Halorubrum among the family Halobacteriaceae, with a close relationship to Hrr. xinjiangense (99.77%of identity). However, value for DNA-DNA hybridization between strain ETD6 and Hrr.xinjiangense were about 24.5%. The G+C content of the genomic DNA was 65.1 mol%(T(m)). Strain ETD6 grew in 15–35%(w/v) NaCl. The temperature and pH ranges for growth were 20–55°C and 6–9, respectively. Optimal growth occurred at 25%NaCl, 37°C, and pH 7.4. The results of the DNA hybridization against Hrr. xinjiangense and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain ETD6 from other Hrr. species. Therefore, strain ETD6 represents a novel species of the genus Halorubrum, for which the name Hrr. sfaxense sp. nov. is proposed. The Genbank EMBL-EBI accession number is GU724599.An extremely halophilic archaeon, strain ETD6, was isolated from a marine solar saltern in Sfax, Tunisia. Analysis of the 16S rRNA gene sequence showed that the isolate was phylogenetically related to species of the genus  Halobacteriales) that inhabits various hypersaline environments (3–5 M) such as salt lakes, salt ponds, and marine salterns. Previous molecular ecology studies showed that archaeal halophiles dominate these ecosystems [Halobacteriaceae with cultivated representatives in solar salterns. These organisms are known to promote crystal formation of halite [Halorubrum belongs to the family Halobacteriaceae, as first proposed by McGenity and Grant [Halobacteriaceae: They were distributed among different genera of the Halobacteriaceae such as Haloquadratum, Halorubrum, and Halobacter [Halorubrum branch [Halorubrum [Halorubrum aidingense [ Halorubrum aquaticum [, Halorubrum alkaliphilum [Halorubrum arcis [Halorubrum californiense [Halorubrum chaoviator [ Halorubrum cibi [Halorubrum coriense [Halorubrum distributum [Halorubrum ejinorense [ Halorubrum ezzemoulense [ Halorubrum kocurii [ Halorubrum lacusprofundi [Halorubrum lipolyticum [Halorubrum litoreum [ Halorubrum luteum [Halorubrum orientale [Halorubrum saccharovorum [ Halorubrum sodomense [Halorubrum tebenquichense [Halorubrum terrestre [Halorubrum tibetense [Halorubrum trapanicum [Halorubrum vacuolatum [Halorubrum xinjiangense [Extremely halophilic archaea belong into a single euryarchaeotal order  belonging to the genus −1) of the solar saltern of Sfax, located in the central-eastern coast of Tunisia about 34–39°N and 10–42°E   was selected for isolation. Different colonies were purified by at least four rounds of streaking on fresh agar plates until a pure colony was obtained. The isolates were maintained as a glycerol suspension  at −80°C.Isolations were performed on DSC-97 medium containing the following ingredients  : casamino acids—7.5 g, yeast extract—10.0 g, trisodium citrate—3.0 g, KCl—2.0 g, MgSOThe pH, temperature, and NaCl ranges for growth were determined in DSC-97 growth medium. Strain ETD6 grew over a temperature, range of (20–55°C) as determined by using a temperature gradient incubator and over a pH range of 6.0–9.0 as determined with various pH buffers. Routine cultivation was performed at 37°C and pH 7.4. The requirements for NaCl for growth were determined in media with 15–35% (w/v) NaCl.Cell motility and morphology were examined using an automated microscope  equipped with a CoolSNAP HQ 2 camera  and a 100x/1.4 DLL objective. Gram staining was carried out as described by Dussault .μL of DNAase RNAase free water and frozen at −80°C for 10 min then heated at 98°C for 10 min to release the DNA. The sample obtained after the thermic shock was used as a template for PCR amplification of 16S rDNA with specific archaeal 16S rRNA gene primers [μL) contained PCR buffer (1x), 0.2 mM of each dNTP, 0.1 μM of each primer, 2 μL of template DNA, and 1 U of high-fidelity expand DNA polymerase (Roche). After initial denaturation (94°C for 5 min), 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min were performed, followed by a final extension . The PCR products were purified, ligated to pGEM T-easy vector (Promega), and transformed into E. coli DH5α cells. Single colonies containing inserts were selected at random, and the inserts were amplified from cells using the primers Sp6 (5′-ATTTAGGTGACACTATAGAATAC-3′) and T7 (5′-GTAATACGACTCACTATAGGGC-3′). Successful transformants were maintained as 40% glycerol stocks. The plasmids extracted were sent to Genome express for sequencing the 16S rRNA gene of each strain. The sequences were registered in the GenBank Data Library under the accession number GU724599. Homologues of ETD6 sequence were retrieved from the nr database at the NCBI using Blastn (http://blast.ncbi.nlm.nih.gov/) [Cell suspension from each isolate was prepared in 100  primers , 21f (5′ih.gov/)  and aligih.gov/)  with a JHalobacteriales. The physiological characterization of a novel halophilic archaeon was guided by Oren et al.,  with the2 0.01% (w/v) sugar , and 25% NaCl. Production of acid from sugars and other compounds was determined as described by Arahal et al., [The phenotypic tests for nitrate reduction, indole formation, and hydrolysis of casein, starch, and urea, were performed as described by Gerhardt et al. . The ass et al., . μg/disc unless indicated otherwise): ampicillin (10), bacitracin (10 U), chloramphenicol (30), erythromycin (15), gentamicin (10), novobiocin (30), penicillin G (10 U), rifampicin (30), streptomycin (10), sulfamethoxazole (25), and tetracycline (30). Oxidase activity was determined by oxidation of tetramethyl-p-phenylenediamine, and catalase activity was determined spectrophotometrically at 240 nm with 20 mM H2O2 [Antibiotic sensitivity tests were performed by spreading bacterial suspensions on culture plates and applying discs impregnated with the following concentrations , Xanthomonas campestris pv. campestris DSM 3586T (G+C content of 65.1 mol%), and Streptomyces violaceoruber DSM 40783 (G+C content of 72.1 mol%)).The G+C content of DNA was determined at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. The DNA was isolated and purified by chromatography on hydroxyapatite, and the G+C content was determined by high-performance liquid chromatography (HPLC) as described by Mesbah et al. . Calibrain situ temperature probe (Varian).Analysis was performed by the identification service of the DSMZ. DNA was isolated using French press cell (thermo Spectronic) and purified by chromatography on hydroxypatite as described by Cashion et al. . DNA-DNA−1. Water analyses and cell counts are presented in 7 cells mL−1 in TS. This measurement in March 2007 was similar to those reported for other marine solar salterns [At the time of sampling, the water of the pond had a temperature of 25°C, a pH of 7.55, a salinity of 37.76% (w/v), and the conductivity was 680 mS cmsalterns , 51.Cells of strain ETD6 were Gram-negative, nonmotile, and showed pleomorphic shape  occurring individually, in pairs, or irregular clusters . ColonieHalobacteriales order and more precisely with representatives of the Halorubrum genus, suggesting that ETD6 may be a new representative of this group. Among sequences from cultivated organisms, the sequence Hrr. xinjiangense (AY994197) displayed the highest similarity (99.77%) with ETD6, suggesting that they may be closely related  and very diverse physiological features and dynamic genomes [ DNA hybridization is acknowledged as the superior method for the elucidation of relationships between closely related taxa, such as strains and species . The levlorubrum . The low genomes .Halorubrum is in the range of 60.2∼71.2 mol% [The G+C content of the DNA of strain ETD6 as determined by HPLC was 65.1 T(m) mol%. The genomic DNA G+C content in the valid species names of the genus 1.2 mol% , 54.Polar lipid analysis was carried out by the identification service of the DSMZ and Dr. B. J. Tindall, DSMZ, Braunschweig, Germany. 2D TLC of extracts of strain (ETD6) revealed that this organism contained phosphatidylglycerol, methyl-phosphatidylglycerophosphate, phosphatidylglycerosulphate, phospholipids, unknown glycolipid, and two unknown lipids.The isolate was an extreme halophilic archaeon growing in high concentrations of NaCl ranging from 15 to 35% (w/v) NaCl with an optimum at 25% . Halorubrum species isolated up to date. As the use of amylase from halophilic microorganisms in industrial processes have the advantage of the enzymes having optimal activities at high salt concentrations [The strain ETD6 is able to reduce nitrate to nitrite, suggesting that it may be involved in the global nitrogen cycle within the solar saltern. The isolate was catalase-positive and oxidase-negative, and was negative for indole formation, positive for the assimilation of glucose, xylose, mannose, arabinose, and maltose, but not sucrose, galactose, or glycerol. ETD6 is an amylase-positive which is not the case for the other trations , 56, stuHrr. xinjiangense on the basis of several characteristics.As shown in Halorubrum, we propose that it may be classified as a novel species : Halorubrum. sp. nov. The above phenotypic, genotypic, chemotaxonomic, and phylogenetic data indicate that strain ETD6 may represent a novel species within the genus Halorubrum for which the name Halorubrum sfaxense sp. nov. is proposed.Because of significant phenotypic, phylogenetic, and genetic differences between this strain and all the validly published members of the family Halorubrum sfaxense .μm), and motile. Colonies are small, round, 0.5–1 mm in diameter, red orange, smooth, and slightly raised. Growth occurs optimally at 25% NaCl and over the range 15–35% NaCl . The pH range for growth is 6–9, with optimum growth at pH 7.4. The temperature range is 20–55°C; optimal temperature for growth is 37°C. Magnesium is not required for growth. They are strictly aerobic and positive for catalase while Kovac's oxidase negative. Starch is hydrolysed while casein is not hydrolysed. H2S is not produced. Nitrate is reduced to nitrite. Indole is not produced. D-Glucose, maltose, xylose, arabinose, and mannose are used as single carbon sources with acid production. No growth or acid production is observed on sucrose, galactose, and glycerol. They are sensitive to bacitracin, novobiocin, streptomycin, sulfamethoxazole, gentamicin, and tetracycline and resistant to ampicillin, chloramphenicol, rifampicin, erythromycin, and penicillin G. The polar lipids are PG, PGP-Me, PGS, single glycolipid GL, phospholipids PL1-PL2, and lipids L1-L2.Cells are Gram-negative, pleomorphic shaped (2–5 × 0.86


