Microtubule Interaction Site of the Kinesin Motor

AbstractKinesin and myosin are motor proteins that share a common structural core and bind to microtubules and actin filaments, respectively. While the actomyosin interface has been well studied, the location of the microtubule-binding site on kinesin has not been identified. Using alanine-scanning mutagenesis, we have found that microtubule-interacting kinesin residues are located in three loops that cluster in a patch on the motor surface. The critical residues are primarily positively charged, which is consistent with a primarily electrostatic interaction with the negatively charged tubulin molecule. The core of the microtubule-binding interface resides in a highly conserved loop and helix (L12/α5) that corresponds topologically to the major actin-binding domain of myosin. Thus, kinesin and myosin have developed distinct polymer-binding domains in a similar region with respect to their common catalytic cores

Sensitivity Optimization for NV-Diamond Magnetometry

Solid-state spin systems including nitrogen-vacancy (NV) centers in diamond constitute an increasingly favored quantum sensing platform. However, present NV ensemble devices exhibit sensitivities orders of magnitude away from theoretical limits. The sensitivity shortfall both handicaps existing implementations and curtails the envisioned application space. This review analyzes present and proposed approaches to enhance the sensitivity of broadband ensemble-NV-diamond magnetometers. Improvements to the spin dephasing time, the readout fidelity, and the host diamond material properties are identified as the most promising avenues and are investigated extensively. Our analysis of sensitivity optimization establishes a foundation to stimulate development of new techniques for enhancing solid-state sensor performance.Comment: 73 pages, 36 figures, 17 table

Ultralong Dephasing Times in Solid-State Spin Ensembles via Quantum Control

Quantum spin dephasing is caused by inhomogeneous coupling to the environment, with resulting limits to the measurement time and precision of spin-based sensors. The effects of spin dephasing can be especially pernicious for dense ensembles of electronic spins in the solid-state, such as for nitrogen-vacancy (NV) color centers in diamond. We report the use of two complementary techniques, spin bath control and double quantum coherence, to enhance the inhomogeneous spin dephasing time ($T_2^*$) for NV ensembles by more than an order of magnitude. In combination, these quantum control techniques (i) eliminate the effects of the dominant NV spin ensemble dephasing mechanisms, including crystal strain gradients and dipolar interactions with paramagnetic bath spins, and (ii) increase the effective NV gyromagnetic ratio by a factor of two. Applied independently, spin bath control and double quantum coherence elucidate the sources of spin dephasing over a wide range of NV and spin bath concentrations. These results demonstrate the longest reported $T_2^*$ in a solid-state electronic spin ensemble at room temperature, and outline a path towards NV-diamond magnetometers with broadband femtotesla sensitivity.Comment: PRX versio

Law, Liberty and the Rule of Law (in a Constitutional Democracy)

In the hunt for a better--and more substantial--awareness of the “law,” The author intends to analyze the different notions related to the “rule of law” and to criticize the conceptions that equate it either to the sum of “law” and “rule” or to the formal assertion that “law rules,” regardless of its relationship to certain principles, including both “negative” and “positive” liberties. Instead, he pretends to scrutinize the principles of the “rule of law,” in general, and in a “constitutional democracy,” in particular, to conclude that the tendency to reduce the “democratic principle” to the “majority rule” (or “majority principle”), i.e. to whatever pleases the majority, as part of the “positive liberty,” is contrary both to the “negative liberty” and to the “rule of law” itself

Efficient, high-throughput transfection of human embryonic stem cells

INTRODUCTION: Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. METHODS: We investigated the ability of two commercially available electroporation systems, the Nucleofection(® )96-well Shuttle(® )System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. RESULTS: Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection(® )96-well Shuttle(® )System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. CONCLUSIONS: Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC

Quantum Diamond Microscope for Dynamic Imaging of Magnetic Fields

Wide-field imaging of magnetic signals using ensembles of nitrogen-vacancy (NV) centers in diamond has garnered increasing interest due to its combination of micron-scale resolution, millimeter-scale field of view, and compatibility with diverse samples from across the physical and life sciences. Recently, wide-field NV magnetic imaging based on the Ramsey protocol has achieved uniform and enhanced sensitivity compared to conventional measurements. Here, we integrate the Ramsey-based protocol with spin-bath driving to extend the NV spin dephasing time and improve magnetic sensitivity. We also employ a high-speed camera to enable dynamic wide-field magnetic imaging. We benchmark the utility of this quantum diamond microscope (QDM) by imaging magnetic fields produced from a fabricated wire phantom. Over a $270\times270 \hspace{0.08333em} \mu\mathrm{m}$$^2$ field of view, a median per-pixel magnetic sensitivity of $4.1(1)\hspace{0.08333em}\mathrm{nT}$$/\sqrt{\mathrm{Hz}}$ is realized with a spatial resolution $\lesssim\hspace{0.08333em}10\hspace{0.08333em}\mu\mathrm{m}$ and sub-millisecond temporal resolution. Importantly, the spatial magnetic noise floor can be reduced to the picotesla scale by time-averaging and signal modulation, which enables imaging of a magnetic-field pattern with a peak-to-peak amplitude difference of about $300\hspace{0.08333em}\mathrm{pT}$. Finally, we discuss potential new applications of this dynamic QDM in studying biomineralization and electrically-active cells.Comment: 18 Pages, 13 figure