Characterization of SNARE Cleavage Products Generated by Formulated Botulinum Neurotoxin Type-A Drug Products

The study evaluated substrate cleavage product(s) generated by three botulinum neurotoxin serotype A (BoNT/A) medicinal drug products utilizing a novel and highly specific, light-chain activity, high-performance liquid chromatography (LCA-HPLC) method. Samples were reacted with a commercially available BoNT/A fluorescent substrate derived from the SNAP-25 sequence. Reaction products were separated by reversed-phase HPLC. The method detected an atypical cleavage pattern by one of the formulated drug products. IncobotulinumtoxinA produced two cleavage fragments rather than the single fragment typically generated by BoNT/A. Identification confirmed the secondary cleavage at a position corresponding to SNAP-25 Arg198–Ala199 (normal BoNT/A cleavage is Gln197–Arg198). Arg198–Ala199 is also the cleavage site for trypsin and serotype C toxin. Normal cleavage was observed for all other BoNT/A drug product samples, as well as 900-kD and 150-kD bulk toxin BoNT/A. The reason for this unexpected secondary cleavage pattern by one formulated BoNT/A drug product is unknown. Possible explanations include a contaminating protease and/or damage to the 150-kD type-A toxin causing nonspecific substrate recognition and subsequent cleavage uncharacteristic of type-A toxin. The BoNT/A drug products were also analyzed via the LCA-HPLC assay using a commercial BoNT/C fluorescent substrate derived from the syntaxin sequence. Cleavage of the serotype C substrate by incobotulinumtoxinA was also confirmed whilst neither of the other drug products cleaved the syntaxin substrate

Observation of kink instability during small B5.0 solar flare on 04 June, 2007

Using multi-wavelength observations of SoHO/MDI, SOT-Hinode/blue-continuum (4504 \AA), G-band (4305 \AA), Ca II H (3968 \AA) and TRACE 171 \AA, we present the observational signature of highly twisted magnetic loop in AR 10960 during the period 04:43 UT-04:52 UT at 4 June, 2007. SOT-Hinode/blue-continuum (4504 \AA) observations show that penumbral filaments of positive polarity sunspot have counter-clock wise twist, which may be caused by the clock-wise rotation of the spot umbrae. The coronal loop, whose one footpoint is anchored in this sunspot, shows strong right-handed twist in chromospheric SOT-Hinode/Ca II H (3968 \AA) and coronal TRACE 171 \AA\, images. The length and the radius of the loop are $L\sim$80 Mm and $a\sim$4.0 Mm respectively. The distance between neighboring turns of magnetic field lines (i.e. pitch) is estimated as $\approx$ 10 Mm. The total twist angle, $\Phi\sim$12$\pi$ (estimated for the homogeneous distribution of the twist along the loop), is much larger than the Kruskal -Shafranov instability criterion. We detected clear double structure of the loop top during 04:47-04:51 UT on TRACE 171 \AA \ images, which is consistent with simulated kink instability in curved coronal loops (T{\"o}r{\"o}k et al. 2004). We suggest, that the kink instability of this twisted magnetic loop triggered B5.0 class solar flare, which occurred between 04:40 UT and 04:51 UT in this active region.Comment: 24 pages, 5 Figures; The Astrophysical Journa

Nonlinear force-free modeling of the solar coronal magnetic field

The coronal magnetic field is an important quantity because the magnetic field dominates the structure of the solar corona. Unfortunately direct measurements of coronal magnetic fields are usually not available. The photospheric magnetic field is measured routinely with vector magnetographs. These photospheric measurements are extrapolated into the solar corona. The extrapolated coronal magnetic field depends on assumptions regarding the coronal plasma, e.g. force-freeness. Force-free means that all non-magnetic forces like pressure gradients and gravity are neglected. This approach is well justified in the solar corona due to the low plasma beta. One has to take care, however, about ambiguities, noise and non-magnetic forces in the photosphere, where the magnetic field vector is measured. Here we review different numerical methods for a nonlinear force-free coronal magnetic field extrapolation: Grad-Rubin codes, upward integration method, MHD-relaxation, optimization and the boundary element approach. We briefly discuss the main features of the different methods and concentrate mainly on recently developed new codes.Comment: 33 pages, 3 figures, Review articl

Electron-acoustic plasma waves: oblique modulation and envelope solitons

Theoretical and numerical studies are presented of the amplitude modulation of electron-acoustic waves (EAWs) propagating in space plasmas whose constituents are inertial cold electrons, Boltzmann distributed hot electrons and stationary ions. Perturbations oblique to the carrier EAW propagation direction have been considered. The stability analysis, based on a nonlinear Schroedinger equation (NLSE), reveals that the EAW may become unstable; the stability criteria depend on the angle $\theta$ between the modulation and propagation directions. Different types of localized EA excitations are shown to exist.Comment: 10 pages, 5 figures; to appear in Phys. Rev.

Micro-events in the active and quiet solar corona

The content of hot material in the corona is not constant. Soft X-ray and high-temperature EUV line observations show that new material, apparently heated and evaporated from the chromosphere, is frequently injected into the corona both in active and quiet regions. Active regions are found to exhibit transient brightenings, termed here microflares, due to such enhancements in emission measure. They appear at a rate of up to 10 per hour in RHESSI observations of 3--15 keV X-rays, occurring even during the periods of lowest solar activity so far in the mission. The RHESSI observations combined with measurements at other wavelengths yield estimates of the energy input into the corona. These observations suggest that the models for coronal heating must be complemented with respect to continuous replenishing the lower corona by chromospheric material heated to coronal temperatures. The observed micro-events are secondary phenomena and do not represent the primary energy release, nor its total amount. Nevertheless, they are an interesting source of information on the heating process(es) of the corona. The micro-events are compared to events in quiet regions, termed here nanoflares, which seem to be a different population, well separated in temperature and emission measure from microflares.Comment: COSPAR meeting Houston 2002, PASP proceedings, in pres

Genome-wide discovery and characterization of maize long non-coding RNAs

BACKGROUND Long non-coding RNAs (lncRNAs) are transcripts that are 200 bp or longer, do not encode proteins, and potentially play important roles in eukaryotic gene regulation. However, the number, characteristics and expression inheritance pattern of lncRNAs in maize are still largely unknown. RESULTS By exploiting available public EST databases, maize whole genome sequence annotation and RNA-seq datasets from 30 different experiments, we identified 20,163 putative lncRNAs. Of these lncRNAs, more than 90% are predicted to be the precursors of small RNAs, while 1,704 are considered to be high-confidence lncRNAs. High confidence lncRNAs have an average transcript length of 463 bp and genes encoding them contain fewer exons than annotated genes. By analyzing the expression pattern of these lncRNAs in 13 distinct tissues and 105 maize recombinant inbred lines, we show that more than 50% of the high confidence lncRNAs are expressed in a tissue-specific manner, a result that is supported by epigenetic marks. Intriguingly, the inheritance of lncRNA expression patterns in 105 recombinant inbred lines reveals apparent transgressive segregation, and maize lncRNAs are less affected by cis- than by trans-genetic factors. CONCLUSIONS We integrate all available transcriptomic datasets to identify a comprehensive set of maize lncRNAs, provide a unique annotation resource of the maize genome and a genome-wide characterization of maize lncRNAs, and explore the genetic control of their expression using expression quantitative trait locus mapping

The Imaging Magnetograph eXperiment (IMaX) for the Sunrise balloon-borne solar observatory

The Imaging Magnetograph eXperiment (IMaX) is a spectropolarimeter built by four institutions in Spain that flew on board the Sunrise balloon-borne telesocope in June 2009 for almost six days over the Arctic Circle. As a polarimeter IMaX uses fast polarization modulation (based on the use of two liquid crystal retarders), real-time image accumulation, and dual beam polarimetry to reach polarization sensitivities of 0.1%. As a spectrograph, the instrument uses a LiNbO3 etalon in double pass and a narrow band pre-filter to achieve a spectral resolution of 85 mAA. IMaX uses the high Zeeman sensitive line of Fe I at 5250.2 AA and observes all four Stokes parameters at various points inside the spectral line. This allows vector magnetograms, Dopplergrams, and intensity frames to be produced that, after reconstruction, reach spatial resolutions in the 0.15-0.18 arcsec range over a 50x50 arcsec FOV. Time cadences vary between ten and 33 seconds, although the shortest one only includes longitudinal polarimetry. The spectral line is sampled in various ways depending on the applied observing mode, from just two points inside the line to 11 of them. All observing modes include one extra wavelength point in the nearby continuum. Gauss equivalent sensitivities are four Gauss for longitudinal fields and 80 Gauss for transverse fields per wavelength sample. The LOS velocities are estimated with statistical errors of the order of 5-40 m/s. The design, calibration and integration phases of the instrument, together with the implemented data reduction scheme are described in some detail.Comment: 17 figure

Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-?-D-glucoside (?-OG), n-dodecyl-?-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR

Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles.

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a "profiling" cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions