Analysis of Mechanically Activated Ion Channels at the Cell-Substrate Interface: Combining Pillar Arrays and Whole-Cell Patch-Clamp

Ionic currents can be evoked by mechanical inputs applied directly at the cell-substrate interface. These ionic currents are mediated by mechanically activated ion channels, where the open probability increases with increasing mechanical input. In order to study mechanically activated ion channels directly at the interface between cells and their environment, we have developed a technique to simultaneously monitor ion channel activity whilst stimuli are applied via displacement of cell-substrate contacts. This technique utilizes whole-cell patch-clamp electrophysiology and elastomeric pillar arrays, it is quantitative and appropriate for studying channels that respond to stimuli that are propagated to an adherent cell via the physical substrate. The mammalian channels PIEZO1, PIEZO2 have been shown to be activated by substrate deflections, using this technique. In addition, TRPV4 mediated currents can be evoked by substrate deflections, in contrast to alternate stimulation methods such as membrane stretch or cellular indentation. The deflections applied at cell-substrate points mimic the magnitude of physical stimuli that impact cells in situ

Controlling the properties of the micellar and gel phase by varying the counterion in functionalised-dipeptide systems

The micellar aggregates formed at high pH for dipeptide-based gelators can be varied by using different alkali metal salts to prepare the solutions. The nature of the micellar aggregates directly affects the properties of the resulting gels

Evaluation of the effectiveness and cost-effectiveness of Families for Health V2 for the treatment of childhood obesity : study protocol for a randomized controlled trial

Background: Effective programs to help children manage their weight are required. Families for Health focuses on a parenting approach, designed to help parents develop their parenting skills to support lifestyle change within the family. Families for Health V1 showed sustained reductions in overweight after 2 years in a pilot evaluation, but lacks a randomized controlled trial (RCT) evidence base. Methods/design: This is a multi-center, investigator-blind RCT, with parallel economic evaluation, with a 12-month follow-up. The trial will recruit 120 families with at least one child aged 6 to 11 years who is overweight (≥91st centile BMI) or obese (≥98th centile BMI) from three localities and assigned randomly to Families for Health V2 (60 families) or the usual care control (60 families) groups. Randomization will be stratified by locality (Coventry, Warwickshire, Wolverhampton). Families for Health V2 is a family-based intervention run in a community venue. Parents/carers and children attend parallel groups for 2.5 hours weekly for 10 weeks. The usual care arm will be the usual support provided within each NHS locality. A mixed-methods evaluation will be carried out. Child and parent participants will be assessed at home visits at baseline, 3-month (post-treatment) and 12-month follow-up. The primary outcome measure is the change in the children’s BMI z-scores at 12 months from the baseline. Secondary outcome measures include changes in the children’s waist circumference, percentage body fat, physical activity, fruit/vegetable consumption and quality of life. The parents’ BMI and mental well-being, family eating/activity, parent–child relationships and parenting style will also be assessed. Economic components will encompass the measurement and valuation of service utilization, including the costs of running Families for Health and usual care, and the EuroQol EQ-5D health outcomes. Cost-effectiveness will be expressed in terms of incremental cost per quality-adjusted life year gained. A de novo decision-analytic model will estimate the lifetime cost-effectiveness of the Families for Health program. Process evaluation will document recruitment, attendance and drop-out rates, and the fidelity of Families for Health delivery. Interviews with up to 24 parents and children from each arm will investigate perceptions and changes made. Discussion: This paper describes our protocol to assess the effectiveness and cost-effectiveness of a parenting approach for managing childhood obesity and presents challenges to implementation. Trial registration: Current Controlled Trials ISRCTN4503220

The Human Cytomegalovirus Latency-Associated Gene Product Latency Unique Natural Antigen Regulates Latent Gene Expression

Human cytomegalovirus (HCMV) infection can lead to either lytic or latent infection, which is dependent on the regulation of the viral major immediate early promoter (MIEP). Suppression of the MIEP is a pre-requisite for latency and is driven by repressive epigenetic modifications at the MIEP during latent infection. However, other viral genes are expressed during latency and this is correlated with activatory epigenetic modifications at latent gene promoters. Yet the molecular basis of the differential regulation of latent and lytic gene expression by epigenetics is unclear. LUNA, a latent viral transcript, has been suggested to be important for HCMV latency and has also been shown to be important for efficient reactivation likely through its known deSUMOylase activity. Intriguingly, we and others have also observed that LUNA enhances latency-associated expression of the viral UL138 gene. Here, we show that in the absence of LUNA, the expression of multiple latency-associated transcripts is reduced during latent infection, which is correlated with a lack of activatory marks at their promoters. Interestingly, we also show that LUNA interacts with the hematopoietic transcription factor GATA-2, which has previously been shown to bind to a number of latency-associated gene promoters, and that this interaction is dependent on the deSUMOylase domain of LUNA. Finally, we show that the deSUMOylase activity of LUNA is required for the establishment and/or maintenance of an open chromatin configuration around latency-associated gene promoters. As such, LUNA plays a key role in efficient latency-associated viral gene expression and carriage of viral genome during latent carriage

Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons

A complex of stomatin-family proteins and acid-sensing (proton-gated) ion channel (ASIC) family members participate in sensory transduction in invertebrates and vertebrates. Here, we have examined the role of the stomatin-family protein stomatin-like protein-3 (STOML3) in this process. We demonstrate that STOML3 interacts with stomatin and ASIC subunits and that this occurs in a highly mobile vesicle pool in dorsal root ganglia (DRG) neurons and Chinese hamster ovary cells. We identify a hydrophobic region in the N-terminus of STOML3 that is required for vesicular localization of STOML3 and regulates physical and functional interaction with ASICs. We further characterize STOML3-containing vesicles in DRG neurons and show that they are Rab11-positive, but not part of the early-endosomal, lysosomal or Rab14-dependent biosynthetic compartment. Moreover, uncoupling of vesicles from microtubules leads to incorporation of STOML3 into the plasma membrane and increased acid-gated currents. Thus, STOML3 defines a vesicle pool in which it associates with molecules that have critical roles in sensory transduction. We suggest that the molecular features of this vesicular pool may be characteristic of a ‘transducosome’ in sensory neurons

Continuity of care for people with psychotic illness: its relationship to clinical and social functioning.

Background: The relationship between continuity of care and user characteristics or outcomes has rarely been explored. The ECHO study operationalized and tested a multi-axial definition of continuity of care, producing a seven-factor model used here. Aims: To assess the relationship between user characteristics and established components of continuity of care, and the impact of continuity on clinical and social functioning. Methods: The sample comprised 180 community mental health team users with psychotic disorders who were interviewed at three annual time-points, to assess their experiences of continuity of care and clinical and social functioning. Scores on seven continuity factors were tested for association with user-level variables. Results: Improvement in quality of life was associated with better Experience & Relationship continuity scores (better user-rated continuity and therapeutic relationship) and with lower Meeting Needs continuity factor scores. Higher Meeting Needs scores were associated with a decrease in symptoms. Conclusion: Continuity is a dynamic process, influenced significantly by care structures and organizational change

Why do we need a new journal about writing for wellbeing?

A discussion among practitioners and researchers, forming an editorial article for the first issue of LIRI

Abrogation of Stem Loop Binding Protein (Slbp) function leads to a failure of cells to transition from proliferation to differentiation, retinal coloboma and midline axon guidance deficits

Through forward genetic screening for mutations affecting visual system development, we identified prominent coloboma and cell-autonomous retinal neuron differentiation, lamination and retinal axon projection defects in eisspalte (ele) mutant zebrafish. Additional axonal deficits were present, most notably at midline axon commissures. Genetic mapping and cloning of the ele mutation showed that the affected gene is slbp, which encodes a conserved RNA stem-loop binding protein involved in replication dependent histone mRNA metabolism. Cells throughout the central nervous system remained in the cell cycle in ele mutant embryos at stages when, and locations where, post-mitotic cells have differentiated in wild-type siblings. Indeed, RNAseq analysis showed down-regulation of many genes associated with neuronal differentiation. This was coincident with changes in the levels and spatial localisation of expression of various genes implicated, for instance, in axon guidance, that likely underlie specific ele phenotypes. These results suggest that many of the cell and tissue specific phenotypes in ele mutant embryos are secondary to altered expression of modules of developmental regulatory genes that characterise, or promote transitions in, cell state and require the correct function of Slbp-dependent histone and chromatin regulatory genes

The Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) Binder Rasa3 Regulates Phosphoinositide 3-kinase (PI3K)-dependent Integrin α IIb β 3 Outside-in Signaling

The class I PI3K family of lipid kinases plays an important role in integrin αIIbβ3 function, thereby supporting thrombus growth and consolidation. Here, we identify Ras/Rap1GAP Rasa3 (GAP1IP4BP) as a major phosphatidylinositol 3,4,5-trisphosphate-binding protein in human platelets and a key regulator of integrin αIIbβ3 outside-in signaling. We demonstrate that cytosolic Rasa3 translocates to the plasma membrane in a PI3K-dependent manner upon activation of human platelets. Expression of wild-type Rasa3 in integrin αIIbβ3-expressing CHO cells blocked Rap1 activity and integrin αIIbβ3-mediated spreading on fibrinogen. In contrast, Rap1GAP-deficient (P489V) and Ras/Rap1GAP-deficient (R371Q) Rasa3 had no effect. We furthermore show that two Rasa3 mutants (H794L and G125V), which are expressed in different mouse models of thrombocytopenia, lack both Ras and Rap1GAP activity and do not affect integrin αIIbβ3-mediated spreading of CHO cells on fibrinogen. Platelets from thrombocytopenic mice expressing GAP-deficient Rasa3 (H794L) show increased spreading on fibrinogen, which in contrast to wild-type platelets is insensitive to PI3K inhibitors. Together, these results support an important role for Rasa3 in PI3K-dependent integrin αIIbβ3-mediated outside-in signaling and cell spreading