Moreover, patients with this mutation, treated with a CHOP regimen , had a poorer OS (p = 0.019). In addition, such patients were prone to have a more intensive infiltration of tumors . Thus, we speculated that patients with a TBL1XR1 mutation have poorer prognosis, partly due to greater invasion and infiltration of tumors. Our results suggest that the TBL1XR1 mutation can be used as an indicator to predict the prognosis of PTL and can be employed as a promising new target for treatment of PTL in the future.Primary testicular lymphoma (PTL), often appearing as focal masses in the scrotum and epididymides, is the most frequent testicular tumor in aged men. Although MYD88 and CD79B mutations were the most common genetic alterations observed, the gene mutation landscape of PTL remains poorly defined. In this study, we identified 1326 mutations involving 311 genes or regions in 90 PTL patients through next-generation sequencing (NGS). PTL patients with the TBL1XR1 mutation, irrespective of treatment therapy, had an inferior overall survival (OS) than TBL1XR1 WT (wild type) patients ( To the editor:Primary testicular lymphoma (PTL) is a rare, clinically aggressive type of extra nodal lymphoma . Approxip < 0.05. Statistical analyses were performed using SPSS 23.0. The follow-up was updated on August 31, 2019, with a median follow-up time of 36 (1–120) months. Fourteen patients (15.5%) were lost to follow-up  received an anthracyline-based chemotherapy, usually CHOP regimen. Twenty-four patients (27%) were simultaneously treated with rituximab. The median chemotherapy course was six courses. Twelve patients received irradiation aimed at the contralateral testis, and no patient received head irradiation. OS was estimated using the Kaplan-Meier method. The two-sided level of significance was r = 0.244 and r = 0.247, respectively)    Table . PTL patTBL1XR1, also known as TBLR1, is an evolutionarily conserved protein that has high structural and functional similarities. It plays an important role in activation of multiple intracellular signaling pathways, such as Wnt-β-catenin, NF-κB, and Notch signaling pathways . Dysregup = 0.025, x2 = 4.890). This finding is consistent with a previous study, which reported that abnormal regulation of TBL1XR1 is associated with advanced tumor stage, metastasis, and poor prognosis in most solid tumors [p = 0.002, HR 2.568, 95%CI 1.382–4.772) (Fig. In addition, patients with TBL1XR1 mutation were prone to have more intensive infiltration of tumors (d tumors . PatientIn conclusion, we found that TBL1XR1 is commonly mutated in PTL. Patients with TBL1XR1 mutations have lower OS, partly due to greater invasion and infiltration of tumors. Therefore, TBL1XR1 mutation can be used as an indicator to predict the prognosis of PTL and a promising new target for treatment of PTL in future.Additional file 1:Table S1. 446 known or putative mutational gene targets in hematologic malignancies detected by the next generation sequencing. Table S2. Characteristics of 90 PTL patients. Table S3. Mutated characteristics of 34 TBL1XR1 mutation PTL patients


In the present study, the seeds of two fragrant rice varieties i.e., Yuxiangyouzhan and Xiangyaxiangzhan under normal and Cd stress conditions i.e., 0 and 100 mg Lp > 0.05) however, it substantially improved the seedling growth and other related physiological attributes under the Cd stress. The mean fresh weight of the shoot, and whole seedling was increased by 16.92–27.88% and by 16.92–27.88% after ZnO NPs application. The root fresh weight, root-shoot length was also substantially improved under ZnO NPs treatment. Moreover, application of ZnO NPs induced modulations in physiological and biochemical attributes e.g., the superoxide dismutase (SOD) activity in root and shoot, the peroxidase (POD) activity and metallothionein contents in root were increased under low levels of ZnO NPs. The α-amylase and total amylase activity were improved by ZnO NPs application under Cd Stress. Besides, modulation in Zn concentration and ZnO NPs uptake in the seedling were detected. The metabolomic analysis indicated that various pathways such as alanine, aspartate and glutamate metabolism, phenylpropanoid biosynthesis, and taurine and hypotaurine metabolism were possibly important for rice response to ZnO NPs and Cd.Seed priming with ZnO NPs had no significant effect on the seed germination (Overall, application of ZnO NPs substantially improved the early growth and related physio-biochemical attributes in rice. Our findings provide new insights regarding the effects of ZnO NPs on seed germination, and early growth of rice, and its potential applications in developing crop resilience against Cd contaminated soils.The online version contains supplementary material available at 10.1186/s12951-021-00820-9. Rice is the staple food for more than four billion people globally and it is cultivated worldwide across different climatic conditions. Among heavy metals, cadmium (Cd) is recognized as the most toxic heavy metal, and its concentration in arable lands has increased due to excessive use of agro-chemical and anthropogenic activities –3.Rice generally has high bioaccumulation for various toxic metals such as arsenic (As), mercury (Hg), lead (Pb), and cadmium (Cd), respectively , 5. DietAlthough, Cd is a non-essential element, it is easily absorbed by plant roots, and competes with other bivalent ions such as Ca, Fe, Mn, and Zn to accumulate in plants –10, withRecently, nanotechnology has been extensively employed in the field of plant sciences to explore its potential impacts in improving crop yields under metal toxic conditions , 23. DueThe ZnO NPs can be applied to the plants in different ways, however, seed priming is considered an easy method which may be beneficial in the early growth stages of plants in environment with adverse conditions . AlthougSeeds of two fragrant rice varieties i.e., Xiangyaxiangzhan and Yuxiangyouzhan, were obtained from College of Agriculture, South China Agricultural University, China. These two varieties are commercially popular in South China as fragrant rice varieties.The ZnO NPs  were purchased from Aladdin Bio-Chem Technology Co. Ltd., Shanghai, China. The characterization of the ZnO NPs was reported earlier in a previous study, the particles were described to have an average size of 36 nm, as assessed by scanning electron microscopy (SEM), and 28 nm as determined by transmission electron microscopy (TEM), matching the specifications mentioned by the manufacturer (~ 30 nm) [− 1, respectively and then exposed to two Cd levels i.e., 0 and 100 mg L− 1. The concentrations of ZnO NPs and Cd used in this study were based on previous reports [−1) may have positive effect of plant growth whilst the Cd treatment with 100 mg L−1 could lead to germination and growth inhibition. There were 16 treatments in total and each treatment has 6 repeats with total 96 glass petri dishes in total.The experiments were conducted at the College of Agriculture, South China Agricultural University, Guangzhou, China. The seeds of the two rice cultivars i.e., Xiangyaxiangzhan and Yuxiangyouzhan were treated with four ZnO NPs treatments at 0, 25, 50 and 100 mg L reports , 36, 37,2+ in the ZnO NPs solutions and the Zn concentration in the seed after priming were detected  with a low concentration of Zn ions is obvious (the dissolution of the ions is about 1.2–1.5 mg L−1)  and stirred for 5 min, then followed by their dispersion in deionized water by water bath with ultrasonic treatment for 30 min . The see− 2 s−1 at daytime was maintained, and the relative humidity was set at 70%.Total 100 seeds were allowed to germinate on two layers of filter paper placed in a 150 mm diameter glass petri dishes. The filter paper in petri dishes were keep wet condition by covered with approximately 20 ml of Cd solution. The solution in the petri dishes was changed daily by replacing with fresh Cd solution in order to keep the concentration of the Cd constant. The petri dishes were placed in an incubator with a 14 h/28 ℃ day, and a 10 h/25 ℃ night, photosynthetically active radiation with a light intensity of 400 µmol of photons mThe germination was monitored every day after sowing, and the morphological traits were recorded from 4 randomly selected glass petri dishes in each treatment at seven days after sowing. Sample of 60 seedlings were randomly selected from each of the 4 randomly selected glass petri dishes of each treatment, and then placed immediately in liquid nitrogen, and stored at − 80 °C for subsequent assessment of physiological and metabolites profiles.Germination traits of seeds were recorded daily after the radicle length exceeded 2 mm. The germination rate is the ratio of the number of germinated seeds to the total number of seeds sown and is expressed as a percentage. Seed vigor index was calculated by multiplying the germination index with the shoot length as described previously .Ten seedlings from each of four randomly selected glass petri dishes were randomly harvested at seven days after sowing, and the length of the prophyll, leaf, leaf sheath, leaf blade, root, and shoot were measured. Another ten seedlings from each of four randomly selected glass petri dishes were randomly harvested for measurement of the fresh weight of the root, shoot, and seedling by using the electronic balance .g for 15 min at 4 °C. The supernatant consisted of the crude enzyme extract which was subjected to subsequent assays for the determination of the activities of antioxidants and the MDA content.The antioxidant enzyme activities and malondialdehyde (MDA) content were quantified according to method reported previously . Fresh s2 buffer, and 0.3 ml of 20 µmol lactoflavin in 5 ml beaker, and allowed to react under 4000 l × light for 30 min. The absorbance of reacted solution was recorded at 560 nm by using a benchtop spectrophotometer . One SOD active unit (U) is defined as the amount of the enzyme that inhibits 50% photochemical reduction of substrate NBT, and SOD activity was defined in the unit U g−1 fresh weight (FW) min−1.For superoxide dismutase (SOD) activity, crude enzyme extract (0.05 ml) was mixed with 1.75 ml of sodium phosphate buffer (pH = 7.8), 0.3 ml of 130 mM methionine buffer, 0.3 ml of 750 µmol nitroblue tetrazolium (NBT) buffer, 0.3 ml of 100 µmol EDTA-Na2O2, 0.95 ml of 0.2% guaiacol, and 1 ml of 50 mM sodium phosphate buffer (pH = 7.0). The absorbance was recorded at 470 nm by using a benchtop spectrophotometer at an interval of 30 s, with 5 replicates. One POD active unit (U) was defined as the amount of enzyme that catalyzed the reaction such that there was a reduction of 1 absorbance per min, and the POD activity was defined as U g−1 FW min−1.For peroxidase (POD) activity, 0.05 ml crude enzyme extract was mixed with 1 ml of 0.3% H2O2, and 1.95 ml of sodium phosphate buffer. The absorbance was recorded at 240 nm every 30 s, with 4 replicates. One CAT active unit (U) was defined the amount of enzyme that catalyzed the reaction such that there was a reduction of 0.1 absorbance per min, and the CAT activity was defined as U g−1 FW min−1.For catalase (CAT) activity, 0.05 ml crude enzyme extract was mixed with 1 ml of 0.3% H−1 FW.For assessing the MDA content, crude extract was reacted with thiobarbituric acid (TBA) in a boiling water bath for 30 min, and the absorbance of the supernatant was recorded at 532, 600, and 450 nm, respectively using a benchtop spectrophotometer, and the MDA content was expressed as µmol g2 with mortar and pestle along with 3.75 ml citrate buffer solution (pH = 5.6), and the homogenized contents were transferred to a 10 ml centrifuge tube and subjected to centrifugation at 12000 × g for 10 min at 4 ℃. The supernatant was transferred to a 25 ml volumetric flask, and diluted with deionized water to 25 ml, and mixed thoroughly. This enzyme extract (1 ml) was pipetted into a 10 ml centrifuge tube. The subsequent steps were the same for quantification of α-amylase activity as well as the total amylase activity, with the exception that the enzyme for α-amylase activity needed to be heated in water bath at 70 ℃ for 15 min. The enzyme extract (1 ml) and citrate buffer solution  were pipette into a 10 ml centrifuge tube and heated in water bath at 40 ℃. After 15 min of heating, 2 ml of 1% soluble starch solution was added into the tube, followed by the addition of 4 ml of 4 mol L−1 NaOH solution to end the reaction. The reaction solution (1 ml) as well as 1 ml of 3,5-dinitrosalicylic acid were added to a 10 ml centrifuge tube, and heated in boiling water for 5 min, and then cooled to room temperature, and diluted with 8 ml deionized water. The absorbance was recorded at 520 nm by using a benchtop spectrophotometer. The amylase activity was expressed as mg g−1 FW min−1. The β-amylase activity value was equal to α-amylase activity value subtracted out of the total amylase activity value.The amylase enzyme activity was estimated as previously described . Samples2 with mortar and pestle, and homogenized with 1 ml of buffer (20 µmol Tris-HCl (pH = 8.6), and 0.5 µmol phenylmethylsulfonyl fluoride), and 3 ml of 0.01% β-mercaptoethanol, and the resulting sample was transferred to a 10 ml centrifuge tube, and centrifuged at 10000 × g for 60 min at 4 °C. The supernatant was pipetted into a 10 ml centrifuge tube, and heated at 70 ℃ in water bath for 10 min, and centrifuged at 10000 × g for 60 min at 4 °C. The supernatant was the MT extract [Samples of fresh germinated seedlings (0.3 g) were crushed in liquid N extract , and the extract . The MT  extract .g for 10 min at 4 ℃. The absorbance of the supernatant was recorded at 665, 649 and 470 nm and the chlorophyll and carotenoids content were estimated [Fresh shoot samples (0.1 g) were extracted with 6 ml of 95% ethanol and put in the dark place for at 4 ℃. After 24 h, the samples were centrifuged at 700 × stimated .3:HClO4 (4:1 v/v) and resultant solutions were diluted to 25 ml and then filtered. The Zn 2+ in the solution was measured after filtration. The Zn and Cd content were determined using the Atomic Absorption Spectrophotometer  [Dry seedling and seeds samples of 0.50 g were digested with 12 ml HNO, Japan) .Fresh roots and shoots of germinated seedlings were thoroughly washed with deionized water, and then immersed in PBS (0.1 m) for 20 min, then centrifuge and pour off the supernatant, repeat three times, soaked with 4 ℃ osmic acid (1%) for 3 h, then immersed in PBS (0.1 m) for 20 min, centrifuged to remove the supernatant, then soaked with a series of gradient alcohol  for 15 min, immersed in acetone: epoxy resin (2:1), acetone: epoxy resin (1:1) and epoxy resin with 37 ℃ for 12 h, respectively. And then put the sample in a small capsule, added epoxy and waited for 48 h at 60 ℃, and sliced on ultra-thin slicer . After staining with lead and uranium, transmission electron microscopy  was performed . To furt2 for 10 min, and stored at − 80 ℃ in triplicate. The metabolite extraction and metabolite profiling analysis were performed at Wuhan MetWare Biotechnology Co., Ltd., China (www.metware.cn) following their standard procedures. In brief, the shoot was crushed using a mixer mill. The samples in their powdered form were weighed (100 mg), and extracted overnight in 0.6 ml of 70% aqueous methanol at 4 ℃ followed by centrifugation at 10000 × g for 10 min. The extract was absorbed using a CNWBOND Carbon-GCB SPE Cartridge , and filtered using a 0.22 µm pore size, and analyzed using an LC-ESI-MS/MS system . The mobile phase consisted of pure water with 0.04% acetic acid (solvent A), and acetonitrile with 0.04% acetic acid (solvent B). The sample measurements were performed with a gradient program that employed starting conditions of 95% A and 5% B. A linear gradient to 5% A and 95% B was programmed to be achieved within the first 10 min, and this level was maintained for 1 min and then, a composition of 95% A and 5% B was achieved within a rapid time frame of 0.1 min which was maintained for 2.9 min. The column oven was set to 40 °C, and the injected volume was 4 µl. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS. On a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), LIT and triple quadrupole (QQQ) scans were performed. The instrument was equipped with an ESI Turbo Ion-Spray interface, and the API 4500 Q TRAP UPLC/MS/MS System was controlled by Analyst 1.6.3 software (AB Sciex).Fresh shoot samples (1.0 g) were cut from rice seedlings, washed with deionized water, and snap-frozen in liquid Np < 0.05 level. For multivariate analysis, data were imported into the MetaboAnalyst software , and the heat map for the investigated parameters was established. The principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were performed to examine the intrinsic variation in the parameters, and to reduce the dimensionality of the data [Analysis of Variance (ANOVA) and correlation analyses were performed by using Statistix version 8  followed by comparisons of the sample means using Tukey’s HSD test at the data .p < 0.05) were noted across varieties (V), and Cd treatments, while differential doses of ZnO NPs treatments did not induce any significant difference in seed germination in both rice varieties. The germination dynamics showed that Cd treatment reduced the germination of rice seeds particularly at 3–5 days after sowing. Cd and V × ZnO NPs × Cd markedly affected seed vigor index. On an average, Cd stress decreased the germination rate, and the vigor index by 5.59% and 52.68%, respectively, as compared with no Cd treatment  for Xiangyaxiangzhan, and at ZnO NPs doses of 50 and 100 mg L−1, respectively for Yuxiangyouzhan under no Cd treatment. ZnO NPs treatments increased the total fresh weight of seedling in Xiangyaxiangzhan by 70.74, 79.48, and 99.56% at treatment of 25, 50, and 100 mg L−1, respectively, when exposed to Cd. Similarly, the total fresh weight of seedling in Yuxiangyouzhan markedly increased by 52.92% and 39.16% at 25, 50, and 100 mg L−1 ZnO NPs, respectively . The shoot length of rice seedlings improved significantly in Xiangyaxiangzhan exposed to ZnO NPs application at 50 mg L−1 (27.20%), and 100 mg L−1 (26.84%). In Yuxiangyouzhan, the shoot length was increased by 29.90–34.79% under the different ZnO NPs treatment  significantly increased after exposure to ZnO NPs treatment at 25, and 100 mg L−1, respectively. The activity of α-amylase in Xiangyaxiangzhan rice seedlings increased after ZnO NPs treatment at concentrations of 50 and 100 mg L−1, respectively under normal conditions. The α-amylase activity in Cd stressed seedlings significantly increased by 17.45%-26.56% and 10.56%-13.71% in both rice varieties under all ZnO NPs treatments , and total amylase (in the seedling) . The MDA content was decreased by 26.94, 33.60, and 15.88% at ZnO NPs dosages of 25, 50, and 100 mg L− 1, respectively, under Cd stress. The MDA content in the seedling was not significantly affected by ZnO NPs treatments for Xiangyaxiangzhan (in absence of Cd), and Yuxiangyouzhan (with or without Cd stress). The MDA content reduced by 16.63% and 26.83 at ZnO NPs at 50 and 100 mg L− 1, respectively as compared to untreated control (0 mg L− 1 ZnO NPs) under Cd exposure  under Cd stress. Moreover, the MT concentration in the shoot of Yuxiangyouzhan was increased by 25.43 and 19.68%, at 25, and 50 mg L− 1 ZnO NPs, respectively, under non-stress conditions. The ZnO NPs at 50 mg L− 1 exhibited significant decrease in MT concentration in the seedlings of Xiangyaxiangzhan under non-stressed conditions, however, the concentration of MT in seedlings of Yuxiangyouzhan significantly increased at 100 mg L− 1 ZnO NPs. Moreover, the concentration of MT in seedling of Yuxiangyouzhan was increased by 21.77 and 15.18% at 50 and 100 mg L− 1 ZnO NPs dosages, respectively under Cd exposure  were sampled for analysis of metabolites  and pmb2922 (uridine 5'-diphospho-d-glucose), while treatment D resulted in the highest values of pmf0585 (dl-proline), pme2634 , mws0256 , mws1712 (d-(+)-phenylalanine), pmb2940 (1-o-β-d-glucopyranosyl sinapate), mws0180 , pmn001578 (hexadecanoic acid), mws0230 (l-(−)-threonine), pmn001695 (trihydroxycinnamoylquinic acid), and mws0281 (citric acid) ’, ‘starch and sucrose metabolism’, ‘pantothenate and CoA biosynthesis’, ‘butanoate metabolism’, ‘glycine, serine and threonine metabolism’, ‘phenylalanine metabolism’, ‘C5-branched dibasic acid metabolism’, ‘biosynthesis of secondary metabolites – unclassified’, and ‘cysteine and methionine metabolism’ were identified from the detected metabolites Fig. .Fig. 8ThThe PCA of metabolic profiles in shoot of rice seedling of Xiangyaxiangzhan and Yuxiangyouzhan under all experimental treatments were constructed  adenosine, guanosine 5'-monophosphate, cytidine 5'-monophosphate (cytidylic acid), and uridine 5'-monophosphate, and upregulated metabolites such as adenine, uridine 5’-diphosphate, adenine, xanthine, uridine 5’-diphosphate, γ-glu-cys, l-ascorbic acid, eicosenoic acid, eicosadienoic acid, and xanthine. The ZnO NPs 50 + Cd 0 treatment resulted in reduction in xanthine. Moreover, ZnO NPs 50 + Cd 100 treatment upregulated metabolites such as adenine, xanthine,γ-Glu-Cys, l-ascorbic acid, eicosenoic acid, and eicosadienoic acid, which led to inhibition in the metabolites such as 5'-deoxy-5'-(methylthio)adenosine and duanosine 5'-monophosphate   Fig. . Previou culture . In thisStarch is an important storage polysaccharide for energy involved in the growth of rice seedlings. A previous study showed that α-amylases could facilitate both seed germination and seedling growth by mobilizing nutrients in the endosperm, however, starch would be immobilized under the Cd toxicity, which resulted in growth inhibition . The exp2− to H2O2 and O2, whereas CAT scavenges the H2O2 generated during the photorespiration and β-oxidation of fatty acids [2O2 in the oxidation of various inorganic and organic substrates. Cd toxicity enhances the production levels of ROS, which increases lipid peroxidation, resulting in an increased generation of MDA as a byproduct [Moreover, seed priming with ZnO NPs regulated the antioxidant enzyme activity i.e., SOD, POD, CAT, as well as MT and MDA contents in both rice varieties. Normally, SOD reduces Oty acids . The PODyproduct , 51. Besyproduct . In thisyproduct . In thisyproduct , and is yproduct . MT cont−1) whilst the Zn concentration in the seed after priming were very high (up to 50 µg g− 1 DW) at ZnO NPs seed priming treatments  were assessed . The AsA-GSH cycle system may use AsA/DHA, GSH/GSSG, and NAD(P)H/NAD(P) to maintain an appropriate redox environment in plants [Moreover, according to the analysis of the differential metabolites, high levels of L-Ascorbic acid, γ-glu-cys, and oxidized glutathione were detected in A vs B and A vs D for both varieties, while no significant change was observed in A vs C , modulation in Zn concentration and ZnO NPs uptake in the seedling, and other metabolites in rice seedlings under Cd stress. Future in-depth of field studies are still needed to explore the exact mechanisms involved in ZnO NPs -induced improvements in early growth of rice under Cd toxic conditions. Overall, present findings may assist in developing feasible strategies for growing economically important crops in agricultural lands contaminated with high concentrations of heavy metals.Additional file 1: Figure S1. The change of pH and Zn2+ in the ZnO NPs solutions and the Zn concentration in the seed after priming. pH in the ZnO NPs solutions (A), Zn2+ in the ZnO NPs solutions (B), and the Zn concentration in the seed after priming (C). ZnO NPs 0, ZnO NPs 25, ZnO NPs 50 and ZnO NPs 100: 0 mg L− 1, 25 mg L− 1, 50 mg L− 1 and 100 mg L− 1 of ZnO NPs. Cd 0 and Cd 100: 0 mg L− 1 and 100 mg L− 1. Values were represented as mean ± SD (n = 4). Different low case letters among the treatments within a variety shows the statistically significant at p < 0.05 according to least significant different test.Figure S2. The chlorophyll and carotenoids content in shoot. The chlorophyll a content in shoot. (A), the chlorophyll b content in shoot. (B), the total chlorophyll content in shoot (C), and the carotenoids content in shoot (D). ZnO NPs 0, ZnO NPs 25, ZnO NPs 50 and ZnO NPs 100: 0 mg L− 1, 25 mg L− 1, 50 mg L− 1 and 100 mg L− 1 of ZnO NPs. Cd 0 and Cd 100: 0 mg L− 1 and 100 mg L− 1. Values were represented as mean ± SD (n = 4). Different low case letters among the treatments within a variety shows the statistically significant at p < 0.05 according to least significant different test. Figure S3. TEM images of the rice roots and shoot in germinated seedlings. Red arrows nanoparticles.Figure S4. Analysis of the metabolic profiles in shoot of rice seedling. Principal component analysis (PCA) of metabolic profiles in shoot of rice seedling of Xiangyaxiangzhan and Yuxiangyouzhan under control and treatments (A). The Identified total significant different metabolites and up- and down-regulated metabolites (B). The Venn diagram of the significant different metabolites among the treatments (C). KEGG enrichment analyses of the identified significant different metabolites (D) and ranking of the identified significant differential metabolites (E) in Xiangyaxiangzhan and Yuxiangyouzhan under different treatments. X_A: ZnO NPs 0 + Cd 0 for Xiangyaxiangzhan, X_B: ZnO NPs 0 + Cd 100 for Xiangyaxiangzhan, X_C: ZnO NPs 50 + Cd 0 for Xiangyaxiangzhan, X_D: ZnO NPs 50 + Cd 100 for Xiangyaxiangzhan; Y_A: ZnO NPs 0 + Cd 0 for Yuxiangyouzhan, Y_B: ZnO NPs 0 + Cd 100 for Yuxiangyouzhan, Y_C: ZnO NPs 50 + Cd 0 for Yuxiangyouzhan, Y_D: ZnO NPs 50 + Cd 100 for Yuxiangyouzhan. The abscissa indicates that the rich factor, ordinate corresponding to each pathway is the path name, and the color of the point is p-value, the redder the enrichment is more significant. The size of the points represents the number of enriched differential metabolites.Figure S5. Metabolic pathways network.Additional file 2: Table S1. ANOVA analysis of the growth and physiological parameters.Additional file 3:Table S2. Metabolites detected in the rice varieties.Additional file 4:Table S3. The identified significant different metabolites in Xiangyaxiangzhan and Yuxiangyouzhan under different treatments.Additional file 5:Table S4. Fold change of the detected metabolites involved in the metabolic pathways.


Saccharomyces cerevisiae expressing heterologous pathways for xylose, arabinose, and galacturonic acid metabolism has been constructed by a Cas9-based genome editing technology [1]. The fermentation performance of the final strain (YE9) was tested under various substrate conditions, and the fermentation parameters were calculated. The dataset can be used for designing bioprocesses for pectin-rich biomass. In This dataset contains 1) the construction of engineered 22.11)Guide RNA (gRNA) plasmid constructionTo construct the YE9 strain, four consecutive transformations were performed as summarized in DpnI and used to transform E. coli TOP10 . The transformants were selected on an LBA  agar plate. The gRNA sequence of the resulting plasmid was confirmed by Sanger sequencing using a universal primer for the T3 promoter. All other gRNA plasmids were constructed using the same procedure but different primers, as listed in 2)Donor DNA preparationgRNA sequences are designed to be target cut site-specific and 20-bp long, as listed in in vivo assembly and genome integration through homologous recombination. Each assembly was an expression cassette of a heterologous gene as described in 3)Yeast transformationDonor DNA fragments were prepared by PCR using the primers listed in For yeast transformation, a gRNA plasmid (4 μg) and donor DNA fragments (4 μg each) were used to transform a designated strain harboring pRS41N-Cas9 . The res2.2oC and 250 rpm. Cells were centrifuged, washed twice, and re-suspended in YP medium supplemented with desired carbon sources. The initial cell density of fermentation was 25 g/L dry weight, which corresponds to approximately 125 g/L wet weight, and this conversion factor was obtained from a prior study [For fermentation of the YE9 strain, one colony was pre-cultured in YP medium (10 g/L yeast extract and 20 g/L peptone) supplemented with 20 g/L of glucose for 36 h at 30or study . In the 2.32SO4 at 0.6 mL/min and 50oC [Quantitation of glucose, fructose, galactose, xylose, arabinose, galacturonic acid, glycerol, and ethanol was performed by high-performance liquid chromatography  device equipped with a RI detector and a Rezex-ROA Organic Acid H+ (8%) (150 mm × 4.6 mm) column . The column was eluted with 0.005 N H


Activity and expression of the phosphoinositide 3-kinase (PI3K) catalytic isoform, PIK3CD/p110δ, is increased in schizophrenia, autism, and intellectual delay and pro-cognitive preclinical efficacy of p110δ-inhibition has been demonstrated in pharmacological, genetic, and developmental rodent models of psychiatric disorders. Although PI3K signaling has been implicated in the development and function of neurons and glia; isoform-specific roles of the individual PI3Ks are less clear and the biological effects of increased p110δ on neuronal development are unknown. Since the pathobiological direction of p110δ changes in neurodevelopmental disorders are increased expression and activity, we hypothesized that overexpression of p110δ would impact measures of neuronal development and maturation relevant to connectivity and synaptic transmission. p110δ overexpression in primary rat hippocampal cultures significantly reduced dendritic morphogenesis and arborization and increased immature and mature dendritic spine densities, without impacting cell viability, soma size, or axon length. Together, our novel findings demonstrate the importance of homeostatic regulation of the p110δ isoform for normative neuronal development and highlight a potential pathophysiological mechanism of association to disorders of neurodevelopment. PI3K is a heterodimer formed from a regulatory and catalytic subunit, both of which have multiple isoforms. Class I catalytic isoforms are the most commonly studied class of PI3Ks and are termed p110α, -β, -γ, and -δ  are essential lipid kinases that signal up- and down-stream of critical cell signaling molecules including multiple growth factors, cytokines and their cognate receptors  have begun to be uncovered  were sacrificed at embryonic day 18 (E18) and primary dissociated cultures were prepared from dissected embryonic hippocampi, as previously described  and the sequence was verified. Empty vector, with no DNA insert was used as a control for comparison.Human full-length PIK3CD (NM_005026) N-terminal c-Myc tagged DNA construct (Agilent) was generated as previously described  1, 7, and 17, cells were co-transfected using Lipofectamine2000 (Thermo-Fisher), per the manufacturer’s instructions with an incubation time of 1 h, with 0.5 μg plasmid DNA pVenus-GFP  and calcein-AM (1 μM) in neurobasal media was added to cell culture wells and incubated with neurons for 30 min at 37°C. Three images were taken from each of two culture wells per treatment on a Zeiss Axiovert epifluorescent microscope under green and red filters. Images were co-localized using ImageJ software (NIH) and cells were counted. Viability was calculated as the total number of living cells (green) divided by the total number of cells (green + red).n = 17 control and n = 22 p110δ-overexpression (OE) neurons. To calculated the CTCF, the Integrated Density of the p110δ signal was measured from the area around each cell soma in ImageJ; the Mean Fluorescent Intensity was then averaged from three background selections in each image, multiplied by the measured soma area, and subtracted from the Integrated Density  and n = 13 p110δ-OE neurons . Dendritic spines were subdivided into previously established categories based on morphology , DIV10 (transfected DIV7), and DIV20 (transfected DIV17) cells were fixed using PBS with 4% paraformaldehyde/4% sucrose. Cultures used for the detection of phosphorylated protein were additionally treated with PhosSTOP  during fixation. Cells were immunostained using antibodies against GFP  to enhance the fluorescent signal for morphological assessment, against myc  for identification of neurons overexpressing PIK3CD, and against p110δ  and phospho-Akt Ser473  for verification of overexpression and activity. Secondary antibodies were Goat anti-Rabbit 488 and Goat anti-Mouse 555 . Only neurons positive for both myc and GFP were selected for analysis. Z-stack images were taken with a Zeiss inverted LSM700 confocal with 10× (axons), 20× (dendrites), 40× (p110δ and pAkt) and 63× (spines) objectives, and 2D projections were compressed and analyzed in ImageJ software (NIH). The corrected total cell fluorescence (CTCF) was used as a measurement of p110δ immunofluorescent intensity from t-tests to analyze the effect of treatment (control vs. p110δ-OE). Homogeneity of variance was tested using Levene’s Test of Equality of Variances, in SPSS. Where Levene’s Test was statistically significant an adjustment to the degrees of freedom using the Welch-Satterthwaite method, was performed in SPSS and the adjusted t-value, p-value and dfs are presented. Sholl analysis results were compared using repeated-measures ANOVA (RM-ANOVA) with independent samples t-tests at each radius used for post hoc analyses. P < 0.05 considered statistically significant.SPSS (version 24.0) was used to perform all statistical comparisons. Viability, axon length, soma size, dendrite number and length, and spine number results were compared using independent samples t(58) = −1.225, P = 0.226) nor soma size (t(58) = 0.656, P = 0.515) were significantly altered in the context of p110δ overexpression (t(10) = 0.460, P = 0.655; To assess the impact of p110δ overexpression on hippocampal neuronal development we examined axon length and soma size in hippocampal neurons following acute p110δ overexpression from DIV1 to DIV3 . Neitherpression . HippocaF = 13.835, p < 0.001). p110δ overexpression resulted in a dramatic decrease in dendritic complexity  = 5.102, p < 0.001) and a significant effect of treatment  = 15.837, p < 0.001). This remarkable simplification in dendritic complexity was evident across the entire dendritic tree, with a significant reduction in the number of intersections observed at distances >35 μm from the soma (p < 0.05 − <0.001).To assess dendritic growth following p110δ overexpression we performed Sholl analysis as a measure of dendritic complexity on hippocampal neurons following acute p110δ overexpression from DIV7 to DIV10 . As expemplexity  manifestt(27.93) = 3.214, p = 0.003) and tertiary (t(26.1) = 2.393, p = 0.024) but not primary (t(44.58) = 0.303, p = 0.763) dendrites (t(264.1) = 3.319, p = 0.001) and secondary (t(533.9) = 3.929, p < 0.0001) but not tertiary (t(249) = −0.461, p = 0.645) dendrites  = −3.272, P = 0.003) confirming overexpression of p110δ in the experimental condition compared to empty vector transfection. PI3K signaling is often assessed by activation of the downstream target AKT via phosphorylation at Serine 473  for myc-tag and p110δ protein under control and overexpression conditions . p110δ it(66.12) = −3.80, p < 0.001). Strikingly, this increased density in response to p110δ expression was evident across all categories of spines, including filopodia spines (t(63.79) = −3.54, p = 0.001), mushroom spines (t(66.76) = −2.48, p = 0.016) and stubby spines (t(82) = −2.70, p = 0.009; We assessed the impact of p110δ overexpression from DIV17 to DIV20 on dendritic spine dynamics . Hippocaex vivo neuronal primary culture. We report that p110δ overexpression reduced dendritic morphogenesis and arborization as well as increased spine number, without affecting neuronal viability, axon length or soma size. Given that altered dendritic complexity and synaptic spine number are common neuropathy for a number of severe neurodevelopmental disorders including schizophrenia, autism and intellectual disability (Raymond et al., PI3K signaling is essential for a wide range of neurobiological functions, with an emerging focus on the individual roles of PIK3 catalytic subunits in disorders of neurodevelopment (Gross and Bassell, Several studies exemplify that the complexity of dendritic arborizations is critical to neuronal communication and that alterations in dendritic complexity lead to changes in neuronal signaling (Mainen and Sejnowski, In addition to abnormalities of dendritic arborization, growth, and branching, we also find that p110δ overexpression significantly increases the density of dendritic spines, including both immature and mature spine types. While we are the first to report that p110δ levels regulate synaptic spine development and maturation, our findings are consistent with previous work exploring the role of Class I PI3Ks in synaptogenesis, which has revealed that PI3K-overexpression increases synapse numbers in drosophila (Martín-Peña et al., via the AKT/mTOR signaling pathway, the present study failed to demonstrate an effect of p110δ overexpression on hippocampal neuron viability. While a global reduction in PI3K signaling leads to increased cell death (Carpenter and Cantley, While PI3K signaling is commonly associated with cell survival In conclusion, our results demonstrate that temporally elevated levels of p110δ alter neuronal development, with direct implications for altered connectivity and synaptic transmission, consistent with the association of PIK3CD/p110δ to neurodevelopmental disorders including schizophrenia, autism, and intellectual disability. These novel findings provide initial evidence as to how dysregulation of p110δ may mechanistically contribute to the etiology of diverse neurodevelopmental disorders through its effects on neuronal morphogenesis and synaptic connectivity.All datasets generated for this study are included in the article.The animal study was reviewed and approved by University of Colorado Anschutz Medical Campus Institutional Animal Care and Use Committee.VH, CP, and AL designed all experiments, authored the manuscript, performed and oversaw the statistical analysis of results. VH and CP performed all experiments.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.


During the first 100 min of cycling, athletes consumed amylopectin starch (AP), maltodextrin+sucrose (MD+SUC), or maltodextrin+fructose hydrogel (MD+FRU) drinks providing 95 g carbohydrate·h−1, followed by water intake only at 120 and 160 min. Fuel use was determined using indirect calorimetry and stable-isotope techniques. Additionally, dental biofilm pH was measured using the microtouch method in a subsample of participants (n = 6) during resting conditions before, and at different time intervals up to 45 min following a single bolus of drink. Exogenous carbohydrate oxidation (CHOEXO) during the 2nd hour of exercise was significantly (P < 0.05) different between all three drinks: MD+FRU (1.17 ± 0.17 g·min−1), MD+SUC (1.01 ± 0.13 g·min−1), and AP (0.84 ± 0.11 g·min−1). At the end of exercise, CHOEXO and blood glucose concentrations  were significantly lower post MD+FRU consumption than post MD+SUC and AP consumption (P < 0.05). Biofilm acidogenicity at rest demonstrated a less pronounced pH fall for MD+FRU compared to the acidulant-containing MD+SUC and AP (P < 0.05). In conclusion, while total intake of MD+FRU showed signs of completed uptake before end of monitoring, this was less so for MD+SUC, and not at all the case for AP. Thus, this study showed that despite carbohydrates being encapsulated in a hydrogel, a higher CHOEXO was observed following MD+FRU drink ingestion compared to AP and MD+SUC consumption upon exposure to the acidic environment of the stomach. This finding may be related to the higher fructose content of the MD+FRU drink compared with the MD+SUC and AP drinks. Furthermore, a carbohydrate solution without added acidulants, which are commonly included in commercial sport drinks, may have less deleterious effects on oral health.The purpose of this study was to evaluate the substrate oxidation of three commercially available, 14%-carbohydrate sports drinks with different compositions, osmolality, and pH for their impact on dental exposure to low pH. In a cross-over, randomized double-blinded design, 12 endurance athletes (age 31. 2 ± 7.7 years,  It is well-established that carbohydrate-electrolyte drink ingestion can enhance exercise performance and capacity , 2. Prop−1), thereby limiting applicability of the results.However, considering that sweat rate and fluid requirements are dependent on exercise intensity, duration, and environmental conditions , intake of a more concentrated carbohydrate solution may also provide a practical strategy to sustain performance and high carbohydrate oxidation rates during exercise. On the other hand, increasing carbohydrate content of the ingested fluid has been shown to delay gastric emptying rate . Hyperto13C in exhaled CO2) is a common method to track and quantify exogenous carbohydrate fuel use during exercise  commercial sports drinks of different carbohydrate composition, osmolality, and pH, for substrate oxidation and their impact on GI symptoms and dental biofilm acidogenicity. At the time of designing the study (2017), only a limited number of highly concentrated sports drinks specifically marketed for use during exercise were commercially available. Based on previous research using a dual-isotope approach , we hypo−1; body mass: 76.6 ± 3.3 kg−1; and 2max: 65.6 ± 5.0 mL·kg−1) were recruited in the Gothenburg region, Sweden via local contacts. One subject withdrew after completing two of the three experimental exercise trials for reasons unrelated to the study. A subsample (n = 6) of the participants was randomly selected for dental biofilm acidogenicity assessments performed under resting conditions. All participants provided written informed consent and the study was approved by the local ethics committee at Gothenburg University (Dnr. 1088-17).Twelve trained endurance cyclists/triathletes aged 31.2 ± 7.7 years (height: 183.3 ± 7.0 cm2max) and maximum power output (Wmax).All study participants were required to attend four separate visits to the exercise laboratory  see . At firs2max test, the participants performed the first of three exercise trials in randomized order. The following drinks were administered in a double-blind manner at each respective trial: AP , MD+SUC , or MD+FRU . Vitargo Electrolyte was reinforced with pure amylopectin according to the manufacturer's recommendations in order to achieve an increased carbohydrate content without increasing the concentration of other drink components. Drink characteristics are shown in 13C in the three drinks: AP = −11.71%0, MD+SUC = −14.67%0, and MD+FRU = −11.50%0 vs. Vienna Pee Dee Belemnite (VPDB), respectively.Within 2 weeks following the −1 of carbohydrates (95 g·h−1) interspersed as six boluses provided every 20 min during a 100 min period with first feeding at time = 0 . The study design  of participants made three separate visits in randomized order to the cariology lab for dental measurements during resting conditions following a bolus intake of one of the three sports drinks  and under the same environmental conditions (19.5 ± 0.6.C and 41.7 ± 14.1% relative humidity). Additionally, a subsample (s drinks .−1). Maximal oxygen consumption (2max) was considered to be reached when one of the following three criteria were met: (1) a leveling off of 2 with increasing workload; (2) a plateau of 2 with increasing workload; and (3) a respiratory exchange ratio (RER) of ≥1.08. 2max calculated as the average oxygen uptake during the last 60 s of the test. Heart rate  and work load (W) were continuously recorded, and 2 and VCO2 were measured using an online automated gas analysis system  calibrated according to manufacturer's instructions. The Wmax (372 ± 33 W) was predicted from the 2 of each submaximal workload by extrapolation up to 2max using linear regression.The incremental exercise test consisted of a 5 min warm up and 3, 5 min, steady state work-loads, followed by a 22 W increase of load every minute until the participants reached volitional exhaustion or could not maintain pedal speed  and the remaining 180 min at 55% Wmax (60.3 ± 2.5% 2max). Respiratory gases were collected for 5 min periods on 12 occasions: at every 10 min interval for the first 30 min of exercise, and then at 5 min intervals every 20 min interval . Prior to exercise, height, and nude body mass were recorded  and a resting blood sample was collected. Participants were subsequently mounted onto the cycle ergometer; the first 30 min of cycling was performed at 45% interval . Mean va13C/12C in expired CO2, expired gas was withdrawn into two 65 mL syringes  connected to the mixing chamber  via a three-way valve. Samples were emptied from the syringe into 12 mL Exetainer vials  in duplicate for later analysis.For the measurement of Wmax exercise bout.Psychometric scales including overall rating of perceived exertion (RPE)  and severity of GI symptoms , where symptoms were rated on a scale from 0 to 20 (no symptoms to worst conceivable symptoms) and level of digestive comfort (extremely uncomfortable to extremely comfortable) , were reFingertip blood sampling, drink ingestion, and psychometric scales (in that order) were performed immediately following the respiratory gas measurement.The dental biofilm pH was assessed after a single exposure using the microtouch method, where a microelectrode  connected to an Orion SA720 pH/ISE Meter  was inserted into the dental biofilm . Each dr13CO2/12CO2 enrichment (δ13C) using a Thermo Scientific Delta Ray isotope ratio infrared spectrometer (IRIS) with a Universal Reference Interface (URI) and a Teledyne CETAC ASX-7100 Autosampler. Every two samples were bracketed by calibrating gas (δ13C 27.8%0 VPDB). The 13C enrichment of drink contents was determined using a Costech Elemental Analyzer  in continuous flow mode coupled to a Thermo Scientific Delta V plus  isotope ratio mass spectrometer . All isotope ratio data were normalized to the VPDB scale.Blood samples were analyzed for glucose and lactate . Breath samples were analyzed for −1) during the experimental trials were calculated from 2 and VCO2 (L·min−1) using stoichiometric equations . The rate of exogenous carbohydrate oxidation was calculated using the formula of Mosora et al.  is the amount of CO2 (L−1) produced for the complete oxidation of one gram of glucose.where δExp is the isotopic composition of expired CO13C/12C ratio in breath air, the participants exercised for the first 30 min at 45% Wmax in a fasted state (n = 12) performed a 60 min exercise trial in a fasted state (30 min at 45% Wmax followed by 30 min exercise at 55% Wmax), showed no mean difference in δ13C despite differences in work load between time = 30 and 60 min, respectively. Time = 0 min denotes the first carbohydrate feeding  0.40 g/g; SUC (n = 2) 0.421 g/g; MD  0.436 g/g; and AP  0.444 g/g, respectively.The carbon content of a serving  was calcEXO) and the total intake of carbohydrates, both expressed as the corresponding amount of carbon.Oxidation efficiency was calculated as the ratio between the total exogenous carbohydrate oxidation during the trial (AUC_CHOn) was estimated via G* software . Assuming a power of 90% and an α-level of 5%, n = 9 participants was determined to be adequate to detect differences in mean 0–120 min exogenous carbohydrate oxidation between trials. However, since this estimation was predicted from a limited sample size pilot study , we decided to include 12 participants in this study. Furthermore, based on previous research by Hans et al.  when the analysis of variance yielded a significant F-ratio. All data were analyzed using SPSS 22.0 software . All data, unless otherwise stated, are presented as mean ± SD. Reproducibility of isotope enrichment measurements was assessed by comparing δ13C values (X1 and X2) from duplicate series of samples analyzed on different occasions, each series containing thirteen samples. For each series of duplicate samples, standard deviation of first measurement minus second measurements (σX2X1−) was calculated. Reproducibility of single measurements was estimated as σ = σX2X1− × √2−1.All data were checked for normality using a Shapiro–Wilk test. Two-way repeated measures ANOVA (treatment × time) with Bonferroni TOT) between the three experimental trials. A higher mean oxygen uptake was observed in the MD+FRU trial compared to that of the MD+SUC during the 1st hour of exercise and between AP and MD+SUC at 60–120 min and 0–180 min. These differences were significant (P < 0.05), but small  for each participant (13CO2 of breath samples taken during the 3.5 h cycling exercise (13CO2 measurements was at an average 0.06‰ (range 0.03–0.09‰). Mean values for CHOEXO oxidation are presented in EXO across trials . The CHOEXO was significantly higher for MD+FRU compared with both MD+SUC and AP . The CHOEXO for MD+SUC was also significantly higher than for AP.Exogenous carbohydrate oxidation  compared to MD+SUC and MD+FRU, and there was a tendency (P = 0.053) toward MD+FRU generating higher CHOEXO oxidation than MD+SUC. In addition, peak CHOEXO oxidation occurred ~40 min earlier for MD+FRU than MD+SUC and AP  = 15.4; P < 0.001] and mean CHOEXO oxidation was significantly lower in MD+FRU trial than MD+SUC and AP at the end of exercise . As shown in EXO oxidation did not level-off in the AP trial despite the withdrawal of carbohydrate feedings, nor was it attenuated at 140 and 160 min in the MD+SUC trial. However, the total amount of the ingested carbohydrates that was oxidized during the entire 180 min exercise was significantly less following AP than MD+FRU and MD+SUC ingestion  , a significant time × treatment interaction was also evident [ctively] . In summENDO) during exercise was similar across trials . This may have been related to the significantly lower CHOEXO oxidation in the AP trial during this time period .Endogenous carbohydrate oxidation  = 7.40; P = 0.003]. Between 140 and 180 min of exercise, glucose concentrations decreased more in the MD+FRU trial than in the MD+SUC and AP trials, reaching statistical significance at 140, 160, and 180 min for MD+SUC  and for AP at 160 and 180 min . Glucose concentrations at the end of exercise were 3.54 ± 0.50, 4.07 ± 0.67, and 4.28 ± 0.47 mmol·L−1 for MD+FRU, MD+SUC, and AP, respectively.Blood glucose concentrations were similar between MD+FRU, MD+SUC, and AP trials before and during exercise when subjects were fed carbohydrates . During Wmax exercise bout  and reached peak values across trials synchronously with the last carbohydrate feeding at 100 min  and for MD+SUC at 40, 60, and 80 min .Blood lactate concentrations did not differ between drink conditions at the onset of the 55%  100 min . BetweenF = 24.9; P < 0.001] with no difference between drink conditions. Mean RPE in the final hour of exercise was 13.1 ± 0.1, 13.2 ± 0.2, and 13.3 ± 0.2 for MD+FRU, MD+SUC, and AP, respectively  with mean values of respective measures < 4, and no observed differences across the three trials . Subjectectively . The relF = 58.5; P = 0.001], 5 min  and 10 min , and between MD+FRU and AP at 2 min . The pH gradually recovered over a 10–15 min period, and from 15 min onwards no significant differences were seen between the three drinks. No statistically significant differences were found when comparing the AP and MD+SUC drinks. A larger maximum pH-drop  and a lower minimum pH  was found for MD+SUC compared to MD+FRU.At 2–10 min after ingestion, dental biofilm pH was lowered to approximately pH 6.0 for the MD+SUC and AP drinks, while a less pronounced fall in pH was seen for MD+FRU . StatistThe present study investigated the effects of three highly concentrated, commercially available sports drinks differing in carbohydrate composition, osmolality, and acidity (pH) on substrate utilization and dental biofilm pH. As hypothesized, during the 2nd hour of carbohydrate feeding (60–120 min), the MD+FRU drink generated significantly higher exogenous carbohydrate oxidation than both the multiple transportable MD+SUC drink, and the single carbohydrate source drink (AP). Furthermore, biofilm-pH measurements at resting conditions demonstrated a significantly less pronounced fall for MD+FRU compared to that of both MD+SUC and AP.Previous research has indicated that there is a dose-response relationship between carbohydrate ingestion rate, exogenous carbohydrate oxidation, and performance during prolonged exercise , 34, 35.EXO oxidation rate than AP and a tendency (P = 0.053) toward a 9% higher peak CHOEXO oxidation rate compared to MD+SUC. The peak CHOEXO rate observed following AP ingestion is in line with the well-accepted maximal absorption rate (~1 g·min−1) of ample glucose or glucose polymer solutions when fed in excess of 1.2 g·min−1 , where subjects received a 2:1 glucose-sucrose mixture providing a total of 1.8 g of carbohydrates·min−1.The ingestion of MD+FRU resulted in a significantly (30%) higher peak CHO g·min−1 , 36. Whi g·min−1 , 38. Cons et al.   is comparable to oxidation rates achieved in other studies with similar intake levels and ratio of multiple transportable carbohydrate solutions  approach, O'Brien et al.  toward higher peak CHOEXO oxidation rate for MD+FRU than for MD+SUC in the present study could be explained by the MD:SUC solution (1:0.25) providing a smaller amount of fructose (~0.17 g·min−1) for intestinal absorption and subsequent oxidation. For MD+FRU intake, it might be hypothesized that formation of a hydrogel in the stomach could affect gastric emptying rate and/or attenuate intestinal carbohydrate absorption.The peak CHOolutions , 11. Usin et al.  demonstrn et al. , a recenn et al. . Thus, tAttenuating effects have been shown when supplementing meals with 5 g of sodium alginate with the aim of increasing satiety and decreasing postprandial glucose response in subjects with type-2 diabetes . The amoIt is well-established that compared with placebo or water intake, exogenous carbohydrate provision during prolonged exercise increases total carbohydrate oxidation, decreases fat oxidation and reduces the oxidation of endogenous carbohydrate, and that these alterations in substrate metabolism are entirely attributed to the oxidation of ingested  carbohydrates , 13, 14.For the majority of the 12 participants exercising with intake of MD+FRU, a distinct decrease in exogenous carbohydrate oxidation rate was observed after 120 or 140 min , presumaThe relative total amount of the ingested carbohydrate that was oxidized during the 180 min exercise was significantly less for AP compared to MD+SUC and MD+FRU . Not only exogenous carbohydrate oxidation, but also lactate concentrations were consistently higher following ingestion of the fructose-containing drinks (MD+FRU and MD+SUC) than in the AP trial until cessation of carbohydrate feedings (100 min). This finding is most likely explained by the fact that fructose, after intestinal uptake, undergoes a two-step metabolic conversion, resulting in release of glucose and lactate into the circulation, both of which will be taken up and utilized as fuel in the working muscles . Thus, n−1 and even to completely block hepatic glucose output at very large glucose intakes (3 g·min−1)  every 30 min up to 210 min. During the final 30 min of exercise a more pronounced fall in blood glucose concentrations was evident with the lower glucose intake rate , compared to the high glucose trial, indicating that some glucose remained in the GI tract in the latter condition. Significant differences between trials in plasma FFA, glycerol, and insulin concentrations were also noted. In the present study, oxidation efficiency and exogenous carbohydrate oxidation data suggest that MD+FRU is rapidly oxidized, proposing that after cessation of carbohydrate intake a gradual switch from exogenous to endogenous carbohydrate oxidation is initiated. Indeed, endogenous carbohydrate oxidation increased from 120 to 180 min in the MD+FRU trial , a development not seen for the other two trials. The drop in plasma glucose toward the end of the MD+FRU trial indicates that hepatic glucose output was not sufficient to maintain plasma glucose homeostasis during increased reliance on endogenous carbohydrate, probably due to insufficient hepatic glycogenolysis , is indicative of residual carbohydrates in the GI tract progressively entering the circulation. In line with this assumption, McConell et al.  demonstrg·min−1) . Furtherg·min−1)  also indenolysis . In futuAlthough low oxidation efficiency/residual carbohydrates in the GI tract are suggestive of increasing the risk for GI symptoms during prolonged exercise , no adveResearch over the last decades has shown that the damage to the mineralized structures of the teeth is, for both dental caries and erosion, caused by pH-dependent mechanisms . A singlIn this study, 6 × 31.7 g of carbohydrates  was served during the trials. Since monosaccharides , disaccharides (sucrose), and polysaccharide (amylopectin) differ in carbon and thus energy content, the carbon content per serving of the three drinks varied from 13.3 g (MD+FRU) to 13.7 g (MD+SUC) and 14.1 g (AP) . Whilst 2 arising from oxidation of endogenous substrates. Thus, calculation methods using reference δ13C data from a separate trial without carbohydrate ingestion for each time point, or using δ13C measured in the same trial prior to start of exercise  as a reference (δExpref in Equation 4) both suffer from the limitation that endogenous substrates are not utilized in the same proportions when δExp and δExpref are measured. While absolute values of calculated oxidation rates may be affected by choice of the reference values δExpref (Equation 4), mean differences between trials will not be affected, as long as the reference values are determined under conditions when carbohydrates have not been ingested and are therefore independent of type of carbohydrate intake.Peronnet et al.  have hig13C data were obtained in each trial after 30 min of exercise before first intake of carbohydrates, thereby minimizing changes in isotopic composition of CO2 derived from endogenous fat oxidation, which progressively increases over time when no carbohydrates are ingested  and hypotonic AP (no fructose) drinks. Moreover, a less pronounced fall in dental biofilm pH with MD+FRU usage compared to that of MD+SUC and AP was evident, which may be favorable with respect to caries development. The long-term effects on dental health of frequent intake of commercially available sport drinks with differing acidulant content during both resting and active conditions warrants further research.The raw data supporting the conclusions of this article will be made available by the authors on request.The studies involving human participants were reviewed and approved by the study received local ethics committee approval  prior to any testing, and participants were informed about study details, both verbally and in writing, prior to providing written informed consent in accordance with the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.SP, MA, and UA-H: conceptualization, data curation, and visualization. SP, FE, MA, PL, and CS: methodology. SP, FE, MA, and UA-H: formal analysis. SP and MA: writing–original draft preparation. SP, FE, MA, PL, CS, and UA-H: writing–review and editing. SP: project administration.MA was employed by Maurten AB. The remaining 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.


Bone disease severely affects the quality of life of over 70% of multiple myeloma (MM) patients, which daily experience pain, pathological fractures, mobility issues and an increased mortality. Recent data have highlighted the crucial role of the endoplasmic reticulum-associated unfolded protein response (UPR) in malignant transformation and tumor progression; therefore, targeting of UPR-related molecules may open novel therapeutic avenues. Endoplasmic reticulum (ER) stress and UPR pathways are constitutively activated in MM cells, which are characterized by an increased protein turnover as a consequence of high production of immunoglobulins and high rates of protein synthesis. A great deal of scientific data also evidenced that a mild activation of UPR pathway can regulate cellular differentiation. Our previous studies revealed that MM cell-derived small extracellular vesicle (MM-EV) modulated osteoclasts (OCs) function and induced OCs differentiation. Here, we investigated the role of the UPR pathway, and in particular of the IRE1α/XBP1 axis, in osteoclastogenesis induced by MM-EVs. By proteomic analysis, we identified UPR signaling molecules as novel MM-EV cargo, prompting us to evaluate the effects of the MM-EVs on osteoclastogenesis through UPR pathway. MM-EVs administration in a murine macrophage cell line rapidly induced activation of IRE1α by phosphorylation in S724; accordingly, Xbp1 mRNA splicing was increased and the transcription of NFATc1, a master transcription factor for OCs differentiation, was activated. Some of these results were also validated using both human primary OC cultures and MM-EVs from MM patients. Notably, a chemical inhibitor of IRE1α (GSK2850163) counteracted MM-EV-triggered OC differentiation, hampering the terminal stages of OCs differentiation and reducing bone resorption. Multiple myeloma (MM) is a plasma cell dyscrasia characterized by the accumulation of monoclonal plasma cells in the bone marrow. Bone disease is a hallmark of MM due to the alteration of the dynamic balance between osteoclasts (OCs) and osteoblasts (OBs) activity, resulting in the formation of osteolytic lesions which lead, in over 70% of patients, to pathological fractures or compression fractures of the spine, pain, hypercalcemia and renal failure ,2. GrowtSmall extracellular vesicles (EVs), with ranges defined <100 nm or <200 nm of diameter, can modify local tumor microenvironment and support the establishment of the pre-metastatic niche, which sustains tumor progression. They act by specifically transferring miRNAs, lncRNAs, circRNAs and proteins to the cells of the tumor microenvironment, transforming both their proteomic and transcriptomic repertoire and reorganizing a large number of signaling pathways ,8. Our pα)/X box-binding protein 1(XBP1) axis, in myeloma cell–induced OC differentiation. UPR is initiated by three major transducers, the three transmembrane sensors, i.e., protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), IRE1 and activating transcription factor 6 (ATF6), which activate signaling pathways leading to the expression of specific transcription factors and chaperones. Particularly, IRE1α is an ER transmembrane glycoprotein, which contains both kinase and RNase activities in the cytoplasmic domain. ER stress leads to its autophosphorylation and the subsequent activation of its RNase activity. The direct substrate of IRE1α is XBP1 mRNA, which encodes a basic leucine-zipper-containing transcription factor. Splicing of XBP1 mRNA by the RNase activity of IRE1 leads to the mature XBP1 mRNA. Finally, nuclear translocation of XPB1 activates the transcription of the specific target genes, thus ensuring cell survival [In this study, we investigated the role of the unfolded protein response (UPR) pathway, and in particular of the inositol-requiring enzyme-1 alpha -induced osteoclastogenesis in physiological conditions, through nuclear factor-activated T cells c1 (NFATc1) transcription, a pivotal regulator of OCs activation and maturation [In this regard, MM is defined by chronic endoplasmic reticulum (ER) stress induced by a continuous and high production of monoclonal immunoglobulin; in order to maintain ER homeostasis and survival, MM cells express high levels of ER chaperones, such as GRP78, BiP and Grp94, and activate the UPR pathway. It is already known that the pathogenesis of MM is closely linked to dysregulated UPR in the ER, and some authors showed that stably-induced expression of the UPR transcription factor XBP1s in mice causes myeloma . Accordituration ,18,19. Fturation ; in partturation . Other rturation .α/XBP1 pathway. To our knowledge, these are the first experimental data demonstrating a close correlation between MM-EVs and OC differentiation, which occurred through the IRE1α/XBP1 axis.On the basis of our findings and taking into account the literature in the field, we hypothesized an involvement of the UPR pathway also in MM bone disease, and evaluated if the effects induced by MM-EVs on OCs differentiation occurred also through the IRE1Homo sapiens (Human) dataset by using ProteinPilot 4.5 at a 1% critical false discovery rate (FDR) at both protein and peptide levels, allowing the identification of 516 proteins (the lists of identified proteins are shown in https://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0036498) within the domain “Biological Process”, we focused on the term ‘GO:0036498_IRE1-Mediated Unfolded Protein Response’ and its direct parent term ‘GO:0030968_Endoplasmic Reticulum Unfolded Protein Response’. Then, we extrapolated a unique list of proteins implicated in the regulation of the unfolded protein response related to the endoplasmic reticulum stress (UPR_ER) visualized as a complex STRING network imported into Cytoscape  (We first characterized the proteome of EVs isolated from the human MM cell line MM1.s by utilizing mass spectrometry (MS)-based proteomics, as described in materials and methods. A peptide mixture of proteins from MM1.s-derived EVs (MM1.s-EVs) was run in triplicate using a Data-Dependent Acquisition (DDA) method. The integrated DDA data sets from three runs were searched against the UniProtKB Proteomes—Interestingly, STRING network analysis , as described in materials and methods ,24. MM1.Western blotting analysis of IRE1α tot and pS724-IRE1α and the relative densitogram revealed phosphorylation of IRE1α after 6 and 24 h , when MMNext, we evaluated the effects of MM-EVs also on human primary OCs. Both pre-osteoclast cells (pOCs) and mature OCs were prepared from human peripheral blood mononuclear cells (PBMCs) obtained from two healthy donors . As showWe hypothesized that MM-EVs can rapidly activate the IRE1α/XBP1 axis and that the long-term effect of the early activation of the IRE1α/XBP1 axis can promote terminal OC differentiation. To address this issue, we decided to chemically inhibit the IRE1α/XBP1pathway by the IRE1α selective inhibitor GSK2850163, which inhibits both IRE1α kinase activity and RNase activity .We first treated pre-OC cells for short times to verify the inhibition of IRE1α phosphorylation, and then we analyzed the late stages of the MM-EV-induced OC differentiation, concomitantly with the inhibition of the IRE1α/XBP1 axis. To exclude effects of the IRE1α inhibitor on cellular viability, we performed WST-1 assay on Raw264.7 cells; as showed in Then, we co-treated for 6 h Raw264.7 cells with 25 μg/mL of MM-EVs and 200 nM GSK2850163 (IRE1ι); notably, at 6 h we observed that EV/IRE1ι treatment compared to EV treatment (EVs from both MM1.s and U266 cell lines), induced a strong inhibition of IRE1α phosphorylation, . We alsoι was carried on until 6 days in Raw264.7 cells and 14 days in cultures of human primary OCs. Osteoclastogenesis was assessed by performing TRAP staining assay, which allows to visualize the formation of multinucleated, giant and terminally differentiated OCs. As shown in ι. qRT-PCR analysis of Raw264.7 cells co-treated for 6 days with MM-EVS and IRE1ι showed a reduction of Trap and CathK mRNAs  and grown in R.P.M.I. high glucose  supplemented with 10% Fetal Bovine Serum . Murine macrophage Raw264.7 cells were purchased from ATCC®  and cultured in Dulbecco’s modified Eagle’s medium , supplemented with 10% Fetal Bovine Serum . Several aliquots of low passage cell lines were frozen in liquid nitrogen, at −196 °C, for subsequent assays.MM1.s and U266 cell lines were purchased from ATCCEach aliquot was passed for a maximum of 2 months. To induce differentiation, cells were treated with 25 ng/mL of human recombinant RANK Ligand (hrRANKL)  for 6 days in DMEM, supplemented with 10% FBS, previously ultracentrifugated. To study the effects of MM cell-derived exosomes on OCs differentiation through UPR pathway, Raw264.7 cells were treated for 6 and 24 h with 25 μg/mL of MM cell-derived EVs, in DMEM, supplemented with 10% of ultracentrifugated FBS (see below). We choose the concentration of 25 μg/mL of MM-EVS because we previously observed a dose dependent effect of MM-EVs on the mRNA levels of OCs markers and we choose the most responsive one . To inhig, 15 min at 3000× g, 30 min at 10,000× g and ultracentrifuged 90 min at 100,000× g in a Type 70 Ti, fixed angle rotor. The supernatant was carefully removed, and the resulting EV pellets were resuspended in 100 µL of ice-cold PBS. EV protein content was determined by the Bradford assay [EVs released by MM cells (MM1.s and U266) were isolated from the conditioned culture medium after a 48 h culture period in the presence of EV-free FBS, by differential centrifugations as previously described . Brieflyrd assay .n = 3). All patients provided written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the Hospital of the University of Palermo . Extracellular vesicles were isolated from human plasma and prepared as described above. EV pellets were washed and suspended in PBS, and vesicle protein content was determined by the Bradford assay.EVs were also isolated from the bone marrow blood samples of patients affected by MM  were isolated using the Ficoll-Paque  separation technique.6 cells/mL α-Minimum Essential Media (MEM) supplemented with 10% FBS previously ultracentrifuged, 25 ng/mL of human recombinant RANK Ligand , 25 ng/mL of human M-CSF , and 10 nM dexamethasone  (Human OC medium). After 3 days, the culture were washed with α-MEM medium to remove non adherent cells. The remaining cells were mononucleated, expressed TRAP and were considered committed pre-osteoclast cells. For human osteoclastogenesis assays, human OC medium was added and the cultures were continued for additional 6–10 days, at the end of the period they contained large mature multinucleated OCs. The culture period was 14 days for both TRAP staining assay, bone resorption assay and ELISA assay.PBMCs were cultured at 2.5 × 10g ultracentrifugation, were incubated with PKH26 for 10 min at room temperature. Labeled EVs were washed in PBS, centrifugated, resuspended in low serum medium and incubated with Raw264.7 cells for 6 h at 37 °C. After incubation, cells were processed as previously described [MM cell-derived exosomes (isolated from MM1.s and U266) were labeled with PKH26 , according to the manufacturer’s instructions. Briefly, EVs collected after the 100,000× escribed . Raw264.v/v) containing 0.1% FA, and were dried, to be then resuspended in 5% ACN/H2O  containing 0.1% FA. Approximately 1.2 μg of tryptic peptides of MM1.s EVs sample were run in triplicate with Data Dependent Acquisitions (DDA) mode via reverse-phase high-pressure liquid chromatography electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS), using a TripleTOF® 5600 Plus System  equipped with an Eksigent Nanoflow binary gradient HPLC system . RP-HPLC was performed with a trap and elution configuration using a Nano cHiPLC Trap column 200 µm × 0.5 mm ChromXP C18-CL 3 µm 120Å and a Nano cHiPLC column 75 µm × 15 cm ChromXP C18-CL 3 µm 120Å. The reverse-phase LC solvents were: solvent A (0.1% FA in water) and solvent B (0.1% FA in 98% acetonitrile). Each replicate was eluted at a flow rate of 300 nL/min using a gradient method according to which solvent B is linearly increased from 10% to 28% within 120 min and then to 60% within 30 min; afterwards, phase B is further increased to 95% within 2 min. Then, phase B is maintained at 95% for 10 min to rinse the column. Finally, B is lowered to 10% over 2 min and the column equilibrated for 36 min . For these experiments, the mass range for MS scan was set to m/z 350–1250 and the MS/MS scan mass range was set to m/z 230–1500. Using the mass spectrometer, a 250 ms survey scan (MS) was performed, and the top 25 ions were selected for subsequent MS/MS experiments employing an accumulation time of 150 ms per MS/MS experiment for a total cycle time of 4.0504 s. Precursor ions were selected in high resolution mode , tandem mass spectra were recorded in high sensitivity mode . The selection criteria for parent ions included an intensity of greater than 50 cps and a charge state ranging from +2 to +5. A 15 s dynamic exclusion was used. The ions were fragmented in the collision cell using rolling collision energy, and collision energy spread (CES) was set to 2.EVs released by MM1.s cells were subjected to in-solution digestion using 50% 2,2,2-trifluoroethanol (TFE) in PBS, as described in Schillaci et al. , with soHomo sapiens (Human), downloaded in February 2019 . A false discovery rate analysis was performed.The DDA MS raw files generated by the three runs were combined and subjected to database search in unison using ProteinPilot™ 4.5 software  with the Paragon algorithm. The samples were input with the following parameters: iodoacetamide cysteine alkylation, digestion by trypsin, no special factors and biological modifications as ID focus. The searches were conducted through identification efforts in a UniProtKB Proteomes—http://amigo.geneontology.org/amigo) was used to query the GO database in order to obtain a complete list of gene products belong to the term GO:0036498_IRE1-mediated unfolded protein response and to its direct parent term GO:0030968_Endoplasmic Reticulum Unfolded Protein Response. STRING v. 11 (http://string-db.org/) was used to analyzed protein network that was then visualized by using the Cytoscape STRING app [AmiGO browser interface (RING app .v/v) was added to the cell monolayer in each well and incubate for 4 h at 37 °C and 5% CO2; the formazan dye produced by viable cells was quantified spectrophotometrically at 450 nm by Bio-Rad Microplate Reader .Water Soluble Tetrazolium Salts (WST-l) colorimetric reagent  was used to evaluate cell viability. Briefly, WST-1 reagent  or both, in DMEM high glucose medium, supplemented with 10% of ultracentifuged FBS until day 6 when cells were stained for detection of tartrate-resistant acid phosphatase (TRAP) activity, according to the manufacturer’s protocol  Kit; Sigma-Aldrich, St. Louis, MO, USA) and evaluated by light microscopy. Multinucleated TRAP+ cells containing more than three nuclei were scored as mature osteoclasts. Three independent experiments were performed in triplicate; cells from 5 different fields were counted for each condition. As control, Raw264.7 cells were cultured with hrRANKL (25 ng/mL).4 cells/mL in 96-well plates on organic dentine discs  and cultured with GSK2850163, with MM1.s-EVs (25 µg/mL) or both, in DMEM high glucose medium supplemented with 10% FBS, previously ultracentrifuged. The dentine discs, after 6 days of culture, were rinsed with 70% sodium hypochlorite for 5 min and fixed in 4% glutaraldehyde for 3 min. The resorption pits were stained using 1% toluidine blue and observed with a light microscope  at a 20× magnification. Three fields of each dentin disc for each experimental point were scored in three independent experiments. The number of the pits was calculated by NIH image J software analysis (htttp:/rsbweb.nih.gov/ij/). Surface of dentin samples have been observed using Zeiss EVO DH15 SEM .Raw264.7 cells were seeded at a density of 5 × 10Murine Matrix Metalloproteinase 9 (MMP9) levels secreted by Raw264.7 cells were quantified by ELISA Kit for Mus musculus MMP9 Sandwich ELISA . Briefly, Raw264.7 cells were cultured with GSK2850163, with MM1.s-EVs (25 µg/mL) or both, in DMEM high glucose supplemented with 10% FBS, previously ultracentrifugated. Supernatants were harvested after 6 days and analyzed, according to the manufacturer’s protocol. Human Matrix Metalloproteinase 9 (MMP9) levels secreted by human primary OC cultures were quantified by ELISA Kit for Quantikine Human MMP-9 ELISA Kit (DMP900) .SDS-PAGE Electrophoresis and Western Blotting were performed as described below; briefly, cells were washed in PBS and lysed for an 1 h in lysis buffer containing 15 mM Tris/HCl pH7.5, 120 mM NaCl, 25 mM KCl, 1mM EDTA, 0.5% Triton X100 and Protease Inhibitor Cocktail . Cell lysates (30 µg per lane) were separated using Bolt Bis-Tris gel 4–12%  and transferred on Nitrocellulose membranes ; the membrane was incubated in blocking solution  and probed overnight with the specific antibodies. The antibodies against the following proteins were used: IRE1 alpha Antibody Pack  (NBP2-50067) from Novus Biologicals ; antibody CD63 (sc-15363), Calnexin (sc-23954), α Tubulin TU-02 (sc-8035) from Santa-Cruz Biotechnology ; antibody Alix (2171S) from Cell Signaling . The membranes were incubated with secondary antibody Dylight 488  and signal was detected by Chemidoc .Total RNA from both Raw264.7 cell line was extracted using TRIzol Reagent,  according to the manufacturer’s protocol. qRT-PCR was used to confirm the expression levels of mRNAs. For mRNA detection, oligo-dT-primed cDNA was obtained using the High-Capacity cDNA Reverse Transcription Kits  and then used as template to quantify mRNA levels by Fast SYBRR Green Master Mix . Gene primers used to study gene expression profiling in murine Raw264.7 cells and human (Hu) primary OCs were reported in the p < 0.05. After having verified normal distribution (Shapiro–Wilk test) and homogeneity of variance (Levene test), data were analyzed with one-way repeated measures ANOVA by considering the between-subject factor ‘group’  and the within-subject factor ‘experiment ID’ . Dunnett’s test was used to compare Treated Groups with Untreated Group.Statistical analysis was performed using R v.3.6.2 software . Data are reported as mean ± standard error (SE) at a significant level of Overall, our results showed that MM-EVs can induce osteoclastogenesis by early activating the IRE1α/XBP1 axis. Induction of the UPR response might represent a further way used by MM cells to regulate bone remodeling and foster bone disease. These results could be clinically relevant, since ER stress-modulating agents could target not only cancer cells, but also inhibit bone disease development.We can hypothesize that MM cells control bone disease also through the secretion of vesicles containing specific UPR-related molecules. In addition, it is known that MM cells produce misfolded proteins and it is plausible that a part of this cargo may be eliminated through vesicles (as evidenced in other cellular systems: ). The tr


Recent studies have identified cyclic GMP-AMP synthase (cGAS) as an important target for treating autoimmune diseases, and several inhibitors of human cGAS (hcGAS) and their structures in complexation with hcGAS have been reported. However, the mechanisms via which these inhibitors interact with hcGAS are not completely understood. Here, we aimed to assess the performance of molecular mechanics/Poisson–Boltzmann solvent-accessible surface area (MM/PBSA) in evaluating the binding affinity of various hcGAS inhibitors and to elucidate their detailed interactions with hcGAS from an energetic viewpoint. Using molecular dynamics (MD) simulation and MM/PBSA approaches, the estimated free energies were in good agreement with the experimental ones, with a Pearson’s correlation coefficient and Spearman’s rank coefficient of 0.67 and 0.46, respectively. In per-residue energy decomposition analysis, four residues, K362, R376, Y436, and K439 in hcGAS were found to contribute significantly to the binding with inhibitors via hydrogen bonding, salt bridges, and various π interactions, such as π· · ·π stacking, cation· · ·π, hydroxyl· · ·π, and alkyl· · ·π interactions. In addition, we discussed other key interactions between specific residues and ligands, in particular, between H363 and JUJ, F379 and 9BY, and H437 and 8ZM. The sandwiched structures of the inhibitor bound to the guanidinium group of R376 and the phenyl ring of Y436 were also consistent with the experimental data. The results indicated that MM/PBSA in combination with other virtual screening methods, could be a reliable approach to discover new hcGAS inhibitors and thus is valuable for potential treatments of cGAS-dependent inflammatory diseases. Free DNA in the cytosol is detected by a type of nucleotidyltransferase called cyclic GMP-AMP (cGAMP) synthase (cGAS) . Upon bi−/−Trex1 mice dramatically reduces tissue inflammation and hinders autoantibody production [50 = 0.11 µM) of mcGAS that can regulate the levels of interferon; however, it binds to hcGAS with low affinity (IC50 = 2.94 µM) in spite of approximately 60% sequence identity between hcGAS and mcGAS [50 values of 10.2 nM and 27.5 nM, respectively, they were both inactivated during assessment with mcGAS [Kd) of 200 nM (IC50 = 4.9 µM) using high-throughput screening assays [50 = 29.88 ± 3.20, 13.1 ± 0.09, and 4.9 ± 0.26 µM), with highly consistent binding modes, were identified by Zhao and co-workers [Experimental studies have shown that knockout of the cGAS gene in oduction ,9. Thus,oduction ,11,12,13nd mcGAS . A high-To complement and validate the results obtained using an experimental approach, computational modeling, molecular dynamics (MD) simulations, and molecular mechanics/Poisson–Boltzmann solvent-accessible surface area (MM/PBSA) can be used for analyzing the real-time dynamic behavior of biomolecules and predicting the binding free energy. These methods have been widely used for elucidating the binding mechanism and interaction modes of protein-inhibitor systems ,16,17. RIn this study, we aimed to assess the performance of MM/PBSA in evaluating the binding affinity of 10 hcGAS inhibitors . This was well predicted from the calculated binding free energies of −103.8 ± 5.5 kJ/mol and −73.4 ± 5.3 kJ/mol. MM/PBSA also predicted the order of the binding free energy of the inhibitors with diazolo-/tetrazolo-pyrimidine aromatic core to be KHM > KKM > KKP > 8ZM, which is consistent with the IC50 values of 2.0, 8.1, 69.0, and 78.0 μM, respectively. Although the IC50 (13.1 μM) of ER9 in the enzyme activity assay was stronger than that of KKP (69 μM) determined using a fluorescence polarization assay [Kd of 64 ± 3 and 80 ± 4, respectively. The experimental data suggested that the affinity of 9BY was weaker than those of 9BS and 9BV, although the calculated binding free energy of the hcGAS–9BY interaction (−114.1 ± 4.3 kJ/mol) was predicted to be more than that of 9BS and 9BV. We further evaluated the correlation of the binding free energy between the pIC50/∆Gexp and MM/PBSA calculation. It should note that the IC50 or Kd values for these three groups of ligands were obtained from different experimental methods, more specifically, high-throughput screening assay for ligand group I [50 was dependent on the enzyme concentration used in the experiments, the correlation between pIC50 and the predicted ∆GMM/PBSA was thus discussed separately for inhibitors in different groups. As shown in Gexp and ∆GMM/PBSA for groups II and III was as high as 0.61. Note that the RMSD of both ER9 and 9BY  are generally overestimated, similar to the results of other studies on protein-ligand interactions [r) was 0.52 at the early simulation stage (0−4 ns), with Spearman’s rank correlation coefficient (ρ) of 0.32, while the studies on dynamics predicted relatively stronger correlations and fluctuation in the vicinity of 0.66 for r. Furthermore, the rankings of the binding free energies remained constant at ρ = 0.46 after 8 ns. In addition, we inspected the various energy distributions as a function of time, as shown in EvdW and ∆Gnp/solv were generally stabilized during the entire trajectories. However, the electrostatic and polar solvation energies showed larger fluctuations in hcGAS binding with JUJ, KHM, and KKM than the other inhibitors, leading to binding free energies with stronger disturbance. Despite the limitations of MM/PBSA studies, which were mainly due to force field accuracy, polar contribution to solvation, entropy estimation, and sampling conditions, this method was used as a potential tool for predicting the relative binding free energy of protein-ligand interactions [The averaged binding free energies at different time scales from three parallel trajectories for the 10 hcGAS-inhibitor systems obtained using MM/PBSA analysis are shown in ractions ,20. The ractions ,22. As lractions ,24.EvdW), electrostatic energy (∆Eele), and nonpolar solvation energy (∆Gnp/solv) contributed to attractive interactions, while polar solvation energy (∆Gpb/solv) contributed repulsively to the total binding free energies. Similar to the findings of Zhao and co-workers [Gnp) were stronger than the total polar energies (∆Gpb), indicating that total nonpolar components in the hcGAS-inhibitor interactions contributed more significantly than total polar interactions. In terms of attractive contributions, vdW and electrostatic interactions contributed the most to binding free energies, especially for the inhibitors KHM, 9BY, KKM, and ER9. For the hcGAS-9BV and hcGAS-8ZM interactions, the vdW contributions exceeded −110 kJ/mol, while the electrostatic interactions provided moderate contributions under −30 kJ/mol, comparable to the nonpolar solvation energies. The nonpolar energy of 9BY (∆Gnp = −145.3 ± 1.5 kJ/mol) established stronger affinities than those of 9BS and 9BV mentioned above.Energy decompositions for 10 hcGAS-inhibitor interactions are shown in -workers , the totThe binding free energy of single amino acids in each system was determined to identify the residues that contributed considerably to the binding and the similarities and differences in the binding of the 10 inhibitors to the hcGAS protein. We evaluated the residues distributed within 0.6 nm from any atom in the inhibitor, the per-residue binding free energies of which are listed in The predicted binding free energies of K362 with the inhibitors JUJ, KHM, 8ZM, KKP, KKM, 9BY, and 9BS were significantly more attractive than –8.0 kJ/mol see . Among tThe hcGAS-bound inhibitors contain multiple aromatic rings see , which lThe binding modes of K439 with the 10 inhibitors are shown in Aromatic amino acids, H363, F379, and H437, which evidently affected binding with the individual inhibitors, were also considered see , althougStudies have reported that the sandwiched structures formed by the inhibitors with R376 and Y436 play vital roles in cGAS activity ,11,12,13EMM) and weak polar solvation energy between K362 and JUJ. This also confirmed the C−H · · · π interaction between K362 and JUJ, and the absence of direct contacts with JUM  are shown in with JUM . In catewith JUM . Figure with JUM . The binwith JUM . For thewith JUM , which wwith JUM . Figure with JUM . Despitewith JUM , distincwith JUM . Althougδ atom; the other charged amino acids were treated as default based on H++ analysis.The crystal structures of hcGAS inhibitors were retrieved from the RCSB Protein Data Bank (PDB) from the entries of 6MJW, 6MJU, 6NAO, 6NFO, 5VDU, 6NFG, 5VDV, 5V8O, 5VDW, and 6LRL . The che3 volume and then solvated with 23,640 to 25,860 water molecules using the transferable intermolecular potential with 3 points (TIP3P) water model [+ and Cl− counterions were added to neutralize the system with salt concentrations consistent with the ones in experimental studies. The AMBER14SB force field was used to calculate the hcGAS-inhibitor energies for the simulations [2) in the NVT and NPT ensembles at 300 K and 1 bar. Finally, 3 separate 20 ns MD runs were performed for equilibrium with a time step of 2 fs. Temperature coupling was performed using a velocity-rescaling thermostat [The Gaussian 16 program was used to calculate the electrostatic potential (ESP) charges for each inhibitor at the HF/6–31G* level . All boner model . Na+ andulations . After permostat ,37. The ermostat ,39. The ermostat . The parermostat . The cutermostat .Gbind of the hcGAS-inhibitor interaction can be evaluated using the following 3 functions:EMM represents the sum of the bonded (∆Ebonded) and nonbonded (∆EvdW and ∆Eele) terms. In the single-trajectory setup, ∆Ebonded is always assumed to be zero owing to its accuracy and simplicity, which are close to those of the multi-trajectory approach [Gsol includes polar solvation energy (∆Gpb/solv) and nonpolar solvation energy (∆Gnp/solv), which were estimated using the PB Equation and the solvent-accessible surface area model [T and ∆S are the absolute temperature and entropy, respectively; the entropy calculations were ignored in the current calculation process, as they were time-consuming and improved the experimental data only slightly [g_mmpbsa is a fast and reliable tool for estimating the binding free energy based on GROMACS and APBS programs, especially for protein-ligand interactions . Here, tapproach . The solea model ,46. The ea model ,47. T anslightly ,23,48.The calculations regarding per-residue binding free energy decompositions were also performed to assess the effect of each residue on the hcGAS-inhibitor interaction. The single residue-free energy was evaluated using Equation (4):50 or Kd. We determined the binding free energies at different time scales for every 2 ns, showing correlation coefficients of 0.67 (r) and 0.46 (ρ). Furthermore, energy decomposition revealed that total nonpolar energy contributed significantly to the total binding energy of each system, where the vdW interaction was stronger than that of the electrostatic interactions. To identify the residue distinctly affecting the inhibitor, we performed per-residue binding energy analysis and investigated the binding modes of the hcGAS-inhibitor complexes. In addition to K362, R376, and Y436 were the major residues that interacted with the inhibitors, which was consistent with the experimental data. New potential roles of the polar residue K439 and aromatic residues H363, F379, and H437 in various interactions, such as hydrogen bonding, salt bridge, π· · ·π stacking, cation· · ·π, O−H· · ·π, and C−H· · ·π interactions, were also revealed. In addition, we analyzed the sandwiched structures of the inhibitor bound to the guanidinium group of R376 and the phenyl ring of Y436; our results suggested that the mechanism of binding mainly involved the impacts from nonbonded interaction and polar solvation energy. Our study indicated that MM/PBSA, in combination with high-throughput virtual screening methods, could be a promising method to screen new inhibitors from the existing database of small molecules. Furthermore, the vital binding modes of hcGAS to inhibitors could be helpful in selecting inhibitors by visual inspection. However, in the current study, the entropy involved in hcGAS-inhibitor interactions was not considered; a potential improvement with the inclusion of entropy should be tested in the future. Moreover, recently, machine-learning algorithms were successfully employed together with MM/PBSA results to achieve a better prediction of the binding free energy of three kinases [MD simulations and MM/PBSA analyses were performed to analyze the binding of various inhibitors  with hcGAS. RMSD analysis at approximately 0.25 nm indicated the convergence of all the hcGAS inhibitors. Following this, the binding free energies of hcGAS-inhibitor interactions were estimated using MM/PBSA calculations, which were generally consistent with the experimental affinities, IC kinases . A speci