A detailed knowledge about spatial and temporal gene expression is important for understanding both the function of genes and their evolution. For the vast majority of species, transcriptomes are still largely uncharacterized and even in those where substantial information is available it is often in the form of partially sequenced transcriptomes. With the development of next generation sequencing, a single experiment can now simultaneously identify the transcribed part of a species genome and estimate levels of gene expression.Picea abies) was sequenced using next generation sequencing technology. In total, close to 70 million fragments with a length of 76 bp were sequenced resulting in 5 Gbp of raw data. A de novo assembly of these reads, together with publicly available expressed sequence tag (EST) data from Norway spruce, was used to create a reference transcriptome. Of the 38,419 PUTs (putative unique transcripts) longer than 150 bp in this reference assembly, 83.5% show similarity to ESTs from other spruce species and of the remaining PUTs, 3,704 show similarity to protein sequences from other plant species, leaving 4,167 PUTs with limited similarity to currently available plant proteins. By predicting coding frames and comparing not only the Norway spruce PUTs, but also PUTs from the close relatives Picea glauca and Picea sitchensis to both Pinus taeda and Taxus mairei, we obtained estimates of synonymous and non-synonymous divergence among conifer species. In addition, we detected close to 15,000 SNPs of high quality and estimated gene expression differences between samples collected under dark and light conditions.mRNA from actively growing needles of Norway spruce (−09and 1.1 × 10−09) is an order of magnitude smaller than values reported for angiosperm herbs. However, if one takes generation time into account, most of this difference disappears. The estimates of the dN/dS ratio (non-synonymous over synonymous divergence) reported here are in general much lower than 1 and only a few genes showed a ratio larger than 1.Our study yielded a large number of single nucleotide polymorphisms as well as estimates of gene expression on transcriptome scale. In agreement with a recent study we find that the synonymous substitution rate per year (0.6 × 10 A detailed characterization of the transcriptome is not only a prerequisite for genome annotation, but is also crucial to relate gene expression and function to phenotypic variation, to estimate evolutionary rates or to identify genes under selection -5. WhileMus musculus), for example, despite a fully sequenced genome and millions of EST sequences in public databases, MPS of expressed genes from three different tissues revealed several thousand new features of known genes and close to 600 novel gene transcripts [Drosophila melanogaster[Until recently microarrays, based on either cDNA, or in the case of model organisms, oligonucleotides, were the main tool to assess global patterns of gene expression. These microarray studies provided a first characterization of temporal and spatial gene expression patterns in many different organisms, but they also had obvious limitations since they were dependent on already available sequence information from the organism of interest. Furthermore, at least with cDNA arrays, most transcripts were only represented by the most common splice variant making the gene expression estimates unspecific . The emeanogaster. In plananogaster,9. In adanogaster-20).Picea) and pine (Pinus) genus. By April 2012 there were 313,110 and 186,637 EST sequences deposited at NCBI from Picea glauca and P. sitchensis, respectively. Efforts in the European Picea abies (Norway spruce) have been less intensive and only some 14,000 ESTs had been deposited at NCBI by April 2012. Based on sequence similarity, the data obtained in the North American species have been collapsed into 22,472 putative unique transcripts  in P. glauca and 19,828 unigenes in P. sitchensis (see Unigene assembly on NCBI). In addition, more focused experimental and bioinformatic efforts have been made to obtain full-length cDNAs (FL-cDNA) in both P. sitchensis and P. glauca and have so far lead to the identification of 27,720  unique transcribed genes in P. glauca and around 8,000 validated FL-cDNA have been characterized in P. sitchensis[Most efforts have so far focused on angiosperms, less attention being paid to gymnosperms in spite of the availability of several very large EST libraries in the spruce  and during night (in the dark) can we detect differentially expressed transcripts and if so does the inferred pattern differ compared to angiosperms? Fourth, since recent studies of divergence between conifer species have suggested that there might be major differences in evolutionary rates between conifers and angiosperms [P. glauca, P. sitchensis, Pinus taeda and Taxus mairei to evaluate this statement with a larger and more diverse data set. By comparing not only our, but also two other spruce transcriptome sequences created by traditional sequencing methods, to both Pinus taeda and Taxus mairei we could minimize biases that might exist in any single assembly. Furthermore, since inferring pattern of divergence between species can be biased when closely related species (e.g. incomplete lineage sorting) or highly divergent species (saturation on synonymous sites) are used, the use of species that diverged at two different time points should reduce these problems.In this study we present results from deep sequencing of the iosperms  we used P. abies tree growing outside Uppsala, Sweden  was used to produce 6 different adapter-ligated cDNA libraries. In total, close to 70 million 76 bp long fragments were sequenced from both ends and close to 50% of the reads originated from the samples collected in the dark (Table de novo assembly (∼15 million where paired end sequences were retained and ∼18 million when only one of the paired reads was retained). For mapping purposes we created a second data set where reads containing adaptor sequence were trimmed rather than removed and retained if they were longer than 30 bp after trimming. This created a data set of 55,417,522 fragments  of length between 30 and 76 bp . In all downstream analyses we excluded PUTs shorter than 151 bp, leaving 38,419 PUTs with a mean length of 471.5 bp. The total assembly length of PUTs longer than 150 bp was just over 18 Mbp and the mean sequence depth of these PUTs as measured by RPKM (reads per kilobase of transcripts per million of mapped reads) was 281  to plant proteins when translated to protein sequences. Of the 38,419 PUTs 32,082 show a significant hit against either P. glauca or P. sitchensis and 23,450 of these were similar over more than 90% of the sequence length of the P. abies PUT. Of the 882 transcripts longer than 1,500 bp only 57 PUTs did not show any similarity to Arabidopsis or other plant proteins. Taken together this left only 4,167 P. abies PUTs with no or limited similarity to any plant protein sequence or any EST sequence from other spruce species. For complete annotation of PUTs longer than 150 bp against A. thaliana, P. glauca, P. sitchensis, plant proteins, PFAM databases and repeat databases see Additional file 55% of the assembled P. sitchensis[P. glauca[P. sitchensis and P. glauca  of at least 10 amino acids , read direction for alternate alleles (DP4) and coverage 9,394 SNPs were found. Less stringent criteria obviously led to a higher number of identified SNPs and if we only filter on quality 20,781 potential SNPs were identified. From published P. abies population genetic data set the observed transition to transversion ratio is around 1.3, which is very similar to the ratio observed with the criteria that includes both quality and observed minor allele frequency above 0.25 , a yew (Taxus mairei) and a pine (Pinus taeda) to estimate phylogenetic trees and study the rate of molecular evolution in conifers. In all five species we identified putative coding frames and aligned orthologous sequences from all species. This resulted in a data set of 5,246 aligned ORFs between the three Picea species. This data set was reduced to 1,404 when adding pine and further reduced to 244 when adding yew. Phylogenetic inference on this latter data set confirms the very close relationship between all three spruce species and we cannot with any confidence resolve their phylogeny . Mean synonymous divergences between the three spruce species and pine and yew were 0.175  and 0.598 , respectively. Assuming a divergence time between pine and spruce of 160 to 120 million years [−09 to 0.73 × 10−09 and 1 × 10−09 to 1.24 × 10−09, respectively. The ratio of non-synonymous to synonymous divergence between the spruce species and pine and yew show very few genes with ratios larger than one, irrespective if the analysis were performed on only full-length ORFs or on all identified ORFs . 2,076 transcripts were detected as significantly differentially expressed between the two sampling points using BaySeq and a cut-off posterior probability at 0.5, but among these transcripts only few showed a large fold change Figure . We did P. glauca, which has one of the best characterized transcriptome among conifer species, has a transcriptome assembly length of 30.15 Mbp, i.e. 12 Mbp longer than the the P. abies assembly presented here. In addition, the length of individual PUTs is on average shorter in the P. abies assembly than in both P. glauca and P. taeda . This indicates that the P. abies transcriptome assembly only comprises a fraction of the total transcriptome and that a large proportion of the assembled PUTs corresponds to partial transcripts. There are a number of possible reasons for this difference in assembly length despite of the large amount of short reads collected. First, the assembly of short reads from transcriptome sequence is still far from optimal [In spite of the remaining challenges associated to the assembly of short reads to full-length transcripts  RNA-seq  optimal , somethi optimal ,22,43,44P. glauca transcriptome suggest that as much as 11.6 Mbp of the transcriptome is still missing [P. abies, but this seems less likely as both this data and previous studies have shown that spruce species are very closely related and even share many polymorphic sites [P. abies transcriptome contains a large fraction of partial transcripts and is far from complete, the assembly presented here is of a high enough quality and size to be informative on the general properties of the transcriptome.In total over 34,000 transcripts showed significant hits when compared to other spruce species, either at the protein level to plant proteins or at the nucleotide level, leaving only 4,167 PUTs without any annotation. This set of PUTs with low or no similarity to other species could represent erroneously assembled PUTs, but since estimates from the  missing , they coic sites ,39. Stilic sites ), which P. abies with properties that suggest that they are full-length transcripts. A direct comparison of these PUTs towards full-length transcripts from P. glauca indicate that neither ours nor the P. glauca PUT assembly contains a comprehensive set of full-length transcripts and that despite millions of ESTs from spruce species there is still room for efforts aiming at capturing and sequencing full-length transcripts, such as oligo-capping, something that is of special importance in relation to upcoming genome annotations [In fact, ORF predictions identified 6,194 PUTs from otations .P. abies, where the data have been collected with Sanger sequencing [In many of the PUTs with high enough sequence coverage we could extract potentially variable sites, but since sequencing technologies are evolving at a rapid pace, the understanding of the error profiles for a given technique is still sparse making this process nontrivial and prone to false positives. This process is, in the present case, further complicated by the fact that the majority of tools developed to extract SNPs assumes that genome data is available and that sequence depth is even for the underlying genotypes, meaning that one expects around half the reads from each allele at heterozygous sites. In addition, many tools make use of uneven coverage as a tool to detect copy number variants or duplicated regions making standard methods to discover SNPs difficult to use as the uneven coverage and allele specific gene expression will cause deviations from these expectations. By merging population genetics sequence data sets from both unpublished and published projects in quencing ,47 we coquencing -50.P. sitchensis and P. taeda[−09 and 1.1 × 10−09, values that are also in line with previous estimates of 0.7 − 1.31 × 10−09[The observed mean synonymous divergence of 0.175 between spruce and pine obtained here, is similar to the 0.22 reported as a mean over a phylogenetic tree of four conifer species  and the  P. taeda. In the 1 × 10−09. As prev1 × 10−09,34,51. S1 × 10−09,53 one o1 × 10−09,54 and wP. glauca and the branch leading to P. menziesii, respectively), but lower than the pairwise estimate between P. taeda and P. sitchensis ) [et al.[et al.[Arabidopsis thaliana and Populus trichocarpa and obtained a value of 0.0924. They therefore concluded that dN/dS was higher in conifers than in angiosperms. While our results for the conifer data are within the same order of magnitude, it is worth noting that the estimate they obtained between A. thaliana and P. trichocarpa tends to be much lower than previous estimates from angiosperms. For example, in estimates comparing A. thaliana and A. lyrata the mean over more than 5000 genes was 0.203 and within the poplar lineage P. trichocarpa and P. tremula the mean over almost 600 genes was 0.175 [Arabidopsis and Populus lineages and since synonymous divergence seem to have a strong impact in estimates of dN/dS [The ratio of non-synonymous to synonymous divergence at full length ORFs between spruce species and either pine or yew reported here, 0.236  and 0.167 , respectively, are higher than estimates from previous analyses in conifer species [ 0.329)) . The dev) [et al. 100 compl.[et al. also estas 0.175 ,55. It iof dN/dS  comparinEven when the correct orthologous sequences have been aligned, individual estimates of dN/dS values should not be directly used to infer positive selection as recent work have highlighted several problems in using dN/dS larger than one as indicative of positive selection -58. For In model organisms large scale studies of gene expression have identified numerous properties of genes and proteins that significantly correlate with selective constraints. Here we investigated whether level of gene expression in needles correlated with selective constraints, but found no significant pattern. In animals, fungi and plants a correlation between expression and selective constraint, such that highly expressed genes tend to have lower dN/dS, has been reported ,59,60. IP. abies assembly to other gymnosperm species our analysis suggests that evolutionary constraints might not, as some previous work have suggested, be very dissimilar between gymnosperm and angiosperm species. More importantly, our analysis does, together with the analysis of Buschiazzo et al.[Our study highlights the versatility of next generation sequencing technology in generation of full-length expressed genes, identification of polymorphic sites and estimation of gene expression levels. The available data from Norway spruce have with this study been leveraged with available data from North American spruce species. This allowed the comparison of sequence evolution in this group of plants for thousands of full-length genes. By comparing our zo et al., highligzo et al.) it willzo et al.. Furtherzo et al.,61 will μg of total RNA was obtained of which close to 1% were retained as mRNA after two rounds of oligo-dT selection using the MicroPoly(A)PuristTM(AppliedBiosystems).Needles from a single adult individual of Norway spruce growing naturally outside Uppsala, Sweden were collected at two different time points, at 1 pm during day light conditions and at 1 am the following night in the dark (20090527). The collected needles were actively growing and at stage 5 to 6 according to Krutzsch scale . Extractμl of purified ligation reaction in a 50 μl PCR for 18 cycles. A final purification of the fragment libraries was done by separating the samples on agarose and performing a gel extraction with GE health cares Illustra kit. Before sequencing, the fragment sizes and concentrations were evaluated using Bioanalyzer 2100 and equimolar quantities of the library were used for sequencing on Illumina cluster station. A detailed description of the library preparation method can be found in [A high concentration of random hexamer in relation to mRNA was used to synthesize cDNA with a short average fragment size using Invitrogen Supercript III First-Strand Synthesis kit. Following second strand synthesis, end-repairing of fragments, adaptors for short read pair-end sequencing supplied by Illumina corporation were attached to newly synthesized cDNA. The obtained cDNA library had an average size of 150-300 bp  and were used in ligation of adaptors for cluster sequencing. Enrichment of fragments with two attached adaptors was done using 2 found in .E. coli. In total, less than 5% of the reads mapped against these sequence libraries. Following this we created two sets of filtered sequence libraries, one for de novo assembly and one for mapping purposes. For both libraries we retained only high quality reads . For the de novo libraries we removed all reads showing traces of adaptor sequences, whereas for the mapping libraries we trimmed reads showing similarity to adaptor sequences and retained the read if it was longer than 30 bp. For the de novo library we also filtered artifact reads and removed any read with homopolymeric regions longer than 20 bp. We further removed the first 10 and last 11 bp of the reads, since the start of reads showed a different nucleotide composition compared to the rest of the sequence and the end of reads showed a decrease in quality (data not shown). This led to a de novo assembly with 33,185,901 reads of 55 bp length (∼15 million sequenced in both ends and ∼18 million from only one end), whereas mapping was done with 55,417,522 fragments  of length between 30 and 76 bp.The raw sequence data were obtained using the Illumina python pipeline v. 1.3. The obtained sequences were then compared to a number of different potential sources of contamination, including the human transcriptome, rRNA data sets, chloroplast and mitochondrial genomes of spruce, and the genome of de novo assembly of the obtained short read data was performed using a combination of two different assemblers followed by a step where putative unique contigs (PUTs) were created. We used two different programs and approaches for the de novo assembly since different assemblers often outcompete each other at different tasks [Two different types of analyses of the read data were performed. First, nt tasks . The paint tasks  using a nt tasks . In totant tasks  if the int tasks  aligningP. abies PUTs.In order to identify differentially expressed genes we used the BaySeq package , which iP. abies created with BWA. Since there is very limited knowledge about allele specific expression patterns we chose to only present SNPs with very high quality and having a estimated minor allele frequency in our short reads of at least 0.25.Finally, single nucleotide polymorphisms were detected using the mpileup function in SAMtools  on the sAll raw sequences is available at the NCBI SRA at the accession number SRA053572. A fasta file with with all PUTs as well as SNP information and PAML tables are available at DRYAD under DOI 10.5061/dryad.ds2gp.http://plantgdb.org[Taxus mairei[P. sitchensis were downloaded from Genbank with the key word ’FLI’ and merged with Putative Unique Transcripts (PUTs) from plantgdb. P. glauca sequences came from the 27,720 transcripts from the P. glauca gene catalogue [Pinus taeda consisted of PUTs from plantgdb. The phylogenetic relationship between species was inferred using the program BEST [The transcriptome sequence data used for inference of phylogenetic relationship and calculation of synonymous and non-synonymous divergence were collected from the following sources: EST sequences available at NCBI Genbank, PUTs assembled at ntgdb.org, our ownus mairei. Sequencatalogue  and mergram BEST  largely ram BEST , but runWe used the program getorf from the Emboss software suite to get all possible ORF predictions falling between two stop codons (hereafter: stop1 for 5’ stop codon and stop2 for 3’ stop codon) . SequencTo evaluate our ORF prediction method, we tested our method on TAIR10 cDNA representative gene model (released on 2011-10-03) and compared our predictions to the peptide sequence database that consisted of 27,416 protein sequences. A total of 27,384 ORF predictions were found with good blastn quality against the protein database . Among those, 26,867 ORFs have the best blast hit with their own proteins, which means that our procedure predicted the correct ORF in more than 98% of times.Picea species  and Pinus taeda and Taxus mairei using blastn with threshold combination of e-value = 1×10−05, score = 200 and percentage of identity = 90% (for Picea vs Pinus) and 80% (for Picea vs Taxus). Peptides of predicted ORFs from both species were aligned using the program kalign [Picea vs Pinus and Picea vs Taxus, respectively. This procedure resulted in a data set of putative orthologues between the different Picea species and either Pinus taeda or Taxus with all loci having E-values lower than 3 × 10−50 and 1 × 10−50, respectively. Nucleotide alignment was performed based on the protein alignments using revtrans [Picea and Pinus, and Picea and Taxus were estimated using codeml from PAML v. 4.4 [Pairwise Reciprocal Blast hit approach was used to infer putative 1:1 orthologues between m kalign . To avoirevtrans . FinallyThe authors declare no competing interests.TK, NG and UL designed the experiment. TK collected the needle material and did the wet-lab procedures. SU, JC and TK performed the bioinformatic analysis of the data. TK, JC, UL and ML drafted the manuscript. All authors contributed to and approved the final version of the manuscript.Picea abies PUTs. Details on the meaning of the different columns can be found on Sheet1.Annotation file. Annotation and level of gene expression for the Click here for fileBoxplot showing silent diversity estimates from different group of plant species.Click here for fileORF length and dN/dS. Comparison of the relationship between dN/dS and alignment length with Buschiazzo  data on Click here for file


Hookah cafes are an increasingly popular venue for socializing. In addition to beverages, appetizers, and desserts, habitués can order different flavors of tobacco that they smoke through waterpipes. Many patrons of hookah cafes believe smoking a waterpipe is safer than smoking cigarettes—an unsubstantiated belief “as old as the waterpipe itself,” according to the World Health Organization.Tracey Barnett, a social and behavioral scientist at the University of Florida, Gainesville, and colleagues measured CO levels of 173 patrons leaving three local hookah cafes and 198 patrons leaving five traditional bars that allow smoking. Hookah cafe patrons had an average CO level of 30.8 ppm compared with 8.9 ppm for traditional bar patrons. Even hookah cafe patrons who did not smoke from the waterpipe had average elevated CO levels of 11.5 ppm, similar to cigarette smokers.,,The Occupational Safety and Health Administration established a cutoff of 50 ppm for CO exposure over an 8-hour period;Hookah smoke contains toxicants not only from burning tobacco but also from the charcoal used to heat the tobacco in the pipe’s bowl, including CO, heavy metals, and polycyclic aromatic hydrocarbons.Hookah cafes are popular in university towns and large cities. By one 2005 estimate, up to 20% of some U.S. populations of young adults engage in hookah smoking.


Men who have sex with men (MSM) especially those who are HIV positive are at risk for HPV-associated anal cancer. We systematically reviewed studies with data on the prevalence of vaccine preventable anal HPV among men who have sex with men aged 25 or younger and identified 6 studies. None of these studies were specifically designed to determine the prevalence of HPV in this population. Available data, albeit limited, suggest many young MSM may not already be HPV infected. Further studies using representative sampling focused on teenage MSM are required to confirm this. Genital human papillomavirus (HPV) infection is widespread -3 and usThe prophylactic quadrivalent HPV vaccine is effective in preventing infection with HPV types 6, 11, 16 and 18 and in reducing the incidence of anogenital warts and anal intraepithelial neoplasia in males ,15. HoweWe systematically reviewed studies by searching MEDLINE and abstracts from the European Research Organisation on Genital Infection and Neoplasia (EUROGIN) Conferences and the International Papillomavirus (IPV) Conferences. Key words used in the search included: “men who have sex with men” or “MSM” or “homosexual” or “bisexual” or “gay” and “human papillomavirus” or “HPV”.The following data were extracted: author, year of publication, study setting, number of MSM, number of MSM aged 25 or younger, sample collection method, HPV DNA testing method, and HPV type tested. HPV 6/11/16/18 prevalence were extracted, and where possible, HPV 6/11/16/18 prevalence stratified by age group .The outcome of the review is summarized in Figure The 6 studies included provided data on anal HPV for a total of 698 MSM aged ≤ 25. Among men aged ≤ 20, the prevalence of anal HPV 6, 11, 16 and 18 ranged from 0 to 17%, 0 to 17%, 0 to 14% and 0 to 20%, respectively. Among men aged 21 to 25, the prevalence of anal HPV 6, 11, 16 and 18 ranged from 8 to 19%, 0 to 8%, 0 to 18% and 3 to 8%, respectively.Through this review, we found that there have been no published studies that have been designed to specifically determine the prevalence of HPV among young MSM using a representative sample of young MSM. Of the six studies identified, all had potential biases because of the way the studies were designed. These could have led to either an overestimate or an underestimate of the true HPV prevalence among young MSM. Several studies recruited men from clinics including STD clinics ,17,18 whDespite these important limitations, the available data suggest HPV infection may still be uncommon among teenage same sex attracted males, although the data are currently insufficient to draw this conclusion with confidence. A community-based, representative sample is needed to determine the prevalence of HPV infection, defined rigorously, among mainly teenage same sex attracted males.Given that HPV 16 and 18 are responsible for around 80% of anal cancers  and HPV HPV: Human papillomavirus; MSM: Men who have sex with men; EUROGIN: European Research Organisation on Genital Infection and Neoplasia; IPV: The International Papillomavirus Society; STD: Sexually transmitted diseases.CF has received honoraria from CSL Biotherapies and Merck and research funding from CSL Biotherapies. CF owns shares in CSL Biotherapies the manufacturer for Gardasil. JH has received an honorarium from CSL Biotherapies and is an investigator on an Australian Research Council funded project (LP0883831) that includes CSL Biotherapies as a research partner. AG has received honoraria and untied research funding from CSL biotherapies, and has received honoraria from Merck. SG has received advisory board fees and grant support from CSL and GlaxoSmithKline, and lecture fees from Merck, GSK and Sanofi Pasteur; in addition, she has received funding through her institution to conduct HPV vaccine studies for MSD and GSK. SG is a member of the Merck Global Advisory Board as well as the Merck Scientific Advisory Committee for HPV. None of this relates to this specific work. MC and HZ have no conflicts of interest.HZ and MC carried out the literature review and drafted the article. JH, SG, KF and AG participated in the coordination and editing of the article. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/341/prepub


Liolaemus pictus is the southernmost distributed lizard in the Chilean temperate forest, whose genetic structure has likely been influenced by Pleistocene glaciations. We conducted a phylogeographic study of L. pictus in Chile and Argentina based on one mitochondrial and two nuclear genes recovering two strongly divergent groups, Northern and Southern clades. The first group is distributed from the northernmost limit of the species to the Araucanía region while the second group is distributed throughout the Andes and the Chiloé archipelago in Southern Chile. Our results suggest that L. pictus originated 751 Kya, with divergence between the two clades occurring in the late Pleistocene. Demographic reconstructions for the Northern and Southern clades indicate a decrease in effective population sizes likely associated with Pleistocene glaciations. Surprisingly, patterns of genetic variation, clades age and historical gene flow in populations distributed within the limits of the Last Glacial Maximum (LGM) are not explained by recent colonization. We propose an “intra-Andean multiple refuge” hypothesis, along with the classical refuge hypothesis previously proposed for the biota of the Chilean Coastal range and Eastern Andean Cordillera. Our hypothesis is supported by niche modelling analysis suggesting the persistence of fragments of suitable habitat for the species within the limits of the LGM ice shield. This type of refuge hypothesis is proposed for the first time for an ectothermic species.Historical climate changes and orogenesis are two important factors that have shaped intraspecific biodiversity patterns worldwide. Although southern South America has experienced such complex events, there is a paucity of studies examining the effects on intraspecific diversification in this part of the world.  Nothofagus tree species and their associated communities, are formally recognized as a Chilean Biodiversity Hot Spot The landscape of southern South America has been shaped by several climatic and geological processes, of which two are especially important because of the magnitude of their evolutionary consequences: orogenic changes associated to the uplift of Andes The Pleistocene epoch was characterized by several global glacial cycles that deeply impacted polar and temperate regions. The well-studied Last Glacial Maximum (LGM), which occurred approximately 23,000–18,000 years ago (ya) Liolaemus. This clade is endemic to temperate South America and includes approximately 240 described species, of which 70 occur in Chile Liolaemus pictusNothofagus forests Liolaemus species L. pictus ranges from sea level to 1600 m, along over 800 km. Its western distribution covers Chile from the Región del Maule (∼36°S) to the Región de Los Lagos including some islands in the Chiloé archipelago (43°S) while its eastern distribution covers part of the Northern Argentinean Patagonia To date there is no strong evidence for intraglacial refugia throughout the Andes for heterothermic species. During the LGM, direct and periglacial effects may have been stronger for poikilothermic than for homeothermic species because lower temperatures would exclude the former from broad areas Liolaemus species have shown no significant reduction of genetic variation, effective population size (Ne) and distributional ranges in areas far from the LGM effects. In contrast, southern populations and those near the Andes exposed to direct and periglacial effects do show signatures of range reductions and loss of genetic variation L. pictus that during the LGM some mainland localities could have acted as refugia. Remarkably, none of the Liolaemus studies examined the timing of the inferred demographic changes, either retractions or expansions, putatively associated to the Quaternary glaciations. These scenarios may be further tested by assessing the congruence between past stability areas inferred by using ecological niche modeling Previous studies on some L. pictus, based on dense geographic sampling, niche modeling, and genetic information from one cytoplasmic and two nuclear loci, in order to test two hypotheses. First, we predict a pattern of stronger influences of recent glaciations (LGM), both in populations in the eastern (Andean) and southern distribution of this species. If so, we should expect to see signals of disequilibrium between mutation and genetic drift, with a predominance of derived haplotypes in glaciated and recolonized areas, evidence of reductions in effective population size (Ne) during the LGM, and increases in Ne during glacial retreat. In addition, we hypothesize that if the effects of the glaciations in the Andes and within the large ice sheet limits were strong, most of population migrations during a glacial retreats would have been directional from the western refugia towards the Andes. Because phylogeographic histories recovered from mtDNA sequences alone might be discordant with nuclear gene histories L. pictus. We also tested the congruence between the inferred evolutionary lineages and the patterns of genetic differentiation throughout their distributions, and discussed these in the context of the recognized subspecies of L. pictus.The objective of this study was therefore to assess the phylogeographic structure of L. pictus collected at 46 localities across the entire latitudinal range of the species, between 37°47′ and 43°02′ South  and the Ethics Committee of the Universidad de Concepción , as part of the review process for the Fondecyt Research Grant 1090664. Voucher specimens were catalogued in the Museo de Zoología at Universidad de Concepción (MZUC (UCCC), Chile). No specimen was found in the Chilean “Longitudinal Valley”, very likely due to the almost complete replacement of Nothofagus forests by non-native Pinus dominated agro-forestry systems, which has probably eradicated L. pictus populations from this region L. pictus codoceae and L. pictus talcanensis, whose status has been questioned We analyzed 196 specimens of 2′ South , Fig. 1.5′-TGACTTGAARAACCAYCGTTG-3′), F1 (5′-TGAGGACARATATCHTTYTGRGG-3′) y cytb3 (5′-GGCAAATAGGAARTATCATTC-3′). PCR amplifications of cyt-b were carried out as described in Victoriano et al. Of the 196 mitochondrial sequences analyzed, 59 were retrieved from Genbank  and the other 137 were generated in this study. Total genomic DNA was extracted from muscle tissue preserved in ethanol using the Wizard SV Genomic DNA Purification kit (Promega). A fragment of the mitochondrial cytochrome b (cyt-b) gene was amplified using primers GluDGL , number of haplotypes (h), haplotype diversity (Hd) and number of segregating sites (s), were calculated using DnaSP 4.5 For the mtDNA dataset, population diversity estimates, including nucleotide diversity  plot using BEAST 1.4.7 The magnitude and spatial arrangement of gene flow was assessed with the coalescence approach implemented in Migrate 3.27 L. pictus during the LGM and determine if these predicted areas of past occurrence were concordant with those inferred in the phylogeographic inferences. Ecological niches and potential geographic distributions were modeled using the maximum entropy method http://www.worldclim.org. Maxent generates Ecological Niche Models (ENM) using presence-only records, contrasting them with pseudo-absence data sampled from the remainder of the study area. The present-day ENM were developed based on the 123 confirmed occurrence points for L. pictus−5) and maximum number of iterations (500), using 25% of the localities for model testing. As suggested by Waltari et al. We conducted an ecological niche modelling analysis in order to identify potential refugia for http://dx.doi.org/10.5061/dryad.853t3)The inputs of all the analyses were deposited at Dryad  was a weakly supported polytomy of five subclades. The smallest of these, S2B1, was composed of one haplotype shared by individuals from the Andean locality of Malalcahuello (Western Andes), an area geographically very close to some localities harboring haplotypes from the Northern clade. Subclade S2B2 included five haplotypes with the longest branch lengths of the whole sample, most of them from a small region of the Coastal range corresponding to localities 16 (MN Alerce Costero) and 20 (Close to Heicolla). The two subclades S2B3 and S2B4 were composed of haplotypes broadly distributed between western Argentina, the southern Chilean Andes and the Coastal range. Finally, subclade S2B5 was well supported and composed exclusively of haplotypes from Chiloé Island. Only two haplotypes collected at localities 40 and 41 in the north of the island were not in this clade; both were part of clade S2B4. Thus, subclades S2B4 and S2B5 overlap on northern Chiloé Island.The southern phylogroup (Clade S) was composed of two strongly differentiated groups (S1 and S2), the smallest of which (S1) was strongly supported statistically and included only haplotypes from a restricted area from the Chilean Coastal range located at 41°30′S. The larger clade (S2) was composed of two subclades (S2A and S2B) having a moderate probabilistic support. Subclade S2A was well supported and included haplotypes from the Western Andes (e.g. Antillanca and Puyehue) from 39°S to 41°S. These localities are within the Chilean distributional range of the subspecies The AMOVA showed that 55.5% of the total cyt-b variation was explained by differences between the two main clades . The numIn general, all pairwise p-distance values involving comparisons between Northern and Southern clades are higher than values from comparisons among the Southern subclades. In addition, the among-Northern subclade values are higher than among-Southern subclade values .L. pictus (N+S) is estimated to have started approximately 751,000 ya  in the mid Pleistocene. Clade N is estimated to have diverged approximately 523,000 ya , and clade S about 580,000 ya , during the second half of Pleistocene  and three within Clade S . The general distributions of these clusters are detailed in the r = 0.716, p<0.001). Despite the strong correlation for the total pairwise comparisons, three clouds of spots could be detected  to the Chiloé Archipelago and nearby mainland in Southern Chile, as well as the temperate forest of the Argentinean Rio Negro and Chubut Provinces  distributed from the northern extreme of the species range at ∼37°S to 39°S, and a Southern group (clade S) with a broader distribution that includes most of Chiloé Island. Both clades include Coastal and Andean localities and overlap in a small area close to Malalcahuello in the western Andean piedmont , from Northern Chiloé, are recovered in an otherwise mainland phylogroup within subclade S2B4. Within S2B4, other closely related haplotypes are from Argentina (Aluminé and Bariloche), and from both Coastal and Andean Chilean regions in southern Chile. Some of these haplotypes are shared by more than one locality and with a broad distribution , which suggests an historical connection between populations from the east and west of the Andes and Chiloé Island. Our results disagree with Vidal et al. L. pictus dispersal could have been limited by the extensive ice cover during the LGM, especially on the east side of the island. Retreat of this ice shield may have also produced abundant meltwater, and such geologically recent fluvioglacial barriers may have prevented island-mainland gene flow. Therefore, this process would have contributed to reduce the connectivity between Chiloé Island and the continent for L. pictus rather than to increase it. This highlights the importance of considering the temporal dynamics throughout each glacial cycle if one wants to understand the effects of the glaciations on gene flow.The deepest splits between subclades were observed in Clade N; the MRCA of the phylogroup N1A from the northernmost locality in Las Trancas was 72,000 ya , but the split from its sister clade N1B was 440,000 ya . Similarly, clade N2 from the coastal range in Purén diverged 55,000 ya , but its split from sister clade N1 was 523,000 yr . These divergence levels suggest strong fragmentation, low connectivity and reductions of Ne, especially in clades N1A and N2. Although the effects of the glacial cycles were less prominent in the North, Nothofagus forest, due to different physical and chemical properties of the soils which were impacted by marine sediments for a long time. Although L. pictus inhabits the borders of Nothofagus forests, it needs high density, high connectivity, and large size patches of arboreal vegetation Eupsophus calcaratus, a species also associated to the temperate Nothofagus forest, and recovered a monophyletic group for the haplotypes from Chiloé Island which was the sister clade of samples from Alerce Andino in the continent , suggesting historical dispersal between both areas. For larger vertebrates such as the southern deer Pudu pudu, there is evidence for high divergence, low gene flow and reciprocal monophyly between Chiloé Island and the mainland L. pictus . Independent of the presence of small or medium sized fluvial barriers during glacial periods, this species should have been able to move between the island and the continent if the forest had colonized continuous formations over the exposed shelf, thereby erasing the pattern of reciprocal monophyly detected in our study. For Chiloé Island, we found no evidence for recent or post-LGM migration from the northwestern non-glaciated area to the south of the “Chiloé ice diagonal”  and Puyehue (site 22) share three haplotypes in the Western Andes. However, we found evidence for recent gene flow between localities more than 100 km apart, such as between Río Puelo and Bariloche (sites 27 and 35 respectively), Huerquehue and Los Llolles (11 and 17), and Chaitén and Canal Garrado (28–30). The last example suggests a more recent connection between mainland localities surrounding Chiloé Island than between this island and the continent. Prior evidence for post-LGM dispersal in the glaciated Andes has been found in other species like the freshwater crab L. pictusNothofagusL. pictus in the BioBío and Araucanía regions and almost certainly led to the extinction of L. pictus populations in these regions Despite the exhaustive sampling effort over the entire range of Co-distribution of haplotypes from highly divergent clades suggests older migration between different areas. For example, sites 23 and 24 from the Estaquillas area (close to the mainland coast in southern Chile) included S1 haplotypes which are endemic to that area, and haplotypes from the broadly distributed clade S2B4. Similarly, sites 40 and 41 along the northern margin of Chiloé Island included haplotypes from the same clade S2B4 and from clade S2B5, which includes only haplotypes endemic to the island. If the secondary contact hypothesis is valid, and excluding the explanation of ancestral polymorphism due to incomplete lineage sorting, populations would have dispersed over long distances after an old lineage diversification. Evidence for multiclade presence was obtained previously by Vidal et al. for the localities of Las Cascadas and Los Muermos, close to Estaquillas L. pictus phylogroups persisted within the limits of the LGM ice shield. Other clades include haplotypes from both Andean and coastal regions (S2B3 and S2B4). However, each of these clades harbors two subclades that are reciprocally monophyletic, with one each in the Andes and the Coastal range. Gene flow estimates between these areas suggest an exchange of individuals between the coast and the Andes predating the LGM. Pleistocene glaciations that occurred in southern South America are well documented L. pictus should be associated with the coldest Pleistocene glaciations approximately 700,000 ya and the last southern Patagonia glaciations 180,000 ya. Liolaemus pictus is one of the youngest species within the genus, and our molecular divergence estimates  suggest that most of the species, both in the “Chilean group” and in species of the subgenus Eulaemus in Argentina L. pictus.Several clades in the South of the mainland predate the LGM and show a restricted distribution, suggesting a “relict population” with low rates of expansion. For example, clades S1 and S2B2, which are 187,000 and 305,000 ya respectively, are restricted to a small region of the southern coast. Clade S2A, which diverged approximately 300,000 ya, has a broader distribution but is restricted to the Western Andes, suggesting that some e and longer sorting time to reach monophyly for the nuclear loci Although the levels of spatial structure were deeper for the mitochondrial than for both nuclear genes, the spatial trends of the genealogical relationships were similar among them and not in total conflict. The general mitochondrial pattern was strongly suggested by both nuclear genes, but the southern range showed a mixed distribution of related nuclear haplotypes and incomplete lineage sorting. Nevertheless, both nuclear genes suggest differentiation between the Northern and the Southern groups, thus corroborating the mtDNA gene tree showing the oldest split between lineages at approximately 39°S, and a more recent weaker structure among the southern populations. These kinds of differences between the patterns of nuclear and mitochondrial differentiation are expected given the larger NL. pictus corroborate the hypothesis of population size decreases during Pleistocene glaciations L. pictus distribution. The more extensive ice sheets in the South produced extensive exclusion zones for animal and plant populations L. pictus by rapid population growth after the ice sheet melted, but with much greater expansion in the southern group. This population expansion was likely associated with the rapid colonization of Nothofagus forests from southern refugia during the same period L. pictus in the northern part of its distribution is most parsimoniously explained by local population reductions followed by modest post-LGM population expansion with limited dispersal, as found previously in another lizard species Our demographic analyses of L. pictus. This is supported by several lines of evidence, including the presence of private haplotypes within glacial limits, several clades older than the LGM and distributed throughout the Andes, and high genetic diversity for many populations across the species distribution. On the other hand, the presence of probable ancestral haplotypes within LGM limits and signals of gene flow from the Andes to neighboring areas suggest the past occurrence of multiple source populations within the boundaries of the LGM ice shield. Considering the glacial history of the study area, our data suggests that during recurring glacial phases, and in addition to the Pacific Coastal refugia, L. pictus likely persisted in multiple Andean refugia, where genetic variation has accumulated and from where populations expanded during interglacial periods L. pictus, which indicate that during the LGM appropriate habitat persisted in the Andes. In general terms, ENM predicts the occurrence of refugia on Chiloé Island, in the central valley, adjacent Pacific coast, and several isolated valleys within the Andes. As such, many populations of L. pictus could have persisted in these areas, as isolated pockets within the presumed boundaries of the LGM ice shield. Similarly, this same scenario has also likely occurred during older glaciations.Although research is beginning to characterize LGM refugia in southern South America Aegla alacalufi. Similarly, Nuñez et al. Eupsophus calcaratus. Liolaemus pictus haplotypes collected from the Malalcahuello area  were highly divergent and fell in both Southern and Northern clades, Malalcahuello being the area where both groups overlapped. The same pattern was found in the mouse Abrothrix longipilis; in this case highly divergent haplotypes co-occur in the area of Malalcahuello . In addition, Ruiz et al. Araucaria araucana. Future research will clarify the roles of Malalcahuello and Ñuble either as refugia for L. pictus or as areas of secondary contact of variants that differentiated elsewhere. We note that the repetition of the pattern for unrelated taxa seems to suggest that the region was probably a refugium.Independent evidence corroborates the occurrence of ice free areas during the LGM within the