CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both ...genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.
The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic ...dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest Kras
levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous Kras
in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfβ-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.
Optimal wiring between rectangles Dolev, Danny; Karplus, Kevin; Siegel, Alan ...
Proceedings of the thirteenth annual ACM symposium on Theory of computing,
05/1981
Conference Proceeding
Recenzirano
Odprti dostop
We consider the problem of wiring together two parallel rows of points under a variety of conditions. The options include whether we allow the rows to slide relative to one another, whether we use ...only rectilinear wires or arbitrary wires, and whether we can use wires in one layer or several layers. In almost all of these combinations of conditions, we can provide a polynomial-time algorithm to minimize the distance between the parallel rows of points. We also compare two fundamentally different wiring approaches, where one and two layers are used. We show that although the theoretical model implies that there can be great gains for the two-layer strategy, even in cases where no crossovers are required, when we consider typical design rules for laying out VLSI circuits there is no substantial advantage to the two-layer approach over the one-layer approach.
The results of a theoretical investigation of the low-velocity stopping power of ions in a magnetized collisional and classical plasma are reported. The stopping power for an ion is calculated ...through the linear-response (LR) theory. The collisions, which lead to a damping of the excitations in the plasma, are taken into account through a number-conserving relaxation time approximation in the LR function. In order to highlight the effects of collisions and magnetic field, we present a comparison of our analytical and numerical results obtained for nonzero damping or magnetic field with those for vanishing damping or magnetic field. It is shown that the collisions remove the anomalous friction obtained previously Nersisyan et al., Phys. Rev. E 61, 7022 (2000) for the collisionless magnetized plasmas at low ion velocities. One of the major objectives of this paper is to compare and to contrast our theoretical results with those obtained through a diffusion coefficient formulation based on the Dufty-Berkovsky relation evaluated for a magnetized one-component plasma modeled with target ions and electrons.
The results of a theoretical investigation of the energy loss of charged particles in a magnetized classical plasma due to the electric-field fluctuations are reported. The energy loss for a test ...particle is calculated through the linear-response theory. At vanishing magnetic field, the electric-field fluctuations lead to an energy gain of the charged particle for all velocities. It has been shown that in the presence of strong magnetic field, this effect occurs only at low velocities. In the case of high velocities, the test particle systematically loses its energy due to the interaction with a stochastic electric field. The net effect of the fluctuations is the systematic reduction of the total energy loss (i.e., the sum of the polarization and stochastic energy losses) at vanishing magnetic field and reduction or enhancement at strong field, depending on the velocity of the particle. It is found that the energy loss of the slow heavy ion contains an anomalous term that depends logarithmically on the projectile mass. The physical origin of this anomalous term is the coupling between the cyclotron motion of the plasma electrons and the long-wavelength, low-frequency fluctuations produced by the projectile ion. This effect may strongly enhance the stochastic energy gain of the particle.
Neurite Orientation Dispersion and Density Imaging (NODDI) is a diffusion MRI (dMRI) technique used to characterize tissue microstructure by compartmental modelling of neural water fractions. ...Intra-neurite, extra-neurite, and cerebral spinal fluid volume fractions are measured. The purpose of this study was to determine the reproducibility of NODDI in the rat brain at 9.4 Tesla.
Eight data sets were successfully acquired on adult male Sprague Dawley rats. Each rat was scanned twice on a 9.4T Agilent MRI with a 7 ± 1 day separation between scans. A multi-shell diffusion protocol was implemented consisting of 108 total directions varied over two shells (b-values of 1000 s/mm2 and 2000 s/mm2). Three techniques were used to analyze the NODDI scalar maps: mean region of interest (ROI) analysis, whole brain voxel-wise analysis, and targeted ROI analyses (voxel-wise within a given ROI). The coefficient of variation (CV) was used to assess the reproducibility of NODDI and provide insight into necessary sample sizes and minimum detectable effect size.
CV maps for orientation dispersion index (ODI) and neurite density index (NDI) showed high reproducibility both between and within subjects. Furthermore, it was found that small biological changes (<5%) may be detected with feasible sample sizes (n < 6-10). In contrast, isotropic volume fraction (IsoVF) was found to have low reproducibility, requiring very large sample sizes (n > 50) for biological changes to be detected.
The ODI and NDI measured by NODDI in the rat brain at 9.4T are highly reproducible and may be sensitive to subtle changes in tissue microstructure.
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