UA-treated VSMCs showed an increased migratory rate as compared to control cells (p < 0.001) and a re-arrangement of F-actin. Probenecid, proteasome inhibition and ARBs prevented the development of dysfunctional VSMC. This study shows, for the first time, that UA-induced cytoskeleton changes determine an increase in VSMC migratory rate, suggesting UA as a key player in vascular remodeling.The mechanisms by which hyperuricemia induces vascular dysfunction and contributes to cardiovascular disease are still debated. Phenotypic transition is a property of vascular smooth muscle cells (VSMCs) involved in organ damage. The aim of this study was to investigate the effects of uric acid (UA) on changes in the VSMC cytoskeleton, cell migration and the signals involved in these processes. MOVAS, a mouse VSMC line, was incubated with 6, 9 and 12 mg/dL of UA, angiotensin receptor blockers (ARBs), proteasome and MEK-inhibitors. Migration property was assessed in a micro-chemotaxis chamber and by phalloidin staining. Changes in cytoskeleton proteins (Smoothelin B (SMTB), alpha-Smooth Muscle Actin (αSMA), Smooth Muscle 22 Alpha (SM22α)), Atrogin-1 and MAPK activation were determined by Western blot, immunostaining and quantitative reverse transcription PCR. UA exposition modified SMT, αSMA and SM22α levels ( Patients with hyperuricemia (HU) are at high risk for developing hypertension , as wellVSMCs form the medial layer of major arteries and their contractile ability controls the vessel tone. In physiological condition, VSMCs are quiescent but retain remarkable plasticity . On the Although several studies have highlighted the importance of UA as a promoter of cell changes ,26,27, aIn this study, we explore the effects of UA on the VSMCs phenotype. With this purpose, we evaluated UA-induced changes in the expression and arrangement of cytoskeleton components and in the migratory capacity in MOVAS cells, an immortalized VSMC line from murine aorta. We also investigated the pathways involved in these processes by pre-treatment of cultured VSMCs with different inhibitors. First, we tested urate transporter blocker (Probenecid) and angiotensin receptor blockers (ARBs), valsartan and losartan. Secondly, bearing in mind that Atrogin-1, a ubiquitin ligase 3 which controls the VSMC phenotype and survival signaling , has beep < 0.05) and a similar increment was observed after 48-h of treatment .To examine the effects of UA on cell viability, we treated MOVAS with different UA concentrations (6 to 12 mg/dL) for 24–48 h. Cells showed an increase in proliferation regardless of UA concentration. As graphed in 2); UA 6 mg/dL 1018 ± 61–1017 ± 100 (974–1010 μm2) UA 9 mg/dL 1014 ± 50–1016 ± 110 (841–1010 μm2); UA 12 mg/dL 1078 ± 78–905 ± 29 (980–700 μm2); median (interquartile range); for each condition p < 0.0001 vs. CTR)  B. The efp < 0.05; SMT B: +30%, p < 0.01; SM 22-α: +240%, p < 0.01, p < 0.05, respectively) (p < 0.01) , Smoothelin B (SMT B), and smooth muscle 22α (SM22α) mRNA levels. We observed that after 24 h they were significantly increased by 9 mg/dL UA  A. On the < 0.01) B.p < 0.05), but dropped out after 48 h  as graphed in We found the α-SMA protein was upregulated after 24 h UA 9 mg/dL treatment . Accordingly, when cells were stained with Alexa-Fluor 594-conjugated phalloidin, UA induced the F-actin re-arrangement in thinner and poorly oriented fibers localized at cortical level, while in CTR, its compact polymerization in stress fibers along the major cell axis was observed . As a next step, we observed that UA-induced migration was stopped by the MEK-1 and -2 inhibitor U0196  (p < 0.05)  A. In add < 0.05) B,C.Then, we performed co-localization studies of Atrogin 1 and α-SMA and we observed that they were oppositely expressed: in CTR, α-SMA was highly expressed (green) and Atrogin-1 (red) was faintly expressed, whereas in UA-treated cells, the red fluorescence was predominant C. Probenp < 0.01–0.001 vs. UA)  in VSMC phenotypic modification, we pretreated MOVAS cells with 5 µm MG132 which, at 24 h, inhibited the overexpression of α-SMA, SMT B and SM 22 α ( vs. UA) A and res vs. UA) B.p 0.05–0.0001 vs. UA treated cells) (We examined whether Losartan (L) and Valsartan (V) could prevent the development of the dysfunctional VSMC phenotype induced by UA. As depicted in d cells) B–D.However, L and V had an inhibitory effect on UA-induced migration A and thiSeveral novel findings were made in this study: (1) UA treatment induced structural and functional transition into a proliferative/migratory phenotype of VSMCs; (2) UA determined an increase in the cellular migration rate of VSMCs; (3) Blocking the internalization of UA and the inhibition of proteasome, RAAS or MEK activity abolished the UA-induced cellular changes in VSMCs and their increase in migration rate. The mechanisms by which UA induces VSMCs changes are summarized in In human cells, UA has emerged as a major cause of damage in different pathophysiological conditions ,25,26,27VSMCs mainly has a contractility phenotype, which is responsible for the modulation of vascular tone. They also possess extensive plasticity such that they can be stimulated to transform from a contractile state into proliferative/migratory state . The matThe emerging role of cell migration as a necessary process in vessel wall remodeling has stimulated strong interest in cellular events and molecular mechanisms of VSMC migration. The migratory–proliferative phenotype is characterized by certain VSMC cytoskeletal structural changes that enable it to migrate to the source of initiating stimuli . For theMuch research exists about the role of biochemical signaling in VSMC migration, which remains challenging for our limited knowledge of biology complexity. Major goals of these studies include the definition of the proximal signals , as well as the signal transduction pathways that affect cell movement. Intracellular signal-transduction pathways such as mitogen-activated protein kinase/extracellular signal-regulated kinase MAPK p44/42), leading to VSMC proliferation and migration have been demonstrated to be activated by several factors ,42. Our , leadingThe UPS is central for protein turnover as the main pathway for the degradation of proteins related to cell-cycle regulation, signaling and differentiation in eukaryotic cells, including VSMC . UbiquitAngiotensin II (Ang II) plays a vital role in arterial remodeling through multiple pathways . It acceThis study has some limitations. A shift in cell phenotype is observed when VSMCs are removed from their native environment and placed in a culture, presumably due to the absence of the physiological signals that maintain and regulate the VSMC phenotype. Nevertheless, the inhibition both of UPS and RAAS , signifiThe procedures were in accordance with the Declaration of Helsinki. The study was approved by the Ethical Committee of Regione Liguria  on 28 June 2021 .v/v) FBS, 0.2 mg/mL G-418 (Euroclone S.p.A.). Cells were grown at 37 °C in a humidified 5% CO2 conditions.MOVAS cells, an immortalized smooth-muscle cell line from murine aorta, were obtained from ATCC (CRL-2797™). Cells were grown in DMEM  medium supplemented with 10% (MOVAS were grown to sub-confluence and then cultured in starving medium (with 1% FBS) in the presence of Uric Acid (UA) (6–12 mg/dL)  for 24–48 h. Control cells (CTR) were not treated with UA. Firstly, we evaluated the effects of different concentrations of UA on cell viability and then, on the cell dimensions, the migratory capability and the expression of cytoskeleton proteins . Furthermore, the role of ubiquitin–proteasome (UPS) on UA effects was studied, evaluating atrogin-1 (a F-box protein or Ubiquitin-protein ligase E3) expression and exposing cells to MG132 (5 µM) , a potent, reversible and cell-permeable proteasome inhibitor. Probenecid (5–10 µM) (Merck group) was added to the cell culture to test its capability to blunt migration and atrogin-1 expression.To understand if UA effects were direct or mediated by RAS activation, Losartan (L) or Valsartan (V) (both 10 µM)  were added 1 h earlier than 9 mg/dL AU. Lastly, the UA-induced activation of p44/42 MAPK was studied and its effects on cell migration through pretreatment with U0126 (10 µM) (Merck group).This assay for cell viability is based on the reduction of 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) (Merck Group) by mitochondrial dehydrogenase in viable cells to produce a purple formazan product. Each experiment was performed according to the protocol as previously described .MOVAS cells were incubated for 24–48 h with or without 9 mg/dL UA. Total RNA was isolated using the Tri-Reagent . A total of 1 µg RNA was used for cDNA synthesis with SCRIPT RT Supermix . PCR amplification was carried out in a total volume of 10 µL, containing 1 µL cDNA solution, 5 µL SensiFAST SYBR no ROX mix , 0.08 µL of each primer  and 3.84 µL of nuclease-free water. β-actin was quantified, and used for the normalization of expression values of the other genes.Assays were run in triplicate on MasterCycler realplex PCR system . The primer sequences are reported in v/v) glycerol, 0.5% (v/v) NP-40, 1 mM EDTA) supplemented with Protease inhibitor cocktail (Merck Group). Protein concentration was determined by using the Bicinchonic Protein assay kit (Euroclone S.p.A.) and 20–30 µg was resolved on SDS-polyacrylamide gels and electro-transferred to Amersham™ Hybond™ PVDF membrane (Euroclone S.P.A.). Blots were incubated o.n. at 4 °C with monoclonal anti α-smooth muscle actin (α-SMA) antibody (clone1A4) (Merck group), re-probed with anti H3 Histone antibody ,) or p44/42 MAPK  and then incubated in horseradish peroxidase secondary antibodies . Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Merck Group) and band intensities were determined using Alliance imaging system .The cell layers were lysed in cold buffer . After a five minute incubation in cold methanol, cells were exposed to anti α-SMA (Merck Group) and/or anti-atrogin-1  . For immunocytochemistry, cells were probed with an ultra-polymer goat anti-mouse IgG conjugated to HRP  and developed with DAB detection kit . Slides were counterstained with hematoxylin and examined by light microscopy.tm 488 and/or 594 were used . Nuclei were stained with DAPI.For immunofluorescence, secondary antibodies conjugated to Alexa Fluorhttps://theolb.readthedocs.io/en/latest/index.html, accessed on 20 August 2021).The levels of cell fluorescence were determined by ImageJ and they were calculated as corrected total cell fluorescence (CTCF) using this formula: CTCF = Integrated Density–(Area of selected cell × Mean fluorescence of background readings) (® NX software (Molecular Devices): the areas of randomly selected single cells were defined using the cursor and automatically estimated. The median values were calculated upon measurement of 100 cells/condition.The area of MOVAS cells was evaluated after 24 and 48 h exposure to UA (6–12 mg/dL) and compared with that of CTR. In brief, cells grown on chamber slides  were fixed with 2% paraformaldehyde, stained with Hematoxylin–Eosin and photographed with a Leica Microsystems microscope  (40× magnification). Images were then analyzed using MetaMorphCells grown on EZ- chamber slides (Merck group) were exposed to UA (9 mg/dL) with or without inhibitors for 24–48 h. Cells were fixed with 2% paraformaldehyde, permeabilized with 0.05%Triton × 100 and stained with 5 U/mL Alexa-Fluor 594-conjugated phalloidin . Nuclei were counterstained with DAPI. Cells were washed three times with PBS and analyzed by fluorescent microscopy.MOVAS were incubated for 24–48 h in the presence or absence of UA 9 mg/dL and/or Probenecid, Losartan, Valsartan and MG-132. Then, cells were tested for migration in a micro-chemotaxis Boyden chamber  using a 8 μm-pore size, polycarbonate polyvinylpyrrolidone-free filters (Millipore). The lower wells of the chemotaxis chambers were filled with medium alone. After incubation , the filters were removed from the chambers, fixed and stained with May Grunwald–Giemsa stain . Each condition was performed in duplicate. The cells of five random oil-immersion fields were counted and the chemotaxis index was calculated from the number of cells migrated to the test samples divided by the number of cells migrated to the control .t-test or one-way Anova with Bonferroni’s post-test for multiple group comparison. Statistical significance was set at p < 0.05. All statistical analyses were performed using Graph Pad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA.Data are given as mean ± SEM. Statistical analysis was performed by Student’s We found that even a slight increase in UA levels promotes VSMCs’ proliferation and their shift towards synthetic phenotype through proteasome and RAAS activation. These results provide the evidence of the cellular mechanisms that induce those vascular remodelings and damage  which, wFurthermore, our results suggest a novel role of UPS which can modulate cytoskeleton proteins in UA-stimulated VSMCs. UPS-signaling inhibition might serve as a therapeutic target in the management of vascular damage, and RAAS inhibition might be thought to prevent UA-induced atherosclerosis even in the absence of hypertension.


Uric acid is commonly known for its bad reputation. However, it has been shown that uric acid may be actively involved in neurotoxicity and/or neuroprotection. These effects could be caused by oxidative stress or inflammatory processes localized in the central nervous system, but also by other somatic diseases or systemic conditions. Our interest was to summarize and link the current data on the possible role of uric acid in cognitive functioning. We also focused on the two putative molecular mechanisms related to the pathological effects of uric acid—oxidative stress and inflammatory processes. The hippocampus is a prominent anatomic localization included in expressing uric acid's potential impact on cognitive functioning. In neurodegenerative and mental disorders, uric acid could be involved in a variety of ways in etiopathogenesis and clinical presentation. Hyperuricemia is non-specifically observed more frequently in the general population and after various somatic illnesses. There is increasing evidence to support the hypothesis that hyperuricemia may be beneficial for cognitive functioning because of its antioxidant effects but may also be a potential risk factor for cognitive dysfunction, in part because of increased inflammatory activity. In this context, gender specificities must also be considered. In vitro experiments showed that UA is a potent scavenger of singlet oxygen, peroxyl radicals (RO−) (Uric acid (UA) is the final oxidation product of adenine and guanine metabolism . It is fradicals . It alsoradicals  and acticals (−) . Becausecals (−) . On the cals (−) . UA, as cals (−) . In rececals (−) .5H4N4O3) is a heterocyclic organic compound that is the end product of the oxidation of two purine nucleic acids, adenine and guanine. The enzymatic pathway for the degradation of purines is complex and involves numerous enzymes. Briefly, adenosine monophosphate (AMP) is converted to inosine, while guanine monophosphate (GMP) is converted to guanosine by nucleotidase. The nucleosides, inosine and guanosine, are further converted by purine nucleoside phosphorylase (PNP) to the purine bases hypoxanthine and guanine, respectively. Hypoxanthine is then oxidized to xanthine by xanthine-oxidase (XO) and guanine is deaminated to form xanthine by guanine deaminase. Xanthine is again oxidized by xanthine oxidase to form the final product, UA  measurement. Abstracts of potentially relevant titles were assessed, and the full text of potentially eligible studies was reviewed. We performed a forward and backward search for relevant papers and repeated this process until no new titles were found. Letters, comments, editorials, practice guidelines, conference proceedings, theses, case studies and unpublished data were excluded.+ and Cu++) and lipid hydroperoxidases  and hydrogen peroxide (H2O2). Hink et al. demonstrated that UA effectively preserves and enhances extracellular SOD activity in mice at concentrations approaching physiological levels in humans (−), a very potent oxidant implicated in the pathogenesis of several CNS diseases. Peroxynitrite can interact with almost all cellular structures, causing severe cellular damage  model in mice, which is a model of multiple sclerosis (MS) (−) mediated tyrosine nitration and apoptotic cell death in inflamed areas of the spinal cord tissue.UA is a very potent free radical scavenger and is considered one of the most important antioxidants in human plasma  Figure . It has n humans . Removalr damage , 47. Squwith CO2 . The prosis (MS) . In micein vitro in a dose-dependent manner and also inhibited both the decline in mitochondrial respiration and the enhanced release of lactate dehydrogenase (LDH)  . In a mose (LDH) . These rUA is thought to have a pro-inflammatory effect by triggering interleukin (IL)-1β-mediated inflammation via activation of the nucleotide-binding and oligomerization domain (NOD)-like receptor protein (NLRP) 3 inflammasome, a multimolecular complex whose activation appears to play a central role in many pathological inflammatory conditions , 52. It Elevated UA levels induced by a high-UA diet (HUAD) triggered the expression of pro-inflammatory cytokines, activated the Toll-like receptor 4 (TLR4)/NF-κB pathway, and increased gliosis in the hippocampus  and mediThe protective role of UA has also been observed in CNS inflammatory processes. In the EAE model, exogenous treatment with UA prevented disruption of BBB integrity and reduced its permeability to inflammatory cells  Figure . Pre-treCognition as a higher brain function consists of major cognitive domains: memory, attention, language, executive functions and visuospatial functions . All of As a potent antioxidant in the human body, but also as a mediator in inflammatory processes, UA is the subject of increasing research focused on its influence on cognitive functioning , depending on the levels of exposure of cells to UA , and numerous studies have shown that these patients are at higher risk for cognitive impairment –88. It iThe association between UA and subsequent cognitive performance in patients that carry a high vascular burden showed that low UA levels were associated with poorer cognitive performance, manifested by lower global cognitive scores, memory scores, executive scores, and visuospatial scores . A stronThe potential contribution of UA to cognitive reserve could be attributed to its potent antioxidant properties. Euser et al. examined the association between serum UA and lower dementia risk and better cognitive function later in life in a large prospective population-based cohort study over an 11-year follow-up period . Higher There is growing evidence that UA may exert neuroprotective properties by suppressing neuroinflammation and inhibiting oxidative stress in neurodegenerative disorders , 149. InBased on the results of experimental and clinical studies, UA seems to play a dual role as a pro- and antioxidant. The balance between the two effects reflects a very complex interplay of factors that include the concentration of UA, the nature and concentration of free radicals, the presence and concentration of other antioxidant molecules, and the various cascades involved . HoweverRecently, however, there has been a growing body of evidence from clinical and basic research supporting the hypothesis that hyperuricemia, in part through increased inflammatory activity, may be a potential risk factor for cognitive dysfunction. Taking these lines of evidence together, UA appears to exert a protective effect on brain tissue and neurons during the initial stage of elevated UA levels, primarily through its potent antioxidant activity, but long-term elevation appears to trigger an inflammatory response that leads to brain tissue damage. Thus, UA metabolism may be a so-called double-edged sword in terms of the inflammatory and/or oxidative responses it induces in brain tissue, although, its harmful effects appear to outweigh the benefits of UA in most cases.Although numerous factors contributing to cognitive impairment have been identified to date, UA appears to be an important participant in the onset and/or progression of cognitive decline in various disorders. There is still conflicting evidence about UA's pathophysiological role and its clinical significance in influencing cognitive dysfunction. This may be partly explained by UA's dual nature and different properties, but also by a variety of distinct pathologies that can lead to many constellations of cognitive domain's dysfunctions.The UA impact on cognitive abilities may have a long evolution course, suggesting that the effects of UA on cognition should be explored in the terms of long, chronic exposure. The more informative study design would be a prospective long-term follow-up cohort study with a relatively large sample of older adults and measurement of sUA fluctuations concurrently with cognitive testing. Cognitive functions should be assessed with a wider range of domain-specific neurological tests. This would allow us to understand global patterns of cognitive fluctuation over time. In this context, it would also be important to investigate accompanying neuroanatomical and neurophysiological changes. Identification of modifiable risk factors is also important, as this will provide greater insight into pathophysiology, risk stratification and potential interventions. Collection of data on dietary habits, medication and comorbidity is necessary to fully exclude the influence of confounding factors. Further research on these complex topics is needed to help design and implement interventions to preserve cognitive capacities in health and various diseases.MB presented the idea and initial structuration of this review article. NM and KV drew a figure and a table. All authors have searched the literature and given some new insights into specific fields of their competencies, read, discussed, and accepted responsibility for the entire content of this submitted manuscript and approved its submission.This work was supported by the Ministry of Science and Technological Development of the Republic of Serbia, No. 175069 and the Faculty of Medical Sciences, University of Kragujevac, No. JP 03/16.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Drosophila melanogaster and discuss some of the open questions across the different cell and tissue types that have not yet been fully explored.Cellular trafficking through the endosomal–lysosomal system is essential for the transport of cargo proteins, receptors and lipids from the plasma membrane inside the cells and across membranous organelles. By acting as sorting stations, vesicle compartments direct the fate of their content for degradation, recycling to the membrane or transport to the trans-Golgi network. To effectively communicate with their neighbors, cells need to regulate their compartmentation and guide their signaling machineries to cortical membranes underlying these contact sites. Endosomal trafficking is indispensable for the polarized distribution of fate determinants, adaptors and junctional proteins. Conversely, endocytic machineries cooperate with polarity and scaffolding components to internalize receptors and target them to discrete membrane domains. Depending on the cell and tissue context, receptor endocytosis can terminate signaling responses but can also activate them within endosomes that act as signaling platforms. Therefore, cell homeostasis and responses to environmental cues rely on the dynamic cooperation of endosomal–lysosomal machineries with polarity and signaling cues. This review aims to address advances and emerging concepts on the cooperative regulation of endocytosis, polarity and signaling, primarily in  Cellular trafficking and the endosomal–lysosomal system regulate multiple developmental processes from phagocytosis, nutrient uptake and plasma membrane lipid or protein homeostasis, to signaling from cell-surface receptors and membrane trafficking ,2. By acDrosophila neuroblasts, polarity relies more on the differential distribution of cell fate determinants, while classical epithelia polarity involves the differential localization of transmembrane and adaptor proteins or targeting of the transport machinery across apical vs. basolateral membranes . EG. EG49]. Drosophila eye discs, the loss of ESCRT-I, -II or -III components leads to increased EGFR signaling, probably from endocytic vesicles [Along this line of evidence, loss of ESCRT components in some tissues results in increased EGFR signaling while in others in EGFR downregulation ,14,17,50vesicles . Interesvesicles .stam and hrs cause the accumulation of Rhomboid in abnormal endosomal compartments and silence the EGFR before ligand binding [On the other hand, the loss of ESCRT-0 components, such as Hrs, leads to the recycling of the EGFR receptor, which is not trapped in MVBs ,17. In d binding .Drosophila trachea, EGFR signaling levels control the length of the tracheal tubes, by regulating the organization of endosomes in which Crumbs and Serpent proteins are loaded. EGFR loaded on those endosomes acts as a critical hub for the correct delivery of Crumbs  and Serpent  to their final destinations [Drosophila follicle stem cells [Drosophila macrophage-like hemocytes), revealed differential endocytosis of the EGFR based on ligand levels driving EGFR receptor activation [Several pieces of evidence show that the EGFR-containing endosomal compartments can follow different or alternative trafficking routes depending on the specific cell type, the concentration of the ligand, the developmental stage and the cellular conditions ,17. In Dinations . Moreoveem cells . The polem cells ,58,59. Aem cells . βA3/A1-em cells . Anothertivation . More prtivation .In HeLa cells, analysis of EGFR internalization following activation with different ligands has shown that EGFR endocytosis after treatment with all ligands could be inhibited to a certain degree by ablation of clathrin, which confirms the existence of an alternative CIE pathway . HoweverTaken together, EGFR endocytosis and subsequent trafficking through endocytic organelles, form a dynamic network of subcellular compartments, which actively control the timing, amplitude and specificity of the signaling. Furthermore, the distribution of EGFR in apical and/or basolateral cell surfaces can have important biological consequences that affect EGFR downregulation efficiency and endocytic turnover, and influence paracrine vs. autocrine activation by different EGF ligands ,58,59,63The cooperative action of endocytosis and polarity in signaling regulation is very well-illustrated in the case of Notch signaling. Research over the past decades not only revealed the regulatory patterns and alternative pathways of Notch regulation but shed light on the diversity of these networks in specific cell and tissue contexts, and across organisms , Serrate (Ser) or Lag2 (the DSL family) from the neighboring signaling cell. This interaction initiates a cascade of proteolytic cleavages by γ-secretase that releases the intra-cellular domain of Notch (NICD). NICD can now translocate to the nucleus and activate transcription together with the transcription factor Suppressor of Hairless (Su(H)) and the nuclear effector Mastermind ,82,83. ADrosophila. SOPs are polarized epithelial cells that divide asymmetrically to give rise to two daughter cells, the posterior pIIa and anterior pIIb, which in turn divide asymmetrically to generate the four cells that will form the sensory organs: a neuron, a sheath, a shaft, and a socket [numb can no longer inhibit Notch-Spdo recycling in the anterior pIIb, Notch gets activated in the anterior precursor cell which adapts the pIIa fate, and cells finally adapt the socket fate  in the pupal notum of a socket ,65,66,67ket fate C. Conver neurons D [64,65,numb mutants, the increased numbers of Notch signaling endosomes recycle to the membrane in the Rab11-dependent way [numb permits Spdo internalization and recycling back to the plasma membrane [The regulated trafficking of Notch and Spdo plays an important role in this process. Numb interacts with internalized Spdo-Notch oligomers at sorting pIIb endosomes and inhibits the recycling of Notch, thereby creating an asymmetry in Notch distribution along the pIIa–pIIb interface . Numb aldent way . Basolatdent way . Loss ofmembrane . On the membrane .The Actin-related protein (Arp) 2/3 complex and its activators, Scar/WAVE and Wiskott–Aldrich Syndrome protein (WASp), promote actin polymerization and influence cell shape and motility. During SOP cytokinesis, Arp2/3 and WASp are required for the recycling of Delta . On the Notch also participates in a ligand-independent, noncanonical activation through the activity of the ring finger ubiquitin ligase protein Deltex (Dx), which transports the Notch receptor from the cell surface towards the late endosomes ,71,81,82Drosophila ovary stem cell niche and S2 cells [Drosophila homologue of the scaffolding zonula occludens-1 (ZO-1), junctional proteins that localize at tight junctions (TJs) in mammals. Pyd can bind and reduce Dx-dependent Notch trafficking, and thereby attenuate Notch signaling. Another interesting mechanism linking Notch to adaptor junctional components comes from the role of Crumbs in limiting ligand-independent endocytosis of Notch activation. In Drosophila developing wings, the apical polarity determinant Crumbs binds Notch directly and prevents its activation through the non-canonical Dx-pathway. Crumbs exerts its function by directly binding and regulating Notch localization. Yet, this function is independent of the role of Crumbs in the localization of apical components as in other tissues [Other interesting studies shed light on how adaptor, scaffold or polarity determinants mediate the crosstalk between trafficking and junctional complexes as signaling centers. One example is Polychaetoid (Pyd), a negative regulator of the Dx-mediated Notch activation in the S2 cells . Pyd is  tissues . Taken tDrosophila columnar epithelia since their mutations lead to tumor formation and neoplastic transformation that captured the research interest for many years [Drosophila testis [Drosophila but are rather required for SJ localization [Discs large (Dlg), Scribble (Scrib) and Lethal (2) giant larvae (Lgl) are highly conserved polarity and scaffolding proteins ,94,95,96ny years ,102,103.ny years ,99,104. a testis ,107,108.lization ,110. Simlization ,111,112.Xenopus, MDCK (Madin-Darby canine kidney) epithelial cells and C.elegans. Other critical functions of the Dlg polarity module in Drosophila include planar cell polarity, anterior-posterior patterning of ovarian follicle cells, formation of synapses and neuromuscular junctions (NMJs) together with other scaffolding, cell adhesion or receptor complexes  and the Crumbs–polarity complexes to control polarity in epithelial cells and neuroblast asymmetric cell division in finely balanced cooperative and antagonistic interactions ,99,110. iewed in ,113,114)zipper gene in Drosophila) and the cortex, and promotes binding to the GUK domain of Dlg [The subcellular localization of these proteins and shuttling from the basolateral cortex to other cellular compartments is influenced by their phosphorylation state. Phosphorylation of Dlg by the kinases Par-1 and Calcium/calmodulin-dependent protein kinase II (CaMKII) has been shown to affect cortical Dlg targeting ,116,117.n of Dlg ,118,119.dlg, scrib or lgl mutant animals typically develop tumors, whereas knocking down any of these genes in discrete clonal patches surrounded by normal tissue, leads to cell competition that eliminates the tumorous clones. Elimination of these clones is mediated by Jun N-terminal kinase (JNK)-dependent cell death from the surrounding healthy microenvironment [v12Ras) prevents clonal cell death and reverts clones to invasive metastatic tumors [Dlg, Scrib and Lgl cooperate with signaling pathways in normal situations and in cancer. ironment ,123,124.ironment , while oc tumors ,127,128,Several studies link Dlg, Scrib and Lgl to the EGFR/MAPK pathway, especially in mammalian systems Drosophila, yeast and mammals. Dlg and Strabismus (VanGogh) define sites of membrane deposition that allow membrane growth in cellularizing Drosophila embryos [Dlg, Scrib and Lgl are involved in vesicle and membrane trafficking in  embryos . In epit embryos . Dlg con embryos ,140. The embryos . Mammali embryos .Drosophila sensory organ precursor cells, Lgl controls endocytosis of Sanpodo, a four-pass transmembrane protein and regulator of Notch signaling [lgl mutants result in the accumulation of EE (Rab5 and Avl), RE (Rab11) and MVB markers (Hrs) but not LE markers (Car and Rab7), suggesting a role for Lgl in these later stages of endosomal maturation. In this case, Lgl regulates Notch signaling in a Dynamin/Rab5-mediated endocytosis and vesicle acidification but is independent of ESCRT-0 (Hrs) or the Rab11 REs [Drosophila wing epithelia [Drosophila homologue of the mammalian Tumor Necrosis Factor (TNF) [Numerous studies support the cooperation of the Dlg-module with vesicle and membrane trafficking, including endocytosis, exocytosis, recycling of endosomes to the cell membrane and retrograde trafficking  ,130,131.ignaling . In eye ignaling  and promignaling . Lgl locab11 REs . On the pithelia . Scrib ior (TNF) . Along tor (TNF) .Very interesting studies have also revealed a role for the Dlg-module in regulating the retromer complex that traffics protein cargo from endocytic vesicles to the trans-Golgi network (TGN) for recycling ,148. In Drosophila, squamous epithelial cells are present in the amnioserosa [Drosophila testis [Cells communicate with each other and the local microenvironment by guiding their signaling machineries to discrete cortical membrane domains. Based on the signals they receive, cells adapt their intrinsic features and structural characteristics to achieve a coordinated output and highly ordered organ systems. Thus, elucidating regulatory mechanisms related to cell polarity, endocytosis and signaling, requires a good understanding of apico-basal membrane identities, and the ability to monitor the compartmentation of signaling components, receptors and key regulators with spatial precision. Although this process is straightforward in columnar epithelia, it has not yet been addressed in a systematic way in squamous epithelia C. Squamoioserosa  the follioserosa ,152 and a testis ,154.Drosophila testis, squamous epithelial somatic cyst cells (SCCs) become unusually thin and elongated by following the growth of the dividing and differentiating germ cells they encapsulate, building together an organoid-like cyst (rolled (dpERK) entering the nucleus [In the ike cyst A. Squamoike cyst C. They aike cyst B. EGFR i nucleus . This fi nucleus ,158,159. nucleus ,158, whi nucleus . Higher  nucleus . Presuma nucleus .Drosophila male gonads and larval testes [scrib or dlg mutant flies, suggesting a role in establishing intimate soma-germline contacts [dlg, scrib, lgl or CME components  specifically in the squamous SCCs, mimics the effect of EGFR overactivation, resulting in germ cell death and increased signal transduction via the EGFR. Lowering EGFR or downstream signaling components rescues those defects [A recent study revealed that the cortical polarity proteins Dlg, Scrib, Lgl and clathrin-mediated endocytosis (CME) downregulate EGFR signaling levels in SCCs  to prevel testes ,163,164.contacts ,164. Kno defects . Interes defects .Drosophila testis and how EGFR is distributed to apical vs. basal cyst cell membranes is not yet established. Dlg, Scrib or Lgl could act to attenuate EGFR signaling in SCCs by potentiating endocytosis of the EGFR receptor together with CME. Alternatively, the Dlg module could attenuate EGFR/MAPK signaling independent of endocytosis, by binding and inactivating signal transduction components downstream of the EGFR  ,166 or oDrosophila testis, the EGFR stimulates basal autophagy to maintain the identity of the somatic cyst stem cells (CySCs) and to control the lipid levels through lipophagy (the autophagy induced breakdown of lipid droplets)  A. When Ceviously ,168. Unceviously ,170,171.Drosophila testis SCCs provided more insights on the importance of endocytic components in SCC signaling regulation. For example, the early endosome GTPase Rab5 is critically required for proper SCCs and germline differentiation. Knockdown of Rab5 in SCCs results in tumor-like overgrowths with (1) germ cells that become arrested at the spermatogonial-stage (the mitotic division stage) and (2) SCCs that show elevated levels of ectopic Hedgehog (Hh) and Janus kinase signal transducer and activator of transcription (JAK-STAT) activation, increased BMP ligands and an expansion of the Zfh-1 marker  in SCCs away from the niche [A screen in he niche . Thus, Rhe niche .Drosophila testis with the squamous epithelial cyst cells and the cross-communication to the encapsulating germ cells, have provided new insights into the dynamic regulation of a wide range of endosomal–lysosomal trafficking components with short-range signaling and cortical polarity cues during spermatogenesis.Taken together, the Drosophila intestine, asymmetric division of intestinal stem cells (ISCs) gives rise to a new ISC and a daughter progenitor that becomes either an enteroblast (EB) that will differentiate into enterocytes  or an enteroendocrine precursor (EEP) that will differentiate into secretory enteroendocrine cells  [In the EE, 10%) ,173. ThiEE, 10%) . Under nEE, 10%) .Drosophila midgut, the nexin SH3PX1 was shown to be critical for restraining ISC proliferation