Bottom-Up Assembly of Hydrogels from Bacteriophage and Au Nanoparticles: The Effect of Cis- and Trans-Acting Factors

Hydrogels have become a promising research focus because of their potential for biomedical application. Here we explore the long-range, electrostatic interactions by following the effect of trans-acting (pH) and cis-acting factors (peptide mutation) on the formation of Au-phage hydrogels. These bioinorganic hydrogels can be generated from the bottom-up assembly of Au nanoparticles (Au NP) with either native or mutant bacteriophage (phage) through electrostatic interaction of the phage pVIII major capsid proteins (pVIII). The cis-acting factor consists of a peptide extension displayed on the pVIII that mutates the phage. Our results show that pH can dictate the direct-assembly and stability of Au-phage hydrogels in spite of the differences between the native and the mutant pVIII. The first step in characterizing the interactions of Au NP with phage was to generate a molecular model that identified the charge distribution and structure of the native and mutant pVIII. This model indicated that the mutant peptide extension carried a higher positive charge relative to the native pVIII at all pHs. Next, by monitoring the Au-phage interaction by means of optical microscopy, elastic light scattering, fractal dimension analysis as well as Uv-vis and surface plasmon resonance spectroscopy, we show that the positive charge of the mutant peptide extension favors the opposite charge affinity between the phage and Au NP as the pH is decreased. These results show the versatility of this assembly method, where the stability of these hydrogels can be achieved by either adjusting the pH or by changing the composition of the phage pVIII without the need of phage display libraries

Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries

Phage display of combinatorial antibody libraries is a very efficient method for selecting recombinant antibodies against a wide range of molecules. It has been applied very successfully for the generation of therapeutic antibodies for more than a decade. To increase robustness and reproducibility of the selection procedure, we developed a semi-automated selection method for the generation of recombinant antibodies from phage display libraries. In this procedure, the selection targets are specifically immobilised to magnetic particles which can then by automatically handled by a magnetic particle processor. At present up to 96 samples can be handled simultaneously. Applying the processor allows standardisation of panning parameters such as washing conditions, incubation times, or to perform parallel selections on same targets under different buffer conditions. Additionally, the whole protocol has been streamlined to carry out bead loading, phage selection, phage amplification between selection rounds and magnetic particle ELISA for confirmation of binding activity in microtiter plate formats. Until now, this method has been successfully applied to select antibody fragments against different types of target, such as peptides, recombinant or homologous proteins, or chemical compounds

Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis)

<p>Abstract</p> <p>Background</p> <p>The cotton boll weevil (<it>Anthonomus grandis</it>) is a serious insect-pest in the Americas, particularly in Brazil. The use of chemical or biological insect control is not effective against the cotton boll weevil because of its endophytic life style. Therefore, the use of biotechnological tools to produce insect-resistant transgenic plants represents an important strategy to reduce the damage to cotton plants caused by the boll weevil. The present study focuses on the identification of novel molecules that show improved toxicity against the cotton boll weevil. <it>In vitro </it>directed molecular evolution through DNA shuffling and phage display screening was applied to enhance the insecticidal activity of variants of the Cry8Ka1 protein of <it>Bacillus thuringiensis</it>.</p> <p>Results</p> <p>Bioassays carried out with <it>A. grandis </it>larvae revealed that the LC₅₀of the screened mutant Cry8Ka5 toxin was 3.15-fold higher than the wild-type Cry8Ka1 toxin. Homology modelling of Cry8Ka1 and the Cry8Ka5 mutant suggested that both proteins retained the typical three-domain Cry family structure. The mutated residues were located mostly in loops and appeared unlikely to interfere with molecular stability.</p> <p>Conclusions</p> <p>The improved toxicity of the Cry8Ka5 mutant obtained in this study will allow the generation of a transgenic cotton event with improved potential to control <it>A. grandis</it>.</p

Characterization of a Human Antibody Fragment Fab and Its Calcium Phosphate Nanoparticles that Inhibit Rabies Virus Infection with Vaccine

Recombinant antibody phage display technology has been used to mimic many aspects of the processes that govern the generation and selection of high-affinity natural human antibodies in the human immune system, especially for infectious disease prophylaxis. An anti-rabies virus immunized phage-display Fab library was constructed from peripheral blood lymphocytes from vaccinated volunteers. The immunized antibody library, with a diversity of 6.7×108, was used to select and produce antibodies that bound to rabies virus glycoprotein. After five rounds of immobilized fixed rabies virion panning, four unique DNA sequences were found in the higher binding clones, and only one, Fab094, showed neutralization activity. Fab094 components were analyzed by ELISA, immunoprecipitation and immunofluorescent staining. ELISA and immunofluorescence showed that Fab094 bound specifically to rabies virions. Immunoprecipitation and mass spectrometry showed that Fab094 reacted with rabies virus glycoprotein. To improve the penetration power of Fab094 antibodies, we developed Fab094 calcium phosphate nanoparticles (Fab094-CPNPs) and tested their efficacy. The rapid fluorescent focus inhibition test indicated that the neutralizing antibody titers of Fab094 and Fab094-CPNPs were reached at 200.17 IU/Kg and 246.12 IU/Kg, respectively. These findings were confirmed in vivo in a Kunming mouse challenge model. Our results demonstrate that human Fab094 and Fab094-CPNPs are efficacious candidate drugs to replace rabies immunoglobulin in post-exposure prophylaxis (PEP)

Selection of Diethylstilbestrol-Specific Single-Chain Antibodies from a Non-Immunized Mouse Ribosome Display Library

Single chain variable fragments (scFvs) against diethylstilbestrol (DES) were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM) complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3) for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of one clone (30-1). The measured KD was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies

Produção de memórias falsas com listas de associados : análise do efeito do nível de processamento e da natureza da prova de memória

As memórias falsas têm sido amplamente estudadas com base num procedimento experimental designado paradigma DRM (Deese/Roediger/McDermott). Esse paradigma promove a criação de ilusões de memória a partir da apresentação de listas de palavras associadas a um item que não consta da lista. Uma das linhas de investigação com o paradigma DRM visa identificar o momento da criação das falsas memórias e explicar os mecanismos que estão na sua origem. Neste artigo, pretendemos fazer uma revisão da investigação sobre o efeito do nível de processamento e da natureza da tarefa de memória na facilitação ou inibição da produção de memórias falsas com listas de associados semânticos.False memories have been widely studied using an experimental procedure called DRM paradigm (Deese/Roediger/McDermott). This paradigm produces memory illusions due to the presentation of lists of words associated to a critical nonpresented word. One line of research on this topic aims at identifying the moment when the false memories are created and the explanation of the mechanisms underling false memories. In this paper we present a review about the effect of level-of-processing and the nature of memory task for the boost or inhibition of false memories created by means of lists of semantic associates.Le paradigme DRM (Deese/Roediger/McDermott) est un des plus connus et plus robustes parmi les études des faux mémoires dans le contexte du laboratoire. Ce paradigme permet la création d illusions de mémoire à partir des mots sémantiquement associés à un item qui n a pas été présenté. Au milieu des investigations basées sur le paradigme DRM il y a des études dont l objectif est d identifier e d´expliquer les mécanismes qui sont à l origine de la production des faux mémoires. Plus spécifiquement, on a pour but de faire une révision de la recherche sur l effet du niveau de codification et de la nature des tâches de mémoire sur la facilitation ou l´inhibition de la production de faux mémoires à partir des mots sémantiquement associés.Los falsos recuerdos han sido muy estudiados mediante la aplicación del paradigma DRM (Deese/Roediger/McDermott). El paradigma permite producir ilusiones de memoria tras la presentación de listas de palabras asociadas a una palabra que no se incluye en la lista. Una de las líneas de investigación que utilizan el paradigma DRM busca identificar el preciso momento de la creación de falsos recuerdos y explicar los mecanismos que originan ese efecto. El objetivo de este artículo es hacer una revisión de la investigación sobre el efecto de los niveles de procesamiento y la naturaleza de la tarea de memoria en la facilitación y inhibición de la producción de falsos recuerdos con listas de asociados semánticos.Fundação para a Ciência e a Tecnologia (FCT)Centro de Investigação em Psicologia da Universidade do Minho (CIPsi

The Use of Phage-Displayed Peptide Libraries to Develop Tumor-Targeting Drugs

Monoclonal antibodies have been successfully utilized as cancer-targeting therapeutics and diagnostics, but the efficacies of these treatments are limited in part by the size of the molecules and non-specific uptake by the reticuloendothelial system. Peptides are much smaller molecules that can specifically target cancer cells and as such may alleviate complications with antibody therapy. Although many endogenous and exogenous peptides have been developed into clinical therapeutics, only a subset of these consists of cancer-targeting peptides. Combinatorial biological libraries such as bacteriophage-displayed peptide libraries are a resource of potential ligands for various cancer-related molecular targets. Target-binding peptides can be affinity selected from complex mixtures of billions of displayed peptides on phage and further enriched through the biopanning process. Various cancer-specific ligands have been isolated by in vitro, in vivo, and ex vivo screening methods. As several peptides derived from phage-displayed peptide library screenings have been developed into therapeutics in current clinical trials, which validates peptide-targeting potential, the use of phage display to identify cancer-targeting therapeutics should be further exploited

Alchemy, enzymes, and the blind-watchmaker

Since the beginning of the modern era of chemistry, a recurring dream of chemists has been to possess a knowledge and understanding sufficient to allow them to create new enzymes at will. The road toward acquiring this knowledge is arduous, but promises to be a rewarding path for many years to come. To date, activities in this field have been divided into research to create truly new catalysts and research directe d at coaxing Nature's wonderful enzymes into accepting slightly different substrates or working under modified conditions. The article by Ikuo Fujii et al. in this issue reports one of the first successful couplings of these strategies

On the use of combinatorial antibody libraries to clone the "fossil record" of an individual's immune response

For about the last century the record of immunological events could only be obtained from serum proteins. We suggest that in the future this will change and the far more detailed nucleic acid record will provide new insights into immunological processes as well as selective access to the complete repertoire