Identification and characterization of fragment binding sites for allosteric ligand design using the site identification by ligand competitive saturation hotspots approach (SILCS-Hotspots)

BackgroundFragment-based ligand design is used for the development of novel ligands that target macromolecules, most notably proteins. Central to its success is the identification of fragment binding sites that are spatially adjacent such that fragments occupying those sites may be linked to create drug-like ligands. Current experimental and computational approaches that address this problem typically identify only a limited number of sites as well as use a limited number of fragment types.MethodsThe site-identification by ligand competitive saturation (SILCS) approach is extended to the identification of fragment bindings sites, with the method termed SILCS-Hotspots. The approach involves precomputation of the SILCS FragMaps following which the identification of Hotspots, performed by identifying of all possible fragment binding sites on the full 3D structure of the protein followed by spatial clustering.ResultsThe SILCS-Hotspots approach identifies a large number of sites on the target protein, including many sites not accessible in experimental structures due to low binding affinities and binding sites on the protein interior. The identified sites are shown to recapitulate the location of known drug-like molecules in both allosteric and orthosteric binding sites on seven proteins including the androgen receptor, the CDK2 and Erk5 kinases, PTP1B phosphatase and three GPCRs; the β2-adrenergic, GPR40 fatty-acid binding and M2-muscarinic receptors. Analysis indicates the importance of considering all possible fragment binding sites, and not just those accessible to experimental methods, when identifying novel binding sites and performing ligand design versus just considering the most favorable sites. The approach is shown to identify a larger number of known binding sites of drug-like molecules versus the commonly used FTMap and Fpocket methods.General significanceThe present results indicate the potential utility of the SILCS-Hotspots approach for fragment-based rational design of ligands, including allosteric modulators.     

Inferring gene regulatory relationships by combining target-target pattern recognition and regulator-specific motif examination

Although microarray data have been successfully used for gene clustering and classification, the use of time series microarray data for constructing gene regulatory networks remains a particularly difficult task. The challenge lies in reliably inferring regulatory relationships from datasets that normally possess a large number of genes and a limited number of time points. In addition to the numerical challenge, the enormous complexity and dynamic properties of gene expression regulation also impede the progress of inferring gene regulatory relationships. Based on the accepted model of the relationship between regulator and target genes, we developed a new approach for inferring gene regulatory relationships by combining target-target pattern recognition and examination of regulator-specific binding sites in the promoter regions of putative target genes. Pattern recognition was accomplished in two steps: A first algorithm was used to search for the genes that share expression profile similarities with known target genes (KTGs) of each investigated regulator. The selected genes were further filtered by examining for the presence of regulator-specific binding sites in their promoter regions. As we implemented our approach to 18 yeast regulator genes and their known target genes, we discovered 267 new regulatory relationships, among which 15% are rediscovered, experimentally validated ones. Of the discovered target genes, 36.1% have the same or similar functions to a KTG of the regulator. An even larger number of inferred genes fall in the biological context and regulatory scope of their regulators. Since the regulatory relationships are inferred from pattern recognition between target-target genes, the method we present is especially suitable for inferring gene regulatory relationships in which there is a time delay between the expression of regulating and target genes.     

LD-SPatt: large deviations statistics for patterns on Markov chains.

Statistics on Markov chains are widely used for the study of patterns in biological sequences. Statistics on these models can be done through several approaches. Central limit theorem (CLT) producing Gaussian approximations are one of the most popular ones. Unfortunately, in order to find a pattern of interest, these methods have to deal with tail distribution events where CLT is especially bad. In this paper, we propose a new approach based on the large deviations theory to assess pattern statistics. We first recall theoretical results for empiric mean (level 1) as well as empiric distribution (level 2) large deviations on Markov chains. Then, we present the applications of these results focusing on numerical issues. LD-SPatt is the name of GPL software implementing these algorithms. We compare this approach to several existing ones in terms of complexity and reliability and show that the large deviations are more reliable than the Gaussian approximations in absolute values as well as in terms of ranking and are at least as reliable as compound Poisson approximations. We then finally discuss some further possible improvements and applications of this new method.     

Capacitive sensor for environmental monitoring based on thin films of molecularly imprinted polymers. Computer modeling for optimization of the composition of synthetic analogs of bioreceptors

A capacitive sensor for environmental monitoring based on thin films of desmetryn-selective molecularly imprinted polymer (MIP) was developed. The method of modification of gold electrodes with the thin film of herbicide-selective MIP using the grafting polymerization approach was developed. The method of computational modeling was used to optimize the composition of desmetryn-selective MIPs. It was shown that 2-acrylamido-2-methyl-1-propan-sulfonic acid is the optimal functional monomer for desmetryn. Formation of synthetic binding sites in MIPs was demonstrated to be determined by the binding energy between the template and functional monomers as well as the number of functional groups taking part in the recognition of the template molecule. Electrochemical processes occurring at the MIP-modified electrode were analyzed. The detection limit for desmetryn comprised 100 nM. High selectivity of the capacitive sensor towards structural analogues of desmetryn as well as high operational and storage stabilities was demonstrated.     

Hydrogen Bonding Interactions of Liposomes Simulating Cell-Cell Recognition. A Review

Amphiphiles bearing polar heads with the property to form hydrogen bond(s) exhibit unique organizational and aggregational behaviour. Thus appropriate amphiphilic molecules assemble and form liposomes, which further interact through hydrogen bonding with complementary molecules or liposomal counterparts affording larger and more elaborated aggregates. A number of examples are demonstrating the interaction mode of liposomes and of associated phenomena as related to the structural features of the supramolecular aggregates obtained. The recognition between cells incorporating recognizable amphiphiles in their membranes has shown similarities to the analogous interactions between liposomes. Thus molecular recognition between liposomes can be used in modeling recognitions occurring between cells. Designed experiments in this area can support the Lipid World Model proposed for the origin of life.     

Influence of recognition errors of computerised analysis of 24-hour electrocardiograms on the measurement of spectral components of heart rate variability

Spectral methods for the assessment of heart rate variability (HRV) in 24-h electrocardiogram (ECG) are believed to require visual verification and manual editing of the computerised recognition of the ECG. This study investigated the effect of the recognition errors of computerised ECG recognition on two methods providing spectral HRV indices: (a) Fast Fourier Transformation (FFT); and (b) peak-to-trough analysis (PTA). Both methods were used to measure HRV spectra in 24-h ECGs recorded in 557 survivors of acute myocardial infarction. Each ECG was analysed using the Marquette 8000 Holter system and spectral HRV analyses were performed both prior to and after manual verification of the automatic ECG analysis. The FFT and PTA methods were used to calculate the low (0.04-0.15 Hz), medium (0.15-0.40 Hz) and high (0.40-1.00 Hz) HRV spectral components. For each method and for each spectral component, the rank correlations between the results obtained from unedited and edited ECG recognition were calculated. The correlations between the corresponding spectral components provided by the FFT and PTA methods applied to the edited recognitions were also calculated. Both methods were substantially affected by recognition errors. The FFT method was more sensitive to the misrecognition than the PTA method. The inter-method correlations were higher for the high and medium spectral components than for the low spectral component. The study suggests that spectral HRV analysis should be performed only on carefully verified and manually corrected recognitions of long-term electrocardiograms.     

A pairwise alignment algorithm which favors clusters of blocks.

Pairwise sequence alignments aim to decide whether two sequences are related and, if so, to exhibit their related domains. Recent works have pointed out that a significant number of true homologous sequences are missed when using classical comparison algorithms. This is the case when two homologous sequences share several little blocks of homology, too small to lead to a significant score. On the other hand, classical alignment algorithms, when detecting homologies, may fail to recognize all the significant biological signals. The aim of the paper is to give a solution to these two problems. We propose a new scoring method which tends to increase the score of an alignment when "blocks" are detected. This so-called Block-Scoring algorithm, which makes use of dynamic programming, is worth being used as a complementary tool to classical exact alignments methods. We validate our approach by applying it on a large set of biological data. Finally, we give a limit theorem for the score statistics of the algorithm.     

Oligonucleotide frequency matrices addressed to recognizing functional DNA sites.

