Characterizing and inferring quantitative cell cycle phase in single-cell RNA-seq data analysis.

Cellular heterogeneity in gene expression is driven by cellular processes, such as cell cycle and cell-type identity, and cellular environment such as spatial location. The cell cycle, in particular, is thought to be a key driver of cell-to-cell heterogeneity in gene expression, even in otherwise homogeneous cell populations. Recent advances in single-cell RNA-sequencing (scRNA-seq) facilitate detailed characterization of gene expression heterogeneity and can thus shed new light on the processes driving heterogeneity. Here, we combined fluorescence imaging with scRNA-seq to measure cell cycle phase and gene expression levels in human induced pluripotent stem cells (iPSCs). By using these data, we developed a novel approach to characterize cell cycle progression. Although standard methods assign cells to discrete cell cycle stages, our method goes beyond this and quantifies cell cycle progression on a continuum. We found that, on average, scRNA-seq data from only five genes predicted a cell's position on the cell cycle continuum to within 14% of the entire cycle and that using more genes did not improve this accuracy. Our data and predictor of cell cycle phase can directly help future studies to account for cell cycle-related heterogeneity in iPSCs. Our results and methods also provide a foundation for future work to characterize the effects of the cell cycle on expression heterogeneity in other cell types.

Discovery and characterization of variance QTLs in human induced pluripotent stem cells.

Quantification of gene expression levels at the single cell level has revealed that gene expression can vary substantially even across a population of homogeneous cells. However, it is currently unclear what genomic features control variation in gene expression levels, and whether common genetic variants may impact gene expression variation. Here, we take a genome-wide approach to identify expression variance quantitative trait loci (vQTLs). To this end, we generated single cell RNA-seq (scRNA-seq) data from induced pluripotent stem cells (iPSCs) derived from 53 Yoruba individuals. We collected data for a median of 95 cells per individual and a total of 5,447 single cells, and identified 235 mean expression QTLs (eQTLs) at 10% FDR, of which 79% replicate in bulk RNA-seq data from the same individuals. We further identified 5 vQTLs at 10% FDR, but demonstrate that these can also be explained as effects on mean expression. Our study suggests that dispersion QTLs (dQTLs) which could alter the variance of expression independently of the mean can have larger fold changes, but explain less phenotypic variance than eQTLs. We estimate 4,015 individuals as a lower bound to achieve 80% power to detect the strongest dQTLs in iPSCs. These results will guide the design of future studies on understanding the genetic control of gene expression variance.

Impact of regulatory variation across human iPSCs and differentiated cells.

Induced pluripotent stem cells (iPSCs) are an essential tool for studying cellular differentiation and cell types that are otherwise difficult to access. We investigated the use of iPSCs and iPSC-derived cells to study the impact of genetic variation on gene regulation across different cell types and as models for studies of complex disease. To do so, we established a panel of iPSCs from 58 well-studied Yoruba lymphoblastoid cell lines (LCLs); 14 of these lines were further differentiated into cardiomyocytes. We characterized regulatory variation across individuals and cell types by measuring gene expression levels, chromatin accessibility, and DNA methylation. Our analysis focused on a comparison of inter-individual regulatory variation across cell types. While most cell-type-specific regulatory quantitative trait loci (QTLs) lie in chromatin that is open only in the affected cell types, we found that 20% of cell-type-specific regulatory QTLs are in shared open chromatin. This observation motivated us to develop a deep neural network to predict open chromatin regions from DNA sequence alone. Using this approach, we were able to use the sequences of segregating haplotypes to predict the effects of common SNPs on cell-type-specific chromatin accessibility.

Reprogramming LCLs to iPSCs Results in Recovery of Donor-Specific Gene Expression Signature.

Renewable in vitro cell cultures, such as lymphoblastoid cell lines (LCLs), have facilitated studies that contributed to our understanding of genetic influence on human traits. However, the degree to which cell lines faithfully maintain differences in donor-specific phenotypes is still debated. We have previously reported that standard cell line maintenance practice results in a loss of donor-specific gene expression signatures in LCLs. An alternative to the LCL model is the induced pluripotent stem cell (iPSC) system, which carries the potential to model tissue-specific physiology through the use of differentiation protocols. Still, existing LCL banks represent an important source of starting material for iPSC generation, and it is possible that the disruptions in gene regulation associated with long-term LCL maintenance could persist through the reprogramming process. To address this concern, we studied the effect of reprogramming mature LCL cultures from six unrelated donors to iPSCs on the ensuing gene expression patterns within and between individuals. We show that the reprogramming process results in a recovery of donor-specific gene regulatory signatures, increasing the number of genes with a detectable donor effect by an order of magnitude. The proportion of variation in gene expression statistically attributed to donor increases from 6.9% in LCLs to 24.5% in iPSCs (P < 10-15). Since environmental contributions are unlikely to be a source of individual variation in our system of highly passaged cultured cell lines, our observations suggest that the effect of genotype on gene regulation is more pronounced in iPSCs than in LCLs. Our findings indicate that iPSCs can be a powerful model system for studies of phenotypic variation across individuals in general, and the genetic association with variation in gene regulation in particular. We further conclude that LCLs are an appropriate starting material for iPSC generation.

Examining the potential for detecting simultaneous noble gas and aerosol samples in the international monitoring system radionuclide network.

The purpose of the Comprehensive Nuclear-Test-Ban Treaty (CTBT) is to establish a legally binding ban on nuclear weapon test explosions or any other nuclear explosions. The Preparatory Commission for the CTBT Organization (CTBTO PrepCom) is developing the International Monitoring System (IMS) that includes a global network of 80 stations to monitor for airborne radionuclides upon entry into force of the CTBT. All 80 radionuclide stations will monitor for particulate radionuclides and at least half of the stations will monitor for radioxenon. The airborne radionuclide monitoring is an important verification technology both for the detection of a radionuclide release and in the determination of whether the release event originates from a nuclear explosion as opposed to an industrial use of nuclear materials. Nuclear power plants and many medical isotope production facilities release radioxenon into the atmosphere. Low levels of a few particulate isotopes, such as iodine, may also be released. Detections of multiple isotopes are useful for screening the radionuclide samples for relevance to the Treaty. This paper examines the anticipated joint detections in the IMS of noble gas and particulate isotopes from underground nuclear explosions where breaches in the underground containment vents from low levels to up to 1% of the radionuclide inventory of the resulting fission products to the atmosphere. Detection probabilities are based on 844 simulated release events spaced out at 17 release locations and one year in time. Six different release (venting) scenarios, including two fractionated scenarios, were analyzed. When ranked by detection probability, 11 particulate isotopes and one noble gas isotope (133Xe) appear in the top 20 isotopes for all six release scenarios. Using the 11 particulate isotopes and the one noble gas isotope, the IMS has nearly the same detection probability as when 45 particulate and 4 noble gas isotopes are used. Thus, a limited list of relevant radionuclides may be sufficient for treaty verification purposes. The probability that at least one particulate and at least one radioxenon isotope would be detected in the IMS from the release events ranged from 0.15 to 0.86 depending on the release scenario.

Projected network performance for next generation aerosol monitoring systems.

Aerosol monitoring for radioactivity is a mature and proven technology. However, by improving key specifications of aerosol monitoring equipment, more samples per day can be collected and analyzed with the same minimum detectable concentrations as current systems. This work models hypothetical releases of 140Ba and 131I over a range of magnitudes corresponding to the inventory produced from the fission of about 100 g to 1 kiloton TNT-equivalent of 235U. The releases occur over an entire year to incorporate the natural variability in atmospheric transport. Sampling equipment located at the 79 locations for radionuclide stations identified in the Comprehensive Nuclear-Test-Ban Treaty (CTBT) for the International Monitoring System are used to determine the detections of the individual releases. Alternative collection schemes in next generation equipment that collect 2, 3, or 4 samples per day, rather than the current 1 sample per day, would result in detections in many more samples at more stations with detections for a given release level. The authors posit that next generation equipment will result in increased network resilience to outages and improved source-location capability for lower yield source releases. The application of dual-detector and coincidence measurements to these systems would significantly boost sensitivity for some isotopes and would further enhance the monitoring capability.

Engineering the microwave to infrared noise photon flux for superconducting quantum systems.

Electromagnetic filtering is essential for the coherent control, operation and readout of superconducting quantum circuits at milliKelvin temperatures. The suppression of spurious modes around transition frequencies of a few GHz is well understood and mainly achieved by on-chip and package considerations. Noise photons of higher frequencies - beyond the pair-breaking energies - cause decoherence and require spectral engineering before reaching the packaged quantum chip. The external wires that pass into the refrigerator and go down to the quantum circuit provide a direct path for these photons. This article contains quantitative analysis and experimental data for the noise photon flux through coaxial, filtered wiring. The attenuation of the coaxial cable at room temperature and the noise photon flux estimates for typical wiring configurations are provided. Compact cryogenic microwave low-pass filters with CR-110 and Esorb-230 absorptive dielectric fillings are presented along with experimental data at room and cryogenic temperatures up to 70 GHz. Filter cut-off frequencies between 1 to 10 GHz are set by the filter length, and the roll-off is material dependent. The relative dielectric permittivity and magnetic permeability for the Esorb-230 material in the pair-breaking frequency range of 75 to 110 GHz are measured, and the filter properties in this frequency range are calculated. The estimated dramatic suppression of the noise photon flux due to the filter proves its usefulness for experiments with superconducting quantum systems.

Source Term Analysis of Xenon (STAX): An effort focused on differentiating man-made isotope production from nuclear explosions via stack monitoring.

An overview of the hardware and software developed for the Source Term Analysis of Xenon (STAX) project is presented which includes the data collection from two stack monitoring systems installed at medical isotope production facilities, infrastructure to transfer data to a central repository, and methods for sharing data from the repository with users. STAX is an experiment to collect radioxenon emission data from industrial nuclear facilities with the goal of developing a better understanding of the global radioxenon background and the effect industrial radioxenon releases have on nuclear explosion monitoring. A final goal of this work is to utilize collected data along with atmospheric transport modeling to calculate the contribution of a peak or set of peaks detected by the International Monitoring System (IMS) to provide desired discriminating information to the International Data Centre (IDC) and National Data Centers (NDCs). Types of data received from the STAX equipment are shown and collected data was used for a case study to predict radioxenon concentrations at two IMS stations closest to the Institute for RadioElements (IRE) in Belgium. The initial evaluation of results indicate that the data is very valuable to the nuclear explosion monitoring community.

First STAX detector installation at the National Institute for Radioelements (IRE).

The Source Term Analysis of Xenon (STAX) project has been installing stack detectors at medical isotope production facilities to measure radioxenon emissions to investigate the effect of radioxenon releases on nuclear explosion monitoring. This paper outlines the installation of the first STAX detection system at the National Institute for Radioelements (IRE) in Fleurus, Belgium which has been operating for over three years and transferring collected data to the STAX repository. Information about the equipment installed, the data flow established, and calculations for determination of radioxenon releases from the facility are presented. Data quality was investigated to confirm values reported by STAX automated data processing and in a comparison of collected STAX data with data collected by IRE for regulatory reporting.

UPRLIMET: UPstream Regional LiDAR Model for Extent of Trout in stream networks.

Predicting the edges of species distributions is fundamental for species conservation, ecosystem services, and management decisions. In North America, the location of the upstream limit of fish in forested streams receives special attention, because fish-bearing portions of streams have more protections during forest management activities than fishless portions. We present a novel model development and evaluation framework, wherein we compare 26 models to predict upper distribution limits of trout in streams. The models used machine learning, logistic regression, and a sophisticated nested spatial cross-validation routine to evaluate predictive performance while accounting for spatial autocorrelation. The model resulting in the best predictive performance, termed UPstream Regional LiDAR Model for Extent of Trout (UPRLIMET), is a two-stage model that uses a logistic regression algorithm calibrated to observations of Coastal Cutthroat Trout (Oncorhynchus clarkii clarkii) occurrence and variables representing hydro-topographic characteristics of the landscape. We predict trout presence along reaches throughout a stream network, and include a stopping rule to identify a discrete upper limit point above which all stream reaches are classified as fishless. Although there is no simple explanation for the upper distribution limit identified in UPRLIMET, four factors, including upstream channel length above the point of uppermost fish, drainage area, slope, and elevation, had highest importance. Across our study region of western Oregon, we found that more of the fish-bearing network is on private lands than on state, US Bureau of Land Mangement (BLM), or USDA Forest Service (USFS) lands, highlighting the importance of using spatially consistent maps across a region and working across land ownerships. Our research underscores the value of using occurrence data to develop simple, but powerful, prediction tools to capture complex ecological processes that contribute to distribution limits of species.

Effects of vessel traffic and ocean noise on gray whale stress hormones.

Human use of marinescapes is rapidly increasing, especially in populated nearshore regions where recreational vessel traffic can be dense. Marine animals can have a physiological response to such elevated human activity that can impact individual health and population dynamics. To understand the physiological impacts of vessel traffic on baleen whales, we investigated the adrenal stress response of gray whales (Eschrichtius robustus) to variable vessel traffic levels through an assessment of fecal glucocorticoid metabolite (fGC) concentrations. This analysis was conducted at the individual level, at multiple temporal scales (1-7 days), and accounted for factors that may confound fGC: sex, age, nutritional status, and reproductive state. Data were collected in Oregon, USA, from June to October of 2016-2018. Results indicate significant correlations between fGC, month, and vessel counts from the day prior to fecal sample collection. Furthermore, we show a significant positive correlation between vessel traffic and underwater ambient noise levels, which indicates that noise produced by vessel traffic may be a causal factor for the increased fGC. This study increases knowledge of gray whale physiological response to vessel traffic and may inform management decisions regarding regulations of vessel traffic activities and thresholds near critical whale habitats.

Investigating the detection of underground nuclear explosions by radon displacement.

A novel approach is proposed to detect underground nuclear explosions (UNEs) through the displacement of natural radon isotopes (222Rn and 220Rn). Following an explosion, it is hypothesized that the disturbance and pressurization of the sub-surface would facilitate the movement of radon from the depth of the UNE towards the surface resulting in increased soil gas activity. The resulting signal may be magnified by a factor of 2.0-4.9 by the decay of radon to its short-lived progeny. Increases in background activity may be useful for identifying locations to perform additional measurements, or as a detectable signal at monitoring stations. To validate this hypothesis, radon detection instrumentation was deployed at the Dry Alluvium Geology (DAG) site of the Source Physics Experiment (SPE) at the Nevada National Security Site (NNSS). Natural fluctuations in the soil gas activity due to barometric pumping, and the lower yield of the chemical explosions (1-50 t) made it difficult to confirm a displacement of radon from the explosions, and further study to validate the proposed hypothesis is recommended.

Stability of superconducting resonators: Motional narrowing and the role of Landau-Zener driving of two-level defects.

Frequency instability of superconducting resonators and qubits leads to dephasing and time-varying energy loss and hinders quantum processor tune-up. Its main source is dielectric noise originating in surface oxides. Thorough noise studies are needed to develop a comprehensive understanding and mitigation strategy of these fluctuations. We use a frequency-locked loop to track the resonant frequency jitter of three different resonator types­one niobium nitride superinductor, one aluminum coplanar waveguide, and one aluminum cavity­and we observe notably similar random telegraph signal fluctuations. At low microwave drive power, the resonators exhibit multiple, unstable frequency positions, which, for increasing power, coalesce into one frequency due to motional narrowing caused by sympathetic driving of two-level system defects by the resonator. In all three devices, we identify a dominant fluctuator whose switching amplitude (separation between states) saturates with increasing drive power, but whose characteristic switching rate follows the power law dependence of quasi-classical Landau-Zener transitions.

Modeling of fission and activation products in molten salt reactors and their potential impact on the radionuclide monitoring stations of the International Monitoring System.

Molten Salt Reactors (MSRs) are one of six Generation IV reactor designs currently under development around the world. Because of the unique operating conditions of MSRs, which include molten fuel and the continuous removal of gaseous fission products during operation, work was performed to model the production of activation and fission products and analyze the potential impact of emissions on the International Monitoring System (IMS) of the Comprehensive Nuclear-Test-Ban Treaty (CTBT). Simulations were performed to predict the production of IMS-relevant radionuclides in four MSR designs operating under two scenarios: (1) a sealed reactor with releases only during operational shutdown, and (2) continuous reprocessing or sparging of the fuel salt. From these production estimates the radioxenon and radioiodine signatures were extracted and compared to three current reactor designs (Boiling Water Reactor, Pressurized Water Reactor, High-Power Channel-Type Reactor). In cases where continuous reprocessing of the fuel salt occurred, both the radioxenon and radioiodine signatures were nearly indistinguishable from a nuclear explosion. Estimates were also made of the potential emission rate of radioxenon for three reactor designs and it was found that MSRs have the potential to emit radioxenon isotopes at a rate of 1015-8×1016 Bq/d for 133Xe, which may adversely affect nuclear explosion monitoring, if no abatement is used. An assessment was made of activation products using a candidate fuel salt (FLiBe) mixed with corrosion products for the Thorium Molten Salt Reactor (TMSR-LF1).

Possible impacts of molten salt reactors on the International Monitoring System.

Molten salt reactors (MSRs) are gaining support as many countries look for ways to increase power generation and replace aging nuclear energy production facilities. MSRs have inherently safe designs, are scalable in size, can burn transuranic wastes from traditional solid fuel nuclear reactors, can store excess heat in thermal reservoirs for water desalination, and can be used to produce medical isotopes as part of the real-time liquid-fuel recycling process. The ability to remove 135Xe in real time from the fuel improves the power production in an MSR because 135Xe is the most significant neutron-absorbing isotope generated by nuclear fission. Xenon-135, and other radioactive gases, are removed by sparging the fuel with an inert gas while the liquid fuel is recirculated from the reactor inner core through the heat exchangers. Without effective abatement technologies, large amounts of radioactive gas could be released during the sparging process. This work examines the potential impact of radioxenon releases on samplers used by the International Monitoring System (IMS) to detect nuclear explosions. Atmospheric transport simulations from seven hypothetical MSRs on different continents were used to evaluate the holdup time needed before release of radioxenon so IMS samplers would register few detections. Abatement technologies that retain radioxenon isotopes for at least 120 d before their release will be needed to mitigate the impacts from a molten salt breeder reactor used to replace a nuclear power plant. A holdup time of about 150 d is needed to reduce emissions to the average level of current nuclear power plants.

Insight into the kinematics of blue whale surface foraging through drone observations and prey data.

To understand how predators optimize foraging strategies, extensive knowledge of predator behavior and prey distribution is needed. Blue whales employ an energetically demanding lunge feeding method that requires the whales to selectively feed where energetic gain exceeds energetic loss, while also balancing oxygen consumption, breath holding capacity, and surface recuperation time. Hence, blue whale foraging behavior is primarily driven by krill patch density and depth, but many studies have not fully considered surface feeding as a significant foraging strategy in energetic models. We collected predator and prey data on a blue whale (Balaenoptera musculus brevicauda) foraging ground in New Zealand in February 2017 to assess the distributional and behavioral response of blue whales to the distribution and density of krill prey aggregations. Krill density across the study region was greater toward the surface (upper 20 m), and blue whales were encountered where prey was relatively shallow and more dense. This relationship was particularly evident where foraging and surface lunge feeding were observed. Furthermore, New Zealand blue whales also had relatively short dive times (2.83 ± 0.27 SE min) as compared to other blue whale populations, which became even shorter at foraging sightings and where surface lunge feeding was observed. Using an unmanned aerial system (UAS; drone) we also captured unique video of a New Zealand blue whale's surface feeding behavior on well-illuminated krill patches. Video analysis illustrates the whale's potential use of vision to target prey, make foraging decisions, and orient body mechanics relative to prey patch characteristics. Kinematic analysis of a surface lunge feeding event revealed biomechanical coordination through speed, acceleration, head inclination, roll, and distance from krill patch to maximize prey engulfment. We compared these lunge kinematics to data previously reported from tagged blue whale lunges at depth to demonstrate strong similarities, and provide rare measurements of gape size, and krill response distance and time. These findings elucidate the predator-prey relationship between blue whales and krill, and provide support for the hypothesis that surface feeding by New Zealand blue whales is an important component to their foraging ecology used to optimize their energetic efficiency. Understanding how blue whales make foraging decisions presents logistical challenges, which may cause incomplete sampling and biased ecological knowledge if portions of their foraging behavior are undocumented. We conclude that surface foraging could be an important strategy for blue whales, and integration of UAS with tag-based studies may expand our understanding of their foraging ecology by examining surface feeding events in conjunction with behaviors at depth.

Pulsed electron spin resonance of an organic microcrystal by dispersive readout.

We establish a testbed system for the development of high-sensitivity Electron Spin Resonance (ESR) techniques for small samples at cryogenic temperatures. Our system consists of a NbN thin-film planar superconducting microresonator designed to have a concentrated mode volume to couple to a small amount of paramagnetic material, and to be resilient to magnetic fields of up to 400mT. At 65mK we measure high-cooperativity coupling (C≈19) to an organic radical microcrystal containing 1012 spins in a pico-litre volume. We detect the spin-lattice decoherence rate via the dispersive frequency shift of the resonator. Techniques such as these could be suitable for applications in quantum information as well as for pulsed ESR interrogation of very few spins to provide insights into the surface chemistry of, for example, the material defects in superconducting quantum processors.

Systematic Comparison of High-throughput Single-Cell and Single-Nucleus Transcriptomes during Cardiomyocyte Differentiation.

A comprehensive reference map of all cell types in the human body is necessary for improving our understanding of fundamental biological processes and in diagnosing and treating disease. High-throughput single-cell RNA sequencing techniques have emerged as powerful tools to identify and characterize cell types in complex and heterogeneous tissues. However, extracting intact cells from tissues and organs is often technically challenging or impossible, for example in heart or brain tissue. Single-nucleus RNA sequencing provides an alternative way to obtain transcriptome profiles of such tissues. To systematically assess the differences between high-throughput single-cell and single-nuclei RNA-seq approaches, we compared Drop-seq and DroNc-seq, two microfluidic-based 3' RNA capture technologies that profile total cellular and nuclear RNA, respectively, during a time course experiment of human induced pluripotent stem cells (iPSCs) differentiating into cardiomyocytes. Clustering of time-series transcriptomes from Drop-seq and DroNc-seq revealed six distinct cell types, five of which were found in both techniques. Furthermore, single-cell trajectories reconstructed from both techniques reproduced expected differentiation dynamics. We then applied DroNc-seq to postmortem heart tissue to test its performance on heterogeneous human tissue samples. Our data confirm that DroNc-seq yields similar results to Drop-seq on matched samples and can be successfully used to generate reference maps for the human cell atlas.

The detectability of the Wigwam underwater nuclear explosion by the radionuclide stations of the International Monitoring System.

The Comprehensive Nuclear-Test-Ban Treaty (CTBT) bans all nuclear explosions, including those detonated from an underwater nuclear explosion. To improve the understanding of the radionuclide signatures of such an event, and whether it would be detectable under the verification regime of the CTBT, the 1955 Wigwam underwater nuclear explosive test has been modelled. Inventory calculations and atmospheric transport modelling has been performed to estimate the activity at the radionuclide stations (RN) of the International Monitoring System (IMS). This has utilized reported release values (0.92%) and meteorological data from the event. The research shows that there is a high probability that Wigwam would have been detectable at U.S. IMS stations at Wake Island (RN77) at 8.4 d, Upi, Guam (RN80) at 10.7 d and Sand Point, AK (RN71) at 13.7 d. At these locations, the majority of IMS relevant radionuclides were fission products, such that additional radionuclides from the seawater activation had largely decayed before reaching the stations.

Design considerations for future radionuclide aerosol monitoring systems.

Pacific Northwest National Laboratory (PNNL) staff developed the Radionuclide Aerosol Sampler Analyzer (RASA) for worldwide aerosol monitoring in the 1990s. Recently, researchers at PNNL and Creare, LLC, have investigated possibilities for how RASA could be improved, based on lessons learned from more than 15 years of continuous operation, including during the Fukushima Daiichi Nuclear Power Plant disaster. Key themes addressed in upgrade possibilities include having a modular approach to additional radionuclide measurements, optimizing the sampling/analyzing times to improve detection location capabilities, and reducing power consumption by using electrostatic collection versus classic filtration collection. These individual efforts have been made in a modular context that might constitute retrofits to the existing RASA, modular components that could improve a manual monitoring approach, or a completely new RASA. Substantial optimization of the detection and location capabilities of an aerosol network is possible and new missions could be addressed by including additional measurements.