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OBJECTIVES: To compare the microRNAs (miRNAs) contained within serum exosomes isolated from patients with Raynaud's phenomenon (RP) and negative antinuclear antibodies (ANA) to the miRNA contained in serum exosomes isolated from patients with RP and positive ANA. METHODS: Serum exosomes were isolated employing a polymer precipitation procedure. Next Generation Sequencing (NGS) was used to identify the miRNAs contained in the exosomes isolated from the two clinical cohorts and to analyse the differences in their contents. RESULTS: The NGS results identified six miRNAs that displayed significant differences in their content between serum exosomes from patients with RP with negative serum ANA compared to miRNAs contained in serum exosomes from patients with ANA-positive RP. CONCLUSIONS: A comparative analysis of miRNAs contained within serum exosomes of patients with RP and negative ANA vs. samples from patients with RP and positive ANA identified several differentially expressed miRNAs that may represent non-invasive biomarkers to assist in the identification of patients with RP at risk of evolving into systemic sclerosis.
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Anticuerpos Antinucleares , Exosomas , MicroARNs , Enfermedad de Raynaud , Humanos , Enfermedad de Raynaud/sangre , Enfermedad de Raynaud/genética , Enfermedad de Raynaud/inmunología , Enfermedad de Raynaud/diagnóstico , Anticuerpos Antinucleares/sangre , Femenino , Exosomas/genética , Persona de Mediana Edad , MicroARNs/sangre , MicroARNs/genética , Masculino , Adulto , Biomarcadores/sangre , Secuenciación de Nucleótidos de Alto Rendimiento , MicroARN Circulante/sangre , MicroARN Circulante/genética , Anciano , Valor Predictivo de las PruebasRESUMEN
Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) are two emerging research technologies that uniquely characterize gene expression microenvironments on a cellular or subcellular level. The skin, a clinically accessible tissue composed of diverse, essential cell populations, serves as an ideal target for these high-resolution investigative approaches. Using these tools, researchers are assembling a compendium of data and discoveries in healthy skin as well as a range of dermatologic pathophysiologies, including atopic dermatitis, psoriasis, and cutaneous malignancies. The ongoing advancement of single-cell approaches, coupled with anticipated decreases in cost with increased adoption, will reshape dermatologic research, profoundly influencing disease characterization, prognosis, and ultimately clinical practice.
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In mature bone, NGF is produced by osteoblasts following mechanical loading and signals through resident sensory nerves expressing its high affinity receptor, neurotrophic tyrosine kinase receptor type 1 (TrkA), to support bone formation. Here, we investigated whether osteoblastic expression of Toll-like receptor 4 (TLR4), a key receptor in the NF-κB signaling pathway, is required to initiate NGF-TrkA signaling required for load-induced bone formation. Although Tlr4 conditional knockout mice have normal skeletal mass and strength in adulthood, the loss of TLR4 signaling significantly reduced lamellar bone formation following loading. Inhibition of TLR4 signaling reduced Ngf expression in primary osteoblasts and RNA sequencing of bones from Tlr4 conditional knockout mice and wild-type littermates revealed dysregulated inflammatory signaling three days after osteogenic mechanical loading. In total, our study reveals an important role for osteoblastic TLR4 in the skeletal adaptation of bone to mechanical forces.
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GM-CSF-producing T helper (Th) cells play a crucial role in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Recent studies have identified a distinct population of GM-CSF-producing Th cells, named ThGM cells, that also express cytokines TNF, IL-2, and IL-3, but lack expression of master transcription factors (TF) and signature cytokines of commonly recognized Th cell lineages. ThGM cells are highly encephalitogenic in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Similar to Th17 cells, in response to IL-12, ThGM cells upregulate expression of T-bet and IFN-γ and switch their phenotype to Th1. Here we show that in addition to T-bet, TF RUNX3 also contributes to the Th1 switch of ThGM cells. T-bet-deficient ThGM cells in the CNS of mice with EAE had low expression of RUNX3, and knockdown of RUNX3 expression in ThGM cells abrogated the Th1-inducing effect of IL-12. Comparison of ThGM and Th1 cell transcriptomes showed that ThGM cells expressed a set of TFs known to inhibit the development of other Th lineages. Lack of expression of lineage-specific cytokines and TFs by ThGM cells, together with expression of TFs that inhibit the development of other Th lineages, suggests that ThGM cells are a non-polarized subset of Th cells with lineage characteristics.
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Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Citocinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-12/metabolismo , Ratones , Fenotipo , Células TH1 , Células Th17 , Factores de Transcripción/metabolismoRESUMEN
Severe viral infections of the skin can occur in patients with inborn errors of immunity (IEI). We report an all-in-one whole-transcriptome sequencing-based method by RNA-Seq on a single skin biopsy for concomitantly identifying the cutaneous virome and the underlying IEI. Skin biopsies were obtained from healthy and lesional skin from patients with cutaneous infections suspected to be of viral origin. RNA-Seq was utilized as the first-tier strategy for unbiased human genome-wide rare variant detection. Reads unaligned to the human genome were utilized for the exploration of 926 viruses in a viral genome catalog. In 9 families studied, the patients carried pathogenic variants in 6 human IEI genes, including IL2RG, WAS, CIB1, STK4, GATA2, and DOCK8. Gene expression profiling also confirmed pathogenicity of the human variants and permitted genome-wide homozygosity mapping, which assisted in identification of candidate genes in consanguineous families. This automated, online, all-in-one computational pipeline, called VirPy, enables simultaneous detection of the viral triggers and the human genetic variants underlying skin lesions in patients with suspected IEI and viral dermatosis.
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Enfermedades de la Piel , Transcriptoma , Consanguinidad , Homocigoto , Humanos , Enfermedades de la Piel/genética , Secuenciación del ExomaRESUMEN
MicroRNAs (miRNAs/miRs) are small, endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling, we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8+ T cells. We observed that out of 362 measurable mature miRNAs, 120 were differentially expressed by at least 2-fold in influenza-specific effector CD8+ CTLs compared with naive CD8+ T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139-mediated regulation of CTL effector responses, we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139-/- mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8+ T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8+ T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet, miR-139-/- CD8+ T cells are outcompeted by wild-type CD8+ T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes Using an in vitro model of T cell exhaustion, we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation, miR-139 expression is dispensable for influenza-specific CTL responses.
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Virus de la Influenza A/inmunología , Listeria monocytogenes/inmunología , MicroARNs/genética , Infecciones por Orthomyxoviridae/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Regulación hacia Abajo/genética , Femenino , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/inmunologíaRESUMEN
Despite treatment with intensive chemotherapy, acute myelogenous leukemia (AML) remains an aggressive malignancy with a dismal outcome in most patients. We found that AML cells exhibit an unusually rapid accumulation of the repressive histone mark H3K27me3 on nascent DNA. In cell lines, primary cells and xenograft mouse models, inhibition of the H3K27 histone methyltransferase EZH2 to decondense the H3K27me3-marked chromatin of AML cells enhanced chromatin accessibility and chemotherapy-induced DNA damage, apoptosis, and leukemia suppression. These effects were further promoted when chromatin decondensation of AML cells was induced upon S-phase entry after release from a transient G1 arrest mediated by CDK4/6 inhibition. In the p53-null KG-1 and THP-1 AML cell lines, EZH2 inhibitor and doxorubicin cotreatment induced transcriptional reprogramming that was, in part, dependent on derepression of H3K27me3-marked gene promoters and led to increased expression of cell death-promoting and growth-inhibitory genes.In conclusion, decondensing H3K27me3-marked chromatin by EZH2 inhibition represents a promising approach to improve the efficacy of DNA-damaging cytotoxic agents in patients with AML. This strategy might allow for a lowering of chemotherapy doses, with a consequent reduction of treatment-related side effects in elderly patients with AML or those with significant comorbidities. SIGNIFICANCE: Pharmacological inhibition of EZH2 renders DNA of AML cells more accessible to cytotoxic agents, facilitating leukemia suppression with reduced doses of chemotherapy.See related commentary by Adema and Colla, p. 359.
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Cromatina/metabolismo , Histonas/metabolismo , Leucemia Mieloide Aguda/genética , Animales , Humanos , RatonesRESUMEN
PURPOSE: Heritable ectopic mineralization disorders comprise a group of conditions with a broad range of clinical manifestations in nonskeletal connective tissues. We report the genetic findings from a large international cohort of 478 patients afflicted with ectopic mineralization. METHODS: Sequence variations were identified using a next-generation sequencing panel consisting of 29 genes reported in association with ectopic mineralization. The pathogenicity of select splicing and missense variants was analyzed in experimental systems in vitro and in vivo. RESULTS: A total of 872 variants of unknown significance as well as likely pathogenic and pathogenic variants were disclosed in 25 genes. A total of 159 distinct variants were identified in 425 patients in ABCC6, the gene responsible for pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder. The interpretation of variant pathogenicity relying on bioinformatic predictions did not provide a consensus. Our in vitro and in vivo functional assessment of 14 ABCC6 variants highlighted this dilemma and provided unambiguous interpretations to their pathogenicity. CONCLUSION: The results expand the ABCC6 variant repertoire, shed new light on the genetic heterogeneity of heritable ectopic mineralization disorders, and provide evidence that functional characterization in appropriate experimental systems is necessary to determine the pathogenicity of genetic variants.
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Heterogeneidad Genética , Seudoxantoma Elástico , Estudios de Cohortes , Tejido Conectivo/patología , Humanos , Mutación Missense , Seudoxantoma Elástico/genéticaRESUMEN
There has been a significant increase in basal cell carcinoma (BCC) incidence, the most common cancer in humans and the age of presentation with the first diagnosis of BCC has decreased in past decades. In this study, we investigated the possibility of genetic markers that can lead to earlier and closer observation of patients at high risk for development of multiple BCCs. The overall goal is to decrease the morbidity and the economic burden of diagnosis and treatment of recurring and/or advanced BCCs. Four patients with numerous BCCs, some of them exceptionally large, were included in this study. A sample of representative BCCs, normal non-sun-exposed skin and blood samples were obtained from each patient. Whole-exome sequencing of DNA was conducted on all samples, and a series of bioinformatics filtering was performed to identify potentially pathogenic sequence variants. The analysis of the data resulted in detection of oncogenic mutations in PTCH1, two of which being novel, and concurrent mutations in TP53 in BCC tumours of all four patients. Such mutations may explain the numerous and postexcision recurring nature of the BCCs of exceptionally large size observed in all these patients, and they can be suggested to serve as a genetic marker for high-risk patients for early detection, prognostication and close follow-up.
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Carcinoma Basocelular , Neoplasias Cutáneas , Carcinogénesis , Carcinoma Basocelular/genética , Carcinoma Basocelular/patología , Humanos , Mutación , Recurrencia Local de Neoplasia , Receptor Patched-1/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND & AIMS: Sporadic colorectal cancers arise from initiating mutations in APC, producing oncogenic ß-catenin/TCF-dependent transcriptional reprogramming. Similarly, the tumor suppressor axis regulated by the intestinal epithelial receptor GUCY2C is among the earliest pathways silenced in tumorigenesis. Retention of the receptor, but loss of its paracrine ligands, guanylin and uroguanylin, is an evolutionarily conserved feature of colorectal tumors, arising in the earliest dysplastic lesions. Here, we examined a mechanism of GUCY2C ligand transcriptional silencing by ß-catenin/TCF signaling. METHODS: We performed RNA sequencing analysis of 4 unique conditional human colon cancer cell models of ß-catenin/TCF signaling to map the core Wnt-transcriptional program. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, chromatin immunoprecipitation sequencing, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) knockout, and CRISPR epigenome editing, which were cross-validated with human tissue chromatin immunoprecipitation sequencing datasets, to identify functional gene enhancers mediating GUCY2C ligand loss. RESULTS: RNA sequencing analyses reveal the GUCY2C hormones as 2 of the most sensitive targets of ß-catenin/TCF signaling, reflecting transcriptional repression. The GUCY2C hormones share an insulated genomic locus containing a novel locus control region upstream of the guanylin promoter that mediates the coordinated silencing of both genes. Targeting this region with CRISPR epigenome editing reconstituted GUCY2C ligand expression, overcoming gene inactivation by mutant ß-catenin/TCF signaling. CONCLUSIONS: These studies reveal DNA elements regulating corepression of GUCY2C ligand transcription by ß-catenin/TCF signaling, reflecting a novel pathophysiological step in tumorigenesis. They offer unique genomic strategies that could reestablish hormone expression in the context of canonical oncogenic mutations to reconstitute the GUCY2C axis and oppose transformation.
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Neoplasias Colorrectales , beta Catenina , Carcinogénesis/genética , Cateninas/genética , Cateninas/metabolismo , Neoplasias Colorrectales/patología , Humanos , Ligandos , Región de Control de Posición , Receptores de Enterotoxina/genética , Receptores de Enterotoxina/metabolismo , Factores de Transcripción TCF/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
OBJECTIVE: Well-defined germ-line mutations in the PTCH1 gene are associated with syndromic multiple basal cell carcinomas (BCCs). Here, we used whole exome sequencing (WES) to identify the role of patched-1 in patients with multiple, unusually large BCCs. METHODS: A 72-year old patient presenting with numerous BCCs progressing to large ulcerating lesions was enrolled. WES was used to identify the pathogenic gene locus. RESULTS: Genetic work-up by WES identified a homozygous PTCH1 nonsense mutation in the tumor tissue but not present in her blood cells or in non-lesional skin. In addition, heterozygous missense mutations were identified in three cancer-associated genes (EPHB2, RET, and GALNT12) in blood cells as well as in lesional and non-lesional skin. We also tested systemic immune therapy as a potentially beneficial approach to treat patients with numerous large BCCs on scatted areas of involvement. A rapid and sustained response to nivolumab was noted, suggesting that it is an efficacious drug for long-term therapeutic outcome. CONCLUSION: PTCH1, EPHB2, RET, and GALNT12 may potentially contribute to the synergistic oncogene driven malignant transformation manifesting as multiple, unusually large BCCs.
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IFN-ß has been the treatment for multiple sclerosis (MS) for almost three decades, but understanding the mechanisms underlying its beneficial effects remains incomplete. We have shown that MS patients have increased numbers of GM-CSF+ Th cells in circulation, and that IFN-ß therapy reduces their numbers. GM-CSF expression by myelin-specific Th cells is essential for the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. These findings suggested that IFN-ß therapy may function via suppression of GM-CSF production by Th cells. In the current study, we elucidated a feedback loop between monocytes and Th cells that amplifies autoimmune neuroinflammation, and found that IFN-ß therapy ameliorates central nervous system (CNS) autoimmunity by inhibiting this proinflammatory loop. IFN-ß suppressed GM-CSF production in Th cells indirectly by acting on monocytes, and IFN-ß signaling in monocytes was required for EAE suppression. IFN-ß increased IL-10 expression by monocytes, and IL-10 was required for the suppressive effects of IFN-ß. IFN-ß treatment suppressed IL-1ß expression by monocytes in the CNS of mice with EAE. GM-CSF from Th cells induced IL-1ß production by monocytes, and, in a positive feedback loop, IL-1ß augmented GM-CSF production by Th cells. In addition to GM-CSF, TNF and FASL expression by Th cells was also necessary for IL-1ß production by monocyte. IFN-ß inhibited GM-CSF, TNF, and FASL expression by Th cells to suppress IL-1ß secretion by monocytes. Overall, our study describes a positive feedback loop involving several Th cell- and monocyte-derived molecules, and IFN-ß actions on monocytes disrupting this proinflammatory loop.
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Autoinmunidad , Comunicación Celular , Interferón beta/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Autoinmunidad/efectos de los fármacos , Comunicación Celular/genética , Comunicación Celular/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Interferón beta/farmacología , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/efectos de los fármacosRESUMEN
BACKGROUND: Among the approximately 8000 Mendelian disorders, >1000 have cutaneous manifestations. In many of these conditions, the underlying mutated genes have been identified by DNA-based techniques which, however, can overlook certain types of mutations, such as exonic-synonymous and deep-intronic sequence variants. Whole-transcriptome sequencing by RNA sequencing (RNA-seq) can identify such mutations and provide information about their consequences. METHODS: We analyzed the whole transcriptome of 40 families with different types of Mendelian skin disorders with extensive genetic heterogeneity. The RNA-seq data were examined for variant detection and prioritization, pathogenicity confirmation, RNA expression profiling, and genome-wide homozygosity mapping in the case of consanguineous families. Among the families examined, RNA-seq was able to provide information complementary to DNA-based analyses for exonic and intronic sequence variants with aberrant splicing. In addition, we tested the possibility of using RNA-seq as the first-tier strategy for unbiased genome-wide mutation screening without information from DNA analysis. RESULTS: We found pathogenic mutations in 35 families (88%) with RNA-seq in combination with other next-generation sequencing methods, and we successfully prioritized variants and found the culprit genes. In addition, as a novel concept, we propose a pipeline that increases the yield of variant calling from RNA-seq by concurrent use of genome and transcriptome references in parallel. CONCLUSIONS: Our results suggest that "clinical RNA-seq" could serve as a primary approach for mutation detection in inherited diseases, particularly in consanguineous families, provided that tissues and cells expressing the relevant genes are available for analysis.
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Perfilación de la Expresión Génica , Enfermedades de la Piel , Consanguinidad , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Análisis de Secuencia de ARN/métodos , Enfermedades de la Piel/diagnóstico , Enfermedades de la Piel/genética , Secuenciación del ExomaRESUMEN
Elevation of granulocyte-macrophage colony-stimulating factor (GM-CSF)producing T helper (TH) cells has been associated with several autoimmune diseases, suggesting a potential role in the pathogenesis of autoimmunity. However, the identity of GM-CSFproducing TH cells has not been closely examined. Using single-cell RNA sequencing and high-dimensional single-cell mass cytometry, we identified eight populations of antigen-experienced CD45RA−CD4+ T cells in blood of healthy individuals including a population of GM-CSFproducing cells, known as THGM, that lacked expression of signature transcription factors and cytokines of established TH lineages. Using GM-CSF-reporter/fate reporter mice, we show that THGM cells are present in the periphery and central nervous system in a mouse model of experimental autoimmune encephalomyelitis. In addition to GM-CSF, human and mouse THGM cells also expressed IL-2, tumor necrosis factor (TNF), IL-3, and CCL20. THGM cells maintained their phenotype through several cycles of activation but up-regulated expression of T-bet and interferon-γ (IFN-γ) upon exposure to IL-12 in vitro and in the central nervous system of mice with autoimmune neuroinflammation. Although T-bet was not required for the development of THGM cells, it was essential for their encephalitogenicity. These findings demonstrate that THGM cells constitute a distinct population of TH cells with lineage characteristics that are poised to adopt a TH1 phenotype and promote neuroinflammation.
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Encefalomielitis Autoinmune Experimental/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Esclerosis Múltiple/inmunología , Proteínas de Dominio T Box/metabolismo , Células TH1/inmunología , Animales , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Femenino , Regulación de la Expresión Génica/inmunología , Voluntarios Sanos , Humanos , Masculino , Ratones , Ratones Transgénicos , Esclerosis Múltiple/genética , Esclerosis Múltiple/patología , RNA-Seq , Análisis de la Célula Individual , Células TH1/metabolismoRESUMEN
The ability of small extracellular vesicles (sEVs) to reprogram cancer cells is well established. However, the specific sEV components able to mediate aberrant effects in cancer cells have not been characterized. Integrins are major players in mediating sEV functions. We have previously reported that the αVß3 integrin is detected in sEVs of prostate cancer (PrCa) cells and transferred into recipient cells. Here, we investigate whether sEVs from αVß3-expressing cells affect tumour growth differently than sEVs from control cells that do not express αVß3. We compared the ability of sEVs to stimulate tumour growth, using sEVs isolated from PrCa C4-2B cells by iodixanol density gradient and characterized with immunoblotting, nanoparticle tracking analysis, immunocapturing and single vesicle analysis. We incubated PrCa cells with sEVs and injected them subcutaneously into nude mice to measure in vivo tumour growth or analysed in vitro their anchorage-independent growth. Our results demonstrate that a single treatment with sEVs shed from C4-2B cells that express αVß3, but not from control cells, stimulates tumour growth and induces differentiation of PrCa cells towards a neuroendocrine phenotype, as quantified by increased levels of neuroendocrine markers. In conclusion, the expression of αVß3 integrin generates sEVs capable of reprogramming cells towards an aggressive phenotype.
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Emerging technologies are set to play an important role in our response to the COVID-19 pandemic. This paper explores three prominent initiatives: COVID-19 focused datasets (e.g., CORD-19); Artificial intelligence-powered search tools (e.g., WellAI, SciSight); and contact tracing based on mobile communication technology. We believe that increasing awareness of these tools will be important in future research into the disease, COVID-19, and the virus, SARS-CoV-2.
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Understanding the aberrant transcriptional landscape of neuroblastoma is necessary to provide insight to the underlying influences of the initiation, progression and persistence of this developmental cancer. Here, we present chromatin immunoprecipitation sequencing (ChIP-Seq) data for the oncogenic transcription factors, MYCN and MYC, as well as regulatory histone marks H3K4me1, H3K4me3, H3K27Ac, and H3K27me3 in ten commonly used human neuroblastoma-derived cell line models. In addition, for all of the profiled cell lines we provide ATAC-Seq as a measure of open chromatin. We validate specificity of global MYCN occupancy in MYCN amplified cell lines and functional redundancy of MYC occupancy in MYCN non-amplified cell lines. Finally, we show with H3K27Ac ChIP-Seq that these cell lines retain expression of key neuroblastoma super-enhancers (SE). We anticipate this dataset, coupled with available transcriptomic profiling on the same cell lines, will enable the discovery of novel gene regulatory mechanisms in neuroblastoma.
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Epigenómica , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Línea Celular Tumoral , Cromatina/genética , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Histonas/genética , HumanosRESUMEN
Dysregulated Th17 cell differentiation is associated with autoimmune diseases such as multiple sclerosis, which has no curative treatment. Understanding the molecular mechanisms of regulating Th17 cell differentiation will help find a novel therapeutic target for treating Th17 cell-mediated diseases. In this study, we investigated the cell-intrinsic processes by which RNA-binding protein HuR orchestrates Th17 cell fate decisions by posttranscriptionally regulating transcription factors Irf4 and Runx1 and receptor Il12rb1 expression, in turn promoting Th17 cell and Th1-like Th17 cell differentiation in C57BL/6J mice. Knockout of HuR altered the transcriptome of Th17 cells characterized by reducing the levels of RORγt, IRF4, RUNX1, and T-bet, thereby reducing the number of pathogenic IL-17+IFN-γ+CD4+ T cells in the spleen during experimental autoimmune encephalomyelitis. In keeping with the fact that HuR increased the abundance of adhesion molecule VLA-4 on Th17 cells, knockout of HuR impaired splenic Th17 cell migration to the CNS and abolished the disease. Accordingly, targeting HuR by its inhibitor DHTS inhibited splenic Th17 cell differentiation and reduced experimental autoimmune encephalomyelitis severity. In sum, we uncovered the molecular mechanism of HuR regulating Th17 cell functions, underscoring the therapeutic value of HuR for treatment of autoimmune neuroinflammation.
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Diferenciación Celular , Proteína 1 Similar a ELAV/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Inflamación/inmunología , Células Th17/inmunología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proteína 1 Similar a ELAV/antagonistas & inhibidores , Proteína 1 Similar a ELAV/deficiencia , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Furanos , Inflamación/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fenantrenos/farmacología , Quinonas , Células Th17/efectos de los fármacosRESUMEN
Expression of the cell cycle regulatory gene CDK6 is required for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cell growth, whereas expression of the closely related CDK4 protein is dispensable. Moreover, CDK6 silencing is more effective than treatment with the dual CDK4/6 inhibitor palbociclib in suppressing Ph+ ALL in mice, suggesting that the growth-promoting effects of CDK6 are, in part, kinase-independent in Ph+ ALL. Accordingly, we developed CDK4/6-targeted proteolysis-targeting chimeras (PROTACs) that inhibit CDK6 enzymatic activity in vitro, promote the rapid and preferential degradation of CDK6 over CDK4 in Ph+ ALL cells, and markedly suppress S-phase cells concomitant with inhibition of CDK6-regulated phospho-RB and FOXM1 expression. No such effects were observed in CD34+ normal hematopoietic progenitors, although CDK6 was efficiently degraded. Treatment with the CDK6-degrading PROTAC YX-2-107 markedly suppressed leukemia burden in mice injected with de novo or tyrosine kinase inhibitor-resistant primary Ph+ ALL cells, and this effect was comparable or superior to that of the CDK4/6 enzymatic inhibitor palbociclib. These studies provide "proof of principle" that targeting CDK6 with PROTACs that inhibit its enzymatic activity and promote its degradation represents an effective strategy to exploit the "CDK6 dependence" of Ph+ ALL and, perhaps, of other hematologic malignancies. Moreover, they suggest that treatment of Ph+ ALL with CDK6-selective PROTACs would spare a high proportion of normal hematopoietic progenitors, preventing the neutropenia induced by treatment with dual CDK4/6 inhibitors.
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Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes de Fusión/farmacología , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 6 Dependiente de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Perfilación de la Expresión Génica , Genes cdc , Humanos , Ratones , Estructura Molecular , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/uso terapéutico , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Quality Control in any high-throughput sequencing technology is a critical step, which if overlooked can compromise an experiment and the resulting conclusions. A number of methods exist to identify biases during sequencing or alignment, yet not many tools exist to interpret biases due to outliers. RESULTS: Hence, we developed iSeqQC, an expression-based QC tool that detects outliers either produced due to variable laboratory conditions or due to dissimilarity within a phenotypic group. iSeqQC implements various statistical approaches including unsupervised clustering, agglomerative hierarchical clustering and correlation coefficients to provide insight into outliers. It can be utilized through command-line (Github: https://github.com/gkumar09/iSeqQC) or web-interface (http://cancerwebpa.jefferson.edu/iSeqQC). A local shiny installation can also be obtained from github (https://github.com/gkumar09/iSeqQC). CONCLUSION: iSeqQC is a fast, light-weight, expression-based QC tool that detects outliers by implementing various statistical approaches.