RESUMEN

Congenital Zika syndrome (CZS) is characterized by a diverse group of congenital malformations induced by ZIKV infection during pregnancy. Type III interferons have been associated with placental immunity against ZIKV and restriction of vertical transmission in mice, and non-coding single-nucleotide polymorphisms (SNPs) on these genes are well known to influence susceptibility to other viral infections. However, their effect on ZIKV pathogenesis has not yet been explored. To investigate whether maternal non-coding SNPs at IFNL genes are associated with CZS, 52 women infected with ZIKV during pregnancy were enrolled in a case-control association study. A total of 28 women were classified as cases and 24 as controls based on the presence or absence of CZS in their infants, and seven Interferon-λ non-coding SNPs (rs12980275, rs8099917, rs4803217, rs4803219, rs8119886, rs368234815, rs12979860) were genotyped. The results of logistic regression analyses show an association between the G allele at rs8099917 and increased susceptibility to CZS under a log-additive model (adjustedOR = 2.80; 95%CI = 1.14-6.91; p = 0.02), after adjustment for trimester of infection and genetic ancestry. These results provide evidence of an association between Interferon-λ SNPs and CZS, suggesting rs8099917 as a promising candidate for further studies on larger cohorts.

Asunto(s)

Interferones/genética , Complicaciones Infecciosas del Embarazo/genética , Infección por el Virus Zika/congénito , Infección por el Virus Zika/genética , Alelos , Brasil , Estudios de Casos y Controles , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Polimorfismo de Nucleótido Simple , Embarazo , Virus Zika

RESUMEN

PURPOSE: To investigate the presence of the variants of lysyl oxygenase (LOX) and superoxide dismutase 1 (SOD1) genes in Brazilian patients with advanced keratoconus. METHODS: Donor genomic DNA extracted from blood samples was screened for 5'UTR, exonic LOX, and SOD1 variants in a subset of 26 patients presenting with advanced keratoconus (KISA > 1000% and I-S > 2.0) by Sanger sequencing. The impact of non-synonymous amino acid changes was evaluated by SIFT, PMUT, and PolyPhen algorithms. The Mutation Taster tool was used to evaluate the potential impact of formation of new donor and acceptor splice sites in the promoter region of affected volunteers carrying sequence variants. A 7-base SOD1 deletion (IVS2 + 50del7bp) previously associated with keratoconus was screened in 140 patients presenting classical keratoconus by gel fragment analysis, and positive samples were sequenced for confirmation. RESULTS: We found an unreported missense variant in LOX exon 6 in one heterozygous patient, leading to substitution of proline with threonine at residue 392 (p. Thr392Pro) of LOX protein sequence. This mutation was predicted to be potentially damaging to LOX protein. Another LOX variant, Arg158Gln, was also detected in another patient but predicted to be non-pathogenic. Two additional new polymorphisms in LOX 5'UTR region (-116C > T and -58C > T) were found in two patients presenting with advanced keratoconus and were predicted to modulate or create donor/acceptor splice sites in LOX transcripts. Additionally, SOD1 deletion was detected in one patient presenting with severe keratoconus, not in control samples. CONCLUSION: We described three novel LOX polymorphisms identified for the first time in Brazilian patients with advanced keratoconus, as well as a previously described SOD1 deletion strongly associated with keratoconus. A possible role of these variants in modulating transcript levels in the cornea of affected individual requires further investigation.

RESUMEN

PURPOSE: To screen visual system homeobox 1 (VSX1) gene in Brazilian subjects affected with keratoconus (KCN). METHODS: Seventy-three patients with KCN and 106 healthy controls were enrolled in this study. Patients were diagnosed with KCN based on eye examination and corneal topographic features according to Rabinowitz's criteria (K > 47.2, I-S > 1.4 and KISA > 100%). DNA from blood samples was extracted from donors, and the exons and exon-intron boundaries of VSX1 were sequenced. The potential impact of the identified amino acid changes was assessed with Poly-Phen2, SIFT, and PMUT analysis tools. Genotyping was confirmed by RLFP technique, which was also applied to genotype non-affected individuals. RESULTS: We found three non-synonymous substitutions (L68H, R131S, and D105E) in VSX1 exon 1, with L68H mutation as a novel variation in this gene. In silico analysis indicated that all variations found were predicted to be probably damaging to VSX1 structure and function. Examination of R131S and L68H variations segregating in one family suggested a strong effect of these variations in increasing disease severity in the proband, which presented bilateral KCN leading to corneal grafting before the age of sixteen. We found a novel synonymous substitution (P79P) and two previously described exonic polymorphisms, with unknown roles in VSX1 pathogenesis. CONCLUSION: VSX1 polymorphisms found in the Brazilian population support a genetic component in KCN pathogenesis. L68H is a novel mutation, and the phenotypic data suggest that this mutation might enhance disease severity when combined with other polymorphisms. However, further investigations are needed.

