Hypoxia modulates the stem cell population and induces EMT in the MCF-10A breast epithelial cell line

A common feature among pre-malignant lesions is the induction of hypoxia through increased cell propagation and reduced access to blood flow. Hypoxia in breast cancer has been associated with poor patient prognosis, resistance to chemotherapy and increased metastasis. Although hypoxia has been correlated with factors associated with the latter stages of cancer progression, it is not well documented how hypoxia influences cells in the earliest stages of transformation. Using the immortalized MCF-10A breast epithelial cell line, we used hypoxic culture conditions to mimic reduced O2 levels found within early pre-malignant lesions and assessed various cellular parameters. In this non-transformed mammary cell line, O2 deprivation led to some changes not immediately associated with cancer progression, such as decreased proliferation, cell cycle arrest and increased apoptosis. In contrast, hypoxia did induce other changes more consistent with an increased metastatic potential. A rise in the CD44+CD24-/low-labeled cell sub-population along with increased colony forming capability indicated an expanded stem cell population. Hypoxia also induced cellular and molecular changes consistent with an epithelial-to-mesenchymal transition (EMT). Furthermore, these cells now exhibited increased migratory and invasive abilities. These results underscore the contribution of the hypoxic tumour microenvironment in cancer progression and dissemination

The roles of the APC proteins in homeostasis and tumourigenesis

The adenomatous polyposis coli (APC) gene encodes a multifunctional tumour suppressor protein that is essential for normal development. The most characterised role of APC is its ability to mediate the Wnt signaling pathway, a pathway disrupted in the majority of human cancers. The identification of a second adenomatous polyposis coli gene (APC2) which possesses many shared structural characteristics with APC and potentially comparable functions raises the possibility that APC2 also functions both in development and in tumour suppression, and that some redundancy may exist between the two proteins. Analysis of these proteins in the mouse has been hampered due to the lethality of the Apc mutation and the lack of a suitable Apc2 mutation. However, to circumvent the first of these difficulties, Cre-lox technology was employed to conditionally delete Apc in adult mouse tissues and so study its function in vivo. To circumvent the second difficulty, a novel Apc2 null allele had become available from the laboratory of Professor Hans Clevers. Remarkably, constitutive deletion of Apc2 does not lead to embryonic lethality, permitting study of the effects of Apc2 deficiency within adult tissues. In this thesis I aimed to characterise the consequences of Apc2 loss alone, and in the context of tissue specific Apc loss, in a range of tissues. Apc2 deficiency led to subtle changes in Wnt signaling in the intestines and liver however, no detectable differences of this pathway were apparent within the mammary gland. Phenotypically, altered homeostasis was only observed within the intestines. Apc2 deficiency led to an increase in epithelial cell division, an increase in markers of intestinal stemness and increases in intestinal cell migration. However, loss of Apc2 failed to induce tumourigenesis in the intestines or indeed any other tissue. In the context of Apc loss, the effect was dependent upon the tissue. Within the intestines, additional loss of Apc2 altered the immediate phenotype of Apc loss but failed to modify Apc induced tumourigenesis. Within the mammary gland, whilst either Apc protein alone was dispensable, combined loss synergised to disrupt homeostasis and drive tumourigenesis. Contrary to this, in the liver the additional loss of Apc2 attenuated tumourigenesis induced by reduced levels of Apc. Together, these studies highlight the importance of these proteins and their interactions and redundancies in homeostasis and tumourigenesis

Coronary sinus sampling of cytokines after heart transplantation: Evidence for macrophage activation and interleukin-4 production within the graft

AbstractObjectives. This study was undertaken to evaluate the organ-specific release of cytokines after heart transplantation and to assess any correlation with transplant rejection. This cytokine profile should document the relative activation of mononuclear cell subsets within the graft.Background. Up to 60% of mononuclear cell infiltrating the cardiac allograft during rejection are macrophages, but their rote is undetermined. The T lymphocytes are activated, but the activity of specific T cell subsets is not known. We sought to assess for the first time in humans the in vivo activation of mononuclear cell subsets by measuring coronary sinus cytokine levels after heart transplantation.Methods. Palred superior vena cava and coronary sinus serum samples were assayed for interkeukin (IL)-2, IL-4 and IL-6, soluble IL-2 receptors, tumor necrosis factor-alpha and neopterin in 10 patients at the time of 40 routine endomyocardial biopsy procedures. All cytokine measurements were made by using enzyme-linked immunosorbent assay; neopterin was measured by using radioimmunoassy.Results. Interleukin-2 levels were not detectable (<0.8 U/ml) in either the superior vena cava or the coronary sinus in the presence or absence of rejection. Interleukin-2 receptor levels were uniformly elevated to 1,283 U/ml in the superior vena cava and to 1,232 U/ml in the coronary sinus, with no correlation with rejection severity. Interleukin-4 levels were consistently higher in coronary sinus serum than in peripheral blood (229 vs. 61 pg/ml, p < O.0005), but there was no relation with rejection. Interleukin-6 levels were higher in the coronary sinus than in me superior vena cava (200 vs. 120 pg/mi, p < 0.05). Tumor necrosis factor-alpha showed consistently elevated levels in coronary sinus serum (68 vs. 17 pg/ml, p < 0.0005), with no relation with rejection. Neopterin, which is produced only by activated macro-phages, was also consistently elevated in the coronary sinus (2.5 vs. 2.2 nmol, p = 0.08).Conclusions. The cardiac allograft is a major source of cyto-kines after heart transplantation. The cytokine profile allows the activity of subsets of the mononuclear cell infiltrate to be investigated. Elevated coronary sinus activity of the macrophage specific metabolite neopterin suggests macrophage activation within the allograft. This possibility is supported by elevated coronary sinus levels of tumor necrosis factor-alpha and IL-6. The T lymphocytes are activated, as evidenced by high soluble IL-2 receptor levels, but IL-2 production was suppressed by conventional immunosup-pressive therapy. Coronary sinus IL-4 levels represent T helper-2 cell activation within the graft despite immunosuppression. We could find no temporal relation between the coronary sinus or superior vena cava cytokine concentration or profile and severity of rejection on concurrent biopsy studies

Subtle deregulation of the Wnt-signalling pathway through loss of Apc2 reduces the fitness of intestinal stem cells

The importance of the Wnt-signaling pathway on the regulation and maintenance of the intesti- nal stem cell (ISC) population is well recognized. However, our current knowledge base is founded on models using systems of gross deregulation of the Wnt-signaling pathway. Given the importance of this signaling pathway on intestinal homeostasis, there is a need to explore the role of more subtle alterations in Wnt-signaling levels within this tissue. Herein, we have used a model of Apc2 loss to meet this aim. Apc2 is a homolog of Apc which can also form a destruction complex capable of binding b-catenin, albeit less efficiently than Apc. We show that systemic loss of Apc2 results in an increase in the number of cells displaying nuclear b-catenin at the base of the intestinal crypt. This subsequently impacts the expression levels of several ISC markers and the fitness of ISCs as assessed by organoid formation efficiency. This work pro- vides the first evidence that the function and fitness of ISCs can be altered by even minor mis- regulation of the Wnt-signaling pathway. Our data highlights the importance of correct maintenance of this crucial signaling pathway in the maintenance and function of the ISC popu- lation

Changes in plasma itaconate elevation in early rheumatoid arthritis patients elucidates disease activity associated macrophage activation

Objective. To characterize changes in the plasma metabolic profile in newly diagnosed rheumatoid arthritis (RA) patients upon commencement of conventional disease modifying anti-rheumatic drug (cDMARD) therapy. Methods. Plasma samples collected in an early RA randomized strategy study (NCT00920478) that compared clinical (DAS) disease activity assessment with musculoskeletal ultrasound assessment (MSUS) to drive treatment decisions were subjected to untargeted metabolomic analysis. Metabolic profiles were collected at pre- and 3 months post commencement of non-biologic cDMARD. Metabolites that changed in association with changes in the DAS44 score were identified at the 3 month timepoint. Results. A total of ten metabolites exhibited a clear correlation with reduction in DAS44 score following cDMARD commencement, particularly itaconate, its derived anhydride and a derivative of itaconate coA. Increasing itaconate correlated with improved DAS44 score and decreasing levels of CRP. Conclusion. cDMARD treatment effects invoke consistent changes in plasma detectable metabolites, that in turn implicate clinical disease activity with macrophages. Such changes inform RA pathogenesis and reveal for the first time a link between itaconate production and resolution of an inflammatory disease in humans. Quantitative metabolic biomarker based tests of clinical change in state are feasible and should be developed around the itaconate pathway

A Framework for the practical application of the concepts of critical natural capital and strong sustainability

This paper develops a methodology for identifying that natural capital—called critical natural capital (CNC)—the maintenance of which is essential for environmental sustainability. By consideration of the characteristics of natural capital, of the environmental functions that these characteristics enable natural capital to perform and of the importance of these functions to humans and the biosphere, it shows how sustainability standards in respect of these environmental functions may be derived. The difference between the current situation and these standards is termed the sustainability gap. The methodology that emerges from bringing these ideas together into a single analytical framework enables policy makers to identify the extent of current unsustainability, the principal causes of it, the elements and processes of natural capital (the CNC) which need to be maintained or restored to close the sustainability gap and the costs of so doing. The framework should therefore be of use in identifying priorities and policies for moving towards environmental sustainability

Froude Efficiency and Velocity Fluctuation in Forearm-Amputee Front Crawl: Implications for Para Swimming Classification

Purpose: The impact of physical impairment on Froude efficiency and intra-cyclic velocity fluctuation in Para swimmers is not well documented. Identification of differences in these variables between disabled and non-disabled swimmers could help develop a more objective system for assigning Para swimmers to classes for competition. This study quantifies Froude efficiency and intra-cyclic velocity fluctuation in unilateral forearm-amputee front crawl swimmers, and evaluates associations between these variables and performance. Methods: Ten unilateral forearm-amputee swimmers completed front crawl trials at 50 m and 400 m pace; three-dimensional video analysis provided mass centre, wrist and stump velocities. Intra-cyclic velocity fluctuation was calculated as: 1) maximum - minimum mass centre velocity, expressed as % of mean velocity, and 2) coefficient of variation in mass centre velocity. Froude efficiency was the ratio between mean swimming velocity and wrist plus stump velocity during each segment’s respective: 1) underwater phase, and 2) propulsive underwater phase. Results: Forearm-amputees’ intra-cyclic velocity fluctuation (400 m: 22 ± 7%; 50 m: 18 ± 5%) was similar to published values for non-disabled swimmers, whilst Froude efficiencies were lower. Froude efficiency was higher at 400 m (0.37 ± 0.04) than 50 m pace (0.35 ± 0.05; p < .05) and higher for the unaffected limb (400 m: 0.52 ± 0.03; 50 m: 0.54 ± 0.04) than the residual limb (400 m: 0.38 ± 0.03; 50 m 0.38 ± 0.02; p < .05). Neither intra-cyclic velocity fluctuation nor Froude efficiency were associated with swimming performance. Conclusions: Froude efficiency may be a valuable measure of activity limitation in swimmers with an upper limb deficiency and a useful metric for comparing swimmers with different types and severity of physical impairment

Differential expression of the BCAT isoforms between breast cancer subtypes

© 2020, The Author(s). Background: Biological characterisation of breast cancer subtypes is essential as it informs treatment regimens especially as different subtypes have distinct locoregional patterns. This is related to metabolic phenotype, where altered cellular metabolism is a fundamental adaptation of cancer cells during rapid proliferation. In this context, the metabolism of the essential branched-chain amino acids (BCAAs), catalysed by the human branched-chain aminotransferase proteins (hBCAT), offers multiple benefits for tumour growth. Upregulation of the cytosolic isoform of hBCAT (hBCATc), regulated by c-Myc, has been demonstrated to increase cell migration, tumour aggressiveness and proliferation in gliomas, ovarian and colorectal cancer but the importance of the mitochondrial isoform, hBCATm has not been fully investigated. Methods: Using immunohistochemistry, the expression profile of metabolic proteins (hBCAT, IDH) was assessed between breast cancer subtypes, HER2 + , luminal A, luminal B and TNBC. Correlations between the percentage and the intensity of protein expression/co-expression with clinical parameters, such as hormone receptor status, tumour stage, lymph-node metastasis and survival, were determined. Results: We show that hBCATc expression was found to be significantly associated with the more aggressive HER2 + and luminal B subtypes, whilst hBCATm and IDH1 associated with luminal A subtype. This was concomitant with better prognosis indicating a differential metabolic reliance between these two subtypes, in which enhanced expression of IDH1 may replenish the α-ketoglutarate pool in cells with increased hBCATm expression. Conclusion: The cytosolic isoform of BCAT is associated with tumours that express HER2 receptors, whereas the mitochondrial isoform is highly expressed in tumours that are ER + , indicating that the BCAT proteins are regulated through different signalling pathways, which may lead to the identification of novel targets for therapeutic applications targeting dysregulated cancer metabolism

Food biodiversity : quantifying the unquantifiable in human diets

Dietary diversity is an established public health principle, and its measurement is essential for studies of diet quality and food security. However, conventional between food group scores fail to capture the nutritional variability and ecosystem services delivered by dietary richness and dissimilarity within food groups, or the relative distribution (i.e., evenness or moderation) of e.g., species or varieties across whole diets. Summarizing food biodiversity in an all-encompassing index is problematic. Therefore, various diversity indices have been proposed in ecology, yet these require methodological adaption for integration in dietary assessments. In this narrative review, we summarize the key conceptual issues underlying the measurement of food biodiversity at an edible species level, assess the ecological diversity indices previously applied to food consumption and food supply data, discuss their relative suitability, and potential amendments for use in (quantitative) dietary intake studies. Ecological diversity indices are often used without justification through the lens of nutrition. To illustrate: (i) dietary species richness fails to account for the distribution of foods across the diet or their functional traits; (ii) evenness indices, such as the Gini-Simpson index, require widely accepted relative abundance units (e.g., kcal, g, cups) and evidence-based moderation weighting factors; and (iii) functional dissimilarity indices are constructed based on an arbitrary selection of distance measures, cutoff criteria, and number of phylogenetic, nutritional, and morphological traits. Disregard for these limitations can lead to counterintuitive results and ambiguous or incorrect conclusions about the food biodiversity within diets or food systems. To ensure comparability and robustness of future research, we advocate food biodiversity indices that: (i) satisfy key axioms; (ii) can be extended to account for disparity between edible species; and (iii) are used in combination, rather than in isolation