Discrimination of healthy and cancer cells of the bladder by metabolic state, based on autofluorescence

Bladder cancer is among the most common cancers worldwide (4th in men). It is responsible for high patient morbidity and displays rapid recurrence and progression. Lack of sensitivity of gold standard techniques (white light cystoscopy, voided urine cytology) means many early treatable cases are missed. The result is a large number of advanced cases of bladder cancer which require extensive treatment and monitoring. For this reason, bladder cancer is the single most expensive cancer to treat on a per patient basis. In recent years, autofluorescence spectroscopy has begun to shed light into disease research. Of particular interest in cancer research are the fluorescent metabolic cofactors NADH and FAD. Early in tumour development, cancer cells often undergo a metabolic shift (the Warburg effect) resulting in increased NADH. The ratio of NADH to FAD ("redox ratio") can therefore be used as an indicator of the metabolic status of cells. Redox ratio measurements have been used to differentiate between healthy and cancer breast cells and to monitor cellular responses to therapies. Here, we have demonstrated, using healthy and bladder cancer cell lines, a statistically significant difference in the redox ratio of bladder cancer cells, indicative of a metabolic shift. To do this we customised a standard flow cytometer to excite and record fluorescence specifically from NADH and FAD, along with a method for automatically calculating the redox ratio of individual cells within large populations. These results could inform the design of novel probes and screening systems for the early detection of bladder cancer.

Dispersal, establishment and survival of soredia and fragments of the lichen, Hypogymnia physodes (L.) Nyl.

Dispersal, establishment and survival were studied in a population of Hypogymnia physodes (L.) Nyl. growing on an isolated tree (Prunus blireiana L.) at a site in North Seattle, U.S.A. Lichen propagules were trapped daily on adhesive strips pinned to various sites on the tree over a period of 36 days. Both soredia and fragments were deposited on the strips, particularly on the upper branches of the tree, with soredia being considerably more numerous than fragments. Daily variation in total soredia deposited did not correlate with 10 climatic variables including wind speed, relative humidity and average temperature. Establishment and survival of propagules were studied by introducing soredia and fragments into various sites on the bark and by observing the distribution of small thalli in different microsites. Microtopography of bark significantly influenced establishment and survival. Survival was poor on smooth bark compared with survival on algal or lichen crusts and on rough bark. Survival of soredia did not vary significantly at different locations on the tree. It is likely that colonization of the tree by H. physodes occurs largely by soredia. Colonization appears to be limited more by the range of dispersal over the tree than by differential survival over different parts of the tree.

A new experimental protocol for preferential differentiation of mouse embryonic stem cells into insulin-producing cells

Mouse embiyonic stem (ES) cells have the potential to differentiate into insulin-producing cells, but efficient protocols for in vitro differentiation have not been established. Here we have developed a new optimized four-stage differentiation protocol and compared this with an established reference protocol. The new protocol minimized differentiation towards neuronal progeny, resulting in a population of insulin-producing cells with ß-cell characteristics but lacking neuronal features. The yield of glucagon and somatostatin cells was negligible. Crucial for this improved yield was the removal of a nestin selection step as well as removal of culture supplements that promote differentiation towards the neuronal lineage. Supplementation of the differentiation medium with insulin and fetal calf serum was beneficial for differentiation towards monohor-monal insulin-positive cells. After implantation into diabetic mice these insulin-producing cells produced a time-dependent improvement of the diabetic metabolic state, in contrast to cells differentiated according to the reference protocol. Using a spinner culture instead of an adherent culture of ES cells prevented the differentiation towards insulin-producing cells. Thus, prevention of cell attachment in a spinner culture represents a means to keep ES cells in an undifferentiated state and to inhibit differentiation. In conclusion, this study describes a new optimized four-stage protocol for differentiating ES cells to insulin-producing cells with minimal neuronal cell formation. Copyright © 2008 Cognizant Comm. Corp.

Saccharomyces Cerevisiae as a biotechnological tool for ageing research:studies on translation and metabolism

The yeast Saccharomyces cerevisiae is an important model organism for the study of cell biology. The similarity between yeast and human genes and the conservation of fundamental pathways means it can be used to investigate characteristics of healthy and diseased cells throughout the lifespan. Yeast is an equally important biotechnological tool that has long been the organism of choice for the production of alcoholic beverages, bread and a large variety of industrial products. For example, yeast is used to manufacture biofuels, lubricants, detergents, industrial enzymes, food additives and pharmaceuticals such as anti-parasitics, anti-cancer compounds, hormones (including insulin), vaccines and nutraceuticals. Its function as a cell factory is possible because of the speed with which it can be grown to high cell yields, the knowledge that it is generally recognized as safe (GRAS) and the ease with which metabolism and cellular pathways, such as translation can be manipulated. In this thesis, these two pathways are explored in the context of their biotechnological application to ageing research: (i) understanding translational processes during the high-yielding production of membrane protein drug targets and (ii) the manipulation of yeast metabolism to study the molecule, L-carnosine, which has been proposed to have anti-ageing properties. In the first of these themes, the yeast strains, spt3?, srb5?, gcn5? and yTHCBMS1, were examined since they have been previously demonstrated to dramatically increase the yields of a target membrane protein (the aquaporin, Fps1) compared to wild-type cells. The mechanisms underlying this discovery were therefore investigated. All high yielding strains were shown to have an altered translational state (mostly characterised by an initiation block) and constitutive phosphorylation of the translational initiation factor, eIF2a. The relevance of the initiation block was further supported by the finding that other strains, with known initiation blocks, are also high yielding for Fps1. A correlation in all strains between increased Fps1 yields and increased production of the transcriptional activator protein, Gcn4, suggested that yields are subject to translational control. Analysis of the 5´ untranslated region (UTR) of FPS1 revealed two upstream open reading frames (uORFs). Mutagenesis data suggest that high yielding strains may circumvent these control elements through either a leaky scanning or a re-initiation mechanism. In the second theme, the dipeptide L-carnosine (ß-alanyl-L-histidine) was investigated: it has previously been shown to inhibit the growth of cancer cells but delay senescence in cultured human fibroblasts and extend the lifespan of male fruit flies. To understand these apparently contradictory properties, the effects of L-carnosine on yeast were studied. S. cerevisiae can respire aerobically when grown on a non-fermentable carbon source as a substrate but has a respiro-fermentative metabolism when grown on a fermentable carbon source; these metabolisms mimic normal cell and cancerous cell metabolisms, respectively. When yeast were grown on fermentable carbon sources, in the presence of L-carnosine, a reduction in cell growth and viability was observed, which was not apparent for cells grown on a non-fermentable carbon source. The metabolism-dependent mechanism was confirmed in the respiratory yeast species Pichia pastoris. Further analysis of S. cerevisiae yeast strains with deletions in their nutrient-sensing pathway, which result in an increase in respiratory metabolism, confirmed the metabolism-dependent effects of L-carnosine.

The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture

The adult human intervertebral disc (IVD) is normally avascular. Changes to the extracellular matrix in degenerative disc disease may promote vascularisation and subsequently alter cell nutrition and disc homeostasis. This study examines the influence of cell density and the presence of glucose and serum on the proliferation and survival of IVD cells in 3D culture. Bovine nucleus pulposus (NP) cells were seeded at a range of cell densities (1.25 × 10(5)-10(6) cells/mL) and cultured in alginate beads under standard culture conditions (with 3.15 g/L glucose and 10 % serum), or without glucose and/or 20% serum. Cell proliferation, apoptosis and cell senescence were examined after 8 days in culture. Under standard culture conditions, NP cell proliferation and cluster formation was inversely related to cell seeding density, whilst the number of apoptotic cells and enucleated "ghost" cells was positively correlated to cell seeding density. Increasing serum levels from 10% to 20% was associated with increased cluster size and also an increased prevalence of apoptotic cells within clusters. Omitting glucose produced even larger clusters and also more apoptotic and senescent cells. These studies demonstrate that NP cell growth and survival are influenced both by cell density and the availability of serum or nutrients, such as glucose. The observation of clustered, senescent, apoptotic or "ghost" cells in vitro suggests that environmental factors may influence the formation of these phenotypes that have been previously reported in vivo. Hence this study has implications for both our understanding of degenerative disc disease and also cell-based therapy using cells cultured in vitro.

Antimicrobial efficacy of copper surfaces against spores and vegetative cells of Clostridium difficile: the germination theory

OBJECTIVES: Persistent contamination of surfaces by spores of Clostridium difficile is a major factor influencing the spread of C. difficile-associated diarrhoea (CDAD) in the clinical setting. In recent years, the antimicrobial efficacy of metal surfaces has been investigated against microorganisms including methicillin-resistant Staphylococcus aureus. This study compared the survival of C. difficile on stainless steel, a metal contact surface widely used in hospitals, and copper surfaces. METHODS: Antimicrobial efficacy was assessed using a carrier test method against dormant spores, germinating spores and vegetative cells of C. difficile (NCTC 11204 and ribotype 027) over a 3 h period in the presence and absence of organic matter. RESULTS: Copper metal eliminated all vegetative cells of C. difficile within 30 min, compared with stainless steel which demonstrated no antimicrobial activity (P < 0.05). Copper significantly reduced the viability of spores of C. difficile exposed to the germinant (sodium taurocholate) in aerobic conditions within 60 min (P < 0.05) while achieving a >or=2.5 log reduction (99.8% reduction) at 3 h. Organic material did not reduce the antimicrobial efficacy of the copper surface (P > 0.05).

Nrf2 activation supports cell survival during hypoxia and hypoxia/reoxygenation in cardiomyoblasts; the roles of reactive oxygen and nitrogen species

Adaptive mechanisms involving upregulation of cytoprotective genes under the control of transcription factors such as Nrf2 exist to protect cells from permanent damage and dysfunction under stress conditions. Here we explore of the hypothesis that Nrf2 activation by reactive oxygen and nitrogen species modulates cytotoxicity during hypoxia (H) with and without reoxygenation (H/R) in H9C2 cardiomyoblasts. Using MnTBap as a cell permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger and L-NAME as an inhibitor of nitric oxide synthase (NOS), we have shown that MnTBap inhibited the cytotoxic effects of hypoxic stress with and without reoxygenation. However, L-NAME only afforded protection during H. Under reoxygenation, conditions, cytotoxicity was increased by the presence of L-NAME. Nrf2 activation was inhibited independently by MnTBap and L-NAME under H and H/R. The increased cytotoxicity and inhibition of Nrf2 activation by the presence of L-NAME during reoxygenation suggests that NOS activity plays an important role in cell survival at least in part via Nrf2-independent pathways. In contrast, O2 -• scavenging by MnTBap prevented both toxicity and Nrf2 activation during H and H/R implying that toxicity is largely dependent on O2 -.To confirm the importance of Nrf2 for myoblast metabolism, Nrf2 knockdown with siRNA reduced cell survival by 50% during 4h hypoxia with and without 2h of reoxygenation and although cellular glutathione (GSH) was depleted during H and H/R, GSH loss was not exacerbated by Nrf2 knockdown. These data support distinctive roles for ROS and RNS during H and H/R for Nrf2 induction which are important for survival independently of GSH salvage. © 2013 The Authors.

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.