Dissecting heterogeneous cell populations across drug and disease conditions with PopAlign

Single-cell measurement techniques can now probe gene expression in heterogeneous cell populations from the human body across a range of environmental and physiological conditions. However, new mathematical and computational methods are required to represent and analyze gene expression changes that occur in complex mixtures of single cells as they respond to signals, drugs, or disease states. Here, we introduce a mathematical modeling platform, PopAlign, that automatically identifies subpopulations of cells within a heterogeneous mixture, and tracks gene expression and cell abundance changes across subpopulations by constructing and comparing probabilistic models. We apply PopAlign to analyze the impact of 42 different immunomodulatory compounds on a heterogeneous population of donor-derived human immune cells as well as patient-specific disease signatures in multiple myeloma. PopAlign scales to comparisons involving tens to hundreds of samples, enabling large-scale studies of natural and engineered cell populations as they respond to drugs, signals or physiological change

Star Formation at 4<z<64 < z < 6 From the Spitzer Large Area Survey with Hyper-Suprime-Cam (SPLASH)

Using the first 50% of data collected for the Spitzer Large Area Survey with Hyper-Suprime-Cam (SPLASH) observations on the 1.8 deg$^2$ Cosmological Evolution Survey (COSMOS) we estimate the masses and star formation rates of 3398 $M_*>10^{10}M_\odot$ star-forming galaxies at $4 < z < 6$ with a substantial population up to $M_* \gtrsim 10^{11.5} M_\odot$. We find that the strong correlation between stellar mass and star formation rate seen at lower redshift (the "main sequence" of star-forming galaxies) extends to $z\sim6$. The observed relation and scatter is consistent with a continued increase in star formation rate at fixed mass in line with extrapolations from lower-redshift observations. It is difficult to explain this continued correlation, especially for the most massive systems, unless the most massive galaxies are forming stars near their Eddington-limited rate from their first collapse. Furthermore, we find no evidence for moderate quenching at higher masses, indicating quenching either has not occurred prior to $z \sim 6$ or else occurs rapidly, so that few galaxies are visible in transition between star-forming and quenched.Comment: ApJL, accepte

Reduction Algorithms for the Multiband Imaging Photometer for Spitzer

We describe the data reduction algorithms for the Multiband Imaging Photometer for Spitzer (MIPS) instrument. These algorithms were based on extensive preflight testing and modeling of the Si:As (24 micron) and Ge:Ga (70 and 160 micron) arrays in MIPS and have been refined based on initial flight data. The behaviors we describe are typical of state-of-the-art infrared focal planes operated in the low backgrounds of space. The Ge arrays are bulk photoconductors and therefore show a variety of artifacts that must be removed to calibrate the data. The Si array, while better behaved than the Ge arrays, does show a handful of artifacts that also must be removed to calibrate the data. The data reduction to remove these effects is divided into three parts. The first part converts the non-destructively read data ramps into slopes while removing artifacts with time constants of the order of the exposure time. The second part calibrates the slope measurements while removing artifacts with time constants longer than the exposure time. The third part uses the redundancy inherit in the MIPS observing modes to improve the artifact removal iteratively. For each of these steps, we illustrate the relevant laboratory experiments or theoretical arguments along with the mathematical approaches taken to calibrate the data. Finally, we describe how these preflight algorithms have performed on actual flight data.Comment: 21 pages, 16 figures, PASP accepted (May 2005 issue), version of paper with full resolution images is available at http://dirty.as.arizona.edu/~kgordon/papers/PS_files/mips_dra.pd

On-orbit performance of the MIPS instrument

The Multiband Imaging Photometer for Spitzer (MIPS) provides long wavelength capability for the mission, in imaging bands at 24, 70, and 160 microns and measurements of spectral energy distributions between 52 and 100 microns at a spectral resolution of about 7%. By using true detector arrays in each band, it provides both critical sampling of the Spitzer point spread function and relatively large imaging fields of view, allowing for substantial advances in sensitivity, angular resolution, and efficiency of areal coverage compared with previous space far-infrared capabilities. The Si:As BIB 24 micron array has excellent photometric properties, and measurements with rms relative errors of 1% or better can be obtained. The two longer wavelength arrays use Ge:Ga detectors with poor photometric stability. However, the use of 1.) a scan mirror to modulate the signals rapidly on these arrays, 2.) a system of on-board stimulators used for a relative calibration approximately every two minutes, and 3.) specialized reduction software result in good photometry with these arrays also, with rms relative errors of less than 10%

Mechanisms and applications of Cas13 mediated RNA targeting

The rapid evolutionary arms race between bacteria and mobile genetic elements has generated a large repertoire of bacterial defense strategies. CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR associated) systems are adaptive immune systems that protect hosts from invading nucleic acids. Cas proteins use RNA guides that target them to foreign sequences, leading to their destructions. A diverse set of Cas effector proteins exist, which differ in the molecular mechanisms behind target recognition, and nucleic acid cleavage. The programmable cleavage of a target DNA by CRISPR-Cas systems has greatly advanced the field of genome editing, both as a research tool and a potential therapeutic. While most Cas single effectors proteins target DNA, the discovery of RNA targeting Type VI CRISPR systems (Cas13) opened the door for programmable engineering of the transcriptome. Nevertheless, the collateral cleavage induced by the Higher Eukaryote Prokaryote Nucleotide binding domain (HEPN) of Cas13 enzymes leads to a high degree of toxicity in certain cell types, and the molecular mechanisms behind target recognition and binding remain unclear. In this work, we first profiled the ability of various catalytically inactive Cas13 enzymes to inhibit translation of an mRNA in E. coli when targeted to the proximity of a ribosome binding site (RBS). While Cas13d enzymes possessed the ability to interfere with the translation machinery, all Cas13a enzymes tested showed a lack of translational repression. To this end, we performed a high throughput directed evolution screen on Cas13a and identified a series of stabilizing mutations that greatly increased both protein expression and translational repression. Stabilization of Cas13a led to altered cleavage kinetics in vitro, and altered binding properties, shedding light into the kinetic mechanism of Cas13a. To further investigate the molecular mechanism behind Cas13 activator binding, we performed high throughput mismatch profiling for three different Lbu Cas13a mutants isolated above and show that these stabilizing mutations provide insight into the coupling of Cas13a RNA-binding and HEPN nuclease activity. Combining sets of mutations together led to a complete abolishment of single mismatch sensitivity with respect to binding but had no effect on mismatch sensitivity for HEPN activation, suggesting a link between protein stabilization and mismatch sensitivity. We propose a model in which initial seed binding, followed by duplex propagation, leads to an unstable intermediate, lacking stabilizing binary and/or ternary contacts and displaying a high activator off-rate. Once duplex propagation has completed, ternary contacts can fully stabilize the complex, and the HEPN senses proper base pairing, which leads to nuclease activation. Finally, we sought to increase the detection limit of conventional Cas13 based RNA detection assay using feedforward loops encoded by RNA structures, in a process termed Nuclease Chain Reaction. We present our initial trials in developing efficient feedback systems driven by Cas13 nuclease activity. We attempted to design various “caged” activator sequences that are ineffective at switching on the nuclease domain, unless cleaved by an upstream RNAse. Such substrates can be supplemented to normal Cas13 detection at a high concentration, along with a secondary Cas13 RNP which targets the uncaged activator, in hopes of increasing the time to detection, as well as the limit of detection. Testing these various caged RNA structures also shed light into non-specific RNA association mediated by Cas13, as well as requirements for HEPN activation

Voting and contributing when the group is watching

Abstract Members of groups and organizations often have to decide on rules that regulate their contributions to common tasks. They typically differ in their propensity to contribute and often care about the image they project, in particular want to be perceived by other group members as being high contributors. In such environments we study the interaction between the way members vote on rules and their subsequent contribution decisions. We show that multiple norms can emerge. We draw surprising policy implications, on the effect of group size, of supermajority rules and of the observability of actions