Nox4 promotes endothelial differentiation through chromatin remodeling.

RATIONALE: Nox4 is a constitutively active NADPH oxidase that constantly produces low levels of H2O2. Thereby, Nox4 contributes to cell homeostasis and long-term processes, such as differentiation. The high expression of Nox4 seen in endothelial cells contrasts with the low abundance of Nox4 in stem cells, which are accordingly characterized by low levels of H2O2. We hypothesize that Nox4 is a major contributor to endothelial differentiation, is induced during the process of differentiation, and facilitates homeostasis of the resulting endothelial cells. OBJECTIVE: To determine the role of No×4 in differentiation of murine inducible pluripotent stem cells (miPSC) into endothelial cells (ECs). METHODS AND RESULTS: miPSC, generated from mouse embryonic wildtype (WT) and Nox4-/- fibroblasts, were differentiated into endothelial cells (miPSC-EC) by stimulation with BMP4 and VEGF. During this process, Nox4 expression increased and knockout of Nox4 prolonged the abundance of pluripotency markers, while expression of endothelial markers was delayed in differentiating Nox4-depleted iPSCs. Eventually, angiogenic capacity of iPSC-ECs is reduced in Nox4 deficient cells, indicating that an absence of Nox4 diminishes stability of the reached phenotype. As an underlying mechanism, we identified JmjD3 as a redox target of Nox4. iPSC-ECs lacking Nox4 display a lower nuclear abundance of the histone demethylase JmjD3, resulting in an increased triple methylation of histone 3 (H3K27me3), which serves as a repressive mark for several genes involved in differentiation. CONCLUSIONS: Nox4 promotes differentiation of miPSCs into ECs by oxidation of JmjD3 and subsequent demethylation of H3K27me3, which forced endothelial differentiation and stability.

Refraction effects in soft x-ray multilayer blazed gratings.

A 2500 lines/mm Multilayer Blazed Grating (MBG) optimized for the soft x-ray wavelength range was fabricated and tested. The grating coated with a W/B4C multilayer demonstrated a record diffraction efficiency in the 2nd blazed diffraction order in the energy range from 500 to 1200 eV. Detailed investigation of the diffraction properties of the grating demonstrated that the diffraction efficiency of high groove density MBGs is not limited by the normal shadowing effects that limits grazing incidence x-ray grating performance. Refraction effects inherent in asymmetrical Bragg diffraction were experimentally confirmed for MBGs. The refraction affects the blazing properties of the MBGs and results in a shift of the resonance wavelength of the gratings and broadening or narrowing of the grating bandwidth depending on diffraction geometry. The true blaze angle of the MBGs is defined by both the real structure of the multilayer stack and by asymmetrical refraction effects. Refraction effects can be used as a powerful tool in providing highly efficient suppression of high order harmonics.

Multilayer-coated blazed grating with variable line spacing and a variable blaze angle.

The blazing ability of multilayer-coated blazed gratings (MBGs) was systematically investigated via numerical calculation of the diffraction efficiency with a rigorous electromagnetic simulation code. It was found that the blazing condition is not exact and allows significant deviation from the ideal situation for ultra-dense MBGs. A mismatch of the interfaces of the multilayer (ML) stacks of adjacent grooves results in a modified effective blaze angle, which gives the opportunity to control and tune precisely the blaze angle via a proper choice of ML d-spacing. Also this allows a new kind of x-ray gratings that have a variable line spacing (VLS) as well as a variable blaze angle. Precise adjustment of a local blaze angle of a VLS MBG can be achieved with a laterally graded ML, providing very high diffraction efficiency for the whole area of the grating.

Enhancement of diffraction efficiency via higher-order operation of a multilayer blazed grating.

Imperfections in the multilayer stack deposited on a saw-tooth substrate are the main factor limiting the diffraction efficiency of extreme ultraviolet and soft x-ray multilayer-coated blazed gratings (MBGs). Since the multilayer perturbations occur in the vicinity of antiblazed facets of the substrates, reduction of the groove density of MBGs is expected to enlarge the area of unperturbed multilayer and result in higher diffraction efficiency. At the same time the grating should be optimized for higher-order operation in order to keep high dispersion and spectral resolution. In this work we show the validity of this approach and demonstrate significant enhancement of diffraction efficiency of MBGs using higher-order diffraction. A new record for diffraction efficiency of 52% in the second diffraction order was achieved for an optimized MBG with groove density of 2525 lines/mm at the wavelength of 13.4 nm.

Ultra-high efficiency multilayer blazed gratings through deposition kinetic control.

Diffraction efficiency of multilayer-coated blazed gratings (MBG) strongly depends on the perfection of the sawtooth-shaped layers in the overall composite structure. Growth of multilayers on sawtooth substrates should be carefully optimized to reduce groove profile distortion and, at the same time, to avoid significant roughening of multilayer interfaces. In this work, we report on a way to optimize growth of sputter-deposited Mo/Si multilayers on sawtooth substrates through variation of the sputtering gas pressure. We believe a new record for diffraction efficiency of 44% was achieved for an optimized MBG with groove density of 5250 lines/mm at the wavelength of 13.1 nm.

A 10,000 groove/mm multilayer coated grating for EUV spectroscopy.

Ultra-high spectral resolution in the EUV and soft x-ray energy ranges requires the use of very high line density gratings with optimal design resulting in use of a Blazed Multilayer Grating (BMG) structure. Here we demonstrate the production of near-atomically perfect Si blazed substrates with an ultra-high groove density (10,000 l/mm) together with the measured and theoretical performance of an Al/Zr multilayer coating on the grating. A 1st order absolute efficiency of 13% and 24.6% was achieved at incidence angles of 11° and 36° respectively. Cross-sectional TEM shows the effect of smoothing caused by the surface mobility of deposited atoms and we correlate this effect with a reduction in peak diffraction efficiency. This work shows the high performance that can be achieved with BMGs based on small-period anisotropic etched Si substrates, but also the constraints imposed by the surface mobility of deposited species.

Interferometer-controlled scanning transmission X-ray microscopes at the Advanced Light Source.

Two new soft X-ray scanning transmission microscopes located at the Advanced Light Source (ALS) have been designed, built and commissioned. Interferometer control implemented in both microscopes allows the precise measurement of the transverse position of the zone plate relative to the sample. Long-term positional stability and compensation for transverse displacement during translations of the zone plate have been achieved. The interferometer also provides low-distortion orthogonal x, y imaging. Two different control systems have been developed: a digital control system using standard VXI components at beamline 7.0, and a custom feedback system based on PC AT boards at beamline 5.3.2. Both microscopes are diffraction limited with the resolution set by the quality of the zone plates. Periodic features with 30 nm half period can be resolved with a zone plate that has a 40 nm outermost zone width. One microscope is operating at an undulator beamline (7.0), while the other is operating at a novel dedicated bending-magnet beamline (5.3.2), which is designed specifically to illuminate the microscope. The undulator beamline provides count rates of the order of tens of MHz at high-energy resolution with photon energies of up to about 1000 eV. Although the brightness of a bending-magnet source is about four orders of magnitude smaller than that of an undulator source, photon statistics limited operation with intensities in excess of 3 MHz has been achieved at high energy resolution and high spatial resolution. The design and performance of these microscopes are described.

Development of scanning X-ray microscopes for materials science spectromicroscopy at the Advanced Light Source.

The development of two zone-plate microscopes for X-ray spectroscopic analysis of materials is described. This pair of instruments will provide imaging NEXAFS analysis of samples in transmission at atmospheric pressure and imaging XPS and NEXAFS analysis of sample surfaces in a UHV environment.

Soft X-ray Monochromators for Third-Generation Undulator Sources.

Over the past ten years, the resolving power from new designs of bending-magnet-based soft X-ray monochromators has increased by more than an order of magnitude. This has led to a revolution in soft X-ray spectroscopy, but the limited flux at this high resolution has allowed only relatively efficient measurements to be made, such as photo-absorption. Application of this new tool of high-resolution spectroscopy to photoemission, energy-resolved fluorescence spectroscopy and microscopy has now been made possible with the advent of undulator sources of soft X-rays. Here we have reviewed the recent development of undulator-based soft X-ray monochromators, the special features of undulators in general and the resulting benefits and problems, and describe the state of the art undulator beamline, 7.0 at the Advanced Light Source. In addition, we offer some speculation as to the possible routes to the next or ultimate generation of soft X-ray monochromator.

Utilization of parenterally administered glucose oligosaccharides when infused with glucose and amino acids in postsurgical patients.

Utilization of intravenously administered oligosaccharides was tested in postsurgical patients by infusing them simultaneously with glucose and amino acids. Thirty two patients were infused with one of two parenteral regimens for four-day periods. Twenty-two patients received a nutritional regimen providing 46 g glucose, 32.5 g amino acids, and 45 g oligosaccharides per liter, while the remaining ten patients received the same solution without oligosaccharides. Patients infused with the test solution received an overall four-day mean +/- SD of 106 +/- 24.2 g oligosaccharide per day. The mean overall four-day excretion of total glucose (free plus oligosaccharide bound) was significantly higher (P less than 0.001) in patients infused with oligosaccharides (46.1 +/- 29.0 g/d) than in reference patients (1.19 +/- 1.20 g/d). Of the total carbohydrate excreted in patients receiving oligosaccharides, 11.3 +/- 13.1 g/d were glucose, 14.8 +/- 13.1 g/d were maltose plus maltotriose, and 20.8 +/- 19.3 g/d were oligosaccharides of maltotetraose size or larger. Although overall utilization of infused oligosaccharide for all patients was only 58.5% +/- 23.1%, three of the patients showed unexpectedly good utilization (94.6% +/- 1.24%).