| 1. |
- Gafar, Fajri, et al.
(författare)
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Global estimates and determinants of antituberculosis drug pharmacokinetics in children and adolescents : a systematic review and individual patient data meta-analysis
- 2023
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Ingår i: European Respiratory Journal. - : European Respiratory Society. - 0903-1936 .- 1399-3003. ; 61:3
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Forskningsöversikt (refereegranskat)abstract
- Background Suboptimal exposure to antituberculosis (anti-TB) drugs has been associated with unfavourable treatment outcomes. We aimed to investigate estimates and determinants of first-line anti-TB drug pharmacokinetics in children and adolescents at a global level.Methods We systematically searched MEDLINE, Embase and Web of Science (1990–2021) for pharmacokinetic studies of first-line anti-TB drugs in children and adolescents. Individual patient data were obtained from authors of eligible studies. Summary estimates of total/extrapolated area under the plasma concentration–time curve from 0 to 24 h post-dose (AUC0–24) and peak plasma concentration (Cmax) were assessed with random-effects models, normalised with current World Health Organization-recommended paediatric doses. Determinants of AUC0–24 and Cmax were assessed with linear mixed-effects models.Results Of 55 eligible studies, individual patient data were available for 39 (71%), including 1628 participants from 12 countries. Geometric means of steady-state AUC0–24 were summarised for isoniazid (18.7 (95% CI 15.5–22.6) h·mg·L−1), rifampicin (34.4 (95% CI 29.4–40.3) h·mg·L−1), pyrazinamide (375.0 (95% CI 339.9–413.7) h·mg·L−1) and ethambutol (8.0 (95% CI 6.4–10.0) h·mg·L−1). Our multivariate models indicated that younger age (especially <2 years) and HIV-positive status were associated with lower AUC0–24 for all first-line anti-TB drugs, while severe malnutrition was associated with lower AUC0–24 for isoniazid and pyrazinamide. N-acetyltransferase 2 rapid acetylators had lower isoniazid AUC0–24 and slow acetylators had higher isoniazid AUC0–24 than intermediate acetylators. Determinants of Cmax were generally similar to those for AUC0–24.Conclusions This study provides the most comprehensive estimates of plasma exposures to first-line anti-TB drugs in children and adolescents. Key determinants of drug exposures were identified. These may be relevant for population-specific dose adjustment or individualised therapeutic drug monitoring.
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| 2. |
- Gibney, A., et al.
(författare)
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A Click Chemistry-Based Artificial Metallo-Nuclease
- 2023
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Ingår i: Angewandte Chemie-International Edition. - 1433-7851 .- 1521-3773. ; 62:38
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Tidskriftsartikel (refereegranskat)abstract
- Artificial metallo-nucleases (AMNs) are promising DNA damaging drug candidates. Here, we demonstrate how the 1,2,3-triazole linker produced by the Cu-catalysed azide-alkyne cycloaddition (CuAAC) reaction can be directed to build Cu-binding AMN scaffolds. We selected biologically inert reaction partners tris(azidomethyl)mesitylene and ethynyl-thiophene to develop TC-Thio, a bioactive C-3-symmetric ligand in which three thiophene-triazole moieties are positioned around a central mesitylene core. The ligand was characterised by X-ray crystallography and forms multinuclear Cu-II and Cu-I complexes identified by mass spectrometry and rationalised by density functional theory (DFT). Upon Cu coordination, Cu-II-TC-Thio becomes a potent DNA binding and cleaving agent. Mechanistic studies reveal DNA recognition occurs exclusively at the minor groove with subsequent oxidative damage promoted through a superoxide- and peroxide-dependent pathway. Single molecule imaging of DNA isolated from peripheral blood mononuclear cells shows that the complex has comparable activity to the clinical drug temozolomide, causing DNA damage that is recognised by a combination of base excision repair (BER) enzymes.
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| 3. |
- McStay, Natasha, et al.
(författare)
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Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 49:18, s. 10289-10308
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Tidskriftsartikel (refereegranskat)abstract
- Metallodrugs provide important first-line treatment against various forms of human cancer. To overcome chemotherapeutic resistance and widen treatment possibilities, new agents with improved or alternative modes of action are highly sought after. Here, we present a click chemistry strategy for developing DNA damaging metallodrugs. The approach involves the development of a series of polyamine ligands where three primary, secondary or tertiary alkyne-amines were selected and 'clicked' using the copper-catalysed azide-alkyne cycloaddition reaction to a 1,3,5-azide mesitylene core to produce a family of compounds we call the 'Tri-Click' (TC) series. From the isolated library, one dominant ligand (TC1) emerged as a high-affinity copper(II) binding agent with potent DNA recognition and damaging properties. Using a range of in vitro biophysical and molecular techniques-including free radical scavengers, spin trapping antioxidants and base excision repair (BER) enzymes-the oxidative DNA damaging mechanism of copper-bound TC1 was elucidated. This activity was then compared to intracellular results obtained from peripheral blood mononuclear cells exposed to Cu(ll)-TC1 where use of BER enzymes and fluorescently modified dNTPs enabled the characterisation and quantification of genomic DNA lesions produced by the complex. The approach can serve as a new avenue for the design of DNA damaging agents with unique activity profiles.
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| 4. |
- Müller, Vilhelm, 1990, et al.
(författare)
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Enzyme-free optical DNA mapping of the human genome using competitive binding
- 2019
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 47:15
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Tidskriftsartikel (refereegranskat)abstract
- Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized.
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| 5. |
- Obi, Ikenna, et al.
(författare)
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Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication
- 2020
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 48:19, s. 10998-11015
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Tidskriftsartikel (refereegranskat)abstract
- G-quadruplex (G4) structures are stable noncanonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancerassociated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.
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| 6. |
- Singh, Vandana, 1985, et al.
(författare)
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Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging
- 2022
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 594, s. 57-62
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Tidskriftsartikel (refereegranskat)abstract
- DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.
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| 7. |
- Singh, Vandana, 1985, et al.
(författare)
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Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging
- 2020
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Ingår i: Translational Oncology. - : Elsevier BV. - 1936-5233. ; 13:10
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Tidskriftsartikel (refereegranskat)abstract
- Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent “spot” at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents. © 2020 The Authors
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| 8. |
- Singh, Vandana, 1985, et al.
(författare)
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Shining light on single-strand lesions caused by the chemotherapy drug bleomycin
- 2021
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 105
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Tidskriftsartikel (refereegranskat)abstract
- Quantification of the DNA damage induced by chemotherapy in patient cells may aid in personalization of the dose used. However, assays to evaluate individual patient response to chemotherapy are not available today. Here, we present an assay that quantifies single-stranded lesions caused by the chemotherapeutic drug Bleomycin (BLM) in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. We use base excision repair (BER) enzymes to process the DNA damage induced by BLM and then extend the processed sites with fluorescent nucleotides using a DNA polymerase. The fluorescent patches are quantified on single DNA molecules using fluorescence microscopy. Using the assay, we observe a significant variation in the in vitro induced BLM damage and its repair for different individuals. Treatment of the cells with the BER inhibitor CRT0044876 leads to a lower level of repair of BLM-induced damage, indicating the ability of the assay to detect a compromised DNA repair in patients. Overall, the data suggest that our assay could be used to sensitively detect the variation in BLM-induced DNA damage and repair in patients and can potentially be able to aid in personalizing patient doses.
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