MOTIVATION: Recognition of functional sites remains a key event in the course of genomic DNA annotation. It is well known that a number of sites have their own specific oligonucleotide content. This pinpoints the fact that the preference of the site-specific nucleotide combinations at adjacent positions within an analyzed functional site could be informative for this site recognition. Hence, Web-available resources describing the site-specific oligonucleotide content of the functional DNA sites and applying the above approach for site recognition are needed. However, they have been poorly developed up to now. RESULTS: To describe the specific oligonucleotide content of the functional DNA sites, we introduce the oligonucleotide alphabets, out of which the frequency matrix for a given site could be constructed in addition to a traditional nucleotide frequency matrix. Thus, site recognition accuracy increases. This approach was implemented in the activated MATRIX database accumulating oligonucleotide frequency matrices of the functional DNA sites. We have demonstrated that the false-positive error of the functional site recognition decreases if the oligonucleotide frequency matrixes are added to the nucleotide frequency matrixes commonly used. AVAILABILITY: The MATRIX database is available on the Web, http://wwwmgs.bionet.nsc.ru/Dbases/MATRIX/ and the mirror site, http://www.cbil.upenn.edu/mgs/systems/c onsfreq/.     

Current noise around steady states in discrete transport systems

Subject of this paper is the transport noise in discrete systems. The transport systems are given by a number (n) of binding sites separated by energy barriers. These binding sites may be in contact with constant outer reservoirs. The state of the system is characterized by the occupation numbers of particles (current carriers) at these binding sites. The change in time of the occupation numbers is generated by individual “jumps” of particles over the energy barriers, building up the flux matter (for charged particles: the electric current). In the limit n → ∞ continuum processes as e.g. usual diffusion are included in the transport model. The fluctuations in occupation numbers and other quantities linearly coupled to the occupation numbers may be treated with the usual master equation approach. The treatment of the fluctuations in fluxes (current) makes necessary a different theoretical approach which is presented in this paper under the assumption of vanishing interactions between the particles. This approach may be applied to a number of different transport systems in biology and physics (ion transport through porous channels in membranes, carrier mediated ion transport through membranes, jump diffusion e.g. in superionic conductors). As in the master equation approach the calculation of correlations and noise spectra may be reduced to the solution of the macroscopic equations for the occupation numbers. This result may be regarded as a generalization to non-equilibrium current fluctuations of the usual Nyquist theorem relating the current (voltage) noise spectrum in thermal equilibrium to the macroscopic frequency dependent admittance.The validity of the general approach is demonstrated by the calculation of the autocorrelation function and spectrum of current noise for a number of special examples (e.g, pores in membrances, carrier mediated ion transport).     

Communally breeding Bechstein's bats have a stable social system that is independent from the postglacial history and location of the populations

Investigating macro-geographical genetic structures of animal populations is crucial to reconstruct population histories and to identify significant units for conservation. This approach may also provide information about the intraspecific flexibility of social systems. We investigated the history and current structure of a large number of populations in the communally breeding Bechstein's bat ( Myotis bechsteinii ). Our aim was to understand which factors shape the species' social system over a large ecological and geographical range. Using sequence data from one coding and one noncoding mitochondrial DNA region, we identified the Balkan Peninsula as the main and probably only glacial refugium of the species in Europe. Sequence data also suggest the presence of a cryptic taxon in the Caucasus and Anatolia. In a second step, we used seven autosomal and two mitochondrial microsatellite loci to compare population structures inside and outside of the Balkan glacial refugium. Central European and Balkan populations both were more strongly differentiated for mitochondrial DNA than for nuclear DNA, had higher genetic diversities and lower levels of relatedness at swarming (mating) sites than in maternity (breeding) colonies, and showed more differentiation between colonies than between swarming sites. All these suggest that populations are shaped by strong female philopatry, male dispersal, and outbreeding throughout their European range. We conclude that Bechstein's bats have a stable social system that is independent from the postglacial history and location of the populations. Our findings have implications for the understanding of the benefits of sociality in female Bechstein's bats and for the conservation of this endangered species.     

Chromosome synapsis in hexaploids

Theoretical relationships between pachytene multivalent and bivalent frequencies in hexaploids are deduced from a model, based on chromosomes showing sequential association at equidistant pairing sites and uniform propensities for partner exchange throughout their lengths. These relationships approach a limit as the number of pairing sites tends to infinity and the intervals between them tend to zero; at the limit pairing is continuous and the quadrivalent/sexivalent ratio is at a minimum. A maximum of 34·3% of the complement is expected to form quadrivalents when there are two pairing sites per chromosome but this peak is reduced by increasing numbers of pairing sites to a limit of 29·6% when pairing is continuous. Where chromosome length is proportional to the number of pairing sites there will be a log/linear relationship between bivalent frequency and chromosome length otherwise a log/log relationship is expected. In the light of these conclusions, observations on experimental hexaploids could be used to provide estimates of the number of pairing sites on each chromosome.     

Modelling the mechanical effect of muscles with large attachment sites: Application to the shoulder mechanism

A general theory is described for deriving the mechanical effect of muscles with large attachment sites. In a cadaver experiment the complete attachment sites and bundle distribution of 16 muscles of the shoulder mechanism were recorded. These data were used to calculate the mechanical effect, i.e. the resulting force and moment vector, for a large number (200) and a reduced number (maximal 6) of muscle lines of action. The resulting error between both representations is small. The number of muscle lines of action in the reduced representation depends on the shape of the attachment site and muscle architecture. An important feature of this method is that the necessary number of muscle lines of action is determined afterwards. In the often used centroid line approach the number of muscle lines of action and partitioning of muscles is determined before recording the geometry, leading to unverifiable results.     

基于支持向量机(SVM)的剪接位点识别

剪接位点的识别作为基因识别中的一个重要环节, 一直受到研究人员的关注。考虑到剪接位点附近存在的序列保守性,已有一些基于统计特性的方法被用于剪接位点的识别中,但效果仍有待进一步改进。支持向量机（Ｓｕｐｐｏｒｔ Ｖｅｃｔｏｒ Ｍａｃｈｉｎｅｓ） 作为一种新的基于统计学习理论的学习机,近几年有了很大的发展,已被应用在模式识别的许多问题中。文中将其用于剪接位点的识别中,并针对满足ＧＴ－ ＡＧ 规则的序列样本中虚假剪接位点的样本数远大于真实位点这一特性, 提出了一种基于ＳＶＭ 的平衡取小法以获得更好的识别效果。实验结果表明,应用支持向量机进行剪接位点的识别能更好地提取位点附近保守序列的统计特征,对测试集具有更好的推广能力,并且使用上更加简单。这一结果为剪接位点的识别提供了一种新的方法,同时也为生物大分子研究中结构和位点的识别问题的解决提供了新的线索。     

Site-specific labeling of supercoiled DNA

Visualization of site-specific labels in long linear or circular DNA allows unambiguous identification of various local DNA structures. Here we describe a novel and efficient approach to site-specific DNA labeling. The restriction enzyme SfiI binds to DNA but leaves it intact in the presence of calcium and therefore may serve as a protein label of 13 bp recognition sites. Since SfiI requires simultaneous interaction with two DNA recognition sites for stable binding, this requirement is satisfied by providing an isolated recognition site in the DNA target and an additional short DNA duplex also containing the recognition site. The SfiI/DNA complexes were visualized with AFM and the specificity of the labeling was confirmed by the length measurements. Using this approach, two sites in plasmid DNA were labeled in the presence of a large excess of the helper duplex to compete with the formation of looped structures of the intramolecular synaptic complex. We show that the labeling procedure does not interfere with the superhelical tension-driven formation of alternative DNA structures such as cruciforms. The complex is relatively stable at low and high pH (pH 5 and 9) making the developed approach attractive for use at conditions requiring the pH change.     

Some properties of an estimator for the basic reproduction number of the general epidemic model.

In this paper some properties of a convenient estimator, derived from a martingale estimating function, for the basic reproduction number of the general epidemic model are given for both finite and large samples. These properties give some guidelines for using this convenient estimator. It is shown that it underestimates the parameter and that the bias tends to zero when the population size and the initial number of infectives are increased simultaneously. The bias cannot be removed for a fixed number of introductory infectives. However, the estimator is asymptotically unbiased, conditional on a major outbreak. A simulation study shows that the central limit theorem applies for moderate population sizes.     

DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition. In order to elucidate the connection between the mechanics and the chemistry of DNA recognition and cleavage, we used a single-molecule approach to measure rate changes in the reaction pathway of EcoRV and BamHI as a function of DNA tension. We show that the induced-fit rate of EcoRV is strongly reduced by such tension. In contrast, BamHI is found to be insensitive, providing evidence that both substrate binding and hydrolysis are not influenced by this force. Based on these results, we propose a mechanochemical model of induced-fit reactions on DNA, allowing determination of induced-fit rates and DNA bend angles. Finally, for both enzymes a strongly decreased association rate is obtained on stretched DNA, presumably due to the absence of intradomain dissociation/re-association between non-specific sites (jumping). The obtained results should apply to many other DNA-associated proteins.