Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles

We previously reported that the incorporation of tissue factor (TF) into cell-derived microvesicles (MVs) is regulated by the phosphorylation of the cytoplasmic domain of TF. Since the cytoskeletal protein filamin-A is known to bind to the cytoplasmic domain of TF in a phosphorylation-dependent manner, the involvement of filamin-A in the incorporation of TF into MVs was examined. Endothelial cells were transfected to express TF, whereas MDA-MB-231 cells were used to examine endogenously expressed TF. MV release was induced by activating protease-activated receptor-2 (PAR2). Partial suppression of filamin-A expression using two different filamin-A siRNA sequences resulted in significant reductions in the incorporation of TF antigen into MVs as determined by TF-ELISA and western blot analysis, and was reflected in reduced thrombin-generation and FXa-generation capacities of these MVs. Deletion of the cytoplasmic domain of TF also resulted in reduced incorporation of TF into MVs, whereas the suppression of filamin-A expression had no additional effect on the incorporation of truncated TF into MVs. Partial suppression of filamin-A expression had no effect on the number and size distribution of the released MVs. However, >90 % suppression of filamin-A expression resulted in increased MV release, possibly as a result of increased instability of the plasma membrane and underlying cytoskeleton. In conclusion, the presence of filamin-A appears to be essential for the incorporation of TF into MVs following PAR2 activation, but is not required for the process of MV formation and release following PAR2 activation

Accumulation of tissue factor in endothelial cells induces cell apoptosis, mediated through p38 and p53 activation

We previously reported that high levels of tissue factor (TF) can induce cellular apoptosis in endothelial. In this study, TF-mediated mechanisms of induction of apoptosis were explored. Endothelial cells were transfected to express wild-type TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued TF to enhance or prevent TF release respectively. Alternatively, cells were pre-incubated with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein were only increased in cells pre-incubated with TF-rich microvesicle and in cells expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53 using pifithrin-α suppressed the expression of Bax. Finally, siRNA–mediated suppression of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels, p53 nuclear localisation and transcriptional activity, suppressed Bax expression and prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial cell, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53

Evaluation of a Web-Based Research Course

A web-based research course for graduate nursing students was developed, implemented, and evaluated for academic and satisfactions outcomes. A collaborative paradigm was utilized in designing the course to increase successful attainment of the graduate nursing program’s goal of a complete online graduate nursing MSN program. This web-based research course with 24 enrolled students was compared to an identical classroom based research course with 20 enrolled students. Areas of evaluation were academic outcomes of overall numerical course grades, satisfaction with course scores, and qualitative data on satisfaction of course. Results of this descriptive exploratory study demonstrated no statistically significant differences between the academic outcomes of these two groups of students. Both groups were satisfied with the course, however there was a statistically significant difference in mean satisfaction scores for the research courses. Further investigation of environmental factors will need to be done to determine the significance of differences in satisfaction outcomes

STD observations in the southwest Atlantic from cruise 16, leg 9 of the R/V Conrad and cruise 7-75 of the Ara Islas Orcadas

Includes Errata page dated 1 February 1980Continuous salinity-temperature-depth (STD) data from two cruises in the South Atlantic are presented in both tabular and graphic form. Thirty-seven of the stations were made during the R/V CONRAD cruise 16, leg 9, March 29 to April 23, 1973 and 20 stations on the ARA ISLAS ORCADAS cruise 7-75, October 30 to December 20, 1975

Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation

The planar cell polarity (PCP) pathway is conserved throughout evolution, but it mediates distinct developmental processes. In Drosophila, members of the PCP pathway localize in a polarized fashion to specify the cellular polarity within the plane of the epithelium, perpendicular to the apicobasal axis of the cell. In Xenopus and zebrafish, several homologs of the components of the fly PCP pathway control convergent extension. We have shown previously that mammalian PCP homologs regulate both cell polarity and polarized extension in the cochlea in the mouse. Here we show, using mice with null mutations in two mammalian Dishevelled homologs, Dvl1 and Dvl2, that during neurulation a homologous mammalian PCP pathway regulates concomitant lengthening and narrowing of the neural plate, a morphogenetic process defined as convergent extension. Dvl2 genetically interacts with Loop-tail, a point mutation in the mammalian PCP gene Vangl2, during neurulation. By generating Dvl2 BAC (bacterial artificial chromosome) transgenes and introducing different domain deletions and a point mutation identical to the dsh1 allele in fly, we further demonstrated a high degree of conservation between Dvl function in mammalian convergent extension and the PCP pathway in fly. In the neuroepithelium of neurulating embryos, Dvl2 shows DEP domain-dependent membrane localization, a pre-requisite for its involvement in convergent extension. Intriguing, the Loop-tail mutation that disrupts both convergent extension in the neuroepithelium and PCP in the cochlea does not disrupt Dvl2 membrane distribution in the neuroepithelium, in contrast to its drastic effect on Dvl2 localization in the cochlea. These results are discussed in light of recent models on PCP and convergent extension

Descriptive Profiles of the MMPI-2-Restructured Form (MMPI-2-RF) across a National Sample of Four Veteran Affairs Treatment Settings

This investigation provides descriptive information on substantive scale scores from the Minnesota Multiphasic Personality Inventory-2-Restructured Form (MMPI-2-RF) across four common service locations within Veterans Affairs (VA): PTSD clinical team, individual substance use treatment, primary medical care, and residential polytrauma rehabilitation. Test protocols for these four service settings are drawn from a national sample of all MMPI-2-RF and converted MMPI-2 administrations between January 1, 2008 and May 31, 2015 using the VA Mental Health Assist system at any VA across the United States. Frequency of substantive scale elevation and descriptive findings are examined. Results of this investigation suggest that there are differences between VA service locations on the MMPI-2-RF substantive scales, the magnitude of difference depends on the substantive scale examined, and the pattern of elevation within service location follows common clinical concerns for the settings. Implications for the clinical use, and research with, the MMPI-2-RF within the VA and with the veteran population are discussed. The views expressed in this manuscript do not reflect those of the Department of Veteran Affairs or of the United States Government

Patterns of MMPI-2-Restructured Form (MMPI-2-RF) Validity Scale Scores Observed Across Veteran Affairs Settings

The purpose of this investigation is to provide descriptive information on veteran response styles for a variety of VA referral types using the Minnesota Multiphasic Personality Inventory (MMPI)-2- Restructured Form (MMPI-2-RF), which has well-supported protocol validity scales. The sample included 17,640 veterans who were administered the MMPI-2-RF between when it was introduced to the VA system in 2013 until May 31, 2015 at any VA in the United States. This study examines frequencies of protocol invalidity based on the MMPI-2-RF’s validity scales and provides comprehensive descriptive findings on validity scale scores within the VA. Three distinct trends can be seen. First, a majority of the sample did not elevate any of the validity scales beyond their recommended interpretive cut-scores, indicating that scores on the substantive scales would be deemed valid and interpretable in those cases. Second, elevation rates are higher for the overreporting scales in comparison to the underreporting and non-content-based invalid responding scales. Lastly, a majority of those with an elevation on one overreporting validity indicator also had an elevation on at least one other overreporting scale. Implications for practice and the utility of the MMPI-2-RF within the VA are discussed

Investigation of the filamin A-Dependent mechanisms of tissue factor incorporation into microvesicles

We have previously shown that phosphorylation of tissue factor (TF) at Ser253 increases the incorporation of TF into microvesicles (MVs) following protease-activated receptor 2 (PAR2) activation through a process involving filamin-A, whereas Ser258 phosphorylation suppresses this process. Here we examined the contribution of the individual phosphorylation of these serine residues to the interaction between filamin-A and TF, and further examined how filamin-A regulates the incorporation of TF into MVs. In vitro binding assays using recombinant filamin-A C-terminal repeats 22-24 with biotinylated phospho-TF cytoplasmic domain peptides as bait, showed that filamin-A had the highest binding affinities for phospho-Ser253 and double-phosphorylated TF peptides, whilst the phospho-Ser258 TF peptide had the lowest affinity. Analysis of MDA-MB-231 cells using an in situ proximity ligation assay revealed increased proximity between the C-terminus of filamin-A and TF following PAR2 activation, which was concurrent with Ser253 phosphorylation and TF-positive MV release from these cells. Knock-down of filamin-A expression suppressed PAR2-mediated increases in cell surface TF procoagulant activity without reducing cell surface TF antigen expression. Disrupting lipid rafts by pre-incubation with methyl-beta cyclodextrin (MβCD) prior to PAR2 activation reduced TF-positive MV release and cell surface TF procoagulant activity to the same extent as filamin-A knock-down. In conclusion, this study shows that the interaction between TF and filamin-A is dependent on the differential phosphorylation of Ser253 and Ser258. Furthermore the interaction of TF with filamin-A may translocate cell surface TF to cholesterol-rich lipid rafts, increasing cell surface TF activity as well as TF incorporation and release into MVs