Cynoglossus semilaevis is an important economic fish species and has long been cultivated in China. Since the completion of its genome and transcriptome sequencing, genes relating to C. semilaevis development have been extensively studied. R-spondin 3 (Rspo3) is a member of the R-spondin family. It plays an important role in biological processes such as vascular development and oncogenesis. In this study, we cloned and characterized the expression patterns and functions of C. semilaevisRspo3. Initial structural and phylogenetic analyses revealed a unique FU3 domain that exists only in ray-finned fish RSPO3. Subsequent embryonic expression profile analysis showed elevating expression of Rspo3 from gastrulation to the formation of the eye lens, while, in tail bud embryos, Rspo3 expression was significantly high in the diencephalon and mesencephalon. The overexpression of C. semilaevis Rspo3 in Danio rerio embryos resulted in a shortened rostral–caudal axis, edema of the pericardial cavity, stubby yolk extension, and ecchymosis. Vascular anomalies were also observed, which is consistent with Rspo3 role in vascular development. Drug treatment and a dual-luciferase reporter assay confirmed the inhibitory role of C. semilaevis Rspo3 in D. rerio Wnt/β-catenin signaling pathway. We further concluded that the FU2, FU3, and TSP1 domains regulate the maternal Wnt/β-catenin signaling pathway, while the FU1 domain regulates the zygotic Wnt/β-catenin signaling pathway. This study enriches Rspo3 research in non-model animals and serves as the basis for further research into the interactions between Rspo and the Wnt/β-catenin signaling pathway. Cynoglossus semilaevis is one of the important economic fish species in China and has long been the subject of large-scale breeding projects. With the completion of its genome and transcriptome sequencing [C. semilaevis has undergone intensive studies mainly focusing on the molecular mechanisms of growth, sex determination, and immunity.quencing ,2, C. seR-spondin (Rspo) proteins are a family of secreted proteins that a share similar domain organization, with all four members characterized by a signal peptide at the N-terminus, followed by two or three furin-like cysteine-rich domains (FU), a thrombospondin-type 1 domain (TSP1), and a basic amino acid-rich C-terminal domain [Rspo1–4 are involved in embryonic development, cell proliferation, and tumorigenesis by activating the canonical Wnt/β-catenin pathway [The l domain ,4,5. Rsp pathway ,7,8, and pathway .Rspo3, the first known member of the Rspo family, was identified in a high-throughput sequencing study of a human fetal brain cDNA library [Xenopus laevis [Danio rerio [Sdc4) [ library  and was  library ,12,13, Xs laevis , Danio rio rerio , and otho [Sdc4) . Though o [Sdc4) .Rspo3 has mainly focused on its role in development. Rspo3 is prominently expressed in blood-forming organs, and its deficiency leads to lethal vessel remodeling defects in mouse embryos [Rspo3 could also promote vascular development in X. laevis by upregulating the expression of VEGF through the activation of the Wnt/β-catenin signaling pathway. Since inhibiting the Wnt/β-catenin signaling pathway could strengthen hematopoiesis, Rspo3 plays a role in keeping a balance between angiogenesis and hematopoiesis [Rspo3 is a crucial regulator of coronary artery formation in the developing heart [Rspo3 can also maintain adult vessels. Overexpression of Rspo3 tightens endothelium junctions and prevents vascular leakage, thereby mitigating the inflammatory events and associated tissue damage in a mouse model of mesenteric ischemia–reperfusion [Rspo3 is implied in limb, heart, and liver development [The functional study of  embryos . Rspo3 copoiesis . Moreoveng heart . Furthererfusion . In addielopment ,20,21.Rspo3 in oncogenesis has also become a hotspot. Rspo3 was identified as a protooncogene in a high-throughput study of retrovirus-induced mutant screening [Rspo3 was found to be correlated to childhood leukemia, secondary thyroid carcinoma, and colon cancer [Rspo2 and Rspo3 chromosome rearrangements can initiate and maintain tumor development [Recently, the role of creening . Later, n cancer ,24. In aelopment .Rspo3 were recognized with the expansion of genome-wide association studies (GWAS). These studies suggest that Rspo3 is involved in skeletal development and maintenance [More potential biological functions of ntenance ,27,28, fntenance ,30,31, antenance .Rspo3 have been conducted in model organisms, but its role in non-model animals such as C. semilaevis has been poorly studied. In this research, we characterized the temporal and spatial expression profiles of Rspo3 in the developmental stages of C. semilaevis and studied its function by overexpressing C. semilaevis Rspo3 mRNA in zebrafish embryos. Furthermore, we studied the effect of C. semilaevis Rspo3 on the Wnt/β-catenin signaling pathway and explored the functions of different protein domains. This research is a detailed study of Rspo3 in non-model animals and provides insight for future C. semilaevis breeding.To date, most studies of C. semilaevis Rspo3 gene by TA cloning and determined its gene structure by searching the NCBI database. C. semilaevis Rspo3 gene contains six exons and five introns  and display a faster evolutionary rate.To elucidate the evolutionary history of the FU3 domain, a phylogenetic tree and a domain structure chart were constructed based on the amino acid sequences of RSPO3 from 35 species . Major vC. semilaevis Rspo3 mRNA was weakly expressed in the early developmental stages before 50%-epiboly and was highly expressed in the subsequent stages  was carried out in tail bud-forming-stage embryos, and strong antisense signals were observed in the diencephalon, metencephalon, and dorsal stripe  zebrafish embryos, with anomalous and undifferentiated LDA, absent Se, chaotic CV and CA , a marker gene of mesoderm, to assess the impact of C. semilaevis Rspo3 on the Wnt/β-catenin signaling pathway and gastrulation. Three groups of zebrafish embryos were incubated with the Wnt/β-catenin signaling pathway activator 6-bromoindirubin-3′-oxime (BIO) or with the inhibitor XAV-939 or were microinjected with capped C. semilaevis Rspo3 mRNA, and their ntl expression levels were analyzed by RT-PCR and WISH.We chose ntl expression patterns and phenotype, the mRNA-injected embryos were closer to XAV-939-treated embryos than to embryos treated with BIO. The overall expression of ntl was significantly downregulated during the epiboly phase in both mRNA-injected and XAV-939-treated embryos but was upregulated in BIO-treated embryos  were constructed by seamless cloning  [Rspo3 was initially detected at the gastrulation stage in Xenopus [Rspo3 is expressed at a relative high level during gastrulation in all these species, which indicates that the role of Rspo3 as a regulator of gastrulation is highly conserved.The analysis of the embryo expression profiles showed elevating mRNA expression in mination . This in Xenopus  and in t Xenopus . HoweverC. semilaevis Rspo3 mRNA was detected in tail bud embryos. Positive signals were detected in the diencephalon and mesencephalon. The spatial expression patterns of Rspo3 displayed both similarities and differences among the aforementioned species. D. rerio Rspo3 displayed a tissue specific expression pattern after 12 hpf, with strong signals detected in brain  and other tissues [Rspo3 mRNA was detected in the posterior primitive streak and allantois at E8.0 and in the forebrain, dorsal neural tube, migrating neural crest cells, limb bud, and developmental heart from E9.0 to E10.5 [X. laevis Rspo3 mRNA was detected in the neural plate at stage 17 and in the forebrain, branchial arches, pronephric sinus, dorsal neural tube, dorsal plate, ventral blood island, and tail-bud mesoderm at stage 28 [Rspo3 in the brain among these species suggests that Rspo3 is involved in the development of the central nervous system. Regrettably, we did not get more spatial expression information for C. semilaevis because only tail bud embryos were analyzed by WISH.After gastrulation, the expression of  tissues . Mouse Rto E10.5 ,14,35. XC. semilaevis embryos and considering the similarity of protein structure and expression patterns between C. semilaevis and D. rerio Rspo3, a gain-of-function analysis was conducted by overexpressing C. semilaevis Rspo3 mRNA in D. rerio embryos. The forced expression of Rspo3 caused phenotypic malformations in D. rerio, including a shorter axis, edema of the pericardial cavity, stubby yolk extension, and ecchymosis in the pericardial cavity or tail. Vascular anomalies were identified by fluorescence and indicated by the decreased of VEGFa in 50%-epiboly embryos. The mechanisms of such abnormal development were studied by using molecular markers. Since gastrulation is regulated by the Wnt/β-catenin signaling pathway, and previous studies have implied the involvement of Rspo3 in regulating the Wnt/β-catenin signaling pathway [C. semilaevis Rspo3 could also interact with this pathway in zebrafish. The expression of ntl in D. rerio was downregulated by overexpressing C. semilaevis Rspo3 or treating with XAV-939. This indicated that C. semilaevis Rspo3 regulates the formation and patterning of mesoderm via regulating the Wnt/β-catenin signaling pathway.Because of the infeasibility of microinjection in  pathway ,5,9, we Rspo3 expression is related to vasculogenesis and angiogenesis in both Xenopus and mouse. Xenopus Rspo3 is expressed in the dorsal lateral plate and ventral blood islands [Rspo3 is expressed in the posterior primitive streak, allantois, and blood vessels [Rspo3 regulates the balance between blood and endothelial differentiation by triggering Wnt/β-catenin signaling to activate the immediate early target gene VEGF [D. rerio embryos by overexpressing C. semilaevis Rspo3 may result from the downregulation of VEGFa. This speculation was substantiated by observations in transgenic strain (flila: EGFP) zebrafish embryos. Based on the above results, the abnormal phenotype and downregulation of VEGFa suggest that C. semilaevis Rspo3 inhibits the Wnt/β-catenin signaling pathway. islands , while m vessels ,35. All ene VEGF . ConsequXenopus and other vertebrates revealed an activatory role of Rspo in regulating the Wnt/β-catenin signaling pathway [Rspo3 inhibits the Wnt/β-catenin signaling pathway in D. rerio embryos [C. semilaevis. Contrary to Wnt3a, Rspo3 significantly reduced TOPFlash reporter expression induced by endogenous Wnt/β-catenin signaling pathway. In addition, the expression of the maternal β-catenin target gene boz and zygotic Wnt/β-catenin signaling target gene sp5l were downregulated at 50%-epiboly by overexpressing C. semilaevis Rspo3, supporting the conclusion that C. semilaevis Rspo3 inhibits the Wnt/β-catenin signaling pathway. Taken together, our results suggest that C. semilaevis Rspo3 suppresses Wnt/β-catenin signaling pathway in D. rerio.Although previous research conducted in  pathway ,12,36,37 embryos . We emplD. rerio embryos, maternal β-catenin accumulates in dorsal margin blastomeres and establishes dorsal patterning by activating the expression of boz after the mid-blastula transition, while zygotic Wnt/β-catenin signaling is essential for the specification of the ventral and posterior fates [boz is not affected significantly by overexpressing Rspo3 in D. rerio at the sphere stage [C. semilaevis Rspo3 in D. rerio embryos, which indicates that the zygotic Wnt/β-catenin signaling pathway is functional at the 50%-epiboly stage but not at the sphere stage. Regardless of the cause, the downregulation of boz and sp5l indicates that C. semilaevis Rspo3 inhibits the Wnt/β-catenin signaling pathway in zebrafish.In or fates ,38. Thoure stage , our stuC. semilaevis Rspo3 and the Wnt/β-catenin signaling pathway. LGR4-6 and ZNRF3/RNF43 have been identified as RSPO receptors [C. semilaevis Rspo3. This provides a new perspective to understand the inhibitory role of C. semilaevis Rspo3 in regulating the Wnt/β-catenin signaling pathway.We studied the expression of ZNRF3, one of the RSPO receptors, in order to explore the mechanisms of the interaction between eceptors ,40,41,42eceptors ,40,43,44eceptors . Howevereceptors . ZNRF3 aboz and sp5l in 50%-epiboly D. rerio embryos microinjected with the corresponding domain-deletion Rspo3 mRNA was detected. We concluded that the FU2, FU3, and TSP1 domains are important for regulating the maternal Wnt/β-catenin signaling pathway, while the FU1 domain is important for regulating the zygotic Wnt/β-catenin signaling pathway. It was reported that RSPO1-2 can bind to RNF3 and ZNRF3 via the FU1 domain and to LGR via the FU2 domain and that the ability of binding ZNRF3/RNF3 differs significantly among RSPO1–4 [C. semilaevis Rspo3 regulated the maternal and zygotic Wnt/β-catenin signaling pathway depending on different domains.We further constructed different types of domain-deletion plasmids to explore the contribution of different domains regulating the Wnt/β-catenin signaling pathway. The expression of  RSPO1–4 ,48. A re RSPO1–4 . Our stuC. semilaevis Rspo3 domains need to be studied further. In addition, phylogenetic and physicochemical analyses showed that the FU3 domain is closer to the FU2 domain, while the FU1 domain has an extra peptide containing eight consecutive and miscellaneous amino acids. Whether the structural and physicochemical differences among the FU domains play a role in regulating interactions with other proteins also needs further study.However, the interactions between receptors and specific C. semilaevis Rspo3 were studied in this research. Our results enrich the study of Rspo3 in non-model animals and provide insight for C. semilaevis breeding.In conclusion, the expression and function of C. semilaevis fish and embryos were collected from Laizhou Mingbo Aquatic Co., Ltd., in Yantai, China. Tissues and organs, including brain, gill, heart, intestine, kidney, liver, muscle, spleen, and gonads were collected from three one-year-old male and female individuals for RNA extraction. Adult tissues and embryos for RNA extraction were stored in RNAwait (Solarbio), and embryos for WISH were stored in 4% PFA.flila: EGFP)) were cultured under a 14 h light–10 h dark cycle. Embryos were obtained by natural crossing, kept in Holtfreter buffer at 28.5 °C, and staged according to standards proposed by Kimmel et al. [Zebrafish, 1–1.5-year-old, .Total RNA from adult tissues and embryos was extracted with Trizol Reagent  according to the manufacturer’s protocol. Adult tissue cDNA synthesis was performed with Reverse Transcriptase M-MLV Kit , while embryo cDNA was synthesized by PrimeScript™ RT reagent Kit with gDNA Eraser  (TaKaRa). cDNA purity and concentration were determined by 1.5% agarose gel electrophoresis and UV spectrophotometry using NanoPhotometer Pearl .C. semilaevis Rspo3 was amplified using the TaKaRa Ex-Taq PCR kit and ligated to vector of pMD18-T (Takara). The template was mixed adult tissue cDNA, the pair of primers consisted of CS-Rspo3-ORF-Fw and CS-Rspo3-ORF-Rv (The ORF sequence of 3-ORF-Rv . The aduRspo3 were retrieved from NCBI (available online: http://www.ncbi.nlm.gov) and Ensemble (available online: www.ensembl.org). The sequences cover 35 species, including Homo sapiens (NP_116173.2), Mus musculus (NP_082627.3), Pan troglodytes (XP_001166327.1), Sus scrofa (NP_001302585.1), Orcinus orca (XP_004263897.1), Neomonachus schauinslandi (XP_021549174.1), Ornithorhynchus anatinus (XP_007656158.1), Gallus gallus (AGG55029.1), Columba livia (XP_005507484.1), Anas platyrhynchos (XP_005009653.1), Alligator mississippiensis (XP_006272521.2), Pygoscelis adeliae (XP_009323912.1), Pogona vitticeps (XP_020646342.1), Anolis carolinensis (XP_008120556.1), Python bivittatus (XP_007421067.1), Pelodiscus sinensis (XP_006139494.1), Alligator sinensis (XP_006027792.1), Chelonia mydas (XP_007064459.1), Xenopus tropicalis (NP_001123245.1), Rhincodon typus (XP_020372626.1), Callorhinchus milii (XP_007897453.1), Latimeria chalumnae (XP_006006810.1), Lipotes vexillifer (XP_007448028.1), Lepisosteus oculatus (XP_015205296.1), Danio rerio (NP_001017358.1), Takifugu rubripes (XP_003966018.1), Oryzias latipes (NP_001239178.1), Astyanax mexicanus (XP_007251874.1), Esox lucius (XP_012994802.1), Hippocampus comes (XP_019750648.1), Oreochromis niloticus (ENSONIP00000009097), Lates calcarifer (XP_018540154.1), Clupea harengus (XP_012692909.1), Paralichthys olivaceus , and Cynoglossus semilaevis . A phylogenetic tree was constructed by MrByes software (V.3.2.2), mcmc = 200,000 generations [http://smart.embl-heidelberg.de) and NCBI CD-search (available online: http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), and the schematic diagram was constructed by the online tool IBS (available online: http://ibs.biocuckoo.org/online.php).Homologous amino acid sequences of i XP_0215174.1, Or® Green I kit  on Roche Lightcycler 480. B2M was used as a reference gene in C. semilaevis, and β-actin was used as a reference gene in D. rerio. The primers containing “RT-PCR” in their names were used for RT-PCR  and was accepted when p < 0.05.RT-PCR was employed to analyze the mRNA expression of different target genes and was executed using SYBRr RT-PCR . The expRspo3-WISH-Fw/Rv and DR-ntl-WISH-Fw/Rv) were used for probe synthesis . The primers were CS--mRNA-Rv . Microin® Reporter Assay System , following the manufacturer’s instructions. TOPFlash was used as a report plasmid, and Renilla was used as a reference plasmid. D. rerio embryos at one- to four-cell stage were injected with a mixture of target gene mRNA, TOPFlash plasmid, and Renilla plasmid, and raised until shield stage. Three independent samples (each with 30 embryos) of each group were lysed, and the ratio of firefly luciferase to Renilla luciferase was used as relative luciferase activity.Luciferase report assays were performed with the Dual-LuciferaseD. rerio embryos were incubated in corresponding solutions until phenotype observation and statistics or sampling for RNA extraction and WISH were performed.The activator BIO and the inhibitor XAV-939 were used to regulate the Wnt/β-catenin signaling pathway. RSPO3) and mutant plasmids  were constructed to study the function of the protein domains. PRSPO3 was constructed by inserting C. semilaevis Rspo3 ORF sequence into PCS2+EGFP plasmid using EcoRI and BamHI restriction sites. The pair of primers was CS-Rspo3-ORF-EC-Fw and CS-Rspo3-ORF-EC-Rv . Pfu DNA polymerase  was used for PCR, and DpnI  was used to digest the template plasmid.Wild-type (PRF-EC-Rv . The mut


Poor, Good, and Excellent cases identified and distinguished by the use of the universal Flame Retardancy Index (FRI). Moreover, evaluations were rechecked by considering the UL-94 test data in four groups as V0, V1, V2, and nonrated (NR). The dimensionless FRI allowed for comparison between flame retardancy performances of epoxy composites. The results of this survey can pave the way for future innovations in developing flame-retardant additives for epoxy.Nowadays, epoxy composites are elements of engineering materials and systems. Although they are known as versatile materials, epoxy resins suffer from high flammability. In this sense, flame retardancy analysis has been recognized as an undeniable requirement for developing future generations of epoxy-based systems. A considerable proportion of the literature on epoxy composites has been devoted to the use of phosphorus-based additives. Nevertheless, innovative flame retardants have coincidentally been under investigation to meet market requirements. This review paper attempts to give an overview of the research on flame retardant epoxy composites by classification of literature in terms of phosphorus (P), non-phosphorus (NP), and combinations of P/NP additives. A comprehensive set of data on cone calorimetry measurements applied on P-, NP-, and P/NP-incorporated epoxy systems was collected and treated. The performance of epoxy composites was qualitatively discussed as  Innovations are mainly born in a very disciplined manner, but sometimes they arise from serendipity. Regardless of the origin of innovative materials and systems, the identification and classification of systems in terms of explanatory variables requires the use of universal, well-accepted criteria. Nowadays, epoxy-based composites are elements of advanced systems ,2,3. The3O4 nanocomposites [Poor, Good, or Excellent) can be correlated to the performance of flame retardancy . The FRI was also powerful in exploring the complementary actions of mineral and organic additives in polymer systems in terms of the peak of HRR (pHRR), the total heat release (THR), and the time to ignition (TTI) of neat polymer and polymer composites [In a previous work, we used two dimensionless indexes to correlate cure state with corrosion inhibition and flame-retardant properties of epoxy/Femposites . By the mposites  and dimemposites , it was mposites . In thisAccording to the literature, a variety of phosphorus-based flame retardants have been used in epoxy resins. Poor, Good, and Excellent flame-retardant performance, are achieved. This is the characteristic of dependency of flame retardancy performance on both the type and the content of the P type of FR. It can be observed that the majority of epoxy systems contains less than 20 wt.% of phosphorus flame retardants. For instance, a compromise between FRI and FR loading percentage was achieved by incorporation of encapsulated ammonium polyphosphate (APP-) at 15 wt.% with an FRI value of 19. Detailed information about the type of phosphorus flame retardants was provided to the reader in the caption of A brief yet informative view of the effect of the used P family of FRs on the flame retardancy performance of epoxy resins is given in Poor category of flame retardancy performance in terms of FRI. It appears that it is not possible to roughly correlate the obtained results in UL94 to those obtained in cone calorimetry tests.Although variation of FRI values according to the composition reflects the flame retardancy of epoxy composites from cone calorimetry angle , other types of flame tests would give more insights into the real effect of one or complementary actions of two or more P type FR additives in epoxy. Based on available data, a brief view of the effect of the used P-based FRs on the flame retardancy performance of epoxy resins as a function of UL94 results is given in Good zone of FRI. The collection of data with FRI values below 5, where LOI% varies depending on the type of phosphorus additive and undoubtedly the content, is hidden behind these symbols.Another test of importance is the limiting oxygen index (LOI), which is demonstrative of flammability. A self-extinguishing behavior is expected when the LOI value is higher than 28. A brief overview of the effect of the used phosphorus-type flame retardants on the flame retardancy performance of epoxy resins as a function of LOI results is given in According to the literature, a variety of nonphosphorus FRs have been used in epoxy resins. ) has been used in a wide range of content in development of flame-retardant epoxy nanocomposites. It can be seen that at high loading rate (up to 30 wt.%), it gives the best results, Excellent in terms of FRI. It can be concluded that the NP class of additives are not individually responsible for high fire resistance of epoxy.From the comparison between A brief overview of the effect of the NP used as FR in epoxy composite preparation and on the flame retardancy performance of epoxy resins as a function of UL-94 results is given in Poor zone of FRI. On the other hand, Excellent flame retardancy seen at high FRI values has LOI of about 22%. From this perspective, it can be concluded that cone calorimetry is not monotonically representative of the character of FR when used in epoxy.A brief overview of the effect of NP-type FR on the flame retardancy performance of epoxy resins as a function of LOI results is given in Assessing the flame retardancy performance of P- and NP-incorporated epoxy systems unraveled the inadequacy of using one FR additive alone when a high performance is required. The antagonism or synergism may be the result of using two or more FR systems in a given polymer matrix. In the case of epoxy, there have been some attempts towards combinatorial use of P and NP additives for the sake of higher performance. To give a more meaningful overview of the effect of combined P and NP additives on flame retardancy performance of epoxy, FRI values are calculated by using calorimetric data given in Poor and Good labels based on FRI values. It is also interesting to note that for a given category, e.g., V-0, the amount of additive changes the FRI, and UL-94 testing does not make sense of such variations.When looking at the UL-94 test results  meet the requirements of highly flame-retardant epoxy composites?”.In previous sections, we categorized the flame-retardant properties of epoxy resins in terms of the universal FRI criterion and the content of flame retardants of three families. We also attempted to find possible correlations between cone calorimetry (reflected in FRI variations), UL-94, and LOI analyses. Since cone calorimetry is the best way to simulate real state combustion of polymers, here, we give a general picture of flame retardancy of epoxy resins . The Poo


Aronia melanocarpa are a rich source of anthocyanins that have received considerable interest for their relations to human health. In this study, the anti-adipogenic effect of cyanidin-3-O-galactoside-enriched Aronia melanocarpa extract (AM-Ex) and its underlying mechanisms were investigated in an in vivo system. Five-week-old male C57BL/6N mice were randomly divided into five groups for 8-week feeding with a control diet (CD), a high-fat diet (HFD), or a HFD with 50 (AM-Ex 50), 100 (AM-Ex 100), or 200 AM-Ex (AM-Ex 200) mg/kg body weight/day. HFD-fed mice showed a significant increase in body weight compared to the CD group, and AM-Ex dose-dependently inhibited this weight gain. AM-Ex significantly reduced the food intake and the weight of white fat tissue, including epididymal fat, retroperitoneal fat, mesenteric fat, and inguinal fat. Treatment with AM-Ex (50 to 200 mg/kg) reduced serum levels of leptin, insulin, triglyceride, total cholesterol, and low density lipoprotein (LDL)-cholesterol. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that AM-Ex suppressed adipogenesis by decreasing CCAAT/enhancer binding protein α, peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor gamma coactivator-1α, acetyl-CoA carboxylase 1, ATP-citrate lyase, fatty acid synthase, and adipocyte protein 2 messenger RNA (mRNA) expressions. These results suggest that AM-Ex is potentially beneficial for the suppression of HFD-induced obesity by modulating multiple pathways associated with adipogenesis and food intake.  The increased prevalence of obesity is a very serious public health problem . ObesityPolyphenol is a common component in vegetables and fruits, and its variety and abundance are very diverse. Polyphenols exhibit various physiological effects, including antioxidant activity. Since oxidative stress is associated with various diseases, such as insulin resistance, diabetes, cardiovascular disease, and some cancers, plants containing polyphenols are seen as a therapeutic alternative for major diseases in modern people ,12. StudAronia melanocarpa, or chokeberry, is a traditional medicinal herb indigenous to eastern North America and eastern Canada. It is effective for colds and has been used as a hypertension and an arteriosclerosis remedy in Europe and Russia. In Japan, these red-purple fruits have been made into juice, wine, and jam /405 . The epididymal adipose tissues were fixed with 4% paraformaldehyde solution and treated according to a general tissue-processing procedure. The tissue samples were stained with hematoxylin and eosin (H&E) and examined under a light microscope. Photographs were obtained using an AxioImager microscope . The size and the number of adipocytes were quantified using an AxioVision Imaging System (Carl Zeiss) with a magnifying power of 200×, and the slides were examined in a blinded manner. TM SYBR Green kit  according to the manufacturer’s instruction. The sequences of the primers used in this study are shown in Total RNA from the epididymal adipose tissue was extracted with Trizol  according to the manufacturer’s instruction. The content and the purity of the total RNA were estimated using a micro-volume spectrophotometer . Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was conducted using a Rotor-gene 3000 PCR  and a Rotor-GeneP < 0.05 was considered to be statistically significant.All results are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using the Student’s t-test to test the differences between the CD group and the HFD group. An ANOVA test followed by Duncan’s multiple comparison test were performed to compare the means between the HFD, the AM-Ex 50, the AM-Ex 100, and the AM-Ex 200 groups. P < 0.001). The body weights in the AM-Ex 200 group were significantly lower than those in the HFD group after the second week of treatment (P < 0.05). Meanwhile, the body weights in the AM-Ex 50 and AM-Ex 100 groups were statistically lower than those in the HFD group after the third week of treatment . The body weight gains in the three AM-Ex-treated groups were dose-dependently reduced compared to the HFD group (P < 0.05). Consistent with the final body weight results, the fat mass percentage by dual energy X-ray absorptiometry (DEXA) analysis revealed that the HFD group showed markedly higher body fat compared to the CD group, and the body fat in the three AM-Ex-treated groups was decreased compared to the HFD group (P < 0.05) in a dose-dependent manner (P < 0.001). The food efficiency ratios in the three AM-Ex-treated groups were significantly lower than in the HFD group (P < 0.001) but did not differ significantly among the three AM-Ex-treated groups . In addition, the HFD group showed significantly higher weights of axillary adipose tissues, known as typical brown adipose tissue, compared to the CD group (P < 0.001). The AM-Ex treatment significantly decreased the weights of white adipose tissues. However, the axillary adipose tissue weights in the AM-Ex 50, 100, and 200 groups did not differ significantly compared to the HFD group (P < 0.001). The AM-Ex treatment significantly decreased the number of adipocytes over 120 μm in the epididymal adipose tissues  weights were dramatically increased in the HFD group . AM-Ex treatment did not affect serum glucose levels, which were elevated by the HFD treatment. Higher insulin levels were observed in the HFD group compared to the CD group. The insulin levels were significantly lowered in the three AM-Ex treated groups. The HOMA-IR index was calculated on the basis of the serum glucose and insulin levels. The HFD group had a dramatically higher HOMA-IR index than the CD group (P < 0.01), whereas the AM-Ex treatment markedly decreased the HOMA-IR index at 16.8%, 26.4%, and 38.9% of AM-Ex 50, AM-Ex 100, and AM-Ex 200, respectively, compared to the HFD group (P < 0.05). The HFD group had the highest triglyceride, total cholesterol, LDL-cholesterol, and HDL-cholesterol levels. The AM-Ex treatment resulted in a remarkable decrease in triglyceride, total cholesterol, and LDL-cholesterol levels. However, HDL-cholesterol levels did not differ significantly between the HFD group and the three AM-Ex-treated groups . The AM-Ex treatment significantly decreased serum leptin levels in a dose-dependent manner (P < 0.05). However, there was no significant difference in serum adiponectin levels between the HFD group and the three AM-Ex-treated groups . AM-Ex treatment noticeably reduced the mRNA expression of C/EBPα, PPARγ, and SREBP-1c elevated by HFD treatment. In addition, the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), a transcriptional coactivator and a key regulator of energy metabolism, was up-regulated in the HFD group compared to the CD group, while it was down-regulated in the three AM-Ex-treated groups , peroxisome proliferator-activated receptor γ (PPARγ), and sterol regulatory element-binding protein-1c (SREBP-1c) and their target genes, in epididymal adipose tissue. Compared to the CD group, the HFD group exhibited dramatically increased C/EBPα, PPARγ, and SREBP-1c messenger RNA (mRNA) expression  and growth conditions , processing, and storage [Aronia melanocarpa extract also varies greatly depending on the extraction method. To increase the antioxidant capacity of Aronia melanocarpa, a method that extracts the maximum amount of anthocyanin is desirable. As previously reported, we obtained Aronia melanocarpa extracts with high cyanidin-3-O-galactoside content (AM-Ex) by adding pressure condition manipulations to existing extraction methods. When extracted with the new extraction method, the content of cyanidin-3-O-galactoside was 2408 mg/100 g, which was about 2.4 times higher than that of conventional methods. Our in vitro studies using 3T3-L1 cells showed that this AM-Ex inhibited adipogenesis [O-galactoside-enriched AM-Ex in an in vivo system.The anthocyanin content of  storage . The conogenesis . This stP < 0.001), but supplementation with varying concentrations of AM-Ex did not affect the axillary fat mass . However, supplementation with 50-200 mg/kg BW of AM-Ex did not reduce blood glucose levels in HFD fed mice . Supplementation of HFD with AM-Ex decreased both transcription and transcription coactivators. C/EBPs was significantly reduced by 50 mg/kg BW AM-Ex supplementation, and the effect remained the same for supplementation amounts of 100 and 200 mg/kg BW AM-Ex. SREBP-1c, PPAR-γ, and PGC-1α were not significantly different at 50 mg/kg BW AM-Ex treatment, and mRNA expression was significantly decreased at 100 and 200 mg/kg BW AM-Ex supplementation -epigallocatechin, genistein, and resveratrol [O-glycosides are also worth including here. Adipogenesis is regulated by transcription factors such as C/EBPs, PPAR-γ, and SREBP-1c, and transcription coactivators, such as PGC-1α. They act as key regulators of glucose and energy metabolism as well as regulation of preadipocyte differentiation and lipid biosynthesis. PGC-1α interacts with the nuclear receptor PPAR-γ, which permits the interaction of this protein with multiple transcription factors ,45,46,47entation . Specifientation . aP2 is entation . ACC proveratrol . Based oO-glucoside [Adipose tissue is a dynamic organ that secretes adipokines. Adipokines play a crucial role in regulating insulin sensitivity, adipogenesis, and obesity. The dysregulated secretion of adipokines results in obesity and insulin resistance. Leptin is as an obese gene product in control of energy balance by inhibiting hunger—generally in proportion to fat mass—with rises in serum levels resulting in a decrease in food intake and an increase in energy expenditure ,57. In olucoside . In contlucoside ,60. In aIn our study, adipogenesis-related transcription factors and coactivators and their associated gene expression were significantly reduced by AM-Ex supplementation, whereas the genes associated with lipolysis were only partially altered . We examO-galactoside-enriched AM-Ex had an anti-obesity effect in C57BL/6 mice. AM-Ex supplementation reduced insulin resistance, serum triglyceride, LDL- and total cholesterol levels, white fat tissue weights, and leptin levels. These changes were related to adipogenesis-related transcription factors and coactivators and the down-regulation of specific adipogenesis-related genes affected by these transcription factors. These changes were also related to decreased food intake due to changes in leptin levels. Our results suggest that cyanidine-3-O-galactoside-enriched Aronia melanocarpa may be used as an anti-obesity agent. More research is needed to find effective and safe doses to apply these results to humans in the future.To summarize the results of this study, we found that cyanidine-3-


Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595. PRMT5 methylation of the spliceosome is a key event in spliceosome assembly, and the attenuation of PRMT5 activity through knockdown or genetic knockout leads to the disruption of cellular splicing3. In addition, PRMT5 methylation of histone arginine residues  is associated with transcriptional silencing, and symmetric dimethylation of H2AR3 has been further implicated in the repression of differentiation genes in embryonic stem cells4.Protein arginine methyltransferases (PRMTs) are enzymes that methylate arginine side chains to generate monomethylation (MMA), asymmetric (ADMA) and symmetric dimethylation (SDMA) on target proteins. PRMT5 activity is responsible for the vast majority of cellular SDMA9. In breast cancer, high PRMT5 expression, together with high PDCD4 (programmed cell death 4) levels predict overall poor survival7. High expression of PRMT5 in glioma is associated with high tumour grade and overall poor survival and PRMT5 knockdown provides a survival benefit in an orthotopic glioblastoma model8. Increased PRMT5 expression and activity contribute to silencing of several tumour suppressor genes in glioma cell lines.Increasing evidence suggests that PRMT5 is involved in tumourigenesis. PRMT5 protein is overexpressed in many cancer types, including lymphoma, glioma, breast and lung cancer. PRMT5 overexpression alone is sufficient to transform normal fibroblasts, while knockdown of PRMT5 leads to a decrease in cell growth and survival in cancer cell lines5. Recent studies uncovered the role of miRNAs in the upregulation of PRMT5 expression in MCL. It was reported that miR-92b and miR-96 levels inversely correlate with PRMT5 levels in MCL and that the downregulation of these miRNAs in MCL cells results in the upregulation PRMT5 protein levels5. Cyclin D1, the oncogene that is translocated in most MCL patients, associates with PRMT5 and increases its activity through a CDK4-dependent mechanism10. PRMT5 mediates the suppression of key genes that negatively regulate DNA replication allowing for cyclin D1-dependent neoplastic growth. PRMT5 knockdown inhibits cyclin D1-dependent cell transformation causing death of tumour cells. Additionally, PRMT5 has been implicated as a key regulator of p53 activity in lymphoma models11. Increased activity of PRMT5 leads to the methylation and inactivation of p53 in cyclin D1 driven lymphoma models, escaping the need of mutational inactivation of p5311. These data suggest that high PRMT5 activity leads to inactivation of p53 in certain genetic and phenotypic contexts, indicating that PRMT5 inhibition could lead to activation of p53 activity and its transcriptional programs in some p53 wild-type cancers.Recent studies highlighted PRMT5 as a key regulator of lymphomagenesis. The strongest mechanistic link currently described between PRMT5 and cancer is in mantle cell lymphoma (MCL). PRMT5 is frequently overexpressed in MCL and is highly expressed in the nuclear compartment where it increases the levels of histone methylation and silences a subset of tumour suppressor genesHere we describe the cellular activity of two potent and selective inhibitors of PRMT5, GSK3203591 and GSK3326595. We demonstrate that PRMT5 inhibition attenuated growth and survival across solid and hematologic cancer cell lines. Lymphoma and breast cancer cell lines were among the most sensitive cell lines tested. Treatment of lymphoma cells with PRMT5 inhibitor induced G1 arrest and subsequent apoptosis in a subset of cell lines. Mechanistic studies demonstrated that PRMT5 inhibition alters gene expression and the splicing phenotype of cells. Alternative splicing events that occur in response to PRMT5 inhibition are enriched in genes that regulate cell cycle progression, suggesting that the splicing phenotype could potentially contribute to the anti-proliferative activity of PRMT5 inhibitors. Importantly, PRMT5 inhibition activated p53 activity in cancer cells through the induction of alternative splicing of the p53 regulator, MDM4. Genome-wide association studies suggest that p53 mutations are among the most highly correlated mutations with the anti-proliferative activity of PRMT5 inhibitors. These data suggest that PRMT5 inhibitors can potentially target many tumour types at least partially due to their ability to regulate cancer relevant pathways, such as p53 and cell cycle. Our data highlight the potential of PRMT5 inhibition as a treatment approach for human cancers and supports the progression of GSK3326595 to clinical trials in cancer patients with solid tumours and lymphoma (NCT02783300).12. GSK3235025 was further optimized through the medicinal chemistry efforts to improve the cellular potency, physicochemical properties and pharmacokinetics parameters to yield more drug-like molecules. Here we describe the activity of two highly selective PRMT5 inhibitors, GSK3203591 (in vitro tool molecule) and GSK3326595  )nt) Fig. .Figure 1Kiapp value of 43 ± 1 nM  yielding a 50 values) with extended preincubation time revealing slow binding inhibition  to provide an estimate of the true potency (Ki*app). GSK3326595 was found to be a potent inhibitor of PRMT5/MEP50 with an IC50 of 6.2 ± 0.8 nM  values and net cell growth/death values were calculated. gIC50 values for GSK3203591 ranged from 7.6 nM to over 30 µM, but the majority (147/276) of cell lines exhibited values below 1 µM. Lymphoma, including MCL and diffuse large B-cell lymphoma (DLBCL), breast, and multiple myeloma cell lines were among the most sensitive tumour types and had gIC50 values in the low nM range  from control- and PRMT5 inhibitor-treated cells by immunoprecipitation with the SDMA antibody followed by mass-spectrometric analysis (MethylscanTM). Most of the SDMA-containing proteins identified were factors that are involved in cellular splicing and RNA processing , transcription (FUBP1), and translation, highlighting the role of PRMT5 as an important regulator of cellular RNA homeostasis . Nearly complete inhibition of SDMA was observed at concentrations similar to that required for complete inhibition of net cell growth in Z-138 cells (gIC100 = 80.7 nM in a 6-day growth/death assay). These data suggest that complete inhibition of net cell growth and the induction of net cell death in Z-138 cells require near-complete suppression of global SDMA levels. The cellular potency of GSK3326595 (SDMA EC50) was further assessed in a panel of breast and lymphoma cell lines representing a range of sensitivities to PRMT5 inhibition . Overall, GSK3326595 inhibited SDMA in all cancer cell lines tested. A trend of more robust inhibition of SDMA was observed in cell lines that are sensitive to PRMT5 inhibition in a proliferation assay.To explore the potential utility of SDMA to be used as a pharmacodynamic biomarker for PRMT5 inhibition, GSK3326595- and GSK3203591-dependent inhibition of cellular SDMA were measured by western and ELISA. First, Z-138 MCL cells were treated with increasing concentrations of GSK3326595 and GSK3203591 to determine the cellular ECent Fig. . An ELISion Fig. . SDMA le50  < 100 nM) while having no effect on cell cycle distribution in the less sensitive MCL cell lines, JEKO-1, MINO, and REC-1 (gIC50 > 100 nM). When tested in breast cancer cell lines, GSK3326595 treatment resulted in an accumulation of MCF-7 cells in the 2 N population after 2 and 7 days of treatment, while in MDA-MB-468 cells the treatment resulted in a decrease in 2 N, and an increase in sub-2N, 4 N and >4 N with 7 and 10 days of treatment. In both MDA-MB-468 and MCF-7, an accumulation of the sub-2N population was observed after 7 and 10 days of treatment, respectively  in a panel of MCL cell lines. Treatment of Z-138 cells led to a dose-dependent increase in early apoptotic and dead cells Fig. . A dose-3  analysis in a panel of lymphoma cell lines with a range of sensitivities to PRMT5 inhibition. In these datasets, PRMT5 inhibition induced alternative splicing events in thousands of genes, supporting the reported splicing defect3 Fig. . Of note3 Fig. –11, sugg14. The loss of exon 6 in MDM4 is proposed to be a sensing mechanism for splicing defects, whereby the skipping of this exon results in loss of the p53 interacting domain, releasing p53 from MDM4 inhibition15. MDM4 exon skipping was observed in our RNA-seq experiment of PRMT5 inhibitor-treated cells regardless of p53 status  induction were evaluated in Z-138 (p53 wild-type) cells following a dose response of GSK3326595 treatment with concentrations ranging from 1.6 nM to 1 μM. p53 and p21 protein expression were induced in a dose-dependent manner in Z-138 and JVM-2 cells Fig. . In Z-13Next, we determined whether PRMT5 inhibition induces an MDM4 isoform switch and p53 activation in a larger panel of lymphoma and breast cell lines Fig. . An MDM417. These data suggest that p53 protein levels are induced by PRMT5 inhibition independently of the MDM4 isoform switch. While it is unclear why p53 protein levels are induced, the observed phenotype could be a result of direct methylation of p53 by PRMT518. Additionally, expression of MDM4-FL significantly attenuated G1 cell cycle arrest induced by GSK3203591, most notably at 500 nM of GSK3203591 treatment  but was improved by later time points   and the mutation profiles of individual genes in 185 human cancer cell lines for which both proliferation and exome-seq data were available. Wilcoxon rank sum tests revealed TP53 mutations as having one of the most significantly different sensitivities between the wild type and mutant cell lines by either gIC50 (2nd most significant) or activity area (10th most significant)  Fig.  and 13.19. In order to reproduce this experiment in an expanded cell line panel, the Pearson correlation was calculated for the expression of each gene relative to sensitivity for 178 cell lines, and gene set enrichment analysis (GSEA) was performed on this ranked gene list . In our lymphoma dataset, the levels of PRMT5 and SDMA did not correlate with anti-proliferative activity , the efficacy was tested in once daily (QD) and twice daily (BID) schedules. Following 21 days of oral dosing  TGI at the 100 mg/kg BID dosing schedule . Our data suggest that cancer cells treated with PRMT5 inhibitors exhibit defects in cellular splicing early, followed up by significant changes in gene expression, highlighting the splicing phenotype as an early event in the response to PRMT5 inhibition. Our numerous attempts to detect reproducible global changes in histone arginine methylation  were unsuccessful (data not shown), and therefore the contribution of histone methylation to the growth/death phenotype induced by PRMT5 inhibition remains untested.Emerging studies have shown that PRMT5 plays an important role in tumourigenesis through the regulation of epigenetic mechanisms . Future translational studies are needed to further understand biological mechanisms of action of PRMT5 inhibitors and refine additional patient populations that could benefit from this novel agent.28. Antibody and gene expression reagents are as described in Supplementary Table Two PRMT5 inhibitors, GSK3203591 and GSK3326595, were obtained from GSK Medicinal Chemistry or Epizyme. Cell lines were obtained from ATCC or DSMZ and were grown in recommended medium, which was most frequently RPMI-1640 containing 10% FBS. Cell line proliferation assays, Western blotting, qRT-PCR, and cell cycle analyses were performed as described previouslyMethylScan technology  was used to evaluate the cellular targets of PRMT5 using a symmetric dimethyl arginine antibody .SDMA ELISA was performed on homogenized cells and frozen tumour lysates. Fluorescence intensity generated by addition of Luminata Forte (Millipore #WBKUF0500) substrate was measured on a PerkinElmer Envision plate reader.An SDMA Imaging Assay was used to evaluate the potency of GSK3326595 in breast cancer cell lines. Cells were seeded in optical bottom cell culture plates and targets were probed with fluorescent secondary antibodies and DAPI stain. Quantification of nuclear SDMA and SMD3 levels was performed with a Molecular Devices MetaExpress high-content fluorescence imager and image analysis was performed with ImageExpress software.RNA was extracted from cell pellets with Trizol using Qiagen’s RNeasy Mini Kit (74104). Libraries were prepared using Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold Library Prep and sequenced on Illumina HiSeq.2500 to 60–80 million PE 2 × 101 bp.3. Reaction products were separated using agarose ethidium bromide gel electrophoresis and visualized using a Bio-Rad VersaDoc Model 5000 imaging system.RNA was isolated from cell pellets as described above and was converted to cDNA using ABI High Capacity Reverse Transcription Reagents (4274966) per manufacturer’s instructions and PCR was performed as described elsewhere2 for 48 hours. Puromycin selection was used to generate and maintain stable cell lines.Lentiviral particles were added at a multiplicity of transduction (MOT) of 20 to Z-138 cells with 50 ug/mL polybrene and cells were centrifuged at 1,000×g for 90 minutes at 37 °C. The virus was washed off and cells were incubated at 37 °C and 5% COIHC detection was performed using the HRP/DAB IHC detection kit  and an anti-p53 antibody (Thermo MA5-16387), following the manufacturer’s protocol. Images were taken with AxioVision software, and cell staining intensity was quantified using the Meta Imaging Series software from Molecular Devices.All data generated or analyzed during this study are included in this published article (and its Supplementary Information files) or are available from the corresponding author on reasonable request. RNA-Seq data is publicly available under Geo accession #GSE108651.Supplementary Dataset 1


Bcl9/9l deletion has no impact upon normal intestinal homeostasis. Loss of BCL9/9l suppressed many features of acute APC loss and subsequent Wnt pathway deregulation in vivo. This resulted in a level of Wnt pathway activation that favoured tumour initiation in the proximal small intestine (SI) and blocked tumour growth in the colon. Furthermore, Bcl9/9l deletion completely abrogated β-catenin driven intestinal and hepatocellular transformation. We speculate these results support the just-right hypothesis of Wnt–driven tumour formation. Importantly, loss of BCL9/9l is particularly effective at blocking colonic tumourigenesis and mutations that most resemble those that occur in human cancer.Different thresholds of Wnt signalling are thought to drive stem cell maintenance, regeneration, differentiation and cancer. However, the principle that oncogenic Wnt signalling could be specifically targeted remains controversial. Here we examine the requirement of BCL9/9l, constituents of the Wnt-enhanceosome, for intestinal transformation following loss of the tumour suppressor APC. Although required for Lgr5+ intestinal stem cells and regeneration,  Bcl9/9l KO mice, the authors of this study show that the BCL9/9l complex is required for intestinal stem cells to drive tissue regeneration and that loss of BCL9/9l suppresses Wnt-driven transformation.Whether the Wnt enhanceosome’ components BCL9/9l can affect intestinal homeostasis and tumorigenesis is still unclear. Using conditional  APC), which is found in 80% of the patients1. APC is a negative regulator of the canonical Wnt signalling pathway, forming part of the β-catenin destruction complex, with frequent mutation in CRC resulting in the hyperactivation of the pathway3. In the absence of Wnt signalling, APC associates with AXIN, Casein Kinase 1 (CK1) and glycogen synthase kinase 3 beta (GSK3β), which are required for the phosphorylation of β-catenin—marking it for ubiquitination and degradation4. Following Wnt activation or APC mutation, the complex is inactivated, whereby phosphorylated β-catenin can no longer be ubiquitinated, saturates the destruction complex and allows de novo synthesised β-catenin to translocate to the nucleus5. Nuclear β-catenin interacts with T-cell factor-1/lymphoid enhancer factor-1 (TCF/LEF1) transcription factors to drive target gene expression7. Additional transcriptional co-activators of β-catenin such as B-cell lymphoma 9 (BCL9)8 and Pygopus9 co-operate in β-catenin-mediated transcription, forming part of the Wnt enhanceosome10.Deregulated Wnt signalling is a hallmark of colorectal cancer (CRC). This predominantly results from mutations in the tumour suppressor gene adenomatous polyposis coli 11. The MCR encodes the 20 amino acid repeats (20AARs) which are required for β-catenin binding and degradation12 and are truncated in CRC, leading to hyperactivated Wnt signalling. Interestingly, colon tumours retain on average two 20AARs13, thought to result in a ‘just-right’ level of Wnt signalling, which may be sub-maximal14. There is evidence that the number of retained 20AARs influences CRC tumour location: proximal colonic tumours retained more than distal colonic tumours16. This tumour distribution could be influenced by the decreasing Wnt gradient that runs from the proximal to distal colon15. Leedham and colleagues proposed where tumours have high pathological Wnt signalling, proximal colonic tumour formation is unfavourable due to high underlying basal Wnt signalling levels in that region, instead distal colonic tumorigenesis is favoured15. Moreover, we recently showed that pharmacological reduction of Wnt signalling reduced intestinal stem cell (ISC) number, ISC competition and increased proximal small intestinal tumour formation in mice where Apc was deleted in the ISCs17 These studies suggest that colon tumours select for APC mutations providing the optimal level of Wnt signalling and that Wnt signalling influences the size of the ISC pool as well as ISC competition.The majority of RNF43 mutations or RSPO amplifications, appear sensitive to suppression of extracellular Wnt signalling using LRP6 blocking antibodies or Porcupine inhibition19, these mutations are rare in CRC. Importantly, as the majority of CRCs carry APC mutations and are Wnt-ligand independent, there is a need to develop strategies that inhibit Wnt signalling in a ligand-independent manner20. This said, Tankyrase inhibitors, which stabilise AXIN, while exhibiting efficacy in CRC cell lines, have severe intestinal toxicity in vivo22. Additionally, cells that experience chronic Wnt signalling, including APC-mutant CRC cells are refractory to Tankyrase inhibition, due to the significant expression of BCL9l and LEF1 which shield β-catenin from AXIN-mediated destruction23. These studies highlight that successful Wnt-based therapies in CRC must act downstream of the destruction complex; disrupting binding of β-catenin to transcriptional activators. Importantly, proof of concept studies has shown that restoration of APC in aggressive carcinoma of mice causes tumour regression24.There has been limited success in targeting Wnt signalling in CRC. Whilst some Wnt-driven cancers, such as those with 25. However, significant interest exists in targeting the β-catenin–BCL9 association in the Wnt enhanceosome. BCL9 and BCL9l, functionally redundant mammalian homologues of the Drosophila gene legless, play a role in nuclear shuttling of β-catenin and promotion of β-catenin-dependent transcription27. While constitutive deletion of Bcl9 and Bcl9l is embryonically lethal28, conditional deletion in the murine intestine is tolerated29. Deletion of Bcl9 and Bcl9l reduces colonic regeneration following acute colitis and decreases expression of Wnt target genes and ISC markers in colonic tumours generated by chemical carcinogenesis29. Hence, BCL9 and BCL9l have been proposed to regulate stemness within the intestinal crypts30. Furthermore, both are upregulated in human CRC32 and overexpression of BCL9l significantly increased tumour formation in ApcMin/+ mice33. A number of small molecules targeting the β-catenin BCL9 interface have shown promise in APC-deficient cells both in vitro and in vivo34, revealing that targeting of BCL9 and BCL9l may offer a therapeutic window in CRC.The β-catenin–TCF interface is large and dynamic, making it difficult to targetApc gene deletion or β-catenin stabilisation. We also sought to identify differences in the activation of oncogenic Wnt signalling when compared to homeostatic Wnt signalling to determine whether there was a therapeutic window for Wnt pathway inhibition following a mutation in the pathway. We report that deletion of Bcl9/9l sensitises the murine epithelium to perturbation of the Wnt pathway and impacts the Lgr5-ISC population. We show that BCL9/9l are required for the acute transformation of the intestine following homozygous deletion of Apc and for Wnt-driven transcriptional programmes associated with APC loss. Unexpectedly, we found that deletion of Bcl9/9l accelerated an APC-driven model of intestinal tumorigenesis and favoured adenoma formation within the proximal SI, but suppressed colonic tumour growth. However, if the β-catenin destruction complex is intact, BCL9/9l are absolutely required for mutant β-catenin-driven intestinal and hepatic transformation driven by mutant β-catenin. Moreover, Mieszczanek et al. (co-submitted manuscript) show that if mice carry a truncating mutation in Apc that is equivalent to human CRC, loss of Bcl9/9l makes these mice resistant to tumorigenesis. Crucially, we show that it is possible to reduce Wnt signalling to a level which prevents transformation in cancer cells which carry ligand-independent Wnt activating mutations, without disrupting normal homeostasis.As previous studies investigating BCL9 and BCL9l (BCL9/9l) were performed in colitis-associated cancer models, we wished to investigate the effect in models of cancer directly driven by activation of Wnt signalling either by 35, with the highest levels defining the number of Lgr5 positive ISCs. Given that BCL9/9l are implicated in Wnt signalling and are required for colonic regeneration following acute colitis, we sought to determine whether Wnt signalling is perturbed in BCL9/9l-deficient intestines. To do this we generated VillinCreERBcl9fl/flBcl9lfl/fl mice. Mice were injected with tamoxifen to induce intestinal Cre-mediated recombination, and harvested 4 days post-induction. Immunohistochemical staining confirmed accumulation of nuclear β-catenin at the base of small intestinal crypts following deletion of Bcl9/9l  revealed a negative enrichment for the Lgr5+ ISC gene signature . Histological analysis revealed that there was a significant reduction in the number of regenerating small intestinal crypts following deletion of Bcl9/9l , immunohistochemical staining and qPCR . Deletion of Apc disrupts the destruction complex, stabilising β-catenin, allowing translocation to the nucleus. Immunohistochemical staining confirmed accumulation of nuclear β-catenin within the small intestinal crypts of both Cre-induced VillinCreERApcfl/fl and VillinCreERApcfl/flBcl9fl/flBcl9lfl/fl mice . We examined the abundance of γH2AX, a marker of DNA damage through IHC staining of intestinal tissue or tumours either proficient or deficient in BCL9/9l and found no difference  throughout the murine intestine and colon transformed the SI leading to a crypt-progenitor phenotype reminiscent of Apcfl/fl intestines, after approximately 21 days post Cre-induction. Deletion of Bcl9/9l significantly extended survival , compared to VillinCreERApcfl/fl   system to express Cre recombinase specifically in hepatocytes to induce recombination of target genes45. We chose a titre of AAV8-TBG-Cre virus that gives near constitutive recombination of the adult liver. Acute deletion of Bcl9/9l did not perturb liver homeostasis or proliferation  is characterised by activating mutations of β-catenin in approximately 30% of patientssed Fig. . InteresBcl9/9l, we chose to compare livers from AAV8-TBG-Cre Ctnnb1ex3/+ and AAV8-TBG-Cre Ctnnb1ex3/+Bcl9fl/flBcl9lfl/fl mice sampled at day 14. Here, there was a significant reduction in liver-to-body weight ratio of AAV8-TBG-Cre Ctnnb1ex3/+Bcl9fl/flBcl9lfl/fl mice compared with AAV8-TBG-Cre Ctnnb1ex3/+ mice , and reviewed by local ethical review committee at the University of Glasgow. Male and female C57BL/6J >20 g mice were induced  with tamoxifen from 6 to 12 weeks of age. The alleles used were as follows: VillinCreER  49, Apc580S  41, Bcl9fl, Bcl9lfl28, Lgr5CreER  50, R26R-LSL-tdTomato (tdTomfl)51 and Ctnnb1ex3  52. Recombination in the acute models was induced using a single intraperitoneal injection of 80 mg/kg tamoxifen for 2 consecutive days and mice sacrificed 4 days post-induction. VillinCreERApcfl/+ mice were aged until they showed clinical signs .Intracolonic Cre inductions were administered under general anaesthesia—a single 70 µl 100 nM dose of 4-hydroxy tamoxifen  was injected into the colonic sub-mucosa via a colonoscope. Colonic tumour growth was then monitored via a colonoscope. Tumour volume was measured relative to lumen size using ImageJ.−1.For regeneration experiments, mice were exposed to γ-irradiation from a caesium-137 source. This delivered γ-irradiation at 0.423 Gy minThe Porcupine inhibitor LGK974 was administered in a concentration of 5 mg/kg BID  in a vehicle of 0.5% Tween-80/0.5% methylcellulose.53. Briefly, viral particles (2 × 1011 genetic copies/mouse) of AAV8.TBG.PI.Cre.rBG  were injected via tail vein in 100 µl PBS. Mice were sacrificed and analysed at the indicated timepoints or aged until clinical endpoint—weight loss, hunching and a swollen abdomen.AAV mediated recombination was performed as previously describedApcfl/fl model, and our prediction that BCL9 and BCL9l were essential, the minimum sample size assuming no overlap in control vs. experimental is three animals. No randomisation was used and the experimenter was blinded to genotypes.In accordance with the 3Rs, the smallest sample size was chosen that could give a significant difference. Given the robust phenotypes of the IHC was performed on formalin-fixed intestinal sections. Standard IHC techniques were used throughout this study. Primary antibodies used for immunohistochemistry were as follows: BrdU , SOX9 , β-catenin , BCL9 , Glutamine Synthetase (1:200 BD Biosciences #610518), γ-H2AX  and CD44 (1:50 BD Biosciences #550538). For each antibody, staining was performed on at least three mice of each genotype, representative images are shown for each staining. For nuclear β-catenin staining Tris-EDTA based antigen retrieval was used.IF was performed on formalin-fixed intestinal sections. Citrate buffer antigen retrieval was used on all sections. Primary antibodies used for IF were as follows: β-catenin , E-cadherin . Sections were stained with DAPI before mounting. Sections were imaged on Zeiss LSM confocal microscope with a ×40 objective.Lgr5 (312178), Olfm4 (311838), Axin2 (400338), Bcl9 (529268) and Bcl9l (466698) mRNA (All Advanced Cell Diagnostics) was performed using RNAscope 2.5 LS (Brown) Detection Kit (Advanced Cell Diagnostics) on a Bond Rx autostainer (Leica) strictly according to the manufacturer’s instructions. Basescope (Advanced Cell Diagnostics) ApcEx14 #701641 (detects wild-type Apc exon 14) was used according to the manufacturer’s instructions.In situ hybridisation detection for 54. The number of regenerating crypts per circumference of a small intestinal section was scored from at least 5 different sections per mouse. A minimum of three mice per genotype was scored. For liver sections, the number of BrdU positive fields of view were quantified, at least 10 views were scored per mouse from at least three mice of each genotype.Proliferation levels were assessed by measuring BrdU incorporation. Mice were injected with 250 µl of BrdU (Amersham Biosciences) 2 hours before being sacrificed. Immunohistochemical staining for BrdU was then performed using an anti-BrdU antibody. For each analysis, 25 half crypts were scored per mouse from at least three mice of each genotype. Regenerating crypts were scored from H&E-stained sections as previously describedGapdh was used to normalise for differences in RNA input. Primer sequences are described in Supplementary Table Whole pieces of small intestinal tissue or liver were used for RNA purification using RNeasy Mini Kit  according to the manufacturer’s instructions. 1 µg of RNA was reverse transcribed using DyNAmo cDNA Synthesis Kit  according to the manufacturer’s instructions, cDNA was diluted 1:10 in RNase-free water. qPCR was performed on each sample in technical duplicate, and with at least three biological replicates per genotype, in a 20 µl reaction mixture containing 10 µl of 2XDyNAmo HS master mix (Thermo Scientific), 0.5 µM of each of the primers (detailed later) and 3 µl cDNA generated previously. The reaction mixture without a template was run in duplicate as a control. The reaction conditions were as follows: 95 °C for 15 min, followed by 40 cycles of three steps consisting of denaturation at 95 °C for 15 s, primer annealing at 60 °C for 30 s, and primer extension at 72 °C for 30 s. A melting curve analysis was performed from 65 to 95 °C in 0.5 °C intervals. VillinCreERBcl9fl/flBcl9lfl/fl mice sacrificed 4 days post tamoxifen injection, and opened longitudinally and washed with PBS. Crypts were isolated as previously described55. Isolated crypts were mixed with 20 µl of Matrigel (BD Bioscience), plated in 24-well plates in Advanced DMEM/F12 supplemented with penicillin–streptomycin, 10 mM HEPES, 2 mM glutamine, N2, B27 , 100 ng ml−1 Noggin and 50 ng ml−1 EGF (both Peprotech). Wild-type crypts were also supplemented with R-spondin conditioned medium. Growth factors were added every 2 days.Mouse small intestines were isolated from wildtype and Cre-induced 56.Whole tissue from the small intestine was used for RNA purification. RNA integrity was analysed with a NanoChip (Agilent RNA 6000 Nanokit #5067-1511). A total of 2 µg of RNA was purified via Poly-A selection. The libraries were run on the Illumina Next Seq 500 using the High Output 75 cycles kit . Analysis of the RNAseq data was carried out as previously described in ref. Lgr5 high vs low study A, Lgr5 cross-platform57, progenitor cluster58, genes upregulated following APC-KO39, Wnt target genes that are increased in human CRC43, direct and functional β-catenin targets in SW480 cells59.GSEA analysis was performed using the GSEA v2.0 software (Broad Institute). The comparison gene sets were obtained from published sources; PLA was performed on tissue samples fixed at 4 °C for <24 h in 10% formalin prior to processing using the Duolink Detection kit (Sigma) according to the manufacturer’s instructions. Briefly, after citrate buffer-mediated antigen retrieval, the slides were incubated with goat E-cadherin  and mouse β-catenin  overnight. Detection was performed with PLA probes (anti-goat and anti-mouse) conjugated to oligonucleotides. After ligation, amplification detection with a fluorescent probe, slides were imaged on a Zeiss LSM confocal microscope. Z-stacks with ×40 objectives were taken. PLA dots in crypts were analysed with ImageJ calculated as area fraction.Lgr5-EGFP-CreERtdTomfl/+ and Lgr5-EGFP-CreERBcl9fl/flBcl9lfl/fl mice were induced with 0.15 mg tamoxifen and then aged for 4, 10 and 21 days. The proximal small intestine was isolated flushed and opened longitudinally and then fixed in 4% paraformaldehyde at room temperature for 4 hours. Tissue was subsequently stored in PBS at 4 °C ahead of processing. Tissue was incubated with DAPI overnight and imaged lumen side down using a Zeiss LSM confocal microscope on a ×10 objective. On average 20 images were acquired from each mouse and the proportion of Tomato positive crypts was determined and a minimum of 200 crypts scored per mouse.Plasma was separated from blood obtained via cardiac puncture after collection into heparin. Liver biochemistry was performed using the Siemens Dimension Expand clinical chemistry system and compatible kits .Statistical analysis was performed with GraphPad Prism V6 Software  using one-tailed Mann–Whitney tests or otherwise stated. For individual value plots, data displayed as mean  ±standard error of the mean (SEM).Further information on experimental design is available in the Supplementary InformationPeer Review FileReporting Summary


The pace of fracture healing slows with age, associated with a transient increase in β-catenin during the initial phase of the repair process. Here we examined the ability of pharmacologic agents that target β-catenin to improve the quality of fracture repair in old mice. 20 month old mice were treated with Nefopam or the tankyrase inhibitor XAV939 after a tibia fracture. Fractures were examined 21 days later by micro-CT and histology, and 28 days later using mechanical testing. Daily treatment with Nefopam for three or seven days but not ten days improved the amount of bone present at the fracture site, inhibited β-catenin protein level, and increased colony forming units osteoblastic from bone marrow cells. At 28 days, treatment increased the work to fracture of the injured tibia. XAV939 had a more modest effect on β-catenin protein, colony forming units osteoblastic, and the amount of bone at the fracture site. This data supports the notion that high levels of β-catenin in the early phase of fracture healing in old animals slows osteogenesis, and suggests a pharmacologic approach that targets β-catenin to improve fracture repair in the elderly. Effective bone regeneration relies on the inflammatory response to tissue injury and the physiology of bone development. Various environmental and biological factors can hinder this regenerative process2. With age, the pace of fracture repair slows, and the risk of non-union increases4. This slower pace of repair in older individuals is responsible for increased morbidity and even mortality5.Almost a third of humans will fracture a bone, and most often these injuries go on to successfully heal. However, a lack of healing (fracture nonunion) is estimated to occur in over 5% of injuries, depending on the anatomic location6. Recent data suggests that factors produced by young macrophage cells pay a critical role in the rejuvenation process7. While these factors regulate the pace of repair, their effects on mesenchymal cells differentiating to osteoblasts are mediated by signaling pathways such as β-catenin. β-catenin signaling plays different roles in mesenchymal differentiation at different repair stages. In the initial phase of repair, the level of β-catenin needs to be precisely regulated as levels that are too high or too low will prevent undifferentiated mesenchymal cells from becoming osteochondral progenitors and will inhibit fracture healing. Once the cells differentiate to osteochondral progenitors, higher levels of β-catenin stimulate osteogenesis and enhance fracture repair9.While there are many factors that could impair fracture healing with aging, the pace of fracture repair can be rejuvenated by circulating factors present in young animals6. This is in contrast to data from young mice, in which inhibition of β-catenin impairs fracture healing8. Since β-catenin protein level regulates the capacity of undifferentiated mesenchymal cells to become osteochondral progenitors, an elevated protein level in older animals will prevent this differentiation and instead cells will maintain a fibroblastic like phenotype. Such a cell type will not contribute to normal fracture repair. Thus, in older animals but not younger animals lowering β-catenin protein level at the initial phases of repair will increase the proportion of undifferentiated mesenchymal cells at the fracture that can go on to become osteoblasts and heal the injury. In the later phases of the repair process, β-catenin is required for osteoblastic differentiation. Targeting β-catenin to effectively improve healing in old animals would need to focus on the earlier phases of the repair process.Data from studies in mice shows that older animals have a higher level of β-catenin protein during the early phase of fracture repair. Genetic inhibition of β-catenin activation rejuvenates the fracture phenotype in older mice10. It is a non-opioid analgesic of the benzoxazolcine family of compounds. This agent has minimal side effects and is used in clinical settings across Europe and Asia11. Although the mechanisms of action of Nefopam is not fully understood, its use decreased β-catenin levels in hypertrophic scar and desmoid tumors10. Another class of agents that can inhibit β-catenin are tankyrase inhibitors. XAV939 binds the tankyrase catalytic poly-ADP-ribose polymerase (PARP) domain, and subsequently increases β-catenin destruction through stabilization of Axin. It has been utilized as an inhibitor of β-catenin in-vivo15. Here we examined the ability of Nefopam and XAV939 in the early phases following a fracture to improve fracture repair in elderly mice.The ability of pharmacologic agents to improve fracture repair by targeting β-catenin in is not known. Nefopam is one agent that is capable of targeting β-catenin in mesenchymal cells9, The 3 and 7 day time frame is also the period in which β-catenin protein levels are even higher in older animals6. Thus, we selected time frames to test the drug when we knew β-catenin protein levels were elevated, as well as one longer duration time point (treatment with nefopam from 3 to 10 days). Treatment was carried out using a dose known to inhibit β-catenin in mesenchymal cells16. Control mice were treated with carrier alone for the same time interval. At 21 days after fracture, the mice were sacrificed and bones analyzed histologically and by μCT. There were no significant differences in μCT parameters, histomorphometric parameters, or mechanical properties for the carrier controls treated for 3, 7, or 10 days. Treatment with Nefopam for 3 or 7 days improved the bone volume/total volume and the relative density at the fracture site, while mice treated with Nefopam for 10 days displayed little difference compared to controls .To determine if other agents that inhibit β-catenin might regulate fracture healing in old mice, we investigated the tankyrase inhibitor XAV939. Mice were treated for 3 or 7 days after fracture with a previously established effective doseion Fig. . Histomoion Fig. . We thenion Fig. . This daThe ultimate goal of fracture repair is to reconstitute the mechanical properties of the injured bone. To determine if Nefopam treatment would improve the mechanical properties of the fractured bone, we measured the work to failure 4 weeks after injury and compared this to unfractured bone. There was a significant decline in work to failure in the healing control bone compared to uninjured bone. However, there was an increase in the work to failure in healing fractures in bone from Nefopam treated mice  and dissolved in normal saline as carrier control, then delivered by intraperitoneal injection (i.p.) (30 mg/kg). XAV939 was purchased from Selleck  and dissolved in 4% dimethyl sulfoxide (DMSO). Corn oil was used for vehicle or carrier control. XAV939 was subsequently delivered by i.p. injection (20 mg/kg). Mice were randomly selected to either receive treatment or carrier. Treatment was started from the day of surgery and progressed at a duration of 3, 7, or 10 days. Doses were selected from prior studies. Animals were observed for any deleterious symptoms and their weight was recorded before, during and after treatment.Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Duke University. Experiments were performed in compliance with NIH guidelines on the use and care of laboratory and experimental animals. C57BL6 mice were provided from Charles River Laboratories  and housed under a 12-hour light, 12-hour dark cycle. Food and water were provided 8. Briefly after anesthesia was induced, the surgical area was shaved and disinfected. A 3-mm longitudinal incision was made in the patella tendon and 0.5mm hole was drilled by a 25G syringe needle above the tibial tuberosity. Intramedullary fixation was achieved using a 0.7 mm insect pin then the mid-tibial diaphysis was transected with small surgical scissors. Osteotomies were made parallel to the tibial plateau. The incision was then closed using sutures. Analgesia was provided to all mice. As in our prior work20. Slow release buprenorphine hydrochloride analgesia (0.03 mg/kg). was provided as an analgesic during the generation of a tibial injury and the initial painful period after the fracture was generated until animals returned to their usual activity level.A tibia fracture with intramedullary stabilization was used to study bone repair3). Morphometric parameters were quantified21 at the site of fracture which included 1mm proximal and distal from the fracture location. The original grayscale image datasets of the tibia region of interest were imported into the manufacturer software and the cortical and callus regions were separated using an automated contouring method. Calcified tissues were segmented from soft tissues using a global thresholding procedure with a threshold of 480 mg HA/cm3, which represents 45% of the attenuation of mature cortical bone . Callus maturity was measured using differential thresholding in which immature bone was defined by a threshold of 169 to 400 mg HA/cm3. As an additional analysis, the unfractured tibia was also examined using μCT. Parameters such as Bone volume/total volume in the tibia were analyzed using ASBMR guidelines22.The fractured tibia, as well as, and the intact, contralateral tibiae were studied after mice were sacrificed at postoperative 21 days. Tibiae were then fixed in 10% formalin for 48 h at room temperature (25 °C) and then transferred to 70% ethanol. Implants were removed from the tibiae prior to scanning. The quality of fracture repair was assessed using the Faxitron MX20 X-ray system . Quantitative three-dimensional evaluation of the fracture callus and healing was undertaken by μCT using a Viva CT 80  at 55 kVp and 145 μA with a resolution of 15.6 μm voxel size. A hydroxyapatite calibration phantom was used to scale values of linear attenuation for the calcified tissues to bone density values  for 7 days at room temperature. Decalcified samples were dehydrated in a graded ethanol series, embedded into paraffin. Sections 5 µm thick were prepared and stained using Safranin-O (to stain proteoglycans/cartilage red) and counterstained with Fast Green (to stain bone green/blue). For evaluation of osteoclastogenesis, the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells was visualized by TRAP staining (Sigma-Aldrich). The sections were examined and photographed using a Leica digital imaging system . Histomorphometry measurements were quantified using ImageJ software .Bone fragments were collected from the tibia from mice treated with carrier, Nefopam, or XAV939, harvested on the day 3 of treatment. After dissection, the harvested tissues were immediately frozen in liquid nitrogen, pulverized then homogenized in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific). Total protein was quantified with Bradford reagent using a bovine serum albumin (BSA) standard curve as a reference. SDS-polyacrylamide gels at 8% concentration were loaded with 25 µg of protein lysate. Anti-β-catenin antibody (AB19022) was purchased from Millipore, an antibody that recognizes activated (unphosphorylated at Ser-37 and Thr-41) β-catenin (clone 8E7) was also obtained from Millipore and an anti-β-actin (13E5) antibody was purchased from Cell Signaling and was probed as a loading control. Indicated antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and ECL chemiluminescence (Bio-Rad). Protein expression was analyzed by measuring the intensity of detected bands compared to intact tibia samples and normalized to β-actin with ImageJ software. RNA was extracted from CFU cell cultures using Trizol (Invitrogen) and cDNA was generated with Superscript II (Invitrogen). For real-time reverse transcriptase PCR (RT-PCR), mouse–specific Taq-Man fluorogenic probes for alkaline phosphatase, collagen type I, osteocalcin, and osteopontin were used. Asparagine synthetase and glyceraldehyde-3-phosphate were used as internal control genes.5 cells/ml in plating medium  or 1 × 106 cells/ml for seven days. To determine the colony forming units-fibroblastic after14 days with plating media; wells were then washed with PBS, fixed with 10% formalin, and stained with 0.05% crystal violet. Cells were cultured in osteogenic differentiation media  for next 14 days. Cultures were incubated for 21 days at 37 °C with 5% CO2 and media were replenished every 2 days. Wells were washed with PBS, fixed using 10% formalin, and stained for alkaline phosphatase (ALP) using NBT/BCIP and mineralization using 2.5% silver nitrate solution (Von Kossa) on a light box and 2% Alizarin Red S (pH 4.3). Colonies were defined by containing at least 25 cells.Unfractured tibia of 20-month old mice was isolated and contents of medullary cavity (bone marrow) were flushed using αMEM culture medium (Gibco) on POD 21 days. Cell suspensions were passed through an 18G needle to dissociate cell aggregates, and cells were plated in 24-well plates with a density of 5 × 1025. Tibia were tested to failure in four-point bending on a Precision Materials testing machine  with a 500N force transducer . The supports of the four-point bend fixture were cylindrical and 2 mm in diameter. The distance between the midpoints of the lower load supports was set to 8.9 mm, and the distance between the midpoints of the upper load supports was set to 4 mm, spanning the fracture callus and centered over the lower supports. Tibiae were loaded by a flexion moment with the flat anteromedial surface down26 on the lower supports at a constant displacement rate of 0.03 mm/s27 to failure. The data acquisition rate for load, displacement, and time was 20 Hz. Failure loads were calculated using a peak-prominence calculation in MatLab. A prominence of 0.25 was used for intact contralateral tibia and 0.4 for fractured tibia. Failure loads were then confirmed visually as the primary departure of a linear trend of the load-displacement data. Bending stiffness was calculated as the slope of the load v. displacement data between 30–70% of the load at failure to exclude the nonlinear toe-regions. Energy to failure (mJ) was calculated using a trapezoidal algorithm of the area under the load-displacement curves.Tibiae for mechanical testing were harvested four weeks following the generation of a Fracture. Intact tibiae were harvested, soft tissue removed, and immediately wrapped in PBS soaked gauze and then frozen at −20 °C. The limbs were thawed prior to mechanical testing. This process has been shown to be the best method of preservation of mechanical properties in bone. The inherent variability in mechanical testing was found to be greater than any loss in bone properties due to the processing29. The data shown in Figs A priori power analysis to obtain statistical significance  between the two groups. Sample sizes were determined using a power calculation based on variances from prior investigations: Micro-computed-tomography (μCT) imaging, n ≥ 7 per group on day 21; histology, n ≥ 7 per group on day 21; colony forming unit (CFU), n ≥ 3 per group on days 21; western blot, n ≥ 3 per group on days 3. μCT and histology data were analyzed in a blinded manner. Data are expressed as the means ± 95% confidence intervals. The Bartlett test was used to determine if the comparison groups followed a normal distribution. If the Bartlett test showed that the variances equal statistical significance was evaluated through a Student’s t-test for all comparisons in which there were two groups; or a one-way analysis of variance (ANOVA) followed by a Turkey post hoc testing for analyses in which there were three or more comparisons being made. If Bartlett test showed that the variances were not parametric, the Kruskal-Wallis test to test for differences in population means instead of ANOVA and the Steel-Dwass test was used instead of Turkeysupplementary information


Pseudomonas aeruginosa is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). We undertook Biolog Phenotype Microarray testing of P. aeruginosa CF isolates to investigate their catabolic capabilities compared to P. aeruginosa laboratory strains PAO1 and PA14. One strain, PASS4, displayed an unusual phenotype, only showing strong respiration on adenosine and inosine. Further testing indicated that PASS4 could grow on DNA as a sole carbon source, with a higher biomass production than PAO1. This suggested that PASS4 was specifically adapted to metabolize extracellular DNA, a substrate present at high concentrations in the CF lung. Transcriptomic and proteomic profiling of PASS4 and PAO1 when grown with DNA as a sole carbon source identified a set of upregulated genes, including virulence and host-adaptation genes. PASS4 was unable to utilize N-Acetyl-D-glucosamine, and when we selected PASS4 mutants able to grow on this carbon source, they also displayed a gain in ability to catabolize a broad range of other carbon sources. Genome sequencing of the mutants revealed they all contained mutations within the purK gene, encoding a key protein in the de novo purine biosynthesis pathway. This suggested that PASS4 was a purine auxotroph. Growth assays in the presence of 2 mM adenosine and the complementation of PASS4 with an intact purK gene confirmed this conclusion. Purine auxotrophy may represent a viable microbial strategy for adaptation to DNA-rich environments such as the CF lung. Pseudomonas aeruginosa is an opportunistic pathogen associated with complications in cystic fibrosis (CF) and is a major cause of death among CF patients. We found that P. aeruginosa CF isolate PASS4 has very limited carbon catabolism capabilities and may have become specialized at utilizing nucleic acids as a source of carbon. The CF lung contains viscous sputum which includes an abundance of DNA from epithelial and bacterial cells, and therefore provides an excellent niche for such an adaptation. We found that many P. aeruginosa virulence genes have increased expression in response to external DNA. Characterization of P. aeruginosa PASS4 revealed that the molecular basis of its metabolic specialization is a defect in biosynthesis of purines, precursors for DNA synthesis. Better understanding of P. aeruginosa adaptations in the CF lung will help in the development of specialized treatment regimes aimed at eradication of P. aeruginosa infections.CFTR) gene which encodes a cAMP-dependent chloride channel (Cystic fibrosis (CF) is a genetic disorder most common among Caucasian populations . It is c channel . Dysfunc channel . The thi channel . This hy channel .Pseudomonas aeruginosa is one of the primary causes of acute and chronic lung infections in CF patients, resulting in significant morbidity and mortality  that showed significant variability in colonization and virulence-related traits  with two P. aeruginosa model strains (PAO1 and PA14). Strains were screened for their ability to utilize 190 sole carbon sources, including a range of carbohydrates, amino acids, carboxylic acids and miscellaneous compounds  displayed reduced capabilities for carbon utilization  as a sole carbon source. Based on optical density measurement at ODP. aeruginosa strains PASS4 and PAO1 grown to an OD600 = 0.6 with DNA as a sole carbon source was assessed using RNA sequencing , with 322 genes upregulated and 254 genes downregulated , with 359 genes upregulated and 64 genes downregulated  , with an increase in abundance of 232 and decrease in abundance of 61 proteins  . A totalproteins . A corref 0.4072 . Only sif 0.4072 , among tpvd) and pyochelin (pch) biosynthetic gene clusters encode the two major P. aeruginosa siderophores (pch gene cluster and pyochelin receptor (fptA) was significantly induced by exposure to DNA in both PAO1 and PASS4 , FecA ferric citrate receptor (PA3901) and FiuR iron transport protein.The pyoverdine  is known to be regulated by sulfate starvation  gene encoding a sensor kinase was upregulated 5-fold in PASS4. The MxtR protein showed 8-fold and 16-fold increase in abundance in PAO1 and PASS4, respectively. MxtR has been shown to modulate the production of interbacterial 2-alkyl-4(1H)-quinolone (AQ) signal molecule via the LysR-type transcriptional regulator MexT  and is required for AQ biosynthesis  transcripts invasion , biofilminvasion .aceA gene encoding isocitrate lyase , a carbon source that PASS4 cells were unable to utilize  in these PASS4 mutants had apparently circumvented the genetic specialization in the parental PASS4 isolate that allowed it to grow well on DNA, but not on most other carbon sources.The eight mutants displayed much broader carbon catabolic profiles than the parental PASS4 strain when tested on Biolog Phenotype Microarray plates. All of these mutants had gained the ability to utilize a wide range of carbon sources including amino acids, carbohydrates and carboxylic acids . The subpurK gene, located at codon 214 (for PASS4 mutants 2 and 3) or codon 354 (for PASS4 mutants 1 and 4 to 8) (pctB) and a hypothetical protein, respectively. The purK gene encodes a subunit of the phosphoribosylaminoimadazole carboxylase enzyme, a critical step in the de novo synthesis of purines  between the mutants and the PASS4 genome. All of the PASS4 mutants contained a mutation in the  4 to 8) . Additio purines . This supurK enabled PASS4 to grow on a wide range of carbohydrates, amino acids, and carboxylic acids that it previously was unable to utilize  media, solid or liquid, from the frozen stock, allowing minimum passages during cultivation.ere PAO1 , PA14 . Briefly, as per manufacturer’s instructions, the strains were streaked on a DNase test agar plate and incubated at 37°C for 24 h. The DNase test agar contained tryptose, DNA and NaCl. The production of DNase leads to the hydrolysis of DNA in the media. Therefore, following incubation, the plates were flooded with 1N HCl to observe for a clearing/hydrolysis of DNA by the bacterium.The test for the production of exoenzyme DNase by Pseudomonas aeruginosa strains PAO1 and PASS4 were simultaneously inoculated into 5 ml of LB liquid medium from the frozen stock and grown overnight. Overnight cultures were used to inoculate 15 ml of M9 minimal medium supplemented with either L-Asparagine (20 mM) or DNA (1.5 mg/ml) , both in biological triplicates. Cultures were grown until mid-exponential phase (OD600 = 0.6). RNA was extracted from these cultures using the miRNEasy RNA extraction kit (Qiagen) according to the manufacturer’s protocol. To remove any residual genomic DNA, the samples were treated with DNAse using the TURBO DNAse kit . The quality and quantity of extracted RNA was assessed on a NanoDrop spectrophotometer. To remove highly abundant ribosomal RNA from the RNA extracts before sequencing, the samples were treated using RiboZero GN Magnetic rRNA depletion kit (Epicenter). The rRNA depleted samples were purified using RNeasy MinELute Cleanup kit  and re-assessed on NanoDrop and submitted to the Ramaciotti Centre for Genomics for paired-end RNA Sequencing on the HiSeq 2000 platform. Paired-end RNA Sequence files obtained from Ramaciotti were assessed for quality using FastQC software  and processed by trimming the first 10 nucleotides using Fastx Toolkit. Trimmed sequences were tiled against the complete genome of PAO1 via EdgePro software  and 2 mM magnesium sulfate (MgSO4) containing either L-Asparagine (20 mM) as a control condition or DNA (1.5 mg/ml) as an experimental condition. Cultures were grown aerobically (n = 3) at 37°C with constant shaking at 200 rpm. Overnight cultures were subcultured into the same respective medium, prior to harvesting cells in logarithmic phase (OD600 = 0.6). For proteomic analysis, cells were harvested from 60 ml of culture by centrifugation at 3220 g, 10 min at 4°C . Cells were washed with phosphate buffered saline (pH 7.4) and stored at -80°C until further processing. Proteins were extracted by lysing cell pellets with sodium dodecyl sulfate (SDS) lysis buffer . Followed by four 30 s rounds of bead beating at 5.5 m/sec , with intermittent cooling. Cellular debris was removed by centrifugation at for 10,000 × g for 10 min  and the supernatants were collected and stored at -20°C for further processing. The extracted proteins were precipitated using a methanol/chloroform/water protocol  (50%)/100 mM NH4HCO3 (50%) for 10 min. Finally, gel pieces were dehydrated with 100% ACN and air-dried. Proteins were reduced with 10 mM dithiothreitol (DTT) in NH4HCO3 (50 mM) at 37°C for 1 h, followed by alkylation with 50 mM iodoacetamide in NH4HCO3 (50 mM), in the dark at RT for 1 h. Samples were then washed with 100 mM NH4HCO3, followed by ACN (50%)/100 mM NH4HCO3 (50%) for 10 min, dehydrated with 100% ACN and then air-dried. Finally, samples were digested with 20 μL of trypsin (12.5 ng/mL 50 mM NH4HCO3), overnight at 37°C. Proteolytic peptides were extracted twice with ACN (50%)/formic acid (2%), dried using a vacuum centrifuge and reconstituted to 10 μL with 2% formic acid for LC-MS/MS analysis.protocol . ResultaPeptides were analyzed using a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer coupled to a high pressure liquid chromatography unit . Peptides were separated on a 60-min reverse phase gradient of 1–50% solvent B (acetonitrile in 0.1% formic acid) gradient. In each data collection cycle, one full MS scan (350–2000 m/z) was recorded in the Orbitrap. Subsequently, MS2 analysis was conducted for top 10 most intense ions and were fragmented by higher-energy collisional dissociation (HCD) with following settings; normalized collision energy of 30%, isolation window 3.0 m/z, maximum ion accumulation time 60 ms with a dynamic exclusion for 10 s.P. aeruginosa strain PAO1 protein sequence database retrieved from GenBank (January 2013) and Pseudomonas Genome Database1 and in-silico translated genome databases of PASS4 (Protein identification and quantification was performed on Proteome Discoverer 1.3 (Thermo Fisher Scientific) using Mascot search engine . P. aeruof PASS4  were useThe mass spectrometry proteomics data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository  with thet-tests comparing protein expression of PASS4 or PAO1 in DNA relative to L-Asparagine using the in-house developed program based on R modules  categories obtained from the Pseudomonas Database .P. aeruginosa strain PASS4 were inoculated into 10 ml of LB liquid medium, in triplicate, and grown overnight with constant shaking of 200 rpm at 37°C. Overnight cultures were centrifuged at 4000 × g for 7 min at 4°C in an Eppendorf centrifuge Model 5430. Once the supernatant was discarded the cells were washed with phosphate buffered saline and spun at 4000 g for 7 min at 4°C. The cells were then resuspended in 5 ml phosphate buffered saline and 2.04 × 108 washed cells were spread on 20 mM GlcNAc. Once spontaneous mutant colonies were obtained, colonies were cultured into M9 minimal salts medium with 100 μM calcium chloride (CaCl2) and 2 mM magnesium sulfate (MgSO4) containing 20 mM GlcNAc. This culture was consecutively subcultured till cells were able to reach an OD600 = 0.7 within ∼35 h.Single colonies of P. aeruginosa strain PASS4 mutants and the parental PASS4 strain were inoculated into 10 ml of LB liquid medium, in triplicates, from LB agar and grown overnight with constant shaking of 200 rpm at 37°C. DNA was extracted from the samples according to DNA Isolation DNeasy Blood and Tissue Kit protocol for Gram-negative bacteria . Total DNA was quantified using the Nanodrop and submitted to the Ramaciotti Centre for Genomics for paired-end DNA Sequencing on MiSeq v2 platform. Paired-end DNA sequence files obtained from Ramaciotti were assessed for quality using FastQC software . The pair-end reads for each sample were merged by FLASH-1.2.11 (Single colonies of 8 H-1.2.11 . The merH-1.2.11 .2) and 2 mM magnesium sulfate (MgSO4) supplemented with 0.02 mM – 20 mM adenosine using a broth dilution method essentially as previously described  with P. aeruginosa PAO1 genomic DNA as a template, and the primer pair 2_purK_F/R  according to the manufacturer’s protocol. Petri dishes containing solid LB medium supplemented with 25 μg/ml of tetracycline were used to select and maintain the E. coli transformants. The vector with purK gene insertion was purified from the culture of E. coli cells using the Wizard Plus SV Minipreps DNA Purification System (Promega) and the manufacturer’s protocol and used to transform the wild type PASS4 strain via electroporation. Overnight culture of PASS4 was inoculated in 2 × 5 ml fresh LB broth in 50 ml Falcon tubes and incubated overnight, with shaking, at 42°C, following a previously described method (purK plasmid was added (60 ng). The mixture was transferred to cold electroporation cuvette  and electroporated using Bio-Rad MiniPulser (2.5 kV pulse). Immediately after applying the pulse, 1 ml fresh LB was added to the electroporation mixture, mixed, and incubated at 37°C for 3 h. After the incubation the mixture was serially diluted and plated on Petri dishes containing solid LB medium supplemented with 120 μg/ml of tetracycline. The presence of the purK gene in the PASS4 transformants was confirmed using a PCR with the insert_F/R primer pair  containing a total of 190 substrates  and a negative control for each plate , which were subjected to Biolog Phenotype Microarray analysis as described above.Biolog Phenotype analysis was carried out for ch plate . BacteriThe datasets generated for this study can be found in GENE EXPRESSION OMNIBUS, GSE100287.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.