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1.
Sci Rep ; 14(1): 23134, 2024 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-39379474

RESUMO

De novo mutations (DNMs) are drivers of genetic disorders. However, the study of DNMs is hampered by technological limitations preventing accurate quantification of ultra-rare mutations. Duplex Sequencing (DS) theoretically has < 1 error/billion base-pairs (bp). To determine the DS utility to quantify and characterize DNMs, we analyzed DNA from blood and spermatozoa from six healthy, 18-year-old Swedish men using the TwinStrand DS mutagenesis panel (48 kb spanning 20 genic and intergenic loci). The mean single nucleotide variant mutation frequency (MF) was 1.2 × 10- 7 per bp in blood and 2.5 × 10- 8 per bp in sperm, with the most common base substitution being C > T. Blood MF and substitution spectrum were similar to those reported in blood cells with an orthogonal method. The sperm MF was in the same order of magnitude and had a strikingly similar spectrum to DNMs from publicly available whole genome sequencing data from human pedigrees (1.2 × 10- 8 per bp). DS revealed much larger numbers of insertions and deletions in sperm over blood, driven by an abundance of putative extra-chromosomal circular DNAs. The study indicates the strong potential of DS to characterize human DNMs to inform factors that contribute to disease susceptibility and heritable genetic risks.


Assuntos
Taxa de Mutação , Mutação , Espermatozoides , Humanos , Masculino , Espermatozoides/metabolismo , Adolescente , Análise Mutacional de DNA/métodos , Polimorfismo de Nucleotídeo Único , Sequenciamento de Nucleotídeos em Larga Escala , Suécia , Análise de Sequência de DNA/métodos
2.
Pestic Biochem Physiol ; 205: 106164, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39477617

RESUMO

It is urgent to solve insecticide resistance issues for fall armyworm (FAW), Spodoptera frugiperda. Some acetylcholinesterase-1 (Ace-1) mutations (A201S, G227A and F290V) have been identified as a cause of FAW resistance to organophosphates (OPs) and carbamates insecticides (CXs). However, the structural biological mechanisms on the relationship between the Ace-1 mutations and resistance to OPs and CXs still remain elusive. In this study, the A201S and F290V mutaions were found in eight fields populations of FAW except the G227A. Molecular docking revealed that the four Ace-1 proteins (Ace1-WT, Ace1-A201S, Ace1-G227A and Ace1-F290V) had the same binding modes and the same binding energies with acetylcholine (Ach), trichlorfon, chlorpyrifos, methomyl, carbaryl and chlorpyrifos oxide. The structural biological analysis revealed that the A201S mutations can enhance enzyme catalytic efficiency by introducing the hydroxyl group (-OH) from serine which performed the same function as the main-chain -NH and enhanced the interaction with the carboxy oxygen of acetylcholine (Ach), and the F290V mutation can effectively improve FAW resistance to insecticides by increasing the likelihood of Ach to enter the enzyme's active center for phenylalanine replaced by smaller valine under insecticide inhibition conditions. The bioassays and age-stage-specific life table analysis of FAW-SS and FAW-F290V populations revealed that F290V mutation effectively contributed to FAW resistance with a low fitness cost. This study provides a theoretical basis for future pest resistance management.


Assuntos
Acetilcolinesterase , Resistência a Inseticidas , Inseticidas , Simulação de Acoplamento Molecular , Mutação , Spodoptera , Animais , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Inseticidas/farmacologia , Resistência a Inseticidas/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/química
3.
Environ Mol Mutagen ; 65(8): 234-242, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39267335

RESUMO

Regulatory genetic toxicology testing is essential for identifying potentially mutagenic hazards. Duplex Sequencing (DS) is an error-corrected next-generation sequencing technology that provides substantial advantages for mutation analysis over conventional mutagenicity assays including: improved accuracy of mutation detection, ability to measure changes in mutation spectrum, and applicability across diverse biological models. To apply DS for regulatory toxicology testing, power analyses are required to determine suitable sample sizes and study designs. In this study, we explored study designs to achieve sufficient power for various effect sizes in chemical mutagenicity assessment. We collected data from MutaMouse bone marrow and liver samples that were analyzed by DS using TwinStrand's Mouse Mutagenesis Panel. Average duplex reads achieved in two separates studies on liver and bone marrow were 8.4 × 108 (± 7.4 × 107) and 9.5 × 108 (± 1.0 × 108), respectively. Baseline mean mutation frequencies (MF) were 4.6 × 10-8 (± 6.7 × 10-9) and 4.6 × 10-8 (± 1.1 × 10-8), with estimated standard deviations for the animal-to-animal random effect of 0.15 and 0.20, for liver and bone marrow, respectively. We conducted simulation analyses based on these empirically derived parameters. We found that a sample size of four animals per group is sufficient to obtain over 80% power to detect a two-fold change in MF relative to baseline. In addition, we estimated the minimal total number of informative duplex bases sequenced with different sample sizes required to retain power for various effect sizes. Our work provides foundational data for establishing suitable study designs for mutagenicity testing using DS.


Assuntos
Medula Óssea , Sequenciamento de Nucleotídeos em Larga Escala , Fígado , Testes de Mutagenicidade , Animais , Medula Óssea/efeitos dos fármacos , Fígado/efeitos dos fármacos , Testes de Mutagenicidade/métodos , Camundongos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutagênicos/toxicidade , Mutação/efeitos dos fármacos , Taxa de Mutação , Projetos de Pesquisa , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Masculino
4.
Heliyon ; 10(17): e37387, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-39296094

RESUMO

Gladiolus is a highly allogamous flower plant, but owing to the prolonged juvenile phase, asexual propagation is preferred, which acts as a barrier for the induction of natural genetic variability in gladiolus. Therefore, the induced mutagenesis could be utilized for the creation of desirable genotypes, without altering their basic agronomic features. An analysis of the optimum doses of γ radiation for the induction of fruitful mutations could be achieved in short period of time, compared with the conventional method of breeding. The objectives of this study were to perform radiosensitivity tests on various gladiolus genotypes using different doses of gamma rays and to determine the optimal dose of radiation dose for obtaining the greatest number of mutants. The present experiment was carried out during the winter-spring seasons, for the four consecutive years of 2017-18, 2018-19, 2019-20, and 2020-21. The seven genotypes of gladiolus were exposed to seven doses of gamma rays (60Cobalt). Plants irradiated with radiation doses lower than 4.5 Kr (G1) had greater plant survivability than the higher doses of gamma rays (≥5.0 Kr). The radiation of G0 (0 Kr) result in highest plant survivability, while radiation dose of G6 (6.5 Kr) resulted lowest survivability. LD25 and BD50 for all the genotypes were achieved except for V5 and V7, similarly the median lethal doses (LD50) for V3 and V4 genotypes had been achieved. The highest flower blindness percent and percent abnormal plants were observed at G5 and G6 and between the 4.0 Kr (G1) and 5.5 Kr (G4) gamma ray doses, respectively. The flower colour mutation frequency was recorded highest in genotypes Tiger Flame at 5.0 Kr (V7G3), while the Flower colour mutation spectrum was identified between 4.0 Kr (G1) to 5.5 Kr (G4) in all the genotypes except for genotypes V5 and V7. For the generation of higher phenotypic variations, radiation dose between 4.0 Kr (G1) and 5.5 Kr (G4) were found the most prominent. Specifically the gamma rays radiation dose of 5.5 Kr (G4) resulted in the highest flower colour mutation frequency. These isolated mutant lines will broaden the gladiolus gene pool and support future gladiolus breeding experiments.

5.
Discov Oncol ; 15(1): 457, 2024 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-39292326

RESUMO

BACKGROUND: The study aims to comprehensively combine colorectal cancer data cohorts in order to analyze the effects of various DNA methylation-coding genes on colorectal cancer patients. The annual incidence and mortality of colorectal cancer are very high, and there are no effective treatments for advanced colorectal cancer. DNA methylation is a method widely used to regulate epigenetics in the molecular mechanism study of tumors. METHOD: Three single-cell cohorts GSE166555, GSE146771, and EMTAB8107, and five transcriptome cohorts GSE17536, GSE39582, GSE72970, and TCGA-CRC (TCGA-COAD and TCGA-READ) were applied in this study. 2 erasers (ALKBH5 and FTO), There are 7 writers (METTL3, METTL14, WTAP, VIRMA, RBM15, RBM15B, and ZC3H13) and 11 readers (YTHDC1, IGF2BP1, IGF2BP2, IGF2BP3, YTHDF1, YTHDF3, YTHDC2, and HNRNPA2B1, YTHDF2, HNRNPC and RBMX), a total of 20 M6A regulators, were used as the basis of the dataset in this study and were applied to the construction of molecular typing and prognostic models. Drugs that are differentially sensitive in methylation-regulated gene-related prognostic models were identified using the ConsensusClusterPlus package, which was also used to identify distinct methylation regulatory expression patterns in colorectal cancer and to model the relationship between tissue gene expression profiles and drug IC50 values. Finally, TISCH2 assessed which immune cells were significantly expressed with M6A scores. The immunosuppression of M6A methylation is spatially explained. RESULTS: This study used data from 583 CRC patients in the TCGA-CRC cohort. Firstly, the mutation frequency and CNV variation frequency of 20 m6A modification-related factors were analyzed, and the corresponding histogram and heat map were drawn. The study next analyzed the expression variations between mutant and wild forms of the VIRMA gene and explored differences in the expression of these variables in tumor and normal tissues. In addition, the samples were divided into different subgroups by molecular clustering method based on m6A modification, and each subgroup's expression and clinicopathological characteristics were analyzed. Finally, we compared prognostic differences, tumor microenvironment (TME) characteristics, immune cell infiltration, and gene function enrichment among different subpopulations. We also developed a colorectal cancer m6A-associated gene signature and validated its prognostic effects across multiple cohorts. Finally, using single-cell RNA sequencing data, we confirmed that tumor cells show elevated expression of m6A-related gene signatures. DISCUSSION: This study explored the mutation frequency, expression differences, interactions, molecular clustering, prognostic effect, and association with tumor characteristics of m6A modification-related factors in CRC and validated them at the single-cell level. These results clarify the association between m6A alteration and colorectal cancer (CRC) and offer important insights into the molecular recognition and management of cancer.

6.
DNA Repair (Amst) ; 139: 103693, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38776712

RESUMO

MutT proteins belong to the Nudix hydrolase superfamily that includes a diverse group of Mg2+ requiring enzymes. These proteins use a generalized substrate, nucleoside diphosphate linked to a chemical group X (NDP-X), to produce nucleoside monophosphate (NMP) and the moiety X linked with phosphate (XP). E. coli MutT (EcoMutT) and mycobacterial MutT1 (MsmMutT1) belong to the Nudix hydrolase superfamily that utilize 8-oxo-(d)GTP (referring to both 8-oxo-GTP or 8-oxo-dGTP). However, predominant products of their activities are different. While EcoMutT produces 8-oxo-(d)GMP, MsmMutT1 gives rise to 8-oxo-(d)GDP. Here, we show that the altered cleavage specificities of the two proteins are largely a consequence of the variation at the equivalent of Gly37 (G37) in EcoMutT to Lys (K65) in the MsmMutT1. Remarkably, mutations of G37K (EcoMutT) and K65G (MsmMutT1) switch their cleavage specificities to produce 8-oxo-(d)GDP, and 8-oxo-(d)GMP, respectively. Further, a time course analysis using 8-oxo-GTP suggests that MsmMutT1(K65G) hydrolyses 8-oxo-(d)GTP to 8-oxo-(d)GMP in a two-step reaction via 8-oxo-(d)GDP intermediate. Expectedly, unlike EcoMutT (G37K) and MsmMutT1, EcoMutT and MsmMutT1 (K65G) rescue an E. coli ΔmutT strain, better by decreasing A to C mutations.


Assuntos
Nucleotídeos de Desoxiguanina , Proteínas de Escherichia coli , Escherichia coli , Mycobacterium smegmatis , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Especificidade por Substrato , Nucleotídeos de Desoxiguanina/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Substituição de Aminoácidos , Pirofosfatases/metabolismo , Pirofosfatases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/análogos & derivados
7.
Methods Mol Biol ; 2797: 1-12, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38570448

RESUMO

RAS research has entered the world of translational and clinical science. Progress has been based on our appreciation of the role of RAS mutations in different types of cancer and the effects of these mutations on the biochemical, structural, and biophysical properties of the RAS proteins themselves, particularly KRAS, on which most attention has been focused. This knowledge base, while still growing, has enabled creative chemical approaches to targeting KRAS directly. Our understanding of RAS signaling pathways in normal and cancer cells plays an important role for developing RAS inhibitors but also continues to reveal new approaches to targeting RAS through disruption of signaling complexes and downstream pathways.


Assuntos
Antineoplásicos , Neoplasias , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Mutação , Neoplasias/metabolismo , Transdução de Sinais , Antineoplásicos/farmacologia
8.
Food Chem Toxicol ; 185: 114512, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38342231

RESUMO

Duplex sequencing (DS) is an error-corrected next-generation sequencing (NGS) method that can overcome notorious high error rate from the process of NGS and detect ultralow-frequency mutations. In this study, we evaluated the mutagenicity of aristolochic acid, a known genotoxic carcinogen, and methapyrilene, a known nongenotoxic carcinogen using DS. Four male Fisher 344 rats were treated with aristolochic acid, methapyrilene, or the vehicle control for 6 weeks, liver tissues were collected one day after the treatment, and the DNA was isolated for analysis. The mutation frequency for the aristolochic acid-treated group was significantly increased over the vehicle control (44-fold), whereas no significant difference in the mutation frequency was observed between the methapyrilene-treated and the control groups. The primary type of mutation induced by aristolochic acid was A:T > T:A transversion, which occurred frequently at ApT sites, whereas the major type of mutation in the control and methapyrilene-treated groups was G:C > A:T transition, which occurred frequently at CpG sites. These findings are consistent with previously published data obtained with other in vivo mutation assays. Thus, our results suggest that the DS mutation assay is a promising technology for assessing mutagenicity of chemicals in vivo.


Assuntos
Ácidos Aristolóquicos , Metapirileno , Ratos , Animais , Masculino , Mutagênicos/toxicidade , Ácidos Aristolóquicos/toxicidade , Carcinógenos/toxicidade
9.
Chromosome Res ; 31(4): 32, 2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37910282

RESUMO

This review investigates the role of aneuploidy and chromosome instability (CIN) in the aging brain. Aneuploidy refers to an abnormal chromosomal count, deviating from the normal diploid set. It can manifest as either a deficiency or excess of chromosomes. CIN encompasses a broader range of chromosomal alterations, including aneuploidy as well as structural modifications in DNA. We provide an overview of the state-of-the-art methodologies utilized for studying aneuploidy and CIN in non-tumor somatic tissues devoid of clonally expanded populations of aneuploid cells.CIN and aneuploidy, well-established hallmarks of cancer cells, are also associated with the aging process. In non-transformed cells, aneuploidy can contribute to functional impairment and developmental disorders. Despite the importance of understanding the prevalence and specific consequences of aneuploidy and CIN in the aging brain, these aspects remain incompletely understood, emphasizing the need for further scientific investigations.This comprehensive review consolidates the present understanding, addresses discrepancies in the literature, and provides valuable insights for future research efforts.


Assuntos
Aneuploidia , Neoplasias , Animais , Humanos , Instabilidade Cromossômica , Aberrações Cromossômicas , Encéfalo , Cromossomos , Neoplasias/genética , Mamíferos/genética
10.
Hemoglobin ; 47(4): 147-151, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37548174

RESUMO

Beta Thalassemia is the most prevalent and well-studied single gene disorder in Iran. Here, we investigated the spectrum of HBB gene mutations, identified among 2315 patients, referred to a reference thalassemia clinic in Tehran, on the basis of suspicion to thalassemia major or intermedia. The patients were homozygous or compound heterozygous for HBB mutations, and were referred from various Iranian provinces, during 15 years (2001- 2016). The HBB mutations were classified based on their frequency, and the result was compared to a meta-analysis of 14,293 beta thalassemia cases in the Iranian population, within the same time period. The mutation spectrum in this study contained 43 HBB mutations, compared to the 90, presented by the meta-analysis. Similar to the meta-analysis, IVSII-1 (G > A) and IVSI-5 (G > C) were the most common mutations in this study. These two comprised 62.40% of the total HBB mutant alleles in the studied population, comparable to 51.92% of that in the meta-analysis. IVSII-1 (G > A) and IVSI-5 (G > C), followed by 17 other mutations that had frequencies ranging from 0.15% to 5.44%, were among the 20 common HBB mutations in Iran and neighboring countries, according to the meta-analysis. This study provided further evidence to support the spectrum of the most common HBB mutations in the Iranian population.


