Produktion von Glykoproteinen zur Strukturanalyse: Sequenz-spezifische Rekombination in CHO-Zellen

Structures of mammalian glycoproteins attracted increasing interest, as these proteins include important drug targets. Excellent recombinant expression systems for these proteins are stable mammalian cell lines. Their development, however, remains time-consuming. Another challenge for X-ray structure analysis is the heterogeneous and flexible nature of protein glycosylation which often hampers the formation of crystals. Mutant cell lines such as CHO Lec3.2.8.1 produce glycoproteins with truncated carbohydrate chains. These products can be deglycosylated and crystallized efficiently. To find well crystallisable protein constructs, structural projects often necessitate the production of a variety of different constructs per protein. Thus, a fast expression system is of essential interest for structural biology. This work describes the integration of Flp recombination strategies in order to optimise cell line development in CHO Lec3.2.8.1. Firstly, stable and high-producing loci in the host genome were tagged by a GFP gene flanked by Flp recombination target sites. Two rounds of preparative cell sorting enabled the isolating of top fluorescent cells. Depending on the strategy, the GFP gene of these cells was either exchanged against another gene by recombinase-mediated cassette exchange (RMCE) or excised (FLEx). Upon GFP deletion, the gene of interest was put under control of the promoter and thus expressed. A comparison of both recombination systems proved a higher flexibility and speed of RMCE. Altogether, 10 different production cell lines were established in a minimal amount of time. As an example, one of these proteins, a domain of human lysosome associated membrane protein 3, was produced in a bioreactor, purified, deglycosylated and crystallized. Its structure was solved by X-ray structure analysis.Das Interesse an Raumstrukturen glykosylierter Proteine nimmt stetig zu, da unter diesen Proteinen viele wichtige pharmakologische Zielstrukturen sind. Ein hervorragendes System für die Produktion der Glykoproteine stellen stabile Säugerzelllinien dar. Ihre konventionelle Etablierung ist immer noch sehr zeitaufwendig. Ein weiteres Problem für die Röntgenstrukturanalyse ist die Heterogenität und Flexibilität von Glykosylierungen, die häufig die Bildung von Kristallen verhindern. Mutierte Zelllinien wie CHO Lec3.2.8.1 produzieren Glykoproteine mit verkürzten Zuckerseitenketten. Diese Produkte können deglykosyliert und effizient kristallisiert werden. Um zu gut kristallisierbaren Proteinkonstrukten zu gelangen, müssen oft unterschiedliche Varianten von einem Protein hergestellt werden. Daher ist ein schnelles Expressionssystem gerade für die Strukturbiologie von großem Interesse. Diese Arbeit beschäftigt sich mit der Integration von Flp-Rekombinationsstrategien zur Optimierung der rekombinanten Zelllinienentwicklung in CHO Lec3.2.8.1 Zellen. Hierfür wurden genetisch stabile Loci mit starker Genaktivität im Wirtszellgenom mit einem GFP Gen und Flp-Erkennungssequenzen markiert. Zwei Zellsortierungsschritte ermöglichten die Isolierung der am stärksten fluoreszierenden Zellen. Je nach Strategie wurde das GFP entweder über Rekombinase-vermittelten Kassettenaustausch (RMCE) gegen ein anderes Gen ausgetauscht oder es wurde ausgeschnitten (FLEx). Nach der GFP Deletion wurde das zu exprimierende Gen unter die Kontrolle des Promoters gebracht und damit exprimiert. Ein Vergleich der Rekombinationssysteme zeigte die höhere Flexibilität und Schnelligkeit des RMCE Systems. Insgesamt wurden in kurzer Zeit 10 unterschiedliche Produktionszelllinien generiert. Eines der Zielproteine, eine Domäne des humanen lysosomal-assoziierten Membranprotein 3, wurde im Bioreaktor produziert, gereinigt, deglykosyliert und kristallisiert. Seine Struktur wurde mittels Röntgenstrukturanalyse aufgeklärt

Streamlining Homogeneous Glycoprotein Production for Biophysical and Structural Applications by Targeted Cell Line Development

Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP ‘master’ cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP)

