Mannosylated and non-mannosylated nanocarriers demonstrated biocompatibility with slow release of coumarin-6 at pH 6.8 and 7.4 over 24 h. Adherence of the non-mannosylated nanocarriers (50 and 150 µg/mL) to Caco-2 cells was ~10% over 24 h, whereas cellular uptake of 25–30% was noted at 4 h. The mannosylated nanocarriers showed a similar adherence to non-mannosylated nanocarriers after 24 h, but a lower cellular uptake (~20%) at 1 h, comparable uptake at 4 h, and a higher uptake (~25–30%) at 24 h. Overall, the nanocarriers did not affect the integrity of Caco-2 monolayers. Mannosylated nanocarriers elicited higher Papp of 1.6 × 10−6 cm/s (50 µg/mL) and 1.2 × 10−6 (150 µg/mL) than the non-mannosylated ones: 9.8 × 10−7 cm/s (50 µg/mL) and 1.0 × 10−6 (150 µg/mL) after 2 h. Non-mannosylated chitosan nanocarriers elicited enhanced adhesion to porcine gut mucin via mucin-filled microchannels due to higher cationic charge density. These results underpin the importance of surface chemistry in the biological interactions of nanocarriers, while highlighting the role of surface hydrophilicity in mucopermeation due to mannosylation.Crosslinked chitosan nanocarriers (140–160 nm) entrapping coumarin-6 ( Low bioavailability of orally administered drugs has long impeded translation, but nano-drug delivery systems (nano-DDSs) can potentially be used to improve bioavailability . DownsizThe mucus layer above the small intestinal epithelial brush border presents a challenge for oral drug delivery . The proGut mucus presents a formidable barrier against the transport of NCs trying to reach the epithelial layer to either release the encapsulated drug or be endocytosed by epithelia. It has evolved into a highly selective sieve that is difficult to permeate or mimic, either In Vitro or ex vivo. Gut mucus intercepts NCs as foreign entities and rapidly filters them out by first adhering to the NCs and removing them with recycled mucus. Such mucoadhesion is driven by polyvalent interactions, hydrophobic bonding, formation of hydrogen bonds, Van der Waal forces, and π–π stacking ,10.Ideally, NCs made up of mucoadhesive materials might adhere to the gut epithelial layer, where systemic drug absorption is initiated. The mechanism of NC uptake, with the encapsulated cargo of drug molecules, nevertheless remains unclear. It probably occurs as a combination of multiple processes, from the uptake of intact NCs to a complete dissolution at the vicinity of the gut wall followed by an uptake of free drug. In both cases, a mucoadhesive drug-entrapped nanoformulation helps in facilitating the transport of drug molecules across the gut epithelia .Chitosan is a cationic, natural, and biocompatible biopolymer , with itp-coumaric-acid-conjugated CNCs elicited 1.6-fold higher mucoadhesion when compared to native CNCs [stealth NCs that can efficiently permeate a layer of mucus [N-succinyl chitosan have been explored as oral nano-DDSs with beneficial features, including increased mucoadhesion and decreased sensitivity to metabolic enzymes in gut [CNCs have emerged as a nano-DDS with delivery potential in the GI tract . Furtherive CNCs . On the of mucus . Such stof mucus  or cycloof mucus . Similars in gut .Mannose, a C2 epimer of glucose, is an important sugar that participates in metabolism, including glycosylation . Its hydSynthesis of M-CNCs has been reported , albeit N-morpholino)ethanesulfonic acid (MES) buffer, 0.25% (w/v) trypsin, ethylenediaminetetraacetic acid (EDTA) solution (0.02% in 0.5 mM DPBS), Dulbecco’s modified eagle medium , penicillin-streptomycin (product code P4333), L-glutamine, D-(+)-mannose (product code M8574), sodium pentatripolyphosphate , sodium triacetoxyborohydride (product code 316393), coumarin-6 , and Triton®-X-100 were purchased from Sigma Aldrich .Medium molecular weight chitosan  was obtained from ChitOcean Inc. . Fetal bovine serum (FBS), phosphate-buffered saline (PBS), non-essential amino acid , Hank’s balanced salt solution , 2- under 5% CO2 (37 °C) and passaged at 70–80% confluency. Before seeding, cell counting  was performed using Countess® automated cell counter. Passaging was carried out using 0.05% trypsin-EDTA followed by centrifugation at 300× g and resuspension in complete media before counting.Human colonic adenocarcinoma-derived Caco-2 cells were obtained from the American Type Culture Collection. The cells were grown in 75 cmv/v) to the chitosan solution (pH 5–6). Solutions were left stirring for 48–72 h (100–200 rpm on a plate stirrer) for conjugation , and the mixture was stirred for another 30 min to prepare a crosslinked matrix. Only C6 (1 mg/mL in 1:9 chloroform:ethanol) was added before TPP and stirred for 20 min at 100 rpm on a plate stirrer for fluorophore encapsulation. The colloidal dispersion was sonicated for 30–40 min in an ultraprobe sonicator followed by freeze-drying and analyses. CNCs and M-CNCs without encapsulated C6 were prepared as controls.Chitosan solution (4.7 mg/mL) in 2% acetic acid was left stirring overnight in a flask A. Solutijugation B. The maThe loading efficiency (LE) was calculated as:1H NMR. Chitosan samples were dissolved in D2O, while mannosylated chitosan was dissolved in 1% d4-acetic acid (CD3COOD) in D2O. Mannose was dissolved in D2O. The NMR spectra were recorded in a Varian VnmrS spectrometer (600 MHz) at 25 °C. Receiver gain was set to automatic and was between 32 and 42. The relaxation delay was 2 s while the acquisition time was 5 s. The spectra were analyzed using MastreNova 14.2.0 software (Mastrelab Research). All peaks were referenced to the residual solvent peak. A first-order Bernstein polynomial fit was used as the baselining method. No apodization or zero filling was utilized.Chitosan, with or without mannosylation, was characterized with Drops of C6@CNC and C6@M-CNC dispersions (1 mg/mL) were deposited on an aluminum foil and left for overnight drying. The foil with deposited NCs was then fixed on a stub, followed by sputter-coating with gold. The imaging was performed using a Hitachi S-4300 scanning electron microscope.exλ = 633 nm) instrument. Two independent measurements were performed in a clear disposable sizing cuvette at 25 °C and 173° backscatter angles for each sample.Hydrodynamic diameters of the NC dispersions (100 µg/mL) were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (ex/emλ = 455/508 nm) using serial dilutions (0.78 μg/mL–0.76 ng/mL) of C6 in a 9:1 mixture of ethanol/chloroform  and subjected to centrifugation . The free C6 content in supernatant was quantified by spectrofluorometry (ex/emλ = 455/508 nm), while cumulative release was estimated after 5–24 h.In Vitro C6 release studies from C6@CNCs and C6@M-CNCs at 0.78 µg/mL concentration in HBSS-HEPES (pH 7.5) and HBSS-MES (pH 6.8) were conducted. The NCs were suspended in the buffers (1:1) at 37 °C and under constant stirring of 100 rpm. Aliquots were collected at predefined intervals  and incubated for 24 h at 37 °C and 5% CO2. The medium was discarded next day, and the cells were washed twice with PBS followed by equilibration for 30 min with HBSS (2.5 M glucose and buffered with 25 mM MES). Afterward, the cells were treated with the NCs in HBSS/MES for 4 h. Later, the fluid in each well was aspirated, followed by washing (2×) with PBS. The MTS reagent ; product number G3582) was then added to the wells according to the manufacturer’s instructions, and the cells were incubated for a further 4 h. Cells in media without NCs, cells in media, and cells exposed to Triton-X (0.001%) were used as controls. The plate was then read in a microplate reader  at 490 nm wavelength. The data were expressed as a % of the negative control.The Caco-2 cells were seeded in a 96-well plate  after coating with type II rat tail collagen to enhance cell adhesion. Upon seeding, the culture media were replaced every other day for 21 days, and the plate was incubated  to allow the cells to form monolayers. Dispersions of C6@M-CNCs and C6@CNCs with a loading of ~0.64 ng C6/µg NCs (50 µg/mL and 150 µg/mL) and free C6 suspensions were prepared in HBSS (2.5 M glucose and buffered with 25 mM MES). The monolayers were rinsed and equilibrated with HBSS for 30–60 min at 37 °C and 5% CO2 before the exposure. The buffer was later replaced with test samples in HBSS, and the monolayers were incubated for different time intervals  at 37 °C in a shaking incubator. The supernatant was collected at each time point and analyzed with spectrofluorometry (ex/emλ = 455/508 nm). Furthermore, the cells were treated with 0.2% Triton-X 100 to cause cell lysis for 2 h, and cell debris was removed by centrifugation  followed by the spectrofluorometric analysis. The obtained data were used to calculate % cellular uptake, while the adherence (µg/cm2) was calculated from the difference of spectrofluorometric reading of total NCs, and the sum of free and internalized NCs. Experimental controls were as follows: free C6 (same concentration as in NCs), HBSS (buffered), cells with and without test sample. The fluorescence intensity of the wells with cells alone was used as a negative control, while those with the test sample were used as a positive control. The following equation was used to measure cellular uptake:The Caco-2 cells were seeded on six-well plates  with 0.5 and 1.5 mL medium in apical and basolateral compartments, respectively, followed by incubation  for 21 days. The viability of the cells was checked by epifluorescence after staining the nuclei with Hoechst stain (ex/em =λ 470/525 nm). The media were replaced on the apical side 24 h after seeding, and then both the apical and basal media were replaced every two days. The integrity of the cell monolayers on filters was determined by transepithelial electrical resistance (TEER), and transport experiments were conducted on Caco-2 monolayers with TEER ≥ 2000 Ω·cm2. Before the experiment, the cell culture medium  was replaced with HBSS and incubated at 37 °C under 5% CO2 for 30 min. The buffer in the apical side of the filters was then replaced with HBSS containing test samples while permeability studies were conducted at 37 °C and 5% CO2 for 2 h, with samples collected from the basolateral side at t = 0, 0.5, 1, 1.5, and 2 h. The TEER was monitored 30 min before and following equilibrium in HBSS before exposure to ensure the stability of the TEER, and later recorded at t = 0, 0.5, 1, 1.5, 2, 4, and 24 h. The apparent permeability coefficient (Papp) of C6 was calculated by the following equation:t, A = surface area of the transwell membrane, and C0 = initial concentration of C6 in the apical compartment.Transport of the test samples (C6@M-CNCs and C6@CNCs) and free C6 was measured across filter-grown Caco-2 monolayers . Cells wMucin was harvested from abattoir-sourced samples of porcine gut within 6 h of dispatch and was mixed with an equal volume of 8M guanidine hydrochloride (Sigma Aldrich) to solubilize and homogenize the mucus samples. Dithiothreitol (Sigma Aldrich) was added to a final concentration of 10 mM and incubated at 37 °C for 5 h. Sodium iodoacetamide (Sigma Aldrich) was added to a final concentration of 25 mM, and the samples were incubated at room temperature overnight. Isopycnic density gradient centrifugation was carried out by adjusting the density of the sample to 1.40 g/mL with solid cesium chloride, and then placing the sample in Beckman Ultra-clear tubes which were centrifuged at 65,000 rpm × 18 h at 10 °C in an Optima L-100 XP  ultracentrifuge using the 70 Ti rotor without any break. The density gradient created was unpacked in 1 mL fractions from the top to the bottom of the tube. Duplicate 5 μL aliquots from each fraction were then blotted onto a polyvinylidene difluoride membrane using a Whatman manifold Slot blot apparatus  and stained with periodic acid-Schiff stain  to assess the relative intensity of carbohydrate. The carbohydrate-rich fractions observed from the slot blot, which had a density profile of between 1.35 to 1.45 g/mL, were pooled and separated by size exclusion chromatography. The samples were loaded on a Sepharose CL-4B column (Sigma Aldrich) and eluted with 50 mM Tris/100 mM KCl, pH 7.5, as mobile phase. The elute was collected in fractions of 4 mL using a fraction collector, and then a sample of all fractions was slot blotted and stained with the PAS stain. Mucin-rich fractions were pooled and freeze-dried, then resuspended in water and desalted on a Sephadex G-25 column (Sigma Aldrich). Fractions collected with the fraction collector were analyzed by PAS staining (~40 mL of sample), then carbohydrate-rich fractions were pooled and freeze-dried before being stored (1 mg/mL) at −20 °C in sterile water.l × b × h = 17 × 1 × 0.1 mm3; volume 1.7 µL) with reservoirs (60 µL) attached at both ends of each microchannel was used. A 90 µL aliquot of mucin in water (1 mg/mL) was mixed with 10 µL of C6@M-CNCs or C6@CNCs in buffer (150 µg/mL), and 30 µL of the mixture was deposited into an end-reservoir of the microchannels. Gentle suction pressure with the help of a syringe was applied to the opposite end-reservoir to fill the microchannels with the mucin-NC mixture. The filled-out microchannels were then imaged (ex/emλ = 470/525 nm) in an inverted Zeiss Axiovert 200 M epifluorescence microscope (NA0.25 lens and Zeiss filter set 43) at 37 °C and for 100 cycles of 5.5 s exposure at 10 s intervals (n = 3).An optically translucent microslide  and data presented as mean ± standard error of the mean (SEM). Data points were expressed as a % of negative control, and unpaired Student’s t-tests were performed. Data points were considered to be significantly different at the p < 0.05 level.All experiments were done in triplicates  using the calibration curve (The C6 was quantified (at 0.78 µg/mL–0.76 ng/mL) in a microplate reader  for both types of CNCs A. Howevet = 1, 4, and 24 h), which indicated that its cellular uptake would only be possible with the assistance of NCs. Interestingly, C6@CNCs displayed a 20–30% cellular uptake while the C6@M-CNCs demonstrated a higher uptake at 24 h than C6@CNCs at 50 µg/mL  and 1.0 × 10−6 (150 µg/mL). On the contrary, the C6@M-CNCs elicited a higher Papp of 1.6 × 10−6 cm/s (50 µg/mL) and 1.2 × 10−6 cm/s (150 µg/mL) at 2 h, suggesting transcellular permeation. Available literature has established the permeation-enhancing role of chitosan and its derivatives—both as a free polymer and NCs [app ≥ 14.0 × 10−6 cm/s are regarded as being highly permeable whereas a Papp of < 5.0 × 10−6 cm/s depicts low permeability. Thus, both prototypes under investigation demonstrated low permeability. Increased surface hydrophilicity is known to facilitate the transport of NCs across Caco-2 monolayers [app for C6@M-CNCs.Both the C6@CNCs and C6@M-CNCs demonstrated a low flux across monolayers . It is w and NCs . Despitenolayers . Thus, tex/emλ = 470/525 nm). On the contrary, the C6@M-CNCs exhibited lesser binding with the mucin, and particle tracking experiments affirmed the flow of these C6@M-CNCs in (mostly) unbound state  in chitosan [hydrophilic brush effect [The disparate mucoadhesion noted for C6@CNCs and C6@M-CNCs can be attributed to a varied electrostatic interaction between the mucin mesh and NCs . It is kchitosan . Thus, th effect ,47. The h effect . It is happ over the non-mannosylated chitosan. Moreover, the C6@M-CNCs exhibited negligible mucoadhesion due to increased hydrophilicity, whereas the CNCs elicited rapid binding to mucin. The C6@M-CNC emerged as a more suitable nano-DDS compared to non-mannosylated C6@CNC. The integrated data highlight the challenges in achieving a delicate balance between stability and optimized release kinetics of CNCs with an entrapped payload. Further optimization of the synthesis and encapsulation of theranostic molecules within modified CNCs are currently being investigated in our labs.Fluorescent crosslinked chitosan and mannosylated chitosan NCs with an encapsulated fluorophore (C6) were prepared. The stability of these NCs in conjunction with their adequate biocompatibility and capacity to entrap a hydrophobic macromolecule make them potentially exciting agents for  drug delivery. The flux across Caco-2 monolayers for C6 from both these NC prototypes was evident, with the mannosylated prototype having an increased P


Human Papillomavirus is responsible for a wide range of mucosal lesions and tumors. The immune system participate in tumorigenesis in different ways. For example, signaling pathways triggered by Toll-like receptors (TLR) play a role in chemotherapy resistance in several tumor types and are candidates for contributing to the development of HPV-induced tumors. Here, we studied the receptor TLR4 and the adaptor molecule SARM1 in HeLa cells, an HPV-positive cervical cancer cell line. Knocking out of these genes individually proved to be important for maintaining cell viability and proliferation. TLR4 knock out cells were more sensitive to cisplatin treatment, which was illustrated by an increased frequency of apoptotic cells. Furthermore, TLR4 and SARM1 modulated ROS production, which was induced by cell death in response to cisplatin. In conclusion, TLR4 and SARM1 are important for therapy resistance and cervical cancer cell viability and may be relevant clinical targets. HPV persistence leads to increased risk of tumor development despite adaptive or innate immunity against the virus4. More than 500,000 woman are diagnosed every year with cervical cancer worldwide and half of them will die from the disease5.The relationship between Human Papillomavirus (HPV) and cervical cancer is well known and established6. In addition, these viral proteins also deregulate the innate immune system by modifying Toll Like Receptors (TLR) expression, interfering with other antiviral responses, triggering persistence and leading to lesion progression7.High-risk HPV E6 and E7 proteins are essential to generate a series of changes in the cell, increasing mutations that will contribute to tumor development8. Despite being present in normal cells, TLRs can also be expressed and activated in tumors, where they can either unleash antitumor effects or increase tumor progression and metastasis11. TLR signaling contributes to tumor cell proliferation, cytokines and chemokines release, angiogenesis, cell survival, chemoresistance and hence, tumor progression13. As such, TLRs are interesting targets for immunotherapy.TLRs play a pivotal role in innate immunity responses against pathogens and in recognizing cellular stressTLR2, TLR3, TLR7, TLR8 and TLR914. On the other hand, TLR4 has higher expression levels in cervical cancer cells compared to normal cells and its expression levels correlates with increased risk of lymph node metastasis16. Also, down-regulation of TLR9 was also observed in HPV16 infected cell lines17. Our group reported modulation of NF-kB by high-risk HPV E6 oncoprotein through TLR pathway-related proteins , IRAK2 and IKKε), which potentiated TLR pathway activation18.The interaction between HPV and TLR has been previously described. Clearance of HPV16 infection is related to increased expression of 19. SARM1 works as a negative TLR regulator, inhibiting the interaction between TRIF adaptor and TLR4 and TLR320. Despite its role in neuronal degeneration, SARM1 has been recently associated with prostate cancer cell line growth and metastasis progression, and has also been found to be overexpressed in HPV-positive tumor cell lines22.There are seven described TLR adaptors of which three are classified as regulatory adaptors: SARM (SARM1), BCAP (PIK3AP1) and SCIM P15. Here, we sought to further dissect the function of the TLR pathway in a cervical cancer cell line focusing on the roles of TLR4 and SARM1 on cell survival and cisplatin resistance.We have previously reported that cervical cancer cell lines expressing E6 and E7 from high-risk HPV types have several alterations on the TLR pathway, such as increased expression of HMGB1, SARM1 and TLR4TLR4 and SARM1 in the malignant phenotype of cervical cancer cells, we knocked out these genes individually in HeLa cells using CRISPR/Cas9 and isolated individual clones of the edited cells. Lack of protein expression was confirmed by western blot  and SARM1KO (clone 3).To study the role of First, we compared cell viability of TLR4KO and SARM1KO cells to the unedited parental HeLa cells with or without exposure to Lipopolysaccharide (LPS) treatment. Depletion of TLR4 or SARM1 induced approximately 25% reduction of cell viability when compared to the parental HeLa cells , which induces cell damage and stimulates apoptosis. Therefore, we measured ROS production in parental, TLR4KO and SARM1KO HeLa cells exposed to the same treatments performed before Fig. a. We notSince cisplatin can induce apoptosis through ROS production, we exposed cells to a ROS inhibitor, N acetyl L cysteine (NAC) to determine if the chemotherapy sensitivity of knockouts were related to ROS levels , Superoxide Dismutase 2 , Cyclins D1 and E1 , and Ki-67 (a marker of cell proliferation) in HeLa cells depleted of TLR4 or SARM1, in comparison to the parental HeLa cells  are responsible for cell cycle progression. The oncoprotein E6 of HPV can activate the transcription factor eIF4E, and its inhibition in cervical cancer cell lines leads to CCND1 downregulation50. Interestingly, cyclin D1 leads to the release of E2F by inducing phosphorylation of pRB, a protein that is already targeted by HPV oncoprotein E752. Despite the role of CCNE1 in the proliferation of normal cell lines, it has been reported as a biomarker for chemotherapy resistance in ovarian cancer and for prognosis in breast cancer54. Both CCND1 and CCNE1 are upregulated in squamous cervical carcinomas55. Our data showed modulation of all three genes, Ki67, CCND1 and CCNE1, by TLR4 and SARM1.We also observed a decrease in proliferation after knocking out SARM1 drastically reduced cell viability and proliferation while lack of TLR4 significantly increased apoptosis and ROS production after cisplatin treatment. Further studies are warranted to explore the potential use of these findings in the clinic.In conclusion, we demonstrate here that, in HeLa cells, knock out of 23, pX330S23 and PX459 (pSpCas9(BB)-2A-Puro)24 .The following plasmids were used for CRISPR/Cas9 editing: pX330ATLR4 editing, while the pX459 vector was used alone for SARM1 editing as previously described24.The pX330A and pX330S vectors were used in combination to build a multiplex system for Guide sequences for gene editing were designed using a specific software (crispor.tefor.net): SARM1 G # F–CACCGCGCCTGCTTCAGCGCGGACA; SARM1 G # R–AAACTGTCCGCGCTGAAGCAGGCGC; TLR4 G # 1F–CACCGATGCCCCATCTTCAATTGTC; TLR4 G # 1R–AAACGACAATTGAAGATGGGGCATC; TLR4 G # 2F–CACCGGGGTACAGGATGCAATTGGC; TLR4 G # 2R–AAACGCCAATTGCATCCTGTACCCC.2 and transfected using the FuGENE HD reagent  according to the manufacturer's protocol at a 3:1 ratio between reagent and plasmids, followed by selection with 2 µg/ml of puromycin. Individual clones were isolated by serial dilution of selected cells in 96-well plates. Alterations were confirmed by Sanger sequencing  in December 2012. ATCC certified cells through several tests, including: mycoplasma contamination, COI assay, STR analysis, Sterility and Human pathogenic virus presence. Upon receipt HeLa cells were divided, frozen and kept in low passage for all subsequent experiments. Cells were grown in MEM culture medium supplemented with 10% fetal bovine serum (M10 medium) at 37ºC with 5% COTo evaluate whether the genes of interest were knocked out, protein extracts were analyzed by western blotting using polyclonal antibodies specific for SARM1  and TLR4 . Cells were lysed with a buffer containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCI pH 8.0, 1% NP-40, 0.5% Sodium deoxycholate and 0.1% SDS supplemented with protease inhibitors . Protein concentration was determined using Bio-Rad protein assay .Cell viability analysis was performed using the PrestoBlue ™ Cell Viability Reagent kit . Cells were seeded in 96-well plates, and 100 μl of complete medium plus 10 μl of PrestoBlue reagent was added to each well. The plates were incubated for 1 h at 37 °C protected from light. Absorbance was measured at 570 and 600 nm. For viability analysis, cells were previously treated with Lipopolysaccharide (LPS)  at 10 µg/mL with and without cisplatin at 4.7 µM  and analyzed after 72 h of treatment.6 cells were seeded in 6-well plates; 24 h later, cells were treated with 10 µg/ml of mitomycin C  for 2 h at 37ºC. A wound healing assay was performed to observe cell migration, after the mitomycin C treatment. Photos were taken at 0 h, 24 h and 48 h, and the area not occupied by cells was measured using the ImageJ software.For clonogenic assays, cells were seeded at low density (1000 cells per well) in 6-well plates. After 2 weeks, cells were fixed with formalin and stained with crystal violet (0.05% w/v). The number of colonies formed was counted automatically using the ImageJ software . For proliferation assays, 5000 cells were seeded per well in 24-well plates and counted every day in triplicates. For migration assays, 3 × 10Apoptosis was determined using the CellEvent™ Caspase-3/7 Green Detection Reagent kit  following the manufacturer's protocol. Apoptosis and necrosis were also determined using double staining with the eBioscience™ 7-AAD Viability Staining Solution and eBioscience™ Annexin V-FITC Apoptosis Detection Kit  and analyzed by flow cytometry.For reactive oxygen species (ROS) determination, cells were cultured in 6-well plates with 10 µg/mL of LPS, 4.7 µM of cisplatin and 100 µM N-acetyl-l-cysteine (NAC) for 72 h, followed by 30 min of incubation with the DCFDA/H2DCFDA reagent  and analysis by flow cytometry.5 cells were seeded per well in 6-well plates and, after 24 h, cells were treated with 10 µg/ml mitomycin for 2 h. Cells were fixed with 70% ethanol, washed with PBS and treated with 5 µl of RNAse , 0.5 µl of 7-AAD and 0.5 µl of Triton X-100  diluted in 500 µl of PBS for 30 min at 37 °C. Cells were then analyzed by flow cytometry.For cell cycle analysis, 10All experiments were performed on a BD Accuri™ C6 cytometer  and the results analyzed with FlowJo software .25.RNA was extracted with TRIzol as described by the manufacturer . After DNase treatment , 1 µg of RNA was used for cDNA synthesis and qPCR with GoTaq® 2-Step RT-qPCR System  following the manufacturer’s protocol. Primers used were: IL18F TGGCTGTAACTATCTCTGTGAA; IL18R TGTCCTGGGACACTTCTCTG; SOD2 F TGAACAACCTGAACGTCACC; SOD2RGTAGTAAGCGTGCTCCCACA; IL6 F CCACACAGACAGCCACTCAC; IL6 R AGGTTGTTTTCTGCCAGTGC; CCND1 F TGAAGGAGACCATCCCCCTG; CCND1 R TGTTCAATGAAATCGTGCGG; CCNE1 F AAATGGCCAAAATCGACAGG; CCNE1 R CGAGGCTTGCACGTTGAGTT; MKI67 F TGTGCCTGCTCGACCCTACA; MKI67 R TGAAATAGCGATGTGACATGTGCT. Expression analysis was performed using the ΔΔCt methodAll experiments were performed at least in triplicates. Results were analyzed using two-way ANOVA with Prism software .Supplementary Information.


ALD1 affects numbers of nodules produced by Lotus japonicus, but AGD2′s role in leguminous plants remains unclear. A combination of enzymatic analysis and biological characterization of genetic materials was used to study the function of AGD2 (LjAGD2a and LjAGD2b) in L. japonicus. Both LjAGD2a and LjAGD2b could complement dapD and dapE mutants of Escherichia coli and had aminotransferase activity in vitro. ljagd2 plants, with insertional mutations of LjAGD2, had delayed flowering times and reduced seed weights. In contrast, overexpression of LjAGD2a in L. japonicus induced early flowering, with increases in seed and flower sizes, but reductions in pollen fertility and seed setting rates. Additionally, ljagd2a mutation resulted in increased expression of nodulin genes and corresponding increases in infection threads and nodule numbers following inoculation with Rhizobium. Changes in expression of LjAGD2a in L. japonicus also affected endogenous SA contents and hence resistance to pathogens. Our results indicate that LjAGD2a functions as an LL-DAP aminotransferase and plays important roles in plant development. Moreover, LjAGD2a activates defense signaling via the Lys synthesis pathway, thereby participating in legume–microbe interaction.Arabidopsis AGD2 (Aberrant Growth and Death2) and its close homolog ALD1 (AGD2-like defense response protein 1) have divergent roles in plant defense. We previously reported that modulation of salicylic acid (SA) contents by  N-hydroxypipecolic acid (NHP) pathways , 0.5 mM K4[Fe(CN)6], 1 mM EDTA, pH 8.0, 0.1 mM PBS buffer, pH 7.0) at 37 °C for 6–8 h after vacuum infiltration for 15 min. For nodule sectioning, 2-week-old nodules were harvested after inoculation with DsRed-labeled M. loti, and sections (50 μm) of fresh nodules were cut with a Leica VT1200 S vibrating-blade microtome.For histochemical assays, seedlings of 4, 0.24 g/L KH2PO4, pH 8.0), using a Leica VT1200 S vibrating-blade microtome.For the analysis of leaf thickness, leaves were fixed with 2% agar, and 50 μm slices were obtained, in 0.1× PBS buffer 2·4H2O. After incubation at 37 °C for 30 min, germinated pollen grains were viewed with a bright-field microscope [For observations of pollen morphology and fertility, pollen grains were harvested from fully expanded flowers. After staining with an iodine–potassium iodide solution, they were viewed with a bright-field microscope. To assess the pollen germination rate, pollen grains were dispersed in a solution of 10% sucrose, 0.01% Hcroscope . Ralstonia solanacearum strain FQY_4 was cultured in SMSA liquid medium  to OD600 = 0.5–0.6, then 100 μL bacterial liquid was coated on SMSA solid medium and grown for 2 days [2 to OD600 = 0.001. Portions (100 μL) of R. solanacearum suspension were injected into the back of fresh leaves (n > 15) of selected plant lines by a 1 mL sterile syringe. After inoculation, the leaves were stored in agar medium containing 10 ppm imidazole. After cultivation in the dark at 28 °C for 2 days, the leaves were exposed to light for 16 h at 22 °C and dark for 8 h at 18 °C for 2 days. Leaf discs were made with a puncher, washed with sterile water five times and ground with a pestle in 10 mM MgCl2. The resulting homogenates were fully mixed and continuously diluted. A 100 μL diluted bacterial solution was spread on SMSA solid medium, and the number of R. solanacearum colonies was counted 48 h later.r 2 days . The bacg for 30 min, and then absolute ethanol was added to the supernatant to a final concentration of 80% to precipitate protein. After further centrifugation at 3000× g for 30 min, the supernatant was transferred to another tube and dried under vacuum. The dry matter was dissolved in 400 μL of 0.01 N HCl, and the resulting solution was filtered using a 0.45 μm filter membrane. Finally, 50 μL portions of the filtrate were subjected to analysis with an automatic amino acid analyzer  using the C4 free 41AA standard procedure, an LCA K07/Li 4.6 × 150 mm chromatographic column and analyte detection wavelengths of 570 and 440 nm.Salicylic acid was extracted as previously described . For theThe numbers of independent repetitions of each experiment are given in the corresponding figures and tables. Statistical significance was tested as described in the given figures and tables. For all assays, differences between samples or time points with probabilities < 0.05 and 0.01 are indicated in figures by single and double asterisks, respectively. SPSS Statistics V17.0 software was used for all statistical analyses.LjAGD2a and overexpression of LjAGD2a affected shoot, leaf, flower, pod, seed and pollen development, which suggests that LjAGD2a plays important roles in plant development. LjAGD2a, like LjALD1, has a positive role in plant–pathogen interaction and a negative role in legume–rhizobia symbiosis. Our results also indicate that LjAGD2a activates defense signaling via the Lys synthesis pathway, thereby participating in legume–microbe interaction.Our results indicate that LjAGD2a functions as an LL-DAP aminotransferase. We examined how mutation of


N-glycans, we show that galactosylation at a specific location of N-glycans in an afucosylated anti-viral mAb is responsible for FcγRIIIA binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. We report a facile method to obtain purified asymmetric mono-galactosylated biantennary complex N-glycans, and their influence on bioactivity upon incorporation into an afucosylated mAb. Using ELISA, surface plasmon resonance and flow cytometry, we show that galactosylation of the α6 antenna, but not the α3 antenna, consistently increases FcγRIIIA binding affinity. We confirm its relevance in an anti-viral model of respiratory syncytial virus (RSV) using an adapted ADCC reporter assay. We further correlate this structure-function relationship to the interaction of the galactose residue of the α6 antenna with the protein backbone using 2D-1H-15N-NMR, which showed that galactosylation of at this location exhibited chemical shift perturbations compared to glycoforms lacking this galactose residue. Our results highlight the importance of identifying and quantifying specific glycan isomers to ensure adequate quality control in batch-to-batch and biosimilar comparisons.Monoclonal antibodies (mAbs) comprise an essential type of biologic therapeutics and are used to treat diseases because of their anti-cancer and anti-inflammatory properties, and their ability to protect against respiratory infections. Its production involves post-translational glycosylation, a biosynthetic process that conjugates glycans to proteins, which plays crucial roles in mAb bioactivities including effector functions and pharmacokinetics. These glycans are heterogeneous and have diverse chemical structures whose composition is sensitive to manufacturing conditions, rendering the understanding of how specific glycan structures affect mAb bioactivity challenging. There is a need to delineate the effects of specific glycans on mAb bioactivity to determine whether changes in certain glycosylation profiles (that can occur during manufacturing) will significantly affect product quality. Using enzymatic transglycosylation with chemically-defined  N-glycosylation site at N297 of each of the two heavy chains. Protein glycosylation is a post-translational modification that is dynamically regulated by numerous glycosyltransferase and glycosidase enzymes during protein synthesis in the endoplasmic reticulum and Golgi bodies, and by these enzymes that may be in the cell or in the extracellular matrix  are an important class of glycoprotein-based biotherapeutics that are used to treat cancer, inflammation and viral disease, of which many marketed products are of the IgG1 class . The Fabr matrix . As suchr matrix .N-glycan structures can greatly influence mAb bioactivity by altering its binding affinity to Fc receptors (FcR) on the surface of immune cells. Amongst the most widely studied glycan structure is the core fucose residue in N-glycans, where its presence decreases ADCC activity  and RSV-G to host cell surfaces (Nicotiana benthamiana) yields afucosylated agalactosylated complex  and high mannose glycans , which increased FcγRIIIa binding affinity and decreased viral titre in the lungs of Cotton Rats  by HPAEC-PAD analysis  was first characterized using high pH anionic exchange chromatography-pulsed amperometric detection (HPAEC-PAD). Palivizumab was treated with the endoglycosidase PNGaseF to release free N-glycans (i.e. between the two GlcNAc residues closest to the N297 position of the protein backbone) with EndoS D233Q leaves the palivizumab protein backbone with a single GlcNAc and a core-1,6-fucose residue, on each heavy chain , With our goal to perform glycosylation-based structure-function studies on palivizumab, we used a glycoengineering strategy involving enzymatic transglycosylation , 32. Alt+, m/z 366.07) and its complementary fragment  and its complementary fragment  was first extracted from egg yolk  and treaPurified glycans were reacted with 2-chloro-1,3-dimethylimidazolinium chloride (DMC) and trimethylamine to obtain the glycan oxazoline precursors that were then used with EndoS D233Q for transglycosylation. Glycan-remodelled palivizumab were purified using Protein A resin, which included washing with 0.5 M sucrose solutions prior to mAb elution to remove unreacted glycan starting materials.Completion of the reactions were monitored by SDS-PAGE and intact mass spectrometry, To assess the role of varying glycan structures on palivizumab, we first examined whether changes in Fc glycosylation affects the binding of the Fab region to its targeted RSV F-protein, using RSV-F recombinant protein-immobilized ELISA. Consistent with previous reports, varying galactosylation or fucosylation levels by enzymatic transglycosylation did not affect Fab binding, as EC50 values were not statistically significant between commercial (ie. most fucosylated), deglycosylated and afucosylated glycans with varying galactosylation  of FcγRIIIa (CD16A), a receptor primarily involved in ADCC. Using ELISA (D) of glycoforms bearing the galactose at the α6 antenna is twice as low as glycans lacking a galactose at this position . A similar case is observed with the binding affinity of the various galactosylated afucosylated glycoforms to the low-binding F176 isoform, albeit at an approximate five-fold decrease of each glycoform.We further confirm these observations using surface plasmon resonance (SPR) by immobilizing the mAbs to Protein A-bound slides. With respect to binding to the higher-affinity V176 isoform, the dissociation constant 2SO4 as the sole source of nitrogen, yielding a fragment that is over 98% labelled. Yeast expresses afucosylated high mannose glycans, and cleavage with Endo H produced the peptide bearing only a single GlcNAc residue at the N297 position. Treatment with EndoS D184M and the purified glycan oxazolines with variable galactosylation was performed, and completion was monitored by SDS PAGE . Since terminal galactose residues do not have proton-nitrogen pairs, chemical shift perturbations (CSPs) of the Fc protein backbone amides produced by the proximity of the galactose moiety were measured. The advantage of this approach is that the chemical shift of a single backbone amide pair is an absolute measurement of the magnetic environment surrounding this chemical group. In contrast, the intensity of a given NMR resonance can carry more variability due to many experimental factors , this difference in primary sequence is sufficiently distant from residues surrounding the glycans that assignment could be transposed.Two dimensional (2D) i et al.  for a moi et al.  were tra1H-15N-HSQC NMR of the Endo H-deglycosylated 15N-Gn-NISTmAb-Fc with the agalactosylated G0 glycoform -NISTmAb-Fc) induces greater CSPs of the peptide backbone  (i.e.15N-G1 (3)-NISTmAb-Fc, i.e. G2). Monogalactosylated 15N-G1 (6)-NISTmAb-Fc shows significant overlap and few CSPs compared to the digalactosylated 15N-G2-NISTmAb-Fc (15N-G1 (3)-NISTmAb-Fc  and G2) are less than 0.02 ppm . As a control peak that showed minimal CSPs between samples, D312 was used, which had CCSDs of less than 0.01 ppm, regardless of the terminal galactose position.We initially examined the 2D H3 domain, while the fewer and weaker CSPs for the G1 (3) are consistent with a galactose moiety more exposed to the solvent, away from the protein backbone (Mapping of twelve of these CSPs on the X-Ray structure (PDBID: 4byh)  highlighbackbone , which wOur method to generate asymmetrical mono-galactosylated glycans provides a facile alternative procedure to that previously reported , 39. Alti.e. G2) of the anti-viral mAb palivizumab increases binding, corroborating a recent report with polyclonal serum-derived IgG (IVIG) used for anti-inflammation (i.e. G1 (6)) is required to impart this increased binding affinity. In its absence (i.e. G0 and G1 (3)), the binding affinity is comparable to the agalactosylated afucosylated (G0) glycan. Our KD values for both high- (V176) and low- (F176) affinity FcγRIIIA obtained by SPR analysis are within the same magnitude as those previously reported; for example, our digalactosylated afucosylated (G2) palivizumab glycoform had KD values of 5.9 ± 0.8 nM and 25.6 ± 1.3 nM with the high- and low-affinity receptors respectively, compared to 2 nM and 25 nM for G2-rituximab (i.e. lacking the Fab region) comprising G2 (64 nM) (16). Our observed decrease in binding affinity by two-fold upon removal of the galactose on the α3-antenna (KD = ~ 6 nM for G1 (6) and G2 vs ~ 11-12 nM for G0 and G1 (3) for high-affinity FcγRIIIA, and ~ 25-31 nM for G1 (6) and G2 vs ~ 60 nM for G0 and G1 (3) for low-affinity FcγRIIIA) is similar to the 1.5- to 1.7-fold decrease previously reported between agalactosylated and digalactosylated afucosylated glycoforms (i.e. G0 vs G2)  . Our obs0 vs G2) . However0 vs G2) . Interes4 nM . OuN. benthamiana changed the glycoprofile from predominantly fucosylated glycans into one that comprised 79% agalactosylated, afucosylated (G0) glycans, and 17% of high mannosylated (Man 7/9) glycans (i.e. G0) glycoform. However, it is important to note that our comparison between the afucosylated glycoforms and commercial product (that is mostly fucosylated) is consistent with previous literature that removal of the core fucose residue remains a dominant feature to increase FcγRIIIA binding. Rather, our results provide further corroborating evidence with more recent studies that galactosylation further increases ADCC via FcγRIIIA binding , and show that the terminal galactose moiety of the α6-antenna is far from any Fc- FcγRIIIa contacts  compared to the agalactosylated glycoform, indicating that the α6 antenna is in close proximity to the protein backbone and is consistent with reduced mobility; our use of 15N-labeled Fc with defined monogalactosylation at the α6-antenna shows that this occurs independently of galactosylation at the α3 antenna. Conversely, the spectrum of 15N-labeled Fc comprising monogalactosylation at the α3 antenna show small and weak CSPs which is consistent with a solvent-exposed galactose with greater mobility, and occurs independently of the presence of an α6-antennal galactose. It was not useful to measure CCSD for the perturbations observed in G1 (3) relative to G0 since the changes that are observed are either a change of peak shape (narrowing or broadening) or simply disappearance, depending of the frequency of the change in the local magnetic environment. The only residue that shows a clear CSP is T335 with a CCSD of 0.05 ppm where the peak shape has also changed (narrowing). The fact that the spectral overlay of G0 and G1 (3) is mainly showing changes in peak shape is consistent with a mobile galactose moiety that is mainly exposed to the solvent with little to no interactions with the protein backbone. The presence of the afucosylated α6-antennal galactose causes CSPs in a region closer to the CH3 domain , and are in line with a previous study showing that residues F241 and F243 are in close proximity with this galactose in fucosylated digalactosylated glycans (G2F), and mutation of these residues increase glycan mobility and reduce FcγRIIIA receptor affinity (i.e. G1 (6) or G2) reduces mobility of afucosylated Fc-glycans by interacting with the Fc protein backbone, which enhances FcγRIIIA receptor affinity, similarly to previous reports with fucosylated glycans . Therefmobility . Mutatiomobility , 54. Theaffinity . It is t glycans . HoweverWhile afucosylation remains the predominant factor for FcγRIIIa binding and ADCC activity, this two-fold increase in activity based on the location of the terminal galactose residue highlights the importance to carefully characterize glycan structures in biotherapeutics. From a regulatory perspective, depending on the mechanism of action, this may warrant attention when evaluating physicochemical data to assess batch-to-batch variability and comparing biosimilars to existing innovator mAb products.2O) over 10 min at 30°C was used to monitor glycan products. Following reaction completion, resin was removed by passing through a solid phase extraction tube and rinsed with MQ water. The reaction lyophilized, resuspended in MQ water and filtered through a 0.22 µm PVDF filter before it was purified by PGC-HPLC to isolate G1 (3)T and G2T glycans . Following an initial mobile phase composition of 1% MP-D: MP-E , a gradient of 6.0% to 13.7% MP-D: MP-E was used from 1.0 min to 42.0 min at 5 mL/min, at 40°C. Glycans were detected at 214 nm using a UV detector, collected using a fraction collector, analyzed by HPAEC-PAD prior to fraction combination, and lyophilized. Impure samples were re-purified as above. The β- and α- anomers of G1 (3)T eluted at approximately 23.0 and 28.0 min, while the anomers of G2T eluted at approximately 32.0 min and 37.0 min.Sialoglycoprotein (SGP) was isolated from commercially available egg yolk as previously described . Glycans2 (pH 5.7), and 4.55 mL MQ water. 150 uL β-1,4 galactosidase  was then added, and the reaction was incubated at 30°C overnight. The reaction was monitored by HPAEC-PAD and purified by PGC-HPLC as above. The β- and α-anomers of G0T eluted at approximately 17.0 and 22.0 min, while the anomers of G1 (6)T eluted at approximately 26.0 min and 31.5 min.To prepare glycans containing zero or one terminal galactose residue (at the α6 antenna only) T), G2T was diluted to 5 mg/mL (1.0 mL) with 150 µL of KCl (500 mM), 150 µL of a buffer containing 500 mM sodium acetate and 50 mM CaClWith each purified glycan in hand, each was converted to their respective oxazoline intermediates by reaction with 2-chloro-1,3-dimethylimidazolinium chloride  and trimethylamine  at 4°C, and purified using 0.1% ammonium hydroxide in a column packed with G25 resin . Collected fractions were monitored by HPAEC-PAD, and those containing purified oxazolines were combined, lyophilized and stored at -80°C until use.2PO4, 100 mM NaCl, pH 8.0). To the solution, 250 µL rinsed Protein A resin slurry  was added and incubated at 4°C with gentle rocking for 5 min. The solution was then passed through a solid phase extraction tube and washed with cold Protein A binding buffer (0.75 mL x 2), followed by a 50 mM sucrose solution in PBS (1.0 mL x 2) and then cold Protein A binding buffer (0.75 mL x 2). Palivizumab was then eluted from the Protein A column using 50 mM sodium citrate buffer (pH 3.5), and neutralized with 200 µL of Protein A binding buffer. Eluted palivizumab was monitored using SDS PAGE and fractions were combined and passed through Amicon centrifugal filters (30 kDa MWCO) to buffer exchange with Citrate Buffered Saline  and concentrate palivizumab. Solutions were stored at 4°C at approximately 0.3 – 1.0 mg/mL, as quantified by nanodrop (at Absorbance of 280 nm).Methods were adapted from previously reported work , 32, witReaction progress for glycan remodeling of palivizumab was monitored and characterized using reducing SDS PAGE and intact protein mass spectrometry. For reducing SDS PAGE, samples reduced in β-mercaptoethanol for 5 mins at 100°C, followed by loading onto 10% polyacrylamide gels which were then run at 200 V for 70 mins. To ensure accurate analysis of band migration at various glycosylation states, a lane was run comprising both starting material and final product within the same lane.For mass spectrometry, samples were analyzed with an Orbitrap Fusion Lumos Tribrid Mass Spectrometer coupled to an Easy-nLC 1200 (Thermo Scientific) that was calibrated by infusion prior to analysis with a mixture of caffeine, MRFA, and Ultramark 1621. 0.5 µL of each sample was analyzed by loading onto a C4 Pepmap300 PS-DVB trap column (300A x 5µm x 5mm) and desalting with 0.1% formic acid in water (solvent A) before separating on a PepSwift Monolithic PS-DVB analytical column (200 µm x 25 cm) at 50°C. Chromatographic separation was achieved at a flow rate of 1 µl/min over 25 min in five linear steps as follows (solvent B was 0.1% formic acid in 80% acetonitrile): initial, 0% B; 5 min, 50% B; 10 min, 50% B; 20 min, 90% B; 25 min, 90% B. The eluting proteins were analyzed in Intact Protein mode with Orbitrap MS at 15000 resolutions, from 500-4000 m/z with auto maximum injection time, normalized AGC target of 50%, and 10 microscans. BiopharmaFinder3.0 (Thermo) was used to process the data. Protein deconvolution was performed with the ReSpect algorithm, using mass output of 130 – 160 kDa, Model Mass Range of 145000 – 150000, Charge State Range 40-80.Following palivizumab transglycosylation reactions, remodelled glycans were characterized using PNGaseF  to release glycans, followed by HPAEC-PAD analysis. For HPAEC-PAD analysis, a Dionex IC-3000 instrument was used, equipped with a Dionex CarboPac PA200 IC column  and a Gold Standard PAD waveform with an AgCl electrode. For glycoprofile analyses, a flow rate of 0.4 mL/min, and the following gradient was used: 20%/0%/80% to 25%/5%/70% of MP-A/MP-B/MP-C over 25 mins at 30°C, followed by an increase to 0/80/20 over the next 3 mins, and then re-equilibration to 20/0/80 over the next 10 min.2SO4. Plates were immediately read on a BioTek Plate reader at 450 nm.Recombinant human FcγRIIIA proteins were immobilized onto MaxiSorp plates  overnight at 4°C, at 100 µL of 0.33 µg/mL for FcγRIIIA V176 , or at 0.67 µg/mL for FcγRIIIA V176F . Following washing with 0.05% Tween 20 in PBS pH 7.4 (PBS-T) and blocking with 1% bovine serum albumin  in PBS-T, each palivizumab glycoform was serially diluted 2-fold in 1% BSA/PBS-T and 100 µL was added to each well. Plates were gently rocked for 20 min at room temperature, followed by incubation at 4°C overnight. The next day, wells were thoroughly washed with PBS-T, followed by addition of goat anti-Human IgG F(ab’)2 Secondary Antibody-HRP . The plate was then gently rocked at room temperature in the dark for 45 mins, followed by thorough washing with PBS-T. 50 µL of Ultra-TMB  was added, and the plate was gently rocked in the dark for 10 mins, before quenching with 50 µL of 2M HSamples were performed by the National Research Council Human Health Therapeutics Research Centre. Experiments were carried out using a Biacore T200 SPR instrument  at 25°C with a running buffer comprising PBS with 0.05% Tween 20 and 3.4 mM EDTA. Protein A  was immobilized onto two sequential CM-5 sensorchip surfaces at 2000 RUs each using standard amine coupling within the Biacore Immobilization Wizard. Palivizumab analogues were captured onto the second protein A surface at 2.5 µg/mL for 30 s, with the first protein A surface remaining as a blank control surface. FcγRIIIA CD16A isoforms (high binding (V) and low binding (F) variants) were produced and purified by the National Research Council (Canada), as described previously . Sensorg6 cells/mL, and 40 µL was added into each well and mixed gently with a pipette, followed by gentle rocking at 4°C for 30 mins. Plates were then washed with 0.5 mL of cold 1% BSA/PBS, centrifuged at 1700 x rpm (7 min) and supernatant removed.NK92-GFP-CD16A cell lines were purchased from American Type Culture Collection  and cultured according to manufacturer protocols. 2.5-fold serial dilutions of palivizumab glycoforms were prepared (80 µL per well) in cold deep-well 96-well plates and kept at 4°C, using cold 1% bovine serum albumin  in PBS as the diluent. To prepare cells for palivizumab-FcγRIIIa binding experiments, NK92 were harvested, and washed with 1% BSA/PBS. Following re-suspension in 1% BSA/PBS with 2 mM EDTA, cells were counted using a haemocytometer. NK92 cells were mixed thoroughly, diluted to 0.625 x 10® 647-conjugated Goat Anti-Human IgG - F(ab’)2 specific (AffiniPure F(ab’)2 Fragment)  was then added, and incubated with gentle rocking at 4°C for 30 mins. As a positive control, APC-CD16 Ab, anti-human, REA423  at 1/50 dilution was used. As a negative control, the aforementioned AlexaFluor-647 AffiniPure F(ab’’)2 Fragment was added, in the absence of any palivizumab. Then cells were washed with 1 mL of cold 1% BSA/PBS with 2 mM EDTA and centrifuged as above, followed by re-suspension in 135 µL of 1% BSA/PBS, and transferred into a shallow 96-well plate containing 45 µL of 4% PFA/PBS. Shallow plates were incubated with gentle rocking at room temperature for 30 min, followed by centrifugation at 100 xg (1 min) and removal of 140 µL of supernatant. Cell pellets were re-suspended in 80 µL of 0.4 M glycine  in PBS. For each biological replicate , technical duplicates were performed.To each well, 150 µL of a 1/300 dilution of Alexa FluorPlates were then read using a BD LSR II Flow cytometer , equipped with a multi-well plate reader and the following laser/filter sets: Blue laser 488nm (20mW) with GFP filter set: 505 Long Pass 525/50 Band Pass and SSC filter set: 488/10 Band Pass and FSC; Red Laser 640nm (40mW) with APC/AF647 filter set: 660/20 Band Pass. Voltages were optimized in initial experiments and saved as application settings. Application settings were used in FACS DIVA for all voltages in all subsequent experiments to keep measurements consistent between experiments. Data was analyzed using FlowJo (v 10.8.1), and the gating hierarchy is as follows: (i) FSC-A vs SSC-a, (ii) FSC-H vs HSC-W, (iii) SSC-H vs SSC-W, (iv) GFP+, (v) AF647+/-.4 cells/well in 100 µL of DMEM media with 10% low IgG FBS , and incubated at 37°C for 24 h. The next day, cells were infected with 100µL/well of RSV in DMEM containing 10% low IgG FBS, at a MOI of 5, with followed by incubation at 37°C for 24 h. RSV was removed, and wells were washed with PBS (200 µL). 25 µL of RPMI 1640 containing 10% low IgG FBS was immediately added to each well. Solutions of palivizumab glycoforms were diluted in RPMI 1640 with 10% low IgG FBS and 25 µL were added into each well, and incubated for 37°C for 30 min. A vial of effector cells was thawed according to manufacturer protocols, and 25 µL were added into each well and incubated for 37°C for 4 h 45 min. 75 µL Bio-Glo luciferase reagent was then added to each well and the plate was immediately read on a BioTek plate reader.ADCC assays were performed using the ADCC Reporter Bioassay, V Variant . To adapt it to the RSV model, Hep2 cells were first cultured in a 96-well plate at a cell density of 2.0 x 102PO4 at pH 6.0, 10 g/L of 15N-ammonium sulfate, 10 g/L D-glucose, 20 µg/L of biotin, was inoculated with a single colony of Pichia X33-NIST-mAb-Fc and incubated at 29°C until an optical dispersion (OD600) of 0.4 is obtained. An aliquot of 22 mL of this culture was used to inoculate a 450 mL of YNB growth media and incubated at 29°C until an optical dispersion (OD600) of 6.6 is obtained. Optical density is accurately measured with a 1:10 dilution of an aliquot from the culture with water. In order to remove unspent glucose that would inhibit protein synthesis, cells were harvested by centrifugation @ 3000 X g for 10 minutes then re-suspended in i-wash media  and incubated at 25°C for 1 h. Cells were then centrifuged and re-suspended in 25 mL in wash media, and sufficient amount of the slurry (18 mL) to obtain an OD600 of 1.0 was used to inoculate 2 X 1 L of induction media (same composition to YNB growth media with the glucose replaced by 10 mL/L of methanol). Protein induction was carried out for 72 hours at 29°C and 10 mL of methanol 5% were added per liter at 24 h and 48 h post-induction. Cells were separated from media containing 15N-NIST-mAb-Fc by centrifugation at 3000 X g for 10 minutes at 4 °C. To inhibit proteolytic cleavage of the target protein, benzamidine (5mM) and EDTA (0.5 mM) were added, then the pH of the media at 5.84 was adjusted to 7.3 with the addition of potassium hydroxide (10 N). After pH adjustment, a white precipitate formed. After 30 minutes, the precipitate was separated by centrifugation.A starting culture in 50 mL yeast nitrogen based (YNB) media, containing 3.4 g/L of YNB powder without amino acids (Sigma), 100 mM KH15N-NIST-mAb-Fc were dialysed against 50 mM sodium citrate pH 5.5 prior to glycan removal. The high mannose rich glycan was cleaved using 20000 units of Endo H  per 5 mg of protein for 2 hours at 37°C. The reaction mixture was then purified using the Protein A affinity chromatography, then fractions containing the target protein were dialyzed against 50 mM sodium acetate pH 5.0.The target protein was purified by affinity chromatography by loading the solution on a 15 mL protein A column at 1 mL/min. Elution of the protein was carried out with 0.1 M glycine-HCl at pH 3.0 after a wash of the resin with 1x PBS (with protease inhibitors). Fractions containing the Data acquisition was acquired as previously reported . All NMR1H and 15N with respect to a reference cross peak . Non-linear regression  was used for curve-fitting, and differences amongst two treatments were assessed using one-way ANOVA with Tukey 10.6084/m9.figshare.20171174.v1.Mass Spectrometry data presented in the study are deposited in the FigShare repository, doi: 15N-NISTmAb-Fc proteins, and data analysis. LT performed binding and cell-based assays. GG expressed 15N-NISTmAb-Fc proteins. AS performed flow cytometry experiments and analyzed data. XL conceptualized experiments provided resources for cell culture experiments and edited manuscript. YA performed NMR experiments, experimental conceptualization, data interpretation, wrote and edited manuscript. RT conceptualized experiments, synthesized glycans, performed cell-based assays, data analysis and interpretation, wrote and edited manuscript. All authors contributed to the article and approved the submitted version.GH synthesized glycans, performed glycan remodeling on monoclonal antibodies/This work was funded by the Government of Canada.We thank Yi-Min She, Lisa Walrond and Marybeth Creskey of the Mass Spectrometry lab at the Regulatory Research Division at Health Canada for performing mass spectrometry sample analyses. We thank Drs. Jessie Lavoie, Michael Johnston, Simon Sauvé and Michael Rosu-Myles for critical review of this manuscript and thoughtful comments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Sedation in standing procedures is commonly performed in elephants. To successfully and safely initiate standing in elephants, chemical restraint drugs should provide sufficient sedation with minimal complications . This study investigated the sedative effects of dexmedetomidine in Asian elephants. Results suggest dexmedetomidine 1–2 µg/kg provides effective sedation. However, we suggest a single intramuscular dexmedetomidine injection of 2 µg/kg for approximately 70 min of sedation. This is the first study to demonstrate dexmedetomidine use in Asian elephants.2) and clinical observations were monitored during the study and for 3 days post drug administration. D2 provided the longest sedation (approximately 70 min), compared to D1 and D1.5. After Ta, each elephant’s sedative stage lessened within 10–15 min without complications. No significant abnormal clinical observations were noted throughout and during the 3-days post study period. These data suggest that a single 2 µg/kg IM dexmedetomidine injection provides sufficient standing sedation for approximately 70 min in Asian elephants.This study investigated the sedative effects of dexmedetomidine in Asian elephants. We hypothesized that 2 µg/kg dexmedetomidine would provide sufficient standing sedation. A crossover design study was performed in three Asian elephants. Each elephant was assigned to 1 of 3 treatment groups—1 (D1), 1.5 (D1.5) or 2 (D2) µg/kg dexmedetomidine  with a two-week ‘washout period’ between doses. Elephants were monitored for 120 min. At 120 min (Ta), atipamezole was administered IM. Sedation and responsiveness scores were evaluated. Physiological parameters (pulse rate, respiratory rate, and %SpO Due to the large size of elephants, and to minimize general anesthesia-related complications, standing procedures are commonly performed. To successfully perform standing procedures, restraint is crucial. Correct restraint methods should provide safety to both humans and animals. However, it is challenging to execute elephant restraint safely. Chemical immobilization is one of the most common restraint methods. Ideal chemical restraint drugs should provide sufficient standing sedation with minimal complications . To chemically immobilize elephants, α2 adrenergic agonists, i.e., xylazine , detomidDexmedetomidine, an α2 adrenergic agonist, has been used widely in domestic and exotic animals including wildlife, dogs ,6, cats The aim of this study was to investigate the sedative effects of dexmedetomidine in elephants. We hypothesized that 2 µg/kg dexmedetomidine would provide moderately heavy sedation for a longer duration than 1 or 1.5 µg/kg dexmedetomidine in Asian elephants. To our knowledge, this is the first report evaluating dexmedetomidine’s sedative effects in Asian elephants.Three healthy, non-musth male Asian elephants (elephant 1 body weight (BW) 2870 kg, age 28 years; elephant 2 BW 2900 kg, age 20 years; elephant 3 BW 3865 kg, age 27 years, average age, 25.7 ± 5.1 years old, average BW 2800 ± 900.5 kg) from the National Elephant Institute, Forest Industry Organization, Lampang, Thailand, were used in this study. All animal procedures were approved by the Animal Ethics Committee, Faculty of Veterinary Medicine, Chiang Mai University, Thailand (S23/2562). All elephants served as tourist trekking animals or performed in shows for no more than 3 h each day (between 08.00 and 15.00 h). During the day, and when not working, bulls were maintained in a shed among different male groups and provided grass, bananas, sugar cane, hay, rice grains, and water ad libitum. Later on in the afternoon, they were separately tethered with a 5 m long chain near a caretaker’s (mahout) house overnight. The animals received a semiannual physical examination conducted by a veterinarian. Blood from each bull was collected at the National Elephant Institute, where hematological and biochemical profiles were performed. All elephants used in this study were deemed healthy. On the day of the study, animals were kept fasted  and weighed beforehand. Fifteen minutes before drug administration, elephants were brought into a stall; an intravenous catheter was used  which administered normal saline (0.9% NaCl), 5 mL/kg/hr.®; Orion Corporation, Espoo, Finland); (2) dexmedetomidine 1.5 µg/kg (D1.5); or (3) dexmedetomidine 2 µg/kg (D2). Dexmedetomidine was injected (T0) intramuscularly  into the triceps brachii muscle. All elephants were supported with chest belts hanging from a pulley to avoid an accidental fall. This study employed a crossover experimental design, with each drug dose administered according to a 14-day washout interval; thus, sedation was conducted three times in each bull (14 days apart) as outlined in Elephants were randomly assigned to 1 of the following 3 treatment groups: (1) dexmedetomidine 1 µg/kg , was administered  into the ipsilateral triceps brachii muscle, but at different injection sites. Elephants recovered in the sedation stall. Once fully recovered, they were returned to their stalls and monitored daily for 3 consecutive days.At the end of each experiment , atipamezole  and post-dexmedetomidine injection (T0) every 5 min for 120 min. A responsiveness assessment was performed at pre- and post-drug administration every 15 min until min 120; then, an atipamezole was administered (Ta). They were also monitored post atipamezole administration for 30 min, as illustrated in Sedation assessment criteria  are shown in The criteria  are shown in Three blinded, experienced elephant veterinarians evaluated sedation and responsive assessment.Vital sign parameters  were assessed every 5 min from −15 to 150 min , as presented in Elephants were monitored for other clinical signs  throughout the study and at 3 days post-injection. Data are presented as mean ± standard error of the mean (SEM). The differences in the means of sedation score and vital sign values among drug dose groups were compared using ANOVA. Since the Latin square design is employed, the ANOVA model corresponds to the design as follows:Elephants’ weights were not significantly different from week 1 to week 6 throughout the study.Sedation scores (scores 0–5) were observed in all three groups immediately after dexmedetomidine administration and lasted for 10 ± 1.0 (D1), 10 ± 0.1 (D1.5), and 10 ± 0.2 (D2) min. A mild sedation (scores 6–11) duration among the three groups was also observed for 10 ± 0.1 (D1), 10 ± 0.9 (D1.5), and 5 ± 0.4 (D2) min. The onset of moderate-heavy sedation (scores 12–18) was observed for 20 ± 1.5 (D1), 20 ± 2.4 (D1.5), 15 ± 0.6 (D2) min. The duration of moderate-heavy sedation was longer in D2 (70 ± 2.5 min) vs. D1 (50 ± 2.0 min) and D1.5 (55 ± 1.9 min).To lighten the sedative plane, atipamezole was administered. After atipamezole administration (Ta), there was no difference between elephants regarding the onset of slight sedation (lightened sedation plane towards recovery) in the three groups , as shown in A lower responsiveness score (slight responsive) correlates with heavier sedation. The duration of elephants presenting with a continuously low responsiveness score [0–6 (slightly responsive)] was longer in D2 (60 ± 3.0 min) vs. the D1.5 (45 ± 1.1 min) and D1 (15 ± 2.2 min) elephants. D1 elephants returned to a responsiveness score of 12–18  as early as 90 min after a dexmedetomidine injection. Immediately after Ta at 120 min, the D1.5 and D2 elephants returned to a responsive score of 12–18 (lightened sedation plane), as shown in At T0, the pulse rates among the three groups were 37 ± 0.4 (D1), 30 ± 1.2 (D1.5), and 25 ± 2.2 (D2) bpm. The respiratory rate of each group was 7 ± 0.1 (D1), 8 ± 1.0 (D1.5), and 6 ± 2.3 (D2) tpm. The pulse rate and the respiratory rate in three groups were measured throughout the study as presented in 2 of all three groups exceeded 95% throughout the study (data not shown).The %SpOClinical observation: Hypersalivation and periodic snoring were observed in all elephants at all doses, and they were not noticeable by the end of the study. There were no other abnormal clinical signs  observed throughout the study or during the 3 day post-study period.2 among the three groups. There were no significant abnormal clinical observations during the study and post-study period. The current data support our hypothesis that 2 µg/kg dexmedetomidine provides moderate to heavy sedation for a longer period than 1 or 1.5 µg/kg doses in Asian elephants.This study demonstrated that 2 µg/kg dexmedetomidine successfully provided moderate to heavy sedation for 70 ± 2.5 min, which was longer than for the 1 µg/kg (50 ± 2.0 min) or 1.5 µg/kg (55 ± 1.9 min) doses in Asian elephants. There was no difference in recovery time (return to slight sedative stage) among the three groups. There was no difference in pulse rate, respiratory rate, and %SpO2). Due to dexmedetomidine’s high α2 receptor selectivity, it was expected to provide sufficient sedation with minimal side effects. In this study, D2 provided the longest moderate–heavy sedation (70 min) without significant abnormal clinical observations. Only mild hypersalivation and snoring were observed in the three groups and these effects subsided by the end of the study and did not reoccur in the 3-day post-procedure period.To our knowledge, this is the first study to examine the effects of dexmedetomidine use in Asian elephants. The aim of this study was to investigate the effectiveness of a relatively new sedative drug, dexmedetomidine, using three different dosages in elephants. This study included modified criteria for sedative scores, responsiveness scores, and vital sign parameters  elephant’s doses are 2–4 µg/kg [Because ideal chemical restraint drugs should provide sufficient sedation with minimal side effects, choosing the doses and the route of administration requires strong consideration of potential side effects and practicality. Most sedatives provide sedation in a dose-dependent manner; therefore, a higher dose should elicit heavier sedation, and possibly, a higher incidence of side effects. Because the actual dexmedetomidine dose in Asian elephants is unknown, we chose doses based on the closely related α–4 µg/kg ,17.2), under clinical observation, extending into a 3-day post drug administration period. Like other α2 adrenergic agonists, dexmedetomidine can cause dose-dependent cardiorespiratory depression [2 remained above 95% in room air throughout the study. Although 100% O2 supplementation is strongly encouraged when possible, it was not provided because we aimed to mimic the clinical conditions of the farm or field where 100% O2 may be unavailable. Neither apnea nor penile prolapse were observed. Hypersalivation and snoring were observed, although neither were noticeable at the study’s end or during the three-day post-procedure period. Although blood pressure was not recorded, dexmedetomidine has been reported to cause hypertension followed by hypotension [When evaluating sedative drugs, it is vital to observe and assess potential drug complications. This current study included such assessments of physiological parameters  require moderate–heavy sedation. Therefore, the moderate–heavy sedation level, together with low responsiveness scores  was selected to achieve sufficient sedation. The D2 group provided the longest sedation with continuous low responsiveness scores (4–6 or lower). This current study confirms that these scores were useful to evaluate dexmedetomidine’s effectiveness in sedating elephants. During the experiment, we tried to avoid all stimulants, e.g., noise, human voice, movement, etc. However, the snoring sound of another elephant under sedation occurred, which stimulated the response of another elephant during the experiment. The snoring did not affect the results, as it occurred for a second and then stopped.In general, the neuroleptanalgesia concept (a combination of sedatives and opioids) is encouraged to elicit profound sedation, muscle relaxation, and analgesia. The combination requires lower doses of sedatives and opioids, thus minimizing possible side effects. Because the study’s aim was to exclusively evaluate dexmedetomidine’s sedative effects, a neuroleptanalgesia combination was not included. Although this current study did not evaluate dexmedetomidine-induced analgesia, dexmedetomidine reportedly provides analgesia in several animal models ,27,28,29There were several limitations to our study. First, only healthy male Asian elephants were used. Anesthetics have been reported to have different effects in males than in females ,31. It i2 adrenergic agonist for procedures requiring moderate-heavy sedation in elephants. Its benefits are as follows: (1) continuous 70 min sedation with uneventful recovery (within 10 min) after atipamezole administration; (2) unaltered physiological parameters; and (3) insignificant clinical side effects. Further evaluation is needed to titrate the dose when combined with other anesthetics. In conclusion, our data indicate that 2 µg/kg dexmedetomidine (IM) provides an effective sedation lasting approximately 70 min in Asian elephants.Dexmedetomidine is an effective α


MY11 also inhibited tumor growth in an orthotopic breast cancer mouse model by inducing apoptosis through the NF-κB signaling pathway, importantly, with minimal toxicity. In addition, MY11 was found by docking analysis to bind to p65, which might enhance the stability of the p65 protein. Taken together, our findings indicate that MY11 exerts a significant anticancer effect in breast cancer and that it may be a potential candidate for the treatment of breast cancer.Breast cancer is the most common malignancy in women worldwide, and the discovery of new effective breast cancer therapies with lower toxicity is still needed. We screened a series of chalcone derivatives and found that MY11 ((E)-1--3-(4-piperazinylphenyl) prop-2-en-1-one) had the strongest anti-breast cancer activity. MY11 inhibited the growth of MDA-MB-231 and MCF-7 breast cancer cells by arresting the cell cycle and promoting apoptosis, through regulation of the cell cycle and apoptosis-related proteins. PDTC (Pyrrolidinedithiocarbamate ammonium), a specific inhibitor of the NF-κB pathway, abolished the inhibitory effect of MY11 treatment. NF-κB has been shown to regulate PUMA-dependent apoptosis. Our The online version contains supplementary material available at 10.1007/s10637-022-01272-0. Breast cancer is the most commonly diagnosed cancer worldwide, accounting for approximately 24.5% of new cancer cases and 15.5% of deaths in women . There iEffective elimination of cancer cells by promoting apoptosis is a strategy for tumor therapy . The BclThe NF-κB signaling pathway regulates important physiological processes, such as cell growth, differentiation and apoptosis , 16. TheCleistocalyx operculatus is a traditional Chinese herb that has a long history of use in tonic drinks [The bud of c drinks . DMC -1--3-(4-piperazinylphenyl) prop-2-en-1-one) showed the greatest inhibitory potency against breast cancer cells in vitro. Hence, we explored the underlying anticancer mechanism of MY11. Further studies showed that MY11 not only induced cell cycle arrest and apoptosis in breast cancer cells but also activated the NF-κB/PUMA signaling pathway. More importantly, we demonstrated that MY11 also inhibited the growth of breast cancer in an orthotopic breast cancer mouse model by activating the NF-κB/PUMA signaling pathway. Our results indicate that MY11 could be a promising candidate drug for breast cancer treatment.MY11 (purity 95%) was synthesized and provided by pharmaceutical chemistry laboratory, East China University of Science and Technology, China . RPMI 162.Human breast cancer cell lines MDA-MB-231, MCF-7 and mouse breast cancer cell line 4T1 were purchased from the Cell Bank of the Chinese Academy of Sciences . MDA-MB-231 cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/mL Penicillin/Streptomycin. MCF-7 cells were maintained in MEM supplemented with 10% FBS, 1% sodium pyruvate, 0.01 mg/ml human recombinant insulin and 100 U/ml Penicillin/Streptomycin. 4T1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 100 U/mL Penicillin/Streptomycin. All the cells were maintained at 37 °C in a humidified incubator containing 5% CO3 cells each well and then treated with different concentrations of MY11 for 12 h. Subsequently, 20 µL MTT solution (5 mg/mL) was added into each well and the plates were incubated at 37 ℃. After 4 h, the medium was discarded, and the dark blue crystal on the bottom was dissolved completely with 150 µL DMSO. The absorbance was measured at the wavelength of 490 nm using Synergy H1 Hybird Reader . The IC50 values of MY11 in MDA-MB-231, MCF-7 and 4T1 cells were calculated using the SPSS 21.0 software .Cells were seeded into 96-well plates at a density of 4 × 103 cells each well. Cells were treated with different doses of MY11 for 7 h after attachment and incubated for another 24 h. The resultant cell colonies were stained with 1% crystal violet for 10 min and the numbers of colonies were counted for analysis.MCF-7 and MDA-MB-231 cells were seeded into 6-well plates at a density of 1 × 10After treated with different concentrations of MY11 for 12 h, cells were fixed with 75% ethanol overnight, and stained with Sytox Red staining buffer  for 15 min at room temperature. Cell cycle was detected by the D × FLEX Flow Cytometer.After treated with different concentrations of MY11 for 12 h, cells were harvested and washed twice by PBS. Fluorescence of MY11 was detected to overlap with PI and 7AAD. Therefore, Annexin V-FITC and Sytox Red (excitation and emission same as APC) were used to stain cells for 15 min in the dark at room temperature and the apoptosis rates of cells were detected by the D × FLEX Flow Cytometer .Protein was extracted from cells using RIPA lysis buffer  containing protease inhibitor cocktail and phosphatase inhibitor cocktail  and its concentration was detected with BCA assay . 20 μg of total protein from each sample or PageRuler Prestained Protein Ladder  was diluted in loading buffer  for immunoblot. Proteins were separated with SDS-PAGE and transferred to a nitrocellulose blotting membrane. Membranes were blocked in 5% BSA for 1.5 h, and incubated with primary antibodies overnight. Then membranes were washed by PBST and continued to be incubated with HRP-conjugated secondary antibodies for 1 h. The protein bands were visualized by ECL Enhanced Kit  and detected by Bio-Rad ChemiDoc™ Touch Imaging System. Antibodies against p21, Cyclin D1, PUMA, Bcl-2, Bax, Caspase-9, NF-κB p65 and Phospho-NF-κB p65-S536 were used at a dilution of 1: 1,000. β-Actin and secondary antibodies were used at a dilution of 1: 8,000.The cells were harvested after treatment and total RNA was extracted with RNAiso Plus . Total RNA was used for cDNA synthesis using HiScript II Q RT SuperMix for qPCR  according to the manufacturer’s protocol. Real-time PCR was performed in triplicate using SYBR green master mix on a QuantStudio 3 System . The samples with low yield of RNA were pre-determined and excluded. Quantitative analysis was performed using 2-ΔΔCt method for quantification of the relative mRNA expression.https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and all primers are shown as follows. hGAPDH ; hCyclin D1 ; hp21 ; hBcl-2 ; hBax ; mGAPDH ; mCyclin D1 ; mp21 ; mBcl-2 ; mBax .Real-time PCR specific primers were designed and validated by NCBI Primer designing tool (https://www.rcsb.org/structure/). The processing and optimization of virtual screening is executed using Glide module in Schrödinger Maestro software. Protein Preparation Wizard module is used for protein processing. The receptor was pretreated, optimized and minimized (constraint minimization using OPLS3e force field). All compounds were prepared according to the default settings of the LigPre module. When screening in Glide module, the prepared receptor is introduced to specify the appropriate location in receptor grid generation. The protoplast of the protein was selected as the centroid of the 10 Å box. OPlS3e is selected for the relay field, and SP (Standard Precision) is selected as the docking method. Finally, analyze the interaction mode of compounds and proteins, extract the docking score of compounds to speculate whether the compounds to be screened have certain activity.Molecular docking was employed to analyze the binding potential between p65 and MY11. MY11 was constructed by ChemDraw, and optimized by Chem3D. The structure of the target protein p65 was downloaded from the protein database, the RCSB database ; si-p65-2 ; si-p65-3 .4 4T1 cells resuspended in 100μL of PBS and Matrigel (1:1) were injected into the right mammary fat pad of each mouse. One week later, mice were divided into five groups: normal group, vehicle control group (ultrapure water mixed with Solutol HS-15 in a 9:1 ratio), MY11 20 mg/kg group, MY11 30 mg/kg group, MY11 40 mg/kg group. MY11 were diluted in ultrapure water containing Solutol HS-15 (9:1). Treatment for all groups were administered by intraperitoneal injection every day. The tumor size and body weight were measured every 3 days. Tumor volumes were calculated by the formula: Length × Diameter2)/2. After three weeks of treatment, mice were sacrificed, and all tumors and organs were harvested, measured and subjected to following experiments. The expression of Cyclin D1, p21, PUMA, Bcl-2, Bax, Caspase-9, NF-κB p65 and Phospho-NF-κB p65-S536 in the tumors were detected by Western blot.Six weeks old female BALB/c mice were bought from Zhejiang Vital River Laboratory Animal Technology. Mice were housed in a pathogen-free (SPF) environment at 22–23 ℃ with food and water supplied ad libitum throughout the experimental period. All experiments were performed under the ARRIVE Guideline  approved by Institutional Animal Care and Use Committee (IACUC) of Longhua Hospital affiliated to Shanghai University of Traditional Chinese Medicine. After mice were anaesthetized with Isoflurane, 2 × 10Serum ALT and AST levels were measured using the ALT Kit and AST Kit , respectively. Briefly, 10 μL serum was added into ALT or AST reagent. The optical density was measured at the wavelength of 505 nm using Synergy H1 Hybird Reader . Each sample was measured in triplicate.Tissue morphology was analyzed by H&E staining. 4% PFA was used to fix the tumor for 24 h. Sections (5 μm) of paraffin-embedded tumor tissue specimens were prepared on glass slides. The sections were subsequent stained with Hematoxylin and eosin. For immunohistochemical of Ki67 in tumors, five micrometers of formalin-fixed, paraffin-embedded tissue sections were mounted on glass slides. The sections were performed deparaffinization, rehydration, antigen retrieval and endogenous peroxidase inactivation. After blocking, the sections were stained with anti-mouse Ki67 antibody  overnight, then incubated with the HRP-labeled Goat anti-Rabbit IgG (H + L) antibody . Ki67 antibody were used at a dilution of 1: 200. After DAB staining and hematoxylin counterstaining, sections were dehydrated with graded alcohol, hyalinized in xylene, and finally sealed with neutral gum. Histologic analyses were observed and photoed by Olympus Microscope. , the positive cells were stained brown.P < 0.05 was considered statistically significant.Statistical analyses were carried out using the SPSS 21.0 software and GraphPad Prism 7 software. One-way or two-way analysis of variance (ANOVA) was used to analyze the significance between the groups. All values were mean ± standard error of the mean and were representative of three independent experiments, and 50) of MY11 was 8 μg/mL (22 μM) in MDA-MB-231 cells and 9 μg/mL (24 μM) in MCF-7 cells when they were treated for 12 h. In addition, MY11 significantly decreased the number of colonies formed by both cell lines when applied at 4 and 8 μg/mL , with a short hydrogen bond distance (average 2.4 Å) and strong binding ability, promoting the formation of stable complexes between MY11 and the p65 protein. In addition, MY11 had a high matching degree with the protein pocket, and each region could form a strong interaction with the groove of the pocket . Moreover, MY11 upregulated the expression of p21 and Bax and downregulated the expression of Cyclin D1 and Bcl-2 in 4T1 cells  spectrum of MY11. (B) 13C NMR  spectrum of MY11 (TIF 1241 KB)Supplementary file1 Fig. 1 NMR spectra chromatograms of MY11. (A) Supplementary file2 Fig. 2 Screening chalcone derivatives with antiproliferation activity of breast cancer cells. (A) Chemical structure of MY2, MY3, MY6 and MY11. (B) MTT assay for cell viability of MDA-MB-231 cells treated with different concentrations of MY2, MY3, MY6 and MY11 for 24 h, respectively. (C) Chemical structure of MC2-5, MC7. (D) MTT assay for cell viability of MDA-MB-231 cells treated with different concentrations of MC2-5 and MC7 for 24 h, respectively. Data are mean ± standard error of the mean and are representative of three independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001 vs. control (TIF 932 KB)*p< 0.05, **p< 0.01, ***p< 0.001 vs. control (TIF 1023 KB)Supplementary file3 Fig. 3 Inhibitors screen for signaling pathways that MY11 acts on. (A-D) FH535 inhibits Wnt/β-catenin signaling pathway. LY294002 and Wortmannin are PI3K inhibitors. Rapamycin is a specific mTOR inhibitor. MDA-MB-231 cells were treated with or without MY11 (8 μg/mL) for 12 h in the absence or presence of a series of signaling pathway inhibitors after which the cell viability was examined by MTT assay. Data are mean ± standard error of the mean and are representative of three independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001 vs. control (TIF 851 KB)Supplementary file4 Fig. 4 Effect of MY11 on 4T1 cells. (A-D) Relative genes expression in MY11 treated 4T1 cells. Data are mean ± standard error of the mean and are representative of three independent experiments. Below is the link to the electronic supplementary material.