Cluster size dependence of high-order harmonic generation

We investigate high-order harmonic generation (HHG) from noble gas clusters in a supersonic gas jet. To identify the contribution of harmonic generation from clusters versus that from gas monomers, we measure the high-order harmonic output over a broad range of the total atomic number density in the jet (from 3*10^16 cm^{-3} to 3x10^18 cm{-3}) at two different reservoir temperatures (303 K and 363 K). For the firrst time in the evaluation of the harmonic yield in such measurements, the variation of the liquid mass fraction, g, versus pressure and temperature is taken into consideration, which we determine, reliably and consistently, to be below 20% within our range of experimental parameters. By comparing the measured harmonic yield from a thin jet with the calculated corresponding yield from monomers alone, we find an increased emission of the harmonics when the average cluster size is less than 3000. Using g, under the assumption that the emission from monomers and clusters add up coherently, we calculate the ratio of the average single-atom response of an atom within a cluster to that of a monomer and find an enhancement of around 10 for very small average cluster size (~200). We do not find any dependence of the cut-off frequency on the composition of the cluster jet. This implies that HHG in clusters is based on electrons that return to their parent ions and not to neighbouring ions in the cluster. To fully employ the enhanced average single-atom response found for small average cluster sizes (~200), the nozzle producing the cluster jet must provide a large liquid mass fraction at these small cluster sizes for increasing the harmonic yield. Moreover, cluster jets may allow for quasi-phase matching, as the higher mass of clusters allows for a higher density contrast in spatially structuring the nonlinear medium.Comment: 16 pages, 6 figure

Single-shot fluctuations in waveguided high-harmonic generation

For exploring the application potential of coherent soft x-ray (SXR) and extreme ultraviolet radiation (XUV) provided by high-harmonic generation, it is important to characterize the central output parameters. Of specific importance are pulse-to-pulse (shot-to-shot) fluctuations of the high-harmonic output energy, fluctuations of the direction of the emission (pointing instabilities), and fluctuations of the beam divergence and shape that reduce the spatial coherence. We present the first single-shot measurements of waveguided high-harmonic generation in a waveguided (capillary-based) geometry. Using a capillary waveguide filled with Argon gas as the nonlinear medium, we provide the first characterization of shot-to-shot fluctuations of the pulse energy, of the divergence and of the beam pointing. We record the strength of these fluctuations vs. two basic input parameters, which are the drive laser pulse energy and the gas pressure in the capillary waveguide. In correlation measurements between single-shot drive laser beam profiles and single-shot high-harmonic beam profiles we prove the absence of drive laser beam-pointing-induced fluctuations in the high-harmonic output. We attribute the main source of high-harmonic fluctuations to ionization-induced nonlinear mode mixing during propagation of the drive laser pulse inside the capillary waveguide

Recruitment of Eph receptors into signaling clusters does not require ephrin contact

Eph receptors and their cell membrane–bound ephrin ligands regulate cell positioning and thereby establish or stabilize patterns of cellular organization. Although it is recognized that ephrin clustering is essential for Eph function, mechanisms that relay information of ephrin density into cell biological responses are poorly understood. We demonstrate by confocal time-lapse and fluorescence resonance energy transfer microscopy that within minutes of binding ephrin-A5–coated beads, EphA3 receptors assemble into large clusters. While remaining positioned around the site of ephrin contact, Eph clusters exceed the size of the interacting ephrin surface severalfold. EphA3 mutants with compromised ephrin-binding capacity, which alone are incapable of cluster formation or phosphorylation, are recruited effectively and become phosphorylated when coexpressed with a functional receptor. Our findings reveal consecutive initiation of ephrin-facilitated Eph clustering and cluster propagation, the latter of which is independent of ephrin contacts and cytosolic Eph signaling functions but involves direct Eph–Eph interactions

The Autodepalmitoylating activity of APT maintains the spatial organization of Palmitoylated membrane proteins

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.Fil: Vartak, Nachiket. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Papke, Bjoern. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Grecco, Hernan Edgardo. Institut Max Planck Fur Molekulare Physiologie; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rossmannek, Lisaweta. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Waldmann, Herbert. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Hedberg, Christian. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Bastiaens, Philippe I. H.. Institut Max Planck Fur Molekulare Physiologie; Alemani

siRNA cell arrays for high-content screening microscopy

RNA interference (RNAi) is a recent advance that provides the possibility to reduce the expression of specific target genes in cultured mammalian cells with potential applications on a genome-wide scale. However, to achieve this, robust methodologies that allow automated and efficient delivery of small interfering RNAs (siRNAs) into living cultured cells and reliable quality control of siRNA function must be in place. Here we describe the production of cell arrays for reverse transfection of tissue culture cells with siRNA and plasmid DNA suitable for subsequent high-content screening microscopy applications. All the necessary transfection components are mixed prior to the robotic spotting on noncoated chambered coverglass tissue culture dishes, which are ideally suited for time-lapse microscopy applications in living cells. The addition of fibronectin to the spotting solution improves cell adherence. After cell seeding, no further cell culture manipulations, such as medium changes or the addition of 7serum, are needed. Adaptation of the cell density improves autofocus performance for high-quality data acquisition and cell recognition. The co-transfection of a nonspecific fluorescently labeled DNA oligomer with the specific siRNA helps to mark each successfully transfected cell and cell cluster. We demonstrate such an siRNA cell array in a microscope-based functional assay in living cells to determine the effect of various siRNA oligonucleotides against endogenous targets on cellular secretion. </jats:p

Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background&#xd;&#xa;The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.&#xd;&#xa;&#xd;&#xa;Methodology/Principal Findings&#xd;&#xa;When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength &#x2013; a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.&#xd;&#xa;&#xd;&#xa;Conclusions/Significance&#xd;&#xa;Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions

Early detection of cervical cancer in western Kenya : determinants of healthcare providers performing a gynaecological examination for abnormal vaginal discharge or bleeding

Background In western Kenya, women often present with late-stage cervical cancer despite prior contact with the health care system. The aim of this study was to predict primary health care providers' behaviour in examining women who present with abnormal discharge or bleeding. Methods This was a cross-sectional survey using the theory of planned behaviour (TPB). A sample of primary health care practitioners in western Kenya completed a 59-item questionnaire. Structural equation modelling was used to identify the determinants of providers' intention to perform a gynaecological examination. Bivariate analysis was conducted to investigate the relationship between the external variables and intention. Results Direct measures of subjective norms (DMSN), direct measures of perceived behavioural control (DMPBC), and indirect measures of attitude predicted the intention to examine patients. Negative attitudes toward examining women had a suppressor effect on the prediction of health workers' intentions. However, the predictors of intention with the highest coefficients were the external variables being a nurse (beta = 0.32) as opposed to a clinical officer and workload of attending less than 50 patients per day (beta = 0.56). In bivariate analysis with intention to perform a gynaecological examination, there was no evidence that working experience, being female, having a lower workload, or being a private practitioner were associated with a higher intention to conduct vaginal examinations. Clinical officers and nurses were equally likely to examine women. Conclusions The TPB is a suitable theoretical basis to predict the intention to perform a gynaecological examination. Overall, the model predicted 47% of the variation in health care providers' intention to examine women who present with recurrent vaginal bleeding or discharge. Direct subjective norms (health provider's conformity with what their colleagues do or expect them to do), PBC (providers need to feel competent and confident in performing examinations in women), and negative attitudes toward conducting vaginal examination accounted for the most variance. External variables in this study also contributed to the overall variance. As the model in this study could not explain 53% of the variance, investigating other external variables that influence the intention to examine women should be undertaken

Dynamic Recruitment of Licensing Factor Cdt1 to Sites of DNA Damage

For genomic integrity to be maintained, the cell cycle and DNA damage responses must be linked. Cdt1, a G1-specific cell-cycle factor, is targeted for proteolysis by the Cul4-Ddb1Cdt2 ubiquitin ligase following DNA damage. Using a laser nanosurgery microscope to generate spatially restricted DNA damage within the living cell nucleus, we show that Cdt1 is recruited onto damaged sites in G1 phase cells, within seconds of DNA damage induction. PCNA, Cdt2, Cul4, DDB1 and p21Cip1 also accumulate rapidly to damaged sites. Cdt1 recruitment is PCNA-dependent, whereas PCNA and Cdt2 recruitment are independent of Cdt1. Fitting of fluorescence recovery after photobleaching profiles to an analytic reaction-diffusion model shows that Cdt1 and p21Cip1 exhibit highly dynamic binding at the site of damage, whereas PCNA appears immobile. Cdt2 exhibits both a rapidly exchanging and an apparently immobile subpopulation. Our data suggest that PCNA provides an immobile binding interface for dynamic Cdt1 interactions at the site of damage, which leads to rapid Cdt1 recruitment to damaged DNA, preceding Cdt1 degradation

A self-organized synthetic morphogenic liposome responds with shape changes to local light cues

Reconstituting artificial proto-cells capable of transducing extracellular signals into cytoskeletal changes can reveal fundamental principles of how non-equilibrium phenomena in cellular signal transduction affect morphogenesis. Here, we generated a Synthetic Morphogenic Membrane System (SynMMS) by encapsulating a dynamic microtubule (MT) aster and a light-inducible signaling system driven by GTP/ATP chemical potential into cell-sized liposomes. Responding to light cues in analogy to morphogens, this biomimetic design embodies basic principles of localized Rho-GTPase signal transduction that generate an intracellular MT-regulator signaling gradient. Light-induced signaling promotes membrane-deforming growth of MT-filaments by dynamically elevating the membrane-proximal tubulin concentration. The resulting membrane deformations enable recursive coupling of the MT-aster with the signaling system, which generates global self-organized morphologies that reorganize towards local external cues in dependence on prior shape. SynMMS thereby signifies a step towards bio-inspired engineering of self-organized cellular morphogenesis