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Find video protocols related to scientific articles indexed in Pubmed.
Promiscuity and selectivity in covalent enzyme inhibition: a systematic study of electrophilic fragments.
J. Med. Chem.
PUBLISHED: 09-05-2014
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Covalent ligand-target interactions offer significant pharmacological advantages. However, off-target reactivity of the reactive groups, which usually have electrophilic properties, must be minimized, and the selectivity of irreversible inhibitors is a crucial requirement. We therefore performed a systematic study to determine the selectivity of several electrophilic groups that can be used as building blocks for covalently binding ligands. Six reactive groups with modulated electrophilicity were combined with 11 nonreactive moieties, resulting in a small combinatorial library of 72 fragment-like compounds. These compounds were screened against a group of 11 enzyme targets to assess their selectivity and their potential for promiscuous binding to proteins. The assay results showed a considerably lower degree of promiscuity than initially expected, even for those members of the screening collection that contain supposedly highly reactive electrophiles.
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Residence times and boundary-following behavior in animals.
Phys Rev E Stat Nonlin Soft Matter Phys
PUBLISHED: 05-30-2014
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Many animals in heterogeneous environments bias their trajectories displaying a preference for the vicinity of boundaries. Here we propose a criterion, relying on recent invariance properties of residence times for microreversible Boltzmann's walks, that permits detecting and quantifying boundary-following behaviors. On this basis we introduce a boundary-following model that is a nonmicroreversible Boltzmann's walk and that can represent all kinds of boundary-following distributions. This allows us to perform a theoretical analysis of field-resolved boundary following in animals. Two consequences are pointed out and are illustrated: A systematic procedure can now be used for extraction of individual properties from experimental field measurements, and boundary-curvature influence can be recovered as an emerging property without the need for individuals perceiving the curvature via complex physiological mechanisms. The presented results apply to any memoryless correlated random walk, such as the run-and-tumble models that are widely used in cell motility studies.
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Engineered proteins with desired specificity: DARPins, other alternative scaffolds and bispecific IgGs.
Curr. Opin. Struct. Biol.
PUBLISHED: 03-23-2014
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Specific binding proteins have become essential for diagnostic and therapeutic applications, and traditionally these have been antibodies. Nowadays an increasing number of alternative scaffolds have joined these ranks. These additional folds have raised a lot of interest and expectations within the last decade. It appears that they have come of age and caught up with antibodies in many fields of applications. The last years have seen an exploration of possibilities in research, diagnostics and therapy. Some scaffolds have received further improvements broadening their fields of application, while others have started to occupy their respective niche. Protein engineering, the prerequisite for the advent of all alternative scaffolds, remains the driving force in this process, for both non-immunoglobulins and immunoglobulins alike.
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A universal approach to prepare reagents for DNA-assisted protein analysis.
PLoS ONE
PUBLISHED: 01-01-2014
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The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.
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Structural basis for eliciting a cytotoxic effect in HER2-overexpressing cancer cells via binding to the extracellular domain of HER2.
Structure
PUBLISHED: 05-29-2013
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Human epidermal growth factor receptor-2 (HER2) is a receptor tyrosine kinase directly linked to the growth of malignancies from various origins and a validated target for monoclonal antibodies and kinase inhibitors. Utilizing a new approach with designed ankyrin repeat proteins (DARPins) as alternative binders, we show that binding of two DARPins connected by a short linker, one targeting extracellular subdomain I and the other subdomain IV, causes much stronger cytotoxic effects on the HER2-addicted breast cancer cell line BT474, surpassing the therapeutic antibody trastuzumab. We determined crystal structures of these DARPins in complex with the respective subdomains. Detailed models of the full-length receptor, constrained by its rigid domain structures and its membrane anchoring, explain how the bispecific DARPins connect two membrane-bound HER2 molecules, distorting them such that they cannot form signaling-competent dimers with any EGFR family member, preventing any kinase dimerization, and thus leading to a complete loss of signaling.
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Do ants need to estimate the geometrical properties of trail bifurcations to find an efficient route? A swarm robotics test bed.
PLoS Comput. Biol.
PUBLISHED: 03-28-2013
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Interactions between individuals and the structure of their environment play a crucial role in shaping self-organized collective behaviors. Recent studies have shown that ants crossing asymmetrical bifurcations in a network of galleries tend to follow the branch that deviates the least from their incoming direction. At the collective level, the combination of this tendency and the pheromone-based recruitment results in a greater likelihood of selecting the shortest path between the colonys nest and a food source in a network containing asymmetrical bifurcations. It was not clear however what the origin of this behavioral bias is. Here we propose that it results from a simple interaction between the behavior of the ants and the geometry of the network, and that it does not require the ability to measure the angle of the bifurcation. We tested this hypothesis using groups of ant-like robots whose perceptual and cognitive abilities can be fully specified. We programmed them only to lay down and follow light trails, avoid obstacles and move according to a correlated random walk, but not to use more sophisticated orientation methods. We recorded the behavior of the robots in networks of galleries presenting either only symmetrical bifurcations or a combination of symmetrical and asymmetrical bifurcations. Individual robots displayed the same pattern of branch choice as individual ants when crossing a bifurcation, suggesting that ants do not actually measure the geometry of the bifurcations when travelling along a pheromone trail. Finally at the collective level, the group of robots was more likely to select one of the possible shorter paths between two designated areas when moving in an asymmetrical network, as observed in ants. This study reveals the importance of the shape of trail networks for foraging in ants and emphasizes the underestimated role of the geometrical properties of transportation networks in general.
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DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety.
Mol. Ther.
PUBLISHED: 02-05-2013
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Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancers. Many naturally occurring viruses have a preferential, although nonexclusive, tropism for tumors and tumor cells. In addition, specific targeting of cancer cells can be achieved at the virus entry level. We optimized retargeting of cell entry by elongating the measles virus attachment protein with designed ankyrin repeat proteins (DARPins), while simultaneously ablating entry through the natural receptors. DARPin-targeted viruses were strongly attenuated in off-target tissue, thereby enhancing safety, but completely eliminated tumor xenografts. Taking advantage of the unique properties of DARPins of being fused without generating folding problems, we generated a virus simultaneous targeting two different tumor markers. The bispecific virus retained the original oncolytic efficacy, while providing proof of concept for a strategy to counteract issues of resistance development. Thus, DARPin-targeting opens new prospects for the development of personalized, targeted therapeutics.
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Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2.
PLoS ONE
PUBLISHED: 01-01-2013
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Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.
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Cancer prevention in ulcerative colitis: long-term outcome following fluorescence-guided colonoscopy.
Inflamm. Bowel Dis.
PUBLISHED: 02-09-2011
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Patients with long-standing ulcerative colitis require repeated endoscopies for early detection of neoplasias, which, however, are frequently missed by standard colonoscopy. Fluorescence-guided colonoscopy is known to improve the detection rate but the long-term effects of fluorescence-guided colonoscopy are unknown.
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DARPins: an efficient targeting domain for lentiviral vectors.
Mol. Ther.
PUBLISHED: 01-11-2011
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We have recently developed a retargeting system for lentiviral vectors (LVs) that relies on the pseudotyping of LVs with engineered measles virus (MV) glycoproteins (hemagglutinin (H) and fusion protein (F)). Specificity is provided through display of a single-chain antibody (scFv) as targeting domain by fusion to the MV-H protein. As an alternative to scFv, designed ankyrin repeat proteins (DARPins) can be selected to become high-affinity binders to any kind of target molecule. In this study six HER2/neu-specific DARPins exhibiting different affinities and binding to different HER2/neu epitopes were applied as targeting domains. All H-DARPin fusion proteins were efficiently expressed on the cell surface. Upon coexpression with F, syncytia formation was observed in HER2/neu positive cells only and correlated directly with the HER2/neu receptor density. All H-DARPin proteins incorporated into LVs, albeit at different levels. The vectors only transduced HER2/neu-positive cells, while HER2/neu-negative cells remained untransduced. Highest titers were observed with one particular DARPin binding to the membrane distal domain of HER2/neu with medium affinity. When applied in vivo systemically, HER2/neu-targeted LVs showed exclusive gene expression in HER2/neu positive tumor tissue, while vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors mainly transduced cells in spleen and liver. Thus, DARPins are a promising alternative to scFvs for retargeting of LVs.
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Analyzing fish movement as a persistent turning walker.
J Math Biol
PUBLISHED: 03-14-2009
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The trajectories of Kuhlia mugil fish swimming freely in a tank are analyzed in order to develop a model of spontaneous fish movement. The data show that K. mugil displacement is best described by turning speed and its auto-correlation. The continuous-time process governing this new kind of displacement is modelled by a stochastic differential equation of Ornstein-Uhlenbeck family: the persistent turning walker. The associated diffusive dynamics are compared to the standard persistent random walker model and we show that the resulting diffusion coefficient scales non-linearly with linear swimming speed. In order to illustrate how interactions with other fish or the environment can be added to this spontaneous movement model we quantify the effect of tank walls on the turning speed and adequately reproduce the characteristics of the observed fish trajectories.
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Direct identification of ligand-receptor interactions on living cells and tissues.
Nat. Biotechnol.
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Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties--one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.
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Emergency cricothyroidotomy performed by inexperienced clinicians--surgical technique versus indicator-guided puncture technique.
Emerg Med J
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To improve the ease and safety of cricothyroidotomy especially in the hand of the inexperienced, new instruments have been developed. In this study, we compared a new indicator-guided puncture technique (PCK) with standard surgical technique (ST) regarding success rate, performance time and complications.
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Modeling collective animal behavior with a cognitive perspective: a methodological framework.
PLoS ONE
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The last decades have seen an increasing interest in modeling collective animal behavior. Some studies try to reproduce as accurately as possible the collective dynamics and patterns observed in several animal groups with biologically plausible, individual behavioral rules. The objective is then essentially to demonstrate that the observed collective features may be the result of self-organizing processes involving quite simple individual behaviors. Other studies concentrate on the objective of establishing or enriching links between collective behavior researches and cognitive or physiological ones, which then requires that each individual rule be carefully validated. Here we discuss the methodological consequences of this additional requirement. Using the example of corpse clustering in ants, we first illustrate that it may be impossible to discriminate among alternative individual rules by considering only observational data collected at the group level. Six individual behavioral models are described: They are clearly distinct in terms of individual behaviors, they all reproduce satisfactorily the collective dynamics and distribution patterns observed in experiments, and we show theoretically that it is strictly impossible to discriminate two of these models even in the limit of an infinite amount of data whatever the accuracy level. A set of methodological steps are then listed and discussed as practical ways to partially overcome this problem. They involve complementary experimental protocols specifically designed to address the behavioral rules successively, conserving group-level data for the overall model validation. In this context, we highlight the importance of maintaining a sharp distinction between model enunciation, with explicit references to validated biological concepts, and formal translation of these concepts in terms of quantitative state variables and fittable functional dependences. Illustrative examples are provided of the benefits expected during the often long and difficult process of refining a behavioral model, designing adapted experimental protocols and inversing model parameters.
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New laser soldering-based closures: a promising method in natural orifice transluminal endoscopic surgery.
Gastrointest. Endosc.
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Complete closure of gastrotomy is the linchpin of safe natural orifice transgastric endoscopic surgery.
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Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation.
N Biotechnol
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While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-mediated affinity-based assays, such as immuno-PCR and proximity ligation assays (PLA), use oligonucleotides attached to affinity reagents as reporter molecules. Conjugation of oligonucleotides to affinity reagents generally employs chemistries that target primary amines or cysteines. Because of the random nature of these processes neither the number of oligonucleotides conjugated per molecule nor their sites of attachment can be accurately controlled for affinity reagents with several available amines and cysteines. Here, we present a straightforward and convenient approach to functionalize recombinant affinity reagents for PLA by expressing the reagents as fusion partners with SNAP protein tags. This allowed us to conjugate oligonucleotides in a site-specific fashion, yielding precisely one oligonucleotide per affinity reagent. We demonstrate this method using designed ankyrin repeat proteins (DARPins) recognizing the tumor antigen HER2 and we apply the conjugates in different assay formats. We also show that SNAP or CLIP tags, expressed as fusion partners of transfected genes, allow oligonucleotide conjugations to be performed in fixed cells, with no need for specific affinity reagents. The approach is used to demonstrate induced interactions between the fusion proteins FKBP and FRB by allowing the in situ conjugated oligonucleotides to direct the production of templates for localized rolling circle amplification reactions.
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Synthesis and biological activity of cymantrene and cyrhetrene 4-aminoquinoline conjugates against malaria, leishmaniasis, and trypanosomiasis.
Dalton Trans
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Organometallic analogues of chloroquine show promise as new antimalarial agents capable of overcoming resistance to the parent drug chloroquine. Here, the synthesis and characterization of three new cymantrene (CpMn(CO)(3)) and cyrhetrene (CpRe(CO)(3)) 4-aminoquinoline conjugates with either an amine or amide linker are reported. The antimalarial activity of the new organometallic conjugates N-(2-(7-chloroquinolin-4-ylamino)ethyl)-4-cymantrenylbutanamide (3), N-(2-(7-chloroquinolin-4-ylamino)ethyl)-4-cyrhetrenylbutanamide (4) and N-(7-chloroquinolin-4-yl)-N-(cymantrenylmethyl)ethane-1,2-diamine (6) was evaluated against a chloroquine-sensitive (CQS) and a chloroquine-resistant strain (CQR) of the malaria parasite Plasmodium falciparum. The cymantrene complex with an amine linker (6) showed good activity against the CQS strain but was inactive against the CQR strain. In contrast, cymantrene and cyrhetrene compounds with an amide linker were active against both the CQS and the CQR strain. In addition, the antibacterial, anti-trypanosomal and anti-leishmanial activity of the compounds was evaluated. Compound 6 showed submicromolar activity against Trypanosoma brucei at a concentration where the toxicity to normal human cells is low. No significant effect was noticed on the exchange of manganese for rhenium in the CpM(CO)(3) moiety in any of the biological assays.
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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.

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

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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.