Microbial enrichment and gene functional categories revealed on the walls of a spent fuel pool of a nuclear power plant.

Microorganisms developing in the liner of the spent fuel pool (SFP) and the fuel transfer channel (FTC) of a Nuclear Power Plant (NPP) can form high radiation resistant biofilms and cause corrosion. Due to difficulties and limitations to obtain large samples from SFP and FTC, cotton swabs were used to collect the biofilm from the wall of these installations. Molecular characterization was performed using massively parallel sequencing to obtain a taxonomic and functional gene classification. Also, samples from the drainage system were evaluated because microorganisms may travel over the 12-meter column of the pool water of the Brazilian Nuclear Power Plant (Angra1), which has been functioning since 1985. Regardless of the treatment of the pool water, our data reveal the unexpected presence of Fungi (Basidiomycota and Ascomycota) as the main contaminators of the SFP and FTC. Ustilaginomycetes (Basidiomycota) was the major class contributor (70%) in the SFP and FTC reflecting the little diversity in these sites; nevertheless, Proteobacteria, Actinobacteria, Firmicutes (Bacilli) were present in small proportions. Mapping total reads against six fungal reference genomes indicate that there is, in fact, a high abundance of fungal sequences in samples collected from SFP and FTC. Analysis of the ribosomal internal transcribed spacer (ITS) 1 and 2 regions and the protein found in the mitochondria of eukaryotic cells, cytochrome b (cytb) grouped our sample fungi in the clade 7 as Ustilago and Pseudozyma. In contrast, in the drainage system, Alphaproteobacteria were present in high abundances (55%). The presence of Sphingopyxis, Mesorhizobium, Erythrobacter, Sphingomonas, Novosphingobium, Sphingobium, Chelativorans, Oceanicaulis, Acidovorax, and Cyanobacteria was observed. Based on genomic annotation data, the assessment of the biological function found a higher proportion of protein-coding sequences related to respiration and protein metabolism in SFP and FTC samples. The knowledge of this biological inventory present in the system may contribute to further studies of potential microorganisms that might be useful for bioremediation of nuclear waste.

Expression of the stress-response regulators CtsR and HrcA in the uropathogen Staphylococcus saprophyticus during heat shock.

The uropathogen Staphylococcus saprophyticus is an ubiquitous bacterium but little is known about mechanisms that allow its persistence in diverse environments. Here we evaluated S. saprophyticus growth and survival during heat shock, the expression of stress response regulators ctsR and hrcA through qRT-PCR and heat shock protein synthesis through 35S-Met metabolic labeling. S. saprophyticus does not tolerate temperatures much higher than the optimal 37 °C, as its growth is greatly affected at 42 °C, though viability is maintained up to 48 °C. At 42 °C, the expression of ctsR and hrcA repressor genes approximately triple when compared to 37 °C and continue to increase together with temperature till 48 °C. Expression of hrcA peaks after 20 min of heat shock and decreases significantly after 30 min, indicating that heat stress response regulated by this gene may last 20-30 min. An increase in temperature is accompanied by the synthesis of at least eight proteins, three of which are likely the chaperones DnaK, GroEL and ClpB. In silico analysis indicate that the groEL gene may be regulated by HrcA, clpB by CtsR and dnaK by both repressors. This is the first work to discuss heat stress response in S. saprophyticus and a step forward in the understanding of mechanisms that make this a widespread and emergent pathogen.

Symbiont modulates expression of specific gene categories in Angomonas deanei

Trypanosomatids are parasites that cause disease in humans, animals, and plants. Most are non-pathogenic and some harbor a symbiotic bacterium. Endosymbiosis is part of the evolutionary process of vital cell functions such as respiration and photosynthesis. Angomonas deanei is an example of a symbiont-containing trypanosomatid. In this paper, we sought to investigate how symbionts influence host cells by characterising and comparing the transcriptomes of the symbiont-containing A. deanei (wild type) and the symbiont-free aposymbiotic strains. The comparison revealed that the presence of the symbiont modulates several differentially expressed genes. Empirical analysis of differential gene expression showed that 216 of the 7625 modulated genes were significantly changed. Finally, gene set enrichment analysis revealed that the largest categories of genes that downregulated in the absence of the symbiont were those involved in oxidation-reduction process, ATP hydrolysis coupled proton transport and glycolysis. In contrast, among the upregulated gene categories were those involved in proteolysis, microtubule-based movement, and cellular metabolic process. Our results provide valuable information for dissecting the mechanism of endosymbiosis in A. deanei.

Symbiont modulates expression of specific gene categories in Angomonas deanei.

Trypanosomatids are parasites that cause disease in humans, animals, and plants. Most are non-pathogenic and some harbor a symbiotic bacterium. Endosymbiosis is part of the evolutionary process of vital cell functions such as respiration and photosynthesis. Angomonas deanei is an example of a symbiont-containing trypanosomatid. In this paper, we sought to investigate how symbionts influence host cells by characterising and comparing the transcriptomes of the symbiont-containing A. deanei (wild type) and the symbiont-free aposymbiotic strains. The comparison revealed that the presence of the symbiont modulates several differentially expressed genes. Empirical analysis of differential gene expression showed that 216 of the 7625 modulated genes were significantly changed. Finally, gene set enrichment analysis revealed that the largest categories of genes that downregulated in the absence of the symbiont were those involved in oxidation-reduction process, ATP hydrolysis coupled proton transport and glycolysis. In contrast, among the upregulated gene categories were those involved in proteolysis, microtubule-based movement, and cellular metabolic process. Our results provide valuable information for dissecting the mechanism of endosymbiosis in A. deanei.

No correspondence between resistance mutations in the HCV-NS3 protease at baseline and early telaprevir-based triple therapy.

Direct-acting antiviral (DAA)-based therapy is the new standard treatment for chronic hepatitis C virus (HCV) infection. However, protease inhibitor (PI)-resistant viral variants have been often described. This study aimed to examine HCV-NS3 protease variants at baseline and at 4 weeks under triple therapy. To this end, we analyzed the presence of variants in HCV-NS3 protease region from peripheral blood samples of 16 patients infected with HCV-1 at baseline and at 4 weeks of combined therapy with telaprevir, pegylated interferon, and ribavirin, using next-generation sequencing. Several variants with synonymous and non-synonymous amino acid substitutions were detected at both time points. Variants detected at low frequency corresponded to 74% (HCV-1a) and 35% (HCV-1b) of non-synonymous substitutions. We found nine PI-resistance-associated variants (V36A, T54S, V55I, Q80K, Q80R, V107I, I132V, D168E, M175L) in HCV-NS3 of 10 patients. There was no correspondence of resistance-associated variant profile between baseline and at 4 weeks. Moreover, these resistance variants at baseline and short-term treatment are not good predictors of outcome under triple therapy. Our study also shows a large number of others minor and major non-synonymous variants in HCV-NS3 early in telaprevir-based therapy that can be important for further drug resistance association studies with newly developed PI agents.

Genome of the avirulent human-infective trypanosome--Trypanosoma rangeli.

BACKGROUND: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts. METHODOLOGY/PRINCIPAL FINDINGS: The T. rangeli haploid genome is ∼ 24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins. CONCLUSIONS/SIGNIFICANCE: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.

Evidences for the involvement of cell surface glycans in stem cell pluripotency and differentiation.

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development, several issues regarding their pluripotency, differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study, we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that, upon spontaneous differentiation, iPS-MBMC present high amounts of terminal ß-galactopyranoside residues, pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation, prompting an ectoderm commitment. Exposed ß-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface, which could modulate intercellular or intracellular interactions. Together, our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and, therefore, the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation.

Dynamics of resistance mutations to NS3 protease inhibitors in a cohort of Brazilian patients chronically infected with hepatitis C virus (genotype 1) treated with pegylated interferon and ribavirin: a prospective longitudinal study.

UNLABELLED: About sixty thousand new cases of Hepatitis C virus (HCV) infection are recorded in Brazil each year. These cases are currently treated with pegylated interferon (PEG-IFN) and ribavirin (RBV) with an overall success rate of 50%. New compounds for anti-HCV therapy targeted to the HCV NS3 protease are being developed and some already form the components of licensed therapies. Mapping NS3 protease resistance mutations to protease inhibitors or anti-viral drug candidates is important to direct anti-HCV drug treatment. METHODS: Sequence analysis of the HCV NS3 protease was conducted in a group of 68 chronically infected patients harboring the HCV genotype 1. The patients were sampled before, during and after a course of PEG-IFN-RBV treatment. RESULTS: Resistance mutations to the protease inhibitors, Boceprevir and Telaprevir were identified in HCV isolated from three patients (4.4%); the viral sequences contained at least one of the following mutations: V36L, T54S and V55A. In one sustained virological responder, the T54S mutation appeared during the course of PEG-IFN and RBV therapy. In contrast, V36L and V55A mutations were identified in virus isolated from one relapsing patient before, during, and after treatment, whereas the T54S mutation was identified in virus isolated from one non-responding patient, before and during the treatment course. CONCLUSIONS: The incidence and persistence of protease resistance mutations occurring in HCV from chronically infected patients in Brazil should be considered when using protease inhibitors to treat HCV disease. In addition, patients treated with the current therapy (PEG-IFN and RBV) that are relapsing or are non-responders should be considered candidates for protease inhibitor therapy.

A single nucleotide polymorphism, rs129679860, in the IL28B locus is associated with the viral kinetics and a sustained virological response in a chronic, monoinfected hepatitis C virus genotype-1 Brazilian population treated with pegylated interferon-ribavirin.

Single nucleotide polymorphisms (SNPs) in the interleukin (IL)28B locus have been associated with a sustained virological response (SVR) in interferon-ribavirin (IFN-RBV)-treated chronic hepatitis C virus (HCV)-infected patients in European and African populations. In this study, the genotype frequency of two IL28B SNPs (rs129679860 and rs8099917) in a cohort of chronic HCV-monoinfected patients in Brazil was evaluated and the SNP sufficient to predict the treatment response outcome was determined. A total of 66 naïve genotype-1 chronic HCV-infected patients were genotyped and the associated viral kinetics and SVR were assessed. The overall SVR was 38%. Both the viral kinetics and SVR were associated with rs129679860 genotypes (CC = 62% vs. CT = 33% vs. TT = 18%, p = 0.016). However, rs8099917 genotypes were only associated with SVR (TT = 53% vs. TG = 33% vs. GG = 18%; p = 0.032). In this population, the analysis of a single SNP, rs12979860, successfully predicts SVR in the IFN-RBV treatment of HCV.

A single nucleotide polymorphism, rs129679860, in the IL28B locus is associated with the viral kinetics and a sustained virological response in a chronic, monoinfected hepatitis C virus genotype-1 Brazilian population treated with pegylated interferon-ribavirin

Single nucleotide polymorphisms (SNPs) in the interleukin (IL)28B locus have been associated with a sustained virological response (SVR) in interferon-ribavirin (IFN-RBV)-treated chronic hepatitis C virus (HCV)-infected patients in European and African populations. In this study, the genotype frequency of two IL28B SNPs (rs129679860 and rs8099917) in a cohort of chronic HCV-monoinfected patients in Brazil was evaluated and the SNP sufficient to predict the treatment response outcome was determined. A total of 66 naïve genotype-1 chronic HCV-infected patients were genotyped and the associated viral kinetics and SVR were assessed. The overall SVR was 38%. Both the viral kinetics and SVR were associated with rs129679860 genotypes (CC = 62% vs. CT = 33% vs. TT = 18%, p = 0.016). However, rs8099917 genotypes were only associated with SVR (TT = 53% vs. TG = 33% vs. GG = 18%; p = 0.032). In this population, the analysis of a single SNP, rs12979860, successfully predicts SVR in the IFN-RBV treatment of HCV.

Association of IL-10, IL-4, and IL-28B gene polymorphisms with spontaneous clearance of hepatitis C virus in a population from Rio de Janeiro.

