Modified SH2 domain to phototrap and identify phosphotyrosine proteins from subcellular sites within cells.

Spatial regulation of tyrosine phosphorylation is important for many aspects of cell biology. However, phosphotyrosine accounts for less than 1% of all phosphorylated substrates, and it is typically a very transient event in vivo. These factors complicate the identification of key tyrosine kinase substrates, especially in the context of their extraordinary spatial organization. Here, we describe an approach to identify tyrosine kinase substrates based on their subcellular distribution from within cells. This method uses an unnatural amino acid-modified Src homology 2 (SH2) domain that is expressed within cells and can covalently trap phosphotyrosine proteins on exposure to light. This SH2 domain-based photoprobe was targeted to cellular structures, such as the actin cytoskeleton, mitochondria, and cellular membranes, to capture tyrosine kinase substrates unique to each cellular region. We demonstrate that RhoA, one of the proteins associated with actin, can be phosphorylated on two tyrosine residues within the switch regions, suggesting that phosphorylation of these residues might modulate RhoA signaling to the actin cytoskeleton. We conclude that expression of SH2 domains within cellular compartments that are capable of covalent phototrapping can reveal the spatial organization of tyrosine kinase substrates that are likely to be important for the regulation of subcellular structures.

Gastrointestinal complications of post-diarrheal hemolytic uremic syndrome.

PURPOSE: Whereas gastrointestinal symptoms such as vomiting, diarrhea and abdominal pain are common in children suffering from the so-called post-diarrheal form (D+) of hemolytic uremic syndrome (HUS), more serious gastrointestinal complications are rare. We tried to define factors predictive of the severity of gastrointestinal complications post D+ HUS. METHODS: We reviewed the files of all children admitted to our hospital for D+ HUS between 1988 and 2000. We retained those cases with gastrointestinal complications and analyzed the consequences of these complications on the evolution of the children's conditions. RESULTS: Sixty-five children with D+ HUS were admitted to our hospital during this period. Sixteen children developed gastrointestinal complications involving one or more digestive organs: necrosis of the colon or ileum, hemorrhagic colitis, pancreatitis, transient diabetes, hepatic cytolysis and cholestasis, peritonitis and prolapse of the rectum. One child died. CONCLUSION: Gastrointestinal complications of D+ HUS are rare, but they can be lethal, and early surgery may sometimes prove necessary. However, we were not able to demonstrate a correlation between the severity of the gastrointestinal manifestations and the clinical or biological signs accompanying D+ HUS.

Results of the PERFORM magnetic resonance imaging study.

The PERFORM MRI Project was an ancillary study of the PERFORM trial. Its aim was to investigate the potential effects of terutroban in patients with atherothrombotic disorders, in comparison to aspirin, on the evolution of magnetic resonance imaging (MRI) lesions after a recent ischemic stroke or transient ischemic attack (TIA). The change in both hypointense and hyperintense lesions on the fluid attenuated inversion recovery (FLAIR) sequence, in the total brain volume and in the hippocampal volume from baseline (M1) to the final visit (M24) was assessed as well as the number of emergent microbleeds. A total of 748 patients had their MRI examination validated both at M1 and M24 during the study. At baseline, the volume of hypointense and hyperintense lesions on FLAIR images, the total brain volume, the hippocampal volume and the number of patients with microbleeds did not differ between the two groups. During follow-up, the mean volumetric increase of lesions hypointense or hyperintense on FLAIR images (from 5 to 8 %), the mean reduction of total brain volume (−0.4 %) and of hippocampal volume (−4 %), did not differ between the two treatment arms. The same parameters analysed ipsilateral to the ischaemic lesion did not differ either between the two groups. In the terutroban group, 16.3 % of patients presented with emergent microbleeds, 10.7 % in the aspirin group; this difference was not significant. In the PERFORM study, the progression of FLAIR lesions, of cerebral or hippocampal atrophy and of microbleeds did not differ between patients treated by terutroban and those treated by aspirin.

Redox dysregulation in the pathophysiology of schizophrenia and bipolar disorder: insights from animal models.

Abstract Significance: Schizophrenia (SZ) and bipolar disorder (BD) are classified as two distinct diseases. However, accumulating evidence shows that both disorders share genetic, pathological, and epidemiological characteristics. Based on genetic and functional findings, redox dysregulation due to an imbalance between pro-oxidants and antioxidant defense mechanisms has been proposed as a risk factor contributing to their pathophysiology. Recent Advances: Altered antioxidant systems and signs of increased oxidative stress are observed in peripheral tissues and brains of SZ and BD patients, including abnormal prefrontal levels of glutathione (GSH), the major cellular redox regulator and antioxidant. Here we review experimental data from rodent models demonstrating that permanent as well as transient GSH deficit results in behavioral, morphological, electrophysiological, and neurochemical alterations analogous to pathologies observed in patients. Mice with GSH deficit display increased stress reactivity, altered social behavior, impaired prepulse inhibition, and exaggerated locomotor responses to psychostimulant injection. These behavioral changes are accompanied by N-methyl-D-aspartate receptor hypofunction, elevated glutamate levels, impairment of parvalbumin GABA interneurons, abnormal neuronal synchronization, altered dopamine neurotransmission, and deficient myelination. Critical Issues: Treatment with the GSH precursor and antioxidant N-acetylcysteine normalizes some of those deficits in mice, but also improves SZ and BD symptoms when given as adjunct to antipsychotic medication. Future Directions: These data demonstrate the usefulness of GSH-deficient rodent models to identify the mechanisms by which a redox imbalance could contribute to the development of SZ and BD pathophysiologies, and to develop novel therapeutic approaches based on antioxidant and redox regulator compounds. Antioxid. Redox Signal. 18, 1428-1443.

Lymphodepletion by short-term chemotherapy does not alter the highly stable and persistent EBV specific CD8 T cell repertoire

Protective T cell responses againstpersistent viruses like Epstein-Barrvirus in healthy individuals are characterizedby a remarkable stability ofthe T cell receptor (TCR) clonotypicrepertoire, with highly preservedclonotype selection and persistenceover time. Here, we extended recentwork to the study of EBV-specificCD8 T cell responses in melanomapatients treated by short-term chemotherapyfor transient lymphodepletion,followed by adoptive cell transfer(ACT) and immune reconstitutionfor cancer therapy. After this treatment,we observed increased proportionsof virus-specific T cells in 3/5patients, accompanied by a more differentiatedphenotype (EMRA/CD28neg), compared to specific cells ofhealthy individuals. Yet, similarly tohealthy donors, clonotype selectionand composition of virus-specific Tcells varied along the pathway of celldifferentiation, with some clonotypesthat were selected with late differentiation,while others were not. Aftertreatment, we did not observe noveldominant clonotypes, likely related toabsence of EBV reactivation measuredas viral load levels by quantitativePCR in PBMCs and antibody levelsin plasma samples. Furthermore,public TCR BV signatures were frequentlyfound within T cell clonotypesthat dominated the repertoiresof patients, in line with those observedin healthy individuals. Ourfindings indicate that even in situationswhere the immune system isstrongly challenged such as followinglymphodepletion and homeostatic repopulation,cytotoxic T cells specificfor EBV remain strikingly stable interms of clonotype selection and com-position along T cell differentiation.We are currently characterizing theclonotype selection and gene expressionprofiles of single EBV-specificCD8 T lymphocytes sorted ex-vivo inone patient who underwent two cyclesof lymphodepletion with escaladingdoses of chemotherapy overone-year interval. Observations madefrom this setting will provide additionalinsight into the degree of stabilityof virus specific T cells, and changesin the expression levels of genesimportant for cytolytic function andlong-term survival of T cells. Thiswork is supported by the Swiss NationalCenter of Competence in Research(NCCR) Molecular Oncology,and the Swiss National Science Foundation.

On the significance of CD8 alpha alpha expression for T cell memory.

Longitudinal studies on the kinetics of viral antigen specific CD8 T cell responses have led to a model whereby a relatively small subset of the primary effector CD8 T cells expanding after the first week of acute viral infection initiate a program of cell survival and differentiation into long lived memory T cells. These T cells are then critical for maintaining protective immunity to subsequent viral infection. Recent observations, using fluorescent tetramers of the MHC class Ib molecule TL, link transient expression of CD8alphaalpha homodimers on expanding primary effector CD8 T cells to the generation of memory cells. At present it is controversial what the role of CD8alphaalpha is in the generation of memory CD8 T cells. The involvement of the high affinity CD8alphaalpha ligand, the TL molecule, is not understood either. However, evidence from two viral infection models in mice, including one paper in this issue of the European Journal of Immunology, suggest a role for CD8alphaalpha in this process and call for additional research focus into these issues.

Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies.

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

Long-term complete responses after 131I-tositumomab therapy for relapsed or refractory indolent non-Hodgkin's lymphoma.

