Quantification of multiple steroid hormones in serum and human breast cancer tissue by liquid chromatography-tandem mass spectrometry analysis

IntroductionSystemic and local steroid hormone levels may function as novel prognostic and predictive biomarkers in breast cancer patients. We aimed at developing a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of multiple, biologically pivotal steroid hormones in human serum and breast cancer tissue.MethodsThe quantitative method consisted of liquid-liquid extraction, Sephadex LH-20 chromatography for tissue extracts, and analysis of steroid hormones by liquid-chromatography-tandem mass spectrometry. We analyzed serum and tissue steroid hormone levels in 16 and 40 breast cancer patients, respectively, and assessed their correlations with clinical parameters.ResultsThe method included quantification of nine steroid hormones in serum [including cortisol, cortisone, corticosterone, estrone (E1), 17β-estradiol (E2), 17α-hydroxyprogesterone, androstenedione (A4), testosterone and progesterone) and six (including cortisone, corticosterone, E1, E2, A4, and testosterone) in cancer tissue. The lower limits of quantification were between 0.003–10 ng/ml for serum (250 µl) and 0.038–125 pg/mg for tissue (20 mg), respectively. Accuracy was between 98%-126%, intra-assay coefficient of variations (CV) was below 15%, and inter-assay CV were below 11%. The analytical recoveries for tissue were between 76%-110%. Tissue levels of E1 were positively correlated with tissue E2 levels (p<0.001), and with serum levels of E1, E2 and A4 (p<0.01). Tissue E2 levels were positively associated with serum E1 levels (p=0.02), but not with serum E2 levels (p=0.12). The levels of tissue E2 and ratios of E1 to A4 levels (an index for aromatase activity) were significantly higher in patients with larger tumors (p=0.03 and p=0.02, respectively).ConclusionsThe method was convenient and suitable for a specific and accurate profiling of clinically important steroid hormones in serum. However, the sensitivity of the profile method in steroid analysis in tissue samples is limited, but it can be used for the analysis of steroids in breast cancer tissues if the size of the sample or its steroid content is sufficient

Toxicity, Bioaccumulation and Depuration of Bromoform in Five Marine Species

Bromoform has been identified as the single most abundant halogenated organic compound produced by the chlorination of marine waters. To determine the potential biological effects of its release into marine waters, short-term toxicity bioassays and 28-day uptake/28-day depuration studies were conducted with five marine species: Protothaca staminea, Mercenaria mercenaria, Crassostrea virginica, Penaeus aztecus, and Brevoortia tyrannus. The bioassay studies indicate that 96-hr LC50s ranged from approximately 7 ppm for B. tyrannus to greater than 40 ppm for P. staminea. Behavioral changes were noted in P. aztecus and B. tyrannus exposed to sublethal concentrations of bromoform. In all species tested, the uptake and depuration of bromoform was rapid. Bromoform was present in all exposed animal tissues within 24 hours and was depurated within 48 hours. In the mollusk species, there was bioaccumulation above water concentrations in the first week of exposure, and then the tissue concentrations fell to levels approximately equal to the water concentrations. The shrimp and menhaden also bioaccumulated bromoform above water concentrations in the first week of exposure, but then the tissue concentrations fell to approximately 0.4 {micro}g/g and remained at this level independent of water concentrations

The Bathypelagic Biome of the Atlantic Ocean: Character and Ecological Discreteness of the Fish Fauna

Recent global synthetic analyses have revealed that marine taxonomic inventories are far from complete, nowhere more so than in the deep-pelagic ocean. At over a billion km3, it is the largest biome on Earth, yet only a tiny fraction of the biogeographic records include the bathypelagic fauna. This data gap served as the impetus for recent deepwater surveys, many of which have altered our perceptions of pelagic ecosystems. Here we examine data from four deep-pelagic (0-5000+ m) sampling programs in the Atlantic (60°N-25°S) in order to assess the character of bathypelagic fish communities with respect to faunal distinctiveness and ecological connectivity. Regions studied include the Gulf of Mexico, Sargasso 702 Sea, eastern North/South Atlantic, and mid-North Atlantic. Quantitative analyses give contrasting pictures with respect to faunal composition and ecosystem operation. The discreteness of the bathypelagic zone is exhibited faunistically by the suite of ―holobathypelagic‖ species found only below 1000 m, most of which are highly modified morphologically. Geometric abundance class analyses reveal that the character of relative species abundance distributions between the meso- and bathypelagic zones is fundamentally dissimilar; the former exhibit a much higher proportion of common species, while the latter exhibit a much higher percentage of rarer species. From a community energetics perspective, however, the bathy- and mesopelagic zones are highly interconnected. Approximately 70% of fish species collected below 1000 m are also found in the mesopelagic zone, and in the far North Atlantic, are also found in the epipelagial. These species comprised 66 to \u3e90% of individuals collected below 1000 m in the regions sampled. In the mid-North Atlantic, these species contribute to the unexpected water-column biomass maximum observed between 1500-2300 m. Thus, the ―transient‖ taxa (primarily mesopelagic migrators and spanner taxa) add considerably to the ichthyofaunal diversity of the world ocean below 1000 m, and appear to be the vectors that support the diverse array of holobathypelagic fishes whose taxonomic composition is dominated by piscivores. Data from the four regions studied suggests that classic pelagic biogeographic boundaries do not apply to bathypelagic realm, as shared species are the rule rather than the exception. Last, cumulative species curves suggest we are far from understanding the true complexity of the bathypelagic zone

Formative evaluation of electricity distribution utilities using data envelopment analysis

The use of Data Envelopment Analysis (DEA) in the electricity distribution sector has been prolific in the number of papers published in research journals. However, while numerous studies have been documented, they have mostly been summative. Their aim has been predominantly descriptive and classificatory. This paper argues that evaluations of a formative nature are more effective than summative studies in promoting a better understanding of the structures and processes of electricity distribution utilities and, consequently, are more appropriate to contribute to performance improvement. To illustrate the use of DEA for formative evaluation, and highlight some of the difficulties of using DEA in practice, this paper compares the cost-efficiency of the Portuguese electricity distribution companies from 2002 to 2006. A dynamic analysis using Malmquist Indices is also conducted in order to evaluate the changes in productivity over this period. Our analysis shows that the application of DEA for formative purposes meets some difficulties. In particular it shows that while the modelling of productivity/efficiency scores using DEA is relatively straightforward, it is comparatively more difficult to develop models that are economically valid and that produce results with face validity. On the basis of the insights derived from this analysis, the paper provides some recommendations regarding the successful application of DEA for performance improvement

Bathypelagic Fish Association with the Mid-Atlantic Ridge

The bathypelagic zone, Earth’s largest living space, is essentially boundless in three dimensions for most of its extent, structured only by fluid features (e.g., salinity, temperature) of the seawater itself. However, near certain topographic features this zone intersects the seafloor. The mid-ocean ridge system is by far the largest of these features. Unlike the ecosystems of the continental margins, the mid-ocean ridge systems do not receive terrigenous nutrient inputs. Thus, the deep-water fauna associated with mid-ocean ridges ultimately depend on the generally limited local surface production. Despite this limited surface production, there is evidence that near-ridge demersal fish biomass is increased above the mid-Atlantic Ridge (MAR). Two processes by which organic matter can be transferred to the benthic boundary layer include: 1) sinking of aggregates and the carcasses of larger animals, and 2) vertical migration of living animals. To understand the dynamics of the latter process, deep-pelagic and demersal fishes were studied during the 2004 G.O. Sars Expedition, a field campaign of MAR-ECO. MAR-ECO, a Census of Marine Life project, is an international study of the animals inhabiting the northern Mid-Atlantic. Utilizing multiple technologies the water column (to 3500 m) and benthic realms were sampled. Taxonomic analysis to date has revealed over 300 fish species, with ongoing analysis expected to reveal more species, some new to science. Pelagic sampling collected 207 species, with typical orders dominating. Bottom trawling collected ca. 175 species, with typical demersal families, but also pelagic families occurring in numbers higher than would be expected by contamination alone. Discrete, near-bottom pelagic trawls confirmed this observation. In all, 84 species were caught in both pelagic and bottom trawls, with some species showing enhanced abundances in the near-bottom boundary layer, suggesting that overlap of deep-pelagic and demersal faunas is likely a key process regulating mid-ocean ridge community structure

GITR signaling potentiates airway hyperresponsiveness by enhancing Th2 cell activity in a mouse model of asthma

<p>Abstract</p> <p>Background</p> <p>Allergic asthma is characterized by airway hyperresponsiveness (AHR) and allergic inflammation of the airways, driven by allergen-specific Th2 cells. The asthma phenotypes and especially AHR are sensitive to the presence and activity of regulatory T (Treg) cells in the lung. Glucocorticoid-induced tumor necrosis factor receptor (GITR) is known to have a co-stimulatory function on effector CD4⁺T cells, rendering these cells insensitive to Treg suppression. However, the effects of GITR signaling on polarized Th1 and Th2 cell effector functions are not well-established. We sought to evaluate the effect of GITR signaling on fully differentiated Th1 and Th2 cells and to determine the effects of GITR activation at the time of allergen provocation on AHR and airway inflammation in a Th2-driven mouse model of asthma.</p> <p>Methods</p> <p>CD4⁺CD25^-cells were polarized <it>in vitro </it>into Th1 and Th2 effector cells, and re-stimulated in the presence of GITR agonistic antibodies to assess the effect on IFNγ and IL-4 production. To evaluate the effects of GITR stimulation on AHR and allergic inflammation in a mouse asthma model, BALB/c mice were sensitized to OVA followed by airway challenges in the presence or absence of GITR agonist antibodies.</p> <p>Results</p> <p>GITR engagement potentiated cytokine release from CD3/CD28-stimulated Th2 but not Th1 cells <it>in vitro</it>. In the mouse asthma model, GITR triggering at the time of challenge induced enhanced airway hyperresponsiveness, serum IgE and <it>ex vivo </it>Th2 cytokine release, but did not increase BAL eosinophilia.</p> <p>Conclusion</p> <p>GITR exerts a differential effect on cytokine release of fully differentiated Th1 and Th2 cells <it>in vitro</it>, potentiating Th2 but not Th1 cytokine production. This effect on Th2 effector functions was also observed <it>in vivo </it>in our mouse model of asthma, resulting in enhanced AHR, serum IgE responses and Th2 cytokine production. This is the first report showing the effects of GITR activation on cytokine production by polarized primary Th1 and Th2 populations and the relevance of this pathway for AHR in mouse models for asthma. Our data provides crucial information on the mode of action of the GITR signaling, a pathway which is currently being considered for therapeutic intervention.</p

Deep-Sea Fishes of the Mid-Atlantic Ridge: Trophic Structure and Interactions

Because deep-sea fisheries are increasing as coastal fisheries decline, fisheries scientists need baseline data on deep-sea ecosystems prior to further development of deep-water fisheries. We present preliminary results and ongoing efforts to characterize the trophic structure and energy flow of the pelagic ecosystems of the northern Mid-Atlantic Ridge, from Iceland to the Azores. This study is one component of the international CoML field project MAR-ECO (www.mar-eco.no). We found a diverse deep-pelagic fish fauna (205 spp.), with unexpectedly high bathypelagic fish biomass and spatial complexity. Based on literature reports of species present, crustacean planktivory is the dominant trophic guild (79% of individuals 47% of species), primarily within the mesopelagial. Gelativory was second (12% ind., 4% spp.), primarily within the bathypelagial. Omnivory (3%, 13%), shrimpivory (2%, 4%), and piscivory (1%, 21%) were the remaining major feeding guilds. The diets of 22 spp., primarily bathypelagic, are unknown. Based on stable isotope analysis of fish tissue, two distinct trophic modes were identified: a mode at trophic level 4 (18%; crustacean and gelatinous planktivory) and a mode at trophic level 4.5-5.0 (58%; crustacean planktivory and piscivory). The top piscivores were bathypelagic fishes. In terms of fish biomass, the gelativorous taxa dominated, followed by crustacean zooplanktivores and piscivores. Quantitative comparisons of the different trophic pathways are not possible at present, given our limited knowledge of feeding rates of most species. However, microscopical and molecular analyses are currently ongoing to fill this void, including development of techniques to identify/quantify gelatinous prey as an alternate trophic pathway

Expression of NDRG2 is down-regulated in high-risk adenomas and colorectal carcinoma

<p>Abstract</p> <p>Background</p> <p>It has recently been shown that <it>NDRG2 </it>mRNA is down-regulated or undetectable in several human cancers and cancer cell-lines. Although the function of NDRG2 is unknown, high <it>NDRG2 </it>expression correlates with improved prognosis in high-grade gliomas. The aim of this study has been to examine <it>NDRG2 </it>mRNA expression in colon cancer. By examining affected and normal tissue from individuals with colorectal adenomas and carcinomas, as well as in healthy individuals, we aim to determine whether and at which stages <it>NDRG2 </it>down-regulation occurs during colonic carcinogenesis.</p> <p>Methods</p> <p>Using quantitative RT-PCR, we have determined the mRNA levels for <it>NDRG2 </it>in low-risk (n = 15) and high-risk adenomas (n = 57), colorectal carcinomas (n = 50) and corresponding normal tissue, as well as control tissue from healthy individuals (n = 15). <it>NDRG2 </it>levels were normalised to <it>β-actin</it>.</p> <p>Results</p> <p><it>NDRG2 </it>mRNA levels were lower in colorectal carcinomas compared to normal tissue from the control group (p < 0.001). When comparing adenomas/carcinomas with adjacent normal tissue from the same individual, <it>NDRG2 </it>expression levels were significantly reduced in both high-risk adenoma (p < 0.001) and in colorectal carcinoma (p < 0.001). There was a trend for <it>NDRG2 </it>levels to decrease with increasing Dukes' stage (p < 0.05).</p> <p>Conclusion</p> <p>Our results demonstrate that expression of <it>NDRG2 </it>is down-regulated at a late stage during colorectal carcinogensis. Future studies are needed to address whether <it>NDRG2 </it>down-regulation is a cause or consequence of the progression of colorectal adenomas to carcinoma.</p

Monocyte Gene Expression Signature of Patients with Early Onset Coronary Artery Disease

The burden of cardiovascular disease (CVD) cannot be fully addressed by therapy targeting known pathophysiological pathways. Even with stringent control of all risk factors CVD events are only diminished by half. A number of additional pathways probably play a role in the development of CVD and might serve as novel therapeutic targets. Genome wide expression studies represent a powerful tool to identify such novel pathways. We compared the expression profiles in monocytes from twenty two young male patients with premature familial CAD with those from controls matched for age, sex and smoking status, without a family history of CVD. Since all patients were on statins and aspirin treatment, potentially affecting the expression of genes in monocytes, twelve controls were subsequently treated with simvastatin and aspirin for 6 and 2 weeks, respectively

Prognostic value of metabolic response in breast cancer patients receiving neoadjuvant chemotherapy

<p>Abstract</p> <p>Background</p> <p>Today's clinical diagnostic tools are insufficient for giving accurate prognosis to breast cancer patients. The aim of our study was to examine the tumor metabolic changes in patients with locally advanced breast cancer caused by neoadjuvant chemotherapy (NAC), relating these changes to clinical treatment response and long-term survival.</p> <p>Methods</p> <p>Patients (n = 89) participating in a randomized open-label multicenter study were allocated to receive either NAC as epirubicin or paclitaxel monotherapy. Biopsies were excised pre- and post-treatment, and analyzed by high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS). The metabolite profiles were examined by paired and unpaired multivariate methods and findings of important metabolites were confirmed by spectral integration of the metabolite peaks.</p> <p>Results</p> <p>All patients had a significant metabolic response to NAC, and pre- and post-treatment spectra could be discriminated with 87.9%/68.9% classification accuracy by paired/unpaired partial least squares discriminant analysis (PLS-DA) (<it>p </it>< 0.001). Similar metabolic responses were observed for the two chemotherapeutic agents. The metabolic responses were related to patient outcome. Non-survivors (< 5 years) had increased tumor levels of lactate (<it>p </it>= 0.004) after treatment, while survivors (≥ 5 years) experienced a decrease in the levels of glycine (<it>p </it>= 0.047) and choline-containing compounds (<it>p </it>≤ 0.013) and an increase in glucose (<it>p </it>= 0.002) levels. The metabolic responses were not related to clinical treatment response.</p> <p>Conclusions</p> <p>The differences in tumor metabolic response to NAC were associated with breast cancer survival, but not to clinical response. Monitoring metabolic responses to NAC by HR MAS MRS may provide information about tumor biology related to individual prognosis.</p