Spearmint targets microtubules by (-)-carvone.

Allelopathy can provide sustainable alternatives to herbicides because it is based on specific signals rather than generic toxicity. We show that the allelopathic activity of Spearmint and Watermint is linked with their main compounds, (-)-carvone and (+)-menthofuran, both deriving from (-)-limonene. Germination of Poppy and Cress, and root growth of Arabidopsis thaliana are inhibited by very low concentrations of (-)-carvone, acting even through the gas phase. (+)-Menthofuran is active as well, but at lower efficacy. Using fluorescently tagged marker lines in tobacco BY-2 cells and Arabidopsis roots, we demonstrate a rapid degradation of microtubules and a remodeling of actin filaments in response to (-)-carvone and, to a milder extent, to (+)-menthofuran. This cytoskeletal response is followed by cell death. By means of a Root Chip system, we can follow the tissue dependent response of the cytoskeleton and show a cell-type dependent gradient of sensitivity between meristem and distal elongation zone, accompanied by programmed cell death.

Influence of the Available Surface Area and Cell Elasticity on Bacterial Adhesion Forces on Highly Ordered Silicon Nanopillars.

Initial bacterial adhesion to solid surfaces is influenced by a multitude of different factors, e.g., roughness and stiffness, topography on the micro- and nanolevel, as well as chemical composition and wettability. Understanding the specific influences and possible interactive effects of all of these factors individually could lead to guidance on bacterial adhesion and prevention of unfavorable consequences like medically relevant biofilm formation. On this way, the aim of the present study was to identify the specific influence of the available surface area on the adhesion of clinically relevant bacterial strains with different membrane properties: Gram-positive Staphylococcus aureus and Gram-negative Aggregatibacter actinomycetemcomitans. As model surfaces, silicon nanopillar specimens with different spacings were fabricated using electron beam lithography and cryo-based reactive ion etching techniques. Characterization by scanning electron microscopy and contact angle measurement revealed almost defect-free highly ordered nanotopographies only varying in the available surface area. Bacterial adhesion forces to these specimens were quantified by means of single-cell force spectroscopy exploiting an atomic force microscope connected to a microfluidic setup (FluidFM). The nanotopographical features reduced bacterial adhesion strength by reducing the available surface area. In addition, the strain-specific interaction in detail depended on the bacterial cell's elasticity and deformability as well. Analyzed by confocal laser scanning microscopy, the obtained results on bacterial adhesion forces could be linked to the subsequent biofilm formation on the different topographies. By combining two cutting-edge technologies, it could be demonstrated that the overall bacterial adhesion strength is influenced by both the simple physical interaction with the underlying nanotopography and its available surface area as well as the deformability of the cell.

Enhancing the soft-tissue integration of dental implant abutments-in vitro study to reveal an optimized microgroove surface design to maximize spreading and alignment of human gingival fibroblasts.

Within this work, we demonstrate the influences of different microgrooved surface topographies on the alignment and spreading of human gingival fibroblast (HGF) cells and present the optimal parameters for an improved soft-tissue integration design for dental implant abutments for the first time. Microgrooves with lateral widths from 2.5 to 75 µm were fabricated by UV-lithography and wet etching on bulk Ti6Al4V ELI material. The microstructured surfaces were compared to polished and ground surfaces as current state of the art. The resulting microtopographies were analyzed using vertical scanning interferometry and scanning electron microscopy. Samples loaded with HGF cells were incubated for 8 and 72 hr and cell orientation, spreading, resulting area, and relative gene expression were analyzed. The effect of contact guidance occurred on all microstructured surfaces yet there is a clear preferable range for the lateral widths of the microgrooves between approx. 11.5 and 13.9 µm and depths between 1.6 and 2.4 µm for an abutment surface design, where cell orientation and spreading maximizes. For structures larger than 30 µm, cell orientation, spreading and even gene expression of intercellular adhesion molecule-1 and yes-associated protein decrease.

Integration of digital microfluidics with whispering-gallery mode sensors for label-free detection of biomolecules.

We present a multi-sensor chip comprising an array of whispering-gallery mode (WGM) micro-goblet lasers integrated into a digital microfluidic (DMF) system. In contrast to earlier demonstrations, the lasers are fabricated from dye-doped poly-methyl methacrylate (PMMA) at low cost using spin-coating, mask-based optical lithography, wet chemical etching, and thermal reflow techniques. Pumping and read-out of the devices is accomplished via simple free-space optics, thereby allowing large-scale sensor arrays to be addressed. We demonstrate the viability of the system by bulk refractive index-sensing and by measuring the specific binding of streptavidin to a biotinylated sensor surface. This is the first time that optical cavities are used for label-free detection of biomolecules in a DMF system. This approach can be extended to a versatile detector platform that targets a wide range of clinically relevant biomolecules.

Time-resolved NMR metabolomics of plant cells based on a microfluidic chip.

The plant secondary metabolism generates numerous compounds harbouring pharmaceutical activity. In plants, these compounds are typically formed by different and specialised cell types that have to interact constituting a metabolic process chain. This interactivity impedes biotechnological production of secondary compounds, because cell differentiation is suppressed under the conditions of a batch bio-fermenter. We present a novel strategy to address this limitation using a biomimetic approach, where we simulate the situation in a real tissue by a microfluidic chamber system, where plant cells can be integrated into a process flow. We show that walled cells of the plant model tobacco BY-2 can be successfully cultivated in this system and that physiological parameters (such as cell viability, mitotic index and division synchrony) can be preserved over several days. The microfluidic design allows to resolve dynamic changes of specific metabolites over different stages of culture development. These results serve as proof-of-principle that a microfluidic organisation of cultivated plant cells can mimic the metabolic flows in a real plant tissue.

Fabrication of polymeric microfluidic devices with tunable wetting behavior for biomedical applications.

We demonstrate the fabrication of microchannels with specific fluidic behavior due to micro- and/or nanostructures on the surfaces. With a combination of hot embossing and microthermoforming it is possible to produce microchannels with specific surface properties. These surface properties are highly dependent on the micro- and nanostructures embossed into the material. Different structure sizes and geometries where examined by contact angle measurements. Here the dependency of diameter and pitch of the structures on the contact angle is examined as well as the material impact. These results enable the fabrication of highly specific surfaces tunable to an application.

Diffusion- and convection-based activation of Wnt/ß-catenin signaling in a gradient generating microfluidic chip.

Stem cells and developing tissues respond to long-range signaling molecules (morphogens), by starting different nuclear programs that decide about the cell fate. Cells sense the local morphogen concentration and the shape of the gradient. We developed a two-chambered microfluidic chip to reproduce the in vivo situation under shear stress free conditions. The gradient is generated in the lower part of our device and recognized by cells grown in the upper part in the microchamber. We tested our device by activating the Wnt/ß-catenin signaling pathway in HeLa cells as proven by nuclear ß-catenin accumulation in response to the Wnt pathway activator 6-bromoindirubin-3'-oxime (BIO). Applying the same readout system to a recombinant Wnt3a and Dkk-1 bipolar gradient we demonstrate that our microfluidic chip is suitable for morphogens as well as small molecules. More interestingly, our microfluidic device is highly flexible. While the generated gradients are stable for several hours and reproducible, we can change the kind and the shape of the gradient actively on demand. We also can switch from diffusion- to convection-based transport, thus applying the morphogen gradient either in a polarized or non-polarized manner.

Microstructuring of multiwell plates for three-dimensional cell culture applications by ultrasonic embossing.

Since three-dimensional (3D) cell culture models better reflect tissues in vivo in terms of cell shape and microenvironment compared to conventional monolayer cultures, 3D tissue culture substrates gain more importance for a wide range of biological applications like drug discovery, toxicological studies, cancer and stem cell research. In this study we developed a method for the fabrication of 3D cell culture substrates in a multiwell plate format by microstructuring the bottom of 96-well cell culture plates using an ultrasonic embossing process. The resulting microstructured area consists of cubic microcavities in which adherent multicellular aggregates can be formed. We performed the biological evaluation of the system with the liver-derived human cell-line HepG2 and compared the novel substrate with a commercially available 3D culture system comprising porous alginate sponges. Metabolic activity (alamarBlue® reduction) and induction of four biotransformation enzymes (EROD, ECOD, UGT, SULT) were determined by fluorimetry or HPLC. Our results revealed that HepG2 cells in microstructured plates showed a higher mitochondrial activity, as well as enzyme activity of ECOD and UGT after treatment with an inducer when compared to cells cultured in alginate sponges at otherwise comparable conditions. Since we have modified standard cell culture plates, the obtained system is adaptable to automated screening and might be useful for all kinds of cultures including adult, progenitor and stem cells which need a 3D culture configuration to restore or maintain the differentiated status.