Screening for Novel LOX and SOD1 Variants in Keratoconus Patients from Brazil

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

DNA polimerase beta: uma proteína envolvida na replicação e reparo do DNA mitocondrial emTrypanosoma cruzi

Exportado OPUSMade available in DSpace on 2019-08-13T07:51:41Z (GMT). No. of bitstreams: 1 tese_bruno_luiz_reis.pdf: 3900651 bytes, checksum: 28c8a4b61cc75e5b64a643229803e204 (MD5) Previous issue date: 19Durante seu ciclo de vida, o protozoário Trypanosoma cruzi causador da Doença de Chagas deve lidar com a ação deletéria de espécies reativas de oxigênio (ROS) no DNA, principalmente com 7,8-diidro-8-oxoguanina (8oxoG). Caso não seja reparada, a 8oxoG pode levar a transversões durante a divisão celular. Em mamíferos, a DNA polimerase beta (pol) está estritamente envolvida no reparo por excisão de bases (BER), que pode reparar danos oxidativos. Contudo, sua função ainda é pouco conhecida em T. cruzi. Ensaios de localização da pol por imunofluorescência revelaram uma localização nos sítios antipodais do DNA mitocondrial (kDNA) das formas epimastigota e amastigota replicativas. Contudo a polimerase se mostrou dispersa na matriz mitocondrial de formas tripomastigotas não-replicativas, sugerindo sua participação na replicação do kDNA. Adicionalmente, verificamos uma localização estrita da pol nos sítios antipodais do kDNA somente durante as fases G1/S e início da fase G2 de epimastigotas. Clones superexpressando a pol mostraram maiorresistência à H2O2 e benzonidazol quando comparado a células controle. A resistência a H2O2 é perdida após tratar as células com metoxiamina, que é um inibidor seletivo da via do BER. Verificamos também um nível reduzido de 8oxoG no cinetoplasto dos clones superexpressores de pol em relação ao controle. Curiosamente, um possível foco de reparo de DNA envolvendo a pol foi identificado nas imediações do kDNA de epimastigotas após tratamento com H2O2, localizado provavelmente na região cinetoflagelar. Em conjunto, os dados experimentais obtidos sugerem que a pol está participando de processos de replicação e reparo de danos oxidativos no DNA mitocondrial de T. cruzi.During its life cycle, the causative agent of Chagas Disease Trypanosoma cruzi must deal with the deleterious action of reactive oxygen species (ROS) on DNA, mainly 7,8-dihydro-8-oxoguanine (8oxoG). If unrepaired, 8oxoG can lead to transversions during cell division. In mammals, DNA polymerase beta (pol) is strictly involved in base excision repair (BER) of oxidative damage. However its biological function in T.cruzi is still uncertain. Immunofluorescent analysis revealed that pol is localized on antipodal sites of kinetoplast (kDNA) of replicative epimastigotes and amastigotes forms. Nevertheless, the polymerase was seen dispersed in non-replicative trypomastigote forms inside the mitochondrion matrix, suggesting its participation in kDNA replication. In addition, we verify that pol is strictly localized in kDNA antipodal sites between G1/S and early G2 phase of replicative epimastigotes. Clonesoverexpressing pol presented increased survival after treatment with H2O2 and benznidazol compared to control. However this resistance is lost after treating cells with methoxiamine, a potent BER inhibitor. We also verified that the overexpressing clones showed a reduced level of detected 8oxoG in kinetoplast when compared to control. Curiously, a possible DNA repair focus of pol was identified in the vicinity ofkinetoplast of epimastigotes wild type CL Brener cells after H2O2 treatment. We believe that this focus is a DNA repair focus probably mounted in kinetoflagellar zone after oxidative damage to kDNA. Taken together the experimental data obtained suggest participation of pol in DNA replication and repair of oxidative damages in kDNA of T. cruzi

Testosterone Depletion Induces Demethylation of Murine Reelin Promoter CpG Dinucleotides: A Preliminary Study

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

UNC93B1 and nucleic acid-sensing Toll-like receptors mediate host resistance to infection with Leishmania major

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 gamma, 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

Analysis of VSX1 variations in Brazilian subjects with keratoconus

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

Association between Maternal Non-Coding Interferon-λ Polymorphisms and Congenital Zika Syndrome in a Cohort from Brazilian Northeast

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

Association between Maternal Non-Coding Interferon-λ Polymorphisms and Congenital Zika Syndrome in a Cohort from Brazilian Northeast

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

Biochemical studies with DNA polymerase beta and DNA polymerase beta-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance

Mammalian DNA polymerase p 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 polymerase beta, TcPol beta and TcPol beta PAK, 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 TcPol beta PAK, in comparison to TcPol beta, conducted DNA synthesis over a much broader pH range. TcPol beta 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 TcPol beta were not observed for TcPol beta PAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPol beta, 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. (C) 2008 Elsevier B.V. All rights reserved.PRONEXFundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed Minas Gerais, ICB, Dept Bioquim & Imunol, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilCNRS, Inst Pharmacol & Biol Struct, Grp Genet Instabil & Canc, Toulouse, FranceUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions

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-8oxoguanine (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 (Pol beta) 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 (Tcpol beta) 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 Tcpol beta 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 Tcpol beta overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpol beta was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpol beta during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle. (C) 2012 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)PRONEXFundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Howard Hughes Medical InstituteUniv Fed Minas Gerais, Dept Biochem & Immunol, ICB UFMG, Inst Biol Sci, BR-31270901 Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniv Fed Rio de Janeiro, Hertha Meyer Cellular Ultra Struct Lab, Inst Biophys Carlos Chagas Filho, BR-21941 Rio de Janeiro, BrazilSuper Learning & Dev Ctr CESED, Sch Med Sci, Campina Grande, Paraiba, BrazilCNRS, Inst Pharmacol & Struct Biol, UMR5089, Toulouse, FranceUniv Toulouse, Toulouse, FranceUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilCNPq: MCT/CNPq/MS-SCTIE-DECIT 25/2006-Estudo de Doencas NegligenciadasWeb of Scienc