The MuSK-BMP pathway regulates synaptic Nav1.4 localization and muscle excitability.

The neuromuscular junction (NMJ) is the linchpin of nerve-evoked muscle contraction. Broadly considered, the function of the NMJ is to transduce a nerve action potential into a muscle fiber action potential (MFAP). Efficient information transfer requires both cholinergic signaling, responsible for the generation of endplate potentials (EPPs), and excitation, the activation of postsynaptic voltage-gated sodium channels (Nav1.4) to trigger MFAPs. In contrast to the cholinergic apparatus, the signaling pathways that organize Nav1.4 and muscle fiber excitability are poorly characterized. Muscle-specific kinase (MuSK), in addition to its Ig1 domain-dependent role as an agrin-LRP4 receptor, is also a BMP co-receptor that binds BMPs via its Ig3 domain and shapes BMP-induced signaling and transcriptional output. Here we probed the function of the MuSK-BMP pathway at the NMJ using mice lacking the MuSK Ig3 domain ('ΔIg3-MuSK'). Synapses formed normally in ΔIg3-MuSK animals, but the postsynaptic apparatus was fragmented from the first weeks of life. Anatomical denervation was not observed at any age examined. Moreover, spontaneous and nerve-evoked acetylcholine release, AChR density, and endplate currents were comparable to WT. However, trains of nerve-evoked MFAPs in ΔIg3-MuSK muscle were abnormal as revealed by increased jitter and blocking in single fiber electromyography. Further, nerve-evoked compound muscle action potentials (CMAPs), as well as twitch and tetanic muscle torque force production, were also diminished. Finally, Nav1.4 levels were reduced at ΔIg3-MuSK synapses but not at the extrajunctional sarcolemma, indicating that the observed excitability defects are the result of impaired localization of this voltage-gated ion channel at the NMJ. We propose that MuSK plays two distinct roles at the NMJ: as an agrin-LRP4 receptor necessary for establishing and maintaining cholinergic signaling, and as a BMP co-receptor required for maintaining proper Nav1.4 density, nerve-evoked muscle excitability and force production. The MuSK-BMP pathway thus emerges as a target for modulating excitability and functional innervation, which are defective in conditions such as congenital myasthenic syndromes and aging.

Upper wide-angle viewing system for ITER.

The Upper Wide Angle Viewing System (UWAVS) will be installed on five upper ports of ITER. This paper shows major requirements, gives an overview of the preliminary design with reasons for some design choices, examines self-emitted IR light from UWAVS optics and its effect on accuracy, and shows calculations of signal-to-noise ratios for the two-color temperature output as a function of integration time and divertor temperature. Accurate temperature output requires correction for vacuum window absorption vs. wavelength and for self-emitted IR, which requires good measurement of the temperature of the optical components. The anticipated signal-to-noise ratio using presently available IR cameras is adequate for the required 500 Hz frame rate.

Conceptual design of the tangentially viewing combined interferometer-polarimeter for ITER density measurements.

One of the systems planned for the measurement of electron density in ITER is a multi-channel tangentially viewing combined interferometer-polarimeter (TIP). This work discusses the current status of the design, including a preliminary optical table layout, calibration options, error sources, and performance projections based on a CO2/CO laser system. In the current design, two-color interferometry is carried out at 10.59 µm and 5.42 µm and a separate polarimetry measurement of the plasma induced Faraday effect, utilizing the rotating wave technique, is made at 10.59 µm. The inclusion of polarimetry provides an independent measure of the electron density and can also be used to correct the conventional two-color interferometer for fringe skips at all densities, up to and beyond the Greenwald limit. The system features five chords with independent first mirrors to reduce risks associated with deposition, erosion, etc., and a common first wall hole to minimize penetration sizes. Simulations of performance for a projected ITER baseline discharge show the diagnostic will function as well as, or better than, comparable existing systems for feedback density control. Calculations also show that finite temperature effects will be significant in ITER even for moderate temperature plasmas and can lead to a significant underestimate of electron density. A secondary role TIP will fulfill is that of a density fluctuation diagnostic; using a toroidal Alfvén eigenmode as an example, simulations show TIP will be extremely robust in this capacity and potentially able to resolve coherent mode fluctuations with perturbed densities as low as δn∕n ≈ 10(-5).

