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

BLM and BRCA1-BARD1 coordinate complementary mechanisms of joint DNA molecule resolution

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors

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

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A blood atlas of COVID-19 defines hallmarks of disease severity and specificity.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19

Evaluación y desarrollo de modelos "in vitro" para la predicción de neurotoxicidad. Aproximación proteómica a la neurotoxicidad inducida por metilmercurio

Tesis doctoral inédita leída el 20 de julio de 2006 en la Universidad de Barcelona.[EN]: In the last years there has been a growing interest in using in vitro methods to detect toxic potentially chemical compounds, contributing in replacement, reduction and refinement animal use in life science research. Several organizations are working together to develop and validate alternative methods. Responding to the necessity to promote alternative methods in the neurotoxicity field, European Centre for the Validation of Alternative Methods (ECVAM) proposed the elaboration of an alternative methods database to detect chemical compounds potentially neurotoxic. The goals of this thesis include both bibliographic and experimental studies to (1) create a database for in vitro methods to detect neurotoxic chemical compounds, (2) to identify toxic biomarkers by using proteomics techniques in primary cultures of mouse cerebellar granule cells exposed to methylmercury (MeHg) and (3) to evaluate cell death and neuroprotection mechanisms involved in long-term MeHg toxicity in primary cultures of mouse cerebellar granule cells. As a result of the bibliographic study, 6 in vitro methods were proposed to evaluate neurotoxic potency of chemical compounds capable to produce axonopaties and delayed neuropaties, to affect neurotransmission through GABAA receptors, voltage dependent sodium channels or ionotropic glutamate receptors, or to produce neuronal and glial neurotoxicity. All this methods supplied useful information to a future method validation (now available in ecvam-dbalm.jrc.cec.eu.int). Methylmercury is an environmental contaminant with special toxicity to Central Nervous System (SNC) and in particular to cerebellar granule cells. A proteomic approach revealed that long-term MeHg exposure to primary cultures of cerebellar granule cells induced a subexpression of succinilCoA:3-ketoacidCoA transferase I (SCOT), enzyme implicated in the ketone body catabolism, and an overexpression of non-phosphorylated cofilin, an actin binding protein involved in the regulation of actin dynamics. On the other hand, long-term MeHg exposure to primary cultures of cerebellar granule neurons produced time- and concentration-dependent apoptotic cell death. This cell death was not an excitotoxic death and was independent of caspasse 3 activation but was partially dependent of lysosomal cathepsin D. Moreover, MeHg exposure produced an increased in lipid peroxidation. In this experimental model, neuronal cell death was protected by antioxidants of wide spectrum, suggesting that this apoptotic cell death could be due to the increased of intracellular reactive oxygen species (ROS).[ES]: En los últimos años ha habido un creciente interés en la utilización de métodos in vitro para la detección del potencial tóxico de compuestos químicos que contribuyan en remplazar, reducir y reformar el uso de animales en experimentación. Distintas organizaciones nacionales e internacionales están trabajando en el desarrollo y la validación de métodos in vitro que proporcionen el mismo nivel de información que los modelos in vivo. Se estima que entre el 328 % de los compuestos químicos existentes son potencialmente neurotóxicos. Respondiendo a la necesidad de impulsar la utilización de métodos in vitro en el campo de la neurotoxicología, el Centro Europeo para la Validación de Métodos Alternativos (ECVAM) propuso la elaboración de una base de datos de métodos alternativos para la detección de compuestos químicos potencialmente neurotóxicos. Los objetivos planteados en este trabajo contemplan estudios bibliométricos y experimentales para (1) la creación de una bases de datos de métodos in vitro para la detección de compuestos químicos potencialmente neurotóxicos, (2) para la caracterización de proteínas y la identificación de marcadores de toxicidad mediante herramientas proteómicas utilizando como modelo celular cultivos primarios de células granulares de cerebelo de ratón y como agente tóxico el metilmercurio y (3) la evaluación de los mecanismos de muerte y neuroprotección de células granulares de cerebelo expuestos a metilmercurio. Tras un estudio bibliométrico, se propusieron 6 métodos in vitro para evaluar el potencial tóxico de compuestos químicos capaces de producir axonopatías y neuropatías retardadas, de alterar la transmisión nerviosa a través de los receptores GABAA, de los canales de sodio dependientes de voltaje y de los receptores ionotrópicos de glutamato, y de la utilización de los cultivos reagreagados de cerebro como modelo celular para la evaluación de neurotoxicidad en general. Estos métodos proporcionan información útil para una futura validación de los mismos (consultable en ecvam-dbalm.jrc.cec.eu.int). El metilmercurio (MeHg) es un contaminante ambiental con toxicidad selectiva para el SNC y en particular para las células granulares de cerebelo. El análisis proteómico de extractos proteicos procedentes de cultivos primarios de células granulares de cerebelo de ratón expuestos de forma prolongada a concentraciones subcitotóxicas de metilmercurio permitió identificar dos proteínas, la succinil CoA:3-cetoácido-CoA-transferasa I y la isoforma no muscular de la cofilina, cuya expresión disminuyó e incrementó en presencia de metilmercurio, respectivamente. La exposición prolongada de los cultivos primarios de células granulares de cerebelo a MeHg produce una muerte neuronal apoptótica tiempo y concentración dependiente que cursa por mecanismos no excitotóxicos, independientes de la activación de caspasa 3 y parcialmente dependientes de la liberación de la proteasa lisosomal catepsina D. Además la exposición a MeHg produjo un incremento de la peroxidación lipídica. En nuestro modelo experimental la muerte neuronal fue protegida por antioxidantes de amplio espectro hecho que sugiere que la muerte podría deberse al incremento de ROS intracelular.Peer reviewe

Evaluación y desarrollo de modelos "in vitro" para la predicción de neurotoxicidad. Aproximación proteómica a la neurotoxicidad inducida por metilmercurio

[spa] En los últimos años ha habido un creciente interés en la utilización de métodos "in vitro" para la detección del potencial tóxico de compuestos químicos que contribuyan en remplazar, reducir y reformar el uso de animales en experimentación. Distintas organizaciones nacionales e internacionales están trabajando en el desarrollo y la validación de métodos "in vitro" que proporcionen el mismo nivel de información que los modelos in vivo. Se estima que entre el 3-28 % de los compuestos químicos existentes son potencialmente neurotóxicos. Respondiendo a la necesidad de impulsar la utilización de métodos "in vitro" en el campo de la neurotoxicología, el Centro Europeo para la Validación de Métodos Alternativos (ECVAM) propuso la elaboración de una base de datos de métodos alternativos para la detección de compuestos químicos potencialmente neurotóxicos. Los objetivos planteados en este trabajo contemplan estudios bibliométricos y experimentales para (1) la creación de una bases de datos de métodos "in vitro" para la detección de compuestos químicos potencialmente neurotóxicos, (2) para la caracterización de proteínas y la identificación de marcadores de toxicidad mediante herramientas proteómicas utilizando como modelo celular cultivos primarios de células granulares de cerebelo de ratón y como agente tóxico el metilmercurio y (3) la evaluación de los mecanismos de muerte y neuroprotección de células granulares de cerebelo expuestos a metilmercurio. Tras un estudio bibliométrico, se propusieron 6 métodos in vitro para evaluar el potencial tóxico de compuestos químicos capaces de producir axonopatías y neuropatías retardadas, de alterar la transmisión nerviosa a través de los receptores GABAA, de los canales de sodio dependientes de voltaje y de los receptores ionotrópicos de glutamato, y de la utilización de los cultivos reagreagados de cerebro como modelo celular para la evaluación de neurotoxicidad en general. Estos métodos proporcionan información útil para una futura validación de los mismos (consultable en ecvam-dbalm.jrc.cec.eu.int). El metilmercurio (MeHg) es un contaminante ambiental con toxicidad selectiva para el SNC y en particular para las células granulares de cerebelo. El análisis proteómico de extractos proteicos procedentes de cultivos primarios de células granulares de cerebelo de ratón expuestos de forma prolongada a concentraciones subcitotóxicas de metilmercurio permitió identificar dos proteínas, la succinil CoA:3-cetoácido-CoA-transferasa I y la isoforma no muscular de la cofilina, cuya expresión disminuyó e incrementó en presencia de metilmercurio, respectivamente. La exposición prolongada de los cultivos primarios de células granulares de cerebelo a MeHg produce una muerte neuronal apoptótica tiempo y concentración dependiente que cursa por mecanismos no excitotóxicos, independientes de la activación de caspasa 3 y parcialmente dependientes de la liberación de la proteasa lisosomal catepsina D. Además la exposición a MeHg produjo un incremento de la peroxidación lipídica. En nuestro modelo experimental la muerte neuronal fue protegida por antioxidantes de amplio espectro hecho que sugiere que la muerte podría deberse al incremento de ROS intracelular.[eng] "Evaluation and development of alternative methods for prediction of neurotoxicity. Proteomic approach to methylmercury induced neurotoxicity": In the last years there has been a growing interest in using "in vitro" methods to detect toxic potentially chemical compounds, contributing in replacement, reduction and refinement animal use in life science research. Several organizations are working together to develop and validate alternative methods. Responding to the necessity to promote alternative methods in the neurotoxicity field, European Centre for the Validation of Alternative Methods (ECVAM) proposed the elaboration of an alternative methods database to detect chemical compounds potentially neurotoxic. The goals of this thesis include both bibliographic and experimental studies to (1) create a database for "in vitro" methods to detect neurotoxic chemical compounds, (2) to identify toxic biomarkers by using proteomics techniques in primary cultures of mouse cerebellar granule cells exposed to methylmercury (MeHg) and (3) to evaluate cell death and neuroprotection mechanisms involved in long-term MeHg toxicity in primary cultures of mouse cerebellar granule cells. As a result of the bibliographic study, 6 "in vitro" methods were proposed to evaluate neurotoxic potency of chemical compounds capable to produce axonopaties and delayed neuropaties, to affect neurotransmission through GABAA receptors, voltage dependent sodium channels or ionotropic glutamate receptors, or to produce neuronal and glial neurotoxicity. All this methods supplied useful information to a future method validation (now available in ecvam-dbalm.jrc.cec.eu.int). Methylmercury is an environmental contaminant with special toxicity to Central Nervous System (SNC) and in particular to cerebellar granule cells. A proteomic approach revealed that long-term MeHg exposure to primary cultures of cerebellar granule cells induced a subexpression of succinilCoA:3-ketoacidCoA transferase I (SCOT), enzyme implicated in the ketone body catabolism, and an overexpression of non-phosphorylated cofilin, an actin binding protein involved in the regulation of actin dynamics. On the other hand, long-term MeHg exposure to primary cultures of cerebellar granule neurons produced time- and concentration-dependent apoptotic cell death. This cell death was not an excitotoxic death and was independent of caspasse 3 activation but was partially dependent of lysosomal cathepsin D. Moreover, MeHg exposure produced an increased in lipid peroxidation. In this experimental model, neuronal cell death was protected by antioxidants of wide spectrum, suggesting that this apoptotic cell death could be due to the increased of intracellular reactive oxygen species (ROS)

GABA released from cultured cortical neurons influences the modulation of t-[35S]butylbicyclophosphorothionate binding at the GABAA receptor: Effects of thymol

El pdf del artículo es la versión pre-print.Thymol is a monoterpene that specifically interacts with synaptic neural functions in neuronal GABA-operated Cl(-) channels. Here we explore the effects of thymol, and propofol as positive control, on t-[(35)S]butylbicyclophosphorothionate ([(35)S]TBPS) binding in primary cultures of cortical neurons. The study includes a meaningful analysis of the effect of various exposure buffers, and their correlation with GABA released from cells, chloride influx through the GABA(A) receptor and GABA transporter activity. Cell viability was also determined. Thymol and propofol inhibited the binding of [(35)S]TBPS to cells exposed to Tris-citrate-NaCl buffer whereas a biphasic effect was observed in HEPES solution. The different effects of the two buffers analysed are due to the higher capacity of Tris-citrate-NaCl buffer to induce the release of endogenous GABA facilitating the binding of [(35)S]TBPS to its recognition site at the GABA(A) receptor. Released GABA in the presence of this buffer was inhibited by the neuronal GABA transporter inhibitor SKF 100330-A. Tris-citrate-NaCl buffer also induced a chloride influx, which was reverted by picrotoxinin. TBPS binding in living cells is facilitated by GABA released from the cells, which in turn activates basal GABA(A) receptor activity. The results deepen on the allosteric mechanism of thymol as positive modulator of the GABA(A) receptor. Furthermore, we corroborate [(35)S]TBPS binding as an important test to verify the capacity of drugs to act on and recognize a site at the GABA(A) receptor.This study was supported by grants PI061212 and 2005-SGR-00826 (Ministry of Health and Generalitat de Catalunya, respectively, Spain), SECYT-UNC (Argentina) and CAEN-ISN. DAG is a member of CONICET (Argentina) and was recipient of a post-doctoral fellowship from the Fundación Carolina (Spain). IV was recipient of a CSIC Fellowship in the I3P program, co-financed with European Social Funds.Peer reviewe

Probucol increases glutathione peroxidase-1 activity and displays long-lasting protection against methylmercury toxicity in cerebellar granule cells

El pdf del artículo es el manuscrito de autor.Methylmercury (MeHg) is an environmental neurotoxicant whose molecular mechanisms underlying toxicity remain elusive. Here we investigated molecular events involved in MeHg-induced neurotoxicity in cultured cerebellar granule cells (CGCs) as well as potential protective strategies for such toxicity. Glutathione peroxidase (GPx-1) activity was significantly (p = 0.0017) decreased at 24 h before MeHg-induced neuronal death (day in vitro 4). This event was related to enhanced susceptibilities to hydrogen- or tert-butyl-peroxides and increased lipid peroxidation. However, intracellular calcium levels, glutamate uptake and glutathione levels, as well as glutathione reductase and catalase activities, were not changed by MeHg exposure at this time-point. Probucol (PB), a lipid-lowering drug, displayed a long-lasting protective effect against MeHg-induced neurotoxicity. The beneficial effects of PB were correlated to increased GPx-1 activity and decreased lipid peroxidation. The protection afforded by PB was significantly higher when compared to the antioxidants ascorbic acid and trolox. In vitro studies with the purified GPx-1 proved that MeHg inhibits and PB activates the enzyme activity. Overexpression of GPx-1 prevented MeHg-induced neuronal death. These data indicate that (i) GPx-1 is an important molecular target involved in MeHg-induced neurotoxicity and (ii) PB, which increases GPx-1 activity in CGCs, induces enduring protection against such toxicity. The results bring out new insights on the potential therapeutic strategies for poisonings to MeHg and other pathological conditions related to increased production and/or decreased detoxification of peroxides.This study was supported by the Grant FIS PI061212 and 2005-SGR-00826 (Ministry of Health and Generalitat de Catalunya, respectively, Spain). Marcelo Farina was recipient of a post- doctoral fellowship (Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq/201362/2007-4) and received financial support from CNPq (479239/2007-0) and FAPESC (Jovens Pesquisadores - FAPESC/CNPq 04/2007).Peer reviewe

The selenoprotein glutathione peroxidase is a primary molecular target of methylmercury-induced neurotoxicity: insights on potential therapeutic strategies

Methylmercury (MeHg) is an environmental neurotoxicant whose molecular mechanisms underlying toxicity remain elusive. Here we investigated potential primary molecular events involved with the neurotoxicity induced by low-dose MeHg exposure in cultured cerebellar granule cells (CGCs), as well as potential protective strategies for such toxicity. We found that MeHg induced a time- and dose-dependent neuronal death. This phenomenon was preceded by a decrease in glutathione peroxidase (GPx) activity, but no changes in glutathione levels, intracellular calcium homeostasis and glutamate uptake were observed prior to cell death. In agreement with the decreased GPx activity, MeHg-induced neurotoxicity was related to an enhanced susceptibility to hydrogen- and tert-butyl-peroxides, as well as to increased lipid peroxidation. The antioxidants ascorbic acid and trolox displayed a transitory protective effect against MeHg-induced cell death. Conversely, probucol, a lipid-lowering agent with antioxidant properties, displayed a complete and long-lasting protection against MeHg-induced neurotoxicity, which was correlated to increased GPx activity and inhibition of MeHg-induced lipid peroxidation. These data indicate that GPx is a primary molecular target involved with MeHg-induced neurotoxicity, pointing to a direct inhibitory effect of MeHg toward this selenoenzyme, which leads to increase sensitivity to peroxides, increased lipid peroxidation and cell death. The data also show that probucol, a lipid-lowering agent currently used in the clinic, induces an enduring protection against MeHg-induced neurotoxicity by increasing GPx activity due to direct activating effects. Taken together, the results presented herein bring out new insights on the mechanisms of MeHg neurotoxicity and potential therapeutic strategies for poisonings to this environmental pollutant.This study was supported by the Grant FIS PI061212 and 2005-SGR-00826 (Ministry of Health and Generalitat de Catalunya, respectively, Spain). M. Farina was a recipient of a post-doctoral fellowship (Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq/201362/2007-4).Peer Reviewe