Phocine Distemper in German Seals, 2002

Approximately 21,700 seals died during a morbillivirus epidemic in northwestern Europe in 2002. Phocine distemper virus 1 was isolated from seals in German waters. The sequence of the P gene showed 97% identity with the Dutch virus isolated in 1988. There was 100% identity with the Dutch isolate from 2002 and a single nucleotide mismatch with the Danish isolate

Unravelling differential Hes1 dynamics during axis elongation of mouse embryos through single-cell tracking

The intricate dynamics of Hes expression across diverse cell types in the developing vertebrate embryonic tail have remained elusive. To address this, we have developed an endogenously tagged Hes1-Achilles mouse line, enabling precise quantification of dynamics at the single-cell resolution across various tissues. Our findings reveal striking disparities in Hes1 dynamics between presomitic mesoderm (PSM) and preneural tube (pre-NT) cells. While pre-NT cells display variable, low-amplitude oscillations, PSM cells exhibit synchronized, high-amplitude oscillations. Upon the induction of differentiation, the oscillation amplitude increases in pre-NT cells. Additionally, our study of Notch inhibition on Hes1 oscillations unveils distinct responses in PSM and pre-NT cells, corresponding to differential Notch ligand expression dynamics. These findings suggest the involvement of separate mechanisms driving Hes1 oscillations. Thus, Hes1 demonstrates dynamic behaviour across adjacent tissues of the embryonic tail, yet the varying oscillation parameters imply differences in the information that can be transmitted by these dynamics

Das Öffnen und Teilen von Daten qualitativer Forschung: eine Handreichung

Die Handreichung ist das Ergebnis eines Workshops der Forschungsgruppe "Digitalisierung der Wissenschaft" am Weizenbaum-Institut in Berlin am 17. Januar 2020. Ziel dieser Handreichung ist es, grundsätzliche Einstiegsfragen zum Thema Öffnen und Teilen von qualitativen Forschungsdaten zu beantworten. Dabei verstehen wir unter Öffnen und Teilen die Zugänglichmachung zur Sekundärnutzung für andere Forschende. In dieser Handreichung werden wir auf die Unterschiede zwischen verschiedenen Forschungsfeldern und deren Eigenheiten im Umgang mit qualitativen Forschungsdaten eingehen und dabei auch die Unterschiedlichkeit verschiedener Datensorten berücksichtigen (z.B. Interviewtranskripte, Videoaufzeichnungen, Beobachtungsprotokolle, Primär- und Kontextdaten)

Functional network resilience to pathology in presymptomatic genetic frontotemporal dementia

© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)The presymptomatic phase of neurodegenerative diseases are characterized by structural brain changes without significant clinical features. We set out to investigate the contribution of functional network resilience to preserved cognition in presymptomatic genetic frontotemporal dementia. We studied 172 people from families carrying genetic abnormalities in C9orf72, MAPT, or PGRN. Networks were extracted from functional MRI data and assessed using graph theoretical analysis. We found that despite loss of both brain volume and functional connections, there is maintenance of an efficient topological organization of the brain's functional network in the years leading up to the estimated age of frontotemporal dementia symptom onset. After this point, functional network efficiency declines markedly. Reduction in connectedness was most marked in highly connected hub regions. Measures of topological efficiency of the brain's functional network and organization predicted cognitive dysfunction in domains related to symptomatic frontotemporal dementia and connectivity correlated with brain volume loss in frontotemporal dementia. We propose that maintaining the efficient organization of the brain's functional network supports cognitive health even as atrophy and connectivity decline presymptomatically.This work was funded by the UK Medical Research Council, the Italian Ministry of Health, and the Canadian Institutes of Health Research as part of a Centres of Excellence in Neurodegeneration grant [grant number CoEN015]. JBR was supported by the Wellcome Trust [grant number 103838]. JBR, RB, TR, and SJ were supported by the NIHR Cambridge Biomedical Research Centre and Medical Research Council [grant number G1100464]. The Dementia Research Centre at UCL is supported by Alzheimer's Research UK, Brain Research Trust, and The Wolfson Foundation, NIHR Queen Square Dementia Biomedical Research Unit, NIHR UCL/H Biomedical Research Centre and Dementia Platforms UK. JDR is supported by an MRC Clinician Scientist Fellowship [grant number MR/M008525/1] and has received funding from the NIHR Rare Disease Translational Research Collaboration [grant number BRC149/NS/MH]. MM is supported by the Canadian Institutes of Health Research, Department of Medicine at Sunnybrook Health Sciences Centre and the University of Toronto, and the Sunnybrook Research Institute. RL is supported by Réseau de médecine génétique appliquée, Fonds de recherche du Québec—Santé [grant number FRQS]. FT is supported by the Italian Ministry of Health. DG is supported by the Fondazione Monzino and Italian Ministry of Health, Ricerca Corrente. SS is supported by Cassa di Risparmio di Firenze [grant number CRF 2013/0199] and the Ministry of Health [grant number RF-2010-2319722]. JvS is supported by The Netherlands Organisation for Health Research and Development Memorable grant [grant number 733050103] and Netherlands Alzheimer Foundation Memorable grant [grant number 733050103].info:eu-repo/semantics/publishedVersio

Disease-related cortical thinning in presymptomatic granulin mutation carriers

© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.Mutations in the granulin gene (GRN) cause familial frontotemporal dementia. Understanding the structural brain changes in presymptomatic GRN carriers would enforce the use of neuroimaging biomarkers for early diagnosis and monitoring. We studied 100 presymptomatic GRN mutation carriers and 94 noncarriers from the Genetic Frontotemporal dementia initiative (GENFI), with MRI structural images. We analyzed 3T MRI structural images using the FreeSurfer pipeline to calculate the whole brain cortical thickness (CTh) for each subject. We also perform a vertex-wise general linear model to assess differences between groups in the relationship between CTh and diverse covariables as gender, age, the estimated years to onset and education. We also explored differences according to TMEM106B genotype, a possible disease modifier. Whole brain CTh did not differ between carriers and noncarriers. Both groups showed age-related cortical thinning. The group-by-age interaction analysis showed that this age-related cortical thinning was significantly greater in GRN carriers in the left superior frontal cortex. TMEM106B did not significantly influence the age-related cortical thinning. Our results validate and expand previous findings suggesting an increased CTh loss associated with age and estimated proximity to symptoms onset in GRN carriers, even before the disease onset.The authors thank all the volunteers for their participation in this study. SBE is a recipient of the Rio-Hortega post-residency grant from the Instituto de Salud Carlos III, Spain. This study was partially funded by Fundació Marató de TV3, Spain (grant no. 20143810 to RSV). The GENFI study has been supported by the Medical Research Council UK, the Italian Ministry of Health and the Canadian Institutes of Health Research as part of a Centres of Excellence in Neurodegeneration grant, as well as other individual funding to investigators. KM has received funding from an Alzheimer’s Society PhD studentship. JDR acknowledges support from the National Institute for Health Research (NIHR) Queen Square Dementia Biomedical Research Unit and the University College London Hospitals Biomedical Research Centre, the Leonard Wolfson Experimental Neurology Centre, the UK Dementia Research Institute, Alzheimer’s Research UK, the Brain Research Trust and the Wolfson Foundation. JCvS was supported by the Dioraphte Foundation grant 09-02-03-00, the Association for Frontotemporal Dementias Research Grant 2009, The Netherlands Organization for Scientific Research (NWO) grant HCMI 056-13-018, ZonMw Memorabel (Deltaplan Dementie, project number 733 051 042), Alzheimer Nederland and the Bluefield project. CG have received funding from JPND-Prefrontals VR Dnr 529-2014-7504, VR: 2015-02926, and 2018-02754, the Swedish FTD Initiative-Schörling Foundation, Alzheimer Foundation, Brain Foundation and Stockholm County Council ALF. DG has received support from the EU Joint Programme – Neurodegenerative Disease Research (JPND) and the Italian Ministry of Health (PreFrontALS) grant 733051042. JBR is funded by the Wellcome Trust (103838) and the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre. MM has received funding from a Canadian Institutes of Health Research operating grant and the Weston Brain Institute and Ontario Brain Institute. RV has received funding from the Mady Browaeys Fund for Research into Frontotemporal Dementia. EF has received funding from a CIHR grant #327387. JDR is an MRC Clinician Scientist (MR/M008525/1) and has received funding from the NIHR Rare Diseases Translational Research Collaboration (BRC149/NS/MH), the Bluefield Project and the Association for Frontotemporal Degeneration. MS was supported by a grant 779257 “Solve-RD” from the Horizon 2020 research and innovation programme.info:eu-repo/semantics/publishedVersio

