N-Maleanilic acid 3 was obtained in 94% yield via the reaction of selenoamine 2 with toxilic anhydride. Subsequent dehydration of N-maleanilic acid 3 using acetic anhydride furnished the unexpected isomaleimide 5-((4-((4-bromophenyl)selanyl)phenyl)imino)furan-2(5H)-one (4) instead of the maleimide 5. The molecular structure of compound 4 was confirmed by mass spectrometry, 1H- and 13C-NMR spectroscopy, and X-ray diffraction analysis. Their cytotoxicity was assessed against two oligodendrocytes, and their respective redox properties were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCFDA) assay. Furthermore, their antiapoptotic potential was also evaluated by flow cytometry. The compound crystallizes in triclinic P-1 space group with unit cell parameters a = 5.7880 (4) Å, b = 9.8913 (6) Å, c = 14.5951 (9) Å, V = 1731.0 (3) Å3 and Z = 2. The crystal packing is stabilized by intermolecular hydrogen bonding, π···π, C-Br···π stacking interactions, and other non-covalent interactions. The mapping of different Hirshfeld surfaces and 2D-fingerprint were used to investigate intermolecular interactions. The interaction energies that stabilize the crystal packing were calculated and graphically represented as framework energy diagrams. We present a computational investigation of compound 4’s molecular structure at the Density Functional Theory level using the B3LYP method and the 6-31G ++ basis set in this paper. The optimized structure matches the experimental outcome. The global reactivity descriptors and molecular electrostatic potential (M.E.P.) map emphasize the molecule’s reactive locations, allowing reactivity prediction. The charge transfer properties of molecules can be estimated by examining Frontier molecular orbitals.Reduction of 4,4′-diselanediyldianiline ( They algression . Recentlgression . They hagression . On the volution . Additioeostasis  (Barbosaeostasis .IV) and ethaselen (VI) are among the most investigated OSe compounds with exciting GPx- and TrxR-like activities, respectively (VI) and farinomalein (VII)  . Moreovein (VII) .N-maleamic acids  and M.E.P. mapping studies.We herein report the accidental synthesis and crystal structure of isomaleimide 4 was synthesized in good yield (77%) over three steps by the reduction of the diselenide 1 using NaBH4 and subsequent reaction with bromo-4-(bromomethyl)benzene followed by reaction with toxilic anhydride and subsequent ring closure using acetic anhydride and sodium acetate , 4-bromobenzyl bromide , aliquat 336  and sodium tetrahydridoborate  in ethylactetae and water mixed solvent  under reflux for 3 h. The formation of compound 2 was followed by TLC (petroleum ether: EtOAc = 6:1). The solvent was evaporated and the product was purified by column chromatography (petroleum ether: EtOAc = 6:1.5) as white solid; Yield: 310.31 mg (91%); mp 156–158°C .Compound 3 was synthesized from the reaction of compound 2  and maleic anhydride  in dry toluene (5 ml) with stirring at r.t. for 3 h. The formation of compound 3 was monitored by TLC (chloroform: methanol = 8:1). The product was purified by column chromatography (chloroform: methanol = 6:1) to give yellow solid; Yield: 412.66 mg (94%); mp 215–217°C .Compound 4 was synthesized from the gentle heating of compound 3 , acetic anhydride (3 ml), and sodium acetate (100 mg) for 2 h at 50–60°C. Water was added and the resulting mixture was extracted with CH2Cl2 (200 ml), dried with Mg2SO4, CH2Cl2 was evaporated and the residue was purified by silica gel chromatography. The progress of the product formation was followed by TLC petroleum ether: EtOAc = 8:1, Rf = 0.45, purified by column silica gel chromatography with petroleum ether: EtOAc = 6:1. Yellow solid; Yield: 324.17 mg (77%), mp 185–187°C. 1H NMR  δ 7.47–7.29 , 7.25–7.14 , 7.04–6.96 , 6.76 , 6.62 , 4.01 ; 13C NMR  δ 166.94, 150.34, 143.16, 142.77, 137.50, 134.29, 134.02, 131.59, 130.49, 127.93, 125.94, 120.83, 31.52; MS (ESI): m/z = found 478.92 [M+ + CH3COO−]; calcd. 420.92 [M+]; HRMS calcd. for C17H12BrNO2Se [M+ + 1]: 475.93688, found 477.93289 [M+ + CH3COO−].Compound 2, 3, and 4 were assessed against two oligodendrocyte cell lines, namely, 158 N and 158 JP, by the M.T.T. assay using 7-ketocholesterol as reference control  radiation (λ = 0.71073 Å). The full data set was used to calculate and refine unit cell parameters. S.A.D.A.B.S. iso(H) = 1.2Ueq(C). The crystal packing and molecular packing were drawn using the program Diamond 3  is divided into electrostatic (Eele), polarization (Epol), dispersion (Edis), and repulsion (Erep) contribution energies, with cylinders representing the relative strength of the molecular packing and fixed at a scale factor of 200 and cutoff energy of 5 kJ/mol.Hirshfeld surfaces  were maplorer 17 . Hirshfelorer 17 , with thgy model , startinμ), electronegativity (χ), electrophilicity index (ω) and chemical hardness (η), and softness (S) were all calculated using the corresponding HOMO and LUMO energies (The Gaussian 09 software  was usedenergies .1) was used as the starting building block to prepare the target isomaleimide 4 benzene to give Ose amine 2 in 85% yield . Surprisingly, gentle heating of compound 3 with Ac2O in the presence of NaOAc afforded the unexpected kinetically controlled isomaleimide 4 instead of thermodynamically controlled 1-(4-((4-bromobenzyl)selanyl)phenyl)-1H-pyrrole-2,5-dione (5)   (Shaabanione (5) .4 was established by 1HNMR and 13CNMR spectroscopy. 1HNMR of isomaleimide 4 showed the characteristic upfield singlet signal of the methylene fragment (CH2) at δ 4.01 ppm with a coupling constant (J) at 5.5 Hz. The characteristic isomaleimide vinylic protons appeared at δ 4.01 ppm δ 6.76 and 6.62 ppm. The rest eight aromatic protons appeared as multiplet signals at δ 7.47, 7.25, and 7.04 ppm. Furthermore, the isomaleimide 4 showed 13 carbon signals in the 13CNMR spectroscopy. The aliphatic methylene carbon appeared upfield at δ 31.52 ppm. On the other hand, one carbonyl signal appeared downfield at δ 166.94 ppm and the azomethine (N=C) at δ 150.34 ppm. The vinylic carbons appeared at δ 137.50 and δ 127.93 ppm. The rest eight aromatic carbons appeared at δ 143.16–120.83 ppm.The structure of isomaleimide 2, 3, and 4 were assessed in 158N and 158 JP cells. Interestingly, OSe compounds 2 and 4 did not show any apparent toxicity (IC50 ≥ 100 µM), whereas the N-maleanilic acid 3 showed moderate-low cytotoxicity , affecting the transmission of the neuronal signal and the proper axon function . OSe com2, 3, and 4 were also evaluated by flow cytometry in 158N cells stained with P.I. using 7Kc as apoptosis stimulator and the positive reference  −x−1, −y+1, −z; (iii) −x, −y+2, −z]), (i hydrogen bonding , (a axis. The centroid-to-centroid separation of Cg1 and Cg3 is 3.564(1) Å (Br···Cg1 = 3.7321(11) Å), Se···Br and Se···O interactions , ; Table 3, −z+1], . The mai564(1) Å ; Table 3ractions ; Table 3ractions .4 is mapped over dnorm. By plotting H.S., we can not only visualize H-bonding interactions but also investigate other non-covalent interaction types, such as stacking interactions. To visualize this interaction, the H.S. is plotted on a shape index and curvedness. The presence of triangular regions of red and blue around the furan and aromatic rings on the shape index , corresponding to C-H···N and C-H···Br interactions, as shown in the 2D fingerprint plot by a pair of pointed spikes representative of strong hydrogen-bonding interactions  can be divided into total electrostatic (Eele = −54.7 kJ/mol), total polarization (Epol = −33.6 kJ/mol), total dispersion (Edis = −327 kJ/mol), and total repulsion (Erep = 137 kJ/mol) . a axis with respect to the selected molecule.The intermolecular interaction energies are calculated using the HF/3-21G energy model with scale factors to determine E kJ/mol) . Obvious4 are depicted in 4.The optimized structural parameters of the isomaleimide X is Xtheo, while the experimental value is Xexp. The average variance of the distances and angles produced by the DFT approach is on the order of less than 2%, indicating that the diverse theoretical conclusions obtained are in excellent agreement with those found experimentally by crystallographic analysis.Furthermore, we believe it is worthwhile, to begin with the results gained from the geometrical parameters of the investigated molecules. In 4. A significant value isomaleimide 4 shows the molecule’s tendency to keep its electrons. A negative chemical potential (μ) reflects the molecule’s molecular stability or difficulty breaking it down into its constituent parts (η). A big HOMO-LUMO energy gap implies a complicated molecule with low polarizability and chemical and biological activities but high kinetic sensitivity  of a molecule measures its stabilization energy or resistance to exchange electrons with the system after the addition of an external electronic charge  is 2.90 eV. Chemical potential, electronegativity, hardness, softness, Global Softness, and electrophilicity for the isomaleimide 4 were determined as −4.52, 4.52, 1.45, 0.73, 0.69, and 7.04 eV, respectively, using The gap energy, which is the energy difference of the two preceding molecular orbitals , H···H (25.4%), O∙∙∙H/H∙∙∙O (13%) and Br∙∙∙H/H∙∙∙Br (9.2%) interactions. The energy-framework analysis reveals that the dispersive energies are the most important forces in the crystal. In parallel with the experimental study, we carried out a theoretical study detail using quantum chemical methods to determine the properties structural of compound 4. We carried out a comparison between the theoretical geometrical parameters and those obtained by X-ray diffraction. It appears significantly that the calculations obtained are in good agreement with the experimental data.Isomaleimide


Brucea antidysenterica is an African medicinal plant traditionally used to treat cancer, dysentery, malaria, diarrhea, stomach aches, helminthic infections, fever, and asthma. The present work was designed to identify the cytotoxic constituents of Brucea antidysenterica on a broad range of cancer cell lines and to demonstrate the mode of induction of apoptosis of the most active samples.Cancer remains a global health concern and constitutes an important barrier to increasing life expectancy. Malignant cells rapidly develop drug resistance leading to many clinical therapeutic failures. The importance of medicinal plants as an alternative to classical drug discovery to fight cancer is well known. Brucea antidysenterica by column chromatography and structurally elucidated using spectroscopic techniques. The antiproliferative effects of the crude extracts and compounds against 9 human cancer cell lines were evaluated by the resazurin reduction assay (RRA). The activity in cell lines was assessed by the Caspase-Glo assay. The cell cycle distribution, apoptosis via propidium iodide (PI) staining, mitochondrial membrane potential (MMP) through 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, and the reactive oxygen species (ROS) via 2´,7´-dichlorodihydrofluoresceine diacetate (H2DCFH-DA) staining, were investigated by flow cytometry.Seven phytochemicals were isolated from the leaves (BAL) and stem (BAS) extract of H indole (1) and hydnocarpin (2), as well as the reference compound, doxorubicin, had antiproliferative activity against 9 cancer cell lines. The IC50 values varied from 17.42 µg/mL (against CCRF-CEM leukemia cells) to 38.70 µg/mL (against HCT116 p53−/− colon adenocarcinoma cells) for BAL, from 19.11 µM (against CCRF-CEM cells) to 47.50 µM (against MDA-MB-231-BCRP adenocarcinoma cells) for compound 1, and from 4.07 µM (against MDA-MB-231-pcDNA cells) to 11.44 µM (against HCT116 p53/++ cells) for compound 2. Interestingly, hypersensitivity of resistant cancer cells to compound 2 was also observed. BAL and hydnocarpin induced apoptosis in CCRF-CEM cells mediated by caspase activation, the alteration of MMP, and increased ROS levels.Phytochemical studies of the botanicals (BAL and BAS) led to the isolation of seven compounds. BAL and its constituents 3, (3-(3-Methyl-1-oxo-2-butenyl))12, are potential antiproliferative products from Brucea antidysenterica. Other studies will be necessary in the perspective of the discovery of new antiproliferative agents to fight against resistance to anticancer drugs.BAL and its constituents, mostly compound The online version contains supplementary material available at 10.1186/s12906-023-03877-1. Cancer remains an incredible human killer and is recognized globally as an important barrier to increasing life expectancy. An incredible number of deaths is linked to tumors in many tissues such as the breast (685 thousand), stomach (769 thousand), liver (830 thousand), colon and rectum (916 thousand), and lung  . FightinThe exploitation of medicinal plants traditionally used under rigorously well-conducted scientific investigations in vitro, in vivo, or in silico, has contributed significantly to the improvement of human health, with many pharmaceuticals deriving from botanicals –7.Ambrosia maritma , , , , , , , , , , , , , , , , , , . Analytic thin layer chromatography (TLC) was used to pooled them into 19 sub-fractions (F1′-F19′) as follows: fractions 5–11 (F1’); 12–29 (F2’); 30–45 (F3’); 46–60 (F4’); 61–81 (F5’); 82–97 (F6’); 98–116 (F7’); 117–133 (F8’); 134–150 (F9’); 151–164 (F10’); 165–185 (F11’); 186–202 (F12’); 203–220 (F13’); 221–234 (F14’); 235–250 (F15’); 251–269 (F16’); 270–282 (F17’); 283–305 (F18’); 306–321 (F19’). Theses fractions afforded compounds 1 , 2 , 3  and 4  after precipitation and filtration.The amounts of 2.2 kg of the air-dried and powdered leaves of Brucea antidysenterica were extracted with MeOH for 72 h. The solution was evaporated in vacuum to yield a brown residue of 24.10 g. The crude extract (22.10 g) was adsorbed on silica (22.10 g) and chromatographed over a silica gel CC  with n-hexane-ethly acetate (AcOEt) and CHCl3-MeOH gradients as eluents. Fractions of 100 ml each were collected as follows: , , , , , , , , , , , , , , , , , , . The analytic thin layer chromatography (TLC) was used to pooled them into 19 sub-fractions (F1-F19) as follows: fractions 7–15 (F1); 18–29 (F2); 32–48 (F3); 57–64 (F4); 66–81 (F5); 83–99 (F6); 101–107 (F7); 110–129 (F8); 133–147 (F9); 152–162 (F10); 166–184 (F11); 187–200 (F12); 203–219 (F13); 225–230 (F14); 234–248 (F15); 250–269 (F16); 273–289 (F17); 291–303 (F18); 307–319 (F19). Compound 1 (25 mg) was isolated by filtration of the precipitates of the sub-from F2. Compounds 5 (13.9 mg) was isolated as beige powder by filtration of the precipitates of the sub-from F4. From F14, compound 6 (10.5 mg) was obtained respectively as yellow powder. From F18, compound 7 (26 mg) was isolated as a white powder.The amounts of 2.3 kg of the air-dried, finely powdered stem of ΔEGFR cells transfected with epidermal growth factor receptor (EGFR) [p53/++ colon adenocarcinoma cells and its knock-out clone HCT116 p53−/− cells [pcDNA3 breast adenocarcinoma cells and breast cancer resistance protein (BCRP)-transfected, and its multidrug-resistant MDA -MB-231-BCRP clone 23 cells [BCRP cells were obtained from Dr. Douglas D. Ross ; HCT116 p53/++ and HCT116 p53−/− cells were a generous gift from Dr. B. Vogelstein and H. Hermeking ; U87MG and U87MG.ΔEGFR cells were kindly provided by Dr. W. K. Cavenee ; HepG2 and AML12 cells were obtained from ATCC (USA). The RPMI-1640 medium was used for the culture of CCRF-CEM and CEM/ADR5000 cells whilst DMEM medium was used for carcinoma cells. The culture media were supplemented (complete medium) with 10% fetal bovine serum and 1% penicillin/streptomycin . The CEM/ADR5000 cell line was maintained in a complete medium containing 5000 ng/ml of doxorubicin to maintain its resistant phenotype. To maintain the resistance phenotype of the resistant carcinoma cell lines such as MDA-MB-231/BCRP and HCT116 p53−/−, and U87MG.ΔEGFR cells, the complete DMEM medium containing geneticin at 800 ng/ml and 400 µg/ml, respectively were used [In this work, 9 cancer cell lines with various drug resistance patterns as well as AML12 hepatocytes as normal control cell line were used. The origins of the leukemia cell lines, CCRF-CEM (drug-sensitive) and CEM/ADR5000 (its subline overexpressing multidrug-resistant (MDR) P-glycoprotein (P-gp)) have been reported by several authors –35; Simir (EGFR) , HCT116 /− cells , and MDA23 cells . The AML23 cells . CCRF-CEere used , 42.1–7) from Brucea antidysenterica, and doxorubicin was monitored by RRA [2 atmosphere and an incubation temperature of 37 °C. The tested concentration ranges were 0.63—80 µg/mL for the botanicals and 0.78—100 µM for phytochemicals. The incubation time of the treated cells was 72 h, and the fluorescence was measured with Infinite M2000 Pro™ plate reader . The wavelengths used were 544 nm for the excitation and 590 nm for the emission. The IC50 values of samples were referred to as the concentrations required to inhibit 50% of the cell proliferation and determined using calibration curve as reported previously [50 values of samples in the resistant cell line vs that of corresponding sensitive cell line. The selectivity index (S.I.) was the ratio of the IC50 value of AML12 hepatocytes vs. that of HepG2 cells [The antiproliferative activity of botanicals (BAL and BAS), phytoconstituents  at their ¼ IC50, ½ IC50, IC50, and 2 × IC50 values. The whole was then incubated for 24 h as earlier indicated followed by the analysis of the cell cycle distribution with a BD Accury C6 Flow Cytometer  [The modification of the cell cycle distribution of CCRF-CEM induced by the botanicals BAL, the flavonolignan nditions . BAL andGermany) , 50. Ass2, doxorubicin, or the solvent (DMSO) [2, as well as doxorubicin were applied to CCRF-CEM cells  at their ¼ IC50, ½ IC50, IC50, and 2 × IC50 values followed by a 24 h incubation as indicated earlier. Afterward, apoptosis detection was undertaken with a fluorescein isothiocynate (FITC)-conjugated annexin V/PI assay kit . The measurement was done using BD Accury C6 Flow Cytometer (BD Biosciences) under experimental conditions previously reported [The annexin V/propidium iodide (PI) staining combined to the flow cytometry was used to detect apoptotic cells in CCRF-CEM cells by BAL, the flavonolignan t (DMSO) , 25, 46.reported , 25. The2 was determined by Caspase-Glo assay [2 were applied to CCRF-CEM cells  at their ½ IC50, IC50, and 2 × IC50. The whole was then incubated for 6 h (standard culture conditions) and the activity of caspases was detected by spectrophotometry as previously described [The activity of caspases in CCRF-CEM cells treated with BAL and the flavonolignan lo assay . BAL andescribed .2 at ¼ IC50, ½ IC50, IC50, and 2 × IC50 to CCRF-CEM cells (1 × 106) was performed by flow cytometry [4 cells) in an LSR-Fortessa FACS analyzer (Becton–Dickinson) [2, hydrogen peroxide , DMSO (solvent control) as in the MMP analysis, then incubated for 24 h. They were further stained with H2DCFH-DA and measured (1 × 104 cells) in an LSR-Fortessa FACS analyzer [The modification of the MMP as well as the ROS levels after the application of BAL, and flavonolignan ytometry . DMSO waytometry  and measckinson) . For ROSanalyzer , 51, 52.p-value < 0.05.The Graph Pad Prism 5 software was used for the statistical analysis. Data from independent assays are presented as mean value ± standard deviation. The difference between the mean values of test samples and the control, One-way Analysis Variance (ANOVA) and post hoc Tukey’s test was used to establish the significance. The significant differences were regarded as 1–4) and stem  of Brucea antidysenterica. Their chemical structures , canthin-6-one (5) and bruceolline F (7), a flavonolignan, hydnocarpin (2), two sterol glycosides: (20R)-O-(3)-α-L-arabinopyranosyl-pregn-5-en-3β,20-diol (3) and (20R)-O-(3)-β-D-glucopyranosyl(1 → 2)-α-L-arabinopyranosyl-pregn -5-en-3β,20-diol (4), and a coumarinolignans, cleomiscosin C (6). The NMR spectra of the isolated phytoconstituents are available as supporting information.Seven phytochemicals were purified from the methanol extracts of the leaves –4 and st50 values of BAL  had selective cytotoxic effects. The obtained IC50 values varied from 17.42 µg/ml (against CCRF-CEM leukemia cells) to 38.70 µg/mL (against HCT116 p53−/− colon adenocarcinoma cells) for BAL (Table BCRP breast adenocarcinoma cells) for compound 1, from 4.07 µM (against MDA-MB-231-pcDNA cells) to 11.44 µM (against HCT116 p53/++ cells) for compound 2, and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells) for doxorubicin. The three most active samples  also displayed lower cytotoxicity (S.I. > 2.12) against AML12 cells vs HepG2 cells (Tables BCRP cells vs. MDA-MB-231-pcDNA cells (D.R. of 0.51), and U87.MGΔEGFR glioblastoma cells vs. U87.MG cells (D.R. of 0.69) to BAL was observed (Table p53−/− cells compared to HCT116 p53/++ cells (D.R. of 0.59), and U87.MGΔEGFR cells compared to U87.MG cells (D.R. of 0.58) vis-a-vis compound 2 (Table p53−/− cells compared to HCT116 p53/++ cells (D.R. of 1.04) to BAL was also noted. The tested cancer cells showed cross-resistance to compound 1  of various phases of the cycle of CCRF-CEM cells in a dose-dependent manner. The cell cycle was arrested in S and G2/M phases as the result of