Gap junctional intercellular communication (GJic) is a vital cellular process required for maintenance of tissue homeostasis. In vitro assessment of GJic represents valuable phenotypic endpoint that could be effectively utilized as an integral component in modern toxicity testing, drug screening or biomedical in vitro research. However, currently available methods for quantifying GJic with higher-throughputs typically require specialized equipment, proprietary software and/or genetically engineered cell models. to overcome these limitations, we present here an innovative adaptation of traditional, fluorescence microscopy-based scrape loading-dye transfer (SL-DT) assay, which has been optimized to simultaneously evaluate GJic, cell density and viability. this multiparametric method was demonstrated to be suitable for various multiwell microplate formats, which facilitates an automatized image acquisition. The assay workflow is further assisted by an open source-based software tools for batch image processing, analysis and evaluation of GJic, cell density and viability. our results suggest that this approach provides a simple, fast, versatile and cost effective way for in vitro high-throughput assessment of GJic and other related phenotypic cellular events, which could be included into in vitro screening and assessment of pharmacologically and toxicologically relevant compounds. Gap junctional intercellular communication (GJIC) allows an exchange of low molecular weight molecules (<1.2 kDa) between adjacent cells in both vertebrates and invertebrates 1. GJIC is mediated through intercellular channels build from connexin proteins in vertebrates and from innexin proteins in invertebrates 1,2. GJIC plays a central role in coordinating cell-to-cell communication and integration of signal transduction pathways controlling gene expression and cell behaviour in order to receive coordinated and collective responses from the cells across a tissue of multicellular organism 3,4. Dysregulation of GJIC and GJIC-dependent signal integration, for example by drugs or environmental contaminants, can disrupt the normal homeostatic control of a cell behaviour and lead to numerous adverse outcomes such as cardiovascular 5 , pulmonary 6 or reproductive system 7,8 diseases and cancer 4,9,10. On the other hand, timed and targeted upregulation or downregulation of GJIC, connexin channel or hemichannel activity, induced by pharmacological agents, is currently widely discussed as potential therapeutic approach for treatment of various diseases 10-12. Accordingly, GJIC can be effectively utilized in the modern toxicological and pharmacological research, and GJIC analysis could be a valuable component of any biological study. However, in vitro assessment of GJIC has been used relatively less frequently in the modern research in comparison to other and more traditional phenotypic assays, such as evaluation of cellular metabolic activity, cell proliferation, cell cycle, autophagy, apoptosis, cell migration, cytoskeletal rearangements 13. This m...

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Ambient air pollution and smoking are well-documented risk factors for male infertility. Prevalent air pollutants and cigarette smoke components, polycyclic aromatic hydrocarbons (PAHs), are environmental and occupational toxicants that act as chemicals disrupting endocrine regulation and reproductive potential in males. Testicular gap junctional intercellular communication (GJIC) is critical for normal development and function of testicular tissue, thus we assessed GJIC as a process potentially targeted by PAHs in testes. Lower MW PAHs with a bay or bay-like region rapidly dysregulated GJIC in Leydig TM3 cells by relocalization of major testicular gap junctional protein connexin 43 (Cx43) from plasma membrane to cytoplasm. This was associated with colocalization between Cx43 and ubiquitin in intracellular compartments, but without any effect on Cx43 degradation rate or steady-state Cx43 mRNA levels. A longer exposure to active PAHs decreased steady-state levels of full-length Cx43 protein and its 2 N-truncated isoforms. Inhibition of GJIC by PAHs, similarly to a prototypic GJIC-inhibitor TPA, was mediated via the MAP kinase-Erk1/2 and PKC pathways. Polycyclic aromatic hydrocarbon-induced GJIC dysregulation in testes was cell-type-specific because neither PAH dysregulated GJIC in Sertoli TM4 cells, despite PAHs were rapidly taken up by both Leydig TM3 as well as Sertoli TM4 cells. Because TPA effectively dysregulated GJIC in both testicular cell types, a unique regulator of GJIC targeted by PAHs might exist in Leydig TM3 cells. Our results indicate that PAHs could be a potential etiological agent contributing to reproductive dysfunctions in males through an impairment of testicular GJIC and junctional and/or nonjunctional functions of Cx43.

Development of therapeutic systems to treat glioblastoma, the most common and aggressive brain tumor, belongs to priority tasks in cancer research. We have synthesized colloidally stable magnetic nanoparticles (Dh=336 nm) coated with doxorubicin (Dox) conjugated copolymers of N,N‐dimethylacrylamide and either N‐acryloylglycine methyl ester or N‐acryloylmethyl 6‐aminohexanoate. The terminal carboxyl groups of the copolymers were reacted with alendronate by carbodiimide formation. Methyl ester groups were then transferred to hydrazides for binding Dox by a hydrolytically labile hydrazone bond. The polymers were subsequently bound on the magnetic nanoparticles through bisphosphonate terminal groups. Finally, the anticancer effect of the Dox‐conjugated particles was investigated using the U‐87 glioblastoma cell line in terms of particle internalization and cell viability, which decreased to almost zero at a concentration of 100 μg of particles per ml. These results confirmed that poly(N,N‐dimethylacrylamide)‐coated magnetic nanoparticles can serve as a solid support for Dox delivery to glioblastoma cells.