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1.
Molecules ; 29(7)2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38611760

RESUMO

A multi-residue UHPLC-MS/MS analytical method, previously developed for monitoring 52 pharmaceuticals in drinking water, was used to analyse these pharmaceuticals in wastewater originating from healthcare facilities in the Czech Republic. Furthermore, the methodology was expanded to include the evaluation of the effectiveness of drug removal in Czech wastewater treatment plants (WWTPs). Of the 18 wastewater samples analysed by the validated UHPLC-MS/MS, each sample contained at least one quantifiable analyte. This study reveals the prevalence of several different drugs; mean concentrations of 702 µg L-1 of iomeprol, 48.8 µg L-1 of iopromide, 29.9 µg L-1 of gabapentin, 42.0 µg L-1 of caffeine and 82.5 µg L-1 of paracetamol were present. An analysis of 20 samples from ten WWTPs revealed different removal efficiencies for different analytes. Paracetamol was present in the inflow samples of all ten WWTPs and its removal efficiency was 100%. Analytes such as caffeine, ketoprofen, naproxen or atenolol showed high removal efficiencies exceeding 80%. On the other hand, pharmaceuticals like furosemide, metoprolol, iomeprol, zolpidem and tramadol showed lower removal efficiencies. Four pharmaceuticals exhibited higher concentrations in WWTP effluents than in the influents, resulting in negative removal efficiencies: warfarin at -9.5%, indomethacin at -53%, trimethoprim at -54% and metronidazole at -110%. These comprehensive findings contribute valuable insights to the pharmaceutical landscape of wastewater from healthcare facilities and the varied removal efficiencies of Czech WWTPs, which together with the already published literature, gives a more complete picture of the burden on the aquatic environment.


Assuntos
Acetaminofen , Cosméticos , Iopamidol/análogos & derivados , Humanos , Cafeína , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em Tandem , Águas Residuárias , Preparações Farmacêuticas
2.
Molecules ; 28(15)2023 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-37570870

RESUMO

(1) The occurrence and accumulation of pharmaceuticals and personal care products in the environment are recognized scientific concerns. Many of these compounds are disposed of in an unchanged or metabolized form through sewage systems and wastewater treatment plants (WWTP). WWTP processes do not completely eliminate all active substances or their metabolites. Therefore, they systematically leach into the water system and are increasingly contaminating ground, surface, and drinking water, representing a health risk largely ignored by legislative bodies. Especially during the COVID-19 pandemic, a significantly larger amount of medicines and protective products were consumed. It is therefore likely that contamination of water sources has increased, and in the case of groundwater with a delayed effect. As a result, it is necessary to develop an accurate, rapid, and easily available method applicable to routine screening analyses of potable water to monitor and estimate their potential health risk. (2) A multi-residue UHPLC-MS/MS analytical method designed for the identification of 52 pharmaceutical products was developed and used to monitor their presence in drinking water. (3) The optimized method achieved good validation parameters, with recovery of 70-120% of most analytes and repeatability achieving results within 20%. In real samples of drinking water, at least one analyte above the limit of determination was detected in each of the 15 tap water and groundwater samples analyzed. (4) These findings highlight the need for legislation to address pharmaceutical contamination in the environment.


Assuntos
COVID-19 , Cosméticos , Água Potável , Poluentes Químicos da Água , Humanos , Água Potável/química , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Poluentes Químicos da Água/análise , Pandemias , Monitoramento Ambiental/métodos , COVID-19/epidemiologia , Cosméticos/análise , Preparações Farmacêuticas
3.
Nucleic Acids Res ; 49(1): 285-305, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33332547

RESUMO

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.


Assuntos
DNA de Cadeia Simples/metabolismo , Recombinação Homóloga , Proteínas Motores Moleculares/metabolismo , RecQ Helicases/metabolismo , Imagem Individual de Molécula , Trifosfato de Adenosina/metabolismo , DNA de Cadeia Simples/ultraestrutura , Humanos , Hidrólise , Cinética , Microscopia de Força Atômica , Proteínas Motores Moleculares/ultraestrutura , Mutação de Sentido Incorreto , Mutação Puntual , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RecQ Helicases/genética , RecQ Helicases/ultraestrutura , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/metabolismo , Especificidade por Substrato
4.
Cell Rep ; 33(12): 108543, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33357432

RESUMO

DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51K133A cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNAK164R increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2H188A, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2H188A associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance.


Assuntos
Replicação do DNA , Exodesoxirribonucleases/metabolismo , Mutação , Fosfoproteínas/metabolismo , Rad51 Recombinase/metabolismo , Animais , Exodesoxirribonucleases/genética , Humanos , Masculino , Camundongos , Fosfoproteínas/genética , Rad51 Recombinase/biossíntese , Rad51 Recombinase/genética , Transfecção
5.
J Cell Sci ; 131(13)2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29898918

RESUMO

The canonical role of cohesin is to mediate sister chromatid cohesion. In addition, cohesin plays important roles in processes such as DNA repair and regulation of gene expression. Mounting evidence suggests that various post-translational modifications, including phosphorylation, acetylation and sumoylation regulate cohesin functions. Our mass spectrometry analysis of cohesin purified from Schizosaccharomyces pombe cells revealed that the cohesin subunit Psm1 is methylated on two evolutionarily conserved lysine residues, K536 and K1200. We found that mutations that prevent methylation of Psm1 K536 and K1200 render sensitivity to DNA-damaging agents and show positive genetic interactions with mutations in genes encoding the Mus81-Eme1 endonuclease. Yeast two-hybrid and co-immunoprecipitation assays showed that there were interactions between subunits of the cohesin and Mus81-Eme1 complexes. We conclude that cohesin is methylated and that mutations that prevent methylation of Psm1 K536 and K1200 show synthetic phenotypes with mutants defective in the homologous recombination DNA repair pathway.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Motivos de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Metilação , Mutação , Ligação Proteica , Schizosaccharomyces/química , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Coesinas
7.
PLoS Genet ; 12(6): e1006102, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27304859

RESUMO

To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species.


Assuntos
Segregação de Cromossomos/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Rad51 Recombinase/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Quebras de DNA de Cadeia Dupla , DNA Cruciforme/genética , DNA Fúngico/metabolismo , Endodesoxirribonucleases/genética , Deleção de Genes , Biblioteca Gênica , Resolvases de Junção Holliday/metabolismo , Meiose/genética
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