A peptide mimic of the chemotaxis inhibitory protein of Staphylococcus aureus: towards the development of novel anti-inflammatory compounds

Complement factor C5a is one of the most powerful pro-inflammatory agents involved in recruitment of leukocytes, activation of phagocytes and other inflammatory responses. C5a triggers inflammatory responses by binding to its G-protein-coupled C5a-receptor (C5aR). Excessive or erroneous activation of the C5aR has been implicated in numerous inflammatory diseases. The C5aR is therefore a key target in the development of specific anti-inflammatory compounds. A very potent natural inhibitor of the C5aR is the 121-residue chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Although CHIPS effectively blocks C5aR activation by binding tightly to its extra-cellular N terminus, it is not suitable as a potential anti-inflammatory drug due to its immunogenic properties. As a first step in the development of an improved CHIPS mimic, we designed and synthesized a substantially shorter 50-residue adapted peptide, designated CHOPS. This peptide included all residues important for receptor binding as based on the recent structure of CHIPS in complex with the C5aR N terminus. Using isothermal titration calorimetry we demonstrate that CHOPS has micromolar affinity for a model peptide comprising residues 7–28 of the C5aR N terminus including two O-sulfated tyrosine residues at positions 11 and 14. CD and NMR spectroscopy showed that CHOPS is unstructured free in solution. Upon addition of the doubly sulfated model peptide, however, the NMR and CD spectra reveal the formation of structural elements in CHOPS reminiscent of native CHIPS

Running in standard versus minimalist shoes

The purpose of this study was to determine if running in a minimalist shoe results in a reduction in ground reaction forces and alters kinematics over standard shoe running. The secondary purpose of this study was to determine if within–session accommodation to a novel minimalist shoe occurs. Running in a minimalist shoe appears to, at least in the short–term, increase loading of the lower extremity over standard shoe running. The accommodation period resulted in less favorable landing mechanics in both shoes. These findings bring into question whether minimal shoes will provide enough feedback to induce an alteration that is similar to barefoot running

Solution structure of the inhibitory phosphorylation domain of myosin phosphatase targeting subunit 1.

Cell motility, such as smooth muscle contraction and cell migration, is controlled by the reversible phosphorylation of the regulatory light chain of myosin II and other cytoskeletal proteins. Mounting evidence suggests that in smooth muscle cells and other types of cells in vertebrates, myosin phosphatase (MP) plays an important role in controlling the phosphorylation of myosin II as well as other cytoskeletal proteins, including ezrin, moesin, and radixin.1 MP is a holoenzyme consisting of a catalytic subunit of a type-1 Ser/Thr phosphatase (PP1C) delta isoform, a myosin phosphatase targeting subunit 1 (MYPT1), and an accessory subunit M21. In this ternary complex, MYPT1 is responsible for regulating the phosphatase activity.1 A recent X-ray crystallographic study revealed an allosteric interaction between PP1C and the N-terminal ankyrin repeat domain of MYPT1 that confers the substrate specificity of the enzyme.2 MP activity is suppressed when Thr696 or Thr853 of MYPT1 is phosphorylated by various kinases, such as ROCK, ZIPK, ILK, and PAK.1,3 However, it is still unclear how the phosphorylation of MYPT1 inhibits MP activity. The amino acid sequence around Thr696 of MYPT1 is highly conserved among MYPT1 family members including MYPT2 and MBS85. Therefore, structural insights into the inhibitory domain of MYPT1 are expected to provide new clues to fully elucidate the mechanism that controls phosphatase activity via the phosphorylation of MYPT1 or other family members involved in kinase-phosphatase crosstalk in cytoskeletal regulation. Here, we prepared a bacterial recombinant fragment of MYPT1 corresponding to residues 658 to 714, including the phosphorylation site Thr696, and determined its three-dimensional structure through the use of computer-assisted distance geometry and a simulated annealing protocol combined with stable-isotope-aided multi-dimensional NMR techniques

Effects of acute fatigue on the volitional and magnetically-evoked electromechanical delay of the knee flexors in males and females

Neuromuscular performance capabilities, including those measured by evoked responses, may be adversely affected by fatigue; however, the capability of the neuromuscular system to initiate muscle force rapidly under these circumstances is yet to be established. Sex-differences in the acute responses of neuromuscular performance to exercise stress may be linked to evidence that females are much more vulnerable to ACL injury than males. Optimal functioning of the knee flexors is paramount to the dynamic stabilisation of the knee joint, therefore the aim of this investigation was to examine the effects of acute maximal intensity fatiguing exercise on the voluntary and magnetically-evoked electromechanical delay in the knee flexors of males and females. Knee flexor volitional and magnetically-evoked neuromuscular performance was assessed in seven male and nine females prior to and immediately after: (i) an intervention condition comprising a fatigue trial of 30-seconds maximal static exercise of the knee flexors, (ii) a control condition consisting of no exercise. The results showed that the fatigue intervention was associated with a substantive reduction in volitional peak force (PFV) that was greater in males compared to females (15.0%, 10.2%, respectively, p < 0.01) and impairment to volitional electromechanical delay (EMDV) in females exclusively (19.3%, p < 0.05). Similar improvements in magnetically-evoked electromechanical delay in males and females following fatigue (21%, p < 0.001), however, may suggest a vital facilitatory mechanism to overcome the effects of impaired voluntary capabilities, and a faster neuromuscular response that can be deployed during critical times to protect the joint system

Methanol maser associated outflows: detection statistics and properties

We have selected the positions of 54 6.7 GHz methanol masers from the Methanol Multibeam Survey catalogue, covering a range of longitudes between 20° and 34° of the Galactic plane. These positions were mapped in the J = 3-2 transition of both the 13CO and C18O lines. A total of 58 13CO emission peaks are found in the vicinity of these maser positions. We search for outflows around all 13CO peaks, and find evidence for high-velocity gas in all cases, spatially resolving the red and blue outflow lobes in 55 cases. Of these sources, 44 have resolved kinematic distances, and are closely associated with the 6.7 GHz masers, a subset referred to as Methanol Maser Associated Outflows (MMAOs). We calculate the masses of the clumps associated with each peak using 870 mum continuum emission from the ATLASGAL survey. A strong correlation is seen between the clump mass and both outflow mass and mechanical force, lending support to models in which accretion is strongly linked to outflow. We find that the scaling law between outflow activity and clump masses observed for low-mass objects, is also followed by the MMAOs in this study, indicating a commonality in the formation processes of low-mass and high-mass stars

Motion and position shifts induced by the double-drift stimulus are unaffected by attentional load.

The double-drift stimulus produces a strong shift in apparent motion direction that generates large errors of perceived position. In this study, we tested the effect of attentional load on the perceptual estimates of motion direction and position for double-drift stimuli. In each trial, four objects appeared, one in each quadrant of a large screen, and they moved upward or downward on an angled trajectory. The target object whose direction or position was to be judged was either cued with a small arrow prior to object motion (low attentional load condition) or cued after the objects stopped moving and disappeared (high attentional load condition). In Experiment 1, these objects appeared 10° from the central fixation, and participants reported the perceived direction of the target's trajectory after the stimulus disappeared by adjusting the direction of an arrow at the center of the response screen. In Experiment 2, the four double-drift objects could appear between 6 ° and 14° from the central fixation, and participants reported the location of the target object after its disappearance by moving the position of a small circle on the response screen. The errors in direction and position judgments showed little effect of the attentional manipulation-similar errors were seen in both experiments whether or not the participant knew which double-drift object would be tested. This suggests that orienting endogenous attention (i.e., by only attending to one object in the precued trials) does not interact with the strength of the motion or position shifts for the double-drift stimulus

A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens

Heteronuclear Decoupling by Multiple Rotating Frame Technique

The paper describes the multiple rotating frame technique for designing modulated rf-fields, that perform broadband heteronuclear decoupling in solution NMR spectroscopy. The decoupling is understood by performing a sequence of coordinate transformations, each of which demodulates a component of the Rf-field to a static component, that progressively averages the chemical shift and dipolar interaction. We show that by increasing the number of modulations in the decoupling field, the ratio of dispersion in the chemical shift to the strength of the rf-field is successively reduced in progressive frames. The known decoupling methods like continuous wave decoupling, TPPM etc, are special cases of this method and their performance improves by adding additional modulations in the decoupling field. The technique is also expected to find use in designing decoupling pulse sequences in Solid State NMR spectroscopy and design of various excitation, inversion and mixing sequences.Comment: 18 pages , 5 figure

Integrating genomic resources to present full gene and putative promoter capture probe sets for bread wheat

BACKGROUND: Whole-genome shotgun resequencing of wheat is expensive because of its large, repetitive genome. Moreover, sequence data can fail to map uniquely to the reference genome, making it difficult to unambiguously assign variation. Resequencing using target capture enables sequencing of large numbers of individuals at high coverage to reliably identify variants associated with important agronomic traits. Previous studies have implemented complementary DNA/exon or gene-based probe sets in which the promoter and intron sequence is largely missing alongside newly characterized genes from the recent improved reference sequences. RESULTS: We present and validate 2 gold standard capture probe sets for hexaploid bread wheat, a gene and a putative promoter capture, which are designed using recently developed genome sequence and annotation resources. The captures can be combined or used independently. We demonstrate that the capture probe sets effectively enrich the high-confidence genes and putative promoter regions that were identified in the genome alongside a large proportion of the low-confidence genes and associated promoters. Finally, we demonstrate successful sample multiplexing that allows generation of adequate sequence coverage for single-nucleotide polymorphism calling while significantly reducing cost per sample for gene and putative promoter capture. CONCLUSIONS: We show that a capture design employing an "island strategy" can enable analysis of the large gene/putative promoter space of wheat with only 2 × 160 Mbp probe sets. Furthermore, these assays extend the regions of the wheat genome that are amenable to analyses beyond its exome, providing tools for detailed characterization of these regulatory regions in large populations

Discovery of Large-Scale Gravitational Infall in a Massive Protostellar Cluster

We report Mopra (ATNF), Anglo-Australian Telescope, and Atacama Submillimeter Telescope Experiment observations of a molecular clump in Carina, BYF73 = G286.21+0.17, which give evidence of large-scale gravitational infall in the dense gas. From the millimetre and far-infrared data, the clump has mass ~ 2 x 10^4 Msun, luminosity ~ 2-3 x 10^4 Lsun, and diameter ~ 0.9 pc. From radiative transfer modelling, we derive a mass infall rate ~ 3.4 x 10^-2 Msun yr-1. If confirmed, this rate for gravitational infall in a molecular core or clump may be the highest yet seen. The near-infrared K-band imaging shows an adjacent compact HII region and IR cluster surrounded by a shell-like photodissociation region showing H2 emission. At the molecular infall peak, the K imaging also reveals a deeply embedded group of stars with associated H2 emission. The combination of these features is very unusual and we suggest they indicate the ongoing formation of a massive star cluster. We discuss the implications of these data for competing theories of massive star formation.Comment: v1: 23 pages single-column, 6 figures (some multipart) at end v2: 14 pages 2-column, 6 figures interspersed v3: edited to referee's comments with new sections and new figures; accepted to MNRAS, 20 pages 2-column, 8 figures (some multipart) intersperse