18 research outputs found
A quantitative metric for the comparative evaluation of optical clearing protocols for 3D multicellular spheroids
3D multicellular spheroids quickly emerged as in vitro models because they represent the in vivo tumor environment better than standard 2D cell cultures. However, with current microscopy technologies, it is difficult to visualize individual cells in the deeper layers of 3D samples mainly because of limited light penetration and scattering. To overcome this problem several optical clearing methods have been proposed but defining the most appropriate clearing approach is an open issue due to the lack of a gold standard metric. Here, we propose a guideline for 3D light microscopy imaging to achieve single-cell resolution. The guideline includes a validation experiment focusing on five optical clearing protocols. We review and compare seven quality metrics which quantitatively characterize the imaging quality of spheroids. As a test environment, we have created and shared a large 3D dataset including approximately hundred fluorescently stained and optically cleared spheroids. Based on the results we introduce the use of a novel quality metric as a promising method to serve as a gold standard, applicable to compare optical clearing protocols, and decide on the most suitable one for a particular experiment
Mono Landmark Localization for an Autonomous Navigation of a Cooperative Mobile Robot Formation
Small extracellular vesicles convey the stress-induced adaptive responses of melanoma cells
Exosomes are small extracellular vesicles (sEVs), playing a crucial role in the intercellular communication in physiological as well as pathological processes. Here, we aimed to study whether the melanoma-derived sEV-mediated communication could adapt to microenvironmental stresses. We compared B16F1 cell-derived sEVs released under normal and stress conditions, including cytostatic, heat and oxidative stress. The miRNome and proteome showed substantial differences across the sEV groups and bioinformatics analysis of the obtained data by the Ingenuity Pathway Analysis also revealed significant functional differences. The in silico predicted functional alterations of sEVs were validated by in vitro assays. For instance, melanoma-derived sEVs elicited by oxidative stress increased Ki-67 expression of mesenchymal stem cells (MSCs); cytostatic stress-resulted sEVs facilitated melanoma cell migration; all sEV groups supported microtissue generation of MSC-B16F1 co-cultures in a 3D tumour matrix model. Based on this study, we concluded that (i) molecular patterns of tumour-derived sEVs, dictated by the microenvironmental conditions, resulted in specific response patterns in the recipient cells; (ii) in silico analyses could be useful tools to predict different stress responses; (iii) alteration of the sEV-mediated communication of tumour cells might be a therapy-induced host response, with a potential influence on treatment efficacy
How does β-adrenergic signalling affect the transitions from ventricular tachycardia to ventricular fibrillation?
AIMS: Ventricular tachycardia (VT) and fibrillation (VF) are the most lethal cardiac arrhythmias. The degeneration of VT into VF is associated with the breakup of a spiral wave of the action potential in cardiac tissue. β-Adrenergic (βAR) signalling potentiates the L-type Ca current (I(CaL)) faster than the slow delayed rectifier potassium current (I(Ks)), which transiently prolongs the action potential duration (APD) and promotes early after depolarizations. In this study, we aimed at investigating how βAR signalling affects the transition from VT to VF. METHODS AND RESULTS: We used a physiologically detailed computer model of the rabbit ventricular myocyte in a two-dimensional tissue to determine how spiral waves respond to βAR activation following administration of isoproterenol. A simplified mathematical model was also used to investigate the underlying dynamics. We found that the spatiotemporal behaviour of spiral waves strongly depends on the kinetics of βAR activation. When βAR activation is rapid, a stable spiral wave turns into small fragments and its electrocardiogram reveals the transition from VT to VF. This is due to the transiently steepened APD restitution induced by the faster activation of I(CaL) vs. I(Ks) upon sudden βAR activation. The spiral wave may also disappear if its transient wavelength is too large to be supported by the tissue size upon sudden strong βAR activation that prolongs APD transiently. When βAR activation is gradual, a stable spiral wave remains such, because of more limited increase in both APD and slope of APD restitution due to more contemporaneous I(CaL) and I(Ks) activation. CONCLUSION: Changes in APD restitution during βAR activation revealed a novel transient spiral wave dynamics; this spatiotemporal characteristic strongly depends on the protocol of isoproterenol application