Scientific publications

On command control of gene expression in time and space is required for the comprehensive analysis of key plant cellular processes. Even though some chemical inducible systems showing satisfactory induction features have been developed, they are inherently limited in terms of spatiotemporal resolution and may be associated with toxic effects. We describe here the first synthetic light-inducible system for the targeted control of gene expression in plants. For this purpose, we applied an interdisciplinary synthetic biology approach comprising mammalian and plant cell systems to customize and optimize a split transcription factor based on the plant photoreceptor phytochrome B and one of its interacting factors (PIF6). Implementation of the system in transient assays in tobacco protoplasts resulted in strong (95-fold) induction in red light (660 nm) and could be instantaneously returned to the OFF state by subsequent illumination with far-red light (740 nm). Capitalizing on this toggle switch-like characteristic, we demonstrate that the system can be kept in the OFF state in the presence of 740 nm-supplemented white light, opening up perspectives for future application of the system in whole plants. Finally we demonstrate the system's applicability in basic research, by the light-controlled tuning of auxin signalling networks in N. tabacum protoplasts, as well as its biotechnological potential for the chemical-inducer free production of therapeutic proteins in the moss P. patens. This journal is © the Partner Organisations 2014.

Light-triggered gene expression systems offer an unprecedented spatiotemporal resolution that cannot be achieved with classical chemically inducible genetic tools. Here we describe a protocol for red light-responsive gene expression in mammalian cells. This system can be toggled between stable ON and OFF states by short pulses of red and far-red light, respectively. In the protocol, CHO-K1 cells are transfected to allow red light-inducible expression of the secreted alkaline phosphatase (SEAP) reporter, and gene expression is tuned by illumination with light of increasing wavelengths. As a starting point for elaborate red light-responsive gene expression, we outline the reversible activation of gene expression and describe how a spatial pattern can be 'printed' on a monolayer of cells by using a photomask. The core protocol requires only 4 d from seeding of the cells to reporter quantification, and other than light-emitting diode (LED) illumination boxes no elaborate equipment is required. © 2014 Nature America, Inc.

Mechanobiology is a scientific interface discipline emerging from engineering and biology. With regard to tissue-regenerative cell-based strategies, mechanobiological concepts, including biomechanics as a target for cell and human mesenchymal stem cell behaviour, are on the march. Based on the periodontium as a paradigm, this mini-review discusses the key role of focal-adhesion kinase (FAK) in mechanobiology, since it is involved in mediating the transformation of environmental biomechanical signals into cell behavioural responses via mechanotransducing signalling cascades. These processes enable cells to adjust quickly to environmental cues, whereas adjustment itself relies on the specific intramolecular phosphorylation of FAK tyrosine residues and the multiple interactions of FAK with distinct partners. Furthermore, interaction-triggered mechanotransducing pathways govern the dynamics of focal adhesion sites and cell behaviour. Facets of behaviour not only include cell spreading and motility, but also proliferation, differentiation and apoptosis. In translational terms, identified and characterized biomechanical parameters can be incorporated into innovative concepts of cell- and tissue-tailored clinically applied biomaterials controlling cell behaviour as desired.

Light-dependent dimerization is the basis for recently developed noninvasive optogenetic tools. Here we present a novel tool combining optogenetics with the control of protein kinase activity to investigate signal transduction pathways. Mediated by Arabidopsis thaliana photoreceptor cryptochrome 2, we activated the protein kinase C-RAF by blue light-dependent dimerization, allowing for decoupling from upstream signaling events induced by surface receptors. The activation by light is fast, reversible, and not only time but also dose dependent as monitored by phosphorylation of ERK1/2. Additionally, light-activated C-RAF controls serum response factor-mediated gene expression. Light-induced heterodimerization of C-RAF with a kinase-dead mutant of B-RAF demonstrates the enhancing role of B-RAF as a scaffold for C-RAF activity, which leads to the paradoxical activation of C-RAF found in human cancers. This optogenetic tool enables reversible control of protein kinase activity in signal duration and strength. These properties can help to shed light onto downstream signaling processes of protein kinases in living cells. © 2013 American Chemical Society.

Aluminum coatings were deposited on glass substrates by chemical vapor deposition using N-methylpiperidine (nmp) stabilized dichloroalane [Cl2AlH•2nmp] as aluminum precursor. With regard to temperature, the experimental conditions were varied between 75 °C and 125 °C for the precursor and between 250 °C and 450 °C for the substrate. Depending on these parameters, highly textured layers could be deposited. The substrates have been consistently covered by a layer of idiomorphic, mostly distorted octahedra of aluminum single crystals. The morphologies of the structures and the degree of orientation of the crystals were investigated by scanning electron microscopy and X-ray diffraction measurements. The high order of [111] orientation was found to decrease with increasing precursor and substrate temperature. We propose a mechanism for the generation of the octahedral structures based on the formation of mesocrystals. On heating, the dichloroalane (stabilized with nmp) loses the nmp ligands together with hydrogen and chlorine. The amine (nmp) seems to trigger the formation of aluminum crystals depending on the temperature and thus influences the texture of the Al-layer and the formation of well-formed octahedron-like structures.

Permanent orthopedic implants are becoming increasingly important due to the demographic development. Their optimal osseointegration is key in obtaining good secondary stability. For anchorage dependent cells, topographic features of a surface play an essential role for cell adhesion, proliferation, differentiation and biomineralization. We studied the topographical effect of nanostructured alumina surfaces prepared by chemical vapor deposition on osteogenic differentiation and growth of human osteoblasts. Chemical vapor deposition of the single source precursor (tBuOAlH2)2 led to synthesis of one dimensional alumina nanostructures of high purity with a controlled stoichiometry. We fabricated different topographic features by altering the distribution density of deposited one dimensional nanostructures. Although the topography differed, all surfaces exhibited identical surface chemistry, which is the key requirement for systematically studying the effect of the topography on cells. Forty-eight hours after seeding, cell density and cell area were not affected by the nanotopography, whereas metabolic activity was reduced and formation of actin-fibres and focal adhesions was impaired compared to the uncoated control. Induction of osteogenic differentiation was demonstrated via up-regulation of alkaline phosphatase, bone sialoprotein, osteopontin and Runx2 at the mRNA level, demonstrating the potential of nanostructured surfaces to improve the osseointegration of permanent implants.

The management of end stage heart failure patients is only possible by heart transplantation or by the implantation of artificial hearts as a bridge for later transplantation. However, these therapeutic strategies are limited by a lack of donor hearts and by the associated complications, such as coagulation and infection, due to the used artificial mechanical circulatory assist devices. Therefore, new strategies for myocardial regenerative approaches are under extensive research to produce contractile myocardial tissue in the future to replace non-contractile myocardial ischemic and scarred tissue. Different approaches, such as cell transplantation, have been studied intensively. Although successful approaches have been observed, there are still limitations to the application. It is envisaged that myocardial tissue engineering can be used to help replace infarcted non-contractile tissue. The developed tissue should later mimic the aligned fibrillar structure of the extracellular matrix and provide important guidance cues for the survival, function and the needed orientation of cardiomyocytes. Nanostructured surfaces have been tested to provide a guided direction that cells can follow. In the present study, the cellular adhesion/alignment of human cardiomyocytes and the biocompatibility have been investigated after cultivation on different laser-patterned nanowires compared with unmodified nanowires. As a result, the nanostructured surfaces possessed good biocompatibility before and after laser modification. The laser-induced scalability of the pattern enabled the growth and orientation of the adhered myocardial tissue. Such approaches may be used to modify the surface of potential scaffolds to develop myocardial contractile tissue in the future.

The process is a simple chemical method and aims to produce nano-structured calcium phosphate powders from natural sources, for biomedical applications. For this purpose, Atlantic Deer Cowrie (ADC) shells (Cypraea cervus Linnaeus, 1771) were collected from a local gift store in Istanbul. The empty shells were cleaned and crushed then were ball milled and sieved under 100μm. The raw powders were suspended on a hotplate stirrer for a simple chemical agitation. The temperature was kept at 80°C for 15 min. and then appropriate amount of H3PO4 was added by titration into the prepared solution to form calcium phosphate precursors. The solution was stirred on a hotplate for 8 hours then dried at 100°C for 24 hours. Afterwards the resulting dried sediments were collected and heat treated between 400-800°C for 4 hours, dependent on the required specific calcium phosphate phase. X-ray diffraction (XRD) analysis and scanning electron microscopy (SEM) were carried out for identifying various hydroxyapatite (HA), tricalcium phosphate (TCP) and other calcium phosphate phases. Various particle sizes ranging from nano to micron, are obtained depending on the chemistry used and the processing technique applied during the production. A range of calcium phosphate phases can be obtained from ADC shells, by using a simple and economic conversion method. Proper cleaning methods developed and appropriate preparation techniques will enable us to use these nano calcium phosphate powders in orthopedic and dental applications.

Pulsed laser deposition (PLD) is one of the well-established physical vapor deposition methods used for synthesis of ultra-thin layers. Especially PLD is suitable for the preparation of thin films of complex alloys and ceramics where the conservation of the stoichiometry is critical. Beside several advantages of PLD, inhomogeneity in thickness limits use of PLD in some applications. There are several approaches such as rotation of the substrate or scanning of the laser beam over the target to achieve homogenous layers. On the other hand movement and transition create further complexity in process parameters. Here we present a new approach which we call Matrix Shaped PLD to control the thickness and homogeneity of deposited layers precisely. This new approach is based on shaping of the incoming laser beam by a microlens array and a Fourier lens. The beam is split into much smaller multi-beam array over the target and this leads to a homogenous plasma formation. The uniform intensity distribution over the target yields a very uniform deposit on the substrate. This approach is used to deposit carbide and oxide thin films for biomedical applications. As a case study coating of a stent which has a complex geometry is presented briefly.

Polyetheretherketone (PEEK) is considered as a substitute for metallic implant materials due to its extremely low elastic modulus (3-4 GPa). Despite its good mechanical properties, PEEK exhibits a slow integration with the bone tissue due to its relatively inert surface and low biocompatibility. We introduced a dual modification method, which combines the laser and plasma surface treatments to achieve hierarchically patterned PEEK surfaces. While the plasma treatment leads to nanotopography, the laser treatment induces microstructures over the PEEK surface. On the other hand, plasma and laser treatments induce inhomogeneity in the surface chemistry in addition to the tailored surface topography. Therefore, we coated the structured PEEK surfaces with a thin alumina layer by pulsed laser deposition (PLD) to get identical surface chemistry on each substrate. Such alumina-coated PEEK surfaces are used as a model to investigate the effect of the surface topography on the wetting independent from the surface chemistry. Prepared surfaces bring advantages of enhanced wetting, multiscaled topography, proven biocompatibility (alumina layer), and low elastic modulus (PEEK as substrate), which together may trigger the use of PEEK in bone and other implant applications.