These reports described a total of 270 families in which more than one relative had HTLV-1-associated diseases. In most families, different family members suffered from the same disease (n = 223). The diseases most frequently reported were ATLL (115 families) and HAM/TSP (102 families). Most families (n = 144) included two to four affected individuals. The proportion of ATLL patients with family history of ATLL ranged from 2 to 26%. The proportion of HAM/TSP patients with family history of HAM/TSP ranged from 1 to 48%. The predominant cluster types for ATLL were clusters of siblings and parent-child pairs and for HAM/TSP, an affected parent with one or more affected children. The evidence in the literature, although weak, does suggest that HTLV-1-associated diseases sometimes cluster in families. Whether familial transmission of HTLV-1 is the only determining factor, or whether other factors are also involved, needs further research.Human T-lymphotropic virus 1 (HTLV-1) is a retrovirus that produces a persistent infection. Two transmission routes  favor familial clustering of HTLV-1. It is yet unknown why most HTLV-1 carriers remain asymptomatic while about 10% of them develop complications. HTLV-1 associated diseases were originally described as sporadic entities, but familial presentations have been reported. To explore what is known about family aggregation of HTLV-1-associated diseases we undertook a systematic review. We aimed at answering whether, when, and where family aggregation of HTLV-1-associated diseases was reported, which relatives were affected and which hypotheses were proposed to explain aggregation. We searched MEDLINE, abstract books of HTLV conferences and reference lists of selected papers. Search terms used referred to HTLV-1 infection, and HTLV-1-associated diseases, and family studies. HTLV-1-associated diseases considered are adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), HTLV-1-associated uveitis, and infective dermatitis. Seventy-four records reported HTLV-1-associated diseases in more than one member of the same family and were included. Most reports came from HTLV-1-endemic countries, mainly Japan ( Human T-lymphotropic virus 1 (HTLV-1) is a retrovirus that causes a lifelong infection. HTLV-1 infects an estimated five to ten million people worldwide , HAM/TSP , and ATLL  , abstract books of HTLV conferences and reference lists of selected papers. To retrieve the abstracts, we hand-searched the abstract books of eleven conferences on HTLV and related viruses organized between 1994 and 2015.The PubMed search was done in January 2015 and combined three types of search terms: terms indicating (1) HTLV-1 infection, (2) HTLV-1-associated diseases, and (3) family studies. The detailed search strategy is given as Family aggregation of HTLV-1-associated diseases was defined as the occurrence of an HTLV-1-associated disease in more than one member of a family. We used an extensive definition of a family, including in-laws as well as blood relatives. For the literature search, the following conditions were considered to be HTLV-1-associated diseases: HAM/TSP, ATLL, HTLV-1-associated uveitis, and infective dermatitis.Two reviewers (CA and KV) independently screened all titles and abstracts of the records retrieved through the PubMed search. One reviewer (CA) then read the full text of the preselected records. Doubts and discrepancies in the selection of records were solved through the discussion among two reviewers (CA and KV). All records that mentioned HTLV-1-associated diseases in more than one member of the same family were included. When the same families were identified in more than one record, we selected the record that contained more information and excluded the other. When both records contained the same amount of information, we selected the earliest report.Data were extracted using a pre-designed form. The following information was extracted from the selected records: study design, year, country, number of families, number of relatives affected by HTLV-1-associated diseases, relationship between affected relatives, and hypotheses proposed to explain family aggregation of HTLV-1-associated diseases.For the analysis, we first assessed the characteristics of the selected records, including study design. Next, we combined all those studies that gave detailed information about concrete families in which more than one person had an HTLV-1-associated disease. We presented this information as the number of families with particular diseases, particular family relationships or both. Finally, for those studies that were designed to describe or explain family aggregation, we summarized the results of the individual records.From planning to reporting this review, the recommendations of the PRISMA statement  and Brazil (n = 10). Reports from non-endemic countries (n = 17) described HTLV-1-associated diseases in migrants from endemic regions or in specific ethnic groups  contained two to four affected individuals. However, we also found reports of more complex families in which there were three or more different associated diseases (5 families), five or more affected individuals (6 families), and/or affected individuals in three or more generations (3 families).The number of affected individuals per family ranged from two to seven. Most of the family clusters (Family aggregation has been reported in blood relatives and in in-laws. The type of family relationship between the cases was given for 121 out of the 270 families included in this review (45%). In 110 families, the diseases affected only blood relatives, in six families only in-laws, and in five families, the diseases affected both blood relatives and in-laws. In the 110 families in which only blood relatives were affected, the diseases that clustered most frequently were ATLL (44 families), HAM/TSP (27 families), HAM/TSP + infective dermatitis (17 families), and HAM/TSP + ATLL 13 families). The six families in which only in-laws developed diseases comprised three pairs of spouses with ATLL and three pairs of spouses in which the husband had ATLL and the wife had HAM/TSP  were women; 55% were born in Southern Japan and the median age at enrollment was 60 years for women and 58 years for men. During follow up, 14 study participants developed ATLL. The incidence of ATLL in this cohort was 7 per 1000 person-years. On multivariable Cox regression analysis, four factors were significantly associated with an increased hazard of developing ATLL: high baseline proviral load, advanced age, first diagnosis of HTLV-1 infection during treatment for other diseases, and family history of ATLL. After adjustment for other associated factors, the hazard of developing ATLL was 12 times higher in those HTLV-1 carriers who had a family history of ATLL compared to those who did not have such a family history   among 318 HTLV-1-positive relatives of 334 HAM/TSP patients compared to 15 HAM/TSP cases (7%) among 204 HTLV-1-positive relatives of 230 asymptomatic HTLV-1 carriers. This difference was not statistically significant. The authors concluded that HAM/TSP is usually sporadic but that in some particular families there are HAM/TSP clusters  than sporadic cases (mean 51.6 years). There was no difference in HTLV-1 proviral load between the two groups , they found that the monozygotic twins with different clinical outcomes carried different viral strains. They also found that different HLA molecules were expressed in the mother and her children  were used to measure this  or the timing of HTLV-1 infection , few studies were specifically designed to investigate family aggregation. Nonetheless, we found descriptions of 270 concrete families in which more than one person had an HTLV-1-associated disease. The specific diseases and the relationships within the affected families varied, but the predominant situation was that several blood relatives suffered from the same HTLV-1-associated disease. The majority of the reports were about ATLL, HAM/TSP, or both.The occurrence of several family members affected by HTLV-1-associated diseases has been reported ever since the discovery of HTLV-1 in the early 1980s  as well as the number of reported family clusters (n = 270) suggest that HTLV-1-associated diseases do occur in families more frequently than would be expected by chance. Moreover, one cohort study showed that having a family history of ATLL increases the risk of developing ATLL  received scholarships from the Belgian Development Cooperation through the Flemish Interuniversity Council (VLIR-UOS) ZEIN2010PR376 and the Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica (CONCYTEC-CIENCIACTIVA) of the Peruvian Government. This research was supported by VLIR-UOS grant (ZEIN2010PR376) and “Vaast Leysen Leerstoel voor Wetenschappelijk onderzoek over infectieziekten in ontwikkelingslanden” from KU Leuven, Belgium.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The epidermis is the outermost layer of the skin and provides a protective barrier against environmental insults. It is a rapidly-renewing tissue undergoing constant regeneration, maintained by several types of stem cells. The Hedgehog (HH) signaling pathway is one of the fundamental signaling pathways that contributes to epidermal development, homeostasis, and repair, as well as to hair follicle development and follicle bulge stem cell maintenance. The HH pathway interacts with other signal transduction pathways, including those activated by Wnt, bone morphogenetic protein, platelet-derived growth factor, Notch, and ectodysplasin. Furthermore, aberrant activation of HH signaling is associated with various tumors, including basal cell carcinoma. Therefore, an understanding of the regulatory mechanisms of the HH signaling pathway is important for elucidating fundamental mechanisms underlying both organogenesis and carcinogenesis. In this review, we discuss the role of the HH signaling pathway in the development and homeostasis epidermis and hair follicles, and in basal cell carcinoma formation, providing an update of current knowledge in this field. Although the thickness of the skin is as little as 2 mm on average, it is a complex stratified organ consisting of an outer epidermis and appendages  separated from the underlying dermis by a basement membrane. It is the largest organ in the human body, by area and weight. Skin is the primary barrier that protects the body from major environmental stresses, such as dehydration, scratches, wounds, and microbial infection . To repaThe HH pathway is one of the most important signal transduction pathways in tissue development, homeostasis, and repair. It regulates the morphogenesis of various organs during embryogenesis . We showGli2 knockout mice die at birth, whereas Gli1 knockout mice show normal development, unless one copy of Gli2 is also defective [Gli1 knock-in into the Gli2 allele can rescue the Gli2-null phenotype [Graded levels of HH signaling trigger the expression of different sets of response genes, depending on the ratio of GLI activator (GLI-A) to repressor (GLI-R) forms. In vertebrates, the GLI family consists of three proteins, GLI1, GLI2, and GLI3 . All GLIefective . Intereshenotype .PTCH1 and SMO have been identified in patients with BCC and medulloblastoma [SUFU mutations in BCC and medulloblastoma [Constitutive activation of HH signaling has been observed in many cancers  and promblastoma ,31,32,33blastoma . Furtherblastoma ,11,35.HH signaling exhibits crosstalk with other signaling pathways, including Wnt ,37,38,39During embryonic development, the epidermis is derived from the ectoderm. In mice, after gastrulation, the embryo surface consists of a single layer of neuroectoderm, which will form the nervous system and skin epithelium from embryonic day 9.5 (E9.5) to E12.5. Mesenchymal cells from the underlying layer transmit signals that instruct the stratification of the epidermis and dictate the positioning of downgrowths that mark the initiation of hair follicle morphogenesis . During The development of hair follicles is intrinsically related to the stratification of the embryonic epidermis. Hair follicle development is the result of epithelial–mesenchymal interactions. Although hair follicle development occurs in eight morphologically distinct stages, it can more simply be divided into three phases: induction, organogenesis and cytodifferentiation . In the Ptch1, Smo, Gli1 and Gli2 are expressed in both the epithelial and dermal components of the early hair follicle, although higher levels of Ptch1 and Gli1 are observed in the follicular dermal mesenchyme. In contrast, Ptch2 expression is restricted to epithelial cells of the hair bud where it is co-expressed with Shh [Shh, hair follicle formation is initiated and specialized fibroblasts are condensed, indicating that the initial events of hair follicle development are independent of HH signaling. However, in the hair follicle maturation phase, the loss of Shh selectively reduces keratinocyte proliferation in the developing follicle epithelium, so that the downgrowth of epithelial cells fails. Therefore, the number of hair follicles is reduced in Shh−/− mice. Thus, HH signaling is necessary for the transition from the induction to the organogenesis phase, and for the downgrowth of epithelial cells after the induction phase. Furthermore, the maturation of the dermal papilla also depends on HH signaling, since Shh−/− mice have smaller dermal papillae [Gli2−/− skin because of suppressed epithelial cell proliferation, similar to Shh−/− skin [Gli2−/− skin. In addition, Shh−/−, as well as Gli2−/−, skin fails to induce GLI1. Together, these results show that the SHH–GLI2 axis is required for the proliferation and subsequent downgrowth of the follicular epithelium, and for the maturation of the dermal papilla.In mouse skin at E15.5, at the beginning of the organogenesis phase of hair follicle development, with Shh ,50. Therpapillae . It is bpapillae . Mill et−/− skin . DevelopPtch1, Smo, Gli1, and Gli2 is seen in the dermal papilla [Shh−/− skin. However, HH signaling in dermal cells does not affect cell survival or epithelial proliferation. Rather, dermal HH signaling regulates the expression of a subset of dermal papilla-specific signature genes. These results suggest that dermal HH signaling regulates genes required to maintain the maturation of the papilla [+/CD133+ dermal papilla cells are associated with guard, awl, and auchene hair follicle formation whereas SOX2−/CD133+ dermal papilla cells are involved in the formation of zigzag hair follicles. Moreover, they demonstrated the HH signaling pathway is activated in SOX2−/CD133+ dermal papilla cells, suggesting that HH signaling regulates hair type fate determination through the maintenance of dermal papilla cell heterogeneity [As described above, the expression of  papilla , indicat papilla . Sex-det papilla ,55. ReceShh-null skin, Merkel cells are absent, and SHH overexpression results in increased proliferation of Merkel cells. HH signaling pathway activation, which is required for Merkel cell formation, is initiated by the production of SHH in developing hair follicles. Therefore, HH signaling pathway is required for Merkel cell formation, resulting in touch dome development [Touch domes are unique epidermal sensory structures that form exclusively around primary hair follicles ,57. Merkelopment ,60. The Ptch1-null skin exhibits hyperplasia and skin neoplasia formation, although Ptch2-null skin is undistinguishable from wild-type skin with respect to embryonic hair follicle development [Ptch1-null and Ptch2-null mice exhibits excessive proliferation of epithelial cells, suggesting that epidermal stem and progenitor cells enter a hyperproliferative state. In addition, progenitor cells fail to differentiate, resulting in the loss of epidermal lineage specification. Therefore, PTCH1- and 2-mediated HH signaling suppression is important for regulating epidermal differentiation [Shh−/−/Gli3−/− skin, in which the repressor form of GLI3 (GLI3R), another negative regulator of HH signaling, is lost. Furthermore, keratinocyte differentiation fails in Shh−/−/Gli3−/− skin, resulting in the failure of hair follicle maturation [On the other hand, negative regulators of HH signaling are also important for hair follicle development. PTCH1 and 2 are known to be negative regulators of HH signaling, and Ptch1 is a GLI target gene ,61. Basaelopment ,63. Howentiation . Anotherturation . Recentlturation . At E17.The hair follicle develops as a result of complex reciprocal signaling between epithelial and mesenchymal cells. Various signaling pathways, such as those activated by Wnt, bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), Notch and ectodysplasin, are involved in hair follicle development, and exhibit signaling crosstalk with the HH pathway. In this section, we discuss the crosstalk between HH and these other signaling pathways in hair follicle development.The Wnt signaling pathway has been implicated in many developmental processes . CanonicShh−/− skin, indicating that Wnt5a is downstream, or even a direct target, of HH signaling [The Wnt and HH signaling pathways are often associated. During hair follicle development in mouse embryos, the canonical Wnt signaling pathway is upstream of HH signaling, since β-catenin activation induces Shh expression in epidermis during skin development ,37. On tignaling . Therefoignaling . TherefoBMPs are members of TGF-β family. BMP signaling has been suggested to regulate hair follicle induction and the patterning of follicles within the skin by repressing the placode fate ,74. Suzunoggin−/− mice, hair follicles arrest at the placode formation stage of embryonic development [Sox2 [Sox18 [noggin. Among these genes, noggin partially rescues dermal SMO knockdown-associated hair regeneration defects by increasing epithelial Shh expression. These results suggest that dermal HH signaling regulates specific dermal papilla gene expression signatures to maintain dermal papilla maturation while maintaining a reciprocal HH–Noggin signaling loop to drive embryonic hair follicle development.The HH signaling pathway also affects dermal cells during hair follicle development. Noggin is known as an antagonist of BMPs, and Noggin signaling is a dermal papilla-specific pathway. In elopment ,77. Oro elopment . Dermal nt [Sox2 ,55, Sox12 [Sox18 ,79, and pdgf-a-null skin exhibits thinner dermis, smaller dermal papillae and abnormal hair follicle shape. Shh and pdgf-a expression overlap during hair follicle development. Moreover, PDGFRα is expressed in dermal cells adjacent to sites of SHH expression  antibodies into newborn mice, which perturbed hair formation . Karlssoed to in ). In Shhormation . As descormation . These r−/CD133+ dermal papilla cells [Notch signaling plays a crucial role in cell fate determination and developmental border formation . During la cells . The Notla cells . Previoula cells  and GLI2la cells ,87 expreEctodysplasin A1 (EdaA1) and ectodysplasin receptor (EdaR) respectively belong to the tumor necrosis factor (TNF) multigene family of ligands and receptors, and are able to activate the nuclear factor-κB (NF-κB) and Jun amino-terminal kinase (JNK)/activator protein 1 (AP1) pathway in vitro ,89,90,91The adult epidermis consists of several layers, each with its own characteristics. Cells in the basal layer proliferate to provide new cells that replace those shed from the cornified exterior of the tissue . This reTreatment of adult mice with an anti-SHH antibody blocks the active phase of the hair growth cycle (anagen) and blocks hair regrowth , indicat+) stem cells co-expresses LGR5 [+/LGR5+) exhibit greater proliferative properties and are localized to the lower portion of the bulge, close to the telogen hair germ. In this phase of the hair cycle, GLI1+/LGR5+ stem cells only partially overlap with other bulge stem cell markers, such as CD34 and keratin 15. However, in anagen hair follicles, GLI1+/LGR5+ stem cells are localized to the lower part of the outer root sheath of the reforming hair follicle, where they lose expression of the bulge stem cell markers [+/LGR5+ stem cells may also be necessary for hair follicle growth in anagen. Another population of GLI1+ stem cells in the dermal papilla is involved in dermal papilla maintenance. These results show that, during anagen, the induction of HH target genes, including Gli1, occurs in both epidermal and dermal papilla cells adjacent to SHH-expressing cells, such as those in the follicle matrix.In adult skin, stem cells in the hair follicle bulge cyclically regenerate the follicle, whereas a distinct stem cell population maintains the epidermis. In telogen hair follicles, GLI2 and GLI3 continue to be broadly expressed in both the follicle and the surrounding dermis. Throughout the hair cycle, GLI1 expression is restricted to two distinct epidermal stem cell domains . One popses LGR5 ,102. Ste markers . This lo+ stem cells that does not express LGR5, CD34, or keratin 15 exists in the upper fringe of the bulge. GLI1+ upper bulge stem cells are induced by bulgeassociated nerve cells releasing SHH. GLI1+ stem cells from the upper bulge stably remain in a wounded area of the skin long after it has been repaired [Another population of GLI1repaired . Thereforepaired . Matrix repaired ,108,109.repaired . The rolThe Wnt signaling pathway is also important for the maintenance of hair follicles in adult skin. As described above, activation of canonical Wnt signaling induces SHH expression, indicating HH signaling activation during hair follicle development in mouse embryos. Previous studies have demonstrated that the Wnt–HH signaling axis is also essential for maintaining the hair cycle. Epidermal basal cell-specific β-catenin activation in adult mouse skin also induces SHH expression, resulting in HH signaling activation ,39, and The Hippo signaling pathway is a master regulator of cell proliferation and organ size. Hippo pathway molecules, such as macrophage-stimulating 1/2 (MST1/2) and large tumor suppressor 1/2 (LATS1/2), regulate a transcriptional coactivator, Yes-associated protein (YAP) ,112. YAPAkladios et al. proposed a novel interplay among HH, YAP, Rho-associated protein kinase (ROCK) and β-catenin in the control of skin homeostasis . HH signThe tumor suppressor, p53, is expressed in the epidermal basal layer and also in the outer root sheath and matrix of the hair follicles. Its expression is increased during the transition from anagen to the regression phase of the hair cycle (catagen), suggesting its role in hair follicle involution . In addiA homolog of p53, p63, has an essential role in development. Mice lacking p63 show extensive defects in epidermal development and die at birth ,120. In BCCs are so named because of their histological resemblance to basal cells of the interfollicular epidermis and epidermal appendages, that is, the keratinocytes adjacent to the stroma ,127. ActPTCH1, 5% exhibit loss of SUFU and 10% have activating mutations of SMO [GLI1, GLI2, and GLI3) are not observed in BCC patients, although GLI overexpression is observed. In addition, NRAS, KRAS, HRAS, BRAF, and CTNNB1 are not known to carry mutations in BCC patients [Although in general, BCCs seem to have relatively stable genomes, 67% of BCCs exhibit loss of s of SMO ,128,129.Ptch1+/− mice develop BCCs, and those BCCs often exhibit a deletion of the wild-type copy of Ptch1, as well as up-regulation of HH signaling. Similarly, mice carrying one inactivated, mutant allele of the HH suppressor, SUFU are also susceptible to BCC development [Based on the molecular genetics analysis of human BCCs, several studies have been performed using genetically-modified mice in which the HH signaling pathway is constitutively activated. Constitutive or conditional overexpression of either GLI1  or GLI2 elopment .Ptch1+/− mice arise predominantly, and likely exclusively, from hair follicle bulge stem cells expressing keratin 15 (a bulge cell marker). Furthermore, upregulation of SMO expression is necessary for BCC formation in X-irradiated Ptch1+/− mice [Ptch1 in different cellular compartments revealed that tumors can arise from multiple hair follicle stem cells and from innervated GLI1-expressing progenitor cells in touch domes. In contrast, tumors do not arise from interfollicular epidermal stem cells [Although these results show that the HH signaling pathway has important roles in BCC formation, the actual cell of origin remains to be elucidated. X-ray-induced BCCs in +/− mice . Using a+/− mice  in the a+/− mice . Recentlem cells .FGF19, TGFA, CXCR4, SOX9, and SOX2. These genes are not induced by activation of the canonical HH signaling pathway alone. These results suggest that cooperation between HH and EGFR signaling is important for BCC development, which is mediated by HH–EGFR cooperation-responsive GLI target genes.BCC-specific signaling crosstalk between the HH and other signaling pathways has been reported. Eberl et al. reported crosstalk between the HH and EGFR signaling pathways in BCCs . Skin-spGLI1 and GLI2 promoters and regulates GLI1 and GLI2 transcription downstream of SMO [GLI1 and GLI2 transcription. In addition, JQ1-mediated downregulation of GLI1 and GLI2 transcription is also observed in SMO inhibitor-resistant BCC cells [Vismodegib  is the fm of SMO . Treatmem of SMO , attenuaCC cells . InfanteCC cells . Ally etCC cells . AlthougCC cells . aPKC-i/The HH signaling pathway is one of the most fundamental in epidermal development and epidermal stem cell maintenance. However, deregulated HH signaling induces BCC. Hence, it is important that the intensity of the HH signal is strictly regulated during skin generation and homeostasis. To achieve precise regulation of HH signaling intensity, negative regulators of HH signaling are necessary, as well as crosstalk between the HH and other signal transduction pathways such as those activated by Wnt and BMP. Recently, it has been reported that low HH signaling activity is important for lung homeostasis . TherefoAs described above, aberrant activation of HH signaling causes BCC formation. A tumor microenvironment would exhibit not only the HH pathway regulatory mechanisms that are common with skin development and homeostasis, but also cancer cell-specific regulatory mechanisms. Therefore, understanding the precise mechanisms of HH pathway regulation in the tumor microenvironment is important to advance new therapeutic strategies for SMO inhibitor-resistant BCC. Moreover, such knowledge may also help to provide new therapeutic strategies for other HH pathway-activated cancers.


Strikingly, development can be fully restored without feedback: Lethal patterning defects in lefty mutants can be rescued by ectopic expression of lefty far from its normal expression domain or by spatially and temporally uniform exposure to a Nodal inhibitor drug. While drug-treated mutants are less tolerant of mild perturbations to Nodal signaling levels than wild type embryos, they can develop into healthy adults. These results indicate that patterning without inhibitory feedback is functional but fragile.Developmental signaling pathways often activate their own inhibitors. Such inhibitory feedback has been suggested to restrict the spatial and temporal extent of signaling or mitigate signaling fluctuations, but these models are difficult to rigorously test. Here, we determine whether the ability of the mesendoderm inducer Nodal to activate its inhibitor Lefty is required for development. We find that zebrafish  During animal development, a single fertilized cell gives rise to different tissues and organs. This ‘patterning’ process depends on signaling molecules that instruct cells in different positions in the embryo to acquire different identities. To avoid mistakes during patterning, each cell must receive the correct amount of signal at the appropriate time.In a process called ‘inhibitory feedback’, a signaling molecule instructs cells to produce molecules that block its own signaling. Although inhibitory feedback is widely used during patterning in organisms ranging from sea urchins to mammals, its exact purpose is often not clear. In part this is because feedback is challenging to experimentally manipulate. Removing the inhibitor disrupts feedback, but also increases signaling. Since the effects of broken feedback and increased signaling are intertwined, any resulting developmental defects do not provide information about what feedback specifically does. In order to examine the role of feedback, it is therefore necessary to disconnect the production of the inhibitor from the signaling process.In developing embryos, a well-known signaling molecule called Nodal instructs cells to become specific types – for example, a heart or gut cell. Nodal also promotes the production of its inhibitor, Lefty. To understand how this feedback system works, Rogers, Lord et al. first removed Lefty from zebrafish embryos. These embryos had excessive levels of Nodal signaling, did not develop correctly, and could not survive. Bathing the embryos in a drug that inhibits Nodal reduced excess signaling and allowed them to develop successfully. In these drug-treated embryos, inhibitor production is disconnected from the signaling process, allowing the role of feedback to be examined. Drug-treated embryos were less able to tolerate fluctuations in Nodal signaling than normal zebrafish embryos, which could compensate for such disturbances by adjusting Lefty levels.Overall, it appears that inhibitory feedback in this patterning system is important to compensate for alterations in Nodal signaling, but is not essential for development. Understanding the role of inhibitory feedback will be useful for efforts to grow tissues and organs in the laboratory for clinical use. The results presented by Rogers, Lord et al. also suggest the possibility that drug treatments could be developed to help correct birth defects in the womb. Drosophila wing  the 8 cell stage, 3 hr before the onset of nodal and lefty expression, (2) sphere stage, when nodal and lefty expression normally begins, and (3) shield stage, 2.5 hr after expression has commenced. Strikingly, exposure of -/-;lefty2-/-lefty1 mutants to inhibitor drug resulted in phenotypically normal embryos that developed into fertile adults  resultepression , but uncpression . Wild ty-/-;lefty2-/-lefty1 mutants, in which inhibition is decoupled from signaling . Genotyping primers were identical to the lefty1 forward/reverse primers described above. Complete digestion by PshAI indicates that both alleles are wild type, partial digestion indicates heterozygosity, and failure to digest indicates homozygosity for the a145lefty1 mutation.lefty2: The 11-base-pair deletion in a146lefty2 was detected using a mutant-specific forward primer that spans the deletion. A forward primer specific to the wild type allele was also designed, as well as a reverse primer that is fully complementary to both alleles. To genotype the lefty2 locus, PCR was carried out using either the wild type- or mutant-specific forward primer and the common reverse primer. A band with the wild type- but not mutant-specific primer indicates that both alleles are wild type, bands with both primer sets indicate heterozygosity, and a band with the mutant- but not wild type-specific primers indicates homozygosity for the a146lefty2 mutation. Optimal PCR conditions: Taq polymerase, 25 cycles, 57°C annealing temperature.lefty2 wild type genotyping forward primer: cattttgaccacagcgatlefty2 mutant genotyping forward primer: gttcattttgaccactcaclefty2 reverse primer described above.The common reverse primer was identical to squint: The cz35squint allele has a ~ 1.9 kb insertion in exon 1, and was detected as in  and purified using a Qiagen PCR clean-up kit. Capped mRNA was generated from linearized plasmid using an SP6 mMessage mMachine kit (Ambion) and purified with a Qiagen RNeasy kit.After purification, mRNA was quantified using a NanoDrop spectrophotometer (ThermoFisher) and diluted to the appropriate concentration. For microinjections, a micrometer was used to adjust the drop volume to 0.5 nl. Depending on the concentration of the injection mix, a total volume of 1–2 nl was injected per embryo.Lefty1-GFP, Lefty2-GFP, and untagged Lefty1 and Lefty2 constructs used in lefty alleles retain Nodal inhibitory activity, wild type and mutant lefty mRNA was injected into wild type embryos and Nodal loss-of-function phenotypes were assessed (Allele activity experiments: To determine whether mutant assessed .a145lefty1 mutation removes part of the endogenous Kozak sequence (gaaaag). Therefore, lefty1 constructs containing this endogenous Kozak were generated, rather than the consensus Kozak sequence gccacc as in  vector. In addition to the 11 bp deletion, the mutant construct contains three silent SNPs at position 184 (T->C), 932 (A->C), and 943 (T->A). Both constructs lack endogenous UTRs and contain the consensus Kozak sequence gccacc immediately preceding the start codon.The  used in  and in tsquint mRNA in the Nodal overexpression experiment , transferred onto a coverglass and oriented manually. All embryos (other than sphere stage) were mounted ‘margin down’ . Shield stage embryos were rolled to ensure that the dorsal/ventral axis was parallel to the coverglass. Sphere stage embryos were mounted with the animal/vegetal axis perpendicular to the coverglass . The agarose drop was then dehydrated with three washes of 100% MeOH, two washes of 50:50 (v/v) BBBA:MeOH, and 3 washes of BBBA. Cleared embryos were then sealed to a microscope slide using fast wells reagent reservoirs (Grace Bio-Labs). Imaging was performed on Sytox Green and Alexa 647 channels using an LSM 700 confocal microscope . Image stacks extended from the embryo margin (adjacent to the coverglass) to beyond the center of the embryo. Z-planes were spaced at 2 μm intervals.Embryos were mounted in agarose and cleared with 2:1 benzyl benzoate:benzyl alcohol (BBBA) . BrieflyQuantification of pSmad2 and Sytox green staining intensity in laterally-mounted embryos was performed using the ten z-slices surrounding the center of the embryo axis (i.e. five slices above and five slices below the embryo center). This region was chosen to minimize artifacts due to light scattering, which causes a decrease in apparent fluorescence intensity in deeper tissue planes. Our procedure—looking only at slices close to the ‘central plane’ of the embryo—allows the entire gradient to be sampled within each slice, and ensured that all data were taken from planes within a narrow range of imaging depths, effectively controlling for signal drop-off with imaging depth.http://homepages.inf.ed.ac.uk/rbf/HIPR2/adpthrsh.htm) of the resulting image. Spurious objects were discarded by morphological filtering (based on object size). Final segmentation boundaries were defined after manual checking and correction with a custom MATLAB script   was diss In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Nodal patterning without Lefty inhibitory feedback is functional but fragile" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this manuscript, Rogers et al. devise an interesting test of the relevance of negative feedback in a signaling circuit during early zebrafish development. Nodal signaling, which is activated by the ligands Cyclops and Squint, is kept under control by two secreted feedback inhibitors, Lefty 1 and Lefty 2. Here, the authors investigate the relevance of this regulatory interaction by assessing the effect of exogenous Nodal inhibitors in embryos lacking Lefty 1/2. One of their most remarkable results is that these mutants can be fully rescued by addition of a chemical inhibitor of Nodal signalling . This result clearly shows that signaling-dependent feedback inhibition is not required for normal development under laboratory conditions. The Lefty proteins are not needed to shut down Nodal signaling through increased production, nor are they absolutely essential for mesendodermal patterning that is compatible with viability. The elegance of the reaction-diffusion model also seems to be dispensable under these circumstances. The authors then suggest that feedback inhibition may nevertheless be important to buffer against fluctuations of signaling activity. The latter argument makes sense but is incompletely investigated; some of the suggested revisions may help to strengthen this argument.Overall, this is a very strong and well-written manuscript, leading to a reconsideration of the concept of feedback loops as exemplified by the roles of Nodal ligands and their induced inhibitors Lefty 1/2. This work will undoubtedly generate a lot of discussion in the field. The large amount of data and the careful performance of the experiments will be greatly appreciated by those who want to carefully evaluate the data in light of various models for pattern formation. Consideration of the issues listed below is likely to strengthen the work and enhance its impact.Essential revisions:1) The authors suggest that there is a sweet spot of signaling activity that is required for normal development. This is defined by the dose of inhibitor that is required to support development of the double mutant. An alternative (quantitative) measure of signaling activity (e.g. western blot with anti-pSMAD) in chemically and genetically rescued embryos would strengthen the authors' argument. This would allow them to estimate the tolerance to variation in signaling activity, which is relevant to their argument that the normal function of feedback is to ensure reliable signaling at the right level.2) The authors predict that signaling activity would be variable in the absence of feedback. Is it possible to strengthen this argument by measuring embryo to embryo variation among a population of drug-rescued Lefty double mutants (no feedback) with a quantitative measure as suggested above? Would such variation fall outside the tolerance measured in the previous experiment?cyclops;squint double mutant. Would they expect a just-right level of global signaling that rescues development?3) One possible extension of the finding described is that the spatial requirement for Nodal signalling is not very stringent. Have the authors attempted a converse rescue experiment, i.e. to activate Nodal signaling uniformly, either with a drug or RNA injection in a 4) Regarding the statement "Thus, an ectopic, Nodal-independent source of Lefty at the animal pole can replace endogenous, Nodal-induced Lefty at the margin": Is this confirmed by fate-mapping (with Alexa 488-dextran) of the transplanted, Lefty-expressing cells? 5) It is somewhat surprising that there is not a graded effect of loss of lefty alleles, i.e. that the embryonic phenotypes and response readouts (in situs and pSmad2) do not show a graded response, and that loss of one, two or three alleles of Lefty1/2 seem to be equivalent. Is it correct that there is no dose response? What are the phenotypes of the genetic combinations with just one functional allele?6) In the Discussion, "While Lefty is not essential for viability per se" is an overstatement. The lefty double mutants are not viable. Although they can be experimentally rescued by drug or resupply of Lefty, this does not alter the requirement for functional Lefty during development (whether provided by expression or downstream pathway inhibition), as demonstrated by the double mutant phenotype. It is important in the interpretation of the results and the discussion of the results to uncouple the concept of dynamic feedback inhibition with the concept of the requirement of Lefty to be in the game, at least to modulate Nodal activity levels for viability. Essential revisions:1) The authors suggest that there is a sweet spot of signaling activity that is required for normal development. This is defined by the dose of inhibitor that is required to support development of the double mutant. An alternative (quantitative) measure of signaling activity (e.g. western blot with anti-pSMAD) in chemically and genetically rescued embryos would strengthen the authors' argument. This would allow them to estimate the tolerance to variation in signaling activity, which is relevant to their argument that the normal function of feedback is to ensure reliable signaling at the right level.lft double mutants exposed to rescuing, sub-rescuing, and excessive doses of inhibitor drug  The authors predict that signaling activity would be variable in the absence of feedback. Is it possible to strengthen this argument by measuring embryo to embryo variation among a population of drug-rescued Lefty double mutants (no feedback) with a quantitative measure as suggested above? Would such variation fall outside the tolerance measured in the previous experiment?lft mutants .We thank the reviewers for raising this possibility. We have performed additional experiments to address this point: Using pSmad2 immunofluorescence, we observed similar levels of signaling gradient variability in wild type and drug-rescued  mutants . This re3) One possible extension of the finding described is that the spatial requirement for Nodal signalling is not very stringent. Have the authors attempted a converse rescue experiment, i.e. to activate Nodal signaling uniformly, either with a drug or RNA injection in a cyclops;squint double mutant. Would they expect a just-right level of global signaling that rescues development?cyc;sqt double mutants (1/16 embryos are double mutants) makes such an experiment extremely challenging. We have instead extended the experiments performed in Gritsman et al. 1999, in which the Nodal co-receptor mutant MZoep, which is completely devoid of Nodal signaling, is injected with activin mRNA. Activin bypasses the requirement for oep and is able to activate the Nodal signaling pathway in a dose-dependent manner . We injected different amounts of activin mRNA into MZoep embryos at the one- to two-cell stage and observed partial rescue of some phenotypes, such as loss of trunk mesendoderm and, in one rare instance, rescue of cyclopia  Regarding the statement "Thus, an ectopic, Nodal-independent source of Lefty at the animal pole can replace endogenous, Nodal-induced Lefty at the margin": Is this confirmed by fate-mapping (with Alexa 488-dextran) of the transplanted, Lefty-expressing cells? lft1 mRNA into lft double mutant embryos and transplanted cells from these donors into membrane RFP-expressing host lft double mutants at sphere stage, as in We thank the reviewers for requesting this control experiment. Previous studies  already showed that transplanted cells tend to remain at the animal pole. To directly determine the location of transplanted cells in our experiments, we injected Alexa 488-dextran, membrane RFP, and 5) It is somewhat surprising that there is not a graded effect of loss of lefty alleles, i.e. that the embryonic phenotypes and response readouts (in situs and pSmad2) do not show a graded response, and that loss of one, two or three alleles of Lefty1/2 seem to be equivalent. Is it correct that there is no dose response? What are the phenotypes of the genetic combinations with just one functional allele?lft allele appeared largely morphologically normal . Moreover, we also observed high sensitivity of lft2 expression to alterations in Nodal levels .We thank the reviewers for pointing out this interesting observation. Embryos with even a single functional y normal , and gavl levels . We ther mutants . In the 6) In the Discussion, "While Lefty is not essential for viability per se" is an overstatement. The lefty double mutants are not viable. Although they can be experimentally rescued by drug or resupply of Lefty, this does not alter the requirement for functional Lefty during development (whether provided by expression or downstream pathway inhibition), as demonstrated by the double mutant phenotype. It is important in the interpretation of the results and the discussion of the results to uncouple the concept of dynamic feedback inhibition with the concept of the requirement of Lefty to be in the game, at least to modulate Nodal activity levels for viability.We agree with the reviewers and removed this statement to change the sentence to: “The ability to dynamically adjust pathway activity may allow the embryo to create reliable patterns in the face of signaling fluctuations and uncertain environmental conditions”.


Binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) makes neutrophils roll on and adhere to inflammatory site. Intracellular calcium bursting of adhered neutrophils is a key event for subsequent arresting firmly at and migrating into the injured tissue. But, it remains unclear how the cytoplasmic calcium signaling of the cells were modulated by the fluid shear stress. Here, we focus on mechanical regulation of P-selectin-induced calcium signaling of neutrophil-like HL-60 cells under flow.HL-60 cells were loaded with Fluo-4 AM for fluorescent detection of intracellular calcium ion, and then perfused over P-selectin-coated bottom of parallel-plate flow chamber. The intracellular calcium concentration of firmly adhered cell under flow was observed in real time by fluorescence microscopy.Force triggered, enhanced and quickened cytoplasmic calcium bursting of HL-60 on P-selectin. This force-dependent calcium signaling was induced by the immobilized P-selectin coated on substrates in absence of chemokine. Increasing of both shear stress and P-selectin concentration made the calcium signaling intensive, through quickening the cytosolic calcium release and upregulating both probability and peak level of calcium signaling.Immobilized P-selectin-induced calcium signaling of HL-60 cells is P-selectin concentration- and mechanical force-dependent. The higher both the P-selectin concentration and the external force on cell, the more intensive the calcium signaling. It might provide a novel insight into the mechano-chemical regulation mechanism for intracellular signaling pathways induced by adhesion molecules. Recruitment of polymorphonuclear leukocytes (PMNs) to vessel endothelium of injury or infection is essential for the inflammatory response process –4. PMN rP-selectin, as one of the main adhesion molecules expressed constitutively at the surface of activated vascular endothelial cells , binds p2) but did not in lower shear flow (0.2 dyn/cm2) [Besides, it was demonstrated that immune functions of leukocytes are influenced by not only chemical but also mechanical factors . Externadyn/cm2) , and T-ldyn/cm2) . HoweverHuman leukaemic HL-60 cells, the neutrophilic promyelocytes, are widely used for studying their rolling behaviour on selectins , 24, sig2 in air. RPMI-1640 medium, FBS and BSA were purchased from Sigma Chemical Co. . Streptomycin, penicillin and phosphate buffer saline (PBS) were obtained from Gibco BRL . Recombinant Human P-Selectin/CD62P Fc Chimera Protein  is a disulfide-linked homodimer, containing the Fc moiety of human IgG and the extracellular domain of human P-selectin.Human promyelocytic leukemia HL-60 cells , constitutively expressed PSGL-1 as a ligand for P-selectin, were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 10 mg/mL streptomycin and 100 units/mL penicillin at 37 °C in a humidified atmosphere of 5% CO3) was functionalized through the procedure described in our previous work [2), which was held by a hollowed silicon gasket and marked in the cover slide center, and incubated overnight at 4 °C. After removal of excessive unabsorbed P-selectin, polystyrene petri dishes were washed with PBS containing 1% BSA 3 times, and incubated in the same solution for 1 h at room temperature to block nonspecific cell adhesion. The site density of P-selectin on substrate was determined by 125I radioiodination method [125I labeled antibody was then added onto the P-selectin coated on substrate. The radiation intensity of 125I was detected by GE infinia Hawkeye 4 SPECT  after removing excessive antibody. The site densities of 0.1, 1.0 and 10 µg/mL P-selectin absorbed on polystyrene petri dish were determined to be 21, 208 and 1359 #/μm2. These P-selectin densities were selected to support the firm adhesion of HL-60 on the substrates.Flow chamber . After 10 min centrifugation at 400×g, cells were re-suspended in loading buffer and incubated further for 30 min at 37 °C until use, which allowed complete de-esterification of intracellular AM ester.Relative intracellular calcium levels were assessed by using the calcium indicator Fluo-4 acetoxymethyl (AM) ester, which was obtained from Invitrogen Life Technologies . 1 μM Fluo-4 AM was loaded into cells by incubating cells at a concentration of 1 × 106/mL HL-60 cell suspensions were perfused into flow chamber with substrates of blank (without BSA and P-selectin) or P-selectin  at 2 dyn/cm2 for 7 min. We tracked about 50 cells rolling on the bottom in the direction of flow at each experiment, and defined a firm adhesion event as the cell displacement is smaller than 10 μm (approximately a cell diameter) in observation time of 1 min.The 106/mL in buffer , 12% Ficoll (w/v) and pH 7.35). With a syringe pump , HL-60 suspension was perfused over P-selectin in a parallel-plate flow chamber at different wall shear stresses  [N = (FIC−FIB)/FIB, where FIN is the normalized cell fluorescence intensity, FIC is the mean fluorescence intensity over the cell, and FIB, the fluorescence intensity of background, is the mean of four fluorescence intensities sampled from four equidistant round domains (of 36π µm2) rung the cell with a distance of 24 µm.The labeled cells were re-suspended at a concentration of 1 × 10p < 0.05.The data were compared by one-way analysis of variance (ANOVA) and Student’s t test, and considered statistically significant if 2, the events of firm adhesion were counted one by one, and the calcium bursting of the firmly adhered HL-60 cells were examined and recorded by the fluorescence camera , which is the time interval between firmly adhering and calcium bursting , which was defined as the difference between maximum fluorescence intensity and the averaged fluorescence intensity over delay time, expressed the total release of cytosolic calcium ion  and P-selectin -coated substrates, and measured the cell activation ratio, which was defined as the percentage of calcium signaling cells in all firmly adhered cells under the field of view, to estimate the probability of calcium signaling. The typical time-courses of calcium signaling demonstrated the significant differences between different treatments Fig. a. The actes Fig. b, sugges2. It said that increasing concentration of P-selectin on substrate enhanced the calcium signaling of HL-60 cells. The peak intensity of calcium signaling increased with the immobilized P-selectin density  had almost same effects on the non-specific calcium signaling of HL-60 (on blank substrate). This phenomenon of force-enhanced calcium signaling may be relevant to the catch bond mechanism of interaction of P-selectin with PSGL-1 [In addition, it was shown that the delay time of calcium signaling of HL-60 was 201 ± 20 s at zero force and became 157 ± 13.5 s at 0.2 dyn/cm0 s Fig. d. It mea0 s Fig. d. The inh PSGL-1 .HL-60 cells are principally a type of neutrophilic promyelocyte (precursor) . The regThe P-selectin-induced calcium bursting of HL-60 might be relevant to either release of intracellular calcium stores or influx of extracellular calcium. Perhaps, the P-selecin-induced calcium bursting was referred to a pathway, along which the binding of PSGL-1 to P-selectin not only made cells be capture to substrate  but also2) made P-selectin-induced calcium signaling remarkable  because of that no tensile force acted on P-selectin/PSGL-1 complex, indicating again that force was required for the P-selectin-induced calcium signaling. High wall shear stress (≥0.6 dyn/cmble Fig. , similar dyn/cm2 . Force-e dyn/cm2 , similar dyn/cm2 . It sugg dyn/cm2 . The preHere, we demonstrated that the calcium signaling of HL-60 firmly adhered on immobilized P-selectin is P-selectin concentration- and mechanical force-dependent. High immobilized P-selectin concentration and/or external force on cell would make the calcium signaling of cell more intensive. These results might exhibit a novel insight in understanding the mechano-chemical regulation mechanism for intracellular signaling pathways induced by adhesion molecules, and be contributed to some new ideas in risk assessment, clinical diagnosis and the efficacy of inflammation and cancer treatment.


The results indicated that most of UA derivatives showed a significant inhibitory activity, especially analogues UA-O-i with the IC50 values of 0.71 ± 0.27 μM, which was more potential than other analogues and the positive control. Furthermore, molecular docking studies were also investigated to verify the in vitro study. Structure modification at the C-3 and C-2 positions of UA was an effective approach to obtain the desired ligand from UA, whose structure was in accordance with the active pocket. Besides, suitable hydrophobic group at the position of C-2 might play an important role for the docking selectivity and binding affinity between the ligand and the homology modelling protein. These results could be helpful for designing more potential α-glucosidase inhibitors from UA in the future.Ursolic acid (UA) is a major pentacyclic triterpenoid in plants, vegetables and fruits, which has been reported to have a potential anti-diabetic activity. Despite various semi-synthetic ursolic acid derivatives already described, new derivatives still need to be designed and synthesized to further improve the anti-diabetic activity. In the present study, two series of novel UA derivatives, were synthesized and their structures were confirmed. The enzyme inhibition activities of semi-synthesized analogues against α-glucosidase were screened  Diabetes mellitus (DM) is a chronic metabolic disorder, which is characterized by hyperglycemia12171S,2R,4aS,6aS,6bR,10S,12aR) -10-hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4a-carboxylic acid, 1) is abundantly present in some medicinal herbs and fruits  was applied as a catalyst. What’s more, another series of some new triterpenic analogues were also prepared from UA involved in two steps, Jones oxidation and Claisen Schmidt condensation at the position of C-3 and C-2 of UA respectively. And the bioactivities of these new analogues against α-glucosidase were screened in vitro. Furthermore, molecular docking studies were also performed with an aim to develop more potential α-glucosidase inhibitory agents. This is the first study associated with the anti-diabetic properties of these new UA analogues, in which α-glucosidase (PDB: 1UOK) was chosen as the docking target.Based on our previous studies, UA and some of its analogues have significant inhibitory activity to α-glucosidase, which might play an important role for treatment of DM and its complications261H NMR, 13C NMR, melting point (mp) and electrospray ionization mass spectrometry (ESI-MS).UA analogue compounds UA-01~UA-05 were synthesized according to 1H NMR, 13C NMR, melting point (mp) and electrospray ionization mass spectrometry (ESI-MS).UA analogue compounds UA-O-a~UA-O-j were synthesized according to p-nitrophenol (PNP) released from p-nitrophenol-α-D-glycopyranoside (PNPG) at 405 nm. All examined samples of UA analogues were dissolved in DMSO to generate the stock solution, with the concentrations ranging from 5 μM to 4000 μM. During the enzyme inhibitory assay, each sample’s stock solution was diluted with 0.1 M phosphate buffer solution (pH = 6.8), and the final concentration of enzyme solution was 0.1 U/mL. Then 10 min pre-incubation at 37 °C was conducted. After that, the substrate  was added to initiate the reaction. The reaction was incubated for 30 min at 37 °C before it was terminated by adding 1 mol/L Na2CO3 solution, and the optical density value was measured at 405 nm by using a Multimodel Plate Reader (Infinite 200). Each assay was performed three times.In this assay, α-glucosidase from baker’s yeast has been selected and used in this model according to the procedure described in the previous study with a slight modification282950 value was expressed as percentage inhibitions relative to a control assay with no inhibitor added. Acarbose was used as positive control in this assay for comparison. The IC50 values and inhibition curves were presented in 50 values of this series of UA ester analogues (UA-01~UA-05) ranged from 2.51 ± 0.02 μM to 15.23 ± 0.47 μM, in which UA-02 (IC50 = 2.51 ± 0.02 μM) and UA-04 (IC50 = 3.94 ± 0.10 μM) were better than the parent compound UA (IC50 = 5.08 ± 0.70 μM) and the positive control. And the inhibitory effect of analogue UA-01 (IC50 = 4.98 ± 0.21 μM) presented similarly with UA. However, the inhibition activity of analogue UA-03 (IC50 = 6.72 ± 0.33 μM) and UA-05 (IC50 = 15.23 ± 0.47 μM) were less than that of UA. What’s more, the results were in accordance with our previous study as wellThe enzyme activity was measured at a fixed substrate concentration, in which a series of sample concentrations were introduced. The ICEach experiment was performed in quadruplicate. The data presented representing the mean (n = 4) ± SD.aIC50 value representing the concentration that caused a 50% loss of activity.bAcarbose, positive control.50 of this series of analogues ranges from 0.71 ± 0.04 to 10.32 ± 0.55 μM. According to the enzyme inhibition results, it could be summarized that the bioactivities of analogues UA-O-b (IC50 = 10.32 ± 0.55 μM) and UA-O-c (IC50 = 7.49 ± 0.29 μM) are lower than that of other types of aldehyde with no α-H. Besides, analogue UA-O-g (IC50 = 7.87 ± 0.34 μM) with the pyrazine group at C-2 position of UA-O might decrease the α-glucosidase inhibition activity. The inhibition activities of other analogues are better than that of UA (IC50 = 5.08 ± 0.70 μM) and the positive control, especially the analogue UA-O-i (IC50 = 0.71 ± 0.04 μM) presenting the best inhibition activity than the others. What’s more, the result also implied that a proper group at the position of C-2 could increase its bioactivity of against α-glucosodase.As showed in Each experiment was performed in quadruplicate. The data presented representing the mean (n = 4) ± SD.aIC50 value representing the concentration that caused a 50% loss of activity.bAcarbose, positive control.50 = 2.47 ± 0.14 μM)50 = 10.32 ± 0.55 μM) and UA-O-c (IC50 = 7.49 ± 0.29 μM) have higher IC50 value than UA-O, implying that the length of the substituent group might have a significant effect on its activity. The longer the substituent group, the less the inhibitory activity. Meanwhile, when the substituent groups at the C-2 position were changed into phenyl group or substituent phenyl groups, the inhibitory activity against α-glucosidase could be increased dramatically. Among them, the analogue UA-O-i (IC50 = 0.71 ± 0.04 μM) could be obtained with the substituent group of p-trifluoromethyl benzaldehyde, showing a potential inhibitory activity against α-glucosidase. This result might be attributed to both the selectivity and affinity of the target site with the target compound.In the study, a total of sixteen analogues of UA  were obtained and their structure activity relationships (SAR) against α-glucosidase were deduced. The enzyme inhibition activities were decreased in varying degrees while the hydroxyl group of UA was esterified with different kinds of acyl chlorides, especially the big size of the ester group, which was according with the results of molecular docking study and our previous SAR study. This might be on account of the size of the active site of the target protein. Moreover, the α-glucosidase inhibition activity was more than doubled while the hydroxyl group of UA was oxidized into ketone  was chosen as the target protein for this docking model. And the sequence similarity is around 62.0% and the sequence identity is around 38.0%, as compared with α-glucosidaseIn this molecular docking model, the reasonable binding mode was identified by applying the homology modelled structure of α-glucosidase, in which the structure of oligo-1, 6-glucosidase from As is indicated in All of the synthesized UA derivatives were docked with the developed homology model of α-glucosidase (PDB: 1UOK). In the study, one potential analogue (UA-O-e) against α-glucosidase was presented in p-trifluoromethyl benzene group and the selectivity of the active site. Furthermore, the MOLCAD hydrogen bonding study of the binding surface revealed that two hydrogen bond donors were presented in the hydrophobic pocket while analogue UA-O-i served as an acceptor by forming two hydrogen bonds, showed in The docking study of analogue UA-O-i was depicted in in vitro α-glucosidase inhibition activity, the predicted binding free energies of all ligands docking with the target protein of α-glucosidase were calculated and presented in in vitro and the binding free energy were lower than that of UA. Furthermore, the result also indicated that the practise inhibition activity was in accordance with the theoretical results. It also suggested that the homology protein of α-glucosidase could be applied in the docking model to provide some guidance for the development of α-glucosidase inhibitor.In order to gain an insight into the relationship between the modelling study and the in vitro have been studied. Most of the UA derivatives showed better potency than UA and the positive control, acarbose. However, the bigger-sized substituent group at the position of C-3 showed a negative effect for their bioactivity. Besides, the heterocyclic substituent group condensed at the C-2 position also decreased their α-glucosidase inhibitory activity. As to analogue UA-O-i, which could be obtained by coupling UA-O with p-trifluoromethyl benzaldehyde, presented a great improvement of enzyme inhibition activity (IC50 = 0.71 ± 0.04 μM). These results could also be confirmed by the molecular docking study. The structures of the ligand and the docking pocket were the key factors for the docking binding model and affinity, which played an important role for their potential activity. In conclusion, UA new derivative condensed with a suitable aldehyde at the position of C-2 with a stable docking model could be a promising agent as potential α-glucosidase inhibitor.In summary, two series of UA derivatives were synthesized at the positions of C-2 and C-3, and their enzyme inhibitory activity assays against α-glucosidase 254 plates ; the starting material and product were detected by either viewed under UV light or treated with an ethanolic solution of p-anisaldehyde spray followed by heating.UA was supplied by Nanjing Zelang Medical Technology Co., Ltd., with a purity of over 98%. Silica gel (100–200 or 200–300 mesh) used in column chromatography was bought from Adamas Reagent Ltd. (Shanghai China) or Tsingtao Marine Chemistry Co., Ltd. Other reagents and solvents were purchased from Adamas Reagent Ltd. (Shanghai China) or other commercial suppliers in their analytically or chemically pure forms and used without purification. TLC was performed on pre-coated silica gel F1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE 400 NMR spectrometers under a standard condition; chemical shifts were measured in ppm downfield from TMS as internal standard. Melting point were tested by microscopic melting point apparatus of X-4 from Beijing Tech Instrument Co., LTD. Mass spectra were determined on an apparatus of LC-MS-2010A and the results were presented as m/z. The enzyme inhibition activity was measured by using a Multimodel Plate Reader (Infinite 200).According to our previous study1S,2R,4R,6aS,6bR,10S,12aR)-10-(benzoyloxy)-1,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA was treated with benzoic anhydride at room temperature overnight, and then purified on a silica gel column with petroleum ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound UA-01(Rf = 0.57). Yield: 60%; white powder; mp: 266–268 °C; 1H NMR  δ 7.98 , 7.48 , 7.37 , 7.19 , 5.19 , 4.68 , 2.13 , 2.02–1.76 , 1.76–1.55 , 1.54–1.40 , 1.39–1.15 , 1.03 , 0.95 , 0.89 , 0.85–0.77 , 0.73 .13C NMR  δ 182.8, 166.5, 138.2, 132.9, 131.2, 129.7, 128.47, 126.0, 81.8, 55.6, 52.8, 48.1, 47.7, 42.2, 39.7, 39.2, 39.0, 38.5, 38.3, 37.1, 36.9, 33.1, 30.8, 29.9, 28.4, 28.2, 24.3, 23.8, 23.5, 21.3, 18.4, 17.3, 17.2, 17.2, 15.7. ESI-MS m/z 559.4 [M-H]−.-10-((furan-2-carbonyl) oxy)-1,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA was treated with 2-Furoyl chloride at room temperature overnight, and then purified on a silica gel column with petroleum ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound UA-02 (Rf = 0.55). Yield: 54%; white powder; mp: 227–228 °C; 1H NMR  δ 7.57 , 7.13 , 6.49 , 5.23 , 4.72 , 2.18 , 2.06–1.81 , 1.80–1.61 , 1.59–1.45 , 1.43–1.24 , 1.08 , 0.99 , 0.94 , 0.86 , 0.77 . 13C NMR  δ 184.4, 158.8, 146.2, 145.4, 138.1, 125.8, 117.5, 111.8, 81.8, 55.5, 52.6, 48.1, 47.6, 42.0, 39.7, 39.1, 39.0, 38.4, 38.2, 37.1, 33.0, 30.7, 29.8, 28.3, 28.1, 24.2, 23.8, 23.7, 23.4, 21.3, 18.3, 17.2, 17.1, 16.9, 15.7. ESI-MS m/z 549.3 [M-H]−.-10--1,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA was treated with 2,2-Dichloroacetyl chloride at room temperature overnight, and then purified on a silica gel column with petroleum ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound UA-03 (Rf = 0.45). Yield: 66%; white powder; mp: 235–237 °C; 1H NMR  δ 5.94 , 5.24 , 4.62 , 2.19 , 2.06–1.87 , 1.76–1.65 , 1.59–1.48 s , 1.36–1.24 , 1.08 , 0.97 , 0.92 , 0.86 , 0.78 . 13C NMR  δ 183.3, 164.5, 138.2, 125.8, 85.1, 65.0, 55.5, 52.7, 48.1, 47.6, 42.1, 39.7, 39.2, 39.0, 38.3, 37.1, 36.9, 33.0, 30.8, 29.9, 28.1, 24.2, 23.8, 23.5, 23.3, 22.8, 21.3, 18.2, 17.3, 17.2, 16.7, 15.7. ESI-MS m/z 565.2 [M-H]−.-10-((4-bromo-2-fluorobenzoyl) oxy)-1,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA was treated with 4-Bromo-2-fluorobenzoylchloride at room temperature overnight, and then purified on a silica gel column with petroleum ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound UA-04 (Rf = 0.48). Yield: 58%; white powder; mp: 276–277 °C; 1H NMR  δ 7.81 , 7.34 , 5.25 , 4.74 , 2.20 , 2.07–1.98 , 1.97–1.86 , 1.81–1.67 , 1.60–1.46 , 1.42 , 1.39–1.29 , 1.26 , 1.10 , 1.00 , 0.96 , 0.87 , 0.80 . 13C NMR  δ 183.2, 163.8, 163.8, 138.2, 133.3, 127.7, 125.9, 121.0, 120.7, 83.0, 55.5, 52.8, 48.1, 47.7, 42.1, 39.7, 39.2, 39.0, 38.5, 38.1, 37.1, 36.9, 33.1, 30.8, 29.9, 28.3, 28.2, 24.3, 23.8, 23.7, 23.5, 21.3, 18.4, 17.3, 17.2, 17.0, 15.7. ESI-MS m/z 655.2 [M-H]−.-1,2,6a,6b,9,9,12a-heptamethyl-10-((4-methylpiperazine-1-carbonyl) oxy)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA was treated with 4-Methyl-1-piperazinecarbonylchloride at room temperature overnight, and then purified on a silica gel column with petroleum ether/ethyl acetate (v/v 10:1) as the eluent to obtain compound UA-04 (Rf = 0.40). Yield: 45%; white powder; mp: 127–128 °C; 1H NMR  δ 5.26 , 3.68–3.47 , 3.47–3.32 , 3.18 , 2.49–2.31 , 2.28 , 2.18 , 2.14–1.97 , 1.96–1.82 , 1.80–1.43 , 1.39–1.22 , 1.18–1.04 , 0.98–0.91 , 0.89 , 0.84 , 0.75 . 13C NMR  δ 173.2, 149.6, 137.9, 126.3, 79.0, 55.4, 54.7, 54.3, 52.9, 49.2, 47.7, 46.0, 45.1, 44.2, 42.3, 39.8, 39.2, 38.9, 38.8, 37.1, 36.0, 33.3, 30.6, 28.3, 28.0, 27.3, 24.4, 23.5, 23.4, 21.2, 18.4, 17.5, 17.0, 15.7, 15.6. ESI-MS m/z 581.4 [M-H]−.-11-((Z)-benzylidene)-1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA-O-a were prepared by Claisen Schmidt condensation of UA-O with benzaldehyde in the presence of ethanolic potassium hydroxide at room temperature in good yield. mp: 160–162 °C; 1H NMR  δ 7.61 , 7.29 , 7.23–7.13 , 6.88 , 5.36 , 2.92 , 2.35 , 2.25 , 2.19–1.95 , 1.88 , 1.78–1.64 , 1.53 , 1.47–1.33 , 1.29–1.20 , 1.12 , 0.98–0.89 , 0.86 .13C NMR  δ 207.3, 183.6, 138.5, 138.3, 137.7, 136.6, 135.8, 132.8, 130.8, 128.8, 126.3, 125.8, 125.7, 124.6, 53.4, 53.0, 48.3, 45.4, 45.3, 42.7, 42.4, 39.6, 39.3, 39.0, 36.9, 36.2, 32.4, 30.8, 29.7, 28.2, 24.3, 23.8, 23.6, 22.8, 21.3, 20.4, 19.9, 17.2, 17.0, 15.7. ESI-MS m/z 541.3 [M-H]−.-1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-11-((E) -3-phenylallylidene)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA-O-b were prepared by Claisen Schmidt condensation of UA-O with phenylacrolein in the presence of ethanolic potassium hydroxide at room temperature in good yield. mp: 170–172 °C; 1H NMR  δ 7.68 , 7.50–7.05 , 6.94 , 5.34 , 2.95 , 2.24 , 2.16 , 2.11–1.96 , 1.88 , 1.79–1.62 , 1.53 , 1.47–1.30 , 1.28–1.21 , 1.16–1.08 , 1.00–0.88 , 0.85 .13C NMR  δ 207.3, 183.8, 138.3, 137.0, 136.3, 134.9, 134.1, 134.0, 130.2, 129.7, 127.0, 125.8, 125.7, 53.4, 52.9, 48.3, 45.4, 45.3, 42.8, 42.4, 39.6, 39.3, 39.0, 36.8, 36.2, 32.3, 30.8, 29.7, 28.2, 24.3, 23.8, 23.6, 22.8, 21.3, 20.4, 17.2, 17.0, 15.7. ESI-MS m/z 567.3 [M-H]−.-11-((E)-3-(4-fluorophenyl) allylidene)-1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA-O-c were prepared by Claisen Schmidt condensation of UA-O with (E)-3-(4-fluorophenyl)acrylaldehyde in the presence of ethanolic potassium hydroxide at room temperature in good yield. mp: 172–173 °C; 1H NMR  δ 7.47 , 7.22 , 7.04 , 6.90–6.84 , 5.33 , 2.91 , 2.25 , 2.14 , 2.11–1.99 , 1.92–1.83 , 1.75–1.66 , 1.53 , 1.45–1.36 , 1.29–1.23 , 1.14–1.09 , 0.97 , 0.92 , 0.85 . 13C NMR  δ 207.3, 183.6, 163.1, 139.4, 138.3, 137.2, 133.1, 132.9, 129.0, 128.9, 125.7, 123.3, 116.1, 115.9, 53.3, 53.0, 48.3, 45.3, 45.2, 42.7, 42.4, 39.6, 39.3, 39.0, 36.8, 36.2, 32.4, 30.8, 29.7, 28.1, 24.3, 23.8, 23.6, 22.8, 21.3, 20.4, 17.2, 17.0, 15.7. ESI-MS m/z 585.4 [M-H]−.-1,2,6a,6b,9,9,12a-heptamethyl-11-((Z)-4-nitrobenzylidene)-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA-O-d were prepared by Claisen Schmidt condensation of UA-O with p-nitrobenzaldehyde in the presence of ethanolic potassium hydroxide at room temperature in good yield. mp: 172–174 °C; 1H NMR  δ 8.27 , 7.56 , 7.53 , 5.27 , 2.96 , 2.29 , 2.21 , 2.07–1.80 , 1.78–1.60 , 1.60–1.46 , 1.45–1.23 , 1.19–1.07 , 0.98–0.93 , 0.90 , 0.87 , 0.82 .13C NMR  δ 207.4, 183.4, 147.4, 142.6, 138.4, 137.4, 134.8, 130.8, 125.4, 123.8, 53.4, 52.9, 48.2, 45.6, 45.4, 44.2, 42.4, 39.6, 39.3, 39.0, 36.8, 36.6, 32.2, 30.8, 29.7, 28.1, 24.2, 23.7, 23.6, 22.9, 21.3, 20.4, 17.2, 16.9, 15.6. ESI-MS m/z 586.4 [M-H]−.-11-((Z)-3-chlorobenzylidene)-1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA-O-e were prepared by Claisen Schmidt condensation of UA-O with 3-Chlorobenzaldehyde in the presence of ethanolic potassium hydroxide at room temperature in good yield. mp: 150–151 °C; 1H NMR  δ 7.46–7.39 , 7.39–7.32 , 7.32–7.27 , 5.29 , 2.97 , 2.30–2.17 , 2.08–1.83 , 1.79–1.61 , 1.59–1.23 , 1.13 , 1.07–1.00 , 0.99–0.93 , 0.90 , 0.86 , 0.82 .13C NMR  δ 207.6, 183.8, 138.1, 135.9, 135.2, 134.4, 131.2, 130.1, 129.7, 128.4, 127.9, 125.5, 53.2, 52.7, 48.1, 45.3, 45.3, 43.8, 42.2, 39.5, 39.1, 38.9, 36.7, 36.5, 32.1, 30.6, 29.5, 28.0, 24.1, 23.6, 23.5, 22.7, 21.1, 20.3, 17.1, 16.7, 15.4. ESI-MS m/z 575.2 [M-H]−.-1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-11-((6-(trifluoromethyl) pyridin-3-yl) methylene)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA-O-f were prepared by Claisen Schmidt condensation of UA-O with 6-(Trifluoromethyl)nicotinaldehydein the presence of ethanolic potassium hydroxide at room temperature in good yield. mp: 168–169 °C; 1H NMR  δ 8.83 , 7.90 , 7.76 , 7.52 , 5.30 , 3.00 , 2.34 , 2.24 , 2.10–1.93 , 1.88 , 1.80–1.64 , 1.59–1.48 , 1.48–1.26 , 1.19 , 1.15 , 1.09–1.03 , 0.98 , 0.93 , 0.90 , 0.84 .13C NMR  δ 207.1, 183.6, 150.8, 147.2, 138.5, 138.4, 138.1, 134.9, 131.9, 125.4, 121.6, 120.4, 53.3, 52.9, 48.3, 45.5, 45.4, 44.5, 42.4, 39.6, 39.3, 39.0, 36.8, 36.5, 32.2, 30.8, 29.7, 28.1, 24.2, 23.8, 23.6, 22.8, 21.3, 20.4, 17.2, 16.9, 15.7. ESI-MS m/z 610.3 [M-H]−.-1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-11-(pyrazin-2-ylmethylene)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA-O-g were prepared by Claisen Schmidt condensation of UA-O with Pyrazine-2-carbaldehyde in the presence of ethanolic potassium hydroxide at room temperature in good yield. mp: 162–163  °C; 1H NMR  δ 8.72–8.60 , 8.43 , 7.40 , 5.29 , 3.54 , 2.43 , 2.23 , 2.13–1.95 , 1.94–1.83 , 1.79–1.62 , 1.58–1.46 , 1.43–1.35 , 1.34–1.23 , 1.17 , 1.13 , 0.96 , 0.91 , 0.89 , 0.83 .13C NMR  δ 208.2, 183.0, 151.8, 147.6, 144.3, 142.7, 141.3, 138.2, 130.4, 125.8, 53.3, 53.0, 48.2, 45.6, 45.3, 44.9, 42.4, 39.6, 39.3, 39.0, 36.9, 36.3, 32.3, 30.8, 29.7, 28.2, 24.3, 23.8, 23.6, 22.7, 21.3, 20.5, 17.2, 16.9, 15.8. ESI-MS m/z 543.3 [M-H]−.-1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-11-(thiazol-2-ylmethylene)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA-O-h were prepared by Claisen Schmidt condensation of UA-O with thiazole-2-carbaldehyde in the presence of ethanolic potassium hydroxide at room temperature in good yield. mp: 148–149 °C; 1H NMR  δ 8.01 , 7.64 , 7.50 , 5.33 , 3.37 , 2.34 , 2.24 , 2.20–2.10 , 2.09–1.96 , 1.88 , 1.81–1.65 , 1.59–1.48 , 1.44–1.24 , 1.17 , 1.12 , 0.96 , 0.92 , 0.84 .13C NMR  δ 207.6, 183.6, 163.9, 144.6, 138.2, 137.7, 127.9, 125.8, 121.6, 53.1, 52.9, 48.2, 45.5, 45.4, 45.4, 42.4, 39.6, 39.3, 39.0, 36.9, 36.3, 32.2, 30.8, 29.9, 28.2, 24.3, 23.8, 23.6, 22.6, 21.3, 20.5, 17.2, 16.8, 16.1. ESI-MS m/z 548.3 [M-H]−.-1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-11-((Z)-4-(trifluoromethyl) benzylidene)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA-O-i were prepared by Claisen Schmidt condensation of UA-O with p-Trifluoromethylbenzaldehyde in the presence of ethanolic potassium hydroxide at room temperature in good yield. mp: 171–172 °C; 1H NMR  δ 7.67 , 7.51 , 5.27 , 2.98 , 2.34–2.14 , 2.09–1.97 , 1.94 , 1.86 , 1.78–1.61 , 1.59–1.46 , 1.46–1.18 , 1.14 , 1.08–0.99 , 0.96 , 0.90 , 0.87 , 0.81 . 13C NMR  δ 207.6, 183.7, 139.6, 139.6, 138.3, 136.0, 135.8, 130.4, 125.6, 125.50, 125.46, 53.4, 52.8, 48.2, 45.5, 45.4, 44.1, 42.4, 39.6, 39.3, 39.0, 36.8, 36.6, 32.2, 30.8, 29.7, 28.1, 24.2, 23.7, 23.6, 22.9, 21.3, 20.4, 17.2, 16.9, 15.6. ESI-MS m/z 609.4 [M-H]−.-1,2,6a,6b,9,9,12a-heptamethyl-11-((Z)-4-methylbenzylidene)-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-carboxylic acid . According to the general procedure, UA-O-j were prepared by Claisen Schmidt condensation of UA-O with p-methyl benzaldehyde in the presence of ethanolic potassium hydroxide at room temperature in good yield. mp: 142–143 °C; 1H NMR  δ 7.53 , 7.35 , 7.23 , 5.28 , 3.03 , 2.38 , 2.31–2.24 , 2.22 , 2.09–1.93 , 1.86 , 1.78–1.61 , 1.59–1.45 , 1.45–1.33 , 1.26 , 1.14 , 0.96 , 0.91 , 0.87 , 0.81 . 13C NMR  δ 207.9, 183.8, 138.8, 138.2, 137.8, 133.3, 133.1, 130.6, 129.4, 125.8, 53.3, 52.9, 48.2, 45.5, 45.3, 44.3, 42.4, 39.6, 39.3, 39.0, 36.9, 36.4, 32.3, 30.8, 29.8, 28.2, 24.3, 23.8, 23.6, 22.8, 21.5, 21.3, 20.5, 17.2, 16.9, 15.6. ESI-MS m/z 555.4 [M-H]−.(p-nitrophenyl-α-D-glucopyranoside) were dissolved in 0.1 M phosphate buffer, pH 6.8. 10 μL of each synthesized analogue (1 mg/mL in DMSO) was pre-incubated with 8 μL of α-glucosidase at 37 °C for 10 min. A 100 μL of substrate solution was then added to the reaction mixture, which was further incubated at 37 °C for 30 min. Then, the reaction was terminated by adding 100 μL of 1 M Na2CO3 solution. Enzymatic activity was quantified by measuring the absorbance at 405 nm with a use of a Multimodel Plate Reader (Infinite 200). The percentage of inhibition was calculated by using [(A0 − A1)/A0] × 100%, where A0 was the absorbance without the sample, and A1 was the absorbance with the sample. The IC50 value was determined from a plot of the percentage of inhibition versus the sample concentration. Acarbose was used as the standard control and the experiment was performed in duplicate.The α-glucosidase inhibition assay was performed according to the method of WorawalaiThe molecular minimization of UA analogues were built by use of the Sybyl molecular modelling package, version 2.0 . All structures of tested analogues were minimized with the Tripos force field, and the hydrogen atoms were added. Powel optimized the energy gradient with the maximum times to be 1000 times when the energy convergence criterion reached 0.005 kcal/mol by using Gasteiger–Hückle charges. Ligand-protein docking was performed by the Surflex-Dock in Sybyl 2.0. The crystal structure of α-glucosidase was retrieved from RCSB Protein Data Bank (PDB: 1UOK). Biopolymer module was then used to repair the crystal structure of the protein termini treatment, fix side chain amides, residues and add charges. The potent UA analogues docking with the α-glucosidase selected catalytic pocket of acarbose as active site. The active pocket was formed by computing, the others being the default settings.How to cite this article: Wu, P.-P. et al. Synthesis and biological evaluation of novel ursolic acid analogues as potential α-glucosidase inhibitors. Sci. Rep.7, 45578; doi: 10.1038/srep45578 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Rapid eye movement (REM) sleep behavior disorder (RBD) is characterized by repeated episodes of REM sleep-related vocalizations and/or complex motor behaviors. Definite diagnosis of RBD is based on history and polysomnography, both of which are less accessible due to the lack of trained specialists and high cost. While RBD may be associated with disorders like narcolepsy, focal brain lesions, and encephalitis, idiopathic RBD (iRBD) may convert to Parkinson’s disease (PD) and other synucleinopathies in more than 80% of patients and it is to date the most specific clinical prodromal marker of PD. Identification of individuals at high risk for development of PD is becoming one of the most important topics for current PD-related research as well as for future treatment trials targeting prodromal PD. Furthermore, concomitant clinical symptoms, such as subtle motor impairment, hyposmia, autonomic dysfunction, or cognitive difficulties, in subjects with iRBD may herald its phenoconversion to clinically manifest parkinsonism. The assessment of these motor and non-motor symptoms in iRBD may increase the sensitivity and specificity in identifying prodromal PD subjects. This review evaluates the diagnostic accuracy of individual rating scales and validated single items for screening of RBD and the role and accuracy of available clinical, electrophysiological, imaging, and tissue biomarkers in predicting the phenoconversion from iRBD to clinically manifest synucleinopathies. Rapid eye movement (REM) sleep behavior disorder (RBD) belongs to the parasomnias, which is a group of disorders characterized by paroxysmal motor and behavioral events occurring exclusively during sleep. RBD is defined by dream enactment and complex motor behaviors during REM sleep and loss of normal REM sleep muscle atonia  as detected by polysomnography (PSG) . It is oSeveral reports have shown, that idiopathic RBD (iRBD) may convert to Parkinson’s disease (PD) or other synucleinopathies, including multiple system atrophy (MSA) and Dementia with Lewy Bodies (DLB), in more than 80% of patients , 7. WhilParkinson’s disease and other synucleinopathies are complex progressive multiorgan disorders affecting different neurotransmitter systems across the central, peripheral, and autonomic nervous systems, with Lewy bodies, Lewy neurites or glial cytoplasmic inclusions as their pathological hallmark , 10. In Thus, the aim of this review is to summarize the current evidence on the diagnostic accuracy of screening instruments for identification of iRBD and to summarize the available data on other symptoms and signs, including presence of subtle motor dysfunction, NMS, neuroimaging findings, electrophysiological and tissue biomarkers, in predicting the phenoconversion of iRBD to PD and other synucleinopathies.We searched PubMed for all relevant articles until October 2017, using main search terms “RBD” or “REM sleep behavior disorder” combined with terms: “idiopathic,” “Parkinson*,” “DLB,” “MSA,” “synuclein*,” “screen*,” “questionnaire,” “scale,” “inventory,” “accuracy,” “sensitivity,” “specificity,” “conversion,” “predict*,” “motor,” “UPDRS,” “speech,” “gait,” “non-motor,” “NMS,” “olfactory,” “hyposmia,” “autonomic,” “cardial,” “urinary,” “constipation,” “sleepiness,” “EDS,” “cognit*,” “imaging,” “DaT,” “PET,” “MRI,” “sonography,” “SPECT,” “substantia nigra,” “serum,” “CSF,” “cerebrospinal,” and “biopsy.” Only studies in English language and related to human studies were included. All titles and abstracts were reviewed for relevance. References were supplemented by selection from the reference lists of identified papers, as well as personal knowledge of emerging literature.per se  and low correlation with the RBD severity scale   , 18.p = 0.019), with more dream-related movements and falling out of bed compared to females (p = 0.029) compared to RBD patients without dementia  that may not be appropriate in non-Chinese populations .The RBDSQ was developed as a simple screening tool for RBD . The scar = 0.51, p < 0.001) (p < 0.001) in the general population over 2 years, however, the consistency of score changes over this period was low .The scale has shown good reliability with Cronbach’s alpha of 0.77–0.89 , 25 and < 0.001) . On the < 0.001) . Sensiti< 0.001)  have shoRBDSQ scores did not correlate with age  or duratThere are nine studies that have evaluated the diagnostic accuracy of the RBDSQ in PSG-confirmed RBD populations with sensitivity ranging from moderate to excellent and specificity ranging from low to excellent based on study population and cutoff used  when correlated with PSG . Thus, tPublished data on reliability, validity, with exception of criterion validity (see diagnostic accuracy below), and sensitivity to change are not available.Question 1 of this questionnaire has been found to have an excellent sensitivity and acceptable to excellent specificity for determining the presence of RBD when compared to PSG results , 37 see. In furtThis questionnaire has been used by the group that developed it in two studies with aging and predominantly male populations  , 37. TheThe questionnaire is simple and easy to complete. However, it is limited to patients with a partner that sleeps in the same room or has slept in the same room. The MSQ has a single question that screens for RBD, clinimetric properties of the questionnaire and this single question require further study.The Innsbruck RBD-I  is a shop = 0.011)  . There ip < 0.05). Five of the 9 items had an AUC > 0.7 and were thus included in the final version of the questionnaire . Only the response patterns to the REM sleep items discriminated between patients and controls   . These fde novo PD patients  of 3.9 among available predictive markers to date . HoweverMultiple prodromal clinical NMS have been studied in iRBD patients to identify subjects with an increased risk and time frame of conversion to synucleinopathy. Most of these NMS are recognized as risk or prodromal markers for development of PD .p < 0.001), with no predictive value in those with normal olfaction   . Mean di = 0.91)  in theirOlfactory loss is significantly higher in iRBD subjects compared to healthy controls, severity is reported to be intermediate between controls and PD patients. Olfactory dysfunction may predict early conversion to clinically manifest synucleinopathy and seems to be independent from other NMS. The time frame of conversion needs to be furtherly investigated.Neurodegenerative synucleinopathies are frequently accompanied by autonomic dysfunction: constipation, urinary frequency, urinary incontinence, erectile dysfunction, and orthostatic blood pressure drop.Patients with iRBD experience significantly more gastrointestinal, urinary, and cardiovascular functioning problems in comparison to healthy controls , 61, 65 In a prospective observational study comparing 32 converted RBD subjects versus 59 non-converted patients , autonomIn a multicenter study of iRBD subjects, the 93 convertors scored significantly higher on SCOPA-AUT than the 186 non-convertors (14.1 ± 6.1 vs 12.0 ± 6.9) . The difFereshtehnejad et al.  calculatThe recent study by Li et al.  confirmeQuestionnaire-assessed autonomic dysfunction is significantly more frequent and severe in iRBD compared to controls. Orthostatic drop, erectile dysfunction, and constipation independently increase the LR for the conversion to synucleinopathy. There is not enough evidence to support the predictive role of urinary dysfunction in iRBD subjects specifically. Severity of autonomic dysfunction in iRBD and PD patients was found comparable in some of the studies.Cardiac autonomic testing during wakefulness showed that only 5/14 patients (36%) with RBD (10 iRBD) had normal results in all traditional autonomic tests during wakefulness, and all had reduced heart rate variability (HRV) during sleep . While rHeart rate variability, peripheral sympathetic system activity, autonomic responses to arousal and leg movements in sleep and orthostatic testing show higher prevalence and severity of autonomic dysfunction in iRBD subjects compared to healthy controls. Autonomic evaluation during sleep could identify impaired cardiac dysautonomia in RBD patients earlier than the traditional tests during wakefulness. There is insufficient evidence to calculate diagnostic accuracy of electrophysiological testing in prediction of phenoconversion to manifest synucleinopathy.p < 0.0001). Those who had ESS scores greater than 8 at the time of RBD diagnosis had higher probability to convert to parkinsonism and dementia, both from RBD onset and from RBD diagnosis within the follow-up of 1–15 years (median 3 years) (p = 0.01). EDS significantly predicted an increased risk of developing neurodegenerative diseases  after adjusting for age, sex, body mass index, current depression, OSA, and PLM. Further analyses demonstrated that EDS predicted the conversion to PD  but not dementia  (p = 0.038) based on the non-motor Symptom Questionnaire (Excessive daytime sleepiness (EDS) is associated with the later stages of PD and there are studies documenting higher risk of PD in men reporting daily napping . Arnulf 3 years) . A more 3 years) . Of thes44–4.97) . Similarionnaire .On the other hand, a recent study by Postuma et al.  did not Excessive daytime sleepiness seems to be more common in iRBD subjects compared to controls. EDS seems to increase the likelihood of phenoconversion to synucleinopathies, although this has not been confirmed in all studies. Further evidence is needed to confirm diagnostic accuracy and time frame of phenoconversion. There is insufficient evidence to consider insomnia as a symptom indicating higher risk of phenoconversion.A case–control study  showed tA prospective cohort study of anxiety predictors showed more symptoms of anxiety at baseline that increased over time in patients with RBD compared to early stage untreated PD .Depressive symptoms do not identify RBD subjects with the risk for conversion to synucleinopathy. There is not enough evidence for depression and anxiety to be considered risk factors for neurodegeneration in RBD patients.The abnormality of color vision is one of the NMS in patients with PD, and the severity of axial motor symptoms is closely related to visual dysfunction .In a 10-year prospective study of an iRBD cohort over the age of 55 years old, color vision deficit (Farnsworth-Munsell 100-hue test) increased the HR of neurodegenerative disease conversion by 3.1-fold . Color vColor vision might be helpful in predicting phenoconversion in iRBD subjects. The data are, however, limited and need confirmation in further studies.Significant differences in cognitive functioning  between iRBD and healthy controls have been found –86. The Cognitive impairment is a sign of future phenoconversion but there is not enough evidence to recommend optimal set of neuropsychological examinations for precise estimation of the disease evolution.Imaging of the nigrostriatal dopaminergic pathway typically consists in measuring dopamine transporter (DAT) density  and posiPresynaptic PET and SPECT have been shown to identify PD and related disorders in very early stages. Striatal DAT uptake has been shown to decrease with expected pattern of disease progression from healthy controls to RSWA on PSG to manifest RBD to PD . Compare18F-fluorodeoxyglucose PET shows altered RBD-related regional cerebral metabolism. Increased metabolism has been found in the pons, thalamus, medial frontal and sensorimotor areas, hippocampus, supramarginal and inferior temporal gyri, and posterior cerebellum, and decreased activity in occipital and superior temporal regions . In anotAn elevated PD-related covariance pattern in resting-state metabolic brain imaging with 18F-fluorodeoxyglucose PET has been found in subjects with iRBD compared to healthy controls. For individual subjects with RBD, final phenoconversion status has been predicted using a logistic regression model based on a PD-related covariance pattern expression and subject age at the time of imaging .Resting-state functional magnetic resonance imaging (rs-fMRI) may detect preclinical alterations in brain network functioning. Connectivity measures within resting-state networks differentiated both iRBD and PD from controls, indicating its potential as an indicator of early basal ganglia dysfunction. iRBD was indistinguishable from PD on rs-fMRI despite obvious differences on DAT-SPECT. Basal ganglia connectivity is a promising biomarker for the detection of early basal ganglia disturbance and may help to identify patients at risk of developing PD in the future .Hyperechogenicity of the substantia nigra (SN) on TCS is reported in the vast majority of PD patients and only in 10% of healthy subjects. Several studies have applied TCS in iRBD and SN hyperechogenicity was found in about 35–55% of iRBD subjects and in about 70% of iRBD patients with comorbid depression .Longitudinal follow-up studies showed that 36% of iRBD subjects with SN hyperechogenicity at baseline phenoconverted within 2.5 years  and 42% Combining TCS with other examinations might increase accuracy of prognosis. Combination of TCS with FP-CIT-SPECT revealed 100% sensitivity and 55% specificity to predict subsequent phenoconversion within 2.5 years . AssessmN = 31), PD (N = 26), DLB (N = 6), and controls  at the time of diagnosis. Also, patients who converted had higher tonic REM% , without any difference in phasic REM% . On Cox regression analysis, adjusting for age and sex, having tonic REM > 50% was associated with a HR of 1.88 for development of neurodegenerative disease (p = 0.039)  .These electrophysiology results are interesting pathophysiologically but, to date, do not have a role for predicting phenoconversion of an individual subject with iRBD.p = 0.039) (p = 0.05) in another recent investigation  (p = 0.004) and the MAPT rs12185268  were associated with RBD in different models. Kaplan–Meier survival analysis in a subset of RBD patients (n = 56), demonstrated that homozygous carriers of the USP25 rs2823357 SNP had progressed to synucleinopathies faster than others .Several gene mutations and single nucleotide polymorphisms (SNPs) have been studied in relation to iRBD. The OR for the GBA p.E326K mutation carriers to have probable RBD (RBDSQ score ≥ 6) was 3.13 in one study (= 0.039) , while ttigation . On the tigation , 116. Thtigation . Melanomtigation . As far ctively) . In anotctively)  evaluatiThere is insufficient evidence to support the role of genetic testing in identifying subjects at higher risk of RBD or for predicting phenoconversion to synucleinopathies.Only a limited number of studies evaluating serum and cerebrospinal fluid (CSF) biomarkers in RBD subjects have been published. Anderson et al.,  found noAs far as serum biomarkers, hypercholesterolemia is possibly a predictor of lower conversion rate in iRBD according to one follow-up study  and highThere is insufficient evidence to support the use of CSF and serum biomarkers as predictors for iRBD conversion to synucleinopathy.in vivo confirmation of Lewy pathology in patients with iRBD. A single study has evaluated the presence of 129-phosphorylated a-synuclein (p-a-syn) in colonic biopsies, with positive findings in 4/17 (24%) iRBD patients and none of the healthy controls, resulting in excellent specificity, but low sensitivity (p < 0.01) was reported in another study  and sensitive to change is the RBDQ-HK. The remaining scales were either not studied appropriately from the clinimetric point or view, or the results were not satisfactory. These limitations mostly regard the validity of the scales and their sensitivity to change.Several clinical, imaging and electrophysiological progression markers have been identified in iRBD subjects. For some of these markers, like subtle motor impairment, time from deviation of normal values to a diagnosis of synucleinopathy has been estimated in the range of 4.2–9.8 years. Several other promising markers of phenoconversion, such as olfactory loss, autonomic dysfunction, cognitive impairment, imaging markers, including presynaptic dopaminergic nuclear medicine imaging, rs-fMRI or TCS, as well as peripheral nerve tissue biopsies have been identified. Although these markers seem to predict future conversion to synucleinopathies, reported results have not always been uniform, and there is not enough evidence to support a specific set of evaluations or procedures for precise estimation of disease conversion or progression. Moreover, it is very likely that a single marker will not be sufficient to predict future conversion of iRBD to a manifest synucleinopathy and that a combination of different clinical, imaging and other markers, such as confirmation of presence of pathological a-synuclein aggregation in peripheral tissues, will be necessary for accurate estimation of future disease progression. In this regard, the recently published MDS research criteria for prodromal PD  have yieFuture studies are needed in order to predict conversion of iRBD into specific types of synucleinopathies. Moreover, prospective studies are necessary to confirm and explore the role of individual prodromal markers and their combination in predicting conversion from iRBD to PD, DLB, or MSA.All authors made substantial contributions to the conception of the work, drafted sessions of the manuscript or revised it critically for important intellectual content, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.The authors declare no financial disclosures or other conflicts of interest in regards to this manuscript. The handling Editor declared a past-authorship with one of the authors SM.