RESUMEN
Ras homolog gene family member (RhoA) is a GTPase and a member of the RAS superfamily of GTPases. RhoA is a master regulator of the actin cytoskeleton. It inhibits axon growth preventing repair and recovery following spinal cord and traumatic brain injuries. Despite decades of research into the biological function of Rho GTPases, there exist no small-molecule Rho inhibitors. Here, we screen a library of cysteine electrophiles to explore whether covalent bond formation at Cys-107 leads to inhibition of RhoA activation by guanine exchange factor Trio. Two fragments, propiolamide 1 (ACR-895) and acrylamide 2 (ACR-917), inhibited RhoA nucleotide exchange by Trio in a time-dependent manner. The fragments formed a covalent bond with wild-type RhoA but not Cys107Ser RhoA mutant. Time- and concentration-dependent studies led to equilibrium constants KIs and reaction rates that correspond to t1/2 values in the single-digit hour range. One fragment was selective for RhoA over Rac1 GTPase and had no effect on KRAS nucleotide exchange by SOS1. The fragments did not inhibit RhoA binding to ROCK effector protein. This work establishes Cys-107 as a suitable site for Rho GTPase inhibition and provides fragment starting points for the future development of Rho GTPase covalent inhibitors that could have profound implications in the treatment of patients with injuries of the central nervous system.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido , Guanina , Humanos , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Nucleótidos/metabolismoRESUMEN
Acute lung injury (ALI) is a severe lung respiratory failure characterized by high morbidity and mortality. Novel findings demonstrated the critical roles of long non-coding RNA (lncRNA) in ALI. Here, we tried to investigate the roles and potential mechanism of lncRNA X-inactive specific transcript (XIST) in ALI. Results illustrated that lncRNA XIST was up-regulated in the lipopolysaccharide (LPS)-induced ALI mice models and pulmonary endothelial cells. Biofunctional assays unveiled that knockdown of XIST repressed the inï¬ammatory response and apoptosis in LPS-induced endothelial cells. Mechanistically, XIST acted as the miR-146a-5p sponge to positively regulate STAT3. Moreover, STAT3 combined the promoter region of XIST to accelerate the transcription, constituting the positive feedback loop of XIST/miR-146a-5p/STAT3 in ALI. Collectively, these findings suggested that XIST knockdown attenuates the LPS-induced ALI, providing a potential therapeutic target.
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Lesión Pulmonar Aguda/genética , MicroARNs/genética , ARN Largo no Codificante/genética , Factor de Transcripción STAT3/genética , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/patología , Animales , Apoptosis/genética , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/toxicidad , Ratones , Transcripción GenéticaRESUMEN
Rationale: Chemoresistance is a major obstacle in prostate cancer (PCa) treatment. We sought to understand the underlying mechanism of PCa chemoresistance and discover new treatments to overcome docetaxel resistance. Methods: We developed a novel phenotypic screening platform for the discovery of specific inhibitors of chemoresistant PCa cells. The mechanism of action of the lead compound was investigated using computational, molecular and cellular approaches. The in vivo toxicity and efficacy of the lead compound were evaluated in clinically-relevant animal models. Results: We identified LG1980 as a lead compound that demonstrates high selectivity and potency against chemoresistant PCa cells. Mechanistically, LG1980 binds embryonic ectoderm development (EED), disrupts the interaction between EED and enhancer of zeste homolog 2 (EZH2), thereby inducing the protein degradation of EZH2 and inhibiting the phosphorylation and activity of EZH2. Consequently, LG1980 targets a survival signaling cascade consisting of signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP binding cassette B 1 (ABCB1) and survivin. As a lead compound, LG1980 is well tolerated in mice and effectively suppresses the in vivo growth of chemoresistant PCa and synergistically enhances the efficacy of docetaxel in xenograft models. Conclusions: These results indicate that pharmacological inhibition of EED-EZH2 interaction is a novel strategy for the treatment of chemoresistant PCa. LG1980 and its analogues have the potential to be integrated into standard of care to improve clinical outcomes in PCa patients.
