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Wounds are prone to infection which may be fatal to the life of the patient. The use of antibiotics is essential for managing bacterial infections in wounds, but the long-term use of high doses of antibiotics may lead to bacterial drug resistance and even to creation of superbacteria. Therefore, the development of targeted antimicrobial treatment strategies and the reduction in antibiotic usage are of utmost urgency. In this study, a multifunctional nanodrug delivery system (Cef-rhEGF@ZIF-8@ConA) for the treatment of bacteriostatic infection was synthesized through self-assembly of Zn2+, cefradine (Cef) and recombinant human epidermal growth factor (rhEGF), then conjugated with concanavalin (ConA), which undergoes pH-responsive degradation to release the drugs. First, ConA can specifically combine with bacteria and inhibit the rapid release of Zn2+ ions, thus achieving a long-acting antibacterial effect. Cef exerts its antibacterial effect by inhibiting the synthesis of bacterial membrane proteins. Finally, Zn2+ ions released from the Zn-metal-organic framework (MOF) demonstrate bacteriostatic properties by enhancing the permeability of the bacterial cell membrane. Furthermore, rhEGF upregulates angiogenesis-associated genes, thereby promoting angiogenesis, re-epithelialization and wound healing processes. The results showed that Cef-rhEGF@ZIF-8@ ConA has good biocompatibility, with antibacterial efficacy against Staphylococcus aureus and Escherichia coli of 99.61â¯% and 99.75â¯%, respectively. These nanomaterials can inhibit the release of inflammatory cytokines and promote the release of anti-inflammatory cytokines, while also stimulating the proliferation of fibroblasts to facilitate wound healing. Taken together, the Cef-rhEGF@ZIF-8@ConA nanosystem is an excellent candidate in clinical therapeutics for bacteriostatic infection and wound healing.
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Hypoxic tumor cell-derived exosomes play a key role in the occurrence, development, and metastasis of tumors. However, the mechanism of hypoxia-mediated metastasis remains unclear. In this study, hypoxic hepatocellular carcinoma cell (HCC-LM3)-derived exosomes (H-LM3-exos) were used to induce hepatocytes (HL-7702) over a long term (40 passages in 120 days). A nude mouse experiment further verified the effect of H-LM3-exos on tumor growth and metastasis. The process of cancer development in hepatocytes induced by H-LM3-exos was analyzed using both biological and physical techniques, and the results showed that the proliferation and soft agar growth abilities of the transformed cells were enhanced. The concentration of tumor markers secreted by transformed cells was increased, the cytoskeleton was disordered, and the migration ability was enhanced and was accompanied by epithelial-mesenchymal transition (EMT). Transcriptome results showed that differentially expressed genes between transformed cells and hepatocytes were enriched in cancer-related signaling pathways. The degree of cancer development in transformed cells was enhanced by an increase in H-LM3-exos-induced passages. Nude mice treated with different concentrations of H-LM3-exos showed different degrees of tumor growth and liver lesions. The physical properties of the cells were characterized by atomic force microscopy. Compared with the hepatocytes, the height and roughness of the transformed cells were increased, while the adhesion and elastic modulus were decreased. The changes in physical properties of primary tumor cells and hepatocytes in nude mice were consistent with this trend. Our study linking omics with the physical properties of cells provides a new direction for studying the mechanisms of cancer development and metastasis.
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Carcinoma Hepatocelular , Exosomas , Neoplasias Hepáticas , Ratones , Animales , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Ratones Desnudos , Exosomas/metabolismo , Línea Celular Tumoral , Hepatocitos/metabolismo , Hipoxia/metabolismoRESUMEN
Overexpression of tumor necrosis factor-α (TNF-α) is implicated in many inflammatory diseases, including septic shock, hepatitis, asthma, insulin resistance and autoimmune diseases, such as rheumatoid arthritis and Crohn's disease. The TNF-α signaling pathway is a valuable target, and anti-TNF-α drugs are successfully used to treat autoimmune and inflammatory diseases. Here, we study anti-inflammatory activity of an anti-TNF-α peptide (SN1-13, DEFHLELHLYQSW). In the cellular level assessment, SN1-13 inhibited TNF-α-induced cytotoxicity and blocks TNF-α-triggered signaling activities (IC50 = 15.40 µM). Moreover, the potential binding model between SN1-13 and TNF-α/TNFRs conducted through molecular docking revealed that SN1-13 could stunt TNF-α mediated signaling thought blocking TNF-α and its receptor TNFR1 and TNFR2. These results suggest that SN1-13 would be a potential lead peptide to treat TNF-α-mediated inflammatory diseases.
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FN-kappa B , Factor de Necrosis Tumoral alfa , Humanos , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Simulación del Acoplamiento Molecular , Inhibidores del Factor de Necrosis Tumoral/uso terapéutico , Inflamación/tratamiento farmacológico , Péptidos/farmacología , Péptidos/uso terapéuticoRESUMEN
Osteosarcoma (OS) is a primary malignant bone tumor that occurs mainly in adolescents. Researchers are devoting to develop combination therapy methods in a multifunctional nanoplatform for the treatment of osteosarcoma. The results of previous research have shown that up-regulation of miR-520a-3p could induce anticancer effects in osteosarcoma. In order to improve the effect of gene therapy (GT), we attempted to carry miR-520a-3p in a multifunctional vector for comprehensive therapy. Fe2O3is a type of magnetic resonance imaging (MRI) contrast that is widely used as a drug delivery agent. When coated with polydopamine (PDA), it can also be used as a photothermal therapy (PTT) agent (Fe2O3@ PDA). To deliver nanoagents targeted to a tumor site, folic acid (FA) conjugated with Fe2O3@PDA was manufactured as FA-Fe2O3@PDA. FA was chosen as the target molecule to enhance utilization and reduce toxicity of nanoparticles. However, the therapeutic efficacy of FA-Fe2O3-PDA combined with miR-520a-3p has not yet been studied. In this study, we synthesized FA-Fe2O3@PDA-miRNA and investigated the potential of combining PDA regulated PTT and miR-520a-3p regulated GT to kill osteosarcoma cells. The results indicated that down-regulation of interleukin 6 receptor (IL6R) by miR-520a-3p and the photothermal ability of PDA could induce satisfactory anticancer effects in osteosarcoma, and the curative ratio was better than that used alone PTT or GT. Moreover, as a kind ofT2magnetic contrast, miRNA-Fe2O3@PDA-FA can be used for MRI. These findings indicated that miRNA-Fe2O3@PDA-FA is an effective anti-tumor nanovector for PTT combined with GT.
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Neoplasias Óseas , MicroARNs , Nanopartículas , Osteosarcoma , Humanos , Adolescente , MicroARNs/genética , Terapia Fototérmica , Ácido Fólico , Imagen por Resonancia Magnética , Osteosarcoma/genética , Osteosarcoma/terapia , Línea Celular TumoralRESUMEN
MicroRNAs (miRNAs) play a very important role in biological processes and are used as biomarkers for the detection of a variety of diseases, including neurodegenerative diseases, chronic cardiovascular diseases, and cancers. A sensitive point-of-care (POC) method is crucial for detecting miRNAs. Herein, CRISPR-Cas12a combined with the click chemistry actuated exponential amplification reaction was introduced into an electrochemical biosensor for detecting miRNA-21. The target miRNA-21 initiated the click chemistry-exponential amplification reaction in the electrochemical biosensor to produce numerous nucleic acid fragments, which could stimulate the trans-cleavage ability of CRISPR-Cas12a to cleave hairpin DNA electrochemical reporters immobilized on the electrode surface. Under optimal conditions, the minimum detection limit for this electrochemical biosensor was as low as 1 fM. Thus, the proposed electrochemical biosensor allows sensitive and efficient miRNA detection and could be a potential analysis tool for POC test and field molecular diagnostics.
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In this study, Lba Cas12a (Cpf1) as one of the CRISPR systems from Lachnospiraceae bacterium was coupled with a hybridization chain reaction (HCR) to develop an electrochemical biosensor for detecting the pathogenic bacterium, Salmonella typhimurium. Autonomous cross-opening of functional DNA hairpin structures of HCR yielded polymer double-stranded DNA wires consisting of numerous single-stranded DNAs, which initiated the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave random single-stranded DNA labeling electrochemical tags on the surface of the electrode. It led to a variation in the electron transfer of electrochemical tags. The polymer double-stranded DNA of HCR was immobilized on dynabeads (DBs) via the S. typhimurium aptamer and released from DBs. The established method could selectively and sensitively quantify S. typhimurium in samples with detection limits of 20 CFU/mL. Our study provides a novel insight for exploring universal analytical methods for pathogenic bacteria based on CRISPR-Cas12a coupled with HCR.
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Técnicas Biosensibles/métodos , Sistemas CRISPR-Cas , Técnicas Electroquímicas/métodos , Salmonella typhimurium/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Salmonella typhimurium/patogenicidadRESUMEN
BACKGROUND: 3,17ß-hydroxysteroid dehydrogenase (3,17ß-HSD) is a key enzyme in the metabolic pathway for steroid compounds catabolism in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17ß-HSD. Previous reports showed that three tetR genes are located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first tetR gene encoded a potential repressor of 3,17ß-HSD by sensing environmental signals. However, whether the other proposed tetR genes act as repressors of 3,17ß-HSD are still unknown. METHODS AND RESULTS: In the present study, we cloned the second tetR gene and analyzed the regulatory mechanism of the protein on 3,17ß-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second tetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17ß-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17ß-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second tetR gene knocked-out mutant expresses good biological genetic stability, and the expression of 3,17ß-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction. CONCLUSIONS: The second tetR gene acts as a negative regulator in 3,17ß-HSD expression, and the mutant has potential application in bioremediation of steroids contaminated environment.
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17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Proteínas Bacterianas , Clonación Molecular , Comamonas testosteroni , Inhibidores Enzimáticos , Transactivadores , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Comamonas testosteroni/química , Comamonas testosteroni/genética , Comamonas testosteroni/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transactivadores/biosíntesis , Transactivadores/química , Transactivadores/genéticaRESUMEN
A novel electrochemical biosensor for detecting pathogenic bacteria was designed based on specific magnetic separation and highly sensitive click chemistry. Instead of enzyme-antibody conjugates, organic-inorganic hybrid nanoflowers [concanavalin A (Con A)-Cu3(PO4)2] were used as the signal probe of the sandwich structure. The inorganic component, the copper ions of hybrid nanoflowers, was first used to amplify signal transduction for enzyme-free detection. Sodium ascorbate could dissolve Cu3(PO4)2 of the signal probe to produce Cu2+, which was subsequently converted to Cu+, triggering the Cu+-catalyzed alkyne-azide cycloaddition (CuAAC) reaction between azide-functionalized ssDNA (a fragment of the DNAzyme-containing sequence) and alkyne-functionalized ssDNA immobilized onto the electrode surface. As a result, the DNAzyme was immobilized onto the gold electrode, which produced a positive and stable electrical signal. An exceptional linear relationship was observed between the electrical signal and the concentration of Salmonella typhimurium (101-107 CFU mL-1) with a detection limit of 10 CFU mL-1. The developed electrochemical biosensor based on dual signal amplification of Cu3(PO4)2-mediated click chemistry and DNAzymes exhibited good results in detecting S. typhimurium in milk samples.
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Técnicas Biosensibles , ADN Catalítico , Bacterias , Química Clic , Cobre , Técnicas Electroquímicas , Oro , Límite de DetecciónRESUMEN
Here, we describe a simple, sensitive, and enzyme-free method for visual point-of-care detection of 16S rRNA of Escherichia coli O157:H7 based on an isothermal strand displacement-hybrid chain reaction (ISD-HCR) and lateral flow strip (LFS). In this study, the secondary structure of 16S rRNA of E. coli O157:H7 was unwound by two helper oligonucleotides to expose the single-strand-specific nucleic acid sequence. The free specific sequence promoted the toehold-mediated strand displacement reaction to output a large number of FITC-labeled single-stranded DNA probes (capture probe [CP]). The 3'-end sequence of the reporter probe propagated a chain reaction of hybridization events between the two hairpin probes modified with biotin to form long nicked DNA polymers with multiple biotins (RP-HCR complexes); the free CP and RP-HCR complexes then form CP/RP-HCR complexes. The biotin-labeled double-stranded DNA CP/RP-HCR polymers then introduced numerous streptavidin (SA)-labeled gold nanoparticles (AuNPs) on the LFS. The accumulation of AuNPs produced a characteristic red band, which enabled visual detection of changes in the signal of 16S rRNA of E. coli O157:H7. The current approach could detect E. coli O157:H7 at concentrations as low as 102 CFU mL-1 without instrumentation. This approach thus provides a simple, sensitive, and low-cost tool for point-of-care detection of pathogenic bacteria, especially in resource-limited countries.
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Escherichia coli O157 , Nanopartículas del Metal , Escherichia coli O157/genética , Oro , ARN Ribosómico 16S/genética , EstreptavidinaRESUMEN
Recently, nanozymes have become a topic of particular interest due to their high activity level, stability and biocompatibility. In this study, a visual, sensitive and selective point-of-care immunosensor was established to test the pathogen Escherichia coli O157:H7 (E. coli O157:H7). Hemin and magainin I (MI) hybrid nanocomposites (Hemin@MI) with peroxidase-mimicking activities were synthesized via a "one-pot" method, involving the simple mixing of an antimicrobial peptide (MI) against E. coli O157:H7 and hemin in a copper sulfate sodium phosphate saline buffer. Hemin@MI nanocomposites integrating target recognition and signal amplification were developed as signal probes for the point-of-care colorimetric detection of pathogenic E. coli O157:H7. Hemin@MI nanocomposites exhibit excellent peroxidase activity for the chromogenic reaction of ABTS, which allows for the visual point-of-care testing of E. coli O157:H7 in the range of 102 to 108 CFU/mL, with a limit of detection of 85 CFU/mL. These data suggest this immunosensor provides accessible and portable assessments of pathogenic E. coli O157:H7 in real samples.
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Técnicas Biosensibles , Escherichia coli O157 , Péptidos Catiónicos Antimicrobianos , Colorimetría , Hemina , Inmunoensayo , Peroxidasas , Pruebas en el Punto de AtenciónRESUMEN
An ultrasmall hydrodynamic diameter is a critical factor for the renal clearance of nanoparticles from the body within a reasonable timescale. However, the integration of diagnostic and therapeutic components into a single ultrasmall nanoparticle remains challenging. In this study, pH-activated nanodots (termed Fe-CPNDs) composed of coordination polymers were synthesized via a simple and scalable method based on coordination reactions among Fe(3+), gallic acid and poly(vinylpyrrolidone) at ambient conditions. The Fe-CPNDs exhibited ultrasmall (5.3 nm) hydrodynamic diameters and electrically neutral surfaces. The Fe-CPNDs also exhibited pH-activatable magnetic resonance imaging contrast and outstanding photothermal performance. The features of Fe-CPNDs greatly increased the tumour-imaging sensitivity and facilitated renal clearance after injection in animal models in vivo. Magnetic resonance imaging-guided photothermal therapy using Fe-CPNDs completely suppressed tumour growth. These findings demonstrate that Fe-CPNDs constitute a new class of renal clearable nanomedicine for photothermal therapy and molecular imaging.
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Técnicas de Química Sintética , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/terapia , Nanocompuestos/química , Eliminación Renal , Nanomedicina Teranóstica , Animales , Ácido Gálico/química , Células Hep G2 , Humanos , Hierro/química , Imagen por Resonancia Magnética , Masculino , Ratones , Nanocompuestos/uso terapéutico , Povidona/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hydrophobic ultrasmall nanoparticles synthesized in nonpolar solvents exhibit great potential in biomedical applications. However, a major challenge when applying these nanomaterials in biomedical research is the lack of a versatile strategy to render them water dispersible while preserving the hydrodynamic diameter (HD) to be less than 8 nm for efficient renal clearance. To address this problem, tryptone is employed as the novel ligand to fabricate a simple, versatile, and inexpensive strategy for transferring hydrophobic NaGdF(4) nanodots (3 nm in diameter) from organic phase into aqueous phase without any complicated organic synthesis. The paramagnetic properties of NaGdF(4) nanodots are well retained after the phase transfer process. In particular, the tryptone-NaGdF(4) nanodots have ultrasmall HD (ca., 7.5 nm), which greatly improves their tumor accumulation and facilitates renal clearance within 24 h postinjection. The as-prepared tryptone-NaGdF(4) nanodots can also be further functionalized with other molecules for extensively biomedical and bioanalytical applications. Furthermore, the proposed strategy can easily be extended to transfer other types of inorganic nanoparticles from hydrophobic to hydrophilic for facilitating biomedical applications.
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Gadolinio/química , Riñón/metabolismo , Imagen por Resonancia Magnética/métodos , Nanopartículas del Metal/química , Neoplasias Experimentales/patología , Peptonas/farmacocinética , Animales , Medios de Contraste/síntesis química , Nanopartículas del Metal/ultraestructura , Ratones , Ratones Desnudos , Neoplasias Experimentales/metabolismo , Peptonas/química , Transición de FaseRESUMEN
Polydopamine nanoparticles (PDA NPs) which combine diagnostic and therapeutic functions are potentially useful in biomedicine. However, it is difficult to synthesize PDA NPs of a relatively small size (≤50 nm in diameter) using the traditional polymerization of dopamine monomers in an alkaline water-ethanol solution at room temperature. Herein, PDA NPs with average diameters ranging from 25 nm to 43 nm are prepared in a way which is similar to the silica-like reverse microemulsion process. The size of the PDA NPs can be modulated by changing the amount of dopamine monomers in the microemulsion. After conjugation with ferric ions (Fe3+), the poly(ethylene glycol) modified Fe-PDA NPs (termed as PEG-Fe-PDA NPs) exhibited pH-activatable magnetic resonance imaging (MRI) contrast and high photothermal performance. The combination of a small dimension and the pH-activatable MRI contrast can greatly facilitate tumor accumulation and increase the tumor imaging sensitivity against animal models in vivo. Completely inhibited tumor growth was achieved by the PEG-Fe-PDA NPs mediated by photothermal therapy with MRI guidance.
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The development of biosafe nanoplatforms with diagnostic and therapeutic multifunction is extremely demanded for designing cancer theranostic medicines. Here, a facile methodology is developed to construct a multifunctional nanotheranostic that gathers five functions, upconversion luminescence (UCL) imaging, T1-weighted magnetic resonance imaging (MRI), X-ray computed tomography (CT) imaging, photothermal therapy (PTT), and chemotherapy, into one single nanoprobe (named as UCNP@PDA5-PEG-DOX). For generating the UCNP@PDA5-PEG-DOX, a near-infrared light (NIR)-absorbing polydopamine (PDA) shell is directly coated on oleic-acid-capped ß-NaGdF4:Yb(3+),Er(3+)@ß-NaGdF4 upconverting nanoparticle (UCNP) core for the first time to form monodisperse, ultrastable, and noncytotoxic core-shell-structured nanosphere via water-in-oil microemulsion approach. When combined with 808 nm NIR laser irradiation, the UCNP@PDA5-PEG-DOX shows great synergistic interaction between PTT and the enhanced chemotherapy, resulting in completely eradicated mouse-bearing SW620 tumor without regrowth. In addition, leakage study, hemolysis assay, histology analysis, and blood biochemistry assay unambiguously reveal that the UCNP@PDA5-PEG has inappreciable cytotoxicity and negligible organ toxicity. The results provide explicit strategy for fabricating multifunctional nanoplatforms from the integration of UCNP with NIR-absorbing polymers, important for developing multi-mode nanoprobes for biomedical applications.
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Doxorrubicina/farmacología , Quimioterapia , Indoles/síntesis química , Imagen Multimodal , Nanopartículas/química , Fototerapia , Polímeros/síntesis química , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Endocitosis/efectos de los fármacos , Humanos , Indoles/química , Indoles/toxicidad , Campos Magnéticos , Imagen por Resonancia Magnética , Ratones , Nanopartículas/toxicidad , Polietilenglicoles/síntesis química , Polietilenglicoles/química , Polímeros/química , Polímeros/toxicidad , Espectroscopía Infrarroja Corta , Temperatura , Tomografía Computarizada por Rayos XRESUMEN
Here, we report the covalent conjugation of lectin on Fe2O3@Au core@shell nanoparticle (lectin-Fe2O3@Au NP) for T2-weighted magnetic resonance (MR) and X-ray computed tomography (CT) dual-modality imaging. The lectin-Fe2O3@Au NPs are prepared by coupling lectins to the Fe2O3@Au NP surfaces through bifunctional PEG NHS ester disulfide (NHS-PEG-S-S-PEG-NHS) linkers. After the nonspecific adsorption sites on the nanoparticle surface are blocked by thiolated PEG (PEG-SH), the lectin-Fe2O3@Au NPs exhibit excellent stability in biological medium and inappreciable cytotoxicity. A series of in vitro and in vivo experiments were then carried out for evaluating the capabilities of three selected lectin (ConA, RCA and WGA)-Fe2O3@Au NPs. The results revealed that the lectin-Fe2O3@Au NPs had a capacity not only for dual mode MR and CT imaging in vitro but also for MR and CT imaging of colorectal cancer in vivo. The experimental results also suggest that lectin could be used as tumor targeting ligand for synthesizing nanoparticle-based contrast agents.
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Neoplasias Colorrectales/diagnóstico , Medios de Contraste , Compuestos Férricos/farmacocinética , Oro/farmacocinética , Lectinas/farmacocinética , Nanopartículas del Metal/química , Animales , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Compuestos Férricos/química , Compuestos Férricos/metabolismo , Oro/química , Oro/metabolismo , Humanos , Lectinas/química , Lectinas/metabolismo , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Imagen Multimodal , Distribución Tisular , Tomografía Computarizada por Rayos X , Células Tumorales CultivadasRESUMEN
Here, we report the covalently conjugation of lanthanide doped NaGdF4:Yb(3+), Er(3+)@NaGdF4 upconverting nanoparticles (UCNPs) on methylphosphonate functionalized silica nanospheres (pSi NPs) for in vivo upconversion luminescence (UCL), T1-weighted magnetic resonance (MR), and X-ray computed tomography (CT) multi-modality imaging. The nanocomposites (pSi@UCNPs) were synthesized by a facile ligand exchange strategy. The hydrophobic pSi@UCNPs were transferred into aqueous solution by surface coating Pluronic F127. The Pluronic F127 coated pSi@UCNPs (pSi@UCNPs@F127) exhibit excellent stability in biological medium, inappreciable cytotoxicity and negligible organ toxicity. The pSi@UCNPs@F127 also shows brighter UCL, and higher CT/MR enhancements than that of Pluronic F127 coated NaGdF4:Yb(3+), Er(3+)@NaGdF4 UCNP. In detail, the capability of pSi@UCNPs@F127 as high performance contrast agents for in vivo multi-modality (UCL/MR/CT) imaging is evaluated successfully through small-animal experiments.
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Medios de Contraste , Diagnóstico por Imagen/métodos , Fluoruros , Gadolinio , Nanosferas , Dióxido de Silicio , Animales , Muerte Celular , Línea Celular Tumoral , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Nanosferas/ultraestructura , Neoplasias/diagnóstico , Neoplasias/patología , Poloxámero/química , Análisis Espectral , Tomografía Computarizada por Rayos XRESUMEN
Virus-induced gene silencing (VIGS) is potentially an attractive reverse-genetics tool for studies of plant gene function, but whether it is effective in silencing mineral nutritional-related genes in roots has not been demonstrated. Here we report on an efficient VIGS system that functions in tomato roots using a modified viral satellite DNA (DNAmbeta) associated with Tomato yellow leaf curl China virus (TYLCCNV). A cDNA fragment of the ferric chelate reductase gene (FROl) from tomato was inserted into the DNAmbeta vector. Tomato roots agro-inoculated with DNAmbeta carrying both a fragment of FRO1 and TYLCCNV used as a helper virus exhibited a significant reduction at the FRO1 mRNA level. As a consequence, ferric chelate reductase activity, as determined by visualization of the pink FeBPDS3 complex was significantly decreased. Our results clearly demonstrated that VIGS system can be employed to investigate gene function associated with plant nutrient uptake in roots.