Vernonia patula.A molecular docking approach was employed to evaluate the binding affinity of six triterpenes, namely epifriedelanol, friedelin, α-amyrin, α-amyrin acetate, β-amyrin acetate, and bauerenyl acetate, towards the cannabinoid type 1 receptor (CB1). Molecular docking studies showed that friedelin, α-amyrin, and epifriedelanol had the strongest binding affinity towards CB1. Molecular dynamics simulation studies revealed that friedelin and α-amyrin engaged in stable non-bonding interactions by binding to a pocket close to the active site on the surface of the CB1 target protein. The studied triterpenes showed a good capacity to penetrate the blood–brain barrier. These results help to provide some evidence to justify, at least in part, the previously reported antinociceptive and sedative properties of  Cannabis sativa and other plant-derived cannabinoid-like molecules called cannabimimetics that can interact with the endogenous cannabinoid system +. The mobile phase used was a gradient of 1% formic acid in water and acetonitrile  at a flow rate of 0.2 mL/min. Elution was performed as follows; Fraction 1 (0 to 30 min—60 to 80% solvent B), fraction 2 (0 to 30 min—70 to 90% solvent B), fraction 3 (0 to 30 min—80 to 100% solvent B), and fraction 4 (0 to 30 min—100% solvent B).The extract (10 mg) was subjected to solid phase extraction (SPE) using a Hypersep C18 cartridge  eluting sequentially with 70, 80, 90, and 100% methanol (MeOH). Fraction 1 (1.5 mg), fraction 2 (1.8 mg), fraction 3 (2.1 mg), and fraction 4 (4.2 mg) were collected, and stock solutions (0.1 mg/mL) were prepared for further analysis. The sample solution and blank control were all stored at 4 °C and filtered through a 0.22 μm PTFE syringe filter  prior to analysis by HPLC-DAD-MS. Analysis was carried out using an Agilent HPLC 1260 binary pump and a Sunfire C18 column  . The diode array detector (DAD) was set at λ = 210, 254, 269, and 310 nm. The known, or suspected, major adducts for all samples were registered in the positive electrospray ionization (ESI) mode either as [M+H]Absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of the selected triterpenes were determined using the online ADMET structure-activity relationship database (admetSAR). The specific pharmacokinetic profile for each compound was obtained using compound specific SMILES strings . MLogP w1), friedelin (2), α-amyrin (3), α-amyrin acetate (4), β-amyrin acetate (5), and bauerenyl acetate (6) , geometry-optimized, and saved in PDB format using GaussView v.5.0 .As triterpenes had been reported as CB1 receptor agonists , we decitate (6) , previou acetate , β-amyri acetate , and bauhttps://pymol.org, accessed on 26 March 2021) [The crystal structure of the human CB1 cannabinoid receptor and its active site ,40 was rch 2021) . The str2 and 331.44 Å3, respectively. The grid box was generated so as to include the CB1 binding site residues, with a center set at 12.5230, 7.2495, and 17.7743 Å and a size set at 81.5, 81.5, and 81.5 Å in x, y, and z directions, respectively. Autodock Vina (v.1.1.2) was used to perform the molecular docking study [https://pymol.org, accessed on 26 March 2021), Drug Discovery Studio (v. 4.1) , and LigPlot+ (v. 1.4.5) .The active binding pocket of CB1 was predicted by CASTp (v. 3.0) . The proThe molecular dynamics (MD) simulations were performed on the CB1, CB1–THC, and three selected triterpene–CB1 complexes, namely CB1–friedelin, CB1–α-amyrin, and CB1–α-amyrin acetate. The first two complexes were chosen based on the highest docking scores obtained in the molecular docking study. The third complex was selected to observe the influence of an acetate group on the protein–ligand interactions. MD simulations were conducted using YASARA Dynamics v. 20.8.1 . The AMBX matrix according to the following equation:kT describes how the samples relate to each other, kP demonstrates how the variables relate to each other, k is the number of factors included in the model, and E is the matrix of residuals. PCA was conducted using the OriginPro 2021  software package.Principal component analysis (PCA) was used to analyze any subtle variability among the structural and energy profile data obtained from MD simulations for the selected triterpene–CB1 complexes and CB1 alone. Bond energies, bond angle energies, dihedral angle energies, planarity energies, Coulomb energies, Van der Waals energies, and RMSD-Cα values were included as the variables ,50,51. MThe root mean square deviations (RMSDs) of Cα atoms on the backbone of the CB1 protein and of the selected CB1–triterpene complexes were calculated during the MD simulation period using YASARA macro file followed by OriginPro 2021. Root mean square fluctuation (RMSF) analysis using YASARA macro script and OriginPro 2021 was used to observe the regions that fluctuated during the MD simulation period.complex = total free energy of the protein–ligand complex in solvent, ΔGligand = total energy of the ligand in solvent, and ΔGprotein = total energy of the protein in solvent. ΔGMM = molecular mechanics interaction energy, where the ΔGelec and ΔGVdW are the electrostatic and Van der Waals interactions, respectively. ΔGPB and ΔGSA represent polar solvation and nonpolar solvation energy, respectively. TΔS (temperature = T and entropy = S) is the contribution of entropy to the free energy.The molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method  was used


EB3 protein is expressed abundantly in the nervous system and transiently enters the dendritic spines at the tip of the growing microtubule, which leads to spine enlargement. Nevertheless, the role of dynamic microtubules, and particularly EB3 protein, in synapse function is still elusive. By manipulating the EB3 expression level, we have shown that this protein is required for a normal dendritogenesis. Nonetheless, EB3 overexpression also reduces hippocampal neurons dendritic branching and total dendritic length. This effect likely occurs due to the speeding neuronal development cycle from dendrite outgrowth to the step when dendritic spines are forming. Implementing direct morphometric characterization of dendritic spines, we showed that EB3 overexpression leads to a dramatic increase in the dendritic spine head area. EB3 knockout oppositely reduces spine head area and increases spine neck length and spine neck/spine length ratio. The same effect is observed in conditions of amyloid-beta toxicity, modeling Alzheimer`s disease. Neck elongation is supposed to be a common detrimental effect on the spine’s shape, which makes them biochemically and electrically less connected to the dendrite. EB3 also potentiates the formation of presynaptic protein Synapsin clusters and CaMKII-alpha preferential localization in spines rather than in dendrites of hippocampal neurons, while its downregulation has an opposite effect and reduces the size of presynaptic protein clusters Synapsin and PSD95. EB3′s role in spine development and maturation determines its neuroprotective effect. EB3 overexpression makes dendritic spines resilient to amyloid-beta toxicity, restores altered PSD95 clustering, and reduces CaMKII-alpha localization in spines observed in this pathological state. The synapse is a contact between two neurons, serving to transmit information from one cell to another. Most synapses in the brain are formed between the axon of one neuron and the small membranous protrusion from the dendrite—the dendritic spine, of another neuron. Dendritic spines have various shapes and sizes, which are constantly changing in response to neuronal activity and part of synaptic plasticity mechanisms ,2,3,4. Tp < 0.0001) and total dendritic length (p < 0.0001). The total dendritic length was 4394.34 ± 380.88 µm in the control group, 5331.25 ± 405.79 μm in shControl group, 2327.50 ± 254.44 μm in EB3, and 1621.97 ± 116.86 μm in shEB3 groups, correspondingly.It was shown that EB3 participates in the regulation of dendrite branching by association with PSD95 protein, which is thought to be the stop signal for dendrite branching . Here wep < 0.0001) and 20.12 ± 1.35% (****: p < 0.0001), respectively. We may conclude that both conditions potentiate a formation of immature dendritic protrusions. EB3 overexpression significantly decreases the filopodia fraction to 6.945 ± 0.83% (***: p < 0.0001) to the level comparable with neurons in the control group. Spine head area is known to correlate with the area of the postsynaptic density (PSD), the number of postsynaptic receptors, and the ready-releasable pool of transmitter PSD area [2 to 0.46 ± 0.01 (****: p < 0.0001) μm2 in wild-type neurons and from 0.22 ± 0.01 μm2 to 0.28 ± 0.01 μm2 (****: p < 0.0001) in conditions of amyloid toxicity. In the EB3 knockdown group, spine head area drops to 0.26 ± 0.01, versus 0.30 ± 0.01 μm2 in the shControl group (**: p < 0.01). Spine neck length is tightly linked to its electrical properties, as well as the biochemical and electrical connection between the spine from its neighboring dendrite [p < 0.0001). Amyloid toxicity also leads to an increase in the spine neck length to 734 ± 20 nm, in comparison with 623 ± 18 in the control group (**: p < 0.01). Finally, the ratio between the neck length and the total spine length was analyzed in order to show what part of overall spine length falls on the neck. EB3 knockdown and amyloid toxicity leads to an increase in this ratio to 52.2 ± 0.91% (****: p < 0.0001) and 51.06 ± 0.72% (***: p < 0.001), in comparison to 44.20 ± 0.75 in the control group, and 43.28 ± 0.91 in the shControl group. EB3 overexpression in the condition of amyloid toxicity leads to a decrease in neck length/spine length ratio to normal levels .In previous studies, we discovered that EB3 overexpression increases the percent of mushroom dendritic spines and protects them from elimination in hippocampal neurons derived from PS1-M146V-KI mice, modeling Alzheimer’s disease in vitro . These tPSD area . EB3 ovedendrite ,42,43. I2 to 0.048 ± 0.004 μm2 (p < 0.0001), and Synapsin clusters from 0.260 ± 0.012 μm2 to 0.146 ± 0.017 μm2 (p < 0.0001), and a decrease in Synapsin cluster density from 0.19 ± 0.01 1/μm to 0.11 ± 0.01 1/μm (p < 0.01). For studies of the EB3 protein neuroprotective potential, oligomeric Aβ42 peptides were added for 72 h before the fixation. Conditions of low amyloid-beta toxicity don’t influence the PSD-95 and Synapsin clusters density in hippocampal neurons but cause a significant decrease in their size—areas of both protein clusters drop to the levels of 0.050 ± 0.003 μm2 (p < 0.0001) for PSD95 and 0.148 ± 0.017 μm2 (p < 0.0001) for Synapsin (p < 0.05) in conditions of amyloid toxicity.To further study the effects of EB3 on neuronal maturation, we analyze the density and the size of PSD-95 and Synapsin 1 clusters, and postsynaptic and presynaptic proteins are shown to be robust general markers of synapses . Cells wSynapsin C,E. Virup < 0.001). Virus-mediated EB3 knockout and application of oligomeric beta-amyloid reduced the CaMKII-alpha spine/dendrite ratio to 1.43 ±0.03 and 1.35 ± 0.03, correspondingly . Virus-mediated EB3 overexpression restores the CaMKII-alpha spine/dendrite ratio in conditions of beta-amyloid toxicity to 1.51 ± 0.05 (*: p < 0.05).To investigate the localization of CaMKII-alpha, neurons were co-transfected with the plasmid