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Precision medicine requires the translation of basic biological understanding to medical insights, mainly applied to characterization of each unique patient. In many clinical settings, this requires tools that can be broadly used to identify pathology and risks. Patients often present to the intensive care unit with broad phenotypes, including multiple organ dysfunction syndrome (MODS) resulting from infection, trauma, or other disease processes. Etiology and outcomes are unique to individuals, making it difficult to cohort patients with MODS, but presenting a prime target for testing/developing tools for precision medicine. Using multitime point whole blood (cellular/acellular) total transcriptomics in 27 patients, we highlight the promise of simultaneously mapping viral/bacterial load, cell composition, tissue damage biomarkers, balance between syndromic biology versus environmental response, and unique biological insights in each patient using a single platform measurement. Integration of a transcriptome workflow yielded unexpected insights into the complex interplay between host genetics and viral/bacterial specific mechanisms, highlighted by a unique case of virally induced genetics (VIG) within one of these 27 patients. The power of RNA-Seq to study unique patient biology while investigating environmental contributions can be a critical tool moving forward for translational sciences applied to precision medicine. [ABSTRACT FROM AUTHOR]

Recent clinical trials demonstrated strong association between lipid abnormalities and progression of diabetic retinopathy (DR); however, whether circulating lipid levels or retinal lipid metabolism, or both, contributes to the pathogenesis of DR is not well understood. Limited amounts of retinal tissue available from animal models, such as mouse models of DR, have proved. Limited amount of retinal tissue was especially challenging for cholesterol and oxysterol detection as it precluded identification of individual isomers of each nonesterified sterol class. To measure cholesterol and oxysterols from limited retinal tissue samples, we developed extremely sensitive electrospray ionization liquid chromatography high-resolution/accurate mass measurements on an LTQ Orbitrap Velos mass spectrometer that are able to resolve sterols and oxysterols separated by reverse-phase HPLC using a gradient of 85-100% methanol containing 0.1% formic acid, with subsequent detection in positive ionization mode. This methodology will aid in our understanding of diabetes-induced changes in retinal cholesterol and oxysterol metabolism., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Several recent studies suggest that C24-C38 very long chain fatty acids (VLCFA) play an important role in vision, and decreased levels of retina VLCFA have been associated with vision disorders including the onset and progression of diabetic retinopathy in animal models. Traditional methods for VLCFA analysis lack the sensitivity and specificity needed to enable detailed characterization of VLCFA incorporation into complex lipids in tissues and subcellular components. To assess whether decreased VLCFA in diabetic retina are directly implicated in diabetes-induced breakdown of the blood-retinal barrier, we demonstrated the utility of performing untargeted lipid analysis via Orbitrap high resolution/accurate mass MS and MS/MS-based shotgun lipidomics to identify structural lipids containing VLCFA substituents. This comprehensive and highly sensitive approach to untargeted lipid identification enabled us to characterize low-abundance sphingolipids containing very long chain fatty acids from isolated retinal tight junction complexes, as well as VLCFA-containing phospholipids in retinal tissues. To facilitate future biochemical and physiological studies of the roles of VLCFA in blood-retina barrier integrity and maintenance of vision, this chapter describes steps to isolate tight junction complexes from a cell culture model of the outer blood-retinal barrier and perform untargeted Orbitrap high resolution/accurate mass-based lipid analysis to identify VLCFA in tight junctions and retina tissue., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Hepatic inflammation and fibrosis are key elements in the pathogenesis of nonalcoholic steatohepatitis (NASH), a progressive liver disease initiated by excess hepatic lipid accumulation. Lipid droplet protein Perilipin 2 (Plin2) alleviates dietary-induced hepatic steatosis when globally ablated; however, its role in the progression of NASH remains unknown. To investigate this further, we challenged Plin2 liver-specific knockout mice (designated L-KO) and their respective wild-type (WT) controls with a methionine-choline-deficient (MCD) diet for 15 days to induce a NASH phenotype of increased hepatic triglyceride levels through impaired phosphatidylcholine (PC) synthesis and very-low-density lipoprotein (VLDL) secretion. Results on liver weights, body weights, fat tissue mass, and histology in WT and L-KO mice fed the MCD diet revealed signs of hepatic steatosis, fibrosis, and inflammation; however, these effects were blunted in L-KO mice. In addition, levels of PC and VLDL were unchanged, and hepatic steatosis was reduced in L-KO mice fed the MCD diet, due in part to an increase in remodeling of PE to PC via the enzyme phosphatidylethanolamine N-methyltransferase (PEMT). These mice also exhibited decreased hepatic expression of proinflammatory markers cyclooxygenase 2, IL-6, TNF-α, IL-1β, and reduced expression of endoplasmic reticulum (ER) stress proteins C/EBP homologous protein and cleaved caspase-1. Taken together, these results suggest that Plin2 liver-specific ablation alleviates diet-induced hepatic steatosis and inflammation via a PEMT-mediated mechanism that involves compensatory changes in proteins involved in phospholipid remodeling, inflammation, and ER stress that work to alleviate diet-induced NASH. Overall, these findings support a role for Plin2 as a target for NASH therapy., (Copyright © 2016 the American Physiological Society.)

An automated tandem mass spectrometry based analysis employing precursor ion and neutral loss scans in a triple quadrupole mass spectrometer has been employed to identify and quantify changes in the abundances of glycerophospholipids extracted from retina and erythrocytes in a rat streptozotocin model of type 1 diabetes, 6 weeks and 36 weeks following induction of diabetes, compared to age matched nondiabetic controls. The utility of an 'internal standard' method compared to an 'internal standard free' method for quantification of differences in the abundances of specific lipid ions was evaluated in both retina and erythrocyte lipid extracts. In retina, equivalent results were obtained by using the internal standard and 'internal standard free' methods for quantification. In erythrocytes, the two methods of analysis yielded significantly different results, suggesting that factors intrinsic to particular sample types may influence the outcome of label-free lipidome quantification approaches.Overall increases (~25% to ~35%) in the abundances of major retina glycerophospholipid classes were demonstrated in rats at 6 weeks of diabetes, relative to control animals. However, at 36 weeks of diabetes, subsequent overall decreases in retina glycerophosphocholine and glycerophosphoethanolamine abundances of 16% and 33%, respectively, were observed. Additionally, retina and erythrocyte glycerophosphocholine lipids at both 6 week and 36 weeks of diabetes exhibited increased incorporation of linoleic acid((18:2n6)) and a decrease in docosahexaenoic acid (DHA((22:6n3))) content. Finally, an approximately 5-fold increase in the abundances of specific glycated glycerophosphoethanolamine (Amadori-GPEtn) lipids were observed in the retina of 36 week diabetic rats, with a corresponding 1.6 fold increase of Amadori-GPEtn lipids in diabetic erythrocytes.

Despite an increasing recognition of the causative and diagnostic role of lipids in the onset and progression of retinal disease, information on the global lipid profile of the normal retina is quite limited. Here, a "shotgun" tandem mass spectrometry approach involving the use of multiple lipid class-specific precursor ion and neutral loss scan mode experiments has been employed to analyze lipid extracts from normal rat retina, obtained with minimal sample handling prior to analysis. Redundant information for the identification and characterization of molecular species in each lipid class was obtained by complementary analysis of their protonated or deprotonated precursor ions, or by analysis of their various ionic adducts (e.g., Na+, NH4+, Cl-, CH3OCO2-). Notably, "alternative" precursor ion or neutral loss scan mode MS/MS experiments are introduced that were used to identify rat retina lipid molecular species that were not detected using "conventional" scan types typically employed in large-scale lipid-profiling experiments. This chapter outlines the principles and advantages of utilizing complementary/redundant identification of lipid species as a strategy to overcome inherent challenges and limitations of shotgun lipid analysis, and provides examples of the application of this strategy in the analysis of the retina lipidome.