Metabolomic profiling of the synergistic effects of melittin in combination with cisplatin on ovarian cancer cells

Melittin, the main peptide present in bee venom, has been proposed as having potential for anticancer therapy; the addition of melittin to cisplatin, a first line treatment for ovarian cancer, may increase the therapeutic response in cancer treatment via synergy, resulting in improved tolerability, reduced relapse, and decreased drug resistance. Thus, this study was designed to compare the metabolomic effects of melittin in combination with cisplatin in cisplatin-sensitive (A2780) and resistant (A2780CR) ovarian cancer cells. Liquid chromatography (LC) coupled with mass spectrometry (MS) was applied to identify metabolic changes in A2780 (combination treatment 5 μg/mL melittin + 2 μg mL cisplatin) and A2780CR (combination treatment 2 μg/mL melittin + 10 μg/mL cisplatin) cells. Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate data analysis models were produced using SIMCA-P software. All models displayed good separation between experimental groups and high-quality goodness of fit (R2) and goodness of prediction (Q2), respectively. The combination treatment induced significant changes in both cell lines involving reduction in the levels of metabolites in the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, purine and pyrimidine metabolism, and the arginine/proline pathway. The combination of melittin with cisplatin that targets these pathways had a synergistic effect. The melittin-cisplatin combination had a stronger effect on the A2780 cell line in comparison with the A2780CR cell line. The metabolic effects of melittin and cisplatin in combination were very different from those of each agent alone

Effect of bee venom and its fractions on the release of pro-inflammatory cytokines in PMA-differentiated U937 cells co-stimulated with LPS

The venom of Apis mellifera (honey bee) has been reported to play a role in immunotherapy, but existing evidence to support its immuno-modulatory claims is insufficient. Four fractions from whole bee venom (BV) were separated using medium pressure liquid chromatography. Their ability to induce the production of cytokines TNFα, IL-1β and IL-6 in phorbol-12-myristate-13-acetate (PMA)-treated U937 cells was assessed. The levels of the three cytokines produced by stimulation with the four fractions and crude BV without LPS were not significantly different from negative control values. However, co-stimulation of the cells with LPS and Fraction 4 (F-4) induced a 1.6-fold increase in TNF-α level (p < 0.05) compared to LPS alone. Likewise, LPS-induced IL-1β production was significantly synergised in the presence of F-1 (nine-fold), F-2 (six-fold), F-3 (four-fold) and F-4 (two-fold) fractions, but was only slightly enhanced with crude BV (1.5-fold) relative to LPS. Furthermore, the LPS-stimulated production of IL-6 was not significantly increased in cells co-treated with F-2 and F-3, but the organic fraction (F-4) showed an inhibitory effect (p < 0.05) on IL-6 production. The latter was elucidated by NMR spectroscopy and found to contain(Z)-9-eicosen-1-ol. The effects observed with the purified BV fractions were more marked than those obtained with the crude sample

Metabolomic profiling of the immune stimulatory effect of eicosenoids on PMA-differentiated THP-1 cells

Honey bee venom has been established to have significant effect in immunotherapy. In the present study, (Z)-11-eicosenol-a major constituent of bee venom, along with its derivations methyl cis-11-eicosenoate and cis-11-eicosenoic acid, were synthesised to investigate their immune stimulatory effect and possible use as vaccine adjuvants. Stimuli that prime and activate the immune system have exerted profound effects on immune cells, particularly macrophages; however, the effectiveness of bee venom constituents as immune stimulants has not yet been established. Here, the abilities of these compounds to act as pro-inflammatory stimuli were assessed, either alone or in combination with lipopolysaccharide (LPS), by examining the secretion of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1β (IL-1β), IL-6 and IL-10 by THP-1 macrophages. The compounds clearly increased the levels of IL-1β and decreased IL-10, whereas a decrease in IL-6 levels suggested a complex mechanism of action. A more in-depth profile of macrophage behaviour was therefore obtained by comprehensive untargeted metabolic profiling of the cells using liquid chromatography mass spectrometry (LC-MS) to confirm the ability of the eicosanoids to trigger the immune system. The level of 358 polar and 315 non-polar metabolites were changed significantly (p < 0.05) by all treatments. The LPS-stimulated production of most of the inflammatory metabolite biomarkers in glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, purine, pyrimidine and fatty acids metabolism were significantly enhanced by all three compounds, and particularly by methyl cis-11-eicosenoate and cis-11-eicosenoic acid. These findings support the proposed actions of (Z)-11-eicosenol, methyl cis-11-eicosenoate and cis-11-eicosenoic acid as immune system stimulators

Ultraviolet Diagnostics for the Emission Line Gas in Active Galaxies

Optical diagnostic diagrams are frequently ambiguous as a test of the photoionization or fast shock models of the narrow line regions of active galaxies. Here, we present a set of UV line ratio diagrams which can discriminate between pure shock and photoionization modes of excitation, and to some extent, also discriminate shocks with ionized precursors from photoionization. These diagrams use relatively bright emission lines and reddening insensitive ratios and provide a practical observational test for separating the excitation mechanisms of the narrow line regions of active galaxies. The most useful diagrams are those involving the various ionization stages of Carbon, [OIII]5007/H-beta vs. CIV 1550/ HeII 1640 and the purely UV ratio pair CII] 2326 / CIII] 1909 vs. CIV 1550 / CIII]909. Temperature sensitive FUV lines CIII 977 and NIII 991 also provide good discriminants. The models are compared to observations of nearby AGN, and also to high redshift objects where the UV lines are shifted into the optical.Comment: 26 pages, 13 figures, used AASTeX macros, tarfile of pp.tex and 13 .eps file

