IgE alone stimulates mast cell adhesion to fibronectin via pathways similar to those used by IgE + antigen but distinct from those used by Steel factor

We recently demonstrated that immunoglobulin E (IgE), in the absence of cross-linking agents, activates signaling pathways in healthy murine bone marrow-derived mast cells (BMMCs) and that this activation enhances BMMC survival, at least in part, via secretion of autocrine-acting cytokines. We report herein that IgE alone also triggers the adhesion of both BMMCs and connective tissue mast cells (CTMCs) to the connective tissue component, fibronectin (FN). This adhesion occurs to the same extent as that triggered by optimal levels of Steel factor (SF) or IgE + antigen (IgE + Ag) and is mediated by an increased avidity of the integrin very late antigen 5 (VLA-5). Moreover, this IgE-induced adhesion, which is prolonged compared with that elicited by SF or IgE + Ag, requires phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLCgamma), and extracellular calcium but not extracellular-regulated kinase (Erk) or p38. Interestingly, we found, using the calcium channel blocker, 2-APB (2-aminoethoxydiphenyl borate) and Lyn-/- BMMCs that both IgE- and IgE + Ag-induced adhesion to FN require extracellular calcium entry, whereas SF does not. Furthermore, our data suggest that FN acts synergistically with IgE to prolong intracellular phosphorylation events and to enhance IgE-induced inflammatory cytokine production and BMMC survival.     

Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in mammals and a central component of the NO-signaling pathway. The NO-signaling pathways mediate diverse physiological processes, including vasodilation, neurotransmission, and myocardial functions. sGC is a heterodimer assembled from two homologous subunits, each comprised of four domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length sGC. We used single-particle electron microscopy to obtain the structure of the complete sGC heterodimer and determine its higher-order domain architecture. Overall, the protein is formed of two rigid modules: the catalytic dimer and the clustered Per/Art/Sim and heme-NO/O₂-binding domains, connected by a parallel coiled coil at two hinge points. The quaternary assembly demonstrates a very high degree of flexibility. We captured hundreds of individual conformational snapshots of free sGC, NO-bound sGC, and guanosine-5′-[(α,β)-methylene]triphosphate-bound sGC. The molecular architecture and pronounced flexibility observed provides a significant step forward in understanding the mechanism of NO signaling.Nitric oxide (NO) has emerged as an integral signaling molecule in biology. Soluble guanylate cyclase (sGC), the primary receptor of NO in mammals, binds NO via an Fe^II heme cofactor leading to a several hundred-fold increase in 3,5-cyclic guanosine monophosphate (cGMP) synthesis. cGMP then acts as a second messenger, targeting phosphodiesterases, ion-gated channels, and cGMP-dependent protein kinases. These target proteins go on to regulate many critical physiological functions including vasodilation, platelet aggregation, neurotransmission, and myocardial functions (1, 2). Disruptions in NO signaling have been linked to hypertension, erectile dysfunction, neurodegeneration, stroke, and heart disease (3, 4). sGC has been the focus of small-molecule modulators of activity for therapeutic advantage. Riociguat, which is a stimulator of sGC, has recently been approved for treatment of pulmonary hypertension (5). However, the mechanistic details underlying the modulation of sGC catalytic activity by NO and other small molecules remain largely unknown. Determining the structure of the full-length sGC, free and in complex with NO, is therefore a prerequisite to understanding its function and for the design and improvement of therapeutics for treatment of diseases involving the NO/cGMP pathway.The most extensively studied and physiologically relevant isoform of sGC is the 150-kDa heterodimer containing one α1 and one β1 subunit. Each subunit is comprised of four modular domains: the N-terminal heme-NO/O₂–binding (H-NOX), the Per/Arnt/Sim (PAS), the helical, and the C-terminal catalytic domain (Fig. 1). No structure of the complete holoenzyme is available to date, and its absence precludes answering key questions such as how NO occupancy of the N-terminal β H-NOX sensor domain is communicated to the C-terminal cyclase domain. Atomic models of isolated sGC domains have been obtained by X-ray crystallography or homology modeling (6–9) (Fig. 1). The arrangement of and interactions between these domains have been further studied using a variety of techniques, including mutational and truncation studies, Förster resonance energy transfer (FRET), resonance Raman spectroscopy, chemical cross-linking, small-angle X-ray scattering (SAXS), and hydrogen deuterium exchange (HDX) (10–14). These studies have been used to propose a variety of models for the mechanisms of action of sGC, but the lack of a comprehensive 3D structure of the sGC holoenzyme has so far impeded a confident assignment of domain hierarchy.Open in a separate windowFig. 1.sGC domain organization and X-ray crystallographic models. Each subunit contains four modular domains; α1 domains are shown in shades of gray, and β1 domains are shown in color. The H-NOX domain of the β1 subunit contains the heme cofactor, shown in red. Structures for Rattus norvegicus are modeled based on previously solved crystal structures of homologous domains (Materials and Methods). The H-NOX structures are modeled from a standalone Nostoc sp. PCC 7120 H-NOX domain (PDB: 2O09) (6). The PAS and helical domains are modeled on individual domain truncations. The PAS domain is based on the PAS domain from Manduca sexta (PDB: 4GJ4) (7), and the helical domain is based on the β1 R. norvegicus structure (3HLS) (8). The catalytic domain is the Homo sapiens α1β1 crystal structure (PDB: 3UVJ) (9).Here, we used EM to determine the first structure of the heterodimeric sGC holoenzyme. Fitting of the domain crystal structures into the EM reconstruction provides a detailed model for the higher-order architecture and quaternary organization of sGC and is consistent with all reported biochemical data. We obtained hundreds of individual 3D reconstructions of full-length Rattus norvegicus sGC using automated high-throughput single-particle electron microscopy (15, 16). The structures correspond to various snapshots of the enzyme and describe the conformational trajectory of this highly flexible protein. sGC is assembled from two ridged units: the smaller unit comprises the dimeric catalytic domain, and the larger unit is built from the clustering of the PAS and H-NOX domains. The helical domains form a dimeric parallel coiled coil that flexibly connects the two modules. These modules swing freely in relation to each other thereby allowing the structure to access a wide range of conformations. Strikingly, some of these conformations allow the N-terminal H-NOX domain to contact the C-terminal catalytic domain indicating the possibility of a direct allosteric control mechanism. We also obtained reconstructions of sGC in complex with NO as well as with guanosine-5′-[(α,β)-methylene]triphosphate (GPCPP), a noncyclizable analog of the natural substrate GTP, both in the presence and absence of NO. The overall domain architecture and range of the accessible conformations of these complexes are similar to the unbound sGC state, suggesting that ligand binding induces small-scale intradomain conformational changes mediated by flexibility transitions at two key linker points.     

Prevalence of blood donation eligibility in Australia: A population survey

Background

Reliable estimates of the population proportion eligible to donate blood are needed by blood collection agencies to model the likely impact of changes in eligibility criteria and inform targeted population-level education, recruitment, and retention strategies. In Australia, the sole estimate was calculated 10+ years ago. With several subsequent changes to the eligibility criteria, an updated estimate is required.

Study Design and Methods

We conducted a cross-sectional national population survey to estimate eligibility for blood donation. Respondents were aged 18+ and resident in Australia. Results were weighted to obtain a representative sample of the population.

Results

Estimated population prevalence of blood donation eligibility for those aged 18–74 was 57.3% (95% CI 55.3–59.3). The remaining 42.7% (95% CI 40.7–44.7) were either temporarily (25.3%, 95% CI 23.5–27.2) or permanently ineligible (17.4%, 95% CI 16.1–18.9). Of those eligible at the time of the survey, that is, with the UK geographic deferral for variant Creutzfeldt-Jakob disease included, (52.9%, 95% CI 50.8–54.9), 14.2% (95% CI 12.3–16.3) reported donating blood within the previous 2 years. Eligibility was higher among men (62.6%, 95% CI 59.6–65.6) than women (52.8%, 95% CI 50.1–55.6). The most common exclusion factor was iron deficiency/anemia within the last 6 months; 3.8% (95% CI 3.2–4.6) of the sample were ineligible due to this factor alone.

Discussion

We estimate that approximately 10.5 million people (57.3% of 18–74-year-olds) are eligible to donate blood in Australia. Only 14.2% of those eligible at the time of survey reported donating blood within the previous 2 years, indicating a large untapped pool of potentially eligible blood donors.     

A Replication and Extension of the PEERS Intervention: Examining Effects on Social Skills and Social Anxiety in Adolescents with Autism Spectrum Disorders

This study aimed to evaluate the Program for the Education and Enrichment of Relational Skills (PEERS: Laugeson et al. in J Autism Dev Disord 39(4):596–606, 2009). PEERS focuses on improving friendship quality and social skills among adolescents with higher-functioning ASD. 58 participants aged 11–16 years-old were randomly assigned to either an immediate treatment or waitlist comparison group. Results revealed, in comparison to the waitlist group, that the experimental treatment group significantly improved their knowledge of PEERS concepts and friendship skills, increased in their amount of get-togethers, and decreased in their levels of social anxiety, core autistic symptoms, and problem behaviors from pre-to post-PEERS. This study provides the first independent replication and extension of the empirically-supported PEERS social skills intervention for adolescents with ASD.