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Find video protocols related to scientific articles indexed in Pubmed.
Bioconjugation of Gold Nanoparticles through the Oxidative Coupling of ortho-Aminophenols and Anilines.
Bioconjug. Chem.
PUBLISHED: 10-02-2014
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While there are a number of methods for attaching gold nanoparticles (AuNPs) to biomolecules, the existing strategies suffer from nonspecific AuNP adsorption, reagents that are unstable in aqueous solutions, and/or long reaction times. To improve upon existing AuNP bioconjugation strategies, we have adapted a recently reported potassium ferricyanide-mediated oxidative coupling reaction for the attachment of aniline-functionalized AuNPs to o-aminophenol-containing oligonucleotides, peptides, and proteins. The aniline-AuNPs are stable in aqueous solutions, show little-to-no nonspecific adsorption with biomolecules, and react rapidly (30 min) with o-aminophenols under mild conditions (pH 6.5, 1 mM oxidant).
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Photoactivated bioconjugation between ortho-azidophenols and anilines: a facile approach to biomolecular photopatterning.
J. Am. Chem. Soc.
PUBLISHED: 08-29-2014
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Methods for the surface patterning of small molecules and biomolecules can yield useful platforms for drug screening, synthetic biology applications, diagnostics, and the immobilization of live cells. However, new techniques are needed to achieve the ease, feature sizes, reliability, and patterning speed necessary for widespread adoption. Herein, we report an easily accessible and operationally simple photoinitiated reaction that can achieve patterned bioconjugation in a highly chemoselective manner. The reaction involves the photolysis of 2-azidophenols to generate iminoquinone intermediates that couple rapidly to aniline groups. We demonstrate the broad functional group compatibility of this reaction for the modification of proteins, polymers, oligonucleotides, peptides, and small molecules. As a specific application, the reaction was adapted for the photolithographic patterning of azidophenol DNA on aniline glass substrates. The presence of the DNA was confirmed by the ability of the surface to capture living cells bearing the sequence complement on their cell walls or cytoplasmic membranes. Compared to other light-based DNA patterning methods, this reaction offers higher speed and does not require the use of a photoresist or other blocking material.
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Hierarchical assembly of plasmonic nanostructures using virus capsid scaffolds on DNA origami templates.
ACS Nano
PUBLISHED: 07-18-2014
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Building plasmonic nanostructures using biomolecules as scaffolds has shown great potential for attaining tunable light absorption and emission via precise spatial organization of optical species and antennae. Here we report bottom-up assembly of hierarchical plasmonic nanostructures using DNA origami templates and MS2 virus capsids. These serve as programmable scaffolds that provide molecular level control over the distribution of fluorophores and nanometer-scale control over their distance from a gold nanoparticle antenna. While previous research using DNA origami to assemble plasmonic nanostructures focused on determining the distance-dependent response of single fluorophores, here we address the challenge of constructing hybrid nanostructures that present an organized ensemble of fluorophores and then investigate the plasmonic response. By combining finite-difference time-domain numerical simulations with atomic force microscopy and correlated scanning confocal fluorescence microscopy, we find that the use of the scaffold keeps the majority of the fluorophores out of the quenching zone, leading to increased fluorescence intensity and mild levels of enhancement. The results show that the degree of enhancement can be controlled by exploiting capsid scaffolds of different sizes and tuning capsid-AuNP distances. These bioinspired plasmonic nanostructures provide a flexible design for manipulating photonic excitation and photoemission.
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N-terminal modification of proteins with o-aminophenols.
J. Am. Chem. Soc.
PUBLISHED: 06-25-2014
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The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.
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RcsB positively regulates the Yersinia?Ysc-Yop type III secretion system by activating expression of the master transcriptional regulator LcrF.
Environ. Microbiol.
PUBLISHED: 04-14-2014
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The Rcs phosphorelay is a complex signaling pathway used by the family Enterobacteriaceae to sense, respond and adapt to environmental changes during free-living or host-associated lifestyles. In this study, we show that the Rcs phosphorelay pathway positively regulates the virulence plasmid encoded Ysc-Yop type III secretion system (T3SS) in the enteropathogen Yesinia pseudotuberculosis. Both the overexpression of the wild-type Rcs regulator RcsB or the constitutive active RscB(D56E) variant triggered more abundant Ysc-Yop synthesis and secretion, whereas the non-phosphorylatable mutant RcsB(D56Q) negated this. Congruently, enhanced Yops expression and secretion occurred in an in cis rscB(D56E) mutant but not in an isogenic rscB(D56Q) mutant. Screening for regulatory targets of RcsB identified the virG-lcrF operon that encodes for LcrF, the Ysc-Yop T3SS master regulator. Protein-DNA binding assays confirmed that RcsB directly bound to this operon promoter, which subsequently caused stimulated lcrF transcription. Moreover, active RcsB enhanced the ability of bacteria to deliver Yop effectors into immune cells during cell contact, and this promoted an increase in bacterial viability. Taken together, our study demonstrates the role of the Rcs system in regulating the Ysc-Yop T3SS in Yersinia and reports on RcsB being the first transcriptional activator known to directly control lcrF transcription.
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Effects of NIPAm polymer additives on the enzymatic hydrolysis of Avicel and pretreated Miscanthus.
Biotechnol. Bioeng.
PUBLISHED: 03-14-2014
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There is currently much interest in the economic use of cellulosic biomass as a source of renewable fuels. This process typically involves the enzymatic hydrolysis of plant matter to afford soluble sugars for subsequent fermentation steps. The cost of cellulase enzymes presents a critical barrier to the commercialization of these processes. In this work, we demonstrate that a new family of polymer additives based on NIPAm can increase enzyme performance substantially. When applied to an industrially relevant combination of enzymes and lignin-containing biomass, polymer additives allow a 60% reduction in enzyme loading to achieve the same level of saccharification. Evidence presented herein suggests that these polymers function through multiple mechanisms, including (1) preventing enzyme denaturation through shear and interfacial interactions, (2) preventing non-productive adsorption to lignin, and (3) altering the cellulose structure. An advantage of these polymers over other additives is their thermoresponsive behavior, enabling their recovery and reuse.
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A cis-encoded sRNA controls the expression of fabH2 in Yersinia.
FEBS Lett.
PUBLISHED: 02-10-2014
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YsrH is a novel cis-encoded sRNA located on the opposite strand to fabH2, which is essential for fatty acid biosynthesis in bacteria. In this study, YsrH-mediated regulation of fabH2 expression was investigated in Yersinia pseudotuberculosis. Constitutive and inducible over-expression of YsrH decreased the mRNA level of fabH2, while expression of downstream fabD and fabG remained unaffected. Polynucleotide phosphorylase (PNPase) also played an important role in this regulation process by mediating YsrH decay in the exponential phase. Thus, our data defines a cis-encoded sRNA that regulates fatty acid synthesis via a regulatory mechanism also involving PNPase.
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'Miracle cure' or 'liquid handcuffs': reporting on naltrexone and methadone in the Australian print media.
Drug Alcohol Rev
PUBLISHED: 02-10-2014
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The news media is an important source of information regarding new developments in medicine and public health interventions. Previous research has indicated that in many cases, reporting on new treatments can be inaccurate or sensationalist. This paper presents analysis of Australian print media reporting on two treatment options for heroin dependence (naltrexone and methadone). The aim of this study was to quantitatively compare the volume and content of Australian print media reporting on these two treatments, one of which had a long history of use in Australia, and the other which was comparatively newer.
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Multivalent viral capsids with internal cargo for fibrin imaging.
PLoS ONE
PUBLISHED: 01-01-2014
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Thrombosis is the cause of many cardiovascular syndromes and is a significant contributor to life-threatening diseases, such as myocardial infarction and stroke. Thrombus targeted imaging agents have the capability to provide molecular information about pathological clots, potentially improving detection, risk stratification, and therapy of thrombosis-related diseases. Nanocarriers are a promising platform for the development of molecular imaging agents as they can be modified to have external targeting ligands and internal functional cargo. In this work, we report the synthesis and use of chemically functionalized bacteriophage MS2 capsids as biomolecule-based nanoparticles for fibrin imaging. The capsids were modified using an oxidative coupling reaction, conjugating ?90 copies of a fibrin targeting peptide to the exterior of each protein shell. The ability of the multivalent, targeted capsids to bind fibrin was first demonstrated by determining the impact on thrombin-mediated clot formation. The modified capsids out-performed the free peptides and were shown to inhibit clot formation at effective concentrations over ten-fold lower than the monomeric peptide alone. The installation of near-infrared fluorophores on the interior surface of the capsids enabled optical detection of binding to fibrin clots. The targeted capsids bound to fibrin, exhibiting higher signal-to-background than control, non-targeted MS2-based nanoagents. The in vitro assessment of the capsids suggests that fibrin-targeted MS2 capsids could be used as delivery agents to thrombi for diagnostic or therapeutic applications.
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Hyperpolarized Xenon-Based Molecular Sensors for Label-Free Detection of analytes.
J. Am. Chem. Soc.
PUBLISHED: 12-18-2013
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Nuclear magnetic resonance (NMR) can reveal the chemical constituents of a complex mixture without resorting to chemical modification, separation, or other perturbation. Recently, we and others have developed magnetic resonance agents that report on the presence of dilute analytes by proportionately altering the response of a more abundant or easily detected species, a form of amplification. One example of such a sensing medium is xenon gas, which is chemically inert and can be optically hyperpolarized, a process that enhances its NMR signal by up to 5 orders of magnitude. Here, we use a combinatorial synthetic approach to produce xenon magnetic resonance sensors that respond to small molecule analytes. The sensor responds to the ligand by producing a small chemical shift change in the Xe NMR spectrum. We demonstrate this technique for the dye, Rhodamine 6G, for which we have an independent optical assay to verify binding. We thus demonstrate that specific binding of a small molecule can produce a xenon chemical shift change, suggesting a general approach to the production of xenon sensors targeted to small molecule analytes for in vitro assays or molecular imaging in vivo.
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Yersinia?Ysc-Yop type III secretion feedback inhibition is relieved through YscV-dependent recognition and secretion of LcrQ.
Mol. Microbiol.
PUBLISHED: 11-28-2013
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Human pathogenic Yersinia species share a virulence plasmid encoding the Ysc-Yop type III secretion system (T3SS). A plasmid-encoded anti-activator, LcrQ, negatively regulates the expression of this secretion system. Under inducible conditions, LcrQ is secreted outside of bacterial cells and this activates the T3SS, but the mechanism of targeting LcrQ for type III secretion remains largely unknown. In this study, we characterized the regulatory role of the export apparatus component YscV. Depletion or overexpression of YscV compromised Yop synthesis and this primarily prevented secretion of LcrQ. It followed that a lcrQ deletion reversed the repressive effects of excessive YscV. Further characterization demonstrated that the YscV residues 493-511 located within the C-terminal soluble cytoplasmic domain directly bound with LcrQ. Critically, YscV-LcrQ complex formation was a requirement for LcrQ secretion, since YscV?493-511 failed to secrete LcrQ. This forced a cytoplasmic accumulation of LcrQ, which predictably caused the feedback inhibition of Yops synthesis. Based on these observations, we proposed a model for the YscV-dependent secretion of LcrQ and its role in regulating Yop synthesis in Yersinia.
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Selective Chromium(VI) Ligands Identified Using Combinatorial Peptoid Libraries.
J. Am. Chem. Soc.
PUBLISHED: 11-06-2013
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Hexavalent chromium [Cr(VI)] is a worldwide water contaminant that is currently without cost-effective and efficient remediation strategies. This is in part due to a lack of ligands that can bind it amid an excess of innocuous ions in aqueous solution. We present herein the design and application of a peptoid-based library of ligand candidates for toxic metal ions. A selective screening process was used to identify members of the library that can bind to Cr(VI) species at neutral pH and in the presence of a large excess of spectator ions. There were 11 sequences identified, and their affinities were compared using titrations monitored with UV-vis spectroscopy. To identify the interactions involved in coordination and specificity, we evaluated the effects of sequence substitutions and backbone variation in the highest affinity structure. Additional characterization of the complex formed between this sequence and Cr(VI) was performed using NMR spectroscopy. To evaluate the ability of the developed sequences to remediate contaminated solutions, the structures were synthesized on a solid-phase resin and incubated with environmental water samples that contained simulated levels of chromium contamination. The synthetic structures demonstrated the ability to reduce the amount of toxic chromium to levels within the range of the EPA contamination guidelines. In addition to providing some of the first selective ligands for Cr(VI), these studies highlight the promise of peptoid sequences as easily prepared components of environmental remediation materials.
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Site-specific protein transamination using N-methylpyridinium-4-carboxaldehyde.
J. Am. Chem. Soc.
PUBLISHED: 11-05-2013
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The controlled attachment of synthetic groups to proteins is important for a number of fields, including therapeutics, where antibody-drug conjugates are an emerging area of biologic medicines. We have previously reported a site-specific protein modification method using a transamination reaction that chemoselectively oxidizes the N-terminal amine of a polypeptide chain to a ketone or an aldehyde group. The newly introduced carbonyl can be used for conjugation to a synthetic group in one location through the formation of an oxime or a hydrazone linkage. To expand the scope of this reaction, we have used a combinatorial peptide library screening platform as a method to explore new transamination reagents while simultaneously identifying their optimal N-terminal sequences. N-Methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoports salt, RS) was identified as a highly effective transamination reagent when paired with glutamate-terminal peptides and proteins. This finding establishes RS as a transamination reagent that is particularly well suited for antibody modification. Using a known therapeutic antibody, herceptin, it was demonstrated that RS can be used to modify the heavy chains of the wild-type antibody or to modify both the heavy and the light chains after N-terminal sequence mutation to add additional glutamate residues.
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Mild Bioconjugation Through the Oxidative Coupling of ortho-Aminophenols and Anilines with Ferricyanide.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 08-22-2013
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Using a small-molecule-based screen, ferricyanide was identified as a mild and efficient oxidant for the coupling of anilines and o-aminophenols on protein substrates. This reaction is compatible with thiols and 1,2-diols, allowing its use in the creation of complex bioconjugates for use in biotechnology and materials applications.
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Antibody-guided photoablation of voltage-gated potassium currents.
J. Gen. Physiol.
PUBLISHED: 08-12-2013
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A family of 40 mammalian voltage-gated potassium (Kv) channels control membrane excitability in electrically excitable cells. The contribution of individual Kv channel types to electrophysiological signaling has been difficult to assign, as few selective inhibitors exist for individual Kv subunits. Guided by the exquisite selectivity of immune system interactions, we find potential for antibody conjugates as selective Kv inhibitors. Here, functionally benign anti-Kv channel monoclonal antibodies (mAbs) were chemically modified to facilitate photoablation of K currents. Antibodies were conjugated to porphyrin compounds that upon photostimulation inflict localized oxidative damage. Anti-Kv4.2 mAb-porphyrin conjugates facilitated photoablation of Kv4.2 currents. The degree of K current ablation was dependent on photon dose and conjugate concentration. Kv channel photoablation was selective for Kv4.2 over Kv4.3 or Kv2.1, yielding specificity not present in existing neurotoxins or other Kv channel inhibitors. We conclude that antibody-porphyrin conjugates are capable of selective photoablation of Kv currents. These findings demonstrate that subtype-specific mAbs that in themselves do not modulate ion channel function are capable of delivering functional payloads to specific ion channel targets.
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Demarcating SurA activities required for outer membrane targeting of Yersinia pseudotuberculosis adhesins.
Infect. Immun.
PUBLISHED: 04-15-2013
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SurA is a periplasmic protein folding factor involved in chaperoning and trafficking of outer membrane proteins across the Gram-negative bacterial periplasm. In addition, SurA also possesses peptidyl-prolyl cis/trans isomerase activity. We have previously reported that in enteropathogenic Yersinia pseudotuberculosis, SurA is needed for bacterial virulence and envelope integrity. In this study, we investigated the role of SurA in the assembly of important Yersinia adhesins. Using genetic mutation, biochemical characterization, and an in vitro-based bacterial host cell association assay, we confirmed that surface localization of the invasin adhesin is dependent on SurA. As a surA deletion also has some impact on the levels of individual components of the BAM complex in the Yersinia outer membrane, abolished invasin surface assembly could reflect both a direct loss of SurA-dependent periplasmic targeting and a potentially compromised BAM complex assembly platform in the outer membrane. To various degrees, the assembly of two other adhesins, Ail and the pH 6 antigen fibrillum PsaA, also depends on SurA. Consequently, loss of SurA leads to a dramatic reduction in Yersinia attachment to eukaryotic host cells. Genetic complementation of surA deletion mutants indicated a prominent role for SurA chaperone function in outer membrane protein assembly. Significantly, the N terminus of SurA contributed most of this SurA chaperone function. Despite a dominant chaperoning role, it was also evident that SurA isomerization activity did make a modest contribution to this assembly process.
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DNA-Mediated Assembly of Protein Heterodimers on Membrane Surfaces.
J. Am. Chem. Soc.
PUBLISHED: 03-26-2013
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We present a method based on self-assembling oligonucleotides to anchor proteins to a supported membrane surface. This anchoring method allows control of the surface density of multiple proteins. By incorporating additional recognition sequences into the DNA linkers, defined heterodimers can be produced upon the addition of a heterospecific DNA cross-linking strand. Characterization by fluorescence cross-correlation spectroscopy (FCCS) confirmed lateral mobility and the formation of specific heterodimers. We further demonstrate that proteins linked in this manner as either monomers or dimers can form functional interfaces with living cells.
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Controlled integration of gold nanoparticles and organic fluorophores using synthetically modified MS2 viral capsids.
J. Am. Chem. Soc.
PUBLISHED: 02-12-2013
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The placement of fluorophores in close proximity to metal nanoparticle surfaces is proposed to enhance several photophysical properties of the dyes, potentially leading to improved quantum yields and decreased photobleaching. It is difficult in practice, however, to establish and maintain the nanoscale distances that are required to maximize these effects. The type of metal, size, and shape of the nanoparticle, the physical distance separating the metal nanoparticle from the organic dye, and the spectral properties of the fluorophore itself are all proposed to influence the quantum yield and lifetime. This results in a complex behavior that can lead to either enhanced or quenched fluorescence in different contexts. In this report, we describe a well-defined system that can be used to explore these effects, while physically preventing the fluorophores from contacting the nanoparticle surfaces. The basis of this system is the spherical protein capsid of bacteriophage MS2, which was used to house gold particles within its interior volume. The exterior surface of each capsid was then modified with Alexa Fluor 488 (AF 488) labeled DNA strands. By placing AF 488 dyes at distances of 3, 12, and 24 bp from the surface of capsids containing 10 nm gold nanoparticles, fluorescence intensity enhancements of 2.2, 1.2, and 1.0 were observed, respectively. A corresponding decrease in fluorescence lifetime was observed for each distance. Because of its well-defined and modular nature, this architecture allows the rapid exploration of the many variables involved in metal-controlled fluorescence, leading to a better understanding of this phenomenon.
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Type III secretion translocon assemblies that attenuate Yersinia virulence.
Cell. Microbiol.
PUBLISHED: 01-21-2013
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Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis?yopD mutations were constructed to disrupt a predicted ?-helix motif at the C-terminus. Mutants YopD(I262P) and YopD(K267P) poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopDA(263P) and YopD(A270P) had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.
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Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis.
PLoS ONE
PUBLISHED: 01-01-2013
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Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.
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Real time assays for quantifying cytotoxicity with single cell resolution.
PLoS ONE
PUBLISHED: 01-01-2013
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A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI) as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.
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Choosing an effective protein bioconjugation strategy.
Nat. Chem. Biol.
PUBLISHED: 11-17-2011
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The collection of chemical techniques that can be used to attach synthetic groups to proteins has expanded substantially in recent years. Each of these approaches allows new protein targets to be addressed, leading to advances in biological understanding, new protein-drug conjugates, targeted medical imaging agents and hybrid materials with complex functions. The protein modification reactions in current use vary widely in their inherent site selectivity, overall yields and functional group compatibility. Some are more amenable to large-scale bioconjugate production, and a number of techniques can be used to label a single protein in a complex biological mixture. This review examines the way in which experimental circumstances influence ones selection of an appropriate protein modification strategy. It also provides a simple decision tree that can narrow down the possibilities in many instances. The review concludes with example studies that examine how this decision process has been applied in different contexts.
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Viral capsids as self-assembling templates for new materials.
Prog Mol Biol Transl Sci
PUBLISHED: 10-18-2011
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The self-assembling protein shells of viruses have provided convenient scaffolds for the construction of many new materials with well-defined nanoscale architectures. In some cases, the native amino acid functional groups have served as nucleation sites for the deposition of metals and semiconductors, leading to organic-inorganic composites with interesting electronic, magnetic, optical, and catalytic properties. Other approaches have involved the covalent modification of the protein monomers, typically with the goal of generating targeting delivery vehicles for drug and imaging cargo. Covalently modified capsid proteins have also been used to generate periodic arrays of chromophores for use in light harvesting and photocatalytic applications. All of these research areas have taken advantage of the low polydispersity, high chemical stability, and intrinsically multivalent properties that are uniquely offered by these biological building blocks.
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Oxidative modification of native protein residues using cerium(IV) ammonium nitrate.
J. Am. Chem. Soc.
PUBLISHED: 10-03-2011
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A new protein modification strategy has been developed that is based on an oxidative coupling reaction that targets electron-rich amino acids. This strategy relies on cerium(IV) ammonium nitrate (CAN) as an oxidation reagent and results in the coupling of tyrosine and tryptophan residues to phenylene diamine and anisidine derivatives. The methodology was first identified and characterized on peptides and small molecules, and was subsequently adapted for protein modification by determining appropriate buffer conditions. Using the optimized procedure, native and introduced solvent-accessible residues on proteins were selectively modified with polyethylene glycol (PEG) and small peptides. This unprecedented bioconjugation strategy targets these under-utilized amino acids with excellent chemoselectivity and affords good-to-high yields using low concentrations of the oxidant and coupling partners, short reaction times, and mild conditions.
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Impact of the N-terminal secretor domain on YopD translocator function in Yersinia pseudotuberculosis type III secretion.
J. Bacteriol.
PUBLISHED: 09-30-2011
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Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5 mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from Yersinia pseudotuberculosis. Mutations in the 5 end of yopD that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for yopD translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the in vitro secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of yopD translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.
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Rapid chemoselective bioconjugation through oxidative coupling of anilines and aminophenols.
J. Am. Chem. Soc.
PUBLISHED: 09-28-2011
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A highly efficient protein bioconjugation method is described involving addition of anilines to o-aminophenols in the presence of sodium periodate. The reaction takes place in aqueous buffer at pH 6.5 and can reach high conversion in 2-5 min. The major product was characterized using X-ray crystallography, which revealed that an unprecedented oxidative ring contraction occurs after the coupling step. The compatibility of the reaction with protein substrates has been demonstrated through attachment of small molecules, polymer chains, and peptides to p-aminophenylalanine residues introduced into viral capsids through amber stop codon suppression. Coupling of anilines to o-aminophenol groups derived from tyrosine residues is also described. The compatibility of this method with thiol modification chemistry is shown through attachment of a near-IR fluorescent chromophore to cysteine residues inside the viral capsid shells, followed by attachment of integrin-targeting RGD peptides to anilines on the exterior surface.
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Examining supply changes in Australias cocaine market.
Drug Alcohol Rev
PUBLISHED: 09-12-2011
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Media attention to cocaine use and supply has increased following some of the largest cocaine seizures in Australias history. Whether there has been an expansion in supply remains unclear. This paper examines the evidence behind assertions of increased supply in Australia and the scale and nature of any apparent increase, using proxy indicators of cocaine importation, distribution and use.
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Multivalent, high-relaxivity MRI contrast agents using rigid cysteine-reactive gadolinium complexes.
J. Am. Chem. Soc.
PUBLISHED: 08-26-2011
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MRI contrast agents providing very high relaxivity values can be obtained through the attachment of multiple gadolinium(III) complexes to the interior surfaces of genome-free viral capsids. In previous studies, the contrast enhancement was predicted to depend on the rigidity of the linker attaching the MRI agents to the protein surface. To test this hypothesis, a new set of Gd-hydroxypyridonate based MRI agents was prepared and attached to genetically introduced cysteine residues through flexible and rigid linkers. Greater contrast enhancements were seen for MRI agents that were attached via rigid linkers, validating the design concept and outlining a path for future improvements of nanoscale MRI contrast agents.
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Using synthetically modified proteins to make new materials.
Acc. Chem. Res.
PUBLISHED: 08-03-2011
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The uniquely diverse structures and functions of proteins offer many exciting opportunities for creating new materials with advanced properties. Exploiting these capabilities requires a set of versatile chemical reactions that can attach nonnatural groups to specific locations on protein surfaces. Over the years, we and others have developed a series of new techniques for protein bioconjugation, with a particular emphasis on achieving high site selectivity and yield. Using these reactions, we have been able to prepare a number of new materials with functions that depend on both the natural and the synthetic components. In this Account, we discuss our progress in protein bioconjugation over the past decade, focusing on three distinct projects. We first consider our work to harness sunlight artificially by mimicking features of the photosynthetic apparatus, with its beautifully integrated system of chromophores, electron transfer groups, and catalytic centers. Central to these photosystems are light-harvesting antennae having hundreds of precisely aligned chromophores with positions that are dictated by the proteins within the arrays. Our approach to generating similar arrangements involves the self-assembly of tobacco mosaic virus coat proteins bearing synthetic chromophore groups. These systems offer efficient light collection, are easy to prepare, and can be used to build complex photocatalytic systems through the modification of multiple sites on the protein surfaces. We then discuss protein-based carriers that can deliver drugs and imaging agents to diseased tissues. The nanoscale agents we have built for this purpose are based on the hollow protein shell of bacteriophage MS2. These 27 nm capsids have 32 pores, which allow the entry of relatively large organic molecules into the protein shell without requiring disassembly. Our group has developed a series of chemical strategies that can install dyes, radiolabels, MRI contrast agents, and anticancer drugs on the inside surface of these capsids. We have also developed methods to decorate the external surfaces with binders for specific proteins on cancer cells. As a third research area, our group has developed protein-polymer hybrid materials for water remediation. To reduce the toxicity of heavy metals in living cells, Nature has evolved metallothioneins, which are sulfur-rich polypeptides that bind mercury, cadmium, and other toxic ions at sub-parts-per-billion concentrations. Unfortunately, these proteins are very difficult to incorporate into polymers, largely because typical protein modification reactions target the very cysteine, lysine, and carboxylate-containing residues that are required for their proper function. To address this challenge, we developed a new way to attach these (and many other) proteins to polymer chains by expressing them as part of an N- and C-terminal modification "cassette". The resulting materials retain their selectivity and can remove trace amounts of toxic metal ions from ocean water. Each of these examples has presented a new set of protein bioconjugation challenges that have been met through the development of new reaction methodology. Future progress in the generation of protein-based materials will require scalable synthetic techniques with improved yields and selectivities, inexpensive purification methods for bioconjugates, and theoretical and dynamical treatments for designing new materials through protein self-assembly.
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Phosphorylated CpxR restricts production of the RovA global regulator in Yersinia pseudotuberculosis.
PLoS ONE
PUBLISHED: 07-15-2011
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RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression.
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Varying dependency of periplasmic peptidylprolyl cis-trans isomerases in promoting Yersinia pseudotuberculosis stress tolerance and pathogenicity.
Biochem. J.
PUBLISHED: 07-06-2011
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Periplasmic PPIases (peptidylprolyl cis-trans isomerases) catalyse the cis-trans isomerization of peptidyl-prolyl bonds, which is a rate-limiting step during protein folding. We demonstrate that the surA, ppiA, ppiD, fkpA and fklB alleles each encode a periplasmic PPIase in the bacterial pathogen Yersinia pseudotuberculosis. Of these, four were purified to homogeneity. Purified SurA, FkpA and FklB, but not PpiD, displayed detectable PPIase activity in vitro. Significantly, only Y. pseudotuberculosis lacking surA caused drastic alterations to the outer membrane protein profile and FA (fatty acid) composition. They also exhibited aberrant cellular morphology, leaking LPS (lipopolysaccharide) into the extracellular environment. The SurA PPIase is therefore most critical for maintaining Y. pseudotuberculosis envelope integrity during routine culturing. On the other hand, bacteria lacking either surA or all of the genes ppiA, ppiD, fkpA and fklB were sensitive to hydrogen peroxide and were attenuated in mice infections. Thus Y. pseudotuberculosis exhibits both SurA-dependent and -independent requirements for periplasmic PPIase activity to ensure in vivo survival and a full virulence effect in a mammalian host.
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Incidence and survival of malignant bone sarcomas in England 1979-2007.
Int. J. Cancer
PUBLISHED: 03-03-2011
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Primary malignant bone sarcomas (MBS) are rare and there are few studies examining their incidence and outcome. Here, the incidence and survival of all subtypes of MBS registered in England between 1979 and 2007 were analysed from patient registry data held by the National Cancer Intelligence Network (NCIN). Over 11,002 new cases of MBS were registered, an average of 379 per year. There was no change in incidence demonstrated over the study period (p = 0.08). Although a peak incidence is observed in adolescence, approximately half of MBS are diagnosed in patients over 50 years. An improvement in outcome of MBS was observed between those patients registered from 1979 to 1983 and 1983 to 1987 (p < 0.0001), but there has been no improvement since. In the most recent period studied (patients diagnosed 1998-2002) 5-year survival was 55% in Ewing sarcoma, 70% in chondrosarcoma, 56% in chordoma and 43% in osteosarcoma. Patients diagnosed with osteosarcoma over the age of 40 years or with a non-extremity tumour have a significantly inferior outcome; 22% 5-year survival >40 years compared with 53% <40 years (p < 0.0001) and 16% non-extremity tumour compared to 48% extremity tumour (p < 0.0001). This population-based study has allowed us to confidently define the English incidence and survival rates of both the commoner bone tumours such as osteosarcoma, and rarer entities such as chordoma as well as groups with inferior outcome. The lack of significant improvement over recent decades for these diseases is cause for concern and further research.
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The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function.
PLoS ONE
PUBLISHED: 01-13-2011
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Many gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated ?1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.
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Reported value of cannabis seizures in Australian newspapers: are they accurate?
Drug Alcohol Rev
PUBLISHED: 01-12-2011
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The news media is often touted as an important, yet inaccurate source of information about drug issues for the general public. This paper investigates the accuracy of reporting in the Australian media regarding the value of cannabis seizures made by the police.
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Identification of highly reactive sequences for PLP-mediated bioconjugation using a combinatorial peptide library.
J. Am. Chem. Soc.
PUBLISHED: 11-10-2010
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Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We have previously reported a pyridoxal 5-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine-lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.
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Dramatic thermal stability of virus-polymer conjugates in hydrophobic solvents.
Langmuir
PUBLISHED: 10-21-2010
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We have developed a method for integrating the self-assembling tobacco mosaic virus capsid into hydrophobic solvents and hydrophobic polymers. The capsid was modified at tyrosine residues to display an array of linear poly(ethylene glycol) chains, allowing it to be transferred into chloroform. In a subsequent step, the capsids could be transferred to a variety of hydrophobic solvents, including benzyl alcohol, o-dichlorobenzene, and diglyme. The thermal stability of the material against denaturation increased from 70 °C in water to at least 160 °C in hydrophobic solvents. With a view toward material fabrication, the polymer-coated TMV rods were also incorporated into solid polystyrene and thermally cast at 110 °C. Overall, this process significantly expands the range of processing conditions for TMV-based materials, with the goal of incorporating these templated nanoscale systems into conductive polymer matrices.
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Dual-surface modified virus capsids for targeted delivery of photodynamic agents to cancer cells.
ACS Nano
PUBLISHED: 09-25-2010
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Bacteriophage MS2 was used to construct a targeted, multivalent photodynamic therapy vehicle for the treatment of Jurkat leukemia T cells. The self-assembling spherical virus capsid was modified on the interior surface with up to 180 porphyrins capable of generating cytotoxic singlet oxygen upon illumination. The exterior of the capsid was modified with ?20 copies of a Jurkat-specific aptamer using an oxidative coupling reaction targeting an unnatural amino acid. The capsids were able to target and selectively kill more than 76% of the Jurkat cells after only 20 min of illumination. Capsids modified with a control DNA strand did not target Jurkat cells, and capsids modified with the aptamer were found to be specific for Jurkat cells over U266 cells (a control B cell line). The doubly modified capsids were also able to kill Jurkat cells selectively even when mixed with erythrocytes, suggesting the possibility of using our system to target blood-borne cancers or other pathogens in the blood supply.
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Incorporation of antifreeze proteins into polymer coatings using site-selective bioconjugation.
J. Am. Chem. Soc.
PUBLISHED: 09-10-2010
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The diverse functional repertoire of proteins promises to yield new materials with unprecedented capabilities, so long as versatile chemical methods are available to introduce synthetic components at specific sites on biomolecule surfaces. As a demonstration of this potential, we have used site-selective strategies to attach antifreeze proteins found in Arctic fish and insects to polymer chains. This multivalent arrangement increases the thermal hysteresis activity of the proteins and leads to materials that can be cast into thin films. The polymer-protein conjugates retain the ability of the proteins to slow ice growth in subzero water and can inhibit ice formation after attachment to glass surfaces. These inexpensive materials may prove useful as coatings for device components that must function at low temperature without ice buildup. The polymer attachment also allows higher thermal hysteresis values to be achieved while using less protein, thus lowering the cost of these additives for biomedical applications.
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Illicit drugs and the media: models of media effects for use in drug policy research.
Drug Alcohol Rev
PUBLISHED: 09-06-2010
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Illicit drugs are never far from the media gaze and although identified almost a decade ago as a new battleground for the alcohol and other drug (AOD) field there has been limited research examining the role of the news media and its effects on audiences and policy.
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Immobilization and one-dimensional arrangement of virus capsids with nanoscale precision using DNA origami.
Nano Lett.
PUBLISHED: 06-26-2010
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DNA origami was used as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. To do this, we first modified the interior surface of bacteriophage MS2 capsids with fluorescent dyes as a model cargo. An unnatural amino acid on the external surface was then coupled to DNA strands that were complementary to those extending from origami tiles. Two different geometries of DNA tiles (rectangular and triangular) were used. The capsids associated with tiles of both geometries with virtually 100% efficiency under mild annealing conditions, and the location of capsid immobilization on the tile could be controlled by the position of the probe strands. The rectangular tiles and capsids could then be arranged into one-dimensional arrays by adding DNA strands linking the corners of the tiles. The resulting structures consisted of multiple capsids with even spacing (approximately 100 nm). We also used a second set of tiles that had probe strands at both ends, resulting in a one-dimensional array of alternating capsids and tiles. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multicomponent systems from biological scaffolds using the power of rationally engineered DNA nanostructures.
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YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis.
J. Biol. Chem.
PUBLISHED: 06-04-2010
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YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.
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Site-Specific Protein Bioconjugation via a Pyridoxal 5-Phosphate-Mediated N-Terminal Transamination Reaction.
Curr Protoc Chem Biol
PUBLISHED: 06-01-2010
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The covalent attachment of chemical groups to proteins is a critically important tool for the study of protein function and the creation of protein-based materials. Methods of site-specific protein modification are necessary for the generation of well defined bioconjugates possessing a new functional group in a single position in the amino acid sequence. This article describes a pyridoxal 5-phosphate (PLP)-mediated transamination reaction that is specific for the N-terminus of a protein. The reaction oxidizes the N-terminal amine to a ketone or an aldehyde, which can form a stable oxime linkage with an alkoxyamine reagent of choice. Screening studies have identified the most reactive N-terminal residues, facilitating the use of site-directed mutagenesis to achieve high levels of conversion. Additionally, this reaction has been shown to be effective for a number of targets that are not easily accessed through heterologous expression, such as monoclonal antibodies. Curr. Protoc. Chem. Biol. 2:125-134 © 2010 by John Wiley & Sons, Inc.
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Impact of assembly state on the defect tolerance of TMV-based light harvesting arrays.
J. Am. Chem. Soc.
PUBLISHED: 04-16-2010
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Self-assembling, light harvesting arrays of organic chromophores can be templated using the tobacco mosaic virus coat protein (TMVP). The efficiency of energy transfer within systems containing a high ratio of donors to acceptors shows a strong dependence on the TMVP assembly state. Rod and disk assemblies derived from a single stock of chromophore-labeled protein exhibit drastically different levels of energy transfer, with rods significantly outperforming disks. The origin of the superior transfer efficiency was probed through the controlled introduction of photoinactive conjugates into the assemblies. The efficiency of the rods showed a linear dependence on the proportion of deactivated chromophores, suggesting the availability of redundant energy transfer pathways that can circumvent defect sites. Similar disk-based systems were markedly less efficient at all defect levels. To examine these differences further, the brightness of donor-only systems was measured as a function of defect incorporation. In rod assemblies, the photophysical properties of the donor chromophores showed a significant dependence on the number of defects. These differences can be partly attributed to vertical energy transfer events in rods that occur more rapidly than the horizontal transfers in disks. Using these geometries and the previously measured energy transfer rates, computational models were developed to understand this behavior in more detail and to guide the optimization of future systems. These simulations have revealed that significant differences in excited state dissipation rates likely also contribute to the greater efficiency of the rods and that statistical variations in the assembly process play a more minor role.
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A xenon-based molecular sensor assembled on an MS2 viral capsid scaffold.
J. Am. Chem. Soc.
PUBLISHED: 04-16-2010
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In MRI, anatomical structures are most often differentiated by variations in their bulk magnetic properties. Alternatively, exogenous contrast agents can be attached to chemical moieties that confer affinity to molecular targets; the distribution of such contrast agents can be imaged by magnetic resonance. Xenon-based molecular sensors are molecular imaging agents that rely on the reversible exchange of hyperpolarized xenon between the bulk and a specifically targeted host-guest complex. We have incorporated approximately 125 xenon sensor molecules in the interior of an MS2 viral capsid, conferring multivalency and other properties of the viral capsid to the sensor molecule. The resulting signal amplification facilitates the detection of sensor at 0.7 pM, the lowest to date for any molecular imaging agent used in magnetic resonance. This amplification promises the detection of chemical targets at much lower concentrations than would be possible without the capsid scaffold.
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Site-selective dual modification of periplasmic binding proteins for sensing applications.
Biosens Bioelectron
PUBLISHED: 03-10-2010
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We have developed three sensitive and specific amino acid sensors based on bacterial periplasmic solute binding proteins. A site-specific amino-terminal transamination reaction provides a useful complement to cysteine chemistry for the covalent modification of biomolecules in this application. We demonstrate this combination to attach two different chromophores to a single biomolecule in two locations. The periplasmic glutamine binding protein from E. coli was modified with a pair of dyes suitable for fluorescence resonance energy transfer, and this conjugate exhibited an l-glutamine dependent optical response. Two periplasmic binding proteins from the thermophilic organism Thermotoga maritima, for arginine and aliphatic amino acids, were modified and evaluated similarly. All three conjugates manifested signal changes mediated by resonant energy transfer upon binding their respective ligands, with nanomolar dissociation constants and stereochemical specificity. This represents a readily generalizable method for construction of reagentless biosensors. The double-labeling strategy was also exploited for the surface attachment of a dye-labeled glutamine binding protein via a biotin-streptavidin interaction.
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Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins.
Biochem. J.
PUBLISHED: 02-11-2010
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14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells that play an important role in a multitude of signalling pathways. 14-3-3 proteins bind either to phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners [Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice and Mackintosh (2004) Biochem. J. 379, 395-408]. These interactions result in cell-cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. One example of a phosphorylation-independent interaction is the binding of 14-3-3 to ExoS (exoenzyme S), a bacterial ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. In the present study, we have utilized additional biochemical and infection analyses to define further the structural basis of the interaction between ExoS and 14-3-3. An ExoS leucine-substitution mutant dramatically reduced the interaction potential with 14-3-3 suggesting that Leu422, Leu423, Leu426 and Leu428 of ExoS are important for its interaction with 14-3-3, its enzymatic activity and cytotoxicity. However, ExoS substitution mutants of residues that interact with 14-3-3 through an electrostatic interaction, such as Ser416, His418, Asp424 and Asp427, showed no reduction in their interaction potential with 14-3-3. These ExoS substitution mutants were also as aggressive as wild-type ExoS at inducing cell death and to modify endogenous ExoS target within the cell. In conclusion, electrostatic interaction between ExoS and 14-3-3 via polar residues (Ser416, His418, Asp424 and Asp427) appears to be of secondary importance. Thus the interaction between the roof of the groove of 14-3-3 and ExoS relies more on hydrophobic interaction forces, which probably contributes to induce cell death after ExoS infection and activation.
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Viral capsid DNA aptamer conjugates as multivalent cell-targeting vehicles.
J. Am. Chem. Soc.
PUBLISHED: 07-17-2009
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Nucleic acid aptamers offer significant potential as convenient and evolvable targeting groups for drug delivery. To attach them to the surface of a genome-free viral capsid carrier, an efficient oxidative coupling strategy has been developed. The method involves the periodate-mediated reaction of phenylene diamine substituted oligonucleotides with aniline groups installed on the outer surface of the capsid shells. Up to 60 DNA strands can be attached to each viral capsid with no apparent loss of base-pairing capabilities or protein stability. The ability of the capsids to bind specific cellular targets was demonstrated through the attachment of a 41-nucleotide sequence that targets a tyrosine kinase receptor on Jurkat T cells. After the installation of a fluorescent dye on the capsid interior, capsids bearing the cell-targeting sequence showed significant levels of binding to the cells relative to those of control samples. Colocalization experiments using confocal microscopy indicated that the capsids were endocytosed and trafficked to lysosomes for degradation. These observations suggest that aptamer-labeled capsids could be used for the targeted drug delivery of acid-labile prodrugs that would be preferentially released upon lysosomal acidification.
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Direct cell surface modification with DNA for the capture of primary cells and the investigation of myotube formation on defined patterns.
Langmuir
PUBLISHED: 06-10-2009
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Previously, we reported a method for the attachment of living cells to surfaces through the hybridization of synthetic DNA strands attached to their plasma membrane. The oligonucleotides were introduced using metabolic carbohydrate engineering, which allowed reactive tailoring of the cell surface glycans for chemoselective bioconjugation. While this method is highly effective for cultured mammalian cells, we report here a significant improvement of this technique that allows the direct modification of cell surfaces with NHS-DNA conjugates. This method is rapid and efficient, allowing virtually any mammalian cell to be patterned on surfaces bearing complementary DNA in under 1 h. We demonstrate this technique using several types of cells that are generally incompatible with integrin-targeting approaches, including red blood cells and primary T-cells. Cardiac myoblasts were also captured. The immobilization procedure itself was found not to activate primary T-cells, in contrast to previously reported antibody- and lectin-based methods. Myoblast cells were patterned with high efficiency and remained undifferentiated after surface attachment. Upon changing to differentiation media, myotubes formed in the center of the patterned areas with an excellent degree of edge alignment. The availability of this new protocol greatly expands the applicability of the DNA-based attachment strategy for the generation of artificial tissues and the incorporation of living cells into device settings.
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Chemoselective tryptophan labeling with rhodium carbenoids at mild pH.
J. Am. Chem. Soc.
PUBLISHED: 04-16-2009
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Significant improvements have been made to a previously reported tryptophan modification method using rhodium carbenoids in aqueous solution, allowing the reaction to proceed at pH 6-7. This technique is based on the discovery that N-(tert-butyl)hydroxylamine promotes indole modification with rhodium carbenoids over a broad pH range (2-7). This methodology was demonstrated on peptide and protein substrates, generally yielding 40-60% conversion with excellent tryptophan chemoselectivity. The solvent accessibility of the indole side chains was found to be a key factor in successful carbenoid addition, as demonstrated by conducting the reaction at temperatures high enough to cause thermal denaturation of the protein substrate. Progress toward the expression of proteins bearing solvent accessible tryptophan residues as reactive handles for modification with rhodium carbenoids is also reported.
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DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array.
Lab Chip
PUBLISHED: 04-15-2009
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A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min(-1), while primary T cells exhibited only 2 milli-pH min(-1). This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties.
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Recyclable thermoresponsive polymer-cellulase bioconjugates for biomass depolymerization.
J. Am. Chem. Soc.
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Here we report the construction and characterization of a recoverable, thermoresponsive polymer-endoglucanase bioconjugate that matches the activity of unmodified enzymes on insoluble cellulose substrates. Two copolymers exhibiting a thermoresponsive lower critical solution temperature (LCST) were created through the copolymerization of an aminooxy-bearing methacrylamide with N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMa). The aminooxy group provided a handle through which the LCST was adjusted through small-molecule quenching. This allowed materials with LCSTs ranging from 20.9 to 60.5 °C to be readily obtained after polymerization. The thermostable endoglucanase EGPh from the hypothermophilic Pyrococcus horikoshii was transaminated with pyridoxal-5-phosphate to produce a ketone-bearing protein, which was then site-selectively modified through oxime linkage with benzylalkoxyamine or 5 kDa-poly(ethylene glycol)-alkoxyamine. These modified proteins showed activity comparable to the controls when assayed on an insoluble cellulosic substrate. Two polymer bioconjugates were then constructed using transaminated EGPh and the aminooxy-bearing copolymers. After 12 h, both bioconjugates produced an equivalent amount of free reducing sugars as the unmodified control using insoluble cellulose as a substrate. The recycling ability of the NIPAm copolymer-EGPh conjugate was determined through three rounds of activity, maintaining over 60% activity after two cycles of reuse and affording significantly more soluble carbohydrates than unmodified enzyme alone. When assayed on acid-pretreated Miscanthus, this bioconjugate increased the amount of reducing sugars by 2.8-fold over three rounds of activity. The synthetic strategy of this bioconjugate allows the LCST of the material to be changed readily from a common stock of copolymer and the method of attachment is applicable to a variety of proteins, enabling the same approach to be amenable to thermophile-derived cellulases or to the separation of multiple species using polymers with different recovery temperatures.
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PET Imaging and biodistribution of chemically modified bacteriophage MS2.
Mol. Pharm.
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The fields of nanotechnology and medicine have merged in the development of new imaging and drug delivery agents based on nanoparticle platforms. As one example, a mutant of bacteriophage MS2 can be differentially modified on the exterior and interior surfaces for the concurrent display of targeting functionalities and payloads, respectively. In order to realize their potential for use in in vivo applications, the biodistribution and circulation properties of this class of agents must first be investigated. A means of modulating and potentially improving the characteristics of nanoparticle agents is the appendage of PEG chains. Both MS2 and MS2-PEG capsids possessing interior DOTA chelators were labeled with (64)Cu and injected intravenously into mice possessing tumor xenografts. Dynamic imaging of the agents was performed using PET-CT on a single animal per sample, and the biodistribution at the terminal time point (24 h) was assessed by gamma counting of the organs ex vivo for 3 animals per agent. Compared to other viral capsids of similar size, the MS2 agents showed longer circulation times. Both MS2 and MS2-PEG bacteriophage behaved similarly, although the latter agent showed significantly less uptake in the spleen. This effect may be attributed to the ability of the PEG chains to mask the capsid charge. Although the tumor uptake of the agents may result from the enhanced permeation and retention (EPR) effect, selective tumor imaging may be achieved in the future by using exterior targeting groups.
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The value of C-reactive protein and comorbidity in predicting survival of patients with high grade soft tissue sarcoma.
Eur. J. Cancer
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The aim of this study was to determine whether C-reactive protein (CRP) levels or patients comorbidity before treatment predicted the overall disease-specific survival and local tumour control in high grade soft tissue sarcoma patients.
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Interactions of the CpxA sensor kinase and cognate CpxR response regulator from Yersinia pseudotuberculosis.
BMC Res Notes
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The CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis.
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Female sex work and international sport events - no major changes in demand or supply of paid sex during the 2010 Soccer World Cup: a cross-sectional study.
BMC Public Health
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Important unanswered questions remain on the impact of international sporting events on the sex industry. Speculation about increased demand and supply of sex work often generates significant attention, but also additional funding for HIV programmes. This study assessed whether changes occurred in the demand and supply of paid sex during the 2010 Soccer World Cup in South Africa.
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Osmolyte-mediated encapsulation of proteins inside MS2 viral capsids.
ACS Nano
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The encapsulation of enzymes in nanometer-sized compartments has the potential to enhance and control enzymatic activity, both in vivo and in vitro. Despite this potential, there is little quantitative data on the effect of encapsulation in a well-defined compartment under varying conditions. To gain more insight into these effects, we have characterized two improved methods for the encapsulation of heterologous molecules inside bacteriophage MS2 viral capsids. First, attaching DNA oligomers to a molecule of interest and incubating it with MS2 coat protein dimers yielded reassembled capsids that packaged the tagged molecules. The addition of a protein-stabilizing osmolyte, trimethylamine-N-oxide, significantly increased the yields of reassembly. Second, we found that expressed proteins with genetically encoded negatively charged peptide tags could also induce capsid reassembly, resulting in high yields of reassembled capsids containing the protein. This second method was used to encapsulate alkaline phosphatase tagged with a 16 amino acid peptide. The purified encapsulated enzyme was found to have the same K(m) value and a slightly lower k(cat) value than the free enzyme, indicating that this method of encapsulation had a minimal effect on enzyme kinetics. This method provides a practical and potentially scalable way of studying the complex effects of encapsulating enzymes in protein-based compartments.
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Coiled-coils in the YopD translocator family: a predicted structure unique to the YopD N-terminus contributes to full virulence of Yersinia pseudotuberculosis.
Infect. Genet. Evol.
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Pathogenic Yersinia all harbor a virulence plasmid-encoded Ysc-Yop T3SS. In this system, translocator function is performed by the hydrophobic proteins YopB and YopD. With the goal to better understand how YopD orchestrates yop-regulatory control, translocon pore formation and Yop effector translocation, we performed an in silico prediction of coiled-coil motifs in YopD and YopD-like sequences from other bacteria. Of interest was a predicted N-terminal coiled-coil that occurred solely in Yersinia YopD sequences. To investigate if this unique feature was biologically relevant, two in cis point mutations were generated with a view to disrupting this putative structure. Both mutants maintained full T3SS function in vitro in terms of environmental control of Yops synthesis and secretion, effector toxin translocation and evasion of phagocytosis and killing by cultured immune cells. However, these same mutants were attenuated for virulence in a murine oral-infection model. The cause of this tardy disease progression is unclear. However, these data indicate that any structural flaw in this element unique to the N-terminus will subtly compromise an aspect of YopD biology. Sub-optimal T3SSs are then formed that are unable to fortify Yersinia against attack by the host innate and adaptive immune response.
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Elevated CpxR~P levels repress the Ysc-Yop type III secretion system of Yersinia pseudotuberculosis.
Res. Microbiol.
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One way that Gram-negative bacteria respond to extracytoplasmic stress is through the CpxA-CpxR system. An activated CpxA sensor kinase phosphorylates the CpxR response regulator to instigate positive auto-amplification of Cpx pathway activation, as well as synthesis of various bacterial survival factors. In the absence of CpxA, human enteropathogenic Yersinia pseudotuberculosis accumulates high CpxR~P levels aided by the action of low molecular weight phosphodonors such as acetyl~P. Critically, these bacteria are also defective for plasmid-encoded Ysc-Yop-dependent type III synthesis and secretion, an essential determinant of virulence. Herein, we investigated whether elevated CpxR~P levels account for lost Ysc-Yop function. Decisively, reducing CpxR?P in Yersinia defective for CpxA phosphatase activity - through incorporating second-site suppressor mutations in ackA-pta or cpxR - dramatically restored Ysc-Yop T3S function. Moreover, the repressive effect of accumulated CpxR?P is a direct consequence of binding to the promoter regions of the T3S genes. Thus, Cpx pathway activation has two consequences in Yersinia; one, to maintain quality control in the bacterial envelope, and the second, to restrict ysc-yop gene expression to those occasions where it will have maximal effect.
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N-Terminal labeling of filamentous phage to create cancer marker imaging agents.
ACS Nano
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We report a convenient new technique for the labeling of filamentous phage capsid proteins. Previous reports have shown that phage coat protein residues can be modified, but the lack of chemically distinct amino acids in the coat protein sequences makes it difficult to attach high levels of synthetic molecules without altering the binding capabilities of the phage. To modify the phage with polymer chains, imaging groups, and other molecules, we have developed chemistry to convert the N-terminal amines of the ~4200 coat proteins into ketone groups. These sites can then serve as chemospecific handles for the attachment of alkoxyamine groups through oxime formation. Specifically, we demonstrate the attachment of fluorophores and up to 3000 molecules of 2 kDa poly(ethylene glycol) (PEG2k) to each of the phage capsids without significantly affecting the binding of phage-displayed antibody fragments to EGFR and HER2 (two important epidermal growth factor receptors). We also demonstrate the utility of the modified phage for the characterization of breast cancer cells using multicolor fluorescence microscopy. Due to the widespread use of filamentous phage as display platforms for peptide and protein evolution, we envision that the ability to attach large numbers of synthetic functional groups to their coat proteins will be of significant value to the biological and materials communities.
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HyperCEST detection of a 129Xe-based contrast agent composed of cryptophane-A molecular cages on a bacteriophage scaffold.
Magn Reson Med
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A hyperpolarized 129Xe contrast agent composed of many cryptophane-A molecular cages assembled on an M13 bacteriophage has been demonstrated. Saturation of xenon bound in the large number of cryptophane cages is transferred to the pool of aqueous-solvated xenon via chemical exchange, resulting in efficient generation of hyperCEST contrast. No significant loss of contrast per cryptophane cage was observed for the multivalent phage when compared with unscaffolded cryptophane. Detection of this phage-based hyperCEST agent is reported at concentrations as low as 230 fM, representing the current lower limit for NMR/MRI-based contrast agents.
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Cellular microfabrication: observing intercellular interactions using lithographically-defined DNA capture sequences.
Langmuir
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Previous reports have shown that synthetic DNA strands can be attached to the plasma membrane of living cells to equip them with artificial adhesion "receptors" that bind to complementary strands extending from material surfaces. This approach is compatible with a wide range of cell types, offers excellent capture efficiency, and can potentially be used to create complex multicellular arrangements through the use of multiple capture sequences. In this work, we apply an aluminum "lift off" lithography method to allow the efficient generation of complex patterns comprising different DNA sequences. The resulting surfaces are then demonstrated to be able to capture up to three distinct types of living cells in specific locations. The utility of this approach is demonstrated through the observation of patterned cells as they communicate by diffusion-based paracrine signaling. It is anticipated that the ability of this technique to create virtually any type of 2D heterogeneous cell pattern should prove highly useful for the examination of key questions in cell signaling, including stem cell differentiation and cancer metastasis.
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Direct attachment of microbial organisms to material surfaces through sequence-specific DNA hybridization.
Adv. Mater. Weinheim
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A new technique is reported for the attachment of synthetic DNA strands to the surfaces of microbial organisms. This gives algal, bacterial, and fungal cells the ability to bind to complementary strands extending from patterned surfaces that can be produced on platforms such as microfluidic devices. The ability of this method to establish complex 2- and 3-dimensional cocultures comprising multiple organism types is also presented.
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Quantifying the dynamics of coupled networks of switches and oscillators.
PLoS ONE
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Complex network dynamics have been analyzed with models of systems of coupled switches or systems of coupled oscillators. However, many complex systems are composed of components with diverse dynamics whose interactions drive the systems evolution. We, therefore, introduce a new modeling framework that describes the dynamics of networks composed of both oscillators and switches. Both oscillator synchronization and switch stability are preserved in these heterogeneous, coupled networks. Furthermore, this model recapitulates the qualitative dynamics for the yeast cell cycle consistent with the hypothesized dynamics resulting from decomposition of the regulatory network into dynamic motifs. Introducing feedback into the cell-cycle network induces qualitative dynamics analogous to limitless replicative potential that is a hallmark of cancer. As a result, the proposed model of switch and oscillator coupling provides the ability to incorporate mechanisms that underlie the synchronized stimulus response ubiquitous in biochemical systems.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.