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

Characterization of protein cross-links in the coats of Bacillus subtilis spores and analysis of spore coat rinds resistant to degradative enzymes*

The Bacillus subtilis spore coat is a multilayered, proteinaceous structure that consists of more than 50 proteins. Located on the surface of the spore, the coat provides resistance to potentially toxic molecules as well as to predation by the protozoan Tetrahymena thermophila. GerQ, the spore coat protein examined in this study is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an &egr;-(γ-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In this work, we have identified the residues in GerQ that are essential for the transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization. ^ In addition, we have also examined the proteins of the spore coat on a more global level. When coat-defective spores are fed to Tetrahymena, the spores are readily digested. However, a residue termed a rind that looks like coat material remains intact. These rinds are transparent, spherical or hemispherical structures that appear devoid of internal contents. Atomic force microscopy and chemical analyses have now shown that: (i) the rinds are composed of insoluble protein largely derived from both outer and inner spore coat layers; and (ii) the amorphous layer of the outer coat is largely responsible for providing spore resistance to protozoal digestion. ^ *Portions of this dissertation were previously published. See appendix for copyright permission.

Protozoal Digestion of Coat-Defective Bacillus subtilis Spores Produces “Rinds” Composed of Insoluble Coat Protein▿

The Bacillus subtilis spore coat is a multilayer, proteinaceous structure that consists of more than 50 proteins. Located on the surface of the spore, the coat provides resistance to potentially toxic molecules as well as to predation by the protozoan Tetrahymena thermophila. When coat-defective spores are fed to Tetrahymena, the spores are readily digested. However, a residue termed a “rind” that looks like coat material remains. As observed with a phase-contrast microscope, the rinds are spherical or hemispherical structures that appear to be devoid of internal contents. Atomic force microscopy and chemical analyses showed that (i) the rinds are composed of insoluble protein largely derived from both outer and inner spore coat layers, (ii) the amorphous layer of the outer coat is largely responsible for providing spore resistance to protozoal digestion, and (iii) the rinds and intact spores do not contain significant levels of silicon

AFM images of decoated <i>B. subtilis</i> wild-type spores.

<p>Surface ridges extending along the entire length of spores are indicated with light blue arrows in height (a, b) and phase (c, d) images. Patches of rodlet structures are indicated with red arrows in (b–d). The green arrows in (a–c) indicate remnants of the amorphous outermost layer. High resolution height (e) and phase (f) images showing coincidental patches of rodlet structures denoted with red arrows.</p

AFM images of <i>cotH</i> spores.

<p>(a) Height image of spores with surface ridges extending along the entire length of spores (light blue arrows). (b) High-resolution height image of a spore surface area showing the upper surface area (green rectangle) covered with an amorphous layer (green arrow) and rodlets (red arrows). The lower part of the outermost layer-free area (black rectangle) is covered with nanodots (black arrow). One of the surface ridges in (b) is indicated with a light blue arrow. In panel (c), a two–layer inner coat structure (two purple arrows noting the two layers) decorated with nanodots can be seen.</p

2D nucleation and growth of inner spore coat layers.

<p>Panel (a) shows two putative 2D nuclei (purple arrows) on the inner coat surface of a <i>cotO</i> spore. Panel (b) shows 2D nuclei (purple arrows) on the surface of a satellite tobacco mosaic virus (STMV) crystal. This illustration is reproduced with permission from Malkin AJ, Kuznetsov YuG, Land TA, DeYoreo JJ, McPherson A (1995) Mechanisms of growth for protein and virus crystals. Nature Struct Biol. 2: 956–959 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108560#pone.0108560-Rogilo1" target="_blank">[48]</a>. © (1995) Nature Publishing Group. (c) At a relatively small impurity (indicated as small balls) density, the average impurity distance <i>d</i>_imp is larger than <i>d</i>_crit and steps are able to advance. (d) At higher impurity densities, <i>d</i>_imp<<i>d</i>_crit, the curvature of step segments between impurities increases and steps are halted. Panels (c) and (d) are reproduced, with permission from Plomp M, McPherson A, Malkin AJ (2003). Repair of impurity-poisoned protein crystal surfaces. Proteins: Struct, Function, Bioinform 50: 486–495 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108560#pone.0108560-Land1" target="_blank">[82]</a>. © (2003) John Wiley and Sons.</p

<i>B. subtilis</i> strains used in this study.

a<p>Abbreviations: Cm^r, chloramphenicol resistant; Kan^r, kanamycin resistant; Tet^r, tetracycline resistant.</p>b<p>DNA from the strain to the left of the arrow was used to transform the strain to the right of the arrow.</p><p><i>B. subtilis</i> strains used in this study.</p

AFM images of <i>cotE</i> spores.

<p>(a,b) Height images of spores that exhibit surface ridges (light blue arrows), and several spores with an oversize sacculus are labeled with green stars. In (a) a spore with no apparent ridges is indicated with a dark blue arrow. (c) Height image of a multilayer inner coat structure. Three layers are indicated with numbers, and a kink on a step edge is marked with a purple arrow. Several holes in the layered structure are also indicated with purple circles. The hole in the middle circle corresponds to a pinning point on the step. (d) Height image of a multilayer layer structure similar to ones seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108560#pone-0108560-g005" target="_blank">Figs. 5b,c</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108560#pone-0108560-g006" target="_blank">6c</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108560#pone-0108560-g007" target="_blank">7c</a>, as seen on the surface of a trypsin crystal. Similar to the spore coat layers in (c), three layers, kinks and several holes are indicated with purple numbers, arrows and circles, respectively. The insert in (d) is a larger area of the crystal surface seen in (d). The same holes and three layers seen in (d) are indicated in the insert. The red line in (d) denotes the step contour, which was utilized for the measurement of the sinuosity index. Panel (d) is reprinted with permission from Plomp M, McPherson A, Larson SB, Malkin AJ (2001). Growth mechanisms and kinetics of trypsin crystallization. J Phys Chem B 105: 542–551. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108560#pone.0108560-DeYoreo1" target="_blank">[52]</a>. © (2001) American Chemical Society.</p

AFM height images of <i>gerE</i> spores.

<p>(a) <i>gerE</i> spores are either completely (black stars) or partially (grey stars) covered with coat material. (b) A spore that is completely encased in the coat material, and (c) a spore with patches of coat material (grey arrow).</p

AFM images of <i>B. subtilis</i> wild-type spores.

<p>(a) Height and (b) phase images of spores with surface ridges (coincidental in both images) extending along the entire length of spores (several surface ridges noted by light blue arrows). (c) High-resolution height image of an area on a surface of a single spore showing surface ridges (light blue arrow), patches of an amorphous outermost layer (green arrows), and a rodlet layer (red arrows) seen beneath the amorphous layer. (d) A cross section line drawn perpendicular to rodlets (indicated with red arrows in (c)) showing a periodicity of ∼8.2 nm. (e) High-resolution height image of an area on the surface of a single spore showing patches of an amorphous outermost layer (green arrow and green rectangle), and a rodlet layer (red arrows) seen beneath the amorphous layer.</p