An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

<p>Abstract</p> <p>Background</p> <p><it>In vitro </it>cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied <it>in vitro </it>but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on <it>in vitro </it>systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the <it>in vitro </it>model system and model toxicant, respectively.</p> <p>Results</p> <p>The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD.</p> <p>Conclusions</p> <p>Untargeted profiling of the polar and apolar metabolites of <it>in vitro </it>cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.</p

Binary and Millisecond Pulsars at the New Millennium

We review the properties and applications of binary and millisecond pulsars. Our knowledge of these exciting objects has greatly increased in recent years, mainly due to successful surveys which have brought the known pulsar population to over 1300. There are now 56 binary and millisecond pulsars in the Galactic disk and a further 47 in globular clusters. This review is concerned primarily with the results and spin-offs from these surveys which are of particular interest to the relativity community.Comment: 59 pages, 26 figures, 5 tables. Accepted for publication in Living Reviews in Relativity (http://www.livingreviews.org

The Evolution of Compact Binary Star Systems

We review the formation and evolution of compact binary stars consisting of white dwarfs (WDs), neutron stars (NSs), and black holes (BHs). Binary NSs and BHs are thought to be the primary astrophysical sources of gravitational waves (GWs) within the frequency band of ground-based detectors, while compact binaries of WDs are important sources of GWs at lower frequencies to be covered by space interferometers (LISA). Major uncertainties in the current understanding of properties of NSs and BHs most relevant to the GW studies are discussed, including the treatment of the natal kicks which compact stellar remnants acquire during the core collapse of massive stars and the common envelope phase of binary evolution. We discuss the coalescence rates of binary NSs and BHs and prospects for their detections, the formation and evolution of binary WDs and their observational manifestations. Special attention is given to AM CVn-stars -- compact binaries in which the Roche lobe is filled by another WD or a low-mass partially degenerate helium-star, as these stars are thought to be the best LISA verification binary GW sources.Comment: 105 pages, 18 figure

An Ultra-Fast Metabolite Prediction Algorithm

Small molecules are central to all biological processes and metabolomics becoming an increasingly important discovery tool. Robust, accurate and efficient experimental approaches are critical to supporting and validating predictions from post-genomic studies. To accurately predict metabolic changes and dynamics, experimental design requires multiple biological replicates and usually multiple treatments. Mass spectra from each run are processed and metabolite features are extracted. Because of machine resolution and variation in replicates, one metabolite may have different implementations (values) of retention time and mass in different spectra. A major impediment to effectively utilizing untargeted metabolomics data is ensuring accurate spectral alignment, enabling precise recognition of features (metabolites) across spectra. Existing alignment algorithms use either a global merge strategy or a local merge strategy. The former delivers an accurate alignment, but lacks efficiency. The latter is fast, but often inaccurate. Here we document a new algorithm employing a technique known as quicksort. The results on both simulated data and real data show that this algorithm provides a dramatic increase in alignment speed and also improves alignment accuracy

Binary and Millisecond Pulsars

We review the main properties, demographics and applications of binary and millisecond radio pulsars. Our knowledge of these exciting objects has greatly increased in recent years, mainly due to successful surveys which have brought the known pulsar population to over 1700. There are now 80 binary and millisecond pulsars associated with the disk of our Galaxy, and a further 103 pulsars in 24 of the Galactic globular clusters. Recent highlights have been the discovery of the first ever double pulsar system and a recent flurry of discoveries in globular clusters, in particular Terzan 5.Comment: 77 pages, 30 figures, available on-line at http://www.livingreviews.org/lrr-2005-

Role of the small intestine, colon and microbiota in determining the metabolic fate of polyphenols

(Poly)phenols are a large group of compounds, found in food, beverages, dietary supplements and herbal medicines. Owing to their biological activities, absorption and metabolism of the most abundant compounds in humans are well understood. Both the chemical structure of the phenolic moiety and any attached chemical groups define whether the polyphenol is absorbed in the small intestine, or reaches the colon and is subject to extensive catabolism by colonic microbiota. Untransformed substrates may be absorbed, appearing in plasma primarily as methylated, sulfated and glucuronidated derivatives, with in some cases the unchanged substrate. Many of the catabolites are well absorbed from the colon and appear in the plasma either similarly conjugated, or as glycine conjugates, or in some cases unchanged. Although many (poly)phenol catabolites have been identified in human plasma and / or urine, the pathways from substrate to final catabolite, and the species of bacteria and enzymes involved, are still scarcely reported. While it is clear that the composition of the human gut microbiota can be modulated in vivo by supplementation with some (poly)phenol-rich commodities, such modulation is definitely not an inevitable consequence of supplementation, it depends on the treatment, length of time and on the individual metabotype, and it is not clear whether the modulation is sustained when supplementation ceases. Some catabolites have been recorded in plasma of volunteers at concentrations similar to those shown to be effective in in vitro studies suggesting that some benefit may be achieved in vivo by diets yielding such catabolites

Quantitative metabolomics based on gas chromatography mass spectrometry: status and perspectives

Metabolomics involves the unbiased quantitative and qualitative analysis of the complete set of metabolites present in cells, body fluids and tissues (the metabolome). By analyzing differences between metabolomes using biostatistics (multivariate data analysis; pattern recognition), metabolites relevant to a specific phenotypic characteristic can be identified. However, the reliability of the analytical data is a prerequisite for correct biological interpretation in metabolomics analysis. In this review the challenges in quantitative metabolomics analysis with regards to analytical as well as data preprocessing steps are discussed. Recommendations are given on how to optimize and validate comprehensive silylation-based methods from sample extraction and derivatization up to data preprocessing and how to perform quality control during metabolomics studies. The current state of method validation and data preprocessing methods used in published literature are discussed and a perspective on the future research necessary to obtain accurate quantitative data from comprehensive GC-MS data is provided

Comparing recent PTA results on the nanohertz stochastic gravitational wave background

The Australian, Chinese, European, Indian, and North American pulsar timing array (PTA) collaborations recently reported, at varying levels, evidence for the presence of a nanohertz gravitational wave background (GWB). Given that each PTA made different choices in modeling their data, we perform a comparison of the GWB and individual pulsar noise parameters across the results reported from the PTAs that constitute the International Pulsar Timing Array (IPTA). We show that despite making different modeling choices, there is no significant difference in the GWB parameters that are measured by the different PTAs, agreeing within $1\sigma$. The pulsar noise parameters are also consistent between different PTAs for the majority of the pulsars included in these analyses. We bridge the differences in modeling choices by adopting a standardized noise model for all pulsars and PTAs, finding that under this model there is a reduction in the tension in the pulsar noise parameters. As part of this reanalysis, we "extended" each PTA's data set by adding extra pulsars that were not timed by that PTA. Under these extensions, we find better constraints on the GWB amplitude and a higher signal-to-noise ratio for the Hellings and Downs correlations. These extensions serve as a prelude to the benefits offered by a full combination of data across all pulsars in the IPTA, i.e., the IPTA's Data Release 3, which will involve not just adding in additional pulsars, but also including data from all three PTAs where any given pulsar is timed by more than as single PTA.Comment: 21 pages, 9 figures, submitted to Ap

Comparing Recent Pulsar Timing Array Results on the Nanohertz Stochastic Gravitational-wave Background

The Australian, Chinese, European, Indian, and North American pulsar timing array (PTA) collaborations recently reported, at varying levels, evidence for the presence of a nanohertz gravitational-wave background (GWB). Given that each PTA made different choices in modeling their data, we perform a comparison of the GWB and individual pulsar noise parameters across the results reported from the PTAs that constitute the International Pulsar Timing Array (IPTA). We show that despite making different modeling choices, there is no significant difference in the GWB parameters that are measured by the different PTAs, agreeing within 1σ. The pulsar noise parameters are also consistent between different PTAs for the majority of the pulsars included in these analyses. We bridge the differences in modeling choices by adopting a standardized noise model for all pulsars and PTAs, finding that under this model there is a reduction in the tension in the pulsar noise parameters. As part of this reanalysis, we "extended" each PTA's data set by adding extra pulsars that were not timed by that PTA. Under these extensions, we find better constraints on the GWB amplitude and a higher signal-to-noise ratio for the Hellings–Downs correlations. These extensions serve as a prelude to the benefits offered by a full combination of data across all pulsars in the IPTA, i.e., the IPTA's Data Release 3, which will involve not just adding in additional pulsars but also including data from all three PTAs where any given pulsar is timed by more than a single PTA