Current issues

Table of contents: 2021

Research Article

Many research areas have datasets that face the challenges of high dimensionality and multilinearity. Although existing methods are efficient for constructing a complete model, it is often necessary to select the most important explanatory variables to obtain more parsimonious models. We evaluated and built models using three methods of selection of variables applied to data of single nucleotide polymorphism (SNP) markers and near-infrared spectroscopy (NIR), in addition to assessing the improvement in prediction quality when compared to the use of complete data. These included ordered predictors selection associated with partial least squares regression (PLS-OPS), sparse partial least squares regression (SPLS), and Supervised BLasso, the latter being an adaptation of the Bayesian Lasso (BLasso) method for variables selection. We used simulated data sets evaluated in two scenarios, and three real data sets, composed of one set of SNPs and two sets of NIR data. The predictive quality of each model was evaluated based on the mean correlation coefficient between predicted and actual values, and the square root mean squared error. In the set of simulated data evaluated in the first scenario, in terms of predictive capacity, the models after variables selection were similar when compared to the use of the complete data model, whereas in the second scenario, on average, the models performed better after the selection of variables, with SPLS being superior to the other methods. In the real SNPs dataset, the PLS-OPS had a good performance, attesting the usefulness of this method for this kind of data. In the NIR datasets, the predictive quality of models after variable selection were close to those obtained with the complete data. In general, when using the selection methods, the models maintained a good predictive capacity and became simpler due to the considerable reduction in the number of variables.

Genet. Mol. Res. 20(3): GMR18909
DOI: 10.4238/gmr18909
Research Article

There are over 7,000 components in cigarette smoke, 70 of which are considered genotoxic and carcinogenic. N-Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of these components. When treated with NNK at concentrations of as low as 0.1 nM, breast and lung cells are known to acquire malignant properties. The liver plays an important role in toxin metabolization, yet little is known about the cytotoxic effects of NNK on hepatocytes. Therefore, we assessed the effects of NNK on immortalized murine hepatocytes (AML12 cell line) using a repeated exposure approach. AML12 cells were subjected to either short- (single exposure for up to 72 h) or long-term (cumulative exposure for 90 days) testing, at various NNK concentrations (0.1, 10, and 1000 nM). DNA damage was analyzed using the comet assay, a gold-standard technique to assess DNA strand breaks in eukaryotic cells. The cells subjected to short-term exposure had a significantly increased proliferation rate in the 0.1 and 10 nM groups when compared to controls. Furthermore, the cells from the 10 nM group exhibited increased migration rate after cumulative exposure to NNK. The clastogenic effect of NNK increased in a concentration-dependent manner up to 10 nM. We conclude that NNK is genotoxic and significantly alters cell viability and migration, contributing to malignant transformation of hepatocytes.

Genet. Mol. Res. 20(3): GMR18832
DOI: 10.4238/gmr18832
Research Article

Helicobacter pylori is a gram-negative bacterium associated with the development of severe gastric pathologies, such as atrophy, metaplasia, and gastric adenocarcinoma. This microorganism is considered a class I carcinogen by the International Agency for Research on Cancer. The virulence genes in the strain causing infection influence the clinical outcome and can be used as specific markers for the severity of gastric diseases. We evaluated H. pylori infection and associations of cagA, vacA and dupA virulence genes with gastric pathologies. Antral and gastric body biopsies of 117 patients with dyspepsia were analyzed by histological and molecular techniques. Screening for H. pylori infection was performed using the hpx gene (16S rRNA). Positive samples were evaluated for vacA, cagA, and dupA virulence genes. The clinical outcomes presented by the patients were stratified according to severity, being considered severe lesions atrophy, metaplasia and adenocarcinoma. Gastritis, duodenitis, esophagitis, ulcer, and xanthelasma were considered non-severe pathologies. The prevalence of infection was 64.1%, with a high frequency of strains positive for cagA (80.0%), dupA (70.7%), and vacA (56.0%). The cagA gene was detected in all isolates from patients with severe pathologies, whereas the vacA gene was not detected in this group. Simultaneous detection of the three genes was observed in 14.3% and 35.8% of the isolates from individuals with severe and non-severe pathologies, respectively. Furthermore, fewer virulence genes were detected in isolates from patients with severe disease (0.7) than in isolates from non-severe cases (1.4). Patients with severe diseases had a higher mean age and greater number of gastric diseases than patients with non-severe pathologies. The circulating H. pylori strains in the Brazilian Midwest exhibit high heterogeneity in the frequencies of virulence genes. Individual virulence factors and their combinations may influence clinical outcomes.

