RESUMO
Innovative nanomaterials offer significant potential for diagnosis of severe diseases of the developing world such as malaria. Small sized silver nanoclusters have shown promise for diagnostics due to their intense fluorescence emission and photo-stabilities. Here, double-stranded DNA-scaffolded silver nanoclusters (AgNCs-dsDNA) were prepared to detect the established malaria biomarker, Plasmodium falciparum lactate dehydrogenase (PfLDH). Significant luminescence enhancement over a wide concentration range of PfLDH was demonstrated. In addition, a low limit of detection at 0.20 nM (7.4 pg µL-1) was achieved for PfLDH in buffer solution, sensitive enough for practical use correlating with the clinical level of PfLDH in plasma from malaria-infected patients. Unique specificity was observed towards Plasmodium falciparum over Plasmodium vivax and human lactate dehydrogenase, as well as other non-specific proteins, by combining the use of AgNCs-dsDNA with a DNA aptamer against PfLDH. Moreover, the intrinsic mechanism was revealed in detail for the two-step luminescence response. The combination of DNA-scaffolded silver nanoclusters coupled to a selective single-stranded DNA aptamer allows for a highly specific and sensitive detection of PfLDH with significant promise for malaria diagnosis in future.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , L-Lactato Desidrogenase/isolamento & purificação , Nanopartículas Metálicas , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/isolamento & purificação , Humanos , Malária/diagnóstico , PrataRESUMO
The aggregation-induced emission (AIE) phenomenon of the metal nanoclusters (NCs) has been discovered quite recently, which is considered to be creative to improve the optical properties of NCs upon ligand aggregation. In the present study, we report an AIE system for double-stranded DNA-templated silver nanoclusters (AgNCs-dsDNA) by using bovine serum albumin (BSA) in solution, which induce a moderate emission enhancement of AgNCs (5-fold) and a blue-shift through a loosen interaction. In addition, significant luminescence enhancement (30-fold) is further stimulated by the addition of digestive enzyme to the system by altering the surface structure of the aggregated particles. The processes are observed directly through transmission electron microscopy (TEM), where the dispersed AgNCs spheres are aggregated in a large incompact sheet-like film by BSA; while further trypsin addition lead them into larger particles (>50nm) with higher density. And the intrinsic mechanism is explained well by UV-vis absorption and time-resolved luminescence spectra. The observed luminescence enhancement in step-II is attributed to the AIE through stronger interaction between the hydrolysates of BSA and the ligand dsDNA. Therefore, the optical properties of AgNCs-dsDNA are regulated well by the addition of BSA and trypsin in different amounts, which relates directly to the degree of aggregation and can be extended to other metal nanoclusters by employing the pairs of protein and related enzyme.
Assuntos
DNA/química , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , Soroalbumina Bovina/química , Prata/química , Tripsina/química , Animais , Bovinos , LuminescênciaRESUMO
Gold nanoclusters (AuNCs) have been widely applied in fluorescence sensing, bioimaging and phototherapy. Although great progress has been made, the relatively low quantum yield (QY, <10%) in most currently reported AuNCs limits their application greatly. In the present study, adenosine monophosphate (AMP) capped gold nanoclusters (AuNC@AMP) are prepared by using a newly developed heating method in a short time (within 60 min), and are found to show a strong and stable luminescence emission in a relative high QY (14.52%). In addition, an in-depth investigation by employing infrared, 1H and 31P NMR spectroscopy has attributed the origin of such a high luminescence to both the binding of the purine ring and/or the phosphate moiety of AMP as well as their orientations at the gold core surface. In addition, electron-rich atoms such as nitrogen and oxygen, or group moieties such as -NH2 in the ligands can largely promote the luminescence emission of AuNCs. The present study reveals the intriguing generation of ultrabright luminescence from metal nanoclusters, and it will stimulate more research both on the fabrication and practical applications of luminescent metal NCs. With regard to the high QY of these AuNCs, they have great potential for biological applications as adenine is crucially important in life sciences.
RESUMO
Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-ß gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Assuntos
Antígenos de Helmintos/farmacologia , Meios de Cultivo Condicionados/farmacologia , Proteínas Hedgehog/genética , Células Estreladas do Fígado/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Pentoxifilina/farmacologia , Schistosoma japonicum/química , Animais , Antígenos de Helmintos/isolamento & purificação , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultivo Condicionados/química , Regulação da Expressão Gênica , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/imunologia , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/parasitologia , Cirrose Hepática/prevenção & controle , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/imunologia , Modelos Biológicos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/imunologia , Zigoto/químicaRESUMO
In vitro experiment showed that HNF3beta was the direct regulator of sonic hedgehog (shh) promoter. To investigate the activity of zebrafish shh promoter in vivo, we constructed the expression vector pShh-EGFP with ligating a 538 bp zebrafish shh promoter,which contained two HNF3beta binding sites, to EGFP. The pShh-EGFP DNA was microinjected into one-cell stage embryos of the zebrafish and the embryos were observed for GFP expression with fluorescent microscopy. GFP expression started during gastrulation in the axial hypoblast layer. During segmentation, GFP was detected in the notochord but not in the foor plate. Our experiment demonstrated that the 538 bp shh promoter containing two HNF3beta binding sites is able to confer the notochord-expressing activity.
Assuntos
Proteínas Luminescentes/genética , Notocorda/metabolismo , Regiões Promotoras Genéticas , Transativadores/genética , Peixe-Zebra/genética , Animais , Proteínas de Fluorescência Verde , Proteínas HedgehogRESUMO
OBJECTIVE: To evaluate the results of Fontan operation with extracardiac conduit on beating hearts. METHODS: Forty-two patients (31 males and 11 females) age ranged from 3 to 19 years old included in this study. There were 19 double inlet-ventricle, 10 tricuspid atresia, and 3 patients with mitral atresia, 10 patients with other complex congenital cardiac malformations. Fontan operations with extracardiac conduit were performed in all patients with the help of cardiopulmonary bypass without hypothermia in this study. Atrial septal fenestration was performed in 8 patients. In one patient, bi-directional cardiopulmonary procedure was performed 2 years before Fontan operation. RESULTS: There was one early death caused by acute hepatic function failure and one late death caused by repeated lung infections. The follow-up of 1 to 4.5 years showed that all patients' cardiac functions were grade I to II, and arterial oxygen saturation was 92% - 96%. CONCLUSIONS: The early and mid-term outcome of Fontan operation with extracardiac conduit on beating hearts is good and the method can be used in the single ventricle repair.