摘要: Objectives Poor oocyte quality is detrimental to fertilization and embryo development, which causes infertility. Cystathionine beta-synthase (CBS) is one of the key enzymes modulating the metabolism of homocysteine (Hcy). Studies have shown that CBS plays an important role in female reproduction. However, the role of CBS in regulating oocyte quality during meiotic maturation still needs further investigation. Materials and Methods Immunohistochemistry, immunofluorescence, drug treatment, western blot, cRNA construct and in vitro transcription, microinjection of morpholino oligo and cRNA were performed for this study. Results We found that CBS was expressed both in human and mouse oocytes of follicles. In mouse oocytes, CBS was distributed in the nucleus at germinal vesicle (GV) stage and localized to spindle from germinal vesicle breakdown (GVBD) to metaphase II (MII). The expression of CBS was reduced in ovaries and oocytes of aged mice. CBS depletion resulted in meiotic arrest, spindle abnormality and chromosome misalignment, disrupted kinetochore-microtubule attachments and provoked spindle assembly checkpoint (SAC). CBS was disassembled when microtubules were disrupted with nocodazole, and co-localized with the stabilized microtubules after taxol treatment. Furthermore, CBS depletion decreased the acetylation of alpha-tubulin. Conclusions These results reveal that CBS is required for the acetylation of alpha-tubulin to ensure proper spindle assembly in regulating oocyte quality during meiotic maturation.

摘要: Objectives Drosophila melanogaster has become an excellent model organism to explore the genetic mechanisms underlying tumour progression. Here, by using well-established Drosophila tumour models, we identified Toll-7 as a novel regulator of tumour growth and invasion. Materials and methods Transgenic flies and genetic epistasis analysis were used. All flies were raised on a standard cornmeal and agar medium at 25 degrees C unless otherwise indicated. Immunostaining and RT-qPCR were performed by standard procedures. Images were taken by OLYMPUS BX51 microscope and Zeiss LSM 880 confocal microscope. Adobe Photoshop 2020 and Zeiss Zen were used to analyse the images. All results were presented in Scatter plots or Column bar graphs created by GraphPad Prism 8.0. Results Loss of Toll-7 suppresses Ras(V12)/lgl(-/-)-induced tumour growth and invasion, as well as cell polarity disruption-induced invasive cell migration, whereas expression of a constitutively active allele of Toll-7 is sufficient to promote tumorous growth and cell migration. In addition, the Egr-JNK signalling is necessary and sufficient for Toll-7-induced invasive cell migration. Mechanistically, Toll-7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. Moreover, Toll-7 activates the EGFR-Ras signalling, which cooperates with the Egr-JNK signalling to promote Yki-mediated cell proliferation and tissue overgrowth. Finally, Toll-7 is necessary and sufficient for the proper maintenance of EGFR protein level. Conclusions Our findings characterized Toll-7 as a proto-oncogene that promotes tumour growth and invasion in Drosophila, which shed light on the pro-tumour function of mammalian Toll-like receptors (TLRs).

摘要: Objectives The expression of transcription factor Hoxb5 specifically marks the functional haematopoietic stem cells (HSC) in mice. However, our recent work demonstrated that ectopic expression of Hoxb5 exerted little effect on HSC but could convert B-cell progenitors into functional T cells in vivo. Thus, cell type- and development stage-specific roles of Hoxb5 in haematopoietic hierarchy await more extensive exploration. In this study, we aim to investigate the effect of Hoxb5 expression in multipotent blood progenitor cells. Materials and Methods A Mx1cre/Rosa(LSL-Hoxb5-EGFP/+) mouse model was used to evaluate the effect of Hoxb5 expression in blood multipotent progenitor cells (MPP). Golden standard serial transplantation experiments were used to test the long-term haematopoiesis potential of Hoxb5-expressing MPP. Single-cell RNA-seq analysis was used to characterize the gained molecular features of Hoxb5-expressing MPP and to compare with the global transcriptome features of natural adult HSC and fetal liver HSC (FL HSC). Results Here, with a mouse strain engineered with conditional expression of Hoxb5, we unveiled that induced expression of Hoxb5 in MPP led to the generation of a de novo Sca1(+)c-kit(+)CD11b(+)CD48(+) (CD11b(+)CD48(+)SK) cell type, which can repopulate long-term multilineage haematopoiesis in serial transplantations. RNA-seq analysis showed that CD11b(+)CD48(+)SK cells exhibited acquired features of DNA replication and cell division. Conclusions Our current study uncovers that Hoxb5 can empower MPP with self-renewal ability and indicates an alternative approach for generating HSC-like cells in vivo from blood lineage cells.

摘要: Objectives Nucleotide oligomerization domain receptor 1 (NOD1) mediates host recognition of pathogenic bacteria in periodontium. However, the specific role of NOD1 in regulating osteogenesis is unclear. Therefore, this study focused on the activation status of NOD1 in periodontitis and its effect on the osteogenic capacity of human periodontal ligament stem cells (hPDLSCs) as well as the underlying mechanism. Methods Histological staining and Western blot were utilized to assess NOD1 expression in the periodontium of people with or without periodontitis. HPDLSCs were cultured under NOD1 agonist or antagonist treatment. Q-PCR and Western blot were employed to assess the expression of osteogenic marker genes and proteins. Alizarin red staining and alkaline phosphatase staining were used to determine the osteogenic capability of hPDLSCs. The activation of downstream signalling was determined and specific inhibitors were utilized to confirm the signalling pathway in NOD1-regulated osteogenesis. Results NOD1 expression is significantly elevated in periodontitis. With NOD1 activated by particular agonist tri-DAP, the osteogenic potential of hPDLSCs was impaired. NOD1 antagonist co-incubation partially restored the decreased osteogenesis in hPDLSCs. P38/MAPK was phosphorylated in tri-DAP-induced NOD1 activation. The inhibitor of p38 rescued the suppression of osteogenesis induced by tri-DAP in hPDLSCs. Conclusions Our study revealed the expression status of NOD1 in periodontitis. Its activation greatly decreased the osteogenic capacity of hPDLSCs which was mediated by the phosphorylation of p38 downstream signalling.

摘要: Objectives Large-scale generation of universal red blood cells (RBCs) from O-negative (O-ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year-round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high-density culture platform for large-scale generation of RBCs in vitro. Materials and Methods We generated 10 O-ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high-density cultures of erythroblasts in vitro. Results Based on their tri-lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small-scale generation of high-density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation. Conclusions This study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume-controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.