A Human Islet Cell-Culture System for High-Throuput screening.

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds

High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines.

Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo

A transformed view of cyclosporine

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62591/1/397471a0.pd

Many-body localization in a quantum simulator with programmable random disorder

When a system thermalizes it loses all local memory of its initial conditions. This is a general feature of open systems and is well described by equilibrium statistical mechanics. Even within a closed (or reversible) quantum system, where unitary time evolution retains all information about its initial state, subsystems can still thermalize using the rest of the system as an effective heat bath. Exceptions to quantum thermalization have been predicted and observed, but typically require inherent symmetries or noninteracting particles in the presence of static disorder. The prediction of many-body localization (MBL), in which disordered quantum systems can fail to thermalize in spite of strong interactions and high excitation energy, was therefore surprising and has attracted considerable theoretical attention. Here we experimentally generate MBL states by applying an Ising Hamiltonian with long-range interactions and programmably random disorder to ten spins initialized far from equilibrium. We observe the essential signatures of MBL: memory retention of the initial state, a Poissonian distribution of energy level spacings, and entanglement growth in the system at long times. Our platform can be scaled to higher numbers of spins, where detailed modeling of MBL becomes impossible due to the complexity of representing such entangled quantum states. Moreover, the high degree of control in our experiment may guide the use of MBL states as potential quantum memories in naturally disordered quantum systems.Comment: 9 pages, 9 figure

A massive, quiescent galaxy at redshift of z=3.717

In the early Universe finding massive galaxies that have stopped forming stars present an observational challenge as their rest-frame ultraviolet emission is negligible and they can only be reliably identified by extremely deep near-infrared surveys. These have revealed the presence of massive, quiescent early-type galaxies appearing in the universe as early as z$\sim$2, an epoch 3 Gyr after the Big Bang. Their age and formation processes have now been explained by an improved generation of galaxy formation models where they form rapidly at z$\sim$3-4, consistent with the typical masses and ages derived from their observations. Deeper surveys have now reported evidence for populations of massive, quiescent galaxies at even higher redshifts and earlier times, however the evidence for their existence, and redshift, has relied entirely on coarsely sampled photometry. These early massive, quiescent galaxies are not predicted by the latest generation of theoretical models. Here, we report the spectroscopic confirmation of one of these galaxies at redshift z=3.717 with a stellar mass of 1.7$\times$10$^{11}$ M$_\odot$ whose absorption line spectrum shows no current star-formation and which has a derived age of nearly half the age of the Universe at this redshift. The observations demonstrates that the galaxy must have quickly formed the majority of its stars within the first billion years of cosmic history in an extreme and short starburst. This ancestral event is similar to those starting to be found by sub-mm wavelength surveys pointing to a possible connection between these two populations. Early formation of such massive systems is likely to require significant revisions to our picture of early galaxy assembly.Comment: 6 pages, 7 figures. This is the final preprint corresponding closely to the published version. Uploaded 6 months after publication in accordance with Nature polic

Mosaic DNA imports with interspersions of recipient sequence after natural transformation of Helicobacter pylori

Helicobacter pylori colonizes the gastric mucosa of half of the human population, causing gastritis, ulcers, and cancer. H. pylori is naturally competent for transformation by exogenous DNA, and recombination during mixed infections of one stomach with multiple H. pylori strains generates extensive allelic diversity. We developed an in vitro transformation protocol to study genomic imports after natural transformation of H. pylori. The mean length of imported fragments was dependent on the combination of donor and recipient strain and varied between 1294 bp and 3853 bp. In about 10% of recombinant clones, the imported fragments of donor DNA were interrupted by short interspersed sequences of the recipient (ISR) with a mean length of 82 bp. 18 candidate genes were inactivated in order to identify genes involved in the control of import length and generation of ISR. Inactivation of the antimutator glycosylase MutY increased the length of imports, but did not have a significant effect on ISR frequency. Overexpression of mutY strongly increased the frequency of ISR, indicating that MutY, while not indispensable for ISR formation, is part of at least one ISR-generating pathway. The formation of ISR in H. pylori increases allelic diversity, and contributes to the uniquely low linkage disequilibrium characteristic of this pathogen

Mapping Dynamic Histone Acetylation Patterns to Gene Expression in Nanog-depleted Murine Embryonic Stem Cells

Embryonic stem cells (ESC) have the potential to self-renew indefinitely and to differentiate into any of the three germ layers. The molecular mechanisms for self-renewal, maintenance of pluripotency and lineage specification are poorly understood, but recent results point to a key role for epigenetic mechanisms. In this study, we focus on quantifying the impact of histone 3 acetylation (H3K9,14ac) on gene expression in murine embryonic stem cells. We analyze genome-wide histone acetylation patterns and gene expression profiles measured over the first five days of cell differentiation triggered by silencing Nanog, a key transcription factor in ESC regulation. We explore the temporal and spatial dynamics of histone acetylation data and its correlation with gene expression using supervised and unsupervised statistical models. On a genome-wide scale, changes in acetylation are significantly correlated to changes in mRNA expression and, surprisingly, this coherence increases over time. We quantify the predictive power of histone acetylation for gene expression changes in a balanced cross-validation procedure. In an in-depth study we focus on genes central to the regulatory network of Mouse ESC, including those identified in a recent genome-wide RNAi screen and in the PluriNet, a computationally derived stem cell signature. We find that compared to the rest of the genome, ESC-specific genes show significantly more acetylation signal and a much stronger decrease in acetylation over time, which is often not reflected in an concordant expression change. These results shed light on the complexity of the relationship between histone acetylation and gene expression and are a step forward to dissect the multilayer regulatory mechanisms that determine stem cell fate.Comment: accepted at PLoS Computational Biolog

Pathophysiology of ANCA-Associated Small Vessel Vasculitis

Antineutrophil cytoplasmic autoantibodies (ANCAs) directed to proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA) are strongly associated with the ANCA-associated vasculitides—Wegener’s granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome. Clinical observations, including the efficacy of B-cell depletion via rituximab treatment, support—but do not prove—a pathogenic role for ANCA in the ANCA-associated vasculitides. In vitro experimental studies show that the interplay of ANCA, neutrophils, the alternative pathway of the complement system, and endothelial cells could result in lysis of the endothelium. A pathogenic role for MPO-ANCA is strongly supported by in vivo experimental studies in mice and rats, which also elucidate the pathogenic mechanisms involved in lesion development. Unfortunately, an animal model for PR3-ANCA–associated Wegener’s granulomatosis is not yet available. Here, cellular immunity appears to play a major role as well, particularly via interleukin-17–producing T cells, in line with granulomatous inflammation in the lesions. Finally, microbial factors, in particular Staphylococcus aureus and gram-negative bacteria, seem to be involved in disease induction and expression, but further studies are needed to define their precise role in disease development