Applying Mass Cytometry to the Analysis of Lymphoid Populations in Transplantation.

Single-cell flow cytometric techniques have been indispensable to improving our understanding of the phenotype and function of immune cell subsets that are important in both rejection and tolerance after transplant. Mass cytometry, or cytometry by time of flight, is a single-cell-based platform that utilizes antibodies conjugated to rare heavy metal ions for analysis of cellular proteins by a time-of-flight mass spectrometer. This new technology allows for the evaluation of >40 simultaneous cellular parameters in a single sample because the limitation of spectral overlap, seen in conventional flow cytometry, is eliminated. In this review, we discuss the current state of mass cytometry, describe the advantages and disadvantages compared with multiparameter flow cytometry, introduce novel methods of high-dimensional data analysis and visualization, and review some recent studies using mass cytometry to profile the immune systems of healthy people and transplant recipients.

Epstein-Barr virus modulates host cell microRNA-194 to promote IL-10 production and B lymphoma cell survival.

Epstein-Barr virus (EBV) is a γ-herpesvirus that is linked to the development of posttransplant lymphoproliferative disorder (PTLD) in solid organ recipients. We previously demonstrated that EBV(+) B cell lymphoma cell lines isolated from patients with PTLD produce human IL-10 as an autocrine growth factor. However, little is known regarding IL-10 regulation in B cells. Here we show that EBV infection markedly alters the expression of host B cell microRNA, a class of small noncoding RNA that is an important regulator of transcriptional and posttranscriptional gene expression. Gene arrays reveal unique microRNA profiles in EBV(+) B cell lymphoma lines from patients with PTLD, compared to normal B cells or in vitro generated EBV(+) lymphoblastoid cell lines. We show that microRNA-194 expression is uniquely suppressed in EBV(+) B cell lines from PTLD patients and that the 3'untranslated region of IL-10 is targeted by microRNA-194. Overexpression of microRNA-194 attenuates IL-10 production and increases apoptosis of EBV(+) B cell lymphoma lines. Together, these data indicate that EBV co-opts the host B cell microRNA network and specifically suppresses microRNA-194 to override control of IL-10 expression. Thus, modulation of microRNA-194 may constitute a novel approach to inhibiting proliferation of EBV(+) B cell lymphomas in PTLD.

Extracting depth information of 3-dimensional structures from a single-view X-ray Fourier-transform hologram.

We demonstrate how information about the three-dimensional structure of an object can be extracted from a single Fourier-transform X-ray hologram. In contrast to lens-based 3D imaging approaches that provide depth information of a specimen utilizing several images from different angles or via adjusting the focus to different depths, our method capitalizes on the use of the holographically encoded phase and amplitude information of the object's wavefield. It enables single-shot measurements of 3D objects at coherent X-ray sources. As the ratio of longitudinal resolution over transverse resolution scales proportional to the diameter of the reference beam aperture over the X-ray wavelength, we expect the approach to be particularly useful in the extreme ultraviolet and soft-X-ray regime.

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.

Exploration of magnetic fluctuations in PdFe films.

We investigated magnetic phase transitions, magnetic anisotropy, and magnetic domains in Pd1-xFex alloys with different Fe concentrations x = 2.2-7.2%. The Curie temperature depends linearly on the Fe concentration in the regime studied. The magnetization is dominantly in-plane with a small out-of-plane remanent contribution. Resonant magnetic small angle scattering with circularly polarized x-rays tuned to the L3 resonance edge of Fe revealed a small angle scattering ring corresponding to magnetic domain fluctuations on a length scale of 100 nm. These fluctuations are isotropically distributed in the film plane and appear to have an out-of-plane component. On increasing the transverse coherence of the incident beam, the scattering ring decomposes in a speckle pattern, indicative of magnetic correlations on a length scale smaller than the x-ray coherence length of about 4 µm.

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.

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.

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.

The soft x-ray instrument for materials studies at the linac coherent light source x-ray free-electron laser.

The soft x-ray materials science instrument is the second operational beamline at the linac coherent light source x-ray free electron laser. The instrument operates with a photon energy range of 480-2000 eV and features a grating monochromator as well as bendable refocusing mirrors. A broad range of experimental stations may be installed to study diverse scientific topics such as: ultrafast chemistry, surface science, highly correlated electron systems, matter under extreme conditions, and laboratory astrophysics. Preliminary commissioning results are presented including the first soft x-ray single-shot energy spectrum from a free electron laser.

Regulation of cAMP-sensitive colonic epithelial Na+ channel in oocyte expression system.

In amphibian epithelia and in cortical collecting duct the antidiuretic peptide arginine-vasopressin (AVP) stimulates activity of epithelial Na+ channels (ENaCs). Generally, the AVP action upon Na+ (re)absorption is believed to be a cAMP/protein-kinase-A mediated mechanism. In the Xenopus oocyte expression system, however, a clear stimulation of ENaC activity by cAMP could not be reproduced with channel subunits cloned from A6 cells or rat colon. We have recently shown that membrane-permeant 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) stimulates activity of a hybrid ENaC in Xenopus oocytes, that consists of an alpha-subunit cloned from guinea-pig colon and the beta- and gamma-subunit originating from rat colon (gpalpharbetagammaENaC). In the present study, we have further investigated the mechanisms by which cpt-cAMP upregulates gpalpharbetagammaENaC activity. Interestingly, we found AVP to stimulate the gpalpharbetagammaENaC in oocytes. Also, treatment with GTP-gamma-S largely activated this channel. In contrast, as a conflicting result, forskolin had no stimulatory effect on the cAMP-sensitive gpalpharbetagammaENaC. Experiments with Brefeldin A (BFA) or nocodazole suggested that only a minor part of cpt-cAMP-induced activation is probably due to an additional translocation of channel proteins into the oocyte membrane. In conclusion, the stimulatory effect of synthetic cpt-cAMP does not seem to be exclusively provided by classical cAMP/PKA-associated transduction mechanisms, i.e., as in A6 cells.