Immobilization and characterization of recombinant esterase enzyme on chitosan nanoparticles

Immobilization of biologically important molecules on myriad nano-sized materials has attracted great attention. Through this study, thermophilic esterase enzyme was obtained using recombinant DNA technology and purified applying one-step His-Select HF nickel affinity gel. The synthesis of chitosan was achieved from chitin by deacetylation process and degree of deacetylation was calculated as 89% by elemental analysis. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. The physicochemical properties of the chitosan and chitosan nanoparticles were determined by several methods including SEM (Scanning Electron Microscopy), FT-IR (Fourier Transform Infrared Spectroscopy) and DLS (Dynamic Light Scattering). The morphology of chitosan nanoparticles was spherical and the nanospheres’ average diameter was 75.3 nm. The purified recombinant esterase was immobilized efficiently by physical adsorption onto chitosan nanoparticles and effects of various immobilization conditions were investigated in details to develope highly cost-effective esterase as a biocatalyst to be utilized in biotechnological purposes. The optimal conditions of immobilization were determined as follows; 1.0 mg/mL of recombinant esterase was immobilized on 1.5 mg chitosan nanoparticles for 30 min at 60°C, pH 7.0 under 100 rpm stirring speed. Under optimized conditions, immobilized recombinant esterase activity yield was 88.5%. The physicochemical characterization of enzyme immobilized chitosan nanoparticles was analyzed by SEM, FT-IR and AFM (Atomic Force Microscopy).

Fabrication of cone-shaped boron doped diamond and gold nanoelectrodes for AFM-SECM

We demonstrate a reliable microfabrication process for a combined atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) measurement tool. Integrated cone-shaped sensors with boron doped diamond (BDD) or gold (Au) electrodes were fabricated from commercially available AFM probes. The sensor formation process is based on mature semiconductor processing techniques, including focused ion beam (FIB) machining, and highly selective reactive ion etching (RIE). The fabrication approach preserves the geometry of the original AFM tips resulting in well reproducible nanoscaled sensors. The feasibility and functionality of the fully featured tips are demonstrated by cyclic voltammetry, showing good agreement between the measured and calculated currents of the cone-shaped AFM-SECM electrodes.

Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis

Mitochondrial F(1)F(o)-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F(1)F(o)-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F(1)F(o)-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F(1)F(o)-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F(1) catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F(1) to the lipid monolayer and the F(o) membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F(o) by electron microscopy and AFM.

The supramolecular assemblies of voltage-dependent anion channels in the native membrane

Voltage-dependent anion channels (VDACs) are major constituents of the outer mitochondrial membrane (OMM). These primary transporters of nucleotides, ions and metabolites mediate a substantial portion of the OMM molecular traffic. To study the native supramolecular organization of the VDAC, we have isolated, characterized and imaged OMMs from potato tubers. SDS-PAGE and mass spectrometry of OMMs revealed the presence of the VDAC isoforms POM34 and POM36, as well as the translocase of the OMM complex. Tubular two-dimensional crystals of the VDAC spontaneously formed after incubation of OMMs for two to three months at 4 degrees C. Transmission electron microscopy revealed an oblique lattice and unit cells housing six circular depressions arranged in a hexagon. Atomic force microscopy of freshly isolated OMMs demonstrated (i) the existence of monomers to tetramers, hexamers and higher oligomers of the VDAC and (ii) its spatial arrangement within the oligomers in the native membrane. We discuss the importance of the observed oligomerization for modulation of the VDAC function, for the binding of hexokinase and creatine kinase to the OMM and for mitochondria-mediated apoptosis.

The pollen tube: a soft shell with a hard core

Plant cell expansion is controlled by a fine-tuned balance between intracellular turgor pressure, cell wall loosening and cell wall biosynthesis. To understand these processes, it is important to gain in-depth knowledge of cell wall mechanics. Pollen tubes are tip-growing cells that provide an ideal system to study mechanical properties at the single cell level. With the available approaches it was not easy to measure important mechanical parameters of pollen tubes, such as the elasticity of the cell wall. We used a cellular force microscope (CFM) to measure the apparent stiffness of lily pollen tubes. In combination with a mechanical model based on the finite element method (FEM), this allowed us to calculate turgor pressure and cell wall elasticity, which we found to be around 0.3 MPa and 20–90 MPa, respectively. Furthermore, and in contrast to previous reports, we showed that the difference in stiffness between the pollen tube tip and the shank can be explained solely by the geometry of the pollen tube. CFM, in combination with an FEM-based model, provides a powerful method to evaluate important mechanical parameters of single, growing cells. Our findings indicate that the cell wall of growing pollen tubes has mechanical properties similar to rubber. This suggests that a fully turgid pollen tube is a relatively stiff, yet flexible cell that can react very quickly to obstacles or attractants by adjusting the direction of growth on its way through the female transmitting tissue.

Mechano-Chemical Aspects of Organ Formation in Arabidopsis thaliana: The Relationship between Auxin and Pectin

How instructive signals are translated into robust and predictable changes in growth is a central question in developmental biology. Recently, much interest has centered on the feedback between chemical instructions and mechanical changes for pattern formation in development. In plants, the patterned arrangement of aerial organs, or phyllotaxis, is instructed by the phytohormone auxin; however, it still remains to be seen how auxin is linked, at the apex, to the biochemical and mechanical changes of the cell wall required for organ outgrowth. Here, using Atomic Force Microscopy, we demonstrate that auxin reduces tissue rigidity prior to organ outgrowth in the shoot apex of Arabidopsis thaliana, and that the de-methyl-esterification of pectin is necessary for this reduction. We further show that development of functional organs produced by pectin-mediated ectopic wall softening requires auxin signaling. Lastly, we demonstrate that coordinated localization of the auxin transport protein, PIN1, is disrupted in a naked-apex produced by increasing cell wall rigidity. Our data indicates that a feedback loop between the instructive chemical auxin and cell wall mechanics may play a crucial role in phyllotactic patterning.

Interactive Diffraction from Biological Nanostructures

We describe a technique for interactive rendering of diffraction effects produced by biological nanostructures such as snake skin surface gratings. Our approach uses imagery from atomic force microscopy that accurately captures the nanostructures responsible for structural coloration, that is, coloration due to wave interference, in a variety of animals. We develop a rendering technique that constructs bidirectional reflection distribution functions (BRDFs) directly from the measured data and leverages precomputation to achieve interactive performance. We demonstrate results of our approach using various shapes of the surface grating nanostructures. Finally, we evaluate the accuracy of our precomputation-based technique and compare to a reference BRDF construction technique.

Tailoring surface nanostructures on polyaryletherketones for load-bearing implants

High-performance thermoplastics including polyetheretherketone (PEEK) are key biomaterials for load-bearing implants. Plasma treatment of implants surfaces has been shown to chemically activate its surface, which is a prerequisite to achieve proper cell attachment. Oxygen plasma treatment of PEEK films results in very reproducible surface nanostructures and has been reported in the literature. Our goal is to apply the plasma treatment to another promising polymer, polyetherketoneketone (PEKK), and compare its characteristics to the ones of PEEK. Oxygen plasma treatments of plasma powers between 25 and 150 W were applied on 60 μm-thick PEKK and 100 μm-thick PEEK films. Analysis of the nanostructures by atomic force microscopy showed that the roughness increased and island density decreased with plasma power for both PEKK and PEEK films correlating with contact angle values without affecting bulk properties of the used films. Thermal analysis of the plasma-treated films shows that the plasma treatment does not change the bulk properties of the PEKK and PEEK films.

Formation of supramolecular polymers by chrysene oligomers

Supramolecular assembly of π-conjugated systems is of large interested due to the possibility to use them in electronic devices.[1] Chrysene is a polyaromatic hydrocarbon which has been studied e.g for organic light-emitting diodes (OLEDs).[2] In continuation of our previous work involving the supramolecular polymerisation of pyrene oligomers [3] an oligomer consisting of three chrysenes linked by phophodiesters was synthesised (Chry3). UV-Vis measurements show that aggregates of Chry3 are formed in aqueous medium. This is illustrated by general hypochromicity, a change in vibronic band intensities and, in particular, the appearance of a red-shifted absorption band in the S0 → S2 transition. The data suggest the formation of J-aggregates. The formation of supramolecular polymers is further studied by temperature-dependent absorption- and fluorescence measurements, and by atomic force microscopy (AFM).

Interactive Diffraction from Biological Nanostructures

We describe a technique for interactive rendering of diffraction effects produced by biological nanostructures, such as snake skin surface gratings. Our approach uses imagery from atomic force microscopy that accurately captures the geometry of the nanostructures responsible for structural colouration, that is, colouration due to wave interference, in a variety of animals. We develop a rendering technique that constructs bidirectional reflection distribution functions (BRDFs) directly from the measured data and leverages pre-computation to achieve interactive performance. We demonstrate results of our approach using various shapes of the surface grating nanostructures. Finally, we evaluate the accuracy of our pre-computation-based technique and compare to a reference BRDF construction technique.

Play in Rats: Association across Contexts and Types , and Analysis of Structure.

Play has been proposed as a promising indicator of positive animal welfare. We aimed to study play in rats across contexts (conspecific/heterospecific) and types (social: pinning, being pinned; solitary: scampering), and we investigated its structure using behavioral sequence analysis. Group-housed (three per cage) adolescent male Lister Hooded rats (n = 21) were subjected to a Play-In-Pairs test: after a 3 hour isolation period, a pair of cage-mates was returned to the home cage and both social and solitary play were scored for 20 min. This procedure was repeated for each pair combination across three consecutive days, and individual play scores were calculated. Heterospecific play was measured using a Tickling test: rats were individually tickled by the experimenter through bouts of gentle, rapid finger movements on their underside, and the number of positive 50 kHz frequency modulated vocalizations and experimenter-directed approach behaviors were recorded. Both of the above tests were compared with social play in the home cage. While conspecific play in both the Play-In-Pairs test and home cage were correlated, both seemed to be unrelated to heterospecific play in the Tickling test. During the Play-In-Pairs test, although both solitary and social play types occurred, they were unrelated, and solitary locomotor play of one rat did not predict the subsequent play behavior of its cage mate. Analysis of play structure revealed that social play occurred more often in bouts of repeated behaviors while solitary play sequences did not follow a specific pattern. If play is to be used as an indicator of positive welfare in rats, context, type and structure differences should be taken into account.

Quantitative analysis of imprint shape and its relation to mechanical properties measured by microindentation in bone

Microindentation in bone is a micromechanical testing technique routinely used to extract material properties related to bone quality. As the analysis of microindentation data is based on assumptions about the contact between sample and surface, the aim of this study was to quantify the topological variability of indentations in bone and examine its relationship with mechanical properties. Indentations were performed in dry human and ovine bone in axial and transverse directions and their topology was measured by atomic force microscopy. Statistical shape modeling of the residual imprint allowed to define a mean shape and to describe the variability in terms of 21 principal components related to imprint depth, surface curvature and roughness. The indentation profile of bone was found to be highly consistent and free of any pile up while differing mostly by depth between species and direction. A few of the topological parameters, in particular depth, showed significant but rather weak and inconsistent correlations to variations in mechanical properties. The mechanical response of bone as well as the residual imprint shape was highly consistent within each category. We could thus verify that bone is rather homogeneous in its micromechanical properties and that indentation results are not strongly influenced by small deviations from an ideally flat surface.

DNA-Grafted Supramolecular Polymers: Helical Ribbon Structures Formed by Self-Assembly of Pyrene-DNA Chimeric Oligomers

The controlled arraying of DNA strands on adaptive polymeric platforms remains a challenge. Here, the noncovalent synthesis of DNA-grafted supramolecular polymers from short chimeric oligomers is presented. The oligomers are composed of an oligopyrenotide strand attached to the 5′-end of an oligodeoxynucleotide. The supramolecular polymerization of these oligomers in an aqueous medium leads to the formation of one-dimensional (1D) helical ribbon structures. Atomic force and transmission electron microscopy show rod-like polymers of several hundred nanometers in length. DNA-grafted polymers of the type described herein will serve as models for the development of structurally and functionally diverse supramolecular platforms with applications in materials science and diagnostics.