Aaron Ingham

The sheep genome illuminates biology of the rumen and lipid metabolism

A genome for ewe and ewe Sheep-specific genetic changes underlie differences in lipid metabolism between sheep and other mammals, and may have contributed to the production of wool. Jiang et al. sequenced the genome of two Texel sheep, a breed that produces high-value meat, milk, and wool. The genome information will provide an important resource for livestock production and aid in the understanding of mammalian evolution. Science, this issue p. 1168 A genomic analysis of sheep explains specializations in digestive system physiology and wool production. Sheep (Ovis aries) are a major source of meat, milk, and fiber in the form of wool and represent a distinct class of animals that have a specialized digestive organ, the rumen, that carries out the initial digestion of plant material. We have developed and analyzed a high-quality reference sheep genome and transcriptomes from 40 different tissues. We identified highly expressed genes encoding keratin cross-linking proteins associated with rumen evolution. We also identified genes involved in lipid metabolism that had been amplified and/or had altered tissue expression patterns. This may be in response to changes in the barrier lipids of the skin, an interaction between lipid metabolism and wool synthesis, and an increased role of volatile fatty acids in ruminants compared with nonruminant animals.

Recombinant production of antimicrobial peptides in heterologous microbial systems

The emergence and rapid horizontal spread of antibiotic‐resistant traits in bacteria of human and veterinary clinical significance has been a driving force in the search for new classes of antibiotics. Recent studies have shown that AMPs (antimicrobial peptides) potentially have a role in addressing this problem. These AMPs are produced naturally by a diverse array of organisms, including bacteria, plants, insects, fish and mammals. Given this diversity, researchers trying to perform comparative studies on AMPs are likely to encounter difficulties in obtaining workable quantities of peptide. Such studies are required for optimization of antimicrobial activity, product stability, mode of delivery and industrial‐scale production, and are vital if these peptides are ever to be brought to the market. Recombinant expression of AMPs is one hope for producing suitable amounts of diverse peptides. Here we review the literature regarding microbial heterologous expression systems for the production of recombinant AMPs.

Simultaneous identification of differential gene expression and connectivity in inflammation, adipogenesis and cancer

MOTIVATION Biological differences between classes are reflected in transcriptional changes which in turn affect the levels by which essential genes are individually expressed and collectively connected. The purpose of this communication is to introduce an analytical procedure to simultaneously identify genes that are differentially expressed (DE) as well as differentially connected (DC) in two or more classes of interest. RESULTS Our procedure is based on a two-step approach: First, mixed-model equations are applied to obtain the normalized expression levels of each gene in each class treatment. These normalized expressions form the basis to compute a measure of (possible) DE as well as the correlation structure existing among genes. Second, a two-component mixture of bi-variate distributions is fitted to identify the component that encapsulates those genes that are DE and/or DC. We demonstrate our approach using three distinct datasets including a human systemic inflammation oligonucleotide data; a spotted cDNA data dealing with bovine in vitro adipogenesis and SAGE database on cancerous and normal tissue samples.

Highly Conserved Alpha-Toxin Sequences of Avian Isolates of Clostridium perfringens

ABSTRACT Clostridium perfringens causes necrotic enteritis in chickens, and alpha-toxin has been suggested to be a key virulence determinant. Analysis of the alpha-toxin of 25 chicken-derived C. perfringens strains demonstrated high homology to mammal-derived strains rather than to the only avian-derived C. perfringens alpha-toxin sequence reported previously.

Selective induction of the Notch ligand Jagged‐1 in macrophages by soluble egg antigen from Schistosoma mansoni involves ERK signalling

Soluble egg antigen (SEA) from the helminth Schistosoma mansoni promotes T helper type 2 (Th2) responses by modulating antigen‐presenting cell function. The Jagged/Notch pathway has recently been implicated in driving Th2 development. We show here that SEA rapidly up‐regulated mRNA and protein expression of the Notch ligand Jagged‐1 in both murine bone marrow‐derived macrophages (BMMs) and human monocyte‐derived macrophages (HMDMs). Another potential Th2‐promoting factor, interleukin (IL)‐33, was not transcriptionally induced by SEA in BMMs. Up‐regulation of Jagged‐1 mRNA by SEA was also apparent in conventional dendritic cells (DCs), although the effect was less striking than in BMMs. Conversely, SEA‐pulsed DCs, but not BMMs, promoted IL‐4 production upon T‐cell activation, suggesting that Jagged‐1 induction alone is insufficient for instructing Th2 development. A comparison of the responses initiated in BMMs by SEA and the bacterial endotoxin lipopolysaccharide (LPS) revealed common activation of extracellular signal‐regulated kinase‐1/2 (ERK‐1/2) and p38 phosphorylation, as well as induction of Jagged‐1 mRNA. However, only LPS triggered IκB degradation, phosphorylation of c‐Jun N‐terminal kinase (Jnk) and signal transducer and activator of transcription 1 (Stat1) Tyr701, and IL‐33 and IL‐12p40 mRNA up‐regulation. Inducible gene expression was modified by the presence of the macrophage growth factor colony‐stimulating factor (CSF)‐1, which inhibited Jagged‐1 induction by SEA and LPS, but enhanced LPS‐induced IL‐12p40 expression. Unlike LPS, SEA robustly activated signalling in HEK293 cells expressing either Toll‐like receptor 2 (TLR2) or TLR4/MD2. Pharmacological inhibition of the ERK‐1/2 pathway impaired SEA‐ and LPS‐inducible Jagged‐1 expression in BMMs. Taken together, our data suggest that Jagged‐1 is an ERK‐dependent target of TLR signalling that has a macrophage‐specific function in the response to SEA.

A versatile system for the expression of nonmodified bacteriocins in Escherichia coli

Aims:  To develop a method and plasmid vectors suitable for expression of class II bacteriocins from Escherichia coli.

Mining tissue specificity, gene connectivity and disease association to reveal a set of genes that modify the action of disease causing genes

BackgroundThe tissue specificity of gene expression has been linked to a number of significant outcomes including level of expression, and differential rates of polymorphism, evolution and disease association. Recent studies have also shown the importance of exploring differential gene connectivity and sequence conservation in the identification of disease-associated genes. However, no study relates gene interactions with tissue specificity and disease association.MethodsWe adopted an a priori approach making as few assumptions as possible to analyse the interplay among gene-gene interactions with tissue specificity and its subsequent likelihood of association with disease. We mined three large datasets comprising expression data drawn from massively parallel signature sequencing across 32 tissues, describing a set of 55,606 true positive interactions for 7,197 genes, and microarray expression results generated during the profiling of systemic inflammation, from which 126,543 interactions among 7,090 genes were reported.ResultsAmongst the myriad of complex relationships identified between expression, disease, connectivity and tissue specificity, some interesting patterns emerged. These include elevated rates of expression and network connectivity in housekeeping and disease-associated tissue-specific genes. We found that disease-associated genes are more likely to show tissue specific expression and most frequently interact with other disease genes. Using the thresholds defined in these observations, we develop a guilt-by-association algorithm and discover a group of 112 non-disease annotated genes that predominantly interact with disease-associated genes, impacting on disease outcomes.ConclusionWe conclude that parameters such as tissue specificity and network connectivity can be used in combination to identify a group of genes, not previously confirmed as disease causing, that are involved in interactions with disease causing genes. Our guilt-by-association algorithm should be useful for the discovery of additional modifiers of genetic diseases, and more generally, for the ability to associate genes of unknown function to clusters of genes with defined functions allowing for novel biological inference that can be subsequently validated.

Nematode challenge induces differential expression of oxidant, antioxidant and mucous genes down the longitudinal axis of the sheep gut

To characterize the role of a range of oxidant, antioxidant and mucous‐related genes in the primary response to gastrointestinal nematodes, groups of genetically resistant sheep were challenged with either Haemonchus contortus or Trichostrongylus colubriformis and necropsied for retrieval of tissue at days 0, 3, 7, 14 and 21. To determine if the response was localized to the site of parasite infection, four different gut tissues were sampled: the abomasum, proximal and distal jejunum and ileum. Basal expression patterns of all candidate genes were determined using the day 0 (pre‐challenge) samples. A conserved innate response involving elevated expression of dual oxidase, glutathione peroxidase and trefoil factor was initiated within 3 days of challenge and extended out to 21 days. An increase in host gene expression levels at the preferred site of infection (the abomasum for H. contortus and the proximal jejunum for T. colubriformis) was also common to both nematodes. However, these increases were concomitant with reduced expression in other areas of the gut suggesting a compartmentalized response. Other aspects of the response were parasite‐specific, with T. colubriformis challenge inducing expression peaks at times corresponding to nematode life‐stage transitions.

Gene expression profiling of Hereford Shorthorn cattle following challenge with Boophilus microplus tick larvae

The ability of cattle to resist tick infestations is partly genetically determined. In order to better define the nature of Bos taurus resistance to the cattle tick Boophilus microplus, skin gene expression was studied using a cattle skin derived cDNA microarray. Expression profiles were determined in skin biopsies sampled from three highly tick resistant animals (HR) and two animals with lower tick resistance (LR) at time 0, immediately before challenge, and again 24 h after challenge. The analysis of the resulting expression data addressed two biological questions: first, for any animal exposed to ticks, which genes are differentially expressed in the 24 h following challenge; and second, which genes are differentially expressed between animals of high and low resistance at 24 h after challenge? In total, 214 genes were found to be differentially expressed in response to larval challenge across all the animals. Seventy-two genes were upregulated and 76 were downregulated at 24 h after challenge. Genes with significantly altered gene expression levels following tick infestation were predominantly keratin genes or mitochondrial genes, as well as odorant binding protein (OBP) and Bos taurus major allergen BDA20. In addition, we identified 66 genes with differential expression between HR and LR animals at 24 h. Of these, genes representing the extracellular matrix and immunoglobulin gene expression pathways were overrepresented. Three differentially expressed genes, OBP, Bos taurus major allergen BDA20 and dendritic cell protein HFL-B5 were further analysed by quantitative reverse transcription PCR (qRT-PCR). The qRT-PCR assay results closely mirrored the expression profiles found in the microarray experiment.

A genomics-informed, SNP association study reveals FBLN1 and FABP4 as contributing to resistance to fleece rot in Australian Merino sheep

BackgroundFleece rot (FR) and body-strike of Merino sheep by the sheep blowfly Lucilia cuprina are major problems for the Australian wool industry, causing significant losses as a result of increased management costs coupled with reduced wool productivity and quality. In addition to direct effects on fleece quality, fleece rot is a major predisposing factor to blowfly strike on the body of sheep. In order to investigate the genetic drivers of resistance to fleece rot, we constructed a combined ovine-bovine cDNA microarray of almost 12,000 probes including 6,125 skin expressed sequence tags and 5,760 anonymous clones obtained from skin subtracted libraries derived from fleece rot resistant and susceptible animals. This microarray platform was used to profile the gene expression changes between skin samples of six resistant and six susceptible animals taken immediately before, during and after FR induction. Mixed-model equations were employed to normalize the data and 155 genes were found to be differentially expressed (DE). Ten DE genes were selected for validation using real-time PCR on independent skin samples. The genomic regions of a further 5 DE genes were surveyed to identify single nucleotide polymorphisms (SNP) that were genotyped across three populations for their associations with fleece rot resistance.ResultsThe majority of the DE genes originated from the fleece rot subtracted libraries and over-representing gene ontology terms included defense response to bacterium and epidermis development, indicating a role of these processes in modulating the sheeps response to fleece rot. We focused on genes that contribute to the physical barrier function of skin, including keratins, collagens, fibulin and lipid proteins, to identify SNPs that were associated to fleece rot scores.ConclusionsWe identified FBLN1 (fibulin) and FABP4 (fatty acid binding protein 4) as key factors in sheeps resistance to fleece rot. Validation of these markers in other populations could lead to vital tests for marker assisted selection that will ultimately increase the natural fleece rot resistance of Merino sheep.