Aspergillus terreus has been reported to produce many bioactive metabolites that possess potential activities including anti-inflammatory, cytotoxic, and antimicrobial activities. In the present study, we report the isolation and identification of A. terreus from a collected soil sample. The metabolites existing in the microbial ethyl acetate extract were tentatively identified by HPLC/MS and chemically categorized into alkaloids, terpenoids, polyketides, γ-butyrolactones, quinones, and peptides. In addition, a new triglyceride (1) and a diketopiperazine derivative namely asterrine (4), together with two known butyrolactone (2–3) were purified from the extract. The chemical skeleton of the purified compounds was established by comprehensive analysis of their ESI/MS, 1 and 2D-NMR data. The extract and compounds 3,4 exhibited a strong inhibitory activity for the binding of ACE2 to SARS-CoV-2 spike-protein receptor with IC50 7.4, 9.5, and 8.5 µg/mL, respectively. In addition, the extract, 1 and 2 displayed a potent anti-inflammatory effect with IC50 51.31 and 37.25 pg/mL (Il-6) and 87.97, 68.22 pg/mL (TNF-α), respectively, in comparison to LPS control. In addition, the extract and compound 4 displayed an antimicrobial effect towards S. aureus by MIC 62.5 and 125 μg/mL, while the extract exhibited a potent effect against C. albicans (MIC of 125 μg/mL). Collectively, our data introduce novel bioactivities for the secondary metabolites produced by the terrestrial fungus Aspergillus terreus.  The online version contains supplementary material available at 10.1186/s13568-023-01612-0. Aspergillus terreus metabolites using HPLC/MS analysis. Identification of Aspergillus terreus extract using different spectroscopic techniques.Structure elucidation of isolated metabolites from The microbial extract and purified biomolecules exhibited significant anti-inflammatory, antimicrobial, and ACE2 inhibitory activities..The online version contains supplementary material available at 10.1186/s13568-023-01612-0. Angiotensin-converting enzyme II (ACE2) is an essential membrane protein found in many cells of the human body  and thin layer chromatography (TLC), respectively . 1H and 13C NMR spectra were recorded on a Bruker Avance spectrometer  and JEOL  at 400 and 500 MHz for 1HNMR, as well as, 100 and 125 MHz for 13CNMR. The results were expressed as δ ppm relative to the internal reference (TMS). HPLC/ESI-MS were recorded in negative and positive ionization modes on an XEVO TQD triple quadruple LC/MS/MS . Chromatographic conditions: ACQUITY UPLC—BEH C18 column , flow rate (0.2 mL\min); mobile phase (water and acetonitrile each containing 0.1% formic acid).Silica gel 60-120 mesh and F–1 to 10–5), followed by inoculation of each dilution in potato dextrose agar plates  . For the preparation of soil suspension, 20 g was mixed with sterilized distilled water (200 mL), shaken by a rotary shaker for 3h, and left to be settled for ½ h. For the fungal strain isolation, the supernatant (500 µL) was transferred to a sterile slant containing sterilized water (5 mL) which was further used for serial dilution preparation (10http://www.ncbi.nlm.nih.gov). The collective results and the phylogenetic tree were made using Molecular Evolutionary Genetics Analysis (MEGA) version 10.0.5 (https://www.megasoftware.net/).Identification of the fungal strains was achieved as mentioned before in our previous work  followed by incubation for 14 days at 30°C under static conditions.Positive and negative modes ESI–MS were applied to detect the metabolites in the fungal ethyl acetate extract. Additional file A. terreus was extracted by ethyl acetate till exhaustion (3 × 250 mL), filtered then evaporated the solvent under vacuum to yield 9.0 g dry residue. About 100 g of silica gel was mixed with 8.0 g of the extract dissolved in a mixture of dichloromethane (DCM) /MeOH  and then dried at 60 °C under vacuum. The dried mixture was subjected to fractionation using silica gel 60 vacuum liquid chromatography (VLC) , and eluted with DCM: MeOH (100:0:100%). Ten fractions (1.0 L) were eluted and further grouped into five main fractions depending on their behavior towards UV-light and p-anisaldehyde/sulfuric acid reagent on TLC. The first fraction (2.0 g) eluted with DCM (100%) was subjected to silica gel CC using n-hexane: DCM (30:70 v/v) for elution to yield a pure sample of 1 (200 mg). Fractionation of the second fraction II  using silica gel CC and n-hexane: ethyl acetate (60:40 v/v) as eluent to yield three subfractions. Two of which (i) and (ii) contain a crude sample of compounds 2 and 3 respectively. A pure sample of 2 (12 mg) was obtained through fractionation on silica gel CC and n-hexane: ethyl acetate (70:30 v/v) as eluent, while pure sample 3 (9 mg) was obtained by silica gel CC and n-hexane: ethyl acetate (60:40 v/v) as eluent. The fourth fraction (59.0 mg) was subjected to successive silica gel CC and DCM: MeOH (94:06 v/v) for elution yielding a pure sample of 4 (15 mg). The isolated samples′ purity was checked using TLC and HPLC techniques.The rice culture media of the purified Compound 1: Colorless oil, its 1H and 13CNMR data  is represented in Table m/z 655.5457 [M-H] −. Spectral data of compound 1 are represented in Additional file Compounds 2 and 3: Isolated as a faint yellow amorphous powder. Negative ESI/MS of 2 at m/z 423.2239 [M-H] ˉand 847.4105 for [2M-H]−, while that of 3 at 439.2230 and 879.4200 for [M-H]− and [2M-H]−, respectively. Their 1HNMR, 13CNMR  and ESI–MS spectra are represented in Additional file Compound 4: White amorphous powder; + ve ESI/MS at m/z. 563.6636 [M + H]+. 1 and 2-D NMR data  were represented in Table 1HNMR, 13CNMR  Additional file A. terreus extract and the pure compounds (1–4) against the ACE2 receptor, the ACE2: SARS-CoV-2 spike inhibitor screening colorimetric assay kit  was used  (5–50 µg/mL), then HRP-labeled anti-Fc and substrate were added. The result color was measured at λ450 nm, and all tests were repeated in triplicates. Quercetin was used as a positive control. Calculation of IC50 was done using a dose–response inhibitory curve using Windows Graph Pad Prism version 5 .For assessment of the inhibitory action of 2 atmosphere  then they sub-cultured every 2 days and the cells of the exponential phase were used all over the procedure.Murine macrophage cells (RAW 264.7), supplied by American Type Culture Collection (ATCC), were grown on Dulbecco′s Modified Eagle Medium (DMEM), accompanied with 10%, 100U/100 mL and 100 μg/mL from bovine serum, penicillin, and streptomycin respectively. After that, cells were incubated in a CO1–4) and the extract were diluted by a serial dilution technique (1000–4 μg/mL) were introduced and the plate was placed in a CO2 incubator (37 °C for 24 h). The cells were washed by DMEM and incubated with MTT solution . DMSO (180 μL) was used to solubilize the crystals of formazan and the produced color was measured at λ570 nm. The results were normalized against the control and the cell viability percentage was estimated as follows:In vitro cytotoxicity assay kit MTT-based was purchased from Sigma- Aldrich, MTT cat No: M-5655, MTT solubilization solution: M-8910). RAW 264.7 cells  were plated in a 96-well plate then the test compounds (50) was used to investigate the inflammatory marker in RAW 264.7 macrophages.A quarter of this concentration (¼ IC5 cells/mL) were seeded for 18 h in a 24-well plate having DMEM medium (1 mL). When the cells reached confluence, they were treated with concentrations equivalent to ¼ IC50 of the selected samples (1–4 and extract) for 2 h. Then, LPS (Sigma-Aldrich Cat No: L2630)  was added to all treated and untreated wells to induce the inflammatory process. After incubation for 24 h, the media were taken and frozen till analysis. The untreated wells represented the LPS control group.RAW 264.7 cells  and Il-6 quantitative assay ELISA kit  were used to quantify IL-6 and TNF- α levels in the cell culture medium following the manufacturer′s instructions . Total RNA from the stimulated RAW 264.7 cells was extracted utilizing Qiagen RNA extraction  following the instructions of the procedure. cDNA was synthesized by iScript cDNA synthesis kit . Rotorgene RT-PCR system  was used for PCR amplification using BioRad cyber green PCR MMX . The primers used for the amplification of IL-6, TNF-α, and  mL. Tota−(ΔΔCt) was used to calculate the relative fold change between samples and control.The Formula 2Staphylococcus aureus ATCC 6538,Escherichia coli ATCC 25922, Candida albicans ATCC 10231and Aspergillus niger NRRL-A326 were supplied from Culture Collection Center. The antimicrobial activity of A. terreus extract and 1–4 was investigated by the agar disc diffusion method . Nutrient agar (bacteria and yeast) and PDA (fungi) plates were seeded with 0.1 mL of 105–106 cells/mL then the filter paper discs (0.5 cm) were loaded with about 0.01mg from the extract and compounds, and put on the inoculated agar or PDA plates surface and stayed for 2–4 h at 4°C to reach maximum diffusion. After that, incubation of the plate at 30°C for 24 and 48 h for bacteria and fungi, respectively, to permit maximum growth then the inhibition zone diameter was measured in millimeters (mm). Experiments were carried out in triplicate.6 cfu/ml. 50µL of broth medium was added to all wells of 96 well sterile microplates, then 50 µL of the tested samples (2 mg/mL MeOH for compounds and 10 mg/mL MeOH for extract as stock concentration) was pipetted into the first well and then two-fold serial dilutions were done. 10µL of resazurin indicator  was added to each well followed by 10µL of bacterial and fungi suspension. Duplicate plates were prepared and incubated . The lowest concentration at which the color is changed is the MIC data, while MBC has been calculated by streaking two concentrations higher than MIC showing no growth . Bacteria and fungi cells (pellet) were collected by centrifugation at 5000 rpm, 4 °C, and under aseptic conditions, then washed with sterile saline till the supernatant became clear. Cell suspension has been performed to achieve an optical density of 0.5 to 1 (at 550 nm) yielding actual colony-forming units of 5 × 10The data were obtained from three individual experiments and the statistical significance was established by GraphPad Prism version 5 for Windows  by one-way variance analysis (ANOVA). P < 0.05 is considered as statistically significant.Aspergillus terreus. Data were represented in GenBank under the accession number MW035847 and its phylogenic tree is represented in Fig. In the current study, a filamentous fungus with long, colorless conidiophores was obtained from a soil sample. Moreover, after a comparison search of the fungus 18SrRNA nucleotide sequence by the NCBI database, the results revealed that there is a 100% similarity between the sequence and A. terreus ethyl acetate extract revealed the tentative identification of forty-four metabolites depending on the comparison of their molecular ion peaks with the previously reported data for different Aspergillus species , sesteterrpenoids , meroterpenoids , polyketides (3), γ-butyrolactones , peptides (28–29), prenylated phenol derivative (11), phenolic compound (6).HPLC/ESI—MS for 1–4  and 172. 5 (C-1′′) indicating the presence of three ester carbonyl carbons , 4.07  and 5.19  corresponding to carbon signals at δC 61.9  and 68.9 (C-2) which established that 1 is a related to triglyceride with C13 and C12 fatty acid esters  with H-2 (δH 5.19). In addition, HMBC spectrum  with carbonyl carbon C-1′′ (δC 172.5) and that between H-2′, H-2′″ (δH 2.25) with carbonyl carbon C-1′, C-1‴ (δC 172.9), also that between H-3′, H-3″ and H-3″′ (δH 1.53) and C-1′, C-1′′ (δC 172.9), C-1′′ (δC 172.5), along with the HMBC correlation between H-1a, H-1b  with C-2 (δC 68.9), C-3 (δC 61.9) and C-1′ (δC 172.9), H-3a, H-3b  with C-2 (δC 68.9), C-1 (δC 61.9) C-1′″ (δC 172.9) and that from H-2 (δH 5.19) with C-1, C-3 (δC 61.9) and C-1″ (δC 172.5), which also established that 1 is a long chain fatty acid ester triglyceride. Furthermore, the 1HNMR spectrum displayed multiplet proton signals at δH 5.26 -5.28 for H-6′, H-7′, H-9′, H-10′, H- 6″, H- 7″, H- 6‴, H-7″′, H-9″′ and H-10″′ which correlated in the HSQC data with olefinic carbon signals at δC 127.8–129.9, which indicated the presence of five olefinic double bonds in the structure of 1. As well as, in the HMBC the correlation from H-6′, H- 6″′, H-7′ and H-7′″ (δH 5.26–5.28) with C-5′, H-5″′ (δC 27.1), also that between H-9′, H-10′ and H-9″′ and H-10″′ with C-8′ and C-8″′ (δC 31.8). Moreover, the correlation between H-8′ and H-8′″ (δH 2.69) with C-10′ and C-10′″ (δC 129.5) and C-7′ and C-7″′ (δC 129.7). In addition, there is a correlation from H-6″ and H-7″ to C-5″ and C-8″ (δC 27.1) which support the presence of five olefinic double bonds in the structure. Moreover, the 1HNMR spectrum showed characteristic two signals at δH 0.83 and 0.80 for the terminal methyl groups H-12′′ and H-13′, H-13‴ respectively. This suggestion was confirmed by the correlation between H-12′′ (δH 0.83) with H-11′′ (δH1.19–1.24) in 1H-1H-COSY and that between H-12″ and C-10′′ (δC 31.5) and C-11′′ (δC 22.5) in HMBC and from H-13′, H-13‴ (δH 0.80) with H-12′, H-12‴ (δH1.19–1.24) and C-12′, C-12‴ (δC 22.5) and C-11′, C-11‴ (δC 31.8) in 1H-1H-COSY and HMBC respectively. Furthermore, two methylene triplet doublets at δH 2.23 (H-2″) and 2.25  and multiplet methylene signals at δH 1.53 for H-3′, H-3″ and H-3′″, 1.96 for H-5′, H-5″ and H-5′″, 1.19 for H-4′, H-4″ and H-4″′, in addition to characteristic triplet methylene signal at δH 2.69 (H-8′/H-8‴) were detected in the 1HNMR. This suggestion was confirmed from 1H-1H-COSY connection between H-2′′ and H-2′, H-2‴ with H-3′′ and H-3′, H-3‴, which further correlated with H-4′′ and H-4′, H-4‴ respectively. Moreover, H-5′, H-5′′ and H-5‴ is correlated to H-4′, H-4′′ and H-4‴ and H-6′, H-6′′ and H-6‴ respectively as well as the correlation from H-8′/8‴ (δH 2.69) to H-7′/7‴ and H-9′/9‴. In addition, the HMBC spectrum displayed a correlation from H-3′, and H-3‴ (δH 1.53) to C-1′, C-1‴ (δC 172.9), and C-4′, C-4″′ (δC 29.6) also that from H-3″ (δH 1.53) to C-1″ (δC 172.5) and C-4″ (δC 29.0). Moreover, H-5′, H-5′″ is correlated to C-4′, C-4′″ (δC 29.6) and C-7′, C-7″′ (δC 129.7) and H-5″ is correlated to C-4″, (δC 29.0) and C-7″ (δC 129.8) as well as H-8′/8″′ (δH 2.69) is correlated to C-10′/10″′ (δC 129.5) and C-7′/7″′ (δC 129.7). The remaining 1H-1H-COSY and HMBC correlations were deposited in Table 13C NMR, HSQC, and APT spectra confirmed the direct correlation of the proton signals with the corresponding carbon signals. The presence of only two carbon signal characteristics for three ester carbonyls at δC 172.5 (C-1″), 172.9  supported the evidence that the structure of 1 is a symmetrical triglyceride - trideca-6,9-dienoyl-2-(6Z) dodec-6-enoyl glycerol. To our knowledge, it was identified for the first time from nature.Negative ESI/MS [M-H]2) and butyrolactone III (3) after comparison with the previously reported data , 43.2 (C-3′), 27.4 (C-10′) and 26.2 (C-7), six sp2 quaternary carbons at δC 153.5 (C-7′), 136.9 (C-12a), 129.3 (C-9′a), 127.4 (C-8a), 122.9 (C-5′a) and 109.7 (C-8) in addition to one sp3 quaternary at δC 119.7 (C-1′′). Eight methine sp2 at δC 123.4 (C-14), 122.0(C-9′), 121.4 (C-11), 118.7 (C-10), 118.6 (C-8′), 118.2 (C-9), 114.2 (C-6′), and 111.2 (C-12), and two methine sp3 at δC 54.2 and 51.4 for C-6 and C-10′a respectively in addition to one thiomethyl carbon at δC 12.8 (C-2′′). 1HNMR data together with HSQC revealed that compound 4 has an indole moiety through the presence of four doublet protons in the aromatic region at δH 7.61, 7.34, 7.09, and 7.03 for H-9, H-12, H-11, and H-10, respectively, together one singlet signal at δH 7.16 for H-14. Moreover, this suggestion was confirmed by 1H-1H-COSY and HMBC data  and C-8 (δC 109.7), C-8a (δC 127.4) and C-12a (δC 136.9). Moreover, the HMBC correlation from H-3a/b (δH 3.94 and 3.63) to C-5 (δC 172.7) and that from H-6 (δH 4.93) to C-5 (δC 172.7) gave the evidence for the presence of diketopiperazine skeleton. Moreover, the presence of correlation between CH2-7a/b  to C-5 (δC 172.7) and C-6 (δC 54.2), C-8(δC 109.7), C-8a (δC 127.4), C-14 (δC 123.4) support that the indole moiety is connected to the diketopiperazine moiety through methylene bridge CH2-7a/b. In addition, the 1HNMR and HSQC (Table H 6.99 (δC 114.2) and δH 8.19 (δC 122.0) together with doublet doublets signal at δH 6.90 (δC 118.6) for H-6′, H-9′ and H-8′, respectively, which suggested the presence of trisubstituted benzene ring, which further supported from the correlations displayed in 1H-1H-COSY and HMBC (Table 4 due to the occurrence of methylene group at δH 4.38 and 3.44 for H-3′a/b and methine proton at δH 4.81 (H-10′a) as well as the correlation between H-3′a/b and C-2′ (δC 171.5) and C-4′ (δC 167.9) and that from H-10′a to C-2′ (δC 171.5) in the HMBC data supported the presence of the diketopiperazine moiety. Moreover, the HMBC correlations between H-10′c/d (δH 1.88 and 2.24) with C-2′ (δC 171.5) and C-10′a (δC 51.4) C-9′a (δC 129.3) and C-6′ (δC 114.2) gave evidence for the connection of the trisubstituted benzene ring to the diketopiperazine moiety. Furthermore, the connection of the indole diketopiperazine part with the trisubstituted benzene ring- diketopiperazine part with each other was done by carbon atom bridge between N-1 and N-1′ which supported from HMBC correlation between H-10′c/d  and H-7a/b  with C-1′′ (δC 119.7). Moreover, the thiomethyl group was established by the presence of a singlet signal at δH 2.13 (δC 12.8) as well as attached to C-1′′ (δC 119.7) through the correlation between them in the HMBC spectrum. So, 4 was identified as asterrine, which was purified based on our knowledge for the first time from nature.The molecular formula of 1–4) and the extract were tested for their ability to inhibit ACE2 to SARS-CoV-2 spike-protein receptor binding domains. The results revealed that all compounds have inhibitory activity with different potencies, but compounds 3 and 4 exhibit the most potent activity with IC50, 9.5, and 8.5 µg/mL, respectively  and 87.97, 68.22 pg/mL (TNF-α) respectively in comparison to LPS control  was used for the evaluation the anti-inflammatory activity of the extract and pure compounds, since LPS encouraged cell imitator in vivo condition  and C. albicans (MIC = 125 μg/mL), while compound 4 displayed a moderate activity toward S. aureus(MIC = 125 and MBC = 250 μg/mL) and C. albicans (MIC of 250 μg/mL)  and the new compound 4, which are related to alkaloids, exert the most inhibitory activity. It was reported that some alkaloids isolated from Aspergillus can prohibit SARS-CoV-2 entry inside the host cells via ACE2 as well as it was reported that butenolides can exhibit SARS-CoV-2 (Uras et al. 2 as previously reported that butenolides can exhibit anti-inflammatory activity (Qia et al. A. terreus showed an antimicrobial activity against different strains of microorganisms (Pinheiro et al. The current study aimed to tentatively identify the secondary metabolites of Additional file 1.Supporting data for chemical analysis of isolated metabolites. Data enclose HPLC/MS spectra of the fungal extract, 1H and 13C NMR data of isolated compounds, chemical structures of tentatively identified metabolites, list of ESI data for tentatively identified metabolites, discussion of compound 2 and 3, and list of supporting references.


The title compound, a charge-neutral bis{5--1,2,4-triazol-3-ato)-6-(pyrazol-1-yl)pyridine}iron(II) dimethanol dichloroform solvate, is a high-spin complex with a distorted pseudooctahedral coordination environment of the metal ion. Due to the tapered shape and polar nature, the molecules stack in one-dimensional columns that are bound by weak hydrogen bonds into layers, which, in turn, are arranged in a three-dimensional network without interlayer interactions below van der Waals radii. 18H15N6O2)2]·2CH3OH·2CHCl3, consists of a charge-neutral complex molecule, two methanol and two chloroform molecules. In the complex, the two tridentate 2--1,2,4-triazol-3-yl)-6-(pyrazol-1-yl)pyridine ligands coordinate to the central FeII ion through the N atoms of the pyrazole, pyridine and triazole groups, forming a pseudo-octahedral coordination sphere. Neighbouring tapered molecules are linked through weak C—H(pz)⋯π(ph) interactions into one-dimensional chains, which are joined into two-dimensional layers through weak C—H⋯N/C/O interactions. Furthermore, the layers stack in a three-dimensional network linked by weak interlayer C—H⋯π interactions of the methoxy and phenyl groups. The intermolecular contacts were quantified using Hirshfeld surface analysis and two-dimensional fingerprint plots, revealing the relative contributions of the contacts to the crystal packing to be H⋯H 32.0%, H⋯C/C⋯H 26.3%, H⋯N/N⋯H 13.8%, and H⋯O/O⋯H 7.5%. The average Fe—N bond distance is 2.185 Å, indicating the high-spin state of the FeII ion. Energy framework analysis at the HF/3–21 G theory level was performed to quantify the interaction energies in the crystal structure.The unit cell of the title compound, [Fe(C The calculated continuous shape measure (CShM) value relative to the ideal hO symmetry is 5.391  rings of the ligand molecule Fig. 1. The chlds Fig. 1. The ave3.Cg(ph) distance is 3.574 (5) Å]. The one-dimensional supramolecular chains formed extend along the b-axis direction with the stacking periodicity equal to 10.281 (3) Å (= cell parameter b) ⋯ N/C interactions in the range 3.115–3.705 (5) Å . The layers stack without any interlayer interactions below the van der Waals radii . The voids between the layers are occupied by solvent molecules, which also participate in the bonding within separate layers. The methanol molecule forms a strong O—H⋯N hydrogen bond with the deprotonated triazole group, and a chloroform molecule located between two methoxy groups of the phenyl substituent forms a five-membered cyclic motif with two C—H⋯O bonds ⋯π(ph) intermolecular contact between the pyrazole (pz) and phenyl (ph) groups respectively (BF4)2 prepared by dissolving L = 2--6-(pyrazol-1-yl)pyridine  and Fe(BF4)2·6H2O  in boiling acetone, to which chloroform (5 ml) was then added. The middle layer was a methanol–chloroform mixture , which was covered by a layer of methanol (10 ml), to which 100 ml of NEt3 were added dropwise. The tube was sealed, and black–orange single crystals appeared after 3–4 weeks (yield ca 60%). Elemental analysis calculated for C40H40Cl6FeN12O6: C, 45.61; H, 3.83; N, 15.96. Found: C, 45.52; H, 3.77; N, 15.77.The synthesis of the title compound is identical to that reported recently for a similar complex (Seredyuk 8.Uiso(H) = 1.2–1.5Ueq(C)]. The O-bound H atom was refined with Uiso(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023008423/tx2075sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023008423/tx2075Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023008423/tx2075Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023008423/tx2075sup5.docPacking drawings and energy framework analysis data and drawings. DOI: 2297496CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Interleukin-32 (IL-32), first reported in 2005, and its isoforms have been the subject of numerous studies investigating their functions in virus infection, cancer, and inflammation. IL-32θ, one of the IL-32 isoforms, has been shown to modulate cancer development and inflammatory responses. A recent study identified an IL-32θ mutant with a cytosine to thymine replacement at position 281 in breast cancer tissues. It means that alanine was also replaced to valine at position 94 in amino acid sequence (A94V). In this study, we investigated the cell surface receptors of IL-32θA94V and evaluated their effect on human umbilical vein endothelial cells (HUVECs). Recombinant human IL-32θA94V was expressed, isolated, and purified using Ni-NTA and IL-32 mAb (KU32-52)-coupled agarose columns. We observed that IL-32θA94V could bind to the integrins αVβ3 and αVβ6, suggesting that integrins act as cell surface receptors for IL-32θA94V. IL-32θA94V significantly attenuated monocyte-endothelial adhesion by inhibiting the expression of Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor (TNF)-α-stimulated HUVECs. IL-32θA94V also reduced the TNF-α-induced phosphorylation of protein kinase B (AKT) and c-jun N-terminal kinases (JNK) by inhibiting phosphorylation of focal adhesion kinase (FAK). Additionally, IL-32θA94V regulated the nuclear translocation of nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1), which are involved in ICAM-1 and VCAM-1 expression. Monocyte-endothelial adhesion mediated by ICAM-1 and VCAM-1 is an important early step in atherosclerosis, which is a major cause of cardiovascular disease. Our findings suggest that IL-32θA94V binds to the cell surface receptors, integrins αVβ3 and αVβ6, and attenuates monocyte-endothelial adhesion by suppressing the expression of ICAM-1 and VCAM-1 in TNF-α-stimulated HUVECs. These results demonstrate that IL-32θA94V can act as an anti-inflammatory cytokine in a chronic inflammatory disease such as atherosclerosis. Previous studies showed that NK4 induced pro-inflammatory cytokines such as IL-8, TNF-α, and MIP-2 in several immune cells through the classical cytokine signaling pathway, but lacked sequence homology with other cytokines. Hence, it was renamed IL-32, a part of one of the interleukin families . NumerouIL-32θ, one of the IL-32 isoforms, is the only isoform with an exon 6 deletion, except for IL-32sm. Recent studies have shown that IL-32θ has tumor suppression and anti-inflammatory effects , 17. We Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are markers of inflammatory responses involved in various diseases, including asthma and rheumatoid arthritis –25. Pro-Previous studies have suggested that IL-32β and IL-32γ act as pro-inflammatory cytokines involved in the upregulation of ICAM-1 and VCAM-1 , 37. In 22.1http://n2t.net/addgene:125194; RRID : Addgene 125194) and confirmed by DNA sequencing . The recombinant TEVSH vectors were transformed into DH5α by heat shock and purified using a mini prep kit . The IL-32θA94V expression vectors were transformed into Rosetta strain of Escherichia coli by heat shock transformation. Successfully transformed single colony was picked up and cultured at 37°C for 4 h in a 200-rpm incubator in Luria–Bertani (LB) media with ampicillin (100 μg/mL). The cultured mixture was transferred to 2.4 L of fresh LB media with ampicillin (100 μg/mL) and grown in a 200-rpm incubator at 37°C until it reached an OD600 of 0.6. IL-32θA94V was induced by adding 0.5 mM IPTG. Cells were grown in a 200-rpm incubator at 16°C for 16 h.IL-32θA94V coding sequence was synthesized by Bioneer  using HT-oligo™ synthesis. The synthesized DNA sequence was cloned into a pTH24 based TEVSH vector  using NdeI and AgeI restriction sites and rapid DNA ligation kit . TEVSH was a gift from Helena Berglund  and incubated on ice for 30 min. The lysed cells were sonicated  for 1 min using a sonicator . The lysed cells were centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatant was collected. Ni-NTA resin (Thermo Fisher Scientific) was loaded into a PD-10 column and balanced with equilibration buffer . The lysate was mixed with 10 mL of equilibration buffer and loaded into the column and washed by washing buffer . After washing, elution buffer  was loaded into the column and the flow-through buffer containing IL-32θA94V was reloaded into IL-32mAb (KU32-52)-coupled CNBr-activated Sepharose 4B column that was prepared using CNBr-activated Sepharose 4B  and monoclonal antibody IL-32 mAb KU32-52 , a protein-protein binding prediction tool. HDOCK predicts the binding complexes between two proteins using a hybrid docking strategy. Structures of extracellular segments of integrin αVβ3 (PDB ID: 1JV2) and αVβ6 (PDB ID: 5FFG) were provided by protein data bank (PDB). The docking scores are calculated by knowledge-based iterative scoring function ITScorePP or ITScorePR , which is a hierarchical approach to protein structure prediction and structure-based function annotation. I-TASSER identifies structural templates with full-length models constructed by template-based fragment assembly simulations. Function insights of the target are then derived by re-threading the 3D models through protein function database. Protein-protein docking prediction was performed using the model with the highest confidence score among the models derived from I-TASSER –41. InteTScorePR –46.2.42SO4. The absorbance/optical density was measured at 450 nm using a microplate reader .MaxiSorp flat-bottomed 96-well plates  were coated with 1 μg/mL of recombinant αVβ3 and αVβ6 integrins  diluted in PBS and incubated overnight at 4°C. Next, the wells were blocked with 1% BSA  in PBS at 37°C for 1 h followed by three washing steps with PBS containing 0.05% Tween 20. Subsequently, some wells were preincubated with 10 μM cyclo-RGDfV peptide  or 10% fetal bovine serum (FBS), whereas others were incubated with PBS at 37°C for 1 h. Next, the wells were incubated with various concentrations of mutant IL-32θA94V diluted in PBS, with or without 10 μM cyclo-(RGDfV) or 10% FBS at 37°C for 1 h. The wells were washed three times with wash buffer and incubated with IL-32 mAb KU32-52 diluted in PBS at a concentration of 0.2 μg/mL at 25°C for 1 h. After the incubation, the wells were washed three times with wash buffer and incubated with mouse-IgGκ light chain binding protein conjugated to HRP (m-IgGκ BP-HRP)  at 25°C for 1 h. Next, the wells were washed three times with wash buffer and incubated with TMB substrate at 25°C for 20 min, and the color reaction was stopped with 2.5 N H2.52-containing chamber at 37°C.HUVEC cells were cultured in Dulbecco modified Eagle medium  supplemented with 10% (v/v) heat-inactivated fetal bovine serum , penicillin (100 U/mL), and streptomycin (100 μg/mL). The cells were incubated in a 5% CO2.65 cells/well) were incubated in 6-well plates for 24 h and starved for 4 h with serum-free media. Subsequently, the cells were pre-treated with IL-32θA94V (10 ng/mL) for 1 h and treated with TNF-α (10 ng/mL) for another 4 h. The treated cells were collected and lysed using the easy-BLUE™ Total RNA extraction kit  according to the manufacturer’s instructions. For reverse-transcription (RT) polymerase chain reaction (PCR), RNA (1 µg) was reverse-transcribed to cDNA using oligo (dT) primers and M-MuLV reverse transcriptase . The synthesized cDNA was amplified using a PCR Thermal Cycler Dice instrument . The following sets of primers were used: integrin αV, 5′-AGGAGAAGGTGCCTACGAAGCT-3′ (forward) and 5′-GCA CAGGAAAGTCTTGCTAAGGC-3′ (reverse); integrin β3, 5′-CATGGATTCCAGCAATGTCCTC C-3′ (forward) and 5′-TTGAGGCAGGTGGCATTGAAGG-3′ (reverse); integrin β6, 5′- TCTCCTGCGTGAGACACAAAGG-3′ (forward), and 5′-GAGCACTCCATCTTCAGAGACG-3′ (reverse); ICAM-1, 5′-AGCGGCTGACGTGTGCAGTAAT-3′ (forward), 5′- TCTGAGACCTCTGGCTTCGTCA-3′ (reverse); VCAM-1,5′-GATTCTGTGCCCACAGTAAGG C-3′ (forward) and 5′-TGGTCACAGAGC CACCTTCTTG-3′ (reverse); glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-AGAACATCATCCCTGCCTCT-3′ (forward), 5′-CTGCTT CACCACCTTCTTGA-3′ (reverse). GAPDH was used as an internal control. The PCR products were separated on a 2% agarose gel.HUVEC cells  and the Sensi FAST™ SYBR NO-ROX Kit . The expression of all target genes was normalized to that of the housekeeping gene, GAPDH. Each sample was run with the following primer sets: ICAM-1, 5′-AGCGGCTGACGTGTGCAGTAAT-3′ (forward), 5′- TCTGAGACCTCT GGCTTCGTCA-3′ (reverse); VCAM-1,5′-GATTCTGTGCCCACAGTAAGG C-3′ (forward), 5′-TGGTCACAGAGCCACCTTCTTG-3′ (reverse); GAPDH, 5′-AGAACATCATCCCTGCCTCT-3′ (forward), 5′-CTGCTTCACCACCTTCTTGA-3′ (reverse). GAPDH was used as an internal control. The mRNA levels of each gene were calculated relative to that of the internal reference, GAPDH using the comparative Ct method .2.85 cells/well) were seeded in 6-well plates for 24 h and pre-treated for 1 h with IL-32θA94V (100 ng/mL), followed by treatment with TNF-α (10 ng/mL). The cells were lysed in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.25% sodium deoxycholate, 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid, 1 mM orthovanadate, aprotinin (10 µg/mL), and 0.4 mM phenyl methylsulfonyl fluoride (PMSF) at 4°C for 2 h. The cells were lysed, and the protein content was estimated using a Bradford assay reagent  and UV spectrophotometer (HUVEC cells (3 × 10otometer . The pro2.95 cells/well) were seeded in an 8-well slide chamber for 24 h and starved overnight, followed by pre-treatment for 1 h with IL-32θA94V (100 ng/mL) and a 6 h treatment with TNF-α (10 ng/mL). After the treatment, cells were fixed using 4% paraformaldehyde for 10 min followed by incubation with ice-cold methanol and blocking using 1% BSA in PBS for 1 h at 25°C. The cells were then incubated with primary antibodies against ICAM-1 and VCAM-1  overnight at 4°C, and with secondary antibodies conjugated to Cy3  for 1 h at 25°C, followed by DAPI staining. Two washing steps with PBS were performed between each step. Thereafter, cells were mounted in mounting buffer (Sigma-Aldrich) and examined under a fluorescence microscope.HUVEC cells (1.0 × 102.105 cells/well) were seeded in 6-well plates for 24 h and changed to fresh media. For silencing ICAM-1 and VCAM-1 expression, cells were transfected with the following siRNA sets : NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; ICAM-1, 5′-UUCUUGUGUAUAAGCUGGCCGTT-3′; VCAM-1, 5′-CCAUUGUUCUCAUGGAGAATT-3′;  were seeded in a 4-well slide chamber for 24 h and starved overnight, pre-treated with IL-32θA94V (100 ng/mL) for 1 h, followed by 6 h treatment with TNF-α (10 ng/mL). The THP-1 cells were labeled with 5 µM calcein-AM  for 30 min in RPMI-1640 without FBS. The THP-1 cells labeled with calcein-AM were added to the 4-well slide chamber containing the HUVECs and incubated for 30 min in RPMI-1640 containing 10% FBS. Subsequently, unbound monocytes were removed by three washes with PBS. Remaining monocytes were determined using a fluorescence microscope. The intensity of fluorescence was measured using ImageJ software version 1.5 (HUVEC cells (1.5 × 10sion 1.5 .2.125 cells/dish) were seeded in a cell culture dish for 24 h, pre-treated with IL-32θA94V (100 ng/mL) for 1 h, and treated with TNF-α (10 ng/mL) for 30 min before harvesting and fractionating using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. Equal quantities of protein from these extracts (50 μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The subsequent steps for the procedure were followed as described for Western blotting above. PARP was used as a nuclear protein marker.HUVEC cells , a well-known integrin-mediated signaling molecule, which is an upstream molecule of protein kinase B (AKT) and c-Jun N-terminal kinase (JNK). IL-32θA94V also downregulated phosphorylation levels of AKT and JNK by inhibiting FAK, as expected Figure 73.6TNF-α stimulation activates the FAK/AKT signaling pathway, leading to nuclear translocation of NF-κB (p65/p50) via phosphorylation of IκB. In addition, activation of JNK accelerates nuclear translocation of AP-1 (c-fos/c-jun). These transcription factors promote the expression of ICAM-1 and VCAM-1 , 53. We 4IL-32 is involved in various cell functions such as apoptosis, differentiation, viral infection, and modulation of inflammatory cytokines, indicating that it is a cytokine that plays key roles in several diseases. However, the role of IL-32 isoforms can vary depending on experimental conditions such as cell lines or diseases. Previous studies on IL-32 suggest the necessity to define the various functions of IL-32 isoforms. For example, IL-32α and IL-32β are cytokines with both pro- and anti-inflammatory properties –57. IL-3Recently, an IL-32θA94V mutant was discovered in the tissues from a patient with breast cancer, and IL-32θA94V was found to suppress the expression of pro-inflammatory cytokines in breast cancer cells . Wild tyRosetta, one of the strains of E. coli, by the heat-shock method. The insert DNA contained a His-tag for purification. We obtained pure IL-32θA94V using sequential purification of Ni-NTA column and an IL-32 mAb KU32-52 coupled CNBr-activated Sepharose 4B column. Purified proteins were confirmed by SDS-PAGE (We designed a recombinant vector using Nde1 and Age1 restriction enzymes (Most IL-32 isoforms, including IL-32θA94V, have an RGD motif, and integrin αVβ3 and αVβ6 are known to bind to the RGD motif. In addition, it was reported that these integrins bind to IL-32α and IL-32β . Thus, wRT-PCR analyses confirmed the expression of integrins αVβ3 and αVβ6 in HUVEC cells but not in THP-1 human monocytic cells. We also demonstrated that TNF-α-induced upregulation of ICAM-1 and VCAM-1 expression was significantly decreased in IL-32θA94V-pretreated HUVECs 5. TheseWe confirmed that monocyte–endothelial adhesion was mediated by ICAM-1 and VCAM-1 using siRNA transfection (FAK is a well-known integrin-mediated signaling molecule, which is closely involved in intracellular signals induced by various cytokines and growth factors . Phosphoin vivo models. Moreover, the possibility of other unidentified cell surface receptors for IL-32 should be investigated. In addition, comparison studies of IL-32θA94V with other isoforms should be performed under various conditions. These studies are important for understanding IL-32θ and its mutant, as well as the overall function of IL-32.Increasing evidence suggests that ICAM-1 and VCAM-1 enhance the inflammatory response and are involved in various diseases –72. It hThe original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.J-YP and H-MP performed the experiments and wrote the main manuscript text. SK and K-BJ contributed to the analysis and interpretation of the data. C-ML reviewed and revised the manuscript. D-YY and JH supervised the experiments, contributed to the interpreted the results, edited the manuscript. All authors contributed to the article and approved the final submitted version.


Gαi1 transcripts’ number in NPC tissues is significantly higher than that in the normal nasal epithelial tissues. Its overexpression correlates with poor survival in certain NPC patients. Moreover, Gαi1 is significantly upregulated in NPC tissues of local primary patients and in different primary human NPC cells. Whereas its expression is relatively low in cancer-surrounding normal tissues and in primary nasal epithelial cells. Genetic silencing (via shRNA strategy) or knockout (via CRISPR-sgRNA method) of Gαi1 substantially suppressed viability, proliferation, cell cycle progression, and migration in primary NPC cells, causing significant caspase-apoptosis activation. Contrarily, ectopic Gαi1 expression exerted pro-tumorigenic activity and strengthened cell proliferation and migration in primary NPC cells. Gαi1 is important for Akt-mTOR activation in NPC cells. Akt-S6K phosphorylation was downregulated after Gαi1 shRNA or KO in primary NPC cells, but strengthened following Gαi1 overexpression. In Gαi1-silenced primary NPC cells, a S473D constitutively-active mutant Akt1 (caAkt1) restored Akt-S6K phosphorylation and ameliorated Gαi1 shRNA-induced proliferation inhibition, migration reduction and apoptosis. Bioinformatics analyses proposed zinc finger protein 384 (ZNF384) as a potential transcription factor of Gαi1. In primary NPC cells, ZNF384 shRNA or knockout (via CRISPR-sgRNA method) decreased Gαi1 mRNA and protein expression, whereas ZNF384 overexpression upregulated it. Importantly, there was an increased binding between ZNF384 protein and the Gαi1 promoter in human NPC tissues and different NPC cells. In vivo studies showed that intratumoral injection of Gαi1-shRNA-expressing adeno-associated virus (AAV) impeded subcutaneous NPC xenograft growth in nude mice. Gαi1 downregulation, Akt-mTOR inactivation, and apoptosis induction were detected in Gαi1-silenced NPC xenograft tissues. Gαi1 KO also effectively inhibited the growth of NPC xenografts in nude mice. Together, overexpressed Gαi1 exerts pro-tumorigenic activity in NPC possibly by promoting Akt-mTOR activation.The current study tested the expression and potential functions of Gαi1 in nasopharyngeal carcinoma (NPC). The Cancer Genome Atlas (TCGA) database results demonstrate that  Nasopharyngeal carcinoma (NPC), a common malignancy in nasopharynx epithelia, demonstrates diverse etiopathogy and histopathology , 2. NPC Gαi family proteins  have thret al., discovered that upregulation of Gαi1 was important for gastric cancer growth  .The primary NPC cells were first infected with the lentivirus encoding the Cas9-expressing construct (from Dr. Cao), and stable cells formed after selection using puromycin. The Cas9-expresing NPC cells were then infected with lentivirus with the CRISPR-Gαi1-KO construct [containing small guide RNA (sgRNA) against human  Dr. Cao ) and staThe lentiviral particles containing the S473D constitutively active mutant Akt1 (caAkt1) were provided by Dr. Chen  and wereVerified siRNAs against different transcription factors  were provided by Genechem  and each (at the concentration of 200 nM) was individually transfected to NPC cells using Lipofectamine 3000. The transfection was repeated once at 24 h and stopped at 48 h. At least 70% reduction of targeted mRNA was achieved by each utilized siRNA. The control cells were transfected with non-sense control siRNA (siC).Gαi1 promoter in the JASPAR database was thereafter tested by quantitative PCR (qPCR).As described , fresh tWestern blotting, quantitative real-time PCR (qRT-PCR), cell viability CCK-8 assay, nuclear EdU staining of cell proliferation, “Transwell” in vitro migration, the Caspase-3 activity assay, JC-1 assaying of mitochondrial depolarization, Histone DNA ELISA and nuclear TUNEL staining of cell apoptosis were described in detail in our previous study .s.c.) injected into the flanks of the nude mice and pNPC-1 xenografts were established after three weeks. The xenograft-bearing mice were then assigned into two groups randomly with ten mice in each group. The mice were then subject to intratumoral injection of the designated adeno-associated virus (aav). Virus was injected twice . The protocols were approved by the Ethics Committee and Institute Animal Ethics Review Board of Soochow University.The nude mice were reported previously . At six P < 0.05 indicates significant difference.The numerical data in this study were distributed normally and were always shown as mean ± standard deviation (SD). The Student’s t-test (Excel 2013) was utilized when comparing two groups. Otherwise, one-way ANOVA plus a Scheffe’ and Tukey Test (SPSS 23.0) were utilized for multiple groups’ comparison. Gαi1 (GNAI1) expression data in NPC tissues was retrieved. As shown, the number of Gαi1 mRNA transcripts in NPC tissues (“Tumor”) was significantly higher than that in normal nasopharynx epithelial tissues   database and l”) Fig. . The subges Fig. . MoreovePCs Fig. , N2-N3 NPCs Fig.  and pathPCs Fig. .Fig. 1Gαn = 20) primary NPC patients. All patients were administrated at authors’ institution [Gαi1 mRNA expression in tumor tissues was over three folds higher than that in adjacent normal tissues   and matched adjacent normal nasopharynx epithelial tissues (“N”) from twenty  Fig. .Fig. 2GαGαi1 mRNA and protein expression was relatively low  Gαi1 shRNA Fig.  and protRNA Fig. . WhereasRNA Fig. . KnockdoRNA Fig. . Cell viRNA Fig. . MoreoveRNA Fig. . In addiRNA Fig.  was signRNA Fig.  and pNPCRNA Fig. .Fig. 3GαGαi1 mRNA expression was downregulated in the primary NPC cells with Gαi1-shRNA-s2  Gαi1, the CRSIPR/Cas9 strategy was employed. Specifically, to the Cas-9-expressing pNPC-1 cells, the CRISPR-Gαi1-KO construct-expressing lentivirus was added. Single stable cells were then formed via puromycin selection and lls Fig. . Whereaslls Fig. . Expresslls Fig. . With Gαlls Fig.  was subslls Fig. . In addills Fig. . TherefoGαi1 mRNA  RNA Fig.  and protRNA Fig.  expressiRNA Fig. . With GαRNA Fig. . MoreoveRNA Fig. . EctopicRNA Fig. . Next thlls Fig. . Importalls Fig. . Moreovells Fig. .Fig. 6EcGαi1 was shown to associate with both RTKs , 21, 22 To understand the relationship between Gαi1-driven NPC cell growth and Akt-mTOR cascade activation, a S473D constitutively-active mutant Akt1 (caAkt1) was stably transduced to Gαi1-silenced pNPC-1 cells  was significantly increased in NPC tissues of local patients   Fig.  and alsoN”) Fig. . These rStable silencing of ZNF460, using the same lentiviral shRNA strategy, led to moderate (less than 50%) but significant Gαi1 protein downregulation Fig. . Notablys.c. to flanks of nude mice [3 (“Day-0”). Next, pNPC-1 xenograft-bearing nude mice were randomly assigned into two groups. For the treatment group, aav-packed Gαi1-shRNA-s2 (“shGαi1-aav”) was intratumorally injected. The control group mice were intratumorally injected with aav-packed scramble control shRNA (“shC-aav”). Virus injection was repeated after 48 h (“Day-2”) and every six days tumor volumes were recorded. As shown, shGαi1-aav injection impeded the growth of pNPC-1 xenografts in nude mice and volumes were significantly lower  and the 24th day (Day-24) after initial virus injection, one pNPC-1 xenograft was isolated from each of group. Four total pNPC-1 xenografts were obtained. Part of the xenografts were cut into pieces and were homogenized. As shown, mRNA Fig.  and protFurther analyzing xenograft tissues showed that the Caspase-3 activity was augmented in shGαi1-aav-injected pNPC-1 xenograft tissues Fig. , where lTo further corroborate the role of Gαi1 in NPC cell growth in vivo, we conducted subcutaneous injections of Gαi1-KO pNPC-1 cells and control Cas9-C pNPC-1 cells into the flanks of nude mice. After an eight-week period, we isolated and measured the resulting xenografts. Gαi1-KO pNPC1 xenografts exhibited significantly smaller sizes and lower weights compared to the Cas9-C pNPC1 xenografts Fig. . The bodNPC is considered as a poor-prognosis malignancy, mainly due to the lack of characteristic clinical symptoms , 8, 9. TGαi1 transcripts’ number in NPC tissues is significantly higher than that in the nasal epithelial tissues. Its overexpression correlates with poor survival of a large number of NPC patients. Moreover, Gαi1 mRNA and protein expression is significantly elevated in NPC tissues of local patients and in primary NPC cells. Whereas its expression is relatively low in cancer-surrounding normal tissues and in nasal epithelial cells. Genetic silencing or KO of Gαi1 substantially suppressed viability, proliferation, cell cycle arrest, and migration, and induced apoptosis activation in primary NPC cells. Contrarily, ectopic Gαi1 expression in primary NPC cells exerted pro-tumorigenic activity and augmented cell proliferation and migration. In vivo studies showed that intratumoral injection of Gαi1-shRNA-expressing aav impeded subcutaneous pNPC-1 xenograft growth in nude mice. Gαi1 KO also effectively inhibited the growth of pNPC1 xenografts in nude mice. Therefore, overexpressed Gαi1 is a novel and promising therapeutic target of NPC.The results of the present study supported a key pro-cancerous role of Gαi1 in NPC. TCGA results demonstrate that Recent studies have proposed a pivotal role of Gαi1 in mediating Akt-mTOR cascade activation by a number of different stimuli , 34, 35.Here we found that Gαi1 is important for Akt-mTOR activation in NPC cells. Akt-S6K phosphorylation was downregulated with Gαi1 shRNA or KO in primary NPC cells, but was augmented after Gαi1 overexpression. In Gαi1-silenced primary NPC cells, caAkt1 restored Akt-S6K phosphorylation and largely ameliorated Gαi1 shRNA-induced proliferation inhibition, migration reduction and apoptosis. Akt-mTOR inactivation was also detected in Gαi1-silenced NPC-1 xenograft tissues. Thus, mediating Akt-mTOR activation could be a primary mechanism of Gαi1-driven NPC cell growth in vitro and in vivo see Fig. .Fig. 10TStudies have explored a possible cancer-promoting activity of the transcription factor ZNF384 in different cancers. Meng et al. showed that ZNF384 is upregulated in breast cancer and is important for cancer cell metastasis . ZNF384 Gαi1 mRNA and protein expression, whereas ZNF384 overexpression upregulated Gαi1 expression. Importantly, there was an increased binding between ZNF384 protein and the proposed Gαi1 promoter in both human NPC tissues and cells. Increased binding between the two could be the primary mechanism of Gαi1 upregulation in NPC (see Fig. One important discovery of this study is that ZNF384 could be a primary transcription factor of Gαi1 in NPC cells see Fig. . In primsee Fig. .Overexpressed Gαi1 exerts pro-tumorigenic activity in NPC possibly by promoting Akt-mTOR activation.Supplementary Figuresaj-checklist form


The development of an integrin αvβ6-selective PDC was achieved by combining the therapeuticefficacy of the cytotoxic drug monomethyl auristatin E with the selectivityof the αvβ6-binding peptide (αvβ6-BP) and with the ability of positron emissiontomography (PET) imaging by copper-64. The [64Cu]PDC-1 was produced efficiently and in high purity. The PDC exhibitedhigh human serum stability, integrin αvβ6-selective internalization, cell binding, and cytotoxicity.Integrin αvβ6-selective tumor accumulationof the [64Cu]PDC-1 was visualized with PET-imagingand corroborated by biodistribution, and [64Cu]PDC-1 showed promising in vivo pharmacokinetics. The [natCu]PDC-1 treatment resulted in prolonged survival ofmice bearing αvβ6 (+) tumors tumor group 49 days, and all other control groups 37 days).Many anticancer drugs exhibit high systemic off-targettoxicitiescausing severe side effects. Peptide–drug conjugates (PDCs)that target tumor-specific receptors such as integrin α Since 2019 only 9 ADCs have been FDA approved,including brentuximab vedotin, enfortumab vedotin, and polatuzumabvedotin, which are conjugated to monomethyl auristatin E (MMAE),6 while no PDC has yet gained regulatory approval.8 AlthoughADCs have demonstrated great promise, several challenges remain, notablythe controlled site-specific chemical conjugation of the drug to theantibody, which often leads to ADC instability, poor antibody targetaffinity, and purification challenges.4 In addition, their largesize can result in poor tumor penetration and long blood residencetimes, thereby further increasing systemic toxicity.4 To overcome some of these limitations, peptides have been investigatedas delivery vehicles for the delivery of cytotoxic agents. Peptidesare relatively easily synthesized by solid-phase peptide synthesis(SPPS), can be prepared in large quantities, and are readily modifiedto fine-tune affinity, selectivity, stability, and pharmacokinetics.9 The ease of modification makes them an idealplatform as a PDC, and their smaller size permits better tumor penetrationand a shorter blood residence time which can reduce systemic toxicity.Many of the current cancer treatment optionsare non-targeted andlack selectivity, affecting both the cancer and normal tissue.11 Recently, the integrin αvβ6 has garnered much attention as a target for boththe detection as well as the treatment of cancers. The integrin αvβ6 is an epithelial-specific cell surfacereceptor with low-to-no expression on healthy adult epithelium, butis highly overexpressed in many cancers, including some of the mostlethal malignancies such as pancreatic cancer.14 Studies haveshown that the integrin αvβ6 playsa key role in carcinogenesis, where it is involved in cellular invasion,migration, angiogenesis, and adhesion to the extracellular matrix.15 Importantly, it has been identified as a prognosticindicator, with high expression level correlating to poor prognosisand overall survival for patients.15 Consequently,our group has developed and extensively studied the integrin αvβ6-binding peptide (αvβ6-BP), a peptide with nanomolar affinity and highly selectivebinding to integrin αvβ6. The fluorine-18-labeledαvβ6-BP was translated into theclinic to detect tumors in patients with breast, colon, lung, andpancreas cancer.16 The αvβ6-BP rapidly binds to and is internalized intoαvβ6-expressing cells,19 and therefore, we now propose to use it as a chaperone for the selectivedelivery of the highly potent cytotoxic agent MMAE.Many tumor-specific cell surface receptors have been identifiedas therapeutic targets, among them the integrins which are a familyof cell surface receptors that are involved in cell migration andinvasion.vβ6 tumor-targeting peptide (αvβ6-BP), (2) the cancer-specific cathepsincleavable linker maleimide-PEG2-valine-citrulline-para-aminobenzylcarbamate ,23 (3) the cytotoxic drug MMAE,4 and (4) a 2,2′,2″,2‴-tetraaceticacid (DOTA) chelator for copper-64 chelation. The PDC was evaluatedfor integrin αvβ6-affinity by ELISA.It was radiolabeled with copper-64 to yield [64Cu]PDC-1 which was tested for stability in mouse and human serum, cell binding and internalization studiesusing the melanoma cell lines DX3puroβ6 (+) and DX3puro (−),and the pancreatic cell lines BxPC-3 (+) and MIA PaCa-2 (−).Cytotoxicity was tested by WST-1 assay, and apoptosis was correlatedto caspase-3/7 activity. In vivo (PET/CT and biodistribution) of [64Cu]PDC-1 was done in a paired DX3puroβ6/DX3puroas well as a BxPC-3 xenograft tumor mouse model. Therapeutic efficacyof [natCu]PDC-1 was evaluated in mice bearingeither DX3puroβ6 or DX3puro xenograft tumors.The designof the PDC 1 incorpovβ6-BP was modified to contain a cysteine for conjugationof the MMAE-maleimide linker , in a 1:1 ratio of MMAE-per-peptide, in >99%purity after HPLC purification. The analytical data are: NH2-2, HPLC retention time (RT) = 17.13 min; matrix assistedlaser desorption ionization time of flight (MALDI-TOF) m/z: calcd for C214H404N37O86S [M + H]+ 4901.8126; found, 4901.8124; DOTA-2, HPLC RT = 17.17 min; MALDI-TOF m/z: calcd for C230H430N41O93S [M + H]+ 5287.9927; found, 5287.9900 ; and [natCu]2,HPLC RT = 17.82 min; MALDI-TOF m/z: calcd for C230H429CuN41NaO93S [M + Na]+ 5373.9076; found, 5373.8769 . The analytical data of thePDCs are: NH2-PDC-1, HPLC RT = 19.13 min;MALDI-TOF m/z: calcd for C286H516N49O104S [M + H]+ 6337.5725; found, 6337.5759 ; DOTA-PDC-1, HPLC RT = 19.02 min; MALDI-TOF m/z: calcd for C302H541N53NaO111S [M + Na]+ 6743.7998;found, 6743.7885 ;and [natCu]PDC-1, HPLC RT = 19.58 min; MALDI-TOF m/z: calcd for C302H541CuN53O111S [M + H]+ 6783.7396; found,6783.7199 . Radiolabelingwith [64Cu]CuCl2 generated [64Cu]2 and [64Cu]PDC-1 in nearly quantitativeyields (≥99%) in a molar activity of 18.5 GBq/μmol withhigh radiochemical purity of ≥98%  .The αe linker 1 and N-t50) of DOTA-PDC-1 against biotinylated latency associated peptide for integrinαvβ6 was evaluated by competitiveELISA and determined to be IC50 = 18 ± 2 nM, demonstratingthat the affinity was not affected by the DOTA-C-MMAE-linker modifications(IC50 [DOTA-αvβ6-BP]= 28 ± 3 nM).26The half-maximum inhibitory concentration ;degradation was more rapid in mouse serum  and BxPC-3 (+) was reducedby adding increasing amounts of DOTA-PDC-1, illustratingthat the αvβ6-selective uptake couldbe blocked . Internalizationof [64Cu]-PDC-1 into cells was high with >50%of the bound radioactivity internalized for all cells expressing theintegrin αvβ6. In comparison, cellbinding of [64Cu]2 to DX3puroβ6 (+)cells was 58.8 ± 2.3, and 3.3 ± 0.4% to the DX3puro (−)cells.Cell bindingof [-3 cells2B. Bindi2-PDC-1 and [natCu]PDC-1 exhibited integrinαvβ6-dependent cytotoxicity, onlyreducing cell viability of the αvβ6-positive cells (natCu]PDC-1 high cytotoxicity was observed in DX3puroβ6 (+) cells(EC50: 0.058 ± 0.003 nM) with no observable cytotoxiceffects in the DX3puro (−) cells, while free MMAE had almostequal cytotoxicity to both DX3puroβ6 (+) and DX3puro cells (−) showed an αvβ6-dependent increase in caspase-3/7 activity:for the DX3puroβ6 (+) and DX3puro (−) pair it resultedin a >5 times higher activity at 24 h for the DX3puroβ6 cells,with no observed change for the DX3puro cells  and [natCu]2 (black), showed no effect on caspase-3/7 activity in all cell linesand were indistinguishable from untreated cells (yellow). Notably,a slight increase in caspase-3/7 activity was observed for the MIAPaCa-2 (−) cells when treated with the [natCu]PDC-1 (2-PDC-1 (red) resultedin no significant increase of caspase-3/7 activity at any time point and BxPC-3 (+) tumors by positron emission tomography (PET) imaging,along with no observable uptake in the DX3puro (−) tumor  tumor at 4 h vs 0.56 ±0.12% ID/g in the DX3puro (−) tumor . The BxPC-3 (+) tumor also exhibited a similarlyhigh accumulation . Moderate tumor washout was observed at later time pointsfor both tumor models; it did reach significance at 48 h for the DX3puroβ6tumor . For the BxPC-3 tumor, the uptake went from4.61 ± 1.44% ID/g at 4 h to 3.73 ± 0.44 and 2.93 ±0.80% ID/g, at 24 and 48 h, respectively  10min prior to administration of [64Cu]PDC-1, resulting in 87–91% reduced uptake in the αvβ6 (+) tumors down to the level of the DX3puro (−)tumor ,thus demonstrating integrin αvβ6-selective targeting in vivo . Clearance from the bloodwas rapid, resulting in αvβ6 (+)tumor/blood ratios of ≥32:1 at 4 h (Table S5). [64Cu]PDC-1 primarily clearedthrough the kidneys, from 50 to 64% ID/g at 4 h to ≤25% ID/gat 48 h ([−) tumor 5. The PE−) tumor 5. The binatCu]PDC-1 had slower tumor growth compared to the groups receivingsaline, non-drug bearing peptide [natCu]2,or free, non-targeted MMAE tumors mean tumor volume was >4timessmaller than all other treatment groups . All mice in the groups treated with saline, non-drugbearing peptide [natCu]2, or free, non-targetedMMAE had met an end point criterion (≥2 cm in any directionand/or tumor ulceration) by 56 days, 70 days, and 64 days from startof treatment, respectively, with all these groups having the samemedian survival of 37 days  tumors treated with [natCu]PDC-1 had a median survival of 77 days, and a 20% survival atthe end of the study .Mice treated with PDC-1 which enabled the quantitative assessment of cell binding,internalization, and in vivo pharmacokinetics.MMAE inhibits tubulin assembly with cytotoxicactivity in the picomolarrange and is extremely lipophilic, preventing its use as a therapydue to high systemic toxicity.64Cu]PDC-1 demonstrated integrin αvβ6 receptor selective binding and internalizationin vitro. [natCu]PDC-1 also demonstrated integrinαvβ6 selective cytotoxicity; forexample, the DX3puroβ6 cells, having the highest integrin αvβ6 expression, had an EC50 = 0.058± 0.003 nM, the intermediate integrin αvβ6-expressing BxPC-3 had an EC50 = 65.1 ± 10.6nM, the low expressing MIA PaCa-2 cells showed low cytotoxicity (EC50 > 250 nM) and the non-expressing DX3puro cells exhibitedno observable cytotoxic effects. In contrast, the free, non-targetedMMAE was highly cytotoxic to all cells, having an EC50 of0.14–0.5 nM. The in vitro efficacy of [natCu]PDC-1 was comparable to the integrin αvβ6-targeting PDC containing the cytotoxic drug tesirine  that was previously reported to have an EC50 =4.19–5.37 nM in αvβ6-expressingcells, including in the engineered melanoma cell line A375Pβ6and the pancreatic Capan-1 .42 Thetesirine-PDC (SG3299), when compared to the non-targeting scrambledPDC, tesirine-PDC (SG3511), provided a 15:1 ratio for selective cytotoxicitytoward A375Pβ6 (+) melanoma cells, but the targeting tesirine-PDC(SG3299) also had relatively high cytotoxicity to αvβ6-null engineered melanoma cells A375Ppuro andPanc-1 cells .42 By comparison,in the present study, NH2-PDC-1 was >31-foldand [natCu]PDC-1 was >86-fold more cytotoxictoward the melanoma DX3puroβ6 (+) than the DX3puro (−)cells. Other integrin αvβ3 and αvβ5 targeting camptothecin (CPT) PDCs haveshown less favorable in vitro efficacy of EC50 = 0.16–27μM,34 with some integrin αvβ3 targeting α-amanitin-PDCs exhibitingnon-selective cytotoxicity.41 Piarulliet al. showed that MMAE-PDCs targeting integrin αvβ3 produced cytotoxicities with EC50 =11–400 nM, concluding they had a promising candidate for invivo experiments to obtain evidence of accumulation at the tumor site.40PDC-1 with PET,which demonstrated integrin αvβ6-selective uptake in tumors that was corroborated by biodistributionstudies  4.46 ± 0.91; BxPC-3(+) 4.61 ± 1.44; DX3puro (−) 0.56 ± 0.12). Wang etal. described a similar radiolabeling approach with copper-64 to imageintegrin αvβ3-targeted deliveryof a bicyclic-RGD peptide, CDCRGDCFC (RGD4C), linked to the proteintumor necrosis factor (TNF) as the therapeutic agent.32 They demonstrated TNF-PDC accumulation in an MDA-MB-435breast cancer xenograft tumor model (3.94 ± 0.48% ID/g at 4 h),and approximately double that uptake in a higher αvβ3-expressing glioblastoma U87MG xenograft mousemodel (8.11 ± 0.88% ID/g at 4 h); however, high liver accumulationof 16.22 ± 1.46% ID/g at 20 h was also observed.32 For [64Cu]PDC-1, minimal liveraccumulation was observed . Although the preliminary results are promising, the [64Cu]PDC-1 pharmacokinetic profile still has its limitationsof fast clearance, moderate tumor accumulation with some washout,and some off-target uptake in the gastrointestinal tract and highkidney accumulation 41.6% ID/g at 24 h).Indeed, few studies show biodistributiondata for the PDCs, withlimited examples including tritium or iodine-125 radiolabeled PDCs;however, these have limitations for noninvasive imaging and tracking.vβ6 targeting,the PDC-1 was further evaluated for therapeutic efficacy.To permit a direct side-by-side comparison, this was done with DX3puroβ6(+) or DX3puro (−) tumor bearing mice. Treatment with [natCu]PDC-1 suppressed DX3puroβ6 (+) tumorgrowth and prolonged median survival to 77 days, compared to 49 daysfor the DX3puro (−) tumor-bearing mice, and >2-fold longerthan other treatment groups . The [natCu]PDC-1 treated DX3puroβ6tumor cohort had 20% remaining alive at the end of the study (122days). Notably, the [natCu]PDC-1 treatmentdid not cause adverse systemic side-effects when administered fourtimes at 6 mg/kg (0.9 μmol/kg), as the mice maintained healthybody weight during the course of the study. This concentration correspondsto 0.64 mg/kg of free MMAE, i.e., close to the LD50 forfree MMAE of 1 mg/kg (1.4 μmol/kg),51 thus highlighting the successful administration of a highly cytotoxicagent safely as part of a targeted PDC at concentrations that wouldbe systemically toxic when administered alone.Building on the encouragingin vitro and in vivo data suggestingselective integrin α64Cu]PDC-1 by combiningthe highly cytotoxic drug MMAE with the highly selective integrinαvβ6-BP, with the goal to reduceoff-target toxicity of the drug whilst retaining therapeutic efficacy.In vitro testing demonstrated integrin αvβ6-dependent binding, internalization, and cytotoxicity withhigh stability in human serum at 37 °C. PET/CT imaging of [64Cu]PDC-1 showed integrin αvβ6-selective tumor accumulation and visualization,and the biodistribution confirmed a favorable pharmacokinetic profilewith rapid blood clearance and renal excretion. In vivo therapeuticefficacy studies displayed >2-fold improved overall survival ofmicebearing DX3puroβ6 (αvβ6 +)tumors compared to the control groups. Different dosing regimens arecurrently under evaluation with the goal to develop a highly effective,integrin αvβ6-targeted PDC therapeuticfor a wide range of carcinomas.We developed the CuCl2 (0.0167GBq) in 1.0 M ammonium acetate buffer , and reacted at 37 °C for 30 min. DOTA-PDC-1  was dissolved in metal free water(120 μL) and added to a solution of [64Cu]CuCl2 (0.333 GBq) in 1.0 M ammonium acetate buffer , and reacted at 37 °C for 30 minat a molar activity of 18.5 GBq/μmol. For analysis, an aliquotof the reaction mixture  was quenchedwith 0.1 M ethylenediaminetetraacetic acid , radiochemicalpurity analyzed by analytical RP-HPLC, and identity confirmed by co-injectionwith non-radioactive [natCu]2 or [natCu]PDC-1, respectively. Both [64Cu]2 and [64Cu]PDC-1 were obtained in≥98% radiochemical purity and used for formulation withoutfurther purification.DOTA-64Cu]PDC-1  and incubated at 37 °C.At each time point , an aliquot (50–200 μL)was taken, proteins precipitated with absolute ethanol and removedby centrifugation at 1500g for 4 min. The ethanolsolution was diluted with water (1 mL) and analyzed by RP-HPLC.Mouse serum or human serum (0.5 mL)was combined with an aliquot of 2, MMAE , NH2-PDC-1, or [natCu]PDC-1 in DX3puroβ6(+) and DX3puro (−) cells at variable concentrations up to5 nM, and in BxPC-3 (+) and MIA PaCa-2 (−) cells at variableconcentrations up to 250 nM, or with MMAE at concentrations up to10 nM. Cells were seeded in a 96 well plate at a density of 6000 cells/wellfor DX3puroβ6 and DX3puro, and at a density of 10,000 cells/wellfor BxPC-3 and MIA PaCa-2. DMEM media was used for all cells exceptfor BxPC-3 (RPMI 1640 media). Cells (n = 6–8wells/cell type/compound) were treated with different concentrationsof NH2-2, [natCu]2, MMAE, NH2-PDC-1, or [natCu]PDC-1 dissolved in the respective media, as well as their respectivemedia (no treatment) for 48 h , afterwhich the media was removed; cells were washed twice with media (200μL) and re-incubated in media  for 24 h. The media was then removed and the WST-1 reagent wasadded to each well, and the cells were incubated for 2 h at 37 °C.The 96 well plates were read at 450 nm by a Multiscan Ascent microplatereader. The percent cell viability was normalized to untreated cells(set as 100% viability) for each cell line.Cell viability was measuredafter treatment with either NH2). DX3puroβ6 and DX3puro cells weretreated with 1 nM of MMAE  or 0.625 nM ofthe other compounds: NH2-2, [natCu]2, NH2-PDC-1, or [natCu]PDC-1. BxPC-3 and MIA PaCa-2 cells were treated with10 nM of MMAE or 250 nM of the other compounds: NH2-2, [natCu]2, NH2-PDC-1, or [natCu]PDC-1. All cells weretreated with 100 nM of staurosporine as a positive control.24 Untreated cells (media) were used as a measureof endogenous caspase-3/7 activity . Cells were treated(n = 4/cell line/compound/time) for 24, 48, or 72h  prior to washing. After treatment,the media was removed, cells were washed, and Caspase-Glo 3/7 reagentwas added, and incubated for 1 h at room temperature. Caspase-3/7activity was analyzed by measuring luminescence with a FluoroskanFL microplate reader according to the manufacturer’s protocol.Caspase-3/7 activity wasanalyzed using an ApoTox-Glo Triplex Assay kit. Cells were seededin a 96 well plate at the same density and using the same respectivemedia as described for the WST-1 assay and incubated overnight . Studies commenced once tumors reached a maximum diameter of∼0.5 cm, approximately 3 weeks after inoculation. Food andwater were available ad libitum. [64Cu]PDC-1 was formulated in isotonic 0.9% saline to pH = 7.2 and administeredintravenously (i.v.) via a catheter into the tail vein.All animal procedures conformed tothe Animal Welfare Act and were approved by the University of CaliforniaDavis Institutional Animal Care and Use Committee. All mice used forin vivo work were female athymic nude mice (6–8 weeks old)purchased from Charles River Laboratories . For PETimaging and biodistribution studies, female athymic nude mice (6–8weeks old) were injected subcutaneously with 3 × 1064Cu]PDC-1 in isotonic 0.9% saline were injected intravenously (i.v.) via a catheter into the tail veinof mice (n = 4/tumor model) anesthetized with 2–3%isoflurane in medical grade oxygen. Following a conscious uptake period,animals were anesthetized with 2–3% isoflurane and imaged twoat a time, side by side. PET scans were acquired using an Inveon DPETscanner and CT scans using an Inveon SPECT/CT and analyzed using Inveon Research Workplace software. The mean weightsand standard deviation (SD) of the imaging mice was 26.6 ± 1.6g for the DX3puroβ6/DX3puro paired tumor model and 27.7 ±3.7 g for the pancreatic BxPC-3 tumor model.Aliquots of the formulated [64Cu]PDC-1 in isotonic 0.9% saline were injected i.v. as described above. Following the conscious uptakeperiod, the mice were anesthetized (5% isoflurane), euthanized, anddissected . Tissues were collected, washed, weighed, andradioactivity measured in a γ-counter. Calibrated, decay-correctedradioactivity was expressed as the percentage of injected dose pergram of tissue (% ID/g). Data are reported as mean ± SD. Themean weights and standard deviation of the DX3puroβ6/DX3puropaired tumor model biodistribution mice were 24.3 ± 1.4 g at4 h, 28.2 ± 2.9 g at 24 h, and 27.7 ± 2.6 g at 48 h. Themean weights and standard deviation of the BxPC-3 tumor model biodistributionmice were 25.1 ± 1.9 g at 4 h, 27.0 ± 1.1 g at 24 h and27.1 ± 3.2 g at 48 h. For in vivo blocking studies, DOTA-2 was injected i.v. into two animals/tumor model 10 min prior to [64Cu]PDC-1. The animals were sacrificed after4 h, tissues collected, washed, weighed, and radioactivity measuredin a γ-counter. The mean weights and standard deviation forthe blocking mice were 24.6 ± 0 and 27.9 ± 0.3 g for theDX3puroβ6/DX3puro paired and BxPC-3 tumor models, respectively.Aliquots of the formulated 2), 6 mg/kg, 1.12μmol/kg), (3) non-targeted drug ,or (4) PDC .Dosing of MMAE was 0.3 mg/kg as per maximum dose with no physiologicalresponse.25 The [natCu]PDC-1 treatment groups consisted of n = 10/tumormodel, while all other groups  consisted of n = 4/tumor model. Allgroups received four doses  via i.v. tailvein injection of the above dose dissolved in saline (100 μL).The mean weights and standard deviation of each group was 25.9 ±1.2 g , 25.6 ± 1.5 g ([natCu]2), 25.8 ± 2.5 g (MMAE), 24.9 ± 1.9 g , and 25.7 ± 2.0 g  at day 0. Tumor volumes andbody weights (to assess possible systemic toxicity) were measuredstarting on day 0, and once a week thereafter until the end of thestudy. Tumor volume (V) was determined according to the equation V = (π/6) × L × W × H, where L isthe longest axis, W is the axis perpendicular to L, and H is perpendicular to the planeof L and W. End point determinationcriteria were: any axis >2 cm, active ulceration, or compromisedhealthof the mouse (>20% loss of body weight from the start of the study).All data are represented as the mean ± SD and are plotted beginningat day 0. Survival curves were determined by Kaplan–Meier method.Tumor xenografts were established bysubcutaneous injection of either DX3puroβ6 or DX3puro cells at 95% confidence interval. A P-value of ≤0.05 was considered statistically significant.Quantitative data are reportedas mean ± SD. Statistical significance was determined with pairedtwo-tailed Student’s


Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) coined by inflammation and neurodegeneration. The actual cause of the neurodegenerative component of the disease is however unclear. We investigated here the direct and differential effects of inflammatory mediators on human neurons. We used embryonic stem cell-derived (H9) human neuronal stem cells (hNSC) to generate neuronal cultures. Neurons were subsequently treated with tumour necrosis factor alpha (TNFα), interferon gamma (IFNγ), granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin 17A (IL-17A) and interleukin 10 (IL-10) separately or in combination. Immunofluorescence staining and quantitative polymerase chain reaction (qPCR) were used to assess cytokine receptor expression, cell integrity and transcriptomic changes upon treatment. H9-hNSC-derived neurons expressed cytokine receptors for IFNγ, TNFα, IL-10 and IL-17A. Neuronal exposure to these cytokines resulted in differential effects on neurite integrity parameters with a clear decrease for TNFα- and GM-CSF-treated neurons. The combinatorial treatment with IL-17A/IFNγ or IL-17A/TNFα induced a more pronounced effect on neurite integrity. Furthermore, combinatorial treatments with two cytokines induced several key signalling pathways, i.e. NFκB-, hedgehog and oxidative stress signalling, stronger than any of the cytokines alone. This work supports the idea of immune-neuronal crosstalk and the need to focus on the potential role of inflammatory cytokines on neuronal cytoarchitecture and function.The online version contains supplementary material available at 10.1007/s11481-023-10059-w. Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by both inflammation and neurodegeneration, which can lead to a variety of clinical disabilities such as motor, sensory and cognitive symptoms  containing 1 × B-27 supplement and 1% Pen Strep (Gibco) for neuronal maintenance. Cells were filtered before reseeding to remove any remaining stem cell conglomerates using a 100 µm filter . Cells were cultivated another 7 days in Neurobasal medium before the experimental stage.We used commercially available embryonic stem cell-derived (H9) human neuronal stem cells  for the differentiation to neuronal cultures. hNSC were seeded on Geltrex™ (Gibco) coated wells in 2% StemPro Serum-Free Human Neural Stem Cell Culture Medium (ThermoFisher) supplemented with 2 mM GlutaMAX (Gibco), 20 ng/ml epidermal growth factor , 20 ng/ml basic fibroblast growth factor  and 2% StemPro Neural Supplement (Gibco) and 1% Pen Strep (Gibco). Medium was changed every day for 5 days. After 5 days, neuronal differentiation was started by adding DMEM/F12 medium (Gibco) with 1 × B-27 supplement (Gibco), 10 ng/ml brain-derived neurotrophic factor  and 10 ng/ml neurotrophin 3  and 1% Pen Strep (Gibco). Medium was changed every other day for 14 days. After two weeks, cells were reseeded at a density of 2.6 × 10Neuronal cell cultures were treated with IL-17, IL-10, IFNγ, TNFα, GM-CSF , l-glutamate  or staurosporine  for 24 h respectively. Concentrations of cytokines were selected based on assumed local concentrations in the CNS of PwMS as described in previous studies  in phosphate-buffered saline (PBS)) and permeabilized  for immunofluorescence staining. Monoclonal antibodies against Class III ß-tubulin , microtubule-associated protein 2 , IL-17RA , IL-10RB , TNFR1  or IFNGR1  were added, and cells were incubated for 1 h at room temperature. Afterwards, matching secondary antibodies (Alexa Fluor™ 594 goat anti-mouse IgG(H + L) A11032, Alexa Fluor™ 488 goat anti-mouse IgG(H + L) A11001, Alexa Fluor™ 488 donkey anti-rabbit IgG(H + L) A21206, Alexa Fluor™ 594 goat anti-rabbit IgG(H + L) A11012, all from Invitrogen) each in a dilution of 1:1000 were applied respectively for 1 h at room temperature and DAPI  was used for nuclear staining. Primary antibodies were used at the following concentrations in 1% BSA/PBS upon application: mouse anti hTUBB3 (1:250), rabbit anti hMAP-2 (1:100), anti hIL-17RA (1:100), anti hIL-10RB (1:100), anti hTNFR1 (1:100), anti hIFNGR1 (1:100).Immunofluorescence images were acquired with 20 × or 40 × magnification with a fluorescence microscope  and LAS X Life science software (Leica) using the same microscope settings  for each experiment or test series. Images were assessed using the FilamentTracer algorithm of the commercially available IMARIS® software, which allows semi-automatic detection, tracing and measurement of neuronal cells and their processes. The software evaluates neurite features such as neurite area, neurite length, neurite diameter and neurite branches. We calculated neurite markers relative to the number of cell nuclei as ratio of a given neurite marker per nucleus.2+ imaging of neuronal cultures was performed on black 96-well plates (Ibidi), loaded with 1 µM Fluo-4AM (ThermoFischer Scientific) in Neurobasal Medium without phenol red (Gibco) for 15 min at 37 °C. Fluorescence imaging was performed on an inverted cell^R microscope (Olympus) within an incubation chamber at 37 °C and 5% CO2. Recording was conducted at 5 Hz for 5 min at 512 × 512 pixel resolution using cellSens Imaging Software. ImageJ was used for further processing.CaCytokine receptor expression analyses on untreated neurons were conducted using rt-qPCR in four to five independent experiments Table . We used2 Profiler™ PCR Array . Total RNA of two independent experiments containing 300,000–500,000 cells per condition and experiment was isolated with Quick-RNA™ MicroPrep (Zymo Research Europe) and isolated RNA was transcribed to cDNA using the RT2 First Strand Kit (Qiagen) according to the manufacturer’s protocol. RT2 Profiler™ PCR Array was performed with RT2 SYBR® Green qPCR Mastermix (Qiagen) according to the instructions using QuantStudio™ 5 Real-Time PCR System (Applied Biosystems). qPCR array data were normalization against five housekeeping genes  and relative quantification (RQ) was calculated using the ΔΔCt-Method. R was used for data presentation as a heatmap. Please note: Expression of the following genes could not be analyzed due to a lack of expression either in control samples or in all samples: CA9 (hypoxia), FABP1 (PPAR), OLR1 (PPAR), BMP2 (hedgehog), WNT1 (hedgehog), WNT3A (hedgehog), WNT6 (hedgehog), SOCS3 (JAK-STAT), IRF1 (JAK-STAT), BCL2A (NFκB), BIRC3 (NFκB), IFNG (NFκB), TNF (NFκB), MMP7 (Wnt), WISP1 (Wnt).Expression of 84 genes associated with ten different signal transduction pathways in neurons incubated with IL-17, TNFα, IFNγ, IL-17/ TNFα and IL-17/IFNγ was analyzed using the Human Signal Transduction PathwayFinder™ RTMAP2, TUBB3 and further genes characteristic for different types of mature neuronal cells such as choline acetyltransferase , solute carrier 6a , tyrosine hydroxylase  and vesicular glutamate transporter . Comparing the differentiated neurons to hNSC, neurons showed a 101-fold upregulation of MAP2 (p ≤ 0.01), eightfold of TUBB3 (p ≤ 0.05) as well as a fourfold up-regulation of CHAT (p ≤ 0.05), 13-fold of SLC6A (p ≤ 0.05) and 11-fold of TH (p ≤ 0.05) suggesting a mixed group of neuronal phenotypes, predominantly of dopaminergic and serotonergic/GABAergic neurons  hNSC to mixed neuronal cell cultures. We confirmed the successful neuronal differentiation by immunofluorescence staining for the neuronal maturation markers MAP2 Fig.  and TUBBons Fig. .Fig. 1Ch2+) imaging by fluorescence microscopy of Fluo-4 AM loaded neuronal cultures was performed. Electrophysiological proof of spontaneous activity confirms the cell cultures’ functional maturity . In order to evaluate the basic potential of neurons to respond to these cytokines, we assessed the expression of cytokine receptors using immunofluorescence staining.IL-17RA (0.4-fold), IL-10RA (0.005-fold), IL-10RB (0.1-fold), TNFRSF1A (0.5-fold), TNFRSF1B (0.2-fold), IFNGR1 (0.4-fold) and IFNGR2 (0.7-fold) were expressed on a lower level and the glutamate receptors GRM1 (6.0-fold) and GRIN1 (2.8-fold) and IL-17RC (2.4-fold) on a higher level than in PBMCs  and IFNGR1 (for IFNγ) on a medium level as well as IL-17RA (for IL-17A) on a low level and IL-10RB (for IL-10) on a very low level Fig. . No distMCs Fig. . The gluFRSF1B 0.-fold, IFNext, we assessed the cellular integrity after incubation of H9-hNSC-derived neuronal cultures with cytokines and neurotoxic substances, e.g. staurosporine (apoptosis-inducing protein kinase inhibitor) or l-glutamate (excitotoxic transmitter). We compared the effect of the classical proinflammatory mediators TNFα, IFNγ and GM-CSF to IL-17A and anti-inflammatory IL-10 on neuronal cell integrity using immunofluorescence staining. For this purpose, we incubated neuronal cultures with the cytokines/mediators  for 24 h. After incubation, we performed immunofluorescence staining for TUBB3 as essential protein of the neuronal cytoskeleton. We analysed neurite integrity using the FilamentTracer algorithm of the commercially available IMARIS® software. We quantified neurite area, neurite length, neurite diameter and number of neurite branches per cell nucleus in a blinded analysis Fig. . StaurosAs a release of proinflammatory mediators is usually an orchestrated process involving several of these mediators, we were interested in whether simultaneous neuronal treatment with IL-17A and one of the well-described proinflammatory cytokines TNFα and IFNγ may synergistically enhance the effect of IL-17A on neurite integrity. Neuronal cultures were incubated with either IL-17A/TNFα or IL-17A/IFNγ  for 24 h. We additionally exposed neurons to a combination of TNFα/IFNγ for comparison. Our results show that combinatorial incubation with IL-17A had an amplified impact on neuronal integrity markers extending distinctly beyond the effect of IL-17A, IFNγ or TNFα alone. Both combinations including IL-17A triggered a significant decrease of all cell integrity markers , 105.08 (IFNγ)), CCL5 , 6.60 (IFNγ)), CSF1 , 3.50 (IFNγ)) and STAT1 ). This target gene up-regulation was further amplified upon combinatorial treatment with IL-17A particularly in combination with TNFα or combination of TNFα and IFNγ: ICAM1 RQ 162.47 (IL-17/TNFα), RQ 2066.08 (TNFα/IFNγ), CCL5 RQ 9.26 (IL-17/TNFα), RQ 3442.26 (TNFα/IFNγ), CSF1 RQ 5.48 (IL-17/TNFα), RQ 21.97 (TNFα/IFNγ) and STAT1 RQ 9.96 (IL-17/TNFα), RQ 84.08 (TNFα/IFNγ). Interestingly, IL-17A alone did not lead to an increase in target gene expression of the NFκB pathway or any other pathway investigated. However, combinatorial exposure of neurons to IL-17A and TNFα caused a target gene up-regulation that exceeded single treatments including those of TNFα alone. Furthermore, other signalling pathways were also activated by IL-17/TNFα or TNFα/IFNγ: oxidative stress (glutathione reductase (GSR) RQ 6.82 (IL-17/TNFα), RQ 1.99 (TNFα/IFNγ)), hypoxia (erythropoietin (EPO) RQ 3.0 (IL-17/TNFα), vascular endothelial growth factor A (VEGFA) RQ 3.11 (TNFα/IFNγ)), hedgehog (growth factors WNT5A RQ 5.53 (IL-17/TNFα), RQ 2.22 (TNFα/IFNγ) and BMP4 RQ 3.85 (IL-17/TNFα), RQ 3.64 (TNFα/IFNγ)) and peroxisome proliferator-activated receptor , RQ 9.51 (TNFα/IFNγ)). These regulation patterns were comparable to those of single IFNγ (oxidative stress (glutathione reductase (GSR) RQ 3.28), hypoxia (erythropoietin (EPO) RQ 4.9), hedgehog , peroxisome proliferator-activated receptor ) and were not triggered by single IL-17A or TNFα exposure alone. Comparable to TNFα, IL-17A alone did not up-regulate signalling pathway target genes except for gsr (RQ 1.87) but rather decreased expression of target genes involved in the TGFb (RQ 0.08), JAK/STAT , hedgehog (WNT5A RQ 0.36) and hypoxia  pathways.To investigate how pro-inflammatory mediators impact signalling pathways in hNSC-derived neurons, we analysed expression regulation of target genes associated with ten different signalling pathways upon neuronal treatment with single or combinatory inflammatory mediators for 24 h using a commercially available rt-qPCR array Fig. . Here, win-vitro neuronal cell culture model. We identified cytokine receptor expression on mature mixed neuronal cultures using immunofluorescence staining and qPCR. Treatment with the respective cytokines and in particular with cytokine combinations had distinct effects on neuronal morphology and intracellular signalling events. This is to our best knowledge the first study on direct effects of inflammatory cytokines and cytokine combinations on human neuronal cytoarchitecture. All data presented were collected from an embryonic stem cell derived hNSC cell line with no reported disease. Therefore, these findings might be relevant not only for MS, but for any CNS disease involving the investigated cytokines.In this study, we show that proinflammatory cytokines frequently found and cited in the pathophysiology of MS have direct effects on neurons and their cellular integrity in a human TNFR1 and TNFR2  were both expressed on mixed neuronal cultures. Incubation with TNFα resulted in distinct structural alterations of neuronal processes presenting a prominent beading. This effect of neurite morphology changes was levered by the cytokine IL-17A, which is relevant in context of EAE and MS. The IL-17 cytokine family, which consists of the six cytokines IL-17A to IL-17F, binds to a receptor complex formed by IL-17RA and IL-17RC Supplementary file2 (PDF 1658 KB)Below is the link to the electronic supplementary material.


However, they did not differ from the individuals with heterozygous β-thalassemia and normal α-globin (αα/αα) in terms of mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) levels. In addition, heterogenous clinical phenotypes were found in two individuals with the same genotype. Our study established a database of Ganzhou α-globin gene triplication and provided practical advice for the clinical diagnosis of α-globin gene triplication.α-globin gene triplication carriers were not anemic in general, while some studies found that α-globin gene triplication coinherited with heterozygous β-thalassemia may cause adverse clinical symptoms, which yet lacks sufficient evidence in large populations. In this study, we investigated the prevalence and distribution of α-globin gene triplication as well as the phenotypic characteristics of α-globin gene triplication coinherited with heterozygous β-thalassemia in Ganzhou city, southern China. During 2021-2022, a total of 73,967 random individuals who received routine health examinations before marriage were genotyped for globin gene mutations by high-throughput sequencing. Among them, 1,443 were α-globin gene triplication carriers, with a carrier rate of 1.95%. The most prevalent mutation was ααα Thalassemia is an autosomal recessive genetic disease with one or more mutations in globin chains. It is highly prevalent in malaria endemic areas as an adaptive response to malaria . Multiplα-globin gene triplication is the result of unequal crossing over between misaligned homologous segments in the α-globin gene cluster on chromosome 16 during meiosis, leading to an increased accumulation of α-globin . In the Previous studies indicated that the frequencies of α-globin gene triplication in different populations varied from 0.4%  in Sardinians, 1.2%  in Netherlands to 5%  in Greek . In an IJiangxi province, located in southern China, is highly prevalent with thalassemia . GanzhouA total of 73,967 subjects  receiving routine health examinations before marriage in Ganzhou city of Jiangxi province in China were genotyped for globin gene mutations including α-globin gene triplication from August 2021 to September 2022. These subjects came from 18 counties of Ganzhou, including Xingguo, Ningdu, Shicheng, Huichang, Yudu, Ganxian, Nankang, Longnan, Zhanggong, Shangyou, Chongyi, Dayu, Xinfeng, Anyuan, Xunwu, Dingnan, Quannan, and Ruijin.After obtaining the informed consent of the subjects, peripheral blood was collected in EDTA tubes. All the blood samples were stored at −20°C before the experiment. The hematological parameters of all the blood samples were automatically measured using an automated XS-1000i Hematology Analyzer System . Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Hemoglobin (Hb) concentration were determined and statistically analyzed using ANOVA. The relative proportions of HbA, HbA2 and HbF were measured by CAPILLARYS 3 OCTA  at Ganzhou Maternal and Child Health Hospital.We utilized the Q Kingfisher Flex system  to extract genomic DNA from whole blood. Subsequently, the GenMag Nucleic Acid Isolation kit (Magnetic bead method)  was used for DNA isolation. To quantify the concentrations of DNA samples, we employed a NanoDrop-8000 spectrophotometer . The samples were subjected to restriction based on the following criteria: a DNA concentration exceeding 20 ng/mL and an A260/A280 ratio range of 1.8–2.0.3.7, -α4.2; β deletions: SEA-HPFH, Chinese Gγ+(Aγδβ)0, Taiwanese deletion) were characterized by the Gap-PCR. Other mutations were detected using NGS. Firstly, the target sequences of HBA1, HBA2, and HBB were amplified and enriched through multiplex PCR. Secondly, sequencing libraries were subsequently prepared following the MGISEQ-2000 sequencing library preparation protocol . Finally, sequencing was carried out using the paired-end tag (PE100) on an MGISEQ-2000 chip . The protocol for bioinformatic analysis of identifying hemoglobin gene mutations was previously described  was conducted with the commercial kit SALSA MLPA P140 HBA probemix  by Beijing Yinda Xin Technology Co., Ltd.According to WHO recommendations , non-anep-value <0.05 was considered as significant .All the statistical analyses were performed using the SPSS 19 software . Continuous variables were expressed as means ± SD, while categorical variables were expressed as numbers or percentages. The Chi-square test and one-way ANOVA were employed for statistical difference analysis. p-value = 0.17) in the carrier rate between males (n = 701) and females  and accounted for 43.10%  of the α-globin gene triplication carriers, followed by αααanti4.2/αα and αααanti4.2/-α3.7, which had carrier rates of 0.74%  and 0.26% , respectively, and accounted for 38.12%  and 13.37%  of the α-globin gene triplication carriers  of all the α-globin gene triplication carriers , Yudu (p = 0.001), Shicheng (p = 0.005), Huichang (p = 0.012) and Dayu (p = 0.026), but not significantly different from Ganxian and Quannan (p = 0.633), Longnan (p = 0.480), Xunwu (p = 0.304), Chongyi (p = 0.222) and Xingguo (p = 0.168). Unexpectedly, in contrast to the overall distribution pattern of thalassemia observed in China, which showed high prevalence in the south and low prevalence in the north, the distribution pattern of α-globin gene triplication in Ganzhou appeared to be random.The carrier rates of 18 counties in Ganzhou ranged from 1.32% to 2.51% . ShichenN/βN, n = 80) or heterozygous β-thalassemia . Since all the individuals of this study were in childbearing age, the average age of each group was between 22.67 and 28.00 , group F with genotype αααanti3.7 or anti4.2/αα  and βmut/βN and group G with genotype αααanti4.2/-α3.7 and βmut/βN  were significantly lower than those of group A  with only one subject were also lower than those of group A  according to the genotypes of α and β globin genes . In addind 28.00 . Compareal range . This re group A . Howeveranti4.2/αα and βN/β−28 (A>G) (1 and 2), the coinheritance of αααanti3.7/αα and βN/βIVS−II−654 (C>T) (5 and 7) and the coinheritance of αααanti4.2/αα and βN/βIVS−II−654 (C>T) (6 and 8). These results suggested that the phenotypic impacts brought by α-globin gene triplication co-inherited with heterozygous β-thalassemia was heterogenous.To gain a further understanding of the effect of α-globin gene triplication, eight subjects with α-globin gene triplication and heterozygous β-thalassemia were successfully follow-up visited. None of them had blood transfusion history. MLPA results validated that they had triplicated α-globin genes, which was consistent with the NGS results . HematolH <27 pg , provingH <27 pg . Of notein utero. However, the α-globin gene triplication was not identified until the 1980s , as well as subjects 6 and 8 with genotype αααanti3.7/αα and βN/βIVS−II−654 (C>T). It is intriguing to note that Our study identified 42 carriers of both heterozygous β-thalassemia and α-globin gene triplication, providing a valuable resource to assess the interaction between heterozygous β-thalassemia and α-globin gene triplication in the Chinese population. The imbalance between α- and β-globin chains is the key to cause thalassemia. Several researchers have proposed that due to the increased α-globin accumulation, the co-inheritance of α-globin gene triplication and heterozygous β-thalassemia could result in poor clinical symptoms, such as β-thalassemia intermedia . In consContrarily, a previous study conducted in an Iranian population suggested that there is no necessity to allocate additional attention to α-globin gene triplication in the carriers of heterozygous β-thalassemia . MoreoveWe speculate that such discrepant findings observed in different studies may be attributed to the subjects analyzed. Some studies only focused on the symptomatic patients who sought medical attentions at hospitals , whereasIn this study, we have discovered that the α-globin gene triplication has a limited impact on the anemia phenotype. Although we thoroughly assessed all the globin gene mutations in triplication carriers, some information has not been covered, such as HBA12 which has garnered substantial attention in the research community S.Q. . AdditioIn conclusion, this study conducted an extensive survey to evaluate the occurrence and distribution of α-globin gene triplication in Ganzhou city. The results revealed that α-globin gene triplication is randomly distributed among various counties, with an overall carrier rate of 1.95%. The study also emphasized the diverse clinical manifestations of β-thalassemia with α-globin gene triplication, underscoring the importance of cautious assessment in clinical practice. These findings enrich the existing genetic data on α-globin gene triplication and offer valuable insights for diagnosing atypical cases in the future.