Andes, specifically the Ñuble glacial gap (36°30′S) and the Malalcahuello Valley which might have acted as refugial areas L. pictus, suggests three kinds of refugia in southern South America, all outside the LGM ice shield boundary: 1) a northern area in the central valley, 2) a Pacific Coastal area including Chiloé Island, and 3) the eastern Andes outside the LGM. Palynological and genetic studies suggest that the eastern Andean refugia, at the latitude proposed in our study (37°S–44°S), would have been delimited by Andean glaciers to the West and the Argentinean steppe towards the East L. pictus south to approximately 41°S, and are characterized by unexpectedly high genetic endemism and diversity.The estimated ages for most clades suggest that their origins predate the LGM, therefore the most probable explanation for the observed distribution of the Andean clades S2A, S2B3 and S2B4 would be the long-term persistence of several refugia within the limits of the LGM, as suggested by the ENM. An alternative explanation would be that during the LGM, populations were driven into the Longitudinal Valley and later recolonized the Andean Cordillera and coastal areas. However, the latter scenario predicts less geographic structure within each clade. Some clades, such as S2B3 and S2B4, include haplotypes from both cordilleras , but haplotypes from each cordillera tend to form reciprocally monophyletic groups within each of these clades. Additional evidence for our interpretation of persistent refugia within the LGM ice sheet comes from the geographic provenance of the inferred ancestral haplotypes of some haplogroups. Six of the 10 haplogroups in the cyt-b network were Andean in distribution and occurred in areas within the LGM ice sheet, which suggests that these phylogroups likely originated in these areas prior to the LGM. Additionally, the frequency with which Andean populations acted as sources and Coastal populations as sinks for gene flow is striking, and such patterns can only be explained by the occurrence of multiple Andean refugia during the LGM. Our estimates suggest several cases of gene flow from the Andes to non Andean areas. For clades S2B3 and S2B4, the predominant migration pattern was from the eastern  to the Chilean coastal mainland. According to these results, it is very likely that eastern Andean populations were a source of variants for Pacific coastal sink populations at similar latitudes. The recent review of Sèrsic et al. taxa, these results constitute the first evidence for the intraglacial persistence of multiple lizard populations during the LGM. The proposal of a more complex scenario of several isolated refugia is further supported, and should replace the over-simplified scenario of a continuous LGM ice shield in the southern South America Andes. Future studies based on other co-distributed species are needed to test the proposed scenario.In agreement with similar findings from other L. pictus, in particular in relation to most of the recognized subspecies L. pictus argentinus were recovered in three well-supported clades. Our extensive sampling shows that haplotypes from specimens collected within the distributional range of L. pictus chiloensis fail to form a monophyletic group; two haplotypes belong to an otherwise mainland clade, but most of the individuals from most of Chiloé Island were recovered in a well supported clade, which should be considered as a different evolutionary unit. Remarkably, Chiloean specimens are morphologically distinct from those collected on the mainland. The nature of the incongruence between the mitochondrial and morphological patterns of geographic variation is for the moment unknown L. pictus. However, the deep divergence (ca. 11%) between the Northern and the Southern clades suggest differences that should be recognized at the species level. The analysis of unlinked loci and morphological characters is necessary to test the mitochondrial based hypothesis Liolaemus septentrionalis, and the name L. pictus should be restricted to the Southern group.Our phylogeographic patterns do not corroborate the current taxonomic arrangement within Table S1Collection localities for Liolaemus pictus, sample sizes (N) and subspecies to which are allocated. Localities were grouped into four geographic zones .(DOC)Click here for additional data file.Table S2Haplotypes composition for each sample site for the mitochondrial region cyt-b and the nuclear genes LDB5B and EXPH5 in Liolaemus pictus. Bold numbers are haplotypes distributed in more than one locality. Geographic coordinates and relative distribution of each locality are detailed in (DOC)Click here for additional data file.Table S3Analysis of molecular variance (AMOVA), for the two main haplogroups in Liolaemus pictus. Each group in the AMOVA corresponds to the deepest splited clades, called northern and southern. Northern phylogroup is distributed from the northern extreme to approximately 37°S, and the Southern phylogroup is from this latitude up the southern extreme of the distribution of L. pictus.(DOC)Click here for additional data file.Table S4Estimates of evolutionary divergence , over sequence pairs between phylogroups (clades) in Liolaemus pictus. Greyscale according to evolutionary divergence values. Values on the diagonal are intra clades p-distances. Standard error estimates are shown above the diagonal. Phylogroups codes as in (DOC)Click here for additional data file.Figure S1Haplotype networks for the nuclear gene EXPH5 in L. pictus. Grey haplotypes correspond to the outgroups L. lemniscatus (L.l), and L. tenuis (L. t). Color codes as in the map of (TIF)Click here for additional data file.


Background. Breast cancer patients receiving adjuvant chemotherapy often experience a loss of quality of life. Moreover chemotherapy may induce neutropenia. Patients report a better quality of life when additionally treated with mistletoe products during chemotherapy. Methods. In this prospective randomized open-label pilot study 95 patients were randomized into three groups. All patients were treated with an adjuvant chemotherapy. The primary objective of the study was quality of life, the secondary objective was neutropenia. Here we report the comparison of HxA (n = 34) versus untreated control (n = 31). Results. In the explorative analysis ten of 15 scores of the EORTC QLQ-C30 showed a better quality of life in the HxA group compared to the control group (P < 0.001 to P = 0.038 in Dunnett-T3 test). The difference was clinically relevant  in eight of the ten scores. Neutropenia occurred in 7/34 HxA patients and in 8/31 control patients (P = 0.628). Conclusions. This pilot study showed an improvement of quality of life by treating breast cancer patients with HxA additionally to CAF. Although the open design may be a limitation, the findings show the feasibility of a confirmatory study using the methods described here. Quality of life of cancer patients is frequently reduced during and after chemotherapy . But phyBecause 70% of cancer patients use mistletoe products in Germany, randomized clinical trials are difficult to conduct: low recruitment rates and noncompliance because of therapy preferences are the consequences –14. TherThis randomized clinical pilot trial was carried out to assess the effects of two different mistletoe products on quality of life and on neutrophil count when administered during CAF chemotherapy for breast cancer. The aim was to identify promising outcome measures and methods for future studies in Serbia and to use the results for sample size calculation for a following confirmative study. Here we present the results with the mistletoe product Helixor A (HxA).The objectives of this pilot study were to determine the clinical response  of breast cancer patients to an additional mistletoe therapy during CAF. Our primary hypotheses were: breast cancer patients receiving mistletoe products during six cycles of consecutive treatment with CAF will show a better quality of life and less neutropenia compared to patients receiving CAF alone.We conducted a prospective randomized open-label pilot study with equal-size randomization into three groups. All three groups received six cycles of CAF. In addition, one group received Helixor A (HxA), another group received Iscador M, and a control group had no additional therapy. The study was not placebo-controlled because currently no active placebo is known that can imitate the typical and time-dependent reactions following subcutaneous injections of mistletoe products . Here we report the comparison of HxA versus control. The other part of this combined pilot study has been published elsewhere .1–3N0–2M0 scheduled to receive six consecutive cycles of CAF after surgery were assessed for eligibility.At the Institute of Oncology and Radiology, National Cancer Research Centre of Serbia in Belgrade (IORS), breast cancer patients in the stages TAdditional inclusion criteria were female gender, age ≥ 18 years, Karnofsky-Index ≥ 60, leukocytes ≥ 3,000/mm³, thrombocytes ≥ 100,000/mm³, serum creatinine ≤ 2 mg%, serum glutamic oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT) ≤ 2.5 × the upper institutional limits.Exclusion criteria were pregnancy or lactation, distant metastases, planned radiation or hormone therapy during the CAF treatment period, use of immunostimulant or immunosuppressive agents  except for nausea and emesis, current use of other investigational agents, clinically relevant physical or mental illness such as serious infections, hepatic, renal or other organ dysfunction, or major depression, alcohol abuse, alcoholism, oral or parenteral drug abuse, and methadone treatment.The chance to be allocated to any of the three groups  was 1 : 1 : 1, for randomization variable block sizes were used. No stratification took place prior to randomization. The randomization sequence was generated by Clinical Research Dr. Tröger (CRDT), using SPSS . Allocation concealment was implemented by using sealed envelopes, prepared by CRDT. Patients were enrolled by investigators at the Outpatient Clinic of the IORS, while the sealed randomization envelopes were stored in the Department of Study Coordination of the IORS and released consecutively for each enrolled patient.CAF was administered in six cycles with a three-week interval between each cycle. The scheduled dosage was 500 mg cyclophosphamide, 50 mg adriamycin, and 500 mg 5 FU per 1 m² skin surface administered at one day. All patients received antiemetic therapy with a single dose of ondansetron chloride 8 mg, dexamethasone 8 mg, and ranitidine 50 mg, respectively, administered prior to each CAF cycle.No other antineoplastic or immunomodulatory therapies were permitted during the study. Patient compliance was examined by account of the questionnaires and patient diaries and at each visit.Viscum album from fir tree , fresh plant material). HxA was manufactured and provided by Helixor Heilmittel GmbH & Co. KG, Rosenfeld, Germany, and prepared in 1 mL ampoules for injection, each ampoule containing aqueous extract of 1, 5, 10, 20, 30, or 50 mg of fresh mistletoe herb, respectively, in isotonic saline solution. HxA was administered by subcutaneous injection of 1 mL HxA into the upper abdominal region three times per week. The patients were instructed to inject HxA themselves. The dosage of HxA followed the guidelines of the manufacturer with a stepwise increase: 3 × 1 mg, 3 × 5 mg, 3 × 10 mg, 3 × 20 mg, 3 × 30 mg, and remaining doses 50 mg. Dose-dependent inflammatory reactions at the injection site  were monitored. If such reactions exceeded 5 cm in diameter, the dosage was decreased or the therapy was paused until the reactions had ceased. Depending on the beginning of the injections in the first week, 52–54 injections of HxA were planned. An average of 52.3 ± 2.8 injections with altogether 2,813 mg  were administered in the HxA group.Patients randomly allocated to the first group with additional therapy received Helixor A  in the official Serbian translation . The EORAdverse events (AE) were assessed by interviewing the patients and by analysing laboratory data at each visit. The Common Terminology Criteria of Adverse Events (v3.0) were used for grading. Local reactions to HxA less than 5 cm in diameter