121 research outputs found

    Patient-derived mutations within the N-terminal domains of p85α impact PTEN or Rab5 binding and regulation

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    The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activit

    Psychometric properties of two physical activity questionnaires, the AQuAA and the PASE, in cancer patients

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    Background: This study aimed to evaluate the reliability and validity of two self-report physical activity (PA) questionnaires - the AQuAA (Activity Questionnaire for Adults and Adolescents) and PASE (Physical Activity Scale for the Elderly) - in cancer patients. Methods: Test-retest reliability was determined by administering the questionnaires twice within 5 days. Intraclass correlation coefficient (ICC), standard error of measurement (SEM) and smallest detectable difference (SDD) were calculated. Construct validity was determined by comparing the questionnaire results with ActiGraph accelerometer scores using Spearman correlation coefficients (r(s)) and ICCs. Content validity was examined using the Three-Step Test-Interview (TSTI). Results: Reliability for the AQuAA scores were fair to excellent (ICC = 0.57 to 0.78). Reliability for the PASE scores ranged from good to excellent (ICC = 0.67 to 0.90). Correlations between the ActiGraph and the AQuAA and the PASE were low (r(s) = 0.05 and 0.16 respectively, and ICC = -0.001 to 0.44). The TSTI showed that participants experienced difficulties with the examples provided with the questions, the perceptions of intensity level of PA, and with recalling the time spent on PA. Conclusions: Both questionnaires showed good to excellent test-retest reliability for most scores. Construct validity of both questionnaires was low, as indicated by the low correlations with the ActiGraph. Except for a few difficulties that participants perceived when filling out the questionnaires, the content validity of both questionnaires was goo

    Does Foreign Direct Investment Stimulate New Firm Creation? In Search of Spillovers through Industrial and Geographical Linkages

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    This paper examines the spillover effects of inward foreign direct investment (FDI) on the entrepreneurial activities of new firm creation through both industrial and geographical linkages. Using a dataset of 44,434 newly created small firms in 234 regions of South Korea in 2000–2004, this study finds that while the spillover impacts of FDI in the low-tech industry are positive and significant across almost all four possible combinations of the intra-/inter-regional and intra-/inter-sectoral channels, the impacts in the high-tech industry are largely intra-sectoral within the host region and across neighboring regions. Moreover, all statistically significant spillover effects follow an inverted ‘U’-shaped curvilinear trend

    L-Type Ca2+ Channel Function Is Linked to Dystrophin Expression in Mammalian Muscle

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    BACKGROUND: In dystrophic mdx skeletal muscle, aberrant Ca2+ homeostasis and fibre degeneration are found. The absence of dystrophin in models of Duchenne muscular dystrophy (DMD) has been connected to altered ion channel properties e.g. impaired L-type Ca2+ currents. In regenerating mdx muscle, 'revertant' fibres restore dystrophin expression. Their functionality involving DHPR-Ca2+-channels is elusive. METHODS AND RESULTS: We developed a novel 'in-situ' confocal immuno-fluorescence and imaging technique that allows, for the first time, quantitative subcellular dystrophin-DHPR colocalization in individual, non-fixed, muscle fibres. Tubular DHPR signals alternated with second harmonic generation signals originating from myosin. Dystrophin-DHPR colocalization was substantial in wt fibres, but diminished in most mdx fibres. Mini-dystrophin (MinD) expressing fibres successfully restored colocalization. Interestingly, in some aged mdx fibres, colocalization was similar to wt fibres. Most mdx fibres showed very weak membrane dystrophin staining and were classified 'mdx-like'. Some mdx fibres, however, had strong 'wt-like' dystrophin signals and were identified as 'revertants'. Split mdx fibres were mostly 'mdx-like' and are not generally 'revertants'. Correlations between membrane dystrophin and DHPR colocalization suggest a restored putative link in 'revertants'. Using the two-micro-electrode-voltage clamp technique, Ca2+-current amplitudes (i(max)) showed very similar behaviours: reduced amplitudes in most aged mdx fibres (as seen exclusively in young mdx mice) and a few mdx fibres, most likely 'revertants', with amplitudes similar to wt or MinD fibres. Ca2+ current activation curves were similar in 'wt-like' and 'mdx-like' aged mdx fibres and are not the cause for the differences in current amplitudes. i(max) amplitudes were fully restored in MinD fibres. CONCLUSIONS: We present evidence for a direct/indirect DHPR-dystrophin interaction present in wt, MinD and 'revertant' mdx fibres but absent in remaining mdx fibres. Our imaging technique reliably detects single isolated 'revertant' fibres that could be used for subsequent physiological experiments to study mechanisms and therapy concepts in DMD

    A Genetically Hard-Wired Metabolic Transcriptome in Plasmodium falciparum Fails to Mount Protective Responses to Lethal Antifolates

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    Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites

    Fluid challenges in intensive care: the FENICE study A global inception cohort study

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    Fluid challenges (FCs) are one of the most commonly used therapies in critically ill patients and represent the cornerstone of hemodynamic management in intensive care units. There are clear benefits and harms from fluid therapy. Limited data on the indication, type, amount and rate of an FC in critically ill patients exist in the literature. The primary aim was to evaluate how physicians conduct FCs in terms of type, volume, and rate of given fluid; the secondary aim was to evaluate variables used to trigger an FC and to compare the proportion of patients receiving further fluid administration based on the response to the FC.This was an observational study conducted in ICUs around the world. Each participating unit entered a maximum of 20 patients with one FC.2213 patients were enrolled and analyzed in the study. The median [interquartile range] amount of fluid given during an FC was 500 ml (500-1000). The median time was 24 min (40-60 min), and the median rate of FC was 1000 [500-1333] ml/h. The main indication for FC was hypotension in 1211 (59 %, CI 57-61 %). In 43 % (CI 41-45 %) of the cases no hemodynamic variable was used. Static markers of preload were used in 785 of 2213 cases (36 %, CI 34-37 %). Dynamic indices of preload responsiveness were used in 483 of 2213 cases (22 %, CI 20-24 %). No safety variable for the FC was used in 72 % (CI 70-74 %) of the cases. There was no statistically significant difference in the proportion of patients who received further fluids after the FC between those with a positive, with an uncertain or with a negatively judged response.The current practice and evaluation of FC in critically ill patients are highly variable. Prediction of fluid responsiveness is not used routinely, safety limits are rarely used, and information from previous failed FCs is not always taken into account

    The transcriptome of Candida albicans mitochondria and the evolution of organellar transcription units in yeasts

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