Simulating the formation of molecular clouds. I. Slow formation by gravitational collapse from static initial conditions

We study the formation of H2 in the ISM, using a modified version of the astrophysical magnetohydrodynamical code ZEUS-MP that includes a non-equilibrium treatment of the formation and destruction of H2. We examine two different approximations to treat the shielding of H2 against photodissociation: a local approximation, which gives us a solid lower bound on the amount of shielding, and a method based on ray-tracing that is considerably more accurate in some circumstances but that produces results that are harder to clearly interpret. Either approximation allows one to perform three-dimensional high-resolution simulations of cloud formation with only modest computational resources. We also include a detailed treatment of the thermal behaviour of the gas. In this paper, we focus on the problem of molecular cloud formation in gravitationally unstable, initially static gas. We show that in these conditions, and for initial densities consistent with those observed in the cold, neutral atomic phase of the interstellar medium, H2 formation occurs on a timescale t > 10 Myr, comparable to or longer than the gravitational free-fall timescale of the cloud. We also show that the collapsing gas very quickly reaches thermal equilibrium and that the equation of state of the gas is generally softer than isothermal. Finally, we demonstrate that although these results show little sensitivity to variations in most of our simulation parameters, they are highly sensitive to the assumed initial density n_i. Reducing n_i significantly increases the cloud formation timescale and decreases the amount of hydrogen ultimately converted to H2. (Abridged).Comment: 89 pages, 40 figures, AASTex. Results section significantly revised and extended. Includes results from a large number of new simulations performed using a treatment of H2 photodissociation based on ray-tracing. This version matches that accepted by ApJ

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Evaluation of a novel method for the identification of coevolving protein residues

A novel method for the identification of correlated pairs in aligned homologous protein sequences is presented and evaluated against a model of simulated protein evolution incorporating covariation. Our method is shown to be capable of identifying all coevolutionary pairs of sites, with minimal interference by background correlations, in aligned sequence sets containing ∼60 sequences with a tree depth of at least 30 accepted point mutations. This result is expected even in the presence of a large degree of neutral and non-correlated evolution. It is postulated that, since naturally occurring protein families may be subject to stronger selection pressures and a lesser degree of neutral evolution, this method of covariation analysis may be generally more robust than the model would indicat

Leisure time physical activity in children and young people with cerebral palsy, a population-based study

Marietta van der Linden - ORCID: 0000-0003-2256-6673 https://orcid.org/0000-0003-2256-6673Kavi C Jagadamma - ORCID: 0000-0003-2011-0744 https://orcid.org/0000-0003-2011-0744Thomas Mercer - ORCID: 0000-0002-5078-4769 https://orcid.org/0000-0002-5078-4769Purpose To describe leisure time physical activity (LTPA) in children and young people with cerebral palsy (CP) and identify barriers and facilitators to participation. Methods. LTPA participation (‘at least one club’, ‘not in club’, ‘any type’) was derived from a national CP register and associated factors were analysed. Barriers and facilitators to participation were investigated through a survey. Results. LTPA participation (‘any type’) was recorded for 54% (n=977) of the total sample. All outcomes of participation decreased with increasing Gross Motor Function Classification System (GMFCS) level. LTPA not in club for 11-18 year-olds was significantly lower than those aged 5-10 for GMFCS level II. The survey (n=55) showed that disability and disliking help were common barriers whilst parental encouragement and enjoyment were common facilitators. Conclusions. Data from the register and survey provide insight into factors influencing LTPA participation in young people with CP and what may help to increase this.This work was supported by funding from Edinburgh and Lothian Health Foundation no. 109/600https://doi.org/10.1097/PEP.000000000000088234pubpub

Metabolomic Profiling of the Effects of Melittin on Cisplatin Resistant and Cisplatin Sensitive Ovarian Cancer Cells Using Mass Spectrometry and Biolog Microarray Technology

In the present study, liquid chromatography-mass spectrometry (LC-MS) was employed to characterise the metabolic profiles of two human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant) in response to their exposure to melittin, a cytotoxic peptide from bee venom. In addition, the metabolomics data were supported by application of Biolog microarray technology to examine the utilisation of carbon sources by the two cell lines. Data extraction with MZmine 2.14 and database searching were applied to provide metabolite lists. Principal component analysis (PCA) gave clear separation between the cisplatin-sensitive and resistant strains and their respective controls. The cisplatin-resistant cells were slightly more sensitive to melittin than the sensitive cells with IC50 values of 4.5 and 6.8 μg/mL respectively, although the latter cell line exhibited the greatest metabolic perturbation upon treatment. The changes induced by melittin in the cisplatin-sensitive cells led mostly to reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+). The effects on energy metabolism were supported by the data from the Biolog assays. The lipid compositions of the two cell lines were quite different with the A2780 cells having higher levels of several ether lipids than the A2780CR cells. Melittin also had some effect on the lipid composition of the cells. Overall, this study suggests that melittin might have some potential as an adjuvant therapy in cancer treatment

The VLT-FLAMES Tarantula survey

The Tarantula Survey is an ESO Large Programme which has obtained multi-epoch spectroscopy of over 1,000 massive stars in the 30 Doradus region of the Large Magellanic Cloud. The assembled consortium will exploit these data to address a range of fundamental questions in both stellar and cluster evolution