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Shotgun metagenomics is a powerful tool to identify antimicrobial resistance (AMR) genes in microbiomes but has the limitation that extrachromosomal DNA, such as plasmids, cannot be linked with the host bacterial chromosome. Here we present a laboratory and bioinformatics pipeline HAM-ART (Hi-C Assisted Metagenomics for Antimicrobial Resistance Tracking) optimised for the generation of metagenome-assembled genomes including both chromosomal and extrachromosomal AMR genes. We demonstrate the performance of the pipeline in a study comparing 100 pig faecal microbiomes from low- and high-antimicrobial use pig farms (organic and conventional farms). We found significant differences in the distribution of AMR genes between low- and high-antimicrobial use farms including a plasmid-borne lincosamide resistance gene exclusive to high-antimicrobial use farms in three species of Lactobacilli.Author SummaryAntimicrobial resistance (AMR) is one of the biggest global health threats humanity is facing. Understanding the emergence and spread of AMR between different bacterial species is crucial for the development of effective countermeasures. In this paper we describe a user-friendly, affordable and comprehensive (laboratory and bioinformatics) workflow that is able to identify, associate and track AMR genes in bacteria. We demonstrate the efficiency and reliability of the method by comparing 50 faecal microbiomes from pig farms with high-antibiotic use (conventional farms), and 50 faecal microbiomes from pig farms with low-antibiotic use (organic farms). Our method provides a novel approach to resistance gene tracking, that also leads to the generation of high quality metagenomic assembled genomes that includes genes on mobile genetic elements, such as plasmids, that would not otherwise be included in these assembled genomes.

The Gambian epauletted fruit bat, Epomophorus gambianus, is widely distributed across sub-Saharan Africa. Its assembled and annotated mitochondrial genome (GenBank accession no. KT963027) is 16,702 bases in length, containing 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and two non-coding regions: the control region (D-loop) and the origin of light-strand replication (OL). The average base composition is 32.2% A; 27.6% C; 14% G; and 26.1% T. The mitogenome presented a structural composition greatly conserved between members of the Pteropodidae family.

Objective: Muscle weakness is an important risk factor for falls in older adults. Intensity and duration of muscle activity are important determinants of exercise effectiveness in combating muscle weakness. The aim of this article was to assess the intensity and duration of muscle activity in virtual reality (VR) balance games. Materials and Methods: Thirty young and 30 healthy older adults played seven different VR balance games. Muscle activity of the vastus lateralis, vastus medialis, soleus, and gluteus medius was obtained using surface electromyography (EMG). The processed EMG signals were divided into 200-ms blocks, after which each block was categorized by its average normalized EMG activity, that is, >80%, 60%-80%, 40%-60%, or 40% MVC, to identify prolonged muscle activity. Results: Muscle activity during game play was mostly

The interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil. Herbaspirillum seropedicae strain SmR1, belonging to the betaproteobacteria, is an endophyte that colonizes crops, including rice, maize, sugarcane, and sorghum. Different approaches have revealed genes and pathways regulated during the interactions of H. seropedicae with its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools. Here we successfully employed a combination of in vivo high-density mariner Tn mutagenesis and targeted Tn insertion site sequencing (Tn-seq) in H. seropedicae SmR1. The analysis of multiple gene-saturating Tn libraries revealed that 395 genes are essential for the growth of H. seropedicae SmR1 in tryptone-yeast extract medium. A comparative analysis with the Database of Essential Genes (DEG) showed that 25 genes are uniquely essential in H. seropedicae SmR1. The Tn mutagenesis protocol developed and the gene-saturating Tn libraries generated will facilitate elucidation of the genetic mechanisms of the H. seropedicae endophytic lifestyle. IMPORTANCE A focal point in the study of endophytes is the development of effective biofertilizers that could help to reduce the input of agrochemicals in croplands. Besides the ability to promote plant growth, a good biofertilizer should be successful in colonizing its host and competing against the native microbiota. By using a systematic Tn-based gene-inactivation strategy and massively parallel sequencing of Tn insertion sites (Tn-seq), it is possible to study the fitness of thousands of Tn mutants in a single experiment. We have applied the combination of these techniques to the plant-growth-promoting endophyte Herbaspirillum seropedicae SmR1. The Tn mutant libraries generated will enable studies into the genetic mechanisms of H. seropedicae -plant interactions. The approach that we have taken is applicable to other plant-interacting bacteria.

In Alzheimer's disease, the hippocampus is characterized by abundant deposition of amyloid peptides (amyloid β [Aβ]) and neuroinflammation. Adult hippocampal neurogenesis (AHN) is a form of plasticity that contributes to cognition and can be influenced by either or both pathology and neuroinflammation. Their interaction has been studied before in rapidly progressing transgenic mouse models with strong overexpression of amyloid precursor protein (APP) and/or presenilin 1. So far, changes in AHN and neuroinflammation remain poorly characterized in slower progressing models at advanced age, which approach more closely sporadic Alzheimer's disease. Here, we analyzed 10- to 26-month-old APP.V717I mice for possible correlations between Aβ pathology, microglia, and AHN. The age-related increase in amyloid pathology was closely paralleled by microglial CD68 upregulation, which was largely absent in age-matched wild-type littermates. Notably, aging reduced the AHN marker doublecortin, but not calretinin, to a similar extent in wild-type and APP.V717I mice between 10 and 26 months. This demonstrates that AHN is influenced by advanced age in the APP.V717I mouse model, but not by Aβ and microglial activation.

International audience; The Gambian epauletted fruit bat (Epomophorus gambianus) is an abundant species that roosts in both urban and rural settings. The possible role of E. gambianus as a reservoir host of zoonotic diseases underlines the need to better understand the species movement patterns. So far, neither observational nor phylogenetic studies have identified the dispersal range or behavior of this species. Comparative analyses of mitochondrial and nuclear markers from 20 localities across the known distribution of E. gambianus showed population panmixia, except for the populations in Ethiopia and southern Ghana (Accra and Ve‐Golokwati). The Ethiopian population may be ancestral and is highly divergent to the species across the rest of its range, possibly reflecting isolation of an ancient colonization along an east–west axis. Mitochondrial haplotypes in the Accra population display a strong signature of a past bottleneck event; evidence of either an ancient or recent bottleneck using microsatellite data, however, was not detected. Demographic analyses identified population expansion in most of the colonies, except in the female line of descent in the Accra population. The molecular analyses of the colonies from Ethiopia and southern Ghana show gender dispersal bias, with the mitochondrial DNA fixation values over ten times those of the nuclear markers. These findings indicate free mixing of the species across great distances, which should inform future epidemiological studies.

