The field of lipidomics has been significantly advanced by mass spectrometric analysis. The distinction and quantitation of the unsaturated lipid isomers, however, remain a long-standing challenge. In this study, we have developed an analytical tool for both identification and quantitation of lipid C=C location isomers from complex mixtures using online Paternò-Büchi reaction coupled with tandem mass spectrometry (MS/MS). The potential of this method has been demonstrated with an implementation into shotgun lipid analysis of animal tissues. Among 96 of the unsaturated fatty acids and glycerophospholipids identified from rat brain tissue, 50% of them were found as mixtures of C=C location isomers; for the first time, to our knowledge, the quantitative information of lipid C=C isomers from a broad range of classes was obtained. This method also enabled facile cross-tissue examinations, which revealed significant changes in C=C location isomer compositions of a series of fatty acids and glycerophospholipid (GP) species between the normal and cancerous tissues.Paternò-Büchi reaction | glycerophospholipids | photochemical reaction | lipid biomarkers | cancerous tissue analysis L ipids play a multitude of crucial roles in biological systems by serving as building blocks of cell membranes, sources for energy storage, and media for signal transduction (1-3). Unveiling the mechanisms and networks behind lipid homeostasis calls for sensitive, quantitative, and molecularly specific lipid analysis (4). The recent advancement in mass spectrometry (MS) for bioanalysis has enabled the field of lipidomics (5, 6) by allowing global identification and quantitation of lipid species at high speed (7-9) and providing information of lipid-lipid (10, 11) and lipidprotein interactions (12, 13) at systems level. These capabilities further expedite research on lipid biomarker discovery and metabolite flux analysis (14-16). Among many analytical figures of merit, high molecular specificity is a distinct feature of the MSbased approaches. Rich structural information of lipids in complex biological samples can now be routinely obtained, including the classes of the lipids, fatty acyl/alkyl composition, and even the sn positions of the fatty acyl/alkyl chains (17-19). The locations of the carbon-carbon double bonds (C=C) in the lipids, however, have rarely been identified using commercial MS systems and therefore have been either assumed or not reported in a large body of literatures for lipid study (20).The MS/MS methods, especially those involving low-energy collision-induced dissociation (CID), have not been effective in locating C=C bond locations, which is due to the high bond dissociation energies associated with cleaving a C=C bond. Without characteristic fragment ions produced, the C=C locations cannot be determined using MS/MS. To tackle this problem, two MS approaches have been explored, each with successes achieved but also with limitations observed. The first one employs C=C specific chemical derivatizations before MS analysis. T...
Tumour-derived extracellular vesicles (EVs) are of increasing interest as a resource of diagnostic biomarkers. However, most EV assays require large samples, are time-consuming, low-throughput and costly, and thus impractical for clinical use. Here, we describe a rapid, ultrasensitive and inexpensive nanoplasmon-enhanced scattering (nPES) assay that directly quantifies tumor-derived EVs from as little as 1 μL of plasma. The assay uses the binding of antibody-conjugated gold nanospheres and nanorods to EVs captured by EV-specific antibodies on a sensor chip to produce a local plasmon effect that enhances tumour-derived EV detection sensitivity and specificity. We identified a pancreatic cancer EV biomarker, ephrin type-A receptor 2 (EphA2), and demonstrate that an nPES assay for EphA2-EVs distinguishes pancreatic cancer patients from pancreatitis patients and healthy subjects. EphA2-EVs were also informative in staging tumour progression and in detecting early responses to neoadjuvant therapy, with better performance than a conventional enzyme-linked immunosorbent assay. The nPES assay can be easily refined for clinical use, and readily adapted for diagnosis and monitoring of other conditions with disease-specific EV biomarkers.
Neurological manifestations are a significant complication of coronavirus disease (COVID-19), but underlying mechanisms aren’t well understood. The development of animal models that recapitulate the neuropathological findings of autopsied brain tissue from patients who died from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are critical for elucidating the neuropathogenesis of infection and disease. Here, we show neuroinflammation, microhemorrhages, brain hypoxia, and neuropathology that is consistent with hypoxic-ischemic injury in SARS-CoV-2 infected non-human primates (NHPs), including evidence of neuron degeneration and apoptosis. Importantly, this is seen among infected animals that do not develop severe respiratory disease, which may provide insight into neurological symptoms associated with “long COVID”. Sparse virus is detected in brain endothelial cells but does not associate with the severity of central nervous system (CNS) injury. We anticipate our findings will advance our current understanding of the neuropathogenesis of SARS-CoV-2 infection and demonstrate SARS-CoV-2 infected NHPs are a highly relevant animal model for investigating COVID-19 neuropathogenesis among human subjects.
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