Phloem-feeding insects can manipulate plant-induced resistance and are able to suppress effective jasmonic acid/ethylene (JA/ET) defenses by the induction of inefficient salicylic acid (SA) based responses. As a result, activation of the phenylpropanoid biosynthesis pathway in transgenic plants is anticipated to cause complex interactions between phloem-feeding insects and their host plants due to predicted contradiction between two defense forces: the toxicity of various phenylpropanoids and the accumulation of SA via a branch of the activated pathway.Nicotiana tabacum, by over-expression of the PAP1 transcription factor, on the whitefly Bemisia tabaci, a phloem-feeding insect model. Our performance assays indicated that the over-expression made the transgenic plants a more suitable host for B. tabaci than wild-type (WT) plants, although these plants accumulated significantly higher levels of flavonoids. Transcription analyses of indicator genes in the SA (PR1a) and JA/ET  pathways followed by quantification of the SA and JA hormone levels, indicated that B. tabaci infestation periods longer than 8 hours, caused higher levels of activity of SA signaling in transgenic plants and higher levels of JA/ET signaling in WT plants.Here, we investigated the effect of activating the phenylpropanoids pathway in B. tabaci and likely other phloem-feeding insects. It also indicates the necessity of phloem feeders to suppress these defenses for efficient utilization of plant hosts. Our data also indicate that the defensive chemistry produced by the phenylpropanoids pathway has only a minor effect on the insect fitness.Taken together, these results emphasize the important role JA/ET-induced defenses play in protecting plants from successful infestation by  Plants are in constant battle with herbivorous insects, and have evolved sophisticated defense systems to cope with insect attack, which include both induced and constitutive mechanisms Pathogenensis-related protein (PR) coding genes i.e. to pathogen attack or wounding) PDF1.2VSP2 in A. thaliana) in the MYC2-branch of the JA pathway Plant induced defenses involve activation of distinct signal-transduction pathways, in which three major plant hormones – salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are involved Plant constitutive defenses include the production of secondary metabolites that have antixenotic or antibiotic effects. One of the most widespread secondary metabolic pathways in plants involves the biosynthesis of phenylpropanoids through the shikimic acid pathway. In the starting point of the pathway, phenylalanine ammonia-lyase (PAL) catalyzes the deamination of phenylalanine to cinnamate. Coumaric acid is built by introducing a hydroxy group in the phenyl ring of the cinnamic acid, a process catalyzed by cinnamate-4-hydroxylase. Further downstream, phenylpropanoids metabolism comprises a complex series of branching biochemical pathways that provide plants with thousands of compounds, which are widely used as: structural cell components , pigments , immunity signals (SA) and toxins (coumarins and furanocoumarins) Arabidopsis thaliana and Nicotiana tabacum that constitutively over-express the PAP1 (Production of Anthocyanin Pigment 1)/AtMYB75 or AtMYB12 regulatory proteins of flavonoids biosynthesis Spodoptera frugiperda, Spodoptera litura and Helicoverpa armigeraThe possibility of manipulating the phenylpropanoid pathway, for producing plants resistant to insects, was recently explored by the development of transgenic Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), which is a generalist (polyphagous) species and an important global agricultural pest In this study, we expanded this line of research by investigating the effect of manipulating the phenylpropanoid pathway on phloem feeding insects. In contrast to the aforementioned lepidopteran species, these insects use highly modified mouthparts (stylets) to navigate, intercellularly, through the plant cuticle, epidermis, and mesophyll and establish feeding sites in phloem sieve elements, causing comparatively little tissue damage B. tabaci will have a more complicated relationships with transgenic plants over-expressing PAP1 than lepidopteran chewing insects, due to an anticipated contradiction between two plant defense forces. On one hand, flavonoids, which are specifically over-produced in PAP1 plants N. tabacum glandular trichomes were characterized both at the transcriptomic and proteomic levels, and several flavonoids biosynthesis-related genes as well as proteins were identified Schizaphis graminum, Diuraphis noxia, Aphis craccivora and Aphis fabaeAphis glycines) damage. Analyses of cassava phloem sap and honeydew excretion samples of the phloem feeding mealybug Phenacoccus manihoti indicated both ingestion and metabolic processing of flavonoids by the insect. In addition, significant negative correlation was observed between rutin contents of cassava infested plants and P. manihoti development Our leading hypothesis was that phloem feeding insect such as B. tabaci infestation was shown to induce the SA-responsive gene transcripts, while JA and ET - responsive gene transcripts were repressed or unchanged. Furthermore, work with A. thaliana mutants indicated that the SA-induced defenses are beneficial to the insect and not to the plant which is better protected by JA/ET-induced defenses On the other hand, PAP1 transgenic plants may accumulate high levels of SA, as one of the SA biosynthesis pathways occur via a branch of phenylpropanoids metabolism, involving side-chain shortening of cinnamic acid N. tabacum plants that constitutively over-express the PAP1/AtMYB75 transcription factor. We focused on two main objectives: First, we determined if activation of the phenylpropanoids pathway in N. tabacum plants alters B. tabaci performance. Second, we examined the effect of activating the phenylpropanoids pathway, on the basal and induced expression levels of the JA/ET and SA defense pathways, before and during 18 days of B. tabaci infestation.Testing our hypothesis was achieved by utilizing transgenic F = 121.64, P<0.0001). qRT-PCR analyses of PALA and PALB, revealed no significant transcriptional changes (2χ(1) = 0.42, P = 0.51 and 2χ(1) = 0.047, P = 0.82, respectively). On the other hand, CHS and DFR  were significantly up-regulated in PAP green plants (2χ(1) = 3.85, P = 0.049 for both genes). Selected genes in other branches of the phenylproapnoids pathway: C4H, TOGT and ICS1, did not show significant differences in their expression level between PAP green and WT plants (2χ(1) = 3.85, P = 0.51 for C4H, 2χ(1) = 0.27, P = 0.27 for TOGT, 2χ(1) = 0.42, P = 0.51 for ICS1)  .F = 6.38, P = 0.0178 at 48 h and F = 8.84, P = 0.0061 at 72 h). In addition, estimations of three independent life-history traits related to reproduction, indicated increased adults and nymphs performance on PAP green plants relative to WT plants: (I) significantly higher number of eggs were oviposited  = 4.76, P = 0.036) on PAP green plants  = 5.77, P = 0.026) on PAP green plants  = 6.787, P = 0.033)  thn plants ; (III) t= 0.033) .PR1a was significantly higher in WT plants than in PAP green plants (t(9) = 2.36, P = 0.042 at 0 h and t(10) = 5.53, P = 0.0003 at 8 h). However, during longer periods of infestation, PR1a transcript levels were higher in PAP green plants than in WT plants: marginally significant after 48 h of infestation (t(14) = −1.93, P = 0.07) and highly significant after 18 days (t(14) = −4.85, P = 0.0003). In contrast, after 5 days of infestation, no difference in PR1a expression was observed between WT and PAP green plants. This finding can be related to the intensive mortality of B. tabaci adults observed at this time point, after which new adults were added to the plants (In non-infested (0 h) plants and during the first 8 h of infestation, the expression level of e plants .ERF1 before infestation (0 h) and during the first 8 h of infestation did not differ between WT and PAP green plants (t(9) = 0.19, P = 0.85 at 0 h and t(10) = 1.36, P = 0.20 at 8 h). However, during longer periods of infestation, ERF1 transcript levels were higher in WT plants than in PAP green plants: marginally significant after 48 h of infestation (t(13) = 1.97, P = 0.07) and significant after 5 and 18 days (t(11) = 2.51, P = 0.029 at 5 days and t(13) = 2.09, P = 0.05 at 18 days) (COI1 levels did not differ between the two plant types before and after infestation (P>0.09). However, in both plant types, a trend of increase in the expression of COI1 was observed until 48 h after infestation, followed by a gradual decrease until 18 days (AOC were not significantly different between the two plant types (P>0.13), except for higher levels of expression in WT plants, 18 days after infestation (t(14) = 2.18, P = 0.046) (The expression level of 18 days) . COI1 le 18 days . The exp= 0.046) .t(7) = −0.31, P = 0.75) and during the first 8 h of the infestation period (t(7) = 0.075, P = 0.97). In contrast, after 5 days of infestation, significantly higher levels of SA were detected in PAP green plants than in WT plants (t(9) = 1.99, P = 0.05) (t(8) = 1.26, P = 0.21) and 18 days of infestation (t(10) = 1.43, P = 0.16). In addition, the overall plant-type effect in the three way ANOVA model  was nearly significant (F(1) = 3.56, P = 0.067).Independent estimations of the JA and SA pathways activity in WT and PAP green plants, before and after infestation, were obtained by gas chromatography-mass spectroscopy (GC-MS) quantification of the hormones levels. SA levels did not differ between the two plant types before infestation ( = 0.05) . A promit(8) = 0.19, P = 0.84), but during the different time points after infestation, the levels were higher in WT plants compared to PAP green plants. These differences were nearly significant after 8 h and 5 days (t(8) = −1.84, P = 0.07 for both time points) and highly significant after 18 days of infestation (t(9) = −2.55, P = 0.014). A non significant similar trend was observed after 48 h of infestation (t(8) = −0.55, P = 0.58) (F(1) = 8.46, P = 0.006). Although induced nicotine synthesis is known to be regulated by the JA pathway t(4) = −1.64, P = 0.17) and also after five days of B. tabaci infestation (t(4) = −1.3, P = 0.26)  . In addiB. tabaci nymphs feeding on MeJA-treated PAP green plants reached the late 4th nymph stage or emerged as adults while only 39% of the nymphs feeding on MeJA-treated WT plants have reached these advanced developmental stages (χ2(1) = 7.882, P = 0.0050)  = 0.055, P = 0.81) (2(1) = 14.863, P<0.0001) (2(1) = 1.394, P = 0.24) (B. tabaci and for the low activity of JA-regulated defenses in PAP green plants (see below). Nymph developmental progression was also found to differ significantly between control and SA-treated WT plants  = 9.536, P = 0.0020) and PAP green plants  = 6.198, P = 0.0128). However, these findings should be interpreted cautiously as the high dose of SA (1 mM) used in our repeated spraying regime, greatly reduced the growth rate of WT and PAP green plants and was also associated with chlorosis and yellowing. As previously suggested, this likely indicates physiological or morphological changes caused by the chemical elicitor that seem not to be related to resistance but that nevertheless conduce strong effect on the herbivore fitness parameters As cross-talk between the JA- and SA-defense pathways is commonly associated with plant response to biotic stress, we further dissected the relative importance of the JA- and SA-defense pathways by spraying infested WT and PAP green plants with SA or MeJA (seven sprays during a three weeks period - see ‘ 0.0050) . In cont = 0.81) . As nymp<0.0001)  but not  = 0.24) , these fN. tabacum, on a phloem feeding model, the whitefly B. tabaci. Our no-choice performance assays indicated that activation of the phenylpropanoids pathway made the transgenic plants a more suitable host for B. tabaci than WT plants, although these plants accumulated significantly higher levels of flavonoids. The phenomenon was exemplified by comparing several variables, related both to the insect's survival and reproductive performance.In this study, we investigated the effect of activating the phenylpropanoids/flavonoids pathway in a Solanaceae model, A. thaliana plants and obtained two plant phenotypes: solid purple leaves and purple-veined leaves with a green background (similar to the phenotype of our PAP green plants). While S. frugiperda larvae that fed on solid-purple leaves showed significant reduced feeding than those fed on WT leaves, larvae that fed on purple-veined leaves weighted significantly more than those fed on WT leaves. It was postulated that phenylpropanoids may be capable of enhancing the growth of insect herbivores up to a certain concentration (like in purple-veined leaves) but are toxic at higher concentrations (like in solid-purple leaves). Wuyts et al. Meloidogyne incognita. In both studies, however, the mechanism leading to the enhanced performance of the studied organism, were not explored.Enhanced herbivore performance on transgenic plants over-expressing PAP transcription factors was previously reported in at least two additional systems. Johnson and Dowd PR1a transcript levels were significantly higher in WT plants before and during the first 8 h of B. tabaci infestation. However, at longer infestation periods, PR1a transcript levels were higher in PAP green plants (ERF1 before infestation (0 h) and during the first 8 h of infestation did not differ between WT and PAP green. However, during longer periods of infestation, ERF1 transcript levels were higher in WT plants than in PAP green plants  showed delayed development relative to WT plants, while SA-regulated genes transcripts levels accumulated similarly in the cev1 and WT plants. Furthermore, when npr1 plants (mutants which lack the ability to activate the SA defenses), were treated with MeJA, the treatment accentuated the npr1 delay in B. tabaci nymph development relative to the untreated npr1 plants. Collectively, these data bring strong evidence not only for the strong negative effects of JA/ET defenses on B. tabaci  strain (B-ref) was collected in Israel during 1987. Since its collection, the strain was reared on cotton plants  under standard greenhouse conditions of 26±2°C, photoperiod of L∶D 14 h∶10 h.The N. tabacum variety Xanthi) were grown under standard greenhouse conditions  in 6 cm diameter round pots. Transgenic tobacco plants were produced by over-expressing PAP1, a MYB transcription factor from A. thalianaTobacco plants , C4H (Cinnamate hydroxylase), CHS , DFR (Dihydroflavonol 4-reductase) and TOGT (UDP Glucose scopoletin glucosyltransferase) as well as ICS1 (Isochorismate synthase1), which is involved in SA biosynthesis from the shikimate pathway intermediate chorismate, were determined by quantitative real-time PCR (qRT-PCR) analyses. qRT-PCR protocol and the list of accession numbers, qRT-PCR primers and product sizes are provided in Protocol S2 and Table S1 in A. thaliana plants over-expressing the PAP1 gene indicated that PAP1 specifically regulates flavonoid biosynthetic genes causing accumulation of cyanidin- and quercetin-type flavonoids in a relatively specific manner Anthocyanins content in wild-type (WT) and PAP green plants were determined by the method previously reported in Alon et al. B. tabaci experiments were conducted in a temperature-controlled room in long day conditions . Twenty adult couples, 48 h after emergence, were collected and spread on single WT or PAP green plants in clip cages, situated in 45×60×40 cm Perspex cage. After two days of oviposition, all adults were removed and eggs were allowed to develop. The total number of progeny, their developmental stages , and viability status (live/dead), were recorded after 18 days. Each treatment was replicated 11–20 times. The proportions of live progeny and their developmental progression on WT and PAP green plants were compared using the log-likelihood ratio test (G-test). All statistical analyses conducted in this study used JMP statistical software version 7.0.1  and significance level of P≤0.05.All t-test.For each replicate, one couple of adults, 48 h after emergence, was collected and placed in a 12×8×6 cm plastic cage. One leaf from each plant, WT or PAP green, was gently placed in the cage. Females were allowed to oviposit for 72 h. At the end of each trial, all eggs on both sides of the leaves were counted using a stereoscope, only from replicates in which the females survived the 72 h oviposition period. 7–12 replicates were conducted. The daily oviposition rate on WT and PAP green plants were compared using the Student's B. tabaci adults' was compared between WT and PAP green plants in a non-choice setting, by counting the number of live and dead individuals at various time points  after insect release. Twelve replicates were conducted in each time point. To test for significant differences, we used a three-way repeated-measures ANOVA in which time-point (fixed) was the repeated (within subjects) factor while plant genotype (fixed) and experiment (random) were the main (between subjects) factors. Specific means in the (plant genotype X time-point) interaction were selected a-priori and orthogonal comparisons were conducted.Survival of B. tabaci infestation by quantifying the expression level of the following SA/JA/ET gene markers by qRT-PCR: PR1a (Pathogenesis related protein 1a) which serves as a common indicator for recording the SA signaling pathway activity ERF1 (Ethylene response factor 1) induced by simultaneous action of the JA and ET signaling pathways AOC (Allene oxide cyclase) involved in the JA biosynthetic pathway COI1 (Coronatine insensitive 1) which codes for an F-box protein essential for the induced responses to jasmonates The level of activity of the SA and JA/ET pathways were determined in WT and PAP green plants prior to and after B. tabaci infestation, one leaf of WT or PAP green plant was placed gently into a 12×8×6 cm plastic cage. About 100 B. tabaci adults were aspirated into a glass tube (5 cm high×2 cm diameter) and transferred immediately to each leaf cage for 8 h, 48 h, 5 days and 18 days infestation period. Control WT and PAP green plants were not infested with B. tabaci. A second batch of individuals was released after five days in the 18 days infestation cages, to maintain effective infestation levels. At the end of each infestation period, adults were removed from leaves and total RNA extraction, purification, cDNA synthesis and qRT-PCR analyses were conduced as described in Protocol S2 in For determining the gene's expression levels before and after B. tabaci infestation by gas chromatography-mass spectroscopy (GC-MS) , 72 h after emergence, were collected into a clip cage, 3 cm in diameter, which was attached to a third true leaf. After a 24 h oviposition period, all adults were collected and eggs were allowed to develop. The developmental rate was estimated by calculating the percentage of late fourth nymphs and empty exuvia (indicating emergence) on each plant, 25 days after infestation. Developmental progression on WT and PAP green plants under the three spraying regimes  were compared using the log-likelihood ratio test (G-test). Sequential Bonferroni a priori (planned) comparisons were performed on four selected pairs, using the conservative Dunn-Sidak method at a specified experimental error rate of α = 0.05 (α′ = 1−[1−0.05]k = 41/). This was done in order to avoid minute α′ values obtained when carrying out unplanned comparisons among all possible pairs of means (k = 6×5/2 = 15 comparisons). The four pairs were selected in order to test for two main differences in developmental performance: (I) between nymph feeding on MeJA- or SA-treated WT and PAP green plants ; (II) between nymph feeding on control or MeJA- treated WT and PAP green plants .WT and PAP green tobacco plants were grown under standard greenhouse conditions  in 6 cm diameter round pots until the third true leaf stage. 1 mM SA or MeJA (Sigma) in 0.015% Tween-20 were applied with a household sprayer. Control plants were sprayed with DDW/0.015% Tween-20 only. Spraying was conducted seven times: 24 h before File S1Contains: Protocol S1. Anthocyanin content of PAP green and WT plants. Protocol S2. Quantitative real-time PCR. Protocol S3. Chemical characterization of SA and JA levels in WT and PAP green plants. Protocol S4. Chemical analyses of nicotine levels in WT and PAP green plants. Table S1. List of analyzed phenylpropanoids pathway genes, and their accession numbers, oligonucleodite primers and product sizes. Table S2. List of analyzed induced defense pathways genes, and their accession numbers, oligonucleodite primers and product sizes. Figure S1. Chemical analyses of nicotine levels in WT and PAP green plants: non-infested (A) or after 5 days of B. tabaci infestation (B).(DOC)Click here for additional data file.


Gentiana triflora and Gentiana scabra) are amongst the most popular floricultural plants in Japan. However, genomic resources for Japanese gentians have not yet been developed, mainly because of the heterozygous genome structure conserved by outcrossing, the long juvenile period, and limited knowledge about the inheritance of important traits. In this study, we developed a genetic linkage map to improve breeding programs of Japanese gentians.Japanese gentians  sequences using the recently developed inter-primer binding site (iPBS) method. We also developed retrotransposon microsatellite amplified polymorphism (REMAP) markers combining retrotransposon and inter-simple sequence repeat (ISSR) markers. In addition to SSR and REMAP markers, modified amplified fragment length polymorphism (AFLP) and random amplification polymorphic DNA (RAPD) markers were developed. Using 93 BC1 progeny from G. scabra backcrossed with a G. triflora double haploid line, 19 linkage groups were constructed with a total of 263 markers . One phenotypic trait (stem color) and 10 functional markers related to genes controlling flower color, flowering time and cold tolerance were assigned to the linkage map, confirming its utility.Enriched simple sequence repeat (SSR) libraries from a This is the first reported genetic linkage map for Japanese gentians and for any species belonging to the family Gentianaceae. As demonstrated by mapping of functional markers and the stem color trait, our results will help to explain the genetic basis of agronomic important traits, and will be useful for marker-assisted selection in gentian breeding programs. Our map will also be an important resource for further genetic analyses such as mapping of quantitative trait loci and map-based cloning of genes in this species. Gentiana is the largest genus in the Gentianaceae and includes over 400 species [Gentiana triflora and Gentiana scabra, the so-called Japanese gentians, have been cultivated in Japan since the 1960s. In addition to Japanese gentians, the important floricultural plant Eustoma (Eustoma grandiflorum), otherwise known as lisianthus, and a pharmaceutical plant, Swertia (Swertia japonica), also belong to the family Gentianaceae. Japanese gentians are among the most popular floricultural plants in Japan, and have unique vivid blue flowers [G. triflora, which usually blooms from July to September in Japan. G. scabra blooms later, from September to November, and has some valuable traits, such as an open corolla and resistance to gentian brown leaf spot caused by Mycochaetophora gentianae[G. triflora and G. scabra are 2n=26, whereas those of European pharmaceutical gentians, including Gentiana lutea and Gentiana punctata, are 2n=40. It is difficult to produce interspecific hybrids among members of the genus Gentiana because of the different basal chromosome numbers. The nuclear DNA content in G. triflora is 9.11 to 9.30 pg/2C, and that in G. scabra is 11.35 to 11.75 pg/2C [9 bp/1C), which are larger than those of some other plants, e.g. Arabidopsis thaliana (1.5×108 bp /1C) [Medicago truncatula (3.8×108 bp /1C) [8 bp /1C) [8 bp /1C) [Gentianaceae , comprises 87 genera and more than 1,500 species that are distributed worldwide. The genus  flowers . The maigentianae,4. The cW14/15 alleles, which encode an esterase involved in cold tolerance of winter buds, provided data that clarified phylogenetic relationships in the genus Gentiana, and allowed analysis of the pedigree and breeding history of cultivars derived from those Gentiana spp. [There has been extensive molecular and physiological research on flower colors, flowering times, and cold tolerance of winter buds of Japanese gentians -14. BaseRosa hybrida) [Dianthus caryophyllus) [Lilium sp.) [Catharanthus roseus) [Gentiana or for any member of the family Gentianaceae, including economically important species.Genetic linkage maps are useful for studies on genomic structure and evolution, and for identification of monogenic traits or Mendelian components of quantitative trait loci (QTLs). Therefore, they are very useful for breeding programs and are the basis for future positional gene cloning ,20. Theyhybrida) ,22, carnphyllus) ,24, Asiaium sp.) , and per roseus) . HoweverLolium perenne) was constructed using a backcross population involving a double haploid (DH) tester [G. triflora using anther culture [The majority of floricultural plants are allogamous, but most linkage maps for crop plants have been obtained from segregating populations derived from crosses between inbred lines. Therefore, genetic linkage maps for allogamous and vegetatively reproducing species have been constructed using a double pseudo testcross strategy ,27. Conv) tester ,29. Japa culture .G. scabra, using a BC1 population backcrossed with G. triflora DH line Aki6PS and 308 genetic markers. This is the first reported genetic linkage map for Japanese gentians and the first for any species belonging to the family Gentianaceae. Our results are important for understanding the genetic basis of traits and to apply molecular markers linked to mapped loci for marker-assisted selection in gentian breeding programs.Here, we report the construction of a genetic linkage map for Japanese gentian, G. triflora) yielded 19,416 clones using Roche 454 pyrosequencing. Of these, 8,932 clones (46.0%) contained SSR motifs. Only 599 clones (6.7%) were suitable for designing primer sets for SSR markers; the rest comprised reads that were too short (<200 bp) to design primers from the proximal sequences of the SSR motif. Therefore, in addition to next generation sequencing, 2,539 clones from the enriched SSR libraries were subjected to Sanger DNA sequencing analysis. As a result, 1,048 clones (41.3%) had SSR motifs within the sequence reads, and 630 clones (60.1%) could be used to design primer sets for SSR markers. These results indicated that the conventional strategy using Sanger sequencing was more efficient than next generation sequencing for developing SSR markers, without considering cost, labor, and time.The enriched SSR libraries from the DH line Aki6PS  has been reported in G. scabra[To increase the number of available genetic markers for Japanese gentian, we developed some retrotransposon microsatellite amplified polymorphism (REMAP markers) using a retrotransposon. A retrotransposable element G. scabra. TherefoG. scabra  amplification using combinations of selective primers with three selective nucleotides resulted in too many faint and overlapping fragments in gentian (data not shown). To solve this problem, we added a fourth selective nucleotide to the n et al.  (Table 4We first screened for reproducible and stable random amplification polymorphic DNA (RAPD) markers using 47 primers, as described by Debener and Mattiesch  and by YCHS) and the flowering locus T1 (FT1) and FT2 genes from both G. triflora Aki6PS and G. scabra SP6A1. Comparative sequencing analysis identified significant in/del polymorphisms with the intron regions of CHS and FT1, and marker primer sets were designed in the proximity of the in/dels of each gene (Table CHI), flavanone 3-hydroxylase (FHT), flavonoid 3′,5′-hydroxylase (F3′5′H), anthocyanidin synthase (ANS), MYB3, basic helix loop helix 1 (GtbHLH1), terminal flowering 1 (TFL1) and W14/15 genes, we used the primer sets reported in previous studies [FT2 and TFL1 genes, but some SNPs were detected using restriction enzymes. Therefore, FT2 and TFL1 markers were distinguished between Aki6PS and SP6A1 as cleaved amplified polymorphic sequence (CAPS) markers, when their amplified fragments were digested by Pst I and Sau3A I, respectively.In Japanese gentians, there has been extensive physiological and molecular research on flower pigmentation, flowering time, and cold tolerance of winter buds, and the nucleotide sequences of some important genes have already been deposited in public databases. In addition, our previous study reported significant insertions/deletions (in/dels) in introns and untranslated regions, representing molecular polymorphisms that could be used to discriminate among cultivars and species of Japanese gentians . Therefo studies ,14,16,17G. triflora breeding line Aki6PS has a green stem color, while stems of the G. scabra breeding line SP6A1 and F1 are red because of the accumulation of anthocyanins. Among BC1 progeny, 44 and 49 individuals showed green- and red-stem color phenotypes, respectively. Segregation of stem color fitted a 1:1 ratio based on a chi-square test at P = 0.05, suggesting monogenetic inheritance of this trait.The 1 from G. scabra SP6A1 backcrossed with G. triflora Aki6PS DH line  were assembled from markers with distorted segregation at the 0.1% level in the genetic map of Japanese gentian.We used a population of 93 BCe Figure . In totaApis mellira) and zebrafish (Danio rerio), only 1.2% to 5.4% sequences obtained by 454 FLX Titanium pyrosequencing could be used to design primers for SSR markers [We used 454 FLX Titanium pyrosequencing of an SSR-enriched library to develop SSR markers from Japanese gentians. Recently, next generation sequencing has been used successfully to develop several genetic markers, including SSRs -37. In oGsTRIM1, Tgt1 (gypsy-Ty3 retrotransposon), and GtMITE1, have been identified [Tgt1 and GtMITE1 in the gentian genome [GsTRIM1 and LTR of Tgt1. We designed primers from these newly identified LTR sequences , the use of selective primers with a fourth selective nucleotide also improved amplification of reproducible fragments [RAPD and AFLP technologies are conventional molecular markers. Their advantages are that they do not require sequence information and have a relatively low start-up cost -43. Howeragments . Therefo1 population. Nineteen linkage groups were obtained for the BC1 population at an LOD value of 9.0, and encompassed 613.7 cM , flowering time  and cold tolerance of winter buds (W14/15), were mapped to the 19 linkage groups of Japanese gentian . These findings will be useful for determining the molecular functions of these genes and to study the genetic regulation of flowering time in gentian, although further research is necessary. The mapping of functional markers corresponding to important agricultural traits will be accelerated by further molecular and physiological studies on Japanese gentians. Our genetic linkage map will be useful for mapping QTLs associated with various traits, and for improving Japanese gentian breeding programs. The map is also applicable to other members of the Gentianaceae family, including several other economically important species.Functional markers are those derived from polymorphic sites within genes responsible for phenotype traits . Ten funB3 locus ,49. Cons1 progeny derived from a DH line and included 274 markers . The map revealed 19 linkage groups that covered 613.7 cM, with an average intermarker distance of 2.2 cM. This map is a starting point for mapping single or quantitative trait loci affecting agronomically important phenotypes, and will be useful for research on gentian genetics and breeding.This study presents the first genetic linkage map for Japanese gentian. The map was constructed using four different types of molecular markers. It was produced from genotypes of 93 BC1 progeny were derived from the interspecific backcross Aki6PS×SP6A1  showed hybrid weakness and it was difficult to obtain sufficient progeny.In total, 93 BC1 Figure . Aki6PS  culture . SP6A1 iGenomic DNA was isolated from young leaves (500 mg) of each individual using Nucleon PhytoPure  and stored at −20°C until use.G. triflora Aki6PS or G. scabra SP6A1, and then digested by either Alu I, Hae III, Mse I, or Rsa I. The digested genomic DNA was hybridized with biotin-labeled (CA)15 or (GA)15 probes. SSR-enriched libraries were mixed and subjected to sequencing analysis using 454 GS FLX Titanium pyrosequencing . The SSR-enriched library was also subcloned into the pGEM-T Easy vector , and subjected to sequencing analysis using BigDye terminator version 1.1 cycle sequencing kit and an ABI PRISM 3130xl Genetic Analyzer . SSR markers were predicted and developed using Read2Marker software with the default parameters [SSR markers were developed as described by Sato-Ushiku et al. . Genomicrameters .Ex Taq polymerase . The PCR conditions were as follows: 94°C for 2 min; 30 cycles at 95°C for 20 s, 60°C for 40 s, and 72°C for 1 min; and a final extension at 72°C for 5 min. The amplified fragments were separated on a high-efficiency genome scanning (HEGS) running system  [PCR amplification was performed in a 20-μL reaction mixture containing 10 ng genomic DNA, 0.5 μM each primer  . In brieEx buffer, 1 μM each primer, 0.2 mM dNTPs, and 1 unit ExTaq DNA polymerase. The PCR program was as follows: 1 cycle at 95°C for 3 min; 30 cycles of 95°C for 15 s, 50–70°C for 1 min, and 72°C for 1 min; and a final extension step of 72°C for 5 min. The sequences and annealing temperatures of iPBS primers are shown in Table The sequences of LTRs of retrotransposons were isolated from gentian genome using the iPBS approach . PCR wasEx buffer, 0.2 mM dNTPs, 0.2 μM each primer, and 1 unit Ex Taq polymerase. The PCR program was as follows: 94°C for 4 min; 30 cycles of 94°C for 40 s, 60°C for 40 s, and 72°C for 2 min; followed by final extension at 72°C for 5 min. The amplified fragments were separated on 1.0 or 1.6% (v/v) agarose gels in TAE buffer, and then photographed and analyzed with an ImageQuant LAS-4000 system after staining with ethidium bromide.The REMAP markers were created by combining LTR primers with ISSR primers, as shown in Table Mae I and 50 units of EcoR I, in a reaction volume of 25 μl with restriction enzyme buffer  bovine serum albumin, pH 7.5) at 37°C for 3 h. The restriction enzyme solution was added to 10 μl adapter ligation solution , and then incubated at 37°C overnight. To stop the reaction, 500 μl T10E0.1 buffer  was added to the ligation solution.AFLP analysis was performed as described by Vos et al.  with somExTaq buffer, and 1.25 units ExTaq DNA polymerase (Takara-bio). The PCR program was as follows: 94°C for 2 min; 20 cycles of 94°C for 30 s, 56°C for 1 min and 72°C for 1 min; followed by final extension at 72°C for 10 min. The PCR products were diluted 100-fold in T10E0.1 buffer. Four labeled-EcoR I selective primers with three selective nucleotides  and 10 Mse I selective primers with four selective nucleotides  were used for selective amplification. We screened 14 primer combinations because they provided the highest number of heterozygous bands  using GeneMapper software (Applied Biosystems).Pre-amplification was conducted in a 25-μl reaction volume containing 2.5 μl diluted adaptor-ligated DNA, 0.4 μM EcoRI+A primer, 1.6μM MseI+C primer, 200 μM dNTPs, 1×Ex buffer, 0.2 mM dNTPs, 0.4 μM 15-mer primer, and 1 unit Ex Taq polymerase. The PCR program consisted of 94°C for 90 s; 40 cycles of 95°C for 30 s, 53°C for 1 min 30 s, and 72°C for 2 min; followed by final extension at 72°C for 5 min. The amplified fragments were separated on 1% (v/v) agarose gels in TAE buffer, and then photographed and analyzed as described in the REMAP markers section.The long RAPD markers were developed as described by Debener and Mattiesch  and YamaCHS, CHI, FHT, F3′5′H, ANS, and the transcriptional factors GtMYB3 and GtbHLH1 exhibited significant polymorphisms among Japanese gentian cultivars [GtFT1, GtFT2, and GtTFL1, which are regulator genes of flowering time [′/15a′ and 14b1/14b1′, respectively [GtFT2 and GtTFL1 have no large insertions or deletions between Aki6PS and SP6A1. Therefore, CAPS markers were used for GtFT2 and GtTFL1 by digesting the amplified fragments with Hae III and Sau3A I, respectively.Our previous research revealed that the intron lengths of several flavonoid biosynthetic genes, including ultivars ,16,17. Ping time , were alEx buffer, and 0.25 units Ex Taq polymerase. The PCR conditions were as follows: 94°C for 2 min; 30 cycles at 95°C for 20 s, 60°C for 40 s, and 72°C for 1 min; and a final extension at 72°C for 5 min. The amplified fragments were separated on the HEGS running system or 2% agarose gels in TBE buffer, and then photographed and analyzed as described above.PCR amplification was performed in a 20-μL reaction mixture containing 20 ng genomic DNA, 0.5 μM each primer (as shown in Table G. triflora (A) and G. triflora × G. scabra hybrid (H) genotypes, respectively.Stem colors were investigated three times in the mapping population. Green and red stem colors were scored as We used 308 markers, including 101 SSRs, 38 REMAPs, 103 AFLPs, 54 long RAPDs, 11 functional markers, and one phenotypic trait to construct a genetic linkage map using JoinMap ver. 4.1 . The Kosambi function was used to convert recombination units into genetic distances. The mapping analysis was conducted by using a minimum LOD score of 9.0. Distorted markers analyzed by the chi-square test were used in the construction of the linkage maps.′5′H: Flavonoid 3′,5′-hydroxylase; FHT: Flavanone 3-hydroxylase; FT: Flowering locus T; ISSR: Inter-simple sequence repeat; LTR: Long terminal repeat; MITE: Miniature inverted-repeat in transposable element; PBS: Primer binding site; QTL: Quantitative trait loci; RAPD: Random amplification polymorphic DNA; REMAP: Retrotransposon microsatellite amplified polymorphism; SCAR: Sequence characterized amplified region; SSR: Simple sequence repeat; TDR: Terminal direct repeat; TFL1: Terminal flowering 1; TRIM: Terminal repeat retrotransposon in miniature.AFLP: Amplified fragment length polymorphism; ANS: Anthocyanidin synthase; bHLH: Basic helix-loop-helix; CAPS: Cleaved amplified polymorphic sequence; CHS: Chalcone synthase; CHI: Chalcone isomerase; DH: Doubled haploid; F3The authors declare that they have no competing interests.TN participated in the design of the experiments, and developed and genotyped the SSR, AFLP and functional markers, performed statistical analysis, data interpretation and wrote the paper. EY developed and genotyped the RAPD and REMAP markers. TH participated in the design of the experiments and produced populations for genetic analysis. MS developed and genotyped the SSR and AFLP markers. YU developed the SSR markers. MN participated in the design of the experiments and wrote the paper. All the authors read and approved the manuscript.


Sphagnum carpets (15 cm diameter) collected in hummock, lawn and pool microsites (origin) at three local conditions  using trenches dug in a peatland. One series of samples was seeded with microorganism extract from all microsites. TA community were analysed at T0: 8–2008, T1: 5–2009 and T2: 8–2009. We analysed the data using conditional inference trees, principal response curves (PRC) and DWT inferred from TA communities using a transfer function used for paleoecological reconstruction. Density declined from T0 to T1 and then increased sharply by T2. Species richness, Simpson diversity and Simpson evenness were lower at T2 than at T0 and T1. Seeded communities had higher species richness in pool samples at T0. Pool samples tended to have higher density, lower species richness, Simpson diversity and Simpson Evenness than hummock and/or lawn samples until T1. In the PRC, the effect of origin was significant at T0 and T1, but the effect faded away by T2. Seeding effect was strongest at T1 and lowest vanished by T2. Local condition effect was strong but not in line with the wetness gradient at T1 but started to reflect it by T2. Likewise, TA-inferred DWT started to match the experimental conditions by T2, but more so in hummock and lawn samples than in pool samples. This study confirmed that TA responds to hydrological changes over a 1-year period. However, sensitivity of TA to hydrological fluctuations, and thus the accuracy of inferred DWT changes, was habitat specific, pool TA communities being least responsive to environmental changes. Lawns and hummocks may be thus better suited than pools for paleoecological reconstructions. This, however, contrasts with the higher prediction error and species’ tolerance for DWT with increasing dryness observed in transfer function models.Peatland testate amoebae (TA) are well-established bioindicators for depth to water table (DWT), but effects of hydrological changes on TA communities have never been tested experimentally. We tested this in a field experiment by placing The online version of this article (doi:10.1007/s00248-014-0367-8) contains supplementary material, which is available to authorized users. Sphagnum-dominated peatlands cover vast surfaces of North America and Eurasia. They are nevertheless among the most vulnerable ecosystems to global warming  of testate amoeba taxa observed in the experimental plots at Le Russey, French Jura in seeded and non-seeded plots over time. (DOCX 26.4 kb)Supplementary Table 4Summary results of the RDA model on testate amoeba community data showing changes in significance of the origin , seeding  and local condition  over the course of the experiment . Significant values are indicated in bold. (DOCX 11.9 kb)Supplementary Figure 1origin) placed at high, intermediate and low water table position  in the experimental trenches of Le Russey bog, French Jura. T0, August 2008; T1, May 2009 and T2, August 2009. (JPEG 378 kb)Changes over time in density [ind/mg], species richness (N0), Simpson diversity (N2) and evenness (E2 = N2/N0) of living testate amoebae of communities seeded vs. non-seeded with mixed extract from hummock, lawn and pool habitats, showing data of samples from hummock, lawn and pool habitats (High Resolution Image (EPS 720 kb)Supplementary Figure 2Changes over time in density [ind/mg], species richness (N0), Simpson diversity (N2) and evenness (E2 = N2/N0) of living testate amoebae for samples collected in hummock, lawn and pool habitats, showing data of samples seeded and not seeded with mixed extract from the three habitats (origin) and placed at high, intermediate and low water table position  in the experimental trenches of Le Russey bog, French Jura. T0, August 2008; T1, May 2009 and T2, August 2009. (JPEG 397 kb)High Resolution Image (EPS 725 kb)Supplementary Figure 3Changes over time in density [ind/mg], species richness (N0), Simpson diversity (N2) and evenness (E2 = N2/N0) of living testate amoebae for samples placed at high, intermediate and low water table position  the experimental trenches of Le Russey bog, French Jura, showing data of samples collected in hummock, lawn and pool habitats (origin) seeded and not seeded with mixed extract from the three habitats. T0, August 2008; T1, May 2009 and T2, August 2009. (JPEG 372 kb)High Resolution Image (EPS 580 kb)Supplementary Figure 4D dry, M moist, W wet). Data from communities seeded with mixed extract from hummock, lawn and pool habitats. See Fig. Depth to water table  inferred (using the transfer function model from Engadine ), from tHigh Resolution Image (EPS 444 kb)Below is the link to the electronic supplementary material.


Andrographis paniculata and widely used in Traditional Chinese Medicine, exhibiting potently anti-inflammatory property by inhibiting NF-κB activity. The purpose of our investigation was designed to reveal the effect of andrographolide on various aspects of LPS induced inflammation in vivo and in vitro.Nuclear factor-κB (NF-κB) is a central transcriptional factor and a pleiotropic regulator of many genes involved in acute lung injury. Andrographolide is found in the plant of In vivo, BALB/C mice were subjected to LPS injection with or without andrographolide treatments to induce ALI model. In vitro, MLE-12 cells were stimulated with LPS in the presence and absence of andrographolide. In vivo, pulmonary inflammation, pulmonary edema, ultrastructure changes of type II alveolar epithelial cells, MPO activity, total cells, neutrophils, macrophages, TNF-α, IL-6 and IL-1β in BALF, along with the expression of VCAM-1 and VEGF were dose-dependently attenuated by andrographolide. Meanwhile, in vitro, the expression of VCAM-1 and VEGF was also reduced by andrographolide. Moreover, our data showed that andrographolide significantly inhibited the ratios of phospho-IKKβ/total IKKβ, phospho-IκBα/total IκBα and phospho-NF-κB p65/total NF-κB p65, and NF-κB p65 DNA binding activities, both in vivo and in vitro.These results indicate that andrographolide dose-dependently suppressed the severity of LPS-induced ALI, more likely by virtue of andrographolide-mediated NF-κB inhibition at the level of IKKβ activation. These results suggest andrographolide may be considered as an effective and safe drug for the potential treatment of ALI. Andrographis panicula a popular herb has been widely used as folk medicine in China for the treatment of viral infection, diarrhoea, dysentery and fever for hundreds of years. Moreover, andrographolide, extracted and purified from Andrographis panicula, is currently prescribed for treatment of inflammation related diseases, such as laryngitis, upper respiratory tract infection and rheumatoid arthritis in China Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), were the diseases induced by many extreme conditions including severe sepsis, severe bacterial pneumonia, trauma and burn. The pathophysiological mechanism of ARDS is believed to be associated with the uncontrolled inflammatory response in lungs Vascular adhesion molecule (VCAM)-1, a member of the immunoglobulin gene superfamily, is up-regulated in inflammatory condition, promoting the inflammatory cells binding to the endothelial cell Therefore, our study was designed to investigate the value of andrographolide on various aspects of LPS induced inflammation in vivo and in vitro.All animal use procedures were approved the Committee on the Ethics of Animal Experiments of Chongqing Medical University. This investigation was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Six weeks old specific pathogen-free male BALB/c mice (16–20 g) were maintained under specific pathogen-free conditions in the facilities of the animal center in our university. The mice were kept in a temperature controlled room with 12 h dark/light cycles, and allowed food and water ad libitum. Animals underwent an acclimatization period of at least 7 days before use in our study.Forty male BALB/c mice were randomly divided into 4 groups (n = 10), control group, LPS group, low dosage andrographolide  treatment group  and high dosage andrographolide treatment group . According to the report, ALI was induced by LPS  via intratracheal injection Seventy two hours later, mice were exsanguinated after pentobarbitone (50 mg/kg i.p.) anesthesia. According to the previous report, BALF was collected by cannulating the upper part of the trachea, by lavage three times with 1.0 ml PBS (pH 7.2). The fluid recovery rate was more than 90%. Lavaged sample from each mouse was kept on ice. BALF was centrifuged at 700 ×g for 5 min at 4°C The BALF supernatant was collected after centrifugation (for 4 minutes at 4000 rpm) and stored at –70°C before cytokine assay. TNF-α, IL-6 and IL-1β in BALF were measured by ELISA . The limit of detection of this method was better than 7.8 pg/ml.4 buffer (PH 6.0) with 0.5% hexadecyltrimethylammonium bromide (HTAB). Then, samples were sonicated and incubated at 60°C for 2 h. Later, samples were assayed for activity in a H2O2/o-dianisidine buffer at 460 nm with a spectrophotometer . Results are expressed as units of MPO activity per gram of lung tissue.After BALF collection, the left upper lobe was removed, washed and kept in –80°C. Then, after weighing, the lungs were homogenized, centrifuged  and resuspended in 50 mM KPOThe severity of pulmonary edema was assessed by the wet to dry ratio (W/D ratio). The right lower lungs weighed and then dehydrated at 60°C for 72 h in an oven.The left lower lung from each mouse was fixed in 10% formalin, embedded in paraffin, cut into 5 µm sections, stained with H&E. Lung injury score was measured by a blinded pathologist with a 0 to 4 point scale according to combined assessments of inflammatory cell infiltration in the airspace or vessel wall, alveolar congestion, hemorrhage, alveolar wall thickness and hyaline membrane formation. A score of 0 represented no damage; l represented mild damage; 2 represented moderate damage; 3 represented severe damage and 4 represented very severe histological changes The fresh left upper lung tissues (2×2×2 mm) were taken for electron microscopy. The specimen was fixed in 2.5% glutaraldehyde and phosphate buffer. The specimen was then rinsed in phosphate buffer, postfixed with 1% osmic tetroxide in phosphate buffer. After graded dehydration in ethyl alcohol and propylene oxide, specimen was embedded in Spurr resin. Then, the embedded tissues were thin-sectioned, mounted on copper grids, and stained with uranyl acetate and lead citrate. The images were taken by electron microscope .3 cells per well in a 96-well plate and grown to 80% confluency in Hites media 10% fetal bovine serum. Then, cells were pretreated with 50 µM andrographolide or vehicle (0.01% DMSO) 4 h before LPS (0.5 µg/ml) stimulation. Total and nuclear proteins and mRNA were extracted from cells at specified time intervals. MTT assay  was used to assess cell viability.MLE-12 cells , a cell line derived from murine alveolar epithelial cells, were plated at a density of 5×105′-TGGATGTCTACCAGCGAAGC-3′ and (reverse) 5′-ACAAGGCTCACAGTGATTTT-3′; VCAM-1 (forward) 5′-CCTCACTTGCAGCACTACGGGCT-3′ and (reverse) 5′-TTTTCC AATATCCTCAATGACGGG-3′ β-actin, (forward) 5′-CGAGCGGGCTACAGCTTC-3′ and (reverse) 5′-GTCACGCACGATTCCCTCT-3′. The mouse β-actin housekeeping gene was used as an internal control.Total RNA was isolated from the lung tissues and MLE-12 cells by Trizol reagent . PCR was performed with a DNA thermal cycler in a 50 µl reaction volume, containing 5 µl 10×Taq Buffer, 4 µl 2.5 mM dNTP, 4 µl 25 mM MgCl2, forward and backward primers 2 µl each, Taq polymerase 0.5 µl and cDNA template 2 µl, for 35 cycles via GeneAmp PCR system 9700 . The primer sequences were as follows: VEGF (forward) The protein expression was measured by western blotting. The lung tissues were kept in −80°C. By the aid of a tissue grinder, the lung tissues were homogenized in PBS containing the protease inhibitor cocktail. The homogenates were centrifuged for 15 min at 14, 000 rpm in 4°C. Meanwhile, cells were washed twice with cold PBS. Then, cells were lysed with SDS sample buffer containing 50 mM Tris (pH 7.4), 2% SDS (wt/vol), 5% 2-mercaptoethanol and 10% glycerol. Cell homogenates were centrifuged at 10,000 rpm at 4°C for 60 min. Supernatants of lung tissues and cells were collected, and protein concentration of each sample was measured with a bicinchoninic acid assay kit using BSA as standard . An equal amount of protein from each sample (150 µg) was resolved in 10% Tris-glycine SDS polyacrylamide gel. Protein band was blotted to nitrocellulose membrane. After incubation for 1 h in blocking solution  at room temperature (RT), the membrane was incubated for 24 h with anti-VEGF , anti-VCAM-1 , anti-β-actin , anti-IKKβ , anti-phospho-IKKβ , anti-IκBα , anti-phospho-IκBα , anti-NF-κB p65  or anti-phospho-NF-κB p65 , at 4°C, respectively. The secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin) was added at 1∶10,000 dilution and incubated at room temperature for 1 h. Peroxidase labeling was detected with the enhanced chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech) and analyzed by a densitometry system. The relative protein level was normalized to β-actin .For detection NF-κB p65 DNA-binding activity, TransAM™ NFκB p65 Chemi Transcription Factor Assay Kit was used .P<0.05 was considered to be statistically significant.Statistical analyses were performed with Sigmaplot software (SPSS). Each point corresponds to mean ± SD Statistical differences were determined by one-way or two-way analysis of variance (ANOVA) and [LOOSEST ]To assess the pathological changes, HE staining and lung injury score system were used in our study. Histological evaluation of lung sections 3 day after the LPS injection revealed evidence of notable inflammatory cells infiltration, interstitial edema, interalveolar septal thickening and intraalveolar and interstitial patchy hemorrhage. However, after andrographolide treatments, the pathological changes in the lung tissues were relieved.  And the After 72 h LPS stimulation, typical changes were found in type II alveolar epithelial cells. These features included cell swollen, reduced and indistinct cell surface microvilli, mitochondria reduced with dilated mitochondrial cristae, along with decreased and vacuolated lamellar bodies.  However,To further identify the anti-inflammatory property of andrographolide, cellular counts in BALF were measured in our study. Three days after LPS injection, total cells, neutrophils and macrophages were remarkably increased in BALF. However, andrographolide treatments dose-dependently reduced total cells, neutrophils and macrophages in BALF. .TNF-α, IL-6 and IL-1β in BALF were measured by ELISA. TNF-α, IL-6 and IL-1β in BALF in the mice with LPS injection were markedly higher than the mice in control group. However, TNF-α, IL-6 and IL-1β in BALF in the mice with andrographolide injections were decreased. .The accumulation of activated of neutrophils was assessed by measuring MPO activity in the lung tissues. MPO activity in model group was markedly higher than control group. However, MPO activity was dose-dependently reduced by andrographolide. .Compared with control group, enhanced mRNA and protein expression of VCAM-1 and VEGF were noticed after LPS injection.  MeanwhilNF-κB is a critical transcription factor for inflammatory genes expression. Activation of NF-κB pathway leads to phophorylation of p50 and p65. Our investigation found a noticeable increased p65 subunit phophorylation 72 h after LPS injection, which was ameliorated by andrographolide, indicating that andrographolide inhibited the activation of NF-κB.  MeanwhilMLE-12 cells, derived from murine alveolar epithelial cells, were used in our study. The cell viabilities were significantly reduced after LPS treatment. Meanwhile, MTT assay also revealed that the cell viabilities were improved in the presence of andgrapholide. .To further explore anti-inflammatory activity of andrographolide, MLE-12 cells were treated with LPS to induce inflammation reaction and NF-κB activation. LPS stimulation led to up-regulation of VCAM-1 and VEGF. Pretreatment with andrographolide (50 µM) exhibited strongly suppression of LPS-induced up-regulation of VCAM-1 and VEGF in vitro. .LPS induced a rapid activation of IKKβ, IΚBα and NF-κB p65 in 6 h. And the maximum phosphorylation levels of IKKβ, IΚBα and NF-κB p65 were observed after 12 h of LPS stimulation. Meanwhile, after 24 h of LPS stimulation, the phophorylation levels of IKKβ, IΚBα and NF-κB p65 were still in maximum. In contrast, after andrographolide treatment, lower phophorylation levels of IKKβ, IΚBα and NF-κB p65 were observed at each time point.  FurthermIn present study, we demonstrated that andrographolide dose-dependently attenuates LPS-induced ALI by inactivation of NF-κB at the level of the phosphorylation of IKKβ.Andrographis paniculata and wildly used in China and other Asia countries. Recently, it was reported that andrographolide was a powerful NF-κB inhibitor in inflammatory diseases Andrographolide was one of major effective component in the plant of Acute respiratory distress syndrome (ARDS) is an acute and progressive respiratory disease without abnormal cardiac filling pressure and is characterized by progressively diffuse bilateral pulmonary edema and inflammation, reduced pulmonary compliance, and hypoxemia LPS, the outer membrane of Gram-negative bacteria, was the one of mainly pro-inflammatory reaction factor in infection diseases, leading to over inflammatory reaction in vivo. Neutrophils and macrophages were the mainly inflammatory cells in ALI. They infiltrated into the lung tissues, releasing enzymes and phagocytizing the pathogen. Meanwhile, these inflammatory cells were the fundamental source of inflammatory mediators in vivo. In our study, the classical ALI pathological changes were found in LPS-induced ALI murine model. However, the pathological changes were dose-dependently reduced by andrographolide. , 3 PulmoNeutrophils arrive quickly at sites of infection and form the first line of defence against invading micro-organisms. Neutrophil and macrophages were the major inflammatory cell types in ALI Various inflammatory mediators were involved in ALI. Among them, TNF-α, IL-6 and IL-1β were considered as the most important inflammatory mediators in innate immune response It is known that amount of inflammatory mediators and adhersion molecules (AM) participate in inflammation. VCAM-1 belongs to the immunoglobulin gene superfamily. Its main function is to mediate epithelial cells, including alveolar epithelial cells, binding to the leukocytes with VLA-4. Otherwise, Qiangsong Tong, et al found that on the upstream of the transcription start site of the VACM-1 gene contains a binding site for NF-κB Then, it is reasonable to explore the condition of NF-κB family members and their relations with andrographolide in our study. The NF-κB family of transcriptional factors (TF) plays a critical role on regulation the expression of a wide variety of genes, particularly in the inflammatory process. The NF-κB family has five cellular members. They are p105/p50 (NF-κB1), p100/p52 (NF-κB2), p65 (RelA), RelB and c-Rel Our data show, to our knowledge for the first time, that andrographolide treatment reduced the severity of LPS-induced ALI, more likely by virtue of andrographolide-mediated NF-κB inhibition at the level of IKKβ activation. These results suggest andrographolide may be considered as an effective and safe drug for the potential treatment of ALI. Nevertheless, animal studies are limited to hours while ARDS patients often are treated for several days or even weeks. Taken together, we believe that andrograophlide may show its beneficial effects over time when it is given at the beginning of ALI.


Triflolium pratense), for use in treating asthma or chronicobstructive pulmonary disease (COPD). Biochanin A )significantly attenuated airway resistance (RL), enhanced pause (Penh), and increased lung dynamic compliance (Cdyn) values induced by methacholine (MCh) in sensitized and challenged mice. It also significantly suppressed an increase in the number of total inflammatory cells, neutrophils, and eosinophils, and levels of cytokines,including interleukin (IL)-2, IL-4, IL-5, and tumor necrosis factor (TNF)-α inbronchoalveolar lavage fluid (BALF) of the mice. However, it did not influenceinterferon (IFN)-γ levels. Biochanin A  also significantlysuppressed the total and ovalbumin (OVA)-specific immunoglobulin E (IgE) levels inthe serum and BALF, and enhanced the total IgG2a level in the serum of these mice. The PDE4H/PDE4L value of biochanin A was calculated as >35. Biochanin A did not influence xylazine/ketamine-induced anesthesia. Biochanin A (10~30 μM) significantly reduced cumulative OVA (10~100 μg/mL)-induced contractions in the isolated guinea pig trachealis, suggesting that it inhibits degranulation of mast cells. In conclusion, red clover containing biochanin A has the potential for treating allergic asthma and COPD.The present study investigated the potential of biochanin A, a phytoestrogenicisoflavone of red clover ( Triflolium pratense L., Fabaceae) contains formononetin (4′-O-methyldaidzein), biochanin A (4′-O-methylgenistein), daidzein, and genistein in relative proportions of 5.46%, 1.97%, 0.43%, and 0.11% -Rolipram was purchased from Amersham Pharmacia Biotech . Other reagents, such as CaCl2, MgCl2, and NaCl, were of analytical grade. Biochanin A, rolipram, and Ro 20-1724 were dissolved in a mixture of ethyl alcohol and DMSO (1 : 1). Other drugs were dissolved in distilled water. The final concentration of ethyl alcohol or DMSO in vitro was ≤0.5%, and did not significantly influence tracheal contractions or the binding of test drugs to high-affinity rolipram binding sites (HARBSs).Biochanin A , ovalbumin (OVA), methacholine (MCh), polyethyleneimine, aluminum sulfate hexadecahydrate, dimethylsulfoxide (DMSO), bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (Bis-Tris), chloralose, urethane, Tris-HCl, benzamidine, phenylmethanesulfonyl fluoride (PMSF), Tween 20, ad libitum. Under a protocol approved by the Animal Care and Use Committee of Taipei Medical University, the following in vivo and in vitro experiments were performed.Male Hartley guinea pigs (500~600 g) and female BABL/c mice at age 8–12 weeks were obtained from the Animal Center of the National Science Council . The animals were housed in ordinary cages at 22 ± 1°C with a humidity of 50%~60% under a constant 12/12-h light/dark cycle and provided with food and water μM) or rolipram (0.1~1000 nM), a reference drug, to bind to HARBSs of the membranes was determined by replacing 2 nM [3H]-rolipram in a reaction buffer at 30°C for 1 h, according to a modified version of method described by previous investigators -rolipram, and 5 μL of biochanin A or rolipram. After 1 h, the reaction was terminated by placing the reaction vessel in crushed ice. The reaction mixture was then transferred onto Whatman GF/B glass-fiber filters soaked in a 0.3% polyethyleneimine solution in a minifunnel. The reaction mixture was filtered by centrifuging it at 90 g for 10 s and the filtrate was collected in a 1.5-mL Eppendorf tube with a top adapted to fit the outlet of the minifunnel. The filters were each washed three times with 300 μL of reaction buffer in the same manner and transferred into a 2 mL cocktail for radiation counting  using a β-scintillation counter . Nonspecific binding, which was defined in the presence of 10 μM Ro 20-1724, was subtracted from the total binding to yield the specific binding. The effective concentrations (EC50) values of biochanin A and rolipram, at which half of the [3H]-rolipram bound with HARBSs of cell membranes was displaced, were defined as the PDE4H values and these may be correlated with adverse effects, such as nausea, vomiting, and gastric hypersecretion -rolipram binding on HARBSs of guinea pig brain cell membranes (μM), however, the percentage displacement by biochanin A was 23.4%  ± 2.9% (n = 4). The concentration cannot exceed 300 μM owing to the solubility of biochanin A. In other words, the EC50 value of biochanin A for the displacement was >300 μM. Rolipram (0.1~1000 nM), a selective PDE4 inhibitor, also concentration-dependently displaced [3H]-rolipram binding on HARBSs (n = 8). The EC50 value of rolipram for displacement was 5.2 ± 1.9 nM (n = 8). As defined in the Methods section, therefore, the PDE4H values of biochanin A and rolipram were >300 μM and 5.2 ± 1.9 nM, respectively. To inhibit PDE4 catalytic activity, the IC50 values of biochanin A and rolipram were reportedly required to be 8.5 μM i). The decrease in [Ca2+]i may be due to a reduction in the influx of Ca2+, enhanced Ca2+ uptake into the sarcoplasmic reticula, or enhanced Ca2+ extrusion through the cell membrane [2+ entry blockade [2+]i and a relaxant effect in isolated guinea pig trachea. Consequently, red clover has been reported to have an antispasmodic effect in previous literature -rolipram bound to HARBSs of brain cell membranes and shortened xylazine/ketamine-induced anesthesia at a dose level of 100 μmol/kg (s.c.), suggesting that higher doses of genistein may induce adverse gastrointestinal side effects [μmol/kg (s.c.), did not shorten this kind of anesthesia. This suggests that biochanin A, 1 h after injection (s.c.), is demethylated slightly to form genistein. In the present in vivo study, biochanin A  significantly enhanced the total serum IgG2a levels, which had probably switched from IgM due to an increase in IFN-γ level [γ levels may have been offset by genistein, which has recently been reported to inhibit IFN-γ levels [Following oral administration of biochanin A, genistein, a demethylated metabolite of biochanin A, may have been produced. Biochanin A and genistein have been reported to inhibit protein tyrosine kinase (PTK) of epidermal growth factor receptor with ICectively . In a guhibition . Moreovehibition . In clintipation . In a mu effects . However-γ level , 41. Theγ levels , when biH value of biochanin A was >300 μM, suggesting that it had a low affinity for HARBSs of brain cell membranes. Thus the PDE4H/PDE4L ratio of biochanin A was >35, which was considerably greater than that of AWD 12-281. In addition, biochanin A did not influence xylazine/ketamine-induced anesthesia. These results are consistent with the low affinity of biochanin A for HARBSs of brain cell membranes. However, Ro 20-1724, a selective PDE4 inhibitor, reversed the anesthetic effects. This reversal may have occurred through presynaptic α2-adrenoceptor inhibition [α2-adrenoceptor antagonist, has been reported to reverse xylazine/ketamine-induced anesthesia in rats [α2-adrenoceptor agonist, prevented emesis induced by PDE4 inhibitors in ferrets [In the present results, the PDE4hibition , because in rats  and trig in rats . In cont ferrets . In the In conclusion, the present results support the traditional use of red clover containing biochanin A for the treatment of allergic asthma and COPD, despite evidence of adverse gastrointestinal effects following long time use. The mechanisms of biochanin A are summarized in


Zingiber officinale Roscoe, known commonly as ginger, is consumed worldwide in cookeries as a spice and flavouring agent. It has been used as the spice and medicine for thousands of years. The present study was undertaken to investigate the potential protective effect of Zingiber officinale Rosc. in a model of oxidative damage to pancreatic β cells. The free radical scavenging activities and composition of the isolated n-hexane and ethanolic extracts were confronted with their protective, antioxidant and cytotoxic effects in INS-1E β cells. Unlike the n-hexane extract , both low cytotoxicity and remarkable protective effects on β cell viability, followed by lowering oxidative stress markers were found for the ethanolic extract Zingiber officinale Rosc. The present study is the first pilot study to assess the protective potential of Zingiber officinale Rosc. in a model of cytotoxic conditions imposed by diabetes in β cells.In traditional medicine, several medicinal plants or their extracts have been used to treat diabetes.  Oxidative stress plays a critical role in diabetes type 1 and 2. With regard to their reduced antioxidant reserves, pancreatic insulin producing β cells represent one of its primary targets.Zingiber officinale Roscoe , known universally as ginger, is commonly used as a spice and food as well as medicinal agent in Indian, Asian and Arabic traditional medicine in the form of a fresh paste, dried powder, candy  or slices preserved in syrup . As confirmed by GC-MS, the most abundant terpenes in ginger essential oil are zingiberene, ar-turmerone, curcumene, tumerone and curlone  showed also a good antioxidant activity with correspondence to its phenolic content  was investigated. Furthermore, their intrinsic antiradical properties with their cytotoxicities and protective effects in a model of oxidative damage to pancreatic INS-1E β cells were compared.In this study, the composition of two different The dried powdered roots of ginger were purchased from the local vegetable and fruit market of Vido, Co., Ltd., Bratislava, Slovak Republic (September 2009).DPPH  radical was purchased from Sigma Co.  and Folin-Ciocalteu′s phenol reagent from Merck KGaA, Darmstadt, Germany).et al., –1 penicillin, 100 µg.l–1 streptomycin, 2 mmol.l–1 L-glutamine, 1 mmol.l–1 Na-pyruvate, 55 µmol.l–1, 2-mercaptoethanol, 10 mmol.l–1 HEPES, pH 7.0–7.4 . For assays, the cells were detached by 5–10-min incubation with 0.05% trypsin/EDTA .INS-1E β cells  was separately extracted with five volumes of n-hexane and 60% aqueous ethanol (v:v), for 48 h, subsequently the solid phase was removed by filtration and combined extracts were concentrated under reduced pressure at 40 °C, to give 10.2 g and 13.2 g crude extract, respectively. The prepared extracts were stored at 4 °C until further analyzed.Air-dried and powdered ginger  gas chromatograph equipped with an Agilent Technologies 5975C inert XL mass selective spectrometer. The mass range was scanned from m/z 29–420 Daltons.-1. Injector temperature was 250 °C, injections 0.5 µl, and split 1:100. The carrier gas (helium) flow was maintained at 0.8 ml.min–1 by an electron control of pressure. Identification of the compounds was based on (i) comparison of substance mass spectra with the GC-MS system data bank (NIST 05 library), (ii) comparison of mass spectra with data in the literature  was mixed with the samples tested (0.1 ml) at different concentrations. After 30 min the absorbance was read at 517 nm (Thermo Electron Corporation Genesis 6 Spectrophotometer (UK)). The percentage of absorbance decrease, relative to non-reduced control DPPH solution was evaluated.Free radical-scavenging ability of both extracts tested was determined as described by Wong et al. . The capet al., –1 of dry extract. The samples were measured in triplicate.The total phenolic content was determined using Folin-Ciocalteu′s method . Trypan blue uptake in situ was determined by exposure of the cells to 0.1% trypan blue in phosphate buffered solution . The cells were washed with PBS and examined by light microscopy.Mitochondrial reduction of MTT  was determined spectrophotometrically at λet al., et al., –1 EB and 100 µg.ml–1 AO, 20 µl) was added to each dish at the end of incubation time and the cells were viewed under the fluorescence microscope. Each image (50–200× magnification) was collected with XDS-2 fluorescence microscope running standard software. Early and late apoptotic cells were detected by their bright green and orange nucleus with condensed or fragmented chromatin, respectively, whereas necrotic cells were detected by their intact red nuclei, and live cells had a normal green nucleus. For detection of caspase 3 activation, luminiscence assay using Caspase-Glo 3/7 Assay kit (Promega) was performed according to the manufacturer’s protocol. Fifty thousand cells were used for each measurement.The apoptotic changes were assessed by ethidium bromide and acridine orange (EB/AO) staining assay (Ribble 2DCF-DA) and dihydroethidin (DHE) (probes with differing specificity to oxidizing species) was followed in the β cells exposed to H2O2 stress  was added to the wells at the incubation time of 1 hour, followed by additional incubation under the same conditions for 20 minutes. Images were collected by fluorescence microscope and analyzed for reaction intensity.Simultaneous oxidation of incorporated 2′,7′-dichlorodihydrofluorescein diacetate . Statistical analysis was performed using unpaired Student's t-test using X-Plot v. 2.81 and statistical significance is expressed as *–1 of dry extract, respectively. The results are represented as miligrams of gallic acid equivalent (GAE) per one gram of dry extract.The determined total phenolic content of n-hexane and ethanolic extract amounted to 50.74 and 28.75 mg.g–1 of both n-hexane and ethanolic extract tested, causing reduction of DPPH by 64.9±0.6% and 36.4±1.3%, respectively , other major compounds were alpha-zingiberene (15.25%), beta-sesquiphellandrene (7.163%), tau-muurolol (5.00%) and 4-gingerol (4.64%).Sample constituents of ethanol extract are given in 2O2 (0.5 mmol.l–1) during 1 hour caused a concentration-dependent decrease of MTT reduction by INS-1E β cells  promoted intracellular oxidation preferably of DHE (with a minor portion of H2DCF oxidation)  .