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Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.

The paper reports on the deposition of iridium oxide thin films by dc reactive magnetron sputtering and their characterization by surface analysis methods (X-ray diffraction, scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy) and electrochemical techniques (cyclic voltammetry, electrochemical impedance spectroscopy, and galvanostatic square waves). The sputtering process was investigated by applying the method of generic curves. An optimal combination of sputtering parameters has been established [40 standard cubic centimeters per minute (sccm) argon/8-12 sccm oxygen/40% effective pump power/100 W dc power/“cold” sputtering] that yields stable microporous amorphous films with a highly extended surface. These films have shown an excellent electrochemical reversibility in physiological saline solution with a “safe” potential range of −1.2 to 1.3 V vs. Ag/AgCl reference electrode, a charge delivery capacity of 90-95 mC/cm2, a constant impedance of 70-80 Ω in the frequency range of 100 kHz down to 0.5-1.0 Hz, and a quick response to bipolar current pulses with a safe charge per phase of 2 mC/cm2/ph. The demonstrated remarkable electrochemical characteristics combined with their established long-term mechanical stability and corrosion resistance allow one to recommend the use of sputtered iridium oxide films as an ideal electrode material for neural stimulation. © 2004 The Electrochemical Society. All rights reserved.

In industrial-scale biotechnological processes, the active control of the pH-value combined with the controlled feeding of substrate solutions (fed-batch) is the standard strategy to cultivate both prokaryotic and eukaryotic cells. On the contrary, for small-scale cultivations, much simpler batch experiments with no process control are performed. This lack of process control often hinders researchers to scale-up and scale-down fermentation experiments, because the microbial metabolism and thereby the growth and production kinetics drastically changes depending on the cultivation strategy applied. While small-scale batches are typically performed highly parallel and in high throughput, large-scale cultivations demand sophisticated equipment for process control which is in most cases costly and difficult to handle. Currently, there is no technical system on the market that realizes simple process control in high throughput. The novel concept of a microfermentation system described in this work combines a fiber-optic online-monitoring device for microtiter plates (MTPs)--the BioLector technology--together with microfluidic control of cultivation processes in volumes below 1 mL. In the microfluidic chip, a micropump is integrated to realize distinct substrate flow rates during fed-batch cultivation in microscale. Hence, a cultivation system with several distinct advantages could be established: (1) high information output on a microscale; (2) many experiments can be performed in parallel and be automated using MTPs; (3) this system is user-friendly and can easily be transferred to a disposable single-use system. This article elucidates this new concept and illustrates applications in fermentations of Escherichia coli under pH-controlled and fed-batch conditions in shaken MTPs.

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