Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Commun Biol ; 4(1): 1218, 2021 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-34686758

RESUMO

Bulk RNA sequencing provides the opportunity to understand biology at the whole transcriptome level without the prohibitive cost of single cell profiling. Advances in spatial transcriptomics enable to dissect tissue organization and function by genome-wide gene expressions. However, the readout of both technologies is the overall gene expression across potentially many cell types without directly providing the information of cell type constitution. Although several in-silico approaches have been proposed to deconvolute RNA-Seq data composed of multiple cell types, many suffer a deterioration of performance in complex tissues. Here we present AdRoit, an accurate and robust method to infer the cell composition from transcriptome data of mixed cell types. AdRoit uses gene expression profiles obtained from single cell RNA sequencing as a reference. It employs an adaptive learning approach to alleviate the sequencing technique difference between the single cell and the bulk (or spatial) transcriptome data, enhancing cross-platform readout comparability. Our systematic benchmarking and applications, which include deconvoluting complex mixtures that encompass 30 cell types, demonstrate its preferable sensitivity and specificity compared to many existing methods as well as its utilities. In addition, AdRoit is computationally efficient and runs orders of magnitude faster than most methods.


Assuntos
Perfilação da Expressão Gênica/métodos , Genoma , Transcriptoma , Sensibilidade e Especificidade
2.
J Biol Chem ; 285(10): 7619-32, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20032460

RESUMO

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid beta (Abeta) known as ADDLs. In primary hippocampal cultures ADDLs bind to a subpopulation of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which Abeta oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Calcineurina/metabolismo , Endocitose/fisiologia , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Animais , Calcineurina/genética , Células Cultivadas , Hipocampo/citologia , Humanos , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/química , Receptores de AMPA/genética , Sinapses/patologia , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo
3.
Biochemistry ; 45(51): 15157-67, 2006 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-17176037

RESUMO

A growing body of evidence suggests that soluble oligomeric forms of the amyloid beta peptide known as amyloid-derived diffusible ligands (ADDLs) are the toxic species responsible for neurodegeneration associated with Alzheimer's disease. Accurate biophysical characterization of ADDL preparations is hampered by the peptide's strong tendency to self-associate and the effect of factors such as ionic strength, temperature, and pH on its behavior. In addition, amyloid peptides are known to interact with common laboratory excipients, specifically detergents, further complicating the results from standard analytical methods such as denaturing polyacrylamide gel electrophoresis. We have studied the solution behavior of various amyloid peptide preparations using analytical ultracentrifugation and size exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that ADDL preparations exist in solution primarily as a binary mixture of a monomeric peptide and high-molecular mass oligomers. We relate our findings to previously described characterizations utilizing atomic force microscopy and electrophoretic methods and demonstrate that low-molecular mass oligomers identified by gel electrophoresis likely represent artifacts induced by the peptide's interaction with detergent, while atomic force microscopy results are likely skewed by differential binding of monomeric and oligomeric peptide species. Finally, we confirm that only the high-molecular mass oligomeric components of an ADDL preparation are capable of binding to subpopulations of primary hippocampal neurons in vitro.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Soluções , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/ultraestrutura , Animais , Células Cultivadas , Cromatografia em Gel , Ligantes , Microscopia de Força Atômica , Peso Molecular , Neurônios/química , Neurônios/metabolismo , Neurônios/ultraestrutura , Fragmentos de Peptídeos/toxicidade , Fragmentos de Peptídeos/ultraestrutura , Ligação Proteica , Ratos
4.
Biol Chem ; 387(2): 195-201, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16497152

