Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9

Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements.

In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3' untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites

RNA systems biology: uniting functional discoveries and structural tools to understand global roles of RNAs

RNAs assume sophisticated structures that are active in myriad cellular processes. In this review, we highlight newly identified ribozymes, riboswitches, and small RNAs, some of which control the function of cellular metabolic and gene expression networks. We then examine recent developments in genome-wide RNA structure probing technologies that are yielding new insights into the structural landscape of the transcriptome. Finally, we discuss how these RNA ‘structomic’ methods can address emerging questions in RNA systems biology, from the mechanisms behind long non-coding RNAs to new bases for human diseases

Constraining Pulsar Magnetosphere Geometry with Gamma-Ray Light Curves

We demonstrate a method for quantitatively comparing gamma-ray pulsar light curves with magnetosphere beaming models. With the Fermi LAT providing many pulsar discoveries and high quality pulsar light curves for the brighter objects, such comparison allows greatly improved constraints on the emission zone geometry and the magnetospheric physics. Here we apply the method to Fermi LAT light curves of a set of bright pulsars known since EGRET or before. We test three approximate models for the magnetosphere structure and two popular schemes for the location of the emission zone, the Two Pole Caustic (TPC) model and the Outer Gap (OG) model. We find that OG models and relatively physical B fields approximating force-free dipole magnetospheres are preferred at high statistical significance. An application to the full LAT pulsar sample will allow us to follow the emission zone's evolution with pulsar spindown.Comment: 14 pages, 16 figures. To appear in the Astrophysical Journal, Vol 71

Temporary Bridging Agents for use in Drilling and Completion of Enhanced Geothermal Systems

CSI Technologies, in conjunction with Alta Rock Energy and the University of Utah have undergone a study investigating materials and mechanisms with potential for use in Enhanced Geothermal Systems wells as temporary diverters or lost circulation materials. Studies were also conducted with regards to particle size distribution and sealing effectiveness using a lab-scale slot testing apparatus to simulate fractures. From the slot testing a numerical correlation was developed to determine the optimal PSD for a given fracture size. Field trials conducted using materials from this study were also successful

Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter™ Assay System

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

The Galactic Population of Young Gamma-ray Pulsars

We have simulated a Galactic population of young pulsars and compared with the Fermi LAT sample, constraining the birth properties, beaming and evolution of these spin-powered objects. Using quantitative tests of agreement with the distributions of observed spin and pulse properties, we find that short birth periods P_0 ~ 50ms and gamma-ray beams arising in the outer magnetosphere, dominated by a single pole, are strongly preferred. The modeled relative numbers of radio-detected and radio-quiet objects agree well with the data. Although the sample is local, extrapolation to the full Galaxy implies a gamma-ray pulsar birthrate 1/(59 yr). This is shown to be in good agreement with the estimated Galactic core collapse rate and with the local density of OB star progenitors. We give predictions for the numbers of expected young pulsar detections if Fermi LAT observations continue 10 years. In contrast to the potentially significant contribution of unresolved millisecond pulsars, we find that young pulsars should contribute little to the Galactic gamma-ray background.Comment: 14 pages, 11 figures; to appear in the Astrophysical Journal. Updated in response to referee comment

Plate versus bulk trolley food service in a hospital: comparison of patients’ satisfaction

Objective The aim of this research was to compare plate with bulk trolley food service in hospitals in terms of patient satisfaction. Key factors distinguishing satisfaction with each system would also be identified. Methods A consumer opinion card (n = 180), concentrating on the quality indicators of core foods, was used to measure patient satisfaction and compare two systems of delivery, plate and trolley. Binary logistic regression analysis was used to build a model that would predict food service style on the basis of the food attributes measured. Further investigation used multinomial logistic regression to predict opinion for the assessment of each food attribute within food service style. Results Results showed that the bulk trolley method of food distribution enables all foods to have a more acceptable texture, and for some foods (potato, P = 0.007; poached fish, P = 0.001; and minced beef, P ≤ 0.0005) temperature, and for other foods (broccoli, P ≤ 0.0005; carrots, P ≤ 0.0005; and poached fish, P = 0.001) flavor, than the plate system of delivery, where flavor is associated with bad opinion or dissatisfaction. A model was built indicating patient satisfaction with the two service systems. Conclusion This research confirms that patient satisfaction is enhanced by choice at the point of consumption (trolley system); however, portion size was not the controlling dimension. Temperature and texture were the most important attributes that measure patient satisfaction with food, thus defining the focus for hospital food service managers. To date, a model predicting patient satisfaction with the quality of food as served has not been proposed, and as such this work adds to the body of knowledge in this field. This report brings new information about the service style of dishes for improving the quality of food and thus enhancing patient satisfaction

Galactic Globular and Open Clusters in the Sloan Digital Sky Survey. I. Crowded Field Photometry and Cluster Fiducial Sequences in ugriz

We present photometry for globular and open cluster stars observed with the Sloan Digital Sky Survey (SDSS). In order to exploit over 100 million stellar objects with r < 22.5 mag observed by SDSS, we need to understand the characteristics of stars in the SDSS ugriz filters. While star clusters provide important calibration samples for stellar colors, the regions close to globular clusters, where the fraction of field stars is smallest, are too crowded for the standard SDSS photometric pipeline to process. To complement the SDSS imaging survey, we reduce the SDSS imaging data for crowded cluster fields using the DAOPHOT/ALLFRAME suite of programs and present photometry for 17 globular clusters and 3 open clusters in a SDSS value-added catalog. Our photometry and cluster fiducial sequences are on the native SDSS 2.5-meter ugriz photometric system, and the fiducial sequences can be directly applied to the SDSS photometry without relying upon any transformations. Model photometry for red giant branch and main-sequence stars obtained by Girardi et al. cannot be matched simultaneously to fiducial sequences; their colors differ by ~0.02-0.05 mag. Good agreement (< ~0.02 mag in colors) is found with Clem et al. empirical fiducial sequences in u'g'r'i'z' when using the transformation equations in Tucker et al.Comment: 30 pages, 25 figures. Accepted for publication in ApJS. Version with high resolution figures available at http://www.astronomy.ohio-state.edu/~deokkeun/AnJohnson.pd