RESUMEN
Non-genetic factors can cause individual cells to fluctuate substantially in gene expression levels over time. It remains unclear whether these fluctuations can persist for much longer than the time of one cell division. Current methods for measuring gene expression in single cells mostly rely on single time point measurements, making the duration of gene expression fluctuations or cellular memory difficult to measure. Here, we combined Luria and Delbrück's fluctuation analysis with population-based RNA sequencing (MemorySeq) for identifying genes transcriptome-wide whose fluctuations persist for several divisions. MemorySeq revealed multiple gene modules that expressed together in rare cells within otherwise homogeneous clonal populations. These rare cell subpopulations were associated with biologically distinct behaviors like proliferation in the face of anti-cancer therapeutics. The identification of non-genetic, multigenerational fluctuations can reveal new forms of biological memory in single cells and suggests that non-genetic heritability of cellular state may be a quantitative property.
Asunto(s)
Análisis de la Célula Individual/métodos , Transcriptoma , División Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Genes Reporteros , Humanos , Hibridación Fluorescente in Situ , Microscopía Fluorescente , Análisis de Secuencia de ARN , Imagen de Lapso de TiempoRESUMEN
Conversion of adenosine to inosine is a frequent type of RNA editing, but important details about the biology of this conversion remain unknown because of a lack of imaging tools. We developed inoFISH to directly visualize and quantify adenosine-to-inosine-edited transcripts in situ. We found that editing of the GRIA2, EIF2AK2, and NUP43 transcripts is uncorrelated with nuclear localization and paraspeckle association. Further, NUP43 exhibits constant editing levels between single cells, while GRIA2 editing levels vary.
Asunto(s)
Adenosina/genética , Hibridación Fluorescente in Situ/métodos , Inosina/genética , Imagen Molecular/métodos , Neuronas/metabolismo , Edición de ARN/genética , Adenosina/química , Línea Celular , Humanos , Inosina/química , Imagen Óptica/métodosRESUMEN
The purpose of this study was to elucidate the role of nucleotide binding oligomerization domain-containing protein 2 (NOD2) signaling in atherosclerosis and periodontal bone loss using an Apolipoprotein E(-/-) (ApoE(-/-)) mouse model based on the proposed role of NOD2 in inflammation. NOD2(-/-)ApoE(-/-) and ApoE(-/-) mice fed a standard chow diet were given an oral gavage of Porphyromonas gingivalis for 15 wk. NOD2(-/-)ApoE(-/-) mice exhibited significant increases in inflammatory cytokines, alveolar bone loss, cholesterol, and atherosclerotic lesions in the aorta and the heart compared with ApoE(-/-) mice. In contrast, ApoE(-/-) mice injected i.p. with Muramyl DiPeptide (MDP) to stimulate NOD2 and given an oral gavage of P. gingivalis displayed a reduction of serum inflammatory cytokines, alveolar bone loss, cholesterol, and atherosclerotic lesions in the aorta and aortic sinus compared with ApoE(-/-) mice orally challenged but injected with saline. A reduction in body weight gain was observed in ApoE(-/-) mice fed a high-fat diet (HFD) and injected with MDP compared with ApoE(-/-) mice fed a high-fat diet but injected with saline. MDP treatment of bone marrow-derived macrophages incubated with P. gingivalis increased mRNA expressions of NOD2, Toll-like receptor 2, myeloid differentiation primary response gene 88, and receptor-interacting protein-2 but reduced the expressions of inhibitor of NF-κB kinase-ß, NF-κB, c-Jun N-terminal kinase 3, and TNF-α protein levels compared with saline control, highlighting pathways involved in MDP antiinflammatory effects. MDP activation of NOD2 should be considered in the treatment of inflammatory processes affecting atherosclerosis, periodontal bone loss ,and possibly, diet-induced weight gain.
Asunto(s)
Pérdida de Hueso Alveolar/patología , Aterosclerosis/patología , Infecciones por Bacteroidaceae/complicaciones , Inflamación/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Porphyromonas gingivalis , Acetilmuramil-Alanil-Isoglutamina/administración & dosificación , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Pérdida de Hueso Alveolar/etiología , Análisis de Varianza , Animales , Apolipoproteínas E/genética , Aterosclerosis/etiología , Peso Corporal , Citocinas/sangre , Dieta Alta en Grasa , Inflamación/etiología , Ratones , Ratones Noqueados , Proteína Adaptadora de Señalización NOD2/deficiencia , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Droplet microfluidic systems have been widely deployed to interrogate biological and chemical systems. The major limitations of these systems are the relatively high error rates from critical droplet manipulation functions. To address these limitations, we describe the development of FIDELITY (Flotation and Interdigitated electrode forces on Droplets to Enable Lasting system IntegriTY), a highly sensitive and accurate size-based droplet bandpass filter that leverages the natural buoyancy of aqueous droplets and highly localized dielectrophoretic force generated by interdigitated electrode arrays. Droplet manipulation accuracies greater than 99% were achieved at a throughput of up to 100 droplets/s and separation of droplets that differed in diameter by only 6 µm was demonstrated. Last, the utility of FIDELITY was demonstrated in a droplet size quality control application and also in a droplet-based in vitro transcription/translation workflow. We anticipate FIDELITY to be integrated into a broad range of droplet microfluidic configurations to achieve exceptional operational accuracy.
RESUMEN
AIM: The purpose of this study was to assess the role of anti-bone resorptive agents and an anti-inflammatory compound in murine Porphyromonas gingivalis (P. gingivalis)-induced periodontitis. MATERIAL AND METHODS: Six randomly assigned groups were administered vehicle (saline, control) (n = 6), P. gingivalis infection only (untreated) (n = 6), human-Fc (n = 4), Kavain (n = 6), OPG-Fc (n = 6) and Receptor activator of nuclear factor-kappa B (RANK)-Fc (n = 6) intraperitoneally at day 0, 3 and 7. Animals were euthanized on day 10 and subjected to comprehensive histomorphometric analysis. To capture the progress of inflammation, serum samples were collected at days 0, 3, 7 and 10 for levels of pro-inflammatory cytokines. RESULTS: Compared with control group, OPG-Fc, RANK-Fc and Kavain treatment showed significant bone loss reduction with OPG-Fc performing better than RANK-Fc or Kavain. Epithelial down-growth showed significant reduction in treatment groups with OPG-Fc performing better than RANK-Fc or Kavain. Finally, Kavain, OPG-Fc and RANK-Fc-treated mice displayed reduced inflammatory cell counts and cytokine expression particularly at day 7 postinfection. CONCLUSIONS: RANKL antagonists and Kavain effectively reduced alveolar bone loss in P. gingivalis-induced periodontitis in our mice model. Compared with RANK-Fc, Kavain-treated animals showed milder improvement of bone and connective tissue inflammation. Therapeutic implications in the prevention of periodontal bone loss are discussed.
Asunto(s)
Pérdida de Hueso Alveolar/prevención & control , Periodontitis/metabolismo , Ligando RANK/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Animales , Quimiocina CCL2/sangre , Interleucina-6/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoprotegerina/metabolismo , Porphyromonas gingivalis , Pironas/farmacología , Distribución Aleatoria , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Although single-cell RNA sequencing can reliably detect large-scale transcriptional programs, it is unclear whether it accurately captures the behavior of individual genes, especially those that express only in rare cells. Here, we use single-molecule RNA fluorescence in situ hybridization as a gold standard to assess trade-offs in single-cell RNA-sequencing data for detecting rare cell expression variability. We quantified the gene expression distribution for 26 genes that range from ubiquitous to rarely expressed and found that the correspondence between estimates across platforms improved with both transcriptome coverage and increased number of cells analyzed. Further, by characterizing the trade-off between transcriptome coverage and number of cells analyzed, we show that when the number of genes required to answer a given biological question is small, then greater transcriptome coverage is more important than analyzing large numbers of cells. More generally, our report provides guidelines for selecting quality thresholds for single-cell RNA-sequencing experiments aimed at rare cell analyses.
Asunto(s)
Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Secuencia de Bases/genética , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Hibridación Fluorescente in Situ/métodos , Melanoma/genética , ARN/análisis , ARN/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Transcriptoma/genética , Secuenciación del Exoma/métodosRESUMEN
Studying the morphology of the arterial response to endovascular stent implantation requires embedding the explanted stented artery in rigid materials such as poly(methyl methacrylate) to enable sectioning through both the in situ stent and the arterial wall, thus maintaining the proper anatomic relationships. This is a laborious, time-consuming process. Moreover, the technical quality of stained plastic sections is typically suboptimal and, in some cases, precludes immunohistochemical analysis. Here we describe a novel technique for dissolution of metallic and plastic stents that is compatible with subsequent embedding of "destented" arteries in paraffin, fine sectioning, major staining protocols, and immunohistochemistry.
Asunto(s)
Adhesión en Parafina/métodos , Poliésteres/química , Acero Inoxidable/química , Stents , Animales , Vasos Coronarios/patología , Inmunohistoquímica , Masculino , Modelos Animales , Ratas , Ratas Sprague-Dawley , Solubilidad , Coloración y Etiquetado , Fijación del TejidoRESUMEN
A microfluidic platform is presented which fully automates all incubation steps of a three-stage, multiplexed magnetic bead immunoassay, such as the Luminex® xMAP technology. Magnetic actuation is used to transfer the microbeads between co-infused adjacent laminar flow streams to transport the beads into and out of incubation and wash solutions, with extended incubation channels to allow sufficient bead incubation times (1-30 min, commonly 5 min per stage) to enable high-sensitivity. The serial incubation steps of the immunoassay are completed in succession within the device with no operator interaction, and the continuous flow operation with magnetic bead transfer defines the incubation sequencing requiring no external fluidic controls beyond syringe pump infusion. The binding kinetics of the assay is empirically characterized to determine the required incubation times for specific assay sensitivities in the range 1 pg/ml to 100 ng/ml. By using a Luminex® xMAP duplex assay, concurrent detection of IL-6 and TNF-α was demonstrated on-chip with a detection range 10 pg/ml to 1 ng/ml. This technology enables rapid automation of magnetic microbead assays, and has the potential to perform continuous concentration monitoring.