RESUMO

Antagonists of the B1 bradykinin receptor (B1R), encoded by the BDKRB1 gene, offer the promise of novel therapeutic agents for inflammatory and neuropathic pain. However, the in vivo characterization of the pharmacodynamics of B1R antagonists is hindered by the low level of B1R expression in healthy tissue and the profound species selectivity exhibited by many compounds for the B1R. To circumvent these issues we generated two genetically engineered rodent models. The first is a transgenic rat over-expressing the human B1R under the control of the neuronal-specific enolase promoter; we previously reported the utility of this model in assessing human B1R receptor occupancy in the central nervous system of the rat. The second model, reported here, utilized gene-targeting by homologous recombination to replace the genomic coding sequence for the endogenous mouse B1R with that of the human B1R. The mRNA expression profile of the humanized Bdkrb1 (hBkdrb1) allele is similar to that of the mouse Bdkrb1 (mBkdrb1) in the wild-type animal. Furthermore, in vitro assays indicate that tissues isolated from the humanized mouse possess pharmacological properties characteristic of the human B1R. Therefore, we have generated a humanized B1R mouse model that is suitable for testing the efficacy of human B1R-selective compounds.


Assuntos
Acetamidas/farmacologia , Receptor B1 da Bradicinina , Sulfonas/farmacologia , Acetamidas/química , Animais , Antagonistas de Receptor B1 da Bradicinina , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Animais , Estrutura Molecular , RNA Mensageiro/genética , Receptor B1 da Bradicinina/genética , Receptor B1 da Bradicinina/fisiologia , Sulfonas/química , Transcrição Gênica
5.
Blood ; 100(1): 120-7, 2002 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12070016

RESUMO

During fetal development, there is a continued demand for large numbers of primitive and mature hematopoietic cells. This demand may require that all potential hematopoietic stem cells (HSCs) migrate effectively to emerging hematopoietic sites and subsequently contribute to blood cell production, regardless of their cell cycle status. We recently established that umbilical cord blood cells in the G(1) phase of the cell cycle have a repopulating potential similar to cells in G(0), suggesting that cycling prenatal and neonatal HSCs may have the same functional capabilities described for quiescent, but not cycling, cells from adult sources. To establish the relationship between cell cycle status and hematopoietic potential at early stages of human ontogeny, the in vivo engraftment potential of mitotically defined fetal liver (FL) and fetal bone marrow (FBM) cells were examined in NOD/SCID recipients. Following transplantation of the same numbers of G(0), G(1), or S/G(2)+M CD34(+) cells from FL, equivalent percentages of recipient mice were chimeric (55%, 60%, and 60%, respectively). FBM-derived CD34(+) cells in all phases of the cell cycle engrafted in conditioned recipients and sustained human hematopoiesis, albeit at lower levels than their FL-derived counterparts. Multilineage differentiation was evident in all transplanted mice independent of the source or cell cycle status of graft cells. In addition, levels of chimerism in mice transplanted with fetal blood-derived G(0) or G(1) CD34(+) lineage-depleted cells were similar. These results support the assertion that mitotically quiescent and cycling fetal hematopoietic cells contain marrow-repopulating stem cells capable of multilineage engraftment in NOD/SCID mouse recipients.


Assuntos
Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas/métodos , Mitose , Animais , Antígenos CD34 , Células Sanguíneas/citologia , Células da Medula Óssea/citologia , Ciclo Celular , Feto/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Fígado/citologia , Camundongos , Camundongos Endogâmicos NOD , Modelos Animais , Transplante Heterólogo
6.
Blood ; 99(5): 1585-93, 2002 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-11861272

RESUMO

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34(+) cells, or their G(0) (G(0)CD34(+)) or G(1) (G(1)CD34(+)) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34(+) cells harbored similar numbers of CD34(+) and CD34(-) cells, indicating that CD34(+) cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G(2)+M than in recipients of G(0)/G(1) cells (34.6% +/- 5.9% and 17.1% +/- 6.3%, respectively; P <.01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.


Assuntos
Ciclo Celular/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34 , Células da Medula Óssea/citologia , Movimento Celular , Sobrevivência de Enxerto/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Interfase/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Baço/citologia , Condicionamento Pré-Transplante , Transplante Heterólogo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...