The rapid proximity labeling system PhastID identifies ATP6AP1 as an unconventional GEF for Rheb.

Rheb is a small G protein that functions as the direct activator of the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate signaling cascades in response to nutrients and growth factors. Despite extensive studies, the guanine nucleotide exchange factor (GEF) that directly activates Rheb remains unclear, at least in part due to the dynamic and transient nature of protein-protein interactions (PPIs) that are the hallmarks of signal transduction. Here, we report the development of a rapid and robust proximity labeling system named Pyrococcus horikoshii biotin protein ligase (PhBPL)-assisted biotin identification (PhastID) and detail the insulin-stimulated changes in Rheb-proximity protein networks that were identified using PhastID. In particular, we found that the lysosomal V-ATPase subunit ATP6AP1 could dynamically interact with Rheb. ATP6AP1 could directly bind to Rheb through its last 12 amino acids and utilizes a tri-aspartate motif in its highly conserved C-tail to enhance Rheb GTP loading. In fact, targeting the ATP6AP1 C-tail could block Rheb activation and inhibit cancer cell proliferation and migration. Our findings highlight the versatility of PhastID in mapping transient PPIs in live cells, reveal ATP6AP1's role as an unconventional GEF for Rheb, and underscore the importance of ATP6AP1 in integrating mTORC1 activation signals through Rheb, filling in the missing link in Rheb/mTORC1 activation.

Inducing aortic aneurysm/dissection in zebrafish: evaluating the efficacy of ß-Aminopropionic Nitrile as a model.

Aortic aneurysm/dissection (AAD) poses a life-threatening cardiovascular emergency with complex mechanisms and a notably high mortality rate. Zebrafish (Danio rerio) serve as valuable models for AAD due to the conservation of their three-layered arterial structure and genome with that of humans. However, the existing studies have predominantly focused on larval zebrafish, leaving a gap in our understanding of adult zebrafish. In this study, we utilized ß-Aminopropionic Nitrile (BAPN) impregnation to induce AAD in both larval and adult zebrafish. Following induction, larval zebrafish exhibited a 28% widening of the dorsal aortic diameter (p < 0.0004, n = 10) and aortic arch malformations, with a high malformation rate of 75% (6/8). Conversely, adult zebrafish showed a 41.67% (5/12) mortality rate 22 days post-induction. At this time point, the dorsal aortic area had expanded by 2.46 times (p < 0.009), and the vessel wall demonstrated significant thickening (8.22 ± 2.23 µM vs. 26.38 ± 10.74 µM, p < 0.05). Pathological analysis revealed disruptions in the smooth muscle layer, contributing to a 58.33% aneurysm rate. Moreover, the expression levels of acta2, tagln, cnn1a, and cnn1b were decreased, indicating a weakened contractile phenotype. Transcriptome sequencing showed a significant overlap between the molecular features of zebrafish tissues post-BAPN treatment and those of AAD patients. Our findings present a straightforward and practical method for generating AAD models in both larval and adult zebrafish using BAPN.

Cardiac disorder-related adverse events for aryl hydrocarbon receptor agonists: a safety review.

BACKGROUND: Although cardiac disorder-related adverse events (AEs) have been reported in patients treated with aryl hydrocarbon receptor (AHR) agonists, their safety profiles remain unknown. Here, we identified significant cardiac disorders associated with AHR agonists and further evaluated their relevance. RESEARCH DESIGN AND METHODS: Database queries were performed using OpenVigil 2.1 and AEs voluntarily submitted to Food and Drug Administration Adverse Event Reporting System (FAERS) between 2004 and 2020 were included. This study based on the Medical Dictionary for Regulatory Activities and the standardized MedDRA Queries to define the preferred terms, and we used reporting odd ratio to detect signals. RESULTS: In the FAERS database, 14,078 cardiac disorder-related AEs were identified in patients receiving AHR agonists. Among all AHR agonists, the number of cardiac disorder-related PTs with positive signals for AHR agonists was 93. Peripheral swelling (n = 1572) and atrial fibrillation (n = 1277) were the most reported cardiac disorder-related AEs among AHR agonists in disproportionately reported PTs. Moreover, several AHR agonists were highly associated with tachyarrhythmia. CONCLUSIONS: By mining the FAERS database, we provided more information on the association between AHR agonist use and cardiac disorder-related AEs.

Downregulation of Cx43 reduces cisplatin-induced acute renal injury by inhibiting ferroptosis.

Ferroptosis is one of the main mechanisms involved in different forms of acute kidney injury (AKI), including cisplatin-induced AKI. However, it is not clear whether Cx43 has a regulatory effect on ferroptosis caused by cisplatin. In this study, we investigate the regulatory effects of Cx43 on cisplatin-induced ferroptosis and its mechanism. In vivo and in vitro studies showed that the expression level of Cx43 was significantly upregulated in the cisplatin-induced kidney injury model. In HK2 cells, cisplatin significantly induced ferroptosis. Adding shRNA-Cx43 and gap27 to the HK2 cells downregulated the expression of Cx43 and blocked the effects of cisplatin, resulting in a significantly improved survival rate of HK2 cells. Our primary data suggested that downregulating Cx43 not only inhibits ferroptosis, but also inhibits apoptosis. Through mechanistic studies, we confirmed that downregulating the expression of Cx43 by increasing SLC7A11 can increase the GSH content to inhibit cisplatin-induced ferroptosis. In vivo experiments showed that downregulation of Cx43 expression by gap27 reduced AKI in the animal model by inhibiting cisplatin-induced ferroptosis. Therefore, our results indicated that downregulation of Cx43 can inhibit ferroptosis by restoring the level of SLC7A11 in the system xc‾ transporter and alleviate cisplatin-induced AKI.

Transcriptome analysis revealed changes of multiple genes involved in muscle hardness in grass carp (Ctenopharyngodon idellus) fed with faba bean meal.

An 8-week feeding trial and transcriptome analysis were conducted to investigate the potential mechanism of muscle-hardening caused by faba bean in grass carp (Ctenopharyngodon idellus). Ordinary grass carp (fed with practical diet) and crisp grass carp (fed with faba bean meal) groups were designed. Lower water holding capacity and higher some texture parameters were observed in the muscle of crisp grass carp compared with another group. 19.62 GB clean reads were generated, and total 1354 genes exhibiting differentially expression were identified (FDR < 0.05). Genes function enrichment revealed up-regulated genes in crisp grass carp mainly in response to myofibroblast proliferation, while down-regulated genes in response to immune regulation. Consistent with this, the tight junction pathway and the NF-κB signaling pathway were likewise significantly enriched. In summary, this study identified several candidate genes and putative signaling pathways deserving further investigation to the mechanism of muscle-hardening in fish fed with faba bean.

A novel undifferentiated spermatogonia-specific surface protein 1 (USSP1) in neonatal mice.

Mammalian spermatogenesis is maintained by a rare population of spermatogonial stem cells (SSCs), which are important for male fertility. SSCs remain a subset of undifferentiated spermatogonia, which can be isolated by a combination of surface markers. Specific markers to identify and isolate undifferentiated spermatogonia are lacking. Ussp1, a transcript previously annotated as long noncoding RNA (RIKEN cDNA 4933427D06, Gene ID: 232217), virtually encodes a membrane protein, USSP1, in a highly testis-specific manner in mouse. We demonstrate its expression on the membrane of undifferentiated spermatogonia by a homemade polyclonal rabbit antibody against the protein. In vivo, USSP1+ clusters consist mainly of As, Apr (GFRα1+) and Aal (PLZF+) cells. USSP1+ cells exhibit enrichment of undifferentiated spermatogonia, as shown by increased expression of SSC self-renewal molecular markers and the potential to form SSC clones in vitro and in vivo. However, Ussp1 knockout did not affect the number of SSCs or spermatogenesis in mice. Thy1+ cells from Ussp1 null mice did not show any defect in the SSC colony formation capacity, indicating that USSP1 is not essential for SSC self-renewal. Our data demonstrate that Ussp1 is specifically expressed in undifferentiated murine spermatogonia, indicating the potential to sort undifferentiated spermatogonia with USSP1 antibodies. Ussp1 might be a good maker for SSC enrichment in neonatal mice.

Single-walled carbon nanotube: One specific inhibitor of cancer stem cells in osteosarcoma upon downregulation of the TGFß1 signaling.

