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1.
Circulation ; 142(21): 2060-2075, 2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-32962412

RESUMO

BACKGROUND: Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive. METHODS: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro. RESULTS: We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability. CONCLUSIONS: Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.


Assuntos
Aterosclerose/genética , Aterosclerose/patologia , Diferenciação Celular/fisiologia , Genômica/métodos , Miócitos de Músculo Liso/patologia , Fenótipo , Animais , Aterosclerose/terapia , Desdiferenciação Celular/fisiologia , Movimento Celular/fisiologia , Transdiferenciação Celular/fisiologia , Células Cultivadas , Feminino , Terapia Genética/tendências , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miócitos de Músculo Liso/fisiologia , Análise de Sequência de RNA/métodos
2.
J Zoo Wildl Med ; 47(1): 341-6, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27010299

RESUMO

Two snakes were presented to the Pennsylvania State University Animal Diagnostic Laboratory with one suffering from external lesions where the scales were raised and discolored, and the other with oral lesions and swelling extending to the left eye, which was opaque. Histopathological analysis revealed multifocal granulomas containing fungal hyphae. Morphological and DNA sequence analyses revealed both suffered from infection by Ophidiomyces ophiodiicola, an emerging pathogen of snakes. This is the first report of this disease in Pennsylvania.


Assuntos
Ascomicetos/isolamento & purificação , Micoses/veterinária , Serpentes/microbiologia , Animais , Micoses/genética , Micoses/microbiologia , Filogenia
3.
bioRxiv ; 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38798673

RESUMO

Tumors frequently harbor isogenic yet epigenetically distinct subpopulations of multi-potent cells with high tumor-initiating potential-often called Cancer Stem-Like Cells (CSLCs). These can display preferential resistance to standard-of-care chemotherapy. Single-cell analyses can help elucidate Master Regulator (MR) proteins responsible for governing the transcriptional state of these cells, thus revealing complementary dependencies that may be leveraged via combination therapy. Interrogation of single-cell RNA sequencing profiles from seven metastatic breast cancer patients, using perturbational profiles of clinically relevant drugs, identified drugs predicted to invert the activity of MR proteins governing the transcriptional state of chemoresistant CSLCs, which were then validated by CROP-seq assays. The top drug, the anthelmintic albendazole, depleted this subpopulation in vivo without noticeable cytotoxicity. Moreover, sequential cycles of albendazole and paclitaxel-a commonly used chemotherapeutic -displayed significant synergy in a patient-derived xenograft (PDX) from a TNBC patient, suggesting that network-based approaches can help develop mechanism-based combinatorial therapies targeting complementary subpopulations.

4.
Cancer Cell ; 41(11): 1972-1988.e5, 2023 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-37922910

RESUMO

When compared to other malignancies, the tumor microenvironment (TME) of primary and castration-resistant prostate cancer (CRPC) is relatively devoid of immune infiltrates. While androgen deprivation therapy (ADT) induces a complex immune infiltrate in localized prostate cancer, the composition of the TME in metastatic castration-sensitive prostate cancer (mCSPC), and the effects of ADT and other treatments in this context are poorly understood. Here, we perform a comprehensive single-cell RNA sequencing (scRNA-seq) profiling of metastatic sites from patients participating in a phase 2 clinical trial (NCT03951831) that evaluated standard-of-care chemo-hormonal therapy combined with anti-PD-1 immunotherapy. We perform a longitudinal, protein activity-based analysis of TME subpopulations, revealing immune subpopulations conserved across multiple metastatic sites. We also observe dynamic changes in these immune subpopulations in response to treatment and a correlation with clinical outcomes. Our study uncovers a therapy-resistant, transcriptionally distinct tumor subpopulation that expands in cell number in treatment-refractory patients.


Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Antagonistas de Androgênios/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Androgênios/uso terapêutico , Imunoterapia , Castração , Microambiente Tumoral
5.
Sci Rep ; 12(1): 5718, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35383235

RESUMO

Protein synthesis is dysregulated in many diseases, but we lack a systems-level picture of how signaling molecules and RNA binding proteins interact with the translational machinery, largely due to technological limitations. Here we present riboPLATE-seq, a scalable method for generating paired libraries of ribosome-associated and total mRNA. As an extension of the PLATE-seq protocol, riboPLATE-seq utilizes barcoded primers for pooled library preparation, but additionally leverages anti-rRNA ribosome immunoprecipitation on whole polysomes to measure ribosome association (RA). We compare RA to its analogue in ribosome profiling and RNA sequencing, translation efficiency, and demonstrate both the performance of riboPLATE-seq and its utility in detecting translational alterations induced by specific inhibitors of protein kinases.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Biossíntese de Proteínas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polirribossomos/genética , Polirribossomos/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Análise de Sequência de RNA/métodos
6.
Cancer Cell ; 39(6): 866-882.e11, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-33930309

RESUMO

Cancer-associated fibroblasts (CAF) are a poorly characterized cell population in the context of liver cancer. Our study investigates CAF functions in intrahepatic cholangiocarcinoma (ICC), a highly desmoplastic liver tumor. Genetic tracing, single-cell RNA sequencing, and ligand-receptor analyses uncovered hepatic stellate cells (HSC) as the main source of CAF and HSC-derived CAF as the dominant population interacting with tumor cells. In mice, CAF promotes ICC progression, as revealed by HSC-selective CAF depletion. In patients, a high panCAF signature is associated with decreased survival and increased recurrence. Single-cell RNA sequencing segregates CAF into inflammatory and growth factor-enriched (iCAF) and myofibroblastic (myCAF) subpopulations, displaying distinct ligand-receptor interactions. myCAF-expressed hyaluronan synthase 2, but not type I collagen, promotes ICC. iCAF-expressed hepatocyte growth factor enhances ICC growth via tumor-expressed MET, thus directly linking CAF to tumor cells. In summary, our data demonstrate promotion of desmoplastic ICC growth by therapeutically targetable CAF subtype-specific mediators, but not by type I collagen.


Assuntos
Neoplasias dos Ductos Biliares/patologia , Fibroblastos Associados a Câncer/patologia , Colangiocarcinoma/patologia , Idoso , Animais , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Fibroblastos Associados a Câncer/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colágeno Tipo I/metabolismo , Feminino , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/patologia , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico/metabolismo , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-met/metabolismo , Microambiente Tumoral
7.
Cell Rep ; 33(10): 108474, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33296649

RESUMO

Bi-species, fusion-mediated, somatic cell reprogramming allows precise, organism-specific tracking of unknown lineage drivers. The fusion of Tcf7l1-/- murine embryonic stem cells with EBV-transformed human B cell lymphocytes, leads to the generation of bi-species heterokaryons. Human mRNA transcript profiling at multiple time points permits the tracking of the reprogramming of B cell nuclei to a multipotent state. Interrogation of a human B cell regulatory network with gene expression signatures identifies 8 candidate master regulator proteins. Of these 8 candidates, ectopic expression of BAZ2B, from the bromodomain family, efficiently reprograms hematopoietic committed progenitors into a multipotent state and significantly enhances their long-term clonogenicity, stemness, and engraftment in immunocompromised mice. Unbiased systems biology approaches let us identify the early driving events of human B cell reprogramming.


Assuntos
Reprogramação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Fatores Genéricos de Transcrição/metabolismo , Animais , Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Reprogramação Celular/fisiologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Feminino , Sangue Fetal/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Multipotentes/metabolismo , Fatores de Transcrição/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/fisiologia
9.
G3 (Bethesda) ; 6(2): 463-74, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26681516

RESUMO

The Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism in eukaryotes. Studies in yeast and human cells have shown that nitrogen/amino acid starvation signals act through Npr2/Npr3 and the small GTPases Gtr1/Gtr2 (Rags in humans) to inhibit TORC1. However, it is unclear how other stress and starvation stimuli inhibit TORC1, and/or act in parallel with the TORC1 pathway, to control cell growth. To help answer these questions, we developed a novel automated pipeline and used it to measure the expression of a TORC1-dependent ribosome biogenesis gene (NSR1) during osmotic stress in 4700 Saccharomyces cerevisiae strains from the yeast knock-out collection. This led to the identification of 440 strains with significant and reproducible defects in NSR1 repression. The cell growth control and stress response proteins deleted in these strains form a highly connected network, including 56 proteins involved in vesicle trafficking and vacuolar function; 53 proteins that act downstream of TORC1 according to a rapamycin assay--including components of the HDAC Rpd3L, Elongator, and the INO80, CAF-1 and SWI/SNF chromatin remodeling complexes; over 100 proteins involved in signaling and metabolism; and 17 proteins that directly interact with TORC1. These data provide an important resource for labs studying cell growth control and stress signaling, and demonstrate the utility of our new, and easily adaptable, method for mapping gene regulatory networks.


Assuntos
Genoma Fúngico , Estudo de Associação Genômica Ampla , Genômica , Complexos Multiproteicos/metabolismo , Pressão Osmótica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Genômica/métodos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Mutação , Estresse Fisiológico/genética , Serina-Treonina Quinases TOR/genética
10.
Cell Rep ; 3(5): 1476-82, 2013 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-23643537

RESUMO

Cells respond to stress and starvation by adjusting their growth rate and enacting stress defense programs. In eukaryotes this involves inactivation of TORC1, which in turn triggers downregulation of ribosome and protein synthesis genes and upregulation of stress response genes. Here we report that the highly conserved inositol pyrophosphate (PP-IP) second messengers (including 1-PP-IP5, 5-PP-IP4, and 5-PP-IP5) are also critical regulators of cell growth and the general stress response, acting in parallel with the TORC1 pathway to control the activity of the class I histone deacetylase Rpd3L. In fact, yeast cells that cannot synthesize any of the PP-IPs mount little to no transcriptional response to osmotic, heat, or oxidative stress. Furthermore, PP-IP-dependent regulation of Rpd3L occurs independently of the role individual PP-IPs (such as 5-PP-IP5) play in activating specialized stress/starvation response pathways. Thus, the PP-IP second messengers simultaneously activate and tune the global response to stress and starvation signals.


Assuntos
Histona Desacetilase 1/metabolismo , Fosfatos de Inositol/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Concentração Osmolar , Estresse Oxidativo , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sistemas do Segundo Mensageiro , Serina-Treonina Quinases TOR/metabolismo , Temperatura
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