Showing papers by "Rowett Research Institute published in 2011"

The inflammatory microenvironment in colorectal neoplasia.

Abstract: Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. Inflammatory activity within the stroma of invasive colorectal tumours is known to be a key predictor of disease activity with type, density and location of immune cells impacting on patient prognosis. To date, there has been no report of inflammatory phenotype within pre-malignant human colonic adenomas. Assessing the stromal microenvironment and particularly, inflammatory activity within colorectal neoplastic lesions is central to understanding early colorectal carcinogenesis. Inflammatory cell infiltrate was assessed by immunohistochemistry in paired colonic adenoma and adjacent normal colonic mucosa samples, and adenomas exhibiting increasing degrees of epithelial cell dysplasia. Macrophage phenotype was assessed using double stain immunohistochemistry incorporating expression of an intracellular enzyme of function. A targeted array of inflammatory cytokine and receptor genes, validated by RT-PCR, was used to assess inflammatory gene expression. Inflammatory cell infiltrates are a key feature of sporadic adenomatous colonic polyps with increased macrophage, neutrophil and T cell (specifically helper and activated subsets) infiltration in adenomatous colonic polyps, that increases in association with characteristics of high malignant potential, namely, increasing degree of cell dysplasia and adenoma size. Macrophages within adenomas express iNOS, suggestive of a pro-inflammatory phenotype. Several inflammatory cytokine genes (CXCL1, CXCL2, CXCL3, CCL20, IL8, CCL23, CCL19, CCL21, CCL5) are dysregulated in adenomas. This study has provided evidence of increased inflammation within pre-malignant colonic adenomas. This may allow potential mechanistic pathways in the initiation and promotion of early colorectal carcinogenesis to be identified.

Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures.

Abstract: The quality and yield of extracted DNA are critical for the majority of downstream applications in molecular biology. Moreover, molecular techniques such as quantitative real-time PCR (qPCR) are becoming increasingly widespread; thus, validation and cross-laboratory comparison of data require standardization of upstream experimental procedures. DNA extraction methods depend on the type and size of starting material(s) used. As such, the extraction of template DNA is arguably the most significant variable when cross-comparing data from different laboratories. Here, we describe a reliable, inexpensive and rapid method of DNA purification that is equally applicable to small or large scale or high-throughput purification of DNA. The protocol relies on a CTAB-based buffer for cell lysis and further purification of DNA with phenol : chloroform : isoamyl alcohol. The protocol has been used successfully for DNA purification from rumen fluid and plant cells. Moreover, after slight alterations, the same protocol was used for large-scale extraction of DNA from pure cultures of Gram-positive and Gram-negative bacteria. The yield of the DNA obtained with this method exceeded that from the same samples using commercial kits, and the quality was confirmed by successful qPCR applications.

Diet, environmental factors, and lifestyle underlie the high prevalence of vitamin D deficiency in healthy adults in Scotland, and supplementation reduces the proportion that are severely deficient

Abstract: Vitamin D deficiency has recently been implicated as a possible risk factor in the etiology of numerous diseases, including nonskeletal conditions. In humans, skin synthesis following exposure to UVB is a potent source of vitamin D, but in regions with low UVB, individuals are at risk of vitamin D deficiency. Our objectives were to describe the prevalence of vitamin D deficiency and to investigate determinants of plasma 25-hydroxyvitamin D (25-OHD) concentrations in a high northern latitude country. Detailed dietary, lifestyle, and demographic data were collected for 2235 healthy adults (21-82 y) from Scotland. Plasma 25-OHD was measured by liquid chromatography-tandem MS. Among study participants, 34.5% were severely deficient (25-OHD 40 nmol/L). Among participants who were taking supplements, 21.3% had a May-standardized 25-OHD concentration >50 nmol/L, 54.2% had 25-50 nmol/L, and 24.5% had <25 nmol/L, whereas this was 15.6, 43.3, and 41%, respectively, among those who did not take supplements (P < 0.0001). The most important sources of vitamin D were supplements and fish consumption. Vitamin D deficiency in Scotland is highly prevalent due to a combination of insufficient exposure to UVB and insufficient dietary intake. Higher dietary vitamin D intake modestly improved the plasma 25-OHD concentration (P = 0.02) and reduced the proportion of severely deficient individuals (P < 0.0001). In regions with low UVB exposure, dietary and supplement intake may be much more important than previously thought and consideration should be given to increasing the current recommended dietary allowance of 0-10 μg/d for adults in Scotland.

Estimation of total leaf area in perennial plants using image analysis

Abstract: One feature of most horticultural crop plants that is biologically relevant to their yield and productivity is total leaf area. However, direct methods of estimation of the leaf area cause damage to the plants, whereas indirect methods such as based on light measurement, demand accuracy in the setup of the measurement procedure, which is specific to each crop. Coffee is one of the most important perennial plants related to worldwide trade, and this demands some ability to estimate the productivity of the crop, as well as all the perennial plants involved in production of agricultural products. This study aims to build a model based on indirect measures to estimate the leaf area in coffee plants using image analysis. Two models were evaluated, one based on the height and width of the canopies, and other based on the area of the digital image of a tree. The results of the models have been compared with the real area of the leaves using the destructive approach with measurement of area of all the leaves using a digital scanner. Comparisons between the models and the real values indicated values of adjusted R2 of about 0.82 with a model using the height and the width values, and about 0.91 in the second model which used the area projection. The robustness of the model using the height and the width values were tested using data presented in the literature to other cultivars and achieved R2 = 0.54 with an outlier point and 0.91 without it.

Antibiotic Resistance in Swine-Manure-Impacted Environments

Abstract: An increasing body of evidence demonstrating entry of antibiotics and antibiotic resistance genes from anthropogenic sources into natural soil and water environments has raised even more questions about the lasting and future impacts consequent to drug resistance development in bacteria. These concerns stem, in part, from emerging knowledge about increased incidences, persistence, and diversity of antibiotic resistance (ABR) genes in soil and water environments with still very limited knowledge about the molecular microbial ecology of ABR occurring in situ in these natural systems. The practice of subtherapeutic doses of antibiotics for use in disease prophylaxis and growth promotion in animal livestock production has been the subject of particularly intense scrutiny, prompting bans of such uses completely in the European Union since 2006 and causing mounting concerns in the United State for more judicious use of antibiotics in food animals. Numerous articles over the last two decades have discussed in detail the various health and environmental aspects concerning the routine use of subtherapeutic antibiotic treatment in animal production (Gustafson and Bowen, 1997; Khachatourians, 1998; USGAO, 1999; Isaacson and Torrence, 2002; Seveno et al., 2002; Kummerer, 2004; Shea, 2004; Chee-Sanford et al., 2009).

The biochemical actions of phentolamine and papaverine on rat perfused skeletal muscle.

Abstract: — The direct actions of the vasodilators, papaverine and phentolamine, on skeletal muscle metabolism were investigated in an isolated perfusion system. Eviscerated male rats were hemisected above the diaphragm and perfused, via the aorta, with a physiological perfusion medium containing erythrocytes. Papaverine, but not phentolamine, reduced vascular resistance throughout the 80 min study. Papaverine caused marked reductions in muscle concentrations of ATP and phosphocreatine, when compared with muscle from preparations without added vasodilators. This was accompanied by elevations in lactate concentrations. Water content of papaverine-treated muscle was also higher than values in unperfused muscle taken in-vivo. Phentolamine, in contrast, had no effect on muscle ATP, phosphocreatine, lactate or water content. The metabolism of the entire preparation was also investigated. Papaverine induced increases in lactate output while phentolamine treatment caused an initial uptake, followed by an increased output of lactate. There was no significant effect of either papaverine or phentolamine on the metabolism of K+ and glucose. Arteriovenous differences in oxygen-saturation of haemoglobin and pH were also unaltered. Investigations on aspects of protein metabolism demonstrated that papaverine and phentolamine caused significant reductions in muscle protein synthesis when compared with control perfusions or in-vivo values. The reductions in synthesis were not due to reductions in cAMP or limitations in branched-chain amino acid supply. However, there was the suggestion that phentolamine caused a decrease in protein breakdown. The overall data indicated that papaverine and phentolamine may cause impairment of skeletal muscle metabolism. This has important implications for their therapeutic or experimental use.