The surge of artificial intelligence (AI) in medicine stands on a lengthy, and frequently reticent, buildout. AI is an all-encompassing term for techniques that enable the software to mimic human intelligence (HI) using logic, convolutional neural networks (CNN), decision trees, search algorithms, machine learning (ML) and deep learning (DL) . ML creaTherefore, of special interest is the paper by Choi et al., who aimed to develop and validate MLAs to predict the 5-year risk of starting biologic agents in IBD patients, published in this Special Issue of Journal of Clinical Medicine (JCM) “AI in Medical Practice/Gastroenterology, Hepatology and Endoscopy” . To attaThe appearance of automated lesion recognition endoscopy software combined with the boost in robotic techniques in surgery  has captAI has already supported wireless capsule endoscopy (CE) platforms in the diagnosis of small bowel (SB) bleeding, as well as medical practitioners in the assessment of patients with liver fibrosis or chronic pancreatitis suspected of cancerous progression. AI has been continuously explored for the last decade to facilitate the detection of pre-cancerous and cancerous mucosal GI lesions. In GI endoscopy, AI has become an even hotter topic in recent years, as the majority of manufacturers started to update their systems to accommodate the new needs. Undoubtedly, AI has come to ’democratize’ GI endoscopy by improving patients’ outcomes irrespective of place/time of care, enhancing the professional experience for clinicians, and assisting healthcare systems with capacity issues. Yang et al.  significAI has been instrumental in big data analyses in the era of -omics technologies, targeting microbiomes, metabolomes and other complex living ecosystems. In an era where information overload has become the norm , the numFollowing the outbreak of the novel COVID-19 disease, there has been a drastic reduction in elective healthcare services, including diagnostic endoscopy services, as these carry the risk of communicable viral disease transmission. These reductions for all but the most urgent clinical work have a detrimental impact not only on patients but also on practicing physicians. As the diagnostic and therapeutic capabilities of GI endoscopy strongly correlate with the skills of the operator, the altered learning curves required to achieve proficiency in endoscopic techniques play roles. Moreover, increasing numbers of physicians/nurses present burnout syndromes. Furthermore, increasing numbers of medical staff in GI units report endoscopy-related muco-skeletal injuries. Last but not least, at a time of the pandemic, there is a constant shortage of human and material resources. These all lead to the worsening of patients’ and physicians’ perspectives on contemporary GI endoscopy practice. Those factors seriously interfere with the successful implementation, adoption and continuation of daily medical routines. They also have a negative effect on the number of diagnostic screening and therapeutic procedures. Therefore, various AI solutions to enhance lesion detection and short-/long-term outcome prediction must be rapidly implemented in daily GI practice. It will benefit both physicians and their patients, as well as supporting the overstrained healthcare systems. Of further relevance are the recent advancements in other medical technologies. With patients’ growing expectations for high-tech precision diagnostics, GI endoscopy has also been successfully utilized for the innovative detection/treatment of GI pathologies. For this purpose, robotic gastroscopes and colonoscopes with advanced functionalities have been developed and mastered ,9. The i


The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity. Drosophila and worm genomes, whereas vertebrate genomes harbor four TCF/LEF genes , thereby inhibiting its DNA-binding affinity  is the substrate recognition component of an E3 ubiquitin ligase complex . Inactivrcinomas , 17. Welrcinomas , 19. In rcinomas , 21. Addrcinomas . pVHL bircinomas . Severalrcinomas . Specifircinomas . Additiorcinomas . Furtherith pVHL . A recenith pVHL . Given tin vitro and in vivo. Unexpectedly, our results demonstrated that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in vitro and in vivo. We revealed that VBP1 directly binds to all four TCF/LEF family members and to pVHL. Intriguingly, either overexpression or knockdown of VBP1 in-creased the interaction between TCF/LEFs and pVHL, which then promoted TCF/LEFs protein degradation via the proteasomal pathway. Using a pVHL deficient cell line, we uncovered that VBP1 regulated TCF/LEF protein stability via pVHL. Together, our study provides insights into the mechanism of protein stability regulation of all TCF/LEF family members by VBP1.In the present study, we investigated the role of VBP1 in regulating Wnt/β-catenin signaling both in vitro and in vivo. We first used the TOPFlash reporter, a well-established Wnt/β-catenin signal-responsive reporter, which contains tandem repeats of the TCF/LEF response element, to examine the role of VBP1 in the Wnt/β-catenin pathway in cultured cell lines. Transfection of HEK293T cells with a plasmid encoding the FLAG-tagged VBP1 protein significantly decreased basal Wnt/β-catenin reporter activity (A). In addition, overexpression of VBP1 decreased the expression level of several Wnt/β-catenin direct target genes including AXIN2, CCND1, and CDK2 in HEK293T cells (B). Interestingly, the expression of DKK1, which is a repressor of the Wnt/β-catenin pathway was not changed by VBP1 overexpression. To determine whether Wnt/β-catenin signaling was affected by VBP1 in vivo, the capped mRNA of VBP1 was generated and injected into 1–2-cell stage zebrafish embryos, and then the Wnt/β-catenin reporter activity was measured at 6 hpf (hours postfertilization). As shown in Fig. 1C, forced expression of VBP1 mRNA inhibited endogenous Wnt/β-catenin activity in zebrafish embryos. Taken together, these results suggested that overexpression of VBP1 inhibited endogenous Wnt/β-catenin signaling activity in vitro and in vivo.Because pVHL regulates the Wnt/β-catenin pathway at multiple levels and VBP1 serves as a binding protein to cooperate with pVHL in many biological processes , 25, 26,activity A. In add3T cells B. IntereD). Overexpression of VBP1 inhibited Wnt/β-catenin reporter activity induced by Wnt3a, BIO, β-CatΔN, and VP16-Tcf7l1ΔN in HEK293T cells . Similarly, overexpression of VBP1 inhibited Wnt/β-catenin reporter activity induced by Wnt3a and VP16-Tcf7l1ΔN in zebrafish embryos . Taken together, the above results implied that VBP1 is likely to inhibit Wnt/β-catenin activity at the TCF/LEF level. To further confirm the inhibitory effect of VBP1 on Wnt/β-catenin signaling in vivo, we performed epistatic analysis between VBP1 and the core components of the Wnt/β-catenin signaling pathway on the formation of the dorsoventral axis in zebrafish embryos. Because the VBP1/Vbp1 amino acid sequence is highly conserved in vertebrates from fish to mammals . Collectively, these results suggested that overexpression of VBP1/Vbp1 inhibits Wnt/β-catenin signaling at TCF/LEF level in vitro and in vivo.To further confirm whether VBP1 indeed regulated the Wnt/β-catenin signaling pathway, we investigated the genetic interaction between VBP1 and the core components of the Wnt/β-catenin signaling pathway in HEK293T cells and zebrafish embryos. Induction of Wnt/β-catenin signals by Wnt3a, 6-Bromoindirubin-3′-oxime (BIO) (GSK3β inhibitor) , 29, conFig. 1 mammals , we co-is report , injectiA). Using these cell lines, we tested whether VBP1 knockdown affected the expression of Wnt/β-catenin target genes. As shown in Fig. 2B, knockdown of VBP1 significantly reduced the expression level of Wnt/β-catenin target genes, including AXIN2, LEF1, CCND1, and CDK2 in HEK293T cells. Similar results were observed in U2OS cells . These results suggested that knockdown of VBP1 reduced endogenous Wnt/β-catenin signaling in vitro. We next determined the effect of Vbp1 knockdown on the Wnt/β-catenin signaling activity in vivo using a transgenic zebrafish Wnt/β-catenin reporter line, also known as the TOPdGFP reporter line. GFP expression in the zebrafish TOPdGFP reporter line was controlled by a promoter with multiple TCF/LEF response elements, which allowed the determination of Wnt/β-catenin activity and the identification of responsive cells through the expression of GFP protein . In contrast, the GFP intensity was dramatically reduced in the Vbp1 MO-injected embryos (D and E). The decrease of fluorescence intensity was not because of morphological changes in the midbrain, which were indicated by the normal expression of midbrain marker otx2 . These results suggested that depletion of Vbp1 in zebrafish embryos decreased the endogenous Wnt/β-catenin activity in vivo. Collectively, depletion of VBP1/Vbp1 reduced endogenous Wnt/β-catenin signaling both in vitro and in vivo.To delineate the role of endogenous VBP1 in Wnt/β-catenin signaling, HEK293T and U2OS cell lines stably expressing VBP1 shRNA were established. The knockdown efficiency of VBP1 was confirmed by Western blotting analysis, and each shRNA greatly reduced the VBP1 protein levels A. Using  protein . To ascee 24 hpf ; howevere 24 hpf . Coincide 24 hpf , suggest embryos . SubsequF). Knockdown of VBP1 in cultured cells dramatically reduced the Wnt/β-catenin activity induced by these molecules . These results implied that depletion of VBP1 reduced Wnt/β-catenin signaling at the TCF/LEF transcriptional level. We further tested whether the effect of knockdown of VBP1/Vbp1 on Wnt/β-catenin signaling is specific. Because the effect of VBP1/Vbp1 on Wnt/β-catenin signaling is evolutionarily conserved between zebrafish and humans, we introduced zebrafish Vbp1 into VBP1-depleted human cells to test whether zebrafish Vbp1 could rescue the reduced Wnt/β-catenin activity in cultured human cells. As expected, expression of shRNA-resistant zebrafish Vbp1 significantly rescued the reduced Wnt/β-catenin activity by VBP1 knockdown in the background of LiCl treatment and β-CatΔN overexpression . These results indicated that the reduction of Wnt signaling by VBP1 knockdown was specific and that the effect of knockdown of VBP1/Vbp1 on Wnt/β-catenin signaling was evolutionarily conserved. Taken together, our results demonstrated that depletion of VBP1/Vbp1 attenuated Wnt/β-catenin signaling at the TCF/LEF transcriptional level.We next investigated at which level VBP1 knockdown reduced Wnt/β-catenin activity. Again, Wnt/β-catenin activity was induced by Wnt3a, LiCl (GSK3β inhibitor) , β-CatΔNFig. 2A). In addition, overexpression of VBP1 reduced the endogenous TCF7L2 protein levels in HEK293T cells (B). This effect was restored after the addition of the proteasome inhibitor MG132 (C), indicating that TCF7L2 degradation promoted by overexpressed VBP1 depended on the proteasomal pathway.Given that either overexpression or knockdown of VBP1 attenuated Wnt/β-catenin signaling at the TCF/LEF level and VBP1 is a binding protein of pVHL, which is a subunit of an E3 ubiquitin ligase complex, we speculated that VBP1 might regulate TCF/LEF protein stability. To this end, we co-overexpressed VBP1 and TCF/LEFs in HEK293T cells. When cells were cotransfected with plasmids containing Myc-tagged TCF/LEFs and FLAG-tagged VBP1, exogenous TCF/LEF protein levels were reduced A. In add3T cells B. This eor MG132 C, indicaVBP1 knockdown on TCF/LEF protein levels. Knockdown of VBP1 decreased the protein levels of endogenous TCF7L1 and TCF7L2 in HEK293T cells (D). In contrast, the expression of TCF7L1 and TCF7L2 mRNA was not changed upon the depletion of VBP1 when measured by qRT-PCR analysis (E). These results implied that TCF7L1 and TCF7L2 protein levels were regulated by VBP1 through a posttranslational mechanism. To determine whether knockdown of VBP1 accelerated degradation of TCF7L2, we measured TCF7L2 t1/2 in HEK293T cells treated with cycloheximide (CHX), a protein synthesis inhibitor. The t1/2 of TCF7L2 was significantly shortened when VBP1 was knocked down (F and G). Because the HEK293T cell line was regarded as a Wnt-off cell line, we want to determine whether knockdown of VBP1 regulates TCF/LEF protein stability in Wnt-activated colon cancer cells. Knockdown of VBP1 also decreased TCF7L1 and TCF7L2 protein levels in Wnt-activated HCT116 colon cancer cells, which harbor an oncogenic mutation in β-catenin (H). These data suggested that knockdown of VBP1 reduced TCF/LEF protein stability in both active and inactive Wnt/β-catenin signaling status. Furthermore, the reduction of TCF7L2 protein levels by VBP1 depletion was restored by MG132 treatment in both HEK293T and HCT116 cells (I and J), indicating that VBP1 depletion–mediated TCF7L2 degradation was also dependent on the proteasomal pathway. Protein degradation by the proteasomal pathway may be ubiquitin-dependent and ubiquitin-independent (K). Taken together, these results implied that VBP1 depletion promoted degradation of the TCF7L2 in both basal and activated Wnt/β-catenin activity status via proteasomal pathway.We next examined the effect of 3T cells D. In conanalysis E. These ked down , F and G-catenin H. These 16 cells , I and Jependent . To addrependent , but faiependent K. Taken A). Additionally, when FLAG-tagged VBP1 was overexpressed in HEK293T cells, it formed a complex with endogenous TCF7L2 and pVHL (B). Furthermore, purified GST–VBP1 protein pulled down all four Myc-tagged TCF/LEF members . Likewise, a direct protein-protein interaction among VBP1, endogenous TCF7L2, and FLAG-tagged pVHL was further confirmed by the GST pulldown assay . These results indicated that VBP1 directly interacted with TCF/LEF and pVHL in HEK293T cells. We next assessed whether VBP1 and Tcf7l1 interact with each other in living HeLa cells using a previously established bimolecular fluorescence complementation (BiFC) assay  in the cytoplasm, suggesting that VBP1 and Tcf7l1 did interact with each other in living cells. Subsequently, we generated a series of TCF7L2 domain-deleted mutants to map the region of TCF7L2 involved in the interaction with VBP1 . By Co-IP assay, we found that the TLE/Groucho binding region–deleted mutant of TCF7L2 exhibited rather weak interactions with VBP1 compared with WT TCF7L2, high mobility group box–deleted, or C-terminal region–deleted mutant . This suggests that the TLE/Groucho binding region in TCF7L2 may play an important role for its interaction with VBP1.To understand the mechanism underlying the regulation of TCF/LEF protein stability by VBP1, we first tested the possible interaction between VBP1 and TCF/LEFs. The co-immunoprecipitation (Co-IP) assay detected VBP1 in the immunoprecipitates of four Myc-tagged TCF/LEF family members in HEK293T cells A. Additiand pVHL B. FurtheC) assay . As showB). In addition, VBP1, as a pVHL binding protein, participates and regulates pVHL-related biological processes. We speculated that VBP1-mediated regulation of TCF/LEF stability was related to pVHL function. We, therefore, overexpressed or knocked down VBP1 protein in VHL-deficient clear-cell renal carcinoma 786-O cells. As expected, TCF7L2 protein levels were not changed in 786-O cells with either overexpression or knockdown of VBP1 (A and B). These data suggested that pVHL is required for VBP1 to regulate the stability of TCF/LEFs. We next performed a series of Co-IP analyses to detect the association between endogenous TCF7L2 and pVHL with VBP1 overexpression or knockdown in different cell lines of distinct Wnt/β-catenin activity. We observed that the association between endogenous TCF7L2 and pVHL increased under the condition of either VBP1 overexpression or knockdown in HEK293T cells (C and D). Similarly, knockdown of VBP1 in Wnt-activated HCT116 colon cancer cells also increased the interaction between TCF7L2 and pVHL (E). These results indicated that the presence of pVHL is required for VBP1 to regulate the stability of TCF/LEFs and that alteration of VBP1 protein levels increases the association between TCF/LEF and pVHL, which may promote the degradation of TCF/LEF proteins.Of note, pVHL was also present in the complex containing VBP1 and TCF/LEF B. In add of VBP1 , A and B3T cells , C and Dand pVHL E. These AXIN2, CCND1, and CDK2, but did not reduce the expression of Wnt/β-catenin signaling pathway negative feedback gene, DKK1 (F and G). These results suggested that VBP1 might have physiological roles in colorectal cancers. It is well-known that activation of Wnt/β-catenin signaling promotes proliferation of colorectal cancer cell. It is interesting to know whether alternation of VBP1 decreases the proliferation of colorectal cancers. We, therefore, examined the effect of VBP1 knockdown on proliferation of HCT116 cells using a colony formation assay. Our results indicated that depletion of VBP1 inhibited the proliferation of HCT116 cells, which was highlighted by the significant reduction in the number of colonies (H and I). These results suggest that VBP1 plays a physiological role in the cell proliferation of colorectal cancers.Because colorectal cancers often display overactivated Wnt/β-catenin signaling and VBP1 regulates the protein stability of TCF/LEFs in HCT116 colon cancer cells, we next determined whether VBP1 has a physiological role in colorectal cancer cells. The alterations of Wnt/β-catenin target genes were measured in HCT116 cells after overexpression or knockdown of VBP1. Similar with the effect of VBP1 in HEK293T cells, either overexpression or knockdown of VBP1 in HCT116 cells reduced the expression of Wnt/β-catenin target genes, including ne, DKK1 , F and Gcolonies , H and Iin vitro and in vivo.In the present study, we show that VBP1 plays an important role in regulating the Wnt/β-catenin signaling pathway, which is known as an important signaling pathway that controls developmental processes in the embryo and regulates adult homeostasis and stem cell self-renewal . Transcrin vivo and in vitro. Likewise, VBP1/Vbp1 antagonized the Wnt/β-catenin–induced dorsalizing activity in zebrafish embryos. Surprisingly, depletion of VBP1 also attenuated Wnt/β-catenin activity in vivo and in vitro. Furthermore, we demonstrated that VBP1 regulated the Wnt/β-catenin signaling pathway at the TCF/LEF-dependent transcriptional level by genetic interaction analysis. These data presented here raised an interesting question regarding lower or excessive VBP1 reducing Wnt/β-catenin signaling activity. To answer this question, we showed that either overexpression or knockdown of VBP1 reduced the stability of TCF/LEFs, which might have resulted from increased association between pVHL and TCF/LEFs when VBP1 was overexpressed or knocked down. Consequently, this reduced the activity of Wnt/β-catenin signaling. Similarly, a recent study revealed that either overexpression or knockdown of SRPK1 decrease the association between Akt and PHLPP1 and then interfere with PHLPP-mediated dephosphorylation of Akt, resulting in the promotion of cancer , β-catenin (sc-7199), VBP1 (sc-390465), and pVHL (sc-135657) were purchased from Santa Cruz Biotechnology. Antibodies against TCF7L2 (2569), GFP (2555), and HA (3724) were obtained from Cell Signaling Technology. The antibody against TCF7L1/L2 (ab12065) was provided by Abcam. The antibody against GAPDH (D110016) was purchased from BBI Life Sciences . The antibody against histone H3 (P30266) was obtained from Abmart. The FLAG M2 antibody (F1804) was provided by Sigma-Aldrich. The antibody against β-actin (abs137975) was obtained from Absin Bioscience. Small molecules CHX (66-81-9) and BIO (667463-62-9) were purchased from Sigma-Aldrich. MG132 (HY-13259) was from MedChemExpress.The plasmids pCS2-6×Myc-Tcf7, pCS2-6×Myc-Tcf7l1, pCS2-6×Myc-Tcf7l2, pCS2-6×Myc-Lef1, pCDNA3-Myc-VN-Tcf7l1, pCDNA3-Myc-VN-Tcf7l1ΔN, and pCDNA3-Myc-VN-Tcf7l1ΔNLS were gifts from Dr. Wei Wu . The plasmid pGEX-2T (28-9546-53) was obtained from GE Healthcare. pCS2-FLAG-VBP1, pCS2-FLAG-VBP1-VC, pGEX-2T-VBP1, pCDNA3.1-TCF7L2-HA, and pCS2-VP16-Tcf7l1ΔN were generated by PCR subcloning. Briefly, the human VBP1 ORF was amplified by PCR from HEK293T cell cDNA using the following primers: F, 5′-CGCGGATCCGCGCCACCAT-GGCGGCCGTTAAGGACAG-3′; R, 5′-CCGGAATTCTTA-TGCTTTGTTCTTGGTAGAGTC-3′. The resultant product was subcloned into the pCS2-GFP, pCS2-FLAG, or pGEX-2T, respectively. pCS2-GFP and pCS2-FLAG were obtained by inserting GFP or FLAG tag into pCS2+ vector . TCF7L2 was amplified by PCR from RD cell cDNA using the following primers: F, 5′-CGGGATCCGCCACCATGCCGCAGCTGAACGGCGGT-GGAG-3′; R, 5′-CGGAATTCCTATTCTAAAGACTTG-GTGACGAGCG-3′. The resultant product and mutant were subcloned into the pCDNA3.1-HA . Vp16 and dnTcf7l1 ORF were amplified by PCR and subcloned into pCS2+ vector. Primers are shown below: dnTcf7l1-F, 5′-CCGGAATTCGATGCAGGATGCAGCA-TTCTTCAAGGG-3′; dnTcf7l1-R, 5′-CGCGTCTAGATCAGTCACTGGATTTGGTCACC-3′; Vp16-F, 5′-CCGG-AATTCGCCGCCATGGCTCCAAAGAAGAAGCGT-3′; Vp16-R, 5′-GGCGAATTCCCACCGTACTCGTCAATTCCAAGGGCAT-3′. All plasmids were verified by sequencing.WT (Tübingen) and TOPdGFP zebrafish strains were used in this study. The embryos were incubated in Embryo Medium at 28.5 °C. The embryos were strictly staged according to standard methods . All exp2. 786-O cells were maintained in RPMI medium (HyClone) with the same supplement mix. The cell lines were analyzed with short tandem repeat profiling by ShCellBank . The contamination by mycoplasmas in culture cells was tested by EZ-PCR Mycoplasmas Detection Kit  every three months. Transfections of cells with plasmids were performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions.HEK293T, HeLa, HCT116, and U2OS cell lines were purchased from ATCC and cultured in DMEM (HyClone) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin at 37 °C in 5% CO5 cells were seeded in a 12-well plate and then transfected with TOPFlash reporter and specific expression constructs. Transfection efficiency was normalized by cotransfection with a Renilla reporter. The cells were lysed with 1× Passive Lysis Buffer (Promega). The in vivo luciferase assay was performed as reported previously  were lysed in Passive Lysis Buffer. TOPFlash/Renilla luciferase assays were performed using the Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer's instructions.For cell-based luciferase assays, 8 × 10eviously , 45. In vbp1. The sequences were MO1 (5′-ATTCAAATGTAGTCCAT-CTGTCTTT-3′) and MO2 (5′-CAATGAGCAGGTGATGA-TTTCATCT-3′). The sequence of control MO was: 5′-CCT-CTTACCTCAGTTACAATTTATA-3′. The target sequence (−216 bp to 381 bp) was fused to GFP-coding sequences and subcloned into pCS2+ to generate a reporter for MO-knockdown efficiency test.Two antisense MOs  were designed for targeting the translation start site of zebrafish Capped mRNAs were synthesized using the mMESSAGE mMACHINE SP6 kit  according to the manufacturer's instructions. All morpholinos and mRNAs were injected into the yolks of zebrafish embryos at the 1–2-cell stage and then embryos were raised to the indicated stages.in situ hybridization was performed as previously described (vbp1 and otx2 riboprobes were synthesized with T7 RNA polymerase (Promega). Stained embryos or live embryos were mounted in glycerol or 5% methylcellulose respectively, and images were taken using a Nikon SMZ1500 microscope.Whole-mount escribed , 45. TheGAPDH, forward (F): 5′-TCGACAGTCAGCCGCATCTTCTT-3′ and reverse (R): 5′-GCGC-CCAATACGACCAAATCC-3; LEF1, F: 5′-CAGTCATCCCGAAGAGGAAG-3′ and R: 5′-AGGGCTCCTGAGAGGTT-TGT-3′; AXIN2, F: 5′-CTGGCTTTGGTGAACTGTTG-3′ and R: 5′-AGTTGCTCACAGCCAAGACA-3′; CCND1, F: 5′-CCATCCAGTGGAGGTTTGTC-3′ and R: 5′-AGCGTATCGTAGGAGTGGGA-3′; CDK2, F: 5′-CTCTTCCCCTCATCAAGAGCTA-3′ and R: 5′-ATTTGCAGCCCAGGAGGATT-3′; DKK1, F: 5′-GTGCAAATCTGTCTCGCCTG-3′ and R: 5′-CTGATGACCGGAGACAAACAG-3′; TCF7L1, F: 5′-GTCAACGAGTCGGAGAACCA-3′ and R: 5′-TCTCACTTCGGCGAAATAGTC-3′; TCF7L2, F: 5′- CCTCGGCAGAGAGGGATTTAGCTG-3′ and R: 5′-GAGCCCTCCATCTTGCCTCTTG-3′. Gene expression analysis was performed using the comparative ΔΔCT method with GAPDH normalization. Template-free negative controls were included in each experiment.Total RNA was isolated using RNAiso plus reagent (Takara Bio), and 2.0 μg of total RNA was used as a template for reverse transcription. The cDNA was synthesized using the M-MLV reverse transcriptase (Promega). Quantitative PCR analyses were carried out with the iTaq SYBR GREEN Supermix and iCycler apparatus (Bio-Rad). Each sample was analyzed in duplicate, and qPCR primers were as follows: 6 cells were seeded in a 35-mm dish and the cells were harvested at 24 h posttransfection; then the cells were lysed with radioimmune precipitation assay buffer  supplemented with protease and phosphatase inhibitors on ice for 15 min. Lysates were then centrifuged at 12,000 × g for 10 min at 4 °C. Protein samples were mixed with 5× Laemmli sample buffer, and proteins were separated by SDS/PAGE, transferred to PVDF membranes, and incubated with primary antibodies overnight at 4 °C after blocking with 5% nonfat milk. Primary antibodies were detected with HRP-conjugated secondary antibody (Beyotime Biotechnology). For Co-IP experiments, 6 × 106 cells were seeded in a 100-mm dish and harvested at 48 h posttransfection. The cells were then lysed in IP lysis buffer containing 50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 10% glycerol, 1% Triton X-100, and protease and phosphatase inhibitors. Cleared cell lysates were incubated with the appropriate antibody (1–2 μg) overnight at 4 °C, followed by a 6-h incubation with protein A/G agarose beads at 4 °C. In all cases, the immune complexes bound to protein A/G beads were washed four times with IP lysis buffer and detached from the agarose using SDS loading buffer for immunoblot analysis.Immunoblot and Co-IP were performed as described previously . In briein vivo ubiquitylation assay was performed with hot lysis-extracted protein lysates based on the protocol previously described  with 2 mmN-ethylmaleimide and protease inhibitors, and lysates were boiled for 10 min and then diluted with nine volumes of dilution buffer . The samples were incubated at 4 °C for 30–60 min with rotation and precleared by centrifugation at 12,000 × g for 5 min before performing immunoprecipitation.The escribed . BrieflyEscherichia coli BL21 competent cells were transformed with GST or GST-VBP1 and then induced with 0.1 mm isopropyl β-d-thiogalactopyranoside at 37 °C for 5 h. The cells were collected by centrifugation, washed once with ice-cold PBS, and then lysed by sonication in lysis buffer  on ice. The GST or GST–VBP1 extract was then mixed with GSH Sepharose 4B Beads  overnight on a rotator at 4 °C. These mixtures were centrifuged, and the precipitates were diluted with an equal amount of indicated cell lysates of HEK293T and rotated for 2 h at 4 °C. After extensive washing with the lysis buffer, the precipitated proteins were eluted using SDS/PAGE. The bound proteins were detected by indicated antibodies or Coomassie Blue staining.Xenopus Tcf7l1, Tcf7l1ΔN, and Tcf7l1ΔNLS were fused, respectively, with the amino  terminal of Venus. For immunofluorescence staining, 1 × 105 cells were grown on coverslips inside a 6-well plate. After transfection, the cells were fixed with 4% paraformaldehyde, then permeabilized for 5 min at room temperature with PBS containing 0.25% Triton X-100 and blocked with 3% BSA in PBS. The cells were then incubated with anti-Myc antibody overnight at 4 °C. Goat anti-mouse Cy3 was used as a secondary antibody. After 4′,6-diamidino-2-phenylindole incubation, the cells were mounted in mounting medium. Images were acquired using a Leica SP8 confocal microscope.BiFC assays were performed as previously described . Venus f6 HEK293T cells were seeded in a 60-mm dish with 4 ml of DMEM containing 10% FBS without antibiotic. Next day, pCMV-dR8.2 dvpr, pCMV-VSV-G, the lentivirus vector GV248 containing the GFP reporter gene and the puromycin resistance gene, and the shRNA sequence were cotransfected into the packaging cell line by Lipofectamine 2000. After 18 h, the transfection medium was replaced with fresh DMEM + 10% FBS, and medium containing the lentivirus was harvested after 72 h. Target cells were infected with filtered lentivirus; the infection was carried out in the presence of polybrene (8 μg/ml). At 72 h postinfection, 1.0 μg/ml of puromycin was added to the infected culture plates. The medium was changed every 2–3 days. Cells without integration of transfected plasmid died, whereas cells that had undergone plasmid integration survived at day 7 posttransfection. The surviving cells were allowed to expand in the culture plate, and when they reached a high confluence, these cells were cryo-preserved as a polyclonal line.For shRNA-transduced experiments, vectors named GV248 were purchased from Genechem . VBP1 shRNA-1 sequence 5′-GGAAGACCTTGACTTTCTTCGAG-3′ and VBP1 shRNA-2 sequence 5′-CAAGGATGACTCTACCAAGAACA-3′ were used. Lentivirus was produced in HEK293T cells. Briefly, 2 × 105) were seeded in 6-well tissue culture plates and then incubated overnight. After 12–18 h of incubation, the cells were cultured with CHX at a concentration of 100 μg/ml and incubated for various hours. The cells were harvested at the indicated time points. The lysates were prepared and subjected to immunoblotting analysis. The protein stability was determined by the percentage of TCF7L2 normalized to that of GAPDH at an indicated point compared with that at the initial point.Cells  and cultured in DMEM-high glucose supplemented with 10% FBS for 14 days. Culture medium was changed every 2 days. After most of the cell clones had expanded to >50 cells, the cells were washed with PBS and then fixed with 4% paraformaldehyde for 15 min, and staining was performed with 0.05% crystal violet for 20 min at room temperature. The excess crystal violet was washed with dHt test or one-way analysis of variance followed by Tukey's post-test. A value of p < 0.05 was considered statistically significant. Unless otherwise indicated, all experiments contained three replicates per condition.All graphs were generated using GraphPad Prism5 Software. Values are presented as the means ± S.D. Statistical analyses were performed using a two-tailed, unpaired Student's jfzhou@ouc.edu.cn).All the data arecontained within this article and in the supporting information. All the data are to be shared upon request (Jianfeng Zhou, Ocean University of China,


Curcuma longa, exhibits anti-tumor activity in various cancers. Although the effects of Curcumin on HCC have been elucidated, the underlying mechanism remains unclear. In the present study, Curcumin was demonstrated to inhibit the proliferation of HCC cells via inducing cell cycle arrest and apoptosis. Several previously reported lncRNAs related to tumorigenesis were chosen for examination of their expression profiles, and lincROR was found to be the most down-regulated in the Curcumin-treated HCC cells. Furthermore, Curcumin was found to decrease β-catenin expression and induce the inactivation of Wnt/β-catenin signaling. Therefore, Curcumin suppressed tumor growth through a lincROR/β-catenin regulatory pattern. In conclusion, our results demonstrated that Curcumin suppressed the cell proliferation via the down-regulation of lincROR and inactivation of Wnt/β-catenin signaling, suggesting that it may be a potential anti-cancer candidate for HCC patients with activated Wnt/β-catenin signaling.As one of the leading causes of cancer-related death in the world, hepatocellular carcinoma (HCC) has continued to attract growing attention in recent decades. The use of traditional Chinese herbs in medicine has been practiced for thousands of years, and holds the potential of being a possible treatment for HCC. Curcumin, a bioactive ingredient derived from  Curcuma longa, which has been used for hundreds of years  is the third most frequent cause of cancer-related death in the world . Althougof years . Mountinof years . HoweverIn recent years, non-coding RNAs, especially long non-coding RNAs (lncRNAs), have attracted more and more attention in research fields. They have been identified as important and powerful regulators in multiple diseases, including carcinogenesis . Series via inducing cell cycle arrest and apoptosis in HCC. Further investigation of underlying mechanisms demonstrated that lincROR was down-regulated and thus induced the inactivation of Wnt/β-catenin signaling. Therefore, our study provides a novel molecular mechanism of Curcumin in liver cancer progression and helps to generate a promising therapeutic candidate for cancer patients with aberrantly activated Wnt/β-catenin signaling.In the present study, Curcumin was found to suppress tumorigenesis Curcumin was purchased from sigma laboratory  with purity over 99%.2. All the following experiments were repeated in triplicate, if no special indication is mentioned.Two HCC cell lines, SMMC7721 and Huh-7, and an immortalized non-tumorigenic cell line, LO2, were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin  in a humidified incubator at 37°C with 5% COThe SMMC7721 and Huh-7 cells were seeded in a 96-well plate and treated with various concentrations of Curcumin for 24, 48, and 72 hours. 10μl methylthiazoletetrazolium (MTT) solution (5mg/ml) was incubated for another four hours. Then the medium was removed, and 100μl dimethylsulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was measured at 570nm with a Multiskan FC plate reader .Cells were seeded in a 6-well plate at 500 cells/well and incubated with 16μM Curcumin for two weeks. After being fixed with methanol, the colonies were stained with Giemsa for 20 min. ImmunoSpot analyzers  were used to quantify the colonies.Cells were incubated with 16μM Curcumin for 48 hours and collected for flow cytometry examination. The apoptotic cells were quantified with an FITC-labeled Annexin V/propidium iodide (PI) Apoptosis Detection Kit , as mentioned before .Cells were treated with 16μM Curcumin for 48 hours, and then were fixed in 70% ethanol. Then, the samples were stained with the dye from the Cell Cycle Detection Kit (KeyGEN), and the cells were subjected to flow cytometry examination .The SMMC-7721 cells with lincROR overexpression were generated using the retrovirus-mediated gene delivery system as previously described . BrieflyHCC cells were seeded in 12-Well plates and co-transfected with a TOPflash luciferase reporter (1.25ug) by Lipofectamine 3000 . With 28-hour Curcumin treatment, luciferase activity was determined using the luciferase reporter assay system  . Each exTotal RNA was extracted by Trizol reagent , and the cDNA was generated with PrimeScript RT Reagent Kit . The qPCR analyses were conducted using a power up SYBR Green PCR master mix (Thermo Fisher Scientific) on a Light-Cycler480 System . The primers were listed in The total proteins were extracted using RIPA solution. Nuclear protein fraction was extracted by the nuclear and cytoplasmic extraction kit . The proteins were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes . After blocking, PVDF membranes were incubated with specific antibodies against β-catenin , GADPH , and Lamin A/C  overnight, followed by the HRP-labelled corresponding IgG . Finally, the chemiluminescence  was used to visualize the bands and the quantitative densitometries were determined with Image J.After treatment with Curcumin for 72 hours, HCC cells were incubated with the anti-β-catenin antibody  overnight. They were then incubated with anti-rabbit IgG-Alexa Fluor 594  at 1:2000 dilution in the dark at 37°C for one hour. After the cell nuclei were labeled with DAPI , images were captured using a Zeiss Axiophot 2 microscope.Data are presented as mean±SEM. Two-tailed unpaired Student's t test was employed to determine the statistical significance of differences. Differences were considered significant when P-values <0.05.Curcumin has been reported to inhibit cell proliferation in multiple cancers . To valivia the induction of cell cycle arrest and apoptosis.In the subsequent experiments, the Curcumin-treated HCC cells were subjected to flow cytometry examination for cell cycle analysis. As shown in Many lncRNAs have been demonstrated to serve as critical regulators in tumorigenesis . To idenThe Wnt/β-catenin signaling is an important mechanism for cell proliferation, while its abnormal activation could lead to the development of various cancers . To deteIn our previous studies, lincROR has been demonstrated to activate Wnt/β-catenin signaling so as to promote osteogenesis in human mesenchymal stem cells . In thisCurcuma longa and has been used as a spice in India for centuries  bring new light for the investigation of TCM's mechanisms. In addition to miRNAs, long or large noncoding RNAs (lncRNAs) with a length of over 200 bases have recently been identified as novel regulators of gene activity . A numbevia inactivating the canonical Wnt pathway.As is well known, the canonical Wnt/β-catenin pathway plays a significant role in many cellular functions including organ formation, stem cell renewal, and cell survival . AberranSeveral lncRNAs have been demonstrated to exert their function through activating/inactivating Wnt/β-catenin signaling. For example, lncRNA H19 could activate the Wnt/β-catenin pathway to mediate the epithelial to mesenchymal transition (EMT) and migration in colorectal cancer . lncRNA-In summary, our results demonstrated that Curcumin plays an anti-tumor function through the linc-ROR/β-catenin regulatory pattern in HCC. This study elucidates a new mechanism of Curcumin in HCC and sheds light on developing a novel therapeutic for HCC patients, especially for patients with high levels of Wnt/β-catenin signaling. But more interestingly, although Curcumin is considered as a nutraceutical in foods and supplement products, intake of Curcumin from daily foods could be not sufficient for the anti-tumor activity because of its poor solubility and chemical instability, which results in low bioavailability. Therefore, we will try to develop a strategy to improve curcumin's bioavailability in the next study, which may facilitate the extension of its usage in clinical practice in the future.The datasets generated for this study are available on request to the corresponding authors.J-FZ and W-MF spearheaded and supervised all the experiments. J-FZ and W-MF designed the experiments. YL, C-JS, JS, and F-WZ conducted the experiments. F-FP provided technical support. W-MF, YL, C-JS, JS, and J-FZ analyzed the data and prepared the manuscript.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.


SBE (MaSBE) and six SBE (MbSBE) genes in the banana A- and B-genomes, respectively, which could be classified into three distinct subfamilies according to genome-wide identification. Systematic transcriptomic analysis revealed that six MaSBEs and six MbSBEs were expressed in the developing banana fruits of two different genotypes, BaXi Jiao  and Fen Jiao , among which MaSBE2.3 and MbSBE2.3 were highly expressed. Transient silencing of MaSBE2.3 expression in banana fruit discs led to a significant decrease in its transcription, which coincides with significant reductions in total starch and amylopectin contents compared to those of empty vector controls. The suggested functional role of MaSBE2.3 in banana fruit development was corroborated by its transient overexpression in banana fruit discs, which led to significant enhancements in total starch and amylopectin contents. A number of transcription factors, including three auxin response factors (ARF2/12/24) and two MYBs (MYB3/308), that interact with the MaSBE2.3 promoter were identified by yeast one-hybrid library assays. Among these ARFs and MYBs, MaARF2/MaMYB308 and MaARF12/MaARF24/MaMYB3 were demonstrated via a luciferase reporter system to upregulate and downregulate the expression of MaSBE2.3, respectively.Starch branching enzyme (SBE) has rarely been studied in common starchy banana fruits. For the first time, we report here the molecular characterization of seven  As the main component of the total starch in the storage organs of the abovementioned plant species, amylopectin is primarily catalyzed by soluble starch synthase, debranching enzyme, and starch branching enzyme (SBE)8. Of these three classes of major enzymes, SBE mainly controls the branching pattern of amylopectin and influences the amylopectin yield and, ultimately, the nutritional value of those plants11.Plant starch, which consists of two glucan polymers, linear amylose and branched amylopectin, is a major supplier of human nutrition and calories. Starch is commonly found in cereal grains and potato tubersSBEs have been isolated in several higher plant species based on genomic data: three have been identified in maize (Zea mays), rice (Oryza sativa), sorghum (Sorghum bicolor), barley (Hordeum vulgare), and potato (Solanum tuberosum); two have been identified in Arabidopsis ; six in cassava (Manihot esculenta); and seven in wheat (Triticum aestivum)15. Furthermore, SBEs can be generally classified into no less than three groups—SBEI, II, and III, each of which typically contains a C-terminal all-beta domain and an alpha-amylase domain16. SBEI condenses amylose to long glucan chains13, while SBEII is mainly associated with the biosynthesis of amylopectin using short glucan chains as a substrate17. However, the functional role of SBEIII remains ambiguous despite reports of its increased activity in the process of wheat grain development18.Variable numbers of SBEs in both amylopectin biosynthesis and plant development. For instance, the temporal expression of SBE1 was found to be responsible for amylopectin accumulation in apple fruits and in maize seedling development19. SBEII expression is involved in nut development in chestnut (Castanea mollissima)20 and seed development in rice21. A study on the kidney bean (Phaseolus vulgaris) mutant pvsbe2 suggested that SBEII activity is important for both amylopectin biosynthesis and maturation during the middle and late stages of seed development22. Further, the expression of OsSBEIIa was found to regulate the sugary endosperm trait during development and maturity in the Sugary Endosperm rice mutant23. In maize, SBEIIa-defective mutants exhibit a leaf-senescence phenotype as the result of the reduction in the proportion of branched starch24. Taken together, all of these studies imply that the expression of SBE is a critical factor in amylopectin biosynthesis and plant growth and development.A number of expression and functional studies have further revealed the multifunctional aspect of SBE expression have been identified. In maize, the expression of numerous SBE genes was upregulated when ZmNAC36 was transiently overexpressed25, while the protein levels of maize SBEs were significantly reduced in the endosperm-specific TF opaque2 mutant and prolamin-box-binding factor (PBF) mutant26. In rice, OsbZIP58 was found to upregulate SBE1 expression through its interaction with the SBE1 promoter27. Taken together, all of these studies indicated that SBE expression is regulated by the coordination of multiple TFs.A number of transcription factors (TFs) regulating 28. Amylopectin accounts for approximately 70–80% of the total starch content on a dry weight basis in unripe banana fruits29. The biosynthesis and accumulation of amylopectin in banana fruit is of crucial importance in terms of fruit quality, yield, and nutritional value. Genome-wide identification of key candidate genes associated with amylopectin biosynthesis is therefore necessary to enhance the yield and improve the quality of banana fruits31. In the current study, we report the identification of seven and six distinct SBE members from the banana A- and B-genomes, respectively, which are henceforth referred to as MaSBE and MbSBE. The molecular characteristics, phylogenetic relationships, conserved domains, gene structures, and spatiotemporal expression patterns were investigated in the two different banana genotypes. The potential functionality of MaSBE2.3 was studied through both transient virus-induced gene silencing (VIGS) and overexpression in banana fruit discs. Three auxin response factors (ARF2/12/24) and two MYBs (MYB3/308) that regulate MaSBE2.3 expression were identified using the yeast one-hybrid (Y1H) method and luciferase (LUC) activity analysis. Together, these studies may enhance our understanding of the members of the SBE gene family involved in amylopectin biosynthesis and fruit development and pave the way for the genetic improvement of the quality, yield, and nutritional traits of banana.Banana is a popular fresh starchy fruit worldwide and constitutes the principal staple food in several African countriesM. acuminata, AAB, cultivar Fenjiao) and BaXi Jiao , which were planted at the Danzhou Banana Germplasm Centre . BX fruits are characterized by their high yield, superior quality, and long shelf-life, whereas FJ fruits are characterized by their good flavor, rapid ripening, and strong disease resistance. According to the published classification of banana fruit developmental stages by Ram et al.32, we selected three representative stages corresponding to pulp initiation at 0 day after inflorescence emergence from the pseudostem (DAF), pulp growth (20 DAF), and maturation (80 DAF) for SBE temporal expression analysis. Following harvest at 80 DAF, BX fruits were stored for 0, 8, and 14 days post-harvest (DPH), while FJ fruits were stored for 0, 3, and 6 DPH prior to expression analysis of SBE during fruit ripening. These fruits can also be distinguished by their color: peel green, peel yellowish green, and peel yellow. A spatial gene expression analysis of SBEs was also performed on select banana tissues, including root, leaf, and fruit tissues, at 80 DAF. These expression studies were conducted for three replicates.The two banana genotypes used in this experiment were Fen Jiao  database5 and Musa balbisiana  database7. The putative amino acid sequences of SBE from A. thaliana, T. aestivum, Z. mays, O. sativa, Solanum lycopersicum, S. tuberosum, and Vitis vinifera were retrieved from The Arabidopsis Information Resource, the wheat genome, the Maize Genetics and Genomics database, the Rice Genome Annotation Project, the Sol Genomics Network, the Potato Genome Sequence Consortium, and the grapevine genome, respectively. All bioinformatic analytical databases and websites used are listed in Table A. thaliana, T. aestivum, Z. mays, O. sativa, S. tuberosum, S. lycopersicum, and V. vinifera searches, were analyzed using the BLAST software. The accession numbers of the SBEs from the abovementioned plant species are presented in Table http://downloads.fyxm.net/Clustal-x-58923.html) and MEGA 5.0 software based on 1000 bootstrap replicates31.SBE amino acid sequences were obtained from the SBEs were calculated using ExPASy, and the data are presented in Table SBE genes was deduced by using the Gene Structure Display Server. The MaSBE promoter was obtained from the banana A-genome database within the Banana Genome Hub. The predicted transcription start sites and cis-acting elements were identified by searching the Berkeley Drosophila Genome Project database and the PlantCARE website, respectively.The isoelectric point and molecular mass of the Various banana plant organs, including the roots, leaves, and fruits, at 80 DAF in both BX and FJ genotypes were collected for spatial transcriptomic analysis. The pulp of banana fruits at various developmental and ripening stages, including that of BX and FJ at 0, 20, and 80 DAF, that of BX at 8 and 14 DPH, and that of FJ at 3 and 6 DPH, were collected for transcriptome sequencing at different time points.RNA-sequencing (RNA-seq) libraries were constructed using total RNA samples prepared by the use of a plant RNAout Kit . Deep sequencing was performed by using the GAII platform  according to the protocol provided by the manufacturer. The adapter sequences and low-quality sequences were removed by using the FASTX toolkit and FastQC, respectively. After mapping the clean reads to both the A- and B-genome banana databases, transcriptome assemblies were constructed by using Cufflinks. The gene expression level was assessed as reads per kilobase pair of transcripts per million reads (RPKM). The DESeq package was applied to screen differentially expressed genes. There were two technical replicates and three biological replicates.SBEs revealed by transcriptome sequencing were further identified through qRT-PCR using an Mx3000P system . The primer pair quality was verified according to melting curve analysis, with amplification efficiencies ranging from 0.9 to 1.1, and agarose gel electrophoresis (Table Actin (GenBank accession No. EF672732) and UBQ2 (GenBank accession No. HQ853254) were used as the internal reference genes. The relative gene expression level was calculated according to the 2CT−ΔΔ method33. There were three replicates included.The spatiotemporal expression patterns of banana is Table . Actin (MaSBE2.3 (2502 bp in length) was amplified by PCR and verified by DNA sequencing  together with pRTV1. Surface-sterilized BX banana fruit (80 DAF) discs that were 1–2 mm thick were then vacuum infiltrated with the Agrobacterium solution (OD600 = 0.6) prior to cocultivation on Murashige and Skoog (MS) media at 30 °C for 0, 1, 2, or 3 days35, followed by iodine–potassium iodide (I2-KI) staining36. In addition to I2-KI staining, assessment of the transcript levels of the downregulated genes and the measurements of total starch and amylopectin contents in these agroinfiltrated banana fruit discs were also carried out. Biologically independent replicates were assessed in triplicate.For VIGS vector construction, the entire coding region of MaSBE2.3 was inserted into a pCAMBIA1304 vector  behind the CaMV 35S promoter in the sense orientation using the restriction enzymes Bgl I and Spe I. Following confirmation by DNA sequencing, the recombinant pCAMBIA1304-MaSBE2.3 plasmid was transformed into A. tumefaciens GV3101 cells. The cells of the A. tumefaciens strain were diluted to an OD600 of 0.60 in infiltration buffer ethanesulfonic acid, 10 mM MgCl2), in which the BX banana fruit discs (80 DAF) were soaked34. Following a period of culture on MS media at 30 °C for 3 days, the agroinfiltrated banana fruit discs were subjected to I2-KI staining, transcription analysis, and total starch and amylopectin content evaluations. The biologically independent transformations were performed in triplicate.For the transient overexpression vector, the entire coding region of 37, after which the material was ground into powder. One hundred milligrams of powder was washed in 5 mL of 80% ethanol and centrifuged for 5 min at 4000 rpm. Following the removal of the supernatant, the pellet was washed in 5 mL of 80% Ca(NO3)2 twice and then centrifuged for 5 min at 4000 rpm. The total starch content was calculated as the percentage per gram of dry sample, which was calculated according to a 100 μg/mL starch standard solution  with an absorbance of 620 nm38. Three replicates were measured.The banana pulp was soaked for 10 min in 1.5% sodium bisulfite solution to prevent browning prior to drying for 24 h at 40 °C2-KI solution, and 1 mL of 1 M acetic acid were mixed together prior to further dilution to 100 mL with distilled water in a new volumetric flask. The amylopectin content was calculated as the percentage per gram of dry sample based on a 1 mg/mL amylopectin standard solution  measured at 533 nm. The measurement was carried out for three replicates.In total, 9 mL of 1 M sodium hydroxide and 1 mL of 95% ethanol were added to a sample of 100 mg of banana powder, after which the mixture was incubated at 40 °C for a period of 24 h. Afterward, 5 mL of the mixture, 2 mL of 0.2 N IMaSBE2.3 promoter, which consists of a genomic DNA fragment 1824 bp in length, immediately upstream of the MaSBE2.3 open reading frame  cells, which were subsequently cultured on SD/-Leu+AbA200 media for 3 days at 30 °C. Selected positive colonies were further verified via PCR and sequence analyses. The identified TF genes were annotated in the banana A-genome database5.Total RNA was extracted from the BX banana pulp samples at 0, 20, and 80 DAF by a plant RNAout Kit (TIANGEN Biotech) and then applied to cDNA library construction by using a Mathchmaker Gold Yeast One-Hybrid Library Screening System Kit . The putative MaSBE2.3 promoter and a single TF, the MaSBE2.3 promoter was inserted into a pAbAi vector to form a bait construct (Clontech). A prey construct was built by inserting the ORFs of candidate TF genes  prior to cocultivation with banana fruit discs by soaking and shaking. The luciferase and REN-LUC activities following cocultivation for a period of 3 days were analyzed by using a dual-LUC reporter assay system . Three replicates were measured.The t tests were applied to test the differences between the means revealed by ANOVA. Each sample consisted of three replicates, and p < 0.05 and p < 0.01 were recognized as statistically significant and highly significant, respectively.SPSS 10.0 software  was used for the statistical analyses. The least significant difference test was used for one-way analysis of variance (ANOVA). Student’s M. acuminata MaSBE  and six M. balbisiana MbSBE  amino acid sequences were obtained from the banana A- and B-genome databases7, respectively. Among the 11 chromosomes, MaSBE1/MbSBE1, MaSBE2.1/2.2/MbSBE2.1/2.2, and MaSBE2.3/2.4/2.5/3/MbSBE2.3/2.4/3 were distributed on chromosomes 4, 5, and 6, respectively . MaSBE1/MbSBE1 and MaSBE3/MbSBE3 were found in group I and group III, respectively. The majority of banana SBEs, including five MaSBE  and four MbSBE  members, were present in group II. It is likely that the banana SBEs in group II have undergone significant gene duplication and possible functional diversification throughout the evolutionary process.To understand the evolution of the SBE proteins in banana, a phylogenetic tree was constructed by comparing all seven MaSEB and six MbSBE amino acid sequences containing GlgB domains Tables  and 2 toBEs Fig. . MaSBEs MaSBE and MbSBE family members were further verified by exon–intron structural analysis , II (10–24 exons), and III (20–22 exons)  Fig. , implyinMaSBEs and MbSBEs were further analyzed in silico. Transit peptides were identified in all putative MaSBE and MbSBE proteins  and FJ (AAB). In BX, six MaSBE genes, MaSBE-1, -2.1, -2.2, -2.3, -2.4, and -3, were expressed in all the tested tissues of BX, whereas the expression of MaSBE-2.5 was not discernible  and six MbSBEs  were expressed in all the tested tissues  but also a high level of tissue specificity in the fruits of both genotypes. The consistently high expression in the fruits of both genotypes may imply that SBE2.3 is a key player in banana fruit development and starch accumulation.ble Fig. . In FJ, ues Fig. ; the expSBEs was analyzed by using developing banana fruits and harvested banana fruits under two different storage durations  and six MbSBEs  exhibited various expression changes during fruit development and ripening, among which MaSBE-1 and -2.3 or MbSBE-1 and -2.3 showed significant upregulation during fruit development from 0 to 80 DAF and downregulation during the ripening process. In both genotypes, MaSBE2.3 and MbSBE2.3 maintained the highest expression levels throughout fruit development (RPKM > 11). This genotype-based temporal expression pattern warrants further functional characterization of SBE2.3 in banana.The temporal expression pattern of banana ons Fig. . In BX, ing Fig. . In FJ, MaSBEs  and six MbSBEs  exhibited variable expression in the different tissues or at various fruit developmental stages. This transcriptomic expression profile was verified by qRT-PCR . As illustrated in Fig. MaSBE2.3-VIGS banana fruit discs showed lighter colors of I2-KI staining  compared with the control fruit discs  compatible with ARFs and a MYB-binding site (TAACCA) compatible with MYBs were identified  and O. sativa (OsSBEs), respectively. In group II, the closest orthologs of the banana SBEs were those from T. aestivum, O. sativa, and Z. mays  and four MbSBEs   caryopses42. Whether low-molecular-mass MaSBEs and MbSBEs are encoded by pseudogenes or have diverse functionalities distinct from classic SBEs remains unknown.The phylogenetic and protein structural features of nes Fig. , which iDa Table . In the SBEs may play a crucial role in the fruit development process have been reported in several plant species, such as M. × domestica, P. vulgaris, and C. mollissima22. The M. × domestica SBE1 was reported to be expressed at a high level in developing fruits, suggesting that it plays regulatory role in fruit development and amylopectin accumulation12. It has also been found that high levels of SBEI and SBEII expression are important to seed development in C. mollissima20 and P. vulgaris22. In the current study, among the seven MaSBEs and six MbSBEs, SBE2.3 was identified as the only gene whose expression was upregulated during fruit development and downregulated during fruit ripening in the BX and FJ genotypes , BX (AAA genotype) is a banana variety known to produce higher yields, and the quality of its fruits is better, with a longer shelf-life31. This finding is in agreement with findings of previous reports suggesting that, in banana, the A-genome contains relatively more functional genes that are associated with fruit quality and yield traits than the B-genome contains; thus the A-genome could be used as a valuable source for potential target genes in banana breeding programs31.The findings that pes Fig. , implyinSBEs are key players in starch biosynthesis13. In pea mutants, the loss of SBEII resulted in a wrinkled phenotype and 50% reduction in starch biosynthesis in the seed compared with those of wild type43. In maize, the loss of SBEIIb caused a 20% reduction in starch content and alterations to the morphology of the starch granules44. The production of high-amylose wheat starch, which comprises >70% amylose, was associated with SBEII suppression45. The simultaneous loss of both SBEII genes resulted in the failure of starch synthesis in A. thaliana17. In the present study, we demonstrated that the genetic downregulation of MaSBE2.3 expression resulted either directly or indirectly in the increased turnover of total starch and amylopectin, as revealed by the reduced I2-KI staining (Fig. 43, maize44, and A. thaliana46. In addition, consistent with previous studies in potato1 and chestnut20, the transient overexpression of MaSBE2.3 in BX banana fruit discs at 80 DAF resulted in significant increases in the contents of total starch and amylopectin (Fig. 13. Further, the unusually high level of MaSBE2.3 observed in the transient expression study may not be able to reflect in planta scenarios, which warrants further exploration in future studies.It has been established that ing Fig. . Such retin Fig. . However47. However, it has never been implicated in starch biosynthesis. In the present study, three members of the banana ARF family, MaARF2/12/24, were found to bind the promoter of MaSBE2.3 in Y1H experiments (Fig. MaSBE2.3 expression in banana fruit, while MaARF12 and MaARF24 suppress the activity of the MaSBE2.3 promoter in banana fruits (Fig. 48 and maize49. In hull-less barley, the coexpression of MYB and SBE2b was found to significantly contribute to starch biosynthesis during grain development48. Furthermore, maize ZmMYB14 could bind directly to the ZmSBE1 promoter region in yeast and promote the expression of ZmSBE1 in maize endosperm49. Here we identified two MaMYB members, MaMYB3 and MaMYB308, both of which were found to bind directly to the MaSBE2.3 promoter in yeast. We demonstrated that MaMYB308 and MaMYB3 could promote and suppress MaSBE2.3 expression, respectively, in banana fruit discs (Fig. MaSBE2.3 expression in the amylopectin biosynthesis process in banana fruits.As an auxin signaling regulator, ARF affects almost every aspect of plant development, including lateral root development, embryogenesis, flower development, and seed settingnts Fig. . For theits Fig. . MYBs coscs Fig. . There iSBE gene family in banana through a genome-wide approach based on A- and B-genome data. The spatial and temporal expression profiles of individual SBE members were investigated in two distinct banana genotypes, and SBE2.3 was shown to play a vital role in amylopectin biosynthesis. Using both transient downregulation and overexpression techniques, we further demonstrated that MaSBE2.3 is essential for amylopectin biosynthesis in banana fruit. Furthermore, the identification of two classes of TFs, MaARFs and MaMYBs, which interact with the MaSBE2.3 promoter provides further evidence of the regulatory mechanism of MaSBE2.3 expression in amylopectin biosynthesis in banana. This study clearly improves our understanding of the molecular mechanism underlying the functionality of SBE in banana and also provides a set of useful tools for quality improvement of banana fruit through molecular breeding and genetic engineering.This report represents the first study on the Figure S1Figure S2Figure S3Figure S4Figure S5Table S1Table S2Table S3Table S4Table S5Table S6Table S7Table S8Table S9Table S10


Ki-67 and beta-catenin in pseudoepitheliomatous hyperplasia and squamous cell carcinoma (SCC) in oral mucosal biopsies. To examine the immunohistochemical expression of In this comparative cross sectional study, 70 cases of each PEH and OSCC were taken from the patients of both genders and in all age groups. Study was conducted at Armed Forces Institute of Pathology (AFIP), Rawalpindi from Dec 2017 to March 2019. Statistical analysis was done with the help of SPSS Version 24.0. We used Chi-Squared test with p value of < 0.05 which was considered as statistically significant. Ki-67 labeling index was observed in all (70) PEH cases, which involved only basal layer of the epithelium. Whereas, Ki-67 labeling index was highly expressed in tumor of high grade malignancy than tumor of low grade malignancy. On the other hand, expression of membranous beta-catenin was higher in PEH and cytoplasmic beta-catenin expression was higher in OSCC. In the current study, 80 (57.1%) male and 60 (42.8%) female patients with the mean age of 51.69 ± 16.121 (mean ± SD) years were included. It was found that 6-25% Ki-67 and beta-catenin showed significant expression in PEH and OSCC in oral mucosal biopsies especially those with intense inflammation or unoriented tissue, helping the clinicians to arrive at a final diagnosis before planning any surgical intervention.It is concluded that  Oral cancer is an important problem of global public health, constituting approximately 5% of all cancers . Out of this 5%, almost 90% constitutes oral squamous cell carcinoma. In Pakistan, it accounts for 10% of all malignancies . According to Pakistan Medical Research Council (PMRC), OSCC accounts for the majority of the cancers among males than in females because of heavier consumption of smoked tobacco and alcohol in males with tongue and lips are the most frequent occupied sites . The major risk factor for OSCC is use of tobacco which may be in the form of cigarettes, cigars, pipes and chewing betel nuts. Other risk factors include heavy consumption of alcohol, nutritional deficiencies, sepsis, genetic mutation involving p53 gene, human papillomavirus (HPV) infection, Epstein-Barr virus (EBV) infection and poor dental hygiene . These risk factors leads to changes in epithelium of oral mucosa causing transformation of normal keratinocytes into abnormal keratinocytes, squamous cell atypical and invasive features .One of the benign mimics of OSCC is pseudoepitheliomatous hyperplasia (PEH) which is defined as a histopathological response to numerous stimuli, such as shock, disease, irritation and neoplasia . Histologically, it is seen as tongue like epithelial propagations penetrating into connective tissue . When the mucosal biopsies are of small size, ulcerated, unoriented and inflamed or when the profound rete pegs are cut obliquely, it become extremely difficult to differentiate between PEH and OSCC. Hence, it is important to differentiate between PEH and OSCC as the whole treatment plan of the surgeons and oncologists relies on correct diagnosis .Ki-67 is a monoclonal antibody for proliferation. It is strictly associated with cell proliferation and aggressiveness of malignant tumours . During cell cycle, the presence of Ki-67 protein is seen during prophase and metaphase  but is absent during resting cells (G0) . Whereas; beta-catenin is a subunit of cadherin protein complex. beta-catenin is a 92 kDa protein normally found in cytoplasm of the cell in sub-membranous location which participate in regulation and organization of cell–cell adhesion and transcriptional regulator . Mutation or overexpression in the beta-catenin gene results in nuclear accumulation of the protein. Reduced expression of membranous beta-catenin and amplified expression of cytoplasmic / nuclear beta- catenin has been associated with increase grading and invasiveness of tumor .Ki-67 and beta-catenin protein has not been completely explained in human PEH and OSCC samples by immunohistochemistry (IHC). The aim of this study was to examine the expression of Ki-67 and beta-catenin by IHC in paraffin-embedded oral mucosal tissues exhibiting PEH and OSCC.So far, the expression of An institutional ethical committee approval  (letter no. MP-ORP16-10/READ-IRB/17/394) was taken before the start of the study. A total of 140 oral mucosal biopsy samples, 70 cases each of PEH and OSCC, along with normal mucosal biopsy as control group were collected from Armed Force Institute of Pathology (AFIP), Rawalpindi, Pakistan from December 2017 to March 2019. Ki-67  from Bio SB ; and monoclonal mouse beta-catenin  from Leica Bio system . Results were interpreted on light microscope  Model CX-21) using high power field objective  and further counter checked by consultant pathologist. Evaluation of Ki-67 and beta-catenin was performed as follow:Along with clinical histories of each case, the data of age, gender and site were noted. Immunohistochemistry was performed on 140 mucosal biopsies along with adjacent controls. Scanty and poorly fixed specimens were excluded from the study. Antibodies used for IHC were: monoclonal rabbit Ki-67 staining which is confined to spinous layer or both basal and parabasal layer or only basal layer of epithelium is graded as  : The intensity of brown colored nuclear Mild……. +light brown colorModerate……. ++ dark brown colorSevere……… +++ very dark brown color The pattern of staining was assessed in percentage by calculating the positive cells per 100 basal cells, parabasal or spinous cell layer of epithelium. The percentage of positive cells or labeling index (LI) was as follows : Negative = 0-5%basal layer staining = 6-25%basal and parabasal layer staining = 26-60%basal, parabasal and spinous layer staining = 61-99%.beta-catenin positive cell is defined as brown staining of nucleus, cytoplasm or cell membrane of epithelium and is expressed in the form of immunoreactivity as follows :On the other hand, The proportion score (P) was interpreted as:0……………. Negative1……………. <10% positive cells2……………. 10-50% positive cells3……………. 50-80% positive cells4……………. >80% positive cellsThe intensity score (I) was defined as: 0……………. Negative / No staining 1……………. Weak staining2……………. Moderate staining3……………. Strong stainingImmunoreactive combined score (T) = proportion score (P) x intensity score (I).beta-catenin were categorized into three groups based on the final score:Immunoreactivity scores (T) of Score 0……………. NegativeScore 1-4……………. Weak StainingScore > 4……………. Strong StainingIn a total of 140 cases, 70 (50%) cases showed positive stained cells ranging from 6-25% Ki -67 labeling index. Whereas, 24 (17.1%) and 46 (32.9%) cases showed positive stained cells ranging from 26-50% and 51-99% Ki -67 labeling index respectively .Ki-67 antibody in the basal layer of the epithelium. The labelling index of Ki-67 for PEH is 6-25%. Among 70 cases of PEH, all 70 (100%) cases showed positive staining with Ki-67 labelling index of 26-50% and 51-99 % respectively. No staining pattern of WDOSCC was observed between Ki-67 labelling index of 6-25%. Similarly, 6 (22%) and 21 (77.8%) cases of moderately differentiated OSCC (MDOSCC) showed Ki-67 labelling index of 26-50% in cases and 51-99 % respectively. While, no staining pattern of MDOSCC was observed between Ki-67 labelling indexes of 6-25%. Interestingly, poorly differentiated OSCC (PDOSCC) showed only 51- 99 % Ki-67 labelling index in 3(100%) cases. Whereas, no staining pattern was observed between index of 6-25% and 26-50% in PDOSCC  and 22 (55%) cases of well-differentiated OSCC (WDOSSC) showed n PDOSCC .Among 140 cases, score 0 was not found in any single case. Whereas, 52(37.1%) cases showed weak staining and 88(62.9%) cases showed strong staining .Out of 140 cases (Table 3), 78 cases of the oral mucosal biopsies exhibited membranous expression of β- catenin in which 7.7% of membranous β- catenin was expressed in WDOSCC, 2.6% in MDOSCC, 0% in PDOSCC. In PEH, 87.7% of membranous β- catenin was expressed.β- catenin. But since, in this case, a little bit of cytoplasmic staining was also found, so, this case was included in cytoplasmic/nuclear expression. Eighteen cases of the oral mucosal biopsies exhibited membranous / cytoplasmic expression of β- catenin in which 64.3% of membranous / cytoplasmic β- catenin was expressed in WDOSCC, and 25.7% in MDOSCC. No membranous β- catenin expression found in PDOSCC and PEH. Nine cases of the oral mucosal biopsies exhibited cytoplasmic / nuclear expression of β- catenin in which 12.5% of cytoplasmic / nuclear β- catenin was expressed in WDOSCC, and 50% in MDOSCC. 37.5% in PDOSCC. In PEH, no cytoplasmic / nuclear β- catenin was expressed (Twenty-five (17.9%) cases of the epithelial cells exhibited cytoplasmic expression of β- catenin in which 56% of cytoplasmic β- catenin expression was seen in WDOSCC, 44 % in MDOSCC, 0% in PDOSCC and PEH. Only 1 case of the PDOSCC exhibited nuclear staining of xpressed . So, theOral squamous cell carcinoma is the most frequent malignant neoplasm of oral region . There are a lot of structural features which help in differentiating oral squamous cell carcinoma from pseudoepitheliomatous hyperplasia on H / E sections. But, there has always been argument whether the mucosal biopsies are diagnosed as OSCC or PEH, when they are of small size, ulcerated, unoriented and inflamed or when the profound rete pegs are cut obliquely, as this will lead to unnecessary removal of tissue or additional therapy such as chemo radiotherapy . Ki-67 proliferative marker and beta-catenin on pseudoepitheliomatous hyperplasia and PEH. Current study investigated the immunohistochemical expression of Ki-67 antibody in the basal layer of the epithelium with a labelling index of 6-25% while in most of the cases of OSCC; the entire epithelium showed continuous and prominent Ki-67 labelling. The results are similar to the study made by Humayun and Prasad, (2011). which showed that Ki-67 labelling index in WDOSCC is low (28 ± 20.82) while in MDOSCC and PDOSCC a significantly higher number of Ki-67 positive cases with a mean of 47 ± 6.3 ( mean ± SD ) and 60.3 ± 2.8 ( mean ± SD ) respectively were seen. Similarly, in another study by Ashraf and his co-workers  studied 54 patients showing Ki- 67 staining increases as the tumor leads towards its severe stage.In our study, PEH showed positive staining with Ki-67 and p53 as tumor markers and revealed that in PDOSCC, Ki-67 expression was more diffuse and intense as the cells were less differentiated than WDOSCC and MDOSCC . Similar finding have been seen in our study in which all PDOSCC cases showed 51-99% Ki-67 expression range implying that high grade tumor showed great proliferative activity which has a poor prognosis.Moreover, in a study of Finland by Luukkaa showed that the beta-catenin antibody was seen in pseudoepitheliomatous hyperplasia exhibiting good prediction of recovery- of the patient. Likewise, in the study by purcell  illustrated that when there was increased expression of cytoplasmic/nuclear beta-catenin in squamous cell carcinoma of pharynx, the prognosis is poor. This was parallel to the present study in which all PDOSCC cases showed cytoplasmic/nuclear staining. On the other hand, beta- catenin is a protein normally found in cytoplasm of the cell in sub-membranous location which participate in regulation and organization of cell–cell adhesion and transcriptional regulator . In a research carried out in China, Jiang and his fellows  showed that increased membranous appearance of beta-catenin and its intensity decreased as the cancer differentiation become poor. In our study, nine number of cases of the oral mucosal biopsies showed cytoplasmic/nuclear beta-catenin expression with all PDOSCC cases having low immunoreactivity score of 1-4. While, 78 number of cases showed membranous beta-catenin expression with all PEH cases having high immunoreactivity score of > 4. Our results are concordant to those of previous studies performed by Yu et al., (2005), who compared expression of beta-catenin in normal tissue to cancerous tissue and concluded that in regular ordinary tissue beta-catenin expression was high while in cancerous tissue its expression was low showing poor prognosis. In the present study, number of positive cases of Ki-67 expression and low beta-catenin expression which is significantly linked with poorly and moderately differentiated carcinoma (p<0.05). A point noteworthy is that the sample size of the current study is huge making the results more reliable and statistically significant. Our current results are parallel to those of Japanese pathologist, , who studied high Ki-67, while in PEH there was increased nuclear staining only in the basal layer of the epithelium. On the other hand, number of positive cases and immunoreactive score of beta-catenin decreased as the grade of oral squamous cell carcinoma increases. On the basis of our study, the present results can be interpreted that in OSCC, the entire thickness of epithelium showed nuclear staining with Ki-67 and beta-catenin play a vital role in forecasting histological grades of differentiation in oral mucosal biopsies and prognosis of the lesion. Therefore, helping clinicians to arrive at a final diagnosis before planning any surgical management.Hence, we concluded that opposite expression of But because of small tissue estimate, the utility of this panel is constrained. One of the limitations of this study is that the size of mucosal biopsies is so tiny or small, if we want more sections of the tissue for further study; it is difficult to choose if the sufficient tissue is cleared out in the block. For this purpose, we had also learnt from our present study to slice the tissue fragment for immunohistochemical analysis at the same time when routine sections were sliced so that there is reduce shaving and we have more left over tissue. One of the greatest limitations of our present study is that the sample size of PDOSCC was scarce owing to the patient turnout in our set up. No follow ups were possible owing to the short duration of present study.


P < 5 × 10−8), 11 of which are novel. We estimate the SNP-based heritability as 13.2% (s.e. = 0.21%), reducing to 8.9% (s.e. = 0.17%) with adjustment for body mass index. Significant genetic correlations are observed with cardiometabolic, respiratory and psychiatric traits, educational attainment and all-cause mortality. Mendelian randomization analyses suggest a potential causal link of increasing walking pace with a lower cardiometabolic risk profile. Given its low heritability and simple measurement, these findings suggest that self-reported walking pace is a pragmatic target for interventions aiming for general benefits on health.Walking is a simple form of exercise, widely promoted for its health benefits. Self-reported walking pace has been associated with a range of cardiorespiratory and cancer outcomes, and is a strong predictor of mortality. Here we perform a genome-wide association study of self-reported walking pace in 450,967 European ancestry UK Biobank participants. We identify 70 independent associated loci ( Iain Timmins et al. identify 70 loci associated with self-reported walking pace in the UK Biobank. Using Mendelian randomization, they find evidence for a causal link between increased walking pace and lower cardiometabolic risk profile, which combined with a low estimated heritability, suggests self-reported walking pace could be a target for healthcare intervention. The public health recommendations for walking focus particularly on increasing the time spent walking and the number of steps walked, with walking at a faster pace receiving less emphasis2.Walking is a simple and convenient form of exercise that is widely promoted for its benefit to physical fitness and overall health3. Most notably, self-reported habitual walking pace has been identified as one of the strongest predictors of all-cause mortality4, even when adjusting for the effects of established risk factors such as body mass index (BMI)5 and other lifestyle behaviours including smoking6.However, recent studies have observed a brisk habitual walking pace, self-reported through questionnaire or verbal interview, to be associated with reduced risk of a range of cardiorespiratory and cancer outcomes8. Genome-wide significant markers of objectively measured gait speed were not identified in these studies, which had a maximum sample size of 31,479.Despite the strong associations of self-reported walking pace with health and survival, it is unclear whether these associations arise from common biological processes, including genetic predisposition, nor whether there are causal effects of walking pace on health outcomes. These questions can be addressed with knowledge of the genetics of walking pace. To date, studies examining the genetic component of walking pace have analysed objectively measured gait speed, where speed is assessed by timing participants to walk a distance of up to 8 m. These studies focussed on older adults, giving insight into the biological mechanisms underlying age-related diseases and physical mobility9. Participants self-reported their walking pace as “slow”, “steady/average” or “brisk”. We aimed to identify associated genetic variants and their possible function, quantify the genetic correlation of walking pace with other complex traits, and assess the potential of self-reported walking pace as a modifiable health-related exposure. Through these analyses we identify 70 genetic loci for self-reported walking pace and show that this trait shares its genetic architecture with other cardiometabolic risk factors, including educational attainment and cognitive outcomes. Using Mendelian randomisation (MR) we find evidence in favour of causal relationships between self-reported walking pace and several traits associated with mortality. This suggests that self-reported walking pace may indeed be a logical target of health interventions.To examine the genetic component of self-reported walking pace, we performed a genome-wide association study (GWAS) in UK Biobank, a prospective study of approximately 450,000 adults of European descent, in addition to approximately 50,000 participants of other ethnicities, aged between 40 and 69 years at baseline10. After quality control 10,061,374 imputed variants were analysed . The phenotype was coded 0, 1 and 2 for self-reported slow, steady/average and brisk walking pace, and the characteristics of participants across these categories are summarised in Supplementary Data λGC = 1.597, mean χ2 = 1.767) but, similarly to other phenotypes analysed in UK Biobank11, the LD score intercept of 1.058 (s.e. = 0.0120) suggests that the inflation is largely due to polygenic signal and the large sample size rather than population substructure.We estimated an inflation in the test statistics . In addition, using LD score regression13 we observed a strong genetic correlation between self-reported walking pace with and without adjustment for BMI , suggesting that much of the genetic component of walking pace is independent of BMI.As there is a clear negative association between BMI and self-reported walking pace  mapping, we identified a total of 535 genes associated with genome-wide significant SNPs .To prevent against the potential pleiotropic effects of adiposity-related SNPs in the gene analysis, we further assessed genes that remained prioritised following adjustment for BMI. Of the 152 genes implicated by both the gene mapping and gene-based analyses, 78 remained significantly associated with self-reported walking pace following BMI adjustment. These genes included 26 were enriched for differences in self-reported walking pace. Genes that were associated with self-reported walking pace had increased expression in the brain (P = 9.6 × 10−4) and pituitary (P = 3.1 × 10−6), with tissue-specific enrichments found in the cerebellar hemisphere (P = 5.4 × 10−7) and cerebellum (P = 2.1 × 10−6)  is related to the observed scale through thresholds t1 and t2 in the equationTo provide an interpretable heritability estimate for an ordinal outcome, we parameterised self-reported walking pace on the liability scale. Self-reported walking pace on the observed scale The heritabilities on the observed and liability scales are related using the result27, where zj is the standard normal density at threshold tj and Vo = ∑3k=1k2πk −(∑3k=1kπk)2 , with results summarised in Fig. We assessed whether self-reported walking pace has a shared genetic basis with other complex traits, which may reflect common biological mechanisms or causal effects in either direction. We examined genetic correlations rg = −0.52, P = 4.7 × 10−179), education and cognition  and longevity . Overall, traits related to cardiometabolic risk, lung function, psychiatric disease and muscular strength show genetic correlations with self-reported walking pace. The genetic correlations also support many of the phenotypic associations that have been observed across categories of walking pace in external cohorts30. Traits that remained genetically correlated with self-reported walking pace after adjusting for BMI included hand grip strength, measures of lung function such as forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1), years of schooling, intelligence, insomnia and depressive symptoms. Genetic correlations with adiposity-related traits and glycemic traits were attenuated following adjustment for BMI.The genetic architecture of self-reported walking pace overlaps highly with traits relating to adiposity  with a range of P-value thresholds, and all-cause mortality. We conducted our analyses using sex-stratified GWAS results for self-reported walking pace (see “Methods”) to control for sample overlap.We explored whether the strong associations that exist between self-reported walking pace and survivalP < 10−2; hazard ratio (HR) = 0.95; 95% CI: 0.92–0.97; P = 1.93 × 10−5) and females   and females , which suggests the effect of the genetic variants on mortality is partly independent of BMI.We observed a significant association between genetic variants associated with self-reported walking pace and all-cause mortality in males , lower risk of coronary artery disease (odds ratio (OR) = 0.34, PIVW = 3.1 × 10−8), higher HDL cholesterol levels  and higher FEV1 . We found no evidence of directional pleiotropy by testing the intercept of MR-Egger analysis  and higher FEV1 , though a weaker effect was observed on lowering BMI . Associations were substantially weakened following the exclusion of adiposity-related SNPs between genetically predicted walking pace and glycemic traits such as fasting insulin, HOMA-IR (homeostasis model assessment of insulin resistance index) and type 2 diabetes, suggesting a contribution of pleiotropy that confounds the MR results in these cases.Firstly, largely similar results were found when we excluded 28 SNPs that were previously associated with BMI  suggests a substantial component of the genetic architecture of self-reported walking pace is independent of BMI. This was supported by genetic correlations between self-reported walking pace and a range of complex traits and diseases that were largely robust to adjustment for BMI. In comparison with the genome-wide correlations, a more marked effect of BMI was noted at the genomic loci associated with self-reported walking pace. Only 15 of the 70 loci survived the adjustment for BMI at the genome-wide significance level, whilst 45 loci in total retained a suggestive level of P < 10−5. The attenuation of top hits may partly reflect a mediated effect of BMI on the causal pathway between genotype and self-reported walking pace. To explore this, we performed multivariable MR which is a valid form of mediation analysis33. Following the inclusion of BMI as a secondary exposure alongside self-reported walking pace, we found that across a range of outcomes there was weak evidence of an indirect causal effect (independent of BMI) of self-reported walking pace. One possible explanation to note however for this finding is the limited statistical power available to accurately detect both direct and indirect causal effects in a multivariable MR setting.To better understand the relationship between self-reported walking pace and BMI we performed several sensitivity analyses. The high genetic correlation between self-reported walking pace with and without adjustment for BMI 46.There are several limitations to note. First, the associated loci must be accepted tentatively until validated in an independent cohort. We were specifically interested in the self-reported phenotype owing to its ease of measurement, but while similar measures are available in some prospective cohorts, we were unable to obtain the relevant data during the course of this study. In particular, it is important to confirm the results in a separate demographic, since the UK Biobank participants are known to be healthier than the general populationTherefore, the genetic associations and possible causal effects we report here may not hold for more specific measures of gait. Nevertheless, the strong association of self-reported walking pace with health outcomes and mortality warrants study in its own right. Despite the inherent limitations described, our results highlight the value of studying subjective, self-reported measures of physical activity. We are able to utilise a simple measure of self-reported walking pace to infer that walking at a speed that is brisk in one’s own estimation has important benefits to health and longevity. Arguably this could provide the basis for health advice that is easier to understand and follow compared to walking at or above a precisely defined speed. Nevertheless, further investigation is needed into the generalisability of our findings to interventions aimed at increasing objectively assessed walking pace.32 to suggest that self-reported walking pace may be a beneficial target for intervention in the general population. Given its ease of measurement, by definition by individuals themselves, it may be entirely feasible to develop pragmatic interventions on walking pace that have beneficial effects on health.In conclusion, we have identified 70 genetic loci associated with self-reported walking pace and shown that its strong associations with cardiorespiratory and mortality outcomes is partly explained by genetic correlations. MR arguments augment the results of trials on cardiovascular patientsThe UK Biobank study is a large cohort of 501,726 British residents aged between 40 and 69 at recruitment. The participants attended assessment visits across 23 study centres in the UK, through which extensive phenotypic data were collected. Participants provided informed consent to participate, and the UK Biobank study has ethics approval from the North West National Research Ethics Committee  or “Prefer not to answer” (n = 519). The low numbers of these exclusions suggest minimal impact of any informative missingness. The responses “slow”, “steady/average” and “brisk” were coded as 0, 1 and 2 for our analyses.Self-reported walking pace was ascertained using the ACE touchscreen question “How would you describe your usual walking pace?” with response options of “slow”, “steady/average”, “brisk”, “None of the above” or “Prefer not to answer”. If the participant activated the “Help” button they were shown the message: “Slow pace is defined as less than 3 miles per hour. Steady average pace is defined as between 3-4 miles per hour. Fast pace is defined as more than 4 miles per hour.” We excluded participants whose answers were “None of the above” .Association analysis was carried out in a set of 450,967 individuals of European ancestry with non-missing phenotypes, where ancestry was defined by the K-means clustering of the first two principal components49. Independent significant SNPs were defined using a genome-wide significance threshold of P < 5 × 10−8, independent from each other at r2 < 0.6. Lead SNPs were further identified as a subset of the independent significant SNPs that are in linkage disequilibrium (LD) at r2 < 0.1. Genomic loci were defined by merging lead SNPs that are located within 250 kb of each other.Genomic risk loci were derived using the Functional Mapping and Annotation of genetic associations (FUMA) platform50. In each 3rd degree related pair, we retained the individual with the lower genotyping missingness rate.Interaction effects for the lead SNPs by sex were investigated by carrying out the BOLT-LMM analyses stratified by sex. The strata were ensured to be approximately independent by excluding individuals related to 3rd degree or above (kinship coefficient <0.044) using the software KING13.The effect of confounding by population structure was estimated using the intercept of the LD score regression, which estimates the inflation in test statistics due to confounding of the association between walking pace and genotypeP-value of association for these SNPs under both the linear and ordinal logistic models  were annotated.The functional annotation of SNPs associated with self-reported walking pace was carried out using FUMA52. MAGMA combines the P-values for SNPs within a gene to create gene-based P-values for 19,834 protein-coding genes. A Bonferroni corrected threshold of P < 2.52 × 10−6 was used to determine significantly associated genes. Finally, we used FUMA to perform tissue-enrichment analysis of 30 broad tissue types and 54 specific tissue types from the GTEx database26.Positional mapping and eQTL mapping were used to link self-reported walking pace genomic loci to genes. We used the prioritised genes from the positional and eQTL mapping to perform gene-set enrichment analysis against gene sets defined by traits in the GWAS catalogue. Additionally, gene-based analysis was performed with MAGMA through the FUMA platformP < 10−5) phenotypic associations in published GWAS in the NHGRI-EBI catalogue which overlap with SNPs in LD (r2 > 0.6) with the independent significant SNPs.We identified SNPs with previously reported  and for females 4546 cases . To test for association with all-cause mortality in males, we computed genetic risk scores weighted by effect sizes estimated from the independent sample of females, and vice versa. The polygenic risk scores were constructed using PRSice v2.2.3 software53 for a range of P-value thresholds between 5 × 10−8 and 10−2, using approximately independent genetic markers obtained by clumping the SNPs with an r2 threshold of 0.1 and a window size of 250 kb. To examine the robustness of these associations to adiposity as a mediator, we included covariate adjustment for BMI.Cox proportional hazard models were used to investigate the association between genetically determined self-reported walking pace with all-cause mortality, using age as time scale. Analyses were stratified by sex. For males there were 7049 all-cause mortality cases  Information Centre and the NHS Central Register, Scotland with detailed information on the data linkage procedure available online.55. MR-Egger requires the InSIDE assumption to hold (Instrument Strength Independent of Direct Effect), whilst the weighted median approach requires no more than 50% of the weighted instruments to be invalid due to horizontal pleiotropy. The 75 independent lead SNPs were used as instrumental variables, using proxies in strong LD (r2 > 0.80) if the SNPs were unavailable in the outcome GWAS.To investigate whether walking pace has a causal effect on different outcomes, we performed two-sample MR analyses testing 21 traits identified in the genetic correlation analysis. We used only GWAS data from large scale, previously published studies of European ancestry that do not include participants from the UK Biobank cohort. The inverse variance weighted approach was used as the primary method to infer causal effect estimates. The potential effect of pleiotropy was evaluated using the MR-Egger and weighted median estimate methods56. Estimates in this case correspond to the direct causal effect of walking pace with BMI being fixed. The summary statistic data on BMI was obtained from The Genetic Investigation of Anthropometric Traits (GIANT) consortium12.We conducted further sensitivity analyses to explore the effect of pleiotropy due to BMI, as several of the SNP associations for self-reported walking pace are shared with BMI. Firstly, we conducted the MR analyses with the 28 lead SNPs previously associated with BMI excluded. Secondly, we performed multivariable MR by including both self-reported walking pace and BMI as exposures57 package implemented in R software.MR analyses were performed using the MendelianRandomisationFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1–11Reporting Summary