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peer-reviewed Bovine mastitis is an inflammation-driven disease of the bovine mammary gland that costs the global dairy industry several billion dollars per annum. Because disease susceptibility is a multi-factorial complex phenotype, an integrative biology approach is required to dissect the molecular networks involved. Here, we report such an approach, using next generation sequencing combined with advanced network and pathway biology methods to simultaneously profile mRNA and miRNA expression at multiple time-points (0, 12, 24, 36 and 48h) in both milk and blood FACS-isolated CD14+ monocytes from animals infected in vivo with Streptococcus uberis. More than 3,700 differentially expressed (DE) genes were identified in milk-isolated monocytes (MIMs), a key immune cell recruited to the site of infection during mastitis. Up-regulated genes were significantly enriched for inflammatory pathways, while down-regulated genes were enriched for non-glycolytic metabolic pathways. Monocyte transcriptional changes in the blood, however, were more subtle but highlighted the impact of this infection systemically. Genes up-regulated in blood-isolated-monocytes (BIMs) showed a significant association with interferon and chemokine signalling. Furthermore, twenty-six miRNAs were differentially expressed in MIMs and three in BIMs. Pathway analysis revealed that predicted targets of down-regulated miRNAs were highly enriched for roles in innate immunity (FDR < 3.4E-8) in particular TLR signalling, while up-regulated miRNAs preferentially targeted genes involved in metabolism. We conclude that during S. uberis infection miRNAs are key amplifiers of monocyte inflammatory response networks and repressors of several metabolic pathways. This study was funded in part by Teagasc RMIS 6018 and United States Department of Agriculture ARS funding 3625-32000-102-00. NL is supported by a Teagasc Walsh Fellowship.

Leaves of the Solanum glaucophyllum (Sg) plant, indigenous to South America, have long been known for their calcinogenic toxicity in ruminant animals. It was determined the leaves contained glycosidic derivatives of 1,25-dihydroxyvitamin D3 (1,25D3) and liberation of the free hormone by rumen bacterial populations elicited a hypercalcemic response. Our interest in the leaves is predicated on the concept that the glycoside forms of 1,25D3 would target release of the active hormone in the lower gut of non-ruminant mammals. This would provide a means of delivering 1,25D3 directly to the colon, where the hormone has been shown to have beneficial effects in models of inflammatory bowel disease (IBD) and colon cancer. We fed mice for 10 days with variable amounts of Sg leaf. Feeding 7-333μg leaf/day produced no changes in plasma Ca(2+) and 1,25D3 concentrations, and only at ≥1000μg leaf/day did these values become significantly elevated compared to controls. Gene expression studies from colon tissue indicated a linear relationship between the amount of leaf consumed and expression of the Cyp24a1 gene. In contrast, Cyp24a1 gene expression in the duodenums and ileums of these mice was unchanged compared to controls. One of the major 1,25D3-glycosides was isolated from leaves following extraction and purification by Sep-Pak cartridges and HPLC fractionation. Ultraviolet absorbance was consistent with modification of the 1-hydroxyl group, and positive ion ESI mass spectrometry indicated a diglycoside of 1,25D3. 2-Dimensional NMR analyses were carried out and established the C1 proton of the A-ring was interacting with a C1' sugar proton, while the C3 proton of the A-ring was linked with a second C1' sugar proton. The structure of the isolated compound is therefore consistent with a β-linked 1,3-diglycoside of 1,25D3. Thus, Sg leaf administered to mice at up to 333 ug/day can elicit colon-specific enhancement of Cyp24a1 gene expression without inducing hypercalcemia, and the 1,3-diglycoside is one of the major forms of 1,25D3 found in the leaf. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.