Determination of Mean Cluster Sizes by Fluorescence Detection upon Site-Specific Photoexcitation.

Electronic excitations in the valence shell of Ne clusters were studied by fluorescence spectroscopy. The measured fluorescence excitation functions contain information about the nature and number of excitonic states and the mean cluster size of the produced size distribution. Mean cluster sizes were determined by comparing surface and bulk contributions using a multidimensional fitting algorithm, with good agreement to commonly used scaling laws. The influence of different size distributions, which were not considered in previous investigations, on homogeneous noble gas cluster jets is implemented in the proposed model. The present work is the first approach using fluorescence spectroscopy for the determination of the mean size of Ne cluster jets created by supersonic expansion.

Recovery of High-Energy Photoelectron Circular Dichroism through Fano Interference.

It is commonly accepted that the magnitude of a photoelectron circular dichroism (PECD) is governed by the ability of an outgoing photoelectron wave packet to probe the chiral asymmetry of a molecule. To be able to accumulate this characteristic asymmetry while escaping the chiral ion, photoelectrons need to have relatively small kinetic energies of up to a few tens of electron volts. Here, we demonstrate a substantial PECD for very fast photoelectrons above 500 eV kinetic energy released from methyloxirane by a participator resonant Auger decay of its lowermost O 1s excitation. This effect emerges as a result of the Fano interference between the direct and resonant photoionization pathways, notwithstanding that their individual effects are negligibly small. The resulting dichroic parameter has an anomalous dispersion: It changes its sign across the resonance, which can be considered as an analogue of the Cotton effect in the x-ray regime.

Angular momentum sensitive two-center interference.

In quantum mechanics the Young-type double-slit experiment can be performed with electrons either traveling through a double slit or being coherently emitted from two inversion symmetric molecular sites. In the latter one the valence photoionization cross sections of homonuclear diatomic molecules were predicted to oscillate over kinetic energy almost 50 years ago. Beyond the direct proof of the oscillatory behavior of these photoionization cross sections σ, we show that the angular distribution of the emitted electrons reveals hitherto unexplored information on the relative phase shift between the corresponding partial waves through two-center interference patterns.

Efficient solid-phase synthesis of pppRNA by using product-specific labeling.

A novel solid-phase synthesis and purification strategy for 5'-triphosphate oligonucleotides by using lipophilic tagging of the triphosphate moiety is reported. This is based on triphosphate synthesis with 5'-O-cyclotriphosphate intermediates, whereby a lipophilic tag, such as decylamine, is introduced during the ring-opening reaction to give a linear gamma-phosphate-tagged species. This method enables the highly efficient synthesis of 5'-triphosphorylated RNA derivatives and their gamma-phosphate-substituted analogues and will especially facilitate the advancement of therapeutic approaches that make use of 5'-triphosphate oligonucleotides as potent activators of the cytosolic immune sensor RIG-I.

Stimulation of TLR7 prior to polymicrobial sepsis improves the immune control of the inflammatory response in adult mice.

OBJECTIVE: The role of Toll-like receptor 7 (TLR7), so far regarded as a receptor for viral RNA, was evaluated in a murine sepsis model. MATERIAL: We used the colon ascendens stent peritonitis model (CASP) in female C57B/6 mice. R-848 (1.5 µg/g body weight) was injected intravenously prior to sepsis induction. METHODS: We determined levels of cytokines by CBA detection kit. Different cell populations were isolated from the spleen by magnetic cell separation and the expression of TLR7 was visualized by immunofluorescence staining. Bacterial load of organs was quantified by incubating suspensions on agar in colony forming units. RESULTS: R-848 application per se led to elevated cytokine levels in serum, spleen and peritoneal cavity. Expression of TLR7 on splenocytes was upregulated following CASP. Bacterial clearance in polymicrobial sepsis was significantly increased in spleen and peritoneum of mice pre-treated with the TLR7-agonist. Cytokine release was regulated in the peritoneum and spleen. Furthermore, apoptosis in thymus and spleen during polymicrobial sepsis was significantly decreased following TLR7 agonist application. CONCLUSIONS: TLR7 seems to be essential for pathogen defence not only in viral but also in bacterial infections. Pharmacological stimulation of this receptor prior to induction of sepsis improves the host's capacity to cope with pathogens.

Effects of an active immunization on the immune response of laying Japanese quail (Coturnix coturnix japonica) fed with or without genetically modified Bacillus thuringiensis-maize.

Potentially adverse effects of diets containing transgenic plants are a concern for many consumers, particularly in Europe. For Bacillus thuringiensis-maize, several studies in livestock and poultry showed that the zootechnical data provide no indication for such adverse effects. These studies were all done in homeostatic situations; it remained open whether a deflection of the regulatory physiological systems might yield divergent dynamic responses in B. thuringiensis-maize-fed animals. We therefore tested the effect of an active immunization using BSA as antigen in a feeding regimen with or without B. thuringiensis-maize using quail as a model organism. Newly hatched Japanese quail were randomly allocated to 2 groups (n=120 per group) fed with diets containing either B. thuringiensis-maize or isogenic maize of the same cultivar. The diets did not differ in concentrations of the mycotoxins deoxynivalenol and zearalenone, which were both far below guidance values. After 16 wk on the experimental diets, one-half of each group was immunized against BSA. The remaining birds were injected with saline. Thirty-six hours after the injection, half of the BSA-injected subgroup (n=30) and half of the saline subgroup (n=30) from B. thuringiensis-maize- and isogenic-fed birds were killed and blood samples were collected and analyzed for serum zinc levels, indicative for acute phase response. For determining IgY-mediated immune responses, eggs were collected every other week for 6 wk after the injections from the remaining birds and total IgY concentrations and BSA-specific IgY titers were measured in egg yolk. The BSA injections did not elicit significant decreases of serum zinc concentrations. The serum zinc levels were significantly higher in B. thuringiensis-maize-fed quail. Expectedly, total IgY as well as BSA-specific IgY titers increased with time in the BSA-immunized quail. The response of both variables to the BSA injection did not differ between the feeding groups. Our results indicate that feeding of B. thuringiensis-maize does not impair the immune system of Japanese quail and thus gives no indication for respective concerns.

[Emotional state variations in rats during recall of passive avoidance reactions after neurotensin administration into nucleus accumbens of the brain].

Behavioral effects of neurotensin administration into the nucleus accumbens were studied in rats with neurotoxic lesions of serotoninergic structures of the dorsal raphe nucleus or periaqueductal grey matter. Changes in recall of passive avoidance conditioned reactions and aftereffects of painful stimulation in the locomotor activity were studied in the "open field" and elevated plus-maze and T-maze tests. The toxin administration into the dorsal raphe nucleus did not impair the recall of the passive avoidance reactions, but enhanced the oppressive aftereffects of painful stimulation, which can specify the development of anxiety in rats. The toxin administration into the periaqueductal grey matter had an opposite effect, which can be considered as a manifestation of the panic state. Neurotensin weakened the above mentioned effects of the toxin and, depending on the evoked emotional disorders, produced the anxiolytic or antipanic effects.

Reconstitution of mammary gland development in vitro: requirement of c-met and c-erbB2 signaling for branching and alveolar morphogenesis.

We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met-mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.

Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland.

We have examined the role of two mesenchymal ligands of epithelial tyrosine kinase receptors in mouse mammary gland morphogenesis. In organ cultures of mammary glands, hepatocyte growth factor (HGF, scatter factor) promoted branching of the ductal trees but inhibited the production of secretory proteins. Neuregulin (NRG, neu differentiation factor) stimulated lobulo-alveolar budding and the production of milk proteins. These functional effects are paralleled by the expression of the two factors in vivo: HGF is produced in mesenchymal cells during ductal branching in the virgin animal; NRG is expressed in the mesenchyme during lobulo-alveolar development at pregnancy. The receptors of HGF and NRG (c-met, c-erbB3, and c-erbB4), which are expressed in the epithelial cells, are not regulated. In organ culture, branching morphogenesis and lobulo-alveolar differentiation of the mammary gland could be abolished by blocking expression of endogenous HGF and NRG by the respective antisense oligonucleotides; in antisense oligonucleotide-treated glands, morphogenesis could again be induced by the addition of recombinant HGF and NRG. We thus show that two major postnatal morphogenic periods of mammary gland development are dependent on sequential mesenchymal-epithelial interactions mediated by HGF and NRG.

Development and validation of a beam model applicable to small fields.

A beam model for dose verification of narrow photon beams in water is presented. This model includes the two main effects affecting dose distributions for small fields: the spatial extension of the radiation source and the lateral non-equilibrium conditions of the energy transfer in the absorber. The spatial extension of the radiation source is determined experimentally using two methods: a 'slit-method' for the inner part and the measurement of relative output factors free in air for the outer part. The lateral non-equilibrium is taken into account using dose deposition kernels obtained with Monte Carlo calculations and subsequent convolution techniques. The model was validated in water for a wide range of field sizes in a 6 MV photon beam. Good agreement between measured and calculated dose profiles and output factors was found for all the cases studied. The model is considered to be especially useful for narrow-beam IMRT such as typically used in TomoTherapy units. Thus, it can be used as a supplemental dosimetry check for dose delivery performance and as an independent treatment verification tool for cases involving small radiation fields.

The determination of beam quality correction factors: Monte Carlo simulations and measurements.

Modern dosimetry protocols are based on the use of ionization chambers provided with a calibration factor in terms of absorbed dose to water. The basic formula to determine the absorbed dose at a user's beam contains the well-known beam quality correction factor that is required whenever the quality of radiation used at calibration differs from that of the user's radiation. The dosimetry protocols describe the whole ionization chamber calibration procedure and include tabulated beam quality correction factors which refer to 60Co gamma radiation used as calibration quality. They have been calculated for a series of ionization chambers and radiation qualities based on formulae, which are also described in the protocols. In the case of high-energy photon beams, the relative standard uncertainty of the beam quality correction factor is estimated to amount to 1%. In the present work, two alternative methods to determine beam quality correction factors are prescribed-Monte Carlo simulation using the EGSnrc system and an experimental method based on a comparison with a reference chamber. Both Monte Carlo calculations and ratio measurements were carried out for nine chambers at several radiation beams. Four chamber types are not included in the current dosimetry protocols. Beam quality corrections for the reference chamber at two beam qualities were also measured using a calorimeter at a PTB Primary Standards Dosimetry Laboratory. Good agreement between the Monte Carlo calculated (1% uncertainty) and measured (0.5% uncertainty) beam quality correction factors was obtained. Based on these results we propose that beam quality correction factors can be generated both by measurements and by the Monte Carlo simulations with an uncertainty at least comparable to that given in current dosimetry protocols.

Setup for multicoincidence experiments of photons in the extreme ultraviolet to visible spectral range and charged particles-The solid angle maximization approach.

The coincident detection of particles is a powerful method in experimental physics, enabling the investigation of a variety of projectile-target interactions. The vast majority of coincidence experiments is performed with charged particles, as they can be guided by electric or magnetic fields to yield large detection probabilities. When a neutral species or a photon is one of the particles recorded in coincidence, its detection probability typically suffers from small solid angles. Here, we present two optical assemblies considerably enhancing the solid angle for photon detection in the extreme ultraviolet to visible spectral range. The efficiency and versatility of these assemblies are demonstrated for electron-photon coincidence detection, where electrons and photons emerge from fundamental processes after photoexcitation of gaseous samples by synchrotron radiation.

Beyond double-stranded RNA-type I IFN induction by 3pRNA and other viral nucleic acids.

Production of type I IFN is the key response to viral infection. Since the discovery of type I IFNs in 1957, long double-stranded RNA formed during replication of many viruses was thought to be responsible for type I IFN induction, and for decades double-stranded RNA-activated protein kinase (PKR) was thought to be the receptor. Recently, this picture has dramatically changed. It now became evident that not PKR but two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, and two cytosolic helicases, RIG-I and MDA-5, are responsible for the majority of type I IFNs induced upon recognition of viral nucleic acids. In this review, we focus on the molecular mechanisms by which those innate immune receptors detect viral infection. Based on the recent progress in the field, we now know that TLR7, TLR9, and RIG-I do not require long double-stranded RNA for type I IFN induction.

The change of response of ionization chambers in the penumbra and transmission regions: impact for IMRT verification.

Significant deviations from the expected dose have been reported in the absolute dosimetry validation of an intensity modulated radiation therapy treatment when individual segments are analyzed. However, when full treatment is considered and all segment doses are added together, these discrepancies fade out, leading to overall dose deviations below a 5% action level. This contradictory behavior may be caused by a partial compensation between detector over-responding and under-responding for measurement conditions far from radiation equilibrium. We consider three treatment verification scenarios that may lead to ionization chamber miss-responding, namely: narrow beam irradiation, field penumbra location and multi-leaf collimator transmission contribution. In this work we have analyzed the response of three different ionization chambers with different active volume under these conditions by means of Monte Carlo (MC) simulation methods. Correction factors needed to convert the detector readout into actual dose to water were calculated by inserting the specific detector geometry (carefully modeled) into the simulations. This procedure required extensive use of parallel computing resources in order to achieve the desired level of uncertainty in the final results. The analysis of the simulations shows the relative contribution of each of the three previously mentioned miss-responding scenarios. Additionally, we provide some evidence on dose deviation compensation in multi-segment radiotherapy treatment verification.

Serotoninergic mechanisms of the effects of neurotensin on passive avoidance behavior in rats.

The aim of the present work was to identify the features of the actions of neurotensin on administration into the substantia nigra or dorsal cervical nucleus on the reproduction of passive avoidance reactions in rats. The results showed that the action of neurotensin administered into the substantia nigra was accompanied by sharp reductions in passive avoidance reactions, while administration into the dorsal cervical nucleus, conversely, led to increases in these reactions and slowing of their extinction. The effects of microinjections of the serotonin 5-HT(1A) receptor agonist 8-hydroxydipropylaminotetraline (8-OH-DPAT) into these brain structures were analogous to the effects of neurotensin. The different behavioral effects of administration of neurotensin corresponded to identifiable changes in the levels of serotonin and its metabolite 5-hydroxyindoleacetic acid in the caudate nuclei of the brain. These data led to the conclusion that the effects of neurotensin on passive avoidance behavior are associated with the regulation of the emotional state of the animals via actions on the functions of brain serotoninergic structures.

Note: Soft X-ray transmission polarizer based on ferromagnetic thin films.

A transmission polarizer for producing elliptically polarized soft X-ray radiation from linearly polarized light is presented. The setup is intended for use at synchrotron and free-electron laser beamlines that do not directly offer circularly polarized light for, e.g., X-ray magnetic circular dichroism (XMCD) measurements or holographic imaging. Here, we investigate the degree of ellipticity upon transmission of linearly polarized radiation through a cobalt thin film. The experiment was performed at a photon energy resonant to the Co L3-edge, i.e., 778 eV, and the polarization of the transmitted radiation was determined using a polarization analyzer that measures the directional dependence of photo electrons emitted from a gas target. Elliptically polarized radiation can be created at any absorption edge showing the XMCD effect by using the respective magnetic element.

Symmetry breakdown of electron emission in extreme ultraviolet photoionization of argon.

Short wavelength free-electron lasers (FELs), providing pulses of ultrahigh photon intensity, have revolutionized spectroscopy on ionic targets. Their exceptional photon flux enables multiple photon absorptions within a single femtosecond pulse, which in turn allows for deep insights into the photoionization process itself as well as into evolving ionic states of a target. Here we employ ultraintense pulses from the FEL FERMI to spectroscopically investigate the sequential emission of electrons from gaseous, atomic argon in the neutral as well as the ionic ground state. A pronounced forward-backward symmetry breaking of the angularly resolved emission patterns with respect to the light propagation direction is experimentally observed and theoretically explained for the region of the Cooper minimum, where the asymmetry of electron emission is strongly enhanced. These findings aim to originate a better understanding of the fundamentals of photon momentum transfer in ionic matter.

Engineered mutants of HGF/SF with reduced binding to heparan sulphate proteoglycans, decreased clearance and enhanced activity in vivo.

BACKGROUND: Although a number of growth factors bind cell-surface heparan sulphate proteoglycans (HSPGs), the role of this interaction is unclear except for fibroblast growth factor which requires HSPG binding for signalling. Hepatocyte growth factor/scatter factor (HGF/SF) plays important roles in mammalian development and tissue regeneration and acts on target cells through a specific receptor tyrosine kinase encoded by the c-met proto-oncogene. This factor also binds HSPGs with high affinity, but conflicting data have been reported on the role of HSPG binding in HGF/SF signalling. RESULTS: To map the binding sites for HSPG and the Met receptor in HGF/SF, we have engineered a number of HGF/SF mutants in which several clusters of solvent-accessible residues in the hairpin structure of the amino-terminal domain or in kringle 2 have been replaced. Two of the mutants (HP1 and HP2) showed greatly decreased (more than 50-fold) affinity for heparin and HSPGs but retained full mitogenic and motogenic activities on target cells in culture. Furthermore, when compared with wild-type HGF/SF, the HP1 mutant exhibited a delayed clearance from the blood, higher tissue levels and a higher induction of DNA synthesis in normal, adult murine liver. CONCLUSIONS: These results establish the following: the binding sites in HGF/SF for Met and for HSPGs can be dissociated by protein engineering; high-affinity binding of HGF/SF to HSPGs is not essential for signalling; one role of HSPG binding in the HGF/SF system appears to be sequestration and degradation of the growth factor; and HGF/SF mutants with decreased affinity for HSPGs exhibit enhanced activity in vivo.

Serum IgG autoantibodies directed against the alpha chain of Fc epsilon RI: a selective marker and pathogenetic factor for a distinct subset of chronic urticaria patients?

While it is well established that acute allergic urticaria is caused by degranulation of skin mast cells occurring after allergen/IgE-dependent cross-linking of high affinity IgE receptors (FcepsilonRI), the pathophysiologic mechanisms operative in chronic urticaria (CU) are less well understood. Some evidence points to the existence of histamine-releasing activity in the serum of CU patients which possibly acts via triggering of FcepsilonRI. In this study, we aimed to better characterize this anti-FcepsilonRIalpha reactivity of CU patients using affinity-purified, IgE-depleted IgG fractions of such individuals (CU-IgG). Using immobilized, recombinant soluble FcepsilonRIalpha as a a reaction target for Western blot studies, we found that 12/32 (37%) CU-IgG serum samples exhibited IgG autoreactivity against FcepsilonRI- alpha. These findings were confirmed by experiments demonstrating that immunoblot-reactive, but not immunoblot-nonreactive, CU-IgG preparations precipitated the FcepsilonRIalpha from FcepsilonRI- alphagamma-transfected cells. No anti-FcepsilonRIalpha reactivity was observed in IgG fractions from atopic dermatitis (AD) patients (0/15) or healthy control individuals (CO:0/15). As opposed to the selective occurrence of IgG anti-Fc epsilon RI alpha autoantibodies in CU patients, IgG anti-IgE antibodies were detected in all groups investigated (CU: 69%; AD: 73%; CO: 26%). While both types of autoantibodies can exhibit histamine-releasing properties, not all of the autoantibodies proved to be functional in vitro. Our results indicate that the occurrence of IgG anti-FcepsilonRIalpha reactivity defines an autoimmune-mediated subentity of CU and provide a basis for the development of new diagnostic procedures and, perhaps, therapeutic strategies for this disease.

Four-dimensional magnetic resonance imaging for the determination of tumour movement and its evaluation using a dynamic porcine lung phantom.

A method of four-dimensional (4D) magnetic resonance imaging (MRI) has been implemented and evaluated. It consists of retrospective sorting and slice stacking of two-dimensional (2D) images using an external signal for motion monitoring of the object to be imaged. The presented method aims to determine the tumour trajectories based on a signal that is appropriate for monitoring the movement of the target volume during radiotherapy such that the radiation delivery can be adapted to the movement. For evaluation of the 4D-MRI method, it has been applied to a dynamic lung phantom, which exhibits periodic respiratory movement of a porcine heart-lung explant with artificial pulmonary nodules. Anatomic changes of the lung phantom caused by respiratory motion have been quantified, revealing hysteresis. The results demonstrate the feasibility of the presented method of 4D-MRI. In particular, it enables the determination of trajectories of periodically moving objects with an uncertainty in the order of 1 mm.