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Table of contents: 2022

Research Article

This study aimed to examine the effects of curcumin, a phytochemical antioxidant, on the treatment and care of diabetic nephropathy and to contribute to alternative treatment strategies for diabetes. Male Wistar albino rats (8–10 weeks old) were divided into five groups of seven. Experimental diabetes was induced in all rats except for those in Group 1 (placebo group) by administration of 110 mg/kg nicotinamide, followed by intraperitoneal administration (after 15 min) of 55 mg/kg streptozotocin. Groups 1, 3, 4, and 5 were treated with 0.1 ml normal saline (0.9% NaCl), 150mg/kg/day metformin, 10 mg/kg/day glycazide (diamicron), and 200 mg/kg/day curcumin, respectively. Group 2 did not receive any treatment. Kidney tissues of rats were collected for histopathological examination.  There were no significant differences in the kidney dimensions of the rats. In the histopathological evaluation of kidney tissues with diabetic nephropathy, glomerular congestion and destruction were observed. Rats treated with curcumin had significantly less kidney damage, based on histopathological analysis, than those treated with the diabetes drugs.  We conclude that curcumin has protective effects in kidneys due to its antioxidant properties. It has potential for use, in addition to antidiabetic drugs, for diabetes treatment.

Genet. Mol. Res. 21(2): GMR19026
Research Article

We investigated single nucleotide polymorphisms of four genes (calpain (CAPN-9 and CAPN-14), calpastatin – CAST, diacylglycerol acyltransferase – DGAT, and leptin - LEP) that are related to beef quality, and we examined changes in the synthesis of proteins that they encode in Nellore cattle. Samples from 95 adult males of commercial origin were analyzed. Genomic DNA was extracted from the longissimus dorsi (sirloin) muscle tissue, identified with PCR-single-stranded conformation polymorphism analysis. For each different pattern identified, the products were sent for sequencing and analyzed using sequence scanner software. The data were analyzed by determining the absolute and relative frequencies of the polymorphisms identified in each gene. For the CAPN-9 gene, sequencing showed five polymorphisms (G/A, T/A, T/C, T/C, and A/G), of which two involved amino acid substitutions (c.5861G>A and c.5498A>G). Sequencing of the CAPN-14 gene revealed four polymorphisms (A/C, G/A, T/C, and C/G), with two involving amino acid substitutions (c.11054 T>C and c.11161C>G). Sequencing of the CAST-5 gene revealed five polymorphisms (C/T, T/C, C/A, C/A, and G/T), four of which involved amino acid substitutions (c.29919C>T, c.29963A>C, c.29978C>A, and c.30019G>T). Sequencing of the DGAT gene revealed six polymorphisms (T/A, G/A, A/T, G/C, A/G, and G/A), four of which involved amino acid substitutions (c.11730A>T, c.11809G>C, c.11858A>G, and c.11927G>A). Sequencing of the LEP gene revealed three polymorphisms (C/T, C/T and T/C), with one involving an amino acid substitution (c.14962T>G). These genes   had a large number of polymorphisms resulting in amino acid differences in Nellore Cattle. These polymorphisms involving amino acid changes may promote functional changes in beef characteristics of Nellore cattle given that these genes are associated with beef quality parameters.

Genet. Mol. Res. 21(2): GMR18983
DOI: 10.4238/gmr18983
Research Article

Cynodon dactylon (Bermudagrass) ise used for forage in Brazilian pastures, as well as in lawns, parks, and sports fields. However, in sugarcane fields, it is a difficult to control weed due to its rapid adaptation and growth mechanisms. It competes with sugarcane plants negatively affecting crop yield. Bermudagrass has polyploidy and easy hybridization, which promote high polymorphism, generating varying responses to the environment. Consequently, understanding the structure and variability of Bermudagrass becomes important for the development of strategies for its management as a weed. We examined the levels of genetic variability and structure of five populations (SP1 to SP5) of Bermudagrass, collected from sugarcane fields in the state of São Paulo (Brazil). Thirteen microsatellites were used. PCoA demonstrated STRUCTURE results (K=4) showing a mixture of SP1 and SP3 populations, with SP2, SP4 and SP5 being the most distant. DAPC also confirmed low genetic differentiation for SP1 and SP3. Genetic variability was found to be greater among than within populations, due to the predominance of vegetative growth of the species, which promotes low diversity, and due to geographic distances, which reduce gene flow or even make it unfeasible. The SSRs showed high resolution in characterizing the genetic diversity and structure of the five populations of Bermudagrass. The findings of this study may help to establish biological control methods against Bermudagrass in sugarcane fields.

Genet. Mol. Res. 21(1): GMR19010
DOI: 10.4238/gmr19010
Research Article

The SARS-CoV-2 pandemic has demonstrated the need for genomic epidemiology surveillance. To date, various methodologies have been applied, including metagenomic approaches and amplicon-based sequencing associated with high-throughput sequencing platforms. We adapted some details in amplicon-based sequencing using a SARS-CoV-2 community panel (Illumina AmpliSeq), with additional modifications for balanced and high-quality sequencing using the MiSeq platform. The modified protocol was used to detect circulating SARS-CoV-2 variants in Goiás state, Brazil. Initially, RNA samples were obtained from swab samples from 15 patients from the state of Goiás, Brazil, in November/2020 and February/2021 to validate protocol steps. The libraries were prepared following AmpliSeq for Illumina workflow with modifications; subsequently, we analyzed 305 positive samples collected from the state of Goiás from December 2020 to July 2021. For protocol improvement, we removed the need to treat samples with DNAse and demonstrated the importance of quantification by qPCR before and after library dilution. No fragmentation pattern was observed in the samples analyzed with Bioanalyzer. The libraries returned sequencing results that were used for genome assembly and variant detection. We were able to assemble SARS-CoV-2 genomes from 318 samples, which were used to identify 13 variants of coronavirus circulating in Goiás throughout those months. Variants of concern, such as Alpha (B.1.1.7), Gamma (P.1) and Delta (B.1.617.2) were detected; the latter was detected at first in Goiás in April 2021. The modifications in the workflow we developed were successfully applied to detect SARS-CoV-2 variants, resulting in high coverage genome assembly, and they can be used to increase the number of genome sequences and aid in epidemiological surveillance in Brazil.

Genet. Mol. Res. 21(1): GMR19018
DOI: 10.4238/gmr19018
Research Article

Nitrogen management distinctly influences the expression and magnitude of relationships between wheat ear components and grain yield. We sought to determine the variables linked to wheat ear that are more responsive to changes in the dose and form of nitrogen supply and to determine the direct and indirect effects on grain yield by single and fractional nitrogen supply in soybean/wheat and corn/wheat succession systems. The study was conducted in 2018 and 2019 in a randomized block experimental design with four replications, in a 3 x 3 factorial, for N-fertilizer doses (30, 60, 120 kg ha-1) and forms of supply [single dose (100 %) in phenological stage V3 (third expanded leaf); fractionated dose (70% and 30%) at the V3/V6 phenological stage (third and sixth expanded leaf) and; fractionated dose (70% and 30%) at phenological stage V3/R1 (expanded third leaf and beginning of grain filling)], respectively, in the soybean/wheat and corn/wheat succession systems. The increase in the nitrogen doses promotes productivity due to the greater contribution of grain mass and ear length in the V3 stage and ear grain mass in V3/V6 and V3/R1, in a soybean/wheat system. In the corn/wheat system, the increase in nitrogen promotes productivity, with a greater contribution of grain mass in the ear, regardless of the form of supply. Nitrogen supply in single dose (V3) and fractionated in theV3/V6 gave similar productivity, with a reduction in V3/R1. The grain mass of the ear showed greater contribution of alteration by the form of single and fractioned nitrogen supply, regardless of the dose and succession system. The grain mass of the ear shows a high correlation with yield, regardless of the dose and form of nitrogen supply in the soybean/wheat system, with a positive indirect effect by the ear mass. In the corn/wheat system, ear grain mass shows a high correlation with yield, when nitrogen is supplied in a single dose at 30 and 60 kg ha-1, with a positive indirect effect due to ear length.

Genet. Mol. Res. 21(1): GMR19008
DOI: 10.4238/gmr19008
Research Article

Glutathione S‐transferases are detoxification enzymes that protect cells from oxidative stress and help maintain genomic integrity. GSTM and GSTT family genes may be deleted, causing reduced or no glutathione S-transferase activity so that electrophilic carcinogens cannot be eliminated efficiently. This genetic alteration affects cancer incidence and prognosis. We genotyped GSTM1 and GSTT1 in 87 Brazilian leukemia patients by multiplex PCR, divided into acute or chronic, and lymphocytic or myeloid type cancers. GSTM1-null and GSTT1-null frequency was significantly higher in chronicmyeloid leukemia (67.65% and 46.20%) and chroniclymphoid leukemia (29.41% and 12.30%) in relation to controls, respectively, than in non-leukemia controls. More than that, double null genotyping was significantly more present in acutelymphoid leukemia than controls. When individual GSTM1 and GSTT1 genotyping were analyzed, again CML and CLL presented significantly diferent in genotyping frequency compared to controls (11.11% and 3.8%). Double null genotypes were significantly more frequent in acute lymphoid leukemia than in controls. Furthermore, CML patients presented a statistically significant higher percentage of double-null and GSTM1-null genotypes, and CLL patients had a significantly higher frequency of GSTT1-null genotyping when compared to controls. We suggest that GST genotypes are an important risk factor for leukemia development in the Brazilian population, especially in chronic leukemia due to inefficient detoxification of oxidative stress products.

Genet. Mol. Res. 21(1): GMR18994
DOI: 10.4238/gmr18994
Research Article

Cardiac fibrosis is common and detrimental in numerous heart diseases, affecting millions of patients worldwide. Cardiac fibrosis is characterized by excessive production of extracellular matrix (ECM) constituents such as fibrillar collagens, produced by activated cardiac fibroblasts, i.e.: myofibroblasts. Therapeutic targeting of cardiac fibrosis is highly attractive; however, it remains a major medical challenge. Fibromodulin is a small leucine-rich proteoglycan localized in the ECM. Fibromodulin binds to collagen fibrils, and plays a critical role in collagen fibrillogenesis, ECM organization, wound healing and regulation of the pro-fibrotic cytokine transforming growth factor beta (TGFβ) in several organs. Fibromodulin is highly upregulated in mice and patients with heart failure, but little is known about its role in cardiac fibrosis. Our recent findings from primary cultures of cardiac fibroblasts from neonatal rat hearts suggest that fibromodulin has anti-fibrotic effects. Here we investigated the translational value of these findings by overexpressing fibromodulin in cultured human fetal and adult cardiac fibroblasts. The effects of fibromodulin overexpression on gene expression were measured by qPCR and gene arrays, whereas protein levels were measured by Western blotting, and collagen synthesis by radioactive proline incorporation. The results support our previous findings and indicate relevance for human disease. We found that fibromodulin reduced the expression levels of the collagen cross-linking enzymes lysyl oxidase (LOX) and transglutaminase 2 (TGM2). Fibromodulin also reduced the levels of connective tissue growth factor (CTGF) and periostin (POSTN), indicating reduced TGFβ activity. Reduced levels of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) suggested reduced potential for immune cell adhesion, and gene arrays indicated altered integrin expression, suggesting altered ECM-cell adhesion. Expression of fibrillar collagens was unaffected. In conclusion, fibromodulin reduced TGFβ activity and down-regulated central collagen-crosslinking enzymes, in line with an anti-fibrotic effect of fibromodulin in human cardiac fibroblasts.

Genet. Mol. Res. 21(1): GMR19007
DOI: 10.4238/gmr19007
Research Article

The study of complex traits using large databases of molecular markers has reshaped genetic breeding programs as it allows the direct incorporation of information from a large number of molecular markers for the prediction of genomic values. However, the large number of markers can lead to problems of computational demand, multicollinearity, and dimensionality. We evaluated the use of Multilayer Perceptron Neural networks to resolve this problem and propose a new dimensionality reduction method called Probe Subset Selection  Methodology, for the prediction of genetic values, in Genome Wide Selection studies. We used a simulated F1 population for 12 quantitative traits, including different modeling structures, average degrees of dominance and heritability. The Multilayer Perceptron Neural Networks, together with the proposed Probe Subset Selection Methodology, provided more accurate predictions than the RR-BLUP methodology and reduced the root mean square error from 577.249 to values below 24. The use of computational intelligence in breeding programs is a promising tool for prediction purposes, since epistasis and dominance were not limiting factors for the proposed Multilayer Perceptron Neural Network method.

Genet. Mol. Res. 21(1): GMR18982
Research Article

Orange rust, caused by the fungus Puccinia kuehnii, results in high productivity losses in sugarcane. Selection of resistant genotypes is one of the aims of sugarcane breeding programs. Phenotypic and molecular characterization of the parents of such crosses is essential to obtain superior varieties. We evaluated the reaction to orange rust in the field, the pattern of disease evolution, the molecular marker G1, and the usefulness of this information for the prediction of resistant phenotypes in the main sugarcane parents of the Brazilian Interuniversity Network for Development of the Sugarcane Energy Sector (RIDESA). For this evaluation, 63 sugarcane parents conserved in the RIDESA germplasm bank and that participated most in crosses from 1970 to 2000 were included. The experiment was carried out in a complete block design with three replications, using the clone RB036145 (susceptible to orange rust) as an infective line. Eleven severity assessments were performed using a diagrammatic scale. From the disease severity data, the relative area under the disease progress curve (rAUDPC) was calculated, and the genotypes were classified as resistant, intermediate, and susceptible, compared with the rAUDPC value of the SP79-2233 variety (susceptible to orange rust) and of the variety RB867515 (resistant to orange rust). Of the 63 sugarcane parents, 43 were classified as resistant, of which 27 were positive for the G1 marker; 10 as intermediate, including seven positives for G1; 10 were susceptible, among which four were positive for this marker. The molecular marker G1 showed an accuracy of 71% in predicting the resistant phenotype and could be used as a tool for the characterization of resistant germplasm.

Genet. Mol. Res. 21(1): GMR18980
DOI: 10.4238/gmr18980
Research Article

The yam bean (Pachyrhizus spp.) is an underutilized leguminous tuber that is well adapted to the Amazonian climate. This bean has 5 to 20% protein in its tuberous roots (dry weight), though it is little known even in Brazil. No improved varieties have been recommended for the Amazon region. We examine the Genotype x Year (GxY) interaction in 20 selected yam bean stocks and selected genotypes based on their performance. These materials were obtained from among a selection of 64 genotypes with natural outcrossing of P. erosus x P. tuberosus. The experiments were carried out in the Central Amazon during a low water period, from April to September in 2017 and 2018, in a completely randomized block design with 20 genotypes, three replicates, and eight plants per plot, 1 x 0.5 m apart between and within rows. Pods and roots were harvested after having been cultivated for six months. Pod and root yield, as well as the latter’s proximate composition, were evaluated. We found no significant GxY interaction, for pod and root yield. However, the carbohydrate, protein, lipid, fiber, and ash content in roots showed significant GxY interaction. These results indicate that selection of genotypes with high pod and root yield can be carried out in a single year in the dry period for cultivation during that season. However, selection to increase its nutritional value must be carried out over several years. The selected genotypes for presenting high root yield (> 3 t ha-1) were P7, P11, P15, P19, P37 and P57. The genotypes selected for high protein content in the roots (> 10% dry weight) were P13, P14, P37 and P62.

Genet. Mol. Res. 21(1): GMR18981
DOI: 10.4238/gmr18981
Research Article

Hepatoblastoma (HB) is the most common malignant liver tumor in children; however, the molecular mechanisms responsible for its progression remain unclear. We previously identified hypermethylation of the ovarian cancer immunoreactive antigen domain containing 2 (OCIAD2) as a poor prognostic factor for HB in a genome-wide methylation analysis of HB excision specimens. As it has been already reported that such hypermethylation of OCIAD2 dysregulates the expression of OCIAD2, to elucidate the function of OCIAD2 in HB, we evaluated altered cellular functions such as cell growth, invasion, and migration abilities using OCIAD2 overexpression cell lines of HB. In the in vitro analysis with OCIAD2 overexpression, no significant difference was observed in cell proliferation between the groups. However, migration and invasion abilities were significantly lower in OCIAD2 overexpressed cell lines. Second, overexpression of OCIAD2 reduced matrix metalloproteinase 2 (MMP2) levels in each cell line. These results suggest that OCIAD2 suppresses HB invasion and migration in relation to MMP2.

Genet. Mol. Res. 21(1): GMR19006
DOI: 10.4238/gmr19006
Research Article

Knowledge of the genetic structure of a trait shapes the entire strategy of a breeding program. In this study, the purpose was to determine the additive and non-additive effects that affect the genetic control of common bean roots. A field experiment, with 75 treatments in a partially balanced incomplete block design, was carried out in the 2018/19 growing season. The treatments consisted of backcross progenies (L1 - P1 x F2, L2 - P2 x F2 and L3 - F1 x F2) resulting from a Triple Test Cross mating design, with the Mesoamerican parents P1-BAF50 (accession of the active germplasm bank) and P2-IPR Uirapuru (commercial cultivar). The trait root distribution was assessed based on the soil excavation method, in situ. To this end, trenches were opened under each plant (two plants per replication, in each treatment) and a grid was inserted in the open profile. Pictures were taken of the grid in the trench, based on which the root distribution (percentage) could be quantitatively assessed.  To compare root and shoot biomass, the numbers of pods and grains were counted at harvest. The treatment factor was partitioned into genetic effects (additive, dominant and epistatic) by the establishment of predictive functions. The additive genetic effect was the most influential in the genetic trait control. On the other hand, additive × additive epistasis caused no significant deviation from the genotypic value of plants, neither for roots (P = 0.7941) nor grain yield components. Among the evaluated progenies, effects of dominance deviation and additive x dominant and dominant x dominant epistasis were observed least often, and the expression in the trait root distribution had opposite directions (positive and negative deviations), whereas for the yield components, the non-additive gene effects had one and the same pattern. In crosses between Mesoamerican genotypes, the genetic value of roots is based on the average genetic effects alone, while the genetic interactions are negligible.

Genet. Mol. Res. 21(1): GMR18986
DOI: 10.4238/gmr18986
Research Article

Type 2 diabetes mellitus (T2DM) is a chronic, complex, multifactorial and polygenic disease, constituting one of the greatest public health challenges worldwide. The genetic background has been shown to strongly influence the disease’s susceptibility. We performed genetic screening of risk-variants for T2DM and complications in the Brazilian population. This systematic review is registered in the PROSPERO platform under number CRD42020153032. The searches were conducted in Virtual health library (BVS), EMBASE, Pubmed/NCBI, Scopus, and Web of Science databases, including only case-control studies that related genetic polymorphisms with the risk of developing the disease in the Brazilian population. Among the search results, we also extracted data regarding the susceptibility of developing macro/microvascular complications. Sixteen case-control studies were included, of which 10 addressed T2DM susceptibility and six the disease complications. A total of 4122 individuals were included in the analyses of susceptibility to T2DM, and 1443 in those of susceptibility to complications of the disease. Our findings demonstrate the participation of four main mechanisms in T2DM pathophysiology: glycolytic dysregulation, lipid metabolism, insulin resistance, and inflammatory processes. Among the eight polymorphisms linked to disease susceptibility, we highlight rs7903146 ​​(C/T) in the TCF7L2 gene and rs1800471 (25C/G) in the TGF-β1 gene for diabetic retinopathy. In haplotype analysis, two sets were associated with T2DM susceptibility (ADIPOQ and DIO2 genes), and two sets in the UCP2 gene were associated with diabetic retinopathy and nephropathy. Data from this study characterize the genetic profile of the Brazilian population for susceptibility to T2DM and its complications. This is the first genetic epidemiology study to screen risk variants associated with this disease in Brazil. With this mapping, we aimed to contribute to the development of molecular diagnostic technologies and precision medicine, such as a genetic panel for T2DM susceptibility in the Brazilian population.

Genet. Mol. Res. 21(1): GMR18969
DOI: 10.4238/gmr18969
Research Article

The genus Corydalis (Papaveraceae), which is distributed in temperate regions of the northern hemisphere, has been taxonomically studied mainly on the basis of morphological and molecular genomic information. In the present study, 14 species that belong to the Korean section Corydalis were collected in South Korea and phylogenetically analyzed using four chloroplast genomic regions, which include matK, rbcL, rpL16 genes and the trnG intron. The author tried to include the nuclear Internal Transcribed Spacer (ITS) region in the phylogenetic analysis; however, multiple PCR bands and various band sizes observed led to the conclusion that the ITS region is not suitable for the phylogenetic study of Corydalis. When the four chloroplast genomic regions were separately analyzed, different levels of resolution for species delimitation were observed, and in most cases the resolution levels were quite low. When matK and rpL16 were concatenated, the highest resolution for species delimitation was observed. However, when other regions were added to this concatenated region to improve the resolution, the resolution decreased, which was in contrast to the author’s expectation and deserves further analysis. At the same time, the author observed inconsistencies between the previously established taxonomy based on morphology and the molecular phylogenies in the present study. This discrepancy needs to be addressed in further detail, so that the taxonomy of the genus Corydalis can fully incorporate both morphology and molecular genomic information. Overall, the present study provides insights into the taxonomy of Corydalis, and clearly demonstrates that proper combinations of chloroplast markers can lead to successful discrimination of the species in this genus. Indeed, this study suggests ways to better utilize phylogenetic analyses and species delimitation in this interesting and complicated taxon. Since Corydalis is taxonomically challenging and widely used as medicinal plants in Asia, this study can be a valuable source of information on this genus.

Genet. Mol. Res. 21(1): GMR18996
DOI: 10.4238/gmr18996
Research Article

Chikungunya virus (CHIKV) is an RNA virus from the family Togaviridae transmitted primarily by Aedes mosquitoes. The first report of CHIKV infection in Brazil dates to 2014; since then, the virus has become a major public health challenge. The main goal of this study was to perform a phylogenetic analysis of CHIKV isolates from febrile patients from Espirito Santo (ES) state during the 2017 outbreak to identify the genetic diversity of circulating CHIKV strains. CHIKV RT-qPCR confirmed cases were sequenced for phylogenetic relationship inferences and molecular characterization. Phylogenetic results showed that the virus in the 2017 ES outbreak belongs to the ECSA genotype. Molecular characterization revealed a new mutation in ES strains (E2-N207D). This variation generated an amino acid substitution by exchanging asparagine (N) for aspartate (D) at position 207 and it is associated with an anchoring groove, possibly interfering in viral envelope assembly and associated interaction with the target cell. Here we report a CHIKV-ECSA IIa outbreak that demonstrates the ES population's vulnerability to an Asian strain of this virus circulating elsewhere in Brazil. Despite the small sample size, this study describes phylogenetic data about CHIKV in ES state that helps expand of the virus genotype database, and reveals a new E2 protein CHIKV variant (E2-N207D). This data helps improve understanding of chikungunya fever in order to design efficient public health control strategies.

Genet. Mol. Res. 21(1): GMR18984
DOI: 10.4238/gmr18984