(Phospho)proteomic profiling of microsatellite unstable CRC cells reveals alterations in nuclear signaling and cholesterol metabolism caused by frameshift mutation of NMD regulator UPF3A

DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the UPF3A gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells

Two-Step Co-Immunoprecipitation (TIP) Enables Efficient and Highly Selective Isolation of Native Protein-Complexes.

Co-immunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of co-precipitated contaminants is a well- recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential co-immunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4+ T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions

Columnar and Equiaxed Solidification of Al-7 wt.% Si Alloys in Reduced Gravity in the Framework of the CETSOL Project

International audienceDuring casting, often a dendritic microstructure is formed, resulting in a columnar or an equiaxed grain structure, or leading to a transition from columnar to equiaxed growth (CET). The detailed knowledge of the critical parameters for the CET is important because the microstructure affects materials properties. To provide unique data for testing of fundamental theories of grain and microstructure formation, solidification experiments in microgravity environment were performed within the European Space Agency Microgravity Application Promotion (ESA MAP) project Columnar-to-Equiaxed Transition in SOLidification Processing (CETSOL). Reduced gravity allows for purely diffusive solidification conditions, i.e., suppressing melt flow and sedimentation and floatation effects. On-board the International Space Station, Al-7 wt.% Si alloys with and without grain refiners were solidified in different temperature gradients and with different cooling conditions. Detailed analysis of the microstructure and the grain structure showed purely columnar growth for nonrefined alloys. The CET was detected only for refined alloys, either as a sharp CET in the case of a sudden increase in the solidification velocity or as a progressive CET in the case of a continuous decrease of the temperature gradient. The present experimental data were used for numerical modeling of the CET with three different approaches: (1) a front tracking model using an equiaxed growth model, (2) a three-dimensional (3D) cellular automaton–finite element model, and (3) a 3D dendrite needle network method. Each model allows for predicting the columnar dendrite tip undercooling and the growth rate with respect to time. Furthermore, the positions of CET and the spatial extent of the CET, being sharp or progressive, are in reasonably good quantitative agreement with experimental measurements

Exploring subsurface fluid flow and active dewatering along the oceanic plate boundary between Africa and Eurasia (Gloria Fault)

R/V Meteor cruise M162 was conducted as a systematic continuation of ongoing work dedicated to understand if and howfluid flow through crust and sedimentscontinues along transform-type plate boundaries and fracture zones away from mid-ocean ridges and continental margins. Central target was the Gloria Fault in the central Northeast Atlantic. Previous findings along the eastern continuation of the Gloria Fault revealed fault-controlled fluid advection and mud volcanism along strike-slip faults in the Horseshoe Abyssal Plain and the Gulf of Cadiz, where fluid geochemistry revealed the admixture of fluids from deeply buried oceanic crust and oldest sediments on top of it. TheGloria Fault itselfis an old, reactivated, and seismically active oceanic fracture zone. During M162 a systematic survey along the main trace of the Gloria Fault between the Azores Plateau and the Madeira-Tore Rise was carried out, including sub-bottom profiler surveys, heat flow transects, gravity corer sampling, as well as video-guided CTD and multicorer deployments. In accordance to recently recorded seismic activity along the fault, there isevidence for tectonic motion both in sub-bottom profiler records and sediment cores. Heat flow measurements revealed values significantly elevated above the background in many places, predominantly along the main fault trace and other active faults.Ina number of placesfluid geochemistry revealed enhanced diagenetic processes in the sediments, implying the potential relation to upward-directed fluid flow. In summary, cruise M162revealed the first complementary data set on heat flow and fluid geochemistry along an oceanic fault zone, which will further our understanding on themes like the alteration of oceanic lithosphere and crust-ocean element exchange

Integrin α7 Is a Functional Marker and Potential Therapeutic Target in Glioblastoma

Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target

Revealing Higher Order Protein Structure Using Mass Spectrometry

International audienceThe development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope

Modelling of High Temperature Oxidation of Alumina-Forming Single-Crystal Nickel-Base Superalloys

Microstructure evolution in single-crystal superalloys is modelled as the interplay between oxide growth and substrate response. This is of particular importance for specimens with reduced wall thicknesses, where the affected substrate constitutes a significant fraction of the material. A model based on thermodynamic and kinetic data only is presented in order to predict the growth kinetics of oxides and the resulting influence on microstructure evolution of the substrate. The present work focuses on alumina (Al 2O 3) growth as it is the most important oxide regarding long-term behaviour. Al 2O 3 growth is described using a dynamic growth parameter which is derived using thermodynamic and kinetic principles. The substrate response model calculates the distribution of the alloying elements as well as the evolution of the phase fractions as a function of depth and oxidation time. The model has been applied on the strong alumina-forming alloy René N5 and the weak alumina-forming alloy SCA425+. Since γ′ fraction is one of the most relevant factors for high temperature creep properties, the present work concentrates on the calculation of the time- and space-dependent γ′ precipitate fraction profile. The model predictions were verified with very good agreement with respect to Al 2O 3 growth, element distribution and γ′ fraction distribution. © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved