Multifunctional Constructs Research Articles

Solid-phase bioconjugation of heterobifunctional adaptors for versatile assembly of bispecific targeting ligands.

High-throughput generation of bispecific molecules promises to expedite the discovery of new molecular therapeutics and guide engineering of novel multifunctional constructs. However, high synthesis complexity and cost have hampered the discovery of bispecific molecules in drug development and biomedical research. Herein we describe a simple solid-phase bioconjugation procedure for preparation of Protein A(G,L)-PEG-Streptavidin heterobifunctional adaptors (with 1:1:1 stoichiometry), which enable self-assembly of unmodified antibodies and biotinylated molecules into bispecific targeting ligands in a versatile mix-and-use manner. Utility of such adaptors is demonstrated by assembly of anti-CD3 and anti-Her2 antibodies into bispecific CD3xHer2 targeting ligands, which efficiently drive T-cell-mediated lysis of Her2-positive cancer cells. In comparison to bioconjugation in solution, the solid-phase procedure described here offers precise stoichiometry control, ease of purification, and high yield of functional conjugates. Simplicity and versatility should prove this methodology instrumental for preparation of bispecific ligands, as well as for high-throughput screening of bispecific combinations, before proceeding to synthesis of lead candidates via recombinant engineering or chemical cross-linking.

Multifunctional linker for orthogonal decoration of gold nanoparticles with DNA and protein

A novel trifunctional linker was prepared to allow multiple orthogonal functionalization of gold nanoparticles (AuNPs) with biomolecules. Thiooctic groups contained within the linker act as AuNP surface anchors, while two additional orthogonal functional groups are used for attachment of heme and DNA using amide and copper catalyzed Huisgen cycloaddition, respectively. We demonstrate that heme can act as a platform for reconstitution of a functional heme protein such as myoglobin. Using multifunctional linker, fully functional Au–DNA–Mb hybrids were obtained and such multifunctional constructs expand the synthetic toolbox for nanomaterial tailoring and design of biosensors and novel catalytic materials.

An electrospun scaffold integrating nucleic acid delivery for treatment of full-thickness wounds

We developed a multi-functional construct capable of controlled delivery of bioactive substances that can improve wound repair by supporting the intrinsic ability of the skin to heal. We synthesized electrospun scaffolds—composed of a blend of the degradable polymers poly(l-lactide) (PLA) or polycaprolactone (PCL)—that produce highly efficient non-viral in vivo gene delivery to cells in the wound bed, provide a protective barrier during early wound healing, and support cell migration and growth. This multi-functional material was tested for its influence on wound healing: scaffolds were loaded with plasmids encoding keratinocyte growth factor (KGF) and applied to full-thickness wounds in mice. Compared to scaffolds with control plasmids, animals receiving the KGF plasmid-loaded scaffold produced significant enhancements in wound healing, which was quantified by improvements in the rate of wound re-epithelialization, keratinocyte proliferation, and granulation response. Further, we quantified the expression level of endogenous and plasmid-derived KGF in wound samples: qRT-PCR on wound sections revealed a correlation between the levels of plasmid-derived protein expression and histological analysis of wound healing, revealing an inverse relationship between the expression level of exogenous KGF and the size of the unhealed epithelial layer in wounds. Our findings suggest that engineered nanofiber PLA/PCL scaffolds are capable of highly efficient controlled DNA delivery and are promising materials for treatment of cutaneous wounds.

Single Shapedand Multi-Functional Construction Of Additionals In Turkish

Single Shapedand Multi-Functional Construction Of Additionals In Turkish

Discerning the unique in the universal: the notion of organisation identity

PurposeThe purpose of this paper is to highlight the importance of the organisation identity construct by briefly considering the intellectual development of the organisation identity research field since “emergence”, and introducing previously unreported empirical studies pursued from within the framework provided by organisation identity theory (OIT).Design/methodology/approachMixed methods, qualitative and quantitative designs characterise the seven empirical (field) studies that explored the definitional parameters, existential nature and relevance of organisation identity. Observations are bolstered through conceptual and methodological triangulation across studies.FindingsOrganisation identity (OI) is usually articulated in fairly general terms and empirical research is scarce. In the seven studies reported here, OI is substantially reified and operationalised as the sense of organisational identity (SoI) and the fact of organisational identity (FoI). The studies consistently affirm the existential nature of OI as defined and demonstrate the relevance of OI for contemporary scholars and managers in the relationship of OI with organisational performance.Practical implicationsApart from being a source of stability for organisations during transition and change, organisation identity will increasingly become a significant consideration in performance, competitive strategy, talent attraction and retention, and organisational sustainability. An identity‐centric managerial approach that suggests that management consciously address OI, is proposed.Originality/valueOver the past two decades, conceptual contributions on OI proliferated while empirical studies were rare and generally lacked theoretical coherence. The paper reports on one of the few coherent and systematic approaches to researching OI. It offers a brief account of a series of purposeful, theory‐informed studies since 1999. Unlike previous research, these studies are all empirical in nature and pursued from within the same theory frame (OIT). The studies consistently reveal organisation identity as a significant multifunctional organisational construct.

Multi-Functional Loader Moving Hydraulic System Model Construction and Simulation Based on Power Bond Graphs

Hydraulic moving system has been extensively applied in small type of loader and other engineering vehicles do to its special superority, how to forecast its dynamic behavior in design stage is most impotant problem. This paper firstly analyse a typical moving hydraulic system of multi-functional loader, and construct its powder bond graphs model and encapsulate these key parts model in soft Mworks. at last, a simpled moving hydraulic dynamaic simulation model was constructed by these components, and some characteristic curves of system can be easily abtained.

Microstructure-linked strength properties and impact response of conventional and recycled concrete reinforced with steel and synthetic macro fibres

The effectiveness of the same volume of steel and synthetic macro fibres on impact resistance and microstructure associated mechanical behaviour is studied for concretes made with two different coarse aggregates-gravel and incinerator bottom ash (IBA). The microstructure–strength relationships were analyzed via electron microscopy and X-ray computed tomography (CT). The analysis indicated that increased paste strength was not so significant for the mix properties of plain IBA concrete as extensive cracks through the aggregate took place likely due to a reduced grain–grain interlocking and inter-particle friction as a result of the distinct pore structure, and fibre orientation in gravel concrete produced a line of weakness for failure to take place along. The results also show that the increase with the steel fibre inclusion in the post-peak stress, absorbed energy values, impact life for IBA concrete is much higher than those for gravel concrete. Overall, the research suggests that fibre-reinforced IBA concrete would be very promising as a multifunctional construction material.

An Engineered Cell Surface Polypeptide for Selection, Tracking and Ablation of Genetically Modified Hematopoietically Derived Cells Based on Human EGFR.

AbstractAbstract 1469Hematopoietic cell-based therapies, including genetically manipulated cell products derived from either hematopoietic stem cells or T cells, is an emerging area in applied biotechnology. In both of these venues, a variety of genetic engineering approaches are being studied to endow cells with novel attributes, to increase their therapeutic potency and/or safety. Common to the field of ex vivo cellular genetic engineering is the need to purify cells that express desired quantities of therapeutic transgene(s) and cull out non-expressing cells that either lack transgene endowed therapeutic activity or safety features. However, current drug selection strategies are associated with prolonged ex vivo culture that drives terminal differentiation of the T cells, which has in turn been found to be associated with impaired antitumor efficacy of adoptively transferred CD8+ T cells in vivo. Thus, we were interested in developing a single transgene encoded polypeptide that can serve both as an ex vivo selection epitope and in vivo tracking marker/target for mAb-mediated cell ablation, while fulfilling the criteria of being functionally inert, non immunogenic, and amenable to commercially available cGMP-grade selection systems appropriate for clinical use. Here we describe a truncated human EGFR polypeptide (huEGFRt) devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase domains. Retained features of huEGFRt include type I transmembrane cell surface localization and a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR mAb, cetuximab/Erbitux™. [Display omitted] Applying this system to cellular immunotherapy, we designed lentiviral vector prototypes housing multifunctional constructs combining huEGFRt with CD19-specific chimeric antigen receptors (CARs), and demonstrate that biotinylated-cetuximab immunomagnetic selection of transduced human T cells results in coordinate enrichment of CAR+ cells from 2% to over 90%. The huEGFRt-mediated selection did not affect the phenotype (i.e., TCR, CD3, CD4, CD8, CD28, and granzyme A expression), the in vitro expansion potential, nor the in vivo engraftment fitness (upon transfer into immunodeficient mice) of the T cells. Direct examination of EGF-binding and phospho-tyrosine analysis confirmed that this selection marker is functionally inert and has no negative effect on the T cell product. In addition, cytotoxicity against B cell malignancies and IFN-g/TNF-a production through the CD19-specific CAR was dramatically enhanced in the huEGFRt-selected population. The utility of huEGFRt in tracking the gene modified, transferred cells in vivo within easily obtained human tissues such as blood, bone marrow and tissue biopsies was then also proven via detection of huEGFRt using multiparameter flow cytometric analysis or FDA approved immunohistochemical techniques/reagents. In addition, we were able to demonstrate that Erbitux™ could mediate ADCC of huEGFRt+ T cells in vitro and inhibit the growth of huEGFRt+ CTLL2 cells in NOD/Scid mice, supporting the use of huEGFRt as a suicide gene via cetuximab-mediated ADCC after adoptive transfer. Together these data suggest that huEGFRt is a superior selection marker for any transduction system that can be applied to the generation of cell products for hematopoietic cell-based medical therapies. Disclosures:No relevant conflicts of interest to declare.

Surface functionalized nanoparticles for proximity-activated detection and imaging of breast cancer.

Abstract Abstract #6002 Site directed therapy of metastatic and primary breast cancer promises to minimize systemic toxicity associated with chemotherapy agents that have no specificity for pathologic tissues. We have developed a multifunctionalized nanoparticle specifically designed to target breast cancer, including metastases.
 Quantum dots (QDs) were functionalized with a matrilysin (MMP-7) cleavable, proximity activated substrate, PEG-peptide-PEG (PA construct), where cleavage occurs at the peptide bridge. The QDs were further functionalized with folic acid, a strong breast cancer cell targeting ligand with well-known binding to some normal tissues. The construct initially conceals the folate ligand and reduces binding to non-target tissues. MMP-7 activated cleavage of the PA construct and subsequent unmasking of the folate ligand occurs only at the site of the breast tumor, resulting in a proximity activated targeting (PAT) system. The activity of QDs functionalized with both the PA construct and folic acid (FA-QD-PA) was compared to QDs functionalized with only folic acid (QD-FA).
 Cellular association of QD-FA nanoparticles with the human breast cancer cell line MDA-MB-231 revealed that 61.9 ± 6.5% of the cells were fluorescently labeled. Addition of the PA construct reduced QD-FA recognition by MDA-MB-231 cells to 2.1 ± 0.4%. MMP-7 cleavage of the protected FA-QD-PA construct restored MDA-MB-231 recognition to 21.9 ± 5.4 %, a significant increase compare to the uncleaved multifunctional nanoparticle construct (one-way ANOVA, p*<0.001, n=3). This in vitro data suggests that the PAT nanoparticle model could function effectively in vivo for specific targeting of breast cancer and metastasis. In the next phase of research, the ability of the FA-QD-PA and FA-QD nanoparticles to detect breast cancer in vivo will be tested using murine models of human breast cancer.
 This work was funded by the US Army Medical Research and Material Command CDMRP under award BC043908. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6002.

Towards new functional nanostructures for medical imaging

Nanostructures represent a promising new type of contrast agent for clinical medical imaging modalities, including magnetic resonance imaging, x-ray computed tomography, ultrasound, and nuclear imaging. Currently, most nanostructures are simple, single-purpose imaging agents based on spherical constructs (e.g., liposomes, micelles, nanoemulsions, macromolecules, dendrimers, and solid nanoparticle structures). In the next decade, new clinical imaging nanostructures will be designed as multi-functional constructs, to both amplify imaging signals at disease sites and deliver localized therapy. Proposals for nanostructures to fulfill these new functions will be outlined. New functional nanostructures are expected to develop in five main directions: Modular nanostructures with additive functionality; cooperative nanostructures with synergistic functionality; nanostructures activated by their in vivo environment; nanostructures activated by sources outside the patient; and novel, nonspherical nanostructures and components. The development and clinical translation of next-generation nanostructures will be facilitated by a combination of improved clarity of the in vivo imaging and biological challenges and the requirements to successfully overcome them; development of standardized characterization and validation systems tailored for the preclinical assessment of nanostructure agents; and development of streamlined commercialization strategies and pipelines tailored for nanostructure-based agents for their efficient translation to the clinic.

Tumor Targeting with Antibody-Functionalized, Radiolabeled Carbon Nanotubes

Single-walled carbon nanotubes (CNT) are mechanically robust graphene cylinders with a high aspect ratio that are comprised of sp(2)-bonded carbon atoms and possessing highly regular structures with defined periodicity. CNT exhibit unique mechanochemical properties that can be exploited for the development of novel drug delivery platforms. We hypothesized that novel prototype nanostructures consisting of biologics, radionuclides, fluorochromes, and CNT could be synthesized and designed to target tumor cells. Tumor-targeting CNT constructs were synthesized from sidewall-functionalized, water-soluble CNT platforms by covalently attaching multiple copies of tumor-specific monoclonal antibodies, radiometal-ion chelates, and fluorescent probes. The constructs were characterized spectroscopically, chromatographically, and electrophoretically. The specific reactivity of these constructs was evaluated in vitro by flow cytometry and cell-based immunoreactivity assays and in vivo using biodistribution in a murine xenograft model of lymphoma. A soluble, reactive CNT platform was used as the starting point to build multifunctional constructs with appended antibody, metal-ion chelate, and fluorescent chromophore moieties to effect specific targeting, to carry and deliver a radiometal-ion, and to report location, respectively. These nanoconstructs were found to be specifically reactive with the human cancer cells they were designed to target in vivo in a model of disseminated human lymphoma and in vitro by flow cytometry and cell-based immunoreactivity assays versus appropriate controls. The key achievement in these studies was the selective targeting of tumor in vitro and in vivo by the use of specific antibodies appended to a soluble, nanoscale CNT construct. The ability to specifically target tumor with prototype-radiolabeled or fluorescent-labeled, antibody-appended CNT constructs was encouraging and suggested further investigation of CNT as a novel delivery platform.

An Approach to Deal With Packaging in Power Electronics

Current packaging technology in power electronics is based on assembling pre-manufactured discrete components. Each component consists of a number of parts, manufactured in a variety of manufacturing processes. This has resulted in a diversity of construction parts and mutually incompatible manufacturing processes in a typical power electronic converter and has brought power electronics to the edge where it becomes extremely difficult to reduce the cost and size of power electronic converters. This also makes integration of power electronic converters difficult. In this paper, we present a way to improve the physical construction of power electronic converters by increasing level of integration and using multifunctional construction parts. integration and packaging are two important aspects of physical construction of power electronic converters. Both of them and their mutual relationship are discussed in the paper. Three quantities intended to evaluate integration level and volumetric utilization namely functional elements integration level, K/sub I/; packaging elements integration level K/sub P/; and volumetric packaging efficiency /spl eta//sub v/ are introduced. Based on these values, a number of techniques to increase the integration level are presented. A design process in the form of a flowchart intended to implement these techniques in concrete design cases is presented.

Converter Concepts to Increase the Integration Level

In the previous work, a way to improve packaging of power electronic converters by increasing integration level and using multifunctional construction parts is presented. The quantities intended to evaluate integration level and volumetric utilization in power converters are introduced. Based on these values, a number of methods to increase the integration level are presented. A design process in the form of a flow chart that implements these methods in concrete design problems is presented. In this paper, the design process is applied to a dc/dc 42/14-V converter for automotive applications. Utilizing commercially available technologies that lend themselves to mass production this design process results in three packaging concepts, namely lead frame converter, printed circuit board embedded converter, and heat conductor converter. These concepts are then compared to a benchmark converter implemented in the conventional, discrete packaging technology. It is shown that by smart use of parts in the converter construction, the high values of integration level and high power densities can be achieved.

Single domain camel antibodies: current status

The antigen-binding capacity of the paired variable domains of an antibody is well established. The observation that the isolated heavy chains of anti-hapten antibodies retain some antigen-binding capacity in the absence of light chains led to attempts to obtain an even smaller antigen-binding unit in a VH format. Unfortunately, the poor solubility, the reduced affinity for the antigen and the irreproducible outcome showed that additional protein engineering would be required to successfully generate single-domain antibody fragments. By serendipity, it was discovered that this engineering is already performed continuously in nature. Part of the humoral immune response of camels and llamas is based largely on heavy-chain antibodies where the light chain is totally absent. These unique antibody isotypes interact with the antigen by virtue of only one single variable domain, referred to as VHH. Despite the absence of the VH–VL combinatorial diversity, these heavy-chain antibodies exhibit a broad antigen-binding repertoire by enlarging their hypervariable regions. Methods are described to tap the VHH repertoire of an immunised dromedary or llama. These VHH libraries contain a high titre of intact antigen-specific binders that were matured in vivo. Synthetic libraries of a ‘camelised’ human VH, a mouse VH or a camelid VHH scaffold with a randomised CDR3 could constitute a valid alternative to immune libraries to retrieve useful single-domain antigen binders. The recombinant VHH that are selected from such libraries are well expressed, highly soluble in aqueous environments and very robust. Some in vivo matured VHH were also shown to be potent enzyme inhibitors, and the low complexity of the antigen-binding site is an asset in the design of peptide mimetics. Because of their smaller size and the above properties, the VHH clearly offer added-value over conventional antibody fragments. They are expected to open perspectives as enzyme inhibitors and intrabodies, as modular building units for multivalent or multifunctional constructs, or as immuno-adsorbents and detection units in biosensors.

Progress in "engineering up" nanotechnology devices utilizing DNA as a construction material

DNA technology is in the process of expanding into microelectronic applications. Due to its unique recognition capabilities, physicochemical stability, and mechanical rigidity, DNA is a promising construction material for frameworks, for example, for the selective positioning of molecular devices. For example, based on the hybridization of DNA oligomers, nanocrystal metal clusters can be assembled to supramolecular aggregates. Further applications include the generation of multifunctional constructs, as well as macroscopic and nano-scaled arrays or scaffolds based on DNA. The latter might be used in the preparation of high-resolution matrices for construction of chips to attain circuit sizes below 100 nm. The fabrication of periodic frameworks or individual geometric objects from DNA is empowered by the convenient accessibility of modular building blocks -- as branched, double-stranded nucleic acid molecules- prepared from synthetic oligodeoxyribonucleotides by self-assembly. Design and strategies for the synthesis of nucleic acid-based nanostructures are discussed.