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Thymidylate synthase (TS) is a target for pemetrexed and the prodrug 5-fluorouracil (5-FU) that inhibit the protein by binding at its active site. Prolonged administration of these drugs causes TS overexpression, leading to drug resistance. The peptide lead, LR (LSCQLYQR), allosterically stabilizes the inactive form of the protein and inhibits ovarian cancer (OC) cell growth with stable TS and decreased dihydrofolate reductase (DHFR) expression. To improve TS inhibition and the anticancer effect, we have developed 35 peptides by modifying the lead. The d-glutamine-modified peptide displayed the best inhibition of cisplatin-sensitive and -resistant OC cell growth, was more active than LR and 5-FU, and showed a TS/DHFR expression pattern similar to LR. Circular dichroism spectroscopy and molecular dynamics studies provided a molecular-level rationale for the differences in structural preferences and the enzyme inhibitory activities. By combining target inhibition studies and the modulation pattern of associated proteins, this work avenues a concept to develop more specific inhibitors of OC cell growth and drug leads.

Subclinical mastitis (SM) is one of the most important diseases affecting dairy ewes worldwide, with negative impact on the animal health, farm income and public health. Animals with SM often remain untreated because the disease may not be revealed. Increase in somatic cell count (SCC) and positive bacteriology for mastitis pathogens in milk samples are indicative of SM but the evidence of only one of these alterations must suggest an uncertain SM (UM). UM is defined when positive bacteriological examination (Latent-SM) or SCC>500 000 cells/ml (non-specific-SM) are detected in milk. Nevertheless, SCC and bacteriological examination are expensive, time consuming and are not yet in use at the farm level in dairy ewes. Recently, a sensitive acute phase protein, amyloid A, displaying multiple isoforms in plasma and different body fluids including mammary secretion (milk amyloid A-MAA), has been investigated as a marker of mastitis in cows and, in a few studies, in sheep. The aim of this trial was to compare the concentration of MAA of single udder-halves in ewes with healthy udder-halves (HU-control group) and naturally occurring subclinical mastitis, both confirmed (SM group) and uncertain (UM groups: Latent-SM and non-specific-SM), for monitoring udder health. The reliability of a specific ELISA kit for the measurement of MAA was also tested. During a 3-month trial period, 153 udder halves were assigned to the experimental groups based on their health status: 25 with SM, 40 with UM (11 with latent-SM and 29 with non-specific-SM) and 88 HU. SCC and bacteriological analysis were performed to establish the control and subclinical mastitis groups. MAA concentrations in milk samples were measured using a specific commercially milk ELISA kit. The data were submitted to statistical analysis. Significant (P<0·05) differences among the groups SM, non-specific-SM and HU were detected with the SM having the highest level and HU the lowest. MAA concentration is affected by the udder health status and is a useful indicator of subclinical mastitis and increased SCC in sheep.

The preclinical study of the mechanism of action of anticancer small molecules is challenging due to the complexity of cancer biology and the fragmentary nature of available data. With the aim of identifying a protein subset characterizing the cellular activity of anticancer peptides, we used differential mass spectrometry to identify proteomic changes induced by two peptides, LR and [d-Gln(4)]LR, that inhibit cell growth and compared them with the changes induced by a known drug, pemetrexed, targeting the same enzyme, thymidylate synthase. The quantification of the proteome of an ovarian cancer cell model treated with LR yielded a differentially expressed protein data set with respect to untreated cells. This core set was expanded by bioinformatic data interpretation, the biologically relevant proteins were selected, and their differential expression was validated on three cis-platinum sensitive and resistant ovarian cancer cell lines. Via clustering of the protein network features, a broader view of the peptides' cellular activity was obtained. Differences from the mechanism of action of pemetrexed were inferred from different modulation of the selected proteins. The protein subset identification represents a method of general applicability to characterize the cellular activity of preclinical compounds and a tool for monitoring the cellular activity of novel drug candidates.