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One of the central issues in the understanding of early cellular evolution is the characterisation of the cenancestor. This includes the description of the chemical nature of its genome. The disagreements on this question comprise several proposals, including the possibility that AlkB-mediated methylation repair of alkylated RNA molecules may be interpreted as evidence of a cenancestral RNA genome. We present here an evolutionary analysis of the cupin-like protein superfamily based on tertiary structure-based phylogenies that includes the oxygen-dependent AlkB and its homologs. Our results suggest that the repair of methylated RNA molecules is the outcome of the enzyme substrate ambiguity, and doesn´t necessarily indicates that the last common ancestor was endowed with an RNA genome.
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DNA , Evolução Molecular , Genoma , Filogenia , RNA , RNA/genética , Genoma/genética , DNA/genética , Enzimas AlkB/genética , Enzimas AlkB/metabolismo , MetilaçãoAssuntos
Epigênese Genética , Pré-Eclâmpsia , Humanos , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/diagnóstico , Gravidez , Feminino , Metilação de DNA , BiomarcadoresRESUMO
Cannabis sativa can be classified in two main types, according to psychotropic cannabinoid ∆9-tetrahydrocannabinol (∆9-THC) content: the drug-type and the fiber-type. According to the European Monitoring Center for Drugs and Drug Addiction, most of the European Union countries consider the possession of cannabis, for personal use, a minor offense with possibility of incarceration. Despite of the model of legal supply (i.e., Spanish cannabis clubs, Netherlands coffee shops) or medical use (i.e., Italy), cannabis remains the most used and trafficked illicit plant in the European Union. Differentiating cannabis crops or tracing the biogeographical origin is crucial for law enforcement purposes. Chloroplast DNA (cpDNA) markers may assist to determine biogeographic origin and to differentiate hemp from marijuana. This research aims: to identify and to evaluate nine C. sativa cpDNA polymorphic SNP sites to differentiate crop type and to provide information about its biogeographical origin. Five SNaPshot™ assays for nine chloroplast markers were developed and conducted in marijuana samples seized in Chile, the USA-Mexico border and Spain, and hemp samples grown in Spain and in Italy. The SNapShot™ assays were tested on 122 cannabis samples, which included 16 blind samples, and were able to differentiate marijuana crop type from hemp crop type in all samples. Using phylogenetic analysis, genetic differences were observed between marijuana and hemp samples. Moreover, principal component analysis (PCA) supported the relationship among hemp samples, as well as for USA-Mexico border, Spanish, and Chilean marijuana samples. Genetic differences between groups based on the biogeographical origin and their crop type were observed. Increasing the number of genetic markers, including the most recently studied ones, and expanding the sample database will provide more accurate information about crop differentiation and biogeographical origin.
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Cannabis , DNA de Cloroplastos , Polimorfismo de Nucleotídeo Único , Cannabis/genética , Marcadores Genéticos , DNA de Cloroplastos/genética , México , Reação em Cadeia da Polimerase , Europa (Continente) , Itália , Chile , EspanhaRESUMO
Diagnosis of pulmonary tuberculosis (TB) relies on a sputum sample, which cannot be obtained from all symptomatic individuals. Mycobacterium tuberculosis (Mtb) transrenal DNA (trDNA) has been detected in urine, an easily obtainable, noninvasive, alternative sample type. However, reported sensitivities have been variable and likely depend on collection and assay procedures and aspects of trDNA biology. We analyzed three serial urine samples from each of 75 adults with culture-confirmed pulmonary TB disease in Lima, Peru for detection of trDNA using short-fragment real-time PCR. Additionally, we examined host, urine, and sampling factors associated with detection. Overall per-sample sensitivity was 38 % (95 % Confidence Interval [CI] 30-45 %). On an individual level (i.e., any of the three samples positive), sensitivity was 73 % (95 % CI: 62-83 %). Sensitivity was highest among samples from patients with smear-positive TB, 92 % (95 % CI: 62-100 %). Specificity from a single sample from each of 10 healthy controls was 100 % (95 % CI: 69-100 %). Adjusting our assay positivity threshold increased individual-level sensitivity to 88 % (95 % CI: 78-94 %) overall without affecting the specificity. We did not find associations between Mtb trDNA detection and individual characteristics or urine sample characteristics. Overall, our results support the potential of trDNA detection for TB diagnosis.
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DNA Bacteriano , Mycobacterium tuberculosis , Tuberculose Pulmonar , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Adulto , Feminino , Peru/epidemiologia , Masculino , DNA Bacteriano/urina , DNA Bacteriano/genética , Tuberculose Pulmonar/urina , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Pessoa de Meia-Idade , Adulto Jovem , Reação em Cadeia da Polimerase em Tempo Real , Valor Preditivo dos Testes , Urinálise/métodos , Estudos de Casos e Controles , Reprodutibilidade dos Testes , IdosoRESUMO
Wounds or chronic injuries are associated with high medical costs so, develop healing-oriented drugs is a challenge for modern medicine. The identification of new therapeutic alternatives focuses on the use of natural products. Therefore, the main goal of this study was to evaluate the healing potential and anti-inflammatory mechanism of action of extracts and the main compounds derived from Myrciaria plinioides D. Legrand leaves. The antimicrobial activity of leaf extracts was analyzed. Cell viability, cytotoxicity and genotoxicity of plant extracts and compounds were also assessed. Release of pro- and anti-inflammatory cytokines and TGF-ß by ELISA, and protein expression was determined by Western Blot. The cell migration and cell proliferation of ethanol and aqueous leaf extracts and p-coumaric acid, quercetin and caffeic acid compounds were also evaluated. The aqueous extract exhibited antibacterial activity and, after determining the safety concentrations in three assays, we showed that this extract induced p38-α MAPK phosphorylation and the same extract and the p-coumaric acid decreased COX-2 and caspase-3, -8 expression, as well as reduced the TNF-α release and stimulated the IL-10 in RAW 264.7 cells. In L929 cells, the extract and p-coumaric acid induced TGF-ß release, besides increasing the process of cell migration and proliferation. These results suggested that the healing properties of Myrciaria plinioides aqueous extract can be associated to the presence of phenolic compounds, especially p-coumaric acid, and/or glycosylated metabolites.
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Anti-Inflamatórios , Movimento Celular , Extratos Vegetais , Folhas de Planta , Cicatrização , Extratos Vegetais/farmacologia , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/isolamento & purificação , Cicatrização/efeitos dos fármacos , Camundongos , Células RAW 264.7 , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/metabolismo , Myrtaceae/química , Ácidos Cumáricos/farmacologia , Ácidos Cumáricos/isolamento & purificação , Antibacterianos/farmacologia , Antibacterianos/isolamento & purificaçãoRESUMO
A new sand fly species, Trichophoromyia jariensis n. sp. Cavalcante, Rodrigues, & Galati, from the state of Amapá, Brazil, is described based on both male and female morphology and cytochrome c oxidase subunit I DNA barcodes. The DNA barcoding analysis clearly associated males and females of this new species.
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Rodents are considered good models for investigating genotoxic damage and mutagenic alterations caused by xenobiotic agents, due to their occupation of a wide variety of habitats. However, relatively few in situ studies have focused on DNA damage in wild rodents associated with environmental exposure. In this review, we investigate trends in the application of the micronucleus test and comet assay in in situ studies of wild rodents. A total of 33 papers were identified, distributed across 14 different countries. Brazil and Spain had the most published studies (six each), followed by Bulgaria (n = 5), Mexico (n = 4) and Italy (n = 3). Only 24 of the 2,652 recognized rodent species have been the subject of in situ studies, which have most frequently focus on species of the genus Mus. The protocols used for the micronucleus test and comet assay varied widely, although blood and bone marrow were the primary types of tissue used. Given the paucity of studies on wild rodents, we recommend further research, particularly focusing on the use of this group as bioindicators of environmental quality and the standardization of protocols.
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Ensaio Cometa , Dano ao DNA , Monitoramento Ambiental , Testes para Micronúcleos , Roedores , Ensaio Cometa/métodos , Testes para Micronúcleos/métodos , Animais , Monitoramento Ambiental/métodos , Animais Selvagens , Poluentes Ambientais/toxicidadeRESUMO
Plants have several mechanisms to adapt or acclimate to environmental stress. Morphological, physiological, or genetic changes are examples of complex plant responses. In recent years, our understanding of the role of epigenetic regulation, which encompasses changes that do not alter the DNA sequence, as an adaptive mechanism in response to stressful conditions has advanced significantly. Some studies elucidated and synthesized epigenetic mechanisms and their relationships with environmental change, while others explored the interplay between epigenetic modifications and environmental shifts, aiming to deepen our understanding of these complex processes. In this study, we performed a systematic review of the literature to analyze the progression of epigenetics studies on plant species' responses to abiotic factors. We also aimed to identify the most studied species, the type of abiotic factor studied, and the epigenetic technique most used in the scientific literature. For this, a search for articles in databases was carried out, and after analyzing them using pre-established inclusion criteria, a total of 401 studies were found. The most studied species were Arabidopsis thaliana and Oryza sativa, highlighting the gap in studies of non-economic and tropical plant species. Methylome DNA sequencing is the main technique used for the detection of epigenetic interactions in published studies. Furthermore, most studies sought to understand the plant responses to abiotic changes in temperature, water, and salinity. It is worth emphasizing further research is necessary to establish a correlation between epigenetic responses and abiotic factors, such as extreme temperatures and light, associated with climate change.
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INTRODUCTION: Despite thromboprophylaxis, women with antiphospholipid syndrome (APS) face high-risk pregnancies due to proinflammatory and prothrombotic states. This highlights the need for new monitoring and prognostic tools. Recent insights into the pathophysiological role of neutrophil activation and extracellular trap (NET) formation in this syndrome led to the exploration of plasma cell-free DNA (cfDNA), a derivative of NETosis, as a promising biomarker. MATERIALS AND METHODS: cfDNA was isolated and quantified from plasma samples of healthy pregnant women (control group, HC) and women with APS (APS group). We assessed the physiological variability of cfDNA across the three trimesters in HC. Levels of cfDNA were compared between APS and HC by gestational trimester. ROC curve analysis was performed to evaluate the efficacy of cfDNA levels for classifying APS patients. Furthermore, cfDNA levels in pregnant women with APS with obstetric complications were compared to those from uncomplicated pregnancies. RESULTS: Among HC, cfDNA significantly increased in the third trimester compared to the first and second. Elevated cfDNA levels in APS compared to HC were observed in the first and second trimesters. First-trimester cfDNA levels demonstrated the highest classification ability to discriminate between APS and HC patients (AUC: 0.906). Among APS, those with complicated pregnancies (fetal growth restriction, preeclampsia, placenta accreta) exhibited significantly elevated cfDNA levels in the second trimester. CONCLUSIONS: Elevated levels of cfDNA in pregnant women with APS, particularly among those with obstetric complications, supports further investigation into the potential of cfDNA as a valuable tool in the obstetric management of women with APS.
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Síndrome Antifosfolipídica , Ácidos Nucleicos Livres , Gravidez de Alto Risco , Humanos , Feminino , Gravidez , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/complicações , Ácidos Nucleicos Livres/sangue , Adulto , Gravidez de Alto Risco/sangue , Biomarcadores/sangue , Complicações na Gravidez/sangueRESUMO
Advances in DNA sequencing technologies, especially next-generation sequencing (NGS), which is the basis for whole-exome sequencing (WES) and whole-genome sequencing (WGS), have profoundly transformed immune-mediated rheumatic disease diagnosis. Recently, substantial cost reductions have facilitated access to these diagnostic tools, expanded the capacity of molecular diagnostics and enabled the pursuit of precision medicine in rheumatology. Understanding the fundamental principles of genetics and diversity in genetic variant classification is a crucial milestone in rheumatology. However, despite the growing availability of DNA sequencing platforms, a significant number of autoinflammatory diseases (AIDs), neuromuscular disorders, hereditary collagen diseases, and monogenic bone diseases remain unsolved, and variants of uncertain significance (VUS) pose a formidable challenge to addressing these unmet needs in the coming decades. This article aims to provide an overview of the clinical indications and interpretation of comprehensive genetic testing in the medical field, addressing the related complexities and implications.
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Testes Genéticos , Doenças Reumáticas , Humanos , Testes Genéticos/métodos , Doenças Reumáticas/genética , Doenças Reumáticas/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala , Reumatologia , Sequenciamento do Exoma , Doenças Neuromusculares/genética , Doenças Neuromusculares/diagnóstico , Doenças Hereditárias Autoinflamatórias/genética , Doenças Hereditárias Autoinflamatórias/diagnóstico , ReumatologistasRESUMO
Chronic hepatitis B virus infection (CHB) remains a global health concern, with currently available antiviral therapies demonstrating limited effectiveness in preventing hepatocellular carcinoma (HCC) development. Two primary challenges in CHB treatment include the persistence of the minichromosome, covalently closed circular DNA (cccDNA) of the hepatitis B virus (HBV), and the failure of the host immune response to eliminate cccDNA. Recent findings indicate several host and HBV proteins involved in the epigenetic regulation of cccDNA, including HBV core protein (HBc) and HBV x protein (HBx). Both proteins might contribute to the stability of the cccDNA minichromosome and interact with viral and host proteins to support transcription. One potential avenue for CHB treatment involves the utilization of therapeutic vaccines. This paper explores HBV antigens suitable for epigenetic manipulation of cccDNA, elucidates their mechanisms of action, and evaluates their potential as key components of epigenetically-driven vaccines for CHB therapy. Molecular targeted agents with therapeutic vaccines offer a promising strategy for addressing CHB by targeting the virus and enhancing the host's immunological response. Despite challenges, the development of these vaccines provides new hope for CHB patients by emphasizing the need for HBV antigens that induce effective immune responses without causing T cell exhaustion.
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Objective: The study delved into the epigenetic factors associated with periodontal disease in two lineages of mice, namely C57bl/6 and Balb/c. Its primary objective was to elucidate alterations in the methylome of mice with distinct genetic backgrounds following systemic microbial challenge, employing high-throughput DNA methylation analysis as the investigative tool. Methods: Porphyromonas gingivalis (Pg)was orally administered to induce periodontitis in both Balb/c and C57bl/6 lineage. After euthanasia, genomic DNA from both maxilla and blood were subjected to bisulfite conversion, PCR amplification and genome-wide DNA methylation analysis using the Ovation RRBS Methyl-Seq System coupled with the Illumina Infinium Mouse Methylation BeadChip. Results: Of particular significance was the distinct methylation profile observed within the Pg-induced group of the Balb/c lineage, contrasting with both the control and Pg-induced groups of the C57bl/6 lineage. Utilizing rigorous filtering criteria, we successfully identified a substantial number of differentially methylated regions (DMRs) across various tissues and comparison groups, shedding light on the prevailing hypermethylation in non-induced cohorts and hypomethylation in induced groups. The comparison between blood and maxilla samples underscored the unique methylation patterns specific to the jaw tissue. Our comprehensive methylome analysis further unveiled statistically significant disparities, particularly within promoter regions, in several comparison groups. Conclusion: The differential DNA methylation patterns observed between C57bl/6 and Balb/c mouse lines suggest that epigenetic factors contribute to the variations in disease susceptibility. The identified differentially methylated regions associated with immune regulation and inflammatory response provide potential targets for further investigation. These findings emphasize the importance of considering epigenetic mechanisms in the development and progression of periodontitis.
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Metilação de DNA , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Porphyromonas gingivalis , Animais , Porphyromonas gingivalis/genética , Camundongos , Periodontite/microbiologia , Epigênese Genética , Doenças Periodontais/microbiologia , Suscetibilidade a Doenças , Infecções por Bacteroidaceae/microbiologia , EpigenomaRESUMO
Ruthenium(II) complexes (Ru1-Ru3) with the general formula [Ru(O-O)(PPh3)2(bipy)]PF6, bearing two triphenylphosphine (PPh3), bipyridine (bipy) and a series of natural and synthetic ß-diketones (O,O) ligands were synthesized and characterized using various analytical techniques. The interaction between the complexes and calf thymus DNA (CT-DNA) was investigated and demonstrated a weak interaction. The cytotoxicity of the complexes was investigated against breast cancer cells (MDA-MB-231 and MCF-7), lung cancer cells (A549), cisplatin-resistant ovarian cancer cells (A2780cis), as well as non-tumour lung (MRC-5) and non-tumour breast (MCF-10A) cell lines. All complexes exhibited cytotoxic activity against all the cell lines studied, with half maximal inhibitory concentration (IC50) values ranging from 0.39 to 13 µM. Notably, the three complexes demonstrated selectivity against the A2780cis cell line, with IC50 ranging from 0.39 to 0.82 µM. Among them, Ru2 exhibited the highest cytotoxicity, with an IC50 value of 0.39 µM. Consequently, this new class of complexes shows good selectivity towards cisplatin-resistant ovarian cancer cells and it is promising for further investigation as anti-cancer agents.
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This commentary provides a retrospective on the Ascona B-DNA Consortium (ABC) initiative and on the conference held in April 2023 at Ascona, Switzerland, where we celebrated 22 years of the consortium, sharing the latest advances in simulations and experiments of the effects of sequence on the mechanical properties of DNA from electrons to nucleosomes.
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Background: Members of the genus Cupiennius Simon, 1891 are categorized as wandering spiders and are part of the family Trechaleidae. The genomics and proteomics of Cupiennius spiders from North America remain uncharacterized. The present study explores for the first time molecular data from the endemic species Cupiennius chiapanensis Medina, 2006, and also presents new data for Cupiennius salei (Keyserling, 1878), both collected in southern Mexico. Methods: In total, 88 Cupiennius specimens were collected from southern Mexico and morphologically identified. DNA was extracted and the mitochondrial COI fragment was amplified. COI sequences were analyzed, and a phylogenetic tree was inferred for species from the Americas. Genetic diversity was analyzed using haplotype networks and gene distances. Venom was obtained from C. chiapanensis and C. salei by electrostimulation. The venom was separated by HPLC, visualized using SDS-PAGE, and quantified for use in toxicity bioassays in mice and insects. Results: Analysis of COI sequences from C. chiapanensis showed 94% identity with C. salei, while C. salei exhibited 94-97% identity with sequences from Central and South American conspecifics. The venom from C. chiapanensis exhibited toxic activity against crickets. Venoms from C. chiapanensis and C. salei caused death in Anastrepha obliqua flies. Analysis of venom fractions from C. salei and C. chiapanensis revealed molecular masses of a similar size as some previously reported toxins and neurotoxic components. We determined the amino acid sequences of ChiaTx1 and ChiaTx2, toxins that are reported here for the first time and which showed toxicity against mice and insects. Conclusion: Our work is the first to report COI-based DNA barcoding sequences from southern Mexican Cupiennius spiders. Compounds with toxic activity were identified in venom from both species.
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Environmental exposures and gene-exposure interactions are the major causes of some diseases. Early-life exposome studies are needed to elucidate the role of environmental exposures and their complex interactions with biological mechanisms involved in childhood health. This study aimed to determine the contribution of early-life exposome to DNA damage and the modifying effect of genetic polymorphisms involved in air pollutants metabolism, antioxidant defense, and DNA repair. We conducted a cohort study in 416 Colombian children under five years. Blood samples at baseline were collected to measure DNA damage by the Comet assay and to determine GSTT1, GSTM1, CYP1A1, H2AX, OGG1, and SOD2 genetic polymorphisms. The exposome was estimated using geographic information systems, remote sensing, LUR models, and questionnaires. The association exposome-DNA damage was estimated using the Elastic Net linear regression with log link. Our results suggest that exposure to PM2.5 one year before the blood draw (BBD) (0.83, 95 %CI: 0.76; 0.91), soft drinks consumption (0.94, 0.89; 0.98), and GSTM1 null genotype (0.05, 0.01; 0.36) diminished the DNA damage, whereas exposure to PM2.5 one-week BBD (1.18, 1.06; 1.32), NO2 lag-5 days BBD (1.27, 1.18; 1.36), in-house cockroaches (1.10, 1.00; 1.21) at the recruitment, crowding at home (1.34, 1.08; 1.67) at the recruitment, cereal consumption (1.11, 1.04; 1.19) and H2AX (AG/GG vs. AA) (1.44, 1.11; 1.88) increased the DNA damage. The interactions between H2AX (AG/GG vs. AA) genotypes with crowding and PM2.5 one week BBD, GSTM1 (null vs. present) with humidity at the first year of life, and OGG1 (SC/CC vs. SS) with walkability at the first year of life were significant. The early-life exposome contributes to elucidating the effect of environmental exposures on DNA damage in Colombian children under five years old. The exposome-DNA damage effect appears to be modulated by genetic variants in DNA repair and antioxidant defense enzymes.
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Poluentes Atmosféricos , Dano ao DNA , Exposição Ambiental , Interação Gene-Ambiente , Humanos , Pré-Escolar , Colômbia , Masculino , Feminino , Lactente , Expossoma , Estudos de Coortes , Glutationa Transferase/genética , Material Particulado , Polimorfismo Genético , Poluição do Ar/efeitos adversos , Poluição do Ar/estatística & dados numéricosRESUMO
The genus Urotrygon comprises small- to medium-sized endemic round rays on the American continent and has undergone several synonymization processes. Here, we used an integrative taxonomic approach, including meristic, morphometric, and mtDNA analyses, to resolve the particularly intricate relationship among Urotrygon munda Gill, 1863, Urotrygon chilensis (Günther, 1872), and Urotrygon asterias (Jordan & Gilbert, 1883). The latter species is currently a synonym of U. munda but is also considered the U. chilensis "northern morphotype." These taxonomic entities have historically been confounded, mainly due to their phenotypical resemblance along their geographic distribution in the eastern Pacific. We assessed 78 specimens (43 "northern" and 30 "southern morphotypes" of U. chilensis, as well as 5 U. munda) using 19 external variables for taxonomic and morphometric analysis. Distinct meristic patterns, including pectoral and pelvic ceratotrichia, vertebrae number, and thorn distribution along the dorsal midline, were observed in the series-type specimens of the three taxonomic entities. Our multivariate morphometric analyses consistently differentiated the three groups as distinct taxonomic entities, with an overall classification accuracy of 66.7%. The meristic results also provided reliable information distinguishing the three entities. Based on the nicotinamide adenine dinucleotide (NADH2) and cytochrome oxidase subunit I (COI) genes, our phylogenetic analysis were consistent with the morphometric and meristic data, supporting these three entities as distinct species having their own evolutionary lineages. Our comprehensive approach confidently demonstrated that the northern U. chilensis morphotype matched and corresponded to the description of the Starry round ray, U. asterias, confirming its taxonomic resurrection as a valid species distinct from U. chilensis and U. munda. The geographic distribution of U. asterias spans from the tropical west coast of Mexico (including the Gulf of California) to Costa Rica, revealing that microevolutionary processes have well-defined population clades within this range. Furthermore, U. chilensis is unequivocally established as the sole Urotrygon species occurring south of the Guayaquil marine ecoregion. In addition, the public COI and NADH2 sequences available for Urotrygon hosted in the ad hoc online databases were found to be misidentified, emphasizing the need for rigorous taxonomic scrutiny in this group. Finally, our research underscores the significance of an integrative approach that combines morphometric, meristic, and molecular techniques with historical data to disentangle the complexities of closely related taxa.
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DNA Mitocondrial , Filogenia , Rajidae , Animais , Rajidae/genética , Rajidae/anatomia & histologia , Rajidae/classificação , DNA Mitocondrial/genética , Análise de Sequência de DNA , Variação GenéticaRESUMO
Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library's breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.
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Clonagem Molecular , Biblioteca Gênica , Mutagênese , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase/métodos , Clonagem Molecular/métodos , Vetores Genéticos/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Plasmídeos/genéticaRESUMO
Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li-Fraumeni (LFS). Variants affecting its activity can drive tumorigenesis altering p53 pathways and their identification is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G > C, c.320A > G, c.417G > T, c.460G > A, c,522G > T, c.589G > A and c.997C > T) identified in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed significantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited effect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classification as pathogenic variants. This underscores the significance of conducting functional studies on germline TP53 missense variants classified as variants of uncertain significance to ensure proper management of LFS-related cancer risks.
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Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Síndrome de Li-Fraumeni , Mutação de Sentido Incorreto , Proteína Supressora de Tumor p53 , Síndrome de Li-Fraumeni/genética , Humanos , Proteína Supressora de Tumor p53/genética , Brasil , Proteínas Proto-Oncogênicas c-mdm2/genética , Feminino , Inibidor de Quinase Dependente de Ciclina p21/genética , Masculino , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ativação Transcricional/genética , Proteínas GADD45RESUMO
This study aimed to investigate the relationship between epigenetic mechanisms and oral mucositis (OM) in paediatric patients with acute lymphoblastic leukaemia. Oral cells were collected from 76 participants, including 15 healthy individuals, 10 patients with acute lymphoblastic leukaemia but without a history of OM and 51 acute lymphoblastic leukaemia patients with a history of OM (35 with active OM and 16 who had recovered from OM). Global DNA methylation in the miR-9-1 and miR-9-3 genes was performed. Seven polymorphisms rs1801131, rs1801133 (MTHFR), rs2228611 (DNMT1), rs7590760, rs1550117 (DNMT3A), rs6087990, rs2424913 (DNMT3B) were genotyped and an analysis of association with global DNA methylation was performed. The global methylation levels were lower in cancer patients recovered from OM than in the other groups. A higher frequency of unmethylated profile for miR-9-1 and partially methylated profile for miR-9-3 was observed in cancer patients regardless of OM history compared to healthy patients. The GG genotype of the rs2228611 (DNMT1) polymorphism was associated with higher levels of global methylation in cancer patients irrespective of OM. It was concluded that global methylation is associated with mucosal recovery. The effect of DNMT1 genotype on the global DNA methylation profile, as well as the methylation profile of miR-9-1 and miR-9-3 in cancer patients is independent of OM.