We present the long-term results of 18 chemotherapy relapsed indolent (N = 12) or transformed (N = 6) NHL patients of a phase II anti-CD20 (131)I-tositumomab (Bexxar) therapy study. The biphasic therapy included two injections of 450 mg unlabelled antibody combined with (131)I-tositumomab once as dosimetric and once as therapeutic activity delivering 75 or 65 cGy whole-body radiation dose to patients with normal or reduced platelet counts, respectively. Two patients were not treated due to disease progression during dosimetry. The overall response rate was 81% in the 16 patients treated, including 50% CR/CRu and 31% PR. Median progression free survival of the 16 patients was 22.5 months. Median overall survival has not been reached after a median observation of 48 months. Median PFS of complete responders (CR/CRu) has not been reached and will be greater than 51 months. Short-term side effects were mainly haematological and transient. Among the relevant long-term side effects, one patient previously treated with CHOP chemotherapy died from secondary myelodysplasia. Four patients developed HAMA. In conclusion, (131)I-tositumomab RIT demonstrated durable responses especially in those patients who achieved a complete response. Six of eight CR/CRu are ongoing after 46-70 months.

Pancreatic-specific expression of the glucose transporter type 2 gene: identification of cis-elements and islet-specific trans-acting factors.

A defect in glucose sensing of the pancreatic beta-cells has been observed in several animal models of type II diabetes and has been correlated with a reduced gene expression of the glucose transporter type 2 (Glut2). In a transgenic mouse model, expression of Glut2 antisense RNA in pancreatic beta-cells has recently been shown to be associated with an impaired glucose-induced insulin secretion and the development of diabetes. To identify factors that may be involved in the specific decrease of Glut2 in the beta-cells of the diabetic animal, an attempt was made to localize the cis-elements and trans-acting factors involved in the control of Glut2 expression in the endocrine pancreas. It was demonstrated by transient transfection studies that only 338 base pairs (bp) of the murine Glut2 proximal promoter are needed for reporter gene expression in pancreatic islet-derived cell lines, whereas no activity was detected in nonpancreatic cells. Three cis-elements, GTI, GTII, and GTIII, have been identified by DNAse I footprinting and gel retardation experiments within these 338 bp. GTI and GTIII bind distinct but ubiquitously expressed trans-acting factors. On the other hand, nuclear proteins specifically expressed in pancreatic cell lines interact with GTII, and their relative abundance correlates with endogenous Glut2 expression. These GTII-binding factors correspond to nuclear proteins of 180 and 90 kilodaltons as defined by Southwestern analysis. The 180-kilodalton factor is present in pancreatic beta-cell lines but not in an alpha-cell line. Mutation of the GTI or GTIII cis-elements decreases transcriptional activity directed by the 338-bp promoter, whereas mutation of GTII increases gene transcription. Thus negative and positive regulatory sequences are identified within the proximal 338 bp of the GLUT2 promoter and may participate in the islet-specific expression of the gene by binding beta-cell specific trans-acting factors.

Analyses of a novel SCN5A mutation (C1850S): conduction vs. repolarization disorder hypotheses in the Brugada syndrome.

AIMS: Brugada syndrome (BrS) is characterized by arrhythmias leading to sudden cardiac death. BrS is caused, in part, by mutations in the SCN5A gene, which encodes the sodium channel alpha-subunit Na(v)1.5. Here, we aimed to characterize the biophysical properties and consequences of a novel BrS SCN5A mutation. METHODS AND RESULTS: SCN5A was screened for mutations in a male patient with type-1 BrS pattern ECG. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in HEK293 cells. Sodium currents (I(Na)) were analysed using the whole-cell patch-clamp technique at 37 degrees C. The electrophysiological effects of the mutation were simulated using the Luo-Rudy model, into which the transient outward current (I(to)) was incorporated. A new mutation (C1850S) was identified in the Na(v)1.5 C-terminal domain. In HEK293 cells, mutant I(Na) density was decreased by 62% at -20 mV. Inactivation of mutant I(Na) was accelerated in a voltage-dependent manner and the steady-state inactivation curve was shifted by 11.6 mV towards negative potentials. No change was observed regarding activation characteristics. Altogether, these biophysical alterations decreased the availability of I(Na). In the simulations, the I(to) density necessary to precipitate repolarization differed minimally between the two genotypes. In contrast, the mutation greatly affected conduction across a structural heterogeneity and precipitated conduction block. CONCLUSION: Our data confirm that mutations of the C-terminal domain of Na(v)1.5 alter the inactivation of the channel and support the notion that conduction alterations may play a significant role in the pathogenesis of BrS.

Fibronectin distribution in the chick embryo during formation of the blastula.

The distribution of the fibronectin-rich extracellular matrix (ECM) in the chick embryo during formation of the blastula has been evaluated semiquantitatively using an electron microscopical immunogold staining technique. During the first 10 h of postlaying development, fibronectin was found in both embryonic area pellucida and extra-embryonic area opaca of the blastoderm. In the area pellucida, the fibronectin was (1) associated with the basal lamina of the epiblast, (2) present between epiblastic and hypoblastic cells and (3) occasionally internalized in hypoblastic cells. Along the embryonic axis, a transient and high density of ECM was associated with the front of the anteriorly and rapidly expanding hypoblast. Very high density of fibronectin was observed in the marginal zone of the area pellucida, where the epiblastic and deeper cell layers show contacts and intense re-arrangements. In the area opaca, fibronectin was at first found only sporadically between contacting cells, but its density increased steadily and markedly during the first day of development. These rapid and significant changes in the regional distribution of fibronectin-rich ECM are discussed with respect to the early morphogenesis of the chick embryo.

Petrology and chemistry of the Chalten Plutonic Complex and implications on the magmatic and tectonic evolution of the Southernmost Andes (Patagonia) during the Miocene

Résumé Scientific:Pétrologie et Géochimie du Complexe Plutonique de Chaltén et les conséquences pour l'évolution magmatique et tectonique du Andes du Sud (Patagonia) pendant le MiocèneLe sujet de cette thèse est le Complexe Plutonique de Chaltén (CHPC), situé à la frontière entre le Chili et l'Argentine, en Patagonie (49°15'S). Ce complexe s'est mis en place au début du Miocène, dans un contexte de changements tectoniques importants. La géométrie et la vitesse de migration des plaques en Patagonie a été modifiée suite l'ouverture de la plaque Farallon il y a 25Ma (Pardo-Casas and Molnar 1987) et la subduction de la ride active du Chili sous la plaque sud-américaine il y a 14Ma (Cande and Leslie 1986). Les effets de cette reconfiguration tectonique sur la morphologie et le magmatisme de la plaque supérieure sont encore sujets à discussion. Dans ce contexte, un groupe d'intrusions miocènes - telle que le CHPC - est particulièrement intriguant, car en position transitionnelle entre le batholithe patagonien et l'arc volcanique cénozoïque et récent à l'ouest, et les laves de plateau de Patagonie à l'est (Fig. 1). A cause de leur position tectonique transitoire, ces plutons isolés hors du batholithe représentent un endroit clé pour comprendre les interactions entre la tectonique à large échelle et le magmatisme en Patagonie. Ici, je présente de nouvelles données de terrain, petrologiques, géochimiques et géochronologiques dans le but de caractériser la nature du CHPC, qui était largement inconnu avant cette étude, dans le but de tester l'hypothèse de migration de l'arc et erosion par subduction.Les résultats de l'investigation géochimique (chapitre 2) montrent que le CHPC n'est qu'un exemple parmi les plutons isolés d'arrière arc ave une composition calco-alcaline caractéristique, c-à-d une signature d'arc. La plupart de ces plutons isolés ont une composition alcaline. Le CHPC, contrairement, a une signature calco-alcaline avec Κ intermédiaire, tel que le batholithe patagonien et la plupart des roches volcaniques quaternaires liées à l'arc le long des Andes.De nouvelles données géochronologiques U-Pb de haute précision sur des zircons, acquis par TIMS, sur le CHPC donnent des âges entre 17.0 et 16.4Ma. Les âges absolus sont en accord avec la séquence intrusive déduite des relations de terrain (chapitre 1). Ces données sont les premières contraintes d'âge U-Pb sur le CHPC. Elles montrent clairement que l'histoire magmatique du CHPC n'a pas de lien direct avec la subduction de la ride à cette latitude (Cande and Leslie 1986), car le complexe est au moins 6Ma plus ancien.Une comparaison en profondeur avec les autres intrusions d'âge Miocène en Patagonie révèlent - pour la première fois - une évolution temporelle intéressante. Il y a une tendance E-W distincte au magmatisme calco-alcalin entre 20-16Ma avec une diminution de l'âge vers l'est - le CHPC est l'expression la plus orientale de cette tendance. Je suggère que la relation espace-temps reflète une migration vers l'est (vers le continent) de l'arc magmatique. Je propose que le facteur principal contrôlant cette migration est la subduction rapide suite à la reconfiguration de la vitesse des plaques tectoniques après l'ouverture la plaque Farallon (à ~26Ma) qui résulterait en une déformation importante ainsi qu'à des taux élevés d'érosion dans la fosse de subduction.Les rapports d'isotopes radiogéniques (Pb, Sr, Nd) élevés, une signature 6018 basse et un rapport Th/La élevé sont des paramètres distinctifs pour les roches mafiques du CHPC. Le modèle isotopique présenté (chapitre 2) suggère que cette signature reflète une contamination de la source, dans le coin de manteau, plutôt