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Summary Cardiac differentiation efficiency is hampered by inconsistencies and low reproducibility. We analyzed the differentiation process of multiple human pluripotent stem cell (hPSC) lines in response to dynamic GSK3β inhibition under varying cell culture conditions. hPSCs showed strong differences in cell-cycle profiles with varying culture confluency. hPSCs with a higher percentage of cells in the G1 phase of the cell cycle exhibited cell death and required lower doses of GSK3β inhibitors to induce cardiac differentiation. GSK3β inhibition initiated cell-cycle progression via cyclin D1 and modulated both Wnt signaling and the transcription factor (TCF) levels, resulting in accelerated or delayed mesoderm differentiation. The TCF levels were key regulators during hPSC differentiation with CHIR99021. Our results explain how differences in hPSC lines and culture conditions impact cell death and cardiac differentiation. By analyzing the cell cycle, we were able to select for highly cardiogenic hPSC lines and increase the experimental reproducibility by predicting differentiation outcomes., Highlights • Lineage variety and cell culture density affect the cell cycle in hPSCs • CHIR99021 is cytotoxic to hPSCs with reduced S/G2/M cell-cycle phases • Cardiac differentiation reproducibility depends on cell-cycle consistency in hPSCs • Cell cycle and TCF protein levels modulate CHIR99021-induced differentiation, In this article, Laco and colleagues show that human pluripotent stem cells are characterized by individual cell-cycle profiles. Changes in cell-cycle profiles, mainly due to cell culture density, affect Wnt pathway-induced cardiac differentiation and cell death when GSK3β inhibitors are applied. We show that cell-cycle validation is crucial for stem cell quality and experimental reproducibility.

The meiotic functions of Emi2, an inhibitor of the APC/C complex, have been best characterized in oocytes where it mediates metaphase II arrest as a component of the cytostatic factor. We generated knockout mice to determine the in vivo functions of Emi2-in particular, its functions in the testis, where Emi2 is expressed at high levels. Male and female Emi2 knockout mice are viable but sterile, indicating that Emi2 is essential for meiosis but dispensable for embryonic development and mitotic cell divisions. We found that, besides regulating cell-cycle arrest in mouse eggs, Emi2 is essential for meiosis I progression in spermatocytes. In the absence of Emi2, spermatocytes arrest in early diplotene of prophase I. This arrest is associated with decreased Cdk1 activity and was partially rescued by a knockin mouse model of elevated Cdk1 activity. Additionally, we detected expression of Emi2 in spermatids and sperm, suggesting potential post-meiotic functions for Emi2.

The cohesin ring, which is composed of the Smc1, Smc3, and Scc1 subunits, topologically embraces two sister chromatids from S phase until anaphase to ensure their precise segregation to the daughter cells. The opening of the ring is required for its loading on the chromosomes and unloading by the action of Wpl1 protein. Both loading and unloading are dependent on ATP hydrolysis by the Smc1 and Smc3 "head" domains, which engage to form two composite ATPase sites. Based on the available structures, we modeled the Saccharomyces cerevisiae Smc1/Smc3 head heterodimer and discovered that the Smc1/Smc3 interfaces at the two ATPase sites differ in the extent of protein contacts and stability after ATP hydrolysis. We identified smc1 and smc3 mutations that disrupt one of the interfaces and block the Wpl1-mediated release of cohesin from DNA. Thus, we provide structural insights into how the cohesin heads engage with each other.

An amendment to this paper has been published and can be accessed via a link at the top of the paper., info:eu-repo/semantics/published

Hepatocellular carcinoma (HCC) is associated with high mortality and the current therapy for advanced HCC, Sorafenib, offers limited survival benefits. Here we assessed whether combining the TLR3 agonist: lysine-stabilized polyinosinic-polycytidylic-acid (poly-ICLC) with Sorafenib could enhance tumor control in HCC. Combinatorial therapy with poly-ICLC and Sorafenib increased apoptosis and reduced proliferation of HCC cell lines in vitro, in association with impaired phosphorylation of AKT, MEK and ERK. In vivo, the combinatorial treatment enhanced control of tumor growth in two mouse models: one transplanted with Hepa 1-6 cells, and the other with liver tumors induced using the Sleeping beauty transposon. Tumor cell apoptosis and host immune responses in the tumor microenvironment were enhanced. Particularly, the activation of local NK cells, T cells, macrophages and dendritic cells was enhanced. Decreased expression of the inhibitory signaling molecules PD-1 and PD-L1 was observed in tumor-infiltrating CD8+ T cells and tumor cells, respectively. Tumor infiltration by monocytic-myeloid derived suppressor cells (Mo-MDSC) was also reduced indicating the reversion of the immunosuppressive tumor microenvironment. Our data demonstrated that the combinatorial therapy with poly-ICLC and Sorafenib enhances tumor control and local immune response hence providing a rationale for future clinical studies.

Piotr Konieczny,1,* Anna Grazyna Goralczyk,1,* Radoslaw Szmyd,1,* Lukasz Skalniak,1,* Joanna Koziel,2 Francesca Larese Filon,3 Matteo Crosera,4 Agnieszka Cierniak,1 Ewa K Zuba-Surma,5 Julia Borowczyk,5 Eliza Laczna,5 Justyna Drukala,5 Elzbieta Pyza,6 Danuta Semik,6 Olga Woznicka,6 Andrzej Klein,1 Jolanta Jura11Department of General Biochemistry, 2Department of Microbiology, Jagiellonian University, Kraków, Poland; 3Department of Public Health Sciences, 4Department of Chemical and Pharmaceutical Sciences, University of Trieste, Trieste, Italy; 5Department of Cell Biology, 6Department of Cell Biology and Imaging, Jagiellonian University, Kraków, Poland*These authors equally contributed to this workAbstract: The platinum (Pt)-group elements (PGEs) represent a new kind of environmental pollutant and a new hazard for human health. Since their introduction as vehicle-exhaust catalysts, their emissions into the environment have grown considerably compared with their low natural concentration in the earth crust. PGE emissions from vehicle catalysts can be also in the form of nanometer-sized particles (Pt nanoparticles [PtNPs]). These elements, both in their metallic form or as ions solubilized in biological media, are now recognized as potent allergens and sensitizers. Human skin is always exposed to toxic particles; therefore, in the present study we addressed the question of whether polyvinylpyrrolidone-coated PtNPs may have any negative effects on skin cells, including predominantly epidermal keratinocytes. In this study, PtNPs of two sizes were used: 5.8 nm and 57 nm, in concentrations of 6.25, 12.5, and 25 µg/mL. Both types of NPs were protected with polyvinylpyrrolidone. Primary keratinocytes were treated for 24 and 48 hours, then cytotoxicity, genotoxicity, morphology, metabolic activity, and changes in the activation of signaling pathways were investigated in PtNP-treated cells. We found that PtNPs trigger toxic effects on primary keratinocytes, decreasing cell metabolism, but these changes have no effects on cell viability or migration. Moreover, smaller NPs exhibited more deleterious effect on DNA stability than the big ones. Analyzing activation of caspases, we found changes in activity of caspase 9 and caspase 3/7 triggered mainly by smaller NPs. Changes were not so significant in the case of larger nanoparticles. Importantly, we found that PtNPs have antibacterial properties, as is the case with silver NPs (AgNPs). In comparison to our previous study regarding the effects of AgNPs on cell biology, we found that PtNPs do not exhibit such deleterious effects on primary keratinocytes as AgNPs and that they also can be used as potential antibacterial agents, especially in the treatment of Escherichia coli, representing a group of Gram-negative species.Keywords: platinum nanoparticles, keratinocytes, migration, signaling pathways, DNA damage, toxicity