| 1. |
- Koczkowska, Magdalena, et al.
(author)
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Personalized health risk assessment based on single-cell RNA sequencing analysis of a male with 45, X/48, XYYY karyotype
- 2022
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 12:1
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Journal article (peer-reviewed)abstract
- Numeric sex chromosome abnormalities are commonly associated with an increased cancer risk. Here, we report a 14-year-old boy with a rare mosaic 45, X/48, XYYY karyotype presenting with subtle dysmorphic features and relative height deficiency, requiring growth hormone therapy. As only 12 postnatal cases have been described so far with very limited follow-up data, to assess the proband's long-term prognosis, including cancer risk, we performed high-throughput single-cell RNA sequencing (scRNA-seq) analysis. Although comprehensive cytogenetic analysis showed seemingly near perfect balance between 45, X and 48, XYYY cell populations, scRNA-seq revealed widespread differences in genotype distribution among immune cell fractions, specifically in monocytes, B- and T-cells. These results were confirmed at DNA level by digital-droplet PCR on flow-sorted immune cell types. Furthermore, deregulation of predominantly autosomal genes was observed, including TCL1A overexpression in 45, X B-lymphocytes and other known genes associated with hematological malignancies. Together with the standard hematological results, showing increased fractions of monocytes and CD4+/CD8+T lymphocytes ratio, long-term personalized hemato-oncological surveillance was recommended in the reported patient.
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| 2. |
- Bentinger, Magnus, et al.
(author)
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Effects of various squalene epoxides on coenzyme Q and cholesterol synthesis
- 2014
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In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - : Elsevier BV. - 1388-1981 .- 1879-2618. ; 1841:7, s. 977-986
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Journal article (peer-reviewed)abstract
- 2,3-Oxidosqualene is an intermediate in cholesterol biosynthesis and 2,3:22,23-dioxidosqualene act as the substrate for an alternative pathway that produces 24(S),25-epoxycholesterol which effects cholesterol homeostasis. In light of our previous findings concerning the biological effects of certain epoxidated all-trans-polyisoprenes, the effects of squalene carrying epoxy moieties on the second and third isoprene residues were investigated here. In cultures of HepG2 cells both monoepoxides of squalene and one of their hydrolytic products inhibited cholesterol synthesis and stimulated the synthesis of coenzyme Q (CoQ). Upon prolonged treatment the cholesterol content of these cells and its labeling with [H-3]mevalonate were reduced, while the amount and labeling of CoQ increased. Injection of the squalene monoepoxides into mice once daily for 6 days elevated the level of CoQ in their blood, but did not change the cholesterol level. The same effects were observed upon treatment of apoE-deficient mice and diabetic GK-rats. This treatment increased the hepatic level of CoQ10 in mice, but the amount of CoQ9, which is the major form, was unaffected. The presence of the active compounds in the blood was supported by the finding that cholesterol synthesis in the white blood cells was inhibited. Since the ratio of CoQ9/CoQ10 varies depending on the experimental conditions, the cells were titrated with substrate and inhibitors, leading to the conclusion that the intracellular isopentenyl-PP pool is a regulator of this ratio. Our present findings indicate that oxidosqualenes may be useful for stimulating both the synthesis and level of CoQ both in vitro and in vivo.
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| 3. |
- Bruhn-Olszewska, Bozena, et al.
(author)
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Loss of Y in leukocytes as a risk factor for critical COVID-19 in men.
- 2022
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In: Genome medicine. - : Springer Science and Business Media LLC. - 1756-994X. ; 14:1
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Journal article (peer-reviewed)abstract
- The COVID-19 pandemic, which has a prominent social and economic impact worldwide, shows a largely unexplained male bias for the severity and mortality of the disease. Loss of chromosome Y (LOY) is a risk factor candidate in COVID-19 due to its prior association with many chronic age-related diseases, and its impact on immune gene transcription.Publicly available scRNA-seq data of PBMC samples derived from male patients critically ill with COVID-19 were reanalyzed, and LOY status was added to the annotated cells. We further studied LOY in whole blood for 211 COVID-19 patients treated at intensive care units (ICU) from the first and second waves of the pandemic. Of these, 139 patients were subject to cell sorting for LOY analysis in granulocytes, low-density neutrophils (LDNs), monocytes, and PBMCs.Reanalysis of available scRNA-seq data revealed LDNs and monocytes as the cell types most affected by LOY. Subsequently, DNA analysis indicated that 46%, 32%, and 29% of critically ill patients showed LOY above 5% cut-off in LDNs, granulocytes, and monocytes, respectively. Hence, the myeloid lineage that is crucial for the development of severe COVID-19 phenotype is affected by LOY. Moreover, LOY correlated with increasing WHO score (median difference 1.59%, 95% HDI 0.46% to 2.71%, p=0.025), death during ICU treatment (median difference 1.46%, 95% HDI 0.47% to 2.43%, p=0.0036), and history of vessel disease (median difference 2.16%, 95% HDI 0.74% to 3.7%, p=0.004), among other variables. In 16 recovered patients, sampled during ICU stay and 93-143 days later, LOY decreased significantly in whole blood and PBMCs. Furthermore, the number of LDNs at the recovery stage decreased dramatically (median difference 76.4 per 10,000 cell sorting events, 95% HDI 55.5 to 104, p=6e-11).We present a link between LOY and an acute, life-threatening infectious disease. Furthermore, this study highlights LOY as the most prominent clonal mutation affecting the myeloid cell lineage during emergency myelopoiesis. The correlation between LOY level and COVID-19 severity might suggest that this mutation affects the functions of monocytes and neutrophils, which could have consequences for male innate immunity.
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| 4. |
- Eriksson, Mina, et al.
(author)
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Analysis of Chromatin Opening in Heterochromatic Non-Small Cell Lung Cancer Tumor-Initiating Cells in Relation to DNA-Damaging Antitumor Treatment
- 2018
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In: International Journal of Radiation Oncology, Biology, Physics. - : Elsevier BV. - 0360-3016 .- 1879-355X. ; 100:1, s. 174-187
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Journal article (peer-reviewed)abstract
- Purpose: We previously reported that sphere-forming non-small cell lung cancer (NSCLC) tumor-initiating cells (TICs) have an altered activation of DNA damage response-and repair proteins and are refractory to DNA-damaging treatments. We analyzed whether chromatin organization plays a role in the observed refractoriness.Methods and Materials: Bulk cells and TICs from the NSCLC H23 and H1299 cell lines were examined using cell viability, clonogenic survival, Western blot, short interfering RNA analysis, and micronucleus assay.Results: NSCLC TICs displayed elevated heterochromatin markers trimethylated lysine 9 of histone H3 and heterochromatin protein 1 gamma relative to bulk cells and reduced cell viability upon histone deacetylase inhibition (HDACi). Vorinostat and trichostatin A increased the euchromatin markers acetylated lysine 9/14 of histone H3 and lysine 8 of histone H4, and HDACi pretreatment increased the phosphorylation of the DNA damage response proteins ataxia telangiectasia mutated and DNA-dependent protein kinase, catalytic subunit, upon irradiation in TICs. HDACi sensitized TICs to cisplatin and to some extent to ionizing irradiation. The protectiveness of a dense chromatin structure was indicated by an enhanced frequency of micronuclei in TICs following irradiation, after knockdown of heterochromatin protein 1 gamma.Conclusions: Although confirmatory studies in additional NSCLC model systems and with respect to analyses of other DNA damage response proteins are needed, our data point toward a heterochromatic structure of NSCLC TICs, such that HDACi can sensitize TICs to DNA damage.
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| 5. |
- Filipowicz, Natalia, et al.
