Aging Differentially Affects Multiple Aspects of Vesicle Fusion Kinetics

How fusion pore formation during exocytosis affects the subsequent release of vesicle contents remains incompletely understood. It is unclear if the amount released per vesicle is dependent upon the nature of the developing fusion pore and whether full fusion and transient kiss and run exocytosis are regulated by similar mechanisms. We hypothesise that if consistent relationships exist between these aspects of exocytosis then they will remain constant across any age. Using amperometry in mouse chromaffin cells we measured catecholamine efflux during single exocytotic events at P0, 1 month and 6 months. At all ages we observed full fusion (amperometric spike only), full fusion preceded by fusion pore flickering (pre-spike foot (PSF) signal followed by a spike) and pure “kiss and run” exocytosis (represented by stand alone foot (SAF) signals). We observe age-associated increases in the size of all 3 modes of fusion but these increases occur at different ages. The release probability of PSF signals or full spikes alone doesn't alter across any age in comparison with an age-dependent increase in the incidence of “kiss and run” type events. However, the most striking changes we observe are age-associated changes in the relationship between vesicle size and the membrane bending energy required for exocytosis. Our data illustrates that vesicle size does not regulate release probability, as has been suggested, that membrane elasticity or flexural rigidity change with age and that the mechanisms controlling full fusion may differ from those controlling “kiss and run” fusion

Improving calculation, interpretation and communication of familial colorectal cancer risk: Protocol for a randomized controlled trial

Contains fulltext : 88114.pdf (publisher's version ) (Open Access)BACKGROUND: Individuals with multiple relatives with colorectal cancer (CRC) and/or a relative with early-onset CRC have an increased risk of developing CRC. They are eligible for preventive measures, such as surveillance by regular colonoscopy and/or genetic counselling. Currently, most at-risk individuals do not follow the indicated follow-up policy. In a new guideline on familial and hereditary CRC, clinicians have new tasks in calculating, interpreting, and communicating familial CRC risk. This will lead to better recognition of individuals at an increased familial CRC risk, enabling them to take effective preventive measures. This trial compares two implementation strategies (a common versus an intensive implementation strategy), focussing on clinicians' risk calculation, interpretation, and communication, as well as patients' uptake of the indicated follow-up policy. METHODS: A clustered randomized controlled trial including an effect, process, and cost evaluation will be conducted in eighteen hospitals. Nine hospitals in the control group will receive the common implementation strategy (i.e., dissemination of the guideline). In the intervention group, an intensive implementation strategy will be introduced. Clinicians will receive education and tools for risk calculation, interpretation, and communication. Patients will also receive these tools, in addition to patient decision aids. The effect evaluation includes assessment of the number of patients for whom risk calculation, interpretation, and communication is performed correctly, and the number of patients following the indicated follow-up policy. The actual exposure to the implementation strategies and users' experiences will be assessed in the process evaluation. In a cost evaluation, the costs of the implementation strategies will be determined. DISCUSSION: The results of this study will help determine the most effective method as well as the costs of improving the recognition of individuals at an increased familial CRC risk. It will provide insight into the experiences of both patients and clinicians with these strategies.The knowledge gathered in this study can be used to improve the recognition of familial and hereditary CRC at both the national and international level, and will serve as an example to improve care for patients and their relatives worldwide. Our results may also be useful in improving healthcare in other diseases. TRIAL REGISTRATION: ClinicalTrials.gov NCT00929097

Differences in pain, function and coping in Multidimensional Pain Inventory subgroups of chronic back pain: a one-group pretest-posttest study

Contains fulltext : 97819.pdf (publisher's version ) (Open Access)BACKGROUND: Patients with non-specific back pain are not a homogeneous group but heterogeneous with regard to their bio-psycho-social impairments. This study examined a sample of 173 highly disabled patients with chronic back pain to find out how the three subgroups based on the Multidimensional Pain Inventory (MPI) differed in their response to an inpatient pain management program. METHODS: Subgroup classification was conducted by cluster analysis using MPI subscale scores at entry into the program. At program entry and at discharge after four weeks, participants completed the MPI, the MOS Short Form-36 (SF-36), the Hospital Anxiety and Depression Scale (HADS), and the Coping Strategies Questionnaire (CSQ). Pairwise analyses of the score changes of the mentioned outcomes of the three MPI subgroups were performed using the Mann-Whitney-U-test for significance. RESULTS: Cluster analysis identified three MPI subgroups in this highly disabled sample: a dysfunctional, interpersonally distressed and an adaptive copers subgroup. The dysfunctional subgroup (29% of the sample) showed the highest level of depression in SF-36 mental health (33.4 +/- 13.9), the interpersonally distressed subgroup (35% of the sample) a modest level of depression (46.8 +/- 20.4), and the adaptive copers subgroup (32% of the sample) the lowest level of depression (57.8 +/- 19.1). Significant differences in pain reduction and improvement of mental health and coping were observed across the three MPI subgroups, i.e. the effect sizes for MPI pain reduction were: 0.84 (0.44-1.24) for the dysfunctional subgroup, 1.22 (0.86-1.58) for the adaptive copers subgroup, and 0.53 (0.24-0.81) for the interpersonally distressed subgroup (p = 0.006 for pairwise comparison). Significant score changes between subgroups concerning activities and physical functioning could not be identified. CONCLUSIONS: MPI subgroup classification showed significant differences in score changes for pain, mental health and coping. These findings underscore the importance of assessing individual differences to understand how patients adjust to chronic back pain

Critical Role of TCF-1 in Repression of the IL-17 Gene

Overwhelming activation of IL-17, a gene involved in inflammation, leads to exaggerated Th17 responses associated with numerous autoimmune conditions, such as experimental autoimmune encephalomyelitis (EAE). Here we show that TCF-1 is a critical factor to repress IL-17 gene locus by chromatin modifications during T cell development. Deletion of TCF-1 resulted in increased IL-17 gene expression both in thymus and peripheral T cells, which led to enhanced Th17 differentiation. As a result, TCF-1-/- mice were susceptible to Th17-dependent EAE induction. Rag1-/- mice reconstituted with TCF-1-/- T cells were also susceptible to EAE, indicating TCF-1 is intrinsically required to repress IL-17. However, expression of wild-type TCF-1 or dominant negative TCF-1 did not interfere with Th17 differentiation in mature T cells. Furthermore, expression of TCF-1 in TCF-1-/- T cells could not restore Th17 differentiation to wild-type levels, indicating that TCF-1 cannot affect IL-17 production at the mature T cell stage. This is also supported by the normal up-regulation or activation in mature TCF-1-/- T cells of factors known to regulate Th17 differentiation, including RORγt and Stat3. We observed hyperacetylation together with trimethylation of Lys-4 at the IL-17 locus in TCF-1-/- thymocytes, two epigenetic modifications indicating an open active state of the gene. Such epigenetic modifications were preserved even when TCF-1-/- T cells migrated out of thymus. Therefore, TCF-1 mediates an active process to repress IL-17 gene expression via epigenetic modifications during T cell development. This TCF-1-mediated repression of IL-17 is critical for peripheral T cells to generate balanced immune responses

Oncogenic Function of DACT1 in Colon Cancer through the Regulation of β-catenin

The Wnt/β-catenin signaling pathway plays important roles in the progression of colon cancer. DACT1 has been identified as a modulator of Wnt signaling through its interaction with Dishevelled (Dvl), a central mediator of both the canonical and noncanonical Wnt pathways. However, the functions of DACT1 in the WNT/β-catenin signaling pathway remain unclear. Here, we present evidence that DACT1 is an important positive regulator in colon cancer through regulating the stability and sublocation of β-catenin. We have shown that DACT1 promotes cancer cell proliferation in vitro and tumor growth in vivo and enhances the migratory and invasive potential of colon cancer cells. Furthermore, the higher expression of DACT1 not only increases the nuclear and cytoplasmic fractions of β-catenin, but also increases its membrane-associated fraction. The overexpression of DACT1 leads to the increased accumulation of nonphosphorylated β-catenin in the cytoplasm and particularly in the nuclei. We have demonstrated that DACT1 interacts with GSK-3β and β-catenin. DACT1 stabilizes β-catenin via DACT1-induced effects on GSK-3β and directly interacts with β-catenin proteins. The level of phosphorylated GSK-3β at Ser9 is significantly increased following the elevated expression of DACT1. DACT1 mediates the subcellular localization of β-catenin via increasing the level of phosphorylated GSK-3β at Ser9 to inhibit the activity of GSK-3β. Taken together, our study identifies DACT1 as an important positive regulator in colon cancer and suggests a potential strategy for the therapeutic control of the β-catenin-dependent pathway

Canonical wnt signaling activity in early stages of chick lung development

Wnt signaling pathway is an essential player during vertebrate embryonic development which has been associated with several developmental processes such as gastrulation, body axis formation and morphogenesis of numerous organs, namely the lung. Wnt proteins act through specific transmembrane receptors, which activate intracellular pathways that regulate cellular processes such as cell proliferation, differentiation and death. Morphogenesis of the fetal lung depends on epithelial-mesenchymal interactions that are governed by several growth and transcription factors that regulate cell proliferation, fate, migration and differentiation. This process is controlled by different signaling pathways such as FGF, Shh and Wnt among others. Wnt signaling is recognized as a key molecular player in mammalian pulmonary development but little is known about its function in avian lung development. The present work characterizes, for the first time, the expression pattern of several Wnt signaling members, such as wnt-1, wnt-2b, wnt-3a, wnt-5a, wnt-7b, wnt-8b, wnt-9a, lrp5, lrp6, sfrp1, dkk1, β-catenin and axin2 at early stages of chick lung development. In general, their expression is similar to their mammalian counterparts. By assessing protein expression levels of active/total β-catenin and phospho-LRP6/LRP6 it is revealed that canonical Wnt signaling is active in this embryonic tissue. In vitro inhibition studies were performed in order to evaluate the function of Wnt signaling pathway in lung branching. Lung explants treated with canonical Wnt signaling inhibitors (FH535 and PK115-584) presented an impairment of secondary branch formation after 48 h of culture along with a decrease in axin2 expression levels. Branching analysis confirmed this inhibition. Wnt-FGF crosstalk assessment revealed that this interaction is preserved in the chick lung. This study demonstrates that Wnt signaling is crucial for precise chick lung branching and further supports the avian lung as a good model for branching studies since it recapitulates early mammalian pulmonary development.Rute S. Moura was supported by a grant of ON.2 SR&TD Integrated Program (N-01-01-0124-01-07), ref: UMINHO/BPD/31/2013. The funders had no role in study design, data collection and analysis

Wnt Signalling Pathway Parameters for Mammalian Cells

Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model