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1.
Nature ; 595(7865): 107-113, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33915569

RESUMEN

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.


Asunto(s)
COVID-19/patología , COVID-19/virología , Riñón/patología , Hígado/patología , Pulmón/patología , Miocardio/patología , SARS-CoV-2/patogenicidad , Adulto , Anciano , Anciano de 80 o más Años , Atlas como Asunto , Autopsia , Bancos de Muestras Biológicas , COVID-19/genética , COVID-19/inmunología , Células Endoteliales , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Fibroblastos , Estudio de Asociación del Genoma Completo , Corazón/virología , Humanos , Inflamación/patología , Inflamación/virología , Riñón/virología , Hígado/virología , Pulmón/virología , Masculino , Persona de Mediana Edad , Especificidad de Órganos , Fagocitos , Alveolos Pulmonares/patología , Alveolos Pulmonares/virología , ARN Viral/análisis , Regeneración , SARS-CoV-2/inmunología , Análisis de la Célula Individual , Carga Viral
2.
Genome Res ; 32(10): 1892-1905, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36100434

RESUMEN

Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene expression. Here, we describe expanding the chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probes targeting the protein-coding genes of the human and mouse transcriptomes, referred to as the human or mouse Whole Transcriptome Atlas (WTA). Human and mouse WTAs were validated in cell lines for concordance with orthogonal gene expression profiling methods in regions ranging from ∼10-500 cells. By benchmarking against bulk RNA-seq and fluorescence in situ hybridization, we show robust transcript detection down to ∼100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. Spatial profiling with WTA detected expected gene expression differences between tumor and tumor microenvironment, identified disease-specific gene expression heterogeneity in histological structures of the human kidney, and comprehensively mapped transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.


Asunto(s)
Perfilación de la Expresión Génica , Neoplasias , Humanos , Animales , Ratones , Hibridación Fluorescente in Situ/métodos , Perfilación de la Expresión Génica/métodos , Transcriptoma , Microambiente Tumoral
3.
Neurobiol Learn Mem ; 153(Pt A): 2-12, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29474956

RESUMEN

Circadian regulation is a conserved phenomenon across the animal kingdom, and its disruption can have severe behavioral and physiological consequences. Core circadian clock proteins are likewise well conserved from Drosophila to humans. While the molecular clock interactions that regulate circadian rhythms have been extensively described, additional roles for clock genes during complex behaviors are less understood. Here, we show that mutations in the clock gene period result in differential time-of-day effects on acquisition and long-term memory of aversive olfactory conditioning. Sleep is also altered in period mutants: while its overall levels don't correlate with memory, sleep plasticity in different genotypes correlates with immediate performance after training. We further describe distinct anatomical bases for Period function by manipulating Period activity in restricted brain cells and testing the effects on specific aspects of memory and sleep. In the null mutant background, different features of sleep and memory are affected when we reintroduce a form of the period gene in glia, lateral neurons, and the fan-shaped body. Our results indicate that the role of the clock gene period may be separable in specific aspects of sleep or memory; further studies into the molecular mechanisms of these processes suggest independent neural circuits and molecular cascades that mediate connections between the distinct phenomena.


Asunto(s)
Encéfalo/fisiología , Relojes Circadianos , Proteínas de Drosophila/fisiología , Memoria/fisiología , Proteínas Circadianas Period/fisiología , Sueño , Animales , Relojes Circadianos/genética , Drosophila/genética , Drosophila/fisiología , Proteínas de Drosophila/genética , Genotipo , Aprendizaje/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Proteínas Circadianas Period/genética , Sueño/genética , Factores de Tiempo
4.
J Neurosci ; 33(17): 7475-87, 2013 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-23616553

RESUMEN

CREB-responsive transcription has an important role in adaptive responses in all cells and tissue. In the nervous system, it has an essential and well established role in long-term memory formation throughout a diverse set of organisms. Activation of this transcription factor correlates with long-term memory formation and disruption of its activity interferes with this process. Most convincingly, augmenting CREB activity in a number of different systems enhances memory formation. In Drosophila, a sequence rearrangement in the original transgene used to enhance memory formation has been a source of confusion. This rearrangement prematurely terminates translation of the full-length protein, leaving the identity of the "enhancing molecule" unclear. In this report, we show that a naturally occurring, downstream, in-frame initiation codon is used to make a dCREB2 protein off of both transgenic and chromosomal substrates. This protein is a transcriptional activator and is responsible for memory enhancement. A number of parameters can affect enhancement, including the short-lived activity of the activator protein, and the time-of-day when induction and behavioral training occur. Our results reaffirm that overexpression of a dCREB2 activator can enhance memory formation and illustrate the complexity of this behavioral enhancement.


Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Proteínas de Drosophila/fisiología , Memoria a Largo Plazo/fisiología , Transactivadores/fisiología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Drosophila , Proteínas de Drosophila/genética , Datos de Secuencia Molecular , Transactivadores/genética
5.
Neurobiol Learn Mem ; 106: 258-67, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24076014

RESUMEN

The transcription factor CREB is an important regulator of many adaptive processes in neurons, including sleep, cellular homeostasis, and memory formation. The Drosophila dCREB2 family includes multiple protein isoforms generated from a single gene. Overexpression of an activator or blocker isoform has been shown to enhance or block memory formation, but the molecular mechanisms underlying these phenomena remain unclear. In the present study, we generate isoform-specific antibodies and new transgenic flies to track and manipulate the activity of different dCREB2 isoforms during memory formation. We find that nuclear accumulation of a dCREB2 activator-related species, p35+, is dynamically regulated during memory formation. Furthermore, various dCREB2 genetic manipulations that enhance or block memory formation correspondingly increase or decrease p35+ levels in the nucleus. Finally, we show that overexpression of S6K can enhance memory formation and increase p35+ nuclear abundance. Taken together, these results suggest that regulation of dCREB2 localization may be a key molecular convergence point in the coordinated host of events that lead to memory formation.


Asunto(s)
Núcleo Celular/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Memoria/fisiología , Neuronas/metabolismo , Isoformas de Proteínas/genética , Animales , Animales Modificados Genéticamente , Conducta Animal/fisiología , Núcleo Celular/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Isoformas de Proteínas/metabolismo
6.
Nat Genet ; 54(8): 1178-1191, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35902743

RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment-refractory cancer. Molecular stratification in pancreatic cancer remains rudimentary and does not yet inform clinical management or therapeutic development. Here, we construct a high-resolution molecular landscape of the cellular subtypes and spatial communities that compose PDAC using single-nucleus RNA sequencing and whole-transcriptome digital spatial profiling (DSP) of 43 primary PDAC tumor specimens that either received neoadjuvant therapy or were treatment naive. We uncovered recurrent expression programs across malignant cells and fibroblasts, including a newly identified neural-like progenitor malignant cell program that was enriched after chemotherapy and radiotherapy and associated with poor prognosis in independent cohorts. Integrating spatial and cellular profiles revealed three multicellular communities with distinct contributions from malignant, fibroblast and immune subtypes: classical, squamoid-basaloid and treatment enriched. Our refined molecular and cellular taxonomy can provide a framework for stratification in clinical trials and serve as a roadmap for therapeutic targeting of specific cellular phenotypes and multicellular interactions.


Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/terapia , Perfilación de la Expresión Génica , Humanos , Terapia Neoadyuvante , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Pronóstico , Transcriptoma/genética , Neoplasias Pancreáticas
7.
Nat Genet ; 53(3): 354-366, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33603233

RESUMEN

The arrangement (syntax) of transcription factor (TF) binding motifs is an important part of the cis-regulatory code, yet remains elusive. We introduce a deep learning model, BPNet, that uses DNA sequence to predict base-resolution chromatin immunoprecipitation (ChIP)-nexus binding profiles of pluripotency TFs. We develop interpretation tools to learn predictive motif representations and identify soft syntax rules for cooperative TF binding interactions. Strikingly, Nanog preferentially binds with helical periodicity, and TFs often cooperate in a directional manner, which we validate using clustered regularly interspaced short palindromic repeat (CRISPR)-induced point mutations. Our model represents a powerful general approach to uncover the motifs and syntax of cis-regulatory sequences in genomics data.


Asunto(s)
Biología Computacional/métodos , Motivos de Nucleótidos , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Inmunoprecipitación de Cromatina , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Aprendizaje Profundo , Ratones , Células Madre Embrionarias de Ratones/fisiología , Proteína Homeótica Nanog/metabolismo , Redes Neurales de la Computación , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Reproducibilidad de los Resultados , Factores de Transcripción SOXB1/metabolismo
8.
bioRxiv ; 2021 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-33655247

RESUMEN

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

9.
Elife ; 52016 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-27661450

RESUMEN

Understanding how the brain captures transient experience and converts it into long lasting changes in neural circuits requires the identification and investigation of the specific ensembles of neurons that are responsible for the encoding of each experience. We have developed a Robust Activity Marking (RAM) system that allows for the identification and interrogation of ensembles of neurons. The RAM system provides unprecedented high sensitivity and selectivity through the use of an optimized synthetic activity-regulated promoter that is strongly induced by neuronal activity and a modified Tet-Off system that achieves improved temporal control. Due to its compact design, RAM can be packaged into a single adeno-associated virus (AAV), providing great versatility and ease of use, including application to mice, rats, flies, and potentially many other species. Cre-dependent RAM, CRAM, allows for the study of active ensembles of a specific cell type and anatomical connectivity, further expanding the RAM system's versatility.

10.
Front Syst Neurosci ; 8: 43, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24744705

RESUMEN

Many biological phenomena oscillate under the control of the circadian system, exhibiting peaks and troughs of activity across the day/night cycle. In most animal models, memory formation also exhibits this property, but the underlying neuronal and molecular mechanisms remain unclear. The dCREB2 transcription factor shows circadian regulated oscillations in its activity, and has been shown to be important for both circadian biology and memory formation. We show that the time-of-day (TOD) of behavioral training affects Drosophila memory formation. dCREB2 exhibits complex changes in protein levels across the daytime and nighttime, and these changes in protein abundance are likely to contribute to oscillations in dCREB2 activity and TOD effects on memory formation.

11.
Science ; 334(6063): 1669-75, 2011 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-22194569

RESUMEN

The rapid encoding of contextual memory requires the CA3 region of the hippocampus, but the necessary genetic pathways remain unclear. We found that the activity-dependent transcription factor Npas4 regulates a transcriptional program in CA3 that is required for contextual memory formation. Npas4 was specifically expressed in CA3 after contextual learning. Global knockout or selective deletion of Npas4 in CA3 both resulted in impaired contextual memory, and restoration of Npas4 in CA3 was sufficient to reverse the deficit in global knockout mice. By recruiting RNA polymerase II to promoters and enhancers of target genes, Npas4 regulates a learning-specific transcriptional program in CA3 that includes many well-known activity-regulated genes, which suggests that Npas4 is a master regulator of activity-regulated gene programs and is central to memory formation.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Región CA3 Hipocampal/fisiología , Regulación de la Expresión Génica , Memoria , Transcripción Genética , Animales , Región CA3 Hipocampal/citología , Condicionamiento Psicológico , Elementos de Facilitación Genéticos , Miedo , Eliminación de Gen , Genes Inmediatos-Precoces , Aprendizaje , Ratones , Ratones Noqueados , Neuronas/fisiología , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Activación Transcripcional
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