RESUMEN
The cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses are key steps. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of the CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. Furthermore, it was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications.
Asunto(s)
Aptámeros de Péptidos , Bromovirus , Nanopartículas , Aptámeros de Péptidos/análisis , Aptámeros de Péptidos/metabolismo , Proteínas de la Cápside/metabolismo , Cápside/metabolismoRESUMEN
Drug development for cancer treatment is a complex process that requires special efforts. Targeting crucial proteins is the most essential purpose of drug design in cancers. Bcl-xl is an anti-apoptotic protein that binds to pro-apoptotic proteins and interrupts their signals. Pro-apoptotic Bcl-xl effectors are short BH3 sequences that form an alpha helix and bind to anti-apoptotic proteins to inhibit their activity. Computational systematic evolution of ligands by exponential enrichment (SELEX) is an exclusive approach for developing peptide aptamers as potential effectors. Here, the amino acids with a high tendency for constructing an alpha-helical structure were selected. Due to the enormous number of pentapeptides, Taguchi method was used to study a selected number of peptides. The binding affinity of the peptides to Bcl-xl was assessed using molecular docking, and after analysis of the obtained results, a final set of optimized peptides was arranged and constructed. For a better comparison, three chemical compounds with approved anti-Bcl-xl activity were selected for comparison with the top-ranked 5mer peptides. The optimized peptides showed considerable binding affinity to Bcl-xl. The molecular dynamics (MD) simulation indicated that the designed peptide (PO5) could create considerable interactions with the BH3 domain of Bcl-xl. The MM/GBSA calculations revealed that these interactions were even stronger than those created by chemical compounds. In silico SELEX is a novel approach to design and evaluate peptide-aptamers. The experimental design improves the SELEX process considerably. Finally, PO5 could be considered a potential inhibitor of Bcl-xl and a potential candidate for cancer treatment.
Asunto(s)
Aptámeros de Péptidos , Secuencia de Aminoácidos , Apoptosis , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Aptámeros de Péptidos/metabolismo , Ligandos , Simulación del Acoplamiento Molecular , Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Proyectos de Investigación , Proteína bcl-X/química , Proteína bcl-X/metabolismoRESUMEN
Short synthetic peptide molecules which bind to a specific target protein with a high affinity to exert its function are known as peptide aptamers. The high specificity of aptamers with small-molecule targets (metal ions, dyes and theophylline; ATP) is within 1 pM and 1 µM range, whereas with the proteins (thrombin, CD4 and antibodies) it is in the nanomolar range (which is equivalent to monoclonal antibodies). The recently identified coronavirus (SARS-CoV-2) genome encodes for various proteins, such as envelope, membrane, nucleocapsid, and spike protein. Among these, the protein necessary for the virus to enter inside the host cell is spike protein. The work focuses on designing peptide aptamer targeting the spike receptor-binding domain of SARS-CoV-2. The peptide aptamer has been designed by using bacterial Thioredoxin A as the scaffold protein and an 18-residue-long peptide. The tertiary structure of the peptide aptamer is modeled and docked to spike receptor-binding domain of SARS CoV2. Molecular dynamic simulation has been done to check the stability of the aptamer and receptor-binding domain complex. It was observed that the aptamer binds to spike receptor-binding domain of SARS-CoV-2 in a similar pattern as that of ACE2. The aptamer-receptor-binding domain complex was found to be stable in a 100 ns molecular dynamic simulation. The aptamer is also predicted to be non-antigenic, non-allergenic, non-hemolytic, non-inflammatory, water-soluble with high affinity toward ACE2 than serum albumin. Thus, peptide aptamer can be a novel approach for the therapeutic treatment for SARS-CoV-2.
Asunto(s)
Aptámeros de Péptidos , Tratamiento Farmacológico de COVID-19 , Antivirales/química , Aptámeros de Péptidos/metabolismo , Humanos , Péptidos/metabolismo , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Affinity-based proteomics has enabled scalable quantification of thousands of protein targets in blood enhancing biomarker discovery, understanding of disease mechanisms, and genetic evaluation of drug targets in humans through protein quantitative trait loci (pQTLs). Here, we integrate two partly complementary techniques-the aptamer-based SomaScan® v4 assay and the antibody-based Olink assays-to systematically assess phenotypic consequences of hundreds of pQTLs discovered for 871 protein targets across both platforms. We create a genetically anchored cross-platform proteome-phenome network comprising 547 protein-phenotype connections, 36.3% of which were only seen with one of the two platforms suggesting that both techniques capture distinct aspects of protein biology. We further highlight discordance of genetically predicted effect directions between assays, such as for PILRA and Alzheimer's disease. Our results showcase the synergistic nature of these technologies to better understand and identify disease mechanisms and provide a benchmark for future cross-platform discoveries.
Asunto(s)
Proteoma/genética , Proteómica/métodos , Sitios de Carácter Cuantitativo , Adulto , Enfermedad de Alzheimer/genética , Anticuerpos/metabolismo , Aptámeros de Péptidos/metabolismo , Estudios de Cohortes , Femenino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Persona de Mediana Edad , Fenotipo , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas/genética , Proteoma/metabolismo , Receptores Inmunológicos/genéticaRESUMEN
BACKGROUND: Esophageal cancer is the fifth most common cancer affecting men in China. The primary treatment options are surgery and traditional radio-chemotherapy; no effective targeted therapy exists yet. Self-assembled RNA nanocarriers are highly stable, easily functionally modified, and have weak off-tumor targeting effects. Thus, they are among the most preferred carriers for mediating the targeted delivery of anti-tumor drugs. miR-375 was found to be significantly down-regulated in esophageal squamous cell carcinoma (ESCC) tissues and its overexpression effectively inhibits the proliferation, migration, and invasion of ESCC cells. Moreover, epidermal growth factor receptor (EGFR) was overexpressed in ESCC cells, and accumulation of RNA nanoparticles in ESCC tumors was enhanced by EGFR-specific aptamer (EGFRapt) modification. RESULTS: Herein, a novel four-way junction RNA nanocarrier, 4WJ-EGFRapt-miR-375-PTX simultaneously loaded with miR-375, PTX and decorated with EGFRapt, was developed. In vitro analysis demonstrated that 4WJ-EGFRapt-miR-375-PTX possesses strong thermal and pH stabilities. EGFRapt decoration facilitated tumor cell endocytosis and promoted deep penetration into 3D-ESCC spheroids. Xenograft mouse model for ESCC confirmed that 4WJ-EGFRapt-miR-375-PTX was selectively distributed in tumor sites via EGFRapt-mediating active targeting and targeted co-delivery of miR-375 and PTX exhibited more effective therapeutic efficacy with low systemic toxicity. CONCLUSION: This strategy may provide a practical approach for targeted therapy of ESCC.
Asunto(s)
Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/metabolismo , MicroARNs , Terapia Molecular Dirigida/métodos , Nanopartículas , Animales , Apoptosis/efectos de los fármacos , Aptámeros de Péptidos/metabolismo , Aptámeros de Péptidos/farmacocinética , Línea Celular Tumoral , Receptores ErbB/metabolismo , Receptores ErbB/farmacocinética , Femenino , Humanos , Ratones , Ratones Desnudos , MicroARNs/química , MicroARNs/farmacocinética , MicroARNs/farmacología , Sistema de Administración de Fármacos con Nanopartículas/química , Sistema de Administración de Fármacos con Nanopartículas/farmacocinética , Sistema de Administración de Fármacos con Nanopartículas/farmacologíaRESUMEN
In the search for improved stool biomarkers for inflammatory bowel disease (IBD), an aptamer-based screen of 1129 stool proteins was conducted using stool samples from an IBD cohort. Here we report that of the 20 proteins subsequently validated by ELISA, stool Ferritin, Fibrinogen, Haptoglobin, Hemoglobin, Lipocalin-2, MMP-12, MMP-9, Myeloperoxidase, PGRP-S, Properdin, Resistin, Serpin A4, and TIMP-1 are significantly elevated in both ulcerative colitis (UC) and Crohn's disease (CD) compared to controls. When tested in a longitudinal cohort of 50 UC patients at 4 time-points, fecal Fibrinogen, MMP-8, PGRP-S, and TIMP-2 show the strongest positive correlation with concurrent PUCAI and PGA scores and are superior to fecal calprotectin. Unlike fecal calprotectin, baseline stool Fibrinogen, MMP-12, PGRP-S, TIMP-1, and TIMP-2 can predict clinical remission at Week-4. Here we show that stool proteins identified using the comprehensive aptamer-based screen are superior to fecal calprotectin alone in disease monitoring and prediction in IBD.
Asunto(s)
Colitis Ulcerosa/patología , Enfermedad de Crohn/patología , Heces/química , Proteínas/análisis , Adolescente , Aptámeros de Péptidos/metabolismo , Biomarcadores/análisis , Niño , Preescolar , Humanos , Complejo de Antígeno L1 de Leucocito/análisis , Proteómica/métodos , Índice de Severidad de la EnfermedadRESUMEN
Chimeric proteins have been widely used to evaluate intracellular protein-protein interactions (PPIs) in living cells with various readouts. By combining an interleukin-3-dependent murine cells and chimeric proteins containing a receptor tyrosine kinase c-kit, we previously established a c-kit-based PPI screening (KIPPIS) system to evaluate and select protein binders. In the KIPPIS components, proteins of interest are connected with a chemically inducible helper module and the intracellular domain of the growth-signaling receptor c-kit, which detects PPIs based on cell proliferation as a readout. In this system, proteins of interest can be incorporated into chimeric proteins without any scaffold proteins, which would be advantageous for evaluating interaction between small peptides/domains. To prove this superiority, we apply KIPPIS to 6 peptide aptamer-polypeptide pairs, which are derived from endogenous, synthetic, and viral proteins. Consequently, all of the 6 peptide aptamer-polypeptide interactions are successfully detected by cell proliferation. The detection sensitivity can be modulated in a helper ligand-dependent manner. The assay results of KIPPIS correlate with the activation levels of Src, which is located downstream of c-kit-mediated signal transduction. Control experiments reveal that KIPPIS clearly discriminates interacting aptamers from non-interacting ones. Thus, KIPPIS proves to be a versatile platform for evaluating the binding properties of peptide aptamers.
Asunto(s)
Aptámeros de Péptidos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas Proto-Oncogénicas c-kit/metabolismo , Aptámeros de Péptidos/química , Humanos , Ligandos , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Ingeniería de Proteínas/métodos , Proteínas Proto-Oncogénicas c-kit/químicaRESUMEN
Intervention in protein-protein interactions (PPIs) has tremendous effects in the molecular therapy of many diseases. To fulfill the requirements for targeting intracellular proteins, here we develop SOS-localization-based interaction screening (SOLIS), which elaborately mimics signaling via the Ras-mitogen-activated protein kinase pathway. SOLIS employs two chimeric proteins in which a membrane localization motif (CaaX) is fused at the C-terminus of a protein of interest and the catalytic domain of SOS is fused at the C-terminus of another protein of interest. Interaction between the two proteins of interest induces membrane localization of the SOS chimera and cell proliferation. Thus, the SOLIS system enables enrichment of superior binders based on cell proliferation in an intracellular PPI-dependent manner. This was verified by three major modalities against intracellular PPIs (small molecules, peptide aptamers, and intrabodies). The system worked over a broad range of affinities (KD = 0.32-140 nM). In a screening of a site-directed randomized library, novel intrabody clones were selected on the basis of the potency of cell proliferation. Three other PPI detection methods (NanoBiT, SPR, and pull-down assays) were employed to characterize the SOLIS system, and several intrabody clones were judged as false negatives in these assays. SOLIS signals would be less sensitive to the orientation/conformation of the chimeric proteins, and this feature emerges as the advantage of SOLIS as a mammalian cytosolic PPI detection system with few false negatives.
Asunto(s)
Espacio Intracelular/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Mapas de Interacción de Proteínas , Transducción de Señal/genética , Proteínas Son Of Sevenless/metabolismo , Animales , Anticuerpos/metabolismo , Antígenos/metabolismo , Aptámeros de Péptidos/metabolismo , Línea Celular , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Interleucina-3/genética , Interleucina-3/metabolismo , Interleucina-3/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Terapia Molecular Dirigida/métodos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción Genética , Transfección , Proteínas ras/metabolismoRESUMEN
Understanding the genetic architecture of host proteins interacting with SARS-CoV-2 or mediating the maladaptive host response to COVID-19 can help to identify new or repurpose existing drugs targeting those proteins. We present a genetic discovery study of 179 such host proteins among 10,708 individuals using an aptamer-based technique. We identify 220 host DNA sequence variants acting in cis (MAF 0.01-49.9%) and explaining 0.3-70.9% of the variance of 97 of these proteins, including 45 with no previously known protein quantitative trait loci (pQTL) and 38 encoding current drug targets. Systematic characterization of pQTLs across the phenome identified protein-drug-disease links and evidence that putative viral interaction partners such as MARK3 affect immune response. Our results accelerate the evaluation and prioritization of new drug development programmes and repurposing of trials to prevent, treat or reduce adverse outcomes. Rapid sharing and detailed interrogation of results is facilitated through an interactive webserver ( https://omicscience.org/apps/covidpgwas/ ).
Asunto(s)
COVID-19/genética , COVID-19/virología , Interacciones Huésped-Patógeno/genética , Proteínas/genética , SARS-CoV-2/fisiología , Sistema del Grupo Sanguíneo ABO/metabolismo , Aptámeros de Péptidos/sangre , Aptámeros de Péptidos/metabolismo , Coagulación Sanguínea , Sistemas de Liberación de Medicamentos , Femenino , Regulación de la Expresión Génica , Factores Celulares Derivados del Huésped/metabolismo , Humanos , Internet , Masculino , Persona de Mediana Edad , Sitios de Carácter Cuantitativo/genéticaRESUMEN
The use of alloplastic materials in periodontal regenerative therapies is limited by their incapacity to establish a dynamic dialog with the surrounding milieu. The aim of the present study was to control biomaterial surface bioactivity by introducing aptamers to induce the selective adsorption of fibronectin from blood, thus promoting platelets activation in vitro and bone regeneration in vivo. A hyaluronic acid/polyethyleneglycole-based hydrogel was enriched with aptamers selected for recognizing and binding fibronectin. In vitro, the capacity of constructs to support osteoblast adhesion, as well as platelets aggregation and activation was assessed by chemiluminescence within 24 h. Matrices were then evaluated in a rat periodontal defect to assess their regenerative potential by microcomputed tomography (µCT) and their osteogenic capacity by Luminex assay 5, 15 and 30 d postoperatively. Aptamers were found to confer matrices the capacity of sustaining firm cell adhesion (p = 0.0377) and to promote platelets activation (p = 0.0442). In vivo, aptamers promoted new bone formation 30 d post-operatively (p < 0.001) by enhancing osteoblastic lineage commitment maturation. Aptamers are a viable surface modification, which confers alloplastic materials the potential capacity to orchestrate blood clot formation, thus controlling bone healing.
Asunto(s)
Aptámeros de Péptidos/metabolismo , Materiales Biocompatibles/metabolismo , Fibronectinas/metabolismo , Periodoncio/fisiología , Animales , Regeneración Ósea/fisiología , Adhesión Celular/fisiología , Diferenciación Celular , Células Cultivadas , Humanos , Técnicas In Vitro , Masculino , Ensayo de Materiales , Osteoblastos/citología , Osteoblastos/fisiología , Osteogénesis/fisiología , Periodoncio/diagnóstico por imagen , Periodoncio/lesiones , Activación Plaquetaria/fisiología , Ratas , Ratas Endogámicas WKY , Propiedades de Superficie , Microtomografía por Rayos XRESUMEN
BACKGROUND: As two important tumor markers, vascular endothelial growth factor (VEGF) and carcinoembryonic antigen (CEA) have a great value for clinical application in the early diagnosis of cancer. Due to the complex composition of biological samples, the results from combined detection of CEA and VEGF are often taken as a comprehensive indicator in order to make an accurate judgment on a disease. However, most of the current methods can only be used to detect the content of one biomarker. Therefore, it is necessary to explore a simple, rapid, low-cost, and highly sensitive method for the simultaneous detection of CEA and VEGF. METHODS: Based on specific aptamers and magnetic separation, a time-resolved chemiluminescence enzyme-linked aptamer assay was developed for the simultaneous detections of CEA and VEGF in serum samples. RESULTS: Under the optimal conditions, the linear range of the calibration curve for VEGF was from 0.5 to 80 ng mL-1, and the limit of detection was 0.1 ng mL-1. The linear range of the calibration curve for CEA was 0.5 to 160 ng mL-1, and the limit of detection was 0.1 ng mL-1. The established method was applied to detect VEGF and CEA in serum samples. The results were consistent with those of commercial kits. CONCLUSION: The method has high sensitivity and can quickly obtain accurate results, which could greatly improve the measurement efficiency, reduce the cost, and also reduce the volume of sample consumed. It can be seen that the method established in this study has important application value and broad application prospect in clinical diagnosis.
Asunto(s)
Aptámeros de Péptidos/metabolismo , Antígeno Carcinoembrionario/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Mediciones Luminiscentes , Factor A de Crecimiento Endotelial Vascular/sangre , Fosfatasa Alcalina/metabolismo , Biocatálisis , Calibración , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Cinética , Mediciones Luminiscentes/métodos , Fenómenos Magnéticos , Factores de TiempoRESUMEN
Peptide aptamers are short amino acid chains that are capable of binding specifically to ligands in the same way as their much larger counterparts, antibodies. Ligands of therapeutic interest that can be targeted are other peptide chains or loops located on the surface of protein receptors (e.g., GCPR), which take part in cell-to-cell communications either directly or via the intermediary of hormones or signalling molecules. To confer on aptamers the same sort of conformational rigidity that characterises an antibody binding site, aptamers are often constructed in the form of cyclic peptides, on the assumption that this will encourage stronger binding interactions than would occur if the aptamers were simply linear chains. However, no formal studies have been conducted to confirm the hypothesis that linear peptides will engage in stronger binding interactions with cyclic peptides than with other linear peptides. In this study, the interaction of a model cyclic decamer with a series of linear peptide constructs was compared with that of a linear peptide with the same sequence, showing that the cyclic configuration does confer benefits by increasing the strength of binding.
Asunto(s)
Aptámeros de Péptidos/metabolismo , Péptidos/metabolismo , Unión Proteica/fisiología , Aminoácidos/metabolismo , Anticuerpos/metabolismo , Sitios de Unión/fisiología , Comunicación Celular/fisiología , Hormonas/metabolismo , Ligandos , Conformación Molecular , Péptidos Cíclicos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
We for the first time report that the activated CRISPR-Cas12a system trans-cleaves DNA G-quadruplexes (G4). The cleavage activity on human telomere G4 and TBA G4 was investigated and verified by FRET, CD, gel electrophoresis and NMR. We believe that this finding will pave a new avenue for advancing the applications of CRISPR-Cas12a and G4 in biosensing and biochemistry.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Endodesoxirribonucleasas/metabolismo , G-Cuádruplex , ARN Guía de Kinetoplastida/metabolismo , Aptámeros de Péptidos/metabolismo , Proteínas Bacterianas/química , Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas/genética , Dicroismo Circular , División del ADN , Endodesoxirribonucleasas/química , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Humanos , Cinética , Telómero/metabolismoRESUMEN
Exosomes hold great potential for cancer treatment to deliver therapeutics due to its inherent low immunogenicity. Exosomes are biocompatible cell-exocytosed secreted vesicles by most cell types, which can be used to construct novel biomanufacturing platform for drug delivery and cancer therapy. In this study, we implemented nano-sized vesicles which were secreted by mesenchymal stem cell (MSC), to encapsulate doxorubicin (DOX) through electroporation method (DOX@exosome). DOX was loaded into exosomes, with an encapsulation efficiency of up to 35% and separated by ultracentrifugation. Subsequently, carboxylic acid-end MUC1 aptamer was used to covalently decorate the surface amine groups of the exosomes via amide bond formation to provide selective guided drug delivery (DOX@exosome-apt). The data showed that the DOX@exosome-apt provided highly efficient DOX transportation to MUC1-positive cancer cells in vitro as confirmed by MTT and flow cytometry experiments. Moreover, in vivo study on ectopic model of C26 (mouse colon adenocarcinoma) in BALB/c mice indicated that the single dose intravenous injection of DOX@exosome-apt significantly suppress tumor growth in comparison with free DOX. Ex vivo fluorescent imaging also verified the desirable biodistribution of DOX@exosome-apt by exhibiting higher tumor accumulation and faster liver clearance in comparison with DOX@exosome and free DOX. It could be concluded that MUC1 aptamer-decorated exosomes can be implemented therapeutically for the safe and versatile delivery of DOX to colon adenocarcinoma, thus offering valuable platform for clinical applications.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Doxorrubicina/uso terapéutico , Exosomas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Animales , Aptámeros de Péptidos/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Doxorrubicina/farmacología , Endocitosis/efectos de los fármacos , Femenino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Alzheimer's disease (AD) causes cognitive impairment and serious social isolation. However, there are no effective treatments and even no established confirmatory diagnostic tools for the disease. Amyloid beta (Aß) aggregation in the brain is the best-known pathognomonic mechanism of AD, so various methods for Aß detection have been developed for the diagnosis of this disease. We synthesized two novel, ultra-sensitive peptide probes specialized in detecting Aß aggregates, and examined their potential for future diagnostic application. The peptides are produced through phage high-throughput screening (HTS) and amplified through a serial process called biopanning, which is a repeating method of elution and amplification of probes. We picked phages specific for amyloid from two kinds of phage display. The synthesized peptides were confirmed to have excellent binding affinity to Aß aggregates, by immunohistochemical staining and western blotting using the brains of 3X transgenic (Tg) AD mice at different stages (5-7, 12-17 months old) of AD severity. In the present study, it was confirmed that newly developed amyloid-binding peptides could be used as novel probes for the detection of Aß aggregates, which can be used for clinical diagnosis of AD in the future.
Asunto(s)
Péptidos beta-Amiloides/análisis , Aptámeros de Péptidos/metabolismo , Fragmentos de Péptidos/análisis , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Aptámeros de Péptidos/química , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Agregado de Proteínas/fisiología , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Electrolyte gated organic transistors can operate as powerful ultrasensitive biosensors, and efforts are currently devoted to devising strategies for reducing the contribution of hardly avoidable, nonspecific interactions to their response, to ultimately harness selectivity in the detection process. We report a novel lab-on-a-chip device integrating a multigate electrolyte gated organic field-effect transistor (EGOFET) with a 6.5 µL microfluidics set up capable to provide an assessment of both the response reproducibility, by enabling measurement in triplicate, and of the device selectivity through the presence of an internal reference electrode. As proof-of-concept, we demonstrate the efficient operation of our pentacene based EGOFET sensing platform through the quantification of tumor necrosis factor alpha with a detection limit as low as 3 pM. Sensing of inflammatory cytokines, which also include TNFα, is of the outmost importance for monitoring a large number of diseases. The multiplexable organic electronic lab-on-chip provides a statistically solid, reliable, and selective response on microliters sample volumes on the minutes time scale, thus matching the relevant key-performance indicators required in point-of-care diagnostics.
Asunto(s)
Técnicas Biosensibles/métodos , Factor de Necrosis Tumoral alfa/análisis , Aptámeros de Péptidos/química , Aptámeros de Péptidos/metabolismo , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/patología , Técnicas Biosensibles/instrumentación , Electrodos , Oro/química , Humanos , Dispositivos Laboratorio en un Chip , Límite de Detección , Transistores Electrónicos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Emerging urinary biomarkers continue to show promise in evaluating lupus nephritis (LN). Here, we screen urine from active LN patients for 1129 proteins using an aptamer-based platform, followed by ELISA validation in two independent cohorts comprised of 127 inactive lupus, 107 active LN, 67 active non-renal lupus patients and 74 healthy controls, of three different ethnicities. Urine proteins that best distinguish active LN from inactive disease are ALCAM, PF-4, properdin, and VCAM-1 among African-Americans, sE-selectin, VCAM-1, BFL-1 and Hemopexin among Caucasians, and ALCAM, VCAM-1, TFPI and PF-4 among Asians. Most of these correlate significantly with disease activity indices in the respective ethnic groups, and surpass conventional metrics in identifying active LN, with better sensitivity, and negative/positive predictive values. Several elevated urinary molecules are also expressed within the kidneys in LN, based on single-cell RNAseq analysis. Longitudinal studies are warranted to assess the utility of these biomarkers in tracking lupus nephritis.
Asunto(s)
Aptámeros de Péptidos/metabolismo , Biomarcadores/orina , Nefritis Lúpica/diagnóstico , Proteínas/análisis , Molécula de Adhesión Celular del Leucocito Activado/orina , Adulto , Negro o Afroamericano/estadística & datos numéricos , Pueblo Asiatico/estadística & datos numéricos , Selectina E/análisis , Femenino , Humanos , Nefritis Lúpica/etnología , Nefritis Lúpica/orina , Properdina/orina , Sensibilidad y Especificidad , Molécula 1 de Adhesión Celular Vascular/orina , Población Blanca/estadística & datos numéricos , Adulto JovenRESUMEN
Implantation of synthetic small-diameter vascular bypass grafts is often associated with an increased risk of failure, due to thrombotic events or late intimal hyperplasia. As one of the causes an insufficient hemocompatibility of the artificial surface is discussed. Endothelialization of synthetic grafts is reported to be a promising strategy for creating a self-renewing and regulative anti-thrombotic graft surface. However, the establishment of a shear resistant cell monolayer is still challenging. In our study, cyto- and immuno-compatible poly(ether imide) (PEI) films were explored as potential biomaterial for cardiovascular applications. Recently, we reported that the initial adherence of primary human umbilical vein endothelial cells (HUVEC) was delayed on PEI-films and about 9 days were needed to establish a confluent and almost shear resistant HUVEC monolayer. To accelerate the initial adherence of HUVEC, the PEI-film surface was functionalized with an aptamer-cRGD peptide based endothelialization supporting system. With this functionalization the initial adherence as well as the shear resistance of HUVEC on PEI-films was considerable improved compared to the unmodified polymer surface. The in vitro results confirm the general applicability of aptamers for an efficient functionalization of substrate surfaces.
Asunto(s)
Aptámeros de Péptidos/metabolismo , Éter/metabolismo , Imidas/metabolismo , Polímeros/metabolismo , Técnicas de Cultivo de Célula , HumanosRESUMEN
The lack of blood-brain barrier (BBB) penetrating ability has hindered the delivery of many therapeutic agents for tauopathy treatment. In this study, we report the synthesis of a circular bifunctional aptamer to enhance the in vivo BBB penetration for better tauopathy therapy. The circular aptamer consists of one reported transferrin receptor (TfR) aptamer to facilitate TfR-aptamer recognition-induced transcytosis across BBB endothelial cells, and one Tau protein aptamer that we recently selected to inhibit Tau phosphorylation and other tauopathy-related pathological events in the brain. This novel circular Tau-TfR bifunctional aptamer displays significantly improved plasma stability and brain exposure, as well as the ability to disrupt tauopathy and improve traumatic brain injury (TBI)-induced cognitive/memory deficits in vivo, providing important proof-of-principle evidence that circular Tau-TfR aptamer can be further developed into diagnostic and therapeutic candidates for tauopathies.
Asunto(s)
Aptámeros de Péptidos/metabolismo , Barrera Hematoencefálica , Receptores de Transferrina/metabolismo , Tauopatías/terapia , Transferrina/metabolismo , Proteínas tau/metabolismo , Animales , Humanos , Ratones , Prueba de Estudio ConceptualRESUMEN
Esophageal squamous cell carcinoma (ESCC) is a predominant cancer type in developing countries such as China, where ESCC accounts for approximately 90% of esophageal malignancies. Lacking effective and targeted therapy contributes to the poor 5-year survival rate. Recent studies showed that about 30% of ESCC cases have high levels of SOX2. Herein, we aim to target this transcription factor with aptamer. We established a peptide aptamer library and then performed an unbiased screening to identify several peptide aptamers including P42 that can bind and inhibit SOX2 downstream target genes. We further found that P42 overexpression or incubation with a synthetic peptide 42 inhibited the proliferation, migration, and invasion of ESCC cells. Moreover, peptide 42 treatment inhibited the growth and metastasis of ESCC xenografts in mouse and zebrafish. Further analysis revealed that P42 overexpression led to alternations in the levels of proteins that are important for the proliferation and migration of ESCC cells. Taken together, our study identified the peptide 42 as a key inhibitor of SOX2 function, reducing the proliferation and migration of ESCC cells in vitro and in vivo, and thereby offering a potential therapy against ESCC.