A Universal Live Cell Barcoding-Platform for Multiplexed Human Single Cell Analysis.

Single-cell barcoding enables the combined processing and acquisition of multiple individual samples as one. This maximizes assay efficiency and eliminates technical variability in both sample preparation and analysis. Remaining challenges are the barcoding of live, unprocessed cells to increase downstream assay performance combined with the flexibility of the approach towards a broad range of cell types. To that end, we developed a novel antibody-based platform that allows the robust barcoding of live human cells for mass cytometry (CyTOF). By targeting both the MHC class I complex (beta-2-microglobulin) and a broadly expressed sodium-potassium ATPase-subunit (CD298) with platinum-conjugated antibodies, human immune cells, stem cells as well as tumor cells could be multiplexed in the same single-cell assay. In addition, we present a novel palladium-based covalent viability reagent compatible with this barcoding strategy. Altogether, this platform enables mass cytometry-based, live-cell barcoding across a multitude of human sample types and provides a scheme for multiplexed barcoding of human single-cell assays in general

New Relativistic Particle-In-Cell Simulation Studies of Prompt and Early Afterglows from GRBs

Nonthermal radiation observed from astrophysical systems containing relativistic jets and shocks, e.g., gamma-ray bursts (GRBs), active galactic nuclei (AGNs), and microquasars commonly exhibit power-law emission spectra. Recent PIC simulations of relativistic electron-ion (or electron-positron) jets injected into a stationary medium show that particle acceleration occurs within the downstream jet. In collisionless, relativistic shocks, particle (electron, positron, and ion) acceleration is due to plasma waves and their associated instabilities (e.g., the Weibel (filamentation) instability) created in the shock region. The simulations show that the Weibel instability is responsible for generating and amplifying highly non-uniform, small-scale magnetic fields. These fields contribute to the electron's transverse deflection behind the jet head. The resulting "jitter" radiation from deflected electrons has different properties compared to synchrotron radiation, which assumes a uniform magnetic field. Jitter radiation may be important for understanding the complex time evolution and/or spectra in gamma-ray bursts, relativistic jets in general, and supernova remnants.Comment: : 4 pages, 1 figure and 1 table, typos are corrected, submitted for the Proceedings of The 4th Heidelberg International Symposium on High Energy Gamma-Ray Astronomy, July 7-11, 2008, in Heidelberg, German

Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy.

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery

The Rapid Outbursting Star GM Cep: An EX-or in Tr 37?

We present optical, IR and millimeter observations of the solar-type star 13-277, also known as GM Cep, in the 4 Myr-old cluster Tr 37. GM Cep experiences rapid magnitude variations of more than 2 mag at optical wavelengths. We explore the causes of the variability, which seem to be dominated by strong increases in the accretion, being similar to EX-or episodes. The star shows high, variable accretion rates (up to ~10$^{-6}$ Msun/yr), signs of powerful winds, and it is a very fast rotator (Vsini~43 km/s). Its strong mid-IR excesses reveal a very flared disk and/or a remnant envelope, most likely out of hydrostatic equilibrium. The 1.3 millimeter fluxes suggest a relatively massive disk (Mdisk~0.1 Msun). Nevertheless, the millimeter mass is not enough to sustain increased accretion episodes over large timescales, unless the mass is underestimated due to significant grain growth. We finally explore the possibility of GM Cep having a binary companion, which could trigger disk instabilities producing the enhanced accretion episodes.Comment: 43 pages, including 10 figures, ApJ in pres

High-Dimensional Single-Cell Mapping of Central Nervous System Immune Cells Reveals Distinct Myeloid Subsets in Health, Aging, and Disease

Individual reports suggest that the central nervous system (CNS) contains multiple immune cell types with diverse roles in tissue homeostasis, immune defense, and neurological diseases. It has been challenging to map leukocytes across the entire brain, and in particular in pathology, where phenotypic changes and influx of blood-derived cells prevent a clear distinction between reactive leukocyte populations. Here, we applied high-dimensional single-cell mass and fluorescence cytometry, in parallel with genetic fate mapping systems, to identify, locate, and characterize multiple distinct immune populations within the mammalian CNS. Using this approach, we revealed that microglia, several subsets of border-associated macrophages and dendritic cells coexist in the CNS at steady state and exhibit disease-specific transformations in the immune microenvironment during aging and in models of Alzheimer's disease and multiple sclerosis. Together, these data and the described framework provide a resource for the study of disease mechanisms, potential biomarkers, and therapeutic targets in CNS disease

Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates >= 98% of peripheral immune cells with >= 4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery

CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets [version 3; peer review: 2 approved]

High-dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high-throughput interrogation and characterization of cell populations. Here, we present an updated R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signalling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models or linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g., multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g., plots of aggregated signals)

Chaotic, memory and cooling rate effects in spin glasses: Is the Edwards-Anderson model a good spin glass?

We investigate chaotic, memory and cooling rate effects in the three dimensional Edwards-Anderson model by doing thermoremanent (TRM) and AC susceptibility numerical experiments and making a detailed comparison with laboratory experiments on spin glasses. In contrast to the experiments, the Edwards-Anderson model does not show any trace of re-initialization processes in temperature change experiments (TRM or AC). A detailed comparison with AC relaxation experiments in the presence of DC magnetic field or coupling distribution perturbations reveals that the absence of chaotic effects in the Edwards-Anderson model is a consequence of the presence of strong cooling rate effects. We discuss possible solutions to this discrepancy, in particular the smallness of the time scales reached in numerical experiments, but we also question the validity of the Edwards-Anderson model to reproduce the experimental results.Comment: 17 pages, 10 figures. The original version of the paper has been split in two parts. The second part is now available as cond-mat/010224

GRIPS - Gamma-Ray Imaging, Polarimetry and Spectroscopy

We propose to perform a continuously scanning all-sky survey from 200 keV to 80 MeV achieving a sensitivity which is better by a factor of 40 or more compared to the previous missions in this energy range. The Gamma-Ray Imaging, Polarimetry and Spectroscopy (GRIPS) mission addresses fundamental questions in ESA's Cosmic Vision plan. Among the major themes of the strategic plan, GRIPS has its focus on the evolving, violent Universe, exploring a unique energy window. We propose to investigate $\gamma$-ray bursts and blazars, the mechanisms behind supernova explosions, nucleosynthesis and spallation, the enigmatic origin of positrons in our Galaxy, and the nature of radiation processes and particle acceleration in extreme cosmic sources including pulsars and magnetars. The natural energy scale for these non-thermal processes is of the order of MeV. Although they can be partially and indirectly studied using other methods, only the proposed GRIPS measurements will provide direct access to their primary photons. GRIPS will be a driver for the study of transient sources in the era of neutrino and gravitational wave observatories such as IceCUBE and LISA, establishing a new type of diagnostics in relativistic and nuclear astrophysics. This will support extrapolations to investigate star formation, galaxy evolution, and black hole formation at high redshifts.Comment: to appear in Exp. Astron., special vol. on M3-Call of ESA's Cosmic Vision 2010; 25 p., 25 figs; see also www.grips-mission.e

Circulating Glucagon 1-61 Regulates Blood Glucose by Increasing Insulin Secretion and Hepatic Glucose Production

Glucagon is secreted from pancreatic a cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in b cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion