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Matrix metalloproteinase 9 (MMP9) plays a pivotal role in mammary ductal morphogenesis, angiogenesis and glandular tissue architecture remodeling. However, the molecular mechanism of MMP9 expression in mammary epithelial cells of dairy cows remains unclear. This study aimed to explore the underlying mechanism of MMP9 expression. In this study, to determine whether the PI3K/AKT/mTORC1/NF-κB signalling pathway participates in the regulation of MMP9 expression, we treated mammary epithelial cells with specific pharmacological inhibitors of PI3K (LY294002), mTORC1 (Rapamycin) or NF-κB (Celastrol), respectively. Western blotting results indicated that LY294002, Rapamycin and Celastrol markedly decreased MMP9 expression and P65 nuclear translocation. Furthermore, we found that NF-κB (P65) overexpression resulted in elevated expression of MMP9 protein and activation of MMP9 promoter. In addition, we observed that Celastrol markedly decreases P65-overexpression-induced MMP9 promoter activity. Moreover, the results of the promoter assay indicated that the core regulation sequence for MMP9 promoter activation may be located at -420 ∼ -80 bp downstream from the transcription start site. These observations indicated that the PI3K/AKT/mTORC1 signalling pathway is involved in MMP9 expression by regulating MMP9 promoter activity via NF-κB in the mammary epithelial cells of dairy cows.

Inducible dimerization systems, such as rapamycin-induced dimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB-FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB-FKBP remains unclear. Here, we report that FRB-FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter-subunit contacts as in the crystal trimer. These findings expand the applications of the FRB-FKBP oligomerization system in diverse biological events., (© 2024 Federation of European Biochemical Societies.)

Limited resources affect an organism's physiology through the conserved metabolic pathway, the mechanistic target of rapamycin (mTOR). Males and females often react differently to nutritional limitation, but whether it leads to differential mTOR pathway expression remains unknown. Recently, we found that dietary restriction (DR) induced significant changes in the expression of mTOR pathway genes in female Japanese quails (Coturnix japonica). We simultaneously exposed 32 male and female Japanese quails to either 20%, 30%, 40% restriction or ad libitum feeding for 14 days and determined the expression of six key genes of the mTOR pathway in the liver to investigate sex differences in the expression patterns. We found that DR significantly reduced body mass, albeit the effect was milder in males compared to females. We observed sex-specific liver gene expression. DR downregulated mTOR expression more in females than in males. Under moderate DR, ATG9A and RPS6K1 expressions were increased more in males than in females. Like females, body mass in males was correlated positively with mTOR and IGF1, but negatively with ATG9A and RS6K1 expressions. Our findings highlight that sexes may cope with nutritional deficits differently and emphasise the importance of considering sexual differences in studies of dietary restriction., (© 2024. The Author(s).)

Background: Increasing evidence suggests that mitochondrial dysfunction plays a significant role in PTSD. However, the exact mechanism is still unclear. Mitochondrial dynamics could be one of the mechanisms, as it is crucial for mitochondrial homeostasis and is widely affected in traumatic situations. Mitochondrial dynamics regulate mitochondrial homeostasis via orexinergic receptors, and it is shown that antagonism of orexinergic receptors attenuates PTSD-like symptoms. Therefore, the present study aimed to determine how orexin antagonists affect mitochondrial dynamics in rats exhibiting PTSD-like symptoms., Methods: Using rats, a stress-re-stress (SRS) model with PTSD-like symptoms was established. On day 2 (D-2), the animals were exposed to variable stressors including 2 h of restraint followed by brief mild foot shock and exposure to 4%halothane. Foot shock was performed as a re-stress from D-8 to D-32 at six-day intervals., Results: SRS exposure caused PTSD-like phenotype, hypothalamic-pituitary-adrenal axis dysfunction, activation of mammalian target of rapamycin (mTOR), and mitochondrial-fission-process-1 (MTFP-1). SRS-subjected rats exhibited enhanced expression of fission-regulating proteins, including dynamin-related protein-1 and mitochondrial-fission-protein-1 and reduced expression of fusion-regulating proteins, including optic-atrophy-1 and mitofusin-2, in the amygdala. TEM analysis revealed that SRS exposure further damaged the mitochondria. Treatment with suvorexant with rapamycin significantly mitigated PTSD-like symptoms and improved mitochondrial dynamics in SRS-exposed rats. However, their combination showed a more pronounced effect. Further, suvorexant in combination with rapamycin significantly mitigated mTOR and MTFP-1 activation. Sertraline attenuated PTSD-like symptoms without affecting SRS-induced activation of mTOR and disparity in mitochondrial dynamics. Suvorexant pharmacological effects on mitochondrial biogenesis also involve the mTOR pathway., Limitation: The role of orexinergic pathway in SRS-induced mitochondrial mitophagy was not explored., Conclusions: Targeting both the orexinergic and mTOR pathways might exert a beneficial synergistic effect for treating PTSD., Competing Interests: Declaration of competing interest Declaration from the author that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Plant growth is intimately linked to the availability of carbon and energy status. The Target of rapamycin (TOR) pathway is a highly relevant metabolic sensor and integrator of plant-assimilated C into development and growth. The cell wall accounts for around a third of the cell biomass, and the investment of C into this structure should be finely tuned for optimal growth. The plant C status plays a significant role in controlling the rate of cell wall synthesis. TOR signaling regulates cell growth and expansion, which are fundamental processes for plant development. The availability of nutrients and energy, sensed and integrated by TOR, influences cell division and elongation, ultimately impacting the synthesis and deposition of cell wall components. The plant cell wall is crucial in environmental adaptation and stress responses. TOR senses and internalizes various environmental cues, such as nutrient availability and stresses. These environmental factors influence TOR activity, which modulates cell wall remodeling to cope with changing conditions. Plant hormones, including auxins, gibberellins, and brassinosteroids, also regulate TOR signaling and cell wall-related processes. The connection between nutrients and cell wall pathways modulated by TOR are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Adeno-associated virus (AAV) vector gene therapy is a promising approach to treat rare genetic diseases; however, an ongoing challenge is how to best modulate host immunity to improve transduction efficiency and therapeutic outcomes. This report presents two studies characterizing multiple prophylactic immunosuppression regimens in male cynomolgus macaques receiving an AAVrh10 gene therapy vector expressing human coagulation factor VIII (hFVIII). In study 1, no immunosuppression was compared with prednisolone, rapamycin (or sirolimus), rapamycin and cyclosporin A in combination, and cyclosporin A and azathioprine in combination. Prednisolone alone demonstrated higher mean peripheral blood hFVIII expression; however, this was not sustained upon taper. Anti-capsid and anti-hFVIII antibody responses were robust, and vector genomes and transgene mRNA levels were similar to no immunosuppression at necropsy. Study 2 compared no immunosuppression with prednisolone alone or in combination with rapamycin or methotrexate. The prednisolone/rapamycin group demonstrated an increase in mean hFVIII expression and a mean delay in anti-capsid IgG development until after rapamycin taper. Additionally, a significant reduction in the plasma cell gene signature was observed with prednisolone/rapamycin, suggesting that rapamycin's tolerogenic effects may include plasma cell differentiation blockade. Immunosuppression with prednisolone and rapamycin in combination could improve therapeutic outcomes in AAV vector gene therapy., (© 2023. The Author(s).)

Retinopathy of prematurity (ROP), a retinal disease that can occur in premature infants, can lead to severe visual impairment. In this study, we examined the preventive and therapeutic effects of mammalian target of rapamycin complex 1 (mTORC1) inhibition on abnormal retinal blood vessels in a rat model of ROP. To induce ROP-like vascular abnormalities, rats were subcutaneously treated with KRN633, an inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, on postnatal day 7 (P7) and P8. KRN633-treated (ROP) rats were treated subcutaneously with the mTORC1 inhibitor rapamycin according to preventive and therapeutic protocols, i.e., from P11 to P13 (P11-P13) and from P14 to P20 (P14-P20), respectively. To compare with the effects of VEGF inhibition, KRN633 was administered according to similar protocols. Changes in retinal vasculature, phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, and the proliferative status of vascular cells were evaluated at P14 and P21 using immunohistochemistry. Rapamycin treatment from P11 to P13 prevented increases in arteriolar tortuosity, capillary density, and the number of proliferating vascular cells, and eliminated pS6 immunoreactivity in ROP rats. KRN633 treatment at P11 and P12 (P11/P12) also prevented the appearance of ROP-like retinal blood vessels. Rapamycin treatment from P14 to P20 failed to attenuate arteriolar tortuosity but prevented increases in capillary density and proliferating vascular cell number at the vascular front, but not at the central zone. KRN633 treatment from P14 to P20 significantly reduced abnormalities in the retinal vasculature; however, the effects were inferior to those of KRN633 treatment on P11/P12. These results suggest that activation of the mTORC1 pathway in proliferating endothelial cells contributes to the appearance and progression of ROP-like retinal blood vessels. Therefore, inhibition of mTORC1 may be a promising approach for selectively targeting abnormal retinal blood vessels in ROP., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Study Question: What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these?, Summary Answer: Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation., What Is Known Already: Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans., Study Design, Size, Duration: Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21-41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 μM in separate experiments, using biopsies from seven women (aged 27-41) and six women (aged 21-34), respectively. Biopsies from four women (aged 28-38) were used to investigate the effect of rapamycin or AMH only., Participants/materials, Setting, Methods: Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively., Main Results and the Role of Chance: Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated., Limitations, Reasons for Caution: These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles., Wider Implications of the Findings: These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring., Study Funding/competing Interest(s): This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

To reveal the molecular mechanism of brain damage induced by chronic fluorosis, expression of PTEN-induced kinase 1 (PINK1)/parkin RBR E3 ubiquitin-protein ligase (Parkin)-mediated mitophagy pathway and activity of mitochondrial superoxide dismutase (SOD) were investigated in rat brains and primary cultured neurons exposed to high level of fluoride. Sprague-Dawley (SD) rats were treated with fluoride (0, 5, 50, and 100 ppm) for 3 and 6 months. The primary neurons were exposed to 0.4 mM (7.6 ppm) fluoride and thereafter treated with 100 nM rapamycin (a stimulator of mitophagy) or 50 μM 3-methyladenine (3-MA, an inhibitor of mitophagy) for 24 h. The expressions of PINK1/Parkin at the protein level and the activity of SOD in mitochondria of rat brains and cultured neurons were determined by Western blotting and biochemical method, respectively. The results showed that the rats exposed to fluoride exhibited different degrees of dental fluorosis. In comparison to controls, the expressions of PINK1 and Parkin were significantly higher in the rat brains and primary neurons exposed to high fluoride. In addition, a declined activity of mitochondrial SOD was determined. Interestingly, rapamycin treatment enhanced but 3-MA inhibited the changes of PINK1/Parkin pathway and SOD activity, and the correlations between the inhibited SOD activity and the elevated PINK1/Parkin proteins were observed. The results suggest that the inhibition of mitochondrial SOD activity induced by fluorosis may stimulate the expressions of mitophagy (PINK1/ Parkin) pathway to maintain the mitochondrial homeostasis., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Many viruses enter host cells by hijacking endosomal trafficking. CapZ, a canonical actin capping protein, participates in endosomal trafficking, yet its precise role in endocytosis and virus infection remains elusive., Results: Here, we showed that CapZ was transiently associated with early endosomes (EEs) and was subsequently released from the matured EEs after the fusion of two EEs, which was facilitated by PI(3)P to PI(3,5)P2 conversion. Vacuolin-1 (a triazine compound) stabilized CapZ at EEs and thus blocked the transition of EEs to late endosomes (LEs). Likewise, artificially tethering CapZ to EEs via a rapamycin-induced protein-protein interaction system blocked the early-to-late endosome transition. Remarkably, CapZ knockout or artificially tethering CapZ to EEs via rapamycin significantly inhibited flaviviruses, e.g., Zika virus (ZIKV) and dengue virus (DENV), or beta-coronavirus, e.g., murine hepatitis virus (MHV), infection by preventing the escape of RNA genome from endocytic vesicles., Conclusions: These results indicate that the temporal association of CapZ with EEs facilitates early-to-late endosome transition (physiologically) and the release of the viral genome from endocytic vesicles (pathologically)., (© 2024. The Author(s).)

The intricate evolutionary dynamics of endosymbiotic relationships result in unique characteristics among the genomes of symbionts, which profoundly influence host insect phenotypes. Here, we investigated an endosymbiotic system in Phenacoccus solenopsis, a notorious pest of the subfamily Phenacoccinae. The endosymbiont, "Candidatus Tremblaya phenacola" (T. phenacola PSOL), persisted throughout the complete life cycle of female hosts and was more active during oviposition, whereas there was a significant decline in abundance after pupation in males. Genome sequencing yielded an endosymbiont genome of 221.1 kb in size, comprising seven contigs and originating from a chimeric arrangement between betaproteobacteria and gammaproteobacteria. A comprehensive analysis of amino acid metabolic pathways demonstrated complementarity between the host and endosymbiont metabolism. Elimination of T. phenacola PSOL through antibiotic treatment significantly decreased P. solenopsis fecundity. Weighted gene coexpression network analysis demonstrated a correlation between genes associated with essential amino acid synthesis and those associated with host meiosis and oocyte maturation. Moreover, altering endosymbiont abundance activated the host mechanistic target of rapamycin pathway, suggesting that changes in the amino acid abundance affected the host reproductive capabilities via this signal pathway. Taken together, these findings demonstrate a mechanism by which the endosymbiont T. phenacola PSOL contributed to high fecundity in P. solenopsis and provide new insights into nutritional compensation and coevolution of the endosymbiotic system., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Background: Spinal cord injury (SCI) is a serious trauma to the central nervous system. M1/M2 microglial polarization as well as the following neuroinflammatory response are crucial factors in SCI. Autophagy plays an important role in SCI, but its neuroprotective or neurodegenerative role remains controversial., Materials and Methods: Here, we majorly examined the properties of autophagy in SCI and uncovered the regulatory relationship between autophagy and microglial polarization in SCI., Results: In our study, the Basso-Beattie-Bresnahan (BBB) score was declined in SCI. The cervical contusion SCI stimulated a sustaining neuropathic pain-linked phenotype characterized by thermal hyperalgesia as well as mechanical allodynia. It was revealed the structural damage to the spinal cord in SCI. Besides, the expression of microglia markers as well as inflammatory factor were promoted in SCI. Cervical contusion SCI induced autophagy inhibition and nuclear factor kappa-B (NF-κB) activation in mice. More importantly, enhanced autophagy induced by rapamycin suppressed the NF-κB pathway and alleviated cervical contusion SCI-induced neurological function damage in mice. Additionally, rapamycin promoted microglia M2 polarization and improved microglia-mediated inflammatory response., Conclusions: In conclusion, our study demonstrated that autophagy played a protective role in cervical SCI by promoting microglia polarization toward M2 through the NF-κB pathway. Our study may provide a novel sight for SCI treatment.

Vocal fold scar formation due to vocal fold injury (VFI) is a common cause of surgery or trauma-induced voice disorders. Severe scar formation can lead to reduced voice quality or even be life-threatening. Here, we investigated the role of autophagy in VFI, focusing on fibrosis as a consequence of autophagy in inducing VFI. A VFI model was constructed in rats by dissecting the lamina propria tissue from the thyroarytenoid muscle. Real-time PCR and Western blot were used to analyze expressions of autophagy markers, including Beclin1 and Atg7, in VFI. Tgfb1 and Col1a1 were assessed to determine the correlation of fibrosis with VFI progression and autophagy levels. Rat vocal fold fibroblasts were also treated with TGF-β1 or rapamycin, which activates and suppresses autophagy respectively, to explore how autophagy regulates fibrosis in VFI. Initially, we observed that autophagy was downregulated in vocal fold mucosa after VFI in rats. This was particularly evident by the time-dependent downregulation of Beclin1 and Atg7 following VFI. Concurrently, levels of Tgfb1 and Col1a1 also surged, hinting at elevated fibrosis levels. Furthermore, our experiments with TGF-β1 stimulation revealed that it inhibited autophagy in rat vocal fold fibroblasts. Interestingly, when we introduced rapamycin, this effect was reversed. Our data suggest that autophagy is a suppressor of VFI by alleviating fibrosis, making targeting autophagy a potential therapeutic route in VFI. NEW & NOTEWORTHY The study has demonstrated that autophagy is a suppressor of VFI by alleviating fibrosis, making autophagy a potential therapeutic target in VFI.

Background and Aim: Autophagy and gut microbiota correlates closely with the inflammatory bowel disease. Herein, we aimed to study the roles of rapamycin on the gut microbiota in inflammatory bowel disease., Methods: Acute colitis was induced with dextran sodium sulfate (DSS) and 2,4,6-trinitrobenzenesulfonic acid solution in mice. Mice were administered with rapamycin or hydroxychloroquine. Weight loss, disease activity index scores, histopathological score, serum inflammatory cytokines, intestinal permeability, and colonic autophagy-related proteins were detected. Cecal content was also preserved in liquid nitrogen and subsequently analyzed following the 16S DNA sequencing. The antibiotic cocktail-induced microbiome depletion was performed to further investigate the relationship between autophagy activation and gut microbiota., Results: Compared with the control group, the colonic autophagy-related proteins of P62, mTOR, and p-mTOR increased significantly, while the levels of LC3B and ATG16L1 decreased (all P < 0.05) in the model group. After rapamycin intervention, the colonic pathology of mice improved, while the disease activity index score decreased substantially; the colon length increased, and the expression of IL-6 and TNF-α decreased. Following hydroxychloroquine treatment, some indicators suggested aggravation of colitis. Principal coordinates analysis showed that the DSS group was located on a separate branch from the rapamycin group but was closer to the hydroxychloroquine group. Compared with the DSS group, the rapamycin group was associated with higher abundances of f&#95;Lactobacillaceae (P = 0.0151), f&#95;Deferribacteraceae (P = 0.0290), g&#95;Lactobacillus (P = 0.0151), g&#95;Mucispirillum (P = 0.0137), s&#95;Lactobacillus&#95;reuteri (P = 0.0028), and s&#95;Clostridium&#95;sp&#95;Culture&#95;Jar-13 (P = 0.0082) and a lower abundance of s&#95;Bacteroides&#95;sartorii (P = 0.0180). Linear discriminant analysis effect size showed that rapamycin increased the abundances of Lactobacillus-reuteri, Prevotellaceae, Paraprevotella, Christensenella and Streptococcus and decreased those of Peptostreptococcaceae and Romboutsia Bacteroides-sartorii. Besides, the improvement effect of autophagy activation on colitis disappears following gut microbiome depletion., Conclusion: The therapeutic effects of rapamycin on extenuating experimental colitis may be related to the gut microbiota., (© 2023 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

The thermodynamic and biophysical implications of the introduction of a co-solvent during protein-ligand binding remain elusive. Using ternary complexes of 12-kDa FK506 binding protein (FKBP12), FKBP-rapamycin binding (FRB) domain of the mammalian/mechanistic target of rapamycin (mTOR) kinase, and rapamycin analogs (rapalogs) in glycerol-water mixtures, the influence of solvent composition on ligand binding dynamics was explored. The pharmaceutical potential of rapalogs and the utility of glycerol as a co-solvent in drug delivery applications were critical in deciding the system to be studied. Consolidation of existing studies on rapamycin modification was first performed to strategically design a new rapalog called T1. The results from 100-ns dual-boost Gaussian accelerated molecular dynamics simulations showed that protein stability was induced in the presence of glycerol. Reweighting of the trajectories revealed that the glycerol-rich solvent system lowers the energy barrier in the conformational space of the protein while also preserving native contacts between the ligand and the residues in the binding site. Calculated binding free energies using MM/GBSA also showed that electrostatic energy and polar contribution of solvation energy are heavily influenced by the changes in solvation. Glycerol molecules are preferentially excluded through electrostatic interactions from the solvation shell which induce complex stability as seen in existing experiments. Hence, using glycerol as a co-solvent in rapamycin delivery has a significant role in maintaining stability. In addition, compound T1 is a potential mTORC1-selective inhibitor with strong affinity for the FKBP12-FRB complex. This study aims to provide insights on the design of new rapalogs, and the applicability of glycerol as co-solvent for FKBP12-rapalog-FRB complexes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Mammary physiology is distinguished in containing adult stem/progenitor cells that are actively amending the breast tissue throughout the reproductive lifespan of women. Despite their importance in both mammary gland development, physiological maintenance, and reproduction, the exact role of mammary stem/progenitor cells in mammary tumorigenesis has not been fully elucidated in humans or animal models. The implications of modulating adult stem/progenitor cells in women could lead to a better understanding of not only their function, but also toward possible breast cancer prevention led us to evaluate the efficacy of rapamycin in reducing mammary stem/progenitor cell activity and malignant progression markers., Methods: We analyzed a large number of human breast tissues for their basal and luminal cell composition with flow cytometry and their stem and progenitor cell function with sphere formation assay with respect to age and menopausal status in connection with a clinical study (NCT02642094) involving a low-dose (2 mg/day) and short-term (5-7 days) treatment of the mTOR inhibitor sirolimus. The expression of biomarkers in biopsies and surgical breast samples were measured with quantitative analysis of immunohistochemistry., Results: Sirolimus treatment significantly abrogated mammary stem cell activity, particularly in postmenopausal patients. It did not affect the frequency of luminal progenitors but decreased their self-renewal capacity. While sirolimus had no effect on basal cell population, it decreased luminal cell population, particularly in postmenopausal patients. It also significantly diminished prognostic biomarkers associated with breast cancer progression from ductal carcinoma in situ to invasive breast cancer including p16INK4A, COX-2, and Ki67, as well as markers of the senescence-associated secretary phenotype, thereby possibly functioning in preventing early breast cancer progression., Conclusion: Overall, these findings indicate a link from mTOR signaling to mammary stem and progenitor cell activity and cancer progression. Trial registration This study involves a clinical trial registered under the ClinicalTrials.gov identifier NCT02642094 registered December 30, 2015., (© 2023. The Author(s).)

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 ( riC9 ) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.

Objective: To investigate the effect of emodin on high glucose (HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)-mediated autophagy in podocytes (MPC5 cells) in vitro., Methods: MPC5 cells were treated with different concentrations of HG (2.5, 5, 10, 20, 40, 80 and 160 mmol/L), emodin (2, 4, 8 µ mol/L), or HG (40 mmol/L) and emodin (4 µ mol/L) with or without rapamycin (Rap, 100 nmol/L) and compound C (10 µ mol/L). The viability and apoptosis of MPC5 cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy marker light chain 3 (LC3) I/II, and AMPK/mTOR signaling pathway-related proteins were determined by Western blot. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy., Results: HG at 20, 40, 80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells, whereas emodin (4 µ mol/L) significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage (P<0.01). Emodin (4 µ mol/L) significantly increased LC3-II protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells (P<0.01). Furthermore, the protective effects of emodin were mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 µ mol/L) reversed emodin-induced autophagy activation., Conclusion: Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway, which might provide a potential therapeutic option for diabetic nephropathy., (© 2022. The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Even if adoptive cell transfer (ACT) has already shown great clinical efficiency in different types of disease, such as cancer, some adverse events consistently occur, and suicide genes are an interesting system to manage these events. Our team developed a new medical drug candidate, a chimeric antigen receptor (CAR) targeting interleukin-1 receptor accessory protein (IL-1RAP), which needs to be evaluated in clinical trials with a clinically applicable suicide gene system. To prevent side effects and ensure the safety of our candidate, we devised two constructs carrying an inducible suicide gene, RapaCasp9-G or RapaCasp9-A, containing a single-nucleotide polymorphism (rs1052576) affecting the efficiency of endogenous caspase 9. These suicide genes are activated by rapamycin and based on the fusion of human caspase 9 with a modified human FK-binding protein, allowing conditional dimerization. RapaCasp9-G- and RapaCasp9-A-expressing gene-modified T cells (GMTCs) were produced from healthy donors (HDs) and acute myeloid leukemia (AML) donors. The RapaCasp9-G suicide gene demonstrated better efficiency, and we showed its in vitro functionality in different clinically relevant culture conditions. Moreover, as rapamycin is not pharmacologically inert, we also demonstrated its safe use as part of our therapy., (© 2023. The Author(s).)

In eukaryotes, a target of rapamycin (TOR) is a well-conserved kinase that controls cell metabolism and growth in response to nutrients and environmental factors. Nitrogen (N) is an essential element for plants, and TOR functions as a crucial N and amino acid sensor in animals and yeast. However, knowledge of the connections between TOR and the overall N metabolism and assimilation in plants is still limited. In this study, we investigated the regulation of TOR in Arabidopsis (Arabidopsis thaliana) by the N source as well as the impact of TOR deficiency on N metabolism. Inhibition of TOR globally decreased ammonium uptake while triggering a massive accumulation of amino acids, such as Gln, but also of polyamines. Consistently, TOR complex mutants were hypersensitive to Gln. We also showed that the glutamine synthetase inhibitor glufosinate abolishes Gln accumulation resulting from TOR inhibition and improves the growth of TOR complex mutants. These results suggest that a high level of Gln contributes to the reduction in plant growth resulting from TOR inhibition. Glutamine synthetase activity was reduced by TOR inhibition while the enzyme amount increased. In conclusion, our findings show that the TOR pathway is intimately connected to N metabolism and that a decrease in TOR activity results in glutamine synthetase-dependent Gln and amino acid accumulation., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hypercholesterolemia can aggravate contrast-induced acute kidney injury, and the exacerbation of renal tubular epithelial cell (RTEC) injury is a major cause. However, the exact mechanisms remain obscure. Mitophagy, a type of autophagy, selectively eliminates damaged mitochondria and reduces mitochondrial oxidative stress, which is strongly implicated in cell homeostasis and acute kidney injury. Oxidized low-density lipoprotein (Ox-LDL) is accumulated in hypercholesterolemia and has a cytotoxic effect. This study aimed to determine whether and how ox-LDL exacerbates contrast-induced injury in RTECs and to further explore whether PINK1/Parkin-dependent mitophagy is involved in this process. Iohexol and ox-LDL were used alone or in combination to treat HK-2 cells. Rapamycin pretreatment was utilized to enhance mitophagy. Cell viability, apoptosis, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) were detected by cell counting kit-8, TUNEL staining, JC-1 kit and MitoSOX fluorescence, respectively. The expression of mitophagy-related proteins (including PINK1, Parkin, and so on) and cleaved caspase-3 was confirmed by western blot. Colocalization of MitoTracker-labeled mitochondria and LysoTracker-labeled lysosomes was observed by fluorescence microscopy to evaluate mitophagy. The results of our study showed that ox-LDL aggravated MMP decline, mtROS release and apoptosis in iohexol-treated HK-2 cells, accompanied by a further increased autophagy level. Enhancement of PINK1/Parkin-dependent mitophagy by rapamycin alleviated apoptosis and mitochondrial injury in HK-2 cells in response to iohexol under ox-LDL condition. Therefore, our findings indicate that ox-LDL aggravates contrast-induced injury of RTECs by increasing mitochondrial damage and mitochondrial oxidative stress, which may be associated with the relative insufficiency of PINK1/Parkin-dependent mitophagy., (© 2023 The Authors. Journal of Biochemical and Molecular Toxicology published by Wiley Periodicals LLC.)

Expression of transmembrane protein 16 A (TMEM16A), a calcium activated chloride channel, is elevated in some human cancers and impacts tumor cell proliferation, metastasis, and patient outcome. Evidence presented here uncovers a molecular synergy between TMEM16A and mechanistic/mammalian target of rapamycin (mTOR), a serine-threonine kinase that is known to promote cell survival and proliferation in cholangiocarcinoma (CCA), a lethal cancer of the secretory cells of bile ducts. Analysis of gene and protein expression in human CCA tissue and CCA cell line detected elevated TMEM16A expression and Cl - channel activity. The Cl - channel activity of TMEM16A impacted the actin cytoskeleton and the ability of cells to survive, proliferate, and migrate as revealed by pharmacological inhibition studies. The basal activity of mTOR, too, was elevated in the CCA cell line compared with the normal cholangiocytes. Molecular inhibition studies provided further evidence that TMEM16A and mTOR were each able to influence the regulation of the other's activity or expression respectively. Consistent with this reciprocal regulation, combined TMEM16A and mTOR inhibition produced a greater loss of CCA cell survival and migration than their individual inhibition alone. Together these data reveal that the aberrant TMEM16A expression and cooperation with mTOR contribute to a certain advantage in CCA. NEW & NOTEWORTHY This study points to the dysregulation of transmembrane protein 16 A (TMEM16A) expression and activity in cholangiocarcinoma (CCA), the inhibition of which has functional consequences. Dysregulated TMEM16A exerts an influence on the regulation of mechanistic/mammalian target of rapamycin (mTOR) activity. Moreover, the reciprocal regulation of TMEM16A by mTOR demonstrates a novel connection between these two protein families. These findings support a model in which TMEM16A intersects the mTOR pathway to regulate cell cytoskeleton, survival, proliferation, and migration in CCA.

In yeast Saccharomyces cerevisiae, the immunosuppressant rapamycin inhibits the TORC1 kinase causing rapid alteration in gene expression and leading to G 1 arrest. We recently reported the isolation and characterization from the histone mutant collection of a histone H2B R95A mutant that displays resistance to rapamycin. This mutant is defective in the expression of several genes belonging to the pheromone response pathway including STE5 encoding a scaffold protein that promotes the activation of downstream MAP kinases. Cells lacking Ste5 cannot arrest the cell cycle in response to rapamycin and as a consequence exhibit similar resistance to rapamycin as the H2B R95A mutant. Herein, we show that the H2B R95A mutation weakens the association of H2B with Spt16 a component of the FACT complex (FAcilitates Chromatin Transcription), and an essential factor that interacts with the histone H2A-H2B dimer to promote transcription and preserve chromatin integrity. From a collection of spt16 mutants, spt16 E857K and spt16-11 showed striking sensitivity to rapamycin as compared to the parent strain. spt16 E857K and spt16-11 expressed distinct forms of Ste5, while a suppressor mutation H2B A84D of the spt16-11 mutant prevents the expression of Ste5 and confers marked resistance to rapamycin. We interpret these findings to suggest that the Arg95 residue of histone H2B is required to recruit Spt16 to maintain the expression of STE5, which performs a role to arrest cells in the G 1 phase in response to rapamycin., (© 2023. The Author(s).)

Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest., Competing Interests: Declaration of interests PSL valorisation has submitted a patent related to the J-DNA (PCT FR2018/053533) with D.K., T.S., and C.G. among the inventors., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Skeletal muscle is a highly dynamic organ that is essential for locomotion as well as endocrine regulation in all populations of horses. However, despite the importance of adequate muscle development and maintenance, the mechanisms underlying protein anabolism in horses on different diets, exercise programs, and at different life stages remain obscure. Mechanistic target of rapamycin (mTOR) is a key component of the protein synthesis pathway and is regulated by biological factors such as insulin and amino acid availability. Providing a diet ample in vital amino acids, such as leucine and glutamine, is essential in activating sensory pathways that recruit mTOR to the lysosome and assist in the translation of important downstream targets. When the diet is well balanced, mitochondrial biogenesis and protein synthesis are activated in response to increased exercise bouts in the performing athlete. It is important to note that the mTOR kinase pathways are multifaceted and very complex, with several binding partners and targets that lead to specific functions in protein turnover of the cell, and ultimately, the capacity to maintain or grow muscle mass. Further, these pathways are likely altered across the lifespan, with an emphasis of growth in young horses while decreases in musculature with aged horses appears to be attributable to degradation or other regulators of protein synthesis rather than alterations in the mTOR pathway. Previous work has begun to pinpoint ways in which the mTOR pathway is influenced by diet, exercise, and age; however, future research is warranted to quantify the functional outcomes related to changes in mTOR. Promisingly, this could provide direction on appropriate management techniques to support skeletal muscle growth and maximize athletic potential in differing equine populations., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The mammalian target of rapamycin (mTOR) signaling pathway is crucial in maintaining cell growth and metabolism. The mTOR protein kinase constitutes the catalytic subunit of two multimeric protein complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). As such, this pathway is indispensable for many organs, including the kidney. Since its discovery, mTOR has been associated with major renal disorders such as acute kidney injury, chronic kidney disease, and polycystic kidney disease. On top of that, emerging studies using pharmacological interventions and genetic disease models have unveiled mTOR role in renal tubular ion handling. Along the tubule, mTORC1 and mTORC2 subunits are ubiquitously expressed at mRNA level. Nevertheless, at the protein level, current studies suggest that a tubular segment-specific balance between mTORC1 and mTORC2 exists. In the proximal tubule, mTORC1 regulates nutrients transports through various transporters located in this segment. On the other hand, in the thick ascending limb of the loop of Henle, both complexes play a role in regulating NKCC2 expression and activity. Lastly, in the principal cells of the collecting duct, mTORC2 determines Na + reabsorption and K + excretion by regulating of SGK1 activation. Altogether, these studies establish the relevance of the mTOR signaling pathway in the pathophysiology of tubular solute transport. Despite extensive studies on the effectors of mTOR, the upstream activators of mTOR signaling remain elusive in most nephron segments. Further understanding of the role of growth factor signaling and nutrient sensing is essential to establish the exact role of mTOR in kidney physiology., (© 2023 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Ribosomes are the basis for protein production, whose biogenesis is essential for cells to drive growth and proliferation. Ribosome biogenesis is highly regulated in accordance with cellular energy status and stress signals. In eukaryotic cells, response to stress signals and the production of newly-synthesized ribosomes require elements to be transcribed by the three RNA polymerases (RNA pols). Thus, cells need the tight coordination of RNA pols to adjust adequate components production for ribosome biogenesis which depends on environmental cues. This complex coordination probably occurs through a signaling pathway that links nutrient availability with transcription. Several pieces of evidence strongly support that the Target of Rapamycin (TOR) pathway, conserved among eukaryotes, influences the transcription of RNA pols through different mechanisms to ensure proper ribosome components production. This review summarizes the connection between TOR and regulatory elements for the transcription of each RNA pol in the budding yeast Saccharomyces cerevisiae . It also focuses on how TOR regulates transcription depending on external cues. Finally, it discusses the simultaneous coordination of the three RNA pols through common factors regulated by TOR and summarizes the most important similarities and differences between S. cerevisiae and mammals.

The advancement in pharmaceutical research has led to the discovery and development of new combinatorial life-saving drugs. Rapamycin is a macrolide compound produced from Streptomyces hygroscopicus. Rapamycin and its derivatives are one of the promising sources of drug with broad spectrum applications in the medical field. In recent times, rapamycin has gained significant attention as of its activity against cytokine storm in COVID-19 patients. Rapamycin and its derivatives have more potency when compared to other prevailing drugs. Initially, it has been used exclusively as an anti-fungal drug. Currently rapamycin has been widely used as an immunosuppressant. Rapamycin is a multifaceted drug; it has anti-cancer, anti-viral and anti-aging potentials. Rapamycin has its specific action on mTOR signaling pathway. mTOR has been identified as a key regulator of different pathways. There will be an increased demand for rapamycin, because it has lesser adverse effects when compared to steroids. Currently researchers are focused on the production of effective rapamycin derivatives to combat the growing demand of this wonder drug. The main focus of the current review is to explore the origin, development, molecular mechanistic action, and the current therapeutic aspects of rapamycin. Also, this review article revealed the potential of rapamycin and the progress of rapamycin research. This helps in understanding the exact potency of the drug and could facilitate further studies that could fill in the existing knowledge gaps. The study also gathers significant data pertaining to the gene clusters and biosynthetic pathways involved in the synthesis and production of this multi-faceted drug. In addition, an insight into the mechanism of action of the drug and important derivatives of rapamycin has been expounded. The fillings of the current review, aids in understanding the underlying molecular mechanism, strain improvement, optimization and production of rapamycin derivatives., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Chagas disease is caused by the parasite Trypanosoma cruzi (T. cruzi) and, among all the chronic manifestations of the disease, Chronic Chagas Cardiomyopathy (CCC) is the most severe outcome. Despite high burden and public health importance in Latin America, there is a gap in understanding the molecular mechanisms that results in CCC development. Previous studies showed that T. cruzi uses the host machinery for infection and replication, including the repurposing of the responses to intracellular infection such as mitochondrial activity, vacuolar membrane, and lysosomal activation in benefit of parasite infection and replication. One common signaling upstream to many responses to parasite infection is mTOR pathway, previous associated to several downstream cellular mechanisms including autophagy, mitophagy and lysosomal activation. Here, using human iPSC derived cardiomyocytes (hiPSCCM), we show the mTOR pathway is activated in hiPSCCM after T. cruzi infection, and the inhibition of mTOR with rapamycin reduced number of T. cruzi 48 h post infection (hpi). Rapamycin treatment also reduced lysosome migration from nuclei region to cell periphery resulting in less T. cruzi inside the parasitophorous vacuole (PV) in the first hour of infection. In addition, the number of parasites leaving the PV to the cytoplasm to replicate in later times of infection was also lower after rapamycin treatment. Altogether, our data suggest that host's mTOR activation concomitant with parasite infection modulates lysosome migration and that T. cruzi uses this mechanism to achieve infection and replication. Modulating this mechanism with rapamycin impaired the success of T. cruzi life cycle independent of mitophagy., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Imaging protein-protein interactions (PPIs) is a hot topic in molecular medicine in the postgenomic sequencing era. In the present study, we report bright and highly sensitive single-chain molecular strain probe templates which embed full-length Renilla luciferase 8.6-535SG (RLuc86SG) or Artificial luciferase 49 (ALuc49) as reporters. These reporters were deployed between FKBP-rapamycin binding domain (FRB) and FK506-binding protein (FKBP) as a PPI model. This unique molecular design was conceptualized to exploit molecular strains of the sandwiched reporters appended by rapamycin-triggered intramolecular PPIs. The ligand-sensing properties of the templates were maximized by interface truncations and substrate modulation. The highest fold intensities, 9.4 and 16.6, of the templates were accomplished with RLuc86SG and ALuc49, respectively. The spectra of the templates, according to substrates, revealed that the colors are tunable to blue, green, and yellow. The putative substrate-binding chemistry and the working mechanisms of the probes were computationally modeled in the presence or absence of rapamycin. Considering that the molecular strain probe templates are applicable to other PPI models, the present approach would broaden the scope of the bioassay toolbox, which harnesses the privilege of luciferase reporters and the unique concept of the molecular strain probes into bioassays and molecular imaging.

The intestines play a crucial role in the development of sepsis. The balance between autophagy and apoptosis in intestinal epithelial cells is dynamic and determines intestinal permeability. The present study focused on the potential role of autophagy in sepsis-induced intestinal barrier dysfunction and explored the mechanisms in vivo and in vitro. Excessive apoptosis in intestinal epithelia and a disrupted intestinal barrier were observed in septic mice. Promoting autophagy with rapamycin reduced intestinal epithelial apoptosis and restored intestinal barrier function, presenting as decreased serum diamine oxidase (DAO) and fluorescein isothiocyanate-dextran 40 (FD40) levels and increased expression of zonula occludens-1 (ZO-1) and Occludin. Polo-like kinase 1 (PLK1) knockdown in mice ameliorated intestinal epithelial apoptosis and the intestinal barrier during sepsis, whereas these effects were reduced with chloroquine and enhanced with rapamycin. PLK1 also promoted cell autophagy and improved lipopolysaccharide-induced apoptosis and high permeability in vitro. Moreover, PLK1 physically interacted with mammalian target of rapamycin (mTOR) and participated in reciprocal regulatory crosstalk in intestinal epithelial cells during sepsis. This study provides novel insight into the role of autophagy in sepsis-induced intestinal barrier dysfunction and indicates that the PLK1-mTOR axis may be a promising therapeutic target for sepsis., (Copyright © 2023 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Along with differences in life histories, metazoans have also evolved vast differences in cellularity, involving changes in the molecular pathways controlling the cell cycle. The extent to which the signalling network systemically determines cellular composition throughout the body and whether tissue cellularity is organized locally to match tissue-specific functions are unclear. We cultured genetic lines of Drosophila melanogaster on food with and without rapamycin to manipulate the activity of target of rapamycin (TOR)/insulin pathways and evaluate cell-size changes in five types of adult cells: wing and leg epidermal cells, ommatidial cells, indirect flight muscle cells and Malpighian tubule epithelial cells. Rapamycin blocks TOR multiprotein complex 1, reducing cell growth, but this effect has been studied in single cell types. As adults, rapamycin-treated flies had smaller bodies and consistently smaller cells in all tissues. Regardless, females eclosed with larger bodies and larger cells in all tissues than males. Thus, differences in TOR activity and sex were associated with the orchestration of cell size throughout the body, leading to differences in body size. We postulate that the activity of TOR/insulin pathways and their effects on cellularity should be considered when investigating the origin of ecological and evolutionary patterns in life histories.

Atractylenolide III (ATL-III) is a major active constituent of the natural plant Atractylodes rhizome. Our previous study has shown that ATL-III may alleviate alveolar macrophage apoptosis via the inhibition of the mammalian target of rapamycin (mTOR)-mediated autophagy of human silicosis. Therefore, we aimed to further explore the function of ATL-III in autophagy, apoptosis, and pulmonary fibrosis by establishing the ATL-III-intervened silicosis mouse model in this study. Meanwhile, we sought and then verified potential autophagy-related signaling pathways by matching differentially expressed genes (attained by RNA sequencing) and the autophagy database. In this study, RNA-sequencing results implied that the epidermal growth factor receptor, the crucial upstream activator of mTOR, was seen as a potential autophagy-regulatory molecule in the ATL-III-intervened silicosis mouse model. The finding of this study was that ATL-III might improve the disorder of autophagic degradation via the activation of epidermal growth factor receptor-mTOR signals in the pulmonary tissue of the silicosis mouse model. ATL-III also alleviated cell apoptosis and silicotic fibrosis. Overall, we supposed that ATL-III might be a potential protective medicine, which had a regulatory effect on autophagy, for the intervention of silicotic fibrosis. In the future, the therapeutic drugs for silicosis should be further focused on the development and application of such natural autophagy agents., (Copyright © 2022 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

In Brief: Fibroblast growth factor-2 (FGF2) is essential for early placenta development in sheep. This study shows that the mechanistic target of rapamycin is the key modulator of trophoblast adaptive response under FGF2 modulation., Abstract: During the early stage of placentation in sheep, normal conceptus development is affected by trophoblast cell functionality, whose dysregulation results in early pregnancy loss. Trophoblast metabolism is supported mainly by histotrophic factors, including fibroblast growth factor-2 (FGF2), which are involved in cell differentiation and function through the modulation of specific cellular mechanisms. The mechanistic target of rapamycin (mTOR) is known as a cellular 'nutrient sensor', but its downstream regulation remains poorly understood. The hypothesis was that during trophoblast development, the FGF2 effect is mediated by mTOR signalling pathway modulation. Primary trophoblast cells from 21-day-old sheep placenta were characterised and subjected to FGF2 and rapamycin treatment to study the effects on cell functionality and gene and protein expression profiles. The model showed mainly mononuclear cells with epithelial cell-like growth and placental morphological properties, expressing typical trophoblast markers. FGF2 promoted cell proliferation and migration under normal culture conditions, whereas mTOR inhibition reversed this effect. When the mTOR signalling pathway was activated, FGF2 failed to influence invasion activity. mTOR inhibition significantly reduced cell motility, but FGF2 supplementation restored motility even when mTOR was inhibited. Interestingly, mTOR inhibition influenced endocrine trophoblast marker regulation. Although FGF2 supplementation did not affect ovine placenta lactogen expression, as observed in the control, interferon-tau was drastically reduced. This study provides new insights into the mechanism underlying mTOR inhibitory effects on trophoblast cell functionality. In addition, as mTOR is involved in the expression of hormonal trophoblast markers, it may play a crucial role in early placenta growth and fetal-maternal crosstalk.

Although the genetic code of the yeast Saccharomyces cerevisiae was sequenced 25 years ago, the characterization of the roles of genes within it is far from complete. The lack of a complete mapping of functions to genes hampers systematic understanding of the biology of the cell. The advent of high-throughput metabolomics offers a unique approach to uncovering gene function with an attractive combination of cost, robustness, and breadth of applicability. Here, we used flow-injection time-of-flight mass spectrometry to dynamically profile the metabolome of 164 loss-of-function mutants in TOR and receptor or receptor-like genes under a time course of rapamycin treatment, generating a dataset with >7000 metabolomics measurements. In order to provide a resource to the broader community, those data are made available for browsing through an interactive data visualization app hosted at https://rapamycin-yeast.ethz.ch. We demonstrate that dynamic metabolite responses to rapamycin are more informative than steady-state responses when recovering known regulators of TOR signaling, as well as identifying new ones. Deletion of a subset of the novel genes causes phenotypes and proteome responses to rapamycin that further implicate them in TOR signaling. We found that one of these genes, CFF1, was connected to the regulation of pyrimidine biosynthesis through URA10. These results demonstrate the efficacy of the approach for flagging novel potential TOR signaling-related genes and highlight the utility of dynamic perturbations when using functional metabolomics to deliver biological insight., Competing Interests: SR, PD, TG, AB, RL, US, DH No competing interests declared, (© 2023, Reichling et al.)

Introduction: Short-term fasting protects against renal ischemia reperfusion injury (IRI). mTOR signaling is downregulated and may be involved in its protective effect. Rapamycin is considered a possible mimetic as it inhibits the mTOR pathway. This study examines the effect of rapamycin on renal IRI., Material and Methods: Mice were divided into four groups: ad libitum (AL), fasted (F), AL treated with rapamycin (AL+R), and F treated with rapamycin (F+R). Rapamycin was administered intraperitoneally 24 h before bilateral renal IRI was induced. Survival was monitored for 7 days. Renal cell death, regeneration, and mTOR activity were determined 48 h after reperfusion. Oxidative stress resistance of human renal proximal tubular and human primary tubular epithelial cells after rapamycin treatment was determined., Results: All F and F+R mice survived the experiment. Although rapamycin substantially downregulated mTOR activity, survival in the AL+R group was similar to AL (10%). Renal regeneration was significantly reduced in AL+R but not in F+R. After IRI (48 h), pS6K/S6K ratio was lower in F, F+R, and AL+R groups compared to AL fed animals (p = 0.02). In vitro, rapamycin also significantly downregulated mTOR activity (p &lt; 0.001) but did not protect against oxidative stress., Conclusion: Rapamycin pretreatment does not protect against renal IRI. Thus, protection against renal IRI by fasting is not exclusively mediated through inhibition of mTOR activity but may involve preservation of regenerative mechanisms despite mTOR downregulation. Therefore, rapamycin cannot be used as a dietary mimetic to protect against renal IRI., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Introduction: Graft-versus-host disease (GvHD) is a life-threatening syndrome commonly associated with hematopoietic stem cell transplantation (HSCT). Preventing the incidence of GVHD after HSCT along with minimizing long-term immunosuppression is currently under investigation with regulatory T cells (Tregs). As Tregs are a low-frequency population and the yield of all memory Tregs is not sufficient for clinical application, an initial Treg expansion is essential., Methods: Thirty milliliters of peripheral blood from the β-thalassemia major (beta-TM) patients and healthy controls were obtained and Tregs were isolated using MACS. Isolated cells were cultured in the presence of rapamycin and rIL-2 followed by activated with anti-CD3/CD28-coated beads. To evaluate Treg plasticity, expanded Tregs were cultured in a medium containing IL1β, IL6, TGFβ, and IL2, with or without 500 nM rapamycin for 72 h. To assess the functional properties of Tregs, CFSE dilution assays were performed to evaluate the ability of in vivo expanded Tregs from beta-TM patients. Statistical analysis was performed using paired t-test and independent t-test, with the aid of SPSS version 12.0. p-value ≤0.05 was considered significant., Results: The percentage of Tregs isolated from the control group was significantly higher than the Tregs isolated from patients (p-value = 0.01), which is probably due to the iron overload in beta-TM patients as a result of continuous blood transfusion. Also, the percentage of Tregs after 5 days of expansion had a significant increase in both groups compared to before expansion (p-value = 0.03). Our results also showed that the expansion of Tregs after 72 h in the presence of inflammatory cytokines and in the absence of rapamycin led to the increase in the intracellular expression of IL-17 (p-value = 0.01), while intracellular expression of IL-17 remained low following the addition of 100 nM rapamycin to the culture medium (pvalue = 0.073). The results of the functional evaluation of expanded Tregs showed relatively differences in both patient and control groups. Thus, expanded Tregs inhibited the proliferation of responder T cells in a dose-dependent manner in the control group (p-value = 0.028), while in the patient group this inhibitory effect was not significant (p-value = 0.055)., Conclusion: Tregs isolated from beta-TM patients have poorer inhibitory performance than Tregs isolated from healthy individuals. Also, we concluded that rapamycin stabilizes the Treg population by inhibiting the production of IL-17, all necessitating the administration of appropriate immunosuppressive drugs in patients receiving Treg therapy., (© 2022 S. Karger AG, Basel.)

Neointimal hyperplasia is a persistent complication after vascular interventions, and it is also the leading cause of vascular graft restenosis and failure after arterial interventions, so novel treatment methods are needed to treat this complication. We hypothesized that adventitial injection of HA/SA hydrogel loaded with PLGA rapamycin nanoparticle (hydrogel-PLGA-rapamycin) could inhibit neointimal hyperplasia in a rat aortic wire injury model. The HA/SA hydrogel was fabricated by the interaction of hyaluronic acid (HA), sodium alginate (SA), and CaCO 3 ; and loaded with PLGA rapamycin nanoparticle or rhodamine uniformly. A SD rat aortic wire injury induced neointimal hyperplasia model was developed, the control group only received wire injury, the adventitial application group received 10 μL hydrogel-PLGA-rapamycin after wire injury, and the adventitial injection group received 10 μL hydrogel-PLGA-rapamycin injected into the aortic adventitia after wire injury. Tissues were harvested at day 21 and analyzed by histology and immunohistochemical staining. Hydrogel loaded with rhodamine can be successfully injected into the aortic adventitia and was encapsuled by the adventitia. The hydrogel could be seen beneath the adventitia after adventitial injection but was almost degraded at day 21. There was a significantly thinner neointima in the adventitial application group and adventitial injection group compared to the control group (p = 0.0009). There were also significantly fewer CD68 + (macrophages) cells (p = 0.0012), CD3 + (lymphocytes) cells (p = 0.0011), p-mTOR + cells (p = 0.0019), PCNA + cells (p = 0.0028) in the adventitial application and adventitial injection groups compared to the control group. The endothelial cells expressed arterial identity markers (Ephrin-B2 and dll-4) in all these three groups. Adventitial injection of hydrogel-PLGA-rapamycin can effectively inhibit neointimal hyperplasia after rat aortic wire injury. This may be a promising drug delivery method and therapeutic choice to inhibit neointimal hyperplasia after vascular interventions., (© 2022. Controlled Release Society.)

