Enhanced insulin sensitivity associated with provision of mono and polyunsaturated fatty acids in skeletal muscle cells involves counter modulation of PP2A

International audienceAims/Hypothesis: Reduced skeletal muscle insulin sensitivity is a feature associated with sustained exposure to excess saturated fatty acids (SFA), whereas mono and polyunsaturated fatty acids (MUFA and PUFA) not only improve insulin sensitivity but blunt SFA-induced insulin resistance. The mechanisms by which MUFAs and PUFAs institute these favourable changes remain unclear, but may involve stimulating insulin signalling by counter-modulation/repression of protein phosphatase 2A (PP2A). This study investigated the effects of oleic acid (OA; a MUFA), linoleic acid (LOA; a PUFA) and palmitate (PA; a SFA) in cultured myotubes and determined whether changes in insulin signalling can be attributed to PP2A regulation. Principal Findings: We treated cultured skeletal myotubes with unsaturated and saturated fatty acids and evaluated insulin signalling, phosphorylation and methylation status of the catalytic subunit of PP2A. Unlike PA, sustained incubation of rat or human myotubes with OA or LOA significantly enhanced Akt-and ERK1/2-directed insulin signalling. This was not due to heightened upstream IRS1 or PI3K signalling nor to changes in expression of proteins involved in proximal insulin signalling, but was associated with reduced dephosphorylation/inactivation of Akt and ERK1/2. Consistent with this, PA reduced PP2Ac demethylation and tyrosine 307 phosphorylation-events associated with PP2A activation. In contrast, OA and LOA strongly opposed these PA-induced changes in PP2Ac thus exerting a repressive effect on PP2A.Conclusions/Interpretation: Beneficial gains in insulin sensitivity and the ability of unsaturated fatty acids to oppose palmitate-induced insulin resistance in muscle cells may partly be accounted for by counter-modulation of PP2A

The use of full-setting non-invasive ventilation in the home care of people with amyotrophic lateral sclerosis-motor neuron disease with end-stage respiratory muscle failure: a case series

<p>Abstract</p> <p>Introduction</p> <p>Little has been written about the use of non-invasive ventilation in the home care of amyotrophic lateral sclerosis-motor neuron disease patients with end-stage respiratory muscle failure. Nocturnal use of non-invasive ventilation has been reported to improve daytime blood gases but continuous non-invasive ventilation dependence has not been studied in this regard. There continues to be great variation by country, economics, physician interest and experience, local concepts of palliation, hospice requirements, and resources available for home care. We report a case series of home-based amyotrophic lateral sclerosis-motor neuron disease patients who refused tracheostomy and advanced non-invasive ventilation to full-setting, while maintaining normal alveolar ventilation and oxygenation in the course of the disease. Since this topic has been presented in only one center in the United States and nowhere else, it is appropriate to demonstrate that this can be done in other countries as well.</p> <p>Case presentation</p> <p>We present here the cases of three Caucasian patients (a 51-year-old Caucasian man, a 45-year-old Caucasian woman and a 57-year-old Caucasian woman) with amyotrophic lateral sclerosis who developed continuous non-invasive ventilation dependence for 15 to 27 months without major complications and were able to maintain normal CO₂and pulse oxyhemoglobin saturation despite a non-measurable vital capacity. All patients were wheelchair-dependent and receiving riluzole 50 mg twice a day. Patient one developed mild-to-moderate bulbar-innervated muscle weakness. He refused tracheostomy but accepted percutaneous gastrostomy. Patient two had two lung infections, acute bronchitis and pneumonia, which were treated with antibiotics and cough assistance at home. Patient three had three chest infections (bronchitis and pneumonias) and asthmatic episodes treated with antibiotics, bronchodilators and cough assistance at home. All patients had normal speech while receiving positive pressure; they died suddenly and with normal oxygen saturation.</p> <p>Conclusions</p> <p>Although warned that prognosis was poor as vital capacity diminished, our patients survived without invasive airway tubes and despite non-measurable vital capacity. No patient opted for tracheostomy. Our patients demonstrate the feasibility of resorting to full-setting non-invasive management to prolong survival, optimizing wellness and management at home, and the chance to die peacefully.</p

Characterising the inhibitory actions of ceramide upon insulin signaling in different skeletal muscle cell models:a mechanistic insight

International audienceCeramides are known to promote insulin resistance in a number of metabolically important tissues including skeletal muscle, the predominant site of insulin-stimulated glucose disposal. Depending on cell type, these lipid intermediates have been shown to inhibit protein kinase B (PKB/Akt), a key mediator of the metabolic actions of insulin, via two distinct pathways: one involving the action of atypical protein kinase C (aPKC) isoforms, and the second dependent on protein phosphatase-2A (PP2A). The main aim of this study was to explore the mechanisms by which ceramide inhibits PKB/Akt in three different skeletal muscle-derived cell culture models; rat L6 myotubes, mouse C2C12 myotubes and primary human skeletal muscle cells. Our findings indicate that the mechanism by which ceramide acts to repress PKB/Akt is related to the myocellular abundance of caveolin-enriched domains (CEM) present at the plasma membrane. Here, we show that ceramide-enriched-CEMs are markedly more abundant in L6 myotubes compared to C2C12 myotubes, consistent with their previously reported role in coordinating aPKC-directed repression of PKB/Akt in L6 muscle cells. In contrast, a PP2A-dependent pathway predominantly mediates ceramide-induced inhibition of PKB/Akt in C2C12 myotubes. In addition, we demonstrate for the first time that ceramide engages an aPKC-dependent pathway to suppress insulin-induced PKB/Akt activation in palmitate-treated cultured human muscle cells as well as in muscle cells from diabetic patients. Collectively, this work identifies key mechanistic differences, which may be linked to variations in plasma membrane composition, underlying the insulin-desensitising effects of ceramide in different skeletal muscle cell models that are extensively used in signal transduction and metabolic studies

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium

Phospholipase D inhibitors reduce human prostate cancer cell proliferation and colony formation

BACKGROUND: Phospholipases D1 and D2 (PLD1/2) hydrolyse cell membrane glycerophospholipids to generate phosphatidic acid, a signalling lipid, which regulates cell growth and cancer progression through effects on mTOR and PKB/Akt. PLD expression and/or activity is raised in breast, colorectal, gastric, kidney and thyroid carcinomas but its role in prostate cancer (PCa), the major cancer of men in the western world, is unclear. METHODS: PLD1 protein expression in cultured PNT2C2, PNT1A, P4E6, LNCaP, PC3, PC3M, VCaP, 22RV1 cell lines and patient-derived PCa cells was analysed by western blotting. PLD1 protein localisation in normal, benign prostatic hyperplasia (BPH), and castrate-resistant prostate cancer (CRPC) tissue sections and in a PCa tissue microarray (TMA) was examined by immunohistochemistry. PLD activity in PCa tissue was assayed using an Amplex Red method. The effect of PLD inhibitors on PCa cell viability was measured using MTS and colony forming assays. RESULTS: PLD1 protein expression was low in the luminal prostate cell lines (LNCaP, VCaP, 22RV1) compared with basal lines (PC3 and PC3M). PLD1 protein expression was elevated in BPH biopsy tissue relative to normal and PCa samples. In normal and BPH tissue, PLD1 was predominantly detected in basal cells as well in some stromal cells, rather than in luminal cells. In PCa tissue, luminal cells expressed PLD1. In a PCa TMA, the mean peroxidase intensity per DAB-stained Gleason 6 and 7 tissue section was significantly higher than in sections graded Gleason 9. In CRPC tissue, PLD1 was expressed prominently in the stromal compartment, in luminal cells in occasional glands and in an expanding population of cells that co-expressed chromogranin A and neurone-specific enolase. Levels of PLD activity in normal and PCa tissue samples were similar. A specific PLD1 inhibitor markedly reduced the survival of both prostate cell lines and patient-derived PCa cells compared with two dual PLD1/PLD2 inhibitors. Short-term exposure of PCa cells to the same specific PLD1 inhibitor significantly reduced colony formation. CONCLUSIONS: A new specific inhibitor of PLD1, which is well tolerated in mice, reduces PCa cell survival and thus has potential as a novel therapeutic agent to reduce prostate cancer progression. Increased PLD1 expression may contribute to the hyperplasia characteristic of BPH and in the progression of castrate-resistant PCa, where an expanding population of neuroendocrine-like cells express PLD1.British Journal of Cancer advance online publication, 14 November 2017; doi:10.1038/bjc.2017.391 www.bjcancer.com

O Governo aberto na cidade de Córdoba, Argentina: uma política de regulação da sensibilidade

This article aims at analyzing the meanings of the so-called open government (OG) from a communication approach articulated with social semiotics tools. Our hypothesis points out the impossibility to study these policies isolated from the urban social context where they are developed, making up a type of city and the citizens for such city. Our theoretical-methodological strategy is focused on approaching the OG as a sensitivity regulation device that establishes new ways of social control. The analysis will be conducted on the city of Córdoba, Argentina, whose urban population matrix and evaluating ideological environment legitimize these OG policies. Thus, from initially reconstructing the OG, we will try to understand its configuration and operation by setting up collective political and action scenes, creating a particular enunciation and visibility regime, establishing some possible practices and relationships, and defining a specific subjective type. The corpus of this study will consist of heterogeneous discursive practices: local media journalistic news and articles; municipal communications through official channels; and the OG website, the Portal de la Ciudad platform. The research period ranges from the launching of the municipal website in 2016 to its relaunch in 2018.Este artículo propone un estudio de los sentidos producidos sobre aquello que se reconoce como gobierno abierto (GA), desde un enfoque comunicacional articulado con herramientas provenientes de la sociosemiótica. Nuestra hipótesis señala que no es posible investigar estas políticas aisladas del contexto sociourbano en el que se despliegan, configurando un tipo de ciudad y un sujeto para dicha ciudad. Nuestra estrategia teórico-metodológica se centra en abordar al GA como un dispositivo de regulación de la sensibilidad que funda nuevos modos de control social. El análisis se desarrollará sobre el caso de la ciudad de Córdoba, Argentina, cuya matriz urbano-poblacional y medio ideológico evaluante legitiman estas políticas de GA. Así, intentaremos, a partir de reconstruir el dispositivo de GA, comprender las líneas que lo conforman y que operan instaurando los horizontes políticos y de acción colectivos, creando un particular régimen de enunciación y visibilidad; estableciendo ciertas prácticas y relaciones posibles; y definiendo un específico tipo subjetivo. Este estudio se realizará sobre un corpus constituido por prácticas discursivas heterogéneas: notas periodísticas de medios locales, comunicaciones del gobierno municipal por canales oficiales y la propia plataforma de GA, el Portal de la Ciudad. El período de investigación comprende desde el inicio del sitio municipal, en el año 2016, hasta su relanzamiento en el 2018.Este artigo propõe um estudo dos sentidos produzidos sobre aquilo que se reconhece como Governo Aberto (GA) com foco na comunicação articulada com ferramentas da socio-semiótica. Nossa hipótese indica que não é possível pesquisar essas políticas isoladas do contexto sócio-urbano em que estão implantadas, performando um tipo de cidade e um sujeito para a referida cidade. Nossa estratégia teórico metodológica centra-se na abordagem do GA como um dispositivo de regulação da sensibilidade que estabelece novos modos de controle social. A análise será desenvolvida no caso da cidade de Córdoba, Argentina, cuja matriz urbano-populacional e meio ideológico avaliativo legitimam essas políticas da GA. Assim, tentaremos compreender, a partir da reconstrução do dispositivo GA, as linhas que o configuram e que operam caracterizando os horizontes políticos e a ação coletiva, criando um regime particular de enunciação e visibilidade; estabelecendo determinadas práticas e relacionamentos possíveis; e definindo um tipo subjetivo específico. Este estudo será realizado sobre um corpus constituído por práticas discursivas heterogêneas: notas jornalísticas da mídia local; comunicações do governo municipal por meio de canais oficiais e plataforma própria da GA, o Portal da Cidade. O período de pesquisa vai desde o início do site municipal em 2016, até o seu relançamento em 2018

Molecular Lesions Associated with Loss of Heterozygosity Identified in CMML

Abstract Abstract 416 Chronic myelomonocytic leukemia (CMML) is characterized by monocyte/monoblast proliferation, dysplastic features, progression to leukemia and poor prognosis. With the exception of rare cases of CMML with balanced PDGFRa or PDGFRb translocations, the molecular pathogenesis of the majority of CMML cases remains elusive. To date, somatic mutations pathogenic for CMML have not been identified, and large tyrosine kinase sequencing projects have been negative. The recent application of SNP-A as a karyotyping tool has allowed for more precise analyses of chromosomal defects and detection of copy-neutral loss of heterozygosity (CN-LOH). Previously, various recurrent areas of CN-LOH have been found in myeloid malignancies, but these lesions were particularly frequent in CMML. We have examined 68 patients with CMML and AML with antecedent CMML for recurrent areas of LOH, mapped mutations in genes contained within corresponding minimally affected regions, and examined their impact on clinical outcomes. We focused on Cbl, TET2 and RAS mutations and the resultant clinical and pathomorphologic phenotypes and outcomes associated with individual mutations. SNP-A-based karyotyping showed the presence of chromosomal aberrations in a larger proportion of cases than did metaphase cytogenetics: recurrent areas of somatic UPD were found in 49% of patients, with the most common shared lesions including UPD4q (n=6), UPD7q (n=6) and UPD11q (n=4). Initial identification of recurrent UPD11q and UPD4q and micro-deletions defining the smallest commonly affected areas led to the discovery of new mutations in CMML, including c-Cbl and TET2. In sum, we identified c-Cbl in 13%, TET2 in 49%, and RAS mutations in 10% of patients with CMML and sAML derived from CMML. LOH involving chromosome 7 was present in 18% of patients. All c-Cbl mutations were somatic and in the RING finger domain or linker sequence; 3/6 mutations were homozygous and one case with del11q harbored a hemizygous mutation. TET2 alterations were identified in 23 patients; in 26% of patients, both alleles were affected. However, no clinical differences were found between heterozygous and homozygous cases of TET2 mutations, suggesting a dominant negative effect. We also identified patients who harbored both TET2 and c-Cbl or RAS mutations demonstrating a multi-step pathogenesis of CMML. In addition, aberrant methylation at the CpG islands of promoters of TET2 (2 sites), c-Cbl (2 sites), b-Cbl (2 sites) and RAS (7 sites) was found to be relatively infrequent among patients. Intricate analysis of clinical and phenotypic features did not reveal pathognomonic phenotypes; however TET2 and c-Cbl mutations were associated with higher WBC (p=.027) or thrombocytopenia (p=.026), respectively and RAS mutations were associated with advanced disease (p=.027). Patients with LOH7q were included in our analysis as a separate group because they likely carried mutations in a putative candidate gene on 7q. In general, megakaryocyte nuclei displayed aberrant STAT5 staining in 31% of CMML cases (n=32) and correlated with morphologic dysplasia. Aberrant pSTAT5 staining suggestive of downstream pathways involving proliferative signals was present in 43% of TET2 mutated patients and 67% of c-Cbl mutants. None of the RAS mutants displayed aberrant STAT5 staining. Thus, aberrant STAT5 activation is not an obligatory feature of TET2 and RAS mutant cases, as opposed to the majority of c-Cbl mutant cases. There was no significant difference in survival between patients with TET2, c-Cbl and RAS mutations or those with LOH7q as compared to those without. Nevertheless, TET2 and c-Cbl mutant cases trended towards less favorable survival within lower-risk (<5% blasts) CMML, while no differences were seen in advanced cases, likely due to the uniformly poor survival in this group. Regardless of the presence of putative mutations, patients with LOH7q, including UPD7, shared a poor prognosis (7 vs. 11mo). In sum, our results characterize CMML as a disorder frequently associated with acquired chromosomal lesions, activated STAT5 signaling, genetic mutations in RAS, ubiquitin modification and TET2 pathways. Mutations present in these patients may help to clarify pathogenic pathways, and thereby develop directed therapies. Disclosures: No relevant conflicts of interest to declare

Risk factors for amyotrophic lateral sclerosis: A regional United States case-control study

Most amyotrophic lateral sclerosis (ALS) cases are considered sporadic, without a known genetic basis, and environmental exposures are thought to play a causal role. To learn more about sporadic ALS etiology, we recruited n = 188 ALS patients from northern New England and Ohio and matched controls 2:1 from the general population of the same regions. Questionnaires evaluated the association between a variety of lifestyle, behavioral (ie, hobbies and activities), and occupational factors and the risk of ALS, including the duration of time between exposure and ALS onset, and exposure frequency. Head trauma was associated with increased ALS risk (adjusted odds ratio [OR] 1.60 95% confidence interval [CI] 1.04-2.45), with significantly greater effects for injuries occurring 10 or more years prior to symptom onset (P = .037). ALS risk was increased for those reporting severe electrical burns (adjusted OR 2.86, 95% CI 1.37-6.03), with odds ratios highest for burns after age 30 (OR 3.14), and for burns 10 or more years prior to symptom onset (OR 3.09). Hobbies involving lead were the most strongly associated with ALS risk (adjusted OR 2.92, 95% CI 1.45-5.91). Exposures to lead 20 or more years prior to diagnosis had larger effect sizes compared to those occurring more recently. Holding a job in mechanics, painting, or construction was associated with ALS. The identification of these specific environmental factors associated with ALS highlight the need for future prospective and laboratory studies to assess causality, biological mechanisms, and find prevention or treatment opportunities