The formation of a functional blood vessel network relies on the ability of endothelial cells (ECs) to dynamically rearrange their adhesive contacts in response to blood flow and guidance cues, such as vascular endothelial growth factor-A (VEGF-A) and class 3 semaphorins (SEMA3s). Neuropilin 1 (NRP1) is essential for blood vessel development, independently of its ligands VEGF-A and SEMA3, through poorly understood mechanisms. Grounding on unbiased proteomic analysis, we report here that NRP1 acts as an endocytic chaperone primarily for adhesion receptors on the surface of unstimulated ECs. NRP1 localizes at adherens junctions (AJs) where, interacting with VE-cadherin, promotes its basal internalization-dependent turnover and favors vascular permeability initiated by histamine in both cultured ECs and mice. We identify a splice variant of tryptophanyl-tRNA synthetase (mini-WARS) as an unconventionally secreted extracellular inhibitory ligand of NRP1 that, by stabilizing it at the AJs, slows down both VE-cadherin turnover and histamine-elicited endothelial leakage. Thus, our work shows a role for NRP1 as a major regulator of AJs plasticity and reveals how mini-WARS acts as a physiological NRP1 inhibitory ligand in the control of VE-cadherin endocytic turnover and vascular permeability. Functional vascular tree formation is a key step in many contexts, such as cancer, and Neuropilin1 (NRP1) has been associated with adhesion receptor endocytic turnover. Here, authors show NRP1 and its mini-WARS ligand play a role in reducing endothelial permeability. Neuropilin-1 (NRP1) gene first emerged in vertebrates concomitantly with the appearance of the cardiovascular apparatus and a more elaborated nervous system1. Consistently, during embryonic development, the expression of NRP1 transmembrane protein is enriched in neurons and vascular endothelial cells (ECs)2. Furthermore, improper cardiac outflow tract (OFT) septation3, along with severe abnormalities of blood vessels3 and peripheral nerves4, are observed in Nrp1 knock-out mouse embryos, revealing key morphogenetic functions of NRP1 receptor during patterning of vertebrate body plan. Yet, differently from neurons5, the molecular mechanisms by which NRP1 signals in ECs to control vascular morphogenesis are still poorly understood6.During evolution, the 7. For this reason, NRP1 is mainly postulated to function as a co-receptor of SEMA3s and VEGF-A ligands, that signal via type A/D plexin (PLXNA/D) receptors9 and VEGF receptor-2 (VEGFR-2)11. In fact, neuronal guidance4 and OFT septation12 defects caused by Nrp1 gene deletion are largely phenocopied in mice lacking Sema3a13 and Sema3c14, respectively. However, different vascular abnormalities are phenotypically observed upon murine Nrp115 or Sema3a17 or Sema3c14 knock-out. Moreover, mice harboring a point mutation disrupting the VEGF-A binding to NRP1 survive to adulthood with normal vasculature18. Altogether, these data hint that in ECs NRP1 may signal and play a functional role(s) without requiring the binding with extracellular ligands. In this respect, even if, as conceivable, VEGF-A and SEMA3A promote NRP1 internalization19, we have previously shown that, in ECs, NRP1 displays the ligand-independent ability to associate with and promoting the endocytosis of active α5β1 integrin20, the main fibronectin (FN) receptor driving embryonic vascular development and cancer angiogenesis21, thanks to the interaction of its short cytosolic tail with trafficking adaptors, such as GIPC1. In addition, the b1 domain-binding carboxy (C)-terminal basic sequence (C-end Rule or CendR) motif-containing peptides are taken up by NRP1-dependent internalization23 and cause NRP1-driven blood vessel leakage, similarly to VEGF-A24. Our recent finding that high-affinity binding to NRP1 is required for SEMA3A to increase integrin internalization and to elicit vascular permeability25 further suggests that NRP1 displays autonomous function(s), such as the constitutive endocytosis of interacting membrane proteins20, and that may be modulated by b1 domain-interacting ligands. Interestingly, the secreted glycyl tRNA synthetase (GARS) mutants implicated in type 2D Charcot-Marie-Tooth (CMT2D) neuropathy compete with VEGF-A for the binding to the extracellular b1 domain of NRP1, which is a CMT2D modifier gene27. Indeed, during evolution aminoacyl-tRNA synthetase (aaRS) proteins have acquired additional domains and motifs that endowed them with nontranslational properties, such as those of being secreted and acting as extracellular proteins that signal through mechanisms that are still incompletely characterized29. For example, vertebrate tryptophanyl-tRNA synthetase (WARS) has acquired, passing from fish to humans, a N-terminal WHEP domain to negatively regulate its anti-angiogenic activity30.The extracellular coagulation factor V/VIII homology domain b1 of NRP1 binds with high affinity to the C-terminal arginine of chemorepulsive secreted class 3 semaphorin proteins (SEMA3s), such as SEMA3A or SEMA3C, and the angiogenic vascular endothelial growth factor-A (VEGF-A)To draw a detailed molecular portrait of the protein interaction network potentially responsible for NRP1 intrinsic biological activities, we have analyzed by quantitative mass spectrometry (MS) its interacting partners both on the surface and in the endosomal compartment of ECs in the absence of exogenously added ligands. Grounding on our unbiased comprehensive analysis, we revealed that, in ECs, NRP1 interacts with and promotes the endocytic turnover of VE-cadherin at cell-to-cell contacts, thus allowing histamine to elicit vascular permeability. Furthermore, we identified NRP1 as a potential receptor for several aaRS family members. Among those, an unconventionally secreted splice variant of WARS without the WHEP domain (mini-WARS), upon binding the extracellular region of NRP1, decreases both NRP1 and VE-cadherin endocytosis at adherens junctions (AJs) and impairs EC permeability in response to histamine in a NRP1-dependent manner.31, and combined it with quantitative mass spectrometry (MS) analysis. To this aim, we generated a NRP1 construct containing a N-terminal modified HT that covalently binds membrane-impermeable chloroalkane containing ligands, such as biotin, thus allowing an effective antibody (Ab)-independent isolation of HT-tagged proteins on streptavidin resin31. We subcloned in a lentiviral vector a construct in which the HT encoding sequence was fused immediately 3’ to that of the signal peptide of silencing-resistant murine Nrp1 (HT-NRP1)  ECs -based approach, which enables effective surface labeling and pull-down of proteinsP1) Fig. . We tranP1) Fig. . ConfocaP1) Fig. . MoreoveP1) Fig. . Next, H 32 Fig. . We haveECs Fig. . Thus, tECs Fig. .Fig. 1Ge33  of the 966 quantified proteins identified components that segregated the three experimental conditions . Of those potential NRP1 interactors, 61 proteins represented the NRP1 surface interactome and 86 the NRP1 endosomal interactome , PECAM1 (CD31), and MCAM (CD146), as potential NRP1 transmembrane partners. In sum, one-third of the transmembrane proteins that we found to associate with NRP1 mediate or regulate EC adhesion  and platelet EC adhesion molecule 1 (PECAM1/CD31). Indeed, cell-to-cell and cell-to-ECM contacts share different adaptor and signaling proteins, and their mechanical crosstalk is critical for FN fibrillogenesis and directional collective cell migration41. Thus, NRP1 may represent a regulatory protein that functions as an endocytic receptor both at ECM and intercellular adhesion sites. To verify this hypothesis, we determined NRP1 subcellular positioning relatively to intercellular adhesion molecules identified in our MS interactome. Confocal microscopy analysis showed that in unstimulated ECs in culture, NRP1 clearly co-localizes at cell-to-cell contacts with VE-cadherin , lysed, immunoprecipitated, and analyzed by western blotting with mAbs recognizing either VE-cadherin or PECAM1. NRP1 co-immunoprecipitated with VE-cadherin  was significantly higher   targeting human NRP1 (siNRP1), which we have previously validated, or control non-targeting siRNA (siCtrl)01) Fig. . Further 20 Fig. , effecti 20 Fig. . Notably 20 Fig. . Next, wECs Fig. . Altoget48, the turnover of AJs largely impinges on cycles of cadherin endocytosis and recycling back to the plasma membrane50. Hence, we sought to biochemically gauge the effect of NRP1 silencing on VE-cadherin internalization in ECs by implementing a well-established method employed to quantitatively monitor integrin endocytosis53. Confluent ECs were surface labeled with cleavable biotin at 4 °C and then incubated at 37 °C for 1 min to allow internalization of biotinylated plasma membrane proteins. Next, the biotin remaining on cell-surface proteins was cleaved and VE-cadherin endocytosis quantified by capture ELISA on cell lysates, employing an anti-VE-cadherin Ab as a catcher. Knockdown of NRP1 diminished by 26 ± 0.004% the quantity of VE-cadherin internalized by ECs and this endocytic defect was rescued by the transduction of silencing-resistant mouse Nrp1  shared by all eukaryotic WARSs, and a WHEP domain, which appeared in vertebrate WARS and is connected to ESE by a 21 amino acid long disordered linker59 Fig. . Mini-WA 59 Fig. .Fig. 5Mi60 and its adenylate pocket mediates an inhibitory interaction with two N-terminal Trp side chains of VE-cadherin61 that is sterically interfered by the presence of the WHEP domain28. Interestingly, CMT2D patients display point mutations in GARS, which make the full-length synthetase, in presence of a metazoan WHEP domain, capable of binding the b1 domain of NRP1, thus outcompeting with ligands, such as VEGF-A27. Therefore, we investigated which WARS isoform may bind NRP1 in ECs. Lysates from unstimulated ECs were immunoprecipitated with anti-NRP1 Ab or control non-immune IgGs and analyzed by western blot with an anti-WARS Ab raised against a recombinant fragment containing both the catalytic Rossmann fold and the anticodon recognition domains. Of note, while in cultured ECs FL-WARS was expressed more abundantly than its two shorter anti-angiogenic isoforms, NRP1 robustly and selectively co-immunoprecipitated with mini-WARS only  construct of NRP1  rely on an S100A10-regulated unconventional mechanism63 to be released in the extracellular environment. In sum, similarly to VEGF-A11, SEMA3A7, and CMT2D GARS mutants27, secreted extracellular mini-WARS may also interact with the b1 domain of the extracellular portion of NRP1 and act as a physiological modulator of NRP1 function.Extracellular T2-WARS localizes at EC intercellular junctionsnly Fig. . Next, wnly Fig. . In vitrnly Fig. . Moreovenly Fig. . To confnly Fig. , lysatesnly Fig. . We founRP1 Fig. . Finallyium Fig. . TherefoP < 0.05) and VE-cadherin  localized at intercellular contacts in mini-WARS overexpressing ECs, compared to control ECs  and VE-cadherin  from the surface of the ECs , in basal conditions. Moreover, both in vitro and in vivo the lack of NRP1 in ECs impairs vascular permeability elicited by histamine that, independently from NRP1, signals via G-protein coupled histamine receptor H157. However, the direct binding of activatory ligands, such as SEMA3A, further increases the ability of NRP1 to promote adhesion receptor endocytosis and to elicit vascular leakage25. Similarly, in ECs VEGF-A19 and CendR peptides23 trigger NRP1 internalization and vascular permeability. In addition, SEMA3A-elicited neuron growth cone collapse also relies on NRP1-dependent endocytosis75 and endosomal signaling76. Hence, both in ECs and neurons, NRP1 appears to behave as an endocytic receptor whose basal activity controls the turnover of associated transmembrane proteins, such as adhesion receptors, and to be increased by activatory ligands. It has been proposed that NRP1-dependent endocytosis resembles macropinocytosis23. Since phagocytosis and macropinocytosis display several mechanistic similarities77, it will be worth investigating if NRP1-elicited endocytosis of adhesion receptors impinges on the activation of key phagosomal signaling components that we have identified in our MS analysis as potential NRP1 interactors, e.g., the Arp2/3 protein machinery that drives actin polymerization, or Ezrin and Moesin adaptors that mediate receptor binding to F-actin77.We have observed that in ECs NRP1 promotes the endocytosis of interacting adhesion receptors, e.g. active α5β1 integrins27. A striking feature of these three aaRSs is their shared N-terminal WHEP domain. In fact, these three aaRSs are the only ones with a WHEP domain at the N-terminus27. Our previous structural analysis indicated that the WHEP domain regulates the conformation of GARS and that deletion of the WHEP domain induces a conformational change in GARS that mimics CMT-causing mutants which have gained a capacity to interact with NRP178. Consistently, we show here that in ECs mini-WARS but not the FL-WARS with a WHEP domain can interact with NRP1, supporting the concept that the WHEP domain may act as a negative regulator for aaRS-NRP1 interaction. The fact that all three NRP1-interacting aaRSs share a WHEP domain suggest that their interaction with NRP1 may be regulated by the inclusion or exclusion of the WHEP domain.Remarkably, we found that in unstimulated ECs NRP1 interacted with at least three different aaRSs, namely GARS, HARS, and WARS, all of which have been causatively linked to CMT disease79 often combined to their unconventional secretion63 in the extracellular environment29. Of note, both HARS80 and WARS82 were characterized for their anti-angiogenic properties. In the case of FL-WARS, removal of the N-terminal WHEP domain-containing portion, either by alternative splicing or proteolytic cleavage, gives rise to mini-WARS and T2-WARS respectively that behave as potent secreted anti-angiogenic factors82. In particular, T2-WARS anti-angiogenic function relies on its tryptophan and adenosine pocket-mediated binding to Trp2 and Trp4 of VE-cadherin N-terminal extracellular domain61. Here, we provide evidence that in ECs mini-WARS, but neither FL nor T2-WARS, effectively binds the extracellular segment of NRP1 , may be under the control of NRP1. In turn, NRP1 pro-endocytic function may be regulated by activating (VEGF-A and SEMA3A) or inhibiting (mini-WARS and CMTD2 GARS mutants) ligands  and the following oligonucleotide primers: 5’-CTGAAGATTTTAAGTGTGCTGGACCAACCACAC-3’ (forward); 5’- GTGTGGTTGGTCCAGCACACTTAAAATCTTCAG-3’ (reverse). The HaloTag encoding sequence (derived from Promega) was fused immediately 3’ to that of the signal peptide of PINCO-NRP1 WT, by standard PCR protocols. FL-WARS, mini-WARS, and T2-WARS constructs were cloned into pcDNA6/V5-His C vector . HaloTag-NRP1 and mini-WARS were subcloned into a third-generation lentiviral vector pCCL.sin.cPPT.PGK.GFP.WPRE87 (pCCL), with In-Fusion 2.0-CF Dry-DownPCR Cloning-Kit . VE-cadherin-mCherry in the lentiviral vector HIV3 was kindly donated by Elisabetta Dejana .PINCO-Nrp1 WT and PINCO-Nrp1 ∆cyto constructs were previously describedg. Cells were first resuspended in Endothelial Cell Growth Basal Medium (EBM-2) supplemented with EGM-2 BulletKit (Lonza) (EGM-2), and subsequently plated in cell culture dishes that had been previously adsorbed with 1% gelatin from porcine skin . Cells were tested for mycoplasma contamination by means of Venor GeM Mycoplasma Detection Kit  and grown in EGM-2 medium. The isolation of primary arterial ECs from human umbilical cords was approved by the Office of the General Director and Ethics Committee of the Azienda Sanitaria Ospedaliera Ordine Mauriziano di Torino hospital  and informed consent was obtained from each patient.Primary human ECs were isolated from the umbilical cords. Briefly, umbilical arteries were cannulated with a blunt 17-gauge needle that was secured by clamping the cord over the needle. The umbilical arteries were then perfused with 50 ml of phosphate-buffered saline (PBS) to wash out the blood. Next, 10 ml of 0.2% collagenase A  diluted in cell culture medium were infused into the umbilical arteries and incubated 30 min at room temperature. The collagenase solution containing the ECs was flushed from the cord by perfusion with 40 ml of PBS, collected in a sterile 50 ml centrifuge tube, and centrifuged 5 min at 800 × Mouse monoclonal antibody (mAb) anti-HaloTag ; mAb anti-α tubulin ; goat polyclonal anti-EEA1 ; rabbit anti VEGFR-2 ; mAb anti-NRP1 ; goat polyclonal anti-VE-cadherin ; rabbit anti-VE-cadherin ; rabbit anti-NRP1 ; mAb anti-PECAM1 ; goat polyclonal anti-NRP1 ; rabbit polyclonal anti-WARS ; mAb anti-vinculin ; rabbit anti-V5 ; rat monoclonal Ab anti-HA ; mAb anti-V5 ; mAb anti S100A10 ; mAb anti-transferrin receptor 1 ; polyclonal sheep anti-NRP1 ; rabbit monoclonal Ab anti-Nrp1 ; rat monoclonal Ab anti-panendothelial cell antigen Meca32 ; goat anti-rabbit secondary Ab ; goat anti-mouse secondary Ab ; goat anti-rat HRP ; Alexa Fluor-488 donkey anti-mouse (A21202) and anti-goat (A11055) IgG (H + L) secondary Ab ; Alexa Fluor-555 donkey anti-mouse (A31570) and anti-goat (A21432) IgG (H + L) secondary Ab ; Alexa Fluor-647 donkey anti-mouse (A31571) and anti-goat (A21447) IgG (H + L) secondary Ab ; DAPI ; normal Goat (NI02-100UG) and mouse (NI03-100UG) IgGs ; mouse monoclonal agarose-conjugated anti-NRP1 antibody ; goat anti-human IgG Fc HRP preadsorbed ; agarose anti-V5 tag antibody ; mAb anti-GAPDH . HaloTag Alexa 660-labeled fluorescent chloroalkane Ligand ; HaloTag PEG-Biotin Ligand for surface labeling ; Streptavidin-Agarose conjugate beads ; Histamine ; Fluorescein isothiocyanate (FITC)-Dextran 70 ; 0.4 µm pore size costar transwell ; Dynabeads™ protein G beads ; Amicon Ultra-30k Centrifugal Filters .Human plasma fibronectin , human NRP1-Fc , human VE-Cadherin-Fc  and human IgG1-Fc ; recombinant human VEGF-A165 .E. coli BL21(DE3) cells with 1 mM isopropyl beta-d-thiogalactopyranoside. Following cell lysis, WARS was purified using Ni-NTA beads (Qiagen). The N-terminal His6-SUMO tag was cleaved with sumo protease ULP1. The resulting tag-free WARS proteins were further purified using HiLoad 16/60 Superdex 200 prep grade column .For the expression and purification of recombinant human WARS isoforms, DNA encoding human FL-WARS, mini-WARS, or T2-WARS with a N-terminal His6-SUMO tag was cloned into pET-28a (+) vector (Novagen). WARS expression was induced in 3 cells/well. Oligofection of siRNA duplexes was performed according to the manufacturer’s protocols. Briefly, human ECs were transfected twice (at 0 and 24 h) with 200 pmol of siGENOME Non-Targeting siRNA Pool #1 (D-001206-13) as control (siCtrl) or a mix of three (in the case of human NRP1 and WARS), or a mix of four (for human S100A10) siRNA oligonucleotides , using Oligofectamine Transfection Reagent (ThermoFisher Scientific). Twenty-four hours (NRP1), 48 h (WARS), or 96 h (S100A10) after the second oligofection, ECs were lysed or tested in functional assays. Knockdown of human S100A10 was achieved through the single oligonucleotide sequences: (1) 5’-GAUAAAGGCUACUUAACAAUU-3’; (2) 5’-GAAAAGGAGUUCCCUGGAUUU-3’; (3) 5’-GAACACGCCAUGGAAACCAUU-3’; (4) 5’-GGACCAGUGUAGAGAUGGCUU-3’. While, in the case of human WARS, the single oligonucleotide sequences were: (1) 5’-GGUUCCAAGUAUACUCUUAUU-3’; (2) 5’-CCAAAUGACCCUAGACCCUUU-3’; (3) 5’-CUGGAAGAGCAAAGCCAAAUU-3’; in the case of human NRP1, the single oligonucleotide sequences were: (1) 5′-AAUCAGAGUUUCCAACAUA-3′; (2) 5′-GAAGGAAGGGCGUGUCUUG-3′; (3) 5′-GUGGAUGACAUUAGUAUUA-3′.For siRNA-mediated silencing, the day before oligofection, ECs were seeded in six-well dishes at a concentration of 10 × 10The day before ECs transduced with pCCL HaloTag-NRP1 were plated on glass coverslips coated with gelatin 1%. Cells were incubated for 15 min at 4 °C (to inhibit endocytosis) with the cell impermeable HaloTag Alexa 660 fluorescent ligand (dilution 1:200), and then shifted or not to 37 °C for 3 min. ECs were washed in warm fresh medium twice, fixed in 1% paraformaldehyde (PFA), permeabilized in 0.01% saponin for 5 min on ice, and were stained with goat anti-EEA1 (dilution 1:200) and Alexa Fluor secondary Ab (dilution 1:400). Cells were analyzed by using a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems).2 in a humidified atmosphere. Cells were surface labeled with the cell impermeable HaloTag PEG-Biotin Ligand 3 µM at 4 °C for 15 min, stimulated or not with VEGF-A165 30 ng/ml for 10 min at 37 °C. ECs were lysed in buffer containing 25 mM Tris–HCl pH 7.6, 100 mM NaCl, 1% Triton X-100, 5% glycerol, 0.5 mM EGTA, 2 mM MgCl2, 1 mM PMSF, 1 mM Na3VO4 and protease inhibitor cocktail. Cellular lysates were incubated for 20 min on wet ice, and then centrifuged at 15,000 × g, 20 min, at 4 °C. The total protein amount was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce). Equivalent amounts (1 mg) of protein were immunoprecipitated for 2 h at 4 °C on streptavidin-conjugated beads. Immunoprecipitates were washed three times with lysis buffer with or without detergent and then separated by SDS–PAGE with Mini PROTEAN TGX precast 7.5% gel (BIO-RAD). Proteins were then transferred to a Trans-Blot Turbo™ PVDF Transfer (BIO-RAD), probed with antibodies of interest and detected by enhanced chemiluminescence technique (PerkinElmer).Transduced ECs were starved for 3 h with EBM-2 medium (Lonza) at 37 °C, 5% COIn all, 6000 transduced and silenced ECs were resuspended in 0.1 ml of EBM-2 medium (Lonza) and plated on 96-well microtiter plates (Costar) that were previously coated with 3 µg/ml FN and then saturated with 3% bovine serum albumin (BSA). Cells were left to adhere for 20 min. Cells were fixed in 8% glutaraldehyde and then stained with 0.1% crystal violet, 20% methanol. Cells were photographed with a QIcam FAST1394 digital color camera (QImaging) and counted by means of Image-ProPlus 6.2 software (Media Cybernetics).2, 1 mM PMSF, 1 mM Na3VO4 and protease inhibitor cocktail. Cellular lysates were incubated for 20 min on wet ice, and then centrifuged at 15000xg, 20 min, at 4 °C. The total protein amount was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce). Equivalent amounts (3 mg) of protein were incubated for 2 h at 4 °C on streptavidin-conjugated beads. Proteins captured on streptavidin-conjugated beads were then washed three times with lysis buffer with and without detergent and eluted with reducing sample buffer.ECs transduced with pCCL lentivirus carrying HaloTag-NRP1 construct were surface labeled or not (Ctrl) with the cell impermeable HaloTag PEG-Biotin Ligand 2.5 µM at 4 °C for 15 min and shifted or not at 37 °C for 3 min. Cells were lysed in buffer containing 25 mM Tris–HCl pH 7.6, 100 mM NaCl, 1% Triton X-100, 5% glycerol, 0.5 mM EGTA, 2 mM MgCl88 with trypsin (Promega). Digested peptides were desalted using StageTip89, and injected on an EASY-nLC system coupled on line to a LTQ-Orbitrap Elite via a nanoelectrospray ion source (Thermo Scientific). Peptides were separated using a 20-cm fused silica emitter (New Objective) packed in house with reversed-phase Reprosil Pur Basic 1.9 μm (Dr. Maisch GmbH), and eluted with a flow of 200 nl/min from 5 to 30% of buffer containing 80% ACN in 0.5% acetic acid, in a 90 min linear-gradient, as previously described90. The mass range acquired for the full MS scan was 300–1650 m/z with a resolution of 120,000 (HCD mode) at 400 Th and the Orbitrap aimed to collet 1 × 106 charges at a time. The top ten most intense peaks in the full MS were isolated for fragmentation with a target of 40,000 ions at a resolution of 15,000 at 400 Th. Ions singly charged were excluded, whilst the ions that have been isolated for MS/MS were subsequently added to an exclusion list. MS data were acquired using the Xcalibur software (Thermo Scientific) and .raw files processed with the MaxQuant computational platform91 version 1.5.0.26 and searched with the Andromeda search engine92 against the human UniProt database35 . To search the parent mass and fragment ions we required a mass deviation of 4.5 ppm and 20 ppm. The minimum peptide length was seven amino acids and maximum of two missed cleavages and strict specificity for trypsin cleavage were required. Carbamidomethylation (cysteine) was set as fixed modification, whereas oxidation (methionine) and N-acetylation were set as variable modifications. The false discovery rates at the protein and peptide levels were set to 1%. The requantification and match between runs features were enabled. For protein quantification, label-free quantification (LFQ) was calculated by MaxQuant34. Unique and razor peptides were used for protein quantification and we required proteins to be quantified with at least two ratio counts.Proteins eluted in sample buffer were boiled 5 min at 95 °C and separated on 4–12% gradient NuPAGE Novex Bis-Tris gel (Life Technologies) and in-gel digested93. Common reverse and contaminant hits (as defined in MaxQuant) were removed, as well as proteins only identified by site. The LFQ Intensity values were used for protein quantification. Only proteins quantified in at least three of the four biological replicates in at least one experimental group were kept for follow-up analysis. LFQ values were log2 scaled and missing values were replaced using the Imputation function . For each protein, its enrichment in the NRP1 surface interactome and NRP1 endosomal interactome was calculated by dividing the LFQ intensity to the LFQ intensity measured in the control sample . A one-sample t test on the calculated ratios  was used to calculate P values and generate the volcano plots. Proteins that had a fold increase of at least 1.5 compared to the control and P value lower than 0.05 were considered potential NRP1 interactors. In Supplementary Data, we also reported the adjusted P values , which were calculated using Benjamini–Hockbert FDR. To exclude that the potential NRP1 interactors were proteins commonly found as background in affinity purification experiments using the HaloTag, we compared the list of NRP1 interactors to those found in the CRAPome of HaloTag affinity purification experiments (www.crapome.org). Potential background proteins have been highlighted in Supplementary Data The proteinGroup.txt output of MaxQuant was analyzed with Perseus35 “Topology” annotation. The “UniProt Annotation Retriever” software tool [https://github.com/gkoulouras/UniProt-annotation-retriever] was used to fetch relevant protein annotation. Custom R scripts were developed, and the following terms were used: “Single-pass type I membrane protein”, “Single-pass type II membrane protein” and “Multi-pass membrane protein”. Based on their “subcellular locations”, as defined by UniProt, the selected transmembrane proteins were grouped into the following groups “Membrane”, “Focal adhesion”, “Cell membrane”, and “Cell Junction”. Endosomal proteins were selected based on the subcellular location classification defined by UniProt, in particular we selected the categories “Early endosome”, “Late endosome”, “Lysosome”, and “Cytoplasmic vesicles”.The selection of transmembrane proteins from the NRP1 interactome was based on the UniProt94 was used for category enrichment analysis  and to generate the interaction network of the NRP1 interactome with transmembrane and endosomal proteins excluded. For Supplementary Fig. STRING96” (source code: https://github.com/jokergoo/circlize) and “igraph97” (https://igraph.org/r/), respectively.The chord diagrams and networks in Fig. 2+) and magnesium (Mg2+) supplemented PBS, fixed in 2% paraformaldehyde (PFA), permeabilized in 0.1% Triton X-100 for 2 min on ice or fixed in Methanol at −20 °C for 10 min. Cells were incubated with different primary Abs for 1 h and revealed by appropriate Alexa Fluor- secondary Ab. Cells were analyzed by using a Leica TCS SPE confocal laser-scanning microscope (Leica Microsystems). We quantified the Mean Fluorescence Intensity in AJs drawing linear regions of interest (ROI) by means of the Leica Confocal Software Quantification Tool (Leica LAS-X). Image acquisition was performed by adopting a laser power, gain, and offset settings that allowed maintaining pixel intensities  within the 0–255 range and hence avoid saturation.ECs were plated on glass coverslips coated with 1% gelatin from porcine skin  and allowed to adhere overnight or plated on fibronectin (3 μg/ml) to reach the required confluence to observe cell-to-cell contacts. Cells were washed in calcium  assay (Pierce). Equivalent amounts (1 mg) of protein were immunoprecipitated for 1 h at 4 °C with the antibody of interest, and immune complexes were recovered on protein A- or G-Sepharose  for 1 h at 4 °C. To co-immunoprecipitate VE-cadherin and PECAM1 with NRP1, ECs were incubated live with the mouse mAb that recognize the extracellular domain of NRP1 or control mouse IgGs for 10 min at 37 °C and immune complexes were recovered on protein A- or G-Sepharose  for 2 h at 4 °C. Immunoprecipitates were washed three times with lysis buffer with or without detergent and then separated by SDS–PAGE. Proteins were then transferred to a Trans-Blot Turbo Nitrocellulose membrane (BIO-RAD), probed with antibodies of interest, and detected by Clarity Western ECL Substrate (BIO-RAD).ECs were seeded in 150 mm dishes coated with 1% gelatin coating. ECs were lysed in buffer containing 25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 5% glycerol, 0.5 mM EGTA, 2 mM MgCl2. For evaluating the direct interaction of NRP1 with WARS proteins, 0.1 μg of the recombinant NRP1-Fc or human IgG1-Fc protein was immobilized on 2 μl of Dynabeads™ protein G beads (ThermoFisher Scientific) for 30 min at 4 °C. Dynabeads coated with NRP1-Fc or Fc control were then incubated with 0.5 μg of recombinant tag-free human FL-, mini-, or T2-WARS for 2 h at 4 °C. Samples were washed three times with wash buffer . Lastly, samples were eluted with 50 μl of 1×LDS containing 5 mM DTT and analyzed by western blotting.For evaluating direct NRP1 and VE-Cadherin interaction, 2 μg of recombinant Fc-tagged whole extracellular portion of human NRP1  were incubated with 2 μg of recombinant Fc-tagged whole extracellular portion of human VE-Cadherin (VE-cadherin-Fc) or with the human IgG1-Fc alone on agarose-conjugated anti-NRP1 antibody (1 μg) for 2 h at 4 °C, in 25 mM Tris–HCl buffer pH 7.4, 100 mM NaCl, 5 mM CaCl2, and hybrid detectors. The 488 nm line of Argon laser at high intensity was used for beaching the selected ROI, and 2 pre-bleach, 2 bleach, and 50 post-bleach frames (every 1.4 s) were acquired.Transduced and silenced ECs, plated in WillCo Glass Bottom Dishes, 1% gelatin from porcine skin -coated, were analyzed by using a Leica TCS SP8 AOBS confocal microscope equipped with ×63 (HC PL APO CS2 ×63/1.40 oil) objective, 37 °C humidified chamber with 5% CO2, 1 mM Na3VO4, 0.5 mM (ethylene glycol tetraacetic acid) EGTA, 1 % Triton X-100, 5% glycerol, protease inhibitor cocktail , and 1 mM phenylmethanesulfonylfluoride (PMSF). Lysates were cleared by centrifugation at 12,000 × g for 20 min. Levels of biotinylated VE-cadherin, NRP1 or transferrin receptor 1 were determined by capture ELISA assay.Cells were transferred to ice, washed twice in cold phosphate-buffered saline (PBS), and surface labeled at 4 °C with 0.2 mg/ml sulfo-NHS-SS-biotin (Pierce) in PBS for 30 min. Labeled cells were washed in cold PBS and transferred to prewarmed EBM-2 containing 2% FBS at 37 °C. At the indicated times, the medium was removed, and dishes were rapidly transferred to ice and washed twice with ice-cold PBS. Biotin was removed from proteins remaining at the cell surface by incubation with a solution containing 20 mM sodium 2-mercaptoethanesulfonate (MesNa) in 50 mM Tris–HCl (pH 8.6), 100 mM NaCl for 1 h at 4 °C. MesNa was quenched by the addition of 20 mM iodoacetamide (IAA) for 10 min, and after other two further washes in PBS, the cells were lysed in 25 mM Tris–HCl, pH 7.4, 100 mM NaCl, 2 mM MgCl2CO3 (pH 9.6) at 4 °C and were blocked in PBS containing 0.01% Tween-20 (PBS-T) with 5% BSA for 1 h at RT. Proteins were captured by overnight incubation of 50 μl cell lysate at 4 °C. Unbound material was removed by extensive washing with PBS-T, and wells were incubated with streptavidin-conjugated horseradish peroxidase  in PBS-T containing 1% BSA for 1 h at 4 °C. Following further washing, biotinylated receptors were detected by a chromogenic reaction with ortho-phenylenediamine.Corning 96 Well Clear Polystyrene High Bind Stripwell Microplate  were coated overnight with 1 μg/ml of appropriate anti-VE-cadherin, NRP1 or transferrin receptor 1 antibodies in 0.05 M NaECs were seeded on 6.5-mm diameter Cell Culture Inserts contain PET membrane with 0.4-μm pore size (Costar) pre-coated with 1% gelatin or fibronectin (3 μg/ml), cultured for 24 h in complete culture medium, starved in EBM 2 % FBS 1% BSA for 3 h and assayed for permeability to fluorescein isothiocyanate (FITC)-dextran (70 kDa) (Sigma-Aldrich). When cells were confluent, 25 mg/ml of FITC-dextran with the stimulus Histamine 100 μM, were added to the medium (EBM 2% FBS) of the insert apical compartment. At 2 h, 4 h, and 6 h, 50 µl aliquots of the medium were collected from the basal compartment and the paracellular flux was measured as the amount of FITC-dextran in the medium using the fluorometer Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments).Nrp1EC−/−) were generated in a C57BL/6 background by crossing floxed conditional Nrp1-null (Nrp1fl/fl) mice [B6.129 (SJL) Nrp1tm2Ddg/J12 from The Jackson Laboratories] with Cdh5(PAC)-CreERT298 mice that carry a tamoxifen-inducible Cre deleter under the control of the VE-cadherin promoter Cdh5(PAC) and were provided from Cancer Research Technology Ltd, according with the Agreement procedures. Gene deletion in adult mice was induced as described previously99. In order to analyze junction integrity after treatment, 10 µg of histamine dissolved in 50 µl PBS was injected subcutaneously in the right flank of Nrp1EC−/− and control Nrp1+/+ mice, while PBS injected in the left flank of the same animal served as a negative control. 