RESUMEN
Cardiomyopathy is a common myocardial disease that can lead to sudden death. However, molecular mechanisms underlying cardiomyopathy remain unclear. Jumonji and AT-rich interaction domain-containing 2 (Jarid2) is necessary for embryonic heart development, but functions of Jarid2 after birth remain to be elucidated. Here, we report that myocardial-specific deletion of Jarid2 using αMHC::Cre mice (Jarid2αMHC) causes dilated cardiomyopathy (DCM) and premature death 6-9 months after birth. To determine functions of Jarid2 in the adult heart and DCM, we analyzed gene expression in the heart at postnatal day (p)10 (neonatal) and 7 months (DCM). Pathway analyses revealed that dysregulated genes in Jarid2αMHC hearts at p10, prior to cardiomyopathy, represented heart development and muscle contraction pathways. At 7 months, down-regulated genes in Jarid2αMHC hearts were enriched in metabolic process and ion channel activity pathways and up-regulated genes in extracellular matrix components. In normal hearts, expression levels of contractile genes were increased from p10 to 7 months but were not sufficiently increased in Jarid2αMHC hearts. Moreover, Jarid2 was also necessary to repress fetal contractile genes such as TroponinI1, slow skeletal type (Tnni1) and Actin alpha 2, smooth muscle (Acta2) in neonatal stages through ErbB2-receptor tyrosine kinase 4 (ErbB4) signaling. Interestingly, Ankyrin repeat domain 1 (Ankrd1) and Neuregulin 1 (Nrg1), whose expression levels are known to be increased in the failing heart, were already elevated in Jarid2αMHC hearts within 1 month of birth. Thus, we demonstrate that ablation of Jarid2 in cardiomyocytes results in DCM and suggest that Jarid2 plays important roles in cardiomyocyte maturation during neonatal stages.
Asunto(s)
Cardiomiopatía Dilatada/genética , Eliminación de Gen , Miocardio/patología , Complejo Represivo Polycomb 2/genética , Animales , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Neurregulina-1/genética , Neurregulina-1/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Receptor ErbB-4/genética , Receptor ErbB-4/metabolismo , Transducción de SeñalRESUMEN
The homologue of human YTHDF2, Ydr374c (Pho92), is the only protein that has a YTH (YT521-B homology) domain in Saccharomyces cerevisiae. Based on microarray analysis, genes involved in the phosphate signal transduction (PHO) pathway were up-regulated in the Δpho92 strain, as were genes regulated by Pho4, which is an important transcription factor in the PHO pathway. To identify the exact mechanism of Pho92 action with respect to phosphate metabolism, we investigated the effect of Pho92 on PHO4 expression. The half-life of PHO4 mRNA was increased in the Δpho92 strain; this phenotype was also observed in the deletion mutants UPF1 and POP2, which are components of the NMD (nonsense-mediated decay) pathway and the Pop2-Ccr4-Not deadenylase complex respectively. Pho92 interacts physically with Pop2 of the Pop2-Ccr4-Not deadenylase complex. Furthermore, Pho92 binding to the 3'-UTR of PHO4 was dependent on the phosphate concentration. Deletion of the PHO4 3'-UTR resulted in PHO4 mRNA resistance to Pho92-dependent degradation. The results of the present study indicate that Pho92 regulates Pho4 expression at the post-transcriptional level via the regulation of mRNA stability. Taken together, Pho92 participates in cellular phosphate metabolism, specifically via the regulation of PHO4 mRNA stability by binding to the 3'-UTR in a phosphate-dependent manner.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas de Transporte de Fosfato/química , Proteínas de Transporte de Fosfato/genética , Factores de Empalme de ARN , Estabilidad del ARN , ARN Bacteriano/química , ARN Mensajero/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ribonucleasas/química , Ribonucleasas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Factores de Escisión y Poliadenilación de ARNm/química , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Ydr374c (Pho92) contains a YTH domain in its C-terminal region and is a human YTHDF2 homologue. Previously, we reported that Pho92 regulates phosphate metabolism by regulating PHO4 mRNA stability. In this study, we found that growth of the ∆pho92 strain on SG media was slower than that of the wild type and that PHO92 expression was up-regulated by non-fermentable carbon sources, such as ethanol and glycerol, but not by fermentable carbon sources. Furthermore, two conserved Gcr1-binding regions were identified in the upstream, untranslated region of PHO92. Gcr1 is an important factor involved in the coordinated regulation of glycolytic gene expression. Mutation of two Gcr1-binding sites of the PHO92 upstream region resulted in a growth defect on SD media. Finally, mutagenesis of the Gcr1-binding sites of the PHO92 upstream region and deletion of GCR1 resulted in up-regulation of PHO92, and this resulted from inhibition of PHO4 mRNA degradation. Based on these results, we suggest that Gcr1 regulates the expression of PHO92, and Pho92 is involved in glucose metabolism.