DC-SIGN engagement promoted autophagy flux which coincided with the recruitment of ATG-related factors. As such, the autophagy initiation factor ATG9 was found to be associated with DC-SIGN very early upon receptor engagement and required for an optimal DC-SIGN-mediated autophagy flux. The autophagy flux activation upon DC-SIGN engagement was recapitulated using engineered DC-SIGN-expressing epithelial cells in which ATG9 association with the receptor was also confirmed. Finally, Stimulated emission depletion (STED) microscopy performed in primary human MoDC revealed DC-SIGN-dependent submembrane nanoclusters formed with ATG9, which was required to degrade incoming viruses and further limit DC-mediated transmission of HIV-1 infection to CD4+ T lymphocytes. Our study unveils a physical association between the Pattern Recognition Receptor DC-SIGN and essential components of the autophagy pathway contributing to early endocytic events and the host’s antiviral immune response.Dendritic cells (DC) are critical cellular mediators of host immunity, notably by expressing a broad panel of pattern recognition receptors. One of those receptors, the C-type lectin receptor DC-SIGN, was previously reported as a regulator of endo/lysosomal targeting through functional connections with the autophagy pathway. Here, we confirmed that DC-SIGN internalization intersects with LC3 Dphocytes ,8.Mycobacterium tuberculosis complex, was reported to be a key DC-SIGN ligand able to influence anti-mycobacterial immunity [trans-infection of CD4+ lymphocytes [Mycobacterium tuberculosis [Among these receptors, the C-type lectin receptor (CLR) dendritic cell-specific ICAM-grabbing non-integrin  was characterized as a high-affinity receptor for mannosylated ligands. In particular, the mannose-capped lipoarabinomannan (ManLAM), a major cell wall component synthesized by the immunity . Furtherimmunity ,11. Whilphocytes ,8,12, thphocytes ,14,15,16phocytes ,18. The rculosis ,20, althrculosis . Interesrculosis ,23,24.Autophagy is a tightly regulated and evolutionary conserved lysosomal catabolic process implicated in many physiological functions, from development, stress regulation, and anti-inflammatory conditions to neurodegeneration ,26,27. T+/ATG9+ intracellular structures related to a non-canonical autophagy pathway. Super-resolution imaging analyses evidenced the formation of intracellular ATG9-containing nanoclusters upon DC-SIGN engagement, suggesting a local and specific induction of autophagy-related organelles. Finally, we demonstrate the critical role of ATG9 in DC-SIGN-mediated autophagy flux activation and in contributing to dampening both HIV-1 capture in human dendritic cells and DC-mediated transfer of infection of CD4+ T lymphocytes. Our study sheds light on a novel autophagy-associated endocytic pathway engaged concomitantly to autophagy flux induction upon DC-SIGN-associated ATG9 complex that could rapidly modulate antiviral host response.Here, we identified the autophagy initiation factor ATG9 complexed to the CLR DC-SIGN upon ligand-driven endocytosis. Engagement of DC-SIGN promotes autophagy flux activation and coincides with an interconnection between the endocytosed CLR DC-SIGN and LC3+ vesicles upon 15 min of receptor engagement with DC-SIGN antibodies (Human primary monocyte-derived dendritic cells (MoDC) were used and treated with ligands previously reported to drive DC-SIGN endocytosis. Flow cytometry analyses of MoDC revealed that 30% to 50% of cell surface DC-SIGN were endocytosed upon 20 to 40 min of challenge with DC-SIGN-specific antibodies  or at 37 °C for 30 min, and the corresponding lysates A, left w+ structures appeared to be also functionally related, as evidenced by an increase in the autophagosomal form of LC3 (LC3-II) observed very early upon treatment of MoDC with anti-DC-SIGN (+-ATPase (V-ATPase)  . The DC--ATPase) B, right.-ATPase) C or HIV--ATPase) D. Collec+ T lymphocytes. We and others have shown that DC-SIGN endocytosis also intersected with the autophagy pathway [+ structures as early as 15 min /Golgi pattern, internalized DC-SIGN was preferentially showing up with sub-membrane endosomal ATG9 in MoDC . Such feWe analyzed the physical association between DC-SIGN and ATG9 using an engineered cell line stably expressing DC-SIGN (HEK-DC-SIGN) and transfected with plasmid constructs expressing HA-ATG9 . HEK-DC-+/ATG9+ amphisomes, we turned to super-resolution imaging of the vesicular structures formed early upon DC-SIGN engagement and internalization on MoDC. We first validated the sub-membrane assembly of endocytosed DC-SIGN with ATG9-containing membranes by confocal microscopy upon 30 min of receptor engagement compared to control cells  microscopy with a resolution of <50 nm clearly revealed polarized ATG9-containing domains clustering and shrouding endocytosed DC-SIGN receptors and forming large intracellular vesicles upon 30 min of anti-DC-SIGN engagement  (The same ligand-mediated receptor endocytosis kinetic assay as before was performed and showed that large intracellular vesicles were formed as early as 15 min upon DC-SIGN engagement B. Cluste (0 min) D. Our daTo further assess ATG9 function in DC-SIGN-mediated autophagy activation, we transfected primary human MoDC with irrelevant control siRNA (siCtrl-MoDC) or ATG9-specific siRNA (siATG9-MoDC) to efficiently downregulate ATG9 expression  or transfected with irrelevant siRNA (MoDC-siCtrl) or siRNA directed against ATG9 (MoDC-siATG9). Cells were then left uninfected (MoDC NI) or challenged with R5-tropic HIV-1 wild-type for a short time point (8 h) before analyzing the anti-HIV1-Gag signal by flow cytometry A. This ktrl-MoDC B. These trl-MoDC .+ vesicles, our current study further demonstrated that all the DC-SIGN ligands used also induced a rapid and specific cellular autophagic response in MoDC.In this study, we revealed the formation of a novel protein complex linked to the pathogen recognition receptor DC-SIGN and the autophagy-related proteins ATG9, known to regulate autophagy initiation. We and others reported that DC-SIGN post-endocytic sorting could intersect with autophagy-related intracellular vesicles. These organelles issued from a merge between endosomal and autophagosomal vesicles (amphisomes) were named immunoamphisomes due to their ability to regulate innate and adaptive immune responses ,23. In a+ vesicles localized closely to the plasma membrane upon engagement of the receptor. Noteworthy, some ATG-related factors were functionally connected to the endosomal pathway, seemingly supplying membranes toward the formation of autophagosomal structures. Accordingly, ATG9 and ATG16L1 proteins were both reported to contribute to the formation of pre-autophagosomal structures with membrane sources from endosomes and cellular plasma membranes [+ vesicles were shown to represent a significant membrane source for autophagosome formation [+/ATG9+ vesicles and the plasma membrane, suggesting a local and rapid initiation of autophagy-related organelles transitioning to LC3+ immunoamphisomes upon receptor engagement. The association with ATG9+ isolation membranes seems, therefore, to initiate a very early trafficking fate of incoming virions towards the endo-lysosomal degradation pathway as previously suggested [+/DC-SIGN+ clusters will be required to state if other components of autophagy-initiation complex, phosphatidylinositol synthase (PIS)-enriched ER subdomains and ATG9 vesicles can act in concert with DC-SIGN-containing endosomes to generate immunoamphisomes.We hypothesized that DC-SIGN engagement could be linked to an early autophagic response with pre-autophagy-related structures formed very early upon engagement of the CLR. In agreement with this hypothesis, we observed a significant co-localization between endocytosed DC-SIGN and ATG9embranes ,35,36,37ormation ,35,38 whormation . Our datuggested ,41,42. Iuggested ,44,45. Fuggested ,43,44,46+ vesicles which appear enlarged upon DC-SIGN engagement. This is not totally unexpected as ATG9+ vesicles required for membrane expansion were shown to be recruited to autophagy initiation sites through their interaction with the scaffolding protein ATG17  while ATG1 kinase (ULK1/2 orthologue)-mediated ATG9 phosphorylation was also reported to control both the localization to the pre-autophagosomal structure (PAS) and the rate of autophagosome formation [+ vesicles and the analysis of ATG9 post-translational modification status at the plasma membrane will be required to further delineate cellular machinery linking ATG9+ vesicles with the endosomal pathway and autophagy vesicles nucleation. Noteworthy, our data with an ATG9 mutant (ATG9-N99D) topologically altered in membranes still showed association with DC-SIGN, suggesting that membrane topology of ATG9 is not required for membrane expansion in agreement with previous studies [While other major ATG-related proteins (such as ULK1) were not significantly found to be associated with internalized DC-SIGN in our model, we could not totally exclude their implication in membrane nucleation and post-endocytic sorting events upon DC-SIGN engagement. Hence, we observed that mTOR inhibitors, and promoters of canonical autophagy, can preset ATG9ormation ,38,47,48 studies .+ vesicles upon receptor engagement and the intersection toward LC3+ intracellular vesicles also raised the question of the endocytic route engaged upon ligand-mediated receptor binding and internalization. Our data suggested that DC-SIGN post-endocytic sorting and routing could be functionally linked to a non-canonical autophagy pathway. Non-canonical forms of autophagy are often characterized by the conjugation of ATG8 to single membranes (CASM) [+/clathrin+ endosomes and appeared critical to regulating immune-mediated aggregate removal and microglial activation in a murine model of Alzheimer’s Disease [The rapid association between DC-SIGN and ATG9s (CASM) , which ds (CASM) ,51,52,53s (CASM)  and regus (CASM) ,56. Intes (CASM) ,56. For s (CASM) . Furthers (CASM) . Becauses (CASM) ,58. Alths (CASM) . Interes Disease ,60,61 as Disease ,63 or DN Disease ,65,66 no Disease ,68.Our data suggest the involvement of a non-canonical endosomal autophagy pathway similar to previous reports ,35,36 bu+. Human MoDC were generated by incubating purified monocytes in IMDM supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, 10 mM Hepes, 1% non-essential amino acids, 1 mM sodium pyruvate and cytokines GM-CSF (500 IU/mL) and IL-4 (500 IU/mL) . The obtained immature MoDC were harvested at days 5–6 and phenotyped by flow cytometry before experimental use. The adherent cell lines HEK293T and HEK-DC-SIGN (engineered in this study) were maintained in supplemented DMEM under puromycin selection for the latter only.Human monocytes from buffy coats were obtained according to institutional guidelines of the ethical committee of the CNRS and EFS. After the Ficoll gradient on PBMC, monocytes were isolated using CD14 MicroBeads . Usual purity was >95% CD14gag on 2.5 × 105 CD4+ HeLa P4-R5 corresponds to an MOI of 1 (multiplicities of infection). Viral physical titers were evaluated by quantification of HIV-1 p24gag by ELISA using Innotest®HIV antigen mAb .Virus stocks were produced as previously described ,72. BrieTM cloning strategy using pDEST15 as destination vectors, as already reported [®  or TurboFect  according to the manufacturer’s instructions.The following reagent was obtained through the NIH AIDS Reagent Program, NIAID, NIH: pcDNA3-DC-SIGN from Drs. S. Pöhlmann, F. Baribaud, F. Kirchhoff and R.W. Doms . DC-SIGNreported . All plaMycobacterium bovis BCG, as previously reported [v/v) Triton X-114, 5 mM EDTA, and 10 mM MgCl2. After phase separation at 37 °C, lipoglycans were collected in the lower phase, precipitated by cold ethanol, and extracted by saturated phenol. ManLAM was then separated from other lipoglycans (lipomannan and phospho-inositol mannosides) by gel filtration on a Sephacryl S-200 column  irrigated with tris deoxycholate buffer . The purity of the eluted fractions was assessed by 13% SDS-PAGE and staining for carbohydrates. The structural integrity of the lipoglycan was checked by multi-nuclear magnetic resonance spectroscopy as previously described [®Gold were obtained from Thermo Fisher Scientific .All chemicals were obtained from Sigma unless stated otherwise. For autophagy flux experiments, Torin 1  and chloroquine were used at 1 μM and 50 μM, respectively. The Protein A/G sepharose reagent used for immunoprecipitation experiments was from Rockland Immunochemicals . Mannose-capped lipoarabinomannan was purified from reported ,76. BrieDC-SIGN engagement and internalization assays were performed using monoclonal anti-DC-SIGN antibodies  and clone DCN46 from BD Biosciences ). Other anti-DC-SIGN Abs were used for flow cytometry  and #9E9A8 from Biolegend  and Western blotting . Additionally, the following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH and used for flow cytometry or Western blotting applications: Anti-Human DC-SIGN/L-SIGN Monoclonal (14E3G7) from Dr. Ralph Steinman; Anti-Human DC-SIGN/DC-SIGNR Monoclonal (DC28) from Drs. F. Baribaud, S. Pöhlmann, J.A. Hoxie and R.W. Doms. Antibodies against P62/SQSTM-1 (#H290) were from Santa Cruz Biotechnology . Anti-LC3B (#L7543), anti-GAPDH-HRP (#G9295), anti-ATG13 (#SAB1307145), anti-HA (#H6908) and anti-GST-HRP (#A7340) were from Sigma-Aldrich . Antibodies against ATG16L1 (ab187671), ULK1 (ab128859), ATG9 (ab108338), Lamp1 (ab24170), anti-P62/SQSTM-1 (ab56416) and HA-tag (ab9110) were from Abcam . Anti-LC3 (PM036) and anti-ATG16L (PM040B) were obtained from MBL International , while monoclonal anti-actin (clone C4) was from Millipore . When not stated otherwise, all FACS antibodies used for primary human cell phenotyping were obtained from BD Biosciences  or Miltenyi Biotec . All coupled secondary antibodies used for Western blotting and immunofluorescence assays were purchased from Sigma-Aldrich  and Thermo Fisher Scientific , respectively. Rabbit polyclonal anti-Clathrin antibodies were a kind gift from Paul Mangeat .4, 50 mM NaF, 10 mM Na4P2O7, protease inhibitor cocktail ) and centrifuged at 26,000× g for 20 min. Post-nuclear Supernatants were submitted to electrophoresis on home-made 10% Prosieve  gels or Bolt 4–12% Bis-Tris Plus pre-cast gels before transfer to a nitrocellulose membrane  and then immunoblotted as indicated. Membranes were stripped in 0.1 M Glycine buffer (PH 2.3) in case of re-blotting and loading control. Membranes were revealed with ClarityTM Western ECL substrate . Chemiluminescence was acquired , followed by densitometry analysis .Cells were lysed for 20 min at 4 °C in lysis buffer  for 3 h at 4 °C. Unbound proteins were removed by washings. Bound proteins were then eluted by boiling for 10 min in SDS gel loading buffer and separated by gel electrophoresis.®  were used for siRNA transfection according to the manufacturer’s recommendations and as previously reported [5 iDC were usually transfected with 100 nM siRNA in 500 μL of IMDM/1% SVF medium in 12-well plates. A second round of transfection was performed 24 h later. Specific gene knockdowns were controlled by Western blotting compared with untransfected and siCtrl conditions and normalized to loading control.SMARTpool: ON-Target Plus human siRNA directed against ATG9a (#L-014294-01-0005) as well as siCtrl (5′-AAATGAACGTGAATTGCTCAA-3); siRNA sequence specific for Luciferase) were obtained from Dharmacon . HiPerFect Transfection Reagent  or INTERFERinreported . Briefly5 cells (corresponding to an MOI of 5) and adapted depending on cell number. Functional infection assays and viral capture were conducted in 96-well plates in which 2.5 × 105 cells were challenged with 1 μg p24 HIV-1 (corresponding to an MOI of 2) for 6–8 h. Cells were analyzed by flow cytometry upon fixation and anti-HIV-1-Gag staining. HIV-1 challenged MoDC were also co-cultured with autologous CD4+ T cells (ratio 1:1) for 72 h before the fixation and analysis of HIV-1-Gag signal in CD3+ cells.For immunofluorescence assays, when not stated, HIV-1 treatment was performed on cells previously plated on glass coverslips at 2.5 μg p24 HIV-1 for 2.5 × 10+ T cells when required) was performed in FACS buffer  at 4 °C for 30 min using mAbs from BD Biosciences  and Miltenyi Biotec  and directed against the following molecules: CD1a, CD3, CD4, CD8, CD14, CD45Ro, CD69, CD83, CD86, DC-SIGN, HLA-DR. For DC-SIGN internalization assays, a cocktail of mouse monoclonal antibodies (DCN46 (BD) and MR1  at 5 μg/mL each) or ManLAM (2 μg/mL) were incubated for 30 min on cells previously stored at 4 °C. Internalization times were starting when cells were switched back to 37 °C for indicated kinetic times. The time 0 corresponds to cells left with reagents at 4 °C as above but processed straight without incubation at 37 °C. DC-SIGN levels in antibody-mediated internalization were measured upon staining with only anti-mouse fluorescently labeled secondary antibodies. For ManLAM-mediated DC-SIGN internalization, receptor levels were analyzed by flow cytometry upon staining with anti-DC-SIGN-APC (Biolegend) or anti-DC-SIGN-PE . Isotype-matched mAbs were used in all experiments as negative controls. Samples were analyzed by flow cytometry using a FacsCalibur  and data processing with FlowJo®software .Surface staining for phenotyping of primary cells . Preliminary epifluorescence acquisition was performed using a Leica microscope, and samples were analyzed using the LAS software . Confocal microscopy acquisitions were conducted on a Zeiss LSM780, and data analyses were performed using ZEN software . When required, Z-series of optical sections were performed at 0.5 μm increments for qualitative analysis. Green, red, and far red fluorescences were acquired sequentially. Colocalization events were calculated using intensity-based Manders’ M coefficient obtained from the JACoP plug-in with ImageJ Software , and statistical quantifications were performed with GraphPad Prism .Samples with DC-SIGN antibody capture were prepared as described above. Captured DC-SIGN antibody was stained with goat anti-mouse Alexa Fluor 594. ATG9 was stained using indirect immunofluorescence with Abberior Star 635P secondary antibody. gSTED microscopy with time-gated detection was performed on a Leica SP8 TCS STED microscope  equipped with a pulsed white light excitation laser . Dual-channel STED imaging was performed by sequentially exciting Abberior Star 635P and Alexa 594 at 646 nm and 598 nm, respectively. Both dyes were depleted with a 775 nm STED laser. A single optical section was acquired with an HC PL APO CS2 100×/1.40-N.A. oil objective , a scanning format of 1024 × 1024 pixel, 8-bit sampling, and 6-fold zoom, yielding a pixel dimension of 18.9 × 18.9 nm. Time-gated detection was set from 0.3–6 ns for all dyes. Fluorescence signals were detected sequentially by hybrid detectors at the appropriate spectral regions separated from the STED laser.t-test for paired observations (differences in the kinetics of receptor engagement compared to 0min for each donor or cell type) or unpaired parameters . Statistical analyses were performed using GraphPad Prism 6.0  and significance was set at p < 0.05 .The One-way ANOVA unpaired analysis was applied for statistics from data regarding DC-SIGN co-localization with ATG-related factors in


Importantly, immunisation with PR8NARBD/WSN viruses effectively protected mice against lethal challenges with H1N1, H3N2 IAV, and SARS-CoV-2 Beta variant and significantly reduced lung viral loads. Overall, our research demonstrates the promising potential of PR8NARBD/WSN as an attractive vaccine against emerging SARS-CoV-2 variants and influenza A virus infections.Current COVID-19 vaccines can effectively reduce disease severity and hospitalisation; however, they are not considerably effective in preventing infection and transmission. In this context, mucosal vaccines are pertinent to prevent SARS-CoV-2 infection and spread. In this study, we generated a replication-competent recombinant chimeric influenza A virus (IAV) expressing the receptor-binding domain (RBD) of a SARS-CoV-2 prototype in the C-terminus of the neuraminidase (NA) of A/Puerto Rico/08/1934 H1N1 (PR8). The remaining seven segments from A/WSN/1933 H1N1 (WSN) were named PR8NARBD/WSN. We observed that the recombinant virus with the WSN backbone demonstrated improved expression of NA and RBD. A single intranasal dose of PR8NARBD/WSN(10 Over a short period of time, several vaccine platforms have been developed globally and used to immunise the majority of individuals after ensuring their safety and effectiveness. Most COVID-19 vaccines are intramuscular injections, which can induce both humoral and cellular immune responses to reduce disease severity, hospitalisation, and mortality  anrm cells G and 35.rm cells H in the 2PFU, 103PFU PR8NARBD/WSN, or 103PFU PR8NA/WSN viruses offered full protection against lethal challenge with the PR8 H1N1 virus, with no weight loss and 100% survival , rgH3N2 (A/X31/H3N2), or rgA/Anhui/H7N9 virus 28 days after a single-dose intranasal vaccination A. Immunisurvival B. No virsurvival B. Furthesurvival C, consissurvival C. In conotection D. A highSN group D. This iHistopathological analysis of lungs from challenged mice collected on day 5 dpi showed that the vaccinated group was largely protected against lung damage caused by H1N1 and H3N2 viral infection E–G. Howe3PFU PR8NARBD/WSN viruses were protected against the lethal SARS-CoV-2 Beta challenge  because of its attenuated phenotype in humans . The A/WThe NA gene segment is a promising candidate for inserting exogenous genes, which can insert about 680 bp of exogenous genes without affecting the structure of NA . The NA + CD8+T cells detected after a single-dose immunisation.A combination of IFN-γ ELISpot assays and flow cytometry were used to characterise SARS-CoV-2 RBD-specific and influenza virus NP-specific T cells induced by PR8NARBD/WSN vaccination. The data indicated that the vaccine induced RBD-specific splenocytes and pneumonocytes IFN-γ T cell response. The expression level was higher than that previously reported for a prime-boost single cycle influenza virus-based SARS-CoV-2 vaccine (scPR8-RBD-M2) and comparable to the prime-boost level previously reported for the dNS1-RBD vaccine ,12. Flow+ Trms in the lungs of both mice and humans [+CD103+ T cell expression levels are similar to those prime-boost level reported previously for the dNS1-RBD vaccine and LAIV-RSV vaccines [Several recent experimental studies have reported the importance of memory T cells in the protection against respiratory viruses ,38. Trm d humans , and ourvaccines ,42.This study has some major limitations. First, only the prototype RBD of SARS-CoV-2 inserted in the NA segment was tested; no other targets were studied . Second, we only tested this recombinant vaccine in mice and did not assess its immunogenicity and protection in other animal models. Third, we only detected immunogenicity in IAV-native mice. Previous studies have shown that pre-existing immunity against vaccine vectors can reduce vaccine effectiveness . These lIn conclusion, our current data provide evidence that PR8NARBD/WSN is a unique mucosal vaccine candidate for single-dose intranasal immunisation that provides broad protection against IAV and SARS-CoV-2. Our research demonstrates that PR8NARBD/WSN is an attractive vaccine target for emerging SARS-CoV-2 variants and IAV infections. Continued work on this vaccine platform will help in present and future responses to pandemic respiratory pathogen infections.


Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication origin binding protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation.The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis.RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter.This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. Megakaryocytes (MK) are derived from hematopoietic stem cells (HSCs) via a differentiation process called megakaryocytic differentiation and are responsible for the production of blood platelets , 2. MKs-The replication origin binding protein RepID, also known as DDB1/CUL4-associated factor 14 (DCAF14) or pleckstrin homology domain-interacting protein (PHIP), exhibits pleiotropic functions, including replication fork stability , replicaHere, we report that RepID represses MK differentiation by recruiting CRL4A to the DAB2 promoter, resulting in heterochromatin formation. RepID dissociates from chromatin in response to megakaryocytic differentiation signals, thereby preventing the loading of CRL4A as well as the histone H3K4 demethylase JARID1A, a novel interaction partner of CRL4A, to the DAB2 promoter. These molecular events correlated with increased transcription of megakaryocyte markers and megakaryocytic morphological changes in a subpopulation of RepID-depleted cells. We also observed that megakaryocytic differentiation is accelerated in RepID-depleted cells upon stimulation by phorbol 12-myristate 13-acetate (PMA). These findings provide an example of gene transcription regulation during megakaryocytic differentiation with the RepID-dependent recruitment of an epigenetic modifier.www.atcc.org) and tested for mycoplasma . Cycloheximide, MG132, and PMA were purchased from Sigma (Cat. C4859), Millipore (Cat. 11134515001), and Santa Cruz (Cat. Sc-3576), respectively. Each compound was added to the medium at the indicated concentrations.Human K562 and DMS114 cells, parental and RepID knockout, were cultured in Dulbecco’s modified Eagle’s medium  or RPMI-1640 medium  supplemented with 10% heat-inactivated fetal bovine serum in a 37°C/5% CO2 humidified incubator , fixed with 4.0% paraformaldehyde (PFA) for 10 min, and permeabilized using PBS-T buffer  for 10 min on ice. Primary antibody staining was performed using anti-DAB2 . Secondary antibody staining was performed using Alexa Fluor™ Plus 488 conjugated anti-rabbit IgG . DNA was counterstained with DAPI . A Zeiss LSM880 confocal microscope was used for imaging.Slides of proliferating or differentiating K562 cells were prepared using TXT3 Cytospin  at 900×™ magnetic ChIP assay kit . Antibodies used in this study included normal rabbit IgG , anti-CUL4A , anti-JARID1A , anti-H3K4me3  and anti-H3K9me3 . Immunoprecipitated DNA and control input DNA was analyzed by quantitative real-time PCR using the human DAB2 promoter-specific primers; forward:5’-CTC GCG GAG CTC AGG GGA G-3,’ reverse:5’-GGT AAC TCC CCC TCA ACG TG-3. ‘Chromatin immunoprecipitation (ChIP) analyses were performed on 1% formaldehyde-fixed proliferating or differentiating K562 cells using a Pierce2 Profiler PCR array  with 84 genes associated with the Wnt signaling pathway was used.Proliferating K562 RepID WT and KO cells were harvested, and RNA was extracted using the RNeasy R Mini Kit  and converted to cDNA by using PrimeScript RT Master Mix  according to the manufacturer’s instructions. An RTTotal RNA extraction, cDNA synthesis, and qRT-PCR were performed according to the manufacturer’s instructions provided by Qiagen , Takara (PrimeScript RT Master Mix RR036A), and Invitrogen (SYBR Green PCR Master Mix 4309155), respectively. The expression levels of human DAB2, CD41, and CD61 in the cells were determined by qRT-PCR, using GAPDH as an internal control. DAB2 primers; forward:5’-CCA GAT GCA AGA GGG GAT AA-3’ and reverse:5’-TCC TCC ACA CAC GTA ACC AA-3,’ CD41 primers; forward:5’-AGG TGA GAG GGA GCA GAA CA-3’ and reverse:5’-TCC ACC TTG AGA GGG TTG AC-3,’ CD61 primers; forward:5’-GAC AAG GGC TCT GGA GAC AG-3’ and reverse:5’-ACT GGT GAG CTT TCG CAT CT-3,’ GAPDH primers; forward:5’-GAG TCA ACG GAT TTG GTC GT-3’ and reverse:5’-TTG ATT TTG GAG GGA TCT CG-3.’g for 3 min and washed three times with PBS. Sixty μl 2×SDS Laemmli sample buffer  was added, and the complexes were boiled for 15 min. Protein binding or each subcellular fraction was immunodetected following SDS-PAGE. Unprocessed original blots are shown in the Supplementary file.Cytosol, membrane, soluble nucleus, and chromatin fractions from proliferating or PMA-mediated differentiating K562 cells were prepared using a subcellular protein fractionation kit  together with PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktail. Cell lysis was performed according to the manufacturer’s protocol. Total cell lysates were immunoprecipitated using an anti-CUL4A , or anti-JARIDIA  at 3 μg of antibody per sample. After rotation 14 h at 4°C, 60 μl of Sepharose beads  were added and samples were incubated for an additional 2 h at 4°C in rotator. Protein-antibody-bead complexes were collected by centrifugation at 2000×The following primary antibodies were used: anti-DAB2 , anti-CUL4A , anti-CUL4B , anti-RepID , anti-DDB1 , anti-JARID1A , anti-WDR5 , anti-RBBP5 , anti-ASH2L , anti-FLAG , anti-histone H3 , and anti-a-tubulin . For secondary antibodies, horseradish peroxidase (HRP)-conjugated anti-mouse IgG  and HRP-conjugated anti-rabbit IgG  were used following the manufacturer’s suggested protocols.The Duolink in situ Red starter mouse/rabbit PLA fluorescence assay  was performed according to the manufacturer’s instructions. Briefly, proliferating or PMA-induced differentiating K562 cells were attached to slides using cytospin. Cells were washed with 1×PBS and fixed for 15 min at 4°C in 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 min at 4°C. The coverslips were blocked with Duolink blocking solution and incubated with CUL4A  and JARID1A  antibodies in Duolink antibody diluent overnight, followed by incubation with PLUS and MINUS PLA probes, ligation, and amplification. The coverslips were then washed and mounted using mounting medium containing DAPI. Images were captured using a wide-field microscope, processed, and analyzed using Zeiss Zen 3.7 and ImageJ software.g for 5 min and stained with Wright-Giemsa staining solution . Cell morphology, including cell size and polynucleation, was analyzed by microscopy and ImageJ software. At least 290 cells in randomly separated fields were counted and expressed as relative cell size or percentage of the total cell number in these fields.Proliferating or differentiating K562 cells were attached to slides using TXT3 cytospin (Nasco Korea) at 900×https://discover.nci.nih.gov/cellminercdb/) and histone H3K4 methyltransferase/demethylase were analyzed using the Cancer Cell Line Encyclopedia (CCLE) dataset from the CellMiner cross-database . The PeaTo determine whether RepID participates in transcriptional changes, we performed a PCR array assay using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Interestingly, the highest observed gene deregulation involved the Wnt/b-catenin pathway with Frizzled 3 receptor (FZD3), Disabled-2 (DAB2), and Van Gogh-like 2 (VANGL2) being upregulated in RepID-deficient K562 cells more than 20 fold . Of inteK562 cells are bipotent progenitor cells, which can be differentiated into megakaryocytes or erythroid lineages following exposure to PMA, 12-O-tetradecanoylphorbol 13-acetate (TPA), and retinoic acid –36. Basevs. 96 hr in WT cells, Next, we tested whether RepID depletion could interfere with the induction of megakaryocyte markers and/or megakaryocytic differentiation. In RepID-expressing cells, DAB2 protein levels gradually increased after PMA treatment  are highly specialized large cells that are crucial precursors of blood platelets, small anucleate cell fragments with crucial roles in wound healing, inflammation, angiogenesis and hemostasis. MKs differentiation leads to polyploidy by endomitosis followed by a maturation process for platelet production, and these processes are governed by intensive changes in gene expression patterns. The data presented here demonstrate that megakaryocytic differentiation requires RepID dissociation from chromatin. The loss of chromatin-bound RepID compromises the loading of the CRL4A-JARID1A complex at the DAB2 promoter, leading to the initiation of DAB2 transcription, a key regulator of platelet signaling. MK differentiation was partially executed in K562 RepID-deficient cells, even in the absence of a differentiation signal, while induced MK differentiation by PMA led to faster megakaryocytes-like cell formation when using K562 RepID-depleted cells. Altogether, these observations identified the RepID-CRL4A-JARID1A complex as a putative factor in the maintenance of K562 proliferation.RepID, known as the replication origin binding protein as well as a structural receptor for CRL4 ubiquitin E3 ligase, initiates a subpopulation of DNA replication origins and prevents over-replication via CRL4 recruitment on chromatin to ubiquitinate CDT1 , 37–39. Our results revealed that the dynamics of chromatin loading differ between CUL4A (decreased loading) and CUL4B (increased loading) during induced megakaryocytic differentiation. Human CUL4A and CUL4B share approximately 82% sequence identity, 90% homology and contribute to redundant functions in cell cycle progression and embryonic development by mediating the ubiquitination of CRL4 targets , 46–48. We propose the following model for the role of RepID in repressing the expression of genes required for megakaryocytic differentiation by recruiting the CRL4A-JARID1A complex . In prol


Cytosine base editors (CBEs), which enable precise C-to-T substitutions, have been restricted by potential safety risks, including DNA off-target edits, RNA off-target edits and additional genotoxicity such as DNA damages induced by double-strand breaks (DSBs). Though DNA and RNA off-target edits have been ameliorated via various strategies, evaluation and minimization of DSB-associated DNA damage risks for most CBEs remain to be resolved. Here we demonstrate that YE1, an engineered CBE variant with minimized DNA and RNA off-target edits, could induce prominent DSB-associated DNA damage risks, manifested as γH2AX accumulation in human cells. We then perform deaminase engineering for two deaminases lamprey LjCDA1 and human APOBEC3A, and generate divergent CBE variants with eliminated DSB-associated DNA damage risks, in addition to minimized DNA/RNA off-target edits. Furthermore, the editing scopes and sequence preferences of APOBEC3A-derived CBEs could be further diversified by internal fusion strategy. Taken together, this study provides updated evaluation platform for DSB-associated DNA damage risks of CBEs and further generates a series of safer toolkits with diversified editing signatures to expand their applications.  Cytosine base editors (CBEs), which are generally composed of cytidine deaminases and nicking or deactivated Cas nucleases (nCas9 or dCas9) , could mRNA off-target editing analysis of base editors was commonly evaluated by GATK HaplotypeCaller tool. However, it has been demonstrated that HaplotypeCaller tool lacked sensitivity to detect A-to-I RNA edits with <10% efficiency, while MuTect2 was a more sensitive tool for such analysis . TherefoIt is also known that cytidine deaminases could induce DNA damages by triggering double-strand breaks (DSBs) , which cHere, we demonstrated that an engineered CBE variant YE1 ,11,26, wUniversal expression vector of base editors was derived from PX461 (Addgene #48140), in which the U6-sgRNA cassette was deleted. Then deaminase insertion sites (containing SpeI-BamHI-XbaI multiple cloning sequences) were created at N-/C- or internal regions of nCas9 via mutagenesis strategy . Variant deaminase templates were amplified using high-fidelity DNA polymerase  and inserted into expression vector by restriction cloning. Templates for rAPOBEC1 and APOBEC3A were derived from pCMV-BE3 (Addgene #73021) and HEK293T cDNAs respectively. Templates for lamprey cytidine deaminases have been described previously . Mutagen2 incubator . HEK293T cells were passaged and plated into 6-well or 48-well plates (Corning) for transfection. To characterize potential transcriptomic changes induced by DNA base editors, plasmid expressing base editor (with 2A-GFP), was co-transfected with plasmid expressing scrambled single guide RNA (sgRNA) and uracil glycosylase inhibitor (UGI) (with 2A-puromycin-resistant gene) into HEK293T cells. Base editor- and sgRNA-expressing plasmid (mole ratio 2:1) were mixed with 2.5 μl (48-well) or 8 μl (6-well) Lipo293TM  for transient transfection. For base editing analysis, 72 h after transfection, cells in 48-well plates were digested and prepared for flow cytometry to collect double positive cells. For transcriptome analysis, 24 h after transfection, medium was replaced with fresh DMEM containing 2 μg/ml puromycin  for cells in 6-well plates. After culturing for another 48 h, cells were collected for RNA extraction and RNA sequencing.HEK293T cells  were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% FBS  at 37°C in 5% COGFP/mcherry double-positive cells collected with flow cytrometry  were treated with DirectPCR reagent . Targeted amplifications were performed using LA Taq , and base editing status was quantified with EditR software after Sanger sequencing  or with CRISPResso2 after targeted amplicon sequencing . Targeted amplicon sequencing was performed using Illumina NovaSeq platform at Shanghai Personalbio Technology. Experiments for amplicon sequencing were performed in biological triplicates.P < 0.05 was considered significant.C sites were initially classified according to their positions, and positions containing at least one C site with an average of >20% C-to-T editing frequency were included for sequence preference analysis. Then the impact of base types upstream or downstream of edited cytosine (NC or CN) was analyzed accordingly. For statistical analysis, one-tailed unpaired t-test was performed for comparison between every two groups and To analyze the DSB number induced by different enzymes, we initially transfected nCas9, Cas9, N-YE1-BE or N-eA3A-RL1-BE with double-stranded oligodeoxynucleotide (dsODNs) into HEK293T cells by Nucleofector (Lonza). Cells were collected 3 days after transfection for genome isolation and GUIDE-seq analysis as described previously . For on-HEK293T cells were transfected with base editor- and sgRNA-expressing plasmids as described above. Here, sgRNA-expressing plasmid containing RNF2-targeting sgRNA and puro puromycin resistance gene. Medium was replaced with fresh DMEM containing 2 μg/ml puromycin  for cells in 6-well plates every day for 4 days, and single cell clone was picked up into 96-well plate for further expansion. After culturing for 3–4 weeks, cellular genome DNA was purified using TGuide Cell/Tissue Genomic DNA Kit (TIANGEN Biotech) for whole genome sequencing using MGIseq platform (Genewiz).Paired-end sequencing reads were aligned to human genome build 38 (GRCh38/hg38) using BWA-MEM (v.0.7.17) . AlignedHEK293T cells were seeded into 48-well plates and transfected 24 h later. After culturing for another 72 h, cells were collected and washed with PBS, and fixed with 4% paraformaldehyde at room temperature (25 °C) for 15 min. Following washes with PBS, cells were permeabilized with 0.5% Triton X-100 in PBS for 2 min. Next, block cells with buffer (10% goat serum in TBS) at room temperature (25 °C) for 1 h. For the detection of DNA damage, stain the cells with γ-H2AX antibody  at 4°C for 2 h and wash with PBS. Cells were gated on fluorescein isothiocyanate and allophycocyanin using the Fortessa Flow Cytometer (BD Biosciences), and results were analyzed by FlowJo.R-loop assay was used to assess the Cas9-independent DNA off-target edits. In brief, Base editor-expressing plasmid was co-transfected with particular dSaCas9-UGI-T2A-mcherry-U6-sgsaRNA and cultured for 72 h. Double positive cells were collected by flow cytometry for targeted amplicon sequencing, and high-throughput sequencing data was analyzed using CRISPResso2.http://broadinstitute.github.io/picard), reads were split at junctions into exon segments and reassigned the mapping qualities from STAR. To remove potential experimental biases, base quality score recalibration (BQSR) was performed as the DNA-seq. HaplotypeCaller tool from GATK (v4.1.4)  and RNA sequencing was performed using Illumina Novaseq (PE 2 ◊150), at a depth of ∼20 million reads per sample. RNA-seq data was analyzed following the long-RNA-seq-pipeline of ENCODE Consortium, and reads were aligned to GRCh38 reference genome using annotation GRCh38.v96 via STAR (v2.4.2a) in 2-pass mode . Sentieo(v4.1.4)  and MuTeC5C (underlying for target cytosine) of EMX site  has been exploited to generate functional CBE BE3, with UGI tethered to nCas9, which displayed obvious DNA and RNA off-target effects. Improved CBE variants such as R33A-BE3, R33A-K34A-BE3  and BE3-e Figure , in agree Figure . We furte Figure , and it e Figure , in agree Figure ,10,11. Ae Figure . Therefoe Figure .As it has been demonstrated MuTect2 was a more sensitive tool for RNA base editing analysis, and could effectively detect RNA A-to-I edits with < 10% efficiency , we re-aDSB-associated DNA damage risks could be detected and quantified by accumulation of γH2AX . To provSeveral groups and our studies demonstrated that TadA deaminases could be reprogrammed to generate improved CBEs with minimized off-target risks . Here weIn summary, these analyses demonstrated that TadA-derived CBEs would possibly display detectable RNA off-target edits and DNA damage risks , indicatLamprey cytidine deaminases (hereafter CDAs) are alternative cytidine deaminases that have been exploited to generate functional CBEs ,13. HoweCT > CA/CC>>CG -BE, with RL2 region grafted from APOBEC3F, displayed significantly lower editing activity with editing scope at C4–C10 and preferred CT/CA > CC>>CG & AC>>CC/GC/TC; N-Lj-RL2 (A3G)-BE, with RL2 region grafted from APOBEC3G, displayed modest editing activity with editing scope at C3–C5 and preferred CT > CA > CC>>CG & AC > CC/GC > TC; N-Lj-RL2 (LpL1)-BE, with RL2 region grafted from LpCDA1L1_1, displayed relative high editing activity with editing scope at C3-C5 and preferred CT > CA > CC>>CG & AC > CC/GC/TC  with comparable editing activity to N-YE1-BE, and significantly higher than N-R33A-K34A-BE Figure . N-Lj-BEC Figure . We notiC Figure , while hE Figure . CombinaE Figure . AdditioE Figure . We furtE Figure , and revE Figure . OverallThen these six LjCDA1-derived CBEs were further analysed with MuTect2 to evaluate C-to-U RNA off-target editing risks. It was revealed that all six LjCDA1-derived CBEs induced similar C-to-U RNA off-target SNVs to nCas9 and significantly fewer SNVs than N-YE1-BE, indicating that LjCDA1-derived CBEs displayed minimized RNA off-target editing risks Figure .Furthermore, DSB-associated DNA damage analysis revealed that LjCDA1-derived CBEs induced different DSB-associated DNA damage risks depending on LjCDA1 mutation types , with N-Overall, here we demonstrated that LjCDA1-derived CBEs displayed minimal DNA and RNA off-target edits. DSB-associated DNA damage risks could also be eliminated through deaminase engineering to replace RL2 regions of LjCDA1, and derived CBEs displayed refined editing signatures, with N-Lj-RL2 (AID)-BE displaying editing scopes at single-base resolution. Nevertheless, in consideration of editing activity, R-loop assay analysis and DNA damage risks Figure , LjCDA1-APOBEC3A (hereafter A3A) is another cytidine deaminase that has been engineered to generate functional CBEs . Though C site ; 1010-eA3A-RL1-BE displayed editing scope at C4–C10 and preferred CG > CC > CA > CT & TC > CC > GC > AC; 1029-eA3A-RL1-BE displayed editing scope at C6–C11 and no obvious sequence preference; 1047–1064-eA3A-RL1-BE displayed editing scope at C5–C11 with no obvious sequence preference; 1249-eA3A-RL1-BE displayed editing scope at C8–C15 with anti-preference for CC and CC sites; 535-NL-eA3A-RL1-BE displayed editing scope at C6–C10 and C15 and slightly preferred CG and GC sites  to nCas9 (average of 16.3%) Figure . TherefoOverall, in consideration of editing activity, R-loop assay analysis and DNA damage risks Figure , most eADSB-associated DNA damage risks of cytidine deaminases and derived CBEs have been demonstrated previously ,20,22,23It has been demonstrated that activation-induced cytidine deaminase (AID) generated genome-wide DSBs promiscuously without obvious target-site specificity . Here weIn this study, we used freely expressed UGI separate from CBE constructs but not the classical constructs with UGI tethered to nCas9. The reasons could be classified as follows: Firstly, as we mainly tried to reduce or minimize the safety risks of cytidine deaminase domain in CBEs here, constructs without tethered UGI would provide more accurate DNA damage quantification for different CBEs as compared to nCas9 and Cas9. N-YE1-BE used in this study was also modified to exclude UGI region. Secondly, as demonstrated previously , free UGIt's well-known that classic CBE BE3 induced robust DNA and RNA off-target edits and various strategies have been developed to minimize such safety risks. As the deaminase component of BE3 is rat APOBEC1, available engineered CBE variants with minimal off-target edits at both DNA and RNA levels were mainly derived from APOBEC1 mutants or orthologues ,11,59. ITaken together, we developed a series of CBE tools with multidimensional safety improvement and diversified editing signatures. These newly engineered CBEs would be valuable for future applications in biomedical and clinical translational researches, especially for researches with stringent security requirement.gkad855_Supplemental_FilesClick here for additional data file.


Eosinophilic esophagitis (EoE) is associated with allergen-driven inflammation of the esophagus and an upregulated Th2 cytokine signature. Recombinant interleukin (IL)-13 (rIL-13) administration to mice induces some of the hallmark features of EoE, including increased eotaxin expression and eosinophil recruitment. Inflammation in EoE has previously been shown to depend on the expression of TRAIL and MID-1, which reduced protein phosphatase 2A (PP2A) activity. The relationship between IL-13 and TRAIL signalling in esophageal eosinophilia is currently unknown.To investigate the interaction between IL-13-driven eosinophil infiltration and TRAIL or MID-1 in the esophagus.Tnsf10−/−) or STAT6-deficient (STAT6−/−) mice and targeted MID-1 with small interfering RNA.We administered rIL-13 to wild type (WT), TRAIL-deficient (rIL-13 administration to mice increased TRAIL and MID-1 expression in the esophagus while reducing PP2A activity. TRAIL deficient, but not STAT6 deficient mice demonstrated increased MID-1 expression and PP2A reduction upon IL-13 challenge which correlated with eosinophil infiltration into the esophagus. Silencing MID-1 expression with siRNA completely ablated IL-13 induced eosinophil infiltration of the esophagus, restored PP2A activity, and reduced eotaxin-1 expression.IL-13-driven eosinophil infiltration of the esophagus induced eosinophilia and eotaxin-1 expression in a STAT6-dependent and MID-1-dependent manner. This study highlights a novel mechanism employed by IL-13 to perpetuate eosinophil infiltration. Eosinophil infiltration of the esophagus is associated with several inflammatory pathologies, including gastroesophageal reflux disease, parasitic infections, and eosinophilic esophagitis (EoE). EoE is an allergic phenomenon associated with food antigens, with a tendency to present primarily in young, atopic males –3. As a in vivo studies modeling EoE. IL-5 plays a core role in eosinophilic trafficking to the esophagus, while IL-13 regulates the transcription of chemokines and cytokines involved in epithelial remodeling  and inflammatory  cytokines through the transcription factor signal transducer and activator of transcription (STAT)-6 (The inflammation in EoE is associated with T helper 2 (Th2) driven eosinophilia via the cytokines IL-5 and IL-13 , 10. Botatory CCL, CCL24 c(STAT)-6 . Indeed,Tnfsf10−/−) mice and compared their inflammatory responses to mice that were STAT6 deficient (STAT6−/−). We also silenced MID-1 with small interfering RNAs (siRNAs) to wild-type (WT) mice to dissect further its role in IL-13-induced eosinophilic infiltration of the esophagus. We demonstrate that IL-13-mediated eosinophil infiltration depends on TRAIL and MID-1 and that MID-1-knockdown completely ablated eosinophilia and eotaxin-1 production, unlike TRAIL deficiency.Previous studies on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling in murine models of EoE have shown that inflammation and remodeling of esophageal tissue are dependent on the expression of TRAIL and downstream signaling through the E3 ubiquitin ligase Midline (MID)-1 , 17. TRATnfsf10−/−, and Stat6−/− mice (on BALB/c background) were obtained from Australian Bioresources . All animal experiments were approved by the Animal Care and Ethics Committee of the University of Newcastle. To induce inflammation of the esophagus, WT, Tnfsf10−/−, and Stat6−/− mice were intranasally challenged with 15 μg of rIL-13  under isoflurane anesthethesia administered in a supine position. Validated MID-1 siRNA (seq-5-AGAGUAAUCUCACCAAUCU-3) or control siRNA (seq-5-UGGUUUACAUGUCGACU AA-3) (3.75 nmol in nuclease-free water) (Dharmacon™) were administered 24 h before rIL-13 (Biolegend®) administration , stration . Mice we2 of the transverse esophageal section. Images were taken using the Aperio Slide Scanner (Leica Biosystems).Esophageal tissues were collected as described previously  and wereEsophageal samples isolated from mice were homogenised in TRIzol®  using the Tissue Tearor® rotor-stator. RNA was then extracted using chloroform phase-separation, followed by isopropanol precipitation ethanol wash and water-elution. RNA was reverse transcribed to cDNA using BioScript  per the manufacturer's instructions. All genes (listed below) were quantified using qPCR SYBR® green  on the Eppendorf Realplex  platform. The primers were used at optimal melting temperature (60°C) and are listed in Snap-frozen esophageal tissue was homogenised in Lysis Buffer consisting of IC#10 diluent  and supplemented with Leupeptin (2.5 mg/ml), Pepstatin (1.25 mg/ml), Aprotinin (1.1 mg/ml), and PMSF. Assays were performed on clarified lysates. PP2A activity was determined by Active PP2A DuoSet IC activity assay as per manufacturer protocols (Active Motif). PP2A activity was normalised to Saline (SAL) vehicle controls. TRAIL protein expression was quantified through ELISA as per manufacturers protocols (R&D Systems).t-tests (single comparison) or one-way ANOVA (Holm-Sidak) (multiple comparisons) in Graphpad Prism 6  as outlined in each figure legend. Data are presented as mean ± SEM.Statistical significance was determined between experimental groups using Student's Tnfsf10−/− (TRAIL-deficient) mice and Stat6−/− . Intranasal administration of rIL-13 to WT mice resulted in an upregulation of TRAIL protein  was determined in WT, Tnfsf10−/−and Stat6−/− mice upon IL-13 exposure . PP2A is a constitutive negative regulator of inflammation. Reduced PP2A resulted in inflammation through subsequent increase ERK-1/JNK inflammatory signalling. In order to suppress MID-1 expression locally we administered MID-1 targting siRNA in the esophagus. As expected MID-1 expression was significantly reduced compared to the nonsense (control) siRNA (NONc) control . FurtherTargeting MID-1 using siRNA (as above) prevented IL-13-induced eosinophilic infiltration compared to the NONc control . There win vivo models, we have shown that IL-13 production was dependent on the expression of TRAIL and MID-1  through Stat6 and then MID-1.Upregulation of MID1 following rIL-13 exposure was reduced in In our previous works, IL-13 and STAT6 were dependent on TRAIL and MID-1 expression in allergen-driven EoE . Now, thWe propose that IL-13, signaling through the E3 ubiquitin ligase MID-1, modulates PP2A activity in a STAT6-dependent manner. Targeting IL-13 with biologics is now a therapeutic option for EoE patients . Our dat


Scientific Reports 10.1038/s41598-022-12586-7, published online 24 May 2022Correction to: The original version of this Article contained an error in the Funding section.“This research was funded by National Centre of Science grant UMO-2020/37/B/NZ7/03380 within call OPUS-19.”now reads:"This research was funded by the National Science Centre, Poland grant within the program OPUS-19 (grant number 2020/37/B/NZ7/03380)."The original Article has been corrected.


Ionizing radiation (IR) enhances the migratory capacity of cancer cells. Here we investigate in non-small-cell-lung-cancer (NSCLC) cells a novel link between IR-enhanced ADAM17 activity and the non-canonical pathway of EphA2 in the cellular stress response to irradiation.Cancer cell migration in dependence of IR, EphA2, and paracrine signaling mediated by ADAM17 was determined using transwell migration assays. Changes of EphA2 pS897 and mRNA expression levels upon different ADAM17-directed treatment strategies, including the small molecular inhibitor TMI-005, the monoclonal antibody MEDI3622, and shRNAs, were mechanistically investigated. ADAM17-mediated release and cleavage of the EphA2 ligand ephrin-A1 was measured using ELISA and an acellular cleavage assay.via auto- and paracrine signaling. Genetic and pharmaceutical downregulation of ADAM17 activity abrogated growth factor (e.g. amphiregulin) release, which reduced MAPK pathway-mediated EphA2 S897 phosphorylation in an auto- and paracrine way  in NCI-H358 and A549 cells. These signaling processes were associated with reduced cell migration towards conditioned media derived from ADAM17-deficient cells. Interestingly, ADAM17 inhibition with the small molecular inhibitor TMI-005 led to the internalization and proteasomal degradation of EphA2, which was rescued by amphiregulin or MG-132 treatment. In addition, ADAM17 inhibition also abrogated ephrin-A1 cleavage and thereby interfered with the canonical EphA2-pathway.Irradiation with 5 Gy enhanced tumor cell migration of NSCLC NCI-H358 cells in dependence of EphA2. At the same time, IR increased growth factor-induced EphA2 S897 phosphorylation We identified ADAM17 and the receptor tyrosine kinase EphA2 as two important drivers for (IR-) induced NSCLC cell migration and described a unique interrelation between ADAM17 and EphA2. We demonstrated that ADAM17 influences both, EphA2 (pS897) and its GPI-anchored ligand ephrin-A1. Using different cellular and molecular readouts, we generated a comprehensive picture of how ADAM17 and IR influence the EphA2 canonical and non-canonical pathway in NSCLC cells. Lung cancer is the second most frequent type of cancer in men and women and has with 18%, the highest cancer-related mortality worldwide . On the The reprogramming of the epithelial-mesenchymal transition (EMT) by IR has been described as one of the hallmarks determining treatment success –7. ThereWe here describe a unique interrelation between EphA2 and the disintegrin and metalloproteinase ADAM17 alone and in response to irradiation. ADAM17 is involved in the processing of the extracellular matrix and the release of various growth factors and cytokines that contribute to the remodeling of the TME and facilitate tumor cell dissemination . We rece2.The human lung adenocarcinoma cell lines H358 and A549  and the human prostatic adenocarcinoma cell line PC-3  were newly purchased and cultured in RPMI1640 media , supplemented with 10% (v/v) fetal bovine serum , 1% (v/v) penicillin-streptomycin , and 1% (v/v) L-glutamine . The cells were cultured as monolayers in culture plates in a 37°C humidified atmosphere containing 5% COCustom lentiviral shRNA expression vectors were obtained from Cellecta. The expression vector used for shRNA directed against ADAM17 (A17) and control (NT) was pRSITPRP-U6Tet-sh-PGK-TetRep-2A-TagRFP-2A-Puro. Insert sequence for shA17 was ACC GGG ATC ATC GCT TCT ATA GAT ACG TTA ATA TTC ATA GCG TAT CTG TAG AAG CGA TGA TCT TTT and for the control cell line shNT was ACC GGC AAC AAG ATG GAG AGC ACT AAG TTA ATA TTC ATA GCT TGG TGC TCT TCA TCT TGT TGT TTT. The expression vector used for shEphA2 and shScr was pLKO-Tet-On. Insert sequence for shEphA2 was 5’-CCG GCG GAC AGA CAT ATA GGA TAT TCT CGA GAA TAT CCT ATA TGT CTG TCC GTT TTT-3’, and for the control cell line shScr was 5’-CCG GGA CAC GTC AAC TCG AAT AAC TCT CGA GAG TTA TTC GAG TTG ACG TGT CTT TTT-3’. The pLVX lentiviruses encoding FLAG-tagged EphA2 wt, and FLAG-tagged EphA2 S897A were kindly provided by E. Pasquale  .The lentiviral constructs were packaged into pseudotyped viral particles in HEK293AD cells, using the psPAX2/pMD2.G packaging plasmid mix  according to the manufacturer’s protocol. Target cells were transduced with the lentiviral supernatants in presence of 5 µg/ml polybrene. Cells were selected in presence of 1 µg/mL puromycin. The induction of the directed shRNA construct always began 72h prior to experiment start with 1 µg/ml Doxycycline .MEDI3622 and IgG1 were directly obtained from AstraZeneca. Cells were pretreated with MEDI3622 or IgG1 24 hours prior to sham treatment or irradiation (5 Gy) with a RS-2000 225kV irradiator at 4.2 Gy/min (Rad Source).The small molecular inhibitors TMI-005, SCH772984, and MG-132 were obtained from Axon Medchem (Axon 1507), Biovision (B1687) and Sigma (M7449), respectively. The human recombinant ligands rhEGF and rhAmphiregulin were obtained from Sigma Aldrich (E9644) and Genescript; (Z03103-50). rhEphrin-A1 Fc was obtained from R&D Systems (#6417-A1-050). Cells were treated as described in the results section. For sham treatment PBS or DMSO were used.5 cells  were plated into a 6-well plate in 2ml full RPMI. Simultaneously, 3 x 104 migrating cells  were seeded into transwell inserts (“upper chamber”). On the next day, cells were starved in either RPMI supplemented with 1% FBS (6-well plates) or 0.5% FBS (cells in the inserts). Six hours later the inserts and plates were sham-irradiated or irradiated with 5Gy. 24h post-irradiation, the CM from the 6-well plates containing sham-irradiated or irradiated H358 NT, or H358 A17 was transferred into 24-well plates (“lower chamber) and the inserts were immediately placed on the wells harboring the attracting media. The co-culture was maintained at 37°C in 5% CO2 for 24hours. For quantification, cells from the upper side of the insert were scraped away with a cotton swab and inserts were then fixed in Methanol/Acetic Acid , dried and stained with DAPI  in 99% MeOH. Fluorescent microscopy pictures were taken (Leica 7000DT) and the migrated cells were counted manually in at least 3 images at 10x magnification per insert.For the transwell migration assay, 24-well transwell units (6.5mm diameter) with 8μm pore size PET membranes  were used according to the manufacturer’s instructions. Briefly, for the generation of the conditioned media (CM) 4.0 x 10Santa Cruz Biotechnology; sc-2357) (1:5000 dilution) and enhanced chemiluminescence . For the loading control mouse anti-alpha Tubulin HRP  (1:5000 dilution) was used.For the detection of proteins in cell lysates, cells were lysed with M-PER™ Mammalian Protein Extraction Reagent  supplemented with 1x Halt™ Protease and Phosphatase Inhibitor Cocktail . 30µg of protein lysate was used per lane in the SDS-PAGE and after western blotting the membrane was blocked for 20’ in EveryBlot Blocking Buffer . For the assessment of protein expression, the following primary monoclonal antibodies (1:1000 dilution) were purchased from Cell Signaling Technology: rabbit anti-EphA2 (#6997), rabbit anti-phospho-EphA2 (Ser897) (#6347), rabbit anti-phospho-EphA2 (Tyr772) (#8244), rabbit anti-ERK1/2 (#4695S), rabbit anti-phospho-ERK1/2 (Tyr202/Tyr204) (#4370S), rabbit anti-AKT (#9272), rabbit anti-phospho-AKT (Ser473) (#9271S). Protein bands were visualized using an anti-rabbit horseradish peroxidase (HRP)-coupled secondary antibody  according to the manufacturer’s instructions. RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit  and cDNA was amplified using SYBR Green Master Mix  with the following primers from Microsynth: (5′-3′): GAPDH forward: AACGGATTTGGTCGTATTGGGC; GAPDH reverse: TTGATTTTGGAGGGATCTCG; EphA2 forward: GGGACCTGATGCAGAACATC; EphA2 reverse: AGTTGGTGCGGAGCCAGT. qRT-PCR was performed on LightCycler 480 (Roche).NCI-H358 cells were seeded 24h prior the experiment onto a 6-well plate with cover glasses. The cells were treated with 25µM TMI-005 or DMSO for 8h, 16h and 24 or with rhEphrin-A1 Fc (1µg/ml) for 20min. After the respective incubation times, the cells were washed with ice-cold PBS and fixed in 2% formaldehyde/PBS for 15min at RT. The cells were washed three times with PBS-T (0.02% Tween-20 in PBS) and permeabilized with 0.5% Triton X-100/PBS for 10 min. After washing, the cells were blocked 2% BSA/PBS-T for 1 h. Then cells were incubated with the primary antibody rabbit anti-EphA2  or an IgG1 control (AstraZeneca) at RT for 1h in a humidified chamber. After six times washing with PBS-T, the cells were incubated with secondary antibody goat anti-rabbit Alexa Fluor488  and DAPI  for 1 h at room temperature, in a dark, humidified chamber. After six times washing with PBS-T and two washings with PBS, the cover glasses were mounted on microscope glass slides with Dako fluorescence mounting medium . The microscope images were acquired on Leica DMi8 microscope using a HC PL APO CS2 63x objective, a Leica digital color camera DMC6200 and Leica monochrome fluorescence DFC9000 GTC using 390 and 475 nm laser lines. Refractive index-matched immersion oil (Nikon Instruments) was used for all experiments. Images were reconstructed and analyzed with FlowJo software.200’000 NCI-H358 cells were seeded 24h prior the experiment onto a 6-well plate. Soluble human Ephrin A1 was collected 24 hours and 48 hours after treatment and detected in 200µl filtered conditioned media using a human Ephrin-A1 ELISA kit and a human Amphiregulin DuoSet ELISA kit according to the manufacturer’s guidelines . The absorbance was determined with a plate reader TECAN Infinite 200 PRO at 450 nm excitation and 540 nm emission and normalized to the total protein amount of the cell lysate.A total of 100 ng human recombinant EphrinA1-Fc was incubated with 100 ng human recombinant ADAM17  in the TACE Activity Kit Assay buffer  in a total volume of 25μl per reaction. The samples were incubated +/- TMI-005 for 5h at 37°C. For Western Blot analysis, the samples were boiled at 95°C in 6x Laemmli Buffer.If not indicated otherwise, data were collated from at least three biological replicates obtained from at least three independent experiments. For all experiments applies; p<0.05: *, p<0.01: **, p<0.001: ***.The data were analyzed with GraphPad Prism v9 and tested for normal distribution using the Shapiro-Wilk normality test. For the direct comparison of two treatment groups, the two-tailed unpaired t-test or Mann-Whitney test was applied. For the comparison of more than two treatment groups one-way ANOVA test with Tukey posttest or Dunn’s test was performed. Error bars indicate SEM. A p-value ≤ 0.05 was considered significant. In order to investigate the relevance of EphA2 for basal and IR-induced lung tumor cell migration, a Boyden-chamber transwell migration assay was developed with NCI-H358 lung adenocarcinoma cells, stably transduced with a doxycycline-inducible EphA2-directed shRNA (shEphA2) vector or an inducible scramble shRNA (shScr) expression vector . Basal cTo differentiate between an auto- and paracrine effect of IR-enhanced migration, non-irradiated NCI-H358 cells were seeded into inserts and attracted by conditioned media (CM) derived from NCI-H358 cells sham-irradiated or irradiated 24 hours prior to CM transfer. A significantly enhanced increase of cell migration could be determined towards the CM derived from irradiated cells. Migration was again strongly abrogated upon downregulation of EphA2 in the migrating cell population  or the small molecular ADAM17 inhibitor TMI-005 (25µM). EphA2 pS897 and total EphA2 protein levels were downregulated in response to cellular incubation with both types of ADAM17 inhibitors S2E. To To support the critical role of ADAM17 for growth factor-induced EphA2 S897 phosphorylation we investigated EphA2 and EphA2 pS897 in sham-/irradiated ADAM17 (NT) wildtype and ADAM17 (A17) knockdown cells. EphA2 pS897 was increased in irradiated NCI-H358 control cells 24 and 48 hours after irradiation, but strongly downregulated in sham-irradiated as well as irradiated ADAM17 knockdown cells . Treatment with SCH772984 strongly abrogated total EphA2 levels (anti-flag) in the wt control compared to the S897A mutant . As control for EphA2 degradation, cells were incubated with the recombinant, homodimeric ligand rhEphrin-A1 Fc, which induces a rapid internalization of the EphA2/ephrin-A1 Fc complex followed by proteasomal degradation  and compared it to their untreated controls. The level of soluble ephrin-A1 was strongly reduced in NCI-H358 . Indeed, we could detect significantly decreased levels of soluble ephrin-A1 in the supernatants, derived from TMI-005 or MEDI3622 treated NCI-H358 cells. These results corroborate that cleaved, soluble and monomeric ephrin-A1 does not result in EphA2 internalization , rather than monotherapies will advance our collective battle against cancer.The original contributions presented in the study are included in the article/VW: Investigation, methodology, writing-original draft, project administration, writing-review and editing. FT: Methodology. RW: Methodology. MP: Conceptualization, resources, supervision, funding acquisition, writing-original draft, project administration, writing-review and editing. All authors contributed to the article and approved the submitted version.


W-nitro-L-arginvine methyl ester (L-NAME), methylene blue, lipid emulsion, and linolenic acid on the amlodipine-induced vasodilation and amlodipine-induced cyclic guanosine monophosphate (cGMP) production were examined. Furthermore, the effects of lipid emulsion, amlodipine, and PP2, either alone or combined, on endothelial nitric oxide synthase (eNOS), caveolin-1, and Src-kinase phosphorylation were examined. Amlodipine-induced vasodilation was higher in endothelium-intact aorta than in endothelium-denuded aorta. L-NAME, methylene blue, lipid emulsion, and linolenic acid inhibited amlodipine-induced vasodilation and amlodipine-induced cGMP production in the endothelium-intact aorta. Lipid emulsion reversed the increased stimulatory eNOS (Ser1177) phosphorylation and decreased inhibitory eNOS (Thr495) phosphorylation induced via amlodipine. PP2 inhibited stimulatory eNOS, caveolin-1, and Src-kinase phosphorylation induced via amlodipine. Lipid emulsion inhibited amlodipine-induced endothelial intracellular calcium increase. These results suggest that lipid emulsion attenuated the vasodilation induced via amlodipine through inhibiting nitric oxide release in isolated rat aorta, which seems to be mediated via reversal of stimulatory eNOS (Ser1177) phosphorylation and inhibitory eNOS (Thr495) dephosphorylation, which are also induced via amlodipine.This study aimed to examine the effect of lipid emulsion on the vasodilation induced by a toxic dose of amlodipine in isolated rat aorta and elucidate its mechanism, with a particular focus on nitric oxide. The effects of endothelial denudation, N Lipid emulsion treats systemic toxicity resulting from local anesthetics . In addi−7 M)-induced vasodilation was higher in endothelium-intact than in endothelium-denuded aorta (p < 0.001 at 10–80 min). Dimethyl sulfoxide , which is the same concentration used to dissolve the amlodipine, did not affect the phenylephrine-induced contraction in the endothelium-intact aorta , non-specific GC inhibitor methylene blue (10−6 M), nitric oxide-sensitive GC inhibitor 1H-oxadiazolo quinoxalin-1-one , and calmodulin-regulated enzyme inhibitor calmidazolium (3 × 10−5 M) inhibited the amlodipine (3 × 10−7 M)-induced vasodilation  in the endothelium-intact aorta. The post-treatment with lipid emulsion inhibited the amlodipine (3 × 10−7 M)-induced vasodilation (p < 0.001 vs. control at 40–80 min) in the endothelium-intact aorta. However, it initially transiently increased the amlodipine-induced vasodilation (p < 0.001 vs. control at 10 min). In addition, the lipid emulsion also inhibited the amlodipine (3 × 10−7 M)-induced vasodilation in endothelium-intact aorta pre-treated with L-NAME . However, the difference in the area (min%) under phenylephrine-induced contraction (%) time (30–80 min) curves between the groups with and without lipid emulsion treatment in the endothelium-intact aorta was greater without L-NAME (10−4 M) than with it (p = 0.010). Linolenic acid (3 × 10−6 and 10−5 M) inhibited the amlodipine (3 × 10−7 M)-induced vasodilation in the endothelium-intact aorta . However, linolenic acid (3 × 10−6 and 10−5 M) did not affect the vasodilation in the endothelium-denuded aorta (Amlodipine (3 × 10ct aorta . Nitric ed aorta B.−7 M) increased cyclic guanosine monophosphate (cGMP) formation in the isolated endothelium-intact rat aorta (p < 0.01 vs. control). However, lipid emulsion (1%) or L-NAME (10−4 M) inhibited the amlodipine (3 × 10−7 M)-induced cGMP formation . In addition, linolenic acid (10−5 M) inhibited amlodipine (3 × 10−7 M)-induced cGMP formation (p < 0.05 vs. amlodipine). However, the lipid emulsion (1%) did not affect the amlodipine (3 × 10−7 M)-induced cGMP formation in the endothelium-intact rat aorta pre-treated with L-NAME (10−4 M) (Amlodipine (3 × 10(10−4 M) B.−7 M) increased stimulatory eNOS (Ser1177) phosphorylation (p < 0.05 vs. control) and decreased inhibitory eNOS (Thr 495) phosphorylation (p < 0.001 vs. control) in human umbilical vein endothelial cells (HUVECs). However, lipid emulsion (1%) reversed the increased stimulatory eNOS (Ser1177) phosphorylation and decreased inhibitory eNOS (Thr495) phosphorylation induced using amlodipine (3 × 10−7 M) . In addition, Src-kinase inhibitor PP2 (2 × 10−5 M) attenuated the amlodipine (3 × 10−7 M)-induced stimulatory eNOS (Ser1177) phosphorylation . Furthermore, amlodipine (3 × 10−7 M) induced caveolin-1 (Tyr14) phosphorylation (p < 0.01 vs. control) in HUVECs. However, Src-kinase inhibitor PP2 inhibited the amlodipine (3 × 10−7 M)-induced caveolin-1 (Tyr14) phosphorylation . Amlodipine (3 × 10−7 M) also induced Src-kinase (Tyr 416) phosphorylation (p < 0.001 vs. control) in HUVECs. However, PP2 inhibited the amlodipine (3 × 10−7 M)-induced Src-kinase (Tyr416) phosphorylation .Amlodipine (3 × 10−6 M) increased the intracellular calcium (p < 0.001 vs. control) in HUVECs. However, lipid emulsion (1%) and L-NAME (10−4 M) inhibited the calcium level increase via amlodipine (10−6 M) .Amlodipine  inhibitors of eNOS-nitric oxide-GC pathway attenuated amlodipine-induced vasodilation of the endothelium-intact rat aorta; (2) lipid emulsion and linolenic acid inhibited amlodipine-induced vasodilation in the endothelium-intact rat aorta; (3) lipid emulsion did not affect the amlodipine-induced cGMP formation in L-NAME-pretreated endothelium-intact aorta; (4) lipid emulsion reversed the increased stimulatory eNOS (Ser1177) phosphorylation and decreased inhibitory eNOS (Thr495) phosphorylation induced via amlodipine; and (5) L-NAME and lipid emulsion inhibited the increased intracellular calcium induced via amlodipine.−8 M) i level after amlodipine treatment [−6 M) to measure intracellular calcium. HUVECs were pre-treated with 1% Intralipid or L-NAME, followed by amlodipine (10−6 M), 1% lipid emulsion, or L-NAME alone. Calcium levels were measured every 2.5 s at excitation and emission wavelengths of 485 and 520 nm, respectively. Intracellular calcium was analyzed using images expressed with Fluo-4 AM. Intracellular calcium was calculated as fluorescence intensity (F) divided by baseline fluorescence intensity (F0) before drug treatment. The net change in calcium ions was expressed as (Fmax-F0)/F0, where Fmax is the maximum calcium level in fluorescence intensity after treatment with amlodipine, lipid emulsion, and L-NAME, either alone or combined. The intracellular calcium was measured for approximately 6 min.A confocal laser microscope  was used, as described previously, to measure intracellular calcium [Careatment . HUVECs All the high-purity chemicals are commercially available. Intralipid (20%) was purchased from Fresenius Kabi . Amlodipine, L-NAME, methylene blue, ODQ, phenylephrine, acetylcholine, linolenic acid, calmidazolium, and anti-β actin were obtained from Sigma-Aldrich . Amlodipine, calmidazolium, and ODQ were dissolved in DMSO . Anti-Src-kinase, anti-phospho-Src-kinase, anti-phospho-eNOS (Ser 1177 and Thr 495), anti-caveolin-1, and anti-phospho-caveolin-1 antibodies were purchased from Cell Signaling Technology . Anti-eNOS antibody was purchased from BD Bioscience .t-test. p < 0.05 was considered statistically significant.The study’s primary outcome was the effect of lipid emulsion, inhibitors, and linolenic acid  on the amlodipine-induced vasodilation in the isolated rat aorta. This primary outcome was analyzed using a linear mixed effect model  . The effIn conclusion, lipid emulsion attenuated endothelium-dependent nitric oxide-medi ated vasodilation induced using a toxic dose of amlodipine via inhibition of endothelial nitric oxide release, which seems to be mediated through reversal of stimulatory eNOS phosphorylation (Ser1177) and inhibitory eNOS (Thr495) dephosphorylation induced using amlodipine.


