Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes

Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.</p

Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types in polyubiquitin co-exist and are independently regulated in cells. This ‘ubiquitin code’ determines the fate of the modified protein1. Deubiquitinating enzymes of the Ovarian Tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin2, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. We here reveal how the deubiquitinase Cezanne/OTUD7B specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with mono- and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable reconstruction of the enzymatic cycle in exquisite detail. An intricate mechanism of ubiquitin-assisted conformational changes activate the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the fascinating plasticity of deubiquitinases, and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site

ENIGMA-anxiety working group : Rationale for and organization of large-scale neuroimaging studies of anxiety disorders

Altres ajuts: Anxiety Disorders Research Network European College of Neuropsychopharmacology; Claude Leon Postdoctoral Fellowship; Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, 44541416-TRR58); EU7th Frame Work Marie Curie Actions International Staff Exchange Scheme grant 'European and South African Research Network in Anxiety Disorders' (EUSARNAD); Geestkracht programme of the Netherlands Organization for Health Research and Development (ZonMw, 10-000-1002); Intramural Research Training Award (IRTA) program within the National Institute of Mental Health under the Intramural Research Program (NIMH-IRP, MH002781); National Institute of Mental Health under the Intramural Research Program (NIMH-IRP, ZIA-MH-002782); SA Medical Research Council; U.S. National Institutes of Health grants (P01 AG026572, P01 AG055367, P41 EB015922, R01 AG060610, R56 AG058854, RF1 AG051710, U54 EB020403).Anxiety disorders are highly prevalent and disabling but seem particularly tractable to investigation with translational neuroscience methodologies. Neuroimaging has informed our understanding of the neurobiology of anxiety disorders, but research has been limited by small sample sizes and low statistical power, as well as heterogenous imaging methodology. The ENIGMA-Anxiety Working Group has brought together researchers from around the world, in a harmonized and coordinated effort to address these challenges and generate more robust and reproducible findings. This paper elaborates on the concepts and methods informing the work of the working group to date, and describes the initial approach of the four subgroups studying generalized anxiety disorder, panic disorder, social anxiety disorder, and specific phobia. At present, the ENIGMA-Anxiety database contains information about more than 100 unique samples, from 16 countries and 59 institutes. Future directions include examining additional imaging modalities, integrating imaging and genetic data, and collaborating with other ENIGMA working groups. The ENIGMA consortium creates synergy at the intersection of global mental health and clinical neuroscience, and the ENIGMA-Anxiety Working Group extends the promise of this approach to neuroimaging research on anxiety disorders

Extensive Cross-Reactivity of CD4(+) Adenovirus-Specific T Cells: Implications for Immunotherapy and Gene Therapy

Adenovirus (Ad)-specific T-cell responses in healthy adult donors were investigated. Ad5, inactivated by methylene blue plus visible light, induced proliferation and gamma interferon (IFN-γ) production in peripheral blood mononuclear cells of the majority of donors. Responding T cells were CD4(+) and produced IFN-γ upon restimulation with infectious Ad5 and Ads of different subgroups. T-cell clones showed distinct cross-reactivity patterns recognizing Ad serotypes from either one subgroup (C), two subgroups (B and C), or three subgroups (A, B, and C). This cross-reactivity of Ad-specific T cells has relevance both for Ad-based gene therapy protocols, as well as for the feasibility of T-cell-mediated adoptive immunotherapy in recipients of an allogeneic stem cell transplantation

Adenovirus-specific CD4 T cell clones recognizing endogenous antigen inhibit viral replication in vitro through cognate interaction

Human adenovirus (HAdV) infection is a frequent and potentially severe complication following allogeneic stem cell transplantation in children. Because treatment with antiviral drugs is often ineffective, adoptive transfer of donor-derived HAdV-specific T cells able to control viral replication of HAdV of multiple serotypes may be an option for therapy. In healthy donors, predominantly HAdV-specific T cells expressing CD4 are detected. In this study, a preclinical in vitro model was used to measure the antiviral effect of HAdV-specific CD4 ؉ T cells. CD4 ؉ HAdV-specific T cell clones restricted by HLA class II molecules were generated and most of these clones recognized conserved peptides derived from the hexon protein. These cross-reactive T cell clones were able to control viral replication of multiple serotypes of HAdV in EBV-transformed B cells (B-LCL), melanoma cells (MJS) and primary bronchial epithelial cells through cognate interaction. The HAdV-specific CD4 ؉ T cell clones were able to specifically lyse infected target cells using a perforin-dependent mechanism. Antigenic peptides were also presented to the CD4 H uman adenoviruses (HAdV) 4 rarely cause severe clinical symptoms in healthy children and adults, because infections in immunocompetent individuals are usually selflimiting. However, HAdV may cause life-threatening complications in immunocompromised patients (1, 2). In recent years, the incidence of HAdV infections in pediatric stem cell transplant (SCT) recipients has increased remarkably (3-7). Recipients of a T cell-depleted or CD34 ϩ -enriched allogeneic stem cell graft, i.e., patients with a non-HLA-identical donor, receiving serotherapy with anti-CD52 mAb (Campath) or anti-thymocyte globulin, have a higher risk of developing HAdV infection and dissemination due to the delayed immune reconstitution in these children after SCT (7-9). Clinical symptoms include gastroenteritis, hemorrhagic cystitis, hepatitis, pneumonia, encephalitis, and multiorgan failure. Dissemination of the infection, documented by a rise of HAdV DNA loads in plasma, is associated with a fatal outcome (7, 10 -14). Currently, 51 serotypes of adenovirus have been identified, distributed among six species (A-F) based on their differential hemagglutination with erythrocytes and their DNA homology (15, 16). Species A, B, and C serotypes are most frequently isolated from pediatric immunocompromised hosts and are the major cause of disease Treatment of adenoviral infections with antiviral medication, such as cidofovir and ribavirin, has not been unequivocally effective (reviewed in (20)). Therefore, new strategies to treat disseminated HAdV infections are needed. A potential approach is adoptive immunotherapy by infusion of HAdV-specific lymphocytes. This strategy has already been successfully pursued for other viral infections or reactivations such as CMV or EBV (21-23). Furthermore, case reports have suggested that donor lymphocyte infusions or tapering of immunosuppression may contribute to clearance of an HAdV infection, illustrating the potential role of T cells in the immune response to HAdV (8, Few reports on HAdV-specific immune responses in healthy donors have been published until recently ϩ , although HAdV-specific CD8 ϩ T cells have been described as well (28 -33). Furthermore, in a previous study, we have shown the presence of CD4 ϩ HAdV-specific T cells in the blood of patients recovering from HAdV infection or viremia (7), suggesting that these cells may be functionally involved in clearance of the virus. As mainly CD4 ϩ HAdV-specific T cells can be detected in healthy donors and patients after SCT, the question arises whether these CD4 ϩ T cells can exert direct antiviral functions, and if so, by which mechanisms. To study this, we generated HAdV-specifi

A European Research Agenda for Geriatric Emergency Medicine: a modified Delphi study

PURPOSE: Geriatric Emergency Medicine (GEM) focuses on delivering optimal care to (sub)acutely ill older people. This involves a multidisciplinary approach throughout the whole healthcare chain. However, the underpinning evidence base is weak and it is unclear which research questions have the highest priority. The aim of this study was to provide an inventory and prioritisation of research questions among GEM professionals throughout Europe. METHODS: A two-stage modified Delphi approach was used. In stage 1, an online survey was administered to various professionals working in GEM both in the Emergency Department (ED) and other healthcare settings throughout Europe to make an inventory of potential research questions. In the processing phase, research questions were screened, categorised, and validated by an expert panel. Subsequently, in stage 2, remaining research questions were ranked based on relevance using a second online survey administered to the same target population, to identify the top 10 prioritised research questions. RESULTS: In response to the first survey, 145 respondents submitted 233 potential research questions. A total of 61 research questions were included in the second stage, which was completed by 176 respondents. The question with the highest priority was: Is implementation of elements of CGA (comprehensive geriatric assessment), such as screening for frailty and geriatric interventions, effective in improving outcomes for older patients in the ED? CONCLUSION: This study presents a top 10 of high-priority research questions for a European Research Agenda for Geriatric Emergency Medicine. The list of research questions may serve as guidance for researchers, policymakers and funding bodies in prioritising future research projects.status: Published onlin

A European Research Agenda for Geriatric Emergency Medicine: a modified Delphi study

Key Summary PointsAim To provide an inventory and prioritisation of research questions amongst GEM professionals throughout Europe. Findings A list of 10 research questions was identified and prioritised. Message The list of research questions may serve as guidance for scientists, policymakers and funding bodies in prioritising future research projects. Purpose Geriatric Emergency Medicine (GEM) focuses on delivering optimal care to (sub)acutely ill older people. This involves a multidisciplinary approach throughout the whole healthcare chain. However, the underpinning evidence base is weak and it is unclear which research questions have the highest priority. The aim of this study was to provide an inventory and prioritisation of research questions among GEM professionals throughout Europe. Methods A two-stage modified Delphi approach was used. In stage 1, an online survey was administered to various professionals working in GEM both in the Emergency Department (ED) and other healthcare settings throughout Europe to make an inventory of potential research questions. In the processing phase, research questions were screened, categorised, and validated by an expert panel. Subsequently, in stage 2, remaining research questions were ranked based on relevance using a second online survey administered to the same target population, to identify the top 10 prioritised research questions. Results In response to the first survey, 145 respondents submitted 233 potential research questions. A total of 61 research questions were included in the second stage, which was completed by 176 respondents. The question with the highest priority was: Is implementation of elements of CGA (comprehensive geriatric assessment), such as screening for frailty and geriatric interventions, effective in improving outcomes for older patients in the ED? Conclusion This study presents a top 10 of high-priority research questions for a European Research Agenda for Geriatric Emergency Medicine. The list of research questions may serve as guidance for researchers, policymakers and funding bodies in prioritising future research projects