Stroke is a cerebrovascular event with cerebral blood flow interruption which is caused by occlusion or bursting of cerebral vessels. At present, the main methods in treating stroke are surgical treatment, statins, and recombinant tissue-type plasminogen activator (rt-PA). Relatively, traditional Chinese medicine (TCM) has widely been used at clinical level in China and some countries in Asia. Xiao-Xu-Ming decoction (XXMD) is a classical and widely used prescription in treating stroke in China. However, the material basis of effect and the action principle of XXMD are still not clear. To solve this issue, we designed a new system pharmacology strategy that combined targets of XXMD and the pathogenetic genes of stroke to construct a functional response space (FRS). The effective proteins from this space were determined by using a novel node importance calculation method, and then the key functional components group (KFCG) that could mediate the effective proteins was selected based on the dynamic programming strategy. The results showed that enriched pathways of effective proteins selected from FRS could cover 99.10% of enriched pathways of reference targets, which were defined by overlapping of component targets and pathogenetic genes. Targets of optimized KFCG with 56 components can be enriched into 166 pathways that covered 80.43% of 138 pathways of 1,012 pathogenetic genes. A component potential effect score (PES) calculation model was constructed to calculate the comprehensive effective score of components in the components-targets-pathways (C-T-P) network of KFCGs, and showed that ferulic acid, zingerone, and vanillic acid had the highest PESs. Prediction and docking simulations show that these components can affect stroke synergistically through genes such as MEK, NFκB, and PI3K in PI3K-Akt, cAMP, and MAPK cascade signals. Finally, ferulic acid, zingerone, and vanillic acid were tested to be protective for PC12 cells and HT22 cells in increasing cell viabilities after oxygen and glucose deprivation (OGD). Our proposed strategy could improve the accuracy on decoding KFCGs of XXMD and provide a methodologic reference for the optimization, mechanism analysis, and secondary development of the formula in TCM. In the treatment of complex diseases, TCM usually work in the form of multicomponents and multi-targets, and these components and targets could form complex network. This network contains effective action, side effective and toxicity networks. How to extract the effective action network and obtain the KFCG is the key goal of the optimization of prescriptions in TCM.At present, methods have been established by decoding the potential mechanisms of prescription in treating complex disease, including collections of Chinese medicine components, active ingredients screening, targets prediction, pathways analyzations, and inference of the mechanism of targets. These analysis methods have successfully parsed the mechanisms of some treatments of prescriptions in TCM on the complex disease. For example, Pan et al. used the network pharmacology model and experimental validation to confirm that quercetin in Huanglian Decoction (HLD) affected the synthesis of glucose transporter 4 (GLUT4) by interfering with the insulin signaling pathway and improved the therapeutic effect of HLD on type 2 diabetes (T2DM) . Xu et aHowever, most of current analyses are based on drug-targets or pathogenetic genes. There is a lack of the quantification of network propagation of the intervention effects between drug-targets and pathogenetic genes. Thus, it is desirable to design a novel strategy to solve these problems.Stroke is a cerebrovascular event with cerebral blood flow interruption which is caused by occlusion or bursting of cerebral vessels. It can cause multiple functional damage of body and sustain with some complications . In stroIn addition, during the animal experiments, XXMD has been proved to keep mitochondrial function , inhibitEphedra alata Decne.  (50 g), Stephania tetrandra S.Moore  (50 g), Ginseng quinquefolium (L.) Alph.Wood  (50 g), Scutellaria baicalensis Georgi  (50 g), Cinnamomum cassia (L.) J.Presl  (50 g), Glycyrrhiza uralensis Fisch.  (50 g), Paeonia lactiflora Pall.  (50 g), Ligusticum striatum DC.  (50 g), Amygdalus communis L.  (50 g), Aconitum wilsonii Stapf ex Veitch , Saposhnikovia divaricata (Turcz.) Schischk.  (75 g), and Zingiber officinale Roscoe  (250 g). In this prescription, MH  and pathway enrichment analysis, docking simulations, and experimental validations. It can be superior in finding the KFCG of XXMD more comprehensively and can explain the relationship between stroke pathogenetic genes and targets more clearly. Our strategy could provide a methodologic reference for prescription optimization and secondary development.We query stroke in DisGeNET and obtain a total of 14 stroke-related IDs that are consistent with the IDs of international classification of diseases (ICD10). The IDs are as follows: C0948008, C1857287, C3178801, C0242129, C0262469, C1112433, C1299567, C1298680, C0265113, C0038454, C0740392, C0751956, C0553692, and C3536593. Among these IDs, C0553692 is related to hemorrhagic stroke. The genes obtained from these above 14 IDs were treated as pathogenetic genes of stroke .To construct comprehensive weighted gene network of stroke, the protein–protein interactions (PPI) data were collected from public web servers CMGRN and PTHGRN . PathogeAll herbal components of XXMD were extracted from literatures and two published natural product databases: traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP)  and the The potential active components of XXMD were selected from the TCMSP database and those detected in herbs. The properties of all components of XXMD were retrieved from TCMSP, including molecular weight (MW), oral bioavailability (O B) , Caco-2 permeability (Caco-2), and drug-likeness  . Three aTo obtain the targets of potential active components in XXMD, Open Babel toolkit (version 2.41) was used in the conversion of all chemical structures into canonical SMILES. After that, the commonly used tools, i.e., Similarity Ensemble Approach (SEA) , HitPickNode importance is an important topological property and can be used to evaluate the influence of nodes among the network. The nodes whose node importance is larger than the average node importance of all nodes are treated as critical roles and hub nodes in the network . Here, wv and node t, v to t; x, v represent nodes (genes); s and x are connected.Nim represents the node importance; After being quantized, The new variable G represented the nodes in the network. Each G responds to its unique Q.g represented a node g.FRS represent the set of genes that were selected with our proposed method from the unite of pathogenetic genes and XXMD targets. N represented natural number. To optimize effective components and get the KFCG, which would be used to illustrate the potential molecular mechanism of XXMD in the therapy of stroke, we designed a components contribution ratio (CCR) model to select KFCG:For kx = Wky = max { Wk1: Wkn } and n represent the nth component; U represent unite; Uk represent unite of component responding to genes in FRS; Ukn represent the targets unite of kn; W represent the coverage of targets in Uj.if To quantify the comprehensive function of topological affect and control affection of KFCGs in the C-T-P network, we designed a component potential effect score calculation model. Usually, higher degree of components represents greater influence. The higher degree of the neighbor nodes of a component represents the higher control affection of this component in the topology of the network. It means that a component has a stronger control affection in the network when this component has much more targets and signaling pathways. Based on the above properties, we designed a component potential effect score calculation model, which was considered with both the network topology importance of KFCGs and the functional control ability of KFCGs. The specific model is as follows:kn, is the size of its neighborhood. The degree of node n is the number of edges reaching n, which is equivalent to kn. kp) represents the length of a shortest path between pathway p and component w. The distp,(w) is equal to infinite if Cp)( ≠ Cw) and PubChem (https://pubchem.ncbi.nlm.nih.gov/) and obtained proteins coded by genes involved in the comprehensive pathways. The affinity method and pyMOL were conducted in docking simulations and graph creation, respectively.Computer-simulated modeling can contribute to the prediction in the likelihood of molecular interactions. We obtained the 3D conformer of XXMD KFCG from ZINC  and RPMI-1640 were purchased from ThermoFisher Biochemical Products (Beijing) Co., Ltd. Hypoxic bags were purchased from Mitsubishi Gas Chemical Company, Inc. (Japanese). Ferulic acid (≥98% purity by HPLC), zingerone (≥98% purity by HPLC), and vanillic acid (≥97% purity by HPLC) were purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd. . Caryophyllene oxide (≥99% purity by HPLC) and methylephedrine hydrochloride (≥98% purity by HPLC) were purchased from TargetMol (United States) and Shenzhen Polymeri Biochemical Technology Co., Ltd. , respectively. Edaravone (≥99% purity by HPLC) was purchased from TargetMol (United States). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Japanese).2 atmosphere.The PC12 cell line was obtained from CHI SCIENTIFIC  and cultured in RPMI-1640 with FBS, penicillin 100 U/mL, streptomycin 100 μg/ml, respectively, at 37°C in a fully humidified 5% COin vitro model in studying the pathology and pharmacology of ischemic damage  is a well-established c damage . Considec damage ; we studfor 18 h .in vitro experiments with PC12 cells. Edaravone that has been shown to reduce cell death was selected as a positive drug  and two non-KFCG components (caryophyllene oxide and methylephedrine hydrochloride) were selected to validate our model ive drug . Effects4 per/well). After 24 h incubation, cells were treated without any components for 18 h in OGD. A control group without the treatments of OGD and components was taken at the same time. After the OGD period, cells were treated with 0.01, 0.1, 1, 10, 100, and 1,000 μM ferulic acid  of XXMD and defined the genes that were included in the FRS as effective proteins. Then we evaluated the effective proteins with three evaluation indicators: 1). the proportion of effective proteins in the number of UETs; 2). the proportion of effective proteins in the number of UETs enriched pathways; 3). the proportion of effective proteins in the number of UETs enriched GO-terms.We used four methods in the optimization of targets and pathogenetic genes. Among these four methods, the best optimal method was method 1, our proposed node importance calculation method . Based oThe numbers of effective protein-enriched pathways and GO-terms were 180 and 3,043, respectively . Before KEGG and GO enrichment analysis of the effective proteins in FRS showed that the effective proteins could play a role in treating stroke. Among the 180 signaling pathways of the effective proteins , PI3K-AkThe CCR model was established to optimize the effective components and get the KFCG that could be used to illustrate the potential molecular mechanism of XXMD in the treatment of stroke. According to the contribution accumulation score ranking, targets of the top seven components including vanillic acid, ferulic acid, L-tryptophan, Dihydrocapsaicin, zingerone, rhamnetin, and Norwogonin could cover 50.00% of effective proteins. For further analysis, targets of the top 56 components could cover 90.00% of effective proteins, and these 56 components were selected as KFCG . The higTo analyze XXMD in the treatment of stroke at the functional level, we performed pathway analysis using KFCG targets. The number of KFCG targets enriched pathways is 166 which can cover 80.43% of pathogenetic genes enriched pathways .These KFCG targets were involved in 166 pathways such as Retrograde endocannabinoid signaling (hsa04723), neuroactive ligand-receptor interaction (hsa04080), and cAMP signaling pathway (hsa04024). Most of these signaling pathways have been reported to be related to the occurrence of stroke or the recovery in poststroke. For example, neuroactive ligand–receptor interaction can affect the expression of genes such as GRM5, GRIK1, GRIK3, GABRA3, ADRA2C, and VIPR2 , therebyStroke is classified into hemorrhagic stroke and IS, and has the characteristics of multisystem cross-effects, mainly involving the cerebrovascular system and nervous system. Studies have shown that the occurrence of stroke is related to cerebral microvessels, neural information transmission, neuronal activity , and infTo further explore the potential mechanism of XXMD in the treatment of stroke, we constructed a comprehensive pathway with these four pathways, VEGF signaling pathway (hsa04370), MAPK signaling pathway (hsa04010), PI3K-Akt signaling pathway (hsa04151), and cAMP signaling pathway (hsa04024) .1) KFCG can benefit for cell survival and DNA repairation.Combining KFCG targets and these four pathways, we speculated the potential mechanism of XXMD to be two important aspects: 1) KFCG can benefit for cell proligeration, migration, survival, and DNA repair. 2) KFCG can regulate the activities of proteins which can interact between multiple pathways to play a role in the treatment of stroke by XXMD.2+, K+, and Na+ in cell membrane and affecting the process of pancreatic secretion, bile secretion, and cardiac muscle contraction.In the cAMP signaling pathway, KFCG can activate the activities of PI3K   and Akt In the VEGF signaling pathway, KFCG can regulate the activities of VEGF (gene VEGFA), VEGFR2 (gene KDR), PLC , and PKC , thereby affecting the calcium signaling pathway. KFCG can regulate the activities of PKC , Ras  , Raf-1 , phosphoIn the MAPK signaling pathway, KFCG can regulate the activities of Ras , MEK1 (gene MAP2K1), MEK2 (gene MAP2K2)  and c-fo2) KFCG can regulate the activities of proteins which can interact between multiple pathways to play a role in the treatment of stroke by XXMD.In the PI3K-AKT signaling pathway, KFCG can regulate the activities of TLR2/4  , Rac1 g, AKT ge and can For example, in the 585 KFCG target enrichments, four proteins existed in three pathways and whose activities could be affected by KFCG, and were as MEK , NFκB , PI3K , and Ras  . MeanwhiThe results of the compressed stroke pathways indicated that the KFCG were important and effective in the stroke treatment, and these effects could be achieved through a multichannel biological process. This result suggested that we need to consider the relationship of components and targets and need to consider the interactions between different pathways in the treatment of stroke.Based on the component potential effect score calculation model we constructed above, we calculated the PESs of 56 KFCG . These PTo validate the effect of KFCG on stroke and further validate whether the three components, ferulic acid, vanillic acid, and zingerone, could combine with targets effectively, we conducted molecular docking by 56 components with 3D conformer and 136 protein structures responding to 49 genes in comprehensive pathways and obtained 66,967 binding relationships in the docking results .According to literatures, the lower value of affinity represents the more stable binding between protein and ligand and represent the better binding energy in protein–ligand interactions . The infAmong the 66,967 binding relationships, the three components, ferulic acid, vanillic acid, and zingerone, which were selected from the component potential effect score calculation model, showed good combinations with proteins.Component COM14 can bind 37 genes and 68 proteins with the average binding affinity of −6.44 kcal/mol. It can bind best with protein structure 5ebz responding to gene CHUK with binding affinity of −7.6 kcal/mol, and be equal to COM14-1t46 and COM14-5m6v  1–F2. CoIn addition, we found all the 56 KFCGs could have good binding ability with proteins. For example, MOL001755 can bind best with protein structure 5m6v responding to gene PRKACA with binding affinity of −11.40 kcal/mol 1,A2, foTo test the predictive power of our proposed model, we defined the 1,489 components that were eliminated by our models as the non-KFCG . Three cin vitro experiments proved that our proposed model could be used in selecting the KFCG of XXMD in treating stroke, effectively and accurately.These results in the The main purpose of formula optimization is to reduce the non-pharmacological factors and improve the curative effect of the formula. According to the theory of TCM, different medicinal components are composed as the formula. But whether all of these components are necessary in treating disease still needs further analysis and verifications. During the optimization of formula, some medicine components with certain drug effects can be screened out, and thus the material composition and the effect of formula will become more simplified and clarified.XXMD has a long history in China. Both clinical data and animal experimental evidence have confirmed that XXMD can effectively promote the treatment of stroke . HoweverTo better optimize the classical formula XXMD for the treatment of stroke, network pharmacology and mathematical methods were employed to investigate relatively optimal KFCG. Network pharmacology has the characteristics of system and integrity, which is in line with the philosophical view of “Chinese medicine is holistic” in Chinese medicine research. At present, network pharmacology has been widely used in the treatment of complex diseases by TCM. It emphasizes the concept of multi-target regulation of signaling pathways and helps to improve the therapeutic effect of drugs and reduce toxic and side effects. For example, network pharmacology can contribute to study the potential molecular mechanisms of TCM prescriptions in the treatment of complex diseases, such as “treating different diseases with the same treatment, treating the same diseases with different treatment.” However, there are a few reports on the optimization of TCM prescriptions based on network pharmacology, especially in the study of the classical formula XXMD.1) In this study, we proposed a new method for calculating the importance of nodes, which takes the connectivity and radiation influence of node in the network into account. Based on this method , we dete2) In recent years, network pharmacology provides a powerful tool for exploring the compatibility and mechanism of the TCM formula but with some limitations. For example, there are still lack researches and recommendations on the optimization of XXMD pharmaceutical components even though wet-lab experiments, which are often used in validating the efficacy and potential mechanism of XXMD in the treatment of stroke. In this study, we obtained better performance in the XXMD components optimization.In our study, we propose an integrative strategy to optimize XXMD, obtain the key components of XXMD, and analyze the potential mechanism of these components with comprehensive pathways and docking simulations. Our approach has two advantages:Based on the new model proposed and used in this study, we deeply optimized the formula and obtained the KFCG of XXMD, which can represent the effect of XXMD in treating stroke. We used comprehensive assessments to confirm that KFCG could represent the molecular effect of XXMD. Based on the traditional functional annotations of targets, pathogenetic genes, and KFCG targets, we calculated the functional coverage obtained in our reverse optimization model. As a result, it was found that KFCG could respond well to the combination effects of different chemical components extracted from various herbs in XXMD. The advantages of this study are that FRS and the component reverse search strategy are applied to find the KFCG of XXMD, providing a methodological reference to the study and development of TCM.1-10I2, Based on the FRS, we optimized the contribution score by using the CCR model, and finally obtained KFCG with 56 components. Enriched pathways of KFCG targets can cover 80.43% of the enriched pathways of pathogenetic genes, respectively , meaningin vitro experiments in this study, we estimated that the three components, zingerone, ferulic acid, vanillic acid, could be effectively absorbed and transferred to the brain mainly based on some reports and their properties provided from the TCMSP database (https://old.tcmsp-e.com/load_intro.php?id=29). For example, zingerone had been detected in XXMD even though its concentration had not been reported , whereas the solubility value of −2∼0 represents very easily soluble and −4∼−2 represents soluble. These three components have been proved to across BBB and into brain  We presume that components can be 100% extracted and 100% absorbed, and presume that the weight of a human is 70 kg. 2) CX, RS, and SJ are 50, 50, and 250 g in a dose of XXMD herbs, respectively. The proportions of ferulic acid in CX and RS are 0.19 mg/g and 32.70 mg/g, respectively . The proto brain . 5) Repoto brain , the ratto brain , and theto brain .in vitro experiments, we can know that the concentration 0.34 μM of vanillic acid is located in the concentration range which has the significant promotion for the survival of PC12 cells  into the brain which were estimated based on literatures could also significantly promote the survival of PC12 cells because of that the concentrations into the brain were estimated based on some literatures and their accuracy needs further studies. Results in PC12 cells can be used as a reference for the further study of the key functional components of XXMD. Relatively, if it is necessary to further study whether the key functional components have clinical effect in treating stoke, these components need to provide with further and in-depth verifications and experiments, which is one of the aspects of our efforts in the future.In our study, there are two original purposes: 1) the main purpose of our study is to put forward new methods and build new models for the compound optimization of XXMD in treating stroke. We hope to provide reference for the study of key functional components of XXMD in treating stroke. 2) After screening the key functional components, the in vitro experiments, the experiment with PC12 cells showed a better dose–response (in vitro experiments with PC12 cells and HT22 cells (During the response . For the22 cells  showed t22 cells . In thisOn the whole, we proposed a reverse optimization model based on the association of pathogenetic genes and component targets to improve the accuracy on decoding KFCG of XXMD, providing reference for the optimization and mechanism analysis of the formula in TCM.


Our findings reveal that ApNL1 from the pea aphid can regulate both bone morphogenic protein and receptor tyrosine kinase signaling pathways, and unexpectedly, that the clip domain is not essential for its antagonism of bone morphogenic protein signaling. Our findings indicate that ancestral noggin/noggin-like genes were multifunctional regulators of signaling that have specialized to regulate multiple cell signaling pathways during the evolution of animals.Noggin is an extracellular cysteine knot protein that plays a crucial role in vertebrate dorsoventral patterning. Noggin binds and inhibits the activity of bone morphogenetic proteins via a conserved N-terminal clip domain. Noncanonical orthologs of Noggin that lack a clip domain (“Noggin-like” proteins) are encoded in many arthropod genomes and are thought to have evolved into receptor tyrosine kinase ligands that promote Torso/receptor tyrosine kinase signaling rather than inhibiting bone morphogenic protein signaling. Here, we examined the molecular function of  In animals, cell-to-cell communication depends on a small number of evolutionarily conserved cell signaling receptor superfamilies, 2 of which are the transforming growth factor-β (TGFβ) and receptor tyrosine kinases (RTK) . These pXenopus laevis, where it is secreted from Spemann’s organizer and antagonizes BMP signaling. This activity generates a morphogen gradient along the dorsal-ventral axis, causing prospective ventral mesoderm to become dorsal mesoderm  and prospective epidermis to become neuroectoderm  cell signaling pathway . Noggin noggin (nog) genes are present in most animal genomes, they were thought to be absent from insects (Acyrthosiphon pisum), first revealed genes similar to nog and noggin-like (nog-like)  patterning and Drosophila terminal patterning. Our findings suggest that pea aphid Noggin-like 1 can repress BMP signaling in Xenopus DV patterning and activate MAPK pathway in Xenopus animal caps, while pea aphid Noggin-like 2 only activates MAPK pathway in Xenopus animal caps and Drosophila terminal patterning. These findings support a model whereby ancient extracellular regulators could have been a multifunctional protein, which was later co-opted into other developmental processes, losing its primary role.Within the Noggin/Noggin-like/Trunk/PTTH family of proteins , at least 2 very different signaling activities occur. Canonical Noggins and some Noggin-like proteins function in BMP signaling, while Trunk and PTTH activate RTKs. This implies that this family of proteins has either switched their function entirely from BMP repression to MAPK activation in the insect lineage or that the ancestral Noggin/Noggin-like proteins may be multifunctional and have both MAPK and BMP signaling activity. To distinguish between these possibilities, we focused on understanding the effect on BMP and MAPK signaling of insect ApNL1 (encoding the amino acids PVPSNDPGVIDLIEMP) was deleted from ApNL1 to synthesize ApNL1ΔClip and inserted into ApNL2 and Drosophila melanogaster trunk  to synthesize ApNL2+Clip and DmTrk+Clip. The candidate genes  as described previously  is 100 ng/ml. Animal caps were dissected as previously described . After 4 days of polymerization, the embedding molds were removed by placing the embedding tray at 60°C for 5 min. Next, animal caps were sectioned at 2 µm using a microtome (Reichert-Jung). The sections were then placed on a water bath at 25°C and transferred to microscope slides, later dried at 60°C for an hour. Microscopic slides were flooded with Polychrome I (Sigma-Aldrich) for 60 s and washed with running water. Then the slides were flooded with Polychrome II (Sigma-Aldrich) for 30 s and later washed with running water, followed by a few dips in 95% ethanol and washing with running water. The slides were then dried in a fume hood. Entellan rapid mounting medium (Merck Millipore) was applied to dried slides, and coverslips were placed on them. The slides were left overnight to dry and imaged using an Olympus BX51 microscope.2O. The mixture was then placed in a thermocycler (Eppendorf), and the following incubation periods were set up to run: 25°C for 5 min, 42°C for an hour, 65°C for 20 min. The concentrations of cDNA samples were measured using a Nanodrop2000 spectrophotometer and then stored at −20°C. Mesoderm and neural markers  was used to isolate genomic RNA (gRNA) from the animal caps. Isolated RNAs were then stored at −80°C. The amount of gRNA used in reverse transcription was consistent for each sample (150 ng). The mixture for first cDNA synthesis consisted of 150 ng of template RNA, 2 μl of Random Primers (Invitrogen), 2 μl of 10 × M-MuLV buffer (NEB), 1 μl of M-MuLV Reverse transcriptase (NEB), 1 μl of 10 mM dNTPs (Invitrogen), 0.2 μl of RNase inhibitor (Invitrogen) and the volume was made up to 20 μl with dH markers  were sel markers .Drosophila stocks were used: Oregon-R (BL5), GAL4::VP16.nos.UTR (BL7253) and trkΔ—a null mutant of trunk (Drosophila were obtained by microinjection of pUASP plasmid containing the gene of interest into PhiC31 source Drosophila—attP docking site 86F6 (24749) (BestGene Inc). Ectopic expression was driven in the adult ovary by crossing pUASP lines (GAL4::VP16.nos.UTR (BL7253) driver line and trkΔ; GAL4::VP16.nos.UTR driver lines. The flies were maintained, and crosses were set up at 25°C.The following SP lines  with theDrosophila adults were allowed to lay eggs on apple juice plates containing yeast paste for 4 h before being removed for in situ hybridization chain reaction (HCR) for Dm-tailless (tll) as described previously   was performed using the established protocol (Flies (2 days old) were left for 24 h in fly food containing Baker’s yeast before dissection. Xenopus, BMP signaling is blocked locally by Noggin, leading to dorsal development . If noggin is overexpressed in Xenopus embryos, it induces dorsal structures in embryos or induces a secondary axis when ventrally expressed (nog/nog-like genes from A. pisum   and expression of the mesoderm marker (actc1.L), indicating that it can activate MAPK signaling activity in Xenopus animal caps, plausibly via Xenopus RTKs  and cement gland (7/21), consistent with BMP repression, but also produced mesenchyme  and mesoderm markers . Our findings imply that when overexpressed, ApNL1 may both repress BMP signaling and activate the MAPK pathway, while ApNL2 and Trunk only activate the MAPK pathway in Xenopus animal caps.Injections of canonical Noggin pus RTKs , Fig. 2cme 9/21, . RT-PCR e 18/18,  and exprDrosophila terminal patterning—a robust model system to study the Torso/MAPK pathway  mutant background , nor cause broadening of the terminal tailless (tll) expression domain  or tll expression  without any rescue of tll expression  and tll expression  than females from other crosses and none in our trkΔ rescue assay. Examination of nos>ApNL1 female ovarioles revealed the arrest of stage 9 egg chambers during oogenesis (Drosophila death caspase 1)  85/85,  and posi) 85/85, , in stag embryos , likely A. pisum have different molecular functions, with ApNL1 acting to inhibit BMP/Dpp signaling and activate MAPK, and ApNL2 acting to activate MAPK/RTK signaling, similar to Trunk and PTTH.The relationship between Noggin proteins, which act to repress BMP signaling, and Trunk/PTTH proteins, which activate MAPK, implies that the function of these proteins has switched in their evolution. The evolution of Trunk/PTTH, presumably from a Noggin/Noggin-like ancestor, involved loss of the clip domain, loss of BMP inhibitory activity, and gain of interaction with Torso. In nonholometabolous insect genomes, a number of Noggin and Noggin-like proteins seem to be present, sometimes with Trunk/PTTH proteins as well (Drosophila over-expression of ApNL1 causes phenotypes in the embryo and ovary, whereas over-expression of Xnog.L does not. It is possible this stronger inhibition of BMP by ApNL1 reflects the fact that it is carried out by a part of the protein other than the clip domain.The clip domain, which has been shown to be required for BMP inhibition by hNoggin (Xenopus implies that this protein may be multifunctional and that the evolutionary history of Noggins and Trunk/PTTH is not a switch from one form of signaling to another, but the specialization of an ancestrally multifunctional protein into either regulating BMP or MAPK signaling. Noggin/Noggin-like proteins have also been shown to have activities on wnt signaling pathways (That ApNL1, while strongly inhibiting BMP, also has weak MAPK activity in All data for this study are presented in the manuscript or in the GENETICS online.iyac049_Supplementary_Figs-TableClick here for additional data file.iyac049_Supplemental_Mateiral_LegendClick here for additional data file.


Urolithin A (UA) is a metabolite produced by gut microbiota from ingested ellagic acid. Although the effect of ellagic acid intake on vascular endothelial function (VEF) improvement has been reported, the effect of UA intake on VEF improvement remains obscure. In addition, UA has been reported to improve the intestinal barrier function, and UA may have improved VEF by gut microbiome alteration.In this study, we conducted a clinical trial to explore and analyze the effects of UA intake on vascular endothelial function (VEF) and characteristics of the intestinal environment, such as gut microbiome profiling and organic acid composition.A placebo-controlled, randomized, double-blinded, parallel group trial was conducted on participants who could metabolize small amounts of UA from ellagic acid (non-UA producers) and had relatively poor VEF. VEF was assessed using the flow-mediated vasodilatation (FMD) score. Participants were administered placebo, UA 10 mg/day, or UA 50 mg/day for 12 weeks. FMD was measured and fecal samples were collected at 0, 4, 8, and 12 weeks of treatment. Gut microbiome analysis and organic acid level measurements were performed to evaluate the effects of UA intake on the intestinal environment. This clinical trial is publicly registered at the UMIN-CTR, trial number: UMIN000042014.p < 0.05, not corrected). Participants whose FMD scores improved with UA intake had poor baseline FMD values as well as a low Bacillota/Bacteroidota ratio.The gut microbiota of the UA 50 mg/day group showed a significant increase in alpha diversity (Faith’s phylogenetic diversity). Four and nine microbial genera were significantly altered in the UA 10 mg/day and UA 50 mg/day groups, respectively (Urolithin A intake alters the gut microbiota and improves their alpha diversity. In addition, the effect of UA on VEF correlated with the individual gut microbiota. Our results have practical implications for a new approach to providing healthcare that focuses on intestinal environment-based diet therapy. Cardiovascular diseases are the leading cause of death worldwide, having caused 17.8 million deaths in 2019. This figure is expected to increase to 23 million by 2030 –7.Bifidobacterium animalis and arginine improves VEF via gut microbiota-derived polyamines  scores.A randomized, double-blinded, placebo-controlled, three-way parallel-group study was conducted on Japanese individuals for 12 weeks . Consent1). The inclusion criteria were as follows:This trial was conducted with the approval of the Clinical Trial Ethics Review Committee of the Chiyoda Paramedical Care Clinic Male or female aged between 40 and 65 years.(2)Individuals whose FMD was below 7.0%.(3)Individuals who are UA non-producer or low producer.(4)Individuals who could understand the study procedure, agreed to participate in the study, and provided informed consent prior to the study.The exclusion criteria were as follows:(1)Individuals who were currently receiving medication.(2)Individuals who planned to or already had taken medication  a month prior to the trial, which would have affected the results.(3)Individuals who regularly consumed food for specified health uses, food with function claims, supplements, and/or health foods that would affect the trial results, more than three times per week.(4)Individuals who were breastfeeding, pregnant, or are planning pregnancy.(5)Individuals who have current medical history/anamnesis of severe cardiac, hepatic, renal, or digestive disease.(6)Individuals with high alcohol consumption.(7)Individuals with a history of smoking. However, if the individual had quit smoking for more than a month prior to trial, the individual could be included.(8)Individuals with irregular lifestyle and diet.(9)Individuals who have allergies to food or medication.(10)Individuals who are currently participating, have participated in other studies on medicine or food a month prior to the trial, or participated in other clinical trials after agreeing to participate in this trial.(11)Individuals who had donated more than 200 ml of blood and/or blood components a month prior to the trial.(12)Males who had donated more than 400 ml of blood and/or blood components 3 months prior to the trial.(13)Females who had donated more than 400 ml of blood and/or blood components 4 months prior to the trial.(14)Males who will receive over 1,200 ml of blood and/or blood components, when the sampling amounts within the last 12 months are added to the planned sampling amounts of this study.(15)Females who will receive over 800 ml of blood and/or blood components when the sampling amounts within the last 12 months are added to the planned sampling amounts of this study.(16)Others who were deemed ineligible by the principal investigator or sub-investigator.A total of 157 individuals participated in the pre-trial, from which 36 were selected for the main trial based on the inclusion and exclusion criteria. To select UA non-producers or low-producers, the participants were instructed to consume a commercially available food containing ellagic acid  before sleeping on the night of the pre-test, and the first urine sample was collected early the next day, which was analyzed to measure the urinary UA. Those with urinary creatinine-corrected UA less than 1.00 nM/mg were categorized as UA non-producers or low producers. Among the initial 157 participants, 73, 14, and 70 participants were designated as UA non-producers, UA low-producers, and UA-producers, respectively. Randomization was performed using the block-stratified randomization method with the participant assignment manager. Participants who met the inclusion criteria were assigned to three groups  by stratification, while accounting for age, sex, pre-tested FMD, and blood pressure values. Subsequently, the symbols “P,” “Q”, and “R” were randomly assigned to each group of participants. A test food assignment table with the test food symbol and participant identification code was prepared. Immediately after assignment to the test food, the table was sealed and concealed by the participant assignment manager. The table was only disclosed to the test analyst, investigator, and test-sharing doctor after data collection. A total of 36 participants were included in the main trial. One participant dropped out due to refusal to visit the hospital. Hence, 35 participants completed the trial.DRA014918).The extraction and measurement of DNA and organic acids from fecal samples were performed as previously described . Brieflyg for 10 min) using a Shake Master . Fecal samples (10 mg) were suspended in 1,000 μl of internal standard (crotonic acid), followed by 500 μl of concentrated HCl and 2,000 μl of ether added to each tube. The tubes were then vigorously shaken  using a Shake Master and centrifuged at 10,000 × g for 10 min. After centrifugation, 80 μl of the top ether layer was collected and added to an Agilent crimp-cap vial containing 16 μl of N-tert-butyldimethylsilyl N-methyltrifluoroacetamide for gas chromatography. The vials were capped with a crimp-top natural rubber/polytetrafluoroethylene seal type seven aluminum silver 11 mm chromacol cap and sealed using a crimper. After tapping and mixing, the vials were tightly sealed with parafilm and heated in a water bath at 80°C for 20 min. The capped vials were then left overnight at room temperature for derivatization. Calibration was achieved using standard solutions of derivatized formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric, lactic, and succinic acids, as described for the test samples. The final concentrations of each standard were 50, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, and 0.01 mM. The derivatized samples were run through a 7,890 series GC–MS system  fitted with a DB-5ms column . Helium was used as the carrier gas and delivered at a flow rate of 1.2 ml/min. The head pressure was set at 10.5 psi with a split ratio of 100:1. The injector, ion source, quadrupole mass spectrometer, and transfer line were set to 250, 230, 150, and 260°C, respectively. Two microliters of each sample were injected with a run time of 30 min. The measurement was controlled using the Agilent MassHunter Workstation Data Acquisition software , and the obtained data were analyzed using the Agilent MassHunter Quantitative Analysis software . Organic acid data are presented in Metabolites were extracted from feces using the following procedure. Freeze-dried feces were disrupted with 3.0 mm zirconia beads by vigorous shaking  was used . Primer All statistical analyses were performed using Python scripts (version 3.7.6). Multidimensional scaling was performed using unweighted/weighted UniFrac distance calculated from the microbiome amplicon sequence variants (ASVs) data (scikit-learn version 0.20.0). For pairwise comparison of bacteria, the Wilcoxon signed-rank test with Benjamini–Hochberg false discovery rate correction was used . A trend test (Jonckheere-Terpstra test) was also performed to identify consistent changes in bacteria between the placebo, UA 10 mg/day and UA 50 mg/day groups (scipy version 1.5.2). Bacteria with mean relative abundance below 0.001 were excluded from the comparison. In the comparison of organic acids, two-way analysis of variance were used (statsmodels version 0.10.0). In this study, the test food effect size for each participant was defined as the responder score and was used to determine whether the effects depended on individual basal characteristics. The response score was defined as the value of each individual after 8 weeks of intake minus the baseline. In the responder feature analysis, the Spearman rank correlation coefficient and the test for no correlation were used (scipy version 1.5.2).p-value in Kruskal-Wallis test: 0.148; A placebo-controlled, randomized, double-blinded, parallel group trial was conducted on 36 Japanese UA non-producers or low-producers . BaselinTo investigate the effect of UA intake on the participants’ gut microbiota, we performed 16S rRNA gene-based microbiome analysis at 0 and 8 weeks following UA intake. No significant improvements in all VEF score  were observed . As therq-value >0.10). In addition, a trend test revealed consistent changes in bacteria in the placebo, UA 10 mg/day, and UA 50 mg/day groups, with changes in the 50 mg/day group consistent with those in the three groups .A correlation analysis was performed to investigate the relationship between the participants’ response to UA intake and their clinical features, such as gut microbiota, organic acids, and VEF score. We defined the difference in FMD scores before and after UA intake as FMD response scores, and responses for other clinical features were defined in a similar fashion. Correlation analysis between FMD response score and baseline clinical features showed that baseline FMD values were negatively correlated with FMD response scores for participants in the placebo and UA 10 mg/day groups . In the relation . None ofrelation . The resrelation . After Fp = 0.078), suggesting that UA intake potentially improves VEF. However, this result did not account for the fact that multiple groups and time periods were involved in this study because of the small sample size. Therefore, a larger interventional study is necessary to demonstrate the effect of UA intake on VEF. Although the UA 50 mg/day group was assumed to be more effective, there was no difference at any time point in this clinical trial. This may be due to higher FMD scores at week 0 in the UA 50 mg/day group  coprostanoligenes group, and (Eubacterium) hallii group was significantly higher in the UA 50 mg/day group than that in the placebo group. Ruminiclostridium 5 enrichment restored circadian rhythm disturbances and decreased body temperature and sleep disturbances in rats . Gordonibacter is a minor gut microbe that may be below the quantification limit. Therefore, a quantitative polymerase chain reaction test is necessary to evaluate the abundance of this genus.Differences in individual responses to the oral intake of food and drugs have been observed, with studies showing a link with gut microbiota , 19. In lithin C . Regardie scores . Assumindetected . GordoniThe limitation of this study was the small sample size (approximately 12 participants per group). Although this study used two dosages, the results were inconsistent. Thus, a large interventional study or an animal model study is necessary to validate these findings.Ruminiclostridium 5 in the 50 mg/day UA group. In addition, correlation analysis suggested that UA supplementation may be effective in UA non-producers. By analyzing the gut microbiota and its association with FMD improvement the results obtained have practical implications for a new approach to providing healthcare that focuses on intestinal environment-based diet therapy.This randomized controlled trial showed that UA has a modest effect on gut microbiota, such as increasing of Faith’s PD and https://ddbj.nig.ac.jp/resource/sra-submission/DRA014918.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by Chiyoda Paramedical Care Clinic. The patients/participants provided their written informed consent to participate in this study.YN and KTF: visualization. YU and MK: methodology. YN: writing—original draft and formal analysis. YN, KTF, YU, MK, KZF, YK, and SF: writing—review and editing. All authors contributed to the article and approved the submitted version.


Despite dogs being one of the main species in the veterinary field and having been used as animal models for human musculoskeletal research, the anatomical information about the palmar region of the canine carpus found in the literature is inconsistent. After the dissection and evaluation of complementary histology of 86 carpi obtained from 43 dogs, we provide precise information about the location, extensions, and anatomic relations of the palmar carpal components, with special reference to the carpal canal. The results of this study show that the anatomy of the palmar region of the canine carpus is more similar to human wrists than was previously thought. Numerous similar studies about the carpal anatomy of other species have been published; however, to our knowledge, this is the first article to clarify and describe reference information about the anatomy of the palmar region of the canine carpus. canalis carpi. For this study, 92 cadaveric specimens were obtained from 46 dogs that had died for reasons unrelated to this study. Of these, 43 medium-to-large-breed dogs were randomly selected for the dissection of transverse slices of the carpus. Samples of the flexor retinaculum and flexor carpi radialis tendon and surrounding tissues were taken for complementary histology. For additional histology of the palmar structures in their anatomical position, three small breed dogs were randomly selected for obtaining transverse slices. The anatomic characteristics of the components of the palmar region of the canine carpus were qualitatively described, with special attention to the following structures: flexor retinaculum, flexor carpi radialis muscle, arteria and vena mediana, nervus medianus, interflexorius muscle, flexor digitorum profundus muscle, canalis carpi, and arteria and nervus ulnaris. The findings from this study provide reference information about the anatomy of the palmar region of the canine carpus.The palmar region of the canine carpus is anatomically complex, and the information found in the literature about its anatomy is inconsistent. The aims of this prospective, descriptive, anatomic study were (1) the clarification and (2) the description of the precise anatomic composition of the palmar region of the canine carpus, with special reference to the  However, based on our review of the literature, the use of this species to investigate specific palmar disorders is scarce.To properly design, plan, and carry out any study on a particular animal model, a correct knowledge of its anatomy is mandatory. Despite the fact that the dog is one of the most studied species in the veterinary field, the information about the anatomy of its carpal region found in the literature is not consistent.flexor carpi radialis, flexor digitorum superficialis, flexor digitorum profundus, and flexor carpi ulnaris), the flexor retinaculum, the palmar carpal ligaments, and the neurovascular structures  of the University of Santiago de Compostela.Nomina Anatomica Veterinaria and veterinary anatomic textbooks [The study’s methodology is based on similar procedures performed in other species such as humans  and equiextbooks ,7,8,9 weFor this study, 92 cadaveric specimens were obtained from 46 dogs that had died for reasons unrelated to this study. The specimens were acquired from the Service of Pathological Anatomy in the HVU-RC.n = 5), mixed breed (n = 5), German Shepherd (n = 12), Golden Retriever (n = 3), Great Dane (n = 1), Labrador Retriever (n = 3), Mastiff (n = 8), Pointer (n = 2), Rottweiler (n = 1), Spanish Scenthound (n = 1), and St. Bernard (n = 2).Of these, 43 medium-to-large-breed dogs were randomly selected for dissection according to inclusion criteria, which were weighing more than 20 kg, the absence of gross musculoskeletal disease in an orthopedic examination, having no history of musculoskeletal or nervous diseases in their medical records, and the lack of radiographic signs of musculoskeletal disorders in the carpal region. Of these 43 canine cadavers, 20 were females (46.5%) and 23 were males (53.5%). The mean age of the specimens was 6.73 years (range 1.5–12 years). Of the 43 carpi in group A, 21 were right and 22 were left. Of the 43 carpi in group B, 22 were right and 22 were left. The breeds of the dogs were Beagle  were obtained. During the design of the protocol, the use of diagnostic techniques such as ultrasound, which is more sensitive to evaluate soft tissues than radiography, was considered. However, given that the normal ultrasonographic anatomy of the components of the palmar region of the canine carpus has not been published, in contrast with the dorsal structures , we deciflexor retinaculum, flexor carpi radialis muscle, nervus medianus, arteria and vena mediana, interflexorius muscle, flexor digitorum profundus muscle, canalis carpi and arteria, and nervus ulnaris. In group B, 3 mm thick slices were cut with a hand saw, and photographs of both faces were taken and correlated with the findings of the dissection. Complementary histology was performed in samples of the flexor retinaculum and flexor carpi radialis tendon and surrounding tissues.In the specimens in group A, the hair was clipped from the middle antebrachial region to the distal metacarpus. The skin was longitudinally incised at the dorsolateral aspect, then reflected, and the overlying fascia, connective tissue, and fat were removed. The dissection was performed sequentially, in a medial to lateral and superficial to deep fashion. The studied region of the included soft tissue structures was the area between the distal part of the antebrachium and the proximal aspect of the metacarpus . The anaFor complementary histology of the palmar structures in their anatomical position, three small breed dogs were randomly selected for obtaining transverse slices. The inclusion criteria were the same as for the other dogs, except for the weight. Therefore, three Yorkshire specimens were included, one male and two females, with a mean age of 12.03 years (range 10.25–13.83 years). The carpi were transected (3 mm thick slices), and the samples were fixed by immersion in 10% buffered formaldehyde for a minimum of 48 h. After decalcification in Shandon TDB-1TM, given the difficulty of cutting the calcified tissue, the samples were embedded in paraffin according to standard laboratory procedures, and sections of 5 μm thickness were mounted onto silanized slides and dried overnight at 37 °C. The sections were stained with H and E for routine histological analyses, Azan trichrome stain, OMSB (combined orcein and Martius scarlet blue) for elastic fibers, and Picro Sirius Red for collagen fibers. Samples were evaluated in terms of the general structure and anatomical relations with an Olympus AX70 microscope and a coupled digital camera Olympus DP74.The anatomy of the palmar region of the carpus was consistent in all canine carpal specimens examined.flexor retinaculum .We found a strong similarity between the canine and human sections , as we higament” , but theo et al.  reviewedflexor retinaculum in dogs is briefly mentioned among the main anatomic reference sources and in the NAV, and a detailed description is lacking. Nevertheless, Nickel et al. [flexor retinaculum corresponds to the transverse carpal ligament that forms the canalis carpi. Therefore, considering our results, we propose to term this structure in the canine carpus as the “deep part of the flexor retinaculum”, understanding that it is more accurate given its function and location.In the veterinary literature, there is no specific terminology for this deep part. As previously explained, the l et al.  state thcanalis carpi in dogs was described as a non-distensible and well-defined oval space delimitated palmarly by the deep part of the flexor retinaculum, laterally by a thin fibrous septum, dorsally by the common palmar ligament that covers the palmar region of the carpal bones, and medially by the fixations of the fused antebrachial fascia to the deep part of the flexor retinaculum and the common palmar ligament. It contained the tendons of the flexor digitorum profundus and the interflexorius muscle, as well as the nervus medianus and the arteria and vena mediana. The inconsistency found in the literature is probably related to the non-differentiation between the superficial and deep parts of the flexor retinaculum. Dyce et al. [flexor retinaculum was only constituted by the superficial part, which led to the inclusion of other tendons and neurovascular structures inside the hypothetical canalis carpi. However, the superficial part of the retinaculum did not generate a true canal below because, by definition, a canal is a tubular duct or channel. The presence of the superficial part of the retinaculum did not create a canal itself, and there is no defined canal beneath the extensor retinaculum of the carpus. Barone [canalis carpi only as the space underlying the deep part of the flexor retinaculum. Our results support this and correlate with human anatomy, where the canalis carpi is a space in the palmar region of the carpus formed between the deep part of the flexor retinaculum—the transverse carpal ligament—and the carpal bones, which contains the tendons of the flexor digitorum superficialis, flexor digitorum profundus, and flexor pollicis longus muscles, as well as the nervus medianus [In this study, the e et al. , Evans ae et al. , and Ture et al. ,11,12 ma. Barone  and Ette. Barone  did descmedianus . In humamedianus  and the medianus . In veteflexor carpi radialis muscle was described as passing through its own fibrocartilaginous tunnel, independent of the canalis carpi. Dyce et al. [canalis carpi, not mentioning this specific tunnel. König and Liebich [canalis carpi, passing into a synovial and tendinous sheath, respectively. Our results support the description of Barone [flexor carpi radialis muscle passes through its own osteofibrous tunnel independent of the canalis carpi [flexor carpi radialis muscle lies in close contact with the shared joint capsule of the scaphotrapeziotrapezoid (STT) or triscaphe articulation, which may lead to the extension of disease from one compartment to the other [flexor carpi radialis tunnel syndrome [In this study, the tendon of the e et al.  and Evane et al.  included Liebich  and Nick Liebich  describef Barone  and alsois carpi , to inseis carpi . In humahe other . Additiohe other  and stenhe other , also knsyndrome , clinicanervus ulnaris inside the canalis carpi. Our results, however, place the nervus ulnaris lateral to it, separated by a septum of connective tissue. These findings also correlate with human anatomy. In the ulnar side of the palmar region of the wrist, the nervus ulnaris also passes outside the canalis carpi, running through a tunnel termed the Guyon’s canal [canalis carpi. Then, it continues deep to the medial accessory-metacarpal ligament, where it is surrounded by fat tissue. To the best of our knowledge, there is no published information about the entrapment of this nerve in canines.Evans and de Lahunta  and Köni’s canal ,29. This’s canal , and the’s canal . In the Dogs have been widely selected as animal models in musculoskeletal research and have been used in multiple studies about osteoarthritis, both naturally and induced-occurring ,33. ThisThe confusing anatomic descriptions published so far in the veterinary literature may have prevented the proper application and interpretation of diagnostic techniques. Further research is needed about all these carpal conditions in dogs in veterinary medicine, and the use of this species as animal models to investigate the aforementioned diseases in human wrists merits promotion.The limitations of the present study are, first, the heterogeneity of the included dogs, which did not permit obtaining measurements of the structures transferable to the whole canine population. Additional studies may be interested in providing these data on particular breeds. Second, our results were mainly obtained from a gross anatomy approach, presenting a complete macroscopic perspective of the location, extensions, and anatomic relations of the main structures of the palmar side of the canine carpus. However, more histologically specific studies may be needed to determine the precise microscopic composition of these elements. Third, radiography is a sensitive technique to evaluate bones, but its sensitivity for the examination of soft tissue is poor. The use of more sensitive techniques such as ultrasound to rule out disease in these structures would have been optimal during the selection process of the cadaveric specimens. However, despite the fact that the normal ultrasonographic anatomy of the dorsal region of the carpus of the dog has recently been published , the samThe findings of this study provide an anatomic description of the palmar side of the canine carpus and show that the anatomy of the carpi of dogs and humans is more similar than was previously considered. These findings may constitute a basis in the research of musculoskeletal injuries, both to improve the use of canine models for human diseases and to expand the information about canine carpal diseases inside the veterinary field.


Recently, accumulating evidence has shown that HH signaling is targeted by viruses to facilitate viral transcription, immune evasion, and uncontrolled growth, leading to effective viral replication and pathogenesis. In this study, we will summarize recent advances in functional interaction between HH signaling and different types of viruses, particularly focusing on the pathological role of HH signaling in viral infections and related diseases.The hedgehog (HH) signaling pathway is one of the key pathways that is indispensable for many developmental processes and postnatal tissue homeostasis. Dysregulated HH signaling could lead to developmental disorders and tumorigenesis in a variety of tissues  Drosophila mutant, was discovered as a segment polarity gene that governs segmental pattern along with other components of HH signaling  as a major repressor in vertebrate (Hedgehog (HH), originally named after the spiny appearance of the cuticle in the ignaling . Since ttebrates . The sigrtebrate . The HH rtebrate . HH protA) and the repressor form (GLIR) of the GLI family of zinc finger transcription factors, and ultimately regulates the specific expression of GLIA/GLIR target genes that defines cell identity /EVC2 upon HH stimulation. EVC/EVC2 activates HH signaling downstream of SMO by facilitating cilia localization of GLI2/3-SUFU complex -mediated degradation, which might be responsible for the termination of HH signaling , inositol polyphosphate 5-phosphatase E (INPP5E), and karyopherin β2 (KAPβ2), SLIT and NTRK-like protein-5 (SLITRK5) . We haveXenopus . In addiXenopus .NK6 homeobox 1 (NKX6.1), oligodendrocyte transcription factor 2 (OLIG2), and NKX2.2. HH signaling is required for the maintenance of stem or progenitor cells in embryonic and adult tissues via targeting cell cycle genes MYC and Cyclin D1 and stemness-associated gene NANOG and SOX2 , a rare developmental disorder that is prone to develop into BCC and medulloblastoma . The mosignaling . Elevateignaling . Loss-ofIn contrast, ligand-dependent activation of HH signaling usually occurs in other types of cancer that rarely contain oncogenic mutations, such as prostate cancer, gastric cancer, and gliomas. In these conditions, elevated HH signaling supports the proliferation and survival of cancer cells in an autocrine way where cancer cells act as both HH-producing and HH-responding cells. Alternatively, a pathway is activated in a paracrine way where HH ligands secreted by cancer-surrounding stromal cells stimulate HH signaling in cancer cells, or HH ligands secreted by cancer cells stimulate HH signaling in stromal cells which in turn release cancer-supporting signals . HH signConsistent with the important role of HH signaling in development and homeostasis, selective populations of immature thymocytes and peripheral mature T cells are responsive to HH ligands, and activation of HH signaling is required for fetal and adult T-cell development and peripheral T-cell activation . In contA in T cells attenuates T-cell signaling and T-cell activation induced by T-cell receptor (TCR) stimulation. GLI2A impairs the activation of pro-inflammatory AP-1 and NF-κB signaling. In contrast, GLI2R-mediated transcription repression increases NF-κB activity following TCR stimulation. Thus, HH signaling might skew pro-inflammatory immune responses during inflammation and tissue repair. Indeed, activation of HH signaling increases CD4+ regulatory T-cells (Treg) populations and their immunosuppressive function by activating transforming growth factor-β (TGF-β) production, thereby preventing skin inflammation in the mouse model of atopic dermatitis , whereas loss of IHH induces a rapid immune response characterized by upregulation of stromal CXCL12 and filtration of immune cells  is one of the leading causes of cancer deaths. About 80% of HCC incidence is associated with chronic infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) . HH signAs a major cause of HCC, HBV is a small, double-stranded DNA virus but similar to retroviruses in the viral life cycle and genome organization. Integration of viral DNA into the host genome is an important step during the pathogenesis of HBV-induced HCC. HBV integrations are more abundant in HCC tissues than in adjacent normal tissues and HBV integration-targeted genes are significantly enriched in the HH signaling pathway . HoweverHCV is another leading cause of liver fibrosis and HCC. Unlike HBV, HCV is a positive sense, single-stranded RNA virus. Infection of hepatocytes with HCV significantly increases the expression of SHH mRNA . HCV-perHepatitis D virus (HDV) is a small RNA virus that requires HBV for infection and replication . CompareEpstein-Barr virus (EBV) is a member of the herpesvirus family that contributes to about 1.5% of all cases of human cancer, including Hodgkin’s lymphoma, nasopharyngeal carcinoma (NPC), and gastric cancer . Dysreguvia LMP2A-mediated activation of HH signaling. IHH gene is transcriptionally downregulated by EBV-associated hypermethylation in gastric cancer cells infected with EBV. Downregulation of IHH increases cell growth and colony formation ability, suggesting a tumor-suppressive potential of IHH in EBV-related gastric cancer  is one of the common strategies deployed by cancer cells and viruses to evade host immune response. EBV-encoded LMP2A protein downregulates the expression of HLA Class I in gastric cancer cells . Mechanic cancer . These sHuman papillomavirus (HPV) is a group of related double-stranded DNA viruses known as the primary cause of cervical cancer. HPV 16 and HPV 18 are responsible for at least 70% of cases of cervical cancer, largely dependent on two oncogenes E6 and E7 . An earlA recent study found that GLI1 and E6 stimulate their transcriptional expression reciprocally, resulting in a high level of GLI1 and E6 in cervical cancer stem cells . The coo+ T cells through activating the human TGF-β1 promoter activity. The HIV-1 encoded viral protein Tat has been implicated as the inducer of TGF-β1 in both in vitro and in vivo. The identified interaction between Tat and GLI2 raises the possibility that they might synergistically activate TGF-β1 transcription after HIV infection and contribute to immune suppression.Human immunodeficiency virus (HIV) infection is linked to the suppression of protective immune responses and causes acquired immune deficiency syndrome (AIDS). An early study has shown that the expression of immunosuppressive cytokine TGF-β1 is elevated after HIV-1 infection and leads to the development of induced Treg . As a mavia damaged BBB. As mentioned earlier, HH signaling is essential for the maintenance of BBB integrity (The HIV-associated neurocognitive disorder (HAND) is characterized by the recruitment of infected leukocytes into CNS ntegrity . Impairentegrity , 2017. Bntegrity . DespiteThe HIV-induced EMT of podocytes is an important mechanism of HIV-associated nephropathy (HIVAN). HH signaling is activated in human podocytes infected with HIV and the mouse model of HIVAN . Treatme+ T cells develop T-cell lymphoma (Adult T-cell leukemia (ATL) is a peripheral T-lymphocyte malignancy caused by infection of the retrovirus human T-cell leukemia virus type I (HTLV-1). HTLV-1 bZIP factor (HBZ) and Tax play a critical role in leukemogenesis of ATL, and double transgenic mice of HBZ and Tax in CD4lymphoma . HTLV-1-lymphoma . GLI2 anvia epigenetic modification at the EVC loci. Knockdown of EVC1, EVC2, GLI1 or GLI2, or treatment with GLI1/2 inhibitor GANT 61 induces apoptosis of ATL cells, indicating that EVC1/2-mediated activation of HH signaling supports the survival of ATL cells. Thus, HH signaling plays a vital role in ATL pathogenesis and represents a potential therapeutic target of ATL.Tax extensively crosstalks with cellular signaling pathways to subvert cellular functions and suppresses host defense. EVC1 and EVC2, positive modulators of HH signaling that act downstream of SMO, are found to be aberrantly overexpressed in ATL and HTLV-1-infected cells . The uprvia direct interaction with transcription factor Ci in Drosophila (Influenza A viruses (IAVs) are the causative pathogens of seasonal influenza epidemics that affect the upper and lower respiratory tract and represent a wide spectrum of subtypes based on surface proteins hemagglutinin (H) and neuraminidase (N). IAV-encoded non-structural protein NS1 is highly expressed in infected cells. NS1 activates HH signaling osophila . The A12Influenza is characterized by the destruction of alveolar epithelial and endothelial, which involves IAV-induced activation of non-canonical HH signaling followed by disruption of the epithelial junctions . GLI1 isMerkel cell carcinoma (MCC) is aggressive neuroendocrine skin cancer and its primary cause is Merkel cell polyomavirus (MCPyV). It has been reported that the expression of HH target genes GLI1 and PTCH1 is increased in MCC . SHH levInfection of Zika virus (ZIKV) increases the incidence of microencephaly in fetuses and Guillain-Barre syndrome in adults. The close association between HH signaling with neurodevelopmental disorders raises the possibility that HH signaling might be implicated in ZIKV-induced microencephaly. Using the developing chicken brains as an animal model, Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is a double-stranded DNA virus linked to Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL). Recently, Veratrum californicum was identified as the teratogen responsible for cyclops lambs in 1968 and as the first HH signaling inhibitor in 1998, searching chemical modulators of HH signaling for the therapeutic purpose have attracted numerous research attentions (Mechanistic insight into HH signaling and the discovery of its chemical modulators are mutually beneficial and co-developing. Since cyclopamine from the plant tentions . These etentions . As HH stentions , a new aThe HH signaling regulates numerous biological processes during embryonic development and postnatal tissue homeostasis. Dysregulation of HH signaling is the important driving force during the initiation and progression of a variety of diseases. In general, HH signaling is aberrantly activated in cancer while reduced in developmental disorders. Recently, accumulating studies have extended the pathological role of HH signaling to viral infections and their related diseases. Similarly, most viral infections that lead to cancer usually activate cellular HH signaling. Viral infections that lead to developmental disorders tend to decrease HH signaling activity, as exampled by ZIKV-induced microencephaly . During All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.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.


In this process, the somatic outcome is actively modified by the morphology of the dendritic branches, the expression of channels along the somato-dendritic axis, and spatiotemporal patterns of activation of the synaptic inputs. Recently, it has been suggested that axonal output is also locally regulated along its arbors contrary to the conventional idea of digital-like uniformity. However, we still do not fully understand the nature or logic of the subcellular computations carried out within a single neuron.A neuron receives thousands of synaptic inputs at its dendritic branches, integrates them both locally and at somatic level, and sends output to downstream neurons The aim of this Research Topic is to bring together publications from a wide range of topics on subcellular computation to facilitate our understanding of how the network activity organizes and modifies the spatiotemporal pattern of synaptic inputs and outputs. Particularly, this Research Topic focuses on the processes by which a single neuron integrates several synaptic inputs from upstream neuron assemblies and transmits a variety of output patterns to downstream neuron assemblies. We successfully gathered two original publications and two review articles written by 10 authors in total. The papers cover the topic of subcellular computation in various species, including mice, rats, ferrets, and birds.Meissner-Bernard et al. proposed a voltage-based plasticity model that can explain the location-dependence of the expression of long-term potentiation and long-term depression along the dendrites. They suggest that the combination of presynaptic signaling and the time course of the postsynaptic voltage can predict the outcome of synaptic plasticity at the stimulated synapses. Yates and Scholl investigated how a variety of synaptic inputs produce selective somatic firing. Using a population coding theory, they suggest that heterogeneity in synaptic weight is critical to induce the somatic selectivity based on diverse synaptic input, which matches the functional heterogeneity of dendritic spines in the ferret visual cortex.Two original papers focused on the subcellular computation conducted in single neurons using computational models. Moberg and Takahashi focused on how two distinct populations of pyramidal neurons in layer 5 of the somatosensory cortex, intratelencepharic (IT) and extratelencepharic (ET), play their unique roles in sensory processing and behavior. The review highlights the differences in network configurations and physiological and morphological properties between IT and ET neurons, which contribute to their distinct roles in subcellular computation and behavior.A single brain region consists of diverse neuron types that use different subcellular computation algorithms. +, Ca2+, and NMDA receptor conductances, and the clustering of synapses amplifies EPSPs to induce supralinear integration by activating these conductances. Contrarily, this amplification is prevented in the nucleus laminaris (NL) dendrites since their active conductances are weaker than pyramidal cells (Yamada and Kuba). The unique characteristics, including morphology and channel distribution, enable the neurons in NL to encode interaural time differences for sound-source localization.Regional variations in dendritic computations are an important issue in understanding the diversity of subcellular computations, but they are often overlooked. Dendrites of pyramidal neurons in the cortex and the hippocampus express active NaYamada and Kuba). However, accumulating evidence suggests that neuromodulators also affect subcellular computations. Moberg and Takahashi summarized the effects of individual neuromodulators on excitability and synaptic input in the specific cortical pyramidal neurons (IT and ET). They hypothesized that spatial and temporal dynamics of neuromodulator release play important roles in synaptic integration because neuromodulators change the gain of excitability in cell-type specific manner.The current understanding of subcellular computation is mainly about glutamatergic and GABAergic inputs, with a recognition of the crucial role played by the spatial distributions of excitatory and inhibitory synapses in shaping firing specificity . Our hope is that the information on this Research Topic will be useful for both experimentalists and modelers. We also hope that the papers collected on this Research Topic stimulate researchers in the field and highlight important questions to be addressed in the coming years.This Research Topic illustrates the diversity of questions, methods, and animal models involved in the study of this field. Computational simulations are also powerful tools to understand subcellular computations as demonstrated in the two original papers (All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Background: Pressure ulcer is a severe disease in the paralyzed and aging populations. Endothelial progenitor cells (EPCs) are able to regulate ulcer healing by modulating angiogenesis, but the molecular mechanism is still obscure. Sonic hedgehog (SHH) signaling contributes to angiogenesis in various diseases and has been identified to modulate EPCs function. Here, we aimed to explore the significance of SHH signaling in EPCs function during pressure ulcers.Methods: The EPCs were isolated and characterized by the expression of DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1. Cell proliferation was detected by cell counting kit 8 (CCK-8). The DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1 were analyzed by immunofluorescent analysis. The angiogenesis of EPCs was analyzed by tube formation assay. The pressure ulcers rat model was constructed, the wound injury was analyzed by H&E staining and angiogenesis was analyzed by the accumulation of CD31 based on immunofluorescent analysis.Results: The expression of patched-1 and Gli-1 was enhanced by SHH activator SAG but reduced by SHH inhibitor cyclopamine in the EPCsThe PI3K, Akt, eNOS expression and the Akt phosphorylation were induced by SAG, while the treatment of cyclopamine presented a reversed result. The proliferation and migration of EPCs were enhanced by SAG but repressed by cyclopamine or PI3K/AKT/eNOS signaling inhibitor Y294002, in which the co-treatment of Y294002 could reverse the effect of SAG.Conclusions: Thus, we found that SHH signaling activated angiogenesis properties of EPCs to improve pressure ulcers healing by PI3K/AKT/eNOS signaling. SHH signaling may serve as the potential target for attenuating pressure ulcers. Pressure ulcer is a prevalent disease in the paralyzed and aging groups. Even though most of cases are preventable, pressure ulcers continue to pose a major burden to individuals and the society, affecting almost 3 million adults annually in the United States alone. The prevalence of pressure ulcers has largely remained unchanged over the past two decades, while the associated costs of care continue to increase. The traditional therapeutic strategy for pressure ulcers is prevention, and the common interferences aim to improve wound healing , 2. The The Hedgehog pathway is crucial for precise morphogenesis and the formation of embryogenesis . The intIn this study, we were interested in the role and the underlying mechanism of SHH signaling in regulating angiogenesis properties of EPCs during pressure ulcers. We identified a crucial function of SHH signaling in promoting angiogenesis properties of EPCs to improve pressure ulcers healing by PI3K/AKT/eNOS signaling.2 and 37° C. Dead cells were removed after three days, and then the culture mediums were refreshed every three days. And the EPCs were identified and characterized by DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1 using immunofluorescent analysis after two weeks. The SAG, cyclopamine, and Y294002 were purchased .EPCs were obtained from mononuclear cells (MNCs) as the previous reports . MNCs we3 cells were plated in 96-well dishes and incubated for the transfection or treatment. The cells were added with a CCK-8 solution  and culture for another 2 hours at 37° C. The proliferation was measured at an absorbance of 450nm by applying the ELISA browser .The proliferation was assessed using CCK-8 assays. About 1×105 cells. Then solidified through 4% paraformaldehyde and dyed with crystal violet. The invaded and migrated cells were recorded and calculated.Transwell assays analyzed the migration of EPCs by using a Transwell plate  according to the manufacturer’s instruction. Briefly, the upper chambers were plated with around 1 × 10The angiogenic capacity was analyzed by tube formation assays . The 24-well plates were coated with 100 μl Matrigel  and incubated at 37° C for 2 hours. After the gel was solidified, HUVEC was suspended in culture medium as single cells and were plated onto the Matrigel in 24-well dishes. After incubation at 37° C for 24 hours, the formed tubes were captured by microscopy and quantified by ImageJ software.RIPA buffer  was used to extract the total protein, followed by the quantification based on the BCA method . The proteins at same concentration were subjected in SDS-PAGE and transferred , followed by the incubation with 5% milk and with the primary antibodies at 4° C overnight. The corresponding second HRP-conjugated anti-mouse or anti-rabbit antibodies  were used for incubating the membranes 1 hour at room temperature, followed by the visualization by using chemiluminescence detection kit . The primary antibodies applied in this study comprise of patched-1 , Gli-1 , PI3K , Akt , eNOS , p-Akt , and β-actin . All antibodies were diluted in PBST solution at 1:2000 (v/v).Sprague-Dawley (SD) rats  were applied to construct the pressure ulcers rat model . The ratThe expression of CD31 was analyzed by immunofluorescence analysis. Slices were solidified at 4% paraformaldehyde for 30 min, treated with Triton X 100 (0.2%) for 10 min and treated with BSA (2%) for 30 minutes. The slides were hatched with the primary antibody overnight at 4° C, then hatched with secondary antibodies  for 1 hour at 37° C. The slides were stained with the Hoechst  for 10 min at 25° C. The Nikon microscope  was utilized to analyze the immunofluorescence.The slices of skin tissues (5 μm thick) were analyzed by Hematoxylin and eosin (H&E) staining. The photographs were captured by an Olympus BX60 microscope  at a magnification x200. The quantitative analysis was performed through a quantitative digital image analysis system (Image-Pro Plus 6.0).p < 0.05 were considered as statistically significant.Data were expressed as mean ± S.D of three independent experiments, and the statistical analysis was conducted by GraphPad Prism 7 . The unpaired Student’s t-test was used for comparing two groups when data confer to parametric distribution. For the analysis of datasets with non-parametric distribution, the Mann–Whitney U test was used for comparisons between two groups. To understand the function of SHH and the underlying mechanism in EPCs, the EPCs were isolated and characterized by the expression of DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1 using the using the immunofluorescent analysis . Then, tWe then observed that the proliferation of EPCs was enhanced by SAG and repressed by cyclopamine or PI3K/AKT/eNOS signaling inhibitor Y294002, in which the co-treatment of Y294002 could reverse SAG-mediated EPC proliferation in the system . SimilarNext, we further investigated the impact of SHH/PI3K/AKT/eNOS signaling on the angiogenesis properties of EPCs. Remarkably, tube formation assays showed that the tube length was promoted by SAG but attenuated by cyclopamine and Y294002, in which the co-treatment of Y294002 significantly reversed the effect of SAG in the EPCs .Next, we assess the function of SHH signaling in the pressure ulcers rat model. Significantly, the treatment of EPCs attenuated the wound injury of the pressure ulcers rats, while the SAG treatment could enhance but cyclopamine treatment repressed this effect in the system . We alsoin vivo pressure ulcers rat model demonstrated that SHH pathway contributes to angiogenesis in the pressure ulcers rat model.Pressure ulcer is a severe disease and commonly affects the paralyzed and aging populations. EPCs contributes to the pressure ulcer healing, but the mechanism is still obscure. SHH signaling has been found to regulate EPCs function, but the role of SHH in pressure ulcer remains unclear. In this investigation, we found that SHH activates PI3K/AKT/eNOS signaling in EPCs. The treatment of SAG induced the proliferation, migration, and angiogenesis of EPCs, while the co-treatment of PI3K/AKT/eNOS signaling inhibitor Y294002 was able to inhibit these effects. Moreover, the results from Previous studies have found the critical role of EPCs in wound healing and angiogenesis. Simvastatin enhances EPCs neovascularization and mobilization to modulate diabetic rats wound healing . StronglOur data displays a critical role of SHH signaling in regulating angiogenesis of EPCs during pressure ulcers healing, uncovering the molecular mechanism of EPCs-mediated ulcers healing.Moreover, PI3K/AKT/eNOS signaling presents important roles in EPCs modulation. Naringin promotes the tube formation and proliferation of EPCs by regulating the PI3K/AKT signaling . LncRNA In summary, we concluded that SHH signaling activated angiogenesis properties of EPCs to improve pressure ulcers healing by PI3K/AKT/eNOS signaling. Our finding provides new insight into the mechanism by which SHH signaling contributes to the EPCs-mediated pressure ulcers healing. SHH signaling may be served as the potential targets for attenuating pressure ulcers.


After administration, serum UA decreased to 5.6 mg/dL and FEUA increased to 9.22%. Dapagliflozin increases FEUA and reduces serum UA levels in patients with advanced CKD.Sodium–glucose cotransporter 2 inhibitors (SGLT2i) exhibit renoprotective effect in patients with chronic kidney disease (CKD) and reduce serum uric acid (UA) in patients with diabetes mellitus. However, it is not clarified whether SGLT2i reduce serum UA levels in patients with advanced CKD. This study aimed to investigate the impact of SGLT2i on change in serum UA levels in patients with advanced CKD. Data of 121 Japanese patients with CKD who were newly administered 10 mg dapagliflozin in our department between August 2021 and August 2022 were analyzed. Changes in UA and fractional excretion of UA (FEUA) were analyzed using multiple regression analysis. Of 75 patients, 21 (28.0%) patients, 24 (32.0%) patients, 29 (38.7%) patients, and 1 (1.3%) patient were categorized as having CKD stage 3a, 3b, 4, and 5, respectively. The median age was 67 years, and 72.0% were male. 23 (30.7%) of patients had diabetes mellitus. The median estimated glomerular filtration rate, serum UA, and FEUA were 35.7 mL/min/1.73 m Renin angiotensin aldosterone system inhibitors have been shown to reduce urinary protein levels and delay CKD progression4. Recently, it has been shown that sodium–glucose cotransporter 2 inhibitors (SGLT2i) have a renoprotective effect7. Although the mechanism of their renoprotective effect is believed to involve the reduction of intra-glomerular pressure by tubular glomerular feedback8, SGLT2i have pleiotropic effects and their mechanism of renal protection has not been fully revealed9.Chronic kidney disease (CKD) is a global concern, affecting more than 10 million patients in Japan and 700 million worldwide11. On the other hand, allopurinol did not improve renal outcomes13. One of the pleiotropic effects of SGLT2i is reduction of serum UA in patients with type 2 diabetes mellitus (DM) whose estimated glomerular filtration rate (eGFR) was maintained at > 60 ml/min/1.73 m217; however, the effect of lowering serum UA levels by SGLT2i in patients with CKD whose renal function reduced to ˂60 ml/min/1.73 m2 is unclear.Serum uric acid (UA) and renal outcomes are not fully understood; however, it has been reported that allopurinol reduces serum UA levels and improves renal outcomesDapagliflozin 10 mg is the only drug approved for the treatment of CKD with or without DM in Japan as SGLT2i and we analyzed patients who were administered dapagliflozin 10 mg.19.Approximately 70% of serum UA is excreted into the urine and the rest is thought to be excreted into the intestinal tract. The renal transport system such as urate transporter 1 (URAT1), adenosine triphosphate-binding cassette transporter G2 (ABCG2), glucose transporter 9 (GLUT9) isoform2 plays an important role in the regulation of serum UA level. SGLT2i increase expression of GLUT9 and ABCG2Thus, this study aimed to investigate the effect of SGLT2i in lowering serum UA levels in patients with CKD. In addition, this study aimed to examine the effect of SGLT2i in the change in urinary UA excretion.This study was conducted in accordance with the Declaration of Helsinki guidelines and approved by institutional review board of Sakai City Medical Center (No. 21-261). All patients were provided with the option to opt out of the study. The need for informed consent was waived using our hospital’s opt-out method.This was a single-center retrospective cohort study. A total of 121 Japanese patients with CKD who were newly administered dapagliflozin 10 mg in our department from August 2021 to August 2022 and were followed-up for at least 2 weeks were enrolled in the study. Thirty-four patients were excluded from the analysis owing to changes from other SGLT2i (n = 3) or dapagliflozin dose (n = 2). In addition, patients with changes in the dose of diuretics (n = 3) and antihyperuricemic agents (n = 3), and those whose serum UA level was not measured before or after dapagliflozin 10 mg administration (n = 17) were also excluded. Twelve patients with CKD stages 1 and 2 were excluded from the study.In study 1, 75 patients were retrospectively studied  was not measured before or after dapagliflozin 10 mg administration (n = 40) were excluded from the study. A total of 35 patients were included in Study 2.20. Patients were divided into four groups according to their renal function and CKD category (G3–G5).We collected the data of 121 patients from the electronic medical charts of Sakai City Medical Center, including demographics (sex and age), medications, comorbidities (history of heart failure and DM), and clinical and laboratory variables . FEUA was calculated as  and were compared using the Kruskal–Wallis test. Categorical variables are presented as numbers and percentages and were compared using the Fisher’s exact test. Changes in UA and FEUA were compared using the Wilcoxon signed-rank test and Kruskal–Wallis test.Multiple regression analysis was used to estimate the independent factors associated with changes in UA and FEUA.Spearman’s rank correlation coefficient was used to evaluate an association between change in FEUA and serum UA.21.All statistical analyses were performed using EZR software At administration of dapagliflozin 10 mg, 21 (28.0%) patients, 24 (32.0%) patients, 29 (38.7%) patients, and 1 (1.3%) patient were categorized as having CKD stages 3a, 3b, 4, and 5, respectively  of patients had diabetes mellitus and all patients were Japanese. The median eGFR was 35.7 mL/min/1.73 mp < 0.001) was observed before and after dapagliflozin administration. Rate of UA change was − 0.12 [− 0.20 to − 0.04]% by SGLT2i administration.Figure Figure Multiple regression analysis did not reveal a relationship between eGFR, change in eGFR and rate of UA change Table .Table 2Ip < 0.001)  and reduce serum UA levels  in patients with type 2 DM whose eGFR was 63.25 ± 24.66 mL/min/1.73 m217. Our results in patients with CKD stage 1 and 2 showed that there was a decrease in serum UA levels and an increase in FEUA after dapagliflozin administration same as previous studies  and FEUA increased (before administration: 6.70 [4.22–8.37]% vs. after administration: 8.77 [6.44–8.37]%) in patients with CKD without DM  at the proximal tubule, and may inhibit uric acid reabsorption mediated by GLUT9 isoform 2 at the collecting duct of the renal tubuleAlthough SGLT2i increased FEUA and decreased serum UA in our study, change in FEUA and change in serum UA was not associated with eGFR. Furthermore, we could not find a significant association between change in FEUA and change in serum UA.Mechanisms other than urinary excretion such as intestinal excretion of UA may be involved in lowering effects of SGLT2i on serum UA levels in patients with advanced CKD.28. In animal studies, intestinal urate transport by ABCG2 compensated for abnormal urate handling in the setting of declining renal function19. Lu et al. reported that empagliflozin promoted ABCG2 expression in the kidneys and ileum of diabetic mice and attenuated hyperuricemia29. Although these reports suggest that SGLT2i increase urinary UA excretion and promote intestinal extrusion of uric acid through UA transporters, no obvious evidence has yet been found.ABCG2 is a high-capacity urate secretion transporterFurther studies are needed to demonstrate the underlying mechanism of SGLT2i’s reduction of serum UA levels.The present study had some limitations. First, this was a single-center retrospective cohort study with a relatively small sample size. Second, we did not investigate the dietary characteristics of each patient. Third, because we did not assess difficult endpoints such as ESRD and CKD progression, we did not show whether reduction of UA by SGLT2i may improve renal outcomes.Our results suggest that dapagliflozin increases FEUA and decreases serum UA levels in patients with advanced CKD.However, the mechanism of the effect of dapagliflozin on lowering serum UA level has not been fully elucidated, and it is not clear whether this effect can improve renal outcomes. Therefore, further studies are needed to identify the mechanism and impact of SGLT2i on renal outcomes by lowering of serum UA levels.Supplementary Information.