RESUMEN
Bats, rodents, and shrews are the most important animal sources of human infectious diseases. However, the evolution and transmission of viruses among them remain largely unexplored. Through the meta-transcriptomic sequencing of internal organ and fecal samples from 2,443 wild bats, rodents, and shrews sampled from four Chinese habitats, we identified 669 viruses, including 534 novel viruses, thereby greatly expanding the mammalian virome. Our analysis revealed high levels of phylogenetic diversity, identified cross-species virus transmission events, elucidated virus origins, and identified cases of invertebrate viruses in mammalian hosts. Host order and sample size were the most important factors impacting virome composition and patterns of virus spillover. Shrews harbored a high richness of viruses, including many invertebrate-associated viruses with multi-organ distributions, whereas rodents carried viruses with a greater capacity for host jumping. These data highlight the remarkable diversity of mammalian viruses in local habitats and their ability to emerge in new hosts.
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Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering development of urgently needed innovative therapies. Here, we show that Twist1+/- mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure, and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.
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Cognición/fisiología , Suturas Craneales/fisiopatología , Craneosinostosis/fisiopatología , Regeneración/fisiología , Cráneo/fisiopatología , Animales , Conducta Animal/efectos de los fármacos , Cognición/efectos de los fármacos , Craneosinostosis/genética , Duramadre/patología , Duramadre/fisiopatología , Gelatina/farmacología , Perfilación de la Expresión Génica , Fuerza de la Mano , Presión Intracraneal/efectos de los fármacos , Presión Intracraneal/fisiología , Locomoción/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Metacrilatos/farmacología , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Regeneración/efectos de los fármacos , Cráneo/patología , Proteína 1 Relacionada con Twist/metabolismo , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.
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Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Receptores de Dopamina D1/metabolismo , Transducción de Señal , Regulación Alostérica , Sitio Alostérico , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Catecoles/metabolismo , Microscopía por Crioelectrón , Fenoldopam/química , Fenoldopam/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Multimerización de Proteína , Receptores de Dopamina D1/química , Receptores de Dopamina D1/ultraestructura , Receptores de Dopamina D2/metabolismo , Homología Estructural de ProteínaRESUMEN
Lungfishes are the closest extant relatives of tetrapods and preserve ancestral traits linked with the water-to-land transition. However, their huge genome sizes have hindered understanding of this key transition in evolution. Here, we report a 40-Gb chromosome-level assembly of the African lungfish (Protopterus annectens) genome, which is the largest genome assembly ever reported and has a contig and chromosome N50 of 1.60 Mb and 2.81 Gb, respectively. The large size of the lungfish genome is due mainly to retrotransposons. Genes with ultra-long length show similar expression levels to other genes, indicating that lungfishes have evolved high transcription efficacy to keep gene expression balanced. Together with transcriptome and experimental data, we identified potential genes and regulatory elements related to such terrestrial adaptation traits as pulmonary surfactant, anxiolytic ability, pentadactyl limbs, and pharyngeal remodeling. Our results provide insights and key resources for understanding the evolutionary pathway leading from fishes to humans.
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Adaptación Biológica , Evolución Biológica , Peces/genética , Secuenciación Completa del Genoma , Aletas de Animales/anatomía & histología , Aletas de Animales/fisiología , Animales , Extremidades/anatomía & histología , Extremidades/fisiología , Peces/anatomía & histología , Peces/clasificación , Peces/fisiología , Filogenia , Fenómenos Fisiológicos Respiratorios , Sistema Respiratorio/anatomía & histología , Vertebrados/genéticaRESUMEN
Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.
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Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Pandemias/prevención & control , Neumonía Viral/prevención & control , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Diseño Universal , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/química , COVID-19 , Vacunas contra la COVID-19 , Línea Celular Tumoral , Chlorocebus aethiops , Infecciones por Coronavirus/virología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Coronavirus del Síndrome Respiratorio de Oriente Medio/química , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/virología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/inmunología , Receptores Virales/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , SARS-CoV-2 , Células Sf9 , Organismos Libres de Patógenos Específicos , Spodoptera , Transfección , Vacunación/métodos , Células Vero , Vacunas ViralesRESUMEN
T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.
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Tratamiento Basado en Trasplante de Células y Tejidos , Edición Génica , Neoplasias , Medicina de Precisión , Receptores de Antígenos de Linfocitos T , Linfocitos T , Transgenes , Humanos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Biopsia , Tratamiento Basado en Trasplante de Células y Tejidos/efectos adversos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Síndrome de Liberación de Citoquinas/complicaciones , Progresión de la Enfermedad , Encefalitis/complicaciones , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Mutación , Neoplasias/complicaciones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapia , Seguridad del Paciente , Medicina de Precisión/efectos adversos , Medicina de Precisión/métodos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transgenes/genética , Antígenos HLA/inmunología , Sistemas CRISPR-CasRESUMEN
The gene encoding PTEN is one of the most frequently mutated tumor suppressor-encoding genes in human cancer. While PTEN's function in tumor suppression is well established, its relationship to anti-microbial immunity remains unknown. Here we found a pivotal role for PTEN in the induction of type I interferon, the hallmark of antiviral innate immunity, that was independent of the pathway of the kinases PI(3)K and Akt. PTEN controlled the import of IRF3, a master transcription factor responsible for IFN-ß production, into the nucleus. We further identified a PTEN-controlled negative phosphorylation site at Ser97 of IRF3 and found that release from this negative regulation via the phosphatase activity of PTEN was essential for the activation of IRF3 and its import into the nucleus. Our study identifies crosstalk between PTEN and IRF3 in tumor suppression and innate immunity.
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Inmunidad Innata/inmunología , Factor 3 Regulador del Interferón/inmunología , Interferón Tipo I/inmunología , Fosfohidrolasa PTEN/inmunología , Infecciones por Respirovirus/inmunología , Infecciones por Rhabdoviridae/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Núcleo Celular , Proliferación Celular , Citocinas/inmunología , Células Dendríticas/inmunología , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Técnicas de Transferencia de Gen , Células HEK293 , Humanos , Immunoblotting , Inmunoprecipitación , Factor 3 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/genética , Células MCF-7 , Macrófagos/inmunología , Espectrometría de Masas , Ratones , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus Sendai , VesiculovirusRESUMEN
The timely degradation of proteins that regulate the cell cycle is essential for oocyte maturation. Oocytes are equipped to degrade proteins via the ubiquitin-proteasome system. In meiosis, anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin-ligase, is responsible for the degradation of proteins. Ubiquitin-conjugating enzyme E2 S (UBE2S), an E2 ubiquitin-conjugating enzyme, delivers ubiquitin to APC/C. APC/C has been extensively studied, but the functions of UBE2S in oocyte maturation and mouse fertility are not clear. In this study, we used Ube2s knockout mice to explore the role of UBE2S in mouse oocytes. Ube2s-deleted oocytes were characterized by meiosis I arrest with normal spindle assembly and spindle assembly checkpoint dynamics. However, the absence of UBE2S affected the activity of APC/C. Cyclin B1 and securin are two substrates of APC/C, and their levels were consistently high, resulting in the failure of homologous chromosome separation. Unexpectedly, the oocytes arrested in meiosis I could be fertilized and the embryos could become implanted normally, but died before embryonic day 10.5. In conclusion, our findings reveal an indispensable regulatory role of UBE2S in mouse oocyte meiosis and female fertility.
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Puntos de Control de la Fase M del Ciclo Celular , Meiosis , Animales , Femenino , Ratones , Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Oocitos/metabolismo , Ubiquitinas/metabolismoRESUMEN
Social interactions among animals mediate essential behaviours, including mating, nurturing, and defence1,2. The gut microbiota contribute to social activity in mice3,4, but the gut-brain connections that regulate this complex behaviour and its underlying neural basis are unclear5,6. Here we show that the microbiome modulates neuronal activity in specific brain regions of male mice to regulate canonical stress responses and social behaviours. Social deviation in germ-free and antibiotic-treated mice is associated with elevated levels of the stress hormone corticosterone, which is primarily produced by activation of the hypothalamus-pituitary-adrenal (HPA) axis. Adrenalectomy, antagonism of glucocorticoid receptors, or pharmacological inhibition of corticosterone synthesis effectively corrects social deficits following microbiome depletion. Genetic ablation of glucocorticoid receptors in specific brain regions or chemogenetic inactivation of neurons in the paraventricular nucleus of the hypothalamus that produce corticotrophin-releasing hormone (CRH) reverse social impairments in antibiotic-treated mice. Conversely, specific activation of CRH-expressing neurons in the paraventricular nucleus induces social deficits in mice with a normal microbiome. Via microbiome profiling and in vivo selection, we identify a bacterial species, Enterococcus faecalis, that promotes social activity and reduces corticosterone levels in mice following social stress. These studies suggest that specific gut bacteria can restrain the activation of the HPA axis, and show that the microbiome can affect social behaviours through discrete neuronal circuits that mediate stress responses in the brain.
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Encéfalo/citología , Encéfalo/fisiología , Microbioma Gastrointestinal/fisiología , Neuronas/metabolismo , Conducta Social , Estrés Psicológico , Animales , Corticosterona/sangre , Hormona Liberadora de Corticotropina/metabolismo , Enterococcus faecalis/metabolismo , Vida Libre de Gérmenes , Glucocorticoides/metabolismo , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Glucocorticoides/metabolismo , Transducción de SeñalRESUMEN
The removal of mis-incorporated nucleotides by proofreading activity ensures DNA replication fidelity. Whereas the ε-exonuclease DnaQ is a well-established proofreader in the model organism Escherichia coli, it has been shown that proofreading in a majority of bacteria relies on the polymerase and histidinol phosphatase (PHP) domain of replicative polymerase, despite the presence of a DnaQ homolog that is structurally and functionally distinct from E. coli DnaQ. However, the biological functions of this type of noncanonical DnaQ remain unclear. Here, we provide independent evidence that noncanonical DnaQ functions as an additional proofreader for mycobacteria. Using the mutation accumulation assay in combination with whole-genome sequencing, we showed that depletion of DnaQ in Mycolicibacterium smegmatis leads to an increased mutation rate, resulting in AT-biased mutagenesis and increased insertions/deletions in the homopolymer tract. Our results showed that mycobacterial DnaQ binds to the ß clamp and functions synergistically with the PHP domain proofreader to correct replication errors. Furthermore, the loss of dnaQ results in replication fork dysfunction, leading to attenuated growth and increased mutagenesis on subinhibitory fluoroquinolones potentially due to increased vulnerability to fork collapse. By analyzing the sequence polymorphism of dnaQ in clinical isolates of Mycobacterium tuberculosis (Mtb), we demonstrated that a naturally evolved DnaQ variant prevalent in Mtb lineage 4.3 may enable hypermutability and is associated with drug resistance. These results establish a coproofreading model and suggest a division of labor between DnaQ and PHP domain proofreader. This study also provides real-world evidence that a mutator-driven evolutionary pathway may exist during the adaptation of Mtb.
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Replicación del ADN , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , MutaciónRESUMEN
Transcriptome-wide association studies (TWAS) have identified many putative susceptibility genes for colorectal cancer (CRC) risk. However, susceptibility miRNAs, critical dysregulators of gene expression, remain unexplored. We genotyped DNA samples from 313 CRC East Asian patients and performed small RNA sequencing in their normal colon tissues distant from tumors to build genetic models for predicting miRNA expression. We applied these models and data from genome-wide association studies (GWAS) including 23 942 cases and 217 267 controls of East Asian ancestry to investigate associations of predicted miRNA expression with CRC risk. Perturbation experiments separately by promoting and inhibiting miRNAs expressions and further in vitro assays in both SW480 and HCT116 cells were conducted. At a Bonferroni-corrected threshold of P < 4.5 × 10-4, we identified two putative susceptibility miRNAs, miR-1307-5p and miR-192-3p, located in regions more than 500 kb away from any GWAS-identified risk variants in CRC. We observed that a high predicted expression of miR-1307-5p was associated with increased CRC risk, while a low predicted expression of miR-192-3p was associated with increased CRC risk. Our experimental results further provide strong evidence of their susceptible roles by showing that miR-1307-5p and miR-192-3p play a regulatory role, respectively, in promoting and inhibiting CRC cell proliferation, migration, and invasion, which was consistently observed in both SW480 and HCT116 cells. Our study provides additional insights into the biological mechanisms underlying CRC development.
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Neoplasias Colorrectales , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Transcriptoma/genética , Estudio de Asociación del Genoma Completo , Neoplasias Colorrectales/metabolismo , Células HCT116 , Regulación Neoplásica de la Expresión Génica/genética , Proliferación Celular/genéticaRESUMEN
Genome-wide association studies (GWASs) have repeatedly reported multiple non-coding single-nucleotide polymorphisms (SNPs) at 2p14 associated with rheumatoid arthritis (RA), but their functional roles in the pathological mechanisms of RA remain to be explored. In this study, we integrated a series of bioinformatics and functional experiments and identified three intronic RA SNPs (rs1876518, rs268131, and rs2576923) within active enhancers that can regulate the expression of SPRED2 directly. At the same time, SPRED2 and ACTR2 influence each other as a positive feedback signal amplifier to strengthen the protective role in RA by inhibiting the migration and invasion of rheumatoid fibroblast-like synoviocytes (FLSs). In particular, the transcription factor CEBPB preferentially binds to the rs1876518-T allele to increase the expression of SPRED2 in FLSs. Our findings decipher the molecular mechanisms behind the GWAS signals at 2p14 for RA and emphasize SPRED2 as a potential candidate gene for RA, providing a potential target and direction for precise treatment of RA.
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Artritis Reumatoide , Sinoviocitos , Humanos , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Proliferación Celular/genética , Células Cultivadas , Cromosomas , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Proteínas Represoras/genética , Sinoviocitos/metabolismo , Sinoviocitos/patología , Proteína 2 Relacionada con la Actina/metabolismoRESUMEN
Most of the single-nucleotide polymorphisms (SNPs) associated with insulin resistance (IR)-relevant phenotypes by genome-wide association studies (GWASs) are located in noncoding regions, complicating their functional interpretation. Here, we utilized an adapted STARR-seq to evaluate the regulatory activities of 5,987 noncoding SNPs associated with IR-relevant phenotypes. We identified 876 SNPs with biased allelic enhancer activity effects (baaSNPs) across 133 loci in three IR-relevant cell lines (HepG2, preadipocyte, and A673), which showed pervasive cell specificity and significant enrichment for cell-specific open chromatin regions or enhancer-indicative markers (H3K4me1, H3K27ac). Further functional characterization suggested several transcription factors (TFs) with preferential allelic binding to baaSNPs. We also incorporated multi-omics data to prioritize 102 candidate regulatory target genes for baaSNPs and revealed prevalent long-range regulatory effects and cell-specific IR-relevant biological functional enrichment on them. Specifically, we experimentally verified the distal regulatory mechanism at IRS1 locus, in which rs952227-A reinforces IRS1 expression by long-range chromatin interaction and preferential binding to the transcription factor HOXC6 to augment the enhancer activity. Finally, based on our STARR-seq screening data, we predicted the enhancer activity of 227,343 noncoding SNPs associated with IR-relevant phenotypes (fasting insulin adjusted for BMI, HDL cholesterol, and triglycerides) from the largest available GWAS summary statistics. We further provided an open resource (http://www.bigc.online/fnSNP-IR) for better understanding genetic regulatory mechanisms of IR-relevant phenotypes.
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Resistencia a la Insulina , Polimorfismo de Nucleótido Simple , Humanos , Polimorfismo de Nucleótido Simple/genética , Estudio de Asociación del Genoma Completo , Resistencia a la Insulina/genética , Factores de Transcripción/genética , Cromatina/genética , Fenotipo , Elementos de Facilitación Genéticos/genéticaRESUMEN
Accurate chromosome segregation, monitored by the spindle assembly checkpoint (SAC), is crucial for the production of euploid cells. Previous in vitro studies by us and others showed that Mad2, a core member of the SAC, performs a checkpoint function in oocyte meiosis. Here, through an oocyte-specific knockout approach in mouse, we reconfirmed that Mad2-deficient oocytes exhibit an accelerated metaphase-to-anaphase transition caused by premature degradation of securin and cyclin B1 and subsequent activation of separase in meiosis I. However, it was surprising that the knockout mice were completely fertile and the resulting oocytes were euploid. In the absence of Mad2, other SAC proteins, including BubR1, Bub3 and Mad1, were normally recruited to the kinetochores, which likely explains the balanced chromosome separation. Further studies showed that the chromosome separation in Mad2-null oocytes was particularly sensitive to environmental changes and, when matured in vitro, showed chromosome misalignment, lagging chromosomes, and aneuploidy with premature separation of sister chromatids, which was exacerbated at a lower temperature. We reveal for the first time that Mad2 is dispensable for proper chromosome segregation but acts to mitigate environmental stress in meiotic oocytes.
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Proteínas de Ciclo Celular , Huso Acromático , Animales , Ratones , Proteínas de Ciclo Celular/metabolismo , Huso Acromático/metabolismo , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Segregación Cromosómica/genética , Oocitos/metabolismo , Cinetocoros/metabolismo , Meiosis/genéticaRESUMEN
As a miniature RNA-guided endonuclease, IscB is presumed to be the ancestor of Cas9 and to share similar functions. IscB is less than half the size of Cas9 and thus more suitable for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells limits its in vivo applications. Here we describe the engineering of OgeuIscB and its corresponding ωRNA to develop an IscB system that is highly efficient in mammalian systems, named enIscB. By fusing enIscB with T5 exonuclease (T5E), we found enIscB-T5E exhibited comparable targeting efficiency to SpG Cas9 while showing reduced chromosome translocation effects in human cells. Furthermore, by fusing cytosine or adenosine deaminase with enIscB nickase, we generated miniature IscB-derived base editors (miBEs), exhibiting robust editing efficiency (up to 92%) to induce DNA base conversions. Overall, our work establishes enIscB-T5E and miBEs as versatile tools for genome editing.
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Sistemas CRISPR-Cas , Desoxirribonucleasa I , Animales , Humanos , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Edición Génica , Citosina , ARN/genética , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
As the evolutionary ancestor of Cas9 nuclease, IscB proteins serve as compact RNA-guided DNA endonucleases and nickases, making them strong candidates for base editing. Nevertheless, the narrow targeting scope limits the application of IscB systems; thus, it is necessary to find more IscBs that recognize different target-adjacent motifs (TAMs). Here, we identified 10 of 19 uncharacterized IscB proteins from uncultured microbes with activity in mammalian cells. Through protein and ωRNA engineering, we further enhanced the activity of IscB ortholog IscB.m16 and expanded its TAM scope from MRNRAA to NNNGNA, resulting in a variant named IscB.m16*. By fusing the deaminase domains with IscB.m16* nickase, we generated IscB.m16*-derived base editors that exhibited robust base-editing efficiency in mammalian cells and effectively restored Duchenne muscular dystrophy proteins in diseased mice through single adeno-associated virus delivery. Thus, this study establishes a set of compact base-editing tools for basic research and therapeutic applications.
RESUMEN
During development, patterned neural activity instructs topographic map refinement. Axons with similar patterns of neural activity converge onto target neurons and stabilize their synapses with these postsynaptic partners, restricting exploratory branch elaboration (Hebbian structural plasticity). On the other hand, non-correlated firing in inputs leads to synapse weakening and increased exploratory growth of axons (Stentian structural plasticity). We used visual stimulation to control the correlation structure of neural activity in a few ipsilaterally projecting (ipsi) retinal ganglion cell (RGC) axons with respect to the majority contralateral eye inputs in the optic tectum of albino Xenopus laevis tadpoles. Multiphoton live imaging of ipsi axons, combined with specific targeted disruptions of brain-derived neurotrophic factor (BDNF) signaling, revealed that both presynaptic p75NTR and TrkB are required for Stentian axonal branch addition, whereas presumptive postsynaptic BDNF signaling is necessary for Hebbian axon stabilization. Additionally, we found that BDNF signaling mediates local suppression of branch elimination in response to correlated firing of inputs. Daily in vivo imaging of contralateral RGC axons demonstrated that p75NTR knockdown reduces axon branch elongation and arbor spanning field volume.
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Factor Neurotrófico Derivado del Encéfalo , Dendritas , Factor Neurotrófico Derivado del Encéfalo/fisiología , Dendritas/fisiología , Células Ganglionares de la Retina/fisiología , Axones/fisiología , Sinapsis/fisiologíaRESUMEN
BACKGROUND: The tumor suppressor and proapoptotic transcription factor P53 is induced (and activated) in several forms of heart failure, including cardiotoxicity and dilated cardiomyopathy; however, the precise mechanism that coordinates its induction with accessibility to its transcriptional promoter sites remains unresolved, especially in the setting of mature terminally differentiated (nonreplicative) cardiomyocytes. METHODS: Male and female control or TRIM35 (tripartite motif containing 35) overexpression adolescent (aged 1-3 months) and adult (aged 4-6 months) transgenic mice were used for all in vivo experiments. Primary adolescent or adult mouse cardiomyocytes were isolated from control or TRIM35 overexpression transgenic mice for all in vitro experiments. Adenovirus or small-interfering RNA was used for all molecular experiments to overexpress or knockdown, respectively, target genes in primary mouse cardiomyocytes. Patient dilated cardiomyopathy or nonfailing left ventricle samples were used for translational and mechanistic insight. Chromatin immunoprecipitation and DNA sequencing or quantitative real-time polymerase chain reaction (qPCR) was used to assess P53 binding to its transcriptional promoter targets, and RNA sequencing was used to identify disease-specific signaling pathways. RESULTS: Here, we show that E3-ubiquitin ligase TRIM35 can directly monoubiquitinate lysine-120 (K120) on histone 2B in postnatal mature cardiomyocytes. This epigenetic modification was sufficient to promote chromatin remodeling, accessibility of P53 to its transcriptional promoter targets, and elongation of its transcribed mRNA. We found that increased P53 transcriptional activity (in cardiomyocyte-specific Trim35 overexpression transgenic mice) was sufficient to initiate heart failure and these molecular findings were recapitulated in nonischemic human LV dilated cardiomyopathy samples. CONCLUSIONS: These findings suggest that TRIM35 and the K120Ub-histone 2B epigenetic modification are molecular features of cardiomyocytes that can collectively predict dilated cardiomyopathy pathogenesis.
Asunto(s)
Insuficiencia Cardíaca , Histonas , Ratones Transgénicos , Miocitos Cardíacos , Proteína p53 Supresora de Tumor , Ubiquitinación , Animales , Femenino , Humanos , Masculino , Ratones , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Células Cultivadas , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Histonas/metabolismo , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Regiones Promotoras Genéticas , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
It has been speculated that brain activities might directly control adaptive immune responses in lymphoid organs, although there is little evidence for this. Here we show that splenic denervation in mice specifically compromises the formation of plasma cells during a T cell-dependent but not T cell-independent immune response. Splenic nerve activity enhances plasma cell production in a manner that requires B-cell responsiveness to acetylcholine mediated by the α9 nicotinic receptor, and T cells that express choline acetyl transferase1,2 probably act as a relay between the noradrenergic nerve and acetylcholine-responding B cells. We show that neurons in the central nucleus of the amygdala (CeA) and the paraventricular nucleus (PVN) that express corticotropin-releasing hormone (CRH) are connected to the splenic nerve; ablation or pharmacogenetic inhibition of these neurons reduces plasma cell formation, whereas pharmacogenetic activation of these neurons increases plasma cell abundance after immunization. In a newly developed behaviour regimen, mice are made to stand on an elevated platform, leading to activation of CeA and PVN CRH neurons and increased plasma cell formation. In immunized mice, the elevated platform regimen induces an increase in antigen-specific IgG antibodies in a manner that depends on CRH neurons in the CeA and PVN, an intact splenic nerve, and B cell expression of the α9 acetylcholine receptor. By identifying a specific brain-spleen neural connection that autonomically enhances humoral responses and demonstrating immune stimulation by a bodily behaviour, our study reveals brain control of adaptive immunity and suggests the possibility to enhance immunocompetency by behavioural intervention.
Asunto(s)
Conducta Animal/fisiología , Encéfalo/fisiología , Inmunidad Humoral/inmunología , Bazo/inmunología , Bazo/inervación , Acetilcolina/metabolismo , Acetilcolina/farmacología , Neuronas Adrenérgicas/metabolismo , Amígdala del Cerebelo/citología , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Colina O-Acetiltransferasa/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Hemocianinas/inmunología , Inmunoglobulina G/inmunología , Activación de Linfocitos , Masculino , Ratones , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/metabolismo , Células Plasmáticas/citología , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Receptores Nicotínicos/deficiencia , Receptores Nicotínicos/metabolismo , Bazo/citología , Bazo/efectos de los fármacos , Estrés Psicológico/inmunología , Estrés Psicológico/metabolismo , Linfocitos T/inmunologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.