New Aspects of an Old Drug – Diclofenac Targets MYC and Glucose Metabolism in Tumor Cells

Non-steroidal anti-inflammatory drugs such as diclofenac exhibit potent anticancer effects. Up to now these effects were mainly attributed to its classical role as COX-inhibitor. Here we show novel COX-independent effects of diclofenac. Diclofenac significantly diminished MYC expression and modulated glucose metabolism resulting in impaired melanoma, leukemia, and carcinoma cell line proliferation in vitro and reduced melanoma growth in vivo. In contrast, the non-selective COX inhibitor aspirin and the COX-2 specific inhibitor NS-398 had no effect on MYC expression and glucose metabolism. Diclofenac significantly decreased glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) gene expression in line with a decrease in glucose uptake and lactate secretion. A significant intracellular accumulation of lactate by diclofenac preceded the observed effect on gene expression, suggesting a direct inhibitory effect of diclofenac on lactate efflux. While intracellular lactate accumulation impairs cellular proliferation and gene expression, it does not inhibit MYC expression as evidenced by the lack of MYC regulation by the MCT inhibitor α-cyano-4-hydroxycinnamic acid. Finally, in a cell line with a tetracycline-regulated c-MYC gene, diclofenac decreased proliferation both in the presence and absence of c-MYC. Thus, diclofenac targets tumor cell proliferation via two mechanisms, that is inhibition of MYC and lactate transport. Based on these results, diclofenac holds potential as a clinically applicable MYC and glycolysis inhibitor supporting established tumor therapies.     

Different Genetic Associations of the IgE Production among Fetus,Infancy and Childhood

Elevation of serum IgE levels has long been associated with allergic diseases. Many genes have been linked to IgE production, but few have been linked to the developmental aspects of genetic association with IgE production. To clarify developmental genetic association, we investigated what genes and gene-gene interactions affect IgE levels among fetus, infancy and childhood in Taiwan individuals. A birth cohort of 571 children with completion of IgE measurements from newborn to 1.5, 3, and 6 years of age was subject to genetic association analysis on the 384-customized SNPs of 159 allergy candidate genes. Fifty-three SNPs in 37 genes on innate and adaptive immunity, and stress and response were associated with IgE production. Polymorphisms of the IL13, and the HLA-DPA1 and HLA-DQA1 were, respectively, the most significantly associated with the IgE production at newborn and 6 years of age. Analyses of gene-gene interactions indentified that the combination of NPSR1, rs324981 TT with FGF1, rs2282797 CC had the highest risk (85.7%) of IgE elevation at 1.5 years of age (P = 1.46×10⁻⁴). The combination of IL13, CYFIP2 and PDE2A was significantly associated with IgE elevation at 3 years of age (P = 5.98×10⁻⁷), and the combination of CLEC2D, COLEC11 and CCL2 was significantly associated with IgE elevation at 6 years of age (P = 6.65×10⁻⁷). Our study showed that the genetic association profiles of the IgE production among fetus, infancy and childhood are different. Genetic markers for early prediction and prevention of allergic sensitization may rely on age-based genetic association profiles.     

Mice Do Not Habituate to Metabolism Cage Housing–A Three Week Study of Male BALB/c Mice

The metabolism cage is a barren, non-enriched, environment, combining a number of recognized environmental stressors. We investigated the ability of male BALB/c mice to acclimatize to this form of housing. For three weeks markers of acute and oxidative stress, as well as clinical signs of abnormality were monitored. Forced swim tests were conducted to determine whether the animals experienced behavioral despair and the serotonergic integrity was tested using an 8-OH-DPAT challenge. The metabolism cage housed mice excreted approximately tenfold higher amounts of corticosterone metabolites in feces throughout the study when compared to controls. Urinary biomarkers confirmed that these mice suffered from elevated levels of oxidative stress, and increased creatinine excretions indicated increased muscle catabolism. Changes in the core body temperature (stress-induced hyperthermia) and the fur state of the mice also indicated impaired well-being in the metabolism cage housed mice. However, monitoring body weight and feed intake was found misleading in assessing the wellbeing of mice over a longer time course, and the forced swim test was found poorly suited for studying chronic stress in mice in the present setup. In conclusion, the mice were found not to acclimatize to the metabolism cages whereby concern for animal welfare would dictate that mice should be housed in this way for as short periods as possible. The elevated degree of HPA axis activity, oxidative stress, and increased overall metabolism warrant caution when interpreting data obtained from metabolism cage housed mice, as their condition cannot be considered representative of a normal physiology.     

Efficacy of Acibenzolar‐S‐Methyl and β‐Aminobutyric Acid for Control of Downy Mildew in Greenhouse Grown Basil and Peroxidase Activity in Response to Treatment with these Compounds

Downy mildew, caused by the oomycete pathogen Peronospora belbahrii, is a devastating foliar disease of basil in the United States and worldwide. Currently there are very few chemistries or organic choices registered to control this disease. In this study, two systemic acquired resistance (SAR) inducers, acibenzolar‐S‐methyl (ASM) and β‐aminobutyric acid (BABA), were evaluated for their in vitro effects on the pathogen, for their potential to control basil downy mildew in greenhouses, and for changes in peroxidase activity in basil plants treated with these two SAR inducers. No significant inhibition of sporangial germination was detected in water agar amended with ASM at concentrations lower than 100 mg/l or with BABA at concentrations lower than 500 mg/l. Efficacy of ASM and BABA in greenhouses varied depending on the rate, method and timing of application. The area under the disease progress curve (AUDPC) of disease severity was significantly reduced compared to the non‐treated control when ASM was sprayed (in all experiments) or drenched (in one out of two experiments) pre‐, or pre‐ + post‐inoculation at rates of 25–400 mg/l. Three weekly post‐inoculation sprays of ASM at the rate of 50 mg/l reduced AUDPC by 93.0 and 47.2% when started 3 and 7 days after inoculation (DAI), respectively. The AUDPC of disease severity was also significantly reduced when BABA was sprayed pre‐ + post‐inoculation at rates of 125–500 mg/l. According to the prediction using a log‐logistic function, 50% maximum disease protection was achieved at a concentration of 27.5 mg/l of ASM. Basil plants treated with these two SAR inducers and challenged with the pathogen showed significantly higher peroxidase activity than the non‐treated control at 8 DAI. Temporally, the highest activity of peroxidase was detected at 8 DAI, decreased at 15 DAI and waned further at 23 DAI.     

Physiological and Pathological Impact of Blood Sampling by Retro-Bulbar Sinus Puncture and Facial Vein Phlebotomy in Laboratory Mice

Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals'' physiological condition, which should be considered whenever blood samples are obtained.     

Leveraging the contribution of thermodynamics in drug discovery with the help of fluorescence-based thermal shift assays

The development of new drugs with better pharmacological and safety properties mandates the optimization of several parameters. Today, potency is often used as the sole biochemical parameter to identify and select new molecules. Surprisingly, thermodynamics, which is at the core of any interaction, is rarely used in drug discovery, even though it has been suggested that the selection of scaffolds according to thermodynamic criteria may be a valuable strategy. This poor integration of thermodynamics in drug discovery might be due to difficulties in implementing calorimetry experiments despite recent technological progress in this area. In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery.     

The human serum metabolome

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca.     

DAT1 polymorphism is associated with risk taking in the Balloon Analogue Risk Task (BART)

Twin-studies suggest that a significant portion of individual differences in the propensity to take risks resides in people's genetic make-up and there is evidence that variability in dopaminergic systems relates to individual differences in risky choice. We examined the link between risk taking in a risk taking task (the Balloon Analogue Risk Task, BART) and a variable number tandem repeat (VNTR) polymorphism in the 3'UTR of the dopamine transporter gene (SLC6A3/DAT1). Behavior in BART is known to be associated with activity in striatal reward-processing regions, and DAT1 is assumed to modulate striatal dopamine levels. We find that carriers of DAT1 alleles, which presumably result in lower striatal dopamine availability, showed more risk taking, relative to carriers of the alleles associated with higher striatal dopamine availability. Our analyses suggest that the mechanism underlying this association is diminished sensitivity to rewards among those who take more risks. Overall, our results support the notion that a behavioral genetic approach can be helpful in uncovering the basis of individual differences in risk taking.     

Joint model selection of marginal mean regression and correlation structure for longitudinal data with missing outcome and covariates

This work develops a joint model selection criterion for simultaneously selecting the marginal mean regression and the correlation/covariance structure in longitudinal data analysis where both the outcome and the covariate variables may be subject to general intermittent patterns of missingness under the missing at random mechanism. The new proposal, termed “joint longitudinal information criterion” (JLIC), is based on the expected quadratic error for assessing model adequacy, and the second‐order weighted generalized estimating equation (WGEE) estimation for mean and covariance models. Simulation results reveal that JLIC outperforms existing methods performing model selection for the mean regression and the correlation structure in a two stage and hence separate manner. We apply the proposal to a longitudinal study to identify factors associated with life satisfaction in the elderly of Taiwan.