MutTMPredictor: Robust and accurate cascade XGBoost classifier for prediction of mutations in transmembrane proteins.
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The levels of platelet-derived extracellular vesicles (pEVs) have been reported as elevated in acute ischemic stroke (IS). However, the results of studies remain equivocal. This prospective, case-control study included 168 patients with IS, 63 matched disease controls (DC), and 21 healthy controls (HC). Total pEVs concentration, the concentration of phosphatidylserine-positive pEVs (PS+pEVs), the percentage of PS+pEVs (%PS+pEVs) and the concentration of pEVs with expression of CD62P+, CD40L+, CD31+, and active form of GPIIb/IIIa receptor (PAC-1+) were assessed on days 1, 3, 10, and 90 with the Apogee A50-Micro flow cytometer. The concentrations of pEVs, PS+pEVs, and %PS+pEVs were significantly higher after IS vs. HC (p < 0.001). PS+pEVs were higher after stroke vs. controls (p < 0.01). The concentrations of pEVs with expression of studied molecules were higher on D1 and D3 after stroke vs. controls. The concentration of pEVs after platelet stimulation with ADP was significantly diminished on D3. IS most notably affects the phenotype of pEVs with a limited effect on the number of pEVs. Ischemic stroke moderately disturbs platelet microvesiculation, most notably in the acute phase, affecting the phenotype of pEVs, with a limited impact on the number of pEVs. The accumulation of α-synuclein (α-syn), an essential step in PD development and progression, is observed not only in neurons but also in glia, including astrocytes. The mechanisms regulating astrocytic α-syn level and aggregation remain unclear. More recently, it has been demonstrated that a part of α-syn spreading occurs through extracellular vesicles (EVs), although it is unknown whether this process is involved in astrocytes of PD. It is known, however, that EVs derived from the central nervous system exist in the blood and are extensively explored as biomarkers for PD and other neurodegenerative disorders. Primary astrocytes were transfected with A53T α-syn plasmid or exposed to α-syn aggregates. The level of astrocyte-derived EVs (AEVs) was assessed by nanoparticle tracking analysis and immunofluorescence. The lysosomal function was evaluated by Cathepsin assays, immunofluorescence for levels of Lamp1 and Lamp2, and LysoTracker Red staining. The Apogee assays were optimized to measure the GLT-1+ AEVs in clinical cohorts of 106 PD, 47 multiple system atrophy (MSA), and 103 healthy control (HC) to test the potential of plasma AEVs as a biomarker to differentiate PD from other forms of parkinsonism. The number of AEVs significantly increased in primary astrocytes with α-syn deposition. The mechanism of increased AEVs was partially attributed to lysosomal dysfunction. The number of α-syn-carrying AEVs was significantly higher in patients with PD than in HC and MSA. The integrative model combining AEVs with total and aggregated α-syn exhibited efficient diagnostic power in differentiating PD from HC with an AUC of 0.915, and from MSA with an AUC of 0.877. Pathological α-syn deposition could increase the astrocytic secretion of EVs, possibly through α-syn-induced lysosomal dysfunction. The α-syn-containing AEVs in the peripheral blood may be an effective biomarker for clinical diagnosis or differential diagnosis of PD. Mitochondrial dysfunction has pleiotropic effects and is frequently caused by mitochondrial DNA mutations. However, factors such as significant variability in clinical manifestations make interpreting the pathogenicity of variants in the mitochondrial genome challenging. Here, we present APOGEE 2, a mitochondrially-centered ensemble method designed to improve the accuracy of pathogenicity predictions for interpreting missense mitochondrial variants. Built on the joint consensus recommendations by the American College of Medical Genetics and Genomics/Association for Molecular Pathology, APOGEE 2 features an improved machine learning method and a curated training set for enhanced performance metrics. It offers region-wise assessments of genome fragility and mechanistic analyses of specific amino acids that cause perceptible long-range effects on protein structure. With clinical and research use in mind, APOGEE 2 scores and pathogenicity probabilities are precompiled and available in MitImpact. APOGEE 2's ability to address challenges in interpreting mitochondrial missense variants makes it an essential tool in the field of mitochondrial genetics. Extracellular vesicles (EVs) in body fluids are explored as disease biomarkers, but EV concentrations measured by flow cytometers (FCMs) are incomparable. To improve data comparability, new reference materials with physical properties resembling EVs and reference procedures are being developed. The validation of new reference materials and procedures requires biological test samples. We developed a human plasma EV test sample (PEVTES) that i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry-compatible, and iv) is stable. The PEVTES was prepared from human plasma of 3 fasting donors. EVs were immunofluorescently stained with antibodies against platelet-specific (CD61) and erythrocyte-specific (CD235a) antigens or lactadherin. To reduce the concentration of soluble proteins, lipoproteins, and unbound reagents, stained EVs were isolated from plasma by size-exclusion chromatography. After isolation, the PEVTES was filtered to remove remnant platelets. PEVTESs were diluted in cryopreservation agents, dimethyl sulfoxide, glycerol, or trehalose and stored at -80 °C for 12 months. After thawing, stained EV concentrations were measured with a calibrated FCM (Apogee A60-Micro). We demonstrate that the developed PEVTES resembles subcellular particles in human plasma when measured using FCM and that the concentrations of prestained platelet-derived, erythrocyte-derived, and lactadherin+ EVs in the PEVTES are stable during storage at -80 °C for 12 months when stored in trehalose. The PEVTES i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry-compatible, and iv) is stable. Therefore, the developed PEVTES is an ideal candidate to validate newly developed reference materials and procedures. Flow cytometry is commonly used to detect cell-derived extracellular vesicles in body fluids such as blood plasma. However, continuous and simultaneous illumination of multiple particles at or below the detection limit may result in the detection of a single event. This phenomenon is called swarm detection and leads to incorrect particle concentration measurements. To prevent swarm detection, sample dilution is recommended. Since the concentration of particles differs between plasma samples, finding the optimal sample dilution requires dilution series of all samples, which is unfeasible in clinical routine. Here we developed a practical procedure to find the optimal sample dilution of plasma for extracellular vesicle flow cytometry measurements in clinical research studies. Dilution series of 5 plasma samples were measured with flow cytometry (Apogee A60-Micro), triggered on side scatter. The total particle concentration between these plasma samples ranged from 2.5 × 109 to 2.1 × 1011 mL-1. Swarm detection was absent in plasma samples when diluted ≥1.1 × 103-fold or at particle count rates <3.0 × 103 events·s-1. Application of either one of these criteria, however, resulted in insignificant particle counts in most samples. The best approach to prevent swarm detection while maintaining significant particle counts was by combining minimal dilution with maximum count rate. To prevent swarm detection in a series of clinical samples, the measurement count rate of a single diluted plasma sample can be used to determine the optimal dilution factor. For our samples, flow cytometer, and settings, the optimal dilution factor is ≥1.1 × 102-