Complete Genome Sequence of Clostridium estertheticum DSM 8809, a Microbe Identified in Spoiled Vacuum Packed Beef

peer-reviewedBlown pack spoilage (BPS) is a major issue for the beef industry. Etiological agents of BPS involve members of a group of Clostridium species, including Clostridium estertheticum which has the ability to produce gas, mostly carbon dioxide, under anaerobic psychotrophic growth conditions. This spore-forming bacterium grows slowly under laboratory conditions, and it can take up to 3 months to produce a workable culture. These characteristics have limited the study of this commercially challenging bacterium. Consequently information on this bacterium is limited and no effective controls are currently available to confidently detect and manage this production risk. In this study the complete genome of C. estertheticum DSM 8809 was determined by SMRT® sequencing. The genome consists of a circular chromosome of 4.7 Mbp along with a single plasmid carrying a potential tellurite resistance gene tehB and a Tn3-like resolvase-encoding gene tnpR. The genome sequence was searched for central metabolic pathways that would support its biochemical profile and several enzymes contributing to this phenotype were identified. Several putative antibiotic/biocide/metal resistance-encoding genes and virulence factors were also identified in the genome, a feature that requires further research. The availability of the genome sequence will provide a basic blueprint from which to develop valuable biomarkers that could support and improve the detection and control of this bacterium along the beef production chain

Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome

peer-reviewedBackground Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Results Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p < 0.05), followed by muscle (601 genes) and adipose (16 genes). Results from modified GSEA showed that the high-CLA beef diet affected diverse biological processes across the three tissues, and that the majority of pathway changes reached significance only with the bi-directional test. Combining the liver tissue microarray results with plasma marker data revealed 110 CLA-sensitive genes showing strong canonical correlation with one or more plasma markers of metabolic health, and 9 significantly overrepresented pathways among this set; each of these pathways was also significantly changed by the high-CLA diet. Closer inspection of two of these pathways - selenoamino acid metabolism and steroid biosynthesis - illustrated clear diet-sensitive changes in constituent genes, as well as strong correlations between gene expression and plasma markers of metabolic syndrome independent of the dietary effect. Conclusion Bi-directional gene set enrichment analysis more accurately reflects dynamic regulatory behaviour in biochemical pathways, and as such highlighted biologically relevant changes that were not detected using a traditional approach. In such cases where transcriptomic response to treatment is exceptionally large, canonical correlation analysis in conjunction with Fisher's exact test highlights the subset of pathways showing strongest correlation with the clinical markers of interest. In this case, we have identified selenoamino acid metabolism and steroid biosynthesis as key pathways mediating the observed relationship between metabolic health and high-CLA beef. These results indicate that this type of analysis has the potential to generate novel transcriptome-based biomarkers of disease.Department of Agriculture and Food, Ireland - Food Institutional Research Measure (project no. 5254); IRCSET postgraduate scholarship scheme (MJM); Science Foundation Ireland Principal Investigator Programme (HMR) Programme

Adaptation to AI therapy in breast cancer can induce dynamic alterations in ER activity resulting in estrogen independent metastatic tumours

PURPOSE: Acquired resistance to aromatase inhibitor therapy is a major clinical problem in the treatment of breast cancer. The detailed mechanisms of how tumour cells develop this resistance remain unclear. Here, the adapted function of ER to an estrogen-depleted environment following AI treatment is reported. EXPERIMENTAL DESIGN: Global ER-ChIPseq analysis of AI resistant cells identified steroid-independent ER target genes. Matched patient tumour samples, collected before and after AI treatment, were used to assess ER activity. RESULTS: Maintained ER activity was observed in patient tumours following neoadjuvant AI therapy. Genome-wide ER-DNA binding analysis in AI resistant cell lines identified a subset of classic ligand dependent ER target genes which develop steroid independence. Kaplan Meier analysis revealed a significant association between tumours which fail to decrease this steroid independent ER target gene set in response to neoadjuvant AI therapy, and poor disease-free and overall survival (n=72 matched patient tumour samples, p=0.00339 and 0.00155 respectively). The adaptive ER response to AI treatment was highlighted by the ER/AIB1 target gene, early growth response 3 (EGR3). Elevated levels of EGR3 were detected in endocrine resistant local disease recurrent patient tumours in comparison to matched primary tissue. However, evidence from distant metastatic tumours demonstrates that the ER signalling network may undergo further adaptations with disease progression as estrogen-independent ER target gene expression is routinely lost in established metastatic tumours. CONCLUSIONS: Overall, these data provide evidence of a dynamic ER response to endocrine treatment which may provide vital clues for overcoming the clinical issue of therapy resistance

High-Throughput Proteomics Detection of Novel Splice Isoforms in Human Platelets

Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS/MS datasets

Complete Genome Sequence of Clostridium estertheticum DSM 8809, a Microbe Identified in Spoiled Vacuum Packed Beef

Blown pack spoilage (BPS) is a major issue for the beef industry. Etiological agents of BPS involve members of a group of Clostridium species, including Clostridium estertheticum which has the ability to produce gas, mostly carbon dioxide, under anaerobic psychotrophic growth conditions. This spore-forming bacterium grows slowly under laboratory conditions, and it can take up to 3 months to produce a workable culture. These characteristics have limited the study of this commercially challenging bacterium. Consequently information on this bacterium is limited and no effective controls are currently available to confidently detect and manage this production risk. In this study the complete genome of C. estertheticum DSM 8809 was determined by SMRT® sequencing. The genome consists of a circular chromosome of 4.7 Mbp along with a single plasmid carrying a potential tellurite resistance gene tehB and a Tn3-like resolvase-encoding gene tnpR. The genome sequence was searched for central metabolic pathways that would support its biochemical profile and several enzymes contributing to this phenotype were identified. Several putative antibiotic/biocide/metal resistance-encoding genes and virulence factors were also identified in the genome, a feature that requires further research. The availability of the genome sequence will provide a basic blueprint from which to develop valuable biomarkers that could support and improve the detection and control of this bacterium along the beef production chain

SETD1A methyltransferase is physically and functionally linked to the DNA damage repair protein RAD18.

SETD1A is a SET domain-containing methyltransferase involved in epigenetic regulation of transcription. It is the main catalytic component of a multiprotein complex that methylates lysine 4 of histone H3, a histone mark associated with gene activation. In humans, six related protein complexes with partly nonredundant cellular functions share several protein subunits but are distinguished by unique catalytic SET-domain proteins. We surveyed physical interactions of the SETD1A-complex using endogenous immunoprecipitation followed by label-free quantitative proteomics on three subunits: SETD1A, RBBP5, and ASH2L. Surprisingly, SETD1A, but not RBBP5 or ASH2L, was found to interact with the DNA damage repair protein RAD18. Reciprocal RAD18 immunoprecipitation experiments confirmed the interaction with SETD1A, whereas size exclusion and protein network analysis suggested an interaction independent of the main SETD1A complex. We found evidence of SETD1A and RAD18 influence on mutual gene expression levels. Further, knockdown of the genes individually showed a DNA damage repair phenotype, whereas simultaneous knockdown resulted in an epistatic effect. This adds to a growing body of work linking epigenetic enzymes to processes involved in genome stability

Oviduct-Embryo Interactions in Cattle: Two-Way Traffic or a One-Way Street?1

This study examined the effect of the presence of single or multiple embryos on the transcriptome of the bovine oviduct. In experiment 1, cyclic (nonbred, n = 6) and pregnant (artificially inseminated, n = 11) heifers were slaughtered on Day 3 after estrus, and the ampulla and isthmic regions of the oviduct ipsilateral to the corpus luteum were separately flushed. Oviductal epithelial cells from the isthmus region, in which all oocytes/embryos were located, were snap-frozen for microarray analysis. In experiment 2, heifers were divided into cyclic (nonbred, n = 6) or pregnant (multiple embryo transfer, n = 10) groups. In vitro-produced presumptive zygotes were transferred endoscopically to the ipsilateral oviduct on Day 1.5 postestrus (n = 50 zygotes/heifer). Heifers were slaughtered on Day 3, and oviductal isthmus epithelial cells were recovered for RNA sequencing. Microarray analysis in experiment 1 failed to detect any difference in the transcriptome of the oviductal isthmus induced by the presence of a single embryo. In experiment 2, following multiple embryo transfer, RNA sequencing revealed 278 differentially expressed genes, of which 123 were upregulated and 155 were down-regulated in pregnant heifers. Most of the down-regulated genes were related to immune function. In conclusion, the presence of multiple embryos in the oviduct resulted in the detection of differentially expressed genes in the oviductal isthmus; failure to detect changes in the oviduct transcriptome in the presence of a single embryo may be due to the effect being local and undetectable under the conditions of this study. © 2015 by the Society for the Study of Reproduction, Inc

Analysis of the Hypervariable Region of the Salmonella enterica Genome Associated with tRNA(leuX)

The divergence of Salmonella enterica and Escherichia coli is estimated to have occurred approximately 140 million years ago. Despite this evolutionary distance, the genomes of these two species still share extensive synteny and homology. However, there are significant differences between the two species in terms of genes putatively acquired via various horizontal transfer events. Here we report on the composition and distribution across the Salmonella genus of a chromosomal region designated SPI-10 in Salmonella enterica serovar Typhi and located adjacent to tRNA(leuX). We find that across the Salmonella genus the tRNA(leuX) region is a hypervariable hot spot for horizontal gene transfer; different isolates from the same S. enterica serovar can exhibit significant variation in this region. Many P4 phage, plasmid, and transposable element-associated genes are found adjacent to tRNA(leuX) in both Salmonella and E. coli, suggesting that these mobile genetic elements have played a major role in driving the variability of this region