Probing the O-glycoproteome of gastric cancer cell lines for biomarker discovery

Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "Simple- Cell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer.This work was supported by The Danish Research Councils, The Mizutani Foundation, The Danish National Research Foundation (DNRF107) and Fundacão para a Ciência e a Tecnologia (FCT) and COMPETE (Programa Operacional Temático Factores de Competitividade, comparticipado pelo fundo comunitário europeu FEDER) in the framework of the projects: PTDC/BBB-EBI/0786/2012; EXPL/CTM-BIO/0762/2013. Grants were received from FCT (SFRH/BD/73717/2010 to DC), (SFRH/BPD/75871/2011 to AM), (SFRH/BPD/96510/2013 to CG) and (SFRH/BPD/66288/2009 to JAF). IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education, and is partially supported by FCT

Structural and biochemical characterization of the exopolysaccharide deacetylase Agd3 required for Aspergillus fumigatus biofilm formation

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes. The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Here, the authors study an A. fumigatus enzyme that deacetylates GAG in a metal-dependent manner and constitutes a founding member of a new carbohydrate esterase family.Bio-organic Synthesi

Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins

Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins

Fatal poisonings in Oslo: a one-year observational study

<p>Abstract</p> <p>Background</p> <p>Acute poisonings are common and are treated at different levels of the health care system. Since most fatal poisonings occur outside hospital, these must be included when studying characteristics of such deaths. The pattern of toxic agents differs between fatal and non-fatal poisonings. By including all poisoning episodes, cause-fatality rates can be calculated.</p> <p>Methods</p> <p>Fatal and non-fatal acute poisonings in subjects aged ≥16 years in Oslo (428 198 inhabitants) were included consecutively in an observational multi-centre study including the ambulance services, the Oslo Emergency Ward (outpatient clinic), and hospitals, as well as medico-legal autopsies from 1st April 2003 to 31st March 2004. Characteristics of fatal poisonings were examined, and a comparison of toxic agents was made between fatal and non-fatal acute poisoning.</p> <p>Results</p> <p>In Oslo, during the one-year period studied, 103 subjects aged ≥16 years died of acute poisoning. The annual mortality rate was 24 per 100 000. The male-female ratio was 2:1, and the mean age was 44 years (range 19-86 years). In 92 cases (89%), death occurred outside hospital. The main toxic agents were opiates or opioids (65% of cases), followed by ethanol (9%), tricyclic anti-depressants (TCAs) (4%), benzodiazepines (4%), and zopiclone (4%). Seventy-one (69%) were evaluated as accidental deaths and 32 (31%) as suicides. In 70% of all cases, and in 34% of suicides, the deceased was classified as drug or alcohol dependent. When compared with the 2981 non-fatal acute poisonings registered during the study period, the case fatality rate was 3% (95% C.I., 0.03-0.04). Methanol, TCAs, and antihistamines had the highest case fatality rates; 33% (95% C.I., 0.008-0.91), 14% (95% C.I., 0.04-0.33), and 10% (95% C.I., 0.02-0.27), respectively.</p> <p>Conclusions</p> <p>Three per cent of all acute poisonings were fatal, and nine out of ten deaths by acute poisonings occurred outside hospital. Two-thirds were evaluated as accidental deaths. Although case fatality rates were highest for methanol, TCAs, and antihistamines, most deaths were caused by opiates or opioids.</p

Zinc Finger Nuclease mediated knockout of ADP dependent Glucokinase in Cancer cell lines: Effects on cell survival and Mitochondrial Oxidative Metabolism

<div><p>Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of <i>ADPGK</i>, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of <i>ADPGK,</i> and were ADPGK-null by immunoblotting. <i>ADPGK</i> knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when <i>ADPGK</i> was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of <i>ADPGK</i> in HCT116 cells caused few changes in global gene expression, knockout of <i>ADPGK</i> in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the <i>ADPGK</i> knockouts, in some cases markedly so. Collectively, the results demonstrate that <i>ADPGK</i> can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of <i>ADPGK</i> is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.</p></div

Searches for lepton-flavour-violating decays of the Higgs boson into eτ and μτ in \sqrt{s} = 13 TeV pp collisions with the ATLAS detector

Abstract This paper presents direct searches for lepton flavour violation in Higgs boson decays, H → eτ and H → μτ, performed using data collected with the ATLAS detector at the LHC. The searches are based on a data sample of proton-proton collisions at a centre-of-mass energy s $$\sqrt{s}$$ = 13 TeV, corresponding to an integrated luminosity of 138 fb−1. Leptonic (τ → ℓνℓντ) and hadronic (τ → hadrons ντ) decays of the τ-lepton are considered. Two background estimation techniques are employed: the MC-template method, based on data-corrected simulation samples, and the Symmetry method, based on exploiting the symmetry between electrons and muons in the Standard Model backgrounds. No significant excess of events is observed and the results are interpreted as upper limits on lepton-flavour-violating branching ratios of the Higgs boson. The observed (expected) upper limits set on the branching ratios at 95% confidence level, B $$\mathcal{B}$$ (H → eτ) < 0.20% (0.12%) and B $$\mathcal{B}$$ (H → μτ ) < 0.18% (0.09%), are obtained with the MC-template method from a simultaneous measurement of potential H → eτ and H → μτ signals. The best-fit branching ratio difference, B $$\mathcal{B}$$ (H → μτ) → B $$\mathcal{B}$$ (H → eτ), measured with the Symmetry method in the channel where the τ-lepton decays to leptons, is (0.25 ± 0.10)%, compatible with a value of zero within 2.5σ