RESUMEN
Interactions between communities of the gut microbiome and with the host could affect the onset and progression of metabolic associated fatty liver disease (MAFLD), and can be useful as new diagnostic and prognostic biomarkers. In this study, we performed a multi-omics approach to unravel gut microbiome signatures from 32 biopsy-proven patients (10 simple steatosis -SS- and 22 steatohepatitis -SH-) and 19 healthy volunteers (HV). Human and microbial transcripts were differentially identified between groups (MAFLD vs. HV/SH vs. SS), and analyzed for weighted correlation networks together with previously detected metabolites from the same set of samples. We observed that expression of Desulfobacteraceae bacterium, methanogenic archaea, Mushu phage, opportunistic pathogenic fungi Fusarium proliferatum and Candida sorbophila, protozoa Blastocystis spp. and Fonticula alba were upregulated in MAFLD and SH. Desulfobacteraceae bacterium and Mushu phage were hub species in the onset of MAFLD, whereas the activity of Fonticula alba, Faecalibacterium prausnitzii, and Mushu phage act as key regulators of the progression to SH. A combination of clinical, metabolomic, and transcriptomic parameters showed the highest predictive capacity for MAFLD and SH (AUC = 0.96). In conclusion, faecal microbiome markers from several community members contribute to the switch in signatures characteristic of MAFLD and its progression towards SH.
Asunto(s)
Aciltransferasas , Microbioma Gastrointestinal , Enfermedad del Hígado Graso no Alcohólico , Fosfolipasas A2 Calcio-Independiente , Humanos , Microbioma Gastrointestinal/genética , Genotipo , Metaboloma , Transcriptoma/genética , Aciltransferasas/genética , Fosfolipasas A2 Calcio-Independiente/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/microbiologíaRESUMEN
mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Mensajero/metabolismo , Algoritmos , Sitios de Unión , Sistema Libre de Células , Cartilla de ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Entropía , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Pliegue del ARN , ARN Mensajero/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Ribosomas/química , Ribosomas/metabolismo , Regiones no TraducidasRESUMEN
Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1-2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higher-affinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency.
Asunto(s)
Vacunas contra el SIDA/administración & dosificación , Anticuerpos Antivirales/biosíntesis , Linfocitos B/efectos de los fármacos , Centro Germinal/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/administración & dosificación , Vacunación/métodos , Adyuvantes Inmunológicos/administración & dosificación , Animales , Afinidad de Anticuerpos , Linfocitos B/citología , Linfocitos B/inmunología , Células CHO , Cricetulus , Esquema de Medicación , Femenino , Centro Germinal/citología , Centro Germinal/inmunología , Células HEK293 , Proteína gp120 de Envoltorio del VIH/biosíntesis , Humanos , Inmunogenicidad Vacunal , Bombas de Infusión Implantables , Lípido A/administración & dosificación , Lípido A/análogos & derivados , Ratones , Ratones Endogámicos C57BL , Presión Osmótica , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/biosíntesis , Vacunación/instrumentaciónRESUMEN
Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)(+) macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE(-/-) mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.
Asunto(s)
Hematopoyesis Extramedular/inmunología , Células Madre Hematopoyéticas/inmunología , Macrófagos/inmunología , Mielopoyesis/inmunología , Bazo/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunología , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/inmunología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Hematopoyesis Extramedular/genética , Células Madre Hematopoyéticas/patología , Macrófagos/patología , Ratones , Ratones Noqueados , Mielopoyesis/genética , Infarto del Miocardio/genética , Infarto del Miocardio/inmunología , Infarto del Miocardio/patología , Nanopartículas , Placa Aterosclerótica/genética , Placa Aterosclerótica/inmunología , Placa Aterosclerótica/patología , Interferencia de ARN , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/inmunología , Lectina 1 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 1 Similar a Ig de Unión al Ácido Siálico/inmunología , Bazo/patología , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3' untranslated region (3'UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3'UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3'UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3'UTR targeting as to harness the activity of several Ago proteins.
Asunto(s)
Proteínas Argonautas/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Fibroblastos/citología , Hígado/metabolismo , ARN Mensajero/genética , Animales , Proteínas Argonautas/genética , Células Cultivadas , Embrión de Mamíferos/citología , Femenino , Fibroblastos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Interferencia de ARNRESUMEN
Integrins play an important role during development, regulating cell differentiation, proliferation and survival. Here we show that knockdown of integrin subunits slows down the progression of hepatocellular carcinoma (HCC). Using nanoparticulate delivery of short interfering RNAs targeting ß1 and αv integrin subunits, we downregulate all integrin receptors in hepatocytes. Short-term integrin knockdown (2 weeks) does not cause apparent structural or functional perturbations of normal liver tissue. Alterations in liver morphology accumulate on sustained integrin downregulation (7 weeks). The integrin knockdown leads to significant retardation of HCC progression, reducing proliferation and increasing tumour cell death. This tumour retardation is accompanied by reduced activation of the MET oncogene as well as expression of its mature form on the cell surface. Our data suggest that transformed proliferating cells from HCC are more sensitive to knockdown of integrins than normal quiescent hepatocytes, highlighting the potential of small interfering RNA-mediated inhibition of integrins as an anti-cancer therapeutic approach.
Asunto(s)
Carcinoma Hepatocelular/genética , Hepatocitos/metabolismo , Cadenas alfa de Integrinas/genética , Cadenas beta de Integrinas/genética , Neoplasias Hepáticas Experimentales/genética , Nanopartículas , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Interferente Pequeño , Animales , Proliferación Celular , Progresión de la Enfermedad , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , RatonesRESUMEN
Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.
Asunto(s)
Células Endoteliales/metabolismo , Nanopartículas/química , Polímeros/química , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Animales , Línea Celular , Humanos , Ratones , Nanopartículas/ultraestructura , Neoplasias/genética , Neoplasias/terapia , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéuticoRESUMEN
An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.
Asunto(s)
Endosomas/metabolismo , Lisosomas/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Animales , Polaridad Celular , Células Cultivadas , Endocitosis , Técnicas de Silenciamiento del Gen , Hepatocitos/citología , Hepatocitos/metabolismo , Isoenzimas/biosíntesis , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/metabolismo , Lipoproteínas LDL/metabolismo , Hígado/citología , Hígado/enzimología , Hígado/metabolismo , Ratones , Cuerpos Multivesiculares/metabolismo , Especificidad de Órganos , Biosíntesis de Proteínas , Interferencia de ARN , ARN Mensajero/análisis , ARN Mensajero/genética , Factores de Tiempo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab5/biosíntesis , Proteínas de Unión al GTP rab5/deficiencia , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.
RESUMEN
Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Cationes/química , Silenciador del Gen , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Lípidos/administración & dosificación , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Animales , Western Blotting , Médula Ósea , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Endocitosis , Citometría de Flujo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Antígenos Comunes de Leucocito/antagonistas & inhibidores , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Liposomas , Hígado/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , LDL-Colesterol/metabolismo , Cromosomas Humanos Par 1/genética , Predisposición Genética a la Enfermedad/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas Adaptadoras del Transporte Vesicular/biosíntesis , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , LDL-Colesterol/sangre , Estudios de Cohortes , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/genética , Europa (Continente)/etnología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Estudio de Asociación del Genoma Completo , Haplotipos/genética , Hepatocitos/metabolismo , Humanos , Lípidos/sangre , Lipoproteínas VLDL/sangre , Lipoproteínas VLDL/metabolismo , Hígado/citología , Hígado/metabolismo , Ratones , Infarto del Miocardio/sangre , Infarto del Miocardio/genética , Fenotipo , Transcripción GenéticaRESUMEN
The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Fluorescente/métodos , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiologíaRESUMEN
Identification of insulators is one of the most difficult problems in functional mapping of genomes. For this reason, up to now only a few insulators have been described. In this article we suggest an approach that allows direct isolation of insulators by a simple positive-negative selection based on blocking enhancer effects by insulators. The approach allows selection of fragments capable of blocking enhancers from mixtures of genomic fragments prepared from up to 1-Mb genomic regions. Using this approach, a 1-Mb human genome locus was analyzed and eight potential insulators were selected. Five of the eight sequences were positioned in intergenic regions and two were within introns. The genes of the alpha-polypeptide H+/K+ exchanging ATPase (ATP4A) and amyloid beta (A4) precursor-like protein 1 (APLP1) within the locus studied were found to be flanked by insulators on both sides. Both genes are characterized by distinct tissue-specific expression that differs from the tissue specificity of the surrounding genes. The data obtained are consistent with the conception that insulators subdivide genomic DNA into loop domains that comprise genes characterized by similar expression profiles. Using chromatin immunoprecipitation assay, we demonstrated also that at least six of the putative insulators revealed in this work could bind the CTCF transcription factor in vivo. We believe that the proposed approach could be a useful instrument for functional analysis of genomes.