BACKGROUND: Cytokines play an important role in the regulation of the immune response. In hepatitis C virus (HCV) infection, cytokine levels may influence the outcome of acute HCV infection. Polymorphisms in cytokine genes have been associated to different expression levels in response to infection. This study was carried out to investigate the association of several cytokine gene polymorphisms with disease outcome in HCV-infected patients. FINDINGS: Patients with chronic or spontaneously resolved HCV infection were included in a cross-sectional study. A comparative analysis was performed between the groups regarding frequency distribution of the following cytokines' gene polymorphisms: IL-10 (-1082 A/G; -819 T/C; -592 A/C), IL-4 (+33C/T), IFN-γ (+874 T/A), TNF-α (-238 G/A and -308 G/A) and IL-28B (rs12979860 C/T and rs8099917 T/G). RESULTS: Eighteen patients with spontaneous viral clearance and 161 with chronic HCV infection were included. In the comparative analysis, the GG genotype of the IL-10 polymorphism -1082A/G was more frequent in patients with spontaneous viral clearance when compared to patients with chronic HCV (41.2% vs 6.2%; p = 0.001). This association was also found for the CC genotype of the IL-4 polymorphism +33C/T (72.2% vs 36.7%; p = 0.017) and the CC and TT genotypes of the IL-28B polymorphisms rs 12979860 and rs 8099917 (88.9% vs 30.3%; p < 0.001 and 88.9% vs 49.6%; p = 0.002). The IL10 (A-1082 G) and IL-28B (Crs12979860T) gene polymorphisms showed odds ratios of 12.848 and 11.077, respectively, and thus may have a greater influence on HCV spontaneous viral clearance. The IFN-γ (+874 T/A), TNF-α (-238 G/A and -308 G/A) polymorphisms did not show significant association with spontaneous viral clearance or chronicity. CONCLUSION: The G allele for IL-10 (-1082 A/G), the C allele for IL-4 (+3 C/T) and the C and T alleles for IL-28B (rs12979860 and rs8099917, respectively) are associated with spontaneous viral clearance in hepatitis C infection.

Human menstrual blood-derived mesenchymal cells as a cell source of rapid and efficient nuclear reprogramming.

Induced pluripotent stem cells (iPSCs) were originally generated by forced ectopic expression of four transcription factors genes-OCT4, KLF4, SOX2, and c-MYC-in fibroblasts. However, the efficiency of iPSCs obtention is extremely low, and reprogramming takes about 20 days. We reasoned that adult cells showing basal expression of core embryonic stem (ES) cell regulator genes could be a better cell source for reprogramming. Menstrual blood-derived mesenchymal cells (MBMCs) are multipotent cells that show detectable levels of some of the core ES cells regulators. The aim of this study was to determine whether reprogramming efficiency could be increased by using MBMCs as a cell source to generate iPSCs. MBMCs were transduced with recombinant retroviruses expressing the coding regions of OCT4, SOX2, and KLF4 genes. Cells with high nucleus/cytoplasm ratio can be detected about 5 days of posttransduction, and colonies of typical ES-like cells begun to appear after 7 days. At day 15, colonies were picked up and expanded for characterization. Most of the clones were morphologically identical to ES cells and positive at the mRNA and protein levels for all pluripotency markers tested. The clones are capable of forming embryoid bodies and to differentiate in vitro into cells of the three germ cell layers. Our results show that the reprogramming was faster and with efficiency around 2-5%, even in the absence of ectopic expression of c-MYC. To date, this is the first study showing MBMCs as a cell source for nuclear reprogramming.