were expected reactions and therefore not classified as adverse events.Neutropenia was one of the study objectives and was not classified as AE.Statistical analysis (SPSS 14.0) was performed on the intention-to-treat population, a population consisting of all patients randomised into one of the three groups. Missing data were not replaced. Because of the pilot character of this study, all results of the analysis are explorative and do not have a confirmatory character. Therefore no sample size calculation was performed. A sample size of 90 patients (30 per group) was considered to be sufficient. For calculation of the EORTC QLQ-C30 scores, missing data within one scale were replaced according to the manual .Quality of life (EORTC QLQ-C30) was analysed as follows: for each EORTC QLQ-C30, the mean change from baseline during follow-up in each group was compared among the HxA group, the group with the other mistletoe product, and the control group, using nonparametric marginal models according to Brunner and Langer  with theFor the incidence of neutropenia, the difference between the HxA and control groups was analysed by chi-square test according to the sequential rejective Holm procedure.The study was approved by the Ethics Committee of the National Cancer Research Centre of Serbia without modifications (date: 3 October 2005) and by the Serbian Drug Agency (date: 01 November 2005). Due to its pilot character, this study was not registered in a public study registry. The study was conducted in compliance with the Declaration of Helsinki, Good Clinical Practice guidelines, and national laws. A patient insurance was provided for all participants. All patients provided signed informed consent prior to inclusion. CRDT was responsible for planning, conduct, monitoring, and analysis of the study. Two audits at the CRDT office and one at the study site were performed by the two sponsors during the study; no violation of Good Clinical Practice was detected.This publication followed the CONSORT-PRO statement for reporting of randomized trials , therefon = 34), CAF and another mistletoe product (n = 30), and CAF without additional therapy (n = 31). One patient in the control group was withdrawn from further CAF therapy after three cycles of CAF because of heart disease  and rhinoconjunctivitis with putative relationship to mistletoe therapy (n = 1). These dropout patients were replaced to achieve at least 30 in both groups. All other patients in the HxA group and all patients in the control group received the six scheduled CAF cycles.In the HxA group, 5 patients dropped out; reasons were withdrawal of informed consent  of planned visits in the HxA group, and for 97.2% (211 of 217) of planned visits in the control group. None of the expected questionnaires were missing in the follow-up phase of the study . The neuThe HxA group and the control group did not differ significantly regarding age, tumour stage, body mass index, physical status, vital signs, previous diseases, EORTC QLQ-C30 scores, and neutrophil counts .During chemotherapy with CAF, a deterioration of the quality of life occurred in 14 of 15 EORTC QLQ-C30 mean scores in the control group of which 4 were clinically relevant; whereas a deterioration occurred in 5 of 15 scores in the HxA group, of which 2 were clinically relevant.In the adjusted analyses, mean differences from baseline were compared between the two groups for each EORTC QLQ-C30 score: 14 of 15 comparisons favoured the HxA group and one comparison  favoured the control group. Ten of 14 comparisons favouring the HxA group showed significant differences Figures  and 3. EIn explorative analyses, the differences from baseline for each of the 15 scores at each of the six follow-up assessments were compared between the two groups. 80 out of 90 differences favoured the HxA group, while 10 differences favoured the control group (data not shown). Furthermore, moderate differences of at least 10 points were found once in nine scores , small differences of 5–10 points were found in three scores , and differences <5 points were found in the remaining three scores.μL) was detected seven times in seven different patients of the HxA group and nine times in eight different patients of the control group (P = 0.628).Neutropenia  of the HxA group reported altogether 45 adverse events (AE). The 45 AE were: local inflammatory reaction at the injection site > 5 cm, with definite causal relation to HxA ; rhinoconjunctivitis at one eye, documented by the investigator to be probably caused by HxA—the patient withdrew informed consent and gave no further information (n = 1); and AEs without any causal relationship to the administration of HxA (n = 2).Altogether 1In the control group, 12 AEs and one serious adverse event  and incidence of neutropenia were investigated in breast cancer patients undergoing adjuvant CAF chemotherapy. Patients receiving HxA in addition to CAF had significantly better quality of life, compared to patients receiving CAF alone. HxA therapy was well tolerated.Strengths of this study include a high recruitment rate, detailed assessments of therapy implementation, high therapy compliance, and very low dropout rates.Due to the open-label design, the study cannot distinguish between direct drug effects on quality of life and possible indirect effects from therapy expectations, therapy administration, and so forth, in the HxA group.Generally, medication trials are blinded to separate pharmacological effects from placebo effects. However, the lack of blinding may not necessarily have had relevant effects on the results of this study: an updated Cochrane review of randomized trials comparing placebo to no treatment found no significant placebo effects on eight out of ten evaluable indications, small effects on self-reported pain, and moderate effects on phobia. Even these effects might have been confounded by biases –23.This study was designed as a pilot study, and the limited sample size of 30 patients per group does not allow for hypothesis confirmation. Moreover dropout patients or patients with missing data were more frequent in the HxA-group. As the results were calculated using data from all included patients (ITT), data of two patients in each group were missing. The remaining differences are additional three patients (ITT) in the HxA-group, which may lead to a biased result. Nevertheless, significant differences in 10 of 15 EORTC QLQ-C30 scores favouring the HxA group were found. All of these scores showed a clinically relevant difference of at least 5 points. The latter scores include role function, emotional function, pain, nausea, emesis, appetite loss, and diarrhoea, which are relevant symptoms in patients during chemotherapy with CAF.These results are consistent with findings from another randomized phase III trial, wherein the administration of HxA to patients undergoing chemotherapy for breast cancer, lung cancer, or ovarian cancer was associated with significant beneficial effects on quality of life . NotablyOn the other hand, in a trial of early breast cancer patients receiving radiochemotherapy (CMF schedule), no influence of adjunctive therapy with HxA on any of the EORTC QLQ-C30 scores was found . This diOn the whole, there is some evidence that mistletoe extracts may have an impact on quality of life and reduction of side effects of chemotherapy especially in case of breast cancer patients as concluded in two recent comprehensive systematic reviews , 25.In this pilot study, the effects of HxA on quality of life increased over time with the largest effects observed at the last follow-up visit. A longer follow-up period might show additional long- term benefits of HxA therapy.In this randomized pilot study of patients receiving adjuvant CAF chemotherapy for breast cancer, additional treatment with mistletoe therapy (Helixor A) was associated with significant and clinically relevant improvements of quality of life. Although the open design may be a limitation, the findings show the feasibility and justification of a phase III study using the methods described here.


Dactylospongia elegans, showed antiproliferative or cytotoxic activities on leukemia cells. In this study, we investigated the effect of smenospongine on solid tumors. Since angiogenesis is well known to be closely involved in growth and metastasis of solid tumors, the antiangiogenic effect of smenospongine was determined. We found that smenospongine inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVEC). Moreover, the inhibitory activity of smenospongine on growth of solid tumor cells was investigated. Smenospongine inhibited the growth of 39 human solid cancer cells in vitro, with a mean Log GI50 value of −5.55. In conclusion, smenospongine exhibits antitumor activity on solid tumors via two mechanisms, an antiangiogenic effect on endothelial cells and direct inhibition of growth of tumor cells.  We previously reported that smenospongine, a sesquiterpene aminoquinone isolated from the marine sponge  After the size reaches 1 mmogenesis ,3.Dactylospongia elegans, showed multifaceted antitumor activities on leukemia cells [We previously reported that smenospongine , a sesquia cells ,5,6. Thuia cells ,6. Here,Angiogenesis is a multi-step process involving basement membrane dissolution, endothelial cell proliferation and migration, and new basement membrane formation . To exam50 value was calculated to be 4.9 μM  by use of GraphPad Prism 4. As shown in To further investigate the antiangiogenic activity of smenospongine, the effect on migration of HUVECs was examined by use of a wound healing assay as reported ,9. Figurin vitro tube formation by HUVECs as reported by us previously [Finally, we examined the effect of smenospongine on the eviously . The HUV50 values (the concentrations of given compounds to cause 50% growth inhibition of cells) for each tumor cell line were obtained and the JFCR39 fingerprint was plotted based on the Log GI50 values [50 for all the 39 tumor cell lines was calculated to be −5.55. Among the cell lines, those of colorectal cancers and most lung cancers showed relatively higher sensitivity while those of most ovarian and breast cancers indicated relative resistance, suggesting such type of compound might be more applicable for treatment of the colorectal and lung cancers, than the ovarian and breast cancers, when approved as an anticancer drug in the future.To investigate the direct activity on solid tumor cells, we determined the inhibitory effect on the cell growth of 39 solid tumor cell lines (JFCR39) by use of the sulforhodamine B (SRB) assay, as described by us previously . The GI50 values ,10,11 (FDactylospongia elegans (collected in Indonesia in 2001) as described previously [Smenospongine was isolated from the marine sponge eviously . The streviously . 2.HUVECs were purchased from Clonetics and maintained in endothelial cell growth medium-2  at 37 °C in a humidified atmosphere containing 5% CO2.A panel of 39 human cancer cell lines termed JFCR39 ,10, whic3 cells/well) was seeded in 96-well plate and incubated in EGM2 medium at 37 °C in a humidified atmosphere containing 5% CO2. Twenty-four hours later, 0.5 μL of various stock solutions of smenospongine or DMSO was added. After further incubation for 72 h, 10 μL of WST-8 was added to each well and further incubated. Three hours later, the absorbance at 450 nm was measured. The number of viable cells after treatment was calculated using the following formula: Cell number (% control) = 100 × (absorbance of a given sample − absorbance of Blank well)/(absorbance of Control well − absorbance of Blank well), where the Blank well contained medium but no cells and the Control well contained cells but no smenospongine. The IC50 value was calculated by fitting the data points to a logistic curve using the GraphPad Prism 4 software. Three independent experiments were carried out.Cell viability was determined using the WST-8 assay kit  as described previously by us . To inve2. Then, the monolayer cells were scratched with a 1 mL pipette tip. Fresh EGM2 media were added with DMSO or with various concentrations  of smenospongine. After further incubation for 18 h, the migration was observed under microscope and photographs were taken. For quantification, the migrated cells were counted manually, and the percentage inhibition was expressed by comparison with the control (treated with DMSO). Two independent experiments were carried out.HUVECs were allowed to grow to full confluence in 6-well plates at 37 °C in a humidified atmosphere containing 5% COin vitro was determined following the method reported by us previously [5 cells/mL) was seeded in each well of the coated plate. 0.5 μL of various stock solutions of smenospongine was added to achieve desired final concentrations and the cells were then incubated for 18 h at 37 °C. The capillary-like tubes formed were visualized under microscope. Representative network of tube structures formed in each well was photographed. The length of the tubes was measured for quantification, and the percentage of tubes formed in each smenospongine-treated well relative to the control well (DMSO-treated cells) was calculated. Representative data from 2 independent experiments, each performed in triplicate, were used for analysis.The effect of smenospongine on tube formation eviously . Briefly50) of cells was calculated. The graphic representation (termed fingerprint), the mean differential growth inhibition for the cells used in the JFCR39 panel, was plotted based on a calculation that uses a set of Log GI50 values [Inhibition of cell growth of cancer cells was assessed by the change in total cellular protein following 48 h of treatment with smenospongine, and was measured by SRB assay as described previously ,11. The 0 values ,11. Smenospongine inhibited proliferation, migration and tube formation of human endothelial cells, suggesting favorable antiangiogenic activity. Moreover, this compound inhibited growth of 39 cell lines of human solid tumors. Such results suggest smenospongine exhibits potential antitumor efficacy on solid tumors. To our knowledge, this is the first report about the antiangiogenic activity of sesquiterpene quinines, while some cytotoxic activities of such compounds were reported . Smenosp


Arbuscular mycorrhizal (AM) fungi may make a contribution to the decrease of organophosphate residues in crops and/or the degradation in soils, but such effects remain unknown.As one of the most widely used organophosphate insecticides in vegetable production, phoxim . Carrot and green onion were grown in a greenhouse for 130 d and 150 d. Phoxim solution (100 ml) was poured into each pot around the roots 14d before plant harvest. Results showed that mycorrhizal colonization was higher than 70%, and phoxim application inhibited AM colonization on carrot but not on green onion. Compared with the nonmycorrhizal controls, both shoot and root fresh weights of these two vegetables were significantly increased by AM inoculations irrespective of phoxim application rates. Phoxim concentrations in shoots, roots and soils were increased with the increase of phoxim application rate, but significantly decreased by the AM inoculations. Soil phosphatase activity was enhanced by both AM inocula, but not affected by phoxim application rate. In general, G. intraradices BEG 141 had more pronounced effects than G. mosseae BEG 167 on the increase of fresh weight production in both carrot and green onion, and the decrease of phoxim concentrations in plants and soils.A greenhouse pot experiment studied the influence of AM fungi and phoxim application on the growth of carrot and green onion, and phoxim concentrations in the two vegetables and their soil media. Treatments included three AM fungal inoculations with Our results indicate a promising potential of AM fungi for enhancing vegetable production and reducing organophosphorus pesticide residues in plant tissues and their growth media, as well as for the phytoremediation of organophosphorus pesticide-contaminated soils. However, the average phoxim residue in vegetable crops has almost doubled that to 0.09 µg g−1 fresh weight −1 bodyweight or the LD50  of phoxim for the rat is about 2 g kg−1 bodyweight Organophosphorus pesticides with higher acute toxicities than the banned organochlorine ones have been major alternatives worldwide for plant pest control in agriculture since 1980’s p,p-DDE in soils 2/CO2G. intraradices hydrolysed both 5-bromo-4-chloro-3-indolyl phosphate and phenolphthalein diphosphate. In addition, AM inoculation did increase plant growth whilst phoxim did not decrease the AM colonization of Cynara cardunculusArbuscular mycorrhizal (AM) fungi are ubiquitous symbiotic associations found in both natural and agricultural ecosystems, including organics-contaminated sites We hypothesize that AM inoculation may make a contribution to plant growth and phoxim degradation in a phoxim-contaminated soil, if the applied AM fungi are not sensitive to phoxim. To test our hypothesis with two AM fungal isolates and four phoxim rates, a pot experiment was carried out (1) to assess the effect of AM inoculation on the growth of carrot and green onion grown in soils with different application rates of phoxim; and (2) to identify whether AM inoculation can decrease the phoxim concentrations in the vegetables and their growth media.Glomus intraradices BEG 141 and G. mosseae BEG 167, kindly provided by China Agricultural University, were propagated with alfalfa (Medicago sativa) in a soil–sand mixture in greenhouse for three successive cycles (four months each). The inocula were air-dried and sieved (2 mm), and each consisted of a mixture of rhizospheric soil containing fine root fragments, hyphae and spores . The nonmycorrhizal control inoculum was also produced by the same procedure to provide a similar other microbes.Two AM fungal inocula, Daucus carota L. cv. Changfeng) and green onion (Allium fistulosum L. cv. Fengwang) were surface-sterilized with 10% (v/v) H2O2 for 10 min, and subsequently washed several times with distilled water.Uniform size seeds of carrot  at the Henan University of Science and Technology, were autoclaved at 121°C for 2 h, and air-dried and then thoroughly mixed as the plant growth media. The soil is classified as Aquic Ustochrepts (US soil taxonomy) and soil texture is loamy, with a pH (1∶2.5 soil/water) 7.9, 1.62% organic matter 65.3 mg kg−1) purchased from Beijing Beihua Hengxin Biotechnology Co. Ltd was used for phoxim analysis. Acetonitrile was HPLC-grade and other reagents were analytical grade.The phoxim was a commercial 40% phoxim emulsifying concentrate widely used in China. Standard substance of phoxim  and three inoculation treatments . The 400 mg l−1 phoxim application rate represents the recommended dose that was widely applied in the vegetable production in China. Mycorrhizal inoculation treatments received 20 g mycorrhizal or nonmycorrhizal inocula, which were placed in the middle layer of growth media in pottery pots . As a result, there were 12 treatments with three replicates for each plant species, giving a total of 72 pots in a randomized block arrangement.This experiment was a bifactorial design consisting of four phoxim rates  analysis.p-nitrophenol (PNP) released by phosphatase when soils were incubated with buffered (pH 7.0) sodium p-nitrophenyl phosphate solution and toluene at 37°C for 24 h Determination of root mycorrhizal colonization was followed by the acid fuchsin staining-grid intersect method −1. The column temperature was set at 30°C, and the injection volume was 4 µl. The recoveries were between 95.0% and 100.4%.Analysis of phoxim concentrations in shoots, roots and soils was followed by the Ministry of Agriculture of China n = 3) were subjected to a two-way ANOVA using the SPSS 13.0 software package . Significances between the means under AM inoculation, phoxim application rate, or interactions of AM inoculation and phoxim application were compared by the Duncan’s multiple range test at p<0.05, 0.01 or 0.001.Data  in the BEG 141 and BEG 167 treatment were 5.2 and 4.1 times, respectively , Table 1Phoxim application did not affect shoot or root fresh weights of green onion , Table 1Two-way ANOVA results show that the interactions between AM inoculation and phoxim application had significant effects on both shoot and root biomass production of carrot, but not on shoot or root biomass production of green onion .−1 phoxim application rate).Phoxim was not detected in shoots and roots of both carrot and green onion under the zero phoxim application, whilst increased with the increase of phoxim application rate . For botAll treatments with AM inoculation or phoxim application rate, and the interactions between AM inoculation and phoxim application all had significant effects on phoxim concentrations in shoots and roots for both carrot and green onion . In addiPhoxim was not detected in the soil growth media under the zero phoxim application, whilst increased as the phoxim application rate increased . Both AMTwo-way ANOVA results show AM inoculation, phoxim application, and the interactions between them all had significant effects on phoxim concentrations in soils from both the carrot and green onion pots .Soil phosphatase activity did not vary with the increase of phoxim application rate . CompareSoil phosphatase activity was significant affected by the AM inoculation, but not by the phoxim application. In addition, no significant interactions were observed between the AM inoculation and phoxim application rate on the soil phosphatase activity .Cynara cardunculus−1 phoxim (2 times of the recommended dosage) inhibited mycorrhizal colonization of carrot. These results indicate that phoxim is generally of low toxicity to AM fungi and the formation of AM symbiosis, and the recommended dosage of phoxim (400 mg l−1) is safe to AM fungi and plant growth.Numerous studies have reported that organophosphorus pesticides at recommended dosages have little or no adverse effects on AM colonization and host plant growth G. intraradices BEG 141 showed more pronounced effects than G. mosseae BEG 167 on both the growth of vegetables and phoxim residues in plants and soils, probably because of a high colonization potential of G. intraradices BEG 141 and a high compatibility between this isolate and the two vegetables tested. Also, the two AM inocula were more effective on carrot than on green onion. This may be due to different mycorrhizal dependency of plants and soil P nutrition needed by plants. Mycorrhizal development is generally reduced under high soil P, and in general the mycorrhizal effects on plant growth occur significantly in low P soils. Although green onion was also easily colonized, the available soil P (21.4 mg kg−1) in the present study might be high for this plant and the mycorrhizal effects were thus decreased. In contrast, carrot is one of the plants with high mycorrhizal dependency, and high soil P (100 mg kg−1) is considered as low for this crop Obviously, the effects of pesticides on AM fungi vary with their toxicity and application rates, the tolerance of AM fungi, host plants, and environmental factors. AM fungi react differently to pesticides The mechanisms behind the decreased phoxim concentrations by AM inoculation may be diverse. One possible reason is the dilution effect caused by an enhanced plant growth due to an improved mineral nutrition especially P uptake, which may partly explain the lower phoxim concentrations in mycorrhizal plants. However, the dilution effect was unlikely to be the only explanation, because mycorrhizal roots had higher biomass and larger surface area, and if they have the same or higher capacity for phoxim uptake, the phoxim concentrations in their tissues should not be lower than in the nonmycorrhizal controls. In our results, phoxim concentrations in roots and shoots were all much lower in the mycorrhizal than in the nonmycorrhizal treatments . Additioin vitro carrot mycorrhiza system free from contaminating microorganisms, Koide and Kabir G. intraradices can hydrolyse organic P, and that the resultant inorganic P can be taken up and transported to host roots. On the other hand, phoxim can be degraded and utilized by bacteria Paecilomyces lilaciunsAM fungi are believed to have no direct catabolism or cooxidation on pollutants such as PAH, because they have very limited saprophytic capacities 14C, 15N or 32P-labelled phoxim should be carried out to study the metabolic pathway and the fate of this pesticide in AM fungal structures including mycelia and spores. Analyses of AM fungal gene sequences coding for pesticide-degrading enzymes may provide direct evidences on whether AM fungi can degrade this pesticide. In addition, it would be interesting to investigate if sulfate (S) could also be released from the phoxim since S plays a role in the pungency of green onion.Phoxim is one of plant-metabolized organophosphorus insecticides Our results have provided the evidence that AM fungi simultaneously increase vegetable yields and decrease the concentrations of organophosphorus pesticides in both plants and soils, but the effects vary with AM fungal species, host plants and pesticide application rate. Our results indicate a promising potential of AM fungi for reducing the accumulation of pesticide in vegetable crops and for the phytoremediation of pesticide-contaminated soils, although such effects are needed to be further studied. It is interesting to note that AM fungi can also enhance the tolerance of host plants to pathogens


Osteoprotegerin (OPG), a key regulatory factor in bone metabolism, was documented also a potential pro-angiogenic factor, which acts an important role in protecting vascular endothelial cells. Since preeclampsia has gradually been employed to be vascular diseases, we speculated that OPG might be associated with preeclampsia. The study was to evaluate the level of OPG protein and mRNA in placenta, and investigate the relationship between OPG and the pathogenesis of preeclampsia.Placental specimens from 30 term normal pregnancy, 30 severe preeclampsia and 30 mild cases were studied. The expression and levels of OPGs’ protein and mRNA were detected by immunohistochemisty, western blot analysis and real-time quantitative PCR analysis respectively. The expression of OPG protein was found in cytoplasm of placenta cytotrophoblasts and syncytiotrophoblasts in three groups. There were no significant differences of OPG protein between the maternal and fetal side in each group. The OPG protein and mRNA levels in severe preeclampsia were significantly higher than those in mild cases and normal pregnancy. However, there were no markedly differences of the OPG protein and mRNA levels between term delivery and preterm delivery in severe cases. In preeclampsia, the OPG protein and mRNA level was positively correlated with systolic blood pressure and 24 h urinary protein respectively.OPG protein and mRNA level in placentas of preeclampsia were found abnormal compared with normal pregnancy. In preeclampsia, the OPG protein and mRNA levels were closely related with its important clinical parameters. Taken together, OPG might be closely correlated with the pathogenesis of preeclampsia. Preeclampsia is a specific disorder known to promote maternal or perinatal mortality and morbidity during pregnancy Hence, in attempting to validate the effects of OPG on vascular to provide a solid foundation for future preeclampsia studies, here, we evaluated the expression of OPG in placenta for its putative properties.All the samples were obtained from the Department of Obstetrics & Gynecology, West China Second University Hospital, Sichuan University, during the period from November 2008 to July 2009. Preeclampsia was defined as blood pressure >140/90 mmHg on 2 separate occasions 6 hours apart or a single recording of a diastolic pressure of ≧110 mmHg, in association with proteinuria ≧1+ on dipstick testing or proteinuria ≧300 mg per 24 hours after 20 weeks’ gestation The study was approved by the Institutional Ethics Committee of West China Second University Hospital, and all patients were provided with written informed consent. All the patients delivered undergoing elective cesarean section. The indications for cesarean section included previous cesarean section, breech presentation and social indications. Exclusion criteria included multiple pregnancy, diabetes, chronic nephritis, chronic hypertension, heart diseases and fetal malformation.Information on demographic characteristics of all the participants was recorded. Gestational age was based on the last menstrual period and/or was confirmed by ultrasound examination conducted in the first trimester.Placental tissues were collected as described previously Placental tissue samples from the maternal side and fetal side were first incubated with the mouse anti-human OPG monoclonal antibody  overnight at 4°C prior to a 20 minutes 5% hydrogen peroxide treatment. Samples were subsequently incubated with Envision/HRP-conjugated goat anti-mouse for 30 min at 37°C followed by a brief 5 minutes of 3,3′-diaminobenzidine incubation. Treated sections were routinely washed by 0.1 M phosphate buffer (pH 7.4) with 5 minutes interval. Finally, sections were washed in distilled water. Counterstaining was performed using hemotoxylin.Adipose tissue from the subcutaneous tissues of the rat was used as a positive control. Negative control was performed by replacing the mouse anti-human OPG monoclonal antibody with phosphate buffer saline (PBS) in the same condition. The Brown-yellowish cytoplasm indicated the positive result under the optical microscope.20–50 µg of total protein was separated by 10%-15% gel gradient SDS-PAGE under reducing conditions For Western blotting protein analysis. Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with blocking buffer (PBS containing 5% non fat milk) for 2 h at room temperature. Primary antibody was mouse anti-osteoprotegerin . Membranes were incubated overnight at 4°C. The membrane was washed twice with TBST for 10 min and incubated with secondary antibodies  for 2 h at room temperature. After washing, osteoprotegerin was detected using a chemiluminescence reaction. The results were analyzed with Quantity One  software. Protein levels were normalized to GAPDH protein.Fifty milligrams of the placental maternal side tissues from each sample were processed. Total RNA was extracted according to manufacture’s instructions  and was described previously 2, 1 µl of each primer, and 1 µl of each probe . The PCR cycling conditions included an initial denaturation at 95°C for 2 minutes, followed by 40 cycles at 95°C for 15 seconds, 64°C for 15 seconds, and 68°C for 20 seconds.Real-time PCR analyses were performed in a fluorescent temperature cycler ) according to the manufacturer’s instructions. Two micrograms of total placental RNA were used for each reaction. A 20-µ l amplification mixture contained 3.0 µl RT reaction products, 25 mM MgCl5′-AGGAAATGCAACACACGACA-3′, reverse primer was 5′-TACTTTGGTGCCAGGCAAAT-3′. For β-actin, forward primer was 5′-TGACGTGGACATCCGCAAAG-3′, reverse primer was 5′-CTGGAAGGTGGACAGCGAGG-3′ . The cycle threshold value was obtained using the software provided by the manufacturer. Relative values of PCR products was normalized with respect to the β-actin level in each sample using the formula (2-ΔΔCt) The oligonucleotide-specific primers for OPG (167 bp) and β-actin (205 bp) were fellows. For OPG, forward primer was P-value was less than 0.05. All statistical tests were two-sided.SPSS16.0 software was used to process data. All data were expressed as the mean ± SD. Differences between groups were evaluated using one-way ANOV, followed by Mann-Whitney U test and paired or unpaired–t test and Wilcoxon signed-rank test. Non normal distribution data was analyzed with Jonckheere-Terpstra test and median test. The associations between OPG and continuous variables were analyzed by Pearson or Spearman’s correlation coefficients. Results were considered statistically significant if the Gestational week of delivery, birth weight and lengths of infants in the severe preeclamptic group were less than those of the normal group and the mild preeclamptic group . There were no significant difference in maternal age and pre-pregnancy body mass index among those three groups .On both the maternal side and the fetal side of placenta, strong expression of OPG protein was observed in severe preeclampsia group (SPE). In Mild preeclampsia group (MPE) a significant expression of OPG protein was also observed. While the expression of OPG protein remained low in normal control group. There is a significant difference between the severe preeclampsia group and the normal control group (P<0.05) .Expressions of OPG protein in severe preeclamptic group (SPE), mild preeclamptic (MPE) and normal control (CTRL) group were shown in  by WestP<0.05) .OPG-mRNA of all placentas was detected in all groups. The level of OPG-mRNA in the severe preeclamptic group was significantly higher than those in the mild group and the normal group . There wThe expression of OPG in preeclamptic group was positively correlated with either systolic blood pressure before delivery or 24-hour urine protein, while negatively correlated with plasma albumin. In addition, the level of OPG-mRNA in preeclamptic group was positively correlated with systolic, diastolic blood pressure before delivery and 24-hour urine protein, while negatively correlated with plasma albumin, gestational weeks, and neonatal birth weight or birth length. However, only OPG-mRNA level was negatively correlated with plasma albumin in normal pregnancy group ,3.OPG was firstly discovered by Simonet in 1997 It is certain that the OPG distributed in all of the organs and tissues, especially in the placentas and gestational tissues As we know, preeclampsia is a specific vascular disease, the endothelial dysfunction maybe a crucial factor in the pathogenesis of preeclampsia and OPG is considered to protect vascular endothelial cells. Therefore, we speculated that OPG might be involved in the pathogenesis of preeclampsia. However, whether OPG is expressed in placenta with preeclampsia is still elusive and the mechanism of OPG in the pathogenesis of preeclampsia is unknown.In our study, we demonstrated that OPG was expressed in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts in both normal term and preeclamptic placental tissues. Our results were, in part, consist with Simonet and Phillips’ studies We further demonstrated OPG protein expressed in the maternal side and fetal side in placental tissues. However, no significant difference of OPG protein level between the maternal side and fetal side of the placenta in each group was observed. This result suggested that both the maternal side and fetal side of OPG might have the same special functions, although all of these cells are of fetal origin.In addition, we assessed OPG protein and mRNA levels in placenta and its correlation with the severity of preeclampsia. Our results showed that the changes of protein and mRNA of OPG in each group were very consistency, both OPG protein and mRNA levels were significantly higher in severe cases of preeclampsia than those mild cases and normal pregnancy. Moreover, OPG protein and mRNA levels were much higher in mild preeclampsia than those in normal pregnancy. These indicated that the changes of protein and mRNA levels of OPG were more obvious in severe preeclampsia, suggesting the expression of OPG were consistent with the severity of preeclampsia.Moreover, our study discovered that, in preeclamptic group, both the OPG protein and mRNA was positively correlated with systolic blood pressure and 24 h urinary protein, while negatively correlated with plasma albumin level; furthermore, OPG-mRNA levels was also positively correlated with diastolic blood pressure, and negatively correlated with gestational weeks, neonatal birth weight and birth length. However, in normal pregnancy, only OPG-mRNA level was negatively correlated with plasma albumin. These results suggested that OPG protein and mRNA levels in preeclampsia were closely associated with the important clinical parameters of preeclampsia, and the association was stronger between the OPG-mRNA and the clinical parameters of the cases of preeclampsia.In summary, OPG protein and mRNA expression in placentas of preeclampsia were obviously increased compared with that of the normal pregnancy. Gestational weeks did not affect the level of OPG protein and mRNA. In preeclamptic group, the levels of OPG protein and mRNA were closely related with its important clinical parameters. The abnormal OPG might be involved in the pathogenesis of preeclampsia.