qu'une contamination crustale. La signature des éléments en trace du CHPC indiquent que le coin de manteau a été contaminé par des composés terrigènes, le plus vraisemblablement par des sédiments paléozoïques.Les travaux de terrain, la pétrographie et la géothermobarométrie ont été utilisés dans le but de comprendre l'histoire interne du CHPC (chapitre 3). Ces données suggèrent deux niveaux distincts de cristallisation : l'un dans la croûte moyenne (6 à 4.5kbar) et l'autre à un niveau peu profond (3.5 à 2kbar). La modélisation isotopique AFC de la contamination crustale indique des taux variables d'assimilation, qui ne sont pas corrélés avec le degré de différenciation. Cela suggère que différents volumes de magma se sont différenciés en profondeur, de façon indépendante. Cela implique que le CHPC se serait formés en plusieurs puises de magmas provenant d'au moins trois sources différentes. Les textures des granodiorites et des granites indiquent des teneurs élevées en cristaux avant la mise en place et, par conséquent, des températures d'emplacement faibles. Les observations de terrain montrent que les roches mafiques sont déformées, alors que ce n'est pas le cas pour les granodiorites et granites (plus jeunes). La déformation des roches mafiques est encore sujet de recherche, afin de savoir si elle est liée à la déformation régionale en régime compressif ou à l'emplacement lui-même. Cependant, la mise en place de grand volume de magma felsique riche en cristaux suggère un régime d'extension.Scientific Abstract:Petrology and chemistry of the Chaltén Plutonic Complex and implications on the magmatic and tectonic evolution of the Southernmost Andes (Patagonia) during the MioceneThe subject of this thesis is the Chaltén Plutonic Complex (CHPC) located at the frontier between Chile and Argentina in Patagonia (at 49° 15 'Southern latitude). This complex intruded during early Miocene in a context of major tectonics changes. The plate geometry of Patagonia has been modified by changes in the plate motions after the break up of the Farallôn plate at 25Ma (Pardo-Casas and Molnar 1987) and by the subduction of the Chile spreading Ridge beneath South-America at 14 Ma (Cande and Leslie 1986). The effects of this tectonic setting on the morphology and the magmatism of the overriding plate are a matter of on-going discussion. Particularly intriguing in this context is a group of isolated Miocene intrusions - like the CHPC - which are located in a transitional position between the Patagonian Batholith and the Cenozoic and Recent volcanic arc in the West, and the Patagonian plateau lavas in the East (Fig. 1). Due to their transient tectonic position these isolated plutons outside the batholith represent a key to understanding the interaction between global-scale tectonics and magmatism in Patagonia. Here, I present new field, penological, geochemical and geochronological data to characterize the nature of the CHPC, which was largely unknown before this study, in order to test the hypothesis of time- transgressive magmatism.The results of the geochemical investigation (Chapter 2) show that the CHPC is only one among these isolated back-arc plutons with a characteristic calc-alkaline composition, i.e. arc signature. Most of these isolated intrusives have an alkaline character. The CHPC, in contrast, has a medium Κ calc-alkaline signature, like the Patagonian batholith and most of the Quaternary arc-related volcanic rocks along the Andes.New high precision TIMS U-Pb zircon dating of the CHPC yield ages between 17.0 to 16.4 Ma. The absolute ages support the sequence of intrusion relations established in the field (Chapter 1). These data are the first U-Pb age constraints on the CHPC, and clearly show that the magmatic history of CHPC has no direct link to the subduction of the ridge, since this complex is at least 6 Ma older than the time of collision of the Chile ridge at this latitude (Cande and Leslie 1986).An in-depth comparison with other intrusion of Miocene age in Patagonia reveals - for the first time - an interesting temporal pattern. There is a distinct E-W trend of calc-alkaline magmatism between 20-16 Ma with the younging of ages in the East - the CHPC is the easternmost expression of this trend. I suggest that this time-space relation reflects an eastward (landward) migration of the magmatic arc. I propose that main factor controlling this migration is the fast rates of subduction after the major reconfigurations of the plate tectonic motions after the break up of the Farallôn Plate (at -26 ) resulting in strong deformation and high rates of subduction erosion.High radiogenic isotope ratios (Pb, Sr, Nd) ratios, low 5018 signature and high Th/La ratios in mafic rocks are distinctive features of the CHPC. The presented isotopic models (Chapter 2) suggest that this signature reflects source contamination of the mantle wedge rather than crustal contamination. The trace element signature of the CHPC indicates that the mantle wedge was contaminated with a terrigenous component, most likely from Paleozoic sediments.Fieldwork, petrography and geothermobarometry were used to further unravel the internal history of the CHPC (Chapter 3). These data suggest two main levels of crystallization: one a mid crustal levels (6 to 4.5 kbar) and other a shallow level (3.5 to 2 kbar). Isotopic AFC modeling of crustal contamination indicate variable rates of assimilation, which are not correlated with the degree of differentiation. This suggests that different batches of magma differentiate independently at depths. This implies that the CHPC would have formed by several pulses of magmas from at least 3 different sources. Textures of granodiorites and granites indicate a high content of crystals previous to the emplacement and consequently low emplacement temperatures. Field observations show that the mafic rocks are deformed, whereas the (younger) granodiorites and granites are not. It is still subject of investigation whether the deformation of the mafic rocks is related to regional deformation during a compressional regime or to the emplacement it self. However, the emplacement of huge amount of crystal rich felsic magmas suggests an extensional regime.Résumé Grand PublicPétrologie et Géochimie du Complexe Plutonique de Chaltén et les conséquences pour l'évolution magmatique et tectonique du Andes du Sud (Patagonia) pendant le MiocèneLe Complexe Plutonique de Chaltén (CHPC) est un massif montagneux situé à 49°S à la frontière entre le Chili et l'Argentine, en Patagonie (région la plus au sud de l'Amérique du Sud). Il est composé de montagnes qui peuvent atteindre plus de 3000 mètres d'altitude, telles que le Cerro Fitz Roy (3400m) et le Cerro Torre (3100m). Ces montagnes sont composées de roches plutoniques, c.-à-d. des magmas qui se sont refroidis et ont cristallisés sous la surface terrestre.La composition chimique de ces roches montre que les magmas, qui ont formé ce complexe plutonique, font partie d'un volcanisme d'arc. Celui-ci se forme lorsqu'une plaque océanique plonge sous une plaque continentale. Les géologues appellent ce processus « subduction ». Dans un tel scénario, le manteau terrestre, qui se fait prendre entre ces deux plaques, fond pour former ainsi du magma. Ce magma remonte à travers la plaque continentale vers la surface. Si celui-ci atteint la surface, il forme les roches volcaniques, comme par exemple des laves. S'il n'atteint pas la surface, le magma se refroidit pour former finalement les roches plutoniques.Le long de la marge ouest d'Amérique du Sud, la plaque Nazca - qui se situe au sud-est de la plaque océanique pacifique - passe en dessous de la plaque d'Amérique du Sud. La bordure ouest du sud de la plaque sud-américaine a également été affectée par d'autres processus tectoniques, tels que des changements dramatiques dans les déplacements de plaques (il y a 25Ma) et la collision de la ride du Chili (depuis 15 Ma jusqu'à aujourd'hui). Ces caractéristiques tectoniques et magmatiques font de cette région un haut lieu pour les géologues. La plaque Nazca, s'est formée suite à l'ouverture d'une plaque océanique plus ancienne, il y a 25Ma. Cette ouverture est liée aux vitesses de subduction les plus rapides jamais connues. La ride du Chili est l'endroit où le sol de l'Océan Pacifique s'ouvre, formant deux plaques océaniques : les plaques Nazca et Antarctique. La ride du Chili subducte sous la plaque sud-américaine depuis 15Ma, en association avec la formation de grands volumes de magma ainsi que des changements morphologiques importants. La question de savoir lequel de ces changements tectoniques globaux affecte la géologie et la géographie de Patagonie a été, et est encore, discutée pendant de nombreuses années. De nombreux chercheurs suggèrent que la plupart des caractéristiques morphologiques et magmatiques en Patagonie sont liés à la subduction de la ride du Chili, mais cette suggestion est encore débattue comme le montre notre étude.Le batholithe de Patagonie du sud (SPB) est un énorme massif composé de roches plutoniques et il s'étend tout au long de la côte ouest de Patagonie (au sud de 47°S). Ces roches correspondent certainement aux racines d'un ancien arc volcanique, qui a été soulevé et érodé. Le CHPC, ainsi que d'autres petites intrusions dans la région, se situe dans une position exotique, à 100km à l'est du SPB. Certains chercheurs suggèrent que ces intrusions pourraient être liées à la subduction de la ride du Chili.Afin de débattre de cette problématique, nous avons utilisé différentes méthodes géochronologiques pour déterminer l'âge du CHPC et le comparer (a) à l'âge des roches intrusives similaires du SPB et (b) à l'âge de la collision de la ride du Chili. Dans ce travail, nous prouvons que le CHPC s'est formé au moins 7Ma avant la collision avec la ride du Chili. Sur la base des âges du CHPC et de la composition chimique de ses roches et minéraux, nous proposons que le CHPC fait partie d'un arc volcanique ancien. La migration de l'arc volcanique plus profondément dans le continent résulte de la grande vitesse de subduction entre 25 et lOMa. Des caractéristiques évidentes pour un tel processus - telles qu'une déformation importante et une vitesse d'érosion élevée - peuvent être rencontrées tout au long de la bordure ouest de l'Amérique du sud.

Complex roles of inflammasomes in carcinogenesis.

The central role of chronic inflammation in the promotion of tumor growth is supported by a broad range of experimental and clinical evidence. However, the molecular mechanisms converting transient inflammatory tissue reactions into a tumor-promoting microenvironment remain largely elusive. Because inflammasomes have been shown to regulate the proinflammatory cytokines interleukin 1β (IL-1β) and IL-18, they have been implicated in the relationship between tumor genesis/progression and inflammation. For instance, many cancers have been directly linked to inflammasome-mediated sterile inflammation, where a blockade of IL-1β and IL-18 has been shown to inhibit tumor growth. On the other hand, inflammasome activation also has potent antitumorigenic effects, where malignant precursor cells are eliminated through pyroptotic cell death. Indeed, inflammasome activity can even increase the efficacy of certain chemotherapies. Here, we review the current understanding on the complex and sometimes contradictory role of inflammasomes in carcinogenesis.

A regulatory network for coordinated flower maturation.

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs.

Role of transcription factor PROX1 in colon cancer metastasis

Initiation and progression of most colorectal cancers (CRCs) are driven by hyper-activation of the canonical Wnt/ß-catenin/TCF signaling pathway. However, a basal level of activation of this pathway is necessary for intestinal cell homeostasis; thus only CRC-specific effectors of this pathway could be exploited as potential clinical targets. PROX1 is an evolutionary conserved transcription factor with multiple roles in several tissues in embryogenesis, and increasing relevance in cancer. PROX1 is a colon cancer-specific Wnt target in the intestine, thus it might represent a therapeutic target. The role of PROX1 in promoting the transition from early to highly-dysplastic adenoma was previously described [1], Importantly, tumor metastasis is a leading cause of cancer-related mortality. Frequently, micrometastases are already present in patients at the time of diagnosis, therefore better understanding of the mechanisms regulating growth of macrometastatic lesions is important for the development of novel treatment approaches. In this study we showed that PROX1 is expressed in colon cancer stem cell and promotes the outgrowth of metastatic lesions. Firstly, we analyzed the expression of PROX1 in advanced CRCs and their metastases. We found that PROX1 over-expression is a feature of microsatellite stable tumors (~85% of microsatellite stable (MSS) CRCs), which generally have worse prognosis in comparison to microsatellite unstable CRCs. Analysis of primary CRCs and corresponding metastatic lesions showed that PROX1 expression is conserved, or increased in metastases. Further bioinformatics analysis of tumor and metastases gene expression profiles showed that PROX1 is co- expressed with stem cell and progenitor markers. Moreover, in inducible ApcmLgr5-EGFP-lres-CreERT2 model, Prox1+ cells marked a sub-population of Lgr5+ stem cells and subsequent transient amplifying cell population. Orthotopic model of CRC and lung colonization assays in mice demonstrated that PROX1 promotes tumor cell outgrowth in metastatic lesions, while it has no effect on primary tumor growth, invasion, and survival in circulation or cell extravasation. In vitro, PROX1 expressing tumor cells demonstrated strongly increased capacity to form spheroids, and increased survival and proliferation under hypoxic or nutrient-deprivation conditions. By monitoring cellular respiration under these conditions, we found that PROX1 expressing cells exhibit a better metabolic adaptation to changes in fuel source. Autophagy inhibitors, prevented growth both in vitro and in vivo of PROX1 expressing cells. Importantly, conditional inactivation of PROX1 after the establishment of metastases prevented further growth of macroscopic lesions resulting in stable disease. In summary, we identified a novel mechanism underlying the ability of metastatic colon cancer stem and progenitor cells to survive and grow in target organs through metabolic adaptation. Our results establish PROX1 as a key factor of CRC metastatic disease where it promotes survival of metastatic colon cancer stem-like cells, through their metabolic adaptation in sub-optimal microenvironments - L'initiation et la progression de la plupart des cancers colorectaux (CRC) sont entraînées par une hyper-activation de la voie métabolique Wnt/ß- caténine/TCF. Toutefois, un niveau d'activation minimal de Wnt est nécessaire pour l'homéostasie des cellules intestinales ; ainsi seuls des effecteurs spécifiques du CRC- de cette voie pourraient être exploités comme des cibles cliniques potentielles. PROX1 est un facteur de transcription évolutif conservé avec de multiples rôles dans plusieurs tissus durant l'embryogenèse et une pertinence croissante dans le cancer. PROX1 est une cible Wnt spécifique dans le cancer de l'intestin, donc il pourrait représenter une cible thérapeutique. Le rôle de PROX1 durant l'évolution de la maladie d'un stade précoce jusqu'à l'adénome hautement dysplasique a été décrit précédemment. Surtout, la métastase des tumeurs est une cause majeure de mortalité liée au cancer. Souvent, les micro-métastases sont déjà présentes chez les patients au moment du diagnostic, c'est pourquoi une meilleure compréhension des mécanismes régulant la croissance des lésions macrométastatiques est importante pour le développement de nouvelles approches thérapeutiques. Dans cette étude, nous avons prouvé que PROX1 est exprimé dans les cellules souches du cancer du côlon et favorise l'apparition de lésions métastatiques. Nous avons d'abord analysé l'expression de PROX1 dans des CRC avancés ainsi que dans leurs métastases. Nous avons constaté que la surexpression de PROX1 est une caractéristique des tumeurs stables microsatellites (~85% du MSS CRC), qui ont généralement un pronostic défavorable par rapport aux microsatellites CRC instables. L'analyse des CRC primaires et de leurs métastases liées a montré que l'expression de PROX1 est conservée, voire augmentée dans les métastases. A l'aide d'une base de données de tumeurs et métastases, nous avons observé une co- régulation de PROX1 entre cellules souches et marqueurs de progéniteurs mais pas avec des cellules différenciées. De plus, en utilisant un modèle Apcm Lgr5-EGFP-IRES-CreERT2 inductible, les cellules Prox1+ ont marqué une sous-population de cellules LGR& capable de produire une lignée. Un modèle orthotopique de cancer colorectal et des essais de colonisation du poumon chez la souris ont démontré que PROX1 favorise l'excroissance des cellules tumorales dans les lésions métastatiques, alors qu'il n'a aucun effet sur la croissance tumorale primaire, l'invasion ou une extravasation des cellules. In vitro, les cellules tumorales exprimant PROX1 ont démontré une forte augmentation de leur capacité à former des sphéroïdes, ainsi qu'une augmentation de la survie et de la prolifération dans des conditions hypoxiques ou lors de privation de nutriments. En contrôlant la respiration cellulaire dans ces conditions, nous avons constaté que les cellules exprimant PROX1 présentent une meilleure adaptation métabolique à l'évolution des sources de carburant. Des inhibiteurs de l'autophagie, suggérant une approche thérapeutique potentielle, ont tué à la fois in vitro et in vivo les cellules exprimant PROX1. Surtout, l'inactivation conditionnelle de PROX1 après l'apparition de métastases a empêché la croissance des lésions macroscopiques résultant en une maladie stable. En résumé, nous avons identifié un nouveau mécanisme mettant en évidence la capacité des cellules souches du cancer du côlon métastatique à survivre et à se développer dans les organes cibles grâce à l'adaptation métabolique. Nos résultats définissent PROX1 comme un facteur clé du cancer colorectal métastatique en favorisant la survie des cellules souches métastatiques apparentées au cancer du colon grâce à leur adaptation métabolique aux microenvironnements défavorables.