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Osteoarthritis (OA) is a common joint degenerative disease which currently lacks satisfactory disease-modifying treatments. Oxidative stress-mediated senescent chondrocytes accumulation is closely associated with OA progression, which abrogates cartilage metabolism homeostasis by secreting senescence-associated secretory phenotype (SASP) factors. Numerous studies suggested mesenchymal stem cells-derived small extracellular vesicles (MSC-sEVs) have been regarded as promising candidates for OA therapy. However, MSC-sEVs were applied before the occurrence of cartilage degeneration or at early-stage OA, while in clinical practice, most OA patients who present with pain are already in non-early-stage. Recently, embryonic stem cells-derived sEVs (ESC-sEVs) have been reported to possess powerful anti-aging effects. However, whether ESC-sEVs could attenuate non-early-stage OA progression remains unknown. In this study, we demonstrated ESC-sEVs ameliorated senescent phenotype and cartilage destruction in both mechanical stress-induced non-early-stage posttraumatic OA and naturally aged mice. More importantly, we found ESC-sEVs alleviated senescent phenotype by rejuvenating aged chondrocytes but not inducing apoptosis. We also provided evidence that the FOXO1A-autophagy axis played an important role in the anti-aging effects of ESC-sEVs. To promote clinical translation, we confirmed ESC-sEVs reversed senescent phenotype in ex-vivo cultured human end-stage OA cartilage explants. Collectively, our findings reveal that ESC-sEVs-based therapy is of high translational value in non-early-stage OA treatment.

Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar-sweetened beverages. Nevertheless, the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. We have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose, resembling the hyperangiogenic characteristics observed in proliferative diabetic retinopathy (PDR). Utilizing this model, we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels, especially intersegmental vessels (ISVs). The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells (ECs) into tip cells. Single-cell transcriptomic sequencing analysis of the ECs in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs, proliferating ECs, and a series of upregulated proangiogenic genes. Further analysis and experiments validated that reduced foxo1a mediated the excessive angiogenesis induced by monosaccharides via upregulating the expression of marcksl1a. This study has provided new evidence showing the negative effects of noncaloric monosaccharides on the vascular system and the underlying mechanisms.

FoxO1 (Forkhead box O1) belongs to the evolutionarily conserved FoxO subfamily and is involved in diverse physiologic processes, including apoptosis, cell cycle, DNA damage repair, oxidative stress and cell differentiation. FoxO1 plays an important role in regulating the hypoxia microenvironment such as cancers, but its role in hypoxia adaptation remains unclear in animals. To understand the function of foxO1 in hypoxia response, we constructed foxO1a and foxO1b mutant zebrafish using CRISPR/Cas9 technology. It was found that foxO1a and foxO1b destruction affected the hematopoietic system in the early zebrafish embryos. Specifically, FoxO1a and FoxO1b were found to affect the transcriptional activity of runx1 , a marker gene for hematopoietic stem cells (HSCs). Moreover, foxO1a and foxO1b had complementary features in hypoxia response, and foxO1a or/and foxO1b destruction resulted in tolerance of zebrafish becoming weakened in hypoxia due to insufficient hemoglobin supply. Additionally, the transcriptional activity of these two genes was demonstrated to be regulated by Hif1α. In conclusion, foxO1a and foxO1b respond to Hif1α-mediated hypoxia response by participating in zebrafish erythropoiesis. These results will provide a theoretical basis for further exploring the function of FoxO1 in hematopoiesis and hypoxia response. [Display omitted] [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a common joint degenerative disease which currently lacks satisfactory disease-modifying treatments. Oxidative stress-mediated senescent chondrocytes accumulation is closely associated with OA progression, which abrogates cartilage metabolism homeostasis by secreting senescence-associated secretory phenotype (SASP) factors. Numerous studies suggested mesenchymal stem cells-derived small extracellular vesicles (MSC-sEVs) have been regarded as promising candidates for OA therapy. However, MSC-sEVs were applied before the occurrence of cartilage degeneration or at early-stage OA, while in clinical practice, most OA patients who present with pain are already in non-early-stage. Recently, embryonic stem cells-derived sEVs (ESC-sEVs) have been reported to possess powerful anti-aging effects. However, whether ESC-sEVs could attenuate non-early-stage OA progression remains unknown. In this study, we demonstrated ESC-sEVs ameliorated senescent phenotype and cartilage destruction in both mechanical stress-induced non-early-stage posttraumatic OA and naturally aged mice. More importantly, we found ESC-sEVs alleviated senescent phenotype by rejuvenating aged chondrocytes but not inducing apoptosis. We also provided evidence that the FOXO1A-autophagy axis played an important role in the anti-aging effects of ESC-sEVs. To promote clinical translation, we confirmed ESC-sEVs reversed senescent phenotype in ex-vivo cultured human end-stage OA cartilage explants. Collectively, our findings reveal that ESC-sEVs-based therapy is of high translational value in non-early-stage OA treatment. [Display omitted] • ESC-sEVs reversed senescent phenotype in posttraumatic OA, naturally aged mice, and cultured human OA cartilage • ESC-sEVs attenuated senescent phenotype by rejuvenating chondrocytes but not inducing apoptosis • The FOXO1A-autophagy axis played an important role in the anti-aging effects of ESC-sEVs • ESC-sEVs-based therapy is of high translational value in intervention for non-early-stage OA. [ABSTRACT FROM AUTHOR]

Proteins are the major and most diverse biomolecules, directing all activities of a cell. For this reason, visualizing protein expression, localization, and dynamics is fundamental in biology. In most cases, protein visualization relies on the overexpression of fluorescently tagged proteins which may not recapitulate endogenous expression pattern and dynamics. Henceforth, tagging proteins in the endogenous locus is the most accurate way to recapitulate physiological gene expression. However, this method is not widely implemented for the FOXO gene family due to its technical inefficiency and difficulty. Here we describe the methodology followed to generate a knock-in reporter line for the Foxo1a transcription factor for the zebrafish model system. We describe insertion of an EGFP-polyA cassette in frame at the C-terminal of Foxo1a, generating a fusion protein. Foxo1a has been involved in the regulation of metabolism, stress response, longevity, and cell differentiation, and its functions are conserved from invertebrates to vertebrates. Using in vivo confocal live microscopy at early developmental stages, we validated the expression of Foxo1a in the cardiovascular network, central nervous system, olfactory epithelium, spinal cord, retina, skeletal muscle, and myocardium. This knock-in line opens the way for imaging studies aiming to characterize the expression and localization (cytoplasmic or nuclear) of this transcription factor in a tissue- and context-specific manner, as well as the dynamics of stress adaptation at a whole organism level. Moreover, the knock-in line can be used in combination with other modern techniques such as Cut&Run to determine the transcriptional targets of Foxo1a, with a GFP-directed proteomic to identify interacting partners, many of which remain largely unknown., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar-sweetened beverages. Nevertheless, the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. We have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose, resembling the hyperangiogenic characteristics observed in proliferative diabetic retinopathy (PDR). Utilizing this model, we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels, especially intersegmental vessels (ISVs). The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells (ECs) into tip cells. Single-cell transcriptomic sequencing analysis of the ECs in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs, proliferating ECs, and a series of upregulated proangiogenic genes. Further analysis and experiments validated that reduced foxo1a mediated the excessive angiogenesis induced by monosaccharides via upregulating the expression of marcksl1a . This study has provided new evidence showing the negative effects of noncaloric monosaccharides on the vascular system and the underlying mechanisms., Competing Interests: XW, JZ, JX, BL, XL, GX, XD, DL No competing interests declared, (© 2024, Wang, Zhao, Xu et al.)

Foxo1, a member of Foxo transcription factor family, is involved in a number of physiological processes including metabolism, cell cycle progression, aging, and apoptosis. In the ovarian granulosa cell of mouse, Foxo1 is implicated to inhibit the expression of Cyp19a1, a gene encoding the aromatase that converts androgens into estrogens. Currently, the information about the expression and physiological relevance of Foxo1 homologues in the ovary of teleosts is scarce. In the present study, cDNAs encoding two forms of Foxo1, Foxo1a and Foxo1b, were isolated from the orange-spotted grouper. Phylogenetic analysis indicated that the orange-spotted groupers Foxo1a and Foxo1b were closely related to the counterparts of the ricefield eel. RT-PCR analysis showed that the orange-spotted groupers foxo1a and foxo1b were expressed in a wide range of tissues, with high levels detected in the brain regions, liver, and intestine. Quantitative real-time PCR analysis showed similar expression profiles for cyp19a1a, foxo1a, and foxo1b in the ovary during development from the primary growth to mature stages, with peak values detected at the vitellogenic stage. In situ hybridization detected mRNA of foxo1a, foxo1b, and cyp19a1a in granulosa cells surrounding vitellogenic oocytes. In vitro transfection showed that both Foxo1a and Foxo1b upregulated the orange-spotted grouper cyp19a1a promoter activities, possibly through the conserved Foxo binding site. Collectively, these results suggest that both Foxo1a and Foxo1b may be involved in the regulation of the ovarian functions in the orange-spotted grouper and the physiological roles of Foxo1 homologues in the ovary may be diversified in vertebrates. [ABSTRACT FROM AUTHOR]

Cadmium (Cd), a type of heavy metal that accumulates in the body because of smoking, mediates the toxic effect of smoking in many diseases, such as cardiovascular disease, osteoarthritis, and osteoporosis. However, the toxic effect of Cd on intervertebral disc tissues have not been reported. In the current study, we demonstrated that Cd induced the apoptosis of annulus fibrosus (AF) cells, which contributed to intervertebral disc degeneration (IVDD). Specifically, Cd induced the nuclear translocation of FoxO1a, which drives AF cells apoptosis through mitochondrial‐related pathway. Phosphatidylinositol 3‐kinase/protein kinase B (PI3K/AKT) signal pathway is also involved in this process. The combined use of LY29002, an inhibitor of PI3K, and small interfering RNA‐targeting FoxO1a confirmed the relationship between the PI3K/AKT signal pathway and FoxO1a. In summary, present research explores the mechanism behind the contribution of smoking to IVDD and finds a new feasible target for preventing IVDD in smoking. [ABSTRACT FROM AUTHOR]

The skeletal muscle mass reduces 30–60% after spinal cord injury, this is mostly due to protein degradation through ubiquitin–proteasome system. In this work, we propose that the flavanol (−)-epicatechin, due its widespread biological effects on muscle health, can prevent muscle mass decrease after spinal cord injury. Thirty-six female Long Evans rats were randomized into 5 groups: (1) Spinal cord injury 7 days, (2) Spinal cord injury + (−)-epicatechin 7 days, (3) Spinal cord injury 30 days, (4) Spinal cord injury + (−)-epicatechin 30 days and (5) Sham (Only laminectomy). Hind limb perimeter, muscle cross section area, fiber cross section area and ubiquitin–proteasome system protein expression together with total protein ubiquitination were assessed. At 30 days Spinal cord injury group lost 49.52 ± 2.023% of muscle cross section area (−)-epicatechin treated group lost only 24.28 ± 15.45% being a significant difference. Ubiquitin–proteasome markers showed significant changes. FOXO1a increased in spinal cord injury group vs Sham (−)-epicatechin reduced this increase. In spinal cord injury group MAFbx increased significantly vs Sham but decrease in (−)-epicatechin treatment group at 30 days. At 7 and 30 days MuRF1 increased in the spinal cord injury and decreased in the (−)-epicatechin group. The global protein ubiquitination increases after spinal cord injury, epicatechin treatment induce a significant decrease in protein ubiquitination. These results suggest that (−)-epicatechin reduces the muscle waste after spinal cord injury through down regulation of the ubiquitin–proteasome system. [ABSTRACT FROM AUTHOR]

Abstract To investigate the regulatory effect of miR-34a on the sirtuin 1(sirt1) / forkhead box O 1 (foxo1) axis in Megalobrama amblycephala under high carbohydrate metabolism, M. amblycephala fish were fed either a control or a high glucose diet for eight weeks. Metabolic models of the high glucose group, normal glucose group, miR-34a inhibition group, and miR-34a overexpression group were constructed using miR-34a inhibitor and miR-34a mimic injection (where appropriate) in M. amblycephala. The results showed that the hepatic expression of SIRT1 was decreased by the high glucose diet after eight weeks, which promoted the expression of foxo1a. Additionally, hepatic pepck and g6pase expression were upregulated, thereby promoting gluconeogenesis. The upregulation of pparα increased the expression of lipid metabolic enzymes such as fas , lps , and acc ; therefore, excessive glycogen was deposited as lipid in the adipose tissue. Furthermore, we found that SIRT1 expression increased following miR-34a inhibitor injection, activating foxo1a expression levels, reducing hepatic gluconeogenesis, decreasing hepatic glycogen accumulation, and accelerating hepatic fat decomposition and fatty acid synthesis. In a nutrient metabolic homeostatic state, injection of miR-34a mimics promoted the expression of foxo1a and pepeck , enhanced fat metabolism and fatty acid synthesis, and inhibited SIRT1 protein expression. These results revealed that miR-34a mediates regulation of metabolic enzymes related to hepatic glucose and lipid metabolism by targeting SIRT1/FoxO1 in M. amblycephala under high carbohydrate diet. Highlights • MiR-34a silencing and overexpression models were constructed in M. amblycephala. • MiR-34a negatively regulated hepatic SIRT1 expression. • SIRT1 inhibited gluconeogenesis and enhanced lipolysis by increasing foxo1 expression. • MiR-34a regulates hepatic glycolipid metabolism by targeting the Sirt1/Foxo1 pathway. [ABSTRACT FROM AUTHOR]

Abstract We have previously published that miR15a can reduce inflammatory cytokines, which could be key to diabetic retinal pathology. In this work, we wanted to investigate whether miR15a altered NLR pyrin domain 3 (NLRP3) proteins. Whole retinal lysates from both miR15a overexpressing mice and endothelial cell specific miR15a/16 knockout mice were used to investigate protein levels of forkhead box protein O1 (Foxo1), NLRP3, cleaved caspase 1 and interleukin-1 beta (IL-1β). Primary human retinal endothelial cells (REC) were cultured in normal and high glucose followed by transfection with a miR15a mimic for protein analyses. miR15a expression was verified by quantitative PCR, and a luciferase binding assay was used to examine whether miR15a directly bound Foxo1. In mouse retinal lysates, loss of miR15a increased Foxo1, IL-1β, NLRP3, and cleaved caspase 1 levels. REC grown in high glucose transfected with the miR15a mimic had decreased levels of Foxo1 and NLRP3. miR15a directly binds to Foxo1. miR15a regulates NLRP3 actions in the retinal vasculature. Work in mice showed that loss of miR15a increased NLRP3 pathway signaling and Foxo1. miR15a mimics decreased levels of Foxo1 and NLRP3. Taken together, miR15a reduced inflammasome proteins and Foxo1 levels in the retinal vasculature. Highlights • miR15a decreases inflammasome proteins in retinal endothelial cells grown in high glucose. • Loss of miR15a in mice lead to increased NLRP3 inflammasome proteins. • miR15a binds to Foxo1, leading to decreased inflammatory mediators. [ABSTRACT FROM AUTHOR]

After incomplete spinal cord injury (SCI), patients and animals may exhibit some spontaneous functional recovery which can be partly attributed to remodeling of injured neural circuitry. This post-lesion plasticity implies spinal remodeling but increasing evidences suggest that supraspinal structures contribute also to the functional recovery. Here we tested the hypothesis that partial SCI may activate cell-signaling pathway(s) at the supraspinal level and that this molecular response may contribute to spontaneous recovery. With this aim, we used a rat model of partial cervical hemisection which injures the bulbospinal respiratory tract originating from the medulla oblongata of the brainstem but leads to a time-dependent spontaneous functional recovery of the paralyzed hemidiaphragm. We first demonstrate that after SCI the PI3K/Akt signaling pathway is activated in the medulla oblongata of the brainstem, resulting in an inactivation of its pro-apoptotic downstream target, forkhead transcription factor (FKHR/FOXO1A). Retrograde labeling of medullary premotoneurons including respiratory ones which project to phrenic motoneurons reveals an increased FKHR phosphorylation in their cell bodies together with an unchanged cell number. Medulla infusion of the PI3K inhibitor, LY294002, prevents the SCI-induced Akt and FKHR phosphorylations and activates one of its death-promoting downstream targets, Fas ligand. Quantitative EMG analyses of diaphragmatic contractility demonstrate that the inhibition of medulla PI3K/Akt signaling prevents spontaneous respiratory recovery normally observed after partial cervical SCI. Such inhibition does not however affect either baseline contractile frequency or the ventilatory reactivity under acute respiratory challenge. Together, these findings provide novel evidence of supraspinal cellular contribution to the spontaneous respiratory recovery after partial SCI.

Study Question: Can bioinformatics analysis of publically available microarray datasets be utilized in identifying potentially important transcription factors (TF) in the hormonal regulation of the endometrium?Summary Answer: Systems integration and analysis of existing complex (published) datasets, predicted a role for the novel transcription factor, Forkhead Box D3 (FOXD3) in healthy endometrium and in endometriosis, which was followed by the demonstration of decreased levels of the protein upon decidualisation of normal human endometrial stromal cells in vitro and differential endometrial expression in the stroma in endometriosis.What Is Known Already: The reported endometriosis-associated endometrial aberrations are most pronounced in the progesterone-dominant secretory phase and progesterone resistance is a proposed causative factor.Study Design, Size, Duration: The study was initially an 'in silico' study, with confirmatory 'wet lab' data from western blotting (WB), qPCR and Immunohistochemistry (IHC) on endometrial biopsies obtained from 142 women undergoing gynaecological surgery.Participants/materials, Setting, Methods: The study was conducted at a University Research Institute. Bioinformatic analysis of selected published microarray datasets identified differentially regulated genes for the early and mid-secretory phases relative to the proliferative phase. Diseases and Functions categories were identified with Ingenuity (IPA) 'core analysis' software. The key transcription factors controlling secretory phase gene changes were revealed with oPOSSUM software. FOXD3 expression levels were examined in human endometrial samples from women aged 18-55 years by WB, IHC, and qPCR. The progesterone regulation of endometrial FOXD3 levels was examined in vivo and in cultured primary human endometrial stromal cells in vitro.Main Results and the Role Of Chance: Initial data mining and subsequent bioinformatics analysis of human endometrial microarray datasets identified FOXD3 to be a key regulator of gene expression specific to secretory phase/endometriosis. FOXD3 was dynamically expressed in healthy endometrium and differentially expressed in endometriosis. In vitro decidualisation of primary endometrial stromal cells significantly decreased FOXD3 protein (P = 0.0005) and progestagen (Levonorgestrel) treatment also reduced the high endometrial FOXD3 protein (P = 0.0001) and mRNA levels (P = 0.04) seen in untreated women with endometriosis, with a shift of FOXD3 from the nucleus to the cytoplasm.Limitations, Reasons For Caution: The quality of Bioinformatics analysis and results depends on the published micro-array data.Wider Implications Of the Findings: An in depth analysis of FOXD3 function and its relationship with estrogen and progesterone might provide insights into its potential deregulation in proliferative disorders of the endometrium including endometrial cancer where its expression is also deregulated. Further, FOX transcription factors are increasingly seen as novel therapeutic targets in disease.Study Funding/competing Interests: We acknowledge the support by Wellbeing of Women project grant RG1073 (D.K.H., A.J.V.). We also acknowledge the support of Liverpool Women's Hospital Foundation Trust (J.A.D.), Institute of Translational Medicine (D.M., A.J.V., D.K.H.) and the Institute of Integrative Biology (O.V.), University of Liverpool. All authors declare no conflict of interest.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

After incomplete spinal cord injury (SCI), patients and animals may exhibit some spontaneous functional recovery which can be partly attributed to remodeling of injured neural circuitry. This post-lesion plasticity implies spinal remodeling but increasing evidences suggest that supraspinal structures contribute also to the functional recovery. Here we tested the hypothesis that partial SCI may activate cell-signaling pathway(s) at the supraspinal level and that this molecular response may contribute to spontaneous recovery. With this aim, we used a rat model of partial cervical hemisection which injures the bulbospinal respiratory tract originating from the medulla oblongata of the brainstem but leads to a time-dependent spontaneous functional recovery of the paralyzed hemidiaphragm. We first demonstrate that after SCI the PI3K/Akt signaling pathway is activated in the medulla oblongata of the brainstem, resulting in an inactivation of its pro-apoptotic downstream target, forkhead transcription factor (FKHR/FOXO1A). Retrograde labeling of medullary premotoneurons including respiratory ones which project to phrenic motoneurons reveals an increased FKHR phosphorylation in their cell bodies together with an unchanged cell number. Medulla infusion of the PI3K inhibitor, LY294002, prevents the SCI-induced Akt and FKHR phosphorylations and activates one of its death-promoting downstream targets, Fas ligand. Quantitative EMG analyses of diaphragmatic contractility demonstrate that the inhibition of medulla PI3K/Akt signaling prevents spontaneous respiratory recovery normally observed after partial cervical SCI. Such inhibition does not however affect either baseline contractile frequency or the ventilatory reactivity under acute respiratory challenge. Together, these findings provide novel evidence of supraspinal cellular contribution to the spontaneous respiratory recovery after partial SCI. [ABSTRACT FROM AUTHOR]

Abstract: Selenoprotein P (SeP), serving as selenium transporter and extracellular antioxidant, is assumed to have a protective role in the gastrointestinal tract, which is particularly susceptible to oxidative damage. Decreased SeP mRNA levels have been found in colon cancer; however, information on the control of intestinal SeP biosynthesis is scarce. We analyzed SeP biosynthesis in human intestinal epithelial Caco-2 cells subject to differentiation from crypt- to villous-like enterocytes. In the course of Caco-2 cell differentiation, SeP mRNA expression and secretion increased concomitant with three regulators of SeP transcription: hepatocyte nuclear factor-4α, forkhead box class O1a, and peroxisomal proliferator-activated receptor-γ coactivator 1α. Treatment of differentiated Caco-2 cells with the proinflammatory cytokines IL-1β, TNF-α, and IFN-γ caused a down-regulation of SeP biosynthesis, resulting from induction of nitric oxide synthase 2. These observations were corroborated by decreased SeP mRNA levels in the colon of dextran sodium sulfate-treated mice, an animal model of experimental colitis. We conclude that inflammation of the intestinal mucosa causes a decline in locally produced selenoprotein P in the colon that eventually may contribute to the emergence of inflammatory bowel disease-related colorectal cancer. [Copyright &y& Elsevier]

Blood–brain barrier (BBB) is specialized to limit brain drug delivery. Cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin, could act as a diphtheria toxin receptor-specific carrier protein and deliver drugs across the BBB. CRM197 has previously been shown to inhibit phospatidyl-3-inositol-kinase (PI3K)/Akt signaling. Other studies have demonstrated a link between PI3K/Akt signaling and forkhead transcription factors in endothelial cells. We therefore investigated the effects and mechanisms underlying the potential of CRM197, not only as a carrier protein for targeted drug delivery to the brain, but also for inducing signaling to affect endocytosis in endothelial cells. The hCMEC/D3 cell line had been used to establish a BBB in vitro model; the transport efficiency of CRM197 was analyzed both by association and transcytosis experiments. CRM197 was shown to prefer apical-to-basal transcytosis, which involved the caveolae-mediated pathway. The uptake of CRM197 conjugates by endothelial cells reached equilibrium after 60 min of treatment. The caveolin-1 mRNA and protein expression levels were significantly increased by CRM197. The up-regulation of caveolin-1 may be mediated by CRM197 via a PI3K/Akt dependent pathway and reduction of the phospho-FOXO1A (forkhead box O) transcription factor. Our results indicate that carrier protein CRM197-mediated delivery across the BBB is involved in the induction of FOXO1A transcriptional activity and upregulation of caveolin-1 expression. [ABSTRACT FROM AUTHOR]

In vertebrates and invertebrates, relationships between diet and health are controlled by a conserved signalling pathway responsive to insulin-like ligands. In invertebrate models for example, forkhead transcription factor family O (FOXO) transcription factors in this pathway regulate the rate of aging in response to dietary cues, and in vertebrates, obesity and age-induced deficits in the same pathway are thought to contribute to dysregulation of hepatic gluconeogenesis through genes such as phosphoenolpyruvate carboxykinase (PEPCK). Recently, we have begun to screen for dietary constituents capable of regulating this pathway in our cell culture model. Here, we identify three black tea theaflavins, theaflavin 3- O-gallate, theaflavin 3′- O-gallate, theaflavin 3,3′di- O-gallate and thearubigins as novel mimics of insulin/IGF-1 action on mammalian FOXO1a, PEPCK and moreover we provide evidence that the effects on this pathway of the green tea constituent (-)-epigallocatechin gallate depend on its ability to be converted into these larger structures. With the exception of water, tea is the most popular drink globally, but despite this, little is known about the biological availability of black tea polyphenols in vivo or the molecular target(s) mediating the effects presented here. Further investigation in these two areas might provide insight into how age-related metabolic disease may be deferred. [ABSTRACT FROM AUTHOR]

Recently, the Daf-16 gene has been shown to regulate the lifespan of nematodes and flies. In mammals, the Daf-16 homologues are forkhead (FOXO) transcription factors, of which specific functions have been identified for Foxo1a and Foxo3a. Despite that, their influence on human age-related trajectories and lifespan is unknown. Here, we analysed the effect of genetic variance in Foxo1a and Foxo3a on metabolic profile, age-related diseases, fertility, fecundity and mortality. This study was carried out in the prospective population-based Leiden 85-plus Study, which includes 1245 participants, aged 85 years or more. The mean follow-up time was 4.4 years. Haplotype analyses of Foxo1a revealed that carriers of haplotype 3 ‘TCA’ have higher HbA1c levels (P=0.025) and a 1.14-fold higher all-cause mortality risk (P=0.021). This increase in mortality was attributable to death from diabetes, for which a 2.43-fold increase was observed (P=0.025). The analyses with Foxo3a haplotypes revealed no differences in metabolic profile, fertility or fecundity. However, increased risks of stroke were observed for Foxo3a block-A haplotype 2 ‘GAGC’ (P=0.007) and haplotype 4 ‘AAAT’ (P=0.014) carriers. In addition, the haplotype 2 ’GAGC’ carriers had a 1.13-fold increased risk for all-cause mortality (P=0.036) and 1.19-fold increased risk for cardiovascular mortality (P=0.052). In conclusion, this study shows that genetic variation in evolutionarily conserved Foxo1a and Foxo3a genes influences lifespan in our study population.European Journal of Human Genetics (2007) 15, 294–301. doi:10.1038/sj.ejhg.5201766; published online 24 January 2007 [ABSTRACT FROM AUTHOR]

Abstract: Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) cDNA was cloned from the brain of greater horseshoe bat (Rhinolophus ferrumequinum). The deduced amino acid sequence shares strong homology with these PDK4 of other mammals. Moreover, we partially cloned homologues of dual-specificity tyrosine-phosphorylated and regulated protein kinase 1A (DYRK1A), and forkhead box protein O1A (FOXO1A) from greater horseshoe bat. Among five different tissues tested, PDK4 mRNA was highly expressed in the heart, white adipose tissue and muscle, but weakly expressed in the brain and liver, while DYRK1A and FOXO1A were expressed in all five tissues. Moreover, the transcript levels of PDK4, DYRK1A, and FOXO1A were measured in the heart, white adipose tissue and muscle of hibernating and arousal greater horseshoe bats by Northern blot and real time PCR. The results showed that transcript level of PDK4 was significantly higher in white adipose tissue. Expression level of DYRK1A was significantly higher in hibernating state in white adipose tissue, and expression level of FOXO1A was significantly higher in muscle in aroused state. These results suggest that up-regulation of the transcript levels of PDK4 during hibernation were not regulated via DYRK1A and FOXO1A in white adipose tissue and muscle, and the possible presence of another isoenzyme of PDK which is responsible for the tissue-specific regulation of Pyruvate dehydrogenase complex (PDC) activity in the bat heart during hibernation. [Copyright &y& Elsevier]

Alveolar rhabdomyosarcoma is an aggressive childhood muscle cancer for which outcomes are poor when the disease is advanced. Although well-developed mouse models exist for embryonal and pleomorphic rhabdomyosarcomas, neither a spontaneous nor a transgenic mouse model of alveolar rhabdomyosarcoma has yet been reported. We report the first mouse model of alveolar rhabdomyosarcoma using a conditional Pax3:Fkhr knock-in allele whose activation in late embryogenesis and postnatally is targeted to terminally differentiating Myf6-expressing skeletal muscle. In these mice, alveolar rhabdomyosarcomas occur but at low frequency, and Fkhr haploinsufficiency does not appear to accelerate tumorigenesis. However, Pax3:Fkhr homozygosity with accompanying Ink4a/ARF or Trp53 pathway disruption, by means of conditional Trp53 or Ink4a/ARF loss of function, substantially increases the frequencies of tumor formation. These results of successful tumor generation postnatally from a target pool of differentiating myofibers are in sharp contrast to the birth defects and lack of tumors for mice with prenatal and postnatal satellite cell triggering of Pax3:Fkhr. Furthermore, these murine alveolar rhabdomyosarcomas have an immunohistochemical profile similar to human alveolar rhabdomyosarcoma, suggesting that this conditional mouse model will be relevant to study of the disease and will be useful for preclinical therapeutic testing. [ABSTRACT FROM AUTHOR]

To investigate the role of the translocation-associated gene Pax3:Fkhr in alveolar rhabdomyosarcomas, we generated a Cre-mediated conditional knock-in of Pax3:Fkhr into the mouse Pax3 locus. Exploring embryonic tumor cell origins, we replaced a Pax3 allele with Pax3:Fkhr throughout its expression domain, causing dominant-negative effects on Pax3 and paradoxical activation of the Pax3 target gene; c-Met. Ectopic neuroprogenitor cell proliferation also occurs. In contrast, activation later in embryogenesis in cells that express Pax7 results in viable animals with a postnatal growth defect and a moderately decreased Pax7+ muscle satellite cell pool, phenocopying Pax7 deficiency but remarkably not leading to tumors. [ABSTRACT FROM AUTHOR]

WT1 encodes a tissue-specific transcription factor important in early mesenchymal differentiation. Altered expression or mutation of WT1 occurs in malignancies derived from such tissues. These include Wilms tumour, a paediatric kidney cancer that may show heterologous differentiation into primitive skeletal muscle, especially in tumours with WT1 mutation. A putative role for WT1 in inhibiting myogenesis has been suggested by transient transfection of C2C12 myoblasts. However, using a more robust model of stable transfectants of C2C12 expressing inducible WT1 isoforms, we found no inhibition of myogenic differentiation. We also investigated a possible role for WT1 in the disrupted myogenesis seen in rhabdomyosarcoma, a paediatric cancer resembling foetal skeletal muscle. WT1 expression levels measured by quantitative real-time reverse transcription polymerase chain reaction were low or absent in those tumours with a PAX-FKHR fusion gene characteristic of the alveolar subtype, and were higher in cases lacking these fusion genes. Overall, there was a weak positive correlation between expression of myogenic differentiation marker genes and WT1 levels. We conclude that expression of WT1 in C2C12 cells and in rhabdomyosarcoma does not inhibit myogenic differentiation. [Copyright &y& Elsevier]

The skeletal muscle mass reduces 30-60% after spinal cord injury, this is mostly due to protein degradation through ubiquitin-proteasome system. In this work, we propose that the flavanol (-)-epicatechin, due its widespread biological effects on muscle health, can prevent muscle mass decrease after spinal cord injury. Thirty-six female Long Evans rats were randomized into 5 groups: (1) Spinal cord injury 7 days, (2) Spinal cord injury + (-)-epicatechin 7 days, (3) Spinal cord injury 30 days, (4) Spinal cord injury + (-)-epicatechin 30 days and (5) Sham (Only laminectomy). Hind limb perimeter, muscle cross section area, fiber cross section area and ubiquitin-proteasome system protein expression together with total protein ubiquitination were assessed. At 30 days Spinal cord injury group lost 49.52 ± 2.023% of muscle cross section area (-)-epicatechin treated group lost only 24.28 ± 15.45% being a significant difference. Ubiquitin-proteasome markers showed significant changes. FOXO1a increased in spinal cord injury group vs Sham (-)-epicatechin reduced this increase. In spinal cord injury group MAFbx increased significantly vs Sham but decrease in (-)-epicatechin treatment group at 30 days. At 7 and 30 days MuRF1 increased in the spinal cord injury and decreased in the (-)-epicatechin group. The global protein ubiquitination increases after spinal cord injury, epicatechin treatment induce a significant decrease in protein ubiquitination. These results suggest that (-)-epicatechin reduces the muscle waste after spinal cord injury through down regulation of the ubiquitin-proteasome system.

Study Question: How do NR4A receptors drive decidualization of human endometrial stromal cells (hESCs)?, Summary Answer: NR4A receptors modulate endometrial decidualization by transcriptional activation of FOXO1A, and in adenomyosis patients, the reduced expression of NR4A receptors in the eutopic endometrium may represent a novel mechanism to explain impaired decidualization and subfertility., What Is Known Already: A close relationship between impaired decidualization and subfertility has been established. In human endometrial stromal cells, orphan nuclear receptor NR4A is a novel regulator of decidualization., Study Design, Samples/materials, Methods: Eutopic endometrial tissues and hESCs from fertile controls (n = 56) and adenomyosis patients (n = 27) were collected for in vitro analysis. Primary hESCs isolated from eutopic endometrial tissues were used to evaluate the biological function of NR4A receptors. Adenovirus-mediated overexpression of NR4A and small interfering RNAs targeting NR4A, and FOXO1A were used to investigate the molecular mechanisms. Gene expression regulation was examined by real-time-quantitative PCR, immunostaining, and luciferase reporter assay. Artificial decidualization assay was performed to investigate the role of NR4A1 during decidualization in vivo., Main Results and the Role of Chance: NR4A modulates the decidualization of hESCs by upregulating prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) expression and transformation in vitro. Loss of uterine Nr4a1 results in female subfertility due to impaired decidualization. Mechanistically, NR4A binds to the nerve growth factor 1B (NGFI-B) -responsive element (NBRE) (-843 to -813) within the FOXO1A promoter region and regulates FOXO1A expression. Loss of FOXO1A significantly inhibits PRL and IGFBP-1 expression, as induced by NR4A. Moreover, the expression of NR4A and FOXO1A was lower in adenomyosis endometrial tissues compared to fertile controls, especially in stroma compartments. Ectopic NR4A expression rescued PRL and IGFBP-1 expression in adenomyotic hESCs to near-normal levels. Furthermore, PI3K/AKT signaling pathway involved in inducing NR4A expression under decidualization stimuli in hESCs and the level of p(Ser473)-AKT was significantly higher in stroma in endometrium from patients with adenomyosis., Limitations, Reasons for Caution: This is an in vitro study with a small sample size, utilizing stromal cell cultures from endometrial tissues of adenomyosis patients. Furthermore, results obtained should also be confirmed in a larger data set and with adenomyosis mouse models in vivo., Wider Implications of the Findings: Identification of a positive agonist of NR4A receptors will be critical for the improved treatment of patients with conditions of insufficient decidualization-associated infertility, such as adenomyosis and endometriosis., Large Scale Data: N/A., Study Funding and Competing Interests: This study was supported by the National Natural Science Foundation of China (81170570, G.J.Y. 81370683, G.J.Y. 81501251, Y.J. 31571189, H.X.S. and 81571402, G.J.Y.), and a special grant for clinical medicine science of Jiangsu Province (BL2014003, H.X.S.). The authors have no conflicts of interest to declare., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)