A Real Time Metridia Luciferase Based Non-Invasive Reporter Assay of Mammalian Cell Viability and Cytotoxicity via the β-actin Promoter and Enhancer

Secreted reporter molecules offer a means to evaluate biological processes in real time without the need to sacrifice samples at pre-determined endpoints. Here we have adapted the secreted bioluminescent reporter gene, Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for expression in human cells (hMLuc) and placed under the control of the human β-actin promoter and enhancer. Metridia luciferase activity of stably transfected cell models corresponded linearly with cell number over a 4-log dynamic range, detecting as few as 40 cells. When compared to standard endpoint viability assays, which measure the mitochondrial dehydrogenase reduction of tetrazolium salts, the hMLuc viability assay had a broader linear range of detection, was applicable to large tissue culture vessels, and allowed the same sample to be repeatedly measured over several days. Additional studies confirmed that MLuc activity was inhibited by serum, but demonstrated that assay activity remained linear and was measurable in the serum of mice bearing subcutaneous hMLuc-expressing tumors. In summary, these comparative studies demonstrate the value of humanized Metridia luciferase as an inexpensive and non-invasive method for analyzing viable cell number, growth, tumor volume, and therapeutic response in real time

Formation of Sclerotia and Production of Indoloterpenes by Aspergillus niger and Other Species in Section Nigri

Several species in Aspergillus section Nigri have been reported to produce sclerotia on well-known growth media, such as Czapek yeast autolysate (CYA) agar, with sclerotia considered to be an important prerequisite for sexual development. However Aspergillus niger sensu stricto has not been reported to produce sclerotia, and is thought to be a purely asexual organism. Here we report, for the first time, the production of sclerotia by certain strains of Aspergillus niger when grown on CYA agar with raisins, or on other fruits or on rice. Up to 11 apolar indoloterpenes of the aflavinine type were detected by liquid chromatography and diode array and mass spectrometric detection where sclerotia were formed, including 10,23-dihydro-24,25-dehydroaflavinine. Sclerotium induction can thus be a way of inducing the production of new secondary metabolites from previously silent gene clusters. Cultivation of other species of the black aspergilli showed that raisins induced sclerotium formation by A. brasiliensis, A. floridensis A. ibericus, A. luchuensis, A. neoniger, A. trinidadensis and A. saccharolyticus for the first time

Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis

Plant defense against microbial pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene (ET). In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PLANT DEFENSIN1.2 (PDF1.2). The APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression. The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways

Oculomotor Evidence for Top-Down Control following the Initial Saccade

The goal of the current study was to investigate how salience-driven and goal-driven processes unfold during visual search over multiple eye movements. Eye movements were recorded while observers searched for a target, which was located on (Experiment 1) or defined as (Experiment 2) a specific orientation singleton. This singleton could either be the most, medium, or least salient element in the display. Results were analyzed as a function of response time separately for initial and second eye movements. Irrespective of the search task, initial saccades elicited shortly after the onset of the search display were primarily salience-driven whereas initial saccades elicited after approximately 250 ms were completely unaffected by salience. Initial saccades were increasingly guided in line with task requirements with increasing response times. Second saccades were completely unaffected by salience and were consistently goal-driven, irrespective of response time. These results suggest that stimulus-salience affects the visual system only briefly after a visual image enters the brain and has no effect thereafter

Quantitative Proteomics Identify Novel miR-155 Target Proteins

Background: MicroRNAs are 22 nucleotides long non-coding RNAs and exert their function either by transcriptional or translational inhibition. Although many microRNA profiles in different tissues and disease states have already been discovered, only little is known about their target proteins. The microRNA miR-155 is deregulated in many diseases, including cancer, where it might function as an oncoMir. Methodology/Principal Findings: We employed a proteomics technique called ‘‘stable isotope labelling by amino acids in cell culture’ ’ (SILAC) allowing relative quantification to reliably identify target proteins of miR-155. Using SILAC, we identified 46 putative miR-155 target proteins, some of which were previously reported. With luciferase reporter assays, CKAP5 was confirmed as a new target of miR-155. Functional annotation of miR-155 target proteins pointed to a role in cell cycle regulation. Conclusions/Significance: To the best of our knowledge we have investigated for the first time miR-155 target proteins in the HEK293T cell line in large scale. In addition, by comparing our results to previously identified miR-155 target proteins i

Transcriptomic epidemiology of smoking: the effect of smoking on gene expression in lymphocytes

<p>Abstract</p> <p>Background</p> <p>This investigation offers insights into system-wide pathological processes induced in response to cigarette smoke exposure by determining its influences at the gene expression level.</p> <p>Methods</p> <p>We obtained genome-wide quantitative transcriptional profiles from 1,240 individuals from the San Antonio Family Heart Study, including 297 current smokers. Using lymphocyte samples, we identified 20,413 transcripts with significantly detectable expression levels, including both known and predicted genes. Correlation between smoking and gene expression levels was determined using a regression model that allows for residual genetic effects.</p> <p>Results</p> <p>With a conservative false-discovery rate of 5% we identified 323 unique genes (342 transcripts) whose expression levels were significantly correlated with smoking behavior. These genes showed significant over-representation within a range of functional categories that correspond well with known smoking-related pathologies, including immune response, cell death, cancer, natural killer cell signaling and xenobiotic metabolism.</p> <p>Conclusions</p> <p>Our results indicate that not only individual genes but entire networks of gene interaction are influenced by cigarette smoking. This is the largest <it>in vivo </it>transcriptomic epidemiological study of smoking to date and reveals the significant and comprehensive influence of cigarette smoke, as an environmental variable, on the expression of genes. The central importance of this manuscript is to provide a summary of the relationships between gene expression and smoking in this exceptionally large cross-sectional data set.</p

The anti-vaccination movement and resistance to allergen-immunotherapy: a guide for clinical allergists

Despite over a century of clinical use and a well-documented record of efficacy and safety, a growing minority in society questions the validity of vaccination and fear that this common public health intervention is the root-cause of severe health problems. This article questions whether growing public anti-vaccine sentiments might have the potential to spill-over into other therapies distinct from vaccination, namely allergen-immunotherapy. Allergen-immunotherapy shares certain medical vernacular with vaccination (e.g., allergy shots, allergy vaccines), and thus may become "guilty by association" due to these similarities. Indeed, this article demonstrates that anti-vaccine websites have begun unduly discrediting this allergy treatment regimen. Following an explanation of the anti-vaccine movement, the article aims to provide guidance on how clinicians can respond to patient fears towards allergen-immunotherapy in the clinical setting. This guide focuses on the provision of reliable information to patients in order to dispel misconceived associations between vaccination and allergen-immunotherapy, and the discussion of the risks and benefits of both therapies in order to assist patients in making autonomous decisions about their choice of allergy treatment

Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy

Background: Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. Methods: To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. Results: The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1alpha (HIF-1alpha) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. Conclusion: In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways