Analysis of gene evolution and metabolic pathways using the Candida Gene Order Browser

<p>Abstract</p> <p>Background</p> <p><it>Candida </it>species are the most common cause of opportunistic fungal infection worldwide. Recent sequencing efforts have provided a wealth of <it>Candida </it>genomic data. We have developed the <it>Candida </it>Gene Order Browser (CGOB), an online tool that aids comparative syntenic analyses of <it>Candida </it>species. CGOB incorporates all available <it>Candida </it>clade genome sequences including two <it>Candida albicans </it>isolates (SC5314 and WO-1) and 8 closely related species (<it>Candida dubliniensis</it>, <it>Candida tropicalis</it>, <it>Candida parapsilosis</it>, <it>Lodderomyces elongisporus</it>, <it>Debaryomyces hansenii</it>, <it>Pichia stipitis</it>, <it>Candida guilliermondii </it>and <it>Candida lusitaniae</it>). <it>Saccharomyces cerevisiae </it>is also included as a reference genome.</p> <p>Results</p> <p>CGOB assignments of homology were manually curated based on sequence similarity and synteny. In total CGOB includes 65617 genes arranged into 13625 homology columns. We have also generated improved <it>Candida </it>gene sets by merging/removing partial genes in each genome. Interrogation of CGOB revealed that the majority of tandemly duplicated genes are under strong purifying selection in all <it>Candida </it>species. We identified clusters of adjacent genes involved in the same metabolic pathways (such as catabolism of biotin, galactose and N-acetyl glucosamine) and we showed that some clusters are species or lineage-specific. We also identified one example of intron gain in <it>C. albicans</it>.</p> <p>Conclusions</p> <p>Our analysis provides an important resource that is now available for the <it>Candida </it>community. CGOB is available at <url>http://cgob.ucd.ie</url>.</p

Comparative genome analysis and gene finding in Candida species using CGOB.

The Candida Gene Order Browser (CGOB) was developed as a tool to visualize and analyze synteny relationships in multiple Candida species, and to provide an accurate, manually curated set of orthologous Candida genes for evolutionary analyses. Here, we describe major improvements to CGOB. The underlying structure of the database has been changed significantly. Genomic features are now based directly on genome annotations rather than on protein sequences, which allows non-protein features such as centromere locations in Candida albicans and tRNA genes in all species to be included. The data set has been expanded to 13 species, including genomes of pathogens (C. albicans, C. parapsilosis, C. tropicalis, and C. orthopsilosis), and those of xylose-degrading species with important biotechnological applications (C. tenuis, Scheffersomyces stipitis, and Spathaspora passalidarum). Updated annotations of C. parapsilosis, C. dubliniensis, and Debaryomyces hansenii have been incorporated. We discovered more than 1,500 previously unannotated genes among the 13 genomes, ranging in size from 29 to 3,850 amino acids. Poorly conserved and rapidly evolving genes were also identified. Re-analysis of the mating type loci of the xylose degraders suggests that C. tenuis is heterothallic, whereas both Spa. passalidarum and S. stipitis are homothallic. As well as hosting the browser, the CGOB website (http://cgob.ucd.ie) gives direct access to all the underlying genome annotations, sequences, and curated orthology data

Sexually dimorphic gene expression in bovine conceptuses at the initiation of implantation

In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus transcript abundance and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes that were differentially expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X-chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid of male compared to female conceptuses, while female conceptuses had higher transcript abundance of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in transcript abundance and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different transcriptomic response in the endometrium

Hyperglycemia acts in synergy with hypoxia to maintain the pro-inflammatory phenotype of macrophages

International audienceDiabetic foot ulcers (DFUs) are characterized by a chronic inflammation state which prevents cutaneous wound healing, and DFUs eventually lead to infection and leg amputation. Macrophages located in DFUs are locked in an pro-inflammatory phenotype. In this study, the effect of hyperglycemia and hypoxia on the macrophage phenotype was analyzed. For this purpose, a microarray was performed to study the gene expression profile of macrophages cultivated in a high glucose concentration. Hyperglycemia upregulated the expression of pro-inflammatory cytokines such as TNF-α, IL-1, IL-6, chemokines and down-regulated the expression of two receptors involved in phagocytosis (CD 36 and Class B scavenger type I receptors). In addition, eleven anti-apoptotic factors were upregulated whereas three pro-apoptotic genes were downregulated. Subsequently, the contribution of hypoxia and hyperglycemia to chronic inflammation and their potential synergistic effect was evaluated on activated THP-1 derived macrophages. A long term post activation effect (17 hours) was only observed on the upregulation of pro-inflammatory cytokines when hyp-oxia was combined with a high glucose concentration. In contrast, hyperglycemia and hyp-oxia did not have any effect on wound healing molecules such as TGF-β1. Taken together, the results show that hyperglycemia acts in synergy with hypoxia to maintain a chronic inflammation state in macrophages

Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus

Low back pain is a major cause of disability especially for people between 20 and 50 years of age. As a costly healthcare problem, it imposes a serious socio-economic burden. Current surgical therapies fail to replace the normal disc in facilitating spinal movements and absorbing load. The focus of regenerative medicine is on identifying biomarkers and signalling pathways to improve our understanding about cascades of disc degeneration and allow for the design of specific therapies. We hypothesized that comparing microarray profiles from degenerative and non-degenerative discs will lead to the identification of dysregulated signalling and pathophysiological targets. Microarray data sets were generated from human annulus fibrosus cells and analysed using IPA ingenuity pathway analysis. Gene expression values were validated by qRT-PCR, and respective proteins were identified by immunohistochemistry. Microarray analysis revealed 238 differentially expressed genes in the degenerative annulus fibrosus. Seventeen of the dysregulated molecular markers showed log2-fold changes greater than ±1.5. Various dysregulated cellular functions, including cell proliferation and inflammatory response, were identified. The most significant canonical pathway induced in degenerative annulus fibrosus was found to be the interferon pathway. This study indicates interferon-alpha signalling pathway activation with IFIT3 and IGFBP3 up-regulation, which may affect cellular function in human degenerative disc

Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection

Elevated expression of heat-shock proteins (HSPs) can benefit a microbial pathogen struggling to penetrate host defenses during infection, but at the same time might provide a crucial signal alerting the host immune system to its presence. To determine which of these effects predominate, we constructed a mutant strain of Mycobacterium tuberculosis that constitutively overexpresses Hsp70 proteins. Although the mutant was fully virulent in the initial stage of infection, it was significantly impaired in its ability to persist during the subsequent chronic phase. Induction of microbial genes encoding HSPs might provide a novel strategy to boost the immune response of individuals with latent tuberculosis infection

Characterisation and distribution of a cryptic Salmonella typhi plasmid pHCM2

pHCM2 is a 106 kbp cryptic plasmid harboured by Salmonella typhi CT18, originally isolated from a typhoid patient in Vietnam. The genome of S. typhi CT18, including pHCM2, has recently been completely sequenced and annotated. Bioinformatic analysis revealed that 57% of the coding sequences (CDSs) encoded on pHCM2 display over 97% DNA sequence identity to the virulence-associated plasmid of Yersinia pestis, pFra. pHCM2 encodes no obvious virulence-associated determinants or antibiotic resistance genes but does encode a wide array of putative genes directly related to DNA metabolism and replication. PCR analysis of a series of S. typhi isolates from Vietnam detected pHCM2-related DNA sequences in some S. typhi isolated before, but not after, 1994. Similar pHCM2-related sequences were also detected in S. typhi isolated from other regions of South East Asia and Pakistan but not elsewhere in the world

Inter-laboratory Comparison of Human Renal Proximal Tubule (HK-2) Transcriptome Alterations Due to Cyclosporine A Exposure and Medium Exhaustion

There is an acknowledged need to promote and further develop in vitro techniques in order to achieve the 36 goal of improved risk assessment of chemicals and pharmaceuticals to humans. The EU 6th framework 37 project ¿¿PREDICTOMICS¿ was established in order to contribute to the further development of in vitro 38 toxicology, with a particular focus on emerging techniques including toxicogenomics. DNA microarray 39 technology is being used more frequently in the in vitro field, however, only very few studies have 40 assessed the reproducibility of this technique with respect to in vitro toxicology. To this end we conducted an interlaboratory comparison to test the reproducibility of transcriptomic 42 changes induced by the immunosuppressive agent, Cyclosporine A (CsA) on the human renal proximal 43 tubular cell line, HK-2 cell. Four European laboratories took part in this study. Under standardised con- 44 ditions, each laboratory treated HK-2 cells with 5 lM CsA for 12 and 48 h. RNA was isolated and hybri- 45 dised to Affymetrix HGU-133 plus two arrays at three different sites. Analysis of the transcription profiles demonstrated that one laboratory clustered away from the other 47 laboratories, potentially due to an inclusion of a trypsinisation step by this laboratory. Once the genes 48 responsible for this separate clustering were removed all laboratories showed similar expression profiles. 49 There was a major impact of time since feed, due to medium exhaustion in the 48 h arrays compared to 50 the 12 h arrays, regardless of CsA treatment. Biological processes including general vesicle transport, 51 amino acid metabolism, amino acid transport and amino acid biosynthesis were over represented due 52 to time since feed, while cell cycle, DNA replication, mitosis and DNA metabolism were under-repre- 53 sented. CsA responsive genes were involved in cell cycle, the p53 pathway and Wnt signaling. Addition- 54 ally there was an overlap of differentially expressed genes due to CsA and medium exhaustion which is 55 most likely due to CsA induced glycolysis. The glucose deprivation dependent genes HspA5 and GP96 and 56 the Hsp70 chaperones DNAJ/Hsp40, DNAJ/HspB9, DNAJ/HspC3 DNAJ/HspC10 were induced by both CsA 57 and medium exhaustion. We conclude that under standardised conditions the application of Affymetrix DNA microarrays to 59 in vitro toxiciological studies are satisfactorily reproducible. However, confounding factors such as med- 60 ium exhaustion must also be considered in such analyses.JRC.I.3-In-vitro method