An AICD-based functional screen to identify APP metabolism regulators

<p>Abstract</p> <p>Background</p> <p>A central event in Alzheimer's disease (AD) is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP), to generate the β-amyloid (Aβ) peptide and the APP intracellular domain (AICD). Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered.</p> <p>Results</p> <p>To validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis.</p> <p>Conclusion</p> <p>Taken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y.</p

A human somatostatin receptor (SSTR3), located on chromosome 22, displays preferential affinity for somatostatin-14 like peptides

AbstractWe report here on the cloning of a human intronless gene encoding a member of the G-protein linked somatostatin (SST) receptor subfamily, termed SSTR3. Based on the deduced amino acid sequence, this gene encodes a 418 amino acid protein displaying sequence similarity, particularly within putative transmembrane domains, with the recently cloned human SSTR1 (62%), SSTR2 (64%) and SSTR4 (58%) receptors. Membranes prepared from COS-7 cells transiently expressing the human SSTR3 gene bound [125I]Leu8,d-Trp22,-Tyr22 SST-28 in a saturable manner with high affinity (~200 pM) and with rank order of potency (d-Trp8 SST-14 > SST-14 > SMS-201-995 > SST-28) indicative of a somatostatin-14 selective receptor. The pharmacological profile of the expressed human SSTR3 receptor is similar but not identical to that reported for the rat homolog [(1992) J. Biol. Chem. 267,20422] where the peptide selectivity is SST-28 ≧ SST-14 XXX SMS-201-995. Northern blot analysis reveals the presence of an SSTR3 mRNA species of ~5 kb in various regions of the monkey brain, including the frontal cortex, cerebellum, medulla, amygdala, with little or no SSTR3 mRNA detectable in brain regions such as the striatum, hippocampus, and olfactory tubercle. The SSTR3 receptor gene maps to human chromosome 22. The existence of at least four distinct human genes encoding somatostatin-14 selective receptors with diverse pharmacological specificities may help to account for some of the multiple biological actions of somatostatin under normal and pathological conditions

Localisation of somatostatin and somatostatin receptors in benign and malignant ovarian tumours

Somatostatin has been identified as having anti-proliferative, anti-angiogenic and pro-apoptotic actions in many tumour systems, and these effects are mediated through a family of five transmembrane G-protein coupled SRIF receptors. Ovarian cancer is the commonest gynaecological malignancy in the UK and maintenance therapy is urgently required. Native somatostatin expression and its receptors sst1,2,3 and 5 were studied with immunohistochemistry in 63 malignant and 35 benign ovarian tumours of various histological types. Fifty-seven out of 63 (90%) of malignant and 26/35 (74%) benign tumours expressed somatostatin. Receptors sst1,2,3 and 5 were expressed variably in epithelial, vascular and stromal compartments for both benign and malignant tumours. Somatostatin was found to correlate significantly with stromal sst1 (P=0.008), epithelial sst1 (P<0.001), stromal sst2 (P=0.019), vascular sst2 (P=0.026), epithelial sst3 (P=0.026), stromal sst5 (P=0.013) and vascular sst5 (P=0.038). Increased expression of native somatostatin correlating with somatostatin receptors in malignant ovarian tumours raises the possibility that either synthetic somatostatin antagonists or receptor agonists may have therapeutic potential

Cerebrovascular events and outcomes in hospitalized patients with COVID-19: The SVIN COVID-19 Multinational Registry

© 2020 World Stroke Organization.[Background]: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has been associated with a significant risk of thrombotic events in critically ill patients. [Aim]: To summarize the findings of a multinational observational cohort of patients with SARS-CoV-2 and cerebrovascular disease. [Methods]: Retrospective observational cohort of consecutive adults evaluated in the emergency department and/or admitted with coronavirus disease 2019 (COVID-19) across 31 hospitals in four countries (1 February 2020–16 June 2020). The primary outcome was the incidence rate of cerebrovascular events, inclusive of acute ischemic stroke, intracranial hemorrhages (ICH), and cortical vein and/or sinus thrombosis (CVST). [Results]: Of the 14,483 patients with laboratory-confirmed SARS-CoV-2, 172 were diagnosed with an acute cerebrovascular event (1.13% of cohort; 1130/100,000 patients, 95%CI 970–1320/100,000), 68/171 (40.5%) were female and 96/172 (55.8%) were between the ages 60 and 79 years. Of these, 156 had acute ischemic stroke (1.08%; 1080/100,000 95%CI 920–1260/100,000), 28 ICH (0.19%; 190/100,000 95%CI 130–280/100,000), and 3 with CVST (0.02%; 20/100,000, 95%CI 4–60/100,000). The in-hospital mortality rate for SARS-CoV-2-associated stroke was 38.1% and for ICH 58.3%. After adjusting for clustering by site and age, baseline stroke severity, and all predictors of in-hospital mortality found in univariate regression (p < 0.1: male sex, tobacco use, arrival by emergency medical services, lower platelet and lymphocyte counts, and intracranial occlusion), cryptogenic stroke mechanism (aOR 5.01, 95%CI 1.63–15.44, p < 0.01), older age (aOR 1.78, 95%CI 1.07–2.94, p ¼ 0.03), and lower lymphocyte count on admission (aOR 0.58, 95%CI 0.34–0.98, p ¼ 0.04) were the only independent predictors of mortality among patients with stroke and COVID-19. [Conclusions]: COVID-19 is associated with a small but significant risk of clinically relevant cerebrovascular events, particularly ischemic stroke. The mortality rate is high for COVID-19-associated cerebrovascular complications; therefore, aggressive monitoring and early intervention should be pursued to mitigate poor outcomes

Replication Data for Taxicab tipping and sunlight

The replication files for "Taxicab tipping and sunlight

Taxicab tipping and sunlight

<div><p>Does the level of sunlight affect the tipping percentage in taxicab rides in New York City? We examined this question using data on 13.82 million cab rides from January to October in 2009 in New York City combined with data on hourly levels of solar radiation. We found a small but statistically significant positive relationship between sunlight and tipping, with an estimated tipping increase of 0.5 to 0.7 percentage points when transitioning from a dark sky to full sunshine. The findings are robust to two-way clustering of standard errors based on hour-of-the-day and day-of-the-year and controlling for day-of-the-year, month-of-the-year, cab driver fixed effects, weather conditions, and ride characteristics. The NYC cab ride context is suitable for testing the association between sunlight and tipping due to the largely random assignment of riders to drivers, direct exposure to sunlight, and low confounding from variation in service experiences.</p></div