Venus-CaMKII-alpha and mCherry and infected with lentiviruses encoding control RNAi (shControl), RNAi against EB3 (shEB3), or EB3 at DIV 8-9. At DIV 16-17, cultures were fixed, and analysis of fluorescent protein distribution was performed using confocal microscopy A. To assBased on the obtained data, we may conclude that EB3 protein level has a strong impact on both morphology of dendrites and dendritic spines . PreviouWe showed that EB3 overexpression leads to a dramatic increase in the dendritic spine head area. This is consistent with previous findings, indicating increases in mushroom dendritic spine fraction in hippocampal neurons during EB3 overexpression ,23. HoweIn previous studies, we discovered that EB3 overexpression protects mushroom dendritic spines from elimination in primary hippocampal neurons derived from PS1-M146V-KI Alzheimer’s disease mouse model . It thisPrimary hippocampal neuronal cultures of dissociated hippocampal cells were prepared from newborn FVB mice. Briefly, the hippocampus of postnatal day 0–1 mouse pups were digested with papain solution , then dissociated with 5 mg/mL Deoxyribonuclease I  solution. Neurons were plated in a 24-well culture plate on 12 mm glass coverslips precoated with 1% poly-D-lysine  in Neurobasal-A  medium supplemented with 2% B27 , 1% heat-inactivated fetal bovine serum , 0.5 mM L-Glutamine , and maintained at 37 °C in a 5% CO2 incubator at 24-well glass plate. Transfection is performed at DIV 6-7, then at DIV 15-16, when hippocampal neurons reach maturity and form extensive synaptic contact cells, cells are fixed with a solution of 4% formaldehyde and 4% sucrose in PBS, pH 7.4 for 15 min and then extensively washed with PBS to remove fixation solution. Transfection was performed according to  with a cHEK293T line cells with 50–70% of confluency were co-transfected with shuttle lenti-FLAG-EB3, lenti-shRNAi to scrambled RNA sequence (shControl) or against mouse EB3 (shEB3) and two helper plasmids pCMVΔ8.9 and pVSVg using polyethylenimine reagent  in serum-free Opti-Mem medium . After 5–6 h incubation in a CO2 incubator, Opti-Mem was replaced with neuronal culture medium . 48–72 h after transfection, culture medium was collected, centrifuged 5 min at 2000 rpm, filtered through 0.45 um pore, immediately frozen in liquid nitrogen and then stored at −80 °C. Influence of each batch of generated lentiviruses on EB3 protein expression level in hippocampal neuronal culture was tested by Western blot with rabbit anti-EB3 antibodies  and mouse anti-actin  antibodies as loading control A,B. The For assessment of dendrites and dendritic spine morphology, a Z-stack of the optical section was captured with a confocal microscope . For dendritic analysis 2048 × 2048 pixel images with 0.25 μm/pixel resolution were captured with Z interval of 1 μm using a 20× objective lens . For dendritic spine analysis, 2048 × 2048 pixel images with 0.025 μm/pixel resolution were captured with Z interval of 0.1 μm using a 100× objective lens . Quantitative analysis of dendritic length and branching was performed with the help of the Sholl analysis module of a freely available Neurostudio software package . Quantithttp://www.pvv.org/~perchrh/imagej/thresholding.html) (accessed on 20 December 2021) with the following parameters: r = 2, C = 160, local weight = 5%, to identify protein clusters. Further, the density and area of the clusters were calculated using the Synpanal software [Fixed primary hippocampal neurons washed three times with PBS and permeabilized in 0.25% Triton X-100 in PBS solution for 15 min at room temperature. Nonspecific binding was blocked by incubating cells in 5% bovine serum albumin (BSA) in PBS solution for 1 h. Primary antibodies anti-MAP2 , anti-synapsin I , and anti-PSD-95  were diluted in 2.5% BSA in PBS and incubated at +4 °C overnight. After three washes, the hippocampal cultures were incubated in 2.5% BSA in PBS solution with the secondary antibody  for 1 h at room temperature and visualized with a confocal microscope. For PSD95/Synapsin puncta analysis, 2016 × 2016 pixels images with 0.034 μm/pixel resolution were captured with Z interval of 0.2 μm using a 100× objective lens . All images were pre-processed with a rolling ball filter to reduce noise. The MAP2 images were binarized using the Lee minimum cross-entropy method to identify dendrite boundaries. PSD95/Synapsin images were binarized with ImageJ Adaptive 3D Threshold 1.22 plugin  with 0.03 μm/pixel resolution, and 0.1 Z interval using a 100× objective lens . To assess the CaMKII-alpha spine/dendrite ratio we utilized the approach from ,30,31. BThe statistics were analyzed using R Studio and Microsoft Excel programs. Statistical comparison of the data with normal distribution was carried out using the Student’s t-test and ANOVA. Normality was checked using the Shapiro–Wilk test. For the data with non-normal distribution nonparametric comparison Kruskal–Wallis test with post-hoc Dunn’s test was used. Statistical tests are indicated in figure legends. Data is presented as mean ± S.E.M.


CYP2D6 poor metabolizers (PMs) that receive CYP3A4 inhibitors and tamsulosin. However, no specific pharmacogenetic guideline exists for tamsulosin. Furthermore, to date, no pharmacogenetic information is available for dutasteride. Henceforth, we studied the pharmacokinetics and safety of dutasteride/tamsulosin 0.5 mg/0.4 mg capsules according to 76 polymorphisms in 17 candidate pharmacogenes. The study population comprised 79 healthy male volunteers enrolled in three bioequivalence, phase-I, crossover, open, randomized clinical trials with different study designs: the first was single dose in fed state, the second was a single dose in fasting state, and the third was a multiple dose. As key findings, CYP2D6 PMs  and intermediate metabolizers (IMs)  presented higher AUC (p = 0.004), higher t1/2 (p = 0.008), and lower Cl/F (p = 0.006) when compared with NMs  and UMs  after multiple testing correction. Moreover, fed volunteers showed significantly higher tmax than fasting individuals. Nominally significant associations were observed between dutasteride exposure and CYP3A4 and CYP3A5 genotype and between tamsulosin and ABCG2, CYP3A5, and SLC22A1 genotypes. No association between the occurrence of adverse drug reactions and genotype was observed. Nonetheless, higher incidence of adverse events was found in a multiple-dose clinical trial. Based on our results, we suggest that dose adjustments for PMs and UMs could be considered to ensure drug safety and effectiveness, respectively. Further studies are warranted to confirm other pharmacogenetic associations.Dutasteride and tamsulosin are one of the first-line combination therapies for the management of benign prostatic hyperplasia (BPH). Despite being more effective than monotherapies, they produce frequent adverse drug reactions (ADRs). Institutions such as Food and Drug Administration and European Medicines Agency recommend precaution with  Dutasteride and tamsulosin are one of the first-line combination therapies for the management of benign prostatic hyperplasia (BPH). Combination therapy is frequent in BPH patients, due to difficulties in reaching effectiveness with single treatments .α reductase inhibitors (5-ARIs), which prevent dihydrotestosterone production and, consequently, delay prostatic tissue growth. It is administered by oral route. It presents 60% oral bioavailability, and its median time to reach maximum plasma concentration (tmax) is around 3 h (1–10 h range) after the administration of 0.5 mg single dose. Dutasteride shows a volume of distribution (Vd) of 300–500 L and a high plasma protein binding (>99.5%). Its elimination is dose-dependent. At single doses lower than 5 mg, dutasteride clearance is rapidly performed, with a shorter half-life (t1/2) of 3–9 days. However, at 0.5 mg daily doses, the elimination is slower, reaching a t1/2 of 3–5 weeks. It is extensively metabolized by cytochrome P450 (CYP) isoforms CYP3A4 and CYP3A5 into four major metabolites: two of them less active than dutasteride and two other that are similarly active to the parent drug. They are primarily excreted in stools and marginally in urine; only between 1 and 15.4% of the dutasteride dose is excreted unmetabolized in feces. It has been also reported that dutasteride is not metabolized in vitro by human cytochrome P450 isoenzymes CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 (Dutasteride belongs to 5-d CYP2E1 .max is 6 h (2–24 h range). It has a median t1/2 of 10–13 h both in single-dose and multiple-dose regimens. It is 90% metabolized by mainly CYP3A4 and CYP2D6. The remaining unaltered tamsulosin  is excreted in urine . A summary table of the correspondence between diplotypes and phenotypes is provided in Genotypes were used to infer haplotypes which define phenotypes or diplotypes. The genotyping technique used does not allow knowing with complete certainty whether or not two polymorphisms are located on the same chromosome. This is important in order to correctly define alleles. However, the location of these polymorphisms can be inferred with sufficient confidence from the allele frequency data available. Consequently, allele assignment was conducted according to Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for ry drugs , CYP2C19conazole , CYP2D6  opioids , CYP3A5 crolimus , SLC O 1vastatin , 1, and azanavir , 1. The ublished , individDuring hospitalization, volunteers were asked about treatment tolerability in several occasions. Adverse events (AEs) reported after open questions as well as self-reported AEs were registered in volunteers’ data collection logbook. The causality between drug administration and the occurrence of adverse events (AEs) was evaluated following Karch–Lasagna  algorithCYP1A2 alleles were analyzed independently). Race and clinical trial were added as covariates, and dose/weight correction was applied as control confounding variables. Hardy–Weinberg equilibrium was calculated by χ2 test comparing observed and expected allele distributions. Regarding the pharmacokinetics analysis, Vd and Cl were adjusted for bioavailability  becoming Vd/F and Cl/F, respectively. AUC∞, AUCτ, Cmax, and Cmin were adjusted for the dose-weight ratio (DW). Tamsulosin data were obtained both from multiple- and single-dose studies. Since AUC∞ after a single dose and AUCτ are equivalent , both variables were merged into a single “AUC” variable. Normality was analyzed by quantile–quantile plots. Homoscedasticity was tested by Levene’s test. For homoscedastic normal variables, differences in means were studied by t-test (two categories within a variable) or ANOVA (three or more categories within a variable) with logarithmically transformed pharmacokinetic parameters , in order to achieve normal distribution. For those variables with three or more groups, a pairwise comparison Bonferroni post hoc analysis was performed. For heteroscedastic variables, differences in means were studied by Welch’s t-test (two categories within a variable) or Welch’s ANOVA (three or more categories within a variable). A multivariate analysis was performed by means of linear regression. The significant variants from the univariate analysis and the study design were considered the independent variables for the multivariate analysis of all pharmacokinetic parameters, which were established as dependent variables. Benjamini and Hochberg correction for multiple comparisons was performed, i.e., false discovery rate (FDR) after multivariate analysis . For the multivariate analysis, significance (p < 0.05) was indicated with the unstandardized β-coefficient, R2 value, p of multivariate analysis (pMV), and p after FDR (pFDR). All calculations were computed in R version 4.0.3 software  of Hospital Universitario de la Princesa and the Spanish Drug Agency (AEMPS). S1 (EUDRA-CT number: 2017-001592-23), S2 (EUDRA-CT number: 2017-003227-29), and M (EUDRA-CT number: 2017-003244-21) clinical trials were performed according to Spanish regulation, ICH guidelines for Good Clinical Practices, and Revised Declaration of Helsinki .2. The population was composed of 52 (74%) Caucasians and 18 (26%) Latin individuals. No significant differences in demographics were found between these two groups.The study population comprised 79 male healthy volunteers, defined by mean ± standard deviation, with a median age of 24 ± 6.7 years old, mean height of 1.76 ± 0.07 m, mean weight of 76.87 ± 8.72 kg, and body max index (BMI) of 24.86 ± 2.26 m/kgCYP1A2 *1C (rs2069514), CYP2A6 *9 (rs28399433), CYP2B6 *4 (rs2279343), ABCB1 rs2032582, CYP2C8 *8 (rs1058930), and CYP3A4 *22 (rs35599367).All polymorphisms analyzed were in Hardy–Weinberg equilibrium, except for max for CYP2A6 *9 and SLC22A1 *2, t1/2 for CYP1A2 *1B and CYP2C9 phenotype, and Vd/F for CYP2C9 phenotype.All the analyzed variables presented normal distributions after logarithmic adjustment. All variables presented homoscedastic distribution except tmax (pANOVA = 0.002) and higher Vd/F than fasting conditions (pANOVA = 0.006) after univariate analysis. Moreover, CYP3A4*22 allele carriers showed lower Vd/F than *1/*1 individuals (pANOVA = 0.023). Additionally, SLC28A3 rs7853758 A/G and A/A subjects presented lower AUC (pANOVA = 0.012) and higher Cl/F (pANOVA = 0.043) than G/G. SLC28A3 rs7853758  maintained significance in multivariate analysis for AUC. Food , CYP3A4 genotype , and SLC28A3 rs7853758  remained significant after multivariate analysis for Vd/F. Nonetheless, all of these variables lost significance after FDR correction  than fasting and multiple dose. Fasting conditions and multidose administration exhibited lower tmax  and higher Cmax  than fed conditions. Moreover, multivariate analysis also revealed that single-dose administration showed lower Vd/F  and lower t1/2  than single-dose trials. ABCG2 rs2231142 C allele carriers presented higher Vd/F (pANOVA = 0.014) than G/G individuals. Univariate and multivariate analysis also revealed that CYP2D6 UMs and NMs presented lower AUC than PMs and IMs . Additionally, UMs and NMs had lower t1/2 , higher Vd/F , and lower Cl/F  than PMs and IMs. CYP3A5 NMs and IMs presented higher Vd/F (pANOVA = 0.019) and Cl/F (p = 0.027) than PM. Finally, SLC22A1 *1/*3 individuals presented higher AUC (pANOVA = 0.020), higher Cmax (pANOVA = 0.017), higher Cmin (pANOVA = 0.038), and lower Cl/F (pANOVA = 0.026) than *1/*1 volunteers  and retrograde ejaculation (n = 3), followed by libido decrease (n = 2) and ejaculation disorder (n = 2). The remaining ADRs were only observed in one volunteer. Participants in the multiple-dose clinical trial were related to higher incidence of ADR than participants in single-dose . No relationship between polymorphisms or race with ADR occurrence was found.No serious ADR was reported. The ADRs reported comprised dizziness, testicular pain, epididymo-orchitis, headache, ejaculation disorder, hypotension symptomatic, retrograde ejaculation, libido decreased, and abnormal urine odor. Eight volunteers presented at least one ADR. The most frequent ADRs were headache  delay gastric emptying, augmenting the transit time to the small intestine and, subsequently, delaying the absorption into the systemic circulation . As expekinetics .CYP3A4 and CYP3A5 polymorphism . Similar to the explanation for the feeding conditions, these volunteers consistently presented significantly lower Vd/F. Moreover, CYP3A5 NMs and IMs showed a higher tmax compared to PMs. This might reflect a reduced rate of elimination by PMs. As the elimination rate decreases, it requires less time for the drug to accumulate and to reach peak concentration. Consistently, we observed a 30% greater t1/2 in PMs compared to NM + IMs . However, as these results did not remain significant after multiple testing corrections, they might be spurious. Lastly, SLC28A3 rs7853758 A/G and A/A subjects presented lower AUC, lower t1/2, and higher Cl/F than G/G. Nonetheless, none of these associations remained significant after FDR correction. Further studies are warranted to confirm whether CYP3A or SLC28A3 polymorphism affects dutasteride pharmacokinetics.Consistent with the well-known pharmacokinetic profile of dutasteride, pharmacokinetic variability was significantly related to morphism . CYP3A4 max of 11.3 ± 4.44 ng/ml compared to 147.4 ± 72.8 ng·h/ml and 10.35 ± 3.77 ng/ml, respectively (Agencia Española del Medicamento y Productos Sanitarios (AEMPS), 2015) (JALYN). No significant difference was found between the two groups of race and any pharmacokinetic parameter.The observed tamsulosin pharmacokinetic parameters under single-dose after fed conditions were similarly consistent with the literature, for example, AUC of 187.2 ± 95.7 ng·h/ml and Cmax or Cmax). This is likely caused by the different solubility of a drug based on the stomach pH and the transit time to the small intestine. Previous works state that fasting conditions are related to faster and greater tamsulosin absorption (max (15%) compared to fed volunteers. Congruent with literature, a 16% higher Cmax was also observed in fasting volunteers; nonetheless, the association was not significant   .CYP3A4 and CYP2D6, but also by other cytochrome P450 isoforms to a lesser extent  is 0.4 mg, both in Europe and United States  . It is an ATP-binding cassette transporter and plays a major role in multidrug resistance, specially involved in the response to mitoxantrone and anthracycline (acycline . The impacycline . In thisSLC22A1 encodes for the organic cationic transporter 1 (OCT1), one of the three similar polyspecific cationic transporters mediating the uptake of many organic cations from the blood. It has substrate selectivity for a variety of endogenous ligands  as well as cationic drugs, such as metformin, cimetidine, imatinib, oxaliplatin, and tramadol and agmatine. OCT1 carries drugs into the liver and kidneys, where the compound is metabolized and excreted . We are confident with the robustness of our genotyping, but we must note this limitation. Thus, further studies are required to confirm the results here obtained.Nonetheless, this study presents several limitations. First, the sample size is small. To address this issue, three different clinical trials were analyzed. This leads to the second limitation: merging of three different study designs complicates the statistical analysis. The study design was analyzed as a covariate, but despite this, the statistical power is more limited than in a unique study design. Furthermore, the incidence of CYP2D6 phenotype severely affected tamsulosin pharmacokinetics. PMs and IMs presented twice higher exposure to tamsulosin than UMs and NMs. The results were consistent with the literature and the guidelines of regulatory institutions, such as FDA and EMA, which do not include specific dose adjustment recommendations. Here, we suggest that a dose adjustment could improve tamsulosin effectiveness and safety. Further studies are warranted to confirm whether this adjustment would be beneficial for the patient. Alternatively, dutasteride pharmacokinetics was not altered based on genotypes or drug dose regimen. To the best of our knowledge, this is the largest study analyzing tamsulosin pharmacogenetics (n = 79) and the first study of this type for dutasteride. Additionally, new potential associations were proposed regarding ABCG2, CYP3A4, CYP3A5, and SLC22A1. However, the main limitation of this study is the limited sample size. Consequently, further prospective studies should be addressed to confirm such associations.


Vibrio harveyi BB170 as a reporter strain for the preliminary screening of 14 inhibitors and compound Str7410 had higher AI-2 QS inhibition activity (IC50 = 0.3724 ± 0.1091 μM). Then, co-culture of Pseudomonas aeruginosa PAO1 with Staphylococcus aureus ATCC 25923 was used to evaluate the inhibitory effects of Str7410 on multispecies infection in vitro and in vivo. In vitro, Str7410 significantly inhibited the formation of mixed bacterial biofilms. Meanwhile, the combination of Str7410 with meropenem trihydrate (MEPM) significantly improved the susceptibility of mixed-species-biofilm cells to the antibiotic. In vivo, Str7410 significantly increased the survival rate of wild-type Caenorhabditis elegans N2 co-infected by P. aeruginosa PAO1 and S. aureus ATCC 25923. Real-time quantitative PCR analysis showed that Str7410 reduced virulence factor (pyocyanin and elastase) production and swarming motility of P. aeruginosa PAO1 by downregulating the expression of QS-related genes in strain PAO1 in co-culture with S. aureus ATCC 25923. Compound Str7410 is a candidate agent for treating drug-resistant multispecies infections. The work described here provides a strategy for discovering novel antibacterial drugs.Bacterial drug resistance caused by overuse and misuse of antibiotics is common, especially in clinical multispecies infections. It is of great significance to discover novel agents to treat clinical bacterial infections. Studies have demonstrated that autoinducer-2 (AI-2), a signal molecule in quorum sensing (QS), plays an important role in communication among multiple bacterial species and bacterial drug-resistance. Previously, 14 AI-2 inhibited compounds were selected through virtual screening by using the AI-2 receptor protein LuxP as a target. Here, we used  Most bacterial diseases are caused by infection with multiple species of bacteria, for example, cystic fibrosis (CF) and chronic wounds . On co-iPseudomonas aeruginosa and Staphylococcus aureus are the two most important pathogens in CF and chronic wounds  quorum sensing (QS) system (c wounds . Interacin vitro . In a woncreased . Moreovel wounds  and mousl wounds  was sign) system . This invia AI molecules, and this regulates gene expression  research . Bacteria sense the density of the surrounding population pression . AI-2 is harveyi . Bioluminescence . Thereforesearch . Researcresearch . AI-2-meresearch .S. aureus has two types of QS system. One is the autoinducing peptide signaling molecule-mediated agr system, and the other is the LuxS/AI-2 system  has been found by searching for established AI-2 receptors in S. aureus genomes  signaling molecules  production, swarming motility, and the expression of QS-related genes in co-culture of P. aeruginosa and S. aureus. We used Caenorhabditis elegans N2 as an in vivo model to test the effects of Str7410 on survival rates on co-infection of the nematode with P. aeruginosa and S. aureus. Finally, we preliminarily evaluated the inhibitory mechanism of interspecies QS by the compound. This study provides a new strategy for the treatment of clinical multispecies infections.In our previous study, 14 AI-2 QSI compounds were selected through virtual screening. In this study, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy were recorded on a JEOLECA400 spectrometer, with TMS as an internal standard at ambient temperature. All coupling constants were reported in Hertz. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). High-resolution mass spectra (HRMS) were obtained by electrospray ionization (ESI) using an Agilent TOF G6230A mass spectrometer. The synthesis of compound Str7410 was shown in All the chemical reagents used in the synthesis reactions were analytical-grade and available from commercial resources without further purification. All the reactions were monitored by analytical thin-layer chromatography (TLC) using silica gel TLC plates (GF254). Silica gel (200–300 mesh) was used for chromatography. The N-tert-butoxycarbonyl-1,2-ethylenediamine  in CH3CN (60 ml) was added K2CO3 , then the mixture was heated to 65°C. At this temperature, 1-bromoheptane  in CH3CN (10 ml) was added dropwise to the mixture. The mixture was stirred at 80°C for 6 h and cooled to room temperature. The solvent was concentrated in vacuo. The residue was dissolved in CHCl3, washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by flash silica gel chromatography  to afford 1 as colorless oil . 1H NMR  δ 6.69 , 2.98 , 2.53–2.48 , 2.46 , 1.37 , 1.25 , and 0.86 . 13C NMR  δ 156.14, 77.94, 49.52, 31.83, 30.10, 29.21, 28.75, 27.29, 22.59, and 14.46. HRMS (ESI) of C14H30N2O2 [M + H]+ calcd., 259.2381; found 259.2380.To a solution of 2Cl2 (7.5 ml) was added tert-butyl (2-(heptylamino)ethyl)carbamate , then the mixture was warmed to room temperature and stirred for 2 h. The solution was concentrated in vacuo and used directly in the next step without further purification.To a cold (0°C) solution of trifluoroacetic acid (2.5 ml) and CHN1-heptylethane-1,2-diamine  in CH3CN (40 ml) was added K2CO3  and ethyl 2-bromoacetate . Then, the mixture was stirred at 45°C for 3 h and cooled to room temperature. The solvent was concentrated in vacuo. The residue was dissolved in CHCl3, washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by flash silica gel chromatography  to afford 3 as colorless oil . 1H NMR  δ 4.09 , 3.57 , 3.37 , 2.93 , 2.89 , 2.86–2.81 , 1.50 , 1.27 , 1.21 , and 0.87 . 13C NMR  δ 171.47, 169.40, 60.46, 56.23, 55.06, 54.58, 52.49, 50.32, 31.64, 28.90, 26.73, 25.54, 22.53, 14.60, and 14.43. HRMS (ESI) of C21H40N2O6 [M + H]+ calcd., 417.2959; found 417.2960.To a solution of in vacuo. The residue was added to concentrated hydrochloric acid (15 ml), and the mixture was stirred at 100°C for 12 h and concentrated in vacuo. The residue was recrystallized with ethanol (5 ml) and methyl tert-butyl ether , filtered, and washed with MTBE to afford 4 as white solid . 1H NMR  δ 4.23 , 3.63 , 3.37 , 3.26–3.19 , 3.16 , 1.71–1.59 , 1.26 , and 0.89–0.83 . 13C NMR  δ 172.64, 168.12, 55.11, 54.97, 53.39, 51.70, 49.28, 31.51, 28.67, 26.34, 23.68, 22.49, and 14.44. HRMS (ESI) of C15H28N2O6 [M + H]+ calcd., 333.2020; found 333.2019.Diethyl 2,2'-((2-((2-ethoxy-2-oxoethyl)(heptyl)amino)ethyl)azanediyl) diacetate  was added to concentrated hydrochloric acid (15 ml). The mixture was heated to 100°C and stirred for 12 h. Then, the residue was cooled to room temperature and concentrated Vibrio harveyi BB170 (ATCC BAA-1117), S. aureus ATCC 25923, P. aeruginosa PAO1, and E. coli OP50 were used in this study. AB medium contained 0.3 M NaCl, 0.05 M MgSO4, and 2% acid-hydrolyzed casein (without vitamins), adjusted to pH 7.5 with 1 M KOH. After autoclaving at 121°C, 10 ml of 1 M potassium phosphate buffer , 10 ml of 0.1 M sterile arginine solution (L-Arg), and 20 ml of 50% sterile glycerol were added to the medium (per 1 L). LB medium contained 1% tryptone, 1% NaCl, and 0.5% yeast extract. TSB medium contained 1.5% tryptone, 0.5% soy peptone, and 0.5% NaCl (pH = 7.2 ± 0.2). PB medium contained 2% peptone, 1% K2SO4, and 0.3% MgCl2. PTSB medium contained 5% peptone, 1.7% tryptone, 0.5% NaCl, 0.25% K2HPO4, 0.25% glucose, and 0.3% soy peptone. PGS-agar plates contained 1% peptone, 0.15 M sorbitol, 1% NaCl, 1% glucose, and 1.7% agar. Swimming solid medium contained 0.8% nutrient broth, 0.5% glucose, and 0.3% agar. Swarming solid medium contained 0.8% nutrient broth, 0.5% glucose, and 0.5% agar.Test compounds were dissolved in DMSO to a stock concentration of 65 mM. Antibiotics  were purchased from Sigma and stored at 6.4 mg/ml at −20°C.V. harveyi BB170 bioluminescence  was tested as described previously  to OD600 nm = 1.5. The cells were diluted into fresh AB medium  and added (100 μl/well) to serially diluted test compounds in AB medium (100 μl/well); the final DMSO concentration was 0.5%. The plate was incubated at 30°C for 8 h, and then the bioluminescence (OD460 nm) and cell density (OD600 nm) were measured. The values of OD460 nm and OD600 nm were normalized and IC50 values were calculated using GraphPad Prism 5 software.The concentration of compounds that resulted in 50% maximum eviously . Vibrio Pseudomonas aeruginosa PAO1 and S. aureus ATCC 25923 were inoculated into LB medium, respectively, and cultured at 37°C at 150 rpm until OD600 nm = 0.5. Then, the cultures were diluted with fresh LB medium to OD600 nm = 0.05. Next, the bacteria were cultured in the presence of compound Str7410 , and OD600 nm was measured at intervals of 2 h for up to 24 h using a spectrophotometer . All experiments were performed three times, independently . The final test compound concentrations were 80, 40, 20, 10, and 1 μM. The 96-well plate was incubated at 37°C for 24 h, then the bacterial culture was removed, and the plate was washed three times with phosphate-buffered saline (PBS) and dried. Next, 0.1% (w/v) crystal violet solution was added for 10 min. After washing three times with PBS, the crystal violet-stained biofilms were air-dried. To quantify the biofilm biomass, the crystal violet was removed by adding 150 μl of 33% glacial acetic acid, and OD595 nm of the solubilized dye was determined using a Biotek multifunction microplate reader. The biofilm inhibition rate was defined as (OD595 blank − OD595 sample)/OD595 blank × 100%. Each crystal violet assay was run in triplicate, with a minimum of three replicates per assay.Quantitative analysis of biofilms used crystal violet assay . First, Pseudomonas aeruginosa PAO1 and S. aureus ATCC 25923 were, respectively, cultured in LB medium to OD600 nm = 0.8. Pseudomonas aeruginosa PAO1 alone or in co-culture with S. aureus ATCC 25923  were diluted 1,000-fold with fresh LB medium and added to a 96-well plate. According to the broth dilution method, Antibiotics concentrations ranged from 64 to 0.125 μg/ml. Finally, the plates were incubated at 37°C for 18 h, and OD590 nm was determined using a Biotek multifunction microplate reader.The determination of minimal inhibitory concentrations (MICs) was performed as described previously . PseudomDetermination of minimum biofilm inhibitory concentrations (MBICs) of antibiotics . Based o460 nm was measured using a Biotek multifunction microplate reader. The MBEC represents the lowest concentration of constituents for which the biofilm eradication activity was >99%. Biofilm eradication activity (%) was determined using the following formula:For the minimum biofilm eradication concentrations (MBECs) of compound for biofilm cells , 2020, tTreatment of biofilms and quantification of cells was performed as follows. Dual-species biofilms were grown in 96-well plate. The liquid medium was decanted every 2 days leaving the attached cells at the bottom of the plate, and fresh LB medium was added . After 6Pseudomonas aeruginosa PAO1 and S. aureus ATCC 25923 were cultured to OD600 nm = 2.0 at 37°C in TSB medium, respectively. Then, after mixing in equal proportions, 20 μl of culture were spread on PGS-agar plates containing antibiotic or test compound. The plates were placed in an incubator at 37°C and cultured for 18 h to allow the formation of a bacterial lawn. Then, 50 synchronized nematodes (L4 stage of wild-type C. elegans N2) were selected and placed on a plate. The plates were incubated at 20°C and the number of living and dead nematodes was counted every 24 h using a stereomicroscope. A nematode was defined as dead when it no longer responded to touch. Any nematodes that died as a result of getting stuck to the wall of the plate were excluded from the analysis. Survival curves were drawn for analysis  with and without the test compound  were incubated at 37°C and 150 rpm for 16 h. To extract pyocyanin, the culture was centrifuged at 1,665 × g for 10 min, and the supernatant was collected and extracted with 3 ml of chloroform. Then, 1 ml of 0.2 M HCl was mixed with the chloroform layer, and the upper (aqueous) phase was collected by centrifugation at 1,665 × g for 10 min. The absorbance was measured at 520 nm (520 blank − OD520 sample)/OD520 blank × 100%.Pyocyanin was quantified as follows: t 520 nm . The inhPseudomonas aeruginosa PAO1 and S. aureus ATCC 25923 were, respectively, cultured in PTSB medium at 37°C and 150 rpm to the logarithmic growth phase. After mixing in equal proportions, co-cultures were divided into the experimental group and the control group, and then incubated at 37°C and 150 rpm for 18 h. Then, the cultures were centrifuged at 9,590 × g for 5 min and the supernatant was collected. The bacteria were filtered through a 0.22-μm pore-size filter. Then, 200 μl of the filtrate was added to 1 ml of Elastin-Congo red reaction solution , and the mixture was shaken for 18 h at 37°C. Stop-reaction solution  was added and the mixture was incubated on ice for 5 min, and then centrifuged at 9,590 × g for 10 min. The supernatant was collected and OD495nm was determined.Elastase was determined using the Elastin-Congo red assay with modifications, and the final absorbance was read at 495 nm . PseudomPseudomonas aeruginosa PAO1 was shaken in LB medium at 37°C until OD600 nm = 1.0. Bacterial culture (1 μl) was inoculated into the center of cooled swimming solid medium and further cultured at 30°C for 16 h. Staphylococcus aureus ATCC 25923 was shaken at 37°C in LB medium to OD600 nm = 0.5. The culture was centrifuged at 9,590 × g for 5 min, and the supernatant was filtered (0.22-μm pore-size). The filtered supernatant was added to thawed swarming solid medium at a ratio of 1:10, mixed, poured into a plate, and left to stand for 8 h to dry. Finally, colonies were picked from the P. aeruginosa PAO1 swimming solid medium, added to the center of the swarming solid plate, and cultured at 30°C for 16 h  and grown at 37°C and 150 rpm for 10 h. Pseudomonas aeruginosa PAO1 was also grown in monoculture. After centrifugation  to retain the bacterial cells, a one-step cDNA kit was used [5× All-In-One RT MasterMix  for cDNA Extraction]; the DNA was stored at −20°C.2O. The primer sets used for these analyses are listed in sample – ΔΔCt, reference.Quantitative reverse transcriptase PCR (qRT-PCR) was performed with a Roche LightCycler® 480 instrument and EvaGreen 2× qPCR MasterMix. The 20-μl reaction system contained 0.6 μl each of forward and reverse primers, 1 μl template cDNA, 10 μl EvaGreen 2× qPCR MasterMix, and 7.8 μl nuclease-free Hp < 0.05  by one-way ANOVA with t-tests using GraphPad Prism 8 software.All assays were performed with three replicates and the values obtained are expressed as the mean ± SD. Differences in data were compared with the untreated control at each time-point and considered significant when N-tert-butoxycarbonyl-1,2-ethylenediamine with 1-bromoheptane generated intermediate 1. Removal of the Boc amino-protecting group followed by reaction with ethyl 2-bromoacetate afforded intermediate 3. Acidic hydrolysis twice afforded the desired compound Str7410.Synthesis of compound Str7410 was accomplished in four steps from the commercially-available 1-bromoheptane . Single V. harveyi BB170 (a luxN mutant) as a reporter strain to evaluate the AI-2 QS inhibitory activity of test compounds. The IC50 values of compounds were tested in terms of V. harveyi BB170 bioluminescence. Bromo-furanone C56 (V. harveyi BB170 (IC50 = 0.3724 ± 0.1091 μM). Based on this, compound Str7410 was selected for further research.We used none C56  was usedP. aeruginosa PAO1 and S. aureus ATCC 25923. As The main difference between QSIs and antibiotics is that QSIs inhibit the production of virulence factors without killing pathogenic bacteria, so that the bacteria do not develop drug-resistance mutations . TherefoP. aeruginosa PAO1 and S. aureus ATCC 25923 mixed in equal proportion. As shown in p < 0.05)–48.40% (p < 0.001)]. The inhibitory effect of compound Str7410 was concentration-dependent. When the concentration of Str7410 was 40 μM, the inhibition rate of biofilm formation was 40.58% (p < 0.01).Bacterial resistance to antibiotics is related to the formation of biofilms . We usedP. aeruginosa PAO1 was cultured alone or in co-culture with S. aureus ATCC 25923. As shown in P. aeruginosa PAO1 was cultured alone and co-cultured with S. aureus ATCC 25923. Moreover, planktonic cells or biofilm cells from co-cultures of P. aeruginosa PAO1 and S. aureus ATCC 25923 showed significantly increased resistances to some antibiotics  compared with that of P. aeruginosa PAO1 monoculture. These data indicate that cultures of multiple bacterial species and the formation of biofilms can increase resistance to antibiotics. We also evaluated the MBEC of the compound Str7410 against the formed biofilm. As shown in First, the MIC and MBIC values of antibiotics for single and mixed bacteria were determined when P. aeruginosa PAO1 and S. aureus ATCC 25923 cells. Treatment with MEPM alone and MPEM combined with sonication significantly decreased the number of biofilm cells . The combination of MEPM with Str7410 also had a significant inhibitory effect on biofilm cells (p < 0.01). Notably, compound Str7410 increased the inhibitory effect of MEPM toward biofilm cells (p < 0.05), and the level of inhibition by Str7410 + MEPM was greater than that by MEPM + sonication.Next, we evaluated the effects of compound Str7410 on the susceptibility of mixed-species biofilms to antibiotics. We tested the inhibitory effects of MEPM and compound Str7410 alone, and MEPM combined with compound Str7410. MEPM in combination with sonication was used as a reference treatment. As C. elegans N2 as a model to evaluate the effect of compound Str7410 on survival rates following co-infection with P. aeruginosa PAO1 and S. aureus ATCC 25923 in vivo.We used wild-type C. elegans co-infected with P. aeruginosa PAO1 and S. aureus ATCC 25923 compared with that of nematodes infected with P. aeruginosa PAO1 alone  increased by 12.67% (p < 0.05), and the survival rate of the infected group treated with MEPM (0.5 μg/ml) increased by 14.67% (p < 0.001). Furthermore, the survival rate of the group treated with the combination of Str7410 and MEPM increased by 12.67% (p < 0.05) and 10.67% (p < 0.05) compared with the Str7410-alone and MEPM-alone treatments, respectively  when P. aeruginosa PAO1 was co-cultured with S. aureus ATCC 25923 compared with P. aeruginosa PAO1 monoculture. However, treatment with Str7410 significantly reduced the production of pyocyanin and elastase in co-cultures compared with the untreated controls; this effect of Str7410 was concentration dependent. When the concentration of Str7410 was 40 μM, the inhibition of production of pyocyanin and elastase was 22.07% (p < 0.01) and 24.85% (p < 0.05), respectively, compared with no treatment with Str7410.The effect of Str7410 on virulence factors of P. aeruginosa, is mainly regulated by QS-related genes, such as lasB and pvdQ  and virulence factor-related genes  was significantly upregulated in the co-culture group compared with that in P. aeruginosa PAO1 monoculture. When Str7410 (40 μM) was added, the expression of all the strain PAO1 QS genes was significantly downregulated in co-culture compared with that in the absence of Str7410.From the data above, we found that CF, are often caused by infection with multiple bacterial species  through the pqs system to inhibit the aerobic respiration of S. aureus . Thus, we selected compound Str7410 as the test compound to evaluate its inhibitory effect on interspecific QS when P. aeruginosa PAO1 and S. aureus ATCC 25923 were co-cultured.In previous study, we used the AI-2 signal molecule receptor LuxP as a target to screen 8,600 small molecule compounds from an in-house compound library using virtual screening techniques, and identified 14 AI-2 QSI compounds . Wild-tynse AI-2 . As showvia QS, are an important reason for bacterial drug resistance. Pseudomonas aeruginosa can sense AI-2 produced by other bacteria, which regulates its production of virulence factors and formation of biofilms  and some QS target genes such as the virulence factor-related genes lasB and phzH. However, when compound Str7410 was added to the co-cultures, it reduced the pathogenicity of P. aeruginosa PAO1 by inhibiting the sensation of strain PAO1 to interspecific QS, thereby downregulating the expression of QS-related genes and the production of virulence factors.As a general QS system possessed by both Gram-positive and Gram-negative bacteria, AI-2 QS boosts the coordination and communication between bacteria in bacterial infection . Woods eed genes . In thised genes , S. aureS. aureus and Gram-negative bacterium P. aeruginosa in vitro and in vivo. Compound Str7410 was a potent AI-2 QSI. The combination of compound Str7410 with the antibiotic MEPM significantly increased the susceptibility of mixed-species-biofilm cells to the antibiotic. Preliminary mechanistic studies showed that Str7410 inhibited interspecific QS by inhibiting the AI-2 sensation of P. aeruginosa PAO1 and downregulating the expression of QS-related genes. This study provides a new strategy for the discovery of antibiotics in theory and a strategy for the clinical treatment of multidrug-resistant bacterial infections in practice.In conclusion, this study focused on the inhibitory effects of a compound identified by virtual screening on the QS system of co-cultured Gram-positive bacterium The original contributions presented in the study are included in the article/FL and JX conceived and designed the experiments. KJ, YX, BY, YY, MZ, RL, HW, LW, and YZ performed the experiments. KJ and YX created the figures, analyzed the data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Science and Technology Major Project of China (2018ZX09711003-003).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Triarylboranes have drawn much attention in OLEDs owing to their remarkable solid-state luminescence properties. Here two new A-D-A type compounds, 2,7-bis(dimesitylboryl)-N-ethyl-carbazole (BCz) using triarylborane as electron acceptor and carbazole as electron donor while 2,7-bis((4-(dimesitylboryl)phenyl)ethynyl)-9-ethyl-carbazole (BPACz) using phenylacetylene as extra conjugated bridge, have been synthesized and their photoluminescence related properties in various states have been investigated both experimentally and theoretically. Both compounds show blue emission with high quantum yields, being potential candidates for blue OLED materials. It is e2 as electron acceptor have been reported. While the 3,6-bis(BMes2)carbazole has shown strong fluorescence quantum yield and large two-photon absorption cross-sections . High resolution mass spectrometry (HRMS) was obtained by Thermo Fisher Scientific LTQ FTICR-MS and Thermo Scientific Q Exactive HF Orbitrap-FTMS. All photophysical measurements were performed in standard quartz cuvettes (1 cm × 1 cm cross-section). Luminogen-doped PMMA (polymethyl methacrylate) film was prepared by dissolving 2 mg of a luminogen compound and 100 mg of PMMA in 5 ml of specpure DCM followed by casting the resulting solution onto the surface of a quartz cell. UV-visible absorption spectra were recorded on a HITACHI UH5700 UV-visible spectrophotometer. The fluorescence, phosphorescence spectra, lifetimes and fluorescence quantum yields were recorded on a spectrofluorometer Edinburgh FLS1000. Thermogravimetric data were recorded on TGA5500.All reagents were commercially available and used as supplied without further purification. Ethyl ether, triethylamine and dioxane are re-steamed according to the solvent manual. 2BF and (p-Iodophenyl)dimesitylborane were synthesized according to literature  δ 6.86 , 2.33 , 2.30  (p-Iodophenyl)dimesitylborane was obtained as solid with 50% yield,1HNMR  δ = 7.69 , 7.20 , 6.81 , 2.29 , 1.98 .Compounds (Mes)terature  (Mes)2BF2BF  was then added and the mixture was stirred for another 2 h at –78°C before warming to room temperature and stirring overnight. Added a few drops of ethanol to quench the reaction. The obtained solid was dissolved in 200 ml dichloromethane and washed with water for three times. Isolated the organic layer. After the solvent was removed under vacuum, the residual was suspended in 100 ml ethanol, refluxed for 10 min, then filtered and rinsed with hot ethanol giving BCz as greenish solid powder . 1HNMR  δ = 8.09 , 7.61 , 7.42 , 6.86 , 4.26 , 2.34 , 2.03 , 1.26 ; 13C{1H}NMR  d = 144.4, 142.2, 141.0, 140.8, 138.5, 128.2, 127.2, 125.6, 120.4, 116.8, 37.3, 23.6, 21.3, 14.1. HRMS : Calcd for C50H55B2N m/z = 692.4583; Found 692.4593.2,7-dibromo-9-ethyl-carbazole  was placed in a 500 ml three-necked flask and was dried under vacuum and then backfilled with nitrogen before dry diethyl ether (20 ml) was added. The suspension was cooled to –78°C with vigorous stirring, and then n-BuLi in hexane  was added. The suspension was stirred for further 2 h at –78°C. Mes2dppf (80 mg) and CuI (10 mg) were dissolved in dry paraxylene (10 ml) and triethylamine (10 ml) in a Schlenk tube. Then was heated to 100°C and stirred overnight. Reaction mixture was concentrated at reduced pressure, and crude products were purified by chromatography over silica  to obtain the title compound of white solid . 1HNMR  δ = 8.00 , 7.56 , 7.37 , 4.33 , 3.16 , 1.43 ; 13C{1H} NMR  δ = 140.1, 123.3, 123.0, 120.6, 119.4, 112.6, 84.8, 76.9, 37.7, 13.8.In an argon filled glove box, 2,7-dibromo-9-ethyl-carbazole , PdClp-Iodophenyl)dimesitylborane , CuI (4 mg) and Pd(PPh3)4 (7 mg) were dissolved in dry dioxane (10 ml) and triethylamine (1.5 ml) in a Schlenk tube. Stirred overnight at room temperature. Reaction mixture concentrated at reduced pressure, crude products were purified by silica-gel column chromatography using mixture solvent  to obtain a white solid . 1HNMR  δ = 8.05 , 7.63 , 7.61–7.53 , 7.45 , 6.86 , 4.39 , 2.34 , 2.05 , 1.48 ; 13C{1H} NMR  δ = 141.6, 140.9, 140.3, 138.9, 136.2, 131.1, 128.3, 126.8, 123.1, 122.9, 120.6, 120.4, 112.0, 93.0, 89.8, 37.8, 23.5, 21.3, 13.9. HRMS(AP-MALDI Positive Ion Mode): Calcd for C66H63B2N m/z = 891.5135; Found 891.5141.In an argon filled glove box, 9-ethyl-2,7-diethynyl-carbazole   were collected at room temperature, and the phosphorescence emission spectra and phosphorescence lifetime were collected at 77 K. The fluorescence quantum yields (ΦF) for the compounds were determined relative to anthracene using a standard method.NMR spectra were recorded in CDCl0) of BCz and BPACz were optimized under Cs and C1 symmetry restriction in vacuum, respectively. The functional used includes Becke’s three parameter hybrid functional  calculations of two compounds were performed by using Gaussian 16  package.nctional  in conjunctional , which inctional  and the nctional  package We examined the absorption and emission properties of both compounds in three different matrices, from flowing molecule-dispersed solution to the condensed, aggregated state. PMMA is introduced as a transition medium which can exert rigid environment on dispersed molecule, i.e., solid solution.ΦF) and lifetimes (τF) are shown as well.Two aspects of the properties are evaluated, one is the change of the spectral aspect (position and bandshape) and the other is the evolution of photophysics (PL quantum yield and lifetime). The results of both compounds in variable environment will be logically discussed in a systematically comparative way: from unbridged BCz to BPACz, from liquid solution to PMMA and to powder. The UV-Vis Absorption and PL spectra of two compounds are plotted in ΦF = 0.49 and a corresponding lifetime of τF = 3.94 ns. Compared to 3,6-BCz reported earlier, 2,7-BCz in hexane gives slightly bule-shifted absorption but red-shifted fluorescence with similar quantum yield  with a much shorter lifetime (τF = 0.6 ns), which is probably because of the fast radiative decay rate (1.6 × 109 s−1) compared to BCz (1.3 × 108 s−1), while the non-radiative decay rates of the two compounds keep similar.Compared to BCz in hexane, BPACz with PA bridge showed non-shifted PL with more pronounced vibronic feature and slightly red-shifted absorption (maximum peak at 372 nm). It also shows a much higher quantum yield , we observed little dependence in absorption while a general bathochromic shift and substructure washing out in PL for both compounds. The bathochromic shift implies that the polarity in excited state is much larger than that in ground state. The difference in dipole moment Δequation :Δv¯=v¯aΔf) see , which iThe phosphorescence of BCz and BPACz in frozen solutions were also investigated, and the results are listed in When going from fluid solution (hexane) to solid solution (PMMA), the absorption maxima are similar to that in fluid solution, however spectral shape gets blurred; the PL spectra for both compounds show significant redshift and less vibronic features in PMMA against hexane, similar to the case in polarized solvents. The significant redshift of PL indicates that excited state is of much larger polarity than ground state as we concluded from solvent dependency above. Giving that the rigidity of solid matrix should have a significant effect on narrowing the shape of the torsional potential along with giving more pronounced vibronic structure in the corresponding spectrum, the blurring of spectral shape in PMMA against hexane above demonstrates that PMMA can hardly steepen the torsional potentials in S0 and S1 because of free volume existing in PMMA making it different from the really rigid and highly ordered environments such as perhydrotriphenylene , implyinAs seen in Besides the non-aggregated solid state , the “classical” solid state was investigated, where intermolecular interactions can also significantly influence the emission property. In our case, both compounds show red-shifted and blurred PL in powder against hexane, as well as increased lifetimes to different extent. The former is due to the polarizability of the rigid matrix while the latter is resulted from the suppressed nonradiative rates by the rigidity exerting on the molecular motion. Amorphous powder samples of two compounds show decreased quantum yields compared to solutions see , which sThe equilibrium geometries of both compounds are elucidated by DFT (in vacuum) via full optimization under specific symmetry restrictions . Relevant torsional angles are denoted in 2-containing compounds, which however vary upon substitution  and in particular sharply upon the environment change such as from solution to single crystal; in this paper only the torsion in vacuum are described, the one in single crystal are not done because single crystals are hard to obtain probably due to the poor crystallinity of these compounds. It’s known from earlier studies that Cz exhibits a planar equilibrium geometry  and torsional ends (BMes2); the introduction of PA groups retains this end-twisted geometry in BPACz and only expands the planar core with electronic effect but without geometrical effect. From the experimental results that BCz has similar spectral position to BPACz, we suggest PA’s electronic effect is quite trivial. From the fact that two compounds show little spectral shift upon environment changes, we assume that the torsional potentials around the B-C single bonds are not shallow enough for environmental restriction to exert influence.The results of torsional angles suggest that strong twists in the structure is a general signature for the BMesgeometry  while BM2 end is also the case but with less intensity, which explains the appearance of the strong charge transfer band around 407 nm observed in the absorption spectrum of BCz.We also investigated the MO’s topologies of two compounds with the electron density distribution of the frontier molecular orbitals (HOMO and LUMO) illustrated in 2-Cz structure are potential blue emitting materials which can be exploited further in optoelectronic application.Two new A(B)-D (Cz)-A(B) type organoboron compounds, BCz and BPACz have been synthesized and their photoluminescence property has been investigated. Structured PL around 410 nm with high quantum yield and less structured absorption are found for BCz in hexane, which is more pronounced for BPACz. Both compounds in toluene are phosphorescent at 77 K with long lifetime up to 3 s for BPACz. Absorption spectra show little dependence on solvent polarity while PL shows a general redshift and substructure blurring because the polarity in excited state is much larger than that in ground state. Red-shifted and blurred PL in powder/PMMA against hexane, as well as increased lifetimes to different extent are found; the former is due to the polarizability of the matrix affecting on more polarized excited states while the latter is related to the suppressed nonradiative rates by the matrix’s rigidity exerting on the molecular motion. Quantum yields of PMMA are similar to that of solutions while show a decrease in amorphous powder, which is a reflection of rigid environment’s influence on radiative part. From DFT calculation, we know both molecules exhibit an end-twisted geometry and a tendency of charge transfer from N-ethyl-carbazole to boron atoms upon excitation from HOMO to LUMO. In sum, these two compounds of BMes


Acacia nilotica were extracted with hexane and ethyl acetate, and the extracts were subjected to column chromatography for the isolation of potentially bioactive compounds and their screening against kinetoplastid pathogens. NMR and HREI mass spectrometric analyses identified two new diterpenes, characterized as 16, 19-dihydroxycassa-12-en-15-one  and -7,8:7,17:16,17-triepoxy-7,8-seco-cassa-13-ene . The previously reported  -(-)-7,8-seco-7, 8-oxacassa-13,15-diene-7,17-diol (3),  -(-)-7,8-seco-7, 8-oxacassa-13,15-dien-7-ol-17-al (4), and  -(-)-7,8-seco-7, 8-oxacassa-13,15-dien-7-ol (5) a, mixture of stigmasterol (6a) and sitosterol (6b), and lupeol (7) were also isolated. Several column fractions displayed significant activity against a panel of Trypanosoma and Leishmania spp., and from the most active fraction, compound 4 was isolated with high purity. The compound displayed high activity, particularly against T. brucei, T. evansi, and L. mexicana (0.88–11.7 µM) but only a modest effect against human embryonic kidney cells and no cross-resistance with the commonly used melaminophenyl arsenical and diamidine classes of trypanocides. The effect of compound 4 against L. mexicana promastigotes was irreversible after a 5-h exposure, leading to the sterilization of the culture between 24 and 48 h.The powdered roots of the medicinal plant  Trypanosoma spp. and Leishmania spp., belonging to the family Trypanosomatida and the order Kinetoplastida, are among the most important agents of neglected tropical diseases  infections has significantly decreased in recent years, with only 977 cases recorded in 2018 , African America .Leishmaniasis generally affects the poorest of the poor and is associated with malnutrition, population displacement, poor housing, a weak immune system, and a lack of financial resources . The disAcacia nilotica Linn. (Mimosaceae) is a common medicinal plant found in subtropical and tropical Africa from Nigeria to Egypt and South Africa and other parts of the world  (230–400 mesh ASTM). Thin-layer chromatography (TLC) was performed on precoated aluminum sheets coated with silica gel F250 . Nuclear magnetic resonance (NMR) experiments were carried out on a Bruker AVIII (500 MHz) spectrophotometer using CDClAcacia nilotica were collected from trees growing on the campus of the University of Agriculture, Makurdi. The plant was authenticated at the Department of Forestry and Wildlife of the university and a voucher specimen deposited at their herbarium.Roots of 1 and 3 (fractions 76–79), mixture of compounds 2 and 5 (fractions 18–19), compound 4 (fractions 64–67), a mixture of compounds 6a and 6b (fractions 59–60), and compound 7 (fractions 25–28) as white crystalline solids. The compounds were analyzed by NMR (1D and 2D) spectroscopy and mass spectrometry.Dried roots of the plant were ground to powder (250 g) and extracted with hexane and ethyl acetate. The extracts were combined (based on similarity on TLC) and subjected to column chromatography using silica gel in a glass column. The column was packed wet in a hexane: ethyl acetate (95:5) mixture and eluted with ethyl acetate in hexane gradient starting with 5% ethyl acetate in hexane and increasing the amount of ethyl acetate by 5% until 100% ethyl acetate collecting 10-ml vials to obtain 186 fractions. The fractions were examined by TLC, and similar ones were combined and allowed to dry in a fume hood to obtain a mixture of compounds Trypanosoma brucei brucei bloodstream form (BSF) were used in this study: 1) wild-type (WT) T. b. brucei strain Lister 427 (in vitro exposure to pentamidine and lacks both the TbAT1/P2 transporter and the high-affinity pentamidine transporter (HAPT1) (T. b. brucei strains and drug-sensitive (WT) strains of T. evansi and T. equiperdum were used throughout as bloodstream trypomastigotes and cultured in standard Hirumi’s modified Iscove’s medium 9 (HMI9), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 14 μL/L β-mercaptoethanol, and 3.0 g/L sodium hydrogen carbonate (pH 7.4). The parasites were cultured in vented flasks at 37°C in 5% CO2 atmosphere and were passaged every 3 days (T. congolense savannah-type strain IL3000 and T. congolense strain 6C3 [diminazene-resistant (Leishmania mexicana promastigotes (MNYC/BZ/62/M379 strain) were grown in hemoflagellate modified minimal essential medium (HOMEM)  (pH 7.4) supplemented with 10% heat-inactivated FBS at 27°C.Two strains of ster 427  and 2) aster 427  after in (HAPT1) . The twoy 3 days . The bloesistant ] were cu2 atmosphere.Human embryonic kidney (HEK) cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated FBS, 10 ml/L penicillin/streptomycin (Gibco 15140-122), and 10 ml/L of 200 mM glutamine (Gibco 25030-024). The cells were maintained at 37°C in 5% COAll compounds and mixtures were dissolved in DMSO at 10 mg/ml, and the stock solutions were stored at −20°C.T. brucei s427, T. brucei B48, and T. equiperdum, a seeding density of 2 × 104 cells/well was used, whereas cell densities of 4 × 104 and 5 × 104 were used for T. evansi and the T. congolense strains (IL3000 and 6C3), respectively. Trypanosome cultures with the test drugs were incubated for 48 h, followed by the addition of 20 µL of filter-sterilized 125 μg/ml resazurin sodium salt in phosphate-buffered saline (PBS). This was followed by a further 24 h of incubation. Standard drugs including diminazene aceturate and suramin were used as positive control as appropriate for the species. Fluorescence was measured in 96-well plates with a FLUOstar Optima  at wavelengths of 544 nm for excitation and 590 nm for emission. EC50 values were calculated by nonlinear regression using an equation for a sigmoidal dose–response curve with variable slope .The susceptibilities of bloodstream formtrypanosomes to the compounds and mixtures were determined using resazurin -based assay, as described previously . In brieL. mexicana was carried out using a similar method as described above. However, a seeding density of 2 × 105 cells/well was used for this species. The plate containing the cells and drug dilutions was incubated for a period of 72 h at 27°C, followed by the addition of 20 µL 125 μg/ml resazurin and a further 48 h of incubation. Pentamidine was used as a control drug. Fluorescence was measured as above.Drug sensitivity assay in 5 cells/ml. Then, 100 µL of the cell suspension was distributed to each well of a 96-well plate and incubated for 24 h to allow the cells to adhere to the bottom of the wells. Doubling serial dilutions of the test compounds and control drug were prepared in a separate 96-well plate, across 1 row (11 dilutions plus no-drug control). And then, 100 µL of each dilution was transferred to respective wells of the plate containing the cells, and the plate was incubated for another 30 h. This was followed by the addition of 10 µL 125 μg/ml resazurin sodium salt to each well, and a further incubation was done for 24 h. The plates were read and EC50 determined as above. The selectivity index (SI) was also calculated for each compound/mixture as the ratio of the EC50 in HEK cells to the EC50 in a parasite species.HEK cells were harvested at 80–85% confluence using 0.25% Trypsin–EDTA solution (Sigma T-4049). The cells were washed by centrifugation at 1200 rpm for 10 min and reconstituted in fresh medium at 3 × 10L. mexicana cultures were set at 106 cells/ml in a 24-well plate with or without varying concentrations of HEAN-19b and pentamidine. Depending on intended duration of exposure, cells were centrifuged at 5 h and 48 h and resuspended in either fresh medium or medium containing the drug. Cells were counted manually using a hemocytometer, and a growth curve was plotted using cell density at each time recorded.1, a white crystalline solid, was obtained as the minor component of combined fractions 76-79. The molecular formula C20H32O3 was derived from its high resolution mass spectrum , which yielded an [M-H]− ion at m/z = 319.2229 . Its proton spectrum  and two sets of oxymethylene protons at 4.44 , 4.56 , 3.12 , and 3.42 . It also displayed signals for three methyl protons made up of a methyl doublet at 0.86  and two singlets at 0.80  and 0.88 . The rest of the signals were for three methine  and six pairs of methylene protons. Its 13C spectrum  gave signals for 20 carbon atoms including one ketone carbonyl at δC 198.0 and two hydroxyl bearing carbons at 72.1 and 64.4 ppm. There were also two olefinic carbons, one proton bearing at 141.0 and the other a quaternary at 142.0. The rest of the signals were for three methyls, six methylenes, four methines, and two quaternary carbons. Using correlations in its 2D NMR spectra , the structure . The absence of germinal methyl groups usually at C-4, further confirmed C-19 to be a hydroxymethylene carbon and C-18 a methyl carbon. Therefore, correlations from H-19 and H-18 were used to identify C-3, C-4, and C-5 and from H-5 to identify C-1, C-7, C-10, and C-20 and confirm C-9. The compound was identified as 16, 19-dihydroxycassa-12-en-15-one and given the trivial name Sandynone 1, and its chemical shifts compared very well with similar compounds  at m/z = 318.2195 . The 1H NMR spectrum  and 5.26 , a methine geminal to oxygen at δH 4.52 , and two methylene protons H-16 geminal to oxygen at δH 3.77  and 3.61  coupled with the H-15 methylene protons at δH 2.31 and 1.64 ppm. There were three methyl groups observed at δH 0.83 (Me-20), 0.80 (Me-19), and 0.83 (Me-18). In the 13C DEPT-135 spectrum , 20 signals were observed including two quaternary olefinic carbons at δC 135.9 (C-13) and 129.9 (C-14), two acetal carbons at δC 95.1 (C-7) and 88.6 (C-17), one oxymethylene carbon at 56.4 (C-16), and an oxymethine carbon at 67.4 (C-8). The rest of the signals were for two methine , seven methylene, three methyl, and two quaternary carbons. The structure was confirmed using its 2D NMR spectra  as follows: long-range correlations from the acetal proton H-7 identified carbon C-5, C-8, and C-17, while from the second acetal proton H-17, carbons C-7, C-8, C-13, C-14, and C-16. Similarly, long-range correlations from H-18 and H-19 indicated they were germinal and identified C-3, C-4, and C-5, and H-20 also confirmed C-5 as well identifying C-1, C-9, and C-10. The HSQC spectrum confirmed the proton bearing carbons in the compound and their attached protons, while the COSY spectrum confirmed neighbouring protons. Compared to the macrocyclic dimer isolated from Acacia schaffneri (2) and confirmed by literature reports  -(-)-7,8-seco-7, 8-oxacassa-13,15-diene-7,17-diol, compound 4 as 5S,7R,8R,9R,10S) -(-)-7,8-seco-7, 8-oxacassa-13,15-dien-7-ol-17-al and compound 5 as  -(-)-7,8-seco-7, 8-oxacassa-13,15-dien-7-ol  and 3 (20%), showed promising antitrypanosomal activity with lowest EC50 of 4.27 ± 0.06 μg/ml against T. evansi (2 (70%) and 5 (30%), had only modest to poor activity against all the Trypanosoma and Leishmania species assayed. However, crude extract HEAN-1 displayed good activity against all the kinetoplastid species, except T. congolense (EC50 ≤ 10 μg/ml), and was thus selected for further purification, yielding compound 4. This compound showed very potent antiprotozoal activity with EC50 as low as 0.45 ± 0.02 μg/ml, 0.33 ± 0.05 μg/ml, and 0.28 ± 0.05 μg/ml against T. b. brucei, T. evansi, and L. mexicana, respectively.Two compound mixtures and a pure compound were tested for activity against bloodstream forms of four species of sitology . In addisitology . PAN-76,. evansi . Another4, exhibited only moderate to low activity against T. congolense IL3000, suggesting poor prospects for development as agents in the treatment of AAT, where the infecting species is usually not known, although applications outside the African tsetse belt (T. evansi and T. equiperdum) look more promising. It is a very common occurrence for drugs and test compounds to vary in activity between T. congolense and the species in the T. brucei group of species, making drug development for AAT even more challenging as drug candidates need to be active against three different species, that is, T. b. brucei, T. congolense, and T. vivax. In addition, while PAN-76 and HEAN-18, containing various amounts of compounds 1, 2, 3, and 5, demonstrated at best modest antileishmanial activity, whereas the purified compound 4 displayed an EC50 of 0.28 ± 0.05 μg/ml  between EC50 in the T. brucei WT and multidrug-resistant B48 strain, and between the T. congolense WT and the diminazene-resistant 6C3 strain . Although compound 4 in purified form is more toxic to HEK cells than the crude fraction from which it was isolated, it remains low at ∼30 µM. Thus, compound 4 shows some promise as an antiprotozoal compound, especially against Leishmania spp. and will need to be further investigated.The PAN-76 and HEAN-1 mixtures and compound . evansi , compoun4 prompted the investigation of its effect on growth of L. mexicana promastigotes in vitro. At a concentration of 5 × EC50, the compound irreversibly inhibited the growth of L. mexicana even after a short exposure of 5 h (The selective antiparasitic activity of compound e of 5 h . In addiAcacia nilotica yielded two new diterpenes that had limited anti-kinetoplastid activity and one oxacassadiene, compound 4, isolated from a fraction of the extract which displayed promising activity against several Trypanosoma species and Leishmania mexicana. Production of this compound from the widely available plant should now be scaled up for tests with further Leishmania species, intracellular amastigotes, and in vivo studies with relevant disease models.Due to the unprofitable market for kinetoplastid agents, new developments are unlikely to emerge through the regular discovery process of the pharmaceutical industry, especially for veterinary applications. Alternative local solutions are potentially available in the form of medicinal plants, a practice that is ongoing but requires scientific validation in order to delineate which extracts or isolated compounds from which plants work reliably against which pathogens. Here, we find that crude extracts of


Stenotrophomonas maltophilia strain PE591, which was isolated from plastic debris found in savanna soil. The genome was assembled in 16 scaffolds with a length of 4,751,236 bp, a GC content of 66.5%, and 4,432 predicted genes.We report the genome sequence of a polyethylene-degrading bacterial strain identified as  Stenotrophomonas maltophilia strain PE591 was isolated from plastic debris found in the soil of the Brazilian Cerrado biome  and clinical environments  and incubated for 24 h at 28°C with agitation (150 × g). Subsequently, cells were harvested by centrifugation at 5,500 × g for 5 min at 4°C. Bacterial DNA was purified using the GenElute bacterial genomic DNA kit . A sequencing library was created using the MiSeq reagent kit  according to the manufacturer’s instructions, the MiSeq system user guide, revision L , was used in the sequencing protocol, and genome sequencing was performed with the Illumina MiSeq system (2 × 300-bp paired-end reads) . The resultant reads were subjected to quality analysis using FastQC software v. 0.11.3  and IDBA-UD v. 1.1.1 to assemble contigs , which generates the ANIb , and (ii) digital DNA-DNA hybridization (dDDH) obtained through the Type (Strain) Genome Server (TYGS) (https://tygs.dsmz.de)  .S. maltophilia PE591 was assembled in 16 contigs, with a total length of 4.75 Mb and a GC content of 66.5%. Genome coverage, completeness, and contamination were ∼800×, 100%, and 0%, respectively. N50 and L50 values for the assembly were 473,413 bp and 3 contigs, respectively. Genomic functional features include a total of 4,432 genes, 4,350 protein-coding genes, 7 rRNAs, 74 tRNAs, and 1 transfer-messenger RNA.The genome sequencing generated a total of 13,411,628 reads, with a total of 4,019,163,037 bp. The genome of S. maltophilia PE591 in relation to Stenotrophomonas maltophilia strain NCTC10258 (GenBank accession number NZ_LS483377.1) was 97.75% (ANIb). The dDDH (d4) value provided by the TYGS against Stenotrophomonas maltophilia NBRC 14161 (RefSeq assembly accession number GCF_001591205.1) was 80.2%. According to these results and the minimum parameters for taxonomic classification of prokaryotes using genomic data .This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession number