Imaging spin filter for electrons based on specular reflection from iridium (001).

As Stern-Gerlach type spin filters do not work with electrons, spin analysis of electron beams is accomplished by spin-dependent scattering processes based on spin-orbit or exchange interaction. Existing polarimeters are single-channel devices characterized by an inherently low figure of merit (FoM) of typically 10⁻4-10⁻³. This single-channel approach is not compatible with parallel imaging microscopes and also not with modern electron spectrometers that acquire a certain energy and angular interval simultaneously. We present a novel type of polarimeter that can transport a full image by making use of k-parallel conservation in low-energy electron diffraction. We studied specular reflection from Ir (001) because this spin-filter crystal provides a high analyzing power combined with a "lifetime" in UHV of a full day. One good working point is centered at 39 eV scattering energy with a broad maximum of 5 eV usable width. A second one at about 10 eV shows a narrower profile but much higher FoM. A relativistic layer-KKR SPLEED calculation shows good agreement with measurements.

Overview of the beam emission spectroscopy diagnostic system on the National Spherical Torus Experiment.

A beam emission spectroscopy (BES) system has been installed on the National Spherical Torus Experiment (NSTX) to study ion gyroscale fluctuations. The BES system measures D(α) emission from a deuterium neutral heating beam. The system includes two optical views centered at r/a≈0.45 and 0.85 and aligned to magnetic field pitch angles at the neutral beam. f/1.5 collection optics produce 2-3 cm spot sizes at the neutral beam. The initial channel layout includes radial arrays, poloidal arrays, and two-dimensional grids. Radial arrays provide coverage from r/a≈0.1 to beyond the last-closed flux surface. Photodetectors and digital filters provide high-sensitivity, low-noise measurements at frequencies of up to 1 MHz. The BES system will be a valuable tool for investigating ion gyroscale turbulence and Alfvén/energetic particle modes on NSTX.

Electron cyclotron emission diagnostic for ITER.

Electron temperature measurements and electron thermal transport inferences will be critical to the nonactive and deuterium phases of ITER operation and will take on added importance during the alpha heating phase. The diagnostic must meet stringent criteria on spatial coverage and spatial resolution during full field operation. During the early phases of operation, it must operate equally well at half field. The key to the diagnostic is the front end design. It consists of a quasioptical antenna and a pair of calibration sources. The radial resolution of the diagnostic is less than 0.06 m. The spatial coverage extends at least from the core to the separatrix with first harmonic O-mode being used for the core and second harmonic X-mode being used for the pedestal. The instrumentation used for the core measurement at full field can be used for detection at half field by changing the detected polarization. Intermediate fields are accessible. The electron cyclotron emission systems require in situ calibration, which is provided by a novel hot calibration source. The critical component for the hot calibration source, the emissive surface, has been successfully tested. A prototype hot calibration source has been designed, making use of extensive thermal and mechanical modeling.

Development of a spatially resolving x-ray crystal spectrometer for measurement of ion-temperature (T(i)) and rotation-velocity (v) profiles in ITER.

Imaging x-ray crystal spectrometer (XCS) arrays are being developed as a US-ITER activity for Doppler measurement of T(i) and v profiles of impurities (W, Kr, and Fe) with ∼7 cm (a/30) and 10-100 ms resolution in ITER. The imaging XCS, modeled after a prototype instrument on Alcator C-Mod, uses a spherically bent crystal and 2D x-ray detectors to achieve high spectral resolving power (E/dE>6000) horizontally and spatial imaging vertically. Two arrays will measure T(i) and both poloidal and toroidal rotation velocity profiles. The measurement of many spatial chords permits tomographic inversion for the inference of local parameters. The instrument design, predictions of performance, and results from C-Mod are presented.

The ITER core imaging x-ray spectrometer: x-ray calorimeter performance.

We describe the anticipated performance of an x-ray microcalorimeter instrument on ITER. As part of the core imaging x-ray spectrometer, the instrument will augment the imaging crystal spectrometers by providing a survey of the concentration of heavy ion plasma impurities in the core and possibly ion temperature values from the emission lines of different elemental ions located at various radial positions.

Spin-resolved mapping of spin contribution to exchange-correlation holes.

