Resumo em Inglês:Abstract We examined the role of UCP gene polymorphisms as susceptibility markers for premature coronary artery disease (pCAD). The UCP2 Ala55Val (C/T rs660339), UCP2 -866G/A (rs659366), and UCP3 -55C/T (rs1800849) polymorphisms were genotyped in 948 patients with pCAD, and 763 controls. The distribution of the UCP2 A55V (C/T rs660339) and UCP3 -55 (rs1800849) was similar in patients and controls. However, under a recessive model, the UCP2 -866 (rs659366) A allele was associated with increased risk of developing pCAD (OR = 1.43, Pc = 0.003). On the other hand, patients with pCAD and UCP2 A55V (rs660339) TT showed high levels of visceral abdominal fat (VAF) (Pc = 0.002), low levels of subcutaneous abdominal fat (SAF) (Pc = 0.001) and high VAT/SAT ratio (Pc < 0.001). Also, patients with UCP2 -866 (rs659366) AA showed increased levels of VAF (Pc = 0.003), low levels of SAF (Pc = 0.001) and a high VAT/SAT ratio (Pc = 0.002), whereas patients with the UCP3 -55 (rs1800849) TT presented high levels of VAF (Pc = 0.002). The results suggest the association of the UCP2 -866 (rs659366) polymorphism with risk of developing pCAD. Some polymorphisms were associated with abdominal fat levels and cardiovascular risk factors.
Resumo em Inglês:Abstract 3-Methylcrotonylglycinuria is an organic aciduria resulting from deficiency of 3-methylcrotonyl-CoA carboxylase (3-MCC), a biotin-dependent mitochondrial enzym carboxylating 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA during leucine catabolism. Its deficiency, due to mutations on MCCC1 and MCCC2 genes, leads to accumulation of 3-methylcrotonyl-CoA metabolites in blood and/or urine, primarily 3-hydroxyisovaleryl-carnitine (C5-OH) in plasma and 3-methylcrotonyl-glycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA) in the urine. The phenotype of 3-MCC deficiency is highly variable, ranging from severe neurological abnormalities and death in infancy to asymptomatic adults. Here we report the biochemical and molecular characterization of an Italian asymptomatic girl, positive for the newborn screening test. Molecular analysis showed two mutations in the MCCC2 gene, an already described missense mutation, c.691A > T (p.I231F), and a novel splicing mutation, c.1150-1G > A. We characterized the expression profile of the splice mutation by functional studies.
Resumo em Inglês:Abstract Human longevity is a polygenic and multifactorial trait. Pathways related to lifespan are complex and involve molecular, cellular, and environmental processes. In this analytical observational study, we evaluated the relationship between environment factors, oxidative stress status, DNA integrity level, and the association of FOXO3 (rs2802292), SOD2 (rs4880), APOE (rs429358 and rs7412), and SIRT1 (rs2273773) polymorphisms with longevity in oldest-old individuals from southeastern Brazil. We found an association between the FOXO3 GG genotype and gender. While lifestyle, anthropometric, and biochemical characteristics showed significant results, DNA damage and oxidative stress were not related to lifespan. We found that long-lived individuals with FOXO3 GT genotype had low levels of triglycerides. This study is the first to demonstrate that FOXO3 could be a candidate gene for longevity in the Brazilian population. These results are important in terms of provisions of health care for age-related diseases and lifespan, and provide insight for further research on epigenetic, gene regulation, and expression in oldest-old individuals.
Resumo em Inglês:Abstract The fat mass- and obesity-associated gene (FTO) is significantly associated with obesity, but the associations of FTO with obesity-related traits are not fully described. We aimed to investigate the association of the FTO single nucleotide polymorphism (SNP) rs6499640 with lipid levels in Chinese children. A total of 3503 children aged 6-18 years were included in the present study. Lipid levels were analyzed and the SNP rs6499640 was genotyped using the TaqMan Allelic Discrimination Assay. Statistically significant associations were found between rs6499640 and low-density lipoprotein cholesterol (LDL-C) (p = 0.008), total cholesterol (TC) (p = 0.005), and triglycerides (TG) (p < 0.001) in girls under a dominant model adjusted for age and BMI. No statistical significance was found between the SNP and lipid levels in boys. We demonstrated for the first time that the SNP rs6499640 in FTO is associated with LDL-C, TC, and TG in Chinese girls. Our study identified a new risk locus for lipid levels in children.
Resumo em Inglês:Abstract Disruption of endogenous circadian rhythms has been shown to increase the risk of developing myocardial infarction (MI), suggesting that circadian genes might play a role in determining disease susceptibility. We conducted a case-control study on 200 patients hospitalized due to MI and 200 healthy controls, investigating the association between MI and single nucleotide polymorphisms (SNPs) in four circadian genes (ARNTL, CLOCK, CRY2, and PER2). The variants of all four genes were chosen based on their previously reported association with cardiovascular risk factors, which have a major influence on the occurrence of myocardial infarction. Statistically significant differences, assessed through Chi-square analysis, were found in genotype distribution between cases and controls of the PER2 gene rs35333999 (p=0.024) and the CRY2 gene rs2292912 (p=0.028); the corresponding unadjusted odds ratios, also significant, were respectively OR=0.49 (95% CI 0.26-0.91) and OR=0.32 (95% CI 0.11-0.89). Our data suggest that genetic variability in the CRY2 and PER2 genes might be associated with myocardial infarction.
Resumo em Inglês:Abstract Polyploidy does not usually occur in germinal cells of mammals and other higher vertebrates. We describe a unique example of mosaic autotetraploidy in the meiosis of a human male. Although the original observations were made in the late 1960s, we did not publish them at that time, because we expected to detect further examples that could be described together. However, this did not occur and we have now decided to make the observations available to demonstrate that polyploidy in mammalian male meiosis can arise at a higher frequency than expected by random polyploidization of individual meiotic cells, by either DNA duplication or cell fusion prior to synapsis. This is the first description of a population of primary spermatocytes exhibiting multivalent formation at leptotene /diakinesis in human spermatogenesis, with ring, chain, frying pan and other types of quadrivalents, typical of autotetraploidy. As many of the polyploid configurations showed apoptotic breakdown, it is likely that diploid and/or aneuploid spermatozoa would have rarely or never resulted from this mosaic autotetraploid meiosis.
Resumo em Inglês:Abstract We describe the initial results of a neonatal screening program for four lysosomal storage diseases (MPS I, Pompe, Gaucher and Fabry) using the digital microfluidics methodology. The method successfully identified patients previously diagnosed with these diseases and was used to test dried blood spot samples obtained from 10,527 newborns aged 2 to 14 days. The digital microfluidic technology shows potential for a simple, rapid and high-throughput screening for these four diseases in a standard neonatal screening laboratory.
Resumo em Inglês:Abstract Transposable elements are dispersed repetitive DNA sequences that can move within the genome and are related to genome and chromosome evolution, adaptation, and speciation. The aim of this study was to characterize and determine the chromosomal location and accumulation of a Mariner-like element in populations of four phylogenetically related species of the Dichotomius (Luederwaldtinia) sericeus complex. Mapping of the isolated element was performed by fluorescent in situ hybridization in different populations of analyzed species. Characterization of the isolated element revealed a degenerated transposon, named DgmarMITE. This transposon is 496-bp-long, AT rich (57%), and contains 24 bp terminal inverted repeats. In situ mapping revealed presence of this element only in two out of four species analyzed. DgmarMITE sites were located in heterochromatic and euchromatic regions and varied in location and number on the karyotypes of Dichotomius (L.) gilletti and D. (L.) guaribensis across different populations. These results demonstrate differential accumulation of the DgmarMITE in genomes of these species, which is probably due to the occurrence of ectopic recombination and cross-mobilization of the element mediated by the transposase of closely related or unrelated transposable elements.
Resumo em Inglês:Abstract The linkage disequilibrium (LD) between molecular markers affects the accuracy of genome-wide association studies and genomic selection application. High-density genotyping platforms allow identifying the genotype of thousands of single nucleotide polymorphisms (SNPs) distributed throughout the animal genomes, which increases the resolution of LD evaluations. This study evaluated the distribution of minor allele frequencies (MAF) and the level of LD in the Colombian Creole cattle breeds Blanco Orejinegro (BON) and Romosinuano (ROMO) using a medium density SNP panel (BovineSNP50K_v2). The LD decay in these breeds was lower than those reported for other taurine breeds, achieving optimal LD values (r2 ≥ 0.3) up to a distance of 70 kb in BON and 100 kb in ROMO, which is possibly associated with the conservation status of these cattle populations and their effective population size. The average MAF for both breeds was 0.27 ± 0.14 with a higher SNP proportion having high MAF values (≥ 0.3). The LD levels and distribution of allele frequencies found in this study suggest that it is possible to have adequate coverage throughout the genome of these breeds using the BovineSNP50K_v2, capturing the effect of most QTL related with productive traits, and ensuring an adequate prediction capacity in genomic analysis.
Resumo em Inglês:Abstract Alstroemeria L. (Alstroemeriaceae) represents one of the most diverse genera of vascular plants in Chile. It contains approximately 54 taxa, 40 of which are endemic. The “complex” Alstroemeria magnifica is endemic to Chile, and it comprises four varieties: A. magnifica var. magenta, A. magnifica var. magnifica, A. magnifica var. sierrae, and A. magnifica var. tofoensis. It is distributed from Coquimbo to the Valparaíso Region. We analyzed karyotypes of 10 populations along its natural distribution. All the populations presented an asymmetric karyotype, with 2n = 16 chromosomes but with three different karyotypic formulae. Alstroemeria magnifica var. magnifica and A. magnifica var. sierrae presented the same karyotypic fomula, and A. magnifica var. magenta, and A. magnifica var. tofoensis each had a different formula. The scatter plot among CVCL vs. MCA shows different groupings between populations of the four varieties. Based on the results, it is possible to consider raising Alstroemeria magnifica var. magenta to species level (A. magenta) and A. magnifica var. tofoensis to subspecies level (A. magnifica subsp. tofoensis); A. magnifica var. magnifica and A. magnifica var. sierrae should each remain as varieties. Nevertheless, these taxonomic changes should be considered tentative, as additional sources of evidence become available.
Resumo em Inglês:Abstract Jatropha is an important genus of Euphorbiaceae, with species largely used for various purposes, including the manufacturing of soaps and pharmaceutical products and applications in the bioenergetic industry. Although there have been several studies focusing J. curcas in various aspects, the karyotype features of Jatropha species are poorly known. Therefore, we analyzed six Jatropha species through fluorochrome staining (CMA/DAPI), fluorescent in situ hybridization (FISH) with 5S and 45S rDNA probes and genome size estimation by flow cytometry. Our results revealed several chromosome markers by both CMA/DAPI and FISH for the analyzed species. Five Jatropha species (J. curcas, J. gossypiifolia, J. integerrima, J. multifida and J. podagrica) showed four CMA-positive (CMA+) bands associated with the 5S and 45S rDNA sites (one and two pairs, respectively). However, J. mollissima displayed six CMA+/DAPI- bands co-localized with both 5S and 45S rDNA, which showed a FISH superposition. A gradual variation in the genome sizes was observed (2C = 0.64 to 0.86 pg), although an association between evidenced heterochromatin and genome sizes was not found among species. Except for the unique banding pattern of J. mollissima and the pericentromeric heterochromatin of J. curcas and J. podagrica, our data evidenced relatively conserved karyotypes.
Resumo em Inglês:Abstract The successful development of genetically engineered monocots using Agrobacterium-mediated transformation has created an increasing demand for compatible vectors. We have developed a new expression vector, pGVG, for efficient transformation and expression of different constructs for gene overexpression and silencing in sugarcane. The pCAMBIA2300 binary vector was modified by adding Gateway recombination sites for fast gene transfer between vectors and the maize polyubiquitin promoter Ubi-1 (ZmUbi1), which is known to drive high gene expression levels in monocots. Transformation efficiency using the pGVG vector reached up to 14 transgenic events per gram of transformed callus. Transgenic plants expressing the β-glucuronidase (GUS) reporter gene from pGVG showed high levels of GUS activity. qRT-PCR evaluations demonstrated success for both overexpression and hairpin-based silencing cassettes. Therefore, pGVG is suitable for plant transformation and subsequent applications for high-throughput production of stable transgenic sugarcane. The use of an expression cassette based on the ZmUbi1 promoter opens the possibility of using pGVG in other monocot species.
Resumo em Inglês:Abstract Coffee is one of the most valuable agricultural commodities and the plants’ leaves are the primary site of infection for most coffee diseases, such as the devastating coffee leaf rust. Therefore, the use of bacterial microbiota that inhabits coffee leaves to fight infections could be an alternative agricultural method to protect against coffee diseases. Here, we report the leaf-associated bacteria in three coffee genotypes over the course of a year, with the aim to determine the diversity of bacterial microbiota. The results indicate a prevalence of Enterobacteriales in Coffea canephora, Pseudomonadales in C. arabica ‘Obatã’, and an intriguing lack of bacterial dominance in C. arabica ‘Catuaí’. Using PERMANOVA analyses, we assessed the association between bacterial abundance in the coffee genotypes and environmental parameters such as temperature, precipitation, and mineral nutrients in the leaves. We detected a close relationship between the amount of Mn and the abundance of Pseudomonadales in ‘Obatã’ and the amount of Ca and the abundance of Enterobacteriales in C. canephora. We suggest that mineral nutrients can be key drivers that shape leaf microbial communities.
Resumo em Inglês:Abstract Trypanosoma cruzi is the etiological agent of Chagas disease, a public health challenge due to its morbidity and mortality rates, which affects around 6-7 million people worldwide. Symptoms, response to chemotherapy, and the course of Chagas disease are greatly influenced by T. cruzi‘s intra-specific variability. Thus, DNA mutations in this parasite possibly play a key role in the wide range of clinical manifestations and in drug sensitivity. Indeed, the environmental conditions of oxidative stress faced by T. cruzi during its life cycle can generate genetic mutations. However, the lack of an established experimental design to assess mutation rates in T. cruzi precludes the study of conditions and mechanisms that potentially produce genomic variability in this parasite. We developed an assay that employs a reporter gene that, once mutated in specific positions, convert G418-sensitive into G418-insenstitive T. cruzi. We were able to determine the frequency of DNA mutations in T. cruzi exposed and non-exposed to oxidative insults assessing the number of colony-forming units in solid selective media after plating a defined number of cells. We verified that T. cruzi‘s spontaneous mutation frequency was comparable to those found in other eukaryotes, and that exposure to hydrogen peroxide promoted a two-fold increase in T. cruzi‘s mutation frequency. We hypothesize that genetic mutations in T. cruzi can arise from oxidative insults faced by this parasite during its life cycle.
Resumo em Inglês:Abstract The importance of glutathione (GSH) in alternative cellular roles to the canonically proposed, were analyzed in a model unable to synthesize GSH. Gene expression analysis shows that the regulation of the actin cytoskeleton pathway is strongly impacted by the absence of GSH. To test this hypothesis, we evaluate the effect of GSH depletion via buthionine sulfoximine (5 and 12.5 mM) in human neuroblastoma MSN cells. In the present study, 70% of GSH reduction did not induce reactive oxygen species, lipoperoxidation, or cytotoxicity, which enabled us to evaluate the effect of glutathione in the absence of oxidative stress. The cells with decreasing GSH levels acquired morphology changes that depended on the actin cytoskeleton and not on tubulin. We evaluated the expression of three actin-binding proteins: thymosin β4, profilin and gelsolin, showing a reduced expression, both at gene and protein levels at 24 hours of treatment; however, this suppression disappears after 48 hours of treatment. These changes were sufficient to trigger the co-localization of the three proteins towards cytoplasmic projections. Our data confirm that a decrease in GSH in the absence of oxidative stress can transiently inhibit the actin binding proteins and that this stimulus is sufficient to induce changes in cellular morphology via the actin cytoskeleton.
Resumo em Inglês:Abstract The ubiquitin-specific protease 22 (USP22) is an oncogene and its expression is upregulated in many types of cancer. In the nucleus, USP22 functions as one subunit of the SAGA to regulate gene transcription. However, the genome-wide USP22 binding sites and its direct target genes are yet clear. In this study, we characterized the potential genomic binding sites of UPS22 and GCN5 by ChIP-seq using specific antibodies in HeLa cells. There were 408 overlapping putative target genes bound by both USP22 and GCN5. Motif analysis showed that the sequences bound by USP22 and GCN5 shared two common motifs. Gene ontology (GO) and pathway analysis indicated that the genes targeted by USP22 and GCN5 were involved in different physiological processes and pathways. Further RNA-seq, GO and pathway analyses revealed that knockdown of UPS22 induced differential expression of many genes that participated in diverse physiological processes, such as metabolic process. Integration of ChIP-seq and RNA-seq data revealed that UPS22 bound to the promoters of 56 genes. These findings may provide new insights into the regulation of USP22 on gene expression during the development of cervical cancer.
Resumo em Inglês:Abstract Gastric cancer (GC) remains one of the main causes of cancer-related death worldwide. There are two distinct histological types of GC: diffuse and intestinal. The latter is characterized by the presence of pre-neoplastic lesions. One of the most frequently altered enzymes in intestinal GC is COX-2, an important lesion marker. This work aimed to study COX-2 methylation and expression in N-methyl-N-Nitrosurea (MNU)-induced intestinal GC in six Sapajus apella animals. The partial promoter sequence of S. apella COX-2 gene was obtained and used to identify transcription factors and cis-regulatory element binding sites. The COX-2 methylation pattern was assessed using Methylation-Specific PCR (MSP), and expression was analyzed by immunohistochemistry (IHQ). A total of 20 samples were obtained. A 675 bp fragment of the S. apella COX-2 promoter region was obtained, and it was 99.2% and 68.2% similar to H. sapiens and S. boliviensis, respectively. Similar to humans, several transcription factors and cis-regulatory element binding sites were identified in the S. apella sequence. MSP revealed that all samples were methylated. However, IHQ results demonstrated positive COX-2 expression in all pre-neoplastic and tumoral samples. The results suggest that the analyzed fragment is not crucial in COX-2 regulation of GC in S. apella.
Resumo em Inglês:Abstract Lupinus albescens is a resistant cover plant that establishes symbiotic relationships with bacteria belonging to the Bradyrhizobium genus. This symbiosis helps the development of these plants in adverse environmental conditions, such as the ones found in arenized areas of Southern Brazil. This work studied three Bradyrhizobium sp. (AS23, NAS80 and NAS96) isolated from L. albescens plants that grow in extremely poor soils (arenized areas and adjacent grasslands). The genomes of these three strains were sequenced in the Ion Torrent platform using the IonXpress library preparation kit, and presented a total number of bases of 1,230,460,823 for AS23, 1,320,104,022 for NAS80, and 1,236,105,093 for NAS96. The genome comparison with closest strains Bradyrhizobium japonicum USDA6 and Bradyrhizobium diazoefficiens USDA110 showed important variable regions (with less than 80% of similarity). Genes encoding for factors for resistance/tolerance to heavy metal, flagellar motility, response to osmotic and oxidative stresses, heat shock proteins (present only in the three sequenced genomes) could be responsible for the ability of these microorganisms to survive in inhospitable environments. Knowledge about these genomes will provide a foundation for future development of an inoculant bioproduct that should optimize the recovery of degraded soils using cover crops.
Resumo em Inglês:Abstract Cellulose and its associated polymers are structural components of the plant cell wall, constituting one of the major sources of carbon and energy in nature. The carbon cycle is dependent on cellulose- and lignin-decomposing microbial communities and their enzymatic systems acting as consortia. These microbial consortia are under constant exploration for their potential biotechnological use. Herein, we describe the characterization of the genome of Dyella jiangningensis FCAV SCS01, recovered from the metagenome of a lignocellulose-degrading microbial consortium, which was isolated from a sugarcane crop soil under mechanical harvesting and covered by decomposing straw. The 4.7 Mbp genome encodes 4,194 proteins, including 36 glycoside hydrolases (GH), supporting the hypothesis that this bacterium may contribute to lignocellulose decomposition. Comparative analysis among fully sequenced Dyella species indicate that the genome synteny is not conserved, and that D. jiangningensis FCAV SCS01 carries 372 unique genes, including an alpha-glucosidase and maltodextrin glucosidase coding genes, and other potential biomass degradation related genes. Additional genomic features, such as prophage-like, genomic islands and putative new biosynthetic clusters were also uncovered. Overall, D. jiangningensis FCAV SCS01 represents the first South American Dyella genome sequenced and shows an exclusive feature among its genus, related to biomass degradation.