Background The differentiation of human induced pluripotent stem cells (iPSCs) into oocytes, which involves the transformation from mitosis to meiosis, has been a hotspot of biological research for many years and represents a desirable experimental model and potential strategy for treating infertility. At present, studies have shown that most cells stagnate in the oogonium stage after differentiation into primordial germ cells (PGCs) from human iPSCs. Methods iPSCs carrying a SYCP3-mkate2 knock-in reporter were generated by the CRISPR/Cas9 strategy to monitor meiosis status during induced differentiation from iPSCs into oocytes. These induced PGCs/oogonia were activated by small molecules from the Wnt signaling pathway and then cocultured with reconstructed human ovarian nests in vivo for further development. Results First, human PGCs and oogonia were efficiently induced from iPSCs. Second, induced dormant PGCs resumed meiosis and then differentiated into primary oocytes through the in vitro activation of the Wnt signaling pathway. Finally, a new coculture system involving the reconstruction of ovarian nests in vitro could facilitate the differentiation of oocytes. Conclusions Human PGCs/oogonia induced from iPSCs can be activated and used to resume meiosis by molecules of the Wnt signaling pathway. The coculture of activated PGCs and reconstruction of ovarian nests facilitated differentiation into primary oocytes and the generation of haploid human oocytes in vivo. These findings established a new strategy for germline competence in primary oocytes and provided a keystone for human gametogenesis in vitro and in vivo.

The ClpX ATPase is critical for resistance to cell envelope targeting antibiotics in Bacillus anthracis, however, it is unclear whether this is due to its function as an independent chaperone or as part of the ClpXP protease. In this study, we demonstrate that antibiotic resistance is due to formation of the ClpXP protease through construction of a ClpX complementation plasmid that is unable to interact with ClpP. Additionally, we genetically disrupted both clpP genes, clpP1 and clpP2, found in B. anthracis Sterne and find that the loss of either increases susceptibility to cell envelope targeting antimicrobials, although neither has as strong of a phenotype as loss of clpX and neither clpP gene is essential for virulence in a G. mellonella model of infection. Lastly, we looked at changes to cell envelope morphology that could contribute to increased antibiotic sensitivity. We find no difference in cell charge or cell lysis, although we do see increased hydrophobicity in the ΔclpX strain, decreased cellular density and slightly thinner cells walls. We also see significant cell division defects in ΔclpX, although only when cells are grown in the mammalian cell culture medium, RPMI. We conclude that the intrinsic resistance of B. anthracis to cell wall active antimicrobials is dependent on formation of the ClpXP protease and that this could be due, at least in part, to the role of ClpX in regulating cell envelope morphology.

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In this study we demonstrated that ribonuclease A (RNase A) can recognize the thermodynamic asymmetry of siRNA duplexes, similar to other proteins involved in siRNA function such as argonaute 2. RNase A preferentially invades the siRNA duplex through the less stable terminus, i.e., the 3' terminus of the sense strand. As evidence, only phosphorothioate (PS) modification at the sense strand overhang improved serum stability, whereas PS modification at the antisense strand overhang did not affect stability. Moreover, the improvement in stability caused by modification at the sense strand overhang was found to correlate with the terminal base pair composition of the siRNA. Gel electrophoresis and MALDI-TOF MS analysis indicated that modifications at the sense strand overhang improved the serum stability of siRNAs by inhibiting the directional invasion of RNase A. The blocking effect was not brought about by stabilization of the siRNA duplexes because there was no clear difference between the melting temperatures of siRNAs with PS modifications at each 3' overhang.