RESUMEN
Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).
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Leucemia Mieloide Aguda , Proto-Oncogenes , Animales , Inversión Cromosómica , Cromosomas Humanos Par 3/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Leucemia Mieloide Aguda/patología , Proteína del Locus del Complejo MDS1 y EV11/genética , Ratones , Proto-Oncogenes/genética , Factores de Transcripción/metabolismoRESUMEN
High-affinity memory B cells are preferentially selected during secondary responses and rapidly differentiate into antibody-producing cells. However, it remains unknown whether only high-affinity, mutated memory B cells simply expand to dominate the secondary response or if in fact memory B cells with a diverse VH repertoire, including those with no mutations, accumulate somatic mutations to create a new repertoire through the process of affinity maturation. In this report, we took a new approach to address this question by analyzing the VH gene repertoire of IgG1(+) memory B cells before and after antigen re-exposure in a host unable to generate IgG(+) B cells. We show here that both mutated and unmutated IgG1(+) memory B cells respond to secondary challenge and expand while accumulating somatic mutations in their VH genes in a stepwise manner. Both types of memory cells subsequently established a VH gene repertoire dominated by two major clonotypes, which are distinct from the original repertoire before antigen re-exposure. In addition, heavily mutated memory B cells were excluded from the secondary repertoire. Thus, both mutated and unmutated IgG1(+) memory cells equally contribute to establish a new antibody repertoire through a dynamic process of mutation and selection, becoming optimally adapted to the recall challenge.
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Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Memoria Inmunológica , Mutación , Traslado Adoptivo , Animales , Células Productoras de Anticuerpos/inmunología , Células Productoras de Anticuerpos/metabolismo , Antígenos/inmunología , Linfocitos B/citología , Diferenciación Celular/inmunología , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND: Cell- or tissue-based regenerative therapy is an attractive approach to treat heart failure. A tissue patch that can safely and effectively repair damaged heart muscle would greatly improve outcomes for patients with heart failure. In this study, we conducted a preclinical proof-of-concept analysis of the efficacy and safety of clinical-grade human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches. METHODS: A clinical-grade hiPSC line was established using peripheral blood mononuclear cells from a healthy volunteer that was homozygous for human leukocyte antigens. The hiPSCs were differentiated into cardiomyocytes. The obtained hiPSC-CMs were cultured on temperature-responsive culture dishes for patch fabrication. The cellular characteristics, safety, and efficacy of hiPSCs, hiPSC-CMs, and hiPSC-CM patches were analyzed. RESULTS: The hiPSC-CMs expressed cardiomyocyte-specific genes and proteins, and electrophysiological analyses revealed that hiPSC-CMs exhibit similar properties to human primary myocardial cells. In vitro and in vivo safety studies indicated that tumorigenic cells were absent. Moreover, whole-genome and exome sequencing revealed no genomic mutations. General toxicity tests also showed no adverse events posttransplantation. A porcine model of myocardial infarction demonstrated significantly improved cardiac function and angiogenesis in response to cytokine secretion from hiPSC-CM patches. No lethal arrhythmias were observed. CONCLUSIONS: hiPSC-CM patches are promising for future translational research and may have clinical application potential for the treatment of heart failure.
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Insuficiencia Cardíaca , Células Madre Pluripotentes Inducidas , Humanos , Animales , Porcinos , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares , Miocardio , Insuficiencia Cardíaca/terapiaRESUMEN
Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming.
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Reprogramación Celular , Histonas , Células Madre Pluripotentes Inducidas , Factor 4 Similar a Kruppel , Reprogramación Celular/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Histonas/metabolismo , Diferenciación Celular/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB1/genética , Cromatina/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , TranscriptomaRESUMEN
SETBP1 is a potential epigenetic regulator whose hotspot mutations preventing proteasomal degradation are recurrently detected in myeloid malignancies with poor prognosis. It is believed that the mutant SETBP1 exerts amplified effects of wild-type SETBP1 rather than neomorphic functions. This indicates that dysregulated quantitative control of SETBP1 would result in the transformation of hematopoietic cells. However, little is known about the roles of endogenous SETBP1 in malignant and normal hematopoiesis. Thus, we integrated the analyses of primary AML and healthy samples, cancer cell lines, and a newly generated murine model, Vav1-iCre;Setbp1fl/fl. Despite the expression in long-term hematopoietic stem cells, SETBP1 depletion in normal hematopoiesis minimally alters self-renewal, differentiation, or reconstitution in vivo. Indeed, its loss does not profoundly alter transcription or chromatin accessibilities. Furthermore, although AML with high SETBP1 mRNA is associated with genetic and clinical characteristics for dismal outcomes, SETBP1 is dispensable for the development or maintenance of AML. Contrary to the evidence that SETBP1 mutations are restricted to myeloid malignancies, dependency on SETBP1 mRNA expression is not observed in AML. These unexpected results shed light on the unrecognized idea that a physiologically nonessential gene can act as an oncogene when the machinery of protein degradation is damaged.
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Hematopoyesis , Leucemia Mieloide Aguda , Animales , Humanos , Ratones , Proteínas Portadoras/genética , Diferenciación Celular , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/patología , Mutación , Proteínas Nucleares/genéticaRESUMEN
ATP-dependent chromatin remodeling SWI/SNF complexes exist in three subcomplexes: canonical BAF (cBAF), polybromo BAF (PBAF), and a newly described non-canonical BAF (ncBAF). While cBAF and PBAF regulate fates of multiple cell types, roles for ncBAF in hematopoietic stem cells (HSCs) have not been investigated. Motivated by recent discovery of disrupted expression of BRD9, an essential component of ncBAF, in multiple cancers, including clonal hematopoietic disorders, we evaluate here the role of BRD9 in normal and malignant HSCs. BRD9 loss enhances chromatin accessibility, promoting myeloid lineage skewing while impairing B cell development. BRD9 significantly colocalizes with CTCF, whose chromatin recruitment is augmented by BRD9 loss, leading to altered chromatin state and expression of myeloid-related genes within intact topologically associating domains. These data uncover ncBAF as critical for cell fate specification in HSCs via three-dimensional regulation of gene expression and illuminate roles for ncBAF in normal and malignant hematopoiesis.
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Cromatina , Factores de Transcripción , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ensamble y Desensamble de Cromatina , Diferenciación Celular , Células Madre Hematopoyéticas/metabolismoRESUMEN
Cell therapy using induced pluripotent stem cell (iPSC) derivatives may result in abnormal tissue generation because the cells undergo numerous cycles of mitosis before clinical application, potentially increasing the accumulation of genetic abnormalities. Therefore, genetic tests may predict abnormal tissue formation after transplantation. Here, we administered iPSC derivatives with or without single-nucleotide variants (SNVs) and deletions in cancer-related genes with various genomic copy number variant (CNV) profiles into immunodeficient mice and examined the relationships between mutations and abnormal tissue formation after transplantation. No positive correlations were found between the presence of SNVs/deletions and the formation of abnormal tissues; the overall predictivity was 29%. However, a copy number higher than 3 was correlated, with an overall predictivity of 86%. Furthermore, we found CNV hotspots at 14q32.33 and 17q12 loci. Thus, CNV analysis may predict abnormal tissue formation after transplantation of iPSC derivatives and reduce the number of tumorigenicity tests.
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Células Madre Pluripotentes Inducidas , Animales , Pruebas de Carcinogenicidad , Reprogramación Celular , Variaciones en el Número de Copia de ADN , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Mutación , Polimorfismo de Nucleótido SimpleRESUMEN
Naive human induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with Sendai virus (SeV) vectors. However, only dermal fibroblasts have been successfully reprogrammed this way, and the process requires culture on feeder cells. Moreover, SeV vectors are highly persistent and inhibit subsequent differentiation of iPSCs. Here, we report a modified SeV vector system to generate transgene-free naive human iPSCs with superior differentiation potential. The modified method can be applied not only to fibroblasts but also to other somatic cell types. SeV vectors disappear quickly at early passages, and this approach enables the generation of naive iPSCs in a feeder-free culture. The naive iPSCs generated by this method show better differentiation to trilineage and extra-embryonic trophectoderm than those derived by conventional methods. This method can expand the application of iPSCs to research on early human development and regenerative medicine.
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Células Madre Pluripotentes Inducidas , Humanos , Reprogramación Celular/genética , Virus Sendai/genética , Vectores Genéticos , Diferenciación Celular/genéticaRESUMEN
In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.
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Mesenchymal stem cells (MSCs) can show trisomy 7; however, the safety of these cells has not been fully investigated. The purposes of this study were to determine the ratio of patients whose synovial MSCs were transplanted clinically, to intensively investigate MSCs with trisomy 7 from a safety perspective, and to follow up the patients for 5 years after transplantation. Synovial MSCs at passage 0 were transplanted into a knee for degenerative meniscus tears in 10 patients, and the patients were checked at 5 years. The synovial MSCs were evaluated at passages 0 to 15 by G-bands and digital karyotyping, and trisomy 7 was found in 3 of 10 patients. In those three patients, 5% to 10% of the synovial MSCs showed trisomy 7. The mRNA expressions of representative oncogenes and genes on chromosome 7 did not differ between MSCs with and without trisomy 7. Whole-genome sequencing and DNA methylation analysis showed similar results for MSCs with and without trisomy 7. Transplantation of human synovial MSCs with trisomy 7 into eight mouse knees did not result in tumor formation under the skin or in the knees after 8 weeks in any mouse, whereas transplanted HT1080 cells formed tumors. In vitro chondrogenic potentials were similar between MSCs with and without trisomy 7. Five-year follow-ups revealed no serious adverse events in all 10 human patients, including 3 who had received MSCs with trisomy 7. Overall, our findings indicated that synovial MSCs with trisomy 7 were comparable with MSCs without trisomy 7 from a safety perspective.
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Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Animales , Estudios de Seguimiento , Humanos , Articulación de la Rodilla , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/patología , Ratones , Membrana Sinovial , Trasplante Autólogo , Trisomía/genética , Trisomía/patologíaRESUMEN
Induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons are an expected source for cell-based therapies for Parkinson's disease (PD). The regulatory criteria for the clinical application of these therapies, however, have not been established. Here we show the results of our pre-clinical study, in which we evaluate the safety and efficacy of dopaminergic progenitors (DAPs) derived from a clinical-grade human iPSC line. We confirm the characteristics of DAPs by in vitro analyses. We also verify that the DAP population include no residual undifferentiated iPSCs or early neural stem cells and have no genetic aberration in cancer-related genes. Furthermore, in vivo studies using immunodeficient mice reveal no tumorigenicity or toxicity of the cells. When the DAPs are transplanted into the striatum of 6-OHDA-lesioned rats, the animals show behavioral improvement. Based on these results, we started a clinical trial to treat PD patients in 2018.
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Neuronas Dopaminérgicas/citología , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/trasplante , Enfermedad de Parkinson/terapia , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular/genética , Línea Celular , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Macaca fascicularis , Masculino , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Evaluación de Resultado en la Atención de Salud/métodos , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Ratas Desnudas , Trasplante HeterólogoRESUMEN
Immune attacks are key issues for cell transplantation. To assess the safety and the immune reactions after iPS cells-derived retinal pigment epithelium (iPS-RPE) transplantation, we transplanted HLA homozygote iPS-RPE cells established at an iPS bank in HLA-matched patients with exudative age-related macular degeneration. In addition, local steroids without immunosuppressive medications were administered. We monitored immune rejections by routine ocular examinations as well as by lymphocytes-graft cells immune reaction (LGIR) tests using graft RPE and the patient's blood cells. In all five of the cases that underwent iPS-RPE transplantation, the presence of graft cells was indicated by clumps or an area of increased pigmentation at 6 months, which became stable with no further abnormal growth in the graft during the 1-year observation period. Adverse events observed included corneal erosion, epiretinal membrane, retinal edema due to epiretinal membrane, elevated intraocular pressure, endophthalmitis, and mild immune rejection in the eye. In the one case exhibiting positive LGIR tests along with a slight fluid recurrence, we administrated local steroid therapy that subsequently resolved the suspected immune attacks. Although the cell delivery strategy must be further optimized, the present results suggest that it is possible to achieve stable survival and safety of iPS-RPE cell transplantation for a year.
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Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M gene knockout and HLA-homozygous iPSC stocks can address this issue, but the former approach may induce NK cell activity and fail to present antigens, and it is challenging to recruit rare donors for the latter method. Here, we show two genome-editing strategies for making immunocompatible donor iPSCs. First, we generated HLA pseudo-homozygous iPSCs with allele-specific editing of HLA heterozygous iPSCs. Second, we generated HLA-C-retained iPSCs by disrupting both HLA-A and -B alleles to suppress the NK cell response while maintaining antigen presentation. HLA-C-retained iPSCs could evade T cells and NK cells in vitro and in vivo. We estimated that 12 lines of HLA-C-retained iPSCs combined with HLA-class II knockout are immunologically compatible with >90% of the world's population, greatly facilitating iPSC-based regenerative medicine applications.
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Sistemas CRISPR-Cas/genética , Edición Génica , Antígenos HLA/genética , Histocompatibilidad/inmunología , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Línea Celular , Femenino , Antígenos HLA/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos NODRESUMEN
Steroid receptors are evoluted from the common ancestor and have the same structure as steroid receptor superfamily. The nuclear receptors are characterized by a central DNA binding domain with specific DNA sequences known as hormone response element, ligand binding domain in the C terminal and N terminal domain with the tissue specificity. Nuclear receptor superfamily is divided into four classes based on the dimerization and DNA binding properties, homodimers as steroid hormone receptor, heterodimers with RXR, homodimers and monomers. Most of orphan receptors fall into the last 2 classes. The coregulators as coactivators and corepressor are determined. The mechanism of action of nuclear receptor on chromatin in the nucleus are clarified precisely from the stand point of the clasters of coregulators and the complex of nuclear receptor and coregulators. The crosstalk of nuclear receptor and peptide hormone signal in the cell is important in the autocrine and paracrine action. Non-genomic action of steroid hormone is also proposed.
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Receptores de Esteroides/clasificación , Receptores de Esteroides/fisiología , Receptor Cross-Talk/fisiología , Receptores Citoplasmáticos y Nucleares/clasificaciónRESUMEN
Here, we report that MYC rescues early human cells undergoing reprogramming from a proliferation pause induced by OCT3/4, SOX2, and KLF4 (OSK). We identified ESRG as a marker of early reprogramming cells that is expressed as early as day 3 after OSK induction. On day 4, ESRG positive (+) cells converted to a TRA-1-60 (+) intermediate state. These early ESRG (+) or TRA-1-60 (+) cells showed a proliferation pause due to increased p16INK4A and p21 and decreased endogenous MYC caused by OSK. Exogenous MYC did not enhance the appearance of initial reprogramming cells but instead reactivated their proliferation and improved reprogramming efficiency. MYC increased expression of LIN41, which potently suppressed p21 post-transcriptionally. MYC suppressed p16 INK4A. These changes inactivated retinoblastoma protein (RB) and reactivated proliferation. The RB-regulated proliferation pause does not occur in immortalized fibroblasts, leading to high reprogramming efficiency even without exogenous MYC.
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Reprogramación Celular , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína de Retinoblastoma/metabolismo , Antígenos de Superficie/metabolismo , Línea Celular , Proliferación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Fosforilación , Proteoglicanos/metabolismo , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína de Retinoblastoma/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
In order to study the biosynthesis of agatharesinol, a norlignan, l-phenylalanine-[ring-2,3,4,5,6-2H] and trans-cinnamic acid-[ring-13C6] were administered to fresh sapwood sticks of Cryptomeria japonica (sugi, Japanese cedar), that is, the labeled precursors were allowed to be absorbed through the tangential section of the wood sticks. The wood sticks were then maintained in high humidity desiccators for approximately 20 d after which ethyl acetate (EtOAc) extracts of the wood sticks were analyzed by gas chromatography-mass spectrometry (GC-MS). Native agatharesinol (trimethylsilylated) produces an m/z 369 ion and an m/z 484 ion that are characteristic of its structure. Agatharesinol formed in the sapwood sticks treated with the deuterium-labeled l-phenylalanine generated both of these ions together with m/z 373 and 377 ions (m/z 369+4 and +8, respectively), and also m/z 488 and 492 ions (m/z 484+4 and +8, respectively). Generation of m/z 373 and 488 ions is attributed to the substitution by deuterium of the four hydrogen atoms of either of the p-hydroxyphenyl rings of agatharesinol, and that of m/z 377 and 492 ions is attributed to the substitution by deuterium of the eight hydrogen atoms of both p-hydroxyphenyl rings. In the administration of the 13C-labeled trans-cinnamic acid, m/z 375 and 381 ions (m/z 369+6 and +12, respectively), and also m/z 490 and 496 ions (m/z 484+6 and +12, respectively) were found, indicating that either aromatic ring or both aromatic rings of agatharesinol were 13C-labeled. Consequently, assimilation of the labeled precursors into agatharesinol was clearly detected, and an experimental procedure for studies on the biosynthesis was developed.
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Cryptomeria/metabolismo , Lignanos/biosíntesis , Isótopos de Carbono , Cinamatos/metabolismo , Deuterio , Cromatografía de Gases y Espectrometría de Masas , Humedad , Lignanos/química , Fenilalanina/química , Fenilalanina/metabolismoRESUMEN
The biosynthetic relationship between the two norlignans agatharesinol and trans-hinokiresinol was investigated. Fresh sapwood sticks of Cryptomeria japonica were fed with stable isotope-labeled compounds, namely p-coumaryl alcohol-[9,9-(2)H], p-coumaryl alcohol-[9-(18)O] and trans-hinokiresinol-[1-(2)H], and then incubated under high-humidity for approximately 20 days, during which the two norlignans were produced simultaneously. While trans-hinokiresinol was strongly deuterium-labeled after feeding with p-coumaryl alcohol-[9,9-(2)H], agatharesinol was only lightly labeled after feeding with either p-coumaryl alcohol-[9,9-(2)H] or -[9-(18)O]. These results suggest that p-coumaryl alcohol, which is a precursor of hinokiresinol, is not involved in the biosynthesis of agatharesinol. Therefore, the norlignan carbon skeleton of agatharesinol must be framed from different types of phenylpropanoid monomers compared to those utilized by the trans-hinokiresinol pathway. The biosynthesis of these two norlignans seems to branch at an early stage, i.e., before the framing of the norlignan carbon skeleton. Furthermore, agatharesinol was not labeled with deuterium after feeding with (2)H-labeled trans-hinokiresinol, which has the simplest norlignan structure. This result strongly supports the suggestion that the conversion of trans-hinokiresinol to agatharesinol is not part of the biosynthesis of norlignans and that early branching occurs instead.
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Cryptomeria/metabolismo , Lignanos/biosíntesis , Ácidos Cumáricos , Deuterio/metabolismo , Humedad , Isótopos de Oxígeno/metabolismo , Fenoles , Tallos de la Planta/metabolismo , Propionatos/metabolismoRESUMEN
The most typical and well known inhibitory action in the cortical microcircuit is a strong inhibition on the target neuron by axo-somatic synapses. However, it has become clear that synaptic inhibition in the cortex is much more diverse and complicated. Firstly, at least ten or more inhibitory non-pyramidal cell subtypes engage in diverse inhibitory functions to produce the elaborate activity characteristic of the different cortical states. Each distinct non-pyramidal cell subtype has its own independent inhibitory function. Secondly, the inhibitory synapses innervate different neuronal domains, such as axons, spines, dendrites and soma, and their inhibitory postsynaptic potential (IPSP) size is not uniform. Thus, cortical inhibition is highly complex, with a wide variety of anatomical and physiological modes. Moreover, the functional significance of the various inhibitory synapse innervation styles and their unique structural dynamic behaviors differ from those of excitatory synapses. In this review, we summarize our current understanding of the inhibitory mechanisms of the cortical microcircuit.
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Corteza Cerebral/citología , Inhibición Neural/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Humanos , Modelos Biológicos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Variation in the differentiation capacity of induced pluripotent stem cells (iPSCs) to specific lineages is a significant concern for their use in clinical applications and disease modeling. To identify factors that affect differentiation capacity, we performed integration analyses between hematopoietic differentiation performance and molecular signatures such as gene expression, DNA methylation, and chromatin status, using 35 human iPSC lines and four ESC lines. Our analyses revealed that hematopoietic commitment of PSCs to hematopoietic precursors correlates with IGF2 expression level, which in turn depends on signaling-dependent chromatin accessibility at mesendodermal genes. Maturation capacity for conversion of PSC-derived hematopoietic precursors to mature blood associates with the amount and pattern of DNA methylation acquired during reprogramming. Our study therefore provides insight into the molecular features that determine the differential capacities seen among human iPSC lines and, through the predictive potential of this information, highlights a way to select optimal iPSCs for clinical applications.
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Diferenciación Celular/genética , Epigénesis Genética , Células Madre Pluripotentes Inducidas/metabolismo , Activinas/metabolismo , Animales , Secuencia de Bases , Línea Celular , Linaje de la Célula/genética , Reprogramación Celular/genética , Cromatina/química , Metilación de ADN/genética , Células Eritroides/citología , Células Eritroides/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Redes Reguladoras de Genes , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Factor II del Crecimiento Similar a la Insulina/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Ratones SCID , Transducción de Señal/genética , Trasplante de Células Madre , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismoRESUMEN
Inhibitory interneurons target precise membrane regions on pyramidal cells, but differences in their functional effects on somata, dendrites and spines remain unclear. We analyzed inhibitory synaptic events induced by cortical, fast-spiking (FS) basket cells which innervate dendritic shafts and spines as well as pyramidal cell somata. Serial electron micrograph (EMg) reconstructions showed that somatic synapses were larger than dendritic contacts. Simulations with precise anatomical and physiological data reveal functional differences between different innervation styles. FS cell soma-targeting synapses initiate a strong, global inhibition, those on shafts inhibit more restricted dendritic zones, while synapses on spines may mediate a strictly local veto. Thus, FS cell synapses of different sizes and sites provide functionally diverse forms of pyramidal cell inhibition.