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Regiones no Traducidas 5' , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Glucólisis , Mutagénesis Sitio-Dirigida , Mutación , Dominios Proteicos , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinct subtype of T-ALL with a poor prognosis. To find a cure, we examined the synergistic effect of homoharringtonine (HHT) in combination with the BCL-2 inhibitor venetoclax (VEN) in ETP-ALL. METHODS: Using in vitro cellular assays and ETP-ALL xenograft models, we first investigated the synergistic activity of HHT and VEN in ETP-ALL. Next, to explore the underlying mechanism, we employed single-cell RNA sequencing of primary ETP-ALL cells treated with HHT or VEN alone or in combination and validated the results with western blot assays. Based on the promising preclinical results and given that both drugs have been approved for clinical use, we then assessed this combination in clinical practice. FINDINGS: Our results showed that HHT synergizes strongly with VEN both in vitro and in vivo in ETP-ALL. Mechanistic studies demonstrated that the HHT/VEN combination concurrently downregulated key anti-apoptotic proteins, i.e., MCL1, leading to enhanced apoptosis. Importantly, the clinical results were very promising. Six patients with ETP-ALL with either refractory/relapsed (R/R) or newly diagnosed disease were treated with an HHT/VEN-based regimen. All patients achieved complete remission (CR) after only one cycle of treatment. CONCLUSIONS: Our findings demonstrate that a combination of HHT/VEN is effective on ETP-ALL and represents the "backbone" of a promising and safe regimen for newly diagnosed and R/R patients with ETP-ALL. FUNDING: This work was funded by the National Cancer Institute, Gehr Family Foundation, George Hoag Family Foundation, National Natural Science Foundation of China, and Key Research and Development Program of Zhejiang Province of China.
RESUMEN
Kdx1 is known as a stress-responsive protein. To better understand the function of Kdx1, we performed microarray analysis in KDX1 overexpressing cells and found that the overexpression of KDX1 dramatically induced the expression of RCK1, a stress-responsive gene. This result was confirmed by northern blot analysis. Furthermore, the overexpression of RCK1 partially rescued the growth defect caused by zymolyase stress. The expression of RCK1 was regulated independently by Slt2 and Hog1, and Kdx1 failed to induce the expression of RCK1 in a HOG1 deletion strain. The transcriptional factors Smp1, Sko1, Msn2, Msn4, and Hot1, which are regulated by Hog1, did not affect RCK1 expression, but Rlm1 did. Furthermore, the mutation of certain phosphorylation sites in RLM1 inhibited the induction of RCK1 expression by Kdx1. We found a conserved Rlm1 binding site in the 5' untranslated region (UTR) of RCK1, and the mutation of these Rlm1 binding sites also inhibited the induction of RCK1 expression by Kdx1. Finally, we showed that Kdx1 physically interacts with Rlm1 and that this interaction affects the ability of Rlm1 to bind to the RCK1 5' UTR. Taken together, these data suggest that Kdx1 interacts with Rlm1 to activate RCK1 gene expression in response to stress in Saccharomyces cerevisiae.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas de Dominio MADS/metabolismo , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Pared Celular/enzimología , Pared Celular/genética , Pared Celular/metabolismo , Proteínas de Dominio MADS/genética , Sistema de Señalización de MAP Quinasas , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación/genética , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas de Unión al ARN , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico/genéticaRESUMEN
