Single-particle cryo-EM at atomic resolution.

The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the ß3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.

Alternative Splicing of the Flip/Flop Cassette and TARP Auxiliary Subunits Engage in a Privileged Relationship That Fine-Tunes AMPA Receptor Gating.

Alternative splicing of AMPA-type glutamate receptors (AMPARs) and allosteric modulation by auxiliary subunits, such as transmembrane AMPAR regulatory proteins (TARPs), are two important mechanisms that regulate the time course of glutamatergic neurotransmission. Prior work has shown that alternative splicing of the flip/flop cassette profoundly regulates TARP γ2 modulation, where flip receptor gating exhibits robust sensitivity to TARPs while flop isoforms are relatively insensitive to TARP modulation. Whether this splice variant-specific regulation extends to other auxiliary subunit families, such as cornichons (CNIHs), GSG1L, or CKAMPs, remains unknown. Here, we demonstrate that CNIH-3 modulation is unaffected by AMPAR alternative splicing due to inherent differences in how CNIH-3 and TARP γ2 modify channel gating. CNIH-3 slows receptor deactivation from the outset of current decay, consistent with structural evidence showing its point of contact at the level of the pore. In contrast, TARP γ2 acts via the KGK site of the ligand-binding domain (LBD) to slow the onset of desensitization. Although GSG1L and CKAMP44 primarily slow recovery from desensitization, their effects on channel gating are unaffected by alternative splicing, further underlining that structural events leading to the onset and recovery from desensitization are separable. Together, this work establishes that alternative splicing and TARP auxiliary subunits form a unique partnership that governs fast glutamatergic signaling at central synapses. Since proteomic studies suggest that all native AMPARs co-assemble with at least two TARPs, allosteric coupling between the flip/flop cassette and TARPs may represent a common design element in all AMPAR complexes of the mammalian brain.SIGNIFICANCE STATEMENT All fast excitatory neurotransmission in the mammalian brain is mediated by AMPA-type glutamate receptors (AMPARs). The time course of AMPAR gating can be regulated by two distinct mechanisms: alternative splicing of the flip/flop cassette and association with auxiliary subunits. Although these regulatory mechanisms have been well studied individually, it is not clear whether alternative splicing impacts auxiliary protein modulation of AMPARs. Here, we compare the four main families of AMPAR auxiliary subunits, transmembrane AMPAR regulatory proteins (TARPs; γ2), cornichons (CNIH-3), GSG1L and CKAMPs (CKAMP44), and find a privileged relationship between TARPs and the flip/flop cassette that is not shared by others. The flop cassette acts as a master switch to override TARP action, and this coupling represents a way to fine-tune AMPAR signaling.

Supportive oncology care at home interventions: protocols for clinical trials to shift the paradigm of care for patients with cancer.

BACKGROUND: Patients with cancer often endure substantial symptoms and treatment toxicities leading to high healthcare utilization, including hospitalizations and emergency department visits, throughout the continuum of their illness. Innovative oncology care models are needed to improve patient outcomes and reduce their healthcare utilization. Using a novel hospital at home care platform, we developed a Supportive Oncology Care at Home intervention to address the needs of patients with cancer. METHODS: We are conducting three trials to delineate the role of Supportive Oncology Care at Home for patients with cancer. The Supportive Oncology Care at Home intervention includes: (1) a hospital at home care model for symptom assessment and management; (2) remote monitoring of daily patient-reported symptoms, vital signs, and body weight; and (3) structured communication with the oncology team. Our first study is a randomized controlled trial to test the efficacy of Supportive Oncology Care at Home versus standard oncology care for improving healthcare utilization, cancer treatment interruptions, and patient-reported outcomes in patients with cancer receiving definitive treatment of their cancer. Participants include adult patients with gastrointestinal and head and neck cancer, as well as lymphoma, receiving definitive treatment (e.g., treatment with curative intent). The second study is a single-arm trial assessing the feasibility and acceptability of the Supportive Oncology Care at Home intervention for hospitalized patients with advanced cancer. Eligible participants include adult patients with incurable cancer who are admitted with an unplanned hospitalization. The third study is a single-arm trial assessing the feasibility and acceptability of the Supportive Oncology Care at Home intervention to enhance the end-of-life care for patients with advanced hematologic malignancies. Eligible participants include adult patients with relapsed or refractory hematologic malignancy receiving palliative therapy or supportive care alone. DISCUSSION: These studies are approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board and are being conducted in accordance with the Consolidated Standards of Reporting Trials statement for non-pharmacological trials. This work has the potential to transform the paradigm of care for patients with cancer by providing them with the necessary support at home to improve their health outcomes and care delivery. TRIAL REGISTRATIONS: NCT04544046, NCT04637035, NCT04690205.

The impact and feasibility of a brief, virtual, educational intervention for home healthcare professionals on Parkinson's Disease and Related Disorders: pilot study of I SEE PD Home.

BACKGROUND: Individuals with advanced Parkinson's Disease (PD) and Parkinson-related disorders (PRD) are frequently referred for home allied therapies and nursing care, yet home healthcare professionals have limited training in PD/PRD. While recognizing the need for such care, patients and families report home healthcare professionals are unfamiliar with these conditions, which may be driven by neurophobia and may contribute to suboptimal care and early termination of services. We sought to determine the feasibility and effects of a virtual, multimodal educational intervention on PD knowledge, confidence, and empathy among home health professionals. METHODS: Home health nurses, occupational therapists, physical therapists and physical therapy assistants, and speech-language pathologists participated in a daylong, virtual symposium on advanced PD/PRD, combining focused lectures, discipline-specific breakout sessions, immersive virtual reality vignettes, and interactive panels with both patients and families, and movement disorders and home healthcare experts. Participants completed online pre- and post-symposium surveys including: demographics; PD/PRD knowledge (0-10 points possible); empathy (Interpersonal Reactivity Index); and 10-point scales of confidence with and attitudes towards individuals with PD/PRD, respectively. Pre-post intervention changes and effect sizes were evaluated with paired t-tests and Cohen's d. We performed qualitative analyses of post-symposium free-text feedback using a grounded theory approach to identify participants' intentions to change their practice. RESULTS: Participants had a mean improvement of 3.1 points on the PD/PRD knowledge test (p < 0.001, d = 1.97), and improvement in confidence managing individuals with PD/PRD (p = 0.0003, d = .36), and no change in empathy. The interactive, virtual format was rated as effective by 95%. Common themes regarding symposium-motivated practice change included: interdisciplinary collaboration; greater involvement and weighting of the patient and caregiver voice in care plans; attention to visit scheduling in relation to patient function; recognition and practical management of the causes of sudden change in PD/PRD, including infections and orthostatic hypotension. CONCLUSIONS: A virtual, multimodal, brief educational pilot intervention improved PD/PRD-specific knowledge and confidence among home healthcare nurses and allied health professionals. Future studies are necessary to test the short- and long-term effects of this intervention more broadly and to investigate the impact of this education on patient and caregiver outcomes.

Factors affecting the implementation of an outcome measurement feedback system in youth mental health settings.

Objective: Measurement feedback systems provide clinicians with regular snapshots of a client's mental health status, which can be used in treatment planning and client feedback. There are numerous barriers to clinicians using outcome measures routinely. This study aimed to investigate factors affecting the use of a measurement feedback system across youth mental health settings. Methods: The participants were 210 clinicians from headspace youth mental health services across Australia. They were surveyed on predictors and use of MyLifeTracker, a routine outcome measure. This was explored through three processes: looking at MyLifeTracker before session, using MyLifeTracker in treatment planning, and providing feedback of MyLifeTracker scores to clients. Results: Clinicians were more likely to look at MyLifeTracker before session, less likely to use it in treatment planning, and least likely to provide MyLifeTracker scores to clients. Each measurement feedback system process had a distinct group of predictors. Perceptions of MyLifeTracker's practicality was the only significant predictor of all three processes. Conclusion: Practically, organisations and supervisors can increase the use of measurement feedback systems through targeted supports.

