Moreover, we observed that the more unsaturated the hydrocarbon tail, the higher the CC50s in all head groups. High CC50s were also found in HepG-2, THP-1, and HEK-293 cell lines where cardol triene CC50s were 140.27 ± 8.44, 129.77 ± 12.08, and 92.80 ± 3.93 µM, respectively. Cardol triene expressed pan-dengue inhibition with the EC50s of 5.35 to 8.89 µM and kl loops of dengue envelope proteins were major targets. The strong binding energy at T48, E49, A50, P53, K128, V130, L135, M196, L198, Q200, W206, L207, I270, and L277 prevented cellular pH-dependent fusion. Zika virus kl loops were aligned in the closed position preventing cardol triene to bind and inhibit fusion and infectivity. This study showed for the first time that cardol triene had a potential for further development as anti-dengue inhibitors.Dengue virus causes a global burden that specific chemotherapy has not been established. A previous report suggested that anacardic acid inhibited hepatitis C virus infection. Here, we explored structure activity relationship of anacardic acid, cardanol, and cardol homologues with anti-DENV cellular infectivities. Cardol triene showed the highest therapeutic index at 29.07 with the CC Of these statistics, Asia represented approximately 75% of the total reports2. Dengue virus belongs to the family Flaviviridae, genus Flavivirus, and consists of four serotype (DENV1-4). The infectious virion contains lipid envelope surrounding an icosahedral nucleocapsid. The nucleocapsid contains an 11 kilobases, single stranded, positive sense RNA. Envelope (E) protein is an immunogen and a major antigenic cross-reactive agent3. All serotypes of dengue viruses are transmitted by Aedes mosquitoes2. Severe clinical manifestations including plasma leakage, hemorrhage, and shock are caused by robust but incompetent immunological responses to secondary heterotypic infection5. Remarkably, the antibodies responding to those secondary heterotypic infection are unable to perform complete neutralization and the antibody-tagged viruses are opsonized by macrophage. The viral replication is enhanced inside the cell resulting in increasing the risk to severe dengue development6. Moreover, it was clearly showed that the correlation existed between high viral load, prolonged viremia and clinical severity8. Therefore, reducing the viral load should be a direct means to treat acute dengue infection in order to reduce the progression to severe clinical outcomes.Dengue infection causes a burden over worldwide tropicals with 390 million infections per year13 but none of them has been launched as a licensed drug yet. Natural products derived from medicinal plants are rich source of potential antiviral inhibitors14. Phenolic lipids are amphiphilic molecules containing phenolic rings and long aliphatic chains. Recently, accumulated evidences suggested that phenolic lipids showed a broad spectrum antimicrobials15, and anti-cancer16 activities by disturbing the phospholipid membrane17 and inducing apoptosis18. One of the major sources of phenolic lipids was cashew nut shell liquid (CNSL) existing as mixture of anacardic acid, cardanol, and cardol head groups and saturated (C15:0), monoene (C15:1), diene (C15:2), and triene (C15:3) tails. There is a previous study describing saturated anacardic acid (C15:0) as an inhibitor of hepatitis C virus by interfering with the viral entry, translation, replication, and release19. Dengue virus and hepatitis c virus are likewise members of the family Flaviviridae, therefore it is possible that phenolic lipids should also effectively inhibit dengue and Zika viruses. Since there is limited knowledge of phenolic lipids as antivirals, we aimed to characterize the efficacies of all homologues of CNSL-derived phenolic lipids for dengue virus inhibition. Moreover, the potential candidates would be studied for activities against dengue virus serotype 1–4 and Zika virus; as well as cytotoxicity to human-derived cell lines. The mechanism of drug action and molecular target would also be explored to bring insights into further lead optimization on potential targets.Antiviral drug discovery and development have been substantially advanced within a few decades. Five active leads have entered the clinical trialsGingko biloba inhibited several steps of hepatitis C virus replication19. Hepatitis C virus is one of the members of the family Flaviviridae, therefore, it is likely that anacardic acids, cardanol, and cardol should inhibit dengue virus replication. We extracted phenolic lipids from cashew nut shell liquid (CNSL) and primarily characterized by the three head groups into anacardic acid, cardanol, and cardol . Cardanol triene was insoluble in DMSO, therefore the compound was excluded from this study. It could be inferred that the polyunsaturated hydrocarbon may involve in stabilizing the cell membrane. Moreover, CC50s of the mixtures tend to follow those of the saturated homologues suggesting that the presence of saturated homologues, regardless of the proportion, could dominate the cell membrane disruption. Moreover, we further explored the CC50 of cardol triene in other cell lines to verify its protective effect. Three human-derived cell lines  were used to co-incubate with cardol triene and cardol mixture (73% diene and 27% triene). Results showed that CC50s of cardol triene were about 30–50 µM higher than cardol mixture in all cell lines , monoene (C15:1), diene (C15:2), and triene (C15:3) as described in materials and methods. Toxicities were analyzed and results were calculated to percent viability and CC5:0, monones Fig. . In conc50) of the phenolic lipid homologues against DENV2 infectivity in Vero cells. Briefly, compounds at 10 different concentrations were added to DENV2-infected Vero cells (M.O.I. of 0.1) during and post infection and the viral inhibition was determined by plaque titration. Efficacies of anacardic acids varied among homologues so that the structure-activity relationship was inconclusive. The most efficient anacardic acid was the saturated homologue and its result was similar to that of the mixture although only 18% of saturated anacardic acid was found in the total mixture. It is possible that the viral inhibition and cellular response might be predominately influenced by saturated anacardic acid regardless of the percent constituent. Noted that anacardic acids were major components of CNSL extracts and all homologues were purified directly from CNSL itself. However, CC50s of anacardic acids were generally lower than those of cardols  to 5.89 ± 2.83 µM. However, cardanols were the most cytotoxic of all head groups, thus resulted in the least TI values. The group were then excluded from further investigations.Naturally derived cardanol mixture consisted of monoene, diene, and triene hydrocarbon species. The saturated cardanol was later synthesized by hydrogenation. Overall, the efficacy of cardanols were less potent than those of anacardic acids and cardols Table . One of 50 of 3.24 ± 0.51 µM, and therapeutic index (TI) of 18.63  of 29.07 , DENV3 (16562), DENV4 (c0036), and ZIKV (sv0010/15) Fig.  suggeste20. Based on the previous efficacy results, cardol triene was chosen as a representative of all CNSL-derived phenolic lipids. Cardol triene was prepared in DMSO and added to the final concentrations of 10 µM and 20 µM to DENV2 infected Vero cells (M.O.I. of 0.1). The compound was added to the cells at different time-points. DMSO alone was added to the infected cells as a mock treatment. After 72 h incubation, cell lysates and supernatants were collected for analysis of intracellular RNAs and infectious virions by RT-qPCR and plaque titration, respectively. Results showed that intracellular RNAs  proteins were first tested using blind docking technique. The cardol triene was preliminarily docked into DENV and ZIKV E dimers in order to detect the differences of ligand binding sites Fig. . Pairwis21 by SIE method  methods were used to corroborate the binding energy of cardol triene and the kl hydrophobic pockets. The binding energy was achieved from 200 ns-trajectories and delicately computed at K and K’ sites at 297 and 310 K using previously described methods , 128–135 , 191–207 , and 270–281   was slightly more fluctuated than that of the 297 K (2.91 Å)  Fig. . Noted t77) Fig. . However77) Fig. 22–24.Since dengue envelope protein was the key factor to receptor-binding, endocytosis, and fusion, we asked the question whether the compound would mainly neutralize the virion, inhibit the receptor binding, or inhibit endosomal fusion. Cardol triene at 10 µM was then co-incubated with DENV2 before, during, and after infection to Vero cells Fig. . Results27 of DENV and ZIKV were used to confirm the findings. Briefly, cardol triene at 10 µM was added to DENV2 or ZIKV  and synthesized by hydrogenation. The viral inhibition was analyzed and cardol triene was the most effective compound in this group considering from the highest CC50 and TI values. Cardol triene was also similarly effective against the other DENV but no inhibition was found in Zika virus. Moreover, cardol triene was observed with high CC50 values in three human-derived cell lines; HepG2, HEK-293, and THP-1. One of the molecular targets was the kl hydrophobic pockets that would undergo conformational change under acidic pH and performed endosomal fusion to release the viral genome. Our findings showed for the first time that CNSL-derived phenolic lipids had a potential for further development as anti-dengue inhibitors.Phenolic lipids were reported as active ingredients of plants in the 32. Cardol triene was structurally similar to β-OG, the crystalized ligand, and similar binding moieties were identified. Additional structure-activity relationship (SAR) study should focus on optimizing the phenolic lipids into this binding pocket. A previous report on mannoside glycolipid conjugates suggested that the C24 hydrocarbon tail, either saturated or unsaturated, was active DENV2 inhibitor33, whereas the shorter C21 and C19 did not show any inhibition. Therefore, it is interesting to explore whether the tail length (C15-C25), and level of unsaturation would reflect the binding efficiency. Further investigations should also include biophysical experiments such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) to verify the MD simulation results. Moreover, saturated anacardic acid (C15:0), which inhibited DENV2 at the EC50 of 4.82 ± 1.71 µM , with the EC50 of 7.25 µM19. However, cardol triene did not inhibit ZIKV although the virus was closely related to DENV. Based on the findings, we speculated that the similar efficacies of DENV and HCV might be a coincidence of the compound targeting different molecules. Indeed, the viral inhibition (EC50) was a summary of hundreds of viral activities and virus-host interactions.Since all homologues showed similar efficacies against DENV2 at the kl loops Table , Fig. 5A50 of 207.3 ± 5.24, 140.27 ± 8.44, 129.77 ± 12.08, 92.8 ± 3.93 µM, respectively , KATO-III (CC50 of 19.06 ± 0.39 µM), HepG2 (CC50 of 2.23 ± 0.22 µM), Chago I (CC50 of 2.55 ± 0.18 µM) and BT474 (CC50 of 13.46 ± 0.14 µM) cell lines35. Also, the saturated anacardic acid was cytotoxic against K562 and AGS cell lines with the CC50s of 25.4 µM and 41.6 µM, respectively36. Therefore, the saturated and monoene compounds were suitable for further development as anti-cancer chemotherapy. Moreover, we noticed that the increasing unsaturated hydrocarbon tails positively correlated to the higher CC50 values , purified according to a previously reported protocol41 and characterized by 1H NMR (CDCl3) spectroscopy. The products were analyzedas anacardic acid, cardanol, and cardol, 84, 2, and 0.1 percent, respectively. Briefly, 5% aqueous MeOH (300 mL)  was used to dissolve CNSL (50 g). Ca(OH)2 (50 g)  was gradually added to the reaction mixture while stirring at 50 °C for 5 h. The reaction was monitored by Kieselgel 60 PF254 silica-based thin layer chromatography (TLC) . After 5 h reaction, dark brown calcium anacardate was precipitated which was then filtered and washed with MeOH before drying under vacuum evaporator. The product was suspended in HCl solution  and continuously stirred for 1 h before extraction with EtOAc . The organic layer was collected and washed with water, and subsequently dried over anhydrous Na2SO4 . Finally, the product was concentrated under reduced pressure to yield anacardic acid . The leftover filtrates from calcium anacardate precipitation was also evaporated under reduced pressure and subsequently extracted using EtOAc. The organic layer was recovered and dried over anhydrous Na2SO4 before concentration under reduced pressure. The crude product was further purified by silica column with hexane/EtOAc elution to cardanol  and cardol . Each species was further separated into pure homologues of saturated (C15:0), monoene (C15:1), diene (C15:2), and triene (C15:3) using semi-preparative high performance liquid chromatography. The characteristics and signals of all compounds were shown as follows;Phenolic lipids were extracted from cashew nut shell liquid (CNSL) (1H NMR(CDCl3) δH (ppm): 11.01 , 7.36 , 6.87 , 6.78 , 2.98 , 1.59 , 1.26 , and 0.88 .Anacardic acid C15:0 : white solid  1H NMR (CDCl3) δH (ppm): 11.04 , 7.35 , 6.86 , 6.77 , 5.35 , 2.97 , 2.01 , 1.60 , 1.29 , and 0.88 .Anacardic acid C15:1 (6-[8(Z)-pentadecenyl]salicylic acid): yellow liquid  1H NMR (CDCl3) δH (ppm): 11.07 , 7.35 , 6.86 , 6.77 , 5.37 , 2.97 , 2.77 , 2.01 , 1.33 , and 0.90 .Anacardic acid C15:2 (6-salicylic acid): yellow liquid  1H NMR (CDCl3) δH (ppm): 11.09 , 7.35 , 6.86 , 6.76 , 5.81 , 5.39 , 5.01 , 2.97 , 2.79 , 2.05 , 1.59 , and 1.34 .Anacardic acid C15:3 (6-salicylic acid): yellow liquid  1H NMR (CDCl3) δH (ppm): 7.14 , 6.75 , 6.65 , 5.35 , 2.55 , 2.01 , 1.59 , 1.29 , and 0.89 .Cardanol C15:1 (3-[8(Z)-pentadecenyl]phenol): yellow liquid  1H NMR (CDCl3) δH (ppm): 7.14 , 6.75 , 6.65 , 5.36 , 2.78 , 2.55 , 2.04 , 1.58 , and 0.91 .Cardanol C15:2 (3-phenol): yellow liquid  1H NMR (CDCl3) δH (ppm): 7.14 , 6.76 , 6.65 , 5.83 , 5.40 , 5.03 , 2.82 , 2.56 , 2.04 , 1.60 , and 1.29 .Cardanol C15:3 (3-phenol): yellow liquid  1H NMR (CDCl3) δH (ppm): 6.24 ,6.17 ,5.36 , 2.78 , 2.48 , 2.04 , 1.56 , 1.33 , and 0.91 .Cardol C15:2 (5-resorcinol): brown liquid  1H NMR (CDCl3) δH (ppm): 6.25 , 6.17 , 5.82 , 5.39 , 5.02 , 2.80 , 2.46 , 2.04 , 1.55 , and 1.28 .Cardol C15:3 (5-resorcinol): brown liquid  In addition, the saturated homologues (C15:0) of cardanol and cardol were synthesized from hydrogenation reaction and their signals were shown as follows;1H NMR (CDCl3) δH (ppm): 7.15 , 6.77 , 6.68 , 5.91 , 2.56 , 1.60 , 1.30 , 0.91 .Cardanol C15:0: 1H NMR (CDCl3) δH (ppm): 6.24 , 6.18 , 2.47 , 1.55 , 1.25 , 0.87 .Cardol C15:0: All compounds were stored as solid powder at room temperature. The compounds were prepared in dimethyl sulfoxide (DMSO)  to 50 mM stock solutions and stored as aliquots at −20 °C until use.®CCL-81), LLC/MK2 (ATCC® CCL-7), C6/36 (ATCC® CRL-1660), HEK-293 (ATCC® CRL-1573), HepG2 (ATCC® HB-8065), and THP-1 (ATCC® TIB-202) cells were maintained as previously described42. Also, DENV1 (16007), DENV2 (New Guinea C strain), DENV3 (16562), DENV4 (c0036), and Zika virus (SV0010/15) reference strains were propagated in Vero and C6/36 cells as previously described42.Vero (ATCC4) cells were seeded into each well of 24-well plates. After an overnight incubation, cells were infected with DENV2 (M.O.I. of 0.1) and 10 µM and 25 µM phenolic lipids. DMSO at 1% was used as a mock treatment with 100% infectivity. The compounds were co-incubated with cells 37 °C for 3 days before supernatants collection. Viral titers were evaluated by plaque titration43.The efficacies of anacardic acid, cardol, and cardanol mixtures were primarily studied using DENV2 infected Vero cells. Vero (5 × 1050). Vero (5 × 104) cells were seeded, and infected with DENV2 (M.O.I. of 0.1) as previously described42. Phenolic lipid mixtures and homologues were serially diluted in DMSO to the final concentrations as follows; 0, 0.5, 1, 1.5, 2, 2.5, 5, 7.5, 10, 25, and 50 µM. DMSO at the concentration of 1% was used as a mock treatment referring to 100% infection. Infected cells were treated with the designated compounds during and post infection. Supernatants were collected at 3 days after infection for analysis of virion production by plaque titration. Non-linear regression analysis was used to determine the effective concentrations. Three independent experiments were performed and results were as means and standard error of the means (SEM). The therapeutic index (TI) was a ration of CC50/EC50 of the compound.Next, each homologue was analyzed for effective concentration (EC4) cells seeded into each well of 96-well plates. Phenolic lipids at 10 µM and 25 µM were added to the experimental wells after an overnight incubation. DMSO at 1% was used as a control referring to 100% cells viability. Cell viability was accessed at designated time-points using CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit  and analyzed by spectrophotometer (A450nm) . Results were calculated to percent cell death.The primary screening was performed using Vero (1050) were also studied using Vero cells. Briefly, the 104 cells were seeded into each well of 96-well plates. After an overnight incubation, phenolic lipids were serially diluted in DMSO to the final concentrations as follows; 25, 50, 100, 200, 300, 400, and 500 µM. Each concentration was performed in quadruplicates. DMSO at 1% was used as a mock treatment referring to 100% cell viability. Cells were incubated for 48 h before cell viability was accessed using CellTiler 96® Aqueous One Solution Cell Proliferation Assay kit . Non-linear regression analysis was used to determine cytotoxic concentrations (CC50) of each experiment. Results were derived from three independent experiments and reported as means and standard error of the means (SEM).Cytotoxic concentrations (CC4) cells were seeded into each well of 24-well plates. cells were infected with DENV2 (M.O.I. of 0.1) after an overnight incubation45. Cardol triene (C15:3) at 10 and 20 µM were added to the cells during (0 h) and after infections at early time-points as follows; 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h. Moreover, late time-points after were also accessed at 12, 14, 16, 18, 20, 22, 24, 36, 48, 60, and 72 h after infections. DMSO alone was added to the infected cells at respective time-points as a mock treatment referring to 100% infectivities. After 72 h incubation, cell lysates and supernatants were collected for analysis of intracellular RNAs and infectious virions by RT-qPCR and plaque titration, respectively. Results were confirmed by three independent experiments. Data were calculated, assembled, and reported as percent intracellular RNAs and infectious virions under the drug treatment at designated time-points.Vero (5 × 104) cells were seeded into 24-well plates as previously described. DENV2 (M.O.I. of 1) was diluted in ice-cold MEM with 1% fetal bovine serum42 and was allowed to adsorb on the cells for 1 h with continuous rocking. Cardol triene (10 µM) was prepared in DMSO and was mixed with DENV2 (M.O.I. of 1) before adsorption for 1 h (pre-attachment). Cardol triene (10 µM) was also mixed with DENV2 (M.O.I. of 1) and immediately applied to the cells (co-attachment). Last, cardol triene was added to the cells after 1 h DENV2 adsorption (post-attachment). The experiments were incubated for 2 days at 37 °C. Intracellular viral RNA was determined from cell lysate using RT-qPCR and infectious virion was determined using plaque titration of the supernatant. DMSO was added to the experiment as a no-inhibition control. Means and standard error of the means (SEM) of three independent experiments were noted.Vero (5 × 105) cells were seeded into 24-well plates before infection with DENV2 and ZIKV (M.O.I. of 1) as described42. Cardol triene at 10 µM were introduced to the DENV2 and ZIKV infected cells during and after infection. DMSO was used as a 0% inhibition control and mouse anti-Flavivirus envelope protein antibody (4G2) was used as a 100% inhibition control. Cells were incubate at 28 °C for 2 days before addition of 0.5 M 2-(N-morpholino) ethanesulfonic acid (MES) (pH 5.0) . Moreover, cells from selected wells subsequently underwent DAPI staining. Cell-cell fusion was observed under brightfield and fluorescence using an Eclipse TS100 Inverted Routine Microscope .C6/36 (2 × 104) cells expressing DENV-2 replicon (BHK-21/DENV2)44 were seeded as previously described. Cardol triene at 10 µM and 20 µM, DMSO, and ribavirin  were added to the replicon cells and incubated at 37 °C for 3 days. Cells lysates were collected and viral RNA was quantified by RT-qPCR as previously described44. Data were reported as percent inhibition compared with ribavirin. Three biological replicates were performed and results were means and SEM of all replicates.BHK-21  using Molecular Property Explorer and RTECS database.Phenolic lipids were evaluated for druglikeness by scoring their pharmacological characters (cLogP and solubility)47) was obtained to calculate the interaction energy with phenolic lipids using CDOCKER software . Each kl loop was assigned to 10 Å diameter according to n-octyl-beta-D-glucoside (β-OG) originally bound in the crystal structure48. All phenolic lipids were calculated for partial charge distribution and docked into kl loop with 100 replicas with random conformation of ligand. The best score predicted by CDOCKER interaction energy was selected to construct an initial complex structure for molecular dynamics (MD) simulation using 3 different methods; Solvated Interaction Energy (SIE), Molecular Mechanics-generalized Born/Poisson Boltzmann solvent accessible surface area (MM-GB/PBSA), and Quantum mechanics/molecular mechanics generalized Born surface area (QM/MM-GBSA) with density functional theory-based tight binding (SCC-DFTB) methods.To refine the screening result and construct the initial complex structure, the 3D DENV envelope protein  followed by 1,500 steps of conjugated gradients (CG) in the SANDER module by AMBER 16 were used to minimize the interference of water molecules and hydrogen atoms. The systems were subsequently brought up to 297 K and 310 K within 200 ps and was stabilized at the designated temperature until 300 ns. The MD trajectories were collected every 0.2 ps during 200–300 ns production phase56. The stability of the cardol triene/DENV E protein complex at equilibrium was analyzed for the global root-mean-square displacement (RMSD) of binding free energy using CPPTRAJ module. Snapshots, taken every 1 ns within 200–300 ns-MD trajectories, were used as materials for calculating total binding free energy using SIE, MM-GB/PBSA, and QM/MM-GBSA (SCC-DFTB) methods58. Snapshots were also used to identify the per-residue decomposition energy contribution by MM/GBSA method59.The MD simulations were conducted for cardol triene/DENV E protein complex with two temperature systems; 297 K (23.85 °C) and 310 K (36.85 °C) implemented with the GPU accelerated pmemd.cuda program in the AMBER16 package programThis project was approved by Ethical Review board, Faculty of Medicine, Chulalongkorn University (COE 021/2017 and IRB 375/60) as exempted in compliance with the International guidelines for human research protection as Declaration of Helsinki, The Belmont Report, CIOMS guideline, International Conference on Harmonization in Good Clinical Practice (ICH-GCP) and 45CFR 46.101(b).We performed all pathogen-related experiments in bio-containment level 2. BSL-2 standard operating procedure was performed under the regulation of ISO15189 and ISO15190.Supplementary Information


