Mass spectrometry-based proteomic profiling of extracellular vesicle proteins in diabetic and non-diabetic ischemic stroke patients: a case-control study.

Acute ischemic stroke is the most common cause of neurologic dysfunction caused by focal brain ischemia and tissue injury. Diabetes is a major risk factor of stroke, exacerbating disease management and prognosis. Therefore, discovering new diagnostic markers and therapeutic targets is critical for stroke prevention and treatment. Extracellular vesicles (EVs), with their distinctive properties, have emerged as promising candidates for biomarker discovery and therapeutic application. This case-control study utilized mass spectrometry-based proteomics to compare EVs from non-diabetic stroke (nDS = 14), diabetic stroke (DS = 13), and healthy control (HC = 12) subjects. Among 1288 identified proteins, 387 were statistically compared. Statistical comparisons using a general linear model (log2 foldchange ≥0.58 and FDR-p≤0.05) were performed for nDS vs HC, DS vs HC, and DS vs nDS. DS vs HC and DS vs nDS comparisons produced 123 and 149 differentially expressed proteins, respectively. Fibrinogen gamma chain (FIBG), Fibrinogen beta chain (FIBB), Tetratricopeptide repeat protein 16 (TTC16), Proline rich 14-like (PR14L), Inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKE), Biorientation of chromosomes in cell division protein 1-like 1 (BD1L1), and protein PR14L exhibited significant differences in the DS group. The pathway analysis revealed that the complement system pathways were activated, and blood coagulation and neuroprotection were inhibited in the DS group (z-score ≥2; p ≤ 0.05). These findings underscore the potential of EVs proteomics in identifying biomarkers for stroke management and prevention, warranting further clinical investigation.

Human antigen R: Exploring its inflammatory response impact and significance in cardiometabolic disorders.

RNA-binding proteins (RBPs) play a crucial role in the regulation of posttranscriptional RNA networks, which can undergo dysregulation in many pathological conditions. Human antigen R (HuR) is a highly researched RBP that plays a crucial role as a posttranscriptional regulator. HuR plays a crucial role in the amplification of inflammatory signals by stabilizing the messenger RNA of diverse inflammatory mediators and key molecular players. The noteworthy correlations between HuR and its target molecules, coupled with the remarkable impacts reported on the pathogenesis and advancement of multiple diseases, position HuR as a promising candidate for therapeutic intervention in diverse inflammatory conditions. This review article examines the significance of HuR as a member of the RBP family, its regulatory mechanisms, and its implications in the pathophysiology of inflammation and cardiometabolic illnesses. Our objective is to illuminate potential directions for future research and drug development by conducting a comprehensive analysis of the existing body of research on HuR.

Untargeted Metabolomic Profiling Reveals Differentially Expressed Serum Metabolites and Pathways in Type 2 Diabetes Patients with and without Cognitive Decline: A Cross-Sectional Study.

Diabetes is recognized as a risk factor for cognitive decline, but the underlying mechanisms remain elusive. We aimed to identify the metabolic pathways altered in diabetes-associated cognitive decline (DACD) using untargeted metabolomics. We conducted liquid chromatography-mass spectrometry-based untargeted metabolomics to profile serum metabolite levels in 100 patients with type 2 diabetes (T2D) (54 without and 46 with DACD). Multivariate statistical tools were used to identify the differentially expressed metabolites (DEMs), and enrichment and pathways analyses were used to identify the signaling pathways associated with the DEMs. The receiver operating characteristic (ROC) analysis was employed to assess the diagnostic accuracy of a set of metabolites. We identified twenty DEMs, seven up- and thirteen downregulated in the DACD vs. DM group. Chemometric analysis revealed distinct clustering between the two groups. Metabolite set enrichment analysis found significant enrichment in various metabolite sets, including galactose metabolism, arginine and unsaturated fatty acid biosynthesis, citrate cycle, fructose and mannose, alanine, aspartate, and glutamate metabolism. Pathway analysis identified six significantly altered pathways, including arginine and unsaturated fatty acid biosynthesis, and the metabolism of the citrate cycle, alanine, aspartate, glutamate, a-linolenic acid, and glycerophospholipids. Classifier models with AUC-ROC > 90% were developed using individual metabolites or a combination of individual metabolites and metabolite ratios. Our study provides evidence of perturbations in multiple metabolic pathways in patients with DACD. The distinct DEMs identified in this study hold promise as diagnostic biomarkers for DACD patients.

Profiling the autoantibody repertoire reveals autoantibodies associated with mild cognitive impairment and dementia.

Background: Dementia is a debilitating neurological disease affecting millions of people worldwide. The exact mechanisms underlying the initiation and progression of the disease remain to be fully defined. There is an increasing body of evidence for the role of immune dysregulation in the pathogenesis of dementia, where blood-borne autoimmune antibodies have been studied as potential markers associated with pathological mechanisms of dementia. Methods: This study included plasma from 50 cognitively normal individuals, 55 subjects with MCI (mild cognitive impairment), and 22 subjects with dementia. Autoantibody profiling for more than 1,600 antigens was performed using a high throughput microarray platform to identify differentially expressed autoantibodies in MCI and dementia. Results: The differential expression analysis identified 33 significantly altered autoantibodies in the plasma of patients with dementia compared to cognitively normal subjects, and 38 significantly altered autoantibodies in the plasma of patients with dementia compared to subjects with MCI. And 20 proteins had significantly altered autoantibody responses in MCI compared to cognitively normal individuals. Five autoantibodies were commonly dysregulated in both dementia and MCI, including anti-CAMK2A, CKS1B, ETS2, MAP4, and NUDT2. Plasma levels of anti-ODF3, E6, S100P, and ARHGDIG correlated negatively with the cognitive performance scores (MoCA) (r2 -0.56 to -0.42, value of p < 0.001). Additionally, several proteins targeted by autoantibodies dysregulated in dementia were significantly enriched in the neurotrophin signaling pathway, axon guidance, cholinergic synapse, long-term potentiation, apoptosis, glycolysis and gluconeogenesis. Conclusion: We have shown multiple dysregulated autoantibodies in the plasma of subjects with MCI and dementia. The corresponding proteins for these autoantibodies are involved in neurodegenerative pathways, suggesting a potential impact of autoimmunity on the etiology of dementia and the possible benefit for future therapeutic approaches. Further investigations are warranted to validate our findings.

Blood-Based Proteomic Profiling Identifies Potential Biomarker Candidates and Pathogenic Pathways in Dementia.

Dementia is a progressive and debilitating neurological disease that affects millions of people worldwide. Identifying the minimally invasive biomarkers associated with dementia that could provide insights into the disease pathogenesis, improve early diagnosis, and facilitate the development of effective treatments is pressing. Proteomic studies have emerged as a promising approach for identifying the protein biomarkers associated with dementia. This pilot study aimed to investigate the plasma proteome profile and identify a panel of various protein biomarkers for dementia. We used a high-throughput proximity extension immunoassay to quantify 1090 proteins in 122 participants (22 with dementia, 64 with mild cognitive impairment (MCI), and 36 controls with normal cognitive function). Limma-based differential expression analysis reported the dysregulation of 61 proteins in the plasma of those with dementia compared with controls, and machine learning algorithms identified 17 stable diagnostic biomarkers that differentiated individuals with AUC = 0.98 ± 0.02. There was also the dysregulation of 153 plasma proteins in individuals with dementia compared with those with MCI, and machine learning algorithms identified 8 biomarkers that classified dementia from MCI with an AUC of 0.87 ± 0.07. Moreover, multiple proteins selected in both diagnostic panels such as NEFL, IL17D, WNT9A, and PGF were negatively correlated with cognitive performance, with a correlation coefficient (r2) ≤ -0.47. Gene Ontology (GO) and pathway analysis of dementia-associated proteins implicated immune response, vascular injury, and extracellular matrix organization pathways in dementia pathogenesis. In conclusion, the combination of high-throughput proteomics and machine learning enabled us to identify a blood-based protein signature capable of potentially differentiating dementia from MCI and cognitively normal controls. Further research is required to validate these biomarkers and investigate the potential underlying mechanisms for the development of dementia.

Sestrin2 as a Protective Shield against Cardiovascular Disease.

A timely and adequate response to stress is inherently present in each cell and is important for maintaining the proper functioning of the cell in changing intracellular and extracellular environments. Disruptions in the functioning or coordination of defense mechanisms against cellular stress can reduce the tolerance of cells to stress and lead to the development of various pathologies. Aging also reduces the effectiveness of these defense mechanisms and results in the accumulation of cellular lesions leading to senescence or death of the cells. Endothelial cells and cardiomyocytes are particularly exposed to changing environments. Pathologies related to metabolism and dynamics of caloric intake, hemodynamics, and oxygenation, such as diabetes, hypertension, and atherosclerosis, can overwhelm endothelial cells and cardiomyocytes with cellular stress to produce cardiovascular disease. The ability to cope with stress depends on the expression of endogenous stress-inducible molecules. Sestrin2 (SESN2) is an evolutionary conserved stress-inducible cytoprotective protein whose expression is increased in response to and defend against different types of cellular stress. SESN2 fights back the stress by increasing the supply of antioxidants, temporarily holding the stressful anabolic reactions, and increasing autophagy while maintaining the growth factor and insulin signaling. If the stress and the damage are beyond repair, SESN2 can serve as a safety valve to signal apoptosis. The expression of SESN2 decreases with age and its levels are associated with cardiovascular disease and many age-related pathologies. Maintaining sufficient levels or activity of SESN2 can in principle prevent the cardiovascular system from aging and disease.

Increase in repulsive guidance molecule-a (RGMa) in lacunar and cortical stroke patients is related to the severity of the insult.

Repulsive guidance molecule-a (RGMa) inhibits angiogenesis and increases inflammation. Animal models of cerebral ischemia have shown that an increased expression of RGMa leads to larger infarction and its inhibition attenuates effects of ischemia. We report on the relationship of RGMa to stroke types and severity. This is a prospective study in patients admitted to the stroke service in Qatar. We collected the clinical determinants, including NIHSS at admission, imaging and outcome at discharge and 90-days. RGMa levels were determined by measuring mRNA levels extracted from peripheral blood mononuclear cells (PBMCs) within 24 h of onset and at 5 days. There were 90 patients (lacunar: 64, cortical: 26) and 35 age-matched controls. RGMa mRNA levels were significantly higher in the stroke patients: day 1: 1.007 ± 0.13 versus 2.152 ± 0.19 [p < 0.001] and day-5: 3.939 ± 0.36 [p < 0.0001]) and significantly higher in patients with severe stroke (NIHSS ≥ 8) compared to milder symptoms (NIHSS < 8) at day 1 (NIHSS ≥ 8: 2.563 ± 0.36; NIHSS < 8: 1.947 ± 0.2) and day 5 (NIHSS ≥ 8: 5.25 ± 0.62; NIHSS < 8: 3.259 ± 0.419). Cortical stroke patients had marginally higher RGMa mRNA levels compared to lacunar stroke at day 1 (cortical stroke: 2.621 ± 0.46 vs lacunar stroke: 1.961 ± 0.19) and day 5 (cortical stroke: 4.295 ± 0.76 vs lacunar stroke: 3.774 ± 0.39). In conclusion, there is an increase in the level of RGMa mRNA in patients with acute stroke and seen in patients with lacunar and cortical stroke. The increase in RGMa mRNA levels is related to the severity of the stroke and increases over the initial 5 days. Further studies are required to determine the effects of the increase in RGMa on stroke recovery.

Platelet-Neutrophil Association in NETs-Rich Areas in the Retrieved AIS Patient Thrombi.

Histological structure of thrombi is a strong determinant of the outcome of vascular recanalization therapy, the only treatment option for acute ischemic stroke (AIS) patients. A total of 21 AIS patients from this study after undergoing non-enhanced CT scan and multimodal MRI were treated with mechanical stent-based and manual aspiration thrombectomy, and thromboembolic retrieved from a cerebral artery. Complementary histopathological and imaging analyses were performed to understand their composition with a specific focus on fibrin, von Willebrand factor, and neutrophil extracellular traps (NETs). Though distinct RBC-rich and platelet-rich areas were found, AIS patient thrombi were overwhelmingly platelet-rich, with 90% of thrombi containing <40% total RBC-rich contents (1.5 to 37%). Structurally, RBC-rich areas were simple, consisting of tightly packed RBCs in thin fibrin meshwork with sparsely populated nucleated cells and lacked any substantial von Willebrand factor (VWF). Platelet-rich areas were structurally more complex with thick fibrin meshwork associated with VWF. Plenty of leukocytes populated the platelet-rich areas, particularly in the periphery and border areas between platelet-rich and RBC-rich areas. Platelet-rich areas showed abundant activated neutrophils (myeloperoxidase+ and neutrophil-elastase+) containing citrullinated histone-decorated DNA. Citrullinated histone-decorated DNA also accumulated extracellularly, pointing to NETosis by the activated neutrophils. Notably, NETs-containing areas showed strong reactivity to VWF, platelets, and high-mobility group box 1 (HMGB1), signifying a close interplay between these components.

Identification of distinct circulating microRNAs in acute ischemic stroke patients with type 2 diabetes mellitus.

Stroke is the second leading cause of global mortality and continued efforts aim to identify predictive, diagnostic, or prognostic biomarkers to reduce the disease burden. Circulating microRNAs (miRNAs) have emerged as potential biomarkers in stroke. We performed comprehensive circulating miRNA profiling of ischemic stroke patients with or without type 2 diabetes mellitus (T2DM), an important risk factor associated with worse clinical outcomes in stroke. Serum samples were collected within 24 h of acute stroke diagnosis and circulating miRNAs profiled using RNA-Seq were compared between stroke patients with T2DM (SWDM; n = 92) and those without T2DM (SWoDM; n = 98). Our analysis workflow involved random allocation of study cohorts into discovery (n = 96) and validation (n = 94) datasets. Five miRNAs were found to be differentially regulated in SWDM compared to SWoDM patients. Hsa-miR-361-3p and -664a-5p were downregulated, whereas miR-423-3p, -140-5p, and -17-3p were upregulated. We also explored the gene targets of these miRNAs and investigated the downstream pathways associated with them to decipher the potential pathways impacted in stroke with diabetes as comorbidity. Overall, our novel findings provide important insights into the differentially regulated miRNAs, their associated pathways and potential utilization for clinical benefits in ischemic stroke patients with diabetes.

Dysregulated Metabolic Pathways in Subjects with Obesity and Metabolic Syndrome.

Background: Obesity coexists with variable features of metabolic syndrome, which is associated with dysregulated metabolic pathways. We assessed potential associations between serum metabolites and features of metabolic syndrome in Arabic subjects with obesity. Methods: We analyzed a dataset of 39 subjects with obesity only (OBO, n = 18) age-matched to subjects with obesity and metabolic syndrome (OBM, n = 21). We measured 1069 serum metabolites and correlated them to clinical features. Results: A total of 83 metabolites, mostly lipids, were significantly different (p < 0.05) between the two groups. Among lipids, 22 sphingomyelins were decreased in OBM compared to OBO. Among non-lipids, quinolinate, kynurenine, and tryptophan were also decreased in OBM compared to OBO. Sphingomyelin is negatively correlated with glucose, HbA1C, insulin, and triglycerides but positively correlated with HDL, LDL, and cholesterol. Differentially enriched pathways include lysine degradation, amino sugar and nucleotide sugar metabolism, arginine and proline metabolism, fructose and mannose metabolism, and galactose metabolism. Conclusions: Metabolites and pathways associated with chronic inflammation are differentially expressed in subjects with obesity and metabolic syndrome compared to subjects with obesity but without the clinical features of metabolic syndrome.

The impact of enteric coating of aspirin on aspirin responsiveness in patients with suspected or newly diagnosed ischemic stroke: prospective cohort study: results from the (ECASIS) study.

BACKGROUND AND PURPOSE: Uncertainty remains regarding the impact of enteric-coated aspirin (EC-ASA) on secondary prevention of ischemic stroke compared to plain aspirin (P-ASA). Hence, this study was designed to investigate the effect of EC formulation on ASA response via evaluating thromboxane B2 (TXB2) levels in patients with suspected or newly diagnosed stroke. METHODS: A prospective cohort study on suspected or newly diagnosed ischemic stroke patients who are aspirin-naive was conducted. Patients were received either EC aspirin or plain aspirin for at least 3 days. The primary outcome was the proportion of aspirin non-responsiveness between two groups (level of residual serum TXB2 associated with elevated thrombotic risk (< 99.0% inhibition or TXB2 > 3.1 ng/ml) within 72 h after three daily aspirin doses, while secondary outcomes were the incidence of early gastrointestinal tract (GIT) bleeding with the various aspirin preparations. (Trial registration: Clinicaltrials.gov NCT04330872 registered on 02 April 2020). RESULTS: Of 42 patients, ischemic strokes were confirmed in both P-ASA (81%) and EC-ASA (67%) arms. ASA non-responsiveness showed no significant difference between the two formulations (P-ASA vs. EC-ASA; 28.6% vs 23.8%; P = 0.726). Univariate and multivariate logistic regression analysis showed that patients treated with EC-ASA were more likely to have a lower rate of non-responders compared to P-ASA (unadjusted OR 0.78; 95% CI 0.20, 3.11); with the risk highest in type 2 diabetic patients with HBA1c > 6.5% (adjusted OR 6; 95% CI 1.02, 35.27; P = 0.047). No incidence of GIT bleeding observed throughout the study. CONCLUSION: A significant proportion of ASA non-responsiveness was recorded regardless of ASA formulation administered. The increased risk of ASA non-responsiveness in diabetic patients needs further exploration by larger prospective studies.

Characteristic MicroRNAs Linked to Dysregulated Metabolic Pathways in Qatari Adult Subjects With Obesity and Metabolic Syndrome.

Background: Obesity-associated dysglycemia is associated with metabolic disorders. MicroRNAs (miRNAs) are known regulators of metabolic homeostasis. We aimed to assess the relationship of circulating miRNAs with clinical features in obese Qatari individuals. Methods: We analyzed a dataset of 39 age-matched patients that includes 18 subjects with obesity only (OBO) and 21 subjects with obesity and metabolic syndrome (OBM). We measured 754 well-characterized human microRNAs (miRNAs) and identified differentially expressed miRNAs along with their significant associations with clinical markers in these patients. Results: A total of 64 miRNAs were differentially expressed between metabolically healthy obese (OBO) versus metabolically unhealthy obese (OBM) patients. Thirteen out of 64 miRNAs significantly correlated with at least one clinical trait of the metabolic syndrome. Six out of the thirteen demonstrated significant association with HbA1c levels; miR-331-3p, miR-452-3p, and miR-485-5p were over-expressed, whereas miR-153-3p, miR-182-5p, and miR-433-3p were under-expressed in the OBM patients with elevated HbA1c levels. We also identified, miR-106b-3p, miR-652-3p, and miR-93-5p that showed a significant association with creatinine; miR-130b-5p, miR-363-3p, and miR-636 were significantly associated with cholesterol, whereas miR-130a-3p was significantly associated with LDL. Additionally, miR-652-3p's differential expression correlated significantly with HDL and creatinine. Conclusions: MicroRNAs associated with metabolic syndrome in obese subjects may have a pathophysiologic role and can serve as markers for obese individuals predisposed to various metabolic diseases like diabetes.

Biogenesis of Exosomes Laden with Metallic Silver-Copper Nanoparticles Liaised by Wheat Germ Agglutinin for Targeted Delivery of Therapeutics to Breast Cancer.

The anticancer property of silver-copper metallic nanoparticles (AgCu-NPs) is of greater interest in cancer therapeutics; however, its off-target toxicity limits its therapeutic application. Exosomes emerge as one of the leading idiosyncratic nanocarrier choices for cancer therapeutics due to their size, stability, and phenotypic diversity; however, to encapsulate NPs in extracellular vesicles (EVs) without disrupting their inherited functions is far from the expectations. Here, the loading strategy of AgCu-NP conjugated with wheat germ agglutinin (AgCu-NP-WGA) in exosomes during biogenesis for the targeted delivery of anticancer therapeutics to breast cancer is reported. Based on the intrinsic mechanism of endocytosis of WGA, results show that internalization of WGA or AgCu-NP-WGA bypasses the lysosomal pathway and recycles in EVs. On the contrary, the transport of naked AgCu-NPs to lysosomes; mechanistically, an acidic environment causes oxidation of AgCu-NP. Next, the analysis of EVs harvested by differential centrifugation shows that only AgCu-NPs-WGA (Exo-NP) retain their metallic state. Furthermore, Exo-NP cytotoxicity results manifest that MCF10A-derived Exo-NPs are toxic to its homologous breast cancer cells (MCF-7 and MDA-MB 231) and nontoxic to heterologous cancers NC1-1975 and MCF 10A. In conclusion, this study shows the self-assembly of AgCu-NP in exosomes to target and deliver therapeutics for breast cancer.

Corneal nerve loss in patients with TIA and acute ischemic stroke in relation to circulating markers of inflammation and vascular integrity.

Vascular and inflammatory mechanisms are implicated in the development of cerebrovascular disease and corneal nerve loss occurs in patients with transient ischemic attack (TIA) and acute ischemic stroke (AIS). We have assessed whether serum markers of inflammation and vascular integrity are associated with the severity of corneal nerve loss in patients with TIA and AIS. Corneal confocal microscopy (CCM) was performed to quantify corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD) and corneal nerve fiber length (CNFL) in 105 patients with TIA (n = 24) or AIS (n = 81) and age matched control subjects (n = 56). Circulating levels of IL-6, MMP-2, MMP-9, E-Selectin, P-Selectin and VEGF were quantified in patients within 48 h of presentation with a TIA or AIS. CNFL (P = 0.000, P = 0.000), CNFD (P = 0.122, P = 0.000) and CNBD (P = 0.002, P = 0.000) were reduced in patients with TIA and AIS compared to controls, respectively with no difference between patients with AIS and TIA. The NIHSS Score (P = 0.000), IL-6 (P = 0.011) and E-Selectin (P = 0.032) were higher in patients with AIS compared to TIA with no difference in MMP-2 (P = 0.636), MMP-9 (P = 0.098), P-Selectin (P = 0.395) and VEGF (P = 0.831). CNFL (r = 0.218, P = 0.026) and CNFD (r = 0.230, P = 0.019) correlated with IL-6 and multiple regression analysis showed a positive association of CNFL and CNFD with IL-6 (P = 0.041, P = 0.043). Patients with TIA and AIS have evidence of corneal nerve loss and elevated IL6 and E-selectin levels. Larger longitudinal studies are required to determine the association between inflammatory and vascular markers and corneal nerve fiber loss in patients with cerebrovascular disease.

Identification of Novel Circulating miRNAs in Patients with Acute Ischemic Stroke.

Ischemic strokes are associated with significant morbidity and mortality, but currently there are no reliable prognostic or diagnostic blood biomarkers. MicroRNAs (miRNAs) regulate various molecular pathways and may be used as biomarkers. Using RNA-Seq, we conducted comprehensive circulating miRNA profiling in patients with ischemic stroke compared with healthy controls. Samples were collected within 24 h of clinical diagnosis. Stringent analysis criteria of discovery (46 cases and 95 controls) and validation (47 cases and 96 controls) cohorts led to the identification of 10 differentially regulated miRNAs, including 5 novel miRNAs, with potential diagnostic significance. Hsa-miR-451a was the most significantly upregulated miRNA (FC; 4.8, FDR; 3.78 × 10-85), while downregulated miRNAs included hsa-miR-574-5p and hsa-miR-142-3p, among others. Importantly, we computed a multivariate classifier based on the identified miRNA panel to differentiate between ischemic stroke patients and healthy controls, which showed remarkably high sensitivity (0.94) and specificity (0.99). The area under the ROC curve was 0.97 and it is superior to other current available biomarkers. Moreover, in samples collected one month following stroke, we found sustained upregulation of hsa-miR-451a and downregulation of another 5 miRNAs. Lastly, we report 3 miRNAs that were significantly associated with poor clinical outcomes of stroke, as defined by the modified Rankin scores. The clinical translation of the identified miRNA panel may be explored further.

Exosomes Derived Neuronal Markers: Immunoaffinity Isolation and Characterization.

Neuronal exosomes play a crucial role in intercellular communication in the brain and represent a promising biomarker for neurological diseases, including stroke. However, limited techniques are available for isolating neuronal exosomes due to their small number in the serum exosomes. Thus, the development of efficient tools with brain-specific markers is needed. Here, we show the optimization of an immunoaffinity assay-based isolation protocol for specific exosomes or neuronally derived exosomes (NDE). Our results demonstrated that one-micron functionalized magnetic beads successfully separated CD63+ and L1CAM+ exosomes from serum. The size and shape of exosomes or exosomes pulled by beads were confirmed by Dynamic light scattering and Transmission electron microscopy; also, beads were well resolved in conventional flow cytometry analysis, which revealed that CD63-pulled serum exosomes had 5% expression of L1CAM. Furthermore, transmission electron microscopy showed that exosomes eluted from magnetic beads retained their original size, shape, and form without any damage. Furthermore, we showed isolation of NDE using GluR2/3-capturing antibody (α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor) using an optimized immunoaffinity bead assay utilizing 100 µl serum of stroke patients or age-matched healthy group. GluR2/3-captured exosomes were confirmed by western blot analysis. The western blot analysis showed a significant increase in the 35KDa subunit of GluR2/3 receptor protein in the exosomes of stroke patients compared to the healthy group. In addition, the multimeric GluR2/3 receptor protein in exosomes was further validated by the presence of the GluR2 subunit. Thus, our study shows GluR3/2 may be an effective candidate to isolate neuronal exosomes.

Circulating exosomal immuno-oncological checkpoints and cytokines are potential biomarkers to monitor tumor response to anti-PD-1/PD-L1 therapy in non-small cell lung cancer patients.

Immune checkpoint inhibitors (ICIs) including anti-PD-1 and anti-PD-L1 antibodies, have significantly changed the treatment outcomes of NSCLC patients with better overall survival. However, 15-40% of the patients still fail to respond to ICIs therapy. Identification of biomarkers associated with responses are mandated in order to increase the efficacy of such therapy. In this study we evaluated 27 serum-derived exosomal immuno-oncological proteins and 44 cytokines/chemokines before and after ICIs therapy in 17 NSCLC patients to identify surrogate biomarkers for treatment/monitoring patient stratification for maximum therapeutic benefit. We first confirmed the identity of the isolated exosomes to have their specific markers (CD63, CD81, HSP70 and CD91). We have demonstrated that baseline concentration of exosomal-PD-L1 (p<0.0001), exosomal-PD-L2 (p=0.0413) and exosomal-PD-1 (p=0.0131) from NSCLC patients were significantly higher than their soluble-free forms. Furthermore, the exosomal-PD-L1 was present in all the patients (100%), while only 71% of patients expressed tissue PD-L1. This indicates that exosomal-PD-L1 is a more reliable diagnostic biomarker. Interestingly, exosomal-PD-L2 expression was significantly higher (p=0.0193) in tissue PD-L1-negative patients compared to tissue PD-L1-positive patients. We have also shown that immuno-oncological proteins isolated from pre-ICIs treated patients were significantly higher in exosomes compared to their soluble-free counterparts (CD152, p=0.0008; CD80, p=0.0182; IDO, p=0.0443; Arginase, p<0.0001; Nectin-2, p<0.0001; NT5E, p<0.0001; Siglec-7, p<0.0001; Siglec-9, p=0.0335; CD28, p=0.0092; GITR, p<0.0001; MICA, p<0.0001). Finally, the changes in the expression levels of exosomal immuno-oncological proteins/cytokines and their correlation with tumor response to ICIs treatment were assessed. There was a significant downregulation of exosomal PD-L1 (p=0.0156), E-Cadherin (p=0.0312), ULBP1 (p=0.0156), ULBP3 (p=0.0391), MICA (p=0.0391), MICB (p=0.0469), Siglec7 (p=0.0078) and significant upregulation of exosomal PD-1 (p=0.0156) and IFN- γ (p=0.0156) in responding patients. Non-responding patients showed a significant increase in exosomal-PD-L1 (p=0.0078). Furthermore, responding-patients without liver-metastasis showed significant-upregulation of PD-1 (p=0.0070), and downregulation of ULBP1 (p=0.0137) and Siglec-7 (p=0.0037). Non-responding patients had significant-downregulation of ULBP3 (p=0.0317) in patient without brain-metastasis and significant-upregulation/downregulation of PD-L1 and ULBP3 (p=0.0262/0.0286) in patients with pulmonary-metastasis. We demonstrated for the first time that exosomal immuno-oncological proteins/cytokines are potential biomarkers to monitor response to ICIs therapy and can predict the clinical outcomes in NSCLC patients.

Circulating MicroRNA Profiling Identifies Distinct MicroRNA Signatures in Acute Ischemic Stroke and Transient Ischemic Attack Patients.

Transient ischemic attack (TIA) refers to a momentary neurologic deficit caused by focal cerebral, spinal or retinal ischemic insult. TIA is associated with a high risk of impending acute ischemic stroke (AIS), a neurologic dysfunction characterized by focal cerebral, spinal or retinal infarction. Understanding the differences in molecular pathways in AIS and TIA has merit for deciphering the underlying cause for neuronal deficits with long-term effects and high risks of morbidity and mortality. In this study, we performed comprehensive investigations into the circulating microRNA (miRNA) profiles of AIS (n = 191) and TIA (n = 61) patients. We performed RNA-Seq on serum samples collected within 24 hrs of clinical diagnosis and randomly divided the study populations into discovery and validation cohorts. We identified a panel of 11 differentially regulated miRNAs at FDR < 0.05. Hsa-miR-548c-5p, -20a-5p, -18a-5p, -484, -652-3p, -486-3p, -24-3p, -181a-5p and -222-3p were upregulated, while hsa-miR-500a-3p and -206 were downregulated in AIS patients compared to TIA patients. We also probed the previously validated gene targets of our identified miRNA panel to highlight the molecular pathways affected in AIS. Moreover, we developed a multivariate classifier with potential utilization as a discriminative biomarker for AIS and TIA patients. The underlying molecular pathways in AIS compared to TIA may be explored further in functional studies for therapeutic targeting in clinical translation.

Complement C5a and Clinical Markers as Predictors of COVID-19 Disease Severity and Mortality in a Multi-Ethnic Population.

Coronavirus disease-2019 (COVID-19) was declared as a pandemic by WHO in March 2020. SARS-CoV-2 causes a wide range of illness from asymptomatic to life-threatening. There is an essential need to identify biomarkers to predict disease severity and mortality during the earlier stages of the disease, aiding treatment and allocation of resources to improve survival. The aim of this study was to identify at the time of SARS-COV-2 infection patients at high risk of developing severe disease associated with low survival using blood parameters, including inflammation and coagulation mediators, vital signs, and pre-existing comorbidities. This cohort included 89 multi-ethnic COVID-19 patients recruited between July 14th and October 20th 2020 in Doha, Qatar. According to clinical severity, patients were grouped into severe (n=33), mild (n=33) and asymptomatic (n=23). Common routine tests such as complete blood count (CBC), glucose, electrolytes, liver and kidney function parameters and markers of inflammation, thrombosis and endothelial dysfunction including complement component split product C5a, Interleukin-6, ferritin and C-reactive protein were measured at the time COVID-19 infection was confirmed. Correlation tests suggest that C5a is a predictive marker of disease severity and mortality, in addition to 40 biological and physiological parameters that were found statistically significant between survivors and non-survivors. Survival analysis showed that high C5a levels, hypoalbuminemia, lymphopenia, elevated procalcitonin, neutrophilic leukocytosis, acute anemia along with increased acute kidney and hepatocellular injury markers were associated with a higher risk of death in COVID-19 patients. Altogether, we created a prognostic classification model, the CAL model (C5a, Albumin, and Lymphocyte count) to predict severity with significant accuracy. Stratification of patients using the CAL model could help in the identification of patients likely to develop severe symptoms in advance so that treatments can be targeted accordingly.