Impact of the grapevine-shoot extract vineatrol®30 and its constituents on human topoisomerases and DNA integrity

Resveratrol (3, 4`, 5-Trihydroxystilben) wirkt präventiv in unterschiedlichen Stadien der Karzinogenese. Der Weinrebensprösslingsextrakt Vineatrol®30, der bereits als Nahrungsergänzungsmittel zugelassen ist, besteht zu einem Großteil aus Resveratrol sowie Resveratrol Oligo-und Polymeren. In einer aktuellen Studie wurde gezeigt, dass Resveratrol sowohl DNA-Doppelstrangbrüche induziert, als auch die katalytische Aktivität der humanen Topoisomerase IIα hemmt (Leone et al., 2010). Ziel der vorliegenden Arbeit war es, die Wirkung von Resveratrol auf humane Topoisomerase IIα sowie auf die Integrität der DNA zu bestätigen. Außerdem sollte untersucht werden, ob der Extrakt Vineatrol®30 die katalytische Aktivität der humanen Topoisomerase IIα hemmt, oder ob dieser die Bildung von ternären Komplexen mit dem Enzym und der DNA induziert, und somit als Topoisomerase-Gift wirkt. Des Weiteren sollten die bioaktiven Inhaltsstoffe bezüglich des Effekts auf Topoisomerase II in Vineatrol®30 identifiziert werden. Mittels Comet Assay konnte gezeigt werden, dass Resveratrol in den Konzentrationen von 0,1 bis 200 µM mit einer Inkubationszeit von einer Stunde keine DNA-Strangbrüche in der humanen Vulvakarzinomzelllinie A-431 induziert, obwohl die Tendenz dazu in der höchsten Testkonzentration feststellbar war. Des Weiteren hemmte Resveratrol die katalytische Aktivität der humanen Topoisomerase IIα mit einer minimalen Inhibierungskonzentration (MIC) von 50 µM, wie mit dem zellfreien Dekatenierungsassay gezeigt werden konnte. Der Extrakt Vineatrol®30 hemmte die katalytische Aktivität der humanen Topoisomerase IIα mit einer MIC von 5 µg/ml. Ausgewählte Inhaltsstoffe des Extrakts (Hopeaphenol, r-2-Viniferin und ε-Viniferin) hemmten die katalytische Aktivität des Enzyms in einem MIC-Bereich von 10 bis 25 µM, wobei sich r-2-Viniferin als die potenteste Testsubstanz in einem zellfreien System herausstellte. Außerdem wirkt Vineatrol®30 wohl nicht als Topoisomerase II-Gift in A-431-Zellen, da keine ternären Enzym/DNA/ Vineatrol®30 - Komplexe mittels ICE-Bioassays nachgewiesen werden konnten. Abschließend konnte gezeigt werden, dass Resveratrol wohl keine signifikanten DNA- Strangbrüche in den getesteten Konzentrationen induziert, aber die die katalytische Aktivität der humanen Topoisomerase IIα durch diese Substanz gehemmt wird. Der Weinrebensprösslingsextrakt Vineatrol®30 wirkt als starker Inhibitor des Enzyms, obwohl keine enzymvermittelte DNA-Schädigung, als Folge der Bildung permanenter anstatt transienter DNA-Doppelstrangbrüche, der Topoisomerase-Giftung, festgestellt werden konnte, wie es bei den stärksten Topoisomerase II-Krebstherapeutika der Fall ist.Resveratrol (3, 4`, 5-trihydroxystilbene) has shown to exhibit beneficial effects in the prevention of different stages of carcinogenesis. The grapevine-shoot extract vineatrol®30, admitted as a dietary supplement, contains a high amount of resveratrol as well as resveratrol oligomers and –polymers. Recently, it was shown that resveratrol induces DNA double strand breaks and inhibits the catalytic activity of human topoisomerase IIα (Leone et al., 2010). The aim of the current study was firstly to confirm the effect of resveratrol on human topoisomerase IIα and DNA integrity. Secondly, it should be determined whether the extract vineatrol®30 acts as an inhibitor of the catalytic activity of human topoisomerase II, or as a poison by the generation of ternary complexes with the enzyme and DNA. Thirdly, the bioactive constituents in vineatrol®30 concerning the effect on topoisomerase II should be identified. Using the Comet assay, it was shown that resveratrol exposure (0.1-200 µM) for one hour might not be capable of inducing DNA strand breaks in the human vulva carcinoma cell line A 431 although the tendency is observed with the highest tested concentration. Furthermore, resveratrol inhibits the catalytic activity of human topoisomerase IIα with a minimal inhibition concentration (MIC) of 50 µM as tested with the cell-free Decatenation assay. The extract vineatrol®30 was shown to inhibit the catalytic activity of topoisomerase IIα with a MIC of 5 µg/ml. Selected extract-constituents (hopeaphenol, r-2-viniferin and ε-viniferin) inhibited the catalytic activity of the enzyme within the range of MICs between 10 and 25 µM with r-2-viniferin as the most potent test-substance in a cell-free system. Furthermore, it was concluded that vineatrol®30 does not poison human topoisomerase IIα and IIβ in A 431 cells as no ternary enzyme/DNA/vineatrol®30-complexes were detected using the ICE-bioassay. In conclusion, it was shown that resveratrol might not be capable of significantly inducing DNA strand breaks in the tested concentrations, but inhibits the catalytic activity of human topoisomerase IIα. The grapevine-shoot extract vineatrol®30 acts as strong inhibitor of the enzyme, but might not induce enzyme-mediated DNA-damage by the generation of permanent instead of transient DNA double strand breaks, topoisomerase-poisoning, as performed by the most effective topoisomerase II-targeting drugs applied for cancer therapy

Intermittent PI3Kδ inhibition sustains anti-tumour immunity and curbs irAEs

Phosphoinositide 3-kinase δ (PI3Kδ) has a key role in lymphocytes, and inhibitors that target this PI3K have been approved for treatment of B cell malignancies1-3. Although studies in mouse models of solid tumours have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumour immunity4,5, its effect on solid tumours in humans remains unclear. Here we assessed the effects of the PI3Kδi AMG319 in human patients with head and neck cancer in a neoadjuvant, double-blind, placebo-controlled randomized phase II trial (EudraCT no. 2014-004388-20). PI3Kδ inhibition decreased the number of tumour-infiltrating regulatory T (Treg) cells and enhanced the cytotoxic potential of tumour-infiltrating T cells. At the tested doses of AMG319, immune-related adverse events (irAEs) required treatment to be discontinued in 12 out of 21 of patients treated with AMG319, suggestive of systemic effects on Treg cells. Accordingly, in mouse models, PI3Kδi decreased the number of Treg cells systemically and caused colitis. Single-cell RNA-sequencing analysis revealed a PI3Kδi-driven loss of tissue-resident colonic ST2 Treg cells, accompanied by expansion of pathogenic T helper 17 (TH17) and type 17 CD8+ T (TC17) cells, which probably contributed to toxicity; this points towards a specific mode of action for the emergence of irAEs. A modified treatment regimen with intermittent dosing of PI3Kδi in mouse models led to a significant decrease in tumour growth without inducing pathogenic T cells in colonic tissue, indicating that alternative dosing regimens might limit toxicity

Science from an Ultra-Deep, High-Resolution Millimeter-Wave Survey

Opening up a new window of millimeter-wave observations that span frequency bands in the range of 30 to 500 GHz, survey half the sky, and are both an order of magnitude deeper (about 0.5 uK-arcmin) and of higher-resolution (about 10 arcseconds) than currently funded surveys would yield an enormous gain in understanding of both fundamental physics and astrophysics. In particular, such a survey would allow for major advances in measuring the distribution of dark matter and gas on small-scales, and yield needed insight on 1.) dark matter particle properties, 2.) the evolution of gas and galaxies, 3.) new light particle species, 4.) the epoch of inflation, and 5.) the census of bodies orbiting in the outer Solar System.Comment: 5 pages + references; Submitted to the Astro2020 call for science white paper

Metabolic activation and colitis pathogenesis is prevented by lymphotoxin β receptor expression in neutrophils.

Inflammatory bowel disease is characterized by an exacerbated intestinal immune response, but the critical mechanisms regulating immune activation remain incompletely understood. We previously reported that the TNF-superfamily molecule TNFSF14 (LIGHT) is required for preventing severe disease in mouse models of colitis. In addition, deletion of lymphotoxin beta receptor (LTβR), which binds LIGHT, also led to aggravated colitis pathogenesis. Here, we aimed to determine the cell type(s) requiring LTβR and the mechanism critical for exacerbation of colitis. Specific deletion of LTβR in neutrophils (LTβRΔN), but not in several other cell types, was sufficient to induce aggravated colitis and colonic neutrophil accumulation. Mechanistically, RNA-Seq analysis revealed LIGHT-induced suppression of cellular metabolism, and mitochondrial function, that was dependent on LTβR. Functional studies confirmed increased mitochondrial mass and activity, associated with excessive mitochondrial ROS production and elevated glycolysis at steady-state and during colitis. Targeting these metabolic changes rescued exacerbated disease severity. Our results demonstrate that LIGHT signals to LTβR on neutrophils to suppress metabolic activation and thereby prevents exacerbated immune pathogenesis during colitis

Complex inheritance and parent-of-origin effect in juvenile myoclonic epilepsy

BACKGROUND: Juvenile myoclonic epilepsy (JME) is an idiopathic generalized epilepsy (IGE) with complex inheritance. Previous studies have suggested maternal inheritance and female excess in IGEs but have not been specific for JME. We investigated evidence for maternal inheritance, female excess and patterns of familial seizure risk in a well-characterized sample of JME families. METHODS: We ascertained 89 families through a JME proband and 50 families through a non-JME IGE proband. JME families were divided into those with and without evidence of linkage to the EJM1 susceptibility locus on chromosome 6. We analyzed transmission in 43 multigenerational families, calculated the adjusted sex ratio for JME, and looked for evidence of seizure specific risk in 806 family members. RESULTS: We found evidence for preferential maternal transmission in both EJM1-linked and unlinked families (2.7:1), evidence even more marked when potential selection factors were excluded. The adjusted female: male risk ratio was very high in JME (RR=12.5; 95% CI: 1.9–83.7). Absence seizures in JME probands increased the overall risk of seizures in first degree relatives (15.8% vs. 7.0%, P=0.011), as well as first-degree relatives' specific risk of absence seizures (6% vs. 1.6%, P=0.01), but not myoclonic seizures. CONCLUSIONS: We have confirmed the finding of maternal inheritance in JME, which is not restricted to JME families linked to the EJM1 locus. The striking female excess in JME may relate to anatomical and/or endocrine sexual dimorphism in the brain. Evidence for independent inheritance of absence and myoclonic seizures in JME families reinforces a model in which combinations of loci confer susceptibility to the component seizure types of IGE

Malic Enzyme 2 May Underlie Susceptibility to Adolescent-Onset Idiopathic Generalized Epilepsy

Idiopathic generalized epilepsy (IGE) is a class of genetically determined, phenotypically related epilepsy syndromes. Linkage analysis identified a chromosome 18 locus predisposing to a number of adolescent-onset IGEs. We report a single-nucleotide polymorphism (SNP) association analysis of the region around the marker locus with the high LOD score. This analysis, which used both case-control and family-based association methods, yielded strong evidence that malic enzyme 2 (ME2) is the gene predisposing to IGE. We also observed association among subgroups of IGE syndromes. An ME2-centered nine-SNP haplotype, when present homozygously, increases the risk for IGE (odds ratio 6.1; 95% confidence interval 2.9–12.7) compared with any other genotype. Both the linkage analysis and the association analysis support recessive inheritance for the locus, which is compatible with the fact that ME2 is an enzyme. ME2 is a genome-coded mitochondrial enzyme that converts malate to pyruvate and is involved in neuronal synthesis of the neurotransmitter γ-aminobutyric acid (GABA). The results suggest that GABA synthesis disruption predisposes to common IGE and that clinical seizures are triggered when mutations at other genes, or perhaps other insults, are present

Integrative microbiomics in bronchiectasis exacerbations

This is the author accepted manuscript. The final version is available from Nature Research via the DOI in this recordData availability: All of the sequence data described in this study have been uploaded to the NCBI SRA under project accession PRJNA590225. Publicly available taxonomic and functional databases are referenced by short-read sequence classification tools used in this study as further described in the Nature Research Reporting Summary. Other associated data, including bacterial, fungal and viral profiles for all of the patients, as well as patient clinical attributes, are available at https://github.com/translational-respiratory-lab/The_Interactome/tree/master/Data.Code availability: All code required for generation of the presented results, with accompanying documentation, are available at the study’s online code repository (https://github.com/translational-respiratory-lab/The_Interactome).Bronchiectasis, a progressive chronic airway disease, is characterized by microbial colonization and infection. We present an approach to the multi-biome that integrates bacterial, viral and fungal communities in bronchiectasis through weighted similarity network fusion (https://integrative-microbiomics.ntu.edu.sg). Patients at greatest risk of exacerbation have less complex microbial co-occurrence networks, reduced diversity and a higher degree of antagonistic interactions in their airway microbiome. Furthermore, longitudinal interactome dynamics reveals microbial antagonism during exacerbation, which resolves following treatment in an otherwise stable multi-biome. Assessment of the Pseudomonas interactome shows that interaction networks, rather than abundance alone, are associated with exacerbation risk, and that incorporation of microbial interaction data improves clinical prediction models. Shotgun metagenomic sequencing of an independent cohort validated the multi-biome interactions detected in targeted analysis and confirmed the association with exacerbation. Integrative microbiomics captures microbial interactions to determine exacerbation risk, which cannot be appreciated by the study of a single microbial group. Antibiotic strategies probably target the interaction networks rather than individual microbes, providing a fresh approach to the understanding of respiratory infection.Singapore Ministry of Health National Medical Research CouncilClinician-Scientist Individual Research GrantBiological and Environmental Life Sciences (NIMBELS)British Lung FoundationScottish Government Chief Scientist OfficeEngineering and Physical Sciences Research Council (EPSRC