Roberta E. Redfern

PTEN phosphatase selectively binds phosphoinositides and undergoes structural changes.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor that is mutated or deleted in a variety of human tumors, and even loss of only one PTEN gene profoundly affects carcinogenesis. PTEN encodes a phosphatidylinositol phosphate phosphatase specific for the 3-position of the inositol ring. Despite its importance, we are just beginning to understand the regulatory circuits that maintain the correct levels of PTEN phosphatase activity. Several independent studies reported that PI(4,5)P2 enhances PTEN phosphatase activity, but the reasons for this enhancement are currently being debated. In this study, PTEN bound to PI(4,5)P2-bearing vesicles has increased alpha-helicity, providing direct spectroscopic proof of a conformational change. Neither PI(3,5)P2 nor PI(3,4,5)P3 induced this conformational change. On the basis of experiments with two mutant PTEN proteins, it is shown that PI(4,5)P2 induces this conformational change by binding to the PTEN N-terminal domain. Using PTEN protein and a 21-amino acid peptide based on the PTEN N-terminus, we tested all natural phosphatidylinositol phosphates and found preferential binding of PI(4,5)P2. PTEN also binds to phosphatidylserine-bearing vesicles, resulting in a slight increase in beta-sheet content. In addition, PTEN binds synergistically to PI(4,5)P2 and phosphatidylserine, and hence, these anionic lipids do not compete for PTEN binding sites. Collectively, these results demonstrate that PTEN binds to membranes through multiple sites, but only PI(4,5)P2 binding to the N-terminal domain triggers a conformational change with increased alpha-helicity.

A mutant form of PTEN linked to autism.

The tumor suppressor, phosphatase, and tensin homologue deleted on chromosome 10 (PTEN), is a phosphoinositide (PI) phosphatase specific for the 3‐position of the inositol ring. PTEN has been implicated in autism for a subset of patients with macrocephaly. Various studies identified patients in this subclass with one normal and one mutated PTEN gene. We characterize the binding, structural properties, activity, and subcellular localization of one of these autism‐related mutants, H93R PTEN. Even though this mutation is located at the phosphatase active site, we find that it affects the functions of neighboring domains. H93R PTEN binding to phosphatidylserine‐bearing model membranes is 5.6‐fold enhanced in comparison to wild‐type PTEN. In contrast, we find that binding to phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2) model membranes is 2.5‐fold decreased for the mutant PTEN in comparison to wild‐type PTEN. The structural change previously found for wild‐type PTEN upon interaction with PI(4,5)P2, is absent for H93R PTEN. Consistent with the increased binding to phosphatidylserine, we find enhanced plasma membrane association of PTEN‐GFP in U87MG cells. However, this enhanced plasma membrane association does not translate into increased PI(3,4,5)P3 turnover, since in vivo studies show a reduced activity of the H93R PTEN‐GFP mutant. Because the interaction of PI(4,5)P2 with PTENs N‐terminal domain is diminished by this mutation, we hypothesize that the interaction of PTENs N‐terminal domain with the phosphatase domain is impacted by the H93R mutation, preventing PI(4,5)P2 from inducing the conformational change that activates phosphatase activity.

Cholesterol stabilizes fluid phosphoinositide domains

Local accumulation of phosphoinositides (PIPs) is an important factor for a broad range of cellular events including membrane trafficking and cell signaling. The negatively charged phosphoinositide headgroups can interact with cations or cationic proteins and this electrostatic interaction has been identified as the main phosphoinositide clustering mechanism. However, an increasing number of reports show that phosphoinositide-mediated signaling events are at least in some cases cholesterol dependent, suggesting other possible contributors to the segregation of phosphoinositides. Using fluorescence microscopy on giant unilamellar vesicles and monolayers at the air/water interface, we present data showing that cholesterol stabilizes fluid phosphoinositide-enriched phases. The interaction with cholesterol is observed for all investigated phosphoinositides (PI(4)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3) as well as phosphatidylinositol. We find that cholesterol is present in the phosphoinositide-enriched phase and that the resulting phase is fluid. Cholesterol derivatives modified at the hydroxyl group (cholestenone, cholesteryl ethyl ether) do not promote formation of phosphoinositide domains, suggesting an instrumental role of the cholesterol hydroxyl group in the observed cholesterol/phosphoinositide interaction. This leads to the hypothesis that cholesterol participates in an intermolecular hydrogen bond network formed among the phosphoinositide lipids. We had previously reported that the intra- and intermolecular hydrogen bond network between the phosphoinositide lipids leads to a reduction of the charge density at the phosphoinositide phosphomonoester groups (Kooijman et al., 2009). We believe that cholesterol acts as a spacer between the phosphoinositide lipids, thereby reducing the electrostatic repulsion, while participating in the hydrogen bond network, leading to its further stabilization. To illustrate the effect of phosphoinositide segregation on protein binding, we show that binding of the tumor suppressor protein PTEN to PI(5)P and PI(4,5)P2 is enhanced in the presence of cholesterol. These results provide new insights into how phosphoinositides mediate important cellular events.

Elimination of position-change alarms in an Alzheimer's and dementia long-term care facility.

In a long-term care facility, whose residents have been diagnosed with Alzheimer’s disease or dementia, falls are a particularly prominent issue. Technology in health care has continued to evolve and play a larger role in how we care for our patients, even in preventing falls. However, overreliance on these types of technologies may have detrimental effects. In our facility, it was felt that staff reliance on position-change alarms was inappropriate due to the high rate of false alarms associated with these devices. We took a tiered approach to removing position-change alarms from our facility, monitoring the fall incidence rate for a period before, during, and after the elimination of these alarms. After discontinuing their use, we found a decrease in the rate of falls, and a decrease in the percentage of our residents who fell. Staff has easily adapted and reports a calmer, more pleasant environment.

Effects of an Advanced Nursing Job Satisfaction, Turnover Rate, Assistant Education Program on and Clinical Outcomes

Certified nursing assistants (CNAs) have become an integral part of the health care system, spend the most amount of time with residents, and yet have the least amount of training. Recent reports demonstrate that CNAs believe their salary is not commensurate with their workload, and turnover rates in this field have indicated low job satisfaction. In light of these issues, we developed an advanced training program for CNAs in our institution to determine whether investing in our employees would increase job satisfaction and therefore impact turnover rates and clinical outcomes. Although overall job satisfaction improved slightly during the study period, satisfaction with training offered was the only area significantly affected by the intervention; however, significant decreases in turnover rates were observed between the pre- and postintervention periods. Clinical indicators were slightly improved, and the number of resident urinary tract infections decreased significantly. Offering an advanced training program for CNAs may be an effective way to improve morale, turnover rates, and clinical outcomes.

Overuse of Acid-Suppression Therapy at an Urban Tertiary Hospital.

Objectives Acid-suppressive therapy (AST) is widely used for gastrointestinal prophylaxis in hospitalized patients, particularly to prevent stress-related mucosal bleeding in critically ill individuals. Previous reports suggest gross overutilization and continuation of unnecessary therapy, which have been linked to several adverse effects. Methods Retrospective chart review at a large tertiary care hospital, evaluating the use of AST for ulcer prophylaxis in accordance with American Society of Health-System Pharmacists’ guidelines and the less commonly studied nonsteroidal anti-inflammatory drug–related ulcer prophylaxis guidelines. Results A total of 119 (39.3%) patients who received AST met either American Society of Health-System Pharmacists guidelines or nonsteroidal anti-inflammatory drug–related prophylaxis guidelines. Subjects whose AST was appropriate were older, had a higher Charlson Comorbidity Index (P < 0.001), and were more often men (P = 0.005). The rate of discontinuation at discharge was 70.7%; subjects whose prescriptions were not discontinued were older, had a higher Charlson Comorbidity Index, and longer hospital and intensive care unit lengths of stay (P < 0.001). Family medicine physicians, hospitalists, and surgeons prescribed AST similarly; internal medicine physicians demonstrated higher adherence with guidelines than all others (P = 0.02). Adherence varied by etiology; cardiology patients were treated with the highest level of appropriateness (53.6%), whereas those admitted for gastrointestinal diagnoses demonstrated the lowest (17.6%, P = 0.03). Conclusions Inappropriate prescribing of AST for ulcer prophylaxis remains problematic. There may be differences in prescribing habits of physicians of different specialties. Age and comorbidity scores were associated with inappropriate prescribing and continuation of medication at discharge. Interventions to raise prescribers’ awareness of ulcer risk factors in hospitalized patients, both in the intensive care unit and those who are noncritically ill, are needed.