Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells

Acknowledgments This work was funded by the MRC Program grant MC_PC 12001/1 and MC_UU_00001/1 to K.R., and S.K. was supported by the MRC Oxford Institute of Radiation Oncology (OIRO) Cancer Research UK (CRUK) Studentship. We thank Dr. Rhodri Wilson from the microscopy imaging core (University of Oxford, Department of Oncology) for his technical advice and assistance. Graphical abstract was created with BioRender.Peer reviewedPublisher PD

Pojava listerioze u uzgoju činčila (Chinchilla laniger) u Hrvatskoj.

Listeriosis is an infectious disease of domestic and wild animals caused by Listeria monocytogenes, and is transferable to humans. There are two clinical forms of listeriosis: septicemic and encephalitic. This paper deals with two cases of listeriosis in chinchillas (Chinchilla laniger), originating from a farm in the County of Me|imurje in Croatia, and were pathomorphologically and bacteriologically examined. This particular breed consisted of 64 chinchillas, 34 of which have died due to same symptoms. History reveals that animals had suffered loss of appetite, and abortion occurred about 5 days before death. Gross lesions included general anaemia and cachexia. Whitish-grey nodules the size of millet grain or a little larger were noticed along the capsule of liver, serosa of colon and in mesenterial lymph nodes. Numerous variably-sized granulomas in liver, intestines, lymph nodes and kidneys, with no special pattern, were observed histopathologically. They consisted of necrotic cells with somewhat more granulocytes in the centre and scattered individual gigantocytes. Bacteriological examination of liver specimens revealed Listeria monocytogenes in a pure culture in both cases. This is the first description of listeriosis in chinchillas in Croatia.Listerioza je zarazna bolest domaćih i divljih životinja uzrokovana bakterijom Listeria monocytogenes, a može se prenijeti i na čovjeka. Klinički se manifestira u dva oblika: kao septikemija ili kao encefalitis. Rad opisuje dva slučaja listerioze u činčila (Chinchilla laniger), s jedne farme iz Me|imurja u Hrvatskoj, koji su patomorfološki i bakteriološki obrađeni. U uzgoju su uginule 34 od 64 životinje s istim simptomima. Prema anamnestičkim podacima životinje su izgubile apetit i pobacile oko 5 dana prije uginuća. Makroskopski je bila vidljiva opća anemija i izrazita mršavost životinja. Sivo bijeli čvorići veličine zrna prosa ili nešto veći rasprostirali su se po jetri, serozi kolona i mezenterijalnim limfnim čvorovima. Histopatološkom pretragom jetre, crijeva, limfnih čvorova i bubrega nađeni su brojni granulomi, različite veličine i rasporeda. Sastojali su se od nekrotičnih stanica s nešto više granulocita u centru i razbacanim pojedinačnim gigantocitima. Bakteriološkom pretragom jetre obje činčile izolirana je Listeria monocytogenes u čistoj kulturi. Ovo je prvi opisani slučaj pojave listerioze u činčila u Hrvatskoj

DNA protein crosslink proteolysis repair: From yeast to premature ageingand cancer in humans

DNA-protein crosslinks (DPCs) are a specific type of DNA lesion consisting of a protein covalently and irreversibly bound to DNA, which arise after exposure to physical and chemical crosslinking agents. DPCs can be bulky and thereby pose a barrier to DNA replication and transcription. The persistence of DPCs during S phase causes DNA replication stress and genome instability. The toxicity of DPCs is exploited in cancer therapy: many common chemotherapeutics kill cancer cells by inducing DPC formation. Recent work from several laboratories discovered a specialized repair pathway for DPCs, namely DPC proteolysis (DPCP) repair. DPCP repair is carried out by replication-coupled DNA-dependent metalloproteases: Wss1 in yeast and SPRTN in metazoans. Mutations in SPRTN cause premature ageing and liver cancer in humans and mice ; thus, defective DPC repair has great clinical ramifications. In the present review, we will revise the current knowledge on the mechanisms of DPCP repair and on the regulation of DPC protease activity, while highlighting the most significant unresolved questions in the field. Finally, we will discuss the impact of faulty DPC repair on disease and cancer therapy

Pojava listerioze u uzgoju činčila (Chinchilla laniger) u Hrvatskoj.

Listeriosis is an infectious disease of domestic and wild animals caused by Listeria monocytogenes, and is transferable to humans. There are two clinical forms of listeriosis: septicemic and encephalitic. This paper deals with two cases of listeriosis in chinchillas (Chinchilla laniger), originating from a farm in the County of Me|imurje in Croatia, and were pathomorphologically and bacteriologically examined. This particular breed consisted of 64 chinchillas, 34 of which have died due to same symptoms. History reveals that animals had suffered loss of appetite, and abortion occurred about 5 days before death. Gross lesions included general anaemia and cachexia. Whitish-grey nodules the size of millet grain or a little larger were noticed along the capsule of liver, serosa of colon and in mesenterial lymph nodes. Numerous variably-sized granulomas in liver, intestines, lymph nodes and kidneys, with no special pattern, were observed histopathologically. They consisted of necrotic cells with somewhat more granulocytes in the centre and scattered individual gigantocytes. Bacteriological examination of liver specimens revealed Listeria monocytogenes in a pure culture in both cases. This is the first description of listeriosis in chinchillas in Croatia.Listerioza je zarazna bolest domaćih i divljih životinja uzrokovana bakterijom Listeria monocytogenes, a može se prenijeti i na čovjeka. Klinički se manifestira u dva oblika: kao septikemija ili kao encefalitis. Rad opisuje dva slučaja listerioze u činčila (Chinchilla laniger), s jedne farme iz Me|imurja u Hrvatskoj, koji su patomorfološki i bakteriološki obrađeni. U uzgoju su uginule 34 od 64 životinje s istim simptomima. Prema anamnestičkim podacima životinje su izgubile apetit i pobacile oko 5 dana prije uginuća. Makroskopski je bila vidljiva opća anemija i izrazita mršavost životinja. Sivo bijeli čvorići veličine zrna prosa ili nešto veći rasprostirali su se po jetri, serozi kolona i mezenterijalnim limfnim čvorovima. Histopatološkom pretragom jetre, crijeva, limfnih čvorova i bubrega nađeni su brojni granulomi, različite veličine i rasporeda. Sastojali su se od nekrotičnih stanica s nešto više granulocita u centru i razbacanim pojedinačnim gigantocitima. Bakteriološkom pretragom jetre obje činčile izolirana je Listeria monocytogenes u čistoj kulturi. Ovo je prvi opisani slučaj pojave listerioze u činčila u Hrvatskoj

SPRTN protease-cleaved MRE11 decreases DNA repair and radiosensitises cancer cells

Funding Information: This work was funded by CRUK Programme Grant C5255/A23755. Acknowledgements Mass spectrometry analysis was performed in the MS laboratory at the Target discovery institute—NDM (Oxford) led by Benedikt M. Kessler. We thank Drs. Eva McGrowder and Blaz Groselj for processing of primary bladder tumour samples to produce cell-free extracts. Data availability The LC-MS/MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE48 partner repository with the dataset identifier PXD017964 and 10.6019/PXD017964.Peer reviewedPublisher PD

p97/VCP inhibition causes excessive MRE11-dependent DNA end resection promoting cell killing after ionizing radiation

Funding Information: This work was funded by Cancer Research UK (CRUK) program grant C5255/A23755 to A.E.K. Medical Research Council UK (MRC) program grant MC_PC 12001/1 (MC_UU_00001/1) and Breast Cancer Now (Grant No. 2019DecPR1406) to K.R. S.K. was supported by the MRC Oxford Institute of Radiation Oncology (OIRO) CRUK studentship. We thank Dr. Sovan Sarkar (Department of Oncology, University of Oxford) for generously providing DR-GFP U2OS cells. We thank Diogo Dias (Ludwig Cancer Research Institute, University of Oxford) for his technical advice on HR and SSA assays and assistance with the analysis. We thank Dr. Lisa Folkes and Alix Hampson for the high-performance liquid chromatography (HPLC) analysis of CB-5083 concentration in tissue extracts from CD-1 nude mice bearing subcutaneous RT112 tumors. We also thank the Oxford Radcliffe Biobank for providing us with human tissue sections.Peer reviewedPublisher PD

Targeting TOPK sensitises tumour cells to radiation-induced damage by enhancing replication stress

