Besides, we showed that downregulating the key molecules in the signaling pathways by siRNA can inhibit leptin-induced MMP9 expression and further suppress invasion of HTR-8/SVneo cells. In conclusion, our study revealed a new regulatory mechanism of leptin-induced HTR-8/SVneo cell invasiveness and will provide novel insights into the causes and potential therapeutic targets for diseases related to dysregulation of trophoblast invasion in the future.The process of placental invasion is essential for a successful pregnancy. Leptin is involved in trophoblast invasiveness, and its dysregulation is connected with a series of diseases, including preeclampsia. However, the knowledge of the precise mechanisms in leptin-induced trophoblast invasiveness is still limited. According to the present research, transwell assay suggested that leptin is a dose- and time-dependent regulator in inducing HTR-8/SVneo cell invasion. Western blot analysis and immunofluorescence staining revealed that leptin-induced MMP9 expression is essential in the invasion process of HTR-8/SVneo cells. Mechanistically, we demonstrated that leptin activated  At the early stage of pregnancy, the invasion of trophoblast cells into the uterus is essential for implantation and subsequent placental development . The invDiscovered at the end of 1994, Leptin is a 16 kDa polypeptide hormone containing 167 amino acids, which is produced by the obese gene . During As a kind of calcium-dependent, zinc-containing endopeptidases, matrix metalloproteinases (MMPs) are essential to the degradation of the extracellular matrix (ECM) and successful implantation . Studiesβ-catenin regulates proliferation, migration, and differentiation. And there have been studies demonstrated that β-catenin can promote the trophoblast hyperplasia and invasion [β-catenin pathway, noncanonical WNT/Ca2+ pathway, and noncanonical planar cell polarity pathway. Among them, WNT/β-catenin is the most widely studied, and its aberrant activation has been reported in a variety of diseases, including invasion [β-catenin signaling in trophoblast cells remains largely unknown.Acting as a nuclear transcriptional regulator, invasion , 26. In invasion –30. MTA1invasion , 32. Howβ-catenin and PI3K/AKT pathways in trophoblast cell invasion need to be elucidated.Phosphatidylinositol 3-kinase (PI3K) is a kind of enzyme that phosphorylates the 3′-OH of the inositol ring of phosphatidylinositol . When itTaken together, our study manages to investigate the effect of leptin on HTR-8/SVneo cell invasion and the potential underlying mechanisms.2O to 100 μg/ml. HTR-8/SVneo cells were cultured in RPMI 1640 Medium  supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO2 at 37°C and then treated with or without leptin.Recombinant human leptin  was dissolved in ddHβ-catenin siRNA were synthesized by Genomeditech . And transfection was performed according to the manufacturer's protocols. The target sequences were designed as the following: MTA1 siRNA: 5′-GAACAUCUACGACAUCUCC-3′; AKT1 siRNA: 5′-GACGGGCACAUUAAGAUCA-3′; WNT1 siRNA: 5′-GGUUCCAUCGAAUCCU-The MTA1 siRNA, AKT1 siRNA, WNT1 siRNA, and β-catenin: 5′-CCUUCACUAUGGACUACCA-3′; MMP9: 5′-CACGCACGACGUCUUCCAGUA-3′; negative control siRNA: 5′-UUCUCCGAACGUGUCACGU-3′. The transfection was performed by using Lipofectamine 3000 reagent .GCA-3′; μl of the diluted Matrigel into the upper chamber of 24-well transwell inserts  then incubate the transwell at 37°C 3-4 h for gelling. Cells suspended in 100 μl serum-free RPMI 1640 medium were added to the upper chamber. 600 μl RPMI 1640 medium containing 10% FBS was added into the lower chamber. After being treated with different interventions for intended time at 37°C, cells were fixed with 4% paraformaldehyde and penetrated with 0.3% Triton X-100. After removing noninvading cells, dying with hematoxylin, the cells were counted with a light microscope .Transwell Matrigel invasion assay was used to measure the capacity of HTR-8/SVneo cell invasion. Briefly, dilute Matrigel (1 : 4)  in serum-free RPMI 1640 medium and put 50 A − B)/A × 100%. A and B represent the scratch width after cell migration at 0 and 24 h, respectively.HTR-8/SVneo cells were seeded into 6-well plates, and when confluence reached to 90%, cells were scratched vertically with the same width. Then, wash away the scratched cells before putting the rest into the incubator for further culture. Photographs of five random fields in each chamber were obtained at 0 h and 24 h after scratch. And the percentage of wound closure was analyzed by calculating , and cDNA was synthesized using PrimeScript RT Reagent Kit . According to the manufacturer's protocol, the qPCR reaction was performed on LightCycler® 480 Real-Time PCR System . The sequences of the primers used in qRT-PCR are listed in μg) was separated using SDS-PAGE and transferred to PVDF membranes . The membranes were then blocked with 5% milk or BSA for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. Following incubation with the secondary antibody at room temperature for 1 h, the level of the proteins was quantified using ECL reagent  and imaged by the Amersham Imager 600 . β-Actin was used as the control of total proteins. Nuclear protein was extracted with a Nuclear and Cytoplasmic Extraction Kit , and histone 3 was used as the control of nuclear proteins. The following primary antibodies were used: MMP9 (ab76003), MTA1 (ab71153), WNT1 (ab15251), AKT (ab179463), GSK3β (ab32391), β-catenin (ab32572), histone 3 (ab1791), β-actin (ab6276) ; p-AKT (Ser473) (#4060), p-GSK3β (Ser9) (#9322) .HTR-8/SVneo cells grown to confluency were washed with ice-cold PBS three times and lysed in RIPA buffer  containing proteinase inhibitors and phosphatase inhibitors . After being denatured, equal protein  for 1 h at 37°C under dark conditions. Next, cells were washed with PBS in triplicate, followed by incubation with DAPI for 10 min. Finally, cells were observed under a fluorescence microscope .t-test while differences among multiple groups were analyzed by one-way ANOVA. P < 0.05 was considered a statistically significant difference. All experiments were conducted at least in triplicate.Data analysis was performed with GraphPad Prism 8 and Adobe Photoshop. The experimental results were presented as mean ± SD. Differences between the two groups were analyzed by Student's Previous studies have shown that leptin is involved in the cytotrophoblast invasion. To confirm whether leptin stimulation induced the invasion of HTR-8/SVneo cells, we divided the cells into two groups. One was treated with exogenous leptin  for 24 h; the other was treated for 0 h, 12 h, 24 h, and 36 h with 200 ng/ml leptin. To detect the invasiveness of HTR-8/SVneo cells, both of the groups were performed under a transwell assay, and the results revealed that leptin can increase the HTR-8/SVneo cell invasion in a dose-dependent  and timeConsidering that MMP9 is crucial for trophoblast invasion, we explored the function of MMP9 in the process of leptin-induced invasion in HTR-8/SVneo cells. As shown in Figures β-catenin in leptin-induced HTR-8/SVneo cell invasion, we detected the level of β-catenin with the stimulation of leptin. Firstly, treating HTR-8/SVneo cells with leptin (200 ng/ml) for 24 h, and the following Western blot showed that leptin increased the protein levels of nuclear β-catenin  when compared with the control. Then, we silenced the expression of MTA1 and WNT1 in HTR-8/SVneo cells using MTA1 siRNA and WNT1 siRNA, respectively. The knockdown efficiencies were confirmed by Western blot analysis and qRT-PCR (β (Ser9), and nuclear β-catenin expression (β (Ser9) and nuclear β-catenin expression  and nuclear β-catenin, suggesting the presence of an MTA1/WNT1-independent mechanism to regulate leptin-induced p-GSK3β (Ser9) and β-catenin. Recently, there has been reported a “crosstalk” between the WNT/β-catenin and PI3K/AKT pathways by GSK3β. Considering the role of PI3K/AKT signaling pathway in invasion, we further detected the effect of PI3K/AKT in regulating leptin-induced p-GSK3β (Ser9) and activation of β-catenin in HTR-8/SVneo cells. As shown in β (Ser9) and nuclear β-catenin  to mediate the degradation of extracellular matrix (ECM) , 37. A pβ-catenin could enhance the invasion of trophoblast, while inhibited β-catenin could damage the function of trophoblast [β-catenin in leptin-induced HTR-8/SVneo cells was statistically higher than the control group, proving that leptin may induce the invasion of HTR-8/SVneo cells via promoting the nuclear translocation of β-catenin.Increasing evidences have shown that phoblast , 41. In β-catenin is the core molecule to the activation of WNT/β-catenin signaling pathway [β-catenin is phosphorylated by a multiprotein destruction complex which is composed of glycogen synthase kinase 3β (GSK3β), casein kinase 1 (CK1), tumor suppressor adenomatous polyposis coli (APC) gene product, and scaffolding protein Axin, leading to ubiquitination and subsequent degradation by the proteasomal system [β-catenin. Thus, β-catenin accumulates in the cytoplasm and travels to the nucleus to regulate transcription of target genes [β (Ser9) in leptin-induced HTR-8/SVneo cells and found a significant repression of WNT1, p-GSK3β (Ser9), and nuclear β-catenin when knocking down MTA1 by siRNA, suggesting that MTA1 may mediate WNT/β-catenin signaling in HTR-8/SVneo cells. Intriguingly, silencing MTA1 or WNT1 did not totally inhibit the expression of p-GSK3β (Ser9) and nuclear β-catenin in leptin-treated HTR-8/SVneo cells. Thus, we speculate that there may be other MTA1/WNT1-independent mechanisms to regulate leptin-induced p-GSK3β (Ser9) and nuclear β-catenin.Besides, researches demonstrated that the level of  pathway . With thl system . Insteadet genes . A recenet genes , revealiβ through phosphorylating GSK-3β at Ser9, thereby inhibiting the degradation of β-catenin, leading to the accumulation of β-catenin in the cytoplasm [β-catenin and PI3K/AKT pathway via GSK3β in HTR-8/SVneo cells. By performing Western blot, we observed a significant increase of p-AKT (Ser473) in leptin-induced HTR-8/SVneo cells, and inhibition of AKT reduced the expression level of p-GSK3β (Ser9) and nuclear β-catenin. Besides, we revealed that MTA1/WNT/GSK3β/β-catenin and PI3K/AKT/GSK3β/β-catenin pathways promoted the expression of MMP9 and augmented leptin-induced cell invasion by conducting loss-of-function analysis, enriching the exploration of cell invasion.PI3K/AKT pathway, another important pathway to mediate invasion –46, has ytoplasm , 50. In In summary, our study discovered a link between the leptin and MMP9 in leptin-induced HTR-8/SVneo cell invasion and illuminated a potential mechanism that leptin promoted MMP9 upregulation via the crosstalk between MTA1/WNT and PI3K/AKT pathways , which w


Follistatin (FS)-like 1 (FSTL1) is a member of the FS-SPARC  family of secreted and extracellular matrix proteins. The functions of FSTL1 have been studied in heart and lung injury as well as in wound healing; however, the role of FSTL1 in the kidney is largely unknown. Here, we show using single-cell RNA-Seq that Fstl1 was enriched in stromal cells in obstructed mouse kidneys. In addition, immunofluorescence demonstrated that FSTL1 expression was induced in fibroblasts during kidney fibrogenesis in mice and human patients. We demonstrate that FSTL1 overexpression increased renal fibrosis and activated the Wnt/β-catenin signaling pathway, known to promote kidney fibrosis, but not the transforming growth factor β (TGF-β), Notch, Hedgehog, or Yes-associated protein (YAP) signaling pathways in obstructed mouse kidneys, whereas inhibition of FSTL1 lowered Wnt/β-catenin signaling. Importantly, we show that FSTL1 interacted with Wnt ligands and the Frizzled (FZD) receptors but not the coreceptor lipoprotein receptor–related protein 6 (LRP6). Specifically, we found FSTL1 interacted with Wnt3a through its extracellular calcium–binding (EC) domain and von Willebrand factor type C–like (VWC) domain, and with FZD4 through its EC domain. Furthermore, we show that FSTL1 increased the association of Wnt3a with FZD4 and promoted Wnt/β-catenin signaling and fibrogenesis. The EC domain interacting with both Wnt3a and FZD4 also enhanced Wnt3a signaling. Therefore, we conclude that FSTL1 is a novel extracellular enhancer of the Wnt/β-catenin pathway. Kidney fibrosis is the final common pathway of progressive chronic kidney diseases (CKDs) and is characterized by the increase of interstitial fibroblasts and myofibroblasts, and the excessive production and accumulation of extracellular matrix proteins, such as collagens and fibronectins (Fns), within the kidney , 2, 3, 4The Wnt family of ligands controls a variety of cellular activities. Dysregulation of Wnt signaling has been implicated in human diseases, including tissue fibrosis and tumorigenesis. Wnts signal through the Frizzled (FZD) family of proteins as receptors, and low-density lipoprotein receptor–related proteins 5 and 6 (LRP-5 and LRP-6) as coreceptors, to stabilize β-catenin, which translocates into the nuclei to stimulate the transcription of Wnt target genes , 14, 15.Follistatin (FS)-like 1 (FSTL1) is a secreted glycoprotein, which was initially identified as a TGF-β–inducible gene . FSTL1 bThe biological functions of FSTL1 have been studied in heart injury , 33, 36,A).We first measured Fstl1 mRNA levels in different organs of normal adult mice. Fstl1 was expressed in the kidney at an intermediate level when compared with other organs A.http://www.ruuo-kidney-gene-atlas.com/ (A). Interestingly, Fstl1 was also found to be increased in UUO kidneys in another RNA-Seq dataset (B) and protein (C and D) levels in the whole kidney dramatically increased from day 1 to day 7 after UUO surgery.Unilateral ureteral obstruction (UUO) is a well-known mouse model of kidney fibrosis . We anallas.com/ . Bulk RNlas.com/ A. Intere dataset . Consist dataset B and pro protein , C and DE). Fstl1 enrichment in mesenchymal/stromal cells was found at UUO day 2 and increased at day 7 . In contrast, Fstl1 reads in endothelial cells were not enriched until UUO day 7 , whereas it was not detectable in control kidneys (B). FSTL1 was not detected in lymphocytes (A and 3B), macrophages (C), and dendritic cells (D). Interestingly, FSTL1 was colocalized with the fibroblast marker platelet-derived growth factor receptor beta (PDGFRβ) (G).As shown by immunofluorescence, FSTL1 was found in interstitial cells in kidneys with UUO for 3 days G and S1B kidneys B. FSTL1 phocytes A and 3B,rophages C, and deic cells D. Intere(PDGFRβ) G.H and We also examined FSTL1 expression in kidneys from patients with immunoglobulin A nephropathy at grade III, or membranous nephropathy at grade II, which had developed renal fibrosis (data not shown), and found colocalization of FSTL1 with PDGFRβ H and S4.A and 5B). Fstl1 mRNA and protein levels were only slightly increased in kidneys 24 h after ischemia/reperfusion compared with the sham-operated control (C and 5D). These data indicate that FSTL1 expression was not or only slightly altered by cisplatin-induced or ischemia/reperfusion–induced AKI.We then determined whether FSTL1 was induced by acute kidney injury (AKI). Both mRNA and protein levels of FSTL1 in the kidney were not altered by cisplatin treatment for 72 h A and 5B. control C and 5D.via tail veins 2 days after UUO, and mice were sacrificed the next day. Western blotting confirmed FSTL1 overexpression in the kidneys of mice injected with Fstl1-HA plasmid (A). Alpha-smooth muscle actin (α-SMA), Fn-1, and type I collagen α1 levels in UUO kidneys were increased in Fstl1-HA–treated mice compared with control mice (A). Consistently, immunofluorescence also showed increases in the signals for Fn-1 and α-SMA by FSTL1 overexpression in UUO kidneys (B). Masson’s trichrome staining revealed that renal fibrosis was aggravated by FSTL1 (B). Interestingly, mRNA levels of the inflammatory factors Tnf-α , Tgf-β1, Il-6 (interleukin 6), Il-1β (interleukin 1β), Mcp1, and Cxcl1 did not change by FSTL1 overexpression  or pcDNA3.1 plasmids were injected into mice  plasmid A. Alpha-rol mice A. Consis kidneys B. Massonby FSTL1 B. Interepression . These dA), Hedgehog , and YAP  were not altered by FSTL1 overexpression. Interestingly, phospho-LRP6 . Therefore, the Wnt/β-catenin pathway was more activated by FSTL1 overexpression in UUO kidneys.FSTL1 was found to regulate TGF-β  and BMP Fig. S8tivation ), phosphtivation C. Theref2+/calmodulin–dependent protein kinase II (CaMKII) levels in UUO kidneys were not altered by FSTL1 (D). These results indicate that FSTL1 may not regulate noncanonical Wnt signaling in obstructed kidneys.Phospho c-Jun N-terminal kinase (p-JNK) and phospho-Caby FSTL1 D. These D) but did not alter the TGF-β1 levels and the phosphorylation levels of Smad3 and Smad1/5/8 . As a control, mFSTL1 and hFSTL1 proteins inhibited BMP4-induced BRE luciferase reporter activity in IMCD3 cells (B). mFSTL1 and hFSTL1 proteins also promoted WNT3a-induced 8× TOPFlash-luciferase reporter activity in human embryonic kidney 293T (HEK293T) cells (A) and normal rat kidney 49F (NRK-49F) fibroblasts (B). These results indicate that FSTL1 enhanced WNT3a signaling in multiple kidney cell types.To determine whether FSTL1 directly regulates Wnt signaling, we used the β-catenin-responsive TOPflash or 8× TOPflash luciferase reporter. Both mouse and human FSTL1 proteins increased mWNT3a protein-induced TOPflash-luciferase reporter activity in inner medullary collecting duct 3 (IMCD3) cells A. As a cD3 cells B. mFSTL1T) cells A and norroblasts B. These C) or by transfected Wnt3a (D). Transfected Fstl1-HA also promoted Wnt/β-catenin signaling induced by transfected Wnt1, Wnt2, Wnt8a, Wnt9a, and Wnt10b (E) or combinatorially (F), and this inhibition on Wnt3a signaling was fully rescued by cotransfection with Fstl1-HA (G).To corroborate our findings from recombinant FSTL1 proteins, we overexpressed or knocked down FSTL1 in NRK-49F cells. Transfected Fstl1-HA dose-dependently enhanced 8× TOPflash luciferase reporter activity induced by WNT3a protein C or by ted Wnt3a D. Transfd Wnt10b . Conversd Wnt10b  decreased Wnt10b E or combtorially F, and thFstl1-HA G.A). Transfected Fstl1-HA also increased the phospho-LRP6, active β-catenin, and total β-catenin levels induced by transfected Wnt3a (B and C). These data indicate that FSLT1 promoted Wnt/β-catenin activation.To determine whether FSTL1-induced 8× TOPflash activation correlates with β-catenin activation, we examined active β-catenin levels in NRK-49F cells. WNT3a protein increased active β-catenin levels, which were further elevated by cotreatment with FSTL1 protein A. Transfed Wnt3a , B and CB and D). Therefore, FSTL1 stimulated fibroblast activity in vitro.FSTL1 overexpression also promoted the expression of Fn and collagens induced by Wnt3a overexpression , B and DA), Wnt2, Wnt3a (B), Wnt8a, Wnt8b, Wnt9a, and Wnt10b (C). Interactions between FSTL1 and Wnt2, Wnt3a, or Wnt10b were also found in the conditioned media from HEK293T cells cotransfected with Fstl1-HA and Wnt2-V5, Wnt3a-V5, or Wnt10b-V5 (D–F). As a negative control, the BMP type I receptor ALK3 did not interact with Wnt3a (G).To identify the molecular mechanisms used by FSTL1 to enhance Wnt signaling, we first determined whether FSTL1 interacts with Wnts. We cotransfected Fstl1-HA with plasmids for several canonical Wnts in HEK293T cells. Co-IPs on whole lysates indicated that FSTL1 interacted with Wnt1 A, Wnt2, 2, Wnt3a B, Wnt8a,d Wnt10b C. Interant10b-V5 , D–F. Asth Wnt3a G.Figure F) and WNT1 and WNT3a (H).We also examined whether endogenous FSTL1 and Wnts interacted with each other in the kidney. We used kidneys with UUO for 7 days since they expressed high levels of FSTL1 F and WNTnd WNT3a . We had nd WNT3a H.I). These data demonstrate a direct interaction between FSTL1 and WNT3a.To further explore the interactions between FSTL1 and Wnts, recombinant WNT3a protein alone or a combination of WNT3a and FSTL1 proteins were incubated in a solution, and the complexes containing FSTL1 were pulled down with anti-FSTL1. Protein blotting of the elutes showed that WNT3a was precipitated with FSTL1 I. These J) (K and L), indicating that WNT3a interacted with the EC and VWC domains but not the FS domain (I). Together, our results suggest that FSTL1 interacted with various canonical Wnts. Given that Wnt proteins are highly conserved, our data also imply that FSLT1 interacts with different Wnt ligands through conserved structures of Wnt ligands.FSTL1 consists of an FS domain and an EC domain, followed by a C-terminal domain with homology to the von Willebrand factor type C–like (VWC) domain J 52). I. IJ) (52J) , K and LS domain I. TogethA, neither IP nor reciprocal IP detected any interaction between FSTL1 and LRP6. Interestingly, FSTL1-HA and FZD4 were able to pull down each other (B and C). Both FSTL1-HA and FSTL1 that does not have a tag were coprecipitated with SNAP-tagged FZD4 (SNAP-FZD4) (D). Using the constructs for various FSTL1 domains, we found that FZD4 interacted with the EC domain (E) but not with the FS and VWC domains (F), indicating FZD4 interacted with FSTL1 through the EC domain (I).We then asked whether FSTL1 interacted with Wnt receptors. The Wnt receptor FZD4 and the coreceptor LRP6 are highly expressed in the kidney  53, 54), 5453, 5Figure 6ch other , B and CAP-FZD4) . FSTL1 aAP-FZD4) . Pull-doAP-FZD4) D. Using C domain E but not domains F, indicaC domain I.C, compare lanes 4 and 2 for FSTL1). In the presence of FZD4, LRP6 pulled down FZD4 and FSTL1 although LRP6 did not pull down FSTL1 in the absence of FZD4 , indicating that LRP6 pulled down FSTL1 through FZD4, and that FSTL1, FZD4, and LRP6 formed a complex. FSTL1 increased the interaction between FZD4 and LRP6 .LRP6 did not apparently alter the interaction between FSTL1 and FZD4 . To investigate whether FSTL1 increases the association of WNT3a with FZD4 on the plasma membrane, we used SNAP-FZD4, which has been verified to be active in transducing Wnt/β-catenin signaling . Our results collectively show that FSTL1 interacted with Wnts and FZDs and promoted Wnt/β-catenin signaling by enhancing the presentation of Wnts to FZDs .In a cell-free system, FZD4-Fc directly interacted with WNT3a, and FSTL1 enhanced this interaction A. To invignaling . SNAP-FZignaling . StreptaI). This property led us to examine whether the EC domain enhances Wnt signaling. As shown in Figure 7D, the EC domain dose-dependently increased transfected Wnt3a-induced luciferase activity in HEK293T cells. The EC domain also promoted Wnt signaling induced by Wnt3a transfection (E) or Wnt3a conditioned medium in NRK-49F cells (F). These results reinforce the notion that simultaneous binding of FSTL1 with Wnts and FZDs facilitates Wnt signaling.We have shown that the EC domain of FSTL1 interacted with both WNT3a and FZD4 I. This psfection E or Wnt39F cells F. These A and B). Overexpression (C) or inhibition (D) of FSTL1 did not alter TGF-β1-induced CAGA luciferase activity in NRK-49F cells either. FSTL1 also failed to alter TGF-β1-induced Smad3 phosphorylation and expression of Fn and collagen  nephrectomy . These rC). Our study identifies FSTL1 as a novel positive regulator of Wnt/β-catenin signaling.In summary, we have demonstrated that FSTL1 is upregulated in fibroblasts in fibrotic kidneys. FSTL1 interacts with Wnts and enhances Wnt/β-catenin signaling by acting as a scaffold protein that holds Wnts and FZDs together. FSTL1 promotes the Wnt/β-catenin signaling pathway and thus aggravates renal fibrosis C. Our stet al. .Cryosections were used for immunofluorescent staining. Sections were treated with 1% SDS for 4 min. Sections then were incubated with anti-FSTL1 , anti-PDGFR-β , anti-CD45 , anti-CD3 , anti-CD4 , anti-F4/80 , anti-MHC2 , anti-α-SMA , and anti-Fn  antibodies individually or in combinations as indicated, followed by Alexa Fluor 546 or 555-labeled secondary antibodies (Invitrogen). Images were captured with a regular or confocal fluorescent microscope (Olympus FV1200).Fstl1-HA, FLAG-Fzd4, and SNAP-Fzd4 plasmids were generously provided by Drs Cynthia M. Smas , Jorge FPlasmids for Fstl1, EC domain, and EC–VWC domain were already described . PlasmidHEK293T cells and mouse IMCD3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (high glucose) supplemented with 10% fetal bovine serum (FBS). NRK-49 fibroblasts were cultured in DMEM/F12 (1:1) medium supplemented with 10% FBS.HEK293T cells were seeded in 6-well plates and transfected with Fstl1-HA in combination with plasmids for Wnt ligands using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Cells and media were collected for IP.To study the effects of FSTL1 on the Wnt pathway, NRK-49F cells were seeded in a 12-well plate or 24-well plate and transfected with Fstl1-HA in the absence or the presence of plasmids for Wnt ligands. The transfected cells were starved with FBS-free DMEM/F12 medium containing 0.1% bovine serum albumin (BSA) for 20 h before the cells were harvested for analysis.Cell lysates were incubated with antibodies as indicated in specific experiments at 4 °C overnight. The solutions were then incubated at 4 °C for 6 h with protein G or protein A beads (Pierce Biotechnology). Eluted proteins were subjected to Western blotting using antibodies as indicated.To examine the interaction between endogenous FSTL1 and Wnt ligands in kidney, whole lysates from kidneys with UUO for 7 days were preabsorbed with protein G beads at 4 °C overnight. Preabsorbed lysates were incubated with rabbit anti-Wnt1 antibody  and rabbit anti-WNT3a antibody  or normal rabbit immunoglobulin G at 4 °C overnight. Protein A beads were added, and lysates were incubated at 4 °C for 6 h. Eluted proteins were subjected to Western blot analysis using anti-FSTL1 antibody .To determine whether there are direct interactions between FSTL1 and WNT3a, or between FZD4 and FSTL1, recombinant WNT3a , FSTL1 , and FZD4-Fc  proteins were added to Tris-buffered saline/0.1% Triton X-100 at indicated concentrations and incubated overnight at 4 °C. Input samples were collected, and remaining mixtures were incubated either with anti-FSTL1 antibodies  followed by Protein G agarose that had been blocked with 1% BSA for the FSTL1 and WNT3a interaction or with Protein A agarose that also had been blocked with 1% BSA for the FZD4-Fc and FSTL1 interaction. Eluted proteins and inputs were analyzed by Western blotting.For SNAP–biotin pulldowns, HEK293T cells were transfected with plasmids for SNAP-Fzd4 (100 ng/ml) and the chaperone MESD (50 ng/ml). About 24 h after transfection, cells were serum starved overnight before they were incubated for 3 h with Wnt3a conditioned medium in the absence or the presence of Fstl1-HA conditioned medium, which were also derived from HEK293T cells. Cells were incubated with 1 μM cell membrane–impermeable biotinylated SNAP substrate  for 15 min at room temperature. After washing with PBS, lysates were incubated with streptavidin–agarose (Thermo Fisher Scientific) for 6 h, and the precipitates were analyzed by Western blotting for WNT3a and SNAP-FZD4.A total of 25 to 40 μg of protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were probed with anti-Wnt1 , anti-FSTL1 , anti-V5 , anti-HA , anti-WNT3a , anti-Myc , anti-SNAP , anti-p-GSK3β , anti-GSK3β , anti-active (nonphospho) β-catenin , anti-β-catenin , anti-p-LRP6 , anti-LRP6 , anti-α-SMA , anti-Fn , anti-collagen 1 , anti-TGF-β1 , anti-p-Smad3 , anti-Smad3 , anti-p-Smad1/5/8 , anti-Smad1 , anti-p-JNK , anti-JNK , anti-p-CaMKII , and anti-CaMKII  antibodies. The signals were developed using Enhanced Chemiluminescence Western blotting detection reagents .Mouse IMCD3, HEK293T, and NRK-49F cells were transiently transfected with a BMP-responsive reporter , a TGF-β-responsive reporter ((CAGA)12MPL-Luc), or a β-catenin responsive TOPflash or 8× TOPflash luciferase reporter, in combination with pTK-RL (Promega), with or without cotransfection with FSTL1-HA plasmid, in the absence or the presence of plasmids for various Wnt ligands. Approximately 24 h after transfection, the medium was replaced with serum-free medium, with or without mFSTL1  or hFSTL1  protein, in the absence or the presence of mWNT3a protein . About 16 h later, cells were lysed, and luciferase activity was determined with the Dual Reporter Assay kits (Promega). Experiments were performed in triplicate or quadruplicate wells. Relative light units were calculated as ratios of firefly and Renilla luciferase values.For Fstl1 targeting, NRK-49F cells were transfected (110 nM) with scrambled siRNA control  or a mixture of two rat fstl1 siRNA sequences : 5′-GCUGGAAGCCGAGAUCAUUTT-3’; 5′-GCCUCAACCCAUCCUUCAATT-3’.Total RNA was extracted using RNA extraction kit  with DNase I treatment. Reverse transcription was performed using PrimeScript RT reagent kit . Real-time PCR was performed using SYBR Green PCR Master Mix  on ABI Prism 7900 Sequence Detection System (PE Biosystems).t test. A p value of less than or equal to 0.05 was considered statistically significant. Representative results of one of three experiments were presented for luciferase assays, and representative results of one of two or three experiments were presented for IP.All data are represented as mean ± SD of three or more independent replicates. Comparisons between multiple groups were made by one-way ANOVA. Comparisons between two groups were made by Student’s All animal studies were approved by The Chinese University of Hong Kong Animal Experimentation Ethics Committee and were conducted in accordance with The Chinese University of Hong Kong animal care regulations. The use of human kidney samples was approved by Sun Yat-sen University Experimentation Ethics Committee.All data are contained within the article. All original data pertaining to this study will be made available upon request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Does anyone want bad teeth?) to make them more professional, to appear more charismatic, and to convince an audience. However, in oral presentations, it is not only the what that matters in using stylistic devices like RQs, but also the how, i.e., the RQs’ prosodic realization. To date, however, virtually no handbook on the way of giving a good presentation scrutinizes this prosodic how. Therefore, our investigation focuses on the prosodic realization of German RQs in sales pitches. Specifically, we carry out a perception experiment in which 72 listeners rated both the sales pitch and its speaker based on presentations that contained questions that were lexically biased towards a rhetorical interpretation. They were realized with either the prosody of RQs or information-seeking questions (ISQs: What time is it?). An additional baseline condition was constituted by regular declarative statements with the corresponding prosody. More precisely, we investigate whether particular identified prosodic realizations—previously found for German RQs and ISQs—meet the listeners’ expectation in the context of a presentation situation. We found that listeners prefer lexically marked RQs that are produced with a prosody that is characteristic of German ISQs. We therefore suggest that handbooks should provide their readers not only with clear definitions of RQs as a stylistic device in presentations , but also with the respective prosodic realization  to make them a properly implemented stylistic device.Previous literature recommends using stylistic  devices in presentations such as rhetorical questions (RQs:  The important function of stylistic  devices in speeches has already been described and applied by ancient Greek philosophers . Generally, RQs can appear as both wh-questions and polar questions Who likes dogs?(4)Does anyone like football?According to several authors, the interpretation of the examples (1) and (2) as RQs arises from world knowledge  and (4) can be either signaled and interpreted as RQs – e.g., if the answer to the question is clear to all interlocutors –(4) indicate that there are different types of RQs , in this way evoking agreement  that was found to be characteristic for German wh-RQs and polar RQs in the production experiments mentioned above  and (4)), which they had to realize as naturally as possible in its previous context. The participants’ realized questions were recorded. The realized contours of the target questions were analyzed using the GToBI annotation guidelines for German intonation  and H indicates high targets . An additional asterisk (*) marks which tone (H or L) perceptually shapes the accented syllable. In their analysis of string-identical RQs and ISQs, Braun et al. (celery in Who likes celery? where * marks the tone that is associated with the stressed syllable in celery). As a starting point for investigating RQs in oral presentations, the present analysis makes use of those contours that were most often observed in realizations of German RQs . Concerning ISQs, we chose a representative contour for both question types  and (4) showed that German RQs and ISQs differ with respect to their prosodic properties  which returns to a low level towards the end of the utterance (L-%), whereas wh- and polar ISQs show an accented low syllable (L*) followed by a continuous high rise of the pitch towards the end, indicated in GToBI by an additional upstep (^) in the notation (H-^H%). Polar RQs are characterized by the same pitch accent as in wh-RQs. But, unlike the latter, they end in a final plateau (indicated by H-%), with the voice staying almost at the same level until the end of the utterance.For wh-word in wh-questions or on the verb in polar questions), compared to string-identical ISQs .In subsequent perception experiments with German listeners, Neitsch  reportedhow RQs should be realized in presentations in order to provide the listeners with a strong and professional speech that includes an authentic and convincing realization of RQs. Crucially, we ask whether the particular RQ prosodies for wh- and polar questions  were inserted, one in the teaser paragraph, one in the comparison paragraph, two in the main paragraph, and one in the closing paragraph. The two authors of this paper (both are native speakers of German) opted for the question type (wh- vs. polar) whose syntactic structure and rhythm pattern that matched best with the given context.The written script to each of the two presentations consisted of four paragraphs that meet the basic requirements of a standard sales presentation Bird, : a teasedenn and the indefinite subject anyone (Will denn jemand (…) ‘Wants PRT anyone (…)’). Wh-questions were designed such that the lexical string consisted of the wh-pronoun wer ‘who’, followed by the verb. The third constituent of wh-questions was the German modal particle (PRT) denn  ‘Who wants PRT (…)’). If necessary, another constituent such as ‘das’  was inserted between verb and particle in order to create a suitable syntactic and rhythmic structure.In order to get as close as possible to the question strings of previous investigations . The design resulted in three presentation conditions: question condition with RQ prosody, question condition with ISQ prosody, and declarative condition with a typical pitch declination  shows the respective declaratives of (5) and (6).Additionally, we designed a version of each presentation as reference condition with declarative statements instead of all five questions wh-question:Wer will denn schlechte Zähne?Zähneputzen ist wichtig; ‘Brushing the teeth is important; Who wants bad teeth?’(6)Polar question:Möchte denn jemand dem Organismus schaden?Anders als Koffein ist Guarana jedoch leichter bekömmlich […] und ist auch bei regelmäßigem Genuss absolut unbedenklich für den Organismus. ‘Unlike caffeine, guarana is easier to digest [...] and is absolutely harmless to the organism even with regular consumption. Does anyone want to harm the organism?’(7)declaratives:Es will ja keiner schlechte Zähne.