Polyunsaturated fatty acids inhibit a pentameric ligand-gated ion channel through one of two binding sites

Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs) but the mechanism of inhibition is not well understood. The PUFA, docosahexaenoic acid (DHA), inhibits agonist responses of the pLGIC, ELIC, more effectively than palmitic acid, similar to the effects observed in the GAB

Open-channel structure of a pentameric ligand-gated ion channel reveals a mechanism of leaflet-specific phospholipid modulation

Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the activated, open-channel state of a pLGIC by specific, state-dependent binding to this site

Proceedings of the 3rd Biennial Conference of the Society for Implementation Research Collaboration (SIRC) 2015: advancing efficient methodologies through community partnerships and team science

It is well documented that the majority of adults, children and families in need of evidence-based behavioral health interventionsi do not receive them [1, 2] and that few robust empirically supported methods for implementing evidence-based practices (EBPs) exist. The Society for Implementation Research Collaboration (SIRC) represents a burgeoning effort to advance the innovation and rigor of implementation research and is uniquely focused on bringing together researchers and stakeholders committed to evaluating the implementation of complex evidence-based behavioral health interventions. Through its diverse activities and membership, SIRC aims to foster the promise of implementation research to better serve the behavioral health needs of the population by identifying rigorous, relevant, and efficient strategies that successfully transfer scientific evidence to clinical knowledge for use in real world settings [3]. SIRC began as a National Institute of Mental Health (NIMH)-funded conference series in 2010 (previously titled the “Seattle Implementation Research Conference”; $150,000 USD for 3 conferences in 2011, 2013, and 2015) with the recognition that there were multiple researchers and stakeholdersi working in parallel on innovative implementation science projects in behavioral health, but that formal channels for communicating and collaborating with one another were relatively unavailable. There was a significant need for a forum within which implementation researchers and stakeholders could learn from one another, refine approaches to science and practice, and develop an implementation research agenda using common measures, methods, and research principles to improve both the frequency and quality with which behavioral health treatment implementation is evaluated. SIRC’s membership growth is a testament to this identified need with more than 1000 members from 2011 to the present.ii SIRC’s primary objectives are to: (1) foster communication and collaboration across diverse groups, including implementation researchers, intermediariesi, as well as community stakeholders (SIRC uses the term “EBP champions” for these groups) – and to do so across multiple career levels (e.g., students, early career faculty, established investigators); and (2) enhance and disseminate rigorous measures and methodologies for implementing EBPs and evaluating EBP implementation efforts. These objectives are well aligned with Glasgow and colleagues’ [4] five core tenets deemed critical for advancing implementation science: collaboration, efficiency and speed, rigor and relevance, improved capacity, and cumulative knowledge. SIRC advances these objectives and tenets through in-person conferences, which bring together multidisciplinary implementation researchers and those implementing evidence-based behavioral health interventions in the community to share their work and create professional connections and collaborations

In vitro calcite crystal morphology is modulated by otoconial proteins otolin-1 and otoconin-90.

Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia

Effect of Otoconial Proteins Fetuin A, Osteopontin, and Otoconin 90 on the Nucleation and Growth of Calcite

We investigated the roles of three proteins associated with the formation of otoconia including fetuin A, osteopontin (OPN), and otoconin 90 (OC90). In situ atomic force microscopy (AFM) studies of the effects of these proteins on the growth of atomic steps on calcite surfaces were performed to obtain insight into their effects on the growth kinetics. We also used scanning electron microscopy to examine the effects of these proteins on crystal morphology. All three proteins were found to be potent inhibitors of calcite growth, although fetuin A promoted growth at concentrations below about 40 nM and only became an inhibitor at higher concentrations. We then used in situ optical microscopy to observe calcite nucleation on films of these proteins adsorbed onto mica surfaces. By measuring the calcite nucleation rate as a function of supersaturation, the value of the interfacial energy that controls the free energy barrier to heterogeneous nucleation was determined for each protein. OPN and OC90 films led to significantly reduced interfacial energies as compared to the value for homogeneous calcite nucleation in bulk solution. The value for fetuin A was equal to that for bulk solution within experimental error. Zeta potential measurements showed all of the proteins possessed negative surface charge and varied in magnitude according to sequence fetuin A > OC90 > OPN. In addition, the interfacial energies exhibited an inverse scaling with the zeta potential. In analogy to previous measurements on polysaccharide films, this scaling indicates the differences between the proteins arise from the effect of protein surface charge on the solution–substrate interfacial energy

Scanning Electron Microscope Images of calcite modified by rhOtolin-1 alone or in combination with rmOC90.

<p>Varying concentrations of rhOtolin-1 and rmOC90 protein, (A) 0 <u>nM</u> rhOtolin-1 and rmOC90; (B) 1000 <u>nM</u> rmOC90; (C) 667 <u>nM</u> rhOtolin-1; (D) 667 <u>nM</u> rhOtolin-1+500 <u>nM</u> rmOC90 were dissolved in 7.5 <u>mM</u> CaCl₂ growth solution. Crystals were grown for 48 hours by slow evaporation of NH₄HCO₃ into growth solution. Crystals were examined with JEOL JSM 6320F Field Emission scanning electron microscopy. Scale Bar, A, C, and D: 25 µm; B: 10 µm.</p

Polymorph determination of calcium carbonate crystals modified by rhOtolin-1 alone or in combination with rmOC90.

<p>Varying concentrations of rhOtolin-1 or rmOC90 protein, (A and B) 1000 <u>nM</u> rmOC90; (C and D) 667 <u>nM</u> rhOtolin-1; (E and F) 667 <u>nM</u> rhOtolin-1plus 500 <u>nM</u> rmOC90; (G) pure growth solution were dissolved in 7.5 mM CaCl₂ growth solution and crystals were grown for 48 hours by slow evaporation of NH₄HCO₃ into growth solution. Raman spectra were collected on pre-selected crystals of CaCO₃ with a HoloLab 5000 Raman microprobe spectrometer system.</p

In situ AFM height images (3.0×3.0 µm) exhibiting absorption of rmOC90 and rhOtolin-1 on freshly cleaved mica.

<p>(A) rmOC90 sustains cluster formation randomly distributed on a mica surface after 48 hours of incubation. (B) rhOtolin-1 self assembled into a honeycomb network on a mica surface after 48 hours of incubation. rhOtolin-1 fibril height measurements reveal the presence of monomeric (arrow 1), dimeric (arrow 2) and trimeric (arrow 3) components. (C) An enlarged area of the matrix from panel B.</p