Site-directed spin labeling of proteins for distance measurements in vitro and in cells.

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy allows studying the structure, dynamics, and interactions of proteins via distance measurements in the nanometer range. We here give an overview of available spin labels, the strategies for their introduction into proteins, and the associated potentials for protein structural studies in vitro and in the context of living cells.

Probing of chain conformations in conjugated polymer nanoparticles by electron spin resonance spectroscopy.

In the case of conjugated polymer chains usually considered as rigid or stiff, it is an open question how the individual chains adopt their conformation inside nanoparticles. Here, the conformation of such a rigid conjugated polymer chain is elucidated for the first time. For this purpose, electron paramagnetic resonance spectroscopy as a method allowing for a direct observation is established.

Comparison of clinical presentation and laboratory values at admission between PCR-confirmed influenza A H1N1 infection and influenza-like disease, South-East Austria.

PURPOSE: Reliable and rapid diagnosis of influenza A H1N1 is essential to initiate the appropriate antiviral therapy and preventive measures. As PCR assays are time-consuming and rapid antigen tests have a limited sensitivity, official influenza case definitions are used in many clinical settings. These, however, are based exclusively on clinical criteria and have only a moderate potential to differentiate between influenza and other febrile diseases. Only limited data on the differences in clinical and laboratory parameters between influenza and non-influenza febrile diseases are available to date. METHODS: This was a retrospective case-negative control series that was conducted in Styria, southeast Austria. We analyzed the differences in clinical presentation and laboratory admission parameters between patients with PCR-confirmed H1N1 influenza infection (n = 199) and those with influenza-like disease and negative influenza PCR results (ILD group; n = 252). RESULTS: In the multivariable analysis lower C-reactive protein (CRP) level, lower white blood cell (WBC) count, fever, wheezing, cough, and the absence of nausea or sudden onset remained significant predictors of H1N1 influenza in adult patients (n = 263). Lower CRP level, lower WBC count, and cough remained significant predictors in pediatric patients (<16 years; n = 188). CONCLUSION: Lower CRP level, lower WBC count, and cough were significant predictors of H1N1 in both the adult and pediatric patient group. These data may help to develop an improved case definition for suspected H1N1 infection which combines clinical findings and easily available laboratory parameters.

Long-term stabilization of high power optical parametric chirped-pulse amplifiers.

The long-term stability of optical parametric chirped-pulse amplifiers is hindered by thermal path length drifts affecting the temporal pump-to-signal overlap. A kilowatt-pumped burst amplifier is presented delivering broadband 1.4 mJ pulses with a spectral bandwidth supporting sub-7 fs pulse duration. Active temporal overlap control can be achieved by feedback of optical timing signals from cross-correlation or spectral measurements. Using a balanced optical cross-correlator, we achieve a pump-to-signal synchronization with a residual jitter of only (46 ± 2) fs rms. Additionally, we propose passive pump-to-signal stabilization with an intrinsic jitter of (7.0 ± 0.5) fs rms using white-light continuum generation.

Generation of coherent 19- and 38-nm radiation at a free-electron laser directly seeded at 38 nm.

Initiating the gain process in a free-electron laser (FEL) from an external highly coherent source of radiation is a promising way to improve the pulse properties such as temporal coherence and synchronization performance in time-resolved pump-probe experiments at FEL facilities, but this so-called "seeding" suffers from the lack of adequate sources at short wavelengths. We report on the first successful seeding at a wavelength as short as 38.2 nm, resulting in GW-level, coherent FEL radiation pulses at this wavelength as well as significant second harmonic emission at 19.1 nm. The external seed pulses are about 1 order of magnitude shorter compared to previous experiments allowing an ultimate time resolution for the investigation of dynamic processes enabling breakthroughs in ultrafast science with FELs. The seeding pulse is the 21st harmonic of an 800-nm, 15-fs (rms) laser pulse generated in an argon medium. Methods for finding the overlap of seed pulses with electron bunches in spatial, longitudinal, and spectral dimensions are discussed and results are presented. The experiment was conducted at FLASH, the FEL user facility at DESY in Hamburg, Germany.

Attosecond spectroscopy in condensed matter.

