de transaminasas durante los últimos 6 meses, anti-HCV positivo "(Elisa II)", y cuadro histológico compatible. En todos ellos se efectuó biopsia hepática y extracción de sangre en paralelo, inmediatamente antes y después de finalizar el tratamiento con 4.5 millones de IFN Alfa2a administrado 3 veces por semana, durante 6 meses. El tecijo hepático se almacenó a-80§c y el plasma a-20§c hasta su procesamiento. La extracción de RNA se realizó a partir de 100microliter de plasma y de 20mg de tejido hepático con isotiocianato de guanidinio (Chomcznski). La transcripción reversa se llevó a cabo "primers" sense o antisense para detectar la hélice replicativa (-) o genómica (+) respectivamente. El cDNA de la región 5' no codificante fue amplificado por el sistema "nested". Antes del tratamiento los 11 pacientes evidenciaron la presencia de la hélice genómica en tecijo hepático y plasma. En cambio la hélice replicativa se detectó en 5 casos en hígado, 7 pacientes se revelaron como respondedores y 4 como no respondedores. En los pacientes respondedores las hélices genómica y replicativa en hígado desaparecieron en un 43 por ciento y 57 por ciento respectivamente, y en plasma se observó un descenco del 71 por ciento para la hélice genómica y de un 100 por ciento para la hélice replicativa. En los 4 pacientes no respondedores la hélice genómica permaneció en tecijo hepático y plasma, en cambio la replicativa se mantuvo en tejido hepático y se negativizó en un 75 por ciento en plasma. El índice de Knodell, que determina el estadío histológico, disminuyó en 5 de lo 7 respondedores y permaneció igual en 3 de los 4 no respondedores...