Five-year risk of admission to long-term care home and death for older adults given a new diagnosis of dementia: a population-based retrospective cohort study.

BACKGROUND: After diagnosis of a health condition, information about survival and potential transition from community into institutional care can be helpful for patients and care providers. We sought to describe the association between a new diagnosis of dementia and risk of admission to a long-term care home and death at 5 years. METHODS: We conducted a population-based retrospective cohort study using linked health administrative databases. We identified individuals aged 65 years or older, living in the community, with a first documented diagnosis of dementia between Jan. 1, 2010, and Dec. 31, 2012, in Ontario, Canada. Dementia diagnosis was captured using diagnostic codes from hospital discharges, physician billings, assessments conducted for home care and long-term care, and dispensed prescriptions for cholinesterase inhibitors. Our primary outcome measures were 5-year risk of death and placement in a long-term care home, adjusted for sociodemographic and clinical factors. RESULTS: We identified 108 757 individuals in our study cohort. By the end of 5 years, 24.4% remained alive in the community and 20.5% were living in a long-term care home. Of the 55.1% who died, about half (27.9%) were admitted to a long-term care home before death. Three risk factors were associated with increased odds of death: older age (age ≥ 90 yr; odds ratio [OR] 9.5, 95% confidence interval [CI] 8.8-10.2 [reference: age 65-69 yr]), male sex (OR 1.7, 95% CI 1.6-1.7), and the presence of organ failure, including chronic obstructive pulmonary disease (OR 1.7, 95% CI 1.7-1.8), congestive heart failure (OR 2.0, 95% CI 1.9-2.0) and renal failure (OR 1.7, 95% CI 1.6-1.8). Groups formed by combinations of these 3 factors had an observed 5-year risk of death varying between 22% and 91%. INTERPRETATION: Among community-dwelling older adults with newly identified dementia in Ontario, the majority died or were admitted to a long-term care home within 5 years. This information may be helpful for discussions on prognosis and need for admission to long-term care.

Barriers and enablers to a physician-delivered educational initiative to reduce low-acuity visits to the pediatric emergency department.

BACKGROUND: Use of the pediatric emergency department (PED) for low-acuity health issues is a growing problem, contributing to overcrowding, longer waits and higher health system costs. This study examines an educational initiative aimed at reducing low-acuity PED visits. The initiative, implemented at an academic pediatric hospital, saw PED physicians share a pamphlet with caregivers to educate them about appropriate PED use and alternatives. Despite early impacts, the initiative was not sustained. This study analyzes the barriers and enablers to physician participation in the initiative, and offers strategies to improve implementation and sustainability of similar future initiatives. METHODS: Forty-two PED physicians were invited to participate in a semi-structured individual interview assessing their views about low-acuity visits, their pamphlet use, barriers and enablers to pamphlet use, and the initiative's potential for reducing low-acuity visits. Suggestions were solicited for improving the initiative and reducing low-acuity visits. Constant comparative method was used during analysis. Codes were developed inductively and iteratively, then grouped according to the Theoretical Domains Framework (TDF). Efforts to ensure study credibility included seeking participant feedback on the findings. RESULTS: Twenty-three PED physicians were interviewed (55%). Barriers and enablers for pamphlet use were identified and grouped according to five of the 14 TDF domains: social/professional role and identity; beliefs about consequences; environmental context and resources; social influences; and emotions. CONCLUSIONS: The TDF provided an effective approach to identify the key elements influencing physician participation in the educational initiative. This information will help inform behavior change interventions to improve the implementation of similar future initiatives that involve physicians as the primary educators of caregivers.

Dementia Population Risk Tool (DemPoRT): study protocol for a predictive algorithm assessing dementia risk in the community.