Comprehensive knowledge of the dynamic behaviour of electrons in condensed-matter systems is pertinent to the development of many modern technologies, such as semiconductor and molecular electronics, optoelectronics, information processing and photovoltaics. Yet it remains challenging to probe electronic processes, many of which take place in the attosecond (1 as = 10(-18) s) regime. In contrast, atomic motion occurs on the femtosecond (1 fs = 10(-15) s) timescale and has been mapped in solids in real time using femtosecond X-ray sources. Here we extend the attosecond techniques previously used to study isolated atoms in the gas phase to observe electron motion in condensed-matter systems and on surfaces in real time. We demonstrate our ability to obtain direct time-domain access to charge dynamics with attosecond resolution by probing photoelectron emission from single-crystal tungsten. Our data reveal a delay of approximately 100 attoseconds between the emission of photoelectrons that originate from localized core states of the metal, and those that are freed from delocalized conduction-band states. These results illustrate that attosecond metrology constitutes a powerful tool for exploring not only gas-phase systems, but also fundamental electronic processes occurring on the attosecond timescale in condensed-matter systems and on surfaces.

Attosecond real-time observation of electron tunnelling in atoms.

Atoms exposed to intense light lose one or more electrons and become ions. In strong fields, the process is predicted to occur via tunnelling through the binding potential that is suppressed by the light field near the peaks of its oscillations. Here we report the real-time observation of this most elementary step in strong-field interactions: light-induced electron tunnelling. The process is found to deplete atomic bound states in sharp steps lasting several hundred attoseconds. This suggests a new technique, attosecond tunnelling, for probing short-lived, transient states of atoms or molecules with high temporal resolution. The utility of attosecond tunnelling is demonstrated by capturing multi-electron excitation (shake-up) and relaxation (cascaded Auger decay) processes with subfemtosecond resolution.

Successful management of nosocomial ventriculitis and meningitis caused by extensively drug-resistant Acinetobacter baumannii in Austria.

Nosocomial infections caused by the Gram-negative coccobacillus Acinetobacter baumannii have substantially increased over recent years. Because Acinetobacter is a genus with a tendency to quickly develop resistance to multiple antimicrobial agents, therapy is often complicated, requiring the return to previously used drugs. The authors report a case of meningitis due to extensively drug-resistant A baumannii in an Austrian patient who had undergone neurosurgery in northern Italy. The case illustrates the limits of therapeutic options in central nervous system infections caused by extensively drug-resistant pathogens.

Les infections d'origine nosocomiale causées par le coccobacille Acinetobacter baumannii Gram négatif ont considérablement augmenté ces dernières années. Puisque l'Acinetobacter est un genre qui a tendance à devenir rapidement résistant à de multiples agents antimicrobiens, le traitement est souvent compliqué et exige de revenir à des médicaments déjà utilisés. Les auteurs signalent un cas de méningite attribuable à un A baumannii d'une extrême résistance aux médicaments chez un patient autrichien qui a subi une neurochirurgie dans le nord de l'Italie. Le cas illustre les limites des options thérapeutiques aux infections du système nerveux central causées par des pathogènes d'une extrême résistance aux médicaments.

Pulsed operation of a high average power Yb:YAG thin-disk multipass amplifier.

An Yb:YAG thin-disk multipass laser amplifier system was developed operating in a 10 Hz burst operation mode with 800 µs burst duration and 100 kHz intra-burst repetition rate. Methods for the suppression of parasitic amplified spontaneous emission are presented. The average output pulse energy is up to 44.5 mJ and 820 fs compressed pulse duration. The average power of 4.45 kW during the burst is the highest reported for this type of amplifier.

Evidence for chirped Auger-electron emission.

Auger decay carries valuable information about the electronic structure and dynamics of atoms, molecules, and solids. Here we furnish evidence that under certain conditions Auger electrons are subject to an energetic chirp. The effect is disclosed in time-resolved streaking experiments on the Xe NOO and Kr MNN Auger decay using extreme-ultraviolet pulses from the free-electron laser in Hamburg as well as from a high-order harmonic laser source. The origin of this effect is found to be an exchange of energy between the Auger electron and an earlier emitted correlated photoelectron. The observed time-dependent spectral modulations are understood within an analytical model and confirmed by extensive computer simulations.

Yb:YAG Innoslab amplifier: efficient high repetition rate subpicosecond pumping system for optical parametric chirped pulse amplification.

We report on a Yb:YAG Innoslab laser amplifier system for generation of subpicsecond high energy pump pulses for optical parametric chirped pulse amplification (OPCPA) at high repetition rates. Pulse energies of up to 20 mJ (at 12.5 kHz) and repetition rates of up to 100 kHz were attained with pulse durations of 830 fs and average power in excess of 200 W. We further investigate the possibility to use subpicosecond pulses to derive a stable continuum in a YAG crystal for OPCPA seeding.

Photoinduced reconfiguration cycle in a molecular adsorbate layer studied by femtosecond inner-shell photoelectron spectroscopy.

A time-resolved study of core-level chemical shifts in a monolayer of aromatic molecules reveals complex photoinduced reaction dynamics. The combination of electron spectroscopy for chemical analysis and ultrashort pulse excitation in the extreme ultraviolet allows performing time-correlated 4d-core-level spectroscopy of iodine atoms that probe the local chemical environment in the adsorbate molecule. The selectivity of the method unveils metastable molecular configurations that appear about 50 ps after the excitation and are efficiently quenched back to the ground state.

Coherent control of high harmonic generation via dual-gas multijet arrays.

High harmonic generation (HHG) is a central driver of the rapidly growing field of ultrafast science. We present a novel quasiphase-matching (QPM) concept with a dual-gas multijet target leading, for the first time, to remarkable phase control between multiple HHG sources (>2) within the Rayleigh range. The alternating jet structure with driving and matching zones shows perfect coherent buildup for up to six QPM periods. Although not in the focus of the proof-of-principle studies presented here, we achieved competitive conversion efficiencies already in this early stage of development.

Atomic transient recorder.

In Bohr's model of the hydrogen atom, the electron takes about 150 attoseconds (1 as = 10(-18) s) to orbit around the proton, defining the characteristic timescale for dynamics in the electronic shell of atoms. Recording atomic transients in real time requires excitation and probing on this scale. The recent observation of single sub-femtosecond (1 fs = 10(-15) s) extreme ultraviolet (XUV) light pulses has stimulated the extension of techniques of femtochemistry into the attosecond regime. Here we demonstrate the generation and measurement of single 250-attosecond XUV pulses. We use these pulses to excite atoms, which in turn emit electrons. An intense, waveform-controlled, few cycle laser pulse obtains 'tomographic images' of the time-momentum distribution of the ejected electrons. Tomographic images of primary (photo)electrons yield accurate information of the duration and frequency sweep of the excitation pulse, whereas the same measurements on secondary (Auger) electrons will provide insight into the relaxation dynamics of the electronic shell following excitation. With the current approximately 750-nm laser probe and approximately 100-eV excitation, our transient recorder is capable of resolving atomic electron dynamics within the Bohr orbit time.

The mechanism of Hsp90-induced oligomerizaton of Tau.

Aggregation of the microtubule-associated protein Tau is a hallmark of Alzheimer's disease with Tau oligomers suspected as the most toxic agent. Tau is a client of the molecular chaperone Hsp90, although it is unclear whether and how the chaperone massages the structure of intrinsically disordered Tau. Using electron paramagnetic resonance, we extract structural information from the very broad conformational ensemble of Tau: Tau in solution is highly dynamic and polymorphic, although "paper clip"-shaped by long-range contacts. Interaction with Hsp90 promotes an open Tau conformation, which we identify as the molecular basis for the formation of small Tau oligomers by exposure of the aggregation-prone repeat domain to other Tau molecules. At the same time, formation of Tau fibrils is inhibited. We therefore provide the nanometer-scale zoom into chaperoning an amyloid client, highlighting formation of oligomers as the consequence of this biologically relevant interaction.

X-ray pulses approaching the attosecond frontier.

Single soft-x-ray pulses of approximately 90-electron volt (eV) photon energy are produced by high-order harmonic generation with 7-femtosecond (fs), 770-nanometer (1.6 eV) laser pulses and are characterized by photoionizing krypton in the presence of the driver laser pulse. By detecting photoelectrons ejected perpendicularly to the laser polarization, broadening of the photoelectron spectrum due to absorption and emission of laser photons is suppressed, permitting the observation of a laser-induced downshift of the energy spectrum with sub-laser-cycle resolution in a cross correlation measurement. We measure isolated x-ray pulses of 1.8 (+0.7/-1.2) fs in duration, which are shorter than the oscillation cycle of the driving laser light (2.6 fs). Our techniques for generation and measurement offer sub-femtosecond resolution over a wide range of x-ray wavelengths, paving the way to experimental attosecond science. Tracing atomic processes evolving faster than the exciting light field is within reach.

Monitoring Alzheimer Amyloid Peptide Aggregation by EPR.

Plaques containing the aggregated beta-Amyloid (Abeta) peptide in the brain are the main indicators of Alzheimer's disease. Fibrils, the building blocks of plaques, can also be produced in vitro and consist of a regular arrangement of the peptide. The initial steps of fibril formation are not well understood and could involve smaller aggregates (oligomers) of Abeta. Such oligomers have even been implicated as the toxic agents. Here, a method to study oligomers on the time scale of aggregation is suggested. We have labeled the 40 residue Abeta peptide variant containing an N-terminal cysteine (cys-Abeta) with the MTSL [1-oxyl-2,2,5,5-tetramethyl-Delta-pyrroline-3-methyl] methanethiosulfonate spin label (SL-Abeta). Fibril formation in solutions of pure SL-Abeta and of SL-Abeta mixed with Abeta was shown by Congo-red binding and electron microscopy. Continuous-wave 9 GHz electron paramagnetic resonance reveals three fractions of different spin-label mobility: one attributed to monomeric Abeta, one to a multimer (8-15 monomers), and the last one to larger aggregates or fibrils. The approach, in principle, allows detection of oligomers on the time scale of aggregation.

Electronic decay of core-excited HCl molecules probed by THz streaking.

The ultrafast electronic decay of HCl molecules in the time domain after resonant core excitation was measured. Here, a Cl-2p core electron was promoted to the antibonding σ* orbital initiating molecular dissociation, and simultaneously, the electronic excitation relaxes via an Auger decay. For HCl, both processes compete on similar ultrashort femtosecond time scales. In order to measure the lifetime of the core hole excitation, we collinearly superimposed 40 fs soft x-ray pulses with intense terahertz (THz) radiation from the free-electron laser in Hamburg (FLASH). Electrons emitted from the molecules are accelerated (streaked) by the THz electric field where the resulting momentum change depends on the field's phase at the instant of ionization. Evaluation of a time-shift between the delay-dependent streaking spectra of photo- and Auger electrons yields a decay constant of (11 ± 2) fs for LMM Auger electrons. For further validation, the method was also applied to the MNN Auger decay of krypton. Reproduction of the value already published in the literature confirms that a temporal resolution much below the duration of the exciting x-ray pulses can be reached.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) in the cochlea: evidence for specific transcript expression of PAC1-R splice variants in rat microdissected cochlear subfractions.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from the hypothalamus, named for its high potency in stimulating adenylyl cyclase in pituitary cells. PACAP acts through the specific receptor PAC1-R to modulate the action of neurotransmitters, and additionally, to regulate cell viability via autocrine/intracrine mechanisms. Evidence has now been obtained that PACAP and multiple splice variants of PAC1-R are expressed in the rat cochlea. mRNA for PACAP precursor protein is found by reverse transcription-polymerase chain reaction (RT-PCR) in microdissected cochlear lateral wall, organ of Corti, and spiral ganglion subfractions. A specific pattern of expression of mRNA for PAC1-R splice variants, which mediate the response to PACAP, has been revealed by RT-PCR and cloning for the cochlear subfractions. Transcript for the short form of PAC1-R is found in all three subfractions. Four additional splice variants -- hop1, hop2, hip, and a novel hop1 splice variant -- are expressed in the lateral wall. For the amino terminus splice region of PAC1-R, a new splice variant has been detected in the organ of Corti, representing a deletion of the first 7 of 21 amino acids detected in the PAC1-R very-short sequence. Overall, from message determinations in cochlear subfractions, there are five PAC1-R splice variants in the lateral wall, two in the organ of Corti and one in the spiral ganglion, indicating multiple possible responses to PACAP and/or mechanisms to modulate the response to PACAP in the cochlea. The variety of PAC1-R splice variants expressed may reflect the diversity in cell function between subfractions that is modulated by PACAP. The neuropeptide and its specific receptor have been immunolocalized in the lateral wall, the source of the largest number of cochlear PAC1-R splice variants. The receptor was targeted by primary antibodies which would elicit immunoreactivity for all splice variants of PAC1-R detected with RT-PCR, and evidence has been obtained with Western blot analysis suggesting that PAC1-R is glycosylated in vivo. Within the lateral wall, PACAP and PAC1-R were immunolocalized primarily to the stria vascularis, with immunoreactivity for both neuropeptide and receptor increasing from the basal to apical cochlear turns. Within the stria, PACAP immunoreactivity was localized to the basolateral extensions of marginal cells, while PAC1-R was clearly associated with tight junctions between the marginal cells close to the endolymphatic compartment. In addition, evidence was obtained that PAC1-R was associated with endothelial cells of the capillaries in the stria vascularis. The large number of splice variants expressed, coupled to the specificity in linkage between PAC1-R splice variants and G-protein-coupled second messenger pathways, could provide a mechanism to closely modulate tight junction integrity in the stria vascularis, impacting the endolymphatic potential.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) are positioned to modulate afferent signaling in the cochlea.

Pituitary adenylyl cyclase-activating polypeptide (PACAP), via its specific receptor pituitary adenylyl cyclase-activating polypeptide receptor 1 (PAC1-R), is known to have roles in neuromodulation and neuroprotection associated with glutamatergic and cholinergic neurotransmission, which, respectively, are believed to form the primary basis for afferent and efferent signaling in the organ of Corti. Previously, we identified transcripts for PACAP preprotein and multiple splice variants of its receptor, PAC1-R, in microdissected cochlear subfractions. In the present work, neural localizations of PACAP and PAC1-R within the organ of Corti and spiral ganglion were examined, defining sites of PACAP action. Immunolocalization of PACAP and PAC1-R in the organ of Corti and spiral ganglion was compared with immunolocalization of choline acetyltransferase (ChAT) and synaptophysin as efferent neuronal markers, and glutamate receptor 2/3 (GluR2/3) and neurofilament 200 as afferent neuronal markers, for each of the three cochlear turns. Brightfield microscopy giving morphological detail for individual immunolocalizations was followed by immunofluorescence detection of co-localizations. PACAP was found to be co-localized with ChAT in nerve fibers of the intraganglionic spiral bundle and beneath the inner and outer hair cells within the organ of Corti. Further, evidence was obtained that PACAP is expressed in type I afferent axons leaving the spiral ganglion en route to the auditory nerve, potentially serving as a neuromodulator in axonal terminals. In contrast to the efferent localization of PACAP within the organ of Corti, PAC1-R immunoreactivity was co-localized with afferent dendritic neuronal marker GluR2/3 in nerve fibers passing beneath and lateral to the inner hair cell and in fibers at supranuclear and basal sites on outer hair cells. Given the known association of PACAP with catecholaminergic neurotransmission in sympathoadrenal function, we also re-examined the issue of whether the organ of Corti receives adrenergic innervation. We now demonstrate the existence of nerve fibers within the organ of Corti which are immunoreactive for the adrenergic marker dopamine beta-hydroxylase (DBH). DBH immunoreactivity was particularly prominent in nerve fibers both at the base and near the cuticular plate of outer hair cells of the apical turn, extending to the non-sensory Hensen's cell region. Evidence was obtained for limited co-localization of DBH with PAC1-R and PACAP. In the process of this investigation, we obtained evidence that efferent and afferent nerve fibers, in addition to adrenergic nerve fibers, are present at supranuclear sites on outer hair cells and distributed within the non-sensory epithelium of the apical cochlear turn for rat, based upon immunoreactivity for the corresponding neuronal markers. Overall, PACAP is hypothesized to act within the organ of Corti as an efferent neuromodulator of afferent signaling via PAC1-R that is present on type I afferent dendrites, in position to afford protection from excitotoxicity. Additionally, PACAP/PAC1-R may modulate secretion of catecholamines from adrenergic terminals within the organ of Corti.