Garcinia mangostana) whose antitumor medicinal properties are well-established. Nevertheless, the potential toxic effects of α-MG on red blood cells (RBCs) have, as of yet, not been as well studied. Methods: RBCs were exposed to 1–40 μM of α-MG for 24 h at 37 °C. Hemolysis and related markers were measured using colorimetric assays, eryptotic cells were identified through Annexin-V-FITC, Ca2+ was detected with Fluo4/AM, and oxidative stress was assessed through H2DCFDA using flow cytometry. The toxicity of α-MG was also examined in the presence of specific signal transduction inhibitors and in whole blood. Results: α-MG at 10–40 μM caused dose-dependent hemolysis with concurrent significant elevation in K+, Mg2+, and LDH leakage, but at 2.5 μM it significantly increased the osmotic resistance of cells. A significant increase was also noted in Annexin-V-binding cells, along with intracellular Ca2+, oxidative stress, and cell shrinkage. Moreover, acetylcholinesterase activity was significantly inhibited by α-MG, whose hemolytic potential was significantly ameliorated by the presence of BAPTA-AM, vitamin C, NSC23766, and isosmotic sucrose but not urea. In whole blood, α-MG significantly depleted intracellular hemoglobin stores and was selectively toxic to platelets and monocytes. Conclusions: α-MG possesses hemolytic and eryptotic activities mediated through Ca2+ signaling, Rac1 GTPase activity, and oxidative injury. Also, α-MG leads to accelerated cellular aging and specifically targets platelet and monocyte populations in a whole blood milieu.Background: Chemotherapy-related anemia is prevalent in up to 75% of patients, which may arise due to hemolysis and eryptosis. Alpha-mangostin (α-MG) is a polyphenolic xanthonoid found in the mangosteen tree ( Garcinia mangostana). In Ayurveda, tree extracts have been widely used for gastrointestinal, suppurative, and ulcerative symptoms [Alpha-mangostin (α-MG) is a polyphenolic xanthonoid isolated from the bark, dried sap, and fruit pericarp of the mangosteen tree  overload, cell membrane scrambling, disrupted channel trafficking, dehydration and cell shrinkage, and ceramide buildup. A host of signal transduction pathways have also been identified in RBCs whose role in cell death has been described. These include caspases, p38 mitogen-activated protein kinase (MAPK), AMP-activated protein kinase, casein kinase 1α (CK1α), protein kinase C (PKC), cGMP-dependent protein kinase type I, Janus kinase 3, receptor-interacting proteins 1 and 3, and mixed lineage kinase domain like pseudokinase (MLKL) [Molecular mechanisms of eryptosis include intracytoplasmic Cae (MLKL) .Although α-MG has promising potential as an antitumor agent, its interaction with human RBCs remains largely unknown. In this work, we aim to investigate the toxicity of α-MG on RBCs and identify associated mechanisms.p < 0.0001), 20 µM , and 40 µM of α-MG .As shown in + .p = 0.0044) and 8.02 ± 2.25 folds . The percentage of PS-exposing cells also increased from the negative control values of 3.75 ± 1.60% to 16.26 ± 6.83% , 38.80 ± 14.30% , and 69.93 ± 15.13%  as shown in p = 0.0213, p < 0.01, As seen in p < 0.0001), 1.06 ± 0.18 a.u. (p < 0.0001), and 0.20 ± 0.1 a.u. (p < 0.0001), respectively) compared to negative control values of 2.0 ± 0.12 a.u (p = 0.0038) and 86.08 ± 12.48% (p < 0.0001) after treatment with 10 µM and 20 µM of α-MG, respectively (p < 0.0001), 10 µM , and 20 µM of α-MG  in comparison to negative control values of 3.34 ± 0.9793%.Cells treated with 5, 10, and 20 µM of α-MG exhibited significant changes in FSC (1.59 ± 0.14 a.u. (0.12 a.u B. The peectively C. Furthep < 0.0001), 77.63 ± 21.98 a.u. (p < 0.0001), and 71.67 ± 14.85 a.u. (p < 0.0001) in cells treated with 5, 10, and 20 µM of α-MG, respectively. Notably, increasing extracellular KCl to 125 mM failed to prevent α-MG-induced hemolysis , 3.40 ± 0.73 folds , and 2.40 ± 0.98 folds . The percentage of cells with high Ca2+ was 43.37 ± 7.38% (p < 0.0001), 53.25 ± 8.78% (p < 0.0001), and 44.92 ± 11.22% (p < 0.0001), respectively, relative to the negative control which was 3.244 ± 0.86% , 2.158 ± 01.03 folds , and 1.87 ± 0.29 folds . The percentage of oxidized cells (p < 0.0001), 24.62 ± 7.90% (p < 0.0001), and 35.56 ± 5.924% (p < 0.0001), respectively. Congruently, vitamin C (p < 0.0001).The geomean of DCF fluorescence, shown in ed cells C in negaitamin C D but notitamin C E signifip < 0.001), as revealed in p < 0.0001, No statistically significant decrease in the hemolytic rate of α-MG was observed in the presence of SB203580 A, D4476 p < 0.05) was observed following treatment with 40 µM of α-MG (p < 0.01) and MCHC  were significantly reduced in treated whole blood as shown in 3/µL from control values of 121.20 ± 2.93 × 103/µL (p < 0.05).A slight but significant increase in the HCT from 19.0 ± 0.21% to 19.27 ± 0.15% ( of α-MG B. MCH (2p < 0.05), lymphocyte percentage significantly increased from 42.50 ± 0.81% to 51.30 ± 2.44% (p < 0.01), and monocyte percentage significantly decreased from 10.40 ± 0.42% to 8.28 ± 0.34% (p < 0.01). These changes were accompanied by cell shrinkage and increased lobularity as seen in 3/µL, p < 0.0001) and a decrease in monocyte count  were also evident in It was also noted that α-MG caused significant disruption in leukocyte proportions A as neut2HPO4, and 1.8 mM KH2PO4, pH 7.4, while Ringer solution was composed of 125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 32 mM HEPES, 5 mM glucose, and 1 mM CaCl2, pH 7.4. KCl-Ringer was prepared by replacing NaCl and KCl with 125 mM KCl, while Sucrose-Ringer was prepared by replacing NaCl with 250 mM sucrose. Urea was added to standard Ringer solution at 300 mM [All chemicals were of the highest purity and were purchased from Solarbio Life Science  unless otherwise noted. A stock solution of α-MG (CAS #6147-11-1) was prepared by dissolving 5 mg in 1.21 mL of dimethylsulfoxide (DMSO) and stored at −80 °C. Phosphate-buffered saline (PBS) contained 137 mM NaCl, 2.7 mM KCl, 10 mM Nav/v and stored at 4 °C for a maximum of 24 h. The purity of the RBC suspension was validated using the BC-6200 hematology analyzer . Exposure to α-MG (1–40 μM) was performed in Ringer solution at a hematocrit of 5% at 37 °C for 24 h.This study was approved by the Ethical Committee of King Saud University (E-23-7764). Blood was collected from 10 healthy volunteers in lithium heparin and EDTA vacutainer tubes, and RBCs were isolated by centrifugation at 2500 RPM for 15 min at room temperature. Following repeated washing in PBS and removal of the upper 10% of the sediment, cells were finally suspended in Ringer solution at 1:3 2O, was run in parallel and percent hemolysis was expressed as a fold change relative to negative control values [Control and treated cells were sedimented by centrifugation  and the absorbance of the supernatant was measured at 405 nm using the LMPR-A14 microplate reader . A positive control , prepared by suspending the cells in ddH+ content of extracellular space was measured using the Blood Potassium Content Assay Kit (Solarbio). In brief, cells were treated with the vehicle (0.1% DMSO) or 40 µM of α-MG at 37 °C for 24 h and the supernatant was assayed for K+ leakage. Sodium tetraphenylboron in the reaction mixture reacts with K+ to form potassium tetraphenylboron which is insoluble in water. The resultant turbidity (λmax = 520 nm) is proportional to the concentration of K+ in the sample.The K2+ into the supernatant was measured using Solarbio’s Blood Magnesium Content Assay Kit. Under alkaline conditions, Mg2+ combines with hydroxides and turns orange–red upon reacting with the triazene dye, titan yellow (λmax = 540 nm).Release of intracellular Mg+ and lactic acid to NADH and pyruvate, which further reacts with 2,4 dinitrophenylhydrazine to form pyruvate dinitrobenzene. This has a brown–red color in alkaline conditions proportional in intensity to pyruvate content (λmax = 450 nm). One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nM of pyruvate per minute for each mL of supernatant.LDH activity was assayed using Solarbio’s LDH Activity Assay Kit. In a coupled reaction, LDH converts NADCells were added to solutions of NaCl ranging from 0 mM to 160 mM (0–0.9% NaCl) corresponding to 0–320 mOsm with or without 2.5 µM or 5 µM of α-MG, and incubated at 37 °C for 1 h before hemolysis was measured.Cells were stained with 1% Annexin-V-FITC for 10 min at RT away from light, and fluorescence  was then measured at Ex/Em of 488/512 nm with the Northern Lights flow cytometer  .Forward scatter (FSC) and side scatter (SSC) were determined from 10,000 events by flow cytometry. To prepare cells for electron microscopy, the negative control and the treated samples (20 μM) were fixed in 2.5% glutaraldehyde, washed in PBS, stained with 1% osmium tetraoxide, washed again in PBS, and finally dried in 50–100% ethanol. Samples were coated with carbon and visualized using both the JSM-7610F ultra-high resolution Schottky field emission scanning electron microscope and the JEM-1400 transmission electron microscope at 15.0 kV and 100 kV, respectively  [The enzymatic activity of AChE was measured using Solarbio’s AChE Activity Assay Kit. Briefly, AChE in negative control and experimental lysates generates thiocholine from acetylthiocholine, which reacts with 2-nitrobenzoic acid to form 5-mercaptonitrobenzoic acid whose absorbance at 412 nm is directly proportional to AChE activity. One unit of enzyme activity is the amount of enzyme that catalyzes the generation of 1 nM of 5-mercaptonitrobenzoic acid per minute for each mL of hemolysate .2+ probe Fluo4/AM for 30 min at 37 °C in the dark, then washed twice in PBS (5000 RPM for 1 min) to remove excess dye. The stain was excited at 488 nm and emitted light was detected at 520 nm by flow cytometry. A total of 10,000 events were recorded. [Negative control and treated cells were labeled with 2 µM of Caecorded. .General ROS indicator 2′,7′-dichlorodihydrofluorescein diacetate (Ex/Em = 488/533 nm) was incubated with the negative control and the treated cells for 30 min at 37 °C in the dark, washed twice in PBS (5000 RPM for 1 min) to remove excess dye, and the green light was quantified in 10,000 events by flow cytometry .The sedimentation rate (mm/h) of RBCs in whole blood was recorded in Westergren tubes as previously reported .Whole blood collected in EDTA was diluted 1:2 in PBS with and without 40 µM of α-MG, and a CBC was performed after 24 h of incubation at 37 °C using a BC-6200 hematology analyzer as previously reported.2+ chelator BAPTA-AM ; p38 inhibitor SB203580 (100 µM); CK1α inhibitor D4476 (20 µM); PKC inhibitor staurosporin ; MLKL inhibitor necrosulfonamide ; vitamin C (1 mM); nitric oxide synthase (NOS) inhibitor L-NAME (20 µM); cyclooxygenase inhibitor acetylsalicylic acid ; or Rac GTPase inhibitor NSC23766 (100 µM). Hemolysis was then assessed following incubation at 37 °C for 24 h.Cells were either treated with the vehicle or with 40 µM of α-MG in the presence or absence of Can = 9). GraphPad 9.0  was used for statistical analysis. Two groups were analyzed by Student’s t-test while three or more groups were analyzed by one-way ANOVA. A p value of <0.05 was considered statistically significant.Results are shown as means ± SEM , the osmotic resistance of the cells seems to be increased, suggesting membrane expansion which allows the cell to accommodate more water influx before rupture ensues . Many cov et al.  at 1 andv et al.  and Gink extract .This work also presents the pro-eryptotic effects of α-MG for the first time A–C, whicAnother important marker of cellular aging is AChE activity. Inhibition of the enzyme, as induced by α-MG E, is typ2+ in the cytosol (2+-responsive K+ channels leading to KCl (2+ activity, and may thus account for the detected cell shrinkage (2+-dependent, and as such, become dysregulated in response to increased Ca2+ activity [2+ in driving hemolysis, similar to numerous hemolytic and eryptotic inducers such as allicin and β-lapachone. Thus, other mechanisms are indeed required for the full hemolytic activity of α-MG.The observed shrinkage and loss of surface complexity  indicate cytosol A–C, whicg to KCl C and wathrinkage . Likewisactivity , resulti2+-dependent eryptosis [Although α-MG is reported to exert antioxidant and anti-inflammatory effects ,37, our ryptosis . Anotherryptosis . Notably2+ ions and PKC, which was not essential for the hemolytic activity of α-MG (2+-Rac GTPase-ROS molecular axis, especially given its role in cytoskeletal regulation.The role of Rac GTPases is well established in maintaining the hexagonal organization of the cytoskeleton in healthy RBCs . Previou of α-MG C. Thus, A number of mechanisms could explain the antihemolytic properties of sucrose against α-MG G in a si2+. The selective cytotoxicity of α-MG can be attributed to its potential to alter the actin cytoskeletal structures and stiffness, as has been reported previously in various cell types [Exposure to α-MG in a whole blood context revealed augmented selective cytotoxicity toward platelets H and monll types . Also, all types , but furIn conclusion, this work presents novel insights into the in vitro cytotoxic effects of α-MG in erythrocytes. Given that anemia is a common side effect of many anticancer drugs, hematological evaluation of investigational therapeutics such as α-MG is essential for safety assessment and drug development. Our study thus contributes to the current understanding of the potential therapeutic utility of α-MG. Further research in animal models and clinical trials should be directed towards elucidating the detailed mechanisms  through


Bauhinia thonningii is a plant traditionally used against many human diseases such as gastric ulcers, fever, inflammations, coughs, dysentery, diarrhea, and malaria. In the present investigation, the cytotoxicity of methanol extract of Bauhinia thonningii leaves (BTL), fractions and the isolated phytoconstituents was determined in a panel of 9 human cancer cell lines including drug sensitive and multidrug-resistant (MDR) phenotypes. The acute and sub-chronic oral toxicity of BTL was investigated as well.Compounds were isolated using chromatographic techniques while their chemical structures were determined using spectroscopic methods. The resazurin reduction assay (RRA) was used to evaluate the cytotoxicity of samples, propidium iodide (PI) for apoptosis, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining for mitochondrial membrane potential (MMP) analysis, 2´,7´-dichlorodihydrofluoresceine diacetate (H2DCFH-DA) staining for the quantification of reactive oxygen species (ROS), whereas Caspase Glo assays were combined by means of flow cytometry. Furthermore, the toxicological investigations were performed as recommended by the Organization for Economic Cooperation and Development (OECD).O-L-rhamnopyranoside (5), quercetin-3-O-β-glucopyranoside (6), 6,8-C-dimethylkaempferol 3,7-dimethyl ether (7), and 6,8-C-dimethylkaempferol-3-methyl ether (8) had promising cytotoxic effects in the 9 tested cancer cell lines. The IC50 values below 20 µg/mL  or 10 µM (compounds) on at least 1/9 tested cancer cell lines were considered. The best cytotoxic effects with IC50 values below 5 µM were achieved with compounds 7 against CEM/ADR5000 leukemia cells (2.86 µM) and MDA-MB-231-pcDNA breast adenocarcinoma cells (1.93 µM) as well as 8 against CCRF-CEM leukemia cells (3.03 µM), CEM/ADR5000 cells (2.42 µM), MDA-MB-231-pcDNA (2.34 µM), and HCT116 p53−/− cells (3.41 µM). BTL and compound 8 induced apoptotic cell death in CCRF-CEM cells through caspase activation, alteration of MMP, and increased ROS production. BTL did not cause any adverse effects in rats after a single administration at 5000 mg/kg or a repeated dose of 250 mg/kg body weight (b. w.).The botanicals as well as 6-C-methylquercetin-3,7-dimethyl ether (2), quercetin-3-Bauhinia thonningii and its constituents are sources of cytotoxic drugs that deserve more in-depth studies to develop novel antiproliferative phytomedicine to fight cancer including resistant phenotypes.The online version contains supplementary material available at 10.1186/s12906-023-04172-9. Bauhinia thonningii (Schumach.) Milne-Redh.  is a medicinal plant used in the traditional medicine in the treatment of several pathologies such as dysentery, fever, respiratory ailments, snake bites, hookworm, skin diseases, male erectile malfunction, gastro-intestinal track problems, inflammation, bilharzia, eye diseases, malaria, and catarrh [Bauhinia thonningii leaves on ethanol-induced gastric ulceration in Sprague Dawley rats [Escherichia coli, Staphylococcus aureus, Enterobacter species, Streptococcus pneumoniae, Corynebacterium pyogenes, Enterococcus faecalis, Acinetobacter species, and Pseudomonas aeruginosa [Klebsiella pneumoniae and the Gram-positive bacterium Staphylococcus aureus [Cancer remains one of the world’s biggest threats with an alarming mortality rate placing it among the major categories of public health problems. Malignancies were responsible for around 10 million deaths worldwide in 2020 . Medicin catarrh , 13. It  catarrh , 15. Thiley rats . They shley rats . This plley rats . The learuginosa . Moreoves aureus . Variouss aureus . Leaf exs aureus .Bauhinia thonningii (Schumach.) Milne-Redh.  were collected in Bandjoun (5°21′N 10°24′E) in the West region of Cameroon. This plant is not an endangered species, and it was collected in open community field and therefore no prior permission was required. The plant parts  and the whole plant were identified in the Cameroon National Herbarium (HNC) under the voucher code 33,258/HNC, and furthermore, the plant name Bauhinia thonningii (Schumach.) Milne-Redh.  was further authenticated using the plant index database, the World Flora Online, under the reference code WFO-0001054410. The leaves were dried and powdered, and 2.0 kg were soaked in 15 L of methanol (CH3OH) for 48 h. The filtrate obtained using Whatman filter paper grade 1 was evaporated under vacuum to obtain 200.4 g of the crude methanol extract or the botanical (BTL). The botanical was stored at 4 °C for further use.The leaves of n-hexane–EtOAc  and EtOAc-CH3OH  to obtain 168 sub-fractions (sub-frs) of 300 mL each collected as follows: n-hexane–EtOAc 100:0 (sub-frs 1–24); n-hexane–EtOAc 95:5 (sub-frs 25–47); n-hexane–EtOAc 85:15 (sub-frs 48–84); n-hexane–EtOAc 75:25 (sub-frs 85–108); n-hexane–EtOAc 40:60 (sub-frs 109–117); n-hexane–EtOAc 25:75 (sub-frs 118–132); EtOAc-CH3OH 100:0 (sub-frs 133–144); EtOAc-CH3OH 90:10 (sub-frs 145–160), and EtOAc–MeOH 0:100 (sub-frs 161–168). Based on their TLC profiles, these fractions were pooled into seven major sub-fractions (A-G) as follows: sub-fractions A , B , C , D , E , F , and G . Sub-fractions A and B contained mainly fatty acids and were not further investigated. Compounds 4 (15.1 mg) and 9 (19.9 mg) precipitated from fraction C (2.1 g), while compound 10 (25.0 mg) precipitated from fraction E, obtained after simple filtration with EtOAc. Fraction D was subjected to repeated silica gel CC using a gradient elution of n-hexane–EtOAc  to afford 75 sub-frs of 75 mL each as follows: n-hexane–EtOAc 95:5 (sub-frs 1–33), n-hexane–EtOAc 85:15 (sub-frs 34–49), n-hexane–EtOAc 70:30 (sub-frs 50–63), and n-hexane–EtOAc 50:50 (sub-frs 64–75). Based on their TLC profiles, sub-fractions 44–48 and 50–51 were pooled separately and identified as a mixture of compounds, whereas other sub-fractions obtained in a very small amount were not further investigated. The mixture of compounds was treated and purified by CC over Sephadex LH-20 eluting with CH2Cl2-CH3OH (50:50), v/v to yield compound 1 (9.9 mg) from sub-frs 44–48, as well as 2 (12.1 mg) and 8 (6.1 mg) from sub-frs 50–51.Part of BTL (194.0 g) was triturated with ethyl acetate (EtOAc) to obtain 96.2 g of EtOAc fraction (BTLa) and 78.3 g of residue (BTLb). The two fractions displayed cytotoxic effects in cancer cell lines and were further subjected to purification. Ninety-six grams (96.0 g) of the BTLa were subjected to column chromatography (CC) using silica gel and eluted with a gradient of n-hexane–EtOAc  and EtOAc-CH3OH  to afford 67 sub-frs of 200 mL each collected as follows: n-hexane–EtOAc 100:0 (sub-frs 1–7), n-hexane–EtOAc 85:15 (sub-frs 8–24), n-hexane–EtOAc 75:25 (sub-frs 25–30), n-hexane–EtOAc 60:40 (sub-frs 31–40), EtOAc-CH3OH 100:0 (sub-frs 41–55), and EtOAc-CH3OH 90:10 (sub-frs 56–67). Based on their TLC profiles, these sub-frs were pooled together into the five major sub-frs (I-V) as follows: I , II , III , IV , and V . A precipitate in powder form was observed from fractions II-IV. After simple filtration and purification with ethyl acetate, compounds 7 (8.1 mg), 3 (11.1 mg), and mixture were obtained from fractions II, III, and IV, respectively. The separation of a mixture was possible after CC over Sephadex LH-20 eluting with CH2Cl2-CH3OH (50:50), v/v to afford compounds 5 (15.9 mg) and 6 (17.2 mg).The fraction BTLb (77.0 g) was subjected to repeated silica gel CC using a gradient elution of ΔEGFR [p53/++ colon adenocarcinoma cells and its resistant knock-out congener, HCT116 p53−/− [pcDNA3 breast adenocarcinoma cells and its multidrug-resistant breast cancer resistance protein (BCRP)-transfected MDA-MB-231-BCRP clone 23 subline [BCRP, HCT116 p53−/−, and U87MG.ΔEGFR cells were cultured in a complete medium with additional geneticin at 800 ng/mL and 400 µg/mL, respectively [In this study, a panel of 9 cancer cell lines as well as the normal AML12 hepatocytes were used. The source of these cell lines has been elucidated earlier . The druΔEGFR , HCT116 6 p53−/− , MDA-MB- subline  have alsectively , 33.50 values of tested samples defined as the concentration required to inhibit 50% of the cell proliferation. They were deduced from a calibration curve by linear regression from Microsoft Excel [50 value in the resistant cell line versus IC50 value in the corresponding sensitive cell line [50 value of the normal AML12 hepatocytes versus that of HepG2 cells was referred to as the selectivity index (S.I.) [To determine the cytotoxicity of the botanicals , phytoconstituents (1–10) as well as the positive control, doxorubicin, the resazurin reduction-based assay (RRA) was used as earlier described  under exft Excel . The degell line , 37; thex (S.I.) .50. The treated cells  were incubated for 24 h at 37 °C, 5% CO2 and 95% relative humidity referred to as the standard cell culture condition. The cell cycle analysis was further monitored by means of the BD Accury C6 Flow Cytometer  [The distribution of the cell cycle in CCRF-CEM cells as a result of the application of the botanical BTL, compound 8, doxorubicin, or solvent control, dimethyl sulfoxide (DMSO) was evaluated by the combined flow cytometry and propidium iodide staining similarly to the previously reported experimental conditions . The conGermany) , 40. AssTo detect the apoptotic CCRF-CEM cells resulting from the application of BTL, compound 8, doxorubicin, or DMSO, the annexin V/propidium iodide (PI) staining combined with the flow cytometry was used in similar experimental conditions as reported earlier , 41–43. 50. The amount of the treated cells was 1.5 × 104 cells for the caspase 3/7 assay or 3 × 104 cells for the caspase 8 and caspase 9 assays in 100 µL. The treated cells were then incubated for 6 h under the standard cell culture conditions as described above. Subsequently, the activity of caspases was detected by a spectrophotometry measurement of luminescence using 100 µL caspase solution from the Caspase-Glo 3/7, 8, and 9 Assay kits  with an Infinite M2000 ProTM plate reader (Tecan).To detect the activity of caspases in CCRF-CEM cells resulting from the application of BTL and compound 8, the Caspase-Glo assay combined with the spectrophotometric determination of their luminescence was applied in similar experimental conditions as reported earlier . The con2O2 , the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide  staining and the 2´,7´-dichlorodihydrofluorescein diacetate (H2DCFH-DA) (Sigma-Aldrich) staining, respectively for MMP and ROS measurements, were combined with the flow cytometry used in similar experimental conditions, as earlier reported [50. The treated cells  were incubated for 24 h under the standard cell culture condition. Cells were further stained for 30 min with 10 µL of staining solution  according to the manufacturer’s protocol. Using the LSRFortessa FACS analyzer , the amount of 1 × 104 cells was further measured as described earlier [To quantify the modification of the mitochondrial membrane potential (MMP) and the reactive oxygen species (ROS) production in CCRF-CEM cells resulting from the application of BTL, either compound 8 or DMSO (negative control), or the respective positive controls for MMP or ROS evaluations, valinomycin or Hreported . The con earlier , 45–47.Wistar albino rats at 8 weeks of age weighting between 160 and 200 g were investigated. The animal breeding took place in the animal house, University of Dschang, Cameroon. The animals were placed individually in polypropylene cages at 25 ± 1 ºC in a 12 h: 12 h dark: light cycle. Food and tap water were given to them ad libitum. The animals were treated according to the protocol for the care and use of laboratory animals based on the ethical considerations of the Institutional Ethical Committee of the University of Dschang, Cameroon (Project No. BCH1202/FS/UDS/2020).Thirty-eight The acute toxicity study was conducted according to the OECD guideline with six healthy female rats . They were nulliparous  and wereThe sub-chronic oral toxicity study was conducted according to the standard methods  with thiP-values less than 0.05 were considered statistically significant.Statistical analyses were performed using SPSS version 23.0 for Windows. The results were expressed as mean value ± standard deviation (SD), and the comparisons were performed by the analysis of variance using a one-way analysis of variance test. Differences between the averages of control and drug-treated groups where they existed were separated using the Waller–Duncan test. Bauhinia thonningii led to the isolation and identification of 10 compounds. Their chemical structures are shown in Fig. 1H NMR, 13C NMR, and HMBC data are shown in Supplementary Information S18H16O7  [18H16O7  [19H18O7  [15H10O7  [19H18O6  [18H16O6  [O-L-rhamnopyranoside C21H20O11 [O-β-glucopyranoside C21H20O12  [30H48O3 [O-β-D-glucopyranoside of β-sitosterol C35H60O6  [The phytochemical study of –197 °C) , 6-C-met–187 °C) , quercet–317 °C) , 6,8-C-d–287 °C) , and 6,83–285 °C), and one 212 °C) . These c50 values varied from 18.13 µg/mL (against CCRF-CEM cells) to 40.84 µg/mL (against HCT116 p53/++ cells) for BTL, from 12.46 µg/mL (against U87MG cells) to 47.40 µg/mL (against HCT116 p53−/−cells) for BTLa, and from 16.55 µg/mL (against U87MG cells) to 64.74 µg/mL (against MDA-MB-231-BCRP cells) for BTLb. The obtained IC50 values with doxorubicin varied 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells)  and MDA-MB-231-pcDNA breast adenocarcinoma cells (1.93 µM) and as well as compound 8 against CCRF-CEM leukemia cells (3.03 µM), CEM/ADR5000 cells (2.42 µM), MDA-MB-231-pcDNA (2.34 µM), and HCT116 p53−/− cells (3.41 µM) (Table versus CCRF-CEM cells was achieved vis-à-vis BTLa (D. R. of 0.98), compounds 2 (D. R. of 0.76), 5 (D. R. of 0.83), 6 (D. R. of 0.90), 7 (D. R. of 0.43), and 8 (D. R. of 0.80). This was also the case for MDA-MB-231-BCRP cells versus MDA-MB-231-pcDNA cells vis-à-vis BTL (D. R. of 1.14) BTL a (D. R. of 1.13) and compound 3 (D. R. of 1.20), HCT116 p53−/− cells versus HCT116 p53/++ cells vis-à-vis BTL (D. R. of 0.70), BTLa (D. R. of 1.08), BTLb (D. R. of 0.93), compounds 2 (D. R. of 0.75), 5 (D. R. of 0.73), 6 (D. R. of 0.57), 7 (D. R. of 0.50), and 8 (D. R. of 0.36), and U87.MGΔEGFR cells versus U87.MG cells vis-à-vis BTL (D. R. of 1.07), compounds 2 (D. R. of 0.85), and 7 (D. R. of 0.93)  in a concentration-dependent manner upon treatments; within the concentrations ranges of 1/4 × IC50 and 2 × IC50, the number of cells in the sub-G0/G1 phase varied from 3.92 to 27.40% for BTL, 2.36 to 21.51% for compound 8, and from 3.28 to 12.05% for doxorubicin  of the production in CCRF-CEM cells was recorded upon treatment with BTL, compound 8, and H2O2 .Figures The effect of BTL on the relative organ weight of the animals after 28 days of administration is shown in Fig. Table p < 0.05) was observed with the other parameters measured compared to the animals in the control group.The effect of BTL on markers of rat kidney function after 28 days of administration is shown in Table p < 0.05) decrease in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities at 250 and 500 mg/kg in both sexes compared to those in the control group. Further analysis of cardiovascular markers revealed a significant (p < 0.05) increase in LDL cholesterol levels at 500 and 1000 mg/kg in females compared to the control group. However, in males, a significant (p < 0.05) increase in total and high-density lipoprotein HDL cholesterol levels at doses 500 and 1000 mg/Kg b. w. compared to those in the control group was observed. Similarly, sub-acute administration of methanol extract of BTL resulted in a significant decrease in LDL-cholesterol level at 250 mg/kg b. w. in males compared to the control group.The effect of sub-acute oral administration of BTL on biochemical markers of the liver and cardiovascular function is presented in Table Histopathological analysis of the liver and kidneys of both males and females revealed no abnormalities at all doses of BTL used in this test compared to the control animals Fig. .Fig. 10HBCRP cell lines, U87MG.ΔEGFR cells with mutation-activated EGFR gene (ΔEGFR) as well as the p53 knockout HCT116p53−/− cells [50 values below 20 μg/mL for botanicals [50 values below 20 µg/mL were recorded in the present work with BTL, BTLb, and BTLa on 1, 2, and 6 of the 9 tested cancer cell lines, respectively , 6-C-methylquercetin-3,7-dimethyl ether (2), 6-C-methylquercetin 3,7,3'-trimethyl ether (3), quercetin-3-O-L-rhamnopyranoside (5), quercetin-3-O-β-glucopyranoside (6), 6,8-C-dimethylkaempferol 3,7-dimethyl ether (7), 6,8-C-dimethylkaempferol-3-methyl ether (8) in such a broad panel of cancer cell lines including MDR phenotypes has been reported for the first time. However, the cytotoxicity of quercetin (4) was shown in CCRF-CEM cells and CEM/ADR5000 cells, with the IC50 values of 25 µM and 101 µM, respectively [50 value of 27 µM in CCRF-CEM cells and a value above 50 µM in CEM7ADR5000 cells. Furthermore, the data obtained with β-sitosterol-3-O-β-D-glucopyranoside (10) corroborate the previous studies as this compound was found not active in the cancer cell lines tested in the current work with IC50 values above 100 µM [50 value of 44.28 µM. This also corroborates other reports on this compound. For instance, Shyu reported an IC50 value of 75 µM for compound 9 in human hepatocellular carcinoma HuH7 cells [50 value of 7.1 μg/mL (or 15.57 µM) was also reported with compound 9 against MCF-7 cells [50 values recorded in only two of the 9 tested cancer cell lines  instead of hydroxyl substituent (8)  of the aqueous and methanol extracts of Bauhinia thonningii bark harvested in Kenya. The sub-chronic administration of BTL at different doses for 28 days did not lead to any variation (p > 0.05) in food consumption. However, the decrease in weight growth in animals is not a sign of toxicity in view of the overall results observed as it is traditionally used in West Cameroon for its anti-obesity properties. This extract could therefore be beneficial for people suffering from obesity. According to Raji et al. [The toxicological profile of a substance is useful in the assessment of risks to humans , 69, 70.toxicity . This retoxicity  who obtai et al. , a plantp < 0.05) in ALT and AST activity at doses of 250 and 500 mg/kg in animals of both sexes reflects the protective effect of the extract at these doses on the liver. Indeed, the botanicals with beneficial health effects act by reinforcing the selective permeability of the cell membranes. This led to a decrease in the level of enzymes and serum proteins [Hematopoiesis is a vital process for life and can be affected by both conventional and herbal drugs. The assessment of hematological parameters in animal models provides information on the benefits or toxic manifestations of herbal extracts in the blood . The resproteins . The decproteins . The anaproteins . Based oBauhinia thonningii (BTL) as well as fractions (BTLa and BTLb) and their isolated phytoconstituents was evidenced on a panel of human cancer cell lines including MDR phenotypes. The isolated phytochemicals were 6-C-methylquercetin-3,4'-dimethyl ether (1), 6-C-methylquercetin-3,7-dimethyl ether (2), 6-C-methylquercetin 3,7,3'-trimethyl ether (3), quercetin (4), quercetin-3-O-L-rhamnopyranoside (5), quercetin-3-O-β-glucopyranoside (6), 6,8-C-dimethylkaempferol 3,7-dimethyl ether (7), 6,8-C-dimethylkaempferol-3-methyl ether (8), ursolic acid (9), and 3-O-β-D-glucopyranoside of β-sitosterol (10). The botanicals  as well as compounds 2, 5, 6, 7, and 8 had promising cytotoxic effects. BTL and compound 8 induced apoptotic cell death in CCRF-CEM cells through caspase activation, alteration of MMP, and increased ROS production. BTL did not cause any adverse effects in rats after a single administration at 5000 mg/kg as well as the repeated dose of 250 mg/kg b. w. Bauhinia thonningii and its constituents are sources of cytotoxic drugs and deserve more in-depth studies to develop novel antiproliferative phytomedicine to fight cancer including resistant phenotypes.In the present work, the cytotoxicity of the methanol extract from the leaves of Additional file 1: Figure S1-Figure S24.


After pyrolysis at high temperature, 3D porous VOx/N-doped carbon nanosheet hybrids (3D VNCN) were obtained. The unique 3D porous skeleton, abundant doping elements, and presence of VOx 3D VNCN pyrolyzed at 800 °C (3D VNCN-800) ensured excellent electrochemical performance. The 3D VNCN-800 electrode exhibits a maximum specific capacitance of 408.1 F·g−1 at 1 A·g−1 current density and an admirable cycling stability with 96.8% capacitance retention after 5000 cycles. Moreover, an assembled symmetrical supercapacitor based on the 3D VNCN-800 electrode delivers a maximum energy density of 15.6 Wh·Kg−1 at a power density of 600 W·Kg−1. Our study demonstrates a potential guideline for the fabrication of porous carbon materials with 3D structure and abundant heteroatom-doping.Three-dimensional porous carbon materials with moderate heteroatom-doping have been extensively investigated as promising electrode materials for energy storage. In this study, we fabricated a 3D cross-linked chitosan-dicyandiamide-VOSO With the assistance of glutaraldehyde, a 3D cross-linked gel can form through the interaction between amino groups in chitosan molecules and aldehyde functional groups in glutaraldehyde molecules [Chitosan (CS), as an eco-friendly biopolymer, has drawn considerable attention in the fabrication of various materials ,2,3. Theolecules . The uni−1 at 1 A·g−1 [Carbon materials, as considerable supercapacitor electrode materials, have been extensively investigated to improve their specific capacitance ,6,7,8. S 1 A·g−1 . Zhou’s  1 A·g−1 .Heteroatom-doping is another effective method to improve specific capacitance of carbon materials ,20. The x/N-doped carbon nanosheet hybrids (3D VNCN) through the pyrolysis of cross-linked chitosan-dicyandiamide-VOSO4 (CS-DCDA-VOSO4) hydrogel. The aldehyde functional groups in glutaraldehyde can polymerize with -NH2 and -OH functional groups from CS and DCDA, leading to a cross-linked network structure. Meanwhile, VOSO4 uniformly disperses within the hydrogel. After pyrolysis at high temperature, 3D VNCN hybrids were obtained. The unique 3D skeleton, abundant doping elements, porous structure, and presence of VOx ensure the excellent electrochemical performance of 3D VNCN. The 3D VNCN electrode exhibits a maximum specific capacitance of 408.1 F·g−1 at 1 A·g−1 current density. The maintained capacitance retention was about 96.8% after 5000 cycles. Moreover, a 3D VNCN-based symmetrical supercapacitor shows a maximum energy density of 15.6 Wh·Kg−1 at a power density of 600 W·Kg−1. It is worth noting that our study provides a promising method for the fabrication of 3D porous carbon materials used in the energy storage field.Herein, we design a new route to fabricate 3D porous VO4 were dissolved in 20 mL deionized water. A volume of 200 μL of acetic acid was added dropwise into the above solution and stirred for 15 min. Then, 400 μL glutaraldehyde was added into the above homogeneous suspension for polymerization. After standing for 2 h, the resulting jelly-like hydrogel containing CS, DCDA, and VOSO4 (CS-DCDA-VOSO4 hydrogel) was obtained. Followed by freeze–drying for 24 h, the hydrogel transformed into a 3D interconnected porous structure, and then the dried CS-DCDA-VOSO4 gel was transferred to a tube furnace for further carbonization. The carbonization process was adopted using a two-step pyrolysis method under N2 atmosphere at a heating rate of 5 °C·min−1. At first, the temperature was increased to 600 °C and held for 2 h, and then increased to a higher temperature  for 3 h. The products were marked as 3D VNCN-700, 3D VNCN-800, and 3D VNCN-900, respectively. For comparison, 3D N-doped carbon nanosheets derived from CS-DCDA hydrogel at 800 °C were denoted as 3D NCN. CS, DCDA, and VOSO4 with the same ratio were ground into a light blue powder (CS-DCDA-VOSO4 powder). After pyrolyzing at 800 °C, the obtained VOx/N-doped carbon material was marked as VNC.In a typical procedure, 0.2 g of CS, 0.3 g of DCDA, and 0.05 g of VOSO2 adsorption and desorption isotherms were carried out on a Micrometrics ASAP 2020 V3.04 H system with Brunauer–Emmett–Teller measurements (BET). The functional groups on the surface of the samples were obtained using Fourier transform infrared (FT-IR) spectroscopy with a Nicolet 50 spectrometer. Raman measurements were investigated via an inVia confocal micro-Raman spectroscope (RTS2). Thermogravimetric analysis (TGA) was performed on Discovery TGA5500  at a heating rate of 5 °C·min−1 under flowing N2.X-ray diffraction  was conducted with Cu-Ka radiation (λ = 0.1504 nm) to investigate the phase of the as-prepared samples. Morphologies and structures of the samples were characterized using a scanning electron microscope , transmission electron microscope , and X-ray photoelectron spectroscope . NCs, F·g−1) was calculated from the GCD curves according to the equation Cs = (I × ∆t)/(m × ∆V), where I (A) is the discharge current, ∆t (s) is the discharge time, m (g) is the mass of the active material, and ∆V (V) is the voltage change during the discharge process. Furthermore, a symmetric supercapacitor was assembled with two identical electrodes, and the device was characterized using a 6 M KOH aqueous solution as the electrolyte. The energy and power density were characterized using the following equation: Ecell = Ccell × ∆2V/7.2 and Pcell = Ecell × 3600/∆t.The electrochemical performance was characterized using a CHI760E electrochemical workstation. In a three-electrode system, Pt foil and a Hg/HgO electrode were used as counter and reference electrodes, respectively. The working electrode was prepared by coating a slurry on Ni foam. The slurry was prepared by mixing the active material with acetylene black and polytetrafluoroethylene in ethanol solvent at a mass ratio of 8:1:1. After being pressed under 10 MPa for 30 s and dried at 100 °C for 12 h, the working electrode was obtained for further electrochemical characterization. The mass loading of active material in the working electrode was about 2 mg. A 6 M KOH aqueous solution was used as the electrolyte. Cyclic voltammetry (CV) and galvanostatic charge–discharge (GCD) curves were obtained in the potential range of −0.95–0.25 V. Electrochemical impedance spectroscopy (EIS) measurements were tested under open circuit voltage at an amplitude of 5 mV with a frequency range between 0.01 and 100 kHz. The specific capacitance of the active material  also results from the loss of crystal water and small molecule species. During the next stage, CS decomposes to a carbon material, and DCDA successively condenses to melamine, tris-s-triazine, and C3N4 from 220 °C to 600 °C. VOSO4 transforms to VOx at this stage. Then, at higher temperatures (>600 °C), C3N4 further decomposes and leaves N species introduced into carbon materials derived from chitosan. VOx nanoparticles are also decorated on carbon skeleton, producing a VNC composite.TGA curves of CS-DCDA-VOSO4 gel and 3D VNCN-800. It can be observed that the FT-IR spectrum of CS-DCDA-VOSO4 gel exhibits obvious characteristic peaks ranging from 4200 cm−1 to 800 cm−1. Among them, the characteristic peaks from 3600 to 3000 cm−1 are attributed to the stretching vibration absorption peak of the N–H and O–H groups [−1 is ascribed to C≡N stretching modes. The peaks in the wave number range of 1800–1300 cm−1 are caused by C–N heterocyclic stretching vibrations [−1 are indexed to C–O stretching modes [−1 refer to V–O symmetric and stretching vibrations [−1. No obvious peak of V–O bonds can be observed due to the low content of VOx.The FT-IR spectra c were usbrations . The peang modes . The peabrations ,34. Afteθ degree of 3D VNCN gradually decreases, and the intensity of the (002) peak becomes weaker. This phenomenon can be ascribed to the increased temperature, which reduces stacking between carbon sheets, presenting enlarged interlayer spacing and loose structure of carbon materials. The (002) peak of 3D NCN and VNC centers at 21.2° and 26.6°, exhibiting distinct change compared with 3D VNCN. Without adding VOSO4, 3D NCN becomes less dense compared with 3D VNCN; thus, the (002) plane shifts to a low angle. While VNC fabricated from CS-DCDA-VOSO4 powder shows a bulk structure. The highly stacked structure results in the higher position of the (002) plane. Both VNC and 3D VNCN show no obvious VOx diffraction peaks, which may be ascribed to the small quantity of VOx in VNC and 3D VNCN. Raman characterizations were conducted to evaluate the graphitic degree of 3D VNCN, 3D NCN, and VNC in −1 are associated with disordered carbon structure. At the same time, G-bands at around 1600 cm−1 correspond to 2sp-hybridized carbon [ID/IG values of 3D VNCN-700, 3D VNCN-800, 3D VNCN-900, 3D NCN, and VNC are 1.29, 1.23, 1.08, 1.18, and 1.46, respectively. This result demonstrates that these carbon materials are disordered structures with partial graphitization. The elevated temperature of 3D VNCN leads to a higher degree of graphitization. The 3D VNCN-800 product, with moderate defects and graphitic carbon, could provide abundant ion storage sites and facilitate ion/electron transportation. As for 3D NCN, without doping element and VOx, it exhibits an ordered structure compared with 3D VNCN-800. While VNC shows the highest ID/IG ratio, suggesting a highly disordered structure owing to the lack of a 3D skeleton resulting from CS-DCDA hydrogel.d carbon ,36. The 2 adsorption and desorption isotherms of 3D VNCN, 3D NCN, and VNC. A combination of Type-I and Type-IV with a slight hysteresis loop at high relative pressure (0.5–1.0) can be observed, indicating the co-existence of micropores and mesopores [2·g−1, 288.0 m2·g−1, 358.2 m2·g−1, 285.5 m2·g−1, and 17.5 m2·g−1, respectively. The 3D VNCN-800 and 3D NCN products exhibit similar SSAs, while VNC displays a particularly small SSA due to the stacked structure. The SSA of 3D VNCN gradually increases with increasing temperature due to exfoliated carbon sheets and decreased stacking structure at high temperatures. Pore size distribution (PSD) curves in esopores . The spe3+ in the V–O bond (515.2 eV and 522.3 eV) and V5+ in the V–O bond (516.6 eV and 523.8 eV) demonstrate the existence of the V element as vanadium oxide [3+ and V5+ in 3D VNCN could provide abundant faradic reaction sites, trigger fast redox reactions, and enhance specific capacitance. However, at 900 °C, the V species in 3D VNCN mainly comes from V5+. As for N1s, the high-resolution spectra , 365.0 F·g−1 (2 A·g−1), 324.0 F·g−1 (4 A·g−1), 313.3 F·g−1 (5 A·g−1), 290.7 F·g−1 (8 A·g−1), and 282.5 F·g−1 (10 A·g−1), respectively. The cycling stability of the 3D VNCN-800 electrode was evaluated using successive GCD measurements between −0.95 V and 0.25 V at 10 A·g−1. As shown in Rs), which is 0.68 Ω for the 3D VNCN-800 electrode. Moreover, the small semicircle from the Nyquist plot corresponding to charge transfer resistance indicates a fast adsorption/desorption rate for the 3D VNCN-800 electrode. After 5000 cycling tests, the Nyquist plot of the 3D VNCN-800 electrode shows negligible change, demonstrating well-maintained electrochemical performance.EIS analyses were employed to evaluate the electron/ion transport process of the 3D VNCN-800 electrode before and after the cycling test. The Nyquist plot of the 3D VNCN-800 electrode f shows a−1 (1 A·g−1), 72.0 F·g−1 (2 A·g−1), 66.0 F·g−1 (4 A·g−1), 63.7 F·g−1 (5 A·g−1), 58.7 F·g−1 (8 A·g−1), and 56.7 F·g−1 (10 A·g−1), respectively. The Ragone plot of 3D VNCN-800-SC . N-800-SC d was plox nanoparticles provide additional pseudocapacitance. Hence, 3D VNCN-800 shows a maximum specific capacitance of 408.1 F·g−1 at a current density of 1 A·g−1 and admirable cycling stability with 96.8% capacitance retention after 5000 cycles. Moreover, 3D VNCN-800-SC delivers a maximum energy density of 15.6 Wh·Kg−1 at a power density of 600 W·Kg−1. It is hoped that our works could provide guidance for the fabrication of 3D porous carbon materials with excellent electrochemical performance.To sum up, we present an effective approach to fabricating 3D VNCN composites. The 3D skeleton comes from polymerized CS-DCDA hydrogel with the assistance of glutaraldehyde. After pyrolysis at high temperatures, the 3D structure is well maintained. Moreover, abundant N dopants and VO


Self-assembled H-shaped Lewis acid–base adduct 2d proved to be effective in forming thermally stable host–guest complexes, 2d × solvent, with aromatic hydrocarbon solvents such as benzene, toluene, mesitylene, aniline, and m-, p-, and o-xylene. Crystallographic analysis of these solvent adducts revealed host–guest interactions to primarily occur via π···π contacts between the 4,4′-bipyridyl linker and the aromatic solvents, resulting in the formation of 1:1 and 1:2 host–guest complexes. Thermogravimetric analysis of the isolated complexes 2d × solvent revealed their high thermal stability with peak temperatures associated with the loss of solvent ranging from 122 to 147 °C. 2d, when self-assembled in an equimolar mixture of m-, p-, and o-xylene (1:1:1), preferentially binds to o-xylene. Collectively, these results demonstrate the ability of 1,8-dihydroxy naphthalene to serve as an effective building block in the selective self-assembly to supramolecular aggregates through dative covalent N→B bonds.The new Lewis acid–base adducts of general formula X(nad)B←NC The last decades have witnessed increased interest in the rational design of supramolecular architectures based on the self-assembly of various suitable building blocks ,10,11,121a–e), where nad = 1,8-naphthtalenediolate  to form stable Lewis acid–base adducts with 4,4′-bipyridine  showed a signal at around 29 ppm, which is close to the 11B NMR chemical shift of 1a (δ = 27 ppm in CDCl3) [2a. For the system 4,4′-bipyridine/1b (1:2), two distinct signals at ca. 27 ppm and 7 ppm were observed; the former can be assigned to ester 1b, while the latter is due to the desired Lewis acid–base adduct 2b. In contrast, mixtures of 4,4′-bipyridine with 1c, 1d, and 1e, respectively, showed up-field shifted signals at ca. 12 ppm (1c), 6 ppm (1d), and 4 ppm (1e), indicating the formation of the respective adducts 2c, 2d, and 2e.At the outset, we investigated the ability of our recently prepared boronic esters of general formula R-Bnad  , showing2a–e. The results for 2a, 2b, and 2d are shown in 2a and 2b can structurally be described as double-tweezers. 2a exhibits an almost perfect double-tweezer-shaped structure with nearly planar bipyridine units, while 2b is markedly twisted with a twisting angle of the bipyridine moiety of about 35°. Adduct 2d, on the other hand, is composed of a more H-shaped structure with a nearly planar bipyridine unit (twisting angle ~10°). The bond parameters for all three adducts are similar, with B-N and B-O distances ranging from 1.61 to 1.67 Å and 1.45–1.47 Å, respectively. The central boron atoms in 2a, 2b, and 2d have slightly distorted tetrahedral coordination environments, with O-B-O and N-B-O angles ranging from 111–116° to 102–105°. Unfortunately, it was not possible to obtain suitable single crystals of 2c and 2e from acetone as the solvent. Not only was 2e highly soluble in acetone, but it also slowly degraded to a second product exhibiting a relatively sharp signal in the 11B NMR with a chemical shift of ca. 0 ppm  and average B-O and B-N distances of 1.45 and 1.67 Å, respectively. Notably, 2c × benzene co-crystallizes with two molecules of benzene, both being entrapped via π–π stacking with the 4,4′-bipyridine moiety and C-H…π contacts with both the phenyl and the naphthalene substituents.To confirm our structural assignments, efforts were undertaken to grow single crystals suitable for X-ray analysis from concentrated acetone solutions of 4,4′-bipyridine and see also . This nesee also . However benzene . Similar2c and 2d via a one-pot procedure in excellent yields starting from the respective boronic acids, without the need of preparing the respective esters 1c and 1d ethylene gave— upon reaction with 1,8-dihydroxy naphthalene and 1,4-phenylene diboronic acid in acetone—an orange microcrystalline precipitate, from which single crystals suitable for X-ray analysis could be obtained. The results revealed the compound to be polymeric Lewis acid–base adduct  fashion a. Due to4 (yield 94%) was formed as confirmed by single-crystal X-ray analysis  were heated until clear solutions were obtained. Upon slowly cooling to room temperature, crystalline solids were obtained in isolated yields ranging from 34–81%. In addition to being characterized by 1H NMR spectroscopy and combustion analysis, all compounds were analyzed by X-ray crystallography.Encouraged by the ability of dertaken . 2d was 2d is capable of forming various stable host–guest complexes with all aromatic hydrocarbon solvents used in this study via π–π stacking between the 4,4′-bipyridyl unit of 2d and the respective aromatic solvent. Thus, in benzene as a solvent, 2d × benzene is formed . Interestingly, crystallization experiments in meta-xylene gave two crystallographically distinct single crystals of 2d × m-xylene; the results for one revealed a classical 1:1 host–guest complex  associated with the liberation of the guest were found to be 131 °C and 147 °C, respectively, suggesting stronger host–guest interactions in 2d × toluene, and consistent with the results of DSC analysis (2d × m-xylene), 133 °C (2d × o-xylene), and 136 °C (2d × p-xylene). The slightly higher peak temperatures for 2d × o-xylene and 2d × p-xylene suggest somewhat stronger host–guest interactions compared to 2d × m-xylene, which is supported by DSC measurements , 71 °C (o-xylene), 76 °C (p-xylene), 121 °C (toluene), 139 °C (benzene), and 100 °C (mesitylene). Except for benzene, these values are significantly lower than what was measured for our 1,8-dihydroxy naphthalene-based host–guest complexes 2d × solvent . The obtained crystalline material was collected and analyzed by 1H NMR spectroscopy using DMSO-D6 as the solvent (see 2d for o-xylene (ca. 57%), with an m-, p-, to o-xylene ratio of about 2:1:4, respectively, which is similar to those of the respective catechol-based host–guest complexes reported by Hőpfl and Morales-Rojas [p-xylene appears to be surprising but perhaps can be attributed to kinetic effects during the crystallization process.Based on the results from the host–guest chemistry with es-Rojas . Conside5 and 6 could be obtained from acetone solutions and were characterized by single-crystal X-ray crystallography ethane was employed in reactions with 1,8-dihydroxy naphthalene and various boronic acids. Unfortunately, in none of the cases could host–guest complexes with aromatic hydrocarbon solvents be crystallized and structurally determined. Nonetheless, single crystals of the solvent-free double-tweezers lography a,b.2d × solvent, was prepared via reactions of 4,4′-bipyridine, 1,8-dihydroxy naphthalene, and 3,4,5-trifluorophenyl boronic acid with various aromatic hydrocarbons. SCXRD analysis of these solvent adducts revealed host–guest interactions to primarily occur via π···π contacts between the 4,4′-bipyridyl linker and the aromatic solvents to give 1:1 and 1:2 host–guest complexes, which was confirmed by 1H NMR spectroscopic analysis of the bulk samples. TGA measurements of the bulk samples showed well-defined weight losses at peak temperatures ranging from 122 to 147 °C, which in every case could be attributed to the complete loss of solvent (guest). Self-assembled Lewis acid–base adduct 2d, when recrystallized from an equimolar mixture of m-, p-, and o-xylene (1:1:1), preferentially binds to o-xylene, generating 2d × o-xylene as the major component, similar to what was reported for structurally related catechol-based host–guest complexes [We have demonstrated the ability of 1,8-dihydroxy naphthalene to self-assemble with various aryl boronic acids and 4,4′-bipyridine as a linker to give double-tweezer and H-shaped Lewis acid–base adducts in very good isolated yields by means of N→B bond formation. In addition, a series of host–guest complexes, omplexes . Collect


Allogeneic T cell platforms utilizing induced pluripotent stem cell (iPSC) technology exhibit significant promise for the facilitation of adoptive immunotherapies. While mature T cell receptor (TCR) signaling plays a crucial role in generating T cells from iPSCs, the introduction of exogenous mature TCR genes carries a potential risk of causing graft-versus-host disease (GvHD). In this study, we present the development of truncated TCRα and TCRβ chains, termed mini-TCRs, which lack variable domains responsible for recognizing human leukocyte antigen (HLA)-peptide complexes. We successfully induced cytotoxic T lymphocytes (CTLs) from iPSCs by employing mini-TCRs. Combinations of TCRα and TCRβ fragments were screened from mini-TCR libraries based on the surface localization of CD3 proteins and their ability to transduce T cell signaling. Consequently, mini-TCR-expressing iPSCs underwent physiological T cell development, progressing from the CD4 and CD8 double-positive stage to the CD8 single-positive stage. The resulting iPSC-derived CTLs exhibited comparable cytokine production and cytotoxicity in comparison to that of full-length TCR-expressing T lymphocytes when chimeric antigen receptors (CARs) were expressed. These findings demonstrate the potential of mini-TCR-carrying iPSCs as a versatile platform for CAR T cell therapy, offering a promising avenue for advancing adoptive immunotherapies.  Kaneko and colleagues have pioneered the novel artificial T cell receptor, termed mini-TCR. Although lacking antigen-recognition capacity, mini-TCR facilitates the generation of CAR T cells from iPSCs. This innovative technology holds promise for broadening therapeutic avenues in future cancer therapy. These mini-TCRs recruited CD3 proteins to the cell surface and transduced T cell activation signals. Mini-TCR expression induced differentiation to CD8+ T cells from undifferentiated iPSCs and iPSC-derived hematopoietic stem/progenitor cells (HSPCs). The generated CD8+ T cells also demonstrated specific cytotoxicity of target cells when used for engineering CAR T cells. We propose this technology as a promising tool for developing allogeneic, off-the-shelf CAR T cell therapies.,In the immunological synapse (the interface between T cells and APCs), TCRs form complexes with CD3 proteins and recognize HLA-antigen peptide complexes on APCs.,,,To identify the mini-TCRs that efficiently form complexes with CD3 proteins, we created mini-TCR libraries comprising various lengths of the constant regions of the TCRα and TCRβ chains and designed the cell-based screening system to measure the cell surface localization of the CD3 protein B. The N Based on CD3 recruitment capacities, we selected 17 representative pairs of truncated TCRα and TCRβ, including the pairs with the lowest (β6 and α1) and highest (β1 and α7) recruitment activities Figures A. cDNAs To confirm the loss of antigen-recognition abilities due to deletion of the variable regions, we transfected the 17 pairs of mini-TCR vectors and the full-length TCR that recognized mutated Wilms-Tumor1 (WT1) peptide-HLA-A∗24:02 complex as a positive control into CD3-OE-293T cells. In contrast to the binding of WT1-TCR-expressing cells to the WT1-HLA-A∗24:02 tetramer, none of the mini-TCR-expressing cells exhibited binding to the tetramer, confirming that the mini-TCR did not recognize the antigen peptide-HLA complexes D and 2E.TCR/CD3 complexes transduce TCR signaling and activate T cells.To specifically detect transfected TCR proteins, we attempted to disrupt endogenous TCRA and TCRB genes using gene-editing technology. Both TCRα constant (TRAC) locus and TCRβ constant 1 (TRBC1) loci were targeted with previously reported guide RNAs (gRNAs) A. To conSubsequently, to compare CD3 recruitment activity, we transfected all 17 mini-TCR vectors, the full-length TCR control, and the empty vector as a negative control. Neither CD3D nor CD3E proteins were detected in the empty-vector control samples, confirming that CD3D and CD3E proteins did not localize on the cell surface unless the TCRA and TCRB chains were expressed in the Jurkat knockout (KO) cells A–3D. CelTo assess the function of the mini-TCR/CD3 complex, the transfected TCR-KO Jurkat cells were stimulated with an OKT3 anti-CD3E antibody. Subsequently, we observed the highest upregulation of CD69, a T cell activation marker, in the full-length TCR control, and the mini-TCR b1a7 showed comparable upregulation, followed by other mini-TCRs that showed correlation with mean fluorescence intensities (MFIs) of CD3D and CD3E C.As the binding affinity of the anti-CD3E antibody OKT3 to TCR/CD3 complex measured by bio-layer interferometry was approximately 10-fold higher than that of the extracellular domains of CD3γε,Collectively, these data demonstrated that the introduction of mini-TCRs enables endogenous CD3 proteins to localize on the cell surface and mediate TCR signaling via stimulation with the CD3 antibody. Subsequently, we evaluated the functions of mini-TCRs b1a1 and b1a7 in the differentiation of iPSCs to T cells.The TCR/CD3 complex is also critical for T cell development. The T cell lineage commitment of common lymphoid progenitors derived from multipotent HSPCs is triggered by Notch signaling.We utilized cord-blood-derived iPSCs as a model of the hematopoietic system to test the function of mini-TCRs in T cell development, as these iPSCs were derived from myeloid cells and their TCR genes were not rearranged A. First,,5-fold   J. Mini-To iPSCs) J.5 total reads)   using similar NGS-based TCR repertoire analysis with the similar depth of the sequencing (3.3–4.9 × 10l reads) .36 Amongctively) , whereasctively) , and S3.ctively)  and S2, We examined whether mini-TCRs could be used in undifferentiated iPSCs as a cell source for T cell generation A. To add10 cells from an initial 1 × 106 iPSCs   H. Simila × 1010) I.14,38 ITo investigate whether such expanded CTLs could potentially be used for CAR T cell therapy, full-length or mini-TCR-iPSC-T cells were modified by transducing a retroviral vector containing a second-generation anti-CD20 CAR (CD20CAR) A and S6Bin vitro cytotoxicity against NALM6-Luc-KO cells of b1a7 CD19CAR T cells was comparable to that of full-length TCR CD19CAR T cells  mice (day −3)  A. The mi(day −3) C and S7DIn this study, we developed mini-TCRs, gene constructs of truncated TCRs, proficient in inducing T cell differentiation from iPSCs. Furthermore, we substantiated an innovative approach for engineering allogeneic CAR T cell therapies. Mini-TCRs successfully triggered T cell differentiation in all four iPSC lines tested. These compelling findings strongly suggest that mini-TCRs have the potential to generate CD8+ T cells from iPSCs as a promising allogeneic cell source platform to enhance CAR T cell therapies. In this section, we elaborately compare the mini-TCRs with full-length TCR and other iPS-T cell-related technologies. Additionally, we explore the future perspectives of iPS-T cells in the context of our findings.,,In the screening of mini-TCRs, constant regions of TCRα and TCRβ were cleaved in a stepwise manner, and the combination of these TCRs resulted in a bimodal distribution of CD3 recruitment to the cell surface C. One re,,,,,,,,in vivo, Notch signaling and pre-TCR signaling coordinate the proliferation of DN cells and their differentiation into DP cells.,To date, most studies reporting the induction of T cells from iPSCs have been performed using full-length TCR-overexpressing iPSCs or T cell-derived iPSCs.On the other hand, we observed different expression patterns of CD4, CD8a, and CD8b between full-length TCRs and mini-TCRs when T cells were differentiated from both retroviral-transduced HSPCs and iPSCs transfected with transposon. A DN cell population was formed after the maturation process in mini-TCR-introduced iPSC-T cells, indicating that the developmental cues received from mini-TCRs were weaker than those from full-length TCRs . Moreove+ T cells by knocking down EZH1 and demonstrated superior in vivo antitumor efficacy due to the existence of memory-like T cells that gave rise to effector T cells.,,Recent reports to improve the efficiency of T cell induction and antitumor efficacy are worth considering for combination with the mini-TCR. Jing et al. reported efficient induction of iPSC-derived CD8ab,TRAC locus of T cell-derived iPSCs to prevent GvHD.,,,RAG1 or RAG2 would be worth considering.Among hundreds of CAR T-related clinical trials, only a few allogeneic peripheral-blood-derived CAR T cell products are under development, such as Allo-501A and Allo-715 by Allogene Therapeutics and CTX110 by CRISPR Therapeutics.,,Finally, we present a comprehensive summary of the current state of iPS-T cell technology based on published research. Although culture conditions and iPSC lines exhibit variations, iPS-T cells commonly face challenges in maintaining memory T cell phenotypes, such as CCR7 and CD62L, and gene expression profiles during culture. Moreover, they demonstrate weaker cytokine and TCR signaling than clinically established primary CAR T cells. However, recent efforts to enhance iPS-T cell function have shown promising results. For instance, introducing optimized CAR constructs and membrane-bound IL-15 into iPS-T cells has led to comparable antitumor efficacy to primary CAR T cells in mouse tumor models.In conclusion, the development of mini-TCRs and the potential use of iPSCs as a platform for allogeneic immunotherapies represent significant advancements in cellular therapies. This approach holds promise for future clinical applications and contributes to the continued progress in cutting-edge cellular therapies.Full-length TCRα and TCRβ cDNA were amplified using the SMARTer RACE 5′/3′ Kit (Takara Bio) with T cell-derived iPSCs  linker (sequence: GSGSGS) , CD3G (NM_000073.2), CD3D (NM_000732.4), CD3z (NM_198053.2), and EGFP were fused with T2A peptides (CD3E-T2A-CD3G-T2A-CD3D-T2A-CD3Z-T2A-EGFP) and subcloned into the pBApo-EF1α Pur mammalian expression vector  and the T2A peptide B and sub2 atmosphere. Jurkat cells (DSMZ), B-LCL cells (RIKEN Cell Bank), and NALM6 cells (ATCC) were cultured in RPMI-1640 (Sigma) supplemented with 10% FBS and 1% PSG at 37°C in a 5% CO2 atmosphere.293T cells (DSMZ), GP2-293 cells (Takara Bio), and 293FT cells (Invitrogen) were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine-penicillin-streptomycin solution  at 37°C in a 5% CO5 cells/well, 50 μL/well) or 15-mL tubes . Fluorescent-labeled antibodies  were diluted with the buffer  and transferred to the samples. After incubation at 4°C for 30 min, cells were washed with the buffer and resuspend in PBS supplemented with 2% FBS and DAPI  or propidium iodide  at the volumes of 160 μL/well in 96-well plates or 500 μL/tube for 15-mL tubes. FCS files were analyzed on FlowJo v10 software.Cells were washed with phosphate-buffered saline (PBS) supplemented with 2% FBS and transferred into 96-well plates (2 × 105 cells/well) were seeded into 12-well plates. Next day, the cells were washed with PBS to remove the antibiotics and 1 mL of DMEM supplemented with 10% FBS was added. The CD3 expression vector pEF1α-CD3E-T2A-CD3G-T2A-CD3D-T2A-CD3Z-T2A-EGFP (1 μg) and Lipofectamine 2000 (Thermo Fisher Scientific) were diluted in 50 μL of Opti-MEM (Thermo Fisher Scientific), respectively. After 5 min, the two diluted solutions were combined, incubated at room temperature for 5 min to form DNA-liposome complexes, and transferred into the 293T cells. EGFP+ cells were purified using FACS SH800 (SONY) and maintained to establish a stable transfectant (designated as CD3-OE-293T cells).293T cells  using the Alt-R CRISPR-Cas9 System (Integrated DNA Technologies). TCRαTCRβ double negative cells were purified by FACS on SONY SH800. Disruption of TCRA and TCRB genes was confirmed by single transfection with the full-length TCRα or full-length TCRβ expression vector and subsequent FACS analysis using anti-TCRαβ and anti-CD3E (approximately 99% of the cells were negative for TCRαβ and CD3E). The target genomic sequences were as follows: 5 cells/well. The next day, TCRA-Myc or TCRB-FLAG (0.8 μg) and pEF1α-IRES-hKO1 vectors   were mixed in 50 μL of Opti-MEM and cotransfected into CD3-OE-293T cells as described in the “To validate the protein expression from TCRA-Myc or TCRB-FLAG vectors, CD3-OE-293T cells were seeded in 12-well plates at 1.1 × 10(1)TCRA-Myc (0.4 μg), TCRB-FLAG (0.4 μg), and pEF1α-IRES-hKO1 vectors    and S1(2)TCRβ-T2A-TCRα-pEF1α-IRES-hKO1 vectors (1.0 μg) To detect cell surface localization of CD3E protein on CD3-OE-293T cells, the following two combinations of vectors were transfected as described in the “Two to 3 days later, cells were harvested by trypsinization, stained with anti-CD3E-APC , and analyzed using a BD LSRII (BD Bioscience).TCRβ-T2A-TCRα-pEF1α-IRES-hKO1 vectors (1.0 μg) including full-length TCRs6 cells/sample, 100 μL/well) by using the 4D-Nucleofector electroporator  and SE Cell Line 4D-Nucleofector X Kit L (Lonza) according to the manufacturer’s instruction.To detect cell surface expression of CD3E and CD3D proteins on TCR-KO Jurkat cells, TCRβ-T2A-TCRα-pEF1α-IRES-hKO1 vectors (2 μg) were transfected into TCR-KO Jurkat cells . After incubation , cells were washed twice and stained with Alexa 647-labeled streptavidin . Samples were prepared as described in the “Two days later, transfected cells were harvested and stained with anti-CD3E-PE-Cy7  and anti-CD3D-APC . Biotinylated anti-CD3E antibody OKT3 (BioLegend) was diluted to 200 μg/mL and serial dilution was performed (1:5). Diluted antibody solution (100 μL/well) was transferred to a 96-well plate and mixed with equal amount of TCR-transfected TCR-KO Jurkat cells (1 × 105 cells/well and incubated overnight (day 1). Jurkat cells were stained with anti-CD69-Alexa Fluor 647  and analyzed using a BD LSRII (BD Bioscience).Transfection into TCR-KO Jurkat cells was performed as described in the “,Full-length and selected pairs of TCRβ and TCRα (b1a1 and b1a7) described in the section “6 cells/dish) and incubated overnight. pMYs-IG vectors (2 μg) and VSV-G vector  were diluted in Opti-MEM (200 μL) and combined with Lipofectamine 2000 . After 5 min of incubation at room temperature, they were transfected into the GP2-293 cells and incubated for 2 days. The harvested supernatant (10 mL/dish) was filtered and mixed with a Retro-X Concentrator  and incubated overnight at 4°C. After centrifugation , the pellet was resuspended in alpha-MEM (300 μL). The titers were determined by infection with the serial diluted viruses to Jurkat cells and FACS analyses. Aliquots were stored in a −80°C deep freezer until further use.For viral production, GP2-293 cells (Takara Bio) were seeded onto poly-L-lysine 5× (Sigma-Aldrich)-coated 10-cm dishes . hKO1+ NALM6 cells were sorted on BD FACSAria II and expanded.The GFP gene in CMV-Luciferase-EF1a-copGFP-T2A-Puro Lentivector (System Biosciences) was replaced with hKO1 amplified from the phKO1-S1 vector (MBL). Lentivirus was produced as previously described using 293FT cells.,Myeloid-derived iPSCs  were washed with MaxCyte buffer  three times and resuspended in MaxCyte buffer (4 × 106 cells per 100 μL) in an OC-100 cuvette. Then, the transfer and transposase vectors (16 μg each) were electroporated into the cells using MaxCyte STX (MaxCyte) in the Optimization 8 condition. Following the electroporation, the cells were immediately placed in culture medium and incubated at 37°C and 5% CO2 for 30 min. Then, the cells were transferred to iMatrix-511 coated six-well plates with StemFit AK03. After 5–7 days, CD19-positive cells were sorted by FACS (Full-length GPC3-specific TCRs (provided by Shinobi Therapeutics) and selected pairs of TCRβ and TCRα (b1a1 and b1a7) described in section “ by FACS .6 cells/well in a 5% O2 incubator (day 0). The next day, the cells were harvested and resuspended in 2 mL of StemPro-34 (Thermo Fisher Scientific) supplemented with 0.2× PSG, 1× GlutaMAX (Thermo Fisher Scientific), 50 μg/mL ascorbic acid-2-phosphate , 45 mM monothioglycerol (FUJIFILM Wako), and 1× insulin-transferrin-selenium solution (Thermo Fisher Scientific) , 50 ng/mL recombinant human (rh) BMP-4 (Miltenyi Biotec), 50 ng/mL rhVEGF (FUJIFILM Wako), and 50 ng/mL bFGF (FUJIFILM Wako) per well . After 24 h, 6 μM SB431542 (FUJIFILM Wako) was added . After 2 days, the differentiating EBs were collected, washed, and resuspended in 2 mL of EB basal medium supplemented with 50 ng/mL rhVEGF, 50 ng/mL rhbFGF, and 50 ng/mL rhSCF (R&D Systems) per well and cultured for 2 days . After 2 days, the EBs were cultured in 2 mL of EB basal medium supplemented with 50 ng/mL rhVEGF, 50 ng/mL rhbFGF, 50 ng/mL rhSCF (R&D Systems), 30 ng/mL rhTPO (FUJIFILM Wako), and 10 ng/mL rhFLT3L (FUJIFILM Wako) . The medium was changed every 2–3 days until day 13. Harvested cells were resuspended in TC-Protector cell freezing medium (Iwai) and stored in a −150°C deep freezer.HSPCs were induced by using the embryoid body (EB) method as previously reported.4 cells/well. Titrated retrovirus was added to the cell suspension . The next day, cells were seeded into plates coated with RetroNectin (5 μg/mL) and DLL4 protein (5 μg/mL) and incubated in alpha-MEM supplemented with 50 ng/mL rhSCF, 50 ng/mL rhIL-7 (FUJIFILM Wako), 50 ng/mL rhFLT3L, 100 ng/mL rhTPO, 30 nM rhSDF-1α (FUJIFILM Wako), 15 μM SB203580 (FUJIFILM Wako), 50 μg/mL PAA, 15% FBS, and 1% PSG (day 0). The medium was changed every 2–3 days, and cells were reseeded into fresh RetroNectin- and DLL4-coated 48-well plates every 7 days until day 21. Anti-CD3-BV510 (BioLegend) and anti-CD45-APC-Cy7 (BioLegend) were used for FACS analysis.HSPCs were differentiated into T cells as previously described.4 cells/well and stimulated with OKT3 anti-CD3 antibody  in alpha-MEM supplemented with 15% FBS, 50 μg/mL PAA, 1% PSG, 10 ng/mL rhIL-7, and 10 nM dexamethasone  for 3 days (days 21–24).,T cells induced on DLL4 proteins were seeded into 48-well plates at a density of 1–10 × 10CTLs were co-cultured with irradiated PBMCs (40 Gy) as feeder cells in alpha-MEM supplemented with 10% FBS, 1% PSG, IL-7 (5 ng/mL), IL-15 (5 ng/mL), pan-caspase inhibitor Z-VAD-FMK , PAA (50 μg/mL), ITS-G, and phytohemagglutinin  for 14 days. Feeder-free expansion was performed based on the previous report.The NGS to evaluate expression of endogenous and transduced TCR genes was performed as previously described.Anti-CD19 CAR retrovirus was transduced into iPS-T cells. A 96-well plate was coated with RetroNectin (Takara Bio) for 2 h at room temperature or overnight at 4°C. Two days after CD3/CD28 stimulation, iPS-T cells were transduced with concentrated retroviral supernatants by centrifugation on a RetroNectin-coated plate (MOI = 20). Cells were cultured for 11 days from transduction . Transduction was confirmed with flow cytometry with protein-L (GenScript) and streptavadin-APC (BioLegend). The cryopreserved cell stocks were prepared as outlined in “For detection of CD107a and IFNγ expression of iPSC-T cells, T cells were co-cultured with B-LCLs cells at a 1:1 ratio in a 96-well plate for 5 h in 100 μL of RPMI-1640 supplemented with glutamine, 1× Monensin (BioLegend), 10% FBS, and anti-CD107a-APC antibody . Then the cell surface was stained with anti-CD20-PE-Cy7 (BD Pharmingen) and anti-EGFR-PE (BioLegend). Samples were washed and fixed with Fixation Buffer (BioLegend) for 20 min. Samples were washed twice with Permeabilization Wash Buffer (BioLegend) and were stained with IFNγ-APC-Cy7 (BioLegend) for 20 min. The washed and resuspended cells were analyzed using BD FACSAria II.2. Then, 40 μL of the co-culture supernatant was collected and mixed with 160 μL of Eu solution, and time-resolved fluorescence was measured using a NIVO plate reader .A nonradioactive cellular cytotoxicity assay kit  was used for CD20CAR T cells. Briefly, target B-LCLs were pulsed with BM-HT reagent at 37°C for 15 min, washed three times, and seeded into 96-well plates at a density of 5,000 cells/well. Effector cells were added to the wells at effector-to-target (E:T) ratios ranging from 5:1 to 0.625:1 and were co-cultured for 3 h with α-MEM supplemented with 15% FBS and 1% PSG at 37°C with 5% COIn vitro cytotoxicity of the CD19CAR T cells were examined using the standard 51Cr release method. Briefly, target NALM6-Luc-KO cells were loaded with 1.85 MBq 51Cr for 1 h in α-MEM supplemented with 15% FBS and 1% PSG. Target cells  were co-cultured with effector iPSC-derived CAR T cells for 5 h in the medium with various effector-to-target (E:T) ratios ranging from 16:1 to 1:1. Supernatants were harvested and the released 51Cr was measured using a beta counter (PerkinElmer).6 NALM6-Luc-KO cells were intraperitoneally (i.p.) injected to establish a mouse leukemia model (day −3). From day −1, the tumor burden was monitored weekly by in vivo bioluminescence imaging using IVIS SPECTRUM and Living Image 4.7.3 software (PerkinElmer). CAR T cells (1.6 × 106 cells/mouse) were i.p. injected into mice grouped according to the luminescence on day 0. From day −3, body weight was monitored daily, and 20% weight loss compared to that on day −3 was used as the humane endpoint for sacrifice.Male NSG mice  were housed under controlled humidity and light/dark cycle in a specific pathogen-free facility. All animal experiments were performed in accordance with the Animal Review Board of Kyoto University. A total of 1 × 10The sequences identified and designed during this study are available from supplementary tables or from the corresponding author upon reasonable request.


U0Red in [PW11M(H2O)O39]q− M = Mn(III/II),Fe(III/II), Co(III/II), and Ru(III/II). U0Red calculations for [PW11M(H2O)O39]q− were calculated usinga conductor-like screening model to neutralize the charge in thecluster. We explicitly located K+ counterions which inducedpositive shifting of potentials by > 500 mV. This approximationimprovedthe reproduction of redox potentials for Kx[XW11M(H2O)O39]q−x M = Mn(III/II)/Co(III/II). However,uncertainties in U0Red for Kx[PW11M(H2O)O39]q−x M = Fe(III/II)/Ru(III/II) were observed becauseof the over-stabilization of the ion-pairs. Hybrid functionals exceeding25% Hartree–Fock exchange are not recommended because of largeuncertainties in ΔU0Red attributed to exaggerated proximity of the ion-pairs. Our resultsemphasize that understanding the nature of the electrode and electrolyteenvironment is essential to obtain a reasonable agreement betweentheoretical and experimental results.Polyoxometalates have attracted significant interestowing to theirstructural diversity, redox stability, and functionality at the nanoscale.In this work, density functional theory calculations have been employedto systematically study the accuracy of various exchange–correlationfunctionals in reproducing experimental redox potentials,  U0Red in [PW11M(H2O)O39]q− M = Mn(III/II), Fe(III/II),Co(III/II), and Ru(III/II). Furthermore, we have explicitly located(K+) counterions, rendering our systems’ chargeneutral forming Kx[XW11M(H2O)O39]q−x (x = 4 or 5) and assessing the effect ofcomputed U0Red values.Densityfunctional theory calculations have been employedto assess the accuracy of exchange−correlation functionalsin reproducing experimental redox potentials,  These complexes formally loseone or several M = O vertices and possess reactive cavities with highcharge density around the defective region due to the negatively chargedoxygen ligands.8 The defective region canreact with transition-metal cations forming a new class of compounds,an example shown are mono-transition-metal-substituted polyoxotungstates,[XW11M(L)O39]q− , Si(IV), L = H2O, DMSO, etc.)—see 8 Conventionally, hexacoordinate transition-metals are introducedso the sixth coordination site are occupied by solvent ligands fromthe local environment—see 9 in fieldsincluding water oxidation,11 carbon dioxide reduction,13 and nitrogen activation  are a large groupof discrete, polynuclearmetal-oxo clusters comprising early transition-metal (addenda) andoxide atoms.tivation 1.1438 In this regard, Poblet and co-workers reported the relative stabilityof rotational isomers of Keggin heteropolyanions, α/β-[XM12O40]q−.39 These calculations were performed using thelocal-density approximation functional coupled with Vosko–Wilk–Nusairparametrization, in the gas phase.39 Theenhanced stability of the α-[XM12O40]q− isomer was attributed to the higherenergy of the lowest occupied molecular orbital.39 However, it was shown that four-times-reduced clusters[PW12O40]7– and [SiMo12O40]8– demonstrated enhancedstability (ca. 0.4 eV) toward the β-isomer.39 Later, Zhang and co-workers emphasized the importance ofimplicit solvation for reproducing experimental geometries.40 Several generalized gradient approximation (GGA)functionals were tested, in which Perdew–Burke–Ernzerhof(PBE) and Becke 1988 exchange and Perdew 86 (BP86) methods providedthe closest description to the experimental geometries.40 However, GGA functionals often poorly describeelectron delocalization as previously reported by Poblet and co-workers.41 Their work showed GGA functionals incorrectlylocalized an additional electron at the belt region in mono-substitutedWells–Dawson [P2W17MO62]q−  anions.41 On the other hand, employment of hybrid methods, such asB3LYP or M05 correctly localized the electron.41 However, only B3LYP [20% Hartree–Fock (HF) exchange]gave the correct ordering and relative reduction energies with respectto experimental measurements.41Early computational work on POMs have primarily focusedon chargedmolecules using implicit solvation models.11O40]q−  using a conductor–like screening mode.42 This simplification produced large uncertaintiesin absolute reduction potential. However, shifts in potential withrespect to the Keggin [XM12O40]q− anion were reproduced with correspondingexperimental data.42 Recently, Röschand co-workers reported redox potentials for the tri-Mn-substitutedKeggin in which counterions were explicitly located onto the surfaceof POM to neutralize the system.43 Theauthors employed several exchange–correlation (x–c) functionals with the increase in contributionsof HF exchange  which producedredox potentials that increase (in that order) with the exact exchange.43 The closest agreement with experimental literaturewas shown for the GGA–PBE functional attributed to fortuitouserror cancellations. By contrast, hybrid exchange–functionalsoverestimated experimental potentials by 0.6–1.0 V.43 This work was extended to include explicit treatmentof water molecules derived from molecular dynamics simulations.44 The inclusion of explicit solvation significantlyimproved computed redox potentials; however, its contributions wereout-weighted by the choice of exchange–correlation functional.44 In this work, optimal reproduction of experimentalliterature were obtained using the B3LYP method.44 Recently, Falbo and Penfold reported the influence of theself-interaction error could be reduced by neutralizing the chargedspecies by incorporating counterions into the system.45 The authors reported excellent agreement, within 0.1 Vof the experimental literature for the first redox wave in Na3[SiW12O40].45Quantumchemical calculations reproducing redox potentials in POMshave been reported without accounting for electrolyte environmentand counterions, using implicit solvation models. Aparicio and co-workerscomputed the tungsten redox waves for mono-substituted Keggin, [XMW11M(H2O)O39]q− M = Mn(III/II), Fe(III/II), Co(III/II), and Ru(III/II) and theirpotassium salts. For achieving adequate molecular geometries, we presenta structural benchmark for the cobalt(II)-substituted Keggin againstthe crystallographic structure.46 Thereafter,we explore the challenges in computing redox potentials and providean insight into the geometric and electronic factors controlling it.In this work, we have performed systematic density functionaltheory(DFT) calculations to study the redox properties of q−, MSE, and MAE were used to compare against the crystallographicstructure, reported by Cavaleiro and co-workers.46 Four types of oxygen atoms were examined: Oc and Ot which correspond to the heteroatom–oxygenand terminal–oxygen atoms, while Oa1 and Ob2 denote bridging  oxygens bound to the newly incorporatedtransition-metal—see The selection ofthe exchange–correlation (11Co(H2O)O39]q− employed to assess the accuracy of applied functionalsand basis sets. As is evident, equilibrium geometries in [PW11Co(H2O)O39]q− were generally well described by all methods, rarely exceeding discrepanciesof 0.18 Å. t–Co,Oa1–Co, and Ob2–Co by 0.236, 0.059,and 0.035 Å, respectively. c–Co and Ot–Co. The crystallographicstructure of [PW11Co(H2O)O39]q− illustrates the magnitude of Ot–Co surpasses Oc–Co, reporting distances of 2.321 and 2.079 Å,respectively.46 However, our models haveinverted this observation, in which the magnitude of Oc–Co exceeds Ot–Co. Across all methods, theequatorial distances were accurately described with computed errorsrarely exceeding 0.02 Å. The equatorial parameters were insensitiveacross all applied functionals and basis sets, reporting a range of0.059 and 0.085 Å for Oa1–Co and Ob2–Co, respectively.11M(H2O)O39]q− anions, optimized using thePBE/TZP level of theory. Herein, orbitals associated with the newlyincorporated transition-metal are inserted between the occupied oxoand unoccupied addenda band.The electronic structure of a fully oxidized POMconsists of twoidentifiable bands: (i) an oxo-band comprising occupied oxo-ligands;(ii) and a metallic band encompassing unoccupied addenda orbitals. 11M(H2O)O39]q− M = Mn(III/II),20 Fe(III/II),21 Co(III/II),24 and Ru(III/II).27Table-S1 willbe used to account for modifications to molecular geometries of [PW11M(H2O)O39]q− , under electrochemicalprocesses. One-electron oxidation of [PW11Mn(H2O)O39]5– induced constriction of Oa1–Mn and Ob2–Mn by 0.197 and 0.125Å, respectively. Oxidation of [PW11Mn(H2O)O39]5– removes σ*(Oa1–M) and σ*(Ob2–M) interactions derivedfrom the overlap of O2px or O2py orbitals and dx2–y2 orbitals. One-electron oxidation of[PW11M(H2O)O39]q− (M = Fe(II) or Ru(II)) had no significant impact tomolecular geometry. One-electron oxidation of [PW11Co(H2O)O39]5– constricted Oc–M by 0.284 Å attributed to the removal of σ*(Oc–M) derived from the overlap of O2pz and dz2 orbital. Herein,[PW11Co(H2O)O39]4– possesses the electron configuration: (dxz)2(dyz)2(dxy)2 which are (predominantly)non-bonding. Previous work modeling redox processes of POMs have beencalculated using the reduction energy (RE) which assumes entropicand vibrational contributions are negligible.62 However, the significance of σ*(Oc–M), σ*(Oa1–M), and σ*(Ob2–M) interactionsemphasize the importance of enthalpic, entropic, and zero-point contributionsfor redox modeling of [PW11M(H2O)O39]q− [M = Mn(II) and Co(II)] anions.Cyclic voltammetric experiments have explored theredox behaviorfor q−; X = As(V), Si(IV), Ge(IV), B(III), and Zn(II) arereported in U0Red for[PW11M(H2O)O39]q− M = Fe(III/II)/Ru(III/II) closely reproduced the literaturewithin ca. 0.2 V. By contrast, computed values of U0Red for [PW11M(H2O)O39]q− M = Mn(III/II)/Co(III/II)were notably underestimated by ca. 0.8—1.0 V. The discrepancybetween DFT and experimental potential, U0Error increased with overall anionic chargeof [XW11M(H2O)O39]q−; attributed to the increasing contribution of theself-interaction error. For example, [ZnW11Mn(H2O)O39]7–/8– produced U0Error = 1.26 V superseding [PW11Mn(H2O)O39]4–/5– by 0.23 V.Redoxpotentials, 11M(H2O)O39]q−—see U0Error for[PW11Mn(H2O)O39]q− increased from 1.03 to 1.40 V (ΔU0Error = 0.37 V) for TZP and QZ4P basis sets,respectively.The effect of applied exchange–correlationfunctional andbasis set was assessed for the Mn(III/II), Fe(III/II), Co(III/II),and Ru(III/II) couples in [PWx–c functionalsoutperformed GGA methodologies by reducing U0Error for M(III/II) redox couples. Thisis evidenced by U0Error in[PW11Mn(H2O)O39]q− which notably decreased from 1.03 to 0.28 V (ΔU0Error = 0.75 V) by incorporating15% HF exchange (B3LYP*/TZP). However, the optimal x–c functional was not consistent across allM(III/II) couples. For example, Fe(III/II), Co(III/II), and Ru(III/II)couples were optimally reproduced with 20% HF exchange (B3LYP/TZP)calculating U0Error of 0.01,0.10, and 0.23 V respectively. Thus far, U0Red for [PW11Mn(H2O)O39]q− were calculated without neutralizingtheir charge, and therefore, large discrepancies associated with theself-interaction error are expected for these calculations. Previouswork showed explicitly locating counterions on the surface of thePOM, rendering the system charge neutral can reduce this systematicerror.44Generally, hybrid On the surface of the molecule, the counterion can interact with4-fold (pocket A–F) or 3-fold pockets—see Figure S3. The relative stabilityof all isomers was evaluated by the difference in electronic energy, ERel.—see ERel across K5[PW11Co(H2O)O39] isomers were almost negligible at 1.11 kcal mol–1. The discrepancy between pocket A and B was attributed to reducedelectrostatic attractive forces because of shielding by the aqua protons,correlating with previous work.43 Energydifferences across K4[PW11Co(H2O)O39] isomers were moderate, reporting ΔERel of 3.04 kcal mol–1. It is importantto recognize the electronic energy difference between the three lowestenergy isomers of K4[PW11Co(H2O)O39] ranged by >0.4 kcal mol–1. Hence,itis reasonable to assume all arrangements will co-exist in solution.However, for our redox calculations, we focused on the two lowestenergy isomers: A and A,D.To differentiate between cation arrangements, isomerswere identifiedby their unoccupied pockets. For example, isomer-D corresponds tosystems with an absent cation–oxygen interaction at pocketD—see U0Red versusSHE, for Mn(III/II), Fe(III/II), Co(III/II), and Ru(III/II) couplespresent in Kx[XW11M(H2O)O39]q−x; X = As(V), Si(IV), Ge(IV), B(III), and Zn(II) are reportedin U0Red with Kx[XW11M(H2O)O39]q−x M = Mn(III/II)/Co(III/II) demonstrated significantimprovement, by which U0Error rarely exceeded 0.50 V. A comparison with U0Error in Kx[ZnW11Mn(H2O)O39]q−x was reduced from 1.26 to 0.43 V (ΔU0Error = 0.83 V). However, accuracyin computation of U0Red for Kx[XW11M(H2O)O39]q−x M = Fe(III/II)/Ru(III/II) was impaired in which all couplesproduced U0Error exceeding0.35 V.Redox potentials, x[XW11M(H2O)O39]q−x have employed the A and A,D isomers—see U0Error for Kx[PW11Co(H2O)O39]q−x to assess the sensitivity of our model to differentcation arrangements. The current model demonstrated moderate sensitivityacross all cation arrangements ranging up to 0.18 V. Comparativelypoor agreement with the literature was achieved with the energeticallyfavored A + A,D couples, whose redox couple underestimated potentialsby 0.315 V, with respect to the literature. Accurate computation of U0Red was achieved with isomers: D+ B,E and F + B,E couples, who redox couples underestimated potentialsby 0.135 V.Thus far, models of Kx–c functional and basis set on U0Red for Kx[PW11M(H2O)O39]q−x M = Mn(III/II),Fe(III/II),Co(III/II), and Ru(III/II), as shown in x[PW11M(H2O)O39]q−x in which U0Error increased from 0.35 to 1.07 V (ΔU0Error = 0.72 V). A comparison with 11Mn(H2O)O39]q− reduced U0Error by 0.68, 0.24, and 0.33 V for TZP, TZ2P,and QZ4P basis sets, respectively.For comparison with U0Red for Kx[PW11M(H2O)O39]q−x M = Mn(III/II), Fe(III/II),Co(III/II), and Ru(III/II).ΔU0Error usingGGA–PBE functional (0% exchange) reflected in the range of0.32–0.43 V across all redox couples. Those with larger contributionsof HF exchange varied significantly, for example, ΔU0Error computed using BH&H rangedfrom 0.43 to 1.50 V. Hybrid functionals exceeding 25% HF exchangedid not provide any improvement for reducing ΔU0Error. As is evident, increasing HF exchangepositively shifted U0Red whichwas attributed to the over-stabilization of the ion-pairs. Hybridfunctionals exceeding 25% HF are not recommended because of largeCPU times coupled with significant overestimations to ΔU0Red.We have explored the effect of HF exchange on U0Red became positivelyshifted with the increase in contributions of HF exchange. To rationalizethis observation, we plotted counterion–bridging oxygen (betweenpocket D) as a function of HF exchange, see b–K distances leading to the over-stabilizationof the close contact ion-pairs. The significance of these Ob–K distances is emphasized by the computed range (PBE to BH&H)in Ob–K distances in K5[PW11Co(H2O)O39] calculated at ca. 0.2 Å whichreflected in ΔU0Red =1.29 V. Previous work by Kaledin and co-workers reported the averageOt–K and Ob–K distances for hydrated[PW12O40][K(H2O)16]3 complexes were 5.1 and 5.7 Å, respectively.63 To contrast, Ot–K and Ob–K distances in K5[PW11Co(H2O)O39] were ca. 4.1 and 2.8 Å, respectively.Evidently, further improvements should focus on accurately modelingcounterion–bridging oxygen distances to enable more accuratecomputation of U0Red.11M(H2O)O39]q− anions and their corresponding potassiumsalts. We have focused our attention to Mn(III/II), Fe(III/II), Co(III/II),and Ru(III/II) redox couples associated with [XW11M(H2O)O39]q− X =P(V), Si(IV), Ge(IV), B(III), and Zn(II). Our aim has been to explorethe challenges in computing redox potentials and provide an insightinto the geometric and electronic factors controlling it. We employedseveral x–c functionals includingthe hybrid class selected by their contributions to HF exchange .In this work, we have employed DFT calculationsto systematicallystudy the accuracy of various exchange–correlation functionalsfor reproducing experimental redox properties in mono-transition-metal-substitutedKeggin q−x, whereby U0Error was reduced from 1.26 to 0.43 V (ΔU0Error = 0.83 V). However, problems remain as U0Red for Fe(III/II) and Ru(III/II) were excessively(positively) shifted, attributed to the over-stabilization of theion-pairs. Previous work has shown the average Ot–Kand Ob–K distances for hydrated [PW12O40][K(H2O)16]3 complexeswere 5.1 and 5.7 Å, while our charge neutral model of K5[PW11Co(H2O)O39] produced Ot–K and Ob–K distances reporting atca. 4.1 and 2.8 Å, respectively. We have rationalized this overestimationby plotting Ob–K distances as a function of HF,whereby increasing contributions of exchange (0% PBE to 50% BH&H)produced progressively shorter Ob–K distances. Thereported range in Ob–K was only ca. 0.2 Å;however, its significance is highlighted in K5[PW11Co(H2O)O39] which reflected in the range of1.29 V for U0Red.For direct comparison,we explicitly located KU0Red. The presentresults emphasis the current approach requires significant improvementto achieve more reliable modeling with respect to experimental work.Obtainingaccurate redox potentials using implicit solvation modelsremains a challenge. Our results emphasize that understanding thenature of the electrode and electrolyte environment may be essentialto obtaining reasonable agreement between theoretical and experimentalresults. Further improvements to this work by accurately modelingcounterion–bridging oxygen distance will enable more accuratecomputation of