CTNNB1 gene and identified eight missense point-mutations and one in-frame deletion. We describe and functionally characterize a novel CTNNB1 in-frame deletion  found in a pediatric patient with a classic medulloblastoma, WNT-activated grade IV (WHO 2016). To the best of our knowledge, this mutation has not been previously reported in medulloblastoma, and it is uncertain its role in the disease development and progression. Our analysis discloses gain-of-function properties for the new ΔS37 β-catenin variant.We have analyzed a panel of 88 pediatric medulloblastoma tumors for exon 3 mutations from the CTNNB1 mutations and were immunostained for nuclear β-catenin. One novel heterozygous CTNNB1 mutation was found, which resulted in the deletion of β-catenin Ser37 residue (ΔS37). The ΔS37 β-catenin variant ectopically expressed in U2OS human osteosarcoma cells displayed higher protein expression levels than wild-type β-catenin, and functional analysis disclosed gain-of-function properties in terms of elevated TCF/LEF transcriptional activity in cells. Our results suggest that the stabilization and nuclear accumulation of ΔS37 β-catenin contributed to early medulloblastoma tumorigenesis. Medulloblastoma is the primary malignant tumor of the Central Nervous System (CNS) most common in pediatrics. We present here, the histological, molecular, and functional analysis of a cohort of 88 pediatric medulloblastoma tumor samples. The WNT-activated subgroup comprised 10% of our cohort, and all WNT-activated patients had exon 3  TP53 mutated or TP53 wild-type), and non-WNT/SHH (Group 3 and Group 4) [MYC genes led us a precise assignment of patients for risk-adapted stratification [Medulloblastoma (MB) is the primary malignant tumor of the Central Nervous System (CNS) most common in pediatric populations and represents 15–20% of the total primary CNS tumors in this age group [Group 4) ,7,8,9. Rfication .Recent studies have identified new driver mutations assigned to most patients belonging to Group 3 and 4 MB molecular subgroups, whose genetics and biology remain less clear . New molCTNNB1 activating mutations or mutations in APC or other genes encoding components of the WNT signaling pathway [CTNNB1 gene, that maps to the short arm of chromosome three (3p22.1) and consists of 15 exons encoding 781 amino acids, encodes the β-catenin protein which is a subunit of the cadherin/catenin multiprotein complex. Previous studies have found CTNNB1 mutations in association with several types of cancer, with a hot spot region targeting the exon three (encoding residues 5–80 from β-catenin). Mutations at exon three usually decrease the phosphorylation-dependent ubiquitination of β-catenin. This renders more stable β-catenin proteins that accumulate in the nucleus and display pro-tumorigenic properties [Immunohistochemically, WNT-activated MB shows the nuclear accumulation of β-catenin protein as a surrogate biomarker for WNT activation caused by  pathway ,15,16,17operties ,19,20,21operties ,22. WNT-operties ,23. Althoperties ,22,24.ClinicalTrials.gov Identifier: NCT02066220). In the setting of such clinical trials, it is widely recommended to use two independent methods for reliable identification of these patients, such as immunohistochemistry for β-catenin and sequencing of CTNNB1 exon three or alternative molecular methods  [The precise identification of WNT-activated MB tumors is important because of their prognosis in the pediatric age, which provides guidance for the inclusion of patients into ongoing therapeutic trials aiming to prove that reduction of treatment intensity is possible in these patients ,22 (e.g.sifiers) .CTNNB1 mutational status of 88 pediatric MB. We report one novel CTNNB1 MB mutation, c.109-111del . Functional analysis of ΔS37 β-catenin protein variant disclosed gain-of-function properties in cells.Thus, comprehensive evaluation of pediatric MB is necessary to inform about risk stratification and treatment. In this study, we report the histological analysis and Tumor samples were collected from 88 pediatric MB patients between the years 2016 and 2021 from different Spanish Hospitals. Ethics approval was obtained from the Ethics committee of Euskadi (CEIm-E), and the study was conducted in accordance with the Declaration of Helsinki. Haematoxylin and eosin-stained slides were reviewed for confirmation, diagnosis, and assignment of histological type. All patients were aged <18 years at the time of diagnosis.Tumor tissue samples from all the patients were fixed in buffered 4% formalin, embedded in paraffin, and stained according to standard procedures. The immunohistochemistry followed standardized methods in the Hospital Universitario de Cruces Pathology Lab, Spain. We used β-catenin , p53 , YAP1 , GAB1 , and OTX2  as primary antibodies. Immunohistochemical staining was performed in an automated immunostainer .MYC amplification  and NMYC amplification , and the Meta Systems probe was used for monosomy 6 (Triple Colour XL 6q21/6q23/6cen). FISH studies were performed on formalin-fixed, paraffin-embedded (FFPE), 3 μm thick sections. Fluorescence staining was visualized with an Olympus BX61-microscope  equipped with DAPI, SpectrumGreen, and SpectrumOrange filters. At least 50 nonoverlapping nuclei were analyzed. For the amplification, multiple copies of the green signal or green signal clusters should be observed. For monosomy 6, loss of one green and/or one orange signal should be observed.The Fluorescence In Situ Hybridization (FISH) molecular test was performed in all samples using commercially available probes from ZytoVision for ® DNA/RNA Mini kit , and DNA from FFPE tissue sections were extracted with QIAamp® DNA FFPE Tissue kit. Mutations in exon 3 of the CTNNB1 gene were detected in genomic DNA obtained from frozen tumors and FFPE tissues using Sanger direct method. The PCR reactions were carried out with the following primer pair: CTNNB1_F: 5′-GATTTGATGGAGTTGGACATGG and CTNNB1_R: 5′-TGTTCTTGAGTGAAGGACTGAG [2 (KAPA BIOSYSTEMS), 10 mM of dNTPs mix, 10 pmol of each primer, and 0.5 unit of KAPA Taq polymerase (KAPA BIOSYSTEMS). After PCR amplification, products were loaded onto 2% agarose gel. The single and double strands of the PCR products were visualized by Midori Green Advance DNA stain . The resulting PCR products were purified using spin columns , and sequenced on an ABI 373A sequencer .Genomic DNA from frozen tumors was extracted with All PrepGGACTGAG . SamplesVariants were visually inspected and identified using the biological sequence alignment editor BioEdit 7.2 and the Basic Local Alignment Search Tool (BLAST). Once the variant was identified, it was annotated using the ENSENBL Genome Browser 105 (transcript ENST00000349496.1). Variant nomenclature was based on the genome version GRCh38.p13 (hg38).U2OS human osteosarcoma cells were grown in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum  and 2 mM Ala–Gln . Mammalian expression plasmid, pRK5 β-catenin, has been previously reported . pRK5 β-g for 10 min and collection of the supernatant. Whole cell protein extracts were resolved on 4–10% SDS-PAGE  under reducing conditions and transferred to PVDF membranes . Immunoblot analysis was performed as previously reported [Cell were harvested 72 h post-transfection for immunoblot analysis. Cells were lysed in ice cold M-PER lysis buffer  supplemented with phosphatase inhibitor PhosSTOP (Roche) and complete Mini protein inhibitor cocktail (Roche), followed by centrifugation at 15,200× reported . The ant5 cells were plated in 12-well plates and transfected on the following day as explained above. Intracellular luciferase activity was measured 48 h post-transfection according to Luciferase Reporter Assay kit (Promega). Luminescence was measured using TD-20/20 Luminometer . Media was harvested for measuring secreted alkaline phosphatase (SEAP) using Secrete-Pair Dual Luminescence Assay kit  according to the manufacturer’s recommendations. The extracellular SEAP values were used to normalize for differences in transfection efficiency. Absorbance was measured on Victor3 microplate reader .1 × 10p values of p < 0.05 were considered significant and marked in the results with an asterisk (*). Error bars represent data as mean ± standard deviation from at least 3 independent experiments.The two-tailed student t test was used to evaluate statistical significance (GraphPad). Our cohort consisted of 88 pediatric MB aged <18 at diagnosis. We classified this cohort into different molecular subgroups based on the histological and immunohistochemical features of the tumors. Examples of distinct patterns of the histological defined entities and the immunohistochemistry of different markers in each subgroup are represented in The majority of the tumors, 69.3% (61/88), exhibited classic histology, whereas desmoplastic/nodular and anaplastic/LCA accounted for 13.6% (12/88) and 17.1% (15/88), respectively. The WNT-activated subgroup comprised 10.2% (9/88) of the cases. Non-WNT/SHH MBs was enriched in our cohort, making up 70.5% (62/88) of the cases, whereas SHH-MBs with TP53 wt (wild-type) accounted for 17% (15/88), and SHH-MBs with TP53 mut (mutated) accounted for 2.3% (2/88) .The four molecular subgroups varied in histological distribution. Histological type was associated with molecular group: the majority of the non-WNT/SHH MB had classic histology and 10% of all the cases had large cell/anaplastic histology. All the WNT-activated MB subgroup presented classic histology except for one case, which had large cell/anaplastic histology. For the SHH-MB TP53 wt subgroup, all the cases showed desmoplastic/nodular histology except two cases that were classic MB. Finally, all the cases of SHH-MB TP53 mut exhibited large cell/anaplastic histology .CTNNB1 exon three in the tumor DNA from all patients. All WNT-activated MB tumors had CTNNB1 exon three mutations, accounting for seven different mutations . The other two cases showed mutations in c.98C>A (p.Ser33Tyr) and one case in c.98C>G (p.Ser33Cys). Three mutations resulted in amino acid substitutions in codons 32 and 34, c.94G>T (p.Asp32Tyr), c.95A>G (p.Asp32Gly) and c.100G>A (p.Gly34Arg)  .CTNBB1 resulted in the in-frame deletion of a TCT codon and the loss of Ser37 residue in the variant β-catenin protein (c.109-111del (p.Ser37del)) (https://pecan.stjude.cloud (accessed on 1 December 2021). Since the function of β-catenin p.Ser37del (ΔS37) is uncertain, we studied this variant more in detail.One of the mutations found in r37del)) . To the r37del)) . Figure 2. CSF on day +15 showed the presence of atypical cells, suggesting metastatic spread. Frozen and formalin-fixed, paraffin-embedded (FFPE) tumor samples from the patient were included in the analysis. Basic hematoxylin-eosin and reticulin stain, immunohistochemistry and FISH analyses were performed. After MB diagnosis, treatment was administered (similar to per COG ACNS-0332) with initial radiotherapy with concomitant vincristine and carboplatin, and then maintenance chemotherapy with cisplatin, vincristine, and cyclophosphamide, beginning in 21 January 2019 ending on 2 October 2019. In 20 December 2019, another craniospinal MRI was performed, and no contrast enhancement suggestive of relapse was observed. There are no signs suggestive of tumor spread through the CSF.The novel mutation found in codon 37 was from a 9-year-old, previously healthy, male child presented to the hospital emergency room with holocraneal cephalea every day with two weeks of evolution. Evaluation showed bilateral papilledema, without nausea, or visual or auditive alteration. A craniospinal Magnetic Resonance Imaging (MRI) was performed, having a cerebellar tumor centered on the IV ventricle with expansion thereof. The first diagnosis conclusion was a posterior fossa tumor. The affectation of the cranial exit foramen, in this case left Luschka, is more typical of ependymoma, however, its basal hyperdensity on CT suggests that a MB is the first possibility. After surgery there was a post-surgical residual tumor <1.5 cmMYC and MYCN amplifications. These results agree with a grade IV WNT-activated MB (WHO 2016).Histological sections displayed tumoral fragments with large tissue artefact. Valuable areas were densely cellular with a diffuse growth pattern A and no CTNNB1 encoding β-catenin S33 residue inhibits the proteosomal degradation of the protein [In order to functionally characterize the novel p.Ser37del (ΔS37) β-catenin variant, we engineered a c.109-111del β-catenin human cDNA, which was used for transient transfections in U2OS human osteosarcoma cells. Expression of the ΔS37 β-catenin variant was monitored by an immunoblot using anti-β-catenin antibody, in comparison with wild-type β-catenin and the variant S33C, frequently found in pediatric MB A. As sho protein . Next, wCTNNB1 gene, we have classified a cohort of 88 pediatric MB patients according to the consensus MB molecular subgroups WNT-activated, SHH and non-WNT/SHH. The molecular and histological classification of our MB cases fit with the distribution found in previous studies [The adscription of MB tumors to molecular subgroups has become of critical importance for the inclusion/exclusion criteria in the new MB clinical trials. In this study, based on immunohistochemistry, FISH, and the mutational status of  studies ,10, withCTNNB1 mutations, including one novel MB CTNNB1 mutation c.109-111del (p.Ser37del). Tumor-associated mutations in CTNNB1 exon three target the N-terminal phosphorylation domain of β-catenin protein, resulting in impaired phosphorylation by GSK-3β, increased β-catenin protein stabilization, and nuclear β-catenin protein accumulation [The WNT-activated MB subgroup is associated with a good prognosis , is rarely metastatic at diagnosis (5%–10% of cases), and rarely recurs ,23. By cmulation ,20,21,31CTNNB1 gene encodes β-catenin, which is a central component of the cadherin/catenin complex and participates in cell adhesion and gene transcription regulation mediated by the WNT signaling pathway. Previous studies have found the association of CTNNB1 mutations with several cancers [CTNNB1 exon three, and mainly result in substitutions of residues Ser33, Ser37, Thr41, and Ser45 which are phosphorylation targets of GSK3β and CK1α kinases [CTNNB1 mutations and β-catenin nuclear accumulation in MB clinical samples, with CTNNB1 mutations detected in 60–70% of β-catenin immunohistochemistry nucleopositive cases [CTNNB1 and nuclear β-catenin accumulation in all the WNT-activated MB cases analyzed.The  cancers ,34,35,36 kinases ,39. Thes kinases ,41,42,43 kinases ,44. A stve cases ,23,44,45CTNNB1 mutation described in this work (c.109-111del (p.Ser37del)), creates the deletion of a TCT codon, resulting in the loss of Ser37, a residue phosphorylated by GSK3-3β. To the best of our knowledge, the CTNNB1 mutation c.109-111del has not been previously found in MB, and it has only been reported in one case of adult hepatocellular carcinoma .The Phosphorylation of β-catenin N-terminal domain has been postulated to occur in a sequential manner (Ser45-Thr41-Ser37-Ser33), although evidence suggests that phosphorylation at Ser33, Ser37, or Thr41 can occur in the absence of phosphorylation at Ser45 . PhosphoIn this study we found three mutations resulted in amino acid substitutions in codons 32 and 34, c.94G>T (p.Asp32Tyr), c.95A>G (p.Asp32Gly), and c.100G>A (p.Gly34Arg), suggesting an influence on the accessibility of the Ser 33 for the GSK-3β kinase, preventing its phosphorylation.Ser37 mutations comprised about 10% of the known ß-catenin mutations, with Ser37Phe, Ser37Cys, and Ser37Ala being the more frequent substitutions. It has been recently described as small molecules that bind to non-phosphorylated or Ser37Ala mutant β-catenin and enhance their binding to β-TrCP, representing a therapeutic strategy to target proteins defective in ligase binding, ubiquitylation, and degradation ,50. Our By contrast, non-WNT MB  are characterized by metastatic disease, increased rates of recurrence, and intermediate/poor overall survival . WNT-actThe WNT signaling pathway regulates the development and maturation of the CNS, but its activation may also promote the neoplastic transformation of this tissue . A recenCTNNB1 gene from WNT-activated medulloblastoma tumors from a cohort of 88 pediatric medulloblastoma patients. A novel exon three CTNNB1 mutation targeting codon 37 is described, which has not been previously reported in MB ). We disclosed gain-of-function properties in cells for the novel ΔS37 β-catenin protein variant.We report nine mutations in exon three of the


Reliable measurement of heating power of magnetic nanofluids (MNs) to accurately predict the AC heat-induction performance in tumors is highly desirable for clinical magnetic nanofluids hyperthermia (MNFH) application because it can save time for screening the performance of newly developed MNFH agent and minimize the over-use of animals dramatically. Here, a bio-mimicking phantom model, called Pseudo-Tumor Environment System (P-TES), biochemically designed by considering the external and internal critical factors related to the complex biological environments is proposed to provide a highly reliable evaluation method of heating performance of MNs for in-vivo MNFH applications. According to the experimentally analyzed results, the heating power of MNs measured using the P-TES is well accorded with the heating temperature measured in the tumors during in-vivo MNFH. This result strongly demonstrates that the proposed P-TES can be recommended as a standardized measurement method of heating performance of MNs for clinical MNFH application. It uses thermal energy induced by magnetic nanoparticles (MNPs) in response to an AC magnetic field, 5. For the last two decades, a huge effort has been devoted to the development of new MNPs with superior heat induction power by tuning the material’s parameters such as size, shape, and composition. Many studies have reported their successful applications to MNFH agents in biomedical fields8.Magnetic nanofluid hyperthermia (MNFH) has been recently considered as an emerging modality for cancer treatment due to its non-invasiveness, minimized side effects, and high treatment efficacies−1) and experimentally determined from the heating-up rate of MNs  at a given amount of MNPs (m), V: sample volume, C: heat capacity of solvent)9. In the most experimental conditions , an initial slop of the magnetic heating curve  is used for SLP measurement to get rid of the external heat transfer effect4. Although the SLP generally represents the intrinsic heat performance of MN, it is still difficult to accurately predict the heating ability at the biological conditions, specifically, tissue temperature, based on the SLP measured at the optimal laboratory conditions. Therefore, the determination of therapeutically sufficient concentration of MNs is still difficult, thus several in-vivo performance tests are additionally required to evaluate the heating capability in biological medium. This is because the currently used SLP measurement method does not consider the complex biological conditions such as total ionic concentration, pH, and viscosity, etc.  is specific loss power (SLP). It is defined as the heating power generated per unit mass W g− and expe−1 min−1) to reflect the heat transfer effect of human body and accurately estimate the AC heating temperature rise (ΔT) in the biological environments. Thirdly, the effects of internal experimental parameters such as salt concentration, pH, and viscosity on the heating power of MNs were investigated. By introducing glycerol/agarose, pH 5 ~ 7, and 150 mM NaCl to a phantom model, the colloidal stability and the Brownian relaxation time-dependent change of heating power of MNs were simultaneously evaluated. Finally, the technical validity of the proposed bio-mimicking phantom model was verified using the tumor xenografted mouse models by in-vivo MNFH studies.In this study, we investigated the external and internal factors that can critically affect the heat induction performance of MNs in biological conditions and established a bio-mimicking phantom model to provide an accurate and reliable heat induction power evaluation method for clinical MNFH application. The effects of external experimental and environmental factors on the heating power of MNs were first explored. Particularly, various sample containers with different shapes were tested to remove the measurement uncertainty associated with the shape of sample containers and to find an optimal container because the container can be a basic experimental error-making factor in measuring the heating power of MNs. Secondly, the effects of surrounding medium (SM) on the heating power of MNs were systematically investigated in the air, 37 °C water, and 37 °C water with different flow rates 17 was applied to the MNs (10 mg mL−1) to measure the AC heating characteristics. The wide variation of AC heat induction curves depending on the position of temperature probe was observed for the cone-shape container compared to other containers  was observed for the cone-shape container  was observed for the spherical-shape container. The temperature distribution was further investigated using thermal imaging of each container during MNFH. When the surface temperature of sample containers was monitored using a thermal camera at the 18. These results indicate that the shape of sample container should be considered as one of the critical factors in accurately evaluating the heating performance of newly developed MNs in lab-based experiments to estimate its validity for in-vivo MNFH application.For the precise and reproducible heat measurement of MNs, the relationship between the temperature distribution of MNs across the sample container and the shape of the sample container was systematically investigated. To find an optimal sample container for SLP measurement, three representative sample containers with cone, cylindrical, and spherical-shapes were prepared and the local temperature of MNs at the different heights in the containers Fig. b was meamentclass2pt{minimmentclass2pt{minimmentclass2pt{minimppl Fig. , the inh−1 min−1) related to body temperature homeostasis19. Water was used as a SM instead of air which is typically used for SLP measurement because the body is mostly composed of water  was placed in the water bath  were measured from 10 mg mL−1 Mg doped γFe2O3 MNs and the corresponding SLP are shown in Fig. AC,max ≈ 57 °C. There was no obvious dependence of TAC,max on the WFR and the calculated SLPs measured from Fig. −1 regardless of the SM conditions and WFR. The heat transfer effects during the first 30 s would be very small because there is a negligible difference in the initial temperature between the MNs and the SM. Therefore, the initial slope (dT/dt = 0.29 K s−1 (air), 0.31 K s−1 (water), Fig. 2 ~ 103 Wm−2 K−1, air (1 ~ 30 Wm−2 K−1)]20. The heat induction temperature distribution of MNs was further characterized in the water SM  owing to the negligibly small heat sinking. However, there was a 4 °C difference between the center and the peripheral region of MN at 300 s due to the heat sinking through the SM. These results indicate that the ΔT measured at the conventional lab conditions  is not valid in characterizing the heating performance of MN in the biological environment due to the ambient condition-induced heating sinking effects. Therefore, the SLP and ΔT  of MNs measured in the water SM can be more representative for the biological (in-vivo) application. Regarding WFR effects, it caused a minimal temperature difference after saturation in our experiments , which is another external experimental factor, on the determination of ΔT and SLP of MNs, a circulating water bath system (40 mL) was established on the inside of AC magnetic coil Fig. a,b. This21,The heat transfer from MN to water SM was further studied to establish a ΔT prediction model depending on SLP in the biological environments. The heat exchange to an environment is followed by the Newton-Richman law and the thermal energy balance is expressed byt is time and W is power. The MN is considered as an internal heat source and the heat sinks to SM. When the temperature is reached at an equilibrium state, Teq, the heat generation and sinking are assumed to be equal, thus Eq.  and volumes (0.25–1.0 mL) of MNs  was reversely calculated as a function of SLP at the given volume and concentration ..t is timDh) [effective volume (Vh)] of MNs, hence the longer Brownian relaxation time constant, τB, is induced as described in Eq.  during MNFH. To precisely estimate the heating performance of MNs in biological environments, the AC heat induction behavior under the various physiological conditions were systematically explored. The salt concentration and pH are first considered as basic physiological factors since they can cause MNP aggregation. The aggregated MNPs increase the hydrodynamic size  were dispersed in water, 40% (wt.) glycerol solution, and 1% agarose gel with pH 5 and 150 mM NaCl to mimic the representative physiological environments of tumors 24  showed similar heat induction curves  and 1% agarose gel (65 W g−1) compared to water medium (100 W g−1) , but had almost the same heat induction curves independent of the media. For PEG-coated Mg doped γFe2O3 MNs (PEG Dh 28), it showed relatively higher ΔT (22 °C) and SLP (98 W g−1) in the water compared to dextran coated Mg doped γFe2O3 MNs. However, the ΔT and SLP were very small and almost unmeasurable at 40% glycerol solution and 1% agarose gel conditions  which is much smaller than that of dextran. However, they almost lost their heating capability at the salt-contained medium due to the severely increased Dh. Because of the relatively large size of Mg doped γFe2O3 MNs (25 nm), the PEG molecules may not stably enclose the surface and easily aggregated at the salt-contained medium. In contrast, the dextran-110 k coated Mg doped γFe2O3 MNs showed superior heating power since their colloidal stability is outstanding in all the tested dispersion media. The ~ 25% decreased SLP in 40% glycerol solution is understood not by the colloidal stability but the viscosity difference between the water (0.7 mPa‧s at 37 °C) and the 40% glycerol solution (2.7 mPa‧s at 37 °C)25. For the dextran-10 k coated Mg doped γFe2O3 MNs, it may not stabilize the surface, hence aggregate up to 40 nm during the surface modification due to the relatively short length of dextran polymer. Therefore, the Dh was increased up to 30% compared to the dextran-110 k coated MNs directly resulting in the lower SLP. It was clearly demonstrated that the SLP and ΔT of MNs strongly depend on the testing sample container and physiological factors-induced change of colloidal stability in biological environments. Based on all the experimental results, the bio-mimicking model composed of, (1) sphere shape container, (2) 40% glycerol solution with pH 5 and 150 mM NaCl, and (3) water surrounding medium, is found to be the most likely condition to describe the tumor condition in-vivo. Therefore, we propose this condition as “Pseudo-Tumor Environment System (P-TES)” to reliably evaluate the ΔT and SLP behavior of MNs to directly predict the tumor temperature during MNFH.where, −1 of dextran-110 k coated Mg doped γFe2O3 MN were injected to a tumor . For comparison, the container volume of bio-mimicking phantom was fixed at 0.3 mL and the same amount of MNs were filled (see experiment conditions). The fappl = 100 kHz, Happl = 140 Oe) was applied for 25 min and the real-time temperature changes were monitored at the central region of the tumor  sphere shape container, (2) 40% glycerol solution with pH 5 and 150 mM NaCl, and (3) water SM, was revealed as the P-TES condition to most accurately predict the ΔT of MNs in tumors during MNFH. Moreover, the ΔT of MNs determined by the ΔT prediction model was coincident well with the ΔT measured in tumors during MNFH. All the experimental results strongly demonstrate the proposed P-TES including ΔT prediction model can be recommended as a standardized measurement method of ΔT of MNs for direct clinical MNFH application. Moreover, the proposed P-TES and ΔT prediction model are expected to save time for screening the performance of newly developed MNFH agent and minimize the over-use of animals dramatically.w = 10 kDa and 110 kDa) and trimethoxysilane PEG (Mw: 591–723 g mol−1) were purchased from Phamacosmos A/S and Gelest for the surface modification. For the purification and concentration of MNPs, Amicon® Stirred Cells  was used. The Hypertheranoid™ 140 AC magnetic field generator  was used for measuring the AC heat induction characteristics. A 4-channel benchtop fiber-optic thermometer  and the FLIR One pro thermal camera (FLIR) were used for the temperature monitoring of magnetic nanofluid. All parts for motorized lifting stages  were purchased from Thorlabs. All glassware was purchased from KJ LAB. Custom-built sample containers, water circulation system, and heat baths were fabricated using a 3D printer .Mg acetate tetrahydrate, Fe acetylacetonate, oleic acid, oleyl amine, benzyl were purchased from MiliporeSigma. Dextran , Fe acetylacetonate (2.0 mmol), oleic acid (1.2 mmol), and benzyl ether (20 mL) were added in a 50 mL round-bottom flask. The mixed reaction solutions were heated up to 200 °C for 30 min (~ 8 °C min−1) and maintained for another 50 min (nucleation step) under N2 gas at the flow rate of ~ 100 mL min−1. Then, the solutions were heated again up to 296 °C for 20 min (~ 5 °C min−1) and maintained for 60 min. After cooling down to room temperature, the product was washed with 100 mL of toluene 3 times. A NdFeB magnet was used for washing and purification of Mg doped γFe2O3 MNs. The final product was dispersed in 100 mL of toluene.The Mg doped γFeHappl and fappl were fixed at 140 Oe and 100 kHz, respectively. The SLP value of all the Mg doped γFe2O3 MNs were calculated based on the following equation of The AC heat induction of MNs was characterized using an AC magnetic field induction system. For measuring the temperature distribution of nanofluid using a fiber-optic thermometer, the AC magnetic field generator with a 50 mm (diameter) vertical AC magnetic coil  and 150 mM, respectively. (2) Agarose phantom: A 400 mg of agarose, a 250 mg of NaCl, and a 1 mL of 1 M acetate buffer solution (pH5) were added to 19 mL of deionized water. A mixture was heated up using a microwave until the agarose is completely dissolved. Rapidly transfer a 0.5 mL of mixture solution to a 0.5 mL of 20 mg mL−1 Mg doped γFe2O3 MN that contained a sample tube. Cooldown to the mixture for 1 h. The final concentration of agarose, and NaCl were 1%, and 150 mM, respectively.The MNs were dispersed in glycerol and agarose gel to test the heat induction power in pseudo biological environments. (1) glycerol phantom: A 75 μL of 2 M NaCl and a 25 μL of 1 M acetate buffer solution (pH5) were added to 0.5 mL of 20 mg mL6 cells of LNCaP were implanted in the right thigh of athymic nude mice . When the tumor volume grew more than 0.25 mL, the mice were divided into 4 groups and 0, 2.5, 5.0. 7.5, and a 10 mg/mLtumor of Mg doped γFe2O3 MNs were injected. The nanofluids were injected into the tumor directly using a syringe pump with an injection speed of 5 µL min−1. The volume of the tumor was 0.25–0.35 mL and the injected volume of MN was around 70 µL. Then, mice were placed on the 37 °C heating bed to keep the body temperature and the fiber-optic thermometer was mounted to the center of the tumor for real-time tumor temperature measurement. MNFH  was carried out for 25 min and the tumor temperature was recorded.All animal experiments were conducted with the guideline of the Association for Assessment and Accreditation of Laboratory Animal Care International  and under the approval of the Institutional Animal Care and Use Committee . All methods used for in vivo studies in mice are in accordance with ARRIVE guidelines. The LNCaP xenograft mice were prepared for in vivo MNFH studies. Briefly, 2 × 10Supplementary Information.


Musa acuminata genome group AAA-EA; hereafter referred to as banana) is critical for Uganda’s food supply, hence our aim to map current distribution and to understand changes in banana production areas over the past five decades. We collected banana presence/absence data through an online survey based on high-resolution satellite images and coupled this data with independent covariates as inputs for ensemble machine learning prediction of current banana distribution. We assessed geographic shifts of production areas using spatially explicit differences between the 1958 and 2016 banana distribution maps. The biophysical factors associated with banana spatial distribution and geographic shift were determined using a logistic regression model and classification and regression tree, respectively. Ensemble models were superior  compared to their constituent algorithms trained with 12 and 17 covariates, respectively: random forests , gradient boosting machines , and neural networks . The logistic regression model (AUC = 0.879) performance was similar to that for the ensemble model and its constituent algorithms. In 2016, banana cultivation was concentrated in the western (44%) and central (36%) regions, while only a small proportion was in the eastern (18%) and northern (2%) regions. About 60% of increased cultivation since 1958 was in the western region; 50% of decreased cultivation in the eastern region; and 44% of continued cultivation in the central region. Soil organic carbon, soil pH, annual precipitation, slope gradient, bulk density and blue reflectance were associated with increased banana cultivation while precipitation seasonality and mean annual temperature were associated with decreased banana cultivation over the past 50 years. The maps of spatial distribution and geographic shift of banana can support targeting of context-specific intensification options and policy advocacy to avert agriculture driven environmental degradation.East African highland banana ( Musa acuminata genome group AAA-EA; hereafter referred to as banana) provides food and income for over 30 million inhabitants of the African Great Lakes Region  Lam.), potato (Solanum tuberosum L.), finger millet (Eleusine coracana Gaertn.), common bean (Phaseolus vulgaris L.), groundnut (Arachis hypogaea L.), sorghum (Sorghum bicolor [L.] Moench), and rice (Oryza sativa L.)) across the country highlight differences in biophysical suitability and socioeconomic importance of specific crops across AEZs. The principal AEZs for banana are characterised by even rainfall distribution with less than three dry months each year ; ε is the error term (mean ≠0 and variance dependent on Xi).Where: Our training dataset with 12 covariates was used for fitting logistic regression models representing different complexities: the null model without covariates (M0-12); the full model with all covariates (M1-12); expansion of M1-12 that includes significant two-way interactions (M2-12). All terms that would not maximise predictive power of M2-12 were iteratively excluded via stepwise regression implemented with the stepAIC function of the R package MASS . Model gAnalysing geographic shifts in the occurrence of banana and potential underlying factors can help different stakeholders along the banana value chain to design strategies to increase productivity and enhance sustainability for the future. In this study, the years 1958 and 2016 were the basis for analysing spatial changes in the distribution of banana in Uganda over a period of 50 years. First, the current (2016) map of banana distribution was resampled from 250 m to 5 km resolution, matching the gridded map of historical (1958) banana distribution. From our assessment, we noticed that if banana presence was due to a single banana field, then it would be much easier to find it in a 250 m × 250 m pixel compared to a 5 km × 5km pixel. The regional share of banana in both 1958 and 2016 were computed using counts of pixels with banana in each region divided by the total number of pixels with banana in Uganda. Second, the gridded maps were overlaid to determine the areas where banana cultivation has decreased (-1), increased (1) and remained stable (2). Third, the geographic shift map was overlaid using spatial polygons of administrative regions, and agroecological zones  [16]. Thz-score from -56.117 to 0.056 indicates that spatial filtering helped to achieve the random point pattern required to ensure that extracted covariates satisfy the assumptions of spatial independence . Average nearest neighbour analysis implemented with the unfiltered 18,949 data points revealed a clustered point pattern with less than 1% likelihood of being due to random chance . Howeverpendence .Resampling of the training data helps to resolve the disparity in the frequencies of presence/absence observations  which can have significant negative impact on model fitting and subsequent predictions. We examined the performance of RF, GBM, and NN trained on the 12 and 17 selected covariates under diverse resampling scenarios Figs ; 5. EffeThe goodness-of-fit statistics indicate that the M2-12 model with significant two-way interactions between covariates included had better performance with respect to the log-likelihood, deviance, AIC, BIC, and ROC AUC . We founThe estimates obtained from fitting the model M2-12 reveals a significant positive influence of BD, RB3BLUE, PH, PREC, SLOPE and SOC and a significant negative influence of AMT and PSEA . SignifiIn 1958, most banana was grown in the central (41%), western (29%) and eastern (27%) regions of Uganda . The curGeographic shifts of banana were classified by locations where cultivation has decreased (8%), increased (46%), or remained stable (46%) . At the -3. Additionally, a series of right-side splits that terminate at node 31 show significant increase in the probability of increased cultivation from 62% to 78% when PSEA ≥ 40%, AMT < 23°C, BD < 1398 kg m-3 and PREC < 938 mm. The left-side split at node 15 coupled with a right-side split at node 30 that terminated at node shows a 66% probability of increased cultivation. Moreover, the left-side split at node 30 that terminated at nodes 120 and 121 indicates a 65% and 74% probability of stable and increased cultivation, respectively. The above evidence intimates that factors associated with crop water requirements have facilitated geographic shift (or lack of it) in the banana cropping system over the past five decades.We performed the CART analysis using the geographic shift classes and the eight significant covariates of the model M2-12  for the dependent variable and explanatory variables, respectively. CART selected PSEA, AMT, BD, PREC, and RB3BLUE for tree construction . The rooWe mapped the distribution of banana by collecting presence/absence data from high-resolution satellite imagery. Coupling the presence/absence information with multiple gridded covariates resulted in a robust geospatial dataset for predicting the spatial distribution of banana. Performing data pre-processing was prudent to deal with general issues often associated with large spatial datasets for prediction modelling. Multicollinearity detected among the acquired 71 covariates underscored the need to select relevant and independent covariates so that built models are not subject to overfitting. Other issues we identified were spatial autocorrelation and class imbalance of response variables. These two problems are often ignored in spatial prediction modelling, yet they can increase spatial bias and uncertainty especially when model predictions must be made outside the scope of learned relationships between covariates and response outcomes .Our second objective was to identify approaches for predicting the spatial distribution of banana using remote sensing data. Spatial filtering helped to ensure randomness of the outcome but could not eliminate class imbalance and residual spatial autocorrelation. We accounted for class imbalance and residual autocorrelation by resampling. The effects of oversampling and undersampling on the accuracy of random forests (RF), gradient boosting machines (GBM) and neural networks (NN) were compared. Unlike oversampling which caused fluctuations in performance between the algorithms, the results from undersampling were more consistent which favoured its selection to resolve class imbalance. Robinson et al.  found thThe third objective focused on determining biophysical covariates that influenced the spatial distribution of banana. Logistic regression facilitated making inferential statements about the relationship the 12 subjectively chosen covariates and the presence or absence of banana. Model estimates revealed a positive influence of blue reflectance, soil organic carbon annual precipitation, slope gradient, soil pH and soil bulk density, and a negative relationship with mean annual temperature and precipitation seasonality. The reasons associated with crop growth and development that guided subjective selection of 12 covariates helped to rationalize the importance of these covariates. Unlike other covariates that have direct influence on banana growth and development, we incorporated blue reflectance among covariates primarily to correct for atmospheric disturbances due to smoke and cloudiness that occasionally obstructed visual interpretation of satellite images . Banana For the fourth objective, we assessed geographic shifts in banana production areas using spatially explicit differences between maps of banana distribution in 1958 and 2016. Most bananas were grown in the central region in 1958 but the western region is now the main hub of banana cultivation. Reports on geographic shift of banana have mostly provided details of production timelines, magnitudes of changes, and hypothetical causal factors but limited spatial evidence of the changes . The disIn the fifth objective, we used the classification and regression tree (CART) to evaluate the biophysical factors associated with geographic shifts in banana distribution over the past five decades. Our results indicate that the expansion of banana cropping system has largely occurred in either marginal areas with relatively poor soils and low rainfall or in ecologically sensitive areas like the tropical rain forests of western Uganda and erosion-prone steep foot slopes of Mount Ruwenzori. Main drivers of decline in banana cropping system are variables linked to soil water supply. Uganda’s banana cultivation is rain-fed  and highClimatic and edaphic factors alone cannot account for geographic shifts in banana. The role of various biotic and cultural factors cannot be ignored. Banana weevil and nematodes have for several decades contributed to the reduced lifespan of banana plantations in the central and eastern regions. The most damaging and costly diseases are Black Sigatoka, Fusarium wilt and Xanthomonas wilt. Also, transitions in dietary habits of people have influenced the crops cultivated as banana is a staple food in the central, eastern, and western region.We set out to map the current distribution of banana in Uganda and to understand how this has changed over the past five decades. The resulting map revealed that banana cultivation was concentrated in the western (44%) and central (36%) regions, with small proportions in the eastern (18%) and northern (2%) regions. A logistic regression model showed that banana distribution was positively influenced by annual precipitation, bulk density, soil organic carbon, soil pH and slope gradient, but negatively influenced by mean annual temperature and precipitation seasonality. Geographic shifts in banana cultivation were defined by areas where the crop has decreased (8%), increased (46%), or remained stable (46%). About 60% of increased cultivation was in the western region, a half of decreased cultivation in the eastern region, and 44% of stable cultivation in the central region. The key variables associated with geographic shifts were biophysical factors related to soil water supply, which signifies the importance of irrigation and soil water conservation to mitigate impacts of climate change induced temperature rises and dry spells. The nature of the geographic shifts emphasises the need to enhance resilience to climate change in the agenda for sustainable intensification of banana.S1 FigZone 1: West Nile Farmlands; 2: Northwestern Farmlands-Wooded-Savanna; 3: Northern Moist Farmlands; 4: Northeastern Central Grass-Bush Farmlands; 5: Northeastern Semi-arid Short Grass Plains; 6: Western Mid-Altitude Farmlands and the Semiliki Flats; 7:Central Wooded Savanna; 8: Southern and Eastern Lake Kyoga Plains; 9: Mountt Elgon Farmlands; 10: Western Medium High Farmlands; 11: Southwestern Grass Farmlands; 12: Lake Victoria Crescent and Mbale Farmlands; 13: Ssese Islands and Sango Plains; 14: Southwestern Highlands.(TIF)Click here for additional data file.S2 FigEach algorithm was trained under three different sampling scenarios: Oversampling (OS), undersampling (US) and without sampling (WS). A) RF–OS; B) RF–US; C) RF–WS; D) GBM–OS; E) GBM–US; F) GBM–WS; G) NN–OS; H) NN–US; I) NN–WS.(TIF)Click here for additional data file.S3 FigEach algorithm was trained under three different sampling scenarios: Oversampling (OS), undersampling (US) and without sampling (WS). A) RF–OS; B) RF–US; C) RF–WS; D) GBM–OS; E) GBM–US; F) GBM–WS; G) NN–OS; H) NN–US; I) NN–WS.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


A high plasma level of the choline-derived metabolite trimethylamine N-oxide (TMAO) is closely related to the development of cardiovascular disease. However, the underlying mechanism remains unclear. In the present study, we demonstrated that a positive correlation of protein arginine methyltransferase 5 (PRMT5) expression and TMAO-induced vascular inflammation, with upregulated vascular cell adhesion molecule-1 (VCAM-1) expression in primary rat and human vascular smooth muscle cells (VSMC) in vitro. Knockdown of PRMT5 suppressed VCAM-1 expression and the adhesion of primary bone marrow-derived macrophages to TMAO-stimulated VSMC. VSMC-specific PRMT5 knockout inhibited vascular inflammation with decreased expression of VCAM-1 in mice. We further identified that PRMT5 promoted VCAM-1 expression via symmetrical demethylation of Nuclear factor-κB p65 on arginine 30 (R30). Finally, we found that TMAO markedly induced the expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) and production of reactive oxygen species, which contributed to PRMT5 expression and subsequent VCAM-1 expression. Collectively, our data provide novel evidence to establish a Nox4-PRMT5-VCAM-1 in mediating TMAO-induced VSMC inflammation. PRMT5 may be a potential target for the treatment of TMAO-induced vascular diseases. Cardiovascular diseases, the major cause of morbidity and mortality worldwide, consist of a group of disorders of the heart and blood vessels . AccumulEpigenetic modulators have been shown to play a major role in the regulation of vascular, immune, and tissue-specific gene expressions within atherosclerotic lesions . Protein2O2. Elucidating the role of TMAO and its underlying mechanisms during VSMC inflammation may provide new insights into the prevention and treatment of TMAO-related cardiovascular events.Here, we reported for the first time that aberrant PRMT5 overexpression positively correlated with TMAO-induced vascular inflammation. Mechanically, PRMT5 post-translationally methylated nuclear factor-κB (NF-κB) p65 subunit, a step imperative to the VCAM-1 induced by TMAO. Furthermore, we found that the TMAO-induced PRMT5 expression was mediated at least in part through upregulation of Nox4 and subsequent production of HTo evaluate potential cytotoxic effect of TMAO on primary rat VSMC, the cells were incubated with TMAO (100–2 000 μM) for 24 h or incubated with TMAO (600 μM) for the indicated periods and cell viability was evaluated by MTT assay. As shown in Fig. Next, the role of PRMT5 on VCAM-1 expression in TMAO-stimulated VSMC was investigated. As shown in Fig. Activation of NF-κB is crucial for driving VCAM-1 gene expression . To examNext, we investigated whether PRMT5 contributed to vascular inflammation in vivo using a classical wire injury-induced vascular inflammation model on carotid arteries. SMC-specific PRMT5 knockout (SMC-PRMT5-KO) mice were applied to verify the role of PRMT5 in vascular inflammation  resulted in a dramatic decrease of TMAO-induced PRMT5 expression, accompanied with reduction of VCAM-1 expression  resulted in enhanced PRMT5 and VCAM-1 expression  is critical for PRMT5 expression . As showion Fig. . More imion Fig. . Taken tSeveral clinical and metabolic studies have demonstrated an association between increased TMAO level and the development of inflammatory vascular diseases , 21. Howl-carnitine and metabolism by the gut microbiome, has been now recognized as a contributor to inflammatory vascular diseases [TMAO, a complex compound derived from phosphatidyl choline/diseases , 23. In diseases , 25. Nevdiseases , 27. Condiseases .There is an emerging body of evidence that PRMT5 plays an important role in modulating transcription by catalyzing symmetric dimethylation on histones . For exa2O2 was involved in TMAO-induced upregulation of PRMT5. Besides, Nox4 silencing significantly attenuated BMDM adhesion to TMAO-stimulated VSMC. Consistently, a Nox4/RhoA/focal adhesion kinase-dependent VSMC adhesion has also been recently reported [The potential mechanisms of TMAO-induced PRMT5 expression were further investigated in this study. Current advances about epigenetics have revealed a parallel scenario showing the influence of oxidative stress as a major regulator of epigenetic gene regulation via modification of DNA methylation, histones, and microRNAs . Oxidatireported .Taken together, we demonstrate that PRMT5 promotes TMAO-induced inflammatory response in VSMC in vitro and in vivo. This occurs through PRMT5-mediated dimethylation of NF-κB p65 on Arg-30. Overall, our data support targeting PRMT5 as a potential therapeutic strategy for the treatment of TMAO-associated VSMC inflammation and the related diseases.flox/flox mice  with SM22α-Cre mice . PRMT5flox/flox mice were constructed with the CRISPR/Cas9 system. The primers used for SM22-Cre mice genotyping were as follows: F1: 5′-CTAGGCCACAGAATTGAAAGATCT-3′, R1: 5′-GTAGGTGGAAATTCTAGCATCATCC-3′ and F2: 5′-GCGGTCTGGCAGTAAAAACTATC-3′, R2: 5′-GTGAAACAGCATTGCTGTCACTT. PRMT5flox/flox mice were genotyped using the primers 5′-TTCTCTAGGGCCTTGAAGACTGGG-3′ and 5′- GGGCACATCACTTTCACTACACTGT-3′. Their age-matched and gender-matched littermate SMC-Cre mice on a C57BL/6 background were used as control mice. SMC-PRMT5-KO mice were identified through Western blot analysis.All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and were approved by the Animal Ethics Committee of Jiangnan University (JN.NO20180615C1440210). SMC-specific PRMT5-KO (SMC-PRMT5-KO) mice were created by crossing PRMT5n = 6) and vascular injured model + TMAO group . SMC-PRMT5-KO mice were also used in vascular injured model + TMAO group  in this study. TMAO (86 μmol) or saline was administered daily by intraperitoneal injection for 7 days [Male mice  underwent complete carotid artery injury. In brief, the mice were anaesthetized with isoflurane in oxygen. The left carotid artery was carefully exposed, and the external carotid artery was ligated with an 8-0 suture immediately proximal to the bifurcation of the internal and external carotid arteries. Wire-induced denudation injury was achieved by removing the endothelium with a guidewire . The guidewire was withdrawn with a rotating motion, and the process was repeated five times. After the guidewire was carefully removed and blood flow was restored. The skin incision was then closed. The sham littermate control mice underwent the same procedures without the vascular injury. SMC-Cre mice were randomly divided into control group  after removal of the intima and adventitia and then maintained in Dulbecco’s modified Eagle’s medium  supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in humidified air with 5% CO2. The purity of VSMC was confirmed by positive staining with alpha-smooth muscle actin (α-SMA) antibody. VSMC were used in passages 2–6.VSMC were cultured by the explant method as described previously . In brie6 cells/mL and differentiated for 7 days in the DMEM supplemented with 10% FBS containing macrophage-colony stimulating factor . All cells were regularly tested negative for mycoplasma contamination.Primary bone marrow-derived macrophages (BMDM) for adhesion assay were obtained from femoral bone marrow suspensions of C57BL/6 mice, and plated at 3 × 10The 3--2, 5-diphenyl-2H-tetrazolium (MTT) bromide assay kit  was used to measure cell viability as described previously . In brieTo introduce small interfering RNA (siRNA) into VSMC, the cells were plated on 6-well plates at 30–50% confluence before transfection. Transfection of cells with siRNA was performed using Lipofectamine 3000 transfection reagent , following the manufacturer’s instructions. Two siRNA targeting Nox4 siRNA, PRMT5 siRNA, NF-κB p65 siRNA, and NF-YA siRNA were designed by GenePharma . The ones with the highest knockdown efficacy were selected for the following experiments. Sequences of siRNA were described in Suppl. Table WT, pcDNA3.1/Flag-p65R30A  were purchased from GENEWIZ . For transfection, adherent VSMC of 60% confluence in 6-well plates were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.The plasmids pcDNA3.1/Flag-p65Western blot analysis was performed as previously described . Cells wImmunofluorescence staining was performed as described previously . At 7-da® Green RT‐qPCR was performed using RT‐qPCR system . The relative mRNA levels were normalized to mRNA levels of β-actin (housekeeping control), and calculations for fold change of each mRNA were made on comparative cycle threshold method (2−ΔΔCt). The primers used in this study were provided in Supplementary Table Total RNA was isolated using TRIzol reagent  and was subjected to reverse transcription using Prime‐Script RT reagent kit  following the manufacturer’s instructions. SYBRg for 15 min. The supernatant was transferred to a new tube and a portion was reserved for input. Immunoprecipitation was performed by mixing cleared cell lysates with the antibodies or normal rabbit IgG as a negative control overnight at 4 °C on a rotator. Protein A/G plus magnetic beads were then added and incubated at 4 °C for 2 h. After washing three times using lysis buffer supplemented with protease inhibitor, complexes were released by boiling for 5 min in 2× loading buffer and subjected to Western blot analyses.Cells were lysed in RIPA lysate (weak) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail after the corresponding treatment and washed with PBS. The cell lysate was centrifuged at 12,000×5 cells/mL) were seeded onto VSMC monolayers and incubated for 1 h in a CO2 incubator. The nonadherent BMDM were removed by washing with PBS. The cell adhesion was detected using a fluorescence microscope . Six images were taken in each well and the number of bound BMDM was counted per image area.BMDM were labeled by BCECF-AM  at 37 °C for 1 h in DMEM. The cells were then washed with culture medium and centrifuged. VSMC grown on 12-well plates were transfected with siRNA or plasmid and then stimulated with TMAO (600 μM) for 24 h. After washing twice with PBS, the labeled BMDM  was added to plasma samples before protein precipitation and monitored in positive MRM mode at m/z 85 → 66 [Quantification of TMAO in plasma samples was performed using stable isotope dilution liquid chromatography-tandem mass spectrometry  and monitored in positive MRM MS mode using characteristic precursor–product ion transitions:  85 → 66 . VariousP < 0.05 was considered statistically significant. Statistical analyses of differences were performed by the Student’s t-test between two groups and one‐way analysis of variance followed by Tukey’s test when comparing among multiple groups. These statistical tests were selected to be appropriate for the normality of distribution and homogeneity of variance.Sample sizes were determined based on preliminary studies. Animal studies were designed to generate groups of equal size using blinded analyses. No sample was excluded from these analyses. The number of replicates and statistical method were indicated in each figure legend. Quantitative data were expressed as a mean ± standard deviation (SD). All data analyses were performed with GraphPad Prism 8 software . Supplemental MaterialReproducibility Checklist


In Cervical cancer (CC), in addition to HPV infection, the most relevant alteration during CC initiation and progression is the aberrant activation of Wnt/β-catenin pathway. Several inhibitory drugs of this pathway are undergoing preclinical and clinical studies. Long non-coding RNAs (lncRNAs) are associated with resistance to treatments. In this regard, understanding the efficiency of drugs that block the Wnt/β-catenin pathway in CC is of relevance to eventually propose successful target therapies in patients with this disease.in vitro in CC cells.We analyzed the levels of expression of 249 components of the Wnt/β-catenin pathway in a group of 109 CC patients. Three drugs that blocking specific elements of Wnt/β-catenin pathway  by TOP FLASH assays and qRT-PCR were tested 137 genes of the Wnt/β-catenin pathway were up-regulated and 112 down-regulated in CC patient’s samples, demonstrating that this pathway is dysregulated. C59 was an efficient drug to inhibit Wnt/β-catenin pathway in CC cells. NSC668036, was not able to inhibit the transcriptional activity of the Wnt/β-catenin pathway. Strikingly, ICRT14 was neither able to inhibit this pathway in HeLa cells, due to HOTAIR interaction with β-catenin, maintaining the Wnt/β-catenin pathway activated.These results demonstrate a mechanism by which HOTAIR evades the effect of ICRT14, a Wnt/β-catenin pathway inhibitory drug, in HeLa cell line. The emergence of these mechanisms reveals new scenarios in the design of target therapies used in cancer. Cervical cancer (CC) constitutes a major health concern worldwide since it is the fourth most common cancer in women . EpidemiRecent studies have revealed that the dysregulation of multiple pathways by long-non coding RNAs (lncRNAs) results in drug resistance , 15. HOXin vitro assays to determine the efficacy of C59, ICRT14 and NSC668036 in order to inhibit Wnt/β-catenin signaling pathway in CC cell lines . C59 was an efficient drug, NSC668036 showed no inhibitory effect while, ICRT14 turned out to have an inhibitory effect in SiHa and CaSki cell lines but did not inhibit the Wnt/β-catenin pathway in the HeLa cell line. HOTAIR overexpression was verified in HeLa cells, and its potential interaction with β-catenin, was associated with Wnt/β-catenin activation, decreasing ICRT14 drug efficiency. These data revealed a new resistance mechanism, hence, some target therapies are not convenient against cancer due to the lncRNAs-mediated regulation in order to promote drug-resistance. The description of these mechanisms provides new insights into further therapeutic opportunities in CC.In the present study, we validated the dysregulation of Wnt/β-catenin pathway signaling in CC patients. Next, since there are several drugs to inhibit this pathway, we performed We included 109 cervical cancer patients from 2010 to 2013 through Instituto Nacional de Cancerología of Mexico City (INCan). This study was approved by INCan’s Review Board and Ethics Committee (015/012/IBI-CEI/961/15). All patients of this study agreed and signed the consent form. In order to identify mRNAs deregulated and involved in the Wnt/β-catenin pathway, gene expression microarray assay was performed for which 89 samples were used and 20 were selected to perform validation by qRT-PCR. After surgical excision, tumor biopsies were segmented into two pieces, one for pathological confirmation and another for nucleic acid separation. Twenty non-pathological cervical tissues were obtained from patients who had undergone a hysterectomy due to uterine myomas.http://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE56303.RNA was extracted from 89 cervical cancer samples and 6 cervical non-tumor tissues to perform the microarray assay and it´s quality was measured using the 18S:28S ratio. Microarray was performed as previously reported and the raw data are publicly available at the GEO database  according to the manufacture’s protocol. The total cDNA was generated by reverse transcription from 1µg of total RNA using the kit High-Capacity cDNA Reverse Transcription (Applied Biosystems) with a final volume of 20 µl. To amplify, c-Jun, c-Myc, MMP7, Cox2, CyD1, MMP10, CTNNB, CSNK1, FZD5, DVL, LRP5, NKD2 Klotho (KL), Cerberus (CER1), NKD1, Wnt11 and HOTAIR, a Luminaris Color HiGreen qPCR Master Mix was used along specific primers and specific amplification conditions for each gene  and SiHa (ATCC HTB-35) were cultured in DMEMF12 (Gibco) medium while CaSki cell line (ATCC CRL-1550) was cultured in RPMI (Gibco) medium, both mediums were supplemented with 10% (v/v). All cells were maintained at 37°C in a humidified atmosphere with 5% CO2. The drugs C59 (Bio-vision 2063-5) and ICRT14  were purchased from Sigma Aldrich and were solubilized in dimethyl sulfoxide . NSC668036 (TOCRIS BIOSCENSE 5813) was purchased from TOCRIS and was dissolved in water. All reagents were stored at -20°C. The IC50 (concentration resulting in a 50% inhibition of cell growth) for each drug is provided by the supplier.3 cells were seeded in a 96-well plate. After 24 hrs of incubation, the cells were exposed to different concentrations close to those given by the supplier of C59, NSC668036, ICRT14 and DMSO as a control in fresh medium for 24 hrs. Cells were washed with PBS and were exposed to MTT  for 3 hrs at 37°C. Then, cells were washed and incubated with 100 µL of DMSO for 10-15 min. Finally, the optical density (OD) was recorded at 540 nm in an Epoch Microplate Spectophotometer (Bioteck).In order to verify the half maximal inhibitory concentration (IC50) provided by the supplier for C59, NSC668036 and ICRT14 in HeLa, SiHa and CaSki cell lines, an MTT assay was employed to determine cell viability. Briefly, 4x 105 cells were seeded in a 6-well plate and co-transfected with 2.5 µg of TOP and FOP plasmids. After 24 hrs, cells were incubated with the IC50 of each inhibitor  or 30 µM DsiHOTAIR  and a scramble sequence (scramble silencer negative control Ambion AM4611); using Lipofectamine 2000 transfection agent (Invitrogen). After incubation for 24 hrs, the cells of each group were collected, and then the activity of Wnt/β-catenin signaling pathway was measured by Dual Luciferase Reporter Assay Kit (Promega) in GloMax® 96 Microplate Luminometer .To determine the activity of Wnt/β-catenin pathway, TOP/FOP flash assay (TCF Reporter Plasmid Kit Merck Millipore) was performed following the manufacturer’s instructions. Briefly, 4x105 cells/well. After 24 hrs, the cells were incubated either with the scramble (30 µM for 48 hrs), Dsi HOTAIR (30 µM for 48 hrs), ICRT14 (IC50 12.9 µM for 24 hrs) or the combination DsiHOTAIR (30 µM for 48 hrs) plus ICRT14 (IC50 12.9 µM incubated for this condition for 24 hrs). Then, the cells were washed with PBS. The level of annexin V binding was determined by using a commercially available annexin V apoptosis detection kit , according to the manufacturer´s instructions. The cells were subsequently analyzed by a flow cytometer . Approximately 10,000 events were collected for each sample. The percentage distributions were calculated by Expo32 ADC software . Cells were classified as apoptotic (positive annexin V and negative PI), late apoptotic/necrotic (positive annexin V and positive PI) or viable (negative annexin V and PI). Unstained HeLa cells were used as negative fluorescence controls. The same procedures were performed for 30 μg/ml etoposide treated cells. Moreover, we captured a photography for each condition (Microcopy leica 090-135.002).Apoptosis was assessed by staining cells with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). Briefly, HeLa cells were washed with PBS, and suspended in serum-free, phenol red-free medium. HeLa cells were seeded in 6-well plates at a density of 3x10RNA immunoprecipitation (RIP) was performed using Magna RIP RNA-Binding Protein Immunoprecipitation kit  according to the manufacturer´s instructions. HeLa cells were lysed in complete RIP lysis Buffer, after the lysate was incubated with RIP buffer containing magnetic beads conjugated with 2.5 µg to human Anti- β-catenin  and negative control normal rabbit IgG (Millipore). Samples were incubated with proteinase K and the inmunoprecipitated RNA was isolated. Finally, HOTAIR was amplified by qRT-PCR as mentioned before.Protein extracts from cultured cells were achieved by homogenization in RIPA buffer (Santa Cruz Biotechnology), later dissipated by centrifugation at 12,000 rpm for 20 min. For immunodetection, 50 µg total protein from cultured cells were mixed with Laemmli sample buffer, boiled, separated in 12 or 15% SDS-PAGE, and transferred in a PVDF membrane . Membranes were incubated overnight using a 1:1,000 (v/v) dilution of the anti-caspase 3 , anti-PARP46D11 , anti-c-Jun  and anti-c-Myc . For detection, 1:2,500 (v/v) dilutions of HRP anti-rabbit or anti-mouse conjugate antibodies (Santa Cruz Biotechnology) were used. Finally, using the Super Signal West Femto chemiluminescent substrate (Thermo Scientific), the membranes were scanned in the C-Digit blot scanner (Li-Cor) and the images were analyzed for densitometry in the associated Image Studio software (LiCor). Membranes were stripped and re-probed for detection of actin  as a loading control. A representative image from three independent experiments is shown.http://pridb.gdcb.iastate.edu/RPISeq/) was used to predict the interaction probability between HOTAIR and β-catenin protein. The interaction probability accepted was ≥ 0.8 in both classifiers of random forest (RF) and support vector machine (SVM).RPISeq software  software. This software assigned a score based on the change of expression relative to the standard deviation of repeated measurements of each Wnt pathway-dysregulated components. Genes with scores higher than the threshold are considered potentially significant, in this way, we contemplated as positively or negatively regulated genes those with a delta score >0.3 and less than − 2.0, respectively . All patients were diagnosed with CC and the most common histologic subtype was squamous cell carcinoma (90.8%). According to the clinical stage classification (FIGO), patients’ specimens were categorized as follows; 60.5% stage IIB, 24.7% stage IIIB, 12% stage IB2 and 0.91% stage IIA and IIIA.https://statweb.stanford.edu/~tibs/SAM/), which detects genes with important expression changes using Delta Score (Score (d) ≥ 1.5 and ≤ 1.5 and a false discovery rate (FDR) < 10%). Thus, we analyzed 249 genes and isoforms involved in Wnt signaling pathway which expression was significantly altered in CC specimens compared to normal cervical tissues (137 were up-regulated and 112 down-regulated)  .To confirm the microarray data, we perform RT-qPCR to validate the expression of key upregulated genes such as: NKD2, c-Jun, DVL, FZD5 and c-Myc as well as key downregulated genes such as: Cerberus (CER1), Klotho (KL), NKD1 and Wnt11. The expression was evaluated in an independent cohort of 20 cervical cancer specimens and 10 normal cervical tissues. As shown in One of our main goals was to probe the efficacy of three drugs  to inhibit the Wnt/β-catenin pathway at three different levels  into the CC cells Figure 3In order to determine if C59, NSC668036 and ICRT14 inhibit the Wnt/β-catenin pathway in CC cell lines, we conducted a TOP-flash assay, the standard assay for assessing Wnt/β-catenin pathway activity. The transcriptional activity of Wnt/β-catenin pathway was determined in HeLa, SiHa and CaSki using the IC50 of C59, NSC668036 and ICRT14. As we expected, the results demonstrated that C59, was able to significantly inhibit the transcriptional activity of Wnt/β-catenin in HeLa, SiHa and CaSki cell lines  can be found in the article/The studies involving human participants were reviewed and approved by Instituto Nacional de Cancerología. The patients/participants provided their written informed consent to participate in this study.AC-P and CP-P conceived and designed the study. ST-C, JC-H, ID-W, and OM-C performed the experiments. AC-P wrote manuscript. DH-S and CL-C contributed to the discussion and analysis of results. DC recollected samples and were responsible for all clinical data of the patients. All authors contributed to the article and approved the submitted version.This research was funded by Instituto Nacional de Cancerología, “Institutional Funds”.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.


WNT/β-catenin signaling is a highly complex pathway that plays diverse roles in various cellular processes. While WNT ligands usually signal through their dedicated Frizzled receptors, the decision to signal in a β-catenin-dependent or -independent manner rests upon the type of co-receptors used. Canonical WNT signaling is β-catenin-dependent, whereas non-canonical WNT signaling is β-catenin-independent according to the classical definition. This still holds true, albeit with some added complexity, as both the pathways seem to cross-talk with intertwined networks that involve the use of different ligands, receptors, and co-receptors. β-catenin can be directly phosphorylated by various kinases governing its participation in either canonical or non-canonical pathways. Moreover, the co-activators that associate with β-catenin determine the output of the pathway in terms of induction of genes promoting proliferation or differentiation. In this review, we provide an overview of how protein phosphorylation controls WNT/β-catenin signaling, particularly in human cancer. WNT/β-catenin signaling is a tightly controlled and highly conserved pathway that regulates cell fate during embryogenesis, hepatobiliary development, liver homeostasis, repair in adulthood, cell proliferation, differentiation, and cell polarity , 2. The CTNNB1 gene in humans and is the vertebrate homolog of the Drosophila Armadillo , and two serine/threonine kinases: casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β) –29. BesiThe destruction complex is ultimately responsible for the phosphorylation of β-catenin, thereby priming it for ubiquitin-mediated proteasomal degradation . HoweverInhibition of CK1, WNT signaling activation, and DVL overexpression suppresses Ser45 phosphorylation . MoreoveIt was observed in Drosophila that upon canonical WNT signaling, Arrow (LRP5/6) recruits AXIN to the membrane, which leads to degradation of AXIN. Thus, the scaffolding member of the destruction complex, AXIN, is no longer present to form the destruction complex . A more c-MYC, CCND1, CDKN1A, and BIRC5 , and lymphoid enhancing factor (LEF). The repressor of the TCF/LEF complex, Groucho, is displaced upon this interaction, whose function is to compact chromatin . Thereafnd BIRC5 . C-MYC iferation , 49.After having understood the regulation of β-catenin in the canonical WNT pathway, we now know that mutations in any of the components of this pathway can lead to its deregulation, which can contribute to a variety of diseases. Herein, we will focus on such mutations contributing to cancer. APC plays an important role in β-catenin degradation, and mutation in APC impairs destruction complex formation. Over 70% of colorectal adenocarcinoma patients carry mutations in the APC gene . Other cCTNNB1, accounting for 54% of cases , and ROR1/ROR2 (receptor tyrosine kinase-like orphan receptor) or RYK acts as co-receptors to initiate non-canonical signaling Figure 4pathways , 62.Cyclin D1  activation that increases cytoplasmic Ca2+ levels. Several Ca2+-responsive enzymes such as protein kinase C (PKC), calcineurin, and calcium/calmodulin-dependent protein kinase II (CaMKII) are activated upon sensing the intracellular Ca2+ flux  is required for centrosome function and spindle assembly during mitosis that, once activated, phosphorylates Polo-like kinase 1 (PLK1) at Thr210 in presence of the co-factor Bora –85. PLK12+-responsive while novel PKC isoforms are dependent on DAG. While several PKC family members have been implicated in tumorigenesis, PKCδ acts as a tumor suppressor, as its main function is to induce apoptosis, apart from regulating β-catenin degradation , which is independent of GSK3β and β-TRCP . Howeverradation , 108–114radation . The claradation . Furtherradation . CollectThe cyclic AMP (cAMP)-dependent protein kinase, protein kinase A (PKA), has diverse cellular functions including cell proliferation, differentiation, cell cycle regulation, and apoptosis. The cAMP/PKA pathway plays a highly complex and cell-specific role in regulating cell growth, as it stimulates growth for some cell types while inhibiting others , 119. PKc-MYC and cyclin (Receptor tyrosine kinase (RTK) consists of a family of around 60 mammalian protein tyrosine kinases , 107. Thd cyclin –141. Thid cyclin . The PTBd cyclin . β-catend cyclin , 144. Sod cyclin , 145.γ chain of several cytokine receptors upon stimulation with cytokines  is a family of six protein serine/threonine kinases that acts mainly as the effector proteins for the Rho GTPases CDC42 and RAC . PAK-famSeveral tyrosine phosphorylation sites have been identified on β-catenin and have been shown to play important roles. In the presence of a disrupted cellular contact and active TGF-β1, E-cadherin and the epithelial integrin α3β1 associate with TGF-β1 receptors, where Tyr654 in β-catenin gets phosphorylated by the epithelial integrin in primary alveolar epithelial cells (AECs) and the phosphorylated β-catenin forms a complex with phospho-SMAD2, resulting in EMT initiation in idiopathic pulmonary fibrosis (IPF) , 162. ThThe BCR-ABL fusion protein, which is frequently found in chronic myeloid leukemia (CML), physically interacts with β-catenin and phosphorylates it at Tyr86 and Tyr654 . PhosphoCasein kinase 2 (CK2) is ubiquitously expressed in the nucleus and cytoplasm of eukaryotic cells . CK2 phoProtein kinase D1 (PKD1) lies downstream of the signaling pathways initiated by diacylglycerol and PKC. Diacylglycerol dictates the intracellular localization of PKD1, while PKC activates it by phosphorylation . PKD1 seIn summary, β-catenin activity is tightly controlled by a series of tyrosine and serine/threonine kinases , the Crafoord Foundation (JK), Magnus Bergvalls Stiftelse (JK), the Swedish Cancer Society (JK), and the Swedish Childhood Cancer Foundation (JK).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.


Chronic kidney disease (CKD) imposes a strong and independent risk for peripheral artery disease (PAD). While solutes retained in CKD patients (uremic solutes) inflict vascular damage, their role in PAD remains elusive. Here, we show that the dietary tryptophan-derived uremic solutes including indoxyl sulfate (IS) and kynurenine (Kyn) at concentrations corresponding to those in CKD patients suppress β-catenin in several cell types, including microvascular endothelial cells (ECs), inhibiting Wnt activity and proangiogenic Wnt targets in ECs. Mechanistic probing revealed that these uremic solutes downregulated β-catenin in a manner dependent on serine 33 in its degron motif and through the aryl hydrocarbon receptor (AHR). Hindlimb ischemia in adenine-induced CKD and IS solute–specific mouse models showed diminished β-catenin and VEGF-A in the capillaries and reduced capillary density, which correlated inversely with blood levels of IS and Kyn and AHR activity in ECs. An AHR inhibitor treatment normalized postischemic angiogenic response in CKD mice to a non-CKD level. In a prospective cohort of PAD patients, plasma levels of tryptophan metabolites and plasma’s AHR-inducing activity in ECs significantly increased the risk of future adverse limb events. This work uncovers the tryptophan metabolite/AHR/β-catenin axis as a mediator of microvascular rarefaction in CKD patients and demonstrates its targetability for PAD in CKD models.  Peripheral artery disease (PAD) is a global health problem affecting 200 million adults worldwide , 2. ChroCompared with the general population, patients with chronic kidney disease (CKD) exhibit several unique aspects of PAD . Among 1CKD is characterized by the retention of a host of solutes called uremic solutes/toxins . A groupThe pathophysiological response to limb ischemia is complex, with a singular goal of compensatory increase in blood flow with the development of macro- and microvasculature (postischemic angiogenesis). This is a dynamic process orchestrated by different signaling processes, of which the Wnt pathway is critical . The binhttps://doi.org/10.1172/JCI142260DS1). See complete unedited blots in the supplemental material. ESKD patients showed no differences in age, sex, ethnicity, and blood pressure compared with controls. However, the groups differed in other comorbidities  patients on hemodialysis (uremic serum). The pooled sera were used first to examine the class effect of CKD. Control sera consisted of age-, sex-, and ethnicity-matched controls without CKD  compared with those exposed to control serum  showed 30% reduced survival  and VEGF-A (P = 0.009) compared with controls  and nuclear β-catenin to a greater extent .CKD patients retain a host of solutes (nonprotein). Water-soluble solutes such as urea (19.98 mM), creatinine (530 μM), oxalic acid (55.53 μM), and homocysteine (19.97 μM) at concentrations seen in CKD patients had no effect on β-catenin . In cont2R = 0.542 and P < 0.001; Wnt-on phase, 2R = 0.768 and P < 0.001).We further confirmed these findings on Wnt activity using immunofluorescence (IF) assays. ECs were pretreated with Wnt3a (Wnt-on phase) or vehicle (Wnt-off phase) along with IS . IS suppy axis scale of Wnt3a- and vehicle-treated cells). The effect on nuclear β-catenin was quantified in randomly selected ECs. Their nuclei were delineated as regions of interest and normalized to the surface area of ECs. This value is presented as the integrated density. Integrated density is a composite of pixel number and intensity and is calculated for the area (μm2), as done previously . Collectively, these results suggested that IS downregulated Wnt/β-catenin in ECs.The effect of IS on nuclear β-catenin was analyzed in an IF assay using profile plots. Profile plots were used to examine pixel intensity throughout the length of cells in microns . IS redueviously , 20. WntWe assessed whether this effect of IS was EC specific or observed in other cells. In fibroblasts (NIH 3T3) and epithelial cells (human kidney HK-2), IS at 10 μM showed a trend toward increases in cytosolic β-catenin in these cells. However, nuclear β-catenin was consistently downregulated by IS , A–D. Sk2. ECs exposed to 21% O2 (normoxia) served as controls. As anticipated, in control samples with IS = 0 μM, nuclear β-catenin levels were higher in hypoxia compared with normoxia . These data suggested that IS and uremic serum augmented β-catenin ubiquitination and degradation in ECs.IS-induced β-catenin downregulation can be at the transcriptional or posttranscriptional level. ECs treated with IS showed no changes in β-catenin mRNA , suggestβ-Catenin N terminus contains a “degron” motif that regulates its protein stability. This motif is characterized by a series of serine and threonine, which in Wnt-off phase undergoes sequential phosphorylation by casine kinases (CK1 and CKD2) and GSK-3β . The phoECs expressing these Myc-tagged constructs  were treP < 0.0001; in the IS-treated group, compared with IS = 0 μM, P = 0.043 and P = 0.003). IS suppressed Wnt activity and secreted IL-8 and VEGF-A in EC media expressing WT β-catenin, but not β-catenin S33A (P = 0.001). A combination of 0.4 μg of S33A along with β-catenin WT activity increased Wnt activity compared with β-catenin WT alone (P = 0.002). No difference in Wnt activity was noted between β-catenin WT with DMSO and 0.4 μg of S33A plus β-catenin WT treated with IS, suggesting that β-catenin S33A restored Wnt activity to baseline in IS-treated ECs. The levels of IL-8 and VEGF-A followed Wnt activity  pathway in various cells , 24, 29.P < 0.001) compared with controls, while AHR KO MEFs showed a nonsignificant trend toward reduction in nuclear β-catenin with uremic serum. Importantly, an approximately 2.4-fold reduction in nuclear β-catenin . This effect of uremic serum was abrogated in AHR CRISPR cells (P = 0.003). A similar pattern was observed in AHR CRISPR ECs with IS (P = 0.037 for IS = 1 μM and P < 0.001 for 10 μM). These results suggested that AHR activity in ECs is necessary for IS- and uremic serum–induced Wnt/β-catenin suppression.We examined mesenchymal embryonic fibroblasts (MEFs) from AHR KO mice and compared them with MEFs with restored AHR expression . AHR inhibition restored IS-induced suppression of Wnt activity . A similar increase in Wnt activity was noted with Kyn in its highest concentration . In fact, there was no significant  and by 90% at 100 μM . In this model, ECs are genetically engineered to express EGFP . The thiP < 0.001; Human PAD is induced in responses to arterial occlusion, and this pathology is not well emulated in the zebrafish model. We investigated the effect of IS in a hindlimb ischemia (HLI) model, which is a widely used murine model of PAD. Mice exposed to IS , 31 unde+ for probenecid-exposed controls, an approximately 30% reduction was observed in IS-exposed mice , β-catenin levels in the capillaries , and VEGF-A expression  with the features of postischemia angiogenesis. The levels of IS correlated inversely with the capillary density (P < 0.001) and approximately 2-fold upregulation of AHR activity (P = 0.002) were observed in response to sera from mice exposed to IS compared with the control group  or the xenobiotic-responsive element (XRE) promoter tethered to the luciferase reporter (to measure AHR activity). A 3-fold downregulation in EC Wnt activity  and remained reduced at 14 (P < 0.001) and 21 days after HLI (P = 0.002)  . ConsistP < 0.001; P = 0.006; The mice on an adenine diet showed 80% reduction in β-catenin protein compared with the control , β-catenin in the capillaries , and VEGF-A expression in the capillaries  (P < 0.001) and an approximately 1.8-fold upregulation of AHR activity (P = 0.002) in response to sera from CKD mice . AHR activity in ECs induced by the sera of CKD mice correlated negatively with the capillary density in such mice  . We corrCKD mice , H and In = 16/group) (n = 8) or vehicle  once a day by i.p. injection for 5 days on and 2 days off for 17 days on adenine or normal diet. Seven days of adenine diet resulted in BUN of 47.3 ± 5.8 mg/dl, corresponding to early stage CKD. This experimental strategy mimics PAD initiation in humans at the early stages of CKD and its progression with deterioration of renal function  . After 7function .P = 0.012) and capillary density as measured by normalized CD31+SMA+ cells (P = 0.041). The mice on a normal diet plus CH223191 also demonstrated an upregulation of capillary β-catenin in the capillaries by approximately 30% (P = 0.026), VEGF-A by 27% (P = 0.053), and skeletal muscle β-catenin by approximately 50% . Notably, an increase in the perfusion ratio in CKD mice treated with CH223191 was comparable to that in mice on a normal diet (non-CKD) (shown as a dotted line P = 0.004), a 3-fold increase in β-catenin expression in the capillaries (P = 0.002), a 2-fold increase in β-catenin expression in skeletal muscles (P = 0.007), and an approximately 1.8-fold increase in VEGF-A expression in the capillaries   (We further examined the correlation between EC AHR and Wnt activities induced by sera from CKD mice with their angiogenic parameters . Sera of= 0.002) . There w= 0.030) . These sP < 0.001). Exposure of ECs to uremic sera resulted in greater AHR activity (P < 0.001) and suppressed Wnt activity (P = 0.017) compared with those treated with control sera. EC Wnt activity inversely correlated with the levels of IS  and Kyn  in ESKD patients  and compared with sera derived from age-, sex-, and ethnic background–matched controls . We had patients , A and Bn = 9), CLTI (n = 8), limb loss (n = 2), graft or stent thrombosis (n = 2), any amputation (n = 6), stenosis/restenosis (n = 7), and new revascularization procedures (n = 8). Patients may have had more than one event, but first event date was used for analysis. The events were adjudicated by a panel of 3 physicians using prespecified definitions. Plasma samples collected at baseline were stored at –80°C. There were no differences in the clinical characteristics of PAD patients who experienced a limb event compared with those without limb events  who were followed prospectively for 2 years for PAD clinical events. Of the entire cohort of 348 patients, 28 patients who experienced an adverse limb event had adequate remaining plasma samples for metabolite measurement. These patients were matched by age, sex, and estimated glomerular filtration rate (eGFR) to 35 PAD patients without adverse limb events (control group). The PAD patients with metabolite measurements were older, but otherwise were like the patients without metabolite measurements. Of the 28 patients with adverse limb events, the events included new or worsening claudication (b events .P = 0.028). Similarly, Kyn levels were 2.2-fold higher in PAD patients with adverse limb event , KA , and XA . There were no differences in the levels of Trp, AA, and QA between the 2 groups. Notably, the concentration range of IS and Kyn found in PAD patients with events downregulated Wnt/β-catenin activity in ECs to a greater extent (The plasma of PAD patients who subsequently developed adverse limb events showed higher IS levels (5.8 ± 5.3 μM) than that of PAD patients without adverse limb events  and suppressed Wnt activity in ECs to a greater extent compared with plasma derived from PAD patients without adverse limb events (P = 0.032). Cox’s proportional hazards model was developed for the incidence of adverse limb events adjusting for age, sex, and eGFR. Higher plasma levels of IS, Kyn, KA, XA, and the extent of EC AHR induction and Wnt suppression by these plasma samples were associated with greater risk of adverse limb events  in different cell types. These observations along with other examples , 24, 30 These data suggest that IS and Kyn increase the risk of PAD initiation and progression up to complications with renal deterioration, a notion supported in clinical studies . Not allThe adenine mouse model was selected for high IS and Kyn levels and its ability to recapitulate uremic features. However, this model is not characterized by proteinuria, a known risk factor of PAD . PostiscThis work provides what we believe is the first mechanistic link among a group of uremic toxins, Wnt signaling, and PAD and paves the way for delineating risk-specific mediators of PAD. This study serves as a foundation for future investigation to examine the predictive power of Trp metabolites for refining the stratification of CKD patients at risk for PAD and explores AHR inhibitors as a therapeutic target for PAD complications.Human subjects, laser doppler analysis, IHC, the HLI model, the adenine CKD model, the IS-specific solute model, cell culture and chemical treatment, generation of the AHR CRISPR cell line, subcellular fractionation, Wnt activity, immunoblotting, 3[H] thymidine incorporation assay, quantitative reverse-transcriptase PCR (qRT-PCR), and ELISAs and zebrafish angiogenesis assays are described in the Supplemental Methods.t test was performed for group comparisons. For multiple groups, overall group comparison was first examined using ANOVA. With the rejection of the null hypothesis, post hoc analysis using Bonferroni’s correction or the pairwise comparisons with Tukey’s multiple comparison procedure was performed for in vitro and in vivo studies. Linear regression was performed to correlate 2 parameters. Cox’s proportional hazard model was developed after adjusting for confounders for human study. P ≤ 0.05 was considered statistically significant.Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software) or SAS, version 9.3 (SAS Institute). Results are expressed as the mean ± SEM or median, range, and 25th and 75th percentiles in box-and-whisker plots. Two-tailed Student’s Both the human and animal studies were conducted with the approval of the IRB at Boston University (H-26367) and the IACUC (PROTO201800298) and Institutional Biosafety Committee (IBC). Human subjects provided written, informed consent.NVA is the first author in the list of shared first authors as she initiated the project, developed the animal models of PAD and completed key experiments. WY performed all the mechanistic cell-based studies and completed remaining experiments to bring this manuscript to completion. VCC conceived of the hypothesis. VCC, NR, NVA, and WY designed the research. NVA, MB, and SL performed the HLI. WY, MAN, SR, SL, and JAW performed the experiments. VCC, MAN, and MB performed IF studies. JAW, MHK, DS, JDR, JJS, SA, AF, and JF provided scientific input and data analyses. SAW and NL performed the metabolomics analysis and provided their interpretation. AF and NMH performed the prospective human study. NMH performed the analysis of the BMC PAD cohort. VCC performed the analysis of in vitro and in vivo studies. VCC, NVA, and WY prepared the manuscript. All authors edited the manuscript.