Identification of the key compounds responsible for Cheddar cheese flavour

End of Project ReportThere is a poor understanding of the relationship between organoleptic assessment of cheese and quantitative analysis of flavour compounds. Further, the contribution of particular cheese-making parameters such as ripening temperature and starter culture has not been fully elucidated. During the ripening of most cheese varieties complex chemical conversions occur within the cheese matrix. In most cheese varieties breakdown of protein is the most important flavour development pathway. The primary cheese protein, casein, is degraded enzymatically to short peptides and free amino acids. The agents primarily responsible for these conversions are the residual rennet that is retained in the cheese curd at the end of the manufacturing phase and the proteinases and peptidases that are associated with the starter bacteria. While the rate and degree of proteolysis are of vital significance for desired flavour development, the direct products of proteolysis do not fully define cheese flavour. Much research is now demonstrating that the further biochemical and chemical conversions of the products of proteolysis, in particular the amino acids, are necessary for full flavour development. The products produced by these pathways are volatile at low boiling points and are thus released during mastication of the cheese in the mouth. Many of these volatile compounds contribute to the flavour sensation experienced by the consumer. A very wide spectrum of such compounds have been isolated from cheese, in excess of two hundred in some cheese varieties. It is now generally accepted that there is no individual compound which defines cheese flavour completely and that the flavour sensation is the result of numerous compounds present in the correct proportions. This has become known as the Component Balance Theory . The application of modern analytical techniques as proposed in this project would provide a greater understanding of the significant flavour compounds in Cheddar cheese and help to identify the impact of specific cheese-making parameters such as starter flora and ripening temperature on the production of volatile flavour compounds. This data would assist the general programme on flavour improvement of cheese which should ultimately benefit the cheese manufacturer. Hence this project set out to develop methods to identify the key flavour compounds in Cheddar cheese. These techniques would then be applied to experimental and commercial cheeses during ripening in an effort to identify key compounds and the influence of starter cultures and ripening temperature on their production.Department of Agriculture, Food and the Marin

Fate and Transport of Polycyclic Aromatic Hydrocarbons in Upland Irish Headwater Lake Catchments

Polycyclic aromatic hydrocarbons (PAHs) are a concern due to their carcinogenicity and propensity for transboundary atmospheric transport. Ireland is located on the western periphery of Europe and assumed to receive clean Atlantic air. As such, it has been used as an atmospheric reference for comparison to other regions. Nonetheless, few studies have evaluated concentrations of PAHs within the Irish environment. In the current study, PAHs were measured at five upland (500–800 masl) headwater lake catchments in coastal regions around Ireland, remote from industrial point source emissions. Semipermeable membrane devices were deployed in lakes for a 6-month period in July 2009, and topsoils were sampled from each catchment during October 2010. The concentrations of PAHs were low at most study sites with respect to other temperate regions. Homologue groups partitioned between lake and soil compartments based on their molecular weight were: “lighter” substances, such as Phenanthrene and Fluorene, were found in higher proportions in lakes, whereas “heavier” compounds, such as Chrysene and Benz[a]anthracene, were more prominent in soils. Concentrations of PAHs were highest at the east coast sites, potentially due to contributions from historical transboundary and regional combustion sources

Assessing recovery from acidification of European surface waters in the year 2010: evaluation of projections made with the MAGIC model in 1995

In 1999 we used the MAGIC (Model of Acidification of Groundwater In Catchments) model to project acidification of acid-sensitive European surface waters in the year 2010, given implementation of the Gothenburg Protocol to the Convention on Long-Range Transboundary Air Pollution (LRTAP). A total of 202 sites in 10 regions in Europe were studied. These forecasts can now be compared with measurements for the year 2010, to give a “ground truth” evaluation of the model. The prerequisite for this test is that the actual sulfur and nitrogen deposition decreased from 1995 to 2010 by the same amount as that used to drive the model forecasts; this was largely the case for sulfur, but less so for nitrogen, and the simulated surface water [NO3–] reflected this difference. For most of the sites, predicted surface water recovery from acidification for the year 2010 is very close to the actual recovery observed from measured data, as recovery is predominantly driven by reductions in sulfur deposition. Overall these results show that MAGIC successfully predicts future water chemistry given known changes in acid deposition

Deoxyuridine triphosphatase (dUTPase) expression and sensitivity to the thymidylate synthase (TS) inhibitorD9331

Uracil DNA misincorporation and misrepair of DNA have been recognized as important events accompanying thymidylate synthase (TS) inhibition. dUTPase catalyses the hydrolysis of dUTP to dUMP, thereby maintaining low intracellular dUTP. We have addressed the relationship between dUTPase expression and cellular sensitivity to TS inhibition in four human lung tumour cell lines. Sensitivity (5-day MTT assay) to the growth inhibitory effects of the non-polyglutamatable, specific quinazoline TS inhibitor ZD9331, varied up to 20-fold (IC 50 3–70 nM). TS protein expression correlated with TS activity (r2= 0.88 P= 0.05). Intracellular concentrations of drug following exposure to ZD9331 (1 μM, 24 h) varied by ~2-fold and dTTP pools decreased by > 80% in all cell lines. No clear associations across the cell lines between intracellular drug concentrations, TS activity/expression, or TTP depletion could be made. dUTPase activity varied 17-fold and correlated with dUTPase protein expression (r2= 0.94 P= 0.03). There was a striking variation in the amount of dUTP formed following exposure to ZD9331 (between 1.3 and 57 pmole 10–6cells) and was in general inversely associated with dUTPase activity. A large expansion in the dUTP pool was associated with increased sensitivity to a 24-h exposure to ZD9331 in A549 cells that have low dUTPase activity/expression. dUTPase expression and activity were elevated (approximately 3-fold) in two variants of a human lymphoblastoid cell line with acquired resistance to TS inhibitors, further suggesting an important role for this enzyme in TS inhibited cells. © 2000 Cancer Research Campaig

Bacteria-instructed synthesis of polymers for self-selective microbial binding and labelling

The detection and inactivation of pathogenic strains of bacteria continues to be an important therapeutic goal. Hence, there is a need for materials that can bind selectively to specific microorganisms, for diagnostic or anti-infective applications, but which can be formed from simple and inexpensive building blocks. Here, we exploit bacterial redox systems to induce a copper-mediated radical polymerisation of synthetic monomers at cell surfaces, generating polymers in situ that bind strongly to the microorganisms which produced them. This ‘bacteria-instructed synthesis’ can be carried out with a variety of microbial strains, and we show that the polymers produced are self-selective binding agents for the ‘instructing’ cell types. We further expand on the bacterial redox chemistries to ‘click’ fluorescent reporters onto polymers directly at the surfaces of a range of clinical isolate strains, allowing rapid, facile and simultaneous binding and visualisation of pathogens

Adiposity and hepatic lipid in healthy full-term, breastfed, and formula-fed human infants: a prospective short-term longitudinal cohort study

Background: The effect of mode of infant feeding on adiposity deposition is not fully understood. Objective: The objective was to test the hypothesis that differences in total and regional adipose tissue content and intrahepatocellular lipid (IHCL) arise in early infancy between breast- and formula-fed infants and to describe longitudinal changes. Design: This prospective longitudinal cohort study was performed in 2 hospitals in the United Kingdom. Healthy, full-term, appropriate weight-for-gestational age infants were recruited; adipose tissue volume and distribution were directly quantified by using whole-body magnetic resonance imaging; IHCL was assessed by in vivo proton magnetic resonance spectroscopy. Measurements were performed after birth (median age: 13 d) and at 6–12 wk of age. Method of infant feeding was recorded prospectively by using maternally completed feeding diaries. Breastfed was defined as >80% of feeds consisting of breast milk at both points; formula-fed was defined as >80% of feeds consisting of formula milk at both points. Results: Longitudinal results were obtained from 70 infants (36 breastfed, 9 mixed-fed, and 25 formula-fed). No differences were found in total or regional adipose tissue or IHCL between breastfed and formula-fed infants. In pooled analyses including all feeding groups, IHCL and total adipose tissue approximately doubled between birth and 6–12 wk: IHCL after birth (median: 0.949; IQR: 0.521–1.711) and at 6–12 wk (1.828; 1.376–2.697; P < 0.001) and total adipose tissue after birth (0.749 L; 0.620–0.928 L) and at 6–12 wk (1.547 L; 1.332–1.790 L; P < 0.001). Increasing adiposity was characterized by greater relative increases in subcutaneous than in internal adipose tissue depots. Conclusions: No differences were detectable in adipose tissue or IHCL accretion between breastfed and formula-fed infants up to 2 mo. The substantial increase in IHCL seen over this period in both breastfed and formula-fed infants is a novel observation, which suggests that hepatic storage of lipids may be physiologic up to 2 mo. This trial was registered at www.clinicaltrials.gov as NCT02033005

The ability to accumulate deoxyuridine triphosphate and cellular response to thymidylate synthase (TS) inhibition

Thymidylate synthase (TS) is an important enzyme catalysing the reductive methylation of dUMP to dTMP that is further metabolized to dTTP for DNA synthesis. Loss of viability following TS inhibition occurs as a consequence of depleted dTTP pools and at least in some cell lines, accumulation of dUTP and subsequent misincorporation of uracil into DNA. The expansion in dUTP pools is largely determined by the expression of the pyrophosphatase, dUTPase. Our previous work has shown that following TS inhibition the ability to accumulate dUTP was associated with an earlier growth inhibitory effect. 3 human lung tumour cell lines and HT29 human colon tumour cells transfected with dUTPase have been used to investigate the relationship between loss of viability following TS inhibition and dUTP accumulation. Cell cycle arrest typical of TS inhibition was an early event in all cell lines and occurred irrespective of the ability to accumulate dUTP or p53 function. However, a large expansion of dUTP pools was associated with mature DNA damage (4 h) and an earlier loss of viability following TS inhibition compared to cells in which dUTP pools were not expanded. In A549 cells damage to mature DNA may have been exacerbated by significantly higher activity of the excision repair enzyme, uracil-DNA glycosylase. Consistent with results using different inhibitors of TS, transfection of dUTPase into HT29 cells significantly reduced the cytotoxicity of a 24 h but not 48 h exposure to ZD9331. Although loss of viability can be mediated through dTTP deprivation alone, the uracil misincorporation pathway resulted in an earlier commitment to cell death. The relevance of this latter pathway in the clinical response to TS inhibitors deserves further investigation. © 2001 Cancer Research Campaign http://www.bjcancer.co

Assessment of inherent fluctuations of mitotic and labelling indices of human tumours.

A method is presented to evaluate the influence of statistical errors and inherent variation on the determination of mitotic and labelling indices of human tumours. In most of the experiments reported here, sufficient cells were counted to yield a statistical error which is small in comparison to the inherent differences in the proliferative indices, both between different sites in the same tumour and between different tumours of the same histological type. These inherent fluctuations are, theefore, a critical factor in cell kinetic studies of human tumours

EGFR interacts with the fusion protein of respiratory syncytial virus strain 2-20 and mediates infection and mucin expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from "mucogenic" strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease