Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs

Identification of novel risk loci and causal insights for sporadic Creutzfeldt-Jakob disease: a genome-wide association study

Background: Human prion diseases are rare and usually rapidly fatal neurodegenerative disorders, the most common being sporadic Creutzfeldt-Jakob disease (sCJD). Variants in the PRNP gene that encodes prion protein are strong risk factors for sCJD but, although the condition has similar heritability to other neurodegenerative disorders, no other genetic risk loci have been confirmed. We aimed to discover new genetic risk factors for sCJD, and their causal mechanisms. Methods: We did a genome-wide association study of sCJD in European ancestry populations (patients diagnosed with probable or definite sCJD identified at national CJD referral centres) with a two-stage study design using genotyping arrays and exome sequencing. Conditional, transcriptional, and histological analyses of implicated genes and proteins in brain tissues, and tests of the effects of risk variants on clinical phenotypes, were done using deep longitudinal clinical cohort data. Control data from healthy individuals were obtained from publicly available datasets matched for country. Findings: Samples from 5208 cases were obtained between 1990 and 2014. We found 41 genome-wide significant single nucleotide polymorphisms (SNPs) and independently replicated findings at three loci associated with sCJD risk; within PRNP (rs1799990; additive model odds ratio [OR] 1·23 [95% CI 1·17-1·30], p=2·68 × 10-15; heterozygous model p=1·01 × 10-135), STX6 (rs3747957; OR 1·16 [1·10-1·22], p=9·74 × 10-9), and GAL3ST1 (rs2267161; OR 1·18 [1·12-1·25], p=8·60 × 10-10). Follow-up analyses showed that associations at PRNP and GAL3ST1 are likely to be caused by common variants that alter the protein sequence, whereas risk variants in STX6 are associated with increased expression of the major transcripts in disease-relevant brain regions. Interpretation: We present, to our knowledge, the first evidence of statistically robust genetic associations in sporadic human prion disease that implicate intracellular trafficking and sphingolipid metabolism as molecular causal mechanisms. Risk SNPs in STX6 are shared with progressive supranuclear palsy, a neurodegenerative disease associated with misfolding of protein tau, indicating that sCJD might share the same causal mechanisms as prion-like disorders. Funding: Medical Research Council and the UK National Institute of Health Research in part through the Biomedical Research Centre at University College London Hospitals National Health Service Foundation Trust

A time-resolved proteomic and prognostic map of COVID-19

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease

Clinical and virological characteristics of hospitalised COVID-19 patients in a German tertiary care centre during the first wave of the SARS-CoV-2 pandemic: a prospective observational study

Purpose: Adequate patient allocation is pivotal for optimal resource management in strained healthcare systems, and requires detailed knowledge of clinical and virological disease trajectories. The purpose of this work was to identify risk factors associated with need for invasive mechanical ventilation (IMV), to analyse viral kinetics in patients with and without IMV and to provide a comprehensive description of clinical course. Methods: A cohort of 168 hospitalised adult COVID-19 patients enrolled in a prospective observational study at a large European tertiary care centre was analysed. Results: Forty-four per cent (71/161) of patients required invasive mechanical ventilation (IMV). Shorter duration of symptoms before admission (aOR 1.22 per day less, 95% CI 1.10-1.37, p < 0.01) and history of hypertension (aOR 5.55, 95% CI 2.00-16.82, p < 0.01) were associated with need for IMV. Patients on IMV had higher maximal concentrations, slower decline rates, and longer shedding of SARS-CoV-2 than non-IMV patients (33 days, IQR 26-46.75, vs 18 days, IQR 16-46.75, respectively, p < 0.01). Median duration of hospitalisation was 9 days (IQR 6-15.5) for non-IMV and 49.5 days (IQR 36.8-82.5) for IMV patients. Conclusions: Our results indicate a short duration of symptoms before admission as a risk factor for severe disease that merits further investigation and different viral load kinetics in severely affected patients. Median duration of hospitalisation of IMV patients was longer than described for acute respiratory distress syndrome unrelated to COVID-19

Messungen von dynamischen Tracern und Ozon in der arktischen Stratosphäre: Analyse und Interpretation flugzeuggetragener Submillimeterwellenmessungen

The Airborne Submillimeter Radiometer (ASUR) is a passive heterodyne instrument using a superconducting detector with an instrumental frequency range from 604.3 to 662.3 GHz.Operating onboard an aircraft to avoid absorption by tropospheric water vapor key species of the stratospheric ozone chemistry can be detected for a vertical altitude range from 15 to 55 km. In the winter 1999/2000 the ASUR instrument participated in the THESEO 2000/SOLVE project onboard the NASA research aircraft DC-8. Three deployments were carried out in December 1999, January 2000, and March 2000 with 23 flights total. This last winter was one of the coldest in the last 10 years with sufficiently low temperatures for PSC formation from December 1999 until March 2000. To estimate the chemical ozone loss during the winter chemical and dynamical effects have to be separated. Four different methods have been applied to take diabatic descent into account: isentropes of constant potential temperature, heating rates calculated from ozone measurements, measurements of the dynamical tracer N:sub:2:/sub:O, as well as correlation of ozone and N:sub:2:/sub:O across the vortex edge. Also within this study diabatic descent has been calculated from ASUR N2O measurements and heating rate calculations, using ASUR ozone measurements. Finally the results have been compared to measurements performed by various instruments as well as the SLIMCAT model calculations. An important result of these investigations is that the ozone loss estimated from heating rate calculations as well as from correlation with N2O agree very good with each other. Both methods lead to a chemical loss between 30% and 40% in the lower stratosphere. Taking ASUR´s lower vertical resolution into account, these results also agree very good with sonde measurements and model calculations. The two other methods investigated, isentropes and correlations across the vortex edge tend to underestimate espectively overestimate the chemical ozone loss

Analyse und Interpretation flugzeuggetragener Submillimeterwellenmessungen

Ozone chemistry, tracer measurements, radiometry, airborne,submillimeterwave, Arctic, stratosphere. - The Airborne Submillimeter Radiometer (ASUR) is a passive heterodyne instrument using a superconducting detector with an instrumental frequency range from 604.3 to 662.3 GHz.Operating onboard an aircraft to avoid absorption by tropospheric water vapor key species of the stratospheric ozone chemistry can be detected for a vertical altitude range from 15 to 55 km. In the winter 1999/2000 the ASUR instrument participated in the THESEO 2000/SOLVE project onboard the NASA research aircraft DC-8. Three deployments were carried ...thesi

Measurements of dynamical tracer and ozone in the Arctic stratosphere: Analysis and interpretation of airborne submillimeter measurements

The Airborne Submillimeter Radiometer (ASUR) is a passive heterodyne instrument using a superconducting detector with an instrumental frequency range from 604.3 to 662.3 GHz.Operating onboard an aircraft to avoid absorption by tropospheric water vapor key species of the stratospheric ozone chemistry can be detected for a vertical altitude range from 15 to 55 km. In the winter 1999/2000 the ASUR instrument participated in the THESEO 2000/SOLVE project onboard the NASA research aircraft DC-8. Three deployments were carried out in December 1999, January 2000, and March 2000 with 23 flights total. This last winter was one of the coldest in the last 10 years with sufficiently low temperatures for PSC formation from December 1999 until March 2000. To estimate the chemical ozone loss during the winter chemical and dynamical effects have to be separated. Four different methods have been applied to take diabatic descent into account: isentropes of constant potential temperature, heating rates calculated from ozone measurements, measurements of the dynamical tracer N:sub:2:/sub:O, as well as correlation of ozone and N:sub:2:/sub:O across the vortex edge. Also within this study diabatic descent has been calculated from ASUR N2O measurements and heating rate calculations, using ASUR ozone measurements. Finally the results have been compared to measurements performed by various instruments as well as the SLIMCAT model calculations. An important result of these investigations is that the ozone loss estimated from heating rate calculations as well as from correlation with N2O agree very good with each other. Both methods lead to a chemical loss between 30% and 40% in the lower stratosphere. Taking ASUR´s lower vertical resolution into account, these results also agree very good with sonde measurements and model calculations. The two other methods investigated, isentropes and correlations across the vortex edge tend to underestimate espectively overestimate the chemical ozone loss