The effect of Holstein-Friesian genotype and feeding system on selected performance parameters of dairy cows on grass-based systems of milk production in Ireland

End of project reportThe overall objective of this project was to assess, the effect of strain of Holstein-Friesian dairy cow, pasture-based feed system (FS) and their interaction on animal performance in terms of milk productivity and lactation profile, body weight (BW), body condition score (BCS), feed intake and energy balance (EB), reproductive performance and overall economic profitability

Solvent and thermal stability, and pH kinetics, of proline-specific dipeptidyl peptidase IV-like enzyme from bovine serum

Proline-specific dipeptidyl peptidase-like (DPP IV; EC 3.4.14.5) activity in bovine serum has attracted little attention despite its ready availability and the paucity of useful proline-cleaving enzymes. Bovine serum DPP IV-like peptidase is very tolerant of organic solvents, particularly acetonitrile: upon incubation for 1 h at room temperature in 70% acetonitrile, 47% dimethylformamide, 54% DMSO and 33% tetrahydrofuran (v/v concentrations) followed by dilution into the standard assay mixture, the enzyme retained half of its aqueous activity. As for thermal performance in aqueous buffer, its relative activity increased up to 50 ◦C. Upon thermoinactivation at 71 ◦C, pH 8.0 (samples removed periodically, cooled on ice, then assayed under optimal conditions), residual activities over short times fit a first-order decay with a k-value of 0.071±0.0034 min−1. Over longer times, residual activities fit to a double exponential decay with k1 and k2 values of 0.218±0.025 min−1 (46±4% of overall decay) and 0.040±0.002 min−1 (54±4% of overall decay), respectively. The enzyme’s solvent and thermal tolerances suggest that it may have potential for use as a biocatalyst in industry. Kinetic analysis with the fluorogenic substrate Gly-Pro-7-aminomethylcoumarin over a range of pH values indicated two pK values at 6.18±0.07 and at 9.70±0.50. We ascribe the lower value to the active site histidine; the higher may be due to the active site serine or to a free amino group in the substrate

Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli : purification , biochemical and kinetic characterisation

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl-aminopeptidase I. The amino acid sequence, deduced from the nucleotide sequence, revealed that it consists of 209 amino acid residues and showed to have 98% homology with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary among the two sequences. The bovine enzyme was expressed in XL10-gold Esherichia coli cells. Immobilizied Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 3.3mg of PAP1 per litre culture. The purified enzyme had a specific activity of 1700 units/ml. SDS-PAGE produced a single band for bovine PAP1 with a molecular weight of ~23-24 kDa which is in good agreement with previously reported data on PAP1. Kinetic constants Km and Kcat were 59μΜ and 3.5s-1, respectively. It possessed an optimum pH between 9-9.5, a temperature of 37°C and showed an absolute requirement for a thiol-reducing agent (10mM DTT). EDTA didn’t prove to have an effect on enzyme activity. Competitive inhibition was seen with pyroglutamyl peptides pGlu-His-Pro-NH2 (TRH; Ki= 44.1 uM), pGlu-Ala- OH (Ki=141 uM) and pGlu-Val-OH (Ki=652.17)

Polypeptide-grafted macroporous polyHIPE by surface-initiated N-Carboxyanhydride (NCA) polymerization as a platform for bioconjugation

A new class of functional macroporous monoliths from polymerized high internal phase emulsion (polyHIPE) with tunable surface functional groups was developed by direct polypeptide surface grafting. In the first step, amino-functional polyHIPEs were obtained by the addition of 4-vinylbenzyl or 4-vinylbenzylphthalimide to the styrenic emulsion and thermal radical polymerization. The obtained monoliths present the expected open-cell morphology and a high surface area. The incorporated amino group was successfully utilized to initiate the ring-opening polymer- ization of benzyl-L-glutamate N-carboxyanhydride (BLG NCA) and benzyloxycarbonyl-L-lysine (Lys(Z)) NCA, which resulted in a dense homogeneous coating of polypeptides throughout the internal polyHIPE surfaces as confirmed by SEM and FTIR analysis. The amount of polypeptide grafted to the polyHIPE surfaces could be modulated by varying the initial ratio of amino acid NCA to amino-functional polyHIPE. Subsequent removal of the polypeptide protecting groups yielded highly functional polyHIPE-g-poly(glutamic acid) and polyHIPE-g- poly(lysine). Both types of polypeptide-grafted monoliths responded to pH by changes in their hydrohilicity. The possibility to use the high density of function (−COOH or −NH2) for secondary reaction was demonstrated by the successful bioconjugation of enhanced green fluorescent protein (eGFP) and fluorescein isocyanate (FITC) on the polymer 3D-scaffold surface. The amount of eGFP and FITC conjugated to the polypeptide-grafted polyHIPE was significantly higher than to the amino- functional polyHIPE, signifying the advantage of polypeptide grafting to achieve highly functional polyHIPEs

Dynamic and influential interaction of cancer cells with normal epithelial cells in 3D culture

BACKGROUND: The cancer microenvironment has a strong impact on the growth and dynamics of cancer cells. Conventional 2D culture systems, however, do not reflect in vivo conditions, impeding detailed studies of cancer cell dynamics. This work aims to establish a method to reveal the interaction of cancer and normal epithelial cells using 3D time-lapse. METHODS: GFP-labelled breast cancer cells, MDA-MB-231, were co-cultured with mCherry-labelled non-cancerous epithelial cells, MDCK, in a gel matrix. In the 3D culture, the epithelial cells establish a spherical morphology (epithelial sphere) thus providing cancer cells with accessibility to the basal surface of epithelia, similar to the in vivo condition. Cell movement was monitored using time-lapse analyses. Ultrastructural, immunocytochemical and protein expression analyses were also performed following the time-lapse study. RESULTS: In contrast to the 2D culture system, whereby most MDA-MB-231 cells exhibit spindle-shaped morphology as single cells, in the 3D culture the MDA-MB-231 cells were found to be single cells or else formed aggregates, both of which were motile. The single MDA-MB-231 cells exhibited both round and spindle shapes, with dynamic changes from one shape to the other, visible within a matter of hours. When co-cultured with epithelial cells, the MDA-MB-231 cells displayed a strong attraction to the epithelial spheres, and proceeded to surround and engulf the epithelial cell mass. The surrounded epithelial cells were eventually destroyed, becoming debris, and were taken into the MDA-MB-231 cells. However, when there was a relatively large population of normal epithelial cells, the MDA-MB-231 cells did not engulf the epithelial spheres effectively, despite repeated contacts. MDA-MB-231 cells co-cultured with a large number of normal epithelial cells showed reduced expression of monocarboxylate transporter-1, suggesting a change in the cell metabolism. A decreased level of gelatin-digesting ability as well as reduced production of matrix metaroproteinase-2 was also observed. CONCLUSIONS: This culture method is a powerful technique to investigate cancer cell dynamics and cellular changes in response to the microenvironment. The method can be useful for various aspects such as; different combinations of cancer and non-cancer cell types, addressing the organ-specific affinity of cancer cells to host cells, and monitoring the cellular response to anti-cancer drugs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12935-014-0108-6) contains supplementary material, which is available to authorized users

Climate drives the geography of marine consumption by changing predator communities

Este artículo contiene 7 páginas, 3 figuras, 1 tabla.The global distribution of primary production and consumption by humans (fisheries) is well-documented, but we have no map linking the central ecological process of consumption within food webs to temperature and other ecological drivers. Using standardized assays that span 105° of latitude on four continents, we show that rates of bait consumption by generalist predators in shallow marine ecosystems are tightly linked to both temperature and the composition of consumer assemblages. Unexpectedly, rates of consumption peaked at midlatitudes (25 to 35°) in both Northern and Southern Hemispheres across both seagrass and unvegetated sediment habitats. This pattern contrasts with terrestrial systems, where biotic interactions reportedly weaken away from the equator, but it parallels an emerging pattern of a subtropical peak in marine biodiversity. The higher consumption at midlatitudes was closely related to the type of consumers present, which explained rates of consumption better than consumer density, biomass, species diversity, or habitat. Indeed, the apparent effect of temperature on consumption was mostly driven by temperature-associated turnover in consumer community composition. Our findings reinforce the key influence of climate warming on altered species composition and highlight its implications for the functioning of Earth’s ecosystems.We acknowledge funding from the Smithsonian Institution and the Tula Foundation.Peer reviewe

Acute exercise leads to regulation of Telomere-Associated genes and MicroRNA expression in immune Cells

Telomeres are specialized nucleoprotein structures that protect chromosomal ends from degradation. These structures progressively shorten during cellular division and can signal replicative senescence below a critical length. Telomere length is predominantly maintained by the enzyme telomerase. Significant decreases in telomere length and telomerase activity are associated with a host of chronic diseases; conversely their maintenance underpins the optimal function of the adaptive immune system. Habitual physical activity is associated with longer leukocyte telomere length; however, the precise mechanisms are unclear. Potential hypotheses include regulation of telomeric gene transcription and/or microRNAs (miRNAs). We investigated the acute exercise-induced response of telomeric genes and miRNAs in twenty-two healthy males (mean age = 24.1±1.55 years). Participants undertook 30 minutes of treadmill running at 80% of peak oxygen uptake. Blood samples were taken before exercise, immediately post-exercise and 60 minutes post-exercise. Total RNA from white blood cells was submitted to miRNA arrays and telomere extension mRNA array. Results were individually validated in white blood cells and sorted T cell lymphocyte subsets using quantitative real-time PCR (qPCR). Telomerase reverse transcriptase (TERT) mRNA (P = 0.001) and sirtuin-6 (SIRT6) (P<0.05) mRNA expression were upregulated in white blood cells after exercise. Fifty-six miRNAs were also differentially regulated post-exercise (FDR <0.05). In silico analysis identified four miRNAs (miR-186, miR-181, miR-15a and miR-96) that potentially targeted telomeric gene mRNA. The four miRNAs exhibited significant upregulation 60 minutes post-exercise (P<0.001). Telomeric repeat binding factor 2, interacting protein (TERF2IP) was identified as a potential binding target for miR-186 and miR-96 and demonstrated concomitant downregulation (P<0.01) at the corresponding time point. Intense cardiorespiratory exercise was sufficient to differentially regulate key telomeric genes and miRNAs in white blood cells. These results may provide a mechanistic insight into telomere homeostasis and improved immune function and physical health. Funding NHMR

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The effect of Holstein-Friesian genotype and feeding system on selected performance parameters of dairy cows on grass-based systems of milk production in Ireland

End of project reportThe overall objective of this project was to assess, the effect of strain of Holstein-Friesian dairy cow, pasture-based feed system (FS) and their interaction on animal performance in terms of milk productivity and lactation profile, body weight (BW), body condition score (BCS), feed intake and energy balance (EB), reproductive performance and overall economic profitability