Significance Hatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation.

Bovine embryos are typically cultured at reduced oxygen tension to lower the impact of oxidative stress on embryo development. However, oocyte in vitro maturation (IVM) is performed at atmospheric oxygen tension since low oxygen during maturation has a negative impact on oocyte developmental competence. Lycopene, a carotenoid, acts as a powerful antioxidant and may protect the oocyte against oxidative stress during maturation at atmospheric oxygen conditions. Here, we assessed the effect of adding 0.2 μM lycopene (antioxidant), 5 μM menadione (pro-oxidant), and their combination on the generation of reactive oxygen species (ROS) in matured oocytes and the subsequent development, quality, and transcriptome of the blastocysts in a bovine in vitro model. ROS fluorescent intensity in matured oocytes was significantly lower in the lycopene group, and the resulting embryos showed a significantly higher blastocyst rate on day 8 and a lower apoptotic cell ratio than all other groups. Transcriptomic analysis disclosed a total of 296 differentially expressed genes (Benjamini–Hochberg-adjusted p < 0.05 and ≥ 1-log2-fold change) between the lycopene and control groups, where pathways associated with cellular function, metabolism, DNA repair, and anti-apoptosis were upregulated in the lycopene group. Lycopene supplementation to serum-free maturation medium neutralized excess ROS during maturation, enhanced blastocyst development and quality, and modulated the transcriptomic landscape.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. HCC typically develops on a background of chronic inflammation and fibrosis with tumor associated macrophages (TAMs) playing an important role in chronic inflammation-induced HCC and progression. However, the liver harbors unique macrophages, resident liver Kupffer cells (KCs) and monocyte-derived macrophages (Mo-Mφ), and their contribution to HCC and to the population of TAMs is incompletely known. Here, we characterized the tumor microenvironment and the proportion and transcriptional profile of hepatic macrophages (Mφ) in two commonly used HCC mouse models. A gradually increased expression of inflammatory, immune regulatory, fibrotic and cell proliferation pathways and markers was observed during diethylnitrosamine (DEN)-and non-alcoholic steatohepatitis (NASH)-induced HCC development. The transcriptional phenotypes of isolated hepatic Mφ subsets were clearly distinct and shifted during HCC development, with mixed pro-inflammatory and tumor-promoting expression profiles. There were marked differences between the models as well, with Mφ in NASH-HCC exhibiting a more immunomodulatory phenotype, in conjunction with an upregulation of lipid metabolism genes. Our data show that at least some infiltrated macrophages display expression of pro-tumoral markers, and that Kupffer cells are part of the population of TAMs and enhance tumor progression. These insights are useful to further unravel sequential pathogenic events during hepatocarcinogenesis and direct future development of new treatment strategies for HCC.

Cellular models of induced pluripotent stem cell (iPSC)-derived microglia and macrophages are an emerging toolbox to investigate neuroinflammation in vitro. We previously demonstrated that murine iPSC-microglia and iPSC-macrophages display phenotypical activation properties highly comparable to microglia and macrophages in vivo. Here we extended the characterization of iPSC-microglia and iPSCmacrophages with the analysis of their transcriptome profile. Next, these cellular models were employed to evaluate neuroimmune toxicity in vitro and to investigate the immune-modulatory properties of interleukin 13 (IL13), a cytokine known for its ability to protect against neuroinflammation-induced pathology by modulating microglia and macrophage activation. iPSC-microglia and iPSC-macrophages, in coculture with astrocyte-committed neural stem cells (NSC), were (pre)treated with IL13 and stimulated with lipopolysaccharide (LPS) and interferon γ (IFNγ), to assess how IL13 modulates their inflammatory response. Additionally, the use of luciferaseexpressing NSC (Luc-NSC) allowed real-time monitoring of immune-mediated neurotoxicity. Despite the known anti-inflammatory properties of IL13, iPSC-microglia primed with IL13 before LPS + IFNγ stimulation significantly increased NO secretion. This was associated with a marked reduction of the luminescence signal produced by Luc-NSC. Interestingly, we observed that IL13 signaling has a divergent functional outcome in microglia as compared to macrophages, as for the latter no major alterations in NO release and Luc-NSC viability were observed upon IL13 (pre)treatment. Finally, the striking IL13-induced upregulation of NO secretion by microglia under pro-inflammatory conditions was confirmed in vivo, where intracerebral delivery of IL13 increased inducible nitric oxide synthase mRNA expression. Concluding, we applied iPSC-derived neuroimmune cell culture models to identify distinct neuroimmune (toxicity) responses of microglia and macrophages to IL13-based immune modulation.

Two morphotypes of the cyanobacterial Limnospira indica (formerly Arthrospira sp.) strain PCC 8005, denoted as P2 (straight trichomes) and P6 (helical trichomes), were subjected to chronic gamma radiation from spent nuclear fuel (SNF) rods at a dose rate of ca. 80 Gy.h−1 for one mass doubling period (approximately 3 days) under continuous light with photoautotrophic metabolism fully active. Samples were taken for post-irradiation growth recovery and RNA-Seq transcriptional analysis at time intervals of 15, 40, and 71.5 h corresponding to cumulative doses of ca. 1450, 3200, and 5700 Gy, respectively. Both morphotypes, which were previously reported by us to display different antioxidant capacities and differ at the genomic level in 168 SNPs, 48 indels and 4 large insertions, recovered equally well from 1450 and 3200 Gy. However, while the P2 straight type recovered from 5700 Gy by regaining normal growth within 6 days, the P6 helical type took about 13 days to recover from this dose, indicating differences in their radiation tolerance and response. To investigate these differences, P2 and P6 cells exposed to the intermediate dose of gamma radiation (3200 Gy) were analyzed for differential gene expression by RNA-Seq analysis. Prior to batch normalization, a total of 1553 genes (887 and 666 of P2 and P6, respectively, with 352 genes in common) were selected based on a two-fold change in expression and a false discovery rate FDR smaller or equal to 0.05. About 85% of these 1553 genes encoded products of yet unknown function. Of the 229 remaining genes, 171 had a defined function while 58 genes were transcribed into non-coding RNA including 21 tRNAs (all downregulated). Batch normalization resulted in 660 differentially expressed genes with 98 having a function and 32 encoding RNA. From PCC 8005-P2 and PCC 8005-P6 expression patterns, it emerges that although the cellular routes used by the two substrains to cope with ionizing radiation do overlap to a large extent, both strains displayed a distinct preference of priorities.