Extracting information from imaging cytometry: a review.

The extraction of statistically meaningful quantitative information from microscopy images is increasingly important for modern biological research. Obtaining accurate, quantitative information from biological specimens, however, is a complex process that requires optimization of several parameters. One must consider the number of probes, fluorescent channels required, type of plate to be used, number of fields to be acquired and optimal resolution for image acquisition. The extraction of information from images is dependent on and can be aided greatly by careful consideration of the factors involved in the image acquisition process. I summarize here the general principles behind the imaging and software technology that is used to quantify images and highlight particular issues of concern for critically applying image quantitation techniques for research.

Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

Dysfunction of the mitotic:meiotic switch as a potential cause of neoplastic conversion of primordial germ cells.

Germ cell tumours (GCT) are thought to arise as the result of a defect in early development, probably shortly after arrival of the migrating primordial germ cells (PGC) in the genital ridge when, if in a male genital ridge, the germ cells arrest in mitosis, but in a female genital ridge they enter meiosis. We suggest that dysfunction of the mitotic:meiotic switch, with cells aberrantly co-expressing functions pertinent to both states, might provide the genetic instability that could initiate tumour development. If this hypothesis is correct, GCT could arise because of disruption in the function of any one of a number of different genes involved in controlling mitosis and meiosis, rather than being dependent upon a single prominent susceptibility gene. The Notch signalling system is one candidate system for controlling the switch and we have identified expression of Notch2 and Notch4 in seminomas and carcinoma in situ. Thus those two members of the Notch family are candidates for proto-oncogenes that could play a role in GCT development. We have also identified a human homologue of the synaptonemal complex protein, SCP3, and have found its apparently aberrant expression in some established EC cell lines. One possibility is that abnormal regulation of such proteins involved in the synaptonemal complex could also lead to genetic instability in PGC and so also initiate tumour development.

Brachyury is expressed by human teratocarcinoma cells in the absence of mesodermal differentiation.

Reverse transcription-PCR and Northern and Western blot analyses indicate that mRNA and protein encoded by the Brachyury gene are expressed by the pluripotent human embryonal carcinoma cell line NTERA2 and are only modestly down-regulated during retinoic acid-induced differentiation. This differentiation occurs along a neural lineage, with no obvious evidence of the formation of mesodermal derivatives. Several other human embryonal carcinoma cell lines that do not differentiate, a yolk sac carcinoma cell line and two choriocarcinoma cell lines, also express readily detectable levels of Brachyury mRNA and protein. Thus, in human teratocarcinomas, Brachyury expression is not necessarily an indicator of commitment to mesodermal differentiation.