(author)
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Comprehensive cancer-oriented biobanking resource of human samples for studies of post-zygotic genetic variation involved in cancer predisposition
- 2022
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 17:4
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Journal article (peer-reviewed)abstract
- The progress in translational cancer research relies on access to well-characterized samples from a representative number of patients and controls. The rationale behind our biobanking are explorations of post-zygotic pathogenic gene variants, especially in non-tumoral tissue, which might predispose to cancers. The targeted diagnoses are carcinomas of the breast (via mastectomy or breast conserving surgery), colon and rectum, prostate, and urinary bladder (via cystectomy or transurethral resection), exocrine pancreatic carcinoma as well as metastases of colorectal cancer to the liver. The choice was based on the high incidence of these cancers and/or frequent fatal outcome. We also collect age-matched normal controls. Our still ongoing collection originates from five clinical centers and after nearly 2-year cooperation reached 1711 patients and controls, yielding a total of 23226 independent samples, with an average of 74 donors and 1010 samples collected per month. The predominant diagnosis is breast carcinoma, with 933 donors, followed by colorectal carcinoma (383 donors), prostate carcinoma (221 donors), bladder carcinoma (81 donors), exocrine pancreatic carcinoma (15 donors) and metachronous colorectal cancer metastases to liver (14 donors). Forty percent of the total sample count originates from macroscopically healthy cancer-neighboring tissue, while contribution from tumors is 12%, which adds to the uniqueness of our collection for cancer predisposition studies. Moreover, we developed two program packages, enabling registration of patients, clinical data and samples at the participating hospitals as well as the central system of sample/data management at coordinating center. The approach used by us may serve as a model for dispersed biobanking from multiple satellite hospitals. Our biobanking resource ought to stimulate research into genetic mechanisms underlying the development of common cancers. It will allow all available "-omics" approaches on DNA-, RNA-, protein- and tissue levels to be applied. The collected samples can be made available to other research groups.
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| 6. |
- López Riego, Milagrosa, 1992-, et al.
(author)
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The DNA damage response to radiological imaging : from ROS and γH2AX foci induction to gene expression responses in vivo
- 2023
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In: Radiation and Environmental Biophysics. - 0301-634X .- 1432-2099. ; 62:3, s. 371-393
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Journal article (peer-reviewed)abstract
- Candidate ionising radiation exposure biomarkers must be validated in humans exposed in vivo. Blood from patients undergoingpositron emission tomography–computed tomography scan (PET-CT) and skeletal scintigraphy (scintigraphy) was drawnbefore (0 h) and after (2 h) the procedure for correlation analyses of the response of selected biomarkers with radiation doseand other available patient information. FDXR, CDKN1A, BBC3, GADD45A, XPC, and MDM2 expression was determinedby qRT-PCR, DNA damage (γH2AX) by flow cytometry, and reactive oxygen species (ROS) levels by flow cytometry usingthe 2′, 7′—dichlorofluorescein diacetate test in peripheral blood mononuclear cells (PBMC). For ROS experiments, 0- and2-h samples were additionally exposed to UVA to determine whether diagnostic irradiation conditioned the response tofurther oxidative insult. With some exceptions, radiological imaging induced weak γH2AX foci, ROS and gene expressionfold changes, the latter with good coherence across genes within a patient. Diagnostic imaging did not influence oxidativestress in PBMC successively exposed to UVA. Correlation analyses with patient characteristics led to low correlation coefficientvalues. γH2AX fold change, which correlated positively with gene expression, presented a weak positive correlationwith injected activity, indicating a radiation-induced subtle increase in DNA damage and subsequent activation of the DNAdamage response pathway. The exposure discrimination potential of these biomarkers in the absence of control samples asfrequently demanded in radiological emergencies, was assessed using raw data. These results suggest that the variability ofthe response in heterogeneous populations might complicate identifying individuals exposed to low radiation doses.
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| 7. |
- Olofsson, Dante, et al.
