Factors Affecting Implementation of the California Childhood Obesity Research Demonstration (CA-CORD) Project, 2013.

IntroductionEcological approaches to health behavior change require effective engagement from and coordination of activities among diverse community stakeholders. We identified facilitators of and barriers to implementation experienced by project leaders and key stakeholders involved in the Imperial County, California, Childhood Obesity Research Demonstration project, a multilevel, multisector intervention to prevent and control childhood obesity.MethodsA total of 74 semistructured interviews were conducted with project leaders (n = 6) and key stakeholders (n = 68) representing multiple levels of influence in the health care, early care and education, and school sectors. Interviews, informed by the Multilevel Implementation Framework, were conducted in 2013, approximately 12 months after year-one project implementation, and were transcribed, coded, and summarized.ResultsRespondents emphasized the importance of engaging parents and of ensuring support from senior leaders of participating organizations. In schools, obtaining teacher buy-in was described as particularly important, given lower perceived compatibility of the intervention with organizational priorities. From a program planning perspective, key facilitators of implementation in all 3 sectors included taking a participatory approach to the development of program materials, gradually introducing intervention activities, and minimizing staff burden. Barriers to implementation were staff turnover, limited local control over food provided by external vendors or school district policies, and limited availability of supportive resources within the broader community.ConclusionProject leaders and stakeholders in all sectors reported similar facilitators of and barriers to implementation, suggesting the possibility for synergy in intervention planning efforts

The crystal structure of Pneumolysin at 2.0 Å resolution reveals the molecular packing of the pre-pore complex

Pneumolysin is a cholesterol-dependent cytolysin (CDC) and virulence factor of Streptococcus pneumoniae. It kills cells by forming pores assembled from oligomeric rings in cholesterol-containing membranes. Cryo-EM has revealed the structures of the membrane-surface bound pre-pore and inserted-pore oligomers, however the molecular contacts that mediate these oligomers are unknown because high-resolution information is not available. Here we have determined the crystal structure of full-length pneumolysin at 1.98 Å resolution. In the structure, crystal contacts demonstrate the likely interactions that enable polymerisation on the cell membrane and the molecular packing of the pre-pore complex. The hemolytic activity is abrogated in mutants that disrupt these intermolecular contacts, highlighting their importance during pore formation. An additional crystal structure of the membrane-binding domain alone suggests that changes in the conformation of a tryptophan rich-loop at the base of the toxin promote monomer-monomer interactions upon membrane binding by creating new contacts. Notably, residues at the interface are conserved in other members of the CDC family, suggesting a common mechanism for pore and pre-pore assembly

A structural perspective of how T cell receptors recognise the CD1 family of lipid antigen-presenting molecules

The CD1 family of antigen-presenting molecules adopt a Major Histocompatibility Complex class I (MHC-I) fold. Whereas MHC molecules present peptides, the CD1 family has evolved to bind self- and foreign-lipids. The CD1 family of antigen-presenting molecules comprises four members, CD1a, CD1b, CD1c, CD1d, that differ in their architecture around the lipid-binding cleft, thereby enabling diverse lipids to be accommodated. These CD1-lipid complexes are recognised by T cell receptors (TCRs) expressed on T cells, either through dual recognition of CD1 and lipid or in a new model whereby the TCR directly contacts CD1, thereby triggering an immune response. Chemical syntheses of lipid antigens, and analogues thereof, have been crucial in understanding the underlying specificity of T cell-mediated lipid immunity. This review will focus on our current understanding of how TCRs interact with CD1-lipid complexes, highlighting how it can be fundamentally different from TCR-MHC-peptide co-recognition

A structural perspective of how T cell receptors recognise the CD1 family of lipid antigen-presenting molecules

The CD1 family of antigen-presenting molecules adopt a Major Histocompatibility Complex class I (MHC-I) fold. Whereas MHC molecules present peptides, the CD1 family has evolved to bind self- and foreign-lipids. The CD1 family of antigen-presenting molecules comprises four members, CD1a, CD1b, CD1c, CD1d, that differ in their architecture around the lipid-binding cleft, thereby enabling diverse lipids to be accommodated. These CD1-lipid complexes are recognised by T cell receptors (TCRs) expressed on T cells, either through dual recognition of CD1 and lipid or in a new model whereby the TCR directly contacts CD1, thereby triggering an immune response. Chemical syntheses of lipid antigens, and analogues thereof, have been crucial in understanding the underlying specificity of T cell-mediated lipid immunity. This review will focus on our current understanding of how TCRs interact with CD1-lipid complexes, highlighting how it can be fundamentally different from TCR-MHC-peptide co-recognition

αβ T cell receptor recognition of self-phosphatidylinositol presented by CD1b

CD1 glycoproteins present lipid-based antigens to T-cell receptors (TCRs). A role for CD1b in T-cell–mediated autoreactivity was proposed when it was established that CD1b can present self-phospholipids with short alkyl chains (∼C34) to T cells; however, the structural characteristics of this presentation and recognition are unclear. Here, we report the 1.9 Å resolution binary crystal structure of CD1b presenting a self-phosphatidylinositol-C34:1 and an endogenous scaffold lipid. Moreover, we also determined the 2.4 Å structure of CD1b–phosphatidylinositol complexed to an autoreactive αβ TCR, BC8B. We show that the TCR docks above CD1b and directly contacts the presented antigen, selecting for both the phosphoinositol headgroup and glycerol neck region via antigen remodeling within CD1b and allowing lateral escape of the inositol moiety through a channel formed by the TCR α-chain. Furthermore, through alanine scanning mutagenesis and surface plasmon resonance, we identified key CD1b residues mediating this interaction, with Glu-80 abolishing TCR binding. We in addition define a role for both CD1b α1 and CD1b α2 molecular domains in modulating this interaction. These findings suggest that the BC8B TCR contacts both the presented phospholipid and the endogenous scaffold lipid via a dual mechanism of corecognition. Taken together, these data expand our understanding into the molecular mechanisms of CD1b-mediated T-cell autoreactivity

αβ T cell receptor recognition of self-phosphatidylinositol presented by CD1b

CD1 glycoproteins present lipid-based antigens to T cell receptors (TCRs). A role for CD1b in T cell-mediated autoreactivity was proposed when it was established that CD1b can present self-phospholipids with short alkyl chains (∼C34) to T cells; however, the structural characteristics of this presentation and recognition are unclear. Here we report the 1.9 Å-resolution binary crystal structure of CD1b presenting a self-phosphatidylinositol-C34:1 (PI) and an endogenous scaffold lipid. Moreover, we also determined the 2.4 Å structure of CD1b-PI complexed to an autoreactive αβ TCR, BC8B. We show the TCR docks above CD1b and directly contacts the presented antigen, selecting for both the phosphoinositol headgroup and glycerol neck region via antigen remodelling within CD1b and allowing lateral escape of the inositol moiety through a channel formed by the TCR α-chain. Furthermore, through alanine scanning mutagenesis and surface plasmon resonance, we identified key CD1b residues mediating this interaction, with Glu-80 abolishing TCR binding. We additionally define a role for both CD1b α1 and α2 molecular domains in modulating this interaction. These findings suggest the BC8B TCR contacts both the presented phospholipid and the endogenous scaffold lipid via a dual mechanism of co-recognition. Taken together, these data expand our understanding into the molecular mechanisms of CD1b-mediated T cell autoreactivity

Enzyme-catalyzed mechanism of isoniazid activation in class I and class III peroxidases.

There is an urgent need to understand the mechanism of activation of the frontline anti-tuberculosis drug isoniazid by the Mycobacterium tuberculosis catalase-peroxidase. To address this, a combination of NMR spectroscopic, biochemical, and computational methods have been used to obtain a model of the frontline anti-tuberculosis drug isoniazid bound to the active site of the class III peroxidase, horseradish peroxidase C. This information has been used in combination with the new crystal structure of the M. tuberculosis catalase-peroxidase to predict the mode of INH binding across the class I heme peroxidase family. An enzyme-catalyzed mechanism for INH activation is proposed that brings together structural, functional, and spectroscopic data from a variety of sources. Collectively, the information not only provides a molecular basis for understanding INH activation by the M. tuberculosis catalase-peroxidase but also establishes a new conceptual framework for testing hypotheses regarding the enzyme-catalyzed turnover of this compound in a number of heme peroxidases

CD1a autoreactive T cells recognize natural skin oils that function as headless antigens

CD1a autoreactive T cells are common in human blood and skin, but the search for natural autoantigens has been confounded by background T cell responses to CD1 proteins and self lipids. After capturing CD1a-lipid complexes, we gently eluted ligands, while preserving unliganded CD1a for testing lipids from tissues. CD1a released hundreds of ligands of two types. Inhibitory ligands were ubiquitous membrane lipids with polar headgroups, whereas stimulatory compounds were apolar oils. CD1a autoantigens naturally accumulate in epidermis and sebum, where they were identified as squalene and skin waxes. T cell activation by skin oils suggests that headless mini-antigens nest within CD1a and displace non-antigenic resident lipids with large head groups. Oily autoantigens naturally coat the skin's surface, pointing to a new mechanism of barrier immunity