These plant sources were propagated under greenhouse conditions. The isolated compounds were elucidated by analyzing their spectroscopical data . The antifungal effect on the mycelial growth of Fusarium oxysporum (Fox) of each isolated QA was then evaluated through the amended medium assay. The best antifungal activity was found to be for compounds 8 (IC50 = 16.5 µM), 9 (IC50 = 7.2 µM), 12 (IC50 = 11.3 µM), and 18 (IC50 = 12.3 µM). The inhibitory data suggest that some QAs could efficiently inhibit Fox mycelium growth depending on particular structural requirements deduced from structure–activity relationship scrutinies. The identified quinolizidine-related moieties can be involved in lead structures to develop further antifungal bioactives against Fox.The Genisteae tribe belongs to the Fabaceae family. The wide occurrence of secondary metabolites, explicitly highlighting the quinolizidine alkaloids (QAs), characterizes this tribe. In the present study, twenty QAs (The online version contains supplementary material available at 10.1007/s13659-023-00373-4. Petrosia, Xestospongia, and Oceanapia , and the isolated QA cytisine (12), showed antimicrobial properties at concentrations between 1 and 6 µg/µL against A. solani, Monilia fructicola, Fox, Xanthomonas campestris, Pseudomonas sp., and Erwinia carotovora, involving significant mycelial growth inhibition diameters (> 5 mm at 6 mg/mL) [12, 13, and 15 have also been reported in an alkaloidal extract from G. vuralii and evaluated against E. coli, P. aeruginosa, B. subtilis, S. aureus, C. albicans, and C. krusei, with MIC values within 62.5–500 µg/mL range [15 has no antifungal records as a pure substance; however, extracts of Retama raetam containing compound 9 (43.6%) and 15 (28.5%) mostly showed antimicrobial activity at 75 µg/mL against S. aureus (MIC = 125 µg/mL) [16 and 17 (tetrahydrocytisine-type QAs) had also shown antimicrobial effects against E. coli, B. subtilis, S. aureus, C. albicans, C. krusei, involving a MIC = 128 mg/mL for P. aeruginosa and a MIC = 16 µg/mL for K. pneumoniae [L. angustifolius containing compounds 16 and 17 were tested against strains of the following bacteria: E. coli (MIC = 500 µg/mL), P. aeruginosa (MIC = 62.5 µg/mL), B. subtilis (MIC = 62.5 µg/mL), and S. aureus (MIC = 62.5 µg/mL) as well as the fungi C. albicans (MIC = 500 µg/mL) and C. krusei (MIC = 500 µg/mL) [The highest activity for those QAs structurally related to cytisine was found for 6 mg/mL) . MoreovemL range . Compouneumoniae . Additio0 µg/mL) .18 (matrine-type) showed a high inhibition against the phytopathogen Fox. Compound 18 showed group-I–related antifungal activity (IC50 < 15 µM), including a fungicidal action. Similarly, compounds 19 and 20 also exhibited inhibition results corresponding to group I, but both promoted a fungistatic effect. Matrine-type QAs have been widely referred to in the literature for their antimicrobial properties against phytopathogens. For instance, QAs 18 and 20 showed conidia germination inhibition against Fox , Cladosporium oxysporum , and Marssonina brunnea  [20 more active than 18. However, in our case, compound 18 showed the best inhibitory action on Fox mycelial growth , and this information agrees with the reported antifungal activity against C. albicans [Microsporum lanosum [Fox, Valsa pini, Cladosporium oxysporum, and Marssonina brunnea [20 did not agree with the literature since it has been reported as an antifungal against Fox, involving better EC50 values, i.e., 26 µg/mL, compared to 18 (123 µg/mL) [Finally, QAs structurally related to ctively) , being calbicans , Microsp lanosum , Fox, Va brunnea . Compoun3 µg/mL) . HoweverFox caught our attention. Hence, the relationships between structure and antifungal activity were examined within alkaloid types using the outcome of this mid-size set of isolated QAs. In this regard, for instance, the lupanine-type diazatetracyclic QAs exhibited an antifungal effect at different levels. Such an activity appeared to be influenced by particular structural variations based on substituents and double bonds in different positions, as shown in Fig. 3  and compared to the basic structure related to compound 1 . This observation suggests that unsaturations in the A-ring of the QA and the opposite C7-C8-C9 bridge orientation might be critical to obtaining a better antifungal effect against Fox. However, a double bond in the D-ring did not show any relevant effect since compound 2’s IC50 (ca. 100 µM) was higher than that of 3. These facts showed the particular influence of the relative bridge orientation and the unsaturation location in the QA structure, i.e., neutral and positive influence if a double bond occurs in the D-ring or the A-ring, respectively. Similarly, compound 15 (an anagyrine-type QA) had a conjugated system in the A-ring  and even exhibited an IC50 < 28 µM (group II). In addition, compounds 4 and 5 are structurally related to compound 1 but differ by hydroxyl groups at C4 and C13, respectively. In this way, compound 4 had antifungal activity corresponding to group II, being more active than compound 5, which was moderately active (type III). Therefore, unsaturations or hydroxyl substituents on the A-ring of lupanine-type QAs were relevant structural factors for better Fox growth inhibition. Additionally, a keto group at Cring at C17  and the α-orientation of the H-11  seemed to influence the activity of QAs against Fox negatively, being 6 and 7 the least active QAs in this small test compound set (IC50 > 300 µM). In contrast, a carbonyl group at C10 (B-ring) instead of C2 (A-ring), and even the opposite orientation for H-6 and H-11 compared to 1, exhibited a 15-fold improved activity, constituting QA 9 as the most active compound (IC50 = 7.2 µM). Finally, the opposite orientation of C7-C8-C9 bridge and the absence of the carbonyl group at C2 for the sparteine-type QAs revealed a ca. seven-fold better activity . However, a carbonyl group at C4 and a Δ2(3) unsaturation exhibited a neutral influence on the antifungal activity of 10 (IC50 = 95.5 µM) compared to 1 but a lesser effect than 8.The QA’s structural and bioactivity variations of each compound against 12  showed mycelial growth inhibition changes depending on the substitutions at N12  at N12, but the activity decreased and was situated into group II. In contrast, compound 14 had an electron-withdrawing group  at N12, showing an IC50 value corresponding to the group I. This fact suggested that the substitution nature on the N12 of cytisine-type QAs is a critical feature for the Fox mycelial growth inhibition. Although few molecules related to 12 were isolated in the present study, it was possible to conclude that EWG at N12 can be responsible for group-I-related inhibitory effects (most active). In contrast, EDG at N12 can be considered responsible for decreasing the antifungal activity until lower mycelial growth inhibition .On the other hand, compounds structurally related to 16 and 17) also showed an interesting relationship between antifungal activity and the presence of an alkenyl substituent  at N12 or C11, respectively, as shown in Fig. 16 and 17 belong to group II, but 16 exhibited better activity than 17, indicating a better influence on the antifungal activity if alkenyl group is positioned at N12 but lower than QA 12. To expand this observation, a comparison between 13 and 16, differing by a 2-pyridone moiety and the N12 substituents, can rationalize the structural effect on the antifungal activity of these tricyclic QAs. Compound 13 has a methyl group, while compound 16 has the but-3-enyl moiety. Thus, it was observed that the but-3-enyl substitution increased the inhibition, which suggests that bulkier substitutions at N12 have a positive influence. In contrast, compound 17 contained an allyl substitution at C11, which did not influence the antifungal activity since the IC50 was reduced by more than 50% compared to compound 16. Thus, according to these observations, a short alkenyl chain at C11 does not contribute to the better antifungal activity. Contrarily, a positive impact can be deduced if a but-3-enyl substitution is attached at N12.Furthermore, tetrahydrocytisine-type QAs  and 19 and 20 highlighted some structural differences  to be considered relevant for antifungal activity -( +)-18) , did not have the same fungicidal effects as 18, inferring that unsaturations and/or configuration variations at these positions cause a slight decrease in IC50 values and were also responsible for the fungistatic and non-fungicidal activity of 19. In the case of compound 20, there was a decrease of more than 50% inhibition compared to compound 18. However, the IC50 value was located in group II despite being fungistatic.Finally, a comparison of the structure and antifungal activity between ity Fig. . HomologFox mycelial growth inhibition. QA 12 showed the best antifungal activity (IC50 < 12 µM), and although 13–15 were structurally related to 12 but differed by certain substitutions , the IC50 values were lower (> 15 µM), suggesting an important influence on the antifungal activity depending on the substitution patterns at N12 . In the case of bridged tetracycles, substitutions or unsaturations on the A-ring positively influenced the QA bioactivity, while substitutions or double bonds on the D-ring caused a decrease in the phytopathogen inhibition. Finally, the small set of structural variants of fused tetracyclic QAs did not exhibit substantial activity variations compared to 18, which showed the best antifungal activity for this QA type. All compounds reported in this study (1–20) were isolated from the species under study, tested against a Fox strain, and explored their structure–activity relationships for the first time. Our findings might be considered relevant baseline information to develop QA-based fungicides in further studies oriented to Fox’s management and control.The present study obtained several QAs (n = 20) involving structural differences according to six alkaloid types . In addition, structural variability, substitution patterns, and double bonds were found to be relevant in understanding the outcome changes on Genista monspessulana, L. polyphyllus 'rusell', and L. mutabilis were used to isolated QAs. Lupinus and Genista plants were propagated from seeds under greenhouse conditions (vide infra). Seeds from Lupinus plants were commercially purchased from Sow Right Seeds [G. monspessulana  were collected from wild plants at Choconta municipality, Cundinamarca, Colombia . As described below, leaves were collected to proceed with the alkaloid extraction from the collected plant material.Fresh leaves of ht Seeds . HealthyThe seeds of the above-mentioned fabaceous plants were planted in 72-cell seedbeds containing a combined substrate of loamy-silty soil (LSS) with rice husk (RiH) at a 3:1 ratio. These plants were maintained under greenhouse conditions  = 65 ± 15%, altitude = 2562 masl, total light transmission = 85 ± 5%, total light diffusion = 55 ± 5%, and UV transmission between 290 and 340 nm = 5%) for 30 days. After 10–15 days, the seeds started the germination process. After 20–30 days of seed planting, the first and second pairs of leaves emerged, at which time the seedlings were transplanted into bags of 2 L capacity on a substrate containing a mixture of LSS/RiH 1:3 and were maintained under the same greenhouse conditions. After transplanting, the pot-planted seedlings were irrigated with water (500 mL) every 2 days, and once the elongation of the central axis began to be observed, commercial triple-15 fertilizer at a concentration of 5% was applied. After 70 ± 19 days of development under these conditions, plants reach the flowering stage. At this moment, fresh leaves were collected for QA extraction, as described below.3 solution. Subsequently, liquid–liquid extraction was performed using chloroform to obtain an alkaloid-rich organic phase. Finally, the solvent excess was removed by distillation under reduced pressure at 375 mbar for 5 min, and the respective Lupinus- and Genista-derived quinolizidine-rich extracts (QREs) were obtained to be separated further by chromatographic procedures (vide infra).Fresh leaves (500 g) of each plant mentioned above were extracted using 0.5 M HCl (20 mL) under stirring at 130 rpm in an orbital shaker for 24 h. Subsequently, the acidic solution was filtered and brought to pH 10 with a 15% aqueous NH254. Different solvent mixtures by combining n-hexane (Hex), chloroform (CHCl3), dichloromethane (DCM), toluene (Tol), methanol (MeOH), even ammonia (NH3) and/or formic acid (HCOOH) reagents, were used as mobile phases. Dragendorff's reagent, iodine vapors, and UV at 254 and 366 nm were used as revealers to visualize analytes. Column chromatography (CC) involved gradient elution for separating and purifying target compounds. The stationary phase was silica for column chromatography (0.063–0.200 mm) (Merck), and different mixtures of the solvents mentioned above in different ratios were used as the mobile phases. TLC monitored CC separations.Thin-layer chromatography (TLC) was performed using Silica gel 60 F1–20 were isolated from L. mutabilis, L. polyphyllus 'rusell', G. monspessulana following the procedures described below.Alkaloids L. mutabilis leaves (200 g) were collected, ground, and extracted using the above-mentioned acid–base procedure (vide supra) to obtain the respective QRE (1.3234 g), monitored by TLC (DCM/MeOH/NH3 8.5:1:0.5) and separated by CC (DCM/MeOH/NH3 8.5:1:0.5). Fourteen fractions (Frs.) were gathered after TLC analysis (Frs. Lm1 to Lm14) and analyzed by GC–MS. Chromatographic analysis showed that Frs. Lm1 to Lm4 corresponded to pure 1 (217.2 mg), Frs. Lm5 to Lm7 were a 1, 3 mixture, Frs. Lm8 to Lm11 involved a 1–2 mixture, and, finally, Frs. Lm12 ro Lm14 afforded the pure 8 (103.7 mg). Consecutively, the Frs. Lm5-to-Lm7 were separated by CC using gradient elution starting from the DCM/MeOH/HCOOH 9:1:1 mixture. This separation afforded five gathered fractions (Frs. Lm5-7a to Lm5-7e). In this sense, the Frs. Lm5-7a and Lm5-7b were the pure 1, the Fr. Lm5-7c was a 1, 3 mixture, and Frs. Lm5-7d and Fr. Lm5-7e were the pure 3 (13.5 mg).Fresh L. polyphyllus ‘rusell’ leaves (300 g) were collected, ground, and extracted by the acid–base procedure to obtain the respective QRE (756.4 mg). A portion of this QRE (500 mg) was employed and separated by CC, using gradient elution from a DCM/MeOH/NH3 8:1:1 mixture. The chromatographic separation afforded eight gathered fractions (Lp1 to Lp8). The depuration and purification by CC of fractions Lp2 and Lp4 yielded compounds 2 (9.8 mg) and 7 (15.6 mg), respectively. A new portion of fresh leaves was subsequently obtained after a sixth pruning event of the propagated L. polyphyllus ‘rusell’. These leaves (330 g) were subjected to an acid–base extraction process to obtain another QRE (1254.4 mg). A portion of this QRE (500 mg) was separated by CC using gradient elution from a DCM/MeOH/NH3 8:1:1. Eighteen gathered fractions were obtained (Frs. Lp6-1 to Lp6-18). Frs. Lp6-1-to-Lp6-3 were a 1, 11 mixture, Fr. Lp6-4 was the pure 11 (10.1 mg), Frs. Lp6-5-to-Lp6-7 were the pure 17 (32.5 mg), Frs. Lp6-8-to-Lp6-10 were a 5, 16 mixture, Frs. Lp6-11-to-Lp6-14 were a 5, 16, 18, 19 mixture, and finally, Frs. Lr6-15-to-Lp6-18, were a 5, 18, 19 mixture. A further separation by CC for Frs. Lp6-8-to-Lp6-10 was carried out with isocratic elution (DCM/MeOH/HCOOH 8:1:1) and afforded six gathered fractions (Frs. Lp6-8-10a to Lp6-8-10f). Fr. Lp6-8-10a was the pure 16 (55.8 mg), Frs. Lp6-8-10b-to-Lp6-8-10e were a 5, 16 mixture, and Fr. Lp6-8-10f was the pure 5 (23.6 mg). Similarly, a further CC separation of the Fr. Lp6-15-to-Lp6-18 was performed using a gradient elution starting from a DCM/MeOH/HCOOH 7:2:1 mixture. This separation afforded five gathered fractions (Lp6-15-18a to Lp6-15-18e). Fr. Lp6-15-18a was the pure 18 (15.6 mg), Frs. Lpr6-15–18(b-c) were an 18, 19 mixture, and Frs. Lpr6-15-18d-e were the pure 19 (16.3 mg).Fresh G. monspessulana were used to obtain the respective QRE (10.2132 g). A CC separation was performed using gradient elution starting from a DCM/MeOH/NH3 8:1:1 mixture and affording ten gathered fractions (Frs. Gm1 to Gm10). The resulting fractions were additionally purified by successive CC (gradient elution) to obtain the compounds 9 , 10 , 12 , 13 , 14 , 15 , and 19 , using DCM/MeOH/NH3 mixtures .Fresh leaves (500 g) of split mode, with a flow of 1 mL/min and a split ratio of 30. The transfer line temperature was 250 °C, and the carrier gas was grade-5 helium. The ionization mode was electron impact (EI) at 70 eV. The analyzed extracts were prepared at 1 µg/µL in CH2Cl2 (GC–MS-SupraSolv® grade). Retention indices were calculated using a series of C10-C24n-alkanes from identical GC–MS analysis [The GC–MS analyses were obtained with a Thermo Trace 1300 coupled to an ISQ LT mass spectrometer with a single quadrupole analyzer. For the analysis, a Rxi® 5Sil MS column with 60 m, 0.25 mm ID, and 0.25 μm (5% diphenyl/95% dimethylpolysiloxane) was used. A temperature program was implemented. The starting temperature was 120 °C, which was maintained for 2 min, and then a 6 °C/min ramp was applied until 300 °C was reached, which was maintained for 10 min. The injection volume was 1 μL in analysis .2O) and B (0.1% formic acid in MeOH). The gradient started at 5% B (0 min) and was maintained for 2 min. Then, B was incremented to 100% from 5 to 30 min and maintained for 5 min. The oven temperature was 40 °C, and the wavelength was 254 and 280 nm. The ESI interface was operated in positive ion mode with 4.5 kV in the capillary and 0.5 kV in the endplate offset. The pressure of the nebulization gas was 0.4 Bar; the drying gas was maintained at a flow rate of 8 L/min at 200 °C. The collision and the quadrupole energy were set to 12 and 6 eV, respectively. RF1 and RF2 funnels were programmed to 150 and 200 Vpp, respectively. The mass spectra were calibrated using sodium formate.High-resolution mass spectrometry analyses with electrospray ionization (ESI) were performed using a Bruker micrOTOF–QII mass spectrometer coupled to a Shimadzu Prominence liquid chromatography system, consisting of two analytical pumps model LC-20AD, with SIL-20AHT automatic injector, SPD-20A UV/Vis detector, CTO-20A column oven, CBM-20A controller. Each compound analyzed by this technique was prepared at 1 mg/mL using LCMS-grade methanol. The column used was a Phenomenex Luna C18 . The flow was 0.2 mL/min, and the mobile phase was a mixture of solvents A  were recorded on a DRX 500 spectrometer (Bruker) using CDCl3 or CD3OD as a solvent with tetramethylsilane  as an internal standard at room temperature. Chemical shifts are indicated in δ (ppm) regarding TMS, and coupling constants are expressed in Hertz (Hz). Each spectrum resulted from 128 scans with pulse widths (PW) of 8.0 µs (30 °C) and relaxation delays (RD) of 6.0 s.The 1–20 were recorded by using Jasco P-2000 polarimeter. Spectrophotometric grade methanol was used as a solvent, and the values were reported in terms of specific rotation (The specific rotation data of the QAs Fox LQB-03) obtained from wilting Physalis peruviana plants [Fox in the presence of the test compounds at different concentrations compared to that of a blank (PDA 0.5%), using a 12-well plate amended semi-solid medium assay [The studied phytopathogen was a virulent isolate  by nonlinear regression in GraphPad Prism 5.0 software. The evaluation of each concentration of each treatment was performed in triplicate.Several concentrations of each test compound (0.1–1000 µg/mL) were prepared using serial dilutions by dispersing them in 0.5% PDA medium for the antifungal assays. Subsequently, each concentration of every QA was considered a treatment, and they were randomly placed in a 12-well glass plate (size 79 × 63 × 4 mm). Finally, a 1-mm plug from an 8-day fungal monosporic culture, having a diameter proportional to a borosilicate capillary tube of 32 mm, was taken and placed in the central part of each well containing a treatment. This plate was placed in a humid chamber for 72 h at 25 °C. After incubation, a photograph of the 12-well glass plate was recorded and analyzed in ImageJ software. The growth area was measured, and the inhibition percentage was then calculated. The ICFor the FC or FS activity classification procedure, the plug of the phytopathogen used in the prior treatments was taken at 1 µg/µL and was placed on a fresh, unsupplemented PDA medium for 72 h. After this time, mycelial growth was also observed. In the case of further mycelial growth, the QA was classified as fungistatic (FS), and in the case of no mycelial growth, the QA was classified as fungicidal (FC) .p > 0.05). Once the normal data distribution was verified, an analysis of variance (ANOVA) was then accomplished, followed by a posthoc Tukey test to establish significant differences between treatments (p < 0.05). These analyses were performed in Infostat statistical software [A Shapiro-Wilks normality test was initially carried out to examine the normal distribution of the data . GC–MS of 1; b). EIMS of 1; c). HRESIMS of 1. Figure S3. a). GC–MS of 2; b). EIMS of 2; c). HRESIMS of 2. Figure S4. a). GC–MS of 3; b). EIMS of 3; c). HRESIMS of 3. Figure S5. a). GC–MS of 4; b). EIMS of 4; c). HRESIMS of 4. Figure S6. a). GC–MS of 5; b). EIMS of 5; c). HRESIMS of 5. Figure S7. a). GC–MS of 6; b). EIMS of 6; c). HRESIMS of 6. Figure S8. a). GC–MS of 7; b). EIMS of 7; c). HRESIMS of 7. Figure S9. a). GC–MS of 8; b). EIMS of 8; c). HRESIMS of 8. Figure S10. a). GC–MS of 9; b). EIMS of 9; c). HRESIMS of 9. Figure S11. a). GC–MS of 10; b). EIMS of 10; c). HRESIMS of 10. Figure S12. a). GC–MS of 11; b). EIMS of 11; c). HRESIMS of 11. Figure S13. a). GC–MS of 12; b). EIMS of 12; c). HRESIMS of 12. Figure S14. a). GC–MS of 13; b). EIMS of 13; c). HRESIMS of 13. Figure S15. a). GC–MS of 14; b). EIMS of 14; c). HRESIMS of 14. Figure S16. a). GC–MS of 15; b). EIMS of 15; c). HRESIMS of 15. Figure S17. a). GC–MS of 16; b). EIMS of 16; c). HRESIMS of 16. Figure S18. a). GC–MS of 17; b). EIMS of 17; c). HRESIMS of 17. Figure S19. a). GC–MS of 18; b). EIMS of 18; c). HRESIMS of 18. Figure S20. a). GC–MS of 19; b). EIMS of 19; c). HRESIMS of 19. Figure S21. a). GC–MS of 20; b). EIMS of 20; c). HRESIMS of 20.


Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. This study investigates the therapeutic potential of human Vγ9Vδ2 T cells in GBM treatment. The sensitivity of different glioma specimens to Vγ9Vδ2 T cell-mediated cytotoxicity is assessed using a patient-derived tumor cell clusters (PTCs) model.The study evaluates the anti-tumor effect of Vγ9Vδ2 T cells in 26 glioma cases through the PTCs model. Protein expression of BTN2A1 and BTN3A1, along with gene expression related to lipid metabolism and glioma inflammatory response pathways, is analyzed in matched tumor tissue samples. Additionally, the study explores two strategies to re-sensitize tumors in the weak anti-tumor effect (WAT) group: utilizing a BTN3A1 agonistic antibody or employing bisphosphonates to inhibit farnesyl diphosphate synthase (FPPS). Furthermore, the study investigates the efficacy of genetically engineered Vγ9Vδ2 T cells expressing Car-B7H3 in targeting diverse GBM specimens.The results demonstrate that Vγ9Vδ2 T cells display a stronger anti-tumor effect (SAT) in six glioma cases, while showing a weaker effect (WAT) in twenty cases. The SAT group exhibits elevated protein expression of BTN2A1 and BTN3A1, accompanied by differential gene expression related to lipid metabolism and glioma inflammatory response pathways. Importantly, the study reveals that the WAT group GBM can enhance Vγ9Vδ2 T cell-mediated killing sensitivity by incorporating either a BTN3A1 agonistic antibody or bisphosphonates. Both approaches support TCR-BTN mediated tumor recognition, which is distinct from the conventional MHC-peptide recognition by αβ T cells. Furthermore, the study explores an alternative strategy by genetically engineering Vγ9Vδ2 T cells with Car-B7H3, and both non-engineered and Car-B7H3 Vγ9Vδ2 T cells demonstrate promising efficacy in vivo, underscoring the versatile potential of Vγ9Vδ2 T cells for GBM treatment.Vγ9Vδ2 T cells demonstrate a robust anti-tumor effect in some glioma cases, while weaker in others. Elevated BTN2A1 and BTN3A1 expression correlates with improved response. WAT group tumors can be sensitized using a BTN3A1 agonistic antibody or bisphosphonates. Genetically engineered Vγ9Vδ2 T cells, i.e.,  Car-B7H3, show promising efficacy. These results together highlight the versatility of Vγ9Vδ2 T cells for GBM treatment.The online version contains supplementary material available at 10.1186/s12967-023-04514-8. Glioblastoma (GBM) is the most common primary malignant brain tumor, accounting for 48.6% of all primary brain malignancies. It is classified as a grade IV glioma according to the World Health Organization (WHO) classification , 2. Desp+ cells, representing the main subset of peripheral γδ T cells [Immunotherapy has shown promising results in the treatment of malignant tumors , 6. Howe T cells . Zoledro T cells . Vγ9Vδ2  T cells , 13. Act T cells . Like na T cells –17. Thus T cells .Despite significant progress in immunotherapy, chimeric antigen receptor (CAR)-T cell therapy based on αβ T cells has yet to achieve a breakthrough in treating solid tumors due to several limitations, such as T cell-associated toxicities, limited efficacy against solid tumors, antigen escape, poor trafficking and tumor infiltration, and the immunosuppressive microenvironment within the tumor , 20. HowThis study investigated the safety and antitumor effects of Vγ9Vδ2 T cells and Car-B7H3-γδT cells derived from healthy human peripheral blood mononuclear cells (PBMCs), and tested them against GBM cell lines, primary GBM cells, patient-derived tumor cell clusters (PTCs), and a mouse GBM model. We also conducted a preliminary investigation into the mechanism underlying the different responses of PTCs to Vγ9Vδ2 T cell therapy. Several methods were explored to enhance the anti-glioma ability of Vγ9Vδ2 T cells. The findings suggest that Car-B7H3-γδT cell immunotherapy has significant potential as a well-tolerated therapeutic approach for treating GBM.9/L and activated partial thromboplastin time and prothrombin time within the normal range at Beijing Tiantan Hospital, China. Exclusion criteria: hematological diseases, steroid use within one month, and all malignancies. All participants provided signed informed consent. Vγ9Vδ2 T cells were expanded using a previously described method [6 cells/ml. The cells were incubated at 37 ℃, 5% CO2. The cell density was modified to 1 × 106 cells/ml during the culture process every 2–3 days. On day 10, the number of Vγ9Vδ2 T cells was detected. The purity of Vγ9Vδ2 T cells was determined by flow cytometry. The cells were stained with anti-human TCR Vδ2-PerCP antibodies .PBMCs were obtained from healthy subjects aged 18–50 years with a platelet count of ≥ 100 × 10d method . Briefly3 linker, was inserted between the mouse Ig Kappa signal sequence and the CD8α hinge domain, resulting in the B7-H3-CD28/4-1BB/CD3z-CAR construct. A truncated version of the epidermal growth factor receptor (EGFRt) was co-expressed with a P2A cleavage peptide on the C terminal of the CAR construct. The CAR expression was monitored by measuring EGFRt expression. On day 10 after expansion, Car-B7H3-γδT cells were obtained by transfecting Vγ9Vδ2 T cells with B7-H3-CD28/4-1BB/CD3z-CAR, and the purity was characterized using flow cytometry.To generate Car-B7H3-γδT cells, we constructed a CAR from HIV-1-derived lentivirus vectors containing a mouse Ig Kappa leader sequence, CD8α hinge and transmembrane domains, and CD28, 4-1BB, and CD3ζ endo domains (FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). The configuration of the intracellular domain in the third generation of CAR CD28 can enhance the function of CAR-T cells. Additionally, the presence of the 4-1BB intracellular domain structure can contribute to the persistence of CAR-T cells . The scF2 at 37 ºC. All cell lines were stably transfected with lentiviral luciferase vectors to generate U-87MG-Luc, TJ905-Luc, and HTB15-Luc cells.U-87MG, TJ905, and HTB15 cell lines  were maintained in DMEM (Gibco) supplemented with L-glutamine , 10% FBS, and 1% penicillin–streptomycin (Gibco) in a humidified atmosphere with 5% COThis study was approved by the Ethics Committee of Beijing Tiantan Hospital KY2014-021–02). Human glioma tumor specimens (n = 26) were obtained from patients enrolled in the Neurosurgery Clinical Information and Biobank Project of the Department of Brain Oncology, Beijing Tiantan Hospital, during tumor resection surgery. The patients were aged 18–72 years and were diagnosed with glioma based on histopathological examination. The tumor specimens were divided into three parts: one part was used to generate PTCs, an organoid tumor model for preclinical drug testing . PTCs we4-021–02.The human GBM cell surfaces were stained with 10 µg/mL APC-labeled anti-human MICA/B mAb , FITC-labeled anti-human BTN2A1 pAb , FITC-labeled anti-human BTN3A1 mAb , APC-labeled anti-human B7H3 , APC-labeled anti-human EGFR , or corresponding isotype control. The stained cells were analyzed using a FACS Calibur flow cytometer .g and resuspended in DMEM medium. T cells were then cocultured with target cells  at different effective target (E:T) ratios of 0:1, 0.2:1, 0.5:1, 1:1, or 3:1 in the presence of interleukin 2 (IL-2) in a 96-well plate for 18–20 h at 37 °C. The luciferase activity was measured using a Bright-Lite luciferase detection system . The supernatant was collected to measure IFN-γ  and TNF-α  levels by ELISA.To assess the antitumor effects of Vγ9Vδ2 T cells or Car-B7H3-γδT cells, we incubated them with GBM cells or PTCs. Purified Vγ9Vδ2 T cells or Car-B7H3-γδT cells were centrifuged for 5 min at 300 To evaluate the antitumor effect of Vγ9Vδ2 T cells on PTCs (n = 26), Vγ9Vδ2 T cells were cocultured with each sample at a ratio of 3:1. The antitumor effect was assessed by measuring the diameter changes of PTCs at 8 h after incubation using a confocal high-content imaging microscope (Opera Phenix). The antitumor effect was calculated as (the diameter of PTCs after coculture/the diameter of PTCs before coculture) × 100%. Patients were categorized into two types according to the antitumor effect: those with an antitumor effect ≥ 50% were defined as having a stronger antitumor effect (SAT), while those with an antitumor effect < 50% were defined as having a weaker antitumor effect (WAT).To investigate whether the addition of ZOL or BTN3A1 agonistic monoclonal antibody could enhance the killing effect of Vγ9Vδ2 T cells against WAT primary glioma cells , Vγ9Vδ2 T cells were co-cultured with insensitive primary glioma cells at a 1:1 ratio for 18 to 20 h. The co-cultures were supplemented with ZOL , activating BTN3A1 agonistic monoclonal antibody , eBioscience™), or a control. The luciferase activity was then measured.U87-B7H3-KO (B7H3 knocked out) cells stably transfected with lentiviral luciferase vectors were kindly provided by Dr. Xueguang Zhang from Soochow University. To assess whether Car-B7H3-γδT cells retained their function as Vγ9Vδ2 T cells, Car-B7H3-γδT and Vγ9Vδ2 T cells were co-cultured with U87-B7H3-KO cells in 1:1 ratio for 18 to 20 h in the presence or absence of ZOL . The luciferase activity was measured.Immunodeficient NSG mice  were obtained from Sperf Biotech, Beijing, China, and housed under specific-pathogen-free conditions at 22 ± 1 ºC, 50 ± 1% humidity, with a 12/12 h light/dark cycle and free access to water and a Chow diet. All animal procedures were conducted following the guidelines of the Beijing Neurosugical Institute Laboratory Animal Welfare and Ethics Committee  and in accordance with the AAALAC and IACUC Guidelines. The study was approved by the Beijing Regional Ethics Committee.5 U-87MG-Luc cells at a rate of 1 μL/min using a stereotactic tumor establishment device. The coordinates for the injection were 2 mm to the right from the bregma and 3.5 mm depth. Six to nine days after injection, mice were intraperitoneally injected with 150 ng/g D-luciferin . Tumor formation was observed and imaged using an IVIS system  after 10 min of injection. The IVIS data was analyzed using the Living Image software . Tumor formation was also confirmed by MRI examination.Each mouse received an intracranial injection of 5 μL phosphate-buffered saline (PBS) containing 106 Vγ9Vδ2 T cells or Car-B7H3-γδT cells or PBS into the lateral ventricle . The mice were then intraperitoneally injected with 100 μL of IL-2 (5000 IU) twice, at 3 day intervals. The control group was injected with PBS. The mice were weighted twice a week to monitor body weight changes. Bioluminescence intensities of the tumors were measured once a week.The tumor-bearing mice were randomly divided into three groups: Vγ9Vδ2 T cell group, Car-B7H3-γδT cell group, and control group (n = 5/group). Each mouse received an intracranial injection of 5 × 10g for 5 min and stored at − 80 °C until use. Serum cytokine levels were measured using a cytokine array kit  following the manufacturer’s instructions. The fluorescence intensities were measured using an Axon scanner 4000B and GenePix software. The results were analyzed using a RayBio analysis tool. For each cytokine, the mean fluorescence intensity was calculated, and the z-score was generated. A heatmap of the z-scores ranging from − 3 to 3 was generated using ggplot2 v3.2.1.Cytokine multiplex assay was conducted to evaluate the safety of T cell therapy in mice. Blood samples were collected from the retrobulbar sinus, and serum was obtained by centrifuging the blood samples at 5000 Human glioma tissue samples were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 µm-thick sections. The tissue sections were deparaffinized, rehydrated, and underwent heat-induced epitope repair retrieval. Then, the sections were incubated with anti-BTN2A1  or anti-BTN3A1  antibodies at 4 °C, followed by incubation with a secondary antibody  for 1 h. DAB peroxidase substrate solution staining, hematoxylin blue staining, and differentiation reverse blue staining were performed. The slides were scanned using NanoZoomer 2.0 HT . The positive rate was calculated as  × 100%. An IHC score was calculated as the positive rate × staining intensity. An IHC score > 10 was considered positive.™ 7-color Manual IHC kit  following the manufacturer’s protocol. The detailed experimental procedures and conditions are summarized in Additional file MIS was conducted in glioma tissue samples from mice to evaluate the infiltration of Vγ9Vδ2 T cells into tumors using an OPALP-value < 0.05. Gene set enrichment analysis (GSEA) was performed on hallmark gene sets from the Molecular Signatures Database  to analyze the enrichment of biological pathways.RNA-Seq was performed using glioma tissue samples from patients. DESeq2 (version 1.32.0) was used to identify differentially expressed genes (DEGs) with |fold change|> 2 and adjusted P-value < 0.05 was considered significant. The plots were generated using Prism 6  and the R2 package.Statistical analysis was performed using the SPSS software . Categorical data were compared using chi-squared test or Fisher’s exact test. Continuous data were compared using Student’s t-test or non-parametric test. Kaplan–Meier analysis was used to estimate overall survival. A comparison between two groups was conducted using the Mann–Whitney U-test. A two-tailed ). To evaluate the cytotoxicity of Vγ9Vδ2 T cells, we co-cultured Vγ9Vδ2 T cells with GBM-Luc cells at different E:T ratios in the presence of IL-2. As shown in Fig. P < 0.05). The most prominent antitumoral activity of Vγ9Vδ2 T cells was observed in U87-MG-Luc cells. Additionally, Vγ9Vδ2 T cells produced IFN-γ and TNF-α when cocultured with GBM cell lines  , bioluminescence analysis , and hematoxylin and eosin (H and E) staining  and improves survival   at a 3:1 ratio in the presence of IL-2 for 8 h. We observed significant differences in the responses to Vγ9Vδ2 T cells among the PTCs, with SAT responses in six PTCs  and WAT responses in twenty PTCs   , compared to the WAT group . Furthermore, the BTN2A1 and BTN3A1 IHC scores were significantly higher in the SAT group than in the WAT group  . These data suggest that lipid metabolism and glioma inflammatory response may play important roles in the antitumor effect of Vγ9Vδ2 T cells on GBM.To explore the molecular mechanisms involved in the antitumor activity of Vγ9Vδ2 T cells in GBM, we performed RNA-seq to identify DEGs between SAT and WAT tissue samples and used GSEA to analyze the enriched pathways. Our results revealed that Vγ9Vδ2 T cell response was mainly related to fatty acid metabolism, primary bile acid biosynthesis, nicotinate and nicotinamide metabolism, peroxisome, ABC transporters, tryptophan metabolism, arachidonic acid metabolism, glycerolipid metabolism, proximal tubule bicarbonate reclamation, TGF beta signaling pathway, and PPAR signaling pathway To investigate the potential of enhancing the antitumor activity of Vγ9Vδ2 T cells in unsensitive glioma, we co-cultured Vγ9Vδ2 T cells with WAT primary glioma cells  at E:T ratios of 1:1 in the presence or absence of ZOL or BTN3A1 agonistic antibody. Our results showed that the killing of Vγ9Vδ2 T cells against primary glioma cells was significantly enhanced after ZOL  containing the scFv of the anti-B7-H3 antibody and obtained Car-B7H3-γδT cells with a purity of around 69.5%  and three had methylated MGMT promoters . Flow cytometry results showed that B7-H3 was highly expressed in all six primary cell lines (positive rate > 90%) . Additionally, Car-B7H3-γδT cells produced more IFN-γ and TNF-α when cocultured with primary WAT cells    . Both Vγ9Vδ2 T and Car-B7H3-γδT cell treatments significantly suppressed GBM tumor growth in mice compared to the vehicle control  , and the survival of the Car-B7H3-γδT group was longer than that of the Vγ9Vδ2 T group   Fig. B. Moreov+ and Granzyme B+) and Car-B7H3-γδT cells (B7H3+ and Granzyme B+) peaked on day 7, with more activated Car-B7H3-γδT cells infiltrating the tumor tissue. The infiltrations of both cell types decreased by day 14  Fig. A, B. On  14 Fig. C, indica5 cells/5 μL PBS) or high-dose (5 × 106 cells/5 μL PBS) Vγ9Vδ2 T cells into the lateral ventricle of healthy NSG mice. H&E staining at 7 days after injection showed that Vγ9Vδ2 T cell therapy did not induce any significant histological changes in the liver, lung, ovary, brain, spleen, kidney, stomach, heart, or uterus of the mice compared to control treatment   ratios of 0:1, 0.5:1, 1 :1, or 3:1 in the presence of IL-2. The luciferase activity was measured to determine the cell viability of GBM cells at 18–20 h. E The culture medium was collected. ELISA was performed to measure IFN-γ and TNF-α levels. Data are expressed as the mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001; n = 3. Figure S3. Hematoxylin and eosin staining of the liver, lung, ovary, brain, spleen, kidney, stomach, heart, and uterus tissue samples from NSG mice after injection with PBS A or high-dose (5 × 106 cells/5 μL PBS) Vγ9Vδ2 T cells B. Magnification 10 ×. Figure S4. Evaluation of Vγ9Vδ2 T and Car-B7H3-γδT cell toxicity. A The body weights of mice were measured at different time points after intraventricular injection of control (PBS), low-dose of Vγ9Vδ2 T cells (5 × 105 cells/5 μL PBS), or high-dose of Vγ9Vδ2 T cells (5 × 106 cells/5 μL PBS). B The body weights of GBM tumor-bearing mice were measured at different time points after intraventricular injection. C Cytokine multiplex assay was carried out to examine mouse serum cytokine alterations at 7 days after T cell therapy. Z-score of each cytokine was calculated as the mean fluorescence intensity. A heatmap of the z-scores ranging from − 3 to 3 was generated. Table S1. Basic characteristics of all PTC patients. Table S2.A Primary antibody used in Multiplex staining. B Experimental conditions and procedures of Multiplex staining of TCRδ2 panel. C. Experimental conditions and procedures of Multiplex staining of B7H3 panel. Table S3. Relationship between different grades of glioma and different responses to Vγ9Vδ2 T therapy.


Nelumbo nucifera) seed plumules and leaves exhibit significant tissue specificity for their pharmacological effects and potential nutritional properties. Herein, 46 benzylisoquinoline alkaloids were identified via UPLC-QTOF-HRMS, of which 9 were annotated as glycosylated monobenzylisoquinoline alkaloids concentrated in the seed plumules. The spatial distribution of targeted benzylisoquinoline alkaloids in leaves, seed plumules, and milky sap was determined via MALDI–MSI. Furthermore, 37 Nelumbo cultivars were investigated using targeted metabolomics to provide insights into functional tea development. While aporphine alkaloids comprised the main compounds present in lotus leaves, bisbenzylisoquinoline alkaloids were the main compounds in lotus plumules, where glycosylation primarily occurs. These findings can help understand the distribution of benzylisoquinoline alkaloids in lotus tissue and the directional breeding of varieties enriched with specific chemical functional groups for nutritional and pharmacological applications.Benzylisoquinoline alkaloids in lotus ( •n, 46 alkaloids were identified in lotus.Using UPLC-QTOF-HRMS•Among the 46 alkaloids, 9 were glycosylated monobenzylisoquinolines.•Their tissue distribution was investigated using MALDI-MSI.•Natural monobenzylisoquinoline alkaloid glycosylation enriched the glycosylated alkaloids in a tissue-specific manner.•Our findings provide insights into the biosynthesis of benzylisoquinoline alkaloids. Lotus seeds and underground stems are frequently consumed in East and Southeast Asian countries, owing to their high nutritional properties. Lotus leaf extracts possess antiviral, diuretic, and astringent activities and are also used as an antipyretic +m/z 161.0606 produced by benzyl-group breaks and a neutral loss ion of [M + H–NH3]+m/z 255.1023. Based on the molecular formula C20H25NO3 and a characteristic loss ion of [M + H–C7H11O]+m/z 107.0493, 10 was tentatively identified as 4′-O-methylarmepavine.Compound 11–19 were deduced to have undergone glycosylation with monobenzylisoquinoline alkaloids. Five alkaloid compounds (11–15) exhibited the same molecular formula of C23H29NO8 and [M + H-162Da]+ characteristic ion, indicating loss of glucoside and fragment ions [M + H-162Da-NH3]+m/z 255.0992 and [M + H–C7H11O]+m/z 107.0692. Compounds 11–13 were predicted as N-methylhigenamine-7-O-Glc, N-methylhigenamine-4′-O-Glc, N-methylhigenamine-6-O-Glc, respectively  (16–19) were deduced to have the same molecular formula C24H31NO8 and fragment ion [M + H-162Da]+m/z 300.1584. Owing to the characteristic ion [M + H-162Da-NH3]+m/z 283.1263, 18 was tentatively identified as norarmepavine-Glc. Because of the fragment ions [M + H-162Da-CH3NH2]+m/z 269.1132 and [M + H–C7H7O]+m/z 107.0475, 17 was speculated as N-methylisococlaurine-6-O-Glc. Compound 16 was annotated as N-methylisococlaurine-4′-O-Glc with the same MS/MS spectral behavior of 17. Compound 19 was predicted as N-methylcoclaurine-4′-O-Glc  . Four mo4′-O-Glc .Fig. 1Th2–4) exhibited the protonated ion [M+H]+ at m/z 286.1446. In the MS/MS spectra of 3 and 4, the fragment ions [M + H–C7H11O]+m/z 107.0497, [M + H–NH3]+m/z 269.1171, [M + H–NH3–CH3OH]+m/z 237.092, and [M + H–C7H11O]+m/z 175.0497 were observed; therefore, these compounds were identified as isococlaurine and coclaurine, respectively, and this has been confirmed using the authentic standards. Furthermore, the elution times of the substituent C-7 alkaloids were longer than those of C-6 alkaloids on C18 columns; thus confirming the identities of 3 and 4, which has been previously reported in lotus plumules +m/z 255.1027 was detected, hence, 2 was determined to be N-methylhigenamine.Three monobenzylisoquinoline alkaloids (compounds plumules . In the 5–7) exhibited the same molecular formula C18H19NO. Compound 6 was identified as norarmepavine, with fragment ions [M + H–NH3]+m/z 283.1326 and [M + H–C7H11O]+m/z 189.0912 in the MS/MS spectra. The same characteristic fragments [M + H–CH3NH2]+m/z 269.1176 and [M + H–C8H13O]+m/z 175.0760 were observed in the MS/MS spectra of 5 and 7, and considering the elution order on C18 columns, the two compounds were identified as N-methylisococlaurine and N-methylcoclaurine, respectively +m/z 283.1326, [M + H–C7H7O]+m/z 107.0495 were observed, and according to authentic standards, 8 was identified as armepavine. With the characteristic fragments m/z 58.0658, [M + H–NH3]+m/z 269.1178, and [M + H–C7H7O]+m/z 107.0497, 9 was identified as lotusine.Three monobenzylisoquinoline alkaloids , 10 was tentatively identified as 4′-O-methylarmepavine via comparison with the ion peak intensity of [M + H–C3H8N]+m/z 58.0658 and [M + H–C7H7O]+m/z 107.0493.Compound 3.1.2N. nucifera; among them, 21, 23, 24, 26, 27, and 29 were identified and quantified in lotus leaves, lotus milky sap, and seed plumules. For typical aporphines, the substitution of methoxy (OCH3) or hydroxyl occurs mainly in the A ring, and major methyl substitution occurs in the N linkage +m/z 269.1181, [M + H-(CH2NCH3–CH3)]+m/z 254.0939, and [M − CO]+m/z 283.1335; hence, it was identified as pronuciferine according to a previous report [28 exhibited the protonated ion [M + H-(CH2NCH3)]+m/z 255.1011 and [M + H-(CH2NCH3)–OCH3]+m/z 223.077, and hence, it was tentatively identified as glaziovine, which has been previously reported in lotus leaf stalks +m/z 206.1176 and [M + H–CH3–CH2–C6H5O]+m/z 489.2148, which was confirmed with the authentic standards. Because of the fragment ions [M + H–C26H28NO4]+m/z 192.1006 and [M + H–CH3–CH2–CH2–C6H5O]+m/z 475.2186, 32 was unequivocally identified as isoliensinine, which was confirmed according to the authentic standards. In the MS/MS spectra of 33 and 31, the ions [M + H–C26H28NO4]+m/z 206.1179 and [M + H–CH3–CH2–CH2–C6H5O]+m/z 489.2244 were observed; therefore, 33 was unequivocally identified as neferine based on a previous report [30 was identified as nelumboferine based on the molecular formula C36H40N2O6 and fragment ions [M + H–C26H28NO4]+m/z 192.1044 and [M + H–CH3–CH2–C6H5O]+m/z 475.2186.Two bisbenzylisoquinoline alkaloid compounds, s M + H–CH26NO4+m3.1.4Thirteen other alkaloids, including amine, pyridine, isoquinoline, and indole alkaloids, were identified in lotus leaves, milky sap, and seed plumules. Overall, 21 alkaloids were identified in the sacred lotus, including 9 glycosylated monobenzylisoquinoline alkaloids. Seven glycosylated monobenzylisoquinoline alkaloids were detected in lotus seed plumules, indicating it as the primary site of glycosylation.3.231/32; [M+H]+m/z 611.3108) and neferine  were concentrated and distributed in lotus seed plumules and milky sap  and dehydroanonaine  could be generated in leaves, which has been previously reported in lotus +m/z 312.1599) may be produced by laticifers and transferred to other tissues. It was apparent that very small amounts of bisbenzylisoquinoline were distributed in the center of lotus leaves and widely distributed in the milky sap, primarily around the seed plumules, suggesting that latex affected its transportation, similar to that in opium poppy. Nuciferine  has been previously reported to be the most important alkaloid in all the growth and development stages of lotus leaves , nelumboferine [30], neferine [33]), one aporphine alkaloid (nuciferine [27]), and one monobenzylisoquinoline alkaloid (isococlaurine [3]). Additionally, two glycosylated bisbenzylisoquinoline alkaloids (N-methylhigenamine-6-O-Glc [13] and N-methylisococlaurine-6-O-Glc [17]) were clustered away from the other monobenzylisoquinoline alkaloids and were significantly and positively associated with N-methylhigenamine (2) and armepavine (8). These results suggest that these compounds may be associated with seed development because of the epigenetic modification imparting stress resistant [The accurate masses of 46 BIAs identified in lotus were quantitatively analyzed in lotus leaves, milky sap, and seed plumules. Lotus leaves, milky sap, and seed plumules demonstrated clear separation in the PCA plot by principal component (PC)2 . There w27), N-nornuciferine (26), O-nornuciferine (24), dehydroanonaine (20), and caaverine (22) were the main compounds in lotus leaves, accounting for >60% of the total BIAs in most of the analyzed cultivars. Aporphine and monobenzylisoquinoline chief were the main alkaloids in lotus leaves -corytuberine synthase (CYP80G) catalyzes the conversion of N-methylcoclaurine to generate aporphine alkaloids [O-nornuciferine, N-nornuciferine, and nuciferine) involved in the aporphine synthesis pathway were detected. The same substrate, N-methylcoclaurine, could be used to synthesized bisbenzylisoquinoline alkaloids using berbamunine synthase (CYP80A) [9) exhibited high positive associations with N-methylhigenamine-6-O-Glc  and N-methylisococlaurine-6-O-Glc . Norcoclaurine (1), isococlaurine (3), armepavine (8), and lotusine (9), which are generated by the biosynthetic pathway of monobenzylisoquinoline alkaloids, also exhibit strong positive correlations (0.5 < r < 0.92). In addition, l-tyrosine (40), as the primary synthetic precursor of BIAs, was strongly and negatively correlated with coclaurine  and nuciferine . Notably, O-methyltransferase and N-methyltransferase were involved in the modification of benzylisoquinoline alkaloids of the BIAs of lotus. Methylation is involved in the metabolism of most monobenzylisoquinoline alkaloids, usually only once. Glycosylation is another important modification that primarily occurs in lotus seed plumules, mostly after the first methylation of monobenzylisoquinoline alkaloids at the O- and N-positions.The putative biosynthetic pathway of BIAs was proposed based on the various BIAs detected in lotus leaves, milky sap, and seed plumules . To the synthase ,32,33. Tlkaloids ,34. Furt(CYP80A) ,36. Lotu4Herein, 46 alkaloids, including 9 glycosylated monobenzylisoquinoline alkaloids, were identified using UPLC-QTOF-HRMS and shown to be localized in various tissues  of sacred lotus using MALDI–MSI. These results directly demonstrate the differences in distribution of these alkaloids in various lotus tissues and further evince the role of secondary metabolites in plant stress resistance. In addition, the glycosylated metabolites were differentially localized, being mostly distributed near the vascular bundles of the lotus seed plumules. These findings lay a foundation for the development and application of natural products for glycosylation. While more information is required to identify the sugar moiety of glycosylated monobenzylisoquinoline alkaloid, the discovery of the glycosylation of monobenzylisoquinoline alkaloids explains the enrichment mechanisms of the different types of naturally glycosylated alkaloids in the different tissues and provides useful insights into functional food and tea development. Furthermore, the content correlations of BIAs among the 37 representative lotus cultivars also provided insights into BIA biosynthesis. To the best of our knowledge, herein, the total alkaloid synthetic pathway in lotus was mapped and the known and possible metabolic pathways were labeled based on quantitative data and reported gene functions for the first time, thus providing new ideas for the further analysis of alkaloid synthetic mechanisms in lotus and BIA biosynthesis. Furthermore, the quantitative data serves as a scientific reference for planting selection, and the development and application of different varieties of lotus as well as provides guidance for lotus breeding.Chenyang Hao, Wei Yang, Yuetong Yu, Yan Liu, Xiaolu Wei and Sha Chen: Conceived and designed the experiments; Analyzed and interpreted the data.Gangqiang Dong, Yongping Zhu and Xiaolu Wei: Contributed reagents, materials, analysis tools or data.Chenyang Hao, Jun Zhang and Sha Chen: Performed the experiments; Wrote the Paper.Data included in article/supplementary material/referenced in article.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The title compound was synthesized with 6-oxo-1,6-dihydropyridine-3-carboxylic acid and 3-chloro-2-methylaniline as starting materials. The crystal packing is characterized by two types of N—H⋯O hydrogen bonds. 13H11ClN2O2, the molecules form a three-dimensional network based on two types of hydrogen bonds between NH groups and the carbonyl oxygen atoms and amides. The molecule is highly twisted, as evidenced by the dihedral angle between the 6-oxo-1,6-dihydropyridine and benzene rings [88.1 (2)°].In the crystal structure of the title compound, C N-phenyl-2-hydroxynicotinanilide, which has interesting structural properties  global, I. DOI: 10.1107/S2414314623006028/vm4061Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623006028/vm4061Isup3.cmlSupporting information file. DOI: 2280200CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


We report the molecular characteristics and phenotype-genotype correlation in a large cohort of the β0-thalassemia with 3.4 kb deletion.β-thalassemia patients, and a double heterozygote with α-globin gene triplication, were recruited. Hb and DNA analysis were performed to identify thalassemia mutations and four high Hb F single nucleotide polymorphisms (SNPs) including four base pair deletion (-AGCA) at Aγ-globin promoter, rs5006884 on OR51B6 gene, −158 Gγ-XmnI, BCL11A binding motifs (TGGTCA) between 3´Aγ-globin gene and 5´δ-globin gene.A total of 148 subjects, including 127 heterozygotes, 20 Hb E-β0-thalassemia and Hb E-β0-thalassemia with 3.4 kb deletion had significantly higher Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and Hb F values as compared with those with other mutations. Co-inheritance of heterozygous β0-thalassemia with 3.4 kb deletion and α-thalassemia was associated with even higher MCV and MCH values. The Hb E-β0-thalassemia patients carried a non-transfusion-dependent thalassemia phenotype with an average Hb of around 10 g/dL without blood transfusion. A hitherto undescribed double heterozygous β0-thalassemia with 3.4 kb deletion and α-globin gene triplication presented as a plain β-thalassemia trait. Most of the subjects had wild-type sequences for the four high Hb F SNPs examined. No significant difference in Hb F was observed between those of subjects with and without these SNPs. Removal of the 5´β-globin promoter may likely be responsible for this unusual phenotype.It was found that heterozygous β0-thalassemia with 3.4 kb deletion is a mild β-thalassemia allele. This information should be provided at genetic counseling and prenatal thalassemia diagnosis.The results indicate that  Although some β-globin gene deletions have been reported, most defects are non-deletional mutants of the β-globin gene  and 3–9% of β-thalassemia  (HBB:c.126_129delCTTT), CD17 (A-T) (HBB:c.52A>T), NT-28 (A-G) (HBB:c.