RESUMO
Formamidopyrimidine (Fapyâ¢dG) is a major lesion arising from oxidation of dG that is produced from a common chemical precursor of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). In human cells, replication of single-stranded shuttle vectors containing Fapyâ¢dG is more mutagenic than 8-OxodGuo. Here, we present the first data regarding promoter dependent RNA polymerase II bypass of Fapyâ¢dG. 8-OxodGuo bypass was examined side-by-side. Experiments were carried out using double-stranded shuttle vectors in HeLa cell nuclear lysates and in HEK 293T cells. The lesions do not significantly block transcriptional bypass efficiency. Less than 2% adenosine incorporation occurred in cells when the lesions were base paired with dC. Inhibiting base excision repair in HEK 293T cells significantly increased adenosine incorporation, particularly from Fapyâ¢dG:dC bypass which yielded â¼25% adenosine incorporation. No effect was detected upon transcriptional bypass of either lesion in nucleotide excision repair deficient cells. Transcriptional mutagenesis was significantly higher when shuttle vectors containing dA opposite one of the lesions were employed. For Fapyâ¢dG:dA bypass, adenosine incorporation was greater than 85%; whereas 8-OxodGuo:dA yielded >20% point mutations. The combination of more frequent replication mistakes and greater error-prone Pol II bypass suggest that Fapyâ¢dG is more mutagenic than 8-OxodGuo.
Assuntos
8-Hidroxi-2'-Desoxiguanosina , Dano ao DNA , Desoxiguanosina , Regiões Promotoras Genéticas , RNA Polimerase II , Humanos , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Células HEK293 , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Células HeLa , Reparo do DNA , Transcrição Gênica , Pirimidinas , Dímeros de Pirimidina/metabolismo , Dímeros de Pirimidina/genéticaRESUMO
N6-(2-deoxy-α,ß-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapyâ¢dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). Fapyâ¢dG preferentially gives rise to G â T transversions and G â A transitions. However, the molecular basis by which Fapyâ¢dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase ß (Pol ß), a model mammalian polymerase, bypasses a templating Fapyâ¢dG, inserts Fapyâ¢dGTP, and extends from Fapyâ¢dG at the primer terminus. When Fapyâ¢dG is present in the template, Pol ß incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapyâ¢dGTP is a poor substrate but is incorporated â¼3-times more efficiently opposite dA than dC. Extension from Fapyâ¢dG at the 3'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapyâ¢dG is likely to be the source of the mutagenic effects of the lesion and not Fapyâ¢dGTP. These experiments increase our understanding of the promutagenic effects of Fapyâ¢dG.
Assuntos
DNA Polimerase beta , Replicação do DNA , Formamidas , Furanos , Pirimidinas , Humanos , Cristalografia por Raios X , DNA/química , DNA/metabolismo , DNA Polimerase beta/metabolismo , DNA Polimerase beta/química , Cinética , Modelos Moleculares , Pirimidinas/química , Pirimidinas/metabolismo , Furanos/química , Furanos/metabolismo , Formamidas/metabolismo , MutagêneseRESUMO
Oxidative DNA lesions cause significant detrimental effects on a living species. Two major DNA lesions resulting from dG oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo) and formamidopyrimidine (Fapy·dG), are produced from a common chemical intermediate. Fapy·dG is formed in comparable yields under oxygen-deficient conditions. Replicative bypass of Fapy·dG in human cells is more mutagenic than that of 8-OxodGuo. Despite the biological importance of transcriptional mutagenesis, there are no reports of the effects of Fapy·dG on RNA polymerase II (Pol II) activity. Here we perform comprehensive kinetic studies to investigate the impact of Fapy·dG on three key transcriptional fidelity checkpoint steps by Pol II: insertion, extension, and proofreading steps. The ratios of error-free versus error-prone incorporation opposite Fapy·dG are significantly reduced in comparison with undamaged dG. Similarly, Fapy·dG:A mispair is extended with comparable efficiency as that of the error-free, Fapy·dG:C base pair. The α- and ß-configurational isomers of Fapy·dG have distinct effects on Pol II insertion and extension. Pol II can preferentially cleave error-prone products by proofreading. To further understand the structural basis of transcription processing of Fapy·dG, five different structures were solved, including Fapy·dG template-loading state (apo), error-free cytidine triphosphate (CTP) binding state (prechemistry), error-prone ATP binding state (prechemistry), error-free Fapy·dG:C product state (postchemistry), and error-prone Fapy·dG:A product state (postchemistry), revealing distinctive nucleotide binding and product states. Taken together, our study provides a comprehensive mechanistic framework for better understanding how Fapy·dG lesions impact transcription and subsequent pathological consequences.
Assuntos
Dano ao DNA , Pirimidinas , RNA Polimerase II , Humanos , RNA Polimerase II/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Cinética , Mutagênese , DesoxiguanosinaRESUMO
Tandem lesions, which are defined by two or more contiguously damaged nucleotides, are a hallmark of ionizing radiation. Recently, tandem lesions containing 5-formyl-2'-deoxyuridine (5-fdU) flanked by a 5'-8-OxodGuo or Fapyâ¢dG were discovered, and they are more mutagenic in human cells than the isolated lesions. In the current study, we examined replication of these tandem lesions in Escherichia coli. Bypass efficiency of both tandem lesions was reduced by 30-40% compared to the isolated lesions. Mutation frequencies (MFs) of isolated 8-OxodGuo and Fapyâ¢dG were low, and no mutants were isolated from replication of a 5-fdU construct. The types of mutations from 8-OxodGuo were targeted G â T transversion, whereas Fapyâ¢dG predominantly gave G â T and G deletion. 5'-8-OxodGuo-5-fdU also gave exclusively G â T mutation, which was 3-fold and 11-fold greater, without and with SOS induction, respectively, compared to that of an isolated 8-OxodGuo. In mutY/mutM cells, the MF of 8-OxodGuo and 5'-8-OxodGuo-5-fdU increased 13-fold and 7-fold, respectively. The MF of 5'-8-OxodGuo-5-fdU increased 2-fold and 3-fold in Pol II- and Pol IV-deficient cells, respectively, suggesting that these polymerases carry out largely error-free bypass. The MF of 5'- Fapyâ¢dG-5-fdU was similar without (13 ± 1%) and with (16 ± 2%) SOS induction. Unlike the complex mutation spectrum reported earlier in human cells for 5'- Fapyâ¢dG-5-fdU, with G â T as the major type of errors, in E. coli, the mutations were predominantly from deletion of 5-fdU. We postulate that removal of adenine-incorporated opposite 8-OxodGuo by Fpg and MutY repair proteins is partially impaired in the tandem 5'-8-OxodGuo-5-fdU, resulting in an increase in the G â T mutations, whereas a slippage mechanism may be operating in the 5'- Fapyâ¢dG-5-fdU mutagenesis. This study showed that not only are these tandem lesions more mutagenic than the isolated lesions but they may also exhibit different types of mutations in different organisms.
Assuntos
8-Hidroxi-2'-Desoxiguanosina , Escherichia coli , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Desoxiuridina/farmacologia , Mutagênicos/toxicidade , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Mutação , Mutagênese , Dano ao DNARESUMO
BACKGROUND: Studies have shown an association between medial meniscus posterior root tears (MMPRT) and morphologic characteristics of the bone. However, the association between distal femoral bone morphology and MMPRT, particularly the medial femoral posterior condyle, is poorly understood. Our study aimed to determine the association between the morphologic characteristics of the medial posterior femoral condyle and MMPRT. METHODS: A retrospective case-control study was performed from January 2021 to January 2022. After screening based on the inclusion and exclusion criteria, two matched groups were analyzed: the MMPRT group and the isolated lateral meniscus tears group. The hip-knee-ankle angle (HKA) and Kellgren-Lawrence grade (KLG) were measured on radiographs; the medial tibial slope angle (MTSA), medial tibial plateau depth (MTPD), and radius of the medial femoral posterior condyle (RMFPC) were measured on magnetic resonance imaging (MRI) in both groups. The area under the curve (AUC) and the best cutoff value for predicting MMPRT were calculated by using receiver operating characteristic (ROC) curve analysis. RESULTS: The final analysis included a total of 174 patients (87 MMPRT patients and 87 controls). Significant differences were shown in the RMFPC (17.6 ± 1.0 vs. 16.2 ± 1.0, p < 0.01) and MTSA (6.4 ± 2.0 vs. 4.0 ± 1.3, p < 0.01), which were larger than those of the control group. The MTPD (1.8 ± 0.6 vs. 2.9 ± 0.7, p < 0.01) and HKA (175.4 ± 2.2 vs. 179.0 ± 2.7, p < 0.01) of the injury group were significantly different from the control group, and both were lower than the control group. However, between the MMPRT and control groups on the KLG (2.3 ± 0.6 vs. 2.2 ± 0.6, p = 0.209), there was no statistically significant difference. Among them, the RMFPC cutoff value was calculated to be 16.8 mm by ROC curve analysis, and the sensitivity and specificity were both 81.61%. CONCLUSIONS: This study demonstrated that larger RMFPC, MTSA, smaller MTPD, and HKA were all associated with MMPRT, and RMFPC ≥ 16.8 mm was considered as a significant risk factor for MMPRT.
Assuntos
Fêmur , Imageamento por Ressonância Magnética , Lesões do Menisco Tibial , Humanos , Estudos Retrospectivos , Masculino , Feminino , Lesões do Menisco Tibial/diagnóstico por imagem , Pessoa de Meia-Idade , Estudos de Casos e Controles , Fatores de Risco , Adulto , Fêmur/diagnóstico por imagem , Fêmur/patologia , Rádio (Anatomia)/diagnóstico por imagem , Meniscos Tibiais/diagnóstico por imagem , Meniscos Tibiais/patologiaRESUMO
Heavy metal pollution in farmland soil has become increasingly severe, and multi-element composite pollution has brought enormous harm to human production and life. Environmental changes in cold regions (such as freeze-thaw cycles and dry-wet alternations) may increase the potential physiological toxicity of heavy metals and exacerbate pollution risks. In order to reveal the effectiveness of sepiolite modified biochar in the remediation of the soil contaminated with lead (Pb), cadmium (Cd), and chromium (Cr), the rice husk biochar pyrolyzed at 500 and 800 °C were selected for remediation treatment (denoted as BC500 and BC800). Meanwhile, different proportions of sepiolite were used for modification (biochar: sepiolite = 1: 0.5 and 1: 1), denoted as MBC500/MBC800 and HBC500/HBC800, respectively. The results showed that modified biochar with sepiolite can effectively improve the immobilization of heavy metals. Under natural conservation condition, the amount of diethylenetriaminepentaacetic acid (DTPA) extractable Pb in BC500, MBC500, and HBC500 decreased by 5.95, 12.39, and 13.55%, respectively, compared to CK. Freeze-thaw cycles and dry-wet alternations activated soil heavy metals, while modified biochar increased adsorption sites and oxygen-containing functional groups under aging conditions, inhibiting the fractions transformation of heavy metals. Furthermore, freeze-thaw cycles promoted the decomposition and mineralization of soil organic carbon (SOC), while sepiolite hindered the release of active carbon through ion exchange and adsorption complexation. Among them, and the soil dissolved organic carbon (DOC) content in HBC800 decreased by 49.39% compared to BC800. Additionally, the high-temperature pyrolyzed biochar (BC800) enhanced the porosity richness and alkalinity of material, which effectively inhibited the migration and transformation of heavy metals compared to BC500, and reduced the decomposition of soil DOC.
Assuntos
Carbono , Carvão Vegetal , Argila , Metais Pesados , Poluentes do Solo , Solo , Metais Pesados/química , Carvão Vegetal/química , Solo/química , Argila/química , Poluentes do Solo/química , Carbono/química , Adsorção , Minerais/química , Recuperação e Remediação Ambiental/métodosRESUMO
In 2022, the value of United States (US) beef and beef product exports was $11.7 billion, and the US was the world's largest beef producer and second-largest beef exporter by volume. Therefore, we conducted surveys to evaluate beef purchasing behavior among consumers in important and emerging US beef export markets, including Japan, the United Kingdom (UK), Germany, and Mexico. Results reveal differences in consumers' beef purchasing behavior across countries. Most Mexican consumers purchase beef two-to-three times a week, while consumers in other countries typically purchase it once a week. Using ordered probit models, we examined the factors associated with beef purchase frequency in each country. Japanese consumers who consider price to be an important factor when purchasing beef are less likely to purchase it frequently. German consumers, for whom brands are important when buying beef, are more likely to buy it frequently. British consumers, who consider hormone-free production to be important when purchasing beef, are less likely to buy it frequently. Mexican consumers, who consider grass-fed production to be an important factor when purchasing beef, are less likely to buy it frequently. Across all countries, individuals who purchase beef at supermarkets and from butchers are more likely to purchase it more often. Results also indicate that various consumer demographics are associated with beef purchase frequency across countries. The findings provide valuable insights for stakeholders regarding international consumer beef purchasing behavior.
RESUMO
N6-(2-deoxy-α,ß-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapyâ¢dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). Fapyâ¢dG preferentially gives rise to G â T transversions and G â A transitions. However, the molecular basis by which Fapyâ¢dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase ß (Pol ß), a model mammalian polymerase, bypasses a templating Fapyâ¢dG, inserts Fapyâ¢dGTP, and extends from Fapyâ¢dG at the primer terminus. When Fapyâ¢dG is present in the template, Pol ß incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapyâ¢dGTP is a poor substrate but is incorporated â¼3-times more efficiently opposite dA than dC. Extension from Fapyâ¢dG at the 3'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapyâ¢dG is likely to be the source of the mutagenic effects of the lesion and not Fapyâ¢dGTP. These experiments increase our understanding of the promutagenic effects of Fapyâ¢dG.
RESUMO
Emerging contaminants (ECs) are widely sourced persistent pollutants that pose a significant threat to the environment and human health. Their footprint spans global ecosystems, making their remediation highly challenging. In recent years, a significant amount of literature has focused on the use of biochar for remediation of heavy metals and organic pollutants in soil and water environments. However, the use of biochar for the remediation of ECs in agricultural soils has not received as much attention, and as a result, there are limited reviews available on this topic. Thus, this review aims to provide an overview of the primary types, sources, and hazards of ECs in farmland, as well as the structure, functions, and preparation types of biochar. Furthermore, this paper emphasizes the importance and prospects of three remediation strategies for ECs in cropland: (i) employing activated, modified, and composite biochar for remediation, which exhibit superior pollutant removal compared to pure biochar; (ii) exploring the potential synergistic efficiency between biochar and compost, enhancing their effectiveness in soil improvement and pollution remediation; (iii) utilizing biochar as a shelter and nutrient source for microorganisms in biochar-mediated microbial remediation, positively impacting soil properties and microbial community structure. Given the increasing global prevalence of ECs, the remediation strategies provided in this paper aim to serve as a valuable reference for future remediation of ECs-contaminated agricultural lands.