RNA-seq reveals the pan-transcriptomic impact of attenuating the gliotoxin self-protection mechanism in Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus produces a number of secondary metabolites, one of which, gliotoxin, has been shown to exhibit anti-fungal activity. Thus, A. fumigatus must be able to protect itself against gliotoxin. Indeed one of the genes in the gliotoxin biosynthetic gene cluster in A. fumigatus, gliT, is required for self-protection against the toxin- however the global self-protection mechanism deployed is unclear. RNA-seq was employed to identify genes differentially regulated upon exposure to gliotoxin in A. fumigatus wild-type and A. fumigatus ∆gliT, a strain that is hypersensitive to gliotoxin. RESULTS: Deletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated. Expression of 164 genes was differentially regulated (log2 fold change of 1.5) in A. fumigatus wild-type when exposed to gliotoxin, consisting of 101 genes with up-regulated expression and 63 genes with down-regulated expression. Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin. These consisted of 508 genes with up-regulated expression, and 1192 genes with down-regulated expression. Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism. Specifically, genes involved in gliotoxin biosynthesis, helvolic acid biosynthesis, siderophore-iron transport genes and also nitrogen metabolism genes and ribosome biogenesis genes underwent altered expression. It was confirmed that gliotoxin biosynthesis is induced upon exposure to exogenous gliotoxin, production of unrelated secondary metabolites is attenuated in A. fumigatus ∆gliT, while quantitative proteomic analysis confirmed disrupted translation in A. fumigatus ∆gliT challenged with exogenous gliotoxin. CONCLUSIONS: This study presents the first global investigation of the transcriptional response to exogenous gliotoxin in A. fumigatus wild-type and the hyper-sensitive strain, ∆gliT. Our data highlight the global and extensive affects of exogenous gliotoxin on a sensitive strain devoid of a self-protection mechanism and infer that GliT functionality is required for the optimal biosynthesis of selected secondary metabolites in A. fumigatus

Genome-wide genetic screening with chemically mutagenized haploid embryonic stem cells.

In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mutagenesis and next-generation sequencing, providing a new tool to explore mammalian genetic interactions.Research in the S.P.J. laboratory is funded by Cancer Research UK (CRUK; programme grant C6/A11224), the European Research Council and the European Community Seventh Framework Programme (grant agreement no. HEALTH-F2-2010-259893; DDResponse). Core funding is provided by Cancer Research UK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives salary from the University of Cambridge, supplemented by CRUK. J.V.F. was funded by Cancer Research UK programme grant C6/A11224 and the Ataxia Telangiectasia Society. J.C. was funded by Cancer Research UK programme grant C6/A11224. D.J.A. is supported by CRUK. Research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) and ERC grant agreement no. (311166)

From brand congruence to the ‘virtuous circle’: branding and the commercialization of public service broadcasting

In debates about the commercialization of public service broadcasting little attention has been paid to the ways in which the public might experience the commercial and public service activities of public service broadcasters and the impact that this may have on attitudes towards public service broadcasting. This is despite the fact that public service broadcasters increasingly depend on public support for their continued survival. Using the case study of the BBC, this article examines the ways in which the corporation has adopted strategic brand management to negotiate the relationship between its commercial and public service activities. Focusing on specific examples of the BBC’s commercial and public services, the article reveals a tension between the corporation’s attempts to ensure that all activities support its public purposes and its need to ensure separation between public and commercial work. Focusing on the consumer’s experience of the BBC brand, the article argues that rather than seeing commercial activity and public service broadcasting as inherently contradictory, we should be looking at the ways in which public service broadcasters can better communicate the relationship between their commercial and public service activities while continuing to argue for the social and cultural value of publicly funded broadcasting

Calcisponges have a ParaHox gene and dynamic expression of dispersed NK homeobox genes

This study was funded by the Sars Centre core budget to M. Adamska. Sequencing was performed at the Norwegian High Throughput Sequencing Centre funded by the Norwegian Research Council. O.M.R. and D.E.K.F. acknowledge support from the BBSRC and the School of Biology, University of St Andrews.Sponges are simple animals with few cell types, but their genomes paradoxically contain a wide variety of developmental transcription factors1,2,3,4, including homeobox genes belonging to the Antennapedia (ANTP) class5,6, which in bilaterians encompass Hox, ParaHox and NK genes. In the genome of the demosponge Amphimedon queenslandica, no Hox or ParaHox genes are present, but NK genes are linked in a tight cluster similar to the NK clusters of bilaterians5. It has been proposed that Hox and ParaHox genes originated from NK cluster genes after divergence of sponges from the lineage leading to cnidarians and bilaterians5,7. On the other hand, synteny analysis lends support to the notion that the absence of Hox and ParaHox genes in Amphimedon is a result of secondary loss (the ghost locus hypothesis)8. Here we analysed complete suites of ANTP-class homeoboxes in two calcareous sponges, Sycon ciliatum and Leucosolenia complicata. Our phylogenetic analyses demonstrate that these calcisponges possess orthologues of bilaterian NK genes (Hex, Hmx and Msx), a varying number of additional NK genes and one ParaHox gene, Cdx. Despite the generation of scaffolds spanning multiple genes, we find no evidence of clustering of Sycon NK genes. All Sycon ANTP-class genes are developmentally expressed, with patterns suggesting their involvement in cell type specification in embryos and adults, metamorphosis and body plan patterning. These results demonstrate that ParaHox genes predate the origin of sponges, thus confirming the ghost locus hypothesis8, and highlight the need to analyse the genomes of multiple sponge lineages to obtain a complete picture of the ancestral composition of the first animal genome.PostprintPeer reviewe

MPI-PHYLIP: Parallelizing Computationally Intensive Phylogenetic Analysis Routines for the Analysis of Large Protein Families

Background: Phylogenetic study of protein sequences provides unique and valuable insights into the molecular and genetic basis of important medical and epidemiological problems as well as insights about the origins and development of physiological features in present day organisms. Consensus phylogenies based on the bootstrap and other resampling methods play a crucial part in analyzing the robustness of the trees produced for these analyses. Methodology: Our focus was to increase the number of bootstrap replications that can be performed on large protein datasets using the maximum parsimony, distance matrix, and maximum likelihood methods. We have modified the PHYLIP package using MPI to enable large-scale phylogenetic study of protein sequences, using a statistically robust number of bootstrapped datasets, to be performed in a moderate amount of time. This paper discusses the methodology used to parallelize the PHYLIP programs and reports the performance of the parallel PHYLIP programs that are relevant to the study of protein evolution on several protein datasets. Conclusions: Calculations that currently take a few days on a state of the art desktop workstation are reduced to calculations that can be performed over lunchtime on a modern parallel computer. Of the three protein methods tested, the maximum likelihood method scales the best, followed by the distance method, and then the maximum parsimony method. However, the maximum likelihood method requires significant memory resources, which limits its application to mor

Position and sequence conservation in Amniota of polymorphic enhancer HS1.2 within the palindrome of IgH 3'Regulatory Region

<p>Abstract</p> <p>Background</p> <p>The Immunoglobulin heavy chain (IgH) 3' Regulatory Region (3'RR), located at the 3' of the constant alpha gene, plays a crucial role in immunoglobulin production. In humans, there are 2 copies of the 3'RR, each composed of 4 main elements: 3 enhancers and a 20 bp tandem repeat. The single mouse 3'RR differs from the two human ones for the presence of 4 more regulative elements with the double copy of one enhancer at the border of a palindromic region.</p> <p>Results</p> <p>We compared the 3'RR organization in genomes of vertebrates to depict the evolutionary history of the region and highlight its shared features. We found that in the 8 species in which the whole region was included in a fully assembled contig (mouse, rat, dog, rabbit, panda, orangutan, chimpanzee, and human), the shared elements showed synteny and a highly conserved sequence, thus suggesting a strong evolutionary constraint. In these species, the wide 3'RR (~30 kb in human) bears a large palindromic sequence, consisting in two ~3 kb complementary branches spaced by a ~3 kb sequence always including the HS1.2 enhancer. In mouse and rat, HS3 is involved by the palindrome so that one copy of the enhancer is present on each side. A second relevant feature of our present work concerns human polymorphism of the HS1.2 enhancer, associated to immune diseases in our species. We detected a similar polymorphism in all the studied Catarrhini (a primate parvorder). The polymorphism consists of multiple copies of a 40 bp element up to 12 in chimpanzees, 8 in baboons, 6 in macaque, 5 in gibbons, 4 in humans and orangutan, separated by stretches of Cytosine. We show specific binding of this element to nuclear factors.</p> <p>Conclusions</p> <p>The nucleotide sequence of the palindrome is not conserved among evolutionary distant species, suggesting pressures for the maintenance of two self-matching regions driving a three-dimensional structure despite of the inter-specific divergence at sequence level. The information about the conservation of the palindromic structure and the settling in primates of the polymorphic feature of HS1.2 show the relevance of these structures in the control and modulation of the Ig production through the formation of possible three-dimensional structures.</p

A broad distribution of the alternative oxidase in microsporidian parasites

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of ironsulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosome

Rapid genotype imputation from sequence without reference panels

Inexpensive genotyping methods are essential for genetic studies requiring large sample sizes. In human studies, array-based microarrays and high-density haplotype reference panels allow efficient genotype imputation for this purpose. However, these resources are typically unavailable in non-human settings. Here we describe a method (STITCH) for imputation based only on sequencing read data, without requiring additional reference panels or array data. We demonstrate its applicability even in settings of extremely low sequencing coverage, by accurately imputing 5.7 million SNPs at a mean r(2) value of 0.98 in 2,073 outbred laboratory mice (0.15× sequencing coverage). In a sample of 11,670 Han Chinese (1.7× coverage), we achieve accuracy similar to that of alternative approaches that require a reference panel, demonstrating that our approach can work for genetically diverse populations. Our method enables straightforward progression from low-coverage sequence to imputed genotypes, overcoming barriers that at present restrict the application of genome-wide association study technology outside humans

Angiogenic Activity of Sera from Pulmonary Tuberculosis Patients in Relation to IL-12p40 and TNFα Serum Levels

The role of angiogenesis in the pathogenesis of tuberculosis (TB) is not clear. The aim of this study was to examine the effect of sera from TB patients on angiogenesis induced by different subsets of normal human mononuclear cells (MNC) in relation to IL-12p40 and TNFα serum levels. Serum samples from 36 pulmonary TB patients and from 22 healthy volunteers were evaluated. To assess angiogenic reaction the leukocytes-induced angiogenesis test according to Sidky and Auerbach was performed. IL-12p40 and TNFα serum levels were evaluated by ELISA. Sera from TB patients significantly stimulated angiogenic activity of MNC compared to sera from healthy donors and PBS (p < 0.001). The number of microvessels formed after injection of lymphocytes preincubated with sera from TB patients was significantly lower compared to the number of microvessels created after injection of MNC preincubated with the same sera (p < 0.016). However, the number of microvessels created after the injection of lymphocytes preincubated with sera from healthy donors or with PBS alone was significantly higher (p < 0.017). The mean levels of IL-12p40 and TNFα were significantly elevated in sera from TB patients compared to healthy donors. We observed a correlation between angiogenic activity of sera from TB patients and IL-12p40 and TNFα serum levels (p < 0.01). Sera from TB patients constitute a source of mediators that participate in angiogenesis and prime monocytes for production of proangiogenic factors. The main proangiogenic effect of TB patients’ sera is mediated by macrophages/monocytes. TNFα and IL-12p40 may indirectly stimulate angiogenesis in TB

Ex Vivo Cytokine mRNA Levels Correlate with Changing Clinical Status of Ethiopian TB Patients and their Contacts Over Time

There is an increasing body of evidence which suggests that IL-4 plays a role in the pathogenesis of TB, but a general consensus on its role remains elusive. We have previously published data from a cohort of Ethiopian TB patients, their contacts, and community controls suggesting that enhanced IL-4 production is associated with infection with M. tuberculosis, rather than overt disease and that long-term protection in infected community controls is associated with co-production of the IL-4 antagonist IL-4d2, alongside elevated IL-4. Here, for the first time, we compare data on expression of IFN-γ, IL-4 and IL-4δ2 over time in TB patients and their household contacts. During the follow-up period, the TB patients completed therapy and ceased to display TB-like symptoms. This correlated with a decrease in the relative amount of IL-4 expressed. Over the same period, the clinical status of some of their contacts also changed, with a number developing TB-like symptoms or clinically apparent TB. IL-4 expression was disproportionately increased in this group. The findings support the hypothesis that elevated IL-4 production is generally associated with infection, but that TB disease is associated with a relatively increased expression of IL-4 compared to IFN-γ and IL-4δ2. However, the data also suggest that there are no clear-cut differences between groups: the immune response over time appears to include changes in the expression of IFN-γ, IL-4 and IL-4δ2, and it is the relative, not absolute levels of cytokine expression that are characteristic of clinical status