The Role of Genomic Data in the Discovery, Annotation and Evolutionary Interpretation of the Interferon-Lambda Family

Type-I interferons, type-II interferons, and the IL-10 family are helical cytokines with similar three-dimensional folds. However, their homologous relationship is difficult to detect on the basis of sequence alone. We have previously described the discovery of the human type-III interferons (IFN lambda-1, -2, -3 or IL-29, IL-28A, IL-28B), which required a combination of manual and computational techniques applied to predicted protein sequences.Here we describe how the use of gene structure analysis and comparative genomics enabled a more extensive understanding of these genes early in the discovery process. More recently, additional mammalian genome sequences have shown that there are between one and potentially nine copies of interferon lambda genes in each genome, and that several species have single exon versions of the interferon lambda gene.The variable number of single exon type-I interferons in mammals, along with recently identified genes in zebrafish homologous to interferons allows a story of interferon evolution to be proposed. This model suggests that the gene duplications and single exon retrotransposons of mammalian type-III interferons are positively selected for within a genome. These characteristics are also shared with the fish interferons and could be responsible for the generation of the IL10 family and also the single exon type-I interferons

A comparative study of fragment screening methods on the p38α kinase: new methods, new insights

The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore(™) T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE(™)) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10822-011-9454-9) contains supplementary material, which is available to authorized users

Projected Changes to Growth and Mortality of Hawaiian Corals over the Next 100 Years

BACKGROUND: Recent reviews suggest that the warming and acidification of ocean surface waters predicated by most accepted climate projections will lead to mass mortality and declining calcification rates of reef-building corals. This study investigates the use of modeling techniques to quantitatively examine rates of coral cover change due to these effects. METHODOLOGY/PRINCIPAL FINDINGS: Broad-scale probabilities of change in shallow-water scleractinian coral cover in the Hawaiian Archipelago for years 2000-2099 A.D. were calculated assuming a single middle-of-the-road greenhouse gas emissions scenario. These projections were based on ensemble calculations of a growth and mortality model that used sea surface temperature (SST), atmospheric carbon dioxide (CO(2)), observed coral growth (calcification) rates, and observed mortality linked to mass coral bleaching episodes as inputs. SST and CO(2) predictions were derived from the World Climate Research Programme (WCRP) multi-model dataset, statistically downscaled with historical data. CONCLUSIONS/SIGNIFICANCE: The model calculations illustrate a practical approach to systematic evaluation of climate change effects on corals, and also show the effect of uncertainties in current climate predictions and in coral adaptation capabilities on estimated changes in coral cover. Despite these large uncertainties, this analysis quantitatively illustrates that a large decline in coral cover is highly likely in the 21(st) Century, but that there are significant spatial and temporal variances in outcomes, even under a single climate change scenario

The reliability, validity and sensitivity of a novel soccer-specific reactive repeated-sprint test (RRST).

PURPOSE: The aim of this study was to determine the reliability, validity and sensitivity of a reactive repeated-sprint test (RRST). METHODS: Elite (n = 72) and sub-elite male (n = 87) and elite female soccer players (n = 12) completed the RRST at set times during a season. Total distance timed was 30 m and the RRST performance measure was the total time (s) across eight repetitions. Competitive match running performance was measured using GPS and high-intensity running quantified (≥ 19.8 km h(-1)). RESULTS: Test-retest coefficient of variation in elite U16 and sub-elite U19 players was 0.71 and 0.84 %, respectively. Elite U18 players' RRST performances were better (P < 0.01) than elite U16, sub-elite U16, U18, U19 and elite senior female players (58.25 ± 1.34 vs 59.97 ± 1.64, 61.42 ± 2.25, 61.66 ± 1.70, 61.02 ± 2.31 and 63.88 ± 1.46 s; ES 0.6-1.9). For elite U18 players, RRST performances for central defenders (59.84 ± 1.35 s) were lower (P < 0.05) than full backs (57.85 ± 0.77 s), but not attackers (58.17 ± 1.73 s) or central and wide midfielders (58.55 ± 1.08 and 58.58 ± 1.89 s; ES 0.7-1.4). Elite U16 players demonstrated lower (P < 0.01) RRST performances during the preparation period versus the start, middle and end of season periods (61.13 ± 1.53 vs 59.51 ± 1.39, 59.25 ± 1.42 and 59.20 ± 1.57 s; ES 1.0-1.1). Very large magnitude correlations (P < 0.01) were observed between RRST performance and high-intensity running in the most intense 5-min period of a match for both elite and sub-elite U18 players (r = -0.71 and -0.74), with the best time of the RRST also correlating with the arrowhead agility test for elite U16 and U18 players (r = 0.84 and 0.75). CONCLUSION: The data demonstrate that the RRST is a reliable and valid test that distinguishes between performance across standard, position and seasonal period

IFN-Lambda (IFN-λ) Is Expressed in a Tissue-Dependent Fashion and Primarily Acts on Epithelial Cells In Vivo

Interferons (IFN) exert antiviral, immunomodulatory and cytostatic activities. IFN-α/β (type I IFN) and IFN-λ (type III IFN) bind distinct receptors, but regulate similar sets of genes and exhibit strikingly similar biological activities. We analyzed to what extent the IFN-α/β and IFN-λ systems overlap in vivo in terms of expression and response. We observed a certain degree of tissue specificity in the production of IFN-λ. In the brain, IFN-α/β was readily produced after infection with various RNA viruses, whereas expression of IFN-λ was low in this organ. In the liver, virus infection induced the expression of both IFN-α/β and IFN-λ genes. Plasmid electrotransfer-mediated in vivo expression of individual IFN genes allowed the tissue and cell specificities of the responses to systemic IFN-α/β and IFN-λ to be compared. The response to IFN-λ correlated with expression of the α subunit of the IFN-λ receptor (IL-28Rα). The IFN-λ response was prominent in the stomach, intestine and lungs, but very low in the central nervous system and spleen. At the cellular level, the response to IFN-λ in kidney and brain was restricted to epithelial cells. In contrast, the response to IFN-α/β was observed in various cell types in these organs, and was most prominent in endothelial cells. Thus, the IFN-λ system probably evolved to specifically protect epithelia. IFN-λ might contribute to the prevention of viral invasion through skin and mucosal surfaces

Effects of Blood Products on Inflammatory Response in Endothelial Cells In Vitro

BACKGROUND: Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products. METHODS: The inflammatory response from pre-activated (endotoxin-stimulated) and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC), platelet concentrates (PC) and fresh frozen plasma (FFP) was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated. RESULTS: Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product. CONCLUSION: Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells

Identification of multiple integrin β1 homologs in zebrafish (Danio rerio)

BACKGROUND: Integrins comprise a large family of α,β heterodimeric, transmembrane cell adhesion receptors that mediate diverse essential biological functions. Higher vertebrates possess a single β1 gene, and the β1 subunit associates with a large number of α subunits to form the major class of extracellular matrix (ECM) receptors. Despite the fact that the zebrafish (Danio rerio) is a rapidly emerging model organism of choice for developmental biology and for models of human disease, little is currently known about β1 integrin sequences and functions in this organism. RESULTS: Using RT-PCR, complete coding sequences of zebrafish β1 paralogs were obtained from zebrafish embryos or adult tissues. The results show that zebrafish possess two β1 paralogs (β1–1 and β1–2) that have a high degree of identity to other vertebrate β1 subunits. In addition, a third, more divergent, β1 paralog is present (β1–3), which may have altered ligand-binding properties. Zebrafish also have other divergent β1-like transcripts, which are C-terminally truncated forms lacking the transmembrane and cytoplasmic domains. Together with β1–3 these truncated forms comprise a novel group of β1 paralogs, all of which have a mutation in the ADMIDAS cation-binding site. Phylogenetic and genomic analyses indicate that the duplication that gave rise to β1–1 and β1–2 occurred after the divergence of the tetrapod and fish lineages, while a subsequent duplication of the ancestor of β1–2 may have given rise to β1–3 and an ancestral truncated paralog. A very recent tandem duplication of the truncated β1 paralogs appears to have taken place. The different zebrafish β1 paralogs have varied patterns of temporal expression during development. β1–1 and β1–2 are ubiquitously expressed in adult tissues, whereas the other β1 paralogs generally show more restricted patterns of expression. CONCLUSION: Zebrafish have a large set of integrin β1 paralogs. β1–1 and β1–2 may share the roles of the solitary β1 subunit found in other vertebrates, whereas β1–3 and the truncated β1 paralogs may have acquired novel functions

Unexpected differential metabolic responses of Campylobacter jejuni to the abundant presence of glutamate and fucose

Introduction: Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity. Objectives: For instance, the C. jejuni 11168 strain can utilize both l-fucose and l-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of l-fucose and l-glutamate in the growth medium. Methods: To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h. Results: This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon l-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy. Conclusions: Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon l-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.</p

Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the “RAF paradox”) may have the same effect. BRAF was upregulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a “RAF paradox” effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the “RAF paradox”. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function

Candida glabrata : a review of its features and resistance

Candida species belong to the normal microbiota of the oral cavity and gastrointestinal and vaginal tracts, and are responsible for several clinical manifestations, from mucocutaneous overgrowth to bloodstream infections. Once believed to be non-pathogenic, Candida glabrata was rapidly blamable for many human diseases. Year after year, these pathological circumstances are more recurrent and problematic to treat, especially when patients reveal any level of immunosuppression. These difficulties arise from the capacity of C. glabrata to form biofilms and also from its high resistance to traditional antifungal therapies. Thus, this review intends to present an excerpt of the biology, epidemiology, and pathology of C. glabrata, and detail an approach to its resistance mechanisms based on studies carried out up to the present.The authors are grateful to strategic project PTDC/SAU-MIC/119069/2010 for the financial support to the research center and for Celia F. Rodrigues' grant