Purpose. To examine biomechanical parameters of the cornea in myopic eyes and their relationship with the degree of myopia in a western healthy population. Methods. Corneal hysteresis (CH), corneal resistance factor (CRF), Goldmann correlated intraocular pressure (IOP), and corneal compensated IOP (IOPcc) were measured using the ocular response analyzer (ORA) in 312 eyes of 177 Spanish subjects aged between 20 and 56 years. Refraction was expressed as spherical equivalent (SE), which ranged from 0 to −16.50 diopters (D) (mean: −3.88 ± 2.90 D). Subjects were divided into four groups according to their refractive status: group 1 or control group: emmetropia (−0.50 ≤ SE < 0.50); group 2: low myopia (−0.75 ≤ SE < 3.00 D); group 3: moderate myopia (−3.00 ≤ SE ≤ −6.00 D); and group 3: high myopia (SE greater than −6.00 D). We analyzed the relationship between corneal biomechanics measured with ORA and SE. Results. CH in the emmetropia, low myopia, moderate myopia, and high myopia groups was 11.13 ± 0.98, 11.49 ± 1.25, 10.52 ± 1.54, and 10.35 ± 1.33 mmHg, respectively. CH in the highly myopic group was significantly lower than that in the emmetropic group (P = 0.07) and low myopic group (P = 0.035); however, there were no differences with the moderate myopic group (P = 0.872). There were no statistically significant differences regarding IOP among the four groups (P > 0.05); nevertheless, IOPcc was significantly higher in the moderately myopic (15.47 ± 2.47 mmHg) and highly myopic (16.14 ± 2.59 mmHg) groups than in the emmetropia (15.15 ± 2.06 mmHg) and low myopia groups (14.53 ± 2.37 mmHg). No correlation between age and the measured parameters was found. CH and IOPcc were weakly but significantly correlated with SE . Conclusions. Present study showed only a very weak, but significant, correlation between CH and refractive error, with CH being lower in both moderately and highly myopic eyes than that in the emmetropic and low myopic eyes. These changes in biomechanical properties of the cornea may have an impact on IOP measurement, increasing the risk of glaucoma.  Myopia is the most common ocular disorder. Its worldwide prevalence is about 30% and up to 80% in certain Asian populations –4. CorneIn this observational comparative study, 312 eyes of 177 healthy subjects were analyzed (76 men and 101 women). They were recruited sequentially among patients and healthy volunteers in the Department of Ophthalmology at the Lozano Blesa University Clinic Hospital and Quirón University Hospital, Zaragoza, Spain. The average age of the patients was 33.27 ± 7.65 years . All subjects received a complete ophthalmic examination including measurement of best-corrected visual acuity (BCVA) with ETDRS chart, slit-lamp anterior segment biomicroscopy, fundus examination, and corneal topography (Orbscan II) in order to discard the existence of subclinical corneal pathology. Automated and subjective refractions were performed to determine refractive error in order to use it for the statistical analyses. Their spherical equivalent (SE) of refractive error ranged continuously from 0 to −16.50 D (mean: −3.88 ± 2.90 D). For the purpose of the study, subjects were divided into four groups according to their refractive status: group 1 or control group: emmetropia (−0.50 ≤ SE < 0.50); group 2: low myopia (−0.75 ≤ SE < −3.00 D); group 3: moderate myopia (−3.00 ≤ SE ≤ −6.00 D); and group 4: high myopia (SE greater than −6.00 D). All participants had monocular BCVA of 20/32 (0.20 logMar notation) or better. Subjects who had refractive errors such as hyperopia > 0.5 D or astigmatism > 1 D, IOP > 21 mmHg, signs of glaucomatous optic neuropathy, family history of glaucoma in a first-degree relatives, corneal dystrophy, and myopic macular degeneration, those who had undergone previous eye surgery or trauma, eye infection, diabetes mellitus, corticosteroid use or other acute or chronic diseases, or using any topical eye medication, or subjects that did not meet normal topographic criteria were excluded from the study.Corneal biomechanical properties, such as CH and CRF, were measured by the same masked technician with the Ocular Response Analyzer  using standard technique , 8, 23. r) was used to assess the relationship between spherical equivalent power and the corneal biomechanical properties (CH and CRF) as well as IOP (IOPcc and IOP) and CCT. The level of statistical significance was set to P < 0.05.Values were presented as mean ± SD. Statistical analyses were conducted using a commercial software . The distribution of the measured variables was estimated by the one-sample Kolmogorov-Smirnov test. One-way analysis of variance (ANOVA) and post hoc tests were used for determining whether the values of a particular variable differed between the three diagnostic groups. We combined data from both eyes using the mixed model method , which aP < 0.05; P = 0.77, 0.78, 0.92, 0.42, and 0.51, resp.) (P > 0.05)  and low myopic group . CRF of the emmetropic group was significantly higher than that in the moderately and highly myopic groups . There was no significant difference in IOP ; however, IOPcc was significantly higher in the moderate (15.47 ± 2.47) and highly myopic (16.14 ± 2.59) groups compared to the emmetropia group (15.15 ± 2.06)  and low myopia group (14.53 ± 2.37)   were enrolled in this study. The refraction among all included eyes ranged from 0 to −16.50 D (SE). 45 eyes corresponding to 25 patients (15 women and 10 men) were included as healthy controls (group 1). 71 eyes of 47 patients (20 women and 27 men) were included in group 2. One hundred forty-five eyes of 72 patients were included in group 3. And, finally, 51 eyes of 33 patients (23 women and 10 men) were included in group 4. Significant differences in refraction distribution were found between the four diagnostic groups  . Paramet > 0.05) . CH in t > 0.05) . CH in toc test) .r = 0.171,  P = 0.013 and r = −0.131, P = 0.021, resp., Pearson's correlation coefficient) (No correlations were found in the measured parameters with the age. CH and IOPcc were parameters significantly correlated with SE (ficient) .Corneal hysteresis is the result of viscoelastic properties of the cornea, together with the combined effect of the corneal thickness and rigidity . Low valSeveral studies have used ORA to study the correlation between corneal biomechanics and the degree of myopia. Nevertheless, the results are contradictory. Thus, whereas some authors found a significantly lower CH in highly myopic patients compared with nonmyopic or low myopia subjects –17, otheIt has been suggested that the elastic properties of the cornea may influence the accuracy of IOP measurement . In thatUnlike previous studies, the present study showed that not only high myopic eyes but also moderately myopic ones have a compromised corneal mechanical strength. These inconsistent results could be justified by differences related to other factors such as race, range of age, gender, refractive status, axial length, corneal curvature, and CCT. , 19, 20 The main limitations of this study would include that axial length and corneal curvature were not measured. Because of this, the importance of both factors in the refraction-related mechanical changes of the cornea is unknown.Concluding, the present study shows a very weak but significant correlation between CH and refractive error, with CH being lower in both moderately and highly myopic groups than that in the emmetropic/low myopic ones, indicating that some aspects of corneal biomechanics may need to be interpreted in light of the refraction, especially in myopia. These changes in biomechanical properties of the cornea may have an impact on IOP measurement, increasing the risk of glaucoma. Further studies with larger sample size should be performed.


Stenotrophomonas maltophilia PM102 (NCBI GenBank Acc. no. JQ797560) is capable of growth on trichloroethylene as the sole carbon source. In this paper, we report the purification and characterisation of oxygenase present in the PM102 isolate. Enzyme activity was found to be induced 10.3-fold in presence of 0.7 mM copper with a further increment to 14.96-fold in presence of 0.05 mM NADH. Optimum temperature for oxygenase activity was recorded at 36°C. The reported enzyme was found to have enhanced activity at pH 5 and pH 8, indicating presence of two isoforms. Maximum activity was seen on incubation with benzene compared to other substrates like TCE, chloroform, toluene, hexane, and petroleum benzene. Km and Vmax for benzene were 3.8 mM and 340 U/mg/min and those for TCE were 2.1 mM and 170 U/mg/min. The crude enzyme was partially purified by ammonium sulphate precipitation followed by dialysis. Zymogram analysis revealed two isoforms in the 70% purified enzyme fraction. The activity stain was more prominent when the native gel was incubated in benzene as substrate in comparison to TCE. Crude enzyme and purified enzyme fractions were assayed for TCE degradation by the Fujiwara test. TCE biotransformation products were analysed by FT-IR spectroscopy. Hydrocarbon dioxygenases have broad substrate specificity which makes them attractive candidates for production of industrially and medically important chemicals and development of bioremediation technology. Bacterial aromatic oxygenases are multicomponent enzymes that add oxygen molecules to the aromatic nucleus to form arene cis-diols. For example, toluene dioxygenase that catalyses the following reaction  1]..1].Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations [Toluene 4-monooxygenase (T4MO) of α [cis--1-aminoindan-2-ol. This compound can be synthesized directly from enantiopure cis--dihydroxyindan. Toluene dioxygenase oxidizes indene to produce the desired enantiomer [Current interests in structure and function of dioxygenases are due to their role in bioremediation of contaminants from our environment and in the green synthesis of cis-diols that have significant industrial and medical importance such as development of polyphenylene and prostaglandin E2α . Crixivaantiomer . All members of the dioxygenase family have one or two electron transport proteins preceding their oxygenase components, as shown in Pseudomonas cepacia G4 was the first organism to degrade TCE, reported to contain toluene monooxygenase. Toluene monooxygenase and dioxygenase [α subunits. A novel tce350 gene product from Stenotrophomonas maltophilia PM102 was shown to belong to the dioxygenase family of proteins due to the presence of 71.6% each of alternating α helix and β strand [Bacillus megaterium BM3 that hydroxylates a variety of alkanes, fatty acids, and aromatic compounds [Methylococcus capsulatus. It has been reported that this enzyme contains up to 20 copper ions per heterotrimer, in which the ions are involved in either activation of molecular oxygen or electron transfer [M. capsulatus [xygenase , 9; methxygenase ; propanexygenase ; ammoniaxygenase ; and alsxygenase  identifiβ strand . Monooxyompounds . In all ompounds . CYPs caompounds . Flavin-ompounds . Copper-transfer . Nonheme In this communication, we present the biochemical characterisation of a novel oxygenase induced by copper and benzene from PM102 isolate that utilises trichloroethylene (TCE) as the sole carbon source. A simple spectrophotometric method was used to measure enzyme activity . PurificStenotrophomonas maltophilia PM102 was isolated in our laboratory from soil samples obtained from Asansol and Dhanbad industrial belt, India. It was identified by 16S rDNA sequencing (GenBank Acc. No. JQ797560). The PM102 isolate could grow on TCE as the sole carbon source and degraded TCE efficiently [2HPO4—1 g/L, K2HPO4—3 g/L, NH4Cl—1 g/L, and MgSO4·7H2O—0.4 g/L with 0.1% peptone and 0.2% of different carbon sources: TCE, toluene, chloroform, benzene, hexane, and petroleum benzene.iciently . TCE-indiciently , and TCEiciently . The PM1μL 10 mg/mL lysozyme. The suspension was vortexed and kept at 4°C for 15 minutes followed by temperature shock at 37°C for 1 hour and further incubated at 4°C for 30 minutes. The suspension was centrifuged at 5000 rpm for 5 minutes at 4°C and the supernatant was stored as crude enzyme extract at −20°C for further studies. The supernatant of the cell cultured in media with different carbon sources was also used as the crude enzyme for mono-oxygenase assay.PM102 cells were grown in a shaker incubator (150 rpm), at 34°C, for 24 hours in 200 mL minimal medium with 0.2% of the different carbon sources and 0.1% peptone. Cells were harvested by centrifugation at 10,000 rpm for 10 minutes at 4°C, and cell pellet was suspended in 1.5 mL solution I  with 100 2PO4, pH 5), 3.5 mL substrate—TCE and other organic compounds, 100 μL (90 μg crude enzyme extract intracellular), and 0.05 mM NADH. For extracellular assay, 1 mL of the cell-free medium supernatant (16 μg protein) was added as crude enzyme extract. Protein content in cell lysate and sup was measured by Bradford assay [10 mL reaction mixture contained 5 mL reactant A (25 mg o-Dianisidine + 1 mL methanol + 49 mL phosphate buffer), 1.5 mL phosphate buffer  and supernatant (extracellular) was also studied. Optimum NADH concentration was determined by varying NADH concentration from 0.01 mM to 2 mM in presence of 0.7 mM Cu. Substrate specificity was analysed with TCE, benzene, toluene, chloroform, hexane and petroleum benzine separately to 10 mL reaction mixture setup each. μg of proteins was resolved by 8% native gel that was incubated in 10 mL reactant A, 9 mL 0.1 M phosphate buffer (pH 5), 1 mL substrate (benzene/TCE), and 0.7 mM copper. For zymogram studies, PM102 cells were grown in minimal medium with 0.2% TCE for 24 hours followed by induction with 0.2% of various carbon sources: TCE, toluene, chloroform, benzene, hexane, and petroleum for another 24 hours. Intracellular proteins were extracted by lysozyme, and protein concentration was measured by the Bradford assay. About 100 μL of 10 mM PBS for removal of Ammonium sulphate by dialysis against 200 mL 10 mM PBS for 2 hours at 25°C, followed by another 2 hours after buffer change. A third buffer change was given for overnight dialysis at 4°C. The fractions after dialysis were resolved through 8% native gel. One-half of the gel was stained with Coomassie blue, and the other half was subjected to activity staining.To understand which particular protein band gave colour in activity staining, purification of the crude enzyme extract was done by ammonium sulphate precipitation followed by dialysis. As maximum enzyme activity was recorded in the supernatant, salt precipitation was carried out with 200 mL culture supernatant of PM102 grown in 0.2% TCE with 0.2% benzene induction. The online program ammonium sulphate calculator from EnCor Biotechnology, Inc, Gainesville, Florida , was useμL of enzyme extract was added and incubated with 0.2% TCE added initially. 2 mL aliquots were drawn after respective time intervals, that is, 1, 2, 3, and 24 hours, and Fujiwara assay was done. Absorbance of the red upper aqueous phase was measured at 470 nm. As TCE gets degraded with time, the intensity of the red colour decreases with a corresponding decline in O.D. The experiment was carried out in four sets with crude unpurified enzyme, 30% Ammonium sulphate fraction, 50% fraction and 70% fraction of the extract. TCE degradation studies with the unpurified crude protein extract and with the purified enzyme fractions (extracellular) were carried out by the Fujiwara test. In this reaction, trichloroethylene in presence of pyridine and caustic alkali gives a red colour compound . In 20 m2 at the Instrumentation facility of Indian Association for Cultivation of Science, Kolkata, India. The spectra were recorded in the scan range 6000–500 cm−1 with a resolution of 4 cm−1. The IR spectra obtained were analysed with Origin Pro 8 software .Biomass obtained with TCE or benzene induction and biomass grown in peptone without induction (control) were used for FT-IR spectroscopy. Before analysis, the samples were dried in hot air oven at 50°C for 2 hours. Pellets formed by milling samples with KBr were subjected to FT-IR analysis and performed with a Nicolet impact 400 FT-IR spectrophotometer fitted with a high-linearity room temperature mid-infrared detector (MIR) cooled with liquid NμL supernatant (0.016 mg extracellular proteins) was used for characterisation of enzyme activity. Methylocystis sp. strain M has been reported to have a copper-active site that turned it into a powerful copper oxidase [1000  oxidase . Mangane oxidase , 32. Vmax for benzene was 340 U/mg/min whereas Vmax for TCE was 170 U/mg/min. Km (benzene) was 3.8 mM, and Km (TCE) was 2.1 mM, respectively. Two optimum pH for enzymatic activity were noted at pH 5 (acidic) and pH 8 , respectively. μL cell lysate (0.3 mg crude protein) with 0.7 mM Cu after 4 hours was 5.5%. Thus, percentage of total activity was maximum in the extracellular supernatant with comparatively less activity in intracellular lysate, at pH 5 and pH 8.All the optimisations in enzyme activity were done with 1 mL of culture supernatant  that contained approximately 0.016 mg protein as measured by Bradford assay. To compare the enzyme activity in the intracellular fraction obtained by lysozyme extraction of the 200 mL cell pellet, the reaction was carried out under optimised conditions, that is, 0.7 mM Cu, 0.05 mM NADH, and 36°C, and at pH 5 and pH 8, respectively, as shown in Three 8% native gels were electrophoresed under same conditions. One gel was subjected to the usual staining (Coomassie blue R250) and destaining (30% methanol with 7.5% acetic acid) process , while t−1 corresponding to epoxy ring, tertiary alcohol and carboxylic acid were present in TCE-treated samples only. In control, peaks at 699 and 1416 cm−1 correspond to C–Cl stretch and C–H in plane bend. This evidence points towards the transformation of TCE by the PM102 isolate to TCE epoxide. Previous reports show a soluble methane monooxygenase from Methylosinus trichosporium OB3b converts TCE to chloral hydrate, trichloroacetate, trichloroethanol, and CO2. Toluene monooxygenase from Burkholderia cepacia G4 has been known to convert TCE to TCE epoxide [−1 that corresponds to 1,2-disubstitution (ortho) in aromatic ring which is indicative of benzene-cis-diol or di-hydroxy benzene. Peaks at 1027, 1446, and 2253 cm−1 for benzene-treated cells could be designated as cyclohexane ring, aromatic ring stretch, and isocyanate, respectively.The FT-IR spectra for treated (TCE-induced and benzene-induced biomasses) and control (peptone grown biomass) are shown in Figures  epoxide . Thus, t Isocyanates are known to have specific industrial importance in the manufacture of polyurethanes which have wide known applications in preparation of dental materials, contact lenses, and medical adsorbents and as an ingredient in automobile paints. Medical and industrial importance of cis-diols has been stated previously. Industrial importance of ethylene oxide (epoxide) can be determined from the fact that it is one of the most important raw materials used in the large-scale chemical production, mainly of ethylene glycol that accounts for up to 75% of global consumption. Ethylene glycol is used as antifreeze, in the production of polyester and polyethylene terephthalate , liquid coolants, and solvents. Polyethylene glycols are used in perfumes, cosmetics, pharmaceuticals, lubricants, paint thinners, and plasticizers. Ethylene glycol ethers are part of brake fluids, soaps, detergents, solvents, lacquers and paints . Ethylen


Objective. It has been stated that brain cancers are an increasingly serious issue in many parts of the world. The aim of our study was to determine a possible relationship between Vitamin D receptor (VDR) gene polymorphisms and the risk of glioma and meningioma. Methods. We investigated the VDR Taq-I and VDR Fok-I gene polymorphisms in 100 brain cancer patients (including 44 meningioma cases and 56 glioma cases) and 122 age-matched healthy control subjects. This study was performed by polymerase chain reaction-based restriction fragment length polymorphism (RF LP). Results. VDR Fok-I ff genotype was significantly increased in meningioma patients (15.9%) compared with controls (2.5%), and carriers of Fok-I ff genotype had a 6.47-fold increased risk for meningioma cases. There was no significant difference between patients and controls for VDR Taq-I genotypes and alleles. Conclusions. We suggest that VDR Fok-I genotypes might affect the development of meningioma. The term of brain cancer is a heterogeneous group of neoplasms which occurs in intracranial tissue and the meninges. The incidence of malignant brain tumors is rare and approximately 2% of all cancers in adults , 2. Acco2D is one of potent regulators of cell growth and differentiation, which is an effect on cell death, tumor invasion, and angiogenesis, which makes it a candidate compound for cancer regulation [Vitamin D is a steroid hormone that regulates several endocrine functions and cell functions such as, proliferation and differentiation. , 9. The gulation . The nucgulation . The vitgulation , 13. Thegulation . Variousgulation –17. One gulation . It has gulation , 19. It gulation . Synthetgulation –26. The gulation , 25. SevSubject Selection. We investigated the VDR Taq-I and VDR Fok-I gene polymorphisms in 100 brain cancer patients (including 44 meningioma cases and 56 glioma cases) and 122 age-matched healthy control subjects who were in the follow-up Cerrahpaşa Faculty of Medicine: Department of Neurosurgery in Istanbul University. The mean ages of glioma and meningioma patients and control group were 44.75 ± 15.63, 50.26 ± 12.68, and 47.22 ± 10.63 years, respectively.The specimens were taken after obtaining informed consent, and the study was conducted prospectively. The Medical Ethics Committee of Istanbul Medical Faculty approval was obtained for the study. The protocol followed was consistent with the World Medical Association Declaration of Helsinki .2, 0.2 mM of each dNTP, 0.2 mM of each primer, and Taq polymerase in a 50 μL reaction volume. The PCR conditions were as follows: Initial denaturation step of 94°C for 4 min followed by 5 cycles of 94°C for 45 sec, 64°C for 60 sec, and 72°C for 2 min, and a further 25 cycles of 94°C for 30 sec, 64°C for 30 sec, and 72°C for 45 sec. PCR products were digested with TaqI restriction enzyme at 65°C, electrophoresed on 2% agarose gels, and stained with ethidium bromide. Genotypes were determined as TT , Tt , or tt  for Taq-I polymorphism [2, 0.2 mM of each dNTP, 0.25 mM of each primer, and Taq polymerase  in a 50 μL final volume. The PCR conditions involved an initial denaturation of 4 min at 94°C and followed by 30 cycles of 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min. A final extension step at 72°C for 4 min was also studied. PCR products were digested with FokI restriction enzyme  at 37°C for 3 h followed by electrophoresis in a 2% agarose gel. The FF genotype  shows only one band of 272 bp in agarose gel. The ff genotype  generates two fragments of 198 and 74 bp. The heterozygote displays three fragments  [Blood specimens were collected in tubes containing EDTA, and DNA samples were extracted from the whole blood by a salting out procedure  . Genotypmorphism . The priP < 0.05 were considered statistically significant. Haplotype frequencies D′ and r2 were calculated using Haploview 4.0 programme.Statistical analyses were performed using the SPSS software package . Data are expressed as means + SD. Differences in the distribution of genotypes or alleles between cases and controls were tested using the chi-square statistic. Odds ratios (ORs) and 95% confidence intervals (95% CI) were calculated to estimate the risk of glioma and meningioma. Values of P > 0.05) (P = 0.004) . The VDR Taq-I genotype frequencies for meningioma, glioma, and control cases were not significantly different (P > 0.05). In addition, vitamin D haplotypes were evaluated for association with brain cancers. Haplotype analysis revealed that there was no relationship between VDR polymorphisms and brain cancers   . VDR FokFor the first time, we demonstrated the positive association of VDR Fok-I gene variants with meningioma cases. We also determined that VDR Fok-I ff genotype might affect development of meningioma, but we found that there was no statistically significant difference between VDR polymorphisms with glioma.Vitamin D is an important factor in the regulation of cell division and differentiation. The VDR gene is a member of nuclear receptor superfamily. The VDR gene located on chromosome 12. For a long time, several polymorphisms in VDR gene have been investigated for functional significance and potential effects on disease susceptibility . Many stSadetzki et al. reported that the genotype of KRAS was related to the increased the risk of (nearly 2 fold) meningioma . VDR expDespite an extensive literature study, there was no study that reported the relationship between VDR polymorphisms and brain cancer susceptibility. In conclusion, the present results suggest that the Fok-I polymorphism in the VDR gene may be related to the risk of meningioma. However, further studies with large number of subjects are needed to explain this kind of relationship between VDR-Fok-I genetic variants and meningioma risk.


Drosophila with the aim of testing whether the two species groups suffered differently from inbreeding depression. The traits investigated were egg-to-adult viability, developmental time and resistance to heat, cold and desiccation. Our results showed that levels of inbreeding depression were species and trait specific and did not differ between the species groups for stress resistance traits. However, for the life history traits developmental time and egg-to adult viability, more inbreeding depression was observed in the tropical species. The results reported suggest that for life history traits tropical species of Drosophila will suffer more from inbreeding depression than widespread species in case of increases in the rate of inbreeding e.g. due to declines in population sizes.The evolutionary history of widespread and specialized species is likely to cause a different genetic architecture of key ecological traits in the two species groups. This may affect how these two groups respond to inbreeding. Here we investigate inbreeding effects in traits related to performance in 5 widespread and 5 tropical restricted species of  A species' genetic architecture, referring to the number of segregating functional variants, including dominance and epistatic interactions, underlying phenotypic traits, is formed by phylogenetic, demographic and ecological processes, and determines its biological performance, such as its basal resistance to environmental stressors and the ability to adapt evolutionary and through adaptive phenotypic plasticity to environmental changes Drosophila species are restricted to narrow habitat ranges in the tropics , whereas others occupy habitats ranging from tropic to temperate areas (‘widespread species’). Widespread species are exposed to different, more variable and diverse ecological conditions compared to specialized restricted tropical species, which will likely lead to different selection pressures and subsequent genetic divergence of the two species groups through evolutionary adaptation Drosophila are on average more closely related to each other than to widespread species Drosophila are for those reasons likely to have a different genetic architecture, especially in key ecological traits involved in environmental stress resistance.Some Drosophila species have recently been hypothesized to be more prone to experience reduced population sizes and extinction as compared to more widespread Drosophila species as the environment changes. This is based on evidence from a number of studies showing that widespread species have higher levels of basal resistance to climatic extremes (temperature and humidity) when compared to tropical climate specialists Drosophila have lower adaptive evolutionary potential in the ecological key traits desiccation and cold resistance Tropically restricted Drosophila have a different genetic architecture, it is of interest to investigate if the two species groups respond differently to inbreeding. If the cost of inbreeding differs between the species groups, this may influence the extinction probabilities of these species groups when they experience an increase in inbreeding rates in their natural habitats, due to for instance reductions in population size as may occur under future climate changes. Here, we study the consequences of inbreeding in 5 widespread and 5 tropical restricted species of Drosophila by investigating inbred and outbred lines for the traits egg-to-adult viability, developmental time, and resistance to cold, heat and desiccation stresses. Generally, we found strong line, species and trait specific effects of inbreeding. Tropical species on average suffered more from inbreeding depression compared to widespread species for life history traits, whereas no differences among species groups were observed in stress resistance traits.Based on the hypothesis that tropical and widespread species of Drosophila species was collected during 2007 and 2008 (December to April) in North Queensland, along the Australian east coast. Five of the species are restricted to the tropics, mostly rainforest habitats , while the other 5 are cosmopolitan or widespread species whose habitat includes temperate regions as well . Average latitude of the tropical and widespread species investigated here range from 13.04° to 18.15° and 101.75° to 129.40°, respectively (based on registrations entered in the taxodros database available at http://www.taxodros.uzh.ch). For each species, 18 to 22 inseminated females from a single population were caught and brought to the laboratory to establish mass bred populations, which were maintained for 15 generations with at least 1000 individuals before outbred and inbred lines were established. The expected loss of heterozygosity during this process is calculated according to A population from each of 10 t = 1/4(1+2Ft−1+Ft−2)). Thereafter inbred lines were expanded to about 500 individuals before performing the experiments. Between 3 and 10 inbred lines per species were used for experiments . The experiments were done in the second generation after the last inbreeding round . From prior knowledge of generation times, collection of eggs was timed so that flies from all species emerged over as short a time span as possible. Flies used for experiments were all between 3 and 8 days old.Ten vials (with 7 ml medium) per line each containing 20 eggs were set up as specified above. Emerging flies were counted twice a day (at 8:00 and 20:00 h). Egg-to-adult viability was scored as the number of males and females emerging from each vial and developmental time estimated as the time taken to develop from an egg to an adult female or male fly. Flies were all reared at 20°C degrees since this temperature is considered non-stressful for all species investigated. Emerging flies were used for the temperature and desiccation assays described below.Heat knock down resistance. Ten flies per line and sex were individually put into 5 ml glass vials and submerged into a preheated 38°C water bath. Heat resistance was scored as the time until the flies were unable to move any of their body parts.Chill coma recovery time. Ten flies per line and sex were individually put into 5 ml glass vials and submerged into a 0°C water bath and kept there for 3 hours. The vials were then put at room temperature, and chill coma recovery was scored as the time when the flies were able to stand on their legs.Critical thermal minimum (CTmin). Ten flies per line and sex were individually put into 5 ml glass vials and submerged into a 20°C water bath. The temperature was then decreased by 0.1°C per minute. CTmin was scored as the temperature at which flies were in coma unable to move any body parts , species, breeding regime (inbred or outbred), sex and the interactions between the fixed effects. Inbreeding depression (δ) was estimated for each trait using the formula δ = 1 – mean inbred/mean control 79,1767 = 17.47, P<0.001; species: F9,1767 = 207.95, P<0.001; breeding regime: F1,1767 = 33.52, P<0.001; sex: F1,1767 = 231.53, P<0.001; species×sex: F9,1767 = 5.65, P<0.001; 9,118 = 6.09, P<0.001, 1,118 = 0.80, NS). Levels of inbreeding depression were also not affected by the species×sex interaction .Developmental time: Line (nested within species and breeding regime), species, breeding regime (inbred or outbred), sex and the species×sex interaction all significantly affected developmental time  nor the interaction species×sex impacted on the level of inbreeding depression .Egg-to-adult viability: Line, species, breeding regime (inbred or outbred) and the species×breeding regime interaction all significantly affected egg-to-adult viability . The impact of inbreeding was not affected by species  nor by the interaction species×sexes  (Heat knock down resistance: Line (nested within species and breeding regime), species, and the breeding regime×sex and species×sex interactions significantly affected heat knock down resistance  .77,1704 = 1.97, P<0.001; sex: F1,1704 = 11.18, P<0.001; species: F9,1704 = 116.99, P<0.001; species×breeding regime F9,1704 = 3.38, P<0.01; species×sex: F9,1704 = 5.11, P<0.001; 9,108 = 6.46, P<0.001, 1,108 = 0.36, NS) nor the interaction species×sex impacted on the level of inbreeding depression .Chill coma recovery time: Line (nested within species and breeding regime), sex, species and the species×breeding regime and species×sex interactions all significantly affected egg-to-adult viability in chill coma recovery time , whereas it was the same in males and females of the same species . The interaction species×sex was significant  (CTmin: Line (nested within species and breeding regime), sex, species and breeding regime all significantly affected CTmin (line: F P<0.05) .74,1708 = 6.06, P<0.001; sex: F1,1708 = 46.64, P<0.001; species: F9,1708 = 44.89, P<0.001; breeding regime: F1,1708 = 4.93, P<0.05; species×sex: F9,1708 = 34.30, P<0.001; species×sex×breeding regime: F9,1708 = 2.23, P<0.05; 9,101 = 1.73, NS) nor sexes , but was affected by the interaction species×sex  (Desiccation resistance: Line (nested within species and breeding regime), sex, species, breeding regime (inbred or outbred) and the species×sex and the species×sex×breeding regime interactions all significantly affected desiccation resistance (line: F P<0.05) .1,8 = 9.71, P<0.05; males: F1,8 = 14.86, P<0.01, 1,8 = 1.22, P<0.10; males: F1,8 = 1.07, P<0.20, For developmental time more inbreeding depression was observed in tropical compared to widespread species for both sexes  and stress resistance traits, we found that tropical species suffered on average more from inbreeding depression in life history traits compared to widespread species P<0.001;  whereas Drosophila. The two species groups have a different evolutionary history and they occupy different habitats and thus are likely to have experienced different selection pressures. We tested the hypothesis that, due to likely differences in the genetic architecture of the two species groups, the level of inbreeding depression differs between widespread and tropical species groups. For the stress resistance traits investigated we found no evidence to support this hypothesis  and inbred (‘i’) flies (with standard errors in parentheses) for each sex, species and trait observed in this study. ‘N’ equals the number of inbred lines tested (in parentheses the number of lines available after the 4 generations of full sub mating). N equals 3 for all traits in both males and females in the control lines. The species are: D. bipectinata (bipect), D. birchii (birc), D. bunnanda (bunn), D. hydei (hydei), D. melanogaster (mel), D. pseudoananassae (ps), D. repleta (rep), D. serrata (ser), D. simulans (sim) and D. sulfurigaster (sulf). Symbols: ‘T’ = ‘tropical’ and ‘W’ = ‘widespread’.(DOCX)Click here for additional data file.


Poly(lauryl methacrylate) (PLMA) thin film doped with Mn:ZnSe quantum dots (QDs) was spin-deposited on the front surface of Si solar cell for enhancing the solar cell efficiency via photoluminescence (PL) conversion. Significant solar cell efficiency enhancements (approximately 5% to 10%) under all-solar-spectrum (AM0) condition were observed after QD-doped PLMA coatings. Furthermore, the real contribution of the PL conversion was precisely assessed by investigating the photovoltaic responses of the QD-doped PLMA to monochromatic and AM0 light sources as functions of QD concentration, combined with reflectance and external quantum efficiency measurements. At a QD concentration of 1.6 mg/ml for example, among the efficiency enhancement of 5.96%, about 1.04% was due to the PL conversion, and the rest came from antireflection. Our work indicates that for the practical use of PL conversion in solar cell performance improvement, cautions are to be taken, as the achieved efficiency enhancement might not be wholly due to the PL conversion. Commercial solar cells employ only a small portion of the solar spectrum for photoelectric conversion, with the available wavelengths covering the visible to near-infrared (NIR) regimes . To full2 in size) without AR treatment were offered by the Shanghai Institute of Space Power Supply, Shanghai, China. The QD suspension was firstly mixed within PLMA  and then deposited onto the surface of solar cell with a spin coater. QD concentration (CQD) was determined by adjusting the proportions of QD suspension and PLMA. The thickness of QD-doped PLMA was around 150 nm as measured using a stylus-profiler . Reflectance spectra of Si coated with QD-doped PLMA were obtained with an UV–vis-NIR spectrophotometer . PL spectra were recorded on a fluorescence spectrometer . Monochromatic lights from one He-Cd laser and other three semiconductor lasers with λ = 325, 473, 650, and 980 nm, respectively, were used to investigate the PV responses of short-circuit current (ISC). Also, a simulated all-solar-spectrum (AM0) PV response was measured on a solar simulator  to acquire the PV parameters of photoelectric conversion efficiency (η), fill factor (FF), ISC, and open-circuit voltage (UOC). The EQE measurement of solar cell was performed on a QE/IPCE system of Oriel/Newport.Mn:ZnSe QDs immersed within toluene were purchased from ZKWY Biotech Incorporation Ltd., Beijing, China. Figure I/I’s) under illuminations of four monochromatic light sources  as functions of CQD. ΔI/I is defined as (I1−Ibare)/Ibare, where Ibare and I1 are ISC’s for bare Si solar cell and Si solar cell coated with QD-doped PLMA, respectively. Figure λ = 325 nm), the ΔI/I trends of other three wavelengths can be well explained in terms of their reflectance ones. For instance, for the blue (λ = 473 nm) response, with the increasing CQD, the reflectance increases steadily, and after reaching the maximum around 2.2 mg/ml, it starts to decrease. Due to this evolution of reflectance, ΔI/I decreases steadily at first, and at around 2.2 mg/ml, it starts to increase. As compared to the blue, red (λ = 650 nm), and NIR (λ = 980 nm) ones, the UV response looks like ‘abnormal’; it does not decrease monotonously in terms of the trend of reflectance but shows a raised structure peaking around 1.6 mg/ml. The appearance of such a raised structure should be due to the PL conversion under UV illumination. Since the absorption edge of QDs as indicated in Figure λ = 473, 650, and 980 nm, for the case of UV in Figure pure AR to ΔI/I could then be represented by a monotonously changing curve as indicated by the dashed line, which was drawn through extrapolating the data at CQD < approximately 0.8 mg/ml and CQD > approximately 2.8 mg/ml, where the PL conversion contribution was little. Therefore, at CQD = 1.6 mg/ml, ΔI/I reads 35.07%, among which, approximately 9.66% is from the effect of PL conversion as calculated, and the rest approximately 25.41% due to AR. In the following, we will focus on the cases of CQD = 0 and 1.6 mg/ml only and assess the contribution of PL conversion to Si solar cell efficiency enhancement under two AM0 conditions.Figure ined as I−Ibare/IbCQD = 0 and 1.6 mg/ml (right ordinate), together with the emission spectra of a standard AM0 [η/η0 = (η1− η0)/η0, where η0 and η1 are photoelectric conversion efficiencies of Si solar cell coated with QD-doped PLMA with CQD = 0 and CQD ≠ 0, respectively. It should be noted here that unlike ΔI/I, which is with respect to bare Si solar cell, Δη/η0 is with respect to Si solar cell coated with pure PLMA (CQD = 0). In Table η/η0 could be calculated as follows. The AM0 intensity times EQE yields the modified EQE curve. An example is illustrated in Figure CQD = 0. The modified EQE curve gives the efficiency response for each wavelength in AM0 spectrum. The summation of all the responses, i.e., the area under the modified EQE curve may represent the solar cell efficiency. Δη/η0 can thus be calculated as the area difference between CQD = 1.6 mg/ml and 0, divided by the area for CQD = 0. The calculated Δη/η0 was 5.96%, which is close to the measured value. For λ < approximately 450 nm, the efficiency enhancement could now be regarded as wholly from the contribution of PL conversion, since the reflectance coefficients at CQD = 0 and 1.6 mg/ml are nearly the same as shown in Figure CQD = 1.6 mg/ml and 0 for λ < approximately 450 nm only, divided by the whole area for CQD = 0. It was 1.04%. Therefore, the rest 5.96% − 1.04% = 4.92% was due to AR. In Figure I-V curves for bare Si solar cell and Si solar cell coated with QD-doped PLMA (CQD = 0 and 1.6 mg/ml) are depicted. UOC and FF change slightly; only the ISC varies steadily, leading to a change in η. In Table η/η0 for CQD = 3.0 mg/ml is as high as 9.97%, which is the highest efficiency enhancement achieved in this work. However, from Figure η/η0 at CQD = 3.0 mg/ml is very little. The AR effect contributes dominantly, which could be attributed to the modification of refractive index gradient [Figure dard AM0   were achieved indeed under AM0 conditions. Both the effects of AR and PL conversion contributed to the solar cell efficiency enhancements but that of PL took a small portion. A precise assessment of PL contribution to the efficiency enhancement was made by investigating the PV responses of Si solar cells coated with QD-doped PLMA to monochromatic and AM0 light sources as functions of QD concentration, combined with reflectance and EQE measurements. Our work shows that the real PL contribution might not be all that as reflected by the apparent efficiency enhancement, and cautions are to be taken when applying the PL conversion in this aspect. On the other hand, it indicates again that for practical use of PL conversion, high altitude or/and outer space environments are preferred where the UV proportion is high, and continuing to search for high PL efficiency materials and design efficient optical-coupling structures is still necessary.The authors declare that they have no competing interests.I-V data. HCH measured the EQE data. MZ helped to prepare samples. WS measured the reflectance data. ML designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.DCC prepared all the samples and measured the absorbance, PL, short circuit, and


Study on the accumulation level of heavy metals was conducted on sediment and fishes from estuaries of Bay of Bengal. Heavy metals were determined by using Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) and the results were compared to permissible limits of WHO/USEPA. The accumulation patterns of Fe and Cd were found predominantly in all samples tested when correlated with other metals. It was found that the concentration of metals such as Cd (3.90 ± 0.25 μg/g), Cr (0.44 ± 0.05 μg/g), Ni (0.33 ± 0.01 μg/g), and Mn (1.1 ± 0.11 μg/g) were exceeding the permissible limit, whereas Fe, Co, Pb, and Zn were found within the limit of WHO/USEPA at station 1. In station 2, Cd (16.5 ± 0.4 μg/g), Mn (0.67 ± 0.11 μg/g), and Cr (0.80 ± 0.01 μg/g) were exceeding the permissible limit, whereas Fe, Co, Pb, Ni, and Zn were found within the limit. This study emphasizes that Cd and Mn levels in both stations, are far higher than the acceptable values set by WHO/USEPA and may therefore present human health hazards. It is therefore mandatory to carry out extensive research to evaluate the possible environmental risk factors in the vicinity of both estuaries with respect to heavy metals. Estuarine ecosystems are highly productive because of rich nutrients received from the river runoff. One of the important roles of the estuaries is to provide the nursery ground for many young marine animals, and thus support a unique community of plants and animals, especially adapted for life in the coastal areas.The major environmental problem of estuarine ecosystems is receiving various kinds of contaminants from the land. Majority of the industries at coastal area discharge the chemical effluents into the aquatic environment which in turn cause changes in habitat, species distribution, abundance, and bio-geo chemical cycles . Wastes Fishes are on the top of the aquatic food chain and accumulate large amounts of metals from water and sediment. Sediments are known to be “Trace element traps” , becauseHeavy metals normally occurring in nature are not harmful to the environment, because they play an essential role in tissue metabolism and growth of plants and animals . Metals The estuaries selected for the present study are biologically important and economic hub of the people and expected to be polluted by industries and human activities. This leads to damage of food chain of this environment by those pollutants are toxic to aquatic animals and human those are live nearby area. Comprehensive studies related to the analyses of sediment and fishes around particular estuaries are limited. Therefore, the present study has been undertaken (i) to evaluate the extent of heavy metal contamination in sediment and edible part of fishes, (ii) to investigate the uptake of eight heavy metals in fishes and (iii) to understand the present condition of the ecosystems and to compile the baseline data for future monitoring.Two stations (estuaries) lay in the Bay of Bengal  were selected and the coast has extensive areas of estuaries, mangroves, and brackish water lagoon which all have connection to the Bay of Bengal.Two sampling sites (site 1 and site 2) were chosen in both stations. Site 1 (Stacknend), 2 km away from the mouth of the estuary and site 2 (Bar mouth) at the mouth of the estuary.Mugil cephalus, Oreochromis mossambicus, Arius caelatus, Lutjanus fulviflamma, Terapon jarbua and Chanos chanos. These are commercially and nutritionally important fish species. The samples were collected in sterile sample containers and kept in the laboratory deep freezer (−20°C) to prevent deterioration till further analysis. Total length of fishes was measured and no significant differences were found among the species (P > 0.05)  Table . For eacSediment samples were collected at about 30 cm deep from sediment surface to obtain layer of the sediment according to Norhayati et al., (2007) . TriplicNitric–perchloric acid digestion method was followed for analysis of heavy metal in sediment and edible tissue of fishes ,11. The Sediment samples were air dried in the laboratory at room temperature, ground in fine mixture using mortar and pestle before sieving less than 2 mm mesh. The nitric-perchioric acid digestion method was also followed for the analysis of the sediment.All analysis was performed in triplicate and mean, SD values were calculated by SPSS® (v16) software for windows. Bray Curtis similarity index was carried out by Primer 6 (v6).In the present study, Fe, Zn, Cd, Mn, Pb, Cr, Ni, and Co concentrations were determined in the fishes and sediments of two estuaries. In station 1, accumulation of Fe was found to be higher followed by Zn, Cd, and Mn while Pb, Cr, Ni, and Co were Below Detectable Level (BDL) Table . At StatA. caelatus) to 2.1 ± 0.3 (O. mossambicus) μg/g, for Co 0.008 ± 0.062  to 0.17 ± 0.2 (L. fulviflamma) μg/g, for Cr 0.1 ± 0.07 (L. fulviflamma) to 0.44 ± 0.53 (sst 2) μg/g, for Fe 3.8 ± 0.16 (C. chanos) to 19.8 ± 0.58 (O. mossambicus) μg/g, for Mn 0.20 ± 0.02 (sst 2) to 1.2 ± 0.34 (L. fulviflamma) μg/g, for Ni 0.03 ± 0.01 (A. caelatus) to 0.33 ± 0.40 (O. mossambicus) μg/g, for Pb 0.05 ± 0.03 (A. caelatus) to 1.94 ± 0.01 (sst 2) μg/g, and for Zn 1.24 ± 0.12 (C. chanos) to 7.1 ± 0.9 (L. fulviflamma) μg/g.The metal concentration and the corresponding mean standard deviations all the samples from both estuaries are shown in Tables O. mossambicus) μg/g, for Co 0.006 ± 0.003 (C. chanos) to 0.049 ± 0.05 (O. mossambicus) μg/g, for Cr 0.055 ± 0.005  to 0.21 ± 0.04 (C. chanos) μg/g, for Fe 3.171 ± 0.05 (A. caelatus) to 26.58 ± 2.6 (sst 1) μg/g, for Mn 0.42 ± 0.037 (sst 2) to 1.38 ± 0.28 (A. caelatus) μg/g, for Ni 0.06 ± 0.01 (sst 1) to 0.13 ± 0.012 (sst 2) μg/g, for Pb 0.05 ± 0.003  to 0.78 ± 0.16 (sst 1) μg/g, and for Zn 1.319 ± 0.04  to 4.152 ± 0.073 (O. mossambicus) μg/g.In station 2, the concentration of Cd ranged from 1.12 ± 0.012 (sst 1) to 26.25 ± 0.063  was more polluted than site 2 (bar mouth) because all the waste and industrial effluents were entered through site1 (stacknend).The different forms and concentration of heavy metals in the sediment of an aquatic medium determine their bioavailability and toxicity. Thus, the study of the different fractions of the elements in the sediment was vital because the total concentrations are not representative of the real degree of the potential contamination. Heavy metals can be bound to or occluded in amorphous materials, adsorbed on clay surfaces or iron/manganese oxyhydroxides, co-precipitated in secondary minerals such as carbonates, sulfates, or oxides, complexes with organic matter, or included in the lattice of primary minerals such as silicates .In the current study, heavy metal concentrations in sediment were found to be higher in for fishes at both stations. As such, accumulation of heavy metals in tissue depends mainly on metal concentration in sediment. This shows that Stacknend station was more polluted than the Barmouth. The sediment samples also reflected the same trend.M. cephalus, O. mossambicus, A. caelatus, and C. chanos, T. jarbua and L. fulviflamma collected from station 1, Fe and Zn accumulated highest concentration, whereas Co and Pb had the lowest among all eight metals analyzed. But based on WHO/USEPA reference standard for fish [O. mossambicus and C. chanos, the Cd (2.128 and 0.238 μg/g) was found higher than the standard value of WHO/USEPA. But Mn  and Ni  were found to be in an elevated state (concentration) than the standard value in all fish samples of Station 1. The levels of Fe, Zn, Pb, Cr, and Co were found were found to fall within WHO/EPA safe limits. Fish samples collected from Station 2, concentration of Cd was higher than other metals and Co was at the lowest concentration. These results are indicative that Cd , Mn , and Cr (0.155 μg/g) were relatively higher than the standard value  (safe limits) of WHO distinguishes polluted from unpolluted ecosystems, based on acquired knowledge on metal bio-availability, bio-concentration and bioaccumulation patterns . Resultsfor fish    reportedCyprinus carpio fish. Metal contaminants eventually find their way into the human system through the food chain [Vinodhini and Narayanan 2008)  also pro08  also od chain  reportedEpinephelus chlorostigma, Lutjanus russelli, Terapon jarbua, Cynoglossus arel and Lagocephalus lunaris) varied from 0.274 ± 0.03 to 53.39 ± 14.3 μg/g which led to total environment pollution in Cuddalore, nearest place of our study stations. Nickel is a hazardous element notified by the USFDA (1993) [Mugil cephalus, Oreochromis mossambicus, Arius caelatus, Lutjanus fulviflamma, Terapon jarbua and Chanos chanos.Harvey (1975)  reportedA (1993) , though In our study, the concentrations of Fe, Zn, Pb, and Co of both estuaries were in safe limit. Though these metals are within safe limit at present, there is an increasing chance that it might get increase in the near future, and turn toxic. Cobalt is one of the metal that is present naturally in the body, but in excess concentration, it becomes toxic and lead to harmful potentially permanent side effects like cardiomyopathy, hypothyroidism, and neurological damage as well as impairment of the senses . SimilarThe results of this study provided valuable information about the metal contents in sediment and fish from the two estuaries of Bay of Bengal. Based on the findings, station 2 was more polluted than station 1 because of the high accumulation of Cd. The marginally higher concentrations of Cd, Cr, and Mn in the samples collected from station 2 could be related to industrialization and related anthropogenic activities in these areas. Moreover, these results enable us to understand the chemical quality of fish and to evaluate the possible risk associated with their consumption. This study emphasizes that Cd and Mn levels in both stations, are higher than the acceptable values for human consumption set by various health organizations and presents a greater human health issue in the future. These results insist that station 2 should be monitored on a regular basis for more possible metal toxicity. Besides, further studies are needed to evaluate and to characterize the fishes in the estuary relative to specific metal pollutants because the Cd level in fishes of station 2 and Fe level in fishes of station 1 were higher compared to sediment. This study is an indication of high contamination of water resource due to the anthropogenic sources and the competent monitoring program is an essential adjunct to any attempt of managing the coastal areas in an ecologically sound and sustainable manner.All authors declare that they have no competing interest.SL and TM were the main investigator, designed and performed the study and drafted the manuscript. TV and TTA supervised the study. KVD, JG and TB were advisors of the study. SG helped in the statistical analysis. All authors read and approved the final manuscript.


Euphorbia supina is a weed that has been used in folk medicine in Korea against a variety of diseases. Nine polyphenols were characterized for this plant by using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and the results were compared with the literature data. The individual components were validated using the calibration curves of structurally related external standards and quantified for the first time by using the validated method. Correlation coefficients (r2) were >0.9907. The limit of detection and limit of quantification of the method were >0.028 mg/L and 0.094 mg/L, respectively. Recoveries measured at 50 mg/L and 100 mg/L were 76.1–102.8% and 85.2–98.6%, respectively. The total amount of the identified polyphenols was 3352.9 ± 2.8 mg/kg fresh plant. Quercetin and kaempferol derivatives formed 84.8% of the total polyphenols. The antioxidant activities of the flavonoids were evaluated in terms of 1,1-diphenyl-2-picrylhydrazyl and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation-scavenging activity, and the reducing power showed a dose-dependent increase. Cell viability was effectively suppressed at polyphenol mixture concentrations >250 mg/L.The Korean prostrate spurge  Euphorbia supina is a weed that belongs to the Euphorbiaceae family and is native to North America. It is found in poor, drought-stressed turf and grows well during hot, dry weather in thin soils. It sprouts purple-spotted, up to 0.60-in oval leaves, and blooms very small, inconspicuous flowers in the summer −–hexosyl–H2). It was identified as kaempferol 3-O-hexoside [7 was identified as kaempferol 3-O-pentoside. Its MS/MS consisted of [M+H]+ at m/z 419 and 287 [M+H–pentosyl]+ [8 yielded [M+H]+ at m/z 302, which was fragmented to typical fragment ions 273 [M+H–CO]+, 179 [M–CC6H5O2]+, and 153 . Polyphenol 8 was quercetin [9 was identified as kaempferol and gave [M+H]+ at m/z 287, which produced 257 [M+H–CO]+ and 153  [Polyphenol CO2–CO]− . Polyphe–H–CO2]− . Componeyl–CO2]+ . Polyphehexoside . The MS/hexoside . Polyphentosyl]+ . Polypheragment] , 27.E. supina were quantified for the first time from peak areas of the LC-UV chromatogram obtained at 254 nm. Quantification was validated based on representative external standards from the same group. The validation data are listed in y = ax + b, where y and x were the peak area and the concentration of each standard, respectively. The regression analysis showed correlation coefficients (r2) > 0.9907 for all five standards (n = 5), indicating good linearity. The LODs of the method were 0.028–0.142 mg/L and LOQs were 0.094–0.473 mg/L, indicating good performance limits. Recoveries measured at 50 mg/L and 100 mg/L were 76.1–102.8% and 85.2–98.6%, respectively. Precisions of the method at 50 mg/L and 100 mg/L were 0.1–6.1% and 0.5–14.0%, respectively. Both the accuracy and precision values were acceptable.The nine polyphenols identified in the Korean O-pentoside (5) as the most dominant component, followed by kaempferol 3-O-hexoside (6). Quercetin, kaempferol, and their sugar-bound derivatives are major representatives of the polyphenol subclass that display the antioxidant activity to scavenge reactive oxygen species. As a result, E. supina, which is rich in such components, could be effective for reducing the risk of various chronic diseases resulting from oxidative damage, such as cancer, atherosclerosis, and inflammation [The contents of individual components are listed in ammation , 28.E. supina was evaluated for its antioxidant effects. Contemporary interest in polyphenols focuses on the epidemiological association between their potent antioxidant properties and a low incidence of chronic diseases. Epidemiological studies have shown that oxidative stress plays an important role in the pathogenesis of various chronic diseases, including cancer, cardiovascular disease, atherosclerosis, hypertension, diabetes, neurodegenerative disorders, rheumatoid arthritis, and aging [The polyphenol mixture isolated from Korean nd aging –31. Polynd aging , 33; thein vitro methods. Because the assay results are method dependent, a combined assay involving several methods is often used [E. supina was determined by DPPH∙ and ABTS∙+ scavenging and RP assay at a concentration ranging from 25 to 500 mg/L. In DPPH∙ scavenging tests, antioxidant activity is monitored by measuring the disappearance of purple DPPH∙, which can be detected spectrophotometrically at 517 nm [∙+ scavenging assay, the added antioxidants reduce the deep blue ABTS∙+ to ABTS, and the decrease in absorbance of ABTS∙+ at 414 nm is monitored [3+) to Fe2+. The increase in absorbance of the deep-green Fe2+ solution at ~700 nm is monitored [E. supina polyphenol mixture showed a dose-dependent increase. DPPH∙ or ABTS∙+ scavenging activity can be represented as an EC50 value, which is the antioxidant concentration required to bring about a 50% loss in absorbance at 517 nm for DPPH∙ and 414 nm for ABTS∙+ as determined by linear regression analysis [0.3, which is the reducing activity presented by the sample concentration at 0.3 of the absorbance value at 700 nm. Low EC50 and RP values signify high antioxidant activity [∙ and ABTS∙+ scavenging activity of butylated hydroxytoluene (BHT) as the control were 121.85 ± 0.39 mg/L and 93.85 ± 0.43 mg/L, respectively. The EC0.3 value of BHT was 26.71 ± 0.69 mg/L. The antioxidant capacity values represented in terms of the DPPH∙ and ABTS∙+ scavenging activity and RP value were lower than those of BHT (P < 0.05).Antioxidant capacity can be evaluated by using a number of ten used . In thist 517 nm . In the onitored . The RP onitored . The assanalysis . RP can activity . The DPPE. supina was evaluated for human hepatocellular carcinoma Hep3B cells by MTT assay. The assay is a colorimetric method that measures cancer cell viability, quantifying the activity of the mitochondrial enzyme that reduces the yellow MTT molecule to purple formazan [50 value was 500 mg/L. After treatment with the polyphenol mixtures for 24 h, the morphological changes such as loss of cell adhesion and floating cell debris were also observed, as shown in The anticancer activity of the polyphenol mixture of Korean formazan . The celE. supina were profiled using a single HPLC–MS/MS run. The antioxidant activities of the flavonoids were evaluated in terms of DPPH∙ and ABTS∙+ scavenging activities, and the reducing power showed a dose-dependent increase. Suppression of cell viability was observed at polyphenol mixture concentrations >250 mg/L.Nine polyphenols from the Korean


Ruditapes philippinarum is one of the most important commercial bivalve species consumed in China. Evaluated metal burden in bivalve molluscs can pose potential risks to public health as a result of their frequent consumption. In this study, concentrations of 10 trace elements  were determined in samples of the bivalve Ruditapes philippinarum, collected from nine mariculture zones along the coast of China between November and December in 2010, in order to evaluate the status of elemental metal pollution in these areas. Also, a public health risk assessment was untaken to assess the potential risks associated with the consumption of clams. The ranges of concentrations found for Cu, Zn, Mn, Se, Ni, Cd, Cr, Pb, Hg and As in R. philippinarum were 12.1–38.0, 49.5–168.3, 42.0–68.0, 4.19–8.71, 4.76–14.32, 0.41–1.11, 0.94–4.74, 0.32–2.59, 0.03–0.23 and 0.46–11.95 mg·kg−1 dry weight, respectively. Clear spatial variations were found for Cu, Zn, Cr, Pb, Hg and As, whereas Mn, Se, Ni, and Cd did not show significant spatial variation. Hotspots of trace element contamination in R. philippinarum can be found along the coast of China, from the north to the south, especially in the Bohai and Yellow Seas. Based on a 58.1 kg individual consuming 29 g of bivalve molluscs per day, the values of the estimated daily intake (EDI) of trace elements analyzed were significantly lower than the values of the accepted daily intake (ADI) established by Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives (JFAO/WHO) and the guidelines of the reference does (RfD) established by the United States Environmental Protection Agency (USEPA). Additionally, the risk of trace elements to humans through R. philippinarum consumption was also assessed. The calculated hazard quotients (HQ) of all trace elements were less than 1. Consequently, there was no obvious public risk from the intake of these trace elements through R. philippinarum consumption.The Manila clam  The rapid development of industry and agriculture has been coupled with increasing environmental pollution, especially in developing countries . As a coe-wastes recycling are the main sources contributing to metal pollution in estuarine and coastal waters in China [Among these harmful pollutants, much attention has been focused on heavy metal pollution in the estuarine and coastal waters ,8,9. Heain China . In recein China ,15,16. CRuditapes philippinarum is one of the most important commercial bivalve species consumed in China, and is an important seafood for humans due to its high nutritional value and delicate flavor. It is widely distributed in estuarine and coastal waters and has been cultivated for more than 50 years in China. Clam farming, particularly along the northern coast of China, has become an important component of the marine fishery industry [The Manila clam industry . The proindustry . Furtherindustry ,20,21. Aindustry .This study represents a preliminary investigation of levels of ten trace elements  in Manila clam samples collected along the coast of China. The primary purpose of our study was to examine whether this bivalve species was contaminated with these selected trace elements. Potential human health risk assessment was then conducted to evaluate whether it presented any potential hazard to humans as a result of consumption. From a public health point of view, our study will provide consumers with better knowledge of public health in connection with seafood consumption.Manila clams were collected from nine sampling sites along the coast of China between November and December in 2010 .These collected samples were transferred to the Biological Laboratory of Dalian Ocean University and cultured in filtered seawater for 24 h depuration. 10 individuals from each site, with similar size, weight and shape, were pooled to prepare a composite specimen in order to minimize individual variations in trace elements determined. Differences in the shell length and wet weight of samples among all sampling sites were not statically significant. Three replicates were performed at each sampling site. The whole soft tissues of clams were dissected with a plastic knife, rinsed five times with deionized water , freeze-dried, homogenized and then stored in polyethylene bags at −20 °C until future analysis.3 (65%) and 2 mL of H2O2 (30%) in a microwave digestion system  for 30 min. After cooling, the resultant solution was diluted to 50 mL with deionized water. Concentrations of Cu, Zn, Mn, Se, Ni, Cd, Cr and Pb were determined by inductively coupled plasma atomic emission spectrometer , while Hg and As were determined by atomic fluorescence spectrometer . Standard reference materials  were used for quality assurance and quality control procedures. The results showed good agreement between the certified and the analytical values. All trace elements concentrations were given on a dry weight basis (mg·kg−1 dry weight).Approximated 0.5 g of dry sample was digested with 6 mL of HNOet al. [bivalveC = average trace metal concentrations in R. philippinarum (mg·kg−1 wet weight), bivalvedc = daily bivalve consumption (g·day−1) per capita for the Chinese population as recorded by the FAO [Wb = average body weight (kg) of the target population [The potential human health risk assessment was conducted by considering the following parameters according to Onsanit et al. . The estet al. , and theet al. . The EDI the FAO , and bW pulation .The hazard quotient (HQ) was calculated by dividing the EDI by the established RfD to assess human risk associated with bivalve consumption. There would be no obvious hazard if the value of HQ was less than 1. The HQ was calculated using the following equation:Trace element concentrations in clams collected from sampling sites along the coast of China are shown in R. philippinarum, with the lowest level, of 49.5 mg·kg−1, being found in Ningbo and the highest level, of 168.3 mg·kg−1, occurring in Qinghuangdao. The minimum and maximum Mn levels were found to be 45.0 mg·kg−1 in Xiamen, and 68.0 mg·kg−1 in Dalian, respectively, while the lowest and highest Cu levels were 12.1 mg·kg−1 in Beihai, and 38.0 mg·kg−1 in Qinghuangdao, respectively. The lowest and highest levels of Se were, respectively, 4.19 mg·kg−1 in Dalian, and 8.71 mg·kg−1 in Shenzheng. Ni and Cr have been identified as potential essential metals. The lowest and highest levels of Ni were 4.76 mg·kg−1 in Fuzhou, and 14.32 mg·kg−1 in Qingdao, respectively, while the minimum and maximum Cr levels were found to be 1.12 mg·kg−1 in Shenzheng, and 4.74 mg·kg−1 in Qingdao, respectively.Zn, Mn, Cu, and Se are well-known essential trace elements for all living organisms. As shown in −1 in Shenzheng, and 9.66 mg·kg−1 in Qingdao. The lowest and highest Cd levels were 0.64 mg·kg−1 in Qinghuangdao and 1.11 mg·kg−1 in Xiamen, respectively. The minimum and maximum Pb levels were found to be 0.44 mg·kg−1 in Shenzheng, and 2.59 mg·kg−1 in Fuzhou, respectively, while the lowest and highest Hg levels were, respectively, 0.05 mg·kg−1 in Shenzheng, and 0.23 mg·kg−1 in Qinghuangdao.As, Cd, Pb and Hg have been classified as potentially toxic elements due to their persistence, high bioavailability, and high toxicity. The lowest and highest As levels in clams were, respectively, 0.62 mg·kgR. philippinarum consumption by the Chinese population. JFAO/WHO have set a PTWI of Cu as 3.5 mg·kg−1 bw, a PTWI of Zn as 1.5 mg·kg−1 bw, a PTWI of Mn as 0.98 mg·kg−1 bw, a PTWI of Se as 0.035 mg·kg−1 bw, a PTWI of Cd as 0.007 mg·kg−1 bw, a PTWI of Pb as 0.025 mg·kg−1 bw, a PTWI of Hg as 0.005 mg·kg−1 bw and a PTWI of As as 0.015 mg·kg−1 bw, respectively. Thus the values of ADI (mg·kg−1 bw·day−1) calculated from PTWI were 0.5 for Cu, 0.21 for Zn, 0.14 for Mn, 0.005 for Se, 0.001 for Cd, 0.0036 for Pb, 0.0007 for Hg and 0.0021 for As, respectively. An average weight of 58.1 kg was assumed for a Chinese person based on statistics from 158,666 Chinese people from all provinces [The mean trace element concentrations were used to estimate the daily intake of trace elements through rovinces . On averrovinces . The toxrovinces . About 1rovinces . Thus onR. philippinarum, from the north to the south, by splitting the Chinese coastal areas into four subsections—Bohai Sea, Yellow Sea, East China Sea, and South China Sea  of  the FAO , the ave the FAO . In our


Paeonia lactiflora Pall.) and were followed in three cultivars over different developmental stages as measured by high performance liquid chromatography (HPLC). OA and UA levels in leaves and stems demonstrated an overall upward trend from May 1 to September 15 except for UA in the leaves of “Hong Feng”. The maximum values of OA and UA in leaves of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” were 852.98, 575.60, 290.48 μg/g FW and 924.94, 827.36, 432.67 μg/g FW, respectively. The maximum values of OA and UA in stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” were 359.28, 90.49, 43.90 μg/g FW and 326.86, 82.25, 56.63 μg/g FW, respectively. OA and UA contents in leaves of 47 different herbaceous peony cultivars ranged from 66.73–618.12 and 36.23–665.14 μg/g FW, respectively, with average values of 171.62 and 227.57 μg/g FW, respectively. The results suggested that the aboveground parts of herbaceous peony may be used as an alternative source of OA and UA for medicinal purposes in addition to its ornamental purposes.Oleanolic acid (OA) and ursolic acid (UA) have been proven to possess many biological activities, and much attention is focused on the search for plants which are rich in OA and UA. In this report, the OA and UA accumulation characteristics were investigated in 47 cultivars of Chinese herbaceous peony ( Paeonia lactiflora Pall.), belonging to the Paeoniaceae family [Herbaceous peony  and ursolic acid (UA), which are usually widespread within the plant kingdom in the form of free state or glycosides. Many reports have shown that OA and UA mainly exist in sinensis , Crataegnatifida , Diospyrros kaki ,19, Eriojaponica ,21, Eucaglobules , Ligustr lucidum , Macrocaicinalis , Zizyphus jujuba , and oths jujuba , anti-dis jujuba , anti-ins jujuba , anti-tus jujuba  and hepas jujuba . Previous jujuba ,12,14,31In this study, the OA and UA accumulation characteristics were investigated in 47 cultivars of Chinese herbaceous peony and specifically followed in three cultivars over different developmental stages based on high performance liquid chromatography (HPLC). The results may provide a theoretical basis for herbaceous peony as an alternative source of OA and UA.Herbaceous peony is usually divided into medicinal and ornamental types. Medicinal herbaceous peony is cultivated mainly for harvesting roots, while ornamental herbaceous peony is planted as cut flowers or pot plants. Therefore, investigation of OA and UA contents in herbaceous peony leaves and stems at different developmental stages, and determination of a reasonable harvest time, will be of great significance in ensuring comprehensive utilization of these resources.The tendency for OA and UA in the leaves to change at different developmental stages is shown in The changes of OA and UA contents in the leaves of herbaceous peony were generally similar among the three cultivars. The overall trend was upward from May 1 to September 15 with a small trough on July 15 for “Yangfei Chu Yu” and “Fen Zhu Pan”. For “Hong Feng”, the small trough of OA and UA in the leaves appeared earlier than the two other cultivars on July 1, with very little change in OA content, and by September 15, OA content maintained a relatively stable level, while the UA level even decreased.The OA content in leaves of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” was lowest on May 1, with 9.66, 2.76 and 0.94 μg/g FW, respectively. It reached the highest peaks on September 15, with 852.98, 575.60 and 290.48 μg/g FW, respectively, which was 88.30, 208.55 and 309.02 fold of the lowest value, respectively. The UA content in the leaves of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” was also lowest on May 1, with 109.91, 93.30 and 51.96 μg/g FW, respectively. The highest UA content in leaves of “Yangfei Chu Yu” and “Fen Zhu Pan” appeared on September 15, with 924.94 and 827.36 μg/g FW, respectively, while those of “Hong Feng” reached the highest peak on September 1 with 432.76 μg/g FW. The highest UA content in leaves of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” was 8.42, 8.87 and 8.33 fold of the lowest level, respectively.The ratio of UA to OA in the leaves obviously changed at the different developmental stages. For instance, the UA content in leaves of “Yangfei Chu Yu” was significantly higher (3.5–11.3 fold) than OA in the first three sampling times (May 1 to June 1) and then the ratio of UA to OA decreased to 1.1–2.1 fold, except for the samples on July 15 .The OA and UA contents in stems of the three cultivars significantly changed at different developmental stages as shown in The OA content in stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” was lowest on May 1, with 1.85, 1.89 and 0.25 μg/g FW, respectively. It reached the highest peaks on September 15, with 359.28, 90.49, and 43.90 μg/g FW, respectively, which was 194.21, 47.88 and 175.60 fold of the lowest value, respectively. The UA content in stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” was also lowest on May 1, with 2.14, 8.88 and 0.49 μg/g FW, respectively. The highest UA content in stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” appeared on September 15, with 326.86, 82.25 and 56.63μg/g FW, respectively, which was 152.74, 9.26 and 115.57 fold of the lowest level, respectively.Very little difference in both OA and UA content was observed in the stems as compared with leaves. For instance, the ratio of UA to OA in stems in “Yangfei Chu Yu” was 0.9–2.6 from May 1 to September 15 .Great differences existed between the OA and UA contents of leaves of 47 investigated herbaceous peony cultivars. The contents of OA and UA were 54.26–618.12  and 36.23–665.14  μg/g FW, respectively. The highest contents of OA and UA in leaves were 11.39 and 18.34 fold of the lowest levels, respectively. Among 47 cultivars, “Hong Xiu Qiu” and “Yangfei Chu Yu” contained the highest OA and UA contents, with 618.12 and 665.14 μg/g FW, respectively, while the lowest OA and UA contents were observed in “Fen Zi Lu Jin” and “Jiangshan Ru Hua”, with 54.26 and 36.23 μg/g FW, respectively .The UA content in leaves was generally higher than OA with some exceptions among the 47 cultivars. OA content was mainly concentrated between 100–200 μg/g FW, while the UA content was more than 200 μg/g FW in over half the cultivars. The ratio of UA to OA in leaves of the different herbaceous peony cultivars ranged from 0.52–3.72, with an average value of 1.43.Similarly, the total OA and UA content in leaves of the different cultivars also largely differed with a range of 102.60–1251.38 μg/g FW . From thet al. [Forsythia suspensa (0.5490% and 0.406%) were significantly higher than those in young (0.3805% and 0.3275%) and mature fruits (0.417% and 0.346%). Our study indicated that OA and UA contents of old leaves and stems of herbaceous peony were also higher than those of young leaves and stems. Therefore, a mode of OA and UA content change may exist in plants such that the contents of OA and UA in old organs are higher than those in young ones during development.Lu et al.  found thet al.  reportedet al. [et al. [The contents of OA and UA in the same part of different cultivars differed in varied degrees. Wang et al.  determin [et al.  demonstrThere were some reports about the relationship between flower color and the OA and UA contents in plants. Huang  found siCallicarpa nudiflora leaves [Pterocephalus hookerie underground and aboveground parts [Hedyotis diffusa whole plant [Fructus chaenomeles fruits [Ligustrum lucidum fruits [OA and UA are widely distributed in plants with great variations between different families, genera and species. The results of our experiment showed that the contents of OA and UA in herbaceous peony leaves were in the range of 54.26–618.12 μg/g (0.0054%–0.0618% FW) and 36.32–633.26 μg/g (0.0036%–0.0633%) based on fresh weight, respectively. Considering the roughly 90% water content of plant leaves, OA and UA contents in dry leaves would increase ten-fold over that in fresh samples, with about 0.054%–0.618% and 0.036%–0.633%, respectively; these levels are higher than or similar to those of nd parts , Hedyotile plant , Fructuss fruits , and Ligm fruits .2O) was obtained with a Milli-Q water purification system from Millipore . All other reagents used in the present study were of analytical grade.OA  and UA  standards were purchased from Nanjing TCM Institute of Chinese Materia Medica . HPLC grade methanol was obtained from Caledon Laboratories Co. . The deionized distilled water  was planted in the Germplasm Nursery of Herbaceous Peony at College of Horticulture and Plant Protection, Yangzhou University, Jiangsu Province, China . The leaves and stems of “Yangfei Chu Yu”, “Fen Zhu Pan” and “Hong Feng” were picked every 15 days from May 1 to September 15, 2009. The leaves of 47 cultivars were collected on September 1, 2009. All the samples were frozen in liquid nitrogen immediately after taking back to laboratory, and then stored in refrigerator at −20 °C until analysis.Herbaceous peony . The sample was centrifuged at 8000 g for 10 min, and then the filtrate was collected. Precipitates were extracted again and both filtrates were pooled and evaporated to dryness at 35 °C. The residue was dissolved in 1.5 mL methanol and transferred to a 2 mL tube. The crude extract was filtered through a 0.22 μm micro-filter before HPLC analysis. Each sample was repeated in triplicate.et al. [Quantifications of OA and UA were performed on a Waters-2695 HPLC system  equipped with Waters 2695 pump and 2487 UV detector according to the method of Zhou et al.  with somAll data are means of three replicates with standard deviations. Microsoft Excel  was used to calculate standard deviations.Traditional cultivation of both medicinal and ornamental herbaceous peony often causes a serious waste of resources due to discarding the aboveground biomasses. For medicinal purpose, herbaceous peony roots are usually harvested in August or September. During the same period, the aboveground parts of ornamental herbaceous peony will complete assimilate transportation and start to wilt in August to September. The OA and UA contents in the aboveground parts, mainly including the stems and leaves, reached a higher level from August to September. Therefore, the aboveground parts of herbaceous peony may be used as an alternative medicinal source of OA and UA in addition to its ornamental purposes.