Here we describe a new species of the genus Streptococcus that was isolated from a dairy cow with mastitis in New Zealand. Strain NZ1587T was Gram-positive, coccus-shaped and arranged as chains, catalase and coagulase negative, γ-haemolytic and negative for Lancefield carbohydrates (A-D, F and G). The 16S rRNA sequence did not match sequences in the NCBI 16S rRNA or GreenGenes databases. Taxonomic classification of strain NZ1587T was investigated using 16S rRNA and core genome phylogeny, genome-wide average nucleotide identity (ANI) and predicted DNA–DNA hybridisation (DDH) analyses. Phylogeny based on 16S rRNA was unable to resolve the taxonomic position of strain NZ1587T, however NZ1587T shared 99.4 % identity at the 16S rRNA level with a distinct branch of S. pseudoporcinus . Importantly, core genome phylogeny demonstrated that NZ1587T grouped amongst the ‘pyogenic’ streptococcal species and formed a distinct branch supported by a 100 % bootstrap value. In addition, average nucleotide identity and inferred DNA–DNA hybridisation analyses showed that NZ1587T represents a novel species. Biochemical profiling using the rapid ID 32 strep identification test enabled differentiation of strain NZ1587T from closely related streptococcal species. In conclusion, strain NZ1587T can be classified as a novel species, and we propose a novel taxon named Streptococcus bovimastitidis sp. nov.; the type strain is NZ1587T. NZ1587T has been deposited in the Culture Collection University of Gothenburg (CCUG 69277T) and the Belgian Co-ordinated Collections of Micro-organisms/LMG (LMG 29747).

We present single-contig assemblies for Escherichia coli strain KV7 (serotype O27, phylogenetic group D) and its six plasmids, isolated from a healthy pig, as determined by PacBio RS II and Illumina MiSeq sequencing. The chromosome of 4,997,475 bp and G+C content of 50.75% harbored 4,540 protein-encoding genes.

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, primarily associated with the consumption of contaminated poultry. C. jejuni lineages vary in host range and prevalence in human infection, suggesting differences in survival throughout the poultry processing chain. From 7,343 MLST-characterised isolates, we sequenced 600 C. jejuni and C. coli isolates from various stages of poultry processing and clinical cases. A genome-wide association study (GWAS) in C. jejuni ST-21 and ST-45 complexes identified genetic elements over-represented in clinical isolates that increased in frequency throughout the poultry processing chain. Disease-associated SNPs were distinct in these complexes, sometimes organised in haplotype blocks. The function of genes containing associated elements was investigated, demonstrating roles for cj1377c in formate metabolism, nuoK in aerobic survival and oxidative respiration, and cj1368-70 in nucleotide salvage. This work demonstrates the utility of GWAS for investigating transmission in natural zoonotic pathogen populations and provides evidence that major C. jejuni lineages have distinct genotypes associated with survival, within the host specific niche, from farm to fork.

Infection byCampylobacteris recognised as the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is currently no licenced vaccine and noCampylobacter-resistant chickens. In addition, preventative measures aimed at reducing environmental contamination and exposure of chickens toCampylobacter jejuni(biosecurity) have been ineffective. There is much interest in the factors/mechanisms that driveC. jejunicolonisation and infection of animals, and survival in the environment. It is anticipated that understanding these mechanisms will guide the development of effective intervention strategies to reduce the burden ofC. jejuniinfection. Here we present a comprehensive analysis ofC. jejunifitness during growth and survival within and outside hosts. A comparative analysis of transposon (Tn) gene inactivation libraries in threeC. jejunistrains by Tn-seq demonstrated that a large proportion, 331 genes, of theC. jejunigenome is dedicated to (in vitro) growth. An extensive Tn library inC. jejuniM1cam (~10,000 mutants) was screened for the colonisation of commercial broiler chickens, survival in houseflies and under nutrient-rich and–poor conditions at low temperature, and infection of human gut epithelial cells. We reportC. jejunifactors essential throughout its life cycle and we have identified genes that fulfil important roles across multiple conditions, includingmaf3, fliW, fliD, pflBandcapM, as well as novel genes uniquely implicated in survival outside hosts. Taking a comprehensive screening approach has confirmed previous studies, that the flagella are central to the ability ofC. jejunito interact with its hosts. Future efforts should focus on how to exploit this knowledge to effectively control infections caused byC. jejuni.Author SummaryCampylobacter jejuniis the leading bacterial cause of human diarrhoeal disease.C. jejuniencounters and has to overcome a wide range of “stress” conditions whilst passing through the gastrointestinal tract of humans and other animals, during processing of food products, on/in food and in the environment. We have taken a comprehensive approach to understand the basis ofC. jejunigrowth and within/outside host survival, with the aim to inform future development of intervention strategies. Using a genome-wide transposon gene inactivation approach we identified genes core to the growth ofC. jejuni. We also determined genes that were required during the colonisation of chickens, survival in the housefly and under nutrient-rich and –poor conditions at low temperature, and during interaction with human gut epithelial tissue culture cells. This study provides a comprehensive dataset linkingC. jejunigenes to growth and survival in models relevant to its life cycle. Genes important across multiple models were identified as well as genes only required under specific conditions. We identified that a large proportion of theC. jejunigenome is dedicated to growth and that the flagella fulfil a prominent role in the interaction with hosts. Our data will aid development of effective control strategies.

Purpose: To identify the protein profiles in vitreous associated with retinal fibrosis, angiogenesis, and neurite formation in epiretinal fibrovascular membranes (FVMs) in patients with proliferative diabetic retinopathy (PDR). Methods: Vitreous samples of 5 non-diabetic control patients with vitreous debris and 7 patients with PDR membranes were screened for 507 preselected proteins using the semi-quantitative RayBio® L-series 507 antibody array. From this array, 60 proteins were selected for a custom quantitative antibody array (Raybiotech, Human Quantibody® array), analyzing 7 control patients, 8 PDR patients with FVMs, and 5 PDR patients without FVMs. Additionally, mRNA levels of proteins of interest were measured in 10 PDR membranes and 11 idiopathic membranes and in retinal tissues and cells to identify possible sources of protein production. Results: Of the 507 proteins screened, 21 were found to be significantly elevated in PDR patients, including neurogenic and angiogenic factors such as neuregulin 1 (NRG1), nerve growth factor receptor (NGFR), placental growth factor (PlGF) and platelet derived growth factor (PDGF). Angiopoietin-2 (Ang2) concentrations were strongly correlated to the degree of fibrosis and the presence of FVMs in patients with PDR. Protein correlation analysis showed PDGF to be extensively co-regulated with other proteins, including thrombospondin-1 and Ang2. mRNA levels of glial-derived and brain/derived neurotrophic factor (GDNF and BDNF) were elevated in PDR membranes. These results were validated in a second study of 52 vitreous samples of 32 PDR patients and 20 control patients. Conclusions: This exploratory study reveals protein networks that potentially contribute to neurite outgrowth, angiogenesis and fibrosis in the formation of fibrovascular membranes in PDR. We identified a possible role of Ang2 in fibrosis and the formation of FVMs, and of the neurotrophic factors NRG1, PDGF and GDNF in neurite growth that occurs in all FVMs in PDR.

Background We analyzed the presence of microorganisms at the insertion site of percutaneous endoscopic gastrostomy (PEG) catheters of 100 patients to investigate the microbiologic flora and its possible relation with complaints. Materials and methods Outpatients visiting our hospital PEG-catheter team were included. Symptoms and the local aspect of the skin at the insertion site were described systematically and swabs were taken to analyze the presence of pathogenic microorganisms. Results The mean age of these 100 patients (55 men) was 62 ± 14 years (range, 3-89 years). Oropharyngeal carcinoma (n = 59) and neurologic disorders (n = 27) were the most frequent indications for a PEG catheter. The mean insertion time was 17 ± 20 months (range, 3-99 months). In 15 patients no microorganisms were found. In 85 patients we found Candida albicans (n = 37; 44%), Staphylococcus aureus (n = 28; 33%), Escherichia coli , Klebsiella , Enterobacter , and enterococci (5%-20%). None of the patients reported discomfort. Pain or itching was reported by 24 patients. We did not find any statistically significant relationship between colonization, microorganisms, complaints, and aspect of skin. Conclusions The insertion sites of PEG catheters in outpatients are frequently colonized, especially with C albicans , S aureus , and E coli , without clinical consequences. We did not find a relationship between microorganisms at the insertion sites of PEG catheters and signs and symptoms.

The complement system is an important innate defence mechanism, and the ability to resist complement-mediated killing is considered a key virulence trait of the respiratory tract pathogen M. catarrhalis. We studied the molecular basis of complement resistance by transcriptional profiling and Tn-seq, a genome-wide negative-selection screenings technology. Exposure of M. catarrhalis to human serum resulted in increased expression of 84 genes and reduced expression of 134 genes, among which genes encoding ABC transporter systems and surface proteins UspA1 and McaP. By subjecting a approximately 15 800 transposon mutant library to serum, mutants of 53 genes were negatively selected, including the key complement-resistance factor uspA2H. Validation with directed mutants confirmed Tn-seq phenotypes of uspA2H and 11 newly identified genes, with mutants of MCR_0424, olpA, MCR_1483, and dsbB most severely attenuated. Detailed analysis showed that both components of the disulphide bond formation (DSB) system, DsbB and DsbA, were required for complement-resistance in multiple isolates, and fulfil a critical role in evasion of IgG-dependent classical pathway-mediated killing. Lipooligosaccharide (LOS) structure and membrane stability were severely affected in DeltadsbA strains, suggesting a pivotal role for the DSB system in LOS structure safeguarding and membrane stability maintenance.

Aim of study: The main aim of the work was to summarize availability, quality and comparability of on-going European Research and Monitoring Networks (ERMN), based on the results of a COST FP0903 Action questionnaire carried out in September 2010 and May 2012.Area of study: The COST Action FP0903 involves 29 European countries and 4 non-COST institutions from USA, Morocco and Tunisia. In this study, the total of 22 replies to the questionnaire from 18 countries were included.Materials and methods: Based on the feedback from the Action FP0903 countries, the most popular European Networks were identified. Thereafter, the access to the network database, available quality assurance/quality control procedures and publication were described. Finally, the so-called “Supersites” concept, defined as a “highly instrumented research infrastructure, for both research and monitoring of soil-plant-atmosphere interactions” was discussed.Main results: The result of the survey indicate that the vast majority of the Action FP0903 countries participate in the International Cooperative Programme on Assessment and Monitoring of Air Pollution Effects on Forest (ICP Forest). The multi-disciplinary International Cooperative Programme on Integrated Monitoring of Air Pollution Effects on Ecosystems (ICPIM) is the second most widespread forest programme. Research highlights: To fully understand biochemical cycles in forest ecosystems, long-term monitoring is needed. Hence, a network of “Supersites”, is proposed. The application of the above infrastructure can be an effective way to attain a better integration of research and monitoring networks at forest sites in Europe.Keywords: Supersites; European Research Monitoring Networks; Harmonization; Forest.

Contains fulltext : 172169.pdf (Publisher’s version ) (Open Access) The bacterial speciesMoraxella catarrhalishas been hypothesized as being composed of two distinct lineages (referred to as the seroresistant [SR] and serosensitive [SS]) with separate evolutionary histories based on several molecular typing methods, whereas 16S ribotyping has suggested an additional split within the SS lineage. Previously, we characterized whole-genome sequences of 12 SR-lineage isolates, which revealed a relatively small supragenome when compared with other opportunistic nasopharyngeal pathogens, suggestive of a relatively short evolutionary history. Here, we performed whole-genome sequencing on 18 strains from both ribotypes of the SS lineage, an additional SR strain, as well as four previously identified highly divergent strains based on multilocus sequence typing analyses. All 35 strains were subjected to a battery of comparative genomic analyses which clearly show that there are three lineages-the SR, SS, and the divergent. The SR and SS lineages are closely related, but distinct from each other based on three different methods of comparison: Allelic differences observed among core genes; possession of lineage-specific sets of core and distributed genes; and by an alignment of concatenated core sequences irrespective of gene annotation. All these methods show that the SS lineage has much longer interstrain branches than the SR lineage indicating that this lineage has likely been evolving either longer or faster than the SR lineage. There is evidence of extensive horizontal gene transfer (HGT) within both of these lineages, and to a lesser degree between them. In particular, we identified very high rates of HGT between these two lineages for ss-lactamase genes. The four divergent strains aresui generis, being much more distantly related to both the SR and SS groups than these other two groups are to each other. Based on average nucleotide identities, gene content, GC content, and genome size, this group could be considered as a separate taxonomic group. The SR and SS lineages, although distinct, clearly form a single species based on multiple criteria including a large common core genome, average nucleotide identity values, GC content, and genome size. Although neither of these lineages arose from within the other based on phylogenetic analyses, the question of how and when these lineages split and then subsequently reunited in the human nasopharynx is explored.

Iron sequestration by the human host is a first line defence against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron starvation during colonization. We determined the genetic requirements for M. catarrhalis BBH18 growth during iron starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). By subjecting a large random transposon mutant library to growth under iron-limiting conditions, mutants of the MCR_0996-rhlB-yggW operon, rnd, and MCR_0457 were negatively selected. Growth experiments using directed mutants confirmed the GAF phenotypes with ΔyggW (putative haem-shuttling protein) and ΔMCR_0457 (hypothetical protein) most severely attenuated during iron starvation, phenotypes which were restored upon genetic complementation of the deleted genes. Deletion of yggW resulted in similar attenuated phenotypes in three additional strains. Transcriptional profiles of ΔyggW and ΔMCR_0457 were highly altered with 393 and 192 differentially expressed genes respectively. In all five mutants, expression of nitrate reductase genes was increased and of nitrite reductase decreased, suggesting an impaired aerobic respiration. Alteration of iron metabolism may affect nasopharyngeal colonization as adherence of all mutants to respiratory tract epithelial cells was attenuated. In conclusion, we elucidated the genetic requirements for M. catarrhalis growth during iron starvation and characterized the roles of the identified genes in bacterial growth and host interaction.

Background: Guidelines advise weekly cleansing of spacers, with one of the reasons being to prevent the spacers from becoming colonized with respiratory pathogens. Earlier work in clinical settings showed conflicting results.Methods: Common respiratory pathogens and Candida albicans were applied on Petri dishes with and without inhaled corticosteroids and in 3 brands of spacer devices, with and without inhaled corticosteroids. Growth was measured.Results: After 24 hours, Staphylococcus aureus grew in 7 of 18 spacers (39%); Pseudomonas aeruginosa grew in 12 out of 18 spacers (67%); and C albicans survived in 5 of 18 spacers (28%). Microorganisms survived on Petri dishes with fluticasone and beclomethasone but not when budesonide was applied. One out of 30 metal Nebuhalers (3%) was colonized after 24 hours, whereas of 30 Volumatics 3 (27%) and Aerochambers, 17 (57%) still had viable microorganisms. Application of inhaled steroids did not affect growth in the spacers.Conclusion: The colonization of metal spacers is lower than of spacers made of polycarbonate or polyethylene. C albicans cart survive in spacers. The survival of microorganisms in spacers is not influenced by inhaled corticosteroids. Copyright (c) 2009 by the Association for Professionals in Infection Control and Epidemiology, Inc.

Background Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Result Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept. Conclusion Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-958) contains supplementary material, which is available to authorized users.

Site-specific estimates for various environmentalstress factors were related with measured crowncondition data at a systematic 16 ×: 16 km2 gridover Europe, according to previously statedhypotheses, using a multiple regression approach,including interactions, and lagged effects of stressfactors. Methodological differences among countriesaccounted for >30% of the variation in defoliation.Nevertheless, crown condition was found to varynaturally with tree age, altitude, drought stress and,most likely, also pathogenic fungi and insects.Significant impacts of air pollution (specificallyozone but also NOx, SOx and acid deposition)were found at regional levels in parts of centralEurope, particularly for deciduous species. Impactsseemed less significant for conifers, especially forspruce, but this might be affected by confoundingeffects or strong correlations between (a harsh)climate and (low) atmospheric deposition in the areawhere spruce predominates. National studies indicatethat ozone and acid deposition can have a significanteffect on the defoliation of spruce as well. Weconclude that while forest condition varies naturally,continued emissions will contribute further to forestdecline in the long term.

This paper is the first in a series of four,describing the hypothesis and approach of acorrelative study between observed data on crowncondition in Europe, monitored since 1986 at asystematic 16 × 16 km grid, and site-specificestimations of various natural and anthropogenicstress factors. The study was based on the hypothesisthat forests respond to various natural andanthropogenic stress factors, whose contributiondepend on the geographic region considered. In view ofthis hypothesis, major stand and site characteristics,chemical soil composition, meteorological stressfactors (temperature and drought stress indices) andair pollution stress (concentrations and/ordepositions of SOx, NOy, NHx andO3) were included as predictor variables. Theresponse variables considered were actual defoliationand changes/trends in defoliation for five major treespecies. The spatial distribution of the averagedefoliation during the period 1986–1995 shows highdefoliation in Central Europe and in parts ofScandinavia and of Southern Europe. There are,however, sharp changes at country borders, which aredue to methodological differences between countries.The spatial distribution of the calculated trends showa distinct cluster of large deterioration in parts ofCentral and Eastern Europe and in Spain and a ratherscattered pattern of positive and negative trends for most of Europe, indicating that other factors than airpollution only have a strong impact on defoliation.The limitations of the study are discussed in view ofthe quality of the considered response and predictor variables.

In routine tests to investigate immunological mechanisms as a cause for enhanced destruction of transfused platelets, serum from the patient is tested against a panel of donor lymphocytes and platelets to demonstrate the presence of antibodies against HLA or platelet specific antibodies. Here we describe a flow cytometric technique in which in vivo binding of immunoglobulins (Ig) is measured. By comparing the histograms of the platelet suspensions before and after transfusion, four different patterns were obtained: no Ig binding before and after transfusion (pattern 1), pre-existent Ig binding (patterns 2 and 3) or preferential Ig binding to the transfused platelets (pattern 4). This technique was tested in 164 random platelet transfusions and 34 HLA-matched platelet transfusions. A statistically significant association was found between pattern 1 and a negative result of the indirect platelet immunofluorescence test for the detection of antibodies in the serum taken before the transfusion studied or a good platelet recovery and between pattern 4 and a positive indirect platelet immunofluorescence test or a low platelet recovery.

Contains fulltext : 183574.PDF (Publisher’s version ) (Open Access) Moraxella catarrhalis is a mucosal pathogen that causes childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. During the course of infection, M. catarrhalis needs to adhere to epithelial cells of different host niches such as the nasopharynx and lungs, and consequently, efficient adhesion to epithelial cells is considered an important virulence trait of M. catarrhalis. By using Tn-seq, a genome-wide negative selection screenings technology, we identified 15 genes potentially required for adherence of M. catarrhalis BBH18 to pharyngeal epithelial Detroit 562 and lung epithelial A549 cells. Validation with directed deletion mutants confirmed the importance of aroA (3-phosphoshikimate 1-carboxyvinyl-transferase), ecnAB (entericidin EcnAB), lgt1 (glucosyltransferase), and MCR_1483 (outer membrane lipoprotein) for cellular adherence, with DeltaMCR_1483 being most severely attenuated in adherence to both cell lines. Expression profiling of M. catarrhalis BBH18 during adherence to Detroit 562 cells showed increased expression of 34 genes in cell-attached versus planktonic bacteria, among which ABC transporters for molybdate and sulfate, while reduced expression of 16 genes was observed. Notably, neither the newly identified genes affecting adhesion nor known adhesion genes were differentially expressed during adhesion, but appeared to be constitutively expressed at a high level. Profiling of the transcriptional response of Detroit 562 cells upon adherence of M. catarrhalis BBH18 showed induction of a panel of pro-inflammatory genes as well as genes involved in the prevention of damage of the epithelial barrier. In conclusion, this study provides new insight into the molecular interplay between M. catarrhalis and host epithelial cells during the process of adherence.

Forests are important components of the greenhouse gas balance of Europe. There is considerable uncertainty about how predicted changes to climate and nitrogen deposition will perturb the carbon and nitrogen cycles of European forests and thereby alter forest growth, carbon sequestration and N2O emission. The present study aimed to quantify the carbon and nitrogen balance, including the exchange of greenhouse gases, of European forests over the period 2010–2030, with a particular emphasis on the spatial variability of change. The analysis was carried out for two tree species: European beech and Scots pine. For this purpose, four different dynamic models were used: BASFOR, DailyDayCent, INTEGRATOR and Landscape-DNDC. These models span a range from semi-empirical to complex mechanistic. Comparison of these models allowed assessment of the extent to which model predictions depended on differences in model inputs and structure. We found a European average carbon sink of 0.160 ± 0.020 kgC m−2 yr−1 (pine) and 0.138 ± 0.062 kgC m−2 yr−1 (beech) and N2O source of 0.285 ± 0.125 kgN ha−1 yr−1 (pine) and 0.575 ± 0.105 kgN ha−1 yr−1 (beech). The European average greenhouse gas potential of the carbon sink was 18 (pine) and 8 (beech) times that of the N2O source. Carbon sequestration was larger in the trees than in the soil. Carbon sequestration and forest growth were largest in central Europe and lowest in northern Sweden and Finland, N. Poland and S. Spain. No single driver was found to dominate change across Europe. Forests were found to be most sensitive to change in environmental drivers where the drivers were limiting growth, where changes were particularly large or where changes acted in concert. The models disagreed as to which environmental changes were most significant for the geographical variation in forest growth and as to which tree species showed the largest rate of carbon sequestration. Pine and beech forests were found to have differing sensitivities to environmental change, in particular the response to changes in nitrogen and precipitation, with beech forest more vulnerable to drought. There was considerable uncertainty about the geographical location of N2O emissions. Two of the models BASFOR and LandscapeDNDC had largest emissions in central Europe where nitrogen deposition and soil nitrogen were largest, whereas the two other models identified different regions with large N2O emission. N2O emissions were found to be larger from beech than pine forests and were found to be particularly sensitive to forest growth.

Spacers are used in the treatment of children with asthma. It is advised to clean the spacers regularly. However, cleaning can influence drug delivery. One obvious reason to clean a spacer is prevention of bacterial contamination. Whether spacers are contaminated or not is unknown. We cultured spacers, brought in by children with asthma or recurrent wheeze who visited our outpatient clinic during a 4-month period. The spacers of 64 children were studied, and 24 (38%) were contaminated; most often (13 cases), Bacillus species were found. Only one spacer grew a potential pathogenic bacterium (Pseudomonas aeruginosa). No correlation with type of inhaler, duration of usage, drug, or visual aspect was found. Spacers, cleaned according to national guidelines, were not less contaminated. Bacterial contamination of spacers is frequent, but the bacteria cultured are not pathogenic. Intensive cleaning of spacers does not influence the level of contamination with environmental bacteria.

Here we report the annotated genome sequence of Moraxella catarrhalis strain RH4, a seroresistant-lineage strain isolated from the blood of an infected patient. This genome sequence will allow us to gain further insight into the genetic diversity of clinical M. catarrhalis isolates and will facilitate study of M. catarrhalis pathogenesis.

Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative, human-restricted pathogen. Although this bacterium typically colonizes the nasopharynx in the absence of clinical symptoms, it is also one of the major pathogens causing otitis media (OM) in children. Complement represents an important aspect of the host defense against NTHi. In general, NTHi is efficiently killed by complement-mediated killing; however, various resistance mechanisms have also evolved. We measured the complement resistance of NTHi isolates isolated from the nasopharynx and the middle ear fluids of OM patients. Furthermore, we determined the molecular mechanism of NTHi complement resistance. Complement resistance was strongly increased in isolates from the middle ear, which correlated with decreased binding of IgM. We identified a crucial role for the R2866_0112 gene in complement resistance. Deletion of this gene altered the lipooligosaccharide (LOS) composition of the bacterium, which increased IgM binding and complement-mediated lysis. In a novel mouse model of coinfection with influenza virus, we demonstrate decreased virulence for the R2866_0112 deletion mutant. These findings identify a mechanism by which NTHi modifies its LOS structure to prevent recognition by IgM and activation of complement. Importantly, this mechanism plays a crucial role in the ability of NTHi to cause OM., IMPORTANCE Nontypeable Haemophilus influenzae (NTHi) colonizes the nasopharynx of especially young children without any obvious symptoms. However, NTHi is also a major pathogen in otitis media (OM), one of the most common childhood infections. Although this pathogen is often associated with OM, the mechanism by which this bacterium is able to cause OM is largely unknown. Our study addresses a key biological question that is highly relevant for child health: what is the molecular mechanism that enables NTHi to cause OM? We show that isolates collected from the middle ear fluid exhibit increased complement resistance and that the lipooligosaccharide (LOS) structure determines IgM binding and complement activation. Modification of the LOS structure decreased NTHi virulence in a novel NTHi-influenza A virus coinfection OM mouse model. Our findings may also have important implications for other Gram-negative pathogens harboring LOS, such as Neisseria meningitidis, Moraxella catarrhalis, and Bordetella pertussis.

Background M. catarrhalis is a gram-negative, gamma-proteobacterium and an opportunistic human pathogen associated with otitis media (OM) and exacerbations of chronic obstructive pulmonary disease (COPD). With direct and indirect costs for treating these conditions annually exceeding $33 billion in the United States alone, and nearly ubiquitous resistance to beta-lactam antibiotics among M. catarrhalis clinical isolates, a greater understanding of this pathogen's genome and its variability among isolates is needed. Results The genomic sequences of ten geographically and phenotypically diverse clinical isolates of M. catarrhalis were determined and analyzed together with two publicly available genomes. These twelve genomes were subjected to detailed comparative and predictive analyses aimed at characterizing the supragenome and understanding the metabolic and pathogenic potential of this species. A total of 2383 gene clusters were identified, of which 1755 are core with the remaining 628 clusters unevenly distributed among the twelve isolates. These findings are consistent with the distributed genome hypothesis (DGH), which posits that the species genome possesses a far greater number of genes than any single isolate. Multiple and pair-wise whole genome alignments highlight limited chromosomal re-arrangement. Conclusions M. catarrhalis gene content and chromosomal organization data, although supportive of the DGH, show modest overall genic diversity. These findings are in stark contrast with the reported heterogeneity of the species as a whole, as wells as to other bacterial pathogens mediating OM and COPD, providing important insight into M. catarrhalis pathogenesis that will aid in the development of novel therapeutic regimens.

The complement system is an important part of the innate defense against invading pathogens (Blom et al., 2009; [1]). The ability to resist complement-mediated killing is considered to be an important virulence trait for the human-restricted respiratory tract pathogen Moraxella catarrhalis, as most disease-associated M. catarrhalis isolates are complement-resistant (Wirth et al., 2007; [2]). Here we provide a detailed overview of the experimental methods that we have used to study the molecular basis of M. catarrhalis complement-resistance by transcriptome profiling of the bacterium upon exposure to 10% normal human serum (NHS), associated with the study of de Vries et al. published in Molecular Microbiology in 2014 [3].

Lymphocytes, monocytes, granulocytes, and other blood cells can be distinguished on the basis of their forward (FSC) and sideward (SSC) light scatter properties and their expression of CD45 and CD14. A FSC,SSC gate can be set to include > 95% of the lymphocytes using a “back gating” procedure on the CD45+, CD14− cells. However, nonlymphoid cells such as monocytes have light scattering properties similar to lymphocytes. This problem occurs particularly in patient populations where the light scattering properties of lymphocyte subsets have changed (e.g., due to activation) and are similar to those of the monocytes. Thus, immunophenotyping using antibodies specific for other markers than CD45 and CD14 does not allow a direct assessment of the percentage of all lymphocytes positive for those markers. In order to optimize immunophenotyping we have developed an analytic model in which the FSC,SSC dot plot is partitioned into six nonoverlapping light scatter regions. Each light scatter region contains a mixture population of different cell types, i.e., lymphocytes, monocytes, granulocytes, and other cells. The proportions of each cell type are known from the CD45,CD14 expression within each light scatter region. Under the assumption of independence of fluorescence and scatter properties conditional on cell type, the expression of markers other than CD45 or CD14 are derived from the cell type composition and the fluorescence properties on the other markers of each light scatter region. The underlying statistical model is a latent class model, and maximum likelihood estimates are computed using the expectation-maximization (EM) algorithm. The application of the model for immunophenotyping of lymphocytes of healthy individuals and cancer patients receiving immunotherapy is shown. © 1993 Wiley-Liss, Inc.

