An engineered mammalian band-pass network

Gene expression circuitries, which enable cells to detect precise levels within a morphogen concentration gradient, have a pivotal impact on biological processes such as embryonic pattern formation, paracrine and autocrine signalling, and cellular migration. We present the rational synthesis of a synthetic genetic circuit exhibiting band-pass detection characteristics. The components, involving multiply linked mammalian trans-activator and -repressor control systems, were selected and fine-tuned to enable the detection of ‘low-threshold' morphogen (tetracycline) concentrations, in which target gene expression was triggered, and a ‘high-threshold' concentration, in which expression was muted. In silico predictions and supporting experimental findings indicated that the key criterion for functional band-pass detection was the matching of componentry that enabled sufficient separation of the low and high threshold points. Using the circuitry together with a fluorescence-encoded target gene, mammalian cells were genetically engineered to be capable of forming a band-like pattern of differentiation in response to a tetracycline chemical gradient. Synthetic gene networks designed to emulate naturally occurring gene behaviours provide not only insight into biological processes, but may also foster progress in future tissue engineering, gene therapy and biosensing application

Resonant Photoelectron Diffraction with circularly polarized light

Resonant angle scanned x-ray photoelectron diffraction (RXPD) allows the determination of the atomic and magnetic structure of surfaces and interfaces. For the case of magnetized nickel the resonant L2 excitation with circularly polarized light yields electrons with a dichroic signature from which the dipolar part may be retrieved. The corresponding L2MM and L3MM Auger electrons carry different angular momenta since their source waves rotate the dichroic dipole in the electron emission patterns by distinct angles

Mechanism of Laser-induced Field Emission

We have measured electron energy distribution curves (EDCs) of the laser-induced field emission from a tungsten tip. Field emission from photo-excited nonequilibrium electron distributions were clearly observed, while no enhanced field emission due to optical electric fields appeared up to values of 1.3 V/nm. Thus, we experimentally confirm the emission mechanism. Simulated transient EDCs show that electron dynamics plays a significant role in the laser-induced field emission. The results should be useful to find optimal parameters for defining the temporal and spectral characteristics of electron pulses for many applications based on pulsed field emission.Comment: 4 pages 4 figures 1 table, submitted to Physical Review Letter

Laser-induced Field Emission from Tungsten Tip: Optical Control of Emission Sites and Emission Process

Field-emission patterns from a clean tungsten tip apex induced by femtosecond laser pulses have been investigated. Strongly asymmetric field-emission intensity distributions are observed depending on three parameters: (1) the polarization of the light, (2) the azimuthal and (3) the polar orientation of the tip apex relative to the laser incidence direction. In effect, we have realized an ultrafast pulsed field-emission source with site selectivity of a few tens of nanometers. Simulations of local fields on the tip apex and of electron emission patterns based on photo-excited nonequilibrium electron distributions explain our observations quantitatively. Electron emission processes are found to depend on laser power and tip voltage. At relatively low laser power and high tip voltage, field-emission after two-photon photo-excitation is the dominant process. At relatively low laser power and low tip voltage, photoemission processes are dominant. As the laser power increases, photoemission from the tip shank becomes noticeable.Comment: 12 pages, 12 figures, submitted to Physical Review

The Dynactin Complex Enhances the Speed of Microtubule-Dependent Motions of Adenovirus Both Towards and Away from the Nucleus

