Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of β1,4-galactosyltransferase 1 and α2,6-sialyltransferase 1

Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structur

Whole blood transcriptome analysis of Mycoplasma mycoides subsp mycoides-infected cattle confirms immunosuppression but does not reflect local inflammation

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm), is a severe respiratory disease of cattle responsible for major economic losses in sub-Saharan Africa. Disease control relies mainly on the use of empirically attenuated vaccines that provide limited protection. Thus, understanding the virulence mechanisms used by Mmm as well as the role of the host immune systemin disease development, persistence, and control is a prerequisite for the development of new, rationally designed control strategies. The aim of this study was to assess the use of whole blood transcriptome analysis to study cattle-Mmm interactions, starting by the characterization of the bovine response to Mmm infection during the acute form of the disease. For that purpose, we compared the transcriptome profile of whole blood from six cattle, before challenge by contact with Mmm infected animals and at the appearance of first clinical signs, using a bovine microarray. Functional analysis revealed that 680 annotated genes were differentially expressed, with an overwhelming majority of down-regulated genes characterizing an immunosuppression. The main bio-functions affected were "organismal survival", "cellular development, morphology and functions" and "cell-to cell signaling and interactions". These affected functions were consistent with the results of previous in vitro immunological studies. However, microarray and qPCR validation results did not highlight pro-inflammatory molecules (such as TNF alpha, TLR2, IL-12B and IL-6), whereas inflammation is one of the most characteristic traits of acute CBPP. This global gene expression pattern may be considered as the result, in blood, of the local pulmonary response and the systemic events occurring during acute CBPP. Nevertheless, to understand the immune events occurring during disease, detailed analyses on the different immune cell subpopulations, either in vivo, at the local site, or in vitro, will be required. Whole blood transcriptome analysis remains an interesting approach for the identification of bio-signatures correlating to recovery and protection, which should facilitate the evaluation and validation of novel vaccine formulations

Differential effects of lobe A and lobe B of the conserved oligomeric golgi complex on the stability of β1,4-galactosyltransferase 1 and α2,6-sialyltransferase 1

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch)Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structure

Cellular reprogramming

DNA-methylatie is een epigenetische modificatie. Dit type van modificaties verandert de genetische informatie zelf niet, maar wijzigt de laag erboven. Er worden chemische modificaties toegevoegd of gewijzigd op het DNA of de histonen (waarrond het DNA gewonden is). DNA-methylatie is die modificatie waarbij een methylgroep wordt aangebracht op het cytosine (C) residue en een CG dincucleotide. Als deze wijziging plaats vindt in een zogenaamd CpG eiland (waar deze CG dinucleotiden dens bij elkaar voorkomen) van de promoter van een gen, wordt dit gen niet meer afgeschreven en zal zijn functie verloren gaan. In dit proefschrift worden verschillende aspecten van DNA-methylatie en het belang ervan in de oncologie belicht. In het eerste onderzoeksdeel hebben we verschillende methodes en algoritmes ontwikkeld om methylatiemerkers te identificeren: genen die specifiek in kanker worden gemethyleerd maar niet in normale weefsels of genen die enkel worden gemethyleerd in bepaalde patiëntsubgroepen (bv. die respons vertonen voor een bepaalde chemotherapie). We hebben een databank en methodes gemaakt om kennis uit de literatuur in het snelgroeiende DNA-methylatie veld te halen. Deze databank (PubMeth) laat toe om het verband na te gaan tussen genen en hun methylatiepatroon in verschillende kanker (sub)types. Daarnaast werden verschillende sorterings- en selectiemethodieken ontwikkeld om zo een prioriteit toe te kennen aan de kandidaat DNA-methylatiemerkers. Al deze methodes werden ontwikkeld om data van genoom-wijde experimenten te kunnen verwerken. Nieuwe methylatiemerkers (gemethyleerd in kanker maar niet in normaal) kunnen worden gebruik bij vroegtijdige detectie van kanker. Er werden verschillende computationele oplossingen voorgesteld: - Relaxation ranking: sorteermethodiek gebaseerd op expressie micro-arrays van primair baarmoederhalskanker patiëntmateriaal en re-expressie experimenten op baarmoederhalskanker cellijnen. Deze methode is gebaseerd op lage expressie in kanker en re-expressie na behandeling met het de-methylerend agens DAC en de histondeacetylase inhibitor TSA. De methode gebruikt geen enkele (arbitrair te kiezen) grenswaarde - De deep approach: er werden verschillende DNA-motieven in de promoterregio van genen geïdentificeerd die discrimineren tussen kankerspecifiek gemethyleerde genen en genen die ook gemethyleerd worden in normale weefsels. - De broad approach maakt gebruik van een genoom-wijde alignering van promoterregio’s. Hieruit blijkt dat gekende methylatiemerkers meer dens geclusterd zijn dan verwacht. - Zowel ‘deep’ als ‘broad’ werden gecombineerd met re-expressie studies in cellijnen van verschillende kankertypes. De combinatie van deze experimentele filter en de computationele aanpak verhoogt de succesratio bij het vinden van kankerspecifiek gemethyleerde genen aanzienlijk. - Er werden ook merkers geïdentificeerd die mogelijks de respons op chemotherapie (platinum) kunnen voorspellen in eierstokkanker. Dit opent de weg naar gepersonaliseerde geneeskunde. De methodiek gebruikt een score-schema gebaseerd op zowel primaire kanker stalen (van zowel platinumgevoelige als resistente patiënten) als re-expressie experimenten op kanker cellijnen. De nieuwe identificatiestrategieën werden gevalideerd op primaire kankerstalen en presteren goed: ten opzichte van beschreven priorizatietechnieken is de succesratio verhoogd. Verscheidene nieuwe methylatie biomerkers in verschillende kankertypes (baarmoederhalskanker, eierstok-kanker, hoofd- en nekkanker, neuroblastoom,…) werden succesvol gevalideerd op primaire patiëntenstalen. Vervolgonderzoek op grotere patiëntengroepen zal het mogelijk diagnostische potentieel aantonen. De validatie van sommige studies is momenteel nog lopende. Dit toont de noodzaak aan van snelle en betrouwbare analysetechnieken die geschikt zijn voor validatiedoeleinden. Ten tweede werd er een uniek viraal infectiesysteem gecombineerd met een genoom-wijde methylatiegevoelige detectietechniek om epigenetische ‘herprogrammering’ van humane gastheercellen na infectie met hoge-risico HPV (Humaan Papilloma Virus) te onderzoeken. In dit experi-ment geven we sterke indicaties dat het virus in staat is de gastheercel epigenetisch te wijzigen. Deze hr-HPV virustypes zijn duidelijk geassocieerd met de ontwikkeling van baarmoederhalskanker gezien meer dan 99 % van de patiënten besmet is. Momenteel is het uitstrijkje een wijdverspreide screening methodiek die wordt gebruikt bij vroegdetectie. Onlangs werden er ook vaccins op de markt gebracht die infectie met de meest voorkomende virustypes (ongeveer 80 % van de infecties) moet voorkomen. De vaccinatie zou moeten gebeuren bij meisjes voor het eerste seksueel contact. De ontdekking van biomerkers met hoge specificiteit en sensitiviteit bij baarmoederhalskanker blijft noodzakelijk voor de niet gevaccineerde groep en gezien de vaccins niet tegen alle virustypes bescherming bieden. DNA-methylatiemerkers zijn uitstekende kandida-ten voor vroegdetectie in een screeningsprogramma: ze kunnen op grote schaal en geautomatiseerd gebeuren. Gezien er aangenomen wordt dat verschillende kankertypes verwant zijn met virusinfecties, verhoogt dit onderzoek de kennis bij dit proces en opent dit de weg naar zeer vroege detectie. Momenteel zijn enkel delen van het ‘kanker epigenoom’ bekend. De komst van methodes die grote hoeveelheden data genereren (zoals volgende-generatie sequenering) zou de bestaande kennis in grote mate kunnen laten toenemen. Dit vereist echter grote collecties primaire kankerstalen (liefst nog in verschillende stadia). Een centraal beheerde, goed geannoteerde bibliotheek van patiëntenmateriaal die verschillende kankertypes bevat, zou het onderzoek in een stroomversnelling plaatsen. Bijkomende uitdaging is dat deze technieken het hoogste niveau van precisie hebben (op baseniveau van een enkele DNA-molecule) en tegelijkertijd een hoog aantal stalen verwerken. Dit betekent dat bij het proefopzet sequentiestukken moeten gekozen worden die aangerijkt zijn aan DNA-methylatie of histonmodificaties. De data-analyse strategie moet snel genoeg zijn maar toch nieuwe kennis extraheren uit de terabytes ruwe data die gegenereerd worden. Ons labo werkt zowel op de ontwikkeling van een goed proefopzet als de verwerking van de gegenereerde data.DNA-methylation is an epigenetic modification. These modifications of the DNA do not change the genetic sequence itself, but affect the level ‘above’ it: chemical modifications of the DNA or the histones (where the DNA is wound around) are added or altered. DNA-methylation is the modification where a methyl group is added on cytosine (C) residues in CG dinucleotides. If this takes place in the so-called CpG islands (where CG dinucleotides occur very densely) within the promoter of a gene, this gene is silenced, and will not be transcribed; its function is lost. In this thesis, different aspects of DNA-methylation and its importance in the field of oncology has been dealt with. First, we created different methodologies and algorithms to identify methylation markers: genes that are specifically methylated in cancer but not in normal tissue or genes, methylated in subgroups of patients (e.g. responders of a certain chemotherapy). We built a database and methodologies to discover existing publications in the fast-growing DNA-methylation field. This database (PubMeth) allows to screen which genes are reported methylated in the selected cancer (sub)types and vice versa. Different ranking, sorting and selection techniques were developed to prioritize promising methylation marker candidates. These methodologies were all developed to deal with data from genome-wide approaches. Novel methylation markers (methylated in cancer while not in normal) could be used as early detection markers. Different computational approaches are described here: - Relaxation ranking: ranking strategy based on expression micro-arrays of primary cervical cancer samples and re-expression experiments on cervical cancer cell lines. This methodology is based on low expression in cancer samples and re-expression after treatment with the demethylation agent DAC and the histone de-acetylase inhibitor TSA. The methodology involves no thresholds. - The deep approach: different DNA-patterns in the promoter region of genes were identified that seem to discriminate between cancer-specifically methylated genes and genes that also are me-thylated in normal tissues. - The broad approach makes use of a genome-wide alignment of promoter regions. Apparently, genes described as methylation markers, are more densely clustered together. - Both the deep approach and the broad approach were combined with data from re-expression studies in cancer cell lines. The combination of this experimental filter and the computational approaches drastically improves the success rate in finding cancer-specific methylation markers in various cancer types. - There were also markers discovered, that may be able to predict chemotherapy (platinum) response in ovarian cancer, clearing the roads towards personalized medicine. The methodology uses a score-scheme, based on both primary cancer samples (from patients sensitive and resistant to platinum therapies) as re-expression experiments of ovarium cancer cell lines (both cisplatin resistant and sensitive). The novel identification strategies were validated on primary cancer samples and perform well: the success rate was improved in comparison with other prioritization attempts. Several novel methylation markers were discovered in different cancer types (cervical cancer, ovarian cancer, head-and-neck cancer, neuroblastoma, …) and were validated on primary samples. Follow-up research on larger clinical cohorts will demonstrate their potential diagnostic power. For some studies, the validation effort is still in progress; this illustrates the need for fast and accurate analysis techniques, suitable for validation purposes. Secondly, a unique viral infection system, combined with a ge-nome-wide methylation detection methodology was used to investigate the epigenetic ‘reprogramming’ of human host cells after infection with high-risk HPV (Human Papilloma Virus). In this experiment, we prove that a virus is able to epigenetically program their host cell. The high-risk HPV types are clearly related with the development of cervical cancer as more than 99 % of the patients is infected with such a virus type. For the moment, the cytological Pap-smear screening technique is widespread and used for early detection. Recently, vaccines were developed in order to prevent infection with the most prominent virus types (about 80 % of infections covered). The vaccination strategy must be applied in young girls (before sexual contact). The discovery of cervical cancer biomarkers with high sensitivity and specificity remains necessary for the non-vaccinated group and screening programs remain needed as the vaccines do not cover all hr-HPV virus types. DNA-methylation markers that seem to be related with infection with the virus, are ideal candidates for very early detection in a screening program: large scale analysis can be automated. As it is believed that multiple cancers may occur after viral infec-tion, or at least that these infections plays a key role in the development, this broadens the knowledge in this process and opens ways to very early detection. Currently, only fractions of the cancer epigenome are known. The introduction of methods that generate significantly large amounts of data (such as next generation sequencing) might be able to greatly expand the current knowledge. However, large collections of primary cancer samples (preferentially of different stages) will be needed. A centrally managed, well described and annotated library of patient material containing different cancer types would be extremely beneficial. In addition, the highest level of precision (base pair level of single DNA-molecules) will be reached for a high number of samples at the same time with sequencing techniques. The initial set-up of such an experiment must be chosen so that the sequenced parts of the genome are enriched in DNA-methylation or histone modifications. The data analysis pipeline for the interpretation of the generated data must perform fast and extract new knowledge out of the terabytes of raw data generated. Both the experimental set-up and the downstream data analysis, our laboratory is working on

Blood flow-induced Notch activation and endothelial migration enable vascular remodeling in zebrafish embryos.

Arteries and veins are formed independently by different types of endothelial cells (ECs). In vascular remodeling, arteries and veins become connected and some arteries become veins. It is unclear how ECs in transforming vessels change their type and how fates of individual vessels are determined. In embryonic zebrafish trunk, vascular remodeling transforms arterial intersegmental vessels (ISVs) into a functional network of arteries and veins. Here we find that, once an ISV is connected to venous circulation, venous blood flow promotes upstream migration of ECs that results in displacement of arterial ECs by venous ECs, completing the transformation of this ISV into a vein without trans-differentiation of ECs. Arterial blood flow initiated in two neighboring ISVs prevents their transformation into veins by activating Notch signaling in ECs. Together, different responses of ECs to arterial and venous blood flow lead to formation of a balanced network with equal numbers of arteries and veins

MicroRNA-483 amelioration of experimental pulmonary hypertension.

Endothelial dysfunction is critically involved in the pathogenesis of pulmonary arterial hypertension (PAH) and that exogenously administered microRNA may be of therapeutic benefit. Lower levels of miR-483 were found in serum from patients with idiopathic pulmonary arterial hypertension (IPAH), particularly those with more severe disease. RNA-seq and bioinformatics analyses showed that miR-483 targets several PAH-related genes, including transforming growth factor-β (TGF-β), TGF-β receptor 2 (TGFBR2), β-catenin, connective tissue growth factor (CTGF), interleukin-1β (IL-1β), and endothelin-1 (ET-1). Overexpression of miR-483 in ECs inhibited inflammatory and fibrogenic responses, revealed by the decreased expression of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1. In contrast, inhibition of miR-483 increased these genes in ECs. Rats with EC-specific miR-483 overexpression exhibited ameliorated pulmonary hypertension (PH) and reduced right ventricular hypertrophy on challenge with monocrotaline (MCT) or Sugen + hypoxia. A reversal effect was observed in rats that received MCT with inhaled lentivirus overexpressing miR-483. These results indicate that PAH is associated with a reduced level of miR-483 and that miR-483 might reduce experimental PH by inhibition of multiple adverse responses

Traumatic Brain Injury Induces Genome-Wide Transcriptomic, Methylomic, and Network Perturbations in Brain and Blood Predicting Neurological Disorders.

The complexity of the traumatic brain injury (TBI) pathology, particularly concussive injury, is a serious obstacle for diagnosis, treatment, and long-term prognosis. Here we utilize modern systems biology in a rodent model of concussive injury to gain a thorough view of the impact of TBI on fundamental aspects of gene regulation, which have the potential to drive or alter the course of the TBI pathology. TBI perturbed epigenomic programming, transcriptional activities (expression level and alternative splicing), and the organization of genes in networks centered around genes such as Anax2, Ogn, and Fmod. Transcriptomic signatures in the hippocampus are involved in neuronal signaling, metabolism, inflammation, and blood function, and they overlap with those in leukocytes from peripheral blood. The homology between genomic signatures from blood and brain elicited by TBI provides proof of concept information for development of biomarkers of TBI based on composite genomic patterns. By intersecting with human genome-wide association studies, many TBI signature genes and network regulators identified in our rodent model were causally associated with brain disorders with relevant link to TBI. The overall results show that concussive brain injury reprograms genes which could lead to predisposition to neurological and psychiatric disorders, and that genomic information from peripheral leukocytes has the potential to predict TBI pathogenesis in the brain