Resolvin D2 promotes host defense in a 2 - hit model of sepsis with secondary lung infection.

In the development of sepsis, there is early, massive inflammation which can lead to multiple organ failure. Later there is an immunosuppressed phase where the host is susceptible to secondary infections or is unable to clear existing infection. Specialized Pro-resolving Mediators (SPMs) are endogenously produced lipids which resolve infection by decreasing bacteria load and reducing systemic inflammatory response. There has been little work studying if SPMs given late, can promote host defense. We examined if an SPM, Resolvin D2 (RvD2) could promote host defense in a 2-hit mouse model of cecal ligation and puncture (CLP) sepsis and secondary Pseudomonas aeruginosa lung infection. RvD2 given 48 h after mild CLP (1st hit), increased gene expression of Toll-like receptor-2 (TLR-2) and alveolar macrophage/monocyte phagocytic ability compared to CLP mice given saline vehicle. In this model, RvD2 did not affect plasma IL-6 or IL-10. These effects induced by RvD2, lowered lung bacterial load and decreased mortality after the secondary infection of Pseudomonas aeruginosa (2nd hit). Splenic T-cell numbers were also increased in RvD2 treated mice compared to saline vehicle treated animals. The results suggest that RvD2 promoted mechanisms of host defense in a 2-hit model sepsis and secondary lung infection.

Lipoxin A4 promotes reduction and antibiotic efficacy against Pseudomonas aeruginosa biofilm.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterium commonly found in wound infections and airways of cystic fibrosis patients. P. aeruginosa readily forms biofilms which can reduce the efficacy of antibiotics used to eradicate the pathogen. We have previously shown that a Specialized Pro-resolving Mediator (SPM), Lipoxin A4 (LxA4) is a quorum sensing inhibitor which can reduce P. aeruginosa virulence. In this study, we examined the direct actions of LxA4 and RvD2 on P. aeruginosa biofilm formation and virulence gene expression. The influence of LxA4 on antibiotic efficacy and the combined effects on biofilm formation were also investigated. LxA4 and RvD2 reduced P. aeruginosa biofilm formation and virulence gene expression. LxA4 increased ciprofloxacin inhibition on biofilm formation but did not affect ciprofloxacin's action on non-adherent bacteria. On the other hand, LxA4 increased bacterial killing action of imipenem but did not affect imipenem's action on biofilm. We also found that LxA4 can increase ciprofloxacin's bacterial killing ability in established biofilm. Together these results suggest that LxA4 has direct effects on P. aeruginosa biofilm formation and can increase antibiotic efficacy directly.

Clinical management of Brucella suis infection in dogs and implications for public health.

BACKGROUND: Brucellosis caused by Brucella suis is a notifiable disease that has recently emerged in dogs in New South Wales (NSW). Given the potential for zoonotic transmission, euthanasia of affected dogs is recommended, but this action is not mandatory. We report the clinical management of three dogs that underwent treatment at their owners' request. CASE REPORTS: A 14-month-old spayed female crossbreed originally obtained from an urban animal shelter underwent extensive investigations in 2011-12 for lameness and back pain, culminating in decompressive laminectomy. Diagnosis of multifocal discospondylitis and spinal empyema was made, with B. suis cultured from surgical biopsy specimens. The dog responded to long-term treatment using rifampicin and doxycycline. A second case of B. suis infection was diagnosed in January 2016 in a 3-year-old crossbreed pig-hunting dog with unilateral testicular enlargement. Following serological diagnosis the dog was given preliminary therapy using rifampicin and doxycycline, the affected testis was resected and the patient given a further month of combination therapy. In March 2016 a 7-year-old crossbreed pig-hunting dog with brucellosis was handled similarly, although both testes were removed. CONCLUSION: Brucellosis should be considered in the differential diagnosis of back pain, discospondylitis, lameness, abortion, prostatic abscessation and testicular/epididymal enlargement in dogs, especially if there is exposure to feral pigs or consumption of uncooked feral pig meat. Euthanasia is the only guarantee of reducing the public health risk to zero. However, where treatment is desired by the owner, combination therapy using rifampicin and doxycycline appears to be effective, when combined with surgical resection of infected tissues. Further monitoring of dogs during and after treatment is required to document cure.

Modeling birds on wires.

In this paper we introduce a mathematical model to study the group dynamics of birds resting on wires. The model is agent-based and postulates attraction-repulsion forces between the interacting birds: the interactions are "topological", in the sense that they involve a given number of neighbors irrespective of their distance. The model is first mathematically analyzed and then simulated to study its main properties: we observe that the model predicts birds to be more widely spaced near the borders of each group. We compare the results from the model with experimental data, derived from the analysis of pictures of pigeons and starlings taken in New Jersey: two different image elaboration protocols allow us to establish a good agreement with the model and to quantify its main parameters. We also discuss the potential handedness of the birds, by analyzing the group organization features and the group dynamics at the arrival of new birds. Finally, we propose a more refined mathematical model that describes landing and departing birds by suitable stochastic processes.

HydroCoils, occlusion rates, and outcomes: a large single-center study.

BACKGROUND AND PURPOSE: The HydroCoil is an expansile hydrogel coil designed to produce a greater degree of volumetric packing within cerebral aneurysms when compared with bare platinum coils. This increased packing is, in turn, believed to decrease the risk of recurrence within aneurysms and hence the risk of their rupture in the long term. The aim of this work was to assess whether the use of HydroCoils and the proportion of HydroCoil used have any influence on the subsequent occlusion and recurrence rates of treated aneurysms. MATERIALS AND METHODS: A retrospective study was performed of 328 patients during 5 years at a single institution. The initial angiographic and follow-up angiographic occlusion rates were recorded as were any procedural complications. The proportion of HydroCoil used was described as the relative amount of HydroCoil length to the total coil length used during an aneurysm treatment, thus forming 4 groups: 0%-19%, 20%-49%, 50%-69%, 70%-100%, and the subgroups with 100%. RESULTS: Two hundred seventy patients had angiographic follow-up during an average of 13 months. The overall risk of permanent neurologic deficit and death was 3%. The rate of complete occlusion was 31% immediately postcoiling and 64.8% on follow-up. At the latest follow-up, 25.6% had residual necks and 9.6% had residual aneurysms. There was a statistically significant trend for HydroCoils to produce greater occlusion rates on follow-up when >70% HydroCoil was used (P = .025). The overall rate of recurrence for all aneurysms as a group was 15.5%. The retreatment rate was 6.6%. There has been 1 rebleed in the 328 patients. CONCLUSIONS: The overall results following the use of HydroCoils to occlude aneurysms compare well with those in other reported series. HydroCoils do produce a statistically significantly greater rate of occlusion when >70% of total aneurysm coil length is HydroCoil compared with coiling with <20% HydroCoil. There was no significant difference, however, in the recurrence or retreatment rate when comparing these groups.

Enhanced DNA binding capacity on up-regulated epidermal wild-type p53 in vitiligo by H2O2-mediated oxidation: a possible repair mechanism for DNA damage.

Vitiligo is characterized by a patchy loss of inherited skin color affecting approximately 0.5% of individuals of all races. Despite the absence of the protecting pigment and the overwhelming evidence for hydrogen peroxide (H(2)O(2))-induced oxidative stress in the entire epidermis of these patients, there is neither increased photodamage/skin aging nor a higher incidence for sun-induced nonmelanoma skin cancer. Here we demonstrate for the first time increased DNA damage via 8-oxoguanine in the skin and plasma in association with epidermal up-regulated phosphorylated/acetylated p53 and high levels of the p53 antagonist p76(MDM2). Short-patch base-excision repair via hOgg1, APE1, and polymerasebeta DNA repair is up-regulated. Overexpression of Bcl-2 and low caspase 3 and cytochrome c levels argue against increased apoptosis in this disease. Moreover, we show the presence of high epidermal peroxynitrite (ONOO(-)) levels via nitrotyrosine together with high nitrated p53 levels. We demonstrate by EMSA that nitration of p53 by ONOO(-) (300 x 10(-6) M) abrogates DNA binding, while H(2)O(2)-oxidized p53 (10(-3) M) enhances DNA binding capacity and prevents ONOO(-)-induced abrogation of DNA binding. Taken together, we add a novel reactive oxygen species to the list of oxidative stress inducers in vitiligo. Moreover, we propose up-regulated wild-type p53 together with p76(MDM2) as major players in the control of DNA damage/repair and prevention of photodamage and nonmelanoma skin cancer in vitiligo.

SPINE bioinformatics and data-management aspects of high-throughput structural biology.

SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.

Haplotypes in the CTLA4 region are associated with coeliac disease in the Irish population.

Chromosomal region 2q33 encodes the immune regulatory genes, CTLA4, ICOS and CD28, which are involved in regulation of T-cell activity and has been studied as a candidate gene locus in autoimmune diseases, including coeliac disease (CD). We have investigated whether an association exists between this region and CD in the Irish population using a comprehensive analysis for genetic variation. Using a haplotype-tagging approach, this gene cluster was investigated for disease association in a case-control study comprising 394 CD patients and 421 ethnically matched healthy controls. Several SNPs, including CTLA4_CT60, showed association with disease; however, after correction for multiple-testing, CTLA4-658C/T was the only polymorphism found to show significant association with disease when allele, genotype, or carrier status frequency were analysed (carrier status (Allele C), P = 0.0016). Haplotype analysis revealed a haplotype incorporating the CD28/CTLA4 and two 5' ICOS polymorphisms to be significantly associated with disease (patients 24.1%; controls 31.5%; P = 0.035), as was a shorter haplotype composed of the CTLA4 markers only (30.9 vs 34.9%; P = 0.042). The extended haplotype incorporating CD28/CTLA4 and 5' ICOS is more strongly associated with disease than haplotypes of individual genes. This suggests a causal variant associated with this haplotype may be associated with disease in this population.

HTHquery: a method for detecting DNA-binding proteins with a helix-turn-helix structural motif.

SUMMARY: HTHquery is a web-based service to determine if a protein structure has a helix-turn-helix structural motif which could bind to DNA. It is based on a similarity with a set of structural templates, the accessibility of a putative structural motif and a positive electrostatic potential in the neighbourhood of the putative motif. A set of scores are computed, based on each template, using a linear predictor. From the training set used, the predictor has a true positive rate of 83.5% and a false positive rate of 0.8%. The emphasis for the website is on providing a straightforward interface which can be easily used by a bench-based scientist. AVAILABILITY: HTHquery is implemented using a set of Perl scripts and C program and can be accessed freely on the website http://www.ebi.ac.uk/thornton-srv/databases/HTHquery.

Amino acid architecture and the distribution of polar atoms on the surfaces of proteins.

We propose that a necessary condition for a protein to be soluble is the absence of large hydrophobic patches on its solvent-accessible surface, which can cause aggregation to occur. We note that the polar nature of the backbone of all amino acids guarantees a minimum polar content and hence can interrupt such patches. As a result, a carefully conserved detailed atomic placement of residues on the protein surface is not necessary for solubility. In order to demonstrate this, we construct a measure based on the average hydrophobicity of a simply defined patch. We use this measurement to compare surfaces that exhibit a clear difference in their solubility properties, namely, a) the solvent accessible surfaces for a set of homo-dimers and the surfaces buried in their interfaces and b) for a set of monomers the surfaces of fragments of secondary structure which are solvent accessible/inaccessible. Having demonstrated a difference in the first set of distributions, we characterize the solvent accessible surfaces of monomeric proteins. To test if cooperative behavior occurs between the atoms for these surfaces, we construct a set of randomized surfaces, which obey a very simple stereochemical constraint. We find that the observed and randomized distributions are much more similar than the previous sets we examined. This implies that while surfaces of soluble proteins must have sufficient polar content, the relative placement of atoms of one amino acid with respect to the atoms of neighboring amino acid need not be finely tuned, which provides an innate robustness for protein design and folding.

Chromosome 5q candidate genes in coeliac disease: genetic variation at IL4, IL5, IL9, IL13, IL17B and NR3C1.

Genetic predisposition to coeliac disease (CD) is determined primarily by alleles at the HLA-DQB locus, and evidence exists implicating other major histocompatibility complex-linked genes (6p21) and the CTLA4 locus on chromosome 2q33. In addition, extensive family studies have provided strong, reproducible evidence for a susceptibility locus on chromosome 5q (CELIAC2). However, the gene responsible has not been identified. We have assayed genetic variation at the IL4, IL5, IL9, IL13, IL17B and NR3C1 (GR) loci, all of which are present on chromosome 5q and have potential or demonstrated involvement in autoimmune and/or inflammatory disease, in a sample of 409 CD cases and 355 controls. Thirteen single nucleotide polymorphisms were chosen on the basis of functional relevance, prior disease association and, where possible, prior knowledge of the haplotype variation present in European populations. There were no statistically significant allele or haplotype frequency differences between cases and controls. Therefore, these results provide no evidence that these loci are associated with CD in this sample population.

Haplotype variation at the IBD5/SLC22A4 locus (5q31) in coeliac disease in the Irish population.

In addition to the well-established association of coeliac disease (CD) with HLA-DQ (6p21) and possibly CTLA4 (2q33), there is considerable evidence for a susceptibility locus on chromosome 5q, which contains many potential candidates for inflammatory disease, including a cluster of cytokine genes in 5q31. CD cases and controls were genotyped for four single-nucleotide polymorphism (SNP) markers that together characterize >90% of the haplotype variation at the IBD5 locus encoding, among others, the SLC22A4 gene. IBD5 and SLC22A4 map to 5q31 and have recently been associated with Crohn's disease and rheumatoid arthritis. Haplotype frequencies do not differ significantly between CD cases and controls in the Irish population, and therefore the chromosome 5 CD susceptibility locus most likely lies elsewhere on 5q.

An examination of the conservation of surface patch polarity for proteins.

MOTIVATION: The solubility of a protein is crucial for its function and is therefore an evolutionary constraint. As the solubility of a protein is related to the distribution of polar and hydrophobic residues on its solvent accessible surface, such a constraint should provide a valuable insight into the evolution of protein surfaces. We examine how the surfaces of proteins have evolved by considering how the average hydrophobicities of patches of surface residues vary across homologous proteins. We derive distributions for the average hydrophobicity/philicity of surface patches at a residue-based level-which we refer to as the residue hydrophobic density. This is computed for a set of 28 monomeric proteins and their homologues. The resulting distributions are compared with a set of randomized sequences, with the same residue content. RESULTS: We find that the patches, involving typically more than 10 residues, maintain a more hydrophilic surface than one would expect from a random substitution model, indicating a cooperative behaviour for these surfaces residues in terms of this single variable. SUPPLEMENTARY INFORMATION: Additional plots for all of the proteins examined in this paper can be found at: http://www.ebi.ac.uk/~shanahan/PCon/index.html

The (betaalpha)(8) glycosidases: sequence and structure analyses suggest distant evolutionary relationships.

There are currently at least nine distinct glycosidase sequence families which are all known to adopt a TIM barrel fold [Henrissat,B. and Davies,G. (1997) CURR: Opin. Struct. Biol., 7, 637-644]. To explore the relationships between these enzymes and their evolution, comprehensive sequence and structure comparisons were performed, generating four distinct clusters. The first cluster, S1, comprises the alpha-amylase related enzymes, all with the retention mechanism (axial-->axial). The second cluster, S2, included two functional subgroups, one composed of various kinds of glucosidases all with the retention mechanism (equatorial-->equatorial) (the so-called 4/7 superfamily), and the other subgroup including the beta-amylases with the inversion mechanism (axial--> equatorial). The third cluster, S3, with the retention mechanism (equatorial-->equatorial), could be subdivided, based on the catalytic residues and mechanisms, into two functional subgroups: the chitinase group, catalysed by two acidic residues on the C-termini of beta-4 and beta-6, and the hevamine group, using two acidic residues on the C-termini of beta-4 for catalysis. The fourth cluster, S4, is composed of chitobiase with the retention mechanism (equatorial--> equatorial). These clusters are compared with the sequence families derived by Henrissat and coworkers. PSI-BLAST profiles and multiple-alignments of tertiary structures suggest that S1 and S2 are distantly related, as are S3 and S4, which have N-acetylated substrates. This work highlights the difficulties of untangling distant evolutionary relationships in ubiquitous folds such as the TIM barrel.

On the molecular discrimination between adenine and guanine by proteins.

The molecular recognition and discrimination of adenine and guanine ligand moieties in complexes with proteins have been studied using empirical observations on carefully selected crystal structures. The distribution of protein folds that bind these purines has been found to differ significantly from that across the whole PDB, but the most populated architectures and folds are also the most common in three genomes from the three different domains of life. The protein environments around the two nucleic acid bases were significantly different, in terms of the propensities of amino acid residues to be in the binding site, as well as their propensities to form hydrogen bonds to the bases. Plots of the distribution of protein atoms around the two purines clearly show different clustering of hydrogen bond donors and acceptors opposite complimentary acceptors and donors in the rings, with hydrophobic areas below and above the rings. However, the clustering pattern is fuzzy, reflecting the variety of ways that proteins have evolved to recognise the same molecular moiety. Furthermore, an analysis of the conservation of residues in the protein chains binding guanine shows that residues in contact with the base are in general better conserved than the rest of the chain.

Small-molecule metabolism: an enzyme mosaic.

Escherichia coli has been a popular organism for studying metabolic pathways. In an attempt to find out more about how these pathways are constructed, the enzymes were analysed by defining their protein domains. Structural assignments and sequence comparisons were used to show that 213 domain families constitute approximately 90% of the enzymes in the small-molecule metabolic pathways. Catalytic or cofactor-binding properties between family members are often conserved, while recognition of the main substrate with change in catalytic mechanism is only observed in a few cases of consecutive enzymes in a pathway. Recruitment of domains across pathways is very common, but there is little regularity in the pattern of domains in metabolic pathways. This is analogous to a mosaic in which a stone of a certain colour is selected to fill a position in the picture.

The evolution and structural anatomy of the small molecule metabolic pathways in Escherichia coli.

The 106 small molecule metabolic (SMM) pathways in Escherichia coli are formed by the protein products of 581 genes. We can define 722 domains, nearly all of which are homologous to proteins of known structure, that form all or part of 510 of these proteins. This information allows us to answer general questions on the structural anatomy of the SMM pathway proteins and to trace family relationships and recruitment events within and across pathways. Half the gene products contain a single domain and half are formed by combinations of between two and six domains. The 722 domains belong to one of 213 families that have between one and 51 members. Family members usually conserve their catalytic or cofactor binding properties; substrate recognition is rarely conserved. Of the 213 families, members of only a quarter occur in isolation, i.e. they form single-domain proteins. Most members of the other families combine with domains from just one or two other families and a few more versatile families can combine with several different partners. Excluding isoenzymes, more than twice as many homologues are distributed across pathways as within pathways. However, serial recruitment, with two consecutive enzymes both being recruited to another pathway, is rare and recruitment of three consecutive enzymes is not observed. Only eight of the 106 pathways have a high number of homologues. Homology between consecutive pairs of enzymes with conservation of the main substrate-binding site but change in catalytic mechanism (which would support a simple model of retrograde pathway evolution) occurs only six times in the whole set of enzymes. Most of the domains that form SMM pathways have homologues in non-SMM pathways. Taken together, these results imply a pervasive "mosaic" model for the formation of protein repertoires and pathways.

Amino acid-base interactions: a three-dimensional analysis of protein-DNA interactions at an atomic level.

To assess whether there are universal rules that govern amino acid-base recognition, we investigate hydrogen bonds, van der Waals contacts and water-mediated bonds in 129 protein-DNA complex structures. DNA-backbone interactions are the most numerous, providing stability rather than specificity. For base interactions, there are significant base-amino acid type correlations, which can be rationalised by considering the stereochemistry of protein side chains and the base edges exposed in the DNA structure. Nearly two-thirds of the direct read-out of DNA sequences involves complex networks of hydrogen bonds, which enhance specificity. Two-thirds of all protein-DNA interactions comprise van der Waals contacts, compared to about one-sixth each of hydrogen and water-mediated bonds. This highlights the central importance of these contacts for complex formation, which have previously been relegated to a secondary role. Although common, water-mediated bonds are usually non-specific, acting as space-fillers at the protein-DNA interface. In conclusion, the majority of amino acid-base interactions observed follow general principles that apply across all protein-DNA complexes, although there are individual exceptions. Therefore, we distinguish between interactions whose specificities are 'universal' and 'context-dependent'. An interactive Web-based atlas of side chain-base contacts provides access to the collected data, including analyses and visualisation of the three-dimensional geometry of the interactions.

Identification of higher brain centres that may encode the cardiorespiratory response to exercise in humans.

1. Positron emission tomography (PET) was used to identify the neuroanatomical correlates underlying 'central command' during imagination of exercise under hypnosis, in order to uncouple central command from peripheral feedback. 2. Three cognitive conditions were used: condition I, imagination of freewheeling downhill on a bicycle (no change in heart rate, HR, or ventilation, V(I)): condition II, imagination of exercise, cycling uphill (increased HR by 12 % and V(I) by 30 % of the actual exercise response): condition III, volitionally driven hyperventilation to match that achieved in condition II (no change in HR). 3. Subtraction methodology created contrast A (II minus I) highlighting cerebral areas involved in the imagination of exercise and contrast B (III minus I) highlighting areas activated in the direct volitional control of breathing (n = 4 for both; 8 scans per subject). End-tidal P(CO(2)) (P(ET,CO(2))) was held constant throughout PET scanning. 4. In contrast A, significant activations were seen in the right dorso-lateral prefrontal cortex, supplementary motor areas (SMA), the right premotor area (PMA), superolateral sensorimotor areas, thalamus, and bilaterally in the cerebellum. In contrast B, significant activations were present in the SMA and in lateral sensorimotor cortical areas. The SMA/PMA, dorso-lateral prefrontal cortex and the cerebellum are concerned with volitional/motor control, including that of the respiratory muscles. 5. The neuroanatomical areas activated suggest that a significant component of the respiratory response to 'exercise', in the absence of both movement feedback and an increase in CO(2) production, can be generated by what appears to be a behavioural response.