Hakai reduces cell-substratum adhesion and increases epithelial cell invasion

[Abstract] Background. The dynamic regulation of cell-cell adhesions is crucial for developmental processes, including tissue formation, differentiation and motility. Adherens junctions are important components of the junctional complex between cells and are necessary for maintaining cell homeostasis and normal tissue architecture. E-cadherin is the prototype and best-characterized protein member of adherens junctions in mammalian epithelial cells. Regarded as a tumour suppressor, E-cadherin loss is associated with poor prognosis in carcinoma. The E3 ubiquitin-ligase Hakai was the first reported posttranslational regulator of the E-cadherin complex. Hakai specifically targetted E-cadherin for internalization and degradation and thereby lowered epithelial cell-cell contact. Hakai was also implicated in controlling proliferation, and promoted cancer-related gene expression by increasing the binding of RNA-binding protein PSF to RNAs encoding oncogenic proteins. We sought to investigate the possible implication of Hakai in cell-substratum adhesions and invasion in epithelial cells. Methods. Parental MDCK cells and MDCK cells stably overexpressing Hakai were used to analyse cell-substratum adhesion and invasion capabilities. Western blot and immunofluoresecence analyses were performed to assess the roles of Paxillin, FAK and Vinculin in cell-substratum adhesion. The role of the proteasome in controlling cell-substratum adhesion was studied using two proteasome inhibitors, lactacystin and MG132. To study the molecular mechanisms controlling Paxillin expression, MDCK cells expressing E-cadherin shRNA in a tetracycline-inducible manner was employed. Results. Here, we present evidence that implicate Hakai in reducing cell-substratum adhesion and increasing epithelial cell invasion, two hallmark features of cancer progression and metastasis. Paxillin, an important protein component of the cell-matrix adhesion, was completely absent from focal adhesions and focal contacts in Hakai-overexpressing MDCK cells. The expression of Paxillin was found to be regulated by a proteasome-independent mechanism, possibly due to the decreased abundance of E-cadherin. Conclusions. Taken together, these results suggest that Hakai may be involved in two hallmark aspects of tumour progression, the lowering cell-substratum adhesion and the enhancement of cell invasion.Xunta de Galicia; PS09/24Xunta de Galicia; 10CSA916023P

Effects of the information presentation format on project control

In this paper, we investigate the relationship between the information presentation format and project control. Furthermore, the effects of some system conditions, namely the number of projects to be controlled and the level of time pressure, on the quality of the project control decisions are analyzed. Information provided by Earned Value Analysis is used to monitor and control projects, and simulation is applied to replicate and model the uncertain project environments. Software is developed to generate random cost figures, to present the data in different visual forms and to collect users’ responses. Having performed the experiments, the statistical significance of the results is tested.

Alzheimer's disease in the pupil : pupillometry as a biomarker of cognitive processing in Alzheimer's disease

Alzheimer’s disease (AD) is a neurodegenerative disorder and a major public health issue that is rising at an alarming rate as the population ages. Despite all the efforts to develop effective pharmacological therapies, AD is currently incurable. In light of the lack of efficient treatment, a main focus in AD management is the identification of individuals who are at risk of AD; this is to predict disease onset and, ideally, to maintain cognitive and functional abilities before substantial neurological decline. Two main biomarkers can be used to reflect the onset of AD, namely, the presence of amyloid-β (Aβ) and tau pathology in cerebrospinal fluid (CSF) and the reduced volume of the hippocampus as can be observed on structural magnetic resonance imaging (MRI). While these biomarkers are widely used to detect AD, there is still need to identify better markers of the disease, ideally, a noninvasive (as CSF tests require medical intervention) and inexpensive test (as MRI is typically expensive). To tackle this challenge, we investigate whether pupil dilation can offer a noninvasive and inexpensive marker of cognitive decline in AD. More specifically, we offer a case study in which we evaluate whether pupil dilation can index cognitive effort in a patient with AD. By doing so, we aim to reveal whether pupil dilation has the potential to mirror cognitive processing in AD

Time to exacerbation of heart failure is longer in Malaysian population on dipeptidyl peptidase-4 inhibitor

Context: Diabetes mellitus is a recognized risk factor for heart failure. Dipeptidyl peptidase-4 inhibitors (DPP4i) are used in patients with diabetes largely due to its efficacy in glycated hemoglobin (HbA1c) reduction, neutral weight effect, and lower hypoglycemic events. New antidiabetic medications such as the glitazones have been linked with increasing mortality and heart failure exacerbations. The effect of DPP4i in heart failure has not been shown in a heterogenous Asian population. Aims: The aim of this study was to assess incidence of heart failure and cardiovascular (CV) events in patients with diabetes with known coronary artery disease (CAD) treated with DPP4i. Subjects and Methods: This was a single-center, retrospective analysis of patients with diabetes mellitus attending various specialist clinics in Universiti Teknologi MARA treated with available DPP4i agents from January 2013 to July 2015. Medical records were reviewed and data collected for demographic, anthropometric, laboratory, and treatment modalities. Endpoints include changes in body weight, body mass index, lipid, renal profile, and CV events during follow-up. Results: Three hundred and twenty-three patients with diabetes were screened and 307 fulfilled the inclusion criteria. Fifty-four were on linagliptin, 115 were on vildagliptin, and 154 were on saxagliptin. Majority of patients (87.6%) had uncontrolled diabetes at baseline (HbA1c, %) (8.9 ± 2.07). There was a significant reduction in HbA1c from baseline to visit 1 at 3 months (P = 0.000). Similarly, significant improvement in HbA1c seen from baseline to visit 1 (P = 0.000). A higher CV event rate was found between 20 and 30 weeks of therapy with DPP4i. The cumulative survival was 99.5% at 20 weeks and reduced to 98.75% at 30 weeks (P = 0.033). There were seven reported events (0.98%) due to heart failure or acute coronary syndrome. These participants had higher baseline HbA1c and creatinine compared to the overall cohort. Conclusions: Higher CV events were seen in diabetic patients with known CAD treated with DPP4i between 20 and 30 weeks of therapy and occurred earlier in patients with chronic kidney disease. This is later than published data and raises the need to monitor this group of patients for symptoms of heart failure beyond conventional monitoring

Influence of miR-203-regulated Hakai on cell proliferation.

<p>A, levels of Hakai and loading control α-tubulin were tested in whole-cell lysates by Western blotting 48 h after transfecting HeLa cells with two different siRNAs oligos for Hakai relative to the scrambled Ctrl small RNA group. B, 48 h after transfection of HeLa cells with the indicated siRNA oligos, cell numbers were measured by BrdU incorporation assay and represented as percentage of cells. C, measurement of BrdU incorporation 48 h after transfection of HeLa cells with the indicated small RNAs. D, measurement of cell number by hemocytometer after 48 h of transfection of HeLa cells with the indicated small RNAs. E, forty-eight h after transfecting HeLa cells with the indicated small RNAs, Hakai and α-tubulin levels were measured by Western blot analysis. Western blotting data are representative of three independent experiments. The data in panels B–D are the means ± SEM from three experiments. Significant differences in BrdU incorporation (B and C) and in cell number (D), compared to the transfected scrambled (Ctrl) small RNA are indicated (*p<0.05, **p<0.01).</p

Influence of miR-203 on cell proliferation.

<p>A, 48 h after transfection of HeLa cells with the miRNAs shown, cell numbers were measured using a hemocytometer and represented as percentage of cells relative to the scrambled Ctrl miRNA group. B, measurement of BrdU incorporation by 48 h after transfection of HeLa cells with the indicated miRNAs. The data in panels A, and B are the means ± SEM from three experiments. Significant differences in cell number (A) and BrdU incorporation (B) in the indicated transfected miRNA compared to transfected scrambled (Ctrl) miRNA are indicated (*p<0.05, **p<0.01). C, forty-eight h after transfection with scrambled Ctrl miRNA, Anti-miR-203, or Pre-miR-203, HeLa cells were subjected to FACS analysis. D, the relative G1, S, and G2/M compartments shown in C were calculated and represented in left panel, and total cell numbers in every stage cell cycle compartment were included in the right panel. Data (C and D) are representative of three independent experiments.</p

miR-203 influence on Hakai reporter construct.

<p>A, schematic of EGFP reporter constructs bearing either no Hakai mRNA sequences (pEGFP) or miR-203 target sites on the Hakai 3′-UTR (pEGFP-3′UTR). B, 48 h after cotransfection of the plasmids with scrambled control miRNA or Pre-miR-203, the level of EGFP expressed was analyzed by Western blotting. C, quantification of panel B showing the effect of scrambled Ctrl miRNA or pre-miR-203 on pEGFP and pEGFP-3′UTR. Significant differences in EGFP expression from pEGFP-3′UTR cotransfected with scrambled control miRNA (Ctrl) and pEGFP-3′UTR cotransfected with pre-miR-203 are indicated (***p<0.001). Western blotting data are the means ± SEM from three independent experiments.</p

Influence of miR-203 on Hakai levels in several cell lines.

<p>A, the effect of modulating miR-203 levels in 293 cells was studied as described in 1D and 1E; Western blot analysis (<i>left panel</i>) and quantification by densitometry (<i>right panel)</i>. B, the effect of modulating miR-203 levels in A549 cells was studied as described 1D and 1E. Western blot analysis (<i>left panel</i>) and quantification by densitometry (<i>right panel).</i> C, endogenous Hakai expression levels in human colorectal cell lines (SW480 and SW620), as assessed by western blotting (upper panel) and by RT-qPCR (lower panel) analysis. D, endogenous miR-203 levels in human colorectal cell lines (SW480 and SW620) by RT-qPCR (lower panel). Values (A–D) are the means ± SEM from three independent experiments. Significant differences in the indicated transfected miRNA with respect to transfected scrambled control (Ctrl) miRNA for A–B, and the between SW480 and SW620 cell lines for C–D are indicated (*p<0.05, **p<0.01, ***p<0.001, n = 3). The western blotting data are representative of three independent experiments.</p