INTRODUCTION: The burden of disease from dementia is a growing global concern as incidence increases dramatically with age, and average life expectancy has been increasing around the world. Planning for an ageing population requires reliable projections of dementia prevalence; however, existing population projections are simple and have poor predictive accuracy. The Dementia Population Risk Tool (DemPoRT) will predict incidence of dementia in the population setting using multivariable modelling techniques and will be used to project dementia prevalence. METHODS AND ANALYSIS: The derivation cohort will consist of elderly Ontario respondents of the Canadian Community Health Survey (CCHS) (2001, 2003, 2005 and 2007; 18 764 males and 25 288 females). Prespecified predictors include sociodemographic, general health, behavioural, functional and health condition variables. Incident dementia will be identified through individual linkage of survey respondents to population-level administrative healthcare databases (1797 and 3281 events, and 117 795 and 166 573 person-years of follow-up, for males and females, respectively, until 31 March 2014). Using time of first dementia capture as the primary outcome and death as a competing risk, sex-specific proportional hazards regression models will be estimated. The 2008/2009 CCHS survey will be used for validation (approximately 4600 males and 6300 females). Overall calibration and discrimination will be assessed as well as calibration within predefined subgroups of importance to clinicians and policy makers. ETHICS AND DISSEMINATION: Research ethics approval has been granted by the Ottawa Health Science Network Research Ethics Board. DemPoRT results will be submitted for publication in peer-review journals and presented at scientific meetings. The algorithm will be assessable online for both population and individual uses. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT03155815, pre-results.

Functional rescue of a misfolded eukaryotic ATP-binding cassette transporter by domain replacement.

ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP binding/hydrolysis with the transport of hydrophilic substrates across lipid barriers. Deletion of Phe-670 in the first nucleotide-binding domain (NBD1) of the yeast ABC transporter, Yor1p, perturbs interdomain associations, reduces functionality, and hinders proper transport to the plasma membrane. Functionality of Yor1p-ΔF was restored upon co-expression of a peptide containing wild-type NBD1. To gain insight into the biogenesis of this important class of proteins, we defined the requirements for this rescue. We show that a misfolding lesion in NBD1 of the full-length protein is a prerequisite for functional rescue by exogenous NBD1, which is mediated by physical replacement of the dysfunctional domain by the soluble NBD1. This association does not restore trafficking of Yor1p-ΔF but instead confers catalytic activity to the small population of Yor1p-ΔF that escapes to the plasma membrane. An important coupling between the exogenous NBD1 and ICL4 within full-length aberrant Yor1p-ΔF is required for functional rescue but not for the physical interaction between the two polypeptides. Together, our genetic and biochemical data reveal that it is possible to modulate activity of ABC transporters by physically replacing dysfunctional domains.

Dissecting the ER-associated degradation of a misfolded polytopic membrane protein.

It remains unclear how misfolded membrane proteins are selected and destroyed during endoplasmic reticulum-associated degradation (ERAD). For example, chaperones are thought to solubilize aggregation-prone motifs, and some data suggest that these proteins are degraded at the ER. To better define how membrane proteins are destroyed, the ERAD of Ste6p(*), a 12 transmembrane protein, was reconstituted. We found that specific Hsp70/40s act before ubiquitination and facilitate Ste6p(*) association with an E3 ubiquitin ligase, suggesting an active role for chaperones. Furthermore, polyubiquitination was a prerequisite for retrotranslocation, which required the Cdc48 complex and ATP. Surprisingly, the substrate was soluble, and extraction was independent of a ubiquitin chain extension enzyme (Ufd2p). However, Ufd2p increased the degree of ubiquitination and facilitated degradation. These data indicate that polytopic membrane proteins can be extracted from the ER, and define the point of action of chaperones and the requirement for Ufd2p during membrane protein quality control.

Saccharomyces cerevisiae a-factor mutants reveal residues critical for processing, activity, and export.

The Saccharomyces cerevisiae mating pheromone a-factor provides a paradigm for understanding the biogenesis of prenylated fungal pheromones. The biogenesis of a-factor involves multiple steps: (i) C-terminal CAAX modification (where C is cysteine, A is aliphatic, and X is any residue) which includes prenylation, proteolysis, and carboxymethylation (by Ram1p/Ram2p, Ste24p or Rce1p, and Ste14p, respectively); (ii) N-terminal processing, involving two sequential proteolytic cleavages (by Ste24p and Axl1p); and (iii) nonclassical export (by Ste6p). Once exported, mature a-factor interacts with the Ste3p receptor on MATalpha cells to stimulate mating. The a-factor biogenesis machinery is well defined, as is the CAAX motif that directs C-terminal modification; however, very little is known about the sequence determinants within a-factor required for N-terminal processing, activity, and export. Here we generated a large collection of a-factor mutants and identified residues critical for the N-terminal processing steps mediated by Ste24p and Axl1p. We also identified mutants that fail to support mating but do not affect biogenesis or export, suggesting a defective interaction with the Ste3p receptor. Mutants significantly impaired in export were also found, providing evidence that the Ste6p transporter recognizes sequence determinants as well as CAAX modifications. We also performed a phenotypic analysis of the entire set of isogenic a-factor biogenesis machinery mutants, which revealed information about the dependency of biogenesis steps upon one another, and demonstrated that export by Ste6p requires the completion of all processing events. Overall, this comprehensive analysis will provide a useful framework for the study of other fungal pheromones, as well as prenylated metazoan proteins involved in development and aging.

The src homology 2 domain-containing tyrosine phosphatase 2 regulates primary T-dependent immune responses and Th cell differentiation.

The src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) plays an important role in development and in growth factor receptor signaling pathways, yet little is known of its role in the immune system. We generated mice expressing a dominant-negative version of the protein, SHP2(CS), specifically in T cells. In SHP2(CS) mice, T cell development appears normal with regard to both negative and positive selection. However, SHP2(CS) T cells express higher levels of activation markers, and aged mice have elevated serum Abs. This is associated with a marked increase in IL-4, IL-5, and IL-10 secretion by SHP2(CS) T cells in vitro. In addition, primary thymus-dependent B cell responses are deficient in SHP2(CS) mice. We show that whereas TCR-induced linker for activation of T cells phosphorylation is defective, CTLA-4 and programmed death-1 signaling are not affected by SHP2(CS) expression. Our results suggest that a key action of wild-type SHP2 is to suppress differentiation of T cells to the Th2 phenotype.

Inhibiting farnesylation reverses the nuclear morphology defect in a HeLa cell model for Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disease resulting from a mutation in the LMNA gene, which encodes nuclear lamins A and C. Lamin A is synthesized as a precursor (prelamin A) with a C-terminal CaaX motif that undergoes farnesylation, endoproteolytic cleavage, and carboxylmethylation. Prelamin A is subsequently internally cleaved by the zinc metalloprotease Ste24 (Zmpste24) protease, which removes the 15 C-terminal amino acids, including the CaaX modifications, to yield mature lamin A. HGPS results from a dominant mutant form of prelamin A (progerin) that has an internal deletion of 50 aa near the C terminus that includes the Zmpste24 cleavage site and blocks removal of the CaaX-modified C terminus. Fibroblasts from HGPS patients have aberrant nuclei with irregular shapes, which we hypothesize result from the abnormal persistence of the farnesyl and/or carboxylmethyl CaaX modifications on progerin. If this hypothesis is correct, inhibition of CaaX modification by mutation or pharmacological treatment should alleviate the nuclear morphology defect. Consistent with our hypothesis, we find that expression in HeLa cells of GFP-progerin or an uncleavable form of prelamin A with a Zmpste24 cleavage site mutation induces the formation of abnormal nuclei similar to those in HGPS fibroblasts. Strikingly, inhibition of farnesylation pharmacologically with the farnesyl transferase inhibitor rac-R115777 or mutationally by alteration of the CaaX motif dramatically reverses the abnormal nuclear morphology. These results suggest that farnesyl transferase inhibitors represent a possible therapeutic option for individuals with HGPS and/or other laminopathies due to Zmpste24 processing defects.

Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein.

The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane are monitored by ER quality control. Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. Whereas luminal and integral membrane ERAD substrates both require the proteasome for their degradation, the ER quality control machinery for these two classes of proteins likely differs because of their distinct topologies. Here we establish the requirements for the ERAD of Ste6p*, a multispanning membrane protein with a cytosolic mutation, and compare them with those for mutant form of carboxypeptidase Y (CPY*), a soluble luminal protein. We show that turnover of Ste6p* is dependent on the ubiquitin-protein isopeptide ligase Doa10p and is largely independent of the ubiquitin-protein isopeptide ligase Hrd1p/Der3p, whereas the opposite is true for CPY*. Furthermore, the cytosolic Hsp70 chaperone Ssa1p and the Hsp40 co-chaperones Ydj1p and Hlj1p are important in ERAD of Ste6p*, whereas the ER luminal chaperone Kar2p is dispensable, again opposite their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to require the Sec61p translocon pore but, like CPY*, could depend on the Sec61p homologue Ssh1p. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of Ste6p* employs distinct machinery from that of the soluble luminal substrate CPY* and that Ste6p* is a valuable model substrate to dissect the cellular machinery required for the ERAD of multispanning membrane proteins with a cytosolic mutation.

The internalization of yeast Ste6p follows an ordered series of events involving phosphorylation, ubiquitination, recognition and endocytosis.

A general pathway for the internalization of plasma membrane proteins that involves phosphorylation, ubiquitination, recognition and endocytosis has recently emerged from multiple studies in yeast. We refer to this series of events as the PURE pathway. Here we investigate whether the yeast a-factor transporter Ste6p, an ATP-binding cassette protein, utilizes the PURE pathway. Deletion of a 52-amino acid sequence (the 'A box') within the linker region of Ste6p has previously been shown to block ubiquitination and endocytosis (Kolling R, Losko S. EMBO J 1997; 16:2251-2261). Using wild-type and mutant forms of GFP-tagged Ste6p, we identified two residues (T(613) and S(623)) within the A box as likely sites of Ste6p phosphorylation important for internalization. Mutation of these residues to alanine blocked ubiquitination and endocytosis of Ste6p, similar to the effect of deleting the entire A box, while substitution with glutamic acid (to mimic phosphorylation) suppressed the ubiquitination and endocytic defects. Importantly, a translational fusion of monoubiquitin to the C-terminus of Ste6p-T(613)A, S(623)A or Ste6p-DeltaA restored endocytosis, providing strong evidence that the role of phosphorylation is to direct ubiquitination, which in turn is a critical signal for Ste6p internalization. We also identified multiple (five) lysine residues in the linker that are important for Ste6p ubiquitination. Our results demonstrate that Ste6p follows the PURE pathway and that GFP-tagged Ste6p provides a powerful model protein for studies of endocytosis and post-endocytic events in yeast.

A striking quality control subcompartment in Saccharomyces cerevisiae: the endoplasmic reticulum-associated compartment.

The folding of nascent secretory and membrane proteins is monitored by the endoplasmic reticulum (ER) quality control system. Misfolded proteins are retained in the ER and can be removed by ER-associated degradation. As a model for the ER quality control of multispanning membrane proteins in yeast, we have been studying mutant forms of Ste6p. Here, we identify mislocalized mutant forms of Ste6p that induce the formation of, and localize to, prominent structures that are absent in normal cells. We have named these structures ER-associated compartments (ERACs), based on their juxtaposition to and connection with the ER, as observed by fluorescence and electron microscopy. ERACs comprise a network of tubulo-vesicular structures that seem to represent proliferated ER membranes. Resident ER lumenal and membrane proteins are present in ERACs in addition to their normal ER localization, suggesting there is no barrier for their entry into ERACs. However, the forms of Ste6p in ERACs are excluded from the ER and do not enter the secretory pathway; instead, they are ultimately targeted for ER-associated degradation. The presence of ERACs does not adversely affect secretory protein traffic through the ER and does not lead to induction of the unfolded protein response. We propose that ERACs may be holding sites to which misfolded membrane proteins are specifically diverted so as not to interfere with normal cellular functions. We discuss the likelihood that related ER membrane proliferations that form in response to certain other mutant or unassembled membrane proteins may be substantially similar to ERACs.

A region within a lumenal loop of Saccharomyces cerevisiae Ycf1p directs proteolytic processing and substrate specificity.

Ycf1p, a member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette proteins, is a vacuolar membrane transporter that confers resistance to a variety of toxic substances such as cadmium and arsenite. Ycf1p undergoes a PEP4-dependent processing event to yield N- and C-terminal cleavage products that remain associated with one another. In the present study, we sought to determine whether proteolytic cleavage is required for Ycf1p activity. We have identified a unique region within lumenal loop 6 of Ycf1p, designated the loop 6 insertion (L6(ins)), which appears to be necessary and sufficient for proteolytic cleavage, since L6(ins) can promote processing when moved to new locations in Ycf1p or into a related transporter, Bpt1p. Surprisingly, mutational results indicate that proteolytic processing is not essential for Ycf1p transport activity. Instead, the L6(ins) appears to regulate substrate specificity of Ycf1p, since certain mutations in this region lower cellular cadmium resistance with a concomitant gain in arsenite resistance. Although some of these L6(ins) mutations block processing, there is no correlation between processing and substrate specificity. The activity profiles of the Ycf1p L6(ins) mutants are dramatically affected by the strain background in which they are expressed, raising the possibility that another cellular component may functionally impact Ycf1p activity. A candidate component may be a new full-length MRP-type transporter (NFT1), reported in the Saccharomyces Genome Database as two adjacent open reading frames, YKR103w and YKR104w, but which we show here is present in most Saccharomyces strains as a single open reading frame.