We previously reported that the over-expression of KDX1 up-regulates RCK1 gene expression. To further understand the function of Rck1, microarray analysis was performed using a RCK1 over-expressing strain. Based on microarray and Northern blot analyses, we determined that the expression of KDX1 was down-regulated when RCK1 was over-expressed. Furthermore, we determined that phosphorylated forms of Slt2 and Mkk2 were down-regulated by the over-expression of RCK1. Ptp2, a phosphatase that is regulated by the Slt2 MAP kinase pathway, was down-regulated by the over-expression of RCK1. Ptp2 is a negative regulator of Hog1; thus, the phosphorylated form of Hog1 was up-regulated by RCK1 over-expression. A point mutation of lysine 152 to arginine resulted in a failure to up-regulate Hog1 and the subsequent down-regulation of CTT1, which is a Hog1 pathway target gene. Furthermore, using microarray and Northern blot analyses, we determined that genes that are regulated by Msn2/Msn4 were up-regulated by Rck1 and that this was the result of Hog1 activation by RCK1 over-expression. Together, our results suggest that Rck1 inhibits Slt2 MAP kinase pathway activity and then Ptp2, which subsequently activates Hog1.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Mutación Puntual , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Unión al ARN , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Autism spectrum disorder (ASD) is a neurobiological disorder characterized by distinctive impairments in cognitive function, language, and behavior. Linkage and population studies suggest a genetic association between solute carrier family 6 member 4 (SLC6A4) variants and ASD. METHOD: Logistic regression was used to identify associations between single-nucleotide polymorphisms (SNPs) and ASD with 3 alternative models (additive, dominant, and recessive). Linear regression analysis was performed to determine the influence of SNPs on Childhood Autism Rating Scale (CARS) scores as a quantitative phenotype. RESULTS: In the present study, we examined the associations of SNPs in the SLC6A4 gene and the fibrinogen alpha chain (FGA) gene. Logistic regression analysis showed a significant association between the risk of ASD and rs2070025 and rs2070011 in the FGA gene. The gene-gene interaction between SLC6A4 and FGA was not significantly associated with ASD susceptibility. However, polymorphisms in both SLC6A4 and the FGA gene significantly affected the symptoms of ASD. CONCLUSION: Our findings indicate that FGA and SLC6A4 gene interactions may contribute to the phenotypes of ASD rather than the incidence of ASD.
Asunto(s)
Pueblo Asiatico/genética , Trastorno Autístico/genética , Fibrinógeno/genética , Predisposición Genética a la Enfermedad/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Adolescente , Adulto , Estudios de Casos y Controles , Epistasis Genética/genética , Femenino , Humanos , Masculino , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Adulto JovenRESUMEN
Cadmium is a toxic metal, and the mechanism of cadmium toxicity in living organisms has been well studied. Here, we used Saccharomyces cerevisiae as a model system to examine the detailed molecular mechanism of cell growth defects caused by cadmium. Using a plate assay of a yeast deletion mutant collection, we found that deletion of SML1, which encodes an inhibitor of Rnr1, resulted in cadmium resistance. Sml1 protein levels increased when cells were treated with cadmium, even though the mRNA levels of SML1 remained unchanged. Using northern and western blot analyses, we found that cadmium inhibited Sml1 degradation by inhibiting Sml1 phosphorylation. Sml1 protein levels increased when cells were treated with cadmium due to disruption of the dependent protein degradation pathway. Furthermore, cadmium promoted cell cycle progression into the G2 phase. The same result was obtained using cells in which SML1 was overexpressed. Deletion of SML1 delayed cell cycle progression. These results are consistent with Sml1 accumulation and with growth defects caused by cadmium stress. Interestingly, although cadmium treatment led to increase Sml1 levels, intracellular dNTP levels also increased because of Rnr3 upregulation due to cadmium stress. Taken together, these results suggest that cadmium specifically affects the phosphorylation of Sml1 and that Sml1 accumulates in cells.
Asunto(s)
Cadmio/toxicidad , Proteolisis/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Daño del ADN , Fosforilación/efectos de los fármacos , Ribonucleótido Reductasas/antagonistas & inhibidores , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Regulación hacia ArribaRESUMEN
The ATX1 deletion strain of Saccharomyces cerevisiae is more resistant to Cd(2+) than the wild-type. To investigate the function of Atx1 in Cd(2+) toxicity, we used a metal-binding assay to study the interaction between Atx1 and Cd(2+) in vitro. Using circular dichroism and two-hybrid analyses, we found that Atx1 can bind Cd(2+) specifically and that Cd(2+) binding to Atx1 affects the physical interaction between Atx1 and Ccc2. These results imply that Atx1 delivers Cd(2+) to Ccc2 and that this delivery is, at least in part, responsible for Cd(2+) toxicity in S. cerevisiae.
Asunto(s)
Cadmio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión/metabolismo , Cationes Bivalentes/metabolismo , Mapeo de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Dicroismo Circular , Proteínas Transportadoras de Cobre , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
Adoptive therapies with genetically modified somatic T cells rendered HIV resistance have shown promise for AIDS therapy. A renewable source of HIV-resistant human T cells from induced pluripotent stem cells (iPSCs) would further facilitate and broaden the applicability of these therapies. Here, we report successful targeting of the CCR5 locus in iPSCs generated from T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian cynomolgus macaques (MCM), using CRISPR-Cas9 technology. We found that CCR5 editing does not affect hematopoietic and T cell differentiation potentials of fib-iPSCs. However, T-iPSCs with edited CCR5 lost their capacity to differentiate into CD4+CD8+ T cells while maintaining myeloid differentiation potential. T cells and macrophages produced from CCR5-edited MCM iPSCs did not support replication of the CCR5-tropic simian immunodeficiency viruses SIVmac239 (T cell tropic) and SIVmac316 (macrophage-tropic). Overall, these studies provide a platform for further exploration of AIDS therapies based on gene-edited iPSCs in a nonhuman primate model.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , Células Madre Pluripotentes Inducidas , Animales , Linfocitos T CD8-positivos , Macaca fascicularis , Macrófagos , Receptores CCR5/genéticaRESUMEN
Heterogeneity of endothelial cell (EC) populations reflects their diverse functions in maintaining tissue's homeostasis. However, their phenotypic, molecular, and functional properties are not entirely mapped. We use the Tie2-CreERT2;Rosa26-tdTomato reporter mouse to trace, profile, and cultivate primary ECs from different organs. As paradigm platform, we use this strategy to study bone marrow endothelial cells (BMECs). Single-cell mRNA sequencing of primary BMECs reveals that their diversity and native molecular signatures is transitorily preserved in an ex vivo culture that conserves key cell-to-cell microenvironment interactions. Macrophages sustain BMEC cellular diversity and expansion and preserve sinusoidal-like BMECs ex vivo. Endomucin expression discriminates BMECs in populations exhibiting mutually exclusive properties and distinct sinusoidal/arterial and tip/stalk signatures. In contrast to arterial-like, sinusoidal-like BMECs are short-lived, form 2D-networks, contribute to in vivo angiogenesis, and support hematopoietic stem/progenitor cells in vitro. This platform can be extended to other organs' ECs to decode mechanistic information and explore therapeutics.
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Médula Ósea , Células Endoteliales , Ratones , Animales , Células Endoteliales/fisiología , Transcriptoma , Endotelio , Células Madre Hematopoyéticas/metabolismoRESUMEN
The unripe fruits of Rubus coreanus (Rosaceae) are used in traditional Chinese medicine to relieve kidney dysfunction. In the present study, we evaluated the protective effects of the triterpenoid glycoside niga-ichigoside F1 (NIF1) and of its aglycone 23-hydroxytormentic acid (23-HTA) isolated from the unripe fruits of Rubus coreanus (Rosaceae) against cisplatin-induced cytotoxicity in renal epithelial LLC-PK1 cells. Pretreating LLC-PK1 cells with 23-HTA or NIF1 was found to prevent cisplatin-induced cytotoxicity and apoptosis. In addition, 23-HTA or NIF1 pretreatment significantly improved the changes associated with cisplatin toxicity by increasing levels of glutathione (GSH) and decreasing levels of malondialdehyde (MDA) and reactive oxygen species (ROS). The activity of antioxidant enzymes including catalase (CAT) and superoxide dismutase (SOD) was significantly lower in cisplatin-treated LL-PK1 cells, and 23-HTA or NIF1 treatment notably increased the these enzyme activity and protein and mRNA levels of CAT and manganese SOD (MnSOD). Moreover, cisplatin caused a significant decrease in nuclear levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and pretreatment with 23-HTA or NIF1 significantly suppressed the cisplatin-induced translocation of Nrf2 in LLC-PK1 cells. Taken together, these results suggest that 23-HTA ameliorates cisplatin-induced toxicity via modulation of antioxidant enzymes through activation of Nrf2 in LLC-PK1 cells.
Asunto(s)
Antineoplásicos/toxicidad , Antioxidantes/farmacología , Cisplatino/toxicidad , Glicósidos/farmacología , Riñón/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Triterpenos/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Frutas/química , Frutas/crecimiento & desarrollo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Riñón/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , Rosaceae/química , Sus scrofaRESUMEN
Cadmium is a toxic metal and the mechanism of its toxicity has been studied in various model systems from bacteria to mammals. We employed Saccharomyces cerevisiae as a model system to study cadmium toxicity at the molecular level because it has been used to identify the molecular mechanisms of toxicity found in higher organisms. cDNA microarray and Northern blot analyses revealed that cadmium salts inhibited the expression of genes related to copper metabolism. Western blotting, Northern blotting and chromatin immunoprecipitation experiments indicated that CTR1 expression was inhibited at the transcriptional level through direct inhibition of the Mac1 transcriptional activator. The decreased expression of CTR1 results in cellular copper deficiency and inhibition of Fet3 activity, which eventually impairs iron uptake. In this way, cadmium exhibits a negative effect on both iron and copper homoeostasis.
Asunto(s)
Cadmio/toxicidad , Cobre/metabolismo , Homeostasis/efectos de los fármacos , Proteínas Nucleares/antagonistas & inhibidores , Regulón/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/genética , Transactivadores/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Cobre/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes Fúngicos/genética , Hierro/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Regulón/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Scaling up blood cell production from hPSCs is critical to advancing hPSC technologies for blood transfusion, immunotherapy, and transplantation. Here we explored the potential of the HSC agonist pyrimido-indole derivative UM171, to expand hematopoietic progenitors (HPs) derived from hPSCs in chemically defined conditions. We revealed that culture of hPSC-HPs in HSC expansion conditions (SFEM with added TPO, SCF, FLT3L, IL3 and IL6) in the presence of UM171 predominantly expanded HPs with a unique CD34+CD41aloCD45+ phenotype that were enriched in granulocytic progenitors (G-CFCs). In contrast, in lymphoid cultures on OP9-DLL4, in the presence of SCF, FLT3L, and IL7, UM171 selectively expanded CD34+CD45+CD7+ lymphoid progenitors with NK cell potential, and increased NK cell output up to 10-fold. These studies should improve our understanding of the effect of UM171 on de novo generated HPs, and facilitate development of protocols for robust granulocyte and lymphoid cell production from hPSCs, for adoptive immunotherapies.
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Indoles/farmacología , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Pirimidinas/farmacología , Antígenos CD34/metabolismo , Antígenos CD7/metabolismo , Diferenciación Celular/efectos de los fármacos , Citometría de Flujo , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Antígenos Comunes de Leucocito/metabolismo , Leucosialina/metabolismo , FenotipoRESUMEN
Intracellular levels of iron are tightly regulated. Saccharomyces cerevisiae uses well-defined pathways to extract iron molecules from the environment. Once inside the cell, the iron molecules must be transferred to target sites via an intracellular iron transporter. Although analogous carriers have been described for other metals, such as copper, an iron transporter has yet to be identified. We used two-dimensional gel electrophoresis and mass spectrometry techniques to attempt to identify the iron transporter from cytosolic fraction of S. cerevisiae. In this study, we identified the iron-binding activity of thioredoxin reductase, and our data suggest a potential role for this enzyme in intracellular iron transport.
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Proteínas de Unión a Hierro/fisiología , Hierro/metabolismo , Saccharomyces cerevisiae/enzimología , Tiorredoxina Reductasa 1/fisiología , Cromatografía de Afinidad/métodos , Dicroismo Circular , Medios de Cultivo , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/aislamiento & purificación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Espectrofotometría Ultravioleta , Tiorredoxina Reductasa 1/genética , Tiorredoxina Reductasa 1/aislamiento & purificaciónRESUMEN
BACKGROUND: Premature ovarian failure (POF) is a complex and heterogeneous disorder that is influenced by multiple genetic components. Here, we performed a two-stage association study to identify POF-associated genes. METHODS: A first stage linkage disequilibrium (LD)-based genome-wide association study was performed using 24 pairs of patients with POF and matched controls and a high-throughput BeadChip assay with 109365 single-nucleotide polymorphisms (SNPs) that are scattered throughout the genome in an exon-centric and evenly spaced manner. A region that was shown to be strongly associated with POF was then tested again for POF association in the second stage by using a larger sample size (101 cases and 87 controls) and additional putative causal SNPs. RESULTS: The first stage analysis revealed that many regions were associated with POF, with part of chromosome 7p14 that contains the parathyroid hormone responsive-B1 (PTHB1) gene showing the strongest association. A POF-susceptible haplotype of PTHB1 (ht1, 'GAAAG', P = 0.00034) and a POF-resistant haplotype (ht2, 'TGTGC') were also identified. The association between POF and two PTHB1 SNPs (rs3884597 and rs6944723) and part of ht1 was confirmed in the second stage analysis. The additional SNP, rs11773504, was considered as a putative causal variant causing an amino acid change, Ala to Thr. CONCLUSIONS: We showed for the first time that PTHB1 is strongly associated with POF, and ht1 confers susceptibility to POF. While causative SNPs were not identified, the polymorphism of the non-synonymous SNP rs11773504 and the repeated association of ht1 with POF suggest that PTHB1 may contribute to POF pathogenesis.
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Desequilibrio de Ligamiento , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Insuficiencia Ovárica Primaria/genética , Estudios de Casos y Controles , Mapeo Cromosómico , Proteínas del Citoesqueleto , Femenino , Genotipo , HumanosRESUMEN
A novel type strain, Planococcus faecalis AJ003T, isolated from the feces of Antarctic penguins, synthesizes a rare C30 carotenoid, glycosyl-4,4'-diaponeurosporen-4'-ol-4-oic acid. The complete genome of P. faecalis AJ003T comprises a single circular chromosome (3,495,892â¯bp; 40.9% Gâ¯+â¯C content). Annotation analysis has revealed 3511 coding DNA sequences and 99 RNAs; seven genes associated with the MEP pathway and five genes involved in the carotenoid pathway have been identified. The functionality and complementation of 4,4'-diapophytoene synthase (CrtM) and two copies of heterologous 4,4'-diapophytoene desaturase (CrtN) involved in carotenoid biosynthesis were analyzed in Escherichia coli.
Asunto(s)
Proteínas Bacterianas/genética , Carotenoides/genética , Genoma Bacteriano , Planococcus (Bacteria)/genética , Proteínas Bacterianas/metabolismo , Carotenoides/biosíntesis , Planococcus (Bacteria)/metabolismoRESUMEN
The transcriptional factor GATA2 is required for blood and hematopoietic stem cell formation during the hemogenic endothelium (HE) stage of development in the embryo. However, it is unclear if GATA2 controls HE lineage specification or if it solely regulates endothelial-to-hematopoietic transition (EHT). To address this problem, we innovated a unique system, which involved generating GATA2 knockout human embryonic stem cell (hESC) lines with conditional GATA2 expression (iG2-/- hESCs). We demonstrated that GATA2 activity is not required for VE-cadherin+CD43-CD73+ non-HE or VE-cadherin+CD43-CD73- HE generation and subsequent HE diversification into DLL4+ arterial and DLL4- non-arterial lineages. However, GATA2 is primarily needed for HE to undergo EHT. Forced expression of GATA2 in non-HE failed to induce blood formation. The lack of GATA2 requirement for generation of HE and non-HE indicates the critical role of GATA2-independent pathways in specification of these two distinct endothelial lineages.
Asunto(s)
Diferenciación Celular/genética , Factor de Transcripción GATA2/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factor de Transcripción GATA2/metabolismo , Edición Génica , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Marcación de Gen , Hemangioblastos/citología , Hemangioblastos/metabolismo , Humanos , Leucocitos/citología , Leucocitos/metabolismo , Linfocitos/citología , Linfocitos/metabolismoRESUMEN
The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.