The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its “human-like” glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/)

Interpretable Prediction of Urban Mobility Flows with Deep Neural Networks as Gaussian Processes

The ability to understand and predict the flows of people in cities is crucial for the planning of transportation systems and other urban infrastructures. Deep-learning approaches are powerful since they can capture non-linear relations between geographic features and the resulting mobility flow from a given origin location to a destination location. However, existing methods cannot quantify the uncertainty of the predictions, limiting their interpretability and thus their use for practical applications in urban infrastructure planning. To that end, we propose a Bayesian deep-learning approach that formulates deep neural networks as Gaussian processes and integrates automatic variable selection. Our method provides uncertainty estimates for the predicted origin-destination flows while also allowing to identify the most critical geographic features that drive the mobility patterns. The developed machine learning approach is applied to large-scale taxi trip data from New York City

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

A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in Drosophila melanogaster macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion. We characterize for the first time the T and Tn glycoform O-glycoproteome of the Drosophila melanogaster embryo, and determine that Minerva increases the presence of T-antigen on proteins in pathways previously linked to cancer, most strongly on the sulfhydryl oxidase Qsox1 which we show is required for macrophage tissue entry. Minerva’s vertebrate ortholog, MFSD1, rescues the minerva mutant’s migration and T-antigen glycosylation defects. We thus identify a key conserved regulator that orchestrates O-glycosylation on a protein subset to activate a program governing migration steps important for both development and cancer metastasis

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

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

Risk of severe driver injury by driving with psychoactive substances

Driving with alcohol and other psychoactive substances imposes an increased risk of severe injury accidents. In a population-based case-control design, the relative risks of severe driver injury (MAIS ≥ 2) by driving with ten substance groups were approximated by odds ratios (alcohol, amphetamines, benzoylecgonine, cocaine, cannabis, illicit opiates, benzodiazepines and Z-drugs, i.e. zolpidem and zopiclone, medicinal opioids, alcohol-drug combinations and drug-drug combinations). Data from six countries were included in the study: Belgium, Denmark, Finland, Italy, Lithuania and the Netherlands. Case samples (N = 2490) were collected from severely injured drivers of passenger cars or vans in selected hospitals in various regions of the countries. Control samples (N = 15,832) were sampled in a uniform sampling scheme stratified according to country, time, road type and season. Relative risks were approximated by odds ratios and calculated by logistic regression. The estimates were adjusted for age, gender and country. The highest risk of the driver being severely injured was associated with driving positive for high concentrations of alcohol (≥0.8 g/L), alone or in combination with other psychoactive substances. For alcohol, risk increased exponentially with blood alcohol concentration (BAC). The second most risky category contained various drug-drug combinations, amphetamines and medicinal opioids. Medium increased risk was associated with medium sized BACs (at or above 0.5 g/L, below 0.8 g/L) and benzoylecgonine. The least risky drug seemed to be cannabis and benzodiazepines and Z-drugs. For male drivers, the risk of being severely injured by driving with any of the psychoactive substances was about 65% of that of female drivers. For each of the substance groups there was a decrease in the risk of severe driver injury with increasing age. It is concluded that among psychoactive substances alcohol still poses the largest problem in terms of driver risk of getting injured.</p

Positionering eller effektivisering via lean i den kommunale sektor?

I tidens løb er der udviklet en lang række ledelsesfilosofier, som har til formål at øge produktiviteten i såvel fysiske som administrative processer. En del af disse ledelsesfilosofier er udviklet i en industriel kontekst og søges herefter overført til en offentlig kontekst. Dette er bl.a. gældende for tidens populære ledelsesbegreb ’lean’, som en del danske kommuner øjensynligt har kastet sig over. Lean-begrebet er en moderne betegnelse for Toyota’s produktionssystem, som tidligere er benævnt Just-In-Time, og som trækker på elementer fra Total Quality Management. Formålet med artiklen er at afdække, gennem en spørgeskemaundersøgelse blandt landets 98 kommuner, hvad der forstås ved, og hvordan der arbejdes med lean i den kommunale sektor. Dette giver efterfølgende anledning til refleksion over, om det giver mening at overføre lean fra en produktionskontekst til en kommunal kontekst, og hvad konsekvenserne heraf er. Artiklen konkluderer, at det som kommunerne opfatter som lean mere har karakter af fokus på spild gennem anvendelse af værktøjer som arbejdsgangsanalyser og løbende forbedringer. Lean synes mere at fungere som positionering frem for effektivisering.Over the years, a long line of management philosophies have been developed with the purpose of increasing the productivity of both physical and administrative processes. Some of these management philosophies have originated in an industrial context, and attempts have been made to transfer them into a public sector context, as in the case of the ‘lean’ concept, currently in vogue and immensely popular with many Danish local authorities. ‘Lean’ is a modern phrase for Toyota’s production system, previously designated Just-In- Time, which uses elements from Total Quality Management. A questionnaire survey was undertaken among Denmark’s 98 local authorities to identify how they interpret ‘lean’ and how they work with it. Does it make sense to transfer ‘lean’ from a production to a local authority context, and what are the consequences? The conclusion is that what local authorities perceive as ‘lean’ is more in the nature of focus on waste through the application of tools such as analysis of work processes and continuing improvements. ‘Lean’ seems to function as a measure of positioning rather than improved efficiency

Symmetric Lipofibromatous Hamartoma Affecting Digital Nerves

Lipofibromatous hamartoma of the nerve is a benign tumor, which affects the major nerves and their branches in the human body. It is often found in the median nerve of the hand and is commonly associated with macrodactyly, but it is rarely found in the digital nerves at the peripheral level. This tumor is often found in young adults and may go through a self-limiting course. However, operation is indicated when the tumor size is large or when the associated nerve compressive symptoms are present. We have experienced a rare case of lipofibromatous hamartoma that symmetrically involved the volar digital nerves of both index fingers on the ulnar side. With the aid of a microscope, we dissected and removed the tumor as much as possible without sacrificing the nerve. No sensory change occurred in both fingers and no sign of recurrence was observed upon follow-up

Alkohol, medicin og narkotikaforekomst hos alvorligt tilskadekomne bilister

Denne artikel omhandler forekomsten af alkohol, narkotika og medicin hos danske bilister, som er blevet alvorligt skadet i færdselsuheld. Undersøgelsen er baseret på anonyme blodprøver fra 840 førere af person- og varebiler, der blev indbragt til hospitalerne i Odense, Kolding, Vejle, Viborg og Aalborg i perioden oktober 2007 - marts 2010. Blodprøverne blev taget som led i de blodprøver, der rutinemæssigt tages i forbindelse med behandlingen og de blev analyseret for alkohol og 21 misbrugs- og lægemiddelstoffer samt enkelte omdannelsesprodukter (metabolitter). Til brug for undersøgelsen blev der desuden indhentet oplysning om patientens alder og køn, typen af køretøj, uheldsstedet, typen af færdselsuheld, tidspunkt for færdselsuheldet og prøvetagningen samt eventuel medicin, der var blevet givet før udtagning af blodprøven. Alkohol alene eller alkohol sammen med andre stoffer var til stede hos knap 20 % af bilisterne. I alt var 30,5 % af bilisterne positive for én eller flere stofgrupper. Blandt medicinske stoffer alene var forekomsten højest for medicinske opioider (2,5 %) efterfulgt af benzodiazepiner (1,6 %). For de narkotiske stoffer alene var forekomsten højest for cannabis (2,3 %) efterfulgt af amfetamin (1,1 %). I alt var 41,6 % af de tilskadekomne i eneuheld positive for stoffer, heraf 30 % for alkohol alene eller alkohol sammen med andre stoffer. For bilister i flerpartsuheld var i alt 19 % positive, heraf knap 9 % for alkohol alene eller alkohol sammen med andre stoffer. Forekomsten af alkohol og andre stoffer var mere end dobbelt så stor hos de tilskadekomne mænd som hos de tilskadekomne kvinder. For begge køn var forekomsten størst hos aldersgruppen 25-34 år. Forekomsten af alkohol var størst i weekenden, mens den for alle andre stoffer var størst på hverdage. Resultaterne viste, at alkohol var væsentlig hyppigere til stede hos alvorligt skadede bilister i forhold til medicin og narkotika