High content image-based cytometry as a tool for nuclear fingerprinting

Cytomics aims at understanding the functional relationships between cellular phenotypes (cytome) and metabolic pathways (proteome) that result from a combination of genetically defined mechanisms (genome) and environmental conditions [1,2]. Although flow-cytometry is able to measure the optical properties of single cells at a rate of >1000 cells per minute it has a limited capability of mapping individual events. To accurately quantify (sub-) cellular characteristics within a natural context there is a fast-growing need for image-based cytometry. Images, obtained with fluorescence microscopy, provide the exact information on signal intensity, location and distribution of specific molecules within intact cell systems (tissue or monolayers) and allow for investigating cellular properties in relation to the cell-ecological context [3]. Previously, we have developed a cytometric approach for scoring DNA lesion endpoints in confocal images of murine fibroblasts [3]. We now present a generalized approach for multivariate phenotypic profiling of individual nuclei using automated fluorescence mosaic microscopy and optimized digital image processing tools. An indefinite number of fields, z-slices and channels can be analyzed; the only prerequisite is the presence of a nuclear counterstain, which is used for the generation of masks. To anticipate for erroneous segmentation of clustered nuclei in dense cell cultures we implemented an iterative conditional segmentation (ics) algorithm that uses both morphological and intensity information from the image (Figure 1). The method makes use of a priori knowledge about the size and shape of nuclei in stringent feedback selection of correctly segmented nuclei. Depending on the degree of clustering, segmentation performance varies between 95% and 100%. Complete analysis of nuclei and subnuclear features for a region of 25 images of 1000x1000 pixels, 3 z-slices and 3 channels only takes ~ 3 minutes or ~ 0.7sec/nucleus. Our method is insensitive to scaling, illumination heterogeneity and variability or non-uniformity of staining. We have successfully applied our system in cell cycle analysis, scoring of transfection efficiency and assessment of (localized) DNA damage in response to genotoxic stress and ionizing radiation

Multilevel analysis of nuclear dynamics in lamin perturbed fibroblasts

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 [1]. Most studies regarding the mechanical properties of the nucleus in laminopathic conditions are based on the induction of extracellular stress, such as strain or compression, and focus on nuclear integrity and/or nucleo-cytoskeletal interaction [2]. Far less is known about the role of nuclear organization and mobility under basal steady-state conditions. In this study, we quantitatively compared nuclear organization, nuclear deformation and chromatin mobility of fibroblasts from a Hutchinson-Gilford progeria patient with cells from a lamin A/C-deficient patient and wild-type dermal fibroblasts. To this end, we created a toolbox in imageJ for automatically analyzing both nuclear as well as subnuclear dynamics in living cells. Simultaneously, we developed a workflow for comparing cellular morphology and subcellular protein distribution in a high content fashion. 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 seconds time scale. In contrast, progeria cells showed overall reduced nuclear dynamics. In addition, high content analysis revealed marked morphological and topological differences between different culture passages within a cell type and between different pathological variants of culture-age matched laminopathic cell types

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

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

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

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

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

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

Combined exposure to simulated microgravity and acute or chronic radiation reduces neuronal network integrity and survival

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 Xrays 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

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

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

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

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 similar to 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

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

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/mu m) at doses of 0.5, 2 and 5 Gy. DNA damage was measured 2 and 24 h following irradiation by gamma-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)-kappa B, may be involved in these cellular responses