RESUMO
Enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting and quantifying target analytes in clinical and research laboratories. One of the main drawbacks of ELISA is the involvement of multiple washing steps that desorbs the capture antigen/antibody off the polystyrene plate, thereby producing inconsistent and erroneous data. To overcome the problem of desorption, we hypothesized that gelatin nanoparticles (GelNP) could serve as a "plate-adherent" substrate to irreversibly adhere the capture antigen/antibody of interest. We tested our hypothesis using GelNP-based substrate (Gel-BSA-OHG) to adhere 8-hydroxy-2'-deoxyguanosine (8-OHdG) to the polystyrene plate and assayed this molecule using the ELISA technique. The stability and ELISA performance of Gel-BSA-OHG was evaluated in comparison to the conventional substrate (BSA-OHG). Importantly, the Gel-BSA-OHG substrate was found to be more wash-resistant and consequently resulted in improved sensitivity, accuracy, and precision in the ELISA analysis of 8-OHdG. Finally, the scope of Gel-BSA-OHG substrate-based ELISA for clinical application was demonstrated by validating its ability to detect 8-OHdG in an artificial urine sample with high specificity.
Assuntos
8-Hidroxi-2'-Desoxiguanosina/urina , Ensaio de Imunoadsorção Enzimática/métodos , Gelatina/química , Nanopartículas/química , 8-Hidroxi-2'-Desoxiguanosina/química , Adsorção , Animais , Bovinos , Estudo de Prova de Conceito , Soroalbumina Bovina/químicaRESUMO
Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality with the 5-year survival rate at a dismal 16% for the past 40 years. Drug resistance is a major obstacle to achieving long-term patient survival. Identifying and validating molecular biomarkers responsible for resistance and thereby adopting multi-directional therapy is necessary to improve the survival rate. Previous studies indicated ~20% of tyrosine kinase inhibitor (TKI) resistant NSCLC patients overexpress AXL with increase in EMT and decrease in p53 expression. To overcome the resistance, we designed gelatin nanoparticles covalently conjugated with EGFR targeting antibody and siRNA (GAbsiAXL). GAbsiAXL efficiently silences AXL, decreases mTOR and EMT signaling with concomitant increase in p53 expression. Because of the molecular changes, the AXL silencing sensitizes the cells to TKI. Our results show AXL overexpression has an important role in driving TKI resistance through close association with energy-dependent mitochondrial pathways.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Transição Epitelial-Mesenquimal , Nanoconjugados/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Anticorpos/química , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Gelatina/química , Redes Reguladoras de Genes , Inativação Gênica , Humanos , Neoplasias Pulmonares , Metaloproteinases da Matriz/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Tirosina Quinase AxlRESUMO
Due to their antimicrobial properties, silver nanoparticles (AgNPs) are used in a wide range of consumer products that includes topical wound dressings, coatings for biomedical devices, and food-packaging to extend the shelf-life. Despite their beneficial antimicrobial effects, developmental exposure to such AgNPs may lead to gut dysbiosis and long-term health consequences in exposed offspring. AgNPs can cross the placenta and blood-brain-barrier to translocate in the brain of offspring. The underlying hypothesis tested in the current study was that developmental exposure of male and female mice to AgNPs disrupts the microbiome-gut-brain axis. To examine for such effects, C57BL6 female mice were exposed orally to AgNPs at a dose of 3 mg/kg BW or vehicle control 2 weeks prior to breeding and throughout gestation. Male and female offspring were tested in various mazes that measure different behavioral domains, and the gut microbial profiles were surveyed from 30 through 120 days of age. Our study results suggest that developmental exposure results in increased likelihood of engaging in repetitive behaviors and reductions in resident microglial cells. Echo-MRI results indicate increased body fat in offspring exposed to AgNPs exhibit. Coprobacillus spp., Mucispirillum spp., and Bifidobacterium spp. were reduced, while Prevotella spp., Bacillus spp., Planococcaceae, Staphylococcus spp., Enterococcus spp., and Ruminococcus spp. were increased in those developmentally exposed to NPs. These bacterial changes were linked to behavioral and metabolic alterations. In conclusion, developmental exposure of AgNPs results in long term gut dysbiosis, body fat increase and neurobehavioral alterations in offspring.
Assuntos
Comportamento/efeitos dos fármacos , Disbiose/tratamento farmacológico , Microbioma Gastrointestinal/efeitos dos fármacos , Nanopartículas Metálicas , Prata/efeitos adversos , Animais , Feminino , Humanos , Masculino , Aprendizagem em Labirinto , Testes de Estado Mental e Demência , Metagenoma , Metagenômica/métodos , Nanopartículas Metálicas/química , Camundongos , Modelos Animais , Prata/químicaRESUMO
Current methods for capturing circulating tumor cells (CTCs) are based on the overexpression of cytokeratin (CK) or epithelial cell-adhesion molecule (EpCAM) on cancer cells. However, during the process of metastasis, tumor cells undergo epithelial-to-mesenchymal transition (EMT) that can lead to the loss of CK/EpCAM expression. Therefore, it is vital to develop a capturing technique independent of CK/EpCAM expression on the cancer cell. To develop this technique, it is important to identify common secondary oncogenic markers overexpressed on tumor cells before and after EMT. We analyzed the biomarker expression levels in tumor cells, before and after EMT, and found two common proteins-human epidermal growth factor receptor 2 (Her2) and epidermal growth factor receptor (EGFR) whose levels remained unaffected. So, we synthesized immunomagnetic iron nanocubes covalently conjugated with antibodies of Her2 or EGFR to capture cancer cells irrespective of the EMT status. The nanocubes showed high specificity (6-9-fold) in isolating the cancer cells of interest from a mixture of cells spiked in serum. We characterized the captured cells for identifying their EMT status. Thus, we believe the results presented here would help in the development of novel strategies for capturing both primary and metastatic cancer cells from patients' blood to develop an effective treatment plan.
RESUMO
PURPOSE: We sought to assess a smartphone-based, gold nanoparticle-based colorimetric lateral flow immunoassay paper sensor for quantifying urine 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a biomarker for diabetic retinopathy (DR) screening. METHODS: Paper strips incorporate gold nanoparticle-8-OHdG antibody conjugates that produce color changes that are proportional to urine 8-OHdG and that are discernible on a smartphone camera photograph. Paper strip accuracy, precision, and stability studies were performed with 8-OHdG solutions of varying concentrations. Urine was collected from 97 patients with diabetes who were receiving DR screening examinations, including 7-field fundus photographs. DR was graded by standard methods as either low risk (no or mild DR) or high risk (moderate or severe DR). Paper sensor assays were performed on urine samples from patients and 8-OHdG values were correlated with DR grades. The differences in 8-OHdG values between the low- and high-risk groups were analyzed for outliers to identify the threshold 8-OHdG value that would minimize false-negative results. RESULTS: Lateral flow immunoassay paper strips quantitatively measure 8-OHdG and were found to be accurate, precise, and stable. Average urine 8-OHdG concentrations in study patients were 22 ± 10 ng/mg of creatinine in the low-risk group and 55 ± 11 ng/mg of creatinine in the high-risk group. Screening cutoff values of 8-OHdG >50 ng/mg of creatinine or urine creatinine >1.5 mg minimized screen failures, with 91% sensitivity and 81% specificity. CONCLUSIONS: Urinary 8-OHdG is a useful biomarker to screen DR. Quantitative 8-OHdG detection with the lateral flow immunoassay paper sensor and smartphone camera demonstrates its potential in DR screening. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.