Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
1.
Nat Immunol ; 21(1): 54-64, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31819256

RESUMEN

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6∆PMN) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/ß release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6∆PMN mice. Ptpn6∆PMN neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/ß expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/ß release.


Asunto(s)
Apoptosis/inmunología , Caspasa 8/inmunología , Neutrófilos/inmunología , Proteínas Quinasas/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Animales , Caspasa 8/genética , Células Cultivadas , Eliminación de Gen , Inflamación/inmunología , Interleucina-1/inmunología , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Receptores Tipo I de Interleucina-1/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
2.
PLoS Comput Biol ; 18(1): e1009776, 2022 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-35007280

RESUMEN

[This corrects the article DOI: 10.1371/journal.pcbi.1007764.].

3.
PLoS Comput Biol ; 18(8): e1010401, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35939509

RESUMEN

In analyzing the neural correlates of naturalistic and unstructured behaviors, features of neural activity that are ignored in a trial-based experimental paradigm can be more fully studied and investigated. Here, we analyze neural activity from two patients using electrocorticography (ECoG) and stereo-electroencephalography (sEEG) recordings, and reveal that multiple neural signal characteristics exist that discriminate between unstructured and naturalistic behavioral states such as "engaging in dialogue" and "using electronics". Using the high gamma amplitude as an estimate of neuronal firing rate, we demonstrate that behavioral states in a naturalistic setting are discriminable based on long-term mean shifts, variance shifts, and differences in the specific neural activity's covariance structure. Both the rapid and slow changes in high gamma band activity separate unstructured behavioral states. We also use Gaussian process factor analysis (GPFA) to show the existence of salient spatiotemporal features with variable smoothness in time. Further, we demonstrate that both temporally smooth and stochastic spatiotemporal activity can be used to differentiate unstructured behavioral states. This is the first attempt to elucidate how different neural signal features contain information about behavioral states collected outside the conventional experimental paradigm.


Asunto(s)
Electrocorticografía , Electroencefalografía , Mapeo Encefálico , Humanos , Distribución Normal
4.
PLoS Comput Biol ; 18(11): e1010715, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36395331

RESUMEN

Cell-cell interactions shape cellular function and ultimately organismal phenotype. Interacting cells can sense their mutual distance using combinations of ligand-receptor pairs, suggesting the existence of a spatial code, i.e., signals encoding spatial properties of cellular organization. However, this code driving and sustaining the spatial organization of cells remains to be elucidated. Here we present a computational framework to infer the spatial code underlying cell-cell interactions from the transcriptomes of the cell types across the whole body of a multicellular organism. As core of this framework, we introduce our tool cell2cell, which uses the coexpression of ligand-receptor pairs to compute the potential for intercellular interactions, and we test it across the Caenorhabditis elegans' body. Leveraging a 3D atlas of C. elegans' cells, we also implement a genetic algorithm to identify the ligand-receptor pairs most informative of the spatial organization of cells across the whole body. Validating the spatial code extracted with this strategy, the resulting intercellular distances are negatively correlated with the inferred cell-cell interactions. Furthermore, for selected cell-cell and ligand-receptor pairs, we experimentally confirm the communicatory behavior inferred with cell2cell and the genetic algorithm. Thus, our framework helps identify a code that predicts the spatial organization of cells across a whole-animal body.


Asunto(s)
Caenorhabditis elegans , Comunicación Celular , Animales , Ligandos , Comunicación , Fenotipo
5.
BMC Genomics ; 22(1): 69, 2021 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-33478392

RESUMEN

BACKGROUND: Both RNA-Seq and sample freeze-thaw are ubiquitous. However, knowledge about the impact of freeze-thaw on downstream analyses is limited. The lack of common quality metrics that are sufficiently sensitive to freeze-thaw and RNA degradation, e.g. the RNA Integrity Score, makes such assessments challenging. RESULTS: Here we quantify the impact of repeated freeze-thaw cycles on the reliability of RNA-Seq by examining poly(A)-enriched and ribosomal RNA depleted RNA-seq from frozen leukocytes drawn from a toddler Autism cohort. To do so, we estimate the relative noise, or percentage of random counts, separating technical replicates. Using this approach we measured noise associated with RIN and freeze-thaw cycles. As expected, RIN does not fully capture sample degradation due to freeze-thaw. We further examined differential expression results and found that three freeze-thaws should extinguish the differential expression reproducibility of similar experiments. Freeze-thaw also resulted in a 3' shift in the read coverage distribution along the gene body of poly(A)-enriched samples compared to ribosomal RNA depleted samples, suggesting that library preparation may exacerbate freeze-thaw-induced sample degradation. CONCLUSION: The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility.


Asunto(s)
Criopreservación , ARN , Congelación , Humanos , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN
6.
PLoS Comput Biol ; 16(5): e1007764, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32396573

RESUMEN

Diverse algorithms can integrate transcriptomics with genome-scale metabolic models (GEMs) to build context-specific metabolic models. These algorithms require identification of a list of high confidence (core) reactions from transcriptomics, but parameters related to identification of core reactions, such as thresholding of expression profiles, can significantly change model content. Importantly, current thresholding approaches are burdened with setting singular arbitrary thresholds for all genes; thus, resulting in removal of enzymes needed in small amounts and even many housekeeping genes. Here, we describe StanDep, a novel heuristic method for using transcriptomics to identify core reactions prior to building context-specific metabolic models. StanDep clusters gene expression data based on their expression pattern across different contexts and determines thresholds for each cluster using data-dependent statistics, specifically standard deviation and mean. To demonstrate the use of StanDep, we built hundreds of models for the NCI-60 cancer cell lines. These models successfully increased the inclusion of housekeeping reactions, which are often lost in models built using standard thresholding approaches. Further, StanDep also provided a transcriptomic explanation for inclusion of lowly expressed reactions that were otherwise only supported by model extraction methods. Our study also provides novel insights into how cells may deal with context-specific and ubiquitous functions. StanDep, as a MATLAB toolbox, is available at https://github.com/LewisLabUCSD/StanDep.


Asunto(s)
Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Metabolómica/métodos , Algoritmos , Genoma , Humanos , Redes y Vías Metabólicas , Modelos Biológicos , Modelos Teóricos , Transcriptoma
7.
Biotechnol Bioeng ; 117(2): 593-598, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31631317

RESUMEN

Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced. This study demonstrates that CRISPRa can make targeted alterations of CHO cells for desired phenotypes.


Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Glicosiltransferasas/genética , Animales , Células CHO , Cricetinae , Cricetulus , Glicosilación , Fenotipo , Polisacáridos/análisis , Polisacáridos/química
8.
Biotechnol Bioeng ; 116(7): 1813-1819, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30883679

RESUMEN

Chinese hamster ovary (CHO) cells are widely used for biopharmaceutical protein production. One challenge limiting CHO cell productivity is apoptosis stemming from cellular stress during protein production. Here we applied CRISPR interference (CRISPRi) to downregulate the endogenous expression of apoptotic genes Bak, Bax, and Casp3 in CHO cells. In addition to reduced apoptosis, mitochondrial membrane integrity was improved and the caspase activity was reduced. Moreover, we optimized the CRISPRi system to enhance the gene repression efficiency in CHO cells by testing different repressor fusion types. An improved Cas9 repressor has been identified by applying C-terminal fusion of a bipartite repressor domain, KRAB-MeCP2, to nuclease-deficient Cas9. These results collectively demonstrate that CHO cells can be rescued from cell apoptosis by targeted gene repression using the CRISPRi system.


Asunto(s)
Apoptosis/genética , Sistemas CRISPR-Cas , Caspasa 3 , Marcación de Gen , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2 , Animales , Células CHO , Proteína 9 Asociada a CRISPR/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Cricetulus , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo
9.
NAR Genom Bioinform ; 3(3): lqab061, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34268494

RESUMEN

Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

10.
J Clin Invest ; 131(20)2021 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-34464357

RESUMEN

BACKGROUNDMultisystem inflammatory syndrome in children (MIS-C) is a rare but potentially severe illness that follows exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Kawasaki disease (KD) shares several clinical features with MIS-C, which prompted the use of intravenous immunoglobulin (IVIG), a mainstay therapy for KD. Both diseases share a robust activation of the innate immune system, including the IL-1 signaling pathway, and IL-1 blockade has been used for the treatment of both MIS-C and KD. The mechanism of action of IVIG in these 2 diseases and the cellular source of IL-1ß have not been defined.METHODSThe effects of IVIG on peripheral blood leukocyte populations from patients with MIS-C and KD were examined using flow cytometry and mass cytometry (CyTOF) and live-cell imaging.RESULTSCirculating neutrophils were highly activated in patients with KD and MIS-C and were a major source of IL-1ß. Following IVIG treatment, activated IL-1ß+ neutrophils were reduced in the circulation. In vitro, IVIG was a potent activator of neutrophil cell death via PI3K and NADPH oxidase, but independently of caspase activation.CONCLUSIONSActivated neutrophils expressing IL-1ß can be targeted by IVIG, supporting its use in both KD and MIS-C to ameliorate inflammation.FUNDINGPatient Centered Outcomes Research Institute; NIH; American Asthma Foundation; American Heart Association; Novo Nordisk Foundation; NIGMS; American Academy of Allergy, Asthma and Immunology Foundation.


Asunto(s)
COVID-19/complicaciones , Inmunoglobulinas Intravenosas/uso terapéutico , Síndrome Mucocutáneo Linfonodular/inmunología , Síndrome Mucocutáneo Linfonodular/terapia , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/terapia , COVID-19/sangre , COVID-19/inmunología , COVID-19/terapia , Estudios de Casos y Controles , Muerte Celular/inmunología , Linaje de la Célula/inmunología , Niño , Preescolar , Proteína Ligando Fas/inmunología , Femenino , Humanos , Lactante , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/sangre , Recuento de Leucocitos , Masculino , Síndrome Mucocutáneo Linfonodular/sangre , Activación Neutrófila , Neutrófilos/clasificación , Neutrófilos/inmunología , Neutrófilos/patología , Síndrome de Respuesta Inflamatoria Sistémica/sangre
11.
J Neural Eng ; 16(1): 016021, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30523860

RESUMEN

OBJECTIVE: Current brain-computer interface (BCI) studies demonstrate the potential to decode neural signals obtained from structured and trial-based tasks to drive actuators with high performance within the context of these tasks. Ideally, to maximize utility, such systems will be applied to a wide range of behavioral settings or contexts. Thus, we explore the potential to augment such systems with the ability to decode abstract behavioral contextual states from neural activity. APPROACH: To demonstrate the feasibility of such context decoding, we used electrocorticography (ECoG) and stereo-electroencephalography (sEEG) data recorded from the cortical surface and deeper brain structures, respectively, continuously across multiple days from three subjects. During this time, the subjects were engaged in a range of naturalistic behaviors in a hospital environment. Behavioral contexts were labeled manually from video and audio recordings; four states were considered: engaging in dialogue, rest, using electronics, and watching television. We decode these behaviors using a factor analysis and support vector machine (SVM) approach. MAIN RESULTS: We demonstrate that these general behaviors can be decoded with high accuracies of 73% for a four-class classifier for one subject and 71% and 62% for a three-class classifier for two subjects. SIGNIFICANCE: To our knowledge, this is the first demonstration of the potential to disambiguate abstract naturalistic behavioral contexts from neural activity recorded throughout the day from implanted electrodes. This work motivates further study of context decoding for BCI applications using continuously recorded naturalistic activity in the clinical setting.


Asunto(s)
Conducta/fisiología , Interfaces Cerebro-Computador , Corteza Cerebral/fisiología , Electrocorticografía/métodos , Electroencefalografía/métodos , Desempeño Psicomotor/fisiología , Adolescente , Adulto , Electrodos Implantados , Femenino , Humanos , Masculino , Adulto Joven
12.
Curr Opin Biotechnol ; 51: 64-69, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29223005

RESUMEN

To meet the ever-growing demand for effective, safe, and affordable protein therapeutics, decades of intense efforts have aimed to maximize the quantity and quality of recombinant proteins produced in CHO cells. Bioprocessing innovations and cell engineering efforts have improved product titer; however, uncharacterized cellular processes and gene regulatory mechanisms still hinder cell growth, specific productivity, and protein quality. Herein, we summarize recent advances in systems biology and data-driven approaches aiming to unravel how molecular pathways, cellular processes, and extrinsic factors (e.g. media supplementation) influence recombinant protein production. In particular, as the available omics data for CHO cells continue to grow, predictive models and screens will be increasingly used to unravel the biological drivers of protein production, which can be used with emerging genome editing technologies to rationally engineer cells to further control the quantity, quality and affordability of many biologic drugs.


Asunto(s)
Ingeniería Celular/métodos , Proteínas Recombinantes/biosíntesis , Biología de Sistemas/métodos , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Proteínas Recombinantes/genética
13.
Sci Rep ; 7(1): 17380, 2017 12 12.
Artículo en Inglés | MEDLINE | ID: mdl-29234075

RESUMEN

Animal studies support the hypothesis that in slow-wave sleep, replay of waking neocortical activity under hippocampal guidance leads to memory consolidation. However, no intracranial electrophysiological evidence for replay exists in humans. We identified consistent sequences of population firing peaks across widespread cortical regions during complete waking periods. The occurrence of these "Motifs" were compared between sleeps preceding the waking period ("Sleep-Pre") when the Motifs were identified, and those following ("Sleep-Post"). In all subjects, the majority of waking Motifs (most of which were novel) had more matches in Sleep-Post than in Sleep-Pre. In rodents, hippocampal replay occurs during local sharp-wave ripples, and the associated neocortical replay tends to occur during local sleep spindles and down-to-up transitions. These waves may facilitate consolidation by sequencing cell-firing and encouraging plasticity. Similarly, we found that Motifs were coupled to neocortical spindles, down-to-up transitions, theta bursts, and hippocampal sharp-wave ripples. While Motifs occurring during cognitive task performance were more likely to have more matches in subsequent sleep, our studies provide no direct demonstration that the replay of Motifs contributes to consolidation. Nonetheless, these results confirm a core prediction of the dominant neurobiological theory of human memory consolidation.


Asunto(s)
Hipocampo/fisiología , Consolidación de la Memoria , Sueño de Onda Lenta , Adulto , Electroencefalografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven
14.
Elife ; 52016 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-27849520

RESUMEN

The link between the combined action of neuromodulators in the brain and global brain states remains a mystery. In this study, using biophysically realistic models of the thalamocortical network, we identified the critical intrinsic and synaptic mechanisms, associated with the putative action of acetylcholine (ACh), GABA and monoamines, which lead to transitions between primary brain vigilance states (waking, non-rapid eye movement sleep [NREM] and REM sleep) within an ultradian cycle. Using ECoG recordings from humans and LFP recordings from cats and mice, we found that during NREM sleep the power of spindle and delta oscillations is negatively correlated in humans and positively correlated in animal recordings. We explained this discrepancy by the differences in the relative level of ACh. Overall, our study revealed the critical intrinsic and synaptic mechanisms through which different neuromodulators acting in combination result in characteristic brain EEG rhythms and transitions between sleep stages.


Asunto(s)
Corteza Cerebral/fisiología , Red Nerviosa/fisiología , Fases del Sueño/fisiología , Tálamo/fisiología , Acetilcolina/metabolismo , Animales , Gatos , Corteza Cerebral/anatomía & histología , Electroencefalografía , Histamina/metabolismo , Humanos , Ratones , Especificidad de la Especie , Tálamo/anatomía & histología , Vigilia/fisiología , Ácido gamma-Aminobutírico/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA