Plants with altered intracellular Pi compartmentation surprisingly show accelerated dark-induced senescence. We propose that low cytosolic phosphate concentrations are of critical importance for proper plant development. trans-Golgi compartment, and its impaired activity causes altered intracellular phosphate compartmentation, leading to low cytosolic Pi levels, a blockage of Golgi-related processes such as protein glycosylation and hemicellulose biosynthesis, and a dwarf phenotype. However, it was unclear whether altered Pi homeostasis in pht4;6 mutants causes further cellular problems, typically associated with limited phosphate availability. Here we report that pht4;6 mutants exhibit a markedly increased disposition to induce dark-induced senescence. In control experiments, in which pht4;6 mutants and wild-type plants developed similarly, we confirmed that accelerated dark-induced senescence in mutants is not a ‘pleiotropic’ process associated with the dwarf phenotype. In fact, accelerated dark-induced senescence in pht4;6 mutants correlates strongly with increased levels of toxic NH4+ and higher sensitivity to ammonium, which probably contribute to the inability of pht4;6 mutants to recover from dark treatment. Experiments with modified levels of either salicylic acid (SA) or trans-zeatin (tZ) demonstrate that altered concentrations of these compounds in pht4;6 plants act as major cellular mediators for dark-induced senescence. This conclusion gained further support from the notion that the expression of the pht4;6 gene is, in contrast to genes coding for major phosphate importers, substantially induced by tZ. Taken together, our findings point to a critical function of PHT4;6 to control cellular phosphate levels, in particular the cytosolic Pi availability, required to energize plant primary metabolism for proper plant development. Phosphate and its allocation mediated by PHT4;6 is critical to prevent onset of dark-induced senescence.The phosphate transporter PHT4;6 locates to the  Phosphate is a macronutrient present in all types of cells, and its omnipresence is due to its requirement for synthesis of, for example, nucleic acids, various types of nucleotides, a large number of phosphorylated primary and secondary intermediates, or structural lipids necessary for functional biomembranes. Moreover, phosphate is involved in energy transfer, cellular signal transduction processes, and reversible protein modification, which further highlights the critical importance of this compound  as HPO stimuli . BecauseRoot cells import Pi from the soil via plasma membrane-located PHT1-type carriers, catalyzing a proton-coupled symport , which strans-Golgi-located PHT4;6 protein  were used. PHT4;6 loss-of-function plants have already been described at the genetic and molecular level  using the floral-dip method  medium containing 0.05% MES with or without 1% sucrose. Plants used for nitrogen experiments were cultivated on agar plates with 1/2 MS medium (–N) with addition of KNO3, NaNO3, NH4Cl, or NH4PO4 as the sole nitrogen source. Seeds used for sterile culture were surface sterilized in 5% sodium hypochloride and subsequently incubated for 2 d in the dark at 4 °C for stratification.For all studies, p method . ExperimSAG12, SAG12_nor_fwd and SAG12_nor_rev; SAG13, SAG13_nor_fwd and SAG13_nor_rev; NAC029, NAP_nor_fwd and NAP_nor_rev; SGN1, NYE1_nor_fwd and NYE1_nor_rev; ATG7, ATAGP7_nor_fwd and ATAGP7_nor_rev; S3H, S3H_nor_fwd and S3H_nor_rev; and WRKY53, WRKY53_nor_fwd and WRKY53_nor_rev (see Supplementary Table S1 at JXB online). The RNA loading control was performed by ethidium bromide staining.Determination of mRNA abundance via northern blot was carried out as described previously (http://www.retsch.de). Cytokinin extraction, purification, and quantification were carried out as described by Samples for cytokinin measurements were ground under liquid nitrogen with a mortar and pestle, freeze-dried, and ground again with a Retsch ball-mill . A 50mg aliquot of ground plant material was dissolved in 80% (v/v) ethanol, heated for 10min at 95 °C, and centrifuged for 5min at 17 000 g. The supernatant was transferred and the extraction was repeated with the remaining sediment. Both supernatants were pooled and extinction was measured at 652nm in a microplate reader .Chlorophyll quantification was performed according to a modified method  supplemented with 10 µl ml–1 protease inhibitor  and 1mM phenylmethylsulfonyl fluoride (PMSF). After centrifugation , the supernatant was used for determination of soluble protein (http://www.nanodrop.com).Soluble proteins were isolated from 50mg of ground plant material by addition of 500 µl of phosphate-buffered saline , the supernatant was used for ion chromatography quantification. Sugar quantification was performed by ion chromatography on a Metrosep CARB1-150 column  using an 871-IC compact device (Metrohm) followed by amperometric quantification. NaOH (0.1M) was used as the mobile phase with a flow rate of 1ml min–1. The pressure was 9MPa at a temperature of 27 °C. Quantification of cations was performed by using a 761-IC compact device (Metrohm) on a Metrosep Cation column (Metrohm), followed by conductometry at a flow rate of 1ml min–1 and pressure of 7.5MPa, using 0.1N HNO3 and 1.7mM dipicolinic acid as the mobile phase.For isolation of sugars and cations, plant material was ground under liquid Nhttp://www.walz.com) using ImagingWin software.The method was as described by PHT4;6 (At5g44370), ATG7 (At5g45900), NAC029 (At1g69490), SGN (At4g22920), SAG12 (At5g45890), SAG13 (At2g29350), S3H (At4g10500), and WRKY53 (At4g23810).pht4;6 plants exhibit a number of altered cellular properties which might affect the plant senescence program. However, when grown on soil under standard SD conditions (10h light/14h dark), pht4;6 mutant plants did not exhibit strong phenotypic symptoms of altered senescence when compared with wild-type plants  when compared with the mutant line  than the corresponding wild type  completely and showed only a few chlorotic and dead leaves . This redarkness . Alreadyried out .–1 FW  during onset of re-illumination  prevented senescence symptoms in pht4;6 mutants, and also the application of sucrose strongly suppressed senescence in these mutants .We have already reported that pht4;6::nahg mutants exhibited fewer symptoms of dark-induced senescence when compared with pht4;6 plants .6 plants . Even afants see , in pht4SAG12, encoding a cysteine protease, or SAG13, coding for an oxidoreductase expressed in the early phase of senescence.The expression of a large number of genes has to be altered to induce senescence in plants. These SAGs encode, for example, proteases, transcription factors, proteins involved in mobilization of nitrogen and carbon, or transport proteins  revealed that the day four . Surpris mutants  and decrly again . SAG13 my to day . NAC029,ild type . The trae plants .ATG7 mRNA. A weak ATG7 expression was detectable in the wild type until day three of dark treatment, and at day four mRNA accumulated strongly in these plants , as well as dihydrozeatin (DHZ), N6-(Δ2-isopentenyl)adenine (iP), and their glycosylated derivatives (tZ levels (787.42±35.88 pmol g–1 DW) when compared with pht4;6 plants (479.64±48.45 pmol g–1 DW). After onset of dark-induced senescence, tZ in wild-type plants further increased, reaching 1077.36±85.83 pmol g–1 DW, while tZ contents in pht4;6 plants remained low , but in pht4;6 plants cZ concentrations increased further, reaching 722.71±57.99 pmol g–1 DW  and after the fourth day of dark treatment (473.37±79.02 pmol g–1 DW) compared with the wild type (811.25±34.96 pmol g–1 DW and 1083.96±92.03 pmol g–1 DW). In contrast, the concentrations of the O-glycosides were significantly increased in mutant plants , and phosphate-supplemented wild-type and pht4;6 plants showed only a few yellow leaves  altered SA contents; (ii) expression of pathogen-related genes; and (iii) accumulation of H2O2 —delays onset of dark-induced senescence  and ahk2/ahk3 mutants also results in enhanced sugar sensitivity and an impact on the Pi starvation response. In other words, we here provide the first evidence that intracellular Pi homeostasis, rather than the total cellular Pi level, governs zeatin-specific processes in Arabidopsis.To understand this specific effect, we have to consider that ild type –D, indicSupplementary data are available at JXB onlineFigure S1. Analysis of pht4;6::nahG overexpression plants.Figure S2. Chlorophyll fluorescence image of the analyzed plants.Table S1. List of primers used in this work.


A total of four analytical tools, geNorm, Normfinder, BestKeeper, and the tΔC method, were used to evaluate the suitability of these genes as endogenous controls. According to RefFinder, a web-based software tool which integrates all four above-mentioned algorithms to compare and rank the reference genes, SDHB, 16S, and NADH were the three most stable house-keeping genes under different developmental stages and temperatures. This work is intended to establish a standardized qRT-PCR protocol in pea aphid and serves as a starting point for the genomics and functional genomics research in this emerging insect model.To facilitate gene expression study and obtain accurate qRT-PCR analysis, normalization relative to stable expressed housekeeping genes is required. In this study, expression profiles of 11 candidate reference genes, including  It feeds on a wide range of legume plants(family Fabaceae) worldwide, including pea, clover, alfalfa, and broad bean, and is considered as the aphid species of major agronomical importance A. pisumis an emerging model organism for the studies of insect-plant interactions, especially after the release of its genome in 2010 The pea aphid, Acyrthosiphon pisum, as well as in other insects which is the selection of appropriate references genes with stable expression under different experimental conditions. Here, the expression profiles of 11 candidate reference genes, including actin (Actin), elongation factor 1 α (EF1A), TATA-box-binding protein (TATA), ribosomal protein L12 (RPL12), β-tubulin (Tubulin), NADH dehydrogenase (NADH), vacuolar-type H+-ATPase (v-ATPase), succinate dehydrogenase B (SDHB), 28S ribosomal RNA (28S), 16S ribosomal RNA (16S), and 18S ribosomal RNA (18S) from the pea aphid genome The objective of this study was to address an important aspect of gene expression studies in the pea aphid, Acyrthosiphon pisum (Harris) colony was kindly provided by Dr. John Obrycki (University of Kentucky). Aphids were maintained at 20–28°C on seedlings of fava bean, Viciafaba in a greenhouse.Pea aphid, Fifteen adult females were allowed to lay the offspring for 24 h on fava bean leaves resting on wet filter paper in a petri dish (9 cm diameter). Then10 adults as one replicate and 20nymphs (less than 24 h old) as one replicate, respectively were exposed to 10°C, 22°C, and 30°C, respectively for 2 d in a climate chamber with a photoperiod of 14: 10 (L: D) and 50% relative humidity. All collected samples were preserved in 1.5 ml centrifuge tubes and stored at −80°C after being frozen in liquid nitrogen. Each treatment was repeated three times independently, therefore, there are 18 biological samples in total.Total RNA was extracted using TRIzol  following the manufacturer's instruction. First-strand cDNA was synthesized from 1 µg of total RNA using the M-MLV reverse transcription kit  according the manufacturer's recommendations.2+ Plus), 1 µldNTP mix (10 mM of each nucleotide), 5 µl of each primer (10µM each), and 0.25 µl of Go Taq(5 u/µl) (Promega). The PCR parameters were as follows: one cycle of 94°C for 3 min; 35 cycles of 94°C for 30 s, 59°C for 45 s, and 72°C for 1 min; a final cycle of 72°C for 10 min. Amplicons of the expected size were purified and cloned into the pCR4-TOPO vector  for sequencing confirmation.Eleven commonly used reference genes were selected . PCR amp2O, 10 µl of 2×SYBR Green MasterMix (Bio-Rad), 1 µl of each specific primer (10 µM), and 1 µl of first-strand cDNA template. The qPCR program included an initial denaturation for 3 min at 95°C followed by 40 cycles of denaturation at 95°C for 10 s, annealing for 30 s at 55°C, and extension for 30 s at 72°C. For melting curve analysis, a dissociation step cycle  was added. The reactions were set up in 96-well format Microseal PCR plates (Bio-Rad) in triplicates. All experiments were replicated in triplicate.Gene-specific primers  were use[-1/slope] -1)×100.Reactions were performed in a MyiQ single Color Real-Time PCR Detection System (Bio-Rad). Existence of single peaks in melting curve analysis was used to confirm gene-specific amplification and rule out non-specific amplification and primer-dimer generation. The qRT-PCR was determined for each gene using slope analysis with a linear regression model. Relative standard curves for the transcripts were generated with serial dilutions of cDNA . The corresponding qRT-PCR efficiencies (E) were calculated according to the equation: E = (10geNormNormFinderBestKeeperΔCt method RefFinder (http://www.leonxie.com/referencegene.php).All biological replicates were used to calculate the average Ct value. The stability of the ten housekeeping genes were evaluated by algorithms First, 11 candidate reference genes were investigated by reverse transcription polymerase chain reaction (RT-PCR). All genes tested were expressed in pea aphid, and visualized as a single amplicon with expected size on a 1.5% agarose gel. All amplicons were sequenced and displayed 100% identity with their corresponding sequences. Furthermore, gene-specific amplification of these genes was confirmed by a single peak in real-time melting-curve analysis. A standard curve was generated for each gene, using five-fold serial dilution of the pooled cDNAs. The correlation coefficient and PCR efficiency for each standard curve were shown in t values for all the samples together. v-ATPasehad the most variable expression levels reflected in its high SD values. On the contrary, 28S had the least variable expression levels reflected in its low SD values. In addition, TATA (Ctavg = 26.05) had the highest Ct values and was therefore the least expressed among the gene candidates. 18S (Ctavg = 10.72) had the lowest Ct values and was therefore the mostly expressed among the gene candidates.  of the Cdidates. .geNorm. Starting with two genes, the software sequentially adds another gene and recalculates the normalization factor ratio. If the added gene does not increase the normalization factor ratio above the proposed 0.15 cut-off value, then the original pair of genes is enough for normalization. However, if the new ratio is above 0.15, then more genes should be included. The first V-value <0.15 was after V2/3 . The lower the M-value is, the higher the ranking. SDHB and 16S were co-ranked as the most stable genes (M = 0.382). The overall order based on geNorm from most stable to least stable reference genes was: SDHB = 16S, NADH,18S, RPL12,28S, Tublin, Actin, EF1A, v-ATPase, TATA  was the top-ranked gene. The overall order from most stable to least stable reference genes based on the tΔC method was: SDHB, NADH,16S, RPL12,18S, Actin, EF1A,28S, Tublin, v-ATPase, TATA was the gene with the least variation in expression levels; thus SDHB would be the most reliable reference gene. The overall order from most stable to least stable reference genes based on NormFinder was: SDHB, NADH,16S, RPL12,18S, Actin, EF1A,28S, Tublin, v-ATPase, TATA  and 16S (r = 0.852) had the highest[r] value, whereas 28S(SD = 0.321) and EF1A (SD = 0.371) had the least variable expression levels across all the samples Click here for additional data file.Table S2Ranking of 11 reference gene candidates based on BestKeeper. Two criteria are considered: Pearson's correlation coefficient and BestKeeper computed SD values. The stability of a gene is directly proportional to the [r] value, while it is inversely proportional to the SD value.(DOCX)Click here for additional data file.


Coprinus comatus with an isolation procedure including three ion-exchange chromatography steps on DEAE-cellulose, CM-cellulose, and Q-Sepharose and one gel-filtration step by fast protein liquid chromatography on Superdex 75. The purified enzyme was a monomeric protein with a molecular weight of 64 kDa. It possessed a unique N-terminal amino acid sequence of AIGPVADLKV, which has considerably high sequence similarity with that of other fungal laccases, but is different from that of C. comatus laccases reported. The enzyme manifested an optimal pH value of 2.0 and an optimal temperature of 60°C using 2,2′-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) as the substrate. The laccase displayed, at pH 2.0 and 37°C, Km values of 1.59 mM towards ABTS. It potently suppressed proliferation of tumor cell lines HepG2 and MCF7, and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC50 value of 3.46 μM, 4.95 μM, and 5.85 μM, respectively, signifying that it is an antipathogenic protein.A novel laccase was isolated and purified from fermentation mycelia of mushroom  Laccases , belonging to polyphenol oxidases, play a key role in lignin degradation in nature. They can oxidate a variety of phenolic and inorganic compounds, including diphenols, polyphenols, and substituted phenols, using molecular oxygen as the electron acceptor . In receCoprinus comatus, commonly named as lawyer's wig or shaggy mane, is a common fungus often seen growing on lawns in spring or autumn. In China, Japan, and other Asian countries, the species is cultivated as an excellent edible mushroom. It is very unusual because it is edible when young but becomes poisonous when old. The cap is white and closely covers the stipe over; then it turns black and dissolves itself in a matter of hours after being picked or depositing spores. Previous studies on C. comatus mainly focus on its polysaccharide extract, antitumor and immunomodulatory activities, fermentation, and so forth. In recent researches, ethyl acetate extract from fruit bodies of C. comatus manifested antiproliferative activity towards human ovarian cancer cell lines [α and IL-2 [ C. comatus promoted femoral fracture healing in streptozotocin-diabetic rats with a 35.5% increase in the total area of callus [ C. comatus. A fibrin-specific fibrinolytic enzyme was produced by liquid culture of C. comatus [ C. comatus, and functionally heterologously expressed it in Pichia pastoris [ll lines . A waterand IL-2 . Vanadiuf callus . There a comatus . Recentlpastoris . C. comatus showed that laccase extract demonstrated different enzymatic properties from those of the recombinant laccase newly reported [ C. comatus and then study its properties and applications.In the present work, preliminary studies based on mycelia ofreported . Hence,  C. comatus were collected in the campus of China Agricultural University . Strain JT-01 was isolated from fresh fruiting bodies. Strain identification was based on a standard ITS sequence amplification and analysis and also fruiting experiments [Fruiting bodies oferiments . The funμL) was mixed with 1 mM ABTS solution  at 37°C for 5 min, followed by ending the reaction by an addition of 10% TCA (250 μL). The change in the absorbance was monitored at 405 nm for enzyme activity. One enzyme unit (U) was defined as the amount of enzyme required to produce one absorbance increase at 405 nm per minute per millilitre of the reaction mixture under the assay conditions. All determinations were performed in triplicate.Laccase activity was determined by a modified method described by Shin and Lee using 2,2′-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) as the substrate . In brie4)2SO4 was added to the supernatant until 80% saturation to precipitate proteins. The mixture was left 4°C for 4 h, then centrifuged at 10000 rpm and 4°C for 15 min, and dialyzed against distilled water overnight. The crude laccase extract was further purified by three successive steps of ion exchange chromatography: firstly on DEAE-cellulose  with a flow rate of 2 mL/min, secondly on CM-cellulose  with a flow rate of 2 mL/min, and finally on Q-Sepharose  with a flow rate of 1 mL/min. The laccase active fraction was finally purified by fast protein liquid chromatography (FPLC) on a Superdex 75 HR 10/30 gel filtration column  with a flow rate of 0.8 mL/min.After two-week fermentation, mycelia were harvested by centrifugation at 10000 rpm and 4°C for 15 min. Subsequently, the mycelia homogenized and extracted in 0.15 M NaCl  at 4°C overnight, followed by another centrifugation at 10000 rpm and 4°C for 15 min. Then, (NHMr) of the purified laccase, both FPLC-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used. In FPLC-gel filtration, a standard curve based on elution volume and Mr of molecular mass standards  can be obtained. Mr of the present laccase fraction can be calculated [ Mr was calculated based on another curve of relative mobility and log Mr.To determine the molecular mass  and sodium citrate acid buffers (pH 2.2–8.0). In the temperature assay, standard assay mixture was tested in different temperature (20–100°C) instead of 37°C in the standard assay. Km values were obtained from a Lineweaver-Burk plot [The Michaelis-Menten constants of the purified laccase were determined using ABTS as substrate at pH 2.0 in various concentrations  and at 37°C. All determinations were performed in triplicate. Theurk plot .3, CaCl2, CoCl2, CuCl2, FeCl2, HgCl2, KCl, LiCl, MgCl2, MnCl2, ZnCl2, and EDTA. Control samples were assayed without the metal ions. All determinations were performed in triplicate.To estimate metal ions and EDTA on enzyme activity, equal volumes of the purified laccase solution were preincubated with metal ions or EDTA solutions  at 4°C for 1 h before the standard laccase assay was performed. The chemical reagents of metal ions were including AlCl2. Cells were seeded into 96-well plates with a concentration of 8 × 103 cells/well and incubated for 24 h. Different concentrations of C. comatus laccase were added into the wells with serum-free medium and incubated for 72 h. After that, MTT assays were carried out to measure cell viability. Briefly, 20 μL of a 5 mg/mL solution of MTT in serum-free medium was spiked into each well, and the plates were incubated for 4 h. The supernatant was carefully removed, and 200 μL of DMSO was added into each well to dissolve the MTT formazan. The absorbance at 560 nm was measured with a microplate reader. PBS was added into the wells instead of laccase as the control [Antiproliferative activity has been reported for many mushroom proteins . The tum control , 26.The assay for the inhibitory activity on human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from Boehringer  . The assMr) of 64 kDa. In SDS-PAGE, SU1 fraction appeared as a single band with a Mr of 64 kDa  is AIGPVADLKV. An N-terminal amino acid sequence comparison of CCL in the present study and other laccases or laccase-like proteins from C. comatus earlier reported was listed in  Km value of 1.59 mM towards ABTS using Lineweaver-Burk plots  [ C. comatus laccases or laccase-like proteins reported, but they are obviously different with the highest similarity of 80%. It suggests that the purified laccase in the present study is a novel laccase among those C. comatus laccases reported. On the other hand, CCL also manifests considerable similarity with other Polyporaceae laccases at the N-terminal position, which suggests that they might share a similar protein secondary or tertiary structure.The shaggy mane mushroom is a common but unusual mushroom species because it is widely distributed but will turn black and dissolve itself in a matter of hours after maturation. Up to now, only 6 protein sequences from10993.1) . The pre Agrocybe cylindracea [ Russula virescens [ Pleurotus eryngii [ Lepiota ventriosospora is unabsorbed on CM-cellulose [ Clitocybe maxima (16.8-fold) [ Hericium erinaceus (15-fold) [ C. maxima laccase, SP-Sepharose, a kind of strong cation chromatography media, was used instead of CM-cellulose. Parts of laccase active fraction might be irreversibly adsorbed on the gel. On the other hand, when the H. erinaceum laccase was purified, four successive steps of ion exchange chromatography and one gel filtration were used. The more the purification steps were used, the less the objective proteins were gained.During the purification process, CCL was absorbed on DEAE-cellulose, CM-cellulose, and Q-Sepharose, just like other fungal laccases fromindracea , Russulairescens , and Ple eryngii . On the ellulose . The pur.8-fold)  and the 15-fold) . During  Pleurotus nebrodensis (64 kDa) [ Pleurotus nebrodensis (64 kDa) [ H. echinaceus (63 kDa) [ C. comatus laccase is a monomeric protein, while laccases from Phellinus ribis and Gaeumannomyces graminis are dimeric and quadruple proteins, respectively [The present laccase is a monomeric protein with a molecular mass of 64 kDa, which falls well within the range of molecular masses for most of the fungal laccases reported (50–90 kDa) . It is j(64 kDa) . that of(64 kDa) , H. echi(63 kDa) . Just liectively , 35. A. cylindracea (pH 5.2) [ P. nebrodensis (pH 5.0) [ L. ventriosospora (pH 4.0) [ A. blazei (pH 2.3) and R. virescens (pH 2.2) also share a very low pH optimum, while laccase from Pleurotus ostreatus reaches its maximal oxidizing activity towards ABTS at pH 6.9 which is very close to neutral [ R. virescens [ A. placomyces (30°C) [ Inonotus baumii (20°C) [The purified laccase manifests a quite low pH optimum of pH 2.0 towards ABTS, which is much lower than that of laccases from(pH 5.2) , P. nebr(pH 5.0) , and L. (pH 4.0) . Laccase neutral . The preirescens , twice as (30°C) , and thri (20°C) . It sugg Km value of the purified laccase towards ABTS at pH 2.0 and 37°C was 1.59 mM which was 10 times higher than that of R. virescens laccase (0.115 mM) [ C. comatus laccase (Lac1) manifested a much lower Km value towards ABTS of 34 μM at its optimal enzymatic conditions (pH 3.0 and 65°C) [ Km value was assayed at a lower temperature of 37°C but not its optimal temperature of 60°C. That is why Km of CCL was much higher than that of Lac1. The present laccase was not sensitive to the assayed metal ions and EDTA except Fe2+. Fe2+ is a reducing agent and strongly decreases most of the reported laccases, such as Lac1 from C. comatus [ Abortiporus biennis [ I. baumii [ R. virescens [2+ was a special ion for the laccase activity. High concentration of Cu2+ (5–10 mM) slightly increased the laccase activity just like that of laccases for Polyporus sp. [ A. biennis [The.115 mM) . A recom biennis , I. baumirescens . Cu2+ wa biennis . CCL is 50 value towards HepG2, MCF7, and HIV-1 RT of 3.46 μM, 4.95 μM, and 5.85 μM, respectively, indicating that it is also an antipathogenic protein. The laccase from I. baumii, a very famous traditional Chinese medicinal mushroom, also manifests antiproliferative activities towards tumor cell lines HepG2 and L1210 with IC50 values of 2.4 μM and 3.2 μM, respectively, but is devoid of inhibitory activity toward HIV-1 RT. On the other hand, the present laccase is purified from liquid fermentation, which means that the protein is very easy to obtain. It is noteworthy that it possesses further applications of agents for cancer or AIDS therapy.It is remarkable that some of mushroom components including laccases, lectins, polysaccharopeptides, and antifungal proteins exhibit inhibitory activities towards tumor cells or HIV-1 RT. In the present study, CCL manifests both antiproliferative and anti-HIV-1 RT activities with an IC C. comatus obtained from liquid fermentation. Characterization studies show that the enzyme possesses a molecular mass of 64 kDa, a pH optimum at 2.0, a temperature optimum at 60°C, and a Km value of 1.59 mM towards ABTS. The laccase also exhibits antiproliferative activity towards tumor cells and inhibitory activity toward HIV-1RT, suggesting that it is an antipathogenic protein.In summary, a novel laccase (CCL) with a distinctive N-terminal sequence is purified from mycelia of mushroom


A novel fluorescence sensor (BIP) bearing NH and OH subunits displayed a highly selective and sensitive recognition property for fluoride over other anions. Fluoride-driven ESPT, poorly used in anion recognition and sensing, was suggested to be responsible for the fluorescence enhancement with a blue shift of 35 nm in the emission spectrum. Design and synthesis of selective and efficient sensors for various anions involved in biological, industrial and environmental processes have drawn a lot of attention –3. In re15E)-2-(((1H-benzo[d]imidazol-2-yl)imino)methyl)-5-(dimethylamino)phenol (BIP) made up of benzimidazole and phenol units with a C=N linker. Although BIP is weakly fluorescent, its anion-binding adduct is expected to be highly fluorescent by opening up ESPT channel [15During the last few years, various types of artificial sensors have been developed for anion recognition and sensing –1519–20.1515 channel –1519. In1H NMR, mass spectrometry and elemental analysis before its practical application.BIP was synthesized by the condensation of 2-aminobenzimidazole and 4-(dimethylamino)salicylaldehyde according to the literature –29 with 3CN. Among these, the band in the region around 276 and 228 nm is attributed to the excitation of π electrons localized at the C=N bond and at the aromatic system [− , as shown in −, Br−, I−, H2PO4−, NO3−, AcO−, added as their TBA salts). This indicated the poor interaction between BIP and these anions.The anion binding and sensing properties of BIP were first evaluated by UV–vis studies. There were three characteristic bands centered at 410, 276 and 228 nm in CHc system , and thec system . Upon ad−. The association constant, calculated from the UV−vis plot at 410 nm as the function of anion concentration [4 (R = 0.9926) for F− , as displayed in − and OH− , fluorescence measurements were carried out. The weak fluorescence emission at 411 nm, corresponding to the enol form of a Schiff base [− to the CH3CN solution of BIP, the fluorescence intensity at 411 nm increased slowly. This indicated the occurrence of hydrogen-bonding interaction between BIP and F−. Moreover, the acidity of the OH group of BIP was expected to be enhanced by anion binding, and the ESPT channel would be open [10−. The fluoride-driven ESPT displayed a fluorescence enhancement and a dramatically blue-shift with 35 nm in the emission spectrum.To further explore the photophysical sensing properties of BIP with various anions  also indicated the formation of an 1:1 stoichiometric complex of BIP with F−  in the concentration range of 0.04–1.2 μM salicylaldehyde was synthesized according to the literature [3  was added dropwise to dry DMF  containing 3-(dimethylamino)phenol  at 0 °C, and the mixture was stirred for 10 min, slowly warmed to room temperature and stirred for another 30 min. The reaction mixture was heated at 80 °C overnight. After cooling to room temperature, the mixture was poured into ice cold water. The solution was neutralized with saturated Na2CO3. The precipitate was washed several times with distilled water, and dried under vacuum to yield 3.30 g of 1. 1H NMR  δ 3.09 , 6.10–7.29 , 9.53 , 11.61 . This was consistent with the literature.terature –16 with Synthesis of compound BIP: 2-Aminobenzimidazole  and compound 1  were dissolved in 25 mL ethanol. The resulting solution was heated at reflux for 4 h. The solvent was removed by a rotary evaporator at reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate/n-hexane 1:3) and recrystallized with ethanol to yield yellow solid. Yield: 1.03 g (61.2%); 1H NMR  δ 3.05 , 6.16–7.53 , 9.34 , 12.41 , 12.85 ; ESIMS: 280.1408 (M + H+).File 1Data analysis of QP after the addition of fluoride.


The emergence of an infectious viral disease caused by the Chinese giant salamander iridovirus (GSIV) has led to substantial economic losses. However, no more molecular information is available for the understanding of the mechanisms associated with virus–host interaction. In this study, de novo sequencing was used to obtain abundant high-quality ESTs and investigate differentially-expressed genes in the spleen of Chinese giant salamanders that were either infected or mock infected with GSIV. Comparative expression analysis indicated that 293 genes were down-regulated and 220 genes were up-regulated. Further enrichment analysis showed that the most enriched pathway is “complement and coagulation cascades”, and significantly enriched diseases include “inherited thrombophilia”, “immune system diseases”, “primary immunodeficiency”, “complement regulatory protein defects”, and “disorders of nucleotide excision repair”. Additionally, 30 678 simple sequence repeats (SSRs) from all spleen samples, 26 355 single nucleotide polymorphisms (SNPs) from the spleens of uninfected animals and 36 070 SNPs from the spleens of infected animals were detected. The large amount of variation was specific for the Chinese giant salamanders that were infected with GSIV. The results reported herein provided significant and new EST information that could contribute greatly in investigations into the molecular functions of immune genes in the Chinese giant salamander.The online version of this article (doi:10.1186/s13567-015-0279-8) contains supplementary material, which is available to authorized users. Andrias davidianus, is the largest extant amphibian species and is considered a living fossil because it has existed for more than 350 million years [Amphibians are an important evolutionary bridge between aquatic and terrestrial vertebrates . The Chion years . The phyon years . CurrentWith the rapid expansion of Chinese giant salamander farming, emerging infectious diseases have been increasing. Viral diseases, including the iridovirus, have caused major impacts to the Chinese giant salamander industry , 6. The Next-generation sequencing technology, such as de novo transcriptome sequencing, can be used for large-scale efficient and economical production of ESTs, and has become an important method for studying non-model species , 19. TraHypophthalmichthys nobilis) for 2 weeks prior to the experiment. The virus suspension used to infect a portion of the salamanders was obtained from GSIV-infected EPC cells. The fish in infected group (GS_TS) were injected intramuscularly with 0.2 mL of the GSIV suspension (5 × 107 TCID50/mL), and fish in control group (GS_CS) were injected intramuscularly with equal volume of DPBS (Sigma). The spleens from three individual salamanders from each group were collected at 48 h post-injection.Chinese giant salamanders  were obtained from the research farm of the Yangtze River Fisheries Research Institute in Wuhan, China. Prior screening indicated that these animals were free of GSIV. All the salamanders were kept in aerated, tap water supplied tanks at 20 °C and fed with diced bighead carp . Samples of the three individuals from each treatment group were pooled in equal amounts to generate one RNA sample per group. These two RNA samples were sent to Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd.  for the cDNA library construction and Illumina deep sequencing.Total RNA was extracted from the spleens using TRIzolTwo cDNA libraries were prepared using the TruseqTM RNA sample prep Kit  following the manufacturer’s instructions. Briefly, poly (A)+ RNA was purified from 5 μg of pooled total RNA using oligo (dT) magnetic beads, sheared into short fragments, and primed for cDNA library synthesis using the TruSeq RNA sample preparation kit according to the manufacturer’s instructions (Illumina). After quantitation using a TBS-380 minifluorometer (PicoGreen), the samples were clustered  and sequenced on the HiSeq 2000 platform .−5. GO terms were extracted from the best hits obtained from the BLASTx against the nr database using Blast2GO [−6) to predict possible functional classifications and molecular pathways [The raw reads from the images were generated using Solexa GA pipeline 1.6. After the removal of low-quality reads, processed reads with an identity value of 95% and a coverage length of 100 bp were assembled using the Trinity de novo assembler . The isoBlast2GO . These rpathways .p value was computed, and then Benjamini–Hochberg false discovery rate (FDR) was applied to correct the results for p value. The transcripts that were increased or decreased at an estimated absolute log2-fold change of >1 and FDR adjusted p value ≤ 0.05 were considered to be differentially expressed.To identify differentially expressed genes/isogenes between infected and uninfected groups, genes/isogenes expression levels were measured by using numbers of fragments per kilobase of transcript per million fragments sequenced (FPKM) , similarMSATCOMMANDER V. 0.8.2  was used−ΔΔCt method.Quantitative real time PCR was performed using iQ™ SYBR Green Supermix  on a BIO-RAD CFX96 Real-Time System under the following conditions: 3 min at 95 °C, followed by 45 cycles of 15 s at 94 °C, 15 s at 55 °C and 30 s at 72 °C. Different genes including complement component C1R, C1S, C1S-like, C2, C3, C4, C5, C7, C8A and C9 were used for validation. An additional file shows the primer sequences used in this study  was over 98% for both samples (Table Two sequencing libraries were prepared from spleen samples obtained from control (GS_CS) and GSIV-infected (GS_TS) Chinese giant salamanders that were sequenced using an Illumina Hiseq 2000. In total, 122.48 million raw reads were generated from GS_CS and 154.75 million for GS_TS. The data was refined by discarding low-quality reads that contained unknown bases or whose length was lower than 20 nt after removal of the adaptors and low-quality bases. The resulting high-quality reads numbered 113.45 million and 143.78 million for the GS_CS and GS_TS samples, respectively. The total length of these reads was 9.6 × 10De novo assembly was performed using Trinity that resulted in 80 367 genes and 123 440 isogenes. The total length was 182 916 518 bp, with an average length of 1481 bp (Table −5. There were 38 106 (64.6%) isogenes with homologous sequences in at least one of the databases. Among them, 38 106 (64.6%), 15 579 (26.4), and 20 352 (34.5%) isogenes were found in NR, String and Gene databases, respectively. A total of 9629 (16.3%) isogenes were found in all three databases, while 20 873 (35.4%) isogenes were not identified  isogenes contained an ORF. The predicted protein sequences were blasted against NR (non-redundant protein sequences in NCBI), String and Gene databases using BLASTP with a cut-off E value of 10−5. There were 10 138 (15.7%) isogenes with homologous sequences in at least one of above databases. Among them, 10 138 (15.7%), 959 (1.5%), and 3351 (5.2%) isogenes were found in the NR, String and Gene databases, respectively. A total of 516 (0.8%) isogenes were found in all three databases, while 54 323 (84.3%) isogenes were not identified  isogenes could not be predicted, and the DNA sequences of 64 461 isogenes were blasted against the NR, String and Gene databases using BLASTX with a cut-off E value of 10Based on NR annotations, the Gene Ontology (GO) classification system was used to classify the possible functions of the isogenes. A total of 31 356 25.4%) isogenes were successfully assigned to at least one GO term annotation and were classified into three main categories including biological process, cellular component and molecular function . Out of 38 106 NR hits, 7719 20.3%) of the sequences showed a COG classification . Among those, 23 712 isogenes were assigned to 317 KEGG pathways  that were identified. All DEGs were mapped to each term of the GO database, and the GO terms with a corrected Staphylococcus aureus infection”, “tight junction”, “viral myocarditis”, “transcriptional misregulation in cancer” and “allograft rejection”  were the three predominant types among mononucleotide SSR motifs, with frequencies of 30.93, 30.14 and 27.77%, respectively  have been shown to be an efficient tool for performing quantitative trait loci (QTL) analysis and constructing genetic linkage(s) due to their high diversity and abundance. In the present study, 30 678 SSRs were obtained from the transcriptomic information from the Chinese giant salamander. Among them, the most frequent repeat motifs were mononucleotide SSR motifs (76.2%), followed by trinucleotide (11.95%), dinucleotide (10.06%), tetranucleotide (1.6%), pentanucleotide (0.14%) and hexanucleotide (0.02%) , followed by IRF7 (5 motifs) and RIG-I (4 motifs). For the Toll-like receptor signaling pathway, 21 genes that included AKT, CCL3,ERK1, FADD, IKBKE,IKBKG, IL8, IRF7, JUN, MAP2K7, MAP3K7IP2, MYD88, NFKB1, NFKBIA, P38, PIK3C, PIK3R, TBK1, TBK1, TLR1 and TLR5 contained 59 SSR motifs. Among them, AKT contained the most mononucleotide SSRs, and PIK3C had the most trinucleotide SSRs  SNPs were specific for the GSIV-infected Chinese giant salamanders. The antiviral PRRs MDA5, LGP2 and RIG-I also contained SNP motifs. For the Toll-like receptor signaling pathway, 41 genes contained 445 SNPs that consisted of 141 SNPs 31.69%) specific for the GS_CS samples and 228 SNPs (51.24%) specific for the GS_TS samples, and 73 common SNPs (16.4%) for both the GS_CS and GS_TS samples . Except for complement components C1R (comp81391_c0_seq2), C1S (comp72777_c0_seq1) and C7 (comp93878_c1_seq8), the expression of other 7 complement components including C1S-like, C2, C3, C4, C5, C8A and C9 was in agreement with their transcript abundance changes determined by RNA-seq  are present throughout virtually the entire animal kingdom and have important functions in initiating inflammatory responses and shaping adaptive immunity . A typicselegans , 38, how of TLRs , 40. In  of TLRs . Most fi of TLRs , 43.Six major TLR families were identified in all vertebrate taxa designated as TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11 . In the A large family of cytoplasmic NACHT-LRR receptors (NLRs), characterized by the presence of a nucleotide-binding protein (NACHT domain), have important functions in apoptosis, inflammation and innate immune signaling –48. TherThe expansion of NLR-encoding genes has been described in the sea urchin, amphioxus and zebrafish , 50, 51.In the Chinese giant salamander, many NLRs such as APAF1, IPAF, NOD2, NOD3, CIITA and NALP3/NALP12 existed as multiple isoforms. Our previous studies have shown that the isoforms of immune genes regulated positively or negatively antibacterial and antiviral immunity –54. The Besides NLRs, the RIG-I-like receptors (RLRs) represent another crucial family of intracellular pattern recognition receptors, which use C-terminal RNA helicases to recognize viral RNA and N-terminal CARD domains for signaling . The RLRIridoviruses are large double-stranded DNA (dsDNA) viruses that can infect invertebrates and poikilothermic vertebrates, including insects, fish, amphibians and reptiles. A real-time polymerase chain reaction (PCR) assay for a marine fish iridovirus showed that the spleen and kidney contained the largest number of viral particles while no viral DNA was detected in the muscle tissue . A compaUsing 454 pyrosequencing, 755 up-regulated genes and 695 down-regulated genes were identified in the two spleen-complementary DNA libraries, that were constructed from Singapore grouper iridovirus (SGIV) infected and control orange-spotted grouper . AlthougThe vertebrate complement system is a humoral and proteolytic system that is composed of approximately 40 soluble and membrane-bound proteins. It is an integral part of the innate immune system protecting the host against invasion and proliferation of various pathogens. The mammalian complement system has three different activation pathways which include classical, alternative and lectin. Activation of the classical pathway is triggered by the binding of C1q proteins to immune complexes or aggregates containing IgG or IgM . The lecIn the study reported herein, the majority of genes involved in the complement and coagulation cascades  were identified, and the alternative, lectin and classical pathways appeared conserved in the Chinese giant salamander (Additional file


However, very few studies have focused on testicular oxidative stress and/or apoptosis.Due to the increased application of titanium dioxide nanoparticles (TiO2 NPs, male mice were exposed to 2.5, 5, or 10 mg/kg body weight TiO2 NPs for 90 consecutive days. Testicular oxidative stress and apoptosis were then evaluated, and the testicular mRNA expression of several genes and their proteins involved in oxidative stress and/or apoptosis was investigated.In order to understand the possible molecular mechanisms of testicular lesions following exposure to TiO2 NPs entered Sertoli cells and caused severe testicular oxidative damage and/or apoptosis, accompanied by excessive production of reactive oxygen species and peroxidation of lipids, proteins and DNA as well as a significant reduction in antioxidant capacity. Furthermore, exposure to TiO2 NPs resulted in the up-regulation of caspase-3, Nrbp2, and cytochrome c expression, and caused down-regulation of SOD, CAT, GPx, GST, GR, Cyp1b1, Car3, Bcl-2, Acaa2, and Axud1 expression in mouse testis.TiO2 NPs entered Sertoli cells via the blood-testis barrier and were deposited in mouse seminiferous cord and/or Sertoli cells, causing oxidative damage and apoptosis.TiO Ba. Ba2 NPs lesions . Exposurt . SO. SO2 NP-neration . Furtherneration . In the nditions -43]. As. As2 NP-nditions . Oral adnditions . In the se liver , kidney se liver ,48], lu, lu2 NP-se liver ,50], br, br2 NP-se liver , and ovase liver .2 NP deposition in the testis is closely related to seminiferous tubule injury or apoptosis of Sertoli cells following exposure to TiO2 NPs. To understand the mechanisms of oxidative stress and/or apoptosis, the expression of oxidative stress and/or apoptosis-related genes and their proteins in the testis was detected and is listed in Figures 2 NPs induced apoptosis in mouse spleen by activating caspase-3 . In. In2 NP e spleen . Nrbp2 be spleen . Nrbp2 ul damage . Axud1 ipoptosis . Therefoive site . CAT conxidation . GPX alsxidation ,67]. GR. GR2 NP xidation . GST is xidation . Thus, dnt liver . Overexppoptosis . Cyp1b1  in vitro. Therefo2 NPs entered Sertoli cells via the blood-testis barrier and were deposited in mouse testicular tissue and/or Sertoli cells, causing testicular oxidative damage and cell apoptosis. Furthermore, testicular damage following exposure to TiO2 NPs may be related to significant overproduction of ROS, peroxidation of lipids, proteins and DNA, and a significant reduction in antioxidant capacity. Sertoli cell apoptosis caused by TiO2 NPs was associated with up-regulation of caspase-3, Nrbp2, and cytochrome c expression, and down-regulation of SOD, CAT, GPx, GST, GR, Cyp1b1, Car3, bcl-2, Acaa2, and Axud1 expression in mouse testis. Therefore, the application of TiO2 NPs should be performed with caution to reduce potential risks to humans.In summary, TiO2 nanoparticles were prepared via controlled hydrolysis of titanium tetrabutoxide. The details of TiO2 NP synthesis were previously described . Hy. Hy2 nan2 NPs were obtained at room temperature with a charge-coupled device (CCD) diffractometer  using Ni-filtered Cu Kα radiation. The average aggregate or agglomerate size of the TiO2 NPs after incubation in 0.5% w/v HPMC solution (5 mg/L) for 0, 12 and 24 h was measured by dynamic light scattering (DLS) using a Zeta PALS + BI-90 Plus  at a wavelength of 659 nm. The scattering angle was fixed at 90°.The particle sizes of both the powder and nanoparticles suspended in 0.5% w/v HPMC solution (5 mg/L) following incubation were determined using a TecnaiG220 transmission electron microscope (TEM)  operating at 100 kV, respectively. In brief, particles were deposited in suspension onto carbon film TEM grids, and allowed to air-dry. The mean particle size was determined by measuring > 100 randomly sampled individual particles. X-ray-diffraction (XRD) patterns of TiOAll experiments were conducted during the light phase, and were approved by the Animal Experimental Committee of Soochow University (Grant 2111270) in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Guidelines).ad libitum. Before treatment, the mice were acclimated to this environment for five days. All the animals were handled in accordance with the guidelines and protocols approved by the Care and Use of Animals Committee of Soochow University (China).One hundred sixty CD-1 (ICR) male mice (24 ± 2 g) were purchased from the Animal Center of Soochow University (China). The mice were housed in stainless steel cages in a ventilated animal room. The room temperature in the housing facility was maintained at 24 ± 2°C, with a relative humidity of 60 ± 10% and a 12-h light/dark cycle. Distilled water and sterilized food were available 2 NP powder was dispersed onto the surface of 0.5% w/v HPMC and the suspension containing TiO2 NPs was treated ultrasonically for 30 min and mechanically vibrated for 5 min. The mice were randomly divided into four groups (n = 40 in each group), including a control group treated with 0.5% w/v HPMC and three experimental groups treated with 2.5, 5, or 10 mg/kg TiO2 NPs. The mice were weighed, a volume of TiO2 NP suspension was calculated for each mouse, and the fresh TiO2 NP suspension was administered by gavage using a gavage needle each day for 90 days. Symptoms, growth status, eating, drinking, activity and mortality were observed and carefully recorded daily during the 90-day period. After 90 days, the mice were weighed, anesthetized using ether, and then sacrificed. The testes were quickly removed, placed on ice, dissected, and then frozen at −80°C .TiOAll histopathological tests were performed using standard laboratory procedures . Five sen = 5 in each group) were fixed in a fresh solution of 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and 2% formaldehyde followed by a 2 h fixation period at 4°C with 1% osmium tetroxide in 50 mM sodium cacodylate (pH 7.2-7.4). Staining was performed overnight with 0.5% aqueous uranyl acetate. The specimens were dehydrated in a graded series of ethanol , and embedded in Epon 812. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate, and observed with a HITACHI H600 TEM . Testicular apoptosis was determined based on the changes in nuclear morphology .Testes  were embedded in paraffin blocks, sliced into 5-μm thick sections, and placed onto glass slides. The slides were dewaxed, hydrated, and then scanned using the confocal Raman microscope.Raman analysis was performed using backscattering geometry in a confocal configuration at room temperature using a HR-800 Raman microscope system equipped with a 632.817 nm HeNe laser . It was reported that when the size of the TiO48.7 cm−1. Laser p2·− and H2O2) production and levels of malondialdehyde (MDA), protein carbonyl (PC), and 8-hydroxy deoxyguanosine (8-OHdG) in the testicular tissues (n = 5 in each group) were assayed using commercial enzyme-linked immunosorbent assay kits  according to the manufacturer’s instructions.Reactive oxygen species (ROS) (On = 5 in each group) were determined using commercial assay kits  according to the manufacturer’s instructions.The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S epoxide transferase (GST), and glutathione reductase (GR), and the contents of reduced glutathione (GSH) and ascorbic acid in the testicular tissues  according to the manufacturer’s instructions. Probes and cycling condition were optimized in accordance with MIQE guidelines for PCR . SynthesN = 5 in each group) from experimental and control mice was extracted using Cell Lysis Kits (GENMED SCIENTIFICS INC.USA) and quantified using BCA protein assay kits (GENMED SCIENTIFICS INC.USA). ELISA was performed using commercial kits that were selective for each respective protein , following the manufacturer’s instructions. The absorbance was measured on a microplate reader at 450 nm , and the concentrations of SOD, CAT, GPx, GST, GR, Cyp1b1, Car3, caspase-3, Bcl-2, Nrbp2, Acaa2, Axud1, and cytochrome c were calculated from a standard curve for each sample.To determine protein levels of SOD, CAT, GPx, GST, GR, Cyp1b1, Car3, caspase-3, Bcl-2, Nrbp2, Acaa2, Axud1, and cytochrome c in the testes, total protein from the frozen testicular tissues  was carried out to compare the differences of means among the multi-group data using SPSS 19 software . Dunnett’s test was performed when each dataset was compared with the solvent control data. Statistical significance for all tests was judged probability level of 0.05 (P < 0.05).The authors declare that they have no competing interests.Conceived and designed the experiments: FH, XZ, LS, and LW. Performed the experiments: FH, XZ, LS, LW, JH, and XY. Analyzed the data: FH, XZ, LS, LW, JH, XY, YZ, XS, and QS. Contributed reagents/materials/analysis tools: YZ, XS, and QS. Wrote the paper: FH, XZ, LS, and LW. All authors read and approved the final manuscript.


Non-invasive imaging of beta cells is a much-needed development but is one that faces significant biological and technological hurdles. A relevant imaging method should at least allow for an evaluation over time of the mass of beta cells under physiological and pathological conditions, and for an assessment of novel therapies. We, therefore, investigated the ability of a new MRI probe to repeatedly measure the loss of beta cells in a rodent model.We developed an innovative nanoparticle probe that targets the glucagon-like peptide 1 receptor, and can be used for both fluorescence imaging and MRI. Using fluorescence, we characterised the specificity and biodistribution of the probe. Using 1.5T MRI, we longitudinally imaged the changes in insulin content in male and female mice of the RIP-DTr strain, which mimic the changes expected in type 1 and type 2 diabetes, respectively.We showed that this probe selectively labelled beta cells in situ, imaged in vivo native pancreatic islets and evaluated their loss after diphtheria toxin administration, in a model of graded beta cell deletion. Thus, using clinical MRI, the probe quantitatively differentiates, in the same mouse strain, between female animals featuring a 50% loss of beta cells and the males featuring an almost complete loss of beta cells.The approach addresses several of the hurdles that have so far limited the non-invasive imaging of beta cells, including the potential to repeatedly monitor the very same animals using clinically available equipment, and to differentiate graded losses of beta cells.The online version of this article (doi:10.1007/s00125-014-3442-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users. The non-invasive, repeated in vivo imaging of beta cells, which are central players in most forms of diabetes , is highThe position of the pancreas within the abdomen and the dispersion of its small endocrine islets imply the need for a deep-penetrating and highly sensitive approach. Therefore, most of these studies have relied on nuclear medical imaging methods (positron emission computed tomography [PET] and single photon emission computed tomography [SPECT]), in spite of the limited spatial resolution of these methods and their potential safety issues , 3. SpecN-maleimidomethyl) cyclohexane-1-carboxylate  to obtain maleimide-activated Np647, which was reacted with ExCys1  was synthesised by GeneCust Europe . Molday ION C6Amine iron oxide nanoparticles, featuring ~9,000 iron atoms per particle and a 35 nm diameter , were reacted with the succinimidyl ester of Alexa Fluor 647  to obtain a ratio of 20 fluorochrome molecules/nanoparticle (Np647). Np647 was reacted with sulfosuccinimidyl-4- equipped with filters for Alexa 647.Cells of the MIN6, Panc-1 and HeLa lines were cultured as previously reported . At the To assess whether Np647–ExCys1 affected the viability of MIN6 cells, cultures were exposed for 24 h to 50 μg/ml Np647–ExCys1 added directly to DMEM . Cells were then tested in a thiazolyl blue tetrazolium bromide (MTT) assay , and for glucose-induced insulin secretion .Hprt locus of the X chromosome. Thus, in this model, DT administration leads to a parallel loss of insulin content and beta cells, which is partially due to the random X inactivation (on average 50%) in hemizygous female mice and nearly complete ablation in male mice.Rat insulin promoter-diphtheria toxin receptor (RIP-DTr) mice were generated as reported . BrieflyTo evaluate the total insulin content of the pancreas, whole glands were carefully dissected and extracted in acid-ethanol for 24 h, as reported , 35. PanMale RIP-DTr mice  were inj7 [p s–1 cm–2 sr–1]/[μW/cm2]) were quantified on the unmixed image after suppression of the autofluorescence levels.Mice were killed 24 h later, and immediately perfused via the left ventricle, first with 10 ml 0.9% NaCl (154 mmol/l) and then with 10 ml 4% paraformaldehyde in 0.1 mol/l phosphate buffer at 37°C. The pancreas, liver, spleen, kidneys, lung, duodenum and heart were harvested and fixed in paraformaldehyde for 2 h at 4°C. The organs were rinsed for 2 h in phosphate buffer at 4°C, and their fluorescence recorded with an IVIS Spectrum  equipped with filters for Alexa 647. Corresponding organs from mice injected with Np647–ExCys1 and Np647–ExScra, as well as from mice injected with Np647–ExCys1 with and without an excess of exendin-4, were imaged in parallel. To differentiate between tissue autofluorescence and fluorescence due to the A647 fluorochrome, the organs were excited using 535, 570, 605 and 640 nm excitation filters and fluorescence was recorded using a 680 nm emission filter. Spectral unmixing was performed with the Living Image 4.3.1 software  and fluorescence signals , and cryo-sectioned at 7 μm thickness. Sections were mounted and examined by fluorescence microscopy. Pancreas sections were immunolabelled using either guinea pig antibodies against insulin , diluted 1/200, mouse antibodies against glucagon (Sigma-Aldrich), diluted 1/2,000, or rabbit antibodies against exendin-4 , diluted 1/100. Secondary antibodies were anti-guinea pig antibodies coupled to Dylight 405  diluted 1/800, anti-mouse antibodies coupled to tetramethylrhodamine , diluted 1/500, and anti-rabbit antibodies coupled to A488 , diluted 1/500, for insulin, glucagon and exendin-4 immunolabelling, respectively. Coverslips were placed over the sections and the sections were photographed with an Axiophot fluorescence microscope (Zeiss).Three-to-four-months-old male and female mice of the RIP-DTr strain , 36 wereTo test whether the probe accumulation reflected the insulin content of the pancreas, a group of RIP-DTr mice was killed for pancreas sampling and determination of insulin content, immediately after a single MRI session.e (T2 before nanoparticle injection/T2 24 h after nanoparticle injection) [All magnetic resonance images were acquired on a 1.5 T clinical scanner , equipped with a surface coil (Ø = 47 mm). Coronal T2 maps were generated using a three-dimensional turbo spin echo (TSE) sequence with the following parameters: repetition time (TR) = 145 ms; six different echo time (TE) values ranging from 9 to 54 ms; flip angle = 90°; matrix size = 116 × 83; number of average = 1; field of view = 60 × 41; slice thickness = 0.5 mm. A total of 102 sequential coronal images were obtained to cover the entire pancreatic region. All images were analysed with the dicom viewer Osirix . Regions of interest for analyses were manually defined on three consecutive MRI slices of each pancreas. Probe accumulation was given by logjection) , 29.t test, assuming a two-tailed distribution, or two-way ANOVA, followed by the Bonferroni multiple comparison post hoc test . A p value of ≤0.05 was considered statistically significant.Data were analysed using either paired or unpaired Student’s Thirty minutes after the addition of 50 μg/ml Np647–ExCys1 to the culture medium of MIN6 cells, which express insulin and GLP-1r, fluorescence microscopy revealed co-staining of most cells for exendin-4 and Np647–ExCys1 Fig. . Under iTwenty-four hours after intravenous injection, the Np647–ExCys1 probe distributed in the pancreas Fig.  a, b, anTwenty-four hours after the intravenous injection of Np647–ExCys1 in normoglycaemic mice of the RIP-DTr strain, fluorescence microscopy revealed that the nanoparticles labelled most insulin-containing beta cells, but not the glucagon-containing alpha cells, and the surrounding acinar cells Fig. . The Np6t = 0), the T2 of pancreas was significantly reduced compared with the baseline value measured before the injection  and most female mice  in male mice (average 74%) than in female mice  correlation (R = 0.834), between insulin content and the accumulation of the probe in the pancreas  when using nanoparticles tagged with the scrambled peptide, a widely used control in nanoparticle studies [We developed a novel probe which couples USPIOs to a modified exendin peptide in a different manner to that previously reported . The new studies , 41, 42; studies . What ca studies , 29, 30. studies , 36. Thu studies . The dat studies . Such pr studies , 23.Given that the data were generated using widely available MRI equipment, they also open exciting perspectives for the most-needed clinical imaging of patients with type 1 and type 2 diabetes. However, whether the method could improve the present status of human islet imaging , 22, 44 ESM Methods(PDF 80 kb)ESM Fig. 1(PDF 172 kb)ESM Fig. 2(PDF 59 kb)ESM Fig. 3(PDF 411 kb)ESM Fig. 4(PDF 1.05 mb)ESM Fig. 5(PDF 91.2 kb)ESM Fig. 6(PDF 89.5 kb)Below is the link to the electronic supplementary material.


Aspergillus oryzae NSK that possesses a relatively high GABA biosynthesizing capability compared to other reported GABA-producing fungal strains, indicating the presence of an active GAD. This finding has prompted us to explore an effective method to recover maximum amount of GAD for further studies on the GAD’s biochemical and kinetic properties. The extraction techniques examined were enzymatic lysis, chemical permeabilization, and mechanical disruption. Under the GAD activity assay used, one unit of GAD activity is expressed as 1 μmol of GABA produced per min per ml enzyme extract (U/ml) while the specific activity was expressed as U/mg protein.γ-Amino butyric acid (GABA) is a major inhibitory neurotransmitter of the mammalian central nervous system that plays a vital role in regulating vital neurological functions. The enzyme responsible for producing GABA is glutamate decarboxylase (GAD), an intracellular enzyme that both food and pharmaceutical industries are currently using as the major catalyst in trial biotransformation process of GABA. We have successfully isolated a novel strain of ® at 60°C for 30 min was the least effective. It yielded only 0.70 U/mg of GAD. Extraction using sonication was further optimized using a one-variable-at-a-time approach (OVAT). Results obtained show that the yield of GAD increased 176% from 1.99 U/mg to 3.50 U/mg.Mechanical disruption by sonication, which yielded 1.99 U/mg of GAD, was by far the most effective cell disintegration method compared with the other extraction procedures examined. In contrast, the second most effective method, freeze grinding followed by 10% v/v toluene permeabilization at 25°C for 120 min, yielded only 1.17 U/mg of GAD, which is 170% lower than the sonication method. Optimized enzymatic lysis with 3 mg/ml YatalaseA. oryzae NSK, sonication was found to be the best. Under optimized conditions, about 176% of GAD was recovered compared to recovery under non optimized conditions. The high production level of GAD in this strain offers an opportunity to conduct further studies on GABA production at a larger scale.Of the techniques used to extract GAD from  Glutamate decarboxylase (GAD) is a unique enzyme that catalyzes the irreversible conversion of L-glutamic acid (Glu) to γ-aminobutyric acid (GABA) and carbon dioxide through a single step α-decarboxylation pathway. The cofactor that is involved in the pathway is pyridoxal phosphate . GABA, aAspergillus oryzae was frequently chosen for its ability to secrete various desired enzymes that are widely used in the production of fermented foods such as soysauce, vinegar etc -1-propanesulfonate (CHAPS), sodium dodecyl sulfate (SDS), and citric acid monohydrate were purchased from Acros Organics . Trisodium citrate was sourced from Fisher Scientific (United States). All chemicals were of analytical reagent grade.γ-Amino butyric acid (GABA), 5′-pyridoxal phosphate (5′PLP) and calcium chloride dihydrate were obtained from Sigma Aldrich Inc. . Methanol, toluene, hexane, acetone, chloroform, potato dextrose agar (PDA), Tween 80, glucose monohydrate, L-glutamic acid, magnesium sulfate heptahydrate, sodium carbonate, monopotassium phosphate, and Lactophenol Cotton Blue were purchased from Merck (Germany). Bacto™ yeast extract was from Becton, Dickinson & Company (USA). YatalaseA. oryzae NSK were prepared from a 5-day old culture on PDA (incubated at room temperature) by scraping them into 5.0 mL of 5 (w/v) Tween 80 under sterile condition. The number of conidia was determined using a hemocytometer. Conidia at 104 mL-1 cell concentration was inoculated in a medium (pH 5.5) containing 5 (w/v) glucose, 0.4% (w/v) L-glutamic acid, 0.1% (w/v) MgSO47H2O, 0.15% (w/v) KH2PO4, 0.6% (w/v) yeast extract and 0.2% (w/v) CaCl2.2H2O. The medium was then incubated for 3 days in an orbital shaker at 200 rpm and 37°C. Fungal mycelia that germinated from the conidia were harvested by filtration through a Whatman No. 4 nylon membrane filter and the filtrate was collected for the analysis of possible extracellular GAD activity , cell suspension volume , acoustic power  and sonication time  on a discontinuous mode . The distance between the sonicator tip and the base of sample holder was maintained at a distance of 2 cm throughout the experiment. All experiments were carried out in triplicate.2CO3 solution -1-propanesulfonate; SDS: Sodium dodecyl sulfate; PITC: Phenylisothiocyanate.The authors declare that they have no competing interests.Aspergillus oryzae NSK and optimized its fermentation media. HMG, RNZRAR and NS contributed to conception of the study, experimental design, and revision of the manuscript. All authors read and approved the final manuscript.ALYY performed all data acquisition, data analyses, and manuscript writing. MSBA-K isolated the fungal strain of ALYY is master student and MSAK is currently a PhD student. HMG and NS are professors of enzyme technology and food biochemistry respectively at Faculty of Food Science and Technology while RNZRAR is a professor at the Faculty of Biotechnology and Biomolecular Sciences.®.Optimization of the condition of enzymatic lysis on the recovery of GAD by using Yatalase (a) Effect of incubation time (min) of Yatalase® (10 mg/ml) on 10% (w/v) of mycelia at 30°C in 50 mM Citrate buffer, pH 5.5. (b) Effect of concentration of lytic enzyme on mycelia (10% w/v) at 30°C for 60 min. (c) Effect of cell concentration (% w/v) on the enzymatic lysis of Yatalase® (10 mg/ml) at 30°C for 60 min. (d) Effect of incubation temperature on the enzymatic lysis of Yatalase® (10 mg/ml) at 10% (w/v) of mycelia for 60 min. Results represent the mean (± SD) of three experiments.Click here for file


C-X-C motif chemokine 10 (CXCL10) is produced in response to interferon-γ, and tumor necrosis factor-α (TNF-α) triggers the accumulation of activated lymphocytes. CXCL13 is constitutively expressed in secondary lymphoid tissues, and the expression is upregulated by TNF-α, via T cell stimulation. It appears that CXCL10 and CXCL13 could play a potential role in the pathogenesis of adult-onset Still’s disease (AOSD), therefore, we investigated the associations between CXCL10 and CXCL13 levels and clinical manifestations in patients with active AOSD.Blood samples were collected from 39 active AOSD patients, 32 rheumatoid arthritis (RA) patients and 40 healthy controls (HC). Of the AOSD patients, follow-up samples were collected from 15 9.6 ± 9.2 months later. Serum levels of CXCL10 and CXCL13 were determined using enzyme-linked immunosorbent assay. CXCL10, CXCL13, and C-X-C chemokine receptor type 3 (CXCR3) expression levels in biopsy specimens obtained from 26 AOSD patients with skin rashes were investigated via immunohistochemistry.p = 0.008) and HC . Also, the CXCL13 levels of AOSD patients (158.8 ± 151.2 pg/mL) were higher than those of RA  and HC . Serum CXCL10 levels correlated with ferritin and systemic scores. Serum CXCL13 levels correlated with those of hemoglobin, C-reactive protein, ferritin, and albumin, and systemic scores. In follow-up AOSD patients, the levels of CXCL10 and CXCL13 fell significantly . On immunohistochemistry, the percentages of inflammatory cells expressing CXCL10 ranged from 1 to 85 %, CXCL13 from 1 to 72 %, and CXCR3 from 2 to 65 %. The percentage of CXCL10-positive inflammatory cells was higher in skin biopsy samples exhibiting mucin deposition than in those that did not (p = 0.01). CXCL13 levels were correlated with those of CD4 and CD68.The CXCL10 levels in AOSD patients  were higher than in RA  contains supplementary material, which is available to authorized users. Adult-onset Still’s disease (AOSD) is a systemic inflammatory disease exhibiting various clinical manifestations characterized by a high spiking fever, an evanescent skin rash, polyarthralgia, and hepatosplenomegaly , 2. AcutAs is true of other cytokines, the levels of several chemokines have been reported to change under various systemic conditions, and such changes may serve as useful diagnostic and prognostic markers . It appeCXCL10 is produced in response to interferon-γ (IFN-γ), and TNF-α triggers the accumulation of activated lymphocytes via interaction with a specific receptor, C-X-C chemokine receptor type 3 (CXCR3), creating an amplification feedback loop , 14. CXCMycobacterium tuberculosis-specific T cell IFN-γ assays before starting high-dose glucocorticosteroid therapy, and only two patients had latent tuberculosis. The HC was recruited using a screening questionnaire from healthy individuals without history of exposure to Mycobacterium tuberculosis and medical history of autoimmune, rheumatic, and any other diseases through the public announcement. Of the 39 AOSD patients, follow-up samples were collected from 15 9.6 ± 9.2 months later. All blood samples were stored at −70 °C immediately after collection. Information on medical histories, clinical symptoms, and the findings of physical examinations was entered into a database together with serum test results. Each patient underwent a series of laboratory tests, including a complete blood count; ESR evaluation; assessment of CRP, rheumatoid factor (RF), anti-nuclear antibody (ANA), and ferritin levels ; liver function testing; and urinalysis. AOSD disease activity was scored as previously described; scores ranged from 0 to 12 with 1 point being given for each of the following manifestations: fever, a typical rash, pleuritis, pneumonia, pericarditis, hepatomegaly or abnormal liver function test data, splenomegaly, lymphadenopathy, leukocytosis ≥ 15,000/mm2, sore throat, myalgia, and abdominal pain [Thirty-nine active AOSD patients, 32 RA patients and 40 HC were included in the present study, and serum samples were collected from all subjects. AOSD patients were diagnosed using Yamaguchi’s criteria after exclusion of those who had infectious, neoplastic and autoimmune disorders . Twenty-nal pain . This stCXCL10 levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits  according to the manufacturer’s instructions. CXCL13 levels were also determined using an ELISA kit (R & D Systems) according to the manufacturer’s instructions.Skin biopsies were obtained from 26 patients with AOSD, and we examined hematoxylin and eosin-stained sections. All slides were independently examined by three pathologists  with respect to the following skin histological parameters: (1) epidermal change; (2) extent of inflammatory cell infiltration; and the presence of (3) karyorrhexis, (4) vasculitis, and (5) interstitial mucin.Immunohistochemistry were performed on the formalin-fixed paraffin-embedded sections using a Benchmark XT automated staining system . The primary antibodies used were: CD4, 1:10 and CD8, 1:50 ; CD68, 1:200 ; CXCL10, 1:50; CXCL13, 1:50; and CXCR3, 1:20 (R & D Systems). Detection was achieved using a Ventana Optiview DAB kit . Scores were calculated by dividing the numbers of positive inflammatory cells by the numbers of all inflammatory cells, expressed as percentages  or graded on a scale from 1 to 3: 1:1–33 %; 2, 34–66 %; and 3, 67–100 % .t test or the Mann–Whitney U test. Correlations between histological scores and disease activity marker levels were evaluated using Pearson’s correlation test or Spearman’s correlation test. The Wilcoxon signed-rank test was also used to compare CXCL10 and CXCL13 levels in patients who underwent follow-up serum sampling. All statistical analyses were performed using SPSS version 20.0 . A p value <0.05 was regarded as indicative of statistical significance.All data are shown as means ± standard deviations. Differences among CXCL10 and CXCL13 levels were evaluated using the independent p = 0.001). The principal clinical symptoms of AOSD patients included a high spiking fever (87.2 %), a skin rash (82.1 %), arthritis (61.5 %), sore throat (53.8 %), and splenomegaly (25.6 %). In RF-positive patients, a diagnosis of RA was excluded using the 1987 American College of Rheumatology (ACR) criteria [Table criteria . In ANA-criteria . Of the Of the AOSD patients, five (12.8 %) were diagnosed with reactive hemophagocytic syndrome (RHS). These patients were diagnosed to RHS by tissue biopsy or clinical features .p = 0.008) and HC   and HC  . However, the CXCL13 levels did not differ between the two groups .Figure 06) Fig. . Also, t01) Fig. . We compp = 0.039), but serum CXCL10 did not differ between the two groups. When we compared CXCL10 and CXCL13 levels by AOSD clinical course (monocyclic vs. polycyclic vs. chronic articular) of 30 patients with high-level disease activity, those levels did not differ among the three groups (data not shown).Comparing the CXCL10 and CXCL13 levels according to existence of RHS, we found that serum CXCL13 were significantly higher in AOSD patients with RHS (229.6 ± 90.3 pg/mL) than in patients without RHS , aspartate transaminase (AST) levels , and systemic scores . Serum CXCL13 levels correlated with hemoglobin levels , CRP levels , ferritin levels , albumin levels , and the systemic scores . Also, serum CXCL10 levels correlated with those of CXCL13 .The correlations between the levels of pre-existent disease activity markers and those of serum CXCL10 or CXCL13 in AOSD patients are shown in Table r = 0.809) than CRP , ferritin , CXCL10 , and ESR .Furthermore, we compared the correlation coefficient between systemic scores and several inflammatory markers including CXCL10 and CXCL13. Systemic scores have the best correlation coefficient with CXCL13 levels (p = 0.002 and p = 0.001) . The change in CXCL13 was positively correlated with both the change in CRP level  and the change in systemic score .Repeat serum samples were collected from 15 AOSD patients at 9.6 ± 9.2 months after the first samplings. The CXCL10 level was 153.7 ± 130.1 pg/mL, and that of CXCL13 89.1 ± 117.4 pg/mL. The systemic scores were 1.13 ± 1.19. In AOSD patients who were followed up, systemic scores decreased somewhat and serum CXCL10 and CXCL13 levels significantly decreased . Five patients (19.2 %) had persistent pruritic eruptions, and two patients had papulopustular lesions on the trunk. Two patients had painful swelling of the low extremities. Most skin biopsies exhibited mild lymphocytic or histiocytic infiltration in the upper dermis. Nuclear debris was frequently evident in the dermis . Neutrophil infiltrations were shown in some cases , More than half of all cases showed interstitial mucin deposition. Some cases exhibited interface dermatitis with keratinocyte necrosis or basal vacuolization.The skin manifestations of 17 patients were maculopapular eruptions on the upper and lower extremities, and the trunk (p = 0.01).The percentage of cells positive for CD8 was higher than that for CD68 (57.7 %). The number of CD8-positive lymphocytes was higher than that of CD4-positive lymphocytes in almost all cases (88.5 %). To control for CXCL10, CXCL13, and CXCR3 immunohistochemical evaluations, lymphoid cells in the paracortical zone or germinal center of a reactive lymph node were stained. The antibodies revealed a granular pattern of cytoplasmic staining. The staining patterns of inflammatory cells in skin biopsies were similar to those of lymphoid cells in a lymph node  and CD68 .The correlations between CD4, CD8, and CD68 grades, and the percentages of CXCL10-, CXCR3-, and CXCL13-positive inflammatory cells in the skin of AOSD patients, are shown in Additional file To evaluate the clinical utilities of serum CXCL10 and CXCL13 levels, we studied these markers of AOSD patients and compared the levels with those of RA and HC. Moreover, we also confirmed that these markers and their receptor, CXCR3, immunohistochemically stained in skin rash material from AOSD patients. This is the first study to show that serum CXCL13 levels are significantly higher in patients with active AOSD than in RA and HC. Furthermore, the serum levels of CXCL10 and CXCL13 in patients with AOSD correlated with those of several inflammatory markers, and systemic scores, and the serum levels of CXCL10 and CXCL13 fell upon reduction in disease activity in follow-up AOSD patients.CXCL10 has been detected in the serum, synovial fluid, and synovial tissues of RA patients, and CXCR3, the receptor thereof, is expressed in T cell-rich areas of inflamed synovial tissue , 32, 33.Proinflammatory cytokines, such as TNF-α and IL-6, play roles upstream of CXCL13 expression and lymphoid tissue organization, as demonstrated in animal models of lymphoid tissue generation , 39. ManThe limitations of this study include the lack of a normal skin and follow-up skin biopsy in AOSD for comparison with chemokine expression. We did not compare these markers with other febrile disorders for diagnostic value of these markers, and had a relatively small sample size for subgroup analysis and comparison with follow-up samples. Further studies involving larger sample size are required for evaluating usefulness of these markers in AOSD patients with control groups of other febrile disorders such as sepsis.We found significantly higher levels of CXCL10 and CXCL13 in the serum from patients with active AOSD. We also found that most follow-up AOSD patients exhibited reduced CXCL10 and CXCL13 levels after improvement in disease activity. These results indicate that CXCL10 and CXCL13 may play important roles in the pathogenesis of the acute inflammatory process in AOSD, although the levels did not significantly differ among the various AOSD disease courses. This exploration of chemokine levels in AOSD not only improves our understanding of the role played by such materials in immunopathogenesis but also lays the foundation for development of novel therapeutic strategies, such as anti-CXCL10 antibody administration, for AOSD.


Vinpocetine is a safe nootropic agent used for neurological and cerebrovascular diseases. The anti-inflammatory activity of vinpocetine has been shown in cell based assays and animal models, leading to suggestions as to its utility in analgesia. However, the mechanisms regarding its efficacy in inflammatory pain treatment are still not completely understood. Herein, the analgesic effect of vinpocetine and its anti-inflammatory and antioxidant mechanisms were addressed in murine inflammatory pain models. Firstly, we investigated the protective effects of vinpocetine in overt pain-like behavior induced by acetic acid, phenyl-p-benzoquinone (PBQ) and formalin. The intraplantar injection of carrageenan was then used to induce inflammatory hyperalgesia. Mechanical and thermal hyperalgesia were evaluated using the electronic von Frey and the hot plate tests, respectively, with neutrophil recruitment to the paw assessed by a myeloperoxidase activity assay. A number of factors were assessed, both peripherally and in the spinal cord, including: antioxidant capacity, reduced glutathione (GSH) levels, superoxide anion, tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) levels, as well as nuclear factor kappa B (NF-κB) activation. Vinpocetine inhibited the overt pain-like behavior induced by acetic acid, PBQ and formalin (at both phases), as well as the carrageenan-induced mechanical and thermal hyperalgesia and associated neutrophil recruitment. Both peripherally and in the spinal cord, vinpocetine also inhibited: antioxidant capacity and GSH depletion; increased superoxide anion; IL-1β and TNF-α levels; and NF-κB activation. As such, vinpocetine significantly reduces inflammatory pain by targeting oxidative stress, cytokine production and NF-κB activation at both peripheral and spinal cord levels. Modern lifestyle has raised life expectancy, but also increased the incidence of chronic diseases , consequVinca minor, commonly known as the lesser periwinkle [Vinpocetine (ethyl apovincamine-22-oate) is a synthetic ethyl ester of the alkaloid apovincamine, which is isolated from the leaves of riwinkle . Vinpoceriwinkle , as wellriwinkle . Importariwinkle , whilst riwinkle .As well as being a phosphodiasterase-1 inhibitor , the emeData pertinent to the analgesic effect of vinpocetine show that it inhibits neuronal tetrodotoxin-resistant NaV1.8 sodium channel currents and sodium current-induced calcium influx ,19, withMale Swiss mice (25–30 g) from the Universidade Estadual de Londrina, Londrina, Paraná, Brazil, were used in this study. Mice were housed in standard clear plastic cages with free access to food and water with a light/dark cycle of 12/12h at a constant temperature of 21°C. All behavioral testing was performed between 9 a.m. and 5 p.m. in a temperature-controlled (21°C) room. Animal care and handling procedures were approved by the Ethics Committee of the Universidade Estadual de Londrina (process number 13278.2011.3). All efforts were made to minimize animal suffering and to reduce the number of animals used.p-benzoquinone .Materials were obtained from the sources as follows: vinpocetine , carrageenan , acetic acid and formaldehyde , phenyl-Mice received per oral (p.o.) treatment with vinpocetine  1 h before the inflammatory stimulus. The dose of inflammatory stimuli was based on previous studies in our laboratory –25. The The PBQ and acetic acid-induced writhing models were performed as previously described . PBQ  maintained at 55°C. The reaction time was indicated by the time for the animal to jump or lick their paws. A maximum latency (cut-off) was set at 30 s to avoid tissue damage .2HPO4 buffer  containing HTAB (0.5% weight/volume), and the homogenates were centrifuged (16100 g × 2 min × 4°C). The supernatants (30 μL) were mixed with K2HPO4 buffer  containing o-dianisidine dihydrochloride  and hydrogen peroxide . The absorbance was determined after 5 min at 450 nm . The results of MPO activity are expressed as the number of neutrophils per mg of tissue by using a standard curve of neutrophils (196–400000 cells).Myeloperoxidase (MPO) activity, an indicant of neutrophil recruitment to the paw skin, was evaluated by the MPO kinetic-colorimetric assay . Samplesg × 10 min × 4°C), and the supernatants were used in both assays. Diluted ABTS solution (200 μL) was mixed with 10 μL of sample in each well. After 6 min of incubation at 25°C, the absorbance was measured at 730 nm. For FRAP assay, the supernatants (10 μL) were mixed with the freshly prepared FRAP reagent (150 μL). The reaction mixture was incubated at 37°C for 30 min, and the absorbance was measured at 595 nm (Multiskan GO Thermo Scientific). The results of ABTS and FRAP assays were equated against a standard Trolox curve (0.02–20 nmol).The ability of samples to resist oxidative damage was determined by their free radical scavenging  and ferric reducing (FRAP assay) properties. The tests were adapted to a 96-well microplate format as previously described . Plantarg x 4°C). The supernatant was added to 200 μL of 0.2 M TRIS buffer, pH 8.2, and 10 μL of 0.01M DTNB. After 5 min, the absorbance was measured at 412 nm  against a blank reagent with no supernatant. A standard GSH curve was formed. The results are expressed as GSH per mg of protein [Samples of paw skin and spinal cord were collected and maintained at −80°C for at least 48 h. The sample was homogenized with 200 μL of 0.02 M EDTA. The homogenate was mixed with 25 μL of trichloroacetic acid 50% and was homogenized three times over 15 min. The mixture was centrifuged (15 min x 1500  protein .The quantification of superoxide anion production in tissue homogenates (10 mg/mL in 1.15% KCl) was performed using the nitroblue tetrazolium (NBT) assay . BrieflyPaw skin and spinal cord (L4-L6) samples were homogenized in 500 μL of buffer containing protease inhibitors, with IL-1β and TNF-α levels being determined as previously described by an enzyme-linked immunosorbent assay (ELISA) using eBioscience kits . The resg × 10 min × 4°C), with the supernatants used to assess the levels of phosphorylated and total NF-κB p65 subunit by ELISA using PathScan kits  according to the manufacturer’s directions. The results represent the sample ratio  measured at 450 nm (Multiskan GO Thermo Scientific).The paw skin samples were collected and homogenized in ice-cold lysis buffer . The homogenates were centrifuged  or vehicle  1 hour before acetic acid injection. Vinpocetine, at the doses of 3–30 mg/kg, inhibited the acetic acid-induced writhing response . At 1 mgMice were treated with vinpocetine  1 h before carrageenan  i.pl. injection, with mechanical  and therMice were treated with vinpocetine  1 h before injection with carrageenan . Mice were terminally anesthetized 3 h after the injection, with samples from the paw skin and spinal cord (L4-L6) being collected to evaluate the ABTS free radical scavenging ability and FRAP. Carrageenan reduced ABTS scavenging ability  and FRAPMice were treated with vinpocetine  1 h before i.pl. injection of carrageenan. Samples of cutaneous paw skin and spinal cord (L4-L6) were collected 3 h after carrageenan injection for GSH measurement  and spinMice were treated with vinpocetine  1h before carrageenan i.pl. injection. Samples from the paw skin and spinal cord tissue (L4-L6) were collected 3 h after carrageenan injection for IL-1β and TNF-α measurement . CarrageThe levels of total and phosphorylated NF-κB p65 subunits were evaluated in samples from the paw skin and spinal cord (L4-L6). Mice were treated with vinpocetine (30 mg/kg) 1 h before carrageenan i.pl. injection, with the samples being collected 3 h after injection. Vinpocetine  attenuat2 (PGE2) and the superoxide anion [Increased pain sensitivity is a common feature of the inflammatory response and occurs after tissue injury. The peripheral sensitization is triggered by NF-κB-related pro-inflammatory mediators, including the cytokines TNF-α and IL-1β ,33,34, ade anion . Consequde anion ,34. It ide anion ,39, whicVinpocetine is a safe nootropic agent that is widely used to improve memory and to treat neurological diseases. Vinpocetine has a low oral bioavailability, achieving about 0.7% absorption in humans , althougRecent work shows vinpocetine to afford protection in numerous inflammatory conditions, including atherosclerosis ,45, lung2 [NF-κB heterodimer p50/p65 activates the transcription of a large number of genes, many of which are key mediators of the inflammatory response –54. Inhi2 ,60,61. I2 ,60,61. I2 , althoug2 , but als2 ,62, and 2 ,21,45–47Inflammation-induced peripheral nociceptor sensitization leads to increases in nociceptor inputs and in nociceptive stimuli transmission. This enhanced afferent activity, in turn, induces long-lasting increases in the excitability of spinal cord neurons and contributes to inflammatory pain hypersensitivity . This ce3CL1 production by neurons, which activates satellite glial cells to release TNF-α and IL-β around the dorsal root ganglion that contributes to nociceptor sensitization [The antioxidant effects of vinpocetine described in cerebral isolated nerve endings are similar to those of α-tocopherol, a classical antioxidant . Considetization , the perAdditionally, vinpocetine has direct neuronal effects that could also reduce the nociceptive inputs, including blocking the retrograde axoplasmic transport of nerve growth factor, which is proposed as its analgesic mechanism of action in neuropathic pain . Vinpoce2 production, which is ultimately responsible for nociceptor sensitization and hyperalgesia development in the carrageenan model [2 activates EP3 and EP4 receptors increasing cAMP and PKA to induce hyperalgesia [Ca) in neurons and neuroendocrine cells, which contribute to membrane hyperpolarization and thus also represents a possible analgesic mechanism of vinpocetine [Ca channels to the analgesic effect of vinpocetine.Vinpocetine is also known as a phosphodiasterase-1 (PDE-1) inhibitor, which degrades cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) . Increasan model ,81. PGE2ralgesia . Moreoveralgesia . As suchralgesia  and inhiralgesia . Additiopocetine . As suchIt is noteworthy that vinpocetine inhibits TNF-α- and LPS-induced inflammatory mediators and NF-κB activation by targeting IκB kinase (IKK) activity in a variety of cell lines, as well as in models of lung inflammation . In thisFinally, it is important to highlight that vinpocetine exhibits gastric protective  and antiVinpocetine inhibited inflammatory pain by targeting NF-κB activation and the NF-κB-driven transcriptional consequences, including cytokine production and oxidative stress, both peripherally in the paw skin and centrally in the spinal cord. Therefore, given that vinpocetine is already in clinical use as a nootropic drug with a good safety profile, it will be important to further investigate its utility and mechanism(s) of action as an analgesic.


PCSK9 inhibitor therapy has been approved by the FDA as an adjunct to diet-maximal tolerated cholesterol lowering drug therapy for adults with heterozygous familial hypercholesterolemia (HeFH) or clinical atherosclerotic cardiovascular disease (ASCVD) with suboptimal LDL cholesterol (LDLC) lowering despite maximal diet-drug therapy. With an estimated ~24million of US hypercholesterolemic patients potentially eligible for PCSK9 inhibitors, costing ~ $14,300/patient/year, it is important to assess health-care savings arising from PCSK9 inhibitors vs ASCVD cost.In 103 patients with HeFH, and/or ASCVD and/or suboptimal LDLC lowering despite maximally tolerated diet-drug therapy, we assessed pharmacoeconomics of PCSK9 inhibitor therapy with lowering of LDLC. For HeFH diagnosis, we applied Simon Broome’s or WHO Dutch Lipid Criteria (score >8). Estimates of direct and indirect costs for ASCVD events were calculated using American Heart Association (AHA), U.S. DHHS, Healthcare Bluebook, and BMC Health Services Research databases. We used the ACC/AHA 10-year ASCVD risk calculator to estimate 10-year ASCVD risk and estimated corresponding direct and indirect costs. Assuming a 50 % reduction in ASCVD events on PCSK9 inhibitors, we calculated direct and indirect health-care savings.We started 103 patients (58 [56 %] women and 45 [44 %] men), on either alirocumab (62 %) or evolocumab (38 %), median age 63, BMI 29.0, and LDLC 149 mg/dl. Of the 103 patients, 28 had both HeFH and ASCVD, 33 with only ASCVD, 33 with only HeFH, and 9 had neither. Of the 103 patients, 61 had a first ASCVD event at median age 55 and on best tolerated cholesterol-lowering therapy median LDLC was 137 mg/dl. In these 61 patients, total direct costs attributable to ASCVD were $8,904,361 , the median 10-year risk of a new CVD event was calculated to be 13.1 % with total cost $1,654,758. Assuming a 50 % reduction in ASCVD events on PCSK9 inhibitors in our 61 patients, $4,452,180 would have been saved in the past; and future 10-year savings would be $1,123,345.In the 61 CVD patients, net costs/patient/year were estimated to be $7,000 in the past, with future 10-year intervention net costs/patient/year being $12,459, both below the $50,000/year quality adjusted life-year gained by PCSK9 inhibitor therapy. Many patients with elevated LDLC fail to achieve treatment targets –3, becauWhether and to what degree health care insurers will facilitate approval of PCSK9 class of medications , 14, 16 Of 734 patients referred to our Cholesterol Center for diagnosis and treatment of high LDLC and/or CVD, with LDLC ≥70 mg/dl despite maximally tolerated cholesterol lowering therapy, we recently reported  that 30 Given current pricing of $14,000–14,600 per patient per year, annual United States PCSK9 inhibitor costs might approximate $185–$342 billion, reflecting the use of an expensive specialty drug for endemic CVD, the leading cause of mortality in the USA , 21. In Answers are needed for major questions regarding PCSK9 inhibitor therapy including whether the PCSK9 inhibitors will significantly reduce morbid and mortal CVD events in hypercholesterolemic patients beyond the best currently available diet-statin therapy , and wheIn 103 hypercholesterolemic patients, 61 with previous CVD , we estimated direct + indirect costs of CVD, costs of estimated next 10 year CVD events, and PCSK9 inhibitor costs to assess whether PCSK9s would provide an incremental cost-effectiveness ratio  within aThe procedures were in accordance with the ethical standards of the responsible committee on human experimentation, approved by the Jewish Hospital Institutional Review Board. The study was carried out with signed informed consent.n = 31) were those who were on “maximal-tolerated statin therapy,” and also maximum tolerated cholesterol lowering therapy . The second group of patients (n = 72) were those who couldn’t tolerate ANY dose of two or more statins and were on maximal tolerated dose of colesevelam and/or ezetimibe. “Maximal tolerated statin therapy” includes not being able to tolerate any statin dose level.Since the commercialization of PCSK9 inhibitors, starting July 2015, we have started 103 patients on either alirocumab or evolocumab. When considering PCSK9 inhibitor therapy, we had two groups of patients based on FDA and insurance criteria with suboptimal LDLC lowering. The first group of patients , and/or CVD or none of the above.In order to assess for HeFH, we applied Simon Broome’s  or WHO DAt the time of PCSK9 insurance coverage application, before starting PCSK9 therapy, we assessed the following patient characteristics: type and dose of PCSK9 therapy to be started, lipids and lipoprotein cholesterol levels on maximally tolerated diet and lipid lowering drugs, age, gender, BMI (body mass index), previous and current cholesterol lowering therapies, and CVD event age, if applicable. Within the CVD events group, we documented coronary artery disease, acute myocardial infarction, cerebrovascular accidents/stroke, carotid artery disease, and heart failure.For the 61 patients who had a CVD event, the associated direct and indirect costs before starting PCSK9 therapy were calculated using U.S. Department of Health and Human Services, BMC Health Services Research, and Healthcare Bluebook databases –31. For We used the ACC/AHA 10 year cardiovascular disease risk calculator  to estimTo date we have started 103 patients on either alirocumab (62 %) or evolocumab (38 %). Table In the 103 patients, mean ± SD 10-year CVD risk calculated from the AHA/ACC calculator was 14.1 ± 12.3 %, median 11.3 %  to 76 mg/dl, with the median decrement of LDLC on therapy of 79 mg/dl, percent LDLC drop from baseline 54 % (median)  within a society willingness-to-pay threshold , 26, proIn human health-economics there are two major approaches: the human capital approach, and the friction cost method –40. The Approximately 735,000 people in US have a myocardial infarction and 795,000 have a stroke each year [20]. Heidenreich et al. project that approximately 41 % of the United States population will have some form of CVD by 2030 . In the Statins have been used for 3 decades or more, with the CDC estimating that about 55 % of hypercholesterolemic patients are taking cholesterol lowering therapy, of whom ~90 % are taking statins . ConsequMany patients with elevated LDLC cannot achieve treatment targets –3 for map < 0.001 [With the introduction of powerful PCSK9 inhibitors, and as shown in the current study, many patients can regularly meet LDLC targets, <70 mg/dl for those with CVD , reducin < 0.001 . In 2461 < 0.001 . Further < 0.001  beyond s < 0.001 .Statin intolerance, predominantly caused by myalgia, myositis, and myopathy occurs in 5 % to 20 % of statin-treated patients  who willPCSK9 inhibitor therapy is reserved as an adjunct to diet and maximally tolerated statin therapy for adults with HeFH or clinical ASCVD requiring additional lowering of LDLC, at a currently listed cost of $14,000–14,600/patient/year. The cost to the society of this drug will rest heavily on the number of people thought to be at high risk with suboptimal cholesterol lowering despite maximally tolerated cholesterol lowering therapy with history of HeFH –58 or CVStrengths of our study include documented direct costs of CVD events in hypercholesterolemic patients with CVD, as well as calculated indirect costs, both for CVD events in the past, and projected over the forthcoming 10 years. We have used a PVLE model  in orderIn the 61 CVD patients in our study, net costs would be $7,000 /patient/year if PCSK9 had been used in their past, with a 50 % reduction in CVD event rate. For PCSK9 intervention for the future 10 years, net costs were estimated to be $12,459 /patient/year, both below the $50,000/year quality adjusted life-year a benchmark for value of care . As curr