Cafiusbistriatus (Erichson) along the eastern Pacific and the western Atlantic coasts including the Caribbean Sea is presented based on morphological and molecular (COI and 28S) characters. Specimens of the Cafiusbistriatus species complex  are similar to each other, including the form and structure of the aedeagus, and they can be treated as cryptic species. Detailed micromorphological characters (SEM) and molecular analyses support the validity of these three species. Intraspecific genetic divergence of COI using uncorrected p-distance among individuals of Cafiusbistriatus ranged from 0% to 1.56%, while interspecific divergence among three species ranged from 4.90% to 14.59%. All three species were each supported as a single lineage using COI and 28S on both parsimony and maximum likelihood trees. Morphological characters among C.bistriatus, C.rufifrons, and C.sulcicollis are compared. Cafiusbistriatusfulgens Frank is synonymized under C.bistriatus and Cafiusbistriatus is redescribed with illustrations of diagnostic characters.A systematic review of the marine littoral  Cafiusbistriatus (Erichson) occurs along the coasts of eastern Pacific and the western Atlantic including the Caribbean Sea and is known to be predaceous on invertebrates in decaying seaweeds on sandy beaches  may be the same species. They are very similar in external form and internal structure including the male genitalia. Hereafter, we will refer to them as the C.bistriatus species complex  collected from a broad geographic distribution  in order to test the accuracy of the species identifications.We studied about 250 specimens of ion Fig. . We alsoC.bistriatus species complex and redescribed C.bistriatus with illustrations of diagnostic characters.In this paper, we compared morphological and molecular characters among individuals of the SEM) and habitus photographs followed BMNH), London, United Kingdom; Canadian National Collection of Insects(CNC), Ottawa, Canada; Chungnam National University Insect Collection (CNUIC), Daejeon, Korea; Field Museum of Natural History (FMNH), Chicago, USA; Museum für Naturkunde(MFNB), Berlin, Germany; and Naturhistorisches Museum (NMW), Vienna, Austria.For the study of morphological characters, we selected many specimens with different geographic distributions for their intraspecific variation, and dissected and cleared the whole body for examining microstructures . ScanninCOI and 17 for 28S) for DNA extraction in the dataset and the specimens studied are listed in Table COI and nuclear 28S were selected. Primers and amplification strategies are detailed in PA) and maximum likelihood (ML) analyses, and intra- and inter-specific distances were conducted using search strategies described by Cafiushistrio (Sharp).For the study of molecular characters, we included a total of 18 specimens . All new sequences were deposited in GenBank  were generated . Stephens, 1829: 23 384B21D1-2C9E-5431-8610-AD080A03030EPhilonthusbistriatusMFNB]. Erichson, 1840: 502. . Erichson, 1840: 503. . new synonym. See  Frank, 1986: 153 in  ; 1 syntype, “6152 || bistriatus | Typus | Er. f. 502 || Long. 75(2. | Zimmerman | 5.5. || = bilineatus Er. | sec. Fauvel || bistriatus | Er.” (MFNB).1 syntype, “6155 || Canada: 6 exx., Nova Scotia, Cape Breton H. N. P. Presqu’île, 3 m, PG562728, 13.IX.1984\J. M. Campbell & A. Davies, sifting beach wrack; 1 ex., Nova Scotia, Cape Breton H. N. P. Ingonish N. Bay, PG984711\29.VI.1983, L. LeSage, LL54 TP18, seashore wrack; 2 exx., Nova Scotia, Cape Breton H. N. P. Pleasant Bay\27.V.1984, L. Masner, seabeach kelp; 2 exx., Nova Scotia, Cape Breton H. N. P. Pleasant Bay, seashore kelp\29.VII.1983, D.E., J.E. Bright pans; 4 exx., N. S. Cape Breton Highl. N. P. 25 km SE Cap Rouge, 14.VI.84, A. Smetana; 1 ex., C. I.\Schwarz; 1 ex., New Brunswick, Passamaquoddy Bay, Pottery Bch., 29.VII.1976, M.J. Dadswell; 1 ex., New Brunswick, Passamaquoddy Bay, Pottery Bch., 29.VII.1976, M.J. Dadswell; 1 ex., QUE: St. Adelaide 0.5 mi. W. Sandy Beach Stn., 21.VIII.1953, E.L.Bousfield; 3 exx., QUE., 4 mi. S. Riviėre-à-Claude, VII-18-1972, 200’, J.M. Campbell; 5 exx., N. S. Point Aconi, VIII-13-1972, J.M. & BA Campbell; 5 exx., N. S. Big Bras d’Or, VII-25-1972, J.M. Campbell; 1 ex., Kouchibouguac N. P., N. B., 1.VI.1977, S.J. Miller, Code-5195U\Cafiusbistriatus Er. Det. J.M. Campbell 1978; 1 ex., Kouchibouguac N. P., N. B., 13.IX.1977, J.M. Campbell, Code-5953Y. Jamaica: 2 exx., JA., Clarendon P., Jackson’s Bay, 12.XII.1972, J. Peck\Sifting algae on beach; 8 exx., JA., St. Ann Runaway Bay, 22.VIII.1974, S. Peck, beach drift; 10 exx., JA. St. Catherine, 7mi. SE. Sp. town, Hellshire Beach, 27.VII.1974, S. Peck, beach drift; 1 ex., JA., Trelawny Parish, Duncans, VIII.21.1966, Howden & Becker. Mexico: 6 exx., Mulege SEAWEED Baja Cal. Sur, Mex., J. Klink Coll., X.3.66\ERIC; Scheerpeltz; 48 exx., Sonora, NR. PT. Cirio, 29.50-112.40, 28 VIII 1974 V. Roth Coll; 163 exx., Sonora, EI Desemboque, 29.30-112.34, 23 V 1974, Brown & Speich ex flying on beach; 2♂2♀, Sonora, EI Desembogue May 23 1974 Brown & Speich; 1 ex., Veracruz Prov. 8km S Veracruz, Hwy 150, 10 July 1990 J. Ashe, K-J. Ahn, R. Leschen ex: under seaweed on beach; 1 ex., Bahia Magdelena, 1 June 1968 W. G. Evans ex; under turtle carapace\SM0038043 KUNHM-ENT. USA: 1 ex., 698., A30 S. Thomas, bistriatus Er. S. Thomas, coll. L.W. Schaufuß; 1 ex., Marion, Mass. Bowditch., Cafiusbistriatus Er., Coll. Schubert; 7♂6♀ , Puerto Rico, Municipio Fajardo, Seven Seas Beach, 18°22.227'N, 65°38.359'W, 6 VI 2009, Park09-021, JS Park, ex under seaweeds; 4♂10♀ , Florida, Pinellas Co., Anclote Gulf Park, 25 X 2008, KJ Ahn, JH Ahn, under seagrasses; 1♂5♀ , Florida, Brevard Co., Port Canaveral Jetty Park, 14 II 2009, KJ Ahn, under stones with empty barnacles; 2 exx., Florida Tampa, Clearwater Beach, 10 VI 1995, K.-J. Ahn., ex under seaweed; 14 exx. , Florida, Cedar Key, 28 XII 2015, KJ Ahn; 1 ex., FLA., Flamingo, Everglades Nat. Pk., 4.I.1971, L. Masner; 1 ex., CONN, Fairfield Co. Norwalk, 9 Aug 78 Calf Pasture Beach\R.E.Orth family\Wrack-debris on sandy beach; 2 exx., CONN, Fairfield Co. Westport, 11 Aug 78 Sherwood Is. St. Pk\R.E.Orth family\Wrack-debris on sandy beach\M3; 1 ex., CONN, Fairfield Co. Fairfield, 10 Aug 78 Jennings Beach\R.E.Orth family\Wrack-debris on sandy beach; 34 exx. , North Carolina, Dare Co., Oregon Inlet, 6 IV 2009, KJ Ahn, JH Ahn, under seaweeds; 2 exx., Cape Cod, Massachusetts, VII.3.1975, E. J. Kiteley, under clumps seaweed Beach; 1 ex., South Shore S. I., N. Y.\C. L. Pollard, Apr. 11-111, No.; 1 ex., Portland Maine, July 23 1966, E. J. Kiteley, dry full carcass; 1 ex., C. I.\145\Schwarz; 1 ex., Md.; 1 ex., Pt. Isabel, Texas, VI-26-30\JOMartin Collector; 2 exx., Lynn, Mass. Essex Co. V -12; 2 exx., Lynn, Mass. Essex Co.; 1 ex., N.Y. Rockaway L.I., 15.V.1941 W. Spector\C.N.H.M. 1960 Boris Malkin Coleoptera Colln.; 2 exx., Barnegat Bay NJ JW Green VIII.4.28 \C.N.H.M. 1960 Boris Malkin Coleoptera Colln.; 1 ex., Rocwy Bcb. L. L.; 1 ex., Peekskill 4/8.80 NY\Sherman; 1♀\Marion Mass. Bowditch.\Cafiusbistriatus Er.\ex coll. Scheerpeltz\bistriatus Er.; 1 ex., TEXAS: Wilacy Co. Port Mansfield 30 September 1990 J. S. Ashe ex., beach wrack.HeadMale.   3.1–3.7 mm. Body brown to dark brown; head black; metaventrite dark brown to black; anterior part of epipleura usually brighter than elytra. ead Figs , 4A. Dulead Figs  filiformtum Fig.  glossy otum Fig.  with 2 ltum Fig.  long , Mexico, West Indies, Venezuela.Cafiusbistriatus species complex  are very similar in external form and internal structure and so they could be treated as cryptic species. A comparison table of morphological characters among C.bistriatus, C.rufifrons, and C.sulcicollis is presented in Table Members of the Cafiusbistriatus species complex  include sculpture and punctation patterns on the head and pronotum, and form and structure of the male genitalia. C.rufifrons was very similar to C.bistriatus. However, C.bistriatus and C.sulcicollis on the Pacific coast. C.bistriatus and C.rufifrons on the Atlantic coast. They also described Pacific coast populations of Cafiusbistriatus as the separate subspecies, C.b.fulgens Frank, mainly based on the microsculpture of head but it is more similar to that of C.rufifrons. No differences in male genitalia were mentioned. We synonymized this subspecies under Cafiusbistriatus because they are within the variation of C.bistriatus such as the form of the aedeagus and the sculpture patterns on head and pronotum.The diagnostic characters among members of the Cafiusbistriatus from both Atlantic and Pacific coasts were examined, though no sequences were available from the Pacific coast. Members of the Cafiusbistriatus species complex  very closely resembled each other in external and internal form and structure including the aedeagus  but they are fully resolved based on COI data (not shown) and the concatenated data of 28S and COI  . (Sharp) . CafiusbC.rufifrons for COI and 28S sequences were available in this study, and no individuals were available for the Pacific population of C.bistriatus. For a more robust test of species limits within the C.bistriatus complex, we suggest future work to include more intensive and broader sampling from the oceanic beaches along the eastern Pacific and western Atlantic coasts.Unfortunately, only two individuals of


Non-coding RNA (ncRNA) is a type of non-protein-coding RNA molecule transcribed from the genome which performs broad regulation of a variety of biological functions in human cells. The Wnt signaling pathway is highly conserved in multicellular organisms, playing an important role in their growth and development. Increasing evidence suggests that ncRNA can regulate cell biological function, enhance bone metabolism, and maintain normal bone homeostasis by interacting with the Wnt pathway. Studies have also demonstrated that the association of ncRNA with the Wnt pathway may be a potential biomarker for the diagnosis, evaluation of prognosis, and treatment of osteoporosis. The interaction of ncRNA with Wnt also performs an important regulatory role in the occurrence and development of osteoporosis. Targeted therapy of the ncRNA/Wnt axis may ultimately be the preferred choice for the treatment of osteoporosis in the future. The current article reviews the mechanism of the ncRNA/Wnt axis in osteoporosis and reveals the relationship between ncRNA and Wnt, thereby exploring novel molecular targets for the treatment of osteoporosis and providing theoretical scientific guidance for its clinical treatment. As the global population has aged, the incidence of OP has increased incessantly. According to recent statistics, more than 75 million people around the world suffer from osteoporosis [Osteoporosis (OP) is a systemic bone metabolic disease, characterized by a weakening of bone strength, destruction of the bone microstructure, and increased bone fragility, representing a common cause of fracture . Age andoporosis , many remetabolism and regulate bone homeostasis, allowing a novel research direction for the treatment of osteoporosis.Non-coding RNA (ncRNA) is a form of non-protein-coding RNA molecule that is transcribed from the genome, providing regulation for various biological functions in human cells. The principal molecules of ncRNA include microRNA (miRNA), long chain non coding RNA (LncRNA), and circular RNA (CircRNA). These ncRNA molecules mainly participate in cell proliferation, cell differentiation, and cell death . PreviouAs a classical signaling pathway, Wnt signaling has been found to regulate multiple biological processes such as cell proliferation, migration, invasion, and apoptosis . The β-cTherefore, the occurrence and development of osteoporosis are controlled by the interaction between ncRNA and key target molecules in the Wnt signaling pathway, possibly suggesting a new method for diagnosing and treating osteoporosis. The present manuscript reviews the molecular mechanism and function of non-coding RNA associated with the Wnt pathway related to the occurrence and progression of osteoporosis, and seeks to reveal novel molecular targets for the treatment of osteoporosis, so as to provide scientific theoretical guidance for its clinical treatment.Non-coding RNA (ncRNA) refers to RNA molecules transcribed from the genome which do not code for protein, including miRNA, LncRNA, and CircRNA . NcRNA iMiRNA is a class of non-coding single-stranded RNA molecules encoded by endogenous genes with a length of approximately –24 nucleLong-chain non-coding RNA was first discovered in mice (Mus musculus) in 1989 , althougCircRNA was first discovered in viruses in 1976, but due to the low abundance of transcripts, it did not attract great attention from researchers at that time . As rese2+ pathways [In 1982, researchers identified the proto-oncogene, int1, at the integration site of a mouse breast tumor virus, and with the homology of the wingless gene of drosophila as subsequently reported by Sharma, collectively called Wnt , 52. Thepathways , 56. Thupathways –59.. demonstrated that inhibition of the Wnt/β-catenin signaling pathway results in a reduction in the ability of bone marrow mesenchymal stem cells to differentiate and their ability to proliferate negatively regulated [The canonical Wnt/β-catenin signaling pathway is a key pathway that regulates osteoporosis. Where the Wnt ligand exists, β-catenin is not phosphorylated and it aggregates in the nucleus, thus regulating the downstream related target genes of Wnt . A promiegulated . In a poegulated . In cellegulated . In a seegulated . In addiegulated . The res2+ pathway is principally activated by Wnt5a and Wnt11, which may bind to the Fzd and Ror2 receptors on the cell membrane, activate Ca2+-sensitive signal molecules via CaMKII and calcineurin, and cause the intracellular Ca2+ concentration to increase, thereby regulating cell motility and cell adhesion [2+ pathway, regulate the expression of downstream factors, and play an important role in bone formation, absorption, bone loss, and matrix mineralization [2+ signaling in bone marrow mesenchymal stem cells [2+ in mesenchymal cells and activating the Wnt/Ca2+ signaling pathway [The plane cell polarity pathway (Wnt/PCP) is one of the non-canonical Wnt signaling pathways. It mainly activates c-Jun N-terminal kinase (JNK) and causes transcription changes by activating the downstream region of Dsh, Rac, small GTPase, Rho and Cde42  The non-adhesion , 68–70. lization , 72. Wanlization . Li et alization . Lin disem cells . Wu et a pathway . ClarifyTo summarize, the Wnt signaling pathway is a complex protein regulatory network system, comprising principally of a group of downstream signal transduction pathways stimulated by a combination of the Wnt protein ligand and membrane protein receptors Fig. . The WntA large number of studies have confirmed that the pathogenesis of osteoporosis is associated with multiple factors such as inflammation, autophagy, oxidative stress, and an imbalance in bone homeostasis –79, with. [. discovered that miR-483-5p can increase the progression of osteoporosis following the analysis and comparison of the gene expression profiles of blood and bone tissue samples collected from osteoporotic patients and normal people, the mechanism of which was established as the promotion of osteoclast differentiation [. [Autophagy is key to bone homeostasis, and its dysfunction in bone cells is related to the occurrence of osteoporosis. Lu et al.  found th. . Li et antiation . A numbeation [.  observed. [. observed that the LncRNA, WT1-AS, was upregulated during osteoporosis and actively regulated the expression of p53 in osteoblasts, thereby regulating apoptosis in osteoblasts [Over recent years, LncRNA research in osteoporosis has become a focus of research attention. It has been found that the expression levels of the LncRNA, SNHG1, in plasma in postmenopausal osteoporosis patients are considerably lower than in the healthy population, and it has been speculated that its plasma levels could be utilized as a biomarker for the diagnosis and treatment of postmenopausal osteoporosis . It has .  found theoblasts . In vitreoblasts . Chen foeoblasts . From thOsteoporosis is associated with an imbalance in bone resorption compared with bone formation. It has been reported that Circ_0005564 plays a potential role in bone formation and is an active regulator of the osteogenic differentiation of bone marrow mesenchymal stem cells . Zhao foThe interaction between ncRNA molecules is currently a highly active research topic. In the currently published literature, the majority of studies have tended to investigate the regulation of downstream target gene expression using LncRNA and CircRNA via miRNA. It has been reported that in osteoporosis patients, the LncRNA, ROR, is downregulated while miR-145-5p is over-expressed. Knock down of ROR was shown to inhibit osteoblast proliferation and exacerbate the progression of osteoporosis by targeting miR-145-5p . Cong de. [. demonstrated that up-regulation of CircAtp9b occurred after reduced formation of mature miR-17-92a, with the promotion of osteoblast apoptosis, resulting in osteoporosis [Estrogen deficiency is a cause of osteoporosis in elderly women. It has been reported that estrogen can promote the expression of LncRNA H19 and regulate the osteogenic differentiation of bone marrow mesenchymal stem cells via the miR-532-3p/SIRT1 axis, thereby reducing osteoporosis . A numbe.  found thoporosis . Compareoporosis . The expoporosis . In summoporosis . In anotoporosis  ThereforNew evidence indicates that there are a number of clinical features of osteoporosis associated with ncRNA that are related to the Wnt pathway. In addition, the ncRNA/Wnt axis promotes the progression of osteoporosis by the regulation of multiple cellular biological functions. In this section, we will introduce the expression of the ncRNA/Wnt axis, and the corresponding clinical features, function, and mechanisms of action Table .Table 2R. discovered that downregulation of miR-146a promoted osteogenic differentiation and inhibited the expression of DDK1, which was observed following activation of the Wnt/β-catenin signaling pathway. Therefore, downregulation of miR-146a was able to relieve osteoporosis and so represents a potential therapeutic target for osteoporosis [MiRNA and Wnt signaling pathways play key roles in the development of osteoporosis. A study by Li indicated that silencing miR-23b-3p can reduce osteoporosis in mice. In addition, knock-down of miR-23b-3p promoted the osteogenic differentiation of hMSCs by upregulation of the expression of Runx2, OCN, and Osterix, thereby enhancing the activity of ALP. Mechanistically speaking, MRC2 is the downstream target gene of miR-23b-3p, a miRNA that inhibits the Wnt/β-catenin signaling pathway during the process of hMSCs osteogenic differentiation by targeting MRC2, thus promoting their osteogenic differentiation and delaying the manifestation of osteoporosis, providing a novel perspective on a potential treatment for osteoporosis . Liu et oporosis . It has oporosis . In an ooporosis . Other roporosis . In summWith the wide use of gene chips, genomics, and other high-throughput gene sequencing analytic technologies, increasing numbers of LncRNA molecules have been identified that play both positive and negative roles in the regulation of the occurrence and progression of osteoporosis. Shen demonstrated that the LncRNA, HOTAIR, inhibits the osteogenic differentiation of BMSCs by regulating the expression of the osteogenic gene, RUNX2D, via downregulation of the Wnt/β-catenin pathway . Wang deCompared with the miRNA/Wnt and LncRNA/Wnt axes, the role of the CircRNA/Wnt axis in osteoporosis has been studied to a lesser degree, although its role should not be underestimated. Huang observed for the first time that circ_0024097 promoted osteogenic differentiation via the miR-376b-3p/YAP1 axis and Wnt/β-catenin pathway, thus reducing osteoporosis . Silenci. observed that the expression of SNHG1 was downregulated in a time-dependent manner during osteogenic differentiation, while its overexpression was shown to inhibit osteogenic differentiation. Additional studies have confirmed that SNHG1 regulates the Wnt/β-catenin signaling pathway and reduces BMSC osteogenic differentiation via the miR-101/DKK1 axis as competitive endogenous RNA [MiRNA, CircRNA and LncRNA have complex network regulatory relationships. According to current research, the three types of ncRNA not only independently regulate the Wnt signaling pathway to act on osteoporosis, but also regulate crosstalk and together play a biological role Fig. . Studiesnous RNA . Zhao conous RNA . It has nous RNA . Yue hasnous RNA . Circ_00nous RNA . In concBone remodeling is the core of osteoporosis, and the maintenance of bone mass mainly depends on the balance between osteoblastic bone formation and osteoclast bone absorption. As an important physiological process, bone remodeling is regulated by a variety of cytokines. Adil and other scholars have found that miR-200c can promote osteoblast differentiation and bone regeneration by targeting Sox2 and Klf4 to activate Wnt signals, which can be used as an effective gene therapy tool for human clinical bone reconstruction . Liang e. demonstrated that miR-486-3p targets CTNNBIP1, thus activating the Wnt/β-catenin signaling pathway and promoting BMSCs to undergo osteogenic differentiation [In the past, ncRNA has been considered a transcriptional by-product with no particular function and without direct participation in protein coding. However, as epigenetic research has become more sophisticated, ncRNA has been found to be important in the manifestation and development of osteoporosis. Additionally, the Wnt signaling pathway is also involved in cell proliferation, differentiation, apoptosis, and oxidative stress and inflammatory reactions. Recent evidence suggests that the ncRNA/Wnt axis regulates the expression of genes related to bone metabolism and influences the progression of osteoporosis. We have found that ncRNA is the key therapeutic target for OP, and the majority ncRNA modifies osteoblast-related genes via the canonical Wnt/β-catenin pathway, resulting in an imbalance in bone homeostasis that induces osteoporosis. Over recent years, a series of ncRNA/Wnt axis inhibitors have been identified. Catenin-β-interacting protein 1 (CTNNBIP1) is an inhibitor of Wnt/β-catenin signaling, although its role in osteogenesis remains unclear. Zhang et alntiation . ncRNA iAs the global population ages, osteoporosis has become one of the most common and costly diseases around the world. Osteoporosis patients are prone to fracture, and severe cases may even lead to death. In addition, osteoporosis has a considerable effect on the health and quality of life of patients, causing a heavy socio-economic burden with major social problems. The manifestation and development of osteoporosis are regulated by multiple complex factors, and so there is a need to innovate with novel treatment methods and the development of more effective drugs to prevent and treat osteoporosis. By summarizing the mechanisms of the ncRNA/Wnt axis in osteoporosis, we have shown that ncRNA plays an important role in regulating bone homeostasis, and represents a basis for screening new therapeutic targets for osteoporosis, in addition to using it as a key target for new drug research and development.We have also found that the research on ncRNA in osteoporosis is mostly limited to the canonical Wnt/β-catenin signaling pathway and mechanisms of osteoblast metabolic regulation, while the non-canonical Wnt signaling pathway and the mechanisms of the metabolic regulation of osteoclasts and other forms of metabolic regulation are somewhat limited. Therefore, the study of ncRNA on nonclassical Wnt signaling pathways, and mechanisms of osteoclast metabolic regulation and other metabolic regulation in osteoporosis should be more prominent in the future.


The Wnt/β-catenin signaling pathway is highly conserved throughout evolution, playing crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins  or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway’s activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12× or 16×) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterization of TCF/β-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development. The Wnt/β-catenin signaling pathway is a highly conserved pathway, which appeared early in phylogenesis and is common to all metazoan life forms. This pathway plays crucial roles during the entire lifespan of all organisms, from early embryogenesis to homeostasis in the adult. Wnt ligands bind to the transmembranal Frizzled receptor family and along with members of the LRP transmembranal co-receptor family, they mediate a large array of cell responses including cell fate speciﬁcation, polarization, migration, and mitogenic stimulation . The firc-fos promoter driving the expression of the luciferase gene (http://web.stanford.edu/group/nusselab/cgi-bin/wnt/). More sensitive TOPflash reporters were generated in Drosophila and zebrafish by increasing the number of TCF/LEF sites to 8, 12, and 16  to generate transgenic or knock-in mouse lines . While tImportantly, recent evidence suggests that mobilization of β-catenin from the cell membrane pool can also trigger the activation of TCF/LEF reporters in a WNT ligand-independent manner . This inDrosophila) to considerably increase reporter protein yields , we increased transcriptional activity further, using 16 TCF/LEF repeats. This was combined with translational enhancers to drive the expression of a nuclear, destabilized, fast maturing Venus  as reporThis resulted in two transgenic lines in which the characterization of the TCF/β-catenin transcriptional activity during embryogenesis is readily observed, including using in vivo imaging. Particularly remarkable is the 16xTF-VNP line, where intense, yet dynamic, reporter activity unveils unexpected features of TCF/β-catenin-responding cells and tissues.This study underlines the importance of developing novel strategies to generate reporters efficient for monitoring of the spatiotemporal dynamics of signaling pathways and it opens new routes of investigation for correlating TCF/β-catenin transcriptional activities with unique cell behaviors during amniote embryonic development.To generate the 12xTF-d2GFP line (TgT2[12TCF/LEF:d2EGFP]), we modified a TCF/β-catenin transcriptional reporter we previously generated, which was intended for in vivo electroporation in chicken embryos  by inserDrosophila myosin heavy chain, which facilitates mRNA export to the cytoplasm . For this, we synthesized and cloned 16 TCF/LEF repeats upstream of the TK minimal promoter, followed by IVS and Syn21 sequences , directlytoplasm . Syn21 iulovirus . A Crystulovirus . FinallyTo validate the efficiency of this construct, we electroporated one-half of the neural tube of HH15 (E2.5) chicken embryos with a DNA mix containing the 16xTF-VNP construct and a TagBFP protein driven by the CAG ubiquitous promoter as a marker of electroporated cells. Twenty-four hours after electroporation, weak expression of 16xTF-VNP was observed in the dorsal neural tube (NT) of the electroporated half, while a stronger expression of the reporter was seen in the migrating neural crest cell population NC; . The co-To test the added value of translation enhancers in a vertebrate environment, we compared the fluorescence levels obtained with the 16xTF-VNP, the 12xTFd2GFP, and a 16xTF-VNP construct lacking the translation enhancers (referred to here as 16xTF-VNP plain). We co-electroporated the right side of the neural tube of E2.5 chicken embryos with the 12xTFd2GFP, 16xTF-VNP plain, or the 16xTF-VNP, together with CAG-TagBFP as internal control . Twenty-We used the same technique as above to generate transgenic quails with this reporter. One female was selected as the transgenic founder using the CrystallGFP marker and crossed with a WT male to expand the transgenic line. The transmission of the transgene to the offspring presented a Mendelian distribution, suggesting a single insertion.To characterize TCF/β-catenin transcriptional activity during embryonic development, we analyzed and compared the activity of the two reporters in the two lines we generated, in whole mount preparations of immunostained embryos, on immunostained sections and in live tissues at different developmental stages. Whole mount preparations of HH21  and HH20  transgenic quail embryos immunostained for GFP or mVenus, Pax7 and Myosin Heavy Chain (MyHC), clarified by the ‘3DISCO’ technique  and imagen route to their sites of differentiation . We observed that the activity of the reporter was not detected in the dorsal neural tube in the 12xTF-d2GFP  and weakXenopus and chicken have led to the premise that these molecules play important functions in the proliferation and/or differentiation of neuronal precursors within the CNS, opposing a Shh gradient from the notochord and floor plate  of the neural tube in mouse and chicken early embryos while their transcripts are absent from migrating NC . Wnt1 anor plate . Howeveror plate . In neuror plate . Wnt sigor plate . The avaor plate .A similar gradient of reporter activity was observed in somites, since it was active in the DML and the transition zone and rapidly decreased in the myotome . As prevThus, the use of destabilized fluorescent reporters to generate these quail lines allows the detection of dynamic changes (increase and decrease) in reporter activity throughout embryonic development that could not be appreciated with stable reporters, such as the BAT-gal mouse line .To characterize TCF/β-catenin activity during early embryonic development, we performed immunostaining on sections and on whole mount embryos, coupled with classical confocal microscopy. We observed that the reporters were first detected at the posterior end of a gastrulating embryo  in the 16xTF-VNP embryos  and two To gain a cellular resolution of the 16xTF-VNP reporter activity, we prepared transverse sections of HH14 (E2.5) embryos at the level of cervical somites, somite I (the newly formed somite) and the PSM, and stained for mVenus, Acetylated Tubulin and DAPI . These sWe also analyzed the activity of the reporters at 9 days of development HH35; . TissuesTCF/β-catenin spatiotemporal activity is highly dynamic, both on a cellular level and throughout development, a feature that has been difficult to study due to lack of reliable destabilized reporters. By using the 12xTF-d2GFP and the 16xTF-VNP transgenic lines, we followed the dynamics of TCF/β-catenin transcriptional activity in vivo. Observation of early somites showed a strong activation of the 12xTF-d2GFP reporter activity in single cells located in the DML . The timA possible limitation of the TCF/β-catenin reporters we designed resides in the half-life of the chromophores we used (i.e. d2EGFP and VNP), which has been estimated to be about 2 hr . This suAn interesting finding of this study was the intense TCF/β-catenin-response in the fore- and hindlimb AER. The AER remained strongly labeled throughout limb growth , embryos were washed in PBS, incubated in 80% glycerol and directly (i.e. less than 24 hr after fixation) examined. To image native fluorescence expression, the detection spectrum for each fluorophore was set, using a Leica sp5 confocal microscope. The imaging parameters were set according to the sample with the strongest fluorescence to avoid over expression, and sample bleaching. These acquisition parameters were then left the same for the rest of the samples in the same experiment. For section preparations, embryos were embedded in 15% sucrose/7.5% gelatine/PBS solution and sectioned with Leica cryostat at 20 µm. Antibody staining was performed as described in Mann–Whitney two-tailed non-parametric tests were applied on the entire population of counted cells to evaluate significance of each treatment. **p-value 0.001–0.01; * p-value ≤ 0.05.Performed as described in http://quailnet.geneticsandbioinformatics.eu/. Embryos were imaged using Leica SP8 confocal microscope on upright DM6000 stand with HCX APO L 20x/1.00 water dipping (WD 2.00 mm). Z-stack images were taken every 15 min for 9 hr  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Senior Editor and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal their identity: Aixa Victoria Morales (Reviewer #1).Thank you for submitting your article "Transgenic quails reveal dynamic TCF/β-catenin signaling during avian embryonic development" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The reviewers found agree that your paper presents a powerful and improved tool to follow Wnt signalling activation in vivo. However, the consensus is that the paper requires extensive revision to demonstrate that the tool is reliable and robust tool. In particular, more transgenic lines are required to demonstrate that this offers a dynamic view of Wnt activation, close to endogenous dynamics. Essential changes are summarized below and elaborated upon in the individual reviews.1. The authors need to better demonstrate the positive effects of the regulatory elements used in the transgenes. Multiple independent transgenic lines are needed in order to draw firm conclusions. In addition, a demonstration of transgene response to small molecules is needed.2. Additional experiments should be added to quantitate the expression levels in vivo. The lifetimes need to be better characterized by analysis of kinetic effects of induction and inhibition of b-catenin signalling .3. As the quail lines may not be generally accessible to many researchers, the authors should provide more data on the expression profiles at different stages of development and better characterize the constructs for use in electroporation.Reviewer #1 (Recommendations for the authors):In spite of the robustness and usefulness of the new Wnt/β-catenin reporter lines, there are some concerns about the results presented:1. The proof of the fine temporal resolution of the dynamics of Wnt signalling using the best of the two constructs (16xTCF-VNP) does not seem strong enough. It is mostly based in a 9 hours recording (Sup. Video 3) of somitic cells. It is unclear how long does it take to the two somitic cells indicated by magenta arrow to change their EGFP levels and also it is unclear if they are more intense at the end of the video because they were out of focus during the first frames of the video. It would be more convenient to explore the property of temporal resolution of 16xTF-VNP in a highly dynamic context such as the segmentation clock in presomitic mesoderm at E2 (similar to Video 4). In that tissue, cyclic expression with a periodicity of around 90 min (chicken) or 120-140 min (mouse) has been shown for several components of the Notch and Wnt signalling pathway . In fact, this recent important work has not been acknowledged by the authors.2. In relation to Supplementary Video 4 (embryos stage E2), the authors speculate that "the reporter-positive cells were found scattered in a wide region of early limb ectoderm, intermingled with reporter negative cells. As the limbs grew, reporter-positive cells migrated towards and coalesced to form the AER, suggesting that they constitute a population of AER progenitors. ". In fact in sections of embryos stage E3, they show how 16xTCF-VNP drives EGFP expression in limb bud mesoderm (besides the expression of AER). In the video at E2 it is unclear if the migrating cells in the hindlimb bud are mesodermal or ectodermal cells. Authors should clarify these aspects  or at least, tone down their interpretations.Reviewer #2 (Recommendations for the authors):Some data are presented to suggest that the 16x multimerised line is more sensitive than the 12x multimerised line based on the expression level in neural crest cells in transient expression experiments and in somitic tissue in the transgenic lines. These data are suggestive but not very quantitative. Can this be improved, for instance could embryos/tissues be dissociated and fluorescence intensity be measured in flow cytometry to quantify these effects, preferably at more than one time point?It is also suggested that the 16x reporter is more dynamic since it drives expression of a destabilised GFP variant, however there are no direct measurements of lifetimes to support this. It would be good to try to measure this and demonstrate that this is indeed the case, for instance by treating embryos with a small molecule Wnt/β catenin inhibitor and try to measure the dynamics in the response in both strains. It would also be very useful to have an estimate of the time it takes for the reporter to come on, for instance by locally applying a signal and measuring the kinetics of the response. It is somewhat worrying that in the transfection experiments this appears to take a very long time (up to 24 hours?).It would be really useful to have a stable as well as destabilised reporter and it would be useful to know what these half times are in-vivo and whether they vary development.Drosophila, but it is not clear from the data provided that these help or are necessary in the chick. If there is any information on this it would be helpful to provide this, to guide sensible decisions on future construct complexity.The 16x constructs is quite complex, it contains domains known to enhance translation in An initial characterisation of the reporters showed that at stages E3 and E9 of development activity is seen in places where it is expected. It would however be good to see what the activity looks like at some earlier stages n development (gastrulation/neurulation) when Wnt signalling is known to play a critical role in patterning and differentiation. Since the embryo geometry is simpler at these stages, it will also be easier to document its activity, both in fixed and live preparations. Furthermore since in early development changes in signalling occur fast this will is will also provide better insight in the sensitivity and kinetics of the reporters.Reviewer #3 (Recommendations for the authors):For Figure 1, 2 and 3, the figure legends state that anti-GFP antibodies were used on the embryos and sections before imaging. Does this mean that the endogenous fluorescence was not of sufficient intensity to survive fixation and cryopreservation? What do the fluorescent embryos appear under a stereomicroscope? Can the embryos be live imaged during embryogenesis? The Supplementary Video 3 of a somite is of interest. Could more tissues be shown?Other recommendations:1. Continuation of the regulatory element analysis using electorporation in the neural tube.2. If possible, create independent transgenic lines. Essential revisions:The reviewers found agree that your paper presents a powerful and improved tool to follow Wnt signalling activation in vivo. However, the consensus is that the paper requires extensive revision to demonstrate that the tool is reliable and robust tool. In particular, more transgenic lines are required to demonstrate that this offers a dynamic view of Wnt activation, close to endogenous dynamics. Essential changes are summarized below and elaborated upon in the individual reviews.1. The authors need to better demonstrate the positive effects of the regulatory elements used in the transgenes. Multiple independent transgenic lines are needed in order to draw firm conclusions. In addition, a demonstration of transgene response to small molecules is needed.2. Additional experiments should be added to quantitate the expression levels in vivo. The lifetimes need to be better characterized by analysis of kinetic effects of induction and inhibition of b-catenin signalling .3. As the quail lines may not be generally accessible to many researchers, the authors should provide more data on the expression profiles at different stages of development and better characterize the constructs for use in electroporation.Many questions were raised by reviewers on the reliability of the reporters we have used (12XTFd2EGFP and 16xTF-VNP). As explained in the manuscript, the TCF/LEF binding sites (TCF BS) are well-established tools which have been used for decades in literally hundreds of publications to monitor canonical Wnt signaling, starting with the seminal work of Korinek et al., in 1997. Such constructs were shown to efficiently respond to various members of the pathway, in vivo and in vitrohttp://web.stanford.edu/group/nusselab/cgi-bin/wnt/inhibitors; http://web.stanford.edu/group/nusselab/cgi-bin/wnt/activators_detectors), as well as to small activating and inhibiting pharmacological compounds . Altogether, there is a wealth of data showing that TCF BS reliably respond to Wnt signaling., we have built and extensively characterized the 12XTFd2GFP Wnt reporter used here and compared it to existing constructs containing 3 or 8 TCF-BS.We then showed that an increase in the number of TCF-BS led to a significant enhancement of sensitivity to Wnt signaling that was not detrimental to the reporter accuracy, since the 12XTFd2GFP responded positively to an activated form of b-catenin  and was strongly repressed by the Wnt-inhibiting molecule Dkk1 . As the point of this paper was to combine the Wnt reporter to a destabilized fluorophore, we showed that the expression of the destabilized GFP reporter protein was similar to its mRNA, suggesting that 12XTFd2GFP provides a precise view of cells actively engaged in Wnt signaling in vivo. Those important elements have been added to the text  for more clarity.Drosophila , we were concerned that the basal level of 16xTF-VNP activity may be too high to be useful in our system. In the previous version of the manuscript, we showed that the fluorescent signal of 16xTFVNP was strongly repressed by a dominant form of LEF1 (DN LEF). As using a dominant negative form of LEF1 to repress a TCF/LEF binding site may not be the best of all experiments, we now performed an additional experiment showing that 16xTF-VNP fluorescence is also robustly repressed by the Wnt signaling repressor Axin2 . We believe these explanations and additional data should satisfy the reviewers’ concerns on reliability.The 16xTF-VNP construct builds on the same logic of multimerizing the TCF-BS (up to 16) and therefore it should bear the same reliability to Wnt signaling. An important additional point is the use of translation enhancers, which we show are effective in vertebrates (to a point that we now routinely use them in many constructs we generate in the lab). The point of the destabilized fluorophore is discussed below (response 1 to reviewer 1). Even though a multimer of 16 TCF-BS had already been successfully used in To further characterize the effect of the use of regulatory elements to enhance the activity of the reporter, we have performed additional electroporation experiments that allow quantification of the signals obtained in vivo with (i) the 12XTFd2GFP; (ii) a 16xTF-VNP construct without the translation enhancers (this construct was not used in the previous version of the manuscript) and (iii) a 16xTF-VNP construct with the translation enhancers. These experiments are shown in Sup Figure 2. In addition, additional pictures of side-by-side embryos at the same developmental stage  show that the fluorescent signal is very similar in the two lines, but significantly stronger in the 16xTF-VNP than in the 12XTFd2GFP. This observation also supports the premise that there is no significant positional effect of the transgene insertion into the genome.To address the request for more developmental stages, we now provide 3 novel main figures and 2 Sup Figures that illustrate the expression of the reporters in embryos at E2+ in whole mount  and on sections , at E4 in whole mount , during gastrulation  and at E2- .Reviewer #1 (Recommendations for the authors):In spite of the robustness and usefulness of the new Wnt/β-catenin reporter lines, there are some concerns about the results presented:1. The proof of the fine temporal resolution of the dynamics of Wnt signalling using the best of the two constructs (16xTCF-VNP) does not seem strong enough. It is mostly based in a 9 hours recording (Sup. Video 3) of somitic cells. It is unclear how long does it take to the two somitic cells indicated by magenta arrow to change their EGFP levels and also it is unclear if they are more intense at the end of the video because they were out of focus during the first frames of the video. It would be more convenient to explore the property of temporal resolution of 16xTF-VNP in a highly dynamic context such as the segmentation clock in presomitic mesoderm at E2 (similar to Video 4).The tool we have designed is appropriate to explore dynamic events that have a velocity compatible with the half-life of the Venus-PEST, which is about 90-110 minutes (see below). The changes of fluorescence we observed in Sup video 3 are true changes in Wnt activity. We have now added a timeline to this video, which shows that the increase in activity (from lowest to highest) takes about 5 hours. To address a related comment from this reviewer (“(cells) were out of focus during the first frames of the video), as mentioned in the manuscript, this video was taken with a confocal microscope, which means that all cells are in focus along the entire video. Regarding the segmentation clock, it cycles in chicken PSM with a periodicity of 90’, which we believe is too short to be detected with this tool. Accordingly, in the new Figure 4 and Sup Figure 9, we do not see any sign of periodicity of the reporter fluorescence in the PSM. The paper of Sonnen et al., that is mentioned by reviewer 1 is a KI of Venus-Pest in the axin2 locus. While the Venus-Pest they used should have the same half-life than ours, the periodicity of axin2 expression in mouse was estimated in their work to be 144 minutes, i.e. more compatible with the half-life of VenusPEST.To draw a parallel with the design of Notch reporters that allowed the detection of cyclic Notch signaling in the PSM, Auhlela et al., Delaune et al., Masamizu et al., Sorolodoni et al., resorted to various tricks to further destabilize the fluorescent reporter they used, for instance by adding RNA destabilizing sequences in the 3' UTR. A similar strategy was used by Shimojo et al., to detect cyclic variations of Notch signaling in neurons. The transgenic animals that were used in these studies were designed specifically for that purpose and because the signals they generate is so weak, one can see in the literature that these lines are very seldom used.As mentioned in the manuscript, the signal of a reporter is linearly linked to its stability, and further shortening its half-life will significantly reduce the signal. We have chosen a compromise between high signal and short half-life that may not be suitable for all purposes. We have mentioned the limitation of the reporter lines we designed to detect dynamic changes in Wnt signaling with a short periodicity (2nd§ page 9).2. In relation to Supplementary Video 4 (embryos stage E2) , the authors speculate that "the reporter-positive cells were found scattered in a wide region of early limb ectoderm, intermingled with reporter negative cells. As the limbs grew, reporter-positive cells migrated towards and coalesced to form the AER, suggesting that they constitute a population of AER progenitors. ". In fact in sections of embryos stage E3, they show how 16xTCF-VNP drives EGFP expression in limb bud mesoderm (besides the expression of AER). In the video at E2 it is unclear if the migrating cells in the hindlimb bud are mesodermal or ectodermal cells. Authors should clarify these aspects  or at least, tone down their interpretations.We now provide sections at different AP levels of a stage HH14 16xTF-VNP embryo . One clearly sees that there are TF-positive cells in the ectoderm in the region of the future limb (see panels I-L). These are likely the same AER progenitors that are observed in Sup Video 4.Reviewer #2 (Recommendations for the authors):Some data are presented to suggest that the 16x multimerised line is more sensitive than the 12x multimerised line based on the expression level in neural crest cells in transient expression experiments and in somitic tissue in the transgenic lines. These data are suggestive but not very quantitative.We have further quantified the fluorescence that was observed with the 12x and the 16x constructs. To do this, we electroporated the neural tube with 3 constructs: (i) the 12XTFd2GFP; (ii) a 16xTF-VNP construct without the translation enhancers (this construct was not used in the previous version of the manuscript) and (iii) a 16xTF-VNP construct with the translation enhancers.The results are shown in Sup Figure 2. They show that the change from 12 TCF/LEF1 binding sites  to 16 BS , together with a fluorophore change , led to a strong increase in the observed fluorescent signal (by about 11 x). The addition of the translation enhancers  further increased the fluorescent signal by about 60%. Altogether, these data demonstrate the utility of the strategy we followed to improve the TOPflash reporter and it shows that translation enhancers which were developed in invertebrates are also active in vertebrates.It is also suggested that the 16x reporter is more dynamic since it drives expression of a destabilised GFP variant, however there are no direct measurements of lifetimes to support this.The half-life of the d2EGFP and the Venus-NLS-PEST are only slightly different. The d2EGFP was designed by Clontech years ago and its half-life is 2 hours. The half-life of Venus-NLS-PEST is reported to be 1.8 hours . Years ago, we tested d2EGFP and Venus-NLSPEST half-life (HL) and confirmed that d2EGFP HL is indeed 2 hours, while Venus-NLS-PEST HL was in our hands more towards 90' (unpublished). Venus is faster-folding than EGFP , which should slightly decrease the reporter response time. All in all, those differences are rather limited and they should not fundamentally change their response to Wnt signaling.It would also be very useful to have an estimate of the time it takes for the reporter to come on, for instance by locally applying a signal and measuring the kinetics of the response. It is somewhat worrying that in the transfection experiments this appears to take a very long time (up to 24 hours?)We have extensively characterized the 12XTFd2GFP construct years ago, showing that the EGFP fluorescence is observed in cells that activate the Wnt response . In the Rios paper, we also compared a stable and a destabilized TF reporter, demonstrating the importance of the latter to obtain a snapshot of the cells actively responding to Wnt. The 16xTF-VNP construct is a variant of the 12x and it reacts similarly. Even though there is likely a delay between the binding of Wnt to its receptor and an observable fluorescence, it is also the case for a transcriptional response to any signaling pathway. Reviewer 2 seems surprised about the 24 hours delay between electroporation and observation: this delay was just out of convenience , but not necessary.Drosophila, but it is not clear from the data provided that these help or are necessary in the chick.The 16x constructs is quite complex, it contains domains known to enhance translation in See above response 1 to the same reviewer 2. Note that we have added a note about an additional IVS cassette upstream of Syn21 in the text (last § page 4).An initial characterisation of the reporters showed that at stages E3 and E9 of development activity is seen in places where it is expected. It would however be good to see what the activity looks like at some earlier stages n development (gastrulation/neurulation) when Wnt signalling is known to play a critical role in patterning and differentiation.We now provide a picture  that shows the fluorescence in the posterior end of a gastrulating 16xTF-VNP embryo.Reviewer #3 (Recommendations for the authors):For Figure 1, 2 and 3, the figure legends state that anti-GFP antibodies were used on the embryos and sections before imaging. Does this mean that the endogenous fluorescence was not of sufficient intensity to survive fixation and cryopreservation? What do the fluorescent embryos appear under a stereomicroscope? Can the embryos be live imaged during embryogenesis? The Supplementary Video 3 of a somite is of interest. Could more tissues be shown?GFP and its variant Venus do not like fixation and their fluorescence tends to fade quite quickly. In contrast, RFP and variants are resistant to fixation. To observe native GFP/Venus after fixation, we image embryos very fast, typically the next day after a step of glycerol-mediated clarification. This point has been clarified in the Materials and methods section . When we need to apply additional antibodies, for instance in Figures 1, 2 and 3, those experiments take days, and we need to restore the GFP/Venus signal with GFP-specific antibodies. While GFP antibody staining can enhance faint signals, it also tends to level down subtle changes in fluorescence intensity. Evidently, those lines were created to be able to perform live imaging, such as those shown in videos 3 and 4 and it is likely that there will be more to come from our and other labs when the new TOPflash line will be up and running.


The long-term degradation of epoxy as the matrix and adhesive serviced in harsh environments plays a key role in engineering applications. Understanding how to improve the toughness and durability of epoxy through reasonable material replacement and design is significant to prolong the service life of engineering structures. In the present paper, thermoplastic polypropylene and thermosetting epoxy were exposed in a coupling environment of elevated temperature, water immersion and sustained bending loading. The evolutions of mechanical and thermal properties were further analyzed and compared. Long-term life prediction was conducted to evaluate the corrosive resistances of polypropylene and epoxy. It can be found that polypropylene has better hydrophobic behavior compared to epoxy. At 80 °C, the ratios of the diffusion coefficient and saturated water uptake between the two matrices were 114.4 and 2.94. At the longest immersion time of 90 days, the degradation percentages of tensile strength were 4.7% (40 °C), 7.5% (60 °C) and 8.8% (80 °C), respectively, which had the higher strength retention (>90%). The maximum strength increase in the multiples of polypropylene/epoxy and polypropylene/polyurethane was 1.95 and 1.75, respectively. The bending loading led to a maximum increase in tensile strength (~1.47%) owing to the oxygen isolation effect. The degradation mechanism was attributed to the active functional groups from the production process reacting with oxygen, resulting in the fracture of the local chain segment. By comparison, water molecules reacted with the hydroxyl groups or interrupted the intermolecular Van der Waals force/hydrogen bond of the epoxy, resulting in irreversible hydrolysis and property degradation. Through the comparison, it can be found that polypropylene and its composites have outstanding properties compared to epoxy, which can make them achieve great application prospects in engineering applications when considering a complex service environment. Polymers have been widely used as coatings, fiber reinforced polymer (FRP) composite and adhesives to reinforce, strengthen and repair newly built structures. For the coating, the main types are fire-proof coating, corrosion-resistant coating and friction-resistant coating ,2,3,4, sThermosetting and thermoplastic matrices are two main categories of polymers. Generally, thermosetting matrix (such as epoxy and polyurethane) can be used as the matrix of FRP , which hCompared to thermosetting epoxy, the thermoplastic matrix has a more optimal elongation at breaks, fracture toughness, thermal resistance and recyclability ,44. GeneAt present, the related investigation mainly focused on the long-term evolution of water absorption and mechanical properties of fiber-reinforced polypropylene composites. Arbelaiz et al.  investigBased on the above analysis, it is necessary to find a high-performance matrix with superior corrosion/fatigue resistances to replace epoxy for use in civil engineering when considering the complex service environment. Furthermore, the knowledge of the aging behavior of high-performance matrix (such as degradation mechanism and service life) is significant for the design parameters of engineering applications. In the present study, the typical service environment was applied to investigate the long-term evolution of polypropylene and epoxy. At the same time, the comparison of long-term performance between epoxy and polypropylene was also conducted. The immersion temperatures were 40 °C, 60 °C and 80 °C, and the distilled water was adopted as the exposure medium. To simulate the real service condition, a bending fixture was designed to apply the bending loading, and the maximum bending strain in the midspan was 0.54%. Water uptake, mechanical and thermal properties tests were conducted to obtain the long-term evolution. The degradation mechanism of mechanical properties was revealed based on the microstructure analysis. Based on the Arrhenius theory, the long-term life predictions of mechanical properties were conducted. Finally, the application prospect analysis of polypropylene in structural engineering was analyzed and compared with epoxy and polyurethane in terms of failure strain, long-term property and fatigue resistance.3. Bisphenol A epoxy from Dagong Composite Company  was adopted and anhydride was used as a curing agent. The mass ratio of the above two components was 1:0.345. The molecular structure formula of epoxy can be found in the research [3 and 89.8 °C, respectively. The preparation process of the epoxy sheet was as follows. Firstly, the curing agent was added to the epoxy and then evenly stirred for 10 min, followed by oscillation with ultrasonic waves for 15 min to remove the bubbles. Then, the mixed solution was poured into an aluminum mold and cured at 60 °C for 24 h in an oven. After the curing, the resin sheets were cut into different sizes and polished with a polishing machine according to the experimental tests. Thermoplastic polypropylene (PP) and thermosetting epoxy (EP) were adopted to conduct the water uptake, mechanical and thermal tests. For polypropylene, its type was isotactic polypropylene with a molecular weight of ~250,000, which was produced by Taixinlong Plastic Products Co., Ltd.  with a nominal rectangle sheet size of 300 mm × 300 mm × 2 mm. The surface of the matrix sheet was milky white with a density of 0.906 g/cmresearch  with thev is the maximum bending deflection, and it is the sum of midspan bending deflection (20 mm) and specimen thickness (h = 2.0 mm), L is the specimen span (180 mm). Therefore, the maximum bending strain of the matrix plate in the midspan was calculated to be 0.54%.In order to realize the coupling exposure of loading and water immersion, a bending fixture was designed to apply the bending loading and its schematic diagram was as shown in The exposure conditions of two kinds of matrices under the loading and unloading conditions were shown in tW is the wet weight at time t, and W0 is the control weight before the immersion. Two kinds of matrices were put into a temperature-constant water tank with the temperatures of 40, 60 and 80 °C to conduct the water absorption test. Each interval time of the water uptake test was set as 4 h, 12 h, 1 day, 3 days, 7 days, 14 days, 21 days, 30 days, 60 days and 90 days, respectively. For each time interval, the samples were taken out from the water tank and dried with absorbent paper. An electronic balance with an accuracy of 0.1 mg was adopted to obtain the weight. After the weighing, the samples were put back into the water tank for continuous immersion. In order to reduce the experimental error, the whole weighing process was controlled within 15 min. Five samples were weighed and the average value was obtained. The water absorption of two kinds of matrices was obtained according to the following equation [P is the tensile breaking load, b is the sample width, and h is the sample thickness.The tensile test of the polypropylene plate was conducted with the universal tensile machine  according to ASTM D638-2010. The samples with the dumbbell shape were prepared to conduct the tensile test. The crosshead displacement rate of the tensile machine grip was 1 mm/min. Three samples were tested to obtain the average. The calculation of tensile strength was performed as follows:Long-term thermal properties of polypropylene and epoxy before and after the immersions were evaluated by thermogravimetric analysis test (TGA). For polypropylene, the effects of immersion temperature , time (30 and 90 days) and bending loading (0 and 0.54%) were considered. For epoxy, the control and aged for three months at 60 °C were adopted as the compartive group. The mass variations of two matrices were obtained by a TGA analyzer  from room temperature to 600 °C at a heating rate of 10 °C/min in air. One sample was tested with the weight of ~10 mg in powder and 20 mL/min dry air flow was applied during the test. Mass loss curves at elevated temperatures were tracked to analyze the decomposition rate and temperature.The surface morphology of polypropylene and epoxy before and after the immersions were observed through the Scanning Electron Microscope . Before the tests, the samples were vacuumed and sprayed with gold to increase the electrical conductivity. The test frequency, electric current and voltage amplitude were set as 1000 Hz, 0.7 A and 30 kV, respectively. It was noted that the samples before and after the tensile test were adopted to analyze the fracture failure mode.tM is the water uptake at immersion time t, M∞ is the saturated water uptake, D is the diffusion coefficient, and h is the specimen thickness. Equation (4) and Origin 8.1 software were adopted to conduct the nonlinear fitting after assigning the initial values of D and M∞. By fitting, the dependences of water uptake on the immersion time at different temperatures were shown in Immersion in distilled water led to the weight gain of polypropylene and epoxy plates, and the water absorption curves of polypropylene plates for as long as 90 days were shown in Similarly, the water uptake data and fitting curves of epoxy at different temperatures were shown in Water absorption fitting parameters of two kinds of matrices were presented in D-1000/T curve of epoxy was above that of polypropylene, indicating the faster diffusion behavior of water molecules in the matrix. For the same immersion temperature, the water absorption coefficient of epoxy was higher than that of polypropylene. After analyzing the water absorption behavior, the effect of immersion on tensile properties was further investigated. After analyzing the tensile properties of the control sample, the effect of immersion on the tensile strength of polypropylene was shown in The comparisons of tensile strength retention of polypropylene, epoxy and polyurethane immersed in distilled water, alkali or salt solution were conducted ,65 and sAfter analyzing the degradation mechanism of tensile strength, the ratios of tensile strength retention of polypropylene compared to epoxy and polyurethane immersed in distilled water were obtained and shown in The effects of immersion in distilled water on the thermal properties of polypropylene and epoxy were analyzed through the weight variation curves at elevated temperatures as shown in The degradation mechanism of mechanical properties of polypropylene and epoxy after the immersion was revealed by surface morphology analysis as shown in The fracture surface morphology of two kinds of matrices after the tensile tests were shown in Y is the retention of tensile strength, t is the time and τ) and degree of fitting (R2) were shown in R2 implied that the Arrhenius model can well describe the tensile strength evolution. Furthermore, the degradation rate can be expressed through the Arrhenius relationship as follows [Ea is the activation energy of materials, R is the universal gas constant and T is the immersion temperature. Based on Equation (6), Arrhenius plots of tensile strength for two kinds of matrices were obtained and shown in Ea/R) and degree of fitting (R2) of life prediction were shown in R2 were 0.91 (PP) and 0.99 (EP), respectively. After analyzing the degradation mechanism, the long-term life predictions of tensile strength for polypropylene and epoxy were carried out to provide the durability design parameters for engineering applications. Arrhenius model was adopted for the single-factor dominant degradation mechanism during the immersion as follows . Further follows :(6)ln, the time-shift factor (TSF) for tensile strength to reach the same value at temperatures Substituting the fitting parameters from Equation (6) into Equation (7), TSF can be obtained in three typical bridge service environments. It should be pointed out that polypropylene and epoxy were considered to be applied to bridge structures as the matrix and adhesive, etc. The above three typical bridge service environments included different geographical locations in China. Furthermore, the annual average temperatures of the above three bridge environments were 8.8 °C (Shenyang Youth Bridge), 15.9 °C (Jiangsu Yangtze River Bridge) and 26.9 °C (Hainan Century Bridge). Finally, the time-shift factor of life prediction of tensile strength for two kinds of matrices was obtained and shown in Based on After obtaining the mechanical degradation and life prediction of polypropylene, its application prospect in structural engineering was also further analyzed through the comparison with epoxy and polyurethane. Comparative analysis among three kinds of matrices included failure strain, long-term property and fatigue resistance. The tensile stress-strain curves of three kinds of matrices were compared and shown in The long-term properties of three kinds of matrices in the service environment were compared and the predicted retention of tensile strength were presented in After analyzing the toughness and long-term property, the fatigue performance analysis was carried out. In conclusion, in terms of failure strain, long-term performance and fatigue performance, polypropylene and its composites may achieve great application prospects in engineering applications.(1)Simplified Fick’s diffusion model can well describe the water absorption and diffusion behavior of two matrices. Water molecules were easier to diffuse in epoxy than polypropylene at the same immersion temperature, which indicated that polypropylene has better hydrophobic behavior compared to epoxy. For example, the ratios of the diffusion coefficient and saturated water uptake between the two matrices were 114.4 and 2.94 at 80 °C.(2)At the longest immersion time of 90 days, the degradation percentages of tensile strength were 4.7% (40 °C), 7.5% (60 °C) and 8.8% (80 °C), respectively, which had the higher strength retention (>90%). The maximum strength increase multiples of polypropylene/epoxy and polypropylene/polyurethane were 1.95 and 1.75, respectively. The bending loading led to a maximum increase in tensile strength (~1.47%) owing to the oxygen isolation effect.(3)Tensile strength degradation for polypropylene was the active functional groups reacted with oxygen, resulting in local chain segment fracture. For epoxy and polypropylene, water molecules reacted with the hydroxyl groups or interrupted the intermolecular Van der Waals force/hydrogen bond, resulting in irreversible hydrolysis and property degradation.(4)Long-term life prediction of tensile strength showed when the service time was 500 days, the retention of tensile strength for polypropylene was close to 90% at 8.8 °C. In contrast, the retention of tensile strength of epoxy was only ~75% for the service time of 120 days. In terms of failure strain, long-term and fatigue performances, polypropylene and its composites have outstanding advantages compared to epoxy and polypropylene, which can achieve great application prospects in engineering applications when considering the complex service environment.In the present paper, thermoplastic polypropylene was exposed to the coupling condition of elevated temperature, water immersion and sustained bending loading for as long as 90 days. Water uptake, mechanical and thermal properties tests were conducted to obtain the long-term evolution. The degradation mechanism of mechanical properties was revealed and compared with thermosetting epoxy. Arrhenius model was adopted to obtain the long-term life prediction. Finally, the application prospect analysis of polypropylene in structural engineering was compared with epoxy and polyurethane in terms of failure strain, long-term property and fatigue resistance. The following conclusions can be drawn:


Aberrant expressions of RSPOs are detected in various human malignancies and are always correlated with oncogenesis.Although extensive studies of RSPOs have been conducted, their precise molecular mechanism remains poorly understood.Bioinformatic analysis revealed that RSPOs may play a part in the development of the immune composition of the tumor microenvironment.The R-spondin protein family comprises four members (RSPO1-4), which are agonists of the canonical Wnt/β-catenin pathway. Emerging evidence revealed that RSPOs should not only be viewed as agonists of the Wnt/β-catenin pathway but also as regulators for tumor development and progression. Aberrant expression of RSPOs is related to tumorigenesis and tumor development in multiple cancers and their expression of RSPOs has also been correlated with anticancer immune cell signatures. More importantly, the role of RSPOs as potential target therapies and their implication in cancer progressions has been studied in the preclinical and clinical settings. These findings highlight the possible therapeutic value of RSPOs in cancer medicine. However, the expression pattern, effects, and mechanisms of RSPO proteins in cancer remain elusive. Investigating the many roles of RSPOs is likely to expand and improve our understanding of the oncogenic mechanisms mediated by RSPOs. Here, we reviewed the recent advances in the functions and underlying molecular mechanisms of RSPOs in tumor development, cancer microenvironment regulation, and immunity, and discussed the therapeutic potential of targeting RSPOs for cancer treatment. In addition, we also explored the biological feature and clinical relevance of RSPOs in cancer mutagenesis, transcriptional regulation, and immune correlation by bioinformatics analysis. R-spondins  are represented by four secreted glycoproteins RSPO1-4) with a sequence similarity of 40 ∼ 60%. The common structure of RSPO1-4 contains four major functional regions: two adjacent cysteine-rich furin-like domains (CR), a thrombospondin type 1 repeat domain (TSP1) and a basic amino acid-rich domain (BR) with varying length at the C-terminus [-4 with aRSPOs stimulate and synergize the canonical Wnt/β-catenin pathway, which regulates cell development and proliferation, as well as disease pathogenesis. RSPOs cannot directly initiate Wnt signaling, but act as a ligand for LGR4-6 receptors to activate the canonical Wnt/β-catenin pathway and potentiate reactions of low-dose Wnt proteins . Upon biThere is growing evidence implicating RSPOs in cancer development, and the expression of RSPOs is associated with a favorable prognosis. Bioinformatic analyses such as gene enrichment found that RSPOs are widely involved in immune regulation. For example, the latest study reported that RSPO increased tumor sensitivity to anti-PD-1 therapy, highlighting an unprecedented treatment approach ,10. ImmuThe RSPO protein family consists of four members, RSPO1-4, with sequence similarity of 40 ∼ 60% and a common structure. Thus far, all reported RSPO proteins contain four major functional regions: two adjacent cysteine-rich furin-like domains (FU1-FU2), a TSP1 domain, and a BR domain with varying length at the C-terminus. As a typical secretory protein, the RSPO protein has a signal peptide with a length of 20 ∼ 25 amino acids at its N-terminus. After the signal peptide, there are two adjacent furin-like domains, which are rich in cysteine and are the most conserved regions in the RSPO protein structure .The main function of RSPOs is to synergize the canonical Wnt/β-catenin signaling pathway. The operation of the canonical Wnt/β-catenin signaling pathway depends on the activation of the upstream Wnt/FZD/LRP complex . In the It is well established that the furin-like domain plays a decisive role in activating the canonical Wnt/β-catenin signaling pathway via RSPO proteins. Reports from Nam et al. implicated that RSPO could bind to LRP6, FZD, and Wnt1. However, in the absence of two furin-like domains, RSPO is unable to bind to LRP6, FZD, and Wnt1, affecting the activation of the Wnt/β-catenin signaling pathway . AdditioThe TSP1 domain is involved in many signaling pathways that are critical to vascular physiology and disease occurrence. Ayadi et al. conducted three-dimensional structure simulation experiments on the TSP1 domain of human RSPO4 and found that the presence of basic amino acids in this domain formed a large positively charged region, in which tryptophan and arginine were arranged to form a groove-like structure . A similThe RSPO family requires the activity of Wnt ligands and LRP6 to amplify the signal conduction of Wnt3A, Wnt1, and Wnt7A, indicating that RSPO proteins are common modulators of canonical Wnt/β-catenin signaling . Han et RSPO gene was commonly expressed with Wnt, and the expression is formed in Wnt → RSPO → Wnt [Recent studies have also discovered some novel molecular mechanisms of RSPOs. By way of illustration, Hao et al. demonstrated that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homolog ring finger 43 (RNF43) had negative roles in Wnt signaling. ZNRF3 is connected with the Wnt receptor complex, and the Wnt signaling is suppressed due to the turnover of FZD and LRP6. Inhibiting ZNRF3 not only enhances Wnt/β-catenin signaling but also interrupts Wnt/planar cell polarity (PCP) signaling . KazanskPO → Wnt .Although RSPO family proteins can increase the activity of the canonical Wnt/β-catenin signaling pathway, their regulatory capabilities differ due to structural differences. Based on the evidence, a new study has reported that RSPO2 and RSPO3 can enhance Wnt/β-catenin signaling with the deletions of LGR4-6. The need for LGR4-6 is determined by the interplay between RSPOs and the ZNRF3/RNF43 E3 ubiquitin ligases . Lee et RSPOs have been proven to induce the genesis of Wnt-dependent cancers in a variety of malignancies. Based on recent research reports, the authors identified five previously unreported RSPO fusion events (e.g. IFNGR1-RSPO3) [RSPO2 and RSPO3, where the two transcriptions appeared together in patients with colon cancer in colon cancer. After fusion with EIF3E or PTPRK exon 1, the expression of RSPO2 or RSPO3 increased, which activated the Wnt pathway accordingly [EIF3E, RSPO2, PTPRK, RSPO3, TADA2A, and CD4 in HCT116 cells and the expression of fusion transcripts [Gene fusions have been considered to be driver mutations in tumors, and the findings of numerous studies have aided us in developing a better knowledge of the carcinogenic process . Gene fu1-RSPO3) . Seshagiordingly . Zhang enscripts . A more RSPO1 gene was upregulated in the embryonic gonads of each species at the beginning of sex differentiation [Foxl2 gene activation and estrogen synthesis. To study fish sex determination, the research of the RSPO1 gene of zebrafish by Zhang et al. indicated that the expression of RSPO1 in the ovary was stronger than that in the testis, clarifying a differential RSPO1 expression between various sex organs [Rspo1-null (-/-Rspo1) mice as models, then finally observed that -/-Rspo1 female mice exhibited male characteristics, including pseudosexuality in the reproductive ducts and fetuses [The RSPO family has a huge impact on the development of animal reproductive systems . Smith entiation . In chicx organs . Moreove fetuses . It follNobox gene, which plays a significant role in oocyte development, in mice, but RSPO2 was significantly expressed in early oogenesis, indicating that RSPO2 may regulate the development of oocytes downstream of Nobox [Rspo2 (+/−) male mice, Rspo2 (+/−) female mice started to lose their fertility from 4 months of age [RSPO2 can also regulate the development of the female reproductive system. Choi et al. knocked out the of Nobox . De Cianof Nobox . RSPO2 ps of age . In the s of age . Unlike Rspo3 allele and discovered that the homozygous mutant mice died from the damage to the labyrinthine layer of the placenta at about embryonic day 10, suggesting that RSPO3 may affect blood vessel generation and development [Xenopus embryos caused defects in the vascular system, which implied that RSPO3 regulation between angioblast and blood cell specification was essential. Similarly, the targeted destruction of RSPO3 in mice resulted in vascular defects and death. In addition, RSPO signaling can boost proliferation and angiogenesis in human endothelial cells [Wnt/β-catenin signaling is indispensable for the process of angiogenesis. RSPO3 could activate Wnt/β-catenin signaling and have crucial effects on angiogenesis. Aoki et al. constructed mice models carrying a mutant elopment . Kazanskal cells . Caruso al cells . These rRspo2 gene, MYF5 in the developing limbs of mouse embryos exhibited a reduced expression [Rspo2 conditional knockout (Rspo2floxed) mice with the Osteocalcin-Cre mouse line (Ocn- Cre + Rspo2f/f), which sequentially influenced the expression of RSPO2 in osteoblast-lineage cells. They observed lower overall body size and bone mass of Ocn- Cre + Rspo2f/f mice as compared with the control littermates. These results indicated that Rspo2 gene knockout had a significant effect on skeletal growth in mice [Skeletal development is regulated by the Wnt/β-catenin signaling pathway. Wnt/β-catenin signal controls the development of animal bones by promoting the development, differentiation, and activation of osteoprotegerin (OPG) gene expression . Bell etpression . Further in mice . Doherty in mice .in vitro. hRSPO1 is expressed in intestinal endocrine and epithelial cells of assorted tissues, and also displayed effectiveness in the model of chemotherapy-induced intestinal mucositis [in vitro into TOPGAL mice and tested the tongue, stomach, lung, liver, small intestine, large intestine, skin, and gonads. The results specified that the tongue exhibited an RSPO1-specific response. RSPO1 enhanced the proliferation of the basal epithelial cells in the tongue mucosa via Wnt/β-catenin signaling [in vitro, suggesting that RSPO is necessary for the generation of differentiated taste cells. Therefore, it is proposed that RSPO can maintain the steady state of taste tissues.A certain amount of evidence revealed that RSPOs play a crucial role in the development of both the digestive and respiratory systems. For example, recent evidence suggests that recombinant human RSPO1 (rhRSPO1) treatment improved the culture of mouse intestinal organoid units, and increased their size and survival rate ucositis . Zhao etignaling . Interesvia Wnt signaling [Helicobacter pylori (H. pylori) in the gastric glands. The deficiency of Lgr5+ cells yielded similar results, and RSPO3 overexpression in the stroma cleared H. pylori from the gastric glands. Accordingly, RSPO3/LGR5 can regulate the regeneration of the digestive tract mucosa and improve their defensive ability [in vitro led to a significant reduction in organoid growth, while exogenous RSPO3 protein restored this phenotype [LGR5 is RSPOs receptor that regulate both Wnt/β-catenin and Wnt/PCP signaling. Harnack et al. demonstrated that RSPO3 was a key element for epithelial restoration in differentiated cells ignaling . Axin2 i ability . Yan et  ability . A reporhenotype . On the henotype .Rspo2(−/−) homozygous mutant mice had lung development defects [Rspo2 (TgRspo2) and –Lrp6 alleles in mice models and discovered that the mice developed lung hypoplasia and damaged the tracheobronchial ring and laryngeal structures [In the respiratory system, Nam et al. found that  defects . Bell etructures . Wu et aructures . Munguíaructures .A growing body of evidence revealed that the expression of RSPOs in all manner of tumors tends to be abnormal. For instance, the expressions of RSPO1, RSPO2, and RSPO3 were significantly reduced in lung cancer patients as compared with normal tissues . Coussy http://gepia.cancer-pku.cn/). As seen in We analyzed the abnormal expression of RSPOs in different tumors through the GEPIA database (Rspo1-4 gene from the NCBI database (https://www.ncbi.nlm.nih.gov/) and predicted their transcription factors (homo sapiens only) through UCSC-JASPAR . The track scores were over 600 . We utilized NCBI to study the tissue specificity of the predicted transcription factors and selected the tissues with the highest expression of human transcription factors. We predicted the binding sites of Rspo1-4 genes and potential transcription factors through JASPAR and displayed the binding sites with the highest relative score, including the bases and positions . Using the univariate cox regression analysis and the ‘forestplot’ R package, the p-value, hazard ratio (HR) and 95% CI of each variable were determined. The univariate Cox was proportional to the hazards regression analysis    and RSPOGene mutations have been a hot topic in tumor research. It can occur at any stage of development, usually during DNA replication. The mutation is related to DNA replication, damage, and repair, as well as cancer and aging. It is also one of the important factors in biological evolution. Therefore, the study of gene mutation has extensive biological significance in addition to its theoretical significance.http://www.cbioportal.org/), and the results are provided in Genetic mutations of RSPOs were analyzed through the cBioPortal online tool (https://www.mutarget.com/) to analyze samples harboring mutated RSPOs. As displayed in Table S1, some tumors, such as bladder cancer, did not yield consistent results due to insufficient mutated patient samples. RSPO mutations affected the expression of many genes in colon adenocarcinoma, lung adenocarcinoma, and other tumors. These data are useful for studying new drug targets in a cohort of patients with a given mutation.Mutations in one gene may affect both its expression and the expression of other genes. To identify the altered genes, we used the muTarget database  [+ stem cells, and also suppressed cancer progression by restraining the Wnt/β-catenin pathway, thereby reducing cancer cell proliferation and metastasis [RSPO2 contributes to the secretory activation of the canonical Wnt signaling pathway. Gong et al. investigated the influence of RSPO2 on human dental pulp stem/progenitor cells (hDPSCs) and found that RSPO2 mediated the canonical Wnt/β-catenin signaling pathway, leading to the proliferation of hDPSCs . Conboy  (Trp53) . Zhang e (Trp53) . Howevertastasis .RSPO3 gene rearrangements could be considered a potential target in CRC [RSPO3 was linked to a better prognosis. Exogenous expression of RSPO3 in tumors can increase the cytotoxic effector cells infiltration and function, resulting in tumor regression [RSPO3, a vital activator of the Wnt/β-catenin signaling pathway, is involved in the development of a variety of malignancies. Chen et al. overexpressed RSPO3 in JEG-3 cells and detected the expression of biomarkers related to tumorigenicity. The overexpression of RSPO3 accelerated the proliferation of the JEG-3 cells and suppressed their migration, invasion, and mortality . Fischert in CRC . Zhang et in CRC . Nevertht in CRC . Tang etgression .Unlike RSPO1-3, RSPO4 has not been identified in almost all kinds of tumor tissues and normal tissues yet. Wu et al. demonstrated that the expression level of RSPO4 was not directly related to the overall survival (OS) of patients with lung cancer, indicating the functional differences between RSPO4 and RSPO1-3 .Although RSPOs are most recognized for their ability to agonize and synergize the canonical Wnt/β-catenin pathway, recent research has also found that RSPOs can be involved in tumor progression through other signaling pathways.RSPO2, a new cancer inhibitor gene, was able to prevent HCC from proliferating and invading the MAPK signaling pathway. Zheng et al. observed that the MAPK signaling pathway was changed after transfecting the shRNAs against RSPO2. Furthermore, RSPO2 knockout enhanced the tumorigenicity of HCC cells [CC cells .Rspo3 deletion (Rspo3-iECKO) mice had vascular defects, and this phenotype was consistent with that induced by non-canonical Wnt signal. This was due to the deletion of Wnt secretion factor Evi/Wls, inferring that RSPO3 and EVI/WLS might have a co-coordinated role [RSPO3 knockout enhanced the osteogenic capacity of human adipose-derived stem cells (hASCs), which were rescued via suppression of ERK signaling. Besides, the lack of LGR4 abolished RSPO3-induced ERK1/2 signaling suppression [RSPO3 was reported to remodel the endothelial vasculature by activating a non-canonical Wnt signaling pathway. Scholz et al. discovered that inducible endothelial ted role . Furtherpression . On the pression . LGR5/4 pression .The RSPO family is known to activate the canonical Wnt/β-catenin signaling pathway by playing the role of ligand for the LGR4-6 receptors. An increasing amount of studies has reflected that abnormal RSPOs, mainly characterized by gene fusion or abnormal expression, have been recorded in patients with cancer of any kind. At present, most of our knowledge about the molecular activity of RSPO has been gained through intestinal studies. While these provide valuable and pertinent insights, only a small fraction of the mechanism has been uncovered. This would include the unknown role of RSPOs in non-Wnt pathways. Since RSPOs are secreted proteins, this greatly increases the diversity of their molecular mechanisms in tumor progression, despite the lack of available data.In the future, it is of vital importance to comprehend the molecular mechanisms of RSPOs. Tissue specificity should also be considered (since the roles of RSPOs may differ in different tissues) to provide a good direction for clinical studies. The wide range of indications for the carcinogenic effect of RSPOs has led to the exploration of probable treatment methods, and RSPOs have been identified as prospective targets for tumor treatment, especially in tumor immunotherapy. Recent studies have also implied that inhibitors of RSPOs or their receptors (such as LGR5) are being evaluated as candidate targets for cancer therapeutic intervention, which provides a valuable framework for developing new therapeutic strategies.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Scientific Reports 10.1038/s41598-022-10115-0, published online 20 April 2022Correction to: –6. Consequently, numeric results in the original Article were incorrect.The original version of this Article contained errors, where parameters for every participant varied by on average 1 × 10As a result, in the Results under the subheading, ‘Peer presence shifts tolerance for inequity.’,p = 0.0033), indicating greater advantageous inequity aversion than disadvantageous inequity aversion while Alone; Fig. 2D).”“When Alone, adolescents exhibited little disadvantageous inequity aversion (fixed effects Alone α = 0.018) but moderate advantageous inequity aversion (fixed effects Alone β = 0.35). A pairwise comparison of the estimated marginal means for Alone β versus Alone α was statistically significant (now reads:p = 0.0052), indicating greater advantageous inequity aversion than disadvantageous inequity aversion while Alone; Fig. 2D).”“When Alone, adolescents exhibited little disadvantageous inequity aversion (fixed effects Alone α = 0.015) but moderate advantageous inequity aversion (fixed effects Alone β = 0.34). A pairwise comparison of the estimated marginal means for Alone β versus Alone α was statistically significant  and significantly increased aversion to advantageous inequity .”“Next, we evaluated how peer presence altered inequity aversion. When Watched compared to when Alone, adolescents showed marginally decreased aversion to disadvantageous inequity  and significantly increased aversion to advantageous inequity .”“Next, we evaluated how peer presence altered inequity aversion. When Watched compared to when Alone, adolescents showed decreased aversion to disadvantageous inequity .”“We also estimated the influence of performing the Watched condition first or last and found no statistically significant difference in either α or β .”“We also estimated the influence of performing the Watched condition first or last and found no statistically significant difference in either α or β , while the effect of peer presence on disadvantageous inequity aversion was non-significant .”“The finding that peer presence significantly increased advantageous inequity aversion still held among the subset of participants who completed the Watched condition without knowledge of their peers’ choices , while the effect of peer presence on disadvantageous inequity aversion was non-significant .”“The finding that peer presence significantly increased advantageous inequity aversion still held among the subset of participants who completed the Watched condition without knowledge of their peers’ choices . Although this could be due to friends’ shared prosocial attitudes, we note that Alone condition inequity aversion was not correlated within dyads .”“Watchednow reads:2 inequity aversion was correlated with what they had observed for advantageous, but not disadvantageous, inequity aversion . Although this could be due to friends’ shared prosocial attitudes, we note that Alone condition inequity aversion was not correlated within dyads .”“WatchedUnder the subheading, ‘Payouts to self are processed earlier than peer outcomes when alone.’,“To minimize any bias in this estimation that may arise from different amplitudes of the decision weights for these monetary rewards, we normalized all time points to a proportion of each attribute’s full final amplitude . We then estimated a piecewise growth model for each attribute that fits a parameter for attribute latency .”now reads:“To minimize any bias in this estimation that may arise from different amplitudes of the decision weights for these monetary rewards, we normalized all time points to a proportion of each attribute’s full final amplitude . We then estimated a piecewise growth model for each attribute that fits a parameter for attribute latency .”And,p < 0.001).”“In the Alone condition, information about self-outcomes was processed earlier than information about peer outcomes .”“In the Alone condition, information about self-outcomes was processed earlier than information about peer outcomes . Peer presence, in fact, reduced the temporal advantage held by self-outcomes in the alone condition to a non-significant difference. That is, self and peer latencies went from being statistically significantly different in the Alone condition  to not statistically significantly different in the Watched condition . Peer presence did not have a statistically significant effect upon self-reward latency . There was not a significant attribute-by condition interaction  = 3.14, p = 0.08; Watched1 only: F = 0.35, p = 0.56).”“Compared to the Alone condition, peer presence in the Watched condition significantly reduced the latency of processing information about rewards for peers by approximately 200 ms . Peer presence, in fact, reduced the temporal advantage held by self-outcomes in the alone condition to a non-significant difference. That is, self and peer latencies went from being statistically significantly different in the Alone condition  to not statistically significantly different in the Watched condition . Peer presence did not have a statistically significant effect upon self-reward latency . There was not a significant attribute-by condition interaction  = 3.14, p = 0.08; Watched1 only: F = 0.02, p = 0.89).”“Compared to the Alone condition, peer presence in the Watched condition significantly reduced the latency of processing information about rewards for peers by approximately 200 ms  and 91.8% correct for the Watched condition (range 75.6–99.0%).”now reads:“Our resulting parameter estimates were highly accurate at correctly predicting participants’ choices: an average of 90.5% correct for the Alone condition (range 76.6–98.2%) and 91.8% correct for the Watched condition (range 75.6–99.0%).”Lastly, the Data availability section contained an error, where“All data that support the findings of this study, and the stimuli used to collect the data, as well as complete analysis scripts, will be deposited in an OSF repository upon publication.”now reads:https://osf.io/9nxsw/.”“All data that support the findings of this study, and the stimuli used to collect the data, as well as complete analysis scripts, are available here: The original Article has been corrected.