A suspension cell-based interaction platform for interrogation of membrane proteins

The majority of clinically approved therapeutics target membrane proteins (MPs), highlighting the need for tools to study this important category of proteins. To overcome limitations with recombinant MP expression, whole cell screening techniques have been developed that present MPs in their native conformations. Whereas many such platforms utilize adherent cells, here we introduce a novel suspension cell-based platform termed “biofloating” that enables quantitative analysis of interactions between proteins displayed on yeast and MPs expressed on mammalian cells, without need for genetic fusions. We characterize and optimize biofloating and illustrate its sensitivity advantage compared to an adherent cell-based platform (biopanning). We further demonstrate the utility of suspension cell-based approaches by iterating rounds of magnetic-activated cell sorting selections against MP-expressing mammalian cells to enrich for a specific binder within a yeast-displayed antibody library. Overall, biofloating represents a promising new technology that can be readily integrated into protein discovery and development workflows.     

Synthetic Cancer‐Targeting Innate Immune Stimulators Give Insights into Avidity Effects

Multispecific and multivalent antibodies are seen as promising cancer therapeutics, and numerous antibody fragments and derivatives have been developed to exploit avidity effects that result in increased selectivity. Most of these multispecific and multivalent antibody strategies make use of recombinant expression of antigen‐binding modules. In contrast, chemical synthesis and chemoselective ligations can be used to generate a variety of molecules with different numbers and combinations of binding moieties in a modular and homogeneous fashion. In this study we synthesized a series of targeted immune system engagers (ISErs) by using solid‐phase peptide synthesis and chemoselective ligations. To explore avidity effects, we constructed molecules bearing different numbers and combinations of two “binder” peptides that target ephrin A2 and integrin α₃ receptors and an “effector” peptide that binds to formyl peptide receptors and stimulates an immune response. We investigated various strategies for generating multivalent and multispecific targeted innate immune stimulators and studied their activities in terms of binding to cancer cells and stimulation of immune cells. This study gives insights into the influence that multivalency and receptor density have on avidity effects and is useful for the design of potential anticancer therapeutics.     

PBI powder processing to performance parts

A new route (“direct forming”) was developed for forming dense PBI shapes from PBI powder. The new process affords the possibility of automated PBI powder shaping (“cold compaction”) and densification in batches of multiple parts by a “powder-assisted hot isostatic pressing” process. Direct forming is a more productive alternative to hot compression molding. Two developments enable PBI direct forming: (1) the discovery that PBI powders that are porous and plasticized with moisture can be shaped by compaction at ambient temperatures (cold-compacted), and (2) a finding that cold-compacted shapes can be densified in large batches by a powder-assisted hot isostatic pressing. The porous PBI powder is formed from PBI in solution by a spray-precipitation process. When plasticized with moisture, this powder is cold-compactible to PBI shapes with densities up to 94% of that of ultimate density of PBI. These shapes, which have sufficient strength to be handled, are then further consolidated via powder-assisted hot isostatic pressing to shapes with excellent thermal and mechanical properties and densities of about 99% of the ultimate. © 1994 John Wiley & Sons, Inc.     

Novel full-ceramic monoblock acetabular cup with a bioactive trabecular coating: design,fabrication and characterization

Over the last 25 years, the philosophy behind an optimal fixation of orthopaedic implants to hard tissues progressively evolved towards “bone-conservative” solutions in order to minimize bone resection/loss and maximize tissue-implant integration. Hence, the researchers׳ attention moved from “traditional” fixation of the prosthesis to host bone by using screws or acrylic cement to new strategies based on physico-chemical bonding and surface modification of the implant. This research work explores the feasibility of a novel bioceramic monoblock acetabular cup for hip joint prosthesis that can be fixed to the patient׳s bone by means of a bone-like trabecular coating able to promote implant osteointegration. Sponge replica method was properly adapted and optimized to produce hemispherical foam-like bioactive glass-ceramic coatings that were joined to Al₂O₃/ZrO₂ composite cups by the interposition of a glass-ceramic interlayer. Morphological analyses by scanning electron microscopy (SEM) and micro-computed tomography revealed the good quality of joining at the different interfaces. Preliminary investigation of the mechanical properties was carried out to evaluate the suitability of the device for biomedical use. In vitro bioactive behaviour was assessed by immersion studies in simulated body fluid and evaluating the apatite formation on the struts of the trabecular coating. The concepts and findings reported in the present work can have a significant impact in the field of implantable devices, suggesting a valuable alternative to currently-applied but often suboptimal techniques for bone-prosthesis fixation.     

A method for joining individual graphene sheets

A graphene-based device requires the graphene to have an ideal shape, structure, and orientation, and be large enough, to allow them to be formed into a new device. Here the joining of individual single-layer and multi-layer graphene is performed in a transmission electron microscope-scanning tunneling microscope (TEM–STM) holder inside a 200 kV field emission TEM. Attempts have been made to join individual graphene sheets (GSs) with the so-called “top-to-top” and “layer-to-layer” geometries by applying a voltage. In the two geometries, the “top-to-top” form has resulted in a seamless joining for both single-layer and multi-layer GSs. The as-joined GSs show the same excellent electrical and mechanical properties as those of the original GSs. Large Joule heating originating from the field emission current will cause atom diffusion and self-assembly and then rearrangement of carbon networks at the GS edge front. In this way individual GSs could be extended and mended with the so-called “top-to-top” geometries by applying a constant voltage, to meet the required and desired shape, size, configuration, and functions for a variety of the special micro/nano scaled devices.     

Two and three-dimensional pattern recognition of organized surfaces by specific antibodies

Understanding molecular recognition of supramolecules for solid substrates is essential for designing chemical sensors and molecular devices. The rules of molecular recognition are well established at the level of single molecules. However, during the transition from molecular-scale devices to macroscopic devices, issues concerning control over recognition that are well-established at the molecular level become much more complex. Hopefully, the conceptual and practical considerations reported here will clarify some of these issues. The immune system uses antibodies to identify molecular surfaces through molecular recognition. Antibodies are thus appropriate tools to study the rules of macromolecule-surface interactions, and this was done using crystal surfaces as substrates. Crystals can be formed or introduced into organisms and should be thus treated by the organism as any other intruder, by eliciting antibodies specific to their surfaces. A structure-recognizing antibody is defined here as complementary to a certain ordered supramolecular organization. It can be considered as a mold bearing in its binding site memory of the organization against which it was elicited. On the surface of a crystal composed of relatively small organic molecules, an antibody binding site would encompass an array of 10-20 molecular moieties. The antibody binding site would not detect one molecule, but rather a two- or three-dimensional molecular arrangement on the surface, similar to a macromolecular surface. The complementarity between antibody binding site and surface is supported by stereoselective supramolecular interactions to the repetitive structural motifs that are exposed at the surface. A procedure was developed in order to isolate monoclonal antibodies that specifically recognize a certain crystalline surface. The procedure was applied in particular to crystals of cholesterol monohydrate, of 1,4-dinitrobenzene, and of the tripeptide (S)leucine-(S)leucine-(S)tyrosine (LLY). A series of antibodies were selected and studied, three of which provided reliable specific antibody-antigen structural models. The three docking models show an astounding geometrical and chemical match of the antibody binding sites on the respective crystal surfaces. We also showed that antibodies are intrinsically capable of recognition at the length scale necessary for detection of chirality. Once the structural parameters determining the antibody specificity to the target surfaces are characterized, the antibodies may be conceivably used as reporters of the existence and location of target domains with similar structure in biological milieus. In this context, we developed and characterized monoclonal antibodies specific to crystalline mixed monolayers of cholesterol and ceramide, fundamental building blocks of lipid microdomains in cellular membranes. When used on cells, one antibody indeed labels cell membrane domains composed of cholesterol and ceramide. The fundamental contribution of the approach developed here may be in the antibody ability to report on the structural organization of paracrystalline domains that cannot be determined by other means. Alternatively, structure-recognizing antibodies may be conceivably used to carry information or build connections to specific targets, which may offer interesting developments in medicine or electronics.     

Classification of properties of elastomers using the “optimum property concept.” I. Introduction

An attempt is made to distinguish properties of elastomers by types. “Basic properties of materials” or “network properties” in elastomers are properties which either increase or decrease from the liquid to the solid state of materials or over the range of the “elastomeric plateau” of elastomers. From these are distinguished properties that exhibit characteristic maxima and are therefore “maximum properties” or bivalued properties. Mechanical failure properties show the characteristics of “maximum properties.” The maxima in “maximum properties” generally do not coincide. This noncoincidence of the maxima with a change in a “basic property of a material” has major theoretical and practical implications, for example, it is the cause of the crossovers in the relative performance rating of materials under different test conditions. The implications of this noncoincidence of the failure property maxima on the relevance of correlations between these properties are discussed. A change in the testing conditions is reflected in a shift of the optimum value in a “basic property of a material” with respect to a specific “maximum property.” Data and certain conclusions in the literature are interpreted on the basis of this concept. Examples of the limitations of the validity of mathematical relationships are presented. Also, a definition of the term “state of cure” is proposed and a suggestion for the rating of severities of test equipment and applications of elastomeric materials recommended. The effect of increased degrees of crosslinking for a series of polymers and crosslinking agents is assessed. It is suggested that the “mechanisms” of failure properties will remain elusive if their rationalization is attempted on the basis of other failure properties, e.g., the mechanism of abrasion on that of tear strength or cut growth. The main purpose of this proposal is to provide support for a drastic reduction in laboratory testing by identifying those properties which can lead to different relative ratings in routine evaluations and actual applications. A more empirical approach to materials evaluations is recommended based on the calibration of laboratory instrumentation with respect to specific applications. A de-emphasis of routine evaluations of materials on the basis of their “maximum properties” seems to be justified.     

Expression of redesigned mussel silk-like protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Silks have been used widely for human beings due to their several extraordinary properties. Until now, the studies on silk proteins have mainly focused on spiders and silkworms. Because silk properties are organism-dependent, novel silk protein types can be found and developed through investigation of new silk-bearing organisms. We noticed that marine mussel has silk-like domains containing many repeats with abundance of glycine and alanine. In the present work, we redesigned mussel-derived silk-like gene sequence which contains alternating repeated and nonrepeated regions with optimized codons for Escherichia coli. For successful expression of recombinant mussel silk-like protein in E. coli cells, we employed several experimental strategies, including use of strong promoter, cold shock expression, and genetic fusions. We observed significant repression on cell growths by even low expression levels of soluble mussel silk-like proteins in cold shock- and glutathione s-transferase (GST) fusion-based systems. Thus, we finally used baculoviral polyhedrin protein as a fusion partner and successfully expressed insoluble mussel silk-like protein with relatively high expression level and without cell growth repression in E. coli.     

Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin

Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystine-knot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein.     

Properties of electrically responsive hydrogels as a potential dynamic tool for biomedical applications

Hydrogels with electric responsive properties are gaining research focus due to increasing demand for miniaturized devices that can be precisely controlled using an external stimulus. Such systems are well suited due to their ability to expand and contract when in contact with different types of fluid. This study reports on the synthesis of a “smart” electroresponsive network, using a neutral, “non‐smart,” biocompatible hydrogel forming building block, Pluronic F127 (PF127), as a starting molecule. The PEO–PPO–PEO copolymer was modified with telechelic methacrylic end functionalities to form a triblock linear prepolymer with crosslinkable end groups (crosslinker). This bifunctional prepolymer, PF127 bismethacrylate (PF127BMA), was copolymerized covalently with anionic methacrylic acid sodium salt groups into a nonsoluble 3D hydrogel network in the presence of redox initiators. The polyelectrolyte domains in the pluronic hydrogel afforded controllable swelling capabilities with volumetric expansion exceeding 8500% in deionized water or 1400% in Krebs solution. The hydrogels were further assessed for their mechanical and electroactive response as a function of increasing acid salt content. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 41195.     

Development of specific "drug-like property" rules for carboxylate-containing oral drug candidates

The carboxylate moiety is an important pharmacophore in the medicinal chemist's arsenal and is sometimes an irreplaceable functionality in drug–target interactions. Thus, practical guidance on its use in the most optimized manner would be a welcome addition to rational drug design. Key physicochemical and ADMET‐PK properties from a dataset of drugs containing a carboxylate (COOH) moiety were assembled and compared with those of a broader, general drug dataset. Our main objective was to identify features specific to COOH‐containing oral drugs that could be converted into simple rules delineating the boundaries within which prospective COOH‐containing chemical series and COOH‐containing drug candidates would be reasonably expected to possess properties suitable for oral administration. These specific “drug‐like” property rules include molecular weight, the number of rotatable bonds, the number of hydrogen bond donors and acceptors, predictions of lipophilic character (calculated log P and log D values), topological polar surface area (TPSA), and the pK_a value of the carboxylate moiety. Similar to the various sets of criteria that have emerged over the past decade and which have significantly reshaped the way medicinal chemists think about preferred drug chemical space, we propose these specific COOH “drug‐like” property rules as a guide for the design of superior COOH‐containing drug candidates and as a tool to better manage the liabilities generally associated with the presence of a COOH moiety.     

Overcoming Technical Challenges and Moving into the Future with Laser Glass

The long-term commercial potential for solid-state laser gain materials based on glass has only been possible by constant technological developments that have overcome otherwise “market lethal” performance and cost issues. We will discuss a few examples that resulted in the development of completely new manufacturing processes that expanded the laser glass operation window and made possible the construction of large laser systems such as the National Ignition Facility in the United States and the French Laser Mégajoule. In parallel, through compositional modifications and identification of special postprocessing treatments, new active glasses with tailored properties have been continuously developed for specific laser architectures. We will also discuss current research activity directed at finding customized laser glass compositions for the next generation of high-peak-power (e.g., Exawatt class) laser systems.     

Impact of cooling method and incoming sheet quality on final part surface quality in thick sheet paint film thermoformed parts

Thick sheet, “dry paint” film parts were thermoformed using different cooling methods and sheet temperatures to determine whether these two parameters had a direct effect on the surface quality of the final part. Although some thermoformers have claimed that applying chilled air after forming “dry paint” film parts improves the gloss of the parts, the data from this study showed that application of chilled air did not have an effect on either the parts' initial gloss or their gloss after time‐dependent hazing. The critical factor in maintaining surface quality in these parts was the maximum temperature reached by the “dry paint” film during heating. In addition, analysis of the data taken on the sheet prior to forming versus that taken on the part after forming demonstrated the importance of validating the surface quality of the as‐received sheet prior to conducting process versus appearance experiments. On the basis of these findings, a recommendation is made for incoming sheet surface quality levels for both process development studies and production applications. POLYM. ENG. SCI., 2009. © 2008 Society of Plastics Engineers     

Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.     

Large scale atmospheric pressure chemical vapor deposition of graphene

We demonstrate that large scale high quality graphene synthesis can be performed using atmospheric pressure chemical vapor deposition (CVD) on Cu and illustrate how this procedure eliminates major difficulties associated with the low pressure CVD approach while allowing straightforward expansion of this technology to the roll-to-roll industrial scale graphene production. The detailed recipes evaluating the effects of copper foil thicknesses, purity, morphology and crystallographic orientation on the graphene growth rates and the number of graphene layers were investigated and optimized. Various foil cleaning protocols and growth conditions were evaluated and optimized to be suitable for production of large scale single layer graphene that was subsequently transferred on transparent flexible polyethylene terephthalate (PET) polymer substrates. Such “ready to use” graphene–PET sandwich structures were as large as 40″ in diagonal and >98% single layer, sufficient for many commercial and research applications. Synthesized large graphene film consists of domains exceeding 100 μm. Some curious behavior of high temperature graphene etching by oxygen is described that allows convenient visualization of interdomain boundaries and internal stresses.     

Properties and Photoactivity of Rhodopsin Mutants

A variety of spectroscopic and biochemical studies of recombinant site-directed mutants of rhodopsin and related visual pigments have been carried out. These studies have elucidated key structural elements common to visual pigments, such as a conserved disulfide bond. In addition, systematic analysis of the chromophore-binding pocket in rhodopsin and cone pigments has led to an improved understanding of the mechanism of the opsin-shift, and of particular molecular determinants underlying color vision in humans. Identification of conformational changes which occur upon rhodopsin photoactivation has been of particular recent concern. Assignment of these molecular alterations to specific regions in the receptor has been attempted by studying native opsin regenerated with synthetic retinal analogues or recombinant opsins regenerated with 11-cis-retinal. Individual molecular groups that undergo structural alterations during photoactivation have been identified. Analysis of particular mutant pigments in which specific groups are locked into their respective “on” or “off” states has provided a framework to identify determinants of the active conformation as well as the minimal number of intramolecular transitions required to switch between inactive and active conformations. A simple model for the active state of rhodopsin can be compared to structural models of its ground state to localize chromophore-protein interactions that may be important in the photoactivation mechanism.     

Development of Oral Strips Containing Chitosan as Active Ingredient: A Product for Buccal Health

In the last years, the number of products for oral care has been expanded to adapt to consumer needs. Thus, in addition to conventional products, new “pocket products” such as sugar-free chewing gums and oral strips (OS) have been developed for oral care. In the present study, OS were formulated using chitosan as the film-forming polymer in adequate concentrations to also be used as antimicrobial agent. Other strip components, such as the type of plasticizer, were also optimized. Mechanical properties of the optimal OS were evaluated and, due to chitosan's characteristic astringency, the strips were also sensorially evaluated.     

Some factors influencing cooling rates of rotationally molded parts

Recently, rotational molding engineers, concerned with warpage and uneven cooling in parts, have been “pre-cooling” the mold in forced draft air after removal from the oven and prior to water quenching to removal temperature. In this paper, we analyze some of the factors that influence the rate of heat removal from an amorphous plastic in a metal mold. We find that mold thickness and thermal diffusivity, convection heat transfer coefficient of the cooling fluid, the thermal properties of the plastic and the initial, final and “freezing” temperatures of the plastic influence this cooling rate and the corresponding rate of volumetric shrinkage. We illustrate our analysis with several examples and discuss some guidelines in detail.     

Studies on the mechanical,thermal, and morphological properties of poly(ether ether ketone)/poly(ether sulfone)/barium titanate nanocomposites: Correlation of experimental results with theoretical predictive models

In this study, mechanical, thermal, and morphological properties of the nanocomposites fabricated with the optimized blend of poly(ether ether ketone) (PEEK) and poly(ether sulfone) (PES) incorporated with nanobarium titanate (BT) were investigated. The optimized blend was based on the mechanical and thermal properties of the PEEK and PES in the ratio of 75 : 25 wt %. Nanoparticles were incorporated into the optimized blend with the help of twin‐screw extruder. The concentration of nano‐BT was varied from 2 to 6 wt % (0.41–1.28 vol %). With the increase in the nanosized BT concentrations, the tensile strength, tensile modulus, and elongation at break increased, whereas the crystallinity of the nanocomposites calculated by using differential scanning calorimetry method was found to decrease marginally. Morphological studies were carried out using scanning electron microscopy. The nanocomposites were evaluated by using theoretical predictive models according to “Pukanszky model” applicable to tensile strength and “Takayanagi's model” and “Guth and Smallwood model” applicable to tensile modulus. Upper and lower boundary of Hashin–Shtrikman model as well as Paul's model, applicable to tensile modulus, were also used to compare the experimental data. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Iterative Stochastic Elimination for Solving Complex Combinatorial Problems in Drug Discovery

Iterative Stochastic Elimination (ISE) is a novel algorithm that was originally developed to solve extremely complex problems in protein structure and interactions, and has since been applied to diverse topics that share a few general “ingredients”: they are extremely complex, of combinatorial nature, may be presented as large sets of variables that can each have many alternative values, there is some interdependence of the variables on each other, and there is a scoring function that can evaluate each choice of the problems “configuration”; this is the set of single values of each of the variables that constitute its full presentation. Those are picked randomly in a large sample, the analysis of which allows decisions to be made for rejecting some values for each of the variables; thus resulting in a smaller set of potential combinations. This continues in iterations until the number of combinations allows all the remaining options to be computed exhaustively and to order them by their scores. ISE has been mainly applied to problems that are relevant to drug design and discovery. We demonstrate, among others, the use of ISE to determine the properties of molecular ensembles and to pick the best molecules (“focused libraries”) for hitting a specific target. Future ideas for using ISE are discussed, as well as mentioning its contributions to the construction of two start-up companies.