Genet. Mol. Res. 20(3): GMR18883
DOI: 10.4238/gmr18883
Research Article

Neurodegenerative diseases affect nerve cells, causing impairment in mobility and cognitive abilities, characteristics present in two highly relevant neuropathologies, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). The pathogenesis of these diseases, although distinct, share common neurodegenerative mechanisms. Several studies suggest that miRNAs, small molecules of endogenous non-coding RNA, may assume an important regulatory role in neurodegeneration where their differential expression enables the elucidation of the molecular basis of this process, in addition to offering possible therapeutic targets. We performed a systematic review of the literature through a search in Web of Science, Pubmed/NCBI, and virtual health library (BVS) databases, applying terms indexed in MeSH and DeCS, such as “microRNA”, “amyotrophic lateral sclerosis” and “multiple sclerosis”. We included studies in English, Portuguese, or Spanish, published in the last five years, relating miRNAs associated with pathophysiological pathways of ALS and MS. We excluded studies on non-humans or with polymorphisms in pre-miR genes, duplicated data, or with unavailable data, resulting in a final number of 70 studies included. According to systematic review findings, the families miR-9, miR-23, miR-26, miR-125, miR-133, miR-146, miR-181, miR-206, miR-320, and miR-326 are frequently dysregulated in ALS and MS. The miR-155 and let-7 families are commonly associated with both diseases, regulating genes involved in mechanisms such as neuroinflammation, neurogenesis, and cell differentiation. The elucidation of the newest miRNAs and their main pathways may assist in the characterization of the molecular basis of these diseases, mainly involving those associated with pro-inflammatory processes, microglia activation, and neuronal death, mechanisms associated with ALS. They also can involve myelin breakdown mechanisms, astrocyte damage, and neuronal death, which are related to MS. These molecular markers may help determine biomarkers for amyotrophic lateral sclerosis and multiple sclerosis.

Genet. Mol. Res. 20(3): GMR18843
DOI: 10.4238/gmr18843
Research Article

Gaucher Disease (GD) is an autosomal recessive genetic disorder, caused by a deficiency of the enzyme B-glucocerebrosidase (GBA). In Colombia, despite considerable research on GD, the frequency of the GBA gene variants in the population is unknown, making it difficult to determine the risk of occurrence based on carriers. To identify the variants of the GBA gene, a transversal, descriptive, non-experimental study was carried out with the results obtained from the sequencing of the complete exome of 320 patients with complex disease, without clinical suspicion of GD. Bioinformatics software was used to analyze the clinical significance of the different variants. The population frequency of each variant was calculated, and a network of interaction of the GBA gene was developed. As a result, 41 variants associated with the GBA gene were found; 21/41 of the variants reported have a benign significance, 5/41 of the variants reported were classified as pathogenic or probably pathogenic and 7/41 of the variants reported presented uncertain significance. The gene interaction network showed close associations between GBA and genes PSAP, SCARB2, LAMP2, all of them focused on functions of vacuolar locations, lysosomal and vacuolar lumen instructions, vacuolar and lysosomal membranes. We conclude that the impact on the phenotype highly depends on the pathogenicity of the variants. In our sample, a high frequency of benign variants was found; however, pathogenic variants were detected, which should be the object of study in precision medicine associated with GD.

Genet. Mol. Res. 20(2): GMR18818
DOI: 10.4238/gmr18818
Research Article

Aspergillus flavus produces carcinogenic aflatoxins. This species is divided based on the type of sclerotia produced, designated as “S” for numerous small and “L” for fewer and large. “L” strains are concerning since they are prevalent in corn, peanut, rice, and soil samples. Nearly all “S” strains and approximately 70% of “L” strains produce aflatoxins. Host specificity within isolates is not well understood and needs to be better characterized. We isolated A. flavus from commercial peanut samples from Southern Brazil and investigated their genetic variability and their genetic correlation with four A. flavus isolates previously obtained from commercial bulgur wheat in the same region. We also evaluated the antifungal activity of essential oils on some of the isolates. Eleven “L” type A. flavus strains were isolated from peanut kernels. Eight of these isolates were considered aflatoxigenic, based on thin-layer chromatography and culture techniques. The genetic biodiversity of the A. flavus isolates was analyzed using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) analyses, which indicated differences according to host, however, did not regarding aflatoxin production. Essential oils of oregano, thyme, cinnamon, lavender, and fennel inhibited the isolate’s growth. In conclusion, A. flavus isolates differ according to host and essential oils have potential to control this fungus.

Genet. Mol. Res. 20(2): GMR18787
DOI: 10.4238/gmr18787
Research Article

Brazil is the world largest producer and exporter of Coffea arabica. In this country, numerous breeding programs have generated a great multiplicity of cultivars with expressive productivity that are adapted to the different regions. Evaluating genetic diversity is critical since it provides parameters for defining breeding strategies. We estimated the genetic diversity among and within 32 cultivars of the Brazilian Coffee Trial using AFLP markers. This trial is composed of the main cultivars developed and some under development by Empresa De Pesquisa Agropecuaria do Estado de Minas Gerais (EPAMIG), Fundação PROCAFÉ, Instituto Agronômico de Campinas (IAC), and Instituto de Desenvolvimento Rural do Paraná (IDR-Paraná). A total of 982 AFLP fragments were generated, of which 97.35% were polymorphic. The percentage of polymorphic loci ranged from 22.8  to 50.5%, with genetic diversity varying from 0.06 to 0.16. Variable levels of genetic diversity observed among cultivars probably derived from the diverse germplasm sources and methods used in the genetic breeding programs, the number of advanced generations of each cultivar, as well as genetic recombination or cross-fertilization during breeding programs. Bayesian cluster analysis, principal component analysis, and Neighbor-Net showed three divergent genetic groups, with a high genetic differentiation index (FST = 0.46). The pairwise FST also revealed high divergence among cultivars. IDR-Paraná had the cultivars with the highest genetic variability among these four Brazilian coffee breeding centers. We found that AFLP markers allowed us to distinguish the cultivars/progenies in the Brazilian National Trial. We conclude that Brazilian coffee germplasm still has considerable genetic variability for the development of new cultivars with high productivity, resistance to disease, superior beverage quality, and adaptation to diverse edaphoclimatic conditions in the different producing regions.

Genet. Mol. Res. 20(2): GMR18772
DOI: 10.4238/gmr18772
Research Article

Evolution of mitochondrial genomes is essential for the adaptation of yeasts to changes in environmental oxygen levels. Although Saccharomyces cerevisiae mitochondrial DNA lacks all complex I genes, respiration is possible because alternative NADH dehydrogenases are encoded by NDE1 and NDI1 nuclear genes. The apparent whole genome duplication (WGD) in the yeast ancestor 100-150 million years ago caused nuclear gene duplications and secondary losses, although its relation to the loss of mitochondrial complex I is unknown. We produced phylogenomic supertrees and a supermatrix tree of 46 mitochondrial genomes, showing that the loss of complex I predates WGD and occurred independently in the S. cerevisiae group and the fission yeast Schizosaccharomyces pombe. The branching patterns did not differ substantially in supertrees and supermatrix phylogenies. We found consistent relations between conserved mitochondrial chromosomal gene order (synteny) in closely related yeasts. Correlation of mitochondrial molecular clock estimates and atmospheric oxygen variation in the Phanerozoic suggests that the Saccharomyces lineage might have lost complex I during hypoxic periods near Permian-Triassic or Triassic-Jurassic mass extinction events, while the Schizosaccharomyces lineage possibly lost complex I during hypoxic environment periods during the Middle Cambrian until the Lower Devonian. The loss of mitochondrial complex I, as a result of low oxygen levels, might not affect yeast metabolism due to a fermentative switch. The return to increased oxygen periods could have favored adaptations to aerobic metabolism. Additionally, we also show that NDE1 and NDI1 phylogenies indicate evolutionary convergence in yeasts in which mitochondrial complex I is absent.

Genet. Mol. Res. 20(2): GMR18485
DOI: 10.4238/gmr18485
Research Article

We examined the biological roles of microRNAs (miRNAs) in the pathogenesis of bronchopulmonary dysplasia (BPD). Neonatal rats were randomly assigned to hyperoxia (85% O2) and normoxia (21% O2) groups, and each group had eight neonatal. Twenty differentially expressed miRNAs were identified by deep sequencing, of which 10 were up-regulated and 10 were down-regulated in the hyperoxia group. A total of 5,794 molecular related to gene ontology functions were enriched, including cell location and biological processes. rno-miR-29b-3p were up-regulated, and rno-miR-322-5p and rno-miR-335 were down-regulated in the hyperoxia sample based on quantitative real-time PCR. In conclusion, BPD appears to be caused by activation of extracellular matrix -receptor interaction, cytokine-cytokine receptor interaction, RNA transport, cell cycle, and cell adhesion molecule pathways. These miRNAs may play a role in the occurrence and development of BPD. Our study provides new insight into the biological processes of BPD.

Genet. Mol. Res. 20(2): GMR18751
DOI: 10.4238/gmr18751
Research Article

Cleome viscosa (common names, wild mustard, Asian spiderflower, and tick weed) is an annual oilseed crop plant that has a high vitamin E content, with the most active forms being the tocopherols (α, β, γ and δ). These act as antioxidants in plants and are also useful for human health. The gene expression profiles and partial-length cDNA encoding tocopherol cyclase (CvVTE1), homogentisic acid prenyltransferase (CvVTE2), MPBQ methyltransferase (CvVTE3), gamma-tocopherol methyltransferase (CvVTE4), hydroxyphenyl-pyruvate dioxygenase (CvHPPD) and homogentisate solanesyltransferase (CvHST) genes involved with tocopherol biosynthetic enzymes were determined. Partial cDNA sequences of six genes as 694 bp CvVTE1, 375 bp CvVTE2, 387 bp CvVTE3, 402 bp CvVTE4, 334 bp CvHPPD and 461 bp CvHST showed high identity to their homologs in other higher plants. Expression levels of CvVTE1, CvVTE2, CvVTE3, CvVTE4, CvHPPD and CvHST genes were analyzed by real-time quantitative PCR (qPCR) at plant nodes L1-L3 in leaves and 1-4 weeks after fruit set (WAF) of seeds. CvVTE3 and CvHPPD were predominantly expressed in the leaves, while in seeds CvVTE2 was expressed at both 2 and 3 WAF at rates of 231-fold and 224-fold, which coordinated with CvVTE1 and CvVTE3 expression. Co-expression of CvVTE4 and CvHPPD supports high levels of flux toward the synthesis of tocopherols at 4 WAF without any down regulation. We conclude that CvVTE2 plays a key role in tocopherol biosynthesis in C. viscosa seeds.

Genet. Mol. Res. 20(2): GMR18802
DOI: 10.4238/gmr18802
Research Article

Dalbergia cochinchinensis (Fabaceae) is known as Thai rosewood. It is a Thai native and widespread throughout Thailand. Due to it being a hardwood tree, it became a valuable hardwood tree species for its commercial value for luxury furniture and as a first-class prime timber, which has made it a potentially endangered species. For genetic studies of this tree, we tested four protocols of genomic DNA extraction from the inner bark based on the sodium dodecyl sulfate method and two protocols of the cetyltrimethyl ammonium bromide method, . We evaluated the quantity, purity, and integrity of the extracted genomic DNA from 15 genotypes of D. cochinchinensis using PCR amplification and restriction enzyme digestion to develop a protocol for this species. We found that optimal concentrations of lithium chloride and polyvinylpyrrolidone could improve the quantity and quality of DNA in the extraction buffer without using liquid nitrogen. The highest concentration of high-quality DNA was obtained with protocol M5 (392 ng/µL DNA and a purity ratio of A260/A280 equal to 1.96). In contrast, the commercial Nucleospin Plant II Mini Kit provided the lowest yield of 13.94 ng/µL DNA, with a low purity ratio of A260/A280 (1.58). Start codon targeted \ and sequence-related amplified polymorphism fingerprints further demonstrated that protocol M5 developed for inner bark tissue samples gives good DNA quality and quantity for genetic studies of D. cochinchinensis.

Genet. Mol. Res. 20(2): GMR18836
DOI: 10.4238/gmr18836
Research Article

The quality of fit of a multiple linear regression model often encounters multicollinearity and high dimensionality problems, making it impossible to obtain stable estimates through the traditional method of estimation based on ordinary least squares. To overcome such challenges, dimensionality reduction methods have been proposed, because of their simple theory and easy application. We compared three dimensionality reduction methods: Principal Components Regression (PCR), Partial Least Squares (PLS), and Independent Components Regression (ICR). An important step for dimensionality reduction and prediction is selecting the number of components, as it affects the linear combinations of the explanatory variables. The linear combinations are inserted into the model to predict the response based on a reduced number of parameters. We examined the criteria for the selection of the number of components. The dimensionality reduction methods were applied to genomic and phenotype data. We evaluated 370 accessions of Asian rice, Oryza sativa, which were genotyped for 36,901 SNPs markers considered to predict the genomic values for the number of panicles per plant trait. This data set presented multicollinearity and high dimensionality. The computational time for each method was also recorded. Among the methods, PCR and ICR gave the highest accuracy values, with ICR standing out for presenting estimates of the least biased genomic values. However, ICR required more computational time than the other methodologies.

Genet. Mol. Res. 20(2): GMR18877
DOI: 10.4238/gmr18877
Research Article

Soybean cultivars with resistance against Asian soybean rust (ASR) are necessary to maintain plant yield when control methods become ineffective. We examined the influence of temperature (18, 21, 24, 27 and 30°C) and leaf wetness duration (0, 6, 12, 24 and 48 hours) on the penetration and temporal progress of ASR in soybean genotypes with different levels of partial resistance from the Federal University of Uberlandia breeding program. The genotypes were selected in the field from 100 progenies during 15 years under ASR epidemic conditions. Information on inheritance and molecular markers for disease resistance was available for our study. There were significant differences (P=0.001) in the area under the disease progress curve (AUDPC) between genotypes under the interaction of the binomial (temperature x leaf wetness duration - LWD) and for incubation period at different temperatures. No signs or symptoms of rust appeared at temperatures below18°C or above 30°C. The relationship between temperature and LWD affect the AUDPC and temperature affects incubation period. The highest AUDPC values occurred at 24°C and leaf wetting period of 24 hours, and the lowest values were achieved at temperatures above 27ºC. (Cultivar Desafio RR 8473 RSF) showed susceptibility to ASR.ASR lesions started to appear on average at least 15 days after infection in partially resistant genotypesF8 BRSGO Luziânia X Potenza, F8 BRSGO Caiapônia X Potenza, and F8 BRSGO Caiapônia X IAC100, which also had lower AUDPC and severity values, during the monocycle experiment. We conclude that ASR is shifting to a longer incubation period and fast sporulation induction after uredinia formation. These genotypes can be used in the field, with less need for fungicides helping avoid the emergence of new ASR pathotypes and races with low sensitivity to currently used fungicides.

Genet. Mol. Res. 20(2): GMR18781
DOI: 10.4238/gmr18781
Research Article

Lactobacilli are the predominant bacterial species colonizing the vaginal surfaces of healthy women, where they play a protective role against opportunistic and polymicrobial infections, such as bacterial vaginosis. Several Lactobacillus species, especially L. crispatus, have been prospected for probiotic applications due to their potential antimicrobial and anti-inflammatory capacities. During the last decade, several genomic studies have been investigating the genetics of L. crispatus strains in an effort to identify novel probiotic strains and evaluate their potential for improving human and animal health. This mini review highlights the main genes associated with L. crispatus protective mechanisms in four novel strains of this species that we recently isolated from healthy Brazilian women of reproductive age. Among the probiotic features of these strains, the roles of a pyruvate oxidase-encoding gene, lactate synthesis related enzymes, bacteriocin genes, and genomic islands, are reviewed, and the next steps for confirming their activity are indicated.

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Genet. Mol. Res. 20(2): GMR18900
DOI: 10.4238/gmr18900
Research Article

The umbu tree (Spondias tuberosa) is one of the best known plants of the Brazilian semi-arid region; it has great potential for the fruit market due to excellent consumer acceptance. This tree is not presently cultivated; fruit commercialization is based on extrativism. Consequently, there has been little research on the genetics of this species. Our objective was to develop, evaluate and transfer single sequence repeat (SSR) loci to S. tuberosa to support work on genetic resources and agronomic improvement of this species. SSR loci for the umbu tree were developed from a new enriched genomic library and evaluated by PCR.  Fourteen SSR loci developed for S. radlkoferi were evaluated for use in S. tuberosa, as well as 18 SSR loci previously identified for this species. DNA was extracted from leaf tissue of eight umbu trees available that are part of a germplasm collection located in Petrolina, PE, Brazil. Of the 14 pairs of primers that were tested, six yielded amplicons, and two showed polymorphism in the genotyped samples. All SSR loci of S. radlkoferi transferred to the umbu tree species, yielding amplicons; however, only four were polymorphic in this sample. Among the eighteen available species-specific SSR loci, six were polymorphic for these eight trees. Among the 38 loci that we evaluated, 12 were polymorphic in the sample, including two new loci for S. tuberosa. The number of alleles ranged from 2 to 12, and 10 of them were moderately to highly informative (PIC>0.50), while nine had heterozygosity greater than 0.50. The six new SSR loci and the 14 SSR loci transferred from S. radlkoferi increase the number of available loci for population studies, germplasm collection and resource management for the development of new umbu tree cultivars.

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Genet. Mol. Res. 20(2): GMR18778
DOI: 10.4238/gmr18778