Unlike transport vesicles or organelles, human adenovirus (HAdV) directly binds to the microtubule minus end-directed motor dynein for transport to the nucleus. The dynein cofactor dynactin enhances nuclear transport of HAdV and boosts infection. To determine if dynactin has a specific role in cytoplasmic trafficking of incoming HAdV on microtubules, we used live cell spinning disc confocal microscopy at 25 Hz acquisition frequency and automated tracking of single virus particles at 20–50 nm spatial resolution. Computational dissection by machine-learning algorithms extracted specific motion patterns of viral trajectories. We found that unperturbed cells supported two kinds of microtubule-dependent motions, directed motions (DM) and fast drifts (FD). DM had speeds of 0.2 to 2 μm/s and run lengths of 0.4 up to 7 μm, while FD were slower and less extensive at 0.02 to 0.4 μm/s and 0.05 to 2.5 μm. Dynactin interference by overexpression of p50/dynamitin or a coiled-coil domain of p150/Glued reduced the speeds and amounts of both center- and periphery-directed DM but not FD, and inhibited infection. These results indicate that dynactin enhances adenovirus infection by increasing the speed and efficiency of dynein-mediated virus motion to the nucleus, and, surprisingly, also supports a hereto unknown motor activity for virus transport to the cell periphery

Corrugated single layer templates for molecules: From h -BN nanomesh to graphene based quantum dot arrays

Functional nano-templates enable self-assembly of otherwise impossible arrangements of molecules. A particular class of such templates is that of sp 2 hybridized single layers of hexagonal boron nitride or carbon (graphene) on metal supports. If the substrate and the single layer have a lattice mismatch, superstructures are formed. On substrates like rhodium or ruthenium these superstructures have unit cells with ∼3-nm lattice constant. They are corrugated and contain sub-units, which behave like traps for molecules or quantum dots, which are small enough to become operational at room temperature. For graphene on Rh(111) we emphasize a new structural element of small extra hills within the corrugation landscape. For the case of molecules like water it is shown that new phases assemble on such templates, and that they can be used as "nano-laboratories” where many individual processes are studied in parallel. Furthermore, it is shown that the h-BN/Rh(111) nanomesh displays a strong scanning tunneling microscopy-induced luminescence contrast within the 3 nm unit cell which is a way to address trapped molecules and/or quantum dot

Optical Control of Field-Emission Sites by Femtosecond Laser Pulses

We have investigated field emission patterns from a clean tungsten tip apex induced by femtosecond laser pulses. Strongly asymmetric modulations of the field emission intensity distributions are observed depending on the polarization of the light and the laser incidence direction relative to the azimuthal orientation of tip apex. In effect, we have realized an ultrafast pulsed field-emission source with site selectivity on the 10 nm scale. Simulations of local fields on the tip apex and of electron emission patterns based on photo-excited nonequilibrium electron distributions explain our observations quantitatively.Comment: 4 pages, submitted to Physical Review Letter

A quantitative method for separation of living hydra cells

We describe a rapid method for the isolation of large numbers of livingHydra cells of defined cell type in an isotonic cell medium (Gierer et al. 1972). Intact animals are enzymatically dissociated into a single cell suspension and the various cell types separated in less than one hour by counterflow centrifugation elutriation. Cell loss is minimal. RNA isolated from various fractions can be probed with cell type specific cDNA-clones

A synthetic low-frequency mammalian oscillator

Circadian clocks have long been known to be essential for the maintenance of physiological and behavioral processes in a variety of organisms ranging from plants to humans. Dysfunctions that subvert gene expression of oscillatory circadian-clock components may result in severe pathologies, including tumors and metabolic disorders. While the underlying molecular mechanisms and dynamics of complex gene behavior are not fully understood, synthetic approaches have provided substantial insight into the operation of complex control circuits, including that of oscillatory networks. Using iterative cycles of mathematical model-guided design and experimental analyses, we have developed a novel low-frequency mammalian oscillator. It incorporates intronically encoded siRNA-based silencing of the tetracycline-dependent transactivator to enable the autonomous and robust expression of a fluorescent transgene with periods of 26 h, a circadian clock-like oscillatory behavior. Using fluorescence-based time-lapse microscopy of engineered CHO-K1 cells, we profiled expression dynamics of a destabilized yellow fluorescent protein variant in single cells and real time. The novel oscillator design may enable further insights into the system dynamics of natural periodic processes as well as into siRNA-mediated transcription silencing. It may foster advances in design, analysis and application of complex synthetic systems in future gene therapy initiatives

Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanid