The hexagonal structure of the crystalline MAX phase was confirmed by X-ray diffraction. Moreover, the compacted layered structure of the MAX phase was observed via scanning electron microscopy and high-resolution transmission electron microscopy. Under the desired operating conditions, Cr2AlC MAX phase (0.75 g/L) showed suitable potential to activate H2O2 (1 mmol/L) under sonication, thereby allowing a considerable removal efficiency for various organic pollutants, including dimethyl phthalate (69.1%), rifampin (94.5%), hydroxychloroquine (100%), and acid blue 7 (91.5%) with initial concentration of 15 mg/L within 120 min of treatment. Kinetic analysis proved that the degradation reaction followed pseudo-first-order kinetics. Scavenging tests demonstrated that hydroxyl radicals and singlet oxygen were effective species during degradation. Furthermore, a probable mechanism for dimethyl phthalate degradation was suggested according to gas chromatography-mass spectroscopy and nuclear magnetic resonance analyses. The obtained results confirmed the capability of the triple Cr2AlC/H2O2/US process as a promising method for treating contaminated water.This study aims to investigate the sonocatalytic activation of hydrogen peroxide (H Particularly, the Cr2AlC MAX phase as a member of M2AX phase, has a high oxidation resistance compared with that of other MAX phases like Ti3AlC2 or Ti2AlC. This makes it suitable for use in high-temperature applications MAX phases as newfangled nanolaminates are recently gaining interest in various applications owing to their desirable characteristics such as thermal and electrical conductivity, oxidation and corrosion resistance, strength and stiffness, elastic modulus, and lightweight nature 2O2) is based on the heightened generation of reactive species, particularly hydroxyl radicals , scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray spectroscopy (EDS), dot mapping, X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FT-IR), ultraviolet–visible diffuse reflectance spectroscopy (UV–Vis DRS), and Brunauer–Emmett–Teller (BET) analyses. Then, the sonocatalytic activation of H2O2 was studied using the Cr2AlC MAX phase for DMP degradation in water. The effect of the major operating parameters, such as catalyst dosage, oxidant concentration, pH, and pollutant initial concentration was investigated and the synergy factor was evaluated for the triple Cr2AlC/H2O2/US system based on degradation kinetics. Afterwards, the performance of the Cr2AlC/H2O2/US process for degrading diverse organic pollutants, including hydroxychloroquine (HCQ), rifampin (RIF), and acid blue 7 (AB7), was assessed under the identified optimal operating conditions. Furthermore, scavenging tests were implemented to recognize the contribution of radical and non-radical species within the degradation process. A plausible treatment mechanism was suggested according to gas chromatography-mass spectroscopy (GC–MS) and nuclear magnetic resonance (NMR) analyses. To the best of our knowledge, this study is the first attempt to determine the catalytic function of the Cr2AlC MAX phase for the degradation of organic contaminants using the Cr2AlC/H2O2/US method.In the present study, a Cr22.12O2, DMP (C10H10O4), sodium hydroxide (NaOH), hydrochloric acid (HCl), sodium nitrate (NaNO3), ethanol (C2H5OH), l-histidine (C8H13N3O4), tert-butanol (C4H10O), ethylenediaminetetraacetic acid , and o-phenylenediamine (C6H4(NH2)2) were supplied by Merck Co. (Germany). Additionally, HCQ (C18H26ClN3O), RIF (C43H58N4O12), and AB7 (C37H35N2Na O6S2) were supplied by Mofid Pharmaceutical Co. (Iran), Hakim Pharmaceutical Co. (Iran), and Shimi Boyakhsaz Co. (Iran), respectively.Graphite powder  was supplied by Aladdin Reagent Co., ltd. (China). Chromium and aluminum powders (99.95%) with a 200 mesh size were purchased from SCRC of National Medicine Group (China). Moreover, H2.22AlC MAX phase inspired by the literature 2AlC MAX phase powder The modified reactive sintering procedure was employed to prepare the Cr2.32AlC MAX phase powder was analyzed for structural identification using an X-ray diffractometer  including Cu Kα radiation . Furthermore, SEM micrographs, EDS spectrum, and dot mapping patterns were recorded using the Tescan Mira3 microscope (Czech Republic) for analyzing the surface morphology and elemental characterization, respectively. Additionally, HRTEM images were obtained by a JEM-2100 Plus electron microscope  to further assess the surface morphology. The surface compound and oxidation states of the prepared MAX phase were studied by XPS carried out by a Thermo Scientific Escalab 250Xi Plus XPS spectrometer (UK). Surface functional groups were identified by FT-IR spectrum recorded on a Bruker Tensor 27 spectrometer (Germany). The N2 adsorption/desorption analysis was performed using Belsorp Mini II  to determine the specific surface area of the MAX phase by BET method. UV–Vis DRS was acquired to investigate the optical properties of the Cr2AlC MAX phase with an UV–Vis spectrophotometer  while utilizing the reflectance standard of barium sulfate. The 1H NMR signals of DMP and its degradation intermediates were determined by a Bruker-Spectrospin 400 MHz UltraShieldTM NMR spectrometer. Agilent 6890 gas chromatography supported by an Agilent 5973 mass spectrometer  was applied to analyze the intermediates formed during the oxidation of DMP. Total organic carbon (TOC) measurements were carried out by Shimadzu TOC analyzer (Japan).The Cr2.42AlC MAX phase powder (0.75 g/L) was added to DMP solution  in a 250 mL Pyrex glass flask. The H2O2 addition at the first step can affect the degradation efficiency sharply owing to its effect on the hydroxyl radicals production. To observe the effect of H2O2 addition in every step it was decided to add H2O2 at 10 min intervals. Experiments were carried out at the original pH of the DMP solution In this study, DMP degradation was implemented in an ultrasonic bath  operating at a power and frequency of 150 W and 36 kHz, respectively, at 20℃. In a typical run, a defined dose of Cr2.5pzc of the Cr2AlC MAX phase using the pH drift method 3 solutions with a concentration of 0.01 mol/L were prepared and their pH levels were adjusted to 2, 4, 6, 8, 10 using HCl and NaOH solutions. Then, 0.075 g of the catalyst was added to the solutions and all obtained suspensions were stirred in a shaker at the speed of 200 rpm and the temperature of 20 °C for 24 h. Ultimately, the final pH of the solution was plotted versus the initial pH. The point of zero charge (pHpzc) of the Cr2AlC MAX phase was determined from the point at which the initial pH equals the final pH.To determine pH33.12AlC MAX phase is presented in 2AlC MAX phase. Moreover, the observed peaks at 2θ = 14.1°, 28.0°, 36.4°, 37.0°, 42.2°, 46.4°, 51.5°, 57.0°, 63.2°, 65.4°, and 77.3° corresponded to the (0 0 2), (0 0 4), (1 0 0), (1 0 1), (1 0 3), (1 0 4), (1 0 5), (1 0 6), (1 0 7), (1 1 0), and (1 0 9) planes of the so-synthesized sample, respectively, which demonstrate the hexagonal structure of the prepared MAX phase The XRD pattern of the as-prepared Cr2AlC MAX phase  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg>O, and C—H bonds, respectively, which are formed due to the oxidation of end-groups −1 was related to the –OH stretching bond, confirming the presence of water molecules on the MAX phase surface FT-IR analysis was conducted to demonstrate the surface functional groups in CrAX phase b. The pe2 adsorption/desorption isotherm is presented in 2AlC MAX phase was a nonporous material. The specific surface area (SBET) of the Cr2AlC MAX phase was calculated as 0.75326 m2/g by the multipoint BET method 2AlC MAX phase were studied via the UV–Vis DRS method .e is the energy of an electron vs energy of hydrogen (4.5 eV), X corresponds to the absolute electronegativity, and EVB and ECB represent the valence band and conduction band potentials, respectively. Further, a, b, and c refer to the amount of each constituent in the sample 2AlC MAX phase were calculated to be −0.01 eV and −5.33 eV, respectively.The NS method d. The Ku2AlC MAX phase can be observed in SEM image  demonstrated that these systems could not sufficiently remove DMP 2AlC/H2O2, H2O2/US, and Cr2AlC/US processes displayed removal efficiencies of 33.9%, 37.5%, and 44.6%, respectively. The coupled systems could thus enhance the treatment of organic contaminants 2AlC/H2O2/US process showed desired performance for DMP treatment (69.1%). Each degradation processes in different systems were repeated in the same operating condition three times and their error bars were calculated. As can be seen in 2) values higher than 0.9 (2AlC/H2O2/US process was obtained 2.3 using Eq. app)  c [19].(62AlC/H2O2/US process could be explained through the possible reactions mentioned in Eqs. (7–11) Improved DMP removal for the Cr2AlC MAX phase under the US irradiation generated the electron–hole pairs; as indicated in Eq. •2O by the formed holes. Furthermore, the produced electrons could react with H2O2 and O2, thereby leading to the production of hydroxyl and superoxide radicals, respectively (Eqs. (9 and 10)). Additionally, the reaction between superoxide radicals and water molecules produced singlet oxygen  2AlC/H2O2/US process for industrial wastewater treatment.The removal of different organic pollutants, such as HCQ, RIF, and AB7, was investigated via the Cr2AlC/H2O2/US process was evaluated using various scavengers, including tert-butanol (TBA), l-histidine, and EDTA which were applied to suppress •12AlC/H2O2/US process was reported in •l-histidine, indicating the role of 1l-histidine scavenger compared with TBA revealed the high contribution of 1••max = 419 nm) (Eq. (12)), was distinguished using the UV–Vis spectrophotometer. The results shown in •Scavengers are widely used to quench the free active species that participate in the removal of organic pollutants. Their role in the Cr2O2 could be converted to 1•Consequently, the results confirmed that both radical and nonradical species play a role in DMP degradation . O2 and 3.52 and H2O. Additionally, some inorganic compounds may have been produced during the removal process, but not detected because of their limited retention time in the GC–MS. Moreover, TOC removal of 53.3% was obtained for pollutant solution containing 15 mg/L of DMP and 0.75 g/L catalysts within 360 min of the reaction time, confirming the mineralization of DMP by the triple Cr2AlC/H2O2/US process.The GC–MS method was implemented to identify possible pathways for DMP degradation. The molecular structure and analytical properties of identified intermediates are presented in 2AlC/H2O2/US system 1H NMR analysis, a sample was dried (50 °C) and held under vacuum, and 1H NMR spectra were recorded in D2O as a solvent. 1H NMR spectra for the control and degraded DMP, respectively. The signals of aromatic protons for the control DMP were observed in the 6–8 ppm region. After 100% degradation of DMP during a long time process, significant peaks for the aromatic region disappeared, thereby confirming the destruction of the stable aromatic ring. Proton peaks in the shielded region remained in the aliphatic region. This demonstrates the existence of small-chained hydrocarbons produced during the degradation procedure, which are well-supported with the GC–MS results.The NMR investigation also confirmed the degradation of DMP via the triple Cr42AlC MAX phase powder was prepared by the reactive sintering method. The desired characteristics of the hexagonal MAX phase, with high purity, were confirmed by diverse characterization analyses. The XRD pattern confirmed the high crystallinity of the prepared MAX phase. Furthermore, compacted layered morphology of the MAX phase was observed in SEM and HRTEM images. Surface functional groups, oxidation states, and elemental compositions of the MAX phase were assessed by FT-IR, XPS, and EDS analyses. The catalytic activation of H2O2 by the MAX phase under US waves was evaluated for degrading 15 mg/L DMP. Under the obtained desired conditions, the activation of 1 mmol/L H2O2 by 0.75 g/L Cr2AlC MAX phase under US irradiation within 120 min showed the significant removal of organic pollutants, including DMP (69.1%), HCQ (100%), RIF (94.5%), and AB7 (91.5%). The scavenging tests revealed that •112AlC/H2O2/US process is promising for the degradation of different contaminants in the field of water and wastewater treatment.In this investigation, CrMonireh Alimohamadi: Investigation, Visualization, Writing – original draft. Alireza Khataee: Supervision, Writing – review & editing. Samira Arefi-Oskoui: Conceptualization, Writing – review & editing. Behrouz Vahid: Writing – review & editing. Yasin Orooji: Writing – review & editing. Yeojoon Yoon: Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Transforming growth factor β2 (TGF-β2), also known as glioma-derived T-cell suppressor factor, is associated with the impairment of tumor immune surveillance. Therefore, blocking TGF-β2 signaling probably be a feasible strategy to develop a novel type of adjuvant for glioma vaccines to enhance antitumor immunity.A TGF-β2 inhibitory oligodeoxynucleotide, TIO3, was designed with sequences complementary to the 3' untranslated region of TGF-β2 mRNA. The expression of TGF-β2 and MHC-I was detected by qPCR, western and flow cytometry in vitro. All the percentage and activation of immune cells were detected by flow cytometry. Subsequently, TIO3 was formulated with Glioma cell lysate (TCL) and investigated for its antitumor effects in GL261 murine glioma prophylactic and therapeutic models.TIO3 could efficiently downregulate the expression of TGF-β2 while increase the MHC-I's expression in GL261 and U251 glioma cells in vitro. Meanwhile, TIO3 was detected in mice CD4+ T, CD8+ T, B and Ly6G+ cells from lymph nodes after 24 hours incubation. Moreover, TCL+TIO3 vaccination significantly prolonged the survival of primary glioma-bearing mice and protected these mice from glioma re-challenge in vivo. Mechanistically, TCL+TIO3 formulation strongly evoke the antitumor immune responses. 1) TCL+TIO3 significantly increased the composition of CD4+ and CD8+ T cells from draining lymph nodes while promoted their IFN-γ production and reduced the expression of TGF-β2 and PD1. 2) TCL+TIO3 activated the NK cells with the elevation of CD69 or NKG2D expression and PD1 reduction. 3) TCL+TIO3 increased the glioma-specific lysis CTLs from spleen. 4) TCL+TIO3 downregulated PD-L1 expression in glioma tissues and in Ly6G+ cells among glioma-infiltrating immune cells.TIO3 is a promising adjuvant for enhancing TCL-based vaccines to produce a more vigorous and long-lasting antitumor response by interfering with TGF-β2 expression. Currently, the most common therapeutic treatments include surgery, radiation, and chemotherapy; however, the median survival of patients with glioma is just 15 months, emphasizing the need for novel therapeutic approaches .Tumor vaccines are a promising form of immunotherapy that can activate the immune system to combat tumor growth. Over the last two decades, tumor vaccines have attracted increasing attention for their application in tumor prevention and treatments. Remarkably, Provenge, as the first tumor vaccine for the treatment of advanced prostate cancer, was approved by the U.S. Food and Drug Administration (FDA) in April 2010 . Moreove+ T cells into conventional CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) by binding to TβRIII  and antisense transgenes have been used extensively in mediating the knockdown of TGF-β2 expression in cells. An oncolytic adenovirus expressing a TGF-β2 shRNA and granulocyte-macrophage colony stimulating factor (GM-CSF) can significantly enhance the anti-melanoma efficacy of melanoma antigen Melan-A (MART1) in mice . Similarin situ glioma. Furthermore, TIO3 activated T and NK cells in the lymph nodes and inhibited PD1 and PD-L1 expression in mice bearing GL261 glioma. These data support the impact of TIO3 on antitumor immunity, allowing the advancement of TIO3 as a promising adjuvant for glioma vaccines.In a previous study, we designed a TGF-β2 inhibitory oligodeoxynucleotide (TIO3) with sequences complementary to the 3’ UTRs of TGF-β2 mRNA and demonstrated that TIO3 could enhance multiple microbial vaccines to induce strong and persistent antibody responses by suppressing TGF-β2 expression in immune cells . In the 22.1Two ODNs fully phosphonothioate-modified were used in our study: TIO3, 5’-TTACCACTAGAGCACCACA-3’ (19 nt); control ODN (cODN), 5’-ACTTACTCGAGAACCCCAA-3’ (19 nt). All ODNs, including 3’ Alexa Fluor 488-conjugated TIO3 and 3’ Cy3-conjugated TIO3, were synthesized by Sangon Biotech .2.22 incubator at 37°C. In this study, GL261 cells were used for in vitro cell culture experiments and in vivo tumor challenge experiments. For in vivo tumor challenge experiments, GL261 cells were digested with trypsin-EDTA (0.25%), harvested, centrifuged for 5 min at 300 g, resuspended and then adjusted to a concentration of 5 × 106 cells/mL in serum-free RPMI-1640 for inoculating the mice with 2× 104 cells through an intracranial injection.GL261 cells (a murine glioma cell line) and U251 cells (a human glioblastoma cell line) were kindly provided by the Transfusion Research Institute of the Academy of Military Sciences, Beijing, China. Cells were maintained in RPMI-1640 or DMEM  supplemented with 10% heat-inactivated fetal bovine serum , 100 U/mL penicillin and 100 mg/mL streptomycin in a humidified 5% CO2.3Six-week-old female C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. . Experimental procedures and manipulation involving mice were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Scientific Investigation Board of Science & Technology of Jilin Province and the ethics committee of the College of Basic Medical Sciences of Jilin University (number 2019-01).2.4in vitro, then 106 cells were intraperitoneally inoculated into the mice. Glioma mass was formed in about 20 days, then the mice was euthanized to isolate glioma tissue for preparing glioma cell lysate. To acquire high-quality glioma cell lysates, we first minced the glioma tissues with scissors, resuspended in 0.85% pathogen-free saline buffer and then disrupted in a glass homogenizer followed by filtration with a 40 μm nylon cell strainer. Cells were adjusted with the concentration at 107 cells/mL in saline and then disrupted by high pressure homogenizer at 800 Bar 5 cycles. After centrifugation at 10,000 g for 10 mins, the cell supernatant was separated and passed through a 0.2 μm filter. At last, the tumor cell lysate was conducted freeze-dried for storage at -80 °C (The tumor cell lysates were prepared as described previously . Brieflyt -80 °C . TCLs wet -80 °C .2.52 (3 mM), dNTP mix (0.5 μM), 1 μl M-MLV, 1 µg of total RNA, oligo(dT)15 primer (0.5 µg) and reaction buffer. The RT reaction was incubated at 42°C for 1 h, and the RT enzyme was inactivated by incubation at 70°C for 15 min.GL261 and U251 cells were prepared for isolating total RNA with TRIzol™ Reagent . A UV2800 ultraviolet spectrophotometer was used to analyze the RNA concentration and purity. M-MLV reverse transcriptase  was used for reverse transcription reactions. Briefly, the RT reaction mixture, in a volume of 20 µL, contained MgCl2.6GAPDH, the most commonly used gene for normalization of qPCR data in glioma research, was used  relative to the control.To quantify the mRNA expression of TGF-β2 and MHC-I, we used primers synthesized by Sangon Biotech , which are listed in was used . Relativ2.7All anti-mouse or anti-human monoclonal antibodies (mAbs) in this study were purchased from BD Biosciences .®488-conjugated donkey-anti-goat IgG secondary antibody (ab150129) diluted with PBS at 1:1000 for 40 min and then analyzed by flow cytometry .To analyze the surface TGF-β2 (sTGF-β2) and MHC-I expression on GL261 cells, GL261 cells were cultured with PMA (100 μg/mL), PMA+TIO3 (10 μg/mL), or PMA+cODN (10 μg/mL) for 24 h and then stained with an anti-MHC-I mAb  or goat-anti-mouse TGF-β2 antibody  for approximately 40 minutes at 4°C in the dark. After being washed twice with FACS buffer , the cells were incubated with the Alexa Fluor+ T cells and CD8+ T cells, the mice were intramuscularly immunized with TCL, TCL+TIO3, TCL+cODN or PBS at the cervical lymph node area on Day 0 and Day 10. On Day 14, the lymphocytes from drainage lymph nodes were isolated. The cells were first stained with PE-labeled CD4 mAb and goat-anti-mouse TGF-β2 antibody or with PE-labeled CD8 mAb and goat-anti-mouse TGF-β2 antibody for 40 minutes on ice in the dark. After being washed twice, the cells were incubated with Alexa Fluor®488-conjugated donkey-anti-goat IgG secondary antibody diluted with PBS at 1:1000 for 40 min. For intracellular staining, cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% saponin before staining with APC-labeled IFN-γ mAb (554413), followed by flow cytometry analysis. Similarly, to investigate the effect of TIO3 on the expression of CD69, NKG2D and PD1 in NK cells and CD4+ and CD8+ T cells, lymphocytes isolated from immunized mice were first stained with APC-conjugated anti-NK1.1 mAb (561117), PE-conjugated anti-CD4 mAb (553048), PE-conjugated anti-CD8 mAb (553033), PE-conjugated anti-CD314 mAb , FITC-labeled CD279 (PD1) and/or FITC-conjugated anti-CD69 mAb (553236) for 40 minutes on ice in the dark and then analyzed by BD FACS Calibur with Cell Quest software .To analyze the effect of TIO3 on the expression of TGF-β2 and IFN-γ in CD44 GL261 cells intracranially (i.c.). On Day 28, draining lymph nodes were isolated and minced into a single-cell suspension, and the lymphocytes were stained with PE-labeled anti-CD4 (553048) or PE-labeled anti-CD8 mAb (553033) and FITC-labeled CD69 mAb (553236) at 4°C in the dark for 30 min. After being washed twice, the lymphocytes were analyzed by flow cytometry.To explore the effect of TIO3 on glioma-specific T cells in tumor-bearing mice, C57BL/6 mice were intramuscularly immunized with 100 μL of TCL (100 μg TCL in PBS), TCL + TIO3 (100 μg TCL plus 10 μg TIO3 in PBS), TCL+ cODN (100 μg TCL plus 10 μg control ODN in PBS), or PBS on Day 0 and Day 10. On Day 14, the mice were challenged with 2×102.84 per well per 100 μL) cells at a ratio of 100:1, 50:1 or 25:1 for 8 h at 37 °C in a 5% CO2 humidified atmosphere. A methylthiazolyldiphenyl-tetrazolium bromide assay was conducted to determine the CTL activity.The mice were intramuscularly injected with TCL, TCL+TIO3, TCL+cODN or PBS at the cervical lymph node area on Day 0 and Day 10. On Day 14, the spleen was isolated, and then the splenocytes were used to perform the cytotoxicity assay. Splenocytes were cocultured with GL261 (1 × 102.96 cells/ml in RPMI 1640 supplemented with 10% FBS. The cells were cultured with Cy3-labeled-TIO3 (10 μg/ml) in a 5% CO2 incubator for 24 h at 37°C. After washing twice, the cells were fixed with 4% paraformaldehyde for 10 min at 37°C. The cells were washed twice, permeabilized with 0.1% Triton X-100 at 4°C for 10 min, and then blocked with 5% BSA at 37°C for 1 h. The cells were incubated with PE-conjugated CD4 or CD8 mAb at a dilution of 1:50 for 30 min at 4°C. Unbound antibody was washed and removed with PBS. The stained cells were visualized under a laser scanning confocal microscope (Olympus FV1000) and analyzed by AndorIQ2 software.To observe whether TIO3 could enter T cells, lymphocytes isolated from naive mice were seeded on poly-L-lysine-coated 24-well plates at a concentration of 2×102.104 GL261 cells at 2 mm to the right of the bregma and 3 mm deep using a stereotaxic instrument (Kopf Instruments)  were intramuscularly (i.m.) injected with TCL (100 μg/ml), TCL (100 μg/ml) + TIO3 (10 μg/ml), TCL (100 μg/ml) + cODN (10 μg/ml), or PBS in the neck draining lymph node of the mice on Day 0 and Day 10. On Day 14, each mouse was anesthetized and intracranially (i.c.) injected with 2×10ruments) . Any ani4 GL261 cells on Day 0 and then i.m. injected with TCL, TCL+TIO3, TCL+cODN or PBS on Days 1, 8, 15 and 22. The mice were monitored daily. In each group, 3 mice were euthanized on Day 18, and the brain tumor tissues were separated and fixed in formalin solution for 24 hours, embedded in paraffin, sectioned (4 μm), and stained with hematoxylin-eosin (H&E) for tumor histopathological analysis under a microscope.To explore the effect of TIO3 as a therapeutic glioma vaccine adjuvant in brain glioma in situ, 44 female C57BL/6 mice were split into four groups (n=11), i.c. injected with 2×102.11To isolate the infiltrating immune cells in brain glioma tissues, brain tissue pieces were mechanically processed using collagenase. To digest with collagenase, we first transferred glioma tissue pieces (1 g) to a P60 dish and then incubated them with collagenase IA  at 37°C for 30 minutes. A 5 ml pipette was used to resuspend the sample, which was then transferred into a 15 ml tube and incubated for another 30 minutes at 37°C . This st2.12To explore the effect of TIO3 on TGF-β2 and PD-L1 expression in glioma tissues, we first i.m. injected the mice with TCL, TCL+TIO3, TCL+cODN, or PBS on Days 0 and Day 10 and then challenged the mice with GL261 cells intracranially on Day 14. On Day 28, the glioma was isolated from the mice and used to assess TGF-β2 and PD-L1 expression by western blotting. The glioma cells were cut into small pieces by scissors first and then digested with collagenase for 1 h. After centrifugation, the cells were lysed with ice-cold radioimmunoprecipitation (RIPA) buffer  containing the protease inhibitor PMSF at 4°C for 30 min. Then, the cell lysates were centrifuged at 14,500 × g for 10 min at 4°C. The cell supernatant was collected and quantified with a BCA protein assay kit  followed by separation on 12% SDS−PAGE. After running the gel at 120 V for 1.5 h, we transferred the protein to polyvinylidene difluoride (PVDF) membranes  at 120 V for 1 h at 4°C. Then, the membranes were blocked with TBST containing 5% nonfat dried milk, shaken at room temperature (22-25°C) for 2 h, and probed with an anti-TGF-β2 antibody , anti-PD-L1 antibody  or anti-GAPDH antibody  overnight at 4°C. After being washed with TBST  twice for 10 min intervals, the membranes were incubated with the secondary antibody HRP-conjugated anti-goat IgG  at 25°C for 1 h. After washing three times, substrate was added to the membrane, and the target protein bands were visualized by Hyperfilm ECL (Amersham Biosciences). The intensity of the immunoreactive bands was determined by a densitometric analysis program .2.13post hoc test. The Kaplan−Meier method was used to evaluate the survival curve of mice, and the log-rank test was used for comparison. SPSS 19.0 computer software was used for statistical analysis. The data come from four or more independent experimental groups. Data are expressed as the mean ± SEM. Differences in tumor size among the various groups were determined by repeated-measures analysis of variance (ANOVA).A Student’s 2-tailed unpaired t test was used to determine the statistical significance of differences between two groups. For multiple sets of data analysis, 1- or 2-way ANOVA was used, followed by Scheffé’s 33.1p<0.05) (p<0.05)  cells were isolated and incubated with Cy3- or Alexa-488-labeled TIO3 for 24 h and then stained with FITC-labeled mAbs against CD4, CD8, or V450-labeled mAbs against Ly6G, followed by counterstaining with DAPI, a blue-fluorescent DNA dye for staining the nucleus. Confocal microscopy revealed that both TIO3 and cODN could enter CD4ne cells .3.3To explore whether TIO3 could be formulated as an adjuvant with glioma TCL to induce antitumor immunity, mice were immunized twice with PBS, TCL, TCL+TIO3 or TCL+cODN (TCL plus control ODN) on Day 0 and Day 10. On Day 14, the mice were intracranially inoculated with GL261 cells. We found that the tumor-bearing mice treated with TCL or PBS exhibited symptoms, including weight loss, hunched back, irregular breathing, prostration, paresis, and convulsions. More excitingly, none of the eight mice in the TCL+TIO3 group died within 180 days  could induce the generation of effective T cells in mice, C57BL/6 mice were intramuscularly injected with TCL, TCL+TIO3 or TCL+cODN or PBS on Day 0 and Day 10. One day after the second immunization, the lymphocytes were isolated, and the percentage and activation of T cells were immediately determined by flow cytometry . To verify the effect on cytotoxic T cells induced by TCL+TIO3, splenocytes from each group were isolated and cocultured with GL261 cells for 8 h at effector/target ratios of 100:1, 50:1 and 25:1. The results clearly revealed that TCL+TIO3 induced a stronger cytotoxicity against GL261 tumor cells than TCL and PBS in vitro (p <0.001)  in glioma-bearing mice, mice were intramuscularly injected with TCL, TCL+TIO3, TCL+cODN or PBS in the neck on Days 0 and Days 10, and then the mice were i.c. injected with 2×10observed . Consistobserved . Consistobserved . Consistobserved . Consistp=0.0004) (+ or NKG2D+ NK cells in lymphocytes than TCL (p<0.01)  . Our res3.6+ T, CD8+ T and NK cells by flow cytometry analysis  are considered to be important antitumor effector cells, and the accumulation of activated lymphocytes at tumor sites is considered a good indicator of the regulation of tumor development and growth. As reported, TILs are mainly composed of T cells, B cells, and NK cells . Howevers system . Our hiss system .+ T cells, CD8+ T cells and NK cells and inhibited PD-L1 expression in glioma tissues and tumor-infiltrating neutrophils. Immune checkpoint signals such as PD1 and PD-L1 are key modulators of the antitumor T-cell immune response. In a tumor state, the interaction of PD-L1 on tumor cells with PD1 on T cells can inhibit the cytotoxic ability of T cells against tumor cells (in vitro and in vivo (In addition to the activation of antitumor effector cells, TIO3 also reduced the expression of PD1 on CD4 in vivo . Additio in vivo . Thus, i in vivo . Similarin situ prophylactic and therapeutic models. Interestingly, we found that TIO3 also had a monotherapeutic effect on the xenograft and in situ therapeutic mouse models of glioma (+ T cells and NK cells (Overall, TIO3 as a vaccine adjuvant activated antitumor effector T cells and NK cells by inhibiting the immunosuppressive cytokine TGF-β2 and PD1 and PD-L1 expression on immune cells and tumor cells. Importantly, TIO3 formulated with TCL effectively prolonged the survival of glioma-bearing mice in both f glioma S5, but NK cells . TherefoThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by The ethics committee of the College of Basic Medical Sciences of Jilin University.MY and YLY contributed to the conception and design of the study and wrote the manuscript. LT, XS, LY performed experiments, analyzed data, and performed the statistical analysis. ZW, YPY, LW, XZ and PZ contributed to manuscript revision, read and approved the submitted version. All authors contributed to the article and approved the submitted version.


In silico computational modeling, drug affinity responsive target stability (DARTS), and fluorescence polarization (FP) assays altogether determined that 323–1 and 323–2 directly target the STAT3 SH2 domain and inhibited both phosphorylated and non-phosphorylated STAT3 dimerization. Computational docking predicted that compound 323s bind to three subpockets of the STAT3 SH2 domain. The 323s inhibition of STAT3 dimerization was more potent than the commercial STAT3 SH2 domain inhibitor S3I-201 in the co-immunoprecipitation assay, correlating with computational docking data. The fluorescence polarization assay further confirmed that the compound 323s target the STAT3 SH2 domain by competitively abrogating the interaction between STAT3 and the SH2-binding peptide GpYLPQTV. Compared with S3I-201, the 323 compounds exhibited stronger inhibition of STAT3 and reduced the level of IL-6-stimulated phosphorylation of STAT3 (Tyr705) in LNCaP cells over the phosphorylation of STAT1 (Tyr701) induced by IFN-ɣ in PC3 cells or the phosphorylation of STAT1 (Ser727) in DU145 cells. Both compounds downregulated STAT3 target genes MCL1 and cyclin D1. Thus, the two compounds are promising lead compounds for the treatment of cancers with hyper-activated STAT3.Our drug discovery model has identified two novel STAT3 SH2 domain inhibitors 323–1 and 323–2 (delavatine A stereoisomers) in a series of experiments.  Junaid etal. found activation of STAT3 in 67% of bone and 77% of lymph node metastases of prostate cancer patients  and integrates with other signaling pathways to activate the androgen receptor (AR) pathway. STAT3 may promote stem-like cells and the epithelial-to-mesenchymal transition (EMT) and interactions between tumor cells and the microenvironment . Liu C. patients . Don-Donpatients . All thepatients  and may STAT3 was originally described in 1993 as a transcription factor in IL-6-stimulated human hepatoma (HepG2) cells . The fulCytokines  or growth factors  bind to their corresponding cell surface receptors . These bThe function of STAT3 relies significantly on its SH2 domain, which promotes STAT3 homo- or hetero-dimerization, protein–protein interactions, and nuclear translocation of the STAT3 dimers needed for transcription. Thus, the STAT3 SH2 domain mediates the phosphorylation and dimerization of STAT3 due to its association between STAT3 monomers and phospho-tyrosine motifs within relevant receptors . Due to [de]isoquinoline skeleton, was first reported from the medicinal plant Incarvillea delavayi, with subsequently completed total synthesis of -delavatine A and its chiral isomer -delavatine A (named as compound 323–1 or 323–2) -delavatine A (named as compound 323–1 or 323–1), a natural product with novel cyclopentar 323–2) . Herein,Cryptotanshinone and IL-6 were purchased from . S3I-201 was bought from Thermo Fisher Scientific .Human prostate cancer cell lines LNCaP and 22Rv1 were purchased from the American Type Culture Collection  and cultured in RPMI 1640 medium with 10% FCS. HEK 293T and DU145 cells (ATCC) were cultured in a DMEM with 10% FCS. EPT3M1-STAT3  were culLNCaP, 22Rv1, and DU145 cells were seeded in 96-well plates for 24 h, followed by treatment with different doses of drugs for 4 days. The alamarBlue assay was performed by adding 10 µL/well alamarBlue cell viability reagent  for 4 h, and the absorbance at 570 nm was thereafter recorded using 600 nm as a reference wavelength by using the BioTek Synergy H1 plate reader . The absorbance data of each group were normalized to the data of the DMSO-treated group to get the relative cell viability. Data were analyzed by Prism software-log (inhibitor) vs. response-variable slope (four parameters) with the formula Y = bottom + (top-bottom)/(1 + 10^((LogIC50-X) *HillSlope)), X: log of dose or concentration, Y: response, decreasing as X increases, top and bottom: plateaus in same units as Y.HEK 293T cells were transiently transfected with the Cignal STAT3 reporter  using lipofectamine 3000 transfection reagent  for 24 h, following treatment with 20 ng/ml IL-6 and the indicated concentrations of 323–1, 323–2, S3I-201, or cryptotanshinone. Luciferase activity was measured by using the Dual-Luciferase assay kit  using a luminescence microplate reader . Values were normalized to Renilla luciferase activity of the DMSO vehicle.Franklin Lakes, New Jersey, US).DU145 cells were seeded in 6-well plates for 24 h, followed by treatment with various drugs for 72 h. Apoptosis assay was conducted by using the CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit . After removing the media from the cells, 1 µL of CellEvent™ Caspase-3/7 Green Detection Reagent was added to 1 ml of each cell sample to make a final concentration of 500 nM and incubated at RT for 1 h. At the final 5 min of staining, 1 μL of the 1 mM SYTOX™ AADvanced™ dead cell stain solution was added to the samples to a final concentration of 1 μM. The samples were analyzed by using the BD LSRFortessa™ cell analyzer (g for 10 min at 4°C). Lysates were diluted to the same final volume and proteolyzed in TNC buffer . Then, 200 µM 323–1 or 200 µM 323–2 or the same volume of DMSO was added and incubated for 1 h at room temperature (RT). A measure of 1.25 mg/ml of pronase solution was diluted serially using 1x TNC buffer to generate 1:300, 1:1,000, and 1:3,000 pronase stock aliquots. Pronase was added into both the DMSO and drug groups and incubated for 30 min at RT. Digestion was stopped by adding 4X loading buffer and heating to 90°C for 10 min immediately prior to the Western blot assay, according to publications  containing protease  and phosphatase inhibitors  and centrifuged . Images were captured using the Leica DMRBE microscope or Cytation5 Cell Imaging Multi-Mode Reader.EPT3M1-STAT3 cells (grown on coverslips) were treated with DMSO, 20 µM 323–1 or 323–2, 100 µM S3I-201, or 5 µM cryptotanshinone for 24 h. Cells were fixed with 4% paraformaldehyde, washed with PBS, and permeabilized with cold methanol. Primary and secondary antibodies were described, cat. no. and dilutions , and moDU145 cells were seeded and treated with 5–20 µM 323–1 or 323–2 or 100 µM S3I-201 for 24 h. Nuclear extraction was performed according to the STAT Family Transcription Factor Assay Kit  .The levels of expressions of phosphorylated STAT3 , pSTAT1 , and pSTAT1 (S727) proteins were determined by Western blotting, following the procedures described . The fol6 HEK 293T cells were transfected with 10 µg/10 cm dish FLAG-STAT3 and 10 µg/10 cm dish HA-STAT3 plasmids for 24 h, followed by treatment with 20 µM 323–1 or 323–2 and 100 µM S3I-201 for 4 or 24 h Thereafter, 293T cells were collected in the medium and centrifuged at 800 g × 5 min and then washed with cold PBS twice before adding 500 μL Pierce™ IP lysis buffer , containing protease and phosphatase inhibitor cocktail without DTT  into the cells. Cell lysates were passed several times through a 271⁄2-gauge needle to disrupt the nuclei. Then, 1–2 mg extracts were added with a pre-cleaned 50 µL slurry of Pierce™ DYKDDDDK magnetic agarose , and then, supernatants were collected with magnet and immunoprecipitated (IP) at RT on a rotator for 30 min. Beads were boiled at 95°C for 5 min followed by the addition of 100 μL of 1x non-reducing sample buffer  into beads. The supernatants were collected with a magnet and then added 5 μL of 1M DTT  into samples before proceeding with immunoblotting.For this procedure, 7.5 × 10The computational docking of 323s to three-dimensional crystal structures of STAT3, including the phosphorylated (PDB entry 1BG1) and the non-phosphorylated (PDB entry 3CWG) structure, was utilized by the molecular docking tool Maestro 9.0 Glide, as illustrated previously . The lowdrc. Ki was determined from the resulting IC50 using the Cheng–Prusoff equation and the Kd for fluorochrome-labeled peptide binding to STAT3, Ki = IC50/(([L])/Kd) +1. The assays were performed at the high-throughput chemical biology screening platform  Centre for Molecular Medicine Norway (NCMM), University of Oslo.The FP assay was conducted as described in 6 cells were subcutaneously injected into the right flank. Tumor volume was calculated using the following formula: V = (L × W2)/2, where L is the length and W is the width of the tumor nodules measured with vernier calipers. Once the volume of the tumors reached 75–100 mm3, the mice were randomly divided into three groups (n = 6). The mice were treated daily for 3 weeks with IP injections of either vehicle (olive oil) or 323–2 (20 mg/kg or 40 mg/kg in the vehicle). Body weights and tumor volumes were measured before each drug injection. After the 22nd day, the mice were euthanized, and the tumors were isolated, weighed, and photographed.Four-week-old male BALB/c-nu nude mice (15–20 g) were obtained from the Shanghai BiKai Laboratory Animal Co., Ltd. . The animals were maintained under specific pathogen-free conditions with food and water supplied ad libitum in the Laboratory Animal Center of the Second Military Medical University, Shanghai. All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Committee on the Ethics of Animal Experiments of the Second Military Medical University, China. LNCaP cells were harvested and resuspended in PBS. A total of 5 × 10p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.Significance in groups was determined by using one-way ANOVA multiple comparison. *1H NMR, 13C NMR, and chiral HPLC chromatography (see c = 0.05 in CHCl3) and -6.7 (c = 0.05 in CHCl3), respectively -delavatine A (compound 323–1) and -delavatine A (compound 323–2)  were fouaphy see  with opty in the . As showHerein, we sought to determine whether 323–1 and 323–2 were able to suppress STAT3 phosphorylation and activation. First, to confirm the inhibitory effect of 323s on STAT3 activity, luciferase assays were carried out using a STAT3-luciferase reporter vector in 293T cells, following treatment with 323–1, 323–2 or the commercial STAT3 inhibitors S3I-201 and cryptotanshinone in the presence of IL-6 for 24 h . 323–1 aAberrant STAT3 regulates the expression of downstream target genes involved in anti-apoptosis , cell cycle (cyclin D1 and c-Myc), angiogenesis (VEGF and HIF1α), invasion and metastasis , and the inhibition of host immune surveillance . To furtSeveral available STAT3 inhibitors target the DBD or SH2 domains of STAT3 or inhibit activating phosphorylation of Tyr705. Due to the highly conserved region of the SH2 domain, such as in Src, compared with other proteins and the insufficient capability of the available STAT3 SH2 domain inhibitors , the intTo directly address if compounds 323–1 and 323–2 target STAT3, the DARTS assay was used, based on the observation that proteins bound by drugs may be more stable against protease degradation. In Nuclear translocation of the STAT3 dimers mediates STAT3 transcriptional activity. In order to test whether compound 323s are STAT3 dimerization inhibitors, 293T cells were transiently transfected with HA-tagged STAT3 and FLAG-tagged STAT3 expression plasmids, then stimulated with 323s or S3I-201 for 24 h, and next immunoprecipitated with the anti-FLAG antibody to pull down the STAT3-associated proteins, which were finally subjected to Western blot with anti-HA, anti-FLAG, or anti-β-actin antibodies. As exemplified in in silico computational screening assay by docking compounds into the STAT3 SH2 domain, such as S3I-201 (The STAT3 SH2 domain (residues 583–688) contains three sub-pockets: PY, PY+1, and PY + X. The STAT3 SH2 domain facilitates the phosphorylation and dimerization of STAT3 due to its association between STAT3 monomers and phospho-tyrosine motifs within relevant receptors . To date S3I-201 .in silico computational modeling and the fluorescence polarization (FP) assay. Computational docking and molecular dynamics simulation identified potential binding sites for 323–1 and 323–2 in the STAT3 SH2D . The nitrogen of the isoquinoline group in 323–1 forms a hydrogen bond with Glu594, the carbonyl oxygen on the formaldehyde group faces the pY binding pocket and forms a hydrogen bond with Arg609, and Arg609 can form a direct polarity with phosphotyrosine 705. Arg609 can inhibit the binding of SH2 to phosphotyrosine 705. The small-molecule skeleton cyclopenta [de] isoquinoline faces the pY-X binding pocket and interacts hydrophobically with adjacent amino acid residues . Similarly, compound 323–2 forms a bond with PY, PY+1, and PY-X pockets  in the STAT3 SH2 domain (To confirm that 323s bind to STAT3, we used AT3 SH2D . In the 2 domain .de] isoquinoline of 323s associated with the pY-X binding pocket bound by tetrahydro cyclopentane and formed hydrophobic interactions lined by residues Phe588, IIe589, Ser590, and Val637. These data suggest that 323s are STAT3 SH2 domain inhibitors by disrupting STAT3 dimerization and further supporting the results of the DARTS assay.It is reported that non-phosphorylated STAT3 may also form dimers, enter the nucleus from the cytoplasm to bind to DNA, and activate transcription . In dockin silico predictions were further examined by competition binding FP assays, which detected the displacement by drugs of the binding of labeled phosphopeptide, 5-FLU-GpYLPQTV-NH2, to the human recombinant STAT3 protein. The phosphotyrosine peptide GpYLPQTV corresponds to the residues 903–909 within the gp130 subunit of the IL-6 receptor, which has been validated to bind the STAT3-SH2 domain according to the method (The e method . Stratege method .As STAT3 may be recruited to gp130 within phosphotyrosine residues , we utilTaken together, these data confirm that 323–1 and 323–2 are direct STAT3 SH2 inhibitors by disrupting the binding of the STAT3 phosphotyrosine-peptide, corresponding with the computational docking and DARTS assay.via in vitro clonogenic assays, which correspond to tumorigenicity in nude mice (The effect of compounds 323–1 and 323–2 concerning the inhibition of clonogenicity was investigated ude mice . On the in vitro anti-tumor activity by inhibition of cell proliferation, clonogenicity, and apoptosis induction.One of the effects of antitumor drugs is to induce apoptosis in cancer cells. We next evaluated the anti-apoptotic effect of 323s in DU145 cells. Caspases 3 and 7 are regarded as vital modulators of mitochondrial events of apoptosis. As shown in in vivo tumor model. Compound 323s were administrated i.p. daily to validate the effect on tumor growth. The in vivo data supported that 323s significantly inhibited tumor growth by measuring the tumor weight and tumor volume, respectively (As shown in ectively , especiaThe STAT3 SH2 domain is essential for STAT3 dimerization and activity. Strategies to target STAT3 SH2 have been pursued recently by various groups. Several STAT3 inhibitors have been discovered, and some are in clinical trials. STA-21 reportedly exhibited treatment effects against psoriasis and rheumatoid arthritis but remains unclear concerning cancer treatment . LLL12 hWe have shown in this work that two compounds 323–1 and 323–2 were identified as STAT3 SH2 domain inhibitors with better potency than the S3I-201 in terms of drug-target affinity as well as than cryptatanshinone regarding the capability of deactivating STAT3 signaling. Tyr705 phosphorylation, which is vital for STAT3 dimerization and is prevalent in prostate cancer , was demHowever, the short-time 4 h treatment with drugs did not show disruption of STAT3 dimerization in the co-IP assay . One posThe FP assay allows quantitative analysis of molecular interactions based on a single fluorescent label strategy. Fewer proteins and compounds are needed for the experimental steps, and samples might be repetitively measured without being destroyed. These benefits of the FP assay are one important approach in HTS . HoweverAccording to the FP assay, the ligands 323–1 and 323–2 were found to interrupt the binding between GpYLPQTV and STAT3. Notably, both 323–1 and 323–2 exhibited increased polarization at concentrations above 200 µM. These compounds did not appear to be fluorescent at the measured wavelengths, so at high concentrations, some interaction occurs between the drug and fluorophore to decrease the fluorophore’s mobility. This was confirmed by measuring the polarization of peptides in the presence of high concentrations of the drugs and without STAT3. In particular, at concentrations above 200 µM of 323 drugs, GpYLPQTV became significantly polarized in the absence of STAT3. Another reason is due to the autofluorescence of drugs. The 323 drugs have minor emissions at the wavelengths tested for the GpYLPQTV probe. The emission also appears to be polarized, and the polarization increases with an increasing concentration of 323 drugs in the presence of STAT3. This is further evidence for the binding of the drugs to STAT3.via the ubiquitin-proteasome mechanism (323s appear to bind to the STAT3 SH2 domain with favorable affinity and selectivity compared with other STAT3 inhibitors, especially S3I-201. Modification of the 323s structure may be executed to get more analogs and improve potency and selectivity. The small molecule-based PROTAC technology is reported to degrade target proteins selectively with high potency echanism . To achiTaken together, our study identified two STAT3 SH2 domain inhibitors, which also provide diversified biological activities and structural diversity for anti-tumor drug discovery and might be promising in the treatment of cancers with hyper-activated STAT3.


Benefitting from strong light absorption of Cr3+ in the blue waveband and very efficient Cr3+→Yb3+ energy transfer, the as-synthesized Lu0.2Sc0.8BO3:Cr3+,Yb3+ phosphor emits intense SWIR light in the 900–1200 nm from Yb3+ under excitation with blue light at ~460 nm. The optimized phosphor presents an internal quantum yield of 73.6% and the SWIR luminescence intensity at 100 °C can still keep 88.4% of the starting value at 25 °C. SWIR LED prototype device based on Lu0.2Sc0.8BO3:Cr3+,Yb3+ phosphor exhibits exceptional luminescence performance, delivering SWIR radiant power of 18.4 mW with 9.3% of blue-to-SWIR power conversion efficiency and 5.0% of electricity-to-SWIR light energy conversion efficiency at 120 mA driving current. Moreover, under the illumination of high-power SWIR LED, covert information identification and night vision lighting have been realized, demonstrating a very bright prospect for practical applications.The growing demand for spectroscopy applications in the areas of agriculture, retail and healthcare has led to extensive research on infrared light sources. The ability of phosphors to absorb blue light from commercial LED and convert the excitation energy into long-wavelength infrared luminescence is crucial for the design of cost-effective and high-performance phosphor-converted infrared LEDs. However, the lack of ideal blue-pumped short-wave infrared (SWIR) phosphors with an emission peak longer than 900 nm greatly limits the development of SWIR LEDs using light converter technology. Here we have developed a series of SWIR-emitting materials with high luminescence efficiency and excellent thermal stability by co-doping Cr 3+-Yb3+-co-doped phosphors that emit intense short-wave infrared light in the 900–1200 nm upon blue light excitation have been developed.Cr Recent advancement in smart and portable SWIR spectroscopy for product sorting, quality checking, and noninvasive health monitoring also necessitates the development of high-performance and cost-effective SWIR light emitters10. The specific application requirement determines the research interest of illuminator in the SWIR spectral region, which correlates with the unique characteristics of SWIR light12, such as being unlikely to be seen by standard detectors, low absorption and scattering by biological tissue, and high penetration power in complex environmental conditions .Short-wave infrared  light sources have aroused widespread interest for a wide range of applications in the fields of night vision surveillance, optical communications, remote sensing, and biomedical imaging14. However, the inherent limitations of SWIR LED chips such as complex processing requirements, relatively high product price, and a narrow band of SWIR light (<50 nm), also make them difficult to meet the wide application for daily use. Very recently, solution-processed semiconductors emitting in the SWIR , are offering new and exciting possibilities for SWIR LEDs20. Nevertheless, organic and perovskite LEDs usually exhibit poor device performance with low external quantum efficiency at wavelengths >950 nm22. QD LEDs can exhibit longer-wavelength emissions, spanning a large part of the SWIR region with emissions up to 1500 nm, but challenges remain in achieving high and stable power output, reducing the roll-off of external quantum efficiency, extending the operation lifetime, and avoiding the presence of toxic elements22.In contrast to traditional light sources , infrared LEDs based on III-V inorganic semiconductors  are replacing these unwieldy and low-efficiency lamps for use in smart SWIR devices owing to their solid-state nature, compact design, low power consumption and high radiant flux within 1050–1650 nm 26. For example, Wang et al. fabricated efficient infrared LEDs with a peak wavelength at 980 nm by using PbSe QDs as the light converter of blue GaN LED and reached an external quantum efficiency of 5.3%27. Pradhan et al. demonstrated an optical output power of 14 mW with 13% of power conversion efficiency at a driving current of 360 mA from broadband SWIR light emitters based on stacks of multi-bandgap PbS QDs and 595 nm LED28. Fu and co-workers assembled an infrared LED prototype device by integrating SrO:Er3+ phosphors with 380 nm UV-emitting chip, achieving 1.54 μm SWIR luminescence due to the characteristic electron transition of Er3+,29. Pichon et al. designed LED-pumped SWIR luminescent concentrator by combining 940 nm LEDs with Yb,Er co-doped glass, which emits at 1550 nm with an optical conversion efficiency of 1.29% at 600 mA30. Wang et al. reported a SWIR LED device by coating 450 nm blue LED chip with Zn1.5Sn0.5Ga1.0O4:Cr3+,Ni2+ phosphors, which presented a broad emission band over 1100–1650 nm with an emission peak at 1330 nm31. Recently, Miao et al. demonstrated a SWIR output power of 4.78 mW with an 1120 nm emission peak and power conversion efficiency of 4.4% at 60 mA by employing LiScGeO4:Cr3+ phosphors as the blue-light converter32.On the other hand, converter technology has recently become a cost-effective and promising approach to design and develop high-performance solid-state SWIR light sources, which combine commercial high-power LED chips with quantum dots or inorganic phosphors doped with transition metals or trivalent lanthanides, as the device mechanism occurs in phosphor-converted white and near-infrared LEDs3+) is a favorable luminescent center, which can emit SWIR light in the 940–1200 nm with a peak maximum at ~1000 nm due to the 2F5/2 → 2F7/2 electron transition33. As a result, Yb3+-activated luminescent materials have been paid much more attention for optoelectronic applications in the SWIR, including solid-state lasers34, photon upconversion37, spectral converters for Si photovoltaics39 and SWIR LED devices17. Nevertheless, the blue light absorption ability of Yb3+ is significantly weak due to the absence of energy levels in the blue region, making it hard to achieve high-efficient SWIR luminescence by the use of readily available and high-power blue LED chips. Just recently, Yb3+ has been co-doped into Cr3+-activated broadband NIR-emitting phosphors to broaden the spectral bandwidth and improve the NIR luminescence performance in view of the efficient Cr3+→Yb3+ energy transfer under the excitation of blue LED45. However, with regard to this application, Cr3+ emission and Yb3+ emission usually appear simultaneously with comparable luminescence intensities in order to achieve super broadband NIR LED application, while the intense and dominant emission band over 940–1200 nm from Yb3+ for SWIR LEDs and their practical applications have largely gone unnoticed.Trivalent ytterbium  patterns of Lu0.2Sc0.8BO3:xCr3+ (x = 0.005–0.05) and Lu0.2Sc0.8BO3:2%Cr3+,yYb3+ (y = 0.001–0.1) samples are presented in Fig. 3 (JCPDS No. 79–0097), which indicate that introducing Cr3+ and Yb3+ dopants will not cause the formation of the impurity phase. However, in comparison with ScBO3 crystal, the diffraction peaks of the as-synthesized phosphors show a significant shift to the lower angle region, revealing that the Lu3+ ion with a larger ionic radius has been dissolved into the ScBO3 lattice and Lu0.2Sc0.8BO3 solid solution has been formed. Furthermore, as shown in Fig. 3+ concentration increases, which probably results from the fact that the octahedra gets progressively distorted since the ionic radius of Cr3+ with six-coordination number (0.615 Å) is smaller than that of trivalent cation (Lu3+/Sc3+). The Rietveld refinement of Lu0.2Sc0.8BO3:2%Cr3+,5%Yb3+ phosphor is also presented in Fig. Rwp value of 8.84% and Rp value of 6.27%, indicating the refinement results are accurate. Detailed crystal structure parameters and cell parameter values are presented in Table The schematic crystal structure of Lu0.2Sc0.8BO3:Cr3+,Yb3+ phosphors, SEM images and EDS element mapping of the Lu0.2Sc0.8BO3:2%Cr3+ and Lu0.2Sc0.8BO3:2%Cr3+,5%Yb3+ are obtained, as presented in Fig. 3+ and Yb3+ emitters have been introduced into Lu0.2Sc0.8BO3 lattice (Table In order to further study the microstructure and element composition of Luce Table .z1−SczBO3:Cr3+ phosphors (z = 0.2–0.8) with different Lu/Sc ratios are shown in Fig. 3+ content increases, the NIR emission intensity of the as-synthesized phosphor shows a gradual increase and reaches a maximum at z = 0.8. Moreover, Lu0.2Sc0.8BO3:Cr3+ phosphor exhibits the optimum luminescence thermal stability  transition, the 463 nm excitation peak resulting from the Cr3+ 4A2 → 4T1(4F) transition and the 647 nm excitation peak resulting from the Cr3+ 4A2 → 4T2(4F) transition, respectively. Under 463 nm blue light excitation, Lu0.2Sc0.8BO3:Cr3+ phosphors emit NIR light with a peak maximum at ~830 nm and an FWHM of ~155 nm, which can be attributed to the electron transition from the 4T2(4F) exciting level to the 4A2 ground state level. Moreover, the NIR luminescence spectra of Lu0.2Sc0.8BO3:Cr3+ phosphor remain almost unchanged even at 77 K, as depicted in Fig. 3+ located in octahedral site. Therefore, the corresponding crystal field parameters of Lu0.2Sc0.8BO3:Cr3+ are calculated according to the following formulas48:E(4T2) represents the energy position of 4T2 energy level, E(4T1) is the energy position of 4T1 energy level, B is the Racah parameter, Dq is the crystal field parameter. Therefore, the Dq/B value of Lu0.2Sc0.8BO3:Cr3+ is calculated to be 2.51, as shown in Fig. 0.2Sc0.8BO3:Cr3+ are close to the values in the ScBO3:Cr3+ phosphor reported by Fang et al. (Dq/B = ~2.88)49. Generally, when Cr3+ ions reside in a strong crystal field , the narrowband luminescence assigned to the 2E → 4A2 transition occurs in this system. However, even though the obtained Dq/B value in Lu0.2Sc0.8BO3 host is much larger than 2.3, the Cr3+ emitter suffers from a weak crystal field and presents a NIR broadband emission. This inconsistency has also been noted in some other oxide-based NIR phosphors53, which is probably attributed to the distortion of the octahedron, while the classic Tanabe–Sugano diagram is on the basis of the nearly perfect octahedral position of Cr3+54. Actually, an inspection of the crystal structure of Lu1-xScxBO3 crystal shows that trivalent metal cations are located only in one site that is not perfectly octahedral, but is distorted to a lower local S6 symmetry55. Moreover, for Lu1-xScxBO3 solid solution, it is demonstrated by Liang et al. that the distortion ratio of [LuO6]/[ScO6] octahedra becomes larger as the content of Sc3+ increases in Lux1-ScxBO3 lattice and the distortion ratio reaches a maximum when x = 0.7 (Lu0.3Sc0.7BO3)46.The photoluminescence properties of Luity Fig. . The exc0.2Sc0.8BO3:Cr3+ samples with different Cr3+ doping concentrations upon excitation with blue light are depicted in Fig. 3+ content from 0.005 to 0.05. But the NIR luminescence intensity shows an increasing trend from x = 0.005 to 0.02 and then falls off when the Cr3+ content continues to increase  represents the NIR luminescence intensity at time t, I0 is the initial intensity, and τ is the lifetime. The slight deviation from the single exponential term is assigned to the energy transfer between the Cr3+ ions and their random distribution among the octahedral sites, especially under high doping concentrations. The fluorescence lifetime of Lu0.2Sc0.8BO3:Cr3+ sample decreases from 83.3 to 51.1 μs as the Cr3+ concentration increases from 0.005 to 0.05. To investigate the temperature-dependent NIR photoluminescence of Lu0.2Sc0.8BO3:Cr3+, emission spectra at a different temperatures from 25 to 200 °C are depicted in Fig. 0.2Sc0. 8BO3:2%Cr3+ is measured to be 26.1% when exciting with 463 nm light , poor thermal stability, and low absolute quantum efficiency, which will greatly limit its practical application for NIR spectroscopy. On the other hand, Yb3+ ion can generate desirable SWIR emission at ~1000 nm, but it usually does not have characteristic absorption in the blue region. Considering the efficient Cr3+→Yb3+ energy transfer, Yb3+ ions are co-doped into the Lu0.2Sc0.8BO3:2%Cr3+ phosphors to improve the photoluminescence performance and extend their applications.Lu0.2Sc0.8BO3:5%Yb3+, Lu0.2Sc0.8BO3:2%Cr3+ and Lu0.2Sc0.8BO3:2%Cr3+,5%Yb3+ phosphors are shown in Fig. 3+ single-doped phosphor, the excitation spectrum when monitored at 990 nm emission contains one excitation band centered at 303 nm, originating from the absorption of Yb3+-O2− charge transfer state59. When Yb3+ is introduced into Lu0.2Sc0.8BO3:2%Cr3+ phosphors in an appropriate concentration, strong SWIR emission in a wavelength range over 940–1200 nm that is associated with the characteristic 2F5/2 → 2F7/2 transition of Yb3+ is observed upon 463-nm-light excitation. The excitation spectrum of Lu0.2Sc0.8BO3:2%Cr3+,5%Yb3+ phosphor monitored at 990 nm is in accordance with that of Lu0.2Sc0.8BO3:2%Cr3+, suggesting the emergence of efficient Cr3+→Yb3+ energy transfer. The strong absorption in the blue region for Lu0.2Sc0.8BO3:Cr3+,Yb3+ phosphor  and poor thermal stability (31%@95 °C) upon 492 nm excitation.The emission spectra of Lub3+ Fig. . Note th0.2Sc0.8BO3:2%Cr3+,yYb3+ with different Yb3+ concentrations by monitoring 830 nm emission when excited by blue light at 463 nm are depicted in Fig. 0.2Sc0.8BO3:2%Cr3+,yYb3+ phosphors show a significant declining trend from 58.7 to 10.9 μs as the content of Yb3+ increases, further verifying the appearance of efficient Cr3+→Yb3+ energy transfer, as depicted in Fig. 3+ donor and Yb3+ acceptor can be compensated by the emission of phonons, phonon-assisted Cr3+→Yb3+ energy transfer will be responsible for the SWIR emission under the excitation of blue light60. The efficiency of energy transfer (ηET) is determined by the following equation62:τ and τ0 are the Cr3+ lifetimes with and without Yb3+, respectively. The luminescence lifetime and the calculated efficiency of energy transfer at varied concentration of Yb3+ are presented in Fig. 0.2Sc0. 8BO3:2%Cr3+,10%Yb3+ phosphor.The luminescence decay curves of Lu0.2Sc0.8BO3:2%Cr3+,5%Yb3+ phosphor at different temperature over 25–200 °C are shown in Fig. 3+ almost disappears at elevated temperature, while Yb3+ luminescence intensity remains at a relatively high level , as shown below64:IT is the intensity of emission at a given temperature T, I0, k, and A are constants, ΔE refers to the activation energy, which corresponds to the energy gap from the lowest position of the excited level to the cross point of the excitation level and the ground state level. The fitting is plotted as the red curve in Fig. I0 = I(25 °C), the ΔE value is determined to be 0.38 eV, which is consistent with the superior luminescence thermal stability of Lu0.2Sc0.8BO3:2%Cr3+,5%Yb3+ phosphor. In addition, emission spectra of Lu0.2Sc0.8BO3:2%Cr3+,yYb3+ phosphors at different temperatures with varying Yb3+ concentrations are also provided in Fig. 0.2Sc0.8BO3:2%Cr3+,yYb3+ phosphors at varied temperatures. Compared with Cr3+ single-doped phosphor (50%@100 ºC), the luminescence thermal stability shows an obvious increase as Yb3+ concentration increases, suggesting that Yb3+ emission is more thermally stable than Cr3+ emission. It should also be noted that the thermal quenching of Cr3+ and Cr3+→Yb3+ energy transfer are competitive in Cr3+-Yb3+-co-doped material system. The excitation energy will be transferred to thermally stable Yb3+ emitters rapidly to suppress luminescence thermal quenching of the excited Cr3+ ions. With the increase of Yb3+ content, SWIR luminescence of Yb3+ through energy transfer from Cr3+ dominates the total emission.Environment temperature has a great effect on the photoluminescence performance of luminescent materials, thereby further affecting the luminescence properties of the fabricated photoelectric devices. As a result, the luminescence thermal stability of the yielded SWIR emissive material is also investigated in detail. The emission spectra of Luent Fig. . The 9900.2Sc0.8BO3:Cr3+,Yb3+ phosphor to efficiently convert blue photons into SWIR luminescence, we successfully developed SWIR LED prototype devices through a combination of blue InGaN chip and our SWIR-emitting phosphor. The as-synthesized phosphor powders (a1), the fabricated high-power SWIR LED prototype device (a2), a working SWIR LED taken by a standard digital camera in the absence (a3), and presence (a4) of a long-pass filter at 650 nm are shown in Fig. Inspired by the appealing ability of Lu0.2Sc0.8BO3:Cr3+ can not realize the same applications due to the lack of adequate SWIR light in its emission spectrum , Sc2O3 , H3BO3 , Cr2O3 , and Yb2O3  were used as the starting precursors and ground adequately, except that 25% excess of H3BO3 was added to compensate for the evaporation loss of boron element. After that, the obtained mixture was transferred into a muffle furnace and sintered at different temperatures over 1150–1300 °C, and kept for 5 h in the air to form the final products.Lu0.2Sc0.8BO3:2%Cr3+,5%Yb3+ phosphor and blue SMD 2835 LED (452.5−455 nm) were integrated to construct SWIR LED prototype. The phosphor powder and high refractive index encapsulation silicone glue  were thoroughly mixed in a mass ratio of 1:1 and then deposited on the blue LED chips. The phosphor coating thickness is about 0.3 mm. High-power SWIR LED prototype was also constructed through a combination of Lu0.2Sc0.8BO3:2%Cr3+,5%Yb3+ phosphor with high-power blue LED .The optimized Lu32. Utmost care was taken to assure the reproducibility of the spectral measurements. A thermal imaging camera (FLIR A300) was used to record the operating temperature of the SWIR LEDs. A Canon EOS 800D digital camera and a commercial InGaAs SWIR camera (Raptor Owl 640 S) were used to capture visible and SWIR images, respectively.XRD was collected on a DMAX-2500PC X-ray diffractometer. The microstructure and elemental composition of the as-synthesized phosphor were measured on a scanning electron microscope . The phosphor powder was deposited on an aluminum foil substrate for measurement. The element composition and distribution were further identified by inductively coupled plasma mass spectrometry (ICP-MS). UH4150 spectrophotometer was used to measure the diffuse reflection spectra. The photoluminescence measurements of the yielded phosphors and the luminescence properties of the fabricated SWIR LEDs were measured using similar test equipment to that described in our previous workSupplemental material


Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.  •Uncoordinated-5 receptor D (Unc5D) and glypican-3 (GPC3) form an octameric complex•glue, Nanobreak) enhance or weaken Unc5-GPC3 bindingAnti-GPC3 nanobodies  and morphogen receptor glypican-3 (GPC3) reveal how finely balanced Unc5-GPC3 interactions regulate the migration of neuronal and cancer cells. Context-dependent signaling networks formed by different cell surface proteins direct brain development. Guidance receptors of the Uncoordinated-5 family (Unc5A–D) have emerged as key players in navigating cells and axons , where tIn agreement with previous findings , we showin vivo xenograft model, by grafting human neuroblastoma cells arising from the sympathoadrenal lineage of the neural crest into the equivalent site in chick embryos (Mutations in GPC3 cause Simpson-Golabi-Behmel overgrowth syndrome (SGBS), a genetic disorder that presents with visceral and skeletal abnormalities and an increased risk of cancer . SGBS pa embryos . Using t embryos . GPC3 is embryos  and was  embryos .In vitro, we show that Unc5-GPC3 signaling elicits a repulsive cellular response. In the developing mouse cortex, we find that AP cells present GPC3 that acts as a ligand for neuronal Unc5D. We also show that Unc5/GPC3 signaling is essential for the collective migration of neural-crest derived neuroblastoma cells to their target sympathoadrenal derivatives.Here, we present crystal structures that reveal a striking GPC3:Unc5D (4:4) octameric arrangement. Structured glycan-glycan interactions link C-mannosylated Unc5D tryptophans to an N-linked glycan on GPC3. Protein-protein interactions are formed along the concave face of GPC3 and all three N-terminal domains of Unc5D. We use mutagenesis and molecular dynamics simulations to characterize these interfaces, and present mutants that no longer interact. Anti-GPC3 nanobodies disrupt or enhance Unc5-binding. core) and murine GPC3 residues 31–482 (mGPC3core). Human and murine GPC3 sequences are 94% identical and the two structures are similar . We modeled glycans on two predicted N-glycosylation sites  with Rattus norvegicus Unc5D residues 32–307 (rUnc5DIgIgTSP). The complexes crystallized in two different space groups (During an unbiased enzyme-linked immunosorbent assay (ELISA) , we confe groups  with dife groups . The cene groups D–1I.core is shown in shades of blue, rUnc5IgIgTSP is shown in shades of green.hGPC3IgIgTSP, we revealed masses corresponding to the octamer and its subfragments: 1:1 Unc5D-Gpc3 dimers (93 kDa), 2:2 tetramers (185 kDa) and the full 4:4 octamer (370 kDa)  GPC3 protein did not give clean signals using this method. We speculated that this may be due to a mixture of cleaved and uncleaved protein in our samples, which is only partially processed at the conserved furin-like convertase site in the C-lobe. To produce a more homogeneous sample for mass spectrometry, we introduced two-point mutations in the furin cleavage site . Mixed with rUnc5D370 kDa) A. Each p370 kDa) F–S1H. Thcore contacts the three N-terminal domains of Unc5: Ig1, Ig2, and TSP1 (iso1), is unable to bind GPC3  or ∼640 Å2 (hGPC3 complex). Charge complementarity of the interface is provided by hGPC3  and rUnc5D  (2 (mGPC3 complex) or ∼1330 Å2 (hGPC3 complex) of protein surface. Hydrophobic residues line both sides: rUnc5D I170, A281, P282, L283, F288, and hGPC3 L157, L235. Hydrophobic interactions are complemented by hydrogen bonding and charged interactions, such as hGPC3 E238, which interacts with the backbone of rUnc5D A281 (2 (mGPC3 complex) or ∼360 Å2 (hGPC3 complex). Within the octameric arrangement, the antiparallel Unc5D chains also form interactions between themselves, especially at the Ig2 domains: the buried surface amounts to ∼900 Å2 and ∼840 Å2 for the major antiparallel packing interactions within the Unc5 bundle.Interface 1 is located at the C lobe of GPC3 and interfaces with the Ig1 and Ig2 domains of Unc5D, burying a surface of ∼690 Å6, K157) A. The lac5D A281 B. Interfc5D A281 C. The in1 and W2 of the consensus sequence W1xxW2xxW3 , mUnc5CIgIgTSP (residues 40–317), and rUnc5DIgIgTSP from HEK293T cells. Mass spectrometry shows that the first two tryptophan residues (W1 and W2) were C-mannosylated in all Unc5 homologues tested (Recent work showed that the TSP domains of Unc5 receptors are C-mannosylated at tryptophan residues WxxW2xxW3 . We revixW3 (iso  and notis tested F–S2I. Inin vitro to test its function. Attempts to remove it using endoglycosidases (EndoF1 or PNGase F) were not successful, as the glycan remained uncleaved. We therefore mutated the site (N241Q) to produce a hGPC3 protein that lacks a glycan at this position. The resulting mutant protein was readily expressed and secreted by HEK293 cells. A cell-based binding assay and SPR experiments showed that the N241Q mutant had lost affinity for Unc5A–D , while another inhibits it (Nanobreak)  D. We connobreak) A–S4F. Thadhesion G and 4H.core and the mutant GPC3coreUG. In these assays, cortical neurons preferentially migrated on the mutant protein stripes, demonstrating that GPC3core elicits a repulsive effect via an Unc5-dependent mechanism (core (WT or mutant) and neutral Fc protein, the neurons were strongly repelled by both WT and the mutant protein. This suggests that unknown additional GPC3-receptors, who do not depend on the Unc5-GPC3 interaction, also cause repulsion from GPC3core (glue) or decrease (Nanobreak) cell repulsion from WT GPC3-containing stripes (glue led to immobilization of neurons on the stripe surface and we were unable to quantify any migratory behavior (not shown). As for N2A, SY5Y, and HeLa cells, addition of Nanobreak reduced the preference of neurons for GPC3coreUG over GPC3core. We conclude that the GPC3-Unc5 interaction mediates contact-repulsion in these assays and may contribute to cellular navigation in cortical and neuroblastoma cells.Unc5 receptors are known for their repulsive signaling in neuronal cell guidance . To asseechanism I and 4J.GPC3core G and S4HGPC3core J. We als stripes K, consisIn situ hybridization (ISH) for GPC3 showed restricted expression to the germinal layers, predominantly at the ventricular zone (VZ) where AP cells are located from embryonic days (E)13.5 to E17.5  expressed at earlier stages of brain development  and in nto E17.5 A–S5C. Unto E17.5 . These rto E17.5 C. Analysto E17.5 D  at E13.5. We previously showed that the overexpression of signaling-deficient, but otherwise active, receptor fragments is an effective way of interfering with endogenous interactions  receptor:ligand interactions are emerging for a range of important morphogen and guidance receptors, for example, FLRT/Lphn/Teneurin  is challenging and relies on tools such as mutant proteins or functional nanobodies. Although these enable us to target specific protein-binding surfaces, we cannot rule out that other ligands use the same surfaces for binding and therefore could also be affected. We have mitigated this risk by targeting Unc5 and GPC3 individually in different experiments. A further limitation is that over-expression of proteins elena.seiradake@bioch.ox.ac.uk).Further information and request for resources and reagents should be directed to and will be fulfilled by the lead contact, Elena Seiradake .All mice (C57BL/6 background) were housed with a 12h:12h light:dark cycle and food/water available Naked Neck strain embryonated eggs were obtained from a local supplier . Laying hen’s sanitary status was regularly checked by the supplier according to French laws. Eggs were housed at 18°C until use. They were then incubated at 38.5°C in a humidified incubator until the desired developmental stage, i.e, HH14 for the graft step (54 h of incubation).HeLa, N2A, SY5Y and SY5Y:GFP  NB cell in vitro at 37°C, 5% CO2 in Neurobasal medium supplemented with B27. Neurons were used for stripe assays and were fixed with 4% Paraformaldehyde for 10 min followed by immunostaining.Neurons were dissociated from cortices of E15.5 embryos and cultured on stripes. Neurons were cultured for 1 day core, residues 32–483, UG mutant, N241Q), mouse GPC3 (Uniprot ID: Q8CFZ4) , mouse Unc5A (Uniprot ID: Q8K1S4) , mouse Unc5B (Uniprot ID: Q8K1S3) , into the Age1-Kpn1 or EcoR1-Kpn1 cloning site of vectors from the pHLSec family , human Unc5B (Q8IZJ1) (hUnc5Becto), hUnc5A (Uniprot ID: Q6ZN44) , mouse FLRT2 (FLRT2LRR). For cell binding and functional assays, full length constructs were used, either cloned into a pHLSec vector that encodes an intracellular mVenus, or the pCAGIG vector  and human GPC3  were cloned into a modified pCMV6-XL4 vector in frame with an N-terminal FLAG and C-terminal human Fc or alkaline phosphatase fragments.We coned constructs of human GPC3 (cDNA clone BC035972) , sealed and incubated overnight at 4C. The following day, the plate was washed once with PBS and 1% casein was added as a blocking agent, which was removed after 1 h at room temperature using an automated microplate washer . Next, to each well, 20 μL of Unc5D-AP conditioned medium, containing 2 μL of monoclonal mouse anti-human IgG1-HRP was added using an automated plate copier  along with 20 μL of culture medium from 95 different ecto-Fc prey proteins. Plates were sealed and incubated for 4 h at room temperature in the dark. Plates were subsequently washed, and 35 μL 1-Step Ultra TMB-ELISA HRP substrate was added using an automated multichannel pipette ; after 1 h incubation at room temperature, the absorbance at 650 nm was recorded with a microplate Spectramax i3 plate reader (Molecular Devices). Finally, plates were scanned to obtain matching images of the 650 nm reading. A positive control (known interactors NRXN/NLGN1) was used .Recombinant protein expression and purification were performed as described . BrieflyE. coli strain WK6., grown at 37°C in Terrific Broth until an OD600 = 0.8. Expression was induced with 150 μM IPTG and incubation at 21°C for 16 h. Cells were harvested by centrifugation . Cell pellets were resuspended and incubated in ice-cold 20% sucrose, 30 mM Tris-HCl, pH 7.5, 2 mM EDTA buffer for 20 min. The cell suspension was clarified by centrifugation . After the supernatant collection, cell pellets were resuspended and incubated in ice-cold 30 mM Tris- HCl, pH 7.5, 5 mM MgSO4 buffer for 20 min. The cell suspension was clarified by centrifugation . The supernatant was filtered, supplemented with 150 mM NaCl and 2 mM Imidazole and passed through HisTrap HP columns. The column was washed with 200 mL wash buffer  and the protein was eluted in elution buffer . Elution was loaded onto a Superdex200 16/600 HiLoad column in 20 mM Tris-HCL, pH 7.5, 200 mM NaCl.Nanobodies were expressed in Streptomyces avidinii, Sigma-Aldrich) was mixed with an excess of biotinylated nanobodies, incubated overnight at 4°C and subjected to size exclusion chromatography using Superdex 200 16/60  in 20 mM Tris-HCl (pH 7.5) and 200 mM NaCl.For protein biotinylation in HEK293 cells, protein constructs in pHLsec C-terminal Avi-tag were co transfected with a vector encoding BirA (biotin ligase). Cell culture medium was supplemented with 100mM biotin. Proteins were purified as previously, but in ice-cold conditions. For the nanobody-streptavidin complexes, streptavidin  and 0.02 M of amino acids ). Crystals of mGPC3core were obtained by concentrating the protein to 9.9 mg/mL in the presence of 100 mM NDSB256 and using crystallization solution 2 (0.2 M ammonium nitrate and 20% w/v PEG3350). Crystals of the complex hGPC3core and rUnc5DIgIgTSP were obtained by mixing the two proteins in a 1:1 M ratio and concentrating them to 5.8 mg/mL. The protein solution was then mixed with crystallisation solution 1. Crystals of the mGPC3488 and rUnc5DIgIgTSP complex were obtained by mixing the two proteins in a 1:1 M ratio, concentrating to 7.1 mg/mL and mixing with 15% w/v PEG 3000, 20% v/v 1, 2, 4-butanetrol, 1% w/v NDSB 256, 0.1 M Gly-Gly/AMPD (pH 8.5) and 0.2 M of amino acids .Proteins that were expressed in GnTI-deficient HEK293S cells were used for crystallisation trials. Crystals were grown by the vapor diffusion method at 18°C by mixing the protein solution and crystallization solution in a 1:1 ratio. Purified hGPC3core, hGPC3core/rUnc5DIgIgTSP and mGPC3core/rUnc5DIgIgTSP were flash-cooled in their original crystallization condition. Crystals of mGPC3core were cryoprotected by adding 20% v/v glycerol to the original crystallization solution. All diffraction data were collected at the Diamond Light Source synchrotron at 100K. Data was integrated using DIALS (via XIA2), and intvia XIA2). The dattaraniso . The strDIgIgTSP  and PHASDIgIgTSP . Initialructures . We perfructures . Manual ructures , and modructures  and Phenructures . For theructures  and the ructures . Superpoructures .1C4 conformation (an inverted chair) to keep the alpha linkage in a clash-avoiding equatorial conformation. In order to increase the observation to parameter ratio and restrain the mannoside’s ring conformation individually, external restraints for both N- and C-glycosylation were generated using the MKIV version of the Privateer software /(KD + C), where C is analyte concentration calculated as monomer.Equilibrium binding experiments were performed at 25°C using a Biacore T200 instrument  using PBS +0.005% (v/v) polysorbate 20 (pH 7.5) or 20mM Tris +200mM NaCl +0.005% (v/v) polysorbate 20 (pH 7.5) as running buffers. Glypican, Unc5 or nanobody constructs were biotinylated enzymatically at a C-terminal Avi-Tag and coupled to a streptavidin-coated CM5 chip. Protein analytes were injected over the chip in 2-fold dilution series for 500 s, followed by at least 25 s dissociation time. The regeneration buffer used was 2 M MgClhttps://www.graphpad.com). Significance was determined when p < 0.5.HEK293T cells grown on coverslips were transfected using mVenus-tagged construct with 3 μg of DNA and 9 μL of PEI. Eighteen hours after transfections, cells were incubated with buffer (HBSS with 1% BSA and 10 mM HEPES (pH 7.5)) for 30 min on ice, and then with buffer containing 0.5 μg purified His-tagged protein per coverslip that was previously pre-clustered (20 min at room temperature) with anti-His  in a 1:2 (protein:antibody) ratio for 60 min on ice. Cells were then washed with PBS and fixed with 4% PFA for 20 min, and then washed using PBS supplemented with 50 mM ammonium chloride. Cells were then incubated in the dark with anti-mouse-Cy3 in a 1:7.5 (protein:antibody) ratio in buffer for 60 min on ice. The cells were washed with PBS, stained with DAPI (0.1 μg/mL) and mounted using Immu-Mount. Imaging for data analysis was done with a Nikon ECLIPSE TE2000-U inverted fluorescence microscope. Analysis was performed in ImageJ/Fiji  using thcore and rUnc5DIgIgTSP were concentrated separately to 10 μM, dialyzed against 1M ammonium acetate buffer (pH 7.5) overnight at 4°C, and injected at 3 μM concentration. The mGPC3core(R355A/R358A) - rUnc5DIgIgTSP complex was concentrated to 15 μM (assuming a 4:4 stoichiometry) and dialyzed against 200mM ammonium acetate (pH 7.5) and injected at 3.3 μM concentration. The protein samples were loaded into in-house prepared gold-coated capillary needles (Harvard Apparatus) and were injected directly to the mass spectrometer. The experiments were performed using a Q-Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher). Typically, 3 μL of protein solution was electrosprayed from gold-coated capillaries. The instrument parameters for MS are as follows: 1.2 kV capillary voltage, S-lens RF 200%, quadrupole selection from 1,500 to 20,000 m/z range, in-source trapping energy (0-20V), nitrogen UHV pressure of 6.07 × 10−10 mbar and capillary temperature of 100 °C. The resolution of the instrument was 17,500 at m/z = 200 (transient time of 64 ms). The noise level was set at 3 rather than the default value of 4.64. For MS/MS analysis, collisional activation in the HCD cell was provided (0–300 V). Calibration of the instruments was performed using a 10 mg/mL  solution of cesium iodide in water. Data were analyzed using the Xcalibur 4.1 (Thermo Scientific).Unliganded mGPC3m/z 200), respectively. Precursor signals with an intensity >1.0 x 10−4 and charge state between 2 and 7 were isolated with the quadrupole using a 0.7 m/z isolation window (0.5 m/z offset) and subjected to MS/MS fragmentation using higher-energy collision induced dissociation (30% relative fragmentation energy). MS/MS scans were collected at an AGC setting of 1.0 x 104 or a maximum fill time of 100 ms and precursors within 10 ppm were dynamically excluded for 30 s.Tryptophan mannosylation was verified by LC-MS/MS. 5 μg of each protein (1 mg/mL) was diluted to a final volume of 100 μL in denaturing buffer . The sample was reduced with DTT (2 μL of 200 mM solution in denaturing buffer) at 56°C for 25 min, followed by alkylation with iodoacetamide (4 μL of 200 mM solution in denaturing buffer) at room temperature for 30 min. Alkylation was quenched by further addition of 2 μL of DTT solution. The samples were further diluted 3-folds using 50 mM Ammonium Acetate buffer. Trypsin was added to the sample in 1:50 (Enzyme: Protein (w/w)) ratio. The sample was incubated at 37°C for 16 h. Next day, the digested sample was divided into two fractions. One of the fraction was quenched using 10% Formic acid solution. To the other fraction, AspN was added in 1:20 (Enzyme: Protein (w/w)) ratio. The sample was incubated further at 37°C for 4 h after which it was quenched using 10% Formic acid solution. Resulting peptides were analyzed on an UltiMate 3000 UHPLC system (Thermo Fisher) connected to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher). The peptides were trapped on an guard column  using solvent A . The peptides were separated on an Acclaim PepMap analytical column  using a linear gradient , flow rate: 300 nL/min). The separated peptides were electrosprayed directly into the mass spectrometer in the positive ion mode using data-dependent acquisition with a 3 s cycle time. Precursors and products were detected in the Orbitrap analyzer at a resolving power of 60,000 and 30,000 (@ Raw data files were processed using MaxQuant software (Version 1.6.3.4), having in-built Andromeda search engine . The peacore were raised in a llama by intra-muscular immunization with purified protein using Gerbu LQ#3000 as the adjuvant. Immunisations and handling of the llama were performed under the authority of the project license PA1FB163A . Total RNA was extracted from peripheral blood mononuclear cells, and VHH complementary DNAs were generated by RT-PCR. The pool of VHH-encoding sequences was amplified by two rounds of nested PCR and cloned into the SfiI sites of the phagemid vector pADL-23c as previously described . Phage binders were ranked according to the ELISA signal and grouped by CDR3 sequence identity.Antibodies to hGPC3escribed . Electro7 cells/ml were transfected with control pCAGIG/gCAGIC plasmids, or those coding for Unc5 or GPC3 constructs using the Neon transfection system for electroporation . Twenty-four hours after transfection, cells were harvested, passed through a 40 μm cell-strainer and used at a concentration of either 2 x 105 cells/ml or 4 x 105 cells/ml in aggregation media . For the competition experiments, different amounts of nanobodies  were added at this stage. Cells were then left to aggregate at 37°C, 5% CO2 and 250 rpm for 90 min. After the incubation, cells were diluted in 2 mL of PBS and imaged in a 6-well plate using a Nikon ECLIPSE TE2000-U inverted fluorescence microscope. Images presented were obtained using Inverted DeltaVision widefield microscope at 37°C. The total area of cells and the total area of the aggregates for each picture were calculated using the Analyze particle tool in Fiji (2 (>3/4 cells). For the statistical analysis we used a one-way ANOVA test, with a Tukey’s post-hoc test in GraphPad Prism . Significance was determined when p < 0.5.K562 suspension cells were cultured in RPMI-1640 media supplemented with 10% FBS and 5% L-Glutamine. The cells were harvested by a 3 min spin at 200g, washed with PBS, spinned again and resuspended in R buffer (Neon transfection system). Cells at a concentration of 2x10 in Fiji . The thrhttps://www.graphpad.com). Significance was determined when p < 0.5.For surface staining, K562 cells were harvested 24 h after electroporation and cooled to 4°C. The cells were then incubated with blocking buffer: HBSS with 1% BSA and 10 mM HEPES (pH 7.5) for 30 min. For Unc5D expressing cells, cells were incubated with anti-HA  antibody that was pre-clustered with secondary antibody-Cy5 for 40 min (ratio 1:7.5 for primary:secondary antibody). For GPC3 expressing cells, cells were incubated anti-FLAG  antibody that was pre-clustered with secondary antibody conjugated with Alexa 647 for 40 min (1:7.5 ratio). Cells were washed with PBS, fixed with 4% PFA, DAPI-stained, washed and resuspended in 30% sucrose/PBS. Cells were then deposited onto microscope slides using a homemade cytospin . ImagingWhole cell extract was isolated using RIPA buffer  supplemented with protein inhibitor (Roche Diagnostics). The concentration of isolated proteins was determined using Bradford assays (Bio-Rad). Western Blot analysis was performed using the following primary antibodies: anti-GPC3 , anti-Unc5D , anti-g-tubulin . Anti-mouse IgG HRP  and anti-goat IgG HRP  were used as secondary antibodies.GSE137804) public dataset. Raw sequencing data were processed following the partially published method details, explaining the different UMAP obtained in the present study. The R package Seurat (v4.0.1) was used to calculate the quality control metrics. To filter out low quality cells, we kept all cells with at least 200 detected genes and less than 10% of mitochondrial genes. Doublet cells were removed with the R package DoubletFinder (v2.0.3). To merge all samples without biasing the analysis with batch effects, while preserving the biological variation, we applied Seurat integration and re-computed a clustering based on the corrected matrix. Single RNAseq data for cortex samples were obtained from the published NCBI Gene Expression Omnibus with accession numbers GSE65000 .Control siRNA (siRNA scr)  and human GPC3 siRNA  were purchased from Sigma-Aldrich and used at a concentration of 50 nM pCAGIG vectors encoding for nanobodies or Unc5DHuman HPRT: Fwd: for TGACACTGGCAAAACAATGCA/Rev: GGTCCTTTTCACCAGCAAGCT;Human UNC5A: PrimerPCR SYBR Green Assay; qHsaCID0013056Human UNC5B: PrimerPCR SYBR Green Assay; qHsaCID0021074Human UNC5C: PrimerPCR SYBR Green Assay; qHsaCID0016268Human UNC5D: PrimerPCR SYBR Green Assay; qHsaCED0045738Human GPC3: PrimerPCR SYBR Green Assay; qHsaCID0016381For qRT-PCR analysis, total RNA was extracted from cells using the Nucleospin RNAII kit (Macherey-Nagel). One μg of total RNA was reverse-transcribed using the iScript cDNA Synthesis Kit (BioRad). qRT-PCR was performed using the LightCycler480 SYBRGreen I Master1 kit (Roche Life Science) and the CFX Connect Real-Time PCR Detection System (BioRad). The following list of primers was used in the study:Chick embryos of interest were harvested and fixed in 4% paraformaldehyde (PFA). Embryos were embedded in 7.5% gelatin and 15% sucrose in PBS to perform 20 μm transverse cryosections. Permeabilization and saturation of sections were performed in PBS with 3% BSA and 0.5% Triton. The following primary antibodies were applied to sections: anti-HNK1 mouse IgM , anti-GFP rabbit IgG , anti-mitochondria mouse IgG  and the following secondary antibodies: Alexa 647 anti-mouse IgM ; Alexa 488 anti-rabbit IgG , Alexa 555 anti-mouse IgG  and. Nuclei were stained with Hoechst (Thermo Fisher Scientific). Slices were imaged with a confocal microscope  using a 10× objective. The position of isolated cells and tumor masses was pointed on a reference image for each slice in which tumor cells could be detected, using Fiji .4 cells/ml). The cells were then incubated for 60 h in a 37°C, 5% CO2-incubator. Cells retained on the upper face of the membrane were scrubbed using cotton swabs. The transwell culture dishes were then fixed with with 4% PFA for 30 min, before washing with 3 successive PBS-baths and mounting in Mowiol. Migrating cells were counted using a confocal microscope .Transfected SY5Y cells were plated on the porous filter of the upper chamber of transwell culture dishes  in PBS. Proteins were injected into matrices (90 μm width)  and placed on 60 mm dishes, resulting in red fluorescent stripes. After 30 min incubation at 37°C, dishes were washed with PBS and matrices removed. Dishes were coated with 50 μg/mL Fc or GPC3coreUG protein mixed with 120 μg/mL anti-hFc (Jackson ImmunoResearch) for 30 min at 37°C and washed with PBS. Stripes were further coated with 20 μg/mL Laminin in PBS for at least 2 h and washed with PBS. Cortical neurons (E15.5) were cultured on the stripes in Neurobasal medium supplemented with B27 (Gibco). For testing the effects of nanobodies, neurons were cultured in medium containing 50 μg/mL streptavidin Alexa 594 (CN), Strep-Nanobreak or Strep-Nanoglue. After 24 h neurons were fixed with 4% PFA in PBS for 20 min at room temperature (RT). Neurons were washed and incubated with rabbit monoclonal anti-beta-III tubulin antibody (Sigma-Aldrich) after 20 min permeabilization in 1% BSA, 0.1% Triton X-100 in PBS. Cy2 anti-rabbit IgG secondary antibody  was used to visualize the tubulin signal. Nuclei were counterstained with DAPI before mounting. The numbers of beta-III-tubulin-positive (green) pixels on red or black stripes were quantified with ImageJ (version 1.53f)   using a core protein was mixed with Alexa 568-conjugated anti-hIgG antibody (Thermo Fisher Scientific) in PBS. Protein was injected into matrices (90 μm width)  and placed on 60 mm dishes, resulting in red fluorescent stripes. After 30 min incubation at 37°C, dishes were washed with PBS and matrices removed. Dishes were coated with 50 μg/mL GPC3coreUG protein mixed with 120 μg/mL anti-hFc (Jackson) for 30 min at 37°C and washed with PBS. HeLa, SY5Y or N2A cells were cultured on the stripes in 2% FBS medium, with 50 μg/mL Strep-Nanobreak or Strep-Nanoglue, or without nanobody (control). After 16 h cells were fixed with 4% PFA in PBS for 20 min at room temperature and nuclei were counterstained with DAPI before mounting and imaging. The number of DAPI positive pixels on red or black stripes was quantified.For cell lines, 50 μg/mL of GPC3In Situ Hybridizations (ISH) were performed using the RNAscope Fluorescent Multiplex Reagent Kit (Advanced Cell Diagnostics) according to manufacturer’s instructions. The target genes (Unc5D and GPC3) are listed in the Embryonic brains were fixed in 4% PFA overnight. 10 μm Cryo-sections were pre-treated using the RNAscope Universal Pretreatment Kit (Advanced Cell Diagnostics). RNA In utero electroporation was performed at E13.5 with anesthetized C57BL/6 mice as previously described  lysis buffer:PBS, last wash only PBS). Pulled-down samples were processed for mass spectrometry . Volcano plots were generated using the DEP package in R-studio.Pull-down experiments were performed as previously described . Fresh Eglue and Nanobreak on the Unc5 – GPC3 interaction were performed by coupling biotinylated hGPC3core to high-capacity streptavidin agarose resin  after incubation of the beads with the biotinylated protein for 1 h hUnc5BEcto was mixed with nanobody in 1:1, 1:5, and 1:10 M ratios in 20mM Tris-HCL, pH 7.5, 200mM NaCl, 1% BSA and incubated with hGPC3core-coated strep beads for 3 h. Beads were washed with 20mM Tris-HCL, pH7.5, 200mM NaCl and the proteins were eluted with SDS-containing gel-loading buffer supplemented with 5% beta-mercaptoethanol. Samples were analyzed using SDS-PAGE.Pull-down experiments to investigate the effects of Nanoglue and Nanobreak cloned into pCAGIG (Control) plasmids were electroporated at E13.5. After 3 days, embryonic brains were collected, fixed in 4% PFA overnight and vibratome cut into 75μm sections. Single cell morphology was reconstructed and analyzed using ImageJ (version 1.53) as described previously (Nanoeviously . For sint test (Statistical analyses were performed using GraphPad Prism, employing a two-tailed unpaired Student’s t test E or chi-