Statistical analysis plan for the proactive healthcare of older people in care homes (PEACH) study

The Proactive Healthcare for Older People living in Care Homes (PEACH) study aims to evaluate whether Quality Improvement Collaboratives can be an effective way to work with local health and social care stakeholders, including representatives of the care home sector, to implement Comprehensive Geriatric Assessment (CGA) in the care home setting. It will enlist the support of four Area Improvement Collaboratives from South Nottinghamshire, UK to make changes to enable CGA in care homes in their areas. The primary outcome measure is health-related quality of life (HRQoL), measured using the EuroQoL 5-domain 5-level (EQ-5D-5L) index. A cluster-randomised (where care homes represent clusters) approach will be taken. Secondary outcome measures will be Health Service Resource by service category. These will be analysed using an interrupted time series approach. The methodology is challenging and introduces the need to control for multiple sources of contamination, clustering, time effects including lag effect and measurement issues with the primary outcome variable, including the uncertain reliability of care home staff proxy responses. This paper outlines the statistical analysis plan for the study, describing how these challenges have been addressed. It acts as reference point for further publications from the PEACH study

Competing autocrine pathways involving alternative neuropilin-1 Lligands regulate chemotaxis of carcinoma cells

Neuropilin-1 (NP1), in conjunction with plexins, promotes axon repulsion by binding to semaphorin 3A (SEMA3A). Although NP1 is expressed in carcinoma cells, its functions have remained elusive, and neither SEMA3A nor plexin expression has been explored in cancer. Here we provide evidence that breast carcinoma cells support an autocrine pathway involving SEMA3A, plexin-A1, and NP1 that impedes their ability to chemotax. Reducing SEMA3A or NP1 expression by RNA interference or inhibiting plexin-A1 signaling enhanced migration. Conversely, expression of constitutively active plexin-A1 impaired chemotaxis. The paradox of how breast carcinoma cells expressing these endogenous chemotaxis inhibitors are able to migrate is explained by their expression of vascular endothelial growth factor (VEGF), a NP1 ligand that competes with SEMA3A for receptor binding. Finally, we establish that the ratio of endogenous VEGF and SEMA3A concentrations in carcinoma cells determines their chemotactic rate. Our findings lead to the surprising conclusion that opposing autocrine loops involving NP1 regulate the chemotaxis of breast carcinoma cells. Moreover, our data indicate a novel autocrine function for VEGF in chemotaxis

Evidence that bilayer bending rigidity affects membrane protein folding

The regeneration kinetics of the integral membrane protein bacteriorhodopsin have been investigated in a lipid-based refolding system. Previous studies on bacteriorhodopsin regeneration have involved detergent-based systems, and in particular mixed dimyristoylphosphatidylcholine (DMPC)/CHAPS micelles. Here, we show that the short chain lipid dihexanoylphosphatidylcholine (DHPC) can be substituted for the detergent CHAPS and that bacteriorhodopsin can be regenerated to high yield in mixed DMPC/DHPC micelles. Bacteriorhodopsin refolding kinetics are measured in the mixed DMPC/DHPC micelles. Rapid, stopped flow mixing is employed to initiate refolding of denatured bacterioopsin in SDS micelles with mixed DMPC/DHPC micelles and time-resolved fluorescence spectroscopy to follow changes in protein fluorescence during folding. Essentially identical refolding kinetics are observed for mixed DMPC/CHAPS and mixed DMPC/DHPC micelles. Only one second-order retinal/apoprotein reaction is identified, in which retinal binds to a partially folded apoprotein intermediate, and the free energy of this retinal binding reaction is found to be the same in both types of mixed micelles. Formation of the partially folded apoprotein intermediate is a rate-limiting step in protein folding and appears to be biexponential. Both apparent rate constants are found to be dependent on the relative proportion of DMPC present in the mixed DMPC/DHPC micelles as well as on the pH of the aqueous phase. Increasing the DMPC concentration should increase the bending rigidity of the amphiphilic bilayer, and this is found to slow the rate of formation of the partially folded apoprotein intermediate. Increasing the mole fraction of DMPC from 0.3 to 0.6 slows the two apparent rate constants associated with formation of this intermediate from 0.29 and 0.031 to 0.11 and 0.013 s-1, respectively. Formation of the intermediate also slows with increasing pH, from 0.11 and 0.013 s-1 at pH 6 to 0.033 and 0.0053 s-1 at pH 8. Since this pH change has no known effect on the phase behavior of lecithins, this is more likely to represent a direct effect on the protein itself. Thus, it appears to be possible to control the rate-limiting process in bacterioopsin folding through both bilayer bending rigidity and pH

Effects of valproic acid derivatives on inositol (1,4,5)-trisphosphate depletion, teratogenicity, GSK-3 inhibition and viral replication – A screening approach for new bipolar disorder drugs based on the valproic acid core structure

Inositol-1,4,5-trisphosphate (InsP(3)) depletion has been implicated in the therapeutic action of bipolar disorder drugs, including valproic acid (VPA). It is not currently known whether the effect of VPA on InsP(3) depletion is related to the deleterious effects of teratogenicity or elevated viral replication, or if it occurs via putative inhibitory effects on glycogen synthase kinase-3β(GSK-3β). In addition, the structural requirements of VPA-related compounds to cause InsP(3) depletion are unknown. In the current study, we selected a set of 10 VPA congeners to examine their effects on InsP(3) depletion, in vivo teratogenic potency, HIV replication, and GSK-3β activity in vitro. We found four compounds that function to deplete InsP(3) in the model eukaryote Dictyostelium discoideum, and these drugs all cause growth-cone enlargement in mammalian primary neurons, consistent with the effect of InsP(3) depletion. No relationship was found between InsP(3) depletion and teratogenic or elevated viral replication effects, and none of the VPA congeners were found to affect GSK-3β activity. Structural requirements of VPA congers to maintain InsP(3) depletion efficacy greater than that of lithium are a carboxylic-acid function without dependence on side-chain length, branching, or saturation. Noteworthy is the enantiomeric differentiation if a chiral center exists, suggesting that InsP(3) depletion is mediated by a stereoselective mode of action. Thus, the effect of InsP(3) depletion can be separated from that of teratogenic potency and elevated viral replication effect. We have used this to identify two VPA derivatives that share the common InsP(3)-depleting action of VPA, lithium and carbamazepine, but do not show the side effects of VPA, thus providing promising novel candidates for bipolar disorder treatment

Effects of valproic acid derivatives on inositol trisphosphate depletion, teratogenicity, glycogen synthase kinase-3{beta} inhibition, and viral replication: a screening approach for new bipolar disorder drugs derived from the valproic acid core structure

ABSTRACT Inositol-1,4,5-trisphosphate (InsP 3 ) depletion has been implicated in the therapeutic action of bipolar disorder drugs, including valproic acid (VPA). It is not currently known whether the effect of VPA on InsP 3 depletion is related to the deleterious effects of teratogenicity or elevated viral replication, or if it occurs via putative inhibitory effects on glycogen synthase kinase-3␤ (GSK-3␤). In addition, the structural requirements of VPA-related compounds to cause InsP 3 depletion are unknown. In the current study, we selected a set of 10 VPA congeners to examine their effects on InsP 3 depletion, in vivo teratogenic potency, HIV replication, and GSK-3␤ activity in vitro. We found four compounds that function to deplete InsP 3 in the model eukaryote Dictyostelium discoideum, and these drugs all cause growth-cone enlargement in mammalian primary neurons, consistent with the effect of InsP 3 depletion. No relationship was found between InsP 3 depletion and teratogenic or elevated viral replication effects, and none of the VPA congeners were found to affect GSK-3␤ activity. Structural requirements of VPA congers to maintain InsP 3 depletion efficacy greater than that of lithium are a carboxylic-acid function without dependence on side-chain length, branching, or saturation. Noteworthy is the enantiomeric differentiation if a chiral center exists, suggesting that InsP 3 depletion is mediated by a stereoselective mode of action. Thus, the effect of InsP 3 depletion can be separated from that of teratogenic potency and elevated viral replication effect. We have used this to identify two VPA derivatives that share the common InsP 3 -depleting action of VPA, lithium and carbamazepine, but do not show the side effects of VPA, thus providing promising novel candidates for bipolar disorder treatment

Monitoring the effectiveness of HIV and STI prevention initiatives in England, Wales, and Northern Ireland: where are we now?

Primary and secondary prevention are essential components of the response to HIV and sexually transmitted infections (STIs). We present findings from nationally implemented HIV/STI prevention interventions. In 2003, of those attending STI clinics at least 64% of men who have sex with men (MSM) and 55% of heterosexuals accepted a confidential HIV test; 88% of all HIV infections in women giving birth in England were diagnosed before delivery; 85% of MSM eligible for hepatitis B vaccination received a first dose of vaccine at their first STI clinic attendance; 74% of STI clinic attendees for emergency appointments, and 20% of those for routine appointments were seen within 48 hours of initiating an appointment; the National Chlamydia Screening Programme in England found a positivity of 10% and 13% among young asymptomatic women and men, respectively. Prevention initiatives have seen recent successes in limiting further HIV/STI transmission. However, more work is required if current levels of transmission are to be reduced