treatment with BAL and doxorubicin, while that with compound 2 induced G0/G1 cycle arrest. The amounts of cells in sub-G0/G1 phase also change upon treatment with the test samples, with values significantly increasing (p < 0.05) from 2.67 ± 0.2% (1/4 × IC50) to 22.00 ± 1.4% (2 × IC50) with BAL, from 3.41 ± 0.2% (1/4 × IC50) to 38.40 ± 2.6% (2 × IC50) with flavonolignan 2, and from 3.28 ± 1.1% (1/4 × IC50) to 12.05 ± 0.9% (2 × IC50) with doxorubicin. Apoptosis can be suggested by the increase of the cells in sub-G0/G1 phase and can be evidenced by annexin V/PI staining. For instance, the data depicted in Fig. 50) vs. 2.6 ± 0.1% for non-treated control  vs. 6.1% for non-treated control  and its isolate (compound 2) activated the caspases enzymes.The results of the caspase activity resulting in the application of BAL and flavonolignan 2, and valinomycin, the JC-1 staining was applied to analyze the integrity of MMP. The results illustrated in Fig. 2 to CCRF-CEM cells considerably altered the MMP. The amounts of 59.0% and 10.4% intact MMP cells resulted from BAL and flavonolignan 2 applications, respectively, at 2 × IC50vs. 92.6% for non-treated control  while 44.5% cells and 16.8% had MMP loosed or disrupted, respectively.After treatment of CCRF-CEM cells with BAL, flavonolignan 2 to CCRF-CEM cells induced a significant dose-dependent raise  to 17.80% (2 × IC50) and from 6.00% (1/4 × IC50) to 49.00% (2 × IC50) with BAL and phytoconstituent 12, respectively. H2O2 (positive control) caused significant raise in the ROS levels to 94.30% at 50 µM vs. 0.6% in non-treated CCRF.CEM cells.Regarding ROS production, the application of BAL and flavonolignan ise Fig. . In effei.e., collateral sensitivity or hypersensitivity) or as active  on resistant than sensitive cells [i.e., more sensitivity of the resistant counterpart to the applied sample. They resistant model of leukemia cell line include CEM/ADR5000 cells. This cell line overexpressed P-gp (ABCB1/MDR1) which is the adenosine triphosphate-binding-cassette transporter (ABC). The resistant carcinoma cell lines included were MDA-MB231/BCRP breast cancer adenocarcinoma cells, HCT116 p53−/− −/− colon adenocarcinoma cells, and U87.MGΔEGFR glioblastoma multiforme cells. HCT116 p53−/− cells had p53 knockout and represent as a model of many suppressor genes [/BCRP cells bear the breast cancer resistance protein (BCRP) [ΔEGFR cells harbor a mutation-activated EGFR gene (ΔEGFR) [BCRP cells vs. MDA-MB-231-pcDNA cells and U87.MGΔEGFR cells vs. U87.MG cells to the botanical (BAL) were obtained. Hypersensitivity of CEM/ADR5000 cells vs. CCRF-CEM cells, HCT116 p53−/− cells vs HCT116 p53/++ cells, and U87.MGΔEGFR cells vs. U87.MG cells to flavonolignan 2 was also achieved. These are indications that the two most active samples (BAL and compound 2) could help to combat the cancer drug resistance. IC50 values lower than 20 μg/ml for plant extracts or 10 μM for phytochemicals were set as thresholds for good naturally occurring cytotoxic agents [Brucea antidysenterica (BAL) had IC50 ≤ 20 μg/ml on 3/9 and 1/9 cancer cell lines, respectively  . U87.MGΔ (ΔEGFR) , 57. In c agents . The cruBrucea antidysenterica towards various models of drug-resistant cancer cells is reported here for the first time. Nonetheless, other cytotoxic molecules have been isolated from this taxon. The cytotoxicity of the crude extract on sensitive cancer cell lines was also reported. The root and bark extract had cytotoxic effects on A-549 lung carcinoma cells, MCF-7 breast cancer cells, and PC-3 prostate cancer cells  [50 values = 195.5 μM on PC-3) and B , canthin-6-one , 1-methoxycanthin-6-one , 2-methoxycanthin-6-one , 2-hydroxy-1,11-dimethoxycanthin-6-one  and cleomiscosin A  [50 value of 8.7 μM [50 values = 3.65 μg/ml), and Tmolt3  leukemia leukemia cells, KB nasopharynx cancer cells , S-480 colon adenocarcinoma , TE-418 osteosarcoma cells , and HeLa-S3 uterine cells , MB-9812 lung cancer cells  [50 value of 20.3 μM for hydnocarpin toward SW480 colon cancer cells [The cytotoxicity of the crude extract from 5 μg/ml) . This waf 8.7 μM . The gooer cells .2 induced apoptosis in CCRF-CEM cells. Apoptosis was also observed with hydnocarpin (2) derivatives in the lung and melanoma cancer cells via caspase activation [Apoptosis is recognized as one of the modes of cancer cell death induced by plant extracts and their consituents . It was tivation . The acttivation . The cruBrucea antidysenterica towards multifactorial drug-resistant cancer cell lines. The main cytotoxic constituents of the plant identified included hydnocarpin and 3, (3-(3-Methyl-1-oxo-2-butenyl))1H indole. Hydnocarpin was the most active compound and induced, together with the leave extract, the apoptotic cell death in leukemia CCRF-CEM cells. The leave extract of Brucea antidysenterica and hydnocarpin have good antiproliferative activities. Further in-depth studies including the investigation of more molecular targets, western blots, and in vivo anticancer studies are to be performed in way of discovering new anticancer drugs from these samples.This work has demonstrated the antiproliferative potential of the crude extracts and the phytoconstituents of Additional file 1.


Dear Editor,2 However, there are no effective marketed treatments targeting αvβ3. One possible limitation of Arginine-Glycine-Aspartic (RGD)-mimetic αvβ3 antagonists has been shown to cause partial agonism, which could induce major conformational changes that trigger paradoxical cell adhesion and angiogenesis.4The integrin αvβ3 receptor is a promising target for anticancer therapy.In this study, we identified a novel non-RGD small molecule inhibitor against the integrin αvβ3. Via virtual screening, surface plasmon resonance (SPR) assay-based affinity assay, and anti-tumor activity evaluation, C19 was identified as a hit compound with good affinity Fig. . C19 had2+, different from RGD ligands, which was mostly divalent cation-dependent and C19-9 did not depend on Mn2+ induced conformational changes.4  and MIDAS site  simulations were conducted to search for the stable complex structure of C19-9 with integrin avβ3 Fig. . MD simu50 of C19-9 on 22RV1 at 24 h was 0.467 μM. We also tested the inhibitory effect of C19-9 on PCa organoids and the results of the assay showed that C19-9 significantly inhibited organoid proliferation in a dose-dependent manner with IC50 of 0.652 μM  development, we analyzed the changes of differential genes after 22RV1 cells disposed of C19-9. The GO and KEGG enrichment pathway had a high correlation with the signals related to integrin αvβ3, such as cell adhesion, angiogenesis, extracellular matrix organization, PI3K/Akt pathway, tryptophan metabolism, etc.  and high liver microsomes stability (only 5% C19-9 degradation after incubation for 4 h) in different species.In conclusion, we identified C19-9, a novel non-RGD inhibitor of αvβ3 with excellent drug-like characteristics that could overcome enzalutamide-resistance in CRPC via inhibiting EMT and angiogenesis, blocking PI3K/Akt/AR and EKR signaling pathways, and improving the immunosuppressive microenvironment by JAK/STAT3/IDO signaling pathway Fig. . Taken tSupplementary_Materials-clean


Pseudocsikiachouisp. nov. and Pseudocsikiachanjuansp. nov., are described and illustrated from Taiwan, China. Their habitus and diagnostic characters are illustrated. The two species most resemble P.formosana, which is endemic to Taiwan, by the strongly protruding pronotal anterior angles accompanied by pits and the shape of aedeagus. They can be all grouped as the P.formosana-species group. A key to the species of the P.formosana-species group and an updated checklist of Chinese Pseudocsikia with supplementary notes on type localities are provided. The discovery of two new species highlights the potential species-richness of the flightless click-beetles on Taiwan Island.Two new flightless click beetle species,  Pseudocsikia Schimmel & Platia, 1991  is a small genus of click-beetles known from China, Myanmar, Laos, India, and Nepal . The holotype and one paratype of P.formosana are deposited in the Bernice Pauahi Bishop Museum, Honolulu, Hawaii, USA (BPBM). The collecting data is quoted verbatim (in Chinese) in quotation marks. Translation of the data, as well as additional information, is given in square brackets.The specimens were softened in hot water, and genital segments were excised and dissected after treatment in 80 °C 10% KOH for 10 min. Habitus images were photographed using a Canon EOS RP + Mount Adapter EF-EOS R + a Laowa 25 mm F2.8 2.5–5× Ultra Macro Lens (for Canon EF); diagnostic characters were made using a Leica M205A stereomicroscope and a Leica DFC 550. All figures ; Pseudocsikiarustica Schimmel & Platia, 1991.Diagnosis. Anterior angles of pronotum lateroapically protruded, with anterior edge of pronotum mesally concave in dorsal view. Each protrusion with sides almost parallel in dorsal view, with concavity laterally and a deep pit at basal portion , P.choui sp. nov., P.chanjuan sp. nov.Distribution. China (Taiwan).Taxon classificationAnimaliaColeopteraElateridae1E8BE719-5A7D-5507-812A-131B5161B2AAhttp://zoobank.org/9AD84855-59B9-4C47-9734-FC6A2AAC723EHolotype, male, “2017.IX.13,台湾嘉义县阿里山二万坪, 2000m, 周文一” , “Pseudocsikiachoui sp. nov. 周氏伪斯叩甲 HOLOTYPE des. Qiu et Kundrata 2022” (MYNU).Head, pronotum, and elytra dark brown, with paler lateral portions of pronotum and elytra, legs yellow Fig. . AntennoP.fomosana by the pale coloration of its pronotum and elytral sides and by the sparse punctures of pronotum, but it can be easily distinguished from the latter by the larger body length , darker coloration of pronotum and elytra medially, more forwardly protruded anterior angles of pronotum (pointing more outward in the pronotum of P.formosana), and shorter and more divergent posterior angle of pronotum . The shape of aedeagus also readily differentiates these two species. Based on the illustration of P.formosana has four large acute processes laterally, and its apex is somewhat rectangular; and the paramere of P.formosana has the apex rounded and slightly outward.This species superficially resembles . Body smooth, surface covered with curved, semi-erect, and moderately long pubescence. Body length 5.9 mm; width 2.2 mm; antenna length 3.0 mm; pronotum length 1.4 mm, pronotum width 1.9 mm (measured at posterior angles), elytra length 3.5 mm.Body brown, pubescence yellow Fig. . Head daHead including eyes 0.5 times as wide as pronotum. Supra-antennal carinae short, directed mesad and fading medially so that median portion of frontoclypeus is not formed by sharp carina; frontoclypeus overhanging base of labrum in lateral view. Head surface sparsely punctate; punctures small, intervals between punctures mostly equal 2–3 puncture diameters. Maxillary palpus with palpomere III longer than wide. Antenna Fig.  surpassiPronotum Fig.  wider thHypomeron Fig.  more denScutellar shield Fig.  suboval,Elytra Fig.  elongateAedeagus .The specific patronymic epithet is dedicated to Dr Wen-I Chou , the collector of the holotype.Taxon classificationAnimaliaColeopteraElateridae16003A22-D969-5423-B977-AB956F641787http://zoobank.org/835DC83E-6E39-437D-8A81-7C78F81757A9Holotype, male, “2017.IX.16, 台湾台东县金峰乡太麻里山, 1300 m, 周文一” , “Pseudocsikiachanjuan sp. nov. 婵娟伪斯叩甲 HOLOTYPE des. Qiu et Kundrata 2022” (MYNU).Pronotum and elytra almost unicolored brown, but with paler apices of elytra and lateral margins of pronotum (in dry specimen condition), legs yellow Fig. . AntennoP.formosana and P.choui sp. nov. by the denser punctures of pronotum and larger body size (6.3 mm versus 4.0–5.9 mm). This new species can be further distinguished from P.choui sp. nov. by the more outwardly protruded anterior angles of pronotum, and the larger and straight posterior angle of pronotum. The shape of aedeagus also differs from these. The median lobe of P.chanjuan sp. nov. has a slightly enlarged and rounded apex and two small acute processes laterally near midlength, its paramere is rounded at apex but with small process subapically, and the phallobase is less rounded basally than those of the other two species.This species can be distinguished from . Body smooth, surface covered with curved, semi-erect, and moderately long pubescence. Body length 6.3 mm; width 2.3 mm; antenna length 3.3 mm; pronotum length 1.7 mm, width 2.1 mm (measured at hind angles), elytra length 3.7 mm.Body generally brown, pubescence yellow Fig. . Head .Chanjuan is derived from the Chinese 婵娟 [chán juān], which means “beauty”.The specific epithet Taxon classificationAnimaliaColeopteraElateridae93A30EAF-26ED-5558-96D7-C9A1E5BC09A3Csikiaformosana Ôhira, 1972: 8 ; Pseudocsikiaformosana : Holotype of Csikiaformosana Ôhira, 1972, male, “TAIWAN (C.): Bu-kai. 900 m. VI.15–34. Gressitt”, “Csikiaformosana Ôhira, 1972 Det H Ohira 1972”, “HOLOTYPE”, “No. 9486”, “Examined. Det. W. Suzuki, 1982” (BPBM). One paratype of Csikiaformosana Ôhira, 1972, sex unknown, “TAIWAN (C.): Bu-kai. 900 m. VI-15-34. Gressitt”, “Csikiaformosana Ohira, 1972 Det H Ohira 1972”, “PARATYPE” (BPBM).BPBM). Body length 4.0 mm, width 1.7 mm .Pseudocsikiaformosana Chinese common name: 台湾伪斯叩甲.Type locality: “Bukai” ( “Bukai” .Note: Bukai is an old name of Fatyu [法治] (or Wuchieh [武界]) (eh [武界]) , Ren’ai Pseudocsikiagaoligongshana Schimmel, 1996Chinese common name: 高黎贡伪斯叩甲.Type locality: “Yunnan, Gaoligongshan, 100 km westlich von Baoshan [100 km W of Baoshan]” .Pseudocsikiachanjuan Qiu & Kundrata, sp. nov.Chinese common name: 婵娟伪斯叩甲.Type locality: Mount Taimalishan, Taitung County, Taiwan, 1300 m (this work).Dimini is represented not only by the lineages with flying species, but also by flightless ones, such as those from genera Dima Charpentier, 1825, Neodima Schimmel & Platia, 1992, and Sinodima Kundrata, Sormova & Qiu, 2019 (Dima and four spp. of Neodima) (Dima and one sp. of Sinodima) and eastern China (two spp. of Dima)  , with veof Dima) . Previous or not , but basleoptera . The dis


Bacillus tequilensis BL01, and multiple genetic engineering strategies were implemented to improve γ-PGA production.Poly γ-glutamic acid (γ-PGA) is a promising biopolymer for various applications. For glutamic acid-independent strains, the titer of γ-PGA is too low to meet the industrial demand. In this study, we isolated a novel γ-PGA-producing strain, 4)2SO4 at 37 °C, respectively. Second, the sucA, gudB, pgdS, and ggt genes were knocked out simultaneously, which increased the titer of γ-PGA by 1.75 times. Then, the titer of γ-PGA increased to 18.0 ± 0.3 g/L by co-overexpression of the citZ and pyk genes in the mutant strain. Furthermore, the γ-PGA titer reached 25.3 ± 0.8 g/L with a productivity of 0.84 g/L/h and a yield of 1.50 g of γ-PGA/g of citric acid in fed-batch fermentation. It should be noted that this study enables the synthesis of low (1.84 × 105 Da) and high (2.06 × 106 Da) molecular weight of γ-PGA by BL01 and the engineering strain.First, the one-factor-at-a-time method was used to investigate the influence of carbon and nitrogen sources and temperature on γ-PGA production. The optimal sources of carbon and nitrogen were sucrose and (NHB. tequilensis BL01 is reported. The titer of γ-PGA increased 2.17-fold and 1.32-fold compared with that of the wild type strain in the flask and 5 L fermenter. The strain shows excellent promise as a γ-PGA producer compared with previous studies. Meanwhile, different molecular weights of γ-PGA were obtained, enhancing the scope of application in industry.The application of recently published strategies to successfully improve γ-PGA production for the new strain The online version contains supplementary material available at 10.1186/s12934-022-01994-z. Reced groups . The SiOd groups . In agrid groups .Bacillus species, including B. subtilis [B. siamensis [B. licheniformis [B. methylotrophic [B. amyloliquefaciens [B. velezensis [l-glutamic acid as a direct precursor [γ-PGA is synthesized primarily by various subtilis , 13, B. iamensis , 15, B. niformis , 17, B. otrophic , B. amylefaciens , 20, andlezensis . Dependirecursor –15. Supprecursor –18, 20. B. amyloliquefaciens, the γ-PGA titer increased from 3.14 to 5.12 g/L by double deletion of the cwlO gene and the epsA-O cluster, as well as expression of the Vitreoscilla hemoglobin (VHb) gene [pgdS, ggt, gudB, rocG, and proB genes, was also studied [To meet the growing demand for economical γ-PGA, an increasing number of studies have focused on metabolic engineering strategies. In Hb) gene . ImproviHb) gene . FurtherHb) gene , 23, 24. studied . InterespgdS and ggt genes were deleted to reduce γ-PGA degradation. Third, the alsS, sucA, gudB, proB, and rocG genes were deleted to decrease the production of byproducts and improve glutamate production. Fourth, the genes involved in the synthesis of precursor and γ-PGA were overexpressed . γ-PGA-producing strains were preliminarily screened and cultured in basal medium containing 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 20 g/L glucose, and 10 g/L monosodium glutamate. Agar (20 g/L) was added to basal medium agar plates. A strain with a high γ-PGA yield was screened, and the influencing factors were analyzed in the fermentation medium, which contained 30 g/L glucose, 20 g/L monosodium glutamate, 5 g/L (NH4)2SO4, 0.5 g/L K2HPO4, 0.5 g/L MgSO4·7H2O, 0.04 g/L FeCl3·6H2O, 0.104 g/L MnSO4·H2O, 0.15 g/L CaCl2, and 0.5 g/L NaCl. For the fermentation of genetically modified bacteria, 10 g/L sodium citrate was added to the medium instead of 20 g/L monosodium glutamate.The The working concentrations of chloramphenicol, ampicillin, kanamycin and β-d-1-thiogalactopyranoside (IPTG) in this study were 5 μg/mL, 100 μg/mL, 40 μg/mL, and 0.1 mM, respectively.pgdS , ggt , sucA , alsS , proB , gudB  and rocG  were first identified in the Bacillus subtilis subsp. subtilis str. 168 complete genome in NCBI. Related genes were identified in B. tequilensis BL01 by sequence alignment. The pgdS gene deletion was used as an example to describe the construction of the knockout plasmid. First, the gRNA of pgdS was obtained using online software (https://www.atum.bio/eCommerce/cas9/ input). The upstream (pgdS-N20-F) and downstream (pgdS-N20-R) primers of the gRNA were annealed and then inserted into the plasmid pBAC-Cas9 with the Golden Gate cloning method to form pBAC-Cas9-N20-pgdS. Then, the upstream homologous arm with the primer  and the downstream homologous arm with the primer  of the pgdS gene were amplified, each approximately 1000 bp. Subsequently, the two fragments were joined together using the In-Fusion cloning method. Finally, the homologous arm and the plasmid pBAC-Cas9-N20-pgdS were digested by the SfiI restriction enzyme (Thermo Scientific) and ligated to obtain the knockout plasmid pBAC-Cas9-N20-pgdS-up-down. The knockout plasmid was transformed into B. tequilensis BL01 as previously described [The primers used for plasmid construction and gene knockout are listed in Additional file escribed . CorrectcitZ , icd , gltA , pgsBCAE , pycA , pyk  and pdhABCD  in the type strain B. subtilis subsp. subtilis str. 168 complete genomes were first found in NCBI, and then related genes were amplified from B. tequilensis BL01 with the corresponding primers . The phylogenetic tree was reconstructed using the neighbor-joining method in MEGA 7.0 [The strain with the highest γ-PGA production was subjected to 16S rDNA sequence analysis. The 16S rDNA gene sequence was amplified using universal 27F and 1492R primers. Sequencing was performed by GENEWIZ Inc. . The 16S rDNA gene sequence was compared with that of type strains reported in the EzBioCloud database 2SO4 were added to the medium. After optimizing carbon and nitrogen sources, the effect of temperature (28–42 °C) on bacterial growth and γ-PGA production was studied.The main nutritional components, including the sources of carbon and nitrogen and culture temperature, were optimized using the OFAT experimental design. To optimize the carbon sources, 30 g/L (w/v) glucose, sucrose, fructose, glycerol, arabinose, and xylose were added to the fermentation medium. To determine the effect of different nitrogen sources, 5 g/L (w/v) NHB. tequilensis BL01 for the production of γ-PGA, the identification medium contained 30 g/L sucrose, 0.5 g/L K2HPO4, 0.5 g/L MgSO4·7H2O, 0.04 g/L FeCl3·6H2O, 0.104 g/L MnSO4·H2O, 0.15 g/L CaCl2, 0.5 g/L NaCl and (1) 5 g/L yeast extract; (2) 5 g/L (NH4)2SO4; (3) 5 g/L monosodium glutamate; (4) 5 g/L yeast extract and 10 g/L sodium citrate; (5) 5 g/L (NH4)2SO4 and 10 g/L sodium citrate; and (6) 5 g/L monosodium glutamate and 10 g/L sodium citrate.To identify the type of 600 = 5.0 ± 0.1) were inoculated into 250 mL flasks containing 50 mL of fresh fermentation medium and cultured at 37 °C for 48 h at 200 rpm. Cell growth and γ-PGA yield were measured every 6 h.For shake-flask fermentation, a single colony from the agar plate was inoculated into a 250 mL flask containing 50 mL of liquid basal medium and cultured at 37 °C for 12 h at 200 rpm. Subsequently, 1% (v/v) of the precultures (OD3·H2O (25–28%). Sugar and citric acid levels were monitored regularly, and the consumption per hour was speculated to determine the replenishment point. When the sugar level dropped below 10 g/L, and the citric acid dropped below 5 g/L, 150 mL of feed medium containing 700 g/L sucrose and 20 g of sodium citrate dissolved in 50 mL sterile water were fed into the fermenter, individually.For fed-batch fermentation, cells were precultured in 300 mL of the basal medium at 37 °C for 12 h at 200 rpm and then inoculated in a 5 L fermenter  containing 2.7 L of fresh medium. The 5 L fermenter was operated at an aeration rate of 10 NL/min, and dissolved oxygen (DO) was kept above 5% by adjusting the agitation rate to 400–700 rpm. The pH was controlled at pH 6.5 ± 0.2 with 1 M HCl or NH™ linear column . The mobile phase, flow rate, and injection volume were 0.1 N NaNO3, 0.5 mL/min, and 20 µL, respectively. Glutamic acid was measured using an Agilent ZORBAX Eclipse Plus C18 column  with a UV detector (338 nm) (https://www.agilent.com/cs/library/applications/5990-4547EN.pdf).Cell biomass was determined by measuring the absorbance of the fermentation broth at 600 nm using a spectrophotometer. The standard curve relating OD to cell dry weight  was used (1 OD600 = 0.414 g CDW/L). The concentration of γ-PGA was determined by a CTAB-dependent spectrophotometric assay, as previously described [P < 0.05.For statistical analysis of cell growth and γ-PGA titers, the data set from each experiment was treated individually with the Baranyi model fitted to each model. Data from each experiment under all studied conditions were pooled and statistically analyzed using SPSS software (version 11.5), using a two-factor ANOVA, where the factors were biomass and γ-PGA titer, followed by a Tukey test, with significant differences at https://www.ezbiocloud.net/) showed that these strains belonged to different species  were amplified and sequenced. BLAST analysis using EzBioCloud , but the γ-PGA titer normalized to the biomass of the sucrose-based medium was higher than that of the medium with glucose as the carbon source   Fig. a. Carbonnthetase . This coentation . Howeveriamensis . When gliamensis .Fig. 2OnP > 0.05). In general, γ-PGA production was higher with inorganic nitrogen sources than with organic nitrogen sources, but organic nitrogen sources promoted cell growth, consistent with several previous reports [4+ reacts with α-ketoglutarate in the tricarboxylic acid cycle to form glutamate, which contributes to γ-PGA production [Experiments on the effect of nitrogen sources on cell growth and γ-PGA production were conducted in sucrose-based medium. As shown in Fig.  reports . Under toduction , 18.Bacillus strains, the optimal temperature for γ-PGA production is approximately 37 °C [Figure ly 37 °C , 39.B. tequilensis BL01, the strain was inoculated in the medium by adding yeast extract, (NH4)2SO4, sodium citrate, or sodium glutamate as a nitrogen source to test cell growth and γ-PGA production. As shown in Fig. 4)2SO4 to the medium. No γ-PGA was produced by adding only (NH4)2SO4 and yeast extract to the medium. When 5 g/L sodium glutamate was added, 0.6 ± 0.1 g/L γ-PGA was produced in 12 h, which was then rapidly consumed 2SO4 in the medium not only maintains cell growth, but also promotes γ-PGA synthesis.γ-PGA producing strains are divided into glutamic acid-dependent and glutamic acid-independent strains. For glutamic acid-independent strains, the carbon source can synthesize γ-PGA directly , 18, 20.med Fig. b. The re4+ and α-ketoglutaric acid are converted to glutamic acid by the action of aminotransferase [pgsBCAE) [+ in the medium to produce glutamic acid. The results showed that B. tequilensis BL01 was a glutamic acid-independent strain for γ-PGA production. The γ-PGA produced in sodium glutamate medium decreased after 12 h, which is consistent with the idea that PGA depolymerase exists in Bacillus spp. [4+, resulting in insufficient synthesis of glutamate. Therefore, γ-PGA cannot be produced.Free NHnsferase , 23. Glunsferase , and thelus spp. . Yeast eB. tequilensis BL01, the PGA depolymerase genes, including gamma-DL-glutamyl hydrolase (pgdS) and membrane-bound gamma-glutamyltranspeptidase (ggt) were deleted to obtain B. tequilensis BL01ΔpgdS and the double deletion strain B. tequilensis BL01ΔpgdSΔggt. The growth profiles and γ-PGA production of the mutants and the wild-type strain were tested in medium supplemented with sodium citrate, as shown in Fig. B. tequilensis BL01ΔpgdSΔggt, yielding 11.9 ± 0.6 g/L γ-PGA compared with 7.8 ± 0.3 g/L from the wild type (a 52.6% increase). B. tequilensis BL01ΔpgdS showed γ-PGA production similar to that of the wild-type strain. Furthermore, it was found that after the knockout of pgdS and double deletion of pgdS and the ggt gene, the decomposition rate of γ-PGA was significantly reduced by prolonging the culture time. For all strains, lactic acid was consumed at the later stage of fermentation  and the 2-oxoglutarate dehydrogenase E1 component (sucA) were deleted to block the 2,3-BD synthesis pathway and tricarboxylic acid cycle (TCA). It was found that deletion of the alsS gene was not conducive to strain growth, and that there was no γ-PGA production. The titer of acetic acid was increased by 20% compared to the wild-type strain and became the main byproduct  higher than that of BL01, while cell growth was slower than that of BL01. Furthermore, the double deletion strain B. tequilensis BL01ΔsucAΔpgdS and the triple deletion strain B. tequilensis BL01ΔsucAΔpgdSΔggt produced more γ-PGA, and the titers reached 10.3 ± 0.2 g/L and 11.4 ± 0.7 g/L, respectively  was deleted to block the TCA cycle; thus, cell growth was lower than that of the wild-type strain, but the reduced degradation of α-ketoglutarate increased the γ-PGA titer. Interestingly, the growth rate of γ-PGA was much higher in the B. tequilensis BL01ΔsucAΔpgdSΔggt strain than in the B. tequilensis BL01ΔpgdSΔggt strain before 24 h  , cryptic glutamate dehydrogenase (gudB), and NAD-specific glutamate dehydrogenase (rocG) in B. tequilensis BL01 were removed to construct strains B. tequilensis BL01ΔproB, B. tequilensis BL01ΔgudB and B. tequilensis BL01ΔrocG. When glutamate degradation genes were knocked out, there was a significant increase in glutamic acid production compared to the control  compared to the strain B. tequilensis BL01ΔgudB (11.4 ± 0.6 g/L)  [rocG was more prone to glutamate degradation. Therefore, the deletion of rocG can activate glutamate synthesis and improve the titer of γ-PGA. The gudB gene, which harbors an insertion of three amino acids, encodes an inactive glutamate dehydrogenase in B. subtilis [B. tequilensis BL01, the insertion of three amino acids does not exist, suggesting that glutamate degradation occurs. The gudB mutant strain presented a higher γ-PGA production than B. tequilensis BL01ΔrocG, and gudB may play a more important role than rocG. The gudB and rocG double mutant strain presented lower γ-PGA production than the gudB or rocG mutant strains, possibly caused by the obvious growth defect in the fermentation medium  , 46. In subtilis . Howeversubtilis , 49. In gudB, rocG and proB genes), another way is to obtain sufficient precursors through enhanced metabolism. α-Ketoglutaric acid is an important intermediate metabolite in the TCA cycle, and glutamic acid is generated from α-ketoglutaric acid. However, a large amount of byproducts  are converted from pyruvate, which reduces the metabolic flow of carbon sources to glutamate [B. tequilensis BL01, including pyruvate dehydrogenase (pdhABCD), pyruvate carboxylase (pycA), pyruvate kinase (pyk), citrate synthase (citZ), isocitrate dehydrogenase (icd), glutamate synthase (gltA) and polyglutamate synthase (pgsBCAE).Accumulating more precursors (citric acid and glutamic acid) is effective in increasing γ-PGA production. To improve the supply of precursors, in addition to reducing glutamate consumption , but transformation into B. tequilensis BL01 always failed (data not shown). In addition, the overexpression plasmids with significant effects on γ-PGA production were transferred to B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB to obtain different engineered strains. The γ-PGA production of strain B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB:P43-citZ-pyk was increased to 18.0 ± 0.3 g/L, increase of 23.3% compared to that of the host strain (14.6 ± 0.6 g/L). Unfortunately, overexpression of the pdhABCD and pgsBCAE genes in B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB reduced γ-PGA production, and the γ-PGA titers obtained in the two engineered strains were only 11.4 ± 0.9 g/L and 10.3 ± 0.4 g/L, respectively  gene, resulting in a 34.93% increase in the titer of γ-PGA in B. licheniformis WX-02 [pdhABCD gene in B. tequilensis BL01 showed positive results for γ-PGA synthesis. This suggests that for enhanced precursor synthesis, the rate-limiting step is reflected in the direction of pyruvate metabolism toward the branch of the TCA cycle . The highest productivity of γ-PGA reached 0.84 g/L/h after 30 h of cultivation  or high MW (2.06 × 106 Da), respectively.To further increase the level of γ-PGA using BL01 and the engineered strain, a scale-up experiment was carried out on a 5 L fermenter by initially adding 10 g/L sodium citrate to the culture medium. As shown in Fig. ion Fig. b. The ciion Fig. c. 2,3-BDion Fig. d, the 2,B. tequilensis BL01 strain: deleting the γ-PGA degrading enzyme genes, reduces glutamate consumption and enhances precursors generation. Although the reported metabolic engineering strategies were already employed elsewhere alone, there can be some variation in the results, probably due to the differences in strains [B. tequilensis BL01, and the finally obtained BL01ΔpgdSΔggtΔsucA ΔgudB:P43-citZ-pyk strain could produce 25.2 g/L γ-PGA in a 5 L fermenter. During fed-batch fermentation, the production of γ-PGA increases the viscosity of the medium and thus reduces dissolved oxygen and mass transport. Although the remaining high dissolved oxygen (≥ 20%) was beneficial to improve γ-PGA production [B. tequilensis BL01 to produce more energy, accelerating growth and γ-PGA synthesis. It is not feasible to maintain high dissolved oxygen by fermentation alone; future research will employ metabolic engineering strategies to improve dissolved oxygen, such as the expression of Vitreoscilla hemoglobin (VHb) in B. tequilensis BL01 [In this study, the “push-pull-inhibit” strategy was applied to engineer the  strains , 44, 45.oduction , a furthoduction . Dissolvsis BL01 .B. tequilensis BL01 is a glutamic acid-independent strain for γ-PGA production. However, the strain itself cannot synthesize more citric acid to sustain cell growth and γ-PGA synthesis, and we must add a small amount of citric acid as a precursor. Interestingly, the yield reached 1.50 g γ-PGA/g citric acid, because citric acid can be converted from sucrose through the tricarboxylic acid cycle [4–10 × 106 Da [5 Da) is typically used in food, cosmetics, and agriculture [6 Da) is helpful for mediating antitumor immunity and may be a promising candidate for cancer immunotherapy [6 Da) have critical applications as bioadhesive hydrogels [id cycle . Future × 106 Da . Low-moliculture , 4, 11. otherapy . Composiydrogels . Here, wB. tequilensis BL01 for the efficient synthesis of γ-PGA through a systematic genetic engineering strategy. First, the culture conditions were optimized to improve γ-PGA production. Then genes related to the glutamate synthesis pathway were overexpressed or removed to systematically optimize the metabolic network of B. tequilensis BL01 in the synthesis of γ-PGA. The final obtained strain BL01ΔpgdSΔggtΔsucAΔgudB:P43-citZ-pyk could produce 18.0 ± 0.3 g/L and 25.3 ± 0.8 g/L γ-PGA in the flask and 5 L fermenter, respectively, which was 2.17-fold and 1.32-fold higher than that of the strain B. tequilensis BL01 strain. The molecular weight of γ-PGA (2.06 × 106 Da) produced by the mutant strain was more than 10 times higher than that produced by the wild-type strain (1.84 × 105 Da). These strategies have significantly increased the titer of γ-PGA, and more work must be done to improve the γ-PGA economy.This work constructed a strain of Additional file 1: Figure S1. Neighbor-joining tree based on the 16S rDNA gene sequence showing relationships between BL01 and Bacillus species. Bootstrap values greater than 50 are indicated at the branch nodes. Bar, 0.005 substitutions per nucleotide position. T, representative type strain. GenBank accession numbers of 16S rDNA appear in brackets. Figure S2. The γ-PGA titer normalized to the biomass of the individual cultures. (a) Effect of carbon sources; (b) Effect of nitrogen sources; (c) Effect of temperature. Values represent the mean ± SD, n=3. Figure S3. Effect of disruption of pgdS and ggt genes in the B. tequilensis BL01 strain. (a) Change curves of lactic acid with fermentation time, (b) Change curves of acetic acid with fermentation time, (c) Growth curves of 2,3-BD with fermentation time. Values represent the mean ± SD, n=3. Figure S4. Effect of disruption of sucA and alsS genes in the B. tequilensis BL01 strain. (a) Change curves of acetic acid with fermentation time, (b) Change curves of lactic acid with fermentation time. Values represent the mean ± SD, n=3. Figure S5. The glutamic acid curve of B. tequilensis BL01 and mutant strains. Values represent the mean ± SD, n=3. Figure S6. The growth curve of B. tequilensis BL01 and engineered strains in the medium without citric acid. Values represent the mean ± SD, n=3. Figure S7. Time curves of fed-batch fermentation of γ-PGA in a 5 L fermenter by BL01 and BL01ΔpgdSΔggtΔsucAΔgudB:P43-citZ-pyk strain, (a) 2,3-BD growth curves, (b) acetic acid growth curves. Data are given as the mean ± SD, n=3. Aeration rate: 10 mL/min; agitation rate: 400 rpm (0–6 h), 500 rpm (6–12 h), 600 rpm (12–24 h), 700 rpm (24–48 h); the pH was controlled at pH 6.5±0.2. 20g of sodium citrate dissolved in 50 mL sterile water was fed into the fermenter at 24h, and 150 mL of feed medium containing 700 g/L of sucrose was fed at 30 and 36 h, individually. Figure S8. The molecular weight of γ-PGA produced by (a) B. tequilensis BL01 and (b) B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB:P43-citZ-pyk strain. Table S1. Strains and plasmids used in this study. Table S2. Primers used in this study. Table S3. γ-PGA production by the isolated strains.


There are only two types of intermolecular interactions in the crystal structure: strong hydrogen-bonding C—H⋯O interactions and π–π stacking interactions.The hydrazone form is the predominant form in the solid state. The naphthol and benzene fragments attached to the –N=N– moiety adopt the  16H11N3O3, belongs to the family of azo dyes. In the light of a single-crystal X-ray study, it is evident that of the tautomeric forms (azo–hydrazone), the hydrazone form is the predominant form in the solid state, namely, (1E)-1-[2-(3-nitrophenyl)hydrazin-1-ylidene]-1,2-dihydronaphthalen-2-one. The naphthol and benzene fragments attached to the –N=N– moiety adopt the s-trans conformation. Furthermore, the molecules are nearly coplanar, subtending a dihedral angle of 2.63 (5)°. An intramolecular N—H⋯O hydrogen bond occurs. There are only two types of intermolecular interactions in the crystal structure: strong hydrogen-bonding C—H⋯O interactions and π–π stacking interactions. The importance of C—H⋯O interactions in the molecular packing is reflected by the relatively high contributions (28.5%) made by O⋯H/H⋯O contacts to the Hirshfeld surface.The title compound, C Structural information obtained from single-crystal X-ray diffraction analysis including conformation, stereochemistry, intra- and intermolecular interactions is related to the optical properties of azo dyes , 1.394 (5), 1.330 (5) and 1.276 (5) Å, respectively, indicating that the dye compound has crystallized in the hydrazone tautomeric form (i.e. proton transfer from the naphthol group to the azo group); bond lengths and angles are within normal ranges and are comparable to those reported for other azo compounds °, indicating significant electron delocalization within the molecule. The molecular structure is stabilized by an intramolecular N—H⋯O hydrogen bond involving hydrogen atoms from the hydrazo groups  = 3.607 (2) Å where Cg1 and Cg2 are the centroids of the C1–C6 and C7–C12 rings, respectively].In the crystal, molecules are held together by strong intermolecular C—H⋯O hydrogen bonds Table 1, formingon Fig. 2. Cohesioon Fig. 2, the mos4.et al., 2016E)-2-(5-chloro-2 hydroxyphenyl)hydrazin-1-ylidene]naphthalen-2(1H)-one -1-(4-fluorophenyl)-2-diazen-1-ium  naphthalen-2-ol ligand  a.u. to 1.2354 (blue) a.u. The donors and acceptors of intermolecular C—H⋯O closest interactions in the structure are seen as bright-red spots near the benzene-H2, naphthalene-H9, hydroxyl-O1 and nitro-O3 atoms. The Hirshfeld surface mapped over shape-index is shown in Fig. 5The supramolecular interactions in the title structure were investigated quantitatively and visualized with 6.M) and 6 mL of water were added to 3-nitroaniline (0.02 mol) at 273 K. Sodium nitrite solution  was added dropwise to the cooled mixture and stirred for 15 min. To the formed diazonium salt was added dropwise an aqueous solution of 2-naphthol (0.02 mol in 100 mL of water) containing sodium hydroxide (16 mL). The mixture was then allowed to stir for 1 h at 273 K. The resulting red precipitate was filtered and washed several times with distilled water and dried in air. Red needle-shaped crystals suitable for X-ray analysis were obtained by slow evaporation of an ethanol solution at room temperature (yield 85.4%).The title compound was obtained through the diazotization of 3-nitroaniline followed by a coupling reaction with 2-naphthol. A solution of hydrochloric acid (12 7.Uiso(H) = 1.2Ueq(N). The other hydrogen atoms were placed in calculated positions with C—H = 0.93 Å and refined using a riding model with fixed isotropic displacement parameters [Uiso(H) = 1.2Ueq(C)].Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023001068/zn2025sup1.cifCrystal structure: contains datablock(s) global, I. DOI: Click here for additional data file.10.1107/S2056989023001068/zn2025Isup2.cmlSupporting information file. DOI: 2239846CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The co-crystal is sustained by I⋯N halogen bonds where the photoproduct acts as a linear linker while the donor behaves as a bent two-connected node within the zigzag chain.The formation of a halogen-bonded zigzag molecular network based upon 1,2-diiodoperchlorobenzene and the head-to-tail photoproduct  6I2Cl41,2-C) while the acceptor is a head-to-tail photoproduct, namely rctt-1,3-bis(pyridin-4-yl)-2,4-diphenylcyclobutane (ht-PP). In this co-crystal ·(ht-PP), the donor acts as a bent two-connected node while the acceptor behaves as a linear linker to form the extended solid. Neighbouring chains pack in a tongue-and-groove-like pattern that engage in various Cl⋯π interactions to both the phenyl and pyridyl rings resulting in a supramolecular two-dimensional sheet.The formation and crystal structure of a zigzag molecular network held together by I⋯N halogen bonds is reported. In particular, the halogen-bond donor is 1,2-diiodoperchlorobenzene ( In both networks, the halogen-bond donor was 1,4-diiodoperchlorobenzene, which acted as a linear linker due to the para-position of the two I-atoms.A continued area of research within crystal engineering is the design and formation of supramolecular networks that have specific and targeted structures  to act as a halogen-bond donor ·(ht-PP) that has a zigzag topology due to the ortho-position of the I atoms on the halogen-bond donor. This co-crystal is sustained by I⋯N halogen bonds where neighbouring chains pack in a tongue-and-groove-like pattern. These neighbouring chains engage in various Cl⋯π interactions to both the phenyl and pyridyl rings on the photoproduct, resulting in a supramolecular two-dimensional sheet.Using this as inspiration, a research project was undertaken to exploit the ability of 1,2-diiodoperchlorobenzene ·(ht-PP) crystallizes in the centrosymmetric monoclinic space group P21/n. The asymmetric unit contains a full molecule of both 6I2Cl41,2-C and ht-PP  and 70.9 (1)°] and two obtuse [101.9 (1) and 121.0 (1)°] angles between neighbouring aromatic rings within the photoproduct  and 2.927 (6) Å along with bond angles for C27—I1⋯N1 and C28—I2⋯N2i of 177.8 (2) and 175.6 (2)°, respectively  or phenyl rings [3.288 (4) and 3.842 (4) Å]. These distances were measured from the chlorine atom to the centroid of the aromatic ring ·(ht-PP). The polymeric chain is sustained by I⋯N halogen bonds between 6I2Cl41,2-C and the photoproduct ht-PP.These zigzag chains interact with nearest neighbours by various Cl⋯π interactions Fig. 3. In partet al., 2021dnorm cyclobutane-1,3-diyl)dipyridine and 4,4′-cyclobutane-1,3-diyl)dipyridine, respectively.A search of the Cambridge Crystallographic Database , methylene chloride, and toluene were all purchased from Sigma-Aldrich Chemical  and used as received. In addition, 4,6-dichlororesorcinol , 4-stilbazole (SB), and sodium hydroxide pellets were also purchased from Sigma-Aldrich and were used as received. The [2 + 2] cycloaddition reaction was conducted in an ACE Glass photochemistry cabinet using UV radiation from a 450 W medium-pressure mercury lamp. The occurrence and yield of the [2 + 2] cycloaddition reaction was determined by using 1H Nuclear Magnetic Resonance Spectroscopy on a Bruker Avance 400 MHz spectrometer with dimethyl sulfoxide (DMSO-d6) as the solvent. The halogen-bond donor 1,2-diiodoperchlorobenzene  was synthesized utilizing a previously published method ·(SB) was achieved using a previously published approach ·(SB) were formed by dissolving 50.0 mg of SB in 2.0 mL of ethanol, which was then combined with a separate 2.0 mL ethanol solution containing 24.7 mg of 4,6-diCl res . Then the resulting solution was allowed to slowly evaporate. After evaporation of the solvent, the remaining solid was removed and placed between Pyrex glass plates for irradiation. After 20 h of UV exposure, the [2 + 2] cycloaddition reaction occurred with a 100% yield. The formation of ht-PP was confirmed by 1H NMR . The 4,6-diCl res template was then removed by a base extraction with a 5.0 mL of a 0.2 M sodium hydroxide solution that was heated and stirred on a hot plate for 10 minutes. Afterwards, ht-PP was extracted by using three 10 mL aliquots of methylene chloride as the solvent. Then the methylene chloride was removed under vacuum to yield pure ht-PP. The formation of ·(ht-PP) was achieved by dissolving 25.0 mg of 6I2Cl41,2-C in 2.0 mL of toluene and then combined with a 3.0 mL toluene solution containing 19.4 mg of ht-PP . Within two days, single crystals suitable for X-ray diffraction were formed upon loss of some of the solvent by slow evaporation.6.