Traditional solution and gas-phase synthetic pathways do not compete with the practicality and efficiency offered by the mechanochemical synthesis, which now opens up a new reaction space for inducing curvature at a molecular level.The transformation of planar aromatic molecules into π-extended non-planar structures is a challenging task and has not been realized by mechanochemistry before. Here we report that mechanochemical forces can successfully transform a planar polyarene into a curved geometry by creating new C-C bonds along the rim of the molecular structure. In doing so, mechanochemistry does not require inert conditions or organic solvents and provide better yields within shorter reaction times. This is illustrated in a 20-minute synthesis of corannulene, a fragment of fullerene C The transformation of planar aromatic molecules into non-planar structures is a challenging task and has not been realized by mechanochemistry before. Here, the authors demonstrate that mechanochemical forces can transform a planar polyarene into a curved geometry by creating new C-C bonds along the rim of the molecular structure. The angle strain associated with this pyramidalization needs to be overcome in any viable synthesis. This necessitates application of either high-energy reaction conditions or high-energy precursors. For instance, flash vacuum pyrolysis (FVP), in which molecules are subjected to high temperatures (500–1100 °C) in the gas-phase, is the most successful synthetic methodology to create non-planar molecules2. In a landmark publication in 1991, Scott established the utility of FVP as a synthetic method that could bend diethynylfluoranthene, a flat polyarene, into a bowl-shaped aromatic structure, corannulene3. Scott reasoned that the high-energy conditions temporarily populate bent molecular geometries that would be inaccessible under normal circumstances. Only under such folded conformation, the two-carbon side chains could reach across the fluoranthene bay region to intramolecularly generate two new 6-membered rings. Ever since, a large family of molecular bowls have been produced through this fascinating method1. A decade later the technique reached a new high in the chemical synthesis of fullerene C605. This feat of organic synthesis serves as an inspiration to explore new methods that can twist aromatic molecules and create new covalent bonds to lock the curved molecular geometry.The synthesis of strained aromatic molecules from strain-free precursors is a challenging task. This requires the natural trigonal planar geometry of the 21. In this regard, in the past few years, various synthetic techniques such as direct mechanocatalysis25 and liquid assisted grinding31 are developed. New possibilities for heating34, cooling36 and conducting gaseous38 mechanochemical reactions are established. Furthermore, analytical techniques for in situ monitoring of the reactions are described45. Along with these developments, the application of mechanochemistry for the synthesis of various polyarenes has also been increasing51. So far, however, mechanochemistry is not known to transform planar aromatic molecules into non-planar structures. We reasoned that mechanochemistry, similar to FVP, creates extraordinary reaction conditions but through impact and shear forces. Such conditions maybe able to bend the molecules such as fluoranthene and allow them to react at the bay regions to form the curved fused-ring system. Unlike FVP, however, mechanochemistry offers no restrictions on the nature of precursors (flash vacuum pyrolysis requires the precursor to be able to sublime and withstand the sublimation conditions), a simpler experimental set-up, possible scalability, better yields, shorter times and operation under ambient conditions. Therefore, it might be a valuable method to access curved structures. To explore this hypothesis, we focused our efforts on tetrabromofluoranthene (1), as a precursor to corannulene synthesis  as a free-radical source resolves this, generating tetrabromomethylfluoranthene within 3 h  could be obtained upon milling of 1 with NaOH or NaOt-Bu  and tetrabutylammonium fluoride (TBAF) were used. However, this led to a drastic reduction in yield 5%) or a complete failure of reaction (Supplementary Table % or a co8) serve as a precursor for corannulene synthesis? To answer this, we employed the knowledge gained thus far to prepare 8 through subjecting tetrabromomethylfluoranthene to a mechanochemical halide-exchange reaction with the help of TBACl , a bathochromic shift of 34 nm is observed due to extension in π-conjugation. Fluoranthene absorption is known to be highly complex with a number of independent electronic transitions60. This can be observed in the case of halide derivatives 1 and 8 with absorption extending into the blue region of the electromagnetic spectrum. The halide substitution pattern does not alter the absorption characteristics due to a methylene spacer, which interrupts the electronic conjugation between the halides and the aromatic nucleus. Formation of the first aromatic ring in the bay region of fluoranthene, however, results in loss of molecular planarity and a blue shift of ~25 nm is observed for compound 7. Formation of a strained bowl upon second ring-closure results in a further loss in planarity and a further blue shift of 57 nm. The absorption spectrum of corannulene matches well with the previous literature reports62.In UV-Vis spectroscopy Fig. , compoun55 which employs the high-energy octabrominated precursor 9  and a 15 mm ∅ stainless steel ball (13.55 g) were added to a 30 mL stainless steel jar. The jar was tightly sealed using parafilm and milled at 30 Hz for 15 min. The resulting powder was scraped from the jar, dispersed in 10 mL of acetone and filtered. The filtered cake was washed twice with 10 mL of acetone and the filtrate is evaporated to yield 3 . 1H NMR : δ 7.76 , 7.67 , 7.36 , 2.90 – 2.75 , 2.61 , 2.15 , 1.68 – 1.61 . 13C NMR : δ 210.97, 170.65, 139.26, 139.12, 133.36, 131.89, 131.00, 130.92, 130.87, 130.79, 130.78, 130.73, 128.34, 128.06, 125.82, 86.19, 48.82, 22.23, 19.87, 11.21, 10.34. HRMS (EI) calcd for C19H18O2 (M+) 278.1307, found 278.1311.1.0 g 3,8-Dimethylacenaphthenequinone (4.76 mmol), 476 mg NaOH (11.9 mmol), 3-pentanone , MgSO3 (0.36 mmol), 294 µL norbornadiene (2.88 mmol), 131 mg trimethylbenzylammonium chloride (0.72 mmol), 68 µL acetic anhydride (0.72 mmol) and 600 mg of MgSO4 were loaded into a 15 mL stainless steel jar (10 mm Teflon ring) with a 10 mm ∅ stainless steel ball (4.02 g). The jar is heated to 95 °C and milled at 30 Hz for 2 h, with 20 min break in between. The crude mixture is scrapped from the jar and purified using a short silica plug (eluent: 100% hexane) to obtain 4 as a yellow solid . 1H NMR : δ 7.82 , 7.50 , 7.26 , 2.99 , 2.90 . 13C NMR : δ 140.08, 135.05, 133.85, 132.15, 131.99, 130.83, 126.77, 126.31, 25.28, 24.46. HRMS (EI) calcd for C20H18 (M+) 258.1409, found 258.1406.100 mg of 4 (3.88 mmol), 3.1 g (17.4 mmol) of N-bromo succinimide, 127 mg of AIBN (0.78 mmol) and 50 mL of ethyl acetate. A reflux condenser is attached and the mixture is heated to reflux for 3 h. The reaction mixture is cooled to room temperature and the solvent is removed under reduced pressure. The crude solid is then triturated in cold saturated aqueous NaHCO3 solution and filtered to remove succinimide. The residue is washed with saturated aqueous NaHCO3 solution followed by cooled methanol to obtain crude 1 as an orange solid. The crude sample can be used directly in the next step. However, to improve yield of corannulene synthesis, crude 1 is purified by recrystallisation from ethyl acetate to give a white powder . 1H NMR : δ 7.83 , 7.72 , 7.62 , 5.00 , 4.96 . 13C NMR : δ 138.53, 133.66, 133.65, 133.28, 132.81, 132.68, 132.66, 129.41, 128.22, 34.38, 34.21. HRMS (EI) calcd for C20H14Br4 (M+) 573.7789, found 573.7766.A 100 mL round bottom flask was charged with 1 g of 1 (0.17 mmol) is milled with 400 mg of tetrabutylammonium chloride (1.43 mmol) and 400 mg of sodium sulphate decahydrate in a 15 mL zirconia jar with a 10 mm ∅ zirconia ball (3.08 g) at 30 Hz for 5 min. 240 mg of barium hydroxide octahydrate (1.57 mmol) is added and the mixture is milled for 15 min at 30 Hz. The crude mixture was suspended in DCM and dry loaded onto silica gel. Column chromatography was performed (eluent: from 100% hexane to hexane:DCM 30:1) to obtain pure corannulene as a white powder . 1H NMR : δ 7.80 . 13C NMR : δ135.90, 130.95, 127.15. HRMS (EI) calcd for C20H10 (M+) 250.0783, found 250.0782.100 mg of 1 (0.17 mmol), 400 mg of tetrabutylammonium chloride (1.43 mmol), 400 mg of sodium sulphate decahydrate and 240 mg of barium hydroxide octahydrate (1.57 mmol) were loaded into a 15 mL zirconia jar with a 10 mm ∅ zirconia ball (3.08 g) and milled at 30 Hz for 10 min. The crude mixture was suspended in DCM and dry loaded onto silica gel. Column chromatography was performed (eluent: from 100% hexane to hexane:DCM 30:1) to obtain pure corannulene as a white powder .100 mg of 1 (0.34 mmol), 800 mg of tetrabutylammonium chloride (2.86 mmol) and 800 mg of sodium sulphate decahydrate were loaded into a two sets of 15 mL zirconia jars with a 10 mm ∅ zirconia ball (3.08 g) each and milled at 30 Hz for 3 h. The crude mixture was combined, suspended in water, filtered off and the solids were re-dissolved in DCM to dry load onto silica gel. Column chromatography was performed (eluent: from hexane:DCM 30:1 to hexane:DCM 20:1) to obtain pure tetrachloromethylfluoranthene 8 as an off-white solid . 1H NMR : δ 7.86 , 7.73 , 7.62 , 5.06 . 13C NMR : δ 138.73, 133.97, 133.66, 133.22, 132.45, 132.28, 132.17, 132.07, 129.47, 128.19, 45.55, 45.32. HRMS (EI) calcd for C20H14Cl4 (M+) 395.9822, found 395.9832.200 mg of 43 mg of tetrachloromethylfluoranthene (0.11 mmol), 250 mg of tetrabutylammonium chloride (0.9 mmol), 250 mg of sodium sulphate decahydrate and 150 mg of barium hydroxide octahydrate (0.5 mmol) were loaded into a 15 mL zirconia jar with a 10 mm ∅ zirconia ball (3.08 g) and milled at 30 Hz for 10 min. The crude mixture was suspended in DCM and dry loaded onto silica gel. Column chromatography was performed (eluent: from 100% hexane to hexane:DCM 30:1) to obtain pure corannulene as a white powder .Supplementary Information