Here, we studied the spatial organization of TRPV4 and its relationship to the adherens junction component β-catenin using single- and dual-color direct stochastic optical reconstruction microscopy (dSTORM). In non-stimulated endothelial cells, TRPV4 is clustered in small protein islands, as is β-catenin. Using dual-color imaging, we found that TRPV4 and β-catenin reside in similar islands and can be found at both the basolateral and basal membranes. Following shear stress stimulation, TRPV4 molecules formed smaller clusters, with the majority residing outside of clusters. Further shear stress stimulation changed the molecular distribution of TRPV4 molecules, limiting them to the basal membrane. This redistribution and the smaller clusters resulted in the segregation of TRPV4 from β-catenin. Furthermore, TRPV4 trafficking was controlled by focal adhesion kinase and activation of the α5ß1 integrin. These highly differentiated spatial redistributions suggest that mechanotransduction of blood flow is controlled via a more complex hierarchy than previously thought.Localized Ca In endothelium, mechanotransduction acts through conversion of step-like physical forces into biochemical information in a series of rapid switch-like events that control many aspects of development and physiology2. Cell adhesion molecules are strongly implicated in the mechanotransduction of blood flow3. Previous studies suggested that mechanotransduction of blood flow is transmitted through the cell adhesion proteins at adherens junctions to the basal membrane, which stimulate the association and dissociation of mechanosensitive integrins and extracellular matrix (ECM), indirectly and through signaling pathways. New binding between the integrins and ECM is proposed to be essential for long-distance, downstream signaling events4.Fluid shear stress associated with blood flow plays a pivotal role in vascular remodelling, arterial and venous identity and angiogenesis2+ influx through mechanosensitive cation channels plays an important role in endothelial adaptation to flow dynamics2. The Transient Receptor Potential (TRP) family of ion channels is the major class of Ca2+ permeable ion channels in the endothelium5. An increase in [Ca2+]i level following TRP channel gating leads to various effects on vascular function such as change in vascular tone, alteration in vascular permeability, change in blood coagulation, oxidative damage and vascular remodelling6. Within seconds of shear stress stimulation, Ca2+ influx into the cytoplasm through shear stress-dependent Ca2+ channels, such as TRPV4, activates inward-rectifying Ca2+-sensitive K+ channels that co-activate with the outward-rectifying Cl− channels7. These events repolarize the membrane, eventually leading to hyperpolarization, which is transmitted through myoendothelial gap junctions to the adjacent smooth muscle cells9. Furthermore, opening of TRPV4 in endothelial cells and intact endothelium results in localized Ca2+ sparklets11. These sparklets generate subcellular microdomains rich in Ca2+, which can activate a variety of Ca2+-dependent signaling cascades11.In the vascular endothelium, localized Ca2+]i level in a does dependent manner14. Further in bovine aortic endothelial cells and human umbilical cord endothelial cells (HUVECs), we have shown that shear stress sensitizes the response of TRPV4 to its selective agonist16 and in HUVECs, shear stress increases the exocytosis of functional TRPV4 channels to the cell membrane16.We have previously shown that in HEK293 cell stably expressing TRPV4 (TRPV4-HEK293) shear stress activates TRPV4 and leads to increase in i disrupts the adherens junction via activation of myosin light-chain kinase and the RhoA-Rho kinase pathway and induces actin stress fiber formation19. Here, we studied relative molecular distribution and interaction of TRPV4 channels with ß-catenin after shear stress stimulation, using single- and dual-color direct stochastic optical reconstruction microscopy (dSTORM) in HUVECs.On the cell membrane, TRPV4 interacts with ß-catenin at adherens junctions, linking them to the actin cytoskeletonWe found that TRPV4 channels are expressed in preclustered structures, composed of 20–25 molecules per cluster, and in a complex with β-catenin. After exposure to shear stress, we have observed relocation of TRPV4 channels. Upon shear stress stimulation, TRPV4 channels formed smaller clusters, with the majority of them relocated from the basolateral membrane to basal membrane, and TRPV4 lost its interaction with β-catenin. The shear-induced translocation of TRPV4 channels was controlled by focal adhesion kinase and α4ß1/α5ß1 integrin.To determine the molecular distribution of TRPV4 channels at the cell membrane, we used a super-resolution fluorescence microscopy technique, dSTORM. We labeled TRPV4 channels expressed in HUVECs with anti-TRPV4 antibodies directly conjugated to a photoactivatable fluorescent dye, Alexa 647.2 of shear stress and compared them with TRPV4 channels expressed in the absence of shear stress (Control).We analyzed Alexa 647-labeled TRPV4 channels stimulated with 10 dyn/cm21, TRPV4 channels were not randomly distributed on the plasma membrane but clustered in nanometer-scale membrane compartments −r in Ripley’s K-function analysis (Control vs SS), we observed that the degree of clustering decreased by 226.7 ± 73.83-fold (P < 0.01) and 228.5 ± 86.9-fold (P < 0.01) upon 10 dyn/cm2 SS stimulation for 30 sec and 6 hrs, respectively −r has a maximum value was indicative of the size of the analyzed clusters. The rmax values also decreased from 153.7 ± 28.8 nm for the control to 73.6 ± 9.3 nm after 10 dyn/cm2 SS stimulation for 30 sec −r and rmax values, suggesting that SS responses of TRPV4 are stimulus-dependent  in the endothelium, we used dual-color STORM. HUVECs were fixed and permeabilized, and TRPV4 and ß catenin were subsequently labeled with the fluorophore-conjugated monoclonal antibodies anti-TRPV4-Alexa 647 and anti-ß catenin-Alexa 488. Before switching most of the dyes to the dark state for dSTORM acquisition, we used diffraction-limited total internal reflection fluorescence microscopy (TIRFM) and acquired an image of TRPV4. In TIRFM images, TRPV4 were homogeneously distributed in the absence of SS, residing in small nanoclusters. ß catenin also appeared homogeneously distributed, with significant fluctuation of fluorescence signals observed around the basolateral membrane. After shear stress stimulation, the majority of TRPV4 relocated to the basal membrane and was absent from the cell borders, while ß catenin molecules were still homogeneously distributed Fig. .1. This 2+-permeable ion channel and that increase in [Ca2+]i disrupts adherens junctions, we hypothesized that TRPV4 may play a regulatory function in modulating endothelial membrane permeability function. Therefore, we examined the interaction of TRPV4 with ß catenin in endothelial cells by TIRFM and dSTORM after specific fluorescence labeling with polyclonal anti-ß catenin antibody.TRPV4 formed a complex with ß catenin in adherens junctions of keratinocytes. Given that TRPV4 is a CaFirst, in TIRFM imaging, we observed some clustered structures of ß catenin molecules for both Control and SS stimulation cases. In non-activated cells, the majority of TRPV4 and ß catenin molecules resided together in nanoclusters that were composed of 11.6 and 12.6 molecules of TRPV4 and ß catenin, respectively. The interaction between TRPV4 and ß catenin was at both the basolateral and basal membrane sites Fig. 1,2. In Furthermore, we confirmed these findings by quantitative analysis. We used bivariate cross-correlation function analysis and calculated the fraction of TRPV4 molecules that had at least one neighboring ß catenin molecule Fig. . In the Further, we examined the effect of sustained shear stress on spatial redistribution of TRPV4 and its interaction with ß-catenin by stimulating endothelial cells grown inside the microfluidic channel for 6 hrs. Using dual color dSTORM we found that similar to transient stress (30 sec SS stimulation), the sustained stress (6 hrs SS stimulation) also significantly reduces the interaction of TRPV4 with ß-catenin Fig. .2. Mechanotransduction through β1 integrins controls the trafficking of different membrane proteins to different subcellular compartments23. In the endothelium, αvβ3 and α5β1 play important roles in the mechanotransduction of blood flow24. Previously, we showed that shear stress stimulation controls the rapid trafficking of TRPV4 channels to the cell membrane and showed that trafficking was independent of αvβ316. Here, to examine the hypothesis that mechanotransduction through α4β1 or α5β1 integrin controls the cell surface expression of TRPV4, we used HEK293 cells stably expressing TRPV4 channels fused to Venus protein. Venus-TRPV4 fluorescence was measured in real-time at the sarcolemma by TIRFM. First, in TIRFM images, we observed clusters of TRPV4 channels at both the basolateral and the basal membranes. Upon stimulation, using an anti-α5β1 integrin-activating antibody (clone 12G10), we found progressive increases of the evanescent field intensity at both the basal and the basolateral membranes  by 0.8 ± 0.1-fold  at 20 dyn/cm2. We found that pretreatment of endothelial cells with 100 nM of FAK inhibitor blocked the sensitization effect of shear stress on the TRPV4 response to GSK1016790A . Furthermore, activation of α5β1 using the 12G10 antibody to β1 integrin stimulated the effect of shear stress on TRPV4 responses to its agonist by 0.83 ± 0.18-fold  at 10 dyn/cm2. Inhibition of β1 integrins using P5D2 antibody or ATN-161, a non-RGD- based peptide, mimetic of fibronectin25, reduced the sensitization effect of shear stress to the basal level  or 0.79 ± 0.13-fold  respectively)  inhibits the binding of β1 integrin to fibronectin27.To investigate the insertion of functional TRPV4 channels, we used Caly) Fig. . The antFurther, we used cell surface biotinylation assay to investigate the effect of anti-α5β1 on membrane expression of TRPV4 in HUVECs. Using this approach, we found that stimulation of HUVECs with anti-α5β1, clone 12G10, increases the membrane expression of TRPV4 by 0.38 ± 0.14  fold, compared to unstimulated endothelial cells Fig. .20, Kv2.1 channels in neurons and transfected human embryonic kidney cells21, and MscL channels in lipid bilayers18.Here, we examined the molecular distribution of TRPV4 on the membrane using super-resolution microscopy. First, we investigated the distribution of endogenous TRPV4 in human endothelial cells using antibody staining. In the absence of shear stress, TRPV4 channels resided in nanoscale clusters or protein islands, as previously shown for Nav and Kv channels at the nodes of Ranvier in neurons2+ influx via TRPV4 into the cytoplasm16, we hypothesize that TRPV4 channel activity might be modulated by protein interactions within the clusters.Upon shear stress stimulation, these TRPV4 clusters dispersed into individual channels. Since this event coincided with CaThe mechanotrnsduction, mechanisms by which cells sense and respond to mechanical stress involves the conformational changes of protein domains that are subject to such stress.28.β -cathenin is the main adapter protein of the adherens junctions at the point of cell-cell contact that is proposed to transmit the shear stress to the cell interior and cytoskeleton. In particular, it is proposed that tension regulates the protein domains of the β-cathenin that is involved in cell-cell junctions or its cytosolic domain in order to stabilize the cellular adhesion site and regulate the downstream signaling event28. Thus, β-cathenin is proposed to act as mechanical buffer which can preserve a link between the cadherin ectodomain and the actin cytoskeleton by absorbing energy through unfolding. Such conformational changes might affect the interaction of β-cathenin with other signaling proteins and receptors. In keratinocytes, TRPV4 interacts with β catenin via its N-terminus to armadillo repeat domain of β-catenin17.Structurally, β-cathenin is modular protein that contains 12 characteristic armadillo repeats in its central region, R1 to R12, from N- to C-termini that mediates protein-protein interaction. The armadillo repeat domain contains three tyrosine phosphorylation sites that could modulate β-cathenin binding to α-actinin and cadherins. β-cathenin is very sensitive to small changes in mechanical stress, unlike other components of adherens junctions, such as cadherinsBecause TRPV4 molecules bind to β catenin at keratinocytes, we expected that in endothelial cells, they might also form such a complex. Here, using dSTORM imaging, we found that only 23% of TRPV4 molecules were in a complex with β catenin, while the rest were segregated. This finding was quantitatively supported by cross-correlation function analysis and by analysis of the degree of interaction, which showed that the determined probability of co-clustering was higher than for randomized events.2 of shear stress in a transient manner for 30 sec or in a sustained manner for 6 hrs. Previously we showed that this intensity and duration of signalling was sufficient to stimulate the cell surface expression of functional TRPV4 channels24. Shear stress stimulation resulted in segregation of TRPV4 channels from β catenin counterparts and led to the relocation of the channels to the basal membrane. We anticipated that this relocation might be accompanied by formation of new complexes. However, upon analysis of further interactions with other membrane proteins, such as eNOS, Cav-1 and β1-integrins, we could not detect any significant complex formation with those molecules before or after shear stress stimulation.To analyze TRPV4-β catenin pairs in shear stress-stimulated endothelial cells, we treated endothelial cells with 10 dyn/cm2. PECAM-1 mediates homophilic adhesion, and through its cytoplasmic domain, it binds to the β and γ-catenins. Shear stress induces the association of β-catenin and PECAM-1 with the PI(3)K p85 subunit that is dependent on the adaptor protein, VE-cadherin2. The formation of this signaling complex correlates with PI3K and AKT activation and regulates β1-integrin activity in endothelium30.In endothelial cells, the transmembrane immunoglobulin family protein, PECAM-1, in a mechanosensory complex with VE-cadherin and transmembrane tyrosine kinase vascular endothelial growth factor (VEGF) receptor, Flk-1, mediates the endothelial responses to flow18. RhoA activity is stimulated by Ca2+ influx into the cytoplasm19, thereby facilitating cytoskeleton remodeling and formation of adherens junctions. The relocation of TRPV4 from adherens junctions to the basal membrane and the increase in [Ca2+]i via TRPV4 and RhoA activation supports the concept that shear-induced relocation of TRPV4 from the adherens junction to the basal membrane, and localized Ca2+ influx via TRPV4, causes the prolonged activity of Rho family signaling molecules that accelerate cytoskeleton remodeling and control endothelial membrane permeability.Here, the observed relocation of TRPV4 after shear stress stimulation suggests that TRPV4 may have two different functions. First, via interaction with β1-integrins, it may act as a scaffolding protein that controls the localization of β catenin in adherens junctions. Second, TRPV4 relocation and activity may control RhoA activity and cytoskeletal remodeling. Adherens junctions in the endothelium play important roles in maintaining endothelial membrane permeability function and in regulating the mechanotransduction of blood flowIn summary, TRPV4 and β catenin were preclustered in the same protein islands or nanoclusters. Stimulation with shear stress changed the distribution of TRPV4 and confined them to the basal membrane, where channels lost their interaction with the adherens junction component, β catenin. Changes in TRPV4-β catenin localization post-shear stress stimulation suggest that TRPV4 might negatively regulate the membrane permeability of endothelial cells. Furthermore, the relocation of TRPV4 was regulated via β1 integrin in an outside-in signaling pathway.Anti-TRPV4 (LS-Bio) and anti-ß catenin (Abcam) antibodies were labeled with Alexa 647 and Alexa 488 (Invitrogen), respectively, according to the supplier’s instructions. The labeling efficiencies of TRPV4 and ß catenin were determined to be 0.7–1 dye/protein by absorption spectroscopy assay.2, passaged every 3–4 days, and used for up to 5 passages.Human umbilical vein endothelial cells (HUVECs) were cultured in complete endothelial growth medium-2 (EGM-2) according to the supplier’s instructions (Lonza). Cells were incubated at 37 °C in a humidified incubator gassed with 95% air and 5% CO16 and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum (FBS), hygromycin (50 µg/ml), and blasticidin (5 µg/ml).HEK293 Flipin T-Rex cell lines (Life Sciences) stably expressing Venus-TRPV4 were generated as reported elsewhereFor fixed-cell experiments, cells were rinsed in phosphate-buffered saline (PBS), fixed for 10 min in 4% paraformaldehyde (PFA) at room temperature, and subsequently washed three times in PBS before imaging.16.Western blotting and cell surface biotinylation assay as reported before× TIRF objective; a 1.49 numerical aperture; an Andor iXon electron multiplying charge coupled device (EMCCD) camera; 405-, 491-, 561- and 633-nm laser lines; an appropriate quadrant multiband laser filter (Semrock); a programmable mechanized stage; and infrared autofocus (Perfect Focus).All TIRFM experiments were performed on a Nikon Ti microscope controlled by Nikon Elements software. The microscope was fitted with a 1002, 2 mM CaCl2, 2 mM probenecid and 10% FBS, pH 7.4.). Live-cell TIRFM imaging experiments were performed and analyzed as reported in detail elsewhere16.HEK293 cells stably expressing Venus-TRPV4 were plated on chamber slides (Ibidi µ-Slide VI 0.4) and induced with 0.1 µg/ml tetracycline for 12 hours. Before each experiment, the growth medium was replaced with imaging buffer  containing: 140 mM NaCl, 5 mM KCl, 20 mM HEPES, 11 mM D-glucose, 1 mM MgCldSTORM imaging was performed on a Nikon N-STORM system based on a Nikon Eclipse Ti microscope with a 100× /1.46 N.A lens . TRPV4, ß catenin or ß1 integrin samples stained with Alexa 647-TRPV4, Alexa 488 ß catenin or ß1 integrin were excited by a 640-nm and 488-nm lasers using a Quadrant bandpass filter. Photo-conversion rates were accelerated using a 405-nm UV diode laser  in epifluorescence mode. Fluorescence emissions from Alexa 647 and Alexa 488 were separated by a quadrant multiband laser filter (Semrock). The stream acquisitions of images were obtained using a back-illuminated EMCCD camera  at a readout speed of 5 MHz with full electron multiplying (EM) gain, allowing continuous photo conversion and excitation of the samples.2 tissue culture incubator overnight. Subsequently, cells were stimulated with a shear stress of 10 dyn/cm2 and were fixed with ice-cold 4% (vol/vol) formaldehyde and 0.2% (vol/vol) glutaraldehyde in PBS for 30 min, washed and blocked with 2% (vol/vol) bovine serum albumin (BSA) in PBS, and permeabilized with 0.2% (vol/vol) Triton X-100 in PBS for 30 sec. After incubation with antibodies for labeling and rigorous washes, the chamber was exchanged with an oxygen scavenging buffer  glucose, 0.56 mg/mL glucose oxidase, 40 μg/mL catalase) for dSTORM imaging. The images were acquired at a frame rate of 20–50 ms for approximately 5,000–20,000 frames. Drift correction and single-molecule fitting were performed with the N-STORM software in NIS Elements (version 4.13.01).For dSTORM imaging, the endothelial cells in culture medium were loaded into the flow chamber (Ibidi) and incubated in a CO2; events with localization precision worse than 50 nm were discarded. All acquisition frames were corrected for any drift, including mechanical, thermal, chromatic aberrations and frameshifts, using the drift correction function available in NIS Elements software.dSTORM data was analyzed using custom software written in MATLAB (MathWorks) for a region of 5.6 × 5.6 µmStrict thresholds were applied for signal-to-noise ratio, intensity, full width at half maximum, and the positional accuracy to filter the signals determined to represent single molecules. Furthermore, to avoid duplicate detection of the same molecule, all the signals located within the same pixel in consecutive frames were discarded except for the first frame of the particular series. The reconstructions of dSTORM images were performed using NIS Elements software.http://www.colorado.edu/geography/class_homepages/geog_4023_s07/labs/lab10/RipleysK.m].Before computing the heat map, the data were capped for maximum and minimum values so that scaled color bars could be displayed. The heat map was generated with MATLAB’s hist3 function and interpolated with the MATLAB function imresize to smoothen the contours. The contour map was a heat map with two values. Ripley’s K and L functions were calculated using a function provided by the University of Colorado https://au.mathworks.com/matlabcentral/fileexchange/31353-two-point-correlation-function-of-a-finite-2d-lattice]. As a null hypothesis, we used a random labeling model in which all the molecules were randomly labeled for a given number of molecules similar to the test groups and the locations of random events were preserved31. The positive deviation of the data line from the lower confidence line indicated a statistically co-complexion behavior compared with random labeling.The bivariate pair-correlation function was performed with a function provided by the MathWorks community [16.Shear stress experiments were performed as reported previouslySupplemantary material


Controllable preparation of nanopores to promote the performance of electrochemical biosensing interfaces has become one of the researching frontiers in biosensing. A double biocatalysis signal amplification of glucose biosensor for the study of electrochemical behaviors of glucose oxidase (GOx) was proposed by using horseradish peroxidase biosynthesized porous graphene (PGR) as the platform for the biocatalytic deposition of gold nanoparticles (AuNPs). The biosensor showed a linear range from 0.25 to 27.5 μM with a detection limit of 0.05 μM (S/N = 3) towards glucose. Furthermore, the proposed AuNPs/GOx–PGR modified glassy carbon electrode (AuNPs/GOx–PGR/GCE) achieved direct electron transfer of GOx. Nanomaterials can be used to improve the catalytic performance, impart higher sensitivity, and enhance selectivity to an electrochemical measurement of analysts . DiffereThe good stability of carbon nanomaterials (CNMs) make themwidely applied to fabricate biosensors for electroanalytical investigations ,13,14. TThe aim of the present work is to controllably prepare PGR with versatile pore size by HRP-induced process, and then conduct in situ biocatalytic deposition of gold nanoparticles (AuNPs) onto the GOx–embedded PGR film to fabricate a novel biosensor for the determination of glucose level with the growth reaction. PGR is prepared by biocatalysis process of HRP and in situ biocatalytic deposition of AuNPs onto PGR by GOx. The two biocatalysts can play the role of double signal amplification to improve the sensitivity of the glucose biosensor. Herein, PGR can act not only as an enzyme immobilization reagent, but also as the biosensing platform for the determination of glucose level. Furthermore, the electrochemical behaviors of GOx at the proposed AuNPs/GOx–PGR/GCE (glassy carbon electrode) are also studied. It is expected that the proposed method has the advantage of design compatibility ,30,31,32−1, Type X–S from Aspergillusniger). Chloroauric acid tetrahydrate (HAuCl4·4H2O) and HRP were purchased from Sigma . High–purity graphite powder was purchased from the Shanghai Carbon Plant . Glucose stock solution was allowed to mutarotate at room temperature overnight before use. All other chemicals were of analytical reagent grade, and doubly distilled water was used in all the experiments. A 0.1 M pH 7.0 sodium phosphate buffered saline (PBS) solution was used in all electrochemical studies unless otherwise stated.GOx  was used for SEM images . Transmission electron microscope (TEM) images were collected on an E.M. 912 Ω energy–filtering TEM (120 kV) . A DL–180 ultrasonic cleaning machine  was used to dissolve and form a homogeneous solution. All electrochemical experiments were carried out on a CHI660e electrochemical workstation  using a three electrode system. The working electrode was a GCE or a modified GCE. A saturated calomel electrode (SCE) and a platinum electrode served as reference and counter electrodes, respectively. All the electrochemical experiments were conducted at room temperature (25 ± 2 °C).2O2 was added into the above solution, a 24 h interval passed, the addition of H2O2 was repeated and the operation was last for several days. The product was freeze-dried and stored in the refrigerator (4 °C) for later use.First, 80 μL of 5 mg/mL of HRP was added into 1 mg/mL, 2 mg/mL, 4 mg/mL, and 6 mg/mL graphene oxide (GO) solutions and shaken in a rocking incubator at 37 ± 0.2 °C. Then, 20 μL 3 M H2O3 slurry on a polish cloth and rinsed with double–distilled water, then sonicated in ethanol and double-distilled water for 5 min, respectively.Before use, a glassy carbon electrode (GCE) of 3 mm diameter was polished to a mirror-like finish with 1.0, 0.3, and 0.05 μm AlGOx–PGR composite was prepared by dispersing 1 mg PGR and 5 mg GOx into 1 mL PBS by shaking in a rocking incubator at 37 ± 0.2 °C, mixing for 24 h, then casting 10 μL of the mixture onto the surface of the bare GCE by using a syringe to prepare GOx–PGR/GCE. Without use, the biosensor was stored at 4 °C in a refrigerator.4 in 0.1 M PBS with different concentrations of glucose for 25 min, then it was removed from the above solution and transferred into 0.1 M PBS.All growth solutions were purged with highly purified oxygen for 30 min before experiments, and an oxygen environment was kept over the solution during the growth process. The GOx–PGR/GCE was immersed into a growth solution consisting of 0.2 mM HAuClThe pore structure on PGR was adjusted by reaction concentrations of GO and biocatalytic induction time. By comparison, 15 days was the optimal reaction time. At the beginning, there were only a few pores on GR. As the reaction time increased, the number of pore substantially increased. However, the amount of pores did not change further after 15 days. SEM images of products with different reaction concentrations of GO are shown in 2O2 is produced by the oxidization of glucose from GOx. Then, H2O2 reduces [AuCl4]− into Au (0), catalyzed by HRP on the surface of PGR. A double biocatalysis signal amplification was achieved for the determination of glucose. The sensitivity was greatly improved, with a value 1.2 to 1000 times higher than existing methods.The possible mechanism of the biocatalytic growth of AuNPs has been confirmed by our previous work ,34. H2O2The incubation time may affect the growth of AuNPs on the designed GOx–PGR/GCE. Subsequent experiments were carried out to obtain the optimum incubation time for GOx-induced AuNPs formation with a glucose concentration of 100 μM. The dependence of cyclic voltammetry (CV) peak current of the biocatalytic accumulation of AuNPs on accumulation time is displayed in ssI(μA) = −7.557C (μM) − 75.31 (r = 0.9945) with linearity from 0.25 to 27.5 μM  are shown with the formal potential E°′ = −0.272 V, ΔpE = 90 mV at the scan rate of 100 mV/s. The ratio of cathodic to anodic current intensity is about 1, indicating that a fast direct electron transfer reaction occurred. The value of E°′ was close to the values reported previously [pI = n2F2vAΓ*/4RT = nFQv/4RT [Γ* was calculated to be 2.34 × 10−12 mol/cm2, which matched the theoretical value (2.86 × 10−12 mol/cm2) for the monolayer of GOx on the bare electrode surface, indicating that the AuNPs/GOx–PGR/GCE keep the nanostructure of GR.CVs of the proposed AuNPs/GOx–PGR/GCE with different scan rates are shown in eviously ,36. AcconFQv/4RT , the surIn the present work, HRP–induced PGR with adjustable pore size was employed as the platform for in situ biocatalytic deposition of AuNPs, and a novel double biocatalysis (HRP+GOx) signal amplification glucose biosensor was constructed based on the CV response of AuNPs with different concentrations of glucose. The biosensor showed a low determination limit and high sensitivity for glucose detection. The reason can be ascribed to: (1) The PGR provided a benign microenvironment for the biocatalysis deposition process and the porous structure surface of PGR greatly enlarged the specific surface area to effectively increase the amount of GOx immobilized; (2) The advantages of biocatalysis itself, such as the accumulation and amplification effects; (3) Synergistic effects of PGR and AuNPs not only improved the electron transfer rate of the sensing system, but also exhibited the signal amplification of nanosize materials. Furthermore, the proposed AuNPs/GOx–PGR/GCE achieved the direct electrochemistry of GOx and electrocatalysis towards glucose. The present study may pave the way for the synthesis of novel nanomaterials with unique morphology and properties. Moreover, the advantage of the compatibility of the biosensor design can be integrated for other devices and electronics. Further works employing PGR as the biosensor platform are in our schedule.


Protein phosphorylation is a ubiquitous cellular process that allows for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A) is a heterotrimeric serine-threonine phosphatase—composed of a structural, regulatory, and catalytic subunit—that controls a variety of cellular events via protein dephosphorylation. While much is known about PP2A and its basic biochemistry, the diversity of its components—especially the multitude of regulatory subunits—has impeded the determination of PP2A function. As a consequence of this complexity, PP2A has been shown to both positively and negatively regulate signaling networks such as the Wnt pathway. Wnt signaling modulates major developmental processes, and is a dominant mediator of stem cell self-renewal, cell fate, and cancer stem cells. Because PP2A affects Wnt signaling both positively and negatively and at multiple levels, further understanding of this complex dynamic may ultimately provide insight into stem cell biology and how to better treat cancers that result from alterations in Wnt signaling. This review will summarize literature that implicates PP2A as a tumor suppressor, explore PP2A mutations identified in human malignancy, and focus on PP2A in the regulation of Wnt signaling and stem cells so as to better understand how aberrancy in this pathway can contribute to tumorigenesis. Protein phosphorylation is an essential regulator of many cellular processes, including metabolism, transcription, proliferation, cell motility, and apoptosis ,2. NearlWnt signaling is known to regulate patterning and cell fate decisions during embryonic development, and has been implicated in the pathogenesis of cancer . Over 90Early studies using okadaic acid—a serine-threonine phosphatase inhibitor that targets the catalytic PP2Ac subunit—increased tumor formation in a cutaneous carcinogenesis challenge and provided early biochemical support for a tumor suppressive role of PP2A ,28. Howe1PP2A (PHAP), may also be clinically relevant; however, additional studies in cancer are needed [Clinical evidence further supports a role of PP2A in tumor suppression. Cancer-associated mutations in the PP2A Aα scaffolding subunit impair binding to specific B subunits as well as the catalytic Cα subunit . Mutatioe needed ,42. Recie needed . The PP2e needed ,45,46. ITwo PP2A catalytic subunits, cα and cβ, share 97% sequence homology , yet micThe question of how PP2Acα loss alters E-cadherin localization remains unsolved, but E-cadherin is highly phosphorylated within a serine-enriched domain that comprises the β-catenin binding domain . PhosphoPP2A is unlikely to exert its Wnt-inhibitory effects through direct dephosphorylation of β-catenin, as β-catenin dephosphorylation at Ser33, Ser37, and Thr41 removes β-TrCP recognition sites and subsequently stabilizes the protein . ConsequTreatment with ST antigen potentiates Wnt signaling and also disrupts B subunit binding to the holoenzyme , suggestXenopus embryos reveal that the PP2A A, PP2Ac, and B’ PR61α regulatory subunit all have ventralizing activity, indicating Wnt inhibition. In Xenopus, β-catenin levels are higher dorsally than ventrally, and higher Wnt tone leads to dorsalization/secondary axis formation [Xenopus egg extracts, supporting a role for PP2A as a component of the β-catenin degradation complex [Axis duplication experiments in ormation . Ventral complex . Adding  complex . Taken tFor every piece of evidence that PP2A negatively regulates Wnt signaling, there is evidence to the contrary. Teleological thinking would support a positive role for PP2A in regulating Wnt signaling, as dephosphorylation of the main effector (β-catenin) increases its abundance . AccordiThe scaffolding protein APC is also a putative target of PP2A dephosphorylation and subsequent Wnt activation, as GSK3 phosphorylation of APC improves its ability to bind β-catenin ,20. A nuThe scaffolding protein Axin has binding sites for both GSK3β and β-catenin, and acts as a negative regulator of Wnt signaling by promoting β-catenin phosphorylation. Axin phosphorylation within the β-catenin binding domain increases binding to β-catenin, stabilizing Axin and increasing β-catenin degradation . Using aA role for Wnt signaling in the control of stem cells and cancer stem cells has been well established and previously reviewed ,75,76,77Protein dephosphorylation is a complex and nuanced process, and the PP2A family of serine-threonine phosphatases play an important role in regulating multiple signaling pathways implicated in tumorigenesis, stem cell maintenance, and self-renewal. Early studies of PP2A inhibitors and genomic studies identifying mutations in PP2A subunits support its tumor suppressive role. While perturbations in Wnt signaling can help initiate a number of human malignancies, Wnt signaling is also critical for the maintenance of normal tissue and stem cell homeostasis in the non-transformed state. Ample data supports a role for PP2A as a negative regulator of Wnt signaling; however, there is similarly strong data supporting PP2A’s role in potentiating Wnt signaling. PP2A-mediated regulation of Wnt signaling is likely cellular context-specific, and care must be taken to control for these variables. Given the specificity with which each regulatory subunit targets PP2A-mediated dephosphorylation, future studies must continue to identify how individual trimeric complexes function in regulating a target of interest—referring simply to PP2A provides scant biological relevance. Knockdown and overexpression studies must consider compensatory mechanisms, given the high similarity, yet extreme specificity, of individual regulatory subunits and the numerous levels at which they appear to modulate Wnt signaling. Taken together, the PP2A family of serine/threonine phosphatases regulate Wnt signaling and stemness at multiple levels, both positively and negatively, and further understanding of this complex dynamic will aid in identifying key regulators of tumorigenesis and normal tissue homeostasis.


Dishevelled (DVL) proteins are highly conserved in the animal kingdom and are important key players in β-Catenin-dependent and -independent Wnt signaling pathways. Vertebrate genomes typically comprise three DVL genes, DVL1, DVL2, and DVL3. Expression patterns and developmental functions of the three vertebrate DVL proteins however, are only partially redundant in any given species. Moreover, expression and function of DVL isoforms have diverged between different vertebrate species. All DVL proteins share basic functionality in Wnt signal transduction. Additional, paralog-specific interactions and functions combined with context-dependent availability of DVL isoforms may play a central role in defining Wnt signaling specificity and add selectivity toward distinct downstream pathways. In this review, we recapitulate briefly cellular functions of DVL paralogs, their role in vertebrate embryonic development and congenital disease. In addition, a subpopulation of the NC contributes to the cardiac outflow tract (OFT). Cranio-facial malformations are also visible in DVL1 and DVL2 morphant DVL1 mutations in humans affect predominantly cranio-facial development with little or no aberrations in body height, whereas in DVL3 and WNT5A associated ADRS craniofacial malformations are accompanied by short stature  cells (Greco et al., Overall it appears that DVL function in β-Catenin-independent Wnt signaling is more sensitive to the loss or dysfunction of one or two paralogs and accounts for most of the developmental phenotypes in knock-out animal models and also for the features of ADRS.2+ signaling in regeneration (Chen et al., DVL1 knock-out mice, which has been attributed to lower Wnt/β-Catenin activity as well as decreased AKT activation (van de Schans et al., 2+ and CamKII signaling in cardiomyopathy (Malekar et al., Xenopus gastrulation (Gentzel et al., A number of studies indicate a specific role of DVL1 in cardiac remodeling and regeneration. DVL1 and CamKII are upregulated after induced myocardial infarction and heart failure indicating a role of Wnt/CaDVL1 or DVL3 mutations also differ, indicating some degree of divergence but also overlapping functions. In addition, expression of transgenes in a single knock-out background further supported partial redundancy and indicated a dose dependency. If the DVL paralogs would be fully redundant in function it might be speculated that the summed abundance of all paralogs is important for cell survival. But even in cell culture models, any single knock-down is effective and the cells apparently do not sense overall “DVL concentration” and do not compensate the down-regulation of one protein by upregulation of the others (Cervenka et al., Striking differences between different DVLs and species have been observed in temporal and spatial expression patterns. Loss-of-function phenotypes of each single paralog in mouse as well as ADRS features associated with 2+ pathway, which were revealed in intact tissue or tissue models (Park et al., Functional redundancies however, do not connote biochemical identity. The observed differences could reflect differential expression levels, epistatic relations, or differential biochemical properties such as protein-protein interaction affinity and consequently also molecular function, which would also be consistent with dose-dependencies. The developmental phenotypes further indicate that β-Catenin independent Wnt pathways are more sensitive to the dose of individual DVL paralogs than β-Catenin signaling. This hypothesis is further supported by comparison of triple knock-out and triple-RNAi knock-down embryos. Whereas, in triple KO embryos early β-Catenin signaling is strongly reduced resulting in axis and mesodermal mispatterning, in the knock-down, in which ~25–30% of each paralog were retained, early β-Catenin signaling was not affected, but the embryos showed strong morphogenetic defects in the dorsal mesoderm and neuroectoderm. Consistently, specific and different molecular functionality of the three DVL paralogs has been observed in ciliogenesis and the Wnt/CaOverall, the currently available data indicate that DVL expression and function have diverged to some degree apparently and consistent with phylogenetic models mostly between DVL1 and DVL2/3. Thus, depending on the cellular context, DVL paralogs exhibit both redundant and distinct functionality.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Wnt/β-catenin signaling pathway is an evolutionary conserved mitogenic signaling pathway involved in embryonic patterning of body axes, regulation of stem cell fate and tissue homeostasis . DeregulPGAM5 and PARL knockout cells [Recently, we identified a cell intrinsic way to activate the Wnt/β-catenin pathway via a signaling axis from mitochondria to β-catenin : Loss ofut cells . TogetheThat the Wnt/β-catenin pathway can be activated by both extrinsic and intrinsic mechanisms is reminiscent of the way cellular apoptosis is induced. Also apoptosis can be triggered extrinsically by the binding of FAS ligands to FAS cell surface death receptors, and cell intrinsically via the release of cytochrome c from mitochondria . In bothActivation of Wnt/β-catenin signaling has previously been shown to stimulate mitochondrial biogenesis . Since c


Colletotrichum musae and crown rot caused by fusarium spp. are the most important disease causing agents and result in large percentage of fruit loss in Jimma town of bishishe market.Banana fruits are highly perishable and affected by different microbial contaminates because ripe bananas are very perishable. One of the most important factors causing great economical loss of banana fruits is postharvest fungal diseases caused by lack of proper handling along postharvest chains. In line with this, the study was carried out to assess banana fruits handling practices and identify the major causal agents of postharvest fungal disease of banana fruits in Jimma town of bishishe market. Assessment was carried out using purposive sampling to select the study area, followed by simple random sampling to collect information on handling practices through observation and interview using pretested questioners. The survey result revealed that losses of banana fruits due to spoilage and physical injury were common problems for all fruit vendors. Practices such as lack of sanitation, temperature management, and improper packaging and transportation problems were identified among the common causes for observed losses. A total of 48 fruits were purposively selected from open market, wholesales and retailers and fruit damage, disease incidence, disease severity and identification of diseases causing fungal pathogen were conducted under laboratory. The highest fruit damage (56.2%) was recorded in sample taken from retailers’ shop, and the associated disease incidence and severity were 54.2% and 34%, respectively. Morphological identification of pure culture revealed that anthracnose caused by  It is the second largest produced fruit after citrus, contributing about 16% of the world's total fruit production and the fourth most important staple food crops in the world after rice, wheat, and maize Ellyn, . Accordi6, and manganese  were assessed and interviewed. Two‐stage sampling techniques were employed to select specific banana sellers and traders of the study area. In the first stage, Bishishe market was purposively selected based on the fact that it is the only place in Jimma town where banana fruits are sold in large amounts. In the second stage, simple random sampling method was followed, and a total of 50 respondents that represented five of wholesalers, 25 of retailers, and 20 of farmers selling banana fruits in open market were selected using Yamane 2.2.2n = 16), wholesalers (n =16), and retailers (n = 16) with three replications. All the fruits were transported using truck to the laboratory and stored at room temperature for further evaluation of fruit damage and quiescent (latent) infection of the pathogens.To assess percentage of fruit damage and test for fungal pathogen contamination, representative samples  composed of both injured and apparently healthy looking were sampled purposively based on the volume sell from the farmers selling fruits in open market  for 3 min. After three serial washings in sterile distilled water, tissues were placed (four pieces per plate) on Potato Dextrose Agar (PDA) and incubated at 25°C in the incubator for 7 days. The colonies emerged from each plated fruit tissues were purified and subcultured on the PDA media after 7 days. The plates were incubated at 25°C under similar conditions, and the setups were observed until the organisms became fully grown. Single‐spore cultures of the fungus were then prepared on PDA slants in test tubes, and the identification and characterization of the fungal isolates were carried out based on cultural and morphological structures described in Marasas, Miller, Riley, and Visconti .33.1Among 50 banana vendors interviewed, their gender, age, marital status, and educational levels were quantified Table .In gender category, 14 (28%) of respondents were males and rest of them were females and indicated that the females were involved much in banana fruits sale. Among the age groups, the maximum of 19 (38%) members were <25 years of age followed by 25–35 years and 35–45 years of age, and the minimum of 8 (16%) vendors belong to >45 years of age. The level of education varies among the gender and age categories, 23 of them were illiterates, 27 of them were in various levels (elementary to high school) education. More than 75% of literate respondents have awareness on the influence of improper handling practices on the quality of banana fruits. Similar observation was made by Olayemi, Adegbola, Bamishaiye, and Awagu  who stat3.2Actual observation and assessment results indicated a wide range of mishandling practices that favor fungal developments and results in fruit losses. Postharvest banana fruits fungal developments and associated losses could be related to the following mishandling practices.3.2.1Most of the banana fruits sold in Jimma town was brought from distant places using different packing materials and means of transportations  of the respondents interviewed Figure  simply l2% of theThe observed temperatures of the marketing area were two to threefold higher than the optimum postharvest handling temperatures of banana fruits, and hence, shelf‐life of the fruits would be theoretically only one‐half or even one‐quarter of the potential  of them store damaged and spoiled fruits separately Table . During 3.2.4The percentage of fruit damage at the market varied between fruit handlers Figure . The resDifferent reasons were indicated as major causes for damage, and among these, negligence in good handling during harvesting and on farm, lack of proper transportation, less care during loading and unloading and etc. More than 40 (80%) of the respondents told that banana fruits sold in Jimma market are transported from long distance on bumper road and overloaded in the truck. This might induce mechanical damage on the fruits and as a result induce fungal infections. During the study, we observed that bunches are piled high on the floor during ripening process, and there is a possibility of physical damage for bunches due to overloading one on another. Moreover, there is no control mechanism for temperature and humidity in the ripening room. They simply burn gasoline as a source of heat and ethylene for 24 hrs under enclosed condition to trigger ripening process. This may cause deterioration of product trough enhancing physiological process of fruit and creating favorable environment for disease development by weakening the fruit cell wall Eduardo, .3.3The mean percent of disease incidence and severity varied between banana fruit handlers Table . The maxThe mean percentage of disease incidence and severity results are in line with percentage of fruit damage recorded. This indicated that mechanical damage during handling at different stages along postharvest chain predisposes the fruits for fungal pathogens . About 84% of colonies counted were contributed by Collectotrichum spp., and about 16% of colonies counted were accounted by Fusarium spp. In the similar manner, Zakaria, Sahak, Zakaria, and Salleh  of Jimma town market. They were identified on the basis of their cultural and morphological structures such as shapes and sizes of macroconidia and microconidia, and colony color. The cultural and morphological investigations on the samples revealed that d Salleh  stated tFusarium were observed on sample taken from different parts of ripe fruits. This could be associated with high temperature used during ripening which may create more favorable condition for crown rot development, and Fusarium have been occasionally associated with crown rot disease of banana (Marin, Sutton, Blankenship, & Swallow, Growths of light pink colony of 4Colletotrichum spp. and Fusarium spp were identified as the most important fungal pathogens causing fruit loss in Jimma town of Bishishe market. Mechanical injury due to mishandling along supply chains and sanitary problem in the market could be the possible causes for observed fungal pathogens. In order to reduce mechanical injury and associated microbial deterioration, a close integration of all stakeholders along the value chain of banana fruits becomes necessary.Banana fruits are highly susceptible to mechanical injury owing to their tender texture and high moisture content. If they are exposed to undesirable environmental conditions during handling, the tissue will soften and easily bruise, causing rapid microbial deterioration. In the study area, quality and safety assurance problems such as lack of temperature management, uniformity of quality within containers, sanitation problems in the market, transportation‐related problems, careless handling during loading and unloading were identified as the main factors, which favored fungal pathogen development and associated banana fruit losses. Disease intensity had similar trends with percentage of fruit damage with more damage, and infections were recorded in retailers’ shop. The percentages of fruit damage were as high as 56.2%, and the associated disease incidence and severity were 54.2% and 34%, respectively, in retailers’ shop.


Cx3cr1−/− mice, as compared with those in the wild-type (WT) mice. However, intrapulmonary CX3CR1+ macrophages displayed pro-fibrotic M2 phenotypes; lack of CX3CR1 skewed their phenotypes toward M1 in BLM-challenged lungs. Moreover, fibrocytes expressed CX3CR1, and were increased in BLM-challenged WT lungs. The number of intrapulmonary fibrocytes was decreased in Cx3cr1−/− mice. Thus, locally-produced CX3CL1 can promote PF development primarily by attracting CX3CR1-expressing M2 macrophages and fibrocytes into the lungs.The potential role of macrophages in pulmonary fibrosis (PF) prompted us to evaluate the roles of CX3CR1, a chemokine receptor abundantly expressed in macrophages during bleomycin (BLM)-induced PF. Intratracheal BLM injection induced infiltration of leukocytes such as macrophages into the lungs, which eventually resulted in fibrosis. CX3CR1 expression was mainly detected in the majority of macrophages and in a small portion of α-smooth muscle actin-positive cells in the lungs, while CX3CL1 was expressed in macrophages. BLM-induced fibrotic changes in the lungs were reduced without any changes in the number of leukocytes in  Deposition of extracellular matrix (ECM) components, such as collagen, is a crucial but reversible process in most cases of wound healing. However, if the tissue injury is severe or repetitive, or if the inflammatory response becomes dysregulated, progressive and irreversible accumulation of ECM may occur. The subsequent development of fibrosis can lead to organ failure, as seen in pulmonary fibrosis (PF), end-stage liver, kidney diseases, and heart failure1.When tissue injuries occur due to microbial infection, toxic substances, or mechanical damages, inflammatory responses are initiated. Tissue repair occurs through the interplay between various hematopoietic and non-hematopoietic cells3. CX3CL1 is expressed by epithelial cells in the lungs, kidneys, and intestines as a membrane-bound molecule; its chemokine domain attaches to the cell surface through a mucin-like stalk in the resting state. CX3CR1 is abundantly expressed by monocytes/macrophages, T cells, natural killer cells, and smooth muscle cells3.CX3CL1, also known as fractalkine, is a member of the CX3C chemokine family. It can bind to its specific receptor, CX3CR1, in a 1:1 ratio4. The CX3CL1-CX3CR1 system is suggested to promote the development of both atherosclerosis and coronary artery diseases6. It has also been reported that serum CX3CL1 levels correlate positively with disease severity in rheumatoid arthritis patients7. Furthermore, CX3CL1-CX3CR1 interactions could contribute to the pathogenesis of lung diseases, including asthma9 and emphysema11. On the contrary, CX3CL1-CX3CR1 signaling can either be beneficial or detrimental in carcinogenesis and cancer metastasis via alternative pathways4. Interestingly, studies have shown that the CX3CL1-CX3CR1 axis could protect against epilepsy, Parkinson’s disease, and amyotrophic lateral sclerosis12. Furthermore, the CX3CL1-CX3CR1 axis could maintain β cell function to preserve sufficient insulin secretion13. We have previously demonstrated that CX3CL1 protects against polymicrobial sepsis and toxin A-induced enteritis by binding to CX3CR1-expressing macrophages15.Accumulating evidence has demonstrated differential roles of CX3CL1-CX3CR1 interactions in various types of human diseases16. Moreover, PF can develop from various conditions, such as irradiation injury, oxygen toxicity-related pneumonitis, and scleroderma; however, approximately half of the PF cases are idiopathic. Environmental exposures to various substances17, as well as genetic factors18, have been proposed to be implicated in the development of idiopathic PF (IPF). However, their contribution to disease pathogenesis remains to be investigated. Aberrant tyrosine kinase activation in IPF has incited the clinical application of nintedanib, a multi-tyrosine kinase inhibitor. Moreover, pirfenidone, an agent with anti-inflammatory and anti-oxidant activities, was also examined for its effectiveness against IPF19. While both agents can delay the decline in respiratory function in IPF, they are unable to cure the disease. As a consequence, it is necessary to establish a novel treatment strategy based on the molecular and cellular mechanisms of IPF pathogenesis.PF is characterized pathologically by diffuse interstitial inflammation and fibrosis24. The expression of CX3CR1 on monocytes/macrophages prompted us to investigate the roles of the CX3CL1-CX3CR1 axis in BLM-induced lung injury in CX3CR1-deficient (Cx3cr1−/−) mice. We revealed that genetic ablation of Cx3xr1 significantly attenuated BLM-induced lung fibrosis.Bleomycin (BLM) can induce lung injuries that mimic the pathological features observed in human IPF. Analysis of BLM-induced lung injuries revealed the contribution of pulmonary macrophages to disease onsetCx3cl1 and Cx3cr1 in lungs of WT mice following intratracheal BLM administration. Both Cx3cl1 and Cx3cr1 mRNAs were detected in lungs of untreated WT mice. Expression of Cx3cl1 was significantly upregulated at 1 day and 3 days after the BLM challenge, followed by augmentation in Cx3cr1 expression  dominated over CD86+ macrophages (M1-type). However, in BLM-treated Cx3cr1−/− mice, the opposite trend was observed  mouse-derived BM cells. When WT mice bearing GFP+ BM cells were intratracheally injected with BLM, GFP+ cells was detected in the lungs, indicating that BM cells were recruited into the lungs after BLM treatment. In addition, Col-I was also detected in a substantial proportion of GFP+ cells  and alveolar macrophages (AMs), residing in the interstitial and alveolar spaces, respectively47. Several lines of evidence indicated that macrophages with the M2 phenotype could promote tissue fibrosis49. Indeed, CX3CR1-expressing CD68+ cells in the lungs were also positive for CD20651, a M2-specific marker. This indicated that CX3CR1+ macrophages are M2-macrophages. Moreover, M2-macrophages were reduced in BLM-challenged lungs of Cx3cr1−/− mice. This was concomitant with a reciprocal increase in CD68+ M1-macrophages. Therefore, absence of CX3CR1 can have profound effects on macrophage polarization rather than total macrophage numbers in the lungs after BLM challenge.Alternatively, there remains a possibility that CX3CR1 deficiency may have profound impact on macrophage functions. Macrophages can be functionally polarized into two phenotypes, M1 and M2. Skewing of macrophage polarization can contribute to pathogenesis of inflammatory responses, allergic diseases, tissue repair, and tumor progression52. As a result, myofibroblasts have indispensable roles in connective tissue remodeling and the subsequent development of fibrosis in various organs, including the lung. Myofibroblasts can be differentiated from several cell types, including locally residing mesenchymal cells , other local sources such as epithelial and endothelial cells, and BM-derived fibrocytes53. Studies on BM chimeric mice have revealed the crucial role of radiosensitive BM-derived CX3CR1-expressing cells in BLM-induced collagen deposition in the lung. Moreover, injection of BLM into chimeric mice with BM derived from GFP Tg mice resulted in cellular expression of GFP+ and Col-I+ in the lung. Therefore, fibrocytes may be a major source of collagen-producing myofibroblasts in BLM-induced PF.Myofibroblasts display characteristics of both fibroblasts and smooth muscle cells, as represented by their ECM-synthesizing capacity and contractile cytoskeleton, respectively62. BLM was found to increase fibrocyte numbers in the BM, peripheral blood, and lungs, which is consistent with our previous report26. Moreover, fibrocytes were hypothesized to express several chemokine receptors, including CXCR4, CCR3, CCR5, and CCR764, which allow them to migrate to fibrotic organs in response to their cognate ligands. It was found that inhibition of CXCR4 function31, or deficiencies in Ccr365 or Ccr526 attenuated BLM-induced pathologies, together with reduced intrapulmonary fibrocyte numbers. Our present observations suggested that CX3CR1 is one of the chemokine receptors involved in migration of fibrocytes into fibrotic lungs.Fibrocytes are defined as BM-derived cells that are intermediate between hematopoietic and mesenchymal cells, as they simultaneously express leukocyte markers such as CD45 and mesenchymal cell markers such as Col-I. Fibrocytes, once generated in the BM, circulate in the bloodstream, and migrate to organs in response to tissue-specific cuesCcl3−/− and Ccr5−/− mice exhibit reduction in BLM-induced fibrosis and the number of CD45+Col-I+CCR5+ fibrocytes in the lungs, together with suppressed CXCL12 expression26. These observations indicated that the CCL3-CCR5 axis can mediate BLM-induced fibrocyte migration into the lungs, partly via its interactions with the CXCL12-CXCR4 axis. BLM-induced increase in intrapulmonary fibrocyte numbers was not completely nullified in Cx3cr1−/− mice, suggesting the contribution of other chemokines such as CXCL12 and CCL3 to fibrocyte infiltration. The decreased BLM-induced fibrosis and fibrocyte infiltration into the lungs of Cx3cr1−/− mice, however, were not accompanied with changes in intrapulmonary expression of CXCL12 and ligands for CCR5, CCL3, CCL4, and CCL5. Thus, the CX3CL1-CX3CR1 axis can regulate BLM-induced fibrocyte migration and subsequent fibrosis development independently of other chemokines.We have previously demonstrated that 66; this binding is reversible and unstable. On the contrary, CX3CL1 is expressed as a membrane-bound molecule with a mucin-like stalk3. As compared with other chemokines, CX3CL1 can be firmly retained on the cell surface, and can consequently affect its target cells in a more stable manner. Moreover, in addition to chemotactic activities, CX3CL1-CX3CR1 interactions can promote the survival and/or proliferation of non-hematopoietic cells, such as neuronal cells67, and hematopoietic cells, such as monocytes68 and helper T cells9. If CX3CL1 is able to support survival and/or proliferation of fibrocytes as well as non-hematopoietic cells, it can attract CX3CR1-expressing fibrocytes into the lung. This can eventually promote the survival and/or proliferation of fibrocytes, thereby inducing fibrotic changes.CX3CL1 has distinct structural and functional features from other chemokines. Most chemokines are produced as a secretory molecule, and bind to proteoglycan and glycosaminoglycan in the ECM through their carboxy-terminal α-helix portion10. To summarize, the interactions between CX3CL1 and CX3CR1 may provide potential candidate molecule(s) for the treatment of IPF.Collectively, our observations suggested that the CX3CL1-CX3CR1 axis is essential in the development of BLM-induced PF by regulating fibrocytes and M2-macrophags, which can exert pro-fibrotic activities. Absence of CX3CR1 impairs wound healing in the skin with reduced macrophage recruitment and fibroblast accumulationBLM was purchased from Sigma Chemical Co. . The following monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used for immunohistochemical and immunofluorescence analyses in this study: goat anti-mouse CX3CL1 pAbs and goat anti-mouse CX3CR1 pAb ; rabbit anti-human CX3CR1 pAbs, which cross-reacts with mouse CX3CR1 ; rat anti-mouse F4/80 mAb ; mouse anti-human α-smooth muscle actin (α-SMA) mAb , rabbit anti-mouse pan-Cytokeratin pAbs (Santa Cruz Biotechnology), rabbit anti-CD68 , rat anti-CD206 , rabbit anti-human CD3 pAbs, which cross-reacts with mouse CD3 ; rat anti-mouse Ly-6G mAb, ; rat anti-mouse F4/80 mAb ; rabbit anti-mouse collagen type I (Col-I) pAbs ; rat-anti mouse TGF-β1 mAb , Cy3-conjugated donkey anti-rat, -goat, -mouse, and -rabbit IgG pAbs, and FITC-conjugated donkey anti-goat, -rat, and -rabbit IgG pAbs . For flow cytometric analysis, the following antibodies (Abs) were commercially obtained: PerCP-conjugated rat anti-mouse CD45 mAb  and FITC-conjugated rat anti-mouse CD34 mAb ; rabbit anti-mouse Col-I pAbs ; PE-conjugated rabbit anti-mouse CX3CR1 ; FITC-conjugated rat anti-mouse CD68 ; PE-conjugated rat anti-mouse CD86, FITC-conjugated rat anti-mouse CD8, FITC-conjugated Armenian hamster anti-mouse CD3, PE-conjugated mouse anti-mouse NK1.1, FITC-conjugated rat anti-mouse CD11b ; APC-conjugated rat anti-mouse CD206 ; and FITC-conjugated rat anti-mouse Gr-1, PE-conjugated rat anti-mouse CD4 .Cx3cr1−/− mice on the C57BL/6 genetic background were a generous gift from Drs. P.M. Murphy and J.L. Gao 25. Tg mice on the C57BL/6 genetic background were obtained from RIKEN BRC . All animals were housed individually in cages under specific pathogen-free conditions during the experiments. Age- and sex-matched mice were used for the experiments. All animal experiments complied with the standards set out in the guidelines for the Care and Use of Laboratory Animals at the Wakayama Medical University.Pathogen-free 8-week-old male C57BL/6 mice were obtained from Sankyo Laboratories , and were designated as wild-type (WT) mice. Mice were put under deep anesthesia with an intraperitoneal injection of pentobarbital (50 µg/g weight). A cervical midline incision was made, and the trachea was exposed. Thereafter, BLM (0.075 U) in 50 µl sterile saline was intratracheally administered with a 26-guage needle. At the indicated time intervals following BLM administration, mice were sacrificed by pentobarbital overdose, and both lungs were removed for subsequent analyses.26. Briefly, male Cx3cr1−/− BM → female WT mice, male WT BM → female Cx3cr1−/− mice, male WT BM → female WT mice, and male Cx3cr1−/− BM → female Cx3cr1−/− mice. BM cells were collected from femurs of donor mice by aspiration and flushing. Recipient mice were irradiated to 15 Gy with a RX-650 irradiator . The animals then received intravenous injections of 5 × 106 BM cells in 200 μl sterile PBS(−) from donor mice under anesthesia. Thereafter, the mice were housed in sterilized microisolator cages, and were fed normal chow and autoclaved hyperchlorinated water for 60 days. Similarly, BM chimeric mice were generated by transplanting GFP-Tg mouse-derived BM cells into lethally irradiated WT mice. To verify successful engraftment and reconstitution of the BM in transplanted mice, genomic DNA was isolated from peripheral blood and tail tissues of each chimeric mouse at 30 days after BMT with a NucleoSpin tissue kit . Polymerase chain reaction (PCR) was used to detect the Sry gene in the Y chromosome . The amplified PCR products were fractionated on a 2% agarose gel, and were visualized by ethidium bromide staining. After durable BM engraftment was confirmed, mice were treated with BLM, as described above.BM chimeric mice were prepared as previously described26. Cells were quantified in 10 randomly chosen visual fields at 200× magnification, and the average of the 10 selected microscopic fields was calculated. All measurements were performed by an examiner without prior knowledge of the experimental procedures.Lung tissues were fixed in 10% formalin buffered with PBS (pH 7.2), and were embedded in paraffin. Tissues were sectioned (6 µm-thick) and stained with hematoxylin and eosin or Masson’s trichrome to detect collagen deposition. Immunohistochemical analyses were also performed using anti-Ly6G, anti-F4/80, or anti-CD3 Abs, as previously described69. Briefly, deparaffinized sections were incubated with PBS containing 1% normal donkey serum and 1% BSA to reduce nonspecific reactions. Tissue sections were then incubated with a combination of anti-CX3CR1 and anti-F4/80 Abs, anti-CX3CL1 and anti-F4/80 Abs, or anti-CX3CR1 and anti-α-SMA Abs at 1 µg/ml at 4 °C overnight. Similarly, triple-color immunofluorescence analysis was conducted to identify the molecules expressed on BM-derived fibrocytes (GFP+Col-I+ cells), such as TGF-β1 and CX3CR1. Sections containing GFP+ cells were incubated with a combination of anti-Col-I and anti-CD45 Abs, anti-Col-I and anti-TGF-β1 Abs, or anti-Col-I and anti-CX3CR1 Abs at a concentration of 1 µg/ml at 4 °C overnight. In order to identify the subtypes of macrophages recruited into the lungs, tissue sections were incubated with a combination of anti-CD68, anti-CX3CR1, and anti-CD206 Abs, or anti-CD206 and anti-CX3CR1 Abs at a concentration of 1 mg/ml at 4 °C overnight. Following incubation with fluorochrome-conjugated secondary Abs, tissues were imaged under a fluorescence microscope.Double-color immunofluorescence analysis was conducted to identify the types of CX3CL1- or CX3CR1-expressing cells in the lung, as previously described26. Data were expressed as the amount (μg) of Hyp per lung.Lung tissues were removed at 21 days after BLM administration to determine the content of Hyp, a major component of collagen, as previously described26. Briefly, mice were sacrificed by abdominal aorta dissection. The trachea was cannulated, and the airway lumen was washed two times with 1 ml ice-cold PBS. Single cell suspensions were incubated with 25 µg/ml Fc block  for 15 min on ice to prevent nonspecific binding. Cells were stained with anti-CD68, anti-CD86, CD-206, anti-CD3, anti-CD4, anti-CD8, anti-NK1.1, anti-Gr-1, and anti-CD11b. Analysis was performed on a FACScan flow cytometer (BD Biosciences) using the FlowJo software .BALF was collected as previously described with some modifications15 primers. Quantitative PCR was carried out with the resultant cDNA as templates and SYBR® Premix Ex Taq™ II (Takara Bio) with specific primer sets, as previously described69 , according to the manufacturer’s instructions. Expression levels of target genes were analyzed using the ΔΔCt comparative threshold method. The β-actin gene was used as internal control.Total RNA was extracted from lung tissue using ISOGEN , according to the manufacturer’s instructions. Total RNA (3 µg) was reverse transcribed to cDNA using PrimeScript™ Reverse Transcriptase  with Oligo(dT)25. Nonspecific binding was blocked by incubation with 25 µg/ml Fc block  for 15 min at 4 °C. Cells were stained with PerCP-labeled anti-CD45 and FITC-labeled anti-CD34. After extensive washing with PBS(−), cells were permeabilized using cytofix/cytoperm (BD Biosciences Pharmingen), stained with anti-Col-I pAbs, and incubated with PE-conjugated goat anti-rabbit Ig , as previously described26. After two PBS(−) washes, cells were fixed in 2% paraformaldehyde. Isotype-matched control immunoglobulins were used to detect non-specific binding. Stained cells were analyzed on the FACS Calibur flow cytometer. In parallel, BM cells from BLM-treated WT mice were stained with anti-Col-I, anti-CD45, and anti-CX3CR1 to determine the proportion of CX3CR1+ fibrocytes. Flow cytometry was performed on a FACS Calibur flow cytometer, and data were analyzed using the FlowJo software.Mononuclear cells were isolated from the lungs, the BM, and peripheral blood, as previously describedg for 10 min. Supernatants were used to quantify the active form of TGF-β1 with a commercial ELISA kit (R&D Systems), according to manufacturer’s instructions. The detection limits was 7 pg/ml. Total protein in the supernatant was measured with a commercial kit . Data were expressed as TGF-β1 (pg) per total protein (µg) for each sample.Lung samples from WT mice were homogenized with PBS containing complete protease inhibitor cocktail . Homogenates were centrifuged at 10,000 × U-test. P < 0.05 was accepted as being statistically significant.The means and SEMs were calculated for all investigated parameters in the study. Statistical significance was evaluated by ANOVA or the Mann-Whitney’s All animal experiments were approved by the Committee on Animal Care and Use at the Wakayama Medical University. All methods were performed in accordance with relevant guidelines and regulations.Data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary Information


Despite utilizing extended criteria donors, there remains a shortage of livers for transplantation. No data exists on splitting donor livers with concurrent NMP-L.A liver recovered from a donor after circulatory death was subjected to NMP-L using a red cell based fluid. During NMP-L, a ‘classical’ left lateral + right trisegmentectomy split was performed using an integrated bipolar/ultrasonic device. After splitting, blood flow was confirmed using Doppler ultrasound in each lobe.Prior to splitting, flow rates were maintained physiologically. Lactate decreased from 13.9 to 3.0 mmol/L. Lactate before and after splitting were similar in the hepatic arteries, portal veins and IVC. Doppler ultrasound demonstrated arterial and venous waveforms in both lobes after splitting.‘Classical’ liver splitting during NMP-L is feasible, maintaining viability of both lobes. Establishing this procedure may attenuate cold ischaemic injury, allow pre-implantation monitoring of both grafts and facilitate logistics of transplanting two grafts. The most common approach is the ‘classical’ left lateral + right trisegmentectomy split. The less frequently performed full left-right split can provide livers for two adults but this is technically complex and questions remain over vascular and biliary outcomes  mmHg), total hepatic arterial flow remained just below pre-hilar rates (median 429 [424–450] mL/min). Total portal venous flow rate increased to near pre-splitting rates (median 1250 [1230–60] mL/min) whilst maintaining physiological pressures (median 9 [8–10] mmHg).Post-hilar phase lactates were similar in both hepatic arteries, portal veins and IVC Table . Post-paModerate sinusoidal vasodilatation was noted throughout although milder post-splitting Fig. G. MacrovThis is the first study demonstrating that splitting a liver with concurrent NMP-L throughout is feasible and viability is maintained. Furthermore, this has been achieved in a liver unsuitable for transplantation. No studies to date have investigated liver splitting with concurrent NMP-L throughout splitting using either normothermic, subnormothermic or hypothermic strategies.ex situ splitting. Whilst possible during in situ splitting, concurrent NMP-L provides ex situ direct visualization of perfused segments. Throughout splitting the cut surface can be inspected and vessels ligated to ensure haemostasis. Continuation of NMP-L permits continuous viability assessment. This may contribute to recipient selection, aid informed decision-making and facilitate logistics of transplanting two grafts by preventing long CIT. We have shown that ex situ splitting is feasible in ECD livers performed in combination with NMP-L, and results in pre-transplant biochemical, histological and Doppler findings that are compatible with transplantation.This technique presents advantages over existing methods. There is no increasing temperature and rewarming under ischaemic conditions seen with cold metra™ device. Due to the closed circuit, a hanging procedure was implemented to facilitate dissection whilst minimizing fluid loss from the cut surface. Here perfusion was paused for 6 min to separate the grafts and yielded only an extended right segment still perfused [Liver splitting during NMP-L has been described using the OrganOx® perfused . SpecifiEx situ splitting with NMP-L allows exploration of splitting ECD livers and may increase the donor pool enabling two recipients to be transplanted from a single donor, it could also facilitate training in liver splitting and provide controls for research.ex situ splitting, without impacting on organ retrieval. Future work will allow improved monitoring and facilitate logistics.Classical left lateral + right trisegmentectomy liver splitting with concurrent NMP-L is feasible, maintaining viability of both lobes, combining ‘normal’ physiological conditions and reduced CIT with the ease of Supplementary DataClick here for additional data file.


Protein arginine methyltransferase 5 (PRMT5) is able to regulate gene transcription by catalyzing the symmetrical dimethylation of arginine residue of histone, which plays a key role in tumorigenesis. Many efforts have been taken in discovering small-molecular inhibitors against PRMT5, but very few were reported and most of them were SAM-competitive. EPZ015666 is a recently reported PRMT5 inhibitor with a new binding site, which is different from S-adenosylmethionine (SAM)-binding pocket. This new binding site provides a new clue for the design and discovery of potent and specific PRMT5 inhibitors. In this study, the structure-based virtual screening targeting this site was firstly performed to identify potential PRMT5 inhibitors. Then, the bioactivity of the candidate compound was studied. MTT results showed that compound T1551 decreased cell viability of A549 and H460 non-small cell lung cancer cell lines. By inhibiting the methyltransferase activity of PRMT5, T1551 reduced the global level of H4R3 symmetric dimethylation (H4R3me2s). T1551 also downregulated the expression of oncogene FGFR3 and eIF4E, and disturbed the activation of related PI3K/AKT/mTOR and ERK signaling in A549 cell. Finally, we investigated the conformational spaces and identified collective motions important for description of T1551/PRMT5 complex by using molecular dynamics simulation and normal mode analysis methods. This study provides a novel non-SAM-competitive hit compound for developing small molecules targeting PRMT5 in non-small cell lung cancer. Protein arginine methyltransferases (PRMTs) are a class of enzymes that transfer a methyl group from the cofactor S-adenosylmethionine (SAM) to arginine omega nitrogen of substrate protein. Based on product specificity, PRMTs can be divided into three subclasses: type I, II, and III, which asymmetrically dimethylate, symmetrically dimethylate, and monomethylate their substrates, respectively . ProteinStructure-based virtual screening using molecular docking has become a powerful tool in the drug discovery for rapidly enriching hits from large pools of compound databases. Nowadays, it has been successfully applied to discover novel inhibitors of epigenetic targets, such as SET7, KDM4B, and SIRT2 . The sucMolecular docking-based virtual screening was carried out with Schrödinger software package  or various concentrations  of the studied compound for 24, 48, and 72 h. Then, each well was added 10 μL MTT (5 mg/mL) and incubated for 4 h at 3°C, followed by adding 100 μL acidic isopropanol (10% SDS and 0.01 mol/L HCl). Finally, the absorbance at 570 nm was measured by a Microplate Reader . Cell viability was calculated relative to untreated controls, and the results were based on at least three independent experiments.A549 and H460 cells  were purchased from ATCC, cultivated in RPMI 1640 medium supplemented with 10% FBS , 1% penicillin-streptomycin solution, and maintained at 37°C in a CO50 value, T1551 was diluted into 10 concentrations. PRMT5 protein was purchased from BPS bioscience (Cat. No. 51045), and SAM/SAH were purchased from Sigma. Inc. (Cat. No. A7007-100MG and No. A9384-25MG). T1551 was prepared as 10 mM stock in DMSO and diluted to the final concentration in DMSO. PRMT5 and substrates were incubated with indicated concentrations of T1551 in a 384-well plate for 60 min at room temperature. Then, acceptor and donor solutions were added to label the residual substrates of PRMT5. The labeling process was lasting for 60 min at room temperature, followed by reading endpoint with EnSpire with Alpha mode. In the in vitro enzymatic assays, 1% DMSO was used as vehicle control for normalization.PRMT5 enzymatic assay was carried out by Shanghai ChemPartner Company , as did previously by g, 4°C), and the supernatant was collected. Protein concentrations were determined by Bio-Rad protein Assay kit . Equal amounts of protein (50 μg) were separated on a 10% SDS–PAGE gel, and transferred to a nitrocellulose (NC) membrane at 300 mA and 4°C for 1 h. The membrane was incubated with primary antibody (1:1000), and then with a fluorescence-conjugated secondary antibody (1:10000). The primary antibody against PRMT5 was purchased from Merck Millipore Ltd., (Germany); antibodies against H4R3me2s and H4 were purchased from Abcam ; antibodies against FGFR3 and eIF4E were purchased from Santa Cruz Biotechnology ; antibodies against total/phospho-AKT, total/phospho-ERK and total/phospho-mTOR were purchased from Cell Signaling Technology . GAPDH was used as the loading control and for normalization. The signal intensity of the membranes was detected with a LI-COR Odyssey Scanner .Cells were washed twice with cold PBS, and lysed in RIPA lysis buffer containing protease and phosphatase inhibitors to extract total protein. Cell lysates were centrifuged for 5 min  simulations were used for sampling the conformational spaces of PRMT5-T1551 complex. Normal mode analysis was used for identifying important collective motions for the complex. All MD simulations were performed with Amber 16 software . The AmbP < 0.05 was considered statistically significant.Descriptive analytical data were presented as mean ± SEM. Multiple comparisons were evaluated by one-way analysis of variance (ANOVA) using Graph Prim 5.0. In this study, we aim to find the non-SAM mimics, so EPZ015666-binding site, not SAM-binding site, was targeted in our virtual screening. Enrichment factor calculations showed that the 1 and 10% EFs for PRMT5-EPZ015666-SAM model were 44.6 and 8.7, higher than that (38.3 and 6.8) for PRMT5-EPZ015666 model. The area under receiver operating characteristic curve (AUC) for the former (0.96) was also higher than that for the latter (0.92). Both of two parameters suggested that SAM was helpful for enriching active compounds in the compound library. Therefore, SAM was remained as a part of the receptor in the screening.k-means clustering protocol integrated in Canvas 2.4. When selecting the candidate compounds, the following criteria was considered: (1) choosing one compound at most in a group to retain structural diversity; (2) occupying the binding pocket with molecular size neither too big nor too small; (3) choosing the one with smaller molecular weight or/and lower MM/GBSA score if compounds are similar; (4) forming the reported interactions with the key residues of PRMT5  screenings, the top-1706 compounds ranked by glide score were remained and then clustered into 200 groups using of PRMT5 . For ins50 = 5.8 ± 1.0 μM)  and was chosen as the hit, a range of T1551 concentrations  for 24, 48, and 72 h were then used to treat A549 to calculate its IC50 values. As shown in Figure 2, T1551 exhibited significant anti-proliferation on A549 cell at 24 h in a concentration-dependent manner, with the IC50 value of 11.2 ± 2.5 μM. The cytotoxic effects of T1551 were also verified using H460 cell, another NSCLC cell line with PRMT5 overexpression .The obtained 158 candidate compounds were then tested for MTT assay to determine their inhibitory activity. Many recent studies have showed that PRMT5 is upregulated in A549 non-small cell lung cancer cell line . A549 ceFigure 3A, T1551 inhibited PRMT5 enzyme activity in a dose-dependent manner. The corresponding IC50 value was 34.1 ± 2.8 μM, suggesting that T1551 directly inhibited the methyltransferase function of PRMT5. PRMT5-driven methylation of arginine residues can lead to symmetric dimethylation of arginine residue 3 of histone 4 (H4R3me2s), which in turn alters chromatin structure to promote transcriptional repression , entropic contribution was neglected. The predicted ΔGGB for PRMT5-T1551 was higher than that of PRMT5-EPZ015666  in three replicas, exhibiting a consistent ranking with experimental results  predominated the total energy in two systems, while non-polar solvation part (ΔGsol_np_GB) contributed marginally to inhibitor binding. Therefore, the energetic origin of T1551/EPZ015666 inhibiting PRMT5 is mainly derived from ΔEvdw.To investigate the detailed binding modes of PRMT5-inhibitors and compare the interaction features of T1551 and EPZ015666 with PRMT5, a single 100 ns MD simulations for PRMT5-SAM-T1551 and PRMT5-SAM-EPZ015666 systems were performed, respectively. Based on the obtained trajectory, with respect to the initial structure, the root-mean-square deviations (RMSDs) of protein CA atoms in PRMT5-SAM-T1551 and PRMT5-SAM-EPZ015666 systems were monitored to assess the overall stability of simulations. From Table 1; . Among tFigures 6c,d), we could see that both inhibitors located in a hydrophobic pocket composed of Tyr304, Phe327, Ser578, and Phe580 when interacting with PRMT5. For EPZ015666, Figure 6d showed that its THIQ group formed strong cation–π interactions with partial positively charged methyl group of SAM. Actually, this feature has been reported as a key factor for EPZ015666’s efficiency in the previous study , the helix and loop vibrated in the face–face direction, which seemed like to tighten the binding pocket and thus stabilize EPZ015666 into it. From Figure 7b, we also observed that the obviously higher amplitude motion of loop domain made major contributions in it. Nevertheless, in the PRMT5-T1551 system , the helix and loop moved in the back–back direction, which led the pocket not as compact as that in PRMT5-EPZ015666 system. It may be closely associated with that EPZ015666 has better biological activity for PRMT5 than T1551.Finally, in order to see the effect of inhibitors on conformational space of PRMT5, normal mode analysis was carried out. For clear visualization, only the normal modes of T1551 binding domain (10 Å around T1551) were shown here. From PRMT5, as currently the only known type II PRMT, is also a member with the few inhibitors reported in PRMT family. As the relationship of PRMT5 and lung cancer is constantly revealed, it is urgent to search for effective inhibitors targeting PRMT5 for lung cancer therapy. SAM, as the natural substrate of PRMT5, is responsible for providing the methyl group in the process of methyl transfer. To date, most of PRMT5 inhibitors reported were aimed for SAM-binding site and designed to disturb the interaction of SAM and PRMT5 . HoweverIn this study, we identified T1551 as a non-SAM competitive PRMT5 inhibitor by virtual screening method. Subsequently, the anticancer activity of T1551 against NSCLC was studied from three aspects, namely PRMT5 methyltransferase activity, expression of target genes and signaling pathway mediated by target genes. For the former, the “on-target” and direct inhibitory effect of T1551 was reflected from the low PRMT5 enzymatic activity, and indirect effect was from the low expression level of PRMT5’s histone marker (H4R3me2s), which together suggested that T1551 inhibited PRMT5 methyltransferase activity.For the latter, FGFR3 and eIF4E are two target genes of PRMT5 we focused here. We know that PI3K/AKT/mTOR pathway is a prototypic survival pathway in cancers, whose activation is closely related to cellular proliferation, growth, and mobility. FGFR3 promotes the survival of cancer cells just by stimulating the downstream PI3K/AKT/mTOR pathway . Using RPrevious studies emphasized that cation–π interaction between the tetrahydroisoquinoline group of EPZ015666 and partial positively charged methyl group of SAM was essential for EPZ015666’s higher competitive ability for PRMT5 relative to histone substrate . ReplaciIn summary, a novel PRMT5 inhibitor T1551 with the indole scaffold was identified in this study, whose functional influence on PRMT5 was verified by a series of biological assays and theoretical inhibitory basis on PRMT5 was revealed by molecular dynamic simulation method. These results provide a lead compound for the further design of PRMT5 inhibitors, and contribute to the development of PRMT5-targeting cancer treatment.XY, EL, and LL conceived the project. XY, EL, and QW designed the experiments. QW, JX, YL, JH, ZJ, and YW carried out the research and data analysis. XY, EL, LL, and QW wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.