The PERCIVAL detector: first user experiments.

The PERCIVAL detector is a CMOS imager designed for the soft X-ray regime at photon sources. Although still in its final development phase, it has recently seen its first user experiments: ptychography at a free-electron laser, holographic imaging at a storage ring and preliminary tests on X-ray photon correlation spectroscopy. The detector performed remarkably well in terms of spatial resolution achievable in the sample plane, owing to its small pixel size, large active area and very large dynamic range; but also in terms of its frame rate, which is significantly faster than traditional CCDs. In particular, it is the combination of these features which makes PERCIVAL an attractive option for soft X-ray science.

Clinical outcome and global gene expression data support the existence of the estrogen receptor-negative/progesterone receptor-positive invasive breast cancer phenotype.

The presence or absence of estrogen and progesterone steroid hormone receptor expression (ER, PR) is an essential feature of invasive breast cancer and determines prognosis and endocrine treatment decisions. Among the four ER/PR receptor phenotypes, the ER-/PR+ is infrequent, and its clinical relevance has been controversially discussed. Thus, we investigated its clinical significance and gene expression pattern in large datasets. In a retrospective clinical study of 15,747 breast cancer patients, we determined the ER/PR subtype survival probabilities using Kaplan-Meier and Cox regression analyses. From The Cancer Genome Atlas (TCGA) breast cancer dataset, PAM50 expression signature and pathway analyses were performed to test for distinct molecular features. In our cohort, the ER-/PR+ phenotype has been observed at a frequency of 4.1 % and was associated with an improved 10-year survival for stage I cancers compared to the ER+/PR+ reference subtype (median; 95 % CI 88.1 %; 83-93 vs. 84.3 %; 82-86 %, P = 0.024) as was confirmed by multivariate analysis over the entire follow-up (HR 0.59, 95 % CI 0.38-0.92, P = 0.021). This association lacked significance when including all stages. ER-/PR+ patients treated with antihormonal agents (34.5 %) had shorter survival compared to their non-treated counterparts (Log-rank P = 0.0001). PAM50 signatures suggest a distinct configuration for the ER-/PR+ phenotype. This specific phenotype has been further separated by a set of 59 uniquely expressed genes. Our study supports the notion of the existence of an ER-/PR+ phenotype with clinical and molecular features distinct from the large group of ER+/PR+ patients.

Breakdown of the x-ray resonant magnetic scattering signal during intense pulses of extreme ultraviolet free-electron-laser radiation.

We present results of single-shot resonant magnetic scattering experiments of Co/Pt multilayer systems using 100 fs long ultraintense pulses from an extreme ultraviolet (XUV) free-electron laser. An x-ray-induced breakdown of the resonant magnetic scattering channel during the pulse duration is observed at fluences of 5 J/cm(2). Simultaneously, the speckle contrast of the high-fluence scattering pattern is significantly reduced. We performed simulations of the nonequilibrium evolution of the Co/Pt multilayer system during the XUV pulse duration. We find that the electronic state of the sample is strongly perturbed during the first few femtoseconds of exposure leading to an ultrafast quenching of the resonant magnetic scattering mechanism.

Holographically aided iterative phase retrieval.

Fourier transform holography (FTH) is a noise-resistant imaging technique which allows for nanometer spatial resolution x-ray imaging, where the inclusion of a small reference scattering object provides the otherwise missing phase information. With FTH, one normally requires a considerable distance between the sample and the reference to ensure spatial separation of the reconstruction and its autocorrelation. We demonstrate however that this requirement can be omitted at the small cost of iteratively separating the reconstruction and autocorrelation. In doing so, the photon efficiency of FTH can be increased due to a smaller illumination area, and we show how the presence of the reference prevents the non-uniqueness problems often encountered with plane-wave iterative phase retrieval. The method was tested on a cobalt/platinum multilayer exhibiting out of plane magnetized domains, where the magnetic circular dichroism effect was used to image the magnetic domains at the cobalt L3-edge at 780eV.

Magnetostatic twists in room-temperature skyrmions explored by nitrogen-vacancy center spin texture reconstruction.

Magnetic skyrmions are two-dimensional non-collinear spin textures characterized by an integer topological number. Room-temperature skyrmions were recently found in magnetic multilayer stacks, where their stability was largely attributed to the interfacial Dzyaloshinskii-Moriya interaction. The strength of this interaction and its role in stabilizing the skyrmions is not yet well understood, and imaging of the full spin structure is needed to address this question. Here, we use a nitrogen-vacancy centre in diamond to measure a map of magnetic fields produced by a skyrmion in a magnetic multilayer under ambient conditions. We compute the manifold of candidate spin structures and select the physically meaningful solution. We find a Néel-type skyrmion whose chirality is not left-handed, contrary to preceding reports. We propose skyrmion tube-like structures whose chirality rotates through the film thickness. We show that NV magnetometry, combined with our analysis method, provides a unique tool to investigate this previously inaccessible phenomenon.

Truncation of the amino-terminus of the recombinant aggrecan rAgg1mut leads to reduced cleavage at the aggrecanase site. Efficient aggrecanase catabolism may depend on multiple substrate interactions.

Aggrecanase cleavage at the Glu(373)-Ala(374) site in the interglobular domain of the cartilage proteoglycan aggrecan is a key event in arthritic diseases. The observation that substrates representing only the aggrecanase cleavage site are not catabolized efficiently by aggrecanase prompted us to investigate the requirement of aggrecanase for additional structural elements of its substrate other than the actual cleavage site. Based on the recombinant substrate rAgg1mut we constructed deletion mutants with successively truncated N- or C-termini of the interglobular domain. Catabolism by aggrecanase activities induced in rat chondrosarcoma cells, porcine chondrocytes, and by human recombinant ADAMTS4 showed a gradually decreasing catabolism of progressively shortened, N-terminal deletion mutants of the substrate rAgg1mut. A reduction to 32 amino acids N-terminal to the aggrecanase site resulted in a decrease of at least 42% of aggrecanase cleavage products as compared with the wild-type substrate. When only 16 amino acids preceded the Glu(373)-Ala(374) site, aggrecanase cleavage was completely inhibited. In contrast, C-terminal deletions did not negatively affect aggrecanase cleavage up to the reduction to 13 amino acids C-terminal to the cleavage site. Unlike aggrecanase(s), membrane type 1-matrix metalloprotease (MT1-MMP), able to cleave rAgg1mut both at the aggrecanase and the MMP site, was insensitive to N-terminal deletions regarding aggrecanase cleavage, indicating that the importance of the N-terminus is characteristic for aggrecanase(s). Taken together, the results demonstrate that the amino-terminus of rAgg1mut, containing the MMP site, plays an important role for efficient cleavage by aggrecanase(s), possibly by serving as a further site of interaction between the enzyme and its substrate.

[Diagnosis of spondylitis by MR tomography]. / Diagnostik der Spondylitis durch die MR-Tomographie.

The results of MR tomography in 13 patients with specific and nonspecific spondylitis are reported. There were characteristic changes in signal intensity and in the configuration of the vertebral bodies and the intervertebral discs. The extent of intraspinal stenosis can be demonstrated accurately by MR. Paravertebral abscesses can be shown accurately and their position can be clarified by means of multiplanar sections. The early diagnosis of spondylitis by MR tomography is discussed.

Monolithic focused reference beam X-ray holography.

Fourier transform holography is a highly efficient and robust imaging method, suitable for single-shot imaging at coherent X-ray sources. In its common implementation, the image contrast is limited by the reference signal generated by a small pinhole aperture. Increased pinhole diameters improve the signal, whereas the resolution is diminished. Here we report a new concept to decouple the spatial resolution from the image contrast by employing a Fresnel zone plate to provide the reference beam. Superimposed on-axis images of distinct foci are separated with a novel algorithm. Our method is insensitive to mechanical drift or vibrations and allows for long integration times common at low-flux facilities like high harmonic generation sources. The application of monolithic focused reference beams improves the efficiency of high-resolution X-ray Fourier transform holography beyond all present approaches and paves the path towards sub-10 nm single-shot X-ray imaging.

Domain wall transformations and hopping in La(0.7)Sr(0.3)MnO(3) nanostructures imaged with high resolution x-ray magnetic microscopy.

We investigate the effect of electric current pulse injection on domain walls in La(0.7)Sr(0.3)MnO(3) (LSMO) half-ring nanostructures by high resolution x-ray magnetic microscopy at room temperature. Due to the easily accessible Curie temperature of LSMO, we can employ reasonable current densities to induce the Joule heating necessary to observe effects such as hopping of the domain walls between different pinning sites and nucleation/annihilation events. Such effects are the dominant features close to the Curie temperature, while spin torque is found to play a small role close to room temperature. We are also able to observe thermally activated domain wall transformations and we find that, for the analyzed geometries, the vortex domain wall configuration is energetically favored, in agreement with micromagnetic simulations.

