Hans Zoellner

Subtle toxic effects may be masked in traditional assays that average or summate the response of thousands of cells. We overcome this by using the recent method of single cell tracking in time-lapse recordings. This follows the fate and behavior of individual cells and their progeny and provides unambiguous results for multiple simultaneous biological responses. Further, single cell tracking permits correlation between progeny relationships and cell behavior that is not otherwise possible, including disruption by toxins and toxicants of similarity between paired sister cells. Notably, single cell tracking seems not to have been previously used to study biomaterials toxicity. The culture medium was pre-conditioned by 79 days incubation with orthodontic brackets from seven separate commercial sources. Metal levels were determined by Inductively Coupled Plasma Mass Spectrometry. Metal levels varied amongst conditioned media, with elevated Cr, Mn, Ni, and Cu and often Mo, Pb, Zn, Pd, and Ag were occasionally found. The effect on human dermal fibroblasts was determined by single cell tracking. All bracket-conditioned media reduced cell division (p < 0.05), while some reduced cell migration (p < 0.05). Most bracket-conditioned media increased the rate of asynchronous sister cell division (p < 0.05), a seemingly novel measure for toxicity. No clear effect on cell morphology was seen. We conclude that orthodontic brackets have cytotoxic effects, and that single cell tracking is effective for the study of subtle biomaterials cytotoxicity.

We recently described cell-projection pumping as a mechanism transferring cytoplasm between cells. The uptake of fibroblast cytoplasm by co-cultured SAOS-2 osteosarcoma cells changes SAOS-2 morphology and increases cell migration and proliferation, as seen by single-cell tracking and in FACS separated SAOS-2 from co-cultures. Morphological changes in SAOS-2 seen by single cell tracking are consistent with previous observations in fixed monolayers of SAOS-2 co-cultures. Notably, earlier studies with fixed co-cultures were limited by the absence of a quantitative method for identifying sub-populations of co-cultured cells, or for quantitating transfer relative to control populations of SAOS-2 or fibroblasts cultured alone. We now overcome that limitation by a novel Cartesian plot analysis that identifies individual co-cultured cells as belonging to one of five distinct cell populations, and also gives numerical measure of similarity to control cell populations. We verified the utility of the method by first confirming the previously established relationship between SAOS-2 morphology and uptake of fibroblast contents, and also demonstrated similar effects in other cancer cell lines including from melanomas, and cancers of the ovary and colon. The method was extended to examine global DNA methylation, and while there was no clear effect on SAOS-2 DNA methylation, co-cultured fibroblasts had greatly reduced DNA methylation, similar to cancer associated fibroblasts.

We earlier reported that cell-projection pumping transfers fibroblast contents to cancer cells and this alters the cancer cell phenotype. Here, we report on single-cell tracking of time lapse recordings from co-cultured fluorescent fibroblasts and SAOS-2 osteosarcoma cells, tracking 5201 cells across 7 experiments. The fluorescent lipophilic marker DiD was used to label fibroblast organelles and to trace the transfer of fibroblast cytoplasm into SAOS-2 cells. We related SAOS-2 phenotypic change to levels of fluorescence transfer from fibroblasts to SAOS-2 cells, as well as what we term ‘compensated fluorescence’, that numerically projects mother cell fluorescence post-mitosis into daughter cells. The comparison of absolute with compensated fluorescence allowed us to deduct if the phenotypic effects in mother SAOS-2 cells were inherited by their daughters. SAOS-2 receipt of fibroblast fluorescence correlated by Kendall’s tau with cell-profile area and without evidence of persistence in daughter cells (median tau = 0.51, p < 0.016); negatively and weakly with cell circularity and with evidence of persistence (median tau = −0.19, p < 0.05); and very weakly with cell migration velocity and without evidence of persistence (median tau = 0.01, p < 0.016). In addition, mitotic SAOS-2 cells had higher rates of prior fluorescence uptake (median = 64.9 units/day) than non-dividing cells (median = 35.6 units/day, p < 0.016) and there was no evidence of persistence post-mitosis. We conclude that there was an appreciable impact of cell-projection pumping on cancer cell phenotype relevant to cancer histopathological diagnosis, clinical spread and growth, with most effects being ‘reset’ by cancer cell mitosis.