by downregulating the EGFR-Ras-MAPK signaling (highly expressed in ISCs), through an endocytosis-autophagy network [2+ signaling, ISC mitotic divisions and overproliferation. Thus, intracellular vesicle trafficking through the endocytosis-autophagy pathway can restrain ISC proliferation and hyperplasia by counteracting the EGFR activity. This mechanism is critical to maintain gut epithelial homeostasis and can provide an alternate route to EGFR signaling activation in human cancers [Another regulatory mechanism was revealed by analyzing sorting Nexins (SNXs), an evolutionarily conserved family of proteins that regulate intracellular membrane traffic most likely through their phosphoinositide-binding domain . In the  network . This ne cancers .Drosophila RalA from the intestinal stem and progenitor cells in Drosophila leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Thus, in contrast to other systems where receptor endocytosis attenuates signaling [RAL small GTPases are effectors of Ras signaling, widely expressed in the intestinal epithelium ,177. Theignaling ,28,29, hignaling ,177.Drosophila ovary, follicle cells undergo a cuboidal-to-squamous transformation, which involves lateral membrane shortening and apical membrane extension via the action of the Tao protein [tao gene, result in Fas II accumulation on the lateral membrane of those cells that block cuboidal-to-squamous cell remodeling [Endocytosis plays an important role in morphogenetic changes of lateral membrane shortening. In the  protein . Tao normodeling . This stmodeling .Drosophila gastrulation and epithelial morphogenesis, endocytosis and plasma membrane compartmentation differentially regulate Rho1 and Myosin II activation [During tivation . The GPCtivation . In the tivation , which cDrosophila nephrocytes, the equivalent of mammalian podocytes, as part of the filtration barrier in human kidneys [Drosophila nephrocytes are not physically connected to the fly excretory and osmoregulatory system of the Malpighian tubules . Fly nephrocytes filtrate the hemolymph and endocytose proteins and toxins to store and permanently inactivate them [Apical-basal polarity components play a key role in endocytosis and cellular trafficking in  kidneys ,180,181. kidneys ,180,182.ate them ,180,182.Drosophila homologue of the mammalian Nephrin. Apical polarity components of the PAR and Crumbs complexes localize and build the SD, while basolateral components of the Dlg-module localize at the basal region between neighboring SDs. Clathrin-mediated endocytosis (CME), Rab5-dependent EE, Rab11-dependent RE and the exocyst complex are critical for SD maintenance [Although nephrocytes are not of epithelial origin, they resemble epithelial cells as they are polarized. Nephrocytes have an enlarged apical domain and reduced basal surface , which altogether build a cell surface comprised of membrane invaginations that form finger-like labyrinthine channels  Figure . The slintenance ,180,181 dlg, scrib and lgl disrupt the localization of Sns on SDs, yet severe defects on nephrocyte endocytic function are seen only in scrib loss of function nephrocytes [dlg, scrib, lgl and par1 function in nephrocytes, led to the accumulation of SD proteins in Rab5 vesicles which mis-localized, while the integrity of SDs was compromised. Moreover, Vps34 (PI3K59F) has been shown to regulate autophagosome-autolysosome formation in nephrocytes, associated with the accumulation of Rab5 and Rab7 endocytic compartments, suggesting a disruption in vesicular transport [A recent study  has showhrocytes . Crumbs,hrocytes . Anotherhrocytes  confirmeransport . Furtherransport . These sSaccharomyces cerevisiae, adaptor proteins and signaling components work together to regulate the endocytosis of nutrient transporters and thereby modify the yeast’s growth and response to environmental cues [S. cerevisiae are destined to switch in order to match the food availability. This is performed through the membrane addition of the necessary transporters through the secretory pathway and the endocytic removal of those no longer required. For example, yeast growing in a lactate medium would favor the addition of the Jen1 transporter that absorbs this molecule, while the addition of glucose would promote endocytosis of Jen1 and its replacement with a glucose transporter [S. cerevisiae has 14 different ARTs, which are under multilevel control of the major nutrient-sensing systems and become targeted by signaling pathways [In multicellular organisms discussed so far, the interplay of cellular trafficking, polarity and signaling has been actively involved in developmental and morphogenetic decisions that shape tissues and organs. In unicellular organisms such as the budding yeast tal cues ,186,187.nsporter ,186,187.pathways .The target of rapamycin complex 1 (TORC1) signaling is one of the critical signaling pathways that regulate ART proteins . TORC1 sDistinct ART–Rsp5 complexes can act at different yeast internal compartments to regulate the fate of nutrient transporters along the endocytic pathway . Such anExcess of individual nutrients or their deprivation can activate complementary ART-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition. Art1–Rsp5 not only promotes endocytosis of the Can1 arginine transporter, but it also mediates the endocytosis of the methionine transporter Mup1 and the lysine transporter Lyp1 in response to the excess of their respective amino acid substrates . ConversPost-translational modification of ART proteins regulates their switch from active to inactive state. Rod1/Art4 is an ART protein necessary for yeast cells to endocytose the lactate transporter Jen1 in the presence of glucose. Yeast cells preferentially use glucose as an energy nutrient over lactate when possible. Rod1 phosphorylation is regulated by glucose availability, which consequently modulates its activity. More precisely, glucose-induced dephosphorylation of Rod1 promotes its ubiquitylation, a modification that is essential for Jen1 endocytosis . The finTrafficking through the endo–lysosomal system is intimately linked to polarity and signaling, and together they play a central role in regulating fundamental aspects of cell physiology and homeostasis. Research over recent decades revealed the complexity of their cooperation and mutual dependence across multiple pathways where trafficking, polarity and signaling intersect. Along this line, polarity scaffolds are not just static barriers and endosomal compartments are not simple cargo transporters. Compelling evidence shows that they emerge as dynamic organizing centers involved in site-specific protein targeting (or exclusion) and in stabilizing supramolecular signaling complexes that (1) provide guiding cues for targeted membrane insertion, cell compartmentation, communication, and remodeling; and (2) actively control the timing, amplitude and specificity of intracellular signaling.Drosophila, which combines a relatively simple organization with sufficient cellular complexity and conservation, allowed us to “zoom” into different cell types and uncover the regulatory strategies in very diverse contexts. From gut intestinal epithelia, sensory organ precursors (SOPs) and imaginal epithelia of developing wings, legs and ommatidia, to squamous epithelia in testis and ovaries, and non-epithelia nephrocytes, research in the fruit fly has made a major contribution to our understanding of how endosomal–lysosomal processes, polarity and (intracellular and inter-cellular) signaling, coordinate cellular responses and establish homeostasis. Although we are still far from breaking the mysteries of endocytosis across species [ species , we can


Prevalence showed differences between sports categories, and were significantly higher in acrobatic athletes than non-contact athletes (p = 0.03). Conclusions: CAI was highly prevalent among elite athletes in this study, with female athletes and athletes in acrobatic sports being associated with a higher risk of developing CAI in their professional careers. Therefore, extra precautions need to be taken into account when applying ankle protections for these athletes.Background: Ankle sprains are one of the most common injuries in athletic populations. Misdiagnosed and untreated ankle sprains will cause chronic ankle instability (CAI), which can significantly affect the performance of athletes. This study aimed to investigate the prevalence and characteristics of CAI in elite athletes of different sports. Method: This cross-sectional study included 198 elite athletes from Guangdong provincial sports teams. All participants answered a questionnaire about ankle sprains and ankle instability. The severity of their ankle instability was evaluated by the Cumberland Ankle Instability Tool (CAIT). Participants further underwent clinical examinations from sports medicine doctors to determine the presence and characteristics of ankle instability. The datasets were analyzed to determine the differences in prevalence between age, gender, sports teams, and sports categories. Results: In 198 athletes, 39.4% (n = 78) had bilateral CAI while 25.3% (n = 50) had unilateral CAI. Female athletes had a higher prevalence of CAI than male athletes in the study ( Ankle sprain is one of the most common musculoskeletal injuries, with a high incidence in competitive sports ,2. AcuteIn 2011, Hiller et al.  presenteThe International Ankle Consortium (IAC) published a diagnosis standard for CAI in 2014, including: a previous severe lateral ankle sprain (LAS), recurrent LAS, a feeling of the ankle “giving way”, and validated self-reported questionnaires ,13,14, tThe purposes of the study were (1) to discover the positive rate of CAI in elite athletes from different sports based on the IAC criteria; (2) to compare the differences in the demographic and ankle characteristics between athletes with or without CAI; and (3) to investigate if the prevalence is impacted by the category of sport . We hypothesized that the prevalence of CAI in elite athletes is influenced by sex, age, and the category of sport.This study represents a cross-sectional analysis of data on the CAI prevalence of elite athletes from Guangdong provincial sports team. This study was performed in the Guangdong Sport Hospital  from December 2021 to March 2022. All participants signed informed consent forms before the study began.Sanda, trampolining, and gymnastics) and (2) signed the informed consent form. The exclusion criteria included (1) acute injuries in the lower limbs (sprains or fractures); (2) a history of lower limb surgery or fracture; and (3) being unable to complete questionnaires or assessments.Athletes from different sports were diagnosed with or without CAI using questionnaires and clinical assessments based on the criteria recommended by IAC . All athDemographic characteristics of all participants  and questionnaires for CAI were collected individually. The clinical assessments were measured separately for the right and left ankle. The diagnosis criteria of CAI included: (1) a history of significant ankle sprain (more than 12 months); (2) the injured ankle having a history of a feeling of “giving way” and/or recurrent sprain; and (3) a CAIT score ≤ 24. The clinical assessment to determine the severity of ankle instability was performed via anterior drawer tests, talar tilt tests, and other assessments.Foot and ankle characteristics including the arch of the foot and dorsiflexion ranges were measured using the navicular drop test  and dorsp < 0.05 was considered statistically significant and all the analyses were performed using SPSS software (version 21.0).Differences in demographics and ankles characteristics between athletes with CAI and without CAI were examined using the Mann–Whitney U test or independent T test. The Chi-square test was used to determine the difference in the presence of CAI between sports and gender. Sanda, 20 trampolining, and 20 gymnastics).p < 0.05, 2(1) = 9.2, p = 0.010)  had no history of ankle sprain. Of the 170 athletes with previous ankle sprains, 128 athletes met the criteria of having CAI. = 0.010) .p = 0.12; right: p = 0.68, p = 0.82; right: p = 0.88, For foot and ankle characteristics, 206 ankles meet the IAC criteria ; their means (SD) of navicular drop distance were 0.39 (0.17) cm and 0.41(0.19) cm for left and right, respectively, while for toe–wall distance they were 11.40 (3.00) cm and 12.01 (3.37) cm for left and right, respectively. In those 190 ankles which did not meet the CAI inclusion criteria , for navicular drop distance they were 0.42 (0.18) cm and 0.42 (0.19) cm for left and right, respectively, and for toe–wall distance they were 11.51 (3.50) cm and 11.94 (3.28) cm for left and right, respectively. There was no significant difference in the navicular drop tests between the ankles that did and did not meet the CAI inclusion criteria , 67.3% for contact (35/52), and 56.1% for non-contact (46/82) sports (2(1) = 4.674, p = 0.03).) sports . AthleteAdditionally, there was no significant differences in terms of the CAIT value in the 198 athletes among the nine sports teams . Table 5Our study investigated the prevalence of chronic ankle instability in 198 elite athletes from different sports, as well as the differences in demographic and ankle characteristics between athletes with CAI and without CAI. A total of 85.9% of all the elite athletes had previous ankle sprains, and the prevalence of CAI was 64.6% (128/198). The prevalence of CAI was significantly different among genders and sports categories. Female elite athletes had a significantly higher presence of CAI than male elite athletes. Between sports categories, CAI was significantly higher in “acrobatic” athletes than in “non-contact” athletes. However, there was no significant difference in the ankle characteristics between the ankles which did and did not meet the CAI inclusion criteria.The primary aim of our study was to provide persuasive evidence of the prevalence and characteristics of CAI. Considering that most previous CAI articles investigated the general population, we aimed to discover whether there are CAI problems among elite athletes without focusing on identifying the risk factors of CAI. In our study, the prevalence of CAI was 64.6% 128/198) . Previou8 . PreviOur study investigated the prevalence of CAI in elite athletes based on the inclusion and exclusion criteria of the International Ankle Consortium (IAC). In several studies ,20, percOur study found that male athletes had a lower prevalence of CAI than female athletes . Some exIn our study, there was no significant difference in the ankle characteristics (including the arch of foot and dorsiflexion ranges) between participants that did and did not meet the CAI inclusion criteria . For the2(2) = 4.947, p = 0.08) (2(1) = 4.674, p = 0.03). Previous research [It was hypothesized that a significantly higher prevalence of CAI would be present among the contact and acrobatic sports groups. However, in our study no sports team showed a higher prevalence. Surprisingly, we found that diving and trampolining, the acrobatic sports, had the highest percentage of CAI (75%) . We susp = 0.08) , acrobatresearch  showed tresearch  indicateThe CAIT was recommended by the IAC as a validated ankle instability-specific self-reported questionnaire . The ChiA previous study  graded lOur study has several limitations. Firstly, although our study diagnosed CAI based on the IAC criteria, the effect of recall bias is difficult to avoid. Some research suggests that the IAC criteria cannot capture the entire population of CAI. For example, some athletes with CAI are excluded because they have a history of knee surgery, which leads to a misestimation in the prevalence of CAI. Secondly, the number of athletes in each gender was limited and unequal. If the number of the female athletes had been the same as that of the male athletes, the prevalence of CAI in elite athletes might have been different. In consideration of the statistical significance determined in the study, we hope that the data can be useful for further research, even in a limited sample size. Finally, the performance of athletes was not counted in this study, which may have a correlation with the prevalence of CAI. A prospective follow-up study on the changes in athletes’ performance after the first-time ankle sprain and during the development of CAI should be conducted to overcome this limitation.The aim of our study was to identify the prevalence of CAI in elite athletes. Our study found that 85.9% of athletes (170/198) had previous ankle sprains, and the prevalence of CAI was 64.6% (128/198 athletes). Female athletes had a significantly higher prevalence of CAI than male athletes. Prevention measures of CAI should be a focus for acrobatic athletes, as they have a higher risk of developing CAI. When applying ankle protections for athletes, gender and sports categories should be taken into consideration.


Colorectal cancer (CRC), mostly categorized as a low immunogenic microsatellite-stable phenotype bearing complex immunosuppressive tumor microenvironment (TME), is highly resistant to immunotherapy. Seeking safe and efficient alternatives aimed at modulating tumor immunosuppressive TME to improve outcome of CRC is highly anticipated yet remains challenging.Methods: Enlightened from the drug complementary art in traditional Chinese medicine, we designed a self-assembled nanomedicine (termed LNT-UA) by the natural active ingredients of ursolic acid (UA) and lentinan (LNT) through a simple nano-precipitation method, without any extra carriers, for CRC immunotherapy.Results: UA induces immunogenic cell death (ICD), while LNT further promotes dendritic cell (DC) maturation and repolarizes tumor-associated macrophage (TAM) from a protumorigenic M2 to an antitumor M1 phenotype. Co-delivery of UA and LNT by LNT-UA effectively reshapes the immunosuppressive TME and mobilizes innate and adaptive immunity to inhibit tumor progression in the CT26 CRC tumor model. Following the principle of integrative theoretical system of traditional Chinese medicine (TCM) on overall regulation, the further combination of LNT-UA and anti-CD47 antibody (αCD47) would reinforce the antitumor immunity by promoting phagocytosis of dying tumor cells and tumor-associated antigens (TAAs), leading to effective suppression of both primary and distant tumor growth with 2.2-fold longer of median survival time in the bilateral tumor model. Most notably, this combination effect is also observed in the spontaneous CRC model induced by chemical carcinogens, with much less and smaller size of tumor nodules after sequential administration of LNT-UA and αCD47 through gavage and intraperitoneal injection, respectively.Conclusions: This study provides a promising self-assembled traditional Chinese nanomedicine to improve immunotherapy for CRC, which might be applicable for future clinical translation. Indeed, numerous empirical prescriptions, monomers and extracts of TCM have been used in modulating systemic immunity, whether implemented alone or incorporated with western modern therapies Prunella vulgaris L., Hedyotis diffusa Willd. or Plantago asiatica L, has been regarded as a highly effective lead compound for immune-modulation through affecting the JAK/STAT signaling pathway in lymphocytes in vivoIn recent years, growing attentions have been driven toward seeking traditional Chinese medicines (TCMs) as potential modulators of tumor immunotherapy Lentinan (LNT), as a kind of water-soluble polysaccharide consisting of β-(1→3)-glucan backbone with β-(1→6)-glucose branch chain extracted from Lentinus edodes (Berk.) sing, is frequently used in clinic as a host defense potentiator via binding to the signal regulatory protein alpha (SIRPα) receptor Fufang, LNT-UA) based on TCM theory focusing on the overall regulation offers a promising strategy for CRC immunotherapy.Even if there are sufficient TAAs and reprogrammed phagocytes induced by the incorporation role of LNT and UA in the tumor microenvironment (TME), one mechanism that might diminish LNT-UA efficiency is the persistence of a large number of antiphagocytic molecules like CD47, which will send a “don't eat me” signal to inhibit phagocytosis and sequentially attenuate the tumor antigen presentation to T cells @ . Ursolic acid (A1810063) was obtained from Aladdin@ . BCA Protein Assay Kit (DQ111-01) and Cell Counting Kit-8  were purchased from TransGen Biotech Co., Ltd. . Live/Dead Cell Staining Kit (C542) and Annexin V-FITC/PI Apoptosis Detection (AD10) were obtained from Dojindo (JPN). Primary antibodies were purchased from Abcam (USA). Fluorescent secondary antibodies and streaming antibodies were purchased from Thermo Fisher Scientific Inc. (USA). Adenosine triphosphate (ATP) assay kit (S0026) and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay kit (C1088) were obtained from Beyotime Biotech Co., Ltd. . In vivo MAb anti-mouse CD47 (BE0270) was obtained from Bio X Cell (USA). Azoxymethane  was purchased from Sigma (USA). Dextran sulfate sodium  was obtained from MP Biomedicals (USA). Granulocyte macrophage colony-stimulating factors  and interleukin-4  were purchased from R&D Systems (USA). Enzyme-Linked Immunosorbent Assay (ELISA) kits were obtained from Boster Biological Technology Co., Ltd. . All other chemicals were used as obtained unless otherwise specified.Lentinan (DX0085) was obtained from Desite2O with a dialysis bag of molecular weight cut-off (MWCO) of 8000-14000 for 72 h to remove the organic solvent and unloaded the free drug.LNT-UA was prepared by a nanoprecipitation method. Briefly, LNT (10 mg) and UA (5 mg) were co-dissolved in 1 mL of dimethyl sulfoxide (DMSO), followed by dropwise addition into 2 mL of ultrapure water under continuously stirring. Subsequently, the crude product was dialyzed against ultrapure HThe morphology and particle diameter of LNT-UA nanoparticles were observed using a transmission electron microscope (TEM) and scanning electron microscopy (SEM). For TEM, LNT-UA was negatively stained with 1% uranium acetate on a copper grid. The hydrodynamic diameters and zeta potential of LNT-UA were measured by a Zetasizer Nano ZS . The chemical structures of LNT-UA were analyzed by a Fourier transform infrared (FT-IR) spectrometer . The encapsulation efficiency and loading capacity of LNT-UA were analyzed by the high-performance liquid chromatography (HPLC) system , as previously reported The release profiles of LNT and UA from LNT-UA were investigated using the dialysis method in different buffers  containing 0.1% (v/v) Tween 80. Briefly, 0.8 mL of LNT-UA was sealed in dialysis bags (MWCO 8000-14000) with 7.2 mL of different buffers outside and incubated at 37 °C and stirred at 100 rpm. Samples were withdrawn at predetermined time intervals, and the LNT and UA content of each sample were detected by phenol-sulfuric acid method and HPLC, respectively.6 murine colon tumor cell CT26 cells were seeded in a 10 cm culture plate. After 24 h of incubation, the medium was replaced by the fresh one containing free UA or LNT-UA (UA: 22 μmol/L) for 4 or 8 h. As for free UA formulation, it was firstly pre-dissolved in DMSO and then diluted in culture medium. After treatments, a part of cell suspension was lysed by radio-immunoprecipitation assay (RIPA) lysis buffer for determining total protein content by a BCA Protein Assay Kit. The rest of cell suspension was frozen and thawed repeatedly 3 times, followed by the addition of the same volume of methanol to extract UA. The UA concentration was determined by using HPLC. Finally, the cell uptake was quantitatively analyzed by normalizing UA content to the corresponding total protein weight.5×104 cells per well were seeded in a 96-well plate and incubated for 24 h. Then, cells were incubated with the fresh medium containing various concentrations of free UA, LNT, or LNT-UA for 24 h. After removing the supernatant, 10 μL of CCK-8 solution was added to each well followed by incubation for another 1.5 h. Finally, the absorbance was measured using the Spectra Max M5 microplate reader  at 450 nm. With respect to the Live/Dead staining assay, the cells after treatments were stained with both calcein AM and propidium iodide (PI) in a 37 °C incubator for 15 min, and then observed and photographed under a fluorescence microscope . For the Annexin V-FITC/PI assay, Annexin V-FITC and PI were added into the treated cells for staining of 15 min. Finally, the cells were filtered through a 40 μm cell filter before being tested by flow cytometry .Cell viabilities were determined by CCK-8, Live/Dead staining as well as Annexin V-FITC/PI assay. For the CCK-8 assay, 1.5×10in vitro. For CRT analysis, CT26 cells were seeded into 35 mm confocal dishes overnight and then incubated with LNT, UA, and LNT-UA at the equivalent UA concentration of 22 μmol/L UA for 4 h. Afterward, the cells were fixed using 4% paraformaldehyde for 15 min and then sealed with 3% BSA in PBS for 30 min. Subsequently, the cells were incubated with anti-CRT antibody  overnight at 4 °C. Afterward, the cells were washed with PBS and stained with anti-rabbit IgG  and DAPI for 1 h and 10 min, respectively. Finally, the cells were observed by a confocal laser scanning microscope . Intracellular HMGB1 distribution was also monitored by the same immunofluorescence analysis. After 8 h of incubation with LNT, UA or LNT-UA, the cells were fixed, permeabilized using 0.3% Triton X-100 and incubated with anti-HMGB1 antibody  for CLSM observation. The cell surface exposure of CRT and the nucleus level of HMGB1 were also quantitatively analyzed by flow cytometry . Intracellular ATP level was measured by ATP assay kit, and the cells with the aforementioned treatments were collected and lysed for detection according to the manufacturer's instruction. ATP-induced chemiluminescence was measured by a Spectra Max M5 microplate reader .To verify LNT-UA-induced ICD, surface expression of calreticulin (CRT), extracellular release of high mobility group protein 1 (HMGB1), and ATP secretion were determined Bone marrow-derived dendritic cells (BMDCs) were prepared according to the previously reported method 5/per well). Then, the cells were stimulated by LNT or LPS for 48 h at 37 °C. Then, the cells were stained with anti-CD11b-APC  and anti-CD80-PE  and then detected by flow cytometry.To prepare bone marrow-derived macrophages (BMDMs), bone marrow cells obtained from femurs and tibias were cultured on Dulbecco's modified eagle medium (DMEM) complete medium containing recombinant mouse GM-CSF (20 ng/mL) for 7 days. BMDMs were isolated and incubated on a 24-well plate . Animal experiment protocols were approved by the Animal Ethics Committee of Mengchao Hepatobiliary Hospital of Fujian Medical University and carried out according to the institutional guidelines.5 CT26 cells in 100 µL of PBS were subcutaneously injected into the right side of the back of BALB/c mice. Afterward, these CT26 tumor-bearing mice were randomized into four groups with the tumor volume of about 100 mm3. The mice were then treated with saline (control), LNT, UA, or LNT-UA at the equivalent UA dose of 5 mg/kg through intratumoral injection every three days for three times in total. After the 1st administration, the tumor sizes and body weight were monitored every two days. The tumor size was determined as follows: width2×length/2. The tumors of mice were harvested for hematoxylin-eosin (H&E) staining, immunohistochemistry of Ki-67, and immunofluorescence staining for CRT, HMGB1.To establish the animal tumor model, 5×106/mL) were successively injected into the right (primary tumor) and left (distant tumor) flank of BALB/c mice. Afterward, these CT26 tumor-bearing mice were randomized into four groups with the primary tumor volume of about 100 mm3: Control , LNT-UA, αCD47, LNT-UA + αCD47. LNT-UA (UA: 5 mg/kg) was injected intratumorally into the primary tumors on days 1, 5 and 9. The αCD47 antibody (2.5 mg/kg) was injected into the primary tumors intratumorally on days 2, 6 and 10. Body weight and tumor volume were measured every other day, and survival time was also recorded. Animals were euthanized in the presence of impaired health or the size of their tumors exceeded 1500 mm3. The bilateral tumors from different groups of mice were harvested for further study, like immunofluorescence staining for CD47 or CD8, H&E, immunohistochemistry of Ki-67, and TUNEL assay for histopathological analysis.To establish CT26 tumor model with primary and distant tumors, 100 µL and 50 µL of cell suspension  was administrated intragastrically on days 68, 72 and 76, while αCD47 antibody (5 mg/kg) intraperitoneally injected on days 69, 73 and 77. On day 84, the major organs  and the whole blood were collected for H&E staining and blood chemistry examination for biosecurity assay. The colon ending invaded with tumor nodules were harvested from different groups of mice for photograph and histopathological observation.in vivo, the TDLNs from CT26-tumor bearing mice were harvested, ground gently and strained by 40 μm filter net to get cell suspension. Then the cells were stained with anti-CD11c-APC, anti-CD80-PE and anti-CD86-PE-Cy7 antibodies for detecting DC maturation by flow cytometry.To analyze DC maturation To analyze tumor infiltrated immune cells, the tumor tissues were harvested post treatment and cut into small pieces, digested with collagenase IV, hyaluronidase, and DNase. The single-cell suspension was further purified by a density gradient centrifugation (Ficoll solution). Then the cells were stained with T lymphocyte markers  and macrophage markers  before examined by flow cytometry.To determine the intratumor levels of interleukin-10 (IL-10), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), the tumor tissues were homogenized with 200 μL PBS and centrifuged at 14,000×g for 5 min. The cytokines of supernatant were measured by ELISA kits.t test (two-tailed). Data in multiple groups were evaluated using the one-way analysis of variance (ANOVA) followed by the Tukey post hoc test. A log-rank test was applied for the comparison in the survival study. In addition, the Kruskal-Wallis H test was used for the comparison of non-normal distributed data. In this study, P < 0.05 was deemed to indicate statistically significant.Results were shown in this study as mean ± standard deviation (SD). Data between two groups were assessed using independent Student's -1, C—H at 2928 cm-1 and C=O at 1690 cm-1 appeared both in the spectra of UA and LNT-UA. In addition, the characteristic peak of 1045 cm-1 ascribing to the stretching vibration of glucosidic bond (C—OH and C—O—C) in LNT also appeared in the spectrum of LNT-UA The LNT-UA nanodrugs were synthesized by the self-assembly of LNT and UA through the multiple nonbonded interactions such as hydrogen bond and van der Waals force Figure A, and obin vitro, we first detected the cell viability of murine colorectal cancer cell line CT26 cells at different concentrations by CCK-8 assay. After 24 h of treatment, we found that the viability of free UA and LNT-UA gradually decreased with the increase of UA concentration  by CCK-8 assay. As shown in in vivo antitumor effect, more APCs may be recruited into the initial tumor site, and the tumor-associated antigens in tumor debris post treatment may be transported into nearby lymph nodes and then processed by APCs to stimulate immune response, although APCs pre-existing in the tumor might have been killed alongside the tumor Moreover, we further explored the Besides the release of TAAs, tumor cells with immunogenic cell death have other distinct characteristics: CRT exposure on the cell membrane as a “eat me” signal to facilitate phagocytosis, HMGB1 release from nucleus to effectively promote the maturation of APCs, and ATP release as a “find me” signal to recruit more immune cells +CD80+ cells). However, the polarization effect of LNT-UA was less pronounced than LNT  was increased significantly from 8.8% to 31.0%, while M2 phenotype (CD11b+CD206+) decreased from 68.7% to 43.0% after incubation with LNT. Additionally, LNT also significantly promoted the expression of CD80 (35.8%) and CD86 (25.7%) on the BMDC surface, indicating its role to stimulate the maturation of BMDCs. Taken together, LNT not only can serve as a carrier to increase the bioavailability of UA but also has the ability to stimulate the maturation of DCs and induce BMDMs to polarize toward M1, which benefits to enhance the ICD immune response.The immune response triggered by drug-induced immunogenic cell death alone is usually weak, while the combination with an immunomodulator can amplify the antitumor immunity. LNT has been widely used in studies as an immune adjuvant, so we checked the effect of LNT on mouse BMDMs and mouse BMDCs by flow cytometry Figure -7. CompaT Figure , which mT Figure . To furtin vitro, we next sought to investigate the antitumor effect of LNT-UA on subcutaneous transplanted CT26 cells in BALB/c mice. Four days after the 3rd administration, a portion of mice was euthanatized for detecting the relative indexes, as illustrated in Figure in vitro and in vivo anticancer effect of the physical mixture of LNT and UA (LNT+UA) has been investigated and compared with LNT-UA, as shown in Encouraged by the remarkable tumor suppressive effects of LNT-UA in vivo, CRT and HMGB1 expression were analyzed by immunofluorescent staining of tumor slides. As observed in Figure in vivo. Immature DCs would uptake TAAs released from dying tumor cells, and became matured in the TDLNs to present antigen to naïve T cells. Therefore, we analyzed the maturation of DCs in TDLNs by flow cytometry. The results in Figure To explore the underlying mechanism of the antitumor effect of LNT-UA +CD8+ T cells and CD3+CD4+ T cells, which were nearly 2-fold and 4-fold higher in comparison with the control group, respectively. Figure +CD80+ cells) in the tumor tissues of the LNT-UA group were markedly increased, while the percentages of M2 (CD11b+CD206+ cells) were decreased, leading to the upregulated ratio of M1/M2 macrophages  accounting for the largest number of immune cells in tumors tend to be polarized into a tumorigenic M2-like phenotype s Figure E. Furthein situ vaccination response” to suppress distant micro-metastases, we established a bilateral subcutaneous graft tumor model in BALB/c mice with primary tumor treated by various formulations as a tumor vaccine. LNT-UA was injected intratumorally at day 1, 5, 9, while αCD47 was also intratumorally injected into the primary tumor to prevent non-specific binding with platelets and red blood cells in the blood at day 2, 6, 10  still experienced further growth after treatment , middle tumor (≥ 2 mm2 and <4 mm2), and smaller tumor (<2 mm2) To better mimic the microenvironment in which CRC cells grow in the intestinal tract, a spontaneous CRC model was established by administration with AOM + DSS to evaluate the therapeutic efficacy of LNT-UA plus αCD47 in vivo, major organs and serum were collected for histological and biochemical indicator detection at the endpoint. The results of H&E staining demonstrated no significant toxic damage in major organs of the mice in the LNT-UA + αCD47 group  or eliminating evil (Qu Xie)” in traditional Chinese medicine with a long history, to mediate immunotherapy for CRC. The nanodrug displayed spherical morphology with narrow size distribution and superior stability in aqueous solution, with an extremely high drug-loading rate as no extra carriers were used. Compared with free UA, LNT-UA displayed an improved drug bioavailability and anticancer performance, which then induced ICD response to increase tumor immunogenicity. In addition to being a solubilizer for UA, LNT in the nanodrug was demonstrated to further act as an immunologic adjuvant to promote ICD response, thereby activating DCs, polarizing TAMs towards M1, recruiting effector T cells, and increasing antitumor related cytokine levels. Following the principle of an integrative theory in TCM on overall regulation, the further combination of LNT-UA and αCD47 was demonstrated to more efficiently reinforce the antitumor immunity to effectively inhibit tumor growth and metastasis, as proved in multiple tumor models including bilateral and spontaneous CRC tumors. To our knowledge, this is the first report on the use of the co-assembly of LNT and UA for CRC treatment, and our findings may stimulate the development of various TCM-based formulations based on TCM theory. Thus, this relatively facile, scalable, and modernized natural product formulation  based on TCM theory focusing on the overall regulation offers a promising strategy for CRC immunotherapy.In summary, we herein presented a natural product nano-formulation (LNT-UA) by the self-assembly of LNT and UA based on the theory of “strengthening the body (Supplementary figures and table.Click here for additional data file.


Protein kinase CK2 (CK2) is a ubiquitous holoenzyme involved in a wide array of developmental processes. The involvement of CK2 in events such as neurogenesis, cardiogenesis, skeletogenesis, and spermatogenesis is essential for the viability of almost all organisms, and its role has been conserved throughout evolution. Further into adulthood, CK2 continues to function as a key regulator of pathways affecting crucial processes such as osteogenesis, adipogenesis, chondrogenesis, neuron differentiation, and the immune response. Due to its vast role in a multitude of pathways, aberrant functioning of this kinase leads to embryonic lethality and numerous diseases and disorders, including cancer and neurological disorders. As a result, CK2 is a popular target for interventions aiming to treat the aforementioned diseases. Specifically, two CK2 inhibitors, namely CX-4945 and CIBG-300, are in the early stages of clinical testing and exhibit promise for treating cancer and other disorders. Further, other researchers around the world are focusing on CK2 to treat bone disorders. This review summarizes the current understanding of CK2 in development, the structure of CK2, the targets and signaling pathways of CK2, the implication of CK2 in disease progression, and the recent therapeutics developed to inhibit the dysregulation of CK2 function in various diseases. During development, the phosphorylation and dephosphorylation of proteins is essential for the proper formation of organisms ,2,3. Thi−/− mice remain viable, male mice are infertile and incapable of producing offspring, as germ cells do not form properly . To. To45]. The implication of CK2 in many developmental processes is due to its multifunctionality. Specifically, over 400 substates are identified as targets of CK2 ,21. WhilA second binding site of CK2 is the bone morphogenetic protein receptor type Ia (BMPRIa), which regulates Smad and non-Smad signaling pathways. Within cells, such as osteoblasts, chondrocytes, and adipocytes, CK2 typically binds to BMPRIa and prevents the downstream activation of Smad signaling ,59,60,61Aside from Wnts and Bone morphogenetic proteins (BMPs), CK2 is involved in signaling pathways responsible for proliferation and survival, such as Akt/Phosphatidylinositol-3-Kinase (PI3K) signaling. Specifically, Akt is phosphorylated by CK2 at serine 129, which promotes cell viability and proliferation ,76,77. TNext, CK2 activates the nuclear factor kappa B (NF-κB) signaling pathways. In this context, CK2 phosphorylates the RelA/p65 subunit of NF-κB at serine 529, leading to the activation of this protein ,84,85. FIn addition, CK2 activates the Janus Kinase/signal transducer and activator of transcription (JAK/STAT) pathway, leading to enhanced cellular survival, differentiation, and proliferation . Here, CCK2 also has a critical role in mammalian target of rapamycin (mTOR) signaling. Specifically, the inhibition of CK2 expression is associated with an increased activity of Ikaros, which binds to the mTOR gene to suppress its activity and prevent tumor formation ,99,100. Furthermore, CK2 plays a role in signaling pathways governing the DNA damage and the repair response. Specifically, CK2 responds to DNA damage and increases the repair response to accelerate the process ,106. HerFinally, an additional pathway in which CK2 is interestingly involved includes zinc signaling. As zinc can serve as a secondary messenger, it is involved in various signaling pathways. Specifically, CK2 can regulate the concentration of zinc through the phosphorylation of one of its channels, zinc channel ZIP7, located on the Golgi apparatus and endoplasmic reticulum (ER) ,111. Onc2β2, αα’β2, or α’2β2 subunits  is downregulated at the translational level. The ectopic expression of these histone demethylases suppressed SAHF and senescence-associated β-galactosidase activity. The effects of the translational downregulation of LSD1 due to the reduction in CK2 activity are complex, since it has non-histone substrates such as p53. The p53/p21Cip1/WAF1 pathway, which is an upstream regulator of SUV39h1 and H3K9me3, is also upregulated in CK2α knockdown cells. ,194. TheThe implication of CK2 in the progression of many diseases has led to its popularity as a therapeutic target. Specifically, researchers have attempted to prevent the kinase activity of CK2, thereby to prevent pathological activity. In cells, CK2 contains pocket domains for adenosine triphosphate (ATP)-binding . The pocAnother CK2 inhibitor, known as CIBG-300, is also currently in clinical trials as a therapeutic to treat cancer ,203. ThiIn addition, inhibiting the enzymatic activity of CK2 can be used to treat other disorders, such as osteoporosis (OP) and osteoarthritis (OA). CK2 is expressed in bone cells, such as osteoblasts and osteoclasts. Within these cells, CK2 is bound to the intracellular domain of BMPRIa at its phosphorylation sites. As a result, the BMP-signaling pathway is inactive until an extracellular ligand, such as BMP-2, binds to BMPRIa to cause a conformational change of BMPRIa and release CK2. This release allows BMPRII to phosphorylate BMPRIa, leading to the downstream activation of Smad and non-Smad signaling to increase bone and cartilage formation. However, as humans age, bone mineral density and cartilage decrease; these changes lead to osteoporosis and osteoarthritis, even in the absence of extracellular ligands, respectfully. The interaction between CK2 and BMPRIa can be disrupted intracellularly to increase Smad and non-Smad signaling. Here, the Nohe lab constructed mimetic peptides named CK2.1, 2.2, and 2.3, each of which contain a phosphorylation site sequence of BMPRIa. This sequence is flanked by other amino acids, and the peptides contain the antennapedia homeodomain for cellular uptake. Once endocytosed by cells, CK2.X binds to CK2 and prevents it from associating with BMPRIa. CK2.1 (phosphorylation site SYED) leads to chondrogenesis; CK2.2 (phosphorylation site SLYD) leads to adipogenesis, and CK2.3 (SLKD) leads to osteogenesis. Further, CK2.3 upregulates signaling and leads to increased mineralization in primary osteoblasts isolated from OP patients. Further, this peptide enhances bone mineralization in both in vivo and in vitro models in the absence of BMP-2 C. These This review aimed to describe the current roles of CK2 in development and adulthood, as well as describing its role in signaling pathways and disease progression. CK2 is essential for a myriad of developmental processes and the regulation of signaling pathways throughout adulthood. CK2 is involved in neurogenesis, cardiogenesis, spermatogenesis, and limb formation. Further, this enzyme has over 200 known phosphorylation substrates and is involved in pathways including cellular proliferation and survival, osteogenesis, angiogenesis, and chondrogenesis. However, due to the ubiquitous expression and constitutive activity of CK2, it has been well documented to be involved in the progression of many cancers, bone disorders, neurological and neurodegenerative disorders, immune disorders, and infectious diseases. Further, as the role of CK2 in various diseases is established, it has also become a popular target to treat the aforementioned disorders. With the novel findings about the role of the individual subunits of CK2 during disease progression, the targeting of CK2 activity may become more effective. Further, studies with its cell-lineage-specific function in contrast to its ubiquitous expression and constitutive activity can identify specific pathways for therapeutics to target. Specifically, two peptide-derived therapeutics are currently in clinical trials, known as CX-4945 and CIBG-300. These peptides have been successful in inhibiting the proliferation and progression of cancers and tumors. Further, other peptides currently being investigated in research are CK2.1 and CK2.3, which may be utilized to treat osteoarthritis and osteoporosis, respectively. Therefore, targeting CK2 and further elucidating its role in disease progression may be useful in treating many different disorders.


Blautia coccoides, and model intestinal microbes. The metabolites of the cultured broth supplemented with glucosylceramide were significantly different from those of broth not treated with glucosylceramide. The number of Gram-positive bacteria was significantly increased upon the addition of glucosylceramide compared to that in the control. Glucosylceramide endows intestinal microbes with tolerance to secondary bile acid. These results first demonstrated that glucosylceramide plays a role in the modification of intestinal microbes.Glucosylceramide is present in many foods, such as crops and fermented foods. Most glucosylceramides are not degraded or absorbed in the small intestine and pass through the large intestine. Glucosylceramide exerts versatile effects on colon tumorigenesis, skin moisture, cholesterol metabolism and improvement of intestinal microbes in vivo. However, the mechanism of action has not yet been fully elucidated. To gain insight into the effect of glucosylceramide on intestinal microbes, glucosylceramide was anaerobically incubated with the dominant intestinal microbe,  The importance of diet in health has long been recognized worldwide. The Chinese ancient document, the Shurai, written in 800 BC, describes medical doctors who specialized in foods, and the ancient Greek medical doctor, Hippocrates 460–370 BC), described the importance of food in medicine. After the scientific approach was established, the relationship between health and intestinal microbes was studied by Mechnikov, a doctor who, in 1907, proposed that intestinal microbes lower the pH of the intestinal environment and prevent intestinal decay and aging . Fructoo70 BC, deSphingolipids are lipids composed of a sphingoid base, an amide-linked fatty acid moiety, and other sensory bases. Since the discovery by Thudichum in 1874, sphingolipids have attracted attention because of their relevance to several neural disorders, such as Gaucher disease, Niemann–Pick, Fabry, Tay–Sachs, Krabbe, and Sandohoff diseases in the 1930s and their chemical structures were determined . In the koji, Aspergillus oryzae [A. luchuensis [Cryptococcus neoformans [Blautia coccoides, Bacteroides sartorii, and Hathewaya histolytica levels in vivo [Glucosylceramide is one of the sphingolipids composed of a sugar moiety and ceramide. In glucosylceramides from s oryzae , A. luchchuensis ,20,21, aoformans , a simploformans ,24,25. Goformans ,28,29,30oformans  and skinoformans ,33. To eoformans . Glucosyoformans ,36, wher in vivo . Most fo in vivo . Indeed, in vivo ,38. Seve in vivo .Bile acids are synthesized in a glycine- or taurine-conjugated form in the liver, pooled in the bladder, secreted into the duodenum, and degraded into non-ester forms by intestinal microbes . Bile acBlautia coccoides, increases the ratio of Gram-positive bacteria and endows tolerance to deoxycholic acid in certain bacteria such as Lactobacillus delbrueckii, Streptococcus mutans, Clostridium butyricum, Blautia coccoides and Enterococcus faecalis. These novel findings provide information on the mechanism of action of glucosylceramide on intestinal microbes in vivo.To gain insight into the role of glucosylceramide in vivo, glucosylceramide was added to intestinal microbes in vitro and cultured anaerobically, and its effects on intestinal microbes and metabolites were analyzed. We found that glucosylceramide alters the metabolism of and production of lactic acid by https://www.nite.go.jp/data/000107977.pdf, accessed on 5 May 2022). Bacillus subtilis (NBRC13719), Bifidobacterium pseudocatenulatum (NBRC113353), Clostridium butyricum (NBRC13949), Corynebacterium striatum (NBRC15291), Cutibacterium acnes subsp. acnes (NBRC107605), Lactobacillus delbrueckii subsp. delbrueckii (NBRC3202), Streptococcus mutans (NBRC13955), Staphylococcus epidermidis (NBRC100911), Acinetobacter radioresistens (NBRC102413), Bacteroides uniformis (NBRC113350), Enterocluster clostridioforme (NBRC113352), Comamonas terrigena (NBRC13299), Escherichia coli (NBRC3301), Parabacteroides distasonis (NBRC113806), and Pseudomonas putida (NBRC14164). Enterococcus faecalis (NBRC 100481) was commercially purchased from the National Institute of Technology and Evaluation. Blautia coccoides (JCM1395) was commercially purchased from the Japan Collection of Microorganisms, RIKEN BRC .The following 15 strains were used as the cell-mock-1, which are bacteria residing with human beings commercially available from National Institute of Technology and Evaluation, Japan  and glucosylceramide from soybeans was purchased from Nagara Science Co., Ltd. .Lactobacillus delbrueckiii subsp. lactis was cultured in MRS broth . Escherichia coli, Bacteroides uniformis, and Blautia coccoides were cultured in GAM broth . Staphylococcus epidermidis was cultured in No. 702 broth . Clostridium butyricum was cultured in CM0149 broth . Corynebacterium striatum was cultured in Trypic Soy broth . Streptococcus mutans was cultured in broth containing Tryptic soy broth of 30 g/L and yeast extract of 3 g/L. Other bacteria were cultured in the yeast casitone fatty acids (YCFA) medium and vitamin solution prepared as previously described [escribed . BacteriA glass vial  was used for anaerobic culture. Nitrogen gas infusion for 5 min was used to purge air. The vial was then locked using a PTFE/butyl cap and autoclaved. All bacteria were grown at 37 °C for 24 h under anaerobic conditions using resarulin  as an indicator of an anaerobic environment.600 = 0.1) were added individually to the sterilized broth. The total volume of the liquid in the vial was 5 mL.Microbes were inoculated into appropriate media and incubated at 37 °C for total 48 h. Cell-mock-1 was incubated as a mixture of 15 bacteria. Other bacteria were incubated individually. Considering that the deoxycholic acid concentration in the colon is 0.046–0.21 mM , the finThe cultured broth was freeze-dried, and water-soluble metabolites were extracted and analyzed using gas chromatography with flame ionization detection (GC-FID), as previously described .The lactic acid concentration in the cultured broth was analyzed using high-performance liquid chromatography (HPLC), as previously described .Cultured broth (5 mL) was collected and frozen at −27 °C. Next-generation sequencing (NGS) analysis was performed using the Techno Suruga Laboratory . Briefly, bacterial DNA was extracted using the MORA-EXTRACT kit  and FastPrep-24 5G . Conditions for PCR amplification of 16S rDNA including primers are described by Takahashi et al. . The PCRt-test. Hierarchical clustering and principal component analysis were used to group the operational taxonomic units (OTUs) of the NGS analyses. Hierarchical clustering, principal component analysis, and partial least squares-discriminant analysis (PLS-DA) were used to analyze the data obtained by GC-mass spectrometry (MS). Hierarchical clustering was performed using MetaboAnalyst ver. 5.0  [50 values were calculated using the ImageJ software. Curve fitting (four-variable logistic curve) with the ImageJ software was used to create approximate dose-response curve equations. The concentration of deoxycholic acid at a 50% reduction in bacterial concentration was considered the semi-inhibitory concentration of the bacteria. Each experiment was repeated three times, and the average value was calculated.IBM SPSS Statistics  and SIMCA-P+, ver. 13.0  were used for statistical analyses. Normality of the data was verified using the Shapiro–Wilk test. Significance of differences between the means was verified using a one-sided unpaired Student’s ay 2022) . The OTUBlautia coccoides, a major intestinal microbe involved in the decrease in visceral fat accumulation [B. coccoides were incubated with or without the addition of glucosylceramide. Considering that glucosylceramide is contained in crops and fermented foods at a concentration of 0.94–2.2 mg/g [n = 8, t-test revealed a substantial increase in citric acid content in the glucosylceramide group compared with that of the control group (p < 0.01). To further elucidate the effect of glucosylceramide on the physiology of B. coccoides, the short-chain fatty acids produced by B. coccoides were analyzed. Lactic acid levels were significantly increased in the broth supplemented with glucosylceramide compared with that of the control broth . In contrast, the Gram-negative bacterium, Acinetobacter radioresistens (p < 0.05).These results clearly indicate that glucosylceramide alters the metabolism of the intestinal microbes in this system. Next, to determine which bacteria were increased in response to glucosylceramide, NGS analysis was performed after incubation of model intestinal microbes (cell-mock-1), consisting of known ratios of bacteria that reside within human beings with glucosylceramide . Cell-modominant A, maybe brueckii B, Staphydermidis C, Strepts mutans D, Corynestriatum E, and Clutyricum F, were sesistens G, decreaConsidering that certain intestinal bacteria have a tolerance to bile acids , we hypoL. delbrueckii showed increased growth at 1.0–2.0 mM deoxycholic acid relative to that in the control (L. delbrueckii displayed an increased half-maximal inhibitory concentration value compared with that of the control, although the difference was not statistically significant (With the addition of glucosylceramide,  control A. Glucosnificant .S. epidermidis, to deoxycholic acid was also investigated. There was no significant difference in the growth or half-maximal inhibitory concentration between the glucosylceramide-treated and control S. epidermidis (S. mutans, C. striatum and C. butyricum in the presence of deoxycholic acid were also partially rescued with glucosylceramide (50 of S. mutans was significantly increased (p < 0.01, E. coli was not inhibited by deoxycholic acid (Bacteroides uniformis (Blautia coccoides and Enterococcus faecalis, against deoxycholic acid were investigated. The growth of B. coccoides was rescued with glucosylceramide at 1 mM deoxycholic acid concentration (E. faecalis was partially rescued with glucosylceramide (50 value for E. faecalis was also significantly higher after glucosylceramide addition than that in the control (The tolerance of another bacterium, dermidis . Growthsceramide C–E. IC50lic acid F and it niformis G. Furthentration H. Similaceramide I. The IC control . These rB. coccoides has a significant metabolic effect. Furthermore, the addition of glucosylceramide increased the number of Gram-positive bacteria through tolerance to deoxycholic acid. These results provide the first evidence of a mechanism underlying the action of glucosylceramide on intestinal microbes.Although we have long known that glucosylceramide is present in foods such as crops and fermented foods at 0.94–2.2 mg/g ,24,25,53This research has certain limitations and weaknesses. Adding pure components to monocultures does not necessarily reflect what occurs in the complex microbiota of the gut, since the actual gut is filled with various substances and microbes. Moreover, a certain amount of glucosylceramide, although very small , might bSecondary bile acids are formed in the large intestine by intestinal microbes and disrupt the membranes of bacteria, killing them . In the Blautia species are one of the most dominant intestinal epithelial barrier-associated bacteria, which belongs to the Lachnospiraceae family and Firmicutes phylum [Blautia species are strictly anaerobic, non-motile bacteria with spherical or oval morphologies. They can assimilate various carbohydrates such as glucose, fructose, lactose, mannose, arabinose and xylose, CO and H2/CO2, and produces acetic acid, succinic acid, lactic acid and ethanol. It is suggested from gene expression analysis that the tendency of assimilation of carbohydrates of Blautia species is lower than that of lactic acid bacteria. There is a negative correlation between the abundance of Blautia and the markers of obesity-related metabolic disorders when fed with maize-derived non-digestible feruloylated oligo- and polysaccharides [Blautia and Bifidobacterium and a low abundance of Bacteroides [Blautia has a strong taxonomic association with twin inheritance [Blautia species are also linked to abnormal Paneth cell counts [B. coccoides in the intestine increases with glucosylceramide in an animal model [Blautia species produce useful secondary metabolites such as chrysin, apigenin, desmethyllicaritin, 3′desmethyllarctigenin, bisdemethylcurcmin, demethyldemethoxycurcumin and ceramide in the intestine [s phylum . Blautiacharides , potato charides  and soy charides . Intestiteroides ,61. Conseritance , there ml counts , Crohn’sl counts . B. coccal model , but decal model . Blautiantestine .L. brueckii subsp. delbrueckii improves immune health in an animal model [Klebsiella pneumonia [C. butyricum also functions as probiotics by inducing interleukin-10-producing macrophages in inflamed mucosa via the Toll-like receptor 2/myeloid differentiation primary response gene 88 pathway [C. striatum [S. mutans [S. epidermidis [Promoted survival of Gram-positive bacteria elucidated in this study suggests the clinical benefits of glucosylceramide. For example, feeding of al model  and inhineumonia . C. buty pathway  and has  pathway . Althougstriatum , these e. mutans  and S. edermidis  form bioLacticaseibacillus casei was reported to bind glucosylceramide [Lactobacillus johnsonii to asialoganglioside [Propionibacterium freudenreichii to lactosylceramide [This study did not elucidate how or why glucosylceramide increased the ratio of Gram-positive bacteria. Since Gram-positive bacteria have a thick peptidoglycan layer, they may be better suited for the attachment of glucosylceramide and its incorporation into their structures as compared with that of Gram-negative bacteria. Consistent with this hypothesis, ceramide . In addiglioside  and Propceramide . TherefoThis study proposes a potential mechanism for the observed effects in vivo ,28,29,30S. epidermidis did not show significant recovery of tolerance to deoxycholic acid with glucosylceramide, which was inconsistent with the NGS analysis. The tolerance analysis may have lacked sufficient sensitivity to detect the difference in S. epidermidis tolerance towards deoxycholic acid, which can be elucidated using a more precise analysis tool in future studies.Our analysis of bacterial tolerance to deoxycholic acid was generally consistent with our results obtained from NGS analysis. However, Several substances have been proposed as potential prebiotics. However, there are no reports of the effects of glucosylceramide on intestinal microbes. Based on our results, glucosylceramide can be hypothesized as a type of prebiotic.Feeding of fructooligosaccharide and galactooligosaccharide decreased butyrate, increased Actinobacteria, Bifidobacterium, Acidaminococcosu and decreased Salmonella, Coprococcus, Turicbacter, Enterobacter and Phascolactobacterium in vivo . TherefoBacteroides [Sphingolipid synthesis was shown to improve the survival of intestinal teroides . Therefo2 cells treated with lipopolysaccharide in an in vitro intestinal tract model [Bacteroides fragilis toxin [Glucosylceramide in the luminal side of the intestine, which was investigated in this study, might affect that in the epithelial cells. Glucosylceramide is shown to protect CaCOct model . Furtheris toxin . TogetheB. coccoides and increases the number of Gram-positive bacteria in an intestine-simulating environment through tolerance to deoxycholic acid. These results first indicate the prebiotic-like function of glucosylceramide.In conclusion, we demonstrate that the addition of glucosylceramide induces changes in the metabolism of