RESUMEN

Schizophrenia (SZ) is a debilitating mental disorder characterized by psychotic events, abnormal social behavior, false beliefs, and auditory hallucinations. Hypermethylation of the promoter region of reelin (RELN), a gene involved in regulation of neuronal positioning during telencephalic development, is strongly associated with low protein expression in several cortical structures and promoter hypermethylation in brain from postmortem SZ subjects. Recent experimental data suggests that testosterone is able to promote RELN demethylation, although no direct evidence of hormonal influence on reelin promoter methylation was obtained. We investigated if reduced levels of plasma testosterone in adult male mice lead to Reln promoter demethylation. Animals were administered with flutamide, an antiandrogenic compound, and reelin promoter methylation was assessed using methylationspecific PCR using bisulfite DNA from cerebellum. We found that flutamide was able to significantly lower plasma testosterone when compared to control mice, and treatment did not influence animal survival and body weight. We also show that low plasma testosterone was associated with demethylation of a cytosine residue located at -860 in reelin promoter region. These preliminary data suggest that androgenic hormones can influence cerebral reelin demethylation. To our knowledge, this is the first experimental approach directly linking testosterone depletion and RELN promoter methylation.

Asunto(s)

Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Islas de CpG , Proteínas de la Matriz Extracelular/genética , Proteínas del Tejido Nervioso/genética , Serina Endopeptidasas/genética , Testosterona/sangre , Antagonistas de Andrógenos/administración & dosificación , Antagonistas de Andrógenos/metabolismo , Animales , Peso Corporal , Cerebelo/química , Citocinas/genética , Metilación de ADN , Modelos Animales de Enfermedad , Flutamida/administración & dosificación , Masculino , Ratones , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteína Reelina , Esquizofrenia/genética , Sulfitos/química , Testosterona/química

RESUMEN

Benznidazole (BZ) is the most commonly used drug for the treatment of Chagas disease. Although BZ is known to induce the formation of free radicals and electrophilic metabolites within the parasite Trypanosoma cruzi, its precise mechanisms of action are still elusive. Here, we analyzed the survival of T. cruzi exposed to BZ using genetically modified parasites overexpressing different DNA repair proteins. Our results indicate that BZ induces oxidation mainly in the nucleotide pool, as heterologous expression of the nucleotide pyrophosphohydrolase MutT (but not overexpression of the glycosylase TcOgg1) increased drug resistance in the parasite. In addition, electron microscopy indicated that BZ catalyzes the formation of double-stranded breaks in the parasite, as its genomic DNA undergoes extensive heterochromatin unpacking following exposure to the drug. Furthermore, the overexpression of proteins involved in the recombination-mediated DNA repair increased resistance to BZ, reinforcing the idea that the drug causes double-stranded breaks. Our results also show that the overexpression of mitochondrial DNA repair proteins increase parasite survival upon BZ exposure, indicating that the drug induces lesions in the mitochondrial DNA as well. These findings suggest that BZ preferentially oxidizes the nucleotide pool, and the extensive incorporation of oxidized nucleotides during DNA replication leads to potentially lethal double-stranded DNA breaks in T. cruzi DNA.

Asunto(s)

Enzimas Reparadoras del ADN/genética , Resistencia a Medicamentos/genética , Nitroimidazoles/farmacología , Proteínas Protozoarias/genética , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Supervivencia Celular , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/genética , Enfermedad de Chagas/parasitología , ADN Glicosilasas/genética , Reparación del ADN/efectos de los fármacos , ADN Protozoario/efectos de los fármacos , Guanina/análogos & derivados , Guanina/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Trypanosoma cruzi/genética

RESUMEN

The mammalian homolog B1 of Unc-93 Caenorhabditis elegans known as UNC93B1 is a chaperone protein that mediates translocation of the nucleic acid-sensing Toll-like receptors (TLRs) from the endoplasmic reticulum to the endolysosomes. The triple deficient (UNC93B1 mutant) mice have a functional single point mutation in the UNC93B1 that results in non-functional TLR3, TLR7, and TLR9. Herein, we demonstrate that UNC93B1 mutant mice, in the C57BL/6 (resistant) genetic background, are highly susceptible to Leishmania major infection. Enhanced swelling of the footpad was associated with high levels of interleukin 10, decreased levels of interferon γ, and increased parasitism. None of the single TLR3, TLR7, and TLR9 knock-out (KO) mice resemble the UNC93B1 mutant phenotype upon infection with L. major. Whereas the double TLR7/TLR9 KO showed a partial phenotype, the triple TLR3/TLR7/TLR9 KO mice were as susceptible as the UNC93B1 mutant mice, when infected with Leishmania parasites. Finally, we demonstrate that treatment with either anti-interleukin 10 receptor monoclonal antibody or recombinant interleukin 12 restored a robust anti-parasite TH1 response and reverted the susceptible phenotype of UNC93B1 mutant mice. Altogether, our results indicate the redundant and essential role of nucleic acid-sensing TLR3, TLR7 and TLR9 in inducing interleukin 12, development of a TH1 response, and resistance to L. major infection in resistant C57BL/6 mice.

Asunto(s)

Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Proteínas de Transporte de Membrana/inmunología , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 9/inmunología , Receptores Toll-Like/inmunología , Animales , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Células Cultivadas , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Femenino , Interacciones Huésped-Parásitos/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Leishmania major/fisiología , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/parasitología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Ácidos Nucleicos/inmunología , Ácidos Nucleicos/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Factores de Tiempo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo

RESUMEN

Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8-oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. In mammals, DNA polymerase beta (Polß) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpolß) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpolß showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpolß overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolß was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolß during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle.

Asunto(s)

ADN Polimerasa beta/metabolismo , Reparación del ADN , Replicación del ADN , ADN de Cinetoplasto/metabolismo , Trypanosoma cruzi/enzimología , Microscopía Fluorescente , Mitocondrias/química , Mitocondrias/enzimología , Estrés Oxidativo , Trypanosoma cruzi/química , Trypanosoma cruzi/genética

RESUMEN

We report the cloning and characterization of the DNA polymerase eta gene from Trypanosoma cruzi (TcPoleta), the causative agent of Chagas disease. This protein, which can bypass cyclobutane pyrimidine dimers, contains motifs that are conserved between Y family polymerases. In vitro assays showed that the recombinant protein is capable of synthesizing DNA in undamaged primer-templates. Intriguingly, T. cruzi overexpressing TcPoleta does not increase its resistance to UV-light (with or without caffeine) or cisplatin, despite the ability of the protein to enhance UV resistance in a RAD30 mutant of Saccharomyces cerevisiae. Parasites overexpressing TcPoleta are also unable to restore growth after treatment with zeocin or gamma irradiation. T. cruzi overexpressing TcPoleta are more resistant to treatment with hydrogen peroxide (H(2)O(2)) compared to nontransfected cells. The observed H(2)O(2) resistance could be associated with its ability to bypass 8-oxoguanine lesions in vitro. The results presented here suggest that TcPoleta is able to bypass UV and oxidative lesions. However the overexpression of the gene only interferes in response to oxidative lesions, possibly due to the presence of these lesions during the S phase.

Asunto(s)

ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/fisiología , Proteínas Protozoarias/fisiología , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Northern Blotting , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Peróxido de Hidrógeno/farmacología , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/efectos de la radiación , Rayos Ultravioleta

RESUMEN

Mammalian DNA polymerase beta is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymerasebeta, TcPolbeta and TcPolbetaPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolbetaPAK, in comparison to TcPolbeta, conducted DNA synthesis over a much broader pH range. TcPolbeta was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolbeta were not observed for TcPolbetaPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolbeta, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.

Asunto(s)

ADN Polimerasa beta/metabolismo , ADN Mitocondrial/metabolismo , Trypanosoma cruzi/enzimología , Quinasas p21 Activadas/metabolismo , Secuencia de Aminoácidos , Animales , Bioquímica/métodos , Clonación Molecular , Cartilla de ADN/química , Microscopía Confocal , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido