Lampetra morii. Although Lm-cystatin F shares a lower homology with cystatin superfamily members, it is also composed of a signal peptide and three highly conserved motifs, including the G in the N-terminal, QXVXG, as well as the PW in the C-terminal of the sequence. After sequence optimization and recombination, the recombinant protein was expressed as a soluble protein in Escherichia coli with a molecular weight of 19.85 kDa. Through affinity chromatography and mass spectrometry analysis, the purified protein was identified as a recombinant Lm-cystatin F (rLm-cystatin F). Additionally, rLm-cystatin F could inhibit the activity of papain. Based on MTT assay, rLm-cystatin F inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) dose dependently with an IC50 of 5 μM. In vitro studies show that rLm-cystatin F suppressed the adhesion, migration, invasion, and tube formation of HUVECs, suggesting that rLm-cystatin F possesses anti-angiogenic activity, which provides information on the feeding mechanisms of Lampetra morii and insights into the application of rLm-cystatin F as a potential drug in the future.Cystatins are a family of cysteine protease inhibitors which are associated with a variety of physiological and pathological processes in vivo. In the present study, the cDNA sequence of a cystatin F homologue called Lm-cystatin F was cloned from the buccal glands of  Cystatins are a group of inhibitors, which could suppress the activity of C1 cysteine proteases and play important roles in a variety of physiological process to protect our tissues from inappropriate proteolysis . RegardeIn bloodsucking animals, such as ticks, cystatins are usually expressed in their salivary glands or the midgut, which suggests that cystatins might participate in the feeding process of bloodsuckers ,13,14,15Lampetra morii (L. morii), which also suck the blood of fishes to survive, were compared through proteomic assays [L. morii that had been fed on the blood of a catfish for 60 min, which suggests that Lm-cystatin F is closely related to the parasitic mechanisms of the L. morii  were also investigated.To date, cystatin F from the other vertebrates and invertebrates was extensively studied ,26. Howehttp://www.cbs.dtu.dk/services/SignalP/), the signal peptide sequence of Lm-cystatin F is MSRVASLSLLLCGLCYFCCEA, which indicated that Lm-cystatin F might be secreted extracellularly (Lm-cystatin F has been submitted to the GenBank database (accession number: MG902948). Based on the three-dimensional structure of human cystatin F reported in the previous study, the mimetic structure of Lm-cystatin F was performed and it contains three α helixes, 5 β sheets, two loops (L1 and L2), and two disulfide bonds . As shown in Escherichia coli (E. coli) named rLj-26, which is a mutant of rLj-RGD3, and it also migrated as a single protein band on 16.5% Tricine SDS-PAGE [In order to further reveal the functions of cystatin F in SDS-PAGE . The resNaja naja atra) showed anti-angiogenic activity [50) of rLm-cystatin F on HUVECs’ proliferation was 5 μM  and 38 ± 9.8% (p < 0.01), respectively. Similarly, 3 and 7 µM rLm-cystatin F suppressed HUVECs adhered to laminin by 27 ± 5.6% (p < 0.01) and 45 ± 7.6% (p < 0.01), respectively, and thwarted HUVECs adhered to collagen IV by 12 ± 4.3% (p < 0.05) and 31 ± 3.5% (p < 0.01), respectively. Furthermore, 0.1 and 0.2 ng LPS did not affect the adhesion rate of HUVECs when collagen IV, fibronectin, and laminin were used as adhesive molecules (p < 0.05) and 75.6 ± 3.6% (p < 0.01) in the 3 and 7 μM rLm-cystatin F treating groups, respectively (p < 0.05) and 78.2 ± 2.8% (p < 0.01) in the 3 and 7 μM rLm-cystatin F treating groups, respectively  and 58.2 ± 6.23% (p < 0.001) in the 3 and 7 μM rLm-cystatin F treating groups, respectively , cystatins (family 2), and kininogens (family 3) [L. morii) are one of the most primitive vertebrates. Actually, lots of proteins identified from agnathans are clustered as a single group, which is located at the bottom of the phylogenetic tree [In the present study, a cystatin F homologue was cloned from the buccal gland secretion of s family , cystatis family , cystatitic tree .Pichia pastoris (P. pastoris) or insect cells probably due to the relatively complex structures, which might affect their expression and folding [E. coli [E. coli, the recombinant cystatins usually contain a tag with a relatively larger molecular weight that needs to be removed or set in an empty plasmid control to eliminate its effects on the functions of the recombinant cystatins [E. coli (our unpublished data). As Lm-cystatin F contains eight rare codons in its cDNA sequence, we speculated that the rare codons might lead to the difficult expression of Lm-cystatin F in E. coli. Thus, we optimized the sequence of Lm-cystatin F to replace the rare codons without changing its amino acid sequence. With the help of a plasmid named pTf16, which would help the recombinant protein form correct folding, rLm-cystatin F was obtained as a soluble protein with only a His-tag. Importantly, the yield of rLm-cystatin F was also improved and it showed inhibitory effects on papain activity and angiogenesis. This method helped us acquire the rLm-cystatin F in a relatively simple step, which provided novel information for the expression of the other members in the cystatin superfamily in the E. coli system. Actually, the expression of recombinant proteins in E. coli usually encounters aggregation or degradation due to their inability to form correct folding [E. coli to check whether rLm-cystatin F formed the same folding as that expressed in virus or eukaryotic systems.To date, previous studies used affinity chromatography, reverse-phase chromatography, and ion-exchange chromatography to purify the native cystatin from the venom of the snakes to reveal its biological functions ,32. The  folding ,16. Actu[E. coli ,34. Althystatins ,36,37. A folding . At pres folding . In the Lampetra japonica, was expressed in E. coli with a His-tag (unpublished data). Additionally, rLj-GRIM19 was proved not to be able to inhibit novel blood vessel generation in chorioallantoic membrane (CAM) models, suggesting that rLj-GRIM19 did not possess anti-angiogenic activity [E. coli and contained a His-tag, was also proved not to suppress tube formation of HUVECs in vitro. This further indicated that the His-tag would not affect the functions of rLm-cystatin F. Furthermore, the residual imidazole in the purified rLm-cystatin F was removed through ultrafiltration as the molecular weight of imidazole is only 68 Da, which is far smaller than that of rLm-cystatin F (19.85 kDa). During the expression in E. coli, LPS might be accompanied with the production of recombinant proteins. Additionally, it needs to be removed in extremely special conditions. In the present study, we did not remove the residual LPS in the purified rLm-cystatin F, as LPS was reported to promote angiogenesis, which was contrary to the functions of rLm-cystatin F [In the present study, the His-tag in rLm-cystatin F was not removed. According to the previous studies, a recombinant protein named rLj-GRIM19, which was identified in the buccal glands of activity . In our statin F ,41. FurtBitis arietans), Japanese Habu (Trimeresurus flavoviridis), Taiwan cobra (Naja naja atra), pit viper (Bothrops jararaca), Brown Treesnake (Boiga irregularis), elapid, etc. [Eriocheir sinensis) [Naja naja atra was able to suppress the growth, invasion, and metastasis of B16F10 cells and MHCC97H cells by reducing the activity of cathepsin B, matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9), and inhibition of epithelial-mesenchymal transition [L. morii was also able to repress angiogenesis based on our in vitro studies. Although Lm-cystatin F shares 29% identity with sv-cystatin, it also suppressed crucial steps of angiogenesis, including the proliferation, adhesion, migration, invasion, and tube formation of endothelial cells (HUVECs), probably due to the highly-conserved motifs in its amino acid sequence. In addition, previous studies have reported that papain and some cysteine proteases could promote angiogenesis [L. morii inhibit the wound healing process of the host fishes. However, further studies are still required to clarify the detailed mechanisms of Lm-cystatin F on anti-angiogenesis. In addition, previous studies have shown that angiogenesis is closely related to tumor progression as novel blood vessels would provide nutrition for the tumor cells, thus, rLm-cystatin F might also affect the activity of tumors [According to the previous studies, cystatins participate in a variety of physiological activities and are closely related to the initiation of several diseases in abnormal conditions ,7,8,9,10id, etc. ,42,43,44id, etc. . In the id, etc. . At the inensis) . In 2011ansition . Two yeaansition . The autansition . This isogenesis ,47. Thisf tumors . HoweverL. morii were captured in December of 2015 in Yalu River in Liaoning province of China. After collecting the buccal glands of L. morii in an RNase-free tube, total RNA was immediately extracted through a TakaRa MiniBEST Universal RNA extraction Kit . According to the instructions of the manufacturer, the total RNA was used to synthesize cDNA templates by a PrimeScriptTM RT-PCR Kit . Bases on the ORF sequence (Accession number: ENSPMAP00000007215) in the Ensembl database (www.ensembl.org), the primers for Lm-cystatin F, were designed and listed as follows: 5’-ATGTCCCGTGTGGCATCGTTGTC-3’; 5’-TTAGGCGTTTGGCATGGTAGAAGGT-3’. After PCR amplifications, Lm-cystatin F was subcloned into a pMD® 19-T Vector and sequenced by a PRISMTM 3730XL DNA Analyzer .The handling of live animals was approved by the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University (Permit Number: SYXK2004—0029). In the present study, the http://www.expasy.ch/tools/blast). The multiple sequence alignments of cystatin F were performed by ClustalX 1.83 software. A neighbor-joining tree was constructed by MEGA 4.0 software based on the pair-wise deletion of gaps/missing data and a p-distance matrix of an amino acid model with 1000 bootstrapped replicates.The amino acid sequence and bioinformatic analysis of Lm-cystatin F were performed on the website listed in EcoR I and Pst I restriction sites were designed based on the optimized sequence of Lm-cystatin F and listed as follows: 5’-GGAATTCCTGCCGGAAACCCGTTG-3’; 5’-AACTGCAGTTAAGCGTTCGGCATGG TAG-3’. Subsequently, the optimized sequence of Lm-cystatin F was subcloned into a pCold I vector and transformed into chaperone competent cells with a plasmid named pTf16  at the same time. After induction with 0.5 mg/mL L-Arabinose and 0.1 mM IPTG at 15 °C for 24 h, the cells were collected through centrifugation at 4 °C and washed with 10 mM Tris-HCl buffer containing 25 mM NaCl and 10 mM imidazole. The rLm-cystatin F was purified through a HisTrap affinity column  equilibrated with the above Tris-HCl buffer and eluted with the 40–400 mM imidazole in a gradient concentration. Next, 30 mL rLm-cystatin F was firstly concentrated to 2 mL rLm-cystatin F through Amicon® Ultra-15 10K Centrifugal Filter Devices . Then, 10 mL PBS was added into the above filter and further ultrafiltrated to 2 mL. This step was repeated for three times. After ultrafiltration, the concentration and the purity of rLm-cystatin F were, respectively, detected by a BCA Protein Assay kit  and 12% SDS-PAGE. The protein band of rLm-cystatin F on 12% SDS-PAGE was digested in-gel by trypsin  and analyzed by MALDI-TOF/TOF mass spectrometry . Furthermore, a control protein, rLj-26, was obtained based on the methods reported in the previous study [TM Chromogenic LAL Endotoxin Assay Kit, Nanjing, China).Firstly, the sequence of Lm-cystatin F was synthesized by substituting the rare codons in the original sequence of Lm-cystatin F . Then, the primers with us study . The resBased on the previous studies, the inhibitory effect of rLm-cystatin F on the activity of cysteine proteases was analyzed by using casein as a substrate . BrieflyOD595 indicated the absorbance of the reactions which contained casein, papain, and rLm-cystatin F; while OD595’ indicated the absorbance of the reaction, which contained only casein and papain. Triplicate experiments were performed independently.2 incubator . After trypsin digestion, the HUVECs were put into 96-well plates and cultured in the incubator for 24 h. rLm-cystatin F was diluted to different concentrations from 0–11.3 μM with the above medium without FBS. The same volumes of PBS and rLm-cystatin F were incubated with the HUVECs in the incubator for 24 h. Then, 5 mg/mL MTT solution was put into the above HUVECs for 4 h. After removing the medium without drawing the formazan, 100 μL dimethyl sulfoxide (DMSO) was added into the HUVECs and incubated at 37 °C for 10 min. The absorbance at 492 nm of the HUVECs in the presence of PBS and rLm-cystatin F was recorded by a microplate reader . The proliferative rate of HUVECs was calculated according to our previous studies [HUVECs were obtained from Dr. Jihong Wang in Liaoning Normal University. HUVECs were cultured in the medium named RPMI 1640  in the presence of 10% FBS  in a CO studies . At the 2 incubator for 2 h (collagen IV) or 3 h (fibronectin and laminin). Subsequently, the HUVECs without adhesion were washed with PBS buffer and the adhesive HUVECs were measured with MTT assay. During the migration assays, the pretreated HUVECs were put into the upper chamber of the Transwells. Meanwhile, the 1640 medium with 15% FBS was added into the lower chamber of the Transwells. Twenty hours later, the polycarbonate filters were fixed with the fixative. After removal from the Transwells, the filters were put onto the slides and stained with the Wright-Giemsa solution according to the previous studies [After digestion, the HUVECs were collected and resuspended with the FBS-free 1640 medium. PBS, 3, and 7 μM rLm-cystatin F were then added into the HUVECs. During the adhesive assays, fibronectin, laminin, as well as collagen IV, were dissolved in PBS buffer with final concentration of 0.1 mg/mL and then were, respectively, used to coat the 96-well plates at low temperatures. After removal of the extracellular matrix proteins, the pretreated HUVECs were put into the above 96-well plates and incubated in the CO studies . Four vi studies . In addiFirstly, both the 96-well plates and tips were put at 4 °C overnight. Subsequently, the Matrigel  was diluted with the 1640 medium in the absence of FBS and then added into the 96-well plates. After incubation at 37 °C for 40 min, the PBS, 3, and 7 μM rLm-cystatin F were, respectively, mixed with the HUVECs and then added into the above 96-well plates. After 10 h, the inverted fluorescence microscope was used to observe the tube formation from the HUVECs. Four views were randomly selected and captured. The total area of the tubes was analyzed by the NIS-Elements D software. At the same conditions, the effects of LPS and rLj-26 on the tube formation ability of HUVECs were also observed with PBS as a control according to our previous studies .t-test was used to analyze the differences between the PBS and rLm-cystatin F treating groups. A statistical significance was shown as followed: * p < 0.05; ** p < 0.01, and *** p < 0.001.The experiments were performed three times, each in triplicate. Student’s L. morri. After cloning and recombination, Lm-cystatin F was successfully expressed as a soluble protein in the E. coli system with a His-tag. Although Lm-cystatin F shares low sequence identity with the members from the cystatin superfamily, rLm-cystatin F still inhibited papain activity and showed anti-angiogenic activity, which suggested that Lm-cystatin F might be an important protein to suppress the wound healing process of host fishes and might be used as a potential anti-angiogenic drug in the future.This is the first report to show the characterization of a cystatin F homologue (Lm-cystatin F) identified from the buccal glands of


Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a heterotrimeric αβγ complex that functions as a central regulator of energy homeostasis. Energy stress manifests as a drop in the ratio of adenosine triphosphate (ATP) to AMP/ADP, which activates AMPK’s kinase activity, allowing it to upregulate ATP-generating catabolic pathways and to reduce energy-consuming catabolic pathways and cellular programs. AMPK senses the cellular energy state by competitive binding of the three adenine nucleotides AMP, ADP, and ATP to three sites in its γ subunit, each, which in turn modulates the activity of AMPK’s kinase domain in its α subunit. Our current understanding of adenine nucleotide binding and the mechanisms by which differential adenine nucleotide occupancies activate or inhibit AMPK activity has been largely informed by crystal structures of AMPK in different activity states. Here we provide an overview of AMPK structures, and how these structures, in combination with biochemical, biophysical, and mutational analyses provide insights into the mechanisms of adenine nucleotide binding and AMPK activity modulation. AMPK is the primary energy sensor and regulator of energy homeostasis in eukaryotes. It is activated by energy stress in response to increased ATP consumption  or decreased ATP production , which are sensed as low ratios of ATP to AMP and ADP. Upon activation, AMPK phosphorylates downstream targets to directly or indirectly modulate the activities of rate-limiting metabolic enzymes, transcription and translation factors, proliferation and growth pathways, and epigenetic regulators. Collectively, this increases oxidative phosphorylation, autophagy, and uptake and metabolism of glucose and fatty acids, and decreases the synthesis of fatty acids, cholesterol, proteins, and ribosomal RNAs (rRNAs), as well as decreasing cell growth and proliferation ,3,4,5,6.C-terminal domain (β-CTD) that interacts with both the γ subunit, and the α-CTD, and the extended β-linker loop that connects the CBM with the β-CTD , two alternative β subunits (β1 and β2), and three alternative γ subunits  that can form up to 12 different αβγ isoforms . The α she β-CTD A,B. The he β-CTD . CBS1, 3AMPK is a highly dynamic complex with a stable core formed by the γ subunit and the α- and β-CTDs, in which the β-CTD is sandwiched between the α and γ subunits . N-terminal lobe (N-lobe), composed of a β-sheet and the αB and αC helices, and a larger α-helical C-terminal lobe  the activation loop at the entrance of the catalytic cleft; (ii) the αC helix in the N-lobe, which positions the ATP-binding lysine (K47 in human α1) and the Mg2+-binding DFG (Asp-Phe-Gly) loop; and (iii) the peptide substrate-binding catalytic loop in the C-lobe , the αC helix (L70) and the αC-αD loop (L81) are stacked against each other [AMPK belongs to the RD (Arg-Asp) kinases, in many of which phosphorylation stabilizes the activation loop through a charge interaction between the negatively charged activation loop phosphate, and the positively charged residues from the αC helix (K62 in AMPK α1), the activation loop (N164), and the catalytic loop (R140). This conformation in turn stabilizes the αC helix and positions the arginine (R) and adjacent aspartate (D) of the catalytic loop for substrate binding . The halch other ,29,30, aAMPK activity is regulated at three different levels: at the level of (i) activation loop phosphorylation by upstream kinases, (ii) protection against activation loop dephosphorylation by protein phosphatases, and (iii) at the level of phosphorylation-independent, allosteric kinase activation A. Activa2+/calmodulin-dependent protein kinase kinase β (CaMKK2) [2+-dependent AMPK phosphorylation, AMP binding to the γ subunit increases activation loop phosphorylation through LKB1 by inducing a conformation that stabilizes formation of a complex between myristoylated AMPK, Axin, and LKB1/STRAD/MO25 [The two main mammalian AMPK activation loop-phosphorylating kinases are the tumor suppressor LKB1 in complex with STRAD and MO25, and Ca(CaMKK2) ,40,41,42RAD/MO25 ,43. HoweRAD/MO25 ,16,17 anRAD/MO25  and LKB1RAD/MO25 . In addition to adenine nucleotides, glucose, glycogen, and nicotinamide adenine dinucleotides are also important energy metabolites. Glucose has recently been identified as an important AMPK activity regulator, but it does so without direct AMPK binding ,46. In cFinally, a number of pharmacological activators bind AMPK at a unique site at the interface between CBM and KD , as first shown for Merck compound 991 , and derBesides activity regulation, the level of AMPK is regulated by ubiquitination and proteasomal degradation in brown adipose tissue , testis The structure of the yeast and mammalian AMPK core scaffolds revealed a disk-shaped γ subunit composed of four CBS sites. Each CBS consists of a strand-helix-strand-strand-helix fold (β1-α1-β2-β3-α2) with long intervening loops A. β1 is While the structure of the core complexes revealed how adenine nucleotides bind the γ subunit, they did not provide information on how the binding signal is transduced to the KD in the α subunit. In 2011, the Gamblin and Carling groups crystallized an AMPK complex containing rat α1, human β2 CTD, and rat γ1 . While tOf the three functional CBS sites, only CBS3 interacts with the α subunit, an interaction that is directly modulated by AMP and ATP. In contrast, CBS1 and CBS4 do not interact with any part of the α- or β-subunit. Moreover, CBS4 binds AMP very tightly ,58 and iC-terminal helix (α3) directly binds the hinge between the KD N- and C-lobes at the backside of the KD  first demonstrated that the activation loop directly interacts with the core of AMPK [A major regulatory mechanism for AMPK activation by AMP and ADP is the protection of activation loop p-T172 (human α1 T174) against dephosphorylation. p-T172 protection can be demonstrated in a cell-free, reconstituted system independent of the phosphatase used , and AMP does not, or only slightly inhibit the dephosphorylation of a different substrate, casein, by PP2Cα . Therefo of AMPK . Specifi of AMPK . However of AMPK , indicat of AMPK , β1-link of AMPK ), p-T172AMPK is a molecular machine consisting of the adenine nucleotide-binding core (γ subunit plus α- and β-CTDs), the catalytic KD, and at least four dynamic domains . We propose that adenine nucleotides, ADaM site ligands, and CBM phosphorylation affect the conformation of the KD through induced movements of the dynamic domains, while phosphorylation of activation loop and S/T loop modulate the KD conformation directly. Through concerted efforts, the mechanism of direct, allosteric AMPK activation through AID movement and αC stabilization is relatively well understood. However, the structural basis of direct inhibition by ATP, of activation loop accessibility regulation through ligands and possibly phosphorylation, and of the AMP-induced interaction with Axin and the LKB1 complex all remain poorly understood. The most important future challenges in AMPK structural biology will therefore be the determination of the structures of holo-AMPK in its inhibited, ATP-bound conformation, and in complex with Axin and LKB/STRAD/MO25.


Discharge from sliding outlet glaciers controls uncertainty in projections for future sea level. Remarkably, over 90% of glacial area is subject to gravitational driving stresses below 150 kPa (median ∼70 kPa). Longstanding explanations that appeal to the shear-thinning rheology of ice tend to overpredict driving stresses and are restricted to areas where ice sheets only deform (roughly 50%). Over the more dynamic portions that slide, driving stresses must be balanced by thermo-mechanical interactions that control basal strength. Here we show that median bed strength is comparable to a threshold effective stress set by ice–liquid surface energy and till pore size. Above this threshold, ice infiltrates sediment to produce basal layers of debris-rich ice, even where net melting takes place. We demonstrate that the narrow range of inferred bed strengths can be explained by the mechanical resistance to sliding where roughness is enhanced by heterogeneous freeze-on. Across all glaciers, ice caps, and ice sheets, the gravitational driving stress, and therefore the average basal shear stress falls in a narrow range that tops out around 1 bar. Here, the authors show that the mechanical resistance posed by heterogeneous infiltration of ice into sediments governs the peak bed strength. As recognized by Nye and elaborated upon further by Weertman3, when combined with considerations of mass conservation, the shear-thinning rheology of ice yields a plausible explanation. The argument can be summarized as follows: the scale of stresses needed to shear ice at a characteristic velocity [U] over a characteristic depth [d] implies that [τ] ~ [η][U]/[d], where the ice viscosity follows Glen’s law [η] = 1/(2[A][τ]n−1) with temperature-dependent ice softness [A] and n is an empirical exponent. Assuming flow to be close to a steady state so that glacier mass is conserved, this ice motion must evacuate net accumulation [a] over upstream length [l], so [U][d] ~ [a][l]. This balance is likely responsible for the correlation observed in Greenland's outlet glaciers4 between velocity and an inverse power of height above buoyancy . Finally, recognizing that the gravitational driving stress acting down the surface slope [sinα] with weight density [ρig] leads to [τ] ~ [d][ρig][sinα], these considerations lead ton ≈ 3 and [ρig]2/[A] is approximately constant for temperate glacial ice. Equation  ≈ 0.2, and on [sinα] to the power of 2/(n + 2) ≈ 0.4.In his seminal work on the mechanics of glacier flow, Nyeτdeform] = [η][U]/[d] and driving stress [τdrive] = [d][ρig][sinα]. Much of this discrepancy can be attributed to sliding at the bottom of the ice sheet, where ice meets the sediment. Allowing for sliding along this interface reduces the deformation velocity [U], which reduces the estimate for [τdeform]. For sliding ice masses, the average basal shear stress is constrained to match the average bed strength; any connection with ice rheology is indirect. The glacial contribution to eustatic sea-level change is dominated by sliding, which occurs at appreciable rates only where basal ice nears its melting point. Model results by Pattyn5 indicate that 55% of the grounded portion of the Antarctic ice sheet is in such a thermal state, and a recent synthesis of estimates  suggests that melting temperatures prevail along a similar proportion of the bed beneath the Greenland ice sheet. Indeed, Rignot and Mouginot7 report that basal sliding exceeds internal deformation over more than 50% of the Greenland ice sheet; sliding is also common beneath smaller ice caps and valley glaciers9 and governs ice motion for vast regions of Antarctica10. Assessments of geomorphic features generated by major Pleistocene ice sheets  and Greenland (green). The median driving stresses of ~60 and 70 kPa, respectively, for these ice sheets are remarkably modest in comparison with median normal stresses σn = ρig(zsurface − zbed) of ~20 MPa in each case and maximum normal stresses extending to roughly twice that level. Since the average driving stress and resisting basal stress must be approximately in balance over the large areal scales that the constituent data points characterize, these distributions imply that more than three quarters of the basal area of both Greenland and Antarctica is subject to shear stresses below 100 kPa (1 bar); maps of driving stress for these two ice sheets are included in Supplementary Note 6. However, the consensus holds that sliding takes place over sufficiently large areas that the range of basal strengths in those regions alone should be well represented by these comprehensive data sets. Furthermore, the average thicknesses and slopes tabulated in the GlaThiDa data set8 (blue dotted) suggest that despite being subjected to much lower normal stresses , basal stresses beneath alpine glaciers fall within a similar range.Figure τ plotted in Fig. 13 and regions of current fast glacier flow suggests that the discharge from ice sheets is dominated by sliding over beds that are either completely or at least partially mantled by unconsolidated sediments. Even where sliding takes place against exposed bedrock, the ubiquity of striations and moraines in deglaciated terrain and observations of sediment plumes in water bodies adjacent to contemporary ice masses15 testify to the common availability of lithic tools that facilitate the erosive power of glaciers. The load supported by particle contacts in fluid-saturated sediments, referred to as the effective stress N, is defined as the difference between normal stress and the pressure supported by pore fluids. In consequence, the frictional component of sliding resistance that can be sourced to clastic contacts is directly proportional to N. Since rock friction coefficients μ are of the order unity, in circumstances where friction dominates, τ and N are of similar size. These observations suggest pursuing an explanation for the narrow range of τ beneath sliding ice by analyzing constraints on the range of N under which sliding is permitted.Over the vast regions characterized by the narrow range of 17, significant basal motion is limited to conditions that are very close to phase equilibrium. Bulk equilibrium conditions pertain when the free energies of ice and liquid molecules are equal at some particular temperature Tm, which is a function of the pressure to which both are subjected . Two such points are marked with open squares in Fig. Tm(Pm + ΔP) relative to Tm(Pm) that accompanies a differential pressure of magnitude ΔP between the upstream and downstream sides. However, as illustrated by an open circle in Fig. 11. Away from mineral surfaces in the pore throats that separate sediment particles, the difference between the ice and liquid pressures is given by the product of the surface energy γsl ≈ 0.035 J/m2 with the interfacial curvature. For intuition, in an idealized circular pore throat of radius rp that separates glacial ice from the liquid-saturated pores beneath, the interfacial curvature can attain a maximum of 2/rp; higher values imply that the interface curves so sharply that ice can extend downward and infiltrate the underlying pore space. This sets the threshold pressure difference pf between the ice and liquid phases when ice first infiltrates basal sediments to ΔP = pf = 2γsl/rp . More generally, the mean interfacial curvature of the ice–liquid interface at its deepest extent above liquid-saturated pores must attain a uniform value so that ΔP = pf when the equilibrium temperature is Tm − ΔT ≈ Tm[1 − pf/(ρiL)], where ρiL is the latent heat per unit volume. These considerations imply that sliding of “clean” glacial ice, with N = ΔP, can take place only with N < pf, whereas higher effective stresses are accompanied by ice infiltration into underlying pores and entrainment of ice-bound sediments12. Importantly, though particle sizes and hence the characteristic dimensions of the pore throats that separate them vary widely, the mechanical limits on comminution prevent the finest particles from being reduced in diameter below the micron scale18; this ensures that locations with pf exceeding 100 kPa are rare, and is consistent, for example, with observations beneath the Siple Coast ice streams of West Antarctica, where minimum particle sizes are around 1 micron19.Although some sliding can take place at very cold temperaturesN increases towards pf, ice conforms more closely to the geometry of the uppermost clastic surfaces and can drag them over their stationary neighbors while the pore space beneath remains saturated with liquid water. Once N > pf, ice infiltrates into the underlying sediments a distance h that is set by the balance between the gravitational load, the supporting fluid pressure, and the stress transmitted to particle contacts. The characteristic depth h* = ΔT/G, for temperature gradient G and ΔT = pfTm/(ρiL) provides a useful gauge. Steady-state predictions for the scaled freeze-on thickness h/h* as a function of the scaled effective stress N/pf are illustrated in Fig. N/pf are accommodated by an approximately linear increase in h/h* so that the temperature at the top of the ice-infiltrated zone cools enough that wetting interactions can transmit the required increased portion of the gravitational load to sediment contacts. The rightmost solid line has ice melting at N/pf result in comparatively less pronounced increases in h/h* because the higher water pressures that drive melt flow offset some of the change in net gravitational load, thereby enabling thinner ice-infiltrated zones since less stress needs to be transmitted to the sediment. The leftmost dot-dashed line shows a case in which net freezing enhances the glacier thickness by 6 mm/a and requires the liquid pressure gradient to steepen so that water can be drawn upward through the ice-infiltrated sediments. With net freezing, more dramatic changes in h/h* with N/pf are expected at first, but a maximum steady-state value of N/pf is reached at a particular h/h* (here ~0.9)—the resistance to liquid transport through thicker ice-infiltrated zones is so great that only lower values of N/pf can be accommodated. The extension of steady-state curves into the shaded region at larger depths is unstable12. Higher (lower) freezing rates lead to peak stable steady-state values of h/h* that are smaller (larger). While no single set of constitutive behavior is able to characterize the broad range of subglacial materials that host sliding, the model curves in Fig. N, decreases with pf , decreases with melt rate G. Moreover, when the heat balance requires net freezing to take place, no steady-state h is stable beyond a small multiple of h*, as delineated here by the shaded region; instead, transient freezing can continue to large depths and effectively anchor the overlying glacier to its frozen bed, leading to motion by internal ice deformation alone.As h that alter the topography of the basal slip surface. Though the geometrical perturbations along any particular flow line will be dictated by the local hydrology through N, the geology through pf, and the heat transport through G, consideration of the thermo-mechanical feedbacks along an idealized transect demonstrates the basic mechanisms through which ice infiltration limits bed strength. For a planar ice–till interface with N < pf everywhere, the local frictional resistance to sliding is τ = μN (consistent with the absence of a correlation between τ and u on many Greenland outlet glaciers4). With amplified heterogeneities so that N > pf in some regions, the enhanced cohesion that accompanies ice infiltration causes the slip surface to migrate to the base of these zones; hence, the still liquid-saturated sediments beneath regions where ice cannot infiltrate as deeply represent topographic obstacles ([u][h]/[A][λ]2)n1/, where the factor C ≈ 30 depends on geometrical details23. This transition is conceptually akin to a transition from form-to-skin-dominated drag in aerodynamics. For a given driving stress, the sliding velocity u decreases with increasing h in this regime, and in cases where the slowdown also decreases dissipation τu, the associated reduction in melt rate h. Ultimately, h can become sufficiently large to anchor the overlying glacier and halt basal sliding so that internal deformation within the ice column becomes the dominant control on the basal shear stress, which should then follow the scaling given by Eq. , with [a][l][sinα] = 102 m2/a  produce values of [τ] within the observed range illustrated in Fig. Within the sliding regime, the resistance to slip posed by obstacles separated by smaller wavelengths is overcome by ploughing, whereas localized deformation more easily surmounts obstacles with smaller amplitudes. Figure mentclass2pt{minim26. The similarity to expected values of [h*] further supports the viability of thermo-mechanical feedbacks stemming from ice infiltration as a control on sliding strength. For simplicity, and in recognition of the tendency for the most rapid glacial sliding to take place over unconsolidated sediments we have focused on simple cases in which bedrock protrusions are of limited consequence. During such “soft-bedded” sliding, we have argued that surface energy and pore size combine to control the scale of effective stress (pf) beyond which obstacle growth increases the basal roughness enough to retard significant sliding. In the opposite limit where “hard-bedded” conditions prevail, in principle the scaling in the first part of Eq.  at the base of the ice-infiltrated zone, the vertical force balance and Stefan condition provide the additional two boundary conditions needed to determine the unknown thickness h of the ice-infiltrated zone and the temperature Tl at its top. Important sediment constitutive behavior include the porosity ϕ, the variation in ice saturation Si and permeability k with temperature, and the density difference Δρtill between the sediment particles and water ρw (for simplicity the 10% density difference between ice and liquid is neglected). Figure The thickness of ice-infiltrated sediments at the base of a glacier must adjust to satisfy the constraints imposed by conservation considerations for heat, mass, and momentum. RempelG is approximated as a constant  and net conductive heat flux of 60 mW/m2). The first term in the numerator is the effective stress N at the base of the ice-infiltrated layer, which is equivalent to the load supported by the underlying sediment contacts. The second term, pf = ρiL(1 − Tf/Tm) (with temperatures in Kelvin), can be thought of as the load supported by the wetting films at the base of the ice-infiltrated layer. The third term is a small correction to the first term in the denominator, which accounts for the load supported by wetting interactions throughout the ice-infiltrated sediments. The final term in the numerator, involving the liquid viscosity η and melt rate ϕ = 0.35, Δρtill = 1650 kg/m3, and Tm − Tf = 0.031 K, with the ice saturation and permeability modeled using the power lawsβ = 0.53, α = 3.1, and k0 = 4.1 × 10−17 m2 (see Table 2 in Rempel29). Together, these parameter choices yield pf ≈ 35 kPa and h* = (Tm − Tf)/G ≈ 1 m.The terms on the denominator of Eq.  account All of the data used in this paper are publicly available.Supplementary InformationPeer Review File


Furthermore, this IgE response strongly drives tumour outgrowth of epithelial cells harbouring oncogenic mutation. These findings indicate that natural IgE antibodies support skin barrier defences, but that during chronic tissue inflammation this role may be subverted to promote tumour growth.IgE is the least abundant circulating antibody class but is constitutively present in healthy tissues bound to resident cells via its high-affinity receptor, FcεRI. The physiological role of endogenous IgE antibodies is unclear but it has been suggested that they provide host protection against a variety of noxious environmental substances and parasitic infections at epithelial barrier surfaces. Here we show, in mice, that skin inflammation enhances levels of IgE antibodies that have natural specificities and a repertoire, VDJ rearrangements and CDRH3 characteristics similar to those of IgE antibodies in healthy tissue. IgE-bearing basophils are recruited to inflamed skin via CXCL12 and thymic stromal lymphopoietin (TSLP)/IL-3-dependent upregulation of CXCR4. In the inflamed skin, IgE/FcεRI-signalling in basophils promotes epithelial cell growth and differentiation, partly through histamine engagement of H IgE is present at low concentrations in the blood and is the least abundant circulating antibody class. However, all epithelial tissues contain resident cells constitutively binding IgE antibodies. Although the half-life of free IgE in the serum is short (1–2 days), tissue-resident IgE may persists for weeks or months . In the 2). In humans, subsets of dendritic cells, monocytes and eosinophils can express low levels of FcεRI but only as a trimer, lacking the β chain (αγ2). The density of mast cell and basophil FcεR1 expression correlates with serum IgE levels, so an increase in IgE antibody may directly increase FcεRI signalling (Mast cells and basophils are the only two cell types that express the complete high-affinity receptor for IgE (FcεRI) with all four polypeptide subunits   , MC903 (tivator)  and R848tivator) , signifitivator) .+ leukocytes. Basophil numbers further increased at 48 hr after cessation of TPA treatment and then declined as the inflammation subsided, while mast cells returned to the skin (The increase in serum IgE was accompanied by an increase in the number of IgE-secreting plasma cells in the skin-draining lymph nodes (LNs) . IgE levthe skin . Thus, u–/Tcrb– mice  were markedly protected from developing tumours in this two-stage model , we investigated the skin microenvironment following repeated topical application of TPA on its own to the ear skin. This showed that TPA caused substantial de novo infiltration of CD45–/– mice . There wted skin . Similar–/– mice . Neverth–/– mice . In addislinking . A low aslinking . Further WT mice . Consist WT mice  and into WT mice  of TPA-t–/– mice , suggeste dermis , signifie dermis . Togethe–/–Fcer1a mice (–/–Igh7 mice (Cre/+Mcpt8) showed a very limited release of histamine in inflamed skin and an absence of histamine in the serum  antagonists  were grown in vitro and at 70% confluency supplemented with media from IgE-activated basophils or media alone as control. Basophil-derived conditioned media induced significant upregulation of Ki67 in EC, downregulation of the basal keratins K5 and K14, and upregulation of the suprabasal keratins K1 and K10 , suggestagonists . Indeed,in vitro  and in vin vitro . Thus, ICcl2, Ccl5, Ccl8, Ccl11 and Cxcl12 mRNAs when compared with normal UT skin , which differed significantly from the CCRs expressed on mast cells from the same tissue (–/–Ccr2) had reduced recruitment of monocytes but not of basophils  prior to topical TPA significantly reduced the recruitment of basophils to the skin , indicat UT skin . In parae tissue . Therefoe tissue , whereasasophils . Inhibitasophils , suggestasophils ; therefoasophils ,  were significantly less susceptible to tumour development  as well as exposure to noxious xenobiotics, irritants and venoms . The hosFCER1A-expression in human cSCC and good disease prognosis , the lowor Trp53) and currFCER1A and overall survival  were more protected from carcinogenesis than Cre/+Mcpt8 mice . Further, mast cells have previously been shown to support SCC growth in tumour models driven by human papilloma virus , whereas the late-phase response is driven by recruited basophils. In our study, basophils were dominant over mast cells in the inflamed skin, and only basophils appeared to enter the tumours, whilst mast cells remained at the base of the tumours and in the surrounding tissue. Epidermal hyperplasia during tissue inflammation was driven by FcεRI-signalling in basophils, and tumour growth also appeared to be promoted mainly by basophils, although we cannot exclude a role for mast cells because ma virus  and indema virus . Althougma virus . For exama virus . Interesma virus , consist1Rs, however newer findings, including this report, suggest that H4R-signalling strongly affects skin ECs. H4R-deficient mice have thinner epidermis than WT mice at steady state , and HDC-deficient mice have significantly altered myeloid cell composition including a large increase in immature myeloid cells, which are recruited to the tissue during inflammation and promote cancer growth , MC903 and 7,12-dimethylbenz[a]anthracene (DMBA) were purchased from Sigma, R848 from Enzo Life Sciences and they were dissolved in ethanol  or acetone . Skin inflammation was induced by exposing the dorsal sides of the ear skin to a single or repeated dose of 2.5 nM TPA, 1 nM MC903 or 100 μg R848 in 25 μl.For cutaneous carcinogenesis, age-matched female mice were used at 7 weeks of age. The fur of the dorsal back area was removed using hair clippers and mice were rested for 1 week. Applications of chemicals and tumour monitoring were performed as previously described . In brie2 pieces using a scalpel blade and incubated for 2 hr in digestion buffer containing 25 ug/ml liberase (Roche), 250 ug/ml DNAseI (Roche) and 1x DNAse buffer  at 37°. Following digestion, tissue was transferred into C-tubes (Miltenyi Biotech) containing RPMI-1640 media (Thermo Fisher) supplemented with 10% heat-inactivated foetal calf serum, 1% penicillin-streptomycin-glutamine (Thermo Fisher) and physically disrupted using a Miltenyi cell dissociator. Spleen and LN tissue was disaggregated mechanically using a rubber policeman. All cell suspensions were filtered and cells were counted using a CASY cell counter (Roche).Skin and tumour tissues were cut into small 1 mm2. Subsequently, intact dermal sheets were isolated and washed in PBS. They were then fixed in ice-cold acetone at –20°C for 15 min, followed by rehydration in PBS and blocking of nonspecific binding with 2% BSA for 1 hr. IgE in the tissue was visualised by first incubating with rat anti-mouse IgE  followed by Alexa Fluor 555–conjugated goat anti-rat IgE , each for 1 hr at room temperature. After extensive washing in PBS, samples were mounted in VectaShield containing DAPI (Vectashield) and visualised with a Leica SP5 confocal laser-scanning microscope (Leica).Ears were collected, split into dorsal and ventral sides and floated dermis side down in 0.5 M NH4SCN for 40 min, 37°C and 5% v/v CO1R or H4R antagonist was administered by i.p. injection of 20 mg/kg cetirizine dihydrochloride (Tocris) or 20 mg/kg JNJ 7777120 (Tocris), respectively, or of the appropriate vehicle controls.The dorsal sides of the ears were treated topically twice a week for 3 weeks with 2.5 nM TPA and subsequently fixed and paraffin embedded. Ear skin from untreated mice or the untreated contralateral ear was embedded in parallel. 5 µm sections were cut and stained for H&E. Images were obtained using a Leica DM4B microscope (Leica). Epidermal thickness was measured using Fiji software by taking five measurements per section from five sections per ear in a blinded manner. In some experiments, HFor ear skin, ears were removed and a defined central section cut. Tumours were removed from the back of the ears along with a small piece of adjacent skin. All tissues were snap-frozen in OCT on dry ice. 6 μm sections were cut using a Leica JUNG CM1800 cryostat and stored at –80°C.For staining, slides were returned to room temperature before fixation with 4% paraformaldhyde for 15 min. Samples were then blocked in 5% goat serum for 1 hr at room temperature before staining with rat anti-mouse IgE  overnight at 4°C. Following staining, samples were washed and incubated with Alexa Fluor 555–conjugated goat anti-rat IgG . After extensive washing, samples were then either further incubated with Alexa Flour 647-conjugated rat anti-mouse CD49F  or directly mounted in VectaShield containing DAPI (Vectashield). Tissue samples were visualised with a Leica SP5 confocal laser-scanning microscope (Leica).Cell suspensions were blocked for non-specific binding using antibody against FcγR (2.4G2) and 2% normal rat serum (Sigma) prior to any staining protocols. For staining of cell surface markers, cell suspensions were stained with fluorochrome-conjugated antibodies or appropriate isotype control with the addition of a fixable, live/dead discrimination dye (Invitrogen) for 25 min and subsequently washed. Intracellular staining was then carried out using an Intracellular Staining kit as per manufacturer’s instructions (ThermoFisher Scientific). Cells were fixed for 10 min at 4°C, washed with Perm buffer and stained for intracellular markers for 25 min. For intranuclear γH2AX staining, cells were fixed/permeablised in ice-cold 70% ethanol at −20°C for 2 hr, blocked with 2% normal mouse serum (Sigma), Fc-block and 2% fetal calf serum for 15 min, followed by 45 min staining for γH2AX at room temperature. Stained cells were analysed using BD FACSVerse and Fortessa X20  machines. Data analysis was performed using FlowJo 10 for Mac . For cell sorting, a FACS AriaIII High Speed Cell Sorter (BD) was used. For Imagestream analysis, internalisation scores were determined using CD45 staining as a membrane marker and CXCR4 staining as the probe. Analyses were performed using an ImageStream X Mark II imaging flow cytometer and IDEAS v6 software (AMNIS).Antibodies were sourced from Biolegend unless otherwise stated. The following antibodies were used: anti-CD11b (M1/70), anti-Siglec-F (E50-2440), anti-Ly6C (HK1.4), anti-Ly6G (IA8), anti-CD4 (GK1.5), anti-CD8 (53–6.7), anti-CXCR4 (I.276F12), anti-CD138  anti-CD45 , anti-IgE , anti-TCRβ (H57-597), anti-TCRγδ , anti-Vγ5 , anti-Vγ4 (UC3-10Ab), anti-CD117 (2B8), anti-CD41 (MWReg30), anti-FcεRI (MAR-1), anti-CD95 , γH2AX , anti-IL-4 , anti-IL-5 (TRFK5) and anti-IL-13 .+CD117+IgE/FcεRI+CD41–; basophils — CD45midCD117–IgE/FceRI+CD41+; eosinophils — CD45+GR1+CD11b+Siglec-F+; neutrophils — CD45+Ly6G+Ly6C–CD11b+; monocytes/macrophages — CD45+CD11b+Ly6G–Ly6C+; αβT cells — CD45+Tcrb+CD4/CD8+; γδ IEL — CD45+TcrdhiVg5+; dermal γδ T cell — CD45+Tcrd+Vg5-; plasma cell — FSChiCD95+CD138+.Cells subsets were gated on live singlets for all and then as: mast cells — CD45Total IgE was measured by an IgE capture method. Sera to be tested and IgE standard were added to plate wells coated with 1 μg/ml rat monoclonal anti-mouse IgE (PharMingen) and blocked with 1% rat serum. Biotinylated rat monoclonal anti-mouse IgE (PharMingen) at 1 μg/ml were then added and incubated for 2 hr at 37°C. After washing in PBS 0.05% Tween, plates were incubated with alkaline phosphatase-conjugated Streptavidin (PharMingen) for 1 hr at 37°C. After washing, detection of antibody levels was carried out by addition of alkaline phosphatase substrate pNPP (Sigma) and absorbance was measured at 405 nm. For detection of anti-phosphorylcholine (anti-PC) specific IgE, total IgE was captured with anti-mouse IgE (PharMingen), followed by addition of PC-BSA at 50 mg/ml (Sigma). After washing, detection was achieved by incubating first with anti-PC IgM antibody (clone BH8) (Millipore), followed by anti-IgM conjugated to alkaline phosphatase (Southern Biotech) and addition of pNPP substrate (Sigma). Absorbance was measured at 405 nm. Detection limit was 3 ng/ml.For IgG1 and IgG2a antibodies, NUNC Immune Maxisorp 96-well plates (Thermo Scientific) were coated with 5 μg/ml goat anti-mouse IgH+L (Southern Biotech) in borate buffered saline at 37°C for 3 hr. After washing, plates were blocked with PBS containing 0.5% BSA for 1 hr at room temperature, and appropriately diluted sera added and incubated overnight at 4°C. After washing, plates were incubated with alkaline phosphatase-conjugated polyclonal goat anti-mouse IgG1 or IgG2a  (both Southern Biotech) for 5 hr at 4°C. Following further washing, the alkaline phosphatase substrate pNPP (Sigma) was added and absorbance measured at 405 nm.Histamine levels in serum and ear supernatant were measured using an ultra-sensitive histamine ELISA kit (Enzo Life Sciences) following the manufacturer’s instructions. Skin supernatant was acquired by floating the dorsal ear skin on 250 µl of complete media for 16 hr.–ΔCt method. The following primers were used — IL-1α: F (5′- TTGGTTAAATGACCTGCAACA-3′), R (5′-GAGCGCTCACGAACAGTTG-3′), IL-4: F (5′-CATCGGCATTTTGAACGAG-3′), R (5′-CGAGCTCACTCTCTGTGGTG-3′), IL-5: F (5′-GAAAGAGACCTTGACACAGCTG-3′), R (5′-GAACTCTTGCAGGTAATCCAGG-3′), IL-6: F (5′-TGATGGATGCTACCAAACTGG-3′), R (5′-TTCATGTACTCCAGGTAGCTATGG-3′), IL-13: (5′-ACACAAGACCAGACTCCCC-3′), R (5′-CTCCTCATTAGAAGGGGCCG-3′), IL-18: F (5′-ACATCTTCTGCAACCTCCAGCA-3′), R (5′-CATTGTTCCTGGGCCAAGAGG-3′), IL-25: (5′- TGGAGCTCTGCATCTGTGTC-3′), R (5′-GATTCAAGTCCCTGTCCAACTC-3′), IL-31: F (5′-GGCCTTCCTCACTCTCTTAC-3′), R (5′-GTATAGGAACCTGGCTGGC-3′), IL-33: F (5′-CACATTGAGCATCCAAGGAA-3′), R (5′-AACAGATTGGTCATTGTATGTACTCAG-3′), TNFα: F (5′-AGCCCACGTAGCAAACCACCA-3′), R (5′-ACACCCATTCCCTTCACAGAGCAAT-3′), Ki67: F (5′-TCTGATGTTAGGTGTTTGAG-3′), R (5′-CACTTTTCTGGTAACTTCTTG-3′), K1: F (5′-TTTGCCTCCTTCATCGACA-3′), R (5′-GTTTTGGGTCCGGGTTGT-3′), K5: F (5′-CCTGCAGAAGGCCAAGCA-3′), R (5′-TGGTGTTCATGAGCTCCTGGTA-3′), K10: F (5′-GGATGAGCTGACCCTTAGCA-3′), R (5′-CATTTTGAAGGTCTCTCATTTCCT-3′), K14: F (5′-CAGCCCCTACTTCAAGACCA-3′), R (5′-GGCTCTCAATCTGCATCTCC-3′), Cpa3: F (5′-CTACGGCCCAATAGCATCCA-3′), R (5′-TGCCCAGGTCATAAACCCAG-3′), Mcpt8: F (5′-CAGTCTATCGCTGTGGTGGT-3′), R (5′-GAGCTTTGCGTTCCAGCTTC-3′), HDC: F (5′-GAGTGCACAGCACAGACAAAGG-3′), R (5′-TCTAGCTCGGTAGTATTCACT-3′), Ptgds: F (5′-GACACAGTGCAGCCCAACTTTC-3′), R (5′-GGGCTACCACTGTCTTGCACATA-3′), Hpgds: F (5′-ATCCACCAGAGCCTCGCAATAG-3′), R (5′-TCATCCAGCGTGTCCACCA-3′), Ptges1: F (5′-GGATGCGCTGAAACGTGGA-3′), R (5′-CAGGAATGAGTACACGAAGCC-3′), Ptges2: F (5′-CTCATCAGCAAGCGCCTCAA-3′), R (5′-GGTCTTTACCCACGGCTGTCA-3′), Ptges3: F (5′-ATCACATGGGTGGTGATGAGGA-3′), R (5′-AGGCGATGACAACAGCCCTTAC-3′), CCL2: F (5′-CCCAATGAGTAGGCTGGAGA-3′), R (5′-AAAATGGATCCACACCTTGC-3′), CCL5: F (5′-TGCCCACGTCAAGGAGTATTTC-3′), R (5′-AACCCACTTCTTCTCTGGGTTG-3′), CCL8: F (5′-CCCTTCGGGTGCTGAAAAG-3′), R (5′-TCTGGAAAACCACAGCTTCCA-3′), CCL11: F (5′-ATGCACCCTGAAAGCCATAGTC-3′), R (5′-CAGGTGCTTTGTGGCATCCT-3′), CCL24: F (5′-CGGCCTCCTTCTCCTGGTA-3′), R (5′-TGGCCAACTGGTAGCTAACCA-3′), CXCL2: F (5′-CGCTGTCAATGCCTGAAG-3′), R (5′-GGCGTCACACTCAAGCTCT-3′), CXCL8: F (5′-CTCTTGGCAGCCTTCCTGATT-3′), R (5′-TATGCACTGACATCTAAGTTCTTTAGCA-3′), CXCL12: F (5′-GAGCCAACGTCAAGCATCTG-3′), R (5′-CGGGTCAATGCACACTTGTC-3′), CCR1: F (5′-AAGGCCCAGAAACAAAGTCT-3′), R (5′-TCTGTAGTTGTGGGGTAGGC-3′), CCR2: F (5′-ACACCCTGTTTCGCTGTAGG-3′), R (5′-GATTCCTGGAAGGTGGTCAA-3′), CCR3: F (5′-ATGGCATGTGTAAGCTCCTCTCAG-3′), R (5′-TTGCTCCGCTCACAGTCATTTCCCCR-3′), CXCR2: F (5′-AGCAAACACCTCTACTACCCTCTA-3′), R (5′-GGGCTGCATCAATTCAAATACCA-3′), CXCR4: F (5′-TCAACCTCTACAGCAGCGTTCTCTT-3′), R (5′-TGTTGGTGGCGTGGACAAT-3′), CrTH2: F (5′-TCTCAACCAATCAGCACACC-3′), R (5′-CCTCCAAGAGTGGACAGAGC-3′), Cyc: F (5′-CAAATGCTGGACCAAACACAA-3′), R (5′-CCATCCAGCCATTCAGTCTTG-3′).RNA was extracted from isolated cell suspensions or from skin/tumour tissue, and preserved in RNA-later, using RNEasy Mini kits (Qiagen). RNA was dissolved in nuclease-free water, and yield and purity were determined. Complementary DNA (cDNA) was synthesised from RNA with an iScript cDNA synthesis kit (Bio-Rad) as per the manufacturer’s instructions. cDNA was diluted in nuclease-free double-deionized water for qRT–PCR. All primers were single-stranded DNA oligonucleotides (Sigma) that were intron-spanning, as verified by NCBI Primer-Blast tool. Real-time PCR products were detected with SYBR Green (Life) measured continuously with a ViiA 7 Real-Time PCR system . Ct values for genes of interest were normalised against Ct values of the housekeeping gene Cyclophilin (Cyc) using the 2hiCD95hiCD138+) cell sorted on a BD FACS Aria III . RNA was extracted using RNEasy Micro kits (Qiagen) and reverse transcribed using SuperScript III Reverse Transcriptase (ThermoFisher). RNA was also collected from whole spleens of naïve mice and reverse transcribed similarly. Sequences were amplified using Phusion High-Fidelity DNA Polymerase (NewEngland Biolabs) to ensure accuracy and robust performance for large PCR products. PCR with a primer in the constant Cε region 5′-CTAGGGTCATGGAAGCAGTGCC-3′ in combination with a promiscuous V region primer 5′-GAGGTGCAGCTGCAGGAGTCTGG-3′ was performed. Primers were labelled at either end with Multiplex Identifiers (MIDs) for multiplexing. PCR thermal cycling was as follows: 98°C, 30 s; 30x ; and 72°C, 10 min. Amplicons were purified by gel extraction using QIAquick PCR purification kit (Qiagen) and deep-sequenced by long-read 454 pyrosequencing on the Genome Sequencer FLX system (Roche). Raw sequencing data were presented in FASTA format and unproductive sequences were removed  at 0.5 μg/mL for 24 hr and subsequently crosslinked with anti-IgE  at 5 μg/mL. IgE-crosslinked cells were harvested for qRT-PCR analyses and the supernatants stored.Bone marrow was obtained from tibias and femurs of 8–10-week-old BALB/c mice and erythrocytes depleted using Lysis Buffer Hybri-MaxTM (Sigma). Cells were washed and seeded in T25 flasks  in 10 ml RPMI supplemented with 10% heat inactivated foetal bovine serum , 1x penicillin streptomycin glutamine (PSG) (Gibco), 0.1 mM 2-mercaptoethanol (Gibco) and 10 ng/mL rIL-3 . Cells were incubated at 37°C, 5% CO1R antagonist cetirizine dihydrochloride or the H4R antagonist JNJ 7777120 (both from Tocris).Total body wall skin from neonatal mice (<5 days old) was incubated overnight at 4°C in 5 U/ml Dispase (BD) supplemented with 1x antibiotic and antimycotic solution (Sigma). The epidermis was isolated and further digested in TrypLE Express supplemented with 200 μg/ml DNAse I and DNAse buffer. Cell suspensions were filtered and resuspended in defined KC serum-free medium with supplements (Life) and 1x antibiotic–antimycotic solution. Keratinocytes were seeded at an appropriate cell density onto tissue culture vessels coated with rat-tail-derived collagen I (Sigma). Culture vessels were washed with PBS 24 hr following seeding to remove unattached cells, and provided with fresh medium with or without the addition of histamine (Sigma) or conditioned media from IgE-crosslinked basophils. In some experiments, histamine receptors were blocked using 10 μM of the HTo block cell recruitment to the skin, intradermal injections of 20 ng anti-TSLP, 20 ng anti-IL-3, both or isotype controls were injected into the dorsal ear pinnae in a volume of 10 μl. In other experiments, mice received i.v. injections of either 0.4 ug PTX or 0.8 ug PTX (Tocris), 50 ng anti-CXCR2 (R and D), 50 ng anti-CXCR4 (R and D) or suitable controls. Skin inflammation was initiated using a single dose of 2.5 nM of TPA to the dorsal ear skin and the skin was analysed 24 hr later.+cKit–FcεRI+ cells at day 14 of culture and 2 × 105 basophils placed in transwells with a pore size of 5 μm. 100 ng CXCL12 (Peprotech), 100 ng IL-3 (Peprotech) + 1 μg TSLP (R and D), C5a (as positive control) or media alone (negative control) was placed in the bottom chamber and numbers of basophils migrating through the transwell at 37oC was enumerated after 4 hr.Bone-marrow-derived basophils were FACS-sorted as CD45The statistical significance of difference between experimental groups was determined using two-tailed Student’s t-test for unpaired data, one-way ANOVA multiple comparison, Log-rank Mantel-Cox test or linear regression, where appropriate, with results deemed significant at p<0.05. Stars of significance correlate to: *p<0.05; **p<0.01; ***p<0.001 and ****p<0.0001. Statistics were performed with GraphPad Prism 6.00 for Mac . In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This is a very interesting manuscript describing how inflammation drives an increase in IgE locally and systemically together with an influx of basophils into the tissue. The authors nicely show that IgE exacerbates skin inflammation and induces histamine-mediated promotion of epithelial cell proliferation and differentiation, which is primarily mediated through FcεRI signalling in basophils. The authors also show that basophils are recruited to the inflamed skin through the action of CXCL12-CXCR4 and contributes to tumor growth.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Nicola L Harris as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Satyajit Rath as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Marcus Robinson (Reviewer #3).Thank you for submitting your article "Natural IgE promotes epithelial hyperplasia and inflammation-driven tumour growth" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This manuscript describes how administration of the inflammatory agent TPA drives an increase in natural IgE locally and systemically together with an influx of basophils into the tissue. The authors show that IgE exacerbates skin inflammation and induces histamine-mediated promotion of epithelial cell proliferation and differentiation, which is primarily mediated through FceRI signalling in basophils. The authors also show that basophils are recruited to the inflamed skin through the action of CXCL12-CXCR4 whereby CXCR4 is upregulated on basophils by TSLP and IL-3, the latter produced by T cells. The authors then investigated whether IgE was involved in an inflammation-driven model of cutaneous carcinogenesis. In this model, low level mutations are initiated by DMBA and tumour growth is promoted by regular exposure to TPA. They show that both IgE and FceRI are required for tumour development and suggest that it is the FceRI signalling in basophils that promotes the growth of tumour cells.Essential revisions:1) The authors conclude that IgE with "natural specificities" promotes tumour growth in their model (DMBA followed by repeated TPA application). This is in contrast to the autoreactive IgE-mediated protective role that was previously published by the same group using a similar cSCC model (2+ applications of DMBA) , depleting IgE signalling (-/-FcεRIa) or IgE production (-/-Igh7) was substantially more protective than depletion of basophils alone. Thus it appears that an element of redundancy may exist between mast cells and basophils, Perhaps depleting one population gives rise to more of the other at the tumour site? If the authors have data to this effect it would be interesting to include this, otherwise the possible contribution of mast cells should be discussed in the text.2) The authors conclude that basophils mediate the IgE-dependent tumor growth. However it remains entirely possible, and even likely based on their data, that basophils and Mast cell both contribute to tumor growth as depleting both cell types (3) As the recruitment via CXCR4 is a major point of the paper, it would be prudent to demonstrate basophil chemotaxis toward CXCL12 in culture--CXCR4 is important for chemotaxis using in vitro assays of basophils. This point would greatly strengthen the paper, however if such assays cannot be completed within 2 months it would be sufficient to state clearly in the text that but cells can express chemokine receptors without exhibiting chemotaxis toward its ligands . Essential revisions:1) The authors conclude that IgE with "natural specificities" promotes tumour growth in their model (DMBA followed by repeated TPA application). This is in contrast to the autoreactive IgE-mediated protective role that was previously published by the same group using a similar cSCC model (2+ applications of DMBA) . This may indeed contribute to the differing outcomes of IgE engagement in the two models . We show here that topical TPA induces IgE with natural specificities and a similar repertoire to that in resting naïve mice.All three reviewers agreed that although the authors clearly show that TPA increases natural IgE locally and systemically, there was insufficient evidence that this natural IgE drove the tumor growth as it was measured after only 2 weeks. By contrast, TPA was applied twice weekly for >15 weeks in the tumour model. The authors should address this question by providing evidence that the IgE repertoire does not change following the extended TPA application and following DMBA + TPA used in induce tumor growth. Should this evidence not be forthcoming or possible to generate the title and claims should be revised appropriately.We fully acknowledge that ideally the IgE repertoire should have been analysed longitudinally and we would certainly like to extend our knowledge of the IgE repertoire to tumour bearing mice in the future. However, this initial repertoire analysis took a long time to perform as the immunoglobulin repertoire is not as well annotated in mice as it is in human and the heavy chain repertoire is highly variable between inbred strains ). This is one of only a few studies to show a thorough repertoire analysis of IgE in the mouse and we therefore think the data adds substantially to the literature in this field. So, although we fully recognize the value of the reviewer’s suggestion, the proposed experiment would take a lot of time  and resources and therefore is not possible within the time limit.The text has been revised accordingly in appropriate places . The role of IgE in promoting epidermal hyperplasia is measured within the same time-frame as the repertoire analysis is performed, so this text has been left unaltered.Cre/+ mic), depleting IgE signalling (FcεRIa-/-) or IgE production (Igh7-/-) was substantially more protective than depletion of basophils alone. Thus it appears that an element of redundancy may exist between mast cells and basophils, Perhaps depleting one population gives rise to more of the other at the tumour site? If the authors have data to this effect it would be interesting to include this, otherwise the possible contribution of mast cells should be discussed in the text.2) The authors conclude that basophils mediate the IgE-dependent tumor growth. However it remains entirely possible, and even likely based on their data, that basophils and Mast cell both contribute to tumor growth as depleting both cell types  that although depleting one population does not give rise to more of the other, there are other complexities of these models. Notably, the Cre/+Cpa3 and Cre/+Mcpt8 models are not complete knockout models and that the Cre/+Cpa3 mice  also have a significantly reduced number of basophils in inflamed skin and in tumour perilesional skin, although normal proportions of basophils in the tumours. Equally, although the Cre/+Mcpt8 mice lack all basophils in inflamed skin and tumour perilesional skin, some basophils do get through to the tumours . I.e. the intermediate effect on tumour susceptibility that we observe could also be due to incomplete Cre-toxicity in these models and in the case of Cre/+Cpa3 mice, that the basophils are also affected. In the shorter-term skin inflammation models, Cre/+Mcpt8 completely lack basophils and have normal mast cells numbers in the skin – so this data is more straight forward to interpret. A possible contribution of mast cells is discussed in the Discussion.Thank you for your comment. We agree, particularly in the tumour experiments there may indeed be some contribution also from mast cells and some redundancy may occur in the tissue: certainly this cannot be entirely excluded. As the reviewer mention, we routinely see a more complete phenotype in the 3) As the recruitment via CXCR4 is a major point of the paper, it would be prudent to demonstrate basophil chemotaxis toward CXCL12 in culture--CXCR4 is important for chemotaxis using in vitro assays of basophils. This point would greatly strengthen the paper, however if such assays cannot be completed within 2 months it would be sufficient to state clearly in the text that but cells can express chemokine receptors without exhibiting chemotaxis toward its ligands .We have included additional data to strengthen this point. We have performed in vitro chemotaxis experiments toward CXCL12 using bone marrow-derived basophils (that express high levels of CXCR4). We can demonstrate significant basophil chemotaxis toward CXCL12 and that IL-3/TSLP by themselves are not chemotactic . This new data has been added to the manuscript as an additional supplementary figure .


In hMAO-A inhibition, 1–3 showed mild effects (50% inhibitory concentration (IC50): 54‒114 μM). However, 1 displayed moderate inhibition of the hMAO-B isozyme (IC50: 18.14 ± 1.06 μM) followed by mild inhibition by 2 (IC50: 57.71 ± 2.12 μM) and 3 (IC50: 90.59 ± 1.72 μM). Our kinetic study characterized the inhibition mode, and the in silico docking predicted that the moderate inhibitor 1 would have the lowest binding energy. Similarly, cell-based G protein-coupled receptors (GPCR) functional assays in vector-transfected cells expressing dopamine (DA) receptors characterized 1–3 as D1R/D2LR antagonists and D3R/D4R agonists. The half-maximum effective concentration (EC50) of 1–3 on DA D3R/D4R was 15.13/17.19, 20.18/21.05, and 12.63/‒ µM, respectively. Similarly, 1–3 inhibited 50% of the DA response on D1R/D2LR by 6.13/2.41, 16.48/31.22, and 7.16/18.42 µM, respectively. A computational study revealed low binding energy for the test ligands. Interactions with residues Asp110, Val111, Tyr365, and Phe345 at the D3R receptor and Asp115 and His414 at the D4R receptor explain the high agonist effect. Likewise, Asp187 at D1R and Asp114 at D2LR play a crucial role in the antagonist effects of the ligand binding. Our overall results depict 1–3 from M. alba root bark as good inhibitors of hMAO and potent modulators of DA function as D1R/D2LR antagonists and D3R/D4R agonists. These active constituents in M. alba deserve in-depth study for their potential to manage neurodegenerative disorders (NDs), particularly PD and psychosis.In this study, we delineate the human monoamine oxidase ( Monoamine oxidase (MAO) is a flavoenzyme in the outer mitochondrial membrane of neuronal and non-neuronal cells that has a vital role in the etiology of age-regulated neurodegenerative disorders (NDs). MAO catalyzes the oxidative deamination of monoamine neurotransmitters, dietary amines, and xenobiotics and regulates their levels and functions in the brain. During oxidative deamination, MAO liberates hydrogen peroxide, the reactive oxygen species (ROS) most potent in causing oxidative stress and mitochondrial dysfunction . Though 2O2 generation, leading DA deficit and oxidative stress state. And MAO-A inhibition prevents the deamination of neurotransmitters, reduces oxidative stress, and increases the availability of neurotransmitters within noradrenergic and serotonergic neurons of the CNS to regulate neuron signaling via their respective receptors ) for hMAO-A isozyme activity , and 3 was a mixed type inhibitor  of the hMAO-B isozyme (3 with a free enzyme (Kic) and with enzyme-substrate complex (Kiu) identified were 55.19 ± 7.97 and 186.2 ± 10.26 µM, respectively. Likewise, the Ki value of 1 and 2 for hMAO-B inhibition was 17.01 ± 3.31 and 52.09 ± 5.56 µM, respectively.The enzyme inhibition patterns of compounds at different substrate concentrations in the kinetic study are tabulated in ectively a–c. The activity d–f revea isozyme c,f. FromhMAO-A) and safinamide (for hMAO-B) were docked into the active site cavity of the hMAO enzyme, and the ligands were re-docked. The results of the simulation study are tabulated in Computational modeling was performed to obtain insights into the binding affinity between ligands and the enzyme using AutoDock 4.2. To validate the docking result, the reference inhibitor selegiline as well as reversible inhibitor harmine (for 1–3)–hMAO-A complexes showed lower binding energies  than the reference ligand selegiline  and harmine . Ligand 1 posed in the active site by interacting with Gly110, Thr336, Ile207, Gly214, and Ser209 via a hydrogen bond (2 and 3 shared Met300 and Gly404 as common H-bond interacting residues. Reversible inhibitor harmine interacted with flavin adenine dinucleotide (FAD)600, Ile335, and Tyr444 residues at the active site cavity, which were not observed for test ligand-binding. In the case of hMAO-B, ligands 1–3 showed high affinity with binding energies  by forming three and five H-bond interactions, respectively  receptors by measuring their effects on secondary messengers (cAMP modulation or Ca2+ ion mobilization) in transfected cell lines expressing human cloned receptors of interest. The data in 1–3 at 100 µM on the various receptors.To characterize the possible role of compounds 1‒3 exhibited a full antagonist effect on the D1/D2 receptors and a full agonist effect on the D3/D4 receptors. The agonist effects of 1–3 at 100 µM on D3R/D4R were 119.9/86.30, 124.3/90.45, and 102.8/46.10%, respectively. Similarly, at 100 µM, 1‒3 inhibited the DA response on D1R/D2LR by 87.65/101.30, 98.85/99.15, and 67.80/78.55%, respectively. 1–3 on the DA receptor subtypes with corresponding EC50/IC50 values.As shown in the table, 1R/D2LR, with IC50 values in the range of 2.41‒31.22 µM  IDs for 6CM4, 3PBL, and 5WIV, respectively. The results of the docking simulation are tabulated in To validate the results of the functional assays and investigate and identify the ligand–receptor interactions for novel lead discovery, we carried out a computational docking simulation using AutoDock 4.2 . Since tidentity . Subtype1‒D1R complex exhibited four strong H-bond interactions with Lys81, Leu291, Ser188, and Asp314 with low binding energy . The ligand 2‒D1R complex  interacted with Ser202 and Asp103 in H-bonds, similar to the reference antagonist SCH-23390 and agonist DA. Ligand 3, with a binding energy of −9.2 kcal/mol, shared Ser188 and Asp187 with D1R via H-bonds. As shown in 3‒D1R complex . Risperidone and butaclamol are D2LR agonist and antagonist and they bound to the active site of the receptor with binding energies −12.7 and −6.9 kcal/mol, respectively, by forming salt-bridge with Asp114.Furthermore, Asp187, Leu295, Phe306, Pro171, Ala192, and Ala195 were revealed as hydrophobic residues in the ligand  complex . Figure 1–3 did not form a salt-bridge with Asp114, they showed H-bond and π-anion interactions with the residue. Furthermore, the number of H-bond interactions was higher for test ligands compared to resperidone and butaclamol.Though ligands 1 and 3, indicating high affinity with the receptor, whereas Ile184 was a crucially active residue in the second extracellular loop of D2LR, forming a π-alkyl interaction with 2. Phe189 and Val190 are necessary key residues in antagonist-ligand binding, and they were well observed in the 2‒D2LR and 3‒D2LR complexes.Specifically, Asp114 was an H-bond and electrostatic residue for both ligands 3 showed the highest affinity  for ligand‒D3R interactions . Among the test ligands, ractions . Ligand 3R, could be seen in all three ligands‒D3R complexes via electrostatic interaction  than the reference drugs  among the three ligands. Similarly, Val116, His414, and Leu187 were common interaction sites for three ligands at D4R. Other surrounding residues, including Met112, Thr434, Arg186, Phe410, Cys119, and Val193, were involved in hydrophobic interactions with the ligands.One of the most crucial residues in stimulating D helix V . Ligand 1–3 from the root bark of M. alba and found that they exhibit a mild-to-moderate hMAO inhibition effect. The inhibition effect was slightly higher on the hMAO-B isozyme (IC50: 18.14 to 90.59 µM) than on hMAO-A (IC50: 54.79 to 114.31 µM). In particular, ligand 1 demonstrated a moderate inhibition effect on the hMAO-B isozyme, with an IC50 value of 18.14 ± 1.06 µM. Among test compounds 1–3, 1 and 3 are fused benzofurans, and 2 is a mono-isoprenyl substituted flavone. The structural difference between 1 and 3 is the methyl cyclohexene in 1 and methylbenzene in 3. The methyl cyclohexene group of 1 was involved in specific alkyl interactions with Leu337, Ile335, and Met350 and this moiety is facing toward FAD at the hMAO-A active site  did not show a strong correlation with the docking score (binding energies). This variation might be attributed to the physicochemical properties of ligands, especially logP which was predicted high in the range of 6.7 to 7.3 from web-based software PreADMET  (data not reported here). Overall, the results of the hMAO inhibition assay reveal that these Diels–Alder type adducts, especially 1, might have therapeutic value in managing PD. However, this treatment approach is just symptomatic, restoring dopaminergic function in the striatum [In the present study, we tested Diels–Alder type adducts ive site . Howeverstriatum . Therefo1-like (D1R and D5R) and D2-like . These DA receptors have specific anatomical distributions and specifically mediate DA action [1R antagonist (SCH 23390) and D2R antagonists  attenuate heroin-induced reinstatement [3R antagonist (SB-277011A) blocks the acquisition and expression of the conditioned place preference response to heroin [l-tetrahydropalmatine is a D1R/D2R antagonist with an anti-addiction property [1R/D2R antagonist) demonstrated antipsychotic properties in conjunction with pro-cognitive effects in rats [2R binding affinity and antagonizing ability represent the clinical efficacy of antipsychotic drugs [M. alba mediated skin wound healing by upregulating the mRNA levels of chemokine receptor 4, one of the GPCRs [M. alba root extract or its metabolites.Depending on their stimulation or inhibition of adenylyl cyclase and modulation of cAMP levels, DA receptors are categorized into two classes: DA action . Severaltatement ,32, and ist SCH 290 and D2property , and gov in rats . The extic drugs . Previouhe GPCRs . Other t1‒3 on DA  receptors, we conducted a cell-based GPCR functional assay. As shown in 1–3 potently and concentration-dependently inhibited the agonist response of DA at D1R and D2LR. Even at 25 µM, 1–3 inhibited the DA response on D1R/D2LR by 92.32/97.16, 91.09/33.69, and 66.82/81.11%, respectively. Unlike sigmoidal dose–response curves of 1 and 2 at D1R, compound 3 showed an unusual non-sigmoidal curve  in the assay system. This NSI might be attributed to aggregation/colloid formation or chemical reactivity because these are significant sources of nonspecific bioactivity particularly in high throughput screening (HTS) [1–3 with those of a reference agonist (DA) and antagonist (SCH 23390) via a molecular docking simulation. As shown in 1–3 showed a high binding affinity . Test ligands 2 shared common H-bond interaction residues, Asp103 and Ser202 with the reference antagonist (SCH 23390). Furthermore, 1–3 displayed additional H-bond interactions with serine residues  and aspartic acid residues (Asp314 and Asp187). Similarly, at D2R, 1–3 interacted with the key interacting residues Asp114, Trp100, and Phe389 [2LR with high affinity and the binding energy was lower than that of the reference antagonist butaclamol. Interactions with Asp114, Cys118, Phe198, Phe389, Trp386, and Tyr416 were common among the test ligands and butaclamol  . NSI by d Phe389 . All thrtaclamol . Additioperidone , which we of D2R , and thes et al. , Phe389,isorders . Our fin3R and D4R. As shown in 1–3 showed a potent agonist effect on D3R and D4R. D3R is prominently distributed within the limbic system and mediates the psychiatric manifestation of DA receptor stimulation. Therefore, DA receptor agonists with high affinity for D3R have an antidepressant effect [3R-stimulation (rather than D2R-stimulation) might mediate the antiparkinsonian effects of DA receptor agonists with a high preference for D2R [3R >  D2LR  >  D1R  =  D5R  >  D4.4R) developed as a transdermal patch for the treatment of PD [The test compounds showed an agonist effect on Dt effect . Similar for D2R . Rotigotnt of PD .3R ligands [4R agonism has been implicated in the management of cognitive deficits associated with schizophrenia [A previously conducted survey reported that more than 110 patent applications had been submitted concerning selective D ligands . Unfortu ligands . Similarophrenia  and atteophrenia  and alsoophrenia . 1–3 have concentration-dependent agonist effects on D3R and D4R (rank order: D3R > D4R). Even at 25 µM, 1–3 showed potent agonist responses on DA D3R/D4R of 71.92/63.00, 64.99/58.66, and 94.93/‒%, respectively. The agonist effect of 3 on D4R was mild (% stimulation of agonist response of 44.85% at 100 µM). The antagonist effect on these receptor subtypes was negligible. We also used molecular docking simulations to compare the binding affinity and interacting residues between test compounds 1–3 and D3R . In addition to their electrostatic interactions with Asp115 on D4R, 2 and 3 formed H-bond interactions with carboxylic acid group of Asp115.The results of the functional assays in this study show that the ligands  and D3R  with tholozapine . As tabueceptors . In this1 and D5) receptor signaling is mediated chiefly by the heterotrimeric G proteins Gαs/olf, which cause sequential activation of adenylate cyclase, cyclic AMP-dependent protein kinase, and the protein phosphatase-1 inhibitor DARPP-32 [1R is responsible for l-DOPA-induced activation of mTORC1 signaling, and D1R antagonist (SCH23390) blocked the l-DOPA-induced phosphorylation of p70 S6 kinase (S6K), ribosomal protein S6, and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) in 6-OHDA–lesioned mice [1R induces ERK stimulation via a cAMP/protein kinase A (PKA)/Rap1/B-Raf/MAPK/ERK kinase (MEK) pathway and SCH 23,390 completely blocks the p-ERK1/2 levels induced by DA [At a molecular level, D1-like  receptor signaling is mediated by the heterotrimeric G proteins Gαi/o, which causes inhibition of adenylate cyclase thereby decreasing the phosphorylation of PKA substrates. Binding of DA to DA receptors regulate signaling via cAMP response element-binding protein (CREB), glutamate receptors, GABA receptors, and ion channels  [hMAO inhibition with good D1R/D2LR antagonist and D3R/D4R agonist effect. So, what could be the underlying mechanism and in vivo effect is very interesting and need to be studied shortly.Likewise, D2-like (Dtassium) . Previoutassium) ,61. Eventassium) . The tes1), kuwanon G (2), and albanol B (3) were isolated and identified from the root bark of M. alba Linn following a method described previously [hMAO isozymes and reference drugs selegiline, DA, serotonin, butaclamol, SCH 23390, clozapine, and (S)-WAY-100635 were purchased from Sigma-Aldrich .Mulberrofuran G  following the same method of enzyme inhibition. The concentrations of the test compounds for the kinetic study are presented in The potential of the test compounds for human MAO inhibition was evaluated via a chemiluminescence technique using the MAO-Glo kit . Detailed experimental conditions and procedures were reported previously ,65. The 2+ ion mobilization, depending on the receptor type. All assays were performed at Eurofins Cerep  following their in-house protocol, as stated in our previous reports [Cell-based functional GPCR assays were conducted in CHO cells transfected with a plasmid containing the GPCR gene of interest. The functional activity of the test compounds (agonist or antagonist) was evaluated by measuring their effects on cAMP modulation or Ca reports ,67,68.1R, D3R, and D4R was assessed by evaluating the effect on cAMP modulation. For this, stable transfectants  were suspended in HBSS  containing 20 mM HEPES buffer and 500 μM 3-isobutyl-1-methylxanthine, distributed into microplates (5 × 103 cells/well), and incubated for 30 min at room temperature (RT) in the absence (control) or presence of the test compounds  or reference agonist (DA). In the D3R and D4R assays, the adenylyl cyclase activator NKH 477 was added at a final concentration of 1.5 and 0.7 μM and incubated for 30 and 10 min, respectively, at 37 °C. Then, the cells were lysed and a fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody with europium cryptate) were added. The fluorescence transfer was measured at λex = 337 nm and λem = 620 and 665 nm using a microplate reader  after 60 min of incubation at RT. Agonist effects are expressed as the % of the control response to 10 μM DA for D1R and 300 nM DA for D3R/D4R. Similarly, antagonist effects are expressed as the % inhibition of the control response to DA 300 nM for D1R, 10 nM for D3R, and 100 nM for D4R. The reference agonist DA and antagonists SCH 23390, (+)-butaclamol, and clozapine were used to validate the study.The functional activity of the test compounds on D2R was tested by fluorimetrically evaluating their effect on cytosolic Ca2+ ion mobilization. In brief, CHO-D2LR cells were separately suspended in HBSS  complemented with 20 mM HEPES buffer and distributed into microplates (1 × 105 cells/well). Then, a fluorescent probe  mixed with probenecid in HBSS  supplemented with 20 M HEPES (Invitrogen) (pH 7.4) was added to each well, and the cells were allowed to equilibrate for 60 min at 37 °C. Thereafter, the plates were positioned in a microplate reader , and compounds 1–3 , reference agonist, or HBSS  were added. We then measured the fluorescent intensity, which varied in proportion to the free cytosolic Ca2+ ion concentration. Agonist effects are expressed as the % of the control response to 10 μM DA. Similarly, antagonist effects are expressed as the % inhibition of the control response to 700 nM DA. Reference agonist (DA) and antagonist (butaclamol) were used to validate the study.The functional activity of the test compounds on D1 receptor was obtained from UniProt . The β2R (β2 adrenergic receptor) has a higher similarity to DA D1R in the binding site region and sequence identity [2R crystal structure from the RCSB protein data bank (PDB) using ID 2RH1 with SWISS-MODEL. Refining the model was conducted using the ModRefiner sever [The primary sequence of the human DA Der sever .hMAO-A, hMAO-B, hD2LR, hD3R, and hD4R were obtained from the PDB with IDs 2BXR, 2BYB, 6CM4, 3PBL, and 5WIV, respectively. The 3D chemical structures of the three test compounds were obtained from PubChem Compound . The crystal structures of the reference compounds, selegiline, harmine, DA, SCH 23390, risperidone, butaclamol, eticlopride, nemonapride, and clozapine were also obtained from NCBI under CIDs 26758, 5280953, 681, 5018, 5073, 37461, 57267, 156333, and 135398737, respectively. Water and ligand molecules were removed using Discovery Studio . In the case of the hMAO isozymes, the cofactor flavin adenine dinucleotide (FAD) was retained. The Lamarckian genetic algorithm method in AutoDock 4.2 was applied. For the docking calculations, Gasteiger charges were added by default, and all the torsions were allowed to rotate. The grid maps were generated with the AutoGrid program. The docking protocol for rigid and flexible ligand docking consisted of 10 independent genetic algorithms, and other parameters were set using the defaults in the AutoDock Tools. The docking results were visualized using Discovery Studio.Automated single docking simulations were carried out with AutoDock 4.2 . X-ray c1), kuwanon G (2), and albanol B (3) from M. alba root bark in neurodegenerative diseases. Our investigations identified 1–3 as novel multi-target-directed ligands for the management of neurodegenerative diseases via hMAO inhibition and dopaminergic receptor modulation. Specifically, cell-based GPCR functional assays in vector-transfected CHO cells expressing DA receptors characterized 1‒3 as potent D1R/D2LR antagonists and D3R/D4R agonists. The assay results were further supported by molecular docking studies, which predicted tight binding between the test ligands and the receptors. Overall, the results of this study provide evidence that ligands 1‒3 from M. alba could be developed into neuronal drugs targeting DA receptors. Further in vivo studies are warranted to fully and precisely characterize the mechanism of action via a signal transduction pathway.This study is the first to report the therapeutic potential of natural Diels–Alder type adducts, mulberrofuran G (