Structure-based analysis of Bacilli and plasmid dihydrofolate reductase evolution

Dihydrofolate reductase (DHFR), a key enzyme in tetrahydrofolate-mediated biosynthetic pathways, has a structural motif known to be highly conserved over a wide range of organisms. Given its critical role in purine and amino acid synthesis, DHFR is a well established therapeutic target for treating a wide range of prokaryotic and eukaryotic infections as well as certain types of cancer. Here we present a structural-based computer analysis of bacterial (Bacilli) and plasmid DHFR evolution. We generated a structure-based sequence alignment using 7 wild-type DHFR x-ray crystal structures obtained from the RCSB Protein Data Bank and 350 chromosomal and plasmid homology models we generated from sequences obtained from the NCBI Protein Database. We used these alignments to compare active site and non-active site conservation in terms of amino acid residues, secondary structure and amino acid residue class. With respect to amino acid sequences and residue classes, active-site positions in both plasmid and chromosomal DHFR are significantly more conserved than non-active site positions. Secondary structure conservation was similar for active site and non-active site positions. Plasmid-encoded DHFR proteins have greater degree of sequence and residue class conservation, particularly in sequence positions associated with a network of concerted protein motions, than chromosomal-encoded DHFR proteins. These structure-based were used to build DHFR specific phylogenetic trees from which evidence for horizontal gene transfer was identified.     

Molecular dynamics simulations of mutated Mycobacterium tuberculosis l-alanine dehydrogenase to illuminate the role of key residues

l-Alanine dehydrogenase from Mycobacterium tuberculosis (l-MtAlaDH) catalyzes the NADH-dependent interconversion of l-alanine and pyruvate, and it is considered to be a potential target for the treatment of tuberculosis. The experiment has verified that amino acid replacement of the conserved active-site residues which have strong stability and no great changes in biological evolutionary process, such as His96 and Asp270, could lead to inactive mutants [Ågren et al., J. Mol. Biol. 377 (2008) 1161–1173]. However, the role of these conserved residues in catalytic reaction still remains unclear. Based on the crystal structures, a series of mutant structures were constructed to investigate the role of the conserved residues in enzymatic reaction by using molecular dynamics simulations. The results show that whatever the conserved residues were mutated, the protein can still convert its conformation from open state to closed state as long as NADH is present in active site. Asp270 maintains the stability of nicotinamide ring and ribose of NADH through hydrogen bond interactions, and His96 is helpful to convert the protein conformation by interactions with Gln271, whereas, they would lead to the structural rearrangement in active site and lose the catalytic activity when they were mutated. Additionally, we deduce that Met301 plays a major role in catalytic reaction due to fixing the nicotinamide ring of NADH to prevent its rotation, and we propose that Met301 would be mutated to the hydrophobic residue with large steric hindrance in side chain to test the activity of the protein in future experiment.     

Comparative molecular modeling study of the three-dimensional structures of prostaglandin endoperoxide H2 synthase 1 and 2 (COX-1 and COX-2)

To understand the structural features that dictate the selectivity of diverse nonsteroidal antiinflammatory drugs for the two isoforms of the human prostaglandin H₂ synthase (PGHS), the three-dimensional (3D) structure of human COX-2 was assessed by means of sequence homology modeling. The ovine COX-1 structure, solved by X-ray diffraction methods and sharing a 61% sequence identity with human COX-2, was used as template. Both structures were energy minimized using the AMBER 4.0 force field with a dielectric constant of 4r. (S)-Flurbiprofen, a nonselective COX inhibitor, and SC-558, a COX-2-selective ligand, were docked at the cyclooxygenase binding site in both isozymes, evidencing the role of different residues in the ligand–protein interaction. The 3D structures of the constructed four ligand–enzyme complexes were refined by energy minimization. Molecular dynamics simulations were also carried out, to understand more deeply the structural origins of the selectivity. Distances calculated during the dynamics process between the different ligands and the interacting residues of the two PGHS isozymes provided evidence of the flexible nature of the cyclooxygenase active site, permitting the identification of different conserved and nonconserved residues as responsible for ligand selectivity.     

Triangulating multiply-connected polygons: A simple, yet efficient algorithm.

We present a new, simple, yet efficient algorithm for triangulating multiply-connected polygons. The algorithm requires sorting only local concave minima (sags). The order in which triangles are created mimics a flooding process of the interior of the polygon. At each stage, the algorithm analyses the positions and neighborhoods of two vertices only, and possibly checks for active sags, so as to determine which of five possible actions to take. Actions are based on a local decomposition of the polygon into monotonic regions, or gorges (raise the water level in the current gorge, spill into an adjacent gorge, jump to the other bank of a filled gorge, divide a gorge into two, and fill a gorge to its top). The implementation is extremely simple and numerically robust for a large class of polygons. It has been tested on millions of cases as a preprocessing step of a walkthrough and inspection program for complex mechanical and architectural scenes. Extensive experimental results indicate that the observed complexity in terms of the number of vertices, remains under in all cases.     

Molecular dynamics and density functional theory studies of substrate binding and catalysis of human brain aspartoacylase

The active-site dynamics of human brain aspartoacylase (hASPA) complexed with its substrate (N-acetyl-l-aspartate) has been studied using a hybrid quantum mechanical/molecular mechanical (QM/MM) approach based on the self-consistent charge-density functional tight-binding (SCC-DFTB) model. The Michaelis complex, which is constructed from a recent X-ray structure of the human brain aspartoacylase with a stable tetrahedral intermediate analogue, is reproduced in 1 ns molecular dynamics simulations at 300 K. The simulation shows that the substrate is tightly held in the active site by a hydrogen bond network and the putative nucleophilic water molecule is reasonably close to the nucleophilic center. The catalysis is further modeled with the density functional theory (DFT) in a truncated active-site model at the B3LYP/6-31G(d) level of theory. The DFT calculations indicate the reaction proceeds via a water promoted pathway with Glu178 serving as the general base and general acid. Transition state stabilization for nucleophilic addition is achieved by formations of the weak coordination bond between the substrate carbonyl oxygen atom and the zinc ion as well as of the strong hydrogen bonds between the substrate carbonyl oxygen atom and Arg63.     

KeyGraph as Risk Explorer in Earthquake–Sequence

KeyGraph, a document–indexing (keyword–extraction) algorithm, is applied for a new purpose: Extracting active faults with risks of near–future large earthquakes from earthquake–sequences. This paper presents KeyGraph as an extractor of causalities from an event–sequence. This validates KeyGraph as a tool for showing why and which active faults are risky, as well as for showing why and which words abstract a document. The risky faults that are empirically obtained by KeyGraph correspond closely to real earthquake occurrences and seismologists’ risk estimation.     

Molecular modeling study on the effect of residues distant from the nucleotide-binding portion on RNA binding in Staphylococcus aureus Hfq

Hfq is an abundant RNA-binding bacterial protein that was first identified in E. coli as a required host factor for phage Qβ RNA replication. The pleiotrophic phenotype resulting from the deletion of Hfq predicates the importance of this protein. Two RNA-binding sites have been characterized: the proximal site which binds sRNA and mRNA and the distal site which binds poly(A) tails. Previous studies mainly focused on the key residues in the proximal site of the protein. A recent mutation study in E. coli Hfq showed that a distal residue Val43 is important for the protein function. Interestingly, when we analyzed the sequence and structure of Staphylococcus aureus Hfq using the CONSEQ server, the results elicited that more functional residues were located far from the nucleotide-binding portion (NBP). From the analysis seven individual residues Asp9, Leu12, Glu13, Lys16, Gln31, Gly34 and Asp40 were selected to investigate the conformational changes in Hfq–RNA complex due to point mutation effect of those residues using molecular dynamics simulations. Results showed a significant effect on Asn28 which is an already known highly conserved functionally important residue. Mutants D9A, E13A and K16A depicted effects on base stacking along with increase in RNA pore diameter, which is required for the threading of RNA through the pore for the post-translational modification. Further, the result of protein stability analysis by the CUPSAT server showed destabilizing effect in the most mutants. From this study we characterized a series of important residues located far from the NBP and provide some clues that those residues may affect sRNA binding in Hfq.     

Robust reasoning with rules that have exceptions: From second-order probability to argumentation via upper envelopes of probability and possibility plus directed graphs

Rules having rare exceptions may be interpreted as assertions of high conditional probability. In other words, a rule If X then Y may be interpreted as meaning that Pr(Y∣X) 1. A general approach to reasoning with such rules, based on second-order probability, is advocated. Within this general approach, different reasoning methods are needed, with the selection of a specific method being dependent upon what knowledge is available about the relative sizes, across rules, of upper bounds on each rule's exception probabilities Pr(?Y∣X). A method of reasoning, entailment with universal near surety, is formulated for the case when no information is available concerning the relative sizes of upper bounds on exception probabilities. Any conclusion attained under these conditions is robust in the sense that it will still be attained if information about the relative sizes of exception probability bounds becomes available. It is shown that reasoning via entailment with universal near surety is equivalent to reasoning in a particular type of argumentation system having the property that when two subsets of the rule base conflict with each other, the effectively more specific subset overrides the other. As stepping stones toward attaining this argumentation result, theorems are proved characterizing entailment with universal near surety in terms of upper envelopes of probability measures, upper envelopes of possibility measures, and directed graphs. In addition, various attributes of entailment with universal near surety, including property inheritance, are examined.     

Separation and Instabilities in the Viscious Flow over Airfoil Leading edges

The two-dimensional, unsteady, leading-edge flow over stationary, pitching and oscillating airfoils is studied using the Navier–Stokes equations for flow past a parabola. Grid-converged solutions have been obtained for the initial stages of unsteady separation for impulsively-started unsteady flow and for pitch-up at constant rate. The results compare favorably with corresponding full-airfoil computations. It is shown that small perturbations in the flow can generate eddies in the boundary layer before flow reversal occurs in the base flow. Oscillations appear in the skin friction which grow in amplitude and convect downstream with time. Above a certain amplitude, the skin friction becomes negative locally, giving rise to small recirculating eddies. This type of eddy creation prior to reversal in the base flow is in general agreement with the theory of Rayleigh instabilities. The wavelength of these instabilities scales as Re^−0.43 which is in reasonable agreement with the theoretical value of Re^−1/2 for the Rayleigh instability.     

Temporal analysis of forest structural condition at an acid mine site using multispectral digital camera imagery

A large abandoned tailings deposit at a mine site near Timmins, Ontario, Canada has produced significant damage in an adjacent forest due to contamination and wind stress. Significant forest structure changes were measured between 1997 and 1999. A multivariate image-based forest structure index (FSI) was developed using canonical correlation analysis of 1997 field and airborne digital camera data. FSI included decreasing canopy closure and leaf area index, and increasing blown down and standing dead structure measures associated with image spectral, textural and radiometric fraction variables. An image model predicting FSI achieved an R ²=0.66. The model equation was then applied to 1999 airborne imagery to predict FSI for each plot. Comparing the 1999 image predicted FSI to that calculated from field data showed that the model was strong in predicting positive or no forest structure changes, but not increased structure degradation. The latter was due to the presence of herbaceous and shrub vegetation that had developed during the two-year period in open plots near the tailings where blow down was significant. The next research phase will derive means to separate these two signals in forests of open overstory.     

Pattern discovery and change detection of online music query streams

Mining of music data is one of the most important problems in multimedia data mining. In this paper, two research issues of mining music data, i.e., online mining of music query streams and change detection of music query streams, are discussed. First, we proposed an efficient online algorithm, FTP-stream (Frequent Temporal Pattern mining of streams), to mine all frequent melody structures over sliding windows of music melody sequence streams. An effective bit-sequence representation is used in the proposed algorithm to reduce the time and memory needed to slide the windows. An effective list structure is developed in the FTP-stream algorithm to overcome the performance bottleneck of 2-candidate generation. Experiments show that the proposed algorithm FTP-stream only needs a half of memory requirement of original melody sequence data, and just scans the music query stream once. After mining frequent melody structures, we developed a simple online algorithm, MQS-change (changes of Music Query Streams), to detect the changes of frequent melody structures in current user-centered music query streams. Two music melody structures (set of chord-sets and string of chord-sets) are maintained and four melody structure changes (positive burst, negative burst, increasing change and decreasing change) are monitored in a new summary data structure, MSC-list (a list of Music Structure Changes). Experiments show that the MQS-change algorithm is an effective online method to detect the changes of music melody structures over continuous music query streams.

Effects of protein flexibility on the site of metabolism prediction for CYP2A6 substrates

Structure-based prediction for the site of metabolism (SOM) of a compound metabolized by human cytochrome P450s (CYPs) is highly beneficial in drug discovery and development. However, the flexibility of the CYPs’ active site remains a huge challenge for accurate SOM prediction. Compared with other CYPs, the active site of CYP2A6 is relatively small and rigid. To address the impact of the flexibility of CYP2A6 active site residues on the SOM prediction for substrates, in this work, molecular dynamics (MD) simulations and molecular docking were used to predict the SOM of 96 CYP2A6 substrates. Substrates with known SOM were docked into the snapshot structures from MD simulations and the crystal structures of CYP2A6. Compared to the crystal structures, the protein structures obtained from MD simulations showed more accurate prediction for SOM. Our results indicated that the flexibility of the active site of CYP2A6 significantly affects the SOM prediction results. Further analysis for the 40 substrates with definite K_m values showed that the prediction accuracy for the low K_m substrates is comparable to that by ligand-based methods.     

Automatic detection of conserved base pairing patterns in RNA virus genomes.

Almost all RNA molecules--and consequently also almost all subsequences of a large RNA molecule-form secondary structures. The presence of secondary structure in itself therefore does not indicate any functional significance. In fact, we cannot expect a conserved secondary structure for all parts of a viral genome or a mRNA, even if there is a significant level of sequence conservation. We present a novel method for detecting conserved RNA secondary structures in a family of related RNA sequences. The method is based on combining the prediction of base pair probability matrices and comparative sequence analysis. It can be applied to small sets of long sequences and does not require a prior knowledge of conserved sequence or structure motifs. As such it can be used to scan large amounts of sequence data for regions that warrant further experimental investigation. Applications to complete genomic RNAs of some viruses show that in all cases the known secondary structure features are identified. In addition, we predict a substantial number of conserved structural elements which have not been described so far.     

基于状态模式的应用研究

以降低复杂度,增强可扩张性为目标,建立基于传送装置的入口模型,体现出状态模式的优越性,最终通过采用该模式解决了某大型钢铁企业传送装置入口的建模问题。     

Effects of planar inlet plenums on the hydrodynamically developing flows in rectangular microchannels of complementary aspect ratios

The effects of planar inlet plenum geometry on the developing flow fields in two rectangular microchannels of reciprocal aspect ratios (H/W ∼2.75 and ∼0.40) were investigated for Re _D =  1–100 using micro particle image velocimetry (μPIV). These two microchannels were made by a precision sawing and silicon microfabrication techniques. Both the velocity profiles and centerline velocity developments were clearly resolved and extracted along the axial distance from μPIV results. The entrance lengths were found from the centerline velocities using a decaying exponential fitting function where the centerline velocity reaches 99% of the fully developed centerline velocity. The proposed fitting function showed excellent agreement with the experimental results. The planar plenum was shown to cause an upstream predevelopment resulting in the significant reductions in the entrance lengths. Two entrance length correlations were proposed in the forms of Atkinson et al.’s (AIChE J 15:548–553, 1969) and Chen’s (J Fluids Eng 95:153–158, 1973) correlations. The proposed entrance length correlations showed that acquired constant portion and slope of the entrance lengths showed 23–27 and 70–81% reductions for H/W =  2.75 while the entrance length correlation for H/W =  0.40 showed 69–73% increase and 41–63% decrease in the constant portion and slope, respectively.     

An experimental study of pilots' control characteristics for flight of an STOL aircraft in backside of drag curve at approach and landing

Abstract

In general, most vehicles can be modelled by a multi-variable system which has interactive variables. It can be clearly shown that there is an interactive response in an aircraft's velocity and altitude obtained by stick control and/or throttle control. In particular, if the flight conditions fall to backside of drag curve in the flight of an STOL aircraft at approach and landing then the ratio of drag variation to velocity change has a negative value (ΔD/Δu<0) and the system of motion presents a non-minimum phase. Therefore, the interaction between velocity and altitude response becomes so complicated that it affects to pilot's control actions and it may be difficult to control the STOL aircraft at approach and landing.

In this paper, experimental results of a pilot's ability to control the STOL aircraft are presented for a multi-variable manual control system using a fixed ground base simulator and the pilot's control ability is discussed for the flight of an STOL aircraft at backside of drag curve at approach and landing.     

Perspectives for the structure-based design of acetylcholinesterase reactivators

Rational design of active molecules through structure-based methods has been gaining adepts during the last decades due to the wider availability of protein structures, most of them conjugated with relevant ligands. Acetylcholinesterase (AChE) is a molecular target with a considerable amount of data related to its sequence and 3-dimensional structure. In addition, there are structural insights about the mechanism of action of the natural substrate and drugs used in Alzheimer’s disease, organophosphorus compounds, among others. We looked for AChE structural data useful for in silico design of potential interacting molecules. In particular, we focused on information regarding the design of ligands aimed to reactivate AChE catalytic activity. The structures of 178 AChE were annotated and categorized on different subsets according to the nature of the ligand, source organisms and experimental details. We compared sequence homology among the active site from Torpedo californica, Mus musculus and Homo sapiens with the latter two species having the closest relationship (88.9% identity). In addition, the mechanism of organophosphorus binding and the design of effective reactivators are reviewed. A curated data collection obtained with information from several sources was included for researchers working on the field. Finally, a molecular dynamics simulation with human AChE indicated that the catalytic pocket volume stabilizes around 600 ų, providing additional clues for drug design.     

Putative xylosyltransferase genes in <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>

Protein xylosyltransferases are the group of enzymes which are involved in transferring xylose from UDP-d-xylose to serine residue in a protein. These enzymes are commonly found in multicellular organisms and in some unicellular organisms. Previously we had identified the xylosyltransferase (XT) genes in EST sequence of a unicellular organism Trichomonas vaginalis through in silico approach based on the sequence homology. To corroborate if these genes are putative XT genes, we designed a workflow based on the sequence characteristics of xylosyltransferase, to verify if any of the putative XT gene sequences have sequence motifs. The XT genes in T. vaginalis predicted by Hidden Markov Model (HMM) were further analyzed with PfamHMM to identify if each putative sequence belongs to a known protein family, with TMHMM to examine whether the predicted XTs are Golgi xylosyltransferases and with MEME to find out the conserved motifs. The results confirmed our earlier study that these XTs are related to N-linked XTs in plants. To confirm the in silico results further, we analyzed the N-linked glycans of T. vaginalis and the empirical data also confirmed the computational analysis.     

On Dissemination Thresholds in Regular and Irregular Graph Classes

We investigate the natural situation of the dissemination of information on various graph classes starting with a random set of informed vertices called active. Initially active vertices are chosen independently with probability p, and at any stage in the process, a vertex becomes active if the majority of its neighbours are active, and thereafter never changes its state. This process is a particular case of bootstrap percolation. We show that in any cubic graph, with high probability, the information will not spread to all vertices in the graph if p < \frac12p<\frac{1}{2} . We give families of graphs in which information spreads to all vertices with high probability for relatively small values of p.     

Euler angles: conversion of arbitrary rotation sequences to specific rotation sequence

Euler angles have been used to describe the orientation of objects in two‐dimensional and three‐dimensional spaces since its formulation by Leonhard Euler. Many applications intended to represent the rotation of a body have been developed on the basis of Euler angles. Two‐dimensional rotations are combined in sequence to represent three‐dimensional rotations. Because there are three axes in a three‐dimensional Euclidean space (X, Y and Z), 12 rotation sequences in three dimensions are possible: XYZ, XZY, YXZ, YZX, ZXY, ZYX, XYX, ZYZ, ZXZ, YXY, XZX and YZY. Each rotation sequence yields different results, and different applications implement a different rotation sequence. Thus, conversion between different rotation sequences becomes essential to make applications developed in different rotation sequences compatible with each other. In this paper, a new method is introduced to convert arbitrary rotation sequences to a specific rotation sequence of choice. A sample program is also developed in a MATLAB‐Simulink environment to demonstrate the use of the new method in converting an arbitrary Euler rotation sequence to the specific Euler rotation sequence of XYZ. A six‐degrees‐of‐freedom animation block is used in the program to aid users to graphically see the rotation of a body in three‐dimensional space. Copyright © 2013 John Wiley & Sons, Ltd.