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1.
EMBO J ; 43(3): 339-361, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38238476

RESUMO

Hematopoietic stem cell (HSC) divisional fate and function are determined by cellular metabolism, yet the contribution of specific cellular organelles and metabolic pathways to blood maintenance and stress-induced responses in the bone marrow remains poorly understood. The outer mitochondrial membrane-localized E3 ubiquitin ligase MITOL/MARCHF5 (encoded by the Mitol gene) is known to regulate mitochondrial and endoplasmic reticulum (ER) interaction and to promote cell survival. Here, we investigated the functional involvement of MITOL in HSC maintenance by generating MX1-cre inducible Mitol knockout mice. MITOL deletion in the bone marrow resulted in HSC exhaustion and impairment of bone marrow reconstitution capability in vivo. Interestingly, MITOL loss did not induce major mitochondrial dysfunction in hematopoietic stem and progenitor cells. In contrast, MITOL deletion induced prolonged ER stress in HSCs, which triggered cellular apoptosis regulated by IRE1α. In line, dampening of ER stress signaling by IRE1α inihibitor KIRA6 partially rescued apoptosis of long-term-reconstituting HSC. In summary, our observations indicate that MITOL is a principal regulator of hematopoietic homeostasis and protects blood stem cells from cell death through its function in ER stress signaling.


Assuntos
Endorribonucleases , Proteínas Serina-Treonina Quinases , Animais , Camundongos , Apoptose , Células-Tronco Hematopoéticas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
2.
EMBO J ; 41(5): e108899, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35132656

RESUMO

The mechanochemical coupling of ATPase hydrolysis and conformational dynamics in kinesin motors facilitates intramolecular interaction cycles between the kinesin motor and neck domains, which are essential for microtubule-based motility. Here, we characterized a charge-inverting KIF1A-E239K mutant that we identified in a family with axonal-type Charcot-Marie-Tooth disease and also in 24 cases in human neuropathies including spastic paraplegia and hereditary sensory and autonomic neuropathy. We show that Glu239 in the ß7 strand is a key residue of the motor domain that regulates the motor-neck interaction. Expression of the KIF1A-E239K mutation has decreased ability to complement Kif1a+/- neurons, and significantly decreases ATPase activity and microtubule gliding velocity. X-ray crystallography shows that this mutation causes an excess positive charge on ß7, which may electrostatically interact with a negative charge on the neck. Quantitative mass spectrometric analysis supports that the mutation hyper-stabilizes the motor-neck interaction at the late ATP hydrolysis stage. Thus, the negative charge of Glu239 dynamically regulates the kinesin motor-neck interaction, promoting release of the neck from the motor domain upon ATP hydrolysis.


Assuntos
Adenosina Trifosfatases/genética , Cinesinas/genética , Mutação/genética , Neurônios/fisiologia , Idoso , Sequência de Aminoácidos , Axônios/fisiologia , Doença de Charcot-Marie-Tooth , Humanos , Masculino , Microtúbulos/genética , Pessoa de Meia-Idade , Alinhamento de Sequência
3.
EMBO Rep ; 2024 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-39322740

RESUMO

Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells is a principal mechanism for systemic glucose homeostasis, of which regulatory mechanisms are still unclear. Here we show that kinesin molecular motor KIF5B is essential for GSIS through maintaining the voltage-gated calcium channel CaV1.2 levels, by facilitating an Hsp70-to-Hsp90 chaperone exchange to pass through the quality control in the endoplasmic reticulum (ER). Phenotypic analyses of KIF5B conditional knockout (cKO) mouse beta cells revealed significant abolishment of glucose-stimulated calcium transients, which altered the behaviors of insulin granules via abnormally stabilized cortical F-actin. KIF5B and Hsp90 colocalize to microdroplets on ER sheets, where CaV1.2 but not Kir6.2 is accumulated. In the absence of KIF5B, CaV1.2 fails to be transferred from Hsp70 to Hsp90 via STIP1, and is likely degraded via the proteasomal pathway. KIF5B and Hsc70 overexpression increased CaV1.2 expression via enhancing its chaperone binding. Thus, ER sheets may serve as the place of KIF5B- and Hsp90-dependent chaperone exchange, which predominantly facilitates CaV1.2 production in beta cells and properly enterprises GSIS against diabetes.

4.
EMBO J ; 39(1): e101090, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31746486

RESUMO

The transport of N-methyl-d-aspartate receptors (NMDARs) is crucial for neuronal plasticity and synapse formation. Here, we show that KIF3B, a member of the kinesin superfamily proteins (KIFs), supports the transport of vesicles simultaneously containing NMDAR subunit 2A (NR2A) and the adenomatous polyposis coli (APC) complex. Kif3b+/- neurons exhibited a reduction in dendritic levels of both NR2A and NR2B due to the impaired transport of NR2A and increased degradation of NR2B. In Kif3b+/- hippocampal slices, electrophysiological NMDAR response was found decreased and synaptic plasticity was disrupted, which corresponded to a common feature of schizophrenia (SCZ). The histological features of Kif3b+/- mouse brain also mimicked SCZ features, and Kif3b+/- mice exhibited behavioral defects in prepulse inhibition (PPI), social interest, and cognitive flexibility. Indeed, a mutation of KIF3B was specifically identified in human SCZ patients, which was revealed to be functionally defective in a rescue experiment. Therefore, we propose that KIF3B transports NR2A/APC complex and that its dysfunction is responsible for SCZ pathogenesis.


Assuntos
Cinesinas/genética , Cinesinas/fisiologia , Mutação , Neurônios/patologia , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/etiologia , Sinapses/patologia , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Comportamento Animal , Movimento Celular , Humanos , Relações Interpessoais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Fenótipo , Subunidades Proteicas , Transporte Proteico , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Sinapses/metabolismo
5.
J Org Chem ; 2024 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-39436826

RESUMO

Cage-shaped borates, whose Lewis acidity can be precisely modulated by the structural attributes of the triphenolic ligands, were employed as catalysts for glycosylation. Each cage-shaped borate displayed distinctive reactivity; thus, screening of the borate catalysts enabled controllable activation of glycosyl fluorides under mild conditions. Practical glycosylation was achieved by fine-tuning the Lewis acidity tailored to the substrate reactivity, thereby providing a versatile method applicable to the synthesis of complex glycans.

6.
Photochem Photobiol Sci ; 23(7): 1417-1423, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38703275

RESUMO

Double difunctionalization of a vinyl ether tethered hydroxy or carbamoyl group with electron-deficient alkenes such as acrylonitrile or acrylic esters was achieved by visible-light irradiation in a two-molecule photoredox system. Use of anhydrous acetonitrile solution as a solvent promoted both dimerization of the radical cation of electron-rich alkene with electron-rich alkene and intramolecular nucleophilic addition to generate an electron-rich radical that was added to electron-deficient alkene to furnish the double difunctionalized product. A variety of electronically differentiated rich and deficient alkenes were used in the photoreaction; a simple construction of a complex carbon framework containing acetal from simple alkenes was successful under mild conditions.

7.
BMC Pulm Med ; 24(1): 90, 2024 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-38368315

RESUMO

BACKGROUND: The data on bosentan were lacking for the treatment of exercise-induced elevation of pulmonary artery pressure (eePAP) or less severe PH in COPD. This study was conducted to investigate long-term efficacy and safety of bosentan for the treatment of eePAP or less severe PH in COPD. METHODS: COPD patients diagnosed at this hospital as having COPD (WHO functional class II, III or IV) with eePAP or less severe PH whose respiratory symptoms were stable but remained and gradually progressed even after COPD therapy were randomly assigned in a 1:1 ratio to receive either bosentan or no PH treatment for two years and assessed at baseline and every 6 months for respiratory failure, activities of daily living (ADL), lung and heart functions by right heart catheterization (RHC), and other parameters. RESULTS: A total of 29 patients who underwent RHC for detail examination were enrolled in the current study between August 2010 and October 2018.No death occurred in drug-treated group (n = 14) for 2 years; 5 patients died in untreated group (n = 15). Significant differences were noted between the 2 group in hospital-free survival (686.00 ± 55.87 days vs. 499.94 ± 53.27 days; hazard ratio [HR], 0.18; P = 0.026) and overall survival (727 days vs. 516.36 ± 55.38 days; HR, 0.095; P = 0.030) in all causes of death analysis, but not in overall survival in analysis of respiratory-related death. Bosentan was not associated with increased adverse events including requiring O2 inhalation. CONCLUSIONS: This study suggested that the prognosis for COPD patients with eePAP or less severe PH presenting with respiratory symptoms was very poor and that bosentan tended to improve their prognosis and suppress ADL deterioration without worsening respiratory failure. TRIAL REGISTRATION: This study was registered with UMIN-CTR Clinical Trial as UMIN000004749 . First trial registration at 18/12/2010.


Assuntos
Hipertensão Pulmonar , Doença Pulmonar Obstrutiva Crônica , Insuficiência Respiratória , Humanos , Bosentana/uso terapêutico , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/complicações , Artéria Pulmonar , Atividades Cotidianas , Estudos Prospectivos , Antagonistas dos Receptores de Endotelina/uso terapêutico , Sulfonamidas , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Insuficiência Respiratória/complicações , Progressão da Doença , Anti-Hipertensivos/uso terapêutico , Resultado do Tratamento
8.
Gastroenterology ; 162(3): 799-812, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34687740

RESUMO

BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Genes MHC Classe I/genética , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Microglobulina beta-2/genética , Alelos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Metilação de DNA , Epigênese Genética , Expressão Gênica , Antígenos HLA-A/genética , Antígenos HLA-A/metabolismo , Humanos , Imunogenética , Linfócitos do Interstício Tumoral , Instabilidade de Microssatélites , Complexo de Endopeptidases do Proteassoma/genética , Fatores de Transcrição de Fator Regulador X/genética , Taxa de Sobrevida , Microglobulina beta-2/metabolismo
9.
J Asthma ; 60(4): 769-783, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-35759776

RESUMO

Objective: Some of the most common causes of chronic cough include cough variant asthma (CVA), bronchial asthma (BA), and asthma-COPD overlap (ACO). Although there is some overlap in the etiology of these diseases, it is clinically important to attempt an early differential diagnosis due to treatment strategies and prognoses.Methods: Spirometry and impulse oscillometry (IOS) before and after bronchodilator inhalation were analyzed for clinically diagnosed CVA (cCVA, n = 203), BA (cBA, n = 222), and ACO (cACO, n = 61).Results: A significant difference in ΔFEV1 was observed between cBA and cCVA (ΔFEV1 improvement of 122.5 mL/5.4% and 65.7 mL/2.2%, respectively), but no difference was observed in ΔPEF, ΔV50, or ΔV25. Except for R20 (resistance at 20 Hz), significant differences between the three groups were observed in IOS. In IOS, cCVA and cBA showed comparable peripheral airway response to bronchodilator which was thought to be commensurate with changes in V50 and V25. cACO improved ΔFEV1 improvement of 81.0 mL/6.2% and was distinguished by a downward respiratory system reactance (Xrs) waveform with a limited bronchodilator response. FEV1/FVC, %FEV1, and %V25 had relatively strong correlations with the three IOS parameters, X5 (reactance at 5 Hz), resonant frequency (Fres), and low-frequency reactance area (ALX), in the correlation between IOS and spirometers.Conclusion: Changes in IOS parameters were more sensitive in this study than changes in FEV1 or the flow-volume curve. Considering the benefits and relevance of the two different tests, simultaneous IOS and spirometry testing were useful in the diagnosis of asthmatic cough.


Assuntos
Asma , Humanos , Asma/diagnóstico , Asma/tratamento farmacológico , Broncodilatadores/farmacologia , Broncodilatadores/uso terapêutico , Tosse/diagnóstico , Tosse/tratamento farmacológico , Oscilometria , Sistema Respiratório , Espirometria , Suscetibilidade a Doenças , Volume Expiratório Forçado
10.
Nature ; 550(7676): 393-397, 2017 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-29019987

RESUMO

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.


Assuntos
Blastômeros/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Blastocisto/citologia , Blastômeros/metabolismo , Linhagem da Célula , Células Cultivadas , Quimera , Embrião de Mamíferos/citologia , Endoderma/citologia , Epigênese Genética , Epigenômica , Feminino , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Placenta/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Gravidez , Análise de Célula Única , Transcriptoma , Trofoblastos/citologia
11.
Rinsho Ketsueki ; 64(9): 1007-1018, 2023.
Artigo em Japonês | MEDLINE | ID: mdl-37793857

RESUMO

Chronic myeloid leukemia (CML) stem cells have been identified to promote CML relapse due to their quiescent cell cycle and tyrosine kinase inhibitor resistance. Therefore, their eradication is important for the cure of CML. We herein identified the quiescent CML stem cell fraction using a G0 marker that can visualize quiescent cells. Whole-transcriptome analysis of imatinib-resistant, quiescent CML stem cells revealed that NF-κB is activated via inflammatory signals in the same cells. The combination of imatinib and an inhibitor of this inflammatory signal (IRAK1/4 inhibitor) effectively eliminated CML stem cells and attenuated PD-L1 expression in CML stem cells. Furthermore, the combination of anti-PD-L1 antibody and imatinib effectively eliminated CML stem cells in the presence of T-cell immunity, indicating the importance of creating an environment in which T cells can attack CML stem cells. Thus, IRAK1/4 inhibitors exert two effects: blocking CML stem cell survival and proliferation signals by inhibiting NF-κB and blocking T cell immune evasion by reducing PD-L1 expression in CML stem cells. Collectively, their combination may be one of the attractive strategies for achieving a radical cure for CML. Discussions regarding the possibility of future medications seem warranted.


Assuntos
Antígeno B7-H1 , Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , NF-kappa B , Proteínas de Fusão bcr-abl , Apoptose , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Células-Tronco/metabolismo , Células-Tronco Neoplásicas , Resistencia a Medicamentos Antineoplásicos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Quinases Associadas a Receptores de Interleucina-1/farmacologia , Quinases Associadas a Receptores de Interleucina-1/uso terapêutico
12.
Cancer Sci ; 113(4): 1182-1194, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35133065

RESUMO

Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated phenomenon characterized by clonal expansion of blood cells harboring somatic mutations in hematopoietic genes, including DNMT3A, TET2, and ASXL1. Clinical evidence suggests that CHIP is highly prevalent and associated with poor prognosis in solid-tumor patients. However, whether blood cells with CHIP mutations play a causal role in promoting the development of solid tumors remained unclear. Using conditional knock-in mice that express CHIP-associated mutant Asxl1 (Asxl1-MT), we showed that expression of Asxl1-MT in T cells, but not in myeloid cells, promoted solid-tumor progression in syngeneic transplantation models. We also demonstrated that Asxl1-MT-expressing blood cells accelerated the development of spontaneous mammary tumors induced by MMTV-PyMT. Intratumor analysis of the mammary tumors revealed the reduced T-cell infiltration at tumor sites and programmed death receptor-1 (PD-1) upregulation in CD8+ T cells in MMTV-PyMT/Asxl1-MT mice. In addition, we found that Asxl1-MT induced T-cell dysregulation, including aberrant intrathymic T-cell development, decreased CD4/CD8 ratio, and naïve-memory imbalance in peripheral T cells. These results indicate that Asxl1-MT perturbs T-cell development and function, which contributes to creating a protumor microenvironment for solid tumors. Thus, our findings raise the possibility that ASXL1-mutated blood cells exacerbate solid-tumor progression in ASXL1-CHIP carriers.


Assuntos
Hematopoiese Clonal , Neoplasias , Proteínas Repressoras , Animais , Linfócitos T CD8-Positivos/metabolismo , Hematopoiese Clonal/genética , Hematopoese/genética , Camundongos , Mutação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Microambiente Tumoral
13.
Blood ; 136(14): 1670-1684, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32492700

RESUMO

Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knockin (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we used retrovirus-mediated insertional mutagenesis, which exhibited the susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified the hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT-expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and chromatin immunoprecipitation-next-generation sequencing analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicate that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.


Assuntos
Transformação Celular Neoplásica/genética , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Mutação , Células Mieloides/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Biomarcadores Tumorais , Biópsia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Ensaio de Unidades Formadoras de Colônias , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Estudos de Associação Genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Imunofenotipagem , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/mortalidade , Leucemia Mieloide/patologia , Camundongos , Células Mieloides/patologia , Prognóstico , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo
14.
Nature ; 535(7611): 289-293, 2016 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-27383781

RESUMO

In mammals, specification of the three major germ layers occurs during gastrulation, when cells ingressing through the primitive streak differentiate into the precursor cells of major organ systems. However, the molecular mechanisms underlying this process remain unclear, as numbers of gastrulating cells are very limited. In the mouse embryo at embryonic day 6.5, cells located at the junction between the extra-embryonic region and the epiblast on the posterior side of the embryo undergo an epithelial-to-mesenchymal transition and ingress through the primitive streak. Subsequently, cells migrate, either surrounding the prospective ectoderm contributing to the embryo proper, or into the extra-embryonic region to form the yolk sac, umbilical cord and placenta. Fate mapping has shown that mature tissues such as blood and heart originate from specific regions of the pre-gastrula epiblast, but the plasticity of cells within the embryo and the function of key cell-type-specific transcription factors remain unclear. Here we analyse 1,205 cells from the epiblast and nascent Flk1(+) mesoderm of gastrulating mouse embryos using single-cell RNA sequencing, representing the first transcriptome-wide in vivo view of early mesoderm formation during mammalian gastrulation. Additionally, using knockout mice, we study the function of Tal1, a key haematopoietic transcription factor, and demonstrate, contrary to previous studies performed using retrospective assays, that Tal1 knockout does not immediately bias precursor cells towards a cardiac fate.


Assuntos
Embrião de Mamíferos/citologia , Gastrulação/genética , Perfilação da Expressão Gênica , Mesoderma/citologia , Mesoderma/embriologia , Análise de Célula Única , Transcriptoma/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Linhagem da Célula/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Eritropoese/genética , Gástrula/citologia , Gástrula/metabolismo , Mesoderma/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/deficiência , Análise de Sequência de DNA , Proteína 1 de Leucemia Linfocítica Aguda de Células T
15.
Int J Mol Sci ; 23(14)2022 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-35887071

RESUMO

MYC is a major oncogene that plays an important role in cell proliferation in human cancers. Therefore, the mechanism behind MYC regulation is a viable therapeutic target for the treatment of cancer. Comprehensive and efficient screening of MYC regulators is needed, and we had previously established a promoter screening system using fluorescent proteins and the CRISPR library. For the efficient identification of candidate genes, a database was used, for which mRNA expression was correlated with MYC using datasets featuring "Similar" and "Not exactly similar" contexts. INTS14 and ERI2 were identified using datasets featuring the "Similar" context group, and INTS14 and ERI2 were capable of enhancing MYC promoter activity. In further database analysis of human cancers, a higher expression of MYC mRNA was observed in the INTS14 mRNA high-expressing prostate and liver cancers. The knockdown of INTS14 in prostate cell lines resulted in decreased MYC mRNA and protein expression and also induced G0/1 arrest. This study confirmed that CRISPR screening combined with context-matched database screening is effective in identifying genes that regulate the MYC promoter. This method can be applied to other genes and is expected to be useful in identifying the regulators of other proto-oncogenes.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias Hepáticas , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Masculino , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proto-Oncogenes , RNA Mensageiro/genética
16.
Rinsho Ketsueki ; 63(10): 1373-1378, 2022.
Artigo em Japonês | MEDLINE | ID: mdl-36351642

RESUMO

We here present a 33-year-old woman who was referred to our hospital with a complaint of back pain and was found to have elevated IgG and hypercalcemia, as well as osteolytic lesions of pelvis and spines. 18F-FDG-PET/CT scan revealed numerous uptakes in the bones. An examination of the bone marrow revealed increased plasma cells (10.2%). Despite clinical similarities to multiple myeloma, any evidence of plasma cell clonal proliferation, including serum M-protein and light chain restriction, was not found. A reexamination of the bone marrow with a biopsy revealed the proliferation of abnormal cells with chromogranin A and synaptophysin expression but no expression of hematopoietic and epithelial cell markers. Based on these results together with extra-adrenal lesions, a diagnosis of malignant paraganglioma was made. Malignant paraganglioma is known to frequently cause bone metastasis and skeletal related events, whose clinical manifestations are similar to those of multiple myeloma. Since patients with osteolytic lesions, hypercalcemia, and hypergammaglobulinemia are likely to be referred to hematologists, malignant paraganglioma should be considered as a differential diagnosis.


Assuntos
Hipercalcemia , Mieloma Múltiplo , Paraganglioma , Feminino , Humanos , Adulto , Mieloma Múltiplo/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Fluordesoxiglucose F18/metabolismo , Paraganglioma/diagnóstico
17.
Curr Issues Mol Biol ; 43(3): 2098-2110, 2021 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-34940119

RESUMO

Mass spawning in fish culture often brings about a marked variance in family size, which can cause a reduction in effective population sizes in seed production for stock enhancement. This study reports an example of combined pedigree information and gene expression phenotypes to understand differential family survival mechanisms in early stages of Pacific bluefin tuna, Thunnus orientalis, in a mass culture tank. Initially, parentage was determined using the partial mitochondrial DNA control region sequence and 11 microsatellite loci at 1, 10, 15, and 40 days post-hatch (DPH). A dramatic proportional change in the families was observed at around 15 DPH; therefore, transcriptome analysis was conducted for the 15 DPH larvae using a previously developed oligonucleotide microarray. This analysis successfully addressed the family-specific gene expression phenotypes with 5739 differentially expressed genes and highlighted the importance of expression levels of gastric-function-related genes at the developmental stage for subsequent survival. This strategy demonstrated herein can be broadly applicable to species of interest in aquaculture to comprehend the molecular mechanism of parental effects on offspring survival, which will contribute to the optimization of breeding technologies.


Assuntos
Peixes/genética , Expressão Gênica , Estudos de Associação Genética , Linhagem , Fenótipo , Animais , Aquicultura , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Patrimônio Genético , Masculino , Taxa de Sobrevida , Atum/genética
18.
Nat Rev Mol Cell Biol ; 10(10): 682-96, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19773780

RESUMO

Intracellular transport is fundamental for cellular function, survival and morphogenesis. Kinesin superfamily proteins (also known as KIFs) are important molecular motors that directionally transport various cargos, including membranous organelles, protein complexes and mRNAs. The mechanisms by which different kinesins recognize and bind to specific cargos, as well as how kinesins unload cargo and determine the direction of transport, have now been identified. Furthermore, recent molecular genetic experiments have uncovered important and unexpected roles for kinesins in the regulation of such physiological processes as higher brain function, tumour suppression and developmental patterning. These findings open exciting new areas of kinesin research.


Assuntos
Cinesinas/metabolismo , Cinesinas/fisiologia , Proteínas Motores Moleculares/metabolismo , Animais , Transporte Biológico/genética , Dineínas/genética , Dineínas/metabolismo , Humanos , Cinesinas/química , Cinesinas/classificação , Cinesinas/genética , Modelos Biológicos , Proteínas Motores Moleculares/genética , Organelas/genética , Organelas/metabolismo , Filogenia , Proteínas/metabolismo , RNA Mensageiro/metabolismo
19.
BMC Infect Dis ; 21(1): 263, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33722204

RESUMO

BACKGROUND: The Pandemic of coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has critically impacted the spread of infection within nursing facilities. We evaluated the usefulness of genetic and serological tests conducted during a COVID-19 outbreak in a nursing facility in Japan. METHODS: After the first identification of SARS-CoV-2 infection, a comprehensive, facility- and/or unit-wide PCR testing from nasopharyngeal swabs was repeatedly performed in a three-unit facility including 99 residents with dementia and 53 healthcare personnel. Additionally, PCR testing was conducted separately for residents and staff with fever of ≥37.5 °C. Facility-wide serological testing, including rapid kit testing and quantitative assay, was conducted twice over 1 month apart. RESULTS: A total of 322 PCR and 257 antibody tests were performed. 37 (24.3%) of the 152 individuals (25/99 residents, 25.3%; 12/53 staff, 22.6%) were identified as PCR-positive. Seven residents died with a mortality of 7.1% (7/99). Among the 37 individuals, 10 (27.0%) were asymptomatic at the time of testing. PCR positivity was concentrated on one unit (Unit 1) (20/30 residents, 66.7%; 9/14 staff, 64.3%). The other units showed a limited spread of infection. In unit-wide and separate tests, PCR positivity detection was highly prevalent (22.9 and 44.4%, respectively) in Unit 1, compared with that in the other units. Serological testing identified two additional infected residents with a negative PCR result and showed that no staff was newly identified as infected. CONCLUSIONS: Thorough PCR testing, in combination with comprehensive and separate tests, is critical for managing COVID-19 outbreaks in nursing facilities, particularly, in units considered an epicenter. Serological testing is also beneficial for tracing contacts, confirming the number of infected individuals, and authorizing the termination of the outbreak.


Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , Surtos de Doenças , Instituições de Cuidados Especializados de Enfermagem , Idoso , Idoso de 80 Anos ou mais , Busca de Comunicante , Feminino , Febre/virologia , Pessoal de Saúde , Humanos , Japão , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Pandemias
20.
J Org Chem ; 85(8): 5362-5369, 2020 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-32174115

RESUMO

Photoinduced decarboxylative radical reactions of benzoic acids with electron-deficient alkenes, diborane, and acetonitrile under organic photoredox catalysis conditions and mild heating afforded adducts, arylboronate esters, and the reduction product, respectively. The reaction is thought to involve single-electron transfer promoted the generation of aryl radicals via decarboxylation. A diverse range of benzoic acids were found to be suitable substrates for this photoreaction. Only our two-molecule organic photoredox system can work well for the direct photoinduced decarboxylation of benzoic acids.

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