Contaminating transfection complexes can masquerade as small extracellular vesicles and impair their delivery of RNA.

One of the functions of small extracellular vesicles (sEVs) which has received the most attention is their capacity to deliver RNA into the cytoplasm of target cells. These studies have often been performed by transfecting RNAs into sEV-producing cells, to later purify and study sEV delivery of RNA. Transfection complexes and other delivery vehicles accumulate in late endosomes where sEV are formed and over 50% of transfection complexes or delivery vehicles administered to cells are released again to the extracellular space by exocytosis. This raises the possibility that transfection complexes could alter sEVs and contaminate sEV preparations. We found that widely used transfection reagents including RNAiMax and INTERFERin accumulated in late endosomes. These transfection complexes had a size similar to sEV and were purified by ultracentrifugation like sEV. Focusing on the lipid-based transfection reagent RNAiMax, we found that preparations of sEV from transfected cells contained lipids from transfection complexes and transfected siRNA was predominantly in particles with the density of transfection complexes, rather than sEV. This suggests that transfection complexes, such as lipid-based RNAiMax, may frequently contaminate sEV preparations and could account for some reports of sEV-mediated delivery of nucleic acids. Transfection of cells also impaired the capacity of sEVs to deliver stably-expressed siRNAs, suggesting that transfection of cells may alter sEVs and prevent the study of their endogenous capacity to deliver RNA to target cells.

Comprehensive Serum Lipidomics for Detecting Incipient Dementia in Parkinson's Disease.

While a number of methods are available for analyzing lipids, unbiased untargeted lipidomics with high coverage remains a challenge. In this work, we report a study of isotope-standard-assisted liquid chromatography mass spectrometry lipidomics of serum for biomarker discovery. We focus on Parkinson's disease (PD), a neurodegenerative disorder that often progresses to dementia. Currently, the diagnosis of PD is purely clinical and there is limited ability to predict which PD patients will transition to dementia, hampering early interventions. We studied serum samples from healthy controls and PD patients with no clinical signs of dementia. A follow-up 3 years later revealed that a subset of PD patients had transitioned to dementia. Using the baseline samples, we constructed two biomarker panels to differentiate (1) PD patients from healthy controls and (2) PD patients that remained cognitively stable from PD patients with incipient dementia (diagnosed 3 years after sample collection). The proposed biomarker panels displayed excellent performance and may be useful for detecting prodromal PD dementia, allowing early interventions and prevention efforts. The biochemistry of significantly changed lipids is also discussed within the current knowledge of neurological pathologies. Our results are promising and future work using a larger cohort of samples is warranted.

Lipidome Alterations Induced by Cystic Fibrosis, CFTR Mutation, and Lung Function.

Cystic fibrosis is a genetic pathology characterized by abnormal accumulation of mucus in the respiratory, gastrointestinal, and reproductive tracts, caused by mutations in the CFTR gene. Although the classical presentation of the condition is well known, there is still a need for a better characterization of metabolic alterations related to cystic fibrosis and different genotypic mutations. We employed untargeted, comprehensive lipidomics of blood serum samples to investigate alterations in the lipid metabolism related to the pathology, mutation classes, and lung function decline. Six unique biomarker candidates were able to independently differentiate diseased individuals from healthy controls with excellent performance. Cystic fibrosis patients showed dyslipidemia for most lipid subclasses, with significantly elevated odd-chain and polyunsaturated fatty acyl lipids. Phosphatidic acids and diacylglycerols were particularly affected by different genotypic mutation classes. We selected a biomarker panel composed of four lipids, including two ceramides, one sphingomyelin, and one fatty acid, which correctly classified all validation samples from classes III and IV. A biomarker panel of five oxidized lipids was further selected to differentiate patients with reduced lung function, measured as predicted FEV1%. Our results indicate that cystic fibrosis is deeply related to lipid metabolism and provide new clues for the investigation of the disease mechanisms and therapeutic targets.

Development of a NanoLC-MS workflow for high-sensitivity global lipidomic analysis.

Lipidomics focuses on the comprehensive analysis of lipids and their interactions with other molecules. Many biological samples are available in small volumes or limited amounts and display very complex lipid compositions; thus, highly sensitive lipidomic analysis methods are often needed. NanoLC-MS offers extremely high sensitivity, although it is known to be technically more challenging than the conventional LC-MS approach, requiring greater care and maintenance. This work describes the development and optimization of a nanoLC-MS method for routine analysis of the lipidome of small volumes of biological samples. We focused on achieving robust conditions for high sensitivity analysis of complex samples. The nanoLC method, mass spectrometry conditions and sample preparation by liquid-liquid extraction of lipids were fully optimized using serum samples and deuterated lipid standards, including an evaluation of contamination sources. The performance of the method was assessed through the analysis of human and pig sera, as well as cerebrospinal fluid samples from pigs. This method allowed the detection of 9900 to 12,200 features by employing only 1.0-2.5 µL of serum samples and identification of 5842 lipids from 36 subclasses within a 50-min gradient. The method can be easily adapted to other types of biological samples where only limited volumes are available.

Comprehensive Lipidomic and Metabolomic Analysis for Studying Metabolic Changes in Lung Tissue Induced by a Vaccine against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infections in young children. Although the disease may be severe in immunocompromised, young, and elderly people, there is currently no approved vaccine. We previously reported the development and immunological assessment of a novel intranasal vaccine formulation consisting of a truncated version of the RSV fusion protein (ΔF) combined with a three-component adjuvant (TriAdj). Now, we aim to investigate the mechanism of action of the ΔF/TriAdj formulation by searching for metabolic alterations caused by intranasal immunization and the RSV challenge. We carried out untargeted lipidomics and submetabolome profiling (carboxylic acids and amine/phenol-containing metabolites) of lung tissue from ΔF/TriAdj-immunized and nonimmunized, RSV-challenged mice. We observed significant changes of lipids involved in the lung surfactant layer for the nonimmunized animals compared to healthy controls but not for the immunized mice. Metabolic pathways involving the synthesis and regulation of amino acids and unsaturated fatty acids were also modulated by immunization and the RSV challenge. This study illustrates that lipidomic and metabolomic profiling could provide a more comprehensive understanding of the immunological and metabolic alterations caused by RSV and the modulation effected by the ΔF/TriAdj formulation.

Metabolomic and Immunological Profiling of Respiratory Syncytial Virus Infection after Intranasal Immunization with a Subunit Vaccine Candidate.

Respiratory syncytial virus (RSV) is a significant cause of mortality and morbidity in infants, the elderly, immunocompromised individuals, and patients with congenital heart diseases. Despite extensive efforts, a vaccine against RSV is still not available. We have previously reported the development of a subunit vaccine (ΔF/TriAdj) composed of a truncated version of the fusion protein (ΔF) and a polymer-based combination adjuvant (TriAdj). We compared inflammatory responses of ΔF/TriAdj-vaccinated and unvaccinated mice following intranasal challenge with RSV. Rapid and early inflammatory responses were observed in lung samples from both groups but modulated in the vaccinated group 7 days after the viral challenge. The underlying mechanism of action of ΔF/TriAdj was further studied through LC-MS-based metabolomic profiling by using 12C- or 13C-dansyl labeling for the amine/phenol submetabolome. RSV infection predominantly affected the amino acid biosynthesis pathways and urea cycle, whereas ΔF/TriAdj modulated the concentrations of almost all of the altered metabolites. Tryptophan metabolites were significantly affected, including indole, l-kynurenine, xanthurenic acid, serotonin, 5-hydroxyindoleacetic acid, and 6-hydroxymelatonin. The results from the present study provide further mechanistic insights into the mode of action of this RSV vaccine candidate and have important implications in the design of metabolic therapeutic interventions.