Chemical communication between bacteria and cell-free gene expression systems within linear chains of emulsion droplets.

Position-dependent gene expression in gradients of morphogens is one of the key processes involved in cellular differentiation during development. Here, we study a simple artificial differentiation process, which is based on the diffusion of genetic inducers within one-dimensional arrangements of 50 µm large water-in-oil droplets. The droplets are filled with either bacteria or cell-free gene expression systems, both equipped with genetic constructs that produce inducers or respond to them via expression of a fluorescent protein. We quantitatively study the coupled diffusion-gene expression process and demonstrate that gene expression can be made position-dependent both within bacteria-containing and cell-free droplets. By generating diffusing quorum sensing signals in situ, we also establish communication between artificial cell-free sender cells and bacterial receivers, and vice versa.

Microdialysis feasibility investigations with the non-hydrophilic antifungal voriconazole for potential applications in nonclinical and clinical settings.

OBJECTIVE: This study aimed at investigating the feasibility and validity of the microdialysis technique for the non-hydrophilic antifungal voriconazole, in settings for established concentric and new linear catheters. Optimal conditions for application of microdialysis in in vivo studies including steady-state conditions were to be elaborated. MATERIALS AND METHODS: For in vitro microdialysis investigations, a robust and easy-to-handle system was developed permitting standardized physiological-like conditions. Various experiments on the influence of flow-rate (0.4 - 10.0 µl/min), voriconazole concentration (1.0 - 50.0 µg/ml) on relative recovery were performed. Additionally, the mass transfer coefficient r of voriconazole was estimated (WinNonlin™). RESULTS: In vitro microdialysis experiments suggested sufficient voriconazole concentrations in the dialysate for (non-)clinical investigations. The stability of voriconazole was confirmed over 10 h (37 °C). A flow-rate dependency was shown (optimum: 2.0 µl/min) and r was estimated to be 0.09 mm/min and 0.11 mm/min for concentric and linear catheters, respectively. Relative recovery in delivery/recovery experiments was independent of the voriconazole concentration ranging from 96.0% to 97.8%/93.8% to 100.2% (CV 1.7%/ 0.5%) indicating unhampered passage of voriconazole through the catheter. Investigations mimicking steady-state suggested voriconazole concentration of 100 - 200 µg/ml for in vivo catheter calibration solutions. CONCLUSION: The results demonstrate the feasibility and validity of the microdialysis technique in clinically-mimicked settings despite the higher lipophilicity of voriconazole and support the application of the technique in vivo.

Assembly and melting of DNA nanotubes from single-sequence tiles.

DNA melting and renaturation studies are an extremely valuable tool to study the kinetics and thermodynamics of duplex dissociation and reassociation reactions. These are important not only in a biological or biotechnological context, but also for DNA nanotechnology which aims at the construction of molecular materials by DNA self-assembly. We here study experimentally the formation and melting of a DNA nanotube structure, which is composed of many copies of an oligonucleotide containing several palindromic sequences. This is done using temperature-controlled UV absorption measurements correlated with atomic force microscopy, fluorescence microscopy and transmission electron microscopy techniques. In the melting studies, important factors such as DNA strand concentration, hierarchy of assembly and annealing protocol are investigated. Assembly and melting of the nanotubes are shown to proceed via different pathways. Whereas assembly occurs in several hierarchical steps related to the formation of tiles, lattices and tubes, melting of DNA nanotubes appears to occur in a single step. This is proposed to relate to fundamental differences between closed, three-dimensional tube-like structures and open, two-dimensional lattices. DNA melting studies can lead to a better understanding of the many factors that affect the assembly process which will be essential for the assembly of increasingly complex DNA nanostructures.

In vivo efficacy and pharmacokinetics of voriconazole in an animal model of dermatophytosis.

The standard treatment for tinea capitis caused by Microsporum species for many years has been oral griseofulvin, which is no longer universally marketed. Voriconazole has been demonstrated to inhibit growth of Microsporum canis in vitro. We evaluated the efficacy and tissue pharmacokinetics of oral voriconazole in a guinea pig model of dermatophytosis. Guinea pigs (n = 16) were inoculated with M. canis conidia on razed skin. Voriconazole was dosed orally at 20 mg/kg/day for 12 days (days 3 to 14). The guinea pigs were scored clinically (redness and lesion severity) and mycologically (microscopy and culture) until day 17. Voriconazole concentrations were measured day 14 in blood, skin biopsy specimens, and interstitial fluid obtained by microdialysis in selected animals. Clinically, the voriconazole-treated animals had significantly less redness and lower lesion scores than untreated animals from days 7 and 10, respectively (P < 0.05). Skin scrapings from seven of eight animals in the voriconazole-treated group were microscopy and culture negative in contrast to zero of eight animals from the untreated group at day 14. The colony counts per specimen were significantly higher in samples from untreated animals (mean colony count of 28) than in the voriconazole-treated animals (<1 in the voriconazole group [P < 0.0001]). The voriconazole concentration in microdialysate (unbound) ranged from 0.9 to 2.0 microg/ml and in the skin biopsy specimens total from 9.1 to 35.9 microg/g. In conclusion, orally administered voriconazole leads to skin concentrations greater than the necessary MICs for Microsporum and was shown to be highly efficacious in an animal model of dermatophytosis. Voriconazole may be a future alternative for treatment of tinea capitis in humans.

Kinetics of protein-release by an aptamer-based DNA nanodevice.

A recently introduced DNA nanodevice can be used to selectively bind or release the protein thrombin triggered by DNA effector strands. The release process is not well described by simple first or second order reaction kinetics. Here, fluorescence resonance energy transfer and fluorescence correlation spectroscopy experiments are used to explore the kinetics of the release process in detail. To this end the influence of concentration variations and also of temperature is determined. The relevant kinetic parameters are extracted from these experiments and the kinetic behavior of the system is simulated numerically using a set of rate equations. The hydrodynamic radii of the aptamer device alone and bound to thrombin are determined as well as the dissociation constant for the aptamer device-thrombin complex. The results from the experiments and a numerical simulation support the view that the DNA effector strand first binds to the aptamer device followed by the displacement of the protein.

DNA fuel for free-running nanomachines.

We describe kinetic control of DNA hybridization: loop complexes are used to inhibit the hybridization of complementary oligonucleotides; rationally designed DNA catalysts are shown to be effective in promoting their hybridization. This is the basis of a strategy for using DNA as a fuel to drive free-running artificial molecular machines.

Using DNA to construct and power a nanoactuator.

A DNA-based molecular machine is described which has two movable arms that are pushed apart when a strand of DNA, the fuel strand, hybridizes with a single-stranded region of the molecular machine. Through the process of branch migration, a second strand of DNA complementary to the fuel strand is able to remove the fuel strand from the molecular machine, restoring it to its original configuration. Compared with the molecular tweezers we had previously devised, this machine, which we call a nanoactuator, has a reduced tendency to form dimers.

A DNA-fuelled molecular machine made of DNA.

Molecular recognition between complementary strands of DNA allows construction on a nanometre length scale. For example, DNA tags may be used to organize the assembly of colloidal particles, and DNA templates can direct the growth of semiconductor nanocrystals and metal wires. As a structural material in its own right, DNA can be used to make ordered static arrays of tiles, linked rings and polyhedra. The construction of active devices is also possible--for example, a nanomechanical switch, whose conformation is changed by inducing a transition in the chirality of the DNA double helix. Melting of chemically modified DNA has been induced by optical absorption, and conformational changes caused by the binding of oligonucleotides or other small groups have been shown to change the enzymatic activity of ribozymes. Here we report the construction of a DNA machine in which the DNA is used not only as a structural material, but also as 'fuel'. The machine, made from three strands of DNA, has the form of a pair of tweezers. It may be closed and opened by addition of auxiliary strands of 'fuel' DNA; each cycle produces a duplex DNA waste product.