A Comparison of Azacitidine and Decitabine Activities in Acute Myeloid Leukemia Cell Lines

Background: The cytidine nucleoside analogs azacitidine (AZA) and decitabine (DAC) are used for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Few non-clinical studies have directly compared the mechanisms of action of these agents in a head-to-head fashion, and the agents are often viewed as mechanistically similar DNA hypomethylating agents. To better understand the similarities and differences in mechanisms of these drugs, we compared their in vitro effects on several end points in human AML cell lines. Methodology/Principal Findings: Both drugs effected DNA methyltransferase 1 depletion, DNA hypomethylation, and DNA damage induction, with DAC showing equivalent activity at concentrations 2- to 10-fold lower than AZA. At concentrations above 1 mM, AZA had a greater effect than DAC on reducing cell viability. Both drugs increased the sub-G1 fraction and apoptosis markers, with AZA decreasing all cell cycle phases and DAC causing an increase in G2-M. Total protein synthesis was reduced only by AZA, and drug-modulated gene expression profiles were largely non-overlapping. Conclusions/Significance: These data demonstrate shared mechanisms of action of AZA and DAC on DNA-mediated markers of activity, but distinctly different effects in their actions on cell viability, protein synthesis, cell cycle, and gene expression. The differential effects of AZA may be mediated by RNA incorporation, as the distribution of AZA in nucleic aci

Training of Instrumentalists and Development of New Technologies on SOFIA

This white paper is submitted to the Astronomy and Astrophysics 2010 Decadal Survey (Astro2010)1 Committee on the State of the Profession to emphasize the potential of the Stratospheric Observatory for Infrared Astronomy (SOFIA) to contribute to the training of instrumentalists and observers, and to related technology developments. This potential goes beyond the primary mission of SOFIA, which is to carry out unique, high priority astronomical research. SOFIA is a Boeing 747SP aircraft with a 2.5 meter telescope. It will enable astronomical observations anywhere, any time, and at most wavelengths between 0.3 microns and 1.6 mm not accessible from ground-based observatories. These attributes, accruing from the mobility and flight altitude of SOFIA, guarantee a wealth of scientific return. Its instrument teams (nine in the first generation) and guest investigators will do suborbital astronomy in a shirt-sleeve environment. The project will invest $10M per year in science instrument development over a lifetime of 20 years. This, frequent flight opportunities, and operation that enables rapid changes of science instruments and hands-on in-flight access to the instruments, assure a unique and extensive potential - both for training young instrumentalists and for encouraging and deploying nascent technologies. Novel instruments covering optical, infrared, and submillimeter bands can be developed for and tested on SOFIA by their developers (including apprentices) for their own observations and for those of guest observers, to validate technologies and maximize observational effectiveness.Comment: 10 pages, no figures, White Paper for Astro 2010 Survey Committee on State of the Professio

Proteomic identification and characterization of hepatic glyoxalase 1 dysregulation in non-alcoholic fatty liver disease

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. However, its molecular pathogenesis is incompletely characterized and clinical biomarkers remain scarce. The aims of these experiments were to identify and characterize liver protein alterations in an animal model of early, diet-related, liver injury and to assess novel candidate biomarkers in NAFLD patients. Methods: Liver membrane and cytosolic protein fractions from high fat fed apolipoprotein E knockout (ApoE−/−) animals were analyzed by quantitative proteomics, utilizing isobaric tags for relative and absolute quantitation (iTRAQ) combined with nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Differential protein expression was confirmed independently by immunoblotting and immunohistochemistry in both murine tissue and biopsies from paediatric NAFLD patients. Candidate biomarkers were analyzed by enzyme-linked immunosorbent assay in serum from adult NAFLD patients. Results: Through proteomic profiling, we identified decreased expression of hepatic glyoxalase 1 (GLO1) in a murine model. GLO1 protein expression was also found altered in tissue biopsies from paediatric NAFLD patients. In vitro experiments demonstrated that, in response to lipid loading in hepatocytes, GLO1 is first hyperacetylated then ubiquitinated and degraded, leading to an increase in reactive methylglyoxal. In a cohort of 59 biopsy-confirmed adult NAFLD patients, increased serum levels of the primary methylglyoxal-derived advanced glycation endproduct, hydroimidazolone (MG-H1) were significantly correlated with body mass index (r = 0.520, p < 0.0001). Conclusion: Collectively these results demonstrate the dysregulation of GLO1 in NAFLD and implicate the acetylation-ubquitination degradation pathway as the functional mechanism. Further investigation of the role of GLO1 in the molecular pathogenesis of NAFLD is warranted. Keywords: Non-alcoholic fatty liver disease, Glyoxalase, Methylglyoxal, Proteomics, iTRA

Pyruvate: immunonutritional effects on neutrophil intracellular amino or alpha-keto acid profiles and reactive oxygen species production

For the first time the immunonutritional role of pyruvate on neutrophils (PMN), free α-keto and amino acid profiles, important reactive oxygen species (ROS) produced [superoxide anion (O2−), hydrogen peroxide (H2O2)] as well as released myeloperoxidase (MPO) acitivity has been investigated. Exogenous pyruvate significantly increased PMN pyruvate, α-ketoglutarate, asparagine, glutamine, aspartate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Moreover, increases in O2− formation, H2O2-generation and MPO acitivity in parallel with intracellular pyruvate changes have also been detected. Regarding the interesting findings presented here we believe, that pyruvate fulfils considerably the criteria for a potent immunonutritional molecule in the regulation of the PMN dynamic α-keto and amino acid pools. Moreover it also plays an important role in parallel modulation of the granulocyte-dependent innate immune regulation. Although further research is necessary to clarify pyruvate’s sole therapeutical role in critically ill patients’ immunonutrition, the first scientific successes seem to be very promising

Variations in killer-cell immunoglobulin-like receptor and human leukocyte antigen genes and immunity to malaria

Malaria is one of the deadliest infectious diseases in the world. Immune responses to Plasmodium falciparum malaria vary among individuals and between populations. Human genetic variation in immune system genes is likely to play a role in this heterogeneity. Natural killer (NK) cells produce inflammatory cytokines in response to malaria infection, kill intraerythrocytic Plasmodium falciparum parasites by cytolysis, and participate in the initiation and development of adaptive immune responses to plasmodial infection. These functions are modulated by interactions between killer-cell immunoglobulin-like receptors (KIR) and human leukocyte antigens (HLA). Therefore, variations in KIR and HLA genes can have a direct impact on NK cell functions. Understanding the role of KIR and HLA in immunity to malaria can help to better characterize antimalarial immune responses. In this review, we summarize the different KIR and HLA so far associated with immunity to malaria.This work was supported through the DELTAS Africa Initiative (Grant no. 107743), that funded Stephen Tukwasibwe through PhD fellowship award, and Annettee Nakimuli through group leader award. The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Science (AAS), Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust (Grant no. 107743) and the UK government. Francesco Colucci is funded by Wellcome Trust grant 200841/Z/16/Z. The project received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. 695551) for James Traherne and John Trowsdale. Jyothi Jayaraman is a recipient of fellowship from the Centre for Trophoblast Research

Novel Robust Hepatitis C Virus Mouse Efficacy Model

The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line (T7-11). The model was validated by demonstrating that both a small-molecule NS3/4A protease inhibitor (BILN 2061) and human alpha interferon (IFN-α) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. We further showed that protease inhibitor and IFN-α combination therapy was more effective in reducing HCV RNA replication than treatment with each compound alone and supports testing in humans. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo as part of aggressive drug discovery efforts