Competitiveness in a Multipolar Port System: Striving for Regional Gateway Status in Northeast Asia

publisher: Elsevier articletitle: Competitiveness in a Multipolar Port System: Striving for Regional Gateway Status in Northeast Asia journaltitle: The Asian Journal of Shipping and Logistics articlelink: http://dx.doi.org/10.1016/j.ajsl.2016.05.005 content_type: articl

Improved genome editing in human cell lines using the CRISPR method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1

Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens

Advanced three-dimensional tailored RF pulse for signal recovery in T 2 *-weighted functional magnetic resonance imaging

T 2 * -weighted functional MR images are plagued by signal loss artifacts caused by susceptibility-induced through-plane dephasing. We present major advances to the original three-dimensional tailored RF (3DTRF) pulse method that pre-compensates the dephasing using three-dimensional selective excitation. The proposed 3DTRF pulses are designed iteratively with off-resonance incorporation and with a novel echo-volumar trajectory that frequency-encodes in z and phase-encodes in x,y . We also propose a computational scheme to accelerate the pulse design process. We demonstrate effective signal recovery in a 5-mm slice in both phantom and inferior brain, using 3DTRF pulses that are only 15.4 ms long. Compared to the original method, the new approach leads to significantly reduced pulse length and enhancement in slice selectivity. 3D images of the slice volume confirm fidelity of the excited phase pattern and slice profile. Magn Reson Med, 2006. © 2006 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55837/1/21048_ftp.pd

Nonlinear and nonreciprocal transport effects in untwinned thin films of ferromagnetic Weyl metal SrRuO3_3

The identification of distinct charge transport features, deriving from nontrivial bulk band and surface states, has been a challenging subject in the field of topological systems. In topological Dirac and Weyl semimetals, nontrivial conical bands with Fermi-arc surfaces states give rise to negative longitudinal magnetoresistance due to chiral anomaly effect and unusual thickness dependent quantum oscillation from Weyl-orbit effect, which were demonstrated recently in experiments. In this work, we report the experimental observations of large nonlinear and nonreciprocal transport effects for both longitudinal and transverse channels in an untwinned Weyl metal of SrRuO$_3$ thin film grown on a SrTiO$_{3}$ substrate. From rigorous measurements with bias current applied along various directions with respect to the crystalline principal axes, the magnitude of nonlinear Hall signals from the transverse channel exhibits a simple sin$\alpha$ dependent at low temperatures, where $\alpha$ is the angle between bias current direction and orthorhombic [001]$_{\rm o}$, reaching a maximum when current is along orthorhombic [1-10]$_{\rm o}$. On the contrary, the magnitude of nonlinear and nonreciprocal signals in the longitudinal channel attains a maximum for bias current along [001]$_{\rm o}$, and it vanishes for bias current along [1-10]$_{\rm o}$. The observed $\alpha$-dependent nonlinear and nonreciprocal signals in longitudinal and transverse channels reveal a magnetic Weyl phase with an effective Berry curvature dipole along [1-10]$_{\rm o}$ from surface states, accompanied by 1D chiral edge modes along [001]$_{\rm o}$.Comment: 24 pages, 6 figure

A Flagellar A-Kinase Anchoring Protein with Two Amphipathic Helices Forms a Structural Scaffold in the Radial Spoke Complex

A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH–RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs

Bis-tridentate Ir(III) Metal Phosphors for Efficient Deep-Blue Organic Light-Emitting Diodes

Emissive Ir(III) metal complexes possessing two tridentate chelates (bis-tridentate) are known to be more robust compared to those with three bidentate chelates (tris-bidentate). Here, the deep-blue-emitting, bis-tridentate Ir(III) metal phosphors bearing both the dicarbene pincer ancillary such as 2,6-diimidazolylidene benzene and the 6-pyrazolyl-2-phenoxylpyridine chromophoric chelate are synthesized. A deep-blue organic light-emitting diode from one phosphor exhibits Commission Internationale de l'Eclairage (CIE(x,y)) coordinates of (0.15, 0.17) with maximum external quantum efficiency (max. EQE) of 20.7% and EQE = 14.6% at the practical brightness of 100 cd m−2

Parametrically amplified Mathieu-Duffing nonlinear energy harvesters

The steady-state response of a nonlinear piezoelectric energy harvester subjected to external and parametric excitation is investigated based on the Mathieu-Duffing nonlinear oscillator model. The parametric excitation is introduced to amplify the external harmonic excitation and extend the capabilities of the nonlinear piezoelectric energy harvester device. To obtain the approximated solution of the nonlinear periodic responses for displacement and electrical voltage of the energy harvester, the incremental harmonic balance method in combination with the path-following technique is adopted. It is assumed that the proposed nonlinear model consists of cubic and quadratic nonlinearity, where parametric amplification appears in the form of a trigonometric function. The frequency is tuned as one-to-one and the one-to-two ratio between external and parametric excitation. The effects of quadratic and cubic nonlinearity as well as parametric amplification are studied in detail, and their incredible properties to extend harvester application performance is illustrated. It is explicitly demonstrated that for some particular combination of the system parameters, vibration amplitudes and harvested power can be amplified up to three or five times in comparison to the classical broadband nonlinear energy harvester based on the forced Duffing oscillator. This extraordinary amplification shown to be a key motivation to realize the proposed concept in practice. The presence of combined quadratic and cubic nonlinearities resulted in both hardening and softening spring behavior and leading to the appearance of coexisting periodic solutions in the amplitude-frequency responses. Periodic orbits obtained by the proposed methodology are verified with the results from direct numerical integration and fine agreement is demonstrated. Moreover, a significant influence of the parametric amplification on the instantaneous power is revealed in time response diagrams, thus showing better performance of the proposed energy harvester system

Holographic Dark Energy in Braneworld Models with a Gauss-Bonnet Term in the Bulk. Interacting Behavior and the w =-1 Crossing

We apply bulk holographic dark energy in general braneworld models with a Gauss-Bonnet term in the bulk and an induced gravity term and a perfect fluid on the brane. Without making any additional assumptions we extract the Friedmann equation on the physical brane and we show that a $\rho$-$\rho_\Lambda$ coupling arises naturally by the full 5D dynamics. The low-energy (late-time) evolution reveals that the effective 4D holographic dark energy behaves as ``quintom'', that is it crosses the phantom divide $w=-1$ during the evolution. In particular, the Gauss-Bonnet contribution decreases the present value of $w_\Lambda$, while it increases the growing rate of $w_\Lambda(z)$ with $z$, in comparison with the case where such a term is absent.Comment: 16 pages, version published in Phys. Lett.