| 1. |
- Al-Minawi, Ali Z., et al.
(författare)
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The ERCC1/XPF endonuclease is required for completion of homologous recombination at DNA replication forks stalled by inter-strand cross-links
- 2009
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 37:19, s. 6400-6413
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Tidskriftsartikel (refereegranskat)abstract
- Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.
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| 2. |
- Al-Ubaidi, Firas L. T., et al.
(författare)
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CASTRATION THERAPY OF PROSTATE CANCER RESULTS IN DOWNREGULATION OF HIF-1 alpha LEVELS
- 2012
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Ingår i: International Journal of Radiation Oncology, Biology, Physics. - : Elsevier BV. - 0360-3016 .- 1879-355X. ; 82:3, s. 1243-1248
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Tidskriftsartikel (refereegranskat)abstract
- Background and Purpose: Neoadjuvant androgen deprivation in combination with radiotherapy of prostate cancer is used to improve radioresponsiveness and local tumor control. Currently, the underlying mechanism is not well understood. Because hypoxia causes resistance to radiotherapy, we wanted to test whether castration affects the degree of hypoxia in prostate cancer. Methods and Materials: In 14 patients with locally advanced prostate cancer, six to 12 prostatic needle core biopsy specimens were taken prior to castration therapy. Bilateral orchidectomy was performed in 7 patients, and 7 were treated with a GnRH-agonist (leuprorelin). After castrationm two to four prostatic core biopsy specimens were taken, and the level of hypoxia-inducible factor-1 alpha (HIF-1 alpha) in cancer was determined by immunofluorescence. Results: Among biopsy specimens taken before castration, strong HIF-1 alpha expression (mean intensity above 30) was shown in 5 patients, weak expression (mean intensity 10-30) in 3 patients, and background levels of HIF-1 alpha (mean intensity 0-10) in 6 patients. Downregulation of HIF-1 alpha expression after castration was observed in all 5 patients with strong HIF-1 alpha precastration expression. HIF-1 alpha expression was also reduced in 2 of 3 patients with weak HIF-1 alpha precastration expression. Conclusions: Our data suggest that neoadjuvant castration decreases tumor cell hypoxia in prostate cancer, which may explain increased radiosensitivity after castration.
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| 3. |
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| 4. |
- Berglund, Emelie, et al.
(författare)
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Spatial maps of prostate cancer transcriptomes reveal an unexplored landscape of heterogeneity
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Intra-tumor heterogeneity is one of the biggest challenges in cancer treatment today. Here we investigate tissue-wide gene expression heterogeneity throughout a multifocal prostate cancer using the spatial transcriptomics (ST) technology. Utilizing a novel approach for deconvolution, we analyze the transcriptomes of nearly 6750 tissue regions and extract distinct expression profiles for the different tissue components, such as stroma, normal and PIN glands, immune cells and cancer. We distinguish healthy and diseased areas and thereby provide insight into gene expression changes during the progression of prostate cancer. Compared to pathologist annotations, we delineate the extent of cancer foci more accurately, interestingly without link to histological changes. We identify gene expression gradients in stroma adjacent to tumor regions that allow for re-stratification of the tumor microenvironment. The establishment of these profiles is the first step towards an unbiased view of prostate cancer and can serve as a dictionary for future studies.
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| 5. |
- Bryant, Helen E, et al.
(författare)
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PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination.
- 2009
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Ingår i: The EMBO journal. - : Wiley. - 1460-2075 .- 0261-4189. ; 28:17, s. 2601-15
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Tidskriftsartikel (refereegranskat)abstract
- If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea-induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.
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| 6. |
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| 7. |
- Elvers, Ingegerd, et al.
(författare)
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CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation
- 2012
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:17, s. 8440-8448
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Tidskriftsartikel (refereegranskat)abstract
- Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine-inhibiting ATM and ATR amongst other kinases-is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase eta (Pol eta), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.
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| 8. |
- Elvers, Ingegerd, et al.