Polimorfismo do gene da enzima conversora de angiotensina e avaliação cardiovascular em adultos com deficiência do hormônio do crescimento / Angiotensin converting enzyme gene polymorphisms and cardiovascular evaluation in growth hormone deficient adults

Fundamentos: A enzima conversora da angiotensina (ECA) é importante reguladora da pressão arterial (PA). Polimorfismos no gene da ECA estão associados a alterações na PA. Não existem ainda estudos sobre areposição de hormônio do crescimento (GH) em adultos com deficiência do hormônio do crescimento (DGH) deacordo com os genótipos da ECA.Objetivo: Avaliar a resposta cardiovascular ao GH em adultos com DGH de acordo com o seu genótipo da ECA. Métodos: Avaliados 18 pacientes com hipopituitarismo de acordo com o genótipo da ECA no basal e 24 mesesapós reposição com GH de acordo com parâmetros clínicos e cardiovasculares.Resultados: Dez mulheres e 8 homens foram avaliados (média de idade 44,9±10,9 anos). Distribuição genotípicaencontrada: genótipo DD: 7 (38,9%) pacientes; genótipo ID: 11 (61,1%) pacientes. Frequência cardíaca, PA sistólica e diastólica, carga pressórica sistólica e diastólica, e funções sistólica e diastólica foram normais em todos ospacientes. Na avaliação basal, pacientes com genótipo DD demonstraram níveis de PA sistólica, diastólica diurna e nas 24 horas mais altos (p<0,05); carga pressórica diastólica maior (p<0,05). Comparando-se os dois genótipos ao final do estudo, os pacientes com genótipo DD evidenciaram: redução estatisticamente significativa da pressão diastólica diurna e nas 24 horas, da carga pressórica sistólica e diastólica diurna e nas 24 horas; e aumento da frequência cardíaca noturna (p<0,005).Conclusão: Os resultados sugerem que pacientes com DGH e genótipo DD apresentam maiores benefícios coma reposição com GH em relação ao controle da PA.

Efficacy of miltefosine treatment in Leishmania amazonensis-infected BALB/c mice.

Leishmaniasis is one of the most serious worldwide diseases caused by protozoan parasites of the Leishmania genus, affecting millions of people around the world. All currently available treatments present severe toxic side effects, require long-term compliance, cause serious side effects and are uncomfortable for patients. Leishmania amazonensis, a species endemic to Brazil, causes severe localised or diffuse skin lesions in humans. Owing to the unsatisfactory nature of the currently available chemotherapies, new approaches have been assessed for improved therapeutic intervention strategies against leishmaniasis. Miltefosine is an alkylphospholipid analogue that exhibits potent activity against the different clinical manifestations of leishmaniasis. Thus, the aim of this study was to investigate the long-term efficacy of miltefosine in BALB/c mice infected with L. amazonensis owing to the lack of a profound study demonstrating its dose-dependent and long-term effects. It was observed that animals treated with 20-50 mg/kg/day of miltefosine exhibited a significant dose-dependent reduction in lesion size; furthermore, in mice receiving higher doses, lesions disappeared after the end of treatment. To confirm a possible parasitological cure, mice up to 250 days after the end of treatment were analysed. No lesions or presence of parasite DNA were found in mice treated with 30, 40 and 50 mg/kg/day of miltefosine. In summary, these results show that miltefosine may be used to treat cutaneous leishmaniasis caused by L. amazonensis, alone or as combination therapy.

Investigação de variantes gênicas de canais iônicos em pacientes com síndrome do QT longo / Investigation of ion channel gene variants in patients with long QT syndrome / Investigación de variantes génicas de canales iónicos en pacientes con síndrome del QT largo

FUNDAMENTO: A síndrome do QT longo (SQTL) é uma síndrome arrítmica herdada com aumento do intervalo QT e risco de morte súbita. Mutações nos genes KCNQ1, KCNH2 e SCN5A respondem por 90 por cento dos casos com genótipo determinado, e a genotipagem é informativa para aconselhamento genético e melhor manejo da doença. OBJETIVO: Investigação molecular e análise computacional de variantes gênicas de KCNQ1, KCNH2 e SCN5A associadas à SQTL em famílias portadoras da doença. MÉTODOS: As regiões codificantes dos genes KCNQ1, KCNH2 e SCN5A de pacientes com SQTL e familiares foram sequenciadas e analisadas utilizando o software Geneious ProTM. RESULTADOS: Foram investigadas duas famílias com critérios clínicos para SQTL. A probanda da Família A apresentava QTC = 562 ms, Escore de Schwartz = 5,5. A genotipagem identificou a mutação G1714A no gene KCNH2. Foi observado QTC = 521 ± 42 ms nos familiares portadores da mutação contra QTC = 391 ± 21 ms de não portadores. A probanda da Família B apresentava QTc = 551 ms, Escore de Schwartz = 5. A genotipagem identificou a mutação G1600T, no mesmo gene. A análise dos familiares revelou QTC = 497 ± 42 ms nos portadores da mutação, contra QTC = 404 ± 29 ms nos não portadores. CONCLUSÃO: Foram encontradas duas variantes gênicas previamente associadas à SQTL em duas famílias com diagnóstico clínico de SQTL. Em todos os familiares portadores das mutações foi observado o prolongamento do intervalo QT. Foi desenvolvida uma estratégia para identificação de variantes dos genes KCNQ1, KCNH2 e SCN5A, possibilitando o treinamento de pessoal técnico para futura aplicação na rotina diagnóstica.

BACKGROUND: The long QT syndrome (LQTS) is an inherited arrhythmia syndrome with increased QT interval and risk of sudden death. Mutations in genes KCNQ1, KCNH2 and SCN5A account for 90 percent of cases with genotype determined, and genotyping is informative for genetic counseling and better disease management. OBJECTIVE: Molecular investigation and computational analysis of gene variants of KCNQ1, KCNH2 and SCN5A associated with LQTS, in families with the disease. METHODS: The coding regions of genes KCNQ1, KCNH2 and SCN5A in patients with LQTS and their family members were sequenced and analyzed using Geneious ProTM software. RESULTS: Two families with clinical criteria for LQTS were investigated. The proband of Family A had QTC = 562 ms, Schwartz Score = 5.5. The genotyping identified the G1714A mutation in the KCNH2 gene. QTC = 521 ± 42 ms was observed in family members carrying the mutation against QTC = 391 ± 21 ms for non-carriers. The proband of Family B had QTc = 551 ms, Schwartz Score = 5.5. The genotyping identified the G1600T mutation, in the same gene. The analysis of family members revealed QTC = 497 ± 42 ms in mutation carriers, compared with QTC = 404 ± 29 ms in non-carriers. CONCLUSION: Two gene variants previously associated with LQTS were found in two families clinically diagnosed with LQTS. The prolongation of the QT interval was observed in all family members carrying the mutations. A strategy was developed to identify variants of genes KCNQ1, KCNH2 and SCN5A, making it possible to train technical staff for future application to diagnosis routine.

FUNDAMENTO: El síndrome del QT largo (SQTL) es un síndrome arrítmico heredado con aumento del intervalo QT y riesgo de muerte súbita. Mutaciones en los genes KCNQ1, KCNH2 y SCN5A responden por 90 por ciento de los casos con genotipo determinado, y el genotipaje es informativo para aconsejamiento genético y mejor manejo de la enfermedad. OBJETIVO: Investigación molecular y análisis computacional de variantes génicas de KCNQ1, KCNH2 y SCN5A asociadas a la SQTL en familias portadoras de la enfermedad. MÉTODOS: Las regiones codificantes de los genes KCNQ1, KCNH2 y SCN5A de pacientes con SQTL y familiares fueron secuenciadas y analizadas utilizando el software Geneious Pro®. RESULTADOS: Fueron investigadas dos familias con criterios clínicos para SQTL. La probanda de la Familia A presentaba QT C = 562 ms, Escore de Schwartz = 5,5. El genotipaje identificó la mutación G1714A en el gen KCNH2. Fue observado QT C = 521 ± 42 ms en los familiares portadores de la mutación contra QT C = 391 ± 21 ms de no portadores. La probanda de la Familia B presentaba QT C = 551 ms, Escore de Schwartz = 5. El genotipaje identificó la mutación G1600T, en el mismo gen. El análisis de los familiares reveló QT C = 497 ± 42 ms en los portadores de la mutación, contra QT C = 404 ± 29 ms en los no portadores. CONCLUSIÓN: Fueron encontradas dos variantes génicas previamente asociadas a la SQTL en dos familias con diagnóstico clínico de SQTL. En todos los familiares portadores de las mutaciones fue observada la prolongación del intervalo QT. Fue desarrollada una estrategia para identificación de variantes de los genes KCNQ1, KCNH2 y SCN5A, posibilitando el entrenamiento de personal técnico para futura aplicación en la rutina diagnóstica.

