BackgroundPlasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs requires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new techniques are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC.MethodsBy using multiple complementary techniques we assessed the performance of the exoEasy kit in isolating ELVs from 2 ml of human plasma and compared it with the SEC qEV column (Izon Science).ResultsOur data show that exoEasy kit isolates a heterogenous mixture of particles with a larger median diameter, broader size range and a higher yield than the SEC qEV column. The exclusive presence of small RNAs in the particles and the total RNA yield were comparable to the SEC qEV column. Despite being less prone to low density lipoprotein contamination than the SEC qEV column, the overall purity of exoEasy kit EV preparations was suboptimal. The low particle-protein ratio, significant amount of albumin, very low levels of exosome-associated proteins and propensity to triglyceride-rich lipoprotein contamination suggest isolation of mainly non-ELVs and co-isolation of plasma proteins and certain lipoproteins by the exoEasy kit.ConclusionsWe demonstrate that performance of exoEasy kit for the isolation of ELVs for biomarker discovery is inferior to the SEC qEV column. This comprehensive evaluation of a novel EV isolation method contributes to the acceleration of the discovery of EV-associated biomarkers and the development of EV-based diagnostics.Electronic supplementary materialThe online version of this article (10.1186/s12967-017-1374-6) contains supplementary material, which is available to authorized users.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2−infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death globally. In order to develop better diagnostics and more effective treatments, research in the past decades has focused on identification of molecular changes in the genome, transcriptome, proteome, and more recently also the metabolome. Phospholipids, which nevertheless play a central role in cell functioning, remain poorly explored. Here, using a mass spectrometry (MS)-based phospholipidomics approach, we profiled 179 phospholipid species in malignant and matched non-malignant lung tissue of 162 NSCLC patients (73 in a discovery cohort and 89 in a validation cohort). We identified 91 phospholipid species that were differentially expressed in cancer versus non-malignant tissues. Most prominent changes included a decrease in sphingomyelins (SMs) and an increase in specific phosphatidylinositols (PIs). Also a decrease in multiple phosphatidylserines (PSs) was observed, along with an increase in several phosphatidylethanolamine (PE) and phosphatidylcholine (PC) species, particularly those with 40 or 42 carbon atoms in both fatty acyl chains together. 2D-imaging MS of the most differentially expressed phospholipids confirmed their differential abundance in cancer cells. We identified lipid markers that can discriminate tumor versus normal tissue and different NSCLC subtypes with an AUC (area under the ROC curve) of 0.999 and 0.885, respectively. In conclusion, using both shotgun and 2D-imaging lipidomics analysis, we uncovered a hitherto unrecognized alteration in phospholipid profiles in NSCLC. These changes may have important biological implications and may have significant potential for biomarker development.

This study evaluates the performance of the Bruker PET insert combined with a BioSpec 70/30 USR MRI scanner using the manufacturer acceptance protocol and the NEMA NU 4-2008 for small animal positron emission tomographs (PET). The PET insert is made of 3 rings of 8 monolithic LYSO crystals (50 x 50 x 10 mm 3 ) coupled to silicon photomultipliers (SiPM) arrays, conferring an axial and transaxial FOV of 15 cm and 8 cm. The MRI performance was evaluated with and without the insert for the following radiofrequency noise, magnetic field homogeneity and image quality. For the PET performance, we extended the NEMA protocol featuring system sensitivity, count rates, spatial resolution and image quality to homogeneity and accuracy for quantification using several MRI sequences (RARE, FLASH, EPI and UTE). The PET insert does not show any adverse effect on the MRI performances. The MR field homogeneity is well preserved (Diameter Spherical Volume, for 20 mm of 1.98 ± 4.78 without and -0.96 ± 5.16 Hz with the PET insert). The PET insert has no major effect on the radiofrequency field. The SNR measurements also do not show major differences. Image ghosting is well within the manufacturer specifications (<2.5%) and no RF noise is visible. Maximum sensitivity of the PET insert is 11.0% at the center of the FOV even with simultaneous acquisition of EPI and RARE. PET MLEM resolution is 0.87 mm (FWHM) at 5 mm off-center of the FOV and 0.97 mm at 25 mm radial offset. The peaks for true/noise equivalent count rates are 410/240 and 628/486 kcps for the rat and mouse phantoms, and are reached at 30.34/22.85 and 27.94/22.58 MBq. PET image quality is minimally altered by the different MRI sequences. The Bruker PET insert shows no adverse effect on the MRI performance and demonstrated a high sensitivity, sub-millimeter resolution and good image quality even during simultaneous MRI acquisition.