COL3A1 gene that encodes type III collagen (COLLIII), which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures.Vascular Ehlers-Danlos syndrome (vEDS) is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER) homeostasis, COLLs folding and extracellular matrix (ECM) organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition into the ECM of several structural proteins crucial to the integrity of soft connective tissues.To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural  COL3A1 gene encoding the pro-α 1 chain of type III procollagen (COLLIII) [COL3A1, a clinical history of arterial rupture, dissection or aneurysm, rupture of the large intestine, pregnancy complications at young ages, and carotid-cavernous fistula [Vascular Ehlers-Danlos syndrome  is a rare autosomal dominant inherited connective tissue disorder caused by mutations in the COLLIII)  and nowa fistula . Vascula fistula ,5. The f fistula .COL3A1 mutations leads to increased bruising, arterial and bowel rupture, and uterine, cervical, and vaginal fragility during pregnancy and delivery [The predominant expression of COLLIII in the walls of blood vessels and in hollow organs explains why its reduction due to delivery . vEDS padelivery .Current treatment is symptomatic only, since no causal therapy exists. Surgical treatment of vascular disease in vEDS patients has been limited because of friable and fragile tissues . A prospCOL3A1 that disrupt the triple-helix motif constitute a frequent pathogenic mechanism. Indeed, COL3A1 missense mutations altering the glycine codons together with in-frame splice mutations in the triple helical domain account for the majority of disease-causing variants and lead to misfolding of COLLIII in the endoplasmic reticulum (ER) and retention of seven eighths of misfolded procollagen trimers in the cell with consequent enlargement of ER vesicles [COL3A1 mutation influences life expectancy. Splice site mutations that lead to exon skipping have the lowest median survival. Substitutions of bulky residues  for glycine residues in the triple helical domain are usually less severe than splice site mutations, but more consequential than substitutions by smaller residues . Heterozygosity for COL3A1 null alleles typically produces a milder clinical phenotype with a significant delayed onset of complications [COLLIII is a homotrimer formed by the binding of 3 α1(III) chains with a triple-helix structure in the central region. The amino acid sequence of the triple helical domain is characterized by repeated Gly-X-Y motifs that allow the correct folding of α-monomers. As observed in other COLLs-related diseases, i.e., Osteogenesis imperfecta, spondyloepiphyseal dysplasia, achondrogenesis, Stickler syndromes, Ullrich congenital muscular dystrophy, Bethlem myopathy, and other subtypes of EDS , mutatiovesicles –10. Thisvesicles ,12. Someications , 9–12.Col3a1+/− mice present a very mild phenotype, as suggested by a low incidence of vascular events, homozygotes exhibit a severe perinatal mortality owing to arterial ruptures [Although disruption of the COLLIII triple helical structure leads to abnormal protein folding, the molecular mechanism thereby contributing to the etiology of vEDS is poorly studied. Currently, there are two available mouse models for vEDS, both are homozygous or heterozygous nulls, which represent only about 5% or less of all known cases of vEDS patients –15. Whilruptures . Cultureruptures ,17.COL3A1.To gain insights into altered gene expression pattern and dysregulated biological processes underlying molecular pathology of vEDS, we carried out a transcriptome-wide expression profiling in cultured skin fibroblast strains derived from three vEDS patients with different structural mutations in COL3A1 [S1 Table.This study was approved by the local Ethical Committee “Comitato Etico di Brescia, ASST degli Spedali Civili, Brescia, Italia”, registration number NP2658 and performed in accordance with the Declaration of Helsinki Principles. Written informed consent was obtained from all patients and control donors to the study and for skin biopsy according to Italian bioethics laws. Subjects selected for this study were evaluated in the Centre of Heritable Connective tissue disorders and Ehlers-Danlos syndromes of the Spedali Civili of Brescia. vEDS patients were positive for the criteria of the Villefranche nosology . In partCOL3A1 . vEDS paCOL3A1 , respectin vitro at 37° C in a 5% CO2 atmosphere in Earle’s Modified Eagle Medium (MEM) supplemented with 2 mM L-glutamine, 10% FBS, 100 μg/ml penicillin and streptomycin . Cells were expanded until full confluency and then harvested by 0.25% trypsin/0.02% EDTA treatment at the same passage number (from 3rd to 6th).Skin fibroblast cultures from three vEDS patients and nine unrelated age-matched healthy donors were established in our lab from skin biopsies by standard protocols. Fibroblasts were grown Goat anti-COLLIII polyclonal antibody (Ab), and mouse anti-elastin (ELN) (clone 10B8) monoclonal antibody (mAb) were from Millipore-Chemicon Int. . Anti-fibrillin (FBN) mAb (clone 11C1.3) was from NeoMarkers . Anti-EMILINs mAb was kindly provided by Prof. P. Bonaldo . The rabbit anti-versican core protein Ab was from Affinity Bioreagents , the rabbit anti-perlecan core protein Ab and mouse anti-GAPDH mAb (clone 0411) were from Santa Cruz Biotec, Inc. , the anti-decorin (DCN) mAb (clone 115402) was from R&D Systems, Inc. , and the anti-heparan sulfate glycosaminoglycan chains (HS-GAGs) mAb was from USBiological Life Sciences . Rabbit anti-protein disulfide isomerase (PDI) Ab was from Novus Biologicals . Mouse anti-FK506-binding protein 22 (FKBP22) Ab was from Abnova . Rhodamine-conjugated anti-goat IgG and anti-mouse IgM secondary Abs were from Calbiochem–Novabiochem INTL, and the Alexa Fluor 488 anti-rabbit and Alexa Fluor 594 anti-mouse secondary Abs were from Life Technologies. Horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit secondary Abs were from Sigma Chemicals .To analyze the COLLIII-ECM organization, vEDS fibroblasts, grown for 48 h, were immunoreacted as previously reported . In briec Protein Assay  and 30 μg of proteins were separated in reducing conditions by electrophoresis in 8% and 8%-12% SDS-PAGE, for PDI and FKBP22, respectively. After nitrocellulose sheet transfer, membranes were blocked O.N. at 37° C in 5% non-fat dry milk in TBS-0.1% Tween 20 (TBS-T), and immunoreacted at room temperature (RT) with 1:1,000 anti-PDI Ab diluted in TBS-T and 1:500 anti-FKBP22 Ab diluted in 5% dry milk/TBS-T. After washing in TBS-T, membranes were incubated for 2 h at RT with HRP-conjugated anti-rabbit and anti-mouse IgGs, respectively, diluted 1:1,000 in TBS-T and developed using ECL method (Pierce). After stripping, the two filters were reprobed at RT with 1:10,000 anti-GAPDH mAb diluted in TBS-T, as loading control, and with the anti-mouse secondary Ab, as reported above. The semi-quantitative evaluation of the Integrated Optical Density (IOD) of the bands was performed using the Image Pro Plus program (Media Cybernetics). The reported values are the means ± standard error of mean (SEM) of the ratios between the IOD of the sample and control protein bands detected in the same lane, obtained in three independent experiments. Statistical analysis of these data was determined by one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test. Analyses were performed with GraphPad Prism software .To analyze PDI and FKBP22, one control and three vEDS fibroblasts’ strains were cultured in complete MEM for 48 h. In particular, PDI was evaluated in cell extracts obtained by O.N. cell lysis and scraping at +4° C in buffer containing 20 mM Tris-HCl pH 6.8, 135 mM NaCl, 10% glycerol. 1% NP40, 1 μg/ml leupeptin, 4 μg/ml pepstatin, 0.1 UI/ml aprotinin, and centrifuged at 12,000 rpm at +4° C for 15 min. FKBP22 was analyzed after O.N. cell lysis and scraping at +4° C with buffer containing 50 mM Tris-HCl pH 7.4, 1% NP40, 0.5% DOC, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1 μg/ml leupeptin, 4 μg/ml pepstatin, and centrifuged at 12,000 rpm at +4° C for 10 min. Protein concentration was evaluated using detergent compatible Bio-Rad Din vitro transcription, cRNA were reverse transcribed and the corresponding cDNA was fragmented, biotin labeled, and hybridized over night at 45° C onto the arrays. The chips were then washed in the Fluidics station FS 450, scanned using the scanner 3000 7G system, and analyzed with the Affymetrix GeneChip Operating Software. The resulting raw array data were analyzed using Partek Genomics Suite software . Briefly, the microarray raw datasets were reprocessed by the background correction, normalization and summarization of probe intensities using the robust multiarray average analysis to determine the specific hybridizing signal for each probe set. After background correction, the expression data were corrected for perfect match intensity and were transformed in base-2 logarithm. Quality control was performed by investigating principal component analysis to detect grouping patterns in the samples and to identify the outliers and evaluate whether batch effect significantly affected the data. To detect differentially expressed genes (DEGs) between vEDS vs control cells, a one-way ANOVA analysis was used as a criterion for the selection of DEGs. To assess significant differences in the gene expression profile in vEDS fibroblasts, we selected the genes that had more than a 1.5-fold change and p-value <0.05. Multiple testing correction was applied to control the false-discovery rate (FDR) using the Benjamini–Hochberg (BH) procedure [Total RNA was extracted from skin fibroblasts of three patients and nine controls using the Qiagen RNeasy kit, according to manufacturer’s protocol . RNA quality control was assessed on an Agilent 2100 BioAnalyzer . Transcriptome-wide expression profiling was performed using the Affymetrix Gene 1.0 ST platform. Microarray analysis was performed starting from 250 ng of total RNA per sample; labeled targets were prepared using Ambion Whole Transcript Expression Kit (Life Technologies) and GeneChip WT Terminal Labeling and Controls Kit  in accordance with manufacturers’ instructions. Total RNA was primed with synthetic primers containing a T7 promoter sequence, reverse transcribed into first-strand cDNA and converted into double-stranded cDNA. Following the rocedure . Genes wHPRT, GAPDH, ATP5B, and CYC1 reference genes were amplified for normalization of cDNA loading. Amplification plots, dissociation curves, and threshold cycle values were generated by ABI Sequence detection system software version 1.3.1. The list of primers used for qPCR analyses is reported in S2 Table. Relative mRNA expression levels were normalized to the geometric mean of these reference genes and analyzed using the 2-(ΔΔCt) method. Results were expressed as the mean value of relative quantification ± SEM. Statistical significance between groups was determined using unpaired Student’s t-test. Analyses were performed with GraphPad Prism software.Relative expression levels of a series of selected DEGs identified by array analysis were confirmed by quantitative real-time PCR (qPCR) using different RNA extractions obtained from skin fibroblast cultures of patients and healthy subjects. 3 μg of total RNA were reverse-transcribed with random primers by standard procedure. qPCR was performed with SYBR Green qPCR Master Mix (Life Technologies), 10 ng of cDNA, and with 10 μM of each primer set with the following cycling conditions: initial denaturation for 30 s at 95° C, followed by 40 cycles of 95° C for 15 s and 60° C for 60 s, in a ABI PRISM 7500 Real-Time PCR System. COL3A1 mutations, transcriptome-wide expression analysis was carried out comparing gene expression pattern between patients’ and controls’ skin fibroblasts. Gene expression profiling identified a total of 969 DEGs in vEDS patients’ cells when compared to controls: of these, 281 genes were up-regulated, and 688 were down-regulated (S3 Table). Panel A in Fig 1 represents the volcano plot that illustrates the statistical significance (FDR) vs fold-change of DEGs, and Table 1 reports a selection of the most up- and down-regulated DEGs (FDR < 0.05 and fold change threshold ≥ 1.5). To group transcripts with similar expression profiles between patients and controls, hierarchical clustering of DEGs was conducted .To identify genes potentially involved in the molecular mechanisms associated with dominant negative To identify biological processes that were over- or under-represented in patients’ cells, we classified all up- and down-regulated genes according to the GO categories by using the DAVID and ToppGene biological databases. Only GO categories with FDR <20% in each annotation term were considered relevant.Table 2, and S4 Table) were associated with genes having olfactory receptor activity and with functions related to regulation of transcription and involved in the re-organization of actin cytoskeleton.The most enriched GO clusters of the 281 up-regulated DEGs . The most prominent group of down-regulated DEGs was implicated in cell cycle process at several steps, such as telomere organization, separation of sister chromatids, nucleosome assembly, and included cell division cycle protein genes , and cyclin-dependent kinase genes . Another group of significantly down-regulated genes encoded various histone proteins, i.e., HIST1H4L, HIST1H4F, HIST1H3B, HIST1H1B, HIST1H3I. A set of significantly GO enriched terms contained several kinesin family genes, such as KIF14, KIFC1, KIF2C, KIF4A, KIF11, KIF15, KIF18A, KIF20A, which play a pivotal role in cell movements and intracellular trafficking, including chromosome and centrosome positioning during mitosis. Also, processes implicated in DNA damage response and DNA mismatch repair were dysregulated in vEDS, as indicated by low mRNA levels of a number of associated genes, such as MSH2, MSH6, RFC1, RFC2, RFC4, SSBP1, ARMT1, CLSPN, RAD21, FANCI, INTS7, NSMCE2, MGME1, FANCB.Functional analysis of 688 down-regulated genes yielded 28 different GO clusters . Similarly, transcriptome profiling revealed significant expression changes of many genes belonging to the spliceosome machinery, a large RNA–protein complex that catalyzes pre-mRNA splicing . Functional classification of DEGs also indicated in patients’ cells an aberrant expression of several small nucleolar RNAs , which represent a class of non-coding RNAs (ncRNAs) that primarily mediate post-transcriptional modification of ribosomal RNAs.We also found that GO categories related to “ribosome”, “rRNA processing”, and “structural constituent of ribosome” were significantly enriched with many down-regulated genes encoding both large and small ribosomal proteins essential for ribosome biogenesis . Among them, we found low mRNA levels of TXN, PDIA4, PDIA5, and PDIA6 encoding a class of ER-resident enzymes of the thioredoxin proteins superfamily that catalyze the formation and rearrangement of disulphide bonds during protein folding. Functional classification of DEGs also revealed significant expression changes of several members of DnaJ heat shock protein family, i.e., DNAJB11, DNAJB7, DNAJC10, DNAJC24, and DNAJC3, a class of chaperones facilitating protein folding and ER homeostasis (S5 Table).Cell redox balance and homeostasis of protein folding into ER seem to be also perturbed in vEDS cells, as shown by reduced expression of many related genes, such as S6 Table, cell cycle was the most perturbed pathway in patients’ cells, as shown by a significant decreased expression of a number of cell cycle regulating genes. Pathways enrichment analysis also highlighted that DNA replication and mismatch repair processes were impaired in vEDS fibroblasts. This is indicated by low transcriptional levels of different related genes including primase subunit 1 (PRIM1), DNA polymerase epsilon 2 (POLE2), single stranded DNA binding protein 1 (SSBP1), members of replication factor C , belonging to the mini-chromosome maintenance complex , and to the DNA mismatch repair MutS family .To identify differentially expressed pathways in vEDS fibroblasts, an enrichment analysis was carried out on all 969 DEGs by querying both ToppGene and DAVID databases and selecting a threshold of FDR-adjusted p-value <0.05. As reported in S6 Table). Concerning ribosome, vEDS cells showed an impairment of this ribonucleoprotein complex, as shown by low expression levels of many transcripts that encode both large and small subunits of nuclear ribosomal proteins such as RPL21, RPL22, RPL23, RPL27A, RPL36A, RPL9, RPS12, RPS27A, RPS3A. Spliceosome machinery was also disturbed as indicated by impaired expression of a subset of genes encoding members of heterogeneous nuclear ribonucleoproteins family, i.e., HNRNPA1, HNRNPA1L2, factors that enhance pre-mRNA splicing , and belonging to nuclear ribonucleoprotein complex .Similarly, enrichment analysis revealed a dysregulation of proteasome apparatus, as showed by an altered expression pattern of several genes encoding various proteasomal subunits. Moreover, ribosome and spliceosome pathways were also affected in patients’ fibroblasts , ECM organization and COLLs biosynthesis pathway, related to correct protein folding into ER, and belonging to the proteasomal complex . qPCR revealed a statistically significant decrease of a huge range of transcripts involved in the regulation of cell cycle, such as BUB1, CCNA2, CCNB2, CDK1, CDKN1B, CLSPN, PBK, PLK1, TPX2, ANLN . Conversely, CDKN2B, which encodes a cyclin-dependent kinase (CDK) inhibitor, showed an enhanced expression in vEDS cells supporting microarray data . qPCR also confirmed low transcriptional activity of various kinesin family genes, i.e., KIF20A, KIF2C, KIF4A, KIF18A, KIF11, and KIF15 . Significant changes of mRNA levels of several genes associated with ECM homeostasis and COLLs biosynthesis, such as FBN2, ITGA3, HSPG2, MMP24, EDNRA, LOXL3, P4HA2, P4HA3, were also confirmed . We also verified the low transcription of many ER related genes, such as PDIA4, PDIA5, PDIA6, DNAJB1, DNAJC10, TXN, FKBP14 , as well as the low transcript