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The development of a DNA-based cell-responsive biohybrid interface that can be used for spatially confined release of molecular cargo is reported. To this end, tailored DNA–protein conjugates are designed as gatekeepers that can be specifically cleaved by matrix metalloproteases (MMPs), which are secreted by many cancer cells. These gatekeepers can be installed by DNA hybridization on the surface of mesoporous silica nanoparticles (MSNs). The MSNs display another orthogonal DNA oligonucleotide that can be exploited for site-selective immobilization on solid glass surfaces to yield micropatterned substrates for cell adhesion. Using the human fibrosarcoma cell line HT1080 that secretes MMPs, it is demonstrated that the biohybrid surface is specifically modified by the cells to release both MSN-bound gatekeeper proteins and the encapsulated cargo peptide KLA. In view of the enormously high modularity of the system presented here, this approach promising for applications in drug delivery, tissue engineering, or other areas of nanobiotechnology is considered.

A multiscale polydimethylsiloxane (PDMS) chip is presented, which provides an array of mesoscale open wells for cell culturing and, as unique feature, an array of microscale 1 mu m deep channels to fluidically connect neighboring wells. As demonstrated with SH-SY5Y cells, the small dimensions of the channels prevent migration of the cell soma but allow physical contacts established by the outgrowth of protoplasmic protrusions between cells in adjacent wells. Another important feature is the chip's mountability on solid substrates, such as glass. This enables the use of substrates previously patterned with biomolecules, as demonstrated by DNA-directed immobilization of proteins inside the reactor wells. Given the versatile addressability of cells, whether through surface-bound or inkjet-based administration of bioactive substances, it is believed that the reactor could be used for research in cell-cell communication networks, for example, in neurodegenerative diseases such as Alzheimer's disease.

We present a measurement cell that allows simultaneous measurement of second harmonic generation (SHG) and streaming potential (SP) at mineral-water interfaces with flat specimen that are suitable for non-linear optical (NLO) studies. The set-up directly yields SHG data for the interface of interest and can also be used to obtain information concerning the influence of flow on NLO signals from that interface. The streaming potential is at present measured against a reference substrate (PTFE). The properties of this inert reference can be independently determined for the same conditions. With the new cell, for the first time the SHG signal and the SP for flat surfaces have been simultaneously measured on the same surface. This can in turn be used to unambiguously relate the two observations for identical solution composition. The SHG test of the cell with a fluorite sample confirmed previously observed differences in NLO signal under flow vs. no flow conditions in sum frequency generation (SFG) investigations. As a second test surface, an inert ("hydrophobic") OTS covered sapphire-c electrolyte interface was studied to verify the zeta-potential measurements with the new cell. For this system we obtained combined zeta-potential/SHG data in the vicinity of the point of zero charge, which were found to be proportional to each other as expected. Furthermore, on the accessible time scales of the SHG measurements no effects of flow, flow velocity and stopped flow occurred on the interfacial water structure. This insensitivity to flow for the inert surface was corroborated by concomitant molecular dynamics simulations. Finally, the set-up was used for simultaneous measurements of the two properties as a function of pH in automated titrations with an oxidic surface. Different polarization combinations obtained in two separate titrations, yielded clearly different SHG data, while under identical conditions zeta-potentials were exactly reproduced. The polarization combination that is characteristic for dipoles perpendicular to the surface scaled with the zeta-potentials over the pH-range studied, while the other did not. The work provides an advanced approach for investigating liquid/surface interactions which play a major role in our environment. The set-up can be upgraded for SFG studies, which will allow more detailed studies on the chemistry and the water structure at a given interface, but also the combined study of specific adsorption including kinetics in combination with electrokinetics. Such investigations are crucial for the basic understanding of many environmental processes from aquatic to atmospheric systems.

Effects of radiographic contrast media (RCM) application were demonstrated in vitro and in vivo where the injection of RCM into the A. axillaris of patients with coronary artery disease was followed by a significant and RCM-dependent decrease of erythrocyte velocity in downstream skin capillaries. Another study in pigs revealed that the deceleration of erythrocytes coincided with a significant reduction of the oxygen partial pressure in the myocardium--supplied by the left coronary artery--after the administration of RCM into this artery. Further reports showed RCM dependent alterations of erythrocytes like echinocyte formation and exocytosis, sequestration of actin or band 3 and the buckling of endothelial cells coinciding with a formation of interendothelial fenestrations leading to areas devoid of endothelial cells. Key to morphological alterations of erythrocytes is the membrane cytoskeleton, which is linked to the band 3 in the erythrocyte membrane via the junctional complex. Fundamental observations regarding the cell biological and biochemical aspects of the structure and function of the cell membrane and the membrane cytoskeleton of erythrocytes have been reported. This review focuses on recent results gained, e.g., by advanced confocal laser scanning microscopy of different double-stained structural elements of the erythrocyte membrane cytoskeleton.

Different radiographic contrast media (RCM) were shown to induce morphological changes of blood cells (e.g. erythrocytes or thrombocytes) and endothelial cells. The echinocytic shape change of erythrocytes, particularly, affords alterations of the membrane cytoskeleton. The cytoskeleton plays a crucial role for the shape and deformability of the red blood cell. Disruption of the interaction between components of the red blood cell membrane cytoskeleton may cause a loss of structural and functional integrity of the membrane. In this study band4.9 and actin as components of the cytoskeletal junctional complex were examined in human erythrocytes after suspension in autologous plasma or in plasma RCM mixtures (30% v/v Iodixanol-320 or Iopromide-370) followed by a successive double staining with TRITC-/FITC-coupled monoclonal antibodies. After adding Iopromide-370 to the plasma in practically none of the cells the rounded conformation of the membrane cytoskeleton - as it appeared in cells suspended in autologous plasma - was found. In addition, Iopromide-370 induced thin lines and coarse knob-like structures of band4.9 at the cell periphery while most cell centers were devoid of band4.9, and a box-like arrangement of bands of band4.9. A dissociation between colours red (actin) and green (band4.9) occurred as well. In contrast, erythrocytes suspended in a plasma/Iodixanol-320 mixture showed a membrane cytoskeleton comparable to cells suspended in autologous plasma, Similar results were found with respect to the distribution of actin. This study revealed for the first time RCM-dependent differences in band4.9 activities as possible pathophysiological mechanism for the chemotoxicity of radiographic contrast media.

Epilithic lichen (Xanthoria elegans in Canada, Lecanora muralis in Germany) were gathered from 17 locations in Ontario, Canada (from Lake Ontario to James Bay) and 43 locations in Germany (from the Alps to the North Sea and from the Baltic Sea to the Erzgebirge). Sample aliquots were digested in nitric acid and trace elements were analyzed by inductively coupled plasma-mass spectrom-etry techniques. The organohalogens were determined as absorbable organic halogens (AOXs) by coulometry. Concentration ranges from Germany for trace element samples and AOXs were determined. The lichen showed high spatial resolution in their element enrichment patterns. This allowed for differentiation between natural and anthropogenic dominance in ambient air concentrations. This biomonitoring method has proven to be very sensitive, fast, and reliable. No clear relationship could be found between trace element and AOX concentrations. The AOX values may reflect individual metabolic rates of the fungal partner in liche...

In our previous work we described the preparation and characterization of spray dried hydroxyapatite micro granules loaded with 5-fluorouracil (5-FU). These loaded particles are used as a model drug delivery system (DDS). In this study we examined the in vitro response of two cell lines derived from different tissues to 5-FU loaded granules (LG). Both cell lines, either L929 cells of a mouse fibroblast lineage or cells originating from a rat osteosarcoma (ROS 17/2.8) showed a dose dependent decrease in cell proliferation in response to 5-FU-, either dissolved in the culture medium or loaded onto particles. The response of the two cell lines to loaded and nonloaded particles was different. The effect of LG and of a corresponding concentration of free 5-FU was practically the same for the ROS 17/2.8 cells indicating that ROS 17/2.8 cells were not affected by the carrier material. In contrast, L929 cells showed a slight decrease in cell proliferation also in the presence of granules not loaded with 5-FU. This is thought to be attributed to the inhibition of mitogenesis by phosphocitrates, already demonstrated in fibroblasts. In summary, we found that the loaded 5-FU kept its effectivity after the spray drying process and that the response towards the granules varied with cell type. This is the first step towards a tissue specific DDS.

The membrane of red blood cells consists of a phospholipid bilayer with embedded membrane proteins and is associated on the cytoplasmatic side with a network of proteins, the membrane skeleton. Band3 has an important role as centre of the functional complexes e.g. gas exchange complex and as element of attachment for the membrane skeleton maintaining membrane stability and flexibility. Up to now it is unclear if band3 is involved in the morphology change of red blood cells after contact with radiographic contrast media. The study revealed for the first time that Iopromide induced markedly more severe alterations of the membrane skeleton compared to Iodixanol whose effects were similar to erythrocytes suspended in autologous plasma. A remarkable clustering of band3 was found associated with an accumulation of band3 in spicules and also a sequestration of band3 to the extracellular space. This was evidently accompanied by a gross reduction of functional band3 complexes combined with a dissociation of spectrin from band3 leading to a loss of homogeneity of the spectrin network. It could be demonstrated for the first time that RCM not only induced echinocyte formation but also exocytosis of particles at least coated with band3.

Red blood cells demonstrate a unique ability for repeated large deformation. Under the influence of a variety of agents, shapes other than the discocyte - e.g. stomatocytes or echinocytes - can be observed. Some radiographic agents induce shape changes from discocytic to echinocytic cells. Especially the echinocyte formation is associated with a rigidification of the cells bearing the risk of a hindered capillary passage of the echinocytes. The mechanisms leading to the formation of echinocytes are not well understood assuming that the membrane cytoskeleton is a key player. That is why this examination was focused on the participation of components of the membrane cytoskeleton in the formation of echinocytes and the protrusions accompanying the formation of echinocytes. Two radiographic contrast media approved for intra-arterial application were used to study echinocyte formation (Iodix- anol320; Iopromide370). In the in vitro study serious changes in the membrane cytoskeleton were only found in those erythrocytes incubated in plasma supplemented with Iopromide370 (30%v/v). The shape of the spectrin net was completely altered; from the more homogeneous distribution - typical of cells in autologous plasma and also of cells in plasma supplemented with Iodixanol320 - to a distribution of spectrin concentrated in the membrane-near regions with the appearance of spectrin-actin co-localization. Co-localized spectrin with actin was also found around the membranous roots of protrusions which resemble exocytotic processes. In central parts of the cells there was a pronounced dissociation of spectrin and actin; green coloured condensed spectrin bundles originating from the cell membrane reached up to the root of the protrusions. Separate from this there were also fine long actin fibres passing through the whole cell. The incubation of erythrocytes in plasma supplemented with Iopromide370 induced rounded bubble-like protrusions from the cell membrane containing almost completely long bundles of actin fibres. The examination confirmed earlier studies showing that some radiographic contrast media are able to induce echinocyte formation. Furthermore, subcellular mechanisms were revealed explaining the different effects of Iodixanol in comparison to Iopromide.

Patterned two-component, self-assembled monolayers on gold were produced by UV lithography. An oligo(ethylene glycol) terminated disulfide served as inert matrix reducing unspecific protein adsorption and cell adhesion. The second component of the self-assembled monolayer (SAM) presented a benzylguanine moiety for the immobilization of Sonic hedgehog (Shh) fused to a mutant O6-alkylguanine-DNA alkyltransferase (SNAP-tag™). The enzymatic activity of the SNAP-tag allows selective and covalent immobilization of the linked Shh. Time-of-flight secondary ion mass spectrometry verified the correct lateral distribution of the benzylguanine head groups in the patterned SAM. The quantification of unspecific and specific protein binding to mixed SAMs showed increased adsorption of albumin with increasing benzylguanine/(ethylene glycol) ratios. However, the immobilization of SNAP-tagged Shh was not blocked by pre-adsorbed albumin. Furthermore, the obtained micro-patterned substrates permitted direct immobilization of SNAP-tagged Shh even in the presence of many competing proteins from conditioned media of transfected HEK293 cells. Therefore, the presented system is suited for the controlled immobilization of fusion proteins from complex mixtures avoiding purification steps.