Activation peptide of the coagulation factor XIII (AP-F13A1) as a new biomarker for the screening of colorectal cancer

International audienceBackground: Colorectal cancer (CRC) remains a major cause of cancer fatalities in developed countries. The risk of death is correlated to the stage of CRC during the primary diagnosis. Early diagnosis is closely associated with enhanced survival rate. We therefore investigated the AP-F13A1 as a potential protein marker of CRC. Methods: The protein expression of FXIII in 40 serum samples was evaluated by enzyme-linked immunosorbent assays. Additionally, targeted proteomic assays (LC-PRM) were used to evaluate the expression of the activation peptide of F13A1 (AP-F13A1) in a further 113 serum samples. Results were analyzed by the Wilcoxon test and receiver operating characteristic curves generated to assess statistical differences and diagnostic factors between CRC patients and controls. Results: AP-F13A1 was quantified in human serum samples using calibration curves with excellent linearity. AP-F13A1 was reduced in CRC patients using PRM assays from two distinct biobanks. The AUC for AP-F13A1 were 0.95 and 0.93. Sensitivity/specificity values for the two sets of patients were 75%/95% and 71%/95% respectively. Conclusion: We have presented the proof of principle that in vivo release of AP-F13A1 can be measured by PRM-based strategies in CRC serum samples. AP-F13A1 may be an effective serological biomarker as part of a screening program of CRC detection

A simulation framework for rapid prototyping and evaluation of thermal mitigation techniques in many-core architectures

International audienceModern SoCs are characterized by increasing power density and consequently increasing temperature, that directly impacts performances, reliability and cost of a device through its packaging. Thermal issues need to be predicted and mitigated as early as possible in the design flow, when the optimization opportunities are the highest. In this paper, we present an efficient framework for the design of dynamic thermal mitigation schemes based on a high-level SystemC virtual prototype tightly coupled with efficient power and thermal simulation tools. We demonstrate the benefit of our approach through silicon comparison with the SThorm 64-core architecture and provide simulation speed results making it a sound solution for the design of thermal mitigation early in the flow

Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK4/6 inhibitor palbociclib

Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor-positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib-induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib-induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence-like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse-free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells

Killing with proficiency:integrated post-translational regulation of an offensive Type VI secretion system

<div><p>The Type VI secretion system (T6SS) is widely used by bacterial pathogens as an effective weapon against bacterial competitors and is also deployed against host eukaryotic cells in some cases. It is a contractile nanomachine which delivers toxic effector proteins directly into target cells by dynamic cycles of assembly and firing. Bacterial cells adopt distinct post-translational regulatory strategies for deployment of the T6SS. ‘Defensive’ T6SSs assemble and fire in response to incoming attacks from aggressive neighbouring cells, and can utilise the Threonine Protein Phosphorylation (TPP) regulatory pathway to achieve this control. However, many T6SSs are ‘offensive’, firing at all-comers without the need for incoming attack or other cell contact-dependent signal. Post-translational control of the offensive mode has been less well defined but can utilise components of the same TPP pathway. Here, we used the anti-bacterial T6SS of <i>Serratia marcescens</i> to elucidate post-translational regulation of offensive T6SS deployment, using single-cell microscopy and genetic analyses. We show that the integration of the TPP pathway with the negative regulator TagF to control core T6SS machine assembly is conserved between offensive and defensive T6SSs. Signal-dependent PpkA-mediated phosphorylation of Fha is required to overcome inhibition of membrane complex assembly by TagF, whilst PppA-mediated dephosphorylation promotes spatial reorientation and efficient killing. In contrast, the upstream input of the TPP pathway defines regulatory strategy, with a new periplasmic regulator, RtkS, shown to interact with the PpkA kinase in <i>S</i>. <i>marcescens</i>. We propose a model whereby the opposing actions of the TPP pathway and TagF impose a delay on T6SS re-assembly after firing, providing an opportunity for spatial re-orientation of the T6SS in order to maximise the efficiency of competitor cell targeting. Our findings provide a better understanding of how bacterial cells deploy competitive weapons effectively, with implications for the structure and dynamics of varied polymicrobial communities.</p></div

Variations of the Elastic Properties of the CoCrFeMnNi High Entropy Alloy Deformed by Groove Cold Rolling

The variations of the mechanical properties of the CoCrFeMnNi high entropy alloy (HEA) during groove cold rolling process were investigated with the aim of understanding their correlation relationships with the crystallographic texture. Our study revealed divergences in the variations of the microhardness and yield strength measured from samples deformed by groove cold rolling and conventional cold rolling processes. The crystallographic texture analyzed by electron back scattered diffraction (EBSD) revealed a hybrid texture between those obtained by conventional rolling and drawing processes. Though the groove cold rolling process induced a marked strengthening effect in the CoCrFeMnNi HEA, the mechanical properties were also characterized by an unusual decrease of the Young’s modulus as the applied groove cold rolled deformation increased up to about 0.5 before reaching a stabilized value. This decrease of the Young’s modulus was attributed to the increased density of mobile dislocations induced by work hardening during groove cold rolling processing

Architectural effect on 3D elastic properties and anisotropy of cubic lattice structures

This article investigates the elastic properties of a large panel of lattice architectures using a continuous description of geometry. The elastic constants of the orthotropic material are determined, and discussed in terms of specific stiffness and of its density dependence. Different kind of topology families are emerging depending on their specific deformation behavior. For some of them, interesting properties in term of traction-compression were measured, while some other families are predominantly adapted to shear loading. Homogenization technique also allows to quantify the anisotropy of the structures and to compare them. Specific structures having quasi-isotropic properties even at low relative densities were detected. Experimental works demonstrated the validity of the numerical models, and highlighted the necessity to consider carefully the effect of defects on the specific strength, which are of the second-order however not negligible. Finally, this article provides user-friendly maps for selection of optimal architectures for a large variety of specific needs, like a target stiffness or anisotropy

Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

This is the author accepted manuscript. The final version is available from EMBO Press via the DOI in this recordMutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin activators that mimic the effect of ubiquitin(Phospho-Ser65).Wellcome Trust Senior Research Fellowship in Clinical Science101022/Z/13/Z; Medical Research Council; Wellcome Trust; Parkinson's UK; Michael J. Fox Foundation for Parkinson's Disease Research; Tenovus Scotland; Wellcome/MRC; UCL Institute of Neurology; University of Sheffield; MRC‐PPU of University of Dundee; Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer‐Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associ- ated with Parkinson’s disease, chronic inflammation and mycobac- terial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobac- terial control independently of autophagy. In vivo, LRRK2 defi- ciency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2- dependent cellular pathway that controls M. tuberculosis replica- tion by regulating phagosome maturation