Publications by authors named "Patrick Van Oostveldt"

42 Publications

Combined Exposure to Simulated Microgravity and Acute or Chronic Radiation Reduces Neuronal Network Integrity and Survival.

PLoS One 2016 20;11(5):e0155260. Epub 2016 May 20.

Radiobiology Unit, Laboratory of Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre, SCK•CEN, Mol, Belgium.

During orbital or interplanetary space flights, astronauts are exposed to cosmic radiations and microgravity. However, most earth-based studies on the potential health risks of space conditions have investigated the effects of these two conditions separately. This study aimed at assessing the combined effect of radiation exposure and microgravity on neuronal morphology and survival in vitro. In particular, we investigated the effects of simulated microgravity after acute (X-rays) or during chronic (Californium-252) exposure to ionizing radiation using mouse mature neuron cultures. Acute exposure to low (0.1 Gy) doses of X-rays caused a delay in neurite outgrowth and a reduction in soma size, while only the high dose impaired neuronal survival. Of interest, the strongest effect on neuronal morphology and survival was evident in cells exposed to microgravity and in particular in cells exposed to both microgravity and radiation. Removal of neurons from simulated microgravity for a period of 24 h was not sufficient to recover neurite length, whereas the soma size showed a clear re-adaptation to normal ground conditions. Genome-wide gene expression analysis confirmed a modulation of genes involved in neurite extension, cell survival and synaptic communication, suggesting that these changes might be responsible for the observed morphological effects. In general, the observed synergistic changes in neuronal network integrity and cell survival induced by simulated space conditions might help to better evaluate the astronaut's health risks and underline the importance of investigating the central nervous system and long-term cognition during and after a space flight.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155260PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874625PMC
July 2017

Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation.

Int J Mol Med 2014 Oct 11;34(4):1124-32. Epub 2014 Aug 11.

Laboratory of Radiobiology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK‑CEN), Mol, Belgium.

Ionizing radiation can elicit harmful effects on the cardiovascular system at high doses. Endothelial cells are critical targets in radiation-induced cardiovascular damage. Astronauts performing a long-term deep space mission are exposed to consistently higher fluences of ionizing radiation that may accumulate to reach high effective doses. In addition, cosmic radiation contains high linear energy transfer (LET) radiation that is known to produce high values of relative biological effectiveness (RBE). The aim of this study was to broaden the understanding of the molecular response to high LET radiation by investigating the changes in gene expression in endothelial cells. For this purpose, a human endothelial cell line (EA.hy926) was irradiated with accelerated nickel ions (Ni) (LET, 183 keV/µm) at doses of 0.5, 2 and 5 Gy. DNA damage was measured 2 and 24 h following irradiation by γ-H2AX foci detection by fluorescence microscopy and gene expression changes were measured by microarrays at 8 and 24 h following irradiation. We found that exposure to accelerated nickel particles induced a persistent DNA damage response up to 24 h after treatment. This was accompanied by a downregulation in the expression of a multitude of genes involved in the regulation of the cell cycle and an upregulation in the expression of genes involved in cell cycle checkpoints. In addition, genes involved in DNA damage response, oxidative stress, apoptosis and cell-cell signaling (cytokines) were found to be upregulated. An in silico analysis of the involved genes suggested that the transcription factors, E2F and nuclear factor (NF)-κB, may be involved in these cellular responses.
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http://dx.doi.org/10.3892/ijmm.2014.1893DOI Listing
October 2014

Chronic exposure to simulated space conditions predominantly affects cytoskeleton remodeling and oxidative stress response in mouse fetal fibroblasts.

Int J Mol Med 2014 Aug 22;34(2):606-15. Epub 2014 May 22.

Radiobiology Unit, Expert Group of Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK-CEN), Mol, Belgium.

Microgravity and cosmic rays as found in space are difficult to recreate on earth. However, ground-based models exist to simulate space flight experiments. In the present study, an experimental model was utilized to monitor gene expression changes in fetal skin fibroblasts of murine origin. Cells were continuously subjected for 65 h to a low dose (55 mSv) of ionizing radiation (IR), comprising a mixture of high‑linear energy transfer (LET) neutrons and low-LET gamma-rays, and/or simulated microgravity using the random positioning machine (RPM), after which microarrays were performed. The data were analyzed both by gene set enrichment analysis (GSEA) and single gene analysis (SGA). Simulated microgravity affected fetal murine fibroblasts by inducing oxidative stress responsive genes. Three of these genes are targets of the nuclear factor‑erythroid 2 p45-related factor 2 (Nrf2), which may play a role in the cell response to simulated microgravity. In addition, simulated gravity decreased the expression of genes involved in cytoskeleton remodeling, which may have been caused by the downregulation of the serum response factor (SRF), possibly through the Rho signaling pathway. Similarly, chronic exposure to low-dose IR caused the downregulation of genes involved in cytoskeleton remodeling, as well as in cell cycle regulation and DNA damage response pathways. Many of the genes or gene sets that were altered in the individual treatments (RPM or IR) were not altered in the combined treatment (RPM and IR), indicating a complex interaction between RPM and IR.
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http://dx.doi.org/10.3892/ijmm.2014.1785DOI Listing
August 2014

Transcriptomic profiling suggests a role for IGFBP5 in premature senescence of endothelial cells after chronic low dose rate irradiation.

Int J Radiat Biol 2014 Jul;90(7):560-74

Radiobiology Unit, Belgian Nuclear Research Centre, SCK•CEN , Mol , Belgium.

Purpose: Ionizing radiation has been recognized to increase the risk of cardiovascular diseases (CVD). However, there is no consensus concerning the dose-risk relationship for low radiation doses and a mechanistic understanding of low dose effects is needed.

Material And Methods: Previously, human umbilical vein endothelial cells (HUVEC) were exposed to chronic low dose rate radiation (1.4 and 4.1 mGy/h) during one, three and six weeks which resulted in premature senescence in cells exposed to 4.1 mGy/h. To gain more insight into the underlying signaling pathways, we analyzed gene expression changes in these cells using microarray technology. The obtained data were analyzed in a dual approach, combining single gene expression analysis and Gene Set Enrichment Analysis.

Results: An early stress response was observed after one week of exposure to 4.1 mGy/h which was replaced by a more inflammation-related expression profile after three weeks and onwards. This early stress response may trigger the radiation-induced premature senescence previously observed in HUVEC irradiated with 4.1 mGy/h. A dedicated analysis pointed to the involvement of insulin-like growth factor binding protein 5 (IGFBP5) signaling in radiation-induced premature senescence.

Conclusion: Our findings motivate further research on the shape of the dose-response and the dose rate effect for radiation-induced vascular senescence.
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http://dx.doi.org/10.3109/09553002.2014.905724DOI Listing
July 2014

MorphoNeuroNet: an automated method for dense neurite network analysis.

Cytometry A 2014 Feb 12;85(2):188-99. Epub 2013 Nov 12.

Radiobiology Unit, Molecular and Cellular Biology Expert Group, Belgian Nuclear Research Centre, SCK•CEN, Mol, Belgium; Cell Systems and Imaging Research Group (CSI), Department of Molecular Biotechnology, Ghent University, Ghent, Belgium.

High content cell-based screens are rapidly gaining popularity in the context of neuronal regeneration studies. To analyze neuronal morphology, automatic image analysis pipelines have been conceived, which accurately quantify the shape changes of neurons in cell cultures with non-dense neurite networks. However, most existing methods show poor performance for well-connected and differentiated neuronal networks, which may serve as valuable models for inter alia synaptogenesis. Here, we present a fully automated method for quantifying the morphology of neurons and the density of neurite networks, in dense neuronal cultures, which are grown for more than 10 days. MorphoNeuroNet, written as a script for ImageJ, Java based freeware, automatically determines various morphological parameters of the soma and the neurites (size, shape, starting points, and fractional occupation). The image analysis pipeline consists of a multi-tier approach in which the somas are segmented by adaptive region growing using nuclei as seeds, and the neurites are delineated by a combination of various intensity and edge detection algorithms. Quantitative comparison showed a superior performance of MorphoNeuroNet to existing analysis tools, especially for revealing subtle changes in thin neurites, which have weak fluorescence intensity compared to the rest of the network. The proposed method will help determining the effects of compounds on cultures with dense neurite networks, thereby boosting physiological relevance of cell-based assays in the context of neuronal diseases.
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http://dx.doi.org/10.1002/cyto.a.22408DOI Listing
February 2014

Morphological and physiological changes in mature in vitro neuronal networks towards exposure to short-, middle- or long-term simulated microgravity.

PLoS One 2013 16;8(9):e73857. Epub 2013 Sep 16.

Radiobiology Unit, Molecular and Cellular Biology expert group, Belgian Nuclear Research Centre, SCK•CEN, Mol, Belgium ; Laboratory of Biochemistry and Molecular Cytology, Department of Molecular Biotechnology, Ghent University, Ghent, Belgium.

One of the objectives of the current international space programmes is to investigate the possible effects of the space environment on the crew health. The aim of this work was to assess the particular effects of simulated microgravity on mature primary neuronal networks and specially their plasticity and connectivity. For this purpose, primary mouse neurons were first grown for 10 days as a dense network before being placed in the Random Positioning Machine (RPM), simulating microgravity. These cultures were then used to investigate the impact of short- (1 h), middle- (24 h) and long-term (10 days) exposure to microgravity at the level of neurite network density, cell morphology and motility as well as cytoskeleton properties in established two-dimensional mature neuronal networks. Image processing analysis of dense neuronal networks exposed to simulated microgravity and their subsequent recovery under ground conditions revealed different neuronal responses depending on the duration period of exposure. After short- and middle-term exposures to simulated microgravity, changes in neurite network, neuron morphology and viability were observed with significant alterations followed by fast recovery processes. Long exposure to simulated microgravity revealed a high adaptation of single neurons to the new gravity conditions as well as a partial adaptation of neuronal networks. This latter was concomitant to an increase of apoptosis. However, neurons and neuronal networks exposed for long-term to simulated microgravity required longer recovery time to re-adapt to the ground gravity. In conclusion, a clear modulation in neuronal plasticity was evidenced through morphological and physiological changes in primary neuronal cultures during and after simulated microgravity exposure. These changes were dependent on the duration of exposure to microgravity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073857PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774774PMC
May 2014

Differential response to acute low dose radiation in primary and immortalized endothelial cells.

Int J Radiat Biol 2013 Oct 21;89(10):841-50. Epub 2013 Jun 21.

Radiobiology Unit, Belgian Nuclear Research Centre (SCK•CEN) , Mol , Belgium.

Purpose: The low dose radiation response of primary human umbilical vein endothelial cells (HUVEC) and its immortalized derivative, the EA.hy926 cell line, was evaluated and compared.

Material And Methods: DNA damage and repair, cell cycle progression, apoptosis and cellular morphology in HUVEC and EA.hy926 were evaluated after exposure to low (0.05-0.5 Gy) and high doses (2 and 5 Gy) of acute X-rays.

Results: Subtle, but significant increases in DNA double-strand breaks (DSB) were observed in HUVEC and EA.hy926 30 min after low dose irradiation (0.05 Gy). Compared to high dose irradiation (2 Gy), relatively more DSB/Gy were formed after low dose irradiation. Also, we observed a dose-dependent increase in apoptotic cells, down to 0.5 Gy in HUVEC and 0.1 Gy in EA.hy926 cells. Furthermore, radiation induced significantly more apoptosis in EA.hy926 compared to HUVEC.

Conclusions: We demonstrated for the first time that acute low doses of X-rays induce DNA damage and apoptosis in endothelial cells. Our results point to a non-linear dose-response relationship for DSB formation in endothelial cells. Furthermore, the observed difference in radiation-induced apoptosis points to a higher radiosensitivity of EA.hy926 compared to HUVEC, which should be taken into account when using these cells as models for studying the endothelium radiation response.
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http://dx.doi.org/10.3109/09553002.2013.806831DOI Listing
October 2013

Simulated microgravity decreases apoptosis in fetal fibroblasts.

Int J Mol Med 2012 Aug 14;30(2):309-13. Epub 2012 May 14.

Laboratory of Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre, SCK•CEN, Mol, Belgium.

Space travel is a major challenge for human beings. Especially, the mechanisms through which space conditions might alter animal development have been questioned for a long time. The two major physical stress factors that are of relevance in this context are space radiation and weightlessness. While it has been extensively shown that high doses of ionizing radiation induce deleterious effects on embryonic development, so far, little is known about the potential harmful effects of radiation in combination with microgravity on the developing organism. In the present study, we investigated the effects of simulated microgravity on irradiated STO mouse fetal fibroblast cells using a random positioning machine (RPM). Radiation-induced cell cycle changes were not affected when cells were subjected to simulated microgravity for 24 h. Moreover, no morphological differences were observed in irradiated samples exposed to simulated microgravity compared to cells that were exclusively irradiated. However, microgravity simulation significantly decreased the level of apoptosis at all doses as measured by caspase-3 activity and it prevented cells from undergoing radiation-induced size increase up to 1 Gy.
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http://dx.doi.org/10.3892/ijmm.2012.1001DOI Listing
August 2012

X-irradiation induces cell death in fetal fibroblasts.

Int J Mol Med 2012 Jul 10;30(1):114-8. Epub 2012 Apr 10.

Laboratory of Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre, SCK•CEN, Mol, Belgium.

The impact of ionizing radiation on developing organisms has been widely studied for risk assessment purposes. Even though efforts have been made to decrease received doses to as low as reasonably achievable, the possibility of accidental exposure has to be considered as well. Mammalian gestation is usually divided into three periods. Radiation exposure during the 'pre-implantation period' may essentially result in embryonic mortality while exposure during the 'organogenesis period' may characteristically lead to malformations. In humans, the 'fetal period' is one of particular sensitivity to radiation induction of mental retardation, especially if the exposure occurs between weeks 8-15 of gestation. It is also admitted that prenatal irradiation may increase the risk of leukemia and childhood cancer, with an equal risk over the whole pregnancy. The aim of this study was to investigate the effects of moderate to high doses of X-irradiation on mouse skin fetal fibroblasts, one of the cell types subjected to the highest dose of radiation. Exposure of the cells to X-rays led to a rapid and significant increase in γ-H2AX foci, indicative of high levels of DNA double strand breaks. High doses (>2 Gy) also led to a pronounced G2-arrest and a decrease in the number of cells in the S phase, which was followed after 24 h by a decrease in cell survival and an increase in the level of apoptosis and necrosis. This study shows that mouse fetal skin fibroblasts are sensitive to high doses of X-irradiation. Furthermore, we report a better repair for higher doses than lower, which seems to indicate that little DNA damage is not necessarily repaired immediately. However, more sensitive approaches are necessary to identify the risk associated with low doses of radiation.
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http://dx.doi.org/10.3892/ijmm.2012.964DOI Listing
July 2012

Spatiotemporal behavior of nuclear cyclophilin B indicates a role in RNA transcription.

Int J Mol Med 2012 Jun 13;29(6):1031-8. Epub 2012 Mar 13.

Department of Molecular Biotechnology, Ghent University, Ghent, Belgium.

Cyclophilin B (CypB) is an ubiquitously expressed protein, which performs several intra- and extracellular functions. Despite its abundant use as a household protein, little is known about its exact cellular localization and dynamics. In the present study we show that endogenous CypB localizes in one of two distinct compartments, either within the endoplasmic reticulum (ER) or inside the nucleus, accumulating in the fibrillar centers of the nucleoli. By means of a genetic deletion screen, we identified a minimal nucleolar localization signal for efficient relocation to the nucleoli. Within the fibrillar centers, CypB colocalized with RNA polymerase, upstream binding factor-1 (UBF), fibrillarin and dyskerin (DCK1). Even after chemical disruption of the nucleoli, a strong interaction with these proteins remained. Using live cell imaging, we showed a persistent colocalization of CypB with proteins involved in the ribosome biogenesis during the transcriptionally more active phases of the cell cycle. Supported by in silico data, our observations suggest that CypB interacts with these proteins and is involved in ribosome biogenesis and RNA transcription.
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http://dx.doi.org/10.3892/ijmm.2012.937DOI Listing
June 2012

Repeated exposure of human fibroblasts to ionizing radiation reveals an adaptive response that is not mediated by interleukin-6 or TGF-β.

Mutat Res 2011 Oct 18;715(1-2):19-24. Epub 2011 Jul 18.

Bio-imaging and Cytometry Unit, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium.

Exposing cells to a low dose can protect them against a subsequent higher exposure. This phenomenon is known as adaptive response and is frequently observed in a variety of cells. Even though similarities are suspected with other non-targeted effects, such as bystander effects, the exact mechanism behind adaptive response is not fully clarified. In this study human primary fibroblasts were tested for their response to ionizing radiation (IR) after administrating a low priming dose (0.1-0.5Gy). Both the abundance of γH2AX as a marker for double-stranded breaks and the levels of cytokines, secreted in the medium, were monitored in time. Upon challenge, IR-primed cells showed modified γH2AX spot size distributions and altered repair kinetics, consistent with an adaptive response. In addition, 24h after priming with IR, four cytokines were significantly upregulated in the medium - GM-CSF (1.33×); IL6 (4.24×); IL8 (1.33×); TGF-β (1.46×). In order to mimick the protective effect of IR priming, we primed the cells with either IL6 or TGF-β. This did not elicit an altered γH2AX response as observed in IR-primed cells, indicating that the adaptive response in these primary fibroblasts is regulated in an IL-6 and TGF-β independent manner.
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http://dx.doi.org/10.1016/j.mrfmmm.2011.07.002DOI Listing
October 2011

Multiplexed profiling of secreted proteins for the detection of potential space biomarkers.

Mol Med Rep 2011 Jan-Feb;4(1):17-23. Epub 2010 Nov 30.

Bioimaging and Cytometry Unit, Department of Molecular Biotechnology, Ghent University, 9000 Gent, Belgium.

Space travel exposes astronauts to a plethora of potentially detrimental conditions, such as cosmic radiation and microgravity. As both factors are hard to simulate on Earth, present knowledge remains limited. However, this knowledge is of vital importance, making space flight experiments a necessity for determining the biological effects and the underlying biochemical processes, especially when keeping future long-term interplanetary missions in mind. Instead of estimating the long-term effects, which usually implicate severe endpoints (e.g., cancer) and which are often difficult to attribute, research has shifted to finding representative biomarkers for rapid and sensitive detection of individual radiosensitivity. In this context, an appealing set of candidate markers is the group of secreted proteins, as they exert an intercellular signaling function and are easy to assess. We screened a subset of secreted proteins in cells exposed to space travel by means of multiplex bead array analysis. To determine the cell-specific signatures of the secreted molecules, we compared the conditioned medium of normal fibroblast cells to fibroblasts isolated from a patient with Hutchinson-Gilford Progeria syndrome, which are known to have a perturbed nuclear architecture and DNA damage response. Out of the 88 molecules screened, 20 showed a significant level increase or decrease, with a differential response to space conditions between the two cell types. Among the molecules that were retained, which may prove to be valuable biomarkers, are apolipoprotein C-III, plasminogen activator inhibitor type 1, β-2-microglobulin, ferritin, MMP-3, TIMP-1 and VEGF.
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http://dx.doi.org/10.3892/mmr.2010.405DOI Listing
August 2011

A method for visualising polyelectrolyte distribution after polyelectrolyte conditioning of a biotic sludge.

Water Sci Technol 2011 ;63(6):1303-8

Particle and Interfacial Technology Group, Faculty of Bioscience Engineering, Coupure links 653, 9000 Ghent, Belgium.

Charge neutralisation is an important mechanism in (polyelectrolyte) conditioning of biotic sludges and required for efficient sludge dewatering. Based on results from streaming potential and zeta potential measurements, it has been suggested that charge neutralisation is more complete on the outside of the sludge flocs than on the inside. This paper discusses the development of a technique for assessing the spatial distribution of polyelectrolyte (PE) within sludge flocs. After flocculation with a fluorescently labelled PE, fluorescence microscopy can be used to visualise the distribution of the PE in the sludge flocs. Preliminary results indicate that the PE can penetrate relatively deep into the sludge flocs (and flocculi). Inhomogeneity in the PE distribution arises from differences in exposure to PE in different regions, and from differences in the affinity of the PE for different substances.
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http://dx.doi.org/10.2166/wst.2011.375DOI Listing
June 2011

Medium-mediated DNA repair response after ionizing radiation is correlated with the increase of specific cytokines in human fibroblasts.

Mutat Res 2010 May 18;687(1-2):40-48. Epub 2010 Jan 18.

Laboratory for Biochemistry and Molecular Cytology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium.

Radiation induced bystander effects, either protective or adverse, have been identified in a variety of cells and for different endpoints. They are thought to arise from communication between cells through direct cell-cell contacts and via transmissible molecules secreted into the medium by targeted cells. We have investigated medium-mediated damage response in human dermal fibroblasts (HDF) after exposure to ionizing irradiation. We show that HDF experience an elevated level of double stranded DNA damage repair response when incubated with conditioned growth medium of irradiated cells. The magnitude of this response is much lower than observed for directly irradiated cells and is proportional to the radiation dose, as is its persistence across time. Since secretion of cytokines is one of the possible pathways linking targeted and non-targeted cells a multiplex analysis was performed. Four cytokines - IL6, IL8, MCP-1 and RANTES - were identified in the growth medium of irradiated cells after exposure to X-rays (2Gy). These cytokines were significantly upregulated and each cytokine showed differential upregulation kinetics. Finally we performed a functional analysis to see if IL6 and MCP-1 could induce gammaH2AX foci formation. IL6 caused a significant increase in spot occupancy compared to controls. Although only indicative MCP-1 appears to have the opposite effect as it caused a drop in spot occupancy. The combined addition of these two cytokines produced no significant response was observed. Both IL6 and MCP-1 have an effect on the gammaH2AX spot occupancy possibly linking these cytokines to the bystander response.
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http://dx.doi.org/10.1016/j.mrfmmm.2010.01.011DOI Listing
May 2010

Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A-type lamins.

Biochim Biophys Acta 2010 Apr 14;1800(4):448-58. Epub 2010 Jan 14.

Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium.

Background: The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression.

Methods: In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson-Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses.

Results: Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression.

Conclusions: These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics.

General Significance: Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.
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http://dx.doi.org/10.1016/j.bbagen.2010.01.002DOI Listing
April 2010

High content analysis of human fibroblast cell cultures after exposure to space radiation.

Radiat Res 2009 Oct;172(4):423-36

Laboratory for Bio-Imaging and Molecular Cytology, Department of Molecular Biotechnology, Ghent University, 9000 Gent, Belgium.

Space travel imposes risks to human health, in large part by the increased radiation levels compared to those on Earth. To understand the effects of space radiation on humans, it is important to determine the underlying cellular mechanisms. While general dosimetry describes average radiation levels accurately, it says little about the actual physiological impact and does not provide biological information about individual cellular events. In addition, there is no information about the nature and magnitude of a systemic response through extra- and intercellular communication. To assess the stress response in human fibroblasts that were sent into space with the Foton-M3 mission, we have developed a pluralistic setup to measure DNA damage and inflammation response by combining global and local dosimetry, image cytometry and multiplex array technology, thereby maximizing the scientific output. We were able to demonstrate a significant increase in DNA double-strand breaks, determined by a twofold increase of the gamma-H2AX signal at the level of the single cell and a threefold up-regulation of the soluble signal proteins CCL5, IL-6, IL-8, beta-2 microglobulin and EN-RAGE, which are key players in the process of inflammation, in the growth medium.
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http://dx.doi.org/10.1667/RR1682.1DOI Listing
October 2009

Systemic telomere length and preclinical atherosclerosis: the Asklepios Study.

Eur Heart J 2009 Dec;30(24):3074-81

Department of Molecular Biotechnology, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.

Aims: Peripheral blood leucocyte (PBL) telomere length (TL) is a systemic ageing biomarker and has been proposed to be an independent predictor of cardiovascular disease (CVD). We aimed at providing an explanation for this association by the evaluation of the biomarker value of PBL-TL in preclinical atherosclerosis.

Methods And Results: Peripheral blood leucocyte telomere length was assessed by telomere restriction fragment analysis in 2509 volunteers free from established CVD, aged approximately 35-55 years old, from the Asklepios Study cohort. Intima-media thickness (IMT) and plaque presence were determined by ultrasonography in both left and right carotid and femoral arteries. Peripheral blood leucocyte telomere length was not a significant independent determinant of IMT (P > 0.3) or plaque presence (P > 0.05), in either artery or either sex. In women but not in men, PBL-TL was a weak determinant of combined (carotid or femoral) plaque presence, adjusted for other risk factors (women: P = 0.03, men: P > 0.4). However, even in women presenting plaques, PBL-TL was still longer than in men.

Conclusion: Since systemic TL is not a substantial underlying determinant of preclinical atherosclerosis, the association between CVD and TL cannot be explained by the fact that subjects with shorter inherited TL are predisposed to atherosclerosis.
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http://dx.doi.org/10.1093/eurheartj/ehp324DOI Listing
December 2009

Digital titration: automated image acquisition and analysis of load and growth of Chlamydophila psittaci.

Microsc Res Tech 2009 May;72(5):398-402

Faculty of Bioscience Engineering, Department of Molecular Biotechnology (BW14), Ghent University, Coupure Links 653, Gent, Belgium.

Traditionally, the amount of infective chlamydiae in a given sample is determined by inoculating dilution series into cell cultures and physically counting chlamydial inclusions. This approach is time consuming, tedious, and error prone, mainly when dealing with high titers. Therefore, this paper describes a largely automated technique that was developed to standardize the determination of chlamydial load in vitro. Cells are fixed at 36 h post-inoculation and bacteria visualized using standard immunological detection methods. Consequently, for 81 microscopic fields, an image is recorded at the interpolated focal plane. These images are then automatically processed using an ImageJ plugin and the obtained results are imported into Excel to determine the number of inclusion forming units per mL in the sample. The main advantage of this technique is that no or minimal sample dilution is required, thus minimizing dilution errors. In addition, this technique was employed during the early, middle and late growth stages of the chlamydial developmental cycle and results correlated well (P < 0.01) with 16S rRNA values from previous experiments, thereby proving its suitability to follow chlamydial growth in vitro. The method described is highly suitable for high throughput titration of cell culture inoculated samples and assessment of possible antichlamydial effects of novel compounds throughout the chlamydial growth cycle.
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http://dx.doi.org/10.1002/jemt.20694DOI Listing
May 2009

High shear enrichment improves the performance of the anodophilic microbial consortium in a microbial fuel cell.

Microb Biotechnol 2008 Nov;1(6):487-96

Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Ghent, Belgium.

In many microbial bioreactors, high shear rates result in strong attachment of microbes and dense biofilms. In this study, high shear rates were applied to enrich an anodophilic microbial consortium in a microbial fuel cell (MFC). Enrichment at a shear rate of about 120 s(-1) resulted in the production of a current and power output two to three times higher than those in the case of low shear rates (around 0.3 s(-1)). Biomass and biofilm analyses showed that the anodic biofilm from the MFC enriched under high shear rate conditions, in comparison with that under low shear rate conditions, had a doubled average thickness and the biomass density increased with a factor 5. The microbial community of the former, as analysed by DGGE, was significantly different from that of the latter. The results showed that enrichment by applying high shear rates in an MFC can result in a specific electrochemically active biofilm that is thicker and denser and attaches better, and hence has a better performance.
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http://dx.doi.org/10.1111/j.1751-7915.2008.00049.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815290PMC
November 2008

Lower red blood cell counts in middle-aged subjects with shorter peripheral blood leukocyte telomere length.

Aging Cell 2008 Oct 28;7(5):700-5. Epub 2008 Jul 28.

Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, Ghent, Belgium.

Although telomere biology was revealed to play an important role in several hematopoietic disorders, its impact on the age-dependent dynamics of regular hematopoiesis is poorly understood. In vitro results suggest that particularly the erythropoietic capacity might be limited by critically short telomere length (TL). However, it remains unclear whether TL also affects erythropoiesis in healthy individuals in vivo. Therefore, we analyzed the associations between relevant hematopoietic parameters and peripheral blood leukocyte TL in the apparently healthy Asklepios study population, aged approximately 35-55 years (N > 2500). Our data indicate a clear positive, age and paternal age at birth adjusted, correlation between TL and red blood cell count, both in men (p < 0.001) and women (p = 0.011). This association was particularly significant in the older segment of the population (> 45 years old, both sexes: p = 0.003) and in younger men (p = 0.013), but not in younger women (p = 0.521). Further adjustment for known determinants in a general linear model revealed that peripheral blood leukocyte TL is most probably an independent predictor of red blood cell count (p < 0.001), suggesting that critical telomere shortening might also limit erythropoiesis in vivo. While negligible in a middle-aged population, the clinical consequences might be important in the elderly (e.g. in anemia of chronic disease). Further studies are required to confirm the impact of our results.
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http://dx.doi.org/10.1111/j.1474-9726.2008.00419.xDOI Listing
October 2008

Oxidized low-density lipoprotein cholesterol is associated with decreases in cardiac function independent of vascular alterations.

Hypertension 2008 Sep 28;52(3):535-41. Epub 2008 Jul 28.

Department of Cardiovascular Diseases, Ghent University, Ghent, Belgium.

In contrast to the plethora of vasculopathies to which oxidized low-density lipoprotein cholesterol (ox-LDL) can be linked, there are no data linking ox-LDL to myocardial (dys)function in the community. We tested whether ox-LDL, a marker of oxidative stress, was linked to early cardiac structural and functional damage in the general population. The Asklepios Study is a random sample of 2524 male and female volunteers, comparable to the Belgian population between 35 and 55 years free from overt cardiovascular disease. Cardiac morphology, systolic, and early and late diastolic tissue Doppler mitral annulus velocities were recorded during an echocardiography, followed by a vascular examination (carotid and femoral arteries). Serum ox-LDL was measured by sandwich ELISA using the mAb-4E6 monoclonal antibody. Effects of ox-LDL were assessed after adjustment for age, gender, lipid fractions, blood pressure, heart rate, height, weight, glycemia, smoking, and drug treatment. Mean ox-LDL was 96.0+/-38.9 U/L. After adjustment, increasing ox-LDL levels were associated with a more spherical left ventricular cavity (minor/major axis dimensions; P<0.001) and decreasing diastolic (early diastolic tissue Doppler mitral annulus velocity; P<0.001, more pronounced in women) and systolic function (amplitude of systolic tissue Doppler mitral annulus velocity; P=0.008, more pronounced in men). These results remained unaffected when further adjustments were made for inflammatory markers, lifestyle, or vascular damage (atherosclerosis and arterial stiffening). These results are the first "proof of concept" that ox-LDL impacts cardiac structure and function at a community level, independent of classic risk factors, lifestyle, inflammation, and prevalent vascular damage. Our data suggest that ox-LDL is a risk marker for early ventricular remodelling. However, the effect size in the general population is small.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.108.114439DOI Listing
September 2008

Identification and characterization of a type III secretion system in Chlamydophila psittaci.

Vet Res 2008 May-Jun;39(3):27. Epub 2008 Feb 15.

Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium.

Chlamydiaceae are obligate intracellular Gram-negative bacteria replicating in vacuoles inside eukaryotic cells. It has been proven that most of them possess a type III secretion system (T3SS) allowing them to transfer effector molecules in the host cell. We examined the existence of a T3SS in Chlamydophila psittaci by studying the expression of three essential structural proteins SctW, SctC, and SctN, and one putative effector protein IncA. Immunofluorescence assays showed SctW and IncA to be associated with the bacteria and the inclusion membrane, while SctC and SctN were only localized to the bacteria itself. Immuno electron microscopy could confirm these results for SctW, IncA, and SctC. Unfortunately, SctN was not investigated with this technique. Additionally, we sequenced 14 full-length T3S genes (scc1, sctW, sctJ, sctL, sctR, sctS, scc2, copD1, sctN, sctQ, sctC, incA, ca037, and cadd) and examined the transcription of 26 Cp. psittaci T3S genes namely cluster 1 (scc1, sctW, sctV, sctU), cluster 2 (sctJ, sctL, sctR, sctS, sctT, scc2, copB1, copD1), cluster 3 (sctD, sctN, ca037, sctQ, pkn5, sctC) and non-clustered genes (incA, incC, scc3, copD2, cap1, tarp, ca530, cadd). The gene expression study indicated the T3S structural protein encoding genes to be transcribed from mid-cycle (12-18 h post infection (p.i.)) on. Genes encoding effector proteins and putative T3S related proteins were expressed early (1.5 h-8 h p.i.) or late (>24 h p.i.) during the developmental cycle. We hereby provided evidence for the existence of a T3SS and possible effectors in avian Cp. psittaci.
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http://dx.doi.org/10.1051/vetres:2008002DOI Listing
August 2008

Paternal age at birth is an important determinant of offspring telomere length.

Hum Mol Genet 2007 Dec 19;16(24):3097-102. Epub 2007 Sep 19.

Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.

Although evidence supports the function of telomere length (TL) as a marker for biological aging, no major determinants of TL are known besides inheritance, age and gender. Here we validate and, more importantly, assess the impact of paternal age at birth as a determinant for the offspring's peripheral blood leukocyte TL within the Asklepios study population. Telomere restriction fragment length and paternal age information were available for 2433 volunteers (1176 men and 1257 women) aged approximately 35-55 years old. Paternal age at birth was positively associated with offspring TL (offspring age and gender adjusted, P < 10 (-14)). The increase in TL was estimated at 17 base pairs for each supplemental year at birth and was not statistically different between male and female offspring. The effect size of paternal age outweighed the classical TL determinant gender by a factor of 2, demonstrating the large impact. Maternal age at birth was not independently associated with offspring TL. The peculiar interaction between paternal age at birth and inheritance might explain a large part of the genetic component of TL variance on a population level. This finding also provides further proof for the theory that TL is not completely reset in the zygote. Furthermore, as paternal age is subject to demographic evolution, its association with TL might have a substantial impact on the results and comparability of TL within and between epidemiological studies. In conclusion, paternal age is an important determinant for TL, with substantial consequences for future studies.
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http://dx.doi.org/10.1093/hmg/ddm271DOI Listing
December 2007

Telomere length and cardiovascular risk factors in a middle-aged population free of overt cardiovascular disease.

Aging Cell 2007 Oct;6(5):639-47

Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.

Evidence assembled over the last decade shows that average telomere length (TL) acts as a biomarker for biological aging and cardiovascular disease (CVD) in particular. Although essential for a more profound understanding of the underlying mechanisms, little reference information is available on TL. We therefore sought to provide baseline TL information and assess the association of prevalent CVD risk factors with TL in subjects free of overt CVD within a small age range. We measured mean telomere restriction fragment length of peripheral blood leukocytes in a large, representative Asklepios study cohort of 2509 community-dwelling, Caucasian female and male volunteers aged approximately 35-55 years and free of overt CVD. We found a manifest age-dependent telomere attrition, at a significantly faster rate in men as compared to women. No significant associations were established with classical CVD risk factors such as cholesterol status and blood pressure, yet shorter TL was associated with increased levels of several inflammation and oxidative stress markers. Importantly, shorter telomere length was associated with an increasingly unhealthy lifestyle, particularly in men. All findings were age and gender adjusted where appropriate. With these cross-sectional results we show that TL of peripheral blood leukocytes primarily reflects the burden of increased oxidative stress and inflammation, whether or not determined by an increasingly unhealthy lifestyle, while the association with classical CVD risk factors is limited. This further clarifies the added value of TL as a biomarker for biological aging and might improve our understanding of how TL is associated with CVD.
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http://dx.doi.org/10.1111/j.1474-9726.2007.00321.xDOI Listing
October 2007

Effect of ovotransferrin and lactoferrins on Chlamydophila psittaci adhesion and invasion in HD11 chicken macrophages.

Vet Res 2007 Sep-Oct;38(5):729-39. Epub 2007 Jul 10.

Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Gent, Belgium.

The effect of ovotransferrin (ovoTF), human lactoferrin (hLF) and bovine lactoferrin (bLF) on the obligate intracellular pathogen Chlamydophila (Cp.) psittaci was evaluated using a model of Buffalo Green Monkey kidney (BGM) cells and HD11 chicken macrophages as artificial hosts. Firstly, the effect of transferrins on the infectivity of the bacteria was evaluated. Pre-incubation of Cp. psittaci with 0.5 to 5 mg/mL ovoTF prior to infecting BGM cells significantly lowered the infection rate (P < 0.05). For both lactoferrins, the infection rate could only be reduced with 5 mg/mL, albeit not significantly as compared to the infection rate created by the untreated bacteria. Secondly, transferrins were tested for their ability to influence bacterial adhesion and entry in HD11 cells. Maximal non-cytotoxic and non-bactericidal concentrations of 0.05 mg/mL ovoTF and 0.5 mg/mL hLF and bLF were used. Overall, ovoTF was more effective than human and bovine LF in inhibiting bacterial irreversible attachment and cell entry and the latter was accompanied by a dose-dependent reduction of actin recruitment at the bacterial entry site. However, once bacteria had entered HD11 cells, transferrins had apparently no effect on intracellular replication. The present findings suggest a possible role for transferrins and especially ovoTF, in preventing avian Cp. psittaci infections.
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http://dx.doi.org/10.1051/vetres:2007028DOI Listing
November 2007

Methylglyoxal suppresses TNF-alpha-induced NF-kappaB activation by inhibiting NF-kappaB DNA-binding.

Biochem Pharmacol 2007 Aug 3;74(4):579-89. Epub 2007 Jun 3.

VIB Department of Medical Protein Research, Molecular and Metabolic Signaling Unit, VIB, 9000 Ghent, Belgium.

Methylglyoxal is a cytotoxic metabolite that is produced in vivo mainly from glycolysis. Increased production of methylglyoxal can be induced by tumor necrosis factor and occurs in a number of pathological conditions, including diabetes and neurodegenerative disorders. Methylglyoxal is highly reactive and can modify proteins, which results in the formation of advanced glycation end products. Yet, we, and others, have recently proposed a role for methylglyoxal as a signaling molecule. In this study, we show that methylglyoxal inhibits TNF-induced NF-kappaB activation and NF-kappaB-dependent reporter gene expression by inhibiting the DNA binding capacity of NF-kappaB p65. Methylglyoxal slightly delayed, but did not inhibit, TNF-induced degradation of IkappaBalpha and strongly inhibited TNF-induced NF-kappaB-dependent re-synthesis of IkappaBalpha. The TNF-induced nuclear translocation of NF-kappaB p65 was also delayed, but not inhibited, in the presence of methylglyoxal. TNF-induced phosphorylation of p65 was not affected by methylglyoxal. We show that the conserved Cys 38 residue, which is located in the DNA binding loop of NF-kappaB p65 and responsible for the redox regulation of the transcription factor, is involved in the methylglyoxal-mediated inhibition of p65 DNA-binding. Furthermore, overexpression of p65 inhibited TNF-induced cell death; however, in the presence of exogenously added methylglyoxal, overexpression of p65 caused far greater TNF-induced cell death. These findings suggest that methylglyoxal provides another control mechanism for modulating the expression of NF-kappaB-responsive genes and that methylglyoxal may be responsible for tipping the balance towards TNF-induced cell death in cells with constitutive NF-kappaB activation.
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http://dx.doi.org/10.1016/j.bcp.2007.05.026DOI Listing
August 2007

Rationale, design, methods and baseline characteristics of the Asklepios Study.

Eur J Cardiovasc Prev Rehabil 2007 Apr;14(2):179-91

Department of Cardiovascular Diseases, Ghent University Hospital, Ghent, Belgium.

The Asklepios Study is a longitudinal population study focusing on the interplay between ageing, cardiovascular haemodynamics and inflammation in (preclinical) cardiovascular disease. The 2524 participants (1301 women) are a representative cohort of 35-55-year-old individuals, free from overt cardiovascular disease at study initiation, randomly sampled from the twinned Belgian communities of Erpe-Mere and Nieuwerkerken. Baseline examinations (all single-observer, single-device, single-site, single 2-year consecutive timeframe) include: questionnaires, conventional risk factors and biochemistry. Additional phenotypes under study include: (a) vascular structure and function: carotid and femoral atherosclerosis (intima-media thickness, plaque), arterial distension and pressure curves (brachial, carotid, femoral; wall-tracking and applanation tonometry); (b) cardiac structure and function. A novel aspect of the study is 'integrated' non-invasive biomechanical assessment of cardiac, arterial and ventriculovascular function through a combination of modeling, fundamental hydraulical measurements and system identification techniques. Integrated phenotypes result from combining at least two sets of curves (flow/pressure/distension). The value of this 'integrated' haemodynamic phenotype in the detection, prediction and prevention of clinical cardiovascular pathology (atherosclerosis progression, atherothrombosis, development of heart failure) will be tested. A second novel aspect is the systematic determination of peripheral blood leukocyte telomere length as a marker for biological ageing. During follow-up, baseline examinations will be repeated and the incidence of cardiovascular events will be monitored. Sex-specific baseline risk factor and biochemical data are provided in the current analyses. The primary aim is to build a combined dataset that will act as a tool to answer a cluster of questions about ageing, haemodynamics and the emergence of cardiovascular disease, especially the incidence of atherothrombotic events and the development of adverse haemodynamic profiles (arterial stiffening, heart failure). The study will reassess current risk factors and provide a long-term base for the detection of novel (epi)genetic and non-genetic risk factors and for more performant risk stratification modalities. Within these broader goals, a constant will be to strive towards more fundamental mechanistic-haemodynamic insights into cardiovascular disease processes.
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http://dx.doi.org/10.1097/HJR.0b013e328012c380DOI Listing
April 2007

Genotoxicity in wood mice (Apodemus sylvaticus) along a pollution gradient: exposure-, age-, and gender-related effects.

Environ Toxicol Chem 2006 Aug;25(8):2154-62

Department of Biology, Evolutionary Biology Group, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium.

We investigated the effects of environmental pollution on genetic damage in wood mice (Apodemus sylvaticus) by means of the comet assay, with special attention to the role of age and gender as potential confounding variables. The present study was carried out at four sites along a pollution gradient in the vicinity of Antwerp (Belgium), with a nonferrous smelter as the main pollution source. We measured the concentration of heavy metals (Cd, Co, Cr, Cu, Fe, Mn, Pb, and Zn) in mouse liver and kidney and the concentration of organochlorine compounds (polychlorinated biphenyls and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) in mouse muscle tissue to assess individual exposure. Cadmium exposure was very high at the sites closest to the smelter, and exposure to this metal decreased with increasing distance from the smelter. Exposure to the other pollutants was low to moderate at the different sites. Genetic damage was higher in mice from populations in the vicinity of the nonferrous smelter compared with that in the control populations. A significant increase in genetic damage with age was observed at the most polluted sites, but not at the control sites. Genetic damage was higher in male mice than in female mice at the most polluted site, but not at the other areas. Yet, no obvious relationship was found between individual pollutant levels and individual genetic damage levels. We conclude that the comet assay can be used to compare genotoxicity at the population level if the confounding variables of gender and age are taken into account. However, its use for individual health risk assessment remains questionable.
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http://dx.doi.org/10.1897/05-419r.1DOI Listing
August 2006

Telomere attrition as ageing biomarker.

Anticancer Res 2005 Jul-Aug;25(4):3011-21

Department of Molecular Biotechnology, Faculty for Bioscience Engineering, Ghent University, Belgium.

Telomeres, the tandem-repeated hexamers at the termini of mammalian chromosomes, form protective complexes in association with specific proteins that together with telomerase, a specialised telomere-synthesizing enzyme, regulate telomere length. Telomere shortening is associated with cellular senescence and is implicated in tumorigenesis and cancer. Hence, mean telomere length has emerged as a replicative clock within each population of cells and the tissues and organs they build up in vitro and, consequently, as a biomarker for biological ageing in vivo. Chronological ageing per se does not parallel biological ageing, yet accurate and reliable biomarkers are lacking to distinguish between them. The question remains as to whether telomere dynamics is a determinant or merely a predictor of human biological age over and above chronological ageing. Although several reports have suggested a link between telomere attrition and ageing phenotypes and disorders, both reference values and a complete set of determinants are missing. Within this review, current evidence and knowledge on telomere length and telomere erosion rates reported, are summarised.
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September 2005

Telomere shortening is associated with malformation in p53-deficient mice after irradiation during specific stages of development.

DNA Repair (Amst) 2005 Aug;4(9):1028-37

Laboratory for Biochemistry and Molecular Cytology, Department for Molecular Biotechnology, Faculty of Bioscience Engineering FBE, Ghent University, Belgium.

The natural ends of linear chromosomes, the telomeres, recruit specific proteins in the formation of protective caps that preserve the integrity of the genome. Unprotected chromosomes induce DNA damage checkpoint cascades and ultimately lead to senescence both in mouse and man in a p53 dependent manner and initial telomere length setting therefore determines the proliferative capacity of each cell. Yet, only little information is available on telomere biology during embryonic development. We have previously shown that the p53 gene plays a crucial role in the development of malformations (exencephaly, gastroschisis, polydactyly, cleft palate and dwarfism) in control and irradiated mouse embryos. Here, we investigated telomere biology and the outcome of radiation exposure in wild type (p53+/+) and p53-mutant (p53+/-- and--/--) C57BL mouse foetuses irradiated at three different developmental stages. We show that telomeres are significantly shorter in malformed foetuses as compared to normal counterparts. In addition, our results indicate that the observed telomere attrition is primarily associated with p53-deficiency but is also modulated by irradiation, more specifically during the gastrulation and organogenesis stages. In conclusion, we formulate a hypothesis in which telomere shortening is linked to the absence of p53 in mouse foetuses and that when, in the presence of shorter telomeres, these foetuses are irradiated, the chance for the occurrence of developmental defects increases substantially.
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http://dx.doi.org/10.1016/j.dnarep.2005.05.010DOI Listing
August 2005