By means of spin-polarized electron coincidence spectroscopy we explore the fundamental issue of spin-resolved contributions to the exchange-correlation hole in many-electron systems. We present a joint experimental and theoretical study of correlated electron pair emission from a ferromagnetic Fe(001) surface induced by spin-polarized low-energy electrons. We demonstrate that the contribution to the exchange-correlation hole due to exchange is more extended than the contribution due to the screened Coulomb interaction.

Positron-induced emission of electron-positron pairs from solid surfaces.

The collision of a low-energy positron, which impinges on a crystalline surface, with a valence electron may result in the emission of a spatially separated time-correlated electron-positron pair. We present a method for calculating the cross section for this positron surface reaction channel, which we briefly refer to as (p, ep) in analogy to electron-induced pair emission (e, 2e). The two-particle final state is represented by a product of an electron and a positron diffraction state coupled by a 'correlation factor', which accounts for the screened Coulomb interaction. The electron-solid and positron-solid quasi-particle potentials are based on first-principles calculations within density functional theory. Numerical (p, ep) results are presented for Cu(111) and compared to their (e, 2e) counterparts. Energy distributions for constant emission angles reflect, to a large extent, the valence electron density of states. In equal-energy (p, ep) angular distributions, the Coulomb interaction produces a central accumulation zone-in contrast to a depletion zone for (e, 2e)-the relative weight and the extension of which are subject to 'matrix element effects'. At larger angles sharp features arise from single-particle surface resonances.

The NSTX fast-ion D-alpha diagnostic.

A new diagnostic, aimed at energy-resolved measurements of the spatial and temporal dynamics of fast ions in NSTX plasmas, is described. It is based on active charge-exchange recombination spectroscopy. The fast-ion signal is inferred from light emitted in the wavelength range of the D(alpha) line by fast ions recombining with an injected neutral beam. Two complementary systems are operational. The first system, based on a spectrometer coupled to a charge coupled device detector, has 16 channels with space, time, and energy resolution of 5 cm, 10 ms, and 10 keV, respectively. The second system monitors the energy-integrated fast-ion signal on time scales of approximately 20 micros at three different radii. Signals are measured by a multianode photomultiplier tube. For both systems, each channel includes two paired views, intercepting and missing the neutral beam for a direct subtraction of the background signal not associated with fast ions. Examples of signals from the 2008 NSTX run are presented.

Wide-angle point-to-point x-ray imaging with almost arbitrarily large angles of incidence.

The paper describes a new scheme for wide-angle point-to-point x-ray imaging with almost arbitrarily large angles of incidence by a matched pair of spherically bent crystals to eliminate the astigmatism, which is a well-known imaging error of spherical mirrors. In addition to x rays, the scheme should be applicable to a very broad spectrum of the electromagnetic radiation, including microwaves, infrared and visible light, as well as UV and extreme UV radiation, if the crystals are replaced with appropriate spherical reflectors. The scheme may also be applicable to the imaging with ultrasound.

Characterization of cross-section correction to charge exchange recombination spectroscopy rotation measurements using co- and counter-neutral-beam views.

Measurements of rotation using charge exchange recombination spectroscopy can be affected by the energy dependence of the charge exchange cross section. On DIII-D, the associated correction to the rotation can exceed 100 kms at high temperatures. In reactor-relevant low rotation conditions, the correction can be several times larger than the actual plasma rotation and therefore must be carefully validated. New chords have been added to the DIII-D CER diagnostic to view the counter-neutral-beam line. The addition of these views allows determination of the toroidal rotation without depending on detailed atomic physics calculations, while also allowing experimental characterization of the atomic physics. A database of rotation comparisons from the two views shows that the calculated cross-section correction can adequately describe the measurements, although there is a tendency for "overcorrection." In cases where accuracy better than about 15% is desired, relying on calculation of the cross-section correction may be insufficient.

Affinity driven molecular transfer from erythrocyte membrane to target cells.

A wide variety of antimicrobial peptides are known to bind to - and disrupt microbial plasma membranes. Recently, derivatives of the antimicrobial peptide dermaseptin S4 were shown to selectively disrupt the plasma membrane of the intracellular parasite Plasmodium falciparum without harming that of the mammalian host cell. The resulting antimalarial activity is allegedly exerted after the harmless peptide binding to the membrane of the host cell, followed by peptide translocation across a number of intracellular membrane systems and interaction with that of the intraerythrocyte parasite. In this study, we present evidence in support of the ability of a membrane-bound peptide, the dermaseptin S4 derivative K(4)-S4(1-13)a, to transfer from red blood cells (RBCs) to another distant membrane. Binding of K(4)-S4(1-13)a to the plasma membrane of RBCs was assessed in vitro and in vivo, and found to be rapid, spontaneous and receptor independent, as was the transfer of the RBC-bound peptide to the plasma membrane of microorganisms. The present study further provides a basis for the possible use of RBCs as a transport vehicle to deliver drugs to distant targets. This drug delivery system involves the transient "loading" of RBCs with a lipophilic "hook" peptide. Such a peptide has enough affinity for the RBC's plasma membrane to bind to the membrane, but given the opportunity, the peptide will exit its position and transfer to another (target) cell for which it has a greater affinity. The efficacy of such an affinity driven transfer system was demonstrated experimentally by the transfer of K(4)-S4(1-13)a from pre-loaded RBCs to bacteria, yeast and protozoan target cells.

Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas.

PURPOSE: Keratoconus is a disease characterized by thinning of the central and paracentral cornea and scarring in advanced cases. This study was performed to examine the expression of type XII collagen, proteins associated with hemidesmosomes, and beta1 integrin in keratoconus corneas. METHODS: Corneal buttons were collected from normal subjects and patients with keratoconus and other corneal diseases. Immunofluorescence staining was performed on frozen sections for type XII collagen, bullous pemphigoid antigen (BP180), and integrin subunits alpha6, beta4, and beta1. RESULTS: To varying degrees, all proteins examined were expressed in normal human corneas. The staining intensity of type XII collagen was diminished in keratoconus corneas in the epithelial basement membrane zone and the stromal matrix. No significant variation was found in either the staining patterns or intensities for BP180, or integrins alpha6, beta4, and beta1. CONCLUSIONS: The level of type XII collagen was reduced in the epithelial basement membrane zone and stromal matrices in keratoconus corneas. These alterations may affect critical interactions of the corneal epithelium with the under-lying basement membrane, and cell-matrix interactions and matrix organization in the stroma.

Mucolipidosis IV in an African American patient with new findings on electron microscopy.

PURPOSE: We report an unusual case of mucolipidosis IV in a patient of African ancestry, with intracytoplasmic inclusions of the corneal endothelium found on electron microscopy. METHOD: Clinical description with light and electron microscopy. RESULTS: We describe a case of mucolipidosis IV diagnosed in a patient of African ancestry after penetrating keratoplasty. Electron microscopic evaluation revealed intracytoplasmic inclusions in both the corneal epithelium and endothelium. CONCLUSION: The diagnosis of mucolipidosis in a patient of African ancestry is unusual, as this genetic disorder is found predominantly in individuals of Jewish descent. Corneal endothelial involvement in mucolipidosis IV has not previously been reported.

Antimalarial activities of dermaseptin S4 derivatives.

The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC(50)) in the micromolar range. Among these, the substituted S4 analog K(4)K(20)-S4 was the most potent (IC(50) = 0.2 microM), while its shorter version, K(4)-S4(1-13)a, retained a considerable potency (IC(50) = 6 microM). Both K(4)K(20)-S4 and K(4)-S4(1-13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K(4)K(20)-S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K(4)K(20)-S4, the most hemolytic dermaseptin. The same trend was observed with K(4)-S4(1-13)a. The D isomers of K(4)K(20)-S4 or K(4)-S4(1-13)a were as active as the L counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasite's plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.

Structure-activity relationship study of antimicrobial dermaseptin S4 showing the consequences of peptide oligomerization on selective cytotoxicity.

To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K(4)K(20)-S4 was 2-3-fold more potent against protozoa and RBC, yet K(4)K(20)-S4 was more potent by 2 orders of magnitude against bacteria. K(4)-S4 had similar behavior as K(4)K(20)-S4, but K(20)-S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K(20)-S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K(4)-S4 and K(4)K(20)-S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptide's oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptide's activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptide's physico-chemical properties affect potency and selectivity.