FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature.

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease.

Autocrine Action of IGF2 Regulates Adult β-Cell Mass and Function.

Insulin-like growth factor 2 (IGF2), produced and secreted by adult β-cells, functions as an autocrine activator of the β-cell insulin-like growth factor 1 receptor signaling pathway. Whether this autocrine activity of IGF2 plays a physiological role in β-cell and whole-body physiology is not known. Here, we studied mice with β-cell-specific inactivation of Igf2 (βIGF2KO mice) and assessed β-cell mass and function in aging, pregnancy, and acute induction of insulin resistance. We showed that glucose-stimulated insulin secretion (GSIS) was markedly reduced in old female βIGF2KO mice; glucose tolerance was, however, normal because of increased insulin sensitivity. While on a high-fat diet, both male and female βIGF2KO mice displayed lower GSIS compared with control mice, but reduced β-cell mass was observed only in female βIGF2KO mice. During pregnancy, there was no increase in β-cell proliferation and mass in βIGF2KO mice. Finally, β-cell mass expansion in response to acute induction of insulin resistance was lower in βIGF2KO mice than in control mice. Thus, the autocrine action of IGF2 regulates adult β-cell mass and function to preserve in vivo GSIS in aging and to adapt β-cell mass in response to metabolic stress, pregnancy hormones, and acute induction of insulin resistance.

DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport.

The small intestine is a dynamic and complex organ that is characterized by constant epithelium turnover and crosstalk among various cell types and the microbiota. Lymphatic capillaries of the small intestine, called lacteals, play key roles in dietary fat absorption and the gut immune response; however, little is known about the molecular regulation of lacteal function. Here, we performed a high-resolution analysis of the small intestinal stroma and determined that lacteals reside in a permanent regenerative, proliferative state that is distinct from embryonic lymphangiogenesis or quiescent lymphatic vessels observed in other tissues. We further demonstrated that this continuous regeneration process is mediated by Notch signaling and that the expression of the Notch ligand delta-like 4 (DLL4) in lacteals requires activation of VEGFR3 and VEGFR2. Moreover, genetic inactivation of Dll4 in lymphatic endothelial cells led to lacteal regression and impaired dietary fat uptake. We propose that such a slow lymphatic regeneration mode is necessary to match a unique need of intestinal lymphatic vessels for both continuous maintenance, due to the constant exposure to dietary fat and mechanical strain, and efficient uptake of fat and immune cells. Our work reveals how lymphatic vessel responses are shaped by tissue specialization and uncover a role for continuous DLL4 signaling in the function of adult lymphatic vasculature.

Retinoblastoma Protein Knockdown Favors Oxidative Metabolism and Glucose and Fatty Acid Disposal in Muscle Cells.

Deficiency in the retinoblastoma protein (Rb) favors leanness and a healthy metabolic profile in mice largely attributed to activation of oxidative metabolism in white and brown adipose tissues. Less is known about Rb modulation of skeletal muscle metabolism. This was studied here by transiently knocking down Rb expression in differentiated C2C12 myotubes using small interfering RNAs. Compared with control cells transfected with non-targeting RNAs, myotubes silenced for Rb (by 80-90%) had increased expression of genes related to fatty acid uptake and oxidation such as Cd36 and Cpt1b (by 61% and 42%, respectively), increased Mitofusin 2 protein content (∼2.5-fold increase), increased mitochondrial to nuclear DNA ratio (by 48%), increased oxygen consumption (by 65%) and decreased intracellular lipid accumulation. Rb silenced myotubes also displayed up-regulated levels of glucose transporter type 4 expression (∼5-fold increase), increased basal glucose uptake, and enhanced insulin-induced Akt phosphorylation. Interestingly, exercise in mice led to increased Rb phosphorylation (inactivation) in skeletal muscle as evidenced by immunohistochemistry analysis. In conclusion, the silencing of Rb enhances mitochondrial oxidative metabolism and fatty acid and glucose disposal in skeletal myotubes, and changes in Rb status may contribute to muscle physiological adaptation to exercise. J. Cell. Physiol. 231: 708-718, 2016. © 2015 Wiley Periodicals, Inc.

Treatment of pleural malignancies by photo-induction combined to systemic chemotherapy: Proof of concept on rodent lung tumors and feasibility study on porcine chest cavities.

BACKGROUND: Low-dose, Visudyne®-mediated photodynamic therapy (photo-induction) was shown to selectively enhance tumor vessel transport causing increased uptake of systemically administered chemotherapy in various tumor types grown on rodent lungs. The present experiments explore the efficacy of photo-induced vessel modulation combined to intravenous (IV) liposomal cisplatin (Lipoplatin®) on rodent lung tumors and the feasibility/toxicity of this approach in porcine chest cavities. MATERIAL AND METHODS: Three groups of Fischer rats underwent orthotopic sarcoma (n = 14), mesothelioma (n = 14), or adenocarcinoma (n = 12) implantation on the left lung. Half of the animals of each group had photo-induction (0.0625 mg/kg Visudyne®, 10 J/cm(2) ) followed by IV administration of Lipoplatin® (5 mg/kg) and the other half received Lipoplatin® without photo-induction. Then, two groups of minipigs underwent intrapleural thoracoscopic (VATS) photo-induction (0.0625 mg/kg Visudyne®; 30 J/cm(2) hilum; 10 J/cm(2) apex/diaphragm) with in situ light dosimetry in combination with IV Lipoplatin® administration (5 mg/kg). Protocol I (n = 6) received Lipoplatin® immediately after light delivery and Protocol II (n = 9) 90 minutes before light delivery. Three additional animals received Lipoplatin® and VATS pleural biopsies but no photo-induction (controls). Lipoplatin® concentrations were analyzed in blood and tissues before and at regular intervals after photo-induction using inductively coupled plasma mass spectrometry. RESULTS: Photo-induction selectively increased Lipoplatin® uptake in all orthotopic tumors. It significantly increased the ratio of tumor to lung Lipoplatin® concentration in sarcoma (P = 0.0008) and adenocarcinoma (P = 0.01) but not in mesothelioma, compared to IV drug application alone. In minipigs, intrapleural photo-induction combined to systemic Lipoplatin® was well tolerated with no toxicity at 7 days for both treatment protocols. The pleural Lipoplatin® concentrations were not significantly different at 10 and 30 J/cm(2) locations but they were significantly higher in protocol I compared to II (2.37 ± 0.7 vs. 1.37 ± 0.7 ng/mg, P  < 0.001). CONCLUSION: Visudyne®-mediated photo-induction selectively enhances the uptake of IV administered Lipoplatin® in rodent lung tumors. Intrapleural VATS photo-induction with identical treatment conditions combined to IV Lipoplatin chemotherapy is feasible and well tolerated in a porcine model. Lasers Surg. Med. 47:807-816, 2015. © 2015 Wiley Periodicals, Inc.

t(15;21) translocations leading to the concurrent downregulation of RUNX1 and its transcription factor partner genes SIN3A and TCF12 in myeloid disorders.

Through a combined approach integrating RNA-Seq, SNP-array, FISH and PCR techniques, we identified two novel t(15;21) translocations leading to the inactivation of RUNX1 and its partners SIN3A and TCF12. One is a complex t(15;21)(q24;q22), with both breakpoints mapped at the nucleotide level, joining RUNX1 to SIN3A and UBL7-AS1 in a patient with myelodysplasia. The other is a recurrent t(15;21)(q21;q22), juxtaposing RUNX1 and TCF12, with an opposite transcriptional orientation, in three myeloid leukemia cases. Since our transcriptome analysis indicated a significant number of differentially expressed genes associated with both translocations, we speculate an important pathogenetic role for these alterations involving RUNX1.

Le rôle de CRTC1 dans la prise de poids induite par les antipsychotiques

Les antipsychotiques atypiques, de deuxième génération, ont largement contribué à améliorer le traitement des patients souffrant de schizophrénie. Cependant, leur mécanisme d'action reste mal compris et leurs effets secondaires sont importants, notamment la prise de poids. CRTC1 (CREB-regulated transcription coactivator 1), aussi appelé TORC1 (transducers of regulated CREB activity 1), est un coactivateur de CREB. Il régule la transcription de Bdnf1 qui joue un rôle essentiel dans le contrôle de la balance énergétique dépendant du VMH 2, 3, 4. Nous pensons que CRTC1 est impliqué dans la prise de poids induite par certains antipsychotiques. En effet, il a été démontré que les souris Crtc1-/- devenaient hyperphagiques et obèses 5, 6, que la régulation de l'activité de CRTC1 se faisait par l'AMPK et que les antipsychotiques atypiques activaient cette kinase dans l'hypothalamus.7 L'AMPK de l'hypothalamus est liée à la régulation de la prise alimentaire, elle inverse l'action de la leptine, hormone anorexigène. Suite à ces constatations, nous proposons de suivre l'hypothèse de travail suivante : l'activation de l'AMPK par les antipsychotiques atypiques dans l'hypothalamus peut maintenir la phosphorylation de CRTC1 et le bloquer dans le cytoplasme, l'empêchant ainsi d'activer les gènes anorexigènes, comme Bdnf par exemple. En effet, la forme phosphorylée inactive de CRTC1 est séquestrée dans le cytoplasme et sa migration dans le noyau nécessite en même temps l'activation de la phosphatase calcineurine et l'inactivation des kinases de la famille de l'AMPK. Dans le travail que nous avons entamé, nous cherchons donc, par western blot et par immunohistochimie, à comprendre si les antipsychotiques atypiques inactivent CRTC1 en induisant sa phosphorylation par l'AMPK et sa rétention dans le cytoplasme.

Comparison of Griffiths-II and Bayley-II tests for the developmental assessment of high-risk infants.

INTRODUCTION: Two important risk factors for abnormal neurodevelopment are preterm birth and neonatal hypoxic ischemic encephalopathy. The new revisions of Griffiths Mental Development Scale (Griffiths-II, [1996]) and the Bayley Scales of Infant Development (BSID-II, [1993]) are two of the most frequently used developmental diagnostics tests. The Griffiths-II is divided into five subscales and a global development quotient (QD), and the BSID-II is divided into two scales, the Mental scale (MDI) and the Psychomotor scale (PDI). The main objective of this research was to establish the extent to which developmental diagnoses obtained using the new revisions of these two tests are comparable for a given child. MATERIAL AND METHODS: Retrospective study of 18-months-old high-risk children examined with both tests in the follow-up Unit of the Clinic of Neonatology of our tertiary care university Hospital between 2011 and 2012. To determine the concurrent validity of the two tests paired t-tests and Pearson product-moment correlation coefficients were computed. Using the BSID-II as a gold standard, the performance of the Griffiths-II was analyzed with receiver operating curves. RESULTS: 61 patients (80.3% preterm, 14.7% neonatal asphyxia) were examined. For the BSID-II the MDI mean was 96.21 (range 67-133) and the PDI mean was 87.72 (range 49-114). For the Griffiths-II, the QD mean was 96.95 (range 60-124), the locomotors subscale mean was 92.57 (range 49-119). The score of the Griffiths locomotors subscale was significantly higher than the PDI (p<0.001). Between the Griffiths-II QD and the BSID-II MDI no significant difference was found, and the area under the curve was 0.93, showing good validity. All correlations were high and significant with a Pearson product-moment correlation coefficient >0.8. CONCLUSIONS: The meaning of the results for a given child was the same for the two tests. Two scores were interchangeable, the Griffiths-II QD and the BSID-II MDI.

Finding and characterizing Partially Methylated Domains in human haematopoietic cells

DNA cytosine methylation has been demonstrated to be a central epigenetic modification that has essential roles in a myriad of cellular processes. Some examples of these include gene regulation, DNA-protein interactions, cellular differentiation, X-inactivation, maintenance of genome integrity by suppressing transposable elements and viruses, embryogenesis, genomic imprinting and tumourigenesis. This list is increasingly growing thanks to recent advances in genome-wide technologies, like Whole Genome Bisulfite Sequencing (WGBS-Seq). The development of this technology in research has allowed the identification of new features of the DNA methylation landscape that was not possible using previous technologies, like Partially Methylated Domains (PMDs). PMDs have been found in several cell lines, as well as in both healthy and cancer primary samples. They have been described as regions with high variability in methylation levels across individual CpG sites and intermediate methylation levels on average with respect to the genome. Here, we performed an extensive search of PMDs in a big dataset of different haematopoietic primary cells from both myeloid and lymphoid lineages. We found and characterized significant PMDs in plasma B cells, confirming that PMDs are a phenomenon that is restricted to certain differentiated cells. Additionally, we found loci aberrantly hypomethylated in a myeloma sample which overlapped with plasma B cell PMDs. Genome-wide comparison of the myeloma and plasma B cell sample revealed that this is probably also the case for other loci.

The life history of retrocopies illuminates the evolution of new mammalian genes.

New genes contribute substantially to adaptive evolutionary innovation, but the functional evolution of new mammalian genes has been little explored at a broad scale. Previous work established mRNA-derived gene duplicates, known as retrocopies, as models for the study of new gene origination. Here we combine mammalian transcriptomic and epigenomic data to unveil the processes underlying the evolution of stripped-down retrocopies into complex new genes. We show that although some robustly expressed retrocopies are transcribed from preexisting promoters, most evolved new promoters from scratch or recruited proto-promoters in their genomic vicinity. In particular, many retrocopy promoters emerged from ancestral enhancers (or bivalent regulatory elements) or are located in CpG islands not associated with other genes. We detected 88-280 selectively preserved retrocopies per mammalian species, illustrating that these mechanisms facilitated the birth of many functional retrogenes during mammalian evolution. The regulatory evolution of originally monoexonic retrocopies was frequently accompanied by exon gain, which facilitated co-option of distant promoters and allowed expression of alternative isoforms. While young retrogenes are often initially expressed in the testis, increased regulatory and structural complexities allowed retrogenes to functionally diversify and evolve somatic organ functions, sometimes as complex as those of their parents. Thus, some retrogenes evolved the capacity to temporarily substitute for their parents during the process of male meiotic X inactivation, while others rendered parental functions superfluous, allowing for parental gene loss. Overall, our reconstruction of the "life history" of mammalian retrogenes highlights retroposition as a general model for understanding new gene birth and functional evolution.

The role of raptor in cell death

Selon les statistiques, les maladies cancéreuses sont en augmentation dans les pays en développement ainsi que dans les pays industrialisés. Ceci peut s'expliquer largement par les habitudes alimentaires, le tabagisme, les infections, le manque d'activité physique, la pollution et le stress, entre autres. Ainsi, l'Organisation Mondiale de la Santé (OMS) prévoit une augmentation de la fréquence des cancers avec 15 millions de nouveaux cas par an en 2020. La transformation d'une cellule normale en une cellule cancéreuse se déroule en plusieurs étapes avec, au niveau moléculaire, différentes mutations ciblant des protéines régulant la croissance cellulaire. Un des exemples de protéines qui participent au contrôle des voies cellulaires impliquées lors de la prolifération des cellules sont les complexes de protéines mTORCl et mTORC2 (« mammalian target of rapamycin complex 1 and 2 »). Ces complexes mTORCl et mTORC2 activent des processus anaboliques (la synthèse de protéines et de lipides, le métabolisme énergétique, entre autres) et inhibent en même temps des voies de catabolismes cellulaires (autophagie et synthèse de lysosomes). Ils sont souvent mutés dans de nombreux cas de cancers, c'est pourquoi ils sont la cible de nombreux traitements anti-cancéreux. Pour ces raisons, nous nous sommes intéressés aux mécanismes d'actions moléculaires des drogues qui ciblent les complexes mTORCl et mTORC2. Nous avons ainsi découvert qu'une molécule présente uniquement dans le complexe mTORCl, raptor, était clivée en un fragment plus petit lors du traitement de cellules cancéreuses avec des drogues. Des molécules activées durant la mort cellulaire programmée par apoptose, les caspases, se sont révélées responsables du clivage de raptor. Nous avons ensuite décrit de façon précise les sites de clivage de raptor par les caspases durant la mort cellulaire. Il s'est avéré que le clivage de raptor affaiblissait son interaction avec mTOR au sein du complexe mTORCl, ce qui participe à l'inactivation de mTORCl lors de traitements avec des molécules anti-cancéreuses. Ces résultats nous ont permis de mieux comprendre les mécanismes d'actions de différentes drogues anti-cancéreuses au niveau du complexe mTORCl, ce qui peut être utile pour la synthèse de nouvelles molécules ciblant mTORCl ainsi que pour lutter contre les mécanismes de résistance chimiothérapeutiques. -- La protéine « mammalian target of rapamycin » (mTOR) est une sérine/thréonine kinase qui est hautement conservée des protistes à l'être humain. Deux complexes mTOR existent : le complexe 1 mTOR (mTORCl) et le complexe 2 mTOR (mTORC2). Ils régulent positivement des processus anaboliques (synthèse de protéines et de lipides, le métabolisme énergétique, l'organisation du cytosquelette, la survie cellulaire) et négativement des voies cataboliques (autophagic, biogenèse de lysosomes). Les complexes mTORCl et mTORC2 sont sensibles aux signaux mitogéniques tels que les acides aminés, le glucose, les facteurs de croissance, l'état énergétique (ATP) et les niveaux d'oxygène et induisent des voies de croissance cellulaire essentielles. La voie cellulaire regulée par mTORCl peut être hyperactivée dans de nombreux cancers humains. Puisque plusieurs voies cellulaires convergent et régulent les complexes mTORCl et mTORC2, des mutations dans les kinases en amont peuvent mener à une dérégulation de l'activation de mTOR. Des stratégies thérapeutiques ont été développées pour cibler les complexes mTORCl et mTORC2, ainsi que les kinases en amont qui régulent mTOR. Plusieurs drogues ciblant mTORCl, telles que la rapamycine et la curcumine, affectent l'interaction entre mTOR et un composant spécifique de mTORCl, raptor. Dans cette étude, nous nous sommes intéressés aux mécanismes moléculaires des drogues qui ciblent mTORCl, ainsi que leur effet déstabilisant sur l'interaction entre mTOR et raptor dans des lignées cellulaires de lymphomes. Nous avons démontré que raptor était clivé en un fragment de lOOkDa après traitement avec la rapamycine, la curcumine, l'étoposide, la cisplatine, la staurosporine et le ligand Fas (FasL). Etant donné que ces drogues ont été décrites comme induisant I'apoptose, l'utilisation d'un inhibiteur de caspases (z- VAD-fmk) a révélé que le clivage de raptor, lors de la mort cellulaire, était dépendant des caspases. Des essais caspases in vitro ont permis d'identifier la caspase-6 (ainsi que probablement d'autres caspases) comme étant une protéase impliquée dans le clivage de raptor. La séquence protéique de raptor a montré potentiellement plusieurs sites de clivage de caspases aux extrémités amino-terminale et carboxy-terminale. La mutagénèse a permis d'identifier les sites de clivages de raptor par les caspases comme étant DEAD LTD (acides aminés 17-23) et DDADD (acides aminés 939¬943). De plus, le clivage de raptor corrèle avec l'inhibition de l'activité de mTORCl envers ces substrats (S6K et 4E-BP1). Nous avons aussi observé que le clivage de raptor affaiblissait l'interaction entre mTOR et raptor, ce qui indique que ce clivage est une étape critique dans l'inhibition de mTORCl durant I'apoptose. Pour terminer, la mutagénèse du site de clivage de raptor DDADD a montré une résistance à la mort cellulaire de cellules cancéreuses. Notre travail de recherche a révélé un nouveau mécanisme moléculaire qui module l'organisation et l'activité de mTORCl, ce qui peut être d'un grand intérêt pour les recherches dans le domaine de mTOR ainsi que pour la découverte de molécules ciblant mTORCl. -- The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase, which is highly conserved from yeast to humans. Two different mTOR complexes exist: the mTOR complex 1 (mTORCl) and the mTOR complex 2 (mTORC2). They positively regulate anabolic processes (protein and lipid synthesis, energy metabolism, cytoskeleton organization, cell survival) and negatively regulate catabolic pathways (autophagy, lysosome biogenesis). The mTORCl and mTORC2 respond to mitogenic stimuli such as amino acids, glucose, growth factors, energy levels (ATP) and oxygen levels and drive essential cellular growth pathways. The mTORCl pathway can be found hyperactivated in numerous human cancers. As various cellular pathways converge and regulate mTORCl and mTORC2, mutations in upstream protein kinases can lead to a deregulated mTOR activation. Different therapeutic strategies have been developped to target mTORCl, mTORC2, as well as upstream protein kinases regulating mTOR pathways. Various drugs targeting mTORCl, such as rapamycin and curcumin, affect the interaction between mTOR and a specific mTORCl component, raptor. In this study, we investigated the molecular mechanisms of drugs targeting mTORCl, as well as their destabilizing effect on the mTOR-raptor interaction in lymphoma cell lines. We demonstrated that raptor was processed into a lOOkDa fragment after treatment with rapamycin, curcumin, etoposide, cisplatin, staurosporine and FasL. As these drugs were reported to induce apoptosis, the use of a pan-caspase inhibitor (z-VAD-fmk) revealed that the cleavage of raptor under cell death was caspase-dependent. In vitro caspase assays were performed to identify caspases-6 (and probably other caspases) as an important cysteine protease implicated in the cleavage of raptor. Analysis of raptor protein sequence showed several putative caspase-specific cleavage sites at the N-terminal and the C-terminal ends. Mutagenesis studies allowed us to identify the DEADLTD (amino acids 17-23) and the DDADD (amino acids 939-943) as the caspase-dependent cleavage residues of raptor. Furthermore, the cleavage of raptor correlated with inhibition of mTORCl activity towards its specific targets (4E-BP1 and S6K). We also highlighted that raptor processing weakened the interaction between mTOR and raptor, indicating that raptor cleavage is a critical step in the mTORCl inhibition process during apoptosis. Finally, mutagenesis of raptor C-terminal cleavage site (DDADD) conferred resistance to the chemotherapeutic-mediated cell death cascade of cancer cell. Our research work highlighted a new molecular mechanism modulating mTORCl organization and activity, which can be of great interest in the mTOR field research and for designing drugs trageting mTORCl.

In cortical neurons HDAC3 suppresses RD4-dependent SMRT export

The transcriptional corepressor SMRT controls neuronal responsiveness of several transcription factors and can regulate neuroprotective and neurogenic pathways. SMRT is a multi-domain protein that complexes with HDAC3 as well as being capable of interactions with HDACs 1, 4, 5 and 7. We previously showed that in rat cortical neurons, nuclear localisation of SMRT requires histone deacetylase activity: Inhibition of class I/II HDACs by treatment with trichostatin A (TSA) causes redistribution of SMRT to the cytoplasm, and potentiates the activation of SMRT-repressed nuclear receptors. Here we have sought to identify the HDAC(s) and region(s) of SMRT responsible for anchoring it in the nucleus under normal circumstances and for mediating nuclear export following HDAC inhibition. We show that in rat cortical neurons SMRT export can be triggered by treatment with the class I-preferring HDAC inhibitor valproate and the HDAC2/3-selective inhibitor apicidin, and by HDAC3 knockdown, implicating HDAC3 activity as being required to maintain SMRT in the nucleus. HDAC3 interaction with SMRT's deacetylation activation domain (DAD) is known to be important for activation of HDAC3 deacetylase function. Consistent with a role for HDAC3 activity in promoting SMRT nuclear localization, we found that inactivation of SMRT's DAD by deletion or point mutation triggered partial redistribution of SMRT to the cytoplasm. We also investigated whether other regions of SMRT were involved in mediating nuclear export following HDAC inhibition. TSA- and valproate-induced SMRT export was strongly impaired by deletion of its repression domain-4 (RD4). Furthermore, over-expression of a region of SMRT containing the RD4 region suppressed TSA-induced export of full-length SMRT. Collectively these data support a model whereby SMRT's RD4 region can recruit factors capable of mediating nuclear export of SMRT, but whose function and/or recruitment is suppressed by HDAC3 activity. Furthermore, they underline the fact that HDAC inhibitors can cause reorganization and redistribution of corepressor complexes.

The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases.

UNLABELLED: Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus HA for viral activation and infectivityin vitro Recently, we reported that inactivation of a single HA-activating protease gene,Tmprss2, in knockout mice inhibits the spread of H1N1 influenza viruses. However, after infection ofTmprss2knockout mice with an H3N2 influenza virus, only a slight increase in survival was observed, and mice still lost body weight. In this study, we investigated an additional trypsin-like protease, TMPRSS4. Both TMPRSS2 and TMPRSS4 are expressed in the same cell types of the mouse lung. Deletion ofTmprss4alone in knockout mice does not protect them from body weight loss and death upon infection with H3N2 influenza virus. In contrast,Tmprss2(-/-)Tmprss4(-/-)double-knockout mice showed a remarkably reduced virus spread and lung pathology, in addition to reduced body weight loss and mortality. Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virusin vivo IMPORTANCE: Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality. Due to high variability of the virus genome, resistance to available antiviral drugs is frequently observed, and new targets for treatment of influenza are needed. Host cell factors essential for processing of the virus hemagglutinin represent very suitable drug targets because the virus is dependent on these host factors for replication. We reported previously thatTmprss2-deficient mice are protected against H1N1 virus infections, but only marginal protection against H3N2 virus infections was observed. Here we show that deletion of two host protease genes,Tmprss2andTmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection. Thus, TMPRSS4 represents another host cell factor that is involved in cleavage activation of H3N2 influenza virusesin vivo.

Macroautophagic process was differentially modulated by long-term moderate exercise in rat brain and peripheral tissues

The autophagic process is a lysosomal degradation pathway, which is activated during stress conditions, such as starvation or exercise. Regular exercise has beneficial effects on human health, including neuroprotection. However, the cellular mechanisms underlying these effects are incompletely understood. Endurance and a single bout of exercise induce autophagy not only in brain but also in peripheral tissues. However, little is known whether autophagy could be modulated in brain and peripheral tissues by long-term moderate exercise. Here, we examined the effects on macroautophagy process of long-term moderate treadmill training (36 weeks) in adult rats both in brain (hippocampus and cerebral cortex) and peripheral tissues (skeletal muscle, liver and heart). We assessed mTOR activation and the autophagic proteins Beclin 1, p62, LC3B (LC3B-II/LC3B-I ratio) and the lysosomal protein LAMP1, as well as the ubiquitinated proteins. Our results showed in the cortex of exercised rats an inactivation of mTOR, greater autophagy flux (increased LC3-II/LC3-I ratio and reduced p62) besides increased LAMP1. Related with these effects a reduction in the ubiquitinated proteins was observed. No significant changes in the autophagic pathway were found either in hippocampus or in skeletal and cardiac muscle by exercise. Only in the liver of exercised rats mTOR phosphorylation and p62 levels increased, which could be related with beneficial metabolic effects in this organ induced by exercise. Thus, our findings suggest that long-term moderate exercise induces autophagy specifically in the cortex

Macroautophagic process was differentially modulated by long-term moderate exercise in rat brain and peripheral tissues

The autophagic process is a lysosomal degradation pathway, which is activated during stress conditions, such as starvation or exercise. Regular exercise has beneficial effects on human health, including neuroprotection. However, the cellular mechanisms underlying these effects are incompletely understood. Endurance and a single bout of exercise induce autophagy not only in brain but also in peripheral tissues. However, little is known whether autophagy could be modulated in brain and peripheral tissues by long-term moderate exercise. Here, we examined the effects on macroautophagy process of long-term moderate treadmill training (36 weeks) in adult rats both in brain (hippocampus and cerebral cortex) and peripheral tissues (skeletal muscle, liver and heart). We assessed mTOR activation and the autophagic proteins Beclin 1, p62, LC3B (LC3B-II/LC3B-I ratio) and the lysosomal protein LAMP1, as well as the ubiquitinated proteins. Our results showed in the cortex of exercised rats an inactivation of mTOR, greater autophagy flux (increased LC3-II/LC3-I ratio and reduced p62) besides increased LAMP1. Related with these effects a reduction in the ubiquitinated proteins was observed. No significant changes in the autophagic pathway were found either in hippocampus or in skeletal and cardiac muscle by exercise. Only in the liver of exercised rats mTOR phosphorylation and p62 levels increased, which could be related with beneficial metabolic effects in this organ induced by exercise. Thus, our findings suggest that long-term moderate exercise induces autophagy specifically in the cortex