High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility.

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.

Effect of diphenhydramine and cetirizine on immediate and late-phase cutaneous allergic reactions in healthy dogs: a randomized, double-blinded crossover study.

BACKGROUND: Currently, there is insufficient evidence to confirm oral diphenhydramine (DPH) efficacy to prevent mast cell degranulation and histamine release in dogs. HYPOTHESIS/OBJECTIVE: To determine and compare the effects oral of DPH and cetirizine on the immediate- and late-phase cutaneous allergic reactions in healthy dogs. ANIMALS: Twelve healthy laboratory beagle dogs. METHODS AND MATERIALS: The study was designed as a randomized, double-blinded crossover study in which each dog served as its own control; twice-daily oral DPH (2.2 mg/kg) or cetirizine (2 mg/kg) were given for six days with a two week washout period. Intradermal injections of histamine, compound 48/80 (positive control) and saline (negative control) were performed on the right thorax 10 days before drug administration (baseline), during oral antihistamine administration on Day 6 and 10 days after last medication dosage. Global wheal scores (GWS) at 20 min and late-phase reactions (LPR) at 6 h post-injection were evaluated by an investigator blinded to the drug and the interventions. RESULTS: Treatment with cetirizine significantly reduced histamine and compound 48/80 GWS and LPR compared to baseline; there was no significant difference for DPH. In all dogs, oral DPH and cetirizine reached plasma concentrations considered therapeutic in people. No adverse effect or behavioural changes were observed during the study. CONCLUSION AND CLINICAL SIGNIFICANCE: In conclusion, oral cetirizine was effective in preventing cutaneous allergic reactions without any obvious adverse effects in dogs. Oral DPH failed to show an inhibitory effect despite attaining plasma drug concentrations that are considered effective in people.