10 min later, 3 mg of Evans Blue dissolved in 100 µl PBS per 10 g of body weight was injected intravenously. Following 20 min incubation, residual Evans Blue within blood vessels was removed by heart perfusion using saline. Equal-sized skin pieces covering the injected areas were resected using a skin biopsy punching tool and digested in 400 µl formamide for 48 h at 60 °C. Afterward, the amount of extravasated Evans Blue was quantified using a spectrophotometer at a wavelength of 620 nm. Tissue samples were then dried for 96 h at 60 °C, weighted and the amount of extravasated Evans Blue was normalized to (a) its dry weight and (b) the ratio of the sample’s weight and the lightest sample from the same animal.All animal procedures were approved by the ethics committee of the University of Torino and by the Italian Ministry of Health  and also by the Institutional Animal Care and Research Advisory Committee of the KU Leuven. Endothelial-specific Nrp1 knock-out mice  were air-dried, fixed in zinc-fixative 2, 2.5 g Zn(C2H3O2)2, 2.5 g ZnCl, 3.8 ml HCl 37%) for 10 min and blocked in 3% BSA and 5% donkey serum in 1×PBS. Tissue sections were incubated with rabbit monoclonal Ab anti-Nrp1 (ab81321) from Abcam and rat monoclonal Ab anti-panendothelial cell antigen Meca32 (550563) from BD Pharmingen, both diluted 1:100 in saturation solution. Anti-rabbit Alexa Fluor-555 and anti-rat Alexa Fluor-488 were employed as secondary Abs. Nuclei were counterstained with DAPI (Invitrogen). All immunofluorescence images were captured by using a TCS SPE confocal laser-scanning microscope (Leica Microsystems), and by maintaining the same laser power, gain, and offset settings.Endothelial-specific ECs were seeded in six-well dishes (12 wells for condition) and transfected twice with siCtrl or siS100A10 oligonucleotides. 72 h after the second oligofection, ECs were lipofectamine transfected with 1 µg/ml of V5-tagged mini-WARS. After 24 h, culture supernatants were collected, concentrated using Amicon Ultra-30k (Millipore), immunoprecipitated on 10 µl of Agarose Anti-V5 tag antibody (Abcam ab1229) for 2 h at 4 °C and analyzed by western blot analysis.t test was used to assess the statistical significance when two groups of unpaired normally distributed values were compared; when more than two groups were compared, parametric two-tailed analysis of variance (ANOVA) with Bonferroni correction was applied. For each experiment, we performed three independent biological replicates, in which at least three technical replicates were performed. Then, to analyze the reproducibility, we calculated the mean of the three independent biological replicates ± SEM and performed statistical analyses. Statistical differences were considered not significant (ns) = P value > 0.05; significant *P value ≤ 0.05; **P value ≤ 0.01; ***P value ≤ 0.001).For statistical evaluation, data distribution was assumed to be normal. Parametric two-tailed heteroscedastic Student’s Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


GH9 genes (TaGH9s) were identified, which were classified into Class A, Class B and Class C and unevenly distributed on chromosomes. We also investigated the gene duplication and reveled that fragments and tandem repeats contributed to the amplification of TaGH9s. TaGH9s had abundant hormone-responsive elements and light-responsive elements, involving JA–ABA crosstalk to regulate anther development. Ten TaGH9s, which highly expressed stamen tissue, were selected to further validate their function in pollen fertility conversion and anther dehiscence. Based on the cell phenotype and the results of the scanning electron microscope at the anther dehiscence period, we found that seven TaGH9s may target miRNAs, including some known miRNAs (miR164 and miR398), regulate the level of cellulose by light and phytohormone and play important roles in pollen fertility and anther dehiscence. Finally, we proposed a hypothesis model to reveal the regulation pathway of TaGH9 on fertility conversion and anther dehiscence. Our study provides valuable insights into the GH9 family in explaining the male sterility mechanism of the wheat photo-thermo-sensitive genetic male sterile (PTGMS) line and generates useful male sterile resources for improving wheat hybrid breeding. Glycoside hydrolase family 9 (GH9) is a key member of the hydrolase family in the process of cellulose synthesis and hydrolysis, playing important roles in plant growth and development. In this study, we investigated the phenotypic characteristics and gene expression involved in pollen fertility conversion and anther dehiscence from a genomewide level. In total, 74 wheat  GH9s have been reported to be associated with cellulose biosynthesis [Glycoside hydrolase family 9 (GH9) encodes a cellulase gene consisting of endo-β-1,4-glucanase (EGase) . EGases ynthesis . GH9 family genes in plants, including Arabidopsis [GH9 genes were found to be involved in many plant growth processes such as cell elongation, anther dehiscence, pollen tube growth, branch node abscission and fruit ripening [Cel5 gene, a member of endo-β-1,4-D glucanase, is involved in pistil, pedicel and leaf abscission as well as fruit ripening [Arabidopsis, plasma membrane-bound endo-1,4-β-D-glucanase play important roles in normal cell wall growth and cell elongation [OsGLU1 gene leads to reduced cellulose levels in rice, which also affects rice cell elongation [Currently, there are many studies on the bidopsis , rice [8bidopsis  and strabidopsis . In prevripening ,13,14. Iripening . In oranripening . In Arabongation . In addiongation ,19. Arabidopsis, some fibrous thickenings in trinucleate anthers appear on the walls of endothecium cells. Additionally, in the flowering stage, endothecium cells and connective tissue cells shrink after losing water, resulting in anther dehiscence [Anther normal development and dehiscence are key factors for plant fertility. Thus, the normal development of anther (the container of pollen), especially the anther wall, is the key to pollen development. Anther endothecium is composed of cellulose, pectin and endothecium wall proteins . It was hiscence . GH9 may be associated with stamen development and anther dehiscence. It has been found that the glycosyl hydrolase AtGHL is highly expressed in Arabidopsis stamens. The mutation of AtGHL results in stamen deletion, suggesting that the AtGHL gene may be associated with stamen growth [TaBG was higher in the anther dehiscence stage in a sterile environment and was 2.8 times higher in indehiscent anthers than that in normal dehiscent anthers in wheat photo-thermo-sensitive genetic male sterile (PTGMS) line BS366. The results suggest that the increase of TaBG gene expression could make the soluble sugar content increased in anthers, increasing the osmotic potential and slowing down the dehydration activity, resulting in anther indehiscence [Recently, studies have implied that n growth . The exphiscence . GH9 genes were differentially expressed under different fertile environments in BS366. However, few studies have reported how cellulose regulates anther development and is involved in male fertility and how cellulase genes such as GH9 participate in the regulation pathway in these biological processes. In this study, the GH9 family was characterized to reveal the regulation of GH9 on pollen development and anther dehiscence in wheat PTGMS lines. Here, phylogenetic tree, gene structure, conserved motifs and chromosomal localization comprehensively by bioinformatics methods for GH9 gene family were analyzed. In addition, the expression of TaGH9 in BS366 was detected using qRT-PCR. miRNAs such as miR167 and miR172 are associated with anther development in the wheat PTGMS line [GH9 and microRNAs to further understand the roles of the GH9 family, and finally a network regulatory map was constructed. Our study provides a good theoretical basis for further understanding the TaGH9 function and provides a new sight for research on the mechanism of fertility transition in wheat PTGMS lines. Wheat PTGMS line BS366, an important female parent material of two-line hybrid wheat, was developed by the DH (double haploid) population of Jingnong 8121 × E8075-7; its fertility is controlled by light and temperature. The main characteristics of male sterility on BS366 are microspore male sterility and low seed-setting rate due to anther indehiscence . GH9 genGMS line . We alsoGH9 gene, which belonged to 24 TaGH9 genes, were obtained. The coding sequence (CDS) lengths and protein lengths are shown in After removing structurally incomplete genes, 74 copies of the GH9 families in Arabidopsis, rice and poplar, a phylogenetic tree of wheat was constructed. As shown in TaGH9 were classified into three major subclades, Class A, Class B and Class C with 8 (10.8%), 58 (78.4%) and 8 (10.8%) copies of TaGH9, respectively. The Class B subclade is the largest subclade in the GH9 of wheat, which was further divided into three subgroups, Class B1, Class B2 and Class B3, with the numbers 37 (50%), 16 (21.6%) and 5 (6.8%), respectively.Combining the identified GH9 genes on wheat chromosomes, we tagged their relative positions on wheat chromosomes. A total of 74 copies of TaGH9 were distributed on 20 chromosomes, except chromosome 3B. The distribution of genes on chromosomes was scattered and there was no cluster distribution  were highly expressed in the stamens  of fertile and sterile conditions. Compared with pollen under fertile condition, the cell plate at dyad (stage 7) under sterile condition cannot be properly formed in telophase I A, resultnversion B.TaGH9, the expression of these genes was tested during anther dehiscence stages. It was found that TaGH9-2, TaGH9-4 and TaGH9-7 had lower expression under the sterile environment, while TaGH9-9 and TaGH9-15 had higher expression  . The resTaGH9 in the regulation of fertility, seven pollen and/or anther development-related TaGH9s were selected to evaluate the binding association with miRNAs. The results indicate that these seven genes harbored targets for ten different miRNAs , involved in rice pollen germination [TaGH9-2-A/B/D. In addition, other known miRNAs such as tae-miR164 and tae-531 were predicted to target with TaGH9.miRNAs could interact with genes and play an essential function in plant growth and development . In our t miRNAs . Accordit stress . In ricemination . In our mination . In the TaGH9, qRT-PCR was performed. The results show that TaGH9-9, TaGH9-16, TaGH9-15 and TaGH9-19 were targeted with tae-miR164, tae-miR9661-5p, tae-miR5384-5p and tae-miR9676-5p, respectively, involved in pollen fertility conversion , sorghum (Sorghum bicolor (L.) Moench), maize (Zea mays L.), short-stalked grass (Brachypodium distachyon (L.) Beauv.), Arabidopsis thaliana, poplar (Populus) and cotton (Gossypium. L). In our previous study, it was found that GH9 may be involved in male sterility by RNA-seq [GH9 had differential alternative splicing between fertile and sterile condition, involved in fertility conversion. However, there are few reports regarding how GH9 works in pollen and anther development in crop plants. A total of 24 wheat GH9 with 74 copies were isolated in this study, to reveal the function of GH9 in wheat pollen and anther development (Glycoside hydrolase family 9 (GH9) was previously reported as cellulase family E, whose members are spatiotemporal expression differences in plant growth and development and play various roles by encoding various hydrolysis-type genes and binding different substrates . In rece RNA-seq . GH9 hadelopment . cis-regulatory elements could respond to external stimuli, affecting plant growth by regulating gene transcription [TaGH9, the cis-regulatory elements in promoter regions of TaGH9 were analyzed. It was found that four major cis-regulatory elements, including light-responsive elements, stress-related elements, growth and development-related elements and hormone-responsive elements, were identified from TaGH9  and abscisic acid response elements (ABRE element) are enriched in the TaGH9 family  in the stamens were identified based on the results of expression profiles of TaGH9s in tissues. These genes also contained more light and hormone-responsive elements. As molecular switches, cription . To furtom TaGH9 . Hormone9 family . The latnversion . We also tissues  were invOs12BGlu38, a member of GH1, showed shrunken and hollow pollen and deficient mutant pollen. In addition, no inner wall was observed in the pollen of Os12BGlu38 mutants using transmission electron microscopy (TEM) [GH9 have been charactered in Arabidopsis and rice. OsGH9B18, OsGH9B6/14 and OsGH9C2 in rice, At3g43860, AtGH9A3, AtGH9A4 and AtGH9B5/11/14/17 in Arabidopsis, specifically expressed in stamen and pollen [TaGH9-4/7/9 classified into Class C with OsGH9C2 and TaGH9-2/19/15/11 classified into Class B1 with AtGH9B14, AtGH9A3 and OsGH9B18, suggesting that these TaGH9 genes may have a similar function with genes in rice and Arabidopsis , participating in age-related cell death [miR164-CUC1/2 results in the defect in sepals and low fertility in plants [TaGH9-9 genes interacted with miR164, participating in pollen fertility conversion by responding to light  and tapetum . Lipid apy (TEM) . Severald pollen . Genes wbidopsis . TaGH9-1-9 genes . In Arabll death . A lack n plants ,51. In an plants . In our n plants . These rto light . LSSR1, encoding a GH5 cellulase, expressed in the anther microsporogenesis stage of rice. It has been reported that LSSR1 with an N terminal signal peptide is secretory protein stored in pollen grains and plays important roles during fertilization in rice. The results indicate that the process of fertilization was inhibited in the mutant LSSR1, resulting in reduced fertility in rice, indicating that LSSR1 plays an essential role in male fertility in rice [TaGH9-7, TaGH9-15 and TaGH9-19 were highly expressed in the pollen development stage, especially in stage 7, 8 and 10, which were the critical stages of fertility conversion, indicating that highly expressed TaGH9-7, TaGH9-15 and TaGH9-19 during pollen development may be one of the reasons for pollen abortion  line BS366 (winter wheat) was used. Growing conditions: The seeds were planted in October 2020 in the experimental fields in Beijing  and Dengzhou , China, respectively, and managed conventionally.Samples for fertility and anther dehiscence analysis were collected using the following equation reported by ,37.For tissue-specific expression analysis, the root, stem, leaf, petal, pistil, stamen and glume of wheat at the meiosis stage were collected.For anther samples, eight different anther developmental periods in fertile and sterile environments, including microspore mother cells (Stage6), meiotic dyad (Stage7), meiotic tetrad (Stage8), early uninucleate (Stage9) and late uninucleate (Stage10), bicompartmental stage (Stage13), dehiscence stage (Stage14) and recession stage (Stage15), were collected .Anthers at different stages from fertile and sterile conditions were photographed with a ZEISS SteREO Discovery.V20 dissecting microscope . To further analyze pollen fertility conversion, the anthers of BS366 from meiosis to the mature pollen stage under both fertile and sterile conditions were collected and fixed in FAA solution . The pollen grains were released from anthers by tweezers and dyed with improved carbol fuchsin solution  Photograhttps://Plants.ensembl.org/Triticum_aestivum/Info/Index, accessed on 10 January 2022) and the Pfam database . The GH9 protein data of Arabidopsis thaliana (At), Oryza sativa (Os) and poplar were downloaded from TAIR , RAP  and PIR .The protein sequences of the glycoside hydrolase family 9  of wheat were downloaded from the Ensembl Plant database  was used to analyze the basic physicochemical properties of the final obtained wheat GH9 protein, including gene CDS, amino acid number (AA), relative molecular weight (MW), isoelectric point (PI), total mean hydrophilicity value (GRAVY) and aliphatic index (AI). The subcellular localization of the wheat GH9 protein was predicted using the target P 1.1 Server . GH9 subgenomic copies were named A, B and D according to their position on the chromosome.The Ex PASy-ProtParam online tool  [TaGH9 family was carried out by using Circos-0.69  [Positional information of ry 2022) . The visry 2022) .Multiple alignment of GH9 protein sequences was performed by using DNAMAN  with default parameters. The phylogenetic tree was constructed by using MEGA6.0  with thehttp://gsds.cbi.pku.edu.cn/, accessed on 25 February 2022) by comparing the predicted GH9 coding sequences with their corresponding genomic sequences.Gene structure information of the TaGH9 family members were visualized using the Gene Structure Display Server (GSDS) tool   to identify the cis-elements.To identify the cis-elements in the promoter sequences of the GH9 family genes in wheat, 1.5-kb of genomic sequences upstream of the promoter regions of each GH9 genes was chosen and screened against the Plant CARE database  . The TPM (Transcripts Per Kilobase of exon model per million mapped reads) data of GH9 genes were visualized with a heat map using Graphpad Prism 9 software.The RNA-seq data of six tissues  and three developmental periods  of Chinese spring wheat were downloaded and analyzed using expVIP  based on predicted results.miRNA could play an important role in plant development by targeting genes. To investigate whether the candidate ® miRcute Plus miRNA PreMix , respectively, as described previously [–ΔΔCt method for relative expression with three biological replicates and three technical replicates [The total RNA of all samples was extracted using TRIzol reagent  following the manufactures’ instructions, and approximately 1 µg RNA was reverse transcribed with PrimerScript 1st Strand cDNA synthesis kit  and miRcute Plus miRNA cDNA T  for genes and miRNAs, respectively, according to the manufacturer protocol. qRT-RCR assays for TaGH9 and miRNAs were conducted on an Eco Real-time PCR system  using TB Green Premix Ex Taq (Tli RNaseH Plus) kit  and SYBReviously ,75. Wheaplicates . The priTaGH9 gene family in wheat, including gene and protein structures, phylogenetic relationships and expression profiles, was provided. A total 74 copies of TaGH9 were identified and classified into three major subclades, named Class A, Class B and Class C. The TaGH9s in the same subfamily and subclades may own similar functions. There were abundant light-response-related elements and hormone-response-related elements in the promoter region that assist TaGH9s to respond to light and hormones, regulating anther and pollen development. Ten TaGH9s were highly expressed in stamens. Finally, seven TaGH9s may be associated with pollen development and anther dehiscence. A putative pathway of TaGH9s with targeted miRNA regulating to pollen fertility conversion and anther dehiscence was constructed. The potential function of TaGH9 needs to be further verified using more molecular biological techniques and tools. Collectively, these findings establish a foundation for further exploration of TaGH9 and provide novel insights into its biological functions.In this study, a comprehensive overview of the


Hepatocellular carcinoma (HCC) is the predominant type of liver cancer and a leading cause of cancer-related death globally. It is also a sexually dimorphic disease with a male predominance both in HCC and in its precursors, non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of the androgen receptor (AR) in HCC has been well documented; however, AR-targeted therapies have failed to demonstrate efficacy in HCC. Building upon understandings of AR in prostate cancer (PCa), this review examines the role of AR in HCC, non-androgen-mediated mechanisms of induced AR expression, the existence of AR splice variants (AR-SV) in HCC and concludes by surveying current AR-targeted therapeutic approaches in PCa that show potential for efficacy in HCC in light of AR-SV expression. Hepatocellular carcinoma (HCC) is the predominant form of liver cancer making up 90% of cases and is currently the fourth most lethal form of cancer as well as the sixth most common cancer worldwide . HCC exhBased upon the discrepancy in NASH/NAFLD and HCC incidence between the sexes, the role of the male nuclear sex hormone receptor, the androgen receptor (NR3C4/AR), in HCC progression has been investigated. The AR is a transcription factor that operates via ligand-activation by binding to androgens , 5α-dihydrotestosterone (DHT)) resulting in nuclear translocation, dimerization, and binding to androgen response elements (AREs) across the genome allowing regulation of target gene transcription. The AR consists of 3 functional domains; the N-terminal domain (NTD), which contains the binding domains for the transcriptional machinery and cofactors needed for transcriptional regulation, the DNA-binding domain (DBD), which binds to AREs within the genome to regulate target gene transcription, and the ligand-binding domain (LBD) which binds to circulating androgens leading to AR activation and nuclear translocation  [5]. EarThough data are mixed, the balance of early studies on AR levels in HCC tumors found significantly higher AR protein expression when compared with adjacent normal liver tissues and that increased AR protein expression was correlated with increased tumor recurrence and reduced overall survival ,11. HoweThe AR has been implicated in Epithelial–Mesenchymal Transition (EMT) regulation in the early literature on cancer. One study looking at the role of AR and EMT in breast, prostate, and other cancer cells found that AR directly downregulated E-cadherin expression through an ARE leading to EMT and metastases . AdditioBased upon the frank male bias in HCC incidence and mortality along with abundant pre-clinical support for the role of AR in HCC, AR inhibition as a therapy for HCC has been evaluated in a series of clinical trials using combined AR-axis inhibition approaches routinely deployed in prostate cancer (PCa). Grimaldi et al. conducted a double-blind trial with a two-by-two design to test the effectiveness of nilutamide, a first generation anti-androgen , and a lThe contribution of AR signaling to HCC progression has been supported with abundant clinical and pre-clinical evidence showing its correlation with faster disease progression, tumor burden, disease prognosis, overall survival, and as a mediator of metastasis. However, the failure of antiandrogen therapy to improve patient outcomes seems to conflict with an understanding of ligand-dependent AR signaling. To reconcile the clinical failure of androgen-targeted therapy and the overwhelming evidence supporting a role for AR in HCC, there are several explanations to consider. Expression of the AR may be dissociated from circulating androgen levels and could result from feedback from other signaling pathways in HCC. The difference between expression and activity is also a key distinction as increased expression of AR may not necessarily mean an increase in AR activity or vice versa. Finally, examining mechanisms of ligand-independent constitutive activation of AR could decouple AR signaling from androgen dependent AR activation. The examination of these alternative explanations has resulted in a better understanding of the role of AR signaling in HCC and potential therapeutic avenues.Androgen-independent induction of AR expression is one possible resistance mechanism to anti-androgen therapy. AR expression is in part mediated by androgens which can both downregulate AR expression to suppress further AR signaling in the presence of higher levels of androgen or as shown in several HCC cell lines, androgens can upregulate AR expression ,29. HowemTOR engages in a complex signaling pathway that can induce AR expression. Zhang et al. found that mTORC1 plays a role in both inhibiting AR degradation as well as upregulating the translocation of AR to the nucleus A. Once nIn a recent follow-up study from the same group, the mechanism behind mTORC1’s ability to inhibit AR degradation and promote AR nuclear localization was further elucidated. Ren et al. reported that mTORC1 phosphorylates AR at S96, in the amino-terminal domain, which enhances AR stability, translocation to the nucleus, and increased activity of AR independently of ligand C. IncreaLipogenesis also demonstrates a key linkage to AR in HCC progression as demonstrated by Cheng et al. who observed that AR activity and transcription of AR could be affected by dysregulated lipogenesis resulting from high fat diets that induce HCC. Specifically, diacylglycerols (DAGs) were shown to activate AKT which then in turn would activate AR D demonstIn addition to mTOR’s signaling relationship with AR, cell cycle-related kinase (CCRK) has also been shown to play a role in mTOR signaling through AR. In a study conducted by Feng et al., CCRK was established as a direct AR transcriptional target through unbiased ChIP-Chip studies in HCC cells and confirmed to have high AR binding in its regulatory region. They proposed a signaling pathway whereby AR upregulates CCRK which in turn upregulates ß-catenin to exert effects on the cell cycle and on overall cellular proliferation E. This pAnother alternative mechanism for the induction of AR expression was developed by Sun et al. within the context of obesity-related HCC. They advanced an AR, STAT3, and CCRK feedback loop in which CCRK helps to incite STAT3 and AR interactions and their combined localization to androgen response elements (ARE) within the CCRK promoter upregulates CCRK expression. In addition, CCRK indirectly activates mTOR signaling pathways which in turn promotes HCC progression E. This wApart from CCRK related-signaling, another proposed pathway offers a further link between ß-catenin and AR signaling via cooperation with focal adhesion kinase (FAK). The authors, Shang et al., elucidated a direct link between overexpression of FAK paired with ß-catenin mutations and increased levels of AR activation within HCC. They posit that FAK acts by enhancing ß-catenin binding to the AR promoter thereby upregulating AR expression. Notably, they found that carcinogenesis occurring through this pathway is likely AR dependent but androgen independent . This coThese mechanisms of inducing AR expression while not providing a comprehensive overview of the AR interactome and signaling pathways in HCC, provide valuable insight into how AR interacts with other oncogenic signaling pathways and their potential to upregulate the AR-axis. Databases such as BioGRID have compiled a wide array of AR interactors which provides additional support for the need to better understand AR and how it fits into the broader array of dysfunctional signaling pathways within HCC .Several key studies have examined the role of AR in HCC by seeking to differentiate AR expression from activity and androgen-dependent AR signaling from androgen-independent AR signaling. Analyses of the HCC cohort in TCGA have helped illuminate the role of AR activity and expression in hepatocarcinogenesis. Acosta-Lopez et al. showed that while total AR mRNA levels are positively correlated with increased overall survival in this data set, gene regulation resulting from known AR transcriptional activity are negatively correlated with overall survival. This insinuates that AR activation rather than AR expression is key to HCC progression. As a caveat, the gene sets used in these analyses to determine AR activity were derived from PCa and in the future, more HCC specific measures of AR expression may be more valuable .In addition to evidence of induced AR expression, data show that AR activity is not always a direct result of androgen binding in HCC A. At leaOne mechanism for androgen-independent AR signaling involves constitutively active AR splice variants capable of driving AR transcriptional programs in the absence of androgen binding. Lee et al. report that mRNA splicing factors can be expressed differentially within HCC leading to alternative splicing patterns that can then contribute to multiple oncogenic pathways. The hijacking of various splicing factors through differential expression, silencing, or changes in the RNA binding proteins present can replace normative splicing patterns with alternative patterns resulting in proteins with new, similar or even opposite function when compared to the canonical splice form . Given tMost of the current knowledge surrounding the existence of AR splice variants (AR-SVs) and their activity is framed within the context of PCa, in which the androgen receptor and its splice variants play an established role. Within PCa, anti-androgens have been shown to rapidly upregulate splice variants of AR, such as AR variant 7 (AR-V7), which contain cryptic exons and premature stop codons preventing them from expressing the ligand-binding domain and ultimately rendering them androgen independent . Many ARThis understanding of AR-SVs and their role in PCa led to an investigation of a similar role for AR-SVs in HCC. We confirmed the existence of AR-SVs within HCC and determined through analyses of TGCA data that 78% of HCC patients had intra-tumoral AR-SV expression and subsequently discovered that in the patients with the most abundant AR mRNA, AR-SVs accounted for roughly one quarter of expressed AR. Using direct comparisons to well characterized PCa models, these results showed that AR-SVs’ abundance within HCC were comparable to AR-SVs in PCa and AR-SV expression in HCC was higher than in normal liver controls. Additionally, we established both AR-FL and AR-V7 action can promote cell migration and invasion through EMT signaling in agreement with multiple reports showing AR signaling plays a role in cancer metastasis. We found that the SNAI2 gene, whose product is an established mediator of EMT , is likely a direct target of the AR . FinallyInterestingly, despite these similarities between HCC and PCa, there are several striking differences. AR-SV expression is thought to be upregulated in castration-resistant prostate cancer (CRPC) through selective pressure from androgen deprivation therapy (ADT) while HCC expresses AR-SVs in patients absent selective pressure from low levels of circulating androgens or anti-androgen therapy ,44. AddiIt is important to note that in the body of literature surrounding the role of AR in HCC, many immunoblotting techniques used to measure AR protein levels do not account for molecular weight  such that reagents targeting an epitope in the amino terminus of the AR are unable to distinguish AR-FL from lower molecular weight, carboxy-terminus truncated AR-SVs. Many of the most commonly used AR antibodies have amino terminus epitopes , EMD Millipore AR-PG21), Abcam , Dako (AR441)) and have been used to assess AR levels within primary HCC tissue in multiple recent studies ,21,32,36, Abcam  or DNA-binding domain (DBD) to either initiate protein degradation or to block AR transcriptional activity . Due to A very comprehensive overview of therapeutic approaches targeting AR was published recently by Xiang et al., which focused on small molecules that are thought to inhibit either AR-FL only or both AR-FL and AR-SVs . A such,Dysidea sp., and it binds to the AR Activation Function-1 (AF-1) region of the NTD and inhibits the transactivation of both AR-FL and AR-SVs [In addition to the DBD, the N-terminal domain (NTD) is another critical domain for constitutive activity and is located upstream of the DBD ,65. NTD d AR-SVs ,72,73.Another molecule from a marine sponge source called EPI-067 serves as the origin of a well-acknowledged family of AR-NTD inhibitors that covalently bind with the AF-1 region of AR-NTD ,75. EPI-Another group of compounds that interfere with AR-SVs are the UT-series, also known as selective androgen receptor degraders (SARDs). Hwang et al. designed a compound named UT-69 by combing the structural features of an AR antagonist and an AR agonist (Enobosarm), which showed efficacy against Enzalutamide-resistant xenografts and degraded both AR-SVs and AR-FL . FurtherCollectively, there have been a number of therapeutic approaches targeting ligand-independent AR activity. AR-SVs that lack the ligand-binding domain can be deactivated through DBD blockade, NTD inhibition or ubiquitin–proteasome pathway-mediated protein degradation. Given the clinical relevance of AR-SVs to HCC, it is reasonable to believe that these therapeutic approaches would also be applicable to HCC patients.Beyond the novel compounds currently being developed, there have been several proposals to repurpose known compounds. One study found that quercetin , a naturAnother leading candidate for repurposing is the anti-helminthic drug, niclosamide . Lui et Based upon its promising anti-PCa effects, niclosamide has been evaluated in PCa clinical trials. A phase I trial combining niclosamide with enzalutamide in CRPC was halted due to the inability to achieve therapeutically efficacious niclosamide levels in plasma at the maximum tolerated dose . This hiAdditional efforts have been made to improve niclosamide for use in PCa including the exploration of several analogs with the goal of both improving niclosamide’s bioavailability and its anti-AR potency. One analog, ARVib-7 , showed In light of the key role that the mTOR pathways plays in HCC and its interaction with AR signaling, Zheng et al. provide strong support for a therapeutic strategy combining both AR and mTOR inhibition . They shTo conclude, AR signaling plays a key role in HCC progression and the sexual dimorphism of the disease; however, therapeutically targeting the AR-axis has thus far been unable to effectively treat HCC due to the combined effects of induced AR expression, post-translational ligand-independent AR-activation, and the presence of ligand-independent constitutively active AR-SVs. As HCC is a widespread and genetically heterogeneous disease lacking targeted therapy, AR provides a key therapeutic opportunity. The presence of abundant AR-SVs provides an additional explanation for previous failures in anti-androgen or hormone ablative approaches and may also explain, in part, difficulty in targeting key signaling pathways within HCC such as AKT-mTOR. Developing therapeutics that target both AR and AR-SVs could provide an improved therapeutic option for HCC patients with AR positive disease.While considerable work has been carried out to understand the role of AR within HCC, additional work is needed to better understand the influence of AR-SVs on known oncogenic signaling in HCC, their potential for HCC-specific action as compared to PCa, and potentially distinct AR-SV and AR-FL genetic programs in HCC. Unlike PCa, AR-SVs expressed in HCCs are not an adaptive response to therapeutic pressure from anti-androgen treatment as HCC patients are not routinely administered anti-androgens. AR-SVs in HCC are likely distinct in origin such that AR-SV expression profiles differ between PCa and HCC and may even contain novel HCC-specific variants. Better defining the “AR spliceosome” in HCC would facilitate development of biomarkers for AR activity and could help improve patient selection for future AR-targeted therapies. Despite promising recent advances in HCC treatment with immunotherapies , sexual


Neuropilin-1 (NRP1) is a transmembrane protein involved in many physiological and pathological processes, and it functions as a co-receptor to facilitate the entry of SARS-CoV-2 into host cells. Therefore, it is critical to predict the susceptibility to SARS-CoV-2 and prognosis after infection among healthy people and cancer patients based on expression of NRP1. In the current study, we analyzed the conservation and isoform of NRP1 using public databases. NRP1 expression landscape in healthy people, COVID-19 patients, and cancer patients at both bulk and single-cell RNA-seq level was also depicted. We also analyzed the relationship between tissue-specific NRP1 expression and overall survival (OS), as well as tumor immune environment at a pan-cancer level, providing a comprehensive insight into the relationship between the vulnerability to SARS-CoV-2 infection and tumorigenesis. In conclusion, we identified NRP1 as a potential biomarker in predicting susceptibility to SARS-CoV-2 infection among healthy people and cancer patients. AccordiCoronaviruses (CoVs), belonging to the family Coronaviridae, are enveloped viruses with a single-strand, positive-strand RNA . Most CoNRP1, also known as CD304 or BDCA-4, is a member of the type I transmembrane protein family Neuropilins. NRP1 consists of an intracellular cytoplasmic domain, a transmembrane domain and an extracellular domain , and theCancer patients were found to have a higher COVID-19 morbidity and severity rate than individuals without cancer , partialClinical management of COVID-19 patients mainly consisted of supportive therapy and symptoms treatment. Antiviral drugs including remdesivir, favipiravir, lopinavir-ritonavir, and camostat mesilate have already showed apparent efficacy in treating COVID-19 . AntibodIn this study, we demonstrated the landscape of NRP1 expression among different tissue types and did a pan-cancer analysis to figure out potential vulnerable people to COVID-19 using public databases. We further analyzed the relationship between NRP1 and immunity to interpret the immune disorders observed in severe cases of COVID-19. We hope our research could emphasize NRP1 as a reliable biomarker in predicting vulnerability to SARS-CoV-2 infection in cancer patients and provide potential strategy to treat cancer patients combined with COVID-19.https://www.wikipathways.org/index.php/Pathway:WP5065, https://www.wikipathways.org/index.php/Pathway:WP4846). WikiPathways is an open, collaborative platform dedicated to the curation of biological pathways   (inewww/) , which ihttps://www.proteinatlas.org/ENSG00000099250-NRP1)  database (50-NRP1) . The HPAhttp://ualcan.path.uab.edu/index.html) and the ENCORI (https://starbase.sysu.edu.cn/panCancer.php) databases. The UALCAN database is a comprehensive and interactive web resource for analyzing gene expression data based on four databases including TCGA (http://timer.comp-genomics.org/). TIMER2.0 provides tools to compare the differential gene expression between different mutation status . Data were accessed on 10 February 2022.The Tumor Immune Single Cell Hub (TISCH) is an online scRNA-seq database integrating single-cell transcriptomic data of nearly two million cells from 76 tumor datasets across 27 cancer types in 18 organs and tissues, focusing on tumor microenvironment (TME) . The NRPhttp://gepia2.cancer-pku.cn/#isoform) based on the large TCGA and Genotype-Tissue Expression (GTEx) datasets. The impact of NRP1 expression on overall survival (OS) of cancer patients were also determined via GEPIA2 . Overall survival rate is defined as the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for cancer. Patients were classified into low NRP1 group and high NRP1 group based on the median NRP1 expression level for comparison of OS. Data were accessed on 10 February 2022.GEPIA2  is a website providing tools including differential expression analysis, correlation analysis, profiling plotting, similar gene detection, survival analysis, and dimensionality reduction analysis . The NRPhttp://cis.hku.hk/TISIDB/browse.php?gene=NRP1). The Tumor and Immune System Interaction Database (TISIDB) integrated multiple data resources in oncoimmunology to provide analysis on tumor-immune interactions (https://cistrome.shinyapps.io/timer/). Tumor Immune Estimation Resource (TIMER) integrated molecular profiles of tumor-immune interactions across 10,897 tumors from 32 cancer types . For bulk RNA-seq analysis, raw counts data was transformed into log2 (TPM+1) for comparison of gene expression. For snRNA-seq analysis, R package “Seurat” was used to process the raw counts data. Cells were classified into 21 clusters at a resolution of 0.2 after filtering out low low-quality cells.The GEO dataset GSE159585 contains bulk RNA-seq and single nucleus RNA-seq (snRNA-seq) data performed on lungs from seven COVID-19 patients and non-COVID-19 controls. Related data were downloaded from the GEO database  were obtained from the oncology laboratory of Tongji Hospital, Wuhan, China. BEAS-2b and PC-9 were cultured in RPMI-1640 medium , HCT116 was cultured in McCoy’s 5A medium , and the rest cells were cultured in DMEM medium . Medium used contained 10% FBS  and all the cells were maintained in the incubator at 37°C supplemented with 5% COTotal RNA from cells was isolated using TRIzol  and reverse-transcribed to cDNA with Hi Script II QRT SuperMix . Then, qRT-PCR was carried out in Real-Time PCR System  using ChamQ universal SYBR qPCR Master Mix . The relative gene expression levels were calculated using the 2−ΔΔCT method, and the GAPDH mRNA expression levels were used for normalization. The primer sequences were listed as follows: NRP1-forward, 5′- CCCCAAACCACTGATAACTCG -3′ and NRP1- reverse, 5′- AGACACCATACCCAACATTCC -3'; GAPDH-forward, 5′- GACAGTCAGCCGCATCTTCT -3′ and GAPDH-reverse, 5′- GCGCCCAATACGACCAAATC -3'.p < 0.05 was considered statistically significant. R software (version 4.2.0) and GraphPad Prism (version 8.0.1) were used for statistical analyses.Comparison of two normally distributed quantitative data were performed using the Student’s t test. Comparisons of multiple independent ordinal data were performed using Kruskal–Wallis test. Survival data were analyzed using Kaplan-Meier method and log-rank test. Spearman correlation test was applied to calculate the correlation between two normally distributed data. Two-side via NRP1 (WP5065). Both of these two pathways indicated that NRP1 could act as a co-receptor when SARS-CoV-2 infects host human cells. Similar to ACE2, SARS-CoV-2 enters cells through the binding of surface spike glycoprotein (S protein) to NRP1 on the cell membrane  and SARS-CoV-2 altering angiogenesis membrane . Additiovia NRP1 . These rAlignment result of NRP1 homologs in different species including human (NP_003864.5), chimpanzee (XP_001143690.1), Rhesus monkey (NP_001252745.1), cow (NP_001192589.1), dog (XP_005617003.1), mouse (NP_032763.2), rat (NP_659566.1), chicken (NP_990113.1), frog (NP_001093692.1), and zebrafish (NP_852474.2) revealed that it was highly conserved , with a An overview of NRP1 expression profiling at gene transcriptional and translational levels in different normal human tissues and organs was downloaded from the HPA database and was displayed in SARS-CoV-2 is mainly transmitted by droplet and aerosol, leading to severe acute respiratory syndrome . Thus, tp = 0.00067)  . The sam0.00067) . These ivia HPA. NRP1 protein concentration in plasma detected by mass spectrometry was 780 μg/L  were the most abundant cells among them  cells, neutrophils, monocytes/macrophages, mast cells, malignant cells, fibroblasts, endothelial cells, dendritic cells (DCs), and exhausted CD8+ T cells  in the TISCH database were used to analyze the expression of NRP1 in various different cell types. Analysis combining the five datasets showed that NRP1 mRNA expression level was the highest in endothelial cells . We furtong them . Cells iong them , includi T cells . NRP1 mR T cells . This rep = 0.0011) (p = 0.016) (p = 0.037) (p = 0.0086) (p = 0.041) (p = 0.00019) (p = 0.0019)  and OS of patients with GEPIA2. Cancer patients were divided into high- and low-NRP1 group according to median NRP1 expression level. An overview of the result was shown in  0.0011) , cervica= 0.016) , gliobla= 0.037) , low-gra 0.0086) , LUSC (p= 0.041) , STAD (p0.00019) , and uve 0.0019) . However 0.0019) . A previ 0.0019) . Therefop < 2.2e-16) (p < 2.2e-16) (p < 2.2e-16) (p < 2.2e-16)     (p < 2.2e-16) (p < 2.2e-16) (p = 3.27e-13), while positively related with B cells , CD8+ cells , CD4+ cells , macrophages , neutrophils , and DCs  in LUSC tumors (p < 2.2e-16), CTLA4 , TIGIT , PDCD1 , and LAG3    and expr2.2e-16) , NK cell2.2e-16) , activat2.2e-16) , neutrop2.2e-16) , central2.2e-16) , effecto2.2e-16) , central2.2e-16) , effecto2.2e-16) , regulat2.2e-16) , and mye2.2e-16) . Immune C tumors . In addi.05e-08)  in LUSC .05e-08) . Above fp = 2.19e-18), BRCA (p = 3.62e-11), KIRC (p = 1.81e-09), pheochromocytoma/paraganglioma (PCPG) (p = 1.53e-06), LUSC (p = 2.36e-06), COAD (p = 9.13e-06), and STAD (p = 1.57e-05)  . We did .57e-05) , indicatChemokines and their receptors influence tumorigenesis and metastases . The cheThe outbreak of COVID-19 worldwide has been a major challenge for global public health, so figuring out susceptible population to COVID-19 and predicting prognosis is of great importance from the perspective of epidemiology. Previous studies mainly focused on ACE2, a widely accepted receptor of SARS-CoV-2. However, understanding other potential receptors of SARS-CoV-2 may also be helpful in offering novel insights into the pathogenesis and treatment of SARS-CoV-2. In this article, we compared the expression level of NRP1 in different tissues and organs among both healthy people, COVID-19 patients and cancer patients. We also compared the expression of NRP1 with that of ACE2 directly. Besides, we did a pan-cancer analysis of the relationship between NRP1 expression and OS, NRP1 mutation, and TME.We found that NRP1 was highly conserved in different species. Experimental studies have indeed shown that a small number of animals are susceptible to infection with SARS-CoV-2 and can transmit the virus .Our study revealed that NRP1 was highly expressed in female reproductive system, respiratory system, and urinary system. In accordance with the expression profile observed in normal tissues, NRP1 was also highly expressed in ovarian cancer, colorectal cancer, and kidney cancer. This indicated that NRP1 mainly expressed in tissues communicating the outer environment, which was similar with that of ACE2 . It is wPan-cancer analysis revealed that the isoform NRP1-202 might be involved in both tumorigenesis and SARS-CoV-2 infection processes. We found that expression level and mutation rate of NRP1 depended on cancer types. However, in most cancer types, NRP1 functioned as a potential oncogene, which was consistent with previous studies . This inIn most cancer types expression of NRP1 was positively related with expression of immunoinhibitory markers and chemokines/chemokine-receptors. ICIs have gradually became an irreplaceable role in cancer therapeutics in recent years , and inhOur research identified NRP1 as a valuable marker in predicting vulnerability to SARS-CoV-2 infection among normal people, however, the susceptibility to SARS-CoV-2 in cancer patients merits further investigation. The COVID-19 and Cancer Consortium (CCC19) is an international consortium which collects data on patients with cancer and COVID-19 . StudiesIn conclusion, our study figured out susceptible population to SARS-CoV-2 based on NRP1 expression. NRP1 could be a valuable informative marker in predicting susceptibility to SARS-CoV-2 and the severity of COVID-19 patients, and it had a great potential as a therapeutic target especially in cancer patients combined with COVID-19.


To determine the prevalence of allergic rhinitis and associated factors in adolescents and in their parents/guardians.A cross-sectional study, applying a standardized and validated written questionnaire. Adolescents  and their parents/guardians  living in the city of Uruguaiana, southern Brazil, responded to the Global Asthma Network standard questionnaires.The prevalence of allergic rhinitis in adolescents was 28.0%, allergic rhinoconjunctivitis, 21.3%, and severe forms of allergic rhinitis, 7.8%. In the adults, the prevalence of allergic rhinitis was 31.7%. Some associated factors with allergic rhinitis in adolescents include low physical exercise , having only one older sibling  and daily meat consumption . In contrast, consuming sugar  or olive oil  once or twice a week, and eating vegetables daily  were considered factors negatively associated. In adults, exposure to fungi at home  and consumption of meat once or twice a week  were factors associated with the medical diagnosis of allergic rhinitis, while low education  was found to be a factor negatively associated.The prevalence of allergic rhinitis in adolescents is high, as well as its medical diagnosis in adults living in Uruguaiana. Environmental factors, especially food habits, were associated with findings in both groups. Allergic diseases are common at different stages of life, resulting in high costs of healthcare, school absenteeism, and decreased productivity, thus affecting the quality of life of patients and their caregivers. the Global Asthma Network (GAN) was established in 2012 with the objectives of understanding the current state of asthma, preventing it, and improving the overall care of asthmatic patients, especially in low- and middle-income countries.Epidemiological studies on the prevalence of allergic diseases have used standardized measuring tools to achieve importance in the recent decades, which brought more knowledge about them. Following the International Study of Asthma and Allergies in Childhood (ISAAC), study, which provides similar versions for adolescents (13–14 years old) and adults (parents and/or guardians).The GAN standard questionnaire was based on the ISAAC phase III In Latin America, the mean prevalence of AR is 18.5%, ranging from 7.1%  to 45.1% . In Brazil, there is a variation between 8.9% (São Paulo) and 24.4% .Allergic rhinitis (AR) is the most common allergic disease in children; however, it is often underestimated because it is a non-life-threatening disorder. In fact, it is a global health problem. Data obtained from the ISAAC phase III study showed that the prevalence of AR in adolescents ranges from 4.5%  to 45.1% .The ISAAC protocol was retired, showing an increase in the prevalence of allergic rhinitis over 20 years of its application. The GAN protocol opens a new phase of epidemiological assessment of allergic diseases.This study analyzed the prevalence of the symptoms of rhinitis and the factors associated with its occurrence in adolescents and in their parents/guardians living in the city of Uruguaiana, southern Brazil. Between June 2017 and December 2018, 1,200 adolescents from all 17 public schools were invited to participate and answer the GAN phase I standard questionnaire. The selected public schools were the same that participated in previous study, in addition to private schools that did not agree to participate. Inadequately completed or incomplete questionnaires were excluded, and 1,058 adolescents remained. The adolescents answered the questionnaires in their classrooms under the supervision of a researcher.Adolescents and their parents/guardians, living in the city of Uruguaiana (GAN Center No. 504013), participated in a cross-sectional, multicenter, international epidemiological study following the GAN standard protocol.The adolescents included took the questionnaires to their parents/guardians  to be answered at home and returned within one week. A total of 920 questionnaires were returned, and 896 (97.4%) were answered accordingly.The study was approved by the Ethics Committee of the Universidade Federal do Pampa (Unipampa), CAAE no. 62658216.2000.5323, and the Informed Consent and Consent Forms were signed by the adolescents and their parents/guardians, respectively. originally in English, were translated into Portuguese, back translated as recommended, and evaluated for understandability and retention of the contents of the original tool. Demographic data such as age, sex, date of birth, grade of school, date of interview, current height and weight, and birth weight were included. In addition, nasal problems associated with nasal and eye pruritus affecting daily activities; medical diagnosis of AR; previous and current exposure to paracetamol, antibiotics, and pollution; physical activity; and food consumption were also considered in this study. The questionnaires were coded by the center and the school, and participant number was used to ensure confidentiality and to link the questionnaires answered by the adults (parents/guardians) and their adolescents.The GAN standard questionnaires,In the past 12 months, has this nose problem been accompanied by an itchy nose?” identified respondents with AR; an affirmative answer to the question “In the past 12 months, has this nose problem been accompanied by itchy-watery eyes?” identified respondents with allergic rhinoconjunctivitis; and an affirmative answer to the question “In the past 12 months, this nose problem interfered moderately/a lot with your daily activities?” identified respondents with symptoms of severe rhinitis.Among the adolescents, an affirmative answer to the question ““Was your allergic rhinitis confirmed by a doctor?” identified respondents with AR.In adults, an affirmative answer to the question As established by the ISAAC study, the sample size was set between 1,000 and 3,000 participants due to the number of hypotheses investigated, and to get a high response rate, since there are concerns that asthma, rhinitis, or eczema symptoms may result in school absenteeism.® database. On comparison of the two entries, no inconsistencies were found. The data was sent to the GAN Center in Auckland, New Zealand, for initial evaluations, and afterwards, to the GAN Data Center in Spain for further verification.The collected data was entered in an independent double entry into Microsoft Office ExcelCategorical variables were presented as frequency distribution and proportions, and continuous variables were presented as mean and standard deviation. Factors associated with AR in the last 12 months were identified by bivariate and multivariate analysis. The introduction of factors in the multivariate analysis considered p-values lower than 0.20 obtained in bivariate analysis, according to the downward binary logistic regression analysis method. P-values lower than 0.05 were considered statistically significant.Nasal symptoms were indicated by 61.9% of the adolescents, AR by 28.0%, allergic rhinoconjunctivitis by 21.3%, moderate/severe forms of rhinitis by 7.8%, and 29.7% had a medical diagnosis of rhinitis. In adults, the frequency of medical diagnosis of AR was 31.7%.The positively associated factors  with AR in adolescents are presented in The positively associated factors identified for severe rhinitis were: having only one older sibling and using paracetamol at least once a month .In adults, the positively associated factors for a medical diagnosis of AR were: mold exposure in the household and eating meat once or twice a week. Having low educational level was found as a negatively associated factor . The prevalence of AR in adolescents was similar to the medical diagnosis of AR, which was lower than in adults. A phase II ISAAC study conducted 15 years ago in the same city reported a slightly higher prevalence of AR than today. As for the prevalence of allergic rhinoconjunctivitis, the result corroborated another Brazilian study that used the ISAAC protocol in more than 20,000 adolescents from seven Brazilian locations.The Uruguaiana GAN center was the first registered Brazilian center to complete a data collection. which reflected an improvement in the Human Development Index (HDI) from 0.523 to 0.744.The city of Uruguaiana experienced significant changes in the last 15 years, with the increase of basic sanitation from 16.0% to 81.2%, The authors documented a predominance in males aged under 11 years and females aged over 12 years, with no difference in the adults. There were more female respondents among the adult/guardian participants; however, it did not allow any inferences regarding the higher prevalence of AR in women since the result could be a response bias.Although it is a small difference, a higher frequency of AR diagnosis was noticed in male adolescents (data not presented); this is different from the data documented in a recent meta-analysis.Prevalence differences favoring post-pubertal women are attributed to the presence of higher levels of endogenous estrogens that determine an increased Th2 response in women. In contrast, testosterone in men acts by suppressing Th2 response. and approximately 40% in Europeans. The adults evaluated in this study showed a prevalence of AR diagnosis within the mean range.Epidemiological global studies in adults are rare. The prevalence of AR in US adults ranges between 10 and 30%,vs. none or occasional) with increased frequency of asthma, rhinoconjunctivitis, and eczema symptoms. Such apparently antagonistic situations could be explained by the fact that shorter exercise times are usually associated with longer sedentary periods, which leads to less physical conditioning and possibly greater exposure to aeroallergens and aggravated respiratory allergic diseases. In addition, physical activity in a polluted environment may justify its association with respiratory health problems, especially during the practice. Therefore, many authors recommend not to do vigorous physical activities on high-air-pollution days, especially outdoors. Similarly, little physical activity was also positively associated with increase of allergic rhinoconjunctivitis.As for the factors associated with the occurrence of AR symptoms in adolescents, performing minimal exercise was associated with increased AR risk. A study of adolescents using the ISAAC phase III database associated vigorous physical activity (up to three times a week  and corroborated by other authors, and with allergic rhinoconjunctivitis and severe forms of rhinitis. In summary, frequent contact with viral infections would stimulate the development of standard Th1 immune response.In adolescents, having only one older sibling was positively associated with increase of AR, as proposed by Strachan it was not observed in the adolescents evaluated in this study. However, the use of paracetamol at least once a month was identified as a positively associated factor for the occurrence of allergic rhinoconjunctivitis and severe forms of rhinitis.Although the use of paracetamol has been related to the development of allergic diseases such as asthma and AR, These results were also observed in a multicenter study conducted in eight Chinese cities, especially among adults living in buildings constructed after 2005.In adults, mold exposure in the household was identified as a positively associated factor for a medical diagnosis of AR. Household humidity is believed to aggravate AR symptoms and increase susceptibility to common colds and possibly other respiratory infections in young adults.The relationship between diet and occurrence of allergic diseases, asthma, and AR has been extensively studied. This study shows that diet was one of the main factors associated with AR occurrence. The data show that adolescents who consume meat daily, butter, once or twice a week, and milk, regardless of frequency, and adults who consume meat once or twice a week, were positively associated with increase of allergic disease. Analysis of the ISAAC phase III database found that a Western pattern diet, with high margarine, trans fatty acids, animal fats, and fast-food intakes was a risk factor for AR; while a Mediterranean diet, based on vegetables, cereal, fruit, seafood, peanuts, and olive oil was confirmed as a protective factor. The results presented in this study found the Western pattern diet as negatively associated, contrasting with the results reported by other authors.Some studies showed a positive relationship between trans-fat intake, AR, and asthma.Fast-food daily intake was a negatively associated factor, contrasting with the results of ISAAC phase III. Another point to be considered is the difficulty with the use of a food recall survey to assess the consumption of certain foods, especially the processed ones, in terms of presentation diversity, types of preparation, and preservation. Among parents/guardians, the lower educational level was found to be a negatively associated factor for AR occurrence.Having a higher educational level could be related to a better socioeconomic level, better knowledge and understanding of the symptoms and the disease, better understanding of the questions and answers, and better opportunities for diagnosis and medical treatment.One of the limitations of this study is the fact that the information was obtained through a written, self-administered questionnaire focused on symptoms and self-reported AR diagnosis, which was subject to information bias. Verification by physical examination and/or specific laboratory evaluation could reduce such uncertainties. In addition, because it is a cross-sectional study, it is not possible to establish the cause-and-effect relationship with certainty.This is a local study that represents adolescents only from public schools of a single municipality and therefore may not be extrapolated to general population. However, obtaining basic epidemiological data on allergic diseases in adolescents and adults will enable the development of a global map of these diseases that will improve public health planning and provide better management strategies.In conclusion, the prevalence of AR in adolescents is high, as well as its medical diagnosis in adults living in Uruguaina. Environmental factors, especially food habits, were associated with findings in both groups.