8 research outputs found
Chemical Modification Strategies for the Preparation of Bioactive Interfaces
Biomimetic systems and interfaces allow to understand and control cellular behavior in a well defined and reproducible manner. In this study three different strategies are developed to prepare such simplified, well-defined biomimetic materials. Firstly, a combination of click chemistry and gold thiol interactions allows the presentation of two distinct signaling molecules at controlled density and arrangement to investigate the cross-talk between two signaling molecules in cell culture. Secondly, the commonly used Ni2+-NTA interaction with His6- tagged proteins is substantially improved in its stability and inertness for protein immobilization on SAMs by replacing the Ni2+ ions with Co3+ in the complex. Thirdly, His6-tagged proteins are stably tethered on TiO2 nanoparticles
for targeted delivery.
To produce dual functionalized gold nanostructured interfaces, first the presentation of a ligand of interest with azide functionality on glass substrates at controlled density is established. For this pirpose, alkyne terminated poly(ethylene glycol) (PEG) is covalently bound to glass through a silanization reaction and subsequently modified through copper catalyzed azide alkyne cycloaddition (CuAAC). The functionalization density can be statistically tuned through the coimmobilization of a methoxy-terminated PEG. The surface coating and its modification with the CuAAC is analyzed using fluorescence microscopy, XPS, an enzymatic digestion assay for the determination of the ligand density, QCM-D and in cell adhesion studies. This PEG coating is used in combination with the established gold nanostructured surfaces to generate orthogonally dual functionalized biomimetic interfaces where one of the ligands is attached to the PEG coating between the gold nanoparticles using the CuAAC and the second ligand is attached to the gold nanoparticles using the gold thiol interaction. These interfaces, which present two distinct ligands at controlled density and arrangement, are suitable to investigate the mutual influence of two signaling molecules on cell behavior. Exemplarily, the combined effect of the adhesion peptide cRGD and the synergy site PHSRN on REF fibroblast adhesion is investigated. While on neither of the monofunctionalized substrates the cells can attach, the cells adhere on the dual functionalized cRGD and PHSRN presenting interfaces.
The second part of this study deals with the stable immobilization of His-tagged proteins on NTA presenting surfaces using the cobalt(III) mediated interaction. The cobalt(III) complex is generated by first preforming the well established cobalt(II) complex between NTA and His6-tagged proteins and the subsequent chemical oxidation of Co2+ to Co3+ with hydrogen peroxide. A comparison of the Ni(II) and Co(III) mediated interaction between NTA moieties and His6-GFP reveals the lability of the Ni(II) and stability of the Co(III) complexes against high concentrations of competing ligands and washing off overtime. Further, also the resistance of the Co(III) mediated interaction against reducing agents is demonstrated. The oxidation step in this immobilization strategy can potentially harm the protein’s activity and this has to be investigated case by case. To illustrate that this method can be used to immobilize functional protein, the His6-tagged protein A is immobilized through the Co(III) mediated interaction and it is shown that the oxidation step dosen’t influence the immunoglobulin binding activity.
In the third part the Co(III) mediated stable immobilization of His-tagged proteins is used to biofunctionalize TiO2 nanoparticles. Here, the photocatalytic activity of TiO2 is taken advantage of to perform the oxidation of Co(II) complexes between the chelating TETT surface coating on the TiO2 nanoparticles and a His-tagged protein. The Co2+ ion loading capacity of the nanoparticles and their photocatalytic activity is characterized with a colorimetric assay, fluorescence studies using terephtahlic acid as radical detection reagent, absorbance measurements, DLS and zeta potential measurements proving the photo-mediated oxidation of coordinated Co2+ ions to Co3+. Exemplarily, the stable immobilization of the model protein His6-GFP and of the glycoprotein transferrin-His6 is studied
Pregabalin Add-On vs. Dose Increase in Levetiracetam Add-On Treatment: A Real-Life Trial in Dogs With Drug-Resistant Epilepsy
Epilepsy is a common neurological disorder affecting 0.6–0.75% of dogs in veterinary practice. Treatment is frequently complicated by the occurrence of drug-resistant epilepsy and cluster seizures in dogs with idiopathic epilepsy. Only few studies are available to guide treatment choices beyond licensed veterinary drugs. The aim of the study was to compare antiseizure efficacy and tolerability of two add-on treatment strategies in dogs with drug-resistant idiopathic epilepsy. The study design was a prospective, open-label, non-blinded, comparative treatment trial. Treatment success was defined as a 3-fold extension of the longest baseline interseizure interval and to a minimum of 3 months. To avoid prolonged adherence to a presumably ineffective treatment strategy, dog owners could leave the study after the third day with generalized seizures if the interseizure interval failed to show a relevant increase. Twenty-six dogs (mean age 5.5 years, mean seizure frequency 4/month) with drug-resistant idiopathic epilepsy and a history of cluster seizures were included. Dogs received either add-on treatment with pregabalin (PGB) 4 mg/kg twice daily (14 dogs) or a dose increase in levetiracetam (LEV) add-on treatment (12 dogs). Thirteen dogs in the PGB group had drug levels within the therapeutic range for humans. Two dogs in the PGB group (14.3%; 2/14) and one dog in the LEV group (8.3%; 1/12) achieved treatment success with long seizure-free intervals from 122 to 219 days but then relapsed to their early seizure frequency 10 months after the study inclusion. The overall low success rates with both treatment strategies likely reflect a real-life situation in canine drug-resistant idiopathic epilepsy in everyday veterinary practice. These results delineate the need for research on better pharmacologic and non-pharmacologic treatment strategies in dogs with drug-resistant epilepsy
Pregabalin Add-On vs. Dose Increase in Levetiracetam Add-On Treatment: A Real-Life Trial in Dogs With Drug-Resistant Epilepsy
Epilepsy is a common neurological disorder affecting 0.6–0.75% of dogs in veterinary practice. Treatment is frequently complicated by the occurrence of drug-resistant epilepsy and cluster seizures in dogs with idiopathic epilepsy. Only few studies are available to guide treatment choices beyond licensed veterinary drugs. The aim of the study was to compare antiseizure efficacy and tolerability of two add-on treatment strategies in dogs with drug-resistant idiopathic epilepsy. The study design was a prospective, open-label, non-blinded, comparative treatment trial. Treatment success was defined as a 3-fold extension of the longest baseline interseizure interval and to a minimum of 3 months. To avoid prolonged adherence to a presumably ineffective treatment strategy, dog owners could leave the study after the third day with generalized seizures if the interseizure interval failed to show a relevant increase. Twenty-six dogs (mean age 5.5 years, mean seizure frequency 4/month) with drug-resistant idiopathic epilepsy and a history of cluster seizures were included. Dogs received either add-on treatment with pregabalin (PGB) 4 mg/kg twice daily (14 dogs) or a dose increase in levetiracetam (LEV) add-on treatment (12 dogs). Thirteen dogs in the PGB group had drug levels within the therapeutic range for humans. Two dogs in the PGB group (14.3%; 2/14) and one dog in the LEV group (8.3%; 1/12) achieved treatment success with long seizure-free intervals from 122 to 219 days but then relapsed to their early seizure frequency 10 months after the study inclusion. The overall low success rates with both treatment strategies likely reflect a real-life situation in canine drug-resistant idiopathic epilepsy in everyday veterinary practice. These results delineate the need for research on better pharmacologic and non-pharmacologic treatment strategies in dogs with drug-resistant epilepsy
Dual-Functionalized Nanostructured Biointerfaces by Click Chemistry
The
presentation of biologically active molecules at interfaces
has made it possible to investigate the responses of cells to individual
molecules in their matrix at a given density and spacing. However,
more sophisticated methods are needed to create model surfaces that
present more than one molecule in a controlled manner in order to
mimic at least partially the complexity given in natural environments.
Herein, we present dual-functionalized surfaces combining quasi-hexagonally
arranged gold nanoparticles with defined spacings and a newly developed
PEG-alkyne coating to functionalize the glass in the intermediate
space. The PEG-alkyne coating provides an inert background for cell
interactions but can be modified orthogonally to the gold nanoparticles
with numerous azides, including spectroscopically active molecules,
peptides, and biotin at controlled densities by the copperÂ(I)-catalyzed
azide alkyne click reaction. The simultaneous presentation of cRGD
on the gold nanoparticles with 100 nm spacing and synergy peptide
PHSRN in the space between has a striking effect on REF cell adhesion;
cells adhere, spread, and form mature focal adhesions on the dual-functionalized
surfaces, whereas cells cannot adhere on either monofunctional surface.
Combining these orthogonal functionalization methods creates a new
platform to study precisely the crosstalk and synergy between different
signaling molecules and clustering effects in ligand–receptor
interactions
Cobalt Cross-Linked Redox-Responsive PEG Hydrogels: From Viscoelastic Liquids to Elastic Solids
We describe cobalt
cross-linked redox-responsive 4-arm histidine-modified
PEG (4A-PEG-His) hydrogels, which can be switched from self-healing
viscoelastic liquids to form stable elastic solids through a simple
oxidation step from Co<sup>2+</sup> to Co<sup>3+</sup>. The dramatic
change in gel properties is quantified in rheological measurements
and is associated with the altered ligand exchange rate of the cross-linking
cobalt ions. While Co<sup>2+</sup> forms kinetically labile coordination
bonds with low thermodynamic stability, Co<sup>3+</sup> forms kinetically
inert and highly stable coordination bonds. Unlike the Co<sup>2+</sup> cross-linked hydrogels, the Co<sup>3+</sup> cross-linked hydrogels
do not dissolve in buffer and swell overtime, where they remain intact
longer with increasing gel connectivity, increasing polymer concentration
and decreasing temperature. Remarkably, these gels can even resist
the strong chelator EDTA and withstand both low and high pH due to
the low ligand exchange rates in the primary coordination sphere.
Overall, the Co<sup>2+/3+</sup> redox pair provides an attractive
platform to produce redox-responsive materials with big deviations
in mechanical and chemical properties
Geological Mapping of the Ac-H-11 Sintana Quadrangle of Ceres from NASA’s Dawn Mission.
In December 2015, the Dawn spacecraft delivered the first images of the Low Altitude Mapping Orbit (LAMO)
of the dwarf planet Ceres at a resolution of 35 m/pixel. This data will be used to finish the geological mapping
of Ceres’ surface in order to identify composition and surface forming processes. Mapping was already done
using Survey Orbit and High Altitude Mapping Orbit (HAMO) data. With the new images, an updated map will
be presented. To this point, the data material consists of a HAMO clear-filter mosaic (140 m/pixel) [1], a digital
elevation model (DTM) [2] derived from Survey orbit (415 m/pixel) data, color-filter ratios and photometrically
corrected images.
Ceres’ surface has been divided into 15 mapping quadrangles. The Ac-H-11 Sintana quadrangle is located in the
southern hemisphere of Ceres between 21 66�S and 0 90�E. Geological units identified so far are cratered terrain,
which covers most of the area, and a younger unit of relatively smooth material. The latter is characterized by a
low crater density. Material of the same unit was found in adjacent quadrangles as well. Interest is taken in the
diversity of crater shapes. Many craters show different forms of asymmetries. One and the same crater for instance
displays different stages of rim degradation and some crater walls are partly terraced and their slopes’ steepness
is varying alongside the crater rim. Several mass wasting features, which partly cause the observed asymmetries,
have been identified. Next to the multiple collapsed rims, landslides due to later cratering on the primary crater rim
are observed. Whereas collapse structures are mostly blocky, single landslides are characterized by lobate margins.
Occurrence and type of mass wasting feature might hint to subsurface differences. Further, there is a diversity of
inner crater structures, like relaxed crater floors, ridges, central peaks, mounds and smooth plains. Processes like
mass wasting and relaxation have modified many craters. Complex craters for instance often lack central peaks.
Color composite images allow us to identify fresh craters and accompanying ejecta, even though most craters lack
visible ejecta blankets. Secondary crater chains are spread over most of the area, but we were not able to identify
primary craters. On the contrary, at current resolution the Sintana quadrangle lacks linear structures that are of
tectonic origin. LAMO data will help to refine unit boundaries and to distinguish linear structures.
References: [1] Roatsch T. et al. (2015) Planetary and Space Science, in press. [2] Preusker et al. (2016) LPSC
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