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Backgroud CRISPR/Cas is an efficient genome editing system that has been widely used for functional genetic studies and exhibits high potential in biomedical translational applications. Indel analysis has thus become one of the most common practices in the lab to evaluate DNA editing events generated by CRISPR/Cas. Several indel analysis tools have been reported, however, it is often required that users have certain bioinformatics training and basic command-line processing capability. Results Here, we developed CRISPR-GRANT, a stand-alone graphical CRISPR indel analysis tool, which could be easily installed for multi-platforms, including Linux, Windows, and macOS. CRISPR-GRANT offered a straightforward GUI by simple click-and-run for genome editing analysis of single or pooled amplicons and one-step analysis for whole-genome sequencing without the need of data pre-processing, making it ideal for novice lab scientists. Moreover, it also exhibited shorter run-time compared with tools currently available. Conclusion Therefore, CRISPR-GRANT is a valuable addition to the current CRISPR toolkits that significantly lower the barrier for wet-lab researchers to conduct indel analysis from large NGS datasets. CRISPR-GRANT binaries are freely available for Linux (above Ubuntu 16.04), macOS (above High Sierra 10.13) and Windows (above Windows 7) at https://github.com/fuhuancheng/CRISPR-GRANT . CRISPR-GRANT source code is licensed under the GPLv3 license and free to download and use.

Endochondral ossification generates most of the load-bearing bones, recapitulating it in human cells remains a challenge. Here, we report generation of SOX9 + sclerotomal progenitors (scl-progenitors), a mesenchymal precursor at the pre-condensation stage, from human pluripotent stem cells and development of osteochondral induction methods for these cells. Upon lineage-specific induction, SOX9 + scl-progenitors have not only generated articular cartilage but have also undergone spontaneous condensation, cartilaginous anlagen formation, chondrocyte hypertrophy, vascular invasion, and finally bone formation with stroma, thereby recapitulating key stages during endochondral ossification. Moreover, self-organized growth plate-like structures have also been induced using SOX9 + scl-progenitor-derived fusion constructs with chondro- and osteo-spheroids, exhibiting molecular and cellular similarities to the primary growth plates. Furthermore, we have identified ITGA9 as a specific surface marker for reporter-independent isolation of SOX9 + scl-progenitors and established a culture system to support their expansion. Our work highlights SOX9 + scl-progenitors as a promising tool for modeling human skeletal development and bone/cartilage bioengineering. Endochondral ossification is hard to recapitulate with human cells. Here, the authors generated SOX9+ sclerotomal progenitors from hPSCs, modeling key developmental stages of bone development and growth plate-like structures.

NOD-like receptors (NLRs) are traditionally recognized as major inflammasome components. The role of NLRs in germ cell differentiation and reproduction is not known. Here, we identified the gonadspecific Nlrp14 as a pivotal regulator in primordial germ cell-like cell (PGCLC) differentiation in vitro. Physiologically, knock out of Nlrp14 resulted in reproductive failure in both female and male mice. In adult male mice, Nlrp14 knockout (KO) inhibited differentiation of spermatogonial stem cells (SSCs) and meiosis, resulting in trapped SSCs in early stages, severe oligozoospermia, and sperm abnormality. Mechanistically, NLRP14 promoted spermatogenesis by recruiting a chaperone cofactor, BAG2, to bind with HSPA2 and form the NLRP14–HSPA2–BAG2 complex, which strongly inhibited ChIP-mediated HSPA2 polyubiquitination and promoted its nuclear translocation. Finally, loss of HSPA2 protection and BAG2 recruitment by NLRP14 was confirmed in a human nonsense germline variant associated with male sterility. Together, our data highlight a unique proteasome-mediated, noncanonical function of NLRP14 in PGCLC differentiation and spermatogenesis, providing mechanistic insights of gonad-specific NLRs in mammalian germline development.

Sports have traditionally had gendered connotations in society and culture, resulting in solidified gender stereotypes that influence impression evaluations. China has a special gender social culture; however, how sport–gender stereotypes (SGS) influence the gender evaluation of people in China in the Global South is still unknown. This study obtained gender-typed sports and attribute adjectives and proved the existence of SGS through a pilot study (392 college students, n 1  = 207, n 2  = 185) and then used two studies to explore the influence of both explicit and implicit SGS on evaluations and compared the differences between these stereotypes and general gender stereotypes. Study 1 (395 college students, n 1a  = 192, n 1b  = 203) examined the explicit level using a questionnaire experiment. The results of two experiments showed that (1) stereotype-consistent targets were more masculine or feminine in correspondence with their gender, while stereotype-inconsistent targets had higher anti-gender traits; and (2) the inclusion of stereotype-consistent sports activities led targets to be evaluated as more masculine, while stereotype-inconsistent sport activities showed gender evaluation reversal, especially for women. Study 2 (103 college students, n 2a  = 61, n 2b  = 42) measured the implicit attitudes using the Implicit Association Test. The results of two experiments showed that (1) implicit evaluations of stereotype-consistent targets were associated faster than stereotype-inconsistent targets and (2) the inclusion of gender-typed sports weakened implicit gender evaluations. In conclusion, this is the first quantitative study to explore the unique effect of SGS on individual evaluations and how they differ from general gender stereotypes in the Chinese context. These findings could provide valuable insights for research and the application of sports social science and physical education.

It is known for decades that dental epithelium and mesenchyme can reconstitute and regenerate a functional tooth. However, the mechanism of tooth reconstitution remains largely unknown due to the lack of an efficient in vitro model. Here, a chemically defined culture system is established that supports tooth reconstitution, further development with normal anatomy, and prompt response to chemical interference in key developmental signaling pathways, termed as toothoids. By using such a system, it is discovered that, during reconstitution, instead of resetting the developmental clock, dental cells reorganized and restarted from the respective developmental stage where they are originally isolated. Moreover, co‐stimulation of Activin A and Hedgehog/Smoothened agonist (SAG) sustained the initial induction of tooth fate from the first branchial arch, which would be otherwise quickly lost in culture. Furthermore, activation of Bone Morphogenetic Protein (BMP) signaling triggered efficient enamel formation in the late‐stage toothoids, without affecting the normal development of ameloblasts. Together, these data highlight the toothoid culture as a powerful tool to dissect the molecular mechanisms of tooth reconstitution and regeneration. A chemically defined culture is established for tooth reconstitution, where the reconstituted tooth organoids could continuously develop and generate multiple dental lineages. Using such culture, tooth reconstitution is found not to reset the developmental clock, dental fate commitment from the first branchial arch requires co‐stimulation of Activin A and SAG, and enamel formation by ameloblasts requires activation of BMP signaling.

Sperm‐induced Ca 2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca 2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca 2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2‐cell stage. The impaired developmental potential of Nlrp14 ‐deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14 ‐deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin‐like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14 ‐deficient oocytes, and Uhrf1 ‐deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na + /Ca 2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14 mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27‐linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.

Backgroud Here, we developed CRISPR-GRANT, a stand-alone graphical CRISPR indel analysis tool, which could be easily installed for multi-platforms, including Linux, Windows, and macOS. CRISPR-GRANT offered a straightforward GUI by simple click-and-run for genome editing analysis of single or pooled amplicons and one-step analysis for whole-genome sequencing without the need of data pre-processing, making it ideal for novice lab scientists. Moreover, it also exhibited shorter run-time compared with tools currently available. Therefore, CRISPR-GRANT is a valuable addition to the current CRISPR toolkits that significantly lower the barrier for wet-lab researchers to conduct indel analysis from large NGS datasets. CRISPR-GRANT binaries are freely available for Linux (above Ubuntu 16.04), macOS (above High Sierra 10.13) and Windows (above Windows 7) at https://github.com/fuhuancheng/CRISPR-GRANT. CRISPR-GRANT source code is licensed under the GPLv3 license and free to download and use.

The pivotal role of androgen receptor (AR) in regulating male fertility has attracted much research attention in the past two decades. Previous studies have shown that total AR knockout would lead to incomplete spermatogenesis and lowered serum testosterone levels in mice, resulting in azoospermia and infertility. However, the precise physiological role of ar in controlling fertility of male fish is still poorly understood. In this study, we have established an ar knockout zebrafish line by transcription activator-like effectors nucleases. Homozygous ar mutant male fish with smaller testis size were found to be infertile when tested by natural mating. Intriguingly, a small amount of mature spermatozoa was observed in the ar mutant fish. These mature spermatozoa could fertilize healthy oocytes, albeit with a lower fertilization rate, by in vitro fertilization. Moreover, the expression levels of most steroidogenic genes in the testes were significantly elevated in the ar mutants. In contrast, the levels of estradiol and 11-ketotestosterone (11-KT) were significantly decreased in the ar mutants, indicating that steroidogenesis was defective in the mutants. Furthermore, the protein level of LHβ in the serum decreased markedly in the ar mutants when compared with wild-type fish, probably due to the positive feedback from the diminished steroid hormone levels. Summary Sentence In summary, our results provided unequivocal in vivo evidence for the requirement of functional ar in maintaining normal spermatogenesis and steroidogenesis, in ensuring normal fertility in male zebrafish.

It is well established that the luteinizing hormone surge triggers ovulation, a dynamic process leading to the release of the mature oocyte from the ovarian follicle. But how this process controlled by LH signaling remains largely unknown in non-mammalian species. In this study, we investigated the roles of nuclear progesterone receptor ( npr ) in LH-induced ovulation. Our results indicate that the nuclear progesterone receptor serves as an important mediator of LH action on ovulation. This conclusion is based on the following results: (1) the expression level of npr peaks at the full-grown stage of the follicles; (2) the expression of npr is stimulated by LH signaling in vitro and in vivo ; and (3) the npr null females are infertile due to ovulation defects. Moreover, we further show that LH signaling could induce ptger4b expression in an npr -dependent manner and blockage of Ptger4b could also block hCG-induced ovulation. Collectively, our results not only demonstrate that npr serves an indispensable role in mediating the action of LH on ovulation in zebrafish, but also provide insights into the molecular mechanisms of the regulation of ovulation in fish.

MicroRNAs are a group of small non-coding RNAs that play key roles in almost every aspect of mammalian cell. In kidney, microRNAs are required for maintaining normal function of renal cells, disruption of which contributes to pathogenesis of renal diseases. In this study, we investigated the potential role of miRNAs as key regulators of podocyte survival by using a primary cell culture model from non-human primates (NHPs). Through microRNA profile comparison in glomeruli from mouse, rat and NHP, miR-27b was found to be among a list of glomeruli-enriched miRNA conserved across species. In NHP primary podocyte culture, significant downregulation of miR-27b was observed during treatment of puromycin aminonucleoside (PAN), a classic nephrotoxin. Overexpression of miR-27b enhanced PAN-induced apoptosis and cytoskeleton destruction in podocytes while its inhibition had a protective effect. Target identification analysis identified Adora2b as a potential direct target of miR-27b. Ectopic expression of miR-27b suppressed both Adora2b mRNA and protein expression, whereas inhibition of miR-27b increased the transcript and protein expression levels of Adora2B. Dual luciferase assay further confirmed Adora2b as a direct target of miR-27b. Furthermore, knockdown of Adora2b by siRNAs enhanced PAN-induced apoptosis, similar to the phenotypes we had observed with miR-27b overexpression. In addition, stimulating the adenosine signaling by an Adora2b agonist, NECA, improved podocyte survival upon PAN treatment. Taken together, our data identified a novel role of miR-27b-adora2b axis in primary podocyte survival upon injury and suggested a critical role of adenosine signaling pathway in podocyte protection.