Protéomique, Réponse Inflammatoire et Spectrométrie de Masse

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution. Chromosomal instability enables the continuous selection of somatic copy number alterations, which are established as ordered events that often occur in parallel, throughout tumour evolution and metastasis.

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").

Infected cells detect viruses through a variety of receptors that initiate cell-intrinsic innate defense responses. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a cytosolic sensor for many DNA viruses and HIV-1. In response to cytosolic viral DNA, cGAS synthesizes the second messenger 2'3'-cyclic GMP-AMP (cGAMP), which activates antiviral signaling pathways. We show that in cells producing virus, cGAS-synthesized cGAMP can be packaged in viral particles and extracellular vesicles. Viral particles efficiently delivered cGAMP to target cells. cGAMP transfer by viral particles to dendritic cells activated innate immunity and antiviral defenses. Finally, we show that cell-free murine cytomegalovirus and Modified Vaccinia Ankara virus contained cGAMP. Thus, transfer of cGAMP by viruses may represent a defense mechanism to propagate immune responses to uninfected target cells.

Polycomb Group (PcG) proteins maintain transcriptional repression throughout development, mostly by regulating chromatin structure. Polycomb Repressive Complex 2 (PRC2), a component of the Polycomb machinery, is responsible for the methylation of histone H3 lysine 27 (H3K27me2/3). Jarid2 was previously identified as a cofactor of PRC2, regulating PRC2 targeting to chromatin and its enzymatic activity. Deletion of Jarid2 leads to impaired orchestration of gene expression during cell lineage commitment. Here, we reveal an unexpected crosstalk between Jarid2 and PRC2, with Jarid2 being methylated by PRC2. This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2's enzymatic activity. We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation. Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.

Based on a quantitative study of the common fluctuations of 14C and 10Be production rates, we have derived a time series of the solar magnetic variability over the last 1200 years. This record is converted into irradiance variations by linear scaling based on previous studies of sun-like stars and of the sun’s behavior over the last few centuries. The new solar irradiance record exhibits low values during the well-known solar minima centered at about 1900, 1810 (Dalton) and 1690 ad(Maunder). Further back in time, a rather long period between 1450 and 1750 ad is characterized by low irradiance values. A shorter period is centered at about 1200 ad, with irradiance slightly higher or similar to present day values. It is tempting to correlate these periods with the so-called “little ice age” and “medieval warm period” respectively An accurate quantification of the climatic impact of this new irradiance record requires the use of coupled atmosphere−ocean general circulation models (GCMs). Nevertheless, our record is already compatible with a global cooling of about 0.5-1°C during the “little ice age”, and with a general cooling trend during the past millenium followed by global warming during the 20th century (Mann et al., 1999).

Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states.

Abstract Purpose: This study examined the feasibility, efficacy (abscopal effect), and immune effects of TGFβ blockade during radiotherapy in metastatic breast cancer patients. Experimental Design: Prospective randomized trial comparing two doses of TGFβ blocking antibody fresolimumab. Metastatic breast cancer patients with at least three distinct metastatic sites whose tumor had progressed after at least one line of therapy were randomized to receive 1 or 10 mg/kg of fresolimumab, every 3 weeks for five cycles, with focal radiotherapy to a metastatic site at week 1 (three doses of 7.5 Gy), that could be repeated to a second lesion at week 7. Research bloods were drawn at baseline, week 2, 5, and 15 to isolate PBMCs, plasma, and serum. Results: Twenty-three patients were randomized, median age 57 (range 35–77). Seven grade 3/4 adverse events occurred in 5 of 11 patients in the 1 mg/kg arm and in 2 of 12 patients in the 10 mg/kg arm, respectively. Response was limited to three stable disease. At a median follow up of 12 months, 20 of 23 patients are deceased. Patients receiving the 10 mg/kg had a significantly higher median overall survival than those receiving 1 mg/kg fresolimumab dose [hazard ratio: 2.73 with 95% confidence interval (CI), 1.02–7.30; P = 0.039]. The higher dose correlated with improved peripheral blood mononuclear cell counts and a striking boost in the CD8 central memory pool. Conclusions: TGFβ blockade during radiotherapy was feasible and well tolerated. Patients receiving the higher fresolimumab dose had a favorable systemic immune response and experienced longer median overall survival than the lower dose group. Clin Cancer Res; 24(11); 2493–504. ©2018 AACR.

BACKGROUND: Pulmonary involvement in leptospirosis remains poorly recognized in regions where it is endemic, despite reports of recent outbreaks and epidemic disease. METHODS: A prospective, population-based study was carried out to identify febrile patients exposed to Leptospira in urban and rural contexts in Iquitos, Peru. Evidence of exposure to Leptospira was obtained by serologic testing, and diagnosis of leptospirosis was confirmed in pulmonary cases by culture or quantitative real-time PCR assay. RESULTS: Of 633 consecutively enrolled febrile patients, 321 (50.7%) had antileptospiral IgM antibodies or high titers of antileptospiral antibodies. Seven patients with histories of only urban exposure to leptospires had severe pulmonary manifestations; of these, 5 patients died; 4 of the deaths were caused by pulmonary hemorrhage, and 1 was caused by acute respiratory distress syndrome and multiorgan failure. Real-time, quantitative PCR assay showed high levels of leptospiremia (>or=10(4) leptospires/mL) in most fatal cases; 1 patient, from whom tissue specimens were obtained at autopsy, had >or=10(5) leptospires/g of lung, kidney, and muscle tissue. DISCUSSION. This study demonstrates the underdiagnosis of leptospirosis in a region of high endemicity and the underrecognition of grave pulmonary complications. Pulmonary involvement in leptospirosis was present in urban but not rural areas. Presumptive treatment for leptospirosis should be initiated immediately in the appropriate epidemiological and clinical context.

Four studies are reported that examine the status and potentials for local businesses to leverage the Gold Coast Honda Indy. The leveraging efforts of local businesses are identified in Study 1. Most local business managers fail to recognize the event as a leveraging opportunity. Tactics used by businesses that do attempt to leverage the event are examined in Study 2. Businesses that leverage the event obtain benefits through the use of standard promotional and theming tactics. Experts’ views about leveraging the event are obtained in Study 3. The experts conclude that some coordination of local businesses' leveraging efforts would be advantageous. The views of local business leaders are solicited in Study 4. The business leaders favor leveraging but prefer that the coordination come from an existing business organization or association, rather than through government or a new bureaucracy. The studies suggest that the potentials for leveraging are largely unrealized and that some degree of inertia would need to be overcome to realize those potentials. It is argued that event organizers have the most to gain by fostering and coordinating local business leveraging.

Recent efforts at the proteomic level were employed to describe the protein equipment of the plasma membrane of the model plant Arabidopsis thaliana. These studies had revealed that the plasma membrane is rich in extrinsic proteins but came up against two major problems: (i) few hydrophobic proteins were recovered in two-dimensional electrophoresis gels, and (ii) many plasma membrane proteins had no known function or were unknown in the database despite extensive sequencing of the Arabidopsis genome. In this paper, several methods expected to enrich a membrane sample in hydrophobic proteins were compared. The optimization of solubilization procedures revealed that the detergent to be used depends on the lipid content of the sample. The corresponding proteomes were compared with the statistical model AMMI (additive main effects with multiplicative interaction) that aimed at regrouping proteins according to their solubility and electrophoretic properties. Distinct groups emerged from this analysis and the identification of proteins in each group allowed us to assign specific features to several of them. For instance, two of these groups regrouped very hydrophobic proteins, one group contained V-ATPase subunits, another group contained proteins with one transmembrane domain as well as proteins known to interact with membrane proteins. This study provides methodological tools to study particular classes of plasma membrane proteins and should be applicable to other cellular membranes.

Ribosome assembly in Escherichia coli involves 54 ribosomal proteins and three RNAs. Whereas functional subunits can be reconstituted in vitro from the isolated components, this process requires long incubation times and high temperatures compared with the in vivo situation, suggesting that non-ribosomal factors facilitate assembly in vivo. Here, we show that SrmB, a putative DEAD-box RNA helicase, is involved in ribosome assembly. The deletion of the srmB gene causes a slow-growth phenotype at low temperature. Polysome profile analyses of the corresponding cells reveal a deficit in free 50S ribosomal subunits and the accumulation of a new particle sedimenting around 40S. Analysis of the ribosomal RNA and protein contents of the 40S particle indicates that it represents a large subunit that is incompletely assembled. In particular, it lacks L13, one of the five ribosomal proteins that are essential for the early assembly step in vitro. Sucrose gradient fractionation also shows that, in wild-type cells, SrmB associates with a pre50S particle. From our results, we propose that SrmB is involved in an early step of 50S assembly that is necessary for the binding of L13. This step may consist of a structural rearrangement that, at low temperature, cannot occur without the assistance of this putative RNA helicase.

In placental mammals, inactivation of one of the X chromosomes in female cells ensures sex chromosome dosage compensation. The 17 kb non-coding Xist RNA is crucial to this process and accumulates on the future inactive X chromosome. The most conserved Xist RNA region, the A region, contains eight or nine repeats separated by U-rich spacers. It is implicated in the recruitment of late inactivated X genes to the silencing compartment and likely in the recruitment of complex PRC2. Little is known about the structure of the A region and more generally about Xist RNA structure. Knowledge of its structure is restricted to an NMR study of a single A repeat element. Our study is the first experimental analysis of the structure of the entire A region in solution. By the use of chemical and enzymatic probes and FRET experiments, using oligonucleotides carrying fluorescent dyes, we resolved problems linked to sequence redundancies and established a 2-D structure for the A region that contains two long stem-loop structures each including four repeats. Interactions formed between repeats and between repeats and spacers stabilize these structures. Conservation of the spacer terminal sequences allows formation of such structures in all sequenced Xist RNAs. By combination of RNP affinity chromatography, immunoprecipitation assays, mass spectrometry, and Western blot analysis, we demonstrate that the A region can associate with components of the PRC2 complex in mouse ES cell nuclear extracts. Whilst a single four-repeat motif is able to associate with components of this complex, recruitment of Suz12 is clearly more efficient when the entire A region is present. Our data with their emphasis on the importance of inter-repeat pairing change fundamentally our conception of the 2-D structure of the A region of Xist RNA and support its possible implication in recruitment of the PRC2 complex.

Abstract Congruence has long been discussed in the context of brand extensions and advertising, particularly in terms of celebrity endorsement. Although just as relevant for sponsorship, the examination of this concept in that context is more recent, despite evidence that it plays a significant role in determining the effectiveness of this particular communication technique. In this paper, the authors argue in favor of conceptualizing congruence as a bidimensional construct, consistent with previous research. A scale to capture this important construct is subsequently developed and validated in two separate countries. © 2007 Wiley Periodicals, Inc.

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.

Certain characteristic periodicities in the Δ 14 C record from tree rings, such as the well‐known 11‐yr Schwabe cycle, are known to be of solar origin. The origin of longer‐period cycles, such as the 205‐yr de Vries cycle, in the Δ 14 C record was less certain, and it was possible to attribute it either to solar or climatic variability. Here, we demonstrate that the de Vries cycle is present in 10 Be data from the GRIP ice core during the last ice age (25 to 50 kyr BP). Analysis of the amplitude of variation of this cycle shows it to be modulated by the geomagnetic field, indicating that the de Vries cycle is indeed of solar, rather than climatic, origin.

Extracellular ATP is among molecules promoting microglia activation and inducing the release of extracellular vesicles (EVs), which are potent mediators of intercellular communication between microglia and the microenvironment. We previously showed that EVs produced under ATP stimulation (ATP-EVs) propagate a robust inflammatory reaction among astrocytes and microglia in vitro and in mice with subclinical neuroinflammation (1). However, the proteome of EVs released upon ATP stimulation has not yet been elucidated. In this study we applied a label free proteomic approach to characterize the proteome of EVs released constitutively and during microglia activation with ATP. We show that ATP drives sorting in EVs of a set of proteins implicated in cell adhesion/extracellular matrix organization, autophagy-lysosomal pathway and cellular metabolism, that may influence the response of recipient astrocytes to EVs. These data provide new clues to molecular mechanisms involved in microglia response to ATP and in microglia signalling to the environment via EVs.

The microscopic structure of latent tracks in silicates is analyzed using small-angle x-ray scattering methods. Latent tracks are constituted of extended defects, separated by gap zones loaded with point defects. The variation of the linear density of extended defects along the path of the incident ions cannot be scaled with functions previously used, such as the primary rate of ionization. Upon a thermal annealing, the extended defects are much more stable than point defects. These latent tracks are chemically etched and their etching rates are inferred from optical and scanning electron-microscope observations. From these combined studies of latent and etched tracks, a model for the registration of etchable tracks in silicates is developed. In this model, the extended defects dominate the chemical etching and thermal annealing behavior of etchable tracks. The marked differences observed in the sensitivity of various silicates are no longer attributed to a radiation damage mechanism, which would operate much more efficiently in specific silicates, but to the etching behavior of each mineral. Indeed, for a given incident ion, the linear density of extended defects in a given part of the ion residual range appears to be similar in all silicates. This model is used in the framework of a Monte Carlo statistical code to predict etched track-length distributions in silicates, including those relevant to partially annealed tracks. The striking agreement between these predictions and the corresponding observations strongly support our model. It enables us to discuss the most important concepts previously proposed to account for the registration of etchable tracks in silicates, and to suggest a few preliminary guidelines for improving the use of solid-state track detectors.

Three active-site cysteine L,D-transpeptidases can individually anchor the Braun lipoprotein to the Escherichia coli peptidoglycan. We show here that two additional enzymes of the same family form peptide bonds between the third residues of peptidoglycan stems, generating meso-DAP(3)-->meso-DAP(3) unusual cross-links. This activity partially replaces the D,D-transpeptidase activity of penicillin-binding proteins.

We show here that nerve growth factor (NGF), the prototypic neurotrophin, can be targeted in breast cancer to inhibit tumor cell proliferation, survival, and metastasis. Analysis of a series of biopsies revealed widespread expression of NGF in the majority of human breast tumors, with anti-NGF immunoreactivity concentrated in the epithelial cancer cells. Moreover, immunodeficient mice xenografted with human breast cancer cells and treated with either anti-NGF antibodies or small interfering RNA against NGF displayed inhibited tumor growth and metastasis. Such treatments directed against NGF induced a decrease in cell proliferation with a concomitant increase in apoptosis of breast cancer cells and an inhibition of tumor angiogenesis. Together, these data indicate that targeting NGF in breast cancer may have therapeutic ramifications.