Assuntos
Talassemia , Talassemia beta , Humanos , Talassemia beta/diagnóstico , Talassemia beta/epidemiologia , Talassemia beta/genética , Irã (Geográfico)/epidemiologia , Globinas beta/genética , Mutação , Genótipo
11.
Life (Basel) ; 13(7)2023 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-37511853

RESUMO

Somatic mutations in the promoter region of the human telomerase reverse transcriptase (hTERT) gene have been identified in many types of cancer. The hTERT promoter is known to be enriched with sequences that enable the formation of G-quadruplex (G4) structures, whose presence is associated with elevated mutagenicity and genome instability. Here, we used a bioinformatics tool (QGRS mapper) to search for G4-forming sequences (G4 motifs) in the 1000 bp TERT promoter regions of 141 mammalian species belonging to 20 orders, 5 of which, including primates and predators, contain more than 10 species. Groups of conserved G4 motifs and single-nucleotide variants within these groups were discovered using a block alignment approach (based on the Nucleotide PanGenome explorer). It has been shown that: (i) G4 motifs are predominantly located in the region proximal to the transcription start site (up to 400 bp) and are over-represented on the non-coding strand of the TERT promoters, (ii) 11 to 22% of the G4 motifs found are evolutionarily conserved across the related organisms, and (iii) a statistically significant higher frequency of nucleotide substitutions in the conserved G4 motifs compared to the surrounding regions was confirmed only for the order Primates. These data support the assumption that G4s can interfere with the DNA repair process and affect the evolutionary adaptation of organisms and species.

12.
Antimicrob Agents Chemother ; 67(6): e0156722, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37212672

RESUMO

Benzoxaboroles are a new class of leucyl-tRNA synthetase inhibitors. Epetraborole, a benzoxaborole, is a clinical candidate developed for Gram-negative infections and has been confirmed to exhibit favorable activity against a well known pulmonary pathogen, Mycobacterium abscessus. However, according to ClinicalTrials.gov, in 2017, a clinical phase II study on the use of epetraborole to treat complicated urinary tract and intra-abdominal infections was terminated due to the rapid emergence of drug resistance during treatment. Nevertheless, epetraborole is in clinical development for nontuberculous mycobacteria (NTM) disease especially for Mycobacterium avium complex-related pulmonary disease (MAC-PD). DS86760016, an epetraborole analog, was further demonstrated to have an improved pharmacokinetic profile, lower plasma clearance, longer plasma half-life, and higher renal excretion than epetraborole in animal models. In this study, DS86760016 was found to be similarly active against M. abscessus in vitro, intracellularly, and in zebrafish infection models with a low mutation frequency. These results expand the diversity of druggable compounds as new benzoxaborole-based candidates for treating M. abscessus diseases.


Assuntos
Aminoacil-tRNA Sintetases , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Animais , Peixe-Zebra , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Micobactérias não Tuberculosas
13.
Cell Rep ; 42(3): 112199, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36870054

RESUMO

The DNA-alkylating metabolite tilimycin is a microbial genotoxin. Intestinal accumulation of tilimycin in individuals carrying til+ Klebsiella spp. causes apoptotic erosion of the epithelium and colitis. Renewal of the intestinal lining and response to injury requires the activities of stem cells located at the base of intestinal crypts. This study interrogates the consequences of tilimycin-induced DNA damage to cycling stem cells. We charted the spatial distribution and luminal quantities of til metabolites in Klebsiella-colonized mice in the context of a complex microbial community. Loss of marker gene G6pd function indicates genetic aberrations in colorectal stem cells that became stabilized in monoclonal mutant crypts. Mice colonized with tilimycin-producing Klebsiella displayed both higher frequencies of somatic mutation and more mutations per affected individual than animals carrying a non-producing mutant. Our findings imply that genotoxic til+ Klebsiella may drive somatic genetic change in the colon and increase disease susceptibility in human hosts.


Assuntos
Microbiota , Mutagênicos , Humanos , Camundongos , Animais , Mutagênicos/metabolismo , Colo/metabolismo , Mutação/genética , Células-Tronco , Mucosa Intestinal
14.
Microb Pathog ; 176: 106023, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36736799

RESUMO

In the present study 7,7-Dimethyl-4-(4-trifluoromethyl-phenylamino)-2,4,4a,6,7,8-hexahydro-benzo[d] [1,3]thiazin-5-one (DFMBT) was synthesized and evaluated for in vitro activity against Mycobacterium tuberculosis (M.tb) H37RV. Results demonstrated that at 64x MIC, DFMBT completely sterilized the TB culture from day 4 of the incubation whereas at 32 and 16x MIC, it sterilized the TB culture from day 8. The bacterial cultures were completely sterilized by DFMBT at 8x MIC from day 16 of incubation. DFMBT showed 1.5 µg/mL MIC value as compared to the standard anti-tuberculosis drugs using broth macro-dilution method. The MBC value of DFMBT was found to be 6.0 µg/mL whereas for INH, RIF, AMK and LVX the values were found to be 0.312, 0.156, 5.0 and 5.0 µg/mL, respectively. The DFMBT in combination with INH/RIF or AMK showed the ∑FIC value of 0.258, 0.252 and 0.453, respectively indicating synergistic interaction. Moreover, the value of ∑FIC for the combination of DFMBT with LVX was found to be 1.33 suggesting and additive interaction. The post antibiotic effect of DFMBT at 1x and 64x MIC was found to be 29.89 ± 10.12 and 158.75 ± 17.50 h, respectively. The DFMBT showed an MPC value of 150 µg/mL which was intermediate between INH and RIF. In summary, DFMBT exhibits bacteriostatic as well as bactericidal effect on Mycobacterium tuberculosis H37RV. It has synergistic interaction with INH, RIF and AMK anti-TB drugs, descent post antibiotic effect, mutation frequency and mutant prevention concentration. Thus, DFMBT may be developed as an effective agent as anti-TB compound.


Assuntos
Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Antituberculosos/farmacologia , Interações Medicamentosas , Sinergismo Farmacológico , Testes de Sensibilidade Microbiana , Isoniazida/farmacologia
15.
Proc Natl Acad Sci U S A ; 120(2): e2216216120, 2023 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-36595701

RESUMO

The rise of antibiotic-resistant bacterial infections poses a global threat. Antibiotic resistance development is generally studied in batch cultures which conceals the heterogeneity in cellular responses. Using single-cell imaging, we studied the growth response of Escherichia coli to sub-inhibitory and inhibitory concentrations of nine antibiotics. We found that the heterogeneity in growth increases more than what is expected from growth rate reduction for three out of the nine antibiotics tested. For two antibiotics (rifampicin and nitrofurantoin), we found that sub-populations were able to maintain growth at lethal antibiotic concentrations for up to 10 generations. This perseverance of growth increased the population size and led to an up to 40-fold increase in the frequency of antibiotic resistance mutations in gram-negative and gram-positive species. We conclude that antibiotic perseverance is a common phenomenon that has the potential to impact antibiotic resistance development across pathogenic bacteria.


Assuntos
Antibacterianos , Escherichia coli , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Rifampina/farmacologia , Mutação , Bactérias , Farmacorresistência Bacteriana/genética
16.
J Radiat Res ; 64(2): 345-351, 2023 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-36634340

RESUMO

Pluripotent stem cells (PSCs) have the potential to differentiate to any of the other organs. The genome DNA integrity of PSCs is maintained by a high level of transcription for a number of genes involved in DNA repair, cell cycle and apoptosis. However, it remains unclear how high the frequency of genetic mutation is and how these DNA repair factors function in PSCs. In this study, we employed Sup F assay for the measurement of mutation frequency after UV-C irradiation in induced pluripotent stem cells (iPSCs) as PSC models and neural progenitor cells (NPCs) were derived from iPSCs as differentiated cells. iPSCs and NPCs exhibited a lower mutation frequency compared with the original skin fibroblasts. In RNA-seq analysis, iPSCs and NPCs showed a high expression of RAD18, which is involved in trans-lesion synthesis (TLS) for the emergency tolerance system during the replication process of DNA. Although RAD18 is involved in both error free and error prone TLS in somatic cells, it still remains unknown the function of RAD18 in PSCs. In this study we depleted of the RAD18 by siRNA knockdown resulted in decreased frequency of mutation in iPSCs and NPCs. Our results will provide information on the genome maintenance machinery in PSCs.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Reparo do DNA , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Mutação/genética , Mutagênese , Proteínas de Ligação a DNA/metabolismo
17.
Pathology ; 55(3): 329-334, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36428107

RESUMO

Central giant cell granulomas (CGCG) are rare intraosseous osteolytic lesions of uncertain aetiology. Despite the benign nature of this neoplasia, the lesions can rapidly grow and become large, painful, invasive, and destructive. The identification of molecular drivers could help in the selection of targeted therapies for specific cases. TRPV4, KRAS and FGFR1 mutations have been associated with these lesions but no correlation between the mutations and patient features was observed so far. In this study, we analysed 17 CGCG cases of an Italian cohort and identified an interesting and significant (p=0.0021) correlation between FGFR1 mutations and age. In detail, FGFR1 mutations were observed frequently and exclusively in CGCG from young (<18 years old) patients (4/5 lesions, 80%). Furthermore, the combination between ours and previously published data confirmed a significant difference in the frequency of FGFR1 mutations in CGCG from patients younger than 18 years at the time of diagnosis (9/23 lesions, 39%) when compared to older patients (1/31 lesions, 0.03%; p=0.0011), thus corroborating our observation in a cohort of 54 patients. FGFR1 variants in young CGCG patients could favour fast lesion growth, implying that they seek medical attention earlier. Our observation might help prioritise candidates for FGFR1 testing, thus opening treatment options with FGFR inhibitors.


Assuntos
Granuloma de Células Gigantes , Humanos , Adolescente , Granuloma de Células Gigantes/genética , Granuloma de Células Gigantes/diagnóstico , Granuloma de Células Gigantes/patologia , Taxa de Mutação , Mutação , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética
19.
Antibiotics (Basel) ; 11(7)2022 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-35884174

RESUMO

Corallopyronin A (CorA) is active against Gram-positive bacteria and targets the switch region of RNA polymerase. Because of the high frequency of mutation (FoM) leading to rifampicin resistance, we determined the CorA FoM in S. aureus using fluctuation analysis at 4 × minimum inhibitory concentration (MIC). Resistant mutants were characterized. S. aureus strains HG001, Mu50, N315, and USA300 had an MIC of 0.25 mg/L. The median FoM for CorA resistance was 1.5 × 10−8, 4.5-fold lower than the median FoM of 6.7 × 10−8 for rifampicin, and was reflected in a 4-fold lower mutation rate for CorA than rifampicin (6 × 10−9 for CorA vs. 2.5 × 10−8 for rifampicin). In CorA-resistant/rifampicin-sensitive strains, the majority of amino acid exchanges were S1127L in RpoB or K334N in RpoC. S. aureus Mu50, a rifampicin-resistant clinical isolate, yielded two further exchanges targeting amino acids L1131 and E1048 of the RpoB subunit. The plating of >1011 cells on agar containing a combination of 4 × MIC of rifampicin and 4 × MIC of CorA did not yield any growth. In conclusion, with proper usage, e.g., in combination therapy and good antibiotic stewardship, CorA is a potential antibiotic for treating S. aureus infections.

20.
Int J Mol Sci ; 23(12)2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35742840

RESUMO

Monitoring SARS-CoV-2's genetic diversity and emerging mutations in this ongoing pandemic is crucial to understanding its evolution and ensuring the performance of COVID-19 diagnostic tests, vaccines, and therapies. Spain has been one of the main epicenters of COVID-19, reaching the highest number of cases and deaths per 100,000 population in Europe at the beginning of the pandemic. This study aims to investigate the epidemiology of SARS-CoV-2 in Spain and its 18 Autonomous Communities across the six epidemic waves established from February 2020 to January 2022. We report on the circulating SARS-CoV-2 variants in each epidemic wave and Spanish region and analyze the mutation frequency, amino acid (aa) conservation, and most frequent aa changes across each structural/non-structural/accessory viral protein among the Spanish sequences deposited in the GISAID database during the study period. The overall SARS-CoV-2 mutation frequency was 1.24 × 10−5. The aa conservation was >99% in the three types of protein, being non-structural the most conserved. Accessory proteins had more variable positions, while structural proteins presented more aa changes per sequence. Six main lineages spread successfully in Spain from 2020 to 2022. The presented data provide an insight into the SARS-CoV-2 circulation and genetic variability in Spain during the first two years of the pandemic.


Assuntos
COVID-19 , Pandemias , Aminoácidos/genética , COVID-19/epidemiologia , COVID-19/genética , Genoma Viral , Humanos , Mutação , Filogenia , SARS-CoV-2/genética , Espanha/epidemiologia
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