The degradation of the steppe of Inner Mongolia, due to overgrazing, has resulted in ecosystem damage as well as extensive reductions in sheep production. The growth performance of sheep is greatly reduced because of overgrazing, which triggers massive economic losses every year. The liver is an essential organ that has very important roles in multiple functions, such as nutrient metabolism, immunity and others, which are closely related to animal growth. However, to our knowledge, no detailed studies have evaluated hepatic metabolism adaption in sheep due to overgrazing. The molecular mechanisms that underlie these effects remain unclear.In the present study, our group applied isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis to investigate changes in the protein profiles of sheep hepatic tissues when nutrition was reduced due to overgrazing (12.0 sheep/ha), with the goal of characterizing the molecular mechanisms of hepatic metabolism adaption in sheep in an overgrazing condition.The body weight daily gain of sheep was greatly decreased due to overgrazing. Overall, 41 proteins were found to be differentially abundant in the hepatic tissue between a light grazing group and an overgrazing group. Most of the differentially expressed proteins identified are involved in protein metabolism, transcriptional and translational regulation, and immune response. In particular, the altered abundance of kynureninase (KYNU) and HAL (histidine ammonia-lyase) involved in protein metabolic function, integrated with the changes of serum levels of blood urea nitrogen (BUN) and glucose (GLU), suggest that overgrazing triggers a shift in energy resources from carbohydrates to proteins, causing poorer nitrogen utilization efficiency. Altogether, these results suggest that the reductions in animal growth induced by overgrazing are associated with liver proteomic changes, especially the proteins involved in nitrogen compounds metabolism and immunity.This provides new information that can be used for nutritional supplementation to improve the growth performance of sheep in an overgrazing condition.The online version of this article (doi:10.1186/s12953-016-0111-z) contains supplementary material, which is available to authorized users. The Inner Mongolian steppe is the most important region for mutton and milk production in China . HoweverThe liver plays a central role in the regulation of the metabolism of carbohydrates, proteins, lipids and other nutrients in animals. Additionally, the liver has multiple other physiological functions in the body including immune response, regulating inflammation and the removal of xenobiotics , 12. A pStudies have shown lack of correlation between mRNA and protein expression abundance due to RNA editing and posttranslational modifications , 16. ThuThus, we hypothesized that overgrazing can confer negative effects on the hepatic protein expression. Therefore, the objective of this study was to identify and characterize candidate proteins that were differentially induced in the livers of sheep from an overgrazed pasture during the grazing season using a label-based iTRAQ procedure (isobaric tags for relative and absolute quantitation) followed by LC-MS/MS.P = 0.003; P = 0.049). However, the gross energy and nitrogen free extract (NFE) contents were significantly decreased in the OG group than the LG group . There was no significant effect of overgrazing on neutral detergent fibre (NDF) or acid detergent fibre (ADF). However, NDF tended to decrease in the OG group (P = 0.096). A full description of the effects of overgrazing on herbage and animal growth performance are given in Additional file The herbage crude protein (CP) and acid detergent lignin (ADL) contents in the overgrazing (OG) group were 34.4 and 19.6% greater, respectively, than those in the light grazing (LG) group over the grazing period (P = 0.042) and 14.0% reduction in carcass weight (2.8 kg) (P = 0.047)   at the beginning of the grazing experiment. Throughout the experimental grazing period , the OG sheep had a 21.4% reduction in daily weight gain (27 g) . Furthermore, the serum concentration of blood urea nitrogen (BUN) was significantly higher in the OG group than in the LG group (P = 0.046). In contrast, the levels of total protein (TP), glucose (GLU), non-esterified fatty acid (NEFA) and insulin-like growth factor 1 (IGF-1) in the serum were significantly decreased in response to overgrazing compared with light grazing . However, the levels of triglyceride (TG) and cholesterol (CHOL) were similar between the two groups (P > 0.05).The effects of overgrazing on the biochemical parameters of serum in sheep are shown in Table Using iTRAQ analysis, a total of 27,287 peptide spectral matches were found, and 2,153 proteins were identified within the FDR  of 1% , transcriptional and translational regulation (14.6%), immune response, apoptosis and inflammation (14.6%), energy metabolism (12.2%), miscellaneous (12.2%), lipid metabolism (9.8%), stress response and detoxification (9.8%), cell cytoskeleton 7.3%) and cell growth and proliferation (4.9%)  in the identified pathways, which indicates that overgrazing had affected glucose and energy metabolism in the sheep.Furthermore, GO annotation and a KEGG pathway enrichment analysis were performed to determine the overrepresented biological events and to provide a primary overview of the hepatic proteome impacted by overgrazing. The DAVID 6.7 software identified highly overrepresented GO classes including cellular components, molecular functions and biological processes  were selected for validation of proteomic data at the protein level using parallel reaction monitoring (PRM). The results of PRM analysis indicated that candidate proteins show similar trends as the iTRAQ results, which implied the credibility of the proteomics data Fig. .Fig. 3ExP = 0.004; P = 0.007; P = 0.001). While, the levels of IgA, IgG and interferon-γ (IFN-γ) were significantly lower in the OG group than in the LG group .As shown in the list of differentially expressed protein species involved, those related to immune response and inflammation were ranked at one of the top putative protein functions. To further evaluate the effect of overgrazing on the immune response and inflammatory parameters of sheep, we measured the levels of primary immunoglobulins (Ig) and inflammatory cytokines in serum Table . The levThe liver is a vital organ that plays important roles in multiple physiological functions such as nutrient metabolism, detoxification, immune response and others. Any change in dietary components can be closely related to animal growth, which is reflected in variations of hepatic protein expressions in most cases. Whether overgrazing has a direct effect on the functional hepatocytes (thereby causing synthetic and metabolic changes in the sheep’s liver) remains unclear. Substantially less is understood regarding the molecular mechanism of hepatic response in sheep with reduced nutrition due to overgrazing. In the present study, an iTRAQ-based quantitative proteomic analysis integrated with biochemical and immune analyses was applied to investigate the hepatic response in overgrazed sheep. When integrated with the proteomic profiles, biochemical and immune detections, these data suggest that overgrazing triggers a shift in energy resources from carbohydrates to proteins, which results in the impairment of nutrient metabolism (protein and lipid) and immunity, which may be the reasons for reduced growth in sheep.Serum levels of ALT and AST serve as important indicators of hepatic health status and were found to be greatly elevated in OG sheep, indicating that overgrazing had a severely negative effect on hepatic function . Additioin vivo can trigger limited protein deposition and the elevation of urinary N excretion . The crude tissue extracts were centrifuged to remove the remaining debris. The tissue lysates were reduced for 1 h at 37 °C in a water bath through the addition of 10 mM DTT and then alkylated with for 1 h with the addition of 55 mM iodoacetamide in the dark. Afterwards, the lysates were precipitated by adding 4 volumes of pre-chilled acetone. The pellets were then washed three times with pre-chilled pure acetone and resuspended in the buffer (50% TEAB and 0.1% SDS). The centrifugation was repeated to remove the undissolved pellets. Subsequently, protein quantitation was performed using a Bio-Rad Bradford Protein Assay Kit .Each sample was digested with modified sequence grade trypsin  at a 1:30 ratio (3.3 μg trypsin: 100 μg target) overnight at 37 °C. Each isobaric tag   was solubilized in 70 μL isopropanol and then added to its respective sample . Incubation continued for 2 h at room temperature. All labelled peptides were pooled together and separated using SCX chromatography. The eluted peptides were dried under a vacuum and analyzed via LC-MS/MS based on Q-Exactive (Thermo Scientific). All detailed procedures are described in Additional file P-values < 0.05) were considered significant.Peptide and protein identifications were analyzed using Proteome Discover 1.4 (Thermo Fisher Scientific) and searched with the Mascot search engine  against the database Uniprot_Ovis aries _27110_20151123.fasta  with the following parameters: enzyme: trypsin; variable modifications: oxidation (M), gln-pyro-glu (N-term Q), and iTRAQ8plex (Y); fixed modification: carbamidomethyl (C), iTRAQ8plex (N-term), and iTRAQ8plex (K); peptide mass tolerance: ± 20 ppm; fragment mass tolerance: 0.1 Da; maximum missed cleavages: 2. Identified peptides had an ion score above the threshold of peptide identity in Mascot, and protein identifications were accepted as the false discovery rate (FDR) ≤ 0.01 in which at least one such unique peptide match was specific for the protein. Median ratio normalization was performed to obtain the quantitative protein ratios. Proteins with a 1.2-fold change or greater (http://www.blast2go.com/). The Database for Annotation, Visualization and Integrated Discovery (DAVID) 6.7 (http://david.abcc.ncifcrf.gov/) and the Kyoto encyclopedia of genes and genomes (KEGG) data base (http://www.genome.jp/kegg/), were used to classify differentially altered proteins in significantly overrepresented pathways and GO terms.The Gene Ontology (GO) distribution for all proteins that were differentially expressed in the hepatic tissue of overgrazed sheep was classified using Blast2GO software . All results are expressed as the means ± SD. A group difference was analyzed using Student’s


Dendrobium nobile Lindl. increased after infection with mycorrhizal fungus MF23 (Mycena sp.). Large-scale transcriptome sequencing of symbiotic and asymbiotic D. nobile revealed that 30 unigenes encoding proteins were possibly related to the biosynthesis of dendrobine sesquiterpene backbone. A qRT-PCR experiment of 16 unigenes, selected randomly, proved that there were significant changes in the expression levels of AACT, MVD, PMK and TPS21 at 9 weeks after inoculation. These results implied that MF23 might stimulate dendrobine biosynthesis by regulating the expressions of genes involved in the mevalonate (MVA) pathway. The biogenetic pathway of dendrobine was suggested systematically according to the structural features of dendrobine alkaloids and their sesquiterpene precursors, which implied that post-modification enzymes might play a major role in dendrobine biosynthesis. Thus, genes encoding post-modification enzymes, including cytochrome P450, aminotransferase and methyltransferase, were screened for their possible involvement in dendrobine biosynthesis. This study provides a good example of endophytes promoting the formation of bioactive compounds in their host and paves the way for further investigation of the dendrobine biosynthetic pathway.Content determination and microscopic observation proved that dendrobine accumulation in the stem of  Dendrobium nobile Lindl. (Orchidaceae) has been used in Traditional Chinese Medicine (TCM) and as an herbal medicine in many Asian countries for hundreds of years with special pharmacological effects on gastritis, diabetes, cancer and ageing4. It has been officially listed as “Shi Hu” in different versions of the Pharmacopoeia of the People’s Republic of China. However, due to the destruction of wild environments and the increasing market demand, wild D. nobile  is being increasingly depleted.The stem of D. nobile include alkaloids, polysaccharides, and polyphenols7. Among these compounds, sesquiterpene alkaloid dendrobine8 has been regarded as the quality standard of D. nobile. Modern pharmacology has demonstrated that this group of alkaloids has remarkable anti-hypertensive, anti-cancer, analgesic, and antipyretic effects in clinical studies9. Dendrobine is primarily obtained by extraction11 or chemical synthesis13; however, neither method is efficient enough because of the low content in D. nobile14 and technical problems in total synthesis. The high market demand has led to excessive harvesting and exploitation of D. nobile, which has contributed to resource depletion. Considering the advantage of biotechnology and successful examples applied in D. nobile tissue culture, their use for increasing the dendrobine content of D. nobile in large quantities in vitro is becoming possible to not only meet market demand but also protect the wild resource of D. nobile.The major bioactive components of 16. The most direct evidence indicated that mycorrhizal orchids could acquire more P, N and water than those of non-mycorrhizal controls. Thus far, numerous mycorrhizal fungi with growth-promoting effects have been isolated from the roots of wild Dendrobium officinale Kimura et Migo and D. nobile in our laboratory19. Some fungi have been used in actual production to enhance the growth of D. officinale and D. nobile. Our previous reports showed that when D. nobile was inoculated with MF23 (Mycena sp.), a mycorrhizal fungus isolated from the roots of D. officinale, its total alkaloid content significantly increased (18.3%)21, indicating that MF23 might be able to promote dendrobine biosynthesis. However, the relationship between mycorrhizal fungi and their hosts is still not clear, which hinders the application of mycorrhizal fungi. Thus, understanding the regulatory mechanism of MF23 and improving the dendrobine content of D. nobile are of great importance.Mycorrhizal fungi play a critical role in the lifetime of orchids; research has suggested that these fungi can provide inorganic and organic nutrients for seed germination and early seedling developmentD. offcinale24.The development of molecular biology has prompted the use of RNA-seq, a powerful tool in novel gene discovery and prediction. The advances in next-generation sequencing technologies, such as 454 pyrosequencing, have made sequencing cheaper and faster, and obtaining large datasets has become a viable option. Thus far, RNA-seq has been successfully applied to study the regulation of gene expression for many plants, including D. nobile (model group) using RNA-seq technology based on an Illumina HiSeq 4000 platform. qRT-PCR experiments involving key genes, including AACT, MVD, PMK and TPS21, related to the sesquiterpene skeleton formation of dendrobine were performed. These experiments provided an overview of the D. nobile stem transcriptome and candidate genes encoding enzymes involved in the dendrobine biosynthetic pathway. The biogenetic pathway of dendrobine was systematically postulated according to the structural features of dendrobine alkaloid analogues and sesquiterpene precursors. qRT-PCR evaluations of genes encoding cytochrome P450, aminotransferase and methyltransferase suggested that post-modification enzymes might play vital roles in the biogenetic pathway of dendrobine alkaloid. The results in this report provide a good example of endophytes promoting the formation of bioactive compounds in their host and pave the way for further investigations of the dendrobine biosynthetic pathway.In this study, we collected and analysed the transcriptome data for the stems of asymbiotic (control group) and symbiotic D. nobile turned yellow at the same time, suggesting that D. nobile was stressed by MF23 in some way.A comparison of the differences in morphological indexes between the model and control groups  analysis was used to evaluate the dendrobine contents in D. nobile roots revealed that the hyphae of MF23 penetrated the epidermal cells of the host roots and spread from cell to cell. A mass of hyphae also gathered outside the passage cells of the exodermis  raw reads were generated , an international standardized gene functional classification system, 47,372 non-redundant unigenes were classified into three major functional ontologies   database for functional prediction and classification. We subdivided 27,087 non-redundant unigenes into 26 KOG classifications Fig. . Among tUnigene metabolic pathway analysis was also conducted using the Kyoto Encyclopaedia of Genes and Genomes (KEGG) annotation system. According to KEGG, 18,911 unigenes were assigned to 131 pathways Fig. . The patDifferential expression analysis was first performed between the two treatments. DEGs | > 1) were defined as genes that were significantly enriched or depleted in one treatment relative to the other one. In this study, 1,388 DEGs were observed between DF1_S and DC1_S . Moreover, between DF9_S and DC9_S, 2,646 genes were expressed at significantly different levels  Fig. .Figure 7vs. DC1_S and DF9_S vs. DC9_S, respectively , ‘cell part’ (GO:0044464), ‘metabolic process’ (GO:0008152), ‘biosynthetic process’ (GO:0009058), ‘organonitrogen compound metabolic process’ (GO:1901564) and ‘cellular nitrogen compound biosynthetic process’ (GO:0044271). These findings were consistent with the results from the morphology (a 57.69% increase in stem diameter) and metabolome analyses (a threefold increase in dendrobine content). However, for DF9_S vs. DC9_S, the numbers of unigenes associated with DNA metabolic and substance binding were quite high, very different from those of DF1_S vs. DC1_S, implying that MF23 influenced D. nobile in different biological processes at different stages.Among all DEGs, there were 175 and 5 enriched GO terms related to various biological processes for the DF1_S ely Fig. . For DF1vs. DC1_S and DF9_S vs. DC9_S revealed 113 and 125 pathways, respectively. The top 20 pathways of DF1_S vs. DC1_S were mainly involved in amino acid metabolism and respiratory metabolism, indicating that MF23 sped up the metabolism of D. nobile at an early stage. In contrast, in DF9_S vs. DC9_S, the pathway of ‘purine metabolism’ was involved in the largest number of unique transcripts, followed by ones related to ‘phenylpropanoid biosynthesis’. Moreover, unigenes associated with plant-pathogen interactions and biosynthesis of defensive substances such as flavonoids, isoflavones and alkaloids were relatively high in number, suggesting that MF23 might activate the defensive mechanism of D. nobile  pathways. Thus, the unigenes encoding the key enzymes in these two pathways (MVA and MEP) were identified by searching the names of the annotated genes. As a result, 16 types key enzyme genes , was significantly up-regulated (2.12-fold) at 6 weeks and declined sharply (4.99-fold) at 9 weeks, in opposition to dendrobine biosynthesis. Thus, dendrobine biosynthesis was negatively correlated with TPS21 expression. AACT was the first key enzyme-coding gene in the biosynthesis of sesquiterpenes; the marked stimulation (3.14-fold) of AACT at 9 weeks suggested that it might take an active role in the regulation of dendrobine biosynthesis.In this study, 16 key enzyme-coding genes associated with the formation of the backbone of the sesquiterpene alkaloid dendrobine were chosen for qRT-PCR analysis. The expression levels of both way Fig. , were upBy comparing the expression levels of key enzyme-coding genes involved in the MEP pathway between the model group and the control, we found that there were no significant correlations between their expression levels and dendrobine biosynthesis , 13 (aminotransferase) and 49 (methyltransferase) unigenes. Most of them were expressed at high levels, and the ones with top enrichment are listed in Table CYP1D10, METTL23, ATX4 and BCAT2 exhibited higher expression levels at 9 weeks after inoculation than any other stages, implying that these genes might have positive effects on promoting dendrobine biosynthesis. Moreover, the expression of ATX4, which encoded N-methyltransferase was at a low level at 6 weeks (low content of dendrobine) and was activated at 9 weeks (high content of dendrobine). It was postulated that there was a positive relationship between ATX4 expression and dendrobine biosynthesis, which might play an important role in the regulation process of dendrobine biosynthesis influenced by MF23.The expression levels of 9 genes encoding post-modification enzymes were assessed by qRT-PCR Fig. . The exp28. Later research revealed that mycorrhizal fungi could produce some types of phytohormones supplied to their hosts29. Recently, with the development of molecular technology, findings of mycorrhizal fungi promoting Dendrobium growth or metabolism by regulating gene expression have often been reported32. Nevertheless, there is little knowledge of the exact mechanism of how mycorrhizal fungi influence their host Dendrobium. Driven by the positive impact of mycorrhizal fungi on Dendrobium growth and metabolism, we performed morphological, physiological and molecular investigations to obtain substantial knowledge of the interrelationship between mycorrhizal fungus and its host. This report detailed the first possible mechanism for the development of dendrobine content induced by MF23, helping us to understand the relationship between the mycorrhizal fungus and its host.Early in the 1960s, it was demonstrated that mycorrhizal fungi could offer nutrients, such as glucose, directly to the host33, is in some ways considered a nutritional pool and nutritional transformation centre to deliver nutrients from the host and environment to those plant cas34. In this study, morphological observation of the model and the control groups revealed that at 9 weeks, pelotons were formed together with an increase in the dendrobine content. Since dendrobine biosynthesis requires a great deal of energy and nutrition36, these results implied that MF23 might promote dendrobine biosynthesis by forming peloton to supply nutrition to its host plant, D. nobile.Peloton, as a sign of the establishment of typical orchid mycorrhizaeCatharanthus roseus37. Under drought stress, the alkaloid content in the roots of motherwort was increased to a maximum of 1.7 times higher than the control group, with a significant difference (P < 0.01)38. When infected by Ceratocystis fimbriata, several alkaloids in mango increased at different degrees of resistance to infection39. Similarly, increased levels of MF23, as a foreign invader for D. nobile, might elicit plant defence, which is likely to include the alkaloid (dendrobine) biosynthetic pathway.Alkaloids are a type of secondary metabolite of plants that play significant roles in defensive responses to environmental stresses. For instance, binary stress induced an increase in indole alkaloid biosynthesis in D. nobile was formed at the early stage of symbiotic culture, even before the hyphae encounter the root. These results implied that there might exist some fungal elicitor41 or physical contact between MF23 and its host to effect the physiological changes in D. nobile to promote dendrobine biosynthesis.Transcriptome analysis of model and control groups from 1 week revealed that 1388 DEGs were involved in many biological processes, cellular components and molecular functions. Moreover, the effect of MF23 on HMGS and HMGR have been recognized as fairly important key enzymes in the MVA pathway43. Many studies provided evidence of a positive correlation between sesquiterpene yield and the expression of these two genes45. However, unexpectedly, this report does not indicate a specific relationship between these two genes and the dendrobine content, as shown by RNA-seq and qRT-PCR. These results suggested that the genes might not take an active role in the process of dendrobine biosynthesis infected by MF23.46. PMK and MVD are crucial ATP-dependent enzymes in the MVA pathway that directly affect IPP biosynthesis, the building block of sesquiterpene skeletons47. Three genes, AACT, PMK and MVD, encoding the corresponding enzymes were highly expressed with increased in dendrobine content. Previous reports confirmed that product accumulation of downstream reactions in MVA pathway was enhanced in AACT over-expressing transgenic plants48. Therefore, AACT might take a similar role in the dendrobine biosynthesis induced by MF23. In addition, the increases in PMK and MVD expression levels in the model group improved the dendrobine content, which might be due to increased IPP biosynthesis. In recent years, homologous genes of PMK and MVD have been cloned from Amomum villlosum, Ginkgo biloba, and Eleutherococcus senticosus51. A report proved that PMK was highly expressed in the roots of Aconitum heterophyllum Wall where the aconite terpenoid alkaloids were synthesized and accumulated52. Our results were similar to those in the reports mentioned above, but our work investigated the PMK expression level in D. nobile for the first time and was the first report on the effect of the MVD gene on regulating dendrobine biosynthesis, which provided a foundation for further research of PMK and MVD.As the first enzyme of the MVA pathway, AACT condenses two molecules of acetyl-CoA to form acetoacetyl-CoA in terpenoid backbone biosynthesis53. Thus, these enzymes are widely regarded as the rate-determining regulatory steps in MVA pathways. In this study, the expression level of TPS21, a gene encoding sesquiterpene synthase, was negatively correlated with dendrobine biosynthesis. According to the structural features of its catalysate humulene  medium, which was supplemented with potato juice , 3% (w/v) sucrose, and 0.6% (w/v) agar, pH 5.8. Mycelial plugs from 20-day-old MF23 (Mycena sp.) grown on potato dextrose agar medium were placed in the centre of culture bottle, by the side of the roots of D. nobile. Then, the culture bottles were kept in a conventional greenhouse with a 10-h light/14-h dark photoperiod at (24 ± 1) °C and an illumination intensity of 1500 Lx. Un-inoculated plants were maintained as a control.Tissue culture seedlings of Plant samples were collected at different stages: 1, 3, 6 and 9 weeks post-inoculation (model group) and at the same stages for un-inoculation (control group). All samples were divided into two portions; some samples were used for morphological and chemical research, and the rest were frozen using liquid nitrogen and stored until RNA extraction.D. nobile as a repetition, we maintained a total of 12 repetitions. At each point of time, the maximum height and stem diameter of D. nobile were measured. The fresh roots, stems, and leaves from each treatment were weighed and parched at 55 °C. The dry weight was then measured.Using one culture bottle of Stems from each treatment were parched at 55 °C, thoroughly blended and ground to a powder. Standard substances of dendrobine and the internal standard naphthalene were obtained from Sinopharm Chemical Reagent Co, Ltd and Beijing Bei Na Chuang Lian Biotechnology Institute, respectively. The sample preparation for the GC analysis were performed as per the pharmacopoeia of China (2010). Chromatography was performed on an Agilent 6890 GC-FID using an Agilent DB-1 capillary column (0.25 μm × 0.25 mm × 30 m) and nitrogen as a carrier gas.−1. The relative standard deviations (RSDs) of precision, repeatability, stability and recovery rate using this method were 2.1% (n = 5), 2.2% (n = 5), 3.8% (n = 6) and 1.96% (n = 6) . Components of the total ion chromatogram were extracted by the flame ionization detection. Relative correction factors between naphthalene and dendrobine was acquired (f = 1.184849). The linear regression equation y = 0.3135x − 0.022 (r = 0.9994) proved that the dendrobine concentration was linear, with a peak area in the range of 1.1~11 mg · L63. Samples were dehydrated in a graded ethanol series, embedded in paraffin, stained with safranine and fast green, sealed with Gel Damar and then observed and photographed on a light microscope equipped with a camera (ZEISS Axio Imager A1).Fresh root segments were fixed in formalin–acetic acid–alcohol (FAA), an approach described by Feder and O’BrienD. nobile using an RNeasy Plant Mini Kit (cat. nos 74903 and 74904)  following the manufacturer’s instructions. Total RNA degradation and contamination were verified by electrophoresis in a 1.0% agarose gel in 0.5 × TBE . The concentration of total RNA was checked using a Qubit RNA Assay Kit in a Qubit 2.0 Fluorometer . Furthermore, the RNA integrity was assessed using an RNA Nano 6000 Assay Kit with a Bioanalyzer 2100 system .Total RNA was extracted from 1- and 9-week-old stem samples of ® Ultra™ RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was performed using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (NaseH−). Subsequently, the second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. To select cDNA fragments of preferential 150~200 bp in length, the library fragments were purified with the AMPure XP system . Then, 3 μl of USER Enzyme  was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.A total amount of 1.5 μg RNA per sample was used as the input material for RNA sample preparations. Finally, 8 samples with RNA integrity number (RIN) values above 8 were used for library construction. Sequencing libraries were generated using the NEBNext64 with min_kmer_cov set to 2 by default and all other parameters set to defaults. Gene function was annotated based on the following seven databases: Nr (NCBInon-redundant proteinsequences), Nt (NCBI non-redundant nucleotide sequences), Pfam (Protein family), KOG/COG (Clusters of Orthologous Groups of proteins), Swiss-Prot , KO (KEGG Ortholog database) and GO (Gene Ontology), using BLAST with a cutoff E-value of 10−5.Raw data (raw reads) in fastq format were first processed using in-house Perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing poly-N and low-quality reads from raw data. At the same time, the Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. All of the downstream analyses were based on clean data with high quality. The left files (read1 files) from all libraries/samples were pooled into one large left.fq file, and right files (read 2 files) were also pooled into one large right.fq file. Transcriptome assembly was performed based on the left.fq and right.fq using TrinityGene expression levels were estimated using RSEM51 for each sample. Clean data were mapped back onto the assembled transcriptome, and the read count for each gene was then obtained from the mapping results. Differential expression analysis of the two groups was performed using the DESeq R package (1.10.1). DESeq52 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 identified by DESeq were assigned as differentially expressed.65, which can be adjusted for gene length bias in DEGs. KEGG67 is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially from large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). KOBAS68 software was used to test the statistical enrichment of DEGs in KEGG pathways.GO enrichment analysis of the DEGs was implemented with the GOseq R packages based on Wallenius non-central hyper-geometric distributionTM  on an ABI 7500 Real-Time PCR System . The actin gene of D. nobile was used as a reference control69. All primers designed for D. nobile are shown in Supplementary Table −ΔΔCt method70.DEGs identified using the above described methods were validated with qRT-PCR. The qRT-PCR was performed with the SYBR® Premix ExTaqSPSS 19.0  statistical software was used for the statistical evaluation of the results. All results are expressed as the means ± standard deviations (SD) of the number of experiments. An unpaired t-test for the values was performed at P < 0.05.Suppkementary material


Childhood-onset spondyloarthropathies usually start with enthesitis and peripheral arthritis. However, axial disease may develop afterward. Patients are most often classified, following revised (Edmonton 2011) ILAR criteria, as enthesitis-related arthritis, psoriatic arthritis, or unclassified juvenile idiopathic arthritis, particularly in cases of psoriasis in the patient or a first-degree relative. In adults, peripheral spondyloarthritis is classified by ASAS criteria.We retrospectively studied patients with childhood-onset spondyloarthropathies followed for more than one year in our referral centre. We did not exclude patients with a personal or familial history of psoriasis.p <  0.001). Axial disease and sacroiliitis were rare at disease onset. However, they appeared during follow-up in 63% and 47% of cases respectively, after a median disease duration of 2.6 (IC 95% [2.2–4.4]) and 5.3 years (IC 95% [4.1–7.7]), respectively. Multivariable analysis showed that familial history of spondyloarthritis was associated with the presence of sacroiliitis and active disease at the latest follow-up .We included 114 patients followed between January 2008 and December 2015 for a median of 2.5 years (IQR = 2.3). Sixty-nine per-cent of patients fulfilled the revised ILAR classification criteria for enthesitis-related arthritis, and 92% the ASAS criteria for peripheral spondyolarthritis  contains supplementary material, which is available to authorized users. Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory joint diseases of unknown aetiology, defined by the presence of chronic arthritis before the age of 16 years. It is the most common disease treated in paediatric rheumatology clinics with a prevalence of 16 to 150/100,000 children . The lasSpA is a heterogeneous group of inflammatory diseases, first identified in adults. It is characterised by a strong association with HLA-B27 . The disIn adults, the first set of criteria identified only late stage axial SpA, with defined X-ray sacroiliitis (New York criteria) . VariousWe performed a retrospective study of all JSpA patients followed in a paediatric rheumatology tertiary care centre at Necker Hospital in Paris to determine the initial presentation and outcome of patients, including response to treatment, and analyse the risk factors for axial disease or active disease at the last follow up. In addition, we analysed the proportion of patients diagnosed as ERA or SpA according to the revised Edmonton classification criteria and the latest ASAS criteria, respectively.In this retrospective study, we enrolled all patients evaluated at a French national reference centre for juvenile arthritis in Necker-Enfants Malades hospital and the paediatric rheumatology outpatient clinic of Saint-Vincent-de-Paul Hospital between January 2008 and December 2015. Patients were recruited through the “Centre de Référence des Maladies Rares (CEMARA)” database project . The recThe inclusion criteria were having ERA or JSpA, with disease onset at an age of less than 16 years, diagnosed by the attending physician, and confirmed at the latest follow-up visit. We did not exclude patients with a personal or familial history of psoriasis. Patients followed for less than one-year or primarily diagnosed with inflammatory bowel disease were excluded.For each patient, we collected information concerning gender, age at disease onset, age at first visit, personal and familial history of inflammatory and auto-immune disease and psoriasis, presence of the HLA-B27 allele, description of the clinical course, date of first axial involvement, and biological and radiological tests from the medical chart. Data collection was performed two times by one investigator, first from the computerized medical chart and then from the paper chart. We also analysed the response to various treatments and adverse events. We noted any history of uveitis, psoriasis, or IBD to highlight extra-articular manifestation. Imaging data were recorded and analysed as described below.We defined axial disease by the presence of at least one criterion among: 1) inflammatory low-back pain or inflammatory dorsal pain lasting more than one month; 2) limited spine mobility, defined by a Schober index < 10 + 4 cm; 3) sacroiliac pain at examination or alternating buttock pain; or 4) presence of axial disease by any available radiological examination. Active sacroiliitis was defined as sacroiliac pain at examination, alternating buttock pain or the presence of an active lesion by MRI. A joint was considered to be active if at least two criteria among inflammatory pain, limited mobility, and/or swelling were present. Enthesitis was defined as chronic inflammatory pain or tenderness at the site of principal tendon insertion: plantar fascia insertion at the metatarsal heads and calcaneum, Achilles tendon insertion at the calcaneum, quadriceps insertion at the patella and proximal tibia, pelvis , and greater trochanter.An experienced pediatric radiologist (SB) reviewed all available sacroiliac MRI, assessing the presence of acute lesions  and chronic lesions . Active sacroiliitis by MRI was defined by the presence of at least one acute lesion. Our expert radiologist did not systematically review other radiological examinations, such as CT-scans scan or standard radiography. Interrater reliability of MRI evaluations was measured with kappa coefficients. A result of k <  0.40 indicated poor agreement, 0.40–0.59 fair agreement, 0.60–0.74 moderate agreement, and 0.75–1.00 excellent agreement .Clinical response to treatment was evaluated based on the information recorded from the medical chart: reduction of arthritis and clinical symptoms for at least six months, when available, and the treating physician evaluation. Inactive disease was defined as the absence of arthritis and enthesitis during clinical examination. Clinical remission was defined as the absence of symptoms, noted by the treating rheumatologist, for at least six months, with or without treatment.We analysed the following variables; masculine gender, presence of HLA-B27, age of first symptoms under 12 years, hip arthritis, presence of enthesitis, and family history of SpA.p ≤ 0.05) with sacroiliac (SI) involvement in the univariate analysis.All statistical analyses were performed with R (version 3.2.2). Comparisons between patient groups were performed using the chi-squared test and Fisher’s exact test for qualitative variables, and Student’s t-test for quantitative variables. Survival analyses were performed using the Kaplan Meier method. Multivariable analysis, using a logistic model, was performed to determine variables, including disease severity indicators. The predictors used in the final model were those showing a significant correlation  and two patients (1.75%) in the PsA. According to the 2011 ASAS criteria, 83,patients (73%) could be classified with peripheral SpA at the first consultation, with no difference between genders  of our population were classified in the ERA group, according to the Edmonton classification criteria, with a significant difference between boys and girls  developed peripheral arthritis Table , predomi95 = [2.2–4.4], Fig. 95 = [4.1–7.7], Fig. 95 = [54.1–74.6]; Fig. 95 = [44.6–58.2]), respectively. Inflammatory back pain showed the same pattern .Axial disease was active at the latest follow-up in 72 patients 63%). Fifty-four patients (47%) developed sacroiliitis (Table %. Fifty-95 = [0.12–0.86]). Sacroiliitis was confirmed for five patients, based on acute lesions in all five and chronic lesions in two. Acute lesions consisted of synovitis and articular effusion in five cases and bone oedema in four. Chronic lesions consisted of erosion and sclerosis in two cases and degenerative fatty infiltration in one.Thirty-seven of 73 patients with inflammatory back pain (IBP) or sacroiliac tenderness (51%) underwent MRI examination. Axial involvement was confirmed for 18 patients (13/24 boys and 5/13 girls). There were 28 sacroiliac MRI for 21 patients available for review by our expert radiologist. For nine patients, the MRI were not copied on our imaging server and no CD was available for review. Our radiologist disagreed with the initial MRI interpretation for five cases (sacroiliitis had been undiagnosed in one case and overstated in four). The interrater reliability was fair as evaluated by the kappa statistic , 105 patients (92%) met the 2011 ASAS criteria for peripheral SpA, with no difference between genders. Seventy-nine patients (69%) met the Edmonton criteria for ERA and six (5.3%) those for PsA, together identifying 74.3% of the patients as having SpA, witch is significantly less than the ASAS criteria for peripheral SpA.p = 0.06 and OR = 0.24, p = 0.05, respectively). The most frequent adverse effect of NSAIDs was gastralgia in 35% of cases. One serious adverse event was documented .Clinical improvement and clinical remission were recorded for 96 (84.2%) and 52 patients (45.6%), respectively, treated solely with NSAIDs. Polyarthritis during the first years of the disease or axial involvement were predictive of non-response to NSAID therapy  and 68.6% after one year . Boys had a better response than girls to both anti-TNF therapies . Treatment tolerance was good. The most frequent adverse events were allergic reactions (five patients). One patient developed an inflammatory bowel disease after the initiation of etanercept. Another had to stop treatment because of recurrent infection.Anti-TNF therapy was given to 48 patients (42.1%) after a median disease duration of 5.4 years  were in clinical remission, including 23 (20%) off therapy.p = 0.01 and OR = 3.6, p <  0.01, respectively; Table p <  0.01 and OR = 3.1, p = 0.01, respectively). Enthesitis was associated with sacroiliitis . Male gender was the only prognostic factor associated with a lower risk of active disease at last follow up  in univariate, but not multivariable analyses . We did not find any other prognostic factor  and HLA-B27 56%) with JSpA, in accordance with the literature , 13, 14.% with JSOur study confirmed a low rate of symptomatic axial disease at disease onset, with a marked increase, however, within the first years of the disease: 60% of our patients showed axial involvement after five years. This was also true when studying only sacroiliitis or inflammatory lumbar pain. Axial disease has generally been considered to be rare in JSpA since it and sero-negative forms were first described –22. In cMRI results were available for 37 patients in our study and confirmed axial disease in half, calling into question the sensitivity and specificity of clinical examination to determine axial involvement. In one study, the sensitivity of physical examination was 23% and the specificity 67.9% . The lacThe high response rates to NSAID therapy was consistent with that reported for adult SpA. Anti-TNF, mainly etanercept and adalimumab, were the only biological drugs used in our cohort. Most patients treated with TNF antagonists responded rapidly, resulting in inactive disease within 6 to 12 months, as also observed in retrospective studies  and clinAmong several risk factors reported for adult SpA, a familial history of SpA was the only risk factor associated with both axial disease and sacroiliitis during follow-up, whereas the presence of enthesitis was a risk factor for sacroiliitis. Oligo-articular disease at first consultation was a negative predictor of axial disease and sacroiliitis. Published studies on JSpA contain discrepancies regarding the risks due to male gender, age, family history, hip arthritis, or articular counts , 34, 35.Here both univariate and multivariable analyses also showed a familial history of SpA to be the sole risk factor of active disease at the last follow-up after a median disease duration of 4.3 years. Only one study previously analysed risk factors associated with failure to achieve clinical remission: in 2006, B. Flatø et al. showed that female gender, a family history of ankylosis SpA in a first degree-relative, and ankle arthritis within the first six months were associated with failure to achieve remission after 15 years of follow-up [Our study also assessed the 2011 ASAS criteria for peripheral SpA in a paediatric population, showing them to be much more sensitive than the Edmonton classification criteria for ERA, even when associated with the psoriasis JIA criteria. This may be due to several factors. First, the principle of the Edmonton criteria is that all categories are mutually exclusive. Patients who cannot be classified in any of the six types of JIA  are classified as having undefined JIA. In the case of SpA, the fact that PsA is separated from other types of SpA classified in the ERA group is problematic. As discussed in the literature , some paOur study has several limitations. First, our data are retrospective, and although collected from a register, not all data were available for certain time points. Second, we used clinicians’ opinions as a gold standard to diagnose the disease and evaluate its activity because of the lack of good clinical criteria. Third, disease activity and treatment response were evaluated based on the opinion of the treating physician and the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Juvenile Arthritis Disease Activity Score (JADAS), or other parameters of disease activity status were not systematically included. Fourth, the duration of follow-up was heterogeneous, ranging from 1.0 to 7.2 years. Fifth, only one third of patients underwent MRI. MRI examinations in our centre were performed based on the clinical judgement of the treating physician, mostly when lumbosacral pain or limited spinal mobility were present. As discussed earlier, axial disease can be clinically silent. There is no consensus on how to consider axial disease seen only by MRI with no clinical symptoms. Sixth, our population was followed in a tertiary centre, with recruitment likely biased towards more severe cases.We confirm that JSpA is characterised by its peripheral pattern at disease onset, with peripheral arthritis and enthesitis. However, axial disease developed over the next five years in more than half our patients. MRI is clearly a valuable tool to assess axial involvement, but its interpretation may be more difficult in children and require, in many cases, an experienced radiologist. Familial history of SpA is one of the major prognostic factors associated with both sacroiliitis and persistence of active disease over years, favouring the close follow-up of such patients to initiate effective and timely treatment, knowing that TNF inhibitors have proven efficacy in JSpA/ERA patients with an inadequate response to NSAIDs. The current revised Edmonton classification criteria lacked both specificity and sensitivity to classify JSpA/ERA, which merits, among other considerations, the on-going effort to improve classification criteria of JIA. Applying the 2011 ASAS criteria for peripheral SpA may be worth considering for JSpA, as they showed high sensitivity to diagnose our patients both at their first assessment and at the last follow-up.Additional file 1:Table S1. Edmonton criteria for ERA and PsA. (DOCX 13 kb)Additional file 2:Figure S1. Evolution of the prevalence of inflammatory back pain. Inflammatory back pain was defined as proposed by the ASAS in 2009 [ in 2009 . InsidioAdditional file 3:Figure S2. Biological therapy: time to introduction and remission rate. Disease duration before introduction of a first line of biological therapy (years). Remission rate after introduction of a biological therapy (years). Remission was defined as the absence of any articular involvement  and any enthesial involvement for at least six months. (DOCX 104 kb)


Salacia reduces body weight, possibly due to its α-glucosidase inhibitor (α-GI) properties, but this has not been examined previously. In this study, a randomized, placebo-controlled, three-way cross-over design was used to evaluate whether Salacia Chinensis (SC) reduces appetite in healthy overweight/obese individuals . Forty-eight participants were fasted overnight and consumed a dose of SC (300 or 500 mg) or placebo with a fixed breakfast meal at each visit. Appetite sensations, glycemic indices and gastrointestinal peptides were measured. Results indicated that SC had no effect on postprandial appetite. However, in women, hunger was reduced by SC compared to placebo at multiple time points , but not in men. Area under the curve (AUC) for serum glucose, insulin and amylin was attenuated with SC compared to placebo (p < 0.05). Glucagon like peptide-1 had two peaks after the meal, but the AUC did not differ between groups. The AUC of peak areas for peptide YY and ghrelin were greater for SC than placebo (p < 0.05). These findings indicate that Salacia decreases glycemic indices supporting its role as an α-GI, and affects certain gastrointestinal peptides suggesting it may be an appetite modulator.Animal studies indicate  Obesity is a national health problem that has reached epidemic proportions. The prevalence of obese and overweight individuals in the US is 34% and 68%, respectively, and this population is at high risk of developing obesity-related morbidities, such as diabetes mellitus, hypertension, coronary heart disease and hyperlipidemia . While nSalacia, a herbal plant that has α-GI properties, have been used in traditional Asian medicine for therapeutic and/or preventative treatment of various diseases, especially obesity and insulin resistance [Salacia acts as an α-GI to affect glucose metabolism, and that various species of Salacia, including Salacia Chinensis (SC), reduce postprandial plasma glucose and insulin in healthy subjects [Salacia has been shown to reduce body weight in animal studies [Salacia has not been addressed. Thus far, there are no studies examining whether this putative α-GI alters appetite. The primary aim in this study was to determine whether SC affects hunger in healthy overweight/obese individuals after a mixed meal tolerance test. In addition, the effect of SC on glycemic indices, gastrointestinal hormones and taste sensations were also examined.Many animal and clinical studies have shown that α-glucosidase inhibitors (α-GIs), such as miglitol, acarbose and vodagliptin, have antidiabetic effects resulting in decreased blood glucose and insulin levels. In addition, α-GIs also have been found to affect appetite through enhancing satiety and suppressing hunger ,10,11,12sistance . Studiessubjects . In addi studies . However2 were recruited. All participants were recruited at the Department of Nutritional Sciences, Rutgers University through local newspapers and electronic advertisements , and through email list serves every few months between 2015 and 2016. Study screening was recorded using Qualtrics . Individuals were excluded if there was a diagnosis of an eating disorder, gastrointestinal illness, bariatric surgery, hyperparathyroidism, untreated thyroid disease, diabetes, blood pressure > 140/90, significant immune, hepatic, or renal disease, significant cardiac disease, active malignancy or cancer therapy within the past year, current use of obesity medications or dietary supplements or any weight regimen. Telephone interviews were conducted after participants filled out the screening form by trained staff to further determine whether subjects met the experimental criteria. Eligible volunteers underwent biochemical and physical screening including a comprehensive chemistry panel, complete blood count, and brief physical examination to ensure they were healthy and had no evidence of undiagnosed diseases . The study was approved by the Rutgers University Institutional Review Board and all study participants signed an informed consent form before entering the study. The trial is registered at clinicaltrials.gov (NCT02929849). The protocol met the ethical standards in accordance with the Helsinki Declaration.Healthy men and women who were overweight or obese with a body mass index (BMI) between 25–35 kg/mIn this double-blind, placebo-controlled, three-way cross-over trial, participants (aged 21–59 years) were randomly assigned to one of two doses of SC extract (300 mg or 500 mg) or placebo . There wThe VAS was used to assess appetite , and subjective taste perceptions . The scale has a 100 mm horizontal line with the most positive and the most negative rating at each end. Participants chose a point on the line that would be representative of their current perception for satiety. The distance in mm between the leftmost (zero) point and the point marked by participant was measured and used to determine the VAS score. Participants used a Kindle reader (Amazon.com) for the VAS self-assessment. Participants were not allowed to socialize or discuss their ratings during the test or see their previous VAS questionnaire recordings .g for 15 min at 4 °C, and protease inhibitor  was added into the serum. Samples were stored at −80 °C before batch analysis at the end of the trial. Serum glucose was measured using YSI 2300 STAT PLUS Glucose Analyzer . Serum insulin was measured with commercially available enzyme linked immunoassay kits . Serum ghrelin was measured using human ELISA assay kits  were 6.2%–7.8% and 0.9%–1.3%, respectively). Serum peptide YY (PYY), amylin and glucagon-like peptide-1 (GLP-1) were measured using a Multiplex magnetic-bead based immunoassay kits . The inter-assay CV was <20% for amylin and <15% for all others. All assays had an intra-assay CV < 10%.Six blood samples on the 3 study days were collected in serum-separating tubes and allowed to clot at room temperature. Tubes were centrifuged for 1000× p value < 0.05). This sample should have also been adequate to detect differences in other appetite scores . If there is a 30 area under the curve (AUC) difference between groups during the post-prandial period with a standard deviation (SD) of 22 and power of 90%, 11 subjects per group would be needed to detect a difference of treatment compared to placebo, as examined previously with another appetite suppressant food supplement vs. placebo [Salacia vs. placebo) in positive AUC and peak concentration for glucose or insulin indicated that we needed 6 and 16 subjects, respectively at 90% power [Power calculations were made according to previously validated methods ,18. Our 0% power . The ordp value < 0.05 was considered statistically significant.Descriptive statistics of survey respondents and survey results were calculated. Chi-square test was used to examine the relationship between demographic variables. Normality of the data was tested using Shapiro-Wilk′s test of normality. All data obtained in this study were normally distributed. For missing data, last value carried forward was used for the intention to treat (ITT) analysis. Within and between-group comparisons of means from before and at each postprandial time point  were performed using a general linear model for repeated measures ANOVA with Bonferroni correction. In addition, individual values were adjusted for baseline and examined for change at each time point if there were baseline differences between treatment days. The area under the curve (AUC) for peak areas of blood variables was calculated ,20. The A total of 493 participants were screened and 54 participants were eligible for recruitment by meeting the inclusion/exclusion criteria; 54 participants enrolled and 48 successfully completed the mixed-meal tolerance test, participating in all three protocol days. For the 6 participants who were not included in the study, 3 withdrew before any treatment visits, and 3 withdrew after the first treatment visit . The cha2) and 19 obese (32.7 ± 1.9 kg/m2) individuals . There were 28 females (60%) and 20 males (40%), and BMI indicated that there were 29 overweight   compared to placebo ( < 0.05) . Postprap < 0.02) at 30 min. There were no predictors of any of the other appetite measures. When measurements were examined in women, postprandial hunger scores were lower with SC (300 mg) than placebo at 90 and 180 min (p < 0.05). Because the mean age and range of ages in women was greater than men, we examined hunger scores in an age-matched group (< median of 34 years) and found that the lower hunger scores in younger women remained significant, and at more time points   a.There was no significant difference in fasting concentrations of serum glucose, insulin, amylin, GLP-1, PYY and ghrelin between the three study days and mean values are shown in p < 0.05). The meal tolerance curves for serum glucose, insulin and amylin indicated a rise after the meal with peak concentrations at 30 min, as expected , but did not reach significance at the 500 mg dose (p = 0.181) (p < 0.05). GLP-1 had two peaks (60 and 120 min) after the meal that tended to be greater for SC than placebo (p < 0.06); however, SC did not significantly increase GLP-1 AUC compared to placebo  b. Serum  placebo b.p < 0.05; p < 0.05; p < 0.05) (There was an inverse relationship between baseline gut hormones (PYY and GLP-1) and appetite measures (hunger and desire for prospective food consumption) ( < 0.05) .p < 0.02) with a trend for being less full (p = 0.06). Baseline serum glucose, insulin and amylin concentrations, and HOMA-IR were higher in the obese (p < 0.02), whereas QUICKI and the Matsuda index were lower in the obese than overweight (p < 0.001). Postprandial glucose, insulin and amylin total AUC was also greater in the obese than overweight (p < 0.001). With SC treatment, there were no significantly different effects in obese compared to overweight individuals for appetite measures, gut peptides or glycemic indices.In a separate analysis of the obese compared to overweight individuals, appetite measures were not significantly different at baseline (fasting). However, after the meal (180 min), the obese were hungrier, had a greater desire for prospective food intake and were less satiated  tract and play essential roles in the normal regulation of GI function. It is well established that gut peptides could affect appetite and eating behaviors centrally and peripherally. In fact, gut hormone-based therapies are used in the treatment of appetite dysregulation disorders, such as obesity and anorexia-cachexia syndrome ,30,31,32Salacia have focused on glycemic indices due to its α-GI properties, indicating improved glycemic indices in healthy and diabetic subjects, and animals have been reported [Salacia that have an appetite suppressant effect that would be more effective with chronic administration. Furthermore, in this study we used a visual analogue scale to measure appetite. An inherent limitation for measuring appetite with VAS is that there is wide variation between and within participants in this subjective measure that is dependent on individual′s feelings/standards. This limitation of the method has prompted those in the field to propose that VAS measurements with p values of less than 0.1 may be meaningful to avoid type II errors with these measurements [p value was not used in the current study.The primary purpose of this study was to examine the effect of SC on hunger and this is the first trial to examine this outcome as well as gastrointestinal hormones in a randomized controlled crossover design. Previous studies with reported ,41,42,43reported ,9,10. Threported ,45. In aurements . HoweverSalacia Chinensis lowered glycemic indices in response to a meal. There was no overall effect of SC on appetite measures, but there was an effect on gut peptides, and hunger was attenuated in females. Future studies might consider whether these effects on gut peptides can affect appetite under different meal and dosing conditions or subsequent meal size to explain the modest weight loss effects observed with Salacia and α-GIs.In conclusion, we found that


We obtained high-depth whole mitochondrial genome sequences using off target reads from whole exome sequencing to determine the association of mtDNA variation with the development and progression of disease, and to better understand the development of mtDNA mutations and copy number in the aging brain. With this approach, we found a surprisingly high frequency of heteroplasmic mtDNA variants in 32.3% of subjects. However, we found no evidence of an association between rare inherited variants of mtDNA or mtDNA heteroplasmy and disease. In contrast, we observed a reduction in the amount of mtDNA copy in both AD and CJD. Based on these findings, single nucleotide variants of mtDNA are unlikely to play a major role in the pathogenesis of these neurodegenerative diseases, but mtDNA levels merit further investigation.Mitochondria play a key role in common neurodegenerative diseases and contain their own genome: mtDNA. Common inherited polymorphic variants of mtDNA have been associated with several neurodegenerative diseases, and somatic deletions of mtDNA have been found in affected brain regions. However, there are conflicting reports describing the role of rare inherited variants and somatic point mutations in neurodegenerative disorders, and recent evidence also implicates mtDNA levels. To address these issues we studied 1363 The online version of this article (doi:10.1186/s40478-016-0404-6) contains supplementary material, which is available to authorized users. Mitochondria are critical intracellular organelles involved in calcium signaling, lipid biosynthesis, apoptosis , and theThere is emerging evidence that genetic variation of mtDNA contributes to the pathogenesis of neurodegenerative disorders . Common In addition to maternally inherited polymorphisms, acquired somatic mutations of mtDNA have also been associated with neurodegenerative disorders. Unlike the maternally inherited germ line variants (which are ‘homoplasmic’), the somatic mutations are usually present alongside the original wild-type molecules (heteroplasmy), and the proportion of mutated alleles determines whether a biochemical defect is manifest at the cellular level. MtDNA deletions accumulate in the ageing brain, reaching higher levels in regions vulnerable to neurodegeneration , 26, butpost mortem brains with Alzheimer’s disease (AD), Amyotrophic-frontotemporal dementia (ALS-FTD), Creutzfeldt-Jakob Disease (CJD), Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB), and compared them to healthy age-matched control brains. We found no evidence that rare inherited polymorphisms or mtDNA heteroplasmy contributes to the pathogenesis of neurodegenerative diseases, although differences in mtDNA content provide a clue to disease mechanism in AD and CJD.To provide definitive evidence in all three areas, we studied the entire mtDNA sequence and quantity in1363 n = 1190), cerebral cortex in 6.5% of cases (n = 89) with other brain regions in 6.16% (n = 84).Full mitochondrial genome sequence data was extracted from Exome Sequencing data of 1363 case or control brain tissue samples from the Medical Research Council Brain Tissue Resource . Cases fhttp://genome.ucsc.edu) using Burrows-Wheeler Aligner (BWA) v0.7.6 [http://picard.sourceforge.net).Exome sequencing was performed using Nextera 62 Mb Rapid Capture Expended Exome kits on the Illumina HiSeq 2000 with 100 bp paired-end reads. Sequence data were aligned to the human reference sequence (UCSC hg19)  v0.7.6 , formatt) v0.7.6 , and dupMToolBox  recovereKnown insertions or deletions (ins/dels) were defined, and all variants scored in HmtDB  and MITOWe determined the proportion of variant alleles at each site of the mitochondrial genome. We then calculated the heteroplasmic fraction  by dividing the number of variant reads by the total number of reads (for SNVs and deletions) or of the total number of 5′ flanking reads (for insertions). If the HF was <10% or >90%, we conservatively considered the variant site to be homoplasmic. If the HF was between 10 and 90%, the site was considered to be heteroplasmic, and the HF was studied further.http://www.clustal.org/omega). Defined haplogroups were compared to the haplogroups genotyped in 2360 individuals from the 1958 Birth Cohort (WTCCC58C) as previously determined by Hudson et al. [Haplogroup assignment was performed using the mt-classifier tool , and a mn et al. .Minor allele frequencies for each base of the mitochondrial genome were calculated from 30,506 full-length mitochondrial sequences in NCBI-GenBank using custom Python scripts. Rare homoplasmic variations were defined as those alleles present in less than 5% of individuals within their haplogroup using MITOMASTER , and novPathogenicity scores for all possible non-haplogroup defining SNVs were calculated using MutPred  and PolyThe relative amount of mtDNA in each brain (referred to as mtDNA copy number) was calculated as the ratio between the mean mtDNA read depth and the mean exome read depth as previously described .http://CRAN.R-project.org/doc/FAQ/R-FAQ.html), and plots made using Python.Mean variant counts or fractions both within and between groups were performed using Mann-Whitney, Fisher’s exact, or Spearman’s rank test tests as appropriate and as defined at the uncorrected threshold. A Poisson loglinear model was used to test the association between the number of variants and age among individuals within each main group. All statistical analyses were performed using R (v3.0.2)  (range 66–1328) and a mean base quality score of 36.8 (sd = 0.25) (range 36.0–37.5) , MITOMAP  or the 1000 genomes database. Four of the novel variants were in non-coding regions, two were in rRNA genes, and 19 were synonymous  variants per sample. Four hundred sixty-seven variants were defined as ‘common’ , and included known haplogroup defining variants. One thousand, four hundred fifty-six homoplasmic variants were defined as rare (MpAF <0.05 within their haplogroup). Twenty-five of the rare variants were novel, not seen in the NCBI database (0.8 variaMT-TR compared to controls (6/13 vs 9/139) (p = 0.002) , Controls; 16/344 (4.7%)) (p = 0.005)) and again MT-TR , Controls; 0/344) (p = 0.008), although both failed to reach significance at the corrected threshold of p = 0.0014. , Controls (2/65), p = 0.012 (3%) , Controls: 5/213 (2.3%), p = 0.0010  were detected , in 440 cases, with 10 of these variants entirely novel. 55.7% of all heteroplasmic variants occurred within the D-loop, 33.3% in coding regions, 4.8% in rRNA genes and 6.2% in tRNA genes , nor the mean variant pathogenicity score in any disease group Fig. .Fig. 3Thp = 2.85 × 10−7 (AD), p = 3.34 × 10−7 (CJD)), and we observed a strongly positive correlation between age and mtDNA copy number in CJD (p = 2.7 × 10−11). No association with age was seen in other groups  or the FTD-ALS cohort (p = 0.87). However, the CJD cohort did show a greater proportion of cases from the cerebellum compared to all other cohorts .mtDNA copy number was significant lower in AD and CJD compared to controls  is associated with these different disorders, or that different regions of the brain contain a significant burden of mtDNA variants, as recently described [Our most interesting finding was the frequent occurrence of mtDNA heteroplasmy in human brain tissue (HF >10% found in 32.3% of cases and controls), but contrary to previous reports, this did not change with age, nor was it associated with disease. This suggests that, although ongoing replication errors in mtDNA occur with age , 25, modescribed . Howevern = 40, mean mtDNA copy number = 4.14, sd = 2.96) was lower than all other CJD types . Therefore, it is therefore also possible that the trend observed for CJD actually reflects unusually low levels of mtDNA in the brains from patients with vCJD, who died at a younger age than other forms of CJD.Finally we observed a significant reduction in mtDNA copy number in both Alzheimer’s disease and CJD brains. Our data support previous work in AD , but to substantia nigra, but not frontal cortex, raising the possibility that the altered mtDNA levels are a consequence of the underlying pathology, and not the cause. This could be an indirect effect reflecting a change in the proportion of different cell types within a tissue homogenate that is linked to the neurodegeneration. Nonetheless, the observed association between mtDNA copy number and age in CJD brains supports a causal role which requires further investigation. Although this could reflect compensatory mitochondrial biogenesis in older subjects, an alternative explanation is that younger-onset cases have more aggressive pathology, with associated neuronal loss.When taken together, these findings support the role of mtDNA content in the pathogenesis of neurodegeneretive diseases, but with caveats. First, the abnormalities may be restricted to specific brain regions. Previous studies in PD showed a reduction in mtDNA copy number within the Additional file 1:Supplementary Material. (DOCX 6597 kb)


To investigate the effects of combined corneal wavefront-guided transepithelial photorefractive keratectomy (tPRK) and accelerated corneal collagen cross-linking (CXL) after intracorneal ring segment (ICRS) implantation in patients with moderate keratoconus.Medical records of 23 eyes of 23 patients undergoing combined tPRK and CXL after ICRS implantation were retrospectively analyzed. Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), manifest refraction spherical equivalent (MRSE), corneal indices based on Scheimpflug tomography, higher-order aberrations (HOAs), and corneal biomechanical properties were evaluated before and after ICRS implantation, and at 1, 3, and 6 months after combined tPRK and CXL.There were significant improvements in final logMAR UDVA and logMAR CDVA, and reductions in sphere, MRSE, and all corneal indices from baseline. Significant improvements in logMAR UDVA and reductions in sphere, MRSE, maximal keratometry, keratometry at the apex, mean keratometry, and keratoconus index were noted after ICRS implantation. After tPRK and CXL, significant improvements in logMAR UDVA and logMAR CDVA, and reductions in cylinder and all corneal indices were observed. There were significant improvements in final root mean square HOAs and coma aberrations from baseline, but no changes from baseline after ICRS implantation. Significant reductions in final radius and deformation amplitude from baseline were noted.Combined tPRK and accelerated CXL after ICRS implantation in moderate keratoconus appears to be a safe and effective treatment, providing an improvement in visual acuity, corneal indices, and HOAs.NCT03355430). Date registered: 28/11/2017.retrospectively registered (identification no.  Collagen cross-linking (CXL) is known to alter corneal biomechanics and increase mechanical rigidity by strengthening the corneal tissue, consequently resulting in significant increases in stiffness of the anterior corneal stroma . PatientVisual rehabilitation has been accomplished through different combinations of intracorneal ring segment (ICRS) implantations, CXL, and/or photorefractive keratectomy (PRK) for keratoconic patients. ICRS implantations act by flattening the central cornea without affecting the corneal visual axis , 18. TheChanges in a variety of corneal biomechanical properties after PRK, LASIK, small incision lenticule extraction, and CXL can be evaluated using the dynamic Scheimpflug analyzer  –34. ThisBecause of the positive effects achieved by combinations of these surgical modalities in the treatment of keratoconus, we hypothesized that corneal wavefront-guided tPRK and high-fluence accelerated corneal CXL after ICRS implantation would also show clinical improvement in patients with moderate keratoconus. Additionally, the changes in corneal biomechanical properties during combined corneal wavefront-guided tPRK and corneal CXL after ICRS implantation are not yet fully understood. Therefore, the aim of this study was to evaluate the efficacy, safety, higher-order aberrations (HOAs), and corneal biomechanical properties in patients with moderate keratoconus after ICRS implantation, followed by combined corneal wavefront-guided tPRK and corneal CXL.We performed a retrospective, interventional case series of patients with moderate keratoconus who underwent combined corneal wavefront-guided tPRK and high-fluence accelerated CXL at least 1 month after ICRS implantation from January 2010 to December 2015 at the Eyereum Eye Clinic . The study adhered to the tenets of the Declaration of Helsinki and followed good clinical practices with the approval of the Institutional Review Board of Yonsei University College of Medicine . All patients provided informed written consent for their medical information to be included in analysis and for publication. We retrospectively reviewed the medical records of 23 eyes of 23 patients that met the inclusion and exclusion criteria, as defined below.2, systemic autoimmune disease, a history of herpetic corneal disease, pregnancy, lactation, or severe dry eye syndrome.Combined corneal wavefront-guided tPRK and accelerated CXL after ICRS implantation were performed if a patient was intolerant to contact lenses, had moderate keratoconus without apical scarring, and if progression had been noted over the previous 6 months. All included patients underwent combined corneal wavefront-guided tPRK and CXL at least 1 month  after ICRS implantation. We excluded patients with central or para-central corneal scarring, central pachymetry <400 μm, corneal endothelial cell density of less than 2000 cells/mmGrading of keratoconus was based on the Amsler−Krumeich classification . ProgresBefore ICRS implantation (baseline) and after ICRS implantation (before combined tPRK and CXL), and at 1, 3, and 6 months after combined tPRK and CXL, all patients underwent complete ophthalmic examinations, which included examinations for UDVA and CDVA with a Snellen chart , manifest refraction (MR), and autorefraction using the ARK-530A . The safety index was calculated from the final postoperative CDVA/baseline CDVA ratio (in logMAR). The efficacy index was calculated as the final postoperative UDVA/baseline CDVA ratio (in logMAR). Multiple corneal indices were measured at the 8-mm zone using the Scheimpflug tomography system .For measuring changes in corneal aberrations, including HOAs, coma, and spherical aberrations, corneal wavefront analysis was implemented using corneal topographic data obtained with a Keratron Scout topographer . Root mean square (RMS) values of the corneal HOAs, with analysis up to the 7th order by expanding the set of Zernike polynomials, were calculated.Corneal biomechanical properties were measured using the dynamic Scheimpflug analyzer at approximately the same time of day. The dynamic Scheimpflug analyzer automatically calculated corneal deformation amplitude, radius values, and maximal concave power when the cornea is deformed to its greatest curvature by the air puff. The deformation amplitude is defined as the maximum amplitude when the cornea is deformed to its greatest concave curvature and is influenced by corneal stiffness . The radAs a first step, all patients underwent femtosecond laser-enabled  placement of ICRS . Segment sizes were determined according to the nomogram provided by the manufacturer. The depth of the ring channels was set at 75−80% of the thinnest pachymetry reading. After surgery, a bandage contact lens  was placed to be removed the next day. Postoperative medication included topical moxifloxacin 0.5%  and fluorometholone 0.1% .After at least 1 month , all patients were scheduled for combined corneal wavefront-guided tPRK and accelerated CXL treatment. tPRK between the corneal ring segments was performed using an excimer laser . The ablation profile was planned using the integrated Optimized Refractive Keratectomy-Custom Ablation Manager software . Using this software, ablation was planned based on clinical parameters, including manifest refraction, pachymetry, and corneal wavefront data (up to the 7th order) and topography obtained with the Keratron Scout. The optic zone area of tPRK was 5.8 mm−7.5 mm, and the total ablation zone was up to 8.6 mm.2  in a 1:1 pulsatile fashion. The cornea was kept wet at 30-s intervals with additional BSS during the irradiation process.0.1% riboflavin with hydroxypropyl methylcellulose  was soaked onto the corneal surface for 10 min immediately after excimer laser ablation. Additional riboflavin solution was added as needed during the soaking process after which was irrigated with 60 cc of chilled balanced saline solution at completion of soaking. UVA exposure (wavelength: 365 nm) was performed with the KXL system  which was set to provide a uniform circular diameter of 9.0 mm of irradiation for 360 s at a power of 15 mW/cmAt the end of the surgery, topical levofloxacin 0.5%  and fluorometholone 0.1% were administered, a bandage contact lens was placed, and the eye was examined under the slit-lamp. After surgery, topical levofloxacin 0.5% and fluorometholone 0.1% were applied 4 times daily, for 1 month. The dosage was gradually reduced over 3 months.P < .05.Results are expressed as mean ± standard deviation. The Shapiro-Wilk test was used to confirm the normality of data. We performed repeated measures one-way analysis of variance (ANOVA) with Bonferroni-adjusted post-hoc comparison to evaluate the differences between parameters in each follow-up period. All statistical analyses were performed using SPSS software version 20.0 . Statistical significance was defined as P < .001) and reduction in sphere (MR) (P = .002), MRSE (P = .002), maximal keratometry values (Kmax) (P = .001), keratometry values at the apex (Apex K) (P < .001), mean keratometry values (mean K) (P = .001), and keratoconus index (KI) (P = .002)  and logMAR CDVA (P = .024), and reduction in cylinder (P = .020) and all corneal indices  as compared with these values before tPRK and CXL , and reductions in sphere (P = .046), MRSE (P = .005), and all corneal indices from baseline  . The cylinder significantly decreased at 6 months after tPRK-CXL, when compared with 1 month after tPRK-CXL (P = .023). After ICRS implantation, there was no significant reduction in any corneal aberrations. However, after tPRK and CXL, there were statistically significant reductions in RMS HOAs and coma aberrations (both P < .001) as compared with these values before tPRK and CXL. There were also significant improvements in final RMS HOAs and coma aberration values from baseline .This study included 23 eyes of 23 patients 6 women, 17 men). The mean patient age was 27.1 ± 4.4 years (range: 20−38 years). Table 7 men. ThP = .012) and deformation amplitude (P = .012), and an increase in final maximal concave power (P = .005) from baseline. After tPRK and CXL, there was a statistically significant decrease in deformation amplitude (P = .042), as compared with these values before tPRK and CXL, without changes after ICRS implantation from baseline (Table Preoperative and postoperative corneal biomechanical properties, as measured with the dynamic Scheimpflug analyzer, are shown in Table r P = .00 from basIn the present study, we investigated the effects of combined corneal wavefront-guided tPRK and accelerated corneal CXL after ICRS implantation on the visual acuity, refractive outcomes, corneal indices, HOAs, and corneal biomechanical properties in patients with moderate keratoconus. We demonstrated that combined tPRK and CXL after ICRS implantation is beneficial for visual rehabilitation in moderate progressive keratoconus.As a progressive non-inflammatory ectatic disease, keratoconus involves changes in corneal collagen structure and the intercellular matrix, as well as apoptosis and necrosis of keratocytes –42. CXL There have been multiple reports on the combination of ICRS implantation and prophylactic CXL in patients with keratoconus. For example, Chan et al. demonstrated an additive effect of combination of ICRS implantation and CXL on maximal keratometry values and cylindrical error . ImproveIn the present study, after ICRS implantation, there were significant improvements in logMAR UDVA and reduction in MRSE, Kmax, Apex K, mean K, and KI from baseline. These results agreed with a study reporting increased UDVA and CDVA and decreased spherical equivalent and mean keratometry values after ICRS implantation, before CXL . We alsoIn the present study, we demonstrated a significant reduction in final RMS HOAs and coma aberrations as compared with values at baseline and before tPRK and CXL. We reported an improvement in logMAR UDVA attributable to lower order aberration correction by tPRK, and improvement in logMAR CDVA with concomitant decrease in corneal HOAs , 51. TheIn terms of corneal biomechanics, our results showed that final deformation amplitude decreased significantly as compared with that at baseline and before tPRK and CXL. Considering that thinner corneas tend to demonstrate higher deformation amplitudes and that this parameter reflects corneal stiffness, high-fluence accelerated CXL appears to be able to strengthen the cornea in keratoconus . MoreoveThe present study had several limitations, including its retrospective design. Other possible limitations of this study were the relatively small sample size and the lack of a control group. A prospective, controlled long-term, comparative paired-eye study should be performed to validate the current results.A combination of corneal wavefront-guided tPRK and accelerated corneal CXL after ICRS implantation is an effective and safe option for correcting mild refractive errors and improving visual acuity, corneal indices, and HOAs in patients with moderate progressive keratoconus.


Extramural venous invasion (EMVI) is a known adverse prognostic factor in patients with colorectal carcinoma. The prevalence and significance of EMVI in esophageal cancer (EC) patients is still unclear.From a prospectively maintained database, we retrospectively reviewed the resection specimens of patients with pathologic locally advanced (pT3/T4/N0-3) EC who were treated with curative intent between 2000 and 2015. Patients with previous malignancies and gastroesophageal junction (type II/III) tumors were excluded. Included were 81 patients who underwent surgery alone and 37 patients who underwent neoadjuvant chemoradiotherapy (nCRT). EMVI was assessed on hematoxylin and eosin slides and confirmed or excluded by additional Elastica van Gieson staining. Survival was analyzed using a multivariable Cox regression.n = 19) of patients in the surgery-alone group and 21.6% (n = 8) of patients in the nCRT group. The prevalence of EMVI after surgery alone was significantly high in squamous cell carcinomas and among tumors located in the mid-esophagus, as well as those with lymphovascular invasion (p < 0.05). After nCRT, the presence of EMVI was significantly high in tumors with lymphovascular and perineural tumor growth (p = 0.034). EMVI status was an independent adverse prognostic factor for disease-free survival  and overall survival  in the surgery-alone group for node-positive tumors.EMVI was present in 23.5% (In this study of locally advanced > pT3/N0-3 EC patients, EMVI was present in 23.5% of patients in the surgery-alone group and in 21.6% of patients after nCRT. EMVI was an independent adverse prognostic factor in patients after surgery alone. Invasion of tumor cells into blood vessels is an important expression of the metastatic potency of malignant tumors. In esophageal cancer (EC), distant metastases are common and seem to be associated with venous invasion (VI).11The prevalence of EMVI in CRC resection specimens is approximately 28%.Presently, neither the Royal College of Pathologists (RCP) nor the CAP protocol requires differentiation of intramural VI (IMVI), EMVI, and lymphatic invasion in esophageal resection specimens.EMVI was retrospectively analyzed on prospectively collected data of 182 consecutive patients with a curative resectable EC who underwent surgery alone between 2000 and 2014. Excluded were 98 patients with a pathological stage ≤ T1–T2 EC and/or cardia-gastroesophageal junction  tumors, which are frequently considered as gastric cancer. All patients were aged ≥ 18 years and were staged according to a standard protocol with 18F-fluorodeoxyglucose positron emission tomography (FDG-PET), computed tomography (CT), and endoscopic ultrasonography (EUS). To evaluate the impact of EMVI after current treatment with nCRT plus surgery according to the CROSS ,For each patient, archival slides of the tumor resection specimens were reviewed by two gastrointestinal pathologists. EMVI was scored as the presence of tumor cells within venous structures beyond the muscularis propria, as characterized by the ‘protruding tongue’ and the ‘orphan artery’ signs. The ‘protruding tongue’ sign is defined as a tongue-like protrusion of tumor extending from the deepest invasive front into the surrounding peri-esophageal fat,Other examined pathological parameters were T stage (T3/T4), N stage, tumor length, histologic type, tumor differentiation grade (well/moderate vs. poor/signet cell/mucinous), perineural invasion (PNI), and circumferential resection margin (CRM) involvement. PNI was defined as tumor cell infiltration in any layer of the nerve sheath, while both LVI and VI were defined as the presence of tumor cells within an endothelium-lined space without underlying muscular walls. CRM was assessed according to the RCP definition, which defines microscopic tumor cells within 1 mm of the CRM as involved (R1 resection).Patients were evaluated postoperatively every 3 months for the first year, every 6 months for the second and third years, and yearly thereafter. If recurrence was clinically suspected, patients underwent CT or PET/CT scans of the chest/abdomen, and endoscopy for confirmation of locoregional recurrence. Recurrent disease was either proven cytologically/histologically or when unequivocally present on radiologic imaging.p value < 0.2 in univariable analysis were included in the Cox regression multivariable analysis. Survival curves were calculated using the Kaplan–Meier method. Patients were censored at the last point of known contact during follow-up without acquiring the outcomes of interest. The backward conditional Cox regression model was used to delineate significant prognostic factors for survival. Hazard ratios (HR) and 95% confidence intervals (CI) were generated, and a p value < 0.05 was considered significant in the multivariable analysis. Statistical analysis was performed using SPSS 20.0 software .Fisher’s exact test was used to compare characteristics of patients with and without EMVI, and the log-rank test was used for univariable survival analyses of disease-free (DFS) and overall survival (OS). DFS was defined as the time from surgery to the date of recurrence or death. The prognostic value of different factors was first examined in univariable analyses. Clinically relevant factors with a p = 0.003) and in squamous cell carcinomas . Moreover, the presence of EMVI was significantly higher in patients with LVI-positive tumor (p = 0.006). EMVI showed no relationship with pathological (p)T stage and pN stage, tumor length or differentiation grade, or with PNI or CRM involvement  on review of H&E-stained slides. In three suspicious EMVI cases, EVG staining could not be performed due to the loss of the area of interest in additional slides, and these patients were therefore excluded from further analysis. The median age of the remaining 81 patients was 68 years (range 50–85). Tumor-negative CRM of > 1 mm (R0) was seen in 42 (51.9%) patients, and EMVI could be confirmed on EVG-stained slides in 19 (23.5%) cases. The relation of EMVI with the examined tumor characteristics are presented in Table p = 0.034).Of the 37 patients with pathological T3 EC enrolled in this study, EMVI was suspected in 19 51.4%) patients on review of H&E-stained slides Table . Tumor-n% patientp = 0.297) and OS  were not different among patients with or without EMVI. Distant recurrence-free survival was also not different in both groups .Follow-up data were available in 74 of the 81 examined patients. Excluded from survival analysis were six patients who died < 90 days after surgery and one patient with a simultaneous colon carcinoma. In the remaining 74 resected esophageal tumors, EMVI was confirmed in 17 of 37 (46%) suspected cases. Median DFS  and for OS in ACs after surgery alone  [data not shown].When adjusted for histologic type, EMVI was significantly prognostic for DFS in SCCs  and OS  were not different among patients with or without EMVI. Both the univariable analyses and backward conditional multivariable Cox regression analysis have shown no independent prognostic factors associated with DFS  suspected cases. Both DFS  Table . The medIn EC, the prevalence and significance of EMVI has not been well studied. In this study, EMVI was present in approximately one-quarter of patients with a pathologically staged T3 or higher EC after surgery alone, and in 21.6% of patients after nCRT. The rate of EMVI in the surgery-alone group was higher in mid-esophageal carcinomas (42.1% vs. 9.7%) and SCCs (47.4% vs. 19.4%), as well as in tumors with LVI (63.2% vs. 27.4%). In the nCRT group, the numbers regarding type and tumor side were too small to draw clear conclusions. However, EMVI was significantly higher in tumors with perineural tumor growth and those with LVI—both 75% vs. 31%, respectively.More or less the same percentages are found as in CRC, where EMVI is reported in approximately 28% (range 13.4–31.4%) of the resection specimens.15Most reports in EC have only described the presence of VI in general, without further distinction of IMVI and EMVI.21For the identification of VI in esophageal AC, Castonguay et al. used Movat stains, which is more or less equal to EVG staining.24As in CRC, we found that EMVI in EC should be considered as a potential high-risk factor in developing metastatic disease. Consequently, as in CRC, we also observed a significantly shorter DFS in the surgery-alone group  in EMVI-positive EC compared with EMVI-negative tumors (median 25 and 48 months); however, in the nCRT group, the presence of EMVI was not independently associated with survival. The clinical utility in this nCRT group was probably lacking due to a potential bias from case mix with low squamous cell cancers, therefore EMVI should be investigated in a larger study group. Another explanation may be the power of our study group, which was too small or was based on the selection of non-responders to nCRT. Failure of EMVI to regress after nCRT may indicate a lack of response, as shown by the association of EMVI changes on magnetic resonance imaging (MRI) with survival outcomes in CRC, and a potential positive effect of nCRT on microscopic distant disease.To our knowledge, this is the first study comparing outcomes of EMVI among pathological T3 or higher stages in a relatively large group of EC resection specimens in a single, high-volume institute. The usual limitations of retrospective studies and the relatively small sample size of EMVI hinder our ability to draw definite conclusions on the significance of EMVI, especially after nCRT. However, there was no difference in histology type for DFS after surgery alone. The prognostic impact of EMVI after nCRT in SCC is still unclear. EMVI is a histopathologic feature associated with an increased risk of recurrences, and, based on our study, it should be considered as a routine part of pathological reports of resection specimens of EC patients. However, its value should be studied prospectively in larger series and following nCRT in different tumor stages and different histological types.In this study, EMVI was present in 23.5% of patients in the surgery-alone group in 21.6% of patients after nCRT. EMVI was an independently adverse prognostic factor in patients after surgery alone. Therefore, EMVI should be considered as part of the routine histopathology workup, with an accessible use of elastin stains.