Quantitative super-resolution imaging of pathological aggregates reveals distinct toxicity profiles in different synucleinopathies.

Protein aggregation is a hallmark of major neurodegenerative disorders. Increasing data suggest that smaller aggregates cause higher toxic response than filamentous aggregates (fibrils). However, the size of small aggregates has challenged their detection within biologically relevant environments. Here, we report approaches to quantitatively super-resolve aggregates in live cells and ex vivo brain tissues. We show that Amytracker 630 (AT630), a commercial aggregate-activated fluorophore, has outstanding photophysical properties that enable super-resolution imaging of α-synuclein, tau, and amyloid-β aggregates, achieving ∼4 nm precision. Applying AT630 to AppNL-G-F mouse brain tissues or aggregates extracted from a Parkinson's disease donor, we demonstrate excellent agreement with antibodies specific for amyloid-β or α-synuclein, respectively, confirming the specificity of AT630. Subsequently, we use AT630 to reveal a linear relationship between α-synuclein aggregate size and cellular toxicity and discovered that aggregates smaller than 450 ± 60 nm (aggregate450nm) readily penetrated the plasma membrane. We determine aggregate450nm concentrations in six Parkinson's disease and dementia with Lewy bodies donor samples and show that aggregates in different synucleinopathies demonstrate distinct potency in toxicity. We further show that cell-penetrating aggregates are surrounded by proteasomes, which assemble into foci to gradually process aggregates. Our results suggest that the plasma membrane effectively filters out fibrils but is vulnerable to penetration by aggregates of 450 ± 60 nm. Together, our findings present an exciting strategy to determine specificity of aggregate toxicity within heterogeneous samples. Our approach to quantitatively measure these toxic aggregates in biological environments opens possibilities to molecular examinations of disease mechanisms under physiological conditions

A retrospective analysis of adverse events in the elderly in a tertiary referral center in Mumbai (Bombay), India

Background : Adverse events (AEs) account for significant morbidity and mortality in elderly. Inappropriate medication usage has been regarded as an important factor contributing to AEs in them. Beers criteria are a set of standard criteria for guiding drug prescription in elderly. Objective : To estimate the burden of AEs in the elderly in India and use of Beers criteria for assessing appropriateness of drug prescription in them. Materials and Methods : Data on AEs collected by our tertiary referral center for the years 2005 and 2006 was analyzed. The term ′elderly individuals′ was defined as those aged ≥58 years. An AE was defined as any untoward medical occurrence with a medicinal product in a patient or a clinical investigation, whether or not causally related. Results : In 2005, 321 AEs were reported, and in 2006 there were 673. Of them, those in the elderly constituted 60 (18.9%) and 44 (11.8%) AEs in the 2 years, respectively. About 7 (11.6%) of the AEs in elderly in 2005 were due to medications not fulfilling Beers criteria but none in 2006. Two thirds of the AEs in both years were found to be due to antidiabetics, oral anticoagulants and antiplatelets and drugs with a narrow therapeutic index. Warfarin, digoxin and insulin accounted for a quarter of the AEs. Conclusions : Some commonly used medications account for a major proportion of AEs in elderly. Prospective studies of similar nature could further help us assess the burden of AEs in elderly

Lower Endoscopic Diagnostic Yields Observed in Non-hematemesis Gastrointestinal Bleeding Patients

BACKGROUND: Location of bleeding can present a diagnostic challenge in patients without hematemesis more so than those with hematemesis. AIM: To describe endoscopic diagnostic yields in both hematemesis and non-hematemesis gastrointestinal bleeding patient populations. METHODS: A retrospective analysis on a cohort of 343 consecutively identified gastrointestinal bleeding patients admitted to a tertiary care center emergency department with hematemesis and non-hematemesis over a 12-month period. Data obtained included presenting symptoms, diagnostic lesions, procedure types with diagnostic yields, and hours to diagnosis. RESULTS: The hematemesis group (n = 105) took on average 15.6 h to reach a diagnosis versus 30.0 h in the non-hematemesis group (n = 231), (p = 0.005). In the non-hematemesis group, the first procedure was diagnostic only 53% of the time versus 71% in the hematemesis group (p = 0.02). 25% of patients in the non-hematemesis group required multiple procedures versus 10% in the hematemesis group (p = 0.004). Diagnostic yield for a primary esophagogastroduodenoscopy was 71% for the hematemesis group versus 50% for the non-hematemesis group (p = 0.01). Primary colonoscopies were diagnostic in 54% of patients and 12.5% as a secondary procedure in the non-hematemesis group. A primary video capsule endoscopy yielded a diagnosis in 79% of non-hematemesis patients (n = 14) and had a 70% overall diagnostic rate (n = 33). CONCLUSION: Non-hematemesis gastrointestinal bleeding patients undergo multiple non-diagnostic tests and have longer times to diagnosis and then compared those with hematemesis. The high yield of video capsule endoscopy in the non-hematemesis group suggests a role for this device in this context and warrants further investigation

P2X7R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes

This is the final version. Available from Elsevier via the DOI in this record. Availability of data and materials: Raw data and uncropped blots are included as supplementary data files.The purinoceptor P2X7R is a promising therapeutic target for tauopathies, including Alzheimer's disease (AD). Pharmacological inhibition or genetic knockdown of P2X7R ameliorates cognitive deficits and reduces pathological tau burden in mice that model aspects of tauopathy, including mice expressing mutant human frontotemporal dementia (FTD)-causing forms of tau. However, disagreements remain over which glial cell types express P2X7R and therefore the mechanism of action is unresolved. Here, we show that P2X7R protein levels increase in human AD post-mortem brain, in agreement with an upregulation of P2RX7 mRNA observed in transcriptome profiles from the AMP-AD consortium. P2X7R protein increases mirror advancing Braak stage and coincide with synapse loss. Using RNAScope we detect P2RX7 mRNA in microglia and astrocytes in human AD brain, including in the vicinity of senile plaques. In cultured microglia, P2X7R activation modulates the NLRP3 inflammasome pathway by promoting the formation of active complexes and release of IL-1β. In astrocytes, P2X7R activates NFκB signalling and increases production of the cytokines CCL2, CXCL1 and IL-6 together with the acute phase protein Lcn2. To further explore the role of P2X7R in a disease-relevant context, we expressed wild-type or FTD-causing mutant forms of tau in mouse organotypic brain slice cultures. Inhibition of P2X7R reduced insoluble tau levels without altering soluble tau phosphorylation or synaptic localisation, suggesting a non-cell autonomous role of glial P2X7R on pathological tau aggregation. These findings support further investigations into the cell-type specific effects of P2X7R-targeting therapies in tauopathies.Alzheimer’s Research UKAstra ZenecaMedical Research CouncilNational Health and Medical Research CouncilAlzheimer’s Research UKUK Dementia Research InstituteVan Geest Charitable Foundatio

Clinical behavior of second-generation zirconia monolithic posterior restorations: Two-year results of a prospective study with Ex vivo analyses including patients with clinical signs of bruxism.

OBJECTIVES: This study aimed to investigate (1) clinical outcomes of second-generation zirconia restorations, including patients with bruxism clinical signs, and (2) the material wear process. METHODS: A total of 95 posterior monolithic zirconia tooth-elements in 45 patients were evaluated, 85 on implants and 10 on natural teeth, and 20.3% of restorations being fixed partial dentures (FPDs). Occlusal contact point areas were determined and half of those areas were left unglazed and just polished. Restorations were clinically evaluated following criteria of the World Dental Federation and antagonistic teeth were examined at each evaluation time. Wear ex vivo analyses using SEM and 3D laser profilometry were performed at baseline and after 6 months, 1year, and 2 years respectively, temporarily removing the prostheses. RESULTS: The Kaplan-Meier survival rate of restorations was 93.3% (100% for FPDs) and the success rate was 81.8%, with 4 abutment debondings, 3 tooth-supported crown debondings (provisional cement use), 1 restoration fracture, 1minor chipping, 1 core fracture, 1 root fracture, and 2 implant losses. 80% of catastrophic failures occurred in patients with clinical signs of bruxism (61.7% of patients). Complications were also observed on antagonistic teeth (3 catastrophic failures). Clinical evaluation of the restorations showed good results from the aesthetic, functional, and biological perspective. Zirconia wear was inferior to 15mum, while glaze wear was observed on all occlusal contact areas after 1year. CONCLUSIONS: Monolithic zirconia FPDs are promising but the failure rate of single-unit restorations was not as high as expected in this sample including patients with bruxism clinical signs. CLINICAL SIGNIFICANCE: Within study limitations, FPDs showed excellent short-term results but further research is needed for single-unit restorations considering samples, which do not exclude bruxers. The weak link is the restoration support or the antagonist tooth, one hypothesis being that zirconia stiffness and lack of resilience do not promote occlusal stress damping