Actinobacillus pleuropneumoniae were determined in this study. Marbofloxacin (2.50 mg/kg of body weight) was administered, and blood samples were collected with designated time intervals. Plasma-extracted marbofloxacin was injected into the LC-MS/MS system. The in vitro and ex vivo antibacterial activities of marbofloxacin were evaluated against 20 isolates of A. pleuropneumoniae. The mean peak plasma concentrations (Cmax) after i.v., i.m., and p.o administration were 2.60 ± 0.10, 2.59 ± 0.12, and 2.34 ± 0.12 µg/mL at 0.25 ± 0.00, 0.44 ± 0.10, and 1.58 ± 0.40 h, respectively. The area under the plasma concentration-time curves (AUC0–24) and elimination half-lives were 24.80 ± 0.90, 25.80 ± 1.40, and 23.40 ± 5.00 h·μg/mL and 8.60 ± 0.30, 12.80 ± 1.10, and 8.60 ± 0.00 h, for i.v., i.m., and p.o. administration, correspondingly. The AUC0–24/MICs of marbofloxacin after i.v., i.m., and p.o. administration were 253.86 ± 179.91, 264.1 ± 187.16, and 239.53 ± 169.75 h, respectively. The Cmax/MIC values were 26.58 ± 18.84, 26.48 ± 18.77, and 23.94 ± 16.97, and T>MICs were 42.80 ± 1.01, 36.40 ± 1.24, and 38.60 ± 1.18 h, after i.v., i.m., and p.o. administration, respectively. Thus, marbofloxacin dosage of 2.50 mg/kg of body weight by i.v., i.m., and p.o. administration with 24 h dosing interval will provide effective treatment for the infection of pig by A. pleuropneumonia.The pharmacokinetics of marbofloxacin in pigs after intravenous (i.v.), intramuscular (i.m.), and peroral (p.o.) administration and pharmacokinetic/pharmacodynamic indices of this drug against Korean local isolates of Mycoplasma spp. [ Haemophilus parasuis and Actinobacillus pleuropneumoniae, make it a good candidate to deal with a respiratory outbreak caused by any of these pathogens [ A. pleuropneumoniae is the causative agent of porcine pleuropneumonia, a worldwide disease with occasional clinical outbreaks that can have a severe economic impact [ A. pleuropneumoniae in their tonsils for several months without seroconverting [ A. pleuropneumoniae from pig herds has been made with different antibiotics [ A. pleuropneumoniae is gaining resistance against all kinds of antibacterial agents including marbofloxacin [Marbofloxacin is one of the fluoroquinolones that were widely used in veterinary medicine and exhibits concentration-dependent bactericidal activity , 2. Thisathogens . A. pleuc impact . Attemptc impact , 6, thernverting . Eradicaibiotics –7. But, floxacin .It was reported that the resistance rates of fluoroquinolones were increasing in several European countries as well as in Korea, Taiwan, and Japan . InternaCmax/MIC, T > MIC, AUC > MIC, etc.,) which are fundamental to predict efficacy and minimize resistance development [However, there are recent concerns about the emergence of quinolone-resistant bacterial strains and the impact of improper use of these drugs on human and animal; fluoroquinolones are still an important antimicrobial medicine. Thus, there is an important need to use fluoroquinolones with caution to preserve their effectiveness for many years. In veterinary medicine, it is essential to reserve these drugs for cases requiring a powerful antibiotic and to prescribe and/or administer them only under a good clinical assessment and with appropriate regimens . Becauseelopment , 27. A. pleuropneumoniae has not been studied in pigs. Thus, it was intended firstly to develop and validate a sensitive and reliable LC-MS/MS method for attaining the ultimate goal while the prime objective of this study was to characterize the pharmacokinetics of marbofloxacin following i.v., i.m., and p.o. administration at a dose of 2.50 mg/kg of body weight in pigs and to explore in vivo and ex vivo PK/PD indices using Korean local pathogenic strains of A. pleuropneumoniae as a model bacterium.Since there are contradicting values in PK/PD indices which correlate with prevention of resistant mutant selection and efficacy , 29, detMarbofloxacin (Marbocyl 10% solution) was purchased from Vetoquinol Ltd. . Marbofloxacin reference standard was purchased from United States Pharmacopeial Convention . Mueller-Hinton broth (MHB) and chocolate agar media were obtained from Difco Laboratories , and veterinary fastidious medium (VFM) was from Thermo Fisher Scientific . Other reagents and chemicals were of analytical grade. Purified water was prepared using the Milli-Q water purification system from Millipore, Inc. .The study was carried out in 12-week-old castrated cross-bred (Duroc × Landrace × Yorkshire) healthy male pigs at Animal and Plant Quarantine Agency, Gimcheon-si, South Korea. Pigs with body weight of about 30 kg were purchased from the Sunjin CU farm . All animals were kept in a self-contained animal unit. Commercial antibiotic-free feed and fresh water were provided ad libitum throughout the experimental period. The pigs were fasted for overnight (~12 h) and randomly divided into 3 groups, where 4 pigs were assigned in each group. Marbofloxacin  at a dose of 2.5 mg/kg of body weight was administered to different groups of animals through i.v., i.m., and p.o. routes for the determination of the basic pharmacokinetic parameters. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Animal and Plant Quarantine Agency, Gimcheon-si, South Korea .μL) were mixed with 1.5 mL of 0.1% formic acid in acetonitrile to precipitate plasma proteins. After shaking for 20 min, the mixtures were centrifuged at 5000 ×g for 30 min. The solvents of the supernatants were evaporated under nitrogen flow at 50°C to make the volume about 500 μL and then stored in −70°C refrigerator.The blood samples (5 mL) were taken in heparinized Vacutainer tubes by puncturing of jugular vein at 0 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 6 h, 9 h, 12 h, 18 h, 24 h, 30 h, 36 h, 48 h, 60 h, 72 h, and 120 h from all of these animals. The blood samples were centrifuged at 2000 ×g for 10 min at 4°C to obtain the plasma. The plasma samples  column was utilized to determine the content of marbofloxacin in plasma by analyzing 5 μL aliquot of each samples. The mobile phase was a mixture of (A) 0.1% formic acid in distilled water and (B) 0.1% formic acid in acetonitrile, where the ratios of “A” and “B” were different and maintained in a gradient flow. The initial composition of mobile phase was 90% of “A” and 10% of “B” which was linearly changed to 100% of “B” from 0.1 to 3 min and maintained this ratio up to 4.9 min. At 5 min, the ratio was directly returned to its base composition (10% B) and maintained this composition up to the end of the acquisition. The flow rate was 0.6 mL/min, and the injection volume was 5 μL. Tandem mass spectrometry with electrospray ionization was used and maintained in positive mode. The mass to charge ratios (m/z) of precursor ions, quantification ions and confirmation ions, and the collision energies (CE) of marbofloxacin are listed in Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with gradient elution through YMC CSpecificity was determined from three blanks and pooled plasma samples which were analyzed to note the absence of interferences in the elution position of marbofloxacin. Stock solution of marbofloxacin (1 mg/mL) was prepared by dissolving a pure reference standard of marbofloxacin in aqueous solution of 0.1% formic acid to stabilize the pH for establishing the linearity. Further dilutions of the stock solution were prepared, and the drug solutions of different concentrations were spiked into blank plasma to produce calibration curves. Linearity was determined by a series of three injections of six plasma samples spiked with  marbofloxacin. Pooled plasma samples were spiked with known concentrations (10 and 100 ng/mL) of marbofloxacin and deproteinated with acetonitrile for the determination of accuracy and recovery. After extraction of the analyte from the matrix and injection to the analytical instrument, the recovery was determined by comparing the resulting peak response with those of other samples to which the same corresponding concentrations of the drug had been added just before the injection.Repeatability and reproducibility were studied on six injections from spiked plasma samples of three different concentrations. The detection limit and quantitation limit were determined from the calibration curve by analyzing marbofloxacin spiked samples. The limit of detection (LOD) was calculated from the standard deviation of responses and the slope obtained from the calibration curve as stated by the following equation: LOD = (3.3 × SD)/slope , 31. TheTmax), peak plasma concentrations (Cmax), elimination half-life (T1/2), and absolute bioavailability (F) were calculated for pharmacokinetic determination using a noncompartmental analysis. The absolute bioavailability for i.m. administration was determined with the formula F = (AUCim/AUCiv) × 100%. Similarly, absolute bioavailability was determined by F = (AUCpo/AUCiv) × 100%, in the case of p.o. administration. AUCiv, AUCim, and AUCpo are obtained after i.v., i.m., and p.o. administration, respectively. Pharmacokinetic parameters were calculated for each individual animal and are presented as arithmetic means ± SD.The concentrations of marbofloxacin in plasma samples of different time points were analyzed by LC-MS/MS. The pharmacokinetic parameters of marbofloxacin were analyzed by WinNonlin 6.1 software . The area under the curve (AUC), peak times (μL of VFM (veterinary fastidious medium) was added to each well of a 96-well microtitre plate. The bacterial cultures (20 μL) of A. pleuropneumoniae (n = 20) from the stationary-phase of growth were individually added to 10 μL of plasma and mixed with the 170 μL of VFM in each well to give a final concentration of approximately 106 CFU/mL. One hundred microlitre of sample was collected at each time point, serially diluted, and spread over chocolate agar media. The bacteria in plates were further incubated at 35°C with 5% CO2 for 24 h. After incubation, bacteria colonies were manually counted.Plasma samples were collected from pigs after 0, 1/4, 3/4, 2, 9, and 24 h of marbofloxacin administration via i.v., i.m., and p.o., which were used for ex vivo experiment. Controls were prepared from plasma samples collected from those pigs which did not receive any drug. About 170  A. pleuropneumoniae were determined by using Mueller-Hinton broth (MHB) and Mueller-Hinton Agar (MHA) according to the standard broth microdilution method as described in Clinical and Laboratory Standards Institute (CLSI) guideline [μg/mL after inoculation of bacterial culture. Bacterial culture from midlogarithmic phase was diluted and 100 μL of the diluted bacterial suspension was added to serially diluted-drug solutions in 96-well plates where the final inoculum density would be approximately 5 × 105 CFU/mL. The bacteria in presence of drug substances in 96-well plates were incubated at 35°C for 18 h. After incubation, the lowest concentration that inhibited visible growth of microorganism was considered as MIC. Cultures (20 μL) from all microwells in 96-well plates that showed no visible growth were spotted on Tryptic Soy Agar (TSA) plates and incubated for 24 h at 35°C. The lowest concentration that completely inhibited growth on agar plate was considered as minimum bactericidal concentration (MBC). All experimentations were performed in triplicate.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of marbofloxacin against 20 isolates ofuideline . MarboflF-test were performed and P values of less than 0.05 were considered to be statistically significant.Data are presented as mean ± standard deviation of three replicate assays. Analysis of variance (ANOVA) and R2 > 0.999) was observed, and the quantified average recoveries of marbofloxacin were 87−92% at the level of 10 to 100 ng/mL. The within-run precision  for the described method was less than 10% over the range of concentrations studied. The limits of detection (LOD) and the limit of quantification (LOQ) were 2 and 5 ng/mL, respectively. The validation data of the analytical method is presented in The method validation was performed in the sample matrix. Good linearity (Cmax) of marbofloxacin were 2.60 ± 0.10 μg/mL, 2.59 ± 0.12 μg/mL, and 2.34 ± 0.12 μg/mL for i.v., i.m., and p.o. administration, respectively. The areas under the plasma concentration-time curves (AUC0–24) were 24.80 ± 0.90, 25.80 ± 1.40, and 23.40 ± 5.00 h·μg/mL for i.v., i.m., and p.o. administration, correspondingly. The elimination half-life (T1/2) values were 8.60 ± 0.30, 12.80 ± 1.10, and 8.60 ± 0.00 h, correspondingly in i.v., i.m., and p.o. administration. Absolute percentages of bioavailability (F) of the marbofloxacin in pig were 104.60 ± 5.70% and 94.35 ± 8.90%, respectively, in intramuscular and peroral routes compared to intravenous route.The concentrations of marbofloxacin in plasma at different time intervals after i.v., i.m., and p.o. administration are presented in  A. pleuropneumoniae in ex vivo condition were determined in plasma samples which were collected at 0, 1/4, 3/4, 2, 9, and 24 h after i.v., i.m., and p.o. administration of marbofloxacin in pigs  growth, when the plasma samples were collected at 1/4 and 24 h of p.o. administration and the bacteria were incubated in presence of those plasma samples.The time-dependent antibacterial effects of marbofloxacin against in pigs . The add A. pleuropneumonia were determined. The MICs of marbofloxacin against these isolates were ranged from 0.03152 to 1.00 μg/mL. The MBCs of this antimicrobial drug against those strains of A. pleuropneumoniae were from 0.0625 to 8.00 μg/mL. The ratio of MIC and MBC was 1 for 6 strains out of 20. The MBC values were 2- to 16-fold higher than MIC values against most of the strains. The MIC, MBC, and the ratio of MIC and MBC for each strain are presented in The MICs and MBCs of marbofloxacin against 20 isolates of0–24/MIC, AUC0–24/MBC, Cmax/MIC, Cmax/MBC, T > MIC, and AUC0–24 > MIC, which are presented in 0–24/MIC and AUC0–24/MBC ratios of marbofloxacin against A. pleuropneumoniae after i.v., i.m., and p.o. administration were 253.86 ± 179.91, 264.1 ± 187.16, and 239.53 ± 169.75 h and 144.93 ± 143.35, 150.77 ± 149.13, and 136.74 ± 135.26 h, respectively. The time where the plasma concentration exceeds MIC (T > MIC) after i.v., i.m., and p.o. administration was 42.80 ± 1.010, 36.40 ± 1.24, and 38.60 ± 1.18 h, respectively. The AUC0–24/MIC, AUC0–24/MBC, Cmax/MIC, and Cmax/MBC values attained from the application of marbofloxacin through p.o. route are significantly lower than i.v. and i.m. administration values. On the contrary, the AUC0–24 > MIC value with the administration of marbofloxacin through p.o. route is significantly higher than the values obtained from i.v. and i.m. administration. There were no noticeable variations in PK/PD indices of plasma samples collected after i.v. and i.m. administration to pigs.The in vitro MIC and MBC data were integrated with in vivo PK data to determine the PK/PD indices such as AUC0–24/MIC and Cmax/MIC index.In the current study, a sensitive and reliable LC-MS/MS method is developed for the rapid detection of marbofloxacin in plasma. This validated method was applied in determining pharmacokinetics and pharmacokinetic-pharmacodynamic indices of marbofloxacin after i.v., i.m., and p.o. administration in pigs. The pharmacokinetic study of marbofloxacin in this investigation is in accordance with previous studies in horses , beagle m/z) of the spiked marbofloxacin were found to be similar as it was obtained in the solution of pure compound  should not be <0.990 for the least squares method of the analysis of the line [The precision or coefficient of variation (%) of this assay method was within the limits for the tested concentrations according to the guidelines for analytical method development and validation , 37, whithe line . The corThe recovery percentage of this drug compound  may be cThe mean (±SD) plasma concentration versus time curves of marbofloxacin at a dose of 2.5 mg/kg of body weight in pigs following i.v., i.m., and p.o. administration is shown in  t1/2 of the drug. The elimination half-life of marbofloxacin in our study following single p.o. administration in pig was estimated to be 8.6 h, shorter than that in sea turtles (13.33 h) [The longer elimination half-life of marbofloxacin observed in this study reflects the advantage of this drug in maintaining effective concentration in the body thereby allowing longer time for drug-pathogen interaction. The elimination half-life of marbofloxacin in pigs after i.v. administration was comparable with values in pigs, rabbits, and goats , 23, 26 13.33 h)  but simi13.33 h) . The eli13.33 h)  than theTmax of marbofloxacin following single p.o. administration was estimated to be 1.58 h, which is similar to that obtained in chickens (1.48 h) [Tmax after i.m. administration in hanwoo cows (0.95 h) [Tmax value in pigs (0.44 h) in our study. Tmax in sea turtles (0.30 h) [Cmax of 2.59 ± 0.12 μg/mL achieved at 0.44 ± 0.10 h after i.m. administration. The Cmax values of marbofloxacin achieved after i.m. and p.o. administration were higher than the MIC break point of fluoroquinolones recommended against most susceptible bacterial species [μg/mL) [μg/mL) [μg/mL) [Cmax value of marbofloxacin in this study was higher than the previously reported values in pigs (1.81 μg/mL in i.m. and 1.03 μg/mL in p.o.) [The (1.48 h) . The Tma(0.95 h)  was high(0.95 h)  and sea (0.95 h)  was comp(0.30 h)  is simil species . These v [μg/mL) , rabbits [μg/mL) , and she [μg/mL) . Even, tin p.o.) . The relin p.o.)  and in gin p.o.) . The relin p.o.) , 23. In  A. pleuropneumoniae as a model organism. Large AUC0–24 h/MIC and Cmax/MIC ratios were obtained for A. pleuropneumonia isolates following i.v., i.m., and p.o. administration of marbofloxacin in pigs  [ T > MIC should be at least 50% of the dosage interval to ensure an optimal bactericidal effect [ T > MIC at a dose of 2.50 mg/kg of body weight in pigs after i.v., i.m., and p.o. administration.The ex vivo killing study indicates concentration-dependent antibacterial effect of marbofloxacin; increasing the drug concentration led to more-rapid killing of all tested bacterial strains. The values of ex vivo antibacterial effects are rational according to the in vitro MIC and MBC data, and serum concentrations of marbofloxacin at different time points. Roughly, the serum concentrations of marbofloxacin were above the in vitro MBC concentrations of most of the strains at time (0−9) h, (0.25−9) h and (0.5−12) h for i.v., i.m. and p.o. administrations, respectively. Moreover, the elimination half-lives of marbofloxacin from all of these dosage forms in pigs are longer, which also justifies the ex vivo antibacterial effect of marbofloxacin. The integration of PK data with the PD data presents a better approach to dose titration studies for selecting rational dosage regimens in veterinary medicine . Further in pigs . PK/PD iT > MIC) . It is gl effect , 43. DurCmax/MIC, and T > MIC) obtained in this study were compared with the PK/PD values in previous studies. The AUC/MIC and T > MIC of marbofloxacin in serum against Mannheimia haemolytica after i.v. and i.m. administration to calf at a dosage of 2.0 mg/kg were 249.75 ± 25.87 and 252.67 ± 9.16 h and 22.69 ± 1.69 and 22.68 ± 0.79 h, respectively. The ratio of Cmax and MIC in the same situation after i.m. administration was 37.60 ± 1.87 [ Mannheimia haemolytica were 120.2 and 135.5 h, respectively. After i.m. administration T > MIC was 10.5 h, and Cmax/MIC was 21.1 which is comparable with the Cmax/MIC value of this study [Cmax/MIC, and T > MIC of marbofloxacin against Escherichia coli after oral administration (2 mg/kg of body weight) in turkeys were 73.69 ± 25.54 h, 5.35 ± 2.31, and 10.9 h, which are about 4 times lower than the values of corresponding parameters in the present study [24 h/MIC and T > MIC of marbofloxacin against Staphylococcus pseudintermedius after i.v. and i.m administration (2 mg/kg body weight) in beagle dogs were 67.76 ± 1.23 h and 91.18 ± 2.61 h and 9.83 ± 1.72 and 15.50 ± 6.68, respectively, and the Cmax/MIC after i.m. administration in the same situation was 13.04 ± 1.03. All the values of PK/PD parameters in beagle dogs are about half the values obtained in the current study [0–24 h/MIC values 253.86, 264.10, and 239.53 h after i.v., i.m., and p.o. administration, respectively, are likely to provide a good antibacterial outcome. So that, the 2.50 mg/kg dosage may be regarded as appropriate for the strains of A. pleuropneumonia used in this study [0–24 h/MIC ratio of >125 h and a Cmax/MIC ratio of >10 are generally considered the best indicators of activity for agents with concentration-dependent killing and were usually used as a threshold for successful therapeutic outcome of fluoroquinolones against gram-negative bacteria [The values of PK/PD parameters (AUC/MIC, nt study . AUC24 hnt study . The AUCis study , as the bacteria . Neverthbacteria . A. pleuropneumonia. It was also revealed in this study that the marbofloxacin was completely absorbed and slowly eliminated after single i.v., i.m., and p.o. administration in healthy pigs. The mean marbofloxacin plasma concentrations at 24 h after i.v., i.m., and p.o. administration at a dose of 2.50 mg/kg were all higher than 0.25 μg/mL, which are above or very close to the MIC90 against most major pathogenic bacteria [ A. pleuropneumonia. Additional studies may also be necessary to confirm the penetration of marbofloxacin in diseased tissues, so that its potential use in clinical situations could be assessed.The current study demonstrated promising pharmacokinetic profiles of i.v., i.m., and p.o. formulations of marbofloxacin in pigs. Further, the current study established the correlation between the plasma concentration of marbofloxacin and its in vitro activity against an important bacterial pathogen,bacteria , 48. So,


The other crystal of 1,4-bis(5-methylthien-2-yl)-2,3,5,6-tetrafluorobenzene, 2, showed brittle breakage under applied stress . Fibril lamella crystal structure based on criss-cross packed slip-stacked molecular wires and its structural integrity are important factors for the design and production of next generation crystal materials with elastic bending flexibility. Furthermore, mechanical bending–relaxation resulted in reversible change of the morphology and fluorescence (mechanofluorochromism). Such bendable crystals would lead to the next generation solid-state fluorescent and/or semiconducting materials.Organic single crystals with elastic bending flexibility are rare because they are generally brittle. We report here fluorescent organic single crystals based on thiophene-tetrafluorobenzene-thiophene derivatives, mainly 1,4-bis(thien-2-yl)-2,3,5,6-tetrafluorobenzene. Three derivatives were synthesized by Pd-catalyzed cross-coupling reactions (Stille or direct arylation pathways). The crystallization of the derivatives gave large  crystals. Two crystals of 1,4-bis(thien-2-yl)-2,3,5,6-tetrafluorobenzene,  The materials are generally deformed by applied stress, which can lead to expanded, contracted, twisted and bent forms. The deformed shapes are quickly able to return to the original shapes. The elastomers belong to the shape-memory group of materials. Many organic materials consist of polymers5. Most of polymers have both densely-packed  and low-density (amorphous) domains6. The amorphous domains are able to disperse any stress that is applied to a material, and are able to store the external energy. The deformed materials recover their original shapes upon stress reduction, elastic deformation. The amorphous domains in the materials are thus one of the keys for the development of flexible materials6.Elastic materials such as rubbers, which are able to largely and reversibly deform, exhibit entropic elasticity and are termed as elastomers9, and elastically (reversible) bending deformation of organic single crystals by mechanical stress have also been reported in recent years13. Thus, the organic single crystals with mechanical bending flexibility have the potential to be attractive materials but the intentional production of such bendable materials is extremely difficult. The reported organic single crystals with plastic mechanical flexibility are deformed by applied stress to give various shaped crystals. These crystals undergo irreversible deformation, as they are soft but are not elastic. The slip between crystal planes disrupts the ordered structure. Very recently, a few crystals possessing elastic bending flexibility have been discovered14. A cocrystal formed from caffeine and 4-chloro-3-nitrobenzoic acid in methanol was incidentally discovered11. Moreover, crystals of 2,6-dichlorobenzylidine-4-fluoro-3-nitroaniline12, biocrystal13, and terepthalamide14 were also discovered in recent years. Screening of enormous organic molecules may lead to accidental discoveries. We have also succeeded in obtaining the elastic organic single crystal of a fluorescent π-conjugated molecule, 3  and only shows specific fluorescence  behaviour. Fibril lamella10 of slip-stacked (J-aggregated) molecular wires in the single crystal is a key morphological characteristic for the elastic crystals . To advance the field of the intentional production of “elastic” and “fluorescent” organic single crystals, design of molecules based on a tetrafluorophenylene core and thienyl unit18 is promising. Herein, we report the morphologies, optical properties and mechanical characteristics (flexibilities) of organic single crystals based on tetrafluorobenzene–thiophene derivatives.The most densely and regularly packed organic materials, organic single crystals, are not flexible but are hard and brittle because these have no amorphous domains. Plastically (irreversible) bending deformation of organic crystals have been discovered and studied1, 2 and 3: Fig. n-butyltin)thiophene or direct (C-H) arylation reaction20 of 1,4-dibromo-2,3,5,6-tetrafluorobenzene with thiophenes tinthiophene derivatives gave compounds 1, 2 and 3 in 48, 75, and 57% isolated yield , which was optimized to give large  size crystals, was performed with a biphasic methanol–dichloromethane solution containing the compounds, to yield transparent pale-yellow-colored crystal 1, turbid yellow-colored crystal 2 and transparent pale-yellow colored crystal 3 on the millimeter- or centimeter-scale  and F–H (2.220 Å) intramolecular contacts that are significantly shorter than the sums of their van der Waals radii   and F–H (2.228 Å) intramolecular contacts that are significantly shorter than the sums of their van der Waals radii   and F–H (2.218 Å) intramolecular contacts that were significantly shorter than the sums of their van der Waals radii   face similar to that of 1  and 3 (2.347 Å). Moreover, parallel arrangement of the molecules was observed in the (001) face  Fig. . These rbly Fig. . The cenbly Fig.  through bly Fig. . The cry Å) Fig. . These c Å) Fig. . The cen Å) Fig.  through Å)] Fig. . These cÅ)] Fig. . The fibÅ)] Fig.  through f 1 Fig. . The strbly Fig. , but theace Fig. . The cryc-axis had an orientation of 7–10 degrees. According to the Bragg equation, the length was calculated as 11.4, 9.9 and 10.7 Å, for 1, 2 and 3, respectively. The layer length between the slip-stacked wires of 2 was smaller than that of 1 and 3 owing to the parallel packing of the molecules of 2  than 1 (0.24) and 3 (0.22). This result suggests that the packing of 2 in the crystal is of lower quality. The lamella layer orientation of 1, 2 and 3 along the b-axis was 8.9, 9.5 and 11.7 degrees, which corresponds to lengths of 9.9, 9.6 and 8.1 Å, respectively. These lamella distances are in good agreement with the crystal structure.Figure f 2 Figs . It is n23. The signal-to-noise ratio of the 13C NMR spectrum of 1 was significantly worse owing to the extremely long T1H (less efficiency of cross-polarization) compared with those of 2 and 3, indicating complete packing and a rigid molecule   but that of 2 was singlet  indicates the close proximity to 1H nuclei: the 1H- 19F dipolar interaction, which is largely influenced by the distance, makes a 19F signal broad  provides information on the structure and dynamics of solid materialsule Figs  and S5. 1H Fig. . This meing Fig. . On the ts) Fig. . The 19Flet Fig.  and S5H.oad Fig. . There ad 3 Figs . The intits Fig. .24. Differential scanning calorimetry (DSC) and differential thermal analysis (DTA) traces of the crystals show the melting point . However, TGA of 2 did not show any weight loss at approximately 122 °C  is proposed to impart the crystals with structural integrity25.Thermal analyses of the crystals under nitrogen provide information of the melting point, phase transitions and crystal defectsint Fig. . However °C Fig. . Thus, t1, 2 and 3 in tetrahydrofuran, THF. The absorption spectrum for 1  was at a shorter wavelength compared with that of 3 , as a result of the hyperconjugation effect of the methyl group at the 3-position. The peak and edge of the spectrum of 2  was larger than that of the others. The efficiency of the hyperconjugated linkage in 2 between the π-orbital and σC–H in the methyl group is higher than that in 3 because of “through” and “cross” conjugation, respectively. However, the fluorescence spectra of compounds 1, 2 and 3 in THF showed similar π-π* fluorescence bands with peaks at 405–407 nm  a planarization of the conformation and (ii) intermolecular interactions. The dense and regular alignment of the π-conjugated molecules brings (i) the restriction of the molecular motion and (ii) the delocalization of the π-electronic field . The red-shifted absorption peaks of the crystals were proposed to arise from reason (i). The bands of 1 (λmax = 382 nm) and 3 (λmax = 386 nm) were at a slightly shorter wavelength than that of 2 (λmax = 391 nm). This is also because of the hyperconjugation effect of the methyl group. The shoulder bands of the crystals at approximately 425 nm arose from reason (ii): J-aggregation of thiophene–tetrafluorobenzene–thiophene  2  also showed the characteristic blue-shifted band  did not exhibit their blue-shifted band derived from disorder.The absorption spectra of the crystals showed red-shifted bands at approximately 350–450 nm compared with the spectra of the compounds in THF Fig. . In geneene Figs . The fluene Figs . This is) 2 Fig. . This suand Fig. , but the1 exhibited a lower quantum yield in THF (Φ = 15) than in the crystal (Φ = 19). Similarly, compound 3 also exhibited a lower quantum yield in THF (Φ = 15) than in the crystal (Φ = 25). These results are consistent with aggregation-induced emission (AIE) or crystallization-induced emission (CIE)15. The thienyl unit of the molecules undergo dynamic intramolecular rotations, which decrease the Φ values in THF. CIE and a red-shift of the fluorescence band are proposed to be caused by the restriction of intramolecular rotations (RIR) and then slip-stacking (J-aggregate). However, the Φ values of compound 2 were similar in both solution and as a crystal. The Φ value of the crystal 2 was lower than that of 1 and 3. Incorporation of defects or a lower quality of packing in 2 is proposed to result in a low fluorescence efficiency and the observation of the characteristic blue-shifted band  of the compounds in THF or crystal were measured based on an absolute method using an integrating sphere equipped with a multichannel spectrometer Table . Compounand Fig. .Flexible, anisotropic and densely packed molecular materials of delocalized π-systems (π-conjugated molecules) are highly useful compared with those with localized systems (non-conjugated molecules). These thermal stabilities and optical properties of the crystals are extremely important for the development of future elastic crystal devices.1  on a glass plate. The crystal bent without breaking when it was warped. When the crystal was bent back and forth with the tips of a pair of tweezers and a needle, it adopted a bent shape  fixed by a gel  were capable of bending to the c-axis more than 90° under an applied stress and then quickly reverted to their original shapes upon relaxation. In contrast with the elastic bending character of the crystals 1 and 310, the crystal 2 was brittle  crystal morphology and/or (ii) structural integrity  that spoil the co-operative molecular motion under the mechanical bending deformation.It is noteworthy that the crystal gel Fig.  was ablegel Fig. . Reversigel Fig. . The crytle Fig. . There w1  was estimated by using the equation, εn = d/2r26. The tensile ratio of the elastic crystal (εn) was estimated by the original (l0), inner (lin), and outer (lout) lengths, which are illustrated in Fig. l0, lin, and lout can be calculated from the radius of a virtual graph (circle or ellipse) on the image of the bent crystal  in the crystal 1 (83 μm thickness) is 41.5π. Thus, the εn can be estimated by the determination of the radius of circular or elliptical graphs of the bent shape crystal images. The tensile ratios (%) of the crystal 1 are calculated to be 8.1 from the graph shown in Fig. We next focused on the estimation of the tension and the compression in the elastic crystal 1 Fig. . Strain tal Fig. . The lin1 were prepared by (i) fixing both tips (100) of the original crystals using adhesive or (ii) fixing on the straw10. The bent crystals 1 exhibited a slightly high-energy fluorescence band at approximately 420–470 nm compared with the original crystals 1  of the center-to-center separation lengths)27 within the crystal would inhibit efficient energy transfer28. When the bent crystal relaxed upon force removal, the band was identical to the original spectrum. The Φ value of the bent crystals decreased to 11% compared with original crystals (19%). Relaxation of the crystals recovered the Φ value (19%). XRD patterns of these crystals were also recorded  determined by XRD, ssNMR and DSC (or DTA) analyses  of molecule for fibril lamella morphology, is an important for developing next elastic organic single crystal.It is conceivable that the crystal defects or less quality of packing in a fibril lamella spoil the cooperative molecular motion under the mechanical banding deformation. Thus, organic single crystal with structural integrity is an important factor for elastic (or plastic) mechanical deformations of densely packed materials. The crystal ses Figs  and S7. 1. This crystal exhibited an elastic bending motion for many cycles when stress was applied. Fibril lamella crystal structure based on criss-cross packed slip-stacked molecular wires and their structural integrity of the crystals 1 and 3 are important factors for the design and production of next generation crystal materials. On the other hand, the crystal 2 is a brittle material, which arises from a crystal structure including partial crystal defects caused by partially random packing . Further studies for the development of fibrous (elastic) single crystals of π-conjugated molecules and fluorescent device applications are now in progress. Moreover, we have now challenged the simulation of relationships between crystal structure and elastic bending flexibility.We have obtained fluorescent and elastic organic single crystals of 1,4-bis(thienyl)-2,3,5,6-tetrafluorobenzene, 2CO3 (Kanto), palladium chloride (Wako), tetrakis(triphenylphosphine) palladium(0) (Aldrich), and dry N,N-dimethylacetoamide (Wako) were used as received. Other solvents were used as received. 2-Tributylstunnyl-4-methylthiophene and 2-tributylstunnyl-5-methylthiophene were synthesized according to the previous report10.1,4-Dibromo-2,3,5,6-tetrafluorobenzene (TCI), 2-tributylstunnylthiophene (Aldrich), thiophene (Wako), 2-methylthiophene (TCI), 3-methylthiophene (TCI), 1-adamantanecarboxylic acid (TCI), K3)4  were dissolved in 2 mL of dry toluene under argon. After stirred for 24 h at 100 °C, the reaction mixture was passed to silica gel (ca. 100 g) column by hexane then dichloromethane. Crystal was collected by filtration after recrystallization (dichloromethane/methanol).1,4-Dibromo-2,3,5,6-tetrafluorobenzene , 2-tributylstunnylthiophenes (3.00 mmol), Pd and palladium (2.0 mol%) was stirred in dry N,N-dimethylacetoamide (1.0 mL) for 2 h at 120 °C under argon. The reaction mixture was diluted by diethyl ether, rapidly cooled to room temperature, and then filtered to remove insoluble salts. A large amount of water was added to the reaction mixture, and then it was extracted with diethyl ether (5 times). The separated organic phases were dried over MgSO4, evaporated and dried under reduced pressure. The further purification was performed using silica gel chromatography (dichloromethane). Crystal was collected by filtration after recrystallization (dichloromethane/methanol).1,4-Dibromo-2,3,5,6-tetrafluorobenzene , thiophenes (1.00 mmol), 1-adamantanecarboxylic acid , K1. 1H NMR : δ 7.67 , 7.56 , 7.21 . 13C NMR : δ 145.7, 142.4, 130.2, 128.3, 127.8, 127.3. 19F NMR ): δ −140.8. Anal. Calcd. for (C14H6F4S2): C, 53.50; H, 1.92. Found: C, 53.49; H, 1.93.Compound 2. 1H NMR : δ 7.45 , 6.85 , 2.56 . 13C NMR : δ 143.1, 142.3, 142.2, 130.3, 125.7, 125.5, 15.1. 19F NMR ): δ −141.6. Anal. Calcd. for (C16H10F4S2): C, 56.13; H, 2.94. Found: C, 56.11; H, 2.94.Compound 3. 1H NMR : δ 7.46 , 7.13 , 2.38 . 13C NMR : δ 145.6, 142.3, 137.9, 132.4, 127.6, 123.8, 15.6. 19F NMR ): δ −141.2. Anal. Calcd. for (C16H10F4S2): C, 56.13; H, 2.94. Found: C, 56.12; H, 2.92.Compound 1H and 13C NMR spectra were recorded on a JEOL EX-300 spectrometer. 19F NMR spectra were measured using a JEOL EX-270 spectrometer. Elemental analyses were performed on a Thermo Finnigan Flash EA1112 CHN-O analyzer. UV-vis absorption spectra were obtained on an Ocean Optics USB4000-XR1 fiber spectrometer with DH2000-BAL tungsten halogen light source. Fluorescence spectra were obtained on an Ocean Optics USB4000 fiber spectrometer with PX-2 pulsed xenon light source. Absolute quantum yield was obtained by Hamamatsu C9920-02. The single crystal X-ray diffraction data for single crystals were collected at room temperature using SMART APEX II (Bruker AXS) with a CCD detector. The crystal structures were solved by the direct method and refined by full matrix least-squares using SHELXTL. XRD analysis was performed by JEOL JDX-3530 X-ray diffractometer system. Crystal images were obtained by optical microscope, KEYENCE VH-Z500R. DSC analysis was performed by a Shimadzu DS-60, which measured during heating from room temperature to 400 °C at heating rate of 10 °C/min in nitrogen. TGA analysis was performed by a Shimadzu TA-60, which measured during heating from room temperature to 600 °C at heating rate of 20 °C/min in nitrogen. High-resolution solid-state 13C and 19F nuclear magnetic resonance (NMR) spectra were measured using a Varian NMR systems 400WB spectrometer operating at 100.57 MHz for 13C, 376.25 MHz for 19F and 399.94 MHz for 1H. The 13C NMR spectra were obtained by the combined use of cross polarization (CP) from 1H nuclei and magic-angle spinning (MAS) with 1H high-power dipolar decoupling of 110 kHz under the MAS of 20 kHz. The utilized CP contact time was 2 or 4 ms and recycle delay of 200 or 500 s was used. The 19F MAS NMR spectra were measured using the 90 degree pulse of 1.6 μs was used under the MAS rate of 20 kHz. To obtain the 19F spin-lattice relaxation time (T1F), the conventional inversion-recovery method was employed with recycle delay of 5000 or 6000 s. 13C chemical shifts were measured relative to TMS (tetramethylsilane) using the methine carbon signal at 29.47 ppm for solid adamantane as an external standard. 19F chemical shifts were relative to CFCl3 referenced externally to the CF2 signal of Teflon at −122 ppm29.Liquid-state Supporting InformationMove S1Movie S2Movie S3


Acetone–butanol–ethanol fermentation has been studied for butanol production. Alternatively, to achieve acetone-free butanol production, use of clostridium strains producing butanol and 1,3-propanediol  from glycerol, natural and engineered isopropanol–butanol–ethanol (IBE) producers has been attempted; however, residual 1,3-PDO and acetone, low IBE production by natural IBE producers, and complicated gene modification are limitations.Clostridium sp. A1424 capable of producing IB from various substrates with a small residual acetone. Notably, this strain also utilized glycerol and produced butanol and 1,3-PDO. After 46.35 g/L of glucose consumption at pH 5.5-controlled batch fermentation, Clostridium sp. A1424 produced 9.43 g/L of butanol and 13.92 g/L of IB at the productivity of 0.29 and 0.44 g/L/h, respectively, which are the highest values in glucose-based batch fermentations using natural IB producers. More interestingly, using glucose–glycerol mixtures at ratios ranging from 20:2 to 14:8 led to not only acetone-free and 1,3-PDO-free IB fermentation but also enhanced IB production along with a much higher butanol content (butanol/isopropanol ratio of 1.81 with glucose vs. 2.07–6.14 with glucose–glycerol mixture). Furthermore, when the mixture of glucose and crude glycerol at the ratio of 14:8  was used, high butanol/isopropanol ratio (3.44) and butanol titer (9.86 g/L) were achieved with 1.4-fold enhanced butanol yield (0.28 g/g) and productivity (0.41 g/L/h) compared to those with glucose only at pH 5.5.Here, we report an effective isopropanol and butanol (IB) fermentation using a newly isolated Clostridium sp. A1424 was able to produce butanol and isopropanol from various carbon sources. The productivity and titer of butanol and total alcohol obtained in this study were higher than the previously reported results obtained using other natural IB producers. Use of the mixture of glucose and glycerol was successful to achieve acetone-free, 1,3-PDO-free, and enhanced IB production with higher yield, productivity, and selectivity of butanol compared to those with glucose only, providing great advantages from the perspective of carbon recovery to alcohols. This notable result could be accomplished by isolating an effective IB producer Clostridium sp. A1424 as well as by utilizing glucose–glycerol mixtures.A newly isolated The online version of this article (doi:10.1186/s13068-016-0650-7) contains supplementary material, which is available to authorized users. This phenomenon can be explained with a very low carbon flux to acetone and isopropanol, which likely results in a relatively low level of butyric acid re-assimilation and consequently high butyric acid residual.The main metabolic pathways in IB producing clostridia can be divided into net NAD(P)H-generating pathways such as acetate, acetone, and isopropanol producing pathways, while butyrate and butanol producing pathways are net NAD(P)H-neutral or net NAD(P)H-consuming ones. As seen in Fig. els Fig. d. This rC. acetobutylicum [Clostridium butyricum [C. pasteurianum to enhance butanol production [C. beijerinckii NCIMB 8052 for in situ detoxification of furfural [C. pasteurianum and C. beijerinckii, respectively, grown on the mixture of glucose and glycerol. In this study, we investigated the effect of mixture of glucose and glycerol on IB production for the first time. Notably, using a mixture of glucose and glycerol allowed us to achieve acetone-free and 1,3-PDO-free butanol and IB production with Clostridium sp. A1424. More desirably, the fermentation of Clostridium sp. A1424 with glucose and glycerol together was highly efficient for the production of butanol and total alcohol (the sum of butanol and isopropanol) along with high butanol contents. This is the first report of successful increase of butanol content in IB fermentation by using a mixture of glucose and glycerol without genetic engineering.The strategy using glucose and a more reduced carbon source like glycerol has been studied for the control of the metabolic fluxes and NAD(P)H pools using utylicum  and Closutyricum . The useoduction , 17 and furfural . HoweverBecause biodiesel-derived crude glycerol is an attractive cheap resource, we carried out IB fermentation with the mixture of glucose and crude glycerol instead of pure glycerol. Total substrate concentration was about 35.68 g/L with the ratio of glucose to crude glycerol at 14:8. This ratio of mixed substrates yielded the highest butanol production in case of pure glycerol  and that from glucose only at pH 5.5 (9.43 g butanol/L after 32 h)  was successful to achieve acetone-free, 1,3-PDO-free, and enhanced IB production. Particularly, the performance of butanol production  was significantly enhanced using a mixture of glucose and glycerol, compared to using glucose only. Thus, IB fermentation favoring butanol production with Clostridium sp. A1424 is especially valuable for the efficient carbon recovery to IB and butanol, a promising biofuel.A newly isolated 2HPO4, 0.05% KH2PO4, 0.2% (NH4)2SO4, 0.02% MgSO4·7H2O, 0.001% MnSO4·H2O, 0.001% FeSO4·7H2O, 0.001% NaCl, and 1.95% MES (2-(N-morpholino) ethanesulfonic acid) [d-glucose, d-mannose, d-fructose, d-xylose, l-arabinose, d-galactose, d-cellobiose, sucrose, and glycerol were added to MP2 medium as a substrate at a certain concentration as occasion demanded. Crude glycerol containing 81.7% (wt/wt) of glycerol, 10.5% (wt/wt) of water, 5% (wt/wt) of MONG (matter organic non-glycerol), 2.9% (wt/wt) of ash, 2.4% (wt/wt) of sodium, and less than 0.01% (wt/wt) of methanol, magnesium, and potassium was obtained from GS Caltex Corporation (South Korea) [Modified P2 medium (MP2) was used for seed and main cultures. The composition of medium (wt/v) was 0.6% yeast extract, 0.05% Kic acid) . Varioush Korea) . The ini600), the culture broth was serially diluted with 0.85% (wt/v) sterilized saline solution. Then it was plated onto MP2 medium agar plate containing 2% (wt/v) glucose in an anaerobic chamber . After the cultivation for 4 days at 30 °C in the chamber, each colony was cultivated again on MP2 liquid medium with 2% (wt/v) glucose for 48 h and metabolites were analyzed.Diesel-oil-contaminated soil was collected from Baegun Mountain . The soil sample (2 g) was added to 20 mL of sterilized and anaerobic MP2 medium containing 2% (wt/v) glucose; then heated at 80 °C for 30 min. The solution was incubated at 30 °C until cell growth was observed. After confirming the cell growth with the optical density at 600 nm .The 16S rRNA analysis and phylogenetic analysis of e et al. . The 16S600 of 8.0–9.0 in the late-exponential phase, 5% (v/v) culture solution was used to inoculate the main culture medium. Then, the main cultivation was performed at 37 °C and 200 rpm. To achieve anaerobic conditions for the seed and main cultures, the medium in the serum bottle was purged with argon gas for 20 min and the bottle was sealed with a butyl stopper and aluminum crimp seal [The microorganism was stored as a spore solution in sterile distilled water at 4 °C. For the activation of spores, the solution was heated at 80 °C for 10 min. The seed cultivation was started with inoculation of the 5% (v/v) activated spore solution in autoclaved MP2 medium containing 2% (wt/v) glucose at 37 °C and 200 rpm. When cell growth reached ODimp seal .For the pH-controlled fermentations, a 3-L bench fermenter  with a working volume of 1 L was prepared. The MP2 medium containing 6% (wt/v) glucose without MES was used. The fermenter jar was autoclaved and purged with filtered argon gas for 1 h to establish anaerobic conditions. Then, the 5% (v/v) seed culture solution was inoculated. The fermentation was operated at 37 °C and 200 rpm and the pH was controlled with 5 N KOH and HCl at the set point (pH 5.0–6.0). In case of bubble formation on the surface of the culture broth, 25% (v/v) anti-foam solution  was added intermittently.600 and DCW. The amount of carbon sources (except sucrose), 1,3-PDO, acetic acid, and butyric acid were analyzed by HPLC  composed of a refractive index detector (RID) and UV/Vis detector with a Hi-plex H column . A mobile phase was 5 mM sulfuric acid with a flow rate of 0.6 mL/min. Sucrose content was measured using a sucrose test kit . Acetone, isopropanol, ethanol, and butanol were analyzed using a GC  equipped with a flame ionization detector (FID). A DB-624 column  was used to clearly separate each peak of ethanol and isopropanol. The production of ethanol was excluded as the detected concentration was negligible (below 0.1 g/L) in all experiments.Cell growth was monitored with OD at 600 nm using a spectrophotometer  and dry cell weight  was determined using a calibration curve of ODThe generation of NAD(P)H from glucose and glycerol through the glycolytic pathway was calculated by multiplying ‘2’ by the moles of consumed glucose and glycerol, respectively. The requirement of NAD(P)H for the production was calculated by multiplying 4, 2, 1, 0, and 0 by the number of moles of produced butanol, butyric acid, isopropanol, acetone, and acetic acid, respectively  (Additio


Clostridium butyricum and Clostridium baratii species have been known to produce botulinum toxin types E and F, respectively, which can cause botulism, a rare but serious neuroparalytic disease. Here, we present finished genome sequences for two of these clinically relevant strains. Clostridium butyricum and Clostridium baratii carry the botulinum neurotoxin gene cluster , of 120,667 bp, with 25.3% G+C content and 119 coding regions. The chromosome (accession no. CP014204) size is 3,091,050 bp, with 28.4% GC content and 2,885 identified coding regions. The toxin gene was determined to be subtype F7 and was found within the plasmid.C. butyricum serotype E strain CDC51208 was isolated from an infant botulism case. The resulting finished genome includes two plasmids of 9,567 bp  and 820,516 bp . These plasmids consist of 26.8% and 28.2% G+C content, as well as 8 and 763 coding sequences (CDSs), respectively. The chromosome (accession no. CP013239) has 3,809,831 bp, 28.8% G+C content, and 3,334 identified coding regions. The toxin gene was determined to be subtype E4 and was found within the pNPD4_2 plasmid.CP014204 and CP014203 for C. baratii strain CDC51267, and accession numbers CP013238, CP013239, and CP013240 for C. butyricum strain CDC51208.These finished sequences have been deposited in GenBank, with accession numbers


GluN2A subunit levels were concurrently increased following lithium treatment. Similar alterations were also demonstrated in vivo; lithium administration increased GSK3β serine 9 phosphorylation and GluN2A levels, suggesting a reduced GSK3β activity and augmented GluN2A expression. Correspondingly, we observed that the amplitudes of evoked GluN2A-mediated excitatory postsynaptic currents in mPFC pyramidal neurons were significantly increased following lithium administration. Our data suggest that GSK3β activity negatively regulates GluN2A expression, likely by mediating upstream β-catenin phosphorylation, in prefrontal cortical neurons. Furthermore, our biochemical and electrophysiological experiments demonstrate that lithium mediates a specific increase in GluN2A subunit expression, ultimately augmenting GluN2A-mediated currents in the prefrontal cortex.Glycogen synthase kinase 3β (GSK3β) is a highly conserved serine/threonine kinase that has been implicated in both psychiatric and neurodegenerative diseases including schizophrenia, bipolar disorder, and Alzheimer's disease; therefore regulating its activity has become an important strategy for treatment of cognitive impairments in these disorders. This study examines the effects of lithium on GSK3β and its interaction with β-catenin and NMDA receptors within the prefrontal cortex. Lithium, a clinically relevant drug commonly prescribed as a mood stabilizer for psychiatric disorders, significantly increased levels of phosphorylated GSK3β serine 9, an inhibitory phosphorylation site, and decreased β-catenin ser33/37/thr41 phosphorylation  Glycogen synthase kinase 3β (GSK3β) plays an important role in both psychiatric and neurodegenerative disorders , CK (casein kinase), GSK3β, and β-catenin  animal guidelines and the experimental protocols (#20280) were approved by the Institutional Animal Care and Use Committee at Drexel University College of Medicine. Timed pregnant (TP16) and male (200–250 g) Sprague-Dawley (SD) rats were purchased from Charles River Laboratories .in vitro (DIV) four, half of the media was replaced with fresh serum-free media. Cultures were maintained at 37°C for at least 2 weeks before use.Preparation of rat prefrontal cultures was modified from previously described methods  published a dose-response curve  demonstrating lithium's effect on GSK3β activity, comparing phosphorylation of GSK3β serine 9 to total protein levels. Lithium chloride treatment in the dose range of 5–10 mM was shown to inhibit the enzymatic activity of GSK3β by more than 80%. A 5 mM dose of lithium was sufficient to reduce GSK3 kinase activity, while concurrently increasing total β-catenin levels starting at 6 h following treatment  of male SD rats weighing between 200 and 250 g. Control animals were injected with an equal volume of saline. These doses were chosen because serum levels have been reported to fall within the therapeutic range of drug in patients (0.8–1.0 mmol/L) and higher doses were demonstrated to produce adverse side effects  was added to each well, and incubated for 15 min at 4°C with gentle rocking. The RIPA buffer contained the following inhibitors: sodium pyrophosphate tetrabasic , ethylenediaminetetraacetic acid , sodium fluoride , sodium orthovanadate , leupeptin (inhibits serine and cysteine proteases), aprotinin (inhibits serine proteases), PMSF (inhibits serine proteases), and pepstatin A (inhibits aspartic acid proteases). Adherent cells were collected with a cell scraper, and the cell lysate was transferred to a microcentrifuge tube (combining wells of the same treatment). Lysates were homogenized with a mortar and pestle for 20 strokes and then incubated for 45 min on ice. After incubation, samples were centrifuged at 10,000 g for 10 min at 4°C, pellets discarded, and the supernatant collected and stored at −20°C. Western samples (20 μg/15 μl) were prepared, heated at 95°C for 5 min, cooled to room temperature, and stored at −20°C.Plates were set on ice, and all media was removed using vacuum suction. Wells were washed twice with cold 0.1 M PBS, all solution was removed, 50 μl of RIPA buffer  was thawed, weighed, and homogenized in 9 volumes of RIPA buffer containing inhibitors using a mortar and pestle. Lysates were incubated on ice for 30 min. After incubation, samples were centrifuged at 10,000 g for 15 min at 4°C, and the supernatant was collected and stored at −20°C. Western samples (15 μg/15 μl) were prepared, heated at 95°C for 5 min, cooled to room temperature, and stored at −20°C.in vitro; 15 μg/15 μl in vivo) were run on acrylamide gels comprised of a 3.0% acrylamide stacking gel on top of a 7.5% acrylamide separating gel. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane  with a 5% methanol transfer buffer at 100 V for 1 h at 4°C. Membranes were trimmed, rinsed in Tris-buffered saline with 1% Tween-20 (TBST) and blocked for 1 h with 5% milk in TBST. Then, primary antibodies were incubated overnight at 4°C with agitation. The following antibodies were applied at the approximate concentrations listed: GSK3β , pGSK3β serine 9 , GluN1 , GluN2A , GluN3A , GluN2B , β-catenin  and pβ-cateninS33/37/T41 . β-actin  was used as a loading control. After primary antibody incubation, membranes were rinsed with TBST three times for 20 min, blocked for 1 h with 5% milk in TBST, incubated with secondary antibody  for 1 h, and rinsed with TBST three times for 20 min at room temperature with agitation. Membranes were developed with ECL detection kit . After developing, membranes were rinsed with TBST for 5 min and stripped with Restore Western Blot Stripping Buffer  for an additional 15–30 min, depending on the antibody intensity. Membranes were then rinsed with TBST 3 times for 10 min. To probe the membrane for additional proteins the entire process, the described sequence was repeated from primary antibody incubation; membranes were blocked with 5% milk in TBST for 1 h and then incubated with primary antibody overnight at 4°C with agitation. Protein expression was determined using Bio-Sciences, Piscataway, NJ ImageJ64 software.Whole cell fraction samples  or an equivalent dose of physiological saline, and were sacrificed for slice collection after 24 h. Animals were perfused with ice-cold sucrose buffer, decapitated, and the brain was extracted. The mPFC was dissected in ice-cold sucrose solution and glued to a block in a vibratome bath containing the same solution . Slices containing the mPFC (300 μm) were collected and transferred to a slice chamber containing oxygenated ACSF that was bubbled continuously with 95% O+)-based intracellular solution  and in the presence of picrotoxin (50 μM), DNQX (20 μM), and Ro-25-6981 (0.5 μM), to block GABAA, AMPA, and GluN2B-containing NMDA receptor-mediated currents respectively . Recordings were obtained using a Cesium . All data underwent Shapiro-Wilk normality tests and Levene's Test of Equality of Error Variances when appropriate. The data without normal distribution were analyzed with a non-parametric Mann-Whitney U-test using SPSS. All data was presented as a mean ± standard error (S.E). Level for significance was set at p ≤ 0.05 for all comparisons.Western blot protein expression was determined using the ImageJ64 software. Each sample was taken as a ratio to the loading control . In the case of phosphorylation, each sample was taken as a ratio of its respective total protein levels . Total protein in the text refers to an antibody that tags the target protein without specificity for a particular post-translational modification such as phosphorylation, accounting for cellular levels of that protein under all conditions. For example, total GSK3β would encompass phosphorylated as well as unphosphorylated levels. To replicate prior research findings  = 2.163, p = 0.180, or time, F = 0.020, p = 0.891, on total GSK3β protein levels. There was no significant interaction between treatment group and time point on GSK3β protein levels, F = 0.069, p = 0.799. Lithium treatment significantly increased GSK3β ser9 phosphorylation protein levels, F = 40.593, p = 0.000. There was no significant effect of time on GSK3β ser9 phosphorylation, F = 0.467, p = 0.514. There was no significant interaction between treatment group and time point on GSK3β ser9 phosphorylation protein levels, F = 0.037, p = 0.853  = 3.710, p = 0.090, or time, F = 0.017, p = 0.899, on total β-catenin protein levels. There was no significant interaction between treatment group and time point on β-catenin protein levels, F = 0.269, p = 0.618. Lithium treatment significantly decreased β-catenin ser33/37thr41 phosphorylation protein levels, F = 33.341, p = 0.000. There was no significant effect of time on β-catenin ser33/37thr41 phosphorylation, F = 2.652, p = 0.142. There was no significant interaction between treatment group and time point on β-catenin ser33/37thr41 phosphorylation protein levels, F = 1.896, p = 0.206  for two time points, 4 or 24h, and Western blot analysis was used to determine protein expression levels of β-catenin and pβ-catenin ser33/37/thr41, the GSK3β target regulatory site. A two-way ANOVA was conducted to determine the effects of treatment group and time on total β-catenin as well as β-catenin ser33/37thr41 phosphorylation protein levels. There was no significant effect of treatment group, t-test was run to determine if there were differences in total or phosphorylated β-catenin ser33/37/thr41 protein levels between saline and lithium-treated animals. Lithium administration had no effect on either β-catenin, t(10) = 0.518, p = 0.616, or β-catenin ser33/37/thr41 phosphorylation, t(10) = −0. 431, p = 0.675  for two time points, 4 or 24 h and Western blot analysis was used to determine protein expression levels of NMDAR subunits GluN2A, GluN2B, GluN3A, and GluN1.F = 5.343, p = 0.050. There was a significant effect of time on GluN2A expression, F = 13.559, p = 0.006, increasing after 24 h; but no significant interaction between treatment group and time point on GluN2A protein levels, F = 1.627, p = 0.238. There was no significant effect of treatment group, F = 1.728, p = 0.225, or time, F = 4.029, p = 0.080, as well as no significant interaction between treatment group and time point on GluN2B protein levels, F = 0.437, p = 0.527. Additionally, there was also no significant effect of treatment group, F = 0.999, p = 0.347, or time, F = 0.714, p = 0.423, as well as no significant interaction between treatment group and time point on GluN3A protein levels, F = 0.561, p = 0.475  = −0.151, p = 0.883, or GluN1 t(8) = −1.333, p = 0.219, protein levels compared to saline-treated animals  = −2.387, p = 0.038  = −0.227, p = 0.822 , we treated rats with lithium (50 mg/kg) and measured evoked GluN2A-mediated excitatory postsynaptic currents (eEPSCs) in the layer 5 pyramidal neurons of the mPFC. Based on our previous reports . Inositol and GSK3 are the two major targets of lithium, which are both argued to underlie its effects. The inositol depletion model proposes that lithium inhibits IMPase, depleting the cell of endogenous inositol, thereby blocking the production of inositol 1,4,5 triphosphate (InsP3) and preventing extracellular signaling by this pathway. Albeit some researchers suggest inositol depletion mediates the effects of lithium  in cultured neurons reported similar findings regarding the relationship between GSK3β and NMDA receptors. Induced activation of GSK3β, either pharmacologically or genetically, led to decreased GluN2A/B expression that could then be mitigated by the simultaneous inhibition of the kinase. This study demonstrates the negative regulation that GSK3β imparts over GluN2A and GluN2B (Zhu et al.,  in cultuSpecifically, PFC persistent neuronal firing is the foundation of working memory with NMDA receptor activity playing a substantial role in this process Wang, . NMDA anNMDARs and PFC-dependent cognition also share common ties to the pathology observed in schizophrenia. NMDA receptor disruption has been well characterized in animal models of schizophrenia, demonstrating the paramount role these receptors play in disease. Mutations in high-risk gene candidates for schizophrenia such as neuregulin 1, DISC1, and dysbindin have all been demonstrated to disturb NMDA receptor regulation (Geddes et al., Surmounting evidence links NMDA receptor hypofunctioning in the PFC, particularly of GluN2A and GluN2B-containing receptors, as an underlying pathological origin of cognitive impairments (Wang et al., in vitro and in vivo with effects possibly mediated by β-catenin phosphorylation in the PFC. It is important to note that a limitation with our study is that our findings are largely correlational since we do not directly test whether lithium inhibits GSK3β kinase activity or selectively manipulate GSK3β in the mPFC, such as the utilization of shRNA. Therefore, our current study has set an essential framework, but future studies are warranted to further untangle the nature of lithium and its possible therapeutic actions of NMDA receptor augmentation in the PFC and the regulation of GSK3β.In summary, pharmacological inhibition of GSK3β with lithium significantly increased pGSK3β ser9 and decreased pβ-catenin ser33/37/thr41, thereby suggesting GSK3β inhibition and reduced β-catenin degradation. Total GluN2A levels concurrently increased after 24 h following lithium, demonstrating preferential upregulation of the GluN2A gene expression. These changes also corresponded with a physiological change in the mPFC, augmenting GluN2A-mediated eESPCs. We speculate this could be due to an increase in GluN2A expression at the postsynaptic density, which could increase the GluN2A-mediated inward current of mPFC pyramidal neurons, leading to increased amplitude of excitatory events. Conclusively, GSK3β activity influences the expression of GluN2A both The authors have read and have abided by the statement of ethical standards for manuscripts submitted to the Frontiers in Cellular Neuroscience. We declare that submitted manuscript does not contain previously published materials and are not under consideration for publication elsewhere. Each author has made a significant scientific contribution to the study, and is familiar with the primary data. All authors listed have read the complete manuscript and have approved submission of the paper. The manuscript is original work without fabrication, fraud, or plagiarism. All authors declare no conflicts of interest.SM contributed to the design, acquisition, analysis, and interpretation of the data; BF contributed to collection and interpretation of the electrophysiological data; and W-JG contributed to the conception of the work. All authors contributed to writing and editing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA), l-carnitine, and cimetidine, slightly reduced by p-aminohippuric acid (PAH), and unaffected by 1-methyl-4-phenylpyridinium (MPP+), oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs) and organic cation/carnitine transporters (OCTNs), partially transported by organic anion transporters (OATs) and multidrug resistance-associated proteins (MRPs), and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs).This study examined the urinary excretion of tetrodotoxin (TTX) modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK Tetrodotoxin (TTX) is a powerful neurotoxin that binds to voltage-gated sodium channels with very high affinity ,2. TTX i1 cell monolayers) and investigated the effects of several transport inhibitors on the movement of TTX across these monolayers.Previous studies have found that plasma concentrations of TTX decrease rapidly, and are undetectable by 12–24 h after ingestion, although TTX can be detected in the urine four days after ingestion ,20,21,221 cell monolayers) are shown in 2 5 min after incubation, which significantly increased to 1.237 ± 0.229 nmol/mL/cm2 60 min after incubation (p < 0.05). When the incubation temperature was 4 °C, the amount of TTX transported was 0.034 ± 0.008 nmol/mL/cm2 5 min after incubation, which significantly increased to 0.247 ± 0.032 nmol/mL/cm2 60 min after incubation (p < 0.05). At 60 min, the amount of TTX transported at 4 °C was significantly lower (0.20 fold) than the value at 37 °C (p < 0.05), indicating that the urinary excretion of TTX is temperature-dependent.Time course profiles of TTX urinary excretion  are shown in 2 5 min after incubation, which linearly increased to 0.483 ± 0.126 nmol/mL/cm2 60 min after incubation (p < 0.05). At an incubation temperature of 4 °C, the amount of TTX transported was 0.012 ± 0.002 nmol/mL/cm2 5 min after incubation, which increased to 0.091 ± 0.007 nmol/mL/cm2 60 min after incubation (p < 0.05). At 60 min, the amount of TTX transported was significantly lower (0.19 fold) than the value at 37 °C (p < 0.05), indicating that the renal reabsorption of TTX is temperature-dependent. These results indicate that the movement of TTX is both a transcellular and carrier-mediated process. The apparent permeability coefficients of TTX (Papp) for excretion and reabsorption are 1.95 ± 0.40 (10−6 cm/s) and 2.66 ± 0.66 (10−6 cm/s), respectively. There were no statistically significant differences between the two values (p > 0.05).Time course profiles of TTX renal reabsorption  significantly reduced the transport of TTX, with value of 75 ± 4% compared to the control (p < 0.05). No significant inhibitory effects were observed after the addition of probenecid, tetraethylammonium (TEA) and l-carnitine, with values of 85 ± 2%, 96 ± 8% and 84 ± 5%, respectively, compared to the control (p > 0.05).To investigate the transport characteristics of TTX across the LLC-PKexamined , p-aminol-carnitine, and probenecid significantly reduced the movement of TTX, with values of 42 ± 10%, 47 ± 11%, and 52 ± 8%, respectively, compared to the control (p < 0.05). PAH moderately reduced the transport of TTX, with a value of 63 ± 11% compared to the control (p > 0.05). In a separate inhibition assay, cimetidine significantly reduced the transport of TTX, with a value of 60 ± 6% compared to the control (p < 0.05). In contrast, 1-methyl-4-phenylpyridinium (MPP+), oxaliplatin, and cefalexin had no significant effects on transport (p > 0.05), with values of 92 ± 12%, 88 ± 5%, and 85 ± 10%, respectively, compared to the control , a typical OAT substrate, and was significantly reduced by probenecid (p < 0.05), a typical substrate of both OATs and MRPs [1 cells [p < 0.05), while the other inhibitors had no significant effect (p > 0.05). These results indicate that the transporters involved in the movement of TTX differ by the direction of transport; that is, excretion vs. reabsorption.Numerous endogenous metabolites, drugs, and xenobiotics are excreted from the bloodstream into the urine through the renal proximal tubules and/or via glomerular filtration . The rennd OCTNs . It is kse cells ,34. It ase cells ,36. In tand MRPs ,38. Porc[1 cells . These rTEA, which is known to be transported by OCTNs and MATEs ,41,42, mDrugs and metabolic products that bind to plasma proteins are generally not subjected to glomerular filtration. These compounds are known to be secreted into the lumen of the renal proximal tubule from the capillary blood vessel by organic ion transporters located on proximal tubular cell membranes. Indeed, we previously reported that TTX binds to both human alpha-1-acid glycoprotein and bovine serum albumin . Hence, 1 cell line  was purchased from DS Pharma Biomedical company . High-glucose Dulbecco’s modified Eagle’s medium (DMEM), non-essential amino acid (NEAA) solution, and antibiotic-antimycotic solution (penicillin-streptomycin-amphotericin B suspension) were purchased from Wako Pure Chemical Industries . Fetal bovine serum (FBS) was purchased from HyClone Laboratories . Crystalline TTX was purchased from Wako Pure Chemical Industries; in addition to its use in the experiments, it was used as a standard for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Transport inhibitors l-carnitine, tetraethylammonium chloride (TEA), p-aminohippuric acid (PAH), probenecid, 1-methyl-4-phenylpyridinium iodide (MPP+), oxaliplatin, cefalexin, and cimetidine were purchased from Wako Pure Chemical Industries. All other chemicals were of reagent-grade quality.The LLC-PK1 cells were cultured in a 75-cm2 flask  using high-glucose DMEM supplemented with 10% FBS, 1% NEAA, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. The medium was changed every two days. The confluent cells were subcultured weekly by trypsinization with a solution of 0.25% trypsin containing 1 mM EDTA [4 cells/cm2. For the transport experiments, the cells were seeded at a density of 6 × 105 cells/cm2 in 24-well Transwell® inserts containing a polyester membrane . Transwell inserts were placed in a 24-well companion plate , and 0.3 mL and 1 mL of the medium were added to apical and basolateral sides of the Transwell insert chambers, respectively. The medium was changed on 4 days after seeding, and the seeded cells were maintained for 7 days to prepare differentiated cell monolayers.LLC-PK mM EDTA ,51 and s1 cell monolayers (Rtissue) was normalized by subtracting the background of the blank filter (Rblank) from the sample . TEER values of the cell monolayers (TEERtissue) (Ω·cm2) were calculated from the following equations:1 cell monolayers (cm2). The cell monolayers, whose TEER values were above 20 Ω·cm2, were used for the experiments with reference to TEER values reported previously [TEER was measured using a Millicell ERS-2 voltohmmeter . The ohmic resistance of the LLC-PKeviously ,26,52,531 cell monolayers grown in Transwell inserts on day 7 after seeding. The medium was aspirated from both chambers separated by the cell monolayers, and the cell monolayers were washed twice with transport buffer . The cell monolayers were preincubated at 37 °C for 10 min. The time course study of transcellular TTX transport was begun by replacing the buffer in the apical or basolateral side chamber (donor) with transport buffer (pH 7.4) containing 50 µM TTX. Incubation was carried out at 37 °C for up to 60 min. At the designated times, the incubation buffer in the opposite-side chamber (receiver) was rapidly collected. Additionally, the incubation temperature was lowered to 4 °C to examine the temperature-dependence of the transport.Transcellular transport of TTX was examined using LLC-PKl-carnitine, MPP+, oxaliplatin, cefalexin, and cimetidine) were dissolved in transport buffer (pH 7.4) containing 50 µM TTX and 1% methanol to final concentrations of 5 mM. After the preincubation described above, the cell monolayers were incubated at 37 °C for 30 min with transport buffer (pH 7.4) containing 50 µM TTX and 5 mM of each inhibitor in the donor chamber . The transport buffer (pH 7.4) in the receiver chamber was rapidly collected at the end of the incubation period and subjected to LC-MS/MS for determination of TTX levels.The inhibitory effects of the transporter inhibitors on the movement of TTX across the cell monolayers were investigated. The inhibitors . TTX quantification was performed by LC-MS/MS analysis according to the method of Matsumoto et al. . The LC-app) (cm/s) was calculated from the following equation:1 cell monolayers (cm2) and the initial TTX concentration in the donor chamber (µM), respectively. Data are expressed as the mean ± standard error. Dunnett’s test and Student’s t-test were used to analyze the significance of differences among the means at the 5% significance level.The permeation rate of TTX (J) (nmol/min) was defined as the slope of the initial linear portion of the curve that was calculated by linear regression analysis from the scatter plots of the amount of TTX transported (nmol) versus time (min). The apparent permeability coefficient of TTX (P


P21 activated kinase 4 (PAK4), a key regulator of cytoskeletal rearrangement and endothelial microparticles (EMPs), is released after lipopolysaccharide (LPS) stimulation. In addition, it participates in LPS-induced lung injury. In this study, forty-eight Sprague Dawley (SD) rats were divided into two groups, including PAK4 inhibitor (P) and PAK4 inhibitor + simvastatin (P + S) treatment groups. All rats were given PAK4 inhibitor (15 mg/kg/d) orally. Immediately after PAK4 inhibitor administration, simvastatin was injected intraperitoneally to P + S group animals at 20 mg/kg/day. Then, treatment effects on the intestinal mucosal barrier and lung injury caused by PAK4 inhibitor and simvastatin were assessed. The results showed that gut Zonula Occludens- (ZO-) 1, PAK4, mitogen-activated protein kinase 4 (MPAK4), and CD11c protein levels were reduced, while plasma endotoxin levels were increased after administration of PAK4 inhibitor. Furthermore, compared with normal rats, wet-to-dry (W/D) values of lung tissues and circulating EMP levels were increased in the treatment group, while PAK4 and CD11c protein amounts were reduced. Therefore, in this lung injury process induced by PAK4 inhibitor, the protective effects of simvastatin were reflected by intestinal mucosal barrier protection, inflammatory response regulation via CD11c+ cells, and cytoskeleton stabilization. In summary, PAK4 is a key regulator in the pathophysiological process of acute lung injury (ALI) and can be a useful target for ALI treatment. Sepsis is one of the principal causes of death in critical care medicine departments . DespiteALI is associated with pulmonary hypervascular permeability, edema, and high levels of proinflammatory cytokines , 10. DisIn the current study, rats were orally given the PAK4 inhibitor PF-3758309. First, the effects of the PAK4 inhibitor on gut barrier function were assessed, quantifying the protein levels of PAK4, ZO-1, CD11c, and MAP4K4. Meanwhile, a relationship between the effect of the PAK4 inhibitor and the degree of lung injury was determined by plasma endotoxin levels, EMP amounts in circulation, PAK4 and CD11c protein levels, and histological alterations of the lung. Finally, the protective effects of simvastatin on PAK4 inhibitor-induced gut and lung dysfunction were also evaluated.Seven-week-old male Sprague Dawley (SD) rats (250 ± 10 g) were used in this study. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University and performed in strict accordance with the National Institutes of Health guidelines for the use of experimental animals.n = 24), respectively, named PAK4 inhibitor (P) and PAK4 inhibitor + simvastatin (P + S) treatment groups. The PAK4 inhibitor was administered orally once daily at a dose of 15 mg/kg [Forty-eight animals were randomly divided into two groups , ZO-1 , CD11c , and MAP4K4 .At 24, 48, and 72 h after administration of PAK4 inhibitor or PAK4 inhibitor + simvastatin, the animals were exsanguinated by cardiac puncture. Then, the two lungs were harvested from each animal and separated: the right lung was used for subsequent tests , while the left one was homogenized and weighed. Afterward, the homogenate was centrifuged  and desiccated in an oven (70°C for 24 h) for dry weight determination. Lung wet-to-dry weight ratio (W/D) was then derived. Meanwhile, hematoxylin-eosin (H&E) staining was performed for all specimens. Six high power fields were randomly selected in every section, and the Spot Advanced Computer Photo Analysis Microsoft System  was used to measure alveolar wall thickness.μm) were permeabilized with 0.2% Triton X-100 for 20 minutes and stained as described previously [Gut and lung tissue specimens from each rat were obtained after perfusion with cold PBS and 2% paraformaldehyde. After two hours of fixation with 2% paraformaldehyde, the samples were cryopreserved for later use. Gut and lung sections  in PBS containing protease inhibitors, and homogenized (3 × 30 s on ice) on a Polytron . Tissue lysates were cleared by centrifugation  on a Beckman Avanti Centrifuge J-25 I using a JA-25.50 rotor. The subsequent procedures were based on our previous study . PrimaryThe protein content of endotoxins was detected by DC Protein Assay .q test, Student's t-test (between two groups), and two-way analysis of variance (ANOVA) (>2 groups) were used for comparisons. P < 0.05 was considered statistically significant.Data are mean ± standard error (SE) and were statistically analyzed with the SPSS version 16.0 software . Student-Newman-Keuls-μm, respectively, in the P group, and 1.39 ± 0.18, 1.85 ± 0.31, and 2.05 ± 0.37 μm, respectively, in the P + S group; mean alveolar wall thickness was 0.89 ± 0.04 μm in normal rats. Significant differences between the P and P + S groups at 24, 48, and 72 h  were found. W/D values were greatly increased in the lungs of PAK4 inhibitor-treated rats but remained low in those given simvastatin.As shown in P < 0.01 for CD144 at 24–48 h, P < 0.001 for CD144 at 72 h, P < 0.01 for CD62E at 24–72 h, P < 0.05 for CD31 at 72 h, P < 0.01 for CD51 at 24–72 h, and P < 0.05 for CD54 at 72 h, versus control, resp.).EMPs with different phenotypes were counted . FollowiP < 0.05 for CD144 at 72 h, P < 0.05 for CD62E at 72 h, P < 0.05 for CD31 at 24–72 h, P < 0.05 for CD51 at 24–72 h, and P< 0.05 for CD54 at 72 h, resp.).However, compared with the P group, the P + S group showed reduced EMP amplification . Furthermore, significant differences were found between the P and P + S groups at 48 and 72 h, respectively .The expression levels of PAK4 and CD11c in gut tissues from all animals were assessed, and fluorescence intensities are shown in P < 0.001). What is more, significant differences were found between the P and P + S groups at the same time-point . Western blot (Relative CD11c protein expression levels in gut tissues were 3.72 ± 0.64 (Control), 0.49 ± 0.07 , 0.36 ± 0.05 , 0.58 ± 0.09 , 4.15 ± 0.83 , 3.47 ± 0.68 , and 4.34 ± 0.95 , indicating significant differences between control animals and those given the PAK4 inhibitor , while decreasing MAP4K4 protein amounts . However, the protein levels of ZO-1 were much higher after simvastatin administration at 48 and 72 h  compared with P group amounts. Meanwhile, MAP4K4 protein levels were much higher after simvastatin administration at 24, 48, and 72 h .We also detected the protein levels of ZO-1 and MAP4K4 in gut tissues by Western blot . InteresP < 0.01). Furthermore, significant differences between the P and P + S groups were obtained at 24, 48, and 72 h ; however, there was no obvious difference between the control and simvastatin groups at 24 h .P < 0.01 and P < 0.05, resp.). There was also an overt difference between the P and P + S groups at 48 h (P < 0.01), with no significant difference between the control and simvastatin groups at 24 h .Relative expression levels of the CD11c protein in lung tissues were 7.51 ± 1.36 (Control), 7.86 ± 1.59 , 5.92 ± 0.97 , 2.36 ± 0.41 , 4.37 ± 0.93 , 5.37 ± 0.91 , 5.21 ± 1.03 , and 4.34 ± 0.87 , indicating marked differences between control animals and the PAK4 inhibitor-treatment group, particularly at 48 and 72 h . In addition, PAK4 protein levels were much higher than those of the P group after simvastatin treatment at 24, 48, and 72 h . CD11c protein levels were also strikingly decreased after PAK4 inhibitor treatment at 24, 48, and 72 . Compared with P group amounts, CD11c protein levels were much higher after simvastatin treatment at 24, 48, and 72 h .Western blot  directlyP < 0.01 at 24 h, P < 0.001 at 48 h, and P < 0.01 at 72 h) and P + S treated animals at 24 and 48 h .Despite significant investigational efforts, abdominal sepsis is a major cause of mortality in ICU . The intIn this study, an animal model of oral PAK4 inhibitor was established to assess the relationship between the intestinal mucosal barrier and pulmonary vascular cytoskeleton dysfunction. After oral administration of 15 mg/kg/d PAK4 inhibitor to rats, gut PAK4 and ZO-1 protein levels were significantly decreased. ZO-1 is a key component of tight junctions, which seal the paracellular space between adjacent intestinal epithelial cells . AccumulMoreover, MAP4K4 also plays a key regulatory role in inflammation, emerging as a new target for the suppression of LPS-induced macrophage inflammatory responses. In the present study, MAP4K4 levels in gut and lung tissues were increased after administration of the PAK4 inhibitor, suggesting enhanced MAP4K4-associated inflammatory response , 27. FurPAK4 inhibitors, as toxic agents to the gastrointestinal tract, inhibit PAK4 expression and cause gut intestinal mucosa epithelial cell injury. Alterations of ZO-1, PAK4, MAP4K4, and CD11c in gut tissues suggested gut injury, and dysfunction of gut intestinal mucosa epithelial cells directly leads to loss of mucosal integrity and favors bacterial translocation. The increased plasma endotoxin levels after administration of the PAK4 inhibitor also proved loss of mucosal integrity and bacterial translocation.Multiple studies have suggested mechanisms for endotoxin- (LPS-) induced lung injury. We also found that PAK4, which is associated with increased plasma EMP levels, could constitute a useful pulmonary injury marker and an effective therapeutic target in LPS-treated mice [As mentioned above, PAK4 is a key regulator of cytoskeletal rearrangement and EMP release and can be considered a potential target for ALI treatment. In addition, simvastatin has been used in our previous and present studies to alleviate pulmonary injury by enhancing PAK4 expression, reducing PAK4-associated cytoskeletal rearrangement, and decreasing EMP release , while s


Annona muricata and Annona reticulata in vitro against Plasmodium falciparum. Crude ethanolic extracts, methylene chloride fractions, aqueous fractions, subfractions and isolated compounds  were tested for cytotoxicity on erythrocytes and Human Foreskin Fibroblasts cells and against the W2 strain of P. falciparum in culture. Results indicated that none of the extracts was cytotoxic at concentrations up to 10 µg/mL. Most of the extracts, fractions and subfractions inhibited the growth of P. falciparum with IC50 values ranging from 0.07 to 3.46 µg/mL. The most potent was the subfraction 30 from A. muricata stem bark (IC50 = 0.07 µg/mL) with a selectivity index of ˃ 142. Subfraction 3 from A. muricata root also exhibited very good activity (IC50 = 0.09 µg/mL) with a high selectivity index (SI ˃ 111). Amongst the isolated compounds, only gallic acid showed activity with IC50 of 3.32 µg/mL and SI > 10. These results support traditional claims for A. muricata and A. reticulata in the treatment of malaria. Given their limited cytotoxicity profile, their extracts qualify as promising starting points for antimalarial drug discovery.The aim of this work was to screen extracts from  Malaria is a life-threatening disease caused by parasites that are transmitted through the bites of infected mosquitoes. According to the latest estimates, there were about 198 million cases of malaria and an estimated 584,000 deaths from malaria in 2013 -2,5-diphenyltetrazolium bromide/phenazine methosulfate  colorimetric assay , with so2, and 20 µL of MTS/PMS  was added to each well and incubated for 1.5 h at 37 °C. Absorbance was then recorded at 490 nm using a 96-well plate reader . The percent growth inhibition was calculated from the optical densities relative to the negative control, and 50% cell cytotoxicity (CC50) values were determined using GraphPad Prism 5.0. Selectivity indices of plants extracts, defined as the ratio CC50 /IC50 parasites, were determined.To determine selectivity indices of active extracts, a toxicological assessment was carried out with human foreskin fibroblast (HFFs), essentially as previously described . BrieflyPlasmodium falciparum strain W2 was maintained in culture in sealed flasks at 37 °C, in a 3% O2, 5% CO2 and 91% N2 atmosphere in RPMI 1640, 25 mM HEPES, pH 7.4, supplemented with heat inactivated 10% human serum and human erythrocytes to achieve a 2% hematocrit. Parasites were synchronized at the ring stage by serial treatment with 5% sorbitol  [50s were calculated using Prism 5.0 software  with data fitted by non-linear regression to the variable slope sigmoidal dose–response formula, y = 100/1 + 10(logx)HIC50−, where H is the hill coefficient or slope factor [e factor .2O and CH2Cl2 fractions. They were all tested for biological activity, and the CH2Cl2 fractions of A. muricata root and stem bark  were selected as promising  [O-β-d-glucopyranoside (AMsbP3) [O-β-d-glucopyranoside (AMrP1) was reported in the root of Polyalthialongifolia var pendula [Rollinia leptopetala [Guatteria blepharophylla [O-β-d-glucopyranoside (AMsbP3) were recently found in the leaf of Polyalthia longifolia [Annona squamosa [Isolated compounds were identified by comparison of their spectroscopic data with those reported in the literature as stigmasterol-3- (AMrP1) ,19, lich (AMrP1) ,21, gall (AMrP1) ,23 and β(AMsbP3) . Stigmas pendula . Lichexatopetala  and fromrophylla . Gallic ngifolia  and twigsquamosa  respectiThe 79 extract samples prepared from the two investigated plants were screened for antiplasmodial activity.A. muricata and stem bark, root, and fruit of A. reticulata showed antiplasmodial activity, with IC50 values ranging from 0.29 to 1.90 µg/mL (A. reticulata were inactive (IC50 > 10 µg/mL).Crude extracts from the pericarp, root, and stem bark of 90 µg/mL . Crude e2Cl2 and H2O fractions were prepared by liquid-liquid partitions. Overall, none of the H2O fractions showed activity. On the other hand, apart from the ARlCH2Cl2 fraction from A. reticulata leaf, the seven other CH2Cl2 fractions from both plants exhibited potent activity, with IC50 values ranging from 0.19–1.50 µg/mL (2Cl2-IC50 = 0.19 µg/mL). Overall, the fractions AMpCH2Cl2, AMrCH2Cl2, AMsbCH2Cl2, ARtwCH2Cl2, AResbCH2Cl2, ARrCH2Cl2 and ARfrCH2Cl2 were promising, with IC50 values below 5 µg/mL. Fraction AMrCH2Cl2 showed the best selectivity (SI ˃ 52.6).From the crude ethanolic extracts, CH50 µg/mL . The mos50 values greater than 10 μg/mL.All the tested extracts showed no toxicity to erythrocytes and HFF cells at concentrations up to10 μg/mL, indicating IC2Cl2 fractions of root (AMrCH2Cl2) and stem bark (AMsbCH2Cl2) of A. muricata. They were submitted to flash chromatography eluting with solvent systems of increasing polarity  to afford subfractions and four pure compounds (stigmasterol-3-O-β-d-glucopyranoside (AMrP1), lichexanthone (AMsbP1), gallic acid (AMsbp2) and β-sitosterol-3-O-β-d-glucopyranoside (AMsbP3)) that were assessed for antiplasmodial activity. Three of eight subfractions  obtained from the root extract of A. muricata possessed activity, with IC50 values of 0.61 µg/mL, 0.22 µg/mL and 0.09 µg/mL, respectively (50 = 0.79 µg/mL). The isolated compound from the root of A. muricata, stigmasterol-3-O-β-d-glucopyranoside, did not show activity at concentrations up to 10 µg/mL.Further fractionation coupled with biological screening was performed on the two CHectively , with im2Cl2 fraction of A. muricata stem bark extract yielded 43 subfractions among which 18  showed antiplasmodial activity (IC50 < 5 µg/mL), with IC50 values ranging from 0.07 µg/mL (AMsbSF30) to 3.46 µg/mL (AMsbSF16). Among the three isolated compounds from this CH2Cl2 fraction , only gallic acid showed activity, with IC50 3.32 µg/mL. Subfraction AMsbSF30 exerted the best activity (IC50 = 0.07 µg/mL) and also showed the highest selectivity (SI = 142.3).The CHA. muricata that showed IC50 values below 0.2 µg/mL and SI ˃ 100 could be considered as promising starting points for drug development. Furthermore, SI ˃ 10 indicates appropriate pharmacological efficacy and safety of plant extracts [Moreover, according to previously agreed criteria , Subfracextracts . Based oA. muricata, were described. The use of A. reticulata in the traditional cure of malaria was reported [A. muricata and A. reticulata showed antiplasmodial activity, with IC50 values < 10 µg/mL, and did not demonstrate cytotoxicity.In our previous report , Annonacreported . In the A. muricata pericarp, root and stem bark showed potent antiplasmodial activity. Results obtained with A. muricata crude ethanolic extracts from pericarp, root and stem bark and their respective CH2Cl2 fractions corroborated the findings of other authors [A. muricata in a 1:1 chloroform/methanol mixture caused 67% inhibition of P. falciparum [A. muricata leaf exhibited moderate activities against chloroquine sensitive but not chloroquine resistant strains of P. falciparum. However, in their study the most potent ethyl acetate extract was toxic to human monocytes. In a similar approach, A. muricata leaf powder  defatted with hexane and sequentially extracted with dichloromethane, methanol and water showed promising activity against P. falciparum, with negligible toxicity against bovine cells [A. muricata leaf aqueous, ethanol and pentane extracts showed moderate antiplasmodial activity [A. muricata, mainly acetogenins [A. muricata showed antiplasmodial activity [Extracts from  authors . Twenty lciparum . Moreovene cells . In anotactivity . Few restogenins . Anonainactivity  with lowO-β-d-glucopyranoside showed no activity, contrary to the reports of other authors who claimed appreciable potency for the same compound isolated from the bark of Dacryodes edulis (Burseraceae) [Moderate antiplasmodial activity of gallic acid was reported , and auteraceae) .A. reticulata showed activities against P. falciparum. As far as we know, this is the first report on the antiplasmodial activity of extracts from A. reticulata. However, A. reticulata has been investigated for activity against other protozoan parasites. The in vitro anti-leishmanial activity of extracts from the leaf and seed of the plant was studied [L. amazonensis, L. braziliensis and L. guyanensis and against the intracellular amastigote forms of L. amazonensis [Also, extracts and fractions from leaf, twig, root, stem bark and fruit of  studied , and reszonensis .A. muricata and A. reticulata. Detailed studies are ongoing to characterize the active principles and further structure-activity-relationships.Our results highlight the antiplasmodial activity and lack of cytotoxicity of extracts from