Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonella spp. in food.

BACKGROUND: Although a variety of methodologies are available for detection of Salmonella, sensitive, specific, and efficient methods are urgently needed for differentiation of live Salmonella cells from dead cells in food and environmental samples. Propidium monoazide (PMA) can preferentially penetrate the compromised membranes of dead cells and inhibit their DNA amplification, however, such inhibition has been reported to be incomplete by some studies. In the present study, we report an efficient qPCR assay targeting a conserved region of the invA gene of Salmonella in conjunction with PMA treatment for detection of DNA from live Salmonella cells in food samples. RESULTS: We investigated the relationship between amplicon length and inhibitory effect of PMA treatment to prevent DNA amplification from dead cells while allowing for DNA amplification from live cells, and found that the two factors are well correlated with each other. An amplicon that is 130 bp in length was determined to be optimal for PMA treatment and was selected for further PMA-qPCR assay development. A PMA-qPCR assay was established by utilizing this amplicon and adopting a modified PMA-treatment procedure. The PMA-qPCR assay provided excellent inhibition of DNA amplification from dead cells (a 17-CT-value, or 128,000-fold reduction) while only a slight DNA amplification difference (0.5 CT value) was noted between the PMA-treated and untreated live cells. This assay has been validated through stringent inclusivity and exclusivity studies using a large number of (n = 167) Salmonella, including all strains of SARA and SARB collections, and non-Salmonella strains (n = 36). This PMA-qPCR assay is capable of detecting live Salmonella cells in live/dead cell mixtures, or 30 CFU/g live Salmonella cells from enriched spiked spinach samples as early as 4 h. CONCLUSIONS: A 130-bp amplicon in invA gene was demonstrated to be optimal for PMA treatment for selective detection of live Salmonella cells by PCR. This PMA-qPCR assay provides a sensitive, specific, and efficient method for detecting live Salmonella cells in foods and environmental samples and may have an impact on the accurate microbiological monitoring of Salmonella in foods and environment samples.

Dysregulation of glucose transport, glycolysis, TCA cycle and glutaminolysis by oncogenes and tumor suppressors in cancer cells.

A common set of functional characteristics of cancer cells is that cancer cells consume a large amount of glucose, maintain high rate of glycolysis and convert a majority of glucose into lactic acid even in the presence of oxygen compared to that of normal cells (Warburg's Effects). In addition, cancer cells exhibit substantial alterations in several energy metabolism pathways including glucose transport, tricarboxylic acid (TCA) cycle, glutaminolysis, mitochondrial respiratory chain oxidative phosphorylation and pentose phosphate pathway (PPP). In the present work, we focused on reviewing the current knowledge about the dysregulation of the proteins/enzymes involved in the key regulatory steps of glucose transport, glycolysis, TCA cycle and glutaminolysis by several oncogenes including c-Myc and hypoxia inducible factor-1 (HIF-1) and tumor suppressor, p53, in cancer cells. The dysregulation of glucose transport and energy metabolism pathways by oncogenes and lost functions of the tumor suppressors have been implicated as important biomarkers for cancer detection and as valuable targets for the development of new anticancer therapies.

Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker.

The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.

Regulation of mitochondrial respiratory chain biogenesis by estrogens/estrogen receptors and physiological, pathological and pharmacological implications.

There has been increasing evidence pointing to the mitochondrial respiratory chain (MRC) as a novel and important target for the actions of 17beta-estradiol (E(2)) and estrogen receptors (ER) in a number of cell types and tissues that have high demands for mitochondrial energy metabolism. This novel E(2)-mediated mitochondrial pathway involves the cooperation of both nuclear and mitochondrial ERalpha and ERbeta and their co-activators on the coordinate regulation of both nuclear DNA- and mitochondrial DNA-encoded genes for MRC proteins. In this paper, we have: 1) comprehensively reviewed studies that reveal a novel role of estrogens and ERs in the regulation of MRC biogenesis; 2) discussed their physiological, pathological and pharmacological implications in the control of cell proliferation and apoptosis in relation to estrogen-mediated carcinogenesis, anti-cancer drug resistance in human breast cancer cells, neuroprotection for Alzheimer's disease and Parkinson's disease in brain, cardiovascular protection in human heart and their beneficial effects in lens physiology related to cataract in the eye; and 3) pointed out new research directions to address the key questions in this important and newly emerging area. We also suggest a novel conceptual approach that will contribute to innovative regimens for the prevention or treatment of a wide variety of medical complications based on E(2)/ER-mediated MRC biogenesis pathway.

ERalpha-negative and triple negative breast cancer: molecular features and potential therapeutic approaches.

Triple negative breast cancer (TNBC) is a type of aggressive breast cancer lacking the expression of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor-2 (HER-2). TNBC patients account for approximately 15% of total breast cancer patients and are more prevalent among young African, African-American and Latino women patients. The currently available ER-targeted and Her-2-based therapies are not effective for treating TNBC. Recent studies have revealed a number of novel features of TNBC. In the present work, we comprehensively addressed these features and discussed potential therapeutic approaches based on these features for TNBC, with particular focus on: 1) the pathological features of TNBC/basal-like breast cancer; 2) E(2)/ERbeta-mediated signaling pathways; 3) G-protein coupling receptor-30/epithelial growth factor receptor (GPCR-30/EGFR) signaling pathway; 4) interactions of ERbeta with breast cancer 1/2 (BRCA1/2); 5) chemokine CXCL8 and related chemokines; 6) altered microRNA signatures and suppression of ERalpha expression/ERalpha-signaling by micro-RNAs; 7) altered expression of several pro-oncongenic and tumor suppressor proteins; and 8) genotoxic effects caused by oxidative estrogen metabolites. Gaining better insights into these molecular pathways in TNBC may lead to identification of novel biomarkers and targets for development of diagnostic and therapeutic approaches for prevention and treatment of TNBC.

Regulation of energy metabolism pathways by estrogens and estrogenic chemicals and potential implications in obesity associated with increased exposure to endocrine disruptors.

The prevalence of obesity among children, adolescents and adults has been dramatically increasing worldwide during the last several decades. The obesity epidemic has been recognized as one of the major global health problems, because its health hazard is linked to a number of common diseases including breast and prostate cancers. Obesity is caused by combination of genetic and environmental factors. While genetic contribution to obesity has been known to be significant, the genetic factors remain relatively unchanged. Recent studies have highlighted the involvement of environmental "obesogens", i.e. the xenobiotic chemicals that can disrupt the normal development and homeostatic control over adipogenesis and energy balance. Several lines of evidence suggest that increasing exposure to chemicals with endocrine-disrupting activities (endocrine-disrupting chemicals, EDCs) contributes to the increased obesity. The cellular and molecular mechanisms underlying obesogen-associated obesity are just now being appreciated. In this paper, we comprehensively reviewed current knowledge about the role of estrogen receptors alpha and beta (ERalpha and ERbeta) in regulation of energy metabolism pathways, including glucose transport, glycolysis, tricarboxylic acid (TCA) cycle, mitochondrial respiratory chain (MRC), adenosine nucleotide translocator (ANT) and fatty acid beta-oxidation and synthesis, by estrogens; and then examined the disturbance of E(2)/ER-mediated energy metabolism pathways by environmental obesogens; and finally, we discussed the potential implications of disturbance of energy metabolism pathways by obesogens in obesity and pointed out several key aspects of this area that need to be further explored. A better understanding of the cellular and molecular mechanisms underlying obesogen-associated obesity will lead to new approaches for slow down and/or prevention of the increased trend of obesity associated with exposure to obesogens.

Mechanisms of hormone carcinogenesis: evolution of views, role of mitochondria.

CumuIative and excessive exposure to estrogens is associated with increased breast cancer risk. The traditional mechanism explaining this association is that estrogens affect the rate of cell division and apoptosis and thus manifest their effect on the risk of breast cancer by affecting the growth of breast epithelial tissues. Highly proliferative cells are susceptible to genetic errors during DNA replication. The action of estrogen metabolites offers a complementary genotoxic pathway mediated by the generation of reactive estrogen quinone metabolites that can form adducts with DNA and generate reactive oxygen species through redox cycling. In this chapter, we discussed a novel mitochondrial pathway mediated by estrogens and their cognate estrogen receptors (ERs) and its potential implications in estrogen-dependent carcinogenesis. Several lines of evidence are presented to show: (1) mitochondrial localization of ERs in human breast cancer cells and other cell types; (2) a functional role for the mitochondrial ERs in regulation of the mitochondrial respiratory chain (MRC) proteins and (3) potential implications of the mitochondrial ER-mediated pathway in stimulation of cell proliferation, inhibition of apoptosis and oxidative damage to mitochondrial DNA. The possible involvement of estrogens and ERs in deregulation of mitochondrial bioenergetics, an important hallmark of cancer cells, is also described. An evolutionary view is presented to suggest that persistent stimulation by estrogens through ER signaling pathways of MRC proteins and energy metabolic pathways leads to the alterations in mitochondrial bioenergetics and contributes to the development of estrogen-related cancers.

ERbeta shifts from mitochondria to nucleus during estrogen-induced neoplastic transformation of human breast epithelial cells and is involved in estrogen-induced synthesis of mitochondrial respiratory chain proteins.

Both estrogen receptors (ER) alpha (ERalpha) and beta (ERbeta) are localized in the nucleus, plasma membrane, and mitochondria, where they mediate the different physiological effects of estrogens. It has been observed that the relative subcellular localization of ERs is altered in several cancer cells. We have demonstrated that MCF-10F cells, the immortal and non-tumorigenic human breast epithelial cells (HBEC) that are ERalpha-negative and ERbeta-positive, are transformed in vitro by 17beta-estradiol (E(2)), generating highly invasive cells that are tumorigenic in severe combined immunodeficient mice. E(2)-transformed MCF-10F (trMCF) cells exhibit progressive loss of ductulogenesis, invasive (bsMCF) and tumorigenic (caMCF) phenotypes. Immunolocalization of ERbeta by confocal fluorescent microscopy and electron microscopy revealed that ERbeta is predominantly localized in mitochondria of MCF-10F and trMCF cells. Silencing ERbeta expression with ERbeta-specific small interference RNA (siRNA-ERbeta) markedly diminishes both nuclear and mitochondrial ERbeta in MCF-10F cells. The ERbeta shifts from its predominant localization in the mitochondria of MCF-10F and trMCF cells to the nucleus of bsMCF cells, becoming predominantly nuclear in caMCF cells. Furthermore, we demonstrated that the mitochondrial ERbeta in MCF-10F cells is involved in E(2)-induced expression of mitochondrial DNA (mtDNA)-encoded respiratory chain (MRC) proteins. This is the first report of an association of changes in the subcellular localization of ERbeta with various stages of E(2)-induced transformation of HBEC and a functional role of mitochondrial ERbeta in mediating E(2)-induced MRC protein synthesis. Our findings provide a new insight into one of the potential roles of ERbeta in human breast cancer.

Sodium/potassium ATPase (Na+, K+-ATPase) and ouabain/related cardiac glycosides: A new paradigm for development of anti- breast cancer drugs?

Prolonged exposure to 17beta-estradiol (E2) is a key etiological factor for human breast cancer. The biological effects and carcinogenic effects of E2 are mediated via estrogen receptors (ERs), ERalpha and ERbeta. Anti-estrogens, e.g. tamoxifen, and aromatase inhibitors have been used to treat ER-positive breast cancer. While anti-estrogen therapy is initially successful, a major problem is that most tumors develop resistance and the disease ultimately progresses, pointing to the need of developing alternative drugs targeting to other critical targets in breast cancer cells. We have identified that Na+, K+-ATPase, a plasma membrane ion pump, has unique/valuable properties that could be used as a potentially important target for breast cancer treatment: (a) it is a key player of cell adhesion and is involved in cancer progression; (b) it serves as a versatile signal transducer and is a target for a number of hormones including estrogens and (d) its aberrant expression and activity are implicated in the development and progression of breast cancer. There are several lines of evidence indicating that ouabain and related digitalis (the potent inhibitors of Na+, K+-ATPase) possess potent anti-breast cancer activity. While it is not clear how the suggested anti-cancer activity of these drugs work, several observations point to ouabain and digitalis as being potential ER antagonists. We critically reviewed many lines of evidence and postulated a novel concept that Na+, K+-ATPase in combination with ERs could be important targets of anti-breast cancer drugs. Modulators, e.g. ouabain and related digitalis could be useful to develop valuable anti-breast cancer drugs as both Na+, K+-ATPase inhibitors and ER antagonists.

Regulation of mitochondrial respiratory chain structure and function by estrogens/estrogen receptors and potential physiological/pathophysiological implications.

It is well known that the biological and carcinogenic effects of 17beta-estradiol (E2) are mediated via nuclear estrogen receptors (ERs) by regulating nuclear gene expression. Several rapid, non-nuclear genomic effects of E2 are mediated via plasma membrane-bound ERs. In addition, there is accumulating evidence suggesting that mitochondria are also important targets for the action of estrogens and ERs. This review summarized the studies on the effects of estrogens via ERs on mitochondrial structure and function. The potential physiological and pathophysiological implications of deficiency and/or overabundance of these E2/ER-mediated mitochondrial effects in stimulation of cell proliferation, inhibition of apoptosis, E2-mediated cardiovascular and neuroprotective effects in target cells are also discussed.

Estrogen's effects on mitochondrial gene expression: mechanisms and potential contributions to estrogen carcinogenesis.

Estrogen receptor (ER)alpha and ERbeta are localized in the nucleus and involved in the regulation of nuclear estrogen-responsive genes by 17beta estradiol (E2). In addition, recently others have shown that upon E2 binding, ERalpha localizes to the plasma membrane and initiates mitogen-activated protein kinase (MAPK)-mediated signal transduction. Previously, we reported that in liver, cultured rat hepatocytes and human HepG2 cells, estrogen treatment enhanced mitochondrial DNA (mtDNA)-encoded gene transcript levels. These effects were blocked by a specific antiestrogen, suggesting a role for the ER. Others have reported the presence of putative estrogen-responsive elements in mtDNA. These observations suggested the hypothesis that the ER localized in mitochondria and functioned directly to enhance the levels of mtDNA-encoded transcripts, analogous to what has been observed for the glucocorticoid hormone receptor. Using Western blot analysis, confocal immunofluorescence, immunogold electron microscopy, and gel electrophoresis mobility shift assays, we have demonstrated the estrogen-dependent presence of ERbeta and ERalpha within mitochondria of HepG2 and MCF-7 human breast tumor cells. Together, these results suggest that the ERs may act as transcription factors directly involved in the regulation by E2 of mtDNA transcription.