Samuel C. Hess

An efficient strategy for TALEN-mediated genome engineering in Drosophila

In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generation.

Guiding of Electrons in a Few-Mode Ballistic Graphene Channel.

In graphene, the extremely fast charge carriers can be controlled by electron-optical elements, such as waveguides, in which the transmissivity is tuned by the wavelength. In this work, charge carriers are guided in a suspended ballistic few-mode graphene channel, defined by electrostatic gating. By depleting the channel, a reduction of mode number and steps in the conductance are observed, until the channel is completely emptied. The measurements are supported by tight-binding transport calculations including the full electrostatics of the sample.

Template-Particle Stabilized Bicontinuous Emulsion Yielding Controlled Assembly of Hierarchical High-Flux Filtration Membranes

A novel solvent-evaporation-based process that exploits template-particle stabilized bicontinuous emulsions for the formation of previously unreached membrane morphologies is reported in this article. Porous membranes have a wide range of applications spanning from water filtration, pharmaceutical purification, and battery separators to scaffolds for tissue engineering. Different situations require different membrane morphologies including various pore sizes and pore gradients. However, most of the previously reported membrane preparation procedures are restricted to specific morphologies and morphology alterations require an extensive optimization process. The tertiary system presented in this article, which consists of a poly(ether sulfone)/dimethylacetamide (PES/DMAc) solution, glycerol, and ZnO-nanoparticles, allows simple and exact tuning of pore diameters ranging from sub-20 nm, up to 100 nm. At the same time, the pore size gradient is controlled from 0 up to 840%/μm yielding extreme asymmetry. In addition to structural analysis, water flux rates of over 5600 L m(-2) h(-1) are measured for membranes retaining 45 nm silica beads.

Fabrication of ballistic suspended graphene with local-gating

Abstract Herein we discuss the fabrication of ballistic suspended graphene nanostructures supplemented with local gating. Using in situ current annealing, we show that exceptional high mobilities can be obtained in these devices. A detailed description is given of the fabrication of bottom and different top-gate structures, which enable the realization of complex graphene structures. We have studied the basic building block, the p-n junction in detail, where a striking oscillating pattern was observed, which can be traced back to Fabry–Perot oscillations that are localized in the electronic cavities formed by the local gates. Finally we show some examples how the method can be extended to incorporate multi-terminal junctions or shaped graphene. The structures discussed here enable the access to electron-optics experiments in ballistic graphene.

Hollow Silica as an Optically Transparent and Thermally Insulating Polymer Additive

We present an improved synthesis route to hollow silica particles starting from tetramethyl orthosilicate (TMOS) instead of the traditionally used ethyl ester. The silica was first deposited onto polystyrene (PS) particles that were later removed. The here introduced, apparently minor modification in synthesis, however, allowed for a very high purity material. The improved, low density hollow silica particles were successfully implemented into polymer films and permitted maintaining optical transparency while significantly improving the heat barrier properties of the composite. Mechanistic investigations revealed the dominant role of here used methanol as a cosolvent and its role in controlling the hydrolysis rate of the silicic ester, and subsequent formation of hollow silica particles. Systematic experiments using various reaction parameters revealed a transition between regions of inhomogeneous material production at fast hydrolysis rate and reliable silica deposition on the surface of PS as a core-shell structured particle. The shell-thickness was controlled from 6.2 to 17.4 nm by increasing TMOS concentration and the diameter from 95 to 430 nm through use of the different sizes of PS particles. Hollow silica particle with the shell-thickness about 6.2 nm displayed a high light transmittance intensity up to 95% at 680 nm (length of light path ∼ 1 cm). Polyethersulfone (PES)/hollow silica composite films (35 ± 5 μm thick) exhibited a much lower thermal conductivity (0.03 ± 0.005 W m·K(-1)) than pure polymer films. This indicates that the prepared hollow silica is able to be used for cost and energy effective optical devices requiring thermal insulation.

Magnetic superbasic proton sponges are readily removed and permit direct product isolation.

Workup in organic synthesis can be very time-consuming, particularly when using reagents with both a solubility similar to that of the desired products and a tendency not to crystallize. In this respect, reactions involving organic bases would strongly benefit from a tremendously simplified separation process. Therefore, we synthesized a derivative of the superbasic proton sponge 1,8-bis(dimethylamino)naphthalene (DMAN) and covalently linked it to the strongest currently available nanomagnets based on carbon-coated cobalt metal nanoparticles. The immobilized magnetic superbase reagent was tested in Knoevenagel- and Claisen-Schmidt-type condensations and showed conversions of up to 99%. High yields of up to 97% isolated product could be obtained by simple recrystallization without using column chromatography. Recycling the catalyst was simple and fast with an insignificant decrease in catalytic activity.

Direct synthesis of carbon quantum dots in aqueous polymer solution: one-pot reaction and preparation of transparent UV-blocking films

Carbon quantum dots (CQDs) were characterized and their synthesis optimized extensively over recent years. It is becoming more and more important to develop synthesis solutions for CQDs applications to allow simple and cheap applications with composite materials. Here, we present a one-pot CQDs reaction in an aqueous polyvinyl alcohol (PVA) solution and the synthesis of transparent UV protection films. The synthesis solution was tuned in a way that transparent UV-protection films cast from unpurified CQDs–PVA solutions could be obtained without the need of yield consuming purification steps. Measuring the impact of varying PVA, branched polyethylene imine (b-PEI), and citric acid (CA) concentrations on the solutions and films UV/Vis transmission and absorbance allowed elucidating important factors influencing the solution to film property transition. Applying CQDs–PVA films on commercially available PET bottle films resulted in high transparency of the composite film in the visible range (>80%) and UV absorption of over 90%. Lastly, our CQDs–PVA film performed stable UV-blocking and did not show any bleaching effects over several days of UV-exposure.

Tissue mechanics of piled critical size biomimetic and biominerizable nanocomposites: Formation of bioreactor-induced stem cell gradients under perfusion and compression

BACKGROUND Perfusion bioreactors are used to solve problems in critical size bone tissue engineering. Biominerizable and biocompatible nanocomposites are suitable scaffold materials for this purpose because they offer mineral components in organic carriers. Human adipose derived stem cells (ASCs) can potentially be used to increase bone healing. MATERIALS AND METHODS Electrospun nanocomposite disks of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/a-CaP) were seeded with ASCs and eight disks were stacked in a bioreactor running with normal culture. Under perfusion and uniaxial cyclic compression, load-displacement curves as a function of time were assessed. Stiffness and energy dissipation were recorded. Moreover, stem cell densities in the layers of the piled scaffold were determined as well as their morphologies and differentiation status. RESULTS While the stiffness of the cell free constructs increased over time based on the transformation of the a-CaP nanoparticles into flake-like apatite, ASC-seeded constructs showed a constant stiffness. Stem cell density gradients had a linear increase from the bottom to the top of the pile (r(2)>0.95). Stem cells were getting more roundish at higher flow rates. Some osteogenesis was found upon osteopontin immunostaining, while no endothelial cell differentiation and no chondrogenesis was triggered. CONCLUSIONS The fabrication of a critical size bone graft is presented based on a biominerizable bone-biomimetic nanocomposite with preserved stiffness when seeded with ASCs. The cell densities of ASCs inside the piled construct varied with a linear gradient. Beginning osteogenesis was triggered by the dynamic culture conditions including perfusion and compression.

A Bioinspired Ultraporous Nanofiber‐Hydrogel Mimic of the Cartilage Extracellular Matrix

A true biomimetic of the cartilage extracellular matrix (ECM) could greatly contribute to our ability to regenerate this tissue in a mechanically demanding, often inflamed environment. Articular cartilage is a composite tissue made of cells and fibrillar proteins embedded in a hydrophilic polymeric meshwork. Here, a polyanionic functionalized alginate is used to mimic the glycosaminoglycan component of the native ECM. To create the fibrillar component, cryoelectrospinning of poly(ε-caprolactone) on a -78 °C mandrel, subsequently treated by O2 plasma, is used to create a stable, ultraporous and hydrophillic nanofiber network. In this study, cell-laden, fiber-reinforced composite scaffolds thicker than 1.5 mm can be created by infiltrating a chondrocyte/alginate solution into the fiber mesh, which is then physically cross-linked. The fibrillar component significantly reinforces the chondroinductive, but mechanically weak sulfated alginate hydrogels. This allows the production of a glycosaminoglycan- and collagen type II-rich matrix by the chondrocytes as well as survival of the composite in vivo. To further enhance the system, the electrospun component is loaded with dexamethasone, which protected the cells from an IL-1β-mediated inflammatory insult.

Effects of seeding adipose-derived stem cells on electrospun nanocomposite used as chest wall graft in a murine model

OBJECTIVES Malignant neoplasms infiltrating the chest wall often requires resection of the thoracic wall. To replace the defect, Gore-Tex® is usually employed as the gold standard material, however, Gore-Tex® is inert and not degradable. Novel materials are nowadays available which allow a full bio-integration due to their non-toxic degradability. Additionally, stem cell seeding has the capacity to reduce inflammatory response towards such grafts, thus integrating it better into the host organism.