Table of contents: 2020
Sex differentiation in males is a highly complex phenomenon based on synergistic interactions between genes and their coded proteins. The Sox9 gene plays a critical role in embryonic development, cell lineages, and stem cell maintenance during testicular development. Sox9 gene expression is associated with sex reversal, with a 46, XX karyotype in males and regulating normal functioning of Sertoli cells during spermatogenesis. Clinically diagnosed infertile males (n = 40) with non-obstructive azoospermia (NOA) with respective controls (n = 38) were included in the study. Blood samples were collected and after isolation of genomic DNA, the expression of the Sox9 gene (DNA copy number variations) was measured by fluorescence based quantitative PCR, deletion of AZFc locus determined by STS markers, karyotype analysis and SRY using GTG banding and FISH techniques, respectively. Cytogenetics findings revealed that more that 40% of cases had mosaicism with 46, XY/47, XYY karyotypes and a high frequency of deletion of AZFc (>70%) regions as compared to AZFb (>20%) using STS markers in the cases of NOA. The mean relative fluorescence values of Sox9 gene expression differed significantly between cases (0.32) and controls (0.87) (P < 0.001) with a confidence interval at 95% of 0.0310 - 0.787 in the cases of NOA. SRY + ve cases had higher Rfv of Sox9 gene expression when compared to SRY- ve cases. There was a positive correlation between stem cell Sox9 gene expression, mosaicism and AZFc region affecting a non-disjunction event during spermatogenesis and fertility.
Surgery, chemotherapy and radiotherapy, which are known as conventional cancer treatments, have been applied for more than 40 years. Because the target of chemotherapy is tumor cells, treatment agents are used at high doses during application and this creates toxic responses, causing side effects in normal cells. Furthermore, due to the high doses, cancer cells develop resistance to the agent and, as a result, success of the treatment gradually decreases. That is why chemotherapy is combined with various other cancer treatments to increase the therapeutic efficacy while reducing the side effects. In this study, the cytotoxic effect of endostatin, vinorelbine and their combination on breast cancer cell line MDAMB 231 was researched in vitro. In addition, the effects caused by the combination on TRAIL, TNF-α and caspase-2, 3, 6, 8 and -9 proteins, which undertake key roles in apoptosis were investigated. Cytotoxic effects of both agents and their combinations on normal epithelial cells (293 T) were also studied and compared. We found that endostatin is cytotoxic for cancer cells through caspase-8 and TNF-α activation, which affects the extrinsic pathway of apoptosis Combining endostatin as an anti-angiogenic agent with vinorelbine significantly suppressed the cytotoxicity that vinorelbine exerts on normal cells.
Genetic variants of SLC2A9 have been reported to be associated with deficiencies in the excretion of urate and with urate transporter gene 9 (GLUT9) activity. However, the evidence remains inconclusive and no information is available about SLC2A9 genetic variants (rs2280205 and rs6820230) association with hyperuricemia (HUA) in the Thai population. We decided to determine whether rs2280205 and rs6820230 alleles of the SLC2A9 gene are associated with HUA in a cross-sectional study that included 193 subjects in urban Bangkok, Thailand. These single-nucleotide polymorphisms were analyzed using unconditional logistic regression. The prevalence of HUA was 31.1%. Males (HUA defined as serum urate levels >7.0 mg/dL) had a higher prevalence of HUA than females (>6.0 mg/dL) (42.7 versus 24.8%). By SLC2A9 SNP association analysis, the rs2280205 and rs6820230 variants were not significantly associated with HUA (adjusted odds ratio (aOR) = 5.59, 95% CI = 0.50-62.95; aOR = 0.77, 95% CI = 0.11-5.45), respectively). The prevalence of HUA was much higher in this urban Thai population than in similar studies in rural areas of the country and also compared to the mean frequency in developed countries such as the USA (usually 10% or less). The haplotypes rs6820230 and rs2280205 did not influence the risk for HUA in these Bangkok subjects. We concluded that the prevalence of HUA is extremely high in urban Bangkok and that these SNPs are not associated with HUA in this population.
Human papillomavirus (HPV) type 13 is associated with focal epithelial hyperplasia, a benign and self-limited disease of oral mucosa. It is a rare finding, most often seen among young American natives. We carried out a molecular characterization case study of HPV type 13 isolated from a 14-year-old native girl living in Amazonia, in the northern region of Brazil, by describing the E6 and E7 open reading frame (ORF) sequences and comparing them to the only reference sequence in GenBank, which came from a Turkish isolate. The E6 and E7 ORFs were amplified by PCR, and the amplicons were purified and cloned in E. coli using the pCR®-4 TOPO® vector. The purified recombinant plasmids were automatically sequenced, showing 98 and 94% similarity to the E6 and E7 ORFs from the reference sequence, respectively. Six mutations were identified in the E6 ORF, but these changed only three amino acids in the final protein. In the E7 ORF, we found three transversions and one deletion of nine base pairs, which led to three amino acid mutations and three amino acid deletions in the final protein. The mutations in E6 ORF gave no predicted changes in the final protein, while the mutations in E7 were predicted to cause changes in the protein. We conclude that the E6 and E7 ORFs in HPV-13 from the Amazonia and Turkey isolates have a high degree of genetic similarity.
Nanobodies (Nbs) are mini antibodies 10 times smaller than conventional antibodies. They constitute a new class of antibodies devoid of light chains. Their paratope consists of a single variable domain "VHH" (heavy chain variable domain) formed by a single peptide chain. Nevertheless, Nbs maintain the same repertoire of diversity and affinity characteristics as conventional antibodies. Because of their high specificity, small size, and low production cost, Nbs are emerging progressively as a potential alternative to overcome the limitation of conventional antibodies in targeted therapy. Indeed, the therapeutic spectrum of Nbs has been confirmed in animal models and human clinical trials. The present review provides a summary of the technological advances in the production of Nbs and their potential targeted therapeutic biomarkers over the past 10 years.
Pigeon droppings carry microorganisms such as Candida spp., which have high genetic variability and are the most common opportunistic pathogens in humans. There is a need to establish fast, reliable and simple laboratory methods to differentiate pathogenic from non-pathogenic yeast species in pigeon droppings, as they can be a source of contamination for humans. We examined yeasts isolated from domestic pigeon droppings through a proteomic approach using matrix-assisted laser desorption ionization (MALDI-TOF). We also examined genetic characteristics of the isolates by restriction fragment length polymorphism (RFLP) PCR and enterobacterial repetitive intergenic consensus sequences PCR methods. Five colonies isolated from domestic pigeon droppings collected at three different sampling locations on the roof of a Brazilian university building were identified as Candida krusei and Candida rugosa by MALDI-TOF. Phylogenetic studies using the two PCR-based molecular methods were able to differentiate the species C. krusei and C. rugosa into different groups. These genomic methods gave similar results and proved useful for genetic diversity analysis and interspecific differentiation of Candida isolates.
Grapes are an important fruit crop in Kazakhstan. Modern viticulture requires inclusion of traditional morphological and agronomical methods in the development and sustaining of grapevine culture as well as molecular approaches. We used molecular markers and leaf shape analysis to characterize grapevine cultivars developed in Kazakhstan compared with European and Asian varieties. A set of 17 landmarks describing grapevine leaf shape were analyzed using generalized procruste and principal component analyses in order to evaluate shape variation among cultivars. Mean leaf shapes were obtained for 94 cultivars. Outer tip pattern had the most impact on leaf shape variation. Principal component analysis revealed the complex nature of grapevine leaf shape variation and demonstrated the differential impact of leaf landmarks on shape variation. The standard set of six simple sequence repeat markers used for molecular genotyping was sufficient to differentiate each of the cultivars by a distinct allelic profile. Kazakh grapevine cultivars were found to be an admixture of European and Asian varieties. However, for some cultivars, there was no confirmation of identity with respect to pedigree either by bibliography or comparison with the Vitis International Variety Catalogue. Also, 11 markers for resistance-associated alleles in corresponding loci (RUN1, REN1, REN3, Rpv3, Rpv10, and Rpv12) were applied to characterize Kazakhstan and some parent cultivars with different levels of resistance to mildew pathogens.
