RESUMEN
Protein misfolding and aggregation is a characteristic of many neurodegenerative disorders, including Alzheimer's and Parkinson's disease. The oligomers generated during aggregation are likely involved in disease pathogenesis and present promising biomarker candidates. However, owing to their small size and low concentration, specific tools to quantify and characterize aggregates in complex biological samples are still lacking. Here, we present single-molecule two-color aggregate pulldown (STAPull), which overcomes this challenge by probing immobilized proteins using orthogonally labeled detection antibodies. By analyzing colocalized signals, we can eliminate monomeric protein and specifically quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we demonstrate that this approach can specifically detect aggregates with a limit of detection of 5 picomolar. Furthermore, we show that STAPull can be used in a range of samples, including human biofluids. STAPull is applicable to protein aggregates from a variety of disorders and will aid in the identification of biomarkers that are crucial in the effort to diagnose these diseases.
Asunto(s)
Enfermedad de Parkinson , Agregado de Proteínas , Humanos , Enfermedad de Parkinson/metabolismoRESUMEN
BACKGROUND: Ubiquitination is essential for many cellular processes in eukaryotes, including 26S proteasome-dependent protein degradation, cell cycle progression, transcriptional regulation, and signal transduction. Although numerous ubiquitinated proteins have been empirically identified, their cognate ubiquitin E3 ligases remain largely unknown. RESULTS: Here, we generate a complete ubiquitin E3 ligase-encoding open reading frames (UbE3-ORFeome) library containing 98.94% of the 1515 E3 ligase genes in the rice (Oryza sativa L.) genome. In the test screens with four known ubiquitinated proteins, we identify both known and new E3s. The interaction and degradation between several E3s and their substrates are confirmed in vitro and in vivo. In addition, we identify the F-box E3 ligase OsFBK16 as a hub-interacting protein of the phenylalanine ammonia lyase family OsPAL1-OsPAL7. We demonstrate that OsFBK16 promotes the degradation of OsPAL1, OsPAL5, and OsPAL6. Remarkably, we find that overexpression of OsPAL1 or OsPAL6 as well as loss-of-function of OsFBK16 in rice displayed enhanced blast resistance, indicating that OsFBK16 degrades OsPALs to negatively regulate rice immunity. CONCLUSIONS: The rice UbE3-ORFeome is the first complete E3 ligase library in plants and represents a powerful proteomic resource for rapid identification of the cognate E3 ligases of ubiquitinated proteins and establishment of functional E3-substrate interactome in plants.
Asunto(s)
Oryza , Ubiquitina-Proteína Ligasas , Oryza/genética , Oryza/metabolismo , Proteómica , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo , Ubiquitinación , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
α,ß-Deuterated amines are crucial for the development of deuterated drugs. We intend to introduce the novel tandem H/D exchange-single electron transfer (SET) reductive deuteration strategy with high pot- and reagent-economy by the synthesis of α,ß-deuterated amine using nitrile as the precursor. The H/D exchange of the -CH2CN group was achieved by D2O/Et3N, which were also the required reagents in the tandem SmI2-mediated SET reductive deuteration of the α-deuterated nitrile. The potential application of this method was further showcased by the synthesis of bevantolol-d4.
Asunto(s)
Aminas , Deuterio , Transporte de ElectrónRESUMEN
Spatial transcriptomic and proteomic technologies have provided new opportunities to investigate cells in their native microenvironment. Here we present Giotto, a comprehensive and open-source toolbox for spatial data analysis and visualization. The analysis module provides end-to-end analysis by implementing a wide range of algorithms for characterizing tissue composition, spatial expression patterns, and cellular interactions. Furthermore, single-cell RNAseq data can be integrated for spatial cell-type enrichment analysis. The visualization module allows users to interactively visualize analysis outputs and imaging features. To demonstrate its general applicability, we apply Giotto to a wide range of datasets encompassing diverse technologies and platforms.
Asunto(s)
Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Hibridación in Situ , Programas Informáticos , Análisis de Datos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Especificidad de Órganos/genética , Análisis Espacial , TranscriptomaRESUMEN
Osteogenic peptides are often introduced to improve biological activities and the osteogenic ability of artificial bone materials as an effective approach. Covalent bindings between the peptide and the host material can increase the molecular interactions and make the functionalized surface more stable. However, covalent bindings through different functional groups can bring different effects on the overall bioactivities. In this study, carboxyl and amino groups were respectively introduced onto carbon nanotubes, a nanoreinforcement for synthetic scaffold materials, which were subsequently covalently attached to the RGD/BMP-2 osteogenic peptide. MC3T3-E1 cells were cultured on scaffolds containing peptide-modified carbon nanotubes. The results showed that the peptide through the amino group binding could promote cell functions more effectively than those through carboxyl groups. The mechanism may be that the amino group could bring more positive charges to carbon nanotube surfaces, which further led to differences in the peptide conformation, protein adsorption, and targeting osteogenic effects. Our results provided an effective way of improving the bioactivities of artificial bone materials by chemically binding osteogenic peptides.
Asunto(s)
Nanotubos de Carbono , Nanotubos de Péptidos , Huesos , Osteogénesis , PéptidosRESUMEN
This study provided a new method to in vitro evaluate the biocompatibility of nanoscaled scaffolds for tissue engineering with neutrophils other than ordinary cell culture. The neutrophils were separated from human peripheral blood of healthy subjects. In vitro degradation product of nanohydroxyapatite/collagen (nHAC), nanohydroxyapatite/collagen/poly (L-lactic acid) (nHACP), and nHACP reinforced by chitin fibers (nHACP/CF) in the D-Hank's Balanced Salt Solution (D-HBSS) was used as the testing solution, which was thereafter mixed with the neutrophils. It was shown that the cell survival rate in the testing solutions had no significant difference from that in the D-HBSS (control). However, from both gene and protein expression levels, the lactate dehydrogenase and tumor necrosis factor-alpha of the neutrophils in the nHACP/CF testing solution were found lowest during the whole testing period; the main reasons of which might be that the calcium release rate of the scaffold was slowest and that the pH value of its degradation solution was nearest to that of human body. Moreover, in vivo experiments showed that most inflammation reactions happened for nHAC and poly (L-lactic acid) groups, while the least inflammation reactions happened for nHACP/CF group in the subcutaneous dorsum of mice at 2 weeks after the surgery, which confirmed the in vitro findings. These results indicated that the pH value and the certain metal iron concentration of the nanoscaled scaffold degradation solution should be two important factors that significantly affect its biocompatibility. This study provides a simple and effective biocompatibility test method for biodegradable nanoscaled tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2117-2125, 2016.
Asunto(s)
Quitina/química , Colágeno/química , Durapatita/química , Ensayo de Materiales/métodos , Nanopartículas/química , Neutrófilos/metabolismo , Andamios del Tejido/química , Humanos , Neutrófilos/citologíaRESUMEN
As their name suggests, conductive nanomaterials (CNMs) are a type of functional materials, which not only have a high surface area to volume ratio, but also possess excellent conductivity. Thus far, CNMs have been widely used in biomedical applications, such as effectively transferring electrical signals, and providing a large surface area to adsorb proteins and induce cellular functions. Recent works propose further applications of CNMs in biosensors, tissue engineering, neural probes, and drug delivery. This review focuses on common types of CNMs and elaborates on their unique properties, which indicate that such CNMs have a potential to develop into a class of indispensable biomaterials for the diagnosis and therapy of human diseases.
Asunto(s)
Tecnología Biomédica/métodos , Conductividad Eléctrica , Nanoestructuras/química , Animales , Técnicas Biosensibles , Sistemas de Liberación de Medicamentos , Humanos , Ingeniería de TejidosRESUMEN
Bone regeneration is a complicated process that involves a series of biological events, such as cellular recruitment, proliferation and differentiation, and so forth, which have been found to be significantly affected by controlled drug delivery. Recently, a lot of research studies have been launched on the application of nanomaterials in controlled drug delivery for bone regeneration. In this article, the latest research progress in this area regarding the use of bioceramics-based, polymer-based, metallic oxide-based and other types of nanomaterials in controlled drug delivery for bone regeneration are reviewed and discussed, which indicates that the controlling drug delivery with nanomaterials should be a very promising treatment in orthopedics. Furthermore, some new challenges about the future research on the application of nanomaterials in controlled drug delivery for bone regeneration are described in the conclusion and perspectives part.
Asunto(s)
Materiales Biocompatibles/química , Regeneración Ósea/efectos de los fármacos , Preparaciones de Acción Retardada/química , Sistemas de Liberación de Medicamentos/métodos , Nanoestructuras/química , Animales , Materiales Biocompatibles/toxicidad , Preparaciones de Acción Retardada/toxicidad , Humanos , Nanoestructuras/toxicidad , Nanoestructuras/ultraestructura , Nanotecnología/métodosRESUMEN
The overexpression of P-glycoprotein (P-gp) in tumors leads to multidrug resistance (MDR), which is a significant obstacle in clinical cancer chemotherapy. The co-administration of anticancer drugs and MDR modulators is a promising strategy for overcoming this problem. Our study aimed to explore the reversal mechanism and safety of the MDR modulator LBM-A5 in vitro, and evaluate its pharmacokinetics and effects on doxorubicin metabolism in vivo. We evaluated an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay of anticancer agents mediated by LBM-A5, the effect of LBM-A5 on rhodamine123 intracellular accumulation, and the efflux in K562/DOX cells to investigate the reversal mechanisms of LBM-A5. The results showed that LBM-A5 inhibits rhodamine123 efflux and increases intracellular accumulation by inhibiting the efflux pump function of P-gp. Furthermore, the therapeutic index and CYP3A4 activity analysis in vitro suggested that LBM-A5 is reasonably safe to use. Also, LBM-A5 (10 mg/kg body mass) achieved the required plasma concentration in sufficient time to reverse MDR in vivo. Importantly, the LBM-A5 treatment group shared similar doxorubicin (DOX) pharmacokinetics with the free DOX group. Our results suggest that LBM-A5 effectively reverses MDR (EC50 = 483.6 ± 81.7 nmol·L(-1)) by inhibiting the function of P-gp, with relatively ideal pharmacokinetics and in a safe manner, and so may be a promising candidate for cancer chemotherapy research.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacocinética , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Múltiples Medicamentos/fisiología , Animales , Humanos , Células K562 , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
A novel coumarin-based fluorescence probe has been constructed for the selective and sensitive detection of hydrogen sulfide (H2 S). This probe displays high sensitivity and linearity to H2 S in degassed PBS buffers and fetal bovine serum. It reacts with H2 S with high selectivity over Cys, GSH, and other anions. Meanwhile, successful detection of H2 S in living cells was also demonstrated.
Asunto(s)
Cumarinas/química , Colorantes Fluorescentes/química , Sulfuro de Hidrógeno/análisis , Animales , Bovinos , Células/metabolismo , Colorantes Fluorescentes/síntesis química , Células HeLa , Hepatocitos/química , Hepatocitos/metabolismo , Humanos , Sulfuro de Hidrógeno/metabolismo , Especificidad por SustratoRESUMEN
Free fatty acid receptor 1 (FFA1) is a new potential drug target for the treatment of type 2 diabetes because of its role in amplifying glucose-stimulated insulin secretion in pancreatic ß-cell. In the present studies, we identified phenoxyacetic acid derivative (18b) as a potent FFA1 agonist (EC50=62.3 nM) based on the structure of phenylpropanoic acid derivative 4p. Moreover, compound 18b could significantly improve oral glucose tolerance in ICR mice and dose-dependently reduced glucose levels in type 2 diabetic C57BL/6 mice without observation of hypoglycemic side effect. Additionally, compound 18b exhibited acceptable PK profiles. In summary, compound 18b with ideal PK profiles exhibited good activity in vitro and in vivo, and might be a promising drug candidate for the treatment of diabetes mellitus.
Asunto(s)
Acetatos/síntesis química , Acetatos/farmacología , Receptores Acoplados a Proteínas G/agonistas , Acetatos/química , Acetatos/farmacocinética , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Modelos Moleculares , Ratas , Receptores Acoplados a Proteínas G/química , Relación Estructura-ActividadRESUMEN
P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major obstacle for successful cancer chemotherapy. Based on our previous study, 17 novel compounds with the 6,7-dimethoxy-2-{2-[4-(1H-1,2,3-triazol-1-yl)phenyl]ethyl}-1,2,3,4-tetrahydroisoquinoline scaffold were designed and synthesized. Among them, 2-[(1-{4-[2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl]phenyl}-1H-1,2,3-triazol-4-yl)methoxy]-N-(p-tolyl)benzamide (compound 7 h) was identified as a potent modulator of P-gp-mediated MDR, with high potency (EC50 =127.5 ± 9.1 nM), low cytotoxicity (TI>784.3), and long duration (>24 h) in reversing doxorubicin (DOX) resistance in K562/A02 cells. Compound 7 h also enhanced the effects of other MDR-related cytotoxic agents (paclitaxel, vinblastine, and daunorubicin), increased the accumulation of DOX and blocked P-gp-mediated rhodamine 123 efflux function in K562/A02 MDR cells. Moreover, 7 h did not have any effect on cytochrome (CYP3A4) activity. These results indicate that 7 h is a relatively safe modulator of P-gp-mediated MDR that has good potential for further development.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Benzamidas/química , Tetrahidroisoquinolinas/química , Triazoles/química , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Antineoplásicos/farmacología , Benzamidas/síntesis química , Benzamidas/farmacología , Supervivencia Celular/efectos de los fármacos , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/metabolismo , Daunorrubicina/metabolismo , Daunorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Células K562 , Paclitaxel/farmacología , Unión Proteica , Rodamina 123/química , Rodamina 123/metabolismo , Relación Estructura-Actividad , Tetrahidroisoquinolinas/farmacología , Triazoles/síntesis química , Triazoles/farmacología , Vincristina/farmacologíaRESUMEN
A novel series of triazol-N-ethyl-tetrahydroisoquinoline based compounds were designed and synthesized via click chemistry. Most of the synthesized compounds showed P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) reversal activities. Among them, compound 7 with little cytotoxicity towards GES-1 cells (IC50 >80µM) and K562/A02 cells (IC50 >80µM) exhibited more potency than verapamil (VRP) on increasing anticancer drug accumulation in K562/A02 cells. Moreover, compound 7 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 7 in reversing MDR revealed that it could remarkably increase the intracellular accumulation of both rhodamine-123 (Rh123) and adriamycin (ADM) in K562/A02 cells as well as inhibit their efflux from the cells. These results suggested that compound 7 showed more potency than the classical P-gp inhibitor VRP under the same conditions, which may be a promising P-gp-mediated MDR modulator for further development.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/síntesis química , Benzamidas/química , Benzamidas/farmacología , Diseño de Fármacos , Isoquinolinas/química , Isoquinolinas/farmacología , Triazoles/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Benzamidas/síntesis química , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Química Clic , Doxorrubicina/química , Doxorrubicina/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Isoquinolinas/síntesis química , Células K562 , Rodamina 123/química , Rodamina 123/metabolismo , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/farmacologíaRESUMEN
A novel series of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) inhibitors bearing a triazol-phenethyl-tetrahydroisoquinoline scaffold were designed and synthesized via click chemistry. Most of the synthesized compounds showed higher reversal activity than verapamil (VRP). Among them, the most potent compound 5 showed a comparable activity with the known potent P-gp inhibitor WK-X-34 with lower cytotoxicity (IC50s > 100 µm). Compared with VRP, compound 5 exhibited more potency in increasing drug accumulation in K562/A02 MDR cells. Moreover, compound 5 persisted longer chemo-sensitizing effect (>24 h) than VRP (<6 h) with reversibility. Given the low intrinsic cytotoxicity and the potent reversal activity, compound 5 may represent a promising candidate for developing P-gp-mediated MDR inhibitor.