Extending the phenotypic spectrum assessed by the CDR plus NACC FTLD in genetic frontotemporal dementia

INTRODUCTION: We aimed to expand the range of the frontotemporal dementia (FTD) phenotypes assessed by the Clinical Dementia Rating Dementia Staging Instrument plus National Alzheimer's Coordinating Center Behavior and Language Domains (CDR plus NACC FTLD). METHODS: Neuropsychiatric and motor domains were added to the standard CDR plus NACC FTLD generating a new CDR plus NACC FTLD-NM scale. This was assessed in 522 mutation carriers and 310 mutation-negative controls from the Genetic Frontotemporal dementia Initiative (GENFI). RESULTS: The new scale led to higher global severity scores than the CDR plus NACC FTLD: 1.4% of participants were now considered prodromal rather than asymptomatic, while 1.3% were now considered symptomatic rather than asymptomatic or prodromal. No participants with a clinical diagnosis of an FTD spectrum disorder were classified as asymptomatic using the new scales. DISCUSSION: Adding new domains to the CDR plus NACC FTLD leads to a scale that encompasses the wider phenotypic spectrum of FTD with further work needed to validate its use more widely. Highlights: The new Clinical Dementia Rating Dementia Staging Instrument plus National Alzheimer's Coordinating Center Behavior and Language Domains neuropsychiatric and motor (CDR plus NACC FTLD-NM) rating scale was significantly positively correlated with the original CDR plus NACC FTLD and negatively correlated with the FTD Rating Scale (FRS). No participants with a clinical diagnosis in the frontotemporal dementia spectrum were classified as asymptomatic with the new CDR plus NACC FTLD-NM rating scale. Individuals had higher global severity scores with the addition of the neuropsychiatric and motor domains. A receiver operating characteristic analysis of symptomatic diagnosis showed nominally higher areas under the curve for the new scales.</p

Analysis of brain atrophy and local gene expression in genetic frontotemporal dementia.

Frontotemporal dementia is a heterogeneous neurodegenerative disorder characterized by neuronal loss in the frontal and temporal lobes. Despite progress in understanding which genes are associated with the aetiology of frontotemporal dementia, the biological basis of how mutations in these genes lead to cell loss in specific cortical regions remains unclear. In this work we combined gene expression data for 16,772 genes from the Allen Institute for Brain Science atlas with brain maps of gray matter atrophy in symptomatic C9orf72, GRN and MAPT mutation carriers obtained from the Genetic Frontotemporal dementia Initiative study. No significant association was seen between C9orf72, GRN and MAPT expression and the atrophy patterns in the respective genetic groups. After adjusting for spatial autocorrelation, between 1,000 and 5,000 genes showed a negative or positive association with the atrophy pattern within each individual genetic group, with the most significantly associated genes being TREM2, SSBP3 and GPR158 (negative association in C9orf72, GRN and MAPT respectively) and RELN, MXRA8 and LPA (positive association in C9orf72, GRN and MAPT respectively). An overrepresentation analysis identified a negative association with genes involved in mitochondrial function, and a positive association with genes involved in vascular and glial cell function in each of the genetic groups. A set of 423 and 700 genes showed significant positive and negative association, respectively, with atrophy patterns in all three maps. The gene set with increased expression in spared cortical regions was enriched for neuronal and microglial genes, while the gene set with increased expression in atrophied regions was enriched for astrocyte and endothelial cell genes. Our analysis suggests that these cell types may play a more active role in the onset of neurodegeneration in frontotemporal dementia than previously assumed, and in the case of the positively-associated cell marker genes, potentially through emergence of neurotoxic astrocytes and alteration in the blood-brain barrier respectively