Plant breeders need to understand the genetic diversity and relationships between genotypes to choose parents of segregating populations. We evaluated the genetic diversity of 35 soybean genotypes and determined their potential as parent lines in breeding programs by analyzing their agronomic traits and microsatellite markers. Phenotypic analysis was carried out in the field at Fazenda Capim Branco, a research station of the Federal University of Uberlândia. Nine microsatellite markers and seven agronomic traits in were evaluated in 35 conventional soybean genotypes. Genotypes were grouped by UPGMA and Tocher cluster analyses, and molecular analysis was used to calculate polymorphism for each microsatellite locus. Nine microsatellite markers amplified 26 alleles, ranging from two to four. Polymorphism varied between 0.29 and 0.66, and averaged 0.44. The number of days to maturity was the characteristic that contributed the most to yield (29.44%). Seven potential parents (G11, G12, G16, G21, G22, G26 and G33) had average grain yields higher than 5000 kg ha-1. UPGMA and Tocher methods identified groups from the matrix of phenotypic and molecular data. Hybrids of G11, G12, G16, G22, G26 and G33 genotypes segregated promising populations with superior genetic variability.
Signal transducers and activators of transcription (STAT5B), dopamine receptors D2 (DRD2), bone morphogenetic protein (BMP15), and melatonin receptors type C (MTNR1C) play an important role in growth and reproductive traits in chickens. We studied the 9th generation of 93 Pradu Hang Dam and 95 Chee chickens examining PCR-restriction fragment length polymorphisms to genotype for growth and reproductive traits. There was no significant association between other traits, but we confirmed that in Pradu Hang Dam, DRD2 was associated with egg weight at the age at first egg while BMP15 was associated with egg weight at 300 days and body weight at 300 days. Moreover, MTNR1C was associated with egg weight at 300 days and egg number at 300 days. In Chee, MTNR1C was associated with egg number at 300 days. We suggest that DRD2, BMP15, and MTNR1C be used to select for early maturity and growth, while MTNR1C be used for egg number in Pradu Hang Dam. In Chee, MTNR1C can be a marker to improve egg production. For improving the egg production in Thai native chickens, we recommend that MTNR1C be used in marker-assisted selection.
In this study, a modified CTAB method combined with sodium sulphite and silica particles for the extraction of high-quality genomic DNA of recalcitrant plants such as malva, coffee, avocado, mango, olive, jatropha and tillandsia is reported. Maceration with liquid nitrogen, phenol treatment, RNase digestion and the ethanol precipitation step are eliminated using this methodology. The quality of the isolated DNA permits downstream molecular applications including AFLP, restriction, ligation and PCR amplification and sequencing of plant barcode genes (matK and rbcL). The cost per sample (about US$ 0.14) is quite low compared to any commercially available kit. This methodology is reproducible and could be used for a broad spectrum of woody plants with a high content of polyphenols and polysaccharides compounds, without the need for a commercial kit or expensive reagents, providing a viable alternative for laboratories with low budgets.
Various approaches use gene trees to infer species trees produced from incomplete lineage sorting. Generally, one of these approaches is used to deduce the rooted species tree from a rooted gene tree, or another method can be used to determine the unrooted species tree from an unrooted gene tree. Typically, this unrooted species is then rooted through at least one outgroup. However, in theory, the unrooted gene tree can be used consistently and directly to infer the rooted species tree without using an outgroup. We used an unrooted gene tree with the assumption of a multispecies coalescent model to infer the rooted species tree by using the approximate Bayesian computation (ABC). In certain cases, this could be useful, especially when it is hard to locate a fitting outgroup neglected by gene trees. To address the challenges of increasing the taxa number, an ABC was used to gauge the rooted species tree of a large number of taxa, using an unrooted gene tree to develop the rooted species tree. This is the first ABC application that can handle very large numbers of taxa. Based on the results, the Robinson-Foulds (RF) distance is generally equal to 2 when the species tree is in imbalance. When the shape of the species tree is balanced, the RF distance is normally equal to 0. Out of all shapes of species trees, the most recent one is the most appropriate for every clade.
Genome-Wide Selection (GWS) uses molecular markers to predict the genetic merit of animals and plants. Usually, a high density of molecular markers to predict this genetic merit is used. Thus, statistical methods need to deal with problems of high dimensionality, multicollinearity and computational efficiency. We examined a set of machine learning methods, in particular the tree-based regression methods (Regression Tree, Bagging, Random Forest and Boosting) and evaluated them in relation to predictive ability and bias. Moreover, these methods were compared with the Bayesian Least Absolute Shrinkage and Selection Operator (BLASSO) method, which is routinely used in GWS. For this, we used information of 10 carcass traits in Piau x Commercial pigs. The tree-based regression methods were superior to the BLASSO method, presenting shorter computational times to predict the genetic values of the analyses, especially, the Random Forest and Bagging methods. Furthermore, the predictive abilities of tree-based regression methods were competitive with BLASSO. In terms of bias, the BLASSO underestimated the predictions while tree-based regression methods overestimated the predictions. In addition, among the methods, the Random Forest was the one that obtained the bias value closest to ideal for most of the traits, demonstrating the superiority of this method.
The main polymorphisms linked to dyslipidemia include those of the SMARCA4 gene. We evaluated the association between lipidic profile and SMARCA4 gene polymorphism in 200 military police officers. Real time PCR was used to identify SMARCA4 gene polymorphisms. Among the subjects, 116 had dyslipidemia (case group), of which 94% were males, and 84 presented no dyslipidemia (control group), of which 92% were males. For the SMARCA4 gene polymorphism, 66.4% (77/116) presented the GG genotype, 31.0% (36/116), GT and 2.6% (3/116) TT. Individuals with GG, GT and TT genotypes had LDL cholesterol levels higher than 160 mg/dL, respectively, at frequencies of 28.6, 11.1 and 0%, while total cholesterol higher than 190 mg/dL, was at frequencies of 74.0, 58.3 and 33.3%. Logistic regression analysis to determine the p-values, considering the T allele as dominant, suggested that this allele has the ability to protect the individual from high cholesterol levels (OR, 0.488; 95% CI 0.27 - 0.88; P = 0.0163) and high LDL cholesterol levels (OR 0.277; 95% CI 0.09 - 0.84; P = 0.0230). Absence of the T allele was associated with increased susceptibility to dyslipidemia. Individuals who are homozygous or heterozygous for the T allele are approximately two times more likely to have normal cholesterol levels, and about 3.5 times more likely to have normal LDL levels.
The amount of absorbed phosphorus directly impacts on the growth and grain yield of common bean plants. We evaluated dry matter production at different growth stages and grain yield of common bean genotypes in response to phosphorus availability in a nutrient solution and examined possible associations between these characters. The experiment was carried out in a greenhouse, using a completely randomized design in sub-sub-divided plots. The main plots consisted of five phosphorus concentrations (0.5; 0.9; 1.3; 1.9 and 2.3 mmol.L-1) supplied to plants in a nutrient solution in the growth medium. The sub-plots were composed of two common bean genotypes (Pérola and IPR88 Uirapurú, which are commonly used in Rio Grande do Sul state) and the sub-sub-plots by two growing seasons (fall-winter and spring-summer). In the initial stages, at the first trifoliate leaf and flowering stages, the highest dry mass production occurred in the leaves. As the plants developed, they produced more dry matter in the pods during the pod filling stage, and later in grains, at the maturation stage. Phosphorus concentrations in the nutrient solution between 1.33 and 1.84 mmol.L-1 provided the greatest mass of beans at podfilling and at maturation, the largest number of grains and the greatest grain yield in the two genotypes. The characters dry mass of the leaves, stems and pods in pod filling, dry mass of grains at maturity, number of grains and number of pods are promising for indirect selection criteria.
Recently, carbon nanotubes (CNTs) are gaining attention in the field of agriculture as advanced approaches to minimize toxicity of mycotoxins for crop plants. We examined whether MWCNTs can be used to alleviate genotoxicity and DNA damage induced by ochratoxin A (OTA) in the common bean (Phaseolus vulgaris) by comparing the action of three OTA doses, prior and post-adsorption of OTA on the surface of MWCNTs. The phenotypic parameters, ultrastructure of chloroplasts and nuclei using transmission electron microscopy, and status of nuclear DNA (nDNA) using flow cytometry, comet assay and random amplified polymorphic DNA (RAPD) were used as bioassays. Exposure time was 48 hours. The most effective MWCNT dose (optimal) was 50µg/mL; it enhanced the phenotypic parameters (seed germination and seedling growth, tolerance, and vigor indices), induced unexpected modification of size, shape, external and internal ultrastructure of chloroplasts and nuclei, increased the content of nDNA and genome size, reduced the extent of nDNA damage, and produced a larger number of amplified DNA products and new DNA bands more than the control. Lower and higher MWCNT doses had reductions in these parameters. On the other hand, increases in doses in OTA treatments induced major toxicity in the common bean, leading to strong reductions in all parameters of the bioassays. The MWCNTs served as an adsorbent for OTA and led to alleviation of its toxicity. We conclude that optimal and adsorbent MWCNTs dose could be used as nanocarbon-fertilizer and nanocarbon-mycotoxin to protect crop plants in order to increase crop quality and productivity.