Shall I tell my mentor? Exploring the mentor-student relationship and its impact on students' raising concerns on clinical placement.

AIMS: To explore student nurses' and nurse mentors' perceptions and experiences of raising concerns on clinical placement and the influence (if any) of their relationship on this process. A secondary aim is to consider the above, from a regulatory perspective in light of current literature and policy developments. BACKGROUND: Raising concerns whilst on clinical placement has been shown to be challenging for student nurses internationally. Registered nurses in the UK (in this case called "nurse mentors") facilitate learning and assessment in practice. However, limited research exists on the influence of the relationship between the nurse mentor and student nurse on the raising concerns process. DESIGN: A qualitative approach was used to undertake secondary thematic analysis of interview data. The primary data set was generated during a PhD study, focusing on the mentor-student dynamic and the possible influence of this relationship on students' raising concerns. METHODS: 30 individual semi-structured interviews were subjected to concurrent and thematic analysis. Interviews were undertaken with student nurses (n = 16) and nurse mentors (n = 14) between April 2016-January 2018. The COREQ 32-item checklist was used during the preparation of this article. FINDINGS: The following three interrelated analytical themes were generated from the data, "developing a mentor-student relationship," "keeping your mentor sweet" and "the mentor role in the raising concerns process." CONCLUSION: Our analysis of participants' experiences and perceptions offers an original contribution to understanding the factors associated with student nurses raising concerns in practice. Student nurses and most mentors believed that students should be encouraged and supported to raise concerns, but students' decisions were strongly influenced by their perceptions of the immediate interpersonal and educational context. Similar barriers to raising concerns have been shown to exist regardless of geographical boundaries, therefore the findings of this study are nationally and internationally relevant. Relevance to clinical practice This study provides new insight into the role of the nurse mentor in supporting students who raise concerns on clinical placements. The majority of the mentor participants believed that students should be encouraged and supported to speak up if they witness poor care or unprofessional behaviour. Focusing on the compexities around raising concerns in mentorship training and updates would rovide a forum for open discussion amongst mentors and educators.

A Multinational Trial of Prasugrel for Sickle Cell Vaso-Occlusive Events.

BACKGROUND: Sickle cell anemia is an inherited blood disorder that is characterized by painful vaso-occlusive crises, for which there are few treatment options. Platelets mediate intercellular adhesion and thrombosis during vaso-occlusion in sickle cell anemia, which suggests a role for antiplatelet agents in modifying disease events. METHODS: Children and adolescents 2 through 17 years of age with sickle cell anemia were randomly assigned to receive oral prasugrel or placebo for 9 to 24 months. The primary end point was the rate of vaso-occlusive crisis, a composite of painful crisis or acute chest syndrome. The secondary end points were the rate of sickle cell-related pain and the intensity of pain, which were assessed daily with the use of pain diaries. RESULTS: A total of 341 patients underwent randomization at 51 sites in 13 countries across the Americas, Europe, Asia, and Africa. The rate of vaso-occlusive crisis events per person-year was 2.30 in the prasugrel group and 2.77 in the placebo group (rate ratio, 0.83; 95% confidence interval, 0.66 to 1.05; P=0.12). There were no significant differences between the groups in the secondary end points of diary-reported events. The safety end points, including the frequency of bleeding events requiring medical intervention, of hemorrhagic and nonhemorrhagic adverse events that occurred while patients were taking prasugrel or placebo, and of discontinuations due to prasugrel or placebo, did not differ significantly between the groups. CONCLUSIONS: Among children and adolescents with sickle cell anemia, the rate of vaso-occlusive crisis was not significantly lower among those who received prasugrel than among those who received placebo. There were no significant between-group differences in the safety findings. (Funded by Daiichi Sankyo and Eli Lilly; ClinicalTrials.gov number, NCT01794000.).

Nutritional Support in Postcardiotomy Shock Extracorporeal Membrane Oxygenation Patients: A Prospective, Observational Study.

BACKGROUND: Despite the 6000 patients treated with extracorporeal membrane oxygenation (ECMO) annually, there is a paucity of data regarding the nutritional management of these patients. MATERIALS AND METHODS: We performed a prospective, observational study of nutrition in postcardiotomy shock patients at our institution. Over a 3.5-year study period, we identified 50 ECMO patients and 225 non-ECMO patients. We identified type, amount, duration, and disruption of nutritional delivery by cohort. The primary outcome was percent of caloric goal met, and secondary outcome was gastrointestinal complications. RESULTS: ECMO patients met less of their caloric (29% versus 40%, P = 0.017) and protein goals (34% versus 55%, P < 0.001) compared with non-ECMO patients. Tube feeds were administered more slowly (26 versus 37 mL/h, P < 0.001) and held for longer (8.3 versus 4.5 h/d, P < 0.001) in ECMO patients because of procedures (60%) and high-dose pressors (20% versus 7%, P < 0.001). Multivariate analysis demonstrated that ECMO decreased caloric intake by 14%, with no detected increased risk of gastrointestinal complications. CONCLUSIONS: -ECMO patients received significantly less nutrition support compared with a non-ECMO population. Tube feed hold deficits could potentially be avoided by utilizing postpyloric tubes to feed through procedures, by eliminating holds for vasopressors/inotropes in hemodynamically stable patients, or by establishing volume-based feeding protocols. Further clinical studies are needed to establish efficacy of these interventions and to understand the impact of nutrition on outcomes in ECMO patients.

"It's often liberating": consumers discuss causal beliefs in the treatment process.

Background: Causal beliefs are thought to influence consumers' perceptions of their mental illness and self-stigma, and may impact treatment and recovery. Understanding consumers' perspective on causes being addressed in treatment is vital to help guide future research and improve services. Aim: This study aimed to explore consumers' views on causes of mental illness being addressed in treatment, along with their subjective experiences of how causes were focused on in their treatment. Methods: Using a qualitative approach, semi-structured interviews were conducted with 23 consumers who self-identified as having a mental illness. A thematic analytic framework was used to identify and analyse themes that emerged within the data. Results: Consumers believed that causes were important and should be addressed in treatment, and identified several associated benefits including increased insight/personal understanding of their illness, symptom management and relapse prevention and reduced self-blame. Negative consequences and considerations were also identified. Conclusion: Causes help consumers make sense of their illness, and consumers would like causes to be addressed in treatment. More research is needed on how mental health professionals can address causes effectively as consumers are currently dissatisfied with how causes were discussed in their treatment.

A life's work in the Children's Court Clinic: the historical development of the Clinic in the forensic field and the politics impacting on the Court Clinic and now on the law.

Over 50 years' experience at the Children's Court Clinic of Victoria informs this valedictory account of its director. Changes over time to the model of service at the Clinic are detailed; an independent practitioner model (psychologist/psychiatrist) with oversight now endures, while the Clinic works exclusively for the Court. Clinic operations, clinicians, clients, reputation, the nature of referrals and the need for kindness in report writing are discussed. An integral theme is the politics of the child protection field, which can affect the Clinic, and lately - through new child law - has also affected the powers of the Court and the lives of children on care applications (and the purview of the Clinic's protection assessments). New child protection legislation is discussed along with its impacts on the care of a growing number of children, signalling the need to revisit the legislation, promote better engagement of families and greatly increase coordinated expert services to them.

Functional Validation of Heteromeric Kainate Receptor Models.

Kainate receptors require the presence of external ions for gating. Most work thus far has been performed on homomeric GluK2 but, in vivo, kainate receptors are likely heterotetramers. Agonists bind to the ligand-binding domain (LBD) which is arranged as a dimer of dimers as exemplified in homomeric structures, but no high-resolution structure currently exists of heteromeric kainate receptors. In a full-length heterotetramer, the LBDs could potentially be arranged either as a GluK2 homomer alongside a GluK5 homomer or as two GluK2/K5 heterodimers. We have constructed models of the LBD dimers based on the GluK2 LBD crystal structures and investigated their stability with molecular dynamics simulations. We have then used the models to make predictions about the functional behavior of the full-length GluK2/K5 receptor, which we confirmed via electrophysiological recordings. A key prediction and observation is that lithium ions bind to the dimer interface of GluK2/K5 heteromers and slow their desensitization.

Study of LRRK2 variation in tauopathy: Progressive supranuclear palsy and corticobasal degeneration.

BACKGROUND: Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common genetic cause of Parkinson's disease (PD). Unexpectedly, tau pathology has been reported in a subset of LRRK2 mutation carriers. METHODS: To estimate the frequency of pathogenic LRRK2 mutations and to evaluate the association of common LRRK2 variants with risk of primary tauopathies, we studied 1039 progressive supranuclear palsy (PSP) and 145 corticobasal degeneration patients from the Mayo Clinic Florida brain bank and 1790 controls ascertained at Mayo Clinic. Sanger sequencing of LRRK2 exons 30, 31, 35, and 41 was performed in all patients, and genotyping of all 17 known exonic variants with minor allele frequency >0.5% was performed in patients and controls. RESULTS: LRRK2 mutational screening identified 2 known pathogenic mutations (p.G2019S and p.R1441C), each in 1 PSP patient, the novel p.A1413T mutation in a PSP patient and the rare p.R1707K mutation in a corticobasal degeneration patient. Both p.A1413T and p.R1707K mutations were predicted damaging by at least 2 of 3 prediction programs and affect evolutionary conserved sites of LRRK2. Association analysis using common LRRK2 variants only showed nominal association of the p.L153L variant with PSP. CONCLUSIONS: Our study confirms the presence of pathogenic and potentially pathogenic LRRK2 mutations in pathologically confirmed primary tauopathies, albeit with low frequency. In contrast to PD, common LRRK2 variants do not appear to play a major role in determining PSP and corticobasal degeneration risk. © 2016 International Parkinson and Movement Disorder Society.

Successful utilization of an electronic pain diary in a multinational phase 3 interventional study of pediatric sickle cell anemia.

BACKGROUND/AIMS: Patients with sickle cell anemia can experience recurrent pain episodes, which affect quality of life. The reported prevalence of pain is higher in studies using patient diaries than in healthcare facility utilization data. Determining Effects of Platelet Inhibition on Vaso-Occlusive Events was a multinational study that assessed the efficacy and safety of prasugrel in reducing the rate of vaso-occlusive events in children with sickle cell anemia (NCT01794000) and included an electronic patient-reported outcome diary to record pain occurrence. We aimed to capture diary completion rates and compliance in children who used the electronic patient-reported outcome diary during the Determining Effects of Platelet Inhibition on Vaso-Occlusive Events study and examine factors contributing to diary completion rates and compliance. METHODS: Daily electronic patient-reported outcome diary data were collected for up to 9 months in Determining Effects of Platelet Inhibition on Vaso-Occlusive Events participants aged 4 to <18 years in Africa, the Americas, Europe, and the Middle East. The questionnaires were available in 11 languages/dialects for collecting subjective (pain intensity, activity interference) and objective (study drug use, analgesic use, school attendance) data. Pain intensity was measured using the Faces Pain Scale-Revised. Data were entered by participants or caregivers and transferred wirelessly each day to a central database. Diary completion rates were the number of daily diary entries divided by the total number of expected daily diary entries. Percentages of participants who were compliant with the diary (≥80% diary completion) were calculated. RESULTS: A total of 311 participants received a diary; 268 provided diary data through Month 9. Diary completion rates and compliance were high throughout the collection period and across all groups and regions, despite no games being included on the device. For subjective data, the overall completion rate was 94.4%, and 92.6% of participants were compliant. For objective data, the overall completion rate was 93.3%, and 89.7% of participants were compliant. Completion rates and compliance differed significantly by age and region and were higher for 4 to <12 year olds and very much higher for participants from Africa and the Middle East. Caregivers almost always entered data for participants <6 years and rarely entered data for participants ≥12 years. Comparing participant-entered and caregiver-entered data, pain intensity score data were more consistent for 4 to <12 year olds than older children, but pain intensity scores for older children were higher when entered by caregivers. CONCLUSION: With appropriate design, participant training, and sufficient monitoring, an electronic patient-reported outcome diary can capture daily sickle cell-related pain data in large multinational studies. Providing a mechanism for caregiver reporting is particularly valuable for participants <6 years and may also facilitate compliance in older children who experience high levels of pain.

Plasma Levels of Risk-Variant APOL1 Do Not Associate with Renal Disease in a Population-Based Cohort.

Two common missense variants in APOL1 (G1 and G2) have been definitively linked to CKD in black Americans. However, not all individuals with the renal-risk genotype develop CKD, and little is known about how APOL1 variants drive disease. Given the association of APOL1 with HDL particles, which are cleared by the kidney, differences in the level or quality of mutant APOL1­HDL particles could be causal for disease and might serve as a useful risk stratification marker. We measured plasma levels of G0 (low risk), G1, and G2 APOL1 in 3450 individuals in the Dallas Heart Study using a liquid chromatography-MS method that enabled quantitation of the different variants. Additionally, we characterized native APOL1­HDL from donors with no or two APOL1 risk alleles by size-exclusion chromatography and analysis of immunopurified APOL1­HDL particles. Finally, we identified genetic loci associated with plasma APOL1 levels and tested for APOL1-dependent association with renal function. Although we replicated the previous association between APOL1 variant status and renal function in nondiabetic individuals, levels of circulating APOL1 did not associate with microalbuminuria or GFR. Furthermore, the size or known components of APOL1­HDL did not consistently differ in subjects with the renal-risk genotype. Genetic association studies implicated variants in loci harboring haptoglobin-related protein (HPR), APOL1, and ubiquitin D (UBD) in the regulation of plasma APOL1 levels, but these variants did not associate with renal function. Collectively, these data demonstrate that the risk of renal disease associated with APOL1 is probably not related to circulating levels of the mutant protein.

'Old and ill': death anxiety and coping strategies influencing health professionals' well-being and dementia care.

OBJECTIVES: This paper examined the psychological factors that influence the well-being of health professionals who work with people with dementia and the types of care (person-centred or task-oriented) provided to these patients. METHODS: The literature was reviewed to identify the factors influencing the well-being of, and types of care provided by, health professionals working with people experiencing dementia. RESULTS: Based on our review of the literature, we propose that approaches to care and the well-being of health professionals working with dementia patients are influenced by the characterisation of dementia as a terminal illness that typically occurs in older people. Drawing upon terror management theory, we argue that exposure to dementia patients is likely to promote awareness of one's own mortality and death-related anxiety. A theoretical model is presented which posits that health professionals working in dementia care draw on experiential avoidance to manage this anxiety. Both death anxiety, and coping strategies, such as experiential avoidance, used to manage this anxiety may influence health professionals' approaches to care of, and attitudes towards, dementia patients. We also suggest a bi-directional relationship between health professionals' approaches to care and well-being. CONCLUSION: Recommendations are made regarding future directions for research and implications for training of health professionals providing direct service or consultation in dementia care.

α6-Containing GABAA Receptors Are the Principal Mediators of Inhibitory Synapse Strengthening by Insulin in Cerebellar Granule Cells.

Activity-dependent strengthening of central synapses is a key factor driving neuronal circuit behavior in the vertebrate CNS. At fast inhibitory synapses, strengthening is thought to occur by increasing the number of GABAA receptors (GABARs) of the same subunit composition to preexisting synapses. Here, we show that strengthening of mouse cerebellar granule cell GABAergic synapses occurs by a different mechanism. Specifically, we show that the neuropeptide hormone, insulin, strengthens inhibitory synapses by recruiting α6-containing GABARs rather than accumulating more α1-containing receptors that are resident to the synapse. Because α6-receptors are targeted to functionally distinct postsynaptic sites from α1-receptors, we conclude that only a subset of all inhibitory synapses are strengthened. Together with our recent findings on stellate cells, we propose a general mechanism by which mature inhibitory synapses are strengthened. In this scenario, α1-GABARs resident to inhibitory synapses form the hardwiring of neuronal circuits with receptors of a different composition fulfilling a fundamental, but unappreciated, role in synapse strengthening.

Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism.

KEY POINTS: Kainate receptor heteromerization and auxiliary subunits, Neto1 and Neto2, attenuate polyamine ion-channel block by facilitating blocker permeation. Relief of polyamine block in GluK2/GluK5 heteromers results from a key proline residue that produces architectural changes in the channel pore α-helical region. Auxiliary subunits exert an additive effect to heteromerization, and thus relief of polyamine block is due to a different mechanism. Our findings have broad implications for work on polyamine block of other cation-selective ion channels. ABSTRACT: Channel block and permeation by cytoplasmic polyamines is a common feature of many cation-selective ion channels. Although the channel block mechanism has been studied extensively, polyamine permeation has been considered less significant as it occurs at extreme positive membrane potentials. Here, we show that kainate receptor (KAR) heteromerization and association with auxiliary proteins, Neto1 and Neto2, attenuate polyamine block by enhancing blocker permeation. Consequently, polyamine permeation and unblock occur at more negative and physiologically relevant membrane potentials. In GluK2/GluK5 heteromers, enhanced permeation is due to a single proline residue in GluK5 that alters the dynamics of the α-helical region of the selectivity filter. The effect of auxiliary proteins is additive, and therefore the structural basis of polyamine permeation and unblock is through a different mechanism. As native receptors are thought to assemble as heteromers in complex with auxiliary proteins, our data identify an unappreciated impact of polyamine permeation in shaping the signalling properties of neuronal KARs and point to a structural mechanism that may be shared amongst other cation-selective ion channels.

Discovery of novel non-steroidal reverse indole mineralocorticoid receptor antagonists.

Reported herein are a series of reverse indoles that represent novel non-steroidal mineralocorticoid receptor (MR) antagonists. The key structure-activity relationships (SAR) are presented below. This reverse indole series is exemplified by a compound that demonstrated efficacy in an acute natriuresis rodent model comparable to marketed MR antagonists, spironolactone and eplerenone.

Design of the DOVE (Determining Effects of Platelet Inhibition on Vaso-Occlusive Events) trial: A global Phase 3 double-blind, randomized, placebo-controlled, multicenter study of the efficacy and safety of prasugrel in pediatric patients with sickle cell anemia utilizing a dose titration strategy.

BACKGROUND: Sickle cell disease (SCD) is an inherited blood disorder characterized by painful vaso-occlusive crises (VOC) with limited treatment options, particularly for children. Emerging knowledge of the pathophysiology of SCD suggests antiplatelet therapies may hold promise for treatment of VOC. Multiple small studies have evaluated antiplatelet agents on the frequency of VOC with varying results, but there has not been an adequately powered study to definitively determine the effect of antiplatelet agents on VOC. Prasugrel, a third-generation thienopyridine that irreversibly inhibits platelet activation and aggregation, is approved in adults with acute coronary syndrome managed with percutaneous coronary intervention. PROCEDURE: Determining Effects of Platelet Inhibition on Vaso-Occlusive Events (DOVE) is a double-blind, randomized study with planned enrollment of >220 children from 14 countries across the Americas, Europe, Asia, and Africa, designed to test the hypothesis that prasugrel reduces the rate of VOC in children with sickle cell anemia (SCA) (homozygous hemoglobin S [HbSS] and hemoglobin Sß(0) thalassemia [HbSß(0)]). Secondary study endpoints include reductions in rate and intensity of vaso-occlusive pain as recorded in daily electronic diaries. Safety assessments include incidence of hemorrhagic events requiring medical intervention and treatment-emergent adverse events. DOVE incorporates a dose-titration strategy to reduce potential bleeding risks inherent with antiplatelet therapy while maintaining blinded treatment assignment. CONCLUSIONS: DOVE presents a unique opportunity to determine whether antiplatelet therapy reduces frequency of patient-reported VOC and daily vaso-occlusive pain in a global study of children with SCA.