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Spatially targeted proteomics analyzes the proteome of specific cell types and functional regions within tissue. While spatial context is often essential to understanding biological processes, interpreting sub-region-specific protein profiles can pose a challenge due to the high-dimensional nature of the data. Here, we develop a multivariate approach for rapid exploration of differential protein profiles acquired from distinct tissue regions and apply it to analyze a published spatially targeted proteomics data set collected from Staphylococcus aureus-infected murine kidney, 4 and 10 days postinfection. The data analysis process rapidly filters high-dimensional proteomic data to reveal relevant differentiating species among hundreds to thousands of measured molecules. We employ principal component analysis (PCA) for dimensionality reduction of protein profiles measured by microliquid extraction surface analysis mass spectrometry. Subsequently, k-means clustering of the PCA-processed data groups samples by chemical similarity. Cluster center interpretation revealed a subset of proteins that differentiate between spatial regions of infection over two time points. These proteins appear involved in tricarboxylic acid metabolomic pathways, calcium-dependent processes, and cytoskeletal organization. Gene ontology analysis further uncovered relationships to tissue damage/repair and calcium-related defense mechanisms. Applying our analysis in infectious disease highlighted differential proteomic changes across abscess regions over time, reflecting the dynamic nature of host-pathogen interactions.
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Calcio , Proteómica , Animales , Ratones , Proteómica/métodos , Biología Computacional/métodos , Análisis Multivariante , Proteoma/metabolismoRESUMEN
Technological advances have made it feasible to collect multi-condition multi-omic time courses of cellular response to perturbation, but the complexity of these datasets impedes discovery due to challenges in data management, analysis, visualization, and interpretation. Here, we report a whole-cell mechanistic analysis of HL-60 cellular response to bendamustine. We integrate both enrichment and network analysis to show the progression of DNA damage and programmed cell death over time in molecular, pathway, and process-level detail using an interactive analysis framework for multi-omics data. Our framework, Mechanism of Action Generator Involving Network analysis (MAGINE), automates network construction and enrichment analysis across multiple samples and platforms, which can be integrated into our annotated gene-set network to combine the strengths of networks and ontology-driven analysis. Taken together, our work demonstrates how multi-omics integration can be used to explore signaling processes at various resolutions and demonstrates multi-pathway involvement beyond the canonical bendamustine mechanism.
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Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve the sensitivity and depth of coverage for protein and peptide IMS. We demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (MALDI-1) augmented using a second ionizing laser (termed MALDI-2). Proteins were digested in situ using trypsin prior to IMS analysis. For tentative identification of peptides and proteins, a tissue homogenate from the same organ used for IMS was analyzed by LC-MS/MS, and data are available via ProteomeXchange with identifier PXD023877. These identified proteins were then digested in silico to generate a database of theoretical peptides to then match to MALDI IMS data sets. Peptides were tentatively identified by matching the MALDI peak list to the database peptide list based on mass accuracy (5 ppm mass error). This resulted in 1337 ± 96 (n = 3) peptides and 2076 ± 362 (n = 3) unique peptides matched to IMS peaks from MALDI-1 and MALDI-2, respectively. Protein identifications requiring two or more peptides per protein resulted in 276 ± 20 proteins with MALDI-1 and 401 ± 60 with MALDI-2. These results demonstrate that MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and significantly increases the number of proteins identified in IMS experiments.
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Técnicas Histológicas/métodos , Imagen Molecular/métodos , Fragmentos de Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Cromatografía Liquida , Humanos , Riñón/química , Riñón/diagnóstico por imagen , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Tripsina/metabolismoRESUMEN
Here, we describe the preservation and preparation of human kidney tissue for interrogation by histopathology, imaging mass spectrometry, and multiplexed immunofluorescence. Custom image registration and integration techniques are used to create cellular and molecular atlases of this organ system. Through careful optimization, we ensure high-quality and reproducible datasets suitable for cross-patient comparisons that are essential to understanding human health and disease. Moreover, each of these steps can be adapted to other organ systems or diseases, enabling additional atlas efforts.
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Técnica del Anticuerpo Fluorescente/métodos , Riñón/diagnóstico por imagen , Imagen Multimodal/métodos , Manejo de Especímenes/métodos , Animales , Diagnóstico por Imagen , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Riñón/citología , Espectrometría de Masas/métodos , Análisis de la Célula Individual/métodos , Coloración y Etiquetado/métodosRESUMEN
Staphylococcus aureus is a common cause of invasive and life-threatening infections that are often multidrug resistant. To develop novel treatment approaches, a detailed understanding of the complex host-pathogen interactions during infection is essential. This is particularly true for the molecular processes that govern the formation of tissue abscesses, as these heterogeneous structures are important contributors to staphylococcal pathogenicity. To fully characterize the developmental process leading to mature abscesses, temporal and spatial analytical approaches are required. Spatially targeted proteomic technologies such as micro-liquid extraction surface analysis offer insight into complex biological systems including detection of bacterial proteins and their abundance in the host environment. By analyzing the proteomic constituents of different abscess regions across the course of infection, we defined the immune response and bacterial contribution to abscess development through spatial and temporal proteomic assessment. The information gathered was mapped to biochemical pathways to characterize the metabolic processes and immune strategies employed by the host. These data provide insights into the physiological state of bacteria within abscesses and elucidate pathogenic processes at the host-pathogen interface.
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Proteómica , Infecciones Estafilocócicas , Absceso , Proteínas Bacterianas/genética , Humanos , Staphylococcus aureusRESUMEN
Lake Winnipeg (Manitoba, Canada), the world's 12th largest lake by area, is host to yearly cyanobacterial harmful algal blooms (cHABs) dominated by Aphanizomenon and Dolichospermum. cHABs in Lake Winnipeg are primarily a result of eutrophication but may be exacerbated by the recent introduction of dreissenid mussels. Through multiple methods to monitor the potential for toxin production in Lake Winnipeg in conjunction with environmental measures, this study defined the baseline composition of a Lake Winnipeg cHAB to measure potential changes because of dreissenid colonization. Surface water samples were collected in 2013 from 23 sites during summer and from 18 sites in fall. Genetic data and mass spectrometry cyanotoxin profiles identified microcystins (MC) as the most abundant cyanotoxin across all stations, with MC concentrations highest in the north basin. In the fall, mcyA genes were sequenced to determine which species had the potential to produce MCs, and 12 of the 18 sites were a mix of both Planktothrix and Microcystis. Current blooms in Lake Winnipeg produce low levels of MCs, but the capacity to produce cyanotoxins is widespread across both basins. If dreissenid mussels continue to colonize Lake Winnipeg, a shift in physicochemical properties of the lake because of faster water column clearance rates may yield more toxic blooms potentially dominated by microcystin producers.
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Toxinas Bacterianas/análisis , Cianobacterias , Microcistinas/análisis , Saxitoxina/análisis , Uracilo/análogos & derivados , Contaminantes del Agua/análisis , Alcaloides , Animales , Toxinas Bacterianas/genética , Bivalvos , Cianobacterias/genética , Toxinas de Cianobacterias , Monitoreo del Ambiente , Floraciones de Algas Nocivas , Lagos/microbiología , Manitoba , Microcistinas/genética , Filogenia , Saxitoxina/genética , Uracilo/análisisRESUMEN
Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin-fixed paraffin-embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross-links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.
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Proteínas/análisis , Manejo de Especímenes/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Análisis de Matrices Tisulares/métodos , Formaldehído/química , Humanos , Adhesión en Parafina , Proteolisis , Fijación del Tejido , Tripsina/químicaRESUMEN
Zinc (Zn) is an essential trace metal required for all forms of life, but is toxic at high concentrations. While the toxic effects of high levels of Zn are well documented, the mechanism of cell death appears to vary based on the study and concentration of Zn. Zn has been proposed as an anti-cancer treatment against non-small cell lung cancer (NSCLC). The goal of this analysis was to determine the effects of Zn on metabolism and cell death in A549 cells. Here, high throughput multi-omics analysis identified the molecular effects of Zn intoxication on the proteome, metabolome, and transcriptome of A549 human NSCLC cells after 5 min to 24 h of Zn exposure. Multi-omics analysis combined with additional experimental evidence suggests Zn intoxication induces ferroptosis, an iron and lipid peroxidation-dependent programmed cell death, demonstrating the utility of multi-omics analysis to identify cellular response to intoxicants.
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Ferroptosis/efectos de los fármacos , Pulmón/patología , Zinc/toxicidad , Células A549 , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Genómica , Humanos , NAD/biosíntesis , Necrosis , Unión Proteica/efectos de los fármacos , Factores de TiempoRESUMEN
Proteomics, metabolomics, and transcriptomics generate comprehensive data sets, and current biocomputational capabilities allow their efficient integration for systems biology analysis. Published multiomics studies cover methodological advances as well as applications to biological questions. However, few studies have focused on the development of a high-throughput, unified sample preparation approach to complement high-throughput omic analytics. This report details the automation, benchmarking, and application of a strategy for transcriptomic, proteomic, and metabolomic analyses from a common sample. The approach, sample preparation for multi-omics technologies (SPOT), provides equivalent performance to typical individual omic preparation methods but greatly enhances throughput and minimizes the resources required for multiomic experiments. SPOT was applied to a multiomics time course experiment for zinc-treated HL-60 cells. The data reveal Zn effects on NRF2 antioxidant and NFkappaB signaling. High-throughput approaches such as these are critical for the acquisition of temporally resolved, multicondition, large multiomic data sets such as those necessary to assess complex clinical and biological concerns. Ultimately, this type of approach will provide an expanded understanding of challenging scientific questions across many fields.
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Perfilación de la Expresión Génica/métodos , Metabolómica/métodos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteómica/métodos , Genómica/métodos , Células HL-60 , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Biología de Sistemas/métodos , Zinc/farmacologíaRESUMEN
State-of-the-art strategies for proteomics are not able to rapidly interrogate complex peptide mixtures in an untargeted manner with sensitive peptide and protein identification rates. We describe a data-independent acquisition (DIA) approach, microDIA (µDIA), that applies a novel tandem mass spectrometry (MS/MS) mass spectral deconvolution method to increase the specificity of tandem mass spectra acquired during proteomics experiments. Using the µDIA approach with a 10 min liquid chromatography gradient allowed detection of 3.1-fold more HeLa proteins than the results obtained from data-dependent acquisition (DDA) of the same samples. Additionally, we found the µDIA MS/MS deconvolution procedure is critical for resolving modified peptides with relatively small precursor mass shifts that cause the same peptide sequence in modified and unmodified forms to theoretically cofragment in the same raw MS/MS spectra. The µDIA workflow is implemented in the PROTALIZER software tool which fully automates tandem mass spectral deconvolution, queries every peptide with a library-free search algorithm against a user-defined protein database, and confidently identifies multiple peptides in a single tandem mass spectrum. We also benchmarked µDIA against DDA using a 90 min gradient analysis of HeLa and Escherichia coli peptides that were mixed in predefined quantitative ratios, and our results showed µDIA provided 24% more true positives at the same false positive rate.
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Ensayos Analíticos de Alto Rendimiento/métodos , Péptidos/análisis , Proteoma/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Algoritmos , Cromatografía Liquida , Bases de Datos de Proteínas , Escherichia coli/química , Proteínas de Escherichia coli/química , Células HeLa , Humanos , Programas Informáticos , Flujo de TrabajoRESUMEN
The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid. We analyzed the effects of fenretinide, MPR, 3-keto-HPR, and the non-retinoid RBP4 ligand A1120, on the activity of known targets of fenretinide, stearoyl-CoA desaturase 1 (SCD1) and dihydroceramide Δ4-desaturase 1 (DES1) in ARPE-19 cells, and purified recombinant mouse beta-carotene oxygenase 1 (BCO1) in vitro. Lipids and retinoids were extracted and quantified by liquid chromatography-mass spectrometry and reversed phase HPLC, respectively. The data demonstrate that while fenretinide is an inhibitor of the activities of these three enzymes, that 3-keto-HPR is a more potent inhibitor of all three enzymes, potentially mediating most of the in vivo beneficial effects of fenretinide. However, while MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. We conclude that a deeper understanding of the mechanisms of action of fenretinide and its metabolites provides new avenues for therapeutic specificity. For example, administration of 3-keto-HPR instead of fenretinide may be preferential if inhibition of SCD1 or DES1 activity is the goal (cancer), while MPR may be better for BCO1 modulation (carotenoid metabolism). Continued investigation of fenretinide metabolites in the context of fenretinide's various therapeutic uses will begin to resolve the pleotropic nature of this compound.
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Fenretinida/análogos & derivados , Fenretinida/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Terapia Molecular Dirigida , Oxidorreductasas/antagonistas & inhibidores , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Tretinoina/análogos & derivados , beta-Caroteno 15,15'-Monooxigenasa/antagonistas & inhibidores , Animales , Línea Celular , Fenretinida/farmacología , Humanos , Ratones , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
An understanding of how cells respond to perturbation is essential for biological applications; however, most approaches for profiling cellular response are limited in scope to pre-established targets. Global analysis of molecular mechanism will advance our understanding of the complex networks constituting cellular perturbation and lead to advancements in areas, such as infectious disease pathogenesis, developmental biology, pathophysiology, pharmacology, and toxicology. We have developed a high-throughput multiomics platform for comprehensive, de novo characterization of cellular mechanisms of action. Platform validation using cisplatin as a test compound demonstrates quantification of over 10â¯000 unique, significant molecular changes in less than 30 days. These data provide excellent coverage of known cisplatin-induced molecular changes and previously unrecognized insights into cisplatin resistance. This proof-of-principle study demonstrates the value of this platform as a resource to understand complex cellular responses in a high-throughput manner.
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Células/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Redes y Vías Metabólicas , Apoptosis , Línea Celular , Supervivencia Celular , Cisplatino/farmacología , Biología Computacional/métodos , HumanosRESUMEN
In the human ocular lens it is now realized that post-translational modifications can alter protein function and/or localization in fiber cells that no longer synthesize proteins. The specific sites of post-translational modification to the abundant ocular lens membrane proteins AQP0 and MP20 have been previously identified and their functional effects are emerging. To further understand how changes in protein function and/or localization induced by these modifications alter lens homeostasis, it is necessary to determine the spatial distributions of these modifications across the lens. In this study, a quantitative LC-MS approach was used to determine the spatial distributions of phosphorylated AQP0 and MP20 peptides from manually dissected, concentric layers of fiber cells from young and aged human lenses. The absolute amounts of phosphorylation were determined for AQP0 Ser235 and Ser229 and for MP20 Ser170 in fiber cells from the lens periphery to the lens center. Phosphorylation of AQP0 Ser229 represented a minor portion of the total phosphorylated AQP0. Changes in spatial distributions of phosphorylated APQ0 Ser235 and MP20 Ser170 correlated with regions of physiological interest in aged lenses, specifically, where barriers to water transport and extracellular diffusion form.
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Envejecimiento/metabolismo , Acuaporinas/metabolismo , Proteínas del Ojo/metabolismo , Proteínas de la Membrana/metabolismo , Peroxirredoxinas/metabolismo , Adolescente , Adulto , Western Blotting , Cromatografía Liquida , Humanos , Cristalino/metabolismo , Persona de Mediana Edad , Fosforilación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto JovenRESUMEN
BACKGROUND: Imaging mass spectrometry (IMS) generates molecular images directly from tissue sections to provide better diagnostic insights and expand the capabilities of clinical anatomic pathology. Although IMS technology has matured over recent years, the link between microscopy imaging currently used by pathologists and MS-based molecular imaging has not been established. METHODS: We adapted the Vanderbilt University Tissue Core workflow for IMS into a web-based system that facilitates remote collaboration. The platform was designed to perform within acceptable web response times for viewing, annotating, and processing high resolution microscopy images. RESULTS: We describe a microscopy-driven approach to tissue analysis by IMS. CONCLUSION: The Pathology Interface for Mass Spectrometry is designed to provide clinical access to IMS technology and deliver enhanced diagnostic value.
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Visual sensation is fundamental for quality of life, and loss of vision to retinal degeneration is a debilitating condition. The eye is the only part of the central nervous system that can be noninvasively observed with optical imaging. In the clinics, various spectroscopic methods provide high spatial resolution images of the fundus and the developing degenerative lesions. However, the currently utilized tools are not specific enough to establish the molecular underpinnings of retinal diseases. In contrast, mass spectrometric imaging (MSI) is a powerful tool to identify molecularly specific disease indicators and classification markers. This technique is particularly well suited to the eye, where molecular information can be correlated with clinical data collected via noninvasive diagnostic imaging modalities. Recent studies during the last few recent years have uncovered a plethora of new spatially defined molecular information on several vision-threatening diseases, including age-related macular degeneration, Stargardt disease, glaucoma, cataract, as well as lipid disorders. Even though MS inside the eye cannot be performed noninvasively, by linking diagnostic and molecular information, these studies are the first step toward the development of smart ophthalmic diagnostic and surgical tools. Here, we provide an overview of current approaches applying MSI technology to ocular pathology.
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Catarata/diagnóstico por imagen , Glaucoma/diagnóstico por imagen , Degeneración Macular/congénito , Degeneración Macular/diagnóstico por imagen , Imagen Óptica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Catarata/metabolismo , Catarata/patología , Glaucoma/metabolismo , Glaucoma/patología , Humanos , Metabolismo de los Lípidos , Lipofuscina/metabolismo , Degeneración Macular/metabolismo , Degeneración Macular/patología , Imagen Óptica/instrumentación , Retina/metabolismo , Retina/patología , Retina/ultraestructura , Retinoides/metabolismo , Rodopsina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Enfermedad de StargardtRESUMEN
Marine mammals are repeatedly exposed to elevated extra-thoracic pressure and alveolar collapse during diving and readily experience alveolar expansion upon inhalation - a unique capability as compared to terrestrial mammals. How marine mammal lungs overcome the challenges of frequent alveolar collapse and recruitment remains unknown. Recent studies indicate that pinniped lung surfactant has more anti-adhesive components compared to terrestrial mammals, which would aid in alveolar opening. However, pulmonary surfactant composition has not yet been investigated in odontocetes, whose physiology and diving behavior differ from pinnipeds. The aim of this study was to investigate the phosphatidylcholine (PC) composition of lung surfactants from various marine mammals and compare these to a terrestrial mammal. We found an increase in anti-adhesive PC species in harp seal (Pagophilus groenlandicus) and California sea lion (Zalophus californianus) compared to dog (Canus lupus familiaris), as well as an increase in the fluidizing PCs 16:0/14:0 and 16:0/16:1 in pinnipeds compared to odontocetes. The harbor porpoise (a representative of the odontocetes) did not have higher levels of fluidizing PCs compared to dog. Our preliminary results support previous findings that pinnipeds may have adapted unique surfactant compositions that allow them to dive at high pressures for extended periods without adverse effects. Future studies will need to investigate the differences in other surfactant components to fully assess the surfactant composition in odontocetes.
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Perros/metabolismo , Delfines/metabolismo , Phocoena/metabolismo , Surfactantes Pulmonares/química , Leones Marinos/metabolismo , Phocidae/metabolismo , Animales , Pulmón/química , Pulmón/metabolismo , Espectrometría de Masas , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Surfactantes Pulmonares/metabolismo , Especificidad de la EspecieRESUMEN
PURPOSE: Lipofuscin (LF) accumulation within RPE cells is considered pathogenic in AMD. To test whether LF contributes to RPE cell loss in aging and to provide a cellular basis for fundus autofluorescence (AF) we created maps of human RPE cell number and histologic AF. METHODS: Retinal pigment epithelium-Bruch's membrane flat mounts were prepared from 20 donor eyes (10 ≤ 51 and 10 > 80 years; postmortem: ≤4.2 hours; no retinal pathologies), preserving foveal position. Phalloidin-binding RPE cytoskeleton and LF-AF (488-nm excitation) were imaged at up to 90 predefined positions. Maps were assembled from 83,330 cells in 1470 locations. From Voronoi regions representing each cell, the number of neighbors, cell area, and total AF intensity normalized to an AF standard was determined. RESULTS: Highly variable between individuals, RPE-AF increases significantly with age. A perifoveal ring of high AF mirrors rod photoreceptor topography and fundus-AF. Retinal pigment epithelium cell density peaks at the fovea, independent of age, yet no net RPE cell loss is detectable. The RPE monolayer undergoes considerable lifelong re-modeling. The relationship of cell size and AF, a surrogate for LF concentration, is orderly and linear in both groups. Autofluorescence topography differs distinctly from the topography of age-related rod loss. CONCLUSIONS: Digital maps of quantitative AF, cell density, and packing geometry provide metrics for cellular-resolution clinical imaging and model systems. The uncoupling of RPE LF content, cell number, and photoreceptor topography in aging challenges LF's role in AMD.
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Degeneración Macular/patología , Imagen Óptica/métodos , Epitelio Pigmentado de la Retina/patología , Anciano , Anciano de 80 o más Años , Recuento de Células , Progresión de la Enfermedad , Femenino , Humanos , Lipofuscina/farmacocinética , Degeneración Macular/metabolismo , Masculino , Microscopía Fluorescente/métodos , Persona de Mediana Edad , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Euglenophycin is a recently discovered toxin produced by at least one species of euglenoid algae. The toxin has been responsible for several fish mortality events. To facilitate the identification and monitoring of euglenophycin in freshwater ponds, we have developed a specific mass spectrometric method for the identification and quantitation of euglenophycin. The post-extraction stability of the toxin was assessed under various conditions. Euglenophycin was most stable at room temperature. At 8 °C there was a small, but statistically significant, loss in toxin after one day. These methods and knowledge of the toxin's stability will facilitate identification of the toxin as a causative agent in fish kills and determination of the toxin's distribution in the organs of exposed fish.
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Euglena/química , Toxinas Marinas/análisis , Piperidinas/análisis , Contaminantes del Agua/análisis , Espectrometría de Masas en TándemAsunto(s)
Degeneración Macular/metabolismo , Compuestos de Piridinio/análisis , Compuestos de Piridinio/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Retinoides/análisis , Retinoides/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Transportadoras de Casetes de Unión a ATP/genética , Animales , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Lipofuscina/análisis , Lipofuscina/metabolismo , Degeneración Macular/congénito , Degeneración Macular/genética , Degeneración Macular/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Oxidación-Reducción , Epitelio Pigmentado de la Retina/patología , Enfermedad de Stargardt , cis-trans-IsomerasasRESUMEN
Aquaporin-0 (AQP0), the major integral membrane protein in lens fiber cells, becomes highly modified with increasing age. The functional consequences of these modifications are being revealed, and the next step is to determine how these modifications affect the ocular lens, which is directly related to their abundances and spatial distributions. The aim of this study was to utilize matrix-assisted laser desorption ionization (MALDI) direct tissue profiling methods, which produce spatially-resolved protein profiles, to map and quantify AQP0 post-translational modifications (PTMs). Direct tissue profiling was performed using frozen, equatorial human lens sections of various ages prepared by conditions optimized for MALDI mass spectrometry profiling of membrane proteins. Modified forms of AQP0 were identified and further investigated using liquid chromatography tandem mass spectrometry (LC-MS/MS). The distributions of unmodified, truncated, and oleoylated forms of AQP0 were examined with a maximum spatial resolution of 500 µm. Direct tissue profiling of intact human lens sections provided high quality, spatially-resolved, relative quantitative information of AQP0 and its modified forms indicating that 50% of AQP0 is truncated at a fiber cell age of 24 ± 1 year in all lenses examined. Furthermore, direct tissue profiling also revealed previously unidentified AQP0 modifications including N-terminal acetylation and carbamylation. N-terminal acetylation appears to provide a protective effect against N-terminal truncation.