The optimal rabbit anti-thymocyte globulin (rATG) graft-versus-host disease (GVHD) prophylaxis regimen in matched sibling donor peripheral blood stem cell transplantation (MSD-PBSCT) remains to be elucidated. In this prospective study, we used low-dose rATG for GVHD prophylaxis in patients or donors aged ≥ 40 years with hematological malignancies receiving MSD-PBSCT. rATG was administered to 40 patients at an intravenous dose of 5 mg/kg divided over day 5 and day 4 before graft infusion. No graft failure occurred. Median times to leukocyte engraftment and platelet engraftment were 11.0 days and 13.9 days. The cumulative incidence of grades 2–4 and grades 3–4 acute GVHD at day +100 was 30.0% and 2.6%. The 2-year cumulative incidence of extensive chronic GVHD and severe chronic GVHD was 11.4% and 14.7%. 93.5% (29/31) of patients had discontinued immunosuppressive medication within 3 years after transplantation. The 2-year cumulative incidence of transplant-related mortality (TRM) and relapse was 14.0% and 22.6%. The cumulative incidence of cytomegalovirus reactivation, Epstein–Barr virus reactivation, and fungal infection was 22.3%, 12.9%, and 12.5%. Kaplan–Meier estimates for overall survival, disease-free survival, and GVHD-free and relapse-free survival 3 years after transplantation were 68.9%, 68.9%, and 54.0%. rATG for GVHD prophylaxis is tolerable and efficacious at a 5 mg/kg total dose administered over 2 days (days −5 to −4) in patients receiving allogeneic MSD-PBSCT. P = 0.017) and 5.8% (P = 0.049) in patients receiving stem cells from haploidentical donors  patients), Epstein–Barr virus reactivation (20.0% [8/40] patients), and fungal infection (12.5% [5/40] patients). No post-transplantation lympho-proliferative disorder was recorded (Table P = 0.225)  of the 40 patients who received allogeneic MSD-PBSCT. Among these, no patients died of GVHD, 3 patients died of pneumonia and respiratory failure, and 1 patient died of gastrointestinal hemorrhage before engraftment. The 6-month, 1-year, and 2-year cumulative incidence of TRM in the ATG group was 5.8% , 9.5% , and 14.0% , respectively Fig. . The 2-y25) Fig. .Fig. 3CuP = 0.120; Fig. In the ATG group, 5 patients (12.5%) who received allogeneic MSD-PBSCT relapsed at a median of 428 93–682) days after transplantation. The 6-month, 1-year, and 2-year cumulative incidence of relapse was 6.8% , 18.1% , and 22.6% , respectively Fig. . Of the –682 daysIn the ATG group, median follow-up after transplantation among survivors was 861 (271–1622) days. At the time of analysis, 31 (77.5%) patients were still alive in CR. Kaplan–Meier estimates of OS 1 year, 2 years, and 3 years after transplantation were 93.1% ,73.1% , and 68.9%   for patients in the ATG group and 50.2%  for patients in the no-ATG group , 189 (63–488), 686 (483–1355), and 138 (75–250), respectively. CD4+ cell counts were > 200/μL at day +120 and reached 330/μL on day +365. There was no association between CD3+, CD4+, CD8+, and CD56+ cell counts at 1, 2, 3, 6, and 12 months and relapse, the occurrence of GVHD, CMV/EBV reactivation, or TRM.In the ATG group, median lymphocyte counts, stratified into CD3+, CD4+, CD8+, and CD56/CD16+ counts were 1600 (768–2137) and 914 (642–1465), 210 (201–274) and 189 (63–488), 878 (290–1490) and 686 (483–1355), and 315 (111–546) and 238 (75–350)/μL, respectively. There were no differences between the two groups.On day +100 in the no-ATG and ATG groups, median CD3cGVHD is the leading cause of non-relapse morbidity and mortality after allogeneic PBSCT . StrategOptimizing the dose and timing of ATG before and after hemopoietic cell transplantation is essential to enhance patient outcomes. Kroger et al. investigated the use of ATG  at a dose of 10 mg/kg on 3, 2, and 1 days before transplantation of allogeneic peripheral blood stem cells from an HLA-identical donor in patients with acute leukemia . Other A+ T cell immune reconstitution, and clinical outcomes following pediatric cord blood transplantation (CBT), early CD4+ immune reconstitution after CBT improved event-free survival. Early CD4+ immune reconstitution was achieved by reducing exposure to ATG after CBT [+ T cells > 200/μL at 3 months post-transplantation have been associated with superior OS and TRM. In the present study, we observed rapid recovery of CD3+ T cell and CD4+ T cells and a low rate of infectious complications and relapse. On day +100, median CD3+ and CD4+ T cell counts were 1117 and 104/μL. CD4+ cell counts were > 200/μL at day +120 and reached 330/μL on day +365.rATG contains polyclonal immunoglobulins obtained from hyper-immune sera of rabbits immunized with human thymocytes , 23. rATfter CBT . CD4+ T In summary, these data suggest that rATG for GVHD prophylaxis is tolerable and efficacious at a 5 mg/kg total dose administered over 2 days (day −5 to −4) in patients receiving allogeneic MSD-PBSCT. Further studies are required to evaluate the active rATG serum concentrations and the optimal prophylactic strategy in this patient population.


Streptococcus pneumoniae has been linked using the thiol/maleimide coupling chemistry to four different Pneumococcal surface adhesin A (PsaA) mutants, each harboring a single cysteine mutation at a defined position. Humoral response of these 1 to 1 carbohydrate antigen/PsaA conjugates have been assessed in mice. Our results showed that the carbohydrate antigen-PsaA connectivity impacts the anti-carrier response and raise questions about the design of glycoconjugate vaccine whereby the protein plays the dual role of immunogen and carrier.Glycoconjugate vaccines are formed by covalently link a carbohydrate antigen to a carrier protein whose role is to achieve a long lasting immune response directed against the carbohydrate antigen. The nature of the sugar antigen, its length, its ratio per carrier protein and the conjugation chemistry impact on both structure and the immune response of a glycoconjugate vaccine. In addition it has long been assumed that the sites at which the carbohydrate antigen is attached can also have an impact. These important issue can now be addressed owing to the development of novel chemoselective ligation reactions as well as techniques such as site-selective mutagenesis, glycoengineering, or extension of the genetic code. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. A synthetic tetrasaccharide representative of the serotype 14 capsular polysaccharide of  These T helper cells, in turn, stimulate the production of both plasma cells and memory B cells .Surface exposed polysaccharides of bacterial pathogens are perceived as non-self by the host immune system. Therefore, they are often the target of a protective humoral immune response. Immunization using polysaccharides from capsulated bacteria has been introduced by Gotschlich in the late 1960s −β−D−Glcp−(1 → 6)−[β−D−Galp−(1 → 4)]β-d-GlcpNAc 1 (referred to as Pn14TS)  . This ea Pn14TS) , able to Pn14TS) . We obse Pn14TS) . This obin silico MCHII peptide-binding affinity screening helped identifying a panel of 24 putative PsaA T-helper epitopes. Three out of them proved to be able to provoke Th cell proliferation: PsaA67−82, PsaA199−221, and PsaA231−268. The last one was deduced from three potent overlapping 15-mer peptides among which sequence 243–257 was the most potent  of the mPsaA N-terminated by a poly-6-histidine tag sequence to facilitate its purification by immobilized-Ni affinity chromatography plus a Tabacco Etch Protease (TEV) specific cleavage site to remove the tag after purification , was then determined by ELISA assays 1 week after the second and third immunization . The IgG response raised against CP14 by the conjugates was very low and not significant compared to the negative control group (data not shown). This response was equally low when we used Pn14TS as the coating antigen indicating that the observed results were not due to a lack of recognition of CP14 by anti-Pn14TS Abs. Anti-CP14 IgM response was also determined after the second and the third immunizations. Low titers of anti-CP14 IgM Abs could be measured in the sera of mice for any of the tested formulations after the second immunization 197 as the carrier  P < 0.05) but not Pn14TS-mPsaA K242C Streptococcus glycoconjugate vaccines have been prepared using transglutaminase or tyrosine-directed conjugation technology. This coupling strategy was shown to preserve the antigenicity of the Streptococcus proteins used as the carriers 4 introduced at the C-terminus of the maltose-binding protein used as carrier protein model , followed by DpnI digestion using the following primers: K237C forward 5′-GCACCCCGGAGCAAATCTGCACCCTGGTGGAAAAGC, reverse 5′-GCTTTTCCACCAGGGTGCAGATTTGCTCCGGGGTGC; K242C forward 5′-CAAATCAAAACCCTGGTGGAATGCCTGCGTCAGACCAAAGTTCCG, reverse 5′-CGGAACTTTGGTCTGACGCAGGCATTCCACCAGGGTTTTGATTTG; K247C forward 5′- GAAAAGCTGCGTCAGACCTGCGTTCCGAGCCTGTTCGTG, reverse 5′-CACGAACAGGCTCGGAACGCAGGTCTGACGCAGCTTTTC; K309C forward 5′-GGAGGGTCTGGCGTGCTAAGGATCCGGC, reverse 5′-GCCGGATCCTTAGCACGCCAGACC CTCC. The plasmids obtained are then transformed into competent XL1 blue bacteria. For each mutation, extraction of the plasmid (Quiaprep®Miniprep from Quiagen) on three different clones is carried out according to manufacturer protocol, and insertion of the mutations checked by sequencing.E. coli BL21 (DE3) strains for expression of the different mutants. The expression of mPsaA mutant proteins was performed according to the following protocol. Briefly, 250 mL of fresh TB medium with 100 μg/ml ampicillin were inoculated with 0.5 mL of overnight pre-culture and left to grow at 37°C, 180 rpm until they reach the exponential phase (approximately OD of 0.6/0.7 is reached) and at this point the HtTEV-mPsaA expression in the cultures were induced with IPTG . The cultures were left to grow for another 16 h at 25°C and 180 rpm. The cells were harvested at 10,000 rpm. The pellet were re-suspended in 25 ml cold lysis buffer  + 1 μg/mL DNAse, and 1 mg/mL of lysozyme incubated at 4°C under stirring during 30 min and were subjected to sonication  while they were kept in ice, and the cell debris were removed by centrifugation . The obtained supernatant was filtered (0.45 μm) and incubated with 0.5 ml of preconditioned Ni NTA beads for 1 h on rotating disk at 4°C, 12 rpm. Unbound fraction was collected, and the beads were washed with a lysis buffer containing 2–10 mM imidazole. The elution was performed with a lysis buffer containing 300 mM imidazole. The eluted fractions were subjected to SDS-PAGE to identify the fractions containing target protein. Later, the eluted fractions, containing the target protein (100 mg/L of culture), were pooled and dialyzed against water at 4°C.The validated plasmids have been used to transform TM protease ratio in the reaction buffer (50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1 mM DTT), for 16 h at 20°C, without stirring. The crude reaction mixtures were then dialyzed against water and incubated with the Ni NTA beads for the separation of the cleaved mPsaA mutants from the histidine tag, parent mPsaA mutants if not entirely digested and the tagged TEV protease. The eluate was collected and was subjected to SDS-PAGE and western blot analyses to confirm the removal of the histidine tag and assess their purity.The enzymatic cleavage of the histidine tag from mutants was performed using a 5:1 HtTEV-mPsaA/AcTEVFor the western blot, the vertical SDS-PAGE was carried out using 12% acrylamide gels with the loadings of the reaction mixture and tagged mPsaA mutants as controls using the Bio-rad system. The proteins were transferred to a nitrocellulose membrane at 150 mA for 90 min. For the antibody probing, the nitrocellulose membrane was initially blocked with TBS containing 5% skim milk and 0.1% tween 20 for 1 h at RT. A primary antibody to mouse anti-poly histidine , was applied to the membrane and incubated for 16 h at RT with agitation. The membranes were washed thrice with PBS containing 0.1% tween 20 and incubated with secondary antibody (Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 680) for 1 h at RT on a shaker. Membranes were subjected to a final wash with PBS, and the detection was performed using an Odyssey CLx scanner (LI-COR) at 700 nm.galactopyranosyl)−(1 → 4)−−(1 → 6)−[−(1 → 4)]−2−deoxy−2−acetamido−β-d-glucopyranoside 1  in DMF were successively added 3-maleimido-propionic acid succinimidyl ester  and DIEA  at RT. The reaction mixture was stirred at RT overnight, diluted in water and freeze-dried. The crude residue was purified by RP-HPLC to provide 2 ; Rf 0.39 (nBuOH/EtOH/H2O 5:5:3); 1H NMR : δ 6.85 , 4.52 , 4.51 , 4.43 , 4.26 , 3.99–3.88 , 3.84–3.62 , 3.60–3.55 , 3.54–3.48 , 3.36 , 3.28 , 2.48 , 2.00 ; 13C NMR : δ 174.7, 173.5, 172.6, 134.5, 103.0, 102.8, 102.5, 101.2, 78.5, 77.9, 75.4, 75.3, 74.7, 74.3, 73.5, 72.7, 72.6, 72.6, 72.4, 71.0, 71.0, 68.9, 68.6, 68.2, 67.4, 61.1, 61.0, 60.2, 55.1, 39.3, 34.7, 34.5, 22.3; HR-ESI-MS: m/z Calcd for C35H55N3O24[M+Na]+ 924.3073, found 924.3088.To a solution of 2-amino-ethyl (β−D−degassed pH 7 PBS buffer using swing centrifuge. Protein contents are quantified using Nanodrop spectrophotometer at 280 nm prior their conjugation. To a solution of mPsaA mutants (4.8–7.2 mg) in degassed 40 mM PBS, pH 7.0 (1.8–2.4 mg/mL), was added 2 (5 equiv) dissolved in water (1 mg/mL), and the resulting mixture stirred overnight and then purified by gel filtration using a HiLoad 15/600 Superdex™ 75 pg  column and 0.1 M PBS, pH 7.3 as eluent at 0.8 ml/min. Collected fractions were concentrated by centrifugal concentrators (cut-off 3 MWCO)  and then freeze-dried to give the corresponding conjugates which were analyzed for identity and purity by gel electrophoresis and mass spectrometry.mPsaA mutants proteins are rehydrated with ultrapure water to reconstitute 50 mM phosphate buffer. The pH are tested and adjusted to pH 8 with NaOH 0.1 M if necessary. mPsaA solutions are then treated with DTT (1.6 mg/mg of protein). Solutions are mixed first by flush and next let in rotary shaker at room temperature for 15 min. DTT is eliminated with desalting column  prior equilibrated with The conjugates (40 μl of a 5 mg/ml solution in PBS), were first formulated with a mixture of chitosan/poloxamer/TPP (1:1:1) (6 mL) under stirring (600 rpm) for 30 min. The particles were concentrated by centrifugation at 12000 RCF for 12 min at 15°C, using 10 μL of glycerol bed. After the centrifugation, the pellet in the bottom is carefully collected and re-suspended in PBS (50 μL). The Groups of 5 male C57/BL6JRj (5 week old) mice were injected sub-cutaneously (s.c.) with PBS, Pn14TS-mPsaA K237C, Pn14TS-mPsaA K242C, Pn14TS-mPsaA K247C, or Pn14TS-mPsaA K309C (25 μg protein/dose-−0.5 μg tetrasaccharide/dose) diluted with 50 μL of RIBI in PBS. The mice were immunized at day 0, 14, and 28. The sera were collected on days 21 and 35. Sera were stored at −80°C.−1) in 0.1 M sodium citrate (pH 5.2), containing 0.02% hydrogen peroxide, was added to each well and the plate incubated for 20 min at RT in the dark. The reaction was terminated by adding 3 M HCl , and the A492 was read in an Infinite M1000 spectrophotometer (TECAN). The Ab titer was defined as the dilution of immune serum that gave an OD (405 nm) at least twice that observed with pre-immune serum.The Ab responses induced upon immunizations were assessed 1 week after the second and the third injections by ELISA. mPsaA and capsular polysaccharide serotype 14 (CP14) (Alliance Bio Expertise), were used as coated antigens to define the anti-mPsaA or anti-CP14 Ab titers. mPsaA (0.1 μg/well) in 10 mM PBS, pH 7.3 (100 μL/well), was coated on 96 wells microtiter plates Nunc Maxisorp (ThermoFisher Scientific) plates overnight at 4°C. CP14 (1 μg/well) was coated for 48 h at 4°C in 10 mM PBS, pH 7.3 (100 μL/well). Plates were then washed with PBS 0.05% Tween 20 (3 × 200 μL), saturated using PBS containing 10% skimmed milk at 37°C for 2 h, then washed using PBS Tween 20  (3 × 200 μL). Series of dilution of sera in PBS containing 10% skimmed milk (100 μL/well), were incubated at 37°C for 2 h. Plates were then washed with PBST (3 × 200 μL) and then incubated with goat anti-mouse IgG(H+L)-horse radish peroxidase-labeled conjugate (CliniSciences) used as secondary Ab at a dilution of 1/6,000, for 1 h at 37°C and further washed with PBST (5 × 200 μL). The enzyme substrate, o-phenylenediamine dihydrochloride : 7897.CG designed the research and wrote the manuscript with AP, AD, and AL. MPr developed the route for the production of PsaA under EC, NC, and CG supervision. AP and AD produced the PsaA mutants under MPi, TV, and EC guidance. AL and CG carried out the conjugation step and the purification of the conjugates. AP and AF performed the ELISA assays. MF assisted in mass measurements.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The system is isoelectronic and isostructural with [Pd(dddt)2], which is the first example of a single-component molecular crystal that exhibits nodal line semimetallic behavior under high pressure. Systematic high pressure four-probe electrical resistivity measurements were performed up to 21.6 GPa, using a Diamond Anvil Cell (DAC), and high pressure single crystal synchrotron X-ray diffraction was performed up to 11.2 GPa. We found that [Ni(dddt)2] initially exhibits a decrease of resistivity upon increasing pressure but, unlike [Pd(dddt)2], it shows pressure-independent semiconductivity above 9.5 GPa. This correlates with decreasing changes in the unit cell parameters and intermolecular interactions, most notably the π-π stacking distance within chains of [Ni(dddt)2] molecules. Using first-principles density functional theory (DFT) calculations, based on the experimentally-determined crystal structures, we confirm that the band gap decreases with increasing pressure. Thus, we have been able to rationalize the electrical behavior of [Ni(dddt)2] in the pressure-dependent regime, and suggest possible explanations for its pressure-independent behavior at higher pressures.Single-component molecular conductors form an important class of materials showing exotic quantum phenomena, owing to the range of behavior they exhibit under physical stimuli. We report the effect of high pressure on the electrical properties and crystal structure of the single-component crystal [Ni(dddt) For example, as-synthesized [M(tmdt)2]  single crystals exhibit metallic behavior  (ptdt = propylenedithiotetrathiafulva-lenedithiolate) [2]  [2] (hfdt = bis(trifluoro-methyl)tetrathiafulvalenedithiolate) at 8 GPa (Tc = 5.5 K) .π conjugation and ligand flatness can also reduce the solubility of the dithiolene complex anion salt used as a starting material. As a consequence, obtaining large crystals of such single-component molecular conductors is very difficult. By comparison, smaller metal dithiolene complexes have recently attracted considerable attention, owing to their simple synthesis and ease of obtaining large single crystals. Lorcy and co-workers found that the small-sized complex [Au(Me-thiazdt)2]  [2] [2], which features S substituted by Se [2], which becomes metallic at 15.9 GPa [2] , which exhibits temperature-independent resistivity at 12.6 GPa. The results indicated that anisotropic Dirac cones emerge by a closing of the HOMO and LUMO bands originating from crystallographically-independent layers. Furthermore, the combined effect of the intralayer HOMO-HOMO/LUMO-LUMO and interlayer HOMO-LUMO interactions play an important role  has a loop of the Dirac points in the 3-D Brillouin zone, rather than a line [Chemical modifications to increase hiolate)  showed mate) [2]  and [Au2]  would show similar electronic properties to [Pd(dddt)2]. We sought to explain the results using experimental structural data obtained in situ for the first time from high pressure single crystal synchrotron X-ray diffraction, and first-principles band structure calculations based on the high pressure structures. In this way, we could rationalize the high pressure-induced changes in electrical properties, in particular the initial decrease in resistivity above ambient pressure, and suggest reasons for subsequent pressure-independent resistivity above 9.5 GPa.In order to investigate the effects of chemical substitution in single-component Dirac-cone materials, we synthesized and measured the high pressure resistivity of the isoelectronic complex [Ni(dddt)(dddt)2] . Conside2] could be obtained by a recrystallization of powder from benzene, which was prepared by I2 oxidation of [(n-C4H9)4N][Ni(dddt)2] [2] by I2 oxidation in acetone, using an H-shaped diffusion cell sitting on a non-vibration plate at room temperature . It has previously been reported that a single crystal of [Ni(dddt)(dddt)2] ,15. In o2], albeit with slightly smaller cell parameters rameters . Crystalσrt, was 1.6 × 10−3 S cm−1, and the activation energy, Ea, was 0.14 eV. At 10.9 GPa, σrt was 1.2 S cm−1 and Ea was 0.043 eV. At higher pressures, the temperature-dependent data look very similar, indicating resistivity behavior that is independent of pressure; at 21.6 GPa, σrt and Ea were 1.1 S cm−1 and 0.046 eV, respectively. Values for Ea also show pressure independence from 7.3 GPa to 21.6 GPa 2].Resistivity measurements were performed by a DC four-probe Diamond Anvil Cell technique, as described previously , using t GPa see d. Most sa and c decrease reasonably smoothly from 17.777(8) Å to 16.03(6) Å and from 18.405(3) Å to 17.090(7) Å, respectively  Å at 0.1 MPa to 4.1726(5) Å at 3.9 GPa, before plateauing at around 4.1 Å above 5.4 GPa . ectively a. In con 5.4 GPa b. b is by far the most compressible cell parameter: at 5.4 GPa, the length of b is just 0.87 times its ambient pressure value, whilst a and c are 0.94 and 0.95 times their ambient values, respectively ° at 0.1 MPa to 109.77(4)° at 5.4 GPa, before increasing again to 111.82(7)° at 9.6 GPa  Å at 0.1 MPa to 1039(3) Å3 at 9.6 GPa .Overall, the effect of pressure is to broaden the bands of the frontier molecular orbitals, such that the band gap decreases . At low 2] exhibits a semiconducting behavior at high pressure between 0.1 MPa and 21.6 GPa. Resistivity and activation energy decrease as a function of increasing pressure up to around 10.9 GPa, above which they are pressure independent. This behavior contrasts with the isoelectronic, isostructural system [Pd(dddt)2], which exhibits temperature-dependent resistivity, metallization and formation of Dirac cones in the electronic band structure at 12.6 GPa.The DC four-probe resistivity measurements show that the single-component molecular crystal [Ni(dddt)2] (16.9 mg), and the other side was set with I2 (52 mg) and acetic acid (2.5 mL), and then filled with acetone (15 mL). Black needle-shaped single crystals were obtained after about 10 days. Crystal data as follows: [Ni(dddt)2] C8H8NiS8, M 419.37, monoclinic, space group P21/n (no. 14), a = 17.805(3) Å, b = 4.6626(6) Å, c = 18.337(3) Å, β = 111.745(18)°, V = 1413.9(4) Å3, Z = 4.Single crystals of  was placed in a DAC with a culet size of 0.6 mm and a tungsten gasket. 4:1 methanol:ethanol was used as the hydrostatic pressure medium, and the pressure was determined by the shift of Ruby fluorescence R1 lines at room temperature, before and after each measurement. Optical microscope images of the sample prior to measurement at 9.6 GPa, and after measurement at 11.2 GPa, are shown in 2 hydrogen positions were refined with riding coordinates. Crystallographic information files are available from the CCDC, reference numbers 1908148–1908153 8153 see .P21/a. The revised lattice vectors of the unit cell are defined as o + co), b = −bo, c = coa = −, with a gaussian smearing method during self-consistent loops.The present first-principles calculations are based on the generalized gradient approximation (GGA) to the density-functional theory ,28. The


Salvia ballotiflora (Lamiaceae) were tested against the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae). The results demonstrated insecticidal and insectistatical activities against this insect pest with concentrations at 80 µg·mL−1 resulting in 20% larval viability and 10% pupal viability. The larval viability fifty (LV50) corresponded to a concentration of 128.8 µg·mL−1. This oil also increased the duration of the larval phase by 5.5 days and reduced the pupal weight by 29.2% withrespect to the control. The GC-MS analysis of the essential oil of S. ballotiflora showed its main components to be caryophyllene oxide (15.97%), and β-caryophyllene (12.74%), which showed insecticidal and insectistatical activities against S. frugiperda. The insecticidal activity of β-caryophyllene began at 80 µg·mL−1, giving a larval viability of 25% and viability pupal of 20%. The insectistatic activity also started at 80 µg·mL−1 reducing the pupal weight by 22.1% with respect to control. Caryophyllene oxide showed insecticidal activity at 80 µg·mL−1 giving a larval viability of 35% and viability pupal of 20%.The insectistatic activity started at 400 µg·mL−1 and increased the larval phase by 8.8% days with respect to control. The LV50 values for these compounds were 153.1 and 146.5 µg·mL−1, respectively.Essential oils can be used as an alternative to using synthetic insecticides for pest management. Therefore, the insectistatic and insecticidal activities of the essential oil of aerial parts of  Spodoptera frugiperda (Lepidoptera: Noctuidae) is a significant polyphagous insect pest of agricultural importance, not only for the damage it causes, but also due to its control difficulties [The caterpillar of iculties . This spiculties ,3,4. Theiculties ,6,7.et al. [Salvia species and their constituent compounds have been evaluated against insect pests and a number of them have shown considerable promise for the development of natural repellents/insecticides [Salvia genus is the most diverse of Lamiaceae Family, with over 1000 species around the world distributed in tropical and subtropical zones. In Mexico, there are at least 300 species reported [The implementation of Integrated Pest Management Programs appear to be a suitable alternative to managing noctuid insects by combining different methods that include the use of botanical extracts as insecticides . Due to et al.  these sucticides ,12,13,14reported . This gereported .S. frugiperda of the essential oil of the aerial parts of Salvia ballotiflora (Lamiaceae) and its main components.Therefore, the aim of this study was to determine insecticidal and insectistatic activities against S. ballotiflora essential oil, showed 37 different compounds (15 H24) with 12.74% and caryophyllene oxide (C15 H24 O) with 15.97%  [Salvia verbenaca, caryophyllene oxide was the main component with 7.28% [et al. For Salvia umbratica, had a caryophyllene oxide concentration of 8.42% [et al. [E)-caryophyllene (15.35%), α-eudesmol (14.06%), β-eudesmol (8.74%) and γ-eudesmol (7.64%) as a principal components of essential oil of aerial parts of Salvia microphylla.The GC-MS analysis of the aerial parts ompounds , corresph 15.97% . In otheectively ; for Salnd 11.5% . In anotectively . Essentih plant) . In the th 7.28% . Accordi [et al.  found  and Rhyzopertha dominica (Coleoptera: Bostrichidae), after 24 h were 79.17 μL·L−1 and 25.87 μL·L−1 respectively. Moreover, Khiyari et al. [Salvia aucheri, one from a wild plant and another from a cultivated plant against adults of Tribolium castaneum (Coleoptera: Tenebrionidae); the authors determined the CL50 and CL90 for each, and the wild plant the values were 1.00 μL·cm−2, 1.72 μL·cm−2, respectively, and for the cultivated plant the values were 1.25 μL·cm−2 and 2.03 μL·cm−2. Ulukanli et al. [Salvia tomentosa against adults of Acanthoscelides obtectus (Coleoptera: Bruchidae) and T. castaneum; this oil caused 100% mortality at 50 µL·L−1 air and 200 µL·L−1 air for each insect. Liu et al. [50 of 18.12 µg·adult−1 of the essential oil of S. umbratica against Sitophilus zeamais (Coleoptera: Curculionidae). The essential oils from Salvia hydrangea, Salvia numerosa, Salvia multicaulis and Salvia sclarea (Lamiaceae) were also tested against adults of S. granarius. These essential oils caused 67.33%, 39.73%, 55.21% and 41.76% mortality at 153.84 µL·L−1 air concentration [Salvia limbata and Salvia nemorosa (Lamiaceae) resulted in average mortality rates against S. granarius  at 76.91 µL·L−1 air concentration. The mortality rate increased with the concentration of the essential oils and the length of the exposure period [The insecticidal activity of  µg·mL−1 . The insi et al.  showed ti et al.  also repi et al.  evaluateSalvia species, Karahroodi et al. [Salvia multicaulis and Salvia officinalis (Lamiaceae), respectively, against female adults of Plodia interpunctella  at 15.39 µL·L−1 air concentration using an olfactometer. Additionally, Lakshmanan et al. [Spodoptera litura (Lepidoptera: Noctuidae), 45.64% on Helicoverpa armigera (Lepidoptera: Noctuidae) and 79.45% on Achaea janata (Lepidoptera: Noctuidae) with 1000 µg·mL−1 of S. officinalis essential oil. Conti et al. [−2 with the essential oils from Salvia dorisiana, Salvia longifolia, Salvia sclarea against adults of Aedes albopictus (Diptera: Culicidae), and the repellency was 90% after 90 minutes of exposure with 0.4 μL·cm−2 from S. dorisiana.Others studies have reported insectistatic activity with some i et al.  showed 5n et al.  reportedi et al.  describeSalvia species (Lamiaceae) were tested for insectistatic and insecticidal activities against S. frugiperda. All extracts showed both activities. Extracts from Salvia keerlii, and Salvia ballotiflora, had moderate insecticidal activity , and the S. ballotiflora extract increased the larval and pupal phases by 5.2 and 2.9 days, respectively, and reduced the pupal weight by 13.2% [S. ballotiflora had high insecticidal activity . The insectistatic activity increased the larval duration by 30.5, 8.0, 5.5, and 5.5 days, and reduced the pupal weight by 29% a 52% at 600, 400, 120, 80 µg·mL−1 respectively in regards to the control.Specifically, chloroform extracts from the aerial parts of four by 13.2% . In this−1 showed larval viability of 5%, 20%, 20%, 25% and 25% respectively and the pupal viability was 0%, 5%, 5%, 20% and 20% respectively. The LV50 was 153.1 µg·mL−1  of the essential oil of fruits of Illicium pachyphyllum (Schisandraceae). The essential oil of the aerial parts of Saussurea nivea (Asteraceae) used as a fumigant LC50 of 8.89 mg·L−1 and contact LC50 of 10.56 µg·adult−1 against S. zeamais. The main components of this oil were (+)-limonene (15.46%), caryophyllene oxide (7.62%), linalool (7.20%), α-pinene (6.43%), β-pinene (5.66%) and spathulenol (5.02%) [et al. [Piper capense (LD50 26.4 µL·g−1), Piper guineese (LD50 16.1 µL·g−1) and Piper nigrum (LD50 10 µL·g−1) had insecticidal activity against S. zeamais, and that in those oils β-caryophyllene was a compound with 3.4%, 20.8% and 12.8% in each of the species.Liu et al. , reporte (5.02%) . Tchoumbonents 9.% of the Zingiber officinale (Zingiberaceae) and Piper cubeba (Piperaceae) and the main compounds of those oils, that worked against adults and larvae of Tribolium castaneum (Coleoptera: Tenebrionidae) and adults of Sitophilus oryzae (Coleoptera: Curculionidae) were β-caryophyllene and α-pinene. β-caryophyllene showed LD50 values of 0.173 µL cm−2 after 24 h against T. castaneum adults, 0.17 µL cm−2 after 24 h against T. castaneum larvae, and 0.159 µL cm−2 after 24 h against S. oryzae adults. Benelli et al. [Hyptis suaveolens (Lamiaceae) and its principal compounds against Sitophilus granarius (Coleoptera: Curculionidae), β-caryophyllene (11.2%) showed 65% of repellence activity.Chaubey , evaluati et al.  tested tS. ballotiflora were collected in the Municipio of Guadalcázar, San Luis Potosí, México, at 1640 m.a.l.s., in September of 2013, the Taxonomic authentication was performed by José García-Pérez at the Isidro Palacios Herbarium of the Universidad Autónoma de San Luis Potosí. A voucher specimen was stored (SLPM 43013).The aerial parts  of −1 at 20 °C, and refraction index of 1.4095 at 25 °C. The yield was (0.47 w/w). The oil was protected from direct light and stored at 4 °C until its use.Aerial parts of the plant weighing approximately 2 kg, were submitted to hydrodistillation for 3 hours. The mixture obtained was treated with ethyl ether, then the organic phase was separated and concentrated with a rotatory evaporator at 18 °C. The essential oil obtained was dehydrated with anhydrous sodium sulphate, and the ethyl ether residue was eliminated under vacuum, to give a yellow amber essential oil with a density of 0.6836 g·mLβ-caryophyllene and caryophyllene oxide standards were purchased from Sigma-Aldrich .S. ballotiflora essential oil samples (20 µL) were diluted with acetone (1 mL). The essential oil was analyzed on an Agilent Technologies  6890N GC equipped with an HP-5MS column  and an Agilent EM 5973 detector, at 250 °C. The carrier gas was helium, with a flow rate of 1 mL·min−1; the split ratio was 2:1. The column temperature was initially 50 °C (for 3 min) and was gradually increased to 240 °C, at 3 °C·min−1; this temperature was held for 2 min. The injector temperature was 250 °C and 1 μL of essential oil was injected as a duplicate. The spectra were collected at 71 eV ionization voltages and the analyzed mass range was 15–600 m/z. The identification of the components were confirmed by comparison of the retention indices with those of authentic compounds using the Kovats index, based on n-alcanes C6-C26 with the Wiley09/NIST11 library. The identification of the two main components was confirmed by comparison of the retention indices to those of authentic compounds.S. frugiperda) larvae were reared in the Insecticide Natural Compounds Laboratory from the Chemistry Faculty of the Autonomous University of Querétaro, according to the Bergvinson and Kumar [S. frugiperda, the following ingredients were used: 800 mL of distilled water, 60 g diet , 20 g sterile corn spike, 100 g ground corn, 40 g brewer’s yeast, 10 g vitamins , 10 g agar, 1.7 g sorbic acid (dissolved in the ethanol), 17 mL ethanol, 2.5 mL formaldehyde, 1.7 g methyl p-hydroxybenzoate and 0.6 g neomycin sulfate.Fall armyworm . Based on the preliminary screening results, the concentration-dependent levels were selected . The test included a negative control (diet only). The essential oil, β-caryophyllene and caryophyllene oxide were mixed with the larvae diet ingredients during preparation without use of any solvent, according the methodology of Ramos-López et al. [50) corresponded to 50% of the larvae of fall armyworm during all larval phases for each extract.For the bioassay, first instar larvae of z et al. . The eff50 were calculated by Probit analysis, using the SYSTAT statistical analysis program [Statistical analysis was conducted and data was assessed for normality and homoscedasticity prior to analysis. In some cases Kruskal-Wallis non-parametric analysis of variance was used when data violated these assumptions and could not be corrected using a transformation. ANOVA analysis and Tukey test were also performed, and the LV program .S. ballotiflora had insectistatic and insecticidal activities against S. frugiperda. β-caryophyllene and caryophyllene oxide were the main components of S. ballotiflora essential oil and these compounds also showed insectistatic and insecticide activities against the fall armyworm.The essential oil of aerial parts of


Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors—temperature, enzyme lysis time, and concentration of the lysing enzyme—were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 107 protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46–54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.Take-all, caused by  Gaeumannomyces tritici, one of the major soil-borne pathogens, survives saprophytically on crop debris. G. tritici colonizes roots of susceptible cereals, especially wheat, and causes take-all disease in the affected areas [G. tritici mycelium [G. tritici genome sequence is available [ed areas . The typmycelium ,3. This mycelium ,5, resulmycelium ,7,8. Howmycelium ,10. The G. tritici is the production of protoplasts. Transformation of protoplasts has considerable potential for strain improvement and genetic analysis, providing an opportunity to rapidly study a large number of genes. Establishment of protoplast transformation techniques for G. tritici is of crucial importance to biotechnology. Stanway and Buck reported the viral infection of G. tritici protoplasts [2 mediated transformation of protoplasts for G. tritici [G. tritici by 4-nitroquinolene oxide [G. tritici DNA have been reported, we obtained transformants with a low transformation frequency when we used these protocols. Consequently, we developed a new and efficient method for the transformation of G. tritici, and the parameters involved in production of protoplasts were optimized.The polyethylene glycol (PEG)-mediated protoplast transformation technique is the most common approach for transformation of filamentous fungi ,12,13. Ttoplasts . Henson  tritici ,16. Bowyne oxide . AlthougOptimization of conditions for protoplast production using the classical method involves changing one independent variable while the other factors remain constant. The conventional methods for multifactor experimental design are time-consuming and inefficient for detecting the true optimum, especially given the interactions between the factors ,19. The hph) fragment of plasmid pSilent-1 was transformed into the genomic DNA of G. tritici by PEG-mediated transformation of protoplasts, which laid the foundation for establishing a good mutation library of G. tritici.The objective of the present research was to optimize the conditions to maximise the yield of protoplasts using both one-factor-at-a-time optimization and the response surface methodology. The optimal values of these factors were obtained using response surface methodology. Moreover, the hygromycin phosphotransferase gene . The enzyme of 0.25% L was significantly higher than the enzyme of 0.25% D (0.17 × 107 protoplasts/mL) and 0.25% S (0.03 × 107 protoplasts/mL). There was no significant difference between the enzyme of 0.25% L (2.07 × 107 protoplasts/mL), 0.25% S and 0.25% L (2.10 × 107 protoplasts/mL), and 0.25% L and 0.25% D (2.37 × 107 protoplasts/mL) (The results showed that a mixture of 0.25% snailase (S), 0.25% driselase (D), and 0.25% lysing enzyme (L) produced 2.68 × 10asts/mL) .G. tritici inoculated into media of 5× YEG, PDB, and OB were 1.05 × 107, 1.87 × 107, and 4.58 × 107 protoplasts/mL, respectively  and 2.5 h (8.45 × 107 protoplasts/mL). Therefore, the best digesting time for the 0.5% lysing enzyme is between 2.0 and 2.5 h.The mycelia of G. tritici were digested by 0.5% lysing enzyme at 30 °C for 2 h. The yield of protoplasts reached a maximum when 1 mL 0.5% lysing enzyme was reacted with 0.035 g mycelia  and 0.25% lysing enzyme (2.30 × 107 protoplasts/mL). The protoplast yields with different concentrations of lysing enzyme (1% and 2%) were 9.12 × 107 and 8.60 × 107 protoplasts/mL, respectively. There was no significant difference between 1 and 2% lysing enzymes for producing protoplasts  with a very low p value, with (Pmodel > F) = 0.0009  was related to the reliability and precision, and the higher the value of CV, the lower the reliability of the experiment [1 was more significant than the other factors, which implied that the temperature strongly influenced protoplast yield. The interaction coefficients of χ1χ2, χ1χ3, and χ2χ3 seemed to be insignificant, which revealed that the interactions of any two variables insignificantly affected protoplast yield.The analysis of variance (ANOVA) of the quadratic regression model demonstrated that the model was significant due to the Fisher’s = 0.0009 . The fitperiment . In this7 protoplasts/mL would be obtained by using the above optimal conditions.The three-dimensional (3D) response surface plots and the two-dimensional (2D) contour plots described by the regression model were designed to expose the optimal values of the independent variables and the interactive effects of each independent variable on the response . From th7 protoplasts/mL) was almost equal to the maximum predicted yield (9.76 × 107 protoplasts/mL), clearly proving the aptness of model under these conditions. Consequently, the model was considered to be accurate and reliable for predicting the protoplast yield. Moreover, the protoplast vitality was high, reaching 96.27% under the optimized conditions.A verification experiment was performed using the optimized conditions. The experiment demonstrated that the value of the experimental response  plates with 120 μg/mL hygromycin B using toothpicks, and continuously transferred five times onto new PDA plates containing 120 μg/mL hygromycin B. More than 98% transformants were able to grow stably, indicating that we had obtained positive transformants. There were 46–54 transformants/μg NDA .hph into the genome of transformants was verified using PCR and Southern blotting. As showed in G. tritici genomic DNA. Southern blot analyses were evaluated with the probe. The probe detected a single band with an expected size of 514 bp in the four transformants, and no band was found in the wild type   . Taking hph gene from plasmid pSilent-1 using the PEG-mediated protoplast transformation system described above. The present study examined the susceptibility of protoplasts to hygromycin B. The results showed that G. tritici was sensitive to high concentrations of hygromycin B  was provided by the Agricultural Culture Collection of China . The fungus was grown on potato dextrose agar (PDA) at 25 °C for 5–7 days before being used. The strain was stored at −80 °C by cryopreservation in 30% glycerol solution [solution .3, 0.5 g KCl, 1 g MgSO4·7H2O, 1.5 g KH2PO4, and 1000 mL distilled water), STC , 40% PTC (40 g PEG8000 and 100 mL STC), TB3 , bottom agar , and top agar . All the above media were autoclaved at 120 °C for 20 min. Mycelia lysate (0.5%) was sterilized by filtration (200 mg lysing enzyme and 40 mL 1 mol/L sorbitol).The following media were used in this study: PDA , potato dextrose broth (PDB) , oatmeal broth (OB) , 5× yeast extract glucose (5× YEG) , CM . Lysing enzyme (L), driselase (D), and fluorescein diacetate were purchased from Sigma-Aldrich . Snailase (S) and ampicillin (amp) were purchased from Solarbio .PtrpC-hph fragment containing hph under the control of trpC gene promoter was amplified from plasmid pSilent-1 [hph fragment was polymerase chain reaction (PCR)-amplified from pSilent-1 using the primers HygbF (5′-TAACCGTATTACCGCCTTTG-3′) and HygbR (5′-TCGGCATCTACTCTATTCCTTT-3′) in a total reaction volume of 25 μL containing 0.75 μL PCR forward primer (10 μM), 0.75 μL PCR reverse primer (10 μM), 1 μL DNA sample of plasmid pSilent-1, 10 μL RNase free dH2O, and 12.5 μL PrimeSTAR Max . The reaction mixtures were amplified for 32 cycles  in a C1000TM thermal cycler . The PCR products were purified by TIANquick Midi Purification kit .A 1790 bp Silent-1 ,35,36. TG. tritici were harvested from a seven-day-old PDA culture and inoculated into 50 mL of OB and incubated on a rotary shaker (175 rpm) at 25 °C for 2 days. Then 50 mL CM was added and incubation continued on a rotary shaker (175 rpm) at 25 °C for 2 days. The mycelia were filtered and washed twice with 1 mol/L sorbitol, and mixed with mycelia lysate (40 mL), then incubated on a rotary shaker (90 rpm) at 30 °C for 2 h. Protoplasts were filtered with a layer of cloth and two layers of lens cleaning tissue , and centrifuged for 5 min at 3000× g. The supernatant was removed, the protoplasts were suspended with 20 mL STC, and centrifuged for 5 min at 3000× g. The protoplasts were resuspended with 2 mL STC and stored on ice until transformation.Mycelia of Various enzymes and enzyme combinations were used, including 0.25% lysing enzyme (L), 0.25% driselase (D), 0.25% snailase (S), 0.25% L+ 0.25% D, 0.25% L+ 0.25% S, 0.25% D+ 0.25% S, and 0.25% L+ 0.25% D+ 0.25% S. The other operations were the same as in G. tritici were harvested from a seven-day-old PDA culture and inoculated into 50 mL of PDB, OB, and 5× YEG, and incubated on a rotary shaker (175 rpm) at 25 °C for two days. Then, 50 mL CM was added and incubation continued on a rotary shaker (175 rpm) at 25 °C for two days, and the other operations were the same as outlined in Mycelia of G. tritici were tested at digesting temperatures of 26, 28, 30, 32, and 34 °C. The other operations were the same as in Mycelia of G. tritici were digested for 1, 1.5, 2, 2.5, and 3 h. The other operations were the same as in The mycelia of G. tritici mycelial weights of 0.01, 0.02, 0.03, 0.035, 0.04, 0.05, and 0.06 g were digested in 1 mL 0.5% lysing enzyme. The other operations were the same as in G. tritici were digested at rotational speeds of 30, 60, 90, 120, and 150 rpm. The other operations were the same as in On a rotary shaker, mycelia of Use of a proper enzyme system is usually a crucial for preparing highly efficient protoplasts. A series of concentrations of lysing enzyme  were tested. The other operations were the same as in 1), enzyme lysis time (X2), and concentration of lysing enzyme (X3)—were selected as the independent variables. The main purpose of the Box–Behnken design of the experiment was to investigate the relationship between the multiple process variables and protoplast yield in order to determine the optimum conditions for the protoplast production. According to a series of single factor experimental results, three major factors—temperature (X3 (50 µg/mL of amp), incubated on a rotary shaker (100 rpm) at 25 °C for 12 h, and centrifuged for 5 min at 3000× g. Discarded supernatant and suspended 200 µL of STC. Then added 10 mL bottom agar (60 µg/mL hygromycin B), mixed gently and poured into a Petri dish. Cultured 12 h at 25 °C and added 10 mL top agar (120 µg/mL hygromycin B) to the Petri dish. After 4–5 days, the plates were screened for transformants, which were transferred to PDA media.To 200 µL of protoplast in 50 mL tubes were added 4, 6, 8 µg DNA, respectively, mixed gently and incubated at room temperature for 20 min. Then 1.25 mL 40% PTC (filtration sterilization) was added, mixed gently, and incubated at room temperature for 20 min. Added 10 mL of TBG. tritici were grown on petri dishes containing PDA at 25 °C for seven days before they were harvested in sterile distilled water. The mycelia were then transferred to a triangular flask containing 100 mL PDB and incubated on a rotary shaker (175 rpm) at 25 °C for five days. The mycelia were collected by vacuum filtration, and genomic DNA was extracted by Plant Genomic DNA Kit . The primers YzbF (5′-GATCGTTATGTTTATCGGCACT-3′) and YzbR (5′-TGGCGACCTCGTATTGG-3′) were used for testing and verifying PCR amplification of the hph gene fragment.The wild strain and four transformants of Southern blot analyses of transformants were performed with a DIG-High Prime DNA Labeling and Detection Starter Kit I  . For SouStatistical analyses were performed with SPSS 19.0. Statistical differences between treatments were evaluated by Fisher’s least significant difference test at a 5% significance level.


Fasciola hepatica (liver fluke) and Schistosoma mansoni (blood fluke), are responsible for the neglected tropical diseases (NTDs) fasciolosis and schistosomiasis. Due to the absence of prophylactic vaccines, these NTDs are principally managed by the single class chemotherapies triclabendazole (F. hepatica) and praziquantel (S. mansoni). Unfortunately, liver fluke resistance to triclabendazole has been widely reported and blood fluke insensitivity/resistance to praziquantel has been observed in both laboratory settings as well as in endemic communities. Therefore, the identification of new anthelmintics is necessary for the sustainable control of these NTDs in both animal and human populations. Here, continuing our work with phytochemicals, we isolated ten triterpenoids from the mature bark of Abies species and assessed their anthelmintic activities against F. hepatica and S. mansoni larval and adult lifecycle stages. Full 1H and 13C NMR-mediated structural elucidation of the two most active triterpenoids revealed that a tetracyclic steroid-like nucleus core and a lactone side chain are associated with the observed anthelmintic effects. When compared to representative mammalian cell lines (MDBK and HepG2), the most potent triterpenoid  displayed anthelmintic selectivity  and induced severe disruption of surface membranes in both liver and blood flukes. S. mansoni egg production, a process responsible for pathology in schistosomiasis, was also severely inhibited by 700015. Together, our results describe the structural elucidation of a novel broad acting anthelmintic triterpenoid and support further investigations developing this compound into more potent analogues for the control of both fasciolosis and schistosomiasis.Two economically and biomedically important platyhelminth species,   •Abies species contain anthelmintic phytochemical triterpenoids.•The triterpenoid 700015 affects larval, juvenile and adult fluke viabilities.•700015 is moderately selective against both fluke species. Fasciole planet . Howevere planet . Morbidie planet . The lone planet . When coe planet , tremende planet .Fasciola development in the definitive host , it has been the preferred drug of choice since its introduction  on F. hepatica and S. mansoni parasites. Preliminary structural activity relationships (SAR) indicate that a tetracyclic steroid-like nucleus core and a lactone side chain are associated with the observed anthelmintic effects  of the most potent triterpenoids. These results extend the chemical space of plant-based natural products, which display anthelmintic activity against flukes of both veterinary and biomedical importance.Towards this end, we previously have described the anthelmintic effects of a d adults . While td adults . Here, b22.1All procedures performed on mice (project license PPL 40/3700) and sheep  adhered to the United Kingdom Home Office Animals (Scientific Procedures) Act of 1986 as well as the European Union Animals Directive 2010/63/EU and were approved by Aberystwyth University's (AU) and Ridgeway Research Limited's (RRL) Animal Welfare and Ethical Review Bodies (AWERB).2.2Abies species grown in Wales (UK) by normal phase and reverse phase chromatographic systems. Photodiode array and mass spectrophotometry detection to monitor peak separations were also applied during the isolation process. For specific isolation of the most active compound 700015, mature branches (600 g) of Abies procera (the Noble Fir) were collected in January by Forest Research  at Cefn Gethiniog, Talybont-on-Usk, Brecon, Powys (reference N10122) and given PhytoQuest code IG289; this starting material was used to purify 160 mg of pure (>95%) compound. Here, the DCM extraction of the freeze-dried ground material was conducted in a Soxhlett overnight and the extract cleaned using Diaion HP20 resin. Normal phase flash chromatography using Dionex cartridges and heptane:ethyl acetate (70:30) was monitored at 210 nm and the fraction further separated using preparative high performance liquid chromatography  with water: acetonitrile: acetonitrile + 0.1% TFA (26:65:10) giving the pure compound. The second most active compound, 700234, was purified by the same method from 1 kg of mature bark of Abies grandis  supplied by Natural Resources Wales (N10127), yielding 750 mg of pure (>95%) compound. A reference sample was given the PhytoQuest code IG294.A total of ten triterpenoid compounds were isolated from dichloromethane (DCM) extracts of the mature bark of 8 HPLC column  with a flow rate of 1.5 mL/min and a linear gradient that started at 90% water and 10% acetonitrile (containing 0.01% trifluoroacetic acid), rising to 100% acetonitrile over 6 min, which was held for a further 6 min. Structural elucidation was conducted using NMR spectroscopy on a 500 MHz Bruker Avance instrument. Mass spectrometry was performed on an Orbitrap Fusion Thermo Scientific with a Dionex UltiMate 3000 UHPLC system.Compound 700015 gave a distinctive mass spectrum  with major ion 325 atomic mass units (Waters Integrity) while 700234 had distinctive ions at 295 (100%) and 313 (50%) atomic mass units. The UV spectra were end absorbing (200 nm) and not informative. Using an analytical HPLC system, the retention time was 8.4 min for 700015 and 9.5 min for 700234. The analytical method used a C2.31H, 13C and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy. Peak lists for 1H NMR  δ/ppm: 6.83 , 5.43 , 2.41  2.00 , 1.87  1.86 , 1.81 , 1.75 , 1.68 , 1.65 , 1.61, 1.55 , 1.53 , 1.43 , 1.38 , 1.35 , 1.25 , 0.93 , 0.82 , 0.78 , 0.58 . Peak lists for 13C NMR  δ/ppm: 218.4 , 171.4 , 149.0 , 148.9 , 131.8 , 106.1 , 53.9 , 52.7 , 52.7 , 47.3 , 45.9 , 44.0 , 43.7 , 36.2 , 35.7 , 35.7 , 34.8 , 33.3 , 33.3 , 29.1 , 28.4 , 27.7 , 23.5 , 23.5 , 23.4 , 23.3 , 22.7 , 21.7  10.3 . HRMS-ESI m/z: [M + H]+ calculated for C30H45O4 is 469.3318 and experimentally determined to be 469.3317. According to SciFinder (https://www.cas.org/products/scifinder) searches, 700015 has been previously isolated from Abies holophylla  together with lophylla  and Abiesibirica .2.41H, 13C) NMR spectroscopy. Peak lists for 1H NMR  δ/ppm: 6.91 , 5.68 , 5.55 , 3.42 , 2.29 , 2.09 , 2.08 , 2.07 , 2.04 , 1.94 , 1.93 , 1.91 , 1.89 , 1.87 , 1.86 , 1.82 , 1.56 , 1.36, 1.11 , 1.02 , 1.01 , 0.98 , 0.92 . Peak lists for 13C NMR  δ/ppm: 174.6 , 157.8 , 149.9 , 147.9 , 132.3 , 124.9 , 120.9 , 109.4 , 78.2 , 75.2 , 51.7 , 50.6 , 49.9 , 48.8 , 39.8 , 39.7 , 37.2 , 36.9 , 36.4 , 35.1 , 30.3 , 27.5 , 27.4 , 24.9 , 24.7 , 19.8 , 19.1 , 18.9 , 10.3 . HRMS-ESI m/z: [M + H]+ calculated for C30H43O4 is 467.3161 and experimentally determined to be 467.3169. According to searches in PubChem (https://pubchem.ncbi.nlm.nih.gov/search/) and SciFinder (https://www.cas.org/products/scifinder), 700234 represents a novel chemical entity.Colourless oil, structural elucidation involved two steps. Purified 700234 was characterised by HRMS and two-dimensional  and auranofin , which were also diluted in DMSO to a stock concentration of 10 mM and working concentration of 1.6 mM. A positive control for F. hepatica screens included triclabendazole , which was diluted in DMSO to a working concentration of 10 mM.All ten triterpenoids were solubilised in DMSO  to a stock concentration of 10 mM and stored at −20 °C until required. For all fluke screens, compounds were further diluted to a working concentration of 1.6 mM in DMSO. Positive controls for 2.6F. hepatica Italian strain were supplied by RRL and an excystment was performed as previously described  metacercariae and, four and 8 wk later, immature and mature liver fluke were obtained and prepared as previously described  were orally infected with 200 Fescribed . Immaturescribed .2 with 700015 at 40 μM, 13.3 μM and 4.4 μM for 72 h. Every 24 h, 2 mL of fresh media and 700015 was added to each of the cultures; compound-induced motility defects were again scored according to Adult liver flukes (n = 3/condition) were transferred to 15 mL falcon tubes containing 6 mL of the same medium used for cultivating immature flukes and co-cultured at 37 °C in an atmosphere containing 5% CO2.8Biomphalaria glabrata (NMRI strain) snails infected with S. mansoni (Puerto Rican strain) were shed for 2 h under light conditions at 26 °C. Cercariae were collected, mechanically transformed into schistosomula  that were infected percutaneously three weeks earlier with 4000 cercariae. Worms were transferred into 50 mL falcon tubes and centrifuged at 300×g for 2 min. This pellet was collected, washed in phenol-red free DMEM and subjected to repeat centrifugation. This procedure was repeated a further two times, where on the final wash, the parasites were pelleted by gravity. This final, washed pellet was collected and placed in culture media  supplemented with 10% v/v Hepes , 10% v/v Foetal Bovine Serum , 0.7% v/v 200 mM L-Glutamine  and 1X v/v penicillin-streptomycin .Juvenile 2 for 72 h at which time worm motility was scored between 0 and 4: 0 = dead, 1 = movement of the suckers only and slight contraction of the body, 2 = movement at the anterior and posterior regions only, 3 = full body movement but sluggish and 4 = normal movement.A total of 6–19 juvenile worms were transferred to each well of a 96 well plate containing a final volume of 200  μL of media containing 700015 at the following concentrations: 15 μM, 7.5 μM, 3.75 μM and 1.83 μM (in 1.25% DMSO). This was repeated three times (n = 3). Parasites were cultured at 37 °C in an atmosphere containing 5% CO2.10S. mansoni parasites were recovered by hepatic portal vein perfusion from MF-1 mice  that were percutaneously infected seven weeks earlier with 200 cercariae. Three adult worm pairs per well, in duplicate, were transferred into 48 well plates  and cultured at 37 °C in an atmosphere containing 5% CO2 in DMEM media  containing 10% v/v HEPES, 10% v/v Foetal Bovine Serum, 0.7% v/v 200 mM L-Glutamine and 1X penicillin-streptomycin . A total of 3 biological repeats were conducted giving a total of 18 males and 18 females dosed per treatment. Worms were dosed with test compounds at 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM and 0.625 μM (in 1.25% DMSO) for 72 h. Adult worms were scored manually at 72 h using the WHO-TDR metric scoring system as described previously  for 72 h. Afterwards, flukes were prepared for SEM as previously described (Adult liver and blood flukes were cultivated (as described above) in sub lethal concentrations of 700015 (escribed .2.12S. mansoni worms were cultured (as described above) for 72 h in a sub lethal concentration of 700015 (10 μM). After 72 h, a 1  μL aliquot of 10 mM EdU was added to the culture media and incubated for a further 24 h. Worms were subsequently collected and fixed as described previously (3) occupied by each fluorophore.Adult eviously . Anterio2.134 cells/well (HepG2) or 7.5 × 103 cells/well (MDBK) were seeded in black walled 96-well microtiter plates  and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. To each well, 700015 was subsequently added to obtain final concentrations (in 1% DMSO) of 100 μM, 75 μM, 50 μM, 25 μM, 10 μM and 5 μM (HepG2 cells) or 100 μM, 75 μM, 50 μM, 20 μM, 10 μM and 5 μM (MDBK cells); negative (1% DMSO) and positive (1% v/v Triton X-100) control wells were included for both cell types. Following a further incubation for 24 h, the MTT assay was performed as previously described (Overt cytotoxicity of 700015 was assessed on both human HepG2 and bovine MDBK (NBL-1) cells as described previously . Brieflyescribed .2.14All Statistical analyses were conducted using GraphPad Prism 7 software. To determine significant differences amongst population means, a Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test was used.3de novo NTD drug discovery programmes have been developed and include drug repurposing/repositioning, drug rescuing, target repositioning and lead repurposing  anthelmintic activity of ten triterpenoids isolated from the bark of an indigenous coniferous species from Wales, United Kingdom (A. procera and A. grandis).In this regard, terpene/terpenoid phytochemicals have previously been explored and repurposed for their synergistic anti-parasitic capabilities; a well-known example is artemisinin, an endo-peroxide containing sesquiterpenoid lactone derived from feration , artemisferation . Of inteherapies . While therapies , the seaherapies . Here, eherapies , we inveF. hepatica  to the less active triterpenoid (700657) and finally to the completely inactive triterpenoid 700019; all four compounds share a tetracyclic steroid-like nucleus . While tF. hepatica NEJs and S. mansoni schistosomula motility and phenotype metrics after 72 h of co-culture   A and 1.9tosomula B. As thetosomula , we extetosomula .Fig. 4DoF. hepatica, all concentrations of 700015 tested  considerably affected the motility of immature (4 wk old) parasites (50 = 0.7 μM) was equivalent to that seen for immature flukes co-cultured with triclabendazole (40 μM). Due to the limitations in immature fluke numbers, we were unable to titrate 700015 further and, thus, this EC50 is an estimate only. Adult (8 wk old) liver flukes were only significantly affected by higher concentrations of 700015 (40 μM). While this triterpenoid-induced effect (EC50 = 15.6 μM) was equivalent to that seen in adult flukes co-cultivated with triclabendazole (40 μM), a steep drop in activity was seen at 700015 concentrations below 13.3 μM. Nevertheless, as triclabendazole represents the only liver flukicide on the market with activity against NEJs, immature flukes and adults  parasites at all concentrations tested except 1.83 μM  mixed-sex cultures revealed a greater triterpenoid-mediated effect on male (EC50 = 7.4 μM) compared to female (EC50 = 10.3 μM) parasites. Although this gender biased  effect has been observed before for other (di- and sesqui-) terpenes/terpenoids (50s (5 μM - 2.5 μM) calculated for this lifecycle stage  of 700015  and human (CC50 = 32 μM) cell lines; these data indicated a low to moderate degree of general cytotoxicity for this triterpenoid. These findings are similar to those published previously, where moderate 700015 - mediated cytotoxicities on SK-OV-3 , HCT15 , A549  and SK-MEL-2  cell lines were demonstrated (50/IC50) of this triterpenoid ranged between 3 and 46; the exception being adult liver fluke where the SI was 2 (The mechanism of action (MOA) behind these commonly observed anthelmintic phenotypes (i.e. surface membrane defects) is currently unknown, but extrapolating findings from other systems implicates membrane disruption, lipase inhibition, mitochondrial dysfunction and cholesterol homeostasis alterations  as possinstrated . NeverthSI was 2 . These vSI was 2 . CollectA. procera and chemically defined as a triterpenoid containing a tetracyclic steroid-like nucleus and lactone side chain. These core structures were associated with moderately potent and selective anthelmintic activities against larval, immature and mature lifecycle stages of both F. hepatica and S. mansoni flatworm parasites. Further investigations of this novel triterpenoid for the control of both fasciolosis and schistosomiasis are warranted.In summary, a novel phytochemical (700015) was isolated from the bark of The authors indicate that they have a collaborative relationship with Bimeda Ltd, UK, which may have a direct or indirect financial interest in the subject matter discussed in the manuscript.


We report on new Zn–Salen oligomer receptors able to recognize a nerve agent simulant, namely dimethyl methylphosphonate (DMMP), by a supramolecular approach. In particular, three Zn-Salen oligomers , differing by the length distribution, were obtained and characterized by NMR, Gel Permeation Chromatography (GPC), UV-Vis, and fluorescence spectroscopy. Furthermore, we investigated their recognition properties towards DMMP by using fluorescence measurements. We found that the recognition ability depends on the length of the oligomeric chain, and the Zn–Oligo–C shows a binding constant value higher than those already reported in literature for the DMMP detection. The nerve agents (NAs) of G and V series  are the 3 range at 25 °C), while the V-serie shows lower volatility (3–30 mg/m3 at 25 °C). For these reasons, NA can be released in air or in water. These compounds start to be toxic in the 25–400 mg × min/m3 range. For these reasons, a good sensor should be able to detect NA both in solution and in gas phase, with a detection limit of few ppm.G-serie NAs are highly volatile  are forbidden for security reasons, OP compounds, such as Sarin, Soman, and the more recent Novichock  are stilWith the aim to increase human health and safety, many efforts have been made to develop new and more efficient detection systems, able to reveal the presence of OP compounds in low concentrations and to give a measurable and observable response of NA presence .Due to their high toxicity, the use of nerve agents is not even allowed for research activity. For this reason, studies on new molecular receptors are carried out using simulants—less toxic organophosphorus compounds. In this context, recent studies demonstrated that dimethyl methylphosphonate DMMP,  is one oi) A covalent approach, in which the receptor covalently binds the analyte, thus giving a measurable response (such as a change of the fluorescence emission) after the interaction +), that can be assigned to the molecular species reported in the inset of The oligomeric structures of the products were also confirmed by means of MALDI-TOF MS . Similarly, in the absorption spectrum of Zn–Oligo–B in DMSO, this band appeared at 372.0 nm , while in the UV–Vis spectrum of Zn–Oligo–C it lay at 370.0 nm .UV–Vis absorbance spectra of oligomers showed the azomethine transition bands at ca. 325 nm a. AdditiEmission properties of Zn–oligomers were studied in DMSO. Using an excitation wavelength of 365 nm, similar fluorescence spectra were observed for all the three Zn–oligomers, with an intense emission band centered at ca. 480 nm see . The emiRecognition studies of the three receptors towards DMMP were conducted by following the progressive quenching of the emission intensity, probably due to a PET (Photoinduced Electron Transder) mechanism , upon thThe data treatment using the HypSpec software provided the binding constant values for each receptor. Non-linear curve fit plots were performed by using a 1:1 stoichiometry, supported by our previous studies on CWA recognition by Metal–Salen complexes ,35,36,372, 5 ppm NO, and 10 ppm CO) was bubbled for 10 min into a 1 × 10−5 M DMSO solution of Zn–Oligo–C. The emission spectra of the host were acquired before and after air bubbling, thus finding that the emission profile did not change after air exposure . After the exposure to DMMP vapors , a clear decrease of color intensity was observed . This prGeneral experimental methods. The NMR experiments were carried out at 27 °C on a Varian UNITY Inova 500 MHz spectrometer  equipped with pulse field gradient module (Z axis) and a tunable 5 mm Varian inverse detection probe (ID-PFG). A JASCO V-560 UV-Vis spectrophotometer  equipped with a 1 cm path-length cell was used for the UV-Vis measurements. Luminescence measurements were carried out using a Cary Eclipse Fluorescence spectrophotometer  with resolution of 0.5 nm, at room temperature. The emission was recorded at 90° with respect to the exciting line beam using 10:5 slit-widths for all measurements. The fluorescence quantum yields were calculated by usingN-butyl-4-butylamino-1,8-naphthalimide as standard. All chemicals were reagent grade and used without further purification. 13C NMR characterizations of oligomers were precluded due to the scarce solubility of the compounds.Gel permeation chromatography. A PL-GPC 110 (Polymer Laboratories) thermostat system, equipped with three PL-gel 5 mm columns (two Mixed-D and one Mixed-E) attached in series, was used. The analyses were performed at 35 ± 0.1 °C using THF as eluent at a flow rate of 1 mL/min. A differential refractometer  was used as detector.Procedure for epsilon calculation. UV-Vis spectra of four solutions of the hosts at different concentration, from 1 × 10−5 M to 4 × 10−5 M, were measured at 25 °C. Data treatment allowed for the calculation of epsilon for each absorption band.Procedure for fluorescence titrations. Two stock solutions of host and guest (1.0 × 10−3 M) in dry solvent were prepared. From these, progressive amounts of guest solution were added to the host (from 0 to 6 eq.), and emission spectra were recorded at 25 °C. Fluorescence titration with Zn–Oligo–A, –B, and –C were carried out using λex = 365 nm in dry DMSO and recorded at λem= 480 nm at 25 °C. With this data treatment, the apparent binding affinities of receptors with DMMP were estimated using the HypSpec  [2 test (chi-square) was applied, where the residuals follow a normal distribution . In all of the cases, values of χ2 ≤ 10 were found, as obtained by 3 independent measurements sets., Italy) ,46, a soDOSY experiments. Diffusion Ordered SpectroscopY (DOSY) NMR was used to determine the presence of monomeric or higher species in solution. The DOSY technique provides information about the size of the molecular aggregate in solution. Furthermore, the diffusion coefficient value could be associated to the molecular weight by the mathematic treatment recently described [d6 (10 mM) showed a diffusion coefficient of 3.41 × 10−10 m2 s−1, corresponding to a calculated molecular weight of ca. 4650 . While in DMSO-d6, a solution of Oligo–Salen–B (10 mM) showed a diffusion coefficient of 4.23 × 10−10 m2 s−1, . Oligo–Salen–C showed a diffusion coefficient of 5.29 × 10−10 m2 s−1 , relative to a solution of ligand in DMSO–d6 (10 mM).escribed ,43. DOSYSynthesis of Oligo–Salen–A: 5,5’–methylenebis–2–hydroxybenzaldehyde  [meso–1,2–diphenylethylenediamine  were stirred in anhydrous toluene (22 mL) at room temperature for 48 h. Then the solvent was removed under reduced pressure, thus leading to the Oligo-Salen-A (0.380 g) as pale yellow crystals, then washed with hexane: 1H NMR  δ 13.1–12.8 , 8.1–7.9 , 7.4–6.6 , 4.8–4.6 , 3.7  ppm. Anal. Calcd. for C290H260N20O20: C, 80.16; H, 6.03; N, 6.45. Found C, 80.12; H, 6.01; N, 6.41.58 mmol) , 2–hydroSynthesis of Oligo–Salen–B: 5,5’–methylenebis–2–hydroxybenzaldehyde , 2–hydroxy–benzhaldehyde , and meso–1,2–diphenylethylenediamine  were stirred in anhydrous toluene (22 mL) at room temperature for 72 h. Then the solvent was removed under reduced pressure, thus leading to the Oligo–Salen–B (0.230 g) as pale yellow crystals, then washed with hexane: 1H NMR  δ 13.1–12.8 , 8.1–7.9 , 7.4–6.7 , 4.8–4.6 , 3.7  ppm. Anal. Calcd. for C174H156N12O12: Found C, 80.14; H, 6.00; N, 6.39.Synthesis of Oligo–Salen–C: 5,5’–methylenebis–2–hydroxybenzaldehyde , 2–hydroxy–benzhaldehyde , and meso–1,2–diphenylethylenediamine  were stirred in anhydrous toluene (22 mL) at room temperature for 72 h. Then the solvent was removed under reduced pressure, thus leading to the Oligo–Salen–C (40 mg) as yellow crystals, then washed with hexane: 1H NMR  δ 13.1–12.8 , 8.1–7.9 , 7.4–6.7 , 4.8–4.6 , 3.7  ppm. Anal. Calcd. for C116H104N8O8: Found C, 80.11; H, 6.02; N, 6.44.Synthesis of Zn–Oligo–A: To a solution of the Oligo–Salen–A ligand 0.315 g (0.73 mmol) in dry tetrahydrofuran (15 mL), 900 μL (0.90 mmol) of Et2Zn 1 M in hexane was added. The reaction mixture was stirred for 48 h at room temperature, thus affording Zn–Oligo–A (0.350 g) as a brown-yellow precipitate, which was collected by filtration. 1H NMR  δ 8.2–8.0 , 7.2–6.3 , 5.1–5.0 , 3.5  ppm. Anal. Calcd. for C290H240N20O20Zn10: C, 69.96; H, 4.86; N, 5.63. Found C, 69.92; H, 4.81; N, 5.60.Synthesis of Zn–Oligo–B: To a solution of Oligo–Salen–B ligand 0.200 g (0.46 mmol) in dry tetrahydrofuran (19 mL), 680 μL (0.60 mmol) of Et2Zn 1 M in hexane was added. The reaction mixture was stirred for 48 h at room temperature, thus affording Zn–Oligo–B (0.190 g) as a brown-yellow precipitate, which was collected by filtration. 1H NMR  δ 8.2–8.0 , 7.3–6.3 , 5.1–4.9 , 3.5  ppm. Anal. Calcd. for C174H144N12O12Zn6: Found C, 69.93; H, 4.85; N, 5.59.Synthesis of Zn–Oligo–C: Oligo–Salen–C ligand  was dissolved in dry tetrahydrofuran (6 mL). To this solution, 100 μL (0.10 mmol) of Et2Zn 1 M in hexane was added. The reaction mixture was stirred for 24 h at room temperature, thus affording Zn–Oligo–C (23 mg) as a yellow precipitate, which was collected by filtration. 1H NMR  δ 8.2–8.0 , 7.3–6.3 , 5.1–5.0 , 3.5  ppm. Anal. Calcd. for C116H96N8O8Zn4: Found C, 69.91; H, 4.80; N, 5.55.In this manuscript we describe the synthesis and characterization of three new Zn–oligomer–based Salen complexes. The easy synthetic protocols based on the mixing of reagents with a proper stoichiometric ratio allowed for the obtainment of oligomers with four, six, and ten Salen units. These oligomers were tested as receptors for DMMP and we found excellent results with the shorter oligomer. In fact, the shorter oligomer showed the highest binding constant value with respect to the other sensors used for the non-covalent detection of DMMP. These results suggest the possibility to employ the oligomer hosts in the CWA detection, in order to obtain final solid devices with higher affinity for the analytes.