Influence of Intrinsic and Lifestyle Factors on the Development of IgE Sensitization.

BACKGROUND: IgE sensitization is a prerequisite for the development of allergic symptoms. The investigation of factors influencing the development of IgE is therefore crucial for understanding the onset of allergic diseases. METHODS: This epidemiological study investigated personal, intrinsic, and lifestyle factors in a nonselected cohort of 501 Austrian adolescents (aged 12-21 years). IgE levels to 112 allergen molecules were analyzed in the serum of participants using the ImmunoCAP ISAC®. Allergic sensitization, IgE levels to single allergens, and ISAC score sums were correlated with results obtained from a questionnaire. RESULTS: In this adolescent cohort, male participants showed a higher sensitization frequency (56.8%) compared to females (50.9%) and significantly increased IgE levels to profilins. Underweight subjects demonstrated a stronger IgE sensitization. Family size inversely correlated with IgE levels to PR-10 allergens, and predominately paternal allergies were a predictive factor for IgE sensitization in the children. Vaccination, breastfeeding, and delivery mode showed no influence, while a highly protective effect was observed for growing up on a farm. Of all of the investigated lifestyle factors, only smoking significantly influenced the risk for IgE development. Participants with moderate frequencies of colds showed increased sensitization levels. CONCLUSION: A hereditary predisposition and lifestyle factors such as a farming environment, smoking, family size, body weight, or frequency of colds significantly influenced the development of allergen-specific IgE in this cohort of adolescents.

Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein.

The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Šresolution. The hexagonal-shaped crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis.

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open-source ImageJ/Fiji plugin.

Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.

Exposure to Indoor Allergens in Different Residential Settings and Its Influence on IgE Sensitization in a Geographically Confined Austrian Cohort.

BACKGROUND: Exposure to indoor allergens is crucial for IgE sensitization and development of allergic symptoms. Residential settings influence the allergen amount in house dust and hence allergic sensitization. Within this study, we investigated allergen exposure and molecule-based IgE levels in a geographically confined region and evaluated the impact of housing, pets and cleaning. METHODS: 501 adolescents from Salzburg, Austria participated in this cross-sectional study. House dust samples were examined regarding major mite, cat, dog, and mold allergens using a multiplex assay. Serum samples of participants were analyzed for specific IgE to Der p 1, Der p 2, Fel d 1, Can f 1 and Alt a 1 using the multiplex array ImmunoCAP ISAC. Information on allergies, living areas, dwelling form (house, flat, farm), pets, and household cleanliness were obtained by a questionnaire. RESULTS: In investigated house dust samples, the concentration of cat allergen was highest while the prevalence of mold allergens was very low. Participants showed IgE sensitization to Der p 1 (13.2%), Der p 2 (18.2%), Fel d 1 (14.4%), Can f 1 (2.4%) and Alt a 1 (2.0%). In alpine regions, lower mite allergen concentrations were detected which correlated with reduced IgE levels. A trend for increased sensitization prevalence from rural to alpine to urban regions was noted. Living on farms resulted in lower sensitization prevalence to mite and cat allergens, even though exposure to mites was significantly elevated. The presence of cats was associated with a lower sensitization rate and IgE levels to cat and mite allergens, and less frequent allergic diseases. Cleaning did not impact allergen concentrations, while IgE reactivity to mites and allergic diseases were more pronounced when living in cleaner homes. CONCLUSION: Allergen exposure to indoor allergens was influenced by setting of homes. Living in a farm environment and having a cat at home showed a protective effect for IgE sensitization and allergies. This cross-sectional study in combination with hereditary and lifestyle factors enables development of risk schemes for a more efficient management and potential prevention of allergic diseases.

Functional mapping of human dynamin-1-like GTPase domain based on x-ray structure analyses.

Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5'-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains.

Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity.

Huntington's disease is an inherited and incurable neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in huntingtin (encoded by HTT). PolyQ length determines disease onset and severity, with a longer expansion causing earlier onset. The mechanisms of mutant huntingtin-mediated neurotoxicity remain unclear; however, mitochondrial dysfunction is a key event in Huntington's disease pathogenesis. Here we tested whether mutant huntingtin impairs the mitochondrial fission-fusion balance and thereby causes neuronal injury. We show that mutant huntingtin triggers mitochondrial fragmentation in rat neurons and fibroblasts of individuals with Huntington's disease in vitro and in a mouse model of Huntington's disease in vivo before the presence of neurological deficits and huntingtin aggregates. Mutant huntingtin abnormally interacts with the mitochondrial fission GTPase dynamin-related protein-1 (DRP1) in mice and humans with Huntington's disease, which, in turn, stimulates its enzymatic activity. Mutant huntingtin-mediated mitochondrial fragmentation, defects in anterograde and retrograde mitochondrial transport and neuronal cell death are all rescued by reducing DRP1 GTPase activity with the dominant-negative DRP1 K38A mutant. Thus, DRP1 might represent a new therapeutic target to combat neurodegeneration in Huntington's disease.

S-Nitrosylation of DRP1 does not affect enzymatic activity and is not specific to Alzheimer's disease.

Mitochondrial dysfunction and synaptic loss are among the earliest events linked to Alzheimer's disease (AD) and might play a causative role in disease onset and progression. The underlying mechanisms of mitochondrial and synaptic dysfunction in AD remain unclear. We previously reported that nitric oxide (NO) triggers persistent mitochondrial fission and causes neuronal cell death. A recent article claimed that S-nitrosylation of dynamin related protein 1 (DRP1) at cysteine 644 causes protein dimerization and increased GTPase activity and is the mechanism responsible for NO-induced mitochondrial fission and neuronal injury in AD, but not in Parkinson's disease (PD). However, this report remains controversial. To resolve the controversy, we investigated the effects of S-nitrosylation on DRP1 structure and function. Contrary to the previous report, S-nitrosylation of DRP1 does not increase GTPase activity or cause dimerization. In fact, DRP1 does not exist as a dimer under native conditions, but rather as a tetramer capable of self-assembly into higher order spiral- and ring-like oligomeric structures after nucleotide binding. S-nitrosylation, as confirmed by the biotin-switch assay, has no impact on DRP1 oligomerization. Importantly, we found no significant difference in S-nitrosylated DRP1 (SNO-DRP1) levels in brains of age-matched normal, AD, or PD patients. We also found that S-nitrosylation is not specific to DRP1 because S-nitrosylated optic atrophy 1 (SNO-OPA1) is present at comparable levels in all human brain samples. Finally, we show that NO triggers DRP1 phosphorylation at serine 616, which results in its activation and recruitment to mitochondria. Our data indicate the mechanism underlying nitrosative stress-induced mitochondrial fragmentation in AD is not DRP1 S-nitrosylation.