Skeletal muscle weakness has been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and is accompanied by altered mammalian target of rapamycin (mTOR) signalling. We wanted to elucidate the functional role of mTOR in muscle contractility. Most loss-of-function studies for mTOR signalling have used the drug rapamycin to inhibit some of the signalling downstream of mTOR. However, given that rapamycin does not inhibit all mTOR signalling completely, we generated a double knockout for mTOR and for the scaffold protein of mTORC1, raptor, in skeletal muscle. We found that double knockout in mice results in a more severe phenotype compared with deletion of raptor or mTOR alone. Indeed, these animals display muscle weakness, increased fibre denervation and a slower muscle relaxation following tetanic stimulation. This is accompanied by a shift towards slow-twitch fibres and changes in the expression levels of calcium-related genes, such as Serca1 and Casq1. Double knockout mice show a decrease in calcium decay kinetics after tetanus in vivo, suggestive of a reduced calcium reuptake. In addition, RNA sequencing analysis revealed that many downregulated genes, such as Tcap and Fhod3, are linked to sarcomere organization. These results suggest a key role for mTOR signalling in maintaining proper fibre relaxation in skeletal muscle. KEY POINTS: Skeletal muscle wasting and weakness have been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and are accompanied by altered mammalian target of rapamycin (mTOR) signalling. Mammalian target of rapamycin plays a crucial role in the maintenance of muscle mass and functionality. We found that the loss of both mTOR and raptor results in contractile abnormalities, with severe muscle weakness and delayed relaxation following tetanic stimulation. These results are associated with alterations in the expression of genes involved in sarcomere organization and calcium handling and with an impairment in calcium reuptake after contraction. Taken together, these results provide a mechanistic insight into the role of mTOR in muscle contractility., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)

Plants can adjust their growth to specific times of the day and season. Different photoperiods result in distinct growth patterns, which correlate with specific carbon-partitioning strategies in source (leaves) and sink (roots) organs. Therefore, external cues such as light, day length, and temperature need to be integrated with intracellular processes controlling overall carbon availability and anabolism. The target of rapamycin (TOR) pathway is a signalling hub where environmental signals, circadian information, and metabolic processes converge to regulate plant growth. TOR complex mutants display altered patterns of root growth and starch levels. Moreover, depletion of TOR or reduction in cellular energy levels affect the pace of the clock by extending the period length, suggesting that this pathway could participate in circadian metabolic entrainment. However, this seems to be a mutual interaction, since the TOR pathway components are also under circadian regulation. These results strengthen the role of this signalling pathway as a master sensor of metabolic status, integrating day length and circadian cues to control anabolic processes in the cell, thus promoting plant growth and development. Expanding this knowledge from Arabidopsis thaliana to crops will improve our understanding of the molecular links connecting environmental perception and growth regulation under field conditions., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Microalgae constitute a highly diverse group of photosynthetic microorganisms that are widely distributed on Earth. The rich diversity of microalgae arose from endosymbiotic events that took place early in the evolution of eukaryotes and gave rise to multiple lineages including green algae, the ancestors of land plants. In addition to their fundamental role as the primary source of marine and freshwater food chains, microalgae are essential producers of oxygen on the planet and a major biotechnological target for sustainable biofuel production and CO2 mitigation. Microalgae integrate light and nutrient signals to regulate cell growth. Recent studies identified the target of rapamycin (TOR) kinase as a central regulator of cell growth and a nutrient sensor in microalgae. TOR promotes protein synthesis and regulates processes that are induced under nutrient stress such as autophagy and the accumulation of triacylglycerol and starch. A detailed analysis of representative genomes from the entire microalgal lineage revealed that the highly conserved central components of the TOR pathway are likely to have been present in the last eukaryotic common ancestor, and the loss of specific TOR signaling elements at an early stage in the evolution of microalgae. Here we examine the evolutionary conservation of TOR signaling components in diverse microalgae and discuss recent progress of this signaling pathway in these organisms., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

In nature, plants cope with adversity and have established strategies that recall past episodes and enable them to better cope with stress recurrences by establishing a 'stress memory'. Emerging evidence suggests that glucose (Glc) and target of rapamycin (TOR), central regulators of plant growth, have remarkable functions in stress adaptation. However, whether TOR modulates a stress memory response is so far unknown. Global transcriptome profiling identified that Glc, through TOR, regulates the expression of numerous genes involved in thermomemory. Priming of TOR overexpressors with mild heat showed better stress endurance, whereas TOR RNAi showed reduced thermomemory. This thermomemory is linked with histone methylation at specific sites of heat stress (HS) genes. TOR promotes long-term accumulation of H3K4me3 on thermomemory-associated gene promoters, even when transcription of those genes reverts to their basal level. Our results suggest that ARABIDOPSIS TRITHORAX 1 (ATX1), an H3K4 methyltransferase already shown to regulate H3K4me3 levels at the promoters of HS recovery genes, is a direct target of TOR signaling. The TOR-activating E2Fa binds to the promoter of ATX1 and regulates its expression, which ultimately regulates thermomemory. Collectively, our findings reveal a mechanistic framework in which Glc-TOR signaling determines the integration of stress and energy signaling to regulate thermomemory., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Prolonged inactivity of skeletal muscles due to limb immobilization, bedrest, and exposure to microgravity results in a significant muscle atrophy. Inactivity-induced muscle atrophy is caused by a downregulation of protein synthesis (PS) and increased proteolysis. Mechanistic target of rapamycin complex 1 (mTORC1) is considered to be one of the main regulators of translational capacity (quantity of ribosomes), a key determinant of PS. Using a specific mTORC1 inhibitor (rapamycin) we aimed to determine if mTORC1 activity would influence ribosome biogenesis in rat soleus muscle at both early and later stages of mechanical unloading. Wistar rats were subjected to 1- and 7-day hindlimb suspension (HS) with and without rapamycin injections (1.5 mg/kg) and compared to weight-bearing control animals. The key markers of ribosome biogenesis were assessed by RT-PCR or agarose gel electrophoresis. The rate of PS was measured by SUnSET method. Both 1-day and 7-day HS resulted in a significant downregulation of ribosome biogenesis markers (c-Myc, 47S pre-rRNA, 18S + 28S rRNAs) and the rate of PS. Rapamycin administration during 1-day HS fully prevented a decrease in 47S pre-rRNA expression and amount of 18S + 28S rRNAs (without affecting c-Myc mRNA expression) and partially attenuated a decline in PS. Rapamycin treatment during 7-day HS significantly decreased p70S6K phosphorylation but failed to rescue a reduction in both the markers of ribosome biogenesis and the rate of PS. All together, our results suggest that mTORC1 inhibition at the initial (1 day), but not later (7 days) stage of HS can be beneficial for the maintenance of translational capacity (ribosome biogenesis) and the rate of PS in rat soleus muscle., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

To survive and sustain growth, sessile plants have developed sophisticated internal signalling networks that respond to various external and internal cues. Despite the central roles of nutrient and hormone signaling in plant growth and development, how hormone-driven processes coordinate with metabolic status remains largely enigmatic. Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, hormones, and stress signals to promote growth in all eukaryotes. Inspired by recent comprehensive systems, chemical, genetic, and genomic studies on TOR in plants, this review discusses a potential role of TOR as a 'global positioning system' that directs plant growth and developmental programs both temporally and spatially by integrating dynamic information in the complex nutrient and hormonal signaling networks. We further evaluate and depict the possible functional and mechanistic models for how a single protein kinase, TOR, is able to recognize, integrate, and even distinguish a plethora of positive and negative input signals to execute appropriate and distinct downstream biological processes via multiple partners and effectors., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

mRNA translation is the growth rate-limiting step in genome expression. Target of rapamycin (TOR) evolved a central regulatory role in eukaryotes as a signaling hub that monitors nutrient availability to maintain homeostasis and promote growth, largely by increasing the rate of translation initiation and protein synthesis. The dynamic pathways engaged by TOR to regulate translation remain debated even in well-studied yeast and mammalian models, however, despite decades of intense investigation. Recent studies have firmly established that TOR also regulates mRNA translation in plants through conserved mechanisms, such as the TOR-LARP1-5'TOP signaling axis, and through pathways specific to plants. Here, we review recent advances in our understanding of the regulation of mRNA translation in plants by TOR., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Autophagy plays an important role in mammalian oocyte maturation and early embryonic development and rapamycin is well known for inducing autophagy. Although previous studies have reported the effects of rapamycin on oocytes in vitro maturation (IVM) in different species, few studies have been reported on the role of rapamycin in yak oocytes IVM and embryonic development. Therefore, the objective of this study was to examine the effect of rapamycin treatment on yak oocytes IVM and early embryonic development. Specifically, immature yak oocytes during IVM or parthenogenetic (PA) embryos were treated with different rapamycin concentrations to select an optimal dose. Then evaluated its effect on maturation rates, cleavage, and blastocyst formation rates, mitochondrial membrane potential, ROS levels. Related genes and proteins expression in matured oocytes and blastocysts were also evaluated. The results show that 10 nM rapamycin treatment during IVM significantly improved oocyte maturation rates of oocytes and blastocyst formation rates. Treatment with 10 nM rapamycin reduced ROS level but increased mitochondrial membrane potential. Correspondingly, mRNA and protein expressions of LC3, Beclin-1, and Bcl-2 up-regulated while Bax down-regulated in matured yak COCs. When parthenogenetic embryos were treated with different rapamycin concentrations, 10 nM rapamycin treatment showed higher 8-cell and blastocyst formation rates. Also, CDX2, POU5F1, SOX2, and Nanog levels in blastocysts were upregulated. In summary, our findings demonstrate that rapamycin treatment improves oocytes maturation probably by increasing mitochondrial membrane potential, reducing ROS levels, and regulating the apoptosis in mature yak oocytes. Rapamycin treatment also improves embryonic developmental competence in the yak., Competing Interests: Declaration of competing interest The authors hereby declared that there were no commercial or other associations that might have posed a conflict of interest regarding research and publication of the manuscript., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Aim: To investigate cardiac signalling pathways connecting substrate utilization with left ventricular remodelling in a murine pressure overload model., Methods: Cardiac hypertrophy was induced by transverse aortic constriction surgery in 20-week-old C57BL/6J mice treated with or without the sodium-glucose co-transporter 2 (SGLT2) inhibitor ertugliflozin (225 mg kg -1 chow diet) for 10 weeks., Results: Ertugliflozin improved left ventricular function and reduced myocardial fibrosis. This occurred simultaneously with a fasting-like response characterized by improved glucose tolerance and increased ketone body concentrations. While cardiac insulin signalling was reduced in response to SGLT2 inhibition, AMP-activated protein kinase (AMPK) signalling was increased with induction of the fatty acid transporter cluster of differentiation 36 and phosphorylation of acetyl-CoA carboxylase (ACC). Further, enzymes responsible for ketone body catabolism (β-hydroxybutyrate dehydrogenase, succinyl-CoA:3-oxoacid-CoA transferase and acetyl-CoA acetyltransferase 1) were induced by SGLT2 inhibition. Ertugliflozin led to more cardiac abundance of fatty acids, tricarboxylic acid cycle metabolites and ATP. Downstream mechanistic target of rapamycin (mTOR) pathway, relevant for protein synthesis, cardiac hypertrophy and adverse cardiac remodelling, was reduced by SGLT2 inhibition, with alleviation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) providing a potential mechanism for abundant reduced left ventricular apoptosis and fibrosis., Conclusion: SGLT2 inhibition reduced left ventricular fibrosis in a murine model of cardiac hypertrophy. Mechanistically, this was associated with reduced cardiac insulin and increased AMPK signalling as a potential mechanism for less cardiac mTOR activation with alleviation of downstream ER stress, UPR and apoptosis., (© 2022 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

The iron (Fe) metabolism plays important role in regulating systemic metabolism and obesity development. The Fe inside cells can form iron-sulfur (Fe-S) clusters, which are usually assembled into target proteins with the help of a conserved cluster assembly machinery. Family with sequence similarity 96A (FAM96A; also designated CIAO2A) is a cytosolic Fe-S assembly protein involved in the regulation of cellular Fe homeostasis. However, the biological function of FAM96A in vivo is still incompletely defined. Here, we tested the role of FAM96A in regulating organismal Fe metabolism, which is relevant to obesity and adipose tissue homeostasis. We found that in mice genetically lacking FAM96A globally, intracellular Fe homeostasis was interrupted in both white and brown adipocytes, but the systemic Fe level was normal. FAM96A deficiency led to adipocyte hypertrophy and organismal energy expenditure reduction even under nonobesogenic normal chow diet-fed conditions. Mechanistically, FAM96A deficiency promoted mechanistic target of rapamycin (mTOR) signaling in adipocytes, leading to an elevation of de novo lipogenesis and, therefore, fat mass accumulation. Furthermore, it also caused mitochondrial defects, including defects in mitochondrial number, ultrastructure, redox activity, and metabolic function in brown adipocytes, which are known to be critical for the control of energy balance. Moreover, adipocyte-selective FAM96A knockout partially phenocopied global FAM96A deficiency with adipocyte hypertrophy and organismal energy expenditure defects but the mice were resistant to high-fat diet-induced weight gain. Thus, FAM96A in adipocytes may autonomously act as a critical gatekeeper of organismal energy balance by coupling Fe metabolism to adipose tissue homeostasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Many circular RNAs (circRNAs) involved in the osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) have recently been discovered. The role of circHIPK3 in osteogenesis has yet to be determined. Cell transfection was conducted using small-interfering RNAs (siRNAs). Expression of osteogenic markers were detected by quantitative reverse transcription-polymerase chain reaction, western blotting analysis, and immunofluorescence staining. Ectopic bone formation models in nude mice were used to examined the bone formation ability in vivo. The autophagy flux was examined via western blotting analysis, immunofluorescence staining and transmission electron microscopy analysis. RNA immunoprecipitation (RIP) analysis was carried out to analyze the binding between human antigen R (HUR) and circHIPK3 or autophagy-related 16-like 1 (ATG16L1). Actinomycin D was used to determine the mRNA stability. Our results demonstrated that silencing circHIPK3 promoted the osteogenesis of hBMSCs while silencing the linear mHIPK3 did not affect osteogenic differentiation, both in vivo and in vitro. Moreover, we found that knockdown of circHIPK3 activated autophagy flux. Activation of autophagy enhanced the osteogenesis of hBMSCs and inhibition of autophagy reduced the osteogenesis through using autophagy regulators chloroquine and rapamycin. We also discovered that circHIPK3 and ATG16L1 both bound to HUR. Knockdown of circHIPK3 released the binding sites of HUR to ATG16L1, which stabilized the mRNA expression of ATG16L1, resulting in the upregulation of ATG16L1 and autophagy activation. CircHIPK3 functions as an osteogenesis and autophagy regulator and has the potential for clinical application in the future., (© 2022 Federation of American Societies for Experimental Biology.)

Putrescine (Put) has been shown to play an important regulatory role in cell growth in organisms. As the primary center regulating the homeostasis of polyamine (PA) content, ornithine decarboxylase antizyme (AZ) can regulate PA content through feedback. Nevertheless, the regulatory mechanism of Put is poorly understood in fungi. Here, our analysis showed that GlAZ had a modulate effect on intracellular Put content by interacting with ornithine decarboxylase (ODC) proteins and reducing its intracellular protein levels. In addition, GlAZ upregulated the metabolic pathway of ganoderic acid (GA) biosynthesis in Ganoderma lucidum by modulating the intracellular Put content. However, a target of rapamycin (TOR) was found to promote the accumulation of intracellular Put after the GlTOR inhibitor Rap was added exogenously, and unbiased analyses demonstrated that GlTOR may promote Put production through its inhibitory effect on the level of GlAZ protein in GlTOR-GlAZ -cosilenced strains. The effect of TOR on fungal secondary metabolism was further explored, and the content of GA in the GlTOR -silenced strain after the exogenous addition of the inhibitor Rap was significantly increased compared with that in the untreated wild-type (WT) strain. Silencing of TOR in the GlTOR -silenced strains caused an increase in GA content, which returned to the WT state after replenishing Put. Moreover, the content of GA in GlTOR-GlAZ -cosilenced strains was also not different from that in the WT strain. Consequently, these results strongly indicate that GlTOR affects G. lucidum GA biosynthesis via GlAZ. IMPORTANCE Research on antizyme (AZ) in fungi has focused on the mechanism by which AZ inhibits ornithine decarboxylase (ODC). Moreover, there are existing reports on the regulation of AZ protein translation by TOR. However, little is known about the mechanisms that influence AZ in fungal secondary metabolism. Here, both intracellular Put content and GA biosynthesis in G. lucidum were shown to be regulated through protein interactions between GlAZ and GlODC. Furthermore, exploration of upstream regulators of GlAZ suggested that GlAZ was regulated by the upstream protein GlTOR, which affected intracellular Put levels and ganoderic acid (GA) biosynthesis. The results of our work contribute to the understanding of the upstream regulation of Put and provide new insights into PA regulatory systems and secondary metabolism in fungi.