We earlier reported that cell-projection pumping transfers fibroblast contents to cancer cells, and this alters cancer cell phenotype. We now report on single-cell tracking of time lapse recordings from co-cultured fluorescent fibroblasts and SAOS-2 osteosarcoma cells, tracking 5,201 cells across 7 experiments. The fluorescent lipophilic marker DiD was used to label fibroblast organelles, and to trace transfer of fibroblast cytoplasm into SAOS-2. We related SAOS-2 phenotypic change to levels of fluorescence transfer from fibroblasts to SAOS-2, and also to what we term ‘compensated fluorescence’, that numerically projects mother cell fluorescence post-mitosis, into daughter cells. Comparison of absolute with compensated fluorescence, allowed deduction if phenotypic effects in mother SAOS-2, were inherited by their daughters. SAOS-2 receipt of fibroblast fluorescence correlated by Kendall’s tau: with cell-profile area, and without evidence for persistence in daughter cells (median tau = 0.51, p < 0. 016); negatively and weakly with cell circularity, and with evidence for persistence (median tau = −0.19, p < 0.05); and very weakly with cell migration velocity, and without evidence for persistence (median tau = 0.01, p < 0.016). Also, mitotic SAOS-2 had higher rates of prior fluorescence uptake (median = 64.9 units/day), compared with non dividing cells (median = 35.6 units/day, p < 0.016), and there was no evidence for persistence post-mitosis. We conclude there is appreciable impact of cell-projection pumping on cancer cell phenotype, relevant to cancer histopathological diagnosis, clinical spread, and growth, with most effects ‘reset’ by cancer cell mitosis.

Background: Rare diseases may be defined as occurring in less than 1 in 2000 patients. Such conditions are, however, so numerous that up to 5.9% of the population is afflicted by a rare disease. The gambling industry attests that few people have native skill evaluating probabilities. We believe that both students and academics, under-estimate the likelihood of encountering rare diseases. This combines with pressure on curriculum time, to reduce both student interest in studying rare diseases, and academic content preparing students for clinical practice. Underestimation of rare diseases, may also contribute to unhelpful blindness to considering such conditions in the clinic. Methods: We first developed a computer simulation, modelling the number of cases of increasingly rare conditions encountered by a cohort of clinicians. The simulation captured results for each year of practice, and for each clinician throughout the entirety of their careers. Four hundred sixty-two theoretical conditions were considered, with prevalence ranging from 1 per million people through to 64.1% of the population. We then delivered a class with two in-class on-line surveys evaluating student perception of the importance of learning about rare diseases, one before and the other after an in-class real-time computer simulation. Key simulation variables were drawn from the student group, to help students project themselves into the simulation. Results: The in-class computer simulation revealed that all graduating clinicians from that class would frequently encounter rare conditions. Comparison of results of the in-class survey conducted before and after the computer simulation, revealed a significant increase in the perceived importance of learning about rare diseases (p < 0.005). Conclusions: The computer career simulation appeared to affect student perception. Because the computer simulation demonstrated clinicians frequently encounter patients with rare diseases, we further suggest this should be considered by academics during curriculum review and design.

Thiocyanate (SCN⁻) is a pseudohalide anion omnipresent across mammals and is particularly concentrated in secretions within the oral cavity, digestive tract and airway. Thiocyanate can outcompete chlorine anions and other halides (F⁻, Br⁻, I⁻) as substrates for myeloperoxidase by undergoing two-electron oxidation with hydrogen peroxide. This forms their respective hypohalous acids (HOX where X⁻ = halides) and in the case of thiocyanate, hypothiocyanous acid (HOSCN), which is also a bactericidal oxidative species involved in the regulation of commensal and pathogenic microflora. Disease may dysregulate redox processes and cause imbalances in the oxidative profile, where typically favoured oxidative species, such as hypochlorous acid (HOCl), result in an overabundance of chlorinated protein residues. As such, the pharmacological capacity of thiocyanate has been recently investigated for its ability to modulate myeloperoxidase activity for HOSCN, a less potent species relative to HOCl, although outcomes vary significantly across different disease models. To date, most studies have focused on therapeutic effects in respiratory and cardiovascular animal models. However, we note other conditions such as rheumatic arthritis where SCN⁻ administration may worsen patient outcomes. Here, we discuss the pathophysiological role of SCN⁻ in diseases where MPO is implicated.

Background Rare diseases may be defined as occurring in less than 1 in 2,000 patients. Such conditions are, however, so numerous that up to 5.9% of the population is afflicted by a rare disease. The gambling industry attests that few people have native skill evaluating probabilities. We believe that both students and academics, under-estimate the likelihood of encountering rare diseases. This combines with pressure on curriculum time, to reduce both student interest in studying rare diseases, and academic content preparing students for clinical practice. Underestimation of rare diseases, may also contribute to unhelpful blindness to considering such conditions in the clinic. Methods We first developed a computer simulation, modelling the number of cases of increasingly rare conditions encountered by a cohort of clinicians. The simulation captured results for each year of practice, for each clinician throughout the entirety of their careers. 462 theoretical conditions were considered, with prevalence ranging from 1 per million people through to 64.1% of the population. We then delivered a class with two in-class on-line surveys evaluating student perception of the importance of learning about rare diseases, one before and the other after, an in-class real-time computer simulation. Key simulation variables were drawn from the student group, so students could see a projection of their own careers. Results The in-class computer simulation revealed that all graduating clinicians from that class would frequently encounter rare conditions. Comparison of results of the in-class survey conducted before and after the computer simulation, revealed a significant increase in the perceived importance of learning about rare diseases (p < 0.005). Conclusions The computer career simulation appeared to affect student perception. Because the computer simulation demonstrated clinicians frequently encounter patients with rare diseases, we further suggest this should be considered by academics during curriculum review and design.

We recently described a hydrodynamic mechanism for cytoplasmic transfer between cells, termed cell-projection pumping (CPP). Earlier image analysis related altered SAOS-2 osteosarcoma cell morphology, to what we now recognize as CPP uptake of fibroblast cytoplasm. We here examine SAOS-2 phenotype following co-culture with human dermal fibroblasts (HDF) in which organelles were pre-labelled with a fluorescent lipophilic marker. Fluorescence activated cell sorting (FACS) analysis was performed of HDF and SAOS-2, cultured either alone or together. FACS forward scatter is proportionate to cell size, and increased for SAOS-2 with high levels of HDF fluorescence uptake (p < 0.004). FACS side scatter is proportionate to internal cell complexity, and increased in SAOS-2 with increasing uptake of HDF fluorescence (p < 0.004), consistent with uptake of HDF organelles. Scratch migration assays revealed that HDF migrated more quickly than SAOS-2 in both isolated cell culture, and following co-culture (p < 0.004). Notably, SAOS-2 with high levels of HDF labelling migrated faster compared with SAOS-2 with low HDF labelling (p < 0.008). A slight and unconvincing reduction in SAOS-2 proliferation was seen (p < 0.02). Similar results were obtained in single additional experiments with A673 and H312 cancer cells. Forward and side scatter results suggest organellar transfer by CPP increases cancer cell morphological diversity. This may contribute to histological pleomorphism relevant to cancer diagnosis and prognosis. Also, increased migration of sub-populations of cancer cells with high CPP organellar uptake, may contribute to invasion and metastasis in-vivo. We thus suggest relevance of CPP to cancer diagnosis and progression.