The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of phosphoinositide 3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ]. GRP1ʼs pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P 3 with high specificity and affinity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the molecular basis of membrane anchoring by the GRP1 PH domain. Our data reveal a multivalent membrane docking involving PtdIns(3,4,5)P 3 binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P 3 triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P 3 binding and increases membrane penetration as demonstrated by NMR and monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/PtdIns(3,4,5)P 3 vesicles at pH 6.0, but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of which is required for the robust interaction with PtdIns (3,4,5)P 3 and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P 3 -containing vesicles is further amplified (by ?6-fold) by nonspecific electrostatic interactions with phosphatidylserine/phosphatidylinositol. Together, our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.-He, J., R. M. Haney, M. Vora, V. V. Verkhusha, R. V. Stahelin, and T. G. Kutateladze. The signaling lipid phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] is produced in plasma membranes in response to stimulation of cell surface receptors by growth factors and hormones (1). Class I phosphoinositide (PI) 3-kinases phosphorylate the inositol headgroup of the relatively abundant phosphatidylinositol 4,5-bisphosphate [Ptdns(4,5)P 2 ], transiently elevating the level of PtdIns (3,4,5)P 3 from undetectable to nearly 10% of the PtdIns (4,5)P 2 level (2-4). The concentration of PtdIns(3,4,5)P 3 is tightly regulated by the activity of PI 5-and 3-phosphatases, such as SHIP1/2 and PTEN, which dephosphorylate the inositol ring generating PtdIns(3,4)P 2 and PtdIns (4,5)P 2 (5, 6). Despite the transitory accumulation and low concentrations in the plasma membrane, PtdIns (3,4,5)P 3 is implicated in fundamental biological processes including growth, proliferation, migration, and survival of cells (1, 7). The PtdIns(3,4,5)P 3 -mediated signals are primarily recognized and transduced by pleckstrin homology (PH) domain-containing proteins that bind strongly and in some cases specifically to PtdIns(3,4,5)P 3 . Mutations in the PH domains that disrupt or promote PtdIns (3,4,5)P 3 binding cause various signaling disarrays leading to sever...

The FYVE domain associates with phosphatidylinositol 3-phosphate [PtdIns(3)P] in membranes of early endosomes and penetrates bilayers. Here, we detail principles of membrane anchoring and show that the FYVE domain insertion into PtdIns(3)P-enriched membranes and membrane-mimetics is substantially increased in acidic conditions. The EEA1 FYVE domain binds to POPC/POPE/PtdIns(3)P vesicles with a Kd of 49 nM at pH 6.0, however associates ~24 fold weaker at pH 8.0. The decrease in the affinity is primarily due to much faster dissociation of the protein from the bilayers in basic media. Lowering the pH enhances the interaction of the Hrs, RUFY1, Vps27p and WDFY1 FYVE domains with PtdIns(3)P-containing membranes in vitro and in vivo, indicating that pH-dependency is a general function of the FYVE finger family. The PtdIns(3)P binding and membrane insertion of the FYVE domain is modulated by the two adjacent His residues of the R(R/K)HHCRXCG signature motif. Mutation of either His residue abolishes the pH-sensitivity. Both protonation of the His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid-bound form, promoting its penetration and increasing the membrane residence time.

Background Focused cardiac ultrasound (FOCUS) is insensitive for pulmonary embolism (PE). Theoretically, when a clot is large enough to cause vital sign abnormalities, it is more likely to show signs of right ventricular dysfunction on FOCUS, although this has not been well quantified. A rapid bedside test that could quickly and reliably exclude PE in patients with abnormal vital signs could be of high utility in emergency department (ED) patients. We hypothesized that in patients with tachycardia or hypotension, the sensitivity of FOCUS for PE would increase substantially. Methods We performed a prospective observational multicenter cohort study involving a convenience sample of patients from six urban academic EDs. Patients suspected to have PE with tachycardia (heart rate [HR] ≥ 100 beats/min) or hypotension (systolic blood pressure [sBP] < 90 mm Hg) underwent FOCUS before computed tomography angiography (CTA). FOCUS included assessment for right ventricular dilation, McConnell's sign, septal flattening, tricuspid regurgitation, and tricuspid annular plane systolic excursion. If any of these were abnormal, FOCUS was considered positive, while if all were normal, FOCUS was considered negative. We a priori planned a subgroup analysis of all patients with a HR ≥ 110 beats/min (regardless of their sBP). We then determined the diagnostic test characteristics of FOCUS for PE in the entire patient population and in the predefined subgroup, based on CTA as the criterion standard. Inter‐rater reliability of FOCUS was determined by blinded review of images by an emergency physician with fellowship training in ultrasound. Results A total of 143 subjects were assessed for enrollment and 136 were enrolled; four were excluded because they were non–English‐speaking and three because of inability to obtain any FOCUS windows. The mean (±SD) age of enrolled subjects was 56 (±7) years, mean (±SD) HR was 114 (±12) beats/min, and 37 (27.2%) subjects were diagnosed with PE on CTA. In all subjects, FOCUS was 92% (95% confidence interval [CI] = 78% to 98%) sensitive and 64% specific (95% CI = 53% to 73%) for PE. In the subgroup of 98 subjects with a HR ≥ 110 beats/min, FOCUS was 100% sensitive (95% CI = 88% to 100%) and 63% specific (95% CI = 51% to 74%) for PE. There was substantial interobserver agreement for FOCUS (κ = 1.0, 95% CI = 0.31 to 1.0). Conclusions A negative FOCUS examination may significantly lower the likelihood of the diagnosis of PE in most patients who are suspected of PE and have abnormal vital signs. This was especially true in those patients with a HR ≥ 110 beats/min. Our results suggest that FOCUS can be an important tool in the initial evaluation of ED patients with suspected PE and abnormal vital signs.

Interdisciplinary research focused on biological membranes has revealed them as signaling and trafficking platforms for processes fundamental to life. Biomembranes harbor receptors, ion channels, lipid domains, lipid signals, and scaffolding complexes, which function to maintain cellular growth, metabolism, and homeostasis. Moreover, abnormalities in lipid metabolism attributed to genetic changes among other causes are often associated with diseases such as cancer, arthritis and diabetes. Thus, there is a need to comprehensively understand molecular events occurring within and on membranes as a means of grasping disease etiology and identifying viable targets for drug development. A rapidly expanding field in the last decade has centered on understanding membrane recruitment of peripheral proteins. This class of proteins reversibly interacts with specific lipids in a spatial and temporal fashion in crucial biological processes. Typically, recruitment of peripheral proteins to the different cellular sites is mediated by one or more modular lipid-binding domains through specific lipid recognition. Structural, computational, and experimental studies of these lipid-binding domains have demonstrated how they specifically recognize their cognate lipids and achieve subcellular localization. However, the mechanisms by which these modular domains and their host proteins are recruited to and interact with various cell membranes often vary drastically due to differences in lipid affinity, specificity, penetration as well as protein-protein and intramolecular interactions. As there is still a paucity of predictive data for peripheral protein function, these enzymes are often rigorously studied to characterize their lipid-dependent properties. This review summarizes recent progress in our understanding of how peripheral proteins are recruited to biomembranes and highlights avenues to exploit in drug development targeted at cellular membranes and/or lipid-binding proteins.

BackgroundPoint‐of‐care ultrasound (POCUS) competence consists of image acquisition, image interpretation, and clinical integration. Limited data exist on POCUS usage patterns and clinical integration by emergency medicine (EM) residents. We sought to determine actual POCUS usage and clinical integration patterns by EM residents and to explore residents' perspectives on POCUS clinical integration.MethodsWe conducted an explanatory sequential mixed‐methods study at a 4‐year EM residency program. In phase 1, EM ultrasound (US) attendings observed PGY‐4 EM residents' clinical integration of POCUS in real time while on shift in the emergency department (ED). EM US attendings evaluated residents on their intent to perform POCUS, actual POCUS usage, and competence per patient encounter. We used logistic regression to analyze these parameters. In phase 2, we conducted semi‐structured interviews with the observed PGY‐4 residents regarding POCUS usage and clinical integration in the ED. We analyzed qualitative data for themes.ResultsEmergency medicine US attendings observed 10 PGY‐4 EM residents during 254 high‐acuity patient encounters from December 2018 to March 2019. EM US attendings considered POCUS indicated for 26% (66/254) of patients, possibly indicated for 12% (30/254) and not indicated for 62% (158/254). Of the 66 patients for whom EM US attendings considered POCUS indicated, PGY‐4s intended to perform POCUS for patient management 61% (40/66) of the time. PGY‐4s subsequently incorporated POCUS into patient management 73% (48/66) of the time. EM US attendings considered PGY‐4s entrustable to perform POCUS independently 81% (206/254) of the time. We did not find a statistically significant association between shift volume, shift type, or POCUS application, and resident intent to perform POCUS nor competence. Interviews identified three factors that influence PGY‐4's POCUS clinical integration: motivations to use POCUS, barriers to utilization, and POCUS educational methods.ConclusionsThis mixed‐methods study identified a significant gap in POCUS utilization and clinical integration by PGY‐4 EM residents for clinically indicated cases identified by EM US attendings. As clinical integration is a cornerstone of POCUS competence, it is important to ensure that EM resident POCUS curricula emphasize training on clinical utilization and indications for POCUS while on shift in the ED.

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Background Despite the utilization of point‐of‐care ultrasound (POCUS) by trauma surgeons, formal POCUS requirements do not exist for general surgery residents. We sought to evaluate surgery resident comfort with performing and interpreting of Extended‐Focused Assessment for Sonography in Trauma (E‐FAST) scans after a brief educational session. Methods A pre‐survey, sent to PGY‐2 and ‐3 surgical residents before their trauma rotation, evaluated comfort with eight components of the E‐FAST. Residents were then required to watch a 15‐min online video and attend a 1‐h bedside training session moderated by emergency medicine ultrasound fellows during which residents practised E‐FAST image acquisition and interpretation. After the rotation, residents completed a post‐survey evaluating their comfort with the E‐FAST. Results All 27 residents rotating on the trauma service during the 2017–2018 academic year were eligible and, therefore, approached by the study team. Twenty‐one (77.78%) residents completed the pre‐survey, training and post‐survey. Initially, only 52% (13/25) of residents reported feeling confident in performing the E‐FAST. After the session, all (100%) reported feeling confident in their training in E‐FAST. Self‐reported mean comfort with each of the eight components of the E‐FAST showed a statistically significant (P < 0.01) increase from pre–post survey for all residents. Isolating only the residents who initially reported feeling confident in E‐FAST still showed a statistically significant (P < 0.01) increase in mean comfort. Conclusion A single POCUS training programme has been shown to improve surgical residents' comfort in performing and interpreting the E‐FAST. This interdisciplinary approach can enhance collaboration and bridge gaps between emergency medicine and surgery residency programmes.

Point-of-care ultrasound (POCUS) training is well-established in emergency medicine (EM) residency programs in the United States. Demonstration of competency in POCUS has been required for graduates of United States EM residencies since 2001, yet EM resident POCUS education strategies vary widely. 1-4 In 2013, the American College of Emergency Physicians (ACEP), the Society for Academic Emergency Medicine (SAEM), and the Council of Emergency Medicine Residency Directors (CORD) published general guidelines for EM resident POCUS education and assessment. 1 Current EM POCUS education literature suggest that POCUS education and assessment should occur longitudinally and through