‘Nobody wants bad teeth.’Es will ja keiner dem Organismus schaden. ‘Nobody wants to harm the organism.’wh- and polar questions and RQ prosody  and once with wh- and polar questions and ISQ prosody .The phonetically trained speakers  recorded both sales presentations with questions: once with The recorded presentations were cut with Praat . In addition, the recorded RQs showed longer sentence durations than ISQs . The final object noun showed the typical extra lengthening in the RQ realization .Overall, the questions that were recorded in the two different prosodic ways reflected the differences that were previously identified to characterize RQs as opposed to string-identical ISQs in German  containing one presentation in one prosody version of the male speaker and the other presentation of the same prosody version of the female speaker . Prior to the experiment, participants were asked to answer questions concerning their age, education, native language as well as other languages that they had learned before the age of six. Moreover, participants were asked to check the functionality and volume of their loudspeakers. They were asked to set the volume to a comfortable loudness.sympathy, conviction, enthusiasm, determination, visionary, inspiration, attractiveness, confidence, professionalism, charisma, and credibility. During this rating process, they were allowed to listen to the presentation again. Only after a 1-to-6 rating on each of the scales had been made, participants were allowed to proceed. In a second rating block, participants were asked to answer two further questions on the same sales pitch using a slider scale (1 to 10 with 10 indicating the highest probability): ‘How probable is it that you would invest in this product?’ and ‘How probable is it that this product is successful?’. Only if participants moved the slider away from its middle position in the direction of either ‘not at all’ or ‘highly probable’, they were able to proceed with the survey.In the actual experiment, participants used a web player with a play button embedded in the survey. Participants were instructed to listen once to the recorded presentation by clicking on the play button with their computer mouse. While they listened to the presentation, they were presented with a picture that was related to the product. On the next page of the survey, participants were asked to judge the speaker’s performance and perceived traits on 6-point scales ranging from ‘not at all’ (1) to ‘very strong’ (6). Eleven attributes were used, taking into account the MASCharP Scale for rating perceived charisma by D’Errico et al. : sympathSeventy-two monolingual native speakers of German  participated in the experiment . All of them were unaware of the purpose of the study, and none of them reported any speaking or hearing disorders. Overall, the participants showed a large variation regarding their education and profession.sympathy, conviction, enthusiasm, determination, visionary, inspiration, attractiveness, confidence, professionalism, charisma, and credibility) as dependent variables, and speaker  and condition  as independent variables.Participants’ ratings were split into two blocks  and statistically analyzed using R (R Core Team 2013). Mixed effects regression models were calculated by including the rating values for each of the eleven attributes  speaker. The analysis of the subset (a) showed an advantage of the female speaker with respect to the RQ-prosody condition, which obtained overall higher ratings across all eleven attributes compared to that of the male speaker . In contrast, the effect of speaker was absent for the ISQ-prosody condition as well as for declaratives .Participants’ ratings of the speaker attributes along the 6-point scales showed an interaction between speaker and condition  and the declarative condition  were significantly higher across all eleven attributes. The ISQ-prosody condition also obtained significantly higher ratings than the declarative condition across all attributes .Regarding the subset concerning (b), the results of the female speaker indicated that – compared to the RQ condition – ratings for both the ISQ-prosody condition  and the declarative condition  were significantly higher across all eleven attributes. However, the difference between the ISQ condition and the declarative condition only approached significance (p = 0.07), indicating higher ratings for the ISQ condition than for the declarative condition.Results for the male speaker indicated that – compared to the RQ condition – ratings for both the ISQ-prosody condition  or declaratives  in their sales pitches, compared to the RQ prosody. There was no significant difference between pitches with ISQ prosody and declaratives (p = 0.73).To understand the overall picture of these findings, we take a look at the participants’ ratings for each attribute separately  and declaratives  in their presentations, compared to pitches including RQs. Again, there was no significant difference between the ISQ condition and the declarative condition (p = 0.47).Regarding visionary if they realized the target questions with ISQ prosody  rather than RQ prosody in their presentation. There was no difference between the declarative condition and the RQ condition (p = 0.11) or between the declarative condition and the ISQ condition (p = 0.36).The two speakers were rated as more inspiration, we found that speakers were rated as more inspiring if their presentations contained questions with ISQ prosody  or declaratives , compared to when they contained questions that were realized with the RQ prosody. There was no significant difference between the ISQ condition and the declarative condition (p = 0.32).For confidence showed that the speakers were rated as being more confident if they realized questions with the ISQ prosody compared to the RQ prosody . The declarative condition realized in the presentations by the speakers did not differ significantly from both the ISQ condition and the RQ condition .Results concerning professionalism showed that the speakers were generally rated as more professional if they realized their target questions with ISQ prosody  or used declaratives , compared to target questions with RQ prosody. There was no difference between the ISQ condition and the declarative condition .Results for charisma indicated that speakers were perceived as more charismatic if they relied on either the ISQ condition  or the declarative condition , compared to the RQ condition. There was no difference between the ISQ condition and the declarative condition .Results for credibility of the speakers, results showed higher ratings if the presentations contained either question realizations with ISQ prosody  or declaratives , both compared to the RQ condition. There was no additional difference between the ISQ condition and the declarative condition .With respect to the speaker  caused an additional statistical difference in the ratings . Splitting up the data according to speaker, results indicated that the male speaker was rated as significantly more sympathetic if he realized his target questions with an ISQ prosody  or used declaratives , both compared to target questions with RQ prosody. There was no additional difference between the ISQ condition and the declarative condition (p = 0.81). Regarding the female speaker, ratings on sympathy were higher if she realized target questions with ISQ prosody compared to RQ prosody , but there was no additional difference between the ISQ condition and the declarative condition (p = 0.13), or between the declarative condition and the RQ condition (p = 0.58).For instance, regarding enthusiasm showed no interaction between speaker and condition (p > 0.40), but an effect of condition, indicating that presentations were rated as significantly more enthusiastic if the speakers produced target questions with an ISQ prosody rather than an RQ prosody . Additionally, the ISQ condition showed significantly higher ratings than the declarative condition , but ratings related to presentations with declaratives did not differ from presentations containing target questions with an RQ prosody (p = 0.85). The additional effect of speaker showed higher enthusiasm ratings for the male than for the female speaker .Results obtained for attractiveness showed no interaction between speaker and condition (p > 0.70), but an effect of condition, showing that speakers were rated as more attractive if they produced their target questions with ISQ prosody  or if they used declaratives  as compared to target questions with RQ prosody. There was no difference between the ISQ condition and the declarative condition (p = 0.13). An effect of speaker indicated a higher level of attractiveness for the female than for the male speaker . The factor product (chocolate vs. toothpaste) showed no effect on the results  for any of the previous models.Results obtained for probability of product investment), and (ii) if both the speaker and the product would become successful (henceforth: probability of success). To this end, in order to achieve a better specification, product was included as a further variable in the statistical model.As described above, the second part of participants’ task was to assess (i) if they would invest in the product  or used declaratives , both compared to the RQ-prosody condition. However, there was no additional difference between the ISQ condition and the declarative condition (p = 0.62). Regarding the dataset of the female speaker, there was no difference at all . Regarding (i), participants’ ratings showed an interaction between condition, the results for the female speaker regarding presentations containing target questions with RQ prosody showed significantly higher ratings than for the male speaker , an effect which was absent for the ISQ and declarative conditions . Again, there was no effect of product in either of the models .Splitting up the dataset according to condition and speaker (p = 0.08). Therefore, the model was reduced to the possible main effects. Results indicate that both the product as well as the speaker were assumed to become more successful if participants listened to the female speaker compared to the male speaker  or declaratives  as compared to questions with RQ prosody. There was no difference between the ISQ condition and the declarative condition (p = 0.17) and no difference in the models regarding the product that was advertised in the presentation .With respect to (ii), participants’ ratings showed a nearly significant interaction between The present results showed that the different products that were presented did not have any effect on the results, thus underlining the goodness of the designed experimental material. The overall result for participants’ ratings of the attributes concerning the two presenting speakers indicated that the female speaker had an advantage over the male speaker, particularly with respect to the RQ condition. That is, the female speaker achieved higher ratings than the male speaker across all eleven attributes when producing target questions with an RQ prosody. Within each of the speaker-related subsets, the results of the female speaker indicated that – across all attributes – ratings were significantly higher for presentations that relied on declaratives or questions with an ISQ prosody than for presentations that contained target questions in the prosodic RQ shape, with an additional advantage of the ISQs over their declarative counterparts. The same result emerged for the male speaker, apart from the fact that the higher ratings for presentations using target questions with ISQ prosody compared to presentations including declaratives only approached significance.conviction, determination, inspiration, visionary, confident, professionalism, charisma, credibility). For all of them, we found that both speakers achieved higher ratings if they realized their target questions with an ISQ prosody than with an RQ prosody, and that presentations containing target questions with an ISQ prosody never differed significantly from the declarative condition. However, the declarative condition showed an additional difference to the RQ-prosody condition for conviction, determination, inspiration, professionalism, charisma, and credibility. That is, for all of these six attributes, both the ISQ and declarative condition were equivalent with respect to how the speaker was perceived in her/his presentation. The absence of a possible threefold gradation in these six cases indicates that a “genuine question” in terms of a question with ISQ prosody, is about equally as acceptable as a declarative in a sales-pitch setting. A further important point is that speakers using declaratives or target questions with an RQ prosody in their sales pitches sounded comparable in terms of vision and confidence. In other words, to make sure to be perceived as a visionary and confident speaker, declaratives should be avoided (or interspersed with any type of question), since their ratings fall in-between the ratings for presentations containing target questions with ISQ prosody and RQ prosody.Regarding the individual attributes that were rated in the experiment, results indicated no effect of speaker for eight out of eleven attributes  made an additional difference with respect to the ratings for probability of product investment and the probability of success, the findings showed that the male and female presenter were perceived differently depending on the respective experimental condition . For instance, even for the RQ condition that was generally rated lowest, ratings concerning the female speaker showed an advantage over those of the male speaker. Also, regarding the probability of success, both the product and the speaker were assessed as being more successful, if participants listened to the female speaker. Nevertheless, both speakers were always on the “safe side”  if they realized the target questions in their presentations with ISQ prosody.With respect to the In summary, our findings suggest that to be perceived as a good speaker in oral presentations, speakers should realize questions that function as RQs with an ISQ prosody. Using the RQ prosody in presentations like sales pitches did not only cause the lowest ratings across all tested attributes. It also led to the lowest evaluation with respect to the probability of product investment and success of both the speaker and the product. This suggests that questions that are meant to be interpreted as RQs are not automatically acceptable in all settings, such as a presentation situation, if they show the prosodic realization that was previously found to be common for German RQs. Our findings hence indicate that the prosody of RQs seems to be situation-dependent and that a definition of RQs based on their prosody cannot hold for all situations. Instead, researchers working on the investigation of prosody need to focus on the prosody of RQs and the function of prosody in different situations.what RQs are and how they are to be realized prosodically. Based on the present findings suggesting the use of an ISQ prosody  for lexically unambiguous RQs in German, the authors of handbooks should define RQs on the basis of their prosody. Moreover, the results suggest that target questions with an RQ interpretation in presentations are used and realized differently than RQs in other settings. In sales presentations, RQs can be used as openers even though RQs have previously been defined as questions that cannot be realized out of the blue  in sales presentations. However, future studies will also have to investigate the precise prosodic realization of declarative statements as a stylistic tool in presentation situations. Additionally, future investigations should take a closer look at the differences between Generally, we assume that depending on the setting  in which RQs are used, prosodic variation make RQs a highly complex, under-researched phenomenon. Prosody not only signals the communicative function of an utterance , but also the respective affective state of the speaker. If the realized prosody (of RQs) contrasts with the expected aim of a polite invitation of the audience to like or buy a particular product , the speakers receive low ratings for their performance and for the product/idea that they pitch. This suggests that results concerning prosody investigated in lab situations cannot necessarily be generalized to other non-lab situations, which, in turn, stresses the need for (a) investigations in various contexts and (b) for experiments that address the interplay between context, utterance content, and prosody in more detail.Finally, since RQs are not the only stylistic device that make a presentation more professional (cf. the CLT list of Antonakis et al.,


The 1H-NMR analysis results confirmed that epoxy resin was successfully crosslinked with the –NH2 groups of the soy protein molecule to form a water-resistance network structure, and the resulting adhesive contains active epoxy groups. It was found that the addition of PAM can decrease the apparent viscosity, lower curing temperature, and enhancing the wet shear strength of soy protein adhesives effectively, which were capable of facilitating their application as wood adhesives. The resulting soy protein adhesive containing 4% epoxy resin and 0.05% PAM dosage had a reasonable viscosity and lower cure temperature, and showed good water resistance and wet shear strength, which met the requirement for interior use plywood of the Chinese Industrial Standard. These results suggested that waterborne-epoxy resin can be used to prepare high-performance environmentally friendly soy protein adhesives, which might provide a feasible methodology to prepare bio-adhesive adhesives for plywood industrial applications.A simple and useful approach by using non-toxic and water-soluble raw material to improve the bonding properties of soy protein adhesive has attracted much attention recently. The objective of this research was to provide a simple and environmentally friendly approach for preparing a high adhesion performance soy protein adhesive in aqueous solution by using waterborne-epoxy resin, soy protein and water-soluble polyacrylamide (PAM). The chemical structure and curing characteristics, as well as the initial viscosity and adhesion performance of the resulted soy protein adhesive were characterized by  Waterborne epoxy resin mixed with polyacrylamide crosslinked modified soybean protein adhesive. Soy protein, the main byproduct obtained from the production process of soybean oil, contains several functional groups, such as primary amine, hydroxyl and carboxylic groups.3 These reactive function groups make the soy protein molecule available to be modified by chemical approaches for preparing a competitive formaldehyde-free bio-adhesive. Thus, many efforts have focused on improvement in the adhesive properties by modifying the soy protein, mainly including chemical denaturation, crosslink modification, enzyme treatment, and protein molecular modification.2Industrial interest in bio-based polymeric materials from renewable agricultural products has received increased attention over the past decade. Soy proteins are among the most investigated natural compounds for various industrial applications such as wood adhesives, food packaging, bio-based composites and coatings, 4–7 However, the resulting molecule structure network of adhesive is relatively easy to hydrolyze in wet conditions, due to its contained many hydrophilic groups. For soy protein molecular modification, high activity groups were normally grafted onto soy protein molecules through esterification, acylation reaction, etc. to form a cross-linked network structure.8,9 This method can markedly improve the water resistance of soy protein adhesive, however, the main drawback is the complex preparation procedures, which makes it impractical for application in the plywood production.The denaturation treatment of soy protein usually used urea, alkali, sodium dodecyl sulfate, and guanidine hydrochloride to unfold the complex molecule structure of protein and expose the internal hydrophobic groups to enhance the soy protein adhesives water resistance.10–15 In these cross-linkers, epoxy monomer and resulting resins have received special attention, because epoxy groups can freely react with abundant nucleophilic groups such as –NH2, –OH and –COOH on the soy protein molecular. Furthermore, most epoxy resins were produced from the epichlorohydrin, which can be derived from sustainable resource of bio-based glycerol.16 Huang J. et al. synthesized three types of polyepoxides including triglycidylamine (TGA), glycerol polyglycidyl ether (GPE) and trimethylolpropane triglycidyl ether (TTE) as crosslinker for soy-based adhesives.17 The plywood panels bonded by soy-based adhesives with TGA and GPE did meet the interior use requirement.Crosslinking modification is regarded as an effective and facile approach for improving the water resistance of soy protein adhesives. This modification mean use of different chemical species to increase the spatial density of potential crosslinking site. Active polymer and resin appear to be efficient and feasible cross-linkers, since their containing reactive groups can react with the hydroxyl, amino and carboxylic acid groups of soy protein molecular to form a covalent-linkages network within the protein matrix to improve the bonding properties and water resistance of soy protein adhesives. Several researchers have used polymeric methane diphenyl diisocyanate, polyamidoamine-epichlorohydrin (PAE) resin, polyethylene glycol adipate, synthetic latex and epoxide resins as cross-linkers to modify soy protein adhesives, and they have already been proven effective.et al. reported that a soy-based adhesive was prepared by using soybean flour, polyacrylamide, and epoxy resin as cross-linker, which met wet shear strength requirements of interior use panel.18 Enzyme-treated soy-based adhesive was blended with commercial epoxy resin before the application, and a water resistance crosslinked structure was formed during its curing process, resulting in the improvement of plywood bonding strength.19 In addition, the addition of epoxy resins to soy-based adhesives was found to improve its gluability and flowability. These efforts mainly focus on physical mixing of soy-based adhesive directly with cross-linker before its application, whose water-resistant structure was formed during its hot-press curing process. However, taking account of longer curing time of epoxy resin,20 the limited hot-pressing time was not enough for the sufficient crosslinking reaction between epoxy groups and functional groups of soy protein. Furthermore, epoxy resin is oil soluble, which made it difficult to adequately dissolve in water-based soy protein adhesives, thus resulting in the reduction of reinforcement efficiencies of epoxy resin and requiring a large dosage. Waterborne epoxy are dispersed in aqueous solution as colloidal particles, and amine is usually utilized as curing agent. More importantly, many waterborne epoxies are formulated without volatile solvents and use merely water as a diluent, which leading to ultra-low, or even zero, VOC paints and varnishes.21 Therefore, using waterborne-epoxy resin as crosslinking agent added during the preparation process of soy protein adhesive through effective crosslinking provide a simple and useful approach to form uniform and well-distributed bio-adhesive. To our knowledge, this approach to soy protein adhesives modification has not, to date, been reported.Commercial epoxy resin was also an effective cross-linker for enhancing the water resistant and bonding properties of soy protein-based adhesives. Luo 1H nuclear magnetic resonance (NMR). Differential scanning calorimetry (DSC) was also employed to evaluate curing properties of the adhesives.In the present work, a series of soy protein bio-adhesive was developed using waterborne-epoxy resin as cross-linker and soy protein. Because the reactive functional groups on soybean protein molecule are limited, we introduced polyacrylamide (PAM) containing rich amino group into the adhesive matrix to increase crosslinking density through the multiple reaction of epoxy resin, soybean protein, and PAM. Effects of epoxy resin and PAM on the developed adhesive properties were investigated, and by measuring initial viscosity and wet shear strength of plywood verify the performance of soy protein adhesives were enhanced. Crosslinking reaction between waterborne-epoxy resin and soy protein was also confirmed by 2.2.17 molecular weights was obtained from Ruida Purification Materials Co., Ltd., Henan, China. A commercial waterborne-epoxy resin of bisphenol-A  was purchased from Shenzhen Jitian CHEMICAL Co., Ltd. from Guangdong Province, which had a weight per epoxide (WPE) of 463–468, 50% of solid content and viscosity of 820–950 mPa s. Poplar veneer  was supplied by Linyi, Shandong Province, China.Soybean protein isolate  was purchased from Baiwei Biological Technology Co., Ltd, Hebei, China. Defatted soy flour (SF) with about 52.2% of crude protein ground from defatted soy meal was purchased from Shandong San Wei Oil Group Co., Ltd. The remaining components in SPI are mainly moisture (≤4%), ash (≤3%), and fiber (≤1%). The SPI was ground to 200 mesh or more firstly. Sodium hydroxide (NaOH) and other chemical reagents were provided by Xilong Scientific Co., Ltd. Non-ionic polyacrylamide (PAM) with 1.2 × 102.2Soy protein adhesive crosslinked by epoxy resin (EPOXY-SPI) and PAM modified EPOXY-SPI (EPOXY-SPI-PAM) were prepared according to the following procedure:First, for the pure SPI adhesive, 20 g of SPI and 170 g of distilled water were added to into a three-necked flask and stirred rapidly for 20 min at 40 °C water bath. And then the pH value of SPI slurry was adjusted to about 9.0 with 20% NaOH solution (w/w).For the EPOXY-SPI adhesive, various mass fractions of epoxy resin  were added into the pure SPI adhesive and continued stirring 30 min at 45 °C. Final adhesive was then obtained after cooling reaction product temperature to 25–30 °C.For the EPOXY-SPI-PAM adhesive, the same procedure and the same amounts of ingredients as EPOXY-SPI adhesive were used except that different concentrations of PAM aqueous solution were used to substitute for distilled water, and the addition amount of epoxy resin was set to 4%. The used amounts of PAM  were determined by the relative weight percentage of the resulted adhesive.2.3The viscosity of the fresh adhesives was measured with a rotary viscosity meter . The experiments were conducted using NO. 3 rotor at 25 °C with a spinning rate of 12 rpm. There were triplicate replications for each measurement and the average value of viscosity was shown 2.46 for 1H-NMR analysis. All spectra of sample were acquired with a Bruker DRX-400 spectrometer using a 3.4 μs pulse width with a 2.0 s water pre-saturation delay. The chemical shifts of each spectrum were accurate to 0.01 ppm.The adhesive samples (about 20 mg) were dissolved in 0.5 mL DMSO-d2.5−1 under nitrogen gas (20 mL min−1).The DSC analysis were conducted using a DSC 8000  instrument with PYRIS thermal analysis software. Around 5 mg of adhesive sample were sealed in alumina pans and scanning temperature ranged from 30 to 250 °C at a heating rate of 10 °C min2.6−2. The adhesive-coated veneer was stacked between two uncoated veneers with the grain directions of two adjacent veneers perpendicular to each other. The stacked veneers were stored for 10 min at ambient conditions, and then pressed at room temperature at 0.85 MPa for 5.5 min, followed by hot-pressing under 0.95 MPa at 120 °C for 6.5 min. The resulted plywood samples were stored under ambient conditions for at least 48 h before the wet shear strength and water resistance were tested.Three-ply poplar laboratory plywood was prepared using the prepared soy protein adhesives in this study. A certain quality of adhesives were brushed on both faces of the veneer by an adhesive roller with glue spread of 18 mg cm2.7The water resistance of interior use plywood samples were tested according to China National Standards for Type II Plywood (GB/T 9846-2015). More specifically, 6 specimens (75 mm × 75 mm) were cut from two plywood panels, and then soaked in 63 ± 3 °C tap water for 3 h; followed by drying at 63 ± 3 °C for 3 h. After this procedure, the specimens were inspected for delamination. As described in standard, a specimen fails if there is any continuous opening between two layers that is longer than 25 mm. A plywood panel passes water-resistance test for interior applications if 90% of the specimens do not delaminate after the above water-soaking procedure.The test for wet shear strength was also made in accordance with GB/T 9846-2015. The plywood specimens (25 mm × 100 mm) were soaked in tap water at 63 ± 3 °C for 3 h, and then cooled down at room temperature before testing. At least twelve parallel samples from three different panels were measured and their mean value was reported as wet shear strength in 3.3.1etc. during manufacture.22 Generally, the high viscosity of soy protein adhesives may cause excessive amounts of adhesives left at the surface with little penetration into the pores; if low-viscosity adhesives are used, it will easily penetrate deep into wood through the veneer surface, so that little adhesive is left on the surface to bond the wood blocks. Either scenario results in low bond strength. Thus, the recommended viscosity of soy protein adhesive is around 6000 to 20 000 mPa s for sprayline coaters.23 According to the findings shown in 19 which indicates good flowability and penetrability into the veneer, The introduction of epoxy resin led to a marked decrease in the viscosity of SPI adhesive, which agrees with a previous study.18 With the additive amount of epoxy resin, the viscosity of EPOXY-SPI adhesive gradually decreased by 18.1% from 19 753 to 16 159 mPa s, and further decreased by 68.5% to 6229 mPa s when 7 wt% of epoxy resin was loaded, which met the operating viscosity requirements of soy protein adhesive. One reasonable interpretation for this result was that waterborne-epoxy resin acted as a dispersant to reduce the viscosity of SPI adhesive. Due to the limited amino groups of soybean protein molecule,24 PAM was incorporated into the adhesive matrix to increase the amino group content for increasing the crosslinking degree of adhesive networks through the reaction of epoxy group and amino group. As can be seen from Viscosity is very important property for the fabrication of plywood, which mainly governs the adhesive behavior including penetrability, flowability, 3.21H-NMR spectra is powerful evidence regarding whether or not crosslinking reaction between epoxy resin and SPI takes place during the preparation process of EPOXY-SPI adhesive. Therefore, 1H-NMR analysis was performed, and the spectra of the SPI, EPOXY resin and EPOXY-SPI adhesive are shown in 25–27 As shown in δ 3.50–4.27 ppm can be principally assigned to the hydrogens of methylene and methyne on repeating molecular units of the bisphenol A structure. The peaks at δ 6.8 ppm and 7.1 ppm were attributed the hydrogens on the ortho- and meta-carbon of the benzene ring. An obvious peak at around 1.6 ppm was assigned to hydrogen on the methyl group. The signals at δ 2.61, 2.79 and 3.30 ppm were attributed to the protons of –CH2 and –CH in the epoxy group. Meanwhile, the peaks intensity of the epoxy group become much weaker compared with the epoxy resin, which might be a result of the consumption on some amount of epoxy group via the crosslinking reaction of epoxy groups with –NH2 and –COOH groups on the SPI molecules.28 These results above indicated that the epoxy groups of epoxy resin were able to react with the active groups of SPI molecules  during adhesive synthetic process by a ring opening reactions of epoxy groups, and that the resulting adhesive contained a certain amount of epoxy groups, which can further react with hydroxyl group of wood to form better bonding property during its curing process. The incorporation of epoxy resin into macromolecular structure of SPI tended to increase the crosslinking density of EPOXY-SPI adhesive matrix by chemical reaction and enhance its water resistance that was proved by the results in The appearance of new chemical shifts in 3.31H-NMR analysis spectra, EPOXY-SPI adhesive had a certain amount of epoxy groups, which was able to react further with amino groups of soy protein molecule or hydroxyl group of wood during curing. For the samples of EPOXY-SPI and EPOXY-PAM, one distinct exothermic peak appeared at temperatures of approximately 160–180 °C, thus attributed to curing reaction of epoxy groups with amino groups deriving from either the soy protein molecule or PAM polymer. It is also noted that the DSC exothermic thermograms of the EPOXY-SPI-PAM adhesive also displayed one peaks at 160–180 °C. Since SPI would not cure by itself without epoxy resin, this exothermic peak was caused by the cross-linking reaction of both soybean protein and PAM with epoxy resin. In this study, the addition of PAM to EPOXY-SPI caused an increase in the proportion of amide groups in the EPOXY-SPI-PAM adhesive system. As observed from 29 The addition of PAM affected the ΔH value of EPOXY-SPI adhesives significantly. H values based on the mass of liquid adhesive. The EPOXY-SPI-PAM adhesive has much higher ΔH value than the EPOXY-SPI adhesive, which suggested that the addition of PAM made EPOXY-SPI adhesive reacted more strongly because of the higher released ΔH during curing.30The characteristic temperature range of the DSC curves gives valuable information on the cure reaction of resin adhesives. The curing properties of soy protein-based adhesives with or without crosslinker has been studied through DSC analyses, as shown in 3.43.4.119,28 (2) the hydrophilic groups  of soy proteins were consumed by epoxy group to increase relative hydrophobic property. When the addition amounts of epoxy resin were increased to/or more than 4 wt%, the shear strengths were higher than 0.70 MPa, which met the GB/T 9846-2015 standard requirements for interior use plywood. Although the EPOXY-SPI adhesive with 2.5 wt% epoxy resin had not satisfactory wet shear strength (0.65 MPa), it still pass the water resistance test. By increasing the epoxy resin additive amount to 7 wt%, the shear strength reached the maximum value of 1.04 MPa, which about 136% increase compared with pure SPI adhesive. These results show better reaction between epoxy resin and soy protein to form a water resistance structure, which was also supported by 1H-NMR analysis. On the other hand, the viscosity of EPOXY-SPI adhesives obviously reduced , therefore, SF is used to replace SPI for preparing soy protein adhesive (EPOXY-SF-PAM) and the preparation method is similar to that of SPI adhesive except for the raw materials. In this work, the effects of different epoxy resin dosage on bonding properties of EPOXY-SF-PAM adhesives were investigated. The wet shear strength of EPOXY-SF-PAM adhesives are shown in 4.1H-NMR analysis results also indicated that the resulting adhesive contain a certain amount of epoxy groups, which can further react with hydroxyl group of wood to form better bonding property during its curing. It is also seen that the addition of PAM to EPOXY-SPI was capable of decreasing the cure temperature and increasing the wet shear strength of soy protein adhesives. The crosslinked adhesive with the dosage of 4 wt% epoxy resin and 0.05 wt% PAM showed better wet shear strength and water resistance property. When the epoxy resin addition increase from 5 wt% to 10 wt%, the property of EPOXY-SF-PAM adhesive is also meet requirement of interior use plywood. In view of the stringent formaldehyde emission limits imposed on interior-used plywood, the waterborne-epoxy resin crosslinked soy protein adhesive is a better substitute as wood adhesive for formaldehyde-based resins.In this work, an environmentally friendly soy protein bio-adhesive was developed using waterborne-epoxy resin, soy protein, and water-soluble PAM. Epoxy resin was crosslinked with the active groups of soy protein as well as PAM to form a water-resistance network structure through their multiple reactions. There are no conflicts to declare.


We provide a comprehensive overview of sequence variations in Dehong humped cattle genomes with important implications for the future; moreover, we unravel the characteristics of other native cattle in China. In the current study, we investigated a high level of genomic diversities using whole-genome resequencing data in 18 Dehong humped cattle. It is speculated that Dehong humped cattle were influenced by two ancestral segments (Chinese indicine and Indian indicine cattle). Additionally, the selection signatures were detected in genomic regions that are possibly related to economically important traits in Dehong humped cattle. These results will establish a foundation for conservation and breeding programs in the future.FILIP1L, ABHD6) and immune responses . Notably, missense mutations were detected in the candidate gene ABHD6 . The missense mutations may have implications for Dehong humped cattle adaptation to hot environments. This study provides valuable genomic resource data at the genome-wide level and paves the way for future genetic breeding work in the Dehong humped cattle.Dehong humped cattle are precious livestock resources of Yunnan Province, China; they have typical zebu traits. Here, we investigated their genetic characteristics using whole-genome resequencing data of Dehong humped animals (n = 18). When comparing our data with the publicly-available data, we found that Dehong humped cattle have high nucleotide diversity. Based on clustering models in a population structure analysis, Dehong humped cattle had a mutual genome ancestor with Chinese and Indian indicine cattle. While using the RFMix method, it is speculated that the body sizes of Dehong humped cattle were influenced by the Chinese indicine segments and that the immune systems of Dehong humped cattle were affected by additional ancestral segments (Indian indicine). Furthermore, we explored the position selection regions harboring genes in the Dehong humped cattle, which were related to heat tolerance ( Bos taurus) and the humped indicine (Bos indicus) [B. indicus during the cattle domestication period [B. indicus might have been dispersed into East Asia; one important infiltration method to get into China was through Yunnan Province [Domestic cattle can diverge into two subspecies: the humpless taurine (indicus) . They pln period . Later, Province . TherefoDehong humped cattle, bearing typical zebu traits, are precious livestock resources of Yunnan Province. This breed has distinctive characteristics (the hump and pendulous skin). It is adapted to harsh conditions and hot climates. Moreover, 300 years ago, the “Gala” cattle (type of zebu cattle) were introduced through the Myanmar tract and reached the Dehong Autonomous Prefecture in the western Yunnan Province of China. Subsequently, Dehong humped cattle were produced as a result of a cross between the zebu cattle and local yellow cattle . The DehPrevious studies have used Illumina BovineHD BeadChip (777K) for the analysis of Dehong humped cattle, indicating that Dehong humped cattle are pure Zebu cattle ,5. One sHere, we generated the genome resequencing data of 18 Dehong humped cattle and then compared them with reference cattle from different geographical regions in East Asia  . The curThe study was approved by the Institutional Animal Care and Use Committee of Northwest A&F University , following the recommendations by the Regulations for the Administration of Affairs Concerning Experimental Animals of China. Specific consent procedures were not required for this study following the recommendation of the Regulations for the Administration of Affairs Concerning Experimental Animals of China. All operations and experimental procedures complied with the National Standard of Laboratory Animal Guidelines for Ethical Review of Animal Welfare (GB/T 35892-2018) and the Guide for the Care and Use of Laboratory Animals: Eighth Edition.A total of 18 Dehong humped cattle were sampled in China. These samples were collected in Yunnan Province, China. Ear tissues were collected and preserved in 96% ethanol for one day until DNA extraction.Whole genomes were re-sequenced via Illumina NovaSeq 6000 with 2 × 150 bp models at Novogene Bioinformatics Institute, Beijing, China; 150 bp paired-end sequence data were generated. Additionally, whole-genome resequencing (WGS) samples at 12× coverage, representing 4 breeds, were provided by the NCBI sequence Read Archive, including 19 Chinese indicine cattle , 10 India–Pakistan cattle , 9—Yanbian, and 15—Hanwoo. In total, 79 whole genomes of cattle were used for the subsequent analysis.Bos taurus reference genome assembly ARS-UCD1.2 by using BWA-MEM (0.7.13-r1126) [http://broadinstitute.github.io/picard (accessed on 1 September 2020)), and the Genome Analysis Toolkit (GATK version 3.6–0-g89b7209). Based on the latest reference assembly (ARS-UCD1.2), the SNPs were functionally annotated using ANNOVAR [At first, the clean reads were trimmed by using Trimmomatic v0.38) . After t . After 3-r1126) , Picard  ANNOVAR .http://www.r-project.org) (accessed on 1 September 2020).The autosomal SNPs were further filtered for missing genotypes and pruning of genotypes with a parameter (—indep-pairwise 50 5 0.2) using PLINK . To calcTo explore ancestry proportions, we added the genomic data for possible ancestral components and examined the population structures with genetic clusters of K ranging from 2 to 8 using the ADMIXTURE program version 1.3 . We emplp-value < 0.05). Based on the Bos taurus reference genome, a custom Perl script was used to annotate the segments.To infer the haplotype phase and impute the missing allele, we used Beagle v4.1  with defTo identify the selective regions in Dehong humped cattle, the nucleotide diversity (θπ) and composite likelihood ratio (CLR) were performed . BrieflySTF) and cross-population extended haplotype homozygosity (XP-EHH) have effective tools for detecting select elimination regions when strong selection signals are obtained. We separately calculated the STF and XP-EHH among the two cattle breeds using VCFtools (50 kb windows with 20 kb steps) and selscan v1.1 [p < 0.01). It is noteworthy that we calculated Tajima’s D statistic to consolidate our results by using VCFtools.The fixation index (can v1.1 . We choshttp://kobas.cbi.pku.edu.cn/) (accessed on 1 September 2020), including the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) in the present study .To understand the functions and complex pathways of candidate genes, we used KOBAS 3.0 (B. taurus reference genome (ARS-UCD1.2), the population reached ~99.7% genome coverage at a depth of 10.5X  tree A and thep < 0.05)  . The genes were selected , receptor localization to synapse , acylglycerol lipase activity , and negative regulation of transcription by RNA polymerase II   and B cell homeostasis . Genes in inflammatory pathways are also associated with the immune response , the phospholipase D signaling pathway , glutamate receptor activity , and regulation of long-term neuronal synaptic plasticity   A (Supple < 0.05) . A total < 0.05) B. The end , which 0188112) . In addi0188112) C, the mo  .By using two different methods for the signature of selection, we found 539   and 2297STF and XP-EHH) to detect the positive selection characteristics by comparing the differences in ecological adaptability between Dehong humped and Hanwoo cattle (FILIP1L and ABHD6) [ABHD6 enables acylglycerol lipase activity, which is a negative modulator of adaptive thermogenesis [ABHD6 gene in Dehong humped cattle . In addition, we obtained genes  related to the immune response [To further understand the underlying genetic mechanisms in Dehong humped cattle, we implemented two methods (o cattle A (Suppleo cattle . It is wo cattle . The annd ABHD6) ,38. Inted cattle B,C. Two response ,41,42,43Population genetic diversity is an important basis for safeguarding the evolution of species ,45. It iStudies on population structures and phylogenetic relationships are of great importance for understanding historical demographic patterns and tracing ancestral information. Dehong humped cattle consist mainly of Chinese and Indian indicine, which are inextricably linked to the human and geographical environments of their habitats. The home tract of Dehong humped cattle at the border between China and Myanmar  possesses a unique pedigree composition among Chinese native cattle. Thus, the pedigree compositions of Dehong humped cattle provided the context for exploiting the economic effects of the breed.WASF2 and PFN1, two genes belonging to GO terms—actin cytoskeleton organization. The WASF2 gene was a downstream effector molecule that involved signal transductions from tyrosine kinase receptors and small GTPases to the actin cytoskeleton. promoting actin filament formation [PFN1 plays an important role in actin dynamics by regulating actin polymerization in response to extracellular signals. Previous studies have shown that PFN1 could contribute to essential roles in postnatal skeletal homeostasis [LYN and NCKAP1L. LYN plays an important role in the regulation of innate and adaptive immune responses, hematopoiesis, responses to growth factors and cytokines, integrin signaling, as well as responses to DNA damage and genotoxic agents [NCKAP1L controls lymphocyte development, activation, proliferation and homeostasis, erythrocyte membrane stability, as well as phagocytosis and migration by neutrophils and macrophages [To reveal the ancestral proportions of Dehong humped cattle in the local region, the application of RFMix was beneficial to infer local ancestry information. Based on the Kobas 3.0 annotation, the influences of ancestral segments were explored in Dehong humped cattle and we found that Dehong humped cattle received ancestral contributions from Indian indicine and Chinese indicine. Of these, the body sizes of the Dehong humped cattle are influenced by the Chinese indicine segments. The excessive segments inherited from Chinese indicine contained ormation ,49. The ormation . PFN1 pleostasis . Among ec agents . NCKAP1Lrophages ,53. TherABHD6, selected by four methods, has been associated with heat tolerance in Dehong humped cattle. ABHD6 is located on chromosome 22 of cattle at about 0.048 Mbp. This region showed extreme differentiation and distinct haplotype patterns in two populations (Dehong humped and Hanwoo cattle). Tajima’s D analysis exhibited a significantly lower value in Dehong humped cattle. The results verified that the ABHD6 gene, which is associated with heat tolerance, showed strong positive selection in Dehong humped cattle. Additionally, missense mutations in ABHD6 may have crucial effects on heat resistance in Dehong humped cattle. In addition to genes related to heat tolerance, we obtained genes related to immune response, such as GZMM, PRKCZ, STOML2, LRBA, and PIK3CD. LRBA contributes to the secretion of the immune effector molecules. The immune genes in Dehong humped cattle may make them more able to cope with pathogenic challenges in the local environment.Dehong humped cattle, due to the humid and hot living conditions of their home tracts, have remarkable heat-tolerant properties. The gene In this study, WGS data were used to investigate the population structure of Dehong humped cattle, leading to the first in-depth research on gene diversity, phylogenetic relationships, ancestry components, and genomic regions under selection. The identified genes will be helpful to better understand the features of Dehong humped cattle and further unravel the characteristics of other native cattle in China. The revelation of the genetic diversity of Dehong humped cattle will establish a sound foundation for conservation and breeding programs in the future.


Tnfα) mRNA expression was lower in the EX + RES150 group than in the EX group. The energy utilisation of the EX + RES150 group was greater than that of the EX + RES25 group in different muscles. High-dose resveratrol intervention decreased Tnfα mRNA expression and enhanced the mRNA expressions of sirtuin 1, glucose transporter 4, AMP-activated protein kinase α1, and AMP-activated protein kinase α2 in muscles. These results revealed that high-dose resveratrol supplementation can reduce inflammation and oxidation and improve energy utilisation during short-duration high-intensity exercise.Eccentric contraction can easily cause muscle damage and an inflammatory response, which reduces the efficiency of muscle contraction. Resveratrol causes anti-inflammatory effects in muscles, accelerates muscle repair, and promotes exercise performance after contusion recovery. However, whether resveratrol provides the same benefits for sports injuries caused by eccentric contraction is unknown. Thus, we explored the effects of resveratrol on inflammation and energy metabolism. In this study, mice were divided into four groups: a control group, an exercise group (EX), an exercise with low-dose resveratrol group (EX + RES25), and an exercise with high-dose resveratrol group (EX + RES150). The results of an exhaustion test showed that the time before exhaustion of the EX + RES150 group was greater than that of the EX group. Tumour necrosis factor-α ( A habit of exercise contributes to effective metabolism, strong muscles, and low fatigue  and protFor improved sports performance, 500 mg/d of resveratrol has been combined with physical exercise to improve mitochondrial volume density and muscle function and increase total myonuclei in older adults . ResveraMost resting muscles present as contracted. However, in the early stages of muscle excitement, fewer reactive oxidative species (ROS), which promote more muscle contraction to balance energy utilisation, are produced . The mortrans-form polyphenolic compound in nature, commonly found in grape skin, cherries, and peanuts. When consumed orally, trans-resveratrol is rapidly converted to the more biologically active form of dihydroresveratrol [Resveratrol use has been reported to benefit health and has therapeutic effects in humans . Many stveratrol . This reveratrol ,22,23.This study used a downhill running exercise to mimic eccentric contraction injury and assess the efficacy of resveratrol in mice models. We hypothesised that giving mice different doses of resveratrol would protect against the damage caused by short-duration high-intensity eccentric exercise. The mechanisms of resveratrol involved in muscle inflammation, energy utilisation, and exercise performance were also evaluated.Trans-resveratrol was provided by Tokyo Chemistry Industry. All chemicals used in this study were obtained from Sigma-Aldrich .ad libitum. Mice were housed in a regular cycle (12 h light/dark) at room temperature (23 ± 2 °C) and 60–70% humidity in the Taipei Medical University Laboratory Animal Center. All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Taipei Medical University (LAC-2021-0310).C57BL/6J mice (aged 6 weeks) were purchased from the National Laboratory Animal Center . All animals were fed on a chow diet  and distilled water C57BL/6J mice were housed in the animal facility for 1 week for them to adapt to the environment before the study. Twenty-four mice were randomly assigned into four equal groups: control (NC), exercise (EX), exercise with low-dose resveratrol , and exercise with high-dose resveratrol . The mice in the EX, EX + RES25, and EX + RES150 groups underwent 3 days of acclimation running before undertaking the incremental load test (ILT). Acclimation involved running 10 m/min for 15 min with an incline of 0° each day for 3 days. All the mice underwent an exhaustion test the day after the ILT, and the downhill running section of the study started the following week. Resveratrol was dissolved in distilled water. EX-treated mice were gavaged with various amounts  of resveratrol or the vehicle  for C and EX groups for 4 weeks .Mice undertook the ILT on a motorised treadmill . The intensity of exercise was programmed to increase by 3 m/min  in the first 3 min and mice ran on a 0% gradient until exhaustion . Individ2 inhalation overdose and were sacrificed for biopsy, including biopsies of the liver, epididymal adipose tissue (eWAT), gastrocnemius muscle, soleus muscle, and tibialis anterior muscle, which were collected and stored at −80 °C for biochemical analyses. Skin irritation was also evaluated in all animals at the end of the study.After the downhill running test, mice were euthanized by a CORNA was extracted using RNAzol RT  according to manufacturer guidelines. 1 μg of RNA was reverse transcribed using a TOOLSQuant II fast RT kit . Genes were quantified using specific primers with SYBR green using the Applied Biosystems QuantStudio 1 Real-Time PCR System. The final quantification was executed using the 2−∆∆CT method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primer as a control. The sequences of primers are shown in 2 fold change was calculated as log2(sample count 1/sample count 2). The t-test was used to identify significant differences between samples from the two groups (p < 0.05). Gene ontology (GO) enrichment analysis was conducted on the differential genes obtained through screening. When p < 0.05, GO terms were significantly enriched [First, cDNA libraries were collected from four independent samples in each group. The cDNA libraries were assessed using the Agilent 2100 Bioanalyzer system and the Real-Time PCR system. β-Actin served as an internal control to verify the quality and quantity of cDNA in the PCR system. The sequenced libraries underwent fragment size detection using the Agilent Bioanalyzer 2100 system and library concentration determination using the Real-Time PCR system; the quality-confirmed libraries underwent 150-bp paired-end sequencing on the Illumina NovaSeq 6000 sequencer . Raw sequencing reads were filtered using Trimmomatic (version 0.36) . Reads wenriched . The Kyop-value less than 0.05 was considered statistically significant.All results are expressed in terms of the mean ± SEM. The significance of the difference was examined using GraphPad Prism version 9.0 . All data were normally distributed. Significant differences were analyzed using a student’s t-test or one-way analysis of variance with Tukey’s test and Bonferroni’s test for multiple comparisons. A p < 0.05).Average body weight, food intake, and tissue relative weight are shown in p < 0.05); however, the time before exhaustion of the EX and EX + RES25 groups showed no difference. A significant difference was found between the results of the EX and EX + RES150 groups . Compared with the NC group, LDH and CK in the EX + RES25 and EX + RES150 groups were also significantly increased (p < 0.05 for both), but LDH and CK in the EX + RES150 group were significantly lower than those in the EX group  the time before exhaustion decreased due to exercise injury; this was counteracted by the intervention of resveratrol; (2) inflammation in the muscles was reduced by the intervention of resveratrol; (3) the expression of genes related to energy utilisation in the EX + RES150 group was significantly higher than in the EX group, indicating that the intervention of resveratrol increases muscle energy utilisation.The time to exhaustion in the EX group was significantly lower than that in the NC, EX + RES25, and EX + RES150 groups. The results of this experiment demonstrate that resveratrol supplementation is beneficial for endurance exercise. Our study found no difference in endurance performance between the low-dose group and the high-dose group. This difference between this study and ours may have been due to differences in exercise mode (weight-loaded swimming in the previous study).Tnfα mRNA expression in the EX group was significantly higher than that in the NC group, confirming that exercise injury elevated the production of TNF-α [Glut4 mRNA was observed in the gastrocnemius and soleus muscles. This suggests that increased mRNA levels of IL-6 and GLUT4 occur synchronously; thus, IL-6 may trigger GLUT4-induced glucose utilisation in muscle.TNF-α and IL-6 levels are often used to indicate inflammation. When an inflammatory response occurs, the production of cytokines in injured tissues increases significantly. of TNF-α . Resveraof TNF-α . Such phof TNF-α ,33,34. IMany studies have found that during endurance exercise, due to the reduction in available glycogen in the body, more AMPK is activated to facilitate the production of downstream products, providing the body with energy . During Ampkα1 and Ampkα2 mRNA expression in the soleus muscle between the EX and the NC groups were found. These apparently conflicting results were also observed in a 12-week study [Type I muscle fibres support long-duration exercise, type II fibres support explosive short-duration exercise, and type IIx fibres transform into different types of fibres in relation to the intensity of the physical activity at hand . The mRNek study , and we Glut4 mRNA expression. Pereira BC et al. reported that excessive eccentric contraction training impairs insulin signal transduction in mice’s skeletal muscles and reduces the rate of GLUT4 translocation [The mRNA expression of GLUT4 in the gastrocnemius muscle and soleus muscle in the EX + RES150 group was significantly higher than that in the EX group. This result is consistent with the findings of a previous study that the intervention of resveratrol in muscle cells effectively increases the amount of GLUT4 translocated to the plasma membrane . The tiblocation . Thus, mAs an oxidoreductase, SIRT1 regulates various cellular events, including apoptosis, cell survival, endocrine signalling, and gene transcription . SIRT1 aIn this study, we assessed genome expression under resveratrol intervention in short-duration eccentric contraction exercises. We focused on muscle inflammation and energy utilisation–related gene expression. One gene that was intensively activated after high-dose resveratrol intervention was myosin light chain kinase 2 (ENSMUSG00000027470), which is intensively expressed in cardiac and skeletal muscle  and reguTnfα mRNA expression and enhanced the mRNA expressions of SIRT1, GLUT4, AMPK α1, and AMPK α2 in some muscles. These results indicate that high-dose resveratrol supplementation can reduce inflammation and oxidation and improve the utilisation of energy during short-duration high-intensity exercise.High-dose resveratrol intervention prolonged the time before exhaustion for short-duration downhill running. High-dose resveratrol intervention decreased