Cancer stem cells (CSCs) are believed to have a critical role in tumorigenesis, metastasis, therapeutic resistance or recurrence. Therefore, strategies designed to specifically target and eliminate CSCs have become one of the most promising and desirable ways for tumor treatment. Osteosarcoma stem cells (OSCs), the CSCs in osteosarcoma (OS), are critically associated with OS progression. Here, we show that single-walled carbon nanotubes (SWCNTs), including unmodified SWCNT (SWCNT-Raw) and SWCNT-COOH, have the ability to specifically inhibit the process of TGFß1-induced OS cells dedifferentiation, prevent the stem cell phenotypes acquisition in OS cells and reduce the OSC viability under conditions which mimic the OS microenvironment. Concurrently, SWCNT treatment significantly down-regulates the expression of OSC markers in OS, and markedly reduces the tumor microvessel density and tumor growth. Furthermore, we found that SWCNT could suppress the TGFß1-induced activation of TGFß type I receptor and downstream signaling, which are key for the OSC formation and maintenance. Our results reveal an unexpected function of SWCNT in negative modulation of OSCs, and provide significant implications for the potential CSCs-targeted therapeutic applications of SWCNT.

Effective production of recipient male pigs for spermatogonial stem cell transplantation by intratesticular injection with busulfan.

Germ cell transplantation has facilitated spermatogonial stem cell (SSC) and spermatogenesis research and shown great potential in the seed-breeding of domestic livestock. However, little progress has been made in large animals, primarily reflecting the difficulties in preparing sterile recipients. Here, we developed a novel protocol to prepare recipient pigs through the direct injection of busulfan into the cavum vaginale of the scrotums of Landrace-Large bi-crossbreeding male pigs and Seghers male pigs, two economically-important types of pigs, to eliminate endogenous spermatogonia. No severe diseases or weight loss was observed in either pig type after the injection with busulfan. Histologic analysis showed an advanced and dose-dependent germ cell loss, with complete germ cell loss observed in the highest dose group, 3.0 mg/kg in the Landrace-Large bi-crossbreeding pigs and 2.0 mg/kg in the Seghers pigs. A smaller seminiferous tubule diameter, a vacuolized seminiferous epithelium and the overproliferation interstitial cells, frequently observed in mouse germ cell deficiency models, were present in the most of the high-dose busulfan-treated groups. Molecular markers detected in Seghers pigs further confirmed the depletion of endogenous germ cells, providing an accessible niche for exogenous SSCs. This study provides a basis to prepare the transplantation recipients of SSCs in pigs.

Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells.

A recently developed strategy of sequencing alternative polyadenylation (APA) sites (SAPAS) with second-generation sequencing technology can be used to explore complete genome-wide patterns of tandem APA sites and global gene expression profiles. spermatogonial stem cells (SSCs) maintain long-term reproductive abilities in male mammals. The detailed mechanisms by which SSCs self-renew and generate mature spermatozoa are not clear. To understand the specific alternative polyadenylation pattern and global gene expression profile of male germline stem cells (GSCs, mainly referred to SSCs here), we isolated and purified mouse Thy1+ cells from testis by magnetic-activated cell sorting (MACS) and then used the SAPAS method for analysis, using pluripotent embryonic stem cells (ESCs) and differentiated mouse embryonic fibroblast cells (MEFs) as controls. As a result, we obtained 99,944 poly(A) sites, approximately 40% of which were newly detected in our experiments. These poly(A) sites originated from three mouse cell types and covered 17,499 genes, including 831 long non-coding RNA (lncRNA) genes. We observed that GSCs tend to have shorter 3'UTR lengths while MEFs tend towards longer 3'UTR lengths. We also identified 1337 genes that were highly expressed in GSCs, and these genes were highly consistent with the functional characteristics of GSCs. Our detailed bioinformatics analysis identified APA site-switching events at 3'UTRs and many new specifically expressed genes in GSCs, which we experimentally confirmed. Furthermore, qRT-PCR was performed to validate several events of the 334 genes with distal-to-proximal poly(A) switch in GSCs. Consistently APA reporter assay confirmed the total 3'UTR shortening in GSCs compared to MEFs. We also analyzed the cis elements around the proximal poly(A) site preferentially used in GSCs and found C-rich elements may contribute to this regulation. Overall, our results identified the expression level and polyadenylation site profiles and these data provide new insights into the processes potentially involved in the GSC life cycle and spermatogenesis.