[Transvaginal cholecystectomy: results of a randomized study]. / Transvaginale Cholezystektomie: Ergebnisse einer randomisierten Studie.

BACKGROUND: Transvaginal cholecystectomy (TVC) is regarded as a model operation in the newly developed field of natural orifice transluminal endoscopic surgery (NOTES). Randomized, controlled trials to assess TVC as a surgical strategy are largely missing. MATERIALS AND METHODS: The study was a double blind, randomized, controlled, single center trial in female patients > 18 years with symptomatic cholecystolithiasis comparing laparoscopic cholecystectomy (CLC) and TVC. The study investigated pain reduction of ≥ 1 point on a visual-numeric rating scale with a follow-up after 7 days. Secondary endpoints were complications and patient reported outcome. Groups were established using computer-generated randomization and sealed envelopes in the operating theatre. At the end of the surgical procedure all patients received a standard 4-trocar dressing as for CLC and a vaginal tamponade. RESULTS: A total of 426 patients were asked to participate, of which 97 were randomized, 51 in the CLC, 41 in the TVC groups and 5 were excluded from the study. Patients were comparable regarding age, body mass index (BMI) and American Society of Anesthesiologists (ASA) grade. Surgical and anesthesia times were significantly different. There was no difference in postoperative pain. The majority of patients were satisfied with both procedures and TVC was recommended to other patients by 93 % of patients in the TVC group. CONCLUSION: The results did not show superiority of TVC over CLC with regards to postoperative pain. With no differences in postoperative pain and high patient satisfaction, TVC can be recommended to future patients as an alternative method. For confirmation of this evaluation of TVC further randomized trials are needed.

Endstation for ultrafast magnetic scattering experiments at the free-electron laser in Hamburg.

An endstation for pump-probe small-angle X-ray scattering (SAXS) experiments at the free-electron laser in Hamburg (FLASH) is presented. The endstation houses a solid-state absorber, optical incoupling for pump-probe experiments, time zero measurement, sample chamber, and detection unit. It can be used at all FLASH beamlines in the whole photon energy range offered by FLASH. The capabilities of the setup are demonstrated by showing the results of resonant magnetic SAXS measurements on cobalt-platinum multilayer samples grown on freestanding Si(3)N(4) membranes and pump-laser-induced grid structures in multilayer samples.

Ultrafast optical demagnetization manipulates nanoscale spin structure in domain walls.

During ultrafast demagnetization of a magnetically ordered solid, angular momentum has to be transferred between the spins, electrons, and phonons in the system on femto- and picosecond timescales. Although the intrinsic spin-transfer mechanisms are intensely debated, additional extrinsic mechanisms arising due to nanoscale heterogeneity have only recently entered the discussion. Here we use femtosecond X-ray pulses from a free-electron laser to study thin film samples with magnetic domain patterns. We observe an infrared-pump-induced change of the spin structure within the domain walls on the sub-picosecond timescale. This domain-topography-dependent contribution connects the intrinsic demagnetization process in each domain with spin-transport processes across the domain walls, demonstrating the importance of spin-dependent electron transport between differently magnetized regions as an ultrafast demagnetization channel. This pathway exists independent from structural inhomogeneities such as chemical interfaces, and gives rise to an ultrafast spatially varying response to optical pump pulses.

Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris.

A soluble, C-terminal truncated form of human membrane type 1 matrix metalloproteinase (MT1-MMP) containing the hemopexin-like domain was expressed in Pichia pastoris strain KM71. High levels of secreted protein were detected. Although the c-DNA for the proenzyme (Ala(21)-Glu(523) called DeltaTM-MT1-MMP) was cloned, almost only active MT1-MMP (Tyr(112)-Glu(523)) with identical N-terminus as described for the wild-type enzyme was isolated. This active enzyme was highly purified and characterized with respect to its biochemical properties. The recombinant protein showed high stability against autolysis and proteolysis by yeast proteases, although the calculated in vivo half-life is rather low. The biochemical properties of this new MT1-MMP species were compared with the well-characterized catalytic domain (Ile(114)-Ile(318)) of MT1-MMP. The novel form of MT1-MMP exhibited a higher stability against autolysis than the isolated catalytic domain (Ile(114)-Ile(318)).

Utilization of a recombinant substrate rAgg1 to study the biochemical properties of aggrecanase in cell culture systems.

This paper describes the first report of the production and use of an artificial recombinant protein substrate to study "aggrecanase" activity. The substrate (rAgg1) is composed of the complete interglobular domain (IGD) of human aggrecan flanked by the "marker" sequences FLAGTM at the amino terminus and the human immunoglobulin G1 constant region at the carboxyl terminus. The expressed protein occurs as large multimolecular aggregates (>120 kDa) that, upon reduction, consist of a major isoform of 72 kDa (containing the IGD) and a minor 39-kDa species that through alternative splicing has had the IGD deleted. Using this recombinant substrate we developed a novel agarose cell culture system containing either rat chondrosarcoma or bovine chondrocytes that could be used in studies of the biochemical characterization of aggrecanase activities. These studies showed the following. (i) rAgg1 is a suitable substrate for aggrecanase proteolysis. (ii) Aggrecanase activity was specifically induced by exposing chondrocytes to retinoic acid. (iii) A considerable time period was required to synthesize and/or activate aggrecanase, with considerable differences in that found in rat chondrosarcoma versus bovine chondrocyte culture systems. (iv) Aggrecanase cleavage of the aggrecan IGD does not require the presence of the G1 or G2 globular domains or keratan sulfate post-translational modification in the IGD. (v) Aggrecanase is a diffusible activity that does not require association with the chondrocyte plasma membrane or immediate pericellular matrix for its action.

Differential expression of aggrecanase and matrix metalloproteinase activity in chondrocytes isolated from bovine and porcine articular cartilage.

The release of aggrecan catabolites from cartilage is an early event in the pathogenesis of degenerative joint diseases. The enzymes involved in this process are unknown, controversial, and the subject of intense investigation. In this paper we have utilized a recombinant substrate containing the interglobular domain (IGD) of aggrecan to study specifically aggrecanase versus matrix metalloproteinase (MMP) catabolism in this domain of aggrecan. Our studies have shown that (i) there are species differences in the expression of latent versus active MMP activity on the aggrecan IGD; (ii) interleukin-1alpha exposure induces both aggrecanase and MMP activities, whereas retinoic acid induces only aggrecanase activity and inhibits the MMP activity on the aggrecan IGD; (iii) activators of latent MMP activity (p-aminophenylmercuric acetate and trypsin) significantly reduce aggrecanase activity; (iv) the time course of the appearance of aggrecanase versus the MMP catabolism of aggrecan IGD differs; (v) aggrecanase is a protease with metalloprotease characteristics; however (vi) the physiological (tissue) inhibitors of MMPs show weak inhibition (TIMP-1) or no inhibition (TIMP-2) of aggrecanase activity. Collectively, these studies show that aggrecanase and MMP catabolism of the aggrecan IGD are independent and uncoupled.

Photooxygenation of the helimers of (-)-isocolchicine: regio- and facial selectivity of the [4 + 2] cycloaddition with singlet oxygen and surprising endoperoxide transformations.

Photooxygenation of the helimeric mixture of (-)-(M,7S)/(P,7S)-isocolchicine (6) with the superdienophile singlet oxygen has been studied. Cycloaddition occurred with high regioselectivity at the 7a,11-positions of the alkaloid and predominantly at the diene face anti to the amidic substituent at the stereogenic center C-7, leading to two endoperoxides 7 (syn) and 8 (anti) with an 1:7 ratio. The structure of the minor product 7 was established by X-ray analysis. Investigation of the triethylamine induced transformation of the predominant endoperoxide 8 furnished a mixture of two isomers (M,7S)-10a/(M,7S)-10b in a 2:1 ratio possibly with constitutional interconversion and with (M,7S)-9 as plausible intermediate. Treatment of this mixture with silica gel/ethyl acetate at ambient temperature surprisingly led to an atropenantiomerically pure colchicinoid (M,7S)-12 characterized by an eightmembered oxocine B-ring, the structure and absolute configuration of which could be determined by X-ray analysis. For the unprecedented formation of the novel colchicinoid (M,7S)-12 a plausible reaction pathway is suggested, involving a complete transfer of the (M) helical asymmetry of the intermediate (M)-11 into (S) asymmetry of the newly formed carbon center of (M,7S)-12. Prerequisite for such a scenario is the configurational stability of the intermediate pseudobiaryl (M)-11, under the conditions employed, allowing to transmit the axial chirality onto the chiral center of the product (M,7S)-12.