Thomas Corydon - Academia.edu (original) (raw)

Papers by Thomas Corydon

International Journal of Molecular Sciences, 2022

Cancer is a disease exhibiting uncontrollable cell growth and spreading to other parts of the org... more Cancer is a disease exhibiting uncontrollable cell growth and spreading to other parts of the organism. It is a heavy, worldwide burden for mankind with high morbidity and mortality. Therefore, groundbreaking research and innovations are necessary. Research in space under microgravity (µg) conditions is a novel approach with the potential to fight cancer and develop future cancer therapies. Space travel is accompanied by adverse effects on our health, and there is a need to counteract these health problems. On the cellular level, studies have shown that real (r-) and simulated (s-) µg impact survival, apoptosis, proliferation, migration, and adhesion as well as the cytoskeleton, the extracellular matrix, focal adhesion, and growth factors in cancer cells. Moreover, the µg-environment induces in vitro 3D tumor models (multicellular spheroids and organoids) with a high potential for preclinical drug targeting, cancer drug development, and studying the processes of cancer progression a...

Verification of five selected ChIP-seq promoter targets by ChIP-QPCR. From the 251 common promoto... more Verification of five selected ChIP-seq promoter targets by ChIP-QPCR. From the 251 common promotor target genes (PTGs) of BRD1-S and BRD1-L, we selected five for validation with ChIP-QPCR. A The promoter loci at the WD repeat domain 7 (WDR7), digestive organ expansion factor homolog (C1ORF107 or DIEXF), proteasome subunit β type, 2 (PSMB2), ZC3H15, and zinc finger protein 226 (ZNF226) showed BRD1-S and BRD1-L binding, while the promoter loci of the lymphocyte expressed gene granzyme M (GZMM) showed no significant binding of the BRD1 isoforms and was subsequently selected as a negative control. The BRD1-S or BRD1-L binding is shown in black whereas the control ChIP-seq (IP with anti-HA antibody) is shown in pink. B Primers were designed to amplify a 80–150 bp sequence in the promoter region or the intron of all six genes (for primer sequences see Additional file 7). ChIP was performed with either anti-V5 antibody (V5) conjugated beads or anti-HA (HA) conjugated beads using extracts f...

Acta Astronautica, 2019

Endothelial cells (ECs) grow as single layers on the bottom surface of cell culture flasks under ... more Endothelial cells (ECs) grow as single layers on the bottom surface of cell culture flasks under normal (1g) culture conditions. In numerous experiments using simulated microgravity we noticed that the ECs formed three-dimensional, tube-like cell aggregates resembling the intima of small, rudimentary blood vessels. The SPHEROIDS project has now shown that similar processes occur in space. For the first time, we were able to observe scaffoldfree growth of human ECs into multicellular spheroids and tubular structures during an experiment in real microgravity. With further investigation of the space samples we hope to understand endothelial 3D growth and to improve the in vitro engineering of biocompatible vessels which could be used in surgery.

Reproduction, Fertility and Development, 2004

Recently, a zona-free technique for bovine somatic cell nuclear transfer (NT) with no requirement... more Recently, a zona-free technique for bovine somatic cell nuclear transfer (NT) with no requirement for micromanipulation (i.e. hand-made cloning (HMC)) has been described. The present study demonstrates the application of the HMC technique in the production of transgenic porcine blastocysts. In vitro-matured zona-free porcine oocytes were bisected manually using a microblade and halves containing no chromatin (i.e. the cytoplasts) were selected. Two cytoplasts were electrofused with one transgenic fibroblast expressing enhanced green fluorescent protein and reconstructed embryos were activated in calcium ionophore (A23187) followed by 6-dimethylaminopurine. Subsequently, embryos were cultured in NCSU-23 medium supplemented with 4 mg mL–1 bovine serum albumin for 7 days. In five replicates, 93.0 ± 7.0% (mean ± s.e.m.) of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, 37/37 and 28/28). On Day 7 after activation, the respective blastocyst rates (per ...

Reproduction, Fertility and Development, 2005

Activation is a crucial step in mammalian somatic cell nuclear transfer (SCNT). Recently we descr... more Activation is a crucial step in mammalian somatic cell nuclear transfer (SCNT). Recently we described the application of the handmade cloning technique for porcine SCNT that uses oocytes without zonaa pellucidae (zona-free) in a micromanipulation-independent procedure (Kragh et al. 2004 Reprod. Fertil. Dev. 16, 315–18). The purpose of the present study was to investigate the effect of a combined electrical and chemical activation of zona-free porcine oocytes. Cumulus-oocyte complexes were aspirated from ovaries of sows and matured for 41 h. Subsequently, the cumulus cells were removed by the addition of 1 mg/mL hyaluronidase in HEPES-buffered TCM-199. For zonae pellucidae removal, oocytes were incubated in 8 mg/mL pronase in HEPES-buffered TCM-199 supplemented with 20% cattle serum for 10 s. Three independent experiments with four treatments were conducted, and oocytes were activated by a combined electrical and chemical activation. Oocytes were washed once in activation medium (0.3...

Stem cells and development, Jan 15, 2018

Experimental cell research studying three-dimensional (3D) tissues in space and on Earth using ne... more Experimental cell research studying three-dimensional (3D) tissues in space and on Earth using new techniques to simulate microgravity is currently a hot topic in Gravitational Biology and Biomedicine. This review will focus on the current knowledge of the use of stem cells and specialized cells for tissue engineering under simulated microgravity conditions. We will report on recent advancements in the ability to construct 3D aggregates from various cell types using devices originally created to prepare for spaceflights such as the random positioning machine (RPM), the clinostat, or the NASA-developed rotating wall vessel (RWV) bioreactor, to engineer various tissues such as preliminary vessels, eye tissue, bone, cartilage, multicellular cancer spheroids, and others from different cells. In addition, stem cells had been investigated under microgravity for the purpose to engineer adipose tissue, cartilage, or bone. Recent publications have discussed different changes of stem cells wh...

Biomaterials, 2017

Human endothelial cells (ECs) were sent to the International Space Station (ISS) to determine the... more Human endothelial cells (ECs) were sent to the International Space Station (ISS) to determine the impact of microgravity on the formation of three-dimensional structures. For this project, an automatic experiment unit (EU) was designed allowing cell culture in space. In order to enable a safe cell culture, cell nourishment and fixation after a pre-programmed timeframe, the materials used for construction of the EUs were tested in regard to their biocompatibility. These tests revealed a high biocompatibility for all parts of the EUs, which were in contact with the cells or the medium used. Most importantly, we found polyether ether ketones for surrounding the incubation chamber, which kept cellular viability above 80% and allowed the cells to adhere as long as they were exposed to normal gravity. After assembling the EU the ECs were cultured therein, where they showed good cell viability at least for 14 days. In addition, the functionality of the automatic medium exchange, and fixation procedures were confirmed. Two days before launch, the ECs were cultured in the EUs, which were afterwards mounted on the SpaceX CRS-8 rocket. 5 and 12 days after launch the cells were fixed. Subsequent analyses revealed a scaffold-free formation of spheroids in space.

Scientific Reports, 2016

Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are c... more Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. MCS often resemble the organ, from which the cells have been derived. In this study we investigated human MCF-7 breast cancer cells after a 2 h-, 4 h-, 16 h-, 24 hand 5d-exposure to a Random Positioning Machine (RPM) simulating microgravity. At 24 h few small compact MCS were detectable, whereas after 5d many MCS were floating in the supernatant above the cells, remaining adherently (AD). The MCS resembled the ducts formed in vivo by human epithelial breast cells. In order to clarify the underlying mechanisms, we harvested MCS and AD cells separately from each RPM-culture and measured the expression of 29 selected genes with a known involvement in MCS formation. qPCR analyses indicated that cytoskeletal genes were unaltered in short-term samples. IL8, VEGFA, and FLT1 were upregulated in 2 h/4 h AD-cultures. The ACTB, TUBB, EZR, RDX, FN1, VEGFA, FLK1 Casp9, Casp3, PRKCA mRNAs were downregulated in 5d-MCS-samples. ESR1 was upregulated in AD, and PGR1 in both phenotypes after 5d. A pathway analysis revealed that the corresponding gene products are involved in organization and regulation of the cell shape, in cell tip formation and membrane to membrane docking. Breast cancer is the second most common cancer worldwide with 1.7 million cases in 2012 1. Advances in prevention, early diagnosis, surgical treatment and postsurgical therapies enhanced the possibility of a complete cure 2. Known molecular targets (e.g. VEGF, VEGFR, HER2/neu) for approved drugs (e.g. tyrosine kinase inhibitors like sorafenib), or approved therapeutic antibodies (e.g. bevacizumab, ramucirumab, trastuzumab) are proteins, which are predominantly expressed in breast cancer cells and are simultaneously involved in promoting cell growth or apoptosis 3,4. However, it is difficult at the current state of technology to apply the optimal cocktail of drugs to hit all cancer cells of any given patient. Under these circumstances, it is absolutely necessary to find new proteins, which can serve as targets to develop drugs against this cancer type. In earlier studies we proved repeatedly that exposing various cell types like thyroid cells, endothelial cells and chondrocytes to simulated microgravity (s-μ g) results in a scaffold-free production of three-dimensional (3D) aggregates so-called multicellular spheroids (MCS) 5-10. The MCS very often resemble the tissue, from which the cells have been derived. In case of cancer cells, the in vivo structure of tumors appears more closely represented by MCS than by monolayer cell cultures 11-13. A proteomics investigation on thyroid cancer cells had shown that FTC-133 cells express surface proteins binding fibronectin which induces 3D cohesion 5 .

Genome medicine, May 3, 2016

The bromodomain containing 1 (BRD1) gene has been implicated with transcriptional regulation, bra... more The bromodomain containing 1 (BRD1) gene has been implicated with transcriptional regulation, brain development, and susceptibility to schizophrenia and bipolar disorder. To advance the understanding of BRD1 and its role in mental disorders, we characterized the protein and chromatin interactions of the BRD1 isoforms, BRD1-S and BRD1-L. Stable human cell lines expressing epitope tagged BRD1-S and BRD1-L were generated and used as discovery systems for identifying protein and chromatin interactions. Protein-protein interactions were identified using co-immunoprecipitation followed by mass spectrometry and chromatin interactions were identified using chromatin immunoprecipitation followed by next generation sequencing. Gene expression profiles and differentially expressed genes were identified after upregulating and downregulating BRD1 expression using microarrays. The presented functional molecular data were integrated with human genomic and transcriptomic data using available GWAS, ...

PLOS ONE, 2015

In this study we focused on gravity-sensitive proteins of two human thyroid cancer cell lines (ML... more In this study we focused on gravity-sensitive proteins of two human thyroid cancer cell lines (ML-1; RO82-W-1), which were exposed to a 2D clinostat (CLINO), a random positioning machine (RPM) and to normal 1g-conditions. After a three (3d)-or seven-day-culture (7d) on the two devices, we found both cell types growing three-dimensionally within multicellular spheroids (MCS) and also cells remaining adherent (AD) to the culture flask, while 1gcontrol cultures only formed adherent monolayers, unless the bottom of the culture dish was covered by agarose. In this case, the cytokines IL-6 and IL-8 facilitated the formation of MCS in both cell lines using the liquid-overlay technique at 1g. ML-1 cells grown on the RPM or the CLINO released amounts of IL-6 and MCP-1 into the supernatant, which were significantly elevated as compared to 1g-controls. Release of IL-4, IL-7, IL-8, IL-17, eotaxin-1 and VEGF increased time-dependently, but was not significantly influenced by the gravity conditions. After 3d on the RPM or the CLINO, an accumulation of F-actin around the cellular membrane was detectable in AD cells of both cell lines. IL-6 and IL-8 stimulation of ML-1 cells for 3d and 7d influenced the protein contents of ß 1-integrin, talin-1, Ki-67, and betaactin dose-dependently in adherent cells. The ß 1-integrin content was significantly decreased in AD and MCS samples compared with 1g, while talin-1 was higher expressed in MCS than AD populations. The proliferation marker Ki-67 was elevated in AD samples compared with 1g and MCS samples. The ß-actin content of R082-W-1 cells remained unchanged. ML-1 cells exhibited no change in ß-actin in RPM cultures, but a reduction in CLINO samples. Thus, we concluded that simulated microgravity influences the release of cytokines in follicular thyroid cancer cells, and the production of ß 1-integrin and talin-1 and predicts an identical effect under real microgravity conditions.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 13, 2015

Real and simulated microgravity induce a variety of changes in human cells. Most importantly, cha... more Real and simulated microgravity induce a variety of changes in human cells. Most importantly, changes in the cytoskeleton have been noted, and studies on microtubules have shown that they are gravi-sensitive. This study focuses on the effects of short-term real microgravity on gene expression, protein content, and cytoskeletal structure of human chondrocytes. We cultivated human chondrocytes, took them along a parabolic flight during the 24th Deutsches Zentrum für Luft- und Raumfahrt Parabolic (DLR) Flight Campaign, and fixed them after the 1st and the 31st parabola. Immunofluorescence microscopy revealed no changes after the 1st parabola, but disruptions of β-tubulin, vimentin, and cytokeratin networks after the 31st parabola. No F-actin stress fibers were detected even after 31 parabolas. Furthermore, mRNA and protein quantifications after the 31st parabola showed a clear up-regulation of cytoskeletal genes and proteins. The mRNAs were significantly up-regulated as follows: TUBB, ...

Cell Communication and Signaling, 2015

Background: Chondrocytes are the main cellular component of articular cartilage. In healthy tissu... more Background: Chondrocytes are the main cellular component of articular cartilage. In healthy tissue, they are embedded in a strong but elastic extracelluar matrix providing resistance against mechanical forces and friction for the joints. Osteoarthritic cartilage, however, disrupted by heavy strain, has only very limited potential to heal. One future possibility to replace damaged cartilage might be the scaffold-free growth of chondrocytes in microgravity to form 3D aggregates. Results: To prepare for this, we have conducted experiments during the 20th DLR parabolic flight campaign, where we fixed the cells after the first (1P) and the 31st parabola (31P). Furthermore, we subjected chondrocytes to isolated vibration and hypergravity conditions. Microarray and quantitative real time PCR analyses revealed that hypergravity regulated genes connected to cartilage integrity (BMP4, MMP3, MMP10, EDN1, WNT5A, BIRC3). Vibration was clearly detrimental to cartilage (upregulated inflammatory IL6 and IL8, downregulated growth factors EGF, VEGF, FGF17). The viability of the cells was not affected by the parabolic flight, but showed a significantly increased expression of anti-apoptotic genes after 31 parabolas. The IL-6 release of chondrocytes cultured under conditions of vibration was not changed, but hypergravity (1.8 g) induced a clear elevation of IL-6 protein in the supernatant compared with corresponding control samples. Conclusion: Taken together, this study provided new insights into the growth behavior of chondrocytes under short-term microgravity.

Encyclopedia of Life Sciences, 2011

The function of proteins is dependent on other proteins. Proteins function as oligomers, complexe... more The function of proteins is dependent on other proteins. Proteins function as oligomers, complexes, super-complexes or higher order networks, in which they interact with each other, either temporarily when they exert their function or ‘permanently’ in functional units. Genetic defects in single proteins may therefore, in addition to disturbing the specific function of the defective protein, disturb other functions that are dependent on it. In this review we will discuss how the two main types of defects in genetic disease, truncating variations (stop-codon introductions and small out-of-frame deletions/insertions) and in-frame variations (missense variations and small in-frame deletions/insertions), may disturb normal interactions. Depending on the importance (location) of the missing or aberrant protein, the effect on the cellular pathway or interacting network may be severe or mild. Protein interactions and disturbances therein may be determined by protein mass spectrometry after immuno-precipitation or other fractionation and separation methods. Key Concepts: Cellular proteins interact in oligomer and complex structures as well as in higher order networks. Protein interactions may be permanent in the lifetime of proteins or temporary during their function. Genetic defects may disturb interactions between proteins depending on the nature of the defect and type of interaction. Missing proteins due to truncation, comprising big deletions, small out-of-frame deletions/insertions and severe splice alteration, may abolish the function of oligomers, complexes, pathways and networks. Aberrant proteins due to missense variations and in-frame deletions/insertions may disturb interactions with protein partners in oligomers, complexes and networks. Missing and aberrant proteins in branch-points (nodes) have as a role severe consequences, resulting in genotype–phenotype association. Missing and aberrant proteins in redundant pathways have variable consequences, resulting in poor genotype–phenotype association. Missing and aberrant proteins in molecular machines, such as the chaperonin Hsp60, may elicit pleotropic effects due to defective processing of client proteins. Protein interactions and disturbances therein may be determined experimentally by protein mass spectrometry (MS), preceded by immunoprecipitation of target proteins, either directly or after cross-linking, or indicated after mild solubilisation followed by extensive separating procedures and MS. The challenge is to design experiments that can determine qualitative and quantitative disturbances of proteins interactions in cells and tissue from patients, model animals and model cells compared to interactions in normal cells and tissue. Keywords: aberrant protein structures; disturbed protein interaction; protein complexes; protein super-complexes; protein networks; protein quality control; metabolon structures; metabolic channelling; determination of protein interaction; structural mass spectrometry

We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase (SCAD) g... more We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase (SCAD) gene, 625G→A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disease-causing mutations (confirmed by lack of enzyme activity after expression in COS-7 cells) and a new susceptibility variant in the SCAD gene of two patients with SCAD deficiency, and investigated their frequency in patients with elevated EMA excretion. The first SCAD-deficient patient was a compound heterozygote for two mutations, 274G→T and 529T→C. These mutations were not present in 98 normal control alleles, but the 529T→C mutation was found in one allele among 133 patients with elevated EMA excretion. The second patient carried a 1147C→T mutation and the 625G→A polymorphism in one allele, and a single point mutation, 511C→T, in the other. The 1147C→T mutation was not present in 98 normal alleles, but was detected in three alleles of 133 patients with elevated EMA excretion, consistently as a 625A-1147T allele. On the other hand, the 511C→T mutation was present in 13 of 130 and 15 of 67 625G alleles, respectively, of normal controls and patients with elevated EMA excretion, and was never associated with the 625A variant

Theriogenology, 2005

The purpose of our work was to establish an efficient protocol for activation of porcine cytoplas... more The purpose of our work was to establish an efficient protocol for activation of porcine cytoplastfibroblast constructs produced by the handmade cloning technique. Firstly, we investigated a combined electrical and chemical activation protocol for parthenogenetic development of in vitro matured zona-free oocytes. Oocytes were activated by one 80 ms pulse and subsequently cultured in cytochalasin B and cycloheximide. Developmental rates of blastocysts from activated oocytes were 49 AE 1 and 40 AE 2%, when using one 80 ms pulse of 0.85 or 1.25 kV/cm, respectively. The activation procedure was further confirmed by a simultaneous re-fusion and activation of bisected oocytes, resulting in a blastocyst rate of 41 AE 8%. Secondly, the activation protocol was applied in the handmade cloning technique. In vitro matured zona-free porcine oocytes were bisected and halves containing no chromatin, i.e. the cytoplasts, were selected. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused to one fibroblast by one 80 ms pulse of 1.25 kV/cm. After 1 h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously by one 80 ms pulse of 0.85 kV/cm, and subsequently cultured in cytochalasin B and cycloheximide. The development of reconstructed embryos to the blastocyst stage was in average 21 AE 4%, and total blastocyst cell counts were in average 48 AE 3. Thus, the combined www.journals.elsevierhealth.com/periodicals/the

Mammalian Genome, 2000

We have determined the cDNA sequence and exon/intron structure of the human CLPX gene encoding a ... more We have determined the cDNA sequence and exon/intron structure of the human CLPX gene encoding a human ortholog of the E. coli ClpX chaperone and protease subunit. The CLPX gene comprises 14 exons and encodes a 633-amino acid-long precursor polypeptide. The polypeptide contains an N-terminal putative mitochondrial transit peptide, and expression of a full-length ClpX cDNA tagged at its C-terminus (Myc-His) shows that the polypeptide is transported into mitochondria. FISH analysis localized the CLPX gene to human Chromosome (Chr) 15q22.1-22.32. This localization was refined by radiation hybrid mapping placing the CLPX gene 4.6 cR distal to D15S159. Murine ClpX cDNA was sequenced, and the mouse Clpx locus was mapped to a position between 31 and 42 cM offset from the centromere on mouse Chr 9. Experimental observations indicate the presence of a pseudogene in the mouse genome and sequence variability between mouse ClpX cDNAs from different strains. Alignment of the human and mouse ClpX amino acid sequences with ClpX sequences from other organisms shows that they display the typical modular organization of domains with one AAA(+) domain common to a large group of ATPases and several other domains conserved in ClpX orthologs linked by non-conserved sequences. Notably, a C-4 zinc finger type motif is recognized in human and mouse ClpX. This motif of so far unknown function is present only in a subset of the known ClpX sequences.

Journal of Biological Chemistry, 2000

The low density lipoprotein (LDL) receptor is responsible for removing the majority of the LDL ch... more The low density lipoprotein (LDL) receptor is responsible for removing the majority of the LDL cholesterol from the plasma. Mutations in the LDL receptor gene cause the disease familial hypercholesterolemia (FH). Approximately 50% of the mutations in the LDL receptor gene in patients with FH lead to receptor proteins that are retained in the endoplasmic reticulum (ER). Misfolding of mutant LDL receptors is a probable cause of this ER retention, resulting in no functional LDL receptors at the cell surface. However, the specific factors and mechanisms responsible for retention of mutant LDL receptors are unknown. In the present study we show that the molecular chaperone Grp78/BiP co-immunoprecipitates with both the wild type and two different mutant (W556S and C646Y) LDL receptors in lysates obtained from human liver cells overexpressing wild type or mutant LDL receptors. A pulse-chase study shows that the interaction between the wild type LDL receptor and Grp78 is no longer detectable after 2 1 ⁄2 h, whereas it persists for more than 4 h with the mutant receptors. Furthermore, about five times more Grp78 is co-immunoprecipitated with the mutant receptors than with the wild type receptor suggesting that Grp78 is involved in retention of mutant LDL receptors in the ER. Overexpression of Grp78 causes no major alterations on the steady state level of active LDL receptors at the cell surface. However, overexpression of Grp78 decreases the processing rate of newly synthesized wild type LDL receptors. This indicates that the Grp78 interaction is a rate-limiting step in the maturation of the wild type LDL receptor and that Grp78 may be an important factor in the quality control of newly synthesized LDL receptors.