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Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Descubrimiento de Drogas/métodos , Resistencia a Antineoplásicos , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Neoplasias de la Próstata/tratamiento farmacológico , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Docetaxel/farmacología , Docetaxel/uso terapéutico , Sinergismo Farmacológico , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Survivin/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
One of the primary mechanisms of tumor cell immune evasion is the loss of antigenicity, which arises due to lack of immunogenic tumor antigens as well as dysregulation of the antigen processing machinery. In a screen for small-molecule compounds from herbal medicine that potentiate T cell-mediated cytotoxicity, we identified atractylenolide I (ATT-I), which substantially promotes tumor antigen presentation of both human and mouse colorectal cancer (CRC) cells and thereby enhances the cytotoxic response of CD8+ T cells. Cellular thermal shift assay (CETSA) with multiplexed quantitative mass spectrometry identified the proteasome 26S subunit non-ATPase 4 (PSMD4), an essential component of the immunoproteasome complex, as a primary target protein of ATT-I. Binding of ATT-I with PSMD4 augments the antigen-processing activity of immunoproteasome, leading to enhanced MHC-I-mediated antigen presentation on cancer cells. In syngeneic mouse CRC models and human patient-derived CRC organoid models, ATT-I treatment promotes the cytotoxicity of CD8+ T cells and thus profoundly enhances the efficacy of immune checkpoint blockade therapy. Collectively, we show here that targeting the function of immunoproteasome with ATT-I promotes tumor antigen presentation and empowers T cell cytotoxicity, thus elevating the tumor response to immunotherapy.
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Presentación de Antígeno/efectos de los fármacos , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunidad Celular/efectos de los fármacos , Inmunoterapia , Lactonas/farmacología , Neoplasias Experimentales/terapia , Sesquiterpenos/farmacología , Animales , Antígenos de Neoplasias/genética , Células HCT116 , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacocinética , Inmunidad Celular/genética , Lactonas/farmacocinética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Sesquiterpenos/farmacocinéticaRESUMEN
There is substantial interest in the development of small molecules that inhibit the tight and highly challenging protein-protein interaction between the glycophosphatidylinositol (GPI)-anchored cell surface receptor uPAR and the serine protease uPA. While preparing derivatives of a fragment-like compound that previously emerged from a computational screen, we identified compound 5 (IPR-3242), which inhibited binding of uPA to uPAR with submicromolar IC50s. The high inhibition potency prompted us to carry out studies to rule out potential aggregation, lack of stability, reactivity, and nonspecific inhibition. We designed and prepared 16 derivatives to further explore the role of each substituent. Interestingly, the compounds only partially inhibited binding of a fluorescently labeled α-helical peptide that binds to uPAR at the uPAR·uPA interface. Collectively, the results suggest that the compounds bind to uPAR outside of the uPAR·uPA interface, trapping the receptor into a conformation that is not able to bind to uPA. Additional studies will have to be carried out to determine whether this unique inhibition mechanism can occur at the cell surface.
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N6 -methyladenosine (m6 A) and long noncoding RNAs (lncRNAs) are both crucial regulators in non-small-cell lung cancer (NSCLC) tumorigenesis. However, the pathological roles of m6 A and lncRNAs in NSCLC progression are still limited and undefined. Here, lncRNA ABHD11-AS1 was upregulated in NSCLC tissue specimens and cells and the ectopic overexpression was closely correlated with unfavorable prognosis of NSCLC patients. Functionally, ABHD11-AS1 promoted the proliferation and Warburg effect of NSCLC. Mechanistically, m6 A profile was analyzed by methylated RNA immunoprecipitation sequencing (MeRIP-Seq). MeRIP-Seq presented that there was m6 A modification site in ABHD11-AS1. m6 A methyltransferase-like 3 (METTL3) installed the m6 A modification and enhanced ABHD11-AS1 transcript stability to increase its expression. In conclusion, our findings highlight the function and mechanism of METTL3-induced ABHD11-AS1 in NSCLC and inspire the understanding of m6 A and lncRNA in cancer biology.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , ARN Largo no Codificante/metabolismo , Efecto Warburg en Oncología , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Persona de Mediana Edad , Estabilidad del ARN , ARN Largo no Codificante/genética , Transducción de SeñalRESUMEN
Lead smelting slag (LSS) has been identified as general industrial solid waste, which is produced from the pyrometallurgical treatment of the Shuikoushan process for primary lead production in China. The LSS-based geopolymer was synthesized after high-energy ball milling. The effect of unconfined compressive strength (UCS) on the synthesis parameters of the geopolymer was optimized. Under the best parameters of the geopolymer (modulus of water glass was 1-1.5, dosage of water glass (W(SiO2+Na2O)) was 5% and water-to-binder ratio was 0.2), the UCS reached 76.09 MPa after curing for 28 days. The toxicity characteristic leaching procedure (TCLP) leaching concentration of Zn from LSS fell from 167.16 to 93.99 mg/L after alkali-activation, which was below the limit allowed. Meanwhile, C-S-H and the geopolymer of the hydration products were identified from the geopolymer. In addition, the behavior of iron was also discussed. Then, the hydration process characteristics of the LSS-based geopolymer were proposed. The obtained results showed that Ca2+ and Fe2+ occupied the site of the network as modifiers in the glass phase and then dissociated from the glass network after the water glass activation. At the same time, C-S-H, the geopolymer and Fe(OH)2 gel were produced, and then the Fe(OH)2 was easily oxidized to Fe(OH)3 under the air curing conditions. Consequently, the conclusion was drawn that LSS was an implementable raw material for geopolymer production.
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Residuos Industriales/análisis , Plomo , Metalurgia , Dióxido de Silicio , Álcalis , ChinaRESUMEN
Ral (Ras-like) GTPases are directly activated by oncogenic Ras GTPases. Mutant K-Ras (G12C) has enabled the development of covalent K-Ras inhibitors currently in clinical trials. However, Ral, and the overwhelming majority of mutant oncogenic K-Ras, are devoid of a druggable pocket and lack an accessible cysteine for the development of a covalent inhibitor. Here, we report that covalent bond formation by an aryl sulfonyl fluoride electrophile at a tyrosine residue (Tyr-82) inhibits guanine exchange factor Rgl2-mediated nucleotide exchange of Ral GTPase. A high-resolution 1.18-Å X-ray cocrystal structure shows that the compound binds to a well-defined binding site in RalA as a result of a switch II loop conformational change. The structure, along with additional high-resolution crystal structures of several analogs in complex with RalA, confirm the importance of key hydrogen bond anchors between compound sulfone oxygen atoms and Ral backbone nitrogen atoms. Our discovery of a pocket with features found on known druggable sites and covalent modification of a bystander tyrosine residue present in Ral and Ras GTPases provide a strategy that could lead to therapeutic agent targeting oncogenic Ras mutants that are devoid of a cysteine nucleophile.
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Proteínas de Unión al GTP ral/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Factores de Intercambio de Guanina Nucleótido/metabolismo , Bibliotecas de Moléculas Pequeñas , Proteínas de Unión al GTP ral/metabolismoRESUMEN
In recent years, acid rain had a serious negative impact on the leaching behavior of industrial waste residue. Researches were mainly focused on the environmental hazards of heavy metal in the leachate, but ignored the effects of heavy metal speciation on the stability of waste residue in the subsequent stabilization process. In this study, the unstable calcium-arsenic compounds in the arsenic calcium residue were firstly removed by leaching process; subsequently, the crystallization agent was added to treat the remaining calcium-arsenic mixture. The results of the leaching process demonstrated that the decrease in particle size and pH value directly affected the increase in the cumulative leaching amount of arsenic, and the cumulative leaching ratio reached 1.55%. In addition, the concentration of arsenic decreased from 3583 to 49.1 mg L-1. After the crystallization process, the arsenic concentration was lower than the limit value of Identification Standards for Hazardous Wastes (GB 5085.3-2007). The SEM analysis showed the bulk structures, and XRD pattern confirmed that they were the stable compounds. Moreover, the result of XRD and SEM illustrated that acid concentration, chloride ions and sulfate ions were contributed to the transformation and growth of stable calcium arsenate compounds. Therefore, effective control of the acidity of acid rain, the type of anions in acid rain, and the particle size of residues would contribute to adjusting the arsenic speciation to be more stable. The leaching-crystallization process was of great significance to improve the stability of the arsenic-containing residue.
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Lluvia Ácida , Arseniatos/química , Arsénico/química , Compuestos de Calcio/química , Lluvia Ácida/análisis , Arsénico/análisis , Calcio/análisis , Calcio/química , Cristalización , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Rastreo , Tamaño de la Partícula , Difracción de Rayos XRESUMEN
Municipal solid waste incineration fly ash (MSWI FA) and granulated lead smelting slag (GLSS) are toxic industrial wastes. In the present study, granulated lead smelting slag (GLSS) was pretreated as a geopolymer precursor through the high-energy ball milling activation process, which could be used as a geopolymeric solidification/stabilization (S/S) reagent for MSWI FA. The S/S process has been estimated through the physical properties and heavy metals leachability of the S/S matrices. The results show that the compressive strength of the geopolymer matrix reaches 15.32 MPa after curing for 28 days under the best parameters, and the physical properties meet the requirement of MU10 grade fly ash brick. In addition, the toxicity characteristic leaching procedure (TCLP) test results show that arsenic and heavy metals are immobilized effectively in the geopolymer matrix, and their concentrations in the leachate are far below the US EPA TCLP limits. The hydration products of the geopolymer binder are characterized by X-ray diffraction and Fourier transform infrared methods. The results show that the geopolymer gel and Friedel's salt are the main hydration products. The S/S mechanism of the arsenic and heavy metals in the geopolymer matrix mainly involves physical encapsulation of the geopolymer gel, geopolymer adsorption and ion exchange of Friedel's salt.
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Ceniza del Carbón/química , Plomo/química , Polímeros/química , Eliminación de Residuos/métodos , Contaminantes Químicos del Agua/química , Arsénico/química , Incineración , Residuos Industriales , Metales Pesados/químicaRESUMEN
A novel method to activate red mud was proposed in this study. Municipal solid waste incineration fly ash (MSWIFA) and red mud were utilized to prepare red mud-based geopolymer materials (RGM). The hydration characteristics of RGM were studied by X-ray diffraction, scanning electronic microscopy, and Fourier transform infrared spectroscopy. The long-term stability and physical properties of RGM were tested by freeze-thaw cycle, European Community Bureau reference (BCR) and unconfined compressive strength (UCS) tests. Results showed that mechanical activation can not only effectively activate red mud, but also effectively improve the reaction of MSWIFA and red mud. When 14% sodium silicate was added to the binder, the UCS reached 12.75â¯MPa at 28â¯days. In the RGM, aluminosilicate was effectively activated by mechanical activation and reacted with calcium ion to form complex hydration products. The activator reacts adequately with activated aluminum to form a high-strength geopolymer. The freeze-thaw cycles and BCR test results also showed that the RGM had long-term stability and the characteristics satisfied the requirements of MU10 fly ash bricks. This study demonstrated that RGM may be utilized in cement composites.
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Carbono , Ceniza del Carbón , Materiales de Construcción , Incineración , Residuos Sólidos , Difracción de Rayos XRESUMEN
The interactions between Fe2SiO4 and ZnO play an essential role in the recovery of zinc from copper slag. The dissolution and substitution mechanism of ZnO in fayalite were investigated by using TG-DSC, XRD, PPMS DynaCool, XPS, Mossbauer and SEM-EDS analyses and compared with MS calculation results. The results indicate that the dissolution and substitution are actually processes of the penetrating dissolution of Zn(II) ions that can be divided into three steps: 1) ZnO dissociates into Zn1-yO and Zn(II) ions; 2) Zn(II) penetrates the gap of the octahedron outer layer to substitute Fe(II) sites in the internal structure of SiOFe(II) (M2) to form (Fe2-x, Znx)SiO4; 3) Fe(II) is forced to migrate to the surface of (Fe2-x, Znx)SiO4 to form (Zn1-y, Fe(II)y)O. These findings can be derived the occurrence state and distribution of zinc in copper slag theoretically.
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Bone is a common site of metastasis for breast, prostate, lung, kidney and other cancers. Bone metastases are incurable, and substantially reduce patient quality of life. To date, there exists no small-molecule therapeutic agent that can reduce tumor burden in bone. This is partly attributed to the lack of suitable in vitro assays that are good models of tumor growth in bone. Here, we take advantage of a novel ex vivo model of bone colonization to report a series of pyrrolopyrazolone small molecules that inhibit cancer cell invasion and ex vivo tumor growth in bone at single-digit micromolar concentration. We find that the compounds modulated the expression levels of genes associated with bone-forming osteoblasts, bone-destroying osteoclasts, cancer cell viability and metastasis. Our compounds provide chemical tools to uncover novel targets and pathways associated with bone metastasis, as well as for the development of compounds to prevent and reverse bone tumor growth in vivo.
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Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ratones , Estructura Molecular , Embarazo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1 subunit (CaVα1) that is engaged in protein-protein interactions with auxiliary α2/δ and ß subunits. The high-affinity CaV2.2α1â CaVß3 protein-protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1â CaVß3 protein-protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1 Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVß3 Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein-protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking.
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Bloqueadores de los Canales de Calcio/farmacología , Activación del Canal Iónico/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacocinética , Femenino , Células HEK293 , Humanos , Transporte Iónico , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Ratones , Neuralgia/prevención & control , Nocicepción/efectos de los fármacos , Unión Proteica , Ratas , Ratas Sprague-Dawley , Bibliotecas de Moléculas Pequeñas/farmacocinéticaRESUMEN
BACKGROUND: Circulating tumor cells (CTCs) is a promising biomarker for cancer prognosis and monitoring. Molecular characterizing of CTCs could provide beneficial information on the basis of CTCs counting. OBJECTIVE: To investigate the epithelial-mesenchymal transition (EMT) phenotypes and GALC mRNA expression of CTCs in non-small cell lung cancer (NSCLC) patients. METHODS: We analyzed the baseline number, EMT classification, and GALC expression of CTCs in 47 NSCLC patients using CanPatrol platform and RNA in situ hybridization technique. RESULTS: CTCs were detected in 91.5% patients ranging 0-47/5 mL blood. Increased CTCs were associated with advanced tumor stages (6/5 mL) compared with early stages (3.5/5 mL). Patients with effective treatment response presented lower CTCs (3.5/5 mL) than patients with insufficient response (7/5 mL). Epithelial, hybrid and mesenchymal CTCs were detected in 55.4%, 78.7% and 61.7% patients, respectively. Patients with distant metastasis and poor curative outcomes presented higher level of EMT-CTCs. GALC expression was positive in CTCs of 80.6% patients and closely correlated with tumor number and distant metastasis and treatment outcomes. CONCLUSIONS: EMT phenotypes and GALC expression of CTCs are correlated with cancer metastasis and therapeutic outcomes, suggesting them to be potential markers for the prognosis of NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Transición Epitelial-Mesenquimal/genética , Galactosilceramidasa/genética , Expresión Génica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Células Neoplásicas Circulantes , Adulto , Anciano , Biomarcadores de Tumor , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Fenotipo , Pronóstico , ARN Mensajero/genéticaRESUMEN
The aim of the present study was to investigate the novel mechanisms of forkhead box protein P3 (foxp3) in T regulatory (Treg) cells in lung cancer behavior. Treg cells were isolated from the peripheral blood of healthy volunteers and then cocultured with 95D cells. A plasmid overexpressing foxp3 was constructed and transfected into Treg cells and an MTS assay was performed to assess cell viability. Flow cytometry was performed to evaluate cell apoptosis and reverse transcriptionquantitative polymerase chain reaction was used to measure mRNA expression. A Transwell assay was used to assess cell invasion. Treg cells were successfully isolated from peripheral blood with purity of 94.26%. Foxp3 expression in Treg cells was significantly increased following coculture with 95D cells, while matrix metalloproteinase9 expression was upregulated in 95D cells cocultured with Treg cells. The apoptosis, invasion and migration abilities of 95D cells were suppressed by coculture with Treg cells, whereas the adhesive ability was enhanced. Foxp3 overexpression in Treg cells enhanced the viability and invasiveness of 95D cells, whereas cell adhesion and migration were decreased. The results of the present study demonstrate that the viability and invasiveness of 95D cells are enhanced by foxp3 overexpression in Treg cells, indicating that increased levels of foxp3 in the tumor microenvironment may promote tumor cell growth.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Factores de Transcripción Forkhead/genética , Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Adulto , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transcriptoma , Microambiente Tumoral/inmunologíaRESUMEN
A new method in which Pb/Zn smelter waste containing arsenic and heavy metals (arsenic sludge), red mud and lime are utilized to prepare red mud-based cementitious material (RCM) is proposed in this study. XRD, SEM, FTIR and unconfined compressive strength (UCS) tests were employed to assess the physicochemical properties of RCM. In addition, ettringite and iron oxide-containing ettringite were used to study the hydration mechanism of RCM. The results show that the UCS of the RCM (red mud+arsenic sludge+lime) was higher than that of the binder (red mud+arsenic sludge). When the mass ratio of m (binder): m (lime) was 94:6 and then maintained 28days at ambient temperature, the UCS reached 12.05MPa. The red mud has potential cementitious characteristics, and the major source of those characteristics was the aluminium oxide. In the red mud-arsenic sludge-lime system, aluminium oxide was effectively activated by lime and gypsum to form complex hydration products. Some of the aluminium in ettringite was replaced by iron to form calcium sulfoferrite hydrate. The BCR and leaching toxicity results show that the leaching concentration was strongly dependent on the chemical speciation of arsenic and the hydration products. Therefore, the investigated red mud and arsenic sludge can be successfully utilized in cement composites to create a red mud-based cementitious material.
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Flotation waste of copper slag (FWCS), neutralization sludge (NS), and arsenic-containing gypsum sludge (GS), both of which are difficult to dispose of, are major solid wastes produced by the copper smelting. This study focused on the co-treatment of FWCS, NS, and GS for solidification/stabilization of arsenic and heavy metals with minimal cement clinker. Firstly, the preparation parameters of binder composed of FWCS, NS, and cement clinker were optimized to be FWCS dosage of 40%, NS dosage of 10%, cement clinker dosage of 50%, mill time of 1.5 h, and water-to-binder ratio of 0.25. On these conditions, the unconfined compressive strength (UCS) of the binder reached 43.24 MPa after hydration of 28 days. Then, the binder was used to solidify/stabilize the As-containing GS. When the mass ratio of binder-to-GS was 5:5, the UCS of matrix can reach 11.06 MPa after hydration of 28 days, meeting the required UCS level of MU10 brick in China. Moreover, arsenic and other heavy metals in FWCS, NS, and GS were effectively solidified or stabilized. The heavy metal concentrations in leachate were much lower than those in the limits of China standard leaching test (CSLT). Therefore, the matrices were potential to be used as bricks in some constructions. XRD analysis shows that the main hydration products of the matrix were portlandite and calcium silicate hydrate. These hydration products may play a significant role in the stabilization/solidification of arsenic and heavy metals.
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Arsénico/química , Compuestos de Calcio/química , Sulfato de Calcio/química , Materiales de Construcción/análisis , Cobre/química , Metales Pesados/análisis , Aguas del Alcantarillado/análisis , Silicatos/química , Arsénico/análisis , Sulfato de Calcio/análisis , China , Cobre/análisis , Metales Pesados/químicaRESUMEN
Pyrimidine dimers are the most common DNA lesions generated under UV radiation. To reveal the molecular mechanisms behind their formation, it is of significance to reveal the roles of each pyrimidine residue. We thus replaced the 5'-pyrimidine residue with a photochemically inert xylene moiety (X). The electron-rich X can be readily oxidized but not reduced, defining the direction of interbase electron transfer (ET). Irradiation of the XpT dinucleotide under 254â nm UV light generates two major photoproducts: a pyrimidine (6-4) pyrimidone analog (6-4PP) and an analog of the so-called spore photoproduct (SP). Both products are formed by reaction at C4=O of the photo-excited 3'-thymidine (T), which indicates that excitation of a single "driver" residue is sufficient to trigger pyrimidine dimerization. Our quantum-chemical calculations demonstrated that photo-excited 3'-T accepts an electron from 5'-X. The resulting charge-separated radical pair lowers its energy upon formation of interbase covalent bonds, eventually yielding 6-4PP and SP.
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Fosfatos de Dinucleósidos/metabolismo , Electrones , Dímeros de Pirimidina/biosíntesis , Fosfatos de Dinucleósidos/químicaRESUMEN
Target-specific scoring methods are more commonly used to identify small-molecule inhibitors among compounds docked to a target of interest. Top candidates that emerge from these methods have rarely been tested for activity and specificity across a family of proteins. In this study we docked a chemical library into CaMKIIδ, a member of the Ca2+ /calmodulin (CaM)-dependent protein kinase (CaMK) family, and re-scored the resulting protein-compound structures using Support Vector Machine SPecific (SVMSP), a target-specific method that we developed previously. Among the 35 selected candidates, three hits were identified, such as quinazoline compound 1 (KIN-1; N4-[7-chloro-2-[(E)-styryl]quinazolin-4-yl]-N1,N1-diethylpentane-1,4-diamine), which was found to inhibit CaMKIIδ kinase activity at single-digit micromolar IC50 . Activity across the kinome was assessed by profiling analogues of 1, namely 6 (KIN-236; N4-[7-chloro-2-[(E)-2-(2-chloro-4,5-dimethoxyphenyl)vinyl]quinazolin-4-yl]-N1,N1-diethylpentane-1,4-diamine), and an analogue of hit compound 2 (KIN-15; 2-[4-[(E)-[(5-bromobenzofuran-2-carbonyl)hydrazono]methyl]-2-chloro-6-methoxyphenoxy]acetic acid), namely 14 (KIN-332; N-[(E)-[4-(2-anilino-2-oxoethoxy)-3-chlorophenyl]methyleneamino]benzofuran-2-carboxamide), against 337 kinases. Interestingly, for compound 6, CaMKIIδ and homologue CaMKIIγ were among the top ten targets. Among the top 25 targets of 6, IC50 values ranged from 5 to 22⠵m. Compound 14 was found to be not specific toward CaMKII kinases, but it does inhibit two kinases with sub-micromolar IC50 values among the top 25. Derivatives of 1 were tested against several kinases including several members of the CaMK family. These data afforded a limited structure-activity relationship study. Molecular dynamics simulations with explicit solvent followed by end-point MM-GBSA free-energy calculations revealed strong engagement of specific residues within the ATP binding pocket, and also changes in the dynamics as a result of binding. This work suggests that target-specific scoring approaches such as SVMSP may hold promise for the identification of small-molecule kinase inhibitors that exhibit some level of specificity toward the target of interest across a large number of proteins.