The multicohort, phase II, nonrandomized KEYNOTE-059 study evaluated pembrolizumab ± chemotherapy in advanced gastric/gastroesophageal junction cancer. Results from cohorts 2 and 3, evaluating first-line therapy, are presented.2 on day 1 (up to 6 cycles), and 5-fluorouracil 800 mg/m2 on days 1–5 of each 3-week cycle (or capecitabine 1000 mg/m2 twice daily in Japan). Primary end points were safety (combination therapy) and objective response rate per Response Evaluation Criteria in Solid Tumors version 1.1 by central review, and safety (monotherapy).Patients ≥ 18 years old had previously untreated recurrent or metastatic gastric/gastroesophageal junction adenocarcinoma. Cohort 3 (monotherapy) had programmed death receptor 1 combined positive score ≥ 1. Cohort 2 (combination therapy) received pembrolizumab 200 mg on day 1, cisplatin 80 mg/mIn the combination therapy and monotherapy cohorts, 25 and 31 patients were enrolled; median follow-up was 13.8 months (range 1.8–24.1) and 17.5 months (range 1.7–20.7), respectively. In the combination therapy cohort, grade 3/4 treatment-related adverse events occurred in 19 patients (76.0%); none were fatal. In the monotherapy cohort, grade 3–5 treatment-related adverse events occurred in seven patients (22.6%); one death was attributed to a treatment-related adverse event (pneumonitis). The objective response rate was 60.0%  (combination therapy) and 25.8% (95% CI 11.9–44.6) (monotherapy).Pembrolizumab demonstrated antitumor activity and was well tolerated as monotherapy and in combination with chemotherapy in patients with previously untreated advanced gastric/gastroesophageal junction adenocarcinoma.ClinicalTrials.gov NCT02335411The online version of this article (10.1007/s10120-018-00909-5) contains supplementary material, which is available to authorized users. Gastric cancer is the fifth most common malignancy worldwide. In 2012, it led to 723,000 deaths –6. In prTumor cells frequently use the programmed death receptor 1 (PD-1) pathway to evade immune surveillance , 14. BinChemotherapeutic agents such as cisplatin and 5-fluorouracil enhance immunogenicity of cancer cells and increase susceptibility to immune-mediated cytotoxicity –25. CombKEYNOTE-059 is a multicenter, open-label, nonrandomized, 3-cohort, phase II study. Results for cohorts 2 and 3 are presented; results for cohort 1 were published separately . The stuEligibility criteria for both cohorts were age ≥ 18 years, histologically or cytologically confirmed recurrent or metastatic G/GEJ adenocarcinoma, no prior systemic therapy for advanced disease, measurable disease per Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) by central review, HER2/neu-negative status, Eastern Cooperative Oncology Group performance status 0/1, life expectancy ≥ 3 months, and adequate organ function.For cohort 2, patients had to provide new or archival tumor tissue for PD-L1 analysis. For cohort 3, patients had to have PD-L1-positive tumors as per the PD-L1 IHC 22C3 pharmDx assay . PD-L1 positivity was defined as CPS ≥ 1, where CPS is the number of PD-L1-positive cells  divided by the total number of tumor cells multiplied by 100 . Exclusi2 on day 1 (for up to 6 cycles), and 5-fluorouracil 800 mg/m2 by continuous infusion on days 1–5 of each 21-day cycle. In Japan only, patients received capecitabine 1000 mg/m2 twice daily orally on days 1–14 of each 21-day cycle instead of 5-fluorouracil. Up to two dose reductions were allowed per chemotherapy agent (Online Resource Table S2). In cohort 3, patients received pembrolizumab monotherapy 200 mg by intravenous infusion over 30 min on day 1 of each 21-day cycle.In cohort 2, patients received pembrolizumab 200 mg by intravenous infusion over 30 min on day 1, cisplatin 80 mg/mPembrolizumab dose reduction was not permitted. Treatment was continued for up to 35 cycles (~ 2 years) or until confirmed disease progression, unacceptable toxicity, complete response, or patient or investigator decision to withdraw. Clinically stable patients with radiographic progressive disease (PD) could continue treatment at the investigator’s discretion until confirmed PD. Patients in cohort 2 who discontinued chemotherapy could continue pembrolizumab if they had not met criteria for discontinuation.For cohort 2, the primary end points were safety and tolerability. Secondary end points included ORR  per RECIST v1.1 by central imaging review and evaluation of the relationship between PD-L1 expression and efficacy. For cohort 3, primary end points were ORR and safety and tolerability. Secondary end points included disease control rate , duration of response, progression-free survival  per RECIST v1.1 by central imaging review, and OS (time from initiation of study treatment to death from any cause).Tumors were assessed by computed tomography or magnetic resonance imaging at 9 weeks after study treatment initiation, every 6 weeks for year 1, and every 9 weeks thereafter . Response was confirmed by repeat radiographic assessment ≥ 4 weeks after first documentation. Patients were followed up every 12 weeks for survival status until death, withdrawal of consent, or study end.Patients were monitored for adverse events (AEs) until 30 days after the last dose of treatment . AEs were graded using National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0; investigators determined treatment attribution.For cohort 2, the sample size was based on clinical experience. A sample size of ~ 18 patients was planned to provide the reasonable two-sided 95% confidence interval (CI) of AE incidence (Online Resource Table S3). For cohort 3, a sample size of ~ 25 patients with PD-L1 CPS ≥ 1 was estimated using Bayesian sample size methods assuming a uniform prior for the response rate and a true ORR of < 40% (ORR based on standard of care). Safety and efficacy were analyzed in all patients who received ≥ 1 dose of study treatment. Safety was analyzed using descriptive statistics. Analyses were conducted using SAS statistical software version 9.3 . ORR and DCR with 95% CI were calculated using the exact method based on binomial distribution (Clopper–Pearson method ). PatienThe study was conducted at 57 sites in 17 countries. For cohort 2, 25 patients were enrolled at 10 sites in the Republic of Korea (South Korea), Japan, Israel, the United States, and France between March 2, 2015, and December 14, 2015. For cohort 3, 31 patients were enrolled at 18 sites in Canada, Chile, Israel, Japan, South Korea, and the United States between July 23, 2015, and December 2, 2015. Patient characteristics are presented in Table At data cutoff , median follow-up was 13.8 months (range 1.8–24.1) in cohort 2 and 17.5 months (range 1.7–20.7) in cohort 3. In cohort 2, 22 patients (88.0%) had discontinued study treatment and three (12.0%) remained on treatment. In cohort 3, 24 patients (77.4%) had discontinued study treatment, whereas seven patients (22.6%) remained on treatment. In both cohorts, the most common reason for treatment discontinuation was PD .Median duration of pembrolizumab exposure was 7.1 months (range 0.8–23.8), and median number of administrations was 10 (range 2–35). All patients experienced treatment-related AEs (TRAEs) (Table Median duration of exposure to pembrolizumab was 2.8 months (range 0.7–20.3), and median number of administrations was five (range 2–30). Overall, TRAEs occurred in 24 patients (77.4%) (Table Confirmed ORR was 60.0% (95% CI 39.0–79.0), with complete response in one patient 4.0%)  who had measurable disease at baseline and ≥ 1 evaluable postbaseline assessment , median time to response was 2.1 months (range 1.9–3.5), and median duration of response was 4.6 months (range 3.2–18.1+). Among the eight patients with PD-L1 CPS < 1, ORR was 37.5% (95% CI 8.5–75.5%), median time to response was 3.6 months (range 2.1–3.7), and median duration of response was 5.4 months (range 2.8–10.1+).Among 19 patients receiving pembrolizumab, cisplatin, and 5-fluorouracil, ORR was 57.9% (96% CI 33.5–79.7%). Among six Japanese patients receiving pembrolizumab, cisplatin, and capecitabine, ORR was 66.7% (95% CI 22.3–95.7%) (Online Resource Table S11).Confirmed ORR was 25.8% (95% CI 11.9–44.6%), with complete response in two patients (6.5%) Table . Median Of 28 patients with measurable disease at baseline who had ≥ 1 evaluable postbaseline assessment, 24 (85.7%) experienced reduction in target lesion size from baseline , and estimated PFS rate at 6 months was 68.0% Fig. a. MedianMedian PFS was 3.3 months (95% CI 2.0–6.0 months), and estimated PFS rate at 6 months was 34.9%  with pembrolizumab plus cisplatin and 5-fluorouracil or capecitabine in cohort 2 is encouraging relative to response rates observed with cisplatin and fluoropyrimidine doublets (25–48%) . The ORRSixty-four percent of patients in cohort 2 were PD-L1 positive (CPS ≥ 1), compared with an overall prevalence of PD-L1 positivity in advanced gastric cancer of 30–65%, although different methods, antibodies, and cutoff values were used across studies –19. AnalOne patient in cohort 3 who had microsatellite instability-high tumors experienced a partial response. Although this partial response contributed to the ORR in cohort 3, it does not account for the ORR in its entirety. This finding is consistent with that reported for pembrolizumab monotherapy in the cohort of patients who have received ≥ 2 prior therapies for G/GEJ cancer in this KEYNOTE-059 study .Overall, pembrolizumab plus cisplatin and 5-fluorouracil or capecitabine demonstrated manageable safety and promising antitumor activity as first-line therapy in advanced G/GEJ adenocarcinoma, regardless of PD-L1 expression. Pembrolizumab monotherapy also demonstrated encouraging antitumor activity and acceptable safety in patients with PD-L1 CPS ≥ 1. Results support continued evaluation of pembrolizumab in clinical trials.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 1069 KB)


Duguetia lanceolata barks, obtained at 2 (T2) and 4 h (T4), were identified by gas chromatography and gas chromatography/mass spectrometry. β-Elemene (12.7 and 14.9%), caryophyllene oxide (12.4 and 10.7%) and β-selinene (8.4 and 10.4%) were the most abundant components in T2 and T4, respectively. The essential oils inhibited the growth of Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Candida albicans. The essential oils were cytotoxic against brine shrimp. The extraction time influenced the chemical composition and biological activities of essential oils obtained from the barks of D. lanceolata.Essential oils of  Plant and their essential oils are potentially useful sources of bioactive compounds. Some studies have described the biological activities of essential oils against many different types of agents, including important organisms of clinical relevance ,2. AmongDuguetia, Annonaceae family, is composed by around 100 species and possesses as chemical markers alkaloids and acetogenins [Duguetia gardneriana Mart. and Duguetia moricandiana Mart. showed antimicrobial activity [Duguetia glabriuscula R. E. Fries (R.E. Fries) were active against Artemia salina [The genus togenins ,5. Howevtogenins . Consideactivity , while sa salina .Duguetia lanceolata St. Hil., popularly known as pindaíba, beribá or pinhão, is a pereninnial species distributed in several States of Brazil [D. lanceolata presented antinociceptive, anti-inflammatory and antiplasmodial activities [f Brazil . This spf Brazil . Pharmactivities ,10,11.D. lanceolata and in order to provide the scientific basis for the medicinal use of this species, the present study was designed to evaluate the chemical composition and the biological activities of essential oils from barks of this plant. The influence of the extraction time in the obtainment of essential oils in these two propositions for investigation was also analyzed.Considering that we did not find any previously published original articles about essential oils of D. lanceolata is described in the literature. According to the D. lanceolata barks, classified as terpenes (mono- and sesquiterpenes) and hydrocarbons. This total number of detected components (72) was found in the first two hours (T2) and fifty-one in the other four hours (T4). Among those identified in T2, twenty-five were monoterpenes and forty-seven were sesquiterpenes. Considering the time T4, nine were monoterpenes and forty-two were sesquiterpenes. The relation between the monoterpenes and the extraction time was T2 = 2.8 × T4, while for the sesquiterpenes was T2 = 1.1 × T4, demonstrating a relevant loss of monoterpenes.This is the first time that the chemical composition of trans-Pinocarveol (1.4 and 0.4%) and myrtenol (0.8 and 0.5%) were the most abundant monoterpenes observed in both times T2 and T4, with reduction of 71.4% and 37.5%, respectively (ectively . ConsideD. lanceolata barks. β-Elemene (12.7 and 14.9%), caryophyllene oxide (12.4 and 10.7%), β-selinene (8.4 and 10.4%), β-eudesmol (6.8 and 7.9%), humulene epoxide II (7.4 and 6.8%), ledol (3.9 and 3.9%), viridiflorol (3.5 and 3.0%), khusinol (3.6 and 5.0%), β-senensal (3.2 and 5.4) and patchouli alcohol (3.2 and 3.8%) were the most concentrated components in T2 and T4, respectively -aromadendrene, δ-cadinene and viridiflorol were identified in D. glabriuscula [D. eximia were α-eudesmol and spathulenol [D. flagellaris was dominated by spathulenol and α-muurolol, while the bark oil was mainly constituted by germacrene D, cyperene, α-muurolol, humulene epoxide II and spathulenol [D. pycnastera were spathulenol, allo-aromadendrene, germacrene D and elemol. The oil of D. riparia contained spathulenol, caryophyllene oxide and α-pinene as their main compounds [D. trunciflora was dominated by α-pinene, bicyclogermacrene, bulnesol, spathulenol, guaiol, globulol and humulene epoxide II; the bark oil was dominated by β-phellandrene, guaiol and α-cadinol [D. gardneriana oil [D. moricandiana was dominated by germacrene D, α-pinene, viridiflorene, β-pinene and β-caryophyllene [Duguetia may have chemotaxonomic significance to maintain similar morphologic and biochemistry characteristics, which will determine the biosynthesis of their secondary metabolism.Although differences are visualized in the chemical composition of essential oils of the genus rotricha , while  μg/mL and 60.7 (37.1–99.3) μg/mL, respectively (50 = 457.9 μg/mL), the reference substance.The essential oils from barks of ectively . AccordiDuguetia have been studied by using the A. salina bioassay, a simple model applied to the toxicity investigation [D. glabriuscula  presented a potent toxicity on A. salina with LC50 1.6 μg/mL [D. lanceolata, allo-aromadendrene can contribute with the toxicity in this assay because this compound produced LC50 7.8 μg/mL, demonstrating to be one of the active substances responsible for this event [D. glabriuscula were toxic against A. salina [Species of the genus tigation . For exa.6 μg/mL . DespiteDuguetia lanceolata St. Hil. was collected in the city of Juiz de Fora, Minas Gerais State, Southeast region of Brazil. A voucher specimen identified by Dr. Fátima Regina Gonçalves Salimena was deposited in the Herbarium of the Departamento de Botânica of the Universidade Federal de Juiz de Fora (CESJ number 29750). The barks were removed from the discarded branches after pruning, submitted to drying at room temperature and triturated for the extraction of the essential oil.D. lanceolata barks with 5% of humidity and 7% of total ashes by hydrodistillation using a Clevenger apparatus for 2 h (T2) and 4 h (T4) at 100 °C. After, the essential oils were dried with anhydrous sodium sulphate and stored under refrigeration at −18 °C for analysis (the yield was 0.5% v/w).Essential oils were obtained from 100 g Capillary gas chromatography was performed using a Hewlett-Packard 6890 gas chromatograph under the following conditions: fused silica capillary column HP-5 ; helium as carrier gas (1 mL/min); and temperature programming from 70 to 290 °C (2 °C/min); injector temperature 270 °C and detector temperature 300 °C.n-alkanes series), mass spectra and the NIST spectrometer data bank, as well as comparison with literature data [The GC/MS analysis of the essential oils was performed on a Hewlett Packard series 6890 gas chromatograph coupled to HP5972 mass spectrometer under the following analytical conditions: ZB-5MS column (30 m × 0.25 mm × 0.25 μm film thickness); helium (1 mL/min); programmed temperature 60−240 °C (3 °C/min); injector temperature (260 °C) and interface (200 °C); ionization energy, 70 eV; scan range, 30–300 amu; scan time, 1 s. Compound identification was based on the comparison of retention index , Streptococcus pyogenes (ATCC 19615), Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 15442) and Candida albicans (ATCC 10231) which belong to the American Type Culture Collection (ATCC).The essential oils of Streptococcus pyogenes , and Sabouraud Agar for yeast. Overnight broth cultures were prepared, adjusted in peptone-physiological salt solution (1 g peptone and 8.5 g/L NaCl) to yield approximately 106 bacteria/mL and 105 conidia/mL. The agar plates were prepared in 90 mm Petri dishes with 22 mL of agar medium giving a final depth of 4 mm. Sterile cellulose discs, diameter 6 mm, were placed on the inoculated agar surfaces with 100 μL of diluted oil in hexane. Each 100 μL had 5, 10 or 25 mg of essential oil. Hexane (100 μL) was used as negative control. All plates were aerobically incubated at 35 ± 2 °C for 18–24 h (bacteria) and at 22 °C for 48 h (yeast). The antimicrobial activity was estimated by measuring the radius of the inhibition zone (mm). Each test was performed in duplicate and the results were shown as means. 20 μL of chloramphenicol (1 mg/mL) for bacteria and 20 μL of nystatin (1 mg/mL) for the yeast were used as positive controls.The antimicrobial activity was tested through agar diffusion method . Mueller5 CFU/mL and 0.5–2.5 × 103 CFU/mL for bacteria and fungi, respectively. Each essential oil was dissolved in Tween 80 and sterile distilled water. Final two fold concentrations were prepared in the wells of the microtiter plates, between 2,000–10 μg/mL. Chloramphenicol and nystatin were used as reference antibiotics for bacteria and fungi, respectively (64–0.0625 μg/mL). Microtiter plates were incubated at 35 ± 2 °C for 18–24 h for bacteria and at 22 °C for 48 h for yeast. After the incubation period, MIC values were defined as the lowest concentration of the oils that inhibits the visible growth of microorganisms.The broth microdilution method recommended by the Clinical and Laboratory Standards Institute was used to determine the minimum inhibitory concentration (MIC) ,26. Anti2·2H2O 1.5 g/L, KBr 0.1 g/L, KCl 0.7 g/L, Na2SO4 4.0 g/L, NaHCO3 0.3 g/L, MgCl2·6H2O 11 g/L. The essential oils were dissolved in tween 80 and DMSO (1:1) followed by artificial seawater. Ten shrimps  were transferred into test tubes in quadruplicate, containing the following essential oil concentrations: 10, 50, 100, 500 and 1,000 μg/mL. The tubes were maintained under illumination. Survivors were counted 24 h after exhibition to the oil. Thymol was used as standard [50’s and 95% confidence intervals were from the 24-hour counts using the probit analysis method [The artificial seawater used in the experiments presented the following composition: NaCl 24 g/L, CaClstandard . CL50’s s method .Duguetia lanceolata. Both oils (T2 and T4) demonstrated antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes and Candida albicans reference strains tested and were poisonous against Artemia salina. However, even a small variation in the total chemical composition of essential oils may contribute for different levels of biological activities expression.Based on our results we can conclude that β-elemene, caryophyllene oxide and β-selinene were the main constituents of the essential oils from


S1 . Pathological data were retrieved from centrally reviewed local pathological reports. FISH (Treatment groups were classified as follows: 1) “intensive regimens”: incorporating high dose methotrexate: R-CODOX-M/IVAC , n = 23, GMALL-R , n = 9, R-Hyper-CVAD/R-MA , n = 10; 2) “R-CHOP-like”: including also R-COMP, n = 6 , R-miniCHOP, n = 2, R-M/VACOP-B , n = 2, R-mega-CHOP, n = 1; 3) “DA-EPOCH-R”: dose-Adjusted Etoposide, Doxorubicin, Cyclophosphamide, Vincristine, Prednisone and Rituximab. Patients treated with different approaches or palliative care (“others”) were censored for survival analysis, or analyzed separately.The primary end-point was progression-free survival (PFS), defined as the time between lymphoma diagnosis and relapse or progression, lack of response, or death from any cause. Survival curves were estimated using the Kaplan-Meier product limit method. Statistical analyses were performed using the Stata 13.0 software .– MYC+/BCL2+/BCL6+) and for survival analysis were considered among DHLs.All 100 patients presented MYC rearrangement as for inclusion criteria, 57 patients were double hit for BCL2 (DHL-BCL2) and 29 were double hit for BCL6 (DHL-BCL6). The remaining 19 MYC rearranged patients, lacking an additional BCL2 or BCL6 translocation, were defined single hit lymphomas (SHL – MYC+/BCL2-/BCL6-). Five patients carried all the three rearrangements, so called “triple hit lymphoma”  were treated upfront with intensive schedules, followed by R-CHOP like (n = 35) and by DA-EPOCH-R (n = 18). Five patients were treated with other schemes . Eighteen patients received ASCT consolidation. Overall, 70 patients received CNS prophylaxis: intrathecal in 24, systemic MTX in 8, and both in 38. Finally, 20 patients received radiotherapy consolidation. Patients treated with intensive regimens presented with similar baseline features to patients treated with R-CHOP, with exception of more advanced IPI in intensive group (p = 0.006), and similar to DA-EPOCH-R group, with the exception of higher ki67 values (p = 0.04), and advanced stage (p = 0.02). Finally, patients presenting a BCLU histology received more frequently intensive treatment than standard R-CHOP or DA-EPOCH-R, p = 0.001 and p = 0.05, respectively, .Overall response was 71%, with 58 patients achieving complete response (CR) and 13 partial response (PR) at the end of front-line treatment. Three patients died of treatment related complications (infections) during induction, and one did not receive any treatment. CR rate was 68% in SHL and 56% in DHL (p = 0.2), see Supplementary Table S2 . Patients treated with intensive schedules had a CR of 69% (29/42), which was not statistically different from patients treated with R-CHOP , and similar to patients treated with DA-EPOCH-R .After a median follow up for survivors of 33 months, the 24 months PFS and OS of the overall MYC rearranged (MYC-R) population were 54% (95% CI 44–63) and 57% (95% CI 46–66), respectively, Fig. S2A and Fig. S2B . For the 19 patients with SHL and the 81 patients with DHL, PFS at 24 months was 77% (95% CI 51–91) and 49% (95% CI 37–59), respectively (p = 0.05), Fig. S2C , and OS was 77% (95% CI 50–90) vs 52% (95% CI 40–63), respectively (p = 0.1), Fig. S2D. PFS did not vary significantly based on the specific double rearrangements found , Fig. S3 .Analyzing all the MYC-R population, the 24 months PFS was 59% (95% CI 33–78) in patients treated with DA-EPOCH-R , 46% (95% CI 29–62) in those receiving R-CHOP-like schedules (p = 0.04), and 65% (95% CI 49–68) in patients who received intensive treatments, Fig. ASCT consolidation (n = 18) did not give any PFS advantage in patients achieving CR or PR at the end of induction treatment , Fig. S5A and Fig. S5B . Actually, patients in CR at the end of induction retained a good 24 months PFS even without ASCT (82%–95% CI 69–90).Moreover, despite quite aggressive CNS prophylaxis, 5/100 patients experienced a CNS relapse during observation period . Finally, the outcome after relapse was very poor, accounting for a 24 months OS of 13% (95% CI 4–24), Fig. S6 .In the overall MYC-R population, B-symptoms (p = 0.05), ECOG performance score (PS) > 2 (p = 0.003), bone marrow infiltration (p = 0.005), elevated LDH levels (p = 0.03) and poor risk IPIaa (p = 0.05), predicted worse PFS . Restricting analysis for DHL population, only ECOG PS > 2 (p = 0.01) and bone marrow infiltration (p = 0.05) retained their prognostic impact. Finally, after Cox proportional hazard multivariate analysis, only a poor ECOG (> 2) remained predictive of poor PFS, p = 0.009 (95% CI 1.21–3.86), HR 2.16.1)“High-grade B-cell lymphoma with MYC and BCL2/BCL6 rearrangements” show an overall worse prognosis than “single-hit” MYC-R patients in terms of PFS;2)frontline intensive chemotherapy and DA-EPOCH-R are associated with a significant survival benefit if compared with R-CHOP-like treatments;3)restricting the analysis to the group of elderly patients, the advantage for intensive regimens was no longer observed, suggesting that younger patients might benefit the most from these schedules;4)survival is not significantly impacted by ASCT consolidation, regardless of the clinical response observed;5)survival after progression in “high-grade B-cell lymphoma with MYC and BCL2/BCL6 rearrangements” is particularly dismal, suggesting that salvage therapies are ineffective in this setting.Overall, the main results of this study can be summarized as follows:9 As regards CNS relapse, its cumulative incidence was inferior to that of the MDACC retrospective cohort8 (5% at 2 years vs 13% at 3 years). Even if the numbers are too small to drive sound correlations between the received prophylaxis  and the characteristics of CNS relapse , this difference might be due to a more aggressive upfront CNS prophylaxis in our series. Finally, as screening of all DLBCL and BCLU cases for MYC rearrangement was not the routine practice of all participating centers, we cannot exclude the possibility that selection bias influenced the decision to test specific cases.Therefore, with the limits of a retrospective series, we reported a large, National, homogeneous population, where the distribution of the baseline characteristics and the clinical results are very similar to those of previously published unselected cohorts12. Moreover, the complex biological landscape of DLBCL probably cannot be simply resumed by classical FISH or cell of origin analysis: actually, new biological insights, including high-throughput mutational14 and microenvironmental studies,15 are needed for a better classification and a more effective testing of front line targeted agents.In conclusion, the results of our study confirm that R-CHOP is not an appropriate therapeutic choice for “high-grade B-cell lymphoma with MYC and BCL2/BCL6 rearrangements”, and that intensive regimens or DA-EPOCH-R should be preferred, especially for younger patients. Nevertheless, the current diagnostic work-up of aggressive B cell lymphomas is controversial, and the screening strategy to drive FISH testing is not clearly shared.


In order to give the initial evaluation of benign prostatic hyperplasia (BPH) symptoms regarding a patient, physician usually performs the clinical inquiry of the patient, whereas his/her responses may contain hesitant fuzzy and uncertain information. However, existing evaluation/diagnosis approaches of BPH symptoms cannot cope with the hybrid problem of both hesitant and uncertain responses of patients. Furthermore, existing evaluation approaches may lose some useful responses (e.g. hesitant fuzzy information) so as to result in the unreasonable or indeterminate evaluation/diagnosis in the evaluation process of patients. To overcome this insufficiency, this study firstly introduces the concept of a cubic hesitant fuzzy set (CHFS) based on combining uncertain/interval-valued fuzzy information with hesitant fuzzy information so as to express the hybrid fuzzy information and proposes the Dice measure between CHFSs based on the extension method of the least common multiple cardinality/number (LCMC) for the hesitant fuzzy sets in CHFS. Then, the initial evaluation approach of BPH symptoms is developed by using the Dice measure of CHFSs. Lastly, the assessment results of six BPH patients are presented as the clinical actual cases to indicate the applicability and effectiveness of the developed assessment approach in CHFS setting. The comparison with existing common evaluation methods shows that the developed evaluation/diagnosis method is superior to the existing common evaluation methods in the evaluation/diagnosis process of the clinical actual cases. Due to the uncertainty of medical diagnosis information, a medical diagnosis approach was presented based on interval-valued fuzzy sets (IVFSs)4. To express the truth and falsity information, intuitionistic fuzzy sets and interval-valued intuitionistic fuzzy sets were applied to medical diagnoses7. To cope with medical diagnosis problems containing incomplete, uncertainty, and inconsistent information for a disease, simplified neutrosophic sets (SNSs), including single-value and interval neutrosophic sets, were applied to medical assessment/diagnosis problems10. Additionally, single valued neutrosophic multisets (refined neutrosophic sets) were applied in medical diagnosis problems14. Since there exist physicians’ hesitant thinking and representation in medical diagnosis problems, hesitant fuzzy sets (HFSs) were also applied to medical diagnosis problems16.In order to give the initial evaluation/diagnosis of a patient’s disease and symptoms, physician usually carries out the clinical inquiry of the patient, whereas the responses of the patient may contain fuzzy information due to his/her uncertainty and vagueness. Hence, the fuzzy medical diagnosis is an important research topic. It is a medical diagnosis method based on fuzzy relations of diseases and symptoms17 can only represent the hybrid information of both an uncertain/interval-valued fuzzy number (IVFN) and a fuzzy value in real life, but not express the hybrid fuzzy information of both the uncertain/IVFN and the HFS composed of several possible fuzzy values. For instance, when three physicians are required to assess the severe degree of benign prostatic hyperplasia (BPH) symptoms for a patient, the IVFN  is given by one of three experts and the HFS {0.5, 0.6} is given by two of three experts under their uncertain and hesitant situation, and then the hybrid form of both  (the uncertain part) and {0.5, 0.6} (the hesitant fuzzy part) cannot be expressed simultaneously by the aforementioned various fuzzy concepts. Then, Fu et al.18 presented a cubic hesitant fuzzy set (CHFS) so as to express the hybrid fuzzy information and applied it to the evaluation/diagnosis problems of the prostate cancer in CHFS setting. However, the evaluation method in18 can only cope with the evaluation/diagnosis problems of the prostate cancer, but cannot suit the evaluation problems of BPH patients in CHFS setting. Furthermore, existing common evaluation methods of BPH symptoms20 can also cope with evaluation/diagnosis problems of BPH symptoms with SNSs or uncertain information, but cannot handle evaluation/diagnosis problems of BPH symptoms with both uncertain and hesitant fuzzy information in CHFS setting. As the further generalization of the cubic hesitant fuzzy evaluation/diagnosis method18, this study extends it to the evaluation problems of BPH patients. To do so, this paper first proposes the Dice measure of CHFSs, and then develops the initial evaluation/diagnosis of BPH symptoms based on the Dice measure of CHFSs to solve the initial evaluation/diagnosis problems of BPH symptoms in CHFS setting.Since physicians’ thinking and expression may imply uncertain and hesitant evaluation information between a disease and symptoms in medical diagnosis process, however, the aforementioned diagnosis methods cannot cope with the evaluation/diagnosis problems with both uncertain information and hesitant information. Hence, they often lead to diagnostic confusion/uncertainty and puzzle due to losing some useful information. Furthermore, existing (fuzzy) cubic set (CS)As the framework of this study, Section 2 introduces the CHFS concept based on the hybrid form of both IVFN and HFS and proposes the Dice measure between CHFSs based on the extension method of the least common multiple cardinality/number (LCMC) for the HFSs in CHFS. In Section 3, the evaluation method of BPH symptoms is developed based on the Dice measure of CHFSs under CHFS setting. Section 4 presents the evaluation of the BPH symptoms by six BPH patients as the clinical actual cases to show the effectiveness and rationality of the evaluation approach of BPH symptoms based on the Dice measure of CHFSs. Lastly, conclusions and further study are contained in Section 5.et al.17 presented a (fuzzy) CS in a fixed non-empty set U by the following form:a(u) =  is an IVFN and μ(u) is a fuzzy value for u ∈ U.Regarding a hybrid form of an IVFN and a fuzzy value, Jun 22 in a fixed non-empty set U is expressed asμ1, μ2, …, μt} for u ∈ U.Then, a HFS conceptet al.18 gave the definition of CHFS below.Regarding a hybrid form of both a HFS and a CS, Fu U as a fixed non-empty set. A CHFS R is defined as the following form18:u ∈ U is an IVFN for u ∈ U is a HFS, which contains several different fuzzy values in  expressed by μ1, μ2, …, μt} in an ascending order.. Set R is denoted simply as Then, the basic element t = 1, CHFS is reduced to CS, which is a special case of CHFS.Especially when 18μk ∈  for k = 1, 2, …, t;An internal CHFE if every μk ∉  for k = 1, 2, …, t.An external CHFE if every . Set r = 〈, {0.6, 0.7}〉 is called an internal CHFE, where  is its IVFN and {0.6, 0.7} is its HFS.For example, 18:(i)r1 = r2 ⇔ k = 1, 2, …, t;(ii)r1 ⊆ r2 ⇔ μk1 ≤ μk2 for k = 1, 2, …, t;(iii)r1.. Set 18 until both reach the same cardinality (the same number of components) so as to reach reasonable operations of two different CHFEs. Obviously, this LCMC extension method shows the advantage of objectivity and feasibility.Generally speaking, for two different CHFEs t1 and t2 in q. Then both can be extended into the following forms:Assume two CHFEs are For convenient representation, Eqs  and 2)  2) are aThe following numerical example is given to indicate the LCMC extension method.r1 = <, {0.6, 0.7}> and r2 = <, {0.3, 0.4, 0.5}> be two CHFEs. They are extended by the LCMC extension method.Let q = 6 for t1 = 2 and t2 = 3 in r1 and r2. By applying Eqs  are CHFEs. If rn1 and rn2 are considered as the two vectors of qk + 2 dimensions, the Dice measure between R1 and R2 is defined asD indicates the following proposition.Set D contains the following properties:D ≤ 1;0 ≤ D = 1 ⇔ R1 = R2;D = D.The Dice measure Proof:a − b)2 ≥ 0 and a2 + b2 ≥ 2ab, the property (a) is true.Corresponding to the inequalities  = 1. On the contrary, if D = 1, then there are rk1 = rk2, i.e., k = 1, 2, …, n. Hence, R1 = R2 can hold.If It is obvious that the property (c) is true.rk1 and rk2 is taken into account, we set ωk for 0 ≤ ωk ≤ 1 and rk1 and rk2. Thus, the weighted Dice measure between R1 and R2 is presented asWhen the importance of the CHFEs Dω also has the following proposition.Thus, the weighted Dice measure Dω contains the following properties:Dω ≤ 1;0 ≤ Dω = 1 ⇔ R1 = R2;Dω = Dω.The weighted Dice measure By the similar proof manner of Proposition 1, we can prove Proposition 2, which is omitted here.Let us consider two CHFSs:R1 = {r11, r12} = {<, {0.5, 0.6}>, <, {0.3, 0.4, 0.5}>},R2 = {r21, r22} = {<, {0.4, 0.5, 0.6}>, <, {0.7, 0.8}>}.ω =  to calculate the weighted Dice measure between R1 and R2.Then, their weight vector is given as q1 = q2 = 6 from a pair of r11 and r21 and a pair of r12 and r22. Thus, we get the following extension forms:First, we get their LCMC R1 = R2 = R1 and R2 is calculated by the following form:Then, the weighted Dice measure between 20 for BPH, which are scored on a scale from 0 to 5 points so as to use the evaluation/diagnosis of BPH symptoms for clinical patients. An objective documentation of BPH symptoms was offered by the international prostate symptom score (I-PSS)20, In existing common clinical evaluation/diagnosis of BPH symptoms, the common score and evaluation method20 are shown in Tables Aging men commonly encounter the disease of BPH and suffer from obstructive and irritative voiding symptoms. To assess BPH symptoms, the seven questions introduced by the American Urological Association (AUA) are considered as the AUA symptom indicesHowever, this objective evaluation method presented in I-PSS is a traditional and non-fuzzy assessment/diagnosis, whereas the patient’s response to the seven questions of BPH symptoms may imply the hybrid information of both uncertain responses and hesitant responses regarding his/her vague symptoms indicated over the past month. Obviously, CHFS is very fit for the expression of the hybrid information. Thus, the Dice measure-based evaluation/diagnosis can solve the evaluation/diagnosis problems of BPH symptoms with CHFS information. Therefore, this section proposes the Dice measure-based evaluation/diagnosis approach of BPH symptoms in CHFS setting.A = {A1, A2, A3, A4, A5, A6, A7} and the clinical inquiry and answer of a patient Qi  indicate the BPH symptom responses in 5 times over the past month. However, since the patient’s answers may imply his/her uncertainty and hesitancy corresponding to the seven questions, he/she can give the uncertain range and hesitant values in his/her BPH symptom responses in 5 times over the past month.Based on Table Qi.In the clinical actual application, we require that physicians ask the BPH symptoms of patients over the past month by the seven questions in Table 20, we can also sort BPH patients into the three types of symptoms: Mild symptom (R1), Moderate symptom (R2), and Severe symptom (R3), which are constructed as a set of the three types of symptoms R = {R1, R2, R3}, indicating the three symptom patterns, to be used for the initial evaluation/diagnosis of BPH patients, as shown in Table Regarding I-PSSIn Table R1 = {〈A1, , {0, 0.2}〉, 〈A2, , {0, 0.2}〉, 〈A3, , {0, 0.2}〉, 〈A4, , {0, 0.2}〉, 〈A5, , {0, 0.2}〉, 〈A6, , {0, 0.2}〉, 〈A7, , {0, 0.2}〉},R2 = {〈A1, , {0.3,0.4}〉, 〈A2, , {0.3, 0.4}〉, 〈A3, , {0.3, 0.4}〉, 〈A4, , {0.3, 0.4}〉, 〈A5, , {0.3, 0.4}〉, 〈A6, , {0.3, 0.4}〉, 〈A7, , {0.3, 0.4}〉},R3 = {〈A1, , {0.75, 0.85}〉, 〈A2, , {0.75, 0.85}〉, 〈A3, , {0.75, 0.85}〉, 〈A4, , {0.75, 0.85}〉, 〈A5, , {0.75, 0.85}〉, 〈A6, , {0.75, 0.85}〉, 〈A7, , {0.75, 0.85}〉}.t BPH patients are obtained by Table Qi  corresponding to CHFE information, we can present the following evaluation approach.Suppose that the clinical inquiries and answers of Dω for j = 1, 2, 3 and i = 1, 2, …, t can be calculated in order to obtain a fit evaluation/diagnosis of a BPH patient Qi. Then, the fit evaluation Rj* of the BPH patient Qi can be yielded by The Dice measure In this section, we consider the clinical actual cases regarding six BPH patients to show the evaluation process of BPH symptoms by the evaluation/diagnosis method of BPH symptoms based on the Dice measure of CHFSs.Qi  in the clinical actual cases indicate their responses to the clinical inquiries from Table First, the six BPH patients Based on the inquiry results in Table Qi  can be expressed as the extension CHFSs based on the LCMC of HFSs qk = 2 :From Table Q1 = {<A1, , {0.6, 0.6}>, <A2, , {0.8, 0.8}>, <A3, , {0.4, 0.6}>, <A4, , {0.6, 0.6}>, <A5, , {0.8, 0.8}>, <A6, , {0.4, 0.6}>, <A7, , {0.6, 0.6}>},Q2 = {<A1, , {0, 0.2}>, <A2, , {0, 0.2}>, <A3, , {0.2, 0.2}>, <A4, , {0, 0.2}>, <A5, , {0, 0.2}>, <A6, , {0.2, 0.2}>, <A7, , {0, 0.2}>},Q3 = {<A1, , {0.4, 0.4}>, <A2, , {0, 0.2}>, <A3, , {0.2, 0.2}>, <A4, , {0.2, 0.4}>, <A5, , {0.6, 0.6}>, <A6, , {0.4,0.6}>, <A7, , {0.4, 0.6}>},Q4 = {<A1, , {0.6, 0.6}>, <A2, , {0.6, 0.6}>, <A3, , {0.4, 0.4}>, <A4, , {0.6, 0.6}>, <A5, , {0.6, 0.8}>, <A6, , {0.6, 0.6}>, <A7, , {0.6, 0.8}>},Q5 = {<A1, , {0.6, 0.8}>, <A2, , {0.6, 0.8}>, <A3, , {0.8, 0.8}>, <A4, , {0.8, 0.8}>, <A5, , {0.8, 0.8}>, <A6, , {0.8, 0.8}>, <A7, , {0.4, 0.6}>},Q6 = {<A1, , {0.4, 0.6}>, <A2, , {0.4, 0.6}>, <A3, , {0.2, 0.4}>, <A4, , {0.4, 0.6}>, <A5, , {0.2, 0.4}>, <A6, , {0.4, 0.6}>, <A7, , {0.4, 0.6}>}.Ak is ωk = 1/7 for k = 1, 2, …, 7. By using Eq.  and the symptom pattern Rj , which are shown in Table Suppose the weight of each element sing Eq. , we can Q2 has mild BPH symptom, the patients Q1, Q4, and Q5 have severe BPH symptoms, and the patient Q3 and Q6 have moderate BPH symptoms since the patients regarding the largest measure values indicates their fit evaluation results.From Table 20. In this case, if we do not consider the hesitant values in Table To show the effectiveness of the proposed new evaluation method for the six BPH patients, we have to compare the proposed new evaluation method with the existing common evaluation method based on I-PSS20 and the proposed new method are indicated in Table For the convenient comparison, the evaluation/diagnosis results given based on the common evaluation methodQ1, Q3 and Q4 and implies the evaluation/diagnosis indeterminacy so as to difficultly evaluate/diagnose them in this situation; while the latter can clearly indicates its evaluation results and shows its effectiveness and rationality. Therefore, the proposed new evaluation method based on the Dice measure of CHFSs can overcome the insufficiency of the existing simply scoring evaluation method with uncertain values .In Table 10, the proposed evaluation method using the Dice measure of CHFSs contains uncertain and hesitant assessment information of patients, which the evaluation approach in10 cannot carry out. Furthermore, the new evaluation method in this study is very fit for patients’ thinking and expressing habits in their clinical inquiries and answers, which show the main advantage. In the BPH evaluation process, it is obvious that the proposed new evaluation method is more feasible and effective and superior to the existing initial evaluation methods20.Compared with the evaluation approach using exponential similarity measure of SNSs presented inRegarding the hybrid form of both interval-valued fuzzy information and hesitant fuzzy information, a CHFS is very fit for the expression of the hybrid information. Hence, this study firstly proposed the Dice measure of CHFSs based on the LCMC extension method for HFSs in CHFSs. Next, an initial evaluation approach of BPH symptoms was developed by using the Dice measure of CHFSs in CHFS setting. Lastly, the initial evaluations of six BPH patients are presented as the clinical actual cases to show the effectiveness and suitability of the proposed evaluation approach in CHFS setting.20 cannot cope with the evaluation/diagnosis problems along with CHFS information and may lose much useful information (hesitant fuzzy information) in the evaluation process so as to result in uncertain or difficult assessment results. By comparison with existing assessment approaches, the main advantages of this study indicate (1) CHFS is very fit for the expression of uncertain and hesitant fuzzy responses of patients in the clinical assessment process; (2) The Dice measure of CHFSs based on the LCMC extension method shows the objective extension operation without the subjective extension form depending on decision makers’ preference; and (3) the developed initial evaluation approach of BPH symptoms can effectively cope with medical diagnosis problems along with uncertain and hesitant fuzzy information.However, the existing initial evaluation approachesIn the future, this study will be extended to other medical evaluation/diagnosis problems, such as kidney cancer and gastric cancer, in CHFS setting.


This study investigates the effects of long-term ethanol consumption on neuroinflammation in the hippocampus of adolescent rats and the regulatory roles of TLR4 and SENP6. Twenty-one days of ethanol exposure in adolescent rats were used to develop an animal model. The number of microglia, microglial activation, and the expression of TLR4 in the hippocampus of adolescent rats were examined by immunoreactivity. The levels of TLR4, activation of NF-κB including IkB-α and p-NF-κB-p65, and SENP6 were measured by western blotting. Proinflammatory cytokines including TNF-α, IL-1β, and IL-6 were measured by enzyme-linked immunosorbent assay. The NF-κB activation and proinflammatory cytokines released in overexpressed SENP6 and siRNA targeting SENP6 microglial cells after treatment with ethanol were estimated in vitro. This study found that alcohol exposure increased the number of activated microglia and the levels of p-NF-κB-p65 and proinflammatory cytokines, while it decreased the SENP6 level in wild-type rats, but not in TLR4 knockout rats. The ethanol-induced increases of p-NF-κB-p65, TNF-α, and IL-1β were dampened by overxpression of SENP6 and enhanced in SENP6-siRNA microglia. Our data suggest that ethanol exposure during adolescence induces the microglia-mediated neuroinflammation via TLR4 activation, and SENP6 plays an essential role in dampening NF-κB activation and neuroinflammation.The ethanol-induced toll-like receptor 4 (TLR4) signal activation of microglia and neuroinflammation are observed in both adolescent and adult rat brains, but the regulatory mechanisms of some TLR4 signaling-related factors in this process are still unclear. SUMO-specific protease 6 (SENP6) inhibits neuroinflammation by dampening nuclear factor kappa-B (NF- The percentage of alcohol consumption in adolescents is increasing . An adolExcessive ethanol consumption causes neurodegeneration , 12. NeuκB), a pivotal nuclear transcription factor in the TLR4 signal pathway, participates in both the MyD88- and TRIF-dependent pathways and plays an important role in the transcription and synthesis of inflammatory factors [κB depends on the activity of the inhibitor of NF-κB (IkB) kinase (IKK) complex, which makes up an essential regulatory subunit—the NF-κB essential modifier (NEMO/IKKc)—required for NF-κB activation. Intriguingly, NEMO is modified by the polyubiquitin chain, which is also critical for IKK activation [κB activation [κB activation [Nuclear factor kappa-B , with a body weight of 55-70 g and an age of 21-23 days, were obtained from the Animal Experimental Center of Shandong University. Twenty TLR4 knockout rats (KO) from Wistar background with a body weight of 53-71 g and an age of 21 days were produced by transcriptional activator-like effector nuclease- (TALEN-) mediated gene inactivation and sponsored by Dr. Chen's Laboratory of Shandong University  . All ratn = 10), ethanol-treated WT (WT+EtOH) group (n = 10), KO control (KO) group (n = 10), and ethanol-treated KO (KO+EtOH) group (n = 10). Rats in the WT+EtOH and KO+EtOH groups had free access to a 10% aqueous ethanol (v/v), whereas rats in the WT and KO groups received tap water. After 21 days of treatment, all rats were sacrificed.After the 7-day acclimatization period, the 20 WT rats and 20 KO rats were randomly assigned to four groups: WT control (WT) group  with tap water and weighed again. The ethanol consumptions (g/kg body weight/24 h) were calculated for each rat, and values were averaged across 21 days of treatment.2. The medium was replaced every 4 days. Ten days later after shaking at 260 r/min for 2 h at 37°C, the culture media were collected and seeded in new flasks for 1 h. The anchorage-dependent cells, known as microglia, were obtained. Then, the cell culture was stained by immunofluorescence using anti-Iba-1 and DAPI for purity identification. The nuclei in the DAPI staining appeared blue, and Iba-1-positive (Iba-1+) cells appeared red. The merging of blue and red was considered positive cells (microglia) when counting. All counting was performed by researchers who were blinded to the groups. Cells in seven random fields (200x magnification) were quantified and averaged. The microglial purity (%) = number of positive cells/total number of nuclei × 100%. Our data showed that the number of positive cells was 304.43 ± 3.56 cells/field, the total number of nuclei was 319.43 ± 3.43 cells/field, and the microglia purity was 95.30 ± 0.27%. The microglial cells were then treated with ethanol at different doses  for 24 h, and the SENP6 protein levels were measured by the WB to detect the relation of ethanol and SENP6 protein expression. The ethanol concentrations used in the present study were in the range of the blood alcohol levels found among alcoholics (10-120 mM) [Primary rat cultures of mixed glial cells were prepared, and the microglial cells were purified as previously described  with som-120 mM) .μL of each sample was added into a 96-well plate and diluted in Ethanol Assay Buffer to a final volume of 50 μL. Then, after adding 50 μL of the Master Reaction Mix to each of the wells, the solution was mixed well using a horizontal shaker and incubated for 30 minutes at 37°C, and the absorbance was measured at 570 nm. All samples and standards were made in duplicate. The blood ethanol concentrations obtained from the three time points were averaged for each animal.Tail blood samples were collected weekly at two time points, namely, 2 h after the onset of light and dark. Briefly, the blood was centrifuged at 2000 r/min for 5 min. Serum was collected and stored at –80°C until further analysis. The blood ethanol concentrations (BECs) (mg/dL) from the serum were determined with a commercial colorimetric assay . Briefly, 10 n = 4) were sacrificed for fluorescent immunohistochemical staining. Briefly, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (80 mg/kg body weight) and perfused with saline intracardially until the liver turned pale pink, followed by 4% buffered formalin. Then, the brains were removed and fixed in formalin for 24 h. The brain samples were dehydrated and embedded in paraffin wax. Serial sections of the hippocampus were cut at 5 μm intervals. The remaining 6 rats of each group (n = 6) were sacrificed for WB or ELISA analysis. After decapitation, the brains were rapidly removed from the skulls, and the hippocampal formations were dissected, quickly frozen in liquid nitrogen, and stored at –80°C until use.At the end of the treatment, 4 rats from each group (+) or ED-1-positive (ED-1+) cells appeared red, and TLR4-positive (TLR4+) receptors appeared red when simple staining or green when double staining with ED-1. The merging of blue and red  and the merging of blue, red, and green (ED-1+/TLR4+ colabeling cells) were considered when counting. All counting was performed by researchers who were blinded to the groups. Cells in five random fields (400x magnification) across each region of interest  in three consecutive sections of each rat were quantified. The numbers of cells obtained from the three sections were averaged for each animal, and four animals were used for each fluorescent immunohistochemical staining. The results were expressed as the number of cells per mm2.Both quiescent and activated microglia were labeled with the Iba-1 antibody, the phagocytic activated microglia were labeled with ED-1 antibody, and the expressive level of TLR4 was labeled with the TLR4 antibody. After being deparaffinized with xylene, rehydrated with a descending alcohol series, and washed with distilled water, the sections were placed in a repair box full of EDTA antigen repair buffer (pH 8.0) for 15 min antigen retrieval in a microwave. Then, after washing in 0.01 M phosphate-buffered saline (PBS) (pH 7.4) for three bouts of 5 min, spontaneous fluorescence quencher was added to the sections for 5 min, which were then blocked for 30 min in normal goat serum (5% in PBS) in a humid chamber at 37°C and then incubated with polyclonal rabbit anti-Iba-1 primary antibody (1 : 500) , polyclonal rabbit anti-ED-1 primary antibody (1 : 100) , and polyclonal mouse anti-TLR4 primary antibody (1 : 200)  at 4°C overnight. After being diluted three times in PBS containing 0.03% Triton X-100 for 5 min, sections were incubated with the CY3 goat anti-rabbit secondary antibody (1 : 300)  for Iba-1 and ED-1 staining or FITC goat anti-mouse secondary antibody (1 : 400)  for TLR4 staining for 50 min at 37°C and added with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining solution for 5 min at 37°C after washing in PBS (pH 7.4) for three bouts of 5 min. Finally, the sections were mounted with mounting medium containing antiquenching fluorescence and observed using an inverted fluorescence microscope . Image capturing was performed using an imaging system . The nuclei in the DAPI staining appeared blue, Iba-1-positive (Iba-1n = 6 per group) and cultured cells were homogenized in lysis buffer. The homogenate was centrifuged at 14,000 × g for 30 min at 4°C. The supernatant was stored at −80°C until use. The protein concentration was measured using a Protein Quantitative Analysis Kit  from the Bio-Rad DC. Equal amounts of protein (50 μg) from each sample were boiled in 6×Laemmli loading buffer for 5 min, run on 12.5% (for IkB-α and p-NF-κB-p65) or 8% (for TLR4 and SENP6) SDS-polyacrylamide gels, and then transferred to 0.2 μm polyvinylidene fluoride (PVDF) membranes (Millipore Corporation). The blots were blocked for 2 h in blocking solution  at 37°C, then incubated overnight with anti-sera (dilution): β-actin (1 : 2000) , SENP6 (1 : 1000) , IkB-α (1 : 1000) , p-NF-κB-p65 (1 : 1000) , TLR4 (1 : 1000) , GAPDH (1 : 1000) , Lamin A/C (1 : 500)  at 4°C, and visualized using horseradish peroxidase- (HRP-) conjugated goat anti-rabbit , donkey anti-goat , goat anti-mouse  secondary antibodies (1 : 20000), and an ECL detection system (Millipore Corporation). The bands corresponding to β-actin, SENP6, IkB-α, p-NF-κB-p65, TLR4, GAPDH, and Lamin A/C were scanned and densitometrically analyzed using ImageJ software. These quantitative analyses were normalized to β-actin/GAPDH/Lamin A/C (after stripping).The WB assay was modified as previously reported . Brain tα , IL-1β , and IL-6  were determined by ELISA according to the manufacturer's instructions. The standards were run in duplicate and samples in triplicate. Absorbance was measured at 450 nm using the Bio-Rad DC. Cytokine concentrations were normalized to the total protein content and reported as pg/mg of total protein ± S.E.M.The samples of brain tissues and cultured cells were prepared as WB. The protein levels of TNF-μg were transfected into cultured rat microglia using Lipofectamine 2000, according to the manufacturer's instructions (Invitrogen). The plasmids also contained a GFP reporter to allow the determination of the transfection efficiency, which was nearly 100% in the current study. Then, we measured the SENP6 protein level to confirm the transfection effect 48 h later. Half of the Scr group (Scr-E) and the siSENP6 and SENP6 groups were treated with 100 mM ethanol for 24 h, and the remaining Scr group with no treatment as nonethanol control group (Scr-Con). Twenty-four hours later, we measured the SENP6, IkB-α, and p-NF-κB-p65 protein levels by WB, and the TNF-α, IL-1β, and IL-6 protein levels by ELISA to assess the effect of ethanol on activation of NF-κB and associated neuroinflammation in microglial cells and the modulatory role of SENP6 in this process.SENP6-modified plasmids were prepared using routine methods. Briefly, the constructs for overexpressed SENP6 (SENP6), siRNA targeting SENP6 (siSENP6), and control scrambled sequence (Scr) were cloned into pcDNA3.1–EGFP to generate the corresponding plasmids. The sequences were as follows: SENP6: 5′-AACUGCAACUUCUAAACACAAAA-3′, siRNA SENP6: 5′-GGGUGAUAAAGCCUGUAAATT-3′, and Scr: 5′-UUCUCCGAACGUGUCACGUTT-3′. These plasmids of 2 t-test. A result of p < 0.05 was considered to be statistically significant. All statistical analyses were conducted with the SPSS17.0 software.Data were presented as means and standard errors. Data from three or more groups were analyzed using one-way analysis of variance (ANOVA) with treatment as the independent variable. Post hoc analyses were performed whenever appropriate, using the Turkey test when normality assumptions were satisfied. Comparison between two groups was made with an independent-sample t (18) = 0.167, p = 0.869). The blood ethanol concentrations were 34 ± 3.2 mg/dL 2 h after light onset and 105 ± 7.1 mg/dL 2 h after light offset in the WT+EtOH group and 32 ± 2.7 mg/dL 2 h after light onset and 98 ± 7.2 mg/dL 2 h after light offset in the KO+EtOH group. There were no significant differences across the three time points  both at 2 h after light onset  = 0.511, p = 0.484) and at 2 h after light offset  = 0.836, p = 0.373) in the WT+EtOH group, as well as in the KO+EtOH group  = 0.078, p = 0.783; 2 h after light offset: F  = 0.100, p = 0.756). There were no significant differences between the WT+EtOH and KO+EtOH groups across the three time points of both 2 h after light onset (1st week: t (18) = 0.131, p = 0.897; 2nd week: t (18) = 0.346, p = 0.734; 3rd week: t (18) = 0.059, p = 0.954) and 2 h after light offset (1st week: t (18) = 0.096, p = 0.924; 2nd week: t (18) = 0.698, p = 0.494; 3rd week: t (18) = 0.955, p = 0.352).During the 21 days of ethanol treatment, the adolescent rats consumed 9.74 ± 0.67 g of ethanol per kilogram of body weight daily in the WT+EtOH group and 9.57 ± 0.71 g of ethanol per kilogram of body weight daily in the KO+EtOH group. There were no significant differences between the WT+EtOH and KO+EtOH groups in the ethanol consumption (+ microglia in the CA1 (t (6) = 25.354, p < 0.001), CA2/3 (t (6) = 13.594, p < 0.01), and DG (t (6) = 19.424, p < 0.001) of the hippocampus in the WT+EtOH group significantly increased after excessive ethanol exposure compared with the control groups. However, the increased Iba-1+ microglia in the three subfields of the hippocampus were noticeably reduced in the corresponding groups with TLR4 gene knockdown (CA1: t (6) = 22.669, p < 0.001; CA2/3: t (6) = 12.474, p < 0.001; DG: t (6) = 25.811, p < 0.001), and the levels of Iba-1 immunoreactivity were similar in both ethanol-treated and nontreated TLR4-KO rats (CA1: t (6) = 1.667, p = 0.147; CA2/3: t (6) = 0.585, p = 0.580; DG: t (6) = 0.067, p = 0.949). The results indicate that ethanol exposure increases Iba-1 immunoreactivity of microglia in the adolescent rat hippocampus depending on TLR4 activation.As shown in Figures + cells were observed in any subfields of the hippocampus in the WT or TLR4-KO animals without ethanol treatment. However, chronic ethanol treatment markedly increased the number of ED-1+ cells in the WT rats, but not in TLR4-KO animals. The numbers of ED-1+ cells in the WT+EtOH group were 90.00 ± 8.42 and 90.67 ± 7.37 in the CA1 and CA2/3 regions and 95.11 ± 6.92 in the DG region. Furthermore, a large proportion of ED-1+ cells that highly expressed TLR4 was observed in the WT ethanol group; briefly, 80.2% and 70.3% of the ED-1+ cells colocalized with TLR4 in the CA1 and CA2/3 regions, and 53.7% of the ED-1+ cells were TLR4+ cells in the DG region in the WT+EtOH group. The data suggest that alcohol exposure drives microglia to a state of phagocytic activation and supports the key role of TLR4 in regulating ethanol-induced microglial activation.As shown in Figures + cells in immunoreactivity which demonstrated that TLR4 expression only markedly increased in the CA2/3 region (t (6) = 12.810, p < 0.001) of the WT ethanol-treated animals but not in the CA1 (t (6) = 2.420, p = 0.052) or DG (t (6) = 1.895, p = 0.107) subfields. No TLR4+ cells were observed within the hippocampus of TLR4-KO rats. However, the quantification analysis of TLR4 protein level in WB showed no change in the WT groups with or without ethanol treatment (t (10) = 0.226, p = 0.826) (t (10) = 8.942, p < 0.001; WT+EtOH vs. KO+EtOH: t (10) = 7.949, p < 0.001).Figures = 0.826) , suggestt (10) = 3.465, p < 0.01) and IkB-α levels (t (10) = 5.004, p < 0.001), increased levels of p-NF-κB-p65 (t (10) = 7.346, p < 0.001), and increased levels of TNF-α (t (10) = 10.293, p < 0.001) and IL-1β (t (10) = 7.058, p < 0.001) compared with the WT group, while no significant increased IL-6 level was observed in the WT+EtOH group compared with the WT group (t (10) = 0.285, p = 0.789). Conversely, in TLR4-KO rats, no changes were seen in any index after ethanol treatment (SENP6: t (10) = 0.424, p = 0.680; IkB-α: t (10) = 0.288, p = 0.780; p-NF-κB-p65: t (10) = 2.143, p = 0.057; TNF-α: t (10) = 0.209, p = 0.839; IL-1β: t (10) = 0.959, p = 0.360; IL-6: t (10) = 0.453, p = 0.660). Furthermore, the KO+EtOH group displayed significantly increased SENP6 level (t (10) = 3.365, p < 0.01), decreased p-NF-κB-p65 level (t (10) = 6.149, p < 0.001), and lower levels of TNF-α (t (10) = 4.808, p < 0.01), IL-1β (t (10) = 14.915, p < 0.001), and IL-6 (t (10) = 6.437, p < 0.001) compared with the WT+EtOH group. The results indicated that ethanol exposure decreased SENP6 expression and activated NF-κB inflammatory signaling via TLR4 activation.Figures F  = 7.246, p = 0.001) in cultured microglial cells with statistical significance at the dose of 50 mM (p < 0.05) and100 mM (p < 0.001), compared with the nonalcoholic control group.t (10) = 4.579, p < 0.01; Scr vs. siSENP6: t (10) = 3.618, p < 0.05).α and p-NF-κB-p65 levels. The level of p-NF-κB-p65 (t (10) = 12.328, p < 0.001) was markedly increased versus controls when the cells were subjected to 100 mM ethanol. The overexpression of SENP6 significantly dampened the increased effects of ethanol on the level of p-NF-κB-p65 (t (10) = 18.458, p < 0.001). The ethanol-induced activations of the p-NF-κB-p65 (t (10) = 7.003, p < 0.001) was enhanced in the SENP6-siRNA-transfected cells.Figures α, IL-1β, and IL-6 in normal, SENP6, and siSENP6-transfected microglia after treatment with 100 mM ethanol. The levels of TNF-α (t (10) = 8.989, p < 0.001), IL-1β (t (10) = 5.540, p < 0.001), and IL-6 (t (10) = 10.592, p < 0.001) were markedly increased versus controls when the cells were subjected to 100 mM ethanol. The overexpression of SENP6 significantly dampened the increased effects of ethanol on the levels of TNF-α (t (10) = 6.273, p < 0.001) and IL-1β (t (10) = 8.891, p < 0.001). Furthermore, the ethanol-induced activations of the TNF-α (t (10) = 7.354, p < 0.001), IL-1β (t (10) = 2.835, p < 0.05), and IL-6 (t (10) = 6.324, p < 0.001) were further increased in the SENP6-siRNA-transfected cells. These results strongly support that SENP6 contributes to the regulatory mechanism of the ethanol-induced activation of NF-κB and the release of inflammatory cytokines in the microglia.Figures + and ED-1+ cells in the CA1 CA2/3 and DG regions of the hippocampus, decreased IkB-α expression, and increased p-NF-κB-p65, as well as TNF-α and IL-1β levels in the hippocampus. These data suggest that chronic alcohol exposure drives the microglia into a phagocytic activated proinflammatory state.Microglia-mediated neuroinflammation is a hallmark feature of most neurodegenerative diseases , 39. Nump = 0.052). The results indicated that ethanol induced more TLR4 expression in the CA regions of the hippocampus compared with the DG subfield. Meanwhile, the percentage of phagocytic activated microglia in the CA regions (CA1 and CA3) was significantly increased in the ethanol-exposed groups compared to the controls in the hippocampus of the developing brain [κB-p65, TNF-α, and IL-1β levels. Our results demonstrate that TLR4 plays a pivotal role in the ethanol-induced activation of microglia and release of proinflammatory cytokines which is consistent with the previous studies [It is noticed that the present study demonstrated that TLR4 expression only markedly increased in the CA2/3 region of the WT ethanol-treated animals but not in the CA1 and DG subfields. However, the difference of TLR4 expression in the CA1 region between the WT and WT+EtOH groups was marginally significant (ng brain , indicat studies –27.