Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling.

The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling

CO(J = 1-0) Imaging of M51 with CARMA and the Nobeyama 45 m Telescope

We report the CO(J = 1-0) observations of the Whirlpool Galaxy M51 using both the Combined Array for Research in Millimeter Astronomy (CARMA) and the Nobeyama 45 m telescope (NRO45). We describe a procedure for the combination of interferometer and single-dish data. In particular, we discuss (1) the joint imaging and deconvolution of heterogeneous data, (2) the weighting scheme based on the root-mean-square (rms) noise in the maps, (3) the sensitivity and uv coverage requirements, and (4) the flux recovery of a combined map. We generate visibilities from the single-dish map and calculate the noise of each visibility based on the rms noise. Our weighting scheme, though it is applied to discrete visibilities in this paper, should be applicable to grids in uv space, and this scheme may advance in future software development. For a realistic amount of observing time, the sensitivities of the NRO45 and CARMA visibility data sets are best matched by using the single-dish baselines only up to 4-6 kλ (about 1/4-1/3 of the dish diameter). The synthesized beam size is determined to conserve the flux between the synthesized beam and convolution beam. The superior uv coverage provided by the combination of CARMA long baseline data with 15 antennas and NRO45 short spacing data results in the high image fidelity, which is evidenced by the excellent overlap between even the faint CO emission and dust lanes in an optical Hubble Space Telescope image and polycyclicaromatichydrocarbon emission in a Spitzer 8 μm image. The total molecular gas masses of NGC 5194 and 5195 (d = 8.2 Mpc) are 4.9 × 10^9 M_⊙ and 7.8 × 10^7 M_⊙, respectively, assuming the CO-to-H_2 conversion factor of X _(CO) = 1.8 × 10^(20) cm-2(K km s^(–1))^(–1). The presented images are an indication of the millimeter-wave images that will become standard in the next decade with CARMA and NRO45, and the Atacama Large Millimeter/Submillimeter Array

Synthesis and Biological Evaluation of Amidinourea and Triazine Congeners as Inhibitors of MDA-MB-231 Human Breast Cancer Cell Proliferation

A series of novel amidinourea derivatives has been synthesised and the new compounds have been evaluated as inhibitors of MDA-MB-231 human breast cancer cell proliferation. In addition, a second series of triazine derivatives designed as rigid congeners of the amidinoureas was synthesised as well and the compounds were evaluated for their antiproliferative activity. Among the two series, the amidinourea 3d emerged as a potent anticancer hit compound with IC50 = 0.76 micormolar comparable to tamoxifen

The Role of Media and Popular Culture in the Mis/Education of Adults

The purpose of this symposium is to explore multiple perspectives on the role of media in the education and mis-education of adults, and to consider how educators might draw on media in developing a critical public pedagogy

Wheat Stem Maggot (Diptera: Chloropidae): An Emerging Pest of Cover Crop to Corn Transition Systems

The wheat stem maggot (Meromyza americana Fitch) (WSM) is a minor pest of wheat, rye, and other grasses. In 2017, growers in Nebraska reported dead center whorls and excessive tillering in early-season cornfields that followed wheat or rye terminated after planting corn. A survey was conducted to evaluate the risk factors for this insect in cover crop to corn transition systems. In each field, management practices and the percentage of injured plants were recorded. Symptomatic corn plants were collected from each field and dissected to determine larval and plant characteristics. In a few cases, small patches of a field were planted to a cover crop to manage soil erosion, and injured plants were only found where the cover crop was present. From these observations, the hypothesis is that terminating a cover crop after planting corn allowed the WSM larva to move from the dying cover crop to corn to complete its development. Cornfields infested with WSM had a frequency of injured corn plants from 0 to 60% with yield losses estimated at 30 bushels/acre. This paper provides the first detailed documentation of WSM injury in corn and addresses important management practices that may have influenced this uncommon situation

Amygdala and fusiform gyrus temporal dynamics: Responses to negative facial expressions

<p>Abstract</p> <p>Background</p> <p>The amygdala habituates in response to repeated human facial expressions; however, it is unclear whether this brain region habituates to schematic faces (i.e., simple line drawings or caricatures of faces). Using an fMRI block design, 16 healthy participants passively viewed repeated presentations of schematic and human neutral and negative facial expressions. Percent signal changes within anatomic regions-of-interest (amygdala and fusiform gyrus) were calculated to examine the temporal dynamics of neural response and any response differences based on face type.</p> <p>Results</p> <p>The amygdala and fusiform gyrus had a within-run "U" response pattern of activity to facial expression blocks. The initial block within each run elicited the greatest activation (relative to baseline) and the final block elicited greater activation than the preceding block. No significant differences between schematic and human faces were detected in the amygdala or fusiform gyrus.</p> <p>Conclusion</p> <p>The "U" pattern of response in the amygdala and fusiform gyrus to facial expressions suggests an initial orienting, habituation, and activation recovery in these regions. Furthermore, this study is the first to directly compare brain responses to schematic and human facial expressions, and the similarity in brain responses suggest that schematic faces may be useful in studying amygdala activation.</p

Physical Activity in Individuals with Autism Spectrum Disorders (ASD): A Review

Current recommendations indicate that children and youth ages 5–17 should participate in 60 min and adults in 150 min of moderate-to-vigorous physical activity daily. Research suggests that physical activity levels of individuals with autism spectrum disorder (ASD) are lower than typically developing and developed peers. Despite evidence for PA decreasing negative behaviors and promoting positive behaviors, individuals with ASD may be less motivated and less likely to participate. Individuals with ASD may be more likely to be overweight or obese than their typically developing counterparts as a result of decreased activity levels. Conflicting findings regarding PA levels in individuals with ASD have been reported. Given mixed evidence, further inquiry is warranted. The present chapter provides a review of literature pertaining to PA in individuals with ASD. Four databases were searched. Predetermined search terms and inclusion/exclusion criteria were clearly outlined to identify relevant articles which were then critically appraised. This research provides a greater understanding of the status of PA participation of individuals with ASD