This study also investigated the synergic or additive effects of these compounds. Enzymatically isolated primary bovine or human chondrocytes were cultured in monolayer until confluence and then incubated for 24 hours or 48 hours in the absence or in the presence of interleukin-1β and with or without curcuminoids extract, hydrolyzed collagen or green tea extract, added alone or in combination, at different concentrations. Cell viability was neither affected by these compounds, nor by interleukin 1β. In the absence of interleukin-1β, compounds did not significantly affect bovine chondrocytes metabolism. In human chondrocytes and in the absence of interleukin 1β, curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract significantly inhibited matrix metalloproteinase-3 production. In interleukin-1β-stimulated bovine chondrocytes, interleukin-6, inducible nitric oxide synthase, cyclooxygenase2, matrix metalloproteinase 3, a disintegrin and metalloproteinase with thrombospondin type I motifs 4 and a disintegrin and metalloproteinase with thrombospondin type I motifs 5 expressions were decreased by curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract. The combination of the three compounds was significantly more efficient to inhibit interleukin-1β stimulated matrix metalloproteinase-3 expression than curcuminoids extract alone. In interleukin-1β-stimulated human chondrocytes, nitric oxide, interleukin-6 and matrix metalloproteinase 3 productions were significantly reduced by curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract. These findings indicate that a mixture of curcuminoids extract, hydrolyzed collagen and green tea extract has beneficial effects on chondrocytes culture in inflammatory conditions and provide a preclinical basis for the in vivo testing of this mixture.The main objective of this study was to assess the  Osteoarthritis (OA) is a chronic, painful, degenerative and inflammatory condition that affects the joints and leads to functional disability. The most prominent feature of OA is the progressive degradation of articular cartilage. Chondrocytes play a key role in cartilage degradation in OA by producing matrix metalloproteinases (MMP), free radicals and inflammatory cytokines in response to mechanical or biochemical stimuli , 2. ThesNowadays, curative treatments for OA remain missing and the management of OA focuses on the alleviation of symptoms. Current recommendations for the management of OA include a combination of non-pharmacological and pharmacological modalities. Moreover, for patients with severe OA, joint replacement is indicated . Among nCurcuma longa. Anti-catabolic, anti-apoptotic and anti-inflammatory effects of curcumin are largely described in vitro. It is demonstrated that curcumin decreases nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, IL-8, cyclooxygenase2 (COX2), inducible nitric oxide synthase (iNOS), MMP-3 and MMP-9 synthesis through the inhibition of nuclear factor κB (NF-κB) translocation and tumor necrosis factor (TNF)-α signaling pathways in chondrocytes [In this context, safer alternative treatments are needed. Such treatments could come from nutrition and more particularly from nutraceuticals. Some positive beneficial effects are highlighted with nutraceuticals in the course of OA , 8. Amondrocytes . Hydrolydrocytes , 15. Thedrocytes , 17. Prodrocytes , 19. Epidrocytes . Advancedrocytes . The expdrocytes . IL-1β idrocytes . The tradrocytes .It has been reported that the majority of the pro-inflammatory and catabolic mediators linked to OA are regulated by NF-κB . The subFor the first time, this paper compares the effects of curcuminoids extract (C), hydrolyzed collagen (O) and green tea extract (T) in normal bovine and OA human chondrocytes cultured in monolayer. Further, this study investigates the synergic or additive effects of these compounds. Finally, we investigated the effects of these compounds on IL-1β induced NF-κB signaling pathway.Normal bovine articular cartilage was obtained from “Public slaughterhouse of Liege” from the metacarpal/metatarsal-phalangeal joint of 1 to 2 years old steers shortly after death . OA huma6 cells/ml (bovine chondrocytes) or 0.1 x 106 cells/ml (human chondrocytes) of DMEM (with phenol red and 4.5 g/L glucose) supplemented with 10% fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine  and 20 μg/ml proline Full-depth articular cartilage was excised and immersed in Dulbecco’s Modified Eagle Medium (DMEM) (with phenol red and 4.5 g/L glucose) supplemented with N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) 10 mM, penicillin (100 U/ml) and streptomycin (0.1 mg/ml) . After three washings, chondrocytes were released from cartilage by sequential enzymatic digestions with 0.5 mg/ml hyaluronidase type IV S  for 30 min at 37°C, 1 mg/ml pronase E  for 1 h at 37°C and 0.5 mg/ml clostridial collagenase IA  for 16 to 20 h at 37°C. The enzymatically isolated cells were then filtered through a nylon mesh (70 μm), washed three times, counted and filled to the density of 0.25 x 106 cells/well (bovine chondrocytes) or 0.2 x 106 cells/well (human chondrocytes) and cultured in monolayer for 5 days.Cells were seeded in a 6-well plate at the density of 0.5 x 10-10 M) . To investigate their potential synergic or additional effects, nutraceuticals were then tested alone at the final concentration of 12.5 μg/ml or in combination  in the absence or in the presence of recombinant porcine IL-1β (10-10 M). When OA human chondrocytes achieved confluence, the culture medium was removed and replaced by fresh culture medium and tested compounds alone or in combination (1–2–4 μg/ml curcuminoids extract + 1–2–4 μg/ml hydrolyzed collagen + 1–2–4 μg/ml green tea extract), at concentrations ranging between 1 to 4 μg/ml, and in the absence or in the presence of human IL-1β (10-11 M) . To investigate the effects of compounds on IL-1β induced NF-κB signaling pathway, OA human chondrocytes were pre-incubated with 4 μg/ml curcuminoids extract or with the combination 4 μg/ml curcuminoids extract + 4 μg/ml hydrolyzed collagen + 4 μg/ml green tea extract for 24 h and then co-treated with IL-1β (10-11 M) for 5 or 15 minutes or untreated. Curcuma extract is an oleoresin and is obtained from the roots of the plant Curcuma longa. Curcuminoids extract from Naturex is composed of natural extract and methylcellulose, and its content in curcuminoids is about 82% of which 75% are curcumin, 21% are demethoxycurcumin and 4% are bisdemethoxycurcumin. Hydrolyzed collagen from Gelita is a mix of different peptides. In average the peptides are composed by 30 amino acids, meaning a molecular weight of about 3 kDa. Glycine and proline represent more than 35% of total amino acids content. Green tea extract from Naturex, obtained from green tea leaves, contained natural extract and maltodextrin. Total polyphenols content is higher than 25%, catechins content higher than 12.5% and EGCG content higher than 9.3%. Curcuma extract was solubilized in tetrahydrofuran . Tetrahydrofuran was added in each condition to obtain final concentration at 0.1%. Hydrolyzed collagen and green tea extract were dissolved in water and filtered through a sterile mesh (0.20 μm). The effects of compounds were compared to controls consisting in same media without compounds and with or without IL-1β. Each culture condition was tested in triplicate in three independent chondrocytes cultures. Chondrocytes were incubated for 24 or 48 hours with the compounds and/or IL-1β. After 24 h of incubation, cells were scrapped, and RNA extraction was performed using RNeasy mini kit  for quantitative real time polymerase chain reaction using the LightCycler 480 .Chondrocytes were then cultured in monolayer until confluence (for about 24 hours) in DMEM (phenol red-free and containing only 1 g/L glucose)  supplemented with 1% fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 20 μg/ml proline. Only primary cultures were used to ensure the stability of chondrocyte phenotype. When normal bovine chondrocytes achieved confluence, the culture medium was removed and replaced by fresh culture medium containing curcuminoids extract  or hydrolyzed collagen  or green tea extract  at the final concentration of 0.5 μg/ml, 2.5 μg/ml, 12.5 μg/ml or 62.5 μg/ml, with or without recombinant porcine IL-1β  release assay and then stored at-20°C until additional analysis. Cells were scrapped and homogenized in 500 μl of Tris-HCl buffer by ultrasonic dissociation for 20 s at 4°C, to measure desoxyribonucleic acid (DNA) content.Cell viability was estimated by quantifying the release of LDH in the culture supernatant as previously described . A samplDNA content was measured in the cell extracts by a fluorimetric method using Hoechst .RNA from bovine chondrocytes from 3 wells of each condition was isolated using RNeasy mini kit . Then, RNA was reverse transcribed. Quantitative real time Polymerase Chain Reaction (qPCR) was performed by using the SYBR Premix Ex Taq (Tli RNaseH Plus)  as previously described . The PCR2) standard dilutions was mixed with Griess reagent . The absorption was measured at 540 nm.Nitric oxide (NO) production was determined by quantifying its derived product, nitrite, in the culture supernatant using a spectrophotometric method based upon the Griess reaction, as previously described . Briefly2 production was measured in the culture supernatant using the DetectX PGE2 High Sensitivity Immunoassay kit . Briefly, 100 μl of the supernatant or PGE2 standard dilutions were pipetted into a clear microtiter plate coated with an antibody to capture mouse IgG. A PGE2-peroxidase conjugate (25 μl) is added to the standards and supernatants in the wells. The binding reaction was initiated by the addition of 25 μl of a monoclonal antibody to PGE2. After an overnight incubation at 4°C, the plate was washed and 100 μl of substrate was added. After a short incubation, the reaction was stopped and the intensity of the generated colour was detected at 450 nm wavelength.PGEIL-6, MMP-3, and aggrecans were measured by specific enzyme amplified sensitivity immunoassays . IL-6 and MMP-3 productions were measured in culture supernatants, whereas aggrecans production was measured in culture supernatants and in the cell extracts.Cells were collected at 4°C and lysed on ice in 50 μl of buffer  containing protease and phosphatase inhibitors . After incubation at 4°C for 30 minutes, lysates were then centrifugated at 14,000 g for 30 minutes at 4°C to remove insoluble debris. Protein concentrations were determined using the BCA assay . Total protein extracts (20 μg) were fractioned by electrophoresis on a polyacrylamide gel (10%) and transferred onto a PVDF membrane. Membranes were blocked for 1 h in TBS-Tween containing 5% nonfat dried milk (HSC70) or in 2% bovine serum albumin (phospho-NF-κB p65 and phospho-IκBα). Membranes were then incubated overnight at 4°C with primary antibodies. Anti-rabbit phospho-NF-κB p65 (1:1000 dilution), anti-rabbit phospho-IκBα (1:1000 dilution) and anti-rabbit HSC70 (1:1000 dilution)  were used. Horse-radish peroxidase (HRP)-linked anti-rabbit IgG antibody (1:2000 dilution) was used as secondary antibody . The reaction was revealed with the ECL Western blotting substrate .Results were normalized to the HPRT gene expression or to the DNA content of the cells and expressed as the mean ± SEM or reported to the control and expressed in percent. Statistical significance is assessed using a parametric one-way ANOVA, followed by Dunnett’s multiple comparison post-test for statistical significance compared to control, and followed by Tukey’s multiple comparison post-test for statistical significance compared to the mixture. Differences were considered statistically significant at p-value < 0.05.Bovine chondrocytes were cultured for 24 hours  or 48 hours  in the absence or in the presence of IL-1β and with or without curcuminoids extract (C), hydrolyzed collagen (O) or green tea extract (T) added alone or in combination (COT). Cell viability, evaluated by LDH release, and DNA content, was not significantly modified by compounds alone or in combination, with or without IL-1β (data not shown).2 productions were undetectable. However, bovine chondrocytes expressed 0.43 ± 0.23 copies of IL-6 mRNA, 7.83 ± 2.83 copies of iNOS mRNA, 1.62 ± 0.93 copies of COX2 mRNA, 3.00 ± 1.00 copies of MMP-3 mRNA, 0.6 ± 0.4 copies of ADAMTS4 mRNA, 13.13 ± 2.64 copies of ADAMTS5 mRNA and 146.9 ± 82.51 copies of aggrecans mRNA per copy of HPRT. The compounds did not significantly affect the basal expression of these genes.In basal conditions, NO and PGE2 were 5.16 ± 1.42 nmol/μg DNA and 8,372.95 ± 5,187.35 pg/μg DNA, respectively. IL-1β increased the expression of the genes coding for IL-6, iNOS, COX2, MMP-3, ADAMTS4, ADAMTS5 by 178.16 ± 8.04, 17.87 ± 7.72, 11.18 ± 2.48, 88.41 ± 45.19, 25.67 ± 14.73, 3.26 ± 1.03-fold, respectively and decreased aggrecans by 7.00 ± 3.32-fold. In the presence of IL-1β and at the concentrations of 12.5 and 62.5 μg/ml, C significantly and fully inhibited NO production (p = 0.0101), while O and T were without effects   . In contIn the presence of IL-1β and at the concentrations of 12.5 and 62.5 μg/ml, C significantly inhibited IL-6 , COX2  iNOS , MMP-3 , ADAMTS4  and ADAMTS5  gene expressions . In the We have also tested two compounds in combination, CO and CT. CO and CT tended to be more efficient than C on all parameters. The differences between C and CO groups were not statistically significant. In contrast, CT reduced higher IL-1β-stimulated MMP-3 expression than C (p = 0.0386) .2 productions (p<0.0001), but the inhibitory effect was not higher than C alone . Cell viability, evaluated by LDH release, and DNA content, was not affected by these compounds added alone or in combination, with or without IL-1β (data not shown).In basal conditions, NO production was undetectable while the content of IL-6, MMP-3 and aggrecans in culture supernatants were 232.32 ± 85.3 pg/μg DNA, 207.34 ± 15.83 ng/μg DNA and 538 ± 86.48 ng/μg DNA, respectively. The aggrecans content in chondrocytes extract was 28.92 ± 4.61 ng/μg DNA. In basal conditions, MMP-3 production was significantly inhibited by C at 2  and 4 μg/ml  . At the In basal conditions, MMP-3 production was significantly inhibited by the COT mixture at 1:1:1 , 2:2:2  and 4:4:4 μg/ml  . At the -11 M IL-1β, the production of NO was 37.92 ± 6.04 nmol/μg DNA. IL-1β significantly increased the production of IL-6 and MMP-3 by 2,114.91 ± 789.59 and 13.61 ± 2.7-fold, respectively. In contrast, IL-1β inhibited aggrecans content in cells extract and supernatants by 1.37 ± 0.36 and 4.72 ± 1.83-fold, respectively. C significantly inhibited IL-1β stimulated NO production at the concentration of 4 μg/ml  (In the presence of 10<0.0001) , IL-1β s<0.0001)  and IL-1<0.0001) . O signi<0.0001)  but had The COT mixture significantly decreased IL-1β-stimulated NO production at the concentrations of 2:2:2  and 4:4:4 μg/ml  . At the -11 M) for 5 or 15 minutes. C and COT inhibited IL-1β induced phosphorylation and translocation of p65 in nuclear extracts of chondrocytes (-11 M) for 5 or 15 minutes. IL-1β induced IκBα degradation as early as 5 min, but IL-1β could not induce IκBα subunit degradation in C and COT pre-treated chondrocytes  to compare the effects of three nutraceuticals, curcuminoids extract, hydrolyzed collagen and green tea extract on chondrocytes metabolism, 2) to investigate a potential synergic effect between these compounds and 3) to examine whether the effects of curcuminoids extract and the combination of the three compounds on human chondrocytes involved the NF-κB signaling pathway. Numerous mediators contribute to both degradative and nociceptive pathways associated with the progression of OA . Among tynthesis –33. IL-1ynthesis , 33. As We have demonstrated that C had beneficial effects on chondrocytes. In fact, C counteracted the deleterious effects of IL-1β on chondrocytes. More precisely, C reduced the stimulating effect of IL-1β on pro-inflammatory and pro-catabolic mediators like cytokines, NO or MMP. These effects were mediated by inhibiting NF-kB signaling pathway. C inhibited IL-1β induced activation of NF-κB and its translocation to the chondrocyte nucleus. Further, C abolished IL-1β induced degradation of IκBα subunit. Our results were consistent with previously published results showing that curcumin suppressed IL-1β induced NF-κB activation via inhibition of IκBα phosphorylation and p65 phosphorylation , and pre2 and MMP-3 productions, and IL-1β stimulated IL-6 and IL-8 gene expressions in primary bovine normal chondrocytes and human OA chondrocytes [-11 M) for 48 hours. It was already shown that EGCG individually marginally inhibit COX-2 expression and PGE2 production in cytokine-activated equine chondrocytes. Finally, EGCG, in combination with avocado and soya unsaponifiables, reduced COX2 expression close to non-activated control levels and significantly inhibit PGE2 production [These data confirm those of previous studies demonstrating that purified curcumin inhibited IL-1β stimulated NO, PGEdrocytes , 12, by drocytes . In addidrocytes , increasdrocytes  and colldrocytes . EGCG, tdrocytes . Other ddrocytes . These ddrocytes . Furtherdrocytes  or with oduction .For the first time, this paper described the effects of C, O and T in combination on chondrocyte metabolism. In bovine chondrocytes, the three compounds showed additive inhibitory effects on IL-1β stimulated IL-6, iNOS and ADAMTS4 gene expressions. Moreover, the three compounds acted synergically to inhibit IL-1β stimulated COX2, MMP-3 and ADAMTS5 gene expressions. COT showed a higher effect on these parameters than C alone. Further, the inhibitory effect of COT on IL-1β-induced COX-2, iNOS, MMP-3 and ADAMTS4 expressions tended to be greater than that of CO or CT. In human chondrocytes, COT had an additive inhibitory effect on MMP-3 and IL-1β stimulated NO production and acted synergically on IL-1β stimulated IL-6 production.Altogether, these data gave a rationale for combining these compounds in the management of OA.One major limitation of our study is that compounds have been tested on a limited number of cartilage specimens. Additional experiments are needed, for example in order to reach the significance of the effects and identify the best responders among a large panel of specimens.in vitro study (4 μg/ml ~ 10 μM) are superior to those found in plasma after oral administration of high doses of natural curcumin. Therefore, the extrapolation of our in vitro data to human nutrition must be done with caution. However, many effort have been made to increase curcumin bioavaibility. Recently, we performed a Phase I pharmacokinetics study on Flexofytol, a high bioavailable turmeric extract with a water solubility increased 4000 times, that was run on 2 groups of 12 healthy individuals. Each group received orally 1 (42 mg curcumin) or 2 capsules (84 mg of curcumin) of Flexofytol respectively. With 2 capsules administered orally, the mean of Cmax on 12 individuals was 0.9 μM, with a statistical extrapolation at 1.6 μM with 4 capsules (administering 84 mg and 168 mg of curcumin respectively). These values are closer with those used in our in vitro study.Of course, before recommending these natural products in the management of OA, some clinical trials are required to demonstrate the beneficial effects of these nutraceuticals on OA symptoms and structural changes. Another major concern is the absence of information about dietary supplements bioavailability. This point is particularly true for polyphenol like curcumin. In fact, natural curcumin is known for its very low bioavailability. This point is crucial to envisage its administration by oral route. For example, the average peak serum concentrations after oral intake of 4, 6 and 8 g of curcumin per day were 0.51 ± 0.11, 0.63 ± 0.06 and 1.77 ± 1.87 μM, respectively . The serThe challenge is to develop a curcumin with an increased bioavailability, with the aim of reaching plasma concentrations that have demonstrated biological activity. In this purpose, curcumin has been dissolved in oil and then absorbed into chylomicrons, to propose a surface-controlled water-dispersible curcumin, with no modification of its structure , 39. Curin vitro results indicate that the mixture COT might reduce inflammation and pain in OA by reducing the synthesis of inflammatory and catabolic mediators by chondrocytes, probably by inhibiting NF-κB activation. These findings provide a preclinical basis for the in vivo testing of the combinations and suggest that these natural compounds could be helpful to alleviate symptoms in OA patients. These results support firstly the development and use of the mixture COT as an anti-inflammatory / anti-osteoarthritic agent for treatment of OA.Altogether, these


The detection limits for UA and Xa were 3.00 × 10−8 M and 5.00 × 10−8 M, respectively. Finally, the proposed method was applied to simultaneously determine UA and Xa in human urine with good selectivity and high sensitivity.Poly(methylene blue) and electrochemically reduced graphene oxide composite film modified electrode (PMB-ERGO/GCE) was successfully fabricated by electropolymerization and was used for simultaneous determination of uric acid (UA) and xanthine (Xa). Based on the excellent electrocatalytic activity of PMB-ERGO/GCE, the electrochemical behaviors of UA and Xa were studied by cyclic voltammetry (CV) and square wave voltammetry (SWV). Two anodic sensitive peaks at 0.630 V (versus Ag/AgCl) for UA and 1.006 V (versus Ag/AgCl) for Xa were given by CV in pH 3.0 phosphate buffer. The calibration curves for UA and Xa were obtained in the range of 8.00 × 10 Graphene oxide (GO) and graphene are promising materials with many interesting properties for applications ; for exaπ-π noncovalent interaction, some electroactive water-soluble aromatic dyes could absorb on the surface of carbon material, such as the combination of Toluidine blue O and multiwall carbon nanotube  and 1.0 M KCl, followed by sweeping with cyclic voltammetry. The effective area of electrode can be calculated from the Randles-Sevcik plot: Ip = 2.69 × 105n3/2AD1/2v1/2C, wherein ip is current in amps (A), n is number of electrons transferred of K3[Fe(CN)6] in the redox event , A is electrode effective area, D is diffusion coefficient (7.6 × 10−6 cm2·s−1), C is concentration (5 mM), and ν is scan rate (0.05 V·s−1). The effective areas for GCE, PMB/GCE, ERGO/GCE, and PMB-ERGO/GCE were 0.0941 cm2, 0.1906 cm2, 0.2088 cm2, and 0.2291 cm2, respectively. PMB-ERGO/GCE contains the largest effective area, which presumably accounts for its excellent electrocatalytic activity.GCE, PMB/GCE, ERGO/GCE, and PMB-ERGO/GCE were immerged into the solution containing 5.0 × 10−5 M UA and 1.00 × 10−4 M Xa were recorded from pH 2.0 to 8.0. As shown in In most cases, the electrolyte pH is an important condition to the electrochemical reaction. The cyclic voltammograms of 5.00 × 10−5 M) on PMB-ERGO/GCE, cyclic voltammograms were performed by various potential scan rates. To investigate the effect of scan rate on the electrochemical behavior of UA and Xa  RT/. The real reaction electron numbers of UA and Xa were calculated to be 1.964 and 1.913, respectively. Their theoretical reaction electron numbers should be 2. According to literatures [The slope values approximate to 0.5 in equations oftionship ; herein,eratures , 37, 38,Figures The results in −5 M UA and 1.00 × 10−4 M Xa were SWV measured for 20 parallel experiments. The relative standard deviations (RSD) of the peak currents for UA and Xa were 3.1% and 2.7%, respectively, implying remarkable reproducibility. After the modified electrode was stored in humid environment at room temperature for 15 days, it retained 94.5% of its original response and held the similar shape of the original curves, suggesting an acceptable stability of PMB-ERGO/GCE.The mixed solution of 5.00 × 10−5 M UA and 5.00 × 10−5 M Xa. The tolerance limit was set as the maximum concentration of the foreign substances that caused an approximately ±5% relative error in the determination. The results showed that K+, Na+, Ca2+, Fe2+, Mg2+, Ba2+, Zn2+, Al3+, Cl−, NO3−, SO42−, C2O42−, starch, L-arginine, L-threonine, L-serine, L-histidine, dopamine (≥1.0 mg) and L-cysteine (0.6 mg), Cu2+ (0.6 mg), Ag+ (0.05 mg), I− (0.03 mg), and ascorbic acid (0.5 mg) had no interference with the determination of UA and Xa. Therefore, it is possible to simultaneously determine UA and Xa in a sample on PMB-ERGO/GCE.The influences of various foreign species were investigated in a mixture solution containing 2.00 × 10 M PBS (pH 3.0) before measurement. The obtained results are summarized in Human urine sample was selected as the real sample to examine the reliability of the proposed SWV method. 1 mL urine sample was diluted 10 times with 0.1PMB-ERGO/GCE was prepared by electropolymerization. The modified electrode exhibited good conductivity and excellent electrocatalytic activity toward UA and Xa. The introduction of ERGO enhanced the effective surface areas on modified electrode compared with PMB film. The real reaction electron number of UA and Xa were calculated to be 1.964 and 1.913, respectively, in pH 3.0 phosphate buffer solution. The oxidation of UA and Xa were both controlled by diffusion. PMB-ERGO/GCE displayed desirable properties including excellent stability, reproducibility, selectivity, and sensitivity. The results demonstrated that the proposed method is a rapid, sensitive, and reproducible method for determination of UA and Xa in human urine sample. Therefore, PMB-ERGO/GCE would act as a promising sensor for a wide range of electrochemical sensing and biosensing applications.


Hedyosmum sprucei Solms (Chloranthaceae) essential oil (EO). The antioxidant activity, performed through DPPH bioautographic assay and spectrophotometric evaluations (IC50 = 230 ± 10 µg/mL), seemed to be mainly due to α-cadinol and α-muurolol. (HP)TLC bioautography, focused on antimicrobial capacities, pointed out α-cadinol, α-muurolol, τ-muurolol, caryophyllene oxide, and methyleugenol as the most effective compounds against Staphylococcus aureus, considered as testing strain. Moreover, the microdilution method, assessed among a wide panel of microorganisms, revealed Listeria grayi and Staphylococcus aureus as the most sensitive among human tested strains and Clavibacter michiganensis among phytopathogens. GC-MS chemical profile showed that bioactive molecules represented only a small quantity of the whole EO: germacrene D (23.16%), β-caryophyllene (15.53%), δ-cadinene (5.50%), α-copaene (5.08%), and α-phellandrene (3.48%) were the main compounds, highlighting an uncommon composition among the genus Hedyosmum. Finally, H. sprucei EO was checked for cytotoxic potential against A549 (lung cancer) and MCF-7 (breast cancer) cell lines showing promising cytotoxic effects against both cell lines after 48 h  and 72 h . Bioautography has been used as rapid and easy strategy to detect and identify bioactive fractions/molecules in the never before investigated Plants are sources of huge number of bioactive lead/scaffolds with therapeutic and nutraceutical importance. Bioautography is a fast technique that allows us to identify and isolate bioactive fractions/compounds in never previously studied plant extracts by employing a suitable chromatographic process followed by a biological detection system , 2.Hedyosmum sprucei Solms (Chloranthaceae) is an aromatic shrub growing on the eastern versants of the tropical Andes of Ecuador and Peru, in disturbed and undisturbed sites, such as secondary rainforest and streamsides, as well as primary habitats such as cloud forest. The species is found at 500–2000 m.a.s.l. H. sprucei, known in the Pastaza region of Ecuador as “sacha limón panga,” “sacha limón caspa,” or “hoja de monte,” has leaves with a pleasant fragrance similar to a clove-like scent that leaves the mouth slightly numb if chewed   × 100, where A1 is the DPPH absorbance with essential oil and A2 without essential oil. As positive control, commercial thymol  was used, in light of its well-known antioxidant properties [50 (concentration providing DPPH 50% inhibition), determined from inhibition curves obtained by plotting inhibition percentage against essential oil concentration. All experiments were performed in triplicate and values were reported as mean ± SD (standard deviation).An essential oil solution ofoperties . Essenti H. sprucei essential oil was quantified by microdilution method, using 96-well microtiter plates [ Escherichia coli (ATCC 4350) and Pseudomonas aeruginosa (ATCC 27853) and the Gram positive Listeria grayi (DSM 20601) and Staphylococcus aureus (ATCC 29230). Moreover, bioactivity against phytopathogen bacteria was checked versus the Gram negative Agrobacterium tumefaciens (DSM 30207), Agrobacterium vitis (DSM 6583), Pseudomonas syringae pv. syringae (DSM 10604), and Gram positive Clavibacter michiganensis subsp. nebraskensis (DSM 20400).The antibacterial activity ofr plates , in ordeA. tumefaciens and A. vitis) bacterial cultures were incubated in Nutrient Broth , at 37°C overnight, and the remaining strains in Tryptic Soy Broth . Sterile medium (100 μL) was pipetted into all wells together with 100 μL of serial dilutions of H. sprucei essential oil previously dissolved in ethanol (100 mg/mL). Serial dilutions were prepared in order to obtain concentration ranges from 0.03 μL/mL to 2 μL/mL. One hundred μL of bacterial culture standardized to 2 × 107 CFU/mL was added to the wells and incubated at 37°C for 6 h and at 26°C for 24 h, respectively, for human and phytopathogens. After incubation, 40 μL of a 2,3,5-triphenyl-tetrazolium chloride  water solution (20 mg/mL) was added to each well and then incubated. After 30 min, microbial growth was evaluated by microplate reader  at 615 nm. Commercial thymol, due to its known antimicrobial properties [ H. sprucei essential oil. Sterile medium served as growth control.For all the bacterial strains the antibacterial activity was determined in terms of MIC, checked through the microdilution method. Gram negative  and MCF-7 (human breast cancer cell line) were purchased from “Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna,” Brescia, Italy. The cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin, and 2 mM L-glutamine. The cell lines were grown in 75 cm4 cells/well in 200 µL DMEM complete medium and allowed 24 h for attachment. Then the culture medium was replaced with 200 µL medium containing different concentrations (from 1 to 100 µg/mL) of H. sprucei essential oil. Similar assay was carried out with β-caryophyllene standard, in order to evaluate its possible cytotoxic activity considering the high amount in the essential oil. Control culture was exposed to only vehicle (medium containing 2% FBS). After 24, 48, and 72 h, the culture medium was removed and washed with PBS  twice, and 20 μL of MTT (5 mg/mL in PBS) was added in each well and the plates were incubated for 4 h at 37°C. The medium was removed and replaced with 100 μL dimethyl sulphoxide to dissolve the formazan crystals. The extent of MTT reduction was measured spectrophotometrically at 570 nm using a microplate reader . The cytotoxic activity was expressed as the concentration of sample that inhibited 50% of cell growth (IC50).Cytotoxic activity was determined by MTT colorimetric assay  as refle50 values of antioxidant and antiproliferative activities were determined by logarithmic regression curves with 95% confident limits. Relative standard deviations and statistical significance  were calculated using software STATISTICA 6.0 .The experiments were performed in triplicate. IC H. sprucei essential oil is reported in β-caryophyllene (15.53%), α-cadinene (5.50%), and α-copaene (5.08%); among the monoterpenes α-phellandrene was the most abundant (3.48%). Comparing our data to that provided by the related literature on different Hedyosmum species, an interesting result is the high sesquiterpene abundance which characterized the H. sprucei essential oil as compared to that of H. brasiliense [ H. angustifolium, H. scabrum [ H. arborescens [ H. bonplandianum, and H. mexicanum [ Hedyosmum species which is known for its sesquiterpene-rich essential oil is H. costaricense [α-cadinene and α-copaene, found in moderate concentration in H. sprucei, did not occur in H. arborescens, H. mexicanum, H. scabrum, and H. angustifolium, while they were reported in H. brasiliense, H. costaricense, and H. bonplandianum in amount of less than 1%. The sesquiterpenes α-cadinol and α-muurolene were previously observed just in H. costaricense and the 1-epi-cubenol was previously observed just in H. brasiliense; furthermore, the latter species were the only ones to contain methyleugenol. Apart from H. sprucei, none of the species taken into account held compounds as α-muurolol, γ-muurolene, 1,10-di-epi-cubenol, and τ-cadinol. These results, as a first report about the H. sprucei essential oil, represent a further contribution to the phytochemistry of the genus. However, the difference detected between H. sprucei essential oil and those of the other Hedyosmum species could be also related to the Amazonian origin of our samples. In fact, the high biodiversity of Amazonian region is known to drive the plant secondary metabolism to biosynthetic pathways which are particularly diversified in chemical structures [The chemical composition ofhyllene 1.53%, α-c scabrum , H. arboorescens , H. bonppaene 5.0%; among paene 5.0%; among ructures . H. sprucei essential oil, with particular reference to Rf 0.3 and 0.5. Three different areas on the TLC evidenced antioxidant properties, in particular the fractions corresponding to Rf 0.3, 0.5, and 0.9  and α-muurolol (29.44%), as evidenced by GC-MS, seemed to be the most responsible for the bioactivity, given the most evident reactivity to the DPPH radical. Rf 0.5 characterized by methyleugenol (60.23%), caryophyllene oxide (23.48%), 1,8-cineole (6.84%), 1,10-di-epi-cubenol (4.12%), 1-epi-cubenol (2.85%), and humulene 1,2-epoxide (2.48%) showed an intermediate activity, while Rf 0.9, composed of germacrene D (40.28%), β-caryophyllene (23.14%), δ-cadinene (8.78%), β-elemene (6.16%), α-copaene (5.72%), α-caryophyllene (5.55%), α-muurolene (2.00%), γ-muurolene (2.28%), δ-elemene (1.95%), bicyclogermacrene (1.85%), germacrene B (1.17%), and δ-cubenene (1.13%), evidenced the lowest reaction intensity. Given the fact that these results represent the first report concerning H. sprucei essential oil, however, some considerations could be supported in light of the related literature about single compounds characterizing the isolated fractions. In fact, the important involvement of the compounds characterizing Rf 0.3, as α-cadinol and α-muurolol, can be evidenced in antioxidant properties [Rf 0.51 [Rf 0.9 [The DPPH (HP)TLC bioautography showed the antiradical capacity of and 0.9 . Rf 0.3operties , analogoRf 0.51 , 21, and[Rf 0.9 . S. aureus , α-muurolol (13.72%), τ-muurolol (10.78%), selin-11-en-4-α-ol (9.33%), linalool (6.01%), trans-muurola-4(14),5-diene (3.80%), τ-cadinol (3.33%), eudesma-4(15),7-dien-1-β-ol (3.12%), δ-amorphene (2.57%), elemol (2.59%), γ-eudesmol (2.53%), and β-atlantol (2.38%) and Rf 0.5 by caryophyllene oxide (45.30%), methyleugenol (26.33%), 1,10-di-epi-cubenol (18.81%), and humulene 1,2-epoxide (7.77%).(HP)TLC bioautography on. aureus  evidence H. sprucei essential oil were reported to be active as pure compounds: τ-muurolol, τ-cadinol, methyleugenol [α-ol, caryophyllene oxide, α-cadinol, α-muurolol [τ-cadinol, revealed in fraction Rf 0.2– Rf 0.4, is known to exhibit bacteriolytic properties toward S. aureus [Some of the antibacterials detected inleugenol , linalooleugenol , 24, selmuurolol , and elemuurolol . τ-cadin. aureus , since iRf 0.5 fraction, may be responsible for the detected antibacterial activity of H. sprucei essential oil, since the pure compound is pointed out as antimicrobial agent by related literature [Methyleugenol, abundant in terature . Moreoveδ-amorphene [γ-eudesmol [To the best of our knowledge, evidences are reported in the literature of antimicrobial activity of the following remaining compounds, as phytocomplex constituents: morphene , 1,10-dimorphene , and γ-eeudesmol .In conclusion, a lot of sesquiterpenes showed a potential activity. Future investigations on isolated compounds and their mixture could better highlight the lead molecules or synergic active complex. H. sprucei essential oil evidenced an interesting antioxidant activity if compared to standard positive control, not previously reported in the literature. In fact H. sprucei essential oil possessed a DPPH scavenging activity (IC50 = 230 ± 10 μg/mL) about 30% better than thymol (IC50 = 318 ± 7 μg/mL).The H. sprucei essential oil was performed against Gram positive and negative human and plant pathogenic bacteria, through serial microdilution method for MIC (Minimum Inhibitory Concentration) determination. Results were compared to that obtained with thymol, taken as positive control, as suggested by the related literature [ L. grayi and S. aureus, with the latter responsible for important nosocomial infections [Antibacterial activity ofterature  (Table 2fections . Gram ne C. michiganensis subsp. nebraskensis evidenced the lowest detected MIC (62 μg/mL), 87% even better than positive control. Particular relevance should be given to the sensitivity of P. syringae pv. syringae, since the strain is responsible for important plant diseases related to equally important cultivations as kiwi (Actinidia deliciosa (A. Chev.) C. F. Liang & A. R. Ferguson), tobacco (Nicotiana tabacum L.), and mango (Mangifera indica L.).Regarding plant pathogen bacteria, the MIC values were even more performing. In particular,50 values. The results showed that MCF-7 cell line exhibited higher sensitivity than A549 to treatments with H. sprucei essential oil. In particular, MCF-7 cells were affected with interesting growth inhibition with IC50 values of 32.76 ± 4.92 and 33.64 ± 0.43 µg/mL after 48 and 72 h, respectively. According to the previous literature, values lower than 30 µg/mL suggest a good chemopreventive potential for the extract [ H. sprucei essential oil might be a promising source of active compound(s) for innovative therapeutic and/or preventive strategies against breast adenocarcinoma. On the other hand, less interesting were the data on A549 cell line with IC50 values between 44.05 ± 2.35 and 43.55 ± 2.80 µg/mL. The selective toxicity of H. sprucei essential oil toward MCF-7 cells may be due to the sensitivity of the cell line to the active compounds in the phytocomplex or to tissue specific response [β-caryophyllene exhibited cytotoxic activity against breast cancer cells; thus, the high content of these components in the H. sprucei essential oil may explain the cytotoxic activity toward MCF-7 [β-caryophyllene cytotoxicity at 48 h: IC50 values for MCF-7 cell line were 50.85 ± 7.35 µg/mL and for A549 cell line 42.05 ± 0.50 µg/mL. It has been also demonstrated that β-caryophyllene potentiated the cytotoxic activity of α-caryophyllene and other sesquiterpenes on MCF-7 cell line [ H. sprucei essential oil as significant potential source of pure compounds with promising anticancer activity [The cytotoxic activity was evaluated against lung adenocarcinoma cell line (A549) and breast adenocarcinoma cell line (MCF-7) expressed as IC extract . In lighell line . In lighactivity . H. sprucei essential oil and investigated its potential radical scavenging and antibacterial capacities by bioautography and quantitative methods. Moreover, it explored its cytotoxic properties. Preliminary results of this research were presented by our colleague and coauthor of the present paper, Spagnoletti et al., in 2014 [ in vitro and in vivo of H. sprucei essential oil, especially as food and dietary supplements aimed at oxidative stress prevention, representing an alternative to addition of synthetic antioxidant suspected of toxicity [ H. sprucei essential oil are viable for treatment of human and phytopathogenic bacterial diseases.In conclusion, this study characterized for the first time the components of in 2014 . The restoxicity . Moreove


In conclusion, the results indicated that drugs metabolized by CYP2C9 coadministrated with vinpocetine may require attention or dose adjustment.Vinpocetine is a derivative of the alkaloid vincamine, which had been prescribed for chronic cerebral vascular ischemia and acute ischemic stroke or used as a dietary supplement for its several different mechanisms of biological activities. However, information on the cytochrome P450 (CYP) enzyme-mediated drug metabolism has not been previously studied. The present study was performed to investigate the effects of vinpocetine on CYPs activity, and cocktail method was used, respectively. To evaluate the effects of vinpocetine on the activity of human CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, human liver microsomes were utilized to incubate with the mixed CYPs probe substrates and the target components. The results indicate that vinpocetine exhibited weak inhibitory effect on the CYP2C9, where the IC Vinpocetine, propranolol, and gliclazide were obtained from the National Institutes for Food and Drug Control . Acetonitrile of high-performance liquid chromatography grade was obtained from Fisher Co. Ltd. . Milli-Q  water was used throughout the experiments.Chlorzoxazone, dextromethorphan, tolbutamide, testosterone, omeprazole, 6-hydroxychlorzoxazone, dextrorphan, and 5-hydroxyomeprazole were purchased from Sigma-Aldrich Company . 6™, Woburn, USA). The microsomes were frozen and stored at –80°C until used.A pooled sample of human liver microsomes was obtained from BD Gentest Corporation  and vinpocetine at different concentrations [μL) contained 100 mM phosphate buffer (pH 7.4), 3.3 mM MgCl2, 1 mM NADPH, 0.3 mg/mL microsomal protein, five probe substrates, and the tested sample or blank solvent (control). The final concentrations of the probe substrates were 100 mM for tolbutamide, 5 mM for dextromethorphan, and all 50 mM for omeprazole, chlorzoxazone, and testosterone, and the final concentrations of the vinpocetine in the incubation were at serial concentrations of 0.1, 0.3, 1.0, 3.0, 10.0, 30.0, and 100 μM, respectively. The reaction mixture, which includes microsomes, substrates, and the vinpocetine in a final volume of 100 μL, was preincubated for 10 min at 37°C in a shaking water bath, and the reaction was initiated through the addition of NADPH and terminated with 100 μL of ice-cold acetonitrile containing 1 μg/mL propranolol  and 1 μg/mL gliclazide  after 30 min incubation. After vortexing, the mixtures were centrifuged at 15,000 g for 10 min at 4°C. Two 10 μL aliquots of the supernatant were injected directly into the LC-MS/MS system for the determination of the produced metabolites (dextrorphan/6β-hydroxytestosterone/5-hydroxyomeprazole/6-hydroxychlorzoxazone/4-hydroxytolbutamide).Briefly, each mixture  with the column temperature at 40°C. The mobile phase consisted of 0.1% formic acid in water (A) and in acetonitrile (B) with following gradient elution at a flow rate of 0.4 mL/min: 0–0.1 min, 5–90% A; 0.1–4.1 min, 90% A; 4.1–7.0 min, 90–5% A. The ion spray voltage was operated separately in the positive ion mode at 5500 V and the negative ion mode at −4000 V, respectively. The operating conditions were the following: ion source temperature, 400°C; curtain gas, 20 psi; ion source gas 1, 60 psi; ion source gas 2, 60 psi. The quantification was performed by multiple reaction monitoring (MRM) of the molecular ion and the related product ion for each metabolite, ESI+: m/z 258.1 → 157.2 for dextrorphan, m/z 305.1 → 269.3 for 6β-hydroxytestosterone, m/z 362.3 → 214.2 for 5-hydroxyomeprazole, and m/z 261.3 → 116.1 for propranolol; ESI−: m/z 183.8 → 119.9 for 6-hydroxychlorzoxazone, m/z 285.4 → 185.6 for 4-hydroxytolbutamide, and m/z 322.4 → 170.2 for gliclazide.The metabolites of five CYPs probe substrates in all samples were identified by using our previously developed LC-MS/MS method . In brie50) for each CYP isozyme was calculated using a nonlinear regression analysis program in GraphPad Prism 5.0 (GraphPad Software 5.0).The data acquisition and peak integration were performed by utilizing analyst software (Version 1.4.2). The ratios were plotted as a percentage of the relevant control for each metabolic reaction, and the half maximal inhibitory concentration  for dextrorphan, 6β-hydroxytestosterone, 5-hydroxyomeprazole, 6-hydroxychlorzoxazone, and 4-hydroxytolbutamide were 0.50, 5.00, 1.00, 1.00, and 0.50 ng/mL, respectively. The method showed excellent reproducibility with intraday and interday precision less than 12.00%, and the accuracy ranged from 88.96% to 114.73%. The matrix effects were more than 88.59% or less than 112.34%.The concentrations of dextrorphan, 6S method . The cal50 values for five CYPs in human liver microsomes are presented in The IC50 values of 68.91 μM, whereas the IC50 values for CYP3A4, CYP2C19, CYP2D6, and CYP2E1 were all in excess of 100 μM, indicated that vinpocetine did not affect CYP3A4, CYP2C19, CYP2D6, and CYP2E1 activities in vivo.The results indicate that vinpocetine had a weak inhibitory effect on CYP2C9, with ICt0−, AUC0−inf, and Cmax following the single administration of vinpocetine alone and after pretreatment of 5 days with omeprazole  were very similar in both groups [Herbal medicines are often coadministered with therapeutic drugs, raising the potential of DDIs which are frequently caused by induction or inhibition of CYPs and/or P-gp , 22. Up h groups . Accordih groups . in vitro.However, there is a lack of information available regarding the metabolism mechanism of vinpocetine with additional drugs. Therefore, in this study we investigated the effects of vinpocetine on CYPs by using a cocktail method50 values higher than 100 μM are believed to have no inhibitory effect because sufficiently high levels of these compounds are extremely unlikely to be achieved in the clinic, while IC50 values less than 10 μM are considered potent inhibitors, and compounds with IC50 values between 10 and 50 μM are considered moderate inhibitors [50 value is 68.96 μM; in addition, the IC50 values for CYP3A4, CYP2C19, CYP2D6, and CYP2E1 were all over range of 100 μM, which showed that vinpocetine had almost no apparent inhibitory effects on these CYPs.To examine the effects of vinpocetine on CYPs activity, a LC-MS/MS-based cocktail approach was used . Previouhibitors , 24. Our in vitro. However, the needs are required to further in vivo study evaluation.To the best of our knowledge, it is the first time to examine the effects of vinpocetine on CYPs activityIn conclusion, we clearly demonstrated the effect of vinpocetine on the activities of multiple CYP isoforms , 20. It


To determine the keratometric indices calculated based on parameters obtained by Fourier-domain optical coherence tomography (FD-OCT).cal based on the mean calculated keratometric index, K1.3315 calculated by the keratometric index of 1.3315, and K1.3375 calculated by the keratometric index of 1.3375. In addition, the total corneal powers based on Gaussian optics formula  were calculated.The ratio of anterior corneal curvature to posterior corneal curvature (Ratio) and keratometric index (N) were calculated within central 3 mm zone with the RTVue FD-OCT  in 186 untreated eyes, 60 post-LASIK/PRK eyes, and 39 keratoconus eyes. The total corneal powers were calculated using different keratometric indices: Kcal and Kactual, K1.3315 and Kactual, and K1.3375 and Kactual were 0.00 ± 0.11 D, 0.21 ± 0.11 D and 0.99 ± 0.12 D, respectively, in untreated controls; -0.01 ± 0.20 D, 0.85 ± 0.18 D and 1.56 ± 0.16 D, respectively, in post-LASIK/PRK group; and 0.03 ± 0.67 D, 0.56 ± 0.70 D and 1.40 ± 0.76 D, respectively, in keratoconus group.The means for Ratio in untreated controls, post-LASIK/PRK group and keratoconus group were 1.176 ± 0.022 , 1.172–1.179), 1.314 ± 0.042  and 1.229 ± 0.118 , respectively. And the mean calculated keratometric index in untreated controls, post-LASIK/PRK group and keratoconus group were 1.3299 ± 0.00085 , 1.3242 ± 0.00171  and 1.3277 ± 0.0046 , respectively. All the parameters were normally distributed. The differences between KThe calculated keratometric index is negatively related to the ratio of anterior corneal curvature to posterior corneal curvature in untreated, post-LASIK/PRK, and keratoconus eyes, respectively. Using the calculated keratometric index may improve the prediction accuracies of total corneal powers in untreated controls, but not in post-LASIK/PRK and keratoconus eyes. Consideration of the keratometric index is essential for the assessment of total corneal power and the prediction of intraocular lens power.,2 HistorCurrently, there are three kinds of techniques that can detect the information of the anterior and posterior corneal surface and central corneal thickness (CCT) simultaneously, including Slit-scan system (Orbscan Ⅱ) ,11, ScheThe commercially available system RTVue FD-OCT with a speed of 26000 axial scans per second has an axial resolution of 5 μm and a transverse resolution of 15 μm. The corn186 eyes of 186 subjects in the untreated group; the mean age was 26.1 ± 5.6 years; the mean spherical equivalent was -2.53 ± 1.32 D; these subjects were selected from the patients examined before corneal refractive surgery in Wenzhou Eye Hospital  from July 2011 to April 2012.60 eyes of 39 patients in the post-LASIK/PRK group; the mean age was 27.3 ± 6.2 years; the mean spherical equivalent before the laser surgery was -4.81±1.22 D; the mean spherical equivalent after the laser surgery was 0.02±0.33 D; these subjects were selected from the patients who received LASIK at least 3 months previously or PRK at least 6 month previously in Wenzhou Eye Hospital from July 2011 to April 2012.39 eyes of 27 patients in the keratoconus group; the mean age was 34.93 ± 11.41 years; the data were selected from the patients who were diagnosed as keratoconus in Synslaser Clinic from September 2012 to January 2013. All the patients did not receive any eye surgery previously. And 4 of them had apical scars, 5 of them had Vogt striae, and all the others had clean corneas.Inclusion criteria were patients who didn’t receive prior corneal or ocular surgery in the untreated controls, or received uneventful LASIK at least 3 months or PRK at least 6 months previously in the post-LASIK/PRK group, or was diagnosed with keratoconus in the keratoconus group. All the results obtained by RTVue FD-OCT had good reliability (Measurement Reliability Rating GOOD displayed on the Pachymetry + CPwr map). Three groups of subjects were included in this study:The study was conducted at the Eye Hospital of Wenzhou Medical University and Synslaser Clinic . The research was performed in accordance with the principles stated in the Declaration of Helsinki. The untreated controls and post-LASIK/PKR group from Wenzhou Eye Hospital was approved by the Office of Research Ethical Committee, Wenzhou Medical University; and the keratoconus group from Synslaser was approved by the National Committee for Medical and Health Research Ethics in Norway. All participants provided written informed consent after the nature of the study had been explained to them.anterior), posterior corneal curvature (Rposterior) and CCT within central 3 mm zone for each tested eye using RTVue FD-OCT. The ratio of the anterior and posterior corneal curvature in each eye was calculated as follows:We obtained the anterior corneal curvature  within central 3 mm zone by assuming the central cornea as a thick lens. And Kactual in each eye was calculated as follows:0 (1.000), n1 (1.376) and n2 (1.336) are the refractive indices of the air, the cornea and aqueous, respectively. We used the following equation to compute the real keratometric index (n) in each eye:The Gaussian optics formula calculates the actual total corneal power  in each eye as follows:The mean of n in each eye was computed and defined as N, next N was used to estimate the total corneal power  and the conventional keratometric index (1.3375) were calculated as follows:anterior and Rposterior, Ncal and Ratio. Bland-Altman plots were applied to analyze the agreement between Kcal and Kactual, K1.3315 and Kactual, K1.3375 and Kactual. A P value of 0.05 or less was considered statistically significant.All the data were entered into MedCalc Version 11.4.2 for Windows. Correlation coefficient and paired t-test were used to compare values normally distributed in the three groups. Correlation coefficient and regression analysis were used to analyze the correlations between RP > 0.05). The distributions of Ratio and keratometric indices (N) in untreated controls, post-LASIK/PRK group and keratoconus group are shown in posterior, Ratio and Keratometric index (N) in untreated controls, post-LASIK/PRK group and keratoconus group. In posterior from keratoconus group was significantly smaller than those from untreated and post-LASIK/PRK group (both P<0.05). And there was no statistical significance between the mean Rposterior values from untreated controls and post-LASIK/PRK group (P>0.05). In P<0.05), and the mean Ratio from post-LASIK/PRK group was larger than that from keratoconus group (P<0.05). In cal) from post-LASIK/PRK group and keratoconus group were smaller than the mean Ratio from untreated controls (both P<0.05), and the mean N from post-LASIK/PRK group was smaller than that from keratoconus group (P<0.05).anterior and Rposterior, Ratio and N. The regression equations were Rposterior = 0.01021 + 0.8499×Ranterior  and N = 1.3752–0.03852×Ratio  in r = -0.999, r2 = 0.999, P<0.05) in posterior = -2.8393 + 1.218×Ranterior  and N = 1.3759–0.03917×Ratio  in In untreated controls, linear regression revealed that there were good correlations between Rcal and Kactual, K1.3315 and Kactual, K1.3375 and Kactual in the three groups. There were no statistical significances between Kcal and Kactual in all three groups . In untreated controls, K1.3315 and K1.3375 were 0.21 ± 0.11 D and 0.99 ± 0.12 D lager than Kactual, respectively (both P<0.05).cal, K1.3315 and K1.3375) and the actual total corneal power calculated based on Gaussian optics formula  in untreated controls, and the 95% confidence interval (CI) for Kcal vs Kactual, K1.3315 vs Kactual, and K1.3375 vs Kactual were -0.22 to 0.22 D, -0.01 to 0.42 D and 0.76 to 1.21 D, respectively. In post-LASIK/PRK group, K1.3315 and K1.3375 were 0.85 ± 0.18 D and 1.56 ± 0.16 D lager than Kactual, respectively (both P<0.05).cal and Kactual, K1.3315 and Kactual, K1.3375 and Kactual in post-LASIK/PRK group, and the 95% CI for Kcal vs Kactual, K1.3315 vs Kactual, and K1.3375 vs Kactual were -0.40 to 0.38 D, 0.50 to 1.19 D and 1.23 to 1.86 D, respectively. In keratoconus group, K1.3315 and K1.3375 were 0.56 ± 0.70 D and 1.40 ± 0.76 D lager than Kactual, respectively (both P<0.05).cal and Kactual, K1.3315 and Kactual, K1.3375 and Kactual in keratoconus group, and the 95% CI for Kcal vs Kactual, K1.3315 vs Kactual, and K1.3375 vs Kactual were -1.28 to 1.34 D, -0.82 to 1.93 D and -0.09 to 2.88 D, respectively.Keratometric index is critical in both the calculation of total corneal power and the prediction of intraocular lens power. Clinically, the commonly used keratometric indices include the conventional 1.3375 and the 1.3315 based on the Gullstrand schematic eye. However, the keratometric indices calculated in real eyes using Scheimpfulg camera system were smacal) in each group. In untreated controls, it was shown that the Ratio was 1.176 ± 0.022, and Ncal was 1.3299 ± 0.00085. Jin et al.[cal in untreated controls in our study was larger than those calculated by parameters obtained by Pentacam and OrbscanⅡ. The main source for the differences was the different Rposterior obtained. The Rposterior from untreated controls in our study was 6.569 ± 0.265 mm, which is larger than 6.30±0.24 mm from Chinese eyes and 6.42±0.25 mm from German eyes in Jin et al’s study, 6.34±0.28 mm in Ho et al’s study and 6.46±0.26 mm in Fam et al’ study. While the anterior corneal curvatures are comparable, a larger posterior corneal curvature will leads to a smaller ratio of anterior curvature to posterior curvature. Consequently, a larger total corneal power will be calculated based on a larger posterior corneal curvature (Formula 2), thus a larger Ncal will be calculated (Formula 3). The differences of Rposterior obtained by RTVue FD-OCT, Pentacam Scheimpflug camera system and Slit-scan system RTVue maybe caused by distinct measurement principles on which each device is based. RTVue FD-OCT with a speed of 26000 axial scans per second has an axial resolution of 5 μm. The corneal mapping model contained 6.0 mm line scans on 8 meridians with 1019 axial scans centered on the pupil, and the whole scan model was finished within 0.32 seconds. [posterior obtained.[In our study, we used the RTVue FD-OCT to measure the anterior and posterior corneal curvature within central 3 mm zone and central corneal thickness in untreated, post-LASIK/PRK and keratoconus eyes. Then we used the data obtained to calculate the mean ratio of the anterior corneal curvature to the posterior corneal curvature (Ratio) and the mean calculated keratometric index . On the contrary, Ncal from post-LASIK/PRK group (1.3242 ± 0.00171) was the smallest; followed by Ncal from keratoconus group (1.3277 ± 0.0046); whereas Ncal from untreated controls was the largest (1.3299 ± 0.00085). We further analyzed the correlation and linear regression between Ratio and Ncal in each group. It demonstrated that Ncal was negatively correlated to Ratio in each group    , n1 (1.376) and n2 (1.336) are the refractive indices of the air, the cornea and aqueous, respectively.In our study, it was shown that Ratio from post-LASIK/PRK group (1.314 ± 0.042) was the largest; followed by Ratio from keratoconus group (1.229 ± 0.118); whereas Ratio from untreated controls was the smallest (1.176 ± 0.022)  B,N = 1. P<0.05) D,N = 1. P<0.05) F. Here,posterior was very small. We assumed that CCT and Rposterior were 520 um and 6.5mm, respectively, then the value of (0.011×CCT)/Rposterior was calculated as 0.00088, which could be ignored comparing the constant of 1.376 in the same formula. Thus, we obtained a new formula: n = 1.376–0.04 × Ratio, which was very similar to the regression formulas in untreated, post-LASIK/PRK and keratoconus group. It means that Ncal and Ratio obtained from untreated, post-LASIK/PRK and keratoconus eyes have excellent similar linear relationship.The equation can be simplified as: cal in each group , the keratometric index of 1.3315 derived from Gullstrand schematic eye (K1.3315) and the conventional keratometric index of 1.3375 (K1.3375). And we also compared Kcal, K1.3315 and K1.3375 to the total corneal powers calculated based on Gaussian optics formula . In all the 3 groups, Kcal and Kactual were comparable . K1.3315 was 0.21 ± 0.11 D larger than Kactual in untreated controls, and the difference increased to 0.56 ± 0.70 D in keratoconus eyes, and the difference was 0.85 ± 0.18 D. Similarly, K1.3375 were 0.99 ± 0.12D, 1.40 ± 0.76D and 1.56 ± 0.16 D larger than Kactual in untreated, keratoconus and post-LASIK/PRK eyes, respectively. In Ho et al’s study[cal and Kactual were also comparable in untreated controls, but the 95% CI was larger than that in our study . The differences between K1.3315 and Kactual, K1.3375 and Kactual were larger than those in our study . And also the 95% CI were larger than ours . It demonstrated that the agreement between Kcal and Kactual obtained by RTVue FD-OCT was better than that the one obtained by Pentacam Scheimpflug camera system in untreated eyes. In Wang et al’s study[1.3375 was 1.30 D larger than Kactual in untreated eyes, and the difference increased to 1.51 D in eyes after corneal refractive surgery. In Jin et al’s study[1.3375 was 1.26 D larger than Kactual in untreated eyes, and the difference increased to 1.71 D in eyes after corneal refractive surgery. 1.3375 and Kactual obtained by distinct devices in untreated eyes and post-LASIK/PRK eyes. In our study, we found that the trends of the differences between K1.3315 and Kactual, K1.3375 and Kactual coincided with the trend of Ratio, and opposited to the trend of calculated keratometric index in the 3 groups.In our study, the total corneal powers were calculated using Nl’s study using Gal’s study, K1.3375The limitations of this study included: 1) a small number of eyes were included in keratoconus group; 2) the repeatability of corneal parameters obtained by RTVue FD-OCT in keratoconus group has not been proved; 3) For post-LASIK/PRK group, only eyes after myopic-LASIK/PRK were included.In conclusion, the ratio of anterior corneal curvature to posterior corneal curvature obtained by RTVue FD-OCT in untreated eyes is smaller than those obtained from Scheimpflug camera system and Slit-scan system in published studies, and also the calculated keratometric index obtained by RTVue FD-OCT in untreated eyes is larger than those obtained from Scheimpflug camera system and Slit-scan system in published studies. The calculated keratometric index was negatively related to the ratio of anterior corneal curvature to posterior corneal curvature in untreated, post-LASIK/PRK, and keratoconus eyes. Using the calculated keratometric index may improve the prediction accuracies of total corneal powers in untreated eyes, but not in post-LASIK/PRK and keratoconus eyes.


A new imidazole-based zinc complex, synthesized using hydrothermal methods, exhibits luminescent behaviour. 2(C15H12N2)2], was synthesized by reaction of zinc chloride and 1-(biphenyl-4-yl)-1H-imidazole (bpi) under hydrothermal conditions. The ZnII atom is tetrahedrally coordinated by the free imidazole N atoms of two bpi ligands and by two Cl atoms. The bpi ligands are not planar, with dihedral angles of 37.52 (14) and 42.45 (14)° between the phenyl rings and 37.13 (14) and 40.05 (14)° between the phenyl rings and the attached imidazole rings, respectively. Mutual π–π interactions, with a centroid-to-centroid distance of 3.751 (2) Å between the phenyl and imidazole rings of neighbouring ligands, are present, leading to dimers that are arranged in rows parallel to [-211].The mononuclear title compound, [ZnCl The photoluminescence spectrum of compound (I)d10 electron configuration and the bonding interactions with the ligand, such broad emission bands may be assigned to a ligand-to-ligand charge transfer (LLCT), admixing with metal-to-ligand (MLCT) and ligand-to-metal (LMCT) charge transfers °, with a centroid-to-centroid distance of 3.751 (2) Å Fig. 3.2 , bpi , and de-ionized water (9 ml) was loaded into a 20 ml Teflon-lined stainless steel autoclave. The autoclave was sealed and heated at 423 K for 5 d, and then cooled to room temperature by switching off the furnace. Colourless block-shaped crystals were isolated, which were filtered off and washed with de-ionized water. The final product was dried at ambient temperature (yield 75% based on zinc). Analysis calculated (wt%) for ZnCl2(C15H12N2)2: C, 62.47; H, 4.19; N, 9.71. Found: C, 62.45; H, 4.15; N, 9.79.All chemicals were purchased commercially and used without further purification. A mixture of ZnCl−1, both the excitation and the emission slit widths were 5.0 nm.Elemental analyses of C, H, and N were conducted on a Perkin–Elmer 2400 elemental analyser. The photoluminescence (PL) excitation and emission spectra were recorded with an F-7000 luminescence spectrometer equipped with a xenon lamp of 450 W as an excitation light source. The photomultiplier tube voltage was 400 V, the scan speed was 1200 nm minUiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015002807/wm5118sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989015002807/wm5118Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015002807/wm5118Isup3.molSupporting information file. DOI: 1048515CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its known side effects and low efficacy against asymptomatic cyst carriers. E. histolytica is also capable of surviving sub-therapeutic levels of metronidazole in vitro. Novel drugs with different mode of action are therefore urgently needed. The sulfur assimilatory de novo L-cysteine biosynthetic pathway is essential for various cellular activities, including the proliferation and anti-oxidative defense of E. histolytica. Since the pathway, consisting of two reactions catalyzed by serine acetyltransferase (SAT) and cysteine synthase , does not exist in humans, it is a rational drug target against amebiasis. To discover inhibitors against the CS of E. histolytica (EhCS), the compounds of Kitasato Natural Products Library were screened against two recombinant CS isozymes: EhCS1 and EhCS3. Nine compounds inhibited EhCS1 and EhCS3 with IC50 values of 0.31–490 μM. Of those, seven compounds share a naphthoquinone moiety, indicating the structural importance of the moiety for binding to the active site of EhCS1 and EhCS3. We further screened >9,000 microbial broths for CS inhibition and purified two compounds, xanthofulvin and exophillic acid from fungal broths. Xanthofulvin inhibited EhCS1 and EhCS3. Exophillic acid showed high selectivity against EhCS1, but exhibited no inhibition against EhCS3. In vitro anti-amebic activity of the 11 EhCS inhibitors was also examined. Deacetylkinamycin C and nanaomycin A showed more potent amebicidal activity with IC50 values of 18 and 0.8 μM, respectively, in the cysteine deprived conditions. The differential sensitivity of trophozoites against deacetylkinamycin C in the presence or absence of L-cysteine in the medium and the IC50 values against EhCS suggest the amebicidal effect of deacetylkinamycin C is due to CS inhibition.Amebiasis is a common worldwide diarrheal disease, caused by the protozoan parasite,  Entamoeba histolytica. WHO estimates 50 million people are infected worldwide, resulting in 40,000–100,000 deaths annually   and CS (EhCS1-3)  . The SAT complex , which i complex . In addi complex . Thus, ain silico screening of ZINC database studies based on the crystal structure of EhCS1. One compound was discovered and showed moderate inhibitory activity against EhCS1 (74% inhibition at 100 μM) in vitro. A similar virtual screening was also carried out against Trichomonas CS, which yielded some potential inhibitors  cell introduced pET-EhCS  construct was used for protein expression. Each EhCSs protein was expressed by 1 mM isopropyl β-D-thiogalactopyranoside induction for 12 h at 30°C. After induction, the E. coli cell was harvested and re-suspended in the lysis buffer  containing 0.5 mM phenylmethylsulfonyl fluoride. The suspension was sonicated and centrifuged at 24,000 ×g for 30 min at 4°C. The histidine-tagged rEhCS proteins were purified from the supernatant fraction using a Ni2+-NTA column and tagged proteins were eluted with 100–200 mM imidazole in 50 mM Tris-HCl, pH 8.0, and 300 mM NaCl. The purity of the eluted rEhCS proteins was confirmed by SDS-PAGE and then dialyzed in 50 mM Tris-HCl, and 150 mM NaCl, pH 8.0 containing 10% glycerol (v/v) for 24 h at 4°C. The dialyzed proteins were stored at–80°C with glycerol in small aliquots until use, purified proteins retaining activity for more than three months when stored at this temperature.The 2O = 1/1 solution] and 30 μl of reaction mixture, composed of 2 μl of 50 mM O-acetyL-L-serine, 2 μl of 50 mM Na2S, 12.5 μl of 200 mM Tris-HCl (pH 7.5), and 13.5 μl of water, was poured into each well of a flat-bottom 96-well plate. After 10 μl of enzyme solution (containing 0.1 μg of rEhCS1 or rEhCS3 in H2O) was added to each well, plates were incubated for 15 min at 37°C. The final concentrations of reagents in each well are; 2 mM O-acetyL-L-serine, 2 mM Na2S, and 50 mM Tris-HCl. The enzymatic reaction was stopped by adding 50 μl of acetic acid (>99.9%), and subsequently the reaction for cysteine detection was started by adding 50 μl of acid-ninhydrin reagent /H reagent . The mix4 cells/well with 100 μl of MEM medium  containing 10% fetal bovine serum  and 1% penicillin-streptomycin (Life Technologies) and incubated at 37°C with 5% CO2 for 2 days. Test compounds in 5 μl of 50% DMSO–H2O and 100 μl of MEM medium were mixed and added to each well. After 2 days cultivation at 37°C with 5% CO2, cell density and morphological changes were observed under a microscope. After observation, 10 μl of WST-8 solution  was added to the cells and the plate was incubated at 37°C with 5% CO2 for 2 h. Then, absorbance at 450 nm was measured by spectrophotometer . Cytotoxicity was measured in duplicate. Staurosporine  was used as a positive (cytotoxic) compound. The concentration range of test compounds are: 1.6 to 50 μg/ml  for kerriamycin B, kerriamycin C, and aggreticin; 0.075 to 10 μg/ml for deacetylkinamycin C, deoxyfrenolicin, nanaomycin A, and naphthacemycin A9; 3.1 to 200 μg/ml for tetracycline and patulin; 9.4 to 75 μg/ml for exophillic acid. IC50 values were calculated by the equation described in the reference  were prepared. After cultivation, the same amount of EtOH was added to each broth, which were then centrifuged. Obtained supernatants were used for screening as broth extract samples.2O = 1/1 solution per well) were poured into each well of a 96-well microtiter plate to screen for CS inhibitors. For screening microbial broths, 10 μl of the individual broth extract samples (50% EtOH solution) were poured into each well of a 96-well microtiter plate and dried up in vacuo. After drying, 10 μl of DMSO/H2O = 1/1 solution was added and dissolved. Samples showing more than 50% inhibitory activity and having no inhibitory activities against acid ninhydrin reaction were selected for further evaluation. The inhibition values were measured in duplicate or triplicate. The concentration range of test compounds are: 0.15 to 20 μg/ml  for kerriamycin B, kerriamycin C, and aggreticin; 2.0 to 250 μg/ml for deacetylkinamycin C, deoxyfrenolicin, nanaomycin A, tetracycline and patulin; 1.5 to 200 μg/ml for naphthacemycin A9; 3.1 to 400 μg/ml for xanthofulvin, terreinol and citromycetin; 0.06 to 200 μg/ml for exophillic acid. IC50 values were calculated as shown in Section “Evaluation of Cytotoxicity against MRC-5 Cells” with each concentration range.Each 5 μl of DMSO solution  of 316 library compounds and 5 μl of water . Frozen broth of this strain (0.2 ml) was inoculated into a 250-ml Erlenmeyer flask containing 20 ml of medium and incubated in a rotary shaker (220 rpm) at 25°C for 3 days. This seed culture (0.5 ml) was inoculated into a 500-ml Erlenmeyer flask containing 50 ml of medium and the production culture was incubated in a rotary shaker (220 rpm) at 25°C for 4 days.The fungal strain n-BuOH, and the extract was evaporated and dried in vacuo. The dried extract (0.23 g) was dissolved in 50 ml of 50% EtOH–H2O, and after removing EtOH, then applied to an ODS column . The column was eluted with H2O–acetonitrile (MeCN) system to give six fractions . The 40% MeCN fraction (50 mg) was purified by Sephadex LH-20 column chromatography . The eluate was fractionated every 1 ml. Fractions 46-50 were collected and concentrated in vacuo to dryness to afford xanthofulvin (1.9 mg). Fractions 41–43 contained terreinol (2.1 mg). The 20% MeCN fraction (25 mg) of ODS column chromatography was purified by preparative HPLC . The peak eluted at 24–26 min was collected and concentrated in vacuo to dryness to afford citromycetin (7.8 mg).The culture broth (50 ml) was extracted with 50 ml of 1H NMR  δ 7.69 , 6.85 , 6.49 , 4.78 , 2.66 , 2.19 . ESI-MS calcd. for C28H17O14 [M-H]-: 577.0624, found 577.0628 [M-H]-.Xanthofulvin. Yellowish-brown amorphous. 1H NMR  δ 6.97 , 4.92 , 4.75 , 4.06 , 3.97 , 2.66 , 2.13 , 2.13 , 2.04 , 1.89 . ESI-MS calcd. for C13H14O5Na [M+Na]+: 273.0739, found 273.0730 [M+Na]+.Terreinol. Yellowish-brown amorphous. 1H NMR  δ 6.51 , 6.20 , 5.01 , 2.33 . ESI-MS calcd. for C28H21O14 [2M+H]+: 581.0931, found 581.0923 [2M+H]+.Citromycetin. Yellow amorphous. Exophiala sp. FKI-7082 was isolated from a soil sample collected in Kouzu-shima Island, Japan. The producing strain FKI-7082 was classified in the genus Exophiala according to its morphology. This fungal strain was maintained on an LcA slant consisting of 0.1% glycerol, 0.08% KH2PO4, 0.02% K2HPO4, 0.02% MgSO4⋅7H2O, 0.02% KCl, 0.2% NaNO3, 0.02% yeast extract, and 1.5% agar (adjusted to pH 6.0 before sterilization). A loopful of spores was inoculated into two test tubes, each containing 10 ml of a seed medium consisting of 2% glucose, 0.5% Polypepton , 0.2% yeast extract, 0.2% KH2PO4, 0.05% MgSO4⋅7H2O, and 0.1% agar (adjusted to pH 6.0 before sterilization) and incubated on a shaker at 27°C for 3 days. One milliliter of the seed culture was inoculated into each of ten 500-ml Erlenmeyer flasks, each containing 100 ml of a production medium consisting of 3% soluble starch, 1% glycerol, 2% soy bean meal, 0.3% dry yeast, 0.3% KCl, 0.05% KH2PO4, and 0.05% MgSO4⋅7H2O (adjusted to pH 6.5 before sterilization) and incubated on a rotary shaker at 27°C for 6 days.The fungal strain in vacuo. The dried extract (1.2 g) was dissolved in a small amount of chloroform and then applied to a silica gel column . The column was eluted with chloroform–MeOH system to give five fractions . The two active fractions, 90:10 fraction (27 mg) and 50:50 fraction (442 mg), were combined and applied to an ODS column . An H2O–MeCN system  was used as eluent. The active component was eluted in 40–100% MeCN fractions. One of the fractions, 60% MeCN fraction (151 mg), was purified by preparative HPLC . The main peak was collected and concentrated in vacuo to dryness to afford exophillic acid (103.4 mg).The obtained culture broth (1 l) was extracted with 1 l of EtOH and mycelia were then removed by centrifugation and filtration. After evaporation, the residue was extracted three times with 1 l of ethyl acetate, and the organic layer was dried 1H NMR  δ 7.36 , 7.30 , 7.10 , 6.90 , 5.62 , 4.39 , 4.37 , 4.33 , 4.32 , 4.30 , 3.94 , 3.39 , 3.34 , 2.97 , 1.85 , 1.76 , 1.42 , 1.39 , 1.31 , 1.23 , 0.87 , 0.85 . ESI-MS calcd. for C38H55O12 [M-H]-: 703.3694, found 703.3683 [M-H]-.Exophillic acid. White amorphous. E. histolytica clonal strain HM-1:IMSS cl6 were cultured axenically in Diamond’s BI-S-33 medium at 35.5°C  penicillin/streptomycin (Life Technologies) together with test compounds  was added to each well and incubated under anaerobic conditions for 48 h. After incubation, the medium was removed and 90 μl of pre-warmed Opti-MEM I (Life Technologies) and 10 μl of WST-1 solution (Dojindo) were added to each well. Viability of trophozoites was detected with absorbance at 450 nm . Cytotoxicity was measured in triplicate. IC50 values were calculated as shown in Section “Evaluation of Cytotoxicity against MRC-5 Cells” with the concentration range showed above. Metronidazole  was used as positive control.Trophozoites of t 35.5°C . TrophozEntamoeba histolytica has two classes of CS isotypes: EhCS1/EhCS2 and EhCS3. While EhCS1 and EhCS2 are identical except for two amino acid changes . The structures of these inhibitors are shown in Figure 2. Only nephthacemycin A9 inhibited EhCS1 selectively, whereas the other compounds showed inhibitory activity against both EhCS1 and EhCS3. Three compounds, kerriamycins B and C, and aggreticin, showed the most potent inhibition (IC50 = 0.31–1.2 μM). The IC50 values against human fibroblast cells MRC-5 of the nine compounds were also evaluated, as shown in Table 2.The Kitasato Natural Products Library consists of >300 natural or semisynthetic compounds. We initially evaluated the EhCS1/EhCS3 inhibitory activity of all compounds at 100 μg/ml. Nine compounds, kerriamycins B and C, aggreticin, tetracycline, patulin, nanaomycin A, deacetylkinamycin C, deoxyfrenolicin, and naphthacemycin AFigure 2. The most potent EhCS1 and EhCS3 inhibitors, kerriamycins B and C . Despite these structural differences, they inhibited EhCSs at similar concentrations, suggesting that the chromophore structure is essential for inhibitory activity but the sugar moieties may not be important. These compounds are known to have biological activities such as anti-Gram-positive bacteria  and 22 μM (EhCS3), respectively. Kinamycin C displays anti-Gram-positive bacterial activity and anti-tumor activity (Deacetylkinamycin C (kinamycin F) is the perdeacylated compound of kinamycin C, and has the naphthoquinone substructure in its chromophore (activity . Kinamycactivity .50 = 53–65 μM). The structural difference between the two compounds is the length of alkyl chain attached to a chromophore. Therefore, the chromophore containing the naphthoquinone substructure must be important for the inhibition of EhCSs. Unfortunately, cytotoxicities against MRC-5 cells of nanaomycin A and deoxyfrenolicin were also more potent than CS inhibitory activities, as in the case of deacetylkinamycin C.Nanaomycin A, a naphthoquinone antibiotic, has anti-malarial activity  along wi50 values of 170 and 190 μM, respectively. One of the mycotoxins, patulin, is the smallest inhibitor found in this screening. Inhibition of both EhCS1 and EhCS3 by patulin was also weak.Tetracycline also has a naphthoquinone-like substructure in its chromophore. However, inhibition of EhCS1 and EhCS3 by tetracycline was weak, with IC9, isolated from a broth of Streptomyces sp. KB3346-5 was found to be a novel potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus , and selected two fungal broth extracts with no or low cytotoxicity. One extract was obtained from n-BuOH extract of a broth of Penicillium sp. if08054 using an ODS column. CS inhibitory activity was found in the 40% acetonitrile (MeCN) fraction. Subsequently, about 2 mg of xanthofulvin  and the IC50 value in the cysteine-deprived medium (18 μM) were comparable, which suggests the amebicidal effect of deacetylkinamycin C is likely due to CS inhibition. Nanaomycin A exhibited amebicidal activity 15-fold more potent in cysteine-deprived medium, however, the IC50 values against EhCSs were less potent than the amebicidal activity. Though patulin also showed selective amebicidal activity, inhibition of EhCSs was less potent than its anti-amebic activity. Xanthofulvin and exophillic acid did not show amebicidal activities at 170 and 140 μM, respectively. It was reported that xanthofulvin was not likely incorporated into cells (E. histolytica case, it may be difficult to incorporate this compound into cells. The reason why exophillic acid did not exhibit amebicidal activity was unknown.Deacetylkinamycin C, nanaomycin A, and patulin showed cysteine-dependent amebicidal activity. The ICto cells . In an E50 value against MRC-5 cells was lower than those of the IC50 values against E. histolytica cells and against EhCS. Nanaomycin A and deoxyfrenolicin also showed cytotoxicity against MRC-5 cells, at the lower concentrations showing CS inhibition, as in the case of deacetylkinamycin C. Although most compounds that inhibited EhCS in this study were toxic against a human-derived cell line, MRC-5, it is possible to design and produce by organic synthesis their derivatives that possess EhCS-dependent anti-amebic activity but lack cytotoxicity to humans. To design such derivatives, structural studies are needed to determine the moieties required for binding to EhCS.Deacetylkinamycin C, which has DNA- and protein-damaging effects , showed 9 and exophillic acid, from microbial secondary metabolite sources. A naphthoquinone structure seemed to contribute significantly to EhCS inhibition. One EhCS1-selective inhibitor, naphthacemycin A9, has a naphthacene structure with an aryl group attached via biaryl bond. The other EhCS1-selective inhibitor, exophillic acid, has a depside structure which two aryl groups attach to via an ester bond. For EhCS1-selective inhibition, the substituted small aryl group is likely important. Further structural studies using EhCS inhibitors found in this study are needed for developing anti-amebic drugs. We also found EhCS3-selective inhibitors from both fungal and actinomycete broths. These EhCS3-selective inhibitors will be purified and identified in future studies. Since CS is also present in other protozoa, such as Trichomonas vaginalis, Leishmania major, and Trypanosoma cruzi, the newly identified CS inhibitors in this study can also be exploited to develop drugs against other neglected parasitic diseases.In this study, we identified nine general EhCS inhibitors and two isotype-specific EhCS inhibitors, naphthacemycin AThe 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.


Streptococcus pyogenes type emm4 strain MEW427 , a throat isolate from a child with acute-onset neuropsychiatric symptoms meeting clinical criteria for PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus). The genome length is 1,814,455 bp with 38.51% G+C%.We report the complete genome assembly of the  Streptococcus pyogenes is a Gram-positive bacterial pathogen causing a great diversity of human disease manifestations, including acute inflammatory infections and post-infectious, immune-mediated conditions , an emm-type 4.0 throat isolate from a female child meeting clinical criteria for PANDAS.nditions . The clinditions , 3. We pdnaA as the starting point. A preliminary annotation was performed using Prokka v1.11, with a reference library generated from emm4 strain MGAS10750 (NC_008024) , and sequenced using the PacBio RS II Sequencer . Samples were prepared according to manufacturer protocols with the P6 polymerase kit and C4 sequencing reagents, with the exception of an increase to 1-h polymerase binding and 1 h binding to magnetic beads. For library construction, DNA was sheared to fragments of ~22,450 bp, isolated using the BluePippin Electrophoresis System . Sequences were collected using one single-molecule real-time (SMRT) cell. This generated 68,843 reads, each with a length of ~16,050 bp, for a total of 1,104.8 Mb of sequence data (~300- to 500-fold coverage). The sequence was assembled using Celera v8.3rc2 . Genome S. pyogenes MEW427 contains 1,814,455 bp, with a 38.51% G+C% content. The number of coding regions is 1,767 with 15 rRNA and 57 tRNA genes; for comparison, strain MGAS10750 is 1,937,111 bp in length with 1,978 predicted coding regions, and 18 rRNA and 63 tRNA genes (emm4.0 (cluster E1), a common cause of pharyngitis in North America (S. pyogenes molecular biology and potentially help understand the pathogenesis of PANDAS.The genome of NA genes . The webNA genes . By multCP014138. The sequence version referred to is the first version.This genome sequence has been deposited in GenBank under accession number


Heavily involved in the regulation of voluntary movement and habit formation, this structure is of strong importance in Parkinson's disease, obsessive-compulsive disorder, Tourette's syndrome and addiction. The C57/Bl6J mouse strain, the most commonly used strain in preclinical research today, is frequently used as a model organism for analysis of dopaminergic parameters implicated in human pathophysiology. Several components of the dopamine system have been shown to vary with age and sex, however knowledge of the contribution of these factors for dopamine release kinetics in the C57/Bl6J mouse strain is lacking. In the present study, we used an intracranial KCl-stimulation challenge paradigm to provoke release from dopaminergic terminals in the dorsal striatum of anaesthetized C57/Bl6J mice. By high-speed in vivo chronoamperometric recordings, we analyzed DA release parameters in male and female mice of two different ages. Our experiments demonstrate elevated DA amplitudes in adult compared to young mice of both sexes and higher DA amplitudes in females compared to males at both ages. Adult mice exhibited higher recovery capabilities after repeated stimulation than did young mice and also showed a lower variability in the kinetic parameters trise and t80 between stimulations. These results identified age- and sex- dimorphisms in DA release parameters and point to the importance of taking these dimorphisms into account when utilizing the C57/Bl6J mouse strain as model for neurological and neuropsychiatric disorders.The dorsal striatum is the main input structure of the basal ganglia and the major target area of dopaminergic projections originating in the substantia nigra  Spontaneous DA release occur tonically in a nanomolar range but in evoked responses, the release levels increase transiently to a micromolar range . A working electrode/micropipette unit was implanted according to a standard brain atlas The experimental groups consisted of young  and adult (defined age 16–20 weeks) C57/Bl6J mice of both sexes according to: Young males (n = 7); Young females (n = 8); Adult males (n = 6) and Adult females (n = 8). Upon urethane anaesthesia (12.5%), each animal was placed in a digital stereotactic frame  and kept at a body temperature of 37°C on a heating pad . An incision was made over the skull; bregma and lambda were aligned using a dissection microscope  together with the digital stereotactic frame. Anteroposterior ±0.07 mm difference between bregma and lambda was set as a threshold. Single carbon fiber electrodes  were coated with 2–3 layers of a 5% Nafion solution  prior to use in vivo chronoamperometry measurements were performed using the FAST16mkII recording system . A square-wave pulse sufficient to oxidize DA was applied to the coated working electrode  and repeated at a frequency of 5 Hz. An average signal was automatically calculated for every 5 cycles by the recording software  resulting in the temporal resolution of 1 data point per second (1 Hz). DA release was evoked by 7 pressure ejections (stimulations 1–7) of 100–120 nL of 120 mM KCl  applied locally . Stimulations 1–6 were applied in 2-minute intervals followed by a 15-minute resting period after which a final stimulation (7) was given  between injection and maximum peak concentration; and (3) t80, the time (seconds) from maximum peak concentration until 80% decrease of the maximum amplitude as a measure of DA clearance. All parameters are illustrated in Redox ratios were calculated at the peak of every response to identify the analyte contributing to the electrochemical signal. DA typically displays a redox ratio of 0.7–0.9 To account for multiple comparisons, the data was analysed using ANOVA and post-hoc test with Bonferroni correction for stimulations 1–6. For comparison of DA amplitudes over time within each group, 1-way repeated measures ANOVA with KCl injection as repeated measure was applied. For analysis of stimulations 1-6 between each group, 3-way repeated measures ANOVA with sex and age as independent factors and KCl injection as repeated measure. Independently of this, we assessed recovery and regain upon 15-minutes of resting. For this, stimulations 1 and 6 as well as stimulation 6 and 7 were compared using a paired student's t-test.To visualize each measurement value during initial release (stimulation 1) as well as before (stimulation 6) and after resting (stimulation 7) and to allow direct comparison between each group in a single figure, we computed scatter plot matrices. By doing so, we achieved simultaneous comparison of two variables (one on the x and one on the y axis) among all tested groups. Scatter plot matrices showing individual subject values were computed using a custom-made algorithm in Matlab .http://www.r-project.org/). A p-value equal to or less than 0.05 was considered significant .Statistical analysis was performed in Graphpad Prism 6  and R software  evoking a DA release amplitude of 15.33±1.02 µM and the last stimulation (6) an amplitude of 3.77±0.75 µM  = 12.62, p<0.0001; post-hoc test with Bonferroni correction: stimulation 1 compared to stimulation 2: p<0.01; compared to stimulation 3,4,5,6: p<0.0001) . A restirise during stimulation 1 to 6  = 1.16, p = 0.3789)  = 2.89, p<0.05; post-hoc test with Bonferroni correction: stimulation 1 compared to stimulation 2: p<0.05)  or stimulation 6    B or upo 0.3789) . The par p<0.05) . Stimula 0.0922) . Taken tIn adult males, the repeated stimulation in 2-minute intervals led to a significant reduction in DA amplitudes  = 3.85, p<0.05; post-hoc test with Bonferroni correction: stimulation 1 compared to stimulation 3: p<0.01) . DA amplrise did not significantly differ between stimulation 1 to 6  = 0.24, p = 0.9418)  and 6   = 4.64, p<0.01; post-hoc test with Bonferroni correction: stimulation 1 compared to 3: p<0.05; compared to stimulation 6: p<0.01)  and had not increased compared to stimulation 6  (The parameter t 0.9418) . Resting 0.3672) . In cont p<0.01) . The par 0.1712) . Thus, wIn young females, KCl stimulation evoked an initial DA release of 9.55±0.63 µM which decreased significantly over the course of stimulation 1 to 6 reaching a minimum of 1.88±0.50 µM after stimulation 6, directly prior to resting  = 9.028, p<0.0001; post-hoc test with Bonferroni correction: stimulation 1 compared to stimulation 2: p<0.01; compared to stimulation 3,4,5,6: p<0.001) . Followirise were seen during stimulation 1 to 6  = 2.24, p = 0.0721) . No difference was detected between stimulation 1 and 7   = 6.83, p<0.001; post-hoc test with Bonferroni correction: stimulation 1 compared to stimulation 3,4,5: p<0.01; compared to stimulation 2,6: p<0.001) , and in the range of the initial stimulation  (No differences in t 0.0721) . Followi 0.8523) . The parp<0.001) . Followied to 1) . Hence, In young males, the DA amplitudes decreased significantly during the repeated stimulation in 2-minute intervals  = 16.59, p<0.0001; post-hoc test with Bonferroni correction: stimulation 1 compared to stimulation 2,3,4,5,6: p<0.0001) . Initialrise decreased significantly over the course of stimulations 1 to 6  = 4.04, p<0.01; post-hoc test with Bonferroni correction: stimulation 1 compared to 3: p<0.05; compared to stimulation 6: p<0.01)  or stimulation 6   = 5.41, p<0.01; post-hoc test with Bonferroni correction: stimulation 1 compared to 5: p<0.05; compared to stimulation 4: p<0.01; compared to stimulation 6 p<0.001)  or stimulation 6 (paired student's t-test p = 0.0696) (The parameter t p<0.01) . After r 0.2425) . Also, tp<0.001) . Upon st 0.0696) . In summrise and t80 between all four groups of mice. Among adult mice, females showed higher DA amplitudes than males  = 9.88, p<0.01). Following stimulation 1, the amplitude in adult females was twice as high as in adult males (Post-hoc test with Bonferroni correction: p<0.001)   while nop<0.001) . Upon stp<0.001) . Neither1)  = 7.23, p<0.01; Post-hoc test with Bonferroni correction: stimulation 3,5: p<0.05; stimulation 6: p<0.01; stimulation 1: p<0.001). Following stimulation 1, the DA amplitude of young males was only 36% of that of young females (Post-hoc test with Bonferroni correction: p<0.001) . In contrise  = 4.44, p<0.05, Post-hoc test with Bonferroni correction: stimulation 1: p<0.05)  = 8.96, p<0.01; Post-hoc test with Bonferroni correction: stimulation 1: p<0.05)  , young females possessed a significantly higher t p<0.05)  and t80  p<0.05)  than did p<0.01) . In summrise  = 12.03, p<0.01; females: 3-way repeated measure ANOVA, stimulations 1 to 6, F = 22,59, p<0.0001). Following stimulation 1, young males reached 44% of the amplitude displayed by the adult males (Post-hoc test with Bonferroni correction: p<0.05) while young females reached 62% of the maximal amplitude of adult females (Post-hoc test with Bonferroni correction: p<0.01). Similar differences were detected upon stimulation 6 and 7 in both males  and females  . Interesrise  and t80 rise  was followed by additional KCl-ejections in 2-minute intervals (stimulation 2–6). The experiment was finalized by a seventh stimulation after a 15-min long resting period to allow analysis of capacity for recovery and capacity to regain initial release levels. KCl was previously shown to elicit DA release in a µM-range, comparable to electrical stimulation or psychostimulants 80, indicating a more efficient reuptake machinery with increased age. Importantly, sustained depolarization, as in the case of the applied potassium used here, could interfere with DAT function and hence affect the clearance. However, as the amount of KCl used for stimulation is equally high in all animals of this study, possible effects on DAT function should be similar in all animals, thus preserving the intrinsic differences between the groups.DA reuptake is mainly mediated via the electrogenic nature of DAT rise) was not significantly different between C57/Bl6J males and females even though the total amount of DA released varied greatly throughout the groups and individual stimulations. Based on the constant rise time with varying DA amplitudes in both adult males and females, it seems that DA release velocity is adjusted to allow for exact timing of release independently of the actual amount of DA released. In young males, trise was shorter in stimulations 3 and 6, where DA amplitudes lowest. Throughout stimulation 1 to 6, trise in young males significantly decreased with decreasing DA amplitude. In a similar manner, trise increased significantly in young females following resting and was higher in young females compared to young males during initial stimulation and after resting, when their DA release levels are at their highest. Thus, young mice seem to be unable to uphold the precise timing of DA release seen in adult mice during varying DA amplitudes.Further, rise time (tIn any repeated challenging paradigm, newly produced or recycled DA should be loaded into synaptic vesicles for release following the upcoming stimulation. Interestingly, 20-minute, but not 2-minute, intervals between stimulations were previously shown to be sufficient for recovery of DA amplitudes in adult male rats Taken together, the age- and sex-dependent differences in DA release and reuptake capacity observed in our study have several implications for future studies utilizing C57/Bl6J mice to explore the nigrostriatal DA system. Any study carried out without consideration for age or sex could suffer from the group differences identified here. For example, comparing two experimental groups where one randomly consists of mostly relatively young male mice and the other mainly of older female mice, i.e. the two groups exhibiting the highest difference from each other in terms of basal DA amplitudes, could have severe consequences on the interpretation of the data obtained. Further, considering the finding that rise time and clearance differ in young but not adult mice, age differences within an experimental setup of mice have to be considered when performing studies that include manipulations of the DA kinetics. Finally, in view of the finding that DA amplitudes decrease rapidly in response to a potent challenge and young mice seem to need longer resting time for their DA system to regain its initial capacities, any treatment that causes a strong voiding of DA contents should take age into account. In summary, C57/Bl6J is the most commonly used inbred laboratory mouse strain. Data presented here might serve to increase our awareness of sex- and age influences on the release and reuptake capacity of the DA system within this strain which is frequently used as genetic background for the wide array of transgenic mouse lines utilized to model neurological and neuropsychiatric disorders.


Their structures were established by means of spectroscopic analysis. All compounds were evaluated for cytotoxic activity, and compounds 2–6 showed cytotoxicity with the IC50 values of 10.75∼18.87 µg/mL. Three new triterpenoids, 3Supplementary material is available for this article at 10.1007/s13659-012-0038-8 and is accessible for authorized users. Supplementary material, approximately 3.36 MB.


The WGS comprises 2.5 Mb with 2,500 protein-coding sequences, 16rRNA genes, and 60 tRNA genes.We report the whole-genome sequence (WGS) of an  Staphylococcus haemolyticus, a commensal coagulase-negative staphylococcus (CoNS) species from the human skin microbiota, has also been increasingly associated with nosocomial infections and resistance to multiple antimicrobials  of Sh29/312 decreased from 64 to 1 µg/mL.crobials , 2. We rde novo assembled using Mira version 4.0.2 and SPAdes version 3.1.1. The scaffold was obtained using CLC Genomics Workbench version 7.5 and Geneious version 8.0.5 with the genome reference S. haemolyticus strain JCSC1435 (GenBank accession number AP006716.1) (Genome sequencing was performed using the Ion Torrent PGM platform (Life Technologies) and 400-bp kit. The sequences were S. haemolyticus strain Sh29/312/L2 has 2,561.368 bp with 2,417 protein-coding sequences, 16 rRNA genes, 60 tRNA genes, 39 pseudogenes, and 32.72% GC content. Sh29/312/L2 also harbors one intact and other incomplete phages.The sequence analysis showed that S. haemolyticus Sh29/312/L2 genome will provide a reference for a comparative genome analysis with its ancestral linezolid-resistant S. haemolyticus clinical strain, which has some resistance mechanism other than those known target-site modifications that promote elevated MIC for linezolid. This research will help provide insight to the genomic background of an important CoNS species. After the important work of Takeuchi and colleagues (S. haemolyticus has yet been reported.The sequence of the S. haemolyticus strain Sh29/312/L2 has been deposited in DDBJ/ENA/GenBank under the accession number CP011116.The whole-genome sequence of