Larix olgensis or larch is an economically important coniferous tree species with rapid growth in the early stages, strong adaptability, and a short time to harvest. The genetic improvement of larch has garnered considerable attention in recent years for reclaiming timber forests. However, traditional breeding methods are largely ineffective for achieving rapid genetic improvement of L. olgensis. Studies show that the efficiency of plant regeneration can be improved by optimizing somatic embryogenesis. On this basis, we devised a stable, fast and efficient Agrobacterium-mediated genetic transformation method using suspended embryogenic calluses as explants and β-glucuronidase as the reporter. We evaluated the effects of the Agrobacterium load, co-culture period, and addition of acetosyringone and transformant screening antibiotic on the transformation efficiency. In addition, we tested the pCAMBIA 1300-PtHCA 2-1 promoter-GUS binary expression vector, which contains the GUS gene ORF under the control of Populus trichocarpa high cambial activity PtHCA 2-1 promoter, and observed the tissue-specific expression of the GUS gene in the somatic embryos of transgenic larch. This novel technique can not only accelerate the generation of superior transgenic strains of L. olgensis but also aid in future gene functional studies. Larix olgensis is a coniferous tree distributed across northeastern China, Russia, and Korea, and its timber is used in construction and other industries  are distinct. Our preliminary results showed that the expression rate of exogenous gene in embryogenic callus of L. olgensis infected by A. tumefaciens using previously established protocols was either very low (less than 5%) or undetectable. Therefore, it is essential to develop a stable and efficient genetic transformation system for L. olgensis.According to previous reports , the effAgrobacterium-based transformation method for L. olgensis using embryogenic calluses as the explants. Furthermore, the somatic embryos of transgenic larch transformed with GUS gene ORF under the control of Populus trichocarpa HCA 2-1 promoter expressed the gene in a tissue-specific manner. This novel transformation system can greatly accelerate the development of genetically improved L. olgensis strains.In this study, we have described a novel L. olgensis were collected in May 2014 from the Heilongjiang Province. The average size of the embryo proper obtained after peeling the endosperm was 318.04 ± 83.23 μm. The seeds were sterilized using 75% ethanol for 30 s and then 3% sodium hypochlorite for 10 min, cut longitudinally, and then inoculated onto the basic medium , 0.5 mg⋅L–1 6-benzylaminopurine (6-BA), 0.5 mg⋅L–1 kinetin (KT), 1.0 g⋅L–1 glutamine (Gln), 0.5 g⋅L–1 acid hydrolysis casein (CH), 25.0 g⋅L–1 sucrose, and 6.0 g⋅L–1 agar at pH 6 ± 0.02  at the calluses/medium ratio of 1:100 (w/v). The cultures were maintained with constant shaking at 120 rpm and passaged every 14 days.The immature seeds of dium BM;  suppleme6 ± 0.02 . Callus –1 abscisic acid (ABA), 80.0 g⋅L–1 PEG4,000, 5.0 g⋅L–1 silver nitrate (AgNO3), and 60.0 g⋅L–1 sucrose. Somatic embryo maturation was induced for 7 weeks. Normal cotyledon embryo germination was observed after 7 days of inoculation on L&M woody plant basal medium  supplemented with 20.0 g⋅L–1 sucrose, 4.0 g⋅L–1 agar, 3.0 g⋅L–1 vitamin B1 and 2.0 g⋅L–1 activated carbon under a 16 h/day photoperiod (50 μmol⋅m–2⋅s–1). To increase the efficiency of somatic embryogenesis and optimize the synchronization, embryonic calluses were cultured in synchronized culture medium (SM) before the somatic embryo maturation. To determine the optimum culture period, 0.2 g calluses were cultured on medium containing 1/4 BM basic salts with the addition of 60.0 g⋅L–1 sucrose, 10.0 g⋅L–1 inositol, 1.0 g⋅L–1 Gln, 0.5 g⋅L–1 CH, and 6.0 g⋅L–1 agar for 1, 7, 14, 21, or 28 days in the dark. In addition, different concentrations of sucrose , inositol , Gln , CH  and total, 1/2, or 1/4 BM salts were also tested for 14 days. The process is outlined in L. olgensis were examined for each experiment.The embryogenic suspension calluses were isolated from liquid medium and transferred to maturation medium (MM) consisting of the basic salts of BM supplemented with 20.0 mg⋅L–1 cefotaxime (Cef). After culturing for 14 days at 120 rpm and 23 ± 2°C in the dark, the proliferative calluses were separated as above and weighed, and 0.5 g calluses were seeded on solid PM (6 g⋅L–1 agar) supplemented with various concentrations  of kanamycin (Kan) or  hygromycin (Hyg). The plates were incubated at 23 ± 2°C in the dark for 15 days. Each callus was weighed, transferred to SM (with the same respective concentration of Kan or Hyg), and cultured for 14 days and thereafter to antibiotic-supplemented MM. The number of somatic embryos was counted after 8 weeks. The experiment was repeated thrice, and at least 15 embryogenic calluses were examined each time.The embryogenic calluses were filtered out using a 60-mesh cellular sieve (0.3 mm), and the excess amount of water was absorbed on a sterile filter paper. The calluses were weighed, and 0.25 g was inoculated into 25 ml PM supplemented with 0, 100, 200, 300, or 400 mg⋅LAgrobacterium tumefaciens strain GV3101 was transformed with the pBI121, pCAMBIA 1300, and recombinant pCAMBIA-PTHCA 2-1 Promoter-GUS binary vectors (npt II) as selective markers, and Nos terminator. The plasmid pCAMBIA 1301 contains the hygromycin phosphotransferase gene (hpt) and the GUS gene under the control of CaMV35S. The modified p1300-PtHCA 2-1 Promoter-GUS also contains the GUS gene under the control of the P. trichocarpa PtHCA 2-1 promoter. The cryopreserved transformed bacteria were revived, and a single colony was spread on YEB solid medium containing 50 mg⋅L–1 hygromycin and 50 mg⋅L–1 rifampicin. After incubating the plates at 28°C for 2 days, the bacteria were inoculated into 25 ml YEP medium and cultured overnight at 28°C with constant shaking at 200 rpm till the logarithmic phase. The cells were harvested by centrifuging at 8,000 rpm for 15 min at 4°C and resuspended in liquid BM at the appropriate density. vectors . The pBIA. tumefaciens suspension of OD600 0.2, 0.4, 0.6, or 0.8 with/out 100 μM acetosyringone (AS). The calluses were infected for 15–20 min with periodic shaking to allow even dispersion, separated, dried on a sterile filter paper, and seeded into the PM. The callus bacteria were co-cultivated at 25°C in the dark for 1, 2, 3, or 4 days in the presence or absence of 100 μM AS.Freshly proliferated embryogenic calluses were harvested, and ∼5 g callus was inoculated into 20 ml –1 Cef for 5 min each, dried on a sterilized filter paper, and cut into small pieces of diameter 1 ± 0.2 cm. The pieces were placed on PM supplemented with 200 mg⋅L–1 Cef and cultured at 25°C in the dark for 0, 3, 7, 14, or 21 days. The antibiotic-resistant calluses were selected thrice on PM containing 200 mg⋅L–1 Cef and 20 mg⋅L–1 Kan or 4 mg⋅L–1 Hyg at 25°C in the dark for 21 days, and the number of calluses was counted. All treatments were repeated at least three times, and at least 15 calluses were examined from each independent experiment.The cultured calluses were washed sequentially with sterile water and 500 mg⋅LGUS and npt II genes were amplified by PCR using specific primers  according to the manufacturer’s instructions.  primers  and the D-glucuronide), 5 mM ferro-ferricyanide buffer, 100 mM Na-phosphate buffer (pH 7.0), 10 mM EDTA, and 0.1% Triton X-100 and incubated at 37°C for 7 days. The resistant embryogenic calluses stained blue were counted under a microscope, and the staining intensity was evaluated by extracting the chlorophyll of somatic embryos using 75% (v/v) ethanol.Glucuronidase staining was performed as described previously . Brieflyp < 0.05 was considered statistically significant.SPSS 18.0  was used for all data analyses. Data were compared by ANOVA, followed by Fisher’s LSD test, and After 7 days of synchronized culture, the embryogenic calluses surface changed from translucent to opalescent, and the internal areas were browned 28 days later. Following synchronization, the calluses were exposed to maturation stimuli for approximately 7 days. The pro-embryo masses sprouted short, milky-yellow rod-like protuberances on the surface, which is indicative of close to maturation. After 15 days, some embryos differentiated and formed cotyledons . In contp = 0.000) and inositol (p = 0.002) in the synchronization medium . Taken together, these results indicate that synchronization of L. olgensis embryogenic calluses can accelerate somatic embryo maturation.The number of somatic embryos increased gradually for a short period during synchronous culture (day 1–14), peaked on the 14th day, and decreased after the 21st day . Therefon medium . SomaticL. olgensis embryogenic calluses at concentrations exceeding 200 mg⋅L–1 , Kan (p = 0.000), and Hyg (p = 0.000) significantly influenced the growth of embryonic callus, and Kan (p = 0.000) and Hyg (p = 0.000) affected somatic embryogenesis.Cefotaxime inhibited the proliferation of 0 mg⋅L–1 , whereas0 mg⋅L–1 . In addiy 65.66%  and compAgrobacterium suspension of OD600 0.6 and decreased slightly with a lower bacterial load (OD600 0.4). In contrast, low (OD600 0.2) and high (OD600 0.8) density bacterial suspensions decreased the transformation efficiency. Previous reports show that T-DNA transfer and integration from A. tumefaciens to the recipient plant cells require at least 16 h. Consistent with this, the transformation efficiency of the embryogenic calluses was the highest after 3 days of bacterial co-culture, and Kan-resistant calluses were not formed when the bacteria were removed. Extensive bacterial overgrowth was observed with highly dense Agrobacterium suspension (OD600 > 0.8) and co-culture duration longer than 4 days. The multiple sterilization steps required to remove the bacteria led to considerable loss of the transformed embryogenic calluses. Therefore, to ensure maximum transformation efficiency and transformant recovery, we infected the calluses with Agrobacterium suspension of OD600 0.6 for 15–20 min and co-cultured them for 3 days  was only 0.23 ± 0.08  regulates interfascicular cambium formation and vascular tissue development in Arabidopsis , hpt (hygromycin phosphotransferase gene), and bar (glyphosate acetate transferase gene) are commonly used as selection markers for conifer genetic transformation (L. olgensis was highly sensitive to all antibiotics, and 4 mg⋅L–1 Hyg was selected for screening transformants due to the lower false positive rate.Optimal selection pressure, usually an antibiotic, is another determinant of the efficacy of genetic transformation of conifers ; therefoormation . We founAgrobacterium-mediated transformation of synchronized embryonic calluses can rapidly produce a relatively large number of transgenic somatic embryos or plants and can also be scaled up. Compared with the previous studies on larches (L. olgensis was genetically transformed at significantly higher efficiency under appropriate conditions. This approach not only can accelerate the generation of genetically superior L. olgensis varieties but can also potentially aid protein subcellular localization studies and other applications requiring transgene expression. Our future efforts will focus on further enhancing the transformation efficiency of L. olgensis and conducting functional gene studies.Taken together,  larches , L. olgeL. olgensis embryogenic calluses using A. tumefaciens strain GV3101 expressing pCAMBIA 1300, bacterial density OD600 of 0.6, 3 days of co-culture, 100 μM acetosyringone as inducer, and transformant screening with 4 mg⋅L–1 Hyg. Under these optimum conditions, the transformation efficiency was almost 90%, which is higher compared with that reported for other conifers and larch species. This novel method can potentially accelerate genetic improvement of L. olgensis.We improved the protocol of larch somatic embryogenesis and developed a rapid and simple transformation method for The original contributions presented in the study are included in the article/SL and HZ conceived the study. YS and SL designed the study. YS, XB, and NW performed the experiments. YS, SD, YY, and HD contributed to data interpretation. YS and SL wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We then evaluated the anti-inflammatory effects of SQ and HEHSQ on lipopolysaccharide- (LPS-) stimulated RAW264.7 murine macrophages. To determine the effect of SQ and HEHSQ on the viability of RAW264.7 cells, an MTT assay was performed. To quantify the anti-inflammatory effect of SQ and HEHSQ, we measured nitric oxide (NO) production, gene expression, and secretion of the proinflammatory cytokine tumor necrosis factor α (TNF-α) and chemokine C-C motif chemokine 2 (CCL2) in LPS-stimulated RAW264.7 cells using an in vitro inflammatory model. 2,3-Epoxysqualene was prepared according to a reported methodology. The reaction of 2,3-epoxysqualene and ethylene glycol in 2-propanol produced 49% HEHSQ. MTT results showed that 10 and 100 µg/mL HEHSQ treatment decreased cell viability, whereas SQ treatment (1–100 µg/mL) did not have any effect on viability. SQ (100 µg/mL) and HEHSQ (1 µg/mL) treatment significantly reduced the production of LPS-stimulated NO and decreased the expression and secretion of proinflammatory TNF-α and CCL2. Therefore, our results suggested that the anti-inflammatory effects of HEHSQ are 100 times higher than that of unmodified SQ. To the best of our knowledge, this study has demonstrated for the first time that HEHSQ can be potentially used as a safe alternative treatment to anti-inflammatory drugs.Squalene (SQ), a natural precursor of many steroids, can inhibit tumor progression and decrease serum cholesterol levels. However, it is difficult to discern the effect of highly active molecules in the treatment of diseases because not enough active compounds reach the site of pathology in crowded biosystems. Therefore, it is necessary to design artificial probes that work effectively within crowded systems. In this study, to facilitate cell penetration, the ethylene glycol moiety (used as a probe) was chemically added to SQ to form 2-(2-hydroxyethoxy)-3-hydroxysqualene (HEHSQ). HEHSQ was prepared from 2,3-epoxysqualene and characterized by R Acute iammation , 4. For ammation –7. And Cammation . Therefoammation . Thus, mammation . TherefoAurantiochytrium [Aurantiochytrium) had an anti-inflammatory effect on lipopolysaccharide- (LPS-) stimulated mouse macrophage RAW264.7 cells in an in vitro inflammatory model [Squalene (SQ), a natural precursor of many steroids, is so named because of its presence in shark liver oil. It is also prepared from the microalgae chytrium . Becausechytrium , 13, an chytrium , and forchytrium . Moreovery model . UnfortuIn general, however, cells and tissues of living organisms make up a multimolecular system wherein diverse types of molecules get compartmentalized. Therefore, it is not often possible to use highly active molecules in the treatment of diseases, such as cancer, because enough active compounds cannot reach the site of pathology in crowded biosystems. Therefore, to improve pharmacological activity, artificial probes must be designed that effectively work within the crowded tissue architecture.In the present study, we enhanced the pharmacological activity of SQ by increasing its bioavailability. The ethylene glycol moiety (used as a probe) was selectively introduced into SQ, creating HEHSQ, to facilitate tissue permeability. Also, we investigated the anti-inflammatory effect of SQ and HEHSQ on LPS-stimulated RAW264.7 murine macrophages.All solvents used in the study were of reagent-grade quality and used without further purification unless otherwise noted. 2,3-Epoxysqualene was prepared according to a previously described methodology . All colf, FT-IR, 1H NMR, 13C NMR, and high-resolution mass spectrometry. The infrared (IR) spectrum was measured on a JASCO FT-IR-4100. NMR spectra were recorded on a Bruker Avance-400 spectrometer operated at 400 MHz and a JEOL RESONANCE ECX-100 spectrometer operated at 100 MHz at room temperature (20°C) in the Fourier transform mode. 1H NMR spectra were reported in δ units, parts per million (ppm), and calibrated relative to the signal for residual chloroform (7.26 ppm) in chloroform-d1 (CDCl3). 13C NMR data were reported in ppm relative to CDCl3 (77.16 ppm) and obtained with 1H decoupling. High-resolution mass spectrometric analyses were undertaken using an electrospray ionization time of flight based as a reserpine (m/z 609.2812) matrix on a JEOL JMS-100CS instrument.HEHSQ was characterized by Rin vitro experiment.SQ was purchased from Fujifilm Wako Co, Ltd. . SQ and HEHSQ were dissolved in DMSO to obtain the respective stock solutions, which were then diluted in cell culture medium for the Escherichia coli O111:B4) was purchased from EMD Millipore Co. . A total of 5 mg of LPS was dissolved in 2 mL of phosphate-buffered saline without divalent cations (PBS (−)) and stored at −80°C in the dark until subsequent use.LPS . RAW264.7 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin at 37°C in a humidified incubator containing 5% COµg/mL) and HEHSQ . RAW264.7 cells were seeded at 2 × 105 cells/mL in 96-well plates  and incubated for 24 h. Then, the cells were treated with SQ or HEHSQ for 24 h. A solution of 5 mg/mL MTT dissolved in PBS was added (10 µL/well) and incubated for another 24 h. The resulting MTT formazan was dissolved in 100 µL of 10% sodium dodecyl sulfate (w/v), and the absorbance was measured using a microtiter plate reader . The absorbance values were normalized to that of the culture medium, and viability was calculated as a percentage (%) of the untreated cells.Cell viability and mitochondrial activity were determined using the MTT assay to check the cytotoxicity of SQ  and incubated for 24 h. The cells were treated with SQ  or HEHSQ  at 37°C for 24 h. After the treatment, LPS solution  was added to each well and incubated for 12 h at 37°C. Then, the cell supernatant was mixed at a 1 : 1 ratio with the Griess reagent (1% sulfanilic acid and 0.1% N-(1-naphthyl) ethylene diamine dihydrochloride in 2.5% phosphoric acid). After 10 min incubation in the dark, the absorbance was measured at 540 nm using a microtiter plate reader , and nitrite concentration was determined by dilution of sodium nitrite  as a standard.To evaluate the effect of SQ and HEHSQ on NO production in LPS-stimulated RAW264.7 cells, the Griess reaction was used following the methodology described in a previous study . Briefly5 cells/mL in a 10 cm2 dish (BD BioCoat) and incubated at 37°C for 24 h. The RAW264.7 cells were then treated with SQ (100 μg/mL) or HEHSQ (1 μg/mL) at 37°C for 24 h. After the treatment, LPS solution  was added to each well and incubated for a further 12 h at 37°C. Total RNA was isolated using ISOGEN  following the manufacturer's instructions as per a previous study [RAW264.7 cells were seeded at 3.7 × 10us study .α (TNF-α) (Mm00443258_m1), C-C motif chemokine 2 (CCL2) (Mm00441242_m1), and iNOS (Mm00440502_m1) were used.Real-time RT-PCR was performed to evaluate the effect of SQ or HEHSQ on proinflammatory cytokine and chemokine expression in RAW264.7 cells. The TaqMan probe  was used for the quantification of gene expression. Using a superscript III reverse transcriptase kit (Thermo Fisher Scientific), a complementary DNA (cDNA) solution was synthesized following the manufacturer's instructions. For quantification of transcript amounts, TaqMan real-time RT-PCR amplification reactions were performed using the Applied Biosystems 7500 Fast Real-Time System (Thermo Fisher Scientific). All primer sets and the TaqMan Universal PCR Master Mix were obtained from Thermo Fisher Scientific. Specific primers for actin beta (Mm02619580_g1), tumor necrosis factor 5 cells/mL) were seeded in a 10 cm2 dish (BD BioCoat) and treated with SQ (100 μg/mL) or HEHSQ (1 μg/mL) at 37°C for 24 h. After the treatment, LPS solution  was added to each well and incubated for 12 h at 37°C. The cell supernatants were collected by centrifugation at 1000 ×g for 5 min, and the levels of TNF-α and CCL2 were determined using commercial ELISA kits  according to the manufacturer's instructions.The effects of SQ or HEHSQ on cytokine production in RAW 264.7 cells were determined by ELISA. In the assay, cells , and statistical evaluation was performed using the one-way ANOVA followed by 2O (15 mL) and ethyl acetate (30 mL) were added, and the organic layer washed with saturated brine and H2O and dried over Na2SO4. The solvent was evaporated following column chromatography on silica gel, eluting with hexane:ethyl acetate (5 : 1) produced HEHSQ (Rf = 0.08 (hexane:ethyl acetate = 4 : 1). IR (CHCl3): 3451, 2977, 2932, 2877, 1452, 1381, 1219, 1083, 909, 670 cm−1. 1H NMR : δ 5.20–5.08 , 3.75–3.68 , 3.55–3.46 , 2.29 , 2.12–1.95 , 1.68 , 1.62 , 1.60 , 1.52–1.37 , 1.15 , 1.14  ppm. 13C NMR : δ 135.1, 135.1, 134.9, 134.8, 131.2, 124.7, 124.4, 124.3, 124.3 (2C), 77.8, 76.0, 62.4, 62.2, 39.7, 39.7 (2C), 36.8, 29.7, 28.6 (2C), 26.7, 26.7, 26.6, 25.7, 21.6, 19.8, 17.7, 16.0 (3C), 16.0 ppm. HRMS (ESI): m/z 511.4131 . 2,3-Epoxysqualene   or HEHSQ  for 24 h and cell viability was measured using the MTT assay. None of the SQ concentrations tested nor the 1 µg/mL HEHSQ-treated cells showed any cytotoxic effects  treated RAW264.7 cells showed no reduction of cell viability (Supplementary µg/mL of HEHSQ treatments showed a significant (P < 0.01) reduction in cell viability by 31.2 ± 1.3% and 16.5 ± 0.3%, respectively, compared with the untreated group  treated RAW264.7 cells showed the highest increase in NO production compared with the control cells. The treatment with SQ (100 µg/mL) for 24 h significantly reduced LPS-stimulated NO production by approximately 30%, although 1 and 10 µg/mL of SQ-treated cells did not show any significant change  treated RAW264.7 cells did not show any reduction of NO production compared with the LPS-stimulated cells  TNF-α gene expression by approximately 50%  treated RAW264.7 cells did not show any reduction of gene expression of TNF-α  and CCL2 (765.6 ± 19.8 μg/mL) into the culture medium than untreated cells . However, 100 µg/mL SQ (675.2 ± 35.3 μg/mL)- or 1 µg/mL HEHSQ (490.4 ± 18.0 μg/mL)-treated cells showed significantly lower (P < 0.01) TNF-α levels than that of the LPS-stimulated RAW264.7 cells (µg/mL SQ (558.0 ± 63.6 μg/mL)- or 1 µg/mL HEHSQ (524.7 ± 45.8 μg/mL)-treated cells also inhibited CCL2 levels  in macrophages [µg/mL) did not affect NO production, high SQ concentration (100 µg/mL) showed lower NO production than that of the LPS-stimulated RAW cells. Additionally, NO production in RAW cells treated with low concentration HEHSQ (1 µg/mL) was significantly lower than that in the LPS-stimulated RAW cells.In the present study, we evaluated the anti-inflammatory effects of SQ and HEHSQ on LPS-stimulated RAW cells in an rophages , and henrophages . In the α, one of the major proinflammatory cytokines, is released from macrophages in response to inflammation and induces vasodilation and loss of vascular permeability, which facilitates immune cell infiltration including lymphocytes, neutrophils, and monocytes [α helps recruit immune cells into sites of inflammation and hence promotes inflammatory responses. It has been reported previously that excess amounts of TNF-α play a pathological role in several diseases including inflammatory bowel diseases, rheumatoid arthritis, and asthma [α and CCL2, contributes to an anti-inflammatory effect. Moreover, preventing overproduction of inflammatory mediators shifts the balance towards the beneficial effects of chronic inflammation, and it may prevent several diseases. In the present study, the effect of SQ and HEHSQ on TNF-α, CCL2, and iNOS gene expressions (production) and TNF-α and CCL2 levels in the culture supernatant (secretion) were evaluated using LPS-stimulated RAW264.7 cells. Our results showed that LPS treatment significantly increased the expressions of TNF-α, CCL2, and iNOS compared with untreated RAW264.7 cells. Moreover, TNF-α and CCL2 levels in the culture supernatant of LPS-treated cells were also higher than that of untreated cells. However, SQ (100 µg/mL) and HEHSQ (1 µg/mL) treatments significantly lowered the expression and secretion of TNF-α and CCL2 upon LPS stimulation. Therefore, our results indicated that the ethylene glycol modification (HEHSQ) enhanced the anti-inflammatory effect of SQ by 100 fold compared with unmodified SQ. Our results suggest that HEHSQ, which shows anti-inflammatory properties at low concentrations (1/100 concentration of SQ), could be used in dietary supplements, cosmetics, and functional foods for the prevention and treatment of chronic inflammatory diseases.TNF-onocytes . TNF-α hd asthma . Similard asthma . Chemokid asthma –29. Thusβ-activated kinase 1. These receptor and adaptor proteins transmit signals to several pathways, such as nuclear factor-kappa B (NF-κB), one of the key regulators of inflammation and mitogen-activated protein kinase (MAPK) signaling [κB signaling can be activated by various stimulators such as inflammatory cytokines, LPS, microorganisms, and ultraviolet light. Activated NF-κB signaling induces an increase in the expression of proinflammatory cytokines and NO synthase [β-activated kinase 1 activates MAPK kinases, and they subsequently phosphorylate extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. Moreover, phosphorylation of activator protein-1, ETS Like-1 protein Elk-1, and transcription factors initiate the transcription of inflammatory genes [κB and MAPK signaling, which are the key mediators of inflammatory signaling, should be evaluated.In this study, we have uncovered the anti-inflammatory effects of HEHSQ; however, the underlying molecular mechanism effect remains unclear. We used LPS-stimulated RAW264.7 cells to evaluate the anti-inflammatory effect. LPS is a well-known endotoxin that initiates inflammatory responses. LPS binds to TLR4 and effects several adaptor proteins such as toll-IL-1 receptor domain-containing adaptor protein, TNF-receptor-activated factor 6, and transforming growth factor ignaling . NF-κB ssynthase . MAPK siry genes . In the α, and CCL2. Moreover, we also showed that the anti-inflammatory effects of HEHSQ are 100 times more potent than those of natural SQ. Our results in this study could be a great contribution to local pharmacological applications, mainly for the handicap of higher cell toxicity. The results presented here suggest that HEHSQ may be potentially used as a safe alternative treatment to anti-inflammatory drugs. Therefore, we recommend the consumption of foods containing SQ, such as shark liver oil and olive oil, because oral administration of SQ is well absorbed (60–85%) in humans [In conclusion, to the best of our knowledge, our study provides the first evidence that SQ decreases the inflammatory mediators NO, TNF-n humans  and mini


Type-2 diabetes mellitus is one of the most prevalent metabolic diseases in the world, and is characterized by hyperglycemia . Alpha-glucosidases are enzymes in the digestive tract that hydrolyze carbohydrates into glucose. One strategy that has been developed to treat type-2 diabetes is inhibition of the activity of alpha-glucosidases using synthetic drugs. However, these inhibitors are usually associated with gastrointestinal side effects. Therefore, the development of inhibitors from natural products offers an alternative option for the control of hyperglycemia. In recent years, various studies have been conducted to identify alpha-glucosidases inhibitors from natural sources such as plants, and many candidates have transpired to be secondary metabolites including alkaloids, flavonoids, phenols, and terpenoids. In this review, we focus on the alpha-glucosidases inhibitors found in common vegetable crops and the major classes of phytochemicals responsible for the inhibitory activity, and also as potential/natural drug candidates for the treatment of type-2 diabetes mellitus. In addition, possible breeding strategies for production of improved vegetable crops with higher content of the inhibitors are also described. Glycosidases catalyzing the hydrolysis of glycosidic bonds in polysaccharides and glycoconjugates, play critical roles in various biological processes, including carbohydrate digestion, lysosomal catabolism of glycoconjugates, and post-translational modifications of cellular glycoproteins ,2. In paMost AGIs can attach to the carbohydrate binding site of AG due to their similarity with disaccharides or oligosaccharides in molecular structure. Moreover, the complexes have a stronger affinity than the carbohydrate–glucosidase complexes  and plant (Fagopyrum esculentum). Iminosugars play an important role in chemotaxonomy and exhibit antimicrobial properties. Moreover, nojirimycin showed potent AG inhibitory activity, which could be due to its structural resemblance to glucose.A range of chemical compounds identified from various plants shows inhibitory activities against AG enzymes. Most AGIs are secondary metabolites such as alkaloids, phenolic acids, flavonoids, terpenoids, anthocyanins, and their glycosides . AlkaloiSalacia reticulate, used as an antidiabetic, was reported to show potential AGI activity with IC50 values of 0.23 and 0.19 µM against maltase and sucrase enzymes, respectively [Morus alba is another plant species for which AG inhibitory activity has been well-studied [50 value of 91.63 µg/mL compared to that of acarbose (IC50: 402.33 µg/mL) [Mangifera indica L.) were screened for their inhibitory effect against AG and among them arjunolic acid and actinidic acid exhibited inhibitory activity with IC50 values of 239.60 ± 25.00 and 297.37 ± 8.12 μM, respectively [Artocarpus heterophyllus) displayed the strongest inhibitory activity (IC50: 0.05 mg/mL) followed by its seed (IC50: 1.79 ± 0.15 mg/mL), pulp (IC50: 6.81 ± 0.52 mg/mL), and flake (IC50: 10.52 ± 0.73 mg/mL) extracts [50 values of 1.2 ± 0.2 and 0.5 ± 0.1 µg/mL, respectively. These inhibition potencies were much stronger than the inhibitory effect obtained from acarbose (IC50: 91.0 ± 10.8 µg/mL) [Plants are the major source of phytochemicals, and some of these compounds possess different health promoting functions. Various types of these compounds are being isolated from different plant species and studied for their potential to manage type-2 diabetes, and extracts of leaves, roots, barks, and fruits from different medicinal plants, herbs, and other plants are reported to exhibit inhibitory activity against AG ,42. A 13ectively . Morus a-studied , and its3 µg/mL) . A rangeectively . Similarextracts . Grape s8 µg/mL) .O-glucoside flavonoid isolated from pepper leaves showed a similar level of inhibitory activity (IC50: 15 µM) compared to acarbose [50 values ranging from 42.42 ± 0.94 to 78.65 ± 0.48 µg/mL, which is less potent than acarbose (IC50: 15.65 µg/mL) [S. macrocarpon and S. melongena exhibited a moderate inhibitory effect with IC50 values of 71.77 ± 0.50 and 63.24 ± 0.30 µg/mL, respectively [50: 1.14 to 6.48 mg/mL) against AGs than that of acarbose (IC50: 356 ± 20.6 µg/mL) [Allium cepa ascalonicum) peels, peeled bulbs, and bulbs (the whole bulbs) exhibited strong AG inhibitory activity with an IC50 values of 0.012 ± 0.002, 0.035 ± 0.01, and 0.052 ± 0.01 mg/mL, respectively. In the study, the inhibitory activity obtained from the peel was found to be the highest compared to those from different parts of 25 different plant species, including Rheum palmatum roots (IC50: 0.016 ± 0.0002 mg/mL), Cinnamomum zeylanicum bark (IC50: 0.018 ± 0.0006 mg/mL), Brassica juncea leaves (IC50: 0.21 ± 0.02 mg/mL), Capsicum frutescens fruits (IC50: 2.12 ± 0.4 mg/mL), Allium sativum bulbs (IC50: 2.51 ± 0.5 mg/mL), Actinidia deliciosa peels (IC50: 2.77 ± 2.4 mg/mL), and Glycine max beans (IC50: 12.83 ± 4.0 mg/mL) [Major vegetables belonging to the Solanaceae family, such as pepper, tomato, eggplant, and potato, have been studied for their inhibitory activity against AG enzymes. Water extracts from the fruits of several pepper lines were examined for their effects on AG enzymes and the inhibitory percentages from a red sweet pepper variety were found to be 57% and 48% against yeast and rat AG enzymes, respectively . Similaracarbose . Recentl5 µg/mL) . Fruit eectively . Tomato 6 µg/mL) . The inh6 µg/mL) . Ethyl aIpomoea batatas showed strong maltase inhibitory activity with an IC50 value of 0.36 mg/mL [50 value of 3.87 ± 0.12 mg/mL) [50 value of 1.84 mg/mL, while acarbose showed a value of 16.19 μg/mL [Momordica charantia var. charantia and M. charantia var. muricata) displayed 68.8% and 69.2% inhibition on AG activity, respectively. In the study, the IC50 values of M. charantia var. charantia (0.298 ± 0.034 mg/mL) and M. charantia var. muricata (0.292 ± 0.022 mg/mL) were not significantly different from the IC50 value of acarbose (0.28 ± 0.019 mg/mL) [M. charantia showed the highest AG inhibition activity (66.64% ± 2.94%) compared to Trichosanthes cucumerina (Snake gourd: 61.91% ± 1.96%), Lagenaria siceraria (56.04% ± 1.72%), Sechium edule (51.49% ± 2.13%), Benincasa hispida (48.73% ± 0.98%), Luffa acutangula (43.93% ± 1.28%), and Cucurbita maxima (22.11% ± 0.90%) [50: 26.26 ± 0.29 μg/mL), followed by the seed (IC50: 32.50 ± 0.36 μg/mL), the flesh (IC50: 41.38 ± 1.04 μg/mL), and the rind (IC50: 45.44 ± 0.18 μg/mL) [Abelmoschus esculentus) peels and seeds demonstrated inhibitory effect against glucosidase enzymes with an IC50 value of 142.69 ± 0.32 and 150.47 ± 0.28 μg/mL, respectively [Natural acylated anthocyanins extracted from 36 mg/mL . Similar2 mg/mL) . Aqueous2 mg/mL) . Lactuca19 μg/mL . Cucurbi9 mg/mL) . Likewis9 mg/mL)  and ethy± 0.90%) . Extractivity 66.% ± 2.94%ectively .p-nitrophenyl-α-d-glucopyranoside (pNPG) is a synthetic substrate that is hydrolyzed specifically by AGs into a yellow colour product (4-nitrophenol) that is usually quantified at 405 nm. Hence, measuring the amount of 4-nitrophenol produced from pNPG in the presence or absence of inhibitors is used to measure the inhibitory activity of plant compounds against AGs  as a measure of absolute binding affinity would solve this matter as it is not affected either by substrate or enzyme concentrations [Generally, AGI analysis is an enzymatic assay that follows the same basic principles as most enzymatic assays. Mostly, measurement of AG inhibitory activity is based on colorimetric methods. ectively . Howeverectively  and 200 ectively . Therefotrations . In additrations .Very few vegetables have been released/commercialized with approval of ingredients to help in managing type-2 diabetes. “Dangjo” and “Wongi No. 1” are two well-known Korean pepper varieties that have been specifically developed for higher AGI activity ,74. SincS. lycopersicum and S. Chmielowski [PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1), a regulatory gene from Arabidopsis, and the CHALCONE ISOMERASE (CHI) gene, from Allium cepa, into tomato caused 130- and 30-times higher levels of rutin (a bioactive flavonol) and total anthocyanin content, respectively, than those found in wild tomato skin [Recent advances in molecular biology and genomics have given new insight into the biosynthesis pathways of secondary metabolites, including phenolic acids and flavonoids, and allowed the identification of quantitative trait loci (QTLs) involved in the pathways. A number of metabolic quantitative trait loci (mQTLs) and candidate genes responsible for the synthesis of phenolic acids were identified in a population generated from an interspecific cross between ielowski . Based oato skin .Diverse groups of secondary metabolites exist in plants and the current technologies in analytical chemistry as well as biochemistry provide opportunities to develop efficient high-throughput screening methods for compounds with AGI activity. Considerable effort has also been put into producing common vegetables containing higher amounts of AG inhibitory compounds. In addition, the content of AGIs in vegetables can be increased by controlling environmental factors for plant growth and development, such as temperature, light , nutrients, water availability, and so on. A generation of new varieties with higher AG inhibitory compound content is also attainable through breeding with the aid of molecular genetic technologies. Current advances in molecular genetic/genomic technologies, followed by the availability of plant genomic and/or metabolomic information, has made it possible to acquire precise profiles of AGI candidates and even to identify the genes responsible for their biosynthesis. Genome editing (GE) technologies including CRISPR/Cas9 systems are also likely to be utilized for enhanced production of AG inhibitory compounds in common vegetables . We beli


African Swine Fever Virus (ASFV) has tropism for macrophages, which seems to play a crucial role in disease pathogenesis and viral dissemination. Previous studies showed that ASFV developed mechanisms to evade type I interferon (IFN) responses. Hence, we analyzed the ability of ASFV strains of diverse virulence to modulate IFN-β and IFN-α responses. Porcine monocyte-derived macrophages un-activated (moMΦ) or activated with IFN-α (moMΦ + FN-α) were infected with virulent (22653/14) or attenuated (NH/P68) ASFV strains, and expressions of IFN-β and of 17 IFN-α subtypes genes were monitored over time. ASFV strains of diverse virulence induced different panels of IFN genes: infection of moMΦ with either strains caused statistically significant up-regulation of IFN-α3, -α7/11, whereas only attenuated NH/P68 determined statistically significant up-regulation of IFN-α10, -α12, -α13, -α15, -α17, and IFN-β. Infection of activated moMΦ with either strains resulted in up-regulation of IFN-β and many IFN-α subtypes, but statistical significance was found only for IFN-α1, -α10, -α15, -α16, -α17 in response to NH/P68-infection only. These data revealed differences in type I IFNs expression patterns, with differences between strains of diverse virulence. In addition, virulent 22653/14 ASFV seems to have developed mechanisms to suppress the induction of several type I IFN genes. Asfaviridae family. ASFV has tropism for cells of the myeloid lineage, especially monocytes and macrophages, which seems to play a crucial role in disease pathogenesis, viral persistence and dissemination [African Swine Fever virus (ASFV) is the etiological agent of African Swine Fever, a contagious viral disease of domestic pigs and wild boar; currently, it is present in many sub-Saharan African countries, Russian Federation, Trans-Caucasus, Eastern and Central Europe, and South East Asia ,2. The dmination ,2,3. In mination ,6,7,8,9.mination ; deletiomination ,11. Indemination ,13, presmination .IFNs are a family of proteins synthesized and secreted by different cell types. They were named after their capacity to interfere with viral infections ; howeverThe knowledge of this branch of innate immunity would improve our understanding of the early stage of ASFV pathogenesis and might aid the rational design of ASFV vaccines. In addition, a better understanding of ASFV-driven modulation of type I IFN system might help design antiviral agents or a metaphylactic intervention strategy against this virus. In fact, it has been reported that adenovirus-mediated type I interferon expression delayed appearance and reduced clinical signs in pigs infected with another virus, such as classical swine fever virus (CSFV) , and comIn this conceptual framework, the aim of our study was to investigate the pattern of expression of different type I IFN  in response to ASFV infection. We compared an attenuated (NH/P68) and a virulent (22653/14) strains, in order to investigate how the level of attenuation affects the virus ability to induce type I IFN responses.p < 0.05), 9 (p < 0.05), 12 (p < 0.05), 21 (p < 0.001) hours post-infection with respect to time 0. On the contrary, the virulent 22653/14 strain did not modulate IFN-β gene expression with statistical significance (p < 0.05) (p < 0.05), α12 (p < 0.05), -α13 (p < 0.05), -α15 (p < 0.05), -α16 (p < 0.01), -α17 (p < 0.05) 24 h pi (p > 0.05) modulated by this virus, although an increase of IFN-α5/6, -α8, -α14 for both strains was observed.moMΦ were infected with either attenuated NH/P68 or virulent 22653/14 ASFV strains using multiplicity of infection (MOI) 1; in our previous work, intracellular levels of late viral proteins p72 and virus infectious particles in culture supernatants were determined at 21 h pi . We obseificance . Concern < 0.05) . Increas 24 h pi . The othp < 0.01) increase of IFN-β gene expression; in particular, we observed up-regulation, with respect to time 0, at all time points with the exception of 3 h post-infection. In addition, the virulent 22653/14 induced moderate IFN-β induction, with statistical significance at 9 h pi (p < 0.05) (p > 0.05). Both strains induced IFN-α2, -α4, -α5/6, -α8, -α9, -α12, -α13, -α14 induction, but differences were observed between strains: NH/P68 up-regulated -α5/6, -α8, -α12, -α13, -α14 at more times pi compared to virulent 22653/14 (p < 0.05) and 12 (p < 0.01) h pi, IFN-α10 at 9 (p < 0.05), 12 (p < 0.01) and 21 (p < 0.05) h pi, and of -α15, -α16, -α17 at 12 h pi (p < 0.05)  . Regardi22653/14 . NH/P68  < 0.05) .p < 0.05) in IFN-β and IFN-α1 release after infection with both attenuated or virulent ASFV strains .Our ELISA results did not show significant differences , with some of them (like IFN-α) consisting of different subtypes . In pigsWe observed higher induction of IFN-α1 after infection with the attenuated NH/P68 compared to the virulent 22653/14, with statistical significance in moMΦ + IFN-α, and these findings are in accordance with Gil et al. (2008), where researches observed higher induction of IFN-α1 in macrophages after infection with NH/P68 compared to virulent L60 , PRRSV, NH/P68 infection of moMΦ resulted in up-regulation of other IFN genes compared to virulent 22653/14, with statistical significance for IFN-α10, -α12, -α13, -α15, -α16, -α17. Nevertheless, both strains significantly induced IFN-α3 and IFN-α7/11 expression. Interestingly, it was reported that porcine recombinant IFN-α3, -α7, -α11 displayed no antiviral activity against PRV  and bothAbout activated moMΦ + IFN-α, a broader induction of IFN-α subtypes by both strains was observed, but still stronger after infection with NH/P68 compared to 22653/14 ASFV. The virulent Sardinian isolate seems to have developed mechanisms to suppress innate immune responses. NH/P68 only induced gene expression of IFN-α1, -α10, -α15, -α16, -α17 in activated moMΦ. Interestingly, it was described that IFN-α15 and IFN-α16 downregulated expression of MHC class I , and we Nevertheless, information regarding biological activities of diverse IFN-α subtypes are still limited, and future studies should be performed in order to better understand how different IFNs expression pattern can affect host antiviral response. In vitro experiments, like viral yield reduction assays, will help understand the impact of these molecules on ASFV replication. In addition to that, in vivo studies are badly needed to properly characterize the immunomodulatory activities of these molecules, thus revealing the real extent of their protective effects during ASFV infection.Concerning IFN-β, we observed that NH/P68 infection induced higher expression of this gene compared to the virulent 22653/14, in accordance with both Reis et al. (2016) (OURT 88/3 vs. Benin 97/1) and Garcia-Belmonte et al. (2019) (NH/P68 vs. Armenia07) [Although our data showed that the attenuated NH/P68 induced higher induction of type I IFN compared to the virulent 22653/14, we could not detect higher production of either IFN-α1 or IFN-β from infected moMΦ, in accordance with a previous study . The autOverall, our results highlight that the attenuated NH/P68 induced a stronger type I IFN response compared to virulent 22653/14, with statistically significant induction of IFN-β and several IFN-α subtypes in moMΦ. Nevertheless, expression of some IFN-α subtypes (-α3 and -α7/11) in moMΦ was induced by both strains. Regarding activated moMΦ, both IFN-β and diverse IFN-α subtypes were induced by ASFV, but selectively  or with stronger intensity  by NH/P68 compared to the virulent Sardinian isolate. Overall, virulent 22653/14 seems to have developed mechanisms to suppress the induction of several type I IFN genes. In addition, we observed that IFN-α subtypes were differently modulated upon infection, highlighting the importance of future studies to better define the biological properties of these subtypes, and to understand how differences in their expression pattern can affect intensity and duration of host antiviral responses. Data generated in this study could contribute to better understand ASFV-driven modulation of type I IFN system, to improve rational design of vaccines or antiviral agents.Sus scrofa domesticus), housed at the Experiment Station of IZS of Sardinia . The local ethical committee approved all procedures performed, in agreement with the Guide of Use of Laboratory Animals issued by the Italian Ministry of Health. The ASFV-negative status was assessed by real time PCR (EDTA blood samples), ELISA  and Immunoblotting test (serum samples), as described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [Whole blood was obtained from healthy, 6- to 24-month-old, cross-bred pigs , by inoculation in of sub-confluent monolayers of porcine monocytes/macrophages, as previously described [Two ASFV strains of diverse virulence were used in this study: the virulent hemoadsorbing 22653/14   and the attenuated NH/P68 . NH/P68 is non-hemoadsorbing and was isolated in 1968 from a chronically infected pig in Portugal ,26,27,28escribed ,30,31. M2 in Petri dishes in cRPMI , 100 U/mL penicillin and 100 μg/mL streptomycin) supplemented with 50 ng/mL of recombinant hM-CSF  for seven days. Then non-adherent leukocytes were removed, adherent cells were detached by gentle scraping with a pipette, centrifuged at 200× g for 8 min, and number of viable cells were determined using a Countess Automated Cell Counter (Thermo Fisher Scientific). 5 × 105 live cells/mL were seeded in a 12 well plates  in cRPMI (1.5 mL/well). After seeding, macrophages were cultured at 37 °C 5% CO2 for another 24 h: they were left untreated (moMΦ) or stimulated with 100 U/mL recombinant porcine IFN-α  (moMΦ + IFN-α).Porcine leukocytes were obtained from whole blood and heparin was used as an anticoagulant. Macrophages were generated in Petri dishes using human M-CSF (hM-CSF), as previously described ,30,31,322 (adsorption period), viral inoculum was removed, cells were washed with medium and fresh cRPMI was added to the wells [2 and harvested after 3, 6, 9, 12, 21 h post-infection (pi), spanning the approximate life cycle of ASFV replication [g for 3 min and stored at −80 °C until analyzed [moMΦ or moMΦ + IFN-α were infected with virulent 22653/14 or attenuated NH/P68 using a multiplicity of infection (MOI) of 1. After 90 min at 37 °C 5% COhe wells . Cells wlication  At each analyzed .Gene expression of IFN-β and 17 different IFN-α subtypes in ASFV-infected moMΦ at selected time-points  was determined by RT qPCR using primer sets previously described ,35. BrieAfter calculation of 2-ΔΔCt and the Kolmogorov-Smirnov test for normality, data sets were checked for statistically significant differences. We performed six experiments using different blood donor pigs.IFN-α1 and IFN-β levels in culture supernatants were measured by a Swine IFN-α Do-it-Yourself ELISA kit  or a porcine IFN-β ELISA kit , respectively, according to manufacturer’s directions. An Epoch microplate reader  was used to read adsorbance. Experiments were performed in technical duplicate and repeated three (IFN-α) or four (IFN-β) times using different blood donor pigs.p < 0.05. A tendency was declared at p < 0.1.Experiments were performed in technical duplicate, with three/four (ELISA) or six (qPCR) biological replicates using different pis as the source of moMΦ. Data are presented as mean with standard deviations (SD). GraphPad Prism v8.01  and Minitab (version of 2019)  were used to perform graphical and statistical analysis. All data were checked for normality using the Anderson-Darling test; virus effects on IFNs expression and production were analyzed by the parametric one-way ANOVA or the non-parametric Kruskal-Wallis followed by Dunn’s multiple comparison test. The significance threshold was set


Escherichia coli Nudix hydrolase RppH, which triggers 5′-end-dependent RNA degradation by removing orthophosphate from the 5′-diphosphorylated transcripts. Here we show that RppH binds and slowly hydrolyzes NTPs, NDPs and (p)ppGpp, which each resemble the 5′-end of RNA. A series of X-ray crystal structures of RppH-nucleotide complexes, trapped in conformations either compatible or incompatible with hydrolysis, explain the low reaction rates of mononucleotides and suggest two distinct mechanisms for their hydrolysis. While RppH adopts the same catalytic arrangement with 5′-diphosphorylated nucleotides as with RNA, the enzyme hydrolyzes 5′-triphosphorylated nucleotides by extending the active site with an additional Mg2+ cation, which coordinates another reactive nucleophile. Although the average intracellular pH minimizes the hydrolysis of nucleotides by slowing their reaction with RppH, they nevertheless compete with RNA for binding and differentially inhibit the reactivity of RppH with triphosphorylated and diphosphorylated RNAs. Thus, E. coli RppH integrates various signals, such as competing non-cognate substrates and a stimulatory protein factor DapF, to achieve the differential degradation of transcripts involved in cellular processes important for the adaptation of bacteria to different growth conditions.All enzymes face a challenge of discriminating cognate substrates from similar cellular compounds. Finding a correct substrate is especially difficult for the  Accurate substrate recognition is a prerequisite for productive enzymatic function. Many enzymes exploit various structural and chemical peculiarities to ensure discrimination between a cognate substrate and similar compounds from a cellular pool. Other enzymes have evolved to be more promiscuous and recognize multiple substrates that bear common features. Such semi-specific recognition could be challenging for an enzyme if its cognate substrate resembles abundant cellular components.nucleoside diphosphate linked to x (any moiety) , nucleoside diphosphates (NDPs) and related compounds, whose millimolar concentrations in cells far exceed those of mRNAs (E. coli cells.Widespread hydrolases from the Nudix homology clan break a phosphoanhydride bond in compounds containing a  moiety)  and have RNase E ,3. Escheof mRNAs . StressfE. coli RppH avoid hydrolyzing nucleotides? If not, RppH may deplete the cellular pool of nucleotides and force bacteria to waste energy and resources to restore sufficient levels of nucleotides. Previous in vitro studies showed that E. coli RppH cleaves off the γ-phosphate of 5′-triphosphorylated mononucleotide substrates at physiological pH and removes both the γ and β phosphates at elevated enzyme concentrations  (Bdellovibrio bacteriovorus RppH (BdRppH) showed that the nucleobase of GTP specifically binds the enzyme and that the triphosphate moiety seems to be perfectly positioned for cleavage between the α- and β-phosphates yet does not undergo hydrolysis  . These sdrolysis . Howeverchanisms . Unlike bstrates ,13.E. coli RppH, a representative of the most widespread clade of bacterial RppHs , RppHs  and RppHt  were used for biochemical and structural experiments as described in the corresponding subsections of ‘Materials and Methods’ section (E. coli BL21(DE3). The recombinant proteins were purified by affinity chromatography using HisTrap FF column  and the tags were cleaved off by the His-tagged ULP1 protease. The cleavage left an extra serine at the N-terminus of all proteins. The tags and protease were removed by HisTrap FF column. RppH was further purified by ion-exchange chromatography on HiTrap SP column . Finally, proteins were purified by gel filtration on Superdex 75 (RppH) or Superdex 200 (DapF) .A full-length wild-type  in ref. . DapFm cactivity  but do nactivity . This muC18 tips and eluted in 2,4,6-trihydroxyacetophenone-containing matrix for direct spotting, according to Technical Note 225 (Millipore Corporation).RNAs were synthesized by run-off T7 RNA polymerase transcription with DNA templates that comprise a double-stranded T7 promoter and a single-stranded RNA-encoding extension as described in ref. . DNA tems were grown from a solution prepared by mixing 2 μl of 0.26 mM RppHs protein and 2 μl of reservoir solution composed of 0.4 M (NH4)2SO4 and 10% (v/v) PEG4000. For soaking, 0.2 μl of 1 mM GTP was added to the drop for several hours. To determine the ppcpG-bound structure, crystals were grown from a solution prepared by mixing 2 μl of 0.26 mM RppHs, 2 μl of reservoir solution  PEG4000) and 0.5 μl crystal seeds obtained for GTP soaking. ppcpG was soaked into crystals by adding 0.2 μl of a solution containing 1 mM ppcpG, 50 mM HEPES-Na, pH 7.5 and 10 mM MgCl2. For the ppcpA-bound structure, the 4 μl hanging drop was composed of a premixed solution containing 0.25 mM RppHt, 1 mM ppcpA, 0.2 M (NH4)2SO4, 6.25% (v/v) PEG3350 and 5% (v/v) glycerol. The drop was equilibrated against 0.2 ml reservoir solution composed of 0.4 M (NH4)2SO4, 12.25% (v/v) PEG3350 and 10% (v/v) glycerol. For cytidine triphosphate (CTP) and uridine triphosphate (UTP)-bound structures, hanging drops were prepared by mixing 2.5 μl 1.2 mM RppHt, 20 mM Na-acetate, pH 5.0, 50 mM NaCl and 2.5 μl reservoir solution composed of 0.4 M (NH4)2SO4, 10% (v/v) PEG3350, 10% (v/v) glycerol. Soaking was conducted by adding 0.2 μl of a solution containing 1 mM CTP or UTP, 0.1 M Na-cacodylate, pH 6.0, 0.2 M Na2SO4, 15% (v/v) PEG3350 and 10% (v/v) glycerol to the crystal drop and incubating for 30 min. Crystals were cryoprotected by dipping into either reservoir or soaking solution supplemented by either 25% (v/v) glycerol or 25% pentaerythritol propoxylate 5/4 PO/OH as described previously  (or 7 mM β-mercaptoethanol). Crystals were grown in hanging drop format typically against 0.4 ml reservoir solution at 20°C for 2–3 days. Crystals of the GTP-bound RppHm and RppHt at a molar ratio of 1:1.2 in a buffer containing 20 mM Tris–HCl pH 8.0, 500 mM NaCl, and 1 mM DTT. The complex was purified by gel-filtration on Superdex 200 16/600. Crystallization drops of the RppHt–DapFm complex were prepared by mixing 0.5 μl of the complex (0.33 mM) with 0.5 μl of reservoir solution containing 30% (v/v) PEG400 and 0.1 M CHES, pH 9.2. Crystals were grown against 80 μl of the reservoir solution in 96-well plates at 18°C for 5–7 days. To soak GTP, GDP or pppGpp, the drops with crystals were supplemented by 0.2 μl solution containing 1 mM nucleotide, 2 mM (for pppGpp) or 20 mM NaF (for GDP and GTP), and 10 mM MgCl2 and incubated for 1–4 h. Crystals were cryoprotected in the reservoir solution supplemented with 16% (v/v) glycerol.RppH–DapF complex was formed by mixing DapFE. coli RppH or RppH–DapF complex structures (PDB codes: 6D1V and 4S2Y) as search models and PHENIX , FMX of the National Synchrotron Light Source-II  and at the home Rigaku X-ray source. Data were processed using XDS suite  or HKL20d PHENIX . The modd PHENIX  and refi2 and 50 mM buffer with different pH. Buffers were: MES-NaOH, pH 6.0; Na-cacodylate-HCl, pH 6.5; MOPS-NaOH, pH 7.0; HEPES-NaOH, pH 7.5; Tris-HCl, pH 8.0, 8.5 and 9.0. Reactions were incubated at 37°C for 1 h and quenched with 5 μl of 150 mM ethylenediaminetetraacetic acid (EDTA). Samples were analyzed on PEI-cellulose plates  in 0.3 M potassium phosphate buffer, pH 7.5, for adenosine triphosphate (ATP), adenosine diphosphate (ADP), GTP, GDP, pppAG (RNA), ppGpp and pppGpp, and pH 3.3 for CTP and CDP, UTP and UDP. RNAs were visualized by UV254 shadowing. The equimolar amount of wild-type E. coli DapF (Reactions for thin layer chromatography (TLC) were performed with 1 mM nucleotide substrates and 5 μM of the full-length RppH, in a 10-μl solution containing 10 mM MgCl2 , 0.1% Triton X-100, at 37°C. Reactions were incubated for 0–20 min and quenched with 750 μl of 50 mM sodium acetate, pH 4.5. The quenched reaction samples were analyzed by anion-exchange chromatography on a 5 × 50 mm MonoQ column . The reaction products and remaining substrates were eluted with a 0–1.0 M NaCl gradient in 10 mM Tris-HCl, pH 7.5, detected at 260 nm, and the amount of each mono- or oligonucleotide was calculated by using the integration function in UNICORN software . The initial reaction rates were calculated based on the ratio between remaining substrate and reaction products using a linear fit model of GraphPad. Each assay was repeated at least twice.RppH activity on pppAG and ppAG RNAs, NTPs, NDPs, and ppGpp was determined by kinetic analysis, as described in ref. . Each re50 values were determined using an IC50 model of GraphPad.Competition experiments were conducted using the same protocol as cleavage assays with the 0.1 mM substrate RNA concentration, the nucleotide concentration ranging between 0.1-5.0 mM, and 10 min incubation. The initial reaction rates were calculated based on the ratio between reaction products and remaining substrate using a linear fit model of GraphPad software. Because of peak overlap, inhibition of GTP hydrolysis was determined solely based on quantification of the reaction products. ICt in a 20-μL solution containing 10 mM MgCl2, 0.1% Triton X-100 and 50 mM buffer. Buffers were: acetic acid-NaOH, pH 5.0 and 5.5; MES-NaOH, pH 6.0; Na-cacodylate-HCl, pH 6.5; MOPS-NaOH, pH 7.0; HEPES-NaOH, pH 7.5; Tris-HCl, pH 8.0, 8.5, and 9.0. Reactions were quenched with 80 μL of 1% SDS after 1 min incubation at 37 °C. Inorganic pyrophosphate from the quenched reactions was precipitated in the presence of 1 mM CaCl2 and 100 mM NaF. Precipitate collected by centrifugation at 20,000 × g for 10 min was washed with acetone, dried, and re-dissolved in solution containing 1.25 N H2SO4, 10 mM ammonium heptamolybdate and 40 mM β-mercaptoethanol. Intensity of green color developed after 1 h incubation at room temperature was measured at 700 nm and the amount of the pyrophosphate liberated during reaction was calculated from the calibration curve prepared with standard solutions of sodium pyrophosphate.Inorganic pyrophosphate assay was performed colorimetrically according to the published method ,20. Eachd, in a 20-μL solution containing 10 mM divalent cation salt  and 50 mM buffer with different pH . Reactions were incubated at 37 °C for 1 h and quenched with 10 μL 150 mM EDTA. Samples were analyzed on PEI-cellulose plates in 0.3 M potassium phosphate buffer, pH 7.5, at room temperature. RNAs were visualized by UV254 shadowing.Reactions were performed with 5 mM GTP and 5 μM RppHrppH and ΔrppH+pPlacRppH strains were from  cultures. ΔrppH+pPlacRppH (+IPTG) and ΔrppH+pPlacRppH (+10 × IPTG) cultures were induced by 10 and 100 μM isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively, at A600 ≈0.5 and incubation continued up to A600 ≈1.0. 3 ml aliquots of cell cultures were collected by centrifugation and resuspended in 1 ml 20 mM Tris–HCl, pH 8.0, 150 mM NaCl buffer. Cells were centrifuged again and resuspended in 600 μl buffer. ATP concentration was measured by Luminescent ATP Detection Assay Kit . Briefly, 100 μl cells were lysed by 50 μl Detergent Solution by shaking in an orbital shaker at 600–700 rpm in a 96-well plate  at room temperature for 5 min. The 50 μl Substrate Solution was added to the well and shaking continued for 5 more min. The plate was placed in the dark for 10 min and luminescence was measured immediately at room temperature using FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices). The luminescence counts were determined as averages of three–four technical replicates. The final values were determined by averaging data from three biological replicates.WT, Δere from . Fresh cE. coli RppH has the ability to hydrolyze NTPs, which mimic the 5′-triphosphorylated end of RNA  that are comparable to the intracellular pH. However, the enzyme is more reactive at pH 8.0, a value that approaches the upper boundary of the stably maintained intracellular pH. At least at this pH, RppH hydrolyzes NDPs better than NTPs, revealing that the pH dependence of NDP hydrolysis relative to NTP hydrolysis is shifted somewhat toward neutral pH.n = 2, error is SE) than GDP (3.4 ± 0.07 μM/min) and GTP (3.1 ± 0.13 μM/min). As expected, under the same conditions, the reaction rates of the RNA substrates were over 10 times faster than NTP hydrolysis ppGpp to millimolar levels ,34. (p)pdrolases , and it drolases . As obsedrolases .The kinetics of hydrolysis determined by ion-exchange chromatography revealed initial rate of 1.20 ± 0.07 and 1.30 ± 0.10 μM/min for ppGpp and pppGpp, respectively, at pH 7.5 Figure . These rE. coli RppH at pH ≥ 8.0 led us to investigate the optimal pH for the hydrolysis of RNA substrates. To answer this question, we tested the activity of RppH on the RNA dinucleotide pppAG. TLC experiments revealed that RppH activity could be detected at a pH as low as 5.5 and that hydrolysis is most efficient at pH ≥6.5 ppGpp, which have two or three 5′-terminal phosphates. To trap the enzyme–substrate complexes in the catalytically relevant pre-cleavage conformation, we used an alternative approach. First, we used crystals of the RppH-DapF complex  Figure . As in t2+ cations coordinated with octahedral geometry by several amino acids and water molecules  consistent with previously observed Mg-F bond lengths  with this atom and the bridging oxygen atom. We posit that this F− anion replaces a nucleophilic hydroxide ion or water molecule positioned for in-line attack  typically maintained in E. coli cells ppGpp are hydrolyzed by RppH li cells ,23. Morentration  despite E. coli RppH may hydrolyze nucleotides at an increased rate. This problem could be aggravated in alkalophilic bacteria, such as the moderate halophile Vibrio alginolyticus, which possesses an RppH that is very similar in sequence to its E. coli counterpart but maintains a higher average intracellular pH of 7.8  is slightly below its intracellular concentration, while that of GTP (IC50 ∼1.6–4.1 mM) is at the high end of its intracellular concentration.Our biochemical data demonstrate that nucleotides inhibit the reaction of RppH with RNA substrates. As suggested by the crystal structures, such inhibition could be caused by the competition of nucleotides with RNA for binding to the catalytic site and the nucleobase-binding cleft. Remarkably, nucleotides can bind in the cleft without anchoring their phosphates in the catalytic site by Mg of NTPs ,5. Publi of NTPs ,5. Speci of NTPs . Thus, t50 measurements (50–200 μM) suggest that ppGpp and pppGpp can strongly inhibit the reaction of RppH with mRNA substrates under stresses that cause the concentration of (p)ppGpp to surge. Consequently, an RppH-mediated pathway for 5′-end-dependent RNA degradation may become less efficient under these stresses. Such a change may increase the stability of hundreds of mRNAs whose degradation is RppH-dependent.ppGpp concentrations can increase from 40 μM during exponential growth to 0.8 mM during transition to stationary phase, while during the stringent response ppGpp becomes the most abundant nucleotide, accounting for 60% of the cellular nucleotide pool . pppGpp,in vivo. Thus, a competition between cognate and non-cognate substrates likely shapes the specificity of this enzyme in bacterial cells.Interestingly, about 2-3-fold higher concentrations of nucleotides are needed to inhibit the reaction of RppH with diphosphorylated RNA substrates than with the triphosphorylated RNA. Therefore, the more efficient competition of nucleotides with triphosphorylated RNA augments its intrinsically lower reactivity with RppH (E. coli RppH tunes gene expression by integrating two types of signals that affect the reactivity and specificity of the enzyme. In addition to stimulation of RppH by DapF binding, as previously reported (In summary, these studies suggest that reported , RppH lireported , host inreported ,31,48–49reported .2+-F−–RppH-DapF complex; 6VCM, GTP-Mg2+-F−–RppH-DapF complex; 6VCK, GDP-Mg2+-F−–RppH-DapF complex. All relevant data and materials are available upon request.Coordinates of the structures were deposited to the Protein Data Bank with PDB ID codes: 6VCQ, GTP–RppH complex; 6VCN, ppcpG–RppH complex; 6VCO, ppcpA–RppH complex; 6VCR, CTP–RppH complex; 6VCP, UTP–RppH complex; 6VCL, pppGpp-Mggkaa024_Supplemental_FileClick here for additional data file.


We observed that the antigen repertoire displayed on MHC-I molecules at the surface of MCA205 fibrosarcoma is modified upon treatment with IP2. In particular, IP2 enhances the presentation of an exon-derived epitope from the tumor suppressor nischarin. The combination of IP2 with a peptide vaccine targeting the nischarin-derived epitope showed a synergistic antitumor effect in vivo. These findings identify the spliceosome as a druggable target for the development of epitope-based immunotherapies.The success of cancer immunotherapy relies on the induction of an immunoprotective response targeting tumor antigens (TAs) presented on MHC-I molecules. We demonstrated that the splicing inhibitor isoginkgetin and its water-soluble and non-toxic derivative IP2 act at the production stage of the pioneer translation products (PTPs). We showed that IP2 increases PTP-derived antigen presentation in cancer cells in vitro and impairs tumor growth in vivo. IP2 action is long-lasting and dependent on the CD8 + T cell activation and showed greater antitumour activity in vivo. These findings identify the spliceosome as a druggable target for the development of epitope-based immunotherapies.Darrigrand, Pierson et al. study the effect of mRNA splicing inhibitors on the generation of the MHC class I ligands. They developed a derivative of the spliceosome inhibitor isoginkgetin which is less toxic and more effective at enhancing antigen presentation and CD8 Initial clinical trials investigating vaccines targeting tumor antigens (TA) have not met expectations. The main failures have been associated with immunosuppressive mechanisms and with a suboptimal choice of antigens3. One of the important events that drives tumor escape from immunosurveillance and is correlated with a poor prognosis is the loss or the downregulation of MHC-I antigen presentation by tumor cells5. The latter take advantage of defects in components of the MHC-I presentation pathway to escape cytotoxic T lymphocyte (CTL) and natural killer (NK) cell recognition6. Moreover, along with the overall decrease in MHC-I antigen presentation, the very nature and the number of antigens presented at the cell surface, namely the MHC-I immunopeptidome, is of critical importance for immune recognition. For example, the loss of expression at the tumor cell surface of a specific TA identified and targeted with immunotherapy, such as Her/neu in breast cancer or CEA in colon cancer, can lead to immune evasion8. To counter this phenomenon, current strategies aim at enlarging the range of targetable tumor antigens and restoring MHC-I antigen presentation12.All nucleated cells of jawed vertebrates present antigenic peptides (APs) at their surface through the major histocompatibility complex class I (MHC-I) presentation pathway. MHC-I peptides (MIPs) are 8- to 12-amino acid-long and reflect the inherent metabolic cellular activity16, we showed that one of the most important sources of APs is a pioneer translation event that occurs on pre-mRNAs, before introns are spliced out, and independently of the translation of the corresponding full length proteins. The pioneer translation products (PTPs) produced through this non-canonical translation event can therefore derive from intronic sequences, 3’ or 5’ untranslated regions (UTR) as well as alternative reading frames18. The discovery of PTPs emphasizes the existence of a nuclear translation mechanism of precursor mRNAs that participates to the production of MHC-I peptides (MIPs). Moreover, PTPs play a role in the dynamic of cancer development. When inoculated into mice, it has been shown that cancer cells presenting PTP-derived antigens at their surface can be recognized by specific T cells leading to tumor growth reduction19. Besides, purified PTPs encompassing a model immunodominant epitope efficiently promote anticancer immune response when injected as a peptide vaccine into mice.In order to understand the dynamics of the MHC-I immunopeptidome, we focused on the source of APs for the MHC-I presentation pathway. Exploring the concept of defective ribosomal products (DRIPs)21. However, the efficiency of those monoclonal antibodies is limited to a fraction of patients and is often associated with deleterious side effects22. Attention is being paid to small molecules with immunomodulatory properties, which offer a cost-effective production, a reversible action and reduced systemic side effects due to their short half-lives23.Cancer therapies have been constantly evolving with the hope of finding the most effective agents to eradicate tumors with the least toxic effects on healthy tissues. Unlike conventional cancer treatments that focus on tumor cells, immunotherapies target the immune system to strengthen and stimulate the patient’s own defenses against cancer. Breakthrough of the last decade, the immune checkpoint inhibitors against cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) have shown unprecedented antitumor effect in particular against advanced melanoma or non-small cell lung cancerGinkgo biloba tree. Multiple properties of the molecule have been reported and associated with its antitumor activity. Isoginkgetin was first shown to inhibit tumor cell invasion by inhibiting the production of the matrix metalloproteinase 9 (MMP-9)24. Indeed, isoginkgetin-induced downregulation of the NF-κB pathway leads to the upregulation of the MMP-9 inhibitor (TIMP-1) in human fibrosarcoma. More recently, it has been demonstrated that isoginkgetin inhibits 20 S proteasome activity and induces a toxic accumulation of polyubiquitinated proteins25. Eventually, isoginkgetin was described as a general inhibitor of pre-mRNA splicing, which stalls spliceosome assembly at the prespliceosomal A complex26. Pre-mRNA splicing is catalyzed in the nucleus by the spliceosome, a conserved and dynamic multi-RNA/protein complex composed of five small nuclear RNAs (snRNAs) in interaction with over 180 proteins27. A growing number of studies report that the deregulation of the spliceosome complex entails aberrant splicing patterns in many cancers contributing to abnormal tumor cell proliferation and progression31. In a recent study, we observed that splicing inhibition positively modulates the presentation of a PTP-derived model antigen in HEK-293T cells treated with isoginkgetin18.The biflavonoid isoginkgetin is a natural compound extracted from the leaves of the Here we show that the biflavonoid isoginkgetin and its water-soluble derivative IP2 enhance the presentation of PTP-derived antigens at the surface of cancer cells in vitro. In addition, IP2 induces a long-lasting anticancer immune response in vivo. Finally, IP2 was shown to reshape the MHC-I immunopeptidome of MCA205 fibrosarcoma. Our findings shed light on a new immunomodulatory agent whose antitumor activity relies on the induction of immunogenic epitopes that can be targeted in the context of epitope-based immunotherapies.50 determined by MTT assay  were treated with increasing doses of isoginkgetin up to the limit of IC+ T-cell response32. Therefore, we assessed the effect of isoginkgetin treatment on the antitumor response against SL8-expressing tumors in vivo. MCA205 fibrosarcoma and B16F10 melanoma stably expressing the globin-SL8-intron construct were inoculated subcutaneously into C57BL/6 mice. At days 5, 10, and 15 post-tumor inoculation, the mice were injected intraperitoneally with 18 mg/kg of isoginkgetin and the tumor growth was monitored. We observed a 60% decrease in the growth of MCA205-globin-SL8-intron at day 27  and two predicted binders of H-2Db molecules (affinity below 500 nM) were found to be enriched at the surface of MCA205 fibrosarcoma upon treatment with IP2. To refine our selection, we used the IEDB Analysis Resource36 to compute the immunogenicity score of the predicted strong binders, which accounts for peptide preferences in T-cell recognition. Eventually, two H-2Kb epitopes with an affinity for MHC-I molecules lower than 500 nM and a predicted immunogenicity score above 0.1 were shown to be enriched in at least two out of three IP2-treated samples, the SGYEFIHKL 9mer from the tyrosine–tRNA ligase (UniProtKB - E9PX65) and the TNQDFIQRL 9mer (TL9) from the nischarin (UniProtKB–B7ZN33), which has been described as a tumor suppressor in breast and ovarian cancer  encompassing the 9-amino acid-long sequence in central position was produced and its immunogenicity was assessed ex vivo. An irrelevant 39-amino acid-long SLP was also randomly generated and synthesized; both sequences are shared in Supplementary Fig. The TNQDFIQRL epitope shows a good computed immunogenicity score and the TL9 SLP was shown to be immunogenic in two different immunoassays therefore the antitumor potential of the combination between IP2 and the TL9 SLP was assessed in vivo. In a prophylactic setting, mice were vaccinated twice with 100 µg of TL9 SLP, then challenged subcutaneously with MCA205 fibrosarcoma and finally treated intraperitoneally with 24 mg/kg of IP2 Fig. . VaccinaOverall, we show here in the mouse MCA205 fibrosarcoma model that IP2 induces the presentation of immunogenic epitopes that can be targeted in cancer vaccines. This suggests the use of IP2 to build new epitope-based cancer immunotherapies.39. Tumors bearing spliceosome alterations are key targets for treatments with splicing inhibitors some of which are currently under development in acute myeloid leukemia40. Here we observed that treatment with the splicing inhibitor isoginkgetin or its derivative IP2 reduces the growth of MCA205 fibrosarcoma and B16F10 melanoma bearing the PTP-derived SL8 epitope and significantly extends mice survival 1 have the capacity to modify quantitatively and qualitatively the repertoire of epitopes presented at the surface of treated cells. We show here that interfering with the spliceosome using the IP2 compound leads to a remodeling of the MHC-I immunopeptidome of MCA205 fibrosarcoma  and tumor-specific (TSAs) antigens from different regions of the genome in the context of epitope-based immunotherapies.Importantly, IP2 treatment slows down the growth of WT fibrosarcoma and melanoma, which suggests that an immunodominant epitope is not required to observe an impact on tumor growth but however necessary to observe complete tumor regression Fig. . While t56 related to the secretion of exosomes from breast cancer cells57. The nischarin-derived TL9 epitope has been identified throughout multiple proteomic studies referenced in the Immune Epitope Database (IEDB) but has never been further exploited so far. Importantly, the TL9 epitope was found to be expressed in healthy tissues58 however we observed neither an antitumor effect nor any deleterious auto-immune response in mice injected with the TL9 SLP alone. This suggests that the basal level of expression of the antigen at the surface of MCA205 fibrosarcoma is not sufficient to allow CTL recognition, which is no more the case after IP2 treatment. The 39-amino-acid-long TL9 SLP was generated by translating the corresponding sequence identified in the RNA-seq analysis of MCA205 fibrosarcoma. Interestingly, we identified a point mutation in the cancer cell transcriptome, which translates into an amino acid switch, from Alanine (A) to Aspartic acid (D), at the position preceding the epitope. The flanking regions of the epitopes in synthetic long peptides can be optimized to promote epitope generation and presentation59, we hypothesize that this mutation found in the nischarin gene in MCA205 cells could potentially play a role in the increase presentation of the epitope compared with other healthy tissues. Future work will be needed to explore the effect of IP2 treatment on the presentation of the TL9 epitope with and without this mutation within the flanking regions, as well as other IP2-induced MHC ligands in cancerous and noncancerous cells.The exon-derived TL9 epitope from the tumor suppressor nischarin was shown to be immunogenic and to synergize with IP2 in cancer vaccines to inhibit the growth of MCA205 fibrosarcoma. Recent studies highlighted the role of nischarin in the regulation of breast tumorigenesis60. We reaffirm here that we did not observe any toxicity related to the use of the IP2 compound in vivo. While a deeper understanding of isoginkgetin and IP2 effects on splicing is needed, their positive impact on the immune response opens a large field of study on the link between splicing regulation and MHC-I immunopeptidome regulation. This could lead to the development of innovative anticancer immunotherapies targeting the spliceosome.Eventually, this work suggests a role for splicing regulation in the generation of MHC-I peptide for cancer immunosurveillance. The frequent mutations affecting the spliceosome in cancer cells may promote their evasion from T cells and NK cells. This raises the potential of the spliceosome as a druggable target in cancer immunotherapy. Potential side effects inherent to the alteration of such essential cellular mechanisms must be considered in the light of the benefice/risk balance. The use of proteasome inhibitors for myeloma or lymphoma treatment is one pertinent example2 in RPMI 1640 medium (Life Technologies) in the presence of 1% glutamine, 1% sodium pyruvate, 1% nonessential amino-acids, 1% penicillin/streptomycin and 10% FBS (Life Technologies). B16F10 mouse melanoma cell line was cultured at 37 °C under 5% CO2 in DMEM medium (Life Technologies) containing 1% glutamine, 1% penicillin/streptomycin and 10% FBS. The SL8/H-2Kb-specific B3Z CD8+ T-cell hybridoma61 were cultured at 37 °C under 5% CO2 in RPMI 1640 medium supplemented with 1% glutamine, 0.1% 2-mercaptoethanol, 1% penicillin/streptomycin, and 10% FBS. Once a month, mycoplasma contamination in cell cultures was assessed using the Venor®GeM OneStep mycoplasma detection kit (Minerva biolabs). All cells were used within four weeks after thawing (≈10 passages). MCA205 cells were transfected with the reagent jetPRIME (Ozyme) according to the manufacturer protocol. B16F10 cells were transfected with the reagent GeneJuice (Millipore) according to the manufacturer protocol. The plasmids YFP-Globin-SL8-intron, YFP-Globin-SL8-exon and PCDNA3 have been described previously17. The pre-mRNA splicing inhibitor isoginkgetin (Merck Millipore) was resuspended in DMSO. The IP2 and M2P2 derivatives were synthesized from the isoginkgetin powder and resuspended in PBS. Synthetic long peptides (39AA) were synthesized by GL Biochem, resuspended at 5 mg/mL in PBS at delivery and stored at −20 °C before use.MCA205 mouse fibrosarcoma cell line was cultured at 37 °C under 5% CO1H NMR, 31P NMR, and 13C NMR spectra were recorded in either CDCl3, D2O, or DMSO-d6 on Bruker Avance 300 spectrometers, or 200 spectrometers. The chemical shifts of 1H and 13C are reported in ppm relative to the solvent residual peaks. IR spectra were measured on a Bruker Vector 22 spectrophotometer. Melting points were recorded on a Büchi B-450 apparatus and are uncorrected. High resolution mass spectra (HRMS) were recorded on a MicroMass LCT Premier TOF ESI Spectrometer. For the synthesis of compound 1  :100:0:0 to 99:1:0.1) to yield 635 mg of compound 1 as a yellow powder . 1H NMR  δ: 13.00 , 12.79 , 8.02 , 7.97 , 7.46 , 7.18 , 6.99 , 6.95 , 6.84 , 6.65 , 4.32–4.18 , 4.11–3.95 , 3.81 , 1.37 , 1.22 . 13C NMR  δ: 183.0 (CO), 182.7 (CO), 164.4 (Ar), 162.9 (Ar), 162.3 (Ar), 161.9 (Ar), 161.1 (Ar), 157.1 (Ar), 156.1 (Ar), 156.0 (Ar), 154.4 (Ar), 153.6 (Ar), 153.5 (Ar), 131.2 (ArH), 128.6 (ArH), 127.9 (2ArH), 123.1 (Ar), 123.1 (Ar), 121.5 (Ar), 114.7 (2ArH), 111.3 (ArH), 108.3 (Ar), 104.9 (ArH), 104.3 (ArH), 103.9 (ArH), 103.9 (ArH), 103.7 (ArH), 99.1 (ArH), 99.0 (ArH), 65.2 , 64.9 , 56.1 (OCH3), 55.6 (OCH3), 16.3 (CH3), 16.2 (CH3), 16.1 (CH3). 31 P NMR  δ: −7.30, −7.50. IR (neat): 2989 (CH), 1653 and 1604 (CO), 1582 cm−1. HRMS (ESI-TOF) m/z; [M + H] + Calcd. for C40H41O16P2 839.1870; found 839.1865. Melting point = 155 °C. For the synthesis of IP2  δ: 7.64 , 7.51 , 7.24 , 7.00 , 6.81 , 6.58–6.41 , 6.33 , 3.83 , 3.63 . 13 C NMR  δ: 182.5 (CO), 164.5 (Ar), 161.8 (Ar), 160.9 (Ar), 160.8 (Ar), 160.4 (Ar), 159.4 (Ar), 159.3 (Ar), 158.6 (Ar), 156.9 (Ar), 153.7 (Ar), 130.9 (ArH), 128.3 (ArH), 127.3 (2ArH), 122.5 (Ar), 122.1 (Ar), 121.5 (Ar), 114.0 (2ArH), 111.8 (ArH), 107.2 (Ar), 107.1 (Ar), 105.5 (Ar), 104.9 (Ar), 103.6 (ArH), 103.3 (ArH), 102.6 (ArH), 99.8 (ArH), 99.7 (ArH), 56.1 (OCH3), 55.4 (OCH3). 31P NMR  δ: −0.49, −1.59. IR (neat): 3423 (broad), 2989 (CH), 1650 and 1600 (CO), 1563 cm−1. HRMS (ESI-TOF) m/z; [M-H]- Calcd. for C32H19O16P2 725.0467; found 725.0466. Melting point >350 °C. HPLC purity could not be determined due to the hydrolysis of phosphate during analysis. For the synthesis of compound 2, K2CO3  and iodomethane  were successively added to a solution under argon of compound 1  in anhydrous DMF (1.5 mL). The mixture was stirred at room temperature for 20 h and filtered. The solid was washed with ethyl acetate and the filtrate was washed with brine (3 × 30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to yield 110 mg of compound 2 as a yellow powder .1H NMR  δ: 7.99 , 7.93 , 7.38 , 7.16 , 7.11 , 7.04 , 6.80 , 6.67 , 4.31 – 4.18 , 4.16–3.90 , 3.78 , 1.36 , 1.22 . 13 C NMR  δ: 177.9 (CO), 177.4 (CO), 162.3 (Ar), 161.2 (Ar), 161.0 (Ar), 160.9 (Ar), 160.5 (Ar), 160.4 (Ar), 158.9 (Ar), 156.4 (Ar), 154.8 (Ar), 154.8 (Ar), 152.3 (Ar), 152.2 (Ar), 130.7 (ArH), 128.1 (ArH), 127.5 (2ArH), 123.4 (Ar), 123.3 (Ar), 121.6 (Ar), 114.5 (2ArH), 112.0 (Ar), 111.1 (ArH), 108.0 (ArH), 107.3 (ArH), 101.1 (ArH), 101.1 (ArH), 99.9 (ArH), 99.8 (ArH), 99.5 (ArH), 65.2 , 65.1 , 64.9 , 56.9 (OCH3), 56.8 (OCH3), 56.0 (OCH3), 55.5 (OCH3), 16.3 (CH3), 16.2 (CH3), 16.1 (CH3), 16.0 (CH3). 31P NMR  δ: −7.30, −7.50. IR (neat): 3492, 2984–2934 (CH), 1647 and 1605 (CO), 1573 cm−1. HRMS (ESI-TOF) m/z; [M + H] + Calcd. for C42H45O16P2 867.2177; found 867.2177. Melting point = 142 °C. For the synthesis of M2P2, TMSI  was added to a solution under argon of compound 2  in anhydrous dichloromethane (8 mL) and the reaction mixture was stirred at room temperature for 16 h. After evaporation of the solvent under reduced pressure, the yellow solid was dissolved in anhydrous methanol (4 mL) and NaOH  was added. The solution was stirred at room temperature for 30 min and the solvent was evaporated under reduced pressure to yield M2P2  as a yellow solid. 1H NMR  δ: 7.81 , 7.71 , 7.41 , 7.23–6.93 , 6.81 , 6.54 , 6.33 , 4.05 , 3.94 , 3.70 , 3.54 . 13C NMR  δ: 179.9 (CO), 162.4 (Ar), 161.5 (Ar), 160.8 (Ar), 160.4 (Ar), 160.3 (Ar), 160.2 (Ar), 160.1 (Ar), 159.9 (Ar), 159.7 (Ar), 159.4 (Ar), 158.9 (Ar), 157.9 (Ar), 155.6 (Ar), 130.9 (ArH), 128.1 (ArH), 127.1 (2ArH), 126.7 (ArH), 122.8 (Ar), 122.1 (Ar), 113.8 (2ArH), 111.6 (ArH), 108.2 (Ar), 107.6 (Ar), 105.9 (ArH), 104.9 (ArH), 100.9 (ArH), 99.6 (ArH), 56.3 (OCH3), 56.2 (OCH3), 56.2 (OCH3), 56.0 (OCH3). 31P NMR  δ: −0.16, −0.80. IR (neat): 3393 (broad), 2984 (CH), 1631 and 1593 (CO), 1565 cm−1. HRMS could not be determined due to the hydrolysis of phosphate during analysis Melting point >350 °C. HPLC purity could not be determined due to the hydrolysis of phosphate during analysis.Solvents and reagents are obtained from commercial suppliers and were used without further purification. Analytical TLC was performed using Merck silica gel F254 (230–400 mesh) plates and analyzed by UV light. For silica gel chromatography, the flash chromatography technique was used, with Merck silica gel 60 (230–400 mesh) and p.a. grade solvents unless otherwise noted. The 4 cells were co-cultured with 1 × 105 SL8/H-2Kb-specific B3Z CD8+ T-cell hybridoma for 18 h at 37 °C with 5% CO2 overnight. Cells were centrifuged at 1200 rpm for 5 min, washed twice with PBS 1× and lysed for 5 min at 4 °C under shaking. Lysis buffer was composed of 0.2% TritonX-100, 10 mM DTT, 90 mM K2HPO4, 8,5 mM KH2PO4. The lysates were centrifuged at 3000 rpm for 10 min to pellet cell debris and the supernatants were transferred to a 96-well optiplate . A revelation buffer containing 10 mM MgCl2, 11,2 mM β-mercaptoethanol, 0,0015% IGEPAL® CA-630 et 40 µM 4-Methylumbelliferyl β-D-Galactopyranoside (MUG) was added and the plate was incubated at room temperature for 3 h. Finally, β-galactosidase activity was measured using the FLUOstar OPTIMA .MCA205 fibrosarcoma and B16F10 melanoma were transfected with the plasmid YFP-Globin-SL8-intron or with the PCDNA3 empty plasmid as described in the previous section. Twenty-four hours after transfection, cells were treated with different doses of isoginkgetin (Merk Millipore), IP2 or M2P2 for 18 h. Cell were then washed three times with PBS 1X and 5 × 106 cells were incubated with mouse FcR blocking reagent for 10 min at 4 °C according to the manufacturer protocol (Miltenyi Biotech). Cells were stained with anti-mouse MHC class I (H-2Kb)-PerCP-Cy5.5  or with the corresponding PerCp-Cy5.5-conjugated mouse (BALB/c) IgG2a, κ isotype for 30 min at 4 °C. Cells were stained with DAPI prior to acquisition for dead cell exclusion. To study the kinetics of endogenous H-2Kb recovery at the cell surface, cells were treated for 18 h with the different drugs. The day after, cells were washed and treated with ice-cold citric acid buffer  at 1 × 107 cells per milliliter for 120 s, washed three times with PBS, and resuspended in culture medium. At the indicated time point, an aliquot of cells  was collected and stained with anti-mouse H-2Kb PE or anti-mouse SIINFEKL-peptide bound to H-2Kb-PE. All flow cytometry experiments were conducted using the BD LSRII flow cytometer (BD Biosciences) and data are analyzed with the FlowJow software (V10).Mouse cells were treated with drugs for 18 h. Cells were harvested and 1 × 10Mouse cells were treated with drugs for 18 h. Floating and adherent cells were collected, washed in PBS and resuspended in 1× Annexin V Binding Buffer with APC Annexin V (BD Pharmingen) according to the manufacturer protocol. Cells were gently vortexed and incubated 15 min at RT in the dark. Cells were stained with DAPI prior to flow cytometry analysis. Cells were analyzed using the BD LSRII flow cytometer (BD Biosciences) and data were analyzed with the FlowJow software (V10).4 cells per well for 24 h. Cells were then treated with increasing doses of drugs overnight. After removing the medium, the MTT  (Sigma–Aldrich) powder was resuspended in PBS, filtered and added into the plate wells at 2.5 mg/ml. The plate was incubated for 2 h at 37 °C, with 5% CO2. It was then centrifuged, and the supernatant was carefully removed in order not to disturb the precipitated formazan. Crystals were dissolved in DMSO at 200 µL/well and the plate was shaken for 10 min. Absorbance was measured at 544 nm using the FLUOstar OPTIMA.Cells were plated in a 96-well plate at 1 × 10Cells were transfected with the YFP-Globin-SL8-intron as described previously and then treated with drugs for 48 h. Cells were then harvested and total cellular RNA was extracted and purified using the RNeasy Mini kit (Qiagen) according to the manufacturer protocol. The reverse transcription was carried out with 0.5 μg of RNA with the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer protocol. The StepOne real-time PCR system (Applied BioSystems) was used for qPCR and the reaction was performed with the Power SYBR green PCR master mix (Applied BioSystems). Results were analyzed using the StepOne software. Mouse gene specific primers were designed as follow: 18 S mouse forward primer, 5′-GCCGCTAGAGGTGAAATTCTTG-3′; 18 S mouse reverse primer, 5′-CATTCTTGGCAAATGCTTTCG-3′; Glob-SL8-exon forward primer, 5′-AGAAGTCTGCCGTTACTGCC-3′; Glob-SL8-exon reverse primer, 5′-AGGCCATCACTAAAGGCACC-3′; Glob-SL8-intron forward primer, 5′-GTATCAAGGTTACAAGACAG-3′; Glob-SL8-intron reverse primer, 5′-GGGAAAATAGACCAATAGGC-3′.1. Libraries were sequenced on the Illumina HiSeq 4000 sequencer using 50–base pair paired-end read method. All RNA-seq data were aligned to the mouse genome assembly GRCm38 (mm10) with the TopHat2 splice-junction mapper for RNA-Seq reads (version 2.1.1) with default parameters. For the differential gene expression analysis, the Cuffdiff program (version 2.2.1)62 was run using the following parameters: -library-type=fr-first-strand -compatible-hits-norm -library-norm-method geometric -min-reps-for-js-test 2 -dispersion-method per-condition -u -b. In all such analyses, the difference in expression of a gene was considered significant if the q value was less than 0.05 (the Cuffdiff default) and the fold change was superior to 2. Scatter plots were generated using ggplot2 in R environment. For the differential splicing analysis, the SUPPA2 program was run with the default settings64.MCA205 and B16F10 cells were treated with 15 μM of isoginkgetin or 35 μM of IP2 for 18 h. RNA was extracted using QIAGEN RNeasy columns. RNA-seq libraries were prepared from total RNA by poly(A) selection with oligo(dT) beads according to the manufacturer’s instructions (Life Technologies). The resulting RNA samples were then used as input for first-strand stranded library preparation using a custom protocol based on dUTP method as described previously6 MCA205 or B16F10 cells were lysed in 3 mL lysis buffer containing PBS, 0.25% Sodium Deoxycholate and protease inhibitor  during 1 h at 4 °C under agitation. Lysate was sonicated and then cleared by centrifugation for 1 h at 4000 × g at 4 °C. Supernatant was filtered through 0.40 µm filter and applied on affinity columns overnight . Flow was maintained at 10 rpm overnight by a peristaltic pomp. IP resins were prepared as follows: (i) 40 mg CNBr-activated Sepharose were activated in 1 mM HCl for 30 min at RT, (ii) 1 mg of Y-3 or B22-249 antibodies (kindly provided by Prof. Dr. Stefan Stevanović from University of Tübingen) was added in H2O, 0.5 M NaCl, 0.1 M NaHCO3 for 2 h at RT, (iii) resin was blocked in PBS, 0.2 M glycin for 1 h at RT, and finally (iv) resin was washed twice in PBS and resuspended at 1 mg antibody/mL in PBS. Centrifugation at 300 rpm for 4 min (no brake) and removal of the supernatant were performed between each step. On the second day, the peptides were eluted in 100 µL 0.2% trifluoroacetic acid (TFA) for 25 min at 4 °C under agitation. Elution was repeated three times. The four eluates were pooled, cleared by centrifugation for 5 min at 15,000 × g and filtered through Amicon 3 kDa MWCO . Prior to filtration, equilibration of the Omega membrane was performed by successive rinse/centrifugation cycles with MS-grade water, 0.1 N NaOH, MS-grade water and finally 0.2% TFA. Eventually, membrane was rinsed with 32.5% ACN, 0.2% TFA and the sample was concentrated using a Speed Vac Concentrator (Thermo Scientific). Samples were stored at −80 °C prior LC-MS/MS analysis.400 × 1018 pipette tips (Pierce Thermo Scientific), eluted in 40 µL acetonitrile 70% (Fisher Scientific)/0.1% formic acid, vacuum centrifuged and resuspended in 12 µL of 0.1% formic acid. All C18 zip-tipped peptides extracts were analyzed using an Orbitrap Fusion Tribrid equipped with an EASY-Spray Nano electrospray ion source and coupled to an Easy Nano-LC Proxeon 1200 system . Chromatographic separation of peptides was performed with the following parameters: Acclaim PepMap100 C18 pre-column , Pepmap-RSLC Proxeon C18 column , 300 nl/min flow, gradient rising from 95% solvent A  to 28% solvent B  in 105 min and then up to 40%B in 15 min followed by column regeneration for 50 min. Peptides were analyzed in the orbitrap in full ion scan mode at a resolution of 120,000 and with a mass range of m/z 400–650 using quadrupole isolation and an AGC target of 1.5 × 105. Fragments were obtained by Collision Induced Dissociation (CID) activation with a collisional energy of 35%. MS/MS data were acquired in the Orbitrap at a resolution of 30,000 in a top-speed mode, with a total cycle of 3 s with an AGC target of 7 × 104. The maximum ion accumulation times were set to 100 ms for MS acquisition and 150 ms for MS/MS acquisition in parallelization mode.The peptides were desalted using ZipTip C65 using a PWIZ-mzDB (https://github.com/mzdb/pwiz-mzdb) version 0.9.10, and then to MGF peaklists using a mzdb-access (https://github.com/mzdb/mzdb-access) version 0.7.0. Produced MGF files were submitted to Mascot database searches  against a mouse Uniprot protein sequence database released in July 2020 and downloaded from SwissProt website. The database contains 87,954 protein entries. Spectra were searched with a mass tolerance of 10 ppm in MS mode and 20 mmu in MS/MS mode. No missed cleavage was allowed (no enzyme search). Carbamidomethylation of cysteine residues and oxidation of methionine residues were set as variable modifications. For peptide validation, identification results were imported into the Proline software66 version 2.0 (http://proline.profiproteomics.fr) for target/decoy validation and label-free quantification. Peptide Spectrum Matches (PSM) with pretty rank equal to one and a Mascot score above 20 were retained. False Discovery Rate was then optimized to be below 1% at PSM level using an adjusted variant of the Mascot E-value67. For label-free quantification, peptide abundances were extracted by Proline using an m/z tolerance of 5 ppm. Alignment of the LC-MS runs was performed. Cross-assignment (a.k.a. match between runs) of peptide ion abundances was performed between the raw files using an m/z tolerance of 5 ppm and a retention time tolerance of 60 s. Precursor ions abundances were normalized using the quotient normalization method68 implemented in Proline. Fold change was calculated as the ratio of the mean normalized abundances in the IP2-treated and untreated biological replicates.Raw files were converted to mzDB files+ T cells were isolated from spleens of vaccinated mice using magnetic selection with CD8a (Ly-2) MicroBeads (Miltenyi Biotec). CD8+ T cells were co-cultured with BMDCs pulsed with 30 mg/mL of synthetic long peptides for 18 h in ELISpot plates coated with anti-IFNϒ capture antibody (Cell Sciences). Spots were revealed according to manufacturer instructions. IL-2 concentration was measured in the supernatants of the co-culture between CD8+ T cells and peptide-pulsed BMDCs using the Mouse IL-2 ELISA MAX Standard kit (BioLegend).C57BL/6 mice were vaccinated subcutaneously at the tail base with 100 µg of synthetic long peptide mixed with 30 µG ODN 1668. One week after, CD84 MCA205 wild-type (WT) or 7.5 × 104 MCA205-globin-SL8-intron cells, or with 5 × 104 B16F10 wild-type (WT) or B16F10-Globin-SL8-intron cells along with matrigel (VWR). Five days after challenge, mice were treated intraperitonealy with different dose of isoginkgetin, IP2 or M2P2. Intraperitoneal injection was repeated at day 10 and 15. Area of the tumor was recorded every 3–4 days until ethical limit points were reached. Mice which experienced complete tumor regression following treatment were kept for 100 days after tumor challenge and were then rechallenged subcutaneously on the right flank with 7.5 × 104 MCA205-Globin-SL8-intron cells and on the left flank with 5 × 104 B16F10 WT cells.Female wild-type C57BL/6 mice at the age of 6 weeks were obtained from Harlan France. NU/NU nude mice were obtained from Charles River France. Animal experiments were conducted in compliance with the EU Directive 63/2010, and protocols 2016_064_5677 and were approved by the Ethical Committee of the Gustave Roussy Campus Cancer . Mice were challenged subcutaneously in the right flank with 5 × 105 MCA205-globin-SL8-intron cells and thereafter treated with 24 mg/kg of IP2 at days 8 and 11. At day 12, OT-1 CD8+ T cells were isolated from spleens of OT-1 mice using magnetic selection with CD8a (Ly-2) MicroBeads (Miltenyi Biotec) and then intravenously injected into tumor-bearing mice at 3.0 × 106 cells/mouse. Three days after transfer, proliferation of OT-1 cells was measured in spleens by flow cytometry after staining with anti-CD8-FITC and APC-conjugated SL8/H-2Kb Dextramers (Immudex).C57BL/6 mice were challenged subcutaneously with 3.0 × 105 MCA205 WT were inoculated subcutaneously into the right flank. Mice were then treated twice a week with 24 mg/kg of IP2 for 2 weeks. In the therapeutic setting, C57BL/6 mice were injected subcutaneously in the right flank with 1.5 × 105 MCA205 WT and thereafter vaccinated once a week with 100 µg of synthetic long peptide mixed with 30 µG ODN 1668 and treated twice a week with 24 mg/kg of IP2 for 2 weeks.In the prophylactic setting, C57BL/6 mice were vaccinated twice subcutaneously at the tail base with 100 µg of synthetic long peptide mixed with 30 µG ODN 1668, two weeks apart (prime-boost). One week after the booster injection, 1.5 × 10t-test. A one-way analysis of variants (ANOVA) with Tukey’s post-hoc test was run when multiple conditions were tested. All data were processed on the GraphPad Prism (version 5) software.All data from T-cell activation assays were analyzed from at least three biological replicates, each one including three technical replicates. Data were presented as the means ± SEM of biological replicates. Significant changes were analyzed by a one-way analysis of variants (ANOVA) with Dunnett’s post-hoc test. All in vivo experiments were repeated at least twice with a minimum of six mice per group. Significant changes between two conditions were analyzed by a Student’s Further information on research design is available in the Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Description of Additional Supplementary FilesReporting SummaryPeer Review File