RESUMEN
Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.
Asunto(s)
Agammaglobulinemia/inmunología , Linfocitos B/inmunología , Proteínas de Transporte de Catión/inmunología , Zinc/inmunología , Agammaglobulinemia/genética , Agammaglobulinemia/metabolismo , Animales , Linfocitos B/metabolismo , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Preescolar , Citosol/inmunología , Citosol/metabolismo , Modelos Animales de Enfermedad , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Lactante , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Linaje , Zinc/metabolismoRESUMEN
Unique molecular identifiers are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are a source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing unique molecular identifiers using homotrimeric nucleotide blocks provides an error-correcting solution that allows absolute counting of sequenced molecules.
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Secuenciación de Nucleótidos de Alto Rendimiento , Nucleótidos , Análisis de Secuencia de ARN , Oligonucleótidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
Transmembrane proteins are receptors, enzymes, transporters and ion channels that are instrumental in regulating a variety of cellular activities, such as signal transduction and cell communication. Despite tremendous progress in computational capacities to support protein research, there is still a significant gap in the availability of specialized computational analysis toolkits for transmembrane protein research. Here, we introduce TMKit, an open-source Python programming interface that is modular, scalable and specifically designed for processing transmembrane protein data. TMKit is a one-stop computational analysis tool for transmembrane proteins, enabling users to perform database wrangling, engineer features at the mutational, domain and topological levels, and visualize protein-protein interaction interfaces. In addition, TMKit includes seqNetRR, a high-performance computing library that allows customized construction of a large number of residue connections. This library is particularly well suited for assigning correlation matrix-based features at a fast speed. TMKit should serve as a useful tool for researchers in assisting the study of transmembrane protein sequences and structures. TMKit is publicly available through https://github.com/2003100127/tmkit and https://tmkit-guide.herokuapp.com/doc/overview.
Asunto(s)
Biología Computacional , Programas Informáticos , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Biblioteca de GenesRESUMEN
The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell-cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell-cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis.
Asunto(s)
Perfilación de la Expresión Génica , Tendones Isquiotibiales , Transcriptoma , Humanos , Masculino , Adulto , Tendones Isquiotibiales/metabolismo , Fibroblastos/metabolismo , Femenino , Núcleo Celular/metabolismo , Núcleo Celular/genética , Matriz Extracelular/metabolismo , Tendones/metabolismoRESUMEN
BACKGROUND: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers. METHOD: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. RESULTS: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. CONCLUSION: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.
Asunto(s)
Glioblastoma , Proteínas Nucleares , Humanos , Supervivencia Celular , Proteínas Nucleares/metabolismo , Glioblastoma/genética , Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Cinesinas/genética , Cinesinas/metabolismoRESUMEN
Developing B cells can be positively or negatively selected by self-antigens, but the mechanisms that determine these outcomes are incompletely understood. Here, we show that a B cell intrinsic switch between positive and negative selection during ontogeny is determined by a change from Lin28b to let-7 gene expression. Ectopic expression of a Lin28b transgene in murine B cells restored the positive selection of autoreactive B-1 B cells by self-antigen in adult bone marrow. Analysis of antigen-specific immature B cells in early and late ontogeny identified Lin28b-dependent genes associated with B-1 B cell development, including Arid3a and Bhleh41, and Lin28b-independent effects are associated with the presence or absence of self-antigen. These findings identify cell intrinsic and extrinsic determinants of B cell fate during ontogeny and reconcile lineage and selection theories of B cell development. They explain how changes in the balance of positive and negative selection may be able to adapt to meet the immunological needs of an individual during its lifetime.
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Linfocitos B/inmunología , Proteínas de Unión al ARN/inmunología , Animales , Linfocitos B/citología , Proliferación Celular , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/inmunología , Proteínas de Unión al ARN/genéticaRESUMEN
T helper (Th) cells are CD4+ effector T cells that play a critical role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach, we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor RORγt during Th17 differentiation. In mature Th17 cells, GSK-J4 induces an altered transcriptional program with a profound metabolic reprogramming and concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single-cell analysis reveals a specific shift from highly inflammatory cell subsets toward a resting state upon demethylase inhibition. The root cause of the observed antiinflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1. Overall, this leads to reduced mitochondrial biogenesis, resulting in a metabolic switch with concomitant antiinflammatory effects. These data are consistent with an effect of GSK-J4 on Th17 T cell differentiation pathways directly related to proliferation and include regulation of effector cytokine profiles. This suggests that inhibiting KDM6 demethylases may be an effective, even in the short term, therapeutic target for autoimmune diseases, including ankylosing spondylitis.
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Benzazepinas/farmacología , Histona Demetilasas/metabolismo , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Pirimidinas/farmacología , Células Th17/metabolismo , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Benzazepinas/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Código de Histonas/efectos de los fármacos , Histona Demetilasas/antagonistas & inhibidores , Humanos , Interleucina-17/metabolismo , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Cultivo Primario de Células , Pirimidinas/uso terapéutico , RNA-Seq , Espondilitis Anquilosante/tratamiento farmacológico , Espondilitis Anquilosante/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Factores de Transcripción/metabolismoRESUMEN
Fibrotic diseases remain a major cause of morbidity and mortality, yet there are few effective therapies. The underlying pathology of all fibrotic conditions is the activity of myofibroblasts. Using cells from freshly excised disease tissue from patients with Dupuytren's disease (DD), a localized fibrotic disorder of the palm, we sought to identify new therapeutic targets for fibrotic disease. We hypothesized that the persistent activity of myofibroblasts in fibrotic diseases might involve epigenetic modifications. Using a validated genetics-led target prioritization algorithm (Pi) of genome wide association studies (GWAS) data and a broad screen of epigenetic inhibitors, we found that the acetyltransferase CREBBP/EP300 is a major regulator of contractility and extracellular matrix production via control of H3K27 acetylation at the profibrotic genes, ACTA2 and COL1A1 Genomic analysis revealed that EP300 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, and broad transcriptomic and proteomic profiling of CREBBP/EP300 inhibition by the chemical probe SGC-CBP30 identified collagen VI (Col VI) as a prominent downstream regulator of myofibroblast activity. Targeted Col VI knockdown results in significant decrease in profibrotic functions, including myofibroblast contractile force, extracellular matrix (ECM) production, chemotaxis, and wound healing. Further evidence for Col VI as a major determinant of fibrosis is its abundant expression within Dupuytren's nodules and also in the fibrotic foci of idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent a tractable therapeutic target across a range of fibrotic disorders.
Asunto(s)
Proteína de Unión a CREB/genética , Colágeno Tipo VI/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Proteína de Unión a CREB/metabolismo , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Colágeno Tipo VI/fisiología , Proteína p300 Asociada a E1A/genética , Epigénesis Genética/genética , Epigenómica/métodos , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibrosis/genética , Fibrosis/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Miofibroblastos/metabolismo , Miofibroblastos/fisiología , Proteómica , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Aberrant miRNA expression has been associated with a large number of human diseases. Therefore, targeting miRNAs to regulate their expression levels has become an important therapy against diseases that stem from the dysfunction of pathways regulated by miRNAs. In recent years, small molecules have demonstrated enormous potential as drugs to regulate miRNA expression (i.e., SM-miR). A clear understanding of the mechanism of action of small molecules on the upregulation and downregulation of miRNA expression allows precise diagnosis and treatment of oncogenic pathways. However, outside of a slow and costly process of experimental determination, computational strategies to assist this on an ad hoc basis have yet to be formulated. In this work, we developed, to the best of our knowledge, the first cross-platform prediction tool, DeepsmirUD, to infer small-molecule-mediated regulatory effects on miRNA expression (i.e., upregulation or downregulation). This method is powered by 12 cutting-edge deep-learning frameworks and achieved AUC values of 0.843/0.984 and AUCPR values of 0.866/0.992 on two independent test datasets. With a complementarily constructed network inference approach based on similarity, we report a significantly improved accuracy of 0.813 in determining the regulatory effects of nearly 650 associated SM-miR relations, each formed with either novel small molecule or novel miRNA. By further integrating miRNA-cancer relationships, we established a database of potential pharmaceutical drugs from 1343 small molecules for 107 cancer diseases to understand the drug mechanisms of action and offer novel insight into drug repositioning. Furthermore, we have employed DeepsmirUD to predict the regulatory effects of a large number of high-confidence associated SM-miR relations. Taken together, our method shows promise to accelerate the development of potential miRNA targets and small molecule drugs.
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Aprendizaje Profundo , MicroARNs , Neoplasias , Humanos , MicroARNs/metabolismo , Neoplasias/metabolismo , Redes Reguladoras de Genes , Biología ComputacionalRESUMEN
Silencing of the somatic cell type-specific genes is a critical yet poorly understood step in reprogramming. To uncover pathways that maintain cell identity, we performed a reprogramming screen using inhibitors of chromatin factors. Here, we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CREB (cyclic-AMP response element binding protein) binding protein (CBP) and E1A binding protein of 300 kDa (EP300) as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human induced pluripotent stem cells (iPSCs) with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different coactivator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell-type-specific gene expression and maintain cell identity.
Asunto(s)
Bencimidazoles/farmacología , Proteína de Unión a CREB/antagonistas & inhibidores , Reprogramación Celular/efectos de los fármacos , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Isoxazoles/farmacología , Oxazepinas/farmacología , Piperidinas/farmacología , Bencimidazoles/química , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Inhibidores Enzimáticos/química , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Isoxazoles/química , Estructura Molecular , Oxazepinas/química , Piperidinas/química , Dominios Proteicos/efectos de los fármacosRESUMEN
BACKGROUND: Endometriosis is a gynaecological condition characterised by immune cell infiltration and distinct inflammatory signatures found in the peritoneal cavity. In this study, we aim to characterise the immune microenvironment in samples isolated from the peritoneal cavity in patients with endometriosis. METHODS: We applied mass cytometry (CyTOF), a recently developed multiparameter single-cell technique, in order to characterise and quantify the immune cells found in peritoneal fluid and peripheral blood from endometriosis and control patients. RESULTS: Our results demonstrate the presence of more than 40 different distinct immune cell types within the peritoneal cavity. This suggests that there is a complex and highly heterogeneous inflammatory microenvironment underpinning the pathology of endometriosis. Stratification by clinical disease stages reveals a dynamic spectrum of cell signatures suggesting that adaptations in the inflammatory system occur due to the severity of the disease. Notably, among the inflammatory microenvironment in peritoneal fluid (PF), the presence of CD69+ T cell subsets is increased in endometriosis when compared to control patient samples. On these CD69+ cells, the expression of markers associated with T cell function are reduced in PF samples compared to blood. Comparisons between CD69+ and CD69- populations reveal distinct phenotypes across peritoneal T cell lineages. Taken together, our results suggest that both the innate and the adaptive immune system play roles in endometriosis. CONCLUSIONS: This study provides a systematic characterisation of the specific immune environment in the peritoneal cavity and identifies cell immune signatures associated with endometriosis. Overall, our results provide novel insights into the specific cell phenotypes governing inflammation in patients with endometriosis. This prospective study offers a useful resource for understanding disease pathology and opportunities for identifying therapeutic targets.
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Líquido Ascítico/inmunología , Endometriosis/inmunología , Líquido Ascítico/metabolismo , Líquido Ascítico/patología , Endometriosis/metabolismo , Endometriosis/patología , Femenino , Citometría de Flujo , Humanos , Estudios Prospectivos , Linfocitos TRESUMEN
Natural killer (NK) cells are innate lymphocytes, important in immune surveillance and elimination of stressed, transformed, or virus-infected cells. They critically shape the inflammatory cytokine environment to orchestrate interactions of cells of the innate and adaptive immune systems. Some studies have reported that NK cell activation and cytokine secretion are controlled epigenetically but have yielded only limited insight into the mechanisms. Using chemical screening with small-molecule inhibitors of chromatin methylation and acetylation, further validated by knockdown approaches, we here identified Jumonji-type histone H3K27 demethylases as key regulators of cytokine production in human NK cell subsets. The prototypic JMJD3/UTX (Jumonji domain-containing protein 3) H3K27 demethylase inhibitor GSK-J4 increased global levels of the repressive H3K27me3 mark around transcription start sites of effector cytokine genes. Moreover, GSK-J4 reduced IFN-γ, TNFα, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-10 levels in cytokine-stimulated NK cells while sparing their cytotoxic killing activity against cancer cells. The anti-inflammatory effect of GSK-J4 in NK cell subsets, isolated from peripheral blood or tissue from individuals with rheumatoid arthritis (RA), coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggested that histone demethylase inhibition has broad utility for modulating immune and inflammatory responses. Overall, our results indicate that H3K27me3 is a dynamic and important epigenetic modification during NK cell activation and that JMJD3/UTX-driven H3K27 demethylation is critical for NK cell function.
Asunto(s)
Artritis Reumatoide/enzimología , Histonas/inmunología , Histona Demetilasas con Dominio de Jumonji/inmunología , Células Asesinas Naturales/enzimología , Secuencias de Aminoácidos , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Histonas/química , Histonas/genética , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Células Asesinas Naturales/inmunología , Fenotipo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The placenta releases syncytiotrophoblast-derived extracellular vesicles (STB-EV) into the maternal circulation throughout gestation. STB-EV dependent signalling is believed to contribute to the widespread maternal adaptive physiological changes seen in pregnancy. Transfer RNA (tRNA) halves have been identified in vesicles released from other human and murine organ systems, which alter gene expression in target cells. Here, we characterise tRNA-half expression in STB-EV and demonstrate biological activity of a highly abundant tRNA-half. Short RNA from ex-vivo, dual-lobe placental perfusion STB-EV was sequenced, showing that most (>95%) comprised tRNA species. Whole placental tissue contained <50% tRNA species, suggesting selective packaging and export of tRNA into STB-EV. Most tRNA within STB-EV were 5'-tRNA halves cleaved at 30-32 nucleotides. The pattern of tRNA expression differed depending on the size/origin of the STB-EV; this was confirmed by qPCR. Protein synthesis was suppressed in human fibroblasts when they were cultured with a 5'-tRNA half identified from STB-EV sequencing. This study is the first to evaluate tRNA species in STB-EV. The presence of biologically active 5'-tRNA halves, specific to a vesicular origin, suggests a novel mechanism for maternal-fetal signalling in normal pregnancy.
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Vesículas Extracelulares/metabolismo , ARN de Transferencia/metabolismo , Trofoblastos/metabolismo , Vesículas Extracelulares/ultraestructura , Femenino , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Conformación de Ácido Nucleico , Perfusión , Embarazo , ARN de Transferencia/químicaRESUMEN
Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.
RESUMEN
Atherosclerosis is the major cause of cardiovascular disease (CVD), the leading cause of death worldwide. Despite much focus on lipid abnormalities in atherosclerosis, it is clear that the immune system also has important pro- and antiatherogenic functions. The enzyme indoleamine-2,3-dioxygenase (IDO) catalyses degradation of the essential amino acid tryptophan into immunomodulatory metabolites. How IDO deficiency affects immune responses during atherogenesis is unknown and we explored potential mechanisms in models of murine and human atherosclerosis. IDO deficiency in hypercholesterolemic ApoE(-/-) mice caused a significant increase in lesion size and surrogate markers of plaque vulnerability. No significant changes in cholesterol levels were observed but decreases in IL-10 production were found in the peripheral blood, spleen and lymph node B cells of IDO-deficient compared with IDO-competent ApoE(-/-) mice. 3,4,-Dimethoxycinnamoyl anthranilic acid (3,4-DAA), an orally active synthetic derivative of the tryptophan metabolite anthranilic acid, but not l-kynurenine, enhanced production of IL-10 in cultured splenic B cells. Finally, 3,4-DAA treatment reduced lesion formation and inflammation after collar-induced arterial injury in ApoE(-/-) mice, and reduced cytokine and chemokine production in ex vivo human atheroma cell cultures. Our data demonstrate that endogenous production of tryptophan metabolites via IDO is an essential feedback loop that controls atherogenesis and athero-inflammation. We show that the IDO pathway induces production of IL-10 in B cells in vivo and in vitro, suggesting that IDO may induce immunoregulatory functions of B cells in atherosclerosis. The favorable effects of anthranilic acid derivatives in atherosclerosis indicate a novel approach toward therapy of CVD.
Asunto(s)
Aterosclerosis/prevención & control , Indolamina-Pirrol 2,3,-Dioxigenasa/fisiología , Animales , Apolipoproteínas E/genética , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Cinamatos/química , Cinamatos/uso terapéutico , Diseño de Fármacos , Quinurenina/sangre , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , ortoaminobenzoatos/químicaRESUMEN
Since its emergence in 2012, the genome editing technique known as CRISPR-Cas9 and its scientific use have rapidly expanded globally within a very short period of time. The technique consists of using an RNA guide molecule to bind to complementary DNA sequences, which simultaneously recruits the endonuclease Cas9 to introduce double-stranded breaks in the target DNA. The resulting double-stranded break is then repaired, allowing modification or removal of specific DNA bases. The technique has gained momentum in the laboratory because it is cheap, quick, and easy to use. Moreover, it is also being applied in vivo to generate more complex animal model systems. Such use of genome editing has proven to be highly effective and warrants a potential therapy for both genetic and non-genetic diseases. Although genome editing has the potential to be a transformative therapy for patients it is still in its infancy. Consequently, the legal and ethical frameworks are yet to be fully discussed and will be an increasingly important topic as the technology moves towards more contentious issues such as modification of the germline. Here, we review a number of scientific and ethical issues which may potentially influence the development of both the technology and its use in the clinical setting.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/ética , Discusiones Bioéticas , Femenino , Humanos , Embarazo , Diagnóstico Preimplantación/ética , Diagnóstico Preimplantación/métodosRESUMEN
Treg-cell function is compromised in rheumatoid arthritis (RA). As the master regulator of Treg cells, FOXP3 controls development and suppressive function. Stable Treg-cell FOXP3 expression is epigenetically regulated; constitutive expression requires a demethylated Treg-specific demethylated region. Here, we hypothesised that methylation of the FOXP3 locus is altered in Treg cells of established RA patients. Methylation analysis of key regulatory regions in the FOXP3 locus was performed on Treg cells from RA patients and healthy controls. The FOXP3 Treg-specific demethylated region and proximal promoter displayed comparable methylation profiles in RA and healthy-donor Treg cells. We identified a novel differentially methylated region (DMR) upstream of the FOXP3 promoter, with enhancer activity sensitive to methylation-induced silencing. In RA Treg cells we observed significantly reduced DMR methylation and lower DNA methyltransferase (DNMT1/3A) expression compared with healthy Treg cells. Furthermore, DMR methylation negatively correlated with FOXP3 mRNA expression, and Treg cells isolated from rheumatoid factor negative RA patients were found to express significantly higher levels of FOXP3 than Treg cells from RhF-positive patients, with an associated decrease in DMR methylation. In conclusion, the novel DMR is involved in the regulation of Treg-cell FOXP3 expression, but this regulation is lost post-transcriptionally in RA Treg cells.
Asunto(s)
Artritis Reumatoide/inmunología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Anciano , Artritis Reumatoide/genética , Metilación de ADN/genética , Metilación de ADN/inmunología , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Increasing evidence suggests that regulatory T cell (Treg) function is impaired in chronic inflammatory diseases such as rheumatoid arthritis (RA). Here we demonstrate that Tregs are unable to modulate the spontaneous production of TNF-α from RA synovial cells cultured from the diseased synovium site. Cytokine (IL-2, IL-6, TNF-α) activated T cells (Tck), cells we previously demonstrated to mimic the effector function of pathogenic RA synovial T cells, contained Tregs that survived and divided in this cytokine environment; however, the up-regulation of key molecules associated with Treg function (CTLA-4 and LFA-1) was impaired. Furthermore, Tregs were unable to suppress the function of Tcks, including contact-dependent induction of TNF-α from macrophages, supporting the concept that impaired Treg function/responsiveness contributes to chronicity of RA. However, ectopic foxp3 expression in both Tcks and pathogenic RA synovial T cells attenuated their cytokine production and function, including contact-dependent activation of macrophages. This diminished response to cytokine activation after ectopic foxp3 expression involved inhibited NF-κB activity and differed mechanistically from that displayed endogenously in conventional Tregs. These results suggest that diseases such as RA may perpetuate owing to the inability of Tregs to control cytokine-activated T-cell function. Understanding the mechanism whereby foxp3 attenuates the pathogenic function of synovial T cells may provide insight into the mechanisms of chronicity in inflammatory disease and potentially reveal new therapeutic candidates.
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Artritis Reumatoide/inmunología , Factores de Transcripción Forkhead/inmunología , Cápsula Articular/inmunología , Linfocitos T Reguladores/inmunología , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Humanos , Cápsula Articular/citología , Cápsula Articular/metabolismo , Lentivirus , Luciferasas , FN-kappa B/inmunología , FN-kappa B/metabolismo , Transducción Genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
X-ray diffraction crystallography has been most widely used for protein three-dimensional (3D) structure determination for which whether proteins are crystallizable is a central prerequisite. Yet, there are a number of procedures during protein crystallization, including protein material production, purification, and crystal production, which take turns affecting the crystallization outcome. Due to the expensive and laborious nature of this multi-stage process, various computational tools have been developed to predict protein crystallization propensity, which is then used to guide the experimental determination. In this study, we presented a novel deep learning framework, PLMC, to improve multi-stage protein crystallization propensity prediction by leveraging a pre-trained protein language model. To effectively train PLMC, two groups of features of each protein were integrated into a more comprehensive representation, including protein language embeddings from the large-scale protein sequence database and a handcrafted feature set consisting of physicochemical, sequence-based and disordered-related information. These features were further separately embedded for refinement, and then concatenated for the final prediction. Notably, our extensive benchmarking tests demonstrate that PLMC greatly outperforms other state-of-the-art methods by achieving AUC scores of 0.773, 0.893, and 0.913, respectively, at the aforementioned individual stages, and 0.982 at the final crystallization stage. Furthermore, PLMC is shown to be superior for predicting the crystallization of both globular and membrane proteins, as demonstrated by an AUC score of 0.991 for the latter. These results suggest the significant potential of PLMC in assisting researchers with the experimental design of crystallizable protein variants.
RESUMEN
Background: Preeclampsia (PE) is a hypertensive disorder of pregnancy, affecting 2%-8% of pregnancies worldwide, and is the leading cause of adverse maternal and fetal outcomes. The disease is characterized by oxidative and cellular stress and widespread endothelial dysfunction. While the precise mechanisms are not entirely understood, the pathogenesis of PE is closely linked to placental dysfunction and, to some extent, syncytiotrophoblast extracellular vesicle release (STB-EVs). These vesicles can be divided into the less well-studied medium/large EVs (220-1,000â nm) released in response to stress and small EVs (<220â nm) released as a component of intercellular communication. The previously described production of m/lSTB-EVs in response to cellular stress combined with the overwhelming occurrence of cellular and oxidative stress in PE prompted us to evaluate the microRNAome of PE m/lSTB-EVs. We hypothesized that the microRNAome profile of m/lSTB-EVs is different in PE compared to normal pregnancy (NP), which might permit the identification of potential circulating biomarkers not previously described in PE. Methods/study design: We performed small RNA sequencing on medium/large STB-EVs isolated from PE and NP placentae using dual-lobe ex vivo perfusion. The sequencing data was bioinformatically analyzed to identify differentially regulated microRNAs. Identified microRNAs were validated with quantitative PCR analysis. We completed our analysis by performing an in-silico prediction of STB-EV mechanistic pathways. Results: We identified significant differences between PE and NP in the STB-EVs micro ribonucleic acid (microRNA) profiles. We verified the differential expression of hsa-miR-193b-5p, hsa-miR-324-5p, hsa-miR-652-3p, hsa-miR-3196, hsa-miR-9-5p, hsa-miR-421, and hsa-miR-210-3p in the medium/large STB-EVs. We also confirmed the differential abundance of hsa-miR-9-5p in maternal serum extracellular vesicles (S EVs). In addition, we integrated the results of these microRNAs into the previously published messenger RNA (mRNA) data to better understand the relationship between these biomolecules. Conclusions: We identified a differentially regulated micro-RNA, hsa-miR-9-5p, that may have biomarker potential and uncovered mechanistic pathways that may be important in the pathophysiology of PE.