Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
1.
Cytometry A ; 101(1): 27-44, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34390166

RESUMEN

T-cell activation is a key step in the amplification of an immune response. Over the course of an immune response, cells may be chronically stimulated, with some proportion becoming exhausted; an enormous number of molecules are involved in this process. There remain a number of questions about the process, namely: (1) what degree of heterogeneity and plasticity do T-cells exhibit during stimulation? (2) how many unique cell states define chronic stimulation? and (3) what markers discriminate activated from exhausted cells? We addressed these questions by performing single-cell multiomic analysis to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study -with unprecedented depth-how T cells change over the course of chronic stimulation. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, activation downregulated naïve-cell markers and upregulated effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation, defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely up-regulated upon 14 days of stimulation (CD39, ENTPD1, TNFDF10); expression of these markers could be associated with the emergence of short-lived cell types. Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data reveal the high heterogeneity and plasticity of chronically stimulated T cells. Our study demonstrates the power of a single-cell multiomic approach to comprehensively characterize T-cells and to precisely monitor changes in differentiation, activation, and exhaustion signatures during cell stimulation.


Asunto(s)
Linfocitos T CD8-positivos , Activación de Linfocitos , Humanos , Inmunofenotipificación , Cinética , Análisis de la Célula Individual
2.
Immunity ; 36(4): 658-67, 2012 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-22464169

RESUMEN

Innate immunity is highly conserved and relies on pattern recognition receptors (PRRs) such as Toll-like receptors (identified through their homology to Drosophila Toll) for pathogen recognition. Although Drosophila Toll is vital for immune recognition and defense, roles for the other eight Drosophila Tolls in immunity have remained elusive. Here we have shown that Toll-7 is a PRR both in vitro and in adult flies; loss of Toll-7 led to increased vesicular stomatitis virus (VSV) replication and mortality. Toll-7, along with additional uncharacterized Drosophila Tolls, was transcriptionally induced by VSV infection. Furthermore, Toll-7 interacted with VSV at the plasma membrane and induced antiviral autophagy independently of the canonical Toll signaling pathway. These data uncover an evolutionarily conserved role for a second Drosophila Toll receptor that links viral recognition to autophagy and defense and suggest that other Drosophila Tolls may restrict specific as yet untested pathogens, perhaps via noncanonical signaling pathways.


Asunto(s)
Autofagia , Drosophila melanogaster/inmunología , Receptor Toll-Like 7/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular New Jersey/inmunología , Animales , Línea Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Cricetinae , Drosophila melanogaster/virología , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal , Receptor Toll-Like 7/genética , Virus de la Estomatitis Vesicular Indiana/fisiología , Virus de la Estomatitis Vesicular New Jersey/fisiología , Replicación Viral
3.
RNA ; 23(12): 1834-1849, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28851751

RESUMEN

RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the single-celled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation, we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψ's in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψ's being relatively depleted in the 3'-UTR but enriched at position 1 of codons. We show that many Ψ's in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψ's are enriched in developmentally regulated transcripts. RNA-seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites, while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψ's play an important biological role.


Asunto(s)
Fibroblastos/metabolismo , Seudouridina/química , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Toxoplasma/metabolismo , Toxoplasmosis/metabolismo , Células Cultivadas , Fibroblastos/parasitología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , ARN Mensajero/genética , ARN Protozoario/genética , ARN Ribosómico/genética , Toxoplasma/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/genética , Toxoplasmosis/parasitología
4.
J Am Chem Soc ; 140(10): 3583-3591, 2018 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-29505267

RESUMEN

RNA aptamers that generate a strong fluorescence signal upon binding a nonfluorescent small-molecule dye offer a powerful means for the selective imaging of individual RNA species. Unfortunately, conventional in vitro discovery methods are not efficient at generating such fluorescence-enhancing aptamers, because they primarily exert selective pressure based on target affinity-a characteristic that correlates poorly with fluorescence enhancement. Thus, only a handful of fluorescence-enhancing aptamers have been reported to date. In this work, we describe a method for converting DNA libraries into "gene-linked RNA aptamer particles" (GRAPs) that each display ∼105 copies of a single RNA sequence alongside the DNA that encodes it. We then screen large libraries of GRAPs in a high-throughput manner using the FACS instrument based directly on their fluorescence-enhancing properties. Using this strategy, we demonstrate the capability to generate fluorescence-enhancing aptamers that produce a variety of different emission wavelengths upon binding the dye of interest.


Asunto(s)
Aptámeros de Nucleótidos/química , Fluorescencia , Colorantes Fluorescentes/química , Técnica SELEX de Producción de Aptámeros
5.
Anal Chem ; 90(5): 3262-3269, 2018 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-29436820

RESUMEN

Biologic drugs are typically manufactured in mammalian host cells, and it is critical from a drug safety and efficacy perspective to detect and remove host cell proteins (HCPs) during production. This is currently achieved with sets of polyclonal antibodies (pAbs), but these suffer from critical shortcomings because their composition is inherently undefined, and they cannot detect nonimmunogenic HCPs. In this work, we report a high-throughput screening and array-based binding characterization strategy that we employed to generate a set of aptamers that overcomes these limitations to achieve sensitive, broad-spectrum detection of HCPs from the widely used Chinese hamster ovary (CHO) cell line. We identified a set of 32 DNA aptamers that achieve better sensitivity than a commercial pAb reagent set and can detect a comparable number of HCPs over a broad range of isoelectric points and sizes. Importantly, these aptamers detect multiple contaminants that are known to be responsible for therapeutic antibody degradation and toxicity in patients. Because HCP aptamer reagents are sequence-defined and chemically synthesized, we believe they may enable safer production of biologic drugs, and this strategy should be broadly applicable for the generation of HCP detection reagents for other cell lines.


Asunto(s)
Aptámeros de Nucleótidos/química , ADN/química , Contaminación de Medicamentos/prevención & control , Proteínas/análisis , Animales , Anticuerpos/inmunología , Células CHO , Cricetulus , Límite de Detección , Proteínas/inmunología
6.
Acc Chem Res ; 49(9): 1903-10, 2016 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-27526193

RESUMEN

Affinity reagents that specifically bind to their target molecules are invaluable tools in nearly every field of modern biomedicine. Nucleic acid-based aptamers offer many advantages in this domain, because they are chemically synthesized, stable, and economical. Despite these compelling features, aptamers are currently not widely used in comparison to antibodies. This is primarily because conventional aptamer-discovery techniques such as SELEX are time-consuming and labor-intensive and often fail to produce aptamers with comparable binding performance to antibodies. This Account describes a body of work from our laboratory in developing advanced methods for consistently producing high-performance aptamers with higher efficiency, fewer resources, and, most importantly, a greater probability of success. We describe our efforts in systematically transforming each major step of the aptamer discovery process: selection, analysis, and characterization. To improve selection, we have developed microfluidic devices (M-SELEX) that enable discovery of high-affinity aptamers after a minimal number of selection rounds by precisely controlling the target concentration and washing stringency. In terms of improving aptamer pool analysis, our group was the first to use high-throughput sequencing (HTS) for the discovery of new aptamers. We showed that tracking the enrichment trajectory of individual aptamer sequences enables the identification of high-performing aptamers without requiring full convergence of the selected aptamer pool. HTS is now widely used for aptamer discovery, and open-source software has become available to facilitate analysis. To improve binding characterization, we used HTS data to design custom aptamer arrays to measure the affinity and specificity of up to ∼10(4) DNA aptamers in parallel as a means to rapidly discover high-quality aptamers. Most recently, our efforts have culminated in the invention of the "particle display" (PD) screening system, which transforms solution-phase aptamers into "aptamer particles" that can be individually screened at high-throughput via fluorescence-activated cell sorting. Using PD, we have shown the feasibility of rapidly generating aptamers with exceptional affinities, even for proteins that have previously proven intractable to aptamer discovery. We are confident that these advanced aptamer-discovery methods will accelerate the discovery of aptamer reagents with excellent affinities and specificities, perhaps even exceeding those of the best monoclonal antibodies. Since aptamers are reproducible, renewable, stable, and can be distributed as sequence information, we anticipate that these affinity reagents will become even more valuable tools for both research and clinical applications.


Asunto(s)
Aptámeros de Nucleótidos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Técnica SELEX de Producción de Aptámeros/métodos , Aptámeros de Nucleótidos/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Dispositivos Laboratorio en un Chip
7.
Anal Chem ; 88(22): 10842-10847, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27813404

RESUMEN

Sandwich assays are among the most powerful tools in molecular detection. These assays use "pairs" of affinity reagents so that the detection signal is generated only when both reagents bind simultaneously to different sites on the target molecule, enabling highly sensitive and specific measurements in complex samples. Thus, the capability to efficiently screen affinity reagent pairs at a high throughput is critical. In this work, we describe an experimental strategy for screening "aptamer pairs" at a throughput of 106 aptamer pairs per hour-which is many orders of magnitude higher than the current state of the art. The key step in our process is the conversion of solution-phase aptamers into "aptamer particles" such that we can directly measure the simultaneous binding of multiple aptamers to a target protein based on fluorescence signals and sort individual particles harboring aptamer pairs via the fluorescence-activated cell-sorter instrument. As proof of principle, we successfully isolated a high-quality DNA aptamer pair for plasminogen activator inhibitor 1 (PAI-1). Within only two rounds of screening, we discovered DNA aptamer pairs with low-nanomolar sensitivity in dilute serum and excellent specificity with minimal off-target binding even to closely related proteins such as PAI-2.


Asunto(s)
Aptámeros de Nucleótidos/análisis , Bioensayo , Ensayos Analíticos de Alto Rendimiento , Fluorescencia , Inhibidor 1 de Activador Plasminogénico/química
8.
bioRxiv ; 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38766031

RESUMEN

Hematopoietic multipotent progenitors (MPPs) regulate blood cell production to appropriately meet the biological demands of the human body. Human MPPs remain ill-defined whereas mouse MPPs have been well characterized with distinct immunophenotypes and lineage potencies. Using multiomic single cell analyses and complementary functional assays, we identified new human MPPs and oligopotent progenitor populations within Lin-CD34+CD38dim/lo adult bone marrow with distinct biomolecular and functional properties. These populations were prospectively isolated based on expression of CD69, CLL1, and CD2 in addition to classical markers like CD90 and CD45RA. We show that within the canonical Lin-CD34+CD38dim/loCD90CD45RA-MPP population, there is a CD69+ MPP with long-term engraftment and multilineage differentiation potential, a CLL1+ myeloid-biased MPP, and a CLL1-CD69-erythroid-biased MPP. We also show that the canonical Lin-CD34+CD38dim/loCD90-CD45RA+ LMPP population can be separated into a CD2+ LMPP with lymphoid and myeloid potential, a CD2-LMPP with high lymphoid potential, and a CLL1+ GMP with minimal lymphoid potential. We used these new HSPC profiles to study human and mouse bone marrow cells and observe limited cell type specific homology between humans and mice and cell type specific changes associated with aging. By identifying and functionally characterizing new adult MPP sub-populations, we provide an updated reference and framework for future studies in human hematopoiesis.

9.
STAR Protoc ; 1(2)2020 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-33000001

RESUMEN

By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 104 cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling. For complete information on the use and execution of this protocol, please refer to Mair et al. (2020).


Asunto(s)
Perfilación de la Expresión Génica/métodos , Proteínas/análisis , Análisis de la Célula Individual/métodos , Anticuerpos/inmunología , Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas/genética , Proteínas/inmunología , Proteómica/métodos , Análisis de Secuencia de ARN/métodos , Transcriptoma/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA