Delineating the Mechanisms of Misfolded Endoplasmic Reticulum (ER) Luminal Protein Retrotranslocation for ER-Associated Degradation

Secreted, plasma membrane, and resident proteins of the secretory pathway are synthesized in the endoplasmic reticulum (ER) where they undergo post-translational modifications, oxidative folding, and subunit assembly in tightly monitored processes. An ER quality control (ERQC) system oversees protein maturation and ensures that only those reaching their native state will continue trafficking into the secretory pathway to reach their final destinations. Proteins that fail quality control must be recognized and eliminated to maintain ER proteostasis. The ER-associated degradation (ERAD) was discovered nearly 30 years ago and entails the identification of improperly matured secretory pathway proteins and their retrotranslocation to the cytosol for degradation by the ubiquitin-proteasome system. While multiple ER-Associated Degradation (ERAD) components have been identified and their roles elucidated, it remains less clear how folded domains on ERAD clients complicate their extraction from the ER and degradation. To address this, we used several luminal ERAD substrates with well-defined structural properties. Deglycosylation and digitonin permeabilization assays were used to monitor client extraction from the ER. Our fully unfolded clients were released into the cytosol when the proteasome was inhibited. Conversely, ERAD clients possessing a single folded domain were completely retrotranslocated but were not released from the ER membrane without proteasome function. These clients were fully reduced, but still retained structure in their folded domain. Overall, our data argue that ubiquitinated clients with well-folded domains can be dislocated from the ER in a p97-dependent manner, but proteasome activity is required to fully release them from the cytosolic side of the ER membrane and the ERAD extraction machinery.To decipher key steps in the extraction of non-glycosylated ER luminal proteins, and to determine how Hrd1, p97, and the proteasome contribute to the retrotranslocation of clients, we developed a novel in-cell biotin-based reporter system. The bacterial BirA biotin ligase was tethered to FAM8A1, a component of the retrotranslocon that interacts with Hrd1, thus positioning the biotin ligase near the cytosolic exit site. The non-secreted immunoglobulin (Ig) κ light chain (NS1 κ LC), which is composed of a poorly folded N-terminal domain and a well-folded C-terminal domain was used in the biotinylation assays. NS1 was modified at either the N- (BAP-NS1) or C- (NS1-BAP) terminus with a biotin acceptor peptide (BAP) allowing us to detect cytosolic exposure of both ends of this ERAD client. We established that BAP-tagged NS1 constructs were still ERAD substrates and that BirA-tagged FAM8A1 still assembled with the retrotranslocon. We found that both termini of NS1 were readily biotinylated when the proteasome was inhibited by MG132 treatment, or when dominant negative constructs of Hrd1 and p97 were expressed. To differentiate between full extraction of NS1 and partial exposure of the termini to the cytosolic side of the ER, we permeabilize the plasma membrane with digitonin. Cytosolic proteins were released, but both biotinylated and non-biotinylated NS1 remained cell-associated. This argues that the biotinylated light chain was not fully extracted from the ER when NS1 degradation was inhibited with either MG132 or with the Hrd1 and p97 mutants. The digitonin permeabilized cells expressing BAP-NS1 were treated with proteinase K to assess how much of this client was exposed to the cytosolic side of the ER. We found that the NS1 κ light chain was only partially extracted when cytosolic ERAD components (proteasome and p97) were impaired. These data indicate that the termini of this ERAD client can be inserted into the retrotranslocon and “sample” the cytosolic side but require the action of an E3 ligase, the p97 complex and the proteasome to be fully extracted

Gerontechnology: Providing a Helping Hand When Caring for Cognitively Impaired Older Adults—Intermediate Results from a Controlled Study on the Satisfaction and Acceptance of Informal Caregivers

The incidence of cognitive impairment in older age is increasing, as is the number of cognitively impaired older adults living in their own homes. Due to lack of social care resources for these adults and their desires to remain in their own homes and live as independently as possible, research shows that the current standard care provisions are inadequate. Promising opportunities exist in using home assistive technology services to foster healthy aging and to realize the unmet needs of these groups of citizens in a user-centered manner. ISISEMD project has designed, implemented, verified, and assessed an assistive technology platform of personalized home care (telecare) for the elderly with cognitive impairments and their caregivers by offering intelligent home support services. Regions from four European countries have carried out long-term pilot-controlled study in real-life conditions. This paper presents the outcomes from intermediate evaluations pertaining to user satisfaction with the system, acceptance of the technology and the services, and quality of life outcomes as a result of utilizing the services

Report on TID algorithms

This deliverable presents the TID detection algorithms as improved in response to design principles stated in T2.1 and their testing in the lab environment, verification against measurements taken during quiet and disturbed periods of time, benchmarking for their transition to operations, and final validation to the user requirements of accuracy, timeliness, and coverage.TechTIDE project, funded by the European Commission Horizon 2020 research and innovation program [AD-1], will establish a pre-operational system to demonstrate reliability of a set of TID (Travelling Ionospheric Disturbances) detection methodologies to issue warnings of the occurrence of TIDs over the region extending from Europe to South Africa. TechTIDE warning system will estimate the parameters that specify the TID characteristics and the inferred perturbation, with all additional geophysical information to the users to help them assess the risks and to develop mitigation techniques, tailored to their application. This document is TechTIDE D2.2 “Report on the TID algorithms” and it is an output of TechTIDE Task 2.2 (Development of the TID identification algorithms and products) of the WP2 (TID identification methodologies) which has the final goal to release the basic algorithms for the TID identification and to test a first version of the value-added products for implementation in the TechTIDE warning system. The document highlights four aspects of the TID algorithm release process, (1) Developmentbased on the concept, techniques, and algorithms as stated in TechTIDE D2.1, (2) Verification, an internal testing process that ensures algorithm correctness, (3) Benchmarkingneeded to prepare algorithms to transition to operations, and (4) Validation, an external process of ensuring that developed algorithms are compliant with the stated end user expectations.Postprint (published version

Isolation of neural stem and oligodendrocyte progenitor cells from the brain of live rats

Summary Postnatal brain neural stem and progenitor cells (NSPCs) cluster in anatomically inaccessible stem cell niches, such as the subependymal zone (SEZ). Here, we describe a method for the isolation of NSPCs from live animals, which we term “milking.” The intracerebroventricular injection of a release cocktail, containing neuraminidase, integrin-β1-blocking antibody, and fibroblast growth factor 2, induces the controlled flow of NSPCs in the cerebrospinal fluid, where they are collected via liquid biopsies. Isolated cells retain key in vivo self-renewal properties and their cell-type profile reflects the cell composition of their source area, while the function of the niche is sustained even 8 months post-milking. By changing the target area more caudally, we also isolate oligodendrocyte progenitor cells (OPCs) from the corpus callosum. This novel approach for sampling NSPCs and OPCs paves the way for performing longitudinal studies in experimental animals, for more in vivo relevant cell culture assays, and for future clinical neuro-regenerative applications

Consumption of predefined 'Nordic' dietary items in ten European countries - an investigation in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort.

Health-beneficial effects of adhering to a healthy Nordic diet index have been suggested. However, it has not been examined to what extent the included dietary components are exclusively related to the Nordic countries or if they are part of other European diets as well, suggesting a broader preventive potential. The present study describes the intake of seven a priori defined healthy food items (apples/pears, berries, cabbages, dark bread, shellfish, fish and root vegetables) across ten countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) and examines their consumption across Europe

Голодомор 1932 –– 1933 рр. в Україні як геноцид

Given its fundamental role in development and cancer, the Wnt-beta-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively active mutants of beta-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathwa

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection