RESUMEN
Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multiomics datasets into a resource comprising >2.8 million nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550,000 cell type-specific regulatory elements and >1.4 million single-cell expression quantitative trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized ~250 disease-risk genes and drug targets with associated cell types.
Asunto(s)
Encéfalo , Redes Reguladoras de Genes , Trastornos Mentales , Análisis de la Célula Individual , Humanos , Envejecimiento/genética , Encéfalo/metabolismo , Comunicación Celular/genética , Cromatina/metabolismo , Cromatina/genética , Genómica , Trastornos Mentales/genética , Corteza Prefrontal/metabolismo , Corteza Prefrontal/fisiología , Sitios de Carácter CuantitativoRESUMEN
Neuropsychiatric genome-wide association studies (GWASs), including those for autism spectrum disorder and schizophrenia, show strong enrichment for regulatory elements in the developing brain. However, prioritizing risk genes and mechanisms is challenging without a unified regulatory atlas. Across 672 diverse developing human brains, we identified 15,752 genes harboring gene, isoform, and/or splicing quantitative trait loci, mapping 3739 to cellular contexts. Gene expression heritability drops during development, likely reflecting both increasing cellular heterogeneity and the intrinsic properties of neuronal maturation. Isoform-level regulation, particularly in the second trimester, mediated the largest proportion of GWAS heritability. Through colocalization, we prioritized mechanisms for about 60% of GWAS loci across five disorders, exceeding adult brain findings. Finally, we contextualized results within gene and isoform coexpression networks, revealing the comprehensive landscape of transcriptome regulation in development and disease.
Asunto(s)
Empalme Alternativo , Encéfalo , Regulación del Desarrollo de la Expresión Génica , Trastornos Mentales , Humanos , Atlas como Asunto , Trastorno del Espectro Autista/genética , Encéfalo/metabolismo , Encéfalo/crecimiento & desarrollo , Encéfalo/embriología , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sitios de Carácter Cuantitativo , Esquizofrenia/genética , Transcriptoma , Trastornos Mentales/genéticaRESUMEN
RNA splicing is highly prevalent in the brain and has strong links to neuropsychiatric disorders; yet, the role of cell type-specific splicing and transcript-isoform diversity during human brain development has not been systematically investigated. In this work, we leveraged single-molecule long-read sequencing to deeply profile the full-length transcriptome of the germinal zone and cortical plate regions of the developing human neocortex at tissue and single-cell resolution. We identified 214,516 distinct isoforms, of which 72.6% were novel (not previously annotated in Gencode version 33), and uncovered a substantial contribution of transcript-isoform diversity-regulated by RNA binding proteins-in defining cellular identity in the developing neocortex. We leveraged this comprehensive isoform-centric gene annotation to reprioritize thousands of rare de novo risk variants and elucidate genetic risk mechanisms for neuropsychiatric disorders.
Asunto(s)
Trastornos Mentales , Neocórtex , Neurogénesis , Isoformas de Proteínas , Empalme del ARN , Análisis de la Célula Individual , Transcriptoma , Humanos , Empalme Alternativo , Predisposición Genética a la Enfermedad , Trastornos Mentales/genética , Anotación de Secuencia Molecular , Neocórtex/metabolismo , Neocórtex/embriología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Neurogénesis/genéticaRESUMEN
PURPOSE: To evaluate outcomes of intravenous (IV) tocilizumab (TCZ) in patients with pars planitis refractory to conventional immunomodulatory therapy and anti-tumor necrosis factor (TNF) alpha agents. METHODS: Medical records of eight patients diagnosed with pars planitis and treated with monthly 4 or 8 mg/kg IV TCZ were reviewed. The primary objective was to initiate and sustain remission continuously for three consecutive months. Secondary outcome measures were changes in best corrected visual acuity (BCVA), degree of anterior chamber (AC) inflammation, vitreous cell, vitreous haze, presence of vitreous or pars plana exudates, peripheral vasculitis, fluorescein angiography (FA) score and central subfieldthickness (CST) on macular optical coherence tomography (OCT). RESULTS: Fourteen eyes of eight patients were treated with IV TCZ. Seven patients were women. The average age was 31.35 ± 16.42 years. In 6 (75%) out of 8 patients, IV TCZ, either as monotherapy or in combination with another conventional immunomodulatory agent, induced and sustained remission. The average FA score reduced from 11.15 ± 3.52 at the baseline visit to 6.50 ± 2.12 at the one-year follow-up visit (p-value < 0.05). None of the patients experienced any side effects of IV TCZ. CONCLUSION: IV Tocilizumab (TCZ) may represent an effective and safe treatment option for patients diagnosed with pars planitis resistant to conventional immunomodulatory therapy and anti-TNF alpha agents.
RESUMEN
Genomic profiling in postmortem brain from autistic individuals has consistently revealed convergent molecular changes. What drives these changes and how they relate to genetic susceptibility in this complex condition are not well understood. We performed deep single-nucleus RNA sequencing (snRNA-seq) to examine cell composition and transcriptomics, identifying dysregulation of cell type-specific gene regulatory networks (GRNs) in autism spectrum disorder (ASD), which we corroborated using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) and spatial transcriptomics. Transcriptomic changes were primarily cell type specific, involving multiple cell types, most prominently interhemispheric and callosal-projecting neurons, interneurons within superficial laminae, and distinct glial reactive states involving oligodendrocytes, microglia, and astrocytes. Autism-associated GRN drivers and their targets were enriched in rare and common genetic risk variants, connecting autism genetic susceptibility and cellular and circuit alterations in the human brain.
Asunto(s)
Trastorno del Espectro Autista , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Análisis de la Célula Individual , Transcriptoma , Femenino , Humanos , Masculino , Astrocitos/metabolismo , Trastorno del Espectro Autista/genética , Encéfalo/metabolismo , Cromatina/metabolismo , Genómica , Interneuronas/metabolismo , Microglía/metabolismo , Neuronas/metabolismo , Oligodendroglía/metabolismo , RNA-Seq , Análisis de Secuencia de ARN , Preescolar , Niño , Adolescente , Adulto Joven , Adulto , Persona de Mediana EdadRESUMEN
Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet, little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multi-omics datasets into a resource comprising >2.8M nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550K cell-type-specific regulatory elements and >1.4M single-cell expression-quantitative-trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized ~250 disease-risk genes and drug targets with associated cell types.
RESUMEN
PURPOSE: To compare vision acuity outcomes of long-term steroid therapy compared with immunomodulatory therapy for treatment of sympathetic ophthalmia. DESIGN: Single-center, retrospective, comparative clinical study. METHODS: Patients with sympathetic ophthalmia treated from March 2005 to October 2022 with at least 1 year of follow-up were included. Visual acuity outcomes were compared by steroid and immunomodulatory treatment modality. RESULTS: Thirty-five patients with sympathetic ophthalmia were included in the study, with follow-up ranging from 1 to 17 years. Higher rates of vision loss correlated with longer periods of active uveitis and steroid treatment. Lower rates of vision loss correlated with longer periods of uveitis remission on immunomodulatory therapy alone and drug-free remission. Treatment with alkylating agents or combination therapy with an antimetabolite, a biologic-response modifier, and cyclosporine are more likely to result in sympathetic ophthalmia remission. CONCLUSION: Immunomodulatory therapy leads to superior vision outcomes in cases of steroid-resistant or recurrent sympathetic ophthalmia. Steroid therapy may be useful for acute or recalcitrant sympathetic uveitis but is insufficient for long-term inflammatory control. PRéCIS: This manuscript describes a retrospective analysis of vision outcomes in patients with sympathetic ophthalmia. Results indicate that long-term immunomodulatory therapy is associated with better vision outcomes than long-term steroid therapy for sympathetic ophthalmia treatment.
Asunto(s)
Oftalmía Simpática , Humanos , Oftalmía Simpática/diagnóstico , Oftalmía Simpática/tratamiento farmacológico , Estudios Retrospectivos , Inmunosupresores/uso terapéutico , Ciclosporina , Glucocorticoides/uso terapéuticoRESUMEN
Purpose: To assess the possible correlation of anti-retinal antibody titers and number of anti-retinal antibodies with outcome measurements including visual acuity, subjective vision loss, visual field, and electroretinography in patients with autoimmune retinopathy. Design: Single-center, retrospective cross-sectional study. Patients and Methods: Patients with autoimmune retinopathy who underwent anti-retinal antibody testing at least twice during their follow-up were enrolled. Anti-retinal antibody titers and numbers were grouped as improved, stable, or worsened. Outcomes included Snellen visual acuity, patient-reported vision loss, Humphrey visual field mean deviations, and electroretinography parameters. Results: Thirty-one eyes among 16 patients with autoimmune retinopathy were included. Between-group analyses of visual acuity, subjective vision loss, visual field, and electroretinography outcomes did not reveal any significant differences by anti-retinal antibody titer or number group at a 95% confidence interval. Conclusion: Changes in anti-retinal antibody titers or numbers were not associated with any vision outcome. Repeated anti-retinal antibody testing may be unnecessary after diagnosis of autoimmune retinopathy and detection of an anti-retinal antibody.
RESUMEN
Understanding how genetic variation exerts its effects on the human brain in health and disease has been greatly informed by functional genomic characterization. Studies over the last decade have demonstrated robust evidence of convergent transcriptional and epigenetic profiles in post-mortem cerebral cortex from individuals with Autism Spectrum Disorder (ASD). Here, we perform deep single nuclear (sn) RNAseq to elucidate changes in cell composition, cellular transcriptomes and putative candidate drivers associated with ASD, which we corroborate using snATAC-seq and spatial profiling. We find changes in cell state composition representing transitions from homeostatic to reactive profiles in microglia and astrocytes, a pattern extending to oligodendrocytes and blood brain barrier cells. We identify profound changes in differential expression involving thousands of genes across neuronal and glial subtypes, of which a substantial portion can be accounted for by specific transcription factor networks that are significantly enriched in common and rare genetic risk for ASD. These data, which are available as part of the PsychENCODE consortium, provide robust causal anchors and resultant molecular phenotypes for understanding ASD changes in human brain.
RESUMEN
Genomic regulatory elements active in the developing human brain are notably enriched in genetic risk for neuropsychiatric disorders, including autism spectrum disorder (ASD), schizophrenia, and bipolar disorder. However, prioritizing the specific risk genes and candidate molecular mechanisms underlying these genetic enrichments has been hindered by the lack of a single unified large-scale gene regulatory atlas of human brain development. Here, we uniformly process and systematically characterize gene, isoform, and splicing quantitative trait loci (xQTLs) in 672 fetal brain samples from unique subjects across multiple ancestral populations. We identify 15,752 genes harboring a significant xQTL and map 3,739 eQTLs to a specific cellular context. We observe a striking drop in gene expression and splicing heritability as the human brain develops. Isoform-level regulation, particularly in the second trimester, mediates the greatest proportion of heritability across multiple psychiatric GWAS, compared with eQTLs. Via colocalization and TWAS, we prioritize biological mechanisms for ~60% of GWAS loci across five neuropsychiatric disorders, nearly two-fold that observed in the adult brain. Finally, we build a comprehensive set of developmentally regulated gene and isoform co-expression networks capturing unique genetic enrichments across disorders. Together, this work provides a comprehensive view of genetic regulation across human brain development as well as the stage-and cell type-informed mechanistic underpinnings of neuropsychiatric disorders.
RESUMEN
RNA splicing is highly prevalent in the brain and has strong links to neuropsychiatric disorders, yet the role of cell-type-specific splicing or transcript-isoform diversity during human brain development has not been systematically investigated. Here, we leveraged single-molecule long-read sequencing to deeply profile the full-length transcriptome of the germinal zone (GZ) and cortical plate (CP) regions of the developing human neocortex at tissue and single-cell resolution. We identified 214,516 unique isoforms, of which 72.6% are novel (unannotated in Gencode-v33), and uncovered a substantial contribution of transcript-isoform diversity, regulated by RNA binding proteins, in defining cellular identity in the developing neocortex. We leveraged this comprehensive isoform-centric gene annotation to re-prioritize thousands of rare de novo risk variants and elucidate genetic risk mechanisms for neuropsychiatric disorders. One-Sentence Summary: A cell-specific atlas of gene isoform expression helps shape our understanding of brain development and disease. Structured Abstract: INTRODUCTION: The development of the human brain is regulated by precise molecular and genetic mechanisms driving spatio-temporal and cell-type-specific transcript expression programs. Alternative splicing, a major mechanism increasing transcript diversity, is highly prevalent in the human brain, influences many aspects of brain development, and has strong links to neuropsychiatric disorders. Despite this, the cell-type-specific transcript-isoform diversity of the developing human brain has not been systematically investigated.RATIONALE: Understanding splicing patterns and isoform diversity across the developing neocortex has translational relevance and can elucidate genetic risk mechanisms in neurodevelopmental disorders. However, short-read sequencing, the prevalent technology for transcriptome profiling, is not well suited to capturing alternative splicing and isoform diversity. To address this, we employed third-generation long-read sequencing, which enables capture and sequencing of complete individual RNA molecules, to deeply profile the full-length transcriptome of the germinal zone (GZ) and cortical plate (CP) regions of the developing human neocortex at tissue and single-cell resolution.RESULTS: We profiled microdissected GZ and CP regions of post-conception week (PCW) 15-17 human neocortex in bulk and at single-cell resolution across six subjects using high-fidelity long-read sequencing (PacBio IsoSeq). We identified 214,516 unique isoforms, of which 72.6% were novel (unannotated in Gencode), and >7,000 novel exons, expanding the proteome by 92,422 putative proteoforms. We uncovered thousands of isoform switches during cortical neurogenesis predicted to impact RNA regulatory domains or protein structure and implicating previously uncharacterized RNA-binding proteins in cellular identity and neuropsychiatric disease. At the single-cell level, early-stage excitatory neurons exhibited the greatest isoform diversity, and isoform-centric single-cell clustering led to the identification of previously uncharacterized cell states. We systematically assessed the contribution of transcriptomic features, and localized cell and spatio-temporal transcript expression signatures across neuropsychiatric disorders, revealing predominant enrichments in dynamic isoform expression and utilization patterns and that the number and complexity of isoforms per gene is strongly predictive of disease. Leveraging this resource, we re-prioritized thousands of rare de novo risk variants associated with autism spectrum disorders (ASD), intellectual disability (ID), and neurodevelopmental disorders (NDDs), more broadly, to potentially more severe consequences and revealed a larger proportion of cryptic splice variants with the expanded transcriptome annotation provided in this study.CONCLUSION: Our study offers a comprehensive landscape of isoform diversity in the human neocortex during development. This extensive cataloging of novel isoforms and splicing events sheds light on the underlying mechanisms of neurodevelopmental disorders and presents an opportunity to explore rare genetic variants linked to these conditions. The implications of our findings extend beyond fundamental neuroscience, as they provide crucial insights into the molecular basis of developmental brain disorders and pave the way for targeted therapeutic interventions. To facilitate exploration of this dataset we developed an online portal ( https://sciso.gandallab.org/ ).
RESUMEN
Delivery to the proper tissue compartment is a major obstacle hampering the potential of cellular therapeutics for medical conditions. Delivery of cells within biomaterials may improve localization, but traditional and newer void-forming hydrogels must be made in advance with cells being added into the scaffold during the manufacturing process. Injectable, in situ cross-linking microporous scaffolds are recently developed that demonstrate a remarkable ability to provide a matrix for cellular proliferation and growth in vitro in three dimensions. The ability of these scaffolds to deliver cells in vivo is currently unknown. Herein, it is shown that mesenchymal stem cells (MSCs) can be co-injected locally with microparticle scaffolds assembled in situ immediately following injection. MSC delivery within a microporous scaffold enhances MSC retention subcutaneously when compared to cell delivery alone or delivery within traditional in situ cross-linked nanoporous hydrogels. After two weeks, endothelial cells forming blood vessels are recruited to the scaffold and cells retaining the MSC marker CD29 remain viable within the scaffold. These findings highlight the utility of this approach in achieving localized delivery of stem cells through an injectable porous matrix while limiting obstacles of introducing cells within the scaffold manufacturing process.
Asunto(s)
Células Madre/citología , Andamios del Tejido/química , Animales , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Microfluídica/métodos , Ingeniería de Tejidos/métodosRESUMEN
Homeostasis of intraocular pressure (IOP) is important for the maintenance of anterior eye anatomic integrity, minimizing pressure-associated damage to the optic nerve, and maintaining a pressure gradient for blood flow to the eye. IOP is regulated by equilibrium between aqueous humor (AH) production and its outflow. The ciliary body (CB) is thought to actively secrete AH. However, whether AH composition and in particular, its phospholipids are entirely due to CB secretion remains uncertain. Comparison of phospholipids released by cultured CB, phospholipids present within CB tissue, within AH, and within blood and serum are consistent with release of most phospholipids into the AH by the CB. Treatment of CB in culture with timolol, a non-specific beta-adrenergic antagonist, alters the release of phospholipids by CB into the media. However, dorzalamide, a carbonic anhydrase inhibitor that reduces production of AH, does not affect phospholipid release thereby suggesting timolol, which also decreases IOP through decreased AH outflow, affects other physiological homeostatic mechanisms regulating aqueous outflow. These outflow changes also affect the composition of secreted phospholipids. We present evidence that release of lipids by the CB has a prolonged survival effect on cultured primary TM cells and TM tissue.
Asunto(s)
Cuerpo Ciliar/metabolismo , Fosfolípidos/metabolismo , Anciano , Anciano de 80 o más Años , Humor Acuoso/química , Humor Acuoso/metabolismo , Cadáver , Femenino , Humanos , Masculino , Técnicas de Cultivo de Órganos/métodosRESUMEN
PURPOSE: Gray crescents characterized by darkly pigmented regions on the optic nerve head could make assessment of cupping difficult by obscuring the true anatomic disc border. On slit-lamp examination, 2 morphologically distinct types of gray crescents at the curved disc boundary exist. The in vivo histologic characteristics differentiating these 2 types are described using spectral-domain (SD) OCT. METHODS: A prospective interventional case series in a single tertiary referral center. Sixteen eyes of 10 patients with gray crescents determined through clinical examination and fundus photography underwent cross-sectional optic nerve head and parapapillary imaging using high-resolution SD OCT. In vivo histology of gray crescents was characterized. RESULTS: In SD OCT images across the areas of gray crescent, type A gray crescents represent regions where the retinal pigment epithelium (RPE)-Bruch's membrane complex is absent and the dark underlying choroid is directly visible. In contrast, type B gray crescents represent regions of the retina where the RPE is clumped or thickened, typically appearing to be folded upon itself, revealing the embryonic continuity of the RPE and neuroretina. CONCLUSIONS: Shields type A and B gray crescents appear different clinically because histologically they represent differences in RPE. Type A grey crescents lack RPE, whereas type B crescents have thickened, folded RPE. Gray crescents can obscure the true disc border and result in pseudo-cupping of the optic nerve head. Identification of gray crescents is clinically important because failure to recognize pseudo-cupping can contribute to overdiagnosis of glaucoma.
Asunto(s)
Glaucoma/diagnóstico , Disco Óptico/diagnóstico por imagen , Epitelio Pigmentado de la Retina/patología , Tomografía de Coherencia Óptica/métodos , Coroides/patología , Estudios Transversales , Femenino , Estudios de Seguimiento , Humanos , Masculino , Estudios ProspectivosRESUMEN
PURPOSE OF REVIEW: Optical coherence tomography (OCT) has become an integral component of modern glaucoma practice. Utilizing color codes, OCT analysis has rendered glaucoma diagnosis and follow-up simpler and faster for the busy clinician. However, green labeling of OCT parameters suggesting normal values may confer a false sense of security, potentially leading to missed diagnoses of glaucoma and/or glaucoma progression. RECENT FINDINGS: Conditions in which OCT color coding may be falsely negative (i.e., green disease) are identified. Early glaucoma in which retinal nerve fiber layer (RNFL) thickness and optic disc parameters, albeit labeled green, are asymmetric in both eyes may result in glaucoma being undetected. Progressively decreasing RNFL thickness may reveal the presence of progressive glaucoma that, because of green labeling, can be missed by the clinician. Other ocular conditions that can increase RNFL thickness can make the diagnosis of coexisting glaucoma difficult. Recently introduced progression analysis features of OCT may help detect green disease. SUMMARY: Recognition of green disease is of paramount importance in diagnosing and treating glaucoma. Understanding the limitations of imaging technologies coupled with evaluation of serial OCT analyses, prompt clinical examination, and structure-function correlation is important to avoid missing real glaucoma requiring treatment.
Asunto(s)
Glaucoma/diagnóstico por imagen , Fibras Nerviosas/patología , Enfermedades del Nervio Óptico/diagnóstico por imagen , Células Ganglionares de la Retina/patología , Tomografía de Coherencia Óptica/normas , Reacciones Falso Negativas , Humanos , Presión Intraocular , Disco Óptico/patología , Valor Predictivo de las PruebasRESUMEN
PURPOSE: To estimate visual field (VF) sensitivity at which retinal nerve fiber layer (RNFL) thinning reaches the measurement floor and at which RNFL stops thinning (change points), the dynamic range of RNFL thickness, and the number of steps from normal to RNFL floor among three optical coherence tomography (OCT) devices. METHODS: Glaucomatous patients (n = 58) and healthy subjects (n = 55-60) prospectively underwent VF testing and RNFL thickness measurement with Cirrus, Spectralis, and RTVue. Change points and corresponding RNFL thicknesses were estimated with simple linear regression (SLR) and Bayesian change point (BCP) analyses. The dynamic range and number of steps to RNFL floor were determined. RESULTS: The average VF change points and corresponding residual thickness at the time RNFL stopped thinning were -22.2 dB and 57.0 µm (Cirrus), -25.3 dB and 49.2 µm (Spectralis), and -24.6 dB and 64.7 µm (RTVue). The RNFL dynamic ranges derived from SLR values were wider on Spectralis (52.6 µm) than on Cirrus (35.4 µm) and RTVue (35.5 µm); the corresponding number of steps to reach the RNFL floor were 9.0 on Cirrus, 10.6 on Spectralis, and 8.3 on RTVue. CONCLUSIONS: The relative VF sensitivity at which average RNFL thickness reaches the measurement floor, the residual layer thickness, and RNFL dynamic measurement range differ among the three devices. However, the number of steps from normal to the RNFL thickness floor is comparable.
Asunto(s)
Glaucoma de Ángulo Abierto/diagnóstico , Glaucoma de Baja Tensión/diagnóstico , Fibras Nerviosas/patología , Células Ganglionares de la Retina/patología , Tomografía de Coherencia Óptica/instrumentación , Diseño de Equipo , Glaucoma , Humanos , Reproducibilidad de los ResultadosRESUMEN
Obesity, an established risk factor for breast cancer (BC), is associated with systemic inflammation. The breast contains adipose tissue (bAT), yet whether it plays a role in BC progression in obese females is being intensively studied. There is scarce knowledge on the lipid composition of bAT in health and disease. The purpose of this pilot study was: 1) to determine whether obesity and BC are associated with inflammatory changes in bAT 2) to analyze for the first time the lipid profile of bAT in obese and lean mammary tumor-bearing and normal mice. Syngeneic E0771 mammary tumor cells were implanted into the mammary fat pad of lean and diet-induced obese C57BL/6 mice. BATs were analyzed four weeks after tumor cell inoculation by immunohistochemistry and mass spectrometry. Phospholipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan or neutral ion loss scan employing appropriate class specific lipid standards in a two step quantification process. Four main classes of phospholipids were analyzed: phosphatidylcholines phosphatidylserines, phosphatidylethanolamines and phosphatidylinositols. Our results showed that bAT in obese (normal and tumor-bearing) mice contained hypertrophic adipocytes compared with their corresponding samples in lean mice; higher numbers of macrophages and crown-like structures were observed in obese tumor bearers compared to obese normal mice. BAT from normal obese mice revealed higher concentrations of phosphatidylethanolamines. Furthermore, bAT from tumor-bearing mice expressed higher phosphatidylcholines than that from non-tumor bearing mice, suggesting the presence of the tumor is associated with phosphatidylcholines. Conversion of phosphatidylethanolamines to phosphatidylcholines will be investigated in E0771 cells. Additional studies are projected to investigate macrophage activation by these specific classes of phospholipids. Occurrence of triglycerides and free fatty acids will be examined in bAT and similar lipidomic analyses will be carried out visceral adipose tissue, highly inflamed in obesity.
Asunto(s)
Tejido Adiposo/metabolismo , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/patología , Obesidad/patología , Fosfolípidos/química , Fosfolípidos/metabolismo , Animales , Línea Celular Tumoral , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Femenino , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/complicaciones , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos C57BL , Obesidad/complicaciones , Obesidad/metabolismo , Proyectos PilotoRESUMEN
Proteins have important functional roles in the body, which can be altered in disease states. The eye is a complex organ rich in proteins; in particular, the anterior eye is very sophisticated in function and is most commonly involved in ophthalmic diseases. Proteomics, the large scale study of proteins, has greatly impacted our knowledge and understanding of gene function in the post-genomic period. The most significant breakthrough in proteomics has been mass spectrometric identification of proteins, which extends analysis far beyond the mere display of proteins that classical techniques provide. Mass spectrometry functions as a "mass analyzer" which simplifies the identification and quantification of proteins extracted from biological tissue. Mass spectrometric analysis of the anterior eye proteome provides a differential display for protein comparison of normal and diseased tissue. In this article we present the key proteomic findings in the recent literature related to the cornea, aqueous humor, trabecular meshwork, iris, ciliary body and lens. Through this we identified unique proteins specific to diseases related to the anterior eye.
RESUMEN
This study investigated the impact of missing data in the evaluation of artificial neural network (ANN) models trained on complete data for the task of predicting whether breast lesions are benign or malignant from their mammographic Breast Imaging and Reporting Data System (BI-RADS) descriptors. A feed-forward, back-propagation ANN was tested with three methods for estimating the missing values. Similar results were achieved with a constraint satisfaction ANN, which can accommodate missing values without a separate estimation step. This empirical study highlights the need for additional research on developing robust clinical decision support systems for realistic environments in which key information may be unknown or inaccessible.