Table of contents: 2023
Next-generation sequencing (NGS) platforms are now implemented as routine analysis for K-RAS mutation in colon cancer patients before therapy. The DNA used in NGS platforms is extracted from colon cancer formalin-fixed paraffin-embedded (FFPE) blocks. In this study, we utilized 20 FFPE colon cancer blocks. In general, a good quality DNA sample includes compact high molecular weight DNA. The quality of the extracted DNA is checked by agarose gel electrophoresis. Some samples are found to be highly degraded due to natural mechanisms like autolysis and spontaneous depurination, or bacterial contamination and extracted DNA is then routinely fragmented by sonication. In this study, PCR was performed to reconstruct larger DNA fragments rather than to amplify DNA fragments. Primerless PCR relies on the natural power of two segments of the PCR cycle to reconstruct fragmented PCR by: the capability of denatured DNA to anneal randomly to its complementary sequence (annealing), and Taq polymerase to extend the DNA at the 3’ end (extension). By repeating for 150 cycles a larger DNA fragment is generated instead of amplifying the DNA. Fragmented DNA was reconstructed by primerless PCR for 150 cycles. However, 1U of Taq polymerase was added to the PCR reaction every 50 cycles. The samples selected for this study were highly degraded. The degree of degradation of samples was visualized by running the samples on a 1% agarose gel. After running primerless PCR for 50, 100, and 150 cycles, larger fragments of highly intact DNA appeared as sharp, compact bands whereas degraded DNA was a diffuse smear. In conclusion, we have demonstrated that primerless PCR can authentically generate DNA fragments that are larger than the initial template, and the DNA polymerization of self-primed DNA fragments can increase the likelihood of successful regeneration ostensibly by reconstructing the template. Primerless PCR can help in regenerating larger DNA fragments from highly deteriorated samples, such as forensic, and ancient samples.
Essential oils (EO) are substances used by pharmaceutical, cosmetic and food industries. These compounds have useful properties for human health and well-being. Among the species cultivated for production of these substances are those of the genus Cymbopogon. Several species, subspecies, varieties, and sub-varieties are used worldwide, and studies have been conducted to elucidate the chemical features of their EO, mainly for C. citratus and C. winterianus. However, there are still species with great potential that have not yet been fully chemically characterized. In addition, EO composition is influenced by genetic and environmental factors. Along this line, we examined the chemical profile of EO of four species of the genus Cymbopogon (C. citratus, C. distans, C. flexuosus and C. winterianus) grown in southern Brazil. The EO was obtained from fresh leaf tissues by hydrodistillation. Citral was identified as the main component of C. citratus, C. flexuosus, and C. distans, whereas citronellal, citronellol, cis-geraniol and elemol were predominant in C. winterianus. The absence of a large range of compounds was noted in C. distans, which may be useful for the semi-exclusive synthesis of citral and other compounds of interest.
X-chromosome inactivation (XCI), an essential epigenetic event in female embryos, is involved in embryonic development and requires refined control mechanisms. Although studies in cattle have been contributing to the understanding of the dynamic mechanisms of XCI, no research has yet investigated XCI in elongated bovine embryos. We used qPCR to characterize the mRNA levels of five target genes (XIST, JPX, H2AFY, H2AFY2, and EZH2) related to XCI in bovine embryos at stages: 8-16-cells (72 embryos), morula (72 embryos), blastocyst (64 embryos), hatched blastocyst (64 embryos), D11 blastocyst (20 embryos), and D14 (biopsies of 4 biopsies of embryos). Our results showed the same mRNA levels of XIST, JPX, and H2AFY2 at the morula stage, which were higher than those of the other genes. However, JPX declined after the morula stage, whereas XIST and H2AFY2 maintained higher mRNA levels in BL and HB. The expression of XIST and H2AFY2 reached the lowest levels at D11 and D14. We suggest that the changes in mRNA levels of these genes in the initial stages of development are related to the onset of XCI in cattle. This study is relevant to improve our understanding of the dynamic mechanisms that act in XCI in cattle until the elongated embryo stage.
The corn leafhopper, Dalbulus maidis, is the most important pest of maize (Zea mays) in the Neotropics, due to its efficiency in pathogen transmission and close evolutionary association with the crop. This host specialization has enabled D. maidis to spread with maize, from their center of origin in Mesoamerica to all tropical and subtropical America, including Brazil. The population dynamics and survival strategies of D. maidis could be better understood by studying the species’ genetics, which has been little explored thus far. To fill in this knowledge gap, we provide here the complete mitochondrial DNA genome (mitogenome) of D. maidis (GenBank accession number: ON756137). Six adult specimens collected in Southern Brazil (Novo Machado/RS and Salvador das Missões/RS) were used for DNA extraction and sequencing on an Illumina MiSeq, followed by mitogenome assembling using NOVOPlasty v4.3.1, and annotation using MITOS v2.0.8. We confirmed the species identity by molecular diagnostics that showed >98% similarity between our reported partial cytochrome oxidase subunit I (mtCOI) gene and other D. maidis mtCOI sequences reported to date. The mitogenome of D. maidis is 16,488 base pairs long and contains 37 genes: 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), and two ribosomal RNA (rRNA) genes. Similar to other arthropods, the mitogenome of D. maidis is A–T biased (A: 32.3 – 32.9 %, T: 31.9 – 33.0 %). Primers for the mitochondrial COI gene were also designed to assist in future studies. This report of the D. maidis mitogenome will facilitate the development of effective molecular markers, rapid detection of field infestations and identification of migration pathways available for this insect pest in Brazil and neighboring countries.
Alcohol dependence is a multifactorial inherited psychiatric disease that is difficult to diagnose and treat. Recent years have seen an increase in knowledge about molecular pathways and effect of proinflammatory cytokines on alcohol dependence. Understanding this pathology as an inflammatory condition opens the possibility of using new therapeutic agents to treat its harmful effects. The objective of this study was to determine if there was a difference in gene expression of inflammatory response genes between two groups of university Colombians, one with a problem with alcohol (n=25) and one without, as a control (n=25). In previous research conducted by our research group, involving more than 30 single nucleotide variants of more than 10 inflammatory response genes, certain haplotypes, interactions, and gene networks were identified that indicated differences between alcoholics and controls in the genes SNCA, Il6R1, TNFR1, and MIF. Total RNA from blood mononuclear cells was extracted by determining the relative expression by qPCR of these genes; using ELISA, the concentration of their protein products in plasma was determined. There were differences in the relative expression of the TNFR1 and MIF genes, with a decrease in individuals with problematic alcohol consumption. The relative transcription of SNCA increased in men, whereas there were no changes in women. Nevertheless, there were no significant differences observed in the SNCA, IL6R1, and TNFR1 proteins, whereas MIF was decreased in the overall sample. On the other hand, SNCA, IL6R1 and MIF proteins showed differences in men. We observed disparities in the expression of these inflammatory response genes when comparing controls and cases. These findings demonstrate that SNCA, IL6R1, and MIF are candidates for further study as potential therapeutic targets in disease conditions related to alcohol abuse.
Kunitz trypsin inhibitor (KTI), which affects protein digestibility and lipoxygenase isozymes, responsible for the off-flavor associated with soy-based foods, are two undesirable factors present in soybean seeds. These unpleasant factors are usually inactivated by heat treatment. However, heat treatment does not completely eliminate these factors; in addition it may decrease protein solubility and may incur extra energy costs. Genetic elimination of these factors could be an alternative to heat treatment. This study aimed to select soybean lines free of KTI and lipoxygenase isozymes in the seeds. The population under study was obtained by crossing the BRS 213 cultivar, which shows low lipoxygenase activity, with BRS 155, a KTI lacking cultivar. F2:3 hybrid populations were selected and analyzed using DNA markers for the identification of recessive alleles that encode the absence of KTI and the three lipoxygenase enzymes (LOX1, LOX2 and LOX3). F2:3 segregating populations were successfully identified with the KTI specific marker with 100% efficiency. However, the KTi/kti-gene-specific marker does not allow for the identification of heterozygous and homozygous genotypes with dominant KTi alleles; the the simple sequence repeat (SSR) markers Satt228 and Satt409, which bind tightly to the KTi locus, were much more suitable diagnostic marker for screening plants in segregating populations. Satt090 and Satt417 confirmed the presence of the homozygous Lx2 null-allele in the parental cultivar BRS 213 by flanking Lx2 loci at 3.00 and 2.77 cM, respectively. The SSR markers used in this study could be efficiently used in marker assisted selection in a breeding program aimed at improving soybean seed quality.
Fusarium wilt is an important soil-borne disease that affects the common bean. The disease is caused by Fusarium oxysporum f. sp. phaseoli (Fop), a fungus that invades the plant mainly through the roots and colonizes the xylem, causing wilting, vascular discoloration, chlorosis, stunting, and premature plant death. The objective of this study was to analyze Fop disease severity in the Mortiño (tolerant), BAT477 (resistant), and A211 (susceptible) differentiating cultivars of common bean. Another goal was to examine the relationship of the disease severity with the amount of DNA of the pathogen in the vascular system in the stem as well as structural changes in plants affected by Fop infection. Assessment of the level of xylem colonization by Fop isolated in these cultivars grown in an experimental field (plot size of 30 m2) artificially infested with Fop showed in all the cultivars, a gradual increase in the fungus population over time, mainly during the maturation of the plants. The qPCR technique with the diseased genotypes collected at five different times starting in V2 stage (second trifoliolate developed) in the biological replicates proved to be efficient in quantifying the relative amount of DNA of the fungus. In the scanning electronic microscopy and in the evaluation of the disease severity, the structures of the fungus and of the plant were different. In the A211 cultivar, formation of hyphae and microconidia of the fungus and tylose and amyloplasts of the plant were observed. In cv. Mortiño found hyphae of the fungus and tyloses of the plant. In BAT477, no pathogen structure was observed. The disease severity in the differentiating cultivars was correlated with the amount of fungal DNA and plant structures formed in reaction to the presence of the fungus detected mainly in the last collection at the R6 stage of the bean plants.
Knowledge about the genetic variability of pineapple makes it possible to select characteristics of interest and plan crosses to obtain a promising cultivar for the market. This study aimed at the characterization and genetic divergence of pineapple clones from the crosses: IAC Fantástico x Jupi, BRS Imperial x Pérola, BRS Imperial x Smooth Cayenne and BRS Vitória x Smooth Cayenne. The study was carried out at the State University of Mato Grosso, Tangará da Serra campus crop fields. The municipality (Tangará da Serra) markets the Pérola and Jupi cultivars as fresh fruit. The other cultivars were used to obtain traits of interest. A randomized block design with three replications and five plants per plot was used; 42 characteristics (qualitative and quantitative) were analyzed. Vegetative and inflorescence characteristics were measured at inflorescence initiation. Fruit data was collected after harvesting. Variables were analyzed using the Ward-MLM procedure in the SAS program. Three distinct groups were found among the cross progenies. Individuals in group III had the highest averages for fruit mass with and without crown (1600 and 1486 g respectively), length (19.41 cm) and average fruit diameter (12.06 cm). This is an advantage for the producer, as larger fruits have smaller crowns. It also included individuals without thorns, with fruits of cylindrical shape and yellowish pulp, soluble solids and acidity levels within the recommended range, being the most suitable for fresh consumption. Groups I and II had lower means for the characteristics and were similar to each other. The greatest distance was observed between groups II and III; therefore, it is recommended to cross the best individuals of these groups to explore the heterotic effect and expand the existing genetic variability.
Lasiodiplodia theobromae and Neofusicoccum parvum are important fungi affecting mango trees in Northeast Brazil, a prominent region of mango export. Genome-wide association studies in a ‘Haden’ × ‘Tommy Atkins’ mango pseudo-F2 population were performed for symptoms of both fungal diseases to support the development of new cultivars by applying marker-assisted selection. ‘Haden’ is resistant while ‘Tommy Atkins’ is susceptible to both fungal diseases. Single nucleotide polymorphism (SNP) and microsatellite data of 95 progenies were analyzed by allelic and genotypic association and by general (GLM) and mixed linear models (MLM). Artificial pathogen inoculation was performed on 15-year-old progenies by manually spraying a 103 conidia mL-1 suspension on young branches and leaves. The plants were considered resistant when the absence of symptoms was ≥ 90% over three different evaluations. Consensus genomic associations were identified on chromosome 12; position 10.60 Mb (Mi_0096) and 1 (Mango_rep_c1316) position 14.67 Mb, with a significant association with L. theobromae symptoms, accounting for 20% of the total variation. Additional regions identified exclusively by GLM and MLM analysis, in chromosomes 11 and 8 (positions 24.57 Mb and 9.34 MB, respectively), explain 36% of the symptoms variation of this disease. Consensus genomic associations were identified on chromosomes 2, position 20.49 Mb (Mango_rep_c9407) and 9, position 15.01 Mb (Mango_rep_c8984), with a significant association with N. parvum symptoms, accounting for 21% of total resistance to this fungus. An additional region identified exclusively by GLM and MLM analysis, in chromosome 12 (Mango_rep_c7620, position 14.29 MB), explains 29% of the total variation of this disease. Qualitative and quantitative genome association methods run together enabled the identification of consensus chromosomal regions controlling resistance to these diseases. These chromosome regions are candidates for saturation with SNPs or further genome data mining to apply marker-assisted selection in mangoes.
Prader-Willi-Like syndrome (PWLS) is a rare genetic disorder with clinical features that include hypotonia, obesity, short extremities, and developmental delay. As its name suggests, the clinical phenotypes of PWLS overlap with the genetic imprinting disorder of Prader-Willi Syndrome (PWS). The sharing of phenotypes between these syndromes likely indicates that the genomic regions affected in PWLS are involved in the same genetic pathways associated with developing the PWS phenotype. Thus, the genetic heterogeneity of PWLS and the absence of a molecular diagnosis associated with PWS constitute a clinical challenge for health professionals. This review presents phenotypic and genotypic characteristics related to PWLS described in 34 articles, totaling 74 patients, including alterations involving deletion of 1p, 2p, 6q, 15p, duplication of 15q, Xq, Temple Syndrome, Schaaf-Yang Syndrome, Fragile X syndrome, and even associated mutations. Among the most frequent characteristics related to PWLS are global developmental delay (78%), obesity (68%), hypotonia (51%), speech-articulation problems (42%), and behavioral disorders (41%). This review provides an overview of current knowledge of the genetics and phenotypes associated with PWS and PWLS.
We examined the influence of interleukin genetic polymorphisms in individuals with apical periodontitis (AP). Based on the PICO strategy, searches were conducted in PubMed, SciELO, Web of Science, Medline, LILACS and EMBASE databases to answer a guiding question. The quality of studies and methodological rigor were assessed using the Critical Appraisal Skills Program Checklist and classification made according to levels of evidence. The search identified 292 studies, of which six were included based on the criteria. All evaluated the IL-1β polymorphism, of which three found a significant association with a protective and/or potentiating effect of IL-1β on AP. Two studies evaluated the IL-6 polymorphism, one of which identified a significant association. Three studies evaluated TNF-α polymorphism, of which two reported significant associations with AP. An association was also found in one study for IL-8 with AP. In conclusion, polymorphisms of several interleukins have been found to influence the risk and development of AP in humans. Some polymorphisms are more influential and have been more intensively studied than others, such as interleukin 1-β, followed by IL-6 and TNF-α. This influence of genotype can be expressively or mildly marked. Individuals presenting one or two copies of a particular allele appear to be at higher risk of developing periapical lesions. These apparent associations of interleukins with AP merit further study.
The superoxide dismutase (SOD) enzyme initiates the process of neutralization of cytotoxic effects caused by reactive oxygen- species (ROS). In drought-tolerant plants, SOD is rapidly supplied due to their ability to adjust when they sense a lack of water, unlike in drought-sensitive plants. To investigate whether exogenous application of SOD could benefit drought-sensitive plants by ameliorating water stress, we tested two contrasting cultivars, BR 1 and IAC Caiapó, which are drought tolerant and sensitive cultivars, respectively. Plants of both cultivars were grown in pots in a greenhouse and submitted to nine days of water suppression starting at V1 phase, and treated with SOD at different concentrations, 11, 23 and 34 µg mL-1, applied to both adaxial and abaxial surfaces of leaves. The status of water stress was estimated through verification of soil moisture in stressed treatments, in comparison with controls. Additionally, stomatal conductance was estimated to evaluate stomata closing. The plants were assessed for growth, gas exchange, and activity of antioxidative enzymes. Overall, the contribution of exogenous SOD in mitigating the effects of water stress was greater in stressed plants of drought-sensitive cultivars. Gas exchange recovery was observed in the plants even with the lowest applied SOD concentration (11 µg mL-1), whereas higher exogenous SOD supply was required to prevent oxidative damage. More in-depth studies regarding the potential benefits of this type of treatment on the phenology of runner genotypes are merited, since these cultivars are very productive, though highly sensitive to water restriction.
Insertion of nucleic acids into cells unlocks the possibility of modulating gene expression; however, some cells such as primary cells and those that grow in suspension are hard-to-transfect. New therapies for cancer and possible autoimmune diseases, such as those that involve chimeric antigen receptor T cells, rely on the insertion of plasmids into lymphocytes, which fit into the hard-to-transfect category. In such cases virus-based transduction is usually applied, but the carrier vector tends to be incorporated into the cell’s own DNA in a stable manner, with unpredictable consequences. Thus, highly efficient non-viral gene/plasmid delivery is a sought-after technology. We evaluated several commercially available chemical transfection methods as well as electroporation in difficult-to-transfect cells, including a human lymphocyte cell-line (Jurkat) and fresh peripheral blood mononuclear cells (PBMCs), both grown in suspension. The cell-toxicity of the methods was also evaluated. Twenty-four hours after transfection of the plasmid pCMV-GFP, the proportion of GFP positive (GFP+) cells was evaluated by cytometry. The cationic polymer TurboFect yielded ~7.8% of GFP+ Jurkat cells on average, while the other reagents (Lipofectamine 3000, FuGENE HD and X-tremeGENE HP) presented <3% of GFP+ cells. In PBMCs, none of the chemical reagents yielded >3% transfected cells. Electroporation was more efficient, with ~45% of GFP+ in Jurkat and ~15.7% GFP+ in PBMCs. However, it proved to be highly toxic, with ~80% of the cells considered non-viable 24h after the procedure, while TurboFect showed little-to-no toxicity. In conclusion, it was found that despite its high toxicity electroporation was the only method with applicable transfection efficiency in PBMCs, while in Jurkat the reagent TurboFect can be applied with acceptable results. The strategy for insertion of nucleic acids needs to be fine-tuned for each target cell type and experimental condition.
The development of common bean plants with an improved root system can be a strategy for water and nutrient absorption in limiting environmental conditions. The objective of this study was to understand the influence of root phenotyping methods and phenological stages of evaluation on the selection of common bean genotypes for a highly branched root system. In the 2021/22 growing season, this study was initiated with 36 field treatments, consisting of the combination of three genotypes, two parents (Mesoamerican and Andean gene pools) and one progeny, two methods of root phenotyping (Shovelomics and WinRHIZO) and six growth stages (R1-6: four and eight trifoliate leaves, flower bud, full flowering, pod formation and grain filling). The field treatments were randomized in a simple lattice design. Five plants from each experimental unit were evaluated, considering the genotype and phenotyping methods in each developmental stage. The genotype x method x stage interaction was significant. The partitioning of the simple effects of the factors indicated that the root system variables of the parents could be distinguished from those of the progeny at R6. At this stage, the recommended phenotyping method differs according to the genetic origin of the genotypes. Plotting of the standardized canonical scores for the triple interaction showed that the Shovelomics and WinRHIZO phenotyping methods are adequate for the Andean and Mesoamerican genotypes, respectively, in view of their high scores with high discriminative power, allowing treatment discrimination. Specific phenotyping methods were indicated for Mesoamerican versus Andean genotypes in view of the root development trait intrinsic to each gene pool. We conclude that improving root phenotyping for the development of cultivars with a finely branched root system is a useful strategy to maintain common bean yields in environments under stressful conditions.
Approximately 60% of all cases of congenital bilateral sensorineural hearing impairment are due to genetic factors, and about 50% of hearing impairment cases at a later stage are caused by a mutation in a single gene. Because of the high frequency of gap junction beta-2 protein gene (GJB2) mutations, mutation analysis of this gene is widely used in hearing impairment research and diagnosis. This study aimed to determine the prevalence of common GJB2 mutations in patients with profound non-syndromic sensorineural hearing impairment. Sixty-one patients (32 male and 29 female) included in this study had above 90 decibels of bilateral sensorineural hearing impairment. Patient DNA was isolated from buccal cells. The 1st and 2nd exons of the GJB2 gene were amplified with specific primers after gel purification of both regions. Sanger DNA sequencing analysis was used for investigation of changes in these gene regions. The pathological variant was found in nine patients (15%). This variation involved a frameshift mutation in GJB2 (homozygous 35delG) of the 2nd exon; no mutation was detected in the 1st exon. This study is the first report of a genetic investigation of hearing impairment in the Kurdish population in Sulaimani province, northeastern Iraq, near the Iraq-Iran border. The results show that 35delG mutation has a high prevalence in patients with non-syndromic sensorineural hearing impairment.