Depletion of abundant plasma proteins for extracellular vesicle proteome characterization: benefits and pitfalls.

Blood extracellular vesicles (EVs) play essential roles in cell-cell communication and their molecular cargo is a promising source of disease biomarkers. However, proteomic characterization of plasma-derived EVs is challenged by the presence of highly abundant plasma proteins, which limits the detection of less abundant proteins, and by the low number of EVs in biological fluids. The aim of this study was to investigate if the removal of abundant plasma proteins prior to EV isolation could improve plasma-derived EV characterization by LC-MS/MS and expand the proteome coverage. Plasma depletion was performed using a single-use spin column and EVs were isolated from only 100 µL of non-depleted and depleted plasma by size exclusion chromatography. Afterwards, EVs were characterized by nanoparticle tracking analysis and mass spectrometry-based proteomics using a data-independent acquisition approach. Depleted plasma-derived EVs had higher particle concentrations and particle-to-protein ratios. Depletion did increase the protein coverage with a higher number of identifications in EVs from depleted plasma (474 proteins) than from non-depleted (386 proteins). However, EVs derived from non-depleted plasma carried a slightly higher number of common EV markers. Overall, our findings suggest that plasma depletion prior to EV isolation by size exclusion chromatography provides higher yield and protein coverage, but slightly lower identification of EV markers. This study also showed the possibility to characterize the proteome of EVs derived from small plasma volumes, encouraging the clinical feasibility of the discovery of EV biomarkers.

H-FABP as a Biomarker in Transient Ischemic Attack.

The study investigates the utility of heart fatty-acid binding protein (H-FABP) in distinguishing TIA from mimics. Data from 175 patients from the StrokeChip multicenter study was retrospectively analyzed. H-FABP level was measured using a rapid point-of-care test. Findings revealed that H-FABP levels were higher in individuals with TIA compared to mimics [3.10 ng/mL (IQR 2.13-4.78) vs. 1.70 ng/mL (IQR 1.23-2.38)] (p < 0.001). The diagnostic performance of H-FABP, assessed using the area under the curve operating characteristic curve (AUC) was 0. 83 (95% CI = 0.76-0.90) for the final model, indicating good discriminative ability. The PanelomiX determined that a combined cutoff of > 1.85 ng/ml for H-FABP, age > 42.5 years, and baseline NIHSS > 3.5 had a 100% of sensitivity and 23.30% of specificity. The study suggests that H-FABP has potential as a TIA diagnostic biomarker. The rapid application of POCT's for H-FABP measurement supports its potential use in emergency departments and primary care settings.

Neurovascular Unit-Derived Extracellular Vesicles: From Their Physiopathological Roles to Their Clinical Applications in Acute Brain Injuries.

Extracellular vesicles (EVs) form a heterogeneous group of membrane-enclosed structures secreted by all cell types. EVs export encapsulated materials composed of proteins, lipids, and nucleic acids, making them a key mediator in cell-cell communication. In the context of the neurovascular unit (NVU), a tightly interacting multicellular brain complex, EVs play a role in intercellular communication and in maintaining NVU functionality. In addition, NVU-derived EVs can also impact peripheral tissues by crossing the blood-brain barrier (BBB) to reach the blood stream. As such, EVs have been shown to be involved in the physiopathology of numerous neurological diseases. The presence of NVU-released EVs in the systemic circulation offers an opportunity to discover new diagnostic and prognostic markers for those diseases. This review outlines the most recent studies reporting the role of NVU-derived EVs in physiological and pathological mechanisms of the NVU, focusing on neuroinflammation and neurodegenerative diseases. Then, the clinical application of EVs-containing molecules as biomarkers in acute brain injuries, such as stroke and traumatic brain injuries (TBI), is discussed.

Morphine-induced modulation of Nrf2-antioxidant response element signaling pathway in primary human brain microvascular endothelial cells.

Morphine is one of the most potent opioid analgesic used for pain treatment. Morphine action in the central nervous system requires crossing the blood-brain barrier. Due to the controversial relationship between morphine and oxidative stress, the potential pro- or antioxidant effects of morphine in the blood-brain barrier is important to be understood, as oxidative stress could cause its disruption and predispose to neurodegenerative diseases. However, investigation is scarce in human brain endothelial cells. Therefore, the present study evaluated the impact of morphine exposure at three different concentrations (1, 10 and 100 µM) for 24 h and 48 h on primary human brain microvascular endothelial cells. A quantitative data-independent acquisition mass spectrometry strategy was used to analyze proteome modulations. Almost 3000 proteins were quantified of which 217 were reported to be significantly regulated in at least one condition versus untreated control. Pathway enrichment analysis unveiled dysregulation of the Nrf2 pathway involved in oxidative stress response. Seahorse assay underlined mitochondria dysfunctions, which were supported by significant expression modulations of relevant mitochondrial proteins. In conclusion, our study revealed the dysregulation of the Nrf2 pathway and mitochondria dysfunctions after morphine exposure, highlighting a potential redox imbalance in human brain endothelial cells.

Identifying patients with cerebral infarction within the time window compatible with reperfusion therapy, diagnostic performance of glutathione S-transferase-π (GST-π) and peroxiredoxin 1 (PRDX1): exploratory prospective multicentre study FLAG-1 protocol.

INTRODUCTION: Plasma biomarkers may be useful in diagnosing acute cerebral infarction requiring urgent reperfusion, but their performance remains to be confirmed. If confirmed, these molecules could be used to develop rapid and reliable decentralised measurement methods, making it possible to initiate reperfusion therapy before hospital admission. The FLAG-1 large prospective study will constitute a plasma bank to assess the diagnostic performance of two biomarkers: glutathione S-transferase-π and peroxiredoxin 1. These molecules are involved in the oxidative stress response and could identify cerebral infarction within a therapeutic window of less than 4.5 hours following the onset of symptoms. Secondary objectives include assessing performance of these biomarkers within 3-hour and 6-hour windows; identifying additional biomarkers diagnosing cerebral infarction and significant criteria guiding therapeutic decisions: ischaemic features of stroke, presence of diffusion/fluid-attenuated inversion recovery mismatch, volume of cerebral infarction and penumbra on cerebral MRI. METHODS AND ANALYSIS: The exploratory, prospective, multicentre FLAG-1 Study will include 945 patients with acute stroke symptoms (onset ≤12 hours, National Institute of Health Stroke Scale score ≥3). Each patient's 25 mL blood sample will be associated with cerebral MRI data. Two patient groups will be defined based on the time of blood collection (before and after 4.5 hours following onset). Receiver operating characteristic analysis will determine the diagnostic performance of each biomarker, alone or in combination, for the identification of cerebral infarction <4.5 hours. ETHICS AND DISSEMINATION: The protocol has been approved by an independent ethics committee. Biological samples are retained in line with best practices and procedures, in accordance with French legislation. Anonymised data and cerebral imaging records are stored using electronic case report forms and a secure server, respectively, registered with the French Data Protection Authority (Commission Nationale de l'Informatique et des Libertés (CNIL)). Results will be disseminated through scientific meetings and publication in peer-reviewed medical journals. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT03364296).