(författare)
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UV-induced replication fork collapse in DNA polymerase η deficient cells is independent of the MUS81 endonuclease
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The MUS81 endonuclease was initially identified in resonse to UV and MMS lesions, and has been implicated in replication fork collapse after exposure to cross-linking agents. After stalling of replication forks by hydroxyurea treatment, the forks collapse independently of MUS81 but the endonuclease is required for replication fork restart. However in cells deficient in the Werner helicase, MUS81 is needed for collapse of replication forks after hydroxyurea treatment, indicating that the endonuclease might play a role in replication fork collapse in cells with impaired replication. UV irradiation induces DNA damage that physically block replication fork elongation but may be bypassed by translesion synthesis polymerases. Here we have investigated the role of MUS81 after UV irradiation of human fibroblasts deficient in Polη, and restored (wild-type) cells. We show that in wild-type cells, depletion of MUS81 does not affect survival after UV irradiation. However in Polη deficient cells, MUS81 depletion further lowers the survival after exposure to UV. In spite of this, replication forks collapse in UV irradiated Polη deficient cells independently of MUS81.
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| 9. |
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| 10. |
- Erickson, Andrew, et al.
(författare)
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The spatial landscape of clonal somatic mutations in benign and malignant tissue
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. Here, we provide an unsupervised approach to study spatial genome integrity in situ to gain molecular insight into clonal relationships. We employed spatially resolved transcriptomics to infer spatial copy number variations in >120 000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.
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| 11. |
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| 12. |
- Gad, Helge, et al.
(författare)
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MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool
- 2014
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Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 508:7495, s. 215-221
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Tidskriftsartikel (refereegranskat)abstract
- Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bindin the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
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| 13. |
- Gobom, Johan, et al.
(författare)
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Antibody-free measurement of cerebrospinal fluid tau phosphorylation across the Alzheimer's disease continuum.
- 2022
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Ingår i: Molecular neurodegeneration. - : Springer Science and Business Media LLC. - 1750-1326. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer's disease is characterized by an abnormal increase of phosphorylated tau (pTau) species in the CSF. It has been suggested that emergence of different pTau forms may parallel disease progression. Therefore, targeting multiple specific pTau forms may allow for a deeper understanding of disease evolution and underlying pathophysiology. Current immunoassays measure pTau epitopes separately and may capture phosphorylated tau fragments of different length depending on the non-pTau antibody used in the assay sandwich pair, which bias the measurement.We developed the first antibody-free mass spectrometric method to simultaneously measure multiple phosphorylated epitopes in CSF tau: pT181, pS199, pS202, pT205, pT217, pT231, and pS396. The method was first evaluated in biochemically defined Alzheimer's disease and control CSF samples (n=38). All seven pTau epitopes clearly separated Alzheimer's disease from non-AD (p<0.001, AUC=0.84-0.98). We proceeded with clinical validation of the method in the TRIAD (n=165) and BioFINDER-2 cohorts (n=563), consisting of patients across the full Alzheimer's disease continuum, including also young controls (<40years), as well as patients with frontotemporal dementia and other neurodegenerative disorders.Increased levels of all phosphorylated epitopes were found in Alzheimer's disease dementia and Aβ positron emission tomography-positive patients with mild cognitive impairment compared with Aβ-negative controls. For Alzheimer's disease dementia compared with Aβ-negative controls, the best biomarker performance was observed for pT231 (TRIAD: AUC=98.73%, fold change=7.64; BioFINDER-2: AUC=91.89%, fold change=10.65), pT217 (TRIAD: AUC=99.71%, fold change=6.33; BioFINDER-2: AUC=98.12%, fold change=8.83) and pT205 (TRIAD: AUC=99.07%, fold change=5.34; BioFINDER-2: AUC=93.51%, fold change=3.92). These phospho-epitopes also discriminated between Aβ-positive and Aβ-negative cognitively unimpaired individuals: pT217 (TRIAD: AUC=83.26, fold change=2.39; BioFINDER-2: AUC=91.05%, fold change=3.29), pT231 (TRIAD: AUC=86.25, fold change=3.80; BioFINDER-2: AUC=78.69%, fold change=3.65) and pT205 (TRIAD: AUC=71.58, fold change=1.51; BioFINDER-2: AUC=71.11%, fold change=1.70).While an increase was found for all pTau species examined, the highest fold change in Alzheimer's disease was found for pT231, pT217 and pT205. Simultaneous antibody-free measurement of pTau epitopes by mass spectrometry avoids possible bias caused by differences in antibody affinity for modified or processed forms of tau, provides insights into tau pathophysiology and may facilitate clinical trials on tau-based drug candidates.
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| 14. |
- Gottipati, Ponnari, et al.
(författare)
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Poly(ADP-Ribose) Polymerase Is Hyperactivated in Homologous Recombination-Defective Cells
- 2010
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 70:13, s. 5389-5398
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Tidskriftsartikel (refereegranskat)abstract
- Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gamma H2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers. Cancer Res; 70(13); 5389-98. (C) 2010 AACR.
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| 15. |
- Groth, Petra, et al.
(författare)
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Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O-6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 402:1, s. 70-82
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Tidskriftsartikel (refereegranskat)abstract
- Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O-6-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-M-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gamma H2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.
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| 16. |
- Harmon, Matthew B.A., et al.
(författare)
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Microbiological profile of nosocomial infections following cardiac arrest : Insights from the targeted temperature management (TTM) trial
- 2020
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Ingår i: Resuscitation. - : Elsevier BV. - 0300-9572. ; 148, s. 227-233
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Tidskriftsartikel (refereegranskat)abstract
- Aims: Infectious complications frequently occur in intensive care unit patients admitted after out-of-hospital cardiac arrest. There is debate on the effects of temperature management on the incidence of infections, as well as on the efficacy and choice of antibiotic prophylaxis. In this substudy of the targeted temperature management (TTM) trial, we describe the microbiological profile of infectious complications in patients with cardiac arrest and examined the impact of TTM at 33 °C compared to TTM at 36 °C. Furthermore we aimed to determine the association between antibiotic prophylaxis and the incidence of infections. Methods: This is a posthoc analysis of the TTM cohort. Microbiological data was retrospectively collected for the first 14-days of ICU-admission. Logistic regression was used to determine the relationship between antibiotic prophylaxis and pneumonia adjusted for mortality. Results: Of 696 patients included in this analysis, 158 (23%) developed pneumonia and 28 (4%) had bacteremia with a clinically relevant pathogen. Staphylococcus aureus was the most common pathogen isolated in patients with pneumonia (23%) and in patients with bacteremia (24%). Gram-negative pathogens were most common overall. TTM did not have an impact on the microbiological profile. The use of antibiotic prophylaxis was significantly associated with a reduced risk of infection (OR 0.59, 95%CI 0.43-0.79, p = 0.0005). This association remained significant after correcting for confounders (OR 0.64, 95%CI 0.46-0.90; p = 0.01). The association is not present in a model after correction for clustering within centers (aOR 0.55, 95%CI 0.20–1.47, p = 0.22). Adjustment for mortality did not influence the outcome. Conclusion: Gram-negative pathogens are the most common causes of nosocomial infections following cardiac arrest. TTM does not impact the microbiological profile. It remains unclear whether patients in ICUs using antibiotic prophylaxis have a reduced risk of pneumonia and bacteremia that is unrelated to center effects.
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| 17. |
- Harmon, Matthew B.A., et al.
(författare)
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Practice of mechanical ventilation in cardiac arrest patients and effects of targeted temperature management : A substudy of the targeted temperature management trial
- 2018
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Ingår i: Resuscitation. - : Elsevier BV. - 0300-9572. ; 129, s. 29-36
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Tidskriftsartikel (refereegranskat)abstract
- Aims: Mechanical ventilation practices in patients with cardiac arrest are not well described. Also, the effect of temperature on mechanical ventilation settings is not known. The aims of this study were 1) to describe practice of mechanical ventilation and its relation with outcome 2) to determine effects of different target temperatures strategies (33 °C versus 36 °C) on mechanical ventilation settings. Methods: This is a substudy of the TTM-trial in which unconscious survivors of a cardiac arrest due to a cardiac cause were randomized to two TTM strategies, 33 °C (TTM33) and 36 °C (TTM36). Mechanical ventilation data were obtained at three time points: 1) before TTM; 2) at the end of TTM (before rewarming) and 3) after rewarming. Logistic regression was used to determine an association between mechanical ventilation variables and outcome. Repeated-measures mixed modelling was performed to determine the effect of TTM on ventilation settings. Results: Mechanical ventilation data was available for 567 of the 950 TTM patients. Of these, 81% was male with a mean (SD) age of 64 (12) years. At the end of TTM median tidal volume was 7.7 ml/kg predicted body weight (PBW)(6.4–8.7) and 60% of patients were ventilated with a tidal volume ≤ 8 ml/kg PBW. Median PEEP was 7.7cmH2O (6.4–8.7) and mean driving pressure was 14.6 cmH2O (±4.3). The median FiO2 fraction was 0.35 (0.30–0.45). Multivariate analysis showed an independent relationship between increased respiratory rate and 28-day mortality. TTM33 resulted in lower end-tidal CO2 (Pgroup = 0.0003) and higher alveolar dead space fraction (Pgroup = 0.003) compared to TTM36, while PCO2 levels and respiratory minute volume were similar between groups. Conclusions: In the majority of the cardiac arrest patients, protective ventilation settings are applied, including low tidal volumes and driving pressures. High respiratory rate was associated with mortality. TTM33 results in lower end-tidal CO2 levels and a higher alveolar dead space fraction compared to TTTM36.
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| 18. |
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| 19. |
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| 20. |
- Kotova, Natalia, et al.
(författare)
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Genotoxicity of alcohol is linked to DNA replication-associated damage and homologous recombination repair
- 2013
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Ingår i: Carcinogenesis. - : Oxford University Press (OUP). - 0143-3334 .- 1460-2180. ; 34:2, s. 325-330
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Tidskriftsartikel (refereegranskat)abstract
- Although alcohol consumption is related to increased cancer risk, its molecular mechanism remains unclear. Here, we demonstrate that an intake of 10% alcohol for 4 weeks in rats is genotoxic due to induction of micronuclei. Acetaldehyde (AA), the first product of ethanol metabolism, is believed to be responsible for DNA damage induced by alcohol. Here, we observe that AA effectively blocks DNA replication elongation in mammalian cells, resulting in DNA double-strand breaks associated with replication. AA-induced DNA damage sites colocalize with the homologous recombination (HR) repair protein RAD51. HR measured in the hypoxhantineguaninefosforibosyltransferase (HPRT) gene is effectively induced by AA and recombination defective mammalian cells are hypersensitive to AA, clearly demonstrating that HR is essential in the repair of AA-induced DNA damage. Altogether, our data indicate that alcohol genotoxicity related to AA produces replication lesions on DNA triggering HR repair.
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| 21. |
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| 22. |
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| 23. |
- Lundin, Cecilia, et al.
(författare)
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Different Roles for Nonhomologous End Joining and Homologous Recombination following Replication Arrest in Mammalian Cells
- 2002
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Ingår i: Molecular and Cellular Biology. - : American Society of Microbiology. - 0270-7306. ; 22:16, s. 5869-78
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Tidskriftsartikel (refereegranskat)abstract
- Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs.
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| 24. |
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| 25. |
- Lundin, Cecilia, et al.
(författare)
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RAD51 is Involved in Repair of Damage Associated with DNA Replication in Mammalian Cells
- 2003
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836. ; 328:3, s. 521-35
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Tidskriftsartikel (refereegranskat)abstract
- The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than γ-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.
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| 26. |
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| 27. |
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| 28. |
- Marklund, Maja, et al.
(författare)
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Spatio-temporal analysis of prostate tumors in situ suggests pre-existence of treatment-resistant clones
- 2022
-
Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 13:1
-
Tidskriftsartikel (refereegranskat)abstract
- The molecular mechanisms underlying lethal castration-resistant prostate cancer remain poorly understood, with intratumoral heterogeneity a likely contributing factor. To examine the temporal aspects of resistance, we analyze tumor heterogeneity in needle biopsies collected before and after treatment with androgen deprivation therapy. By doing so, we are able to couple clinical responsiveness and morphological information such as Gleason score to transcriptome-wide data. Our data-driven analysis of transcriptomes identifies several distinct intratumoral cell populations, characterized by their unique gene expression profiles. Certain cell populations present before treatment exhibit gene expression profiles that match those of resistant tumor cell clusters, present after treatment. We confirm that these clusters are resistant by the localization of active androgen receptors to the nuclei in cancer cells post-treatment. Our data also demonstrates that most stromal cells adjacent to resistant clusters do not express the androgen receptor, and we identify differentially expressed genes for these cells. Altogether, this study shows the potential to increase the power in predicting resistant tumors. Spatial heterogeneity in prostate cancer can contribute to its resistance to androgen deprivation therapy (ADT). Here, the authors analyse prostate cancer samples before and after ADT using Spatial Transcriptomics, and find heterogeneous pre-treatment tumour cell populations and stromal cells that are associated with resistance.
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| 29. |
- Marklund, Maja, et al.
(författare)
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Spatio-temporal analysis of prostate tumors in situ suggests the pre-existence of ADT-resistance
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The molecular mechanisms by which potentially lethal castration-resistant prostate cancer emerge in advanced metastatic prostate cancer are still poorly understood. Intratumor heterogeneity is believed to contribute to the fact that a majority of affected men succumb to the disease within a few years. In this study, we will challenge the conventional notion that castration-resistant prostate cancer cells evolve as a consequence of treatment. To examine the temporal aspects of resistance, we analyze tumor heterogeneity in core needle biopsies collected pre-and post-treatment. By doing so, we are able to couple clinical responsiveness and morphological information such as Gleason score to transcriptome- wide data. Our data-driven analysis of transcriptomes identified several distinct intratumoral cell populations, characterized by their unique gene expression profiles. Strikingly, certain minor cell populations present before treatment exhibited gene expression profiles that matched those of resistant tumor cell clusters. Such resistant clusters were confirmed by the localization of the androgen receptor to the nuclei in cancer cells present after treatment. Our data also demonstrate that stromal cells adjacent to resistant tumor factors do not express AR before treatment (or after), which can be used to increase the power in predicting resistant tumors.
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| 30. |
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| 31. |
- Mellroth, Peter, et al.
(författare)
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Ligand-induced dimerization of Drosophila peptidoglycan recognition proteins in vitro
- 2005
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 102:18, s. 6455-6460
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Tidskriftsartikel (refereegranskat)abstract
- Drosophila knockout mutants have placed peptidoglycan recognition proteins (PGRPs) in the two major pathways controlling immune gene expression. We now examine PGRP affinities for peptidoglycan. PGRP-SA and PGRP-LCx are bona fide pattern recognition receptors, and PGRP-SA, the peptidoglycan receptor of the Toll/Dif pathway, has selective affinity for different peptidoglycans. PGRP-LCx, the default peptidoglycan receptor of the Imd/Relish pathway, has strong affinity for all polymeric peptidoglycans tested and for monomeric peptidoglycan. PGRP-LCa does not have affinity for polymeric or monomeric peptidoglycan. Instead, PGRP-LCa can form heterodimers with LCx when the latter is bound to monomeric peptidoglycan. Hence, PGRP-LCa can be said to function as an adaptor, thus adding a new function to a member of the PGRP family.
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| 32. |
- Mirzazadeh, Reza, et al.
(författare)
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Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins.
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| 33. |
- Petermann, Eva, et al.
(författare)
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Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.
- 2010
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 37:4, s. 492-502
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Tidskriftsartikel (refereegranskat)abstract
- Faithful DNA replication is essential to all life. Hydroxyurea (HU) depletes the cells of dNTPs, which initially results in stalled replication forks that, after prolonged treatment, collapse into DSBs. Here, we report that stalled replication forks are efficiently restarted in a RAD51-dependent process that does not trigger homologous recombination (HR). The XRCC3 protein, which is required for RAD51 foci formation, is also required for replication restart of HU-stalled forks, suggesting that RAD51-mediated strand invasion supports fork restart. In contrast, replication forks collapsed by prolonged replication blocks do not restart, and global replication is rescued by new origin firing. We find that RAD51-dependent HR is triggered for repair of collapsed replication forks, without apparent restart. In conclusion, our data suggest that restart of stalled replication forks and HR repair of collapsed replication forks require two distinct RAD51-mediated pathways.
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| 34. |
- Pisani, Luigi, et al.
(författare)
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Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome : a pooled analysis of four observational studies
- 2022
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Ingår i: The Lancet Global Health. - 2214-109X. ; 10:2, s. 227-235
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Tidskriftsartikel (refereegranskat)abstract
- Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42·4% vs 44·2%; absolute difference –1·69 [–9·58 to 6·11] p=0·67; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5–8] vs 6 [5–8] cm H2O; p=0·0011). ICU mortality was higher in MICs than in HICs (30·5% vs 19·9%; p=0·0004; adjusted effect 16·41% [95% CI 9·52–23·52]; p<0·0001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0·80 [95% CI 0·75–0·86]; p<0·0001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status. Funding: No funding.
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| 35. |
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| 36. |
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| 37. |
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| 38. |
- Schultz, Niklas, 1962-
(författare)
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Aspects of cytokinesis with particular emphasis on induction of aneuploidy and determination of the cleavage plane
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis focuses primarily on three aspects on cleavage: the relationship between failure of cleavage and aneuploidy; disruption of the cleavage process by hydrophobic chemicals; and the positioning of the spindle and, thus, the cleavage plane. The established Chinese V79 hamster fibroblast cell line was used as the experimental system.Time-lapse video recording of the division of binucleated cells revealed that the daughter cells are often aneuploid, indicating that disturbance of cytokinesis may give rise to aneuploidy.Exposure to a number of alcohols and chlorinated hydrocarbons with different degrees of lipophilicity revealed that cytokinesis can be disrupted by unspecific hydrophobic interactions. This must be taken into account before postulating a specific effect on cleavage. Furthermore, the ratio between multinucleated and binucleated cells allows rough discrimination between mechanisms disturbing the spindle or cleavage, and this ratio might be one valuable endpoint in a test system for aneuploidy.The mechanism underlying positioning of the spindle was investigated using time-lapse video filming, immunostaining and image analyses. Misplaced spindles were found to move to a central position during anaphase, and this translocation involved outgrowth of astral microtubules. Localisation of dynactin suggested that the dynein/dynactin system interact with astral microtubules and the basal layer of the cell to move the spindle. Thus, unpolarised cells appear to contain a system for positioning the spindle similar to that in polarised cells.Finally, even a minor decrease in the level of reduced glutathione (GSH) in primary, non-transformed human fibroblasts by oxidation or conjugation interfered with alignment of the chromosomes in metaphase, suggesting an increased risk for aneuploidy. Although this misalignment did not seem to involve disturbance of cytoskeletal elements such as actin and tubulin, the dynactin/dynein system was altered when the level of GSH was lowered sufficiently to give rise to misarranged metaphase chromosomes. More extensive oxidation of GSH lead to rearrangement of actin and inhibition of cleavage.The present findings increase our understanding of factors which disrupt cleavage and the possible consequences of such disruption. This information will aid in interpreting result obtained with systems designed to screen for agents which induce aneuploidy. Furthermore, the demonstration that unpolarised cells possess a system for positioning the spindle similar to that in polarised cells might facilitate the development of a simple test for screening agents that interfere with this process, which is of fundamental importance for development.
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| 39. |
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| 40. |
- Stoimenov, Ivaylo, 1980-
(författare)
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Interplay between Transcription and Homologous Recombination in the Presence of DNA Damage
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The biochemical processes of DNA repair, replication and recombination compete for the same substrate, the DNA molecule. This competition is natural, as each process requires the same template. In order to resolve possible conflicts between these processes, when they take place on a particular stretch of DNA, certain crosstalk is expected. The complexity is additionally increased by the existence of another DNA dependent process, which occurs in all phases of the cell cycle: transcription. This thesis aims to investigate the link between transcription inhibition and homologous recombination, especially in the context of UV-induced DNA damage. The results show that the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) induces homologous recombination. The DNA damage caused by UVC irradiation is repaired mainly via nucleotide excision repair, however, it is also known to trigger recombinational repair. In the presence of UV-induced damage, transcription inhibition by DRB potentiates the induction of homologous recombination as a necessary mechanism of cell survival. The thesis also focuses on the toxicity mechanisms of the chemotherapeutic compound 6-thioguanine (6TG). The work in the thesis suggests application of 6TG as a treatment for hereditary forms of breast cancer, with genetically altered BRCA1 or BRCA2 functions. Most importantly, the treatment with 6TG is applicable to breast cancers, which have developed resistance to another class of chemotherapeutic drugs, poly-(ADP-ribose) polymerase (PARP) inhibitors. Repair of the DNA damage induced by 6TG treatment is investigated further with a particular focus the pathway of DNA damage avoidance involving DNA polymerase η. The function of DNA polymerase η seems to be an important factor for the outcome of DNA repair after 6TG exposure. The deficiency of DNA polymerase η is also investigated in connection with normal replication and the repair of UV-induced DNA damage. In summary, the work in the thesis sheds more light onto the fundamental connections between DNA replication, recombination, transcription, repair and damage avoidance. On a more practical side, the information obtained about these fundamental connections is used to suggest a possible therapy for several forms of breast cancer.
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| 41. |
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| 42. |
- Stoimenov, Ivaylo, et al.
(författare)
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Transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) causes DNA damage and triggers homologous recombination repair in mammalian cells
- 2011
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Ingår i: Mutation research. - : Elsevier BV. - 0027-5107 .- 1873-135X. ; 706:1-2, s. 1-6
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Tidskriftsartikel (refereegranskat)abstract
- Transcription, replication and homologous recombination are intrinsically connected and it is well established that an increase of transcription is associated with an increase in homologous recombination. Here, we have studied how homologous recombination is affected during transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a compound that prevents activating phosphorylations of the RNA Pol II C-terminal domain. We identify that DRB triggers an increase in homologous recombination within the hprt gene as well as increasing RAD51 foci formation in mammalian cells. Furthermore, we find that DRB-induced transcriptional stress is associated with formation of the nuclear foci of the phosphorylated form of H2AX (γH2AX). We accounted that about 72% of RAD51 foci co-localized with the observed γH2AX foci. Interestingly, we find that XRCC3 mutated, homologous recombination defective cells are hypersensitive to the toxic effect of DRB and fail to form RAD51 foci. In conclusion, we show that DRB-induced transcription inhibition is associated with the formation of a lesion that triggers RAD51-dependent homologous recombination repair, required for survival under transcriptional stress.
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| 43. |
- Stoimenov, Ivaylo, et al.
(författare)
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Transcription Inhibition by DRB Potentiates Recombinational Repair of UV Lesions in Mammalian Cells
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:5, s. e19492-
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Tidskriftsartikel (refereegranskat)abstract
- Homologous recombination (HR) is intricately associated with replication, transcription and DNA repair in all organisms studied. However, the interplay between all these processes occurring simultaneously on the same DNA molecule is still poorly understood. Here, we study the interplay between transcription and HR during ultraviolet light (UV)-induced DNA damage in mammalian cells. Our results show that inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) increases the number of UV-induced DNA lesions (gamma H2AX, 53BP1 foci formation), which correlates with a decrease in the survival of wild type or nucleotide excision repair defective cells. Furthermore, we observe an increase in RAD51 foci formation, suggesting HR is triggered in response to an increase in UV-induced DSBs, while inhibiting transcription. Unexpectedly, we observe that DRB fails to sensitise HR defective cells to UV treatment. Thus, increased RAD51 foci formation correlates with increased cell death, suggesting the existence of a futile HR repair of UV-induced DSBs which is linked to transcription inhibition.
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| 44. |
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| 45. |
- Ström, Cecilia, 1982-, et al.
(författare)
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CK2 phosphorylation of XRCC1 facilitate single-strand break formation during base excision repair
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Casein kinase 2 (CK2) phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express a XRCC1 protein (XRCC1ckm) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification in SSB repair is distinct from its role in base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1ckm (CKM cells) or following inhibition with CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1ckm protein has a higher affinity for nicked DNA substrate than wild type XRCC1 protein and we propose a model whereby the increased affinity for DNA sequesters XRCC1ckm and the repair enzymes associated with it, at the repair site. In conclusion, our results indicate that CK2-phosphorylations of XRCC1 affect the kinetics of SSB repair and BER differentially, and that the modifications on XRCC1 facilitate the BER incision step.
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| 46. |
- Ström, Cecilia E., et al.
(författare)
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CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair
- 2011
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 10:9, s. 961-969
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Tidskriftsartikel (refereegranskat)abstract
- CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the allcylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting.
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| 47. |
- Tarish, Firas L., et al.
(författare)
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Castration radiosensitizes prostate cancer tissue by impairing DNA double-strand break repair
- 2015
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Ingår i: Science Translational Medicine. - : American Association for the Advancement of Science (AAAS). - 1946-6234 .- 1946-6242. ; 7:312
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Tidskriftsartikel (refereegranskat)abstract
- Chemical castration improves responses to radiotherapy in prostate cancer, but the mechanism is unknown. We hypothesized that this radiosensitization is caused by castration-mediated down-regulation of non-homologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). To test this, we enrolled 48 patients with localized prostate cancer in two arms of the study: either radiotherapy first or radiotherapy after neoadjuvant castration treatment. We biopsied patients at diagnosis and before and after castration and radiotherapy treatments to monitor androgen receptor, NHEJ, and DSB repair in verified cancer tissue. We show that patients receiving neoadjuvant castration treatment before radiotherapy had reduced amounts of the NHEJ protein Ku70, impaired radiotherapy-induced NHEJ activity, and higher amounts of unrepaired DSBs, measured by gamma-H2AX foci in cancer tissues. This study demonstrates that chemical castration impairs NHEJ activity in prostate cancer tissue, explaining the improved response of patients with prostate cancer to radiotherapy after chemical castration.
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| 48. |
- Villacampa, Eva Gracia, et al.
(författare)
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Genome-wide Spatial Expression Profiling in Formalin-fixed Tissues
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Formalin-fixed paraffin embedding (FFPE) is the most widespread long-term tissue preservation approach. Here we present a procedure to perform genome-wide spatial analysis of mRNA in FFPE fixed tissue sections. The procedure takes advantage of well-established, commercially available methods for imaging and spatial barcoding using slides spotted with barcoded oligo(dT) probes to capture the 3’ end of mRNA molecules in tissue sections. First, we conducted expression profiling and cell type mapping in coronal sections from the mouse brain to demonstrate the method’s capability to delineate anatomical regions from a molecular perspective. Second, we explored the spatial composition of transcriptomic signatures in ovarian carcinosarcoma samples using data-driven analysis methods, exemplifying the method’s potential to elucidate molecular mechanisms in heterogeneous clinical samples. Finally, we demonstrate the applicability of the assay to characterize organoids and a human lung biopsy specimen infected with SARS-CoV-2.
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| 49. |
- Villacampa, Eva Gracia, et al.
(författare)
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Genome-wide spatial expression profiling in formalin-fixed tissues
- 2021
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Ingår i: Cell Genomics. - : Elsevier BV. - 2666-979X. ; 1:3
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Tidskriftsartikel (refereegranskat)abstract
- Formalin-fixed paraffin embedding (FFPE) is the most widespread long-term tissue preservation approach. Here, we report a procedure to perform genome-wide spatial analysis of mRNA in FFPE-fixed tissue sections, using well-established, commercially available methods for imaging and spatial barcoding using slides spotted with barcoded oligo(dT) probes to capture the 3′ end of mRNA molecules in tissue sections. We applied this method for expression profiling and cell type mapping in coronal sections from the mouse brain to demonstrate the method's capability to delineate anatomical regions from a molecular perspective. We also profiled the spatial composition of transcriptomic signatures in two ovarian carcinosarcoma samples, exemplifying the method's potential to elucidate molecular mechanisms in heterogeneous clinical samples. Finally, we demonstrate the applicability of the assay to characterize human lung and kidney organoids and a human lung biopsy specimen infected with SARS-CoV-2. We anticipate that genome-wide spatial gene expression profiling in FFPE biospecimens will be used for retrospective analysis of biobank samples, which will facilitate longitudinal studies of biological processes and biomarker discovery.
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| 50. |
- Waluk, Dominik P., 1983-, et al.
(författare)
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Identification of glycine N-acyltransferase-like 2 (GLYATL2) as a transferase that produces N-acyl glycines in humans.
- 2010
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 24:8, s. 2795-2803
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Tidskriftsartikel (refereegranskat)abstract
- The discovery of glycine conjugates of long-chain fatty acids (N-acyl glycines) in the brain and other non-neuronal tissues has led to the identification of an emerging class of bioactive lipids. The biological activities of N-acyl glycines include antinociceptive, anti-inflammatory and antiproliferative effects, and activation of G-protein-coupled receptors. However, despite the fact that N-acyl glycines are emerging as a distinct lipid signaling family, pathways for their production are not fully elucidated. Here we report on the characterization of human glycine N-acyltransferase-like 2 (hGLYATL2), a member of a gene family of 4 putative glycine conjugating enzymes, and show that it synthesizes various N-acyl glycines. Recombinantly expressed hGLYATL2 efficiently conjugated oleoyl-CoA, arachidonoyl-CoA, and other medium- and long-chain acyl-CoAs to glycine. The enzyme was specific for glycine as an acceptor molecule, and preferentially produced N-oleoyl glycine. The hGLYATL2 enzyme is localized to the endoplasmic reticulum, and the mRNA shows highest expression in salivary gland and trachea, but is also detected in spinal cord and skin fibroblasts. The expression pattern and the identification of high levels of N-acyl glycines in skin and lung may indicate a role for N-acyl glycines in barrier function/immune response and the potential role of hGLYATL2 in this regard is discussed.
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