Exosomes derived from oral squamous cell carcinoma (OSCC) could modulate OSCC development. This study aimed to explore effects of exosome-mediated lncRNA PART1 on OSCC cells.http://gepia.cancer-pku.cn/). Exosomes isolated from cell lines squamous cell carcinoma 9 (SCC9) and Centre Antoine Lacassagne-27 (CAL27) were identified by transmission electron microscope and western blot. Exosome-mediated lncRNA PART1, microRNA-17-5p(miR-17-5p) and suppressor of cytokine signaling 6(SOCS6) RNA expressions were assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Cell counting kit-8(CCK-8), caspase-3 activity, and flow cytometry were applied to evaluate OSCC cell viabilities and apoptosis. Meanwhile, OSCC cell migratory ability and invasiveness were evaluated using transwell assay. Bindings between miR-17-5p and lncRNA PART1 or SOCS6 were validated using the luciferase reporter test. Western blot was used for detecting the protein levels of SOCS6, phosphorylated signal transducer and activator of transcription 3 (STAT3) and STAT3.This study was performed in Tianjin Medical University Cancer Institute from February 2021 to March 2022. Bioinformatic analysis was performed on the public database GEPIA  patients. After the exosomes from OSCC cells were isolated and verified, PART1 was then confirmed to be secreted by exosomes. Further, overexpression of exosome-mediated lncRNA PART1 inhibited OSCC cell viabilities, migration, and invasiveness but facilitated OSCC cell apoptosis. PART1 upregulated SOCS6 through sponging miR-17-5p. Moreover, exosome-mediated lncRNA PART1 suppressed the phosphorylation of STAT3.Exosome-mediated lncRNA PART1 could mediate the OSCC progression via miR-17-5p/SOCS6 axis in vitro, suggesting that lncRNA PART1 might be a target for treating OSCC. Head and neck squamous cell cancer (HNSCC) mainly occurs in the oral cavity, nasal cavity, pharynx, and larynx, and it ranks as the seventh most common cancer . Oral sqIn recent years, studies have suggested that communication among cells could be achieved through membranes, cell fragments, or extracellular vesicles (EVs) . Exosomehttps://starbase.sysu.edu.cn/), binding sites between miR-17a-5p and lncRNA PART1 were predicted, implying underlying interactions between them. Dysregulation of janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway is closely associated with cancer development [https://www.targetscan.org/vert_72/). Based on our detections, lncRNA PART1, and SOCS6 shared the same binding sites in miR-17-5p. Hence, we hypothesized that LncRNA PART1 modulated OSCC cell progressions via regulating miR-17-5p and SOCS6 axis.MicroRNAs (miRNAs), small noncoding RNAs of 19–24nts, are critical in regulating the biological processes of cancer cells through targeting mRNAs . Moreoveelopment . Suppreselopment . SOCS6 welopment . Beyond Human tongue squamous cell carcinoma cell lines (SCC9 and CAL27) and human normal oral epithelial cells (HOECs) were obtained from American type culture collection  and Procell , respectively. Human embryonic kidney 293(HEK-293T) cells were purchased from Procell, which were used in the luciferase reporter analysis. Thereafter, Roswell Park Memorial Institute 1640 supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin was applied for incubating cells.6 cells/well) were incubated in 6-well plates and transfection was conducted when the confluence reached 85% using the Lipofectamine 3000 . Then, 50 pmol miR-17-5p mimics/inhibitor and negative controls respectively were used in the transfection assays. RNA expressions were then examined 24 h after transfection.MiR-17-5p mimics/inhibitor and negative controls (NC mimics/inhibitor) were bought from GenePharma . LncRNA PART1 overexpressed plasmid (oePART1) and its control (oeNC) were synthesized using lentivirus vectors . Meanwhile, SCC9 and CAL27 cells . First, cell culture was centrifuged for 20 min at 3000 × g to remove cells and cellular debris. Next, the supernatant was filtered to remove particles over 0.8 μm followed by mixing with Buffer XBP. Thereafter, the mixture was bound to exoEasy membrane affinity spin column. Extracted exosomes were rinsed by Buffer XWP and then washed by Buffer XE. Afterwards, exoRNeasy Midi and Maxi Kit(Qiagen) was applied for extracting RNAs.Exosomes were resuspended and fixed by 30 μL 2% paraformaldehyde. Afterwards, glow-discharged copper grid was used to absorb exosomes. Later, glow-discharged copper grids were fixed using 3% glutaraldehyde and then stained by uranyl acetate. Finally, a transmission electron microscope was applied for observing exosomes at 100,000 x.-ΔΔCt method.Trizol  was used for segregating total RNA followed by reverse transcription of lncRNA PART1 and SOCS6 with BeyoRT™II Kit . Reverse transcription of miR-17a-5p was conducted using Mir-X Kit . Then, 7300Plus Real-Time PCR System  with SYBR Green (Applied Biosystem) was applied for RT-qPCR: predenaturation, 95 °C, 30 s and  × 35 cycles. Primers were listed as below, which were: lncRNA PART1 5′-CAATAAGGCAGAAGAAGGTG-3′ (forward), 5′-GGAGAATCTGAAGTCCCAAG-3′ (reverse); miR-17-4 cells/well) were seeded in 96-well plates and 10 μL Cell counting kit 8 was added at 24 h, 48 h, and 72 h. Thereafter, cells were kept culturing for another 1 h. Optical density (OD) values at 450 nm were observed using a lab microplate reader.Transfected SCC9 and CAL27 cells  was applied for evaluating caspase-3 activities in SCC9 and CAL27 cells. Cells were incubated in 96-well plates. Thereafter, detection buffer and Ac-DEVD-pNA (2 mM) were added to the cell culture and the mixture was incubated for 2 h at 37 °C. The absorbance of caspase-3 was measured at 405 nm using a lab microplate reader.Apoptosis of SCC9 and CAL27 cells was evaluated using an Annexin V-FITC assay kit (Beyotime). First, cells after exosome treatment and transfection were collected and resuspended by Annexin V-FITC binding buffer. Then, cells were mixed with 5 μL Annexin V-FITC and 10 μL PI followed by incubation in darkness for 20 min at 23 °C. Finally, Accuri C6 Plus flow cytometer  was used for detecting apoptosis of SCC9 and CAL27 cells.SCC9 and CAL27 cells were seeded into upper chambers  with serum-free medium. Meanwhile, the lower component was added with 600 μL RPMI-1640 medium with 10% FBS. Matrigels  were used for invasion detection. After 24 h, cells in the lower component were stained using crystal violet (Beyotime) and numbers of migrated or invaded cells were checked through a microscope.https://starbase.sysu.edu.cn/) and TargetScan (https://www.targetscan.org/vert_72/), binding sequences of lncRNA PART1 and SOCS6 containing miR-17-5p binding sites and their mutated types were cloned into pmirGLO vector  to create lncRNA PART1-wt/mut and SOCS6-wt/mut. Thereafter, these vectors were cotransfected into HEK-293T cells with NC mimics or miR-17-5p mimics. Fluorescence was examined using the Dual-Luciferase Reporter Assay System (Promega).According to data of ENCORI (RIPA buffer (Beyotime) was applied for lysing SCC9 and CAL27 cells to isolate total protein. Afterwards, proteins were isolated using 10% SDS-PAGE followed by shifting onto PVDF membranes. Then, 5% skim milk was applied for blocking and primary antibodies were used for incubating membranes overnight at 4 °C. Primary antibodies were listed: anti-CD63 , anti-CD9 , anti-TSG101, anti-Calnexin , anti-p-STAT3 , anti-STAT3 , and anti-GAPDH . Thereafter, membranes were cultivated with goat anti-mouse IgG (HRP)  for 1 h at 23 °C. BeyoECL Moon (Beyotime) was used for developing and Image J  was for analyzing bands.http://gepia.cancer-pku.cn/), the boxplot about lncRNA PART1 expressions in HNSCC was generated. Moreover, the survival analysis correlated with lncRNA PART1 expression in HNSCC patients was performed using the Kaplan-Meier method on GEPIA database. All the clinical data were from the public database GEPIA.Based on GEPIA and SPSS 19.0 (USA) were utilized to analyze statistical data. If the data distribution fit a normal distribution with equal variance, differences between the two groups were examined using the student’s t-test. One-way ANOVA was applied for analyzing differences between two groups while two-way ANOVA was carried out to analyze data in the CCK-8 assay. Dunnett’s T3 test was applied for adjusting p values in multiple tests. P < 0.032 was defined to be statistically meaningful.http://gepia.cancer-pku.cn/), lncRNA PART1 expressions were lower in HNSCC tissues  . This cohttps://starbase.sysu.edu.cn/) to predict the bindings with miR-17-5p   . Furtherert_72/) . Later, ert_72/) . Additioert_72/) . Hence, Cancer cells usually produce more exosomes than normal cells and those exosomes provide critical signals for reprogramming stromal cells and cell architectures in tumor microenvironment . Recentl


ITD mutCombination of chemotherapy with FLT3 inhibitors has been shown to improve progression free survival in patients carrying the ITD in the JMD domain of FLT3. On the contrary, chemotherapeutic resistance has been associated with insertion of the ITD in the TKD region, which, therefore, represents an unfavorable prognostic factor for relapse and overall survival (OS) , 4. AccoTo address this question, we aimed at (i) investigating whether the ITD insertion sites influence responsiveness towards cytarabine (Ara-C) treatment in AML cell lines and patient-derived primary AML blasts, at (ii) elucidating the molecular mechanisms controlling Ara-C sensitivity, and at (iii) identifying novel potential therapeutic strategies to revert resistance in ITD-TKD AML cells.ITD-JMD and FLT3ITD-TKD) with Ara-C at clinically-relevant doses. In fact, FLT3ITD-JMD cells were more susceptible towards in vitro Ara-C-mediated killing as compared to their FLT3ITD-TKD counterpart  and 3 FLT3ITD-JMD+ITD-TKD patients (carrying the ITD in the JMD and TKD domain) [ITD-JMD+ITD-TKD blasts showed higher sensitivity to Ara-C in cell death analysis, while induction of apoptosis was barely detected in FLT3ITD-TKD blasts at 1 µM Ara-C     . Given t2AX Fig. . Increaslls Fig. . Importa94) Fig. .ITD-TKD cells showed higher levels of DNA damage than FLT3ITD-JMD cells  [To elucidate the molecular mechanisms underlying the different sensitivity to Ara-C therapy in FLT3-ITD cells, we applied state-of-the-art, high-sensitive, mass spectrometry (MS)-based (phospho)proteomics followed by our recently developed modeling pipeline “SignalingProfiler” Fig.  (https:/rofiler) . This strofiler) .Fig. 2CoITD-TKD and FLT3ITD-JMD cells by employing MS-based (phospho)proteomics. Using this strategy, about 4000 proteins  change upon Ara-C treatment   Table . Intereslls Fig. , panel bITD-TKD cells expression of CDK2 and Cyclin H was significantly increased as compared to their FLT3ITD-JMD counterparts (Fig. ITD-TKD cells are significantly more accumulated in the S phase as compared to FLT3ITD-JMD cells (Fig. ITD-TKD cells responsiveness to Ara-C treatment in both patients-derived FLT3-ITD primary blasts and Ba/F3 cells. As shown in Fig. ITD-TKD cells. Additionally, our data suggest that while in FLT3ITD-JMD cells Ara-C and THZ1 drugs seems to have a synergistic effect on apoptosis, in FLT3ITD-TKD cells they have an additive effect (Fig. First, we confirmed that in Ara-C treated FLT3rts Fig. . Consistlls Fig. . Althougect Fig. .SignalingProfiler pipeline to our phosphoproteomic data revealed that Ara-C responsiveness is associated to the deregulation of the CDK2-CDK7 pathway and consequent accumulation of cells in the S phase. Pharmacological suppression of CDK7 synergistically acts with cytarabine, restoring the FLT3ITD-TKD cells’ sensitivity. This observation is consistent with recent studies reporting that CDK7 inhibitor potentiates the anti-cancer effect of DNA damage agents or tyrosine kinase inhibitor as potent anti-cancer agents [ITD-TKD cells to chemotherapy, however, these pathways need to be investigated more in depth in further pre-clinical studies.Taken together, our findings confirm the clinical relevance of the FLT3-ITD location, which affects disease biology. In our study, ITD-location alters the intrinsic DNA damage level and sensitivity to cytarabine treatment in both AML cell lines and patient-derived primary AML blasts. We recently showed that different FLT3-ITD insertion sites alter sensitivity to TKIs treatment through the modulation of WEE1-CDK1 axis and cell cycle progression . Applyinr agents . FinallySupplementary MaterialTable S1Table S2Table S3Table S4


The SARS-CoV-2 non-Spike (S) structural protein targets on nucleocapsid (N), membrane (M) and envelope (E), critical in the host cell interferon response and memory T-cell immunity, are grossly overlooked in COVID vaccine development. The current Spike-only vaccines bear an intrinsic shortfall for promotion of a fuller T cell immunity. Vaccines designed to target conserved epitopes could elicit strong cellular immune responses that would synergize with B cell responses and lead to long-term vaccine success. We pursue a universal (pan-SARS-CoV-2) vaccine against Delta, Omicrons and ever-emergent new mutants.50, 1,711) and Delta ; and against pseudovirus WT  vs. Omicron BA.1/BA.2/BA.5 variants , respectively. The lower primary neutralizing antibodies in the elderly were uplifted upon boosting to approximately the same high level in young adults. UB-612 also induced potent, durable Th1-oriented (IFN-γ+-) responses  along with robust presence of cytotoxic CD8+ T cells . This UB-612 booster vaccination is safe and well tolerated without SAEs.We explored booster immunogenicity of UB-612, a multitope-vaccine that contains S1-RBD-sFc protein and sequence-conserved promiscuous Th and CTL epitope peptides on the Sarbecovirus N, M and S2 proteins. To a subpopulation  of infection-free participants (aged 18–85 years) involved in a two-dose Phase-2 trial, a UB-612 booster (third dose) was administered 6–8 months after the second dose. The immunogenicity was evaluated at 14 days post-booster with overall safety monitored until the end of study. The booster induced high viral-neutralizing antibodies against live Wuhan WT  vaccines” as exemplified by UB-612, a multitope-vaccine armed with Spike (S1-RBD and S2) and non-Spike (Nucleocapsid N and Membrane M) targets, allowing booster vaccination to elicit broadly-recognizing and durable B-/T-cell memory immunity. Sequence-conserved Th/CTL epitope peptides were designed from S2, N and M proteins to synergistically enhance memory helper and cytotoxic T-cell immunity and RBD targeted B-cell immunity. The SARS-CoV-2 Omicron lineage has swept the globe with a rapid succession of dominating subvariants from BA.1, BA.2 and to the current BA.5 that makes up more than 90% of infection cases with overriding edges in transmissibility and neutralizing antibody escape . RelativOmicron BA.1 is heavily mutated , includiBA.2 exhibits a 1.3- to 1.5-fold higher transmissibility and a 1.3-fold immune evasion than BA.1 ,19, consAmongst double-vaccinated adults, the booster (third dose)-induced neutralization titers against BA.4/BA.5 are notably lower than those against BA.1/BA.2 –5; and, Regardless of vaccination status or hybrid immunity, each reinfection would add risks of mortality, hospitalization and other health hazards including burden of long COVID . The lonrd-dose of mRNA vaccines could compensate Omicron (BA.1)-induced decrease in serum neutralizing antibodies (20- to 30-fold reduction), along with rates of hospitalization and severe disease (80–90% protection)  in Taiwan with 3,844 healthy male or female adults aged >18 to 85 years  which was an extension arm of the Phase-2, placebo-controlled, randomized, observer-blind, multi-center primary 2-dose study  . The goal of the two vaccines in one shot is to deliver durable antibody and T-cell immune protection against severe disease and hospitalization for at least one year.Of interest to note, to enhance vaccine immunity, a T cell vaccine BNT162b4 targeting conserved epitopes on non-Spike proteins, in combination with BNT162b5 BA.5-bivalent or BNT162 BA.1-bivalent vaccine, is being developed  conducted between Oct. 16, 2021 and Apr. 16, 2022. (A) Of the primary series , a total of 1,478 participants (aged at 18–85 years) were enrolled to receive the booster third-dose of 100 μg UB-612; (B) the characteristics of the study participants in the primary and booster series.(DOCX)Click here for additional data file.S2 Fig(A) Solicited local adverse reaction within 7 days after each vaccination. (B) Skin allergic reaction within 14 days after each vaccination. (C) Solicited systemic adverse reaction events 7 days after each vaccination (DOCX)Click here for additional data file.S3 FigWT binding, ACE2:RBDWT binding inhibition, and anti-WT viral-neutralizing activity VNT50 and (B) the summary of geometric mean titer (GMT) with 95% CI. A total of 302 participants  received a booster 3rd-dose. The serum samples of 302 participants were collected at the indicted time points, Days 197 to 242 (the pre-booster day) and Days 211 to 256 (14 days post-booster), and tested for neutralizing antibody levels that inhibit 50% of live SARS-CoV-2 wild-type, expressed as VNT50  , the inhibitory titers against S1-RBD binding to ACE2 by ELISA, expressed as μg/mL , and anti-S1-RBD IgG antibody titers by ELISA (antigenic). Statistical analysis was performed by the Student’s t-test .Immunogenicity overview are presented in (A) antigenic S1-RBD(TIF)Click here for additional data file.S4 Fig50 titer reached at 3,992 against live SARS-CoV-2 Wuhan wild-type, and at 2,358 against live Delta variant. (B) Similarly, unusually high post-booster pVNT50 against Wuhan wild-type pseudovirus at 12,778, and at 2,325 against Omicron BA.1. (C) High post-booster pVNT50 against Omicron BA.2 as well. (D) Summary of geometric mean titer (GMT) with 95% CI are presented for plots shown in panels A—C. *J. Clin. Invest. 2022;132(10):e157707. https://doi.org/10.1172/JCI157707.Geometric mean titers (GMT) at 50% viral-neutralization observed 14 days after the booster third-dose of 100-μg administered at mean Day 286 (Days 255–316) after the primary 2-dose series (Days 0 and 28) of the 196-day Phase-1 trial. (A) In the participants of the 100-μg group (n = 18) with healthy adults aged at 20–55 years, the post-booster VNT(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.S2 Methods(DOCX)Click here for additional data file.S3 Methods(DOCX)Click here for additional data file.S4 Methods(DOCX)Click here for additional data file.S5 Methods(DOCX)Click here for additional data file.S6 Methods(DOCX)Click here for additional data file.S7 Methods(DOCX)Click here for additional data file.S8 Methods(DOCX)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.


NOTCH1 mutations or alterations in its ubiquitin ligase FBXW7. NOTCH signaling has been shown to contribute to chemotherapy resistance in some tumor models. Hence, targeting the NOTCH1 signaling pathway may be an effective option to overcome relapsed and refractory T-ALL.T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic tumor, characterized by several genetic alterations, that constitutes 15% of pediatric and 25% of adult ALL. While with current therapeutic protocols children and adults’ overall survival (OS) rates reach 85–90% and 40–50%, respectively, the outcome for both pediatric and adult T-ALL patients that relapse or are refractory to induction therapy, remains extremely poor, achieving around 25% OS for both patient groups. About 60% of T-ALL patients show increased NOTCH1 activity, due to activating NOTCH1 mutated T-ALL samples. Interestingly, from RNAseq data we observed that anti-NOTCH1 treatment in vivo affects the purine metabolic pathway. In agreement, both in vitro and in vivo, the greatest effect on leukemia growth reduction was achieved by anti-NOTCH1 therapy in combination with antimetabolite drugs. This result was further corroborated by the longer life span of mice treated with the anti-NOTCH1 in combination with antimetabolites, indicating a novel Notch-targeted therapeutic approach that could ameliorate pediatric and adult T-ALL patients outcome with relapse disease for whom so far, no other therapeutic options are available.Here, we focused on the therapeutic activity of the NOTCH1-specific monoclonal antibody OMP-52M51 in combination either with drugs used during the induction, consolidation, or maintenance phase in mice xenografts established from pediatric and adult relapsed The online version contains supplementary material available at 10.1186/s40164-023-00439-6. TP53 mutations and deletions have been shown to occur more frequently at relapse and are adversely associated with second-line therapy survival ,  in all the compartments  as well as a decrease in spleen weight  Fig. , probabl)], Fig. , compare)], Fig. , we starFinally, we performed survival experiments by administrating the anti-NOTCH1 and COMBO2 treatments alone or in combination, and stopped the treatments at 20-40% of circulating blasts in control mice PB  and quantitative PCR (qPCR). Western blot analysis. Preparation of RNA libraries and RNA seq. Bioinformatics analysis. T-ALL Patient Derived Xenografts (PDXs) cells in vitro treatments and  IC50 determination for selected drugs by Caspase-Glo® 3/7 Assay. Statistical analysis. Scientific Image and Illustration software. Additional Tables: Table S1. Characterization of PDXs from T-ALL pediatric and adult relapsed patients. Table S2. Upregulated gene sets in CTRL versus OMP-52M51-treated PDTALL46 cells. Table S3. IC50 determination for selected drugs in T-ALL PDX cells. Table S4. Selected drugs to be combined with anti-NOTCH1 antibody (OMP-52M51). Additional Figures and Legends:Figure S1. NOTCH1 protein and target genes expression in T-ALL PDXs (PDTALL). Figure S2. Anti-NOTCH1 (OMP-52M51) inhibits growth of NOTCH1-driven T-ALL PDXs. Figure S3. RNASeq analysis of OMP-52M51-acute treated PDTALL46 mice. Figure S4. In vitro cell apoptosis determination in T-ALL PDXs cells treated with different drugs alone or in combination with OMP-52M51. Figure S5. In vivo inhibitory effect of OMP-52M51 in combination with COMBO1 and COMBO2 in PDTALL46 model. Figure S6. In vivo inhibitory effect of OMP-52M51 in combination with antimetabolite drugs (COMBO2) in PDTALL39 and PDTALL-AD4 models. Figure S7. Efficacy of Anti-NOTCH1 in combination with antimetabolite drugs in T-ALL PDXs models. Additional References.


Scientific Reports 10.1038/s41598-017-16852-x, published online 07 December 2017Correction to: This Article contains an error in the Title,“Endomucin inhibits VEGF-induced endothelial cell migration, growth, and morphogenesis by modulating VEGFR2 signaling.”should read:“Endomucin knockdown inhibits VEGF-induced endothelial cell migration, growth, and morphogenesis by modulating VEGFR2 signaling.”