Table of Contents | Genet. Mol. Res. 2017 (2)
In this study, we investigated the effects of pingyangmycin (PYM) on the growth inhibition and apoptosis of human umbilical vein endothelial cells (HUVEC). In this study, we aimed to explore the optimal concentration of PYM to induce the apoptosis of HUVEC and to determine its mechanism of action. After treatment of HUVEC with different concentrations of PYM for 24 h, cell counting kit-8 (CCK-8) was used to detect growth inhibiting effects. Annexin V-FITC/propidium iodide stain was used to detect apoptosis, and western blot was used to detect the expression of glucose-related protein 78 (GPR78) and C/EBP homologous protein (CHOP) endoplasmic reticulum stress proteins. With increasing PYM concentration, the growth inhibition of HUVEC increased (P < 0.05), the apoptotic numbers of HUVEC increased (P < 0.05), with higher PYM concentrations inducing necrosis, and the protein expression of GRP78 and CHOP increased (P < 0.05). PYM could obviously inhibit the proliferation and promote the apoptosis of HUVEC. Necrotic cells were more prevalent than apoptotic cells at high PYM concentrations. This study helped to determine the proper concentration of PYM to induce more apoptosis than necrosis, which is critical to minimize inflammation, enhance the healing of the skin, and maintain safety for the patient. PYM might induce HUVEC apoptosis through the endoplasmic reticulum stress pathway.
Rice is a cereal that presents a great ability to adapt to different soil and climate conditions. However, as it is a tropical crop with C3 metabolism, it performs better in warm temperatures with high solar radiation. Tolerance to stress caused by low temperatures is a highly complex process that involves various metabolic pathways and cellular compartments, resulting in general or specific effects on plant growth and development. In order to observe the true effect of a particular stress on genetic expression, reference genes need to be chosen for real-time PCRs, the expression levels of which should remain stable independent of the situation imposed. In this paper, the expression stability was evaluated of the actin 11 (ACT11), ubiquitin-conjugating enzyme 2 (UBC-E2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta tubilin (β-Tubulin), eukaryotic initiation factor 4α (eIF-4-α), eukaryotic initiation factor 1α (eIF-1-α), ubiquitin 10 (UBQ10), ubiquitin 5 (UBQ5), aquaporin (TIP41), and cyclophilin genes, in two rice genotypes cultivated in low temperature (13°C) conditions in vegetative stage (V4). The analysis material (leaves) was collected after 0, 6, 24, 48, and 72 h of exposure to the stress. In this study, the geNorm, BestKeeper, ΔCt, NormFinder, and RefFinder methods were used to evaluate the expression stability of the candidate reference genes. The results revealed that the most indicated genes for all the analysis methods were UBQ10 and UBQ5 for BRS Bojuru and BRS Pampa, respectively. On the other hand, the eIF-1-α gene presents the least expression stability and is not indicated for studies of rice plants subjected to low temperatures. The validation with the antioxidant system genes SODCc1-Cu/Zn, CATC, APX2, and GR2 confirmed the importance of using previously tested normalizing genes for adequate real-time PCR results.
Classification using a scale of visual notes is a strategy used to select erect bean plants in order to improve bean plant architectures. Use of morphological traits associated with the phenotypic expression of bean architecture in classification procedures may enhance selection. The objective of this study was to evaluate the potential of artificial neural networks (ANNs) as auxiliary tools in the improvement of bean plant architecture. Data from 19 lines were evaluated for 22 traits, in 2007 and 2009 winter crops. Hypocotyl diameter and plant height were selected for analysis through ANNs. For classification purposes, these lines were separated into two groups, determined by the plant architecture notes. The predictive ability of ANNs was evaluated according to two scenarios to predict the plant architecture - training with 2007 data and validating in 2009 data (scenario 1), and vice versa (scenario 2). For this, ANNs were trained and validated using data from replicates of the evaluated lines for hypocotyl diameter individually, or together with the mean height of plants in the plot. In each scenario, the use of data from replicates or line means was evaluated for prediction through previously trained and validated ANNs. In both scenarios, ANNs based on hypocotyl diameter and mean height of plants were superior, since the error rates obtained were lower than those obtained using hypocotyl diameter only. Lower apparent error rates were verified in both scenarios for prediction when data on the means of the evaluated traits were submitted to better trained and validated ANNs.
Since there are no studies on the reversal of multidrug resistance by curcumin in the human colorectal cancer cell line HCT-8/5-FU, our aim was to search for highly efficient reversal agents and investigate the underlying mechanisms of this reversal. The cytotoxic effects of curcumin and 5-FU on HCT-8 and HCT-8/5-FU cells and the reversal effects of 5-FU in combination with curcumin on HCT-8/5-FU cells were measured using cell counting kit-8. Apoptosis and the cell cycle were analyzed by flow cytometry. Protein and mRNA expression levels of BCL-2, survivin, P-gp, and HSP-27 were detected by western blotting and quantitative real-time reverse transcription polymerase chain reaction, respectively. Curcumin inhibited the growth of HCT-8 and HCT-8/5-FU cells. It significantly reduced the IC of 5-FU for HCT-8/5-FU cells (P < 0.01) and the expression of BCL-2, survivin, P-gp, and HSP-27 in the cells. Curcumin can effectively reverse multidrug resistance in human colorectal cancer drug-resistant HCT-8/5-FU cells. The mechanism through which this occurs may be associated with decreased expression of BCL-2, survivin, P-gp, and HSP-27. Curcumin may therefore have clinical implications as a new agent for colorectal cancer.
The genetic diversity of epiphytic yeasts from grape carposphere is susceptible to environmental variations that determine the predominant carposphere microbiota. Understanding the diversity of yeasts that inhabit grape carposphere in different environments and their pectinolytic activity is a way to understand the biotechnological potential that surrounds us and help improve winemaking. Therefore, this study aimed to evaluate the pectinolytic activity and characterize the genetic diversity of isolated epiphytic yeasts from grape carposphere. Grapes of the Bordeaux cultivar were collected from different regions of Paraná and Rio Grande do Sul States, in Brazil, and the yeasts were isolated from these grape carpospheres. Monosporic isolates were morphologically and genetically characterized on potato dextrose agar medium and by PCR-RFLP and rep-PCR (BOX-PCR) in the ITS1-5.8S-ITS2 region of rDNA. The index of pectinolytic activity of isolates was also evaluated estimating the ratio between the halo diameter of enzymatic degradation and the diameter of the colony when the isolates were grown in cultivation medium containing 10 g/L pectin, 5 g/L yeast extract, 15 g/L agar, 0.12% (w/v) Congo red, and pH 6.2. We observed that the grape carposphere is an environment with a great genetic diversity of epiphytic yeasts of the following genera: Cryptococcus (31.25%), Pichia (25.0%), Candida (25.0%), Dekkera (12.5%), and Saccharomyces (6.25%). The PCR-RFLP technique allowed analyzing existing polymorphism among individuals of a population based on a more restrict and evolutionarily preserved region, mostly utilized to differentiate isolates at the genus level. Approximately 33% of yeast isolates presented pectinolytic activity with potential biotechnological for wine and fruit juice production. This great genetic variability found indicated that it is a potential reservoir of genes to be applied in viniculture improvement programs.
The use of Y chromosome haplotypes, important for the detection of sexual crimes in forensics, has gained prominence with the use of databases that incorporate these genetic profiles in their system. Here, we optimized and validated an amplification protocol for Y chromosome profile retrieval in reference samples using lesser materials than those in commercial kits. FTA cards (Flinders Technology Associates) were used to support the oral cells of male individuals, which were amplified directly using the SwabSolution reagent (Promega). First, we optimized and validated the process to define the volume and cycling conditions. Three reference samples and nineteen 1.2 mm-diameter perforated discs were used per sample. Amplification of one or two discs (samples) with the PowerPlex Y23 kit (Promega) was performed using 25, 26, and 27 thermal cycles. Twenty percent, 32%, and 100% reagent volumes, one disc, and 26 cycles were used for the control per sample. Thereafter, all samples (N = 270) were amplified using 27 cycles, one disc, and 32% reagents (optimized conditions). Data was analyzed using a study of equilibrium values between fluorophore colors. In the samples analyzed with 20% volume, an imbalance was observed in peak heights, both inside and in-between each dye. In samples amplified with 32% reagents, the values obtained for the intra-color and inter-color standard balance calculations for verification of the quality of the analyzed peaks were similar to those of samples amplified with 100% of the recommended volume. The quality of the profiles obtained with 32% reagents was suitable for insertion into databases.
Breast cancer adversely affects the health status of women; therefore, the prevention and treatment of breast cancer is of critical importance. Lycopene is known to possess several biological effects such as removal of free radicals, alleviation of biological oxidative injury, and inhibition of tumor growth. In this study, we aimed to illustrate the effect of lycopene on tumor cell proliferation and modulation of cancer progression as well as its possible underlying mechanisms in human breast carcinoma cell line MCF-7 in vitro. MCF-7 cells were treated with different lycopene concentrations for 24, 48, and 72 h. Light field microscopy was used to observe cell morphology. MTT assay was used to determine the effect of lycopene on MCF-7 proliferation. Flow cytometry was employed to evaluate cell apoptosis. Real-time quantitative polymerase chain reaction was performed to detect the expression of p53 and Bax. Under microscopic examination, the untreated MCF-7 cells appeared to have a diamond or polygonal shape. Lycopene treatment resulted in cell shrinkage and breakage, whose severity increased in a dose and duration dependent manner. In addition, reduced cell proliferation and increased apoptosis (P < 0.05) were observed using MTT assay and flow cytometry, respectively. Moreover, lycopene could also upregulate the expression of p53 and Bax mRNAs in MCF-7 cells. In conclusion, lycopene inhibits proliferation and facilitates apoptosis of MCF-7 cells in vitro, possibly by regulating the expression of p53 and Bax.
Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by a dynamic mutation due to the expansion of CAG repeats in the HTT gene (4p16.3). The considered normal alleles have less than 27 CAG repeats. Intermediate alleles (IAs) show 27 to 35 CAG repeats and expanded alleles have more than 35 repeats. The IAs apparently have shown a normal phenotype. However, there are some reported associations between individuals that bear an IA and clinical HD signs, such as behavioral disturbs. The association of IAs with the presence of clinical signs gives clinical relevance to these patients. We emphasized the importance of determining the frequency of IA alleles in the general population as well as in HD families. Therefore, the aim of this study was to conduct a systematic review, in order to investigate the frequency of IAs in the overall chromosomes of different ethnic groups and of families with HD history worldwide as well as the frequency of individuals who bear the intermediate alleles. We searched indexed articles from the following electronic databases: U.S. National Library of Medicine and the National Institutes of Health (PubMed), Pubmed Central (PMC) and Virtual Health Library (VHL). Therefore, 488 articles were obtained and, of these, 33 had been published in more than one database. We accepted the article of only one database and ended up with 455 articles for this review. The frequency of IAs within the chromosomes of the general population ranged from 0.45 to 8.7% and of individuals with family history of HD ranged from 0.05 to 5.1%. The higher frequency of IAs in the general population (8.7%) was found in one Brazilian cohort.
The Staphylococcus aureus is the most common isolated microorganism in ruminant animal species diagnostic with clinical or subclinical mastitis. Dairy herds with these diseases can transfer S. aureus into the milk supply, which can lead to food poisoning in humans. The objective of this study was to evaluate the profile of antimicrobial susceptibility, the presence of femA gene, the genetic relationships among isolates of S. aureus obtained from milk originating from flocks diagnosed with subclinical mastitis in nine rural properties in the northern of Minas Gerais State. To this end, 498 samples of bovine milk tested positive for the California mastitis test (CMT) were subjected to morphological methods and biochemical patterns for microbiological presumptive identification of S. aureus. The PCR test with the genetic marker femA was used to confirm the species S. aureus. All the 26 isolates presumptively identified as S. aureus amplified a fragment of 132 bp corresponding to the femA gene. The profile of antimicrobial susceptibility was performed according to the disk-diffusion methodology and two isolates were susceptible to all the antibiotics tested. The drug multiresistence was found in 80.76% of the isolates. The determination of the genetic profile and the clonal relationship among the isolates was performed by the method of DNA RAPD-PCR polymorphism. The S. aureus isolates were divided into two groups with 26 distinct subgroups. The analysis of RAPD-PCR showed no genetic diversity among them, heterogeneous profile and absence of clonality.
The licuri palm Syagrus coronata plays a key role in the ecology and economy of Brazilian semiarid region. Nonetheless, genetic data about populations of this species are absent even though the intensive and uncontrolled exploitation since colonial periods has threatened the sustainability and viability of licuri populations. Therefore, we attempted to test the efficacy of transferability of microsatellite loci isolated from three palm tree species to S. coronata to analyze the population of this species throughout their range. A set of 19 heterologous microsatellite loci was tested in three native populations of S. coronata from the State of Bahia, northeastern Brazil, which amplified using distinct annealing temperatures (50°-60°C). Based on the 10 most polymorphic loci, the selected populations exhibited a mean number of alleles per locus of 9.8, and high genetic diversity values since the expected heterozygosity ranged from 0.573 to 0.754, while the observed heterozygosity varied from 0.785 to 1.000. In conclusion, the tested loci are transferrable and highly efficient to population studies in S. coronata, thus minimizing the lack of species-specific loci to the genetic monitoring of licuri populations.
Sickle cell disease shows several clinical manifestations in distinct levels of severity. This heterogeneity is due to the haplotype variability associated with the HbS gene, levels of fetal hemoglobin and environmental conditions, which modify the disease expression. Science community believes that the presence of a polymorphism in the CCR5 gene, which is related to chronic inflammatory state, could confer a higher survival rate and a lower number of inflammatory events to these patients since the deletion in CCR5Δ32 would knock out the CCR5 gene. Therefore, this study aimed to identify the haplotypes in β and β genes, as well as to investigate the presence of the CCR5Δ32 deletion in patients with sickle cell disease. For this purpose, DNA was isolated with the QIAamp DNA Investigator Kit, and PCR was the method chosen to detect the mutant allele CCR5Δ32. The haplotypes in β and β genes were detected by RFLP with the restriction enzymes XmnI, HindIII, HincII, and HinfI analyzing six polymorphic sites on the β cluster, succeeded by electrophoresis. The atypical haplotype was the most common (54.3%), followed by Benin (28.6%), Bantu (11.5%), Senegal (2.8%), and Cameroon (2.8%). No patients presented CCR5Δ32 deletion. The increase in the frequency of atypical haplotypes suggests that these patients passed by variation in the genetic pattern from ancestral haplotypes throughout the years.
Cytosine DNA methylation is a significant form of DNA modification closely associated with gene expression in eukaryotes, fungi, animals, and plants. Although the reference genomes of cotton (Gossypium hirsutum L.) have been publically available, the salinity-stress-induced DNA methylome alterations in cotton are not well understood. Here, we constructed a map of genome-wide DNA methylation characteristics of cotton leaves under salt stress using the methylated DNA immunoprecipitation sequencing method. The results showed that the methylation reads on chromosome 9 were most comparable with those on the other chromosomes, but the greatest changes occurred on chromosome 8 under salt stress. The DNA methylation pattern analysis indicated that a relatively higher methylation density was found in the upstream2k and downstream2k elements of the CDS region and CG-islands. Almost 94% of the reads belonged to LTR-gspsy and LTR-copia, and the number of methylation reads in LTR-gypsy was four times greater than that in LTR-copia in both control and stressed samples. The analysis of differentially methylated regions (DMRs) showed that the gene elements upstream2k, intron, and downstream2k were hypomethylated, but the CDS regions were hypermethylated. The GO (Gene Ontology) analysis suggested that the methylated genes were most enriched in cellular processes, metabolic processes, cell parts and catalytic activities, which might be closely correlated with response to NaCl stress. In this study, we completed a genomic DNA methylation profile and conducted a DMR analysis under salt stress, which provided valuable information for the better understanding of epigenetics in response to salt stress in cotton.
In several crops, the water deficit is perhaps the main limiting factor in the search for high yields. The objective of this study was to evaluate the phenotypic stability of maize hybrids in environments with and without water restriction using the analytical factor (AF) approach. We evaluated 171 maize hybrids in 14 environments, divided into environments with (A1, A2, A3, A4, A5, A6, and A7) and without (A8, A9, A10, A11, A12, A13, and A14) water restriction, over a period of 7 years. Each year, 36 hybrids were evaluated. A square lattice design (6 x 6) was used, with common treatments between years. The characteristics of grain yield (GY), male flowering (MF) and female flowering (FF), plant height (PH), and ear height (EH) were evaluated. Phenotypic adaptability and stability of the hybrids were also verified. Hybrids G66, G99, G86, and G26 were the most stable and showed potential for use in environments with and without water restriction. The AF models showed to be useful for evaluating hybrids over many years, allowing selection of better hybrids with adaptability, specific and general stability, and correlation of hybrids with their production components, in addition to allowing identification of mega-environments that permit stability in the response of the adapted hybrids.
The recommendation of sugarcane clones depends on several factors, as the response or performance of the clones over different cuts or harvests. The clone by harvest interaction might be difficult to identify superior clones in the final stages of the sugarcane breeding program. Thus, the objective of this study was to investigate and describe the implications of the genotype by harvest interaction in the adaptability and stability of genotypes and delineation of mega-environments from a set of multi-environment trials. Fifteen clones and four checks were evaluated in eight environments. The trait TPH (tons of pol per hectare) was evaluated in two harvests (plant cane and ratoon cane) in 2010 and 2011. The joint analysis showed significance for harvest (H), environment (E), and genotype (G) effects. The interactions GxE, ExH, GxH, and ExGxH were also significant. The last three-way interaction indicated the differential response of the genotypes over environments, and that it depends on the harvests. The overall mean of the trials was 12.77 TPH. The coefficient of variation was 8.70% and the selective accuracy was 98.63%, indicating high experimental precision. The genotypes G4, G14, and G16 were statistically superior to the check varieties used; however, these genotypes did not show high stability as described by the additive main effects and multiplicative interaction method. There was a specific adaptation between the E7 and E5 environments and the G4 and G5 genotypes, respectively. In general, the grouping of the environments was inconsistent throughout the harvests, except for the E1 and E4 environments, which exhibited similarities for the different genotypes.