Investigation of ion channel gene variants in patients with long QT syndrome.

BACKGROUND: The long QT syndrome (LQTS) is an inherited arrhythmia syndrome with increased QT interval and risk of sudden death. Mutations in genes KCNQ1, KCNH2 and SCN5A account for 90% of cases with genotype determined, and genotyping is informative for genetic counseling and better disease management. OBJECTIVE: Molecular investigation and computational analysis of gene variants of KCNQ1, KCNH2 and SCN5A associated with LQTS, in families with the disease. METHODS: The coding regions of genes KCNQ1, KCNH2 and SCN5A in patients with LQTS and their family members were sequenced and analyzed using Geneious ProTM software. RESULTS: Two families with clinical criteria for LQTS were investigated. The proband of Family A had QTC = 562 ms, Schwartz Score = 5.5. The genotyping identified the G1714A mutation in the KCNH2 gene. QTC = 521 ± 42 ms was observed in family members carrying the mutation against QTC = 391 ± 21 ms for non-carriers. The proband of Family B had QTc = 551 ms, Schwartz Score = 5.5. The genotyping identified the G1600T mutation, in the same gene. The analysis of family members revealed QTC = 497 ± 42 ms in mutation carriers, compared with QTC = 404 ± 29 ms in non-carriers. CONCLUSION: Two gene variants previously associated with LQTS were found in two families clinically diagnosed with LQTS. The prolongation of the QT interval was observed in all family members carrying the mutations. A strategy was developed to identify variants of genes KCNQ1, KCNH2 and SCN5A, making it possible to train technical staff for future application to diagnosis routine.

Characterization of RAB-like4, the first identified RAB-like protein from Trypanosoma cruzi with GTPase activity.

RAB proteins, which belong to the RAS superfamily, regulate exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. Few RAB proteins have been identified in parasites. Molecular markers for cellular compartments are important to studies concerning about the protein traffic in Trypanosoma cruzi, the causal agent of Chagas disease. In this work, we describe the characterization of TcRABL4, the first RAB-like gene identified in T. cruzi (GenBank Accession No.: ), present as a single-copy gene. TcRABL4 contains all five consensus RAB motifs but lacks cysteine residues at the C terminus, which are essential to isoprenylation, an absolute prerequisite for membrane association of these proteins. TcRABL4 is a functional GTPase that is able to bind and hydrolyze GTP, and its gene is transcribed as a single 1.2 kb mRNA in epimastigotes. TcRABL4 appears to be differentially regulated in the three cell forms of the parasite, and the protein is not associated to membranes, unlike other RAB proteins. It is possible that TcRABL4 may be a member of a novel family of small GTPases.

Characterization of a RAB5 homologue in Trypanosoma cruzi.

RAB proteins are small GTPases involved in exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. RABs show a remarkable specificity in subcellular localization, so they can be used as molecular markers for studying protein trafficking in Trypanosoma cruzi, the causal agent of Chagas' disease. RAB5 is a component of early endosomes. It has been identified in kinetoplastids such as Trypanosoma brucei and Leishmania donovani. In this work, we describe the characterization of the complete coding sequence of a RAB5 gene homologue in T. cruzi (TcRAB5, GenBank Accession No. AY730667). It is present as a single copy gene, located at chromosomal bands XIII and XIV. TcRAB5 shares the highest degrees of similarity (71%) and identity (63%) with Trypanosoma brucei rhodesiense RAB5a and contains all five characteristic RAB motifs. TcRAB5 is transcribed as a single 1.5kb mRNA in epimastigotes. Its transcript was also detected in the other two forms of the parasite, metacyclic trypomastigotes and spheromastigotes. The recombinant TcRAB5 protein was able to bind and hydrolyze GTP. The identification of proteins involved in T. cruzi endo- and exocytic pathways may generate cellular compartment markers, an invaluable tool to better understand the vesicular transport in this parasite.