Moraxella catarrhalis is an emerging human-restricted respiratory tract pathogen that is a common cause of childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. Here, we report the first completely assembled and annotated genome sequence of an isolate of M. catarrhalis , strain RH4, which originally was isolated from blood of an infected patient. The RH4 genome consists of 1,863,286 nucleotides that form 1,886 protein-encoding genes. Comparison of the RH4 genome to the ATCC 43617 contigs demonstrated that the gene content of both strains is highly conserved. In silico phylogenetic analyses based on both 16S rRNA and multilocus sequence typing revealed that RH4 belongs to the seroresistant lineage. We were able to identify almost the entire repertoire of known M. catarrhalis virulence factors and mapped the members of the biosynthetic pathways for lipooligosaccharide, peptidoglycan, and type IV pili. Reconstruction of the central metabolic pathways suggested that RH4 relies on fatty acid and acetate metabolism, as the genes encoding the enzymes required for the glyoxylate pathway, the tricarboxylic acid cycle, the gluconeogenic pathway, the nonoxidative branch of the pentose phosphate pathway, the beta-oxidation pathway of fatty acids, and acetate metabolism were present. Moreover, pathways important for survival under challenging in vivo conditions, such as the iron-acquisition pathways, nitrogen metabolism, and oxidative stress responses, were identified. Finally, we showed by microarray expression profiling that ∼88% of the predicted coding sequences are transcribed under in vitro conditions. Overall, these results provide a foundation for future research into the mechanisms of M. catarrhalis pathogenesis and vaccine development.

In the climate change discussion, the possibility of carbon sequestration of forests plays an important role. Therefore, research on the effects of environmental changes on net primary productivity is interesting. In this study we investigated the influence of changing temperature, precipitation and deposition of sulphur and nitrogen compounds on forest growth. The database consisted of 654 plots of the European intensive monitoring program (Level II plots) with 5-year growth data for the period 1994–1999. Among these 654 plots only 382 plots in 18 European countries met the requirements necessary to be used in our analysis. Our analysis was done for common beech (Fagus sylvatica), oak (Quercus petraea and Q. robur), Scots pine (Pinus sylvestris) and Norway spruce (Picea abies). We developed an individual tree growth model with measured basal area increment of each individual tree as responding growth factor and tree size (diameter at breast height), tree competition (basal area of larger trees and stand density index), site factors (soil C/N ratio, temperature), and environmental factors (temperature change compared to long-term average, nitrogen and sulphur deposition) as influencing parameters. Using a mixed model approach, all models for the tree species show a high goodness of fit with Pseudo-R2 between 0.33 and 0.44. Diameter at breast height and basal area of larger trees were highly influential variables in all models. Increasing temperature shows a positive effect on growth for all species except Norway spruce. Nitrogen deposition shows a positive impact on growth for all four species. This influence was significant with p

Oxidative stress causes retinal pigment epithelium (RPE) cell dysfunction and is a major risk factor leading to the development of dry-type age-related macular degeneration. Taking pharmacological and genetic approaches, we address the mechanisms by which sublethal oxidative stress inhibits RPE cell phagocytosis. Sublethal oxidative stress dose-dependently inhibited RPE cell phagocytosis of photoreceptor outer segments (POS) and activated AMP-activated protein kinase (AMPK) as determined by increased Thr172 and Ser79 phosphorylation of AMPKalpha and its substrate acetyl-CoA carboxylase, respectively. Similar to oxidative stress, 5-aminoimidazole-4-carboxamide riboside (AICAR), a pharmacological activator of AMPK, inhibited RPE cell phagocytosis of POS in a dose-dependent manner. Inhibition of RPE cell phagocytosis by AICAR was fully reversed by blockade of AICAR translocation into cells by dipyridamole or inhibition of AICAR conversion to ZMP by adenosine kinase inhibitor 5-iodotubercidin. In agreement, AICAR-induced activation of AMPK was abolished by preincubation with dipyridamole or 5-iodotubercidin. Knock-out experiments further revealed that alpha2 but not alpha1 AMPK was involved in RPE cell phagocytosis and that activation of alpha2 AMPK contributed to the inhibition of RPE cell phagocytosis by oxidative stress. Inhibition of RPE cell phagocytosis by activation of alpha2 AMPK was associated with a dramatic increase in acetyl-CoA carboxylase phosphorylation. In comparison, AMPK had no role in oxidative stress-induced breakdown of RPE barrier function. Taken together, reduction in POS load under oxidative stress might direct RPE cells to a self-protected status. Thus, activating AMPK could have therapeutic potential in treating dry macular degeneration.

Item does not contain fulltext The mammalian innate immune system senses viral infection by recognizing viral signatures and activates potent antiviral responses. Besides the interferon (IFN) response, there is accumulating evidence that RNA silencing or RNA interference (RNAi) serves as an antiviral mechanism in mammalian cells. Mammalian viruses encode IFN antagonists to counteract the IFN response in infected cells. A number of IFN antagonists are also capable of blocking RNAi in infected cells and therefore serve as RNA-silencing suppressors. Virus replication in infected cells is restricted by these innate antiviral mechanisms, which may kick in earlier than the viral antagonistic or suppressor protein can accumulate. The yield of virus vaccines and viral gene delivery vectors produced in mammalian producer cells may therefore be suboptimal. To investigate whether blocking of the innate antiviral responses in mammalian cells leads to increased viral vector production, we expressed a number of immunity suppressors derived from plant and mammalian viruses in human cells. We measured that the yield of infectious human immunodeficiency virus-1 particles produced in these cells was increased 5- to 10-fold. In addition, the production of lentiviral and adenoviral vector particles was increased 5- to 10-fold, whereas Sindbis virus particle production was increased approximately 100-fold. These results can be employed for improving the production of viral gene transfer vectors and viral vaccine strains.

Perim Island is an eroded fragment of the southwest flank of a late Miocene (10.5 ± 1.0 Ma) volcano whose centre lay on the southwesternmost tip of Arabia. The volcano is the westernmost of the E–W line of six central vent volcanoes (the Aden Line) that extends 200 km along the south coast of Arabia from Perim to Aden. Major oxide and trace element abundances are given for 35 Perim specimens and these show that the volcano has within-plate trace element characteristics and consists of a petrographically and geochemically simple suite of alumina-poor olivine basalts, andesites, and transitional andesite–trachyandesites. Six specimens, however, are markedly enriched in Al2O3 and CaO, and contain abundant (20–30 mode %) highly calcic (An77–83) plagioclase phenocrysts. Geochemical modelling suggests that the main Perim volcanic sequence was produced by the fractional crystallization (o1 + cpx + Ti-mt + plag) of a silica saturated (SiO2 c. 45%) basic melt. The high A1, high Ca, magmas appear to be mixing products of plagioclase-enriched basic magmas with more evolved melts. Perim is the oldest volcano of the Aden line, which becomes increasingly younger and alkalic eastward. It is suggested that the volcanism is related to an eastwards-propagating rift produced before the most recent stage of sea-floor spreading in the Gulf of Aden (4.5 Ma–present).

Background and Objectives Alloantibodies against platelets can be detected by using different laboratory tests. Most of these tests, which use panel cells or antigens as a target, perform poorly in non-selected haematological patients. In relation to these tests, a crossmatch test of transfused platelets and patient's serum may be viewed as the standard and may be superior in predicting donor platelet destruction by alloimmunization. Materials and Methods In 95 randomly selected thrombocytopenic patients with haematological malignancies, who were receiving leucodepleted blood products, 184 serum samples were studied in an in vitro crossmatch test by using the technique of the platelet immunofluorescence test (crossmatch-PIFT), in an in vivo crossmatch test detecting in vivo binding of immunoglobulins to transfused platelets according to the PIFT technique (in vivo-PIFT), in the indirect PIFT using five random donors as a target (panel-PIFT) and in an enzyme linked immunosorbent assay using immobilized human leucocyte antigens (HLAs) of 100 standardized donors (ELIHLA). The results of all these methods were related to the recovery at 1 and 16 h post-transfusion. Results The results of the crossmatch-PIFT were not associated with platelet recovery at 1 and 16 h after transfusion. Even in a subgroup of patients, in whom predefined clinical factors were excluded, no association with platelet recovery was found. The results of the crossmatch-PIFT correlated with those of the in vivo-PIFT (P = 0·02); however, 35 (19%) discrepant results were identified between these tests. The results of the crossmatch-PIFT were not related to the panel-PIFT (P = 0·25), but did relate to those of the ELIHLA (P = 0·02), still revealing 36 (20%) discrepant results. None of the in vivo-PIFT, the panel-PIFT or the ELIHLA was associated with platelet recovery after 1 h, whilst only a positive panel-PIFT was associated with poor platelet recovery at 16 h after transfusion (P = 0·03). Conclusions In a population at low risk for alloimmunization, the correlation of test outcome and platelet recovery is poor. None of these crossmatch tests or screening tests was identified as superior to any other in this population.

BACKGROUND We developed a technique, in vivo binding of immunoglobulins in the platelet immunofluorescence test (IVBI-PIFT), that detects immunoglobulins bound in vivo to transfused platelets. The visually scored results of this technique, however, are susceptible to interobserver variation. We describe a more objective method to generate results in IVBI-PIFT. METHODS We studied 201 samples in 120 patients with hematologic malignancies in the IVBI-PIFT. Histogram subtraction, i.e., fluorescence (anti–immunoglobulin G and fluorescein isothiocyanate) histogram before platelet transfusion subtracted from the histogram after platelet transfusion, was compared with visual scoring (pattern 1: no enhanced fluorescence before and after transfusion; pattern 2: enhanced fluorescence before and after platelet transfusion; pattern 3: enhanced fluorescence before transfusion; pattern 4: enhanced fluorescence after transfusion, interpreted as alloimmunization). After histogram subtraction, the number of remaining events (events post substraction, EPS) and the mean amount of fluorescence of these remaining events (mean channel post substraction, MCPS) were used and compared with the visual scoring and with platelet survival after transfusion. RESULTS In 26 (13%) of the 201 samples studied in the IVBI-PIFT, fewer than three of five observers agreed on the visually scored pattern. In the 175 (87%) remaining samples, histogram subtraction showed a significant differentiation between pattern 4 and patterns 1 and 2 by using EPS, whereas patterns 4 and 3 were distinguished by using MCPS. The combination of EPS and MCPS differentiated best between pattern 4 and patterns 1, 2, and 3 (73% sensitivity, 96% specificity, 79% positive predictive value, and 95% negative predictive value). In contrast, the predictive value for platelet recovery after 1 and 16 h of pattern 4 from the visual scoring method and the results of histogram subtraction were poor. CONCLUSION This objective method of histogram subtraction correlated well with the visual scoring method of IVBI-PIFT. © 2003 Wiley-Liss, Inc.

Although both vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptors have been shown to be important in the regulation of vascular endothelial cell growth, the roles of phospholipase C (PLC)γ and Ca2+in their downstream signaling cascades are still not clear. We have examined the effects of VEGF and FGF on PLCγ phosphorylation and on changes in intracellular Ca2+levels in primary endothelial cells. VEGF stimulation leads to PLCγ activation and increases in intracellular Ca2+, which are correlated with mitogen-activated protein (MAP) kinase (MAPK) activation and cell growth. Inhibition of Ca2+increases by the Ca2+chelator 1,2-bis(2-aminophenoxy) ethane- N,N,N′,N′-tetraacetic acid (BAPTA)-AM resulted in marked inhibition of MAPK activation, which was shown to be linked to regulation of cell growth in these cells. In contrast, FGF stimulation did not lead to PLCγ activation or to changes in intracellular Ca2+levels, although MAPK phosphorylation and stimulation of cell proliferation were observed. Neither BAPTA-AM nor the PLC inhibitor U-73122 had an effect on these FGF-stimulated responses. These data demonstrate a direct role for PLCγ and Ca2+in VEGF-regulated endothelial cell growth, whereas this signaling pathway is not linked to FGF-mediated effects in primary endothelial cells. Thus endothelial cell-specific factors regulate the ability of VEGF receptors and FGF receptors to couple to this signaling pathway.

Dynamics of C and N in forest soils in the Nutrient Cycling and Soil Acidification Model (NUCSAM) are described by the transformation and decomposition of three organic matter compartments, litter, fermented material and humic material. These three compartments are allocated to the morphological distinguishable L, F and H horizons of the organic layer. Changes in the pools of these organic compartments are described with first order equations for decomposition and transformation. Rate constants for decomposition and transformation were derived by calibrating the model to measured organic matter pools in organic layers of a chrono-sequence of five first succession Scots pine stands between 15 and 120 years old. Simulated pools of organic matter in the organic layers were in agreement with measured pools in the five pine stands, except for the first thirty years of the H-horizon. During this period, an increase in organic matter in the H horizon was simulated while no H horizons were observed in the field. The simulated total pool of organic matter in the organic layer agreed well with values from a field inventory in 20 other Scots pine stands, but the simulated distribution over the three horizons differed from the field measurements which varied among sites. For the Scots pine stands the model was able to simulate the organic matter accumulation in the top 40-cm of the mineral soil; derived almost completely from fine root turnover. The accumulated pool of nitrogen in the organic layer was in agreement with measured pools for the oldest Scots pine stand but was too high for the younger stands. Especially, the accumulation of N in the F-horizon was too fast, presumably due to an overestimated retention of nitrogen.

Gaseous emissions from in-house storage of liquid animal manure remain a major contributor to the environmental impact of manure management. Our aim was to assess the life cycle environmental consequences and reduction potential of segregating fattening pig urine and feces with an innovative V-belt system and to compare it to conventional liquid manure management, that is, the reference. Moreover, we aimed at analyzing the uncertainty of the outcomes related to applied emission factors. We compared a reference with two scenarios: segregation with solid, aerobically, stored feces and with liquid, anaerobically, stored feces. Results showed that, compared to the reference, segregation reduced climate change (CC) up to 82%, due to lower methane emission, reduced terrestrial acidification (TA) and particulate matter formation (PMF) up to 49%, through lower ammonia emission, but increased marine eutrophication up to 11% through nitrogen oxide emission from storage and nitrate leaching after field application. Fossil fuel depletion did not change. Segregation with liquid feces revealed lower environmental impact than segregation with solid feces. Uncertainty analysis supported the conclusion that segregating fattening pig urine and feces significantly reduced CC and additionally segregation with liquid feces significantly reduced TA and PMF compared to the reference.

The influence of forest and soil properties on soil solution concentration and the response of soil solution concentrations upon a reduction in deposition was examined with the dynamic simulation model RESAM. Runs were performed for five different forest-soil combinations in the Netherlands, i.e. for Douglas fir, Scots pine and oak on a Cambic podzol and for Douglas fir on a Carbic podzol and a Gleyic Arenosol. Predicted concentrations generally decreased in the direction Douglas fir > Scots pine > oak. This was mainly caused by differences in filtering of dry deposition (decreasing in the direction Douglas fir > Scots pine > oak) and in precipitation excess (decreasing in the direction oak > Scots pine > Douglas fir). However, concentrations of some elements, especially NH 4 and Ca, were strongly influenced by biocycling. The influence of the considered non calcareous sandy soil types on the soil solution concentrations was relatively small. The influence was mainly caused by differences in N transformation rates. In the Gleyic Arenosol the predicted mineralization and nitrification fluxes were relatively low, whereas the denitrification fluxes were relatively high compared to the podzol soils. Concentrations which are not strongly influenced by biocycling (SO 4 , Cl) were somewhat higher in the Gleyic Arenosol due to a lower precipitation excess. Al concentrations decreased in the direction Carbic podzol > Gleyic Arenosol > Cambic podzol due to a decreasing amount and dissolution rate of Al-(hydr)oxides in that same direction and a lower acidification in the Gleyic Arenosol. In all considered forest-soil combinations, a reduction in deposition level caused an increase in pH and a decrease in SO 4 , NO 3 , NH 4 and Al concentrations. Differences between the rate of de-acidification under the considered three species occurred due to differences in biocycle which result in differences in the ratio between internal and external acid input. In the Gleyic Arenosol a faster de-acidification took place compared to the well-drained podzol soils due to a rise in denitrification upon a rise in pH.

1. Comparison of the rank order of potency of the natural prostanoids prostaglandin E2 (PGE2), PGD2, PGF2 alpha and carbaprostacyclin in stimulating cyclic AMP in Jurkat cells is consistent with the presence of an EP receptor. 2. Lack of responsiveness to the EP1/EP3 selective agonist, sulprostone, and the EP2 agonists, butaprost and AH 13205, indicates that this receptor is not of the EP1, EP2 or EP3 subtypes. 3. Inhibition of PGE2-stimulated cyclic AMP by the EP4 antagonist, AH 23848 is non-competitive, unlike the competitive antagonism reported in the pig saphenous vein EP4 preparation. Furthermore, 16,16-dimethyl PGE2 is 100 fold less potent than PGE2 in Jurkat cells, while these agonists are equipotent in the rabbit jugular vein purported EP4 preparation. In addition, 1-OH PGE1, which also is active in the rabbit jugular vein preparation, is inactive in Jurkat cells at concentrations up to 1 x 10(-4) M. These data are not wholly consistent with any adenylate cyclase coupled EP receptor described to date. 4. It is postulated that an EP receptor, positively coupled to adenylate cyclase, with a unique pharmacological profile is present in Jurkat cells.

Effects of manipulation of litter and humus layers (removal, doubling and control treatments) on the colonization potential of ectomycorrhizal fungi were studied in two secondary stands of Pinus sylvestris (5 and 18 years old) in The Netherlands. Five-mont-hold, sterile-grown Scots pine seedlings, inoculated with Laccaria bicolor, Paxillus involutus or Rhizopogon luteolus and noninoculated seedlings were used as baits. The seedlings were harvested after one growing season. For comparison, sporocarps of ectomycorrhizal fungi were also investigated. Genus composition on the seedlings was independent of initial inoculum, but determined by both treatment and age of the stands. In both stands, removal of litter and humus layers increased, and addition of organic material decreased the number of ectomycorrhizal types on the seedlings. Not all indigenous genera were observed by either outplanting seedlings or sporocarp surveys.

Publisher Summary During the period March to May 1990, the chemical composition of the humus layer, the mineral topsoil (0–30 cm), and the mineral subsoil (60–100 cm) has been determined for 150 forest stands. All stands were part of the national forest vitality inventory. They were all located on noncalcareous sandy soils. Tree species included were Scotch Pine, Black Pine, Douglas Fir, Norway Spruce, Japanese Larch, Oak, and Beech. Measurements for the humus layer and mineral topsoil included total contents of C, N, and P and exchangeable contents of H, Al, Fe, Ca, Mg, K, Na, and NH. Soil solution measurements in the mineral soil included the cations mentioned before and NO, SO, C1, HCO, and DOC. An important aim of the research was to assess the effect of deposition levels, tree species, and site characteristics on the level of A1 mobilization (acidification) and N accumulation (eutrophication) in the soil. Important characteristics of the chemical composition of the humus layer and mineral topsoil are presented.

AGN 190383 is a 5-hydroxy-2(5H)-furanone ring analog of the marine natural product manoalide. When applied topically, AGN 190383 inhibits phorbol ester induced mouse ear edema. It is a potent inhibitor of bee venom phospholipase A2 and blocks the release of arachidonic acid from calcium ionophore A23187 stimulated human neutrophils. AGN 190383 also inhibits both hormone-operated and depolarization-dependent calcium mobilization in GH3 cells, as well as fMLP stimulated increases in free cytosolic calcium in human PMNs. Furthermore, it is also able to block the release of the neutral protease elastase from stimulated neutrophils. The effects of AGN 190383 on arachidonic acid metabolism and leukocyte function may account, in part, for its anti-inflammatory activity in vivo.

Purpose AMP-activated protein kinase (AMPK) has been suggested to be a novel signaling pathway in regulating inflammation. The role of AMPK in retinal pigment epithelial cell inflammatory response is addressed using AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR). Methods Protein expression and activation of signaling molecules were detected by immunoblotting. Cytokines were determined by ELISA kits. AMPKalpha expression was knockdown by siRNAs. Results AICAR inhibited tumor necrosis factor (TNF)-alpha- or interleukin (IL)-1beta-induced production of IL-6, IL-8, and monocyte chemotactic protein (MCP)-1 and of intercellular adhesion molecule (ICAM)-1 expression in human RPE cells. The inhibitory effect on cytokine production and ICAM-1 expression persisted in the RPE cells in which AMPK was knocked down by AMPK siRNA. Moreover, an adenosine kinase inhibitor 5'-iodotubercidin, which effectively abolished AMPK activation caused by AICAR, did not reverse the anti-inflammatory effect of AICAR. In comparison, anti-inflammatory effects of AICAR were mimicked by adenosine but not inosine, the metabolites of AICAR. Finally, with the exception of TNF-alpha-induced IL-6 production, adenosine dialdehyde, an inhibitor of S-adenosylhomocysteine hydrolase, was found to block cytokine production and ICAM-1 expression. Conclusions Regardless of the ability of AICAR to activate AMPK, the inhibitory effects of AICAR on cytokine production and ICAM-1 expression were not associated with AMPK. The mechanism of AICAR inhibition may be attributed to the interference of adenosylmethionine-dependent methylation.