-78A>G), CD71/72 (+A) (HBB:c.217dupA), IVSI-1 (G-T) (HBB:c.92+1G>T), IVSII-654 (C-T) (HBB:c.316-197C>T), 3.4 kb deletion and IVSI-5 (G-C) (HBB:c.92+5G>C) accounted for 97.4% of the total mutations  and mean corpuscular hemoglobin (MCH) with increased Hb A2 to around 5.0% and Hb F 0–3%. Homozygote or compound heterozygote of β-thalassemia with Hb E led to β-thalassemia major and Hb E-β-thalassemia diseases, with variable thalassemia phenotype. The β+-thalassemia has a milder thalassemia phenotype than the β0-thalassemia  modifiers affecting Hb A2 and Hb F expression in a large cohort of β0-thalassemia with 3.4 kb deletion. The molecular basis, and phenotype and genotype interaction observed in both heterozygote and compound heterozygote forms, as well as a hitherto undescribed interaction of the 3.4 kb deletion β0-thalassemia with an α-globin gene triplication, are described.Interestingly, among these common mutations, it was found that the carrier of utations . A high β-thalassemia carriers were selectively recruited from our archival at the Center for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Science, Khon Kaen University and Department of Pathology, Faculty of Medicine, Prince of Songkla University, during January 2008 to December 2021. These included leftover DNA specimens of β-thalassemia carriers with 3.4 kb (n = 148), Filipino (n = 9), and 105 bp (n = 7) deletions. The 454 representative specimens of β+-thalassemia trait (n = 70), β0-thalassemia trait (n = 259), and Hb E- β0-thalassemia disease (n = 125) with non-deletional β-thalassemia mutations were used to compare with the deletional β0-thalassemia mutation. Study was done on leftover DNA specimens; consent was not required. Hematological parameters were recorded on a standard blood cell counter; Sysmex XN-9000 , and Hb analysis was performed using capillary electrophoresis .Ethical approval of the study protocol was obtained from the Institutional Review Board (IRB) of Khon Kaen University, Khon Kaen, Thailand (HE632216). A total of 618 leftover specimens of α0-thalassemia (SEA and THAI deletions), α+-thalassemia (3.7 kb and 4.2 kb deletions), Hb Constant Spring, Hb Paksé, α-globin gene triplication and common β-thalassemia mutations were performed using polymerase chain reaction (PCR)-based techniques  (rs7482144) was determined by PCR-restriction fragment length polymorphism (PCR-RFLP) as described elsewhere . Identification of  chniques . Identifeviously . The Xmnlsewhere .β0-thalassemia with 3.4 kb deletion generated using primers G9 (5′-TCCCCAGTTAACCTCCTATT-3′) and N1 (5′-ACATATGAGCAAGGTTGTGT-3′) on a specific fragment of 456 base pair (bp). To examine three BCL11A binding motifs (TGGTCA) located between +3 kb 3′of Aγ-globin gene and about 1 kb 5′δ-globin gene, the three amplified fragments using primer pairs F29 (5′-CACGAGTCAGCCTTCAGAAA-3′) & G188 (5′- GCAGAGGAGACCAGCATACA-3′) , F18 (5′-GACATGGACTATTGTTCAATGA-3′) & G191 (5′-AGACAACGGGCTAATCCCTC-3′) , and F31 (5′-ACCCACATT GGCATTACAC-3′) & G192 (5′-AGAATGTGTTTGTGAGGGAGGA-3′)  were generated as shown in γ-globin genes, primer F22 (5′-TACTGCGCTGAAACTGTGGC-3′) & γ35 (5′-AGGTAGTTGTTCCCCTTCAA-3′) and SF5 (5′-TTAACGTCTTCAGCCTACAA-3′) & SF6 (5′-CAATCTGCACACTTGAGGGC-3′) were used to amplify DNA fragments of 1,843 and 993 bp, specific for Aγ-globin gene, and primers γ4 (5′-GGCCTAAAACC ACAGAGA-3′) & γ34 (5′-AGGTAGTTGTTGTTGTTGCA-3′) and SF3 (5′-GTTTGTGTGTG TGTGAGAGC-3′) & SF4 (5′-TCTTTAGGCATGCGTCAACA-3′) were used to amplify DNA fragments of 1,787 and 1,121 bp of Gγ-globin gene, followed by DNA sequencing . Specific amplification of Aγ-globin gene promoter was performed using primers F22 and γ35 to produce a fragment of 1,847 bp wild-type or 1,843 bp specific for the four bp deletion. This was confirmed by DNA sequencing and PCR-RFLP assay. The PCR product was digested with a Mse I restriction enzyme (5′-T▾TAA-3′) . After digestion, the 1,021 bp and 167 bp indicate wild type, whereas the 1,184 bp is a specific fragment for the AGCA deletion, as shown in The PCR-RFLP assay was developed to detect four bp deletion (-AGCA) at −225 to −222 of the rs5006884 (NC_000011.10:g.5352021C>T) in the OR51B6 gene on chromosome 11. In this system, primers G197 (5′-AAGACCAGG ATGCAGCAGAT-3′) and G198 (5′-ACCTCGGAGTGACATTGACC-3′) were used to produce an amplified fragment of 544 bp internal control. Specific amplification of the C allele (252 bp) and T allele (331 bp) were obtained using primers G197 & G199 (5′-CCTACTGTCGATCCCATGTAC-3′), and G200 (5′-AGACAGAAAGCATGGGAGAA-3′) & G198, respectively. The PCR reaction mixture (50 µL) contains 50–200 ng of genomic DNA, 200 µM of dNTPs, 30 pmol of each primer, and 1 unit of Taq DNA polymerase  in 10 mM Tris–HCl buffer pH 8.3, 50 mM KCl, 3 mM MgCl2, and 0.01% gelatin. The PCR consists of an initial denaturation at 94 °C for 3 min followed by 30 cycles of amplification at (94 °C for 1 min and 65 °C for 90 s). The PCR product was analyzed on 2% agarose gel electrophoresis and visualized under UV light after ethidium bromide staining. Confirmation of SNP rs5006884 was done using DNA sequencing  . Statistical significance was set at P-value <0.05.Hematological parameters of the subjects were presented as descriptive statistics, mean and standard deviation. The Mann–Whitney β0-thalassemia with 3.4 kb deletion encountered in Thai patients identified the deletion as NG_000007.3:g.69825_73314del3488 or NC_000011.10:g.5224302_5227791del3490 (GenBank Accession no. OQ161123), exactly the same with that reported previously in a Chinese patient  compared to those of the β0-thalassemia trait with other mutations (n = 259) encountered in our series. As shown in the table, significant higher Hb (11.6 ± 1.7 vs. 11.2 ± 1.6 g/dL), hematocrit (Hct) (36.1 ± 5.2 vs. 34.8 ± 5.2%), MCV (66.3 ± 4.3 vs. 63.2 ± 3.8 fL), MCH (21.3 ± 1.3 vs. 20.2 ± 1.4 pg), Hb A2 (6.9 ± 0.8 vs. 5.7 ± 0.6%) and Hb F (6.5 ± 3.4 vs. 1.3 ± 0.9%) values were observed for the former group of subjects. These are also the cases for the patients with compound heterozygous β0-thalassemia and Hb E. We observed significant higher Hb, Hct, MCV, MCH, and Hb F (48.1 ± 8.3 vs. 32.2 ± 15.6%) but lower Hb A2 (3.9 ± 1.1 vs. 6.5 ± 1.9%) values for the patients with 3.4 kb deletion (n = 20) as compared to those with other β0-thalassemia mutations (n = 125). These results indicate that the 3.4 kb deletion β0-thalassemia is associated with high Hb A2 and Hb F in a heterozygote state and a mild hematological phenotype when found in association with Hb E in a Hb E- β0-thalassemia disease. The effect of co-inheritance of α-hemoglobinopathy on the hematological phenotype of heterozygous β-thalassemia with 3.4 kb deletion was summarized in β-thalassemia with 3.4 kb deletion with heterozygous α+-thalassemia . Lower Hb F but not Hb A2 were also observed in double heterozygotes for 3.4 kb deletion β-thalassemia and other deletional forms of α-thalassemia including homozygous α+-thalassemia , αo-thalassemia  and Hb H disease . In contrast, we observed that co-inheritance of a non-deletional α-thalassemia, the Hb Constant Spring , did not lead to a reduction in Hb F expression, although it did have some hematological improvement. However, no statistical analysis was performed due to the small sample sizes of these α-hemoglobinopathies.DNA sequencing of the deletion breakpoint of the  patient . Table 1β-thalassemia with 3.4 kb deletion and an α-globin gene triplication (αααanti−3.7/ αα), a hitherto undescribed condition. He had relatively normal Hb and Hct values with reduced MCV & MCH characteristics of a plain β-thalassemia carrier. Elevated Hb A2 (7.2%) and Hb F (6.8%) were observed. The result indicates that, unlike other β0-thalassemia alleles, the interaction of α-globin gene triplication (αααanti−3.7/ αα) and a β0-thalassemia with 3.4 kb deletion does not lead to a β-thalassemia disease in heterozygotic form.Interestingly, an adult male patient was encountered with a double heterozygote for 2 and Hb F phenotype in our patients. As shown in Aγ-globin promotor, rs5006884 (C>T) of the OR51B6 gene, and Gγ-Xmn I of Gγ-globin gene promoter. In addition, no significant difference in Hb F and other hematological parameters was observed between those of subjects with and without these SNPs. DNA sequencing of the three BCL11A binding motifs (TGGTCA) between the 3′ Aγ-globin gene and the 5′ δ-globin gene , which is synthesized at a low level (2.5−3.5%) in normal adult individuals. Several genetic and non-genetic conditions are associated with Hb A2 expression  and Hb F (6.5 ± 3.4%) levels. Heterozygosity for other β-thalassemia alleles is usually found with elevated Hb A2 (5.7 ± 0.6%) but not Hb F (1.3 ± 0.9%) , the patients with β3.4-thalassemia/Hb E had milder NTDT phenotype with significantly higher Hb, Hct, MCV, MCH, MCHC, and Hb F but lower RDW values. We have also documented recently that the patients with homozygous β0-thalassemia of 3.4 kb deletion were presented with mild NTDT phenotype with Hb ranging from 7–9 g/dL and did not require regular blood transfusion  but lower Hb A2 (3.9 ± 1.1 vs. 6.5 ± 1.9%) as compared to those of other β0-thalassemia mutations  . Hematolngladesh . This 3,nsfusion . It is nutations . This loce versa . It is cβ0-thalassemia and α-thalassemia  could further improve the hematological phenotype of the patients due to the balance between α- and non-α-globin chains. Fortunately, in all cases of these double heterozygotes, the Hb A2 levels are still within the diagnostic range for a β-thalassemia carrier. Interestingly, we have encountered a subject with a hitherto undescribed condition of double heterozygote for the β3.4kb deletion and α-globin gene triplication (αααanti−3.7/αα). This subject presented with β-thalassemia trait phenotype without anemia with Hb 13.0 g/dL and Hct 40.0%, Hb A2 7.2%, and Hb F 6.8%  region where it deletes four functional OR genes and two OR pseudogenes including the OR52A1 that contains a γ-globin gene enhancer , the Filipino β0-thalassemia (n = 9), and 105 bp β0-thalassemia deletion (n = 7), all without α-thalassemia. The 3.4 kb deletion and the Filipino β0-thalassemia are associated with high Hb A2 and high Hb F β-thalassemia trait but not the 105 bp β0-thalassemia. In fact, this is not unexpected. The 105 β0-thalassemia is caused by a DNA deletion of −24 or −25 to +80 or +81 relative to the cap site of β-globin mRNA. Unlike the 3.4 kb deletion and the Filipino β0-thalassemia with 118 kb deletion, this 105 bp deletion does not remove the β-globin gene promoter TATA, CCAAT, and CACCC motifs  on OR51B6 gene, −158Gγ-Xmn I, BCL11A binding motifs between Aγ- and δ-globin genes as well as the whole γ-globin gene sequencing. It has been shown that the four bp deletion (-AGCA) at −225 to −222 of the Aγ-globin gene was associated with reduced Aγ-globin chain and elevation of Gγ chain with slightly increased Hb F level in β-thalassemia subjects (rs5006884 (C>T) on OR51B6 gene located upstream of β-globin gene cluster might intervene in the tetramer formation of α-globin chain with β- and δ-globin chains and regulated Hb A2 level in β-thalassemia carriers Aγ-globin promoter (rs368698783). The polymorphism leads to a decreased Ly-1 antibody reactive clone (LYAR) binding efficiency, which is the repressor of γ-globin genes  have been identified on the β-globin gene cluster at the third hypersensitive site (HS3) of the β-LCR, downstream of Aγ-globin gene and upstream of δ-globin gene , leading to its increased interaction with the γ- and δ-globin genes in cis, increasing their expression resulting in elevation of Hb F and Hb A2  and β3.4kb deletion support that the β3.4kb deletion is mild β0-thalassemia allele and might ameliorate severity when occurring in homozygote or compound heterozygote state. This high Hb A2 and high Hb F β-thalassemia characteristic could also be a useful marker for the β3.4kb deletion before being further confirmed by PCR analysis. This information related to the 3.4 kb deletion β-thalassemia should be provided to the patient’s family for the appropriate management and genetic counseling. This is especially important information for routine prenatal thalassemia screening in a prevention and control program in the regions.Nonetheless, the high Hb, Hct, MCV, MCH, Hb A10.7717/peerj.15308/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15308/supp-2Supplemental Information 2Click here for additional data file.


Hypoxia-inducible factors (HIFs) are best known for their roles in the adaptation to low oxygen environments. Besides hypoxia, HIF-1/2 α-subunits are also regulated by various non-hypoxic stimuli including insulin which can act via the PI3K/protein kinase B (PKB) signaling pathway. However, with respect to insulin little is known about HIF-3α. We aimed to investigate this relationship and found that insulin stimulates HIF-3α expression under both normal and low oxygen conditions. Blocking PKB activity reversed the effects of insulin, indicating that HIF-3α is a direct target of PKB. We identified serine 524, located in the oxygen-dependent degradation domain of HIF-3α, as a phosphorylation site of PKB. Mutating serine 524 impaired binding of PKB to HIF-3α and its ubiquitination, suggesting that PKB regulates HIF-3α stability through phosphorylation, thereby affecting important cellular processes such as cell viability and cell adhesion. Importantly, we discovered that this phosphorylation site also influenced insulin-dependent cell migration. These findings shed light on a novel mechanism by which insulin affects PKB-dependent HIF-3α expression and activity, with potential implications in metabolic diseases and cancer. Transcriptional gene regulation in response to hypoxia is mediated mainly by proteins from the hypoxia-inducible factor (HIF) family. Thereby various cellular functions such as glucose metabolism, hematopoiesis, angiogenesis, cell survival and death are adapted to the changed oxygen availability .HIFs are heterodimeric transcription factors that consist of an α-subunit that is destabilized in the presence of oxygen and a constitutively expressed β-subunit  . Three HHIF3A gene seems to be more complex compared to the HIF1A and EPAS1 (encoding HIF-2α) genes. Several alternative transcription sites can be used, and the transcripts are largely subjected to alternative splicing. As a result, mice mainly express three variants, namely, HIF-3α, inhibitory PAS domain (IPAS), and neonatal embryonic PAS (NEPAS)   whereas  (NEPAS) . Among t (NEPAS) .2-dependent regulation of protein stability. In the presence of O2, this domain is hydroxylated by HIF proline hydroxylases. The hydroxylated prolines then serve as recognition tags for an E3-ubiquitin ligase complex containing the von-Hippel-Lindau protein (pVHL). The resulting polyubiquitylated HIFα subunits are then directed to proteasomal degradation  hydroxylates a certain asparagine residue, which thereby can no longer bind the CBP/p300 co-activators (All full-length HIFα subunits belong to the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) protein family and contain an oxygen-dependent degradation domain (ODDD). The latter is of particular importance for the Oradation . In addiradation . The trativators . In conttivators .In addition to polyubiquitination which is quintessential for preventing HIFα accumulation in normoxia, other post-translational modifications such as phosphorylation, acetylation, methylation or sumoylation constitute another essential regulatory mechanism for the HIF-1α-and HIF-2α subunits .Insulin is a hormone that regulates various cellular processes, including glucose metabolism, cell proliferation and cell migration. The majority of insulin actions are mediated by posttranslational changes, mainly phosphorylation, of key regulators involved. Insulin has been shown to regulate HIF-1α  and HIF-Here we show that insulin stimulates the accumulation of HIF-3α1 under both normoxia and mild hypoxia in a PKB-dependent manner, as this induction could be antagonized by using a specific PKB inhibitor. Furthermore, our data reveal that PKB/AKT interacts with full-length HIF-3α1 and phosphorylates it at serine 524 in the oxygen-dependent degradation domain (ODDD). This phosphorylation has functional consequences as it affects colony formation, adhesion, and migration of cells. Overall, our data provide evidence that HIF-3α1 plays a role in cell growth and suggest that PKB/AKT directly regulates HIF-3α1 activity and function.2O), Wortmannin and Rapamycin were purchased from Sigma Aldrich. BML-257 and MG132 were purchased from Cayman chemicals. Unless otherwise stated, all substances were dissolved in DMSO.All biochemicals and enzymes were of analytical grade and were obtained from commercial suppliers. Insulin (dissolved in 0.9% NaCl), Lithium chloride  in MEM supplemented with 10% FBS. MCF-7 cells were cultured under normoxia  in DMEM containing 10% FBS.HEK 293 and HepG2 cells were cultured under normoxia (16% Omycoplasma negative by using the MycoAlert Detection Kit (Lonza). In all experiments the number of cell passages used was below 10. For protein extraction, cells were seeded into 60 mm dishes and, when necessary, transfections were performed the next day for 12 h. Prior to treatment, cells were starved in serum-free media for 16 h, then treated either alone or in combination with 100 nM insulin, 20 nM wortmannin, 12.5 µM BML-257, 10 nM rapamycin or 500 nM LiCl and further cultured for 4 h under normoxia or mild hypoxia . In all experiments control cells were treated with DMSO.All cell lines were tested The construct for full-length HIF-3α1 was amplified by polymerase chain reaction (PCR) from HepG2 cDNA using the following primer set: human HIF-3α1-F 5′- caccatggactggcaagac-3′ and human HIF-3α1-R 5′- gtcagcctgggctgagcctg-3′. The resulting PCR product was directly cloned into the pcDNA3.1D/V5-His-TOPO vector according to manufacturer’s protocol. The pcDNA6/Myc-His-Gal4-HIF-3α-TAD was generated by inserting a HindIII-BamHI Gal4DBD fragment (1–147) of the pSG424  into the5 cells per 60 mm dish were transfected as previously described  were added for 12 h. Thereafter, the beads were washed five times with lysis buffer and recovered, pellets were dissolved in 2 X Laemmli buffer, loaded onto a 7.5 % SDS gel, blotted and detected with antibodies against ubiquitin and V5- or HA-epitope.For half-life studies, HEK 293 cells were transfected together with expression vectors encoding full-length V5-tagged HIF-3α1 WT or mutant HIF-3α1 S524A. 48 h post transfection, cells were treated with protein synthesis inhibitor cycloheximide ). Afterwards, cells were harvested at the indicated time points and protein levels were measured by immunoblot analysis. For immunoprecipitation and ubiquitylation assays, HEK 293 cells were treated with proteasome inhibitor MG 132 . Four hours later, cells were scraped in lysis buffer ). After scraping, lysates were incubated with continuous shaking at 4°C for 20 min and centrifuged at 12.000 4 cells per well) and cultured under normoxia (16% O2). After 24 h cells were treated with insulin (100 nM) and further cultured in serum-free medium for 8 h under normoxic conditions (16% O2). Then 500 µL of MTT (4 mg/mL) was added to the wells and incubated at 37°C for 2 h. Formation of MTT–formazane was measured with a Tecan plate reader at a wavelength of 570 nm.Cell viability was assessed by a MTT  reduction assay. MCF-7 or HEK 293 cells were transfected with expression vectors for full-length wild-type HIF-3α1 or HIF-3α1 S524A mutant, seeded in 24-well plates (5 × 102) and then seeded into 6-well plates at a density of 2,000 cells/well. After 7 days, cells were fixed with 4% PFA and stained with crystal violet (Applichem). Plates were either scanned or images of the colonies were taken using the Leica MZFLIII microscope at original magnification ×40 and analyzed using the ImageJ software.For colony formation, MCF-7 or HEK 293 cells were transiently transfected with plasmids for full-length V5-tagged HIF-3α1 WT or mutant HIF-3α1 S524A. After transfection, cells were further cultured for 20 h under normoxia (16% O2) until they reached confluency (2–3 days). The 6-well plates were then transferred to an orbital shaker and rotated at 250 rpm for 8 h at 37°C. Control plates remained in the incubator. After rotation, the medium was aspirated from the cells followed by washing with 1x PBS and fixation with 4% PFA for 20 min at room temperature. The cells were briefly washed with 1x PBS and stained with crystal violet solution. Subsequently, the staining solution was aspirated, and the wells were rinsed with water to remove excess stain and air dried. Plates were scanned and analyzed using the ImageJ software for quantification.For adhesion assay, MCF-7 cells were transiently transfected with plasmids for full-length V5-tagged HIF-3α1 WT or mutant HIF-3α1 S524A. 24 h post transfection, cells were seeded into 6-well plates and grown under normoxia (16% O3 cells per well) in DMEM with 10% FBS. The following day the confluent cell monolayer was wounded with the 96 PTFE pin Wound Maker (Essen Bioscience), cells were washed with 1x PBS and further cultured in DMEM supplemented with 0.1% FBS and insulin (100 nM). The live wound closure was phase contrast-imaged for 72 h in 4 h intervals and the confluence analysis was performed using the IncuCyte software.For scratch wound assay, MCF-7 cells were transiently transfected with plasmids for full-length V5-tagged HIF-3α1 WT or mutant HIF-3α1 S524A. 24 h post transfection, cells were seeded onto 96-well Essen ImageLock Plates (Essen Bioscience) (3 × 10Escherichia coli BL21 (DE3) cells transformed with pGEX-GST-HIF-3α-TAD or pGEX-5X1 were grown at 37°C in Luria-Bertani media containing 100 µg of ampicillin/mL and induced with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) for 4 h. Cells were then harvested, and glutathione S-transferase (GST)-HIF-3α-TAD and GST proteins were prepared essentially as described previously (eviously . The int3COO)2] in the presence of active AKT1 (50 mU)  and 1 μCi/μL [γ-33P]ATP . After 30 min of incubation at 30°C, samples were loaded onto a 10% SDS gel, and after electrophoresis and blotting onto a polyvinylidene difluoride membrane, phosphorylated proteins were visualized by phosphorimaging.The purified wild-type or mutant GST-HIF-3α-TAD-N fusion proteins (20 µg) were incubated in kinase buffer [0.2 M MOPS , 0.5 M EDTA, 0.1 M Mg following the manufacturer’s instructions. Total RNA concentration and purity were measured on a NanoDrop spectrophotometer. One μg of total RNA was used for cDNA synthesis using iScript cDNA Synthesis Kit .® green PCR master mix (Life Technologies). The following primer sets were used: human HIF-3α-F (5′-GTCGGAGAGTATCGTCTGTGTC-3′), human HIF-3α-R (5′-TCTGCGAGAGTGTTGCTCCGTT-3′), human β-actin-F (5′-GTTGTCGACGACGAGCG-3′) and human-β-actin-R (5′-GCACAGAGCCTCGCCTT-3′). The experiments for each data point were carried out in triplicate. The relative quantification of gene expression was determined using the ΔΔCt method  by using an Applied Biosystem Power SYBRt method .4P2O7, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, and complete protease inhibitor cocktail tablet (Roche). The lysate was incubated with continuous shaking at 4°C for 20 min and then centrifuged at 12,000 g for 15 min at 4°C. To recover anti-V5, 150 µg of protein was incubated with 2 µg of antibody for 1 h at 4°C, followed by the addition of protein G Sepharose beads  for 12 h. The beads were washed five times with lysis buffer, and the protein was recovered. The pellets were dissolved in 2X Laemmli buffer and loaded onto a 10% SDS gel. Subsequently, the gel was washed briefly with ddH2O and stained with Coomassie Blue for 1 h at room temperature. After overnight destaining, the gel piece containing a ∼72 kDa polypeptide was excised. The gel piece was completely destained with 40 mM ammonium bicarbonate in 40% acetonitrile, treated with 20 mM DTT for 30 min at room temperature, and alkylated by adding 45 mM iodoacetamide (IAA) for 30 min at room temperature. The gel piece was washed once with the same solvent and twice with ddH2O. Trypsin  was added (20 µL) and incubated overnight at room temperature.Co-immunoprecipitation and Mass spectrometry were conducted as follows. HEK 293 cells were transfected with an expression vector encoding full-length V5-tagged HIF-3α1 WT and full-length HA-tagged PKB and incubated for 24 h. Cells were collected by scraping and lysed in lysis buffer containing 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 10 mM NaFor data acquisition, a Nano LC 1000 system  coupled with a Lumos Fusion Orbitrap mass spectrometer  was used. Samples were trapped onto a Symmetry C18 100Å 5 µm 0.180 × 20 mm precolumn and eluted over nanoEaseBEH C18 130Å 1.7 µm 0.075 × 150 mm analytical column. The LC conditions included a flow rate of 0.3 μL/min and a gradient from 97% solvent A (0.1% formic acid in water) to 35% solvent B (0.1% formic acid in acetonitrile) over 90 min. Mass spectrometry data were acquired in data-dependent acquisition (DDA) mode with survey scans at a resolution setting of 120.000 over a mass range of 375–1,500. The automatic gain control (AGC) target was set to 4e5, and the maximum fill time was under 100 ms. MS/MS interrogation was triggered for ions with multiple charges and intensity above 2.5e4 in the Orbitrap, while ions between 1e3 and 2.5e4 were analyzed in the ion trap. AGC for Orbitrap analysis was set to 5e4 with a maximum fill time of 200 ms and a resolution of 15,000. The ion trap was filled for a maximum of 300 ms with AGC set to 10.000. HCD fragmentation with 30% collision energy was used for MS/MS.p values p ≤ 0.05 were considered significant. Luc values presented are means +SEM. Results were compared by ANOVA for repeated Luc measurements followed by the Newman-Keuls test. A probability level p ≤ 0.05 was accepted as significant.Densitometry data were plotted as fold induction of relative density units, with the zero-value absorbance in each figure set arbitrarily to 100%. Statistical comparisons of absorbance differences were performed by the Mann-Whitney test , and HIF3A mRNA levels under both normoxia and mild hypoxia, which is in line with previous studies  or mammalian target of rapamycin (mTOR), are involved in the regulation of HIF-3α1 protein expression. To achieve this, we treated MCF-7 cells with insulin and LiCl both well-known inhibitors of GSK-3 and rapamycin, a mTOR inhibitor alone or in combination under normoxia and mild hypoxia. Consistent with our previous findings, treatment with insulin alone increased HIF-3α1 protein expression under both normoxic and mild hypoxic conditions. However, treatment with LiCl and rapamycin alone slightly reduced HIF-3α1 protein levels compared to the control cells . FurtherTo substantiate the finding that insulin is able to modulate HIF-3α1 protein expression via PKB, we first used computational approaches to search for the presence of putative PKB phosphorylation sites within HIF-3α1. We found that serine 524 matched the consensus motif, which is also highly conserved in mouse and rat HIF-3α . This reTo investigate this, we transfected HepG2 cells with a Gal4 construct expressing a fusion protein with the wild-type TAD of HIF-3α  or a fusion protein in which the respective PKB site has been mutated . The cells were then treated with insulin for 8 h under normoxia and mild hypoxia. We found that mild hypoxia induced Luc activity by about 200% in both the wild-type and mutant construct . TreatmeTo further support the idea that HIF-3α1 might be a direct target of PKB, we performed a phosphorylation assay. When using the GST-HIF-3α-TAD protein as a substrate we found that PKB phosphorylated the HIF-3α-TAD, whereas the GST protein alone was not phosphorylated . Next, wTo validate the notion that HIF-3α1 is a direct target of PKB, we performed coimmunoprecipitation analysis to examine whether PKB forms a complex with HIF-3α1. Indeed, the results demonstrated an interaction between PKB and wild-type HIF-3α1 as they could be found in the same complex. Conversely, when lysates from cells expressing the HIF-3α1 S524A mutant were used, no interaction between PKB and HIF-3α1 could be detected .To determine whether the insulin-mediated HIF-3α1 regulation involves ubiquitination, we first performed coimmunoprecipitation analysis using proteins from HEK 293 cells transfected with vectors for V5-tagged wild-type HIF-3α1 or the HIF-3α1 S524A mutant. Subsequently, the cells were treated with insulin and the proteasome inhibitor MG132 for 4 h before harvesting. Although both wild-type and mutant HIF-3α1 proteins were found to be ubiquitinylated following immunoprecipitation with the V5-tag antibody, the extent of the ubiquitination was different. Treatment of cells with insulin inhibited the ubiquitination of wild-type HIF-3α1 whereas no differences in ubiquitination could be detected with the HIF-3α1 mutant upon insulin treatment . In addiHIF-3α has been proposed to play important roles in regulation of cell proliferation and growth . The proNext, we investigated whether insulin might have an influence on the HIF-3α1 modulated cell viability as well as colony formation. Again, HIF-3α1 WT and the corresponding HIF-3α1 S524A mutant expressing cells were used. We found that application of insulin improved the cell viability of HIF-3α1 WT transfected HEK 293 cells by approximately 180% and increased it by approximately 240% in MCF-7 transfected cells. . In addiSince we observed that overexpression of HIF-3α1 WT or the HIF-3α1 S524A mutant reduced colony number but increased colony size compared to control cells, we next investigated whether HIF-3α1 also affects cell adhesion. To do this, we transfected cells with HIF-3α1 WT or the HIF-3α1 S524A mutant and subjected them to rotation on an orbital shaker for 8 h. After rotation, ∼70% of the control cells were detached. Similarly, cells overexpressing HIF-3α1 WT showed a comparable level of adhesion . By contThe above findings indicated that HIF-3α1 and its insulin-dependent regulation may play a role in cell adhesion as well as colony-forming ability of cells. Next, it was investigated whether HIF-3α1 mediates the mobility of cells in a wound healing assay. The results of the wound healing assay showed that cells overexpressing wild-type HIF-3α1, but not the HIF-3α1 S524A mutant, migrated 2-fold faster than non-transfected cells . As expein-vivo is still pending.The present study aimed to investigate the role of insulin in the regulation of HIF-3α1 and its potential impact on cell adhesion, colony formation, cell viability, and migration. The data revealed involvement of the PI3K/PKB signaling pathway in insulin-dependent HIF-3α1 expression where serine 524 was identified as a crucial phosphorylation site for PKB. The mutation of serine 524 was found to affect the regulation and activity of HIF-3α1, suggesting its importance in cancer-related processes, although evidence in mice Hif-3α was shown to upregulate hypoxia-inducible expression of a large number of genes, including igfbp-1b, mclb, sqrd, and zp3v2 , whereas full-length HIF-3α1 was less robustly ubiquitinated  signaling pathway, which is critical in promoting carcinogenesis in colorectal cancer tissues . Recent Changes in cell adhesion and migration are associated with alterations in the expression of several key proteins involved in cell-cell interactions  and the cytoskeleton . While overexpression of HIF-3α1 WT did not affect cell adhesion significantly, it promoted cell migration in a wound healing assay. This is consistent with a recent study showing that HIF-3α promotes pancreatic cancer cell invasion and migration under both normoxia and hypoxia . InteresIn conclusion, our study provides evidence that HIF-3α1 is regulated by PKB/AKT-mediated phosphorylation, which affects its stability and activity. Furthermore, HIF-3α1 influences colony formation, cell viability, and migration, suggesting its potential role in tumorigenesis. Future investigations should focus on elucidating the HIF-3α1 downstream pathways and their precise contribution to cellular functions.