Desensitization is a property of the cholinergic binding region of the nicotinic acetylcholine receptor, not of the receptor-integral ion channel

The reversible acctylchollne esternse inhibitor (-}.physostiilmine (¢serine) is th© prolotypc of a new class of nie~tinlc ucetylcholinc receptor (nAChR) activating liga,ds: it induces cation fluxes into nAChR.rich membrane vesicl~s from 7~r#eda marmoeata cle~:tric tissue even under condl. lions of antalionist blocked :tcctylcholin~ binding sil~s (Okonjo, Kuhlm~mn. Maelicke. Neuron, in press). This su~tlest's that escrine exerts it~ than. nel.activating proi'~rty via binding sites at the nAChR separate from those of tile natural transmitter. We now report thllt eserine e'-m activate the channel wen when the receptor has t~en preincub~ttcd (des©nsitiz©d) with elevated concentrations of acetylcholi~e, Titus the confornudional state Of the receptor corresponding to de~nsitixation is confined to the transmitter bindinB rclli0n, leaving the ch=tr'4nel fully activatable ,- albeit only from other than the tr~msmitter bindin~ site(s)

A second pathway of activation of the Torpedo acetylcholine receptor channel

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine (D-eserine) with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligandinduced ion flux into nAChR-rich membrane vesicles and of equilibrium binding. We find that (—)physostigmine induces cation flux (and also binds to the receptor) even in the presence of saturating concentrations of antagonists of acetylcholine, such as D-tubocurarine, a-bungarotoxin or antibody WF6, The direct action on the acetylcholine receptor is not affected by removal of the methylcarbamate function from the drug and thus is not due to carbamylation of the receptor. Antibodies FKl and benzoquinonium antagonize channel activation (and binding) of eserine, suggesting that the eserine binding site(s) is separate from, but adjacent to, the acetylcholine binding site at the receptor. In addition to the channel activating site(s) with an affinity of binding in the 50 nM range, there exists a further class of low-affinity (K^ ~ mM) sites from which eserine acts as a direct blocker of the acetylcholine-activatcd channel. Our results suggest the existence of a second pathway of activation of the nAChR channel

Even denominator fractional quantum Hall states in higher Landau levels of graphene

An important development in the field of the fractional quantum Hall effect has been the proposal that the 5/2 state observed in the Landau level with orbital index $n = 1$ of two dimensional electrons in a GaAs quantum well originates from a chiral $p$-wave paired state of composite fermions which are topological bound states of electrons and quantized vortices. This state is theoretically described by a "Pfaffian" wave function or its hole partner called the anti-Pfaffian, whose excitations are neither fermions nor bosons but Majorana quasiparticles obeying non-Abelian braid statistics. This has inspired ideas on fault-tolerant topological quantum computation and has also instigated a search for other states with exotic quasiparticles. Here we report experiments on monolayer graphene that show clear evidence for unexpected even-denominator fractional quantum Hall physics in the $n=3$ Landau level. We numerically investigate the known candidate states for the even-denominator fractional quantum Hall effect, including the Pfaffian, the particle-hole symmetric Pfaffian, and the 221-parton states, and conclude that, among these, the 221-parton appears a potentially suitable candidate to describe the experimentally observed state. Like the Pfaffian, this state is believed to harbour quasi-particles with non-Abelian braid statistic

The c-terminal extension of a hybrid immunoglobulin A/G heavy chain is responsible for its Golgi-mediated sorting to the vacuole

We have assessed the ability of the plant secretory pathway to handle the expression of complex heterologous proteins by investigating the fate of a hybrid immunoglobulin A/G in tobacco cells. Although plant cells can express large amounts of the antibody, a relevant proportion is normally lost to vacuolar sorting and degradation. Here we show that the synthesis of high amounts of IgA/G does not impose stress on the plant secretory pathway. Plant cells can assemble antibody chains with high efficiency and vacuolar transport occurs only after the assembled immunoglobulins have traveled through the Golgi complex. We prove that vacuolar delivery of IgA/G depends on the presence of a cryptic sorting signal in the tailpiece of the IgA/G heavy chain. We also show that unassembled light chains are efficiently secreted as monomers by the plant secretory pathway

Ionospheric electron number densities from CUTLASS dual-frequency velocity measurements using artificial backscatter over EISCAT

Using quasi-simultaneous line-of-sight velocity measurements at multiple frequencies from the Hankasalmi Cooperative UK Twin Auroral Sounding System (CUTLASS) on the Super Dual Auroral Radar Network (SuperDARN), we calculate electron number densities using a derivation outlined in Gillies et al. (2010, 2012). Backscatter targets were generated using the European Incoherent Scatter (EISCAT) ionospheric modification facility at Tromsø, Norway. We use two methods on two case studies. The first approach is to use the dual-frequency capability on CUTLASS and compare line-of-sight velocities between frequencies with a MHz or greater difference. The other method used the kHz frequency shifts automatically made by the SuperDARN radar during routine operations. Using ray tracing to obtain the approximate altitude of the backscatter, we demonstrate that for both methods, SuperDARN significantly overestimates Ne compared to those obtained from the EISCAT incoherent scatter radar over the same time period. The discrepancy between the Ne measurements of both radars may be largely due to SuperDARN sensitivity to backscatter produced by localized density irregularities which obscure the background levels

Nanoscale imaging of equilibrium quantum Hall edge currents and of the magnetic monopole response in graphene

The recently predicted topological magnetoelectric effect and the response to an electric charge that mimics an induced mirror magnetic monopole are fundamental attributes of topological states of matter with broken time reversal symmetry. Using a SQUID-on-tip, acting simultaneously as a tunable scanning electric charge and as ultrasensitive nanoscale magnetometer, we induce and directly image the microscopic currents generating the magnetic monopole response in a graphene quantum Hall electron system. We find a rich and complex nonlinear behavior governed by coexistence of topological and nontopological equilibrium currents that is not captured by the monopole models. Furthermore, by utilizing a tuning fork that induces nanoscale vibrations of the SQUID-on-tip, we directly image the equilibrium currents of individual quantum Hall edge states for the first time. We reveal that the edge states that are commonly assumed to carry only a chiral downstream current, in fact carry a pair of counterpropagating currents, in which the topological downstream current in the incompressible region is always counterbalanced by heretofore unobserved nontopological upstream current flowing in the adjacent compressible region. The intricate patterns of the counterpropagating equilibrium-state orbital currents provide new insights into the microscopic origins of the topological and nontopological charge and energy flow in quantum Hall systems

Method Comparison for Analyzing Wound Healing Rates

Wound healing scratch assay is a frequently used method to characterize cell migration, which is an important biological process in the course of development, tissue repair, and immune response for example. The measurement of wound healing rate, however, varies among different studies. Here we summarized these measurements into three types: I) Direct Rate Average; II) Regression Rate Average; and III) Average Distance Regression Rate. Using Chinese Hamster Ovary (CHO) cells as a model, we compared the three types of analyses on quantifying the wound closing rate, and discovered that type I & III measurements are more resistant to outliers and type II analysis is more sensitive to outliers. We hope this study can help researchers to better use this simple yet effective assay

Alveolar Macrophages Isolated Directly From Human Cytomegalovirus (HCMV)-Seropositive Individuals Are Sites of HCMV Reactivation In Vivo.

Human cytomegalovirus (HCMV) causes significant morbidity in the immunocompromised host. Following primary infection, the virus establishes latent infection in progenitor cells of the myeloid lineage. These cells exhibit limited viral gene transcription and no evidence of de novo virion production. It is well recognized that differentiation of latently infected myeloid progenitor cells to dendritic or macrophage-like cells permits viral reactivation in vitro. This has been used to support the concept that viral reactivation in HCMV carriers routinely occurs from such terminally differentiated myeloid cells in vivo. However, to date this has not been shown for in vivo-differentiated macrophages. This study is the first to demonstrate that alveolar macrophages from HCMV carriers express immediate early lytic genes and produce infectious virus. This supports the view, until now based on in vitro data, that terminally differentiated myeloid cells in vivo are sites of HCMV reactivation and potential centers of viral dissemination in latently infected individuals with no evidence of virus disease or dissemination.This work was supported by the UK Medical Research Council (grant 0701279 to J. S.) and the National Institute for Health Research UK Biomedical Research Centre (to J. S. and E. R. C.).This is the final published version. It first appeared at http://jid.oxfordjournals.org/content/211/12/1936

Automated generation of program translation and verification tools using annotated grammars

Automatically generating program translators from source and target language specifications is a non-trivial problem. In this paper we focus on the problem of automating the process of building translators between operations languages, a family of DSLs used to program satellite operations procedures. We exploit their similarities to semi-automatically build transformation tools between these DSLs. The input to our method is a collection of annotated context-free grammars. To simplify the overall translation process even more, we also propose an intermediate representation common to all operations languages. Finally, we discuss how to enrich our annotated grammars model with more advanced semantic annotations to provide a verification system for the translation process. We validate our approach by semi-automatically deriving translators between some real world operations languages, using the prototype tool which we implemented for that purpose

Reduction of liver macrophage transduction by pseudotyping lentiviral vectors with a fusion envelope from Autographa californica GP64 and Sendai virus F2 domain

<p>Abstract</p> <p>Background</p> <p>Lentiviral vectors are well suited for gene therapy because they can mediate long-term expression in both dividing and nondividing cells. However, lentiviral vectors seem less suitable for liver gene therapy because systemically administered lentiviral vectors are preferentially sequestered by liver macrophages. This results in a reduction of available virus and might also increase the immune response to the vector and vector products.</p> <p>Reduction of macrophage sequestration is therefore essential for efficient lentiviral liver gene therapy.</p> <p>Results</p> <p>Fusions were made of <it>Autographa californica </it>GP64 and the hepatocyte specific Sendai Virus envelope proteins. Lentiviral vectors were produced with either wild type GP64, Sendai-GP64, or both wild type GP64 and Sendai-GP64 and tested <it>in vitro </it>and <it>in vivo </it>for hepatocyte and macrophage gene transfer.</p> <p>Sendai-GP64 pseudotyped vectors showed specific gene transfer to HepG2 hepatoma cells, with no detectable transduction of HeLa cervical carcinoma cells, and a decreased affinity for RAW mouse macrophages. Co-expression of wild type GP64 and Sendai-GP64 resulted in improved viral titers while retaining increased affinity for HepG2 cells.</p> <p>In vivo, the Sendai-GP64 vectors also showed decreased transduction of murine liver macrophages.</p> <p>Conclusion</p> <p>We demonstrate reduced macrophage transduction <it>in vitro </it>and <it>in vivo </it>with GP64/Sendai chimeric envelope proteins.</p