Untargeted metabolomic analysis of Rat neuroblastoma cells as a model system to study the biochemical effects of the acute administration of methamphetamine

Methamphetamine is an illicit psychostimulant drug that is linked to a number of diseases of the nervous system. The downstream biochemical effects of its primary mechanisms are not well understood, and the objective of this study was to investigate whether untargeted metabolomic analysis of an in vitro model could generate data relevant to what is already known about this drug. Rat B50 neuroblastoma cells were treated with 1 mM methamphetamine for 48 h, and both intracellular and extracellular metabolites were profiled using gas chromatography–mass spectrometry. Principal component analysis of the data identified 35 metabolites that contributed most to the difference in metabolite profiles. Of these metabolites, the most notable changes were in amino acids, with significant increases observed in glutamate, aspartate and methionine, and decreases in phenylalanine and serine. The data demonstrated that glutamate release and, subsequently, excitotoxicity and oxidative stress were important in the response of the neuronal cell to methamphetamine. Following this, the cells appeared to engage amino acid-based mechanisms to reduce glutamate levels. The potential of untargeted metabolomic analysis has been highlighted, as it has generated biochemically relevant data and identified pathways significantly affected by methamphetamine. This combination of technologies has clear uses as a model for the study of neuronal toxicology

Repetitive low intensity magnetic field stimulation in a neuronal cell line: a metabolomics study

Low intensity repetitive magnetic stimulation of neural tissue modulates neuronal excitability and has promising therapeutic potential in the treatment of neurological disorders. However, the underpinning cellular and biochemical mechanisms remain poorly understood. This study investigates the behavioural effects of low intensity repetitive magnetic stimulation (LI-rMS) at a cellular and biochemical level. We delivered LI-rMS (10 mT) at 1 Hz and 10 Hz to B50 rat neuroblastoma cells in vitro for 10 minutes and measured levels of selected metabolites immediately after stimulation. LI-rMS at both frequencies depleted selected tricarboxylic acid (TCA) cycle metabolites without affecting the main energy supplies. Furthermore, LI-rMS effects were frequency-specific with 1 Hz stimulation having stronger effects than 10 Hz. The observed depletion of metabolites suggested that higher spontaneous activity may have led to an increase in GABA release. Although the absence of organised neural circuits and other cellular contributors (e.g., excitatory neurons and glia) in the B50 cell line limits the degree to which our results can be extrapolated to the human brain, the changes we describe provide novel insights into how LI-rMS modulates neural tissue

Mimotopes and Proteome Analyses Using Human Genomic and cDNA Epitope Phage Display

In the post-genomic era, validation of candidate gene targets frequently requires proteinbased strategies. Phage display is a powerful tool to define protein-protein interactions by generating peptide binders against target antigens. Epitope phage display libraries have the potential to enrich coding exon sequences from human genomic loci. We evaluated genomic and cDNA phage display strategies to identify genes in the 5q31 Interleukin gene cluster and to enrich cell surface receptor tyrosine kinase genes from a breast cancer cDNA library. A genomic display library containing 2 × 10 6 clones with exon-sized inserts was selected with antibodies specific for human Interleukin-4 (IL-4) and Interleukin-13. The library was enriched significantly after two selection rounds and DNA sequencing revealed unique clones. One clone matched a cognate IL-4 epitope; however, the majority of clone insert sequences corresponded to E. coli genomic DNA. These bacterial sequences act as ‘mimotopes’ (mimetic sequences of the true epitope), correspond to open reading frames, generate displayed peptides, and compete for binding during phage selection. The specificity of these mimotopes for IL-4 was confirmed by competition ELISA. Other E. coli mimotopes were generated using additional antibodies. Mimotopes for a receptor tyrosine kinase gene were also selected using a breast cancer SKBR-3 cDNA phage display library, screened against an anti-erbB2 monoclonal antibody. Identification of mimotopes in genomic and cDNA phage libraries is essential for phage display-based protein validation assays and two-hybrid phage approaches that examine protein-protein interactions. The predominance of E. coli mimotopes suggests that the E. coli genome may be useful to generate peptide diversity biased towards protein coding sequences

Are the black hole masses in Narrow Line Seyfert 1 galaxies actually small?

Narrow Line Seyfert 1 galaxies (NLS1s) are generally considered peculiar objects among the broad class of Type 1 active galactic nuclei, due to the relatively small width of the broad lines, strong X-ray variability, soft X-ray continua, weak [OIII], and strong FeII line intensities. The mass M_BH of the central massive black hole (MBH) is claimed to be lighter than expected from known MBH-host galaxy scaling relations, while the accretion rate onto the MBH larger than the average value appropriate to Seyfert 1 galaxies. In this Letter, we show that NLS1 peculiar M_BH and L/L_Edd turn out to be fairly standard, provided that the broad line region is allowed to have a disc-like, rather than isotropic, geometry. Assuming that NLS1s are rather ``normal'' Seyfert 1 objects seen along the disc axis, we could estimate the typical inclination angles from the fraction of Seyfert 1 classified as NLS1s, and compute the geometrical factor relating the observed FWHM of broad lines to the virial mass of the MBH. We show that the geometrical factor can fully account for the "black hole mass deficit" observed in NLS1s, and that L/L_Edd is (on average) comparable to the value of the more common broad line Seyfert 1 galaxies.Comment: 5 pages, 3 figures. Accepted for publication in MNRAS Letters. Wrong version was uploaded! Check for the correct one in the replacemen

The Spitzer discovery of a galaxy with infrared emission solely due to AGN activity

We present a galaxy (SAGE1CJ053634.78-722658.5) at a redshift of 0.14 of which the IR is entirely dominated by emission associated with the AGN. We present the 5-37 um Spitzer/IRS spectrum and broad wavelength SED of SAGE1CJ053634, an IR point-source detected by Spitzer/SAGE (Meixner et al 2006). The source was observed in the SAGE-Spec program (Kemper et al., 2010) and was included to determine the nature of sources with deviant IR colours. The spectrum shows a redshifted (z=0.14+-0.005) silicate emission feature with an exceptionally high feature-to-continuum ratio and weak polycyclic aromatic hydrocarbon (PAH) bands. We compare the source with models of emission from dusty tori around AGNs from Nenkova et al. (2008). We present a diagnostic diagram that will help to identify similar sources based on Spitzer/MIPS and Herschel/PACS photometry. The SED of SAGE1CJ053634 is peculiar because it lacks far-IR emission and a clear stellar counterpart. We find that the SED and the IR spectrum can be understood as emission originating from the inner ~10 pc around an accreting black hole. There is no need to invoke emission from the host galaxy, either from the stars or from the interstellar medium, although a possible early-type host galaxy cannot be excluded based on the SED analysis. The hot dust around the accretion disk gives rise to a continuum, which peaks at 4 um, whereas the strong silicate features may arise from optically thin emission of dusty clouds within ~10 pc around the black hole. The weak PAH emission does not appear to be linked to star formation, as star formation templates strongly over-predict the measured far-IR flux levels. The SED of SAGE1CJ053634 is rare in the local universe but may be more common in the more distant universe. The conspicuous absence of host-galaxy IR emission places limits on the far-IR emission arising from the dusty torus alone.Comment: Accepted for publication in A&A, 7 pages, 6 figure

Searching for Compton-thick active galactic nuclei at z~0.1

Using a suite of X-ray, mid-IR and optical active galactic nuclei (AGN) luminosity indicators, we search for Compton-thick (CT) AGNs with intrinsic L_X>10^42erg/s at z~0.03-0.2, a region of parameter space which is currently poorly constrained by deep narrow-field and high-energy (E>10keV) all-sky X-ray surveys. We have used the widest XMM-Newton survey (the serendipitous source catalogue) to select a representative sub-sample (14; ~10%) of the 147 X-ray undetected candidate CT AGNs in the Sloan Digital Sky Survey (SDSS) with f_X/f_[OIII]<1; the 147 sources account for ~50% of the overall Type-2 AGN population in the SDSS-XMM overlap region. We use mid-IR spectral decomposition analyses and emission-line diagnostics, determined from pointed Spitzer-IRS spectroscopic observations of these candidate CT AGNs, to estimate the intrinsic AGN emission (predicted L_X,2-10keV (0.2-30)x10^42erg/s). On the basis of the optical [OIII], mid-IR [OIV] and 6um AGN continuum luminosities we conservatively find that the X-ray emission in at least 6/14 (>43%) of our sample appear to be obscured by CT material with N_H>1.5x10^24cm^-2. Under the reasonable assumption that our 14 AGNs are representative of the overall X-ray undetected AGN population in the SDSS-XMM parent sample, we find that >20% of the optical Type-2 AGN population are likely to be obscured by CT material. This implies a space-density of log(Phi) >-4.9Mpc^-3 for CT AGNs with L_X>10^42erg/s at z~0.1, which we suggest may be consistent with that predicted by X-ray background synthesis models. Furthermore, using the 6um continuum luminosity to infer the intrinsic AGN luminosity and the stellar velocity dispersion to estimate M_BH, we find that the most conservatively identified CT AGNs in this sample may harbour some of the most rapidly growing black holes (median M_BH~3x10^7M_o) in the nearby Universe, with a median Eddington ratio of ~0.2.Comment: 16 pages, 2 tables, 6 figures. Accepted for publication in MNRA

The evolving AGN duty cycle in galaxies since z ∼ 3 as encoded in the X-ray luminosity function

We present a new modeling of the X-ray luminosity function (XLF) of active galactic nuclei (AGNs) out to z ~ 3, dissecting the contributions of main-sequence (MS) and starburst (SB) galaxies. For each galaxy population, we convolved the observed galaxy stellar mass (M sstarf) function with a grid of M sstarf-independent Eddington ratio (λ EDD) distributions, normalized via empirical black hole accretion rate (BHAR) to star formation rate (SFR) relations. Our simple approach yields an excellent agreement with the observed XLF since z ~ 3. We find that the redshift evolution of the observed XLF can only be reproduced through an intrinsic flattening of the λ EDD distribution and with a positive shift of the break λ*, consistent with an antihierarchical behavior. The AGN accretion history is predominantly made by massive (1010 44.36 + 1.28 × (1 + z). We infer that the probability of finding highly accreting (λ EDD > 10%) AGNs significantly increases with redshift, from 0.4% (3.0%) at z = 0.5%–6.5% (15.3%) at z = 3 for MS (SB) galaxies, implying a longer AGN duty cycle in the early universe. Our results strongly favor a M sstarf-dependent ratio between BHAR and SFR, as BHAR/SFR ∝ ${M}_{\star }^{0.73[+0.22,-0.29]}$, supporting a nonlinear BH buildup relative to the host. Finally, this framework opens potential questions on super-Eddington BH accretion and different λ EDD prescriptions for understanding the cosmic BH mass assembly

Tol 2240-384 - a new low-metallicity AGN candidate

Active galactic nuclei (AGNs) have typically been discovered in massive galaxies of high metallicity. We attempt to increase the number of AGN candidates in low metallicity galaxies. We present VLT/UVES and archival VLT/FORS1 spectroscopic and NTT/SUSI2 photometric observations of the low-metallicity emission-line galaxy Tol 2240-384 and perform a detailed study of its morphology, chemical composition, and emission-line profiles. We determine abundances of nitrogen, O, Ne, S, Cl, Ar, and Fe by analyzing the fluxes of narrow components of the emission lines using empirical methods. We verify with a photoionisation model that the physics of the narrow-line component gas is similar to that in common metal-poor galaxies. Image deconvolution reveals two high-surface brightness regions in Tol 2240-384 separated by 2.4 kpc.The brightest southwestern region is surrounded by intense ionised gas emission on a spatial scale of ~5 kpc. The profiles of the strong emission lines in the UVES spectrum are asymmetric and all these lines apart from Halpha and Hbeta can be fitted by two Gaussians of FWHM ~75-92 km/s separated by ~80 km/s implying that there are two regions of ionised gas emitting narrow lines. The shapes of the Halpha and Hbeta lines are more complex. In particular, the Halpha emission line consists of two broad components of FWHM ~700 km/s and 2300 km/s, in addition to narrow components of two regions revealed from profiles of other lines. The extraordinarily high luminosity of the broad Halpha line of 3x10e41 erg/s cannot be accounted for by massive stars at different stages of their evolution. The broad Halpha emission persists over a period of 7 years, which excludes supernovae as a powering mechanism of this emission. This emission most likely arises from an accretion disc around a black hole of mass ~10e7 Msun.Comment: 15 pages, 8 figures, accepted for publication in Astronomy and Astrophysic

Toxicological screening and DNA sequencing detects contamination and adulteration in regulated herbal medicines and supplements for diet, weight loss and cardiovascular health

Use of herbal medicines and supplements by consumers to prevent or treat disease, particularly chronic conditions continues to grow, leading to increased awareness of the minimal regulation standards in many countries. Fraudulent, adulterated and contaminated herbal and traditional medicines and dietary supplements are a risk to consumer health, with adverse effects and events including overdose, drug-herb interactions and hospitalisation. The scope of the risk has been difficult to determine, prompting calls for new approaches, such as the combination of DNA metabarcoding and mass spectrometry used in this study. Here we show that nearly 50% of products tested had contamination issues, in terms of DNA, chemical composition or both. Two samples were clear cases of pharmaceutical adulteration, including a combination of paracetamol and chlorpheniramine in one product and trace amounts of buclizine, a drug no longer in use in Australia, in another. Other issues include the undeclared presence of stimulants such as caffeine, synephrine or ephedrine. DNA data highlighted potential allergy concerns (nuts, wheat), presence of potential toxins (Neem oil) and animal ingredients (reindeer, frog, shrew), and possible substitution of bird cartilage in place of shark. Only 21% of the tested products were able to have at least one ingredient corroborated by DNA sequencing. This study demonstrates that, despite current monitoring approaches, contaminated and adulterated products are still reaching the consumer. We suggest that a better solution is stronger pre-market evaluation, using techniques such as that outlined in this study