Showing papers by "Technical University of Denmark published in 2005"

Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids

Abstract: Probablistic models are becoming increasingly important in analyzing the huge amount of data being produced by large-scale DNA-sequencing efforts such as the Human Genome Project. For example, hidden Markov models are used for analyzing biological sequences, linguistic-grammar-based probabilistic models for identifying RNA secondary structure, and probabilistic evolutionary models for inferring phylogenies of sequences from different organisms. This book gives a unified, up-to-date and self-contained account, with a Bayesian slant, of such methods, and more generally to probabilistic methods of sequence analysis. Written by an interdisciplinary team of authors, it is accessible to molecular biologists, computer scientists, and mathematicians with no formal knowledge of the other fields, and at the same time presents the state of the art in this new and important field.

Biomimetic Hydrogen Evolution: MoS2 Nanoparticles as Catalyst for Hydrogen Evolution

Abstract: The electrochemical hydrogen evolution reaction is catalyzed most effectively by the Pt group metals. As H2 is considered as a future energy carrier, the need for these catalysts will increase and alternatives to the scarce and expensive Pt group catalysts will be needed. We analyze the ability of different metal surfaces and of the enzymes nitrogenase and hydrogenase to catalyze the hydrogen evolution reaction and find a necessary criterion for high catalytic activity. The necessary criterion is that the binding free energy of atomic hydrogen to the catalyst is close to zero. The criterion enables us to search for new catalysts, and inspired by the nitrogenase active site, we find that MoS2 nanoparticles supported on graphite are a promising catalyst. They catalyze electrochemical hydrogen evolution at a moderate overpotential of 0.1−0.2 V.

Real-space grid implementation of the projector augmented wave method

Abstract: A grid-based real-space implementation of the projector augmented wave (PAW) method of Bl\"ochl [Phys. Rev. B 50, 17953 (1994)] for density functional theory (DFT) calculations is presented. The use of uniform three-dimensional (3D) real-space grids for representing wave functions, densities, and potentials allows for flexible boundary conditions, efficient multigrid algorithms for solving Poisson and Kohn-Sham equations, and efficient parallelization using simple real-space domain-decomposition. We use the PAW method to perform all-electron calculations in the frozen core approximation, with smooth valence wave functions that can be represented on relatively coarse grids. We demonstrate the accuracy of the method by calculating the atomization energies of 20 small molecules, and the bulk modulus and lattice constants of bulk aluminum. We show that the approach in terms of computational efficiency is comparable to standard plane-wave methods, but the memory requirements are higher.

Handbook of elliptic and hyperelliptic curve cryptography

Abstract: Preface Introduction to Public-Key Cryptography Mathematical Background Algebraic Background Background on p-adic Numbers Background on Curves and Jacobians Varieties Over Special Fields Background on Pairings Background on Weil Descent Cohomological Background on Point Counting Elementary Arithmetic Exponentiation Integer Arithmetic Finite Field Arithmetic Arithmetic of p-adic Numbers Arithmetic of Curves Arithmetic of Elliptic Curves Arithmetic of Hyperelliptic Curves Arithmetic of Special Curves Implementation of Pairings Point Counting Point Counting on Elliptic and Hyperelliptic Curves Complex Multiplication Computation of Discrete Logarithms Generic Algorithms for Computing Discrete Logarithms Index Calculus Index Calculus for Hyperelliptic Curves Transfer of Discrete Logarithms Applications Algebraic Realizations of DL Systems Pairing-Based Cryptography Compositeness and Primality Testing-Factoring Realizations of DL Systems Fast Arithmetic Hardware Smart Cards Practical Attacks on Smart Cards Mathematical Countermeasures Against Side-Channel Attacks Random Numbers-Generation and Testing References

Ammonia synthesis from first-principles calculations.

Abstract: The rate of ammonia synthesis over a nanoparticle ruthenium catalyst can be calculated directly on the basis of a quantum chemical treatment of the problem using density functional theory. We compared the results to measured rates over a ruthenium catalyst supported on magnesium aluminum spinel. When the size distribution of ruthenium particles measured by transmission electron microscopy was used as the link between the catalyst material and the theoretical treatment, the calculated rate was within a factor of 3 to 20 of the experimental rate. This offers hope for computer-based methods in the search for catalysts.

Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

Abstract: O-GalNAc-glycosylation is one of the main types of glycosylation in mammalian cells. No consensus recognition sequence for the O-glycosyltransferases is known, making prediction methods necessary to bridge the gap between the large number of known protein sequences and the small number of proteins experimentally investigated with regard to glycosylation status. From O-GLYCBASE a total of 86 mammalian proteins experimentally investigated for in vivo O-GalNAc sites were extracted. Mammalian protein homolog comparisons showed that a glycosylated serine or threonine is less likely to be precisely conserved than a nonglycosylated one. The Protein Data Bank was analyzed for structural information, and 12 glycosylated structures were obtained. All positive sites were found in coil or turn regions. A method for predicting the location for mucin-type glycosylation sites was trained using a neural network approach. The best overall network used as input amino acid composition, averaged surface accessibility predictions together with substitution matrix profile encoding of the sequence. To improve prediction on isolated (single) sites, networks were trained on isolated sites only. The final method combines predictions from the best overall network and the best isolated site network; this prediction method correctly predicted 76% of the glycosylated residues and 93% of the nonglycosylated residues. NetOGlyc 3.1 can predict sites for completely new proteins without losing its performance. The fact that the sites could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc.

The genome of the protist parasite Entamoeba histolytica

Abstract: Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Proteomic identification of S-nitrosylated proteins in Arabidopsis

Abstract: Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified 63 proteins from cell cultures and 52 proteins from leaves that represent candidates for S-nitrosylation, including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic proteins. Strikingly, many of these proteins have been identified previously as targets of S-nitrosylation in animals. At the enzymatic level, a case study demonstrated NO-dependent reversible inhibition of plant glyceraldehyde-3-phosphate dehydrogenase, suggesting that this enzyme could be affected by S-nitrosylation. The results of this work are the starting point for further investigation to get insight into signaling pathways and other cellular processes regulated by protein S-nitrosylation in plants.

Small Differences in Thyroid Function May Be Important for Body Mass Index and the Occurrence of Obesity in the Population

Abstract: Context: Increasing prevalence of overweight in the population is a major concern globally; and in the United States, nearly one third of adults were classified as obese at the end of the 20th century. Few data have been presented regarding an association between variations in thyroid function seen in the general population and body weight. Objective: The aim of this study was to investigate the association between thyroid function and body mass index (BMI) or obesity in a normal population. Design: A cross-sectional population study (The DanThyr Study) was conducted. Participants: In all, 4649 participants were investigated, and 4082 were eligible for these analyses after exclusion of subjects with previous or present overt thyroid dysfunction. Main Outcome Measures: The study examined the association between category of serum TSH or serum thyroid hormones and BMI or obesity in multivariate models, adjusting for possible confounding. Results: We found a positive association between BMI and category of se...

On the determination of crystallinity and cellulose content in plant fibres

Abstract: A comparative study of cellulose crystallinity based on the sample crystallinity and the cellulose content in plant fibres was performed for samples of different origin. Strong acid hydrolysis was found superior to agricultural fibre analysis and comprehensive plant fibre analysis for a consistent determination of the cellulose content. Crystallinity determinations were based on X-ray powder diffraction methods using side-loaded samples in reflection (Bragg-Brentano) mode. Rietveld refinements based on the recently published crystal structure of cellulose Iβ followed by integration of the crystalline and amorphous (background) parts were performed. This was shown to be straightforward to use and in many ways advantageous to traditional crystallinity determinations using the Segal or the Ruland–Vonk methods. The determined cellulose crystallinities were 90–100 g/100 g cellulose in plant-based fibres and 60–70 g/100 g cellulose in wood based fibres. These findings are significant in relation to strong fibre composites and bio-ethanol production.

Non-classical protein secretion in bacteria.

Abstract: Background We present an overview of bacterial non-classical secretion and a prediction method for identification of proteins following signal peptide independent secretion pathways. We have compiled a list of proteins found extracellularly despite the absence of a signal peptide. Some of these proteins also have known roles in the cytoplasm, which means they could be so-called "moon-lightning" proteins having more than one function.

Uncovering transcriptional regulation of metabolism by using metabolic network topology.

Abstract: Cellular response to genetic and environmental perturbations is often reflected and/or mediated through changes in the metabolism, because the latter plays a key role in providing Gibbs free energy and precursors for biosynthesis. Such metabolic changes are often exerted through transcriptional changes induced by complex regulatory mechanisms coordinating the activity of different metabolic pathways. It is difficult to map such global transcriptional responses by using traditional methods, because many genes in the metabolic network have relatively small changes at their transcription level. We therefore developed an algorithm that is based on hypothesis-driven data analysis to uncover the transcriptional regulatory architecture of metabolic networks. By using information on the metabolic network topology from genome-scale metabolic reconstruction, we show that it is possible to reveal patterns in the metabolic network that follow a common transcriptional response. Thus, the algorithm enables identification of so-called reporter metabolites (metabolites around which the most significant transcriptional changes occur) and a set of connected genes with significant and coordinated response to genetic or environmental perturbations. We find that cells respond to perturbations by changing the expression pattern of several genes involved in the specific part(s) of the metabolism in which a perturbation is introduced. These changes then are propagated through the metabolic network because of the highly connected nature of metabolism.

Screening for Quorum-Sensing Inhibitors (QSI) by Use of a Novel Genetic System, the QSI Selector

Abstract: With the widespread appearance of antibiotic-resistant bacteria, there is an increasing demand for novel strategies to control infectious diseases. Furthermore, it has become apparent that the bacterial life style also contributes significantly to this problem. Bacteria living in the biofilm mode of growth tolerate conventional antimicrobial treatments. The discovery that many bacteria use quorum-sensing (QS) systems to coordinate virulence and biofilm development has pointed out a new, promising target for antimicrobial drugs. We constructed a collection of screening systems, QS inhibitor (QSI) selectors, which enabled us to identify a number of novel QSIs among natural and synthetic compound libraries. The two most active were garlic extract and 4-nitro-pyridine-N-oxide (4-NPO). GeneChip-based transcriptome analysis revealed that garlic extract and 4-NPO had specificity for QS-controlled virulence genes in Pseudomonas aeruginosa. These two QSIs also significantly reduced P. aeruginosa biofilm tolerance to tobramycin treatment as well as virulence in a Caenorhabditis elegans pathogenesis model.

Mass spectrometry in metabolome analysis

Abstract: In the post-genomic era, increasing efforts have been made to describe the relationship between the genome and the phenotype in cells and organisms. It has become clear that even a complete understanding of the state of the genes, messages, and proteins in a living system does not reveal its phenotype. Therefore, researchers have started to study the metabolome (or the metabolic complement of functional genomics). Within this context, mass spectrometry (MS) has increasingly occupied a central position in the methodologies developed for determination of the metabolic state. This review is mainly focused on the status of MS in the metabolome field, trying to direct the reader to the main approaches for analysis of metabolites, reviewing basic methodologies in sample preparation, and the most recent MS techniques introduced. Apart from the description of the different methods, this review will try to state a general comparison between the several different techniques that involve MS and metabolite analysis, and will highlight their limitations and preferred applicability.

Numerical and experimental investigation of flow and scour around a circular pile

Abstract: The flow around a vertical circular pile exposed to a steady current is studied numerically and experimentally. The numerical model is a three-dimensional model. The model validation was achieved against new experimental data (which include two-component laser-Doppler anemometry (LDA) flow measurements and the hot-film bed shear stress measurements, and reported in the present paper) and the data of others, and a k-w turbulence model was used for closure. The model does not have a free-surface facility and therefore is applicable only to cases where the Froude number is small (Fr < 0(0.2)). The flow model was used to study the horseshoe vortex and lee-wake vortex flow processes around the pile. The influence on the horseshoe vortex of three parameters, namely the boundary-layer thickness, the Reynolds number and the bed roughness, was investigated. In the latter investigation, the steady solution of the model was chosen. A study of the influence of the unsteady solution on the previously mentioned flow processes was also carried out. The ranges of the parameters covered in the numerical simulations are: The boundary-layer-thickness-to-pile-diameter ratio is varied from 2 x 10 -2 to 10 2 , the pile Reynolds number from 10 2 to 2 × 10 6 , and the pile diameter-to-roughness ratio from 2 to about 10 3 . The amplification of the bed shear stress around the pile (including the areas under the horseshoe vortex and the lee-wake region) was obtained for various values of the previously mentioned parameters. The steady-state flow model was coupled with a morphologic model to calculate scour around a vertical circular pile exposed to a steady current in the case of non-cohesive sediment. The morphologic model includes (i) a two-dimensional bed load sediment-transport description, and (ii) a description of surface-layer sand slides for bed slopes exceeding the angle of repose. The results show that the present numerical simulation captures all the main features of the scour process. The equilibrium scour depth obtained from the simulation agrees well with the experiments for the upstream scour hole. Some discrepancy (up to 30 %) was observed, however, for the downstream scour hole. The calculations show that the amplification of the bed shear stress around the pile in the equilibrium state of the scour process is reduced considerably with respect to that experienced at the initial stage where the bed is plane.

Searchable encryption revisited: consistency properties, relation to anonymous IBE, and extensions

Abstract: We identify and fill some gaps with regard to consistency (the extent to which false positives are produced) for public-key encryption with keyword search (PEKS). We define computational and statistical relaxations of the existing notion of perfect consistency, show that the scheme of [7] is computationally consistent, and provide a new scheme that is statistically consistent. We also provide a transform of an anonymous IBE scheme to a secure PEKS scheme that, unlike the previous one, guarantees consistency. Finally we suggest three extensions of the basic notions considered here, namely anonymous HIBE, public-key encryption with temporary keyword search, and identity-based encryption with keyword search.

Prediction of twin-arginine signal peptides

Abstract: Background: Proteins carrying twin-arginine (Tat) signal peptides are exported into the periplasmic compartment or extracellular environment independently of the classical Secdependent translocation pathway. To complement other methods for classical signal peptide prediction we here present a publicly available method, TatP, for prediction of bacterial Tat signal peptides. Results: We have retrieved sequence data for Tat substrates in order to train a computational method for discrimination of Sec and Tat signal peptides. The TatP method is able to positively classify 91% of 35 known Tat signal peptides and 84% of the annotated cleavage sites of these Tat signal peptides were correctly predicted. This method generates far less false positive predictions on various datasets than using simple pattern matching. Moreover, on the same datasets TatP generates less false positive predictions than a complementary rule based prediction method. Conclusion: The method developed here is able to discriminate Tat signal peptides from cytoplasmic proteins carrying a similar motif, as well as from Sec signal peptides, with high accuracy. The method allows filtering of input sequences based on Perl syntax regular expressions, whereas hydrophobicity discrimination of Tat- and Sec-signal peptides is carried out by an artificial neural network. A potential cleavage site of the predicted Tat signal peptide is also reported. The TatP prediction server is available as a public web server at http://www.cbs.dtu.dk/services/TatP/.

A comparison study: applying segmentation to array CGH data for downstream analyses

Abstract: Motivation: Array comparative genomic hybridization (CGH) allows detection and mapping of copy number of DNA segments. A challenge is to make inferences about the copy number structure of the genome. Several statistical methods have been proposed to determine genomic segments with different copy number levels. However, to date, no comprehensive comparison of various characteristics of these methods exists. Moreover, the segmentation results have not been utilized in downstream analyses. Results: We describe a comparison of three popular and publicly available methods for the analysis of array CGH data and we demonstrate how segmentation results may be utilized in the downstream analyses such as testing and classification, yielding higher power and prediction accuracy. Since the methods operate on individual chromosomes, we also propose a novel procedure for merging segments across the genome, which results in an interpretable set of copy number levels, and thus facilitate identification of copy number alterations in each genome. Availability: http://www.bioconductor.org Contact: jfridlyand@cc.ucsf.edu Supplementary Information: http://www.cbs.dtu.dk/~hanni/aCGH/

Influence of Environmental Conditions on Methanogenic Compositions in Anaerobic Biogas Reactors

Abstract: The influence of environmental parameters on the diversity of methanogenic communities in 15 full-scale biogas plants operating under different conditions with either manure or sludge as feedstock was studied. Fluorescence in situ hybridization was used to identify dominant methanogenic members of the Archaea in the reactor samples; enriched and pure cultures were used to support the in situ identification. Dominance could be identified by a positive response by more than 90% of the total members of the Archaea to a specific group- or order-level probe. There was a clear dichotomy between the manure digesters and the sludge digesters. The manure digesters contained high levels of ammonia and of volatile fatty acids (VFA) and were dominated by members of the Methanosarcinaceae, while the sludge digesters contained low levels of ammonia and of VFA and were dominated by members of the Methanosaetaceae. The methanogenic diversity was greater in reactors operating under mesophilic temperatures. The impact of the original inoculum used for the reactor start-up was also investigated by assessment of the present population in the reactor. The inoculum population appeared to have no influence on the eventual population.

Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent

Abstract: The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell-cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and H2O2, and are readily phagocytosed by PMNs, in contrast to bacteria with functional QS systems. In contrast to the wild-type, QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently, the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

Evolutionary programming as a platform for in silico metabolic engineering

Abstract: Through genetic engineering it is possible to introduce targeted genetic changes and hereby engineer the metabolism of microbial cells with the objective to obtain desirable phenotypes. However, owing to the complexity of metabolic networks, both in terms of structure and regulation, it is often difficult to predict the effects of genetic modifications on the resulting phenotype. Recently genome-scale metabolic models have been compiled for several different microorganisms where structural and stoichiometric complexity is inherently accounted for. New algorithms are being developed by using genome-scale metabolic models that enable identification of gene knockout strategies for obtaining improved phenotypes. However, the problem of finding optimal gene deletion strategy is combinatorial and consequently the computational time increases exponentially with the size of the problem, and it is therefore interesting to develop new faster algorithms. In this study we report an evolutionary programming based method to rapidly identify gene deletion strategies for optimization of a desired phenotypic objective function. We illustrate the proposed method for two important design parameters in industrial fermentations, one linear and other non-linear, by using a genome-scale model of the yeast Saccharomyces cerevisiae. Potential metabolic engineering targets for improved production of succinic acid, glycerol and vanillin are identified and underlying flux changes for the predicted mutants are discussed. We show that evolutionary programming enables solving large gene knockout problems in relatively short computational time. The proposed algorithm also allows the optimization of non-linear objective functions or incorporation of non-linear constraints and additionally provides a family of close to optimal solutions. The identified metabolic engineering strategies suggest that non-intuitive genetic modifications span several different pathways and may be necessary for solving challenging metabolic engineering problems.

The MAP kinase substrate MKS1 is a regulator of plant defense responses

Abstract: Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two-hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two-hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors.

Identity and effects of quorum-sensing inhibitors produced by Penicillium species.

Abstract: Quorum sensing (QS) communication systems are thought to afford bacteria with a mechanism to strategically cause disease. One example is Pseudomonas aeruginosa, which infects immunocompromised individuals such as cystic fibrosis patients. The authors have previously documented that blockage of the QS systems not only attenuates Ps. aeruginosa but also renders biofilms highly susceptible to treatment with conventional antibiotics. Filamentous fungi produce a battery of secondary metabolites, some of which are already in clinical use as antimicrobial drugs. Fungi coexist with bacteria but lack active immune systems, so instead rely on chemical defence mechanisms. It was speculated that some of these secondary metabolites could interfere with bacterial QS communication. During a screening of 100 extracts from 50 Penicillium species, 33 were found to produce QS inhibitory (QSI) compounds. In two cases, patulin and penicillic acid were identified as being biologically active QSI compounds. Their effect on QS-controlled gene expression in Ps. aeruginosa was verified by DNA microarray transcriptomics. Similar to previously investigated QSI compounds, patulin was found to enhance biofilm susceptibility to tobramycin treatment. Ps. aeruginosa has developed QS-dependent mechanisms that block development of the oxidative burst in PMN neutrophils. Accordingly, when the bacteria were treated with either patulin or penicillic acid, the neutrophils became activated. In a mouse pulmonary infection model, Ps. aeruginosa was more rapidly cleared from the mice that were treated with patulin compared with the placebo group.

The role of the proteasome in generating cytotoxic T-cell epitopes: insights obtained from improved predictions of proteasomal cleavage

Abstract: Cytotoxic T cells (CTLs) perceive the world through small peptides that are eight to ten amino acids long. These peptides (epitopes) are initially generated by the proteasome, a multi-subunit protease that is responsible for the majority of intra-cellular protein degradation. The proteasome generates the exact C-terminal of CTL epitopes, and the N-terminal with a possible extension. CTL responses may diminish if the epitopes are destroyed by the proteasomes. Therefore, the prediction of the proteasome cleavage sites is important to identify potential immunogenic regions in the proteomes of pathogenic microorganisms (or humans). We have recently shown that NetChop, a neural network-based prediction method, is the best method available at the moment to do such predictions; however, its performance is still lower than desired. Here, we use novel sequence encoding methods and show that the new version of NetChop predicts approximately 10% more of the cleavage sites correctly while lowering the number of false positives with close to 15%. With this more reliable prediction tool, we study two important questions concerning the function of the proteasome. First, we estimate the N-terminal extension of epitopes after proteasomal cleavage and find that the average extension is relatively short. However, more than 30% of the peptides have N-terminal extensions of three amino acids or more, and thus, N-terminal trimming might play an important role in the presentation of a substantial fraction of the epitopes. Second, we show that good TAP ligands have an increased chance of being cleaved by the proteasome, i.e., the specificity of TAP has evolved to fit the specificity of the proteasome. This evolutionary relationship allows for a more efficient antigen presentation.

Use of modulated excitation signals in medical ultrasound. Part I: basic concepts and expected benefits

Abstract: This paper, the first from a series of three papers on the application of coded excitation signals in medical ultrasound, discusses the basic principles and ultrasound-related problems of pulse compression. The concepts of signal modulation and matched filtering are given, and a simple model of attenuation relates the matched filter response with the ambiguity function, known from radar. Based on this analysis and the properties of the ambiguity function, the selection of coded waveforms suitable for ultrasound imaging is discussed. It is shown that linear frequency modulation (FM) signals have the best and most robust features for ultrasound imaging. Other coded signals such as nonlinear FM and binary complementary Golay codes also have been considered and characterized in terms of signal-to-noise ratio (SNR) and sensitivity to frequency shifts. Using the simulation program Field II, it is found that in the case of linear FM signals, a SNR improvement of 12 to 18 dB can be expected for large imaging depths in attenuating media, without any depth-dependent filter compensation. In contrast, nonlinear FM modulation and binary codes are shown to give a SNR improvement of only 4 to 9 dB when processed with a matched filter. Other issues, such as depth-dependent matched filtering and use of filters other than the matched filter (inverse and Wiener filters) also are addressed.

Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infections

Abstract: The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis patients. P. aeruginosa colonizes the lungs by forming biofilm microcolonies throughout the lung. Quorum sensing (QS) renders the biofilm bacteria highly tolerant to otherwise lethal doses of antibiotics, and protects against the bactericidal activity of polymorphonuclear leukocytes (PMNs). It has been previously demonstrated that QS is inhibited by garlic extract. In this study, the synergistic effects of garlic and tobramycin, and PMNs activities have been evaluated. P. aeruginosa was grown in vitro in continuous-culture once-through flow chambers with and without garlic extract. The garlic-treated biofilms were susceptible to both tobramycin and PMN grazing. Furthermore, the PMNs showed an increase in respiratory burst activation, when incubated with the garlic-treated biofilm. Garlic extract was administered as treatment for a mouse pulmonary infection model. Mice were treated with garlic extract or placebo for 7 days, with the initial 2 days being prophylactic before P. aeruginosa was instilled in the left lung of the mice. Bacteriology, mortality, histopathology and cytokine production were used as indicators. The garlic treatment initially provoked a higher degree of inflammation, and significantly improved clearing of the infecting bacteria. The results indicate that a QS-inhibitory extract of garlic renders P. aeruginosa sensitive to tobramycin, respiratory burst and phagocytosis by PMNs, as well as leading to an improved outcome of pulmonary infections.