RESUMO
Heart development is a delicate and complex process regulated by coordination of various signaling pathways. In this study, we investigated the role of sox18 in heart development by modulating Wnt/ß-Catenin signaling pathways. Our spatiotemporal expression analysis revealed that sox18 is mainly expressed in the heart, branchial arch, pharyngeal arch, spinal cord, and intersegmental vessels at the tailbud stage of Xenopus tropicalis embryo. Overexpression of sox18 in the X. tropicalis embryos causes heart edema, while loss-of-function of sox18 can change the signal of developmental heart marker gata4 at different stages, suggesting that sox18 plays an essential role in the development of the heart. Knockdown of SOX18 in human umbilical vein endothelial cells suggests a link between Sox18 and ß-CATENIN, a key regulator of the Wnt signaling pathway. Sox18 negatively regulates islet1 and tbx3, the downstream factors of Wnt/ß-Catenin signaling, during the linear heart tube formation and the heart looping stage. Taken together, our findings highlight the crucial role of Sox18 in the development of the heart via inhibiting Wnt/ß-Catenin signaling.
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Fatores de Transcrição SOXF , Proteínas de Xenopus , beta Catenina , Animais , Humanos , beta Catenina/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Via de Sinalização Wnt , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
AIMS: Helicobacter pylori (HP) infection is the most common cause of chronic gastritis worldwide. Due to the small size of HP and limited resolution, diagnosing HP infections is more difficult when using digital slides. METHODS AND RESULTS: We developed a two-tier deep-learning-based model for diagnosing HP gastritis. A whole-slide model was trained on 885 whole-slide images (WSIs) with only slide-level labels (positive or negative slides). An auxiliary model was trained on 824 areas with HP in nine positive WSIs and 446 negative WSIs for localizing HP. The whole-slide model performed well, with an area under the receiver operating characteristic curve (AUC) of 0.9739 (95% confidence interval [CI], 0.9545-0.9932). The calculated sensitivity and specificity were 93.3% and 90.1%, respectively, whereas those of pathologists were 93.3% and 84.2%, respectively. Using the auxiliary model, the highlighted areas of the localization maps had an average precision of 0.5796. CONCLUSIONS: HP gastritis can be diagnosed on haematoxylin-and-eosin-stained WSIs with human-level accuracy using a deep-learning-based model trained on slide-level labels and an auxiliary model for localizing HP and confirming the diagnosis. This two-tiered model can shorten the diagnostic process and reduce the need for special staining.
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Aprendizado Profundo , Gastrite Atrófica , Gastrite , Infecções por Helicobacter , Helicobacter pylori , Humanos , Gastrite/diagnóstico , Gastrite/patologia , Sensibilidade e Especificidade , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/patologiaRESUMO
GIBBERELLIN MYB GENE (GAMYB), UNDEVELOPED TAPETUM1 (UDT1), TDR INTERACTING PROTEIN2 (TIP2/bHLH142), TAPETUM DEGENERATION RETARDATION (TDR), and ETERNAL TAPETUM 1/DELAYED TAPETUM DEGENERATION (EAT1/DTD) are important transcription factors that play a crucial role during pollen development in rice. This study demonstrates that bHLH142 acts downstream of UDT1 and GAMYB and works as a 'hub' in these two pollen pathways. We show that GAMYB modulates bHLH142 expression through specific binding to the MYB motif of the bHLH142 promoter during the early stage of pollen development, while TDR acts as a transcriptional repressor of the GAMYB modulation of bHLH142 by binding to the E-box close to the MYB motif on the promoter. Altered expression of these transcription factors highlights that a tight, precise, and coordinated regulation among them is essential for normal pollen development. Most notably, we show that the regulatory pathways of GAMYB and UDT1 rely on bHLH142 in a direct and indirect manner, respectively, and function in different tissues with distinct biological roles during pollen development. This study advances our understanding of the molecular mechanisms of rice pollen development.
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Oryza , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , Pólen/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Early recognition of adult-onset immunodeficiency associated with neutralizing anti-interferon gamma autoantibodies (anti-IFNγ Abs) remains difficult, and misdiagnoses have been reported. Although febrile lymphadenopathy is among the most common initial manifestations of this disorder, no comprehensive clinicopathologic analysis of lymphadenopathy in patients with anti-IFNγ Abs has been reported. Here, we describe 26 lymph node biopsy specimens from 16 patients. All patients exhibited concurrent disseminated nontuberculous mycobacterial infections, and 31% received a tentative diagnosis of lymphoma at initial presentation. We found 3 distinct histomorphologic patterns: well-formed granuloma (46%), suppurative inflammation or loose histiocytic aggregates (31%), and lymphoproliferative disorder (LPD, 23%). The latter shared some of the features of malignant T-cell lymphoma, IgG4-related disease, and multicentric Castleman disease. Half of the specimens with LPD had monoclonal T cells, and 33.3% were indistinguishable from angioimmunoblastic T-cell lymphoma as per current diagnostic criteria. All lymphadenopathy with LPD features regressed with antibiotics without administration of cytotoxic chemotherapy or immunotherapy. The median follow-up time was 4.3 years. Our study highlights the substantial challenge of distinguishing between lymphoma and other benign lymphadenopathy in the setting of neutralizing anti-IFNγ Abs. Increased vigilance and multidisciplinary discussion among clinicians and pathologists are required to achieve the most appropriate diagnosis and management.
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Síndromes de Imunodeficiência/diagnóstico , Linfadenopatia/diagnóstico , Linfoma/diagnóstico , Linfócitos T/imunologia , Adulto , Idoso , Antibacterianos , Anticorpos Neutralizantes , Autoanticorpos/imunologia , Autoantígenos/imunologia , Proliferação de Células , Feminino , Humanos , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Interferon gama/imunologia , Linfonodos/patologia , Linfadenopatia/imunologia , Linfadenopatia/patologia , Linfoma/imunologia , Linfoma/patologia , Masculino , Pessoa de Meia-Idade , Infecções Oportunistas/imunologia , Infecções Oportunistas/patologiaRESUMO
Vanilla orchid, which is well-known for its flavor and fragrance, is cultivated in tropical and subtropical regions. This shade-loving plant is very sensitive to high irradiance. In this study, we show that vanilla chloroplasts started to have avoidance movement when blue light (BL) was higher than 20 µmol m-2s-1 and significant avoidance movement was observed under BL irradiation at 100 µmol m-2s-1 (BL100). The light response curve indicated that when vanilla was exposed to 1000 µmol m-2s-1, the electron transport rate (ETR) and photochemical quenching of fluorescence (qP) were significantly reduced to a negligible amount. We found that if a vanilla orchid was irradiated with BL100 for 12 days, it acquired BL-acclimation. Chloroplasts moved to the side of cells in order to reduce light-harvesting antenna size, and chloroplast photodamage was eliminated. Therefore, BL-acclimation enhanced vanilla orchid growth and tolerance to moderate (500 µmol m-2s-1) and high light (1000 µmol m-2s-1) stress conditions. It was found that under high irradiation, BL-acclimatized vanilla maintained higher ETR and qP capacity than the control without BL-acclimation. BL-acclimation induced antioxidant enzyme activities, reduced ROS accumulation, and accumulated more carbohydrates. Moreover, BL-acclimatized orchids upregulated photosystem-II-associated marker genes (D1 and PetC), Rubisco and PEPC transcripts and sustained expression levels thereof, and also maximized the photosynthesis rate. Consequently, BL-acclimatized orchids had higher biomass. In short, this study found that acclimating vanilla orchid with BL before transplantation to the field might eliminate photoinhibition and enhance vanilla growth and production.
Assuntos
Clorofila/metabolismo , Cloroplastos/metabolismo , Estiolamento , Luz , Fotossíntese , Vanilla/crescimento & desenvolvimento , Cloroplastos/efeitos da radiação , Fluorescência , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Vanilla/metabolismo , Vanilla/efeitos da radiaçãoRESUMO
Unique tunable aryl imidazolium ionic liquids successfully catalyzed Friedel-Crafts acylation and thioesterification in sealed tubes. These reactions can form a C-C bond and a C-S bond with high atom economy. Ionic liquids exhibited high activity and catalyzed essential reactions with good to excellent yields while retaining their catalytic activities for recycling.
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Imidazóis/química , Líquidos Iônicos/química , Ácidos de Lewis/química , Compostos de Sulfidrila/química , Acilação , Catálise , EsterificaçãoRESUMO
Double-spikes Phalaenopsis orchids have greater market value than those with single-spike. In this study, a gene designated as Spike Activator 1 (SPK1), which encodes a basic helix-loop-helix (bHLH) transcription factor, was isolated and characterized from Phalaenopsis aphrodite (moth orchid). SPK1 was highly expressed in the meristematic tissues. In the axillary bud, SPK1 was highly upregulated by a moderately low temperature of 20 °C but downregulated by a spike inhibition temperature of 30 °C. SPK1 protein is localized in the nucleus. Another bHLH, bHLH35, which is also highly expressed in young tissues in the same way as SPK1 was also identified. In contrast to SPK1, bHLH35 transcripts are downregulated at 20 °C but upregulated at 30 °C. Bimolecular florescence complementation assay and yeast two-hybrid assays indicated that SPK1 interacts with bHLH35 and forms a heterodimer. Virus-induced gene silencing (VIGS) showed that 7 out of 15 vector control plants produced double spikes but that only 1 out of 15 VIGS-spk1 plants produced double spikes. RT-qPCR results indicated that VIGS-spk1 downregulated gene expression levels of SPK1, FT, CYCB, and EXPA8. Overall, we propose that SPK1 plays an essential role in early axillary bud development and spike initiation of P. aphrodite.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Orchidaceae/crescimento & desenvolvimento , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Clonagem Molecular , Temperatura Baixa , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Temperatura Alta , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Orchidaceae/genética , Orchidaceae/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Multimerização Proteica , Ativação TranscricionalRESUMO
Chloroplast movement is important for plants to avoid photodamage and to perform efficient photosynthesis. Phototropins are blue light receptors in plants that function in chloroplast movement, phototropism, stomatal opening, and they also affect plant growth and development. In this study, full-length cDNAs of two PHOTOTROPIN genes, PaPHOT1 and PaPHOT2, were cloned from a moth orchid Phalaenopsis aphrodite, and their functions in chloroplast movement were investigated. Phylogenetic analysis showed that PaPHOT1 and PaPHOT2 orthologs were highly similar to PHOT1 and PHOT2 of the close relative Phalaenopsis equestris, respectively, and clustered with monocots PHOT1 and PHOT2 orthologs, respectively. Phalaenopsis aphrodite expressed a moderate level of PaPHOT1 under low blue light of 5 µmol�m-2�s-1 (BL5) and a high levels of PaPHOT1 at >BL100. However, PaPHOT2 was expressed at low levels at
Assuntos
Orchidaceae/fisiologia , Fototropinas/metabolismo , Fototropismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , DNA Complementar/genética , Expressão Gênica , Inativação Gênica , Hibridização In Situ , Luz , Mutação , Orchidaceae/genética , Orchidaceae/efeitos da radiação , Fotossíntese , Fototropinas/genética , Filogenia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
This work demonstrates the utilization of phosphotungstic acid (PTA) as a novel acidic catalyst for carbohydrate reactions, such as per-O-acetylation, regioselective O-4,6 benzylidene acetal formation, regioselective O-4 ring-opening, and glycosylation. These reactions are basic and salient during the synthesis of carbohydrate-based bioactive oligomers. Phosphotungstic acid's high acidity and eco-friendly character make it a tempting alternative to corrosive homogeneous acids. The various homogenous acid catalysts were replaced by the phosphotungstic acid solely for different carbohydrate reactions. It can be widely used as a catalyst for organic reactions as it is thermally stable and easy to handle. In our work, the reactions are operated smoothly under ambient conditions; the temperature varies from 0 °C to room temperature. Good to excellent yields were obtained in all four kinds of reactions.
RESUMO
BACKGROUND: There are some limitations of standard chemotherapy for acute leukemia. Vincristine and doxorubicin are commonly used for acute leukemia, but they may induce serious side effects such as cardiomyopathy and neurotoxicity. Furthermore, chemotherapy resistance occurs more and more frequently. Therefore, effective treatment strategies are needed. Histone deacetylase 6 inhibition is considered as a potential therapeutic strategy for acute leukemia, since it is observed that HDAC6 is overexpressed in acute leukemia and regulates tumor survival. Combination therapy for cancer is used to minimize adverse drug effects, reduce drug dosage, enhance efficacy, and prevent drug resistance. In order to improve efficacy of chemotherapy agents of acute leukemia, this study will investigate the effects of combination MPT0G211, a novel histone deacetylase 6 inhibitor, with doxorubicin or vincristine on human acute leukemia cells. RESULTS: MPT0G211 combined with doxorubicin induces DNA damage response on human acute myeloid leukemia cells. MPT0G211 can additionally increase Ku70 acetylation and release BAX to mitochondria. Ectopic expression of HDAC6 successively reversed the apoptosis triggered by the combined treatment. Moreover, co-treatment of MPT0G211 and vincristine may alter microtubule dynamics, triggering acute lymphoblastic leukemia cells arrest in mitotic phase followed by induction of the apoptotic pathway. Finally, MPT0G211 plus doxorubicin or vincristine can significantly improve the tumor growth delay in a tumor xenograft model. CONCLUSIONS: Collectively, our data highlighted that MPT0G211 in combination with chemotherapy drugs has significant anticancer activity, suggesting a novel strategy for the treatment of acute leukemia.
Assuntos
Benzamidas/administração & dosagem , Doxorrubicina/administração & dosagem , Inibidores de Histona Desacetilases/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Vincristina/administração & dosagem , Animais , Benzamidas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Células HL-60 , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Humanos , Leucemia Mieloide Aguda/metabolismo , Camundongos , Vincristina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Indoleamine 2,3-dioxygenase is a heme-containing enzyme implicated in the down regulation of the anti-tumor immune response, and considered a promising anti-cancer drug target. Several pharmaceutical companies, including Pfizer, Merck, and Bristol-Myers Squibb, are known to be in pursuit of IDO inhibitors, and Incyte recently reported good results in the phase II clinical trial of the IDO inhibitor Epacadostat. In previous work, we developed a series of IDO inhibitors based on a sulfonylhydrazide core structure, and explored how they could serve as potent IDO inhibitors with good drug profiles. Herein, we disclose the development of the 4-bromophenylhydrazinyl benzenesulfonylphenylurea 5k, a potent IDO inhibitor which demonstrated 25% tumor growth inhibition in a murine CT26 syngeneic model on day 18 with 100â¯mg/kg oral administration twice daily, and a 30% reduction in tumor weight. Pharmacodynamic testing of 5k found it to cause a 25% and 21% reduction in kyn/trp ratio at the plasma and tumor, respectively. In the CT26 tumor model, 5k was found to slightly increase the percentage of CD3+ T cells and lymphocyte responsiveness, indicating that 5k may have potential in modulating anti-tumor immunity. These data suggest 5k to be worthy of further investigation in the development of anti-tumor drugs.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Sulfonas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/isolamento & purificação , Complexo CD3/análise , Complexo CD3/isolamento & purificação , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonas/síntese química , Sulfonas/químicaRESUMO
Male sterility is important for hybrid seed production. Pollen development is regulated by a complex network. We previously showed that knockout of bHLH142 in rice (Oryza sativa) causes pollen sterility by interrupting tapetal programmed cell death (PCD) and bHLH142 coordinates with TDR to modulate the expression of EAT1. In this study, we demonstrated that overexpression of bHLH142 (OE142) under the control of the ubiquitin promoter also leads to male sterility in rice by triggering the premature onset of PCD. Protein of bHLH142 was found to accumulate specifically in the OE142 anthers. Overexpression of bHLH142 induced early expression of several key regulatory transcription factors in pollen development. In particular, the upregulation of EAT1 at the early stage of pollen development promoted premature PCD in the OE142 anthers, while its downregulation at the late stage impaired pollen development by suppressing genes involved in pollen wall biosynthesis, ROS scavenging and PCD. Collectively, these events led to male sterility in OE142. Analyses of related mutants further revealed the hierarchy of the pollen development regulatory gene network. Thus, the findings of this study advance our understanding of the central role played by bHLH142 in the regulatory network leading to pollen development in rice and how overexpression of its expression affects pollen development. Exploitation of this novel functionality of bHLH142 may confer a big advantage to hybrid seed production.
RESUMO
BACKGROUND: Phalaenopsis orchid (Phal. orchid) is visually attractive and it is important economic floriculture species. Phal. orchids have many unique biological features. However, investigation of these features and validation on their biological functions are limited due to the lack of an efficient transformation method. RESULTS: We developed a heritable and efficient Agrobacterium- mediated transformation using protocorms derived from tetraploid or diploid Phal. orchids. A T-DNA vector construct containing eGFP driven by ubiquitin promoter was subjected to transformation. An approximate 1.2-5.2 % transformation rate was achieved. Genomic PCR confirmed that hygromycin selection marker, HptII gene and target gene eGFP were integrated into the orchid genome. Southern blotting indicated a low T-DNA insertion number in the orchid genome of the transformants. Western blot confirmed the expression of eGFP protein in the transgenic orchids. Furthermore, the GFP signal was detected in the transgenic orchids under microscopy. After backcrossing the pollinia of the transgenic plants to four different Phal. orchid varieties, the BC1 progenies showed hygromycin resistance and all surviving BC1 seedlings were HptII positive in PCR and expressed GFP protein as shown by western blot. CONCLUSIONS: This study demonstrated a stable transformation system was generated for Phal. orchids. This useful transformation protocol enables functional genomics studies and molecular breeding.
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Hepatocellular carcinoma (HCC) is a frequent cause of cancer-related death; therefore, more effective anticancer therapies for the treatment of HCC are needed. Histone deacetylase (HDAC) inhibitors serve as promising anticancer drugs because they can induce cell growth arrest and apoptosis. We previously reported that 3-[1-(4-methoxybenzenesulfonyl)-2,3-dihydro-1H-indol-5-yl]-N-hydroxyacrylamide (MPT0G009)-a novel 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines compound-demonstrated potent pan-HDAC inhibition and anti-inflammatory effects. In this study, we evaluated the anti-HCC activity of MPT0G009 in vitro and in vivo. Growth inhibition, apoptosis, and inhibited HDAC activity induced by MPT0G009 were more potent than a marketed HDAC inhibitor SAHA (Vorinostat). Furthermore, MPT0G009-induced apoptosis of Hep3B cells was characterized by an increase in apoptotic (sub-G1) population, loss of mitochondrial membrane potential, activation of caspase cascade, increased levels of pro-apoptotic protein (Bim), and decreased levels of anti-apoptotic proteins (Bcl-2, Bcl-xL, and FLICE-inhibitory protein); the downregulation FLIP by MPT0G009 is mediated through proteasome-mediated degradation and transcriptional suppression. In addition, combinations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with lower concentrations (0.1 µM) of MPT0G009 were synergistic in cell growth inhibition and apoptosis in HCC cells. In the in vivo model, MPT0G009 markedly reduced Hep3B xenograft tumor volume, inhibited HDAC activities, and induced apoptosis in the Hep3B xenografts. Our results demonstrate that MPT0G009 is a potential new candidate drug for HCC therapy.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Ácidos Hidroxâmicos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Sulfonamidas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos Nus , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A one-pot approach has been developed to synthesize copper nanocluster (Cu NC) aggregates from copper nitrate and mercaptobenzoic acid (MBA). Cu NCs prepared separately in the three isomers of MBA exhibit different physical and optical properties.
RESUMO
The signal transduction of acetylated histone can be processed through a recognition module, bromodomain. Several inhibitors targeting BRD4, one of the bromodomain members, are in clinical trials as anticancer drugs. Hereby, we report our efforts on discovery and optimization of a new series of 2-thiazolidinones as BRD4 inhibitors along our previous study. In this work, guided by crystal structure analysis, we reversed the sulfonamide group and identified a new binding mode. A structure-activity relationship study on this new series led to several potent BRD4 inhibitors with IC50 of about 0.05-0.1 µM in FP binding assay and GI50 of 0.1-0.3 µM in cell based assays. To complete the lead-like assessment of this series, we further checked its effects on BRD4 downstream protein c-Myc, investigated its selectivity among five different bromodomain proteins, as well as the metabolic stability test, and reinforced the utility of 2-thiazolidinone scaffold as BET bromodomain inhibitors in novel anticancer drug development.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Descoberta de Drogas , Proteínas Nucleares/antagonistas & inibidores , Tiazolidinas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Antineoplásicos/síntese química , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HT29 , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Tiazolidinas/síntese química , Tiazolidinas/química , Células Tumorais CultivadasRESUMO
The DNA methyltransferases (DNMTs) found in mammals include DNMT1, DNMT3A, and DNMT3B and are attractive targets in cancer chemotherapy. DNMT1 was the first among the DNMTs to be characterized, and it is responsible for maintaining DNA methylation patterns. A number of DNMT inhibitors have been reported, but most of them are nucleoside analogs that can lead to toxic side effects and lack specificity. By combining docking-based virtual screening with biochemical analyses, we identified a novel compound, DC_05. DC_05 is a non-nucleoside DNMT1 inhibitor with low micromolar IC50 values and significant selectivity toward other AdoMet-dependent protein methyltransferases. Through a process of similarity-based analog searching, compounds DC_501 and DC_517 were found to be more potent than DC_05. These three potent compounds significantly inhibited cancer cell proliferation. The structure-activity relationship (SAR) and binding modes of these inhibitors were also analyzed to assist in the future development of more potent and more specific DNMT1 inhibitors.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Humanos , Concentração Inibidora 50 , Relação Estrutura-AtividadeRESUMO
Male sterility plays an important role in F1 hybrid seed production. We identified a male-sterile rice (Oryza sativa) mutant with impaired pollen development and a single T-DNA insertion in the transcription factor gene bHLH142. Knockout mutants of bHLH142 exhibited retarded meiosis and defects in tapetal programmed cell death. RT-PCR and in situ hybridization analyses showed that bHLH142 is specifically expressed in the anther, in the tapetum, and in meiocytes during early meiosis. Three basic helix-loop-helix transcription factors, UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141) are known to function in rice pollen development. bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR1 and EAT1 in pollen development. In vivo and in vitro assays demonstrated that bHLH142 and TDR1 proteins interact. Transient promoter assays demonstrated that regulation of the EAT1 promoter requires bHLH142 and TDR1. Consistent with these results, 3D protein structure modeling predicted that bHLH142 and TDR1 form a heterodimer to bind to the EAT1 promoter. EAT1 positively regulates the expression of AP37 and AP25, which induce tapetal programmed cell death. Thus, in this study, we identified bHLH142 as having a pivotal role in tapetal programmed cell death and pollen development.
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A simple and fast method based on magnetic nanoparticles (MNPs) and capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection was developed for the detection of C-reactive protein (CRP). To optimize the CZE conditions, several factors including buffer compositions, buffer ionic strength, buffer pH, applied voltage and capillary temperature have been examined. The optimal separation buffer selected was a 30 mM sodium phosphate (PB) buffer, pH 8.0. The optimal CE applied voltage and temperature selected were 20 kV and 35°C, respectively. The CZE profile of the MNP-1°Ab-CRP-2°Ab/FITC bioconjugates showed good reproducibility. One major peak was observed for the MNP bioconjugates. The quantitative analysis also showed good results. The coefficient of variation (CV%) for the major peak area was 8.7%, and the CV% for the major peak migration time was 2.5%. The linear range for CRP analysis was 10-150 µg/mL, and the concentration limit of detection (LOD) was 9.2 µg/mL. Non-specific interactions between bovine serum albumin (BSA) and the system can be prevented by including 10% (v/v) of human plasma in the binding buffers. The CE/LIF method might be helpful for analyzing high concentrations of CRP in a patient's plasma after an acute-phase inflammation. This new method demonstrated the possibility of using MNPs and CE/LIF for the detection of proteins, and provided information for the establishment of appropriate CE conditions.
Assuntos
Proteína C-Reativa/análise , Eletroforese Capilar/métodos , Nanopartículas de Magnetita/química , Espectrometria de Fluorescência/métodos , Proteína C-Reativa/química , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Modelos Lineares , Concentração Osmolar , Tamanho da Partícula , Reprodutibilidade dos TestesRESUMO
GPR142 is a G protein-coupled receptor that is predominantly expressed in pancreatic ß-cells. GPR142 agonists stimulate insulin secretion in the presence of high glucose concentration, so that they could be novel insulin secretagogues with reduced or no risk of hypoglycemia. We report here the optimization of HTS hit compound 1 toward a proof of concept compound 33, which showed potent glucose lowering effects during an oral glucose tolerance test in mice and monkeys.