–1 injury of β cells  (–1 (72.9±7.5% of control).Only ethanolic extract ability) . This waability) . The losability) . The max2DCF and DHE oxidation in the stressed cells, appearing as a lack of both ethidium-positive nuclei and dichlorofluorescein fluorescence  in n-hexane extract could be underestimated  has been shown to induce apoptosis in pancreatic carcinoma cells  and ·OH radicals can, as can peroxynitrite  suggesting that superoxide radicals are the major oxidizing species in the present model. The H2DCF can effectively determine the intracellular oxidants only if they are decomposed to radicals, e.g. by means of transition metals. The presence of extracellular iron was shown as necessary for the generation of a signal from H2DCF upon addition of H2O2 to the cells  at its maximum non-toxic concentration (100 µmol.l–1) in INS-1E cells exposed to H2O2 (Račková et al., et al., Only ethanolic extract 2DCF-oxidation in the stressed cells. Accordingly, Z. officinale extracts were shown to exert both superoxide and hydrogen peroxide scavenging activity (Khanom et al., et al., The protective effects of ethanolic extract were accompanied by a significant avoidance of both superoxide-dependent DHE- and superoxide-independent HZingiber officinale Rosc. is a promising substance for the in depth study of its protective effects against cytotoxic conditions imposed by diabetes in pancreatic β cells. A higher content of phenolic constituents and increased intrinsic antiradical capability of diverse ginger extract preparations may not be obviously related to their improved biological efficacy under conditions of oxidative stress, but, paradoxically, accounts for an enhanced cytotoxicity. In view of INS-1E cells as an insulinoma cell line, however, the remarkable proapoptotic effect of Z. officinale constituents may indicate their prospective antitumor efficacies.In conclusion, the present study suggests that ethanolic extract isolated from


Pasteurella multocida B:2 and E:2. Field outbreaks of HS are known to result in localisation of bacteria in the tonsils of surviving buffalo, confirming that animals can become carriers and the role of respiratory tract in the transmission of the disease. This report describes additional sites of localisation of P. multocida B:2 in surviving buffalo following experimental induction of HS.Haemorrhagic septicaemia (HS) is an acute septicaemic disease of buffalo and cattle caused by P. multocida B:2 infection, all calves in group 1 and one calf in group 2 that was allowed to commingle with infected calves from group 1 were euthanised within 48 h. Pasteurella multocida B:2 was detected from the nasal and rectal swab samples on days 5 and 6 from the remaining calves in group 2. The first injection of dexamethasone into the carrier animals resulted in reemergence in samples from the nose, rectum and vagina. However, subsequent dexamethasone injections failed to re-activate P. multocida B:2. When surviving carrier calves in group 2 were euthanised at the end of the experiment, P. multocida B:2 was detected in the lungs and various organs of the respiratory, gastrointestinal and urinary tracts.Following P. multocida B:2 resulted in carriers among surviving buffalo. Pasteurella was found in various organs of the respiratory, gastrointestinal and urinary tracts, suggesting their role in the pathogenesis of HS.Commingling naive buffalo calves with calves acutely infected with  Pasteurella multocida: B:2 (Asian serotype) and E:2 (African serotype). HS has a wide distribution, especially in South East Asia and Africa [P. multocida usually results in rapid disease progression, with typical clinical signs of severe depression, pyrexia, submandibular oedema, dyspnoea, recumbency and death. Transmission is typically by carrier animals [P. multocida B:2 in the tonsil, nasopharynx and upper respiratory tract lymph nodes [Haemorrhagic septicaemia (HS) is an acute, fatal, septicaemic disease of cattle and buffalo caused by two specific serotypes of d Africa . On the d Africa . In rumi animals , which a animals . Previou animals -8 followph nodes ,9,10. Boph nodes ,11.One recent study has indicated gastrointestinal tract involvement in acute HS . HoweverAll calves of group 1 were euthanised following development of advanced clinical signs; two were euthanised at 24 h post-infection, while the rest were euthanised at 48 h post-infection. Affected calves appeared dull and depressed, recumbent, pyrexic, anorexic and dyspnoeic, with nasal discharge and diarrhoea.On days 5, 6 and 7 after being mixed with the calves from group 1, three calves  of group 2 were transiently pyretic, with average body temperatures of 40.4°C (SEM = 0.15), 40.6°C (SEM = 0.32) and 40.3°C (SEM = 0.50), respectively  from group 2 that were euthanised following the development of severe clinical signs had classic lesions of acute HS, including severe congestion and haemorrhage of mucosal surfaces, subcutaneous tissues and respiratory, gastrointestinal and urinary tracts. Fibrin was deposited on the serosal surfaces of all thoracic and abdominal organs.The surviving calves of group 2 had multifocal pulmonary congestion and pneumonia. Fibrin was deposited on the omentum, mesentery, peritoneum and serosal surfaces of the gastrointestinal tract. However, the mucosal surfaces of the gastrointestinal tract appeared normal apart from the rectum, which had mild multifocal haemorrhages.Pasteurella multocida B:2 was successfully isolated from organ samples of all calves that were euthanised because of acute HS.Pasteurella multocida B:2 was cultured from nasal and rectal swabs collected on days 5 and 6 from the surviving buffalo calves  of group 2 on post-exposure days 5–7 and 15–17 (5–7 days after the administration of the first course of dexamethasone). Pasteurella multocida B:2 was also isolated from vaginal swabs of these calves on post-exposure days 15–17. Isolation was unsuccessful between days 7 and 14 and following the second and third courses of dexamethasone  in the tonsils, lung, reticulum, ileum and ureter of calves 2A, 2B and 2C  for 18 h. Bacteria were quantified via serial dilution, and an infective inoculum containing 1.0 × 105 colony forming units (cfu)/mL was prepared [Stock cultures of prepared .All experiments were approved by the Animal Care and Use Committee of Universiti Putra Malaysia .5 cfu/mL of P. multocida B:2 [Calves were divided into three equal groups. Group 1 served as the infected group, group 2  as the commingling group and group 3 as the negative control group. All calves of group 1 were challenged subcutaneously at the shoulder with 5 mL of inoculum containing 1.0 × 10cida B:2 . Calves cida B:2 . Calves Deep nasal, rectal and vaginal swabs were collected daily from all animals, throughout the 43-day study period using sterile cotton swabs. Deep nasal swabs were obtained by cleaning the external nares with 70% alcohol, inserting the swabs 15 cm into the nostrils, and swabbing the nasal and nasopharyngeal mucosa by rotating the swabs several times. The rectal and vaginal swabs were obtained by cleaning the rectal and vaginal regions with 70% alcohol, inserting the swabs 7 cm into the rectum or vagina, and swabbing the vaginal and rectal mucosa by rotating the swabs several times. All swab samples were immediately transported to the laboratory in Cary Blaire medium.Once a week had passed with no positive cultures, calves were injected intramuscularly with dexamethasone  for 3 consecutive days at the rate of 1 mg/kg to induce immunosuppression . After tAfter the final dexamethasone injection, all surviving calves in groups 2 and 3 were euthanised. Samples of nasal passage, tonsil, epiglottis, trachea, all eight lung lobes, liver, rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, mesenteric lymph nodes, caecum, colon, rectum, kidney, ureter and urinary bladder were collected from all euthanised animals in 10% neutral buffered formalin.P. multocida B:2. Tissue samples from the surviving buffalo of group 2 and negative control of group 3 were subjected to culture and identification of P. multocida B:2, histopathologic and immunohistochemical examination.Tissue samples from group 1 were subjected to culture and identification of P. multocida B:2 was based on morphological criteria [All nasal, rectal and vaginal swabs were cultured on blood agar plates incubated at 37°C for 18 h. Identification of criteria  and PCR criteria . Tissue criteria .P. multocida B:2 at 37°C for 1 h, and then incubated with 1:1000 diluted peroxidase conjugated antiserum ; Dako) at 37°C for 30 min. Finally, sections were incubated with 3,3′-diaminbenzidine (DAB) (Dako Liquid DAB + SubstrateChromogen System) at room temperature for 2 min, followed by washing in distilled water and counterstaining with methylene blue. Finally, all sections were examined under a light microscope.Tissue samples were fixed in 10% buffered formalin for at least 24 h before being trimmed, embedded in paraffin and sectioned at 3 μm. Sections were then subjected to immunoperoxidase and methylene blue stainings. Sections were dewaxed by incubation at 60°C for 15 min and immersion into xylene. Rehydration was via successive immersion in absolute alcohol, 90% alcohol, 70% alcohol and 50% alcohol. Sections were then exposed to citrate buffer using a carousel microwave at 50 W for 15 min and then Dako REAL™ Peroxidase Blocking Solution at room temperature for 30 min. Samples were incubated with primary rabbit hyperimmuneserum (1:200) against P. multocida B:2.Adjacent sections were dewaxed and stained with methylene blue for 30 s before being examined under light microscope to detect bipolar t test was used to compare rectal temperatures between the carriers and control calves.The Student’s HS: Haemorrhagic septicaemia; CFU: Colony-forming units; rpm: Revolutions per minute; DNA: Deoxyribonucleic acid; PCR: Polymerase chain reaction; IgG: Immunoglobulin G; DAB: 3,3′-diaminbenzidine.The authors declare that they have no competing interests.AS was involved in experimental design, sampling, sample processing, data analysis and drafting of manuscript. MZS was involved in supervision, research conception and design, data analysis, interpretation and manuscript revision. FFAJ was involved in experimental design, data interpretation and manuscript revision. ZZ was involved in supervision of the experiments and manuscript revision. All authors read and approved the final manuscript.


To assess the prevalence of pseudoexfoliation (PEX) and its associations in a population-based setting.Population-based, cross-sectional study.The Central India Eye and Medical Study included 4711 individuals. All study participants underwent a detailed ophthalmological examination. After medical pupil dilation, PEX was assessed by an experienced ophthalmologist using slit-lamp based biomicroscopy.P<0.001) from 0% in the age group of 30–39 years, to 2.85±0.56% in the age group of 60–69 years, to 6.60±1.21% in the age group of 70–79 years, and to 12.3±4.11% in the age group of 80+ years. In multivariate analysis, PEX prevalence was associated with higher age : 1.11 ), lower body mass index ) and higher diastolic blood pressure ). In the multivariate analysis, PEX was not associated with retinal nerve fiber layer cross section area (P = 0.76) and presence of open-angle glaucoma (P = 0.15).Slit lamp examination results were available for 4646 (98.6%) study participants with a mean age of 49.3±13.3 years (range: 30–100 years). PEX was detected in 87 eyes  of 69 subjects . PEX prevalence increased significantly  was significantly associated with older age, lower body mass index and higher diastolic blood pressure. It was not significantly associated with optic nerve head measurements, refractive error, any ocular biometric parameter, nuclear cataract, early age-related macular degeneration and retinal vein occlusion, diabetes mellitus, smoking, and dyslipidemia. Pseudoexfoliation (PEX) is a disorder which is characterized by the appearance of a fibrillary whitish material on the lens surface, the lens zonules, ciliary body and other parts of the anterior chamber The Medical Ethics Committee of the Medical Faculty Mannheim of the Ruprecht-Karls-University Heidelberg and a similar committee of the Suraj Eye Institute/Nagpur approved the study; all participants gave informed written consent, according to the Declaration of Helsinki.P<0.001) higher in the group of non-participants.The Central India Eye and Medical Study (CIEMS) is a population-based cross-sectional study in Central India. As described in detail recently, the study was performed in 8 villages in Kalmeshwar Tehsil, a rural region of Eastern Maharashtra at a distance of about 40 km from Nagpur in the geographical center of India All examinations were carried out at the hospital. Trained social workers filled out a questionnaire for the participants; this questionnaire included questions regarding socioeconomic background and living conditions, tobacco use and alcohol consumption, and any known diagnosis of major systemic diseases. Pulse, arterial blood pressure, and body height and weight were recorded. One-and-a-half hours after a standardized lunch, blood and urine samples were obtained and biochemically analyzed. Uncorrected visual acuity and visual acuity with the subjectś glasses and after refractive correction were measured using modified Early Treatment of Diabetic Retinopathy Study (ETDRS) charts . Automated refractometry and subjective refraction were performed on all subjects independent of visual acuity. Visual field examinations were performed with frequency-doubling perimetry using the screening program C-20-1 . Intraocular pressure was measured by a slit lamp mounted Goldmann applanation tonometer; if the measurements were higher than 21 mmHg, tonometry was repeated. Slit lamp biomicroscopy was carried out by a fellowship-trained ophthalmologist, and any abnormality of the anterior segment was noted. Corneal pachymetry was performed by sonography using the Pacscan . Ocular biometry was performed by partial coherence interferometry . Anterior chamber depth, lens thickness and axial length were measured for both eyes of all subjects. Using the slit lamp, photographs of the limbal region were taken to assess the limbal anterior chamber depth at the most peripheral part of the cornea, as described by van Herick Gonioscopy was performed for all study participants in dim illumination using the magnaview single mirror gonio lens . The slit beam was brought to its narrowest, and least height on a Haag Streit type slit lamp, to reduce the effect of light on the anatomy of the anterior chamber angle. The chamber angle was estimated as open, if in primary position the posterior pigmented part of the trabecular meshwork was visible without indentation Digital monoscopic photographs of the optic disc  and macula (50 degrees) were taken. Magnification by optic media was corrected for by a built-in algorithm and we measured the area and horizontal diameter and vertical diameter of the optic disc and cup. Glaucoma was defined in two ways. First it was defined according to the criteria of the International Society of Geographical and Epidemiological Ophthalmology (ISGEO) classification scheme P-values were higher than 0.05, starting with the parameters with the highest P-values, until all remaining independent parameters were significantly associated with the presence of PEX. All P-values were two-sided. Odds ratios (OR) and 95% confidence intervals (CI) were presented.Only those subjects with assessment of the presence of pseudoexfoliation were included into the study. Statistical analysis was performed using a commercially available statistical software package . In a first step, we determined the prevalence of pseudoexfoliation (presented as mean ± standard error). In a second step, we performed univariate analyses of the associations between the presence of pseudoexfoliation and other ocular and systemic parameters. In a the third step, we carried out binary regression analyses with the presence of pseudoexfoliation as the dependent parameter and all parameters as independent variables which were associated significantly with the presence of pseudoexfoliation in the univariate analyses. We then removed step by step all independent parameters for which the P<0.001), more myopic (P<0.001), and had longer axial length (P = 0.02). Both groups did not differ significantly in gender (P = 0.78) and intraocular pressure (P = 0.06).Out of the 4711 subjects included in the study, results of the slit lamp examination for PEX were available for 9175 (97.4%) eyes of 4646 (98.6%) persons. The other 65 participants were excluded for assessment for PEX due to refusal for pupillary dilation or due to corneal opacities which rendered a slit examination of the lens surface impossible. The mean age of the 4646 participants (2484 (53.5%) women) was 49.3±13.3 years , and mean refractive error was −0.15±1.78 diopters , and mean axial length was 22.66±0.89 mm . The group of subjects with PEX assessment as compared with the group of subjects excluded for the PEX evaluation were significantly younger (P<0.001) with age (P<0.001) with older age. Taking only the population with an age of 40+ years, 50+ years, and 60+ years, the mean prevalence of PEX per eye and per subject was 1.25±0.13%  and 1.96±0.23% , 1.97±0.21%  and 3.01±0.37% , and 3.00±0.33%  and 4.47±0.56% , respectively.PEX was detected in 87 eyes  in 69 subjects . PEX was found bilateral in 18/69 (26%) participants. There was no significant difference in the frequency of PEX in the right eye versus the left eye in the subjects with unilaterally detected PEX. The prevalence of PEX increased significantly  , lower height , lower body weight , higher diastolic blood pressure , higher systolic blood pressure , higher ocular perfusion pressure , lower level of education , and lower blood hemoglobin concentration ; and with the ocular parameters of more myopic refractive error , higher degree of nuclear cataract , higher intraocular pressure , lower mean retinal nerve fiber layer thickness , and presence of open-angle glaucoma .In univariate analysis, the overall prevalence of pseudoexfoliation was significantly associated with the systemic parameter of higher age ( = 0.17) , lower bP = 0.23), smoking (P = 0.29), smoking package years (P = 0.73), blood concentration of high-density lipoproteins (P = 0.74), creatinine (P = 0.37), cholesterol (P = 0.20), postprandial blood concentration of glucose (P = 0.67), glycosylated hemoglobin HbA1c (P = 0.10), and presence of diabetes mellitus (P = 0.27); and with the ocular parameters of axial length (P = 0.56), central corneal thickness (P = 0.22), anterior corneal curvature radius (P = 0.36), anterior chamber depth (P = 0.12), lens thickness (P = 0.20), presence of angle-closure glaucoma (P = 0.72), optic disc area (P = 0.62), vertical cup/disc ratio (P = 0.20), horizontal cup/disc ratio (P = 0.05), neuroretinal rim area (P = 0.31), neuroretinal rim volume (P = 0.27), presence of diabetic retinopathy (P = 0.75), presence of early age-related macular degeneration (P = 0.84), presence of retinitis pigmentosa (P = 0.81), and presence of retinal vein occlusion (P = 0.61). Side differences in the presence of PEX were not significantly associated in side differences in intraocular pressure (P = 0.40).Presence of PEX was not significantly associated with the systemic parameters of gender ), lower body mass index ), and higher diastolic blood pressure ), while it was no longer significantly associated with systolic blood pressure (P = 0.97), body height (P = 0.81), intraocular pressure (P = 0.80), retinal nerve fiber layer cross section area (P = 0.76), blood concentration of hemoglobin (P = 0.52), level of education (P = 0.98), degree of nuclear cataract (P = 0.39), refractive error (P = 0.32), and presence of open-angle glaucoma (as defined by ISGEO criteria) (P = 0.38) or presence of open-angle glaucoma  (P = 0.15). If visual acuity was added to the analysis, after adjusting for age, diastolic blood pressure and body mass index, PEX was associated with lower best corrected visual acuity (logMAR) .In a multivariate analysis, which included presence of PEX as dependent variable and all those parameters as independent variables, which were significantly associated with PEX in the univariate analysis, presence of PEX remained to be significantly associated only with higher age  than in the remaining eyes without glaucoma  versus 0.6±0.1% ). In eyes with glaucoma  (n = 176 eyes), PEX was significantly more common than in the remaining eyes without glaucoma 4.0±1.5%  versus 0.6±0.1% ; P<0.001; OR: 6.73 . Differentiating within the glaucoma group between open-angle glaucoma and angle-closure glaucoma revealed significant differences in the prevalence of PEX for eyes with open-angle glaucoma versus the remaining eyes , while the difference in the PEX prevalence between eyes with angle closure glaucoma versus the remaining eyes was statistically not significant (P = 1.00). After adjustment for age, the correlation between open-angle glaucoma and PEX was no longer statistically significant P = 0.10).In eyes with glaucoma (as defined by ISGEO criteria) (n = 129 eyes), PEX was more common, however not significantly more common ), lower body mass index ) and higher diastolic blood pressure ), but not with open-angle glaucoma (P = 0.20).If PEX was defined only as the severe stages 4 to 6 , the presence of severe PEX was significantly associated with older age  and 1.96±0.23% , respectively. In multivariate analysis, PEX prevalence was associated with higher age, lower body mass index and higher diastolic blood pressure, while it was not significantly associated with intraocular pressure, retinal nerve fiber layer cross section area, any optic nerve head measurements and presence of open-angle glaucoma or angle-closure glaucoma, refractive error and any ocular biometric parameter, nuclear cataract, early age-related macular degeneration and retinal vein occlusion, diabetes mellitus, smoking, and dyslipidemia. Side differences in the presence of PEX were not significantly associated in side differences in intraocular pressure.The PEX prevalence found in our study population was lower than the PEX prevalence reported from studies from South India  The prevalence of PEX increased significantly with age , in accoP<0.001; OR: 6.73 . This was valid for open-angle glaucoma (P<0.001) but not for angle-closure glaucoma (P = 1.00). In multivariate analysis with adjustment for age, the association between PEX and glaucomatous optic nerve damage was no longer statistically significant. Correspondingly, in multivariate analysis, prevalence of PEX was not significantly associated with parameters of glaucomatous optic neuropathy such as small neuroretinal rim area and high vertical cup/disc diameter ratios. These findings partially agree with, and partially contradict, the results of other studies. In the South Indian Andhra Pradesh Eye Disease Study, the prevalence of PEX in subjects with glaucoma was 4.2% (95% CI: 0.17–8.23) what was not markedly different from the prevalence of PEX in the general study population with a PEX prevalence of 3.01% in subjects aged 40+ years and 6.28% in subjects with an age of 60+ years In univariate analysis, eyes with ophthalmoscopic glaucoma as compared to non-glaucomatous eyes showed significantly more often PEX  versus 0.6±0.1% ; P = 0.004) associated with lower best corrected visual acuity in our study after adjusting for age, diastolic blood pressure and body mass index. It is in agreement with other studies from India such as the Andhra Pradesh Eye Disease Study and the Aravind Comprehensive Eye Survey, in which PEX was significantly associated with blindness after adjustment for age PEX was significantly  was significantly associated with older age, lower body mass index and higher diastolic blood pressure, but not with intraocular pressure, optic nerve head measurements and glaucoma, refractive error and any ocular biometric parameter, nuclear cataract, early age-related macular degeneration and retinal vein occlusion, diabetes mellitus, smoking, and dyslipidemia,.