Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER+ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context

Bcl-2 protein family: Implications in vascular apoptosis and atherosclerosis

Apoptosis has been recognized as a central component in the pathogenesis of atherosclerosis, in addition to the other human pathologies such as cancer and diabetes. The pathophysiology of atherosclerosis is complex, involving both apoptosis and proliferation at different phases of its progression. Oxidative modification of lipids and inflammation differentially regulate the apoptotic and proliferative responses of vascular cells during progression of the atherosclerotic lesion. Bcl-2 proteins act as the major regulators of extrinsic and intrinsic apoptosis signalling pathways and more recently it has become evident that they mediate the apoptotic response of vascular cells in response to oxidation and inflammation either in a provocative or an inhibitory mode of action. Here we address Bcl-2 proteins as major therapeutic targets for the treatment of atherosclerosis and underscore the need for the novel preventive and therapeutic interventions against atherosclerosis, which should be designed in the light of molecular mechanisms regulating apoptosis of vascular cells in atherosclerotic lesions

Structural Elucidation and Functional Characterization of the Hyaloperonospora arabidopsidis Effector Protein ATR13

The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew on the model plant Arabidopsis thaliana and has been adapted as a model system to investigate pathogen virulence strategies and plant disease resistance mechanisms. Recognition of Hpa infection occurs when plant resistance proteins (R-genes) detect the presence or activity of pathogen-derived protein effectors delivered to the plant host. This study examines the Hpa effector ATR13 Emco5 and its recognition by RPP13-Nd, the cognate R-gene that triggers programmed cell death (HR) in the presence of recognized ATR13 variants. Herein, we use NMR to solve the backbone structure of ATR13 Emco5, revealing both a helical domain and a disordered internal loop. Additionally, we use site-directed and random mutagenesis to identify several amino acid residues involved in the recognition response conferred by RPP13-Nd. Using our structure as a scaffold, we map these residues to one of two surface-exposed patches of residues under diversifying selection. Exploring possible roles of the disordered region within the ATR13 structure, we perform domain swapping experiments and identify a peptide sequence involved in nucleolar localization. We conclude that ATR13 is a highly dynamic protein with no clear structural homologues that contains two surface-exposed patches of polymorphism, only one of which is involved in RPP13-Nd recognition specificity

A puzzle form of a non-verbal intelligence test gives significantly higher performance measures in children with severe intellectual disability

<p>Abstract</p> <p>Background</p> <p>Assessment of 'potential intellectual ability' of children with severe intellectual disability (ID) is limited, as current tests designed for normal children do not maintain their interest. Thus a <it>manual puzzle </it>version of the Raven's Coloured Progressive Matrices (RCPM) was devised to appeal to the attentional and sensory preferences and language limitations of children with ID. It was hypothesized that performance on the book and manual puzzle forms would not differ for typically developing children but that children with ID would perform better on the puzzle form.</p> <p>Methods</p> <p>The first study assessed the validity of this puzzle form of the RCPM for 76 typically developing children in a test-retest crossover design, with a 3 week interval between tests. A second study tested performance and completion rate for the puzzle form compared to the book form in a sample of 164 children with ID.</p> <p>Results</p> <p>In the first study, no significant difference was found between performance on the puzzle and book forms in typically developing children, irrespective of the order of completion. The second study demonstrated a significantly higher performance and completion rate for the puzzle form compared to the book form in the ID population.</p> <p>Conclusion</p> <p>Similar performance on book and puzzle forms of the RCPM by typically developing children suggests that both forms measure the same construct. These findings suggest that the puzzle form does not require greater cognitive ability but demands sensory-motor attention and limits distraction in children with severe ID. Thus, we suggest the puzzle form of the RCPM is a more reliable measure of the non-verbal mentation of children with severe ID than the book form.</p

Janus kinase 2 regulates Bcr–Abl signaling in chronic myeloid leukemia

Despite the success of imatinib mesylate (IM) in the early chronic phase of chronic myeloid leukemia (CML), patients are resistant to IM and other kinase inhibitors in the later stages of CML. Our findings indicate that inhibition of Janus kinase 2 (Jak2) in Bcr–Abl+ cells overcomes IM resistance although the precise mechanism of Jak2 action is unknown. Knocking down Jak2 in Bcr–Abl+ cells reduced levels of the Bcr–Abl protein and also the phosphorylation of Tyr177 of Bcr–Abl, and Jak2 overexpression rescued these knockdown effects. Treatment of Bcr–Abl+ cells with Jak2 inhibitors for 4–6 h but not with IM also reduced Bcr–Abl protein and pTyr177 levels. In vitro kinase experiments performed with recombinant Jak2 showed that Jak2 readily phosphorylated Tyr177 of Bcr–Abl (a Jak2 consensus site, YvnV) whereas c-Abl did not. Importantly, Jak2 inhibition decreased pTyr177 Bcr–Abl in immune complexes but did not reduce levels of Bcr–Abl, suggesting that the reduction of Bcr–Abl by Jak2 inhibition is a separate event from phosphorylation of Tyr177. Jak2 inhibition by chemical inhibitors (TG101209/WP1193) and Jak2 knockdown diminished the activation of Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5. These findings suggest that Bcr–Abl stability and oncogenic signaling in CML cells are under the control of Jak2

MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses

A Novel Classification of Lung Cancer into Molecular Subtypes

The remarkably heterogeneous nature of lung cancer has become more apparent over the last decade. In general, advanced lung cancer is an aggressive malignancy with a poor prognosis. The discovery of multiple molecular mechanisms underlying the development, progression, and prognosis of lung cancer, however, has created new opportunities for targeted therapy and improved outcome. In this paper, we define “molecular subtypes” of lung cancer based on specific actionable genetic aberrations. Each subtype is associated with molecular tests that define the subtype and drugs that may potentially treat it. We hope this paper will be a useful guide to clinicians and researchers alike by assisting in therapy decision making and acting as a platform for further study. In this new era of cancer treatment, the ‘one-size-fits-all’ paradigm is being forcibly pushed aside—allowing for more effective, personalized oncologic care to emerge

A Late Form of Nucleophagy in Saccharomyces cerevisiae

Autophagy encompasses several processes by which cytosol and organelles can be delivered to the vacuole/lysosome for breakdown and recycling. We sought to investigate autophagy of the nucleus (nucleophagy) in the yeast Saccharomyces cerevisiae by employing genetically encoded fluorescent reporters. The use of such a nuclear reporter, n-Rosella, proved the basis of robust assays based on either following its accumulation (by confocal microscopy), or degradation (by immunoblotting), within the vacuole. We observed the delivery of n-Rosella to the vacuole only after prolonged periods of nitrogen starvation. Dual labeling of cells with Nvj1p-EYFP, a nuclear membrane reporter of piecemeal micronucleophagy of the nucleus (PMN), and the nucleoplasm-targeted NAB35-DsRed.T3 allowed us to detect PMN soon after the commencement of nitrogen starvation whilst delivery to the vacuole of the nucleoplasm reporter was observed only after prolonged periods of nitrogen starvation. This later delivery of nuclear components to the vacuole has been designated LN (late nucleophagy). Only a very few cells showed simultaneous accumulation of both reporters (Nvj1p-EYFP and NAB35-DsRed.T3) in the vacuole. We determined, therefore, that delivery of the two respective nuclear reporters to the vacuole is temporally and spatially separated. Furthermore, our data suggest that LN is mechanistically distinct from PMN because it can occur in nvj1Δ and vac8Δ cells, and does not require ATG11. Nevertheless, a subset of the components of the core macroautophagic machinery is required for LN as it is efficiently inhibited in null mutants of several autophagy-related genes (ATG) specifying such components. Moreover, the inhibition of LN in some mutants is accompanied by alterations in nuclear morphology

Cognitive ability, parental socioeconomic position and internalising and externalising problems in adolescence: Findings from two European cohort studies

We investigated whether cognitive ability (CA) may be a moderator of the relationship of parental socioeconomic position (SEP) with internalising and externalising problems in adolescents. We used data from two longitudinal cohort studies; the Avon Longitudinal Study of Parents and Children (ALSPAC) and the Tracking Adolescents’ Individual Lives Survey (TRAILS). Indicators of SEP were mother’s education and household income. CA was estimated with IQ scores, derived from the Wechsler Intelligence Scale for Children. Internalising and externalising problems were measured with the Strengths and Difficulties Questionnaire in ALSPAC and with the Child Behavior Checklist in TRAILS. Logistic regression analyses were used to estimate the relative index of inequality (RII) for each outcome; the RII provides the odds ratio comparing the most to least deprived for each measure of SEP. In fully adjusted models an association of mother’s education with externalising problems was observed [ALSPAC RII 1.42 (95%CI: 1.01–1.99); TRAILS RII 2.21 (95%CI: 1.37–3.54)], and of household income with internalising and externalising problems [pooled ALSPAC & TRAILS internalising RII 1.30 (95%CI: 0.99–1.71); pooled ALSPAC & TRAILS externalising RII 1.38 (95%CI: 1.03–1.84)]. No consistent associations were observed between mother’s education and internalising problems. Results of stratified analyses and interaction-terms showed no evidence that CA moderated the association of SEP with internalising or externalising problems