Dramatic Structural Changes Resulting from the Loss of a Crucial Hydrogen Bond in the Hinge Region Involved in C-Terminal Helix Swapping in SurE: A Survival Protein from Salmonella typhimurium

Domain swapping is an interesting feature of some oligomeric proteins in which each protomer of the oligomer provides an identical surface for exclusive interaction with a segment or domain belonging to another protomer. Here we report results of mutagenesis experiments on the structure of C-terminal helix swapped dimer of a stationary phase survival protein from Salmonella typhimurium (StSurE). Wild type StSurE is a dimer in which a large helical segment at the C-terminus and a tetramerization loop comprising two beta strands are swapped between the protomers. Key residues in StSurE that might promote C-terminal helix swapping were identified by sequence and structural comparisons. Three mutants in which the helix swapping is likely to be avoided were constructed and expressed in E. coli. Three-dimensional X-ray crystal structures of the mutants H234A and D230A/H234A could be determined at 2.1 angstrom and 2.35 angstrom resolutions, respectively. Contrary to expectations, helix swapping was mostly retained in both the mutants. The loss of the crucial D230 OD2- H234 NE2 hydrogen bond (2.89 angstrom in the wild type structure) in the hinge region was compensated by new inter and intra-chain interactions. However, the two fold molecular symmetry was lost and there were large conformational changes throughout the polypeptide. In spite of these changes, the dimeric structure and an approximate tetrameric organization were retained, probably due to the interactions involving the tetramerization loop. Mutants were mostly functionally inactive, highlighting the importance of precise inter-subunit interactions for the symmetry and function of StSurE.

MAST: multi-script annotation toolkit for scenic text

This paper describes a semi-automatic tool for annotation of multi-script text from natural scene images. To our knowledge, this is the maiden tool that deals with multi-script text or arbitrary orientation. The procedure involves manual seed selection followed by a region growing process to segment each word present in the image. The threshold for region growing can be varied by the user so as to ensure pixel-accurate character segmentation. The text present in the image is tagged word-by-word. A virtual keyboard interface has also been designed for entering the ground truth in ten Indic scripts, besides English. The keyboard interface can easily be generated for any script, thereby expanding the scope of the toolkit. Optionally, each segmented word can further be labeled into its constituent characters/symbols. Polygonal masks are used to split or merge the segmented words into valid characters/symbols. The ground truth is represented by a pixel-level segmented image and a '.txt' file that contains information about the number of words in the image, word bounding boxes, script and ground truth Unicode. The toolkit, developed using MATLAB, can be used to generate ground truth and annotation for any generic document image. Thus, it is useful for researchers in the document image processing community for evaluating the performance of document analysis and recognition techniques. The multi-script annotation toolokit (MAST) is available for free download.

Isolation of a High Affinity Neutralizing Monoclonal Antibody against 2009 Pandemic H1N1 Virus That Binds at the `Sa' Antigenic Site

Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs) have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (K-D = 2.1 +/- 0.4 pM) murine MAb, MA2077 that binds specifically to the hemagglutinin (HA) surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an in vitro microneutralization assay (IC50 = 0.08 mu g/ml). MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 mu g/ml) against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic) on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein `Sa' and `Sb' sites were independently mutated, localized the binding site of MA2077 within the `Sa' antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus.

Mapping joint ROM on a cube using electromagnetic trackers

This paper presents a unified framework using the unit cube for measurement, representation and usage of the range of motion (ROM) of body joints with multiple degrees of freedom (d.o.f) to be used for digital human models (DHM). Traditional goniometry needs skill and kn owledge; it is intrusive and has limited applicability for multi-d.o.f. joints. Measurements using motion capture systems often involve complicated mathematics which itself need validation. In this paper we use change of orientation as the measure of rotation; this definition does not require the identification of any fixed axis of rotation. A two-d.o.f. joint ROM can be represented as a Gaussian map. Spherical polygon representation of ROM, though popular, remains inaccurate, vulnerable due to singularities on parametric sphere and difficult to use for point classification. The unit cube representation overcomes these difficulties. In the work presented here, electromagnetic trackers have been effectively used for measuring the relative orientation of a body segment of interest with respect to another body segment. The orientation is then mapped on a surface gridded cube. As the body segment is moved, the grid cells visited are identified and visualized. Using the visual display as a feedback, the subject is instructed to cover as many grid cells as he can. In this way we get a connected patch of contiguous grid cells. The boundary of this patch represents the active ROM of the concerned joint. The tracker data is converted into the motion of a direction aligned with the axis of the segment and a rotation about this axis later on. The direction identifies the grid cells on the cube and rotation about the axis is represented as a range and visualized using color codes. Thus the present methodology provides a simple, intuitive and accura te determination and representation of up to 3 d.o.f. joints. Basic results are presented for the shoulder. The measurement scheme to be used for wrist and neck, and approach for estimation of the statistical distribution of ROM for a given population are also discussed.

MAPS: midline analysis and propagation of segmentation

Scenic word images undergo degradations due to motion blur, uneven illumination, shadows and defocussing, which lead to difficulty in segmentation. As a result, the recognition results reported on the scenic word image datasets of ICDAR have been low. We introduce a novel technique, where we choose the middle row of the image as a sub-image and segment it first. Then, the labels from this segmented sub-image are used to propagate labels to other pixels in the image. This approach, which is unique and distinct from the existing methods, results in improved segmentation. Bayesian classification and Max-flow methods have been independently used for label propagation. This midline based approach limits the impact of degradations that happens to the image. The segmented text image is recognized using the trial version of Omnipage OCR. We have tested our method on ICDAR 2003 and ICDAR 2011 datasets. Our word recognition results of 64.5% and 71.6% are better than those of methods in the literature and also methods that competed in the Robust reading competition. Our method makes an implicit assumption that degradation is not present in the middle row.

Constraining large earthquakes along the Andaman trench using deepwater turbidites: prospects and challenges

The 2004 Sumatra-Andaman earthquake was unprecedented in terms of its magnitude (M-w 9.2), rupture length along the plate boundary (1300 km) and size of the resultant tsunami. Since 2004, efforts are being made to improve the understanding of the seismic hazard in the Sumatra-Andaman subduction zone in terms of recurrence patterns of major earthquakes and tsunamis. It is reasonable to assume that previous earthquake events in the Myanmar Andaman segment must be preserved in the geological record in the form of seismo-turbidite sequences. Here we present the prospects of conducting deep ocean palaeoseismicity investigations in order to refine the quantification of the recurrence pattern of large subduction-zone earthquakes along the Andaman-Myanmar arc. Our participation in the Sagar Kanya cruise SK-273 (in June 2010) was to test the efficacy of such a survey. The primary mission of the cruise, along a short length (300 km) of the Sumatra Andaman subduction front was to collect bathymetric data of the ocean floor trenchward of the Andaman Islands. The agenda of our piggyback survey was to fix potential coring sites that might preserve seismo-turbidite deposits. In this article we present the possibilities and challenges of such an exercise and our first-hand experience of such a preliminary survey. This account will help future researchers with similar scientific objectives who would want to survey the deep ocean archives of this region for evidence of extreme events like major earthquakes.

Analysis of designed beta-hairpin peptides: molecular conformation and packing in crystals

The crystal structures of several designed peptide hairpins have been determined in order to establish features of molecular conformations and modes of aggregation in the crystals. Hairpin formation has been induced using a centrally positioned (D)Pro-Xxx segment (Xxx = (L)Pro, Aib, Ac(6)c, Ala; Aib = alpha-aminoisobutyric acid; Ac(6)c = 1-aminocyclohexane-1-carboxylic acid). Structures of the peptides Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (1), Boc-Leu-Tyr-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (2, polymorphic forms labeled as 2a and 2b), Boc-Leu-Val-Val-(D)Pro-(L)Pro-Leu-Val-Val-OMe (3), Boc-Leu-Phe-Val-(D)Pro-Aib-Leu-Phe-Val-OMe (4, polymorphic forms labeled as 4a and 4b), Boc-Leu-Phe-Val-(D)Pro-Ac(6)c-Leu-Phe-Val-OMe (5) and Boc-Leu-Phe-Val-(D)Pro-Ala-Leu-Phe-Val-OMe (6) are described. All the octapeptides adopt type II' beta-turn nucleated hairpins, stabilized by three or four cross-strand intramolecular hydrogen bonds. The angle of twist between the two antiparallel strands lies in the range of -9.8 degrees to -26.7 degrees. A detailed analysis of packing motifs in peptide hairpin crystals is presented, revealing three broad modes of association: parallel packing, antiparallel packing and orthogonal packing. An attempt to correlate aggregation modes in solution with observed packing motifs in crystals has been made by indexing of crystal faces in the case of three of the peptide hairpins. The observed modes of hairpin aggregation may be of relevance in modeling multiple modes of association, which may provide insights into the structure of insoluble polypeptide aggregates.

Investigating proline puckering states in diproline segments in proteins

In the current study, the puckering states of the Proline ring occurring in diproline segments (LPro-LPro) in proteins has been investigated with a segregation made on the basis of cis and trans states for the Pro-Pro peptide bond and the conformational states for the diproline segment to investigate the effects of conformation of the diproline segment on the corresponding puckering state of the Proline ring in the segment if any. The value of the endocyclic ring torsional angles of the pyrrolidine ring has been used for calculating and visualizing various puckering states using a proposed new sign convention (+/-) nomenclature. The results have been compared to that obtained in a previous study on peptides from this group. In this study, quite interestingly, the Planar (G) conformation that was present in 14.3% of the cases in peptides, appears to be nearly a rare conformation in the case of proteins (1.9%). The present study indicates that the (C-exo/C-exo), (C-exo/Twisted C-exo-C-endo) and (Twisted C-endo-C-exo/Twisted C-endo-C-exo) categories are the most preferred combinations. For Proline rings in proteins, the states C-exo, Twisted C-exo-C-endo and Twisted C-endo-C-exo are the most preferred states. Within diproline segments, the pyrrolidine ring conformations do not show a strong co-relation to the backbone conformation in which they are observed. It is likely that five-membered rings have a considerable plasticity of structure and are readily deformed to accommodate a variety of energetically preferred backbone conformations.

Miniaturized Defected Ground High Isolation Crossovers

This letter relates to the design of crossovers for carrying criss crossing signals. Two types of crossovers are proposed in this letter. Both the crossovers are designed using a two layer printed circuit board. An unbroken continuous transmission line is routed in the top layer for carrying signal 1 from one node to another node. Transmission line used for carrying a signal 2 consists of three physically discontinuous, but electrically connected segments. Two end segments of these are located in the top layer while the middle segment is placed in the bottom layer. While Type I crossover offers an isolation of 25 dB, Type II crossover offers isolation better than 35 dB from dc to 10 GHz. These crossovers are compact and measure an actual size of 10 x 10 x 0.78 mm(3).

Automatic speech segmentation using probabilistic latent component modeling

Latent variable methods, such as PLCA (Probabilistic Latent Component Analysis) have been successfully used for analysis of non-negative signal representations. In this paper, we formulate PLCS (Probabilistic Latent Component Segmentation), which models each time frame of a spectrogram as a spectral distribution. Given the signal spectrogram, the segmentation boundaries are estimated using a maximum-likelihood approach. For an efficient solution, the algorithm imposes a hard constraint that each segment is modelled by a single latent component. The hard constraint facilitates the solution of ML boundary estimation using dynamic programming. The PLCS framework does not impose a parametric assumption unlike earlier ML segmentation techniques. PLCS can be naturally extended to model coarticulation between successive phones. Experiments on the TIMIT corpus show that the proposed technique is promising compared to most state of the art speech segmentation algorithms.

Single-step synthesis of internally functionalizable hyperbranched polyethers

Radical catalyzed thiol-ene reaction has become a useful alternative to the Huisgen-type azide-yne click reaction as it helps expand the variability in reaction conditions as well as the range of clickable entities. In this study, the direct generation of a hyperbranched polyether (HBPE) having decyl units at the periphery and a pendant allyl group on every repeat unit of the polymer backbone is described; the allyl groups serve as a reactive handle for postpolymerization modifications and permits the generation of a variety of internally functionalized HBPEs. In this design, the AB(2) monomer carries two decylbenzyl ether units (B-functionality), an aliphatic OH (A-functionality) and a pendant allyl group within the spacer segment; polymerization of the monomer readily occurs at 150 degrees C via melt transetherification process by continuous removal of 1-decanol under reduced pressure. The resulting HBPE has a hydrophobic periphery due to the presence of numerous decyl chains, while the allyl groups that remain unaffected during the melt polymerization provides an opportunity to install a variety of functional groups within the interior; thiol-ene click reaction with two different thiols, namely 3-mercaptopropionic acid and mercaptosuccinic acid, generated interesting amphiphilic structures. Preliminary field emission scanning electron microscope (FESEM) and Atomic Force Microscopy (AFM) imaging studies reveal the formation of fairly uniform spherical aggregates in water with sizes ranging from 200 to 400 nm; this suggests that these amphiphilic HBPs is able to reconfigure to generate jellyfish-like conformations that subsequently aggregate in an alkaline medium. The internal allyl functional groups were also used to generate intramolecularly core-crosslinked HBPEs, by the use of dithiol crosslinkers; gel permeation chromatography traces provided clear evidence for reduction in the size after crosslinking. In summary, we have developed a simple route to prepare core-clickable HBPEs and have demonstrated the quantitative reaction of the allyl groups present within the interior of the polymers; such HB polymeric systems that carry numerous functional groups within the core could have interesting applications in analyte sequestration and possibly sensing, especially from organic media. (c) 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 4125-4135

Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies

Short-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the gamma-phosphate of ATP to propionate during L-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate > acetate approximate to butyrate), nucleotides (ATP approximate to GTP > CTP approximate to TTP; dATP > dGTP > dCTP) and metal ions (Mg2+ approximate to Mn2+ > Co2+). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, alpha-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO4 (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235-290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the gamma-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer. (C) 2013 Elsevier B.V. All rights reserved.

Conformational Diversity in Contryphans from Conus Venom: cis-trans Isomerisation and Aromatic/Proline Interactions in the 23-Membered Ring of a 7-Residue Peptide Disulfide Loop

Conformational diversity or shapeshifting in cyclic peptide natural products can, in principle, confer a single molecular entity with the property of binding to multiple receptors. Conformational equilibria have been probed in the contryphans, which are peptides derived from Conus venom possessing a 23-membered cyclic disulfide moiety. The natural sequences derived from Conus inscriptus, GCV(D)LYPWC* (In936) and Conus loroisii, GCP(D)WDPWC* (Lo959) differ in the number of proline residues within the macrocyclic ring. Structural characterisation of distinct conformational states arising from cis-trans equilibria about Xxx-Pro bonds is reported. Isomerisation about the C2-P3 bond is observed in the case of Lo959 and about the Y5-P6 bond in In936. Evidence is presented for as many as four distinct species in the case of the synthetic analogue V3P In936. The Tyr-Pro-Trp segment in In936 is characterised by distinct sidechain orientations as a consequence of aromatic/proline interactions as evidenced by specific sidechain-sidechain nuclear Overhauser effects and ring current shifted proton chemical shifts. Molecular dynamics simulations suggest that Tyr5 and Trp7 sidechain conformations are correlated and depend on the geometry of the Xxx-Pro bond. Thermodynamic parameters are derived for the cis trans equilibrium for In936. Studies on synthetic analogues provide insights into the role of sequence effects in modulating isomerisation about Xxx-Pro bonds.

D-A-D-structured conducting polymer-modified electrodes for detection of lead(II) ions in water

Donor-acceptor-donor-structured thiophene derivative-based conducting polymer poly(7,9-dithiophene-2yl-8H-cyclopentaa]acenaphthalene-8-one) was chemically synthesized. This polymer was used to modify both glassy-carbon and carbon-paste electrode, which was used to detect lead(II) ions present in water in the range of 1 mM to 0.1 mu M. Cyclic voltammetry confirms the formation of the co-ordination complex between the soft segment of polymer and the dissolved lead ion. Anodic stripping voltammetry was carried out by the modified electrode to determine the lower limit of detection of dissolved lead(II) species in the solution. Differential adsorptive stripping and impedance measurements were also conducted to find the lowest possible response of the as-synthesized polymer to lead(II) ion in water. The electrochemical performance of the modified electrodes at different pH (4, 7 and 9) environments was carried out by stripping voltammetry, to get optimum sensitivity and stability under these conditions. Finally, interference analysis was carried out to detect the modified electrode's sensitivity towards lead ion affinity in water.

Robust Reentry Guidance of a Reusable Launch Vehicle Using Model Predictive Static Programming

A robust suboptimal reentry guidance scheme is presented for a reusable launch vehicle using the recently developed, computationally efficient model predictive static programming. The formulation uses the nonlinear vehicle dynamics with a spherical and rotating Earth, hard constraints for desired terminal conditions, and an innovative cost function having several components with associated weighting factors that can account for path and control constraints in a soft constraint manner, thereby leading to smooth solutions of the guidance parameters. The proposed guidance essentially shapes the trajectory of the vehicle by computing the necessary angle of attack and bank angle that the vehicle should execute. The path constraints are the structural load constraint, thermal load constraint, bounds on the angle of attack, and bounds on the bank angle. In addition, the terminal constraints include the three-dimensional position and velocity vector components at the end of the reentry. Whereas the angle-of-attack command is generated directly, the bank angle command is generated by first generating the required heading angle history and then using it in a dynamic inversion loop considering the heading angle dynamics. Such a two-loop synthesis of bank angle leads to better management of the vehicle trajectory and avoids mathematical complexity as well. Moreover, all bank angle maneuvers have been confined to the middle of the trajectory and the vehicle ends the reentry segment with near-zero bank angle, which is quite desirable. It has also been demonstrated that the proposed guidance has sufficient robustness for state perturbations as well as parametric uncertainties in the model.