RESUMEN
Since establishment of the first embryonic stem cells (ESCs), in vitro culture of totipotent cells functionally and molecularly comparable with in vivo blastomeres with embryonic and extraembryonic developmental potential has been a challenge. Here we report that spliceosomal repression in mouse ESCs drives a pluripotent-to-totipotent state transition. Using the splicing inhibitor pladienolide B, we achieve stable in vitro culture of totipotent ESCs comparable at molecular levels with 2- and 4-cell blastomeres, which we call totipotent blastomere-like cells (TBLCs). Mouse chimeric assays combined with single-cell RNA sequencing (scRNA-seq) demonstrate that TBLCs have a robust bidirectional developmental capability to generate multiple embryonic and extraembryonic cell lineages. Mechanically, spliceosomal repression causes widespread splicing inhibition of pluripotent genes, whereas totipotent genes, which contain few short introns, are efficiently spliced and transcriptionally activated. Our study provides a means for capturing and maintaining totipotent stem cells.
Asunto(s)
Células Madre Totipotentes/citología , Células Madre Totipotentes/metabolismo , Animales , Blastómeros/citología , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Células Madre Embrionarias de Ratones/citología , Células Madre Totipotentes/fisiologíaRESUMEN
It has generally proven challenging to produce functional ß cells in vitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing â¼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. ß cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Receptor de Proteína C Endotelial/metabolismo , Islotes Pancreáticos/citología , Animales , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Femenino , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Islotes Pancreáticos/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Desnudos , Organoides/crecimiento & desarrollo , Organoides/metabolismo , Páncreas/citología , Páncreas/metabolismo , Proteína C/metabolismo , Células Madre/citologíaRESUMEN
There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.
Asunto(s)
ARN Mensajero/genética , ARN Viral/genética , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Sitios de Unión , Vacunas contra la COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Femenino , Células HEK293 , Células HeLa , Humanos , Inmunogenicidad Vacunal , Inyecciones Intramusculares , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos ICR , Nanopartículas/química , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células TH1/inmunología , Potencia de la Vacuna , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Células Vero , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Group 2 innate lymphoid cells (ILC2s) are highly heterogeneous tissue-resident lymphocytes that regulate inflammation and tissue homeostasis in health and disease. However, how these cells integrate into the tissue microenvironment to perform tissue-specific functions is unclear. Here, we show neuropilin-1 (Nrp1), which is induced postnatally and sustained by lung-derived transforming growth factor beta-1 (TGFß1), is a tissue-specific marker of lung ILC2s. Genetic ablation or pharmacological inhibition of Nrp1 suppresses IL-5 and IL-13 production by ILC2s and protects mice from the development of pulmonary fibrosis. Mechanistically, TGFß1-Nrp1 signaling enhances ILC2 function and type 2 immunity by upregulating IL-33 receptor ST2 expression. These findings identify Nrp1 as a tissue-specific regulator of lung-resident ILC2s and highlight Nrp1 as a potential therapeutic target for pulmonary fibrosis.
Asunto(s)
Inmunidad Innata/inmunología , Pulmón/inmunología , Neuropilina-1/inmunología , Animales , Modelos Animales de Enfermedad , Inflamación/inmunología , Interleucina-33/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos ICR , Fibrosis Pulmonar/inmunología , Transducción de Señal/inmunologíaRESUMEN
Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos' switch from "off" to "on" states remains unclear. Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1-8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.
Asunto(s)
Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/ultraestructura , Animales , Axones/metabolismo , Células COS , Caenorhabditis elegans/metabolismo , Proteínas Portadoras , Chlorocebus aethiops , Células HEK293 , Humanos , Ratones , Ratones Endogámicos ICR , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Cultivo Primario de Células , Transducción de Señal , Proteínas RoundaboutRESUMEN
A single mouse blastomere from an embryo until the 8-cell stage can generate an entire blastocyst. Whether laboratory-cultured cells retain a similar generative capacity remains unknown. Starting from a single stem cell type, extended pluripotent stem (EPS) cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. EPS-blastoids resembled blastocysts in morphology and cell-lineage allocation and recapitulated key morphogenetic events during preimplantation and early postimplantation development in vitro. Upon transfer, some EPS-blastoids underwent implantation, induced decidualization, and generated live, albeit disorganized, tissues in utero. Single-cell and bulk RNA-sequencing analysis revealed that EPS-blastoids contained all three blastocyst cell lineages and shared transcriptional similarity with natural blastocysts. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis and pave the way to creating viable synthetic embryos by using cultured cells.
Asunto(s)
Blastocisto/citología , Linaje de la Célula , Implantación del Embrión , Células Madre Pluripotentes Inducidas/citología , Células Madre Embrionarias de Ratones/citología , Creación de Embriones para Investigación/métodos , Animales , Blastocisto/metabolismo , Diferenciación Celular , Línea Celular , Células Cultivadas , Técnicas de Reprogramación Celular/métodos , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Células Madre Embrionarias de Ratones/metabolismo , TranscriptomaRESUMEN
The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.
Asunto(s)
Linaje de la Célula , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Células Dendríticas/metabolismo , Elementos de Facilitación Genéticos , Factores Reguladores del Interferón/metabolismo , Monocitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Células de la Médula Ósea , Células Cultivadas , Subunidades alfa del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/genética , Células Dendríticas/inmunología , Epigénesis Genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/genética , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Monocitos/inmunología , Células Progenitoras Mieloides/inmunología , Fenotipo , Transducción de SeñalRESUMEN
Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROß, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.
Asunto(s)
Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/inmunología , Adulto , Animales , Bencilaminas , Quimiocina CXCL2/farmacología , Ciclamas , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos ICR , Polimorfismo GenéticoRESUMEN
In early mammalian embryos, it remains unclear how the first cell fate bias is initially triggered and amplified toward cell fate segregation. Here, we report that a long noncoding RNA, LincGET, is transiently and asymmetrically expressed in the nucleus of two- to four-cell mouse embryos. Overexpression of LincGET in one of the two-cell blastomeres biases its progeny predominantly toward the inner cell mass (ICM) fate. Mechanistically, LincGET physically binds to CARM1 and promotes the nuclear localization of CARM1, which can further increase the level of H3 methylation at Arginine 26 (H3R26me), activate ICM-specific gene expression, upregulate transposons, and increase global chromatin accessibility. Simultaneous overexpression of LincGET and depletion of Carm1 no longer biased embryonic fate, indicating that the effect of LincGET in directing ICM lineage depends on CARM1. Thus, our data identify LincGET as one of the earliest known lineage regulators to bias cell fate in mammalian 2-cell embryos.
Asunto(s)
Blastocisto/metabolismo , Blastómeros/metabolismo , Linaje de la Célula/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , ARN Largo no Codificante/biosíntesis , Animales , Blastocisto/citología , Blastómeros/citología , Femenino , Histonas/metabolismo , Metilación , Ratones , Ratones Endogámicos ICR , Proteína-Arginina N-Metiltransferasas/biosíntesis , Proteína-Arginina N-Metiltransferasas/genética , ARN Largo no Codificante/genéticaRESUMEN
Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.
Asunto(s)
Quimerismo , Edición Génica , Mamíferos/embriología , Animales , Blastocisto , Sistemas CRISPR-Cas , Bovinos , Embrión de Mamíferos/citología , Femenino , Humanos , Masculino , Mamíferos/clasificación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Células Madre Pluripotentes , Ratas , Ratas Sprague-Dawley , Sus scrofaRESUMEN
Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy.
Asunto(s)
Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Streptomyces/química , Animales , Antibacterianos/química , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , ARN Polimerasas Dirigidas por ADN/química , Farmacorresistencia Bacteriana , Femenino , Células HeLa , Humanos , Ratones , Ratones Endogámicos ICR , Microbiología del Suelo , Infecciones Estreptocócicas/tratamiento farmacológico , Streptococcus pyogenes/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
N6-methyladenosine (m6A) is the most common mRNA modification. Recent studies have revealed that depletion of m6A machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the m6A 'writer' METTL3 or 'reader' YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was m6A modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that m6A-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role m6A serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.
Asunto(s)
Adenosina/análogos & derivados , Interacciones Huésped-Patógeno/genética , Inmunidad Innata/genética , Interferón Tipo I/genética , ARN Mensajero/metabolismo , Adenosina/metabolismo , Animales , Línea Celular Tumoral , Citomegalovirus/inmunología , Modelos Animales de Enfermedad , Femenino , Fibroblastos , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Interacciones Huésped-Patógeno/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Gripe Humana/virología , Interferón Tipo I/inmunología , Masculino , Metilación , Metiltransferasas/genética , Metiltransferasas/inmunología , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Muromegalovirus/inmunología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/metabolismoRESUMEN
Intestinal stem cells (ISCs) are maintained by stemness signaling for precise modulation of self-renewal and differentiation under homeostasis. However, the way in which intestinal immune cells regulate the self-renewal of ISCs remains elusive. Here we found that mouse and human Lgr5+ ISCs showed high expression of the immune cell-associated circular RNA circPan3 (originating from the Pan3 gene transcript). Deletion of circPan3 in Lgr5+ ISCs impaired their self-renewal capacity and the regeneration of gut epithelium in a manner dependent on immune cells. circPan3 bound mRNA encoding the cytokine IL-13 receptor subunit IL-13Rα1 (Il13ra1) in ISCs to increase its stability, which led to the expression of IL-13Rα1 in ISCs. IL-13 produced by group 2 innate lymphoid cells in the crypt niche engaged IL-13Rα1 on crypt ISCs and activated signaling mediated by IL-13âIL-13R, which in turn initiated expression of the transcription factor Foxp1. Foxp1 is associated with ß-catenin in rendering its nuclear translocation, which caused activation of the ß-catenin pathway and the maintenance of Lgr5+ ISCs.
Asunto(s)
Autorrenovación de las Células/inmunología , Interleucina-13/metabolismo , Mucosa Intestinal/inmunología , ARN/metabolismo , Células Madre/fisiología , Animales , Proteínas Portadoras/genética , Diferenciación Celular/inmunología , Autorrenovación de las Células/genética , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/inmunología , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Femenino , Humanos , Interleucina-13/inmunología , Subunidad alfa1 del Receptor de Interleucina-13/genética , Subunidad alfa1 del Receptor de Interleucina-13/inmunología , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , ARN/genética , ARN/inmunología , ARN Circular , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regeneración/genética , Regeneración/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , beta Catenina/inmunología , beta Catenina/metabolismoRESUMEN
Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood. Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR-Cas9-based genetic screens in vivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse KrasG12D-driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and a key component of p53-mediated tumor suppression.
Asunto(s)
Proteínas de Unión al ARN/genética , Proteína p53 Supresora de Tumor/genética , Adenocarcinoma/genética , Empalme Alternativo , Animales , Proteínas de Ciclo Celular/metabolismo , Exones , Perfilación de la Expresión Génica/métodos , Genes Supresores de Tumor , Humanos , Neoplasias Hepáticas/genética , Masculino , Ratones , Ratones Endogámicos ICR , Ratones SCID , Interferencia de ARN , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Vibrio vulnificus causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The most commonly present MARTX effector domain, the Makes Caterpillars Floppy-like (MCF) toxin, is a cysteine protease stimulated by host adenosine diphosphate (ADP) ribosylation factors (ARFs) to autoprocess. Here, we show processed MCF then binds and cleaves host Ras-related proteins in brain (Rab) guanosine triphosphatases within their C-terminal tails resulting in Rab degradation. We demonstrate MCF binds Rabs at the same interface occupied by ARFs. Moreover, we show MCF preferentially binds to ARF1 prior to autoprocessing and is active to cleave Rabs only subsequent to autoprocessing. We then use structure prediction algorithms to demonstrate that structural composition, rather than sequence, determines Rab target specificity. We further determine a crystal structure of aMCF as a swapped dimer, revealing an alternative conformation we suggest represents the open, activated state of MCF with reorganized active site residues. The cleavage of Rabs results in Rab1B dispersal within cells and loss of Rab1B density in the intestinal tissue of infected mice. Collectively, our work describes an extracellular bacterial mechanism whereby MCF is activated by ARFs and subsequently induces the degradation of another small host guanosine triphosphatase (GTPase), Rabs, to drive organelle damage, cell death, and promote pathogenesis of these rapidly fatal infections.
Asunto(s)
Toxinas Bacterianas , Vibrio vulnificus , Proteínas de Unión al GTP rab , Animales , Femenino , Humanos , Ratones , Factores de Ribosilacion-ADP/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/química , Células HEK293 , Ratones Endogámicos ICR , Proteolisis , Proteínas de Unión al GTP rab/metabolismo , Vibriosis/microbiología , Vibriosis/metabolismo , Vibrio vulnificus/metabolismo , Vibrio vulnificus/patogenicidadRESUMEN
During the maternal-to-zygotic transition (MZT), maternal RNAs are actively degraded and replaced by newly synthesized zygotic transcripts in a highly coordinated manner. However, it remains largely unknown how maternal mRNA decay is triggered in early vertebrate embryos. Here, through genome-wide profiling of RNA abundance and 3' modification, we show that uridylation is induced at the onset of maternal mRNA clearance. The temporal control of uridylation is conserved in vertebrates. When the homologs of terminal uridylyltransferases TUT4 and TUT7 (TUT4/7) are depleted in zebrafish and Xenopus, maternal mRNA clearance is significantly delayed, leading to developmental defects during gastrulation. Short-tailed mRNAs are selectively uridylated by TUT4/7, with the highly uridylated transcripts degraded faster during the MZT than those with unmodified poly(A) tails. Our study demonstrates that uridylation plays a crucial role in timely mRNA degradation, thereby allowing the progression of early development.
Asunto(s)
Embrión de Mamíferos/enzimología , Embrión no Mamífero/enzimología , Nucleotidiltransferasas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Transcriptoma , Xenopus laevis/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Gastrulación , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Ratones Endogámicos ICR , Nucleotidiltransferasas/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Proteínas Bacterianas/biosíntesis , ADN Bacteriano/genética , Infecciones por Klebsiella/tratamiento farmacológico , Subunidades Ribosómicas Pequeñas/efectos de los fármacos , Xenorhabdus/metabolismo , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Animales , Antibacterianos/metabolismo , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Modelos Animales de Enfermedad , Femenino , Células Hep G2 , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Masculino , Ratones Endogámicos ICR , Biosíntesis de Proteínas/efectos de los fármacos , Subunidades Ribosómicas Pequeñas/genética , Subunidades Ribosómicas Pequeñas/metabolismoRESUMEN
Enterovirus A71 (EV-A71) can induce severe neurological complications and even fatal encephalitis in children, and it has caused several large outbreaks in Taiwan since 1998. We previously generated VP1 codon-deoptimized (VP1-CD) reverse genetics (rg) EV-A71 viruses (rgEV-A71s) that harbor a high-fidelity (HF) 3D polymerase. These VP1-CD-HF rgEV-A71s showed lower replication kinetics in vitro and decreased virulence in an Institute of Cancer Research (ICR) mouse model of EV-A71 infection, while still retaining their antigenicity in comparison to the wild-type virus. In this study, we aimed to further investigate the humoral and cellular immune responses elicited by VP1-CD-HF rgEV-A71s to assess the potential efficacy of these EV-A71 vaccine candidates. Following intraperitoneal (i.p.) injection of VP1-CD-HF rgEV-A71s in mice, we observed a robust induction of EV-A71-specific neutralizing IgG antibodies in the antisera after 21 days. Splenocytes isolated from VP1-CD-HF rgEV-A71s-immunized mice exhibited enhanced proliferative activities and cytokine production (IL-2, IFN-γ, IL-4, IL-6, and TNF-α) upon re-stimulation with VP1-CD-HF rgEV-A71, as compared to control mice treated with adjuvant only. Importantly, administration of antisera from VP1-CD-HF rgEV-A71s-immunized mice protected against lethal EV-A71 challenge in neonatal mice. These findings highlight that our generated VP1-CD-HF rgEV-A71 viruses are capable of inducing both cellular and humoral immune responses, supporting their potential as next-generation EV-A71 vaccines for combating EV-A71 infection.IMPORTANCEEV-A71 can cause severe neurological diseases and cause death in young children. Here, we report the development of synthetic rgEV-A71s with the combination of codon deoptimization and high-fidelity (HF) substitutions that generate genetically stable reverse genetics (rg) viruses as potential attenuated vaccine candidates. Our work provides insight into the development of low-virulence candidate vaccines through a series of viral genetic editing for maintaining antigenicity and genome stability and suggests a strategy for the development of an innovative next-generation vaccine against EV-A71.
Asunto(s)
Proteínas de la Cápside , Enterovirus Humano A , Infecciones por Enterovirus , ARN Polimerasa Dependiente del ARN , Animales , Ratones , Anticuerpos Antivirales/inmunología , Codón , Enterovirus Humano A/genética , Infecciones por Enterovirus/inmunología , Vacunas Atenuadas , Proteínas de la Cápside/genética , Inmunidad Humoral , Inmunidad Celular , Anticuerpos Neutralizantes/inmunología , Vacunas Virales , Ratones Endogámicos ICR , Ratones Endogámicos BALB C , ARN Polimerasa Dependiente del ARN/genéticaRESUMEN
Dengue fever, an infectious disease prevalent in subtropical and tropical regions, currently lacks effective small-molecule drugs as treatment. In this study, we used a fluorescence peptide cleavage assay to screen seven compounds to assess their inhibition of the dengue virus (DENV) NS2B-NS3 protease. DV-B-120 demonstrated superior inhibition of NS2B-NS3 protease activity and lower toxicity compared to ARDP0006. The selectivity index of DV-B-120 was higher than that of ARDP0006. In vivo assessments of the antiviral efficacy of DV-B-120 against DENV replication demonstrated delayed mortality of suckling mice treated with the compound, with 60-80% protection against life-threatening effects, compared to the outcomes of DENV-infected mice treated with saline. The lower clinical scores of DENV-infected mice treated with DV-B-120 indicated a reduction in acute-progressive illness symptoms, underscoring the potential therapeutic impact of DV-B-120. Investigations of DV-B-120's ability to restore the antiviral type I IFN response in the brain tissue of DENV-infected ICR suckling mice demonstrated its capacity to stimulate IFN and antiviral IFN-stimulated gene expression. DV-B-120 not only significantly delayed DENV-2-induced mortality and illness symptoms but also reduced viral numbers in the brain, ultimately restoring the innate antiviral response. These findings strongly suggest that DV-B-120 holds promise as a therapeutic agent against DENV infection and highlight its potential contribution in addressing the current lack of effective treatments for this infectious disease.IMPORTANCEThe prevalence of dengue virus (DENV) infection in tropical and subtropical regions is escalating due to factors like climate change and mosquito vector expansion. With over 300 million annual infections and potentially fatal outcomes, the urgent need for effective treatments is evident. While the approved Dengvaxia vaccine has variable efficacy, there are currently no antiviral drugs for DENV. This study explores seven compounds targeting the NS2B-NS3 protease, a crucial protein in DENV replication. These compounds exhibit inhibitory effects on DENV-2 NS2B-NS3, holding promise for disrupting viral replication and preventing severe manifestations. However, further research, including animal testing, is imperative to assess therapeutic efficacy and potential toxicity. Developing safe and potent treatments for DENV infection is critical in addressing the rising global health threat posed by this virus.
Asunto(s)
Virus del Dengue , Dengue , Piperidinas , Animales , Ratones , Antivirales/química , Antivirales/uso terapéutico , Enfermedades Transmisibles , Dengue/tratamiento farmacológico , Virus del Dengue/fisiología , Endopeptidasas/farmacología , Ratones Endogámicos ICR , Piperidinas/administración & dosificación , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/químicaRESUMEN
Interleukin 17 (IL-17)-committed γδ T cells (γδT17 cells) participate in many immune responses, but their developmental requirements and subset specific functions remain poorly understood. Here we report that a commonly used CD45.1(+) congenic C57BL/6 mouse substrain is characterized by selective deficiency in Vγ4(+) γδT17 cells. This trait was due to a spontaneous mutation in the gene encoding the transcription factor Sox13 that caused an intrinsic defect in development of those cells in the neonatal thymus. The γδT17 cells migrated from skin to lymph nodes at low rates. In a model of psoriasis-like dermatitis, the Vγ4(+) γδT17 cell subset expanded considerably in lymph nodes and homed to inflamed skin. Sox13-mutant mice were protected from psoriasis-like skin changes, which identified a role for Sox13-dependent γδT17 cells in this inflammatory condition.