(author)
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Biological effectiveness of very high gamma dose rate and its implication for radiological protection
- 2020
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In: Radiation and Environmental Biophysics. - : Springer Science and Business Media LLC. - 0301-634X .- 1432-2099. ; 59, s. 451-460
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Journal article (peer-reviewed)abstract
- Many experimental studies are carried out to compare biological effectiveness of high dose rate (HDR) with that of low dose rate (LDR). The rational for this is the uncertainty regarding the value of the dose rate effectiveness factor (DREF) used in radiological protection. While a LDR is defined as 0.1 mGy/min or lower, anything above that is seen as HDR. In cell and animal experiments, a dose rate around 1 Gy/min is usually used as representative for HDR. However, atomic bomb survivors, the reference cohort for radiological protection, were exposed to tens of Gy/min. The important question is whether gamma radiation delivered at very high dose rate (VHDR-several Gy/min) is more effective in inducing DNA damage than that delivered at HDR. The aim of this investigation was to compare the biological effectiveness of gamma radiation delivered at VHDR (8.25 Gy/min) with that of HDR (0.38 Gy/min or 0.79 Gy/min). Experiments were carried out with human peripheral mononuclear cells (PBMC) and the human osteosarcoma cell line U2OS. Endpoints related to DNA damage response were analysed. The results show that in PBMC, VHDR is more effective than HDR in inducing gene expression and micronuclei. In U2OS cells, the repair of 53BP1 foci was delayed after VHDR indicating a higher level of damage complexity, but no VHDR effect was observed at the level of micronuclei and clonogenic cell survival. We suggest that the DREF value may be underestimated when the biological effectiveness of HDR and LDR is compared.
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| 8. |
- Płódowska, Magdalena, et al.
(author)
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Hypothermia differentially modulates the formation and decay of NBS1, γH2AX and 53BP1 foci in U2OS cells exposed to gamma radiation
- 2022
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12
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Journal article (peer-reviewed)abstract
- In studies on the mechanism of DNA damage response where ionizing radiation is used as the DNA damaging agent, cells are often exposed to ionizing radiation on melting ice (corresponding to 0.8 °C). The purpose of this procedure is to inhibit cellular processes i.e. DNA repair. Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage, but its mechanisms of action are poorly understood. The aim of the study was to analyze the effect of hypothermia at the level of formation and decay of NBS1, γH2AX, and 53BP1 foci, micronuclei, survival, cell cycle progression and oxidative stress in U2OS cells. The results show that hypothermia alone induced oxidative stress and foci. When applied in combination with radiation but only during the exposure time, it potentiated the formation of γH2AX and 53BP1 but not of NBS1 foci. When applied during irradiation and subsequent repair time, 53BP1 and NBS1 foci formed and decayed, but the levels were markedly lower than when repair was carried out at 37 °C. The frequency of micronuclei was elevated in cells irradiated at 0.8 °C, but only when analysed 20 h after irradiation which is likely due to a reduced G2 cell cycle block. Hypothermia reduced cell survival, both with and without radiation exposure. The temperature effect should be considered when cooling cells on melting ice to inhibit DNA repair in the induction of DNA damage.
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| 9. |
- Płódowska, Magdalena, et al.
(author)
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Small is beautiful : low activity alpha and gamma sources for small-scale radiation protection research experiments
- 2021
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In: International Journal of Radiation Biology. - : Informa UK Limited. - 0955-3002 .- 1362-3095. ; 97:4, s. 541-552
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Journal article (peer-reviewed)abstract
- Purpose: Uncertainties regarding the magnitude of health effects following exposure to low dosesof ionizing radiation remain a matter of concern both for professionals and for the public. There isconsensus within the international radiation research community that more research is requiredon biological effects of radiation doses below 100 mGy applied at low dose rates. Moreover, thereis a demand for increasing education and training of future radiation researchers and regulators.Research, education and training is primarily carried out at universities but university-based radiationresearch is often hampered by limited access to radiation sources. The aim of the presentreport is to describe small and cost-effective low activity gamma and alpha sources that can easilybe installed and used in university laboratories.Methods and results: A gamma radiation source was made from an euxenite-(Y) rock(Y,Ca,Ce,U,Th)(Nb,Ta,Ti)2O6) that was found in an abandoned mine in Sweden. It allows exposingcells grown in culture dishes to radiation at a dose rate of 50 μGy/h and lower. Three alpha sourceswere custom-made and yield a dose rate of 1 mGy/h each. The construction, dosimetry andcellular effects of the sources are described.Conclusions: We hope that the report will stimulate research and training activities in the lowdose field by facilitating access to radiation sources.
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| 10. |
- Stankowska, Wiktoria, et al.
(author)
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Tumor Predisposing Post-Zygotic Chromosomal Alterations in Bladder Cancer-Insights from Histologically Normal Urothelium
- 2024
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In: Cancers. - : MDPI. - 2072-6694. ; 16:5
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Journal article (peer-reviewed)abstract
- Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.
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