T-LAK-originated protein kinase (TOPK) overexpression is a feature of multiple cancers, yet is absent from most phenotypically normal tissues. As such, TOPK expression profiling and the development of TOPK-targeting pharmaceutical agents have raised hopes for its future potential in the development of targeted therapeutics. Results presented in this paper confirm the value of TOPK as a potential target for the treatment of solid tumours, and demonstrate the efficacy of a TOPK inhibitor (OTS964) when used in combination with radiation treatment. Using H460 and Calu-6 lung cancer xenograft models, we show that pharmaceutical inhibition of TOPK potentiates the efficacy of fractionated irradiation. Furthermore, we provide in vitro evidence that TOPK plays a hitherto unknown role during S phase, showing that TOPK depletion increases fork stalling and collapse under conditions of replication stress and exogenous DNA damage. Transient knockdown of TOPK was shown to impair recovery from fork stalling and to increase the formation of replication-associated single-stranded DNA foci in H460 lung cancer cells. We also show that TOPK interacts directly with CHK1 and Cdc25c, two key players in the checkpoint signalling pathway activated after replication fork collapse. This study thus provides novel insights into the mechanism by which TOPK activity supports the survival of cancer cells, facilitating checkpoint signalling in response to replication stress and DNA damage

ATXN3 controls DNA replication and transcription by regulating chromatin structure

The deubiquitinating enzyme Ataxin-3 (ATXN3) contains a polyglutamine (PolyQ) region, the expansion of which causes spinocerebellar ataxia type-3 (SCA3). ATXN3 has multiple functions, such as regulating transcription or controlling genomic stability after DNA damage. Here we report the role of ATXN3 in chromatin organization during unperturbed conditions, in a catalytic-independent manner. The lack of ATXN3 leads to abnormalities in nuclear and nucleolar morphology, alters DNA replication timing and increases transcription. Additionally, indicators of more open chromatin, such as increased mobility of histone H1, changes in epigenetic marks and higher sensitivity to micrococcal nuclease digestion were detected in the absence of ATXN3. Interestingly, the effects observed in cells lacking ATXN3 are epistatic to the inhibition or lack of the histone deacetylase 3 (HDAC3), an interaction partner of ATXN3. The absence of ATXN3 decreases the recruitment of endogenous HDAC3 to the chromatin, as well as the HDAC3 nuclear/cytoplasm ratio after HDAC3 overexpression, suggesting that ATXN3 controls the subcellular localization of HDAC3. Importantly, the overexpression of a PolyQ-expanded version of ATXN3 behaves as a null mutant, altering DNA replication parameters, epigenetic marks and the subcellular distribution of HDAC3, giving new insights into the molecular basis of the disease.</p

ATXN3 controls DNA replication and transcription by regulating chromatin structure

The deubiquitinating enzyme Ataxin-3 (ATXN3) contains a polyglutamine (PolyQ) region, the expansion of which causes spinocerebellar ataxia type-3 (SCA3). ATXN3 has multiple functions, such as regulating transcription or controlling genomic stability after DNA damage. Here we report the role of ATXN3 in chromatin organization during unperturbed conditions, in a catalytic-independent manner. The lack of ATXN3 leads to abnormalities in nuclear and nucleolar morphology, alters DNA replication timing and increases transcription. Additionally, indicators of more open chromatin, such as increased mobility of histone H1, changes in epigenetic marks and higher sensitivity to micrococcal nuclease digestion were detected in the absence of ATXN3. Interestingly, the effects observed in cells lacking ATXN3 are epistatic to the inhibition or lack of the histone deacetylase 3 (HDAC3), an interaction partner of ATXN3. The absence of ATXN3 decreases the recruitment of endogenous HDAC3 to the chromatin, as well as the HDAC3 nuclear/cytoplasm ratio after HDAC3 overexpression, suggesting that ATXN3 controls the subcellular localization of HDAC3. Importantly, the overexpression of a PolyQ-expanded version of ATXN3 behaves as a null mutant, altering DNA replication parameters, epigenetic marks and the subcellular distribution of HDAC3, giving new insights into the molecular basis of the disease.Spanish Agencia Estatal de Investigacion´ [PID2019- 109222RB-I00/AEI/10.13039/501100011033]; European Union Regional Funds (FEDER) (to R.F., V.A.J.S.); Agencia Canaria de Investigacion, ´ Innovacion´ y Sociedad de la Informacion´ [ProID2020010109]; FEDER (to R.F.); Agencia Canaria de Investigacion, ´ Innovacion´ y European Social Fund integrated Operational programme of the Canary Islands Sociedad de la Informacion´ de la Consejer´ıa de Econom´ıa, Industria, Comercio y Conocimiento and the 2014–2020, Eje 3 Tema Prioritario 74 (85%) (to E.H.C.); Medical Research Council Programme [MR/X006409/1 to K.R.]; Breast Cancer Now [2019DecPR1406 to K.R.]. Funding for open access charge: Agencia Canaria de Investigacion, ´ Innovacion´ y Sociedad de la Informacion´ [ProID2020010109]; European Union Regional Funds (FEDER)