Characterization of biomimetic surfaces formed from cell membranes

A method for fabricating biomimetic surfaces from intact cell membranes is described. A monolayer of alkanethiol on gold is covered by a second layer derived from the components of erythrocyte membranes either by self-assembly or by Langmuir-Blodgett methods. The resulting asymmetric hybrid layer was characterized by ellipsometry, surface plasmon resonance (SPR), contact angle, capacitance, voltammetry, and electron and atomic force microscopy. The erythrocyte membrane layer was measured to be approximately 30-40 A in thickness. Using SPR, the presence of erythrocyte components on the surface was demonstrated by their selective removal by enzymatic action. The uniform deposition of membranous material on the substrate was shown by electron and atomic force microscopy. Demonstration of acetylcholinesterase (AChase) activity, a membrane-anchored enzyme, on the surface for at least 8 days, suggests that the outer leaflet of the erythrocyte membrane is present in its native form. Cyclic voltammetry demonstrates that enhanced electron transport from a solution redox species accompanies formation of the erythrocyte layer at the surface. This enhanced electron transport is blocked by 4,4'-diisothiocyanate stilbene-2,2'-disulfonic acid, a well known blocker of anion transport, suggesting that an erythrocyte anion transporter protein is incorporated into the surface layer in an active conformation.     

A biomimetic approach to some specifically functionalized cyclic terpenoids

The paper relates on the current advancements in the synthesis of complex cyclic terpenoids by superacidic induced cyclization of open chain precursors. It is shown that functional groups disposal in the initial substrate strongly influences the reaction outcome. Possible variations of the investigated compound structures include particularly alpha-functionalization and alpha,omega-bifunctionalization. This approach allowed a selective initiation of cyclization sequence from an internal double bond or suspending the ring closure cascade to partially cyclized compounds. The reported synthetic schemes are attempts to mimic the biogenetical processes postulated in the living systems.     

Click approach to the discovery of 1,2,3-triazolylsalicylamides as potent Aurora kinase inhibitors

A series of 1,2,3-triazolylsalicylamide derivatives has been developed from the antiproliferative agent 7 and was evaluated for their Aurora kinase inhibitory activity. The novel 1,2,3-triazolylsalicylamide scaffold could be readily assembled by Cu(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition, allowing rapid access to the structurally diverse analogues. The synthesized 1,2,3-triazolylsalicylamide derivatives revealed a significant Aurora kinase inhibitory activity. In particular, 8g inhibited Aurora A with IC₅₀ values of 0.37 μM. The critical role of phenolic –OH in the binding was confirmed by a molecular modeling study.     

Nanoscale engineering of biomimetic surfaces: cues from the extracellular matrix

The ultimate goal in the design of biomimetic materials for use in tissue engineering as permanent or resorbable tissue implants is to generate biocompatible scaffolds with appropriate biomechanical and chemical properties to allow the adhesion, ingrowth, and survival of cells. Recent efforts have therefore focused on the construction and modification of biomimetic surfaces targeted to support tissue-specific cell functions including adhesion, growth, differentiation, motility, and the expression of tissue-specific genes. Four decades of extensive research on the structure and biological influence of the extracellular matrix (ECM) on cell behavior and cell fate have shown that three types of information from the ECM are relevant for the design of biomimetic surfaces: (1) physical properties (elasticity, stiffness, resilience of the cellular environment), (2) specific chemical signals from peptide epitopes contained in a wide variety of extracelluar matrix molecules, and (3) the nanoscale topography of microenvironmental adhesive sites. Initial physical and chemical approaches aimed at improving the adhesiveness of biomaterial surfaces by sandblasting, particle coating, or etching have been supplemented by attempts to increase the bioactivity of biomaterials by coating them with ECM macromolecules, such as fibronectin, elastin, laminin, and collagens, or their integrin-binding epitopes including RGD, YIGSR, and GFOGER. Recently, the development of new nanotechnologies such as photo- or electron-beam nanolithography, polymer demixing, nano-imprinting, compression molding, or the generation of TiO₂ nanotubes of defined diameters (15–200 nm), has opened up the possibility of constructing biomimetic surfaces with a defined nanopattern, eliciting tissue-specific cellular responses by stimulating integrin clustering. This development has provided new input into the design of novel biomaterials. The new technologies allowing the construction of a geometrically defined microenvironment for cells at the nanoscale should facilitate the investigation of nanotopography-dependent mechanisms of integrin-mediated cell signaling.     

The dream of staying clean: Lotus and biomimetic surfaces

The Lotus has been the symbol of purity for thousands of years; contaminations and pathogens are washed off the surfaces of Lotus and some other plants with rain or even dew. After the introduction of scanning electron microscopy, we were able to resolve the mechanism behind this phenomenon. It took some further decades before in-depth studies on self-cleaning with plants were conducted and the effect could be understood in detail. We identified extreme water-repellency ('superhydrophobicity'), characterized by very high contact angles and low sliding angles, as the prerequisite for self-cleaning properties. We could show that the combination of two factors is necessary for obtaining a high degree of water-repellency: (1) low energy surfaces being hydrophobic and (2) surface structures that significantly increase hydrophobicity. It is suggested that this mechanism plays an important role in the protection of plants against pathogens. Our technological application of this effect has resulted in the development of successful, eco-friendly and sustainable industrial products. Another interesting property was found with superhydrophobic surfaces of certain aquatic and semi-aquatic plants and animals: here a layer of air under water is retained. We present a new approach of using this feature for creating structured, air-retaining surfaces for technical underwater applications. It is proposed that such surfaces can reduce significantly the drag of large ships. We conclude that basic biological research is of particular importance for true innovation. Our research on superhydrophobic self-cleaning biological surfaces and the development of similar engineered materials suggests that biomimicry is a matter of multi-stage processes rather than a simple copying of biological developments.     

Tuning cellular responses to BMP-2 with material surfaces

Bone morphogenetic protein 2 (BMP-2) has been known for decades as a strong osteoinductive factor and for clinical applications is combined solely with collagen as carrier material. The growing concerns regarding side effects and the importance of BMP-2 in several developmental and physiological processes have raised the need to improve the design of materials by controlling BMP-2 presentation. Inspired by the natural cell environment, new material surfaces have been engineered and tailored to provide both physical and chemical cues that regulate BMP-2 activity. Here we describe surfaces designed to present BMP-2 to cells in a spatially and temporally controlled manner. This is achieved by trapping BMP-2 using physicochemical interactions, either covalently grafted or combined with other extracellular matrix components. In the near future, we anticipate that material science and biology will integrate and further develop tools for in vitro studies and potentially bring some of them toward in vivo applications.     

A mutational approach to the study of seed development in maize

The maize seed comprises two major compartments, the embryo and the endosperm, both originating from the double fertilization event. The embryogenetic process allows the formation of a well-differentiated embryonic axis, surrounded by a single massive cotyledon, the scutellum. The mature endosperm constitutes the bulk of the seed and comprises specific regions containing reserve proteins, complex carbohydrates, and oils. To gain more insight into molecular events that underlie seed development, three monogenic mutants were characterized, referred to as emp (empty pericarp) on the basis of their extreme endosperm reduction, first recognizable at about 12 d after pollination. Their histological analysis reveals a partial development of the endosperm domains as well as loss of adhesion between pedicel tissues and the basal transfer layer. In the endosperm, programmed cell death (PCD) is delayed. The embryo appears retarded in its growth, but not impaired in its morphogenesis. The mutants can be rescued by culturing immature embryos, even though the seedlings appear retarded in their growth. The analysis of seeds with discordant embryo-endosperm phenotype (mutant embryo, normal endosperm and vice-versa), obtained using B-A translocations, suggests that emp expression in the embryo is necessary, but not sufficient, for proper seed development. In all three mutants the picture emerging is one of a general delay in processes related to growth, as a result of a mutation affecting endosperm development as a primary event.     

A biomimetic approach towards synthesis of zinc oxide nanoparticles

Using natural processes as inspiration, the present study demonstrates a positive correlation between zinc metal tolerance ability of a soil fungus and its potential for the synthesis of zinc oxide (ZnO) nanoparticles. A total of 19 fungal cultures were isolated from the rhizospheric soils of plants naturally growing at a zinc mine area in India and identified on the genus, respectively the species level. Aspergillus aeneus isolate NJP12 has been shown to have a high zinc metal tolerance ability and a potential for extracellular synthesis of ZnO nanoparticles under ambient conditions. UV–visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, transmission electron microscopy, and energy dispersive spectroscopy studies further confirmed the crystallinity, morphology, and composition of synthesized ZnO nanoparticles. The results revealed the synthesis of spherical nanoparticles coated with protein molecules which served as stabilizing agents. Investigations on the role of fungal extracellular proteins in the synthesis of nanoparticles indicated that the process is nonenzymatic but involves amino acids present in the protein chains.     

Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents

Metallodrugs provide important first-line treatment against various forms of human cancer. To overcome chemotherapeutic resistance and widen treatment possibilities, new agents with improved or alternative modes of action are highly sought after. Here, we present a click chemistry strategy for developing DNA damaging metallodrugs. The approach involves the development of a series of polyamine ligands where three primary, secondary or tertiary alkyne-amines were selected and ‘clicked’ using the copper-catalysed azide-alkyne cycloaddition reaction to a 1,3,5-azide mesitylene core to produce a family of compounds we call the ‘Tri-Click’ (TC) series. From the isolated library, one dominant ligand (TC1) emerged as a high-affinity copper(II) binding agent with potent DNA recognition and damaging properties. Using a range of in vitro biophysical and molecular techniques—including free radical scavengers, spin trapping antioxidants and base excision repair (BER) enzymes—the oxidative DNA damaging mechanism of copper-bound TC1 was elucidated. This activity was then compared to intracellular results obtained from peripheral blood mononuclear cells exposed to Cu(II)–TC1 where use of BER enzymes and fluorescently modified dNTPs enabled the characterisation and quantification of genomic DNA lesions produced by the complex. The approach can serve as a new avenue for the design of DNA damaging agents with unique activity profiles.     

Biochemical switching device: biomimetic approach and application to neural network study.

There are many examples of enzymes that share substrates or cofactors in a cyclic manner. Techniques have been developed that use cyclic enzyme systems to assay quantitatively small amounts of biochemical substances (cofactor, substrate), however, only a few studies of the control of these systems have been published. The author previously showed with computer simulations that cyclic enzyme systems have the reliability of ON-OFF types of operation (McCulloch-Pitts' neuronic equation) capable of storing short-memory, and the applicability for a switching circuit in a biocomputer. This paper introduces a unique switching mechanism of cyclic enzyme system (basic switching element), and next, building the integrated biochemical switching system being composed of the basic switching element, shows the physiological phenomenon termed 'selective elimination of synapses' generally produced as a result of low-frequency train of electrical stimuli to the synapses (Kuroda, Y. 1989) Neurochem. Int. 14, 309-319).     

Chemical modification of the surfaces of bacterial cell walls.

The surfaces of the isolated cell walls of four bacterial species were studied by microelectrophoresis following chemical treatments intended to remove specific charged groups. Acid-base titrations of the walls were used to assess specificity and extent of the modifications. Carboxyl groups were specifically and completely modified by activation with a water-soluble carbodiimide and subsequent reaction with a nucleophile, such as glycinamide, to give an uncharged pH-stable product. Aqueous media and mild reaction conditions make the method suitable for modifying carboxyl groups on cell surfaces too labile to withstand the harsh conditions required for conventional esterification reactions. Use of the carbodiimide-mediated reaction for discharging carboxyl groups, along with fluorodinitrobenzene for discharging amino groups and extraction procedures for removing constituents carrying phosphoester groups (teichoic acids), made it possible to obtain information about the spatial arrangement of charged groups on the wall surfaces. Removal of the exterior negative charge dominating wall surfaces allowed underlying amino groups to become electrokinetically effective and, in the case of E. coli, also revealed a lipophilic region with an affinity for a cationic surfactant.     

Cellulose derivatives: An enzymatic approach to their modification

Carboxymethyl cellulose (CMC) with different degrees of substitution (DS) and molecular weights (MW) have been successfully hydrolyzed by cellulases sourced from different microorganisms. The extent of enzymatic hydrolysis of CMC was shown to decrease with increasing DS. According to chromatographic analyses, the best enzymatic degradation by the crude enzymic preparations employed was 47% when cellulase T from Trichoderma species acted on a CMC of DS = 0·7. However, the complete hydrolysis, required for a quantitative analysis, was reached when CMCs with DS up to 0·7 were degraded by cellulase P, a purified form of celluclast from Trichoderma reesei.     

A modular approach to creating large engineered cartilage surfaces

Native articular cartilage has limited capacity to repair itself from focal defects or osteoarthritis. Tissue engineering has provided a promising biological treatment strategy that is currently being evaluated in clinical trials. However, current approaches in translating these techniques to developing large engineered tissues remains a significant challenge. In this study, we present a method for developing large-scale engineered cartilage surfaces through modular fabrication. Modular Engineered Tissue Surfaces (METS) uses the well-known, but largely under-utilized self-adhesion properties of de novo tissue to create large scaffolds with nutrient channels. Compressive mechanical properties were evaluated throughout METS specimens, and the tensile mechanical strength of the bonds between attached constructs was evaluated over time. Raman spectroscopy, biochemical assays, and histology were performed to investigate matrix distribution. Results showed that by Day 14, stable connections had formed between the constructs in the METS samples. By Day 21, bonds were robust enough to form a rigid sheet and continued to increase in size and strength over time. Compressive mechanical properties and glycosaminoglycan (GAG) content of METS and individual constructs increased significantly over time. The METS technique builds on established tissue engineering accomplishments of developing constructs with GAG composition and compressive properties approaching native cartilage. This study demonstrated that modular fabrication is a viable technique for creating large-scale engineered cartilage, which can be broadly applied to many tissue engineering applications and construct geometries.     

Promoting differentiation of cultured myoblasts using biomimetic surfaces that present alpha-laminin-2 peptides

Traditionally, muscle cell lines are cultured on glass coverslips and differentiated to investigate myoblast fusion and differentiation. Efficient differentiation of myoblasts produces a dense network of myotubes with the correct organisation for contraction. Here we have tested the ability of artificially generated, precisely controlled peptide surfaces to enhance the efficiency of myoblast differentiation. We focused on specific short peptides from α-laminin-2 (IKVSV, VQLRNGFPYFSY and GLLFYMARINHA) as well as residues 15–155 from FGF1. We tested if these peptides in isolation, and/or in combination promoted muscle differentiation in culture, by promoting fusion and/or by improving sarcomere organisation. The majority of these peptides promoted fusion and differentiation in two different mouse myogenic cell lines and in primary human myoblasts. The additive effects of all four peptides gave the best results for both mouse cell lines tested, while primary human cell cultures differentiated equally well on most peptide surfaces tested. These data show that a mixture of short biomimetic peptides can reliably promote differentiation in mouse and human myoblasts.     

Radiation-induced biomimetic modification of dual-layered nano/microfibrous scaffolds for vascular tissue engineering

One of the interesting strategies for developing the artificial blood vessels is to generate multi-layered scaffolds for mimicking the structure of native blood vessels such as the intima, media, and adventitia. In this study, we prepared dual-layered poly(L-lactide-co-?-caprolactone) (PLCL) scaffolds with micro- and nanofibers as a basic construct of the vessel using electrospinning methods, which was functionalized using a gelatin through acrylic acid (AAc) grafting by γ-ray irradiation. Based on the microfibrous platform (fiber diameter 5 μm), the thickness of the nanofibrous layer (fiber diameter 700 nm) was controlled from 1.1 ± 0.8 to 32.2 ± 1.7 μm, and the mechanical property of the scaffolds was almost maintained despite the increase in thickness of the nanofibrous layer. The successful AAc graft by γ-ray irradiation could allow the gelatin immobilization on the scaffolds. The proliferation of smooth muscle cells (SMC) on the scaffolds toward a microfibrous layer was approximately 1.3-times greater than in the other groups, and the infiltration was significantly increased, presenting a wide cell distribution in the cross-section. In addition, human umbilical vein endothelial cell (HUVEC) adhesion toward nanofibrous layer was well-managed over the entire surface, and the accelerated proliferation was observed on the gelatin-functionalized scaffolds presenting the well-organized gap-junctions. Therefore, our biomimetic dual-layered scaffolds may be the alternative tools for replacing the damaged blood vessels.     

Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression

Abscisic acid (ABA) is a plant hormone synthesized during seed development that is involved in the induction of seed dormancy. Delayed germination due to seed dormancy allows long-term seed survival in soil but is generally undesirable in crop species. Freshly harvested seeds of wild-type Nicotiana plumbaginifolia plants exhibit a clear primary dormancy that results in delayed germination, the degree of primary dormancy being influenced by environmental culture conditions of the mother plant. In contrast, seeds, obtained either from ABA-deficient mutant aba2-s1 plants directly or aba2-s1 plants grafted onto wild-type plant stocks, exhibited rapid germination under all conditions irrespective of the mother plant culture conditions. The ABA biosynthesis gene ABA2 of N. plumbaginifolia, encoding zeaxanthin epoxidase, was placed under the control of the constitutive 35S promoter. Transgenic plants overexpressing ABA2 mRNA exhibited delayed germination and increased ABA levels in mature seeds. Expression of an antisense ABA2 mRNA, however, resulted in rapid seed germination and in a reduction of ABA abundance in transgenic seeds. It appears possible, therefore, that seed dormancy can be controlled in this Nicotiana model species by the manipulation of ABA levels.     

Systems biology approach to the functional role of enzymatic modification of bacterial ribosome

In this work we describe methodology for studying the role of bacterial ribosome modification in the regulation of gene expression. Ribosomal components modification influences translation efficiencies of certain mRNAs. Proteome analysis allows us to identify cellular protein composition change caused by ribosome modification gene knockout. Particular stage of gene expression responsible for certain protein concentration change could be found using reporter constructs. After identification of mRNA species, whose translation is influenced by ribosome modification we can determine exact mRNA region responsible for the observed changes. The developed methodology can be applied for studying other translational control mechanisms.