Eight japonica rice varieties with significant differences in various eating quality parameters such as palatability and protein content were used in this experiment. Association analysis between nucleotide polymorphism and eating quality showed that S12 and S13 loci in SBE1, S55 in SSS1, S58 in SSS2A were significantly associated with apparent amylose content, alkali digestion value, setback viscosity, consistency viscosity, pasting temperature, which explained most of the variation in apparent amylose content, setback viscosity, and consistency viscosity; and explained almost all variations in alkali digestion value and pasting temperature. Thirty-five SNPs and insertion-deletions from SBE1, SBE3, GBSS1, SSS1, and SSS2A differentiated high or intermediate palatability rice varieties from low palatability rice varieties. Correlation analysis between enzyme activities and eating quality properties revealed that SBE25 and SSS15/W15 were positively correlated with palatability, whereas GBSS10 and GBSS15 were negatively correlated. Gene expressions showed that SBE1 and SBE3 expressions in high palatability varieties tended to be higher than middle and low palatability varieties. Collectively, SBE1, SBE3, SSS1, and SSS2A, especially SBE1 and SBE3 could improve eating quality, but GBSS1 decreased eating quality. The results indicated the possibility of developing high palatability cultivars through modification of key genes related to japonica rice eating quality formation in starch biosynthesis.The composition of amylopectin is the determinant of rice eating quality under certain threshold of protein content and the ratio of amylose and amylopectin. In molecular biology level, the fine structure of amylopectin is determined by relative activities of starch branching enzyme (SBE), granule-bound starch synthase (GBSS), and soluble starch synthase (SSS) in rice grain under the same ADP-Glucose level. But the underlying mechanism of eating quality in molecular biology level remains unclear. This paper reports the differences on major parameters such as SNP and insertion-deletion sites, RNA expressions, and enzyme activities associated with eating quality of  Oryza sativa L.) is the staple food for more than half of the world's population. Each year many new rice varieties are being developed and released with high yield, and high resistance to biotic and abiotic stresses, but their eating quality remains to be improved. Eating quality has now become the primary consideration of final rice-eating consumers and breeding programs. Cultivars with better eating quality are also required for food industry and rice seed producers. Although indica rice varieties are widely planted worldwide, consumers in Northeast Asia preferred japonica rice. Also, japonica rice is becoming more and more popular with worldwide consumers due to its moderate elasticity and stickiness Rice , soluble starch synthase (SSS), granule-bound starch synthase (GBSS), starch branching enzyme (SBE), and starch debranching enzyme (DBE) Waxy (GBSS1) and ALK (SSS2A) are central in determining rice eating and cooking quality by affecting amylose content, gel consistency and gelatinization temperature Waxy functions as the sole major gene for both amylose content and gel consistency but as a minor gene affecting gelatinization temperature. ALK is the sole major gene controlling gelatinization temperature but as a minor gene affecting amylose content and gel consistency. The two genes affect two properties simultaneously: both ISA and SBE3 affect gel consistency and gelatinization temperature. Furthermore, several minor genes are specific for each property: SSS3A, AGPlar (AGP large subunit), PUL, and SSS1 for amylose content, AGPiso (AGP large subunit isoform) for gel consistency, and SSS4B for gelatinization temperature. The correlations among amylose content, gel consistency, and gelatinization temperature were caused by the joint action of these associated genes.The enzymes and genes involved in fine structure of amylopectin play distinct roles, but jointly comprise of the genetic dissection of rice eating quality. Using 70 rice varieties, association analysis between 18 genes related to starch synthesis and amylose content, gel consistency, gelatinization temperature showed that SSS2A of indica cultivars IR36 and Kasalath were replaced by Asp-88 (gac) and Ser-604 (agc), respectively, in both japonica cultivars Nipponbare and Kinmaze SSS2A whereas Val-737 (gtg) and Leu-781 (ctc) in indica SSS2A were replaced by Met-737 (atg) in Nipponbare and Phe-781 (ttc) in Kinmaze SSS2A. These results confirmed that the SSS2A activity determines the type of amylopectin structure of rice starch to be either the typical indica type or japonica type, by playing a specific role in the synthesis of the long B1 chains by elongating short A and B1 chains, notwithstanding the presence of functional two additional SSS2 genes, a single SSS1 gene, two SSS3 genes, and two SSS4 genes in rice plants SSS2A were found in 30 rice varieties and GC/TT polymorphism could differentiate rice varieties with high or intermediate GT from those with low GT at a rate of about 90% correct prediction for 509 varieties In the analysis of sequence variation, Glu-88 (gag) and Gly-604 (ggc) in Activities of AGP at all rice grain filling stages and activities of SBE at the early and middle grain filling stages were not correlated with rice eating quality components including gel consistency, alkali spreading value, and amylose content. SSS activity at the early grain filling stage was negatively correlated with gel consistency and alkali spreading value, and positively correlated with amylose content. Activities of SSS at middle and late grain filling stages and SBE at the late grain filling stage were positively correlated with gel consistency and alkali spreading value, and negatively correlated with amylose content SBE1, SBE3, GBSS1, SSS1, and SSS2A and rice eating quality, between activities of SBE, GBSS1, and SSS at different grain filling stages and rice eating quality, between gene expressions of SBE1 and SBE3 at different grain filling stages and rice eating quality, and to explore the molecular biology aspect of good eating quality formation in japonica rice which provide the basic ideas for breeding good eating quality japonica rice.In this study, our aim was to find the relationships between nucleotide sequence variations of japonica rice instead of sensory test japonica rice varieties were summarized in Palatability from Toyo taste meter served as an important and direct index in estimating eating quality of All traits showed significant differences among varieties and exhibited a wide range of variation, especially palatability (P) with 49.7–70.4, apparent amylose content (AAC) of 14.35–18.67, and protein content (PC) of 5.78–8.54 . The palCorrelation analysis revealed that palatability was negatively correlated with protein content . HoweverSBE1, 10,287 bp for SBE3, 3,444 bp for GBSS1, 6,914 bp for SSS1, and 2,314 bp for SSS2A  products of these genes were sequenced as follows: 8,756 bp for or SSS2A . SequencSBE1 gene, the S6, S8, S12, S13, S16, S18 and S19 sites “AGGGTG-”were the same in Gopum, Koshihikari, Ilpum, and Samgwang, but they were partially replaced by “GAAACAT” in Palgong, Samnam, Singeumo, and Dobong. The S1, S10, S15, and S17 sites “CAT-” were the same in Palgong, Samnam, Singeumo, and Dobong, but they were partially replaced by “TGGG/A” in Gopum, Koshihikari, Ilpum, and Samgwang. For SBE3 gene, the S23, S25–S29, S36, S42, S43, S45, S46 sites “CATGTTAAGCC” were same in Palgong, Samnam, Singeumo, and Dobong, but they were partially replaced by “ACCTG-TG–A” in Gopum, Koshihikari, Ilpum, and Samgwang. Though we detected sequences of GBSS1, SSS1, and SSS2A in some SNP/insertion-deletion sites, we did not find strong evidence as to differences related to eating quality compared to SBE1 and SBE3 in all japonica rice varieties. It indicated that the “AGGGTTG-” linkage sites may be advantageous for high eating quality formation, but the “CATGTTAAGCC” linkage sites may be disadvantageous for high eating quality formation in japonica rice varieties.For SBE1, S55 in SSS1, S58 in SSS2A were significantly associated with apparent amylose content, alkali digestion value, setback viscosity (SBV), consistency viscosity (CTV), and pasting temperature (PT), which explained most of the variation in apparent amylose content, setback viscosity, and consistency viscosity, and almost all variations in alkali digestion value and pasting temperature (2R = 0.99)  and consistency viscosity at P<0.05, which explained about 60% of total variation. S3, S4, and S5 in SBE1 were significantly associated with breakdown viscosity (BDV), indicating that the breakdown viscosity was partially controlled by SBE1. However, palatability was not significantly associated with any single SNP or insertion-deletion locus detected.Association analysis between eating quality properties and SNPs/insertion-deletions showed that S12 and S13 loci in  = 0.99) . S40 in SBE1, SBE3, GBSS1, SSS1, and SSS2A  in developing rice grains were presented in For starch branching enzyme, the peak values of enzyme activity were formed during 10–15 days after flowering in six varieties except for Palgong and Samnam. The activities of SBE at 25 days after flowering were higher in Gopum, Koshihikari, Ilpum, and Samgwang than those in Palgong, Samnam, Singeumo, and Dobong. For GBSS1, the peak values of enzyme activity were formed during 15–20 days after flowering. For soluble starch synthase, the peak values of enzyme activity were showed at 10 days after flowering. The differences were existed among eight varieties, but there were no evident differences for SSS activities between high and inferior palatability groups. In GBSS1, Singeumo and Dobong showed much higher activities than the other varieties during grain filling, but unexpectedly high during 10–15 days after flowering. Also, Palgong was high during 20–25 days after flowering. This implies that the high enzyme activity of GBSS1 was reflected to low palatability based on the negative correlation between GBSS1 activity and palatability .Correlation analyses revealed that SBE25, GBSS10, GBSS15, and SSS15/W15 were significantly correlated with palatability . Among tjaponica varieties, the mRNA expressions of SBE1 and SBE3 tended to increase gradually to peak values, thereafter descended during late grain filling period , middle , and low  palatability. T-tests showed that there were significant differences among high, middle, and low palatability groups. Alkali digestion value and cool paste viscosity were significantly different between middle and low.Since starch branching enzyme was important determinant of eating quality by association analysis, real –time RT-PCR of SBE1 and SBE3 was carried out to elucidate the effect of gene expression to eating quality , respectively (SBE1 mRNA expression at 5 days after flowering (SBE1-5) and SBE3-5 expressions were significantly different between high and low, and between middle and low groups. SBE1-15 and SBE3-15 expressions were significantly different between high and middle groups, and between high and low groups. SBE3-20 expression was significantly different between high and low groups. SBE3-25 expression was significantly different between high and low groups, and between middle and low groups at P<0.05.The differences of ectively . SBE1 mRSBE1 and SBE3 involving SBE1-15 and SBE3-15 played an important role in palatability difference between high and middle groups. Interestingly, mRNA expressions of SBE1-5, SBE1-15, SBE3-5, SBE3-15, SBE3-20, and SBE3-25 contributed a lot to the palatability variation between high and low groups. Among them, SBE1-5, SBE3-5, and SBE3-25 were important in palatability differentiation between middle and low groups. These results suggested that mRNA expressions of SBE1 and SBE3 were important to form good eating quality in japonica varieties, especially SBE1 and SBE3 expressions at both 5 and 15 days after flowering. SBE1-5, SBE3-5, and SBE3-25 also played an important role in alkali digestion value and cool paste viscosity variations between middle and low palatability groups.Correlation analysis between eating quality properties and mRNA expressions of Oryza sativa L.) is the most important staple food of East Asia and the most densely populated landscapes of China, Korea, Japan, Southeast Asia, and South Asia japonica and indica. Molecular and archaeobotanical evidences have shown that japonica and indica rice originated from distinct gene pools in the wild ancestor Oryza rufipogonjaponica rice Rice  as described by Cho et al. SBE1 (LOC_Os06g51084), SBE3 (LOC_Os02g32660), GBSS1 (LOC_Os06g04200), SSS1 (LOC_Os06g06560), and SSS2A (LOC_Os06g12450) were obtained by searching the Gramene database (http://www.gramene.org). Using genomic DNA sequences, primers were designed with Primer3 online software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi), and primer analysis was carried out using Oligo 6 software. The primers used for sequencing were listed in http://clustalw.ddbj.nig.ac.jp/).The nucleotide sequences of 20 primer and SuperScript™ III Reverse Transcriptase .Total RNAs were isolated from rice grains using the RNeasy mini kit (Qiagen) according to the manufacturer's instruction with some modifications. 100 mg of frozen ground seed powder was placed into cold 1.5 ml tube, and then 600 µl of RLC buffer was added. After immediately rapid and strong vortex, centrifugation was carried out at 13,000 rpm for 2 min. The supernatant was rapidly transferred into lilac spin column, and then centrifuged at 12,000 rpm for 2 min. 280 µl of 100% ethanol was added into the flowthrough, mixed 4–5 times by pipetting, and then the sample was transferred into pink RNeasy spin column. After centrifugation at 10,000 rpm for 1 min, 700 µl of RW1 buffer was added into the pink spin column. Centrifugation was carried out at 10,000 rpm for 1 min, and then 500 µl of RPE buffer was used to wash RNA twice at 10,000 rpm for 1 min. After centrifugation for 2 min at 10,000 rpm, 50 µl of RNase-free water was added into the pink spin column in 1.5 ml tube, and centrifuged again at 10,000 rpm for 1 min. The RNA concentration was detected in Nanodrop ND-1000 spectrophotometer , and stored at −80°C freezer. Total RNAs were cleaned using DNase 1 kit , and the first-strand cDNA synthesis was performed by reverse transcription of mRNA using Oligo(dT)SBE1 and SBE3 were blasted at NCBI/BlAST website, and the clustalw  of amino acid sequences from 15 species was carried out, and conserved domains of these amino acid sequences were searched. Using cDNA sequences responsible for the conserved domains of proteins, primers were designed with Primer3 online software, and primer analysis was carried out using Oligo 6 software. Quantitative real-time RT-PCR was performed on the DNA Engine Opticon 2 instrument  in 20 µl reaction volume containing SYBR Green, a fluorophore which binds to all double-strand DNA . The primers used for real-time RT-PCR were shown in Actin-1.The deduced amino acid sequences of 2, 50 mM 2-mercaptoethanol, 12.5% (v/v) glycerol) contained in a precooled mortar with a pestle on ice. The homogenate was transferred into a precooled 40 ml tube, and 6 ml of extraction buffer was added for two times to clean the mortar. 40 ml tubes were centrifuged at 15,000 rpm at 2°C for 20 min, and supernatants were filtered through a filter paper . The filtrates were used for enzyme preparations.Enzyme activity of starch branching enzyme including SBE1, SBE3, and SBE4 together in rice grains was assayed as described by Yamanouchi and Nakamura 2) was added into the solution, and then incubated at 30°C for 30 min in the dark. The SBE activity was determined by measuring the absorbance at 540 nm in UV spectrophotometer (Shimadzu). The controls were prepared as follows: 100 µl of enzyme preparation was added into 50 µl 1 M HCl, mixed well, and 100 µl of cold reaction buffer was added into the mixture. The other steps were same as reaction samples. The blank solution was made using 100 µl of cold extraction buffer to replace 100 µl enzyme preparation and the other steps were same as reaction samples. Three replications were carried out in this experiment.Starch branching enzyme was assayed by monitoring stimulation of α-glucan synthesis from glucose-1-P by rabbit muscle phosphorylase a R×VT/(T×N×d×VS×VS); ΔA, absorbance of samples at 30 min minus absorbance of samples at 0 min; T, time (minute); N, grain number; d, light path in cm; 100, 0.01 optical density as 1 unit which has similar function as ε or %E; VR, reaction volume (enzyme sample volume + working reagent volume + stop solution volume) (ml); VS, enzyme sample volume (ml); VT, total enzyme sample volume (ml). So SBE activity = ΔA×100×1.45×10/(30×15×1×0.1×0.1) = 322.22×ΔA (Units grain−1 min−1).The starch branching enzyme activity was calculated as follows: SBE activity  = ΔA×100×VGBSS1 and SSS enzyme extractions were conducted as described by Umemoto and Terashima 2, and 1.5 units of pyruvate kinase), and incubated at 30°C for 30 min. The ADP produced by the starch synthase reaction was conversed to ATP and the resulting solution was heated in a boiling-water bath for 30 s, and incubated on ice for 10 min, and then subjected to centrifugation at 15,000 rpm and 2°C for 15 min. The supernatant (0.3 ml) was mixed with 0.3 ml of solution C . The GBSS1 activity was measured as the increase in absorbance of 340 nm after the addition of 1 µl each of hexokinase (1.4 units) and G-6-P dehydrogenase (0.35 units) at 25°C for 30 min.The activity of GBSS1 was assayed as described by Nakamura et al. The controls were prepared as follows: 0.15 ml of GBSS1 enzyme preparation was inactivated in a boiling-water bath for 30 s, and incubated on ice for 10 min, and then solution A was added. The other steps were same as reaction samples. The blank solution was made using 0.15 ml of cold extraction buffer to replace 0.15 ml GBSS1 preparation, and the other steps were same as reaction samples. Three replications were carried out in this experiment.R×VT/(T×N×d×VS×VS); ΔA, absorbance of samples at 30 min minus absorbance of samples at 0 min; T, time (minute); N, grain number; d, light path in cm; 100, 0.01 optical density as 1 unit which has similar function as ε or %E; VR, reaction volume (enzyme sample volume + working reagent volume + stop solution volume) (ml); VS, enzyme sample volume (ml); VT, total enzyme sample volume (ml). So GBSS1 activity = ΔA×100×0.602×3/(60×15×0.15×3/4×0.15×3/4) = 15.86×ΔA (Units grain−1 min−1).The enzyme activity of granule-bound starch synthase 1 was calculated as follows: GBSS1 activity = ΔA×100×V2, and 10 µl of 5 mg ml−1 creatine phosphokinase ), and then incubated at 30°C for 30 min. The ADP produced by the soluble starch synthase reaction was conversed to ATP, and the resulting solution was heated in a boiling-water bath for 30 s, and incubated on ice for 10 min, and then subjected to centrifugation at 15,000 rpm and 2°C for 15 min. The supernatant (0.3 ml) was mixed with 0.3 ml of buffer C . The SSS activity was measured as the increase in absorbance of 340 nm after the addition of 1.1 µl of hexokinase  and 1.5 µl of G-6-P dehydrogenase  at 25°C for 30 min.The enzyme activity of SSS was assayed as described by Nishi et al. The controls were prepared as follows: 0.15 ml of soluble starch synthase preparation was inactivated in a boiling-water bath for 30 s, and incubated on ice for 10 min, and then buffer A was added. The other steps were same as reaction samples. The blank solution was made using 0.15 ml of cold extraction buffer to replace 0.15 ml of soluble starch synthase preparation, and the other steps were same as reaction samples. Three replications were carried out in this experiment.R×VT/(T×N×d×VS×VS); ΔA, absorbance of samples at 30 min minus absorbance of samples at 0 min; T, time (minute); N, grain number; d, light path in cm; 100, 0.01 optical density as 1 unit which has similar function as ε or %E; VR, reaction volume (enzyme sample volume + working reagent volume + stop solution volume) (ml); VS, enzyme sample volume (ml); VT, total enzyme sample volume (ml). So SSS activity = ΔA×100×0.6026×3/(20×15×0.15×3/4×0.15×3/4) = 47.61×ΔA (Units grain−1 min−1). SSS10/W10, SSS15/W15, SSS20/W20, and SSS25/W25 were calculated by SSS activities at 10, 15, 20, and 25 days after flowering were divided by 15 grain weight at 10, 15, 20, and 25 days after flowering, respectively.The enzyme activity of soluble starch synthase was calculated as follows: SSS activity = ΔA×100×VFor palatability and physicochemical analyses, rice grains were dehulled and milled to 91% yield. The analyses were performed as described by Lestari et al. RVA pasting properties were detected with a Rapid Visco Analyzer (RVA) according to the manufacture's instruction . Rice starch paste profile was described by seven parameters. They were as follows: peak viscosity (PV), through viscosity or hot paste viscosity (HPV), final viscosity or cool paste viscosity (CPV), breakdown viscosity (BDV = PV-HPV), setback viscosity (SBV = CPV-PV), consistency viscosity (CTV = CPV-HPV), and pasting temperature as described by Bao and Xia http://www.statistix.com/). The general linear model (GLM) was used in the analysis of associations between nucleotide polymorphisms and various rice eating quality parameters, which was performed with Tassel version 2.1 (http://www.maizegenetics.net/tassel). Moreover, phylogenetic analysis was also performed using Tassel 2.1 software.Analysis of variance was performed using SAS version 9.1. Significant differences were determined using t-tests (LSD) for comparison of means. Correlation analyses among rice eating quality parameters and enzyme activities were conducted using Statistix version 8.0 (Table S1Primers used in the sequencing of genes related to rice amylopectin biosynthesis.(DOC)Click here for additional data file.Table S2Primers for SBE1 and SBE3 expressions in developing rice grains.(DOC)Click here for additional data file.Table S3Eating quality parameters for eight japonica rice varieties.(DOC)Click here for additional data file.Table S4Polymorphic sites of genes related to rice amylopectin biosynthesis of eight japonica varieties.(DOC)Click here for additional data file.


The precise function of RecG is poorly understood, although ample evidence shows that it plays critical roles in DNA repair, recombination and replication. We further demonstrate that Mycobacterium tuberculosis RecG (RecGMtb) DNA binding activity had a broad substrate specificity, whereas it only unwound branched-DNA substrates such as Holliday junctions (HJs), replication forks, D-loops and R-loops, with a strong preference for the HJ as a helicase substrate. In addition, RecGMtb preferentially bound relatively long (≥40 nt) ssDNA, exhibiting a higher affinity for the homopolymeric nucleotides poly(dT), poly(dG) and poly(dC) than for poly(dA). RecGMtb helicase activity was supported by hydrolysis of ATP or dATP in the presence of Mg2+, Mn2+, Cu2+ or Fe2+. Like its Escherichia coli orthologue, RecGMtb is also a strictly DNA-dependent ATPase.The RecG enzyme, a superfamily 2 helicase, is present in nearly all bacteria. Here we report for the first time that the  Mycobacterium tuberculosis, the aetiological agent of the re-emerging disease tuberculosis (TB), remains a global health threat, killing at least 1.5 million individuals every year , and D- and R-loops, has been shown to play roles in DNA repair, recombination and replication  for its DNA binding, unwinding and ATPase activities in order to delineate its potential roles in the DNA metabolism of M. tuberculosis.In this study, we have characterized the recombinant RecG enzyme from M. tuberculosis genomic DNA was isolated from a M. tuberculosis H37Rv (ATCC 25618) culture. The M. tuberculosis recG (Rv2973c) gene was PCR-amplified using a forward primer (5′-CGCATATGGCGTCGTTAAGCGATCGGCTC-3′) and reverse primer (5′-CGCTCGAGTCATGACTTATCTAAGTATTCGATGC-3′) which contained NdeI and XhoI restriction sites, respectively (underlined). The PCR product was digested with NdeI and XhoI and ligated into a similarly digested pET28b(+) vector (Novagen). The resulting construct, pET28b–recG with an N-terminal His6-tag, was then transformed into E. coli ER2566 . The nucleotide sequence of the construct was verified by DNA sequencing (ABI).Mtb protein was purified to homogeneity as follows. E. coli ER2566 harbouring pET28b–recG was inoculated into Luria–Bertani broth (Difco) supplemented with 50 µg kanamycin ml−1, 2.5 mM betaine hydrochloride and 0.5 M sorbitol, and grown at 37 °C to OD600 ~0.4. The culture was then transferred to 18 °C and induced at OD600 ~0.6 with 0.5 mM IPTG. After overnight growth, cells were harvested, lysed and purified using a nickel-nitrilotriacetic acid agarose column as described in the QIAexpressionist protocol for native purification of His6-tagged proteins from E. coli , 300 mM NaCl and 1 mM DTT. The N-terminal His6-tag was cleaved off using biotinylated thrombin (Novagen) following the manufacturer’s protocol. Further purification was carried out on a HiTrap Q HP column  after the buffer had been exchanged with 20 mM Bistris (pH 7.2), 100 mM NaCl and 1 mM DTT. Fractions containing pure RecGMtb were pooled and dialysed against storage buffer  and stored at −80 °C until use. RecGMtb protein concentration was determined using the Bradford method (Bio-Rad) using BSA as standard.The recombinant RecG E. coli . β-Merca32P]ATP (PerkinElmer) and T4 PNK enzyme (NEB) for 1 h at 37 °C, followed by enzyme inactivation at 65 °C for 20 min. Unincorporated ATPs were removed using illustra MicroSpin G-25 columns . Labelled and unlabelled complementary oligonucleotides were mixed at a molar ratio of 1 : 2.5 in annealing buffer , denatured at 95 °C for 5 min, and allowed to cool to room temperature for about 3 h. The annealed products were resolved on an 8 % non-denaturing polyacrylamide gel. The bands containing the completely annealed substrates were excised and DNA was eluted into buffer comprising 10 mM Tris/HCl (pH 8.0) and 0.5 mM EDTA by incubating overnight at 4 °C. The concentrations of the eluted DNA substrates were estimated as described by DNA substrates were prepared essentially as described by −1, 6 % (w/v) glycerol, 2 mM DTT, 50 ng poly(dI-dC) µl−1 (Thermo Scientific)] and 0.1 nM of the indicated DNA substrate. Where indicated, poly(dI-dC) was omitted from the binding buffer. Reactions were initiated by adding indicated concentrations of RecGMtb. After 15 min incubation on ice, 4 µl of 60 % (w/v) glycerol was added and immediately loaded onto a pre-cooled and pre-run (30 min) 4 % non-denaturing polyacrylamide gel (29 : 1). Electrophoresis was performed using low-ionic-strength buffer at 200 V for 5 min and at 160 V for an additional 85 min in an ice-water bath with buffer recirculation. Gels were dried, exposed to a phosphorimaging screen, visualized using a phosphorimager  and quantified by ImageQuant TLv 2003.02 software .DNA binding assays were carried out as described by 2, 2 mM ATP, 100 µg BSA ml−1, 2 mM DTT], 0.5 nM DNA substrate and the indicated concentrations of RecGMtb. For divalent metal cofactor studies, MgCl2 in the aforementioned buffer was replaced by 2 mM of the indicated metal chloride. Similarly, in fuel preference studies, 2 mM of the tetrasodium salt of the indicated NTP/dNTP was used in place of ATP in the helicase buffer. Helicase reactions were initiated by adding RecGMtb and, after incubating at 37 °C for 30 min, were terminated with 10 µl of 3× helicase stop solution  containing a 10-fold molar excess of trap oligonucleotide. For helicase time-course assays, the reaction was scaled up to 140 µl and RecGMtb was added into pre-incubated (3 min) reaction mixture at 37 °C. An aliquot (10 µl) of the reaction mixture was withdrawn at the indicated time points and mixed with 5 µl 3× helicase stop solution. All helicase reaction products were resolved by 10 % non-denaturing polyacrylamide (19 : 1) gel electrophoresis at 150 V for 2 h at room temperature using 1× Tris/borate-EDTA buffer. Gels were dried and analysed as described for DNA binding assays. The proportion of helicase substrate unwound (%) was calculated as described by Unless otherwise specified, all helicase assays were conducted in a 20 µl reaction containing helicase buffer ATP, 2 mM DTT] and the indicated DNA cofactor. The reaction was incubated at 37 °C for the indicated times and terminated by adding 5 µl 0.5 M EDTA (pH 8.0). Samples (2 µl) were spotted onto TLC plates  at 1.5 cm intervals and resolved using a solution containing 1 M formic acid and 0.5 M LiCl. The TLC plates were air-dried, exposed to a phosphorimaging screen, imaged and quantified as described above for the DNA binding assays. The proportion of hydrolysed ATP (%) was calculated as {counts for γ-32Pi/(counts for γ-32Pi+counts for [γ-32P]ATP)}×100. The values obtained from samples lacking RecGMtb were subtracted from the samples containing RecGMtb to account for background ATP hydrolysis. An unpaired Student’s t test was used to determine statistical significance.RecG2, 100 µg BSA ml−1, cold ATP, 0.023 nM [γ-32P]ATP, 2 mM DTT], 125 ng plasmid DNA (pET28b) µl−1 and 150 nM RecGMtb. The cold ATP concentration was varied between 100 and 800 µM. The reactions were incubated at 37 °C for 10 min and quenched with 10 µl 0.5 M EDTA (pH 8.0). The concentration of hot ATP was negligible and thus not considered in the calculations. The velocity data points versus cold ATP concentrations were non-linearly fitted to Michaelis–Menten and Hill equations using Prism 5 software (GraphPad).To determine the steady-state kinetic parameters of ATP hydrolysis, a 20 µl reaction was set up with ATPase buffer [20 mM Tris/HCl (pH 7.5), 4 mM MgClE. coli RecG protein sequence was used as a query to search NCBI protein sequence databases , common grape vine (Vitis vinifera), California poplar (Populus trichocarpa) and the lycophyte Selaginella moellendorffii, as well as in green algae such as Ostreococcus tauri and Nannochloris bacillaris. The recG gene was not detected in eukaryote species outside the kingdom Plantae. The plant genes are localized to the nuclear genomes and do not appear to be recently acquired from bacteria. For example, A. thaliana  motif  motif .Mtb bound to a wide variety of model DNA substrates, including partial and complete replication forks, HJ, bubble, and D- and R-loop substrates (Mtb for a linear DNA duplex (49 bp) was very low, and it did not bind DNA substrates containing 20 nt 5′ or 3′ overhangs, in the presence of poly(dI-dC) competitor (Mtb to homopolymeric nucleotides (40 nt) in the absence of poly(dI-dC) competitor, poly(dA) showed very weak binding compared with poly(dC), poly(dG), poly(dT) and random nucleotides (dN) (Mtb to poly(dA) appeared to increase with increasing length of nucleotides as for poly(dT) and poly(dA : dT), yet with less stable protein–DNA complexes .RecGbstrates , Table 2bstrates . In contmpetitor , Table 2des (dN) . HoweverMtb was examined using a variety of DNA substrates, including flayed DNA duplex, and lagging, leading and complete replication fork substrates. RecGMtb did not exert any unwinding activity on flayed DNA duplex, but demonstrated weak and strong unwinding activities on leading and lagging strand replication forks, respectively  of the substrate was converted to flayed duplex by RecGMtb within 1 min , D-loops with 5′ or 3′ tails , D-loops with a hairpin end at the 5′ or 3′ tail , and R-loops with 5′ and 3′ (2′-O-methyl-RNA) tails   . The 2′-Mtb unwound HJ, replication fork and D-loop substrates, and identified HJ as the preferred DNA substrate of RecGMtb helicase, followed by the lagging strand replication fork , 3′- or 5′-tailed DNA duplex , or a bubble substrate . RecGMtb unwound DNA substrates in the presence of ATP or dATP, but was inactive in the presence of other NTP/dNTPs (Mtb unwinding activity was significantly higher in the presence of ATP than dATP (P = 0.023). Furthermore, ADP and the slowly hydrolysable ATP analogue ATPγS did not support the unwinding activity of RecGMtb .The RecG cations . No signTP/dNTPs . RecGMtbMtb was measured in the presence of 50 nM ssDNA (49 nt), dsDNA (49 bp) or an HJ substrate (assembled from four ~49 nt oligos). The efficiency of ATP hydrolysis was 68, 32 and 0 % in the presence of HJ, dsDNA and ssDNA, respectively (The ATPase activity of RecGectively . Moreoveectively . To avoiMtb ATPase activity was performed by titrating ATP in the presence of saturating circular dsDNA (pET28b). Under these conditions, ATP hydrolysis was linear for at least 15 min (data not shown). Michaelis–Menten (hyperbolic) curve-fitting analysis showed that the mean (±se) Vmax, Km and Kcat of RecGMtb were 477 (±27) µM min−1, 202 (±35) µM and 3180 (±59) min−1, respectively. Using the Hill  equation to model the data, the mean (±se) Hill coefficient (H) obtained was 1.0±0.3, which suggests a monomeric state during ATP hydrolysis.Steady-state kinetic analysis of RecGM. tuberculosis genome is susceptible to the effects of genotoxic and general cellular stress, including nitrosative and oxidative damage to DNA, RNA and other biomolecules  of RecG in DNA/nucleic acid metabolism in an intracellular pathogen.The olecules , owing te genome . RecG ise genome , 2002. IMtb binds and unwinds a variety of DNA substrates that mimic intermediates in DNA replication, recombination and repair, like its E. coli orthologue. Among the substrates examined here, RecGMtb had the highest affinity for HJ, while 3′- and 5′-overhang DNA substrates and blunt-end duplex DNA substrates were bound very poorly , and much higher affinity for poly(dT), poly(dG) and poly(dC). Generally, RecGMtb and other superfamily 2 helicases make extensive contact with the sugar-phosphate DNA backbone and this interface is the dominant functional mode of interaction  than for shorter oligomers (20 nt). This suggests that 20 nt/bp DNA substrates are not long enough to form a stable complex with RecGMtb. The relatively more stable binding of RecGMtb ≥40 nt poly(dT) is consistent with the site size determined for E. coli RecG for poly(dT), 36 nt  (as for E. coli RecG) , that its genome has a high G+C content , poly(dG) and poly(dC) than for poly(dA), shed new light on the occurrence and role of RecG in nature. Furthermore, the finding that the preferred helicase substrate for RecGMtb is HJ, a key intermediate in DNA repair, recombination and replication fork restart (Mtb is preferentially involved in such processes in vivo. However, future studies involving M. tuberculosis recG-null mutants are needed to clarify the precise role of RecG in the DNA metabolism, survival, fitness and virulence of M. tuberculosis and possibly of other related mycobacteria.The novel findings presented here that RecG exists in vascular plants and algae, in addition to eubacteria, and that RecG restart , may sug


Objective: To compare the bioavailability of single dose ibuprofen 200 mg and pseudoephedrine hydrochloride 30 mg administered alone or in combination as an oral suspension.Methods: This was a single-center, randomized, single-dose, open-label, 3-period, crossover study. After an overnight fast (≥10 h), 18 healthy male subjects received either ibuprofen 200 mg (reference-A), pseudoephedrine 30 mg (reference-B) or the combination (test-C) as a suspension, on 3 separate visits, with blood sampling up to 36-h post-dose. The primary pharmacokinetic parameters, maximum plasma concentration (Cmax) and area under the plasma concentration–time curve (AUC) from time zero to last measurable concentration (AUC0−t) and extrapolated to infinity (AUC0−∞) were compared by an analysis of variance using log-transformed data. Bioequivalence was concluded if the 90% confidence intervals (CIs) of the adjusted geometric mean (gMean) ratios for Cmax and AUC were within the predetermined range of 80–125%, in accordance with regulatory requirements.Results: For the test formulation, the ibuprofen gMean Cmax was 17.0 μg/mL (vs. 18.1 μg/mL for reference-A), AUC0−t was 57.1 (vs. 60.0 μg·h/mL), and AUC0−∞ was 59.9 μg·h/mL (vs. 63.1 μg·h/mL). The 90% CIs for the ratio (test/reference-A) were 81.0–108.1% for Cmax, 91.5–98.4% for AUC0−t and 91.6–97.9% for AUC0−∞. For pseudoephedrine, the gMean Cmax for the test formulation was 97.2 ng/mL (vs. 98.5 ng/mL for reference-B), AUC0−t was 878.4 (vs. 842.8 ng·h/mL) and AUC0−∞ was 907.8 ng·h/mL (vs. 868.3 ng·h/mL). The 90% CIs for the ratio (test/reference-B) were 92.4–106.9% for Cmax, 97.7–111.0% for AUC0−t and 97.9–111.3% for AUC0−∞. All treatments were well tolerated.Conclusion: This oral suspension containing ibuprofen and pseudoephedrine combined in a new formulation met the regulatory criterion for bioequivalence compared with oral suspensions containing the individual components. The study protocol was approved by an independent ethics committee, and was conducted in accordance with local regulations and the Declaration of Helsinki and Good Clinical Practice. Written informed consent was obtained from all subjects before performing any trial-related activities. The study design was in accordance with the European Medicine Agency note for guidance on the investigation of bioavailability and bioequivalence of fixed combination products , clinically significant medical history, or abnormal ECG, vital signs or laboratory values at screening 21 days before dosing. Additional exclusion criteria included allergy to ibuprofen, pseudoephedrine, or other non-steroidal anti-inflammatory drugs (NSAIDs), history of asthma, nasal polyp or NSAID-induced urticaria or drug or alcohol abuse; subjects who smoked >10 cigarettes per day or could not abstain during the study, who participated in another trial within 90 days, or used any other medication within 14 days of enrolment were also excluded. Subjects were tested for drug and alcohol consumption prior to each study period.Healthy male subjects aged 18–55 years with a body mass index (BMI) of 18–25 kg/mThe study comprised a screening period followed by 3 treatment periods, each separated by a 12-day washout period. Subjects were randomly assigned using a computer-generated list to receive one of 6 possible treatment sequences for single dose ibuprofen , single dose pseudoephedrine hydrochloride  or the combination of ibuprofen 200 mg and pseudoephedrine 30 mg   and 36 h  after dosing. Xanthine-containing food or beverages, alcohol, extreme physical activity, cigarettes and tobacco products were not permitted 48 h before and during post-dose blood sampling.Subjects attended the study center on the evening before dosing. After fasting overnight (≥10 h), subjects were dosed orally (10 mL suspension with 240 mL of water) in the sitting position by study personnel using a syringe. The syringe was then rinsed with water and administered again with any remaining water. Administration was confirmed by a mouth-check. Water was allowed Venous blood samples  for measurement of plasma concentrations of ibuprofen and pseudoephedrine were collected in heparinized vacutainers via an indwelling cannula during each study period before and at 10, 20, 40, 50, and 60 min of the first hour, and thereafter every 20 min for up to 3 h, and 3.5, 4, 6, 8, 10, and 12 h after dosing. Additional samples were collected at 16, 24, 30, and 36 h after dosing with treatments B and C. Samples were centrifuged for 15 min at 10°C (2960 rpm) and plasma aliquots were taken and frozen (−20°C then −60°C) until analysis.Plasma concentrations of ibuprofen and pseudoephedrine were analyzed separately using liquid chromatography mass spectrometry procedures, which were fully validated and developed from methods previously used by Lambda Therapeutic Research Ltd, Ahmedabad, India . Briefly, ibuprofen plasma concentrations were measured following protein precipitation extraction  by high pressure liquid chromatography (HPLC) using a Kromasil® C18 250 × 4.6 mm, 5 μm column (mobile phase 28% acetonitrile and 72% buffer pH 6.8). The limit of quantification was 0.730 μg/mL. Pseudoephedrine plasma concentrations were analyzed following liquid-liquid extraction  by liquid chromatography-mass spectrometry (LC-MS/MS) analysis using a 50 × 4.6 mm Chromolith® SpeedROD RP-18e column . The limit of quantification was 2.053 ng/mL. Assay performance was assessed by back-calculation of calibration standards, tabulation of the standard curve fit function parameters and measurement of quality control samples. Validation data documented adequate accuracy, precision and specificity of the liquid chromatography mass spectrometry assays employed for the study.Clinical examination findings and vital signs  were reported on admission and before discharge from the study center in all 3 study periods. Vital signs were measured 4, 8, and 12 h  and additionally 24, 30, and 36 h (for treatments B and C) after dosing. Subjects were questioned for well-being at the time of clinical assessments. Adverse events were collected during each study period with severity  and investigator assessment of the relationship to the study medication .0−t), from time zero extrapolated to infinity (AUC0−∞) and Cmax. Time of maximum exposure (tmax) was a secondary variable. Non-compartmental analysis of plasma concentration-time data was performed using WinNonlin® Professional software . All values below the limit of quantification were considered as zero for pharmacokinetic analysis.The primary variables were the area under the plasma concentration-time curve from time 0 to the last quantifiable data point  model with sequence, subjects within sequences, period and treatment as sources of variation. The least square means and 90% confidence intervals (CI) were calculated and then back-transformed to the original scale to provide the point estimator and interval estimates for the geometric mean (gMean) of intra-subject test/reference ratio. In accordance with regulatory requirements . AUCNineteen healthy Indian male subjects were enrolled and 18 subjects aged 18–39 years were randomized; one subject withdrew before dosing and was subsequently replaced. The participant demographic characteristics are summarized in Table The ibuprofen and pseudoephedrine plasma concentration-time profiles are shown in Figure max of 50 min  . Bioequivalence was demonstrated as the 90% CIs of the ratios of point estimates (test/reference) for Cmax, AUC0−t, and AUC0−∞ were within the range of 80–125%.Ibuprofen was rapidly absorbed, with a median t7) Table . The pla7) Table . The gMemax of 100 and 120 min for the combination and when given alone, respectively (p = 0.92) . Bioequivalence was demonstrated as the 90% CIs of the point estimates (test/reference) for Cmax, AUC0−t, and AUC0−∞) were in the range 80–125%.Pseudoephedrine was rapidly absorbed, with a median t2) Table . The pla2) Table . The gMeA total of 14 treatment-emergent adverse events were reported by 9 subjects . All adverse events were mild and resolved spontaneously. Two events were considered as possibly related to the test medication (test C). One subject reported forearm itching which was considered definitely related; the event was also reported with pseudoephedrine alone and was also considered possibly related to the study drug. Another subject reported heartburn which was considered as possibly related to the study medication. In addition, itching and generalized body ache were reported as possibly related to reference A . There were no clinically relevant changes in vital signs, ECGs or laboratory findings.max and tmax) and extent (AUC) of absorption of ibuprofen or pseudoephedrine from a suspension formulation were similar when administered as single doses of the individual components and when administered in combination in healthy volunteers. The 90% CIs for the treatment ratios  for Cmax, AUC0−t, and AUC0−∞ of ibuprofen and pseudoephedrine were within the range of 80–125% and therefore satisfied regulatory criteria for bioequivalence  and was fully responsible for the analysis and interpretation of the data.


Memories are not fixed in the brain, but undergo experience-dependent updating and modification through reconsolidation. This occurs when a memory is converted to a labile state, usually involving surprise , which is in turn linked to release of dopamine. Disrupting maladaptive memories may provide a novel form of treatment for neuropsychiatric disorders, but little is known about the neurochemical mechanisms underlying the induction of lability, or destabilization, of a retrieved consolidated memory. Destabilization has been theoretically linked to the violation of expectations during memory retrieval, which, in turn, has been suggested to correlate with prediction error (PE). It is well-established that PE correlates with dopaminergic signaling in limbic forebrain structures that are critical for emotional learning. The basolateral amygdala is a key neural substrate for the reconsolidation of pavlovian reward-related memories, but the involvement of dopaminergic mechanisms in inducing lability of amygdala-dependent memories has not been investigated. Therefore, we tested the hypothesis that dopaminergic signaling within the basolateral amygdala is required for the destabilization of appetitive pavlovian memories by investigating the effects dopaminergic and protein synthesis manipulations on appetitive memory reconsolidation in rats. Intra-amygdala administration of either the D1-selective dopamine receptor antagonist SCH23390 or the D2-selective dopamine receptor antagonist raclopride prevented memory destabilization at retrieval, thereby protecting the memory from the effects of an amnestic agent, the protein synthesis inhibitor anisomycin. These data show that dopaminergic transmission within the basolateral amygdala is required for memory labilization during appetitive memory reconsolidation. Memories are not fixed in the brain, but undergo experience-dependent updating and modification through reconsolidation. This occurs when a memory is converted to a labile state, usually involving surprise , which is in turn linked to release of dopamine. We hypothesized that neurotransmission via dopamine receptors in the amygdala, a region critical for emotional memory processing, is required for memory destabilization. The results show that blocking dopamine receptors in the amygdala protected reward-associated memories from an amnestic treatment. Therefore, dopamine is required for the induction of pavlovian memory lability, supporting a link between destabilization and prediction error. Thus, dopaminergic signaling allows memories to be dynamic and flexible, providing a novel target for the modification of maladaptive memories.Reconsolidation is the process by which memories become destabilized at retrieval and subsequently restabilize in order to persist in the brain. This process has received much interest for its potential as a novel treatment target for neuropsychiatric disorders such as drug addiction  in invertebrates  are expressed within the BLA  antagonist SCH23390, the D2R antagonist raclopride, and the non-subtype selective dopamine receptor antagonist α-flupenthixol on the destabilization of an appetitive CS−sucrose memory, assessed through a procedure that selectively measures the conditioned reinforcing properties of pavlovian CSs. To test whether memory destabilization had occurred, the capacity of the protein synthesis inhibitor anisomycin, given immediately after memory reactivation, to induce subsequent amnesia was assessed, as described previously  Act 1986.Subjects were 77 experimentally naïve male Lister-Hooded rats (Charles River) housed in pairs in a vivarium on a reversed light−dark cycle (lights on at 1900 hours). Subjects weighed at least 290 g prior to surgery. Subjects were food restricted, though not deprived, being maintained at at least 90% of free-feeding weight and fed after training or testing each day. Access to water was Rats were implanted with bilateral guide cannulae  located just dorsal to the BLA  Milton . The coo−1 . One minute of waiting time was imposed from the end of the infusion to the removal of the injectors to allow diffusion of the solution away from the infusion site. Although we did not test whether the infusions were restricted to the BLA, it is the basolateral nucleus, rather than the adjacent central nucleus, that has been implicated in supporting the conditioned reinforcing properties of pavlovian CSs  and 5 μl Hamilton syringes, connected to injectors  by polyethylene tubing. The rats received two infusions; one immediately prior to the memory reactivation session, and one immediately afterwards. All infusions were begun 30 s after the insertion of the injectors and performed over 2 min at a rate of 0.25 μl min−1; Sigma-Aldrich) was dissolved in equimolar HCl and then pH-balanced to pH 7.4 with NaOH. This dose of anisomycin has previously been shown to disrupt memory reconsolidation  or its vehicle (VEH) as their second (post-reactivation) infusion. Anisomycin  was dissolved in PBS at a concentration of 4 μg μl−1 and the D2-selective receptor antagonist raclopride  was dissolved in PBS at a concentration of 10 μg μl−1. These doses of SCH and RAC have been shown to be effective in blocking the consolidation of inhibitory avoidance memory -flupenthixol  was dissolved in PBS at a concentration of 20 μg μlAll behavioral procedures were conducted during the animals’ dark cycle. Rats were trained in conditioning chambers (Med Associates) to make a nosepoke response into a central magazine for presentation of a 0.1 ml of a 10% sucrose reinforcer (Tate & Lyle), which was associated with a 10 s light CS  on a fixed ratio (FR) 1 schedule. Rats were trained over nine sessions, with a maximum of 30 CS−sucrose pairings per session.The day after the completion of training, rats received intra-BLA infusions of either the dopamine receptor antagonists  or VEH, and immediately (within 1 − 2 min) afterwards began a 15 min memory reactivation session. During this session, nosepokes led to the presentation of the light CS and movement of the dipper on an FR1 schedule, but no sucrose was delivered. The rats were limited to a maximum of 30 CS presentations during this session, but the 15 min session normally terminated before this limit was reached. Immediately after the end of the memory reactivation session, the rats received a second intra-BLA infusion of either ANI or its VEH.Acquisition of a new instrumental response for conditioned reinforcement (ANR) testing began 24 h after the memory reactivation session. The rats were returned to the same conditioning chambers, but in this phase they were presented with two novel levers . Depression of the active lever led to an abbreviated (1 s) presentation of the light CS on a variable ratio (VR1-3) schedule, while depression of the inactive lever had no programmed consequence and acted as a control for general activity. The light CS was always presented on the side opposite to the active lever to avoid pavlovian-conditioned approach contributing to lever pressing. No sucrose was available during these sessions. Rats were returned to the chambers for seven 30 min sessions, conducted 1, 2, 5, and 8 d following memory reactivation, and then weekly following day 8 . Lever presses and nosepokes were recorded by computer.Following the end of testing, rats were killed with an overdose of anesthetic  and transcardially perfused with 0.01 M PBS through the ascending aorta, followed by 4% paraformaldehyde. The brains were removed and stored in 4% paraformaldehyde for at least 24 h, before being transferred to a 20% sucrose solution for cryoprotection prior to sectioning. The brains were sectioned at 60 μm, mounted on microscope slides, and stained with Cresyl Violet. Cannulae placements  were verA priori sample size calculations were not conducted but the number of subjects per group was chosen by reference to previous research. Data was collected over an extended period of time, with eight animals being run within a single squad. Since there was no effect of squad on the number of nosepokes at training (TR) or reactivation (React)  = 1.15, p = 0.33, SquadReact: F = 1.54, p = 0.11) or on the number of lever presses during the ANR test  = 1.08, p = 0.39), data from different squads were pooled for analysis. Subjects were pseudo-randomly assigned to experimental groups, such that an individual squad received as a first infusion only a subset of the drugs under investigation . All squads contained animals receiving VEH or ANI for the second infusion. Drug assignments were also made such that training performance was matched across experimental groups.Data were recorded automatically by the Conditioned Reinforcement program , where x is the original data and y the transformed value. Following this transformation, the data satisfied the assumption of normality . The lever pressing and nosepoke data from the ANR phase of the experiment were also not normally distributed, so were transformed using the Box-Cox method with λ = 0.5; i.e. square-root transformed. Following this transformation, the majority of the lever press data satisfied the assumption of normality (p > 0.05).Training and testing data were analyzed using repeated-measures ANOVA, and reactivation data were analyzed using a one-way ANOVA. The normality assumption of ANOVA was checked with the Shapiro-Wilk test, and if this indicated that the data were not normally distributed then they were transformed. This was the case for the nosepoke data from training, which was transformed using the Box-Cox method with λ = −0.5; i.e. using the equation p values are two-tailed. Where appropriate, subsequent ANOVAs and Šidák-corrected pairwise comparisons were conducted to investigate specific a priori hypotheses. For ease of interpretation, we have represented the factor names by the name of the drug infused when reporting the statistics  in If Mauchly’s test indicated that the assumption of sphericity had been violated, then the Greenhouse-Geisser correction was applied where ε < 0.75, and the Huynh-Feldt correction applied where ε > 0.75, as recommended by Cardinal and Aitken . The α lF = 13.0, p < 0.001, η2 = 0.16), though with no differences in instrumental responding between the prospective experimental groups assigned to receive dopamine receptor antagonists or vehicle prior to reactivation (Drug1) and anisomycin or vehicle after reactivation (Drug2)  = 1.59, p = 0.058; Session × Drug2: F = 1.25, p = 0.28; Session × Drug1 × Drug2: F < 1; Drug1: F < 1; Drug2: F < 1; Drug1 × Drug2: F < 1). Furthermore, as the test data for each experiment were analyzed separately, follow-up ANOVAs were conducted and revealed no differences in training performance between groups assigned to receive ANI or its VEH following memory reactivation  for any of the experimental groups receiving a dopamine receptor antagonist  or VEH prior to reactivation . Therefore, all experimental groups were equivalent on the basis of their training performance.Briefly, animals were trained over nine sessions in conditioning chambers to make an instrumental nosepoke response into a magazine to receive presentations of 10% liquid sucrose and a pavlovian light CS, with a maximum of 30 CS−sucrose presentations per day. All animals readily acquired this task, with no differences in the performance of the animals prospectively assigned to different experimental groups  during tF = 1.20, p = 0.297). There were no differences in the number of CS presentations between the prospective experimental groups  = 2.52, p = 0.12; Drug1 × Drug2: F < 1). Due to two rats receiving fewer than 30 CS presentations in one session of training, there were significant interactions of Session × Drug1  = 1.56, p = 0.045, η2 = 0.06) and Session × Drug1 × Drug2  = 1.56, p = 0.045, η2 = 0.06), but it is unlikely that these very small effects are biologically meaningful.All groups also received equal numbers of CS presentations during training, with all except two animals reaching the limit of 30 CS−sucrose presentations in every training session . All aniF < 1) or the number of CSs presented  = 1.45, p = 0.23) during the reactivation session itself. Furthermore, there were no differences in the reactivation experience of those animals receiving VEH or ANI immediately after the memory reactivation session in terms of the number of nosepokes made  or the number of CSs presented , suggesting that these prospective experimental groups were well-matched for behavior.Dopamine receptor antagonists applied to the BLA immediately before the reactivation session affected neither the retrieval of the CS−sucrose memory, nor had any generalized locomotor effects . None ofp’s > 0.37) or the number of CS presentations .Further analysis of the data indicated that for all experimental groups receiving different pre-reactivation infusions , analyzed separately, there were no differences in the performance at reactivation of animals receiving ANI or VEH following reactivation  when assessed through the number of nosepokes made during memory reactivation  = 3.40, p = 0.023, η2 = 0.13; Lever × Drug1 × Drug2: F = 3.11, p = 0.032, η2 = 0.12). Importantly, there were no simple main effects of dopamine receptor antagonism  = 1.24, p = 0.30; Lever × Drug1: F = 1.16, p = 0.33) or protein synthesis inhibition  = 2.67, p = 0.11; Lever × Drug2: F < 1) on the memory; rather, the capacity of the CS to act as a conditioned reinforcer depended upon both treatments, consistent with a blockade of the memory destabilization process  or protein synthesis inhibition  = 1.44, p = 0.23) on nosepoking behavior during testing, and unlike responding for the CS, there was no interaction between the two treatments  = 1.72, p = 0.17). Although the omnibus ANOVA indicated that pre-reactivation treatment with a dopamine receptor antagonist altered nosepoking behavior across the course of ANR testing  = 1.71, p = 0.039, η2 = 0.069), this was a very small effect and there were no differences between animals treated with any of the dopamine receptor antagonists in any of the individual test sessions when Šidák-corrected pairwise comparisons were conducted .As shown previously with NMDA receptor (NMDAR) antagonism at memory reactivation  = 5.33, p = 0.027, η2 = 0.14). Šidák-corrected pairwise comparisons revealed that while the VEH/VEH group showed discriminated responding on the active (CS-producing) lever [p < 0.001] the VEH/ANI group did not discriminate (p = 0.18). Therefore, the pavlovian CS−sucrose memory destabilized in both groups, and was prevented from restabilizing and subsequently persisting in the VEH/ANI group.Consistent with previous data  = 2.05, p = 0.16; Session × ANI: F < 1), indicating that although the pavlovian CS−sucrose memory was rendered sensitive to disruption during the memory reactivation session, the instrumental memory of responding for sucrose was not.The number of nosepoke responses made during the ANR testing sessions  was not 1-selective dopamine receptor antagonist SCH prior to the memory reactivation session prevented the destabilization of the CS−sucrose memory  = 6.75, p = 0.013, η2 = 0.13; ANI: F < 1; Lever × ANI: F < 1; Lever × Session × ANI: F = 1.60, p = 0.21). In contrast to VEH/ANI experimental group, all animals that received SCH prior to memory reactivation showed discriminated responding for conditioned reinforcement on the active lever during subsequent testing, with increased active lever pressing over sessions  = 36.7, p < 0.001, η2 = 0.75; Lever × Session: F = 3.88, p = 0.017, η2 = 0.24). This shows that the CS−sucrose memory remained intact despite the post-reactivation administration of a drug that would otherwise have disrupted memory restabilization. Similarly, protein synthesis inhibition following SCH treatment did not reduce the number of nosepokes  = 1.55, p = 0.24; Session × ANI: F < 1), indicating that the instrumental memory, like the pavlovian memory, remained intact.Administration of the De memory B. A sigosepokes  made dur2-selective dopamine receptor antagonist RAC also prevented the amnestic effect of post-reactivation anisomycin, consistent with a blockade of memory destabilization  = 7.26, p = 0.01, η2 = 0.14)  = 23.1, p < 0.001, η2 = 0.64) and interestingly, though the RAC/VEH and RAC/ANI groups responded differently  = 6.04, p = 0.029, η2 = 0.32), this was due to better discrimination in the RAC/ANI group compared with the RAC/VEH group. While the RAC/ANI group discriminated between the active and inactive lever from the second testing session , the RAC/VEH group did not reliably discriminate between the levers until Session 5 (p’s < 0.042). However, ANI did not produce amnesia (ANI: F < 1), indicating a blockade of memory destabilization by RAC. Furthermore, there were no differences between the RAC/VEH and RAC/ANI groups in the numbers of nosepokes  = 2.64, p = 0.13; Session × ANI: F < 1), indicating that, as for D1-selective dopamine receptor antagonism, D2R antagonism left instrumental responding intact and prevented the destabilization of the pavlovian memory.The D = 0.14) C. Both osepokes  made dur1/D2 dopamine receptor antagonist FLU prior to memory reactivation, by contrast to D1- and D2-subtype-selective dopamine receptor antagonism, did not prevent the destabilization of the CS−sucrose memory  = 2.26, p = 0.14, η2 = 0.05; ANI: F = 7.48, p = 0.019, η2 = 0.41). While the animals in the FLU/VEH group discriminated between the active (CS-producing) and inactive lever during testing (p = 0.018), those in the FLU/ANI group did not (p = 0.12). Thus, pre-reactivation administration of FLU did not prevent the amnestic effect of ANI. Furthermore, as for VEH-treated animals, the number of nosepoke responses  = 1.36, p = 0.27; Session × ANI: F < 1). Thus, in contrast with D1R-selective or D2R-selective antagonism, nonselective dopamine receptor antagonism affected neither destabilization nor restabilization of the pavlovian memory or the instrumental memory.Administration of the mixed De memory D, as poesponses  made durF < 1; Lever × Drug1: F = 1.43, p = 0.25; Session × Drug1: F = 1.11, p = 0.34; Lever × Session × Drug1: F < 1).Dopamine receptor antagonism prior to reactivation did not itself alter subsequent responding for conditioned reinforcement. Comparing groups that received VEH following reactivation, there were no differences in subsequent responding for conditioned reinforcement, regardless of which dopamine receptor antagonist had been given prior to the memory reactivation session  = 3.01, p = 0.049, η2 = 0.26) and a trend towards differences in pressing the active and inactive levers  = 2.53, p = 0.079, η2 = 0.23). Follow-up ANOVAs comparing lever pressing during ANR for individual experimental groups revealed that although the VEH/ANI-treated  = 2.13, p = 0.18) and FLU/ANI-treated  = 3.06, p = 0.12) did not discriminate between the active and inactive levers during ANR testing, the SCH/ANI-treated  = 29.6, p = 0.003, η2 = 0.86) and RAC/ANI-treated  = 39.6, p = 0.001, η2 = 0.87) animals pressed the active lever, which was reinforced by presentation of the previously sucrose-associated CS, more during the test sessions.In contrast, dopamine receptor antagonism prior to memory reactivation did affect the capacity of the protein synthesis inhibitor anisomycin to induce amnesia following reactivation. Comparing groups that received ANI following reactivation, there was a difference in subsequent responding for conditioned reinforcement, depending on which dopamine receptor antagonist had been administered prior to reactivation. This was reflected by differences in overall lever pressing during the ANR sessions (Drug1: 1R with SCH23390 or at the D2R with raclopride prevented the amnesia that normally follows post-reactivation protein synthesis inhibition. Therefore, the CS−sucrose memory, retrieved and behaviorally expressed during the memory reactivation session, did not require protein synthesis in order to restabilize and subsequently persist—consistent with the hypothesis that activity at either D1Rs or D2Rs is required for destabilization of the CS−sucrose memory. That the memory’s persistence was independent of protein synthesis cannot be attributed to the parameters of the memory reactivation session, since rats having received intra-amygdala vehicle infusions prior to reactivation were subsequently amnesic at test following post-reactivation administration of the protein synthesis inhibitor anisomycin. Furthermore, nonselective antagonism at dopamine receptors with α-flupenthixol did not block the subsequent amnestic effect of post-reactivation anisomycin. Although it is possible that a higher dose of α-flupenthixol may have prevented anisomycin-induced amnesia, the dose of α-flupenthixol used in these experiments is sufficiently high to be behaviorally effective, but not so high so as to produce locomotor side effects (Di Ciano and Everitt, 1Rs and D2Rs in the BLA in order to proceed.The data presented here demonstrate that blocking dopaminergic signaling within the BLA either at the DWe speculate that the requirement for dopamine in the destabilization of the CS−sucrose memory is linked to its role in signaling prediction error. It should be noted, however, that in addition to its hypothesized role in prediction error, dopaminergic signaling has also been theoretically implicated in the encoding of hedonic processes (Wise and Rompre, 1R antagonism or D2R antagonism prevented memory destabilization appears counterintuitive, as D1Rs and D2Rs have generally been seen to produce opposing effects on intracellular signaling pathways, at least in the striatum (Stoof and Kebabian, Although prediction error has previously been linked theoretically to reconsolidation (Pedreira et al., 1R and D2R antagonism has been seen previously. Electrophysiological studies of BLA neurons have shown that both D1R and D2R agonists produce an overall reduction in BLA output (Rosenkranz and Grace, 1R antagonists (Lamont and Kokkinidis, 2R antagonists (Guarraci et al., 1Rs being expressed on projection neurons (Pickel et al., 2Rs are mainly expressed on local fast-spiking inhibitory interneurons, both dopamine receptor subtypes are expressed on cells that inhibit amygdala projection neurons. Stimulation of either of these dopamine receptor subtypes leads to a reduction in interneuron activity, and therefore disinhibition of BLA projection neurons (Bissière et al., 1Rs or D2Rs would be predicted to increase interneuron activity, reduce the activity of the BLA projection neuron, and so impair synaptic plasticity processes—we would argue including memory destabilization—within the BLA. Furthermore, the effect of D1R antagonism may be twofold; directly reducing excitation of pyramidal neurons (Kröner et al., 1R or D2R antagonism was sufficient to prevent the destabilization of the reconsolidating memory.The similar effect of intra-amygdala D1R-selective (Kröner et al., 2R-selective (Bissière et al., 1Rs and D2Rs results in an increase in spontaneous inhibition within the BLA inhibitory network (Lorétan et al., The model proposed by Ehrlich et al.  may also1Rs and D2Rs in modulating amygdala activity, and elucidation of the network dynamics, require further study. However, the results of the experiments reported here clearly demonstrate that dopaminergic signaling is a requirement for reactivation-induced memory destabilization. Destabilization and prediction error therefore have a shared dependence upon dopaminergic signaling, as a retrieved memory requires prediction error in order to destabilize (Sevenster et al., The role of D


Pseudomonas aeruginosa were investigated. The results demonstrated that crude enzyme extract exhibited obviously higher degradation efficiency and shorter biodegradation time than Pseudomonas aeruginosa itself. Under the optimum conditions of pH 9.0, 35 °C and protein content of 2000 mg/L, 92.77% of the initial BDE-209 (20 mg/L) was degraded after 5 h. A BDE-209 biodegradation pathway was proposed on the basis of the biodegradation products identified by GC-MS analysis. The biodegradation mechanism showed that crude enzyme extract degraded BDE-209 into lower brominated PBDEs and OH-PBDEs through debromination and hydroxylation of the aromatic rings.The biodegradation effect and mechanism of decabromodiphenyl ether (BDE-209) by crude enzyme extract from  As global environmental contaminants, polybrominated diphenyl ethers (PBDEs) have 209 congeners that are distinguished by the number and location of bromine atoms in the benzene ring . PBDEs aPseudomonas aeruginosa LY11 (“LY11” represented the strain number). Pseudomonas aeruginosa is a common bacteria in the environment, Shi et al. used it and its crude enzyme to degrade BDE-209 .A mineral salt medium (MSM) for degradation of BDE-209 was prepared by using the following composition (1 L): CaClThe pH of the two medium was adjusted to 7.0 by addition of HCl or NaOH before autoclaving at 121 °C for 30 min.−1. Then, the cells were harvested and separated from the medium by centrifugation at 8000 r·min−1 for 10 min. Finally, the harvested cells were weighted.The strain LY11 was cultured in 500 mL Erlenmeyer flasks containing 200 mL beef extract peptone medium at 35 °C for 36 h under a rotary shaker at 200 r·min600  and colony-forming units (CFU) over incubation time. The values of OD600 were determined with an ultraviolet and visible spectrophotometer, CFU was counted via plate counting method [Growth curve of strain LY11 under optimal incubation conditions was described by change of ODg method .2PO4-Na2HPO4 buffer , were subjected to 140 rounds of sonication in an ice-water bath for 3 s followed by cooling for 6 s (the time of 10 rounds was 1 min). The debris was removed by centrifugation at 12000 r·min−1 for 10 min. The supernatant was filtered with 0.22 μm pore-size filters and the filtrate was crude enzyme.1 g LY11 cells (age 36 h) were placed in a 50 mL centrifuge tube, suspended in the ice-cold 12 mL NaHThe protein was measured by Bradford method at 595 nm by UV-vis spectrophotometer using bovine serum albumin (BSA) as a standard . 1 g LY1One hundred milliliters of MSM and 2.0 mg BDE-209 were placed in a 250 mL Erlenmeyer flask and ultrasonically mixed by ultrasonic cleaning, 40 mg LY11 cells were added to the MSM containing BDE-209 (20 mg/L) in a super clean bench, the mixture in the flask was incubated at 35 °C on a rotary shaker at 200 rpm for 5 days.2PO4-Na2HPO4 buffer  and 2.0 mg BDE-209 were placed in a 250 mL Erlenmeyer flask and ultrasonically mixed by ultrasonic cleaning, 200 mg crude enzyme was added to the buffer containing BDE-209 (20 mg/L), the mixture in the flask was incubated at 35 °C on a rotary shaker at 200 rpm for 5 h.One hundred milliliters of NaHEffects of crude enzyme extract were investigated under the following extraction conditions: ultrasonic power (250–550 W), pH value (6.0–9.0), extraction time (4–16 min) and solid-liquid ratio (1:4–1:16 w/v).Biodegradation characteristics of BDE-209 by LY11 were investigated under the following conditions: cell age (6–72 h), inoculation amount (10–120 mg), temperature (15–45 °C), pH value (6.0–9.0), initial concentration of BDE-209 (5–35 mg/L), biodegradation time (1–7 days).Biodegradation characteristics of BDE-209 by crude enzyme extract were investigated under the following conditions: protein content of crude enzyme extract (100–3000 mg/L), temperature (20–50 °C), pH value (6.5–9.5), initial concentration of BDE-209 (5–35 mg/L), biodegradation time (1–7 h).After the specified incubation period, the whole samples were firstly extracted  with toluene in a separating funnel by vigorously shaking for 10 min and allowed to set until phase separation. Then, the organic phase (toluene) was collected and treated with anhydrous sodium sulfate to remove water. The aqueous phase was extracted again as described above. The combined organic phases were concentrated by using a rotary evaporator at 40 °C. Finally, the resulting residue was dissolved in 5 mL toluene for GC-MS analyses.−1. The column temperature program started at 60 °C (held for 1 min), then increased 20 °C·min−1 to 220 °C (held for 1 min), increased 5 °C·min−1 to 250 °C (held for 1 min), increased 20 °C·min−1 to 280 °C (held for 10 min), finally increased 15 °C·min−1 to 300 °C (held for 10min). Mass spectrometer conditions were: electron impact ionization, ionization energy 70 eV, full scan.The quantification of BDE-209 and lower brominated PBDEs were analyzed using 7890-5975c gas chromatography-mass spectrometer  equipped with a DB-5 MS column (60 m × 0.25 mm × 0.25 μm). The injection volume was 1 μL. Helium was used as the carrier gas at a flow rate of 1 mL·min−1. The sample injection volume was 20 μL, and the column temperature was maintained at 30 °C.The concentration of bromide ion was measured via ICS-5000 ion chromatography  using a Dionex Ionpac AS11-HC (4 × 250 mm) analytical column . The mobile phase was 30% NaOH at a flow rate of 1.2 mL·minCC was initial BDE-209 concentration, SC was the BDE-209 concentration in biodegradation test; BrM was the concentration of bromide ions released, TM was the theoretical bromide ion concentration from the complete debromination of the substrate.Biodegradation efficiency of BDE-209 and debromination efficiency were calculated according to the following Equations: All experiments were performed in triplicate flasks and the results presented were the mean values of the three replicates. The standard deviations for measurements ranged from 0.5% to 5.0%.The effect of crude enzyme extracted at ultrasonic power ranging from 250 to 550 W is shown in In general, pH value can affect enzyme activity, as different enzymes have their own optimal pH value. The effect of crude enzyme extracted at pH value ranging from 6.0 to 9.0 is shown in The effect of crude enzyme extracted at different extraction times is presented in In this study, the effect of crude enzyme extracted at above-mentioned solid-liquid ratio is shown in et al. reported that the extraction conditions of crude enzyme from Pseudomonas aeruginosa were ultrasonic power 450 W, pH 7.2 and extraction time 15 min [et al. reported.Therefore, the optimal extraction conditions of crude enzyme from LY11 were ultrasonic power 350 W, pH 8.5, extraction time 14 min and solid-liquid ratio 1:12. Shi e 15 min . It was Growth curve of strain LY11 is shown in As shown in Temperature plays a vital role in organics degradation due to its influence on enzyme activity. At a suitable temperature, cells can grow quickly and produce high amount of enzymes, thus having enzyme activity for organics degradation . Figure In bacteria, pH can influence nutrient absorption and enzyme production . At optiThe effect of BDE-209 biodegradation at different initial concentrations is presented in et al. had reported that the degradation rate of BDE-209 by Pseudomonas aeruginosa was 57%, its biodegradation conditions were cell age 24 h, temperature 30 °C, pH 7.3, BDE-209 1 mg/L and biodegradation time 6 days [et al. [In this part, the optimal conditions for BDE-209 biodegradation by LY11 were cell age 36 h, inoculation amount 40 mg, temperature 35 °C, pH 7.0, BDE-209 20 mg/L and biodegradation time 5 days. Shi e 6 days . In cont [et al. .As mentioned earlier, crude enzyme extract consisted of intracellular enzyme mixtures, and the BDE-209 degradation enzyme in it played a leading role in biodegradation. So far, we have not been able to determine the content of the BDE-209 degradation enzyme accurately, because we have not known which enzyme had the ability of BDE-209 degradation in the crude enzyme extract. Because the content of BDE-209 degradation enzyme was in direct proportion to crude enzyme extract, we determined the content of crude enzyme extract instead of BDE-209 degradation enzyme. So, we determined degradation efficiency and debromination efficiency at amounts of protein of crude enzyme extract, and these results are shown in As a biocatalyst, enzyme exhibits temperature effect just like catalyst. Temperature increment can increase enzymatic reaction velocity. However, on the other hand, it also can accelerate enzyme denaturation velocity. Therefore, in the low temperature range, enzymatic reaction velocity increases with temperature increasing. However, above a certain temperature, enzymatic reaction velocity begins to decrease . As showAs explained above, pH value has a strong influence on enzyme activity. At a certain pH value, enzyme will exhibit the maximum catalytic activity, this pH is the optimum pH value for enzyme. Enzyme activity is low or undetectable at pH value far from the optimum pH value. Already from results presented in At different initial concentrations of BDE-209, the biodegradation behaviour of crude enzyme extract is displayed in As presented in et al. had reported that the degradation rate of BDE-209 by crude enzyme obtained from Pseudomonas aeruginosa was 69%, its biodegradation conditions were temperature 30 °C, pH 7.3, BDE-209 1 mg/L and biodegradation time 12 h [et al. Nevertheless, environmental application of BDE-209 bio-removal by crude enzyme extract will be a key research focus in the future. As shown in Obviously, the optimal conditions for BDE-209 biodegradation by crude enzyme extract from LY11 were: protein content 2000 mg/L, temperature 35 °C, pH 9.0, BDE-209 20 mg/L and biodegradation time 5 h. Shi ime 12 h . These rAt the optimal conditions selected above, the biodegradation effects of LY11 and crude enzyme extract are listed in After being reacted with crude enzyme extract for 5 h, BDE-209 biodegradation products were analyzed using the above method mentioned in It was easy to establish a general rule of BDE-209 biodegradation by crude enzyme extract. First of all, BDE-209 lost one bromine atom to produce BDE-206. Then, BDE-206 successively removed three bromine atoms and was accompanied by hydroxylation reaction to produce lower brominated PBDEs and OH-PBDEs. It was noteworthy that 6-OH-BDE-137 was one of BDE-209’s biodegradation products. Nevertheless, 6-OH-BDE-137 is a common OH-PBDE which has been widely detected in the environment and tissues of wildlife, and has been attracting growing concern by researchers owing to its potential toxicity . FurtherP. aeruginosa LY11. These results may provide substantial support for the potential application of crude enzyme in bioremediation of heavy BDE-209 pollution in the environment. The future work will focus on the genes that encode the BDE-209 degradation enzyme and the complete debromination mechanism of the BDE-209 degradation enzyme.To the authors’ knowledge, this is the first report on the biodegradation of BDE-209 by crude enzyme extract from P. aeruginosa LY11 showed very encouraging results, its degradation efficiency reaching 92.77% after 5 h. By contrast to P. aeruginosa LY11, crude enzyme extract exhibited obviously higher degradation efficiency and shorter biodegradation time. BDE-209 biodegradation products by crude enzyme extract were detected, and its pathway was also tentatively proposed. The biodegradation mechanism was presented that crude enzyme extract could degrade BDE-209 into lower brominated PBDEs and OH-PBDEs through debromination and hydroxylation of the aromatic rings.In the present study, the biodegradation of BDE-209 by crude enzyme extract from


Spartina alterniflora invasion in a chronosequence of 6-, 10-, 17-, and 20-year-old by comparing with bare flat in a coastal wetland of China. S. alterniflora invasion significantly increased soil moisture and salinity, the concentrations of soil water-soluble organic carbon and microbial biomass carbon (MBC), the quantities of total and various types of phospholipid fatty acids (PLFAs), the fungal:bacterial PLFAs ratio and cumulative microbial respiration compared with bare flat. The highest MBC, gram-negative bacterial and saturated straight-chain PLFAs were found in 10-year-old S. alterniflora soil, while the greatest total PLFAs, bacterial and gram-positive bacterial PLFAs were found in 10- and 17-year-old S. alterniflora soils. The monounsaturated:branched PLFAs ratio declined, and cumulative microbial respiration on a per-unit-PLFAs increased following S. alterniflora invasion in the chronosequence. Our results suggest that S. alterniflora invasion significantly increased the biomass of soil various microbial groups and microbial respiration compared to bare flat soil by increasing soil available substrate, and modifying soil physiochemical properties. Soil microbial community reached the most enriched condition in the 10-year-old S. alterniflora community.The role of exotic plants in regulating soil microbial community structure and activity following invasion chronosequence remains unclear. We investigated soil microbial community structure and microbial respiration following  Plant invasion, one component of anthropogenic-induced global change, has caused severe biological impacts on native ecosystems and great economic costs36794et al.178Plant invasion can influence soil microbial community structure and activity by altering the quantity and/or quality of litter entering the soil1113−) bacteria growth and reduce gram-positive (gram+) bacteria biomass2526Plant invasion can modify the soil’s physical and chemical properties, such as soil moisture161618Spartina alterniflora is a perennial C4 grass plant that is native to North America. It has been introduced to China since 1979 for coastal erosion control and sediment stabilization29S. alterniflora invasion in the coastal zone of China has expanded over the past 30 years, from Tianjin in the north to Beihai in the south, by occupying bare flat and/or by replacing native C3 plants , and become one of the dominant plants in China’s coastal wetland16S. alterniflora has a longer growing season, a higher leaf area index and net photosynthetic rate, and a greater net primary production compared with the native plants, Scirpus mariqueter and P. australisS. alterniflora invasion significantly alters soil physicochemical properties14S. alterniflora invasion. We hypothesized that S. alterniflora invasion would alter soil microbial community structure and activity by changing soil C availability and physiochemical properties. To test this hypothesis, we determined soil phospholipid fatty acids (PLFAs) to analyze the soil microbial community structure, and determined cumulative microbial respiration, microbial respiration on a per-unit-PLFAs basis, and the respiration quotient (qCO2) after 30-days of incubation at 25 °C and 35 °C to analyze the soil microbial activity. We measured soil moisture, pH, salinity, soil organic C (SOC), soil organic N (SON), water-soluble organic carbon (WSOC), microbial biomass C (MBC), microbial biomass N (MBN), the MBC:MBN ratio, temperature sensitivity (Q10) of microbial respiration, and the aboveground and root biomass in invasive 6-, 10-, 17-, and 20-year-old S. alterniflora communities and compared these findings with those from a bare flat in a coastal wetland of China.S. alterniflora soils were significantly higher than those in bare flat soil , actinomycete, monounsaturated, branched, and saturated straight-chain (SSC) PLFAs in S. alterniflora soils were significantly higher than those in bare flat soil , WSOC , and salinity   . The big 0.0020) . Meanwhi 0.0020) .S. alterniflora invasion greatly accelerated soil organic C and N accumulation due to greater biomass input14S. alterniflora invasion significantly increased MBC concentration and the quantities of the total and all types of PLFAs compared with bare flat soil  and the microbial biomass  is difficult to be utilized by microbesSoil microbial respiration was highly dependent on soil temperature, moisture, and C inputsare flat , which ware flat  and 3. T biomass , 2, 3, 4 biomass , primarisequence , likely ra soils , cumulatra soils , suggest20 years  and 4c, ra soils  and 4b,cS. alterniflora salt marsh in a invasion chronosequence in a coastal wetland of China. Specifically, S. alterniflora invasion greatly increased the total and various types of soil microbial biomass and cumulative microbial respiration but significantly decreased microbial C utilization efficiency compared to bare flat. 10-year-old S. alterniflora community had the most enriched soil microbial community and the highest microbial C utilization efficiency across 6 to 20 years S. alterniflora invasion. Soil microbial biomass decreased after 17 years S. alterniflora invasion. The variations in the microbial community structure and activity may in turn deeply affect SOM accumulation and ecosystem C and N cycling. This study represents a step forward in our understanding of microbial communities as affected by plant invasion, and it provides valuable insights regarding the better understand the influence mechanism of plant invasion on soil organic C pool.In conclusion, this study highlighted the variations of soil microbial community structure and activity following bare flat was converted to S. alterniflora was introduced to the bare flat of the JYWNNRRB in 1983, and it quickly expanded to form large areas of S. alterniflora salt marshes following mudflat aggradingS. alterniflora salt marshes are located on the low and middle areas of the intertidal zone with semidiurnal tidal periodicityS. alterniflora is a bare flat that had no vegetation prior to S. alterniflora invasionThis study was conducted at the core area of the Jiangsu Yancheng Wetland National Nature Reserve, Rare Birds, China (JYWNNRRB) . This area is characterized as warm temperate with an average annual temperature of 13.8 °C, average annual precipitation of 1000 mm, and average annual sea water salinity of 3.09%S. alterniflora invasion times, was identified based on analyses of Landsat Thematic Mapper 5 (TM5) satellite images and historical records. This chronosequence from seaward to landward contained the bare flat (BF) and the four S. alterniflora communities that were colonized in 2006 (6 years), 2002 (10 years), 1995 (17 years) and 1992 (20 years). In November 2012, three parallel transects (2-km length and 200-m width) were selected along the chronosequence. Within each transect, five locations were marked from the bare flat to the invasive 6, 10, 17, and 20 years S. alterniflora communities. Three 2 m × 2 m plots were randomly established within each location. Three soil samples (5-cm diameter × 30 cm depth) were collected randomly in each plot. The soil samples from each plot were mixed evenly to form a composite sample. Three 50 cm × 50 cm quadrats were established to collect aboveground biomass , and three root sampling blocks (15-cm length × 15-cm width × 30 cm depth) were excavated to collect root biomass in each community of each transect. All soil and plant samples were stored at 4 °C in the field and then transported to the laboratory for subsequent analysis.The sampling region, with its different et al.Each root sampling block was put through a 100 mesh sieve and flushed with water, and the roots remaining in the sieve were collected at the final step2SO4 by shaking for 30 min at 200 rpm and then filtered. Organic C and TN in the K2SO4-extracted solution were determined using a Liqui TOCII analyzer and the Kjeldahl method, respectively. MBC and MBN were calculated according to the equation: MBC = Ec/0.38, MBN = En/0.54, where Ec and En were organic C and TN extracted from fumigated soil subtracted organic C and TN extracted from unfumigated soil, respectively.MBC and MBN were measured using the chloroform fumigation-extraction method3, acetone and methanol, respectively. Subsequently, phospholipids were subjected to a mild-alkali methanolysis to recover fatty acid methyl esters. Samples were then re-dissolved in 200 ml hexane containing nonadecanoic acid methyl ester (19:0) as an internal standard and were analyzed using a Hewlett-Packard 6890 Gas Chromatograph equipped with an Ultra 2-methylpolysiloxane column with N2 as the carrier gas and H2 and air to support the flame. A 2-μL injection of the above dilution with a 1:50 split was employed at 250 °C for the injector and 300 °C for the detector. The oven temperature ramped from 170 °C to 300 °C at 5 °C min−1 and was held for 12 min. Peaks were identified using bacterial fatty acid standards and MIDI peak identification software . The quantity (ng g−1 dry soil) of each PLFAs was calculated based on the 19:0 internal standard (5 μg mL−1). The quantities of the PLFAs in each sample were expressed as ng PLFAs g−1 dry soil and were used to estimate microbial biomass. Bacteria were represented by the sum of the PLFAs: i14:0, i15:0, a15:0, 15:0, i16:0, i17:0, a17:0, 17:0, cy17:0, 14:1ω5c, 15:1ω6c, 16:1ω7c, and 18:1ω7c44525354+ bacteria were identified by the PLFAs: i13:0, i14:0, i15:0, a15:0, i16:0, a16:0, i17:0, and a17:0, and gram− bacteria were identified by the PLFAs: 14:1ω5c, 15:1ω6c, 16:1ω7, 16:1ω9c, 17:1ω8c, 18:1ω7c, 12:0 2OH, 15:0 3OH, 16:1 2OH, cy17:0, cy19:0 ω8c, and 18:1ω7c 11-methyl445444542141414444+ bacterial PLFAs, gram− bacterial PLFAs, AMF PLFAs, actinomycete PLFAs, SSC PLFAs, and 20:4ω6,9,12,15c. The ratios of fungal:bacterial PLFAs, gram−:gram+ and monounsaturated:branched PLFAs were calculated from the above PLFAs. The ratio of cy17:0/16:1ω7c was used as a bacterial stress index, which indicates the growth stage of the gram− bacteria communityThe soil microbial community composition was assessed using PLFAs analysis based on the method of Bossio and Scow2 evolved at 25 °C and 35 °C for 30 days in a laboratory aerobic incubation experiment with soil372 evolved by the soil in the mason jar. The mason jar was sealed and incubated at 25 °C and 35 °C in the dark for 30 days. After incubation for 6, 12, 18, 24, and 30 days, the glass tubes that were equipped with NaOH were removed, and the mason jar was opened for several minutes to maintain sufficient O2 levels. The amount of CO2 was determined by titration of the NaOH solution with 0.1 M HCl in two drops BaCl2.Microbial respiration was measured by alkali absorption of CO10) of microbial respiration was determined using equation (1)48Temperature sensitivity  and T1 (25 °C), which are the temperature levels within 30 days of incubation.where R2) was calculated by dividing the microbial respiration (mg CO2 30 day) per kg by the MBCThe microbial respiration quotient . Soil and plant characteristics were tested for significant contributions to explain the variations in the PLFAs data with redundancy analysis (RDA) using CANOCO software for Windows 4.5. The statistical significance of the RDA was tested using the Monte Carlo permutation test .All of the statistical analyses were performed using SPSS Statistics 19 software. Data not meeting assumptions of normality and homogeneity of variance were log- or cube root-converted prior to statistical testing. One-way analysis of variance (ANOVA) was used to determine the statistical significance of the effect of How to cite this article: Yang, W. et al. Spartina alterniflora invasion alters soil microbial community composition and microbial respiration following invasion chronosequence in a coastal wetland of China. Sci. Rep. 6, 26880; doi: 10.1038/srep26880 (2016).


Host-derived lipids including cholesteryl esters (CEs) such as cholesteryl linoleate have emerged as important antibacterial effectors of innate immunity in the airways and cholesteryl linoleate has been found elevated in the context of inflammation. Cystic fibrosis (CF) patients suffer from chronic infection and severe inflammation in the airways. Here, we identified and quantified CEs in bronchoalveolar lavage fluid (BALF) from CF patients and non-CF disease controls, and tested whether CE concentrations are linked to the disease.CEs in BALF from 6 pediatric subjects with CF and 7 pediatric subjects with non-CF chronic lung disease were quantified by mass spectral analysis using liquid chromatography coupled with tandem mass spectrometry and multiple reaction monitoring. BALFs were also examined for total lipid, total protein, albumin, and, as a marker for inflammation, human neutrophil peptide (HNP) 1–3 concentrations. Statistical analysis was conducted after log 10 transformation of the data.p = 0.018) but the concentrations of CEs, including cholesteryl linoleate, were elevated in the total lipid fraction in CF BALF compared to non-CF disease controls (p < 0.050). In addition, the concentrations of CEs and HNP1-3 correlated with one another (p < 0.050).Total lipid/protein ratio was reduced in CF BALF (The data suggests that the lipid composition of BALF is altered in CF with less total lipid relative to protein but with increased CE concentrations in the lipid fraction, likely contributed by inflammation. Future longitudinal studies may reveal the suitability of CEs as a novel biomarker for CF disease activity which may provide new information on the lipid mediated pathophysiology of the disease. Pseudomonas aeruginosa, as well as chronic neutrophil-driven inflammation ).Raw data were initially analyzed with Excel 2013 and Sigmaplot version 9.0 was used for graphing. IBM SPSS version 20.0 was used for statistical analysis and unless stated otherwise. Statistical significance was determined by first testing for equality of variances using Levene’s test. If there was no evidence of unequal variances, a one-tailed Student’s t-test for independent samples was used; otherwise, Welch's t-test for unequal variances was used. Probability levels less than 0.05 were flagged as statistically significant. With the exception of calculating statistical significance for differences in age, BALF volume, and infection status, statistical tests were run after log transformation of the data  and 7 non-CF disease control pediatric subjects (Non-CF). There was a statistically significant difference between Non-CF and CF subjects in age  and BALF volume , respectively). There was no statistically significant association between disease (Non-CF versus CF) and infection status (p = 0.500 using Fisher’s one-sided exact test for a 2x2 contingency table). Compared to Non-CF, cell counts were elevated in BALF collected from CF subjects, namely nucleated cells 288 ± 78 versus 1732 ± 721, red blood cells 178 ± 117 versus 1331 ± 777, and neutrophils 32 ± 12 versus 1253 ± 618 , and for nucleated cells and neutrophils the differences reached statistical significance after log transformation .Table 2). The CE profile for each patient is shown in Fig 1. The most abundant CE was linoleate, which averaged 88.5 ± 54.5 pmol/mL in Non-CF BALF and 166.9 ± 55.3 pmol/mL in CF BALF (means ± S.E.M.). The next most abundant esters were oleate  and palmitate . Cholesteryl arachidonate concentrations were 16.7 ± 9 pmol/mL in Non-CF and 24.5 ± 8.4 pmol/mL in CF. Cholesteryl linolenate levels averaged 2.8 ± 2 pmol/mL in Non-CF and 5.0 ±1.8 pmol/mL in CF. Of these, the linoleate and arachidonate esters are known to have antimicrobial activity against P. aeruginosa [On the basis of mass concordance and HPLC retention time, the molecular species of CEs detected in BALF were palmitate (16:0), palmitoleate (16:1), oleate (18:1), linoleate (18:2), linolenate (18:3), arachidate (20:0), paullinate (20:1) and arachidonate (20:4) , but the ratio of total lipid to total protein was significantly decreased in CF BALF  and when CE concentrations were adjusted to total lipid, the CEs of palmitate (p = 0.03), oleate (p = 0.028), linoleate (p = 0.021), linolenate (p = 0.016), and arachidonate (p = 0.038), as well as cholesteryl linoleate and cholesteryl arachidonate combined, and all CEs combined, were significantly elevated in CF compared to Non-CF . We then questioned whether this increase correlates with inflammation.To quantify total lipid content, a protocol that employs the fluoroprobe Nile red was adapted . Nile reTable 3), and then their relationship to CEs was determined.Inflammation is typically accompanied by an increase in total protein and increased plasma transudation to deliver additional host defense proteins, and an influx of immune cells, particularly neutrophils in CF. Albumin can be used to assess plasma transudation, and the antimicrobial peptides HNP1-3, major constituents of neutrophil granules, are reliable markers for neutrophil infiltration. In addition to neutrophil counts, these markers were used to assess the lung inflammation status of the subjects (versus 364.2 ± 70.8 μg/mL (p = 0.177) and albumin concentrations were 52.1 ± 26.0 versus 35.0 ± 10.1 μg/mL (p = 0.669), Non-CF versus CF, respectively. When CE concentrations were adjusted to total protein and albumin concentrations, there was no statistically significant difference between CF and Non-CF subjects.Total protein and albumin concentrations did not differ significantly between Non-CF and CF subjects; total protein concentrations were 236.9 ± 51.3 Fig 3 and Table 3). HNP1-3 was present at low levels in Non-CF and significantly elevated in CF patients . Furthermore, HNP1-3 concentrations as well as neutrophil counts correlated with the same CEs that represented an increased fraction of total lipid in CF patients (Table 5). In contrast, lung infection status did not correlate with any of the CEs .Next, HNP1-3 was quantified Click here for additional data file.


Pseudomonas pseudoalcaligenes S1, a marine bacterium, exhibited strong resistance to a high concentration of Hg2+ and remarkable Hg2+ bioaccumulation capacity. Here, we report the 6.9-Mb genome sequence of P. pseudoalcaligenes S1, which may help clarify its phylogenetic status and provide further understanding of the mechanisms of mercury bioremediation in a marine environment. P. pseudoalcaligenes S1 (CGMCC 7.206) from seawater and marine sediment samples in a mangrove conservation district of Shenzhen Bay, China . P. pseudoalcaligenes S1 was preliminarily investigated by us    with optProtein-coding genes were predicted using the NCBI Prokaryotic Genome Annotation Pipeline, which is designed to annotate bacterial and archaeal genomes. Finally, we obtained 6,500 genes, 5,779 protein-coding genes, 78 tRNAs, 8 rRNAs, and 1 noncoding RNA.merR), mercuric transport and binding (merT and merP), and metal ion efflux pump (CzcA) were identified from the P. pseudoalcaligenes S1 complete genome. They may be responsible for the adaptation of mercury contamination through transmembrane transportation and active efflux  was also predicted from our transcriptome data; however, its function remains a mystery.Genes for mercuric regulation (e efflux –13, therJTFL00000000. The version described in this paper is the first version, JTFL01000000.This whole-genome shotgun project has been deposited in DDBJ/EMBL/GenBank under the accession no.