Verification of the PROS timing analysis package

ROSAT observations of known pulsars are used to verify the functions of timing programs. The Crab Pulsar and PSR 0540-69, with 33 and 50 millisecond periods, are used to examine the fast Fourier transform and the epoch-folding task used to search for periodic signals. These fast pulsars provide a more vigorous test of the system than those with periods of a few seconds

Prevention of Borrelia burgdorferi transmission in guinea pigs by tick immunity

We examined the effect of repeated infestation of guinea pigs with Ixodes scapularis on the capacity of ticks to transmit Borrelia burgdorferi infection. Repeated challenges with nymphs or larvae lead to a reduction in duration of nymphal tick attachment and weight of recovered ticks consistent with the development of tick immunity. Only one of 18 I. scapularis-immune guinea pigs challenged with B. burgdorferi-infected nymphal ticks became infected, whereas 10 of 18 naive guinea pigs similarly challenged became infected. We conclude that tick immunity interferes with borrelial transmission

Identification of Proteins Targeted by the Thioredoxin Superfamily in Plasmodium falciparum

The malarial parasite Plasmodium falciparum possesses a functional thioredoxin and glutathione system comprising the dithiol-containing redox proteins thioredoxin (Trx) and glutaredoxin (Grx), as well as plasmoredoxin (Plrx), which is exclusively found in Plasmodium species. All three proteins belong to the thioredoxin superfamily and share a conserved Cys-X-X-Cys motif at the active site. Only a few of their target proteins, which are likely to be involved in redox reactions, are currently known. The aim of the present study was to extend our knowledge of the Trx-, Grx-, and Plrx-interactome in Plasmodium. Based on the reaction mechanism, we generated active site mutants of Trx and Grx lacking the resolving cysteine residue. These mutants were bound to affinity columns to trap target proteins from P. falciparum cell extracts after formation of intermolecular disulfide bonds. Covalently linked proteins were eluted with dithiothreitol and analyzed by mass spectrometry. For Trx and Grx, we were able to isolate 17 putatively redox-regulated proteins each. Furthermore, the approach was successfully established for Plrx, leading to the identification of 21 potential target proteins. In addition to confirming known interaction partners, we captured potential target proteins involved in various processes including protein biosynthesis, energy metabolism, and signal transduction. The identification of three enzymes involved in S-adenosylmethionine (SAM) metabolism furthermore suggests that redox control is required to balance the metabolic fluxes of SAM between methyl-group transfer reactions and polyamine synthesis. To substantiate our data, the binding of the redoxins to S-adenosyl-L-homocysteine hydrolase and ornithine aminotransferase (OAT) were verified using BIAcore surface plasmon resonance. In enzymatic assays, Trx was furthermore shown to enhance the activity of OAT. Our approach led to the discovery of several putatively redox-regulated proteins, thereby contributing to our understanding of the redox interactome in malarial parasites

Liquid-phase mega-electron-volt ultrafast electron diffraction

The conversion of light into usable chemical and mechanical energy is pivotal to several biological and chemical processes, many of which occur in solution. To understand the structure-function relationships mediating these processes, a technique with high spatial and temporal resolutions is required. Here, we report on the design and commissioning of a liquid-phase mega-electron-volt (MeV) ultrafast electron diffraction instrument for the study of structural dynamics in solution. Limitations posed by the shallow penetration depth of electrons and the resulting information loss due to multiple scattering and the technical challenge of delivering liquids to vacuum were overcome through the use of MeV electrons and a gas-accelerated thin liquid sheet jet. To demonstrate the capabilities of this instrument, the structure of water and its network were resolved up to the 3 rd hydration shell with a spatial resolution of 0.6 Å; preliminary time-resolved experiments demonstrated a temporal resolution of 200 fs

Gas Dynamic Virtual Nozzle for Generation of Microscopic Droplet Streams

As shown by Ganan-Calvo and co-workers, a free liquid jet can be compressed in iameter through gas-dynamic forces exerted by a co-flowing gas, obviating the need for a solid nozzle to form a microscopic liquid jet and thereby alleviating the clogging problems that plague conventional droplet sources of small diameter. We describe in this paper a novel form of droplet beam source based on this principle. The source is miniature, robust, dependable, easily fabricated, and eminently suitable for delivery of microscopic liquid droplets, including hydrated biological samples, into vacuum for analysis using vacuum instrumentation. Monodisperse, single file droplet streams are generated by triggering the device with a piezoelectric actuator. The device is essentially immune to clogging

X ray emission spectroscopy of bulk liquid water in no man s land

The structure of bulk liquid water was recently probed by x ray scattering below the temperature limit of homogeneous nucleation TH of amp; 8764;232 K [J. A. Sellberg et al., Nature 510, 381 384 2014 ]. Here, we utilize a similar approach to study the structure of bulk liquid water below TH using oxygen K edge x ray emission spectroscopy XES . Based on previous XES experiments [T. Tokushima et al., Chem. Phys. Lett. 460, 387 400 2008 ] at higher temperatures, we expected the ratio of the 1b1 amp; 8242; and 1b1 amp; 8242; amp; 8242; peaks associated with the lone pair orbital in water to change strongly upon deep supercooling as the coordination of the hydrogen H bonds becomes tetrahedral. In contrast, we observed only minor changes in the lone pair spectral region, challenging an interpretation in terms of two interconverting species. A number of alternative hypotheses to explain the results are put forward and discussed. Although the spectra can be explained by various contributions from these hypotheses, we here emphasize the interpretation that the line shape of each component changes dramatically when approaching lower temperatures, where, in particular, the peak assigned to the proposed disordered component would become more symmetrical as vibrational interference becomes more importan

Genes encoding α-amylase inhibitors are located in the short arms of chromosomes 3B, 3D and 6D of wheat (Triticum aestivum L.)

Three -amylase inhibitors, designated Inh. I, II and III have been purified from the 70% ethanol extract of hexaploid wheat (Triticum aestivum L.) and characterized by amino acid analysis, N-terminal amino acid sequencing and enzyme inhibition tests. Inhibitors I and III have identical N-terminal sequences and inhibitory properties to those of the previously described 0.19/0.53 group of dimeric inhibitors. Inhibitor II has an N-terminal sequence which is identical to that of the previously described 0.28 monomeric inhibitor, but differs from it in that in addition to being active against -amylase from Tenebrio molitor, it is also active against mammalian salivary and pancreatic -amylases. Compensating nulli-tetrasomic and ditelosomic lines of wheat cv. Chinese Spring have been analysed by two-dimensional electrophoresis, under conditions in which there is no overlap of the inhibitors with other proteins, and the chromosomal locations of the genes encoding these inhibitors have been established: genes for Inh. I and Inh. III are in the short arms of chromosomes 3B and 3D, respectively, and that for Inh. II in the short arm of chromosome 6D

Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser

To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach

A data set from flash X-ray imaging of carboxysomes

Citation: Hantke, M. F., Hasse, D., Ekeberg, T., John, K., Svenda, M., Loh, D., . . . Maia, F. R. N. C. (2016). A data set from flash X-ray imaging of carboxysomes. Scientific Data, 3. doi:10.1038/sdata.2016.61Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere