A Generic Platform for Cellular Screening Against Ubiquitin Ligases

Ubiquitin signalling regulates most aspects of cellular life, thus deregulation of ubiquitylation has been linked with a number of diseases. E3 ubiquitin ligases provide substrate selectivity in ubiquitylation cascades and are therefore considered to be attractive targets for developing therapeutic molecules. In contrast to established drug target classes, such as protein kinases, GPCRs, hormone receptors and ion channels, ubiquitin drug discovery is in its early stages. This is, in part, due to the complexity of the ubiquitylation pathways and the lack of robust quantitative technologies that allow high-throughput screening of inhibitors. Here we report the development of a Ubiquitin Ligase Profiling system, which is a novel and generic cellular technology designed to facilitate identification of selective inhibitors against RING type E3 ubiquitin ligases. Utilization of this system requires a single co-transfection of cells with assay vectors, thereby enabling readout of E3 ubiquitin ligase catalytic activity within the cellular environment. Therefore, our robust high-throughput screening platform offers novel opportunities for the development of inhibitors against this difficult-to-target E3 ligase enzyme class

Cosmic rays and molecular clouds

This paper deals with the cosmic-ray penetration into molecular clouds and with the related gamma--ray emission. High energy cosmic rays interact with the dense gas and produce neutral pions which in turn decay into two gamma rays. This makes molecular clouds potential sources of gamma rays, especially if they are located in the vicinity of a powerful accelerator that injects cosmic rays in the interstellar medium. The amplitude and duration in time of the cosmic--ray overdensity around a given source depend on how quickly cosmic rays diffuse in the turbulent galactic magnetic field. For these reasons, gamma-ray observations of molecular clouds can be used both to locate the sources of cosmic rays and to constrain the properties of cosmic-ray diffusion in the Galaxy.Comment: To appear in the proceedings of the San Cugat Forum on Astrophysics 2012, 27 pages, 10 figure

A modified Trastuzumab antibody for the immunohistochemical detection of HER-2 overexpression in breast cancer

The immunohistochemical determination of HER-2 to identify patients with advanced breast cancer candidates for Trastuzumab treatment proved neither accurate nor fully reliable, possibly because none of the current reagents detects the specific antigenic site target of Trastuzumab. To circumvent this problem, we conjugated the NH2 groups of Trastuzumab with biotin, and the compound obtained, designated BiotHER, was added directly to tissue sections. Biotin-labelling was revealed with horseradish peroxidase-conjugated streptavidin. Specificity and sensitivity of BiotHER immunostaining with respect to HER-2 amplification were tested on 164 breast carcinoma samples. BiotHER staining was detected on the tumour cell membrane of 12% of all specimens and in 49% specimens with gene amplification, while absent in nonamplified tumours. Predictivity of BiotHER status with respect to the clinical outcome was analysed in 54 patients with HER-2 amplified advanced breast cancer treated with Trastuzumab plus chemotherapy. BiotHER staining, detected in 50% of tumours with HER-2 amplification, was an independent predictor of clinical outcome. In fact, BiotHER positivity was independently associated with increased likelihood of tumour response and reduced risk of tumour progression and death. Biotinylated Trastuzumab can thus be used for immunohistochemical detection of HER-2 overexpression in breast cancer, and has the potential to identify patients likely to benefit from Trastuzumab treatment

Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation

Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β-integrin tails, F0 and F1 are also required for activation of β1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of β1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins

Ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12 155 patients

The Ki-67 antigen is used to evaluate the proliferative activity of breast cancer (BC); however, Ki-67's role as a prognostic marker in BC is still undefined. In order to better define the prognostic value of Ki-67/MIB-1, we performed a meta-analysis of studies that evaluated the impact of Ki-67/MIB-1 on disease-free survival (DFS) and/or on overall survival (OS) in early BC. Sixty-eight studies were identified and 46 studies including 12 155 patients were evaluable for our meta-analysis; 38 studies were evaluable for the aggregation of results for DFS, and 35 studies for OS. Patients were considered to present positive tumours for the expression of Ki-67/MIB-1 according to the cut-off points defined by the authors. Ki-67/MIB-1 positivity is associated with higher probability of relapse in all patients (HR=1.93 (95% confidence interval (CI): 1.74–2.14); P<0.001), in node-negative patients (HR=2.31 (95% CI: 1.83–2.92); P<0.001) and in node-positive patients (HR=1.59 (95% CI: 1.35–1.87); P<0.001). Furthermore, Ki-67/MIB-1 positivity is associated with worse survival in all patients (HR=1.95 (95% CI: 1.70–2.24; P<0.001)), node-negative patients (HR=2.54 (95% CI: 1.65–3.91); P<0.001) and node-positive patients (HR=2.33 (95% CI: 1.83–2.95); P<0.001). Our meta-analysis suggests that Ki-67/MIB-1 positivity confers a higher risk of relapse and a worse survival in patients with early BC

The ubiquitin-conjugating enzyme CDC34 is essential for cytokinesis in contrast to putative subunits of a SCF complex in Trypanosoma brucei

The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention

The CMS Statistical Analysis and Combination Tool: Combine

Metrics: https://link.springer.com/article/10.1007/s41781-024-00121-4/metricsThis paper describes the Combine software package used for statistical analyses by the CMS Collaboration. The package, originally designed to perform searches for a Higgs boson and the combined analysis of those searches, has evolved to become the statistical analysis tool presently used in the majority of measurements and searches performed by the CMS Collaboration. It is not specific to the CMS experiment, and this paper is intended to serve as a reference for users outside of the CMS Collaboration, providing an outline of the most salient features and capabilities. Readers are provided with the possibility to run Combine and reproduce examples provided in this paper using a publicly available container image. Since the package is constantly evolving to meet the demands of ever-increasing data sets and analysis sophistication, this paper cannot cover all details of Combine. However, the online documentation referenced within this paper provides an up-to-date and complete user guide.CERN (European Organization for Nuclear Research)STFC (United Kingdom)Marie-Curie programme and the European Research Council and Horizon 2020 Grant, contract Nos. 675440, 724704, 752730, 758316, 765710, 824093, 101115353, 101002207, and COST Action CA16108 (European Union); the Leventis Foundation; the Alfred P. Sloan Foundatio