RESUMEN
Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.
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Envejecimiento , Humor Acuoso , Inteligencia Artificial , Biopsia Líquida , Proteómica , Humanos , Envejecimiento/metabolismo , Humor Acuoso/química , Biopsia , Enfermedad de Parkinson/diagnósticoRESUMEN
Purpose: To identify vitreous molecular biomarkers associated with autoimmune retinopathy (AIR). Design: Case-control study. Participants: We analyzed six eyes from four patients diagnosed with AIR and eight comparative controls diagnosed with idiopathic macular holes and epiretinal membranes. Methods: Vitreous biopsies were collected from the participants and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) or multiplex ELISA. Outcome Measures: Protein expression changes were evaluated by 1-way ANOVA (significant p-value <0.05), hierarchical clustering, and pathway analysis to identify candidate protein biomarkers. Results: There were 16 significantly upregulated and 17 significantly downregulated proteins in the vitreous of three AIR patients compared to controls. The most significantly upregulated proteins included lysozyme C (LYSC), zinc-alpha-2-glycoprotein (ZA2G), complement factor D (CFAD), transforming growth factor-beta induced protein (BGH3), beta-crystallin B2, and alpha-crystallin A chain. The most significantly downregulated proteins included disco-interacting protein 2 homolog (DIP2C), retbindin (RTBDN), and amyloid beta precursor like protein 2 (APLP2). Pathway analysis revealed that vascular endothelial growth factor (VEGF) signaling was a top represented pathway in the vitreous of AIR patients compared to controls. In comparison to a different cohort of three AIR patients analyzed by multiplex ELISA, a commonly differentially expressed protein was neuronal cell adhesion molecule (NrCAM) with p-values of 0.027 in the LC-MS/MS dataset and 0.035 in the ELISA dataset. Conclusion: Protein biomarkers such as NrCAM in the vitreous may eventually help diagnose AIR.
RESUMEN
Ophthalmic neurodegenerative diseases encompass a wide array of molecular pathologies unified by calpain dysregulation. Calpains are calcium-dependent proteases that perpetuate cellular death and inflammation when hyperactivated. Calpain inhibition trials in other organs have faced pharmacological challenges, but the eye offers many advantages for the development and testing of targeted molecular therapeutics, including small molecules, peptides, engineered proteins, drug implants, and gene-based therapies. This review highlights structural mechanisms underlying calpain activation, distinct cellular expression patterns, and in vivo models that link calpain hyperactivity to human retinal and developmental disease. Optimizing therapeutic approaches for calpain-mediated eye diseases can help accelerate clinically feasible strategies for treating calpain dysregulation in other diseased tissues.
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Calpaína , Retina , Calcio/metabolismo , Calpaína/metabolismo , Muerte Celular , Humanos , Retina/metabolismoRESUMEN
Venous thromboembolic (VTE) risk assessment remains an important issue in hospitalised, acutely-ill medical patients, and several VTE risk assessment models (RAM) have been proposed. The purpose of this large retrospective cohort study was to externally validate the IMPROVE RAM using a large database of three acute care hospitals. We studied 41,486 hospitalisations (28,744 unique patients) with 1,240 VTE hospitalisations (1,135 unique patients) in the VTE cohort and 40,246 VTE-free hospitalisations (27,609 unique patients) in the control cohort. After chart review, 139 unique VTE patients were identified and 278 randomly-selected matched patients in the control cohort. Seven independent VTE risk factors as part of the RAM in the derivation cohort were identified. In the validation cohort, the incidence of VTE was 0.20%; 95% confidence interval (CI) 0.18-0.22, 1.04%; 95%CI 0.88-1.25, and 4.15%; 95%CI 2.79-8.12 in the low, moderate, and high VTE risk groups, respectively, which compared to rates of 0.45%, 1.3%, and 4.74% in the three risk categories of the derivation cohort. For the derivation and validation cohorts, the total percentage of patients in low, moderate and high VTE risk occurred in 68.6% vs 63.3%, 24.8% vs 31.1%, and 6.5% vs 5.5%, respectively. Overall, the area under the receiver-operator characteristics curve for the validation cohort was 0.7731. In conclusion, the IMPROVE RAM can accurately identify medical patients at low, moderate, and high VTE risk. This will tailor future thromboprophylactic strategies in this population as well as identify particularly high VTE risk patients in whom multimodal or more intensive prophylaxis may be beneficial.
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Medición de Riesgo/métodos , Tromboembolia Venosa/diagnóstico , Anciano , Calibración , Bases de Datos Factuales , Femenino , Fibrinolíticos/uso terapéutico , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Curva ROC , Estudios Retrospectivos , Factores de Riesgo , Sensibilidad y EspecificidadRESUMEN
The accepted mechanism of toxicity of many organophosphorous and carbamate insecticides is inhibition of acetylcholinesterase activity. In mammals, part of the toxicity assessment usually includes monitoring blood and/or brain acetylcholinesterase inhibition. Other tissues, however, contain cholinesterase activity (i.e. acetyl- and butyryl-cholinesterase), and the inhibition of that activity may be informative for a full appraisal of the toxicity profile. The present group of studies first optimized the variables for extraction and solubilization of cholinesterase activity from various rat tissues and then refined an existing automated method, in order to differentially assess acetyl and butyrylcholinesterase activity in those tissues. All these studies were conducted using tissues from untreated, Long-Evans, adult rats. The first studies determined the effect of Triton X-100 or salt (NaCl) on the extraction and solubilization of cholinesterase activity from retina, brain, striated muscle, diaphragm, and heart: phosphate buffer plus detergent (1% Triton X-100) yielded the highest activity for most tissues. For striated muscle, however, slightly more activity was extracted if the phosphate buffer contained both 1% Triton X-100 and 0.5 M NaCl. It was also noted that the degree of homogenization of some tissues (e.g. striated muscle) must be increased for maximal solubilization of all cholinesterase activity. Subsequent studies developed a method for assessing the level of acetylcholinesterase, butyrylcholinesterase and total cholinesterase activity in these tissues using an automated analyzer. In conclusion, automated assay of acetylcholinesterase activity in cholinergically innervated tissues in the rat (other than brain) is achievable and relatively convenient.