| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Gul, Nadia, 1980, et al.
(författare)
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The MTH1 inhibitor TH588 is a microtubule-modulating agent that eliminates cancer cells by activating the mitotic surveillance pathway
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The mut-T homolog-1 (MTH1) inhibitor TH588 has shown promise in preclinical cancer studies but its targeting specificity has been questioned. Alternative mechanisms for the anti-cancer effects of TH588 have been suggested but the question remains unresolved. Here, we performed an unbiased CRISPR screen on human lung cancer cells to identify potential mechanisms behind the cytotoxic effect of TH588. The screen identified pathways and complexes involved in mitotic spindle regulation. Using immunofluorescence and live cell imaging, we showed that TH588 rapidly reduced microtubule plus-end mobility, disrupted mitotic spindles, and prolonged mitosis in a concentration-dependent but MTH1-independent manner. These effects activated a USP28-p53 pathway -the mitotic surveillance pathway -that blocked cell cycle reentry after prolonged mitosis; USP28 acted upstream of p53 to arrest TH588-treated cells in the G1-phase of the cell cycle. We conclude that TH588 is a microtubule-modulating agent that activates the mitotic surveillance pathway and thus prevents cancer cells from re-entering the cell cycle.
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| 3. |
- Dalin, Martin, 1982, et al.
(författare)
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Myocardial KRASG12D expression does not cause cardiomyopathy in mice
- 2014
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Ingår i: Cardiovascular Research. - : Oxford University Press (OUP). - 0008-6363 .- 1755-3245. ; 101:2, s. 229-235
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Tidskriftsartikel (refereegranskat)abstract
- AimsGerm-line mutations in genes encoding components of the RAS/mitogen-activated protein kinase (MAPK) pathway cause developmental disorders called RASopathies. Hypertrophic cardiomyopathy (HCM) is the most common myocardial pathology and a leading cause of death in RASopathy patients. KRAS mutations are found in Noonan and cardio-facio-cutaneous syndromes. KRAS mutations, unlike mutations of RAF1 and HRAS, are rarely associated with HCM. This has been attributed to the fact that germ-line KRAS mutations cause only a moderate up-regulation of the MAPK pathway. Highly bioactive KRAS mutations have been hypothesized to cause severe cardiomyopathy incompatible with life. The aim of this study was to define the impact of KRASG12D expression in the heart.Methods and resultsTo generate mice with endogenous cardiomyocyte-specific KRASG12D expression (cKRASG12D mice), we bred mice with a Cre-inducible allele expressing KRASG12D from its endogenous promoter (Kras2LSL) to mice expressing Cre under control of the cardiomyocyte-specific α-myosin heavy chain promoter (αMHC-Cre). cKRASG12D mice showed high levels of myocardial ERK and AKT signalling. However, surprisingly, cKRASG12D mice were born in Mendelian ratios, appeared healthy, and had normal function, size, and histology of the heart.ConclusionMice with cardiomyocyte-specific KRAS G12D expression do not develop heart pathology. These results challenge the view that the level of MAPK activation correlates with the severity of HCM in RASopathies and suggests that MAPK-independent strategies may be of interest in the development of new treatments for these syndromes. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2013.
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| 4. |
- Nilton, Anna, et al.
(författare)
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Targeting Zfp148 activates p53 and reduces tumor initiation in the gut
- 2016
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Ingår i: OncoTarget. - : Impact Journals, LLC. - 1949-2553. ; 7:35, s. 56183-56192
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Tidskriftsartikel (refereegranskat)abstract
- The transcription factor Zinc finger protein 148 (Zfp148, ZBP-89, BFCOL, BERF1, htβ) interacts physically with the tumor suppressor p53, but the significance of this interaction is not known. We recently showed that knockout of Zfp148 in mice leads to ectopic activation of p53 in some tissues and cultured fibroblasts, suggesting that Zfp148 represses p53 activity. Here we hypothesize that targeting Zfp148 would unleash p53 activity and protect against cancer development, and test this idea in the APCMin/+ mouse model of intestinal adenomas. Loss of one copy of Zfp148 markedly reduced tumor numbers and tumor-associated intestinal bleedings, and improved survival. Furthermore, after activation of β-catenin-the initiating event in colorectal cancer-Zfp148 deficiency activated p53 and induced apoptosis in intestinal explants of APCMin/+ mice. The anti-tumor effect of targeting Zfp148 depended on p53, as Zfp148 deficiency did not affect tumor numbers in APCMin/+ mice lacking one or both copies of Trp53. The results suggest that Zfp148 controls the fate of newly transformed intestinal tumor cells by repressing p53 and that targeting Zfp148 might be useful in the treatment of colorectal cancer.
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| 5. |
- Wilhelmson, Anna S K, et al.
(författare)
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Increased Intimal Hyperplasia After Vascular Injury in Male Androgen Receptor-Deficient Mice
- 2016
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Ingår i: Endocrinology. - : The Endocrine Society. - 1945-7170 .- 0013-7227. ; 157:10, s. 3915-3923
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Tidskriftsartikel (refereegranskat)abstract
- Intimal hyperplasia is a vascular pathological process involved in the pathogenesis of atherosclerosis. Data suggest that T, the most important sex steroid hormone in males, protects men from atherosclerotic cardiovascular disease. T mainly acts via the androgen receptor (AR), and in this study we evaluated formation of intimal hyperplasia in male AR knockout (ARKO) mice using a vascular injury model. Two weeks after ligation of the carotid artery, male ARKO mice showed increased intimal area and intimal thickness compared with controls. After endothelial denudation by an in vivo scraping injury, there was no difference in the reendothelialization in ARKO compared with control mice. Ex vivo, we observed increased outgrowth of vascular smooth muscle cells from ARKO compared with control aortic tissue explants; the number of outgrown cells was almost doubled in ARKO. In vitro, stimulation of human aortic vascular smooth muscle cells with a physiological T concentration inhibited both migration and proliferation of the cells. Analyzing the expression of central regulators of cell proliferation and migration, we found that mRNA and protein levels of p27 were lower in uninjured arteries from ARKO mice and that T replacement to castrated male mice increased p27 mRNA in an AR-dependent manner. In conclusion, AR deficiency in male mice increases intimal hyperplasia in response to vascular injury, potentially related to the effects of androgens/AR to inhibit proliferation and migration of smooth muscle cells.
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| 6. |
- Zou, Zhiyuan V., et al.
(författare)
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Antioxidants promote intestinal tumor progression in mice.
- 2021
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Ingår i: Antioxidants. - : MDPI AG. - 2076-3921. ; 10:2
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Tidskriftsartikel (refereegranskat)abstract
- Dietary antioxidants and supplements are widely used to protect against cancer, even though it is now clear that antioxidants can promote tumor progression by helping cancer cells to overcome barriers of oxidative stress. Although recent studies have, in great detail, explored the role of antioxidants in lung and skin tumors driven by RAS and RAF mutations, little is known about the impact of antioxidant supplementation on other cancers, including Wnt-driven tumors originating from the gut. Here, we show that supplementation with the antioxidants N-acetylcysteine (NAC) and vitamin E promotes intestinal tumor progression in the ApcMin mouse model for familial adenomatous polyposis, a hereditary form of colorectal cancer, driven by Wnt signaling. Both antioxidants increased tumor size in early neoplasias and tumor grades in more advanced lesions without any impact on tumor initiation. Importantly, NAC treatment accelerated tumor progression at plasma concentrations comparable to those obtained in human subjects after prescription doses of the drug. These results demonstrate that antioxidants play an important role in the progression of intestinal tumors, which may have implications for patients with or predisposed to colorectal cancer.
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| 7. |
- Zou, Zhiyuan V., et al.
(författare)
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Genomic profiling of the transcription factor Zfp148 and its impact on the p53 pathway
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Recent data suggest that the transcription factor Zfp148 represses activation of the tumor suppressor p53 in mice and that therapeutic targeting of the human orthologue ZNF148 could activate the p53 pathway without causing detrimental side effects. We have previously shown that Zfp148 deficiency promotes p53-dependent proliferation arrest of mouse embryonic fibroblasts (MEFs), but the underlying mechanism is not clear. Here, we showed that Zfp148 deficiency downregulated cell cycle genes in MEFs in a p53-dependent manner. Proliferation arrest of Zfp148-deficient cells required increased expression of ARF, a potent activator of the p53 pathway. Chromatin immunoprecipitation showed that Zfp148 bound to the ARF promoter, suggesting that Zfp148 represses ARF transcription. However, Zfp148 preferentially bound to promoters of other transcription factors, indicating that deletion of Zfp148 may have pleiotropic effects that activate ARF and p53 indirectly. In line with this, we found no evidence of genetic interaction between TP53 and ZNF148 in CRISPR and siRNA screen data from hundreds of human cancer cell lines. We conclude that Zfp148 deficiency, by increasing ARF transcription, downregulates cell cycle genes and cell proliferation in a p53-dependent manner. However, the lack of genetic interaction between ZNF148 and TP53 in human cancer cells suggests that therapeutic targeting of ZNF148 may not increase p53 activity in humans.
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| 8. |
- Zou, Zhiyuan V.
(författare)
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Mechanisms of intestinal tumor initiation and progression in ApcMin mouse model of colorectal cancer
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Colorectal cancer (CRC) is a cancer that occurs in the colon or rectum and is one of the most common cancer forms and a leading cause of cancer death in the Western world. The majority of CRCs are caused by inherited or somatic mutations in the APC gene. The ApcMin model is driven by a truncating mutation in the Apc gene and is considered to be one of the best mouse models of CRC. The aim of this thesis is to uncover novel mechanisms behind the initiation and progression of intestinal adenomas in ApcMin/+ mice. Clinical studies suggest that the transcription factor Zfp148 may play a role in CRC but the importance of Zfp148 for tumor development has not been properly investigated. We have previously shown that Zfp148 is a potent inhibitor of p53 and hypothesized that Zfp148 deficiency may protect against CRC by increasing p53-activity. In paper I, we show that deletion of one or both copies of Zfp148 markedly reduces tumor formation in ApcMin/+ mice. The result shows that Zfp148 controls the fate of newly transformed tumor cells by repressing p53, and suggests that targeting Zfp148 might be useful in the treatment of colorectal cancer. Previous studies show that Zfp148 inhibits activation of p53; however, the underlying mechanism is not understood. We have previously shown that transcription of Cdkn2a was increased in Zfp148 deficient MEFs. ARF is one of two products of Cdkn2a and is a major activator of the p53-pathway. Therefore, we hypothesized that Zfp148 inhibits p53 by repressing ARF. In paper II, we tested this hypothesis in mouse embryoblasts (MEFs) and found that Zfp148 regulates cell proliferation and p53 activity by repressing the transcription of ARF. Finally, we addressed the role of antioxidants. Clinical studies on the ability of dietary antioxidants to prevent CRC show inconsistent results. To understand the effect of antioxidants, we gave two types of dietary antioxidants to ApcMIn/+ mice and investigated tumor development. Our results indicate that dietary antioxidants have no effect on tumor initiation but accelerate progression of existing tumors. The result raises concerns about the widespread use of dietary antioxidants, especially among high risk populations.
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