κB activation [κB activation and increased release of the proinflammatory cytokines TNF-α and IL-1β in microglial cells. And the ethanol-induced activation of the p-NF-κB-p65 and neuroinflammation were further markedly enhanced in the SENP6-siRNA-transfected cells. These data first highlighted that the SENP6 plays an essential role in dampening ethanol-induced NF-κB activation and neuroinflammation in the microglia. Future in vivo studies should be performed in both adolescent and adult rat brains to improve the completeness of our understanding of the regulatory role of SENP6 in dampening ethanol-induced NF-κB activation and neuroinflammation.Recently, SENP6 was reported to be involved in the regulation of NF-tivation , 33, whitivation . SENP6 ctivation . The ovetivation –47. The κB activation and neuroinflammation in the microglia. The findings may provide important clues regarding SENP6 being involved in neuroinflammation induced by alcohol of the adolescent hippocampus. Further research should be made to investigate the other posttranslational modifications of TLR4 signaling-related factors, other than NEMO, involved in this process.In summary, the current study firstly demonstrates that 21 days of alcohol exposure during adolescence induces a phagocytic activated proinflammatory state of microglia through TLR4 activation. More importantly, we confirmed that SENP6 plays an essential role in dampening ethanol-induced NF-


Villosiclava virens. These metabolites displayed a wide range of bioactivities, such as teratogenic, cytotoxic, phytotoxic, and antibacterial activities. So far 26 ustilaginoidins have been isolated from V. virens, among which 18 compounds contained stereogenic center(s), however, most of them were unknown for the absolute configurations, except that of ustilaginoidin D. In this study, the absolute structures of these ustilaginoidins were constructed for the first time by analysis of the biosynthetic monomers obtained from a gene knockout mutant (ΔUV_2091) of V. virens. The gene UV_2091 was predicted to encode an enzyme that dimerized the monomeric naphtho-γ-pyrones in V. virens. Knockout of this gene led to the accumulation of three monomers, namely hemiustilaginoidin F (1), epihemiustilaginoidin D (2), and hemiustilaginoidin D (3), but the production of ustilaginoidins was completely blocked. The structures of the monomers were deduced by spectroscopic analysis, in combination with TDDFT ECD calculations for determining the absolute configurations. These compounds were tested for their phytotoxic, cytotoxic, antibacterial, and antifungal activities. Compounds 1 and 3 showed inhibition against the radicle and plumule elongation of rice and lettuce seeds at the tested concentrations. Compound 1 was active against the tested five human cancer cells, with half maximal inhibitory concentrations (IC50s) of 13.2~37.3 μM. Compounds 1~3 inhibited the growth of the tested pathogenic bacteria with minimum inhibitory concentrations of 8~32 µg/mL, while compound 3 exhibited antifungal activity against Magnaporthe oryzae . A comparison of these data with those of the ustilaginoidins provided insights into the structure-bioactivity relationships.Ustilaginoidins are a kind of mycotoxins with 9,9′-linked bis-naphtho-γ-pyrones structures produced by the rice false smut pathogen  Villosiclava virens (anamorph: Ustilaginoidea virens)5, which was the causal agent of rice false smut that threatened the production of rice worldwide7. Up to now, 26 ustilaginoidins have been reported from V. virens, including ustilaginoidins A~J3, K~P4, Q~W5, and E14, 2,3-dihydroustilaginoidin T5, and isochaetochromin B25. These compounds were considered to be mycotoxins, as they were teratogenic towards mouse embryo limb bud and midbrain cells8, and cytotoxic against several cancer cells including KB (epidermoid)9, A2780 (ovarian)4, HCT116 (colon), NCI-H1650 (lung), BGC823 (gastric), Daoy (medulloblastoma), and HepG2 (liver) cells5. The toxicities may attributed to their inhibition of ATP synthesis in mitochondrial10. In addition, they showed phytotoxicities on the elongation of radicle or plumule of rice seeds5, and antibacterial activities11.Ustilaginoidins were bis-naphtho-γ-pyrones of 9,9′-linkage produced by the pathogen R12, but also that of the stereogenic centers in the 2,3-dihydropyran-4-one ring when present, which was, however, unknown with regard to the absolute configurations for most of them. Among the 26 reported ustilaginoidins, 18 of them have one or two 2,3-dihydropyran-4-one moiety  of ustilaginoidins. Although Polavarapu, et al. succeeded in determining the absolute configuration of cephalochromin by comparing the experimental and theoretical VCD spectra14, the vibrational bands used to distinguish the diastereomers were of small magnitudes, and the applicability of this method to the bis-naphtho-γ-pyrones with three or four stereogenic centers  still needed to be verified.The chirality of ustilaginoidins included not only the axial chirality, which was reported to be aUV_2091), and were evaluated for their biological activities. In addition, the structure-activity relationships were discussed by comparison with those of the dimers (i.e. ustilaginoidins).In this study, we addressed the stereochemical problem of ustilaginoidins from a biosynthetic point. Three biosynthetic monomers were obtained from a gene knockout mutant (ΔR configuration for the C-9/9′ axis. For those 18 ustilaginoidins that contain stereogenic center(s) in the 2,3-dihydropyran-4-one ring  in Fusarium graminearum17. In this gene cluster, the genes UV_2086, UV_2087, UV_2088 and UV_2091 were highly homologous to PKS12, FG_02325, aurT and gip1 in F. graminearum17, respectively. We particularly interested in UV_2091, a homology of gip1, which was reported to encode a laccase that dimerized two molecules of 9-hydroxyrubrofusarin into the C-7/7′ dimer in F. graminearum17. Thus, we knocked out this gene with the CRISPR-Cas9 system18 and obtained the knockout mutant (ΔUV_2091), whose colony morphology was obviously different from that of the wild type by having less yellow pigment  , conjugated keto (1631 cm−1), and phenyl  groups. Its UV spectrum was similar to that of ustilaginoidins, though not identical  and one singlet (δH 6.348), two methines , and two methyl groups  were observed , ten sp2-hybridized carbons, two methines , and two methyl groups   that showed meta-coupling to H-7  in 3. This allowed us to establish the planar structure of 3 as one half of ustilaginoidin D -3 was randomly selected for conformational search using Molecular Merck force field, followed by geometry optimization using the DFT method (B3LYP/6-31 G(d), CPCM = MeOH). Two stable conformers  were found, with 3a being the dominating one (91.2%) , CPCM = MeOH). Then, the theoretical ECD spectrum of -3 was generated by averaging that of 3a and 3b according to their Boltzmann distributions. The calculated spectrum of -3 showed negative Cotton effect at 325 nm, and strong positive Cotton effects at 223 and 279 nm, which matched well with the experimental spectrum  between H-2 and H-3 revealed their 1 was isolated as the most polar and the second major monomer. Its molecular formula, C14H12O5, as determined by HRESIMS, contained one less CH2 unit than that of 3. Compound 1 has a similar UV, IR, and NMR spectra compared to those of 3, implying they share a similar skeleton. However, the notable differences of the NMR data  in 1 replaced that of the methine in 3. The HMBC experiment revealed correlations from 2-CH3  to C-2 (δC 74.7), and C-3 (δC 44.3), and from H2-3  to C-2, C-4 (δC 199.7), C-4a (δC 103.2), and 2-CH3 (δC 21.2). Thus, 1 was determined as a 3-demethyl derivative of 3  and a shoulder peak was seen for 1 -1, generated by MMFF conformational search, resulted in only one predominant conformer 1a  of 3JH-2, H-3. The CD spectrum of 2 was different from that of 3 mainly in the range of 210~300 nm . Again, the absolute configuration of 2 was established by comparing the predicted ECD spectrum with the experimental one. -2 and its enantiomer -2 were selected for ECD calculations. Two lowest energy conformers were found for -2, with the major conformer accounting for 81.5% of the populations, and the other one 18.5%. In the major one, 2-CH3 was found to be equatorial, while 3-CH3 was axial with regard to the half-chair conformation of the dihydropyranone ring. On the contrary, axial 2-CH3 and equatorial 3-CH3 were seen in the minor conformer -2 -2, and -2 were generated -2 fitted well with the experimental one, while -2 did not. Thus, compound 2 has a 2R, 3S configuration. It is a 3-epimer of 3, and named epihemiustilaginoidin D.Compound ta Table  disclose1~3 revealed similar negative Cotton effects at around 310 and 340 nm, while positive Cotton effects at 210~300 nm though with different peak shape and intensity  seeds (Table 1 inhibited rice radicle growth with inhibition ratio in the range of 7.62~53.37% dependent on the tested concentration and rice cultivation varieties (Lijiang or Zhonghua 11), while compound 3 showed inhibition ratio of 21.88~75.24%. The variety Zhonghua 11 was more susceptible to compound 3 than that of Lijiang, whereas no significant difference was found between both varieties with regard to compound 1. Compound 3 showed stronger inhibition against the plumule elongation for both varieties than that of 1, and this held true for the inhibitory effects on the radicle growth of the Zhonghua 11 variety, but not for that of the Lijiang variety. In the literature, ustilaginoidins B, E, F, I, O, R and U, and isochaetochromin B2 were found to be phytotoxic towards the radicle elongation of rice seeds5. These ustilaginoidins showed more than 50% inhibition ratio against the growth of radicle for the variety Lijiang at 200 μg/mL5, while compounds 1 and 3 exhibited inhibition ratio of 52.2% and 49.35%, respectively, at 400 μg/mL, for the same rice variety. Taking account of the monomeric characters of 1 and 3, they did not showed stronger phytotoxicities than these bioactive dimers (ustilaginoidins). For instance, compound 1  showed weaker inhibition than its homodimer ustilaginoidin F 4, while compound 3  was weaker than its heterodimer isochaetochromin B2 4. However, we could not draw a clear structure-activity relationship regarding the monomers and dimers.Compounds ds Table . They do1 and 3 against the germination of the lettuce (Lactuca sativa L. var. ramose Hort.) seeds  was completely inhibited by compounds 1 and 3 at 400 μg/mL, which was better than the positive control glyphosate. Thus, it was tempting to speculate that ustilaginoidins were phytotoxic against lettuce or the other dicotyledonous plants, though we have not tested yet.Meanwhile, we also tested the phytotoxic activities of ds Table . Similar1 and 3 were evaluated against human carcinoma cells . Compound 1 moderately inhibited the growth of these cells (IC50s 13.2~37.3 μM), while compound 3 was inactive (IC50 > 50.0 μM) (Table 50 0.42~1.94 μM)9, while ustilaginoidins K and L showed inhibition against A2780 cells 4. Ustilaginoidins B, C, H, I, J, R, S, V and W were cytotoxic to several cancer cell lines (the same as in current study) with IC50 values of 4.06~44.1 μM, but none of them was active against all the tested cells5. The structure-activity relationship was elusive regarding the monomers and dimers.The cytotoxicities of 1~3 were screened for antibacterial activities towards pathogenic bacteria including Bacillus subtilis, Staphylococcus haemolyticus, Ralstonia solanacearum, Xanthomonas vesicatoria, Agrobacterium tumefaciens, and Pseudomonas lachrymans . For examples, compound 1 showed inhibitory activity with MIC values ≤ 32 µg/mL, while its homodimer ustilaginoidin F did not exhibit any effect at 128 μg/mL4. Though ustilaginoidin D, the homodimer of compound 3, showed inhibition with MIC values of 32 μg/mL against four tested bacteria, it was inactive against P. lachrymans and R. solanacearum (MIC > 64 μg/mL)4, while compound 3 displayed inhibition against all six tested bacteria (MIC 16~32 μg/mL). It was interesting that the heterodimer of 2 and 3, i.e., isochaetochromin B2, was inactive at 128 μg/mL4, while 2 and 3 were both active with MIC values ≤ 32 µg/mL.Compounds ns Table . All the1 and 3 were evaluated for inhibitory effects towards the rice blast pathogen Magnaporthe oryzae. Compound 3 showed strong inhibition against the spores germination with IC50 value of 5.21 µg/mL, which was comparable to the positive control carbendazim , while compound 1 was moderately active . In literature, the antifungal activities of ustilaginoidins have not been reported. Three related bis-naphtho-γ-pyrones with an aS axial chirality, cephalochromin, isoustilaginoidin A and dihydroisoustilaginoidin A, were found to be inactive against the tested fungi19.In addition, compounds V. virens (ΔUV_2091). These monomers were elucidated as hemiustilaginoidin F (1), epihemiustilaginoidin D (2), and hemiustilaginoidin D (3) by spectroscopic analysis, and their absolute configurations were established by TDDFT ECD computations. The monomeric compounds were evaluated for their phytotoxic, cytotoxic, antibacterial and antifungal effects, and insights were gained for the structure-activity relationship by comparing the bioactivities of the monomeric and dimeric naphtho-γ-pyrones (viz. ustilaginoidins). The monomeric compounds seemed to have less phytotoxicity against rice seeds than the dimers, but with better antimicrobial effects against the tested bacteria and fungus. More structures should be evaluated for a better understanding of the relationships. In this study, neither 2-hydroxymethylated monomer nor the related metabolite was detected from the mutant strain (ΔUV_2091), thus a further investigation on the biosynthesis of ustilaginoidins was necessary and now under progress.The absolute structures of ustilaginoidins were important for studying their structure-activity/toxicity relationship, detoxification, chemical synthesis, biosynthesis, pathogenesis and so on. In the current study, the absolute structures of the ustilaginoidins, whose absolute configurations were unknown regarding the stereogenic centers, were constructed for the first time by analysis of the biosynthetic monomers obtained from a gene knockout mutant of 1H, 13C, and 2D NMR  spectra were recorded on a Bruker Avance 400 NMR spectrometer . Chemical shifts were expressed in δ (ppm) and referenced to tetramethylsilane (the inner standard), while coupling constants in Hertz. HPLC-DAD analysis of the EtOAc extracts was performed on a Shimadzu instrument equipping with a SPD-M20A photodiode array detector  using an analytic C18 column . The column temperature was set at 30 °C. The mobile phase was composed of methanol (B), and water containing 0.02% TFA (A). A gradient elution program eluting from 60% B to 100% B over 40 min was used, and flow rate was 1.0 mL/min. Semipreparative HPLC separation was done on a Lumtech instrument  equipping with a K-501 pump and a K-2501 UV detector using a Luna-C18 column , with flow rate of 3 mL/min.Optical rotations, ultraviolet (UV), and circular dichroism (CD), and infrared (IR) spectra were measured on an automatic polarimeter , UV/vis spectrophotometer , CD spectrometer , and FT-IR spectrometer , respectively. High resolution electrospray ionization mass spectrometry (HRESIMS) spectra were measured on a LC/Q-TOF-MS machine . V. virens P1 was kindly provided by Prof. Wenxian Sun . V. virens strain was cultured on YTD  medium at 28 °C. The vector pCas9-tRp-gRNA was constructed as described18. Preparation of protoplast and transformation of V. virens strains using the PEG-mediated method were performed as described20. For transformation selection, G418  was added to the medium with the final concentration of 700 μg/ml. The primers used in this study were listed in Table The wild strain 22 and analyzed with the Cas9-off program to predict potential off-targets23. The sense and antisense oligonucleotides (Supplementary Table 24. The resulting gRNA spacer was cloned between the two BsmBI sites of pCas9-tRp-gRNA by Golden Gate cloning (New England Biolabs). The 1.17-kb upstream and 1.56-kb downstream flanking sequences of 2091 were amplified with primer pairs of 1 F/2 R and 3 F/4 R, respectively, and fused with the geneticin-resistance (GenR) cassette from pFL225 by double-joint PCR. The resulting PCR products were cloned into the pCas9-tRp-2091-12 vector and then transformed into protoplasts of the wild type strain P1. G418-resistant transformants were screened for deletion of 2091 by PCR with primers 5 F and 6 R, and further verified by PCR with primer pairs 7 F/G855R and 8 R/G856F  was designed using the gRNA designer program for the best on-target scoreUV_2091 was grown on potato dextrose agar  at 25 °C for 10 days. Then, several agar plugs (5 mm × 5 mm) containing mycelia were transferred to the potato dextrose broth medium (100 ml) that was filled in a Erlenmeyer flask (250 mL). The liquid culture was incubated in a rotatory shaker for 10 days  to produce the inoculum, which was used to inoculate the rice medium . The static cultivation was performed on 1.0 kg of rice in total at R.T. in the dark, and last for one month. After harvest, the culture was extracted with EtOAc (5 L × 3) for three times. The EtOAc extract was combined, and the solvent was removed using a rotatory evaporator under reduced pressure to yield a brownish residue (2.5 g).The mutant strain Δ2Cl2-MeOH, 1:1, v/v) to obtain five fractions (Fr. A~E), among which Fr. D (381 mg) was purified by semi-preparative HPLC eluting with MeOH-H2O (0.02% TFA)  to yield compound 1 , compound 2  and compound 3 .The EtOAc extract (2.5 g) was subjected to size-exclusion chromatography over Sephadex LH-20 (CH1). Yellow amorphous powder; [α]27D + 75.6 ; UV(MeOH) λmax (log ε) 235 (4.02), 260 (4.02), 324 (3.68), 338 (3.67), 416 (3.67) nm; ECD  λ (Δε) 416(1.12), 370 (0.32), 358 (0.54), 338 (−3.26), 326 (−1.66), 312 (−2.92), 230 (11.59), 202 (−3.60) nm; 1H NMR , 13C NMR  see Table m/z 259.0611 [M-H]− .Hemiustilaginoidin F . Yellow 2). Yellow amorphous powder; [α]27D + 92.5 ; UV(MeOH) λmax (log ε) 233 (4.03), 266 (4.03), 326 (3.66), 337 (3.65), 423 (3.62) nm; ECD  λ (Δε) 424 (0.43), 362 (0.12), 342 (−0.27), 328 (−0.05), 312 (−0.40), 256 (1.15), 244 (1.07), 232 (1.23), 206 (−0.68) nm; IR νmax 3389, 2981, 1695, 1637, 1507, 1445, 1385, 1358, 1290, 1153, 1085, 994, 908, 851 cm−1; 1H NMR , 13C NMR  see Table m/z 273.0767 [M-H]− .Epihemiustilaginoidin D . Yellow amorphous powder; [α]27D + 78.5 ; UV(MeOH) λmax (log ε) 233 (4.14), 268 (4.15), 324 (3.68), 337 (3.67), 414 (3.65) nm; ECD (c = 1.09 × 10−3 M MeOH) λ (Δε) 430 (1.00), 366 (−0.11), 358 (0.14), 336 (−4.25), 324 (−1.75), 308 (−3.77), 268 (17.64), 244 (8.77), 232 (13.23), 202 (−10.15) nm; IR νmax 3394, 2980, 1699, 1631, 1582, 1498, 1430, 1385, 1343, 1132, 1092, 1068, 998, 930, 841, 730 cm−1; 1H NMR , 13C NMR  see Table m/z 273.0767 [M-H]− .Hemiustilaginoidin D (ε) 233 4.4, 268  of 0.45, 0.4 and 0.3 eV, for 1~3, respectively. The calculated ECD spectra for (2 R)-1, -2, -2, and -3 were generated by summation of the spectra of the lowest energy conformers of each structure using the Boltzmann distributions as weighting factors. The calculated ECD spectra were then compared with the experimental ECD spectra to provide information on the absolute configurations of each structure.The Molecular Merck force field (MMFF) conformational search and DFT/TDDFT calculations were performed as described previouslyLactuca sativa L. var. ramose Hort.) and rice (Oryza sativa L.) as described previously4. The experiment was performed in a 24-well plate. Briefly, five 3d-germinated rice seeds or ten 1d-germinated lettuce seeds were sown onto a well, which contained 200 μL of working solution. Compounds 1 and 3 were tested at the concentrations of 50, 100, 200, and 400 μg/mL in distilled water containing 2.5% DMSO, and experiments were done in triplicate. For comparison purpose, N-(phosphonomethyl)glycine (glyphosate) was used as the positive control. The seeds were grown in a moist chamber at 25 °C in the dark. After 2~3 days, the length of radicle and plumule of each seed was measured. The inhibition ratio (%) was calculated using following equation: [(Ls-Lt)/Ls] × 100, where Ls and Lt was the length of the solvent control and the treated, respectively. Compound 2 was not tested, because of the limited amount.The isolated compounds were tested for their phytotoxic activities against lettuce , non-small-cell lung carcinoma cells (NCI-H1650), gastric cancer cells (BGC-823), medulloblastoma cell (Daoy), and hepatocellular carcinoma cells (HepG2), using the microculture tetrazolium (MTT) assay as described previously5. Taxol was used as the positive control.Compounds 1~3 were evaluated for antibacterial activities against six human/plant pathogenic bacteria, including Bacillus subtilis ATCC 11562, Staphylococcus haemolyticus ATCC 29970, Ralstonia solanacearum ATCC11696, Xanthomonas vesicatoria ATCC 11633, Agrobacterium tumefaciens ATCC 11158, and Pseudomonas lachrymans ATCC 11921, by the modified broth dilution colorimetric assay28. The minimum inhibitory concentration (MIC) and half maximum inhibitory concentration (IC50) were determined. Streptomycin sulfate was used as the positive control.Compounds 1 and 3 were evaluated by testing against the spore germination of the rice blast pathogen Magnaporthe oryzae as described previously28.The antifungal activities of compounds Supplementary information


It could be concluded that α- and β-chitosan nanoparticles had superior superoxide radical scavenging abilities than raw chitosan samples.α- and β-Chitosan nanoparticles were obtained from shrimp shell and squid pen chitosan with different set of deacetylation degree (%DD) and molecular weight (MW) combinations. After nanoparticle formation via ionic gelation with sodium tripolyphosphate (TPP), the % crystallinity index (%CI) of the α- and β-chitosan nanoparticles were reduced to approximately 33% and 43% of the initial %CI of the corresponding α- and β-chitosan raw samples, respectively. Both forms of chitosan and chitosan nanoparticles scavenged superoxide radicals in a dose-dependent manner. The %CI of α- and β-chitosan and chitosan nanoparticles was significantly negatively correlated with superoxide radical scavenging abilities over the range of concentration  studied. High %DD, and low MW β-chitosan exhibited the highest superoxide radical scavenging activity ( Chitosan is a derivative of chitin, which is the second most abundant polymer in Nature after cellulose ,2,3,4. TIt has been reported that the amino and hydroxyl groups on chitosan backbone represent target moieties for chemical modifications to improve the aqueous solubility of chitosan since it is soluble only in an aqueous acidic solution, which is a major hurdle for its application . The uniThe objective of this study was thus to investigate the effects of crystalline structural differences between α- and β-chitosan on their nanoparticle formation via ionic gelation and their superoxide radical scavenging activities. α-and β-Chitosan nanoparticles were obtained from shrimp shell and squid pen chitosan with different sets of %DD and MW combinations, achieved by controlling the factors such as reaction conditions , and extent of the reaction. Physicochemical characteristics of α- and β-chitosan nanoparticles, prepared from corresponding raw α- and β chitosan samples, including particle size, zeta potential, and x-ray diffraction pattern were determined in comparison.N-Acetyl-d-glucosamine, sodium tripolyphosphate (TPP), phenazine methosulfate (PMS), β-nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium, and α-tocopherol were purchased from Sigma Chemical Co. . Acetic acid, hydrochloric acid (HCl) and sodium hydroxide (NaOH) were obtained from VWR . Sodium chloride (NaCl), di-potassium hydrogen orthophosphate, and potassium dihydrogen orthophosphate were purchased from Ajax Finechem . All the chemicals were of analytical grade.Dried shrimp shell and squid pen were obtained from a local food processing plant . w/v) for 2 h at 60 °C and then deproteinized with 2 M NaOH at a ratio of dried shrimp shell to NaOH 1:20 (w/v) for 2 h at 60 °C, and oven dried at 60 °C. Dried squid pen was deproteinized with 2 M NaOH at a ratio of dried squid pen to NaOH 1:20 (w/v) for 2 h at 60 °C, and oven dried at 60 °C.α-Chitin and β-chitin were prepared from dried shrimp shell and squid pen, respectively. Dried shrimp shell was demineralized with 1.0 M HCl at a ratio of 1:20 (w/w) at a range of dried shrimp shell to NaOH ratio 1:30 to 1:20 (w/v), temperature 120–126 °C and the batch process was repeated 1–4 times, resulting in three α-chitosan products with different %DD and MW combinations, that is, aCS1, aCS2, and aCS3, where: a = α-chitosan, CS = chitosan, 1 = low %DD and high MW, 2 = medium %DD and medium MW, and 3 = high %DD and low MW. β-Chitin was deacetylated with 36–50% NaOH (w/w) at a range of dried shrimp shell to NaOH ratio 1:30 to 1:20 (w/v), temperature 120 °C and the batch process was repeated 1–4 times resulting in three β-chitosan products with different %DD and MW combinations, that is, bCS1, bCS2, and bCS3, where: b = β-chitosan, CS = chitosan, 1 = low %DD and high MW, 2 = medium %DD and medium MW, and 3 = high %DD and low MW.α-Chitosan and β-chitosan were prepared from α-chitin and β-chitin respectively. α-Chitin was deacetylated with 50% NaOH (w/v) acetic acid to obtain 1 mg/mL solution, and the pH was adjusted to 4.8 by 2.0 N NaOH. TPP was dissolved in distilled water to obtain a 1 mg/mL solution. Chitosan nanoparticles were assembled when TPP solution was added dropwise into the chitosan solution at a mass ratio of 5:1 under magnetic stirring (750 rpm) at room temperature. The polymer to crosslinker (TPP) ratio of 5:1 was chosen following preliminary experiment results. Ionic gelation of α-chitosan with TPP resulted in three types of α-nanoparticles, that is, aNP1, aNP2, aNP3, where: a = α-chitosan, NP = nanoparticles, 1 = low %DD and high MW, 2 = medium %DD and medium MW, and 3 = high %DD and low MW. Ionic gelation of β-chitosan with TPP resulting in three types of β-nanoparticles, that is, bNP1, bNP2, bNP3, where: b = β-chitosan, NP = nanoparticles, 1 = low %DD and high MW, 2 = medium %DD and medium MW, and 3 = high %DD and low MW.Chitosan nanoparticles were obtained via ionic gelation of chitosan with TPP by a modification of the method of Antoniou et al. . BrieflyN-acetyl-d-glucosamine in 0.01 M acetic acid. The height, H, was measured from the ZCP to the first derivative spectra of the standard solutions. The %DD of chitosan was obtained by scanning absorbance spectra of 0.1 mg/mL chitosan in 0.01 M acetic and the concentration of N-acetyl-d-glucosamine was determined and the %DD was then calculated.The %DD of chitosan samples were determined by the first derivative UV spectrophotometry method of Kiang et al. . Firstlysp/C) against concentration , extrapolating to zero concentration. The MW was assessed using the Mark–Houwink relationship given in Equation (1):−3 and a = 0.93.The MW of chitosan samples were determined according to the method of Jung and Zhao . ChitosaI110 is the maximum intensity of the lattice diffraction pattern at 20° and Iam, the intensity of amorphous diffraction at 16°.X-ray diffraction (XRD) patterns of chitosan and chitosan nanoparticle powder were achieved by X-ray diffractometry  at 40 kV, 40 mA and 2θ with the scan angle from 5° to 60° as described previously . The cryThe particles size, PDI, and zeta potential of chitosan nanoparticles were done at 25 ± 0.5 °C by dynamic light scattering (DLS) technique using a Zetasizer .The superoxide scavenging activity of chitosan and chitosan nanoparticles was assessed by the modified method of Xing et al. . The mixAll data are expressed as means ± standard deviation (SD) from three replicates. One way analysis of variance (ANOVA) were performed, followed by Duncan′s multiple range test for mean comparison. Pearson′s correlation coefficients (r) was used to determine the statistical relationship between two variables.p < 0.05) rather than the chitin crystalline structure. The %DD of α- and β-chitosan were in the range of 82.54–96.16% and 80.12–95.93%, respectively. In general, chitosan product %DD values between 56% and 99%, with an average of 80%, have been reported, dependent on the deacetylation process conditions [p < 0.05) rather than the chitin source of origin. The increase in deacetylation process parameters such as concentration, ratios of chitin to alkali, temperature) and extent of the reaction resulted in lower MW of chitosan end products, accordingly [3) kDa and 0.7–1.5 (×103) kDa, respectively, similar to the reported MW of shrimp shell chitosan of 0.95 to 4.47 (×103) kDa [p < 0.01). It is known that the scission of acetyl moieties occurred at the same time as a reduction of parent chain length during the deacetylation process resulting in a higher %DD of lower MW chitosan products [The %DD and MW of α- and β-chitosan samples with different sets of %DD and MW combinations are illustrated in nditions . The incproducts .d-spacing, and relative intensity  of various planes  of the chitosan and chitosan nanoparticles are summarized in 3” of chitosan), 19°–20° (amine II “–NH2” of chitosan), and a shoulder in 21°, in agreement with previous reports, which correspond to the (110), (020), and (120) planes, respectively [The XRD patterns of chitosan and chitosan nanoparticles are illustrated in ectively ,24,29,33ectively ,34. Simiectively , due to ectively . It was ectively ,23,33.d-spacing values of the N-acetyl-d-glucosamine portion and N-glucosamine portion were in agreement with previous reports [It is known that the crystalline structure of chitosan is destroyed by ionic cross-linking interactions between the amino groups on chitosan and TPP ,30. Depo reports ,36.p < 0.05), as it is known that %CI of α-chitin is higher than β-chitin [p < 0.05), which is in agreement with previous findings [p ≥ 0.05), except for the aNP2 sample which showed little variation. To some extent, the %CI of α- and β-chitosan was positively correlated with the %DD of chitosan, while it was negatively correlated with MW, similar to a previous report [As shown in β-chitin ,8. The %findings . It is os report . The reds report .The particle sizes of chitosan nanoparticles formed via ionic gelation are demonstrated in p < 0.01). The higher %DD of chitosan resulted in the bigger particle size of either α- or β-chitosan nanoparticles. This suggested that the particle size could be related to the −NH2 groups available in the chitosan polymer chains. Higher %DD chitosan may indicate a stronger protonation of the –NH2 moiety leading to stronger molecular repulsion, which made the chitosan polymer chain stretch larger and resulted in a larger particle size [The sizes of α- and β-chitosan nanoparticles were in the range of 165 to 332 nm and 170 to 232 nm, respectively. cle size .p < 0.01), but negatively correlated with %DD  as seen in p < 0.05) as seen in The PDI values of α- and β-chitosan nanoparticles were in the range of 0.168–0.314 and 0.098–0.248, respectively. In case of nanoparticles, a PDI below 0.3 is desired, since values higher than 0.3 indicate low uniformity, being an indication of aggregation . Figure p < 0.01, p < 0.01), as seen in The zeta potentials of α- and β-chitosan nanoparticles were in the range of 23.07–26.33 and 24.83–30.57 mV, respectively. The particle sizes of aNP2 and bNP2 were the smallest, with PDIs lower than 0.3 indicating that there might be an optimal %DD and MW for nanoparticle formation. It is clear that the %DD, and MW of chitosan exerted combined effects on the chitosan nanoparticle characteristics .p < 0.05) and β-chitosan at the highest %DD, that is, bCS3 sample, showed the highest ability to eliminate superoxide radicals. (p < 0.05). Superoxide radical is a zwitterionic radical, which could react with free hydroxyl and amino groups in chitosan, then superoxide radical was scavenged by this reaction [The superoxide radical scavenging activities of α- and β-chitosan and chitosan nanoparticles at concentrations of 0.5, 1, 2 and 3 mg/mL are given in reaction . It coulreaction ,40. Highreaction .p < 0.05). This may result from the higher zeta potential (p < 0.05) as seen in p < 0.05). This may result from the lowest% CI, and smallest particle size (p < 0.05) of the aNP2 sample shown in The superoxide radical scavenging activities of α- and β-chitosan nanoparticles are illustrated in In addition, the superoxide scavenging activities of α- and β-chitosan a were siThe effects of crystalline structure in term of %CI on superoxide radical scavenging activities of α- and β-chitosan and α- and β-chitosan nanoparticles were estimated by the Pearson′s correlation coefficients presented in The highlight of this work was the preparation of α- and β-chitosan nanoparticles from shrimp shell and squid pen chitosan products with different sets of molecular weight (MW) and deacetylation degree (%DD) combinations in order to relate their different crystalline structures to their antioxidant properties on superoxide radicals. The effects of crystalline structural differences between α- and β-chitosan on their nanoparticle formation via ionic gelation were obvious, in terms of %CI, that is, a decrease in %relative intensity of the (110) and (020) planes of chitosan nanoparticles and a shift to the (120) plane reflected that native α- and β-chitosan successfully formed α- and β-chitosan nanoparticles. The characteristics of these chitosan nanoparticles, such as particle size, PDI, zeta potential, and %CI were strongly affected by not only the chitin sources of origin, but also the chitosan raw sample characteristics, such as %DD, and MW. α- and β-Chitosan samples and α- and β-chitosan nanoparticles scavenged superoxide radicals in a concentration-dependent manner. β-Chitosan with higher %DD, and lower MW showed higher superoxide radical scavenging ability. This study suggests that chitosan nanoparticles, either in the α- or β-form, with small particle size, and high zeta potential could be preferentially obtained from chitosan with defined %DD, and MW, in order to achieve high superoxide radical scavenging ability. This study also suggested that more attention should be paid on %DD and MW and chitin source of raw chitosan samples in order to achieve rationally designed nanoparticles.