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989022004200/dx2043sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022004200/dx2043Isup4.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022004200/dx2043sup3.docxNMR Data. DOI: 2133162CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


IL12 or a subunit of its receptor. IL23R deficiency in MSMD has only been reported once, in two pediatric patients from the same kindred with isolated disseminated Bacille Calmette-Guérin disease. We evaluated the impact of a homozygous stop mutation in IL23R (R381X), identified by whole exome sequencing, in an adult patient with disseminated non-tuberculous mycobacterial disease.Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity. The most frequent genetic defects are found in We performed functional validation of the R381X mutation by evaluating IL23R expression and IL-23 signaling  in primary cells  and cell lines (HeLa) with or without back-complementation of wild-type IL23R.+, CD8+ T, and mucosal-associated invariant T (MAIT) cells, and low frequencies of circulating Th17 (CD3+CD45RA−CCR4+CXCR3−RORγT+), Th1* (CD45RA−CCR4−CXCR3+RORγT+), and MAIT (CD3+CD8+Vα7.2+CD161+) cells. Although the patient did not have a history of recurrent fungal infections, impaired Th17 differentiation and blunted IL-23-mediated IL-17 secretion in PBMCs were observed.We report on a 48-year-old male with disseminated non-tuberculous mycobacterial disease. We identified and characterized a homozygous loss-of-function stop mutation underlying IL23R deficiency, resulting in near absent expression of membrane bound IL23R. IL23R deficiency was characterized by impaired IL-23-mediated IFN-γ secretion in CD4IL23R underlies MSMD, corroborating earlier findings with a homozygous p.C115Y IL23R mutation. Our report further supports a model of redundant contribution of IL-23- to IL-17-mediated anti-fungal immunity.1We demonstrate that impaired IL-23 immunity caused by a homozygous R381X mutation in The online version contains supplementary material available at 10.1007/s10875-022-01320-7. CYBB, IFNGR1, IFNGR2, IFNG, IL12RB1, IL12B, IL23R, IL12RB2, ISG15, IRF8, JAK1, NEMO, RORC, SPPL2A, STAT1, TBX21, TYK2, USP18, ZNFX1). The different genetic disorders are further defined by the nature of the causal alleles (null or hypomorphic), protein levels , and the mode of inheritance  and whether they present as isolated MSMD or as a part of a broader spectrum (syndromic MSMD) involving infectious susceptibility to other pathogens and/or autoinflammation [IL12 or a subunit of its receptor (AR complete IL12p40 deficiency or AR IL12RB1 deficiency), followed by defects in the IFN-γ receptor  [IL23R or IL12RB2, only a single kindred with functional validation of the corresponding genetic defect has been described [Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity –3. Patieammation . The mosescribed . This isescribed . In caseFor gDNA extraction from whole blood, Purelink DNA Genomic DNA Mini kit (ThermoFisher Scientific) was used according to the manufacturers’ instructions. Whole exome sequencing was performed using SureSelect Human All Exon V7 (Agilent) for exome capture .Whole blood was diluted 1:1 with RPMI 1640 and layered over lymphocyte separation medium (LSM) . Tubes were centrifuged at 400 G for 25 min and PBMCs were harvested and stored in liquid nitrogen until FACS staining or used immediately for functional assays.mRNA was isolated from PBMCs using TRIzol reagent (Life Technologies). Five hundred microliters of TRIzol was added to PBMCs and frozen at − 80 °C. Later, samples were thawed and mRNA isolated according to manufacturer instructions. Complementary DNA (cDNA) was synthesized from RNA using the GoScript™ Reverse Transcription System (Promega) according to manufacturer instructions.Forward and reverse primers were purchased from Integrated DNA Technologies (IDT). A Taqman probe was designed on exon 2 (ccagacatgaatcaggtcactattcaatgg) with primers located on the span junction of exon1-2 (forward primer tcaaacaggttgaaagagggaaac) and exon 2–3 (reverse primer tcctccatgacaccagctga) and a Taqman probe on exon 8 (tgatcgtctttgctgttatgttgtcaattctttct) with primers on the span junction of exon 7–8 (forward primer acagggcaccttacttctgacaac) and on exon 8 (reverse primer agttcggaatgatctgttaaatatccc) HPRT1 or GAPDH was used as housekeeping gene for all performed qPCRs. The reaction was performed as followed: 0.6 µl of primers (0.30 µM), 0.4 µl of probe (0.25 µM), 6 ng of DNA (2 ng/µL) and 1 × Taqman Fast mastermix (ThermoFisher Scientific) were mixed and plated on a 96-well plate. The plate was run on the StepOneTM Real-Time PCR system (ThermoFisher Scientific) and analyzed with StepOne Software v2.3.5 cells/mL in a 48-well plate and preactivated with plate-bound anti-CD3 antibody  and soluble anti-CD28 antibody  for 3 days, before stimulation with IL-12.STAT3 and 4 phosphorylation was assessed by flow cytometry and Western blot after stimulation. For stimulation, fresh PBMCs or EBV-transformed B cells were plated overnight, and stimulated for 30 min with IL-23 , IL-6 , IFN-α2b , IL-12  in complete RPMI 1640 medium  (GibcoTM). For STAT4-phosphorylation, PBMCs were seeded at 2 × 10PBMCs or EBV-B cells were lysed in lysis buffer  and 1 × phosphatase inhibitor ). Protein concentrations were determined using a Bradford Protein Assay (Bio-Rad). Protein lysate was denaturized in LDS  and DTT (Bio-Rad) at 70 °C for 10 min and was then loaded on a 4–12% Bis–Tris polyacrylamide gel  in MOPS buffer . Separated proteins on the gel were transferred onto a methanol-activated PVDF membrane  in transfer buffer ) using the Tetra Blotting Module (Bio-Rad). The PVDF membrane was blocked in 5% milk Tris-buffered saline with Tween 0.1% (TBS-T) for 30 min at RT and then incubated with primary antibody O/N at 4 °C followed by a wash with TBS-T and incubation with a secondary antibody for 1 h at RT. Primary antibodies used for Western blot: STAT3 , pSTAT3  STAT4 , p-STAT4  and mouse anti-GAPDH (Invitrogen). Secondary antibodies used were conjugated with HRP: goat anti-rabbit (abcam) and anti-mouse (Rockland). Secondary antibodies were visualized using ECLTM15 Prime Western Blotting Detection Reagent (AmershamTM) with the G:box Chemi-XRQ and quantified using ImageJ.pWPXL_EF1a_hIL23R-3HA_Ires_eGFP was generated using Gibson Assembly (NEB). First, hIL23R-3HA gene sequence was PCR amplified from the pCH_SFFV_hIL23R_3HA_tCD34 plasmid  using primers: FW: 5’- acgggatccaggcctaagcttacgcgtgccaccatgaatcaggtcact-3’, RV: 5’-agtcgactcatatatcggaattcctaggcataatcaggcacgtcata-3’. Next, the amplified hIL23R-3HA was inserted into pWPXL_EF1a_Ires_eGFP transfer plasmid, previously digested with EcoRI and MluI restriction enzymes. pWPXL_EF1a_hIL23R-3HA_IRES_eGFP plasmid was transformed into DH5a bacterial competent cells and plasmids extracted by midi-prep (Macherey–Nagel). For IL12RB1 and IL23R (F380-HA) a commercial plasmid (cat nr. SC1200 and OHU25802D) was bought from GeneScript in a pcDNA3.1 vector containing respectively a C-terminal Myc or HA tag.pWPXL_EF1a_hIL23R-3HA_IRES_eGFP was mutagenized to insert the studied hIL23R variants (p.R381Q and p.R381X). pWPXL_EF1a_hIL23R-3HA_IRES_eGFP was PCR amplified by Q5 high fidelity DNA polymerase (NEB) using the following primers set: FW: 5’- atttaacagatcattccaaactgggattaaaag-3’, RV: 5’-cttttaatcccagtttggaatgatctgttaaat-3’ (p.R381Q). FW: 5’-atttaacagatcattctgaactgggattaaaag-3’, RV:5’- cttttaatcccagttcagaatgatctgttaaat-3’ (p.R381X). KLD enzyme Mix (NEB) was used to degrade the DNA template by DpnI. The mutagenized plasmids were transformed into DH5a bacterial competent cells and plasmids extracted by midi-prep (Macherey–Nagel).5 cells/well in DMEM medium supplemented with FBS 5%, HEPES 25 mM and MEM non-essential amino acids) (GibcoTM). When reaching 70% confluency, transfection of C terminal HA tagged plasmids , IL23R-R381Q, IL23R-R381X, IL23R-F380) was performed using Lipofectamine 3000 (ThermoFisher Scientific) according to the manufacturer’s protocol. After 4 h, the medium was changed and cells were rested overnight. Subsequently, HeLa cells were stimulated with IL-23  or IFN-α2b  for 30 min. Afterwards, cell lysis, protein extraction, quantification, and Western blot were performed as described before (section Western Blot for STAT3 phosphorylation).HeLa or HEK293T cells were seeded in a 6-well plate at a density of 1 × 104 cells/well on a 12-mm coverslip precoated with poly-D-lysine (Sigma). Transfection was performed as described above. Coverslips were washed in PBS 1 × and fixed with paraformaldehyde 4% for 20 min. Afterwards, they were permeabilized (Triton 0.2% in PBS 1 ×), blocked , and stained with anti-HA , anti-Myc , anti-GFP  in dilution buffer  overnight. Afterwards, coverslips were washed (Triton 0.05% in PBS 1 ×) and stained for 1 h with secondary antibodies  and AF568 anti-mouse ). Subsequently, coverslips were washed. Images were acquired using a Nikon AXR confocal unit, with a 60 × NA 1.4 oil objective at a 2048 × 2048 pixels resolution.HeLa cells were seeded in a 48-well plate at a density of 1 × 10Lentiviral vectors LV.EF1a:hIL23Rwt-3HA-IRES-eGFP and LV.EF1a: eGFP were generated by co-transfection of HEK293T cells with p.sPAX2, p.MD2.G-VSV.G and the transfer plasmids pWPXL_EF1a_hIL23Rwt-3HA_Ires_eGFP and pWPXL_EF1a_ eGFP by using X-tremeGENE HP Transfection Reagent (Sigma-Aldrich). pMD2.G and psPAX2 were a gift from Didier Trono .5 cells in 200 μL of cRPMI and transduced using LV vectors as described above. After adding the lentiviral vectors, cells were spinoculated for 90 min at 35 °C and 800 G. Afterwards, they were resuspended and medium was changed at day 1 and 3. At day 4, cells were centrifuged and resuspended in stimulation medium  for 60 min. Subsequently, cells were lysed and Western blot was performed as described above (section Western Blot for STAT3 phosphorylation).EBV-B cells were plated in a 96-well plate at a seeding density of 1 × 10PBMCs were stained with fixable viability dye Zombie Aqua at room temperature for 10 min, washed twice in FACS buffer (3% FBS in PBS1 ×) followed by staining for the following surface markers before fixation and permeabilization: CD3 , CD4 , CD8 . Cells were fixed with BD cytofix for 10 min at 37 °C. Afterwards, permeabilization was performed using BD phosflow Perm III  on ice for 30 min followed by intracellular staining with p-STAT3  or p-STAT4  for 60 min at 4 °C.6 for each sample. Cells were plated in a V-bottom 96-well plate, washed once with PBS (Fisher Scientific), and stained with live/dead marker and fluorochrome-conjugated antibodies recognizing surface markers: anti-CD8-BUV805 (clone SK1), anti-CD4-BUV496 (clone SK3), anti-CD95-BUV737 (clone DX2), anti-CD28-BB660-P (clone CD28.2), anti-ICOS-BB630 (clone DX29), anti-CXCR3-BV785 (clone 1C6), anti-PD-1-BV750-P (clone EH12.1), anti-CXCR5-BV650 (clone RF8B2), anti-CCR2-BV605 (clone 1D9) anti-CD31-BV480 (clone WM59), ; anti-CD3-PerCP-Vio700 (clone REA613) (Miltenyi Biotec); anti-CD45RA-FITC (clone HI100), anti-CD14-PE-Cy5.5 (clone TuK4), anti-CCR7-PE-Cy7 (clone 3D12) ; anti-CD25-BV711 (clone BC96), anti-HLA-DR-BV570 (clone L243), anti-CD127-BV421 (clone A019D5), anti-CCR4-PE/Dazzle 594 (clone L291H4) . Samples were stained for 60 min at 4° C, washed twice in PBS/1% FBS (Tico Europe), and then fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Set (eBioscience), according to manufacturer’s instructions. Cells were stained overnight at 4° C with anti-Ki67-BUV615-P (clone B56), anti-CTLA-4-PE-Cy5 (clone BNI3), anti-RORγt-PE (clone Q21-559) , and anti-FOXP3-AF647 (clone 206D) (BioLegend) anti-human intracellular antibody, and were then acquired on a Symphony flow cytometer with Diva software (BD Biosciences). A minimum of 5 × 105 events were acquired for each sample. Compensation Beads (ThermoFisher Scientific) were used to optimize Fluorescence compensation settings for multi-color flow cytometric analysis at a Symphony flow cytometer.Frozen PBMCs were thawed and counted, and cell concentration was adjusted to 5 × 10PBMCs were isolated as described above and plated overnight. On the next day, cells were washed in PBS 1 × and stained for 30 min with anti-CD3 , anti-CD4  , IL23R , and Zombie Aqua fixable viability dye. Cells were washed in FACS buffer (FBS 3% in PBS 1 ×) and stained for 30 min with secondary AF647 anti-rabbit . Data acquisition was performed using the BD FACSCanto II and analyzed using FlowJo software.Naïve T cells were isolated from PBMCs by magnetic purification . They were cultured in Th0 conditions  or Th17 , IL-1β 10 ng/mL (Peprotech), IL-23 10 ng/mL (R&D 1290-IL), IL-6 10 ng/mL (Peprotech), anti-IL-4 µg/mL, anti-IFNγ 20 µg/mL) for 7 days. Supernatant was used for measurement of IL-17 by ELISA according to the manufacturer’s instructions .5 cells/mL with addition of soluble anti-CD28  in RPMI 1640 medium  (GibcoTM). At day 1 of culture, IL-23  and IL-12  were added. At day 7, Brefeldin A  was added for 4 h to the culture to assess intracellular cytokine production by flow cytometry. For NK and iNKT cells, fresh PBMCs were stimulated for 48 h with IL-23 or IL-12 and Brefeldin A was added during the last 8 h of stimulation. In brief, cells were washed with PBS 1 × , stained with fixable viability dye Zombie Aqua at room temperature for 10 min, washed twice in FACS buffer followed by staining for surface markers: anti-CD3 , anti-CD4 ( Pacific Blue), anti-CD8 , anti-Vα7.2 , anti-CD161 , anti-CD56  for 30 min at 4 °C. Later, cells were permeabilized using eBio Fix/Perm, washed twice in Permeabilization buffer, and stained with anti-IFN-γ  for 30 min at 4 °C. Data acquisition was performed using the BD FACSCanto II and analyzed using FlowJo software. For mycobacterial stimulation, cells were plated in the same medium, left unstimulated or co-cultured with Mycobacterium abscessus strain (ATCC19977) (MOI 2) in the presence or absence of IL-23  or IL-12  for 3 or 7 days. As a positive control, PMA/ionomycin (25 ng/mL + 1 µg/mL) without mycobacteria was used. Supernatant was used to measure IFN-γ by ELISA according to the manufacturer’s instruction .IFN-γ was assessed by flow cytometry and ELISA after stimulation. For alpha beta T cell subset and MAIT stimulation, fresh PBMCs were plated on an anti-CD3  coated 48-well plate at a density of 2 × 10t-test  or Mann–Whitney U test  was used. To compare the effects of stimulation in the healthy control or patient group, a paired Student T test, one-way ANOVA, or a Friedman test with post hoc tests was used depending on normality of the data. Significance levels were defined as followed: *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the standard error of the mean (SEM).Statistical analysis was only performed if three biological replicates were available in each group. To compare between healthy controls and patient, an unpaired Student Mycobacterium avium complex, culture, and PCR proven), bone marrow , and gastrointestinal  manifestations  was found in the patient and in heterozygous state in his mother and all of his siblings except for one. He had one brother who died of lung cancer and two sisters who died at an early age , although information on the cause of death could not be retrieved. The other siblings were healthy. The R381X mutation was reported once in heterozygosity in the healthy population database but was not found in homozygosity . In silico tools (9) predict the mutation to be deleterious  lacking an intracellular domain [IL23R transcript level in PBMCs  and ionomycin were used to upregulate its transcription. Compared to healthy controls, the patient had tenfold lower IL23R mRNA levels both unstimulated and after PMA/ionomycin stimulation. Similar results were achieved using a primer–probe set covering the 3’ region, indicative of nonsense-mediated decay  and IL12RB1 to assess the STAT3 phosphorylation response in HeLa cells. The presence of WT and R381Q IL23R on the membrane after transfection was confirmed by confocal microscopy Fig.  and flowNext, we evaluated IL-23 signaling in patient PBMCs and EBV-B cells using flow cytometry Fig. , FigS2 a+ T cells were decreased and CD8+ T cells increased, but absolute numbers remained within normal range  was also severely decreased in the patient  and T effector memory re-expressing CD45RA (TEMRA) cells (CD3+CD4+CD45RA+CCR7−), while naïve T cells (CD3+CD4+CD45RA+CCR7+) were decreased. In the CD4+ memory compartment (CD3+CD45RA−), the number of Th17 cells (CD3+CD45RA−CCR4+CXCR3−RORγT+) and Th1* (CD3+CD45RA−CCR4−CXCR3+RORγT+) were significantly decreased in the patient  upon stimulation with polarizing cytokines in vitro. IL-17 secretion, a surrogate marker for Th17 differentiation, was low at baseline in the patient and did not increase in a Th17 polarizing condition in contrast to the healthy controls  was used. The addition of IL-23 in the patient did not result in an augmented IFN-γ response compared to Mycobacterium abscessus alone, in contrast to healthy controls where a mild increase in IFN-γ production was seen  mutation as a cause of MSMD. IL23R is a heterodimeric receptor consisting of an IL12RB1 and IL23R subunit. After binding to IL-23, it signals downstream through TYK2 (attached to IL12RB1) and JAK2 (attached to IL23Rα) to induce phosphorylation of STAT3. The role of IL-23 signaling is best studied in the context of inflammatory disease. A well-described example is inflammatory bowel disease, where both mouse models, human observational studies and population genetic studies underscore the importance of IL-23 [IL23a−/− mice have impaired long-term control of pulmonary Mycobacterium tuberculosis infection [IL23R [IL23R is reported but the effect of this variant on IL23R expression and signaling was not reported [We report on a novel, homozygous of IL-23 . The connfection , the assL23R p.R31X mutatin [IL23R . In addireported .IL23R mRNA levels and membrane-bound IL23R. The functional impact of this mutation was assessed by determining STAT3 phosphorylation following IL-23 stimulation in primary cells and in HeLa cells transfected with WT IL23R or the partial LOF variant R381Q [+ and CD8+ T cells in our patient differed from healthy controls in contrast to the previously reported IL23R deficient patient [In contrast to the first reported kindred by Martínez-Barricarte et al. where both patients had a homozygous missense mutation p.C115Y) in the extracellular domain resulting in impaired IL-23 signaling, our patient has an adult-onset phenotype and a stop mutation (p.R381X) located in the intracellular domain before the JAK2 binding site. The mutation resides in a well-conserved residue and was predicted to be LOF using in silico prediction tools. Functional testing revealed that our patient had severely decreased total Y in the nt R381Q . The R38nt R381Q , 17. Fur patient . Moreove patient . In addi+, CD8+) and MAIT cells upon IL-23 stimulation. IL-23 is a less potent IFN-γ inducer in T cells compared to IL-12 as described by Martínez-Barricarte et al., characterizing IL-12- and IL-23-specific effects in different immune subsets [Finally, we assessed IFN-γ secretion by alpha–beta T cell subsets  located in the intracellular domain of IL23R. This report illustrates a mechanism of abolished membrane-bound IL23R expression. Functionally, this negatively affects the development of Th17 and MAIT cells and impacts IL-23-stimulated IL-17 and IFN-γ production by alpha–beta T cell subsets and MAIT cells. Our findings together with the report of a previously validated IL23R patient with MSMD and existing data on mycobacterial susceptibility of Supplementary file1 (PDF 1902 KB)Below is the link to the electronic supplementary material.