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Find video protocols related to scientific articles indexed in Pubmed.
Long-term thiol monitoring in living cells using bioorthogonal chemistry.
Chem. Commun. (Camb.)
PUBLISHED: 11-20-2014
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Intracellular thiols play vital roles in living systems, and their in situ monitoring is of great importance. Here, we report on a bioorthogonal chemistry based fluorescent probe, which is capable of monitoring intracellular thiols in living cells for up to 36 hours with an obvious blue-to-green fluorescence change.
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MMP-2 responsive polymeric micelles for cancer-targeted intracellular drug delivery.
Chem. Commun. (Camb.)
PUBLISHED: 10-21-2014
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Multifunctional Biotin-PEG-b-PLL(Mal)-peptide-DOX polymeric micelles were prepared to selectively eliminate cancer cells. The micelles were able to enhance cancer cell uptake via the receptor-mediated endocytosis and respond to the stimulus of cancer cell excessive secreted protease MMP-2 to release the anticancer drug and induce apoptosis of cancer cells in a targeted manner.
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Acetylome analysis reveals diverse functions of lysine acetylation in Mycobacterium tuberculosis.
Mol. Cell Proteomics
PUBLISHED: 09-01-2014
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The lysine acetylation of proteins is a reversible posttranslational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD+-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.
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A pH-responsive prodrug for real-time drug release monitoring and targeted cancer therapy.
Chem. Commun. (Camb.)
PUBLISHED: 08-23-2014
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A novel cancer targeting and pH-responsive prodrug was successfully designed and synthesized. This M-prodrug was demonstrated to have real-time drug release monitoring capability based on the concept of contact-mediated quenching between doxorubicin and a coumarin derivative.
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Stepwise-acid-active multifunctional mesoporous silica nanoparticles for tumor-specific nucleus-targeted drug delivery.
ACS Appl Mater Interfaces
PUBLISHED: 08-18-2014
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In this paper, a novel stepwise-acid-active multifunctional mesoporous silica nanoparticle (MSN-(SA)TAT&(DMA)K11) was developed as a drug carrier. The MSN-(SA)TAT&(DMA)K11 is able to reverse its surface charge from negative to positive in the mildly acidic tumor extracellular environment. Then, the fast endo/lysosomal escape and subsequent nucleus targeting as well as intranuclear drug release can be realized after cellular internalization. Because of the difference in acidity between the tumor extracellular environment and that of endo/lysosomes, this multifunctional MSN-(SA)TAT&(DMA)K11 exhibits a stepwise-acid-active drug delivery with a tumor-specific nucleus-targeted property.
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The S28H mutation on mNeptune generates a brighter near-infrared monomeric fluorescent protein with improved quantum yield and pH-stability.
Acta Biochim. Biophys. Sin. (Shanghai)
PUBLISHED: 07-25-2014
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For living deep-tissue imaging, the optical window favorable for light penetration is in near-infrared wavelengths, which requires fluorescent proteins with emission spectra in the near-infrared region. Here, we report that a single mutant Ser28His of mNeptune with a near-infrared (?650 nm) emission maxima of 652 nm is found to improve the brightness, photostability, and pH stability when compared with its parental protein mNeptune, while it remains as a monomer, demonstrating that there is still plenty of room to improve the performance of the existing near infrared fluorescence proteins by directed evolution.
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Cancer-targeted functional gold nanoparticles for apoptosis induction and real-time imaging based on FRET.
Nanoscale
PUBLISHED: 07-04-2014
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A versatile gold nanoparticle-based multifunctional RB-DEVD-AuNP-DTP has been developed to induce the targeted apoptosis of cancer cells and image in real time the progress of the apoptosis. The multifunctional nanoparticles were demonstrated to have the ability to initiate mitochondria-dependent apoptosis and activate caspase-3 for real-time imaging of the progression of apoptosis.
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High length-diameter ratio nanotubes self-assembled from a facial cyclopeptide.
Soft Matter
PUBLISHED: 07-02-2014
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A six-residue facial cyclopeptide was designed with the following sequence: c-[D-Leu-L-Lys-D-Ala-L-Lys-D-Leu-L-Gln] (CP). Extensive hydrogen bonding between the cyclopeptide backbones mainly regulated CP to self-assemble into single-walled nanotubes. Simultaneously, the hydrophobic interaction among facial hydrophobic side chains of CP was introduced to stabilize the hydrogen bonding, resulting in the formation of the thick-walled nanotubes with high length–diameter ratios.
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Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on "Road Closure"
Biosens Bioelectron
PUBLISHED: 07-01-2014
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Detection of Bacillus anthracis in the field, whether as a natural infection or as a biothreat remains challenging. Here we have developed a new lateral-flow immunochromatographic assay (LFIA) for B. anthracis spore detection based on the fact that conjugates of B. anthracis spores and super-paramagnetic particles labeled with antibodies will block the pores of chromatographic strips and form retention lines on the strips, instead of the conventionally reported test lines and control lines in classic LFIA. As a result, this new LFIA can simultaneously realize optical, magnetic and naked-eye detection by analyzing signals from the retention lines. As few as 500-700 pure B. anthracis spores can be recognized with CV values less than 8.31% within 5min of chromatography and a total time of 20min. For powdery sample tests, this LFIA can endure interference from 25% (w/v) milk, 10% (w/v) baking soda and 10% (w/v) starch without any sample pre-treatment, and has a corresponding detection limit of 6×10(4) spores/g milk powder, 2×10(5) spores/g starch and 5×10(5) spores/g baking soda. Compared with existing methods, this new approach is very competitive in terms of sensitivity, specificity, cost and ease of operation. This proof-of-concept study can also be extended for detection of many other large-sized analytes.
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In vivo imaging of protein-protein and RNA-protein interactions using novel far-red fluorescence complementation systems.
Nucleic Acids Res.
PUBLISHED: 05-09-2014
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Imaging of protein-protein and RNA-protein interactions in vivo, especially in live animals, is still challenging. Here we developed far-red mNeptune-based bimolecular fluorescence complementation (BiFC) and trimolecular fluorescence complementation (TriFC) systems with excitation and emission above 600 nm in the 'tissue optical window' for imaging of protein-protein and RNA-protein interactions in live cells and mice. The far-red mNeptune BiFC was first built by selecting appropriate split mNeptune fragments, and then the mNeptune-TriFC system was built based on the mNeptune-BiFC system. The newly constructed mNeptune BiFC and TriFC systems were verified as useful tools for imaging protein-protein and mRNA-protein interactions, respectively, in live cells and mice. We then used the new mNeptune-TriFC system to investigate the interactions between human polypyrimidine-tract-binding protein (PTB) and HIV-1 mRNA elements as PTB may participate in HIV mRNA processing in HIV activation from latency. An interaction between PTB and the 3'long terminal repeat region of HIV-1 mRNAs was found and imaged in live cells and mice, implying a role for PTB in regulating HIV-1 mRNA processing. The study provides new tools for in vivo imaging of RNA-protein and protein-protein interactions, and adds new insight into the mechanism of HIV-1 mRNA processing.
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Construction of a chimeric lysin Ply187N-V12C with extended lytic activity against staphylococci and streptococci.
Microb Biotechnol
PUBLISHED: 05-05-2014
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Developing chimeric lysins with a wide lytic spectrum would be important for treating some infections caused by multiple pathogenic bacteria. In the present work, a novel chimeric lysin (Ply187N-V12C) was constructed by fusing the catalytic domain (Ply187N) of the bacteriophage lysin Ply187 with the cell binding domain (146-314aa, V12C) of the lysin PlyV12. The results showed that the chimeric lysin Ply187N-V12C had not only lytic activity similar to Ply187N against staphylococcal strains but also extended its lytic activity to streptococci and enterococci, such as Streptococcus?dysgalactiae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium and Enterococcus faecalis, which Ply187N could not lyse. Our work demonstrated that generating novel chimeric lysins with an extended lytic spectrum was feasible through fusing a catalytic domain with a cell-binding domain from lysins with lytic spectra across multiple genera.
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Multifunctional Enveloped Mesoporous Silica Nanoparticles for Subcellular Co-delivery of Drug and Therapeutic Peptide.
Sci Rep
PUBLISHED: 04-30-2014
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A multifunctional enveloped nanodevice based on mesoporous silica nanoparticle (MSN) was delicately designed for subcellular co-delivery of drug and therapeutic peptide to tumor cells. Mesoporous silica MCM-41 nanoparticles were used as the core for loading antineoplastic drug topotecan (TPT). The surface of nanoparticles was decorated with mitochondria-targeted therapeutic agent (Tpep) containing triphenylphosphonium (TPP) and antibiotic peptide (KLAKLAK)2 via disulfide linkage, followed by coating with a charge reversal polyanion poly(ethylene glycol)-blocked-2,3-dimethylmaleic anhydride-modified poly(L-lysine) (PEG-PLL(DMA)) via electrostatic interaction. It was found that the outer shielding layer could be removed at acidic tumor microenvironment due to the degradation of DMA blocks and the cellular uptake was significantly enhanced by the formation of cationic nanoparticles. After endocytosis, due to the cleavage of disulfide bonds in the presence of intracellular glutathione (GSH), pharmacological agents (Tpep and TPT) could be released from the nanoparticles and subsequently induce specific damage of tumor cell mitochondria and nucleus respectively with remarkable synergistic antitumor effect.
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Acs is essential for propionate utilization in Escherichia coli.
Biochem. Biophys. Res. Commun.
PUBLISHED: 04-29-2014
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Bacteria like Escherichia coli can use propionate as sole carbon and energy source. All pathways for degradation of propionate start with propionyl-CoA. However, pathways of propionyl-CoA synthesis from propionate and their regulation mechanisms have not been carefully examined in E. coli. In this study, roles of the acetyl-CoA synthetase encoding gene acs and the NAD(+)-dependent protein deacetylase encoding gene cobB on propionate utilization in E. coli were investigated. Results from biochemical analysis showed that, reversible acetylation also modulates the propionyl-CoA synthetase activity of Acs. Subsequent genetic analysis revealed that, deletion of acs in E. coli results in blockage of propionate utilization, suggesting that acs is essential for propionate utilization in E. coli. Besides, deletion of cobB in E. coli also results in growth defect, but only under lower concentrations of propionate (5mM and 10mM propionate), suggesting the existence of other propionyl-CoA synthesis pathways. In combination with previous observations, our data implies that, for propionate utilization in E. coli, a primary amount of propionyl-CoA seems to be required, which is synthesized by Acs.
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Phosphoproteomic analysis provides novel insights into stress responses in Phaeodactylum tricornutum, a model diatom.
J. Proteome Res.
PUBLISHED: 04-18-2014
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Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification used in signal transduction to control cell growth, proliferation, and stress responses. However, little is known about its extent and function in diatoms. Phaeodactylum tricornutum is a unicellular marine diatom that has been used as a model organism for research on diatom molecular biology. Although more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues have been predicted in P. tricornutum, no phosphorylation event has so far been revealed by classical biochemical approaches. Here, we performed a global phosphoproteomic analysis combining protein/peptide fractionation, TiO(2) enrichment, and LC-MS/MS analyses. In total, we identified 264 unique phosphopeptides, including 434 in vivo phosphorylated sites on 245 phosphoproteins. The phosphorylated proteins were implicated in the regulation of diverse biological processes, including signaling, metabolic pathways, and stress responses. Six identified phosphoproteins were further validated by Western blotting using phospho-specific antibodies. The functions of these proteins are discussed in the context of signal transduction networks in P. tricornutum. Our results advance the current understanding of diatom biology and will be useful for elucidating the phosphor-relay signaling networks in this model diatom.
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Rapid detection of New Delhi metallo-?-lactamase gene and variants coding for carbapenemases with different activities by use of a PCR-based in vitro protein expression method.
J. Clin. Microbiol.
PUBLISHED: 03-26-2014
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New Delhi metallo-?-lactamase (NDM)-producing bacteria are considered potential global health threats. It is necessary to monitor NDM-1 and its variants in clinical isolates in order to understand the NDM-1 epidemic and the impact of its variants on ?-lactam resistance. To reduce the lengthy time needed for cloning and expression of NDM-1 variants, a novel PCR-based in vitro protein expression (PCR-P) method was used to detect blaNDM-1 and its variants coding for carbapenemases with different activities (functional variants). The PCR-P method combined a long-fragment real-time quantitative PCR (LF-qPCR) with in vitro cell-free expression to convert the blaNDM-1 amplicons into NDM for carbapenemase assay. The method could screen for blaNDM-1 within 3 h with a detection limit of 5 copies and identify functional variants within 1 day. Using the PCR-P to analyze 5 recent blaNDM-1 variants, 2 functional variants, blaNDM-4 and blaNDM-5, were revealed. In the initial testing of 23 clinical isolates, the PCR-P assay correctly found 8 isolates containing blaNDM-1. This novel method provides the first integrated approach for rapidly detecting the full-length blaNDM-1 and revealing its functional variants in clinical isolates.
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A pH-responsive drug nanovehicle constructed by reversible attachment of cholesterol to PEGylated poly(l-lysine) via catechol-boronic acid ester formation.
Acta Biomater
PUBLISHED: 01-29-2014
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The present work reports the construction of a drug delivery nanovehicle via a pH-sensitive assembly strategy for improved cellular internalization and intracellular drug liberation. Through spontaneous formation of boronate linkage in physiological conditions, phenylboronic acid-modified cholesterol was able to attach onto catechol-pending methoxypoly(ethylene glycol)-block-poly(l-lysine). This comb-type polymer can self-organize into a micellar nanoconstruction that is able to effectively encapsulate poorly water-soluble agents. The blank micelles exhibited negligible in vitro cytotoxicity, yet doxorubicin (DOX)-loaded micelles could effectively induce cell death at a level comparable to free DOX. Owing to the acid-labile feature of the boronate linkage, a reduction in environmental pH from pH 7.4 to 5.0 could trigger the dissociation of the nanoconstruction, which in turn could accelerate the liberation of entrapped drugs. Importantly, the blockage of endosomal acidification in HeLa cells by NH4Cl treatment significantly decreased the nuclear uptake efficiency and cell-killing effect mediated by the DOX-loaded nanoassembly, suggesting that acid-triggered destruction of the nanoconstruction is of significant importance in enhanced drug efficacy. Moreover, confocal fluorescence microscopy and flow cytometry assay revealed the effective internalization of the nanoassemblies, and their cellular uptake exhibited a cholesterol dose-dependent profile, indicating the contribution of introduced cholesterol functionality to the transmembrane process of the nanoassembly.
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A boronate-linked linear-hyperbranched polymeric nanovehicle for pH-dependent tumor-targeted drug delivery.
Biomaterials
PUBLISHED: 01-21-2014
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Advanced drug delivery systems, which possess post-functionalization feasibility to achieve targetability and traceability, favorable pharmacokinetics with dynamic but controllable stability, and preferable tumor accumulation with prolonged drug residence in disease sites, represent ideal nanomedicine paradigm for tumor therapy. To address this challenge, here we reported a dynamic module-assembly strategy based on reversible boronic acid/1,3-diol bioorthogonality. As a prototype, metastable hybrid nanoself-assembly between hydrophobic hyperbranched diol-enriched polycarbonate (HP-OH) and hydrophilic linear PEG terminated with phenylboronic acid (mPEG-PBA) is demonstrated in vitro and in vivo. The nanoconstruction maintained excellent stability with little leakage of loaded drugs under the simulated physiological conditions. Such a stable nanostructure enabled the effective in vivo tumor accumulation in tumor site as revealed by NIR imaging technique. More importantly, this nanoconstruction presented a pH-labile destruction profile in response to acidic microenvironment and simultaneously the fast liberation of loaded drugs. Accordingly at the cellular level, the intracellular structural dissociation was also proved in terms of the strong acidity in late endosome/lysosome, thus favoring the prolonged retention of remaining drug-loaded HP-OH aggregates within tumor cells. Hence, our delicate design open up a dynamical module-assembly path to develop site and time dual-controlled nanotherapeutics for tumor chemotherapy, allowing enhanced tumor selectivity through prolonged retention of delivery system in tumor cells followed by a timely drug release pattern.
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A FRET-Based Dual-Targeting Theranostic Chimeric Peptide for Tumor Therapy and Real-time Apoptosis Imaging.
Adv Healthc Mater
PUBLISHED: 01-09-2014
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A FRET-based chimeric peptide is reported to mediate specific tumor cell uptaking and apoptosis. Importantly, this chimeric peptide is fluorescence quenched (OFF). Once the apoptosis is initiated and propagated, green fluorescence is lighted up rapidly (ON), achieving simultaneous tumor therapy and real-time apoptosis monitoring.
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Binding pocket alterations in dihydrofolate synthase confer resistance to para-aminosalicylic acid in clinical isolates of Mycobacterium tuberculosis.
Antimicrob. Agents Chemother.
PUBLISHED: 12-23-2013
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The mechanistic basis for resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multi-drug resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor para-aminobenzoic acid, PAS is ultimately bioactivated to hydroxydihydrofolate which inhibits dihydrofolate reductase and disrupts operation of folate-dependent metabolic pathways. As a result, mutation of dihydrofolate synthase, an enzyme needed for bioactivation of PAS, causes PAS resistance in M. tuberculosis H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and M. bovis. From a panel of 85 multi-drug resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in folC within the H2Pte binding pocket resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished bioactivation of hydroxydihydropteroate to hydroxydihydrofolate. Consistent with this model for abolished bioactivation, introduction of a wild type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for development of molecular based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.
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Multi-Functional Envelope-Type Nanoparticles Assembled from Amphiphilic Peptidic Prodrug with Improved Anti-Tumor Activity.
ACS Appl Mater Interfaces
PUBLISHED: 12-20-2013
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A novel multifunctional amphiphilic peptidic prodrug was reported here by conjugating the antitumor drug of doxorubicin (DOX) to the hydrophobic tail of a designed peptide-amphiphile (PA), in which the hydrophilic peptide headgroup comprises a glycine-arginine-glycine-aspartic acid-serine (GRGDS) sequence and octaarginine (R8) sequence. Because of the amphiphilic nature, this peptidic prodrug can spontaneously self-assemble into spherical multifunctional envelop-type nanoparticles (MENPs) with the functional peptide sequences gathered on surface. By means of the multifunctions of RGD-mediated tumor targeting, R8-mediated membrane penetration and intracellular protease-mediated hydrolyzing peptide bonds, the MENPs could targeted deliver doxorubicin (DOX) to tumor cells, showing improved antitumor activity both in vitro and in vivo with much reduced side effects.
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A coumarin derivative as a fluorogenic glycoproteomic probe for biological imaging.
Chem. Commun. (Camb.)
PUBLISHED: 11-26-2013
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Fluorescence imaging in living cells is typically carried out using a functionalized fluorescent dye. But it often causes strong background noise under many conditions where washing is not applicable. Here, we report on a coumarin based fluorogenic probe, which can be used as a bioorthogonal-labeling tool for glycoproteins. The results indicated that the probe was able to image glycoproteins in living cells and it may also be suitable for intracellular imaging.
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Rapid Detection of Mycobacterium tuberculosis and Pyrazinamide Susceptibility Related to pncA Mutations in Sputum Specimens through an Integrated Gene-to-Protein Function Approach.
J. Clin. Microbiol.
PUBLISHED: 11-13-2013
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Testing the pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis isolates is challenging. In a previous paper, we described the development of a rapid colorimetric test for the PZA susceptibility of M. tuberculosis by a PCR-based in vitro-synthesized-pyrazinamidase (PZase) assay. Here, we present an integrated approach to detect M. tuberculosis and PZA susceptibility directly from sputum specimens. M. tuberculosis was detected first, using a novel long-fragment quantitative real-time PCR (LF-qPCR), which amplified a fragment containing the whole pncA gene. Then, the positive amplicons were sequenced to find mutations in the pncA gene. For new mutations not found in the Tuberculosis Drug Resistance Mutation Database (www.tbdreamdb.com), the in vitro PZase assay was used to test the PZA resistance. This approach could detect M. tuberculosis within 3 h with a detection limit of 7.8 copies/reaction and report the PZA susceptibility within 2 days. In an initial testing of 213 sputum specimens, the LF-qPCR found 53 positive samples with 92% sensitivity and 97% specificity compared to the culture test for M. tuberculosis detection. DNA sequencing of the LF-qPCR amplicons revealed that 49 samples were PZA susceptible and 1 was PZA resistant. In the remaining 3 samples, with new pncA mutations, the in vitro PZase assay found that 1 was PZA susceptible and 2 were PZA resistant. This integrated approach provides a rapid, efficient, and relatively low-cost solution for detecting M. tuberculosis and PZA susceptibility without culture.
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Novel Chimeric Lysin with High-Level Antimicrobial Activity against Methicillin-Resistant Staphylococcus aureus In Vitro and In Vivo.
Antimicrob. Agents Chemother.
PUBLISHED: 11-04-2013
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The treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is a challenge worldwide. In our search for novel antimicrobial agents against MRSA, we constructed a chimeric lysin (named as ClyH) by fusing the catalytic domain of Ply187 (Pc) with the non-SH3b-like cell wall binding domain of phiNM3 lysin. Herein, the antimicrobial activity of ClyH against MRSA strains in vitro and in vivo was studied. Our results showed that ClyH could kill all of the tested clinical isolates of MRSA with higher efficacy than lysostaphin as well as its parental enzyme. The MICs of ClyH against clinical S. aureus strains were found to be as low as 0.05 to 1.61 mg/liter. In a mouse model, a single intraperitoneal administration of ClyH protected mice from death caused by MRSA, without obvious harmful effects. The present data suggest that ClyH has the potential to be an alternative therapeutic agent for the treatment of infections caused by MRSA.
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Dual-Targeting Pro-apoptotic Peptide for Programmed Cancer Cell Death via Specific Mitochondria Damage.
Sci Rep
PUBLISHED: 09-17-2013
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Mitochondria are vital organelles to eukaryotic cells. Damage to mitochondria will cause irreversible cell death or apoptosis. In this report, we aim at programmed cancer cell death via specific mitochondrial damage. Herein, a functionalized pro-apoptotic peptide demonstrates a dual-targeting capability using folic acid (FA) (targeting agent I) and triphenylphosphonium (TPP) cation (targeting agent II). FA is a cancer-targeting agent, which can increase the cellular uptake of the pro-apoptotic peptide via receptor-mediated endocytosis. And the TPP cation is the mitochondrial targeting agent, which specifically delivers the pro-apoptotic peptide to its particular subcellular mitochondria after internalized by cancer cells. Then the pro-apoptotic peptide accumulates in mitochondria and causes its serious damage. This dual-targeting strategy has the potential to effectively transport the pro-apoptotic peptide to targeted cancer cell mitochondria, inducing mitochondrial dysfunction and triggering the mitochondria-dependent apoptosis to efficiently eliminate cancer cells.
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Fabrication of Novel Reduction-Sensitive Gene Vectors Based on Three-Armed Peptides.
Macromol Biosci
PUBLISHED: 09-16-2013
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To address the inherent barriers of gene transfection, two reduction-sensitive branched polypeptides (RBPs) are synthesized and explored as novel non-viral gene vectors. The introduced disulfide linkages in RBPs facilitate glutathione-triggered intracellular gene release and reduce polymer degradation-induced cytotoxicity. Furthermore, the highly branched architecture concurrently realizes multivalency for strong DNA binding and elicits conformational flexibility for tight DNA compacting, which are beneficial for cellular entry. To increase the endosomal escape of plasmid DNA, pH-sensitive histidyl residues are incorporated into RBPs to improve buffer capacity in an acidic environment. In vitro study demonstrates that RBPs can efficiently mediate the DNA transfection and avoid apparent cytotoxicity in HeLa and COS7. The present gene delivery system offers a simple and flexible approach to fabricate microenvironment-specific branched gene vectors for gene therapy.
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Acidity-Promoted Cellular Uptake and Drug Release Mediated by Amine-Functionalized Block Polycarbonates Prepared via One-Shot Ring-Opening Copolymerization.
Macromol Biosci
PUBLISHED: 09-09-2013
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This paper reports a drug nanovehicle self-assembled from an amine-functionalized block copolymer poly(6,14-dimethyl-1,3,9,11-tetraoxa-6,14-diaza-cyclohexadecane-2,10-dione)-block-poly(1,3-dioxepan-2-one) (PADMC-b-PTeMC), which is prepared by controlable ring-opening block copolymerization attractively in a "one-shot feeding" pathway. The copolymers display high cell-biocompatibility with no apparent cytotoxicities detected in 293T and HeLa cells. Due to their amphiphilic nature, PADMC-b-PTeMC copolymers can self-assemble into nanosized micelles capable of loading anticancer drugs such as camptothecin (CPT) and doxorubicin (DOX). In particular, the outer PADMC shell endows the PADMC-b-PTeMC nanomicelles with pH-dependent control over the micellar morphology, cell uptake efficiency, and the drug release pattern. Confocal inspection reveals the remarkably enhanced cellular internalization of drug loaded micelles by cancerous HeLa cells at relatively lower pH 5.8 simulating the mildly acid microenvironment in tumors. Along with the acidity-triggered volume expansion of micelles, an accelerated CPT release in vitro occurs. The obtained results adumbrate the possibility of completely biodegradable PADMC-b-PTeMC as pH-sensitive drug carriers for tumor chemotherapy.
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Enhanced cardioprotective effects mediated by plasmid containing the short-hairpin RNA of angiotensin converting enzyme with a biodegradable hydrogel after myocardial infarction.
J Biomed Mater Res A
PUBLISHED: 08-20-2013
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The expression of foreign gene was enhanced and prolonged by sustained releasing a target gene to cells from biodegradable dextran-poly(e-caprolactone)-2-hydroxylethylmethacrylate-poly(N-isopropylacrylamide) (Dex-PCL-HEMA/PNIPAAm) hydrogel in vitro. Moreover, we have demonstrated that injection of the same hydrogel improved post-infarct ventricular remodeling. Therefore, we hypothesized that intramyocardial injection of plasmid containing the short-hairpin RNA (shRNA) of angiotensin converting enzyme (ACE) with the same hydrogel enhances the cardioprotective effects superior to either alone or after rat myocardial infarction (MI). In this study, equal volume of phosphate-buffered solution (PBS), 10 ?g ACE-shRNA plasmids, hydrogel containing 10 ?g negative control ACE-shRNA plasmids and hydrogel containing 10 ?g ACE-shRNA plasmids were shortly injected into the infarct area of rats after MI, respectively. We found that ACE-shRNA plasmid-loaded hydrogel extended the duration of gene expression in vivo. Moreover, it was shown that direct intramyocardial injection of ACE-shRNA plasmid-loaded hydrogel significantly decreased the expression of local ACE expression, inhibited cell apoptosis, reduced infarct size, and improved cardiac function compared with the injection of either alone 30 days after MI in rats. These results suggest that injection of ACE-shRNA plasmid-loaded hydrogel into impaired myocardium obtains more cardioprotective effects than either alone in rat with MI by prolonging the gene silencing of ACE. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.
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One-pot construction of functional mesoporous silica nanoparticles for the tumor-acidity-activated synergistic chemotherapy of glioblastoma.
ACS Appl Mater Interfaces
PUBLISHED: 08-02-2013
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Mesoporous silica nanoparticles (MSNs) have proved to be an effective carrier for controlled drug release and can be functionalized easily for use as stimuli-responsive vehicles. Here, a novel intelligent drug-delivery system (DDS), camptothecin (CPT)-loaded and doxorubicin (DOX)-conjugated MSN (CPT@MSN-hyd-DOX), is reported via a facile one-pot preparation for use in synergistic chemotherapy of glioblastoma. DOX was conjugated to MSNs via acid-labile hydrazone bonds, and CPT was loaded in the pores of the MSNs. At pH 6.5 (analogous to the pH in tumor tissues), a fast DOX release was observed that was attributed to the hydrolysis of the hydrazone bonds. In addition, a further burst release of DOX was found at pH 5.0 (analogous to the pH in lyso/endosomes of tumor cells), leading to a strong synergistic effect. In all, CPT and DOX could be delivered simultaneously into tumor cells, and this intelligent DDS has great potential for tumor-trigged drug release for use in the synergistic chemotherapy of tumors.
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pH Responsive micelle self-assembled from a new amphiphilic peptide as anti-tumor drug carrier.
Colloids Surf B Biointerfaces
PUBLISHED: 07-22-2013
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An acid-responsive amphiphilic peptide that contains KKGRGDS sequence in hydrophilic head and VVVVVV sequence in hydrophobic tail was designed and prepared. In neutral or basic medium, this amphiphilic peptide can self-assemble into micelles through hydrogen bonding and hydrophobic interactions. If changing the solution pH to an acidic environment, the electrostatic repulsion interaction among the ionized lysine (K) residues will prevent the self-assembly of the amphiphilic peptide, leading to the dissociation of micelles. The anti-tumor drug of doxorubicin (DOX) was chosen and loaded into the self-assembled micelles of the amphiphilic peptide to investigate the influence of external pH change on the drug release behavior. As expected, the micelles show a sustained DOX release in neutral medium (pH 7.0) but fast release behavior in acidic medium (pH 5.0). When incubating these DOX-loaded micelles with HeLa and COS7 cells, due to the over-expression of integrins on cancer cells, the micelles can efficiently use the tumor-targeting function of RGD sequence to deliver the drug into HeLa cells. Combined with the low cytotoxicity of the amphiphilic peptide against both HeLa and COS7 cells, the amphiphilic peptide reported in this work may be promising in clinical application for targeted drug delivery.
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A Dual-Responsive Mesoporous Silica Nanoparticle for Tumor-Triggered Targeting Drug Delivery.
Small
PUBLISHED: 06-22-2013
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A novel pH- and redox- dual-responsive tumor-triggered targeting mesoporous silica nanoparticle (TTTMSN) is designed as a drug carrier. The peptide RGDFFFFC is anchored on the surface of mesoporous silica nanoparticles via disulfide bonds, which are redox-responsive, as a gatekeeper as well as a tumor-targeting ligand. PEGylated technology is employed to protect the anchored peptide ligands. The peptide and monomethoxypolyethylene glycol (MPEG) with benzoic-imine bond, which is pH-sensitive, are then connected via "click" chemistry to obtain TTTMSN. In vitro cell research demonstrates that the targeting property of TTTMSN is switched off in normal tissues with neutral pH condition, and switched on in tumor tissues with acidic pH condition after removing the MPEG segment by hydrolysis of benzoic-imine bond under acidic conditions. After deshielding of the MPEG segment, the drug-loaded nanoparticles are easily taken up by tumor cells due to the exposed peptide targeting ligand, and subsequently the redox signal glutathione in tumor cells induces rapid drug release intracellularly after the cleavage of disulfide bond. This novel intelligent TTTMSN drug delivery system has great potential for cancer therapy.
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Recognition and processing of double-stranded DNA by ExoX, a distributive 3-5 exonuclease.
Nucleic Acids Res.
PUBLISHED: 06-14-2013
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Members of the DnaQ superfamily are major 3-5 exonucleases that degrade either only single-stranded DNA (ssDNA) or both ssDNA and double-stranded DNA (dsDNA). However, the mechanism by which dsDNA is recognized and digested remains unclear. Exonuclease X (ExoX) is a distributive DnaQ exonuclease that cleaves both ssDNA and dsDNA substrates. Here, we report the crystal structures of Escherichia coli ExoX in complex with three different dsDNA substrates: 3 overhanging dsDNA, blunt-ended dsDNA and 3 recessed mismatch-containing dsDNA. In these structures, ExoX binds to dsDNA via both a conserved substrate strand-interacting site and a previously uncharacterized complementary strand-interacting motif. When ExoX complexes with blunt-ended dsDNA or 5 overhanging dsDNA, a wedge composed of Leu12 and Gln13 penetrates between the first two base pairs to break the 3 terminal base pair and facilitates precise feeding of the 3 terminus of the substrate strand into the ExoX cleavage active site. Site-directed mutagenesis showed that the complementary strand-binding site and the wedge of ExoX are dsDNA specific. Together with the results of structural comparisons, our data support a mechanism by which normal and mismatched dsDNA are recognized and digested by E. coli ExoX. The crystal structures also provide insight into the structural framework of the different substrate specificities of the DnaQ family members.
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A new anti-cancer strategy of damaging mitochondria by pro-apoptotic peptide functionalized gold nanoparticles.
Chem. Commun. (Camb.)
PUBLISHED: 06-12-2013
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Gold nanoparticles functionalized with pro-apoptotic peptide (PAP-AuNPs) were fabricated, which were able to lead to programmed cell-death by damaging mitochondria.
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Photo-switched self-assembly of a gemini ?-helical peptide into supramolecular architectures.
Nanoscale
PUBLISHED: 06-05-2013
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An azobenzene-linked symmetrical gemini ?-helical peptide was designed and prepared to realize the light-switched self-assembly. With the reversible molecular structure transition between Z- and U-structures, the morphology of the self-assembled gemini ?-helical peptide can reversibly change between nanofibers and nanospheres in acidic medium, and between nanospheres and vesicles in basic medium.
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A novel T-type overhangs improve the enzyme-free cloning of PCR products.
Mol. Biotechnol.
PUBLISHED: 06-05-2013
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PCR product cloning is the foundational technology for almost all fields in the life sciences. Numerous innovative methods have been designed during the past few decades. Enzyme-free cloning is the only one that avoids post-amplification enzymatic treatments, making the technique reliable and cost effective. However, the complementary staggered overhangs used in enzyme-free cloning tend to result in self-ligation of the vector under some circumstances. Here, we describe a "T-type" enzyme-free cloning method: instead of designing the complementary staggered overhangs used in conventional enzyme-free cloning, we create "T-type" overhangs that reduce the possibility of self-ligation and are more convenient for multi-vector cloning. In this study, we systematically optimize "T-type" enzyme-free cloning, compare its cloning background with that in conventional enzyme-free cloning, and demonstrate a promising application of this technique in multi-vector cloning. Our method simplifies post-amplification procedures and greatly reduces cost, offering a competitive option for PCR product cloning.
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Theranostic GO-Based Nanohybrid for Tumor Induced Imaging and Potential Combinational Tumor Therapy.
Small
PUBLISHED: 05-24-2013
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Graphene oxide (GO)-based theranostic nanohybrid is designed for tumor induced imaging and potential combinational tumor therapy. The anti-tumor drug, Doxorubicin (DOX) is chemically conjugated to the poly(ethylenimine)-co-poly(ethylene glycol) (PEI-PEG) grafted GO via a MMP2-cleavable PLGLAG peptide linkage. The therapeutic efficacy of DOX is chemically locked and its intrinsic fluorescence is quenched by GO under normal physiological condition. Once stimulated by the MMP2 enzyme over-expressed in tumor tissues, the resulting peptide cleavage permits the unloading of DOX for tumor therapy and concurrent fluorescence recovery of DOX for in situ tumor cell imaging. Attractively, this PEI-bearing nanohybrid can mediate efficient DNA transfection and shows great potential for combinational drug/gene therapy. This tumor induced imaging and potential combinational therapy will open a window for tumor treatment by offering a unique theranostic approach through merging the diagnostic capability and pathology-responsive therapeutic function.
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Controlled arrays of self-assembled peptide nanostructures in solution and at interface.
Langmuir
PUBLISHED: 05-24-2013
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Controlling the formation of large and homogeneous arrays of bionanostructures through the self-assembly approach is still a great challenge. Here, we report the spontaneous formation of highly ordered arrays based on aligned peptide nanostructures in a solution as well as at an interface by self-assembly. By controlling the time and temperature of self-assembly in the solution, parallel fibrous alignments and more sophisticated two-dimensional "knitted" fibrous arrays could be formed from aligned rod-like fibers. During the formation of such arrays, the "disorder-to-order" transitions are controlled by the temperature-responsible motile short hydrophobic tails of the gemini-like amphiphilic peptides (GAPs) with asymmetric molecular conformation. In addition, the resulting long-range-ordered "knitted" fibrous arrays are able to direct mineralization of calcium phosphate to form organic-inorganic composite materials. In this study, the self-assembly behavior of these peptide building blocks at an interface was also studied. Highly ordered spatial arrays with vertically or horizontally aligned nanostructures such as nanofibers, microfibers, and microtubes could be formed through interfacial assembly. The regular structures and their alignments on the interface are controlled by the alkyl chain length of building blocks and the hydrophilicity/hydrophobicity property of the interface.
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Peptide-based vector of VEGF plasmid for efficient gene delivery in vitro and vessel formation in vivo.
Bioconjug. Chem.
PUBLISHED: 05-21-2013
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Critical limb ischemia is regarded as a potentially lethal disease, and the treatment effects of existing therapies are limited. Here, in order to develop a potential approach to improve the therapy effects, we designed a peptide of TAT-PKKKRKV as the vector for VEGF165 plasmid to facilitate in vivo angiogenesis. In in vitro studies, TAT-PKKKRKV with low cytotoxicity exhibited efficient transfection ability either with or without serum. Additionally, application of TAT-PKKKRKV/VEGF165 complexes in hindlimb ischemia rats obviously promoted the expression of VEGF protein, which further enhanced effective angiogenesis. The results indicated that TAT-PKKKRKV is an efficient gene vector with low toxicity both in vitro and in vivo, which has great potential for clinical gene therapy.
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Therapeutic nanomedicine based on dual-intelligent functionalized gold nanoparticles for cancer imaging and therapy in vivo.
Biomaterials
PUBLISHED: 05-08-2013
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A novel strategy to construct a therapeutic system based on functionalized AuNPs which can specifically respond to tumor microenvironment was reported. In the therapeutic system, doxorubicin was conjugated to AuNPs via thiol-Au bond by using a peptide substrate, CPLGLAGG, which can be specifically cleaved by the protease. In vivo study shows that after injection of the functionalized AuNPs to the tumor-bearing mice, the over-expressed protease of MMP-2 in tumor tissue and intracellular GSH can lead to the rapid release of the anti-tumor drug (doxorubicin) from the functionalized AuNPs to inhibit tumor growth and realize fluorescently imaging simultaneously. The functionalized AuNPs with tumor-triggered drug release property can further improve the efficacy and reduce side effects significantly.
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In situ recognition of cell-surface glycans and targeted imaging of cancer cells.
Sci Rep
PUBLISHED: 05-01-2013
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Fluorescent sensors capable of recognizing cancer-associated glycans, such as sialyl Lewis X (sLe(x)) tetrasaccharide, have great potential for cancer diagnosis and therapy. Studies on water-soluble and biocompatible sensors for in situ recognition of cancer-associated glycans in live cells and targeted imaging of cancer cells are very limited at present. Here we report boronic acid-functionalized peptide-based fluorescent sensors (BPFSs) for in situ recognition and differentiation of cancer-associated glycans, as well as targeted imaging of cancer cells. By screening BPFSs with different structures, it was demonstrated that BPFS? with a FRGDF peptide could recognize cell-surface glycan of sLe(x) with high specificity and thereafter fluorescently label and discriminate cancer cells through the cooperation with the specific recognition between RGD and integrins. The newly developed peptide-based sensor will find great potential as a fluorescent probe for cancer diagnosis.
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Bioreducible polypeptide containing cell-penetrating sequence for efficient gene delivery.
Pharm. Res.
PUBLISHED: 03-27-2013
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To design excellent polypeptide-based gene vectors and determine the gene delivery efficiency.
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Multifunctional Envelope-Type Mesoporous Silica Nanoparticles for Tumor-Triggered Targeting Drug Delivery.
J. Am. Chem. Soc.
PUBLISHED: 03-19-2013
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A novel type of cellular-uptake-shielding multifunctional envelope-type mesoporous silica nanoparticle (MEMSN) was designed for tumor-triggered targeting drug delivery to cancerous cells. ?-Cyclodextrin (?-CD) was anchored on the surface of mesoporous silica nanoparticles via disulfide linking for glutathione-induced intracellular drug release. Then a peptide sequence containing Arg-Gly-Asp (RGD) motif and matrix metalloproteinase (MMP) substrate peptide Pro-Leu-Gly-Val-Arg (PLGVR) was introduced onto the surface of the nanoparticles via host-guest interaction. To protect the targeting ligand and prevent the nanoparticles from being uptaken by normal cells, the nanoparticles were further decorated with poly(aspartic acid) (PASP) to obtain MEMSN. In vitro study demonstrated that MEMSN was shielded against normal cells. After reaching the tumor cells, the targeting property could be switched on by removing the PASP protection layer via hydrolyzation of PLGVR at the MMP-rich tumor cells, which enabled the easy uptake of drug-loaded nanoparticles by tumor cells and subsequent glutathione-induced drug release intracellularly.
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Insights into Stabilization of a Viral Protein Cage in Templating Complex Nanoarchitectures: Roles of Disulfide Bonds.
Small
PUBLISHED: 03-18-2013
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As a typical protein nanostructure, virus-based nanoparticle (VNP) of simian virus 40 (SV40), which is composed of pentamers of the major capsid protein of SV40 (VP1), has been successfully employed in guiding the assembly of different nanoparticles (NPs) into predesigned nanostructures with considerable stability. However, the stabilization mechanism of SV40 VNP remains unclear. Here, the importance of inter-pentamer disulfide bonds between cysteines in the stabilization of quantum dot (QD)-containing VNPs (VNP-QDs) is comprehensively investigated by constructing a series of VP1 mutants of cysteine to serine. Although the presence of a QD core can greatly enhance the assembly and stability of SV40 VNPs, disulfide bonds are vital to stability of VNP-QDs. Cysteine at position 9 (C9) and C104 contribute most of the disulfide bonds and play essential roles in determining the stability of SV40 VNPs as templates to guide assembly of complex nanoarchitectures. These results provide insightful clues to understanding the robustness of SV40 VNPs in organizing suprastructures of inorganic NPs. It is expected that these findings will help guide the future design and construction of protein-based functional nanostructures.
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Oligoamines grafted hyperbranched polyether as high efficient and serum-tolerant gene vectors.
Colloids Surf B Biointerfaces
PUBLISHED: 02-22-2013
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To develop low toxic, high efficient, and excellent serum-tolerant polycation gene delivery systems, a series of oligoamines grafted hyperbranched polyether (oligoamines-g-HBP) were synthesized by conjugating different oligoamines, including triethylenetetramine (TETA) and tetraethylenepentamine (TEPA), onto COOH-functionalized hyperbranched poly(3-ethyl-3-oxetanemethanol). It was found that oligoamines-g-HBP exhibited good buffering capacity, strong DNA binding and high resistance against protein adsorption. In vitro cytotoxicity measurement indicated that oligoamines-g-HBP had much lower cytotoxicity as compared with 25kDa PEI. The transfection efficiency of TEPA-g-HBP/DNA complexes at a certain N/P ratio was significantly higher than that of 25kDa PEI/DNA complexes. Interestingly, it was found that TEPA-g-HBP had much improved serum-tolerant capability as compared with 25kDa PEI even when serum concentration was increased to 30%. Confocal laser images further showed that the amount of YOYO-1 labeled DNA in nuclei got increased with increasing the number of secondary amino ethylene groups in oligoamines-g-HBP. The oligoamines-g-HBP presented great potential as gene delivery vectors for further clinical applications.
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Genome sequencing of 161 Mycobacterium tuberculosis isolates from China identifies genes and intergenic regions associated with drug resistance.
Nat. Genet.
PUBLISHED: 02-07-2013
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The worldwide emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis threatens to make this disease incurable. Drug resistance mechanisms are only partially understood, and whether the current understanding of the genetic basis of drug resistance in M. tuberculosis is sufficiently comprehensive remains unclear. Here we sequenced and analyzed 161 isolates with a range of drug resistance profiles, discovering 72 new genes, 28 intergenic regions (IGRs), 11 nonsynonymous SNPs and 10 IGR SNPs with strong, consistent associations with drug resistance. On the basis of our examination of the dN/dS ratios of nonsynonymous to synonymous SNPs among the isolates, we suggest that the drug resistance-associated genes identified here likely contain essentially all the nonsynonymous SNPs that have arisen as a result of drug pressure in these isolates and should thus represent a near-complete set of drug resistance-associated genes for these isolates and antibiotics. Our work indicates that the genetic basis of drug resistance is more complex than previously anticipated and provides a strong foundation for elucidating unknown drug resistance mechanisms.
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Use of hGluc/tdTomato pair for sensitive BRET sensing of protease with high solution media tolerance.
Talanta
PUBLISHED: 01-25-2013
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Due to the complicated media, monitoring proteases in real physiological environments is still a big challenge. Bioluminescence resonance energy transfer (BRET) is one of the promising techniques but its application is limited by the susceptibility to buffer composition, which might cause serious errors for the assay. Herein we report a novel combination of BRET pair with humanized Gaussia luciferase (hGluc) and highly bright red fluorescence protein tdTomato for sensitive and robust protease activity determination. As a result, the hGluc/tdTomato BRET pair showed much better tolerance to buffer composition, pH and sample matrices, and wide spectral separation (??:~110 nm). With the protease sensor built with this pair, the detection limit for enterokinase reached 2.1 pM in pure buffer and 3.3 pM in 3% serum. The proposed pair would find broad use in both in vitro and in vivo assays, especially for samples with complicated matrix.
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Graphene-based anticancer nanosystem and its biosafety evaluation using a zebrafish model.
Biomacromolecules
PUBLISHED: 01-11-2013
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In this paper, a facile strategy to develop graphene-based delivery nanosystems for effective drug loading and sustained drug release was proposed and validated. Specifically, biocompatible naphthalene-terminated PEG (NP) and anticancer drugs (curcumin or doxorubicin (DOX)) were simultaneously integrated onto oxidized graphene (GO), leading to self-assembled, nanosized complexes. It was found that the oxidation degree of GO had a significant impact on the drug-loading efficiency and the structural stability of nanosystems. Interestingly, the nanoassemblies resulted in more effective cellular entry of DOX in comparison with free DOX or DOX-loaded PEG-polyester micelles at equivalent DOX dose, as demonstrated by confocal microscopy studies. Moreover, the nanoassemblies not only exhibited a sustained drug release pattern without an initial burst release, but also significantly improved the stability of formulations which were resistant to drug leaking even in the presence of strong surfactants such as aromatic sodium benzenesulfonate (SBen) and aliphatic sodium dodecylsulfonate (SDS). In addition, the nanoassemblies without DOX loading showed negligible in vitro cytotoxicity, whereas DOX-loaded counterparts led to considerable toxicity against HeLa cells. The DOX-mediated cytotoxicity of the graphene-based formulation was around 20 folds lower than that of free DOX, most likely due to the slow DOX release from complexes. A zebrafish model was established to assess the in vivo safety profile of curcumin-loaded nanosystems. The results showed they were able to excrete from the zebrafish body rapidly and had nearly no influence on the zebrafish upgrowth. Those encouraging results may prompt the advance of graphene-based nanotherapeutics for biomedical applications.
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Synergistic gene and drug tumor therapy using a chimeric peptide.
Biomaterials
PUBLISHED: 01-06-2013
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Co-delivery of gene and drug for synergistic therapy has provided a promising strategy to cure devastating diseases. Here, an amphiphilic chimeric peptide (Fmoc)2KH7-TAT with pH-responsibility for gene and drug delivery was designed and fabricated. As a drug carrier, the micelles self-assembled from the peptide exhibited a much faster doxorubicin (DOX) release rate at pH 5.0 than that at pH 7.4. As a non-viral gene vector, (Fmoc)(2)KH(7)-TAT peptide could satisfactorily mediate transfection of pGL-3 reporter plasmid with or without the existence of serum in both 293T and HeLa cell-lines. Besides, the endosome escape capability of peptide/DNA complexes was investigated by confocal laser scanning microscopy (CLSM). To evaluate the co-delivery efficiency and the synergistic anti-tumor effect of gene and drug, p53 plasmid and DOX were simultaneously loaded in the peptide micelles to form micelleplexes during the self-assembly of the peptide. Cellular uptake and intracellular delivery of gene and drug were studied by CLSM and flow cytometry respectively. And p53 protein expression was determined via Western blot analysis. The in vitro cytotoxicity and in vivo tumor inhibition effect were also studied. Results suggest that the co-delivery of gene and drug from peptide micelles resulted in effective cell growth inhibition in vitro and significant tumor growth restraining in vivo. The chimeric peptide-based gene and drug co-delivery system will find great potential for tumor therapy.
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Imaging of mRNA-Protein Interactions in Live Cells Using Novel mCherry Trimolecular Fluorescence Complementation Systems.
PLoS ONE
PUBLISHED: 01-01-2013
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Live cell imaging of mRNA-protein interactions makes it possible to study posttranscriptional processes of cellular and viral gene expression under physiological conditions. In this study, red color mCherry-based trimolecular fluorescence complementation (TriFC) systems were constructed as new tools for visualizing mRNA-protein interaction in living cells using split mCherry fragments and HIV REV-RRE and TAT-TAR peptide-RNA interaction pairs. The new mCherry TriFC systems were successfully used to image RNA-protein interactions such as that between influenza viral protein NS1 and the 5 UTR of influenza viral mRNAs NS, M, and NP. Upon combination of an mCherry TriFC system with a Venus TriFC system, multiple mRNA-protein interactions could be detected simultaneously in the same cells. Then, the new mCherry TriFC system was used for imaging of interactions between influenza A virus mRNAs and some of adapter proteins in cellular TAP nuclear export pathway in live cells. Adapter proteins Aly and UAP56 were found to associate with three kinds of viral mRNAs. Another adapter protein, splicing factor 9G8, only interacted with intron-containing spliced M2 mRNA. Co-immunoprecipitation assays with influenza A virus-infected cells confirmed these interactions. This study provides long-wavelength-spectrum TriFC systems as new tools for visualizing RNA-protein interactions in live cells and help to understand the nuclear export mechanism of influenza A viral mRNAs.
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Quantum dot-induced viral capsid assembling in dissociation buffer.
Int J Nanomedicine
PUBLISHED: 01-01-2013
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Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs) are still unknown. In this article, it was found that quantum dots (QDs) can induce simian virus 40 (SV40) capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1) can be assembled into ?25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD=2.19E-10 M), which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles.
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Enhanced nuclear import and transfection efficiency of TAT peptide-based gene delivery systems modified by additional nuclear localization signals.
Bioconjug. Chem.
PUBLISHED: 12-21-2011
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Cellular uptake and nuclear localization are two major barriers in gene delivery. In order to evaluate whether additional nuclear localization signals (NLSs) can improve gene transfection efficiency, we introduced different kinds of NLSs to TAT-based gene delivery systems to form three kinds of complexes, including TAT-PV/DNA, TAT/DNA/PV, and TAT/DNA/HMGB1. The DNA binding ability of different vectors was evaluated by agarose gel electrophoresis. The in vitro transfections mediated by different complexes under different conditions were carried out. The cells treated by different complexes were observed by confocal microscopy. The MTT assay showed that all complexes did not exhibit apparent cytotoxicity in both HeLa and Cos7 cell lines even at high N/P ratios. The luciferase reporter gene expression mediated by TAT-PV/DNA complexes exhibited about 200-fold enhancement as compared with TAT/DNA complexes. Confocal study showed that, except TAT/DNA/PV, all other complexes exhibited enhanced nuclear accumulation and cellular uptake in both HeLa and Cos7 cell lines. These results indicated that the introduction of nuclear localization signals could enhance the transfection efficacy of TAT-based peptides, implying that the TAT peptide-based vectors demonstrated here have promising potential in gene delivery.
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Morphology transformation via pH-triggered self-assembly of peptides.
Langmuir
PUBLISHED: 12-13-2011
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Three flexible peptides (P1: (C(17)H(35)CO-NH-GRGDG)(2)KG; P2: (Fmoc-GRGDG)(2)KG; P3: (CH(3)CO-NH-GRGDG)(2)KG) self-assembled to form a variety of morphologically distinct assemblies at different pHs. P1 formed nanofibers at pH 3, then self-assembled into nanospheres with pH up to 6 and further changed to lamellar structures when the pH value was further increased to 10. P2 aggregated into an entwined network structure at pH 3, and then self-assembled into well-defined nanospheres, lamellar structures, and vesicles via adjusting the pH value. However, P3 did not self-assemble into well-ordered nanostructures, presumably due to the absence of a large hydrophobic group. The varying self-assembly behaviors of the peptides at different pHs are attributed to molecular conformational changes. These self-assembled supramolecular materials might contribute to the development of new peptide-based biomaterials.
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Monofunctionalization of protein nanocages.
J. Am. Chem. Soc.
PUBLISHED: 11-23-2011
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Surface monofunctionalization of protein nanostructures will enable precise topological control over the protein-templated assembly of nanoscale motifs, however, this remains a formidable challenge. Here we demonstrated a novel strategy for this purpose with a protein nanocage, virus-based nanoparticle (VNP) of simian virus 40 as a model system. By simultaneously incorporating a function modality (cysteine) and a purification modality (polyhistidine tag) into the building block (VP1) of VNPs through rational design and genetic engineering, the monofunctionalized cysteine-VNPs are readily obtained through a routine affinity chromatography in virtue of the purification modality of polyhistidine tag, after the coassembly of the functional VP1 and the nonfunctional VP1 at an optimal ratio. This strategy has proved to be highly efficient in constructing monofunctionalized protein nanostructures as highlighted by the monofunctionalized-VNP-guided Au/QD-VNP nanostructures. These nanostructures could be utilized in a wide range of disciplines, including basic biological research, novel nanostructures, and nanodevices fabrication, etc.
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Multifunctional ferritin cage nanostructures for fluorescence and MR imaging of tumor cells.
Nanoscale
PUBLISHED: 11-14-2011
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Bionanoparticles and nanostructures have attracted increasing interest as versatile and promising tools in many applications including biosensing and bioimaging. In this study, to image and detect tumor cells, ferritin cage-based multifunctional hybrid nanostructures were constructed that: (i) displayed both the green fluorescent protein and an Arg-Gly-Asp peptide on the exterior surface of the ferritin cages; and (ii) incorporated ferrimagnetic iron oxide nanoparticles into the ferritin interior cavity. The overall architecture of ferritin cages did not change after being integrated with fusion proteins and ferrimagnetic iron oxide nanoparticles. These multifunctional nanostructures were successfully used as a fluorescent imaging probe and an MRI contrast agent for specifically probing and imaging ?(v)?(3) integrin upregulated tumor cells. The work provides a promising strategy for tumor cell detection by simultaneous fluorescence and MR imaging.
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Comparative analysis of mycobacterial NADH pyrophosphatase isoforms reveals a novel mechanism for isoniazid and ethionamide inactivation.
Mol. Microbiol.
PUBLISHED: 11-03-2011
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NADH pyrophosphatase (NudC) catalyses the hydrolysis of NAD(H) to AMP and NMN(H) [nicotinamide mononucleotide (reduced form)]. NudC multiple sequence alignment reveals that homologues from most Mycobacterium tuberculosis isolates, but not other mycobacterial species, have a polymorphism at the highly conserved residue 237. To elucidate the functional significance of this polymorphism, comparative analyses were performed using representative NudC isoforms from M. tuberculosis H37Rv (NudC(Rv)) and M. bovis BCG (NudC(BCG)). Biochemical analysis showed that the P237Q polymorphism prevents dimer formation, and results in a loss of enzymatic activity. Importantly, NudC(BCG) was found to degrade the active forms of isoniazid (INH), INH-NAD and ethionamide (ETH), ETH-NAD. Consequently, overexpression of NudC(BCG) in Mycobacterium smegmatis mc(2)155 and M. bovis BCG resulted in a high level of resistance to both INH and ETH. Further genetic studies showed that deletion of the nudC gene in M. smegmatis mc(2)155 and M. bovis BCG resulted in increased susceptibility to INH and ETH. Moreover, inactivation of NudC in both strains caused a defect in drug tolerance phenotype for both drugs in exposure assays. Taken together, these data suggest that mycobacterial NudC plays an important role in the inactivation of INH and ETH.
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Amphiphilic star-block copolymers and supramolecular transformation of nanogel-like micelles to nanovesicles.
Chem. Commun. (Camb.)
PUBLISHED: 11-02-2011
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Amphiphilic star-block copolymers based on poly(3-hydroxybutyrate) with adamantyl end-functionalization were synthesized via anionic ring-opening polymerization and alkyne-azide "Click Chemistry" coupling. In aqueous medium, the copolymers self-assembled into nanogel-like large compound micelles, and transformed into vesicular nanostructures under the direction of host-guest interaction between the adamantyl end and dimethyl-?-cyclodextrin.
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Design of a photoswitchable hollow microcapsular drug delivery system by using a supramolecular drug-loading approach.
J Phys Chem B
PUBLISHED: 11-01-2011
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In this study, photoswitchable microcapsules were fabricated based on host-guest interactions between ?-cyclodextrin (?-CD) and azobenzene (Azo). Carboxymethyl dextran-graft-?-CD (CMD-g-?-CD) and poly(acrylic acid) N-aminododecane p-azobenzeneaminosuccinic acid (PAA-C(12)-Azo) were assembled layer by layer on CaCO(3) particles. ?-CD-rhodamine B (?-CD-RhB), used as a model drug, was loaded on PAA-C(12)-Azo layers by host-guest interaction. After removal of CaCO(3) particles by ethylenediaminetetraacetic acid (EDTA), hollow microcapsules loaded with ?-CD-RhB were obtained. Since the interactions between ?-CD and Azo were photosensitive, the capsules could be dissociated with the irradiation of UV light, followed by the release of the model drug, ?-CD-RhB. Compared with traditional drug-loading approaches such as chemical bonding and physical adsorption, our supramolecular drug-loading system has a facile loading process, ideal bonding strength, and photoswitchable behavior. These photosensitive microcapsules exhibit great potential in biomedical applications.
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Impact of epidemic rates of diabetes on the Chinese blood glucose testing market.
J Diabetes Sci Technol
PUBLISHED: 10-27-2011
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China has become the country with the largest diabetes mellitus population in the world since the 1990s. About 100 million diabetes cases have been diagnosed since 2008. Handheld blood glucose meters and test strips are urgently needed for daily patient measurement. The glucose monitor with a screen-printed carbon-based glucose electrode has been in commercial production since 1994. Since then, approximately 20 companies have been involved in manufacturing and marketing meters and test strips in China. The current market and production volume and updates on technology issues are discussed in this article.
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Encapsulation of gold nanoparticles by simian virus 40 capsids.
Nanoscale
PUBLISHED: 08-30-2011
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Viral capsid-nanoparticle hybrid structures constitute a new type of nanoarchitecture that can be used for various applications. We previously constructed a hybrid structure comprising quantum dots encapsulated by simian virus 40 (SV40) capsids for imaging viral infection pathways. Here, gold nanoparticles (AuNPs) are encapsulated into SV40 capsids and the effect of particle size and surface ligands (i.e. mPEG and DNA) on AuNP encapsulation is studied. Particle size and surface decoration play complex roles in AuNP encapsulation by SV40 capsids. AuNPs ?15 nm (when coated with mPEG750 rather than mPEG2000), or ?10 nm (when coated with 10T or 50T DNA) can be encapsulated. Encapsulation efficiency increased as the size of the AuNPs increased from 10 to 30 nm. In addition, the electrostatic interactions derived from negatively charged DNA ligands on the AuNP surfaces promote encapsulation when the AuNPs have a small diameter (i.e. 10 nm and 15 nm). Moreover, the SV40 capsid is able to carry mPEG750-modified 15-nm AuNPs into living Vero cells, whereas the mPEG750-modified 15-nm AuNPs alone cannot enter cells. These results will improve our understanding of the mechanisms underlying nanoparticle encapsulation in SV40 capsids and enable the construction of new functional hybrid nanostructures for cargo delivery.
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A strategy to improve serum-tolerant transfection activity of polycation vectors by surface hydroxylation.
Biomaterials
PUBLISHED: 08-23-2011
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The aim of this contribution is to develop a universal method to promote the serum-tolerant capability of polycation-based gene delivery system. A "hydroxylation camouflage" strategy was put forward by coating the polycation vectors with hydroxyl-enriched "skin". Branched polyethyleneimine (PEI) was herein used as the polycation model and modified via the catalyst-free aminolysis reaction with 5-ethyl-5-(hydroxymethyl)-1,3-dioxan-2-oxo (EHDO). PEI-g-EHDO, PEI and alkylated PEI derivative termed as PEI-g-DPA were comparatively explored with respect to the transfection efficiency in the serum-free and serum-conditioned medium. The resultant data indicate that the serum-tolerant capability largely depended on the surface composition and substitution degree. In addition to the reduced surface charge, the introduced function caused by hydroxyl coating is believed to play a crucial role for the improved properties of PEI-g-EHDOs. The EHDO modification can effectively inhibit the adsorption of BSA proteins onto polyplexes surface. And the polyplexes stability was remarkably enhanced in the presence of DNase and heparin after EHDO modification. Note that the transfection activity of PEI-g-EHDO(34.5%) in the serum-conditioned medium was even higher than that without serum addition. In contrast, serum addition led to appreciable reduction in the transfection efficiency mediated by PEI and PEI-g-DPAs. Specifically, as far as the transfection activity in the presence of serum is concerned, PEI-g-EHDO could be up to 30-fold higher than unmodified PEI25k. PEI-g-EHDO(34.5%) displayed little to no hemolytic effect and high cell-biocompatibility with nearly no cytotoxicity detected in 293T cells and HeLa cells. Taking into account the high biocompatibility and serum-tolerant transfection activity, PEI-g-EHDO(34.5%) holds great potential for the use as efficient gene vector. More importantly, it is expected that such "hydroxylation camouflage" strategy may be universally applicable for a majority of existing polycation vectors.
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Rapid colorimetric testing for pyrazinamide susceptibility of M. tuberculosis by a PCR-based in-vitro synthesized pyrazinamidase method.
PLoS ONE
PUBLISHED: 08-08-2011
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Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug. But PZA susceptibility test is challenging because PZA activity is optimal only in an acid environment that inhibits the growth of M. tuberculosis. For current phenotypic methods, inconsistent results between different labs have been reported. Direct sequencing of pncA gene is being considered as an accurate predictor for PZA susceptibility, but this approach needs expensive sequencers and a mutation database to report the results. An in-vitro synthesized Pyrazinamidase (PZase) assay was developed based on PCR amplification of pncA gene and an in vitro wheat germ system to express the pncA gene into PZase. The activity of the synthesized PZase was used as an indicator for PZA susceptibility. Fifty-one clinical isolates were tested along with pncA sequencing and the BACTEC MGIT 960 methods. The in-vitro synthesized PZase assay was able to detect PZA susceptibility of M. tuberculosis within 24 h through observing the color difference either by a spectrometer or naked eyes. This method showed agreements of 100% (33/33) and 88% (14/16) with the pncA sequencing method, and agreements of 96% (27/28) and 65% (15/23) with the BACTEC MGIT 960 method, for susceptible and resistant strains, respectively. The novel in-vitro synthesized PZase assay has significant advantages over current methods, such as its fast speed, simplicity, no need for expensive equipment, and the potentials of being a direct test, predicting resistance level and easy reading results by naked eyes. After confirmation by more clinical tests, this method may provide a radical change to the current PZA susceptibility assays.
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MutL associates with Escherichia coli RecA and inhibits its ATPase activity.
Arch. Biochem. Biophys.
PUBLISHED: 07-11-2011
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Different DNA repair systems are known to cooperate to deal with DNA damage. However, the regulatory role of the cross-talk between these pathways is unclear. Here, we have shown that MutL, an essential component of mismatch repair, is a RecA-interacting protein, and that its highly conserved N-terminal domain is sufficient for this interaction. Surface plasmon resonance and capillary electrophoresis analyses revealed that MutL has little effect on RecA-ssDNA filament formation, but dose down-regulate the ATPase activity of RecA. Our findings identify a new role for MutL, and suggest its regulatory role in homologous recombination.
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The dimer state of GyrB is an active form: implications for the initial complex assembly and processive strand passage.
Nucleic Acids Res.
PUBLISHED: 07-10-2011
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In a previous study, we presented the dimer structure of DNA gyrase B domain (GyrB C-terminal domain) from Mycobacterium tuberculosis and proposed a sluice-like model for T-segment transport. However, the role of the dimer structure is still not well understood. Cross-linking and analytical ultracentrifugation experiments showed that the dimer structure exists both in the B protein and in the full-length GyrB in solution. The cross-linked dimer of GyrB bound GyrA very weakly, but bound dsDNA with a much higher affinity than that of the monomer state. Using cross-linking and far-western analyses, the dimer state of GyrB was found to be involved in the ternary GyrA-GyrB-DNA complex. The results of mutational studies reveal that the dimer structure represents a state before DNA cleavage. Additionally, these results suggest that the dimer might also be present between the cleavage and reunion steps during processive transport.
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Targeted delivery in breast cancer cells via iodine: nuclear localization sequence conjugate.
Bioconjug. Chem.
PUBLISHED: 07-06-2011
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The nonviral vector with iodine-nuclear localization sequence (namely, NLS-I) targeting breast cancer cells was fabricated. Ternary complexes were formed via charge interactions among NLS-I peptides, PEI 1800, and DNA, and we investigated their cellular internalization, nuclear accumulation as well as transfection efficiency. All the experiments were assessed by employing MCF-7 cells that express sodium/iodide symporter and HeLa cells that lack the expression of the symporter. In MCF-7 cells, cell internalization and nuclear accumulation of NLS-I was markedly increased compared to that in NLS. In addition, compared to that of the PEI1800/DNA complex, PEI1800/DNA/NLS-I complexes exhibited much enhanced luciferase reporter gene expression by up to 130-fold. By contrast, in HeLa cells, the evident improvements of cellular internalization, nuclear accumulation, and transfection efficiency by NLS-I were not observed. This study demonstrates an alternative method to construct a nonviral delivery system for targeted gene transfer into breast cancer cells.
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Facile construction of nanofibers as a functional template for surface boron coordination reaction.
Small
PUBLISHED: 07-04-2011
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A facile strategy to perform the boron coordination reaction on a template of nanofibers is developed. Peptides with phenylboronic acid tails (peptidyl boronic acids) are designed and prepared as building blocks that can self-assemble into nanofibers. After the addition of vicinal diol structural motifs to the self-assembling system, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry indicates that the boron coordination reaction occurs on the template of nanofibers, which results in the increase of the width and roughness of the nanofibers as demonstrated by transmission electron microscopy and atomic force microscopy measurements. Because the surface-bound vicinal diol structural motifs have an ability to form hydrogen bonds with the peptide segments on the nanofibers, which restrain and disturb the hydrogen-bonding interaction among the nanofibers, the network structure formed based on the entanglement of nanofibers via hydrogen-bonding interaction is destroyed, which leads to a gel-sol transition. The novel concept of post-self-assembly modification demonstrated here could lead to a new technique for using self-assembled nanostructures in the emerging fields of nanoscience and nanotechnology.
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Bilayer matrix composed of polycation/DNA complex and sodium alginate gel as a tumor cell catcher.
Macromol Biosci
PUBLISHED: 06-02-2011
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A bilayer matrix consisting of TABP-SS/DNA complexes and sodium alginate gel is formed via electrostatic interaction. In vitro cell adhesion, proliferation and transfection of the bilayer matrix are investigated in HepG2, HeLa and COS7 cells. Results show that this matrix can only promote tumor cell attachment and growth. Compared with normal cells, the bilayer matrix exhibits significantly higher transfection efficacy in tumor cells. Cell co-culture competitive transfection assay shows that the cell uptake of TABP-SS/DNA complexes is significantly enhanced in tumor cells rather than normal cells under the co-culture competitive condition, which confirms that TABP-SS/DNA complexes have strong tumor cell selectivity and tumor targeting transfection ability.
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Interface self-assembly to construct vertical peptide nanorods on quartz template.
Chem. Commun. (Camb.)
PUBLISHED: 05-26-2011
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A rationally designed glycyl-glycine derivative containing a light cleaved pyrenylmethyl ester tail was covalently bound onto the surface of quartz template. The interface self-assembly of this dipeptide building block induced the formation of chemically bound vertically aligned nanorods (CBVANs) with light sensitivity on the template.
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In vitro inhibitory effect of carrageenan oligosaccharide on influenza A H1N1 virus.
Antiviral Res.
PUBLISHED: 05-18-2011
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Carrageenan polysaccharide has been reported to be able to inhibit the infection and replication of many different kinds of viruses. Here, we demonstrated that a 2 kDa ?-carrageenan oligosaccharide (CO-1) derived from the carrageenan polysaccharide, effectively inhibited influenza A (H1N1) virus replication in MDCK cells (selectivity index >25.0). Moreover, the 2 kDa CO-1 inhibited influenza A virus (IAV) replication better than that of 3 kDa and 5 kDa ?-carrageenan oligosaccharides (CO-2 and CO-3). IAV multiplication was suppressed by carrageenan oligosaccharide treatment in a dose-dependent manner. Carrageenan oligosaccharide CO-1 did not bind to the cell surface of MDCK cells but inactivated virus particles after pretreatment. Different to the actions of carrageenan polysaccharide, CO-1 could enter into MDCK cells and did not interfere with IAV adsorption. CO-1 also inhibited IAV mRNA and protein expression after its internalization into cells. Moreover, carrageenan oligosaccharide CO-1 had an antiviral effect on IAV replication subsequent to viral internalization but prior to virus release in one replication cycle. Therefore, inhibition of IAV intracellular replication by carrageenan oligosaccharide might be an alternative approach for anti-influenza A virus therapy.
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Thermo-responsive shell cross-linked PMMA-b-P(NIPAAm-co-NAS) micelles for drug delivery.
Int J Pharm
PUBLISHED: 04-25-2011
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Thermo-responsive amphiphilic poly(methyl methacrylate)-b-poly(N-isopropylacrylamide-co-N-acryloxysuccinimide) (PMMA-b-P(NIPAAm-co-NAS)) block copolymer was synthesized by successive RAFT polymerizations. The uncross-linked micelles were facilely prepared by directly dissolving the block copolymer in an aqueous medium, and the shell cross-linked (SCL) micelles were further fabricated by the addition of ethylenediamine as a di-functional cross-linker into the micellar solution. Optical absorption measurements showed that the LCST of uncross-linked and cross-linked micelles was 31.0°C and 40.8°C, respectively. Transmission electron microscopy (TEM) showed that both uncross-linked and cross-linked micelles exhibited well-defined spherical shape in aqueous phase at room temperature, while the SCL micelles were able to retain the spherical shape with relatively smaller dimension even at 40°C due to the cross-linked structure. In vitro drug release study demonstrated a slower and more sustained drug release behavior from the SCL micelles at high temperature as compared with the release profile of uncross-linked micelles, indicating the great potential of SCL micelles developed herein as novel smart carriers for controlled drug release.
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Quantum dot-aptamer nanoprobes for recognizing and labeling influenza A virus particles.
Nanoscale
PUBLISHED: 04-21-2011
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The fluorescence labeling of viruses is a useful technology for virus detection and imaging. By combining the excellent fluorescence properties of quantum dots (QDs) with the high affinity and specificity of aptamers, we constructed a QD-aptamer probe. The aptamer A22, against the hemagglutinin of influenza A virus, was linked to QDs, producing the QD-A22 probe. Fluorescence imaging and transmission electron microscopy showed that the QD-A22 probe could specifically recognize and label influenza A virus particles. This QD labeling technique provides a new strategy for labeling virus particles for virus detection and imaging.
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