levels of PSMA6, PSMB6, PSMC3, and PSMD2, which encode different catalytic and non-catalytic subunits of the proteasome complex .We validated the differential expression of a selection of DEGs by qPCR. Genes were prioritized based on their expression pattern and GO functional classification that was significantly perturbed in vEDS fibroblasts. Specifically, we focused on genes involved in cell cycle regulation . The consequence of aberrant COLLIII on the organization, expression, and distribution of other structural ECM components, i.e., FBNs, ELN, EMILINs, HS-GAG chains, and the core proteins of the proteoglycans (PGs) perlecan, versican, and DCN was analyzed by IF. As shown in Fig 4A, FBNs were organized in fibrillar networks covering control fibroblasts, whereas these proteins were not assembled into the ECM of vEDS cells that showed rare and sparse cytoplasmic spots. This finding is in line with the strong down-regulation of the FBN2 transcript shown by microarray analysis and confirmed by qPCR. Similarly, ELN was not organized into the ECM of 7 days-grown vEDS fibroblasts, in which the protein was detectable only in the cytoplasm. In addition, vEDS fibroblasts were also unable to produce a fibrillar well-organized ECM network of EMILINs, since only few fibrils were present in intercellular spaces. Moreover, control cells organized HS-GAGs in a rich fibrillar network, whereas in vEDS cells only few fibrils, localized in the extracellular spaces and discrete spots associated with the cell surface, were identified . The HS-specific perlecan, organized by control fibroblasts in a fibrillar ECM meshwork and resembling the HS-GAGs distribution, was highly reduced in patients' fibroblasts. Likewise, the PGs versican and DCN showed a strong reduction of their expression in vEDS cells compared to control cells .In vEDS patients’ skin fibroblasts, we previously demonstrated an abnormal synthesis and deposition of COLLIII into the ECM together with the disarray of FN and the lack of COLLs- and FN-specific integrin receptors ,17. IF aFig 4C). IF of FKBP22 suggested lower cytoplasmic levels of this COLLs-modifying enzyme in patients compared to control cells; WB confirmed this down-regulation demonstrating a 4.1 to 6.4-fold decrease.Based on microarray results, the synthesis and organization of the two ER-related proteins, i.e., PDI and FKBP22, were investigated by IF and WB. Control and vEDS fibroblasts showed comparable levels of PDI, although in mutant cells the IF signals suggested an enlargement of ER vesicles  of COLLs and ELN, including ofibrils . Fibrillofibrils . Fibrillofibrils . Besidesofibrils ,29.in vitro picture consistent with the pathological conditions of arterial walls reported in vEDS patients. Based on these protein findings, it might be reasonable to speculate a possible contribute of the TGFβ signaling in the molecular mechanisms of vEDS, as showed for different connective tissue disorders with cardiovascular complications, i.e., Marfan syndrome, Loyes-Dietz syndrome, and arterial tortuosity syndrome [Our protein data showed a generalized disassembly of all isotypes of fibrillin in vEDS fibroblasts’ ECM, concomitant to the disorganization of EMILINs and ELN networks, providing an syndrome ,30. Nevesyndrome . Anyway,HSPG2 encodes perlecan, a PG present in the basement membranes, which binds to and cross-links many ECM components and cell-surface proteins, such as COLLI, COLLIV, laminin, nidogen, and FN [EDNRA encodes the endothelin receptor type A (ET-A) that interacts with receptor type B to mediate endothelin-1 (ET-1) signaling, a potent endothelium-derived vasoconstrictor peptide that can activate vascular smooth muscle cells, inducing proliferation, and synthesis of ECM proteins [, and FN . It is a, and FN . Our pro, and FN , and the, and FN ,19,33,34proteins . ET-1 caproteins .COL3A1 structural mutations are associated with the alteration of transcript levels of different COLLs-modifying enzymes, such as LOXL3, P4HA2, P4HA3, and FKBP14. LOXL3 encodes a copper-dependent amine oxidase required for the formation of lysine-derived cross-links of COLLs and ELN fibrils [LOXL3 expression in vEDS fibroblasts and the concomitant COLLIII- and ELN-ECM disorganization suggest that in dermal fibroblasts collagenogenesis and elastogenesis should be co-regulated, according to Aziz and colleagues’ findings [P4HA2 and P4HA3 encode two of the three isoforms of the α-subunit of collagen prolyl-4-hydroxylase, a tetrameric enzyme resident in the ER lumen that hydroxylates prolines on the procollagen peptide and serves as a chaperone for procollagen [FKBP14 encodes FKBP22, a peptidyl-prolyl cis-trans isomerase localized in the ER lumen [FKBP14 cause kyphoscoliotic EDS (kEDS-FKBP14) that is complicated by life-threatening cardiovascular complaints. Indeed, aortic rupture, internal carotid artery and celiac artery dissections, and hypogastric artery pseudoaneurysm rupture were reported [in vitro cultured fibroblasts derived from individuals with kEDS and vEDS. In particular, both the FKBP14 inactivation in kEDS-FKBP14 cells and the reduction of FKBP22 in response to the misfolded COLLIII retention in ER of vEDS fibroblasts have a common downstream negative effect on the assembly of several structural proteins into the ECM. Indeed, enhanced storages of FN [Biosynthesis of mature COLL fibrils is a multistep process spanning both intracellular and extracellular PTMs mediated by various enzymes and molecular chaperones that provide specific modifications such as hydroxylation of prolyl and lysyl residues, which are a prerequisite for correct protein folding . In line fibrils . The dowfindings . P4HA2 acollagen . Hydroxycollagen , since ucollagen . FKBP14 ER lumen  that intER lumen ,42. Bialreported ,43–45. Ireported . Moreovereported . The stres of FN  and ELN es of FN ,17,33. FFKBP14, transcriptome profiling also underlined the reduced expression of other genes with functions related to the maintenance of the ER protein folding homeostasis. ER not only provides mechanisms to facilitate proper folding of newly synthesized secretory and membrane proteins, but also harbors molecular machineries that eliminate proteins that fail to fold or assemble correctly [TXN, PDIA4, PDIA5, and PDIA6 belonging to the thioredoxin superfamily that facilitate correct oxidative protein folding, thus ensuring ER-associated proteostasis [PDIA4, PDIA5, and PDIA6 are members of an expanding proteins family known as protein disulfide isomerases that may also act as effectors of protein translocation of terminally un/misfolded proteins to the cytosol for their proteasomal degradation [Besides orrectly . Disturborrectly . About torrectly . Likewiseostasis . Moreoveradation . In thisradation . Althougradation . In lineradation .COL1A1 [Overall our molecular findings may provide further support to early evidences suggesting that the disturbance of ER related homeostasis could be likely associated with the disease mechanism as a result of abnormal protein folding with ensuing intracellular retention of mutant COLLIII chains and ER dilation ,53–55. ICOL1A1 , as wellCOL1A1  and in pCOL1A1 . TherefoCOL1A1 ,63, but COL1A1 . Additioanoikis is driven in vEDS fibroblasts by a survival signaling transduced through the cross-talk between the αvβ3 integrin and EGF receptor [FKBP14 inhibition induces cell cycle arrest and apoptosis [The most down-regulated genes in vEDS patients ‘cells were implicated in processes related to cell division, DNA replication, telomere organization, and associated with nucleosome and chromatin assembly. We also found significant expression changes of a large range of transcripts encoding histone proteins that could reflect a diminished DNA synthesis, given that histones enter in nucleosome formation and proper DNA wrapping after the S phase of cell cycle . Anotherreceptor . Recentlpoptosis . In vEDSCDKN2B, encoding the p15 protein that is an inhibitor of the cyclin D/CDK4 and CDK6 complexes, suggests a possible delay of G1 phase transition to overcome DNA damages and stress conditions, whereas the down-regulation of CDKN1B transcription, which is an inhibitor of cyclin E/CDK2 complex, suggests an opposite response of cells to overcome apoptosis’ activation and go on cell cycle. This cell cycle phases’ perturbation could be a consequence of accumulated DNA mismatches, probably not repaired by not-functioning mismatch repair mechanism. COL3A1 mismatches can be responsible of misfolded COLLIII trimers, which are not processed by proteasome and accumulate in the ER enlarged cisternae blocking vEDS cells’ trafficking. Gene expression profiling also revealed an aberrant expression of many genes implicated in pathways that regulate RNA processing, spliceosome and ribosome assembly. Among these DEGs, vEDS cells showed a differential expression of snoRNAs, a class of ncRNAs involved in the ribosomal RNA maturation and biogenesis [Also, in patients’ fibroblasts the DNA damage response and the cyclins/CDKs/CDK inhibitors machine seem to be perturbed. In particular, the up-regulation of ogenesis . Accumulogenesis ,72. HoweIn conclusion, our approach illustrates global mRNA profiling changes of several genes and related biological processes that could offer novel insights into the molecular pathology of vEDS. Protein data revealed that dominant negative effect triggered by COLLIII misfolding also affects the organization of structural proteins crucial to the integrity of soft connective tissues. Further studies are needed to delineate in detail the functional role of the identified gene expression changes in vEDS fibroblasts and to identify distinct molecular signature(s) that may contribute to the mechanistic events that cause vascular wall weakness of this severe connective tissue disorder.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Transparent conducting oxides (TCOs) are electrical conductive materials with comparably low absorption of electromagnetic waves within the visible region of the spectrum. They are usually prepared with thin film technologies and used in opto-electrical apparatus such as solar cells, displays, opto-electrical interfaces and circuitries. Here, based on a modern database-system, aspects of up-to-date material selections and applications for transparent conducting oxides are sketched, and references for detailed information are given. As n-type TCOs are of special importance for thin film solar cell production, indium-tin oxide (ITO) and the reasonably priced aluminum-doped zinc oxide (ZnO:Al), are discussed with view on preparation, characterization and special occurrences. For completion, the recently frequently mentioned typical p-type delafossite TCOs are described as well, providing a variety of references, as a detailed discussion is not reasonable within an overview publication. Transparent conducting oxides (TCOs) are electrical conductive materials with a comparably low absorption of light. They are usually prepared with thin film technologies and used in opto-electrical devices such as solar cells, displays, opto-electrical interfaces and circuitries. Glass fibers are nearly lossless conductors of light, but electrical insulators; silicon and compound semiconductors are wavelength dependent optical resistors (generating mobile electrons), but dopant dependent electrical conductors. Transparent conducting oxides are highly flexible intermediate states with both these characteristics. Their conductivity can be tuned from insulating via semiconducting to conducting as well as their transparency adjusted. As they can be produced as n-type and p-type conductives, they open a wide range of power saving opto-electrical circuitries and technological applications. A still valuable overview of transparent conductive oxides is given in , basics As transparent conducting oxides are usually compound semiconductors—where the nonmetal part is oxygen—they are discussed along their metal elements. Metals were used as compound materials or dopants (with just a few percent content).yBz, with different opto-electrical characteristics. These opto-electrical characteristics can be changed by doping, AyBz:D (D = dopant), with metals, metalloids or nonmetals. Hence, metals can be part of the compound semiconductor itself, A, or can be a dopant, D. Scanning the periodic table of elements, with a view on the utilization of metals for TCOs, results in In transparent conducting oxides (TCOs), the nonmetal part, B, consists of oxygen. In combination with different metals or metal-combinations, A, they lead to compound semiconductors, AOutstanding good optical characteristics have been provided by tin-, indium- and zinc oxides . Well known is, for example, indium tin oxide (ITO), and the doping of zinc oxide with less than 5% aluminum (ZnO:Al). Moreover, doped delafossite and mayenite compounds are of upcoming interest see . A varie2O3) and tin(IV) oxide (SnO2), with typically 90%wt In2O3, 10%wt SnO2. It is transparent and colorless as a thin film and yellowish to grey as bulk material. Indium tin oxide is the most widely used transparent conducting oxide (TCO [Indium tin oxide (ITO) is a solid solution of indium(III) oxide (Inide (TCO ) becauseide  [wt aluminum and can be produced with spray pyrolysis [Transparent conducting, aluminum doped zinc oxide thin films (Al ZnO:Al) ,38 contayrolysis ,42,43,44yrolysis ,49,50,51yrolysis ,53, vapoyrolysis ,55, magnyrolysis ,58,59,60yrolysis ,62,63,64yrolysis ,80,81,82yrolysis ,84,  further metal(s), A,  and oxygen, O.Commonly, ITO- and ZnO-based TCO thin films are n-doped, as p-doping has been shown to be technologically more difficult. Fortunately, for delafossite compound semiconductors this is Copper may be replaced by silver ,140,141,2/3Sb1/3O2 , respectively AgA2/3Sb1/3O2  [1−xAxO2  delafossites have been investigated [Quaternary semiconductors as for example the Sb-based CuA Ni, Zn) ,140 or tstigated ,178.1−xAgxRh1−yMgyO2 [Ag-Cu and Rh-Mg replacements were for example studied in the quinternary structure Cu1−yMgyO2 . xAyO2+d, has been examined [xAyOzSα, were sputtered  or alreg in H2S .Delafossites have been grown from a melt by a slow cooling-method in air ,183. The2, O2, air [2O4 fraction and formation of highly crystalline films with single-phase delafossite CuCrO2 structure [Annealing in N O2, air ,162,165  O2, air  was examtructure ,164.IIIO2 group shows increasing band gap from AIII = Al, Ga, to In. The largest gap CuInO2can be doped both n- and p-type but not the smaller gaps CuAlO2 and CuGaO2 [2 with Ca results in p-type, doping with Sn in n-type semiconducting TCO thin films [2, too  was discussed in [The CuAd CuGaO2 . Therefoin films ,190. Bidtype: Sn ). In addussed in ,195,196 ussed in . Defect ussed in .2:Ca [2In2O5:Ca [2:Ca [2:Mg [2:Mg [2SrO2:K [2:Sn [2:Sn [Additional p-doping is usually performed with Ca, Mg or occasionally with K, in order to increase the conductivity resulting in e.g., CuInO2:Ca ,187, Cu2Ca [2:Ca ,174, CuCCa [2:Mg ,148,198,Mg [2:Mg ,172 or C[2SrO2:K . N-type :K [2:Sn ,190 or ASn [2:Sn . FurtherSn [2:Sn .2-crystal, anisotropic electrical conductivity was detected in [2 and Cu are reported in [Because of the structural anisotropy of the CuAlOected in . Ohmic corted in .The crystal structures and chemistries are by far the best investigated topics in delafossite (semi)conductor research and systematically discussed in ,193; theReasons for technical advances in transparent conducting oxides are manifold—influencing aspects are: The investigation of adequate novel materials and material-combinations, as for example the first delafossites by Charles Friedel in 1873 ; an increasing financial support for research according to political decisions, as for example the increased financial support of solar cell investigations and therefore of TCOs by the present nuclear power phase-out in Germany; the publication of new results, as research groups in industrial companies often reserve important information; and the efficiency of modern literature data-bases, as only included literature can be found and selected.TCO rises until 2010 by about 100 a year. Until 2011, the number of citations per year decreases—not only because this literature research was done in November 2011.Therefore, technical advances in transparent conducting oxides may be illustrated researching the web of knowledge (Thomson Reuters). Applying e.g., the search item “TCO < name of element > oxide” leads to the carefully selected citation statistics, shown in 2O4 or Cd2SnO4, where H2-annealing is frequently applied to widen the energy gap [Despite these four elements, let us regard the next five metals, which exhibit the most average citations per year in TCO-related publications, see ergy gap ,204,205.2 and delafossites, see above.Copper (Cu) represents the group of doped and undoped CuOat), for ZnO and CdO. On the other hand Ga is the metallic part, A, of Ga2O3. Based on this, gallium zinc oxide (GZO: ZnGa2O4) is produced with 90%wt of Ga2O3 and 10%wt of ZnO. Moreover, aluminum gallium zinc oxide (AGZO) is a combination of aluminum zinc oxide (AZO) and GZO, respectively indium gallium zinc oxide (IGZO) a combination of IZO and GZO [Gallium (Ga) on the one hand is used as dopant, D  is exclusively used as dopant, with an atomic concentration of about 3% SnO2:Nb ,209.2O3 (IMO). MoO is also applied in layer stacks with silver, Ag [Molybdenum (Mo) is usually used in comparatively high conductive TCOs. Mo is a dopant for ZnO (MZO) or Inlver, Ag ,211,212.2O3), ITO and IGZO or lanthanum (La) [xSr1−xSnO3 have been discussed in the last few years.The upcoming importance of transparent conductive materials for thin film solar cells, opto-electrical interfaces, displays and opto-electrical circuitry widens the area of investigation. So, exotic dopants, such as sodium (Na)  and mangnum (La)  for stroFinally, ultra-thin metals without any oxygen content —as for example nickel (Ni)—have been applied as optical transparent conducting materials .Based on a modern database-system, aspects of up-to-date material selections and applications for transparent conducting oxides have been sketched; references for detailed information have been given for the interested reader. As n-type TCOs are of special importance for thin film solar cell production, indium-tin oxide (ITO) and the reasonably priced aluminum-doped zinc oxide (ZnO:Al) have been discussed with view on preparation, characterization and special occurrences. For completion, typical p-type delafossite TCOs have been described the same way, providing a variety of references, as a detailed discussion is not reasonable within an overview-publication. Moreover, absolutely unusual, novel TCO materials have been discussed and their presence and development in the world of science pointed out. Trends have been shown.As transparent conducting oxides are usually compound semiconductors—where the nonmetal part is oxygen—they have been discussed along their metal elements. Metals were used as compound materials or dopants (with just a few percent content).


Gn8.1, BPH-resistant genes Bph6 and Bph9, fertility restorer genes Rf3, Rf4, Rf5, and Rf6 through molecular marker assisted selection. Resistance analysis revealed that the newly developed restorer lines showed stronger BPH-resistance than any of the single-gene donor parent Luoyang-6 and Luoyang-9. Moreover, the three new restorer lines had broad spectrum recovery capabilities for Honglian CMS, Wild abortive CMS and two-line GMS sterile lines, and higher grain yields than that of the recurrent parent 9,311 under nature field conditions. Importantly, the hybrid crosses also showed good performance for grain yield and BPH-resistance. Thus, the development of elite BPH-resistant wide-spectrum restorer lines has a promising future for breeding of broad spectrum BPH-resistant high-yield varieties.Hybrid rice has contributed significantly to the world food security. Breeding of elite high-yield, strong-resistant broad-spectrum restorer line is an important strategy for hybrid rice in commercial breeding programs. Here, we developed three elite brown planthopper (BPH)-resistant wide-spectrum restorer lines by pyramiding big-panicle gene  Rf) genes that are genetically specific to some CMS type and different from each other . Thus, Gn8.1 has great potential in developing elite restorer lines because of its high yield potential and vast pollen for production of more F1 seeds. Brown planthopper (BPH) is one of the severe destructive insect pests in rice production  carrying dy Table . The broA seedling bulk test was conducted to evaluate the brown planthopper resistance of rice populations following previously described methods , plant height (PH), panicle number (PN), number of grain (NG), number of grain per panicle (NGP), spikelet fertility (SF), 1,000-grain weight (GW), and yield per plant (YP). The statistical analyses were performed with SPSS Statistics 20 .O. longistaminata carries big panicle gene Gn8.1, Luoyang-6 and Luoyang-9 carry Bph6 and Bph9, respectively, Shuhui527 carries restoring gene Rf3 and Rf4, and 9,311 carries restoring gene Rf5 and Rf6. During the breeding process, foreground selection was performed from generation F1 to BC3F3 to screen the plants having the desirable alleles of the seven target genes using molecular markers , and further backcrossed as maternal parents with 9,311. In the BC1 generation, 18 out of 890 plants were identified as our expected candidates after careful genotyping and phenotyping. Similarly, the selected BC1, BC2 plants were then backcrossed with 9,311 so as to eliminate the unexpected background. Then, 29 BC3F1 plants were selected for selfing until genetically stable in BC3F3. Finally, 22 stable BC3F3 lines with target genes were developed from 13,580 plants. In consideration of the integrative agronomic traits and the presence of homozygous marker alleles for the seven target genes, 16 lines were selected as potential candidates from the 22 BC3F3 progenies  as resistant control, and 9,311 (recurrent parent) as a susceptible control. After inoculated for 1 week, when all the 9,311 seedlings were dead, the pyramided lines were still green Figure . ResistaImportantly, comparing with the recurrent parent 9,311, these pyramided lines, especially line 05, 06, and 08 all showed excellent performance in the field test. The traits such as growth period, plant height, panicle number and 1,000-grain weight of the three pyramided lines were almost the same as that of 9,311. However, the panicle size and grain number of 05, 06, and 08 were significantly larger than that of 9,311. This is a desirable agronomic trait for increasing yield. As a result, even if the spikelet fertility of 05, 06, and 08 were slightly declined, their yield per plant were still increased by 77.3, 66.1, and 85.1%, respectively than that of 9,311  showed the same pollen fertility phenotype as the typical HL-type hybrid combinations Luohong-4A/9311 and Jiahong-2A/9311. Similarly, all of the WA-type and two-line hybrid combinations showed the same pollen fertility as their respective controls, and reached to almost 100% Figure . MoreoveTo determine if the newly developed BPH-resistant wide-spectrum restorer lines can be well used in hybrid rice production, 30 hybrid rice crosses were constructed by using R345, R346, and R348, and parent restorers 9,311 (for HL-CMS and two-line) and SH527 (for WA-CMS and two-line) to testcross HL-CMS lines Luohong-4A and Jiahong-2A, WA-CMS lines Yuexiang-A and Luofei-A, and two-line sterile lines BPH68S and Guangzhan4S, respectively. Rice yields and agronomic traits including plant height, panicle number, number of grains per panicle, spikelet fertility, and 1,000-grain weight were examined when matured Table . RelativSustainable development of hybrid rice depended greatly on the construction of elite CMS/GMS lines and restorers. Pyramiding multiple favorable traits in a commercial restorer is considered to be one of the most effective approaches to create elite restorer lines. However, it is often difficult to reach such a goal using traditional breeding methods because of the inefficient selection and unexpected linkage drags. Thus, the molecular assisted selection becomes an important strategy in rice breeding programs for the advantage of rapid recovery of the background of the recurrent parents  had higher resistance than Bph3 or Bph27(t) introgression lines (Liu et al., Bph14, Bph15, and Bph18 genes, similarly, showed higher resistance than those two genes pyramided lines, and the two genes pyramided lines also higher than single gene lines (Hu et al., Bph6 and Bph9 were selected as target genes against brown planthopper, due to their advantages in BPH-resistance. Of which, Bph9 shows durable and broad resistance to brown planthopper biotypes 1, 2, and 3 (Zhao et al., Bph6 is also a high BPH-resistant gene, especially against brown planthopper biotype 4 (Qiu et al., Bph6 and Bph9 and even their hybrid rice all showed higher resistance than any of the single-gene donor parents (Figure Bph6 and Bph9 together can confer rice broad, higher and durable resistance to all four brown planthopper biotypes, and these two genes had great potential in future hybrid rice breeding programs.Apart from high fertility restoring ability, strong resistance of the parents to diseases and insect pests are also important requirements for sustainable development of hybrid rice. As for brown planthopper, it is well recognized that the most efficient way to conquer this pest is to breed BPH-resistant rice by pyramiding multiple functional genes against brown planthopper. It is reported that rice carried We developed three elite BPH-resistant wide-spectrum restorer lines by multiple genes pyramiding through MAS. These new restorer lines significantly improved the resistance against BPH, and showed good performance in the two-line and three-line hybrid rice production. Therefore, it can be concluded that the development of elite BPH-resistant wide-spectrum restorer lines had a promising future for breeding of broad spectrum BPH-resistant high-yield varieties.SL, YZ, GH, and FF designed the research; FF, NL, YC, XL, HS, and JW performed molecular and field experiments; FF and SL analyzed data and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glioma-associated oncogene 1 (Gli1) is a critical transcriptional factor of Sonic hedgehog pathway which has been proved to participate in the initiation and progression of tumor in mammalians. However, its clinical value in breast cancer remains unknown. Thus, a meta-analysis was performed to clarify the association of Gli1 over-expression, clinic-pathological characteristics, molecular subtypes and prognosis in breast cancer. According to included criteria, 13 eligible studies containing 2816 patients all around the world were selected in this study. Our results indicated no significant association of Gli1 expression and histological grade , T stage , clinical stage  and lymph node metastasis . In addition, pooled RR showed no correlation of Gli1 expression and progesterone receptor (PR) , estrogen receptor (ER) , human epidermal growth factor receptor 2 (HER-2) . Nonetheless, up-regulated Gli1 expression predicts shorter disease-free survival (DFS) , 3-year survival , 5-year survival  and overall survival (OS) . In conclusion, over-expression of Gli1 tends to progressive stages and is related to unfavorable prognosis of breast cancer, which may become a potential prognosis indicator and therapy target in breast cancer. Worldwide, morbidity of breast cancer has been increased to 1.7 million in 2012. For females, breast cancer has the highest mortality rate among all cancer types, accounting for 15% of all cancer deaths . In metaSonic hedgehog (Shh) signaling pathway, one of the components of the hedgehog (hh) pathway, was originally known to play an important role in embryonic development, cell maturation including differentiation, proliferation and maintenance of tissue polarity –9. RecenHence, to clarify the association of Gli1 over-expression and clinicopathological features, molecular subtypes, and clinical outcomes in breast cancer, a meta-analysis was performed via acquired available published data.n = 387) and EMBASE (n = 319) according to the literature retrieval method mentioned above. Two independent investigators (Bilan Wang and Ting Yu) went through title and abstract of these articles and excluded 535 unrelated citations. Meanwhile, 134 studies were removed from scope because of duplicate data. The remaining 37 candidate studies were reviewed carefully by full text. Among them 12 were conference abstracts and 10 were not exploitable for survival data or clinic-pathological data associated with Gli1. In addition, 1 non-English (Chinese) and 1 review article were excluded as well. Ultimately, our study included 13 articles for further data extraction and analysis , while fixed-effects model was applied in clinical stage parameter analysis (I2 = 0.0%). To sum up, our study revealed Gli1 expression was not associated with histological grade, T stage, clinical stage or lymph node metastasis in breast cancer.We performed pooled analysis on correlations between Gli1 expression and a series of clinic-pathological parameters Figure . FirstlyI2 = 83.8%, I2 = 88.0%, I2 = 55.4%, respectively).Considering breast cancer is a typical hormone-related cancer, we performed pooled analysis on correlations between Gli1 expression and three specific immunohistochemical parameters  in breast cancer patients Figure . Our res2I = 54.2%). In all, our results indicated up-regulated expression of Gli1 was associated with poor survival outcomes involving DFS, 3-year survival, 5-year survival and OS.Survival outcomes of breast cancer with high and low Gli1 expression from 6 studies were extracted and analyzed Figure . Result p = 0.881, Egger: p = 0.994), T stage , clinical stage , lymph node metastasis , PR , ER , HER-2 , DFS , 3-year survival , 5-year survival  and OS .Both Begg's funnel plot and Egger's test were performed to evaluate the publication bias in all studies assessing histological grade, T stage, clinical stage, lymph node metastasis, DFS, 3-year and 5-year survival and OS, respectively  patients had a diagnosis of breast cancer; (2) expression level of Gli1 were determined with method such as IHC or reverse transcription-polymerase chain reaction (RT-PCR); (3) the studies provided direct hazard ratio (HR) and 95% confidence interval (CI) for survival or Kaplan-Meier survival curves at different Gli1 expression level; (4) the studies reported Gli1 expression according to different clinic-pathological parameters of patients. When there is possible duplication of data in several publications, we prefer the most recent or the most integrated study. Studies of letters, case reports, conference abstracts, editorials and reviews without original data; non-English papers; animal or laboratory studies were not in our scope.Two investigators reviewed eligible studies to obtain the following information: first author's name, year of publication, source of patients, sample size, mean age of patients, assay method, cut-off definition, TNM stage, histological grade, immunohistochemical features , Gli1 expression rate and survival data according to Gli1 expression. Disagreements were discussed by the two investigators and settled by consulting a third author to reach consensus. Moreover, quality assessments of all studies included were conducted by Newcastle-Ottawa-Scale (NOS) criteria. High quality studies refer to those scored 5 or above 5.I2 > 50%) or P < 0.10, random-effects model was utilized for data analysis, otherwise the fixed-effects model was utilized. Publication bias was evaluated by funnel plot with a Begg's test [RevMan 5.0 software  was applied to analyze extracted data and calculate RR. To investigate the impact of Gli1 expression on survival outcome, HR and 95% CI from multivariate Cox hazard models were used. If direct data were not available, then Kaplan-Meier survival curves analyses were adopted to yield survival data as what Tierney demonstrated . Furtherg's test . Asymmet


In CEFs, DEFs, and chickens infected intramuscularly, but not in infected ducks, mRNA expression levels of proinflammatory cytokines (interleukin-6 and -8) and interferon-stimulated genes (Mx and OAS) were significantly upregulated. Although some cytokines showed significant upregulation in the oral chickens, most did not change significantly. Finally, the duck retinoic acid-inducible gene I and its caspase activation and recruitment domain both had significant antiviral functions in CEFs, particularly after 24 h postinfection. Taken together, this research provides new insights into the interactions between FAdV-4 and the innate immune systems of studied hosts (chickens and ducks).Hydropericardium syndrome and inclusion body hepatitis, together called hydropericardium-hepatitis syndrome, are acute infectious diseases found in chickens. These diseases are caused primarily by fowl adenovirus serotype 4 (FAdV-4) strains. In this study, we isolated a FAdV-4 strain (SD0828) from clinically diseased chickens and phylogenetically analyzed the L1 loops of the hexon protein sequences in 3-week-old specific pathogen-free chickens and ducks infected intramuscularly and orally, determining differences in the pathogenicity by observing clinical signs and gross and histological lesions. We also detected the viral load in tissue samples. Postinfection necropsy showed that all chickens but no ducks exhibited typical necropsy lesions. Additionally, all chickens infected intramuscularly died within 2 days postinfection (dpi), and all those infected orally died within 5 dpi, whereas no infected ducks died before 28 dpi. Quantitative real-time polymerase chain reaction analysis was used to determine the viral load in the tissues of hearts, livers, spleens, lungs, and kidneys and in cloacal cotton swabs from infected chickens and ducks at 1, 2, 3, 5, 7, 14, 21, and 28 dpi. The greatest number of viral DNA copies was found in the livers of infected chickens, yet no virus was found in any samples from infected ducks. In addition, the viral load increased over time in both chicken and duck embryo fibroblasts ; in the former, replication speed was significantly greater than in the latter. Innate immune responses were also studied, both  Hydropericardium-hepatitis syndrome (HHS) is caused by fowl adenovirus serotype 4 (FAdV-4). It was first seen in the Pakistan Angara Goth in 1987; therefore, it has also been called “Angara Disease” . This seIn recent years, hydropericardium syndrome has been reported in Iraq, Kuwait, India, Mexico, Ecuador, Peru, Chile, Korea, Japan, and South and Central Americas , 11. SinThe first line of defense against microbial infection is a range of pattern recognition receptors (PRRs), consisting primarily of four families: Toll-like receptors (TLRs), nucleotide-binding oligomerization-domain-like receptors (NLRs), retinoic acid-inducible gene I (RIG-I)-like receptors, and C-type lectin families , 19. AftNumerous studies show that duck AdV can cause diseases in ducks. For example, it can induce decreased egg production and egg quality . A speci50) method  method .2.Duck embryo fibroblasts (DEFs), derived from 12-day-old SPF duck embryos, and CEFs were cultured in Dulbecco’s modified Eagle medium  that included 10% fetal bovine serum . They were incubated at 37°C in 5% (v/v) COTo evaluate FAdV-4-SD0828 pathogenicity in chickens and ducks, 91-day-old SPF White Leghorn chickens (Jinan) and an equal number of 1-day-old SPF Shao Xing ducks (Harbin) were used. All animals were housed in isolation and handled in strict accordance with the guidelines of the Shandong Agricultural University Animal Care and Use Committee. The approval number for this study is SDAU-2016-001.The viral DNA load of SD0828 in clinically diseased chicken livers was extracted using viral DNA kits  following manufacturer’s instructions. Specific polymerase chain reaction (PCR) primers for SD0828-F1 and -R1 were designed so that the entire viral hexon gene sequence could be identified Table . All FAdin vivo viral titer was determined to be 106.3 TCID50/mL. After three weeks of normal feeding, SPF chickens were randomly divided into three groups of thirty. Group I was infected with 0.1 mL viral stock via the intramuscular (IM) route. Group II was infected with the same volume of the same stock via the oral route. Controls were inoculated with the same volume of phosphate-buffered saline (PBS) via the IM route. At 1, 2, 3, and 5 days postinfection (dpi), three chickens from each group were euthanized, and the hearts, livers, spleens, lungs, kidneys, brains, and glandular stomachs were collected. Samples were fixed with 4% formalin at room temperature for histopathological examination while other parts were stored at −70°C for DNA and RNA extraction. In addition, the peripheral blood mononuclear cells (PBMCs) were also separation from the uninfected and infected chickens with the peripheral blood lymphocyte separation kit  for the RNA extraction. The cloacal cotton swabs of each group were collected at 1, 2, 3, and 5 dpi to test the viral loads. The treatments of SPF ducks were the same as the chickens except the sample collection times were at 1, 2, 3, 5, 7, 14, 21, and 28 dpi.The in vitro viral titer was determined to be 103.3 TCID50/mL. Both CEFs and DEFs were seeded in 6-well plates and cultured overnight at 37°C in 5% (v/v) CO2. Using 0.5 mL/well viral diluent, CEFs and DEFs were infected, incubated at 37°C for 2 h, then cultured with 2 mL Dulbecco’s modified Eagle medium . At 6, 12, 24, and 48 h postinfection (hpi), cells were harvested, and the RNA and DNA were extracted for detection of cytokine expression and viral load.The ® qPCR Master Mix  and the 7500 Fast Real-Time PCR System . A 20-µL PCR tube was used under these specific conditions: (a) one cycle predenaturation at 95°C for 5 min, (b) 40 cycles denaturation at 95°C for 10 s, and (c) extension at 60°C for 34 s. The dissociation curve was then analyzed, processing each sample in triplicate.The viral DNA loads in the chicken and duck hearts, livers, spleens, lungs, kidneys, and brains as well as CEFs and DEFs and cloacal cotton swabs were extracted as described. Quantitative real-time PCR (qRT-PCR) primers of FAdV-4 were designed as described in a previous study  Table 11, and ithttp://bioinfo.ut.ee/primer3-0.4.0/). The primers for duck β-actin, TLR3, TLR7, MDA5, RIG-I, IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, IL-8, OAS, PKR, and Mx genes were referenced to the previous study . Statistical significance was recognized when P < 0.05 or P < 0.01.The relative mRNA expressions of the target genes in the infected and control groups were calculated based on the 2As the most abundant viral surface protein, the L1 loop of the hexon contains the major antigenic determinants, and in previous studies, AdV was also genotyped by building the phylogenetic tree of this region , 9. In tAt 1 dpi, all chickens from Group I exhibited clinical symptoms including depression, lethargy, ruffling of neck feathers, and decreased feed intake Figure B. DuringAlthough SPF ducks underwent the same treatment (the same infectious doses and routes) as the SPF chickens, no clinical symptoms or gross lesions were found in either treatment group at any sampling point. In addition, ducks in the infected and control groups survived the entire study period (28 days) without clinical symptoms.via light microscopy under oil immersion  could be involved in the antiviral innate immune response, mRNA expressions of TLR3, TLR7, MDA5, RIG-I, IL-1β, IL-6, IL-8, IFN-α, IFN-β, IFN-γ, Mx, PKR, and OAS (ducks)/OASL (chickens) in infected livers, spleens, and PBMCs were examined. In Group I chickens, mRNA expressions of TLR3 and TLR7 showed similar trends in the liver and spleen, both significantly increasing at 1 dpi and returning to the baseline levels at 2 dpi . One possible reason could be the low infection dose; therefore, 3-week-old ducks were infected with a dose 20 times that used with the chickens, but signs of disease and clinical illness remained absent (data not shown). These results are consistent with those found in a previous study . HoweverIn infected chickens, SD0828 could quickly replicate in many tissues. Among them, the liver showed the highest viral load followed by the spleen. Viral loads in various tissues were consistent with those found in a previous study , 39. Alsin vivo and in vitro experiments, we gain a better understanding of SD0828’s pathogenicity. As our results show, the expression trends of PRRs, cytokines, and ISGs are similar between the tissues from IM-infected SPF chickens and CEFs. The most obvious changes were the highly significant upregulations of IL-6, IL-8, Mx, and OASL. As seen with the major proinflammatory cytokines, the highly significant upregulated expressions of IL-6 and IL-8 induced severe inflammatory responses in infected tissues, eventually leading to death at a rapid rate. This is consistent with responses induced by avian influenza and duck Tembusu viral infections  could trigger death. This could explain why the viral titer in CEFs is greater than that in DEFs.All animals used in this study were handled in strict accordance with the guidelines of Shandong Agricultural University Animal Care and Use Committee. The approval number was SDAU-2016-001.RL and GL performed most of the experiments, wrote the manuscript, and made pathological sections. JL and SH collected samples and extracted the tissues RNA and DNA. XH and HW kept animals. MG performed the data calculation with support from ZL and NL. YS reviewed and polished the article. TC and LW designed the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis pumila is an ephemeral plant, and a close relative of the model plant Arabidopsis thaliana, but it possesses higher photosynthetic efficiency, higher propagation rate, and higher salinity tolerance compared to those A. thaliana, thus providing a candidate plant system for gene mining for environmental adaption and salt tolerance. However, A. pumila is an under-explored resource for understanding the genetic mechanisms underlying abiotic stress adaptation. To improve our understanding of the molecular and genetic mechanisms of salt stress adaptation, more than 19,900 clones randomly selected from a cDNA library constructed previously from leaf tissue exposed to high-salinity shock were sequenced. A total of 16,014 high-quality expressed sequence tags (ESTs) were generated, which have been deposited in the dbEST GenBank under accession numbers JZ932319 to JZ948332. Clustering and assembly of these ESTs resulted in the identification of 8,835 unique sequences, consisting of 2,469 contigs and 6,366 singletons. The blastx results revealed 8,011 unigenes with significant similarity to known genes, while only 425 unigenes remained uncharacterized. Functional classification demonstrated an abundance of unigenes involved in binding, catalytic, structural or transporter activities, and in pathways of energy, carbohydrate, amino acid, or lipid metabolism. At least seven main classes of genes were related to salt-tolerance among the 8,835 unigenes. Many previously reported salt tolerance genes were also manifested in this library, for example +-ATPase, NHX1, SOS2, SOS3, NAC, MYB, ERF, LEA, P5CS1VP1, H. In addition, 251 transcription factors were identified from the library, classified into 42 families. Lastly, changes in expression of the 12 most abundant unigenes, 12 transcription factor genes, and 19 stress-related genes in the first 24 h of exposure to high-salinity stress conditions were monitored by qRT-PCR. The large-scale EST library obtained in this study provides first-hand information on gene sequences expressed in young leaves of A. pumila exposed to salt shock. The rapid discovery of known or unknown genes related to salinity stress response in A. pumila will facilitate the understanding of complex adaptive mechanisms for ephemerals. Salinity is a serious problem worldwide, with more than 800 million hectares of land across the world estimated to be affected by high salinity . FurtherAs sessile organisms, plants are subjected to various environmental stresses, such as drought, salinity, and temperature extremes, during their life cycle. Soil salinity is one of the major abiotic factors constraining plant growth and crop production. About 100 million hectares of land in China is exposed to primary salinity or secondary salinity, accounting for approximately 10% of the world’s saline and alkaline land resources. As the largest province in northwest China, Xinjiang occupies one-sixth of the land of China; however, more than one-third of the land in Xinjiang suffers from primary of secondary salinization . Therefo+ and Cl- ions through the plant root system , a brassicaceous spring ephemeral plant, is closely related to the model plant A. thaliana, but is adapted to more stressful environments, and it has a high propagation rate , which are short, single-pass sequence reads from cDNA, have proven to be an efficient and rapid strategy to identify novel genes involved in tolerance to environmental stress. Large-scale cDNA sequencing and EST analyses have been successfully used to identify genes that may be related to stress tolerance in a number of salinity-adapted species such as da salsa , Avicenna marina , Thellunalophila , Salicorrachiata , Suaeda ragoides , and Gosrbadense .A. pumila plants exposed to 500 mM NaCl shock for 14 h by using the gateway technology . The EST database obtained will provide new insights into salt-adaptive mechanisms of Arabidopsis pumila seeds were surface-sterilized by soaking in 70% (v/v) ethanol for 2 min, and then in 2.8% (w/v) sodium hypochlorite solution containing 0.1% surfactant  for 30 min, and finally rinsed five times with sterile distilled water. Seeds were stratified for 3 days at 4°C in darkness to synchronize germination, and then plated on Petri dishes with half-strength Murashige-Skoog (MS) salt mixture , 1% (w/v) sucrose, and 0.8% (w/v) agar. Petri dishes were then placed in a illumination incubator at 22°C under long-day (LD) conditions (16 h light/8 h dark). After 7 days, the seedlings were transplanted into pots containing peat soil and vermiculite (1:1) and kept in a plant growth chamber with a 16-h photoperiod, and the light intensity for A pumila growth is 200 μmol m-2 s-1. To examine gene expression during the salt shock, The 4-week-old plants were watered with 0.5 × MS nutrient solution supplemented with 500 mM NaCl as described previously .All sequences were cleaned by removing the low-quality sequences, such as the chimeric clones, the contaminating sequences, the repeat sequences, and poly(A) tails. Then the vector sequences were eliminated using VecScreen program3, and the largest ORF sequences were used for functional analysis. All the unigenes were then aligned to protein databases like NCBI non-redundant protein (Nr) database, Protein Sequence Database (Swiss-Prot)4, and Cluster of Orthologous Groups of Proteins (COG)5 by blastx , and the NCBI non-redundant nucleotide database (Nt) by blastn .To get their putative functional information, all the unigenes were annotated. First, all the unique sequences were searched for putative open reading frames (ORF) with the program ORFfinder6  to identify putative transcription factors  factors .Total RNA were extracted using RNeasy Plant Mini Kit  and treated with RNase-free DNase (Qiagen) following the manufacturer’s instructions. The quality and quantity of total RNA were monitored using ND-1000 spectrophotometer .Supplementary File S1) were designed using Primer Premier 6.0 software . qRT-PCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System  in a 25-μl volume containing 10 ng of cDNA, 5 pM of each primer, and 25 μl of Fast SYBR Green Master Mixture  according to the manufacturer’s protocol. Primers of Actin2-F (5′-CACCGTGAGTGGCAAAGAAGGGA-3′) and Actin2-R (5′-AACGACCTTAATCTTCATGCTGC-3′) were used to amplify Actin2 gene  according to the manufacturer’s instructions. Gene-specific primers for qRT-PCR analyses  contigs and 6,366 (72.1%) singletons. The average length of the unique sequences was 794 bp, ranged from 200 to 2,610 bp. The detailed information of these ESTs was summarized in Table 1.A total of 19,923 cDNA clones randomly selected were successfully single-pass sequenced from the 5′ terminal, generating 16,014 high-quality ESTs (80.4%) with an average length of 546 bp after cleaning. All of these ESTs sequences have been deposited in GenBank with accession number of Figure 1B. Of the 2,469 contigs, 1,335 (54.1%) contained 2 ESTs, 475 (19.2%) contained 3 ESTs, 209 (8.5%) contained 4 ESTs, 119 (4.8%) contained 5 ESTs, and the remaining 13.4% contained 6 or more ESTs. The results showed that the redundancy in the normalized library was relatively low, confirming that the cDNA library was normalized well for EST generation.Expressed sequence tags per contig averaged at 3.9 in number, while the highest number reached 72. The distribution of EST frequencies after clustering was shown in Table 2 and Supplementary File S2. The abundant transcripts associated with photosynthesis were obtained such as chlorophyll a/b binding protein (CAB) , light-harvesting complex II chlorophyll a/b binding protein (LHC) , ribulose bisphosphate carboxylase (RuBisCO) small chain (RBCS)  (S-transferase 16 (GSTF3)  involved in defense against reactive oxidative species, metallothionein-like protein (MT1C)  involved in response to copper ion (The clusters that contain more than 30 ESTs represent the most abundant unigenes and their annotations were summarized in the 19 ESTs) , suggestpper ion , catalaspper ion . Similarpper ion .Figure 2, of the selected 12 genes, the transcriptions of 11 genes, including CAB1 (Contig5032), LHB1B2 (Contig5030), RBCS1A (Contig5029), CAT3 (Contig5024), LHCA3 (Contig5022), RCA1 (Contig5021), MT1C (Contig5014), CAB2 (Contig5017), LHCA3 (Contig5011), GSTF3 (Contig5012), and RD19 (Contig5007), were truly abundant and significantly up-regulated at 14 h of treatment.To determine whether the putative enriched genes were highly expressed, their expression levels were detected under control and 500 mM NaCl shock conditions by qRT-PCR. As shown in Supplementary File S3), and the average ORF was 558 bp. The translated peptide sequences had a length ranging from 33 to 754 amino acids, with an average size of 185 amino acids.The prediction of ORFs revealed a high amount of putative proteins. Of 8,835 unique sequences, 8,585 (97.2%) were able to detect ORFs that were longer than 100 bp, with the longest ORF of 2,265 bp  of annotated sequences had similarity with other plant species .The blastx search revealed that there were 8,436 (95.5%) unigenes showing significant similarity to proteins in NCBI Nr database. Of the 8,436 unigenes, 8,011 (95%) showed similarities to proteins of known function, and only 425 (5%) showed similarities to predicted proteins of unknown function. In species distribution analysis, 4,255 (50.4%) and 3,960 (46.9%) sequences had best blastx hit with Table 3). The “general function prediction only” category represented the largest group , followed by the categories of “posttranslational modification, protein turnover, chaperones” , “translation, ribosomal structure and biogenesis” , “energy production and conversion” , “carbohydrate transport and metabolism” , “amino acid transport and metabolism” , “lipid transport and metabolism”  and “inorganic ion transport and metabolism” . In addition, 79 (2.5%) unigenes were assigned to the category of “function unknown.” Whereas, only three sequences were assigned into “cell motility,” and one were assigned into “nuclear structure” category, respectively. A number of functional annotations in these 24 COG categories, for example, “energy production and conversion,” “inorganic ion transport and metabolism,” “signal transduction mechanisms,” and “defense mechanisms” are closely related to salt stress.Because the COG database is assumed to be an useful platform for genome-scale analyses for functional annotation of newly sequenced genomes and evolution from the macro , we thenFigure 4). Within the CC category , cell (32.1%) and cell part (32.1%) are most enriched, followed by organelle (14.5%) and macromolecular complex (14%). In the MF categories , binding (40.5%) and catalytic activity (39.8%) are the most enriched; proteins having structural molecular activity (6.9%) and transporter activity (4.8%) are dramatically enriched compared to other terms.A total of 5,500 (62.3%) unique sequences belonged to one or more GO ontologies, of which 1,761 (19.9%), 4,934 (55.8%), and 3,858 (43.7%) unigenes were assigned the GO categories CC, MF, and BP, respectively. A total of 1,256 (14.2%) unigenes were categorized into three ontologies. The GO categories CC, MF, and BP fell predominantly into three or four subcategories , metabolic process (38.3%) and cellular process (31.2%) accounted for the highest proportion, suggesting a high basic metabolic activity existed in the NaCl-stressed tissues; localization (6.6%), establishment of localization (6.5%), biological regulation (5.7%) and pigmentation (5.5%) were dominantly enriched; response to stimuli (2.1%) at the third level were also enriched.Gene Ontology biological process is helpful for the functional classification of the analyzed genes. In the BP category , including metabolism , genetic information processing , environmental information processing , cellular processes , organism system , human diseases . The largest group, for example, metabolism was represented by 3,405 unigenes, including energy metabolism (22% of metabolism), carbohydrate metabolism (21.3% of metabolism), amino acid metabolism (15.9% of metabolism), lipid metabolism (7.5% of metabolism), and biosynthesis of other secondary metabolites (4.2% of metabolism). GIP was the second largest group, with a majority of encoding proteins involved in translation (43.5% of GIP), and folding, sorting, and degradation (31.4% of GIP). In the category of EIP, signal transduction (65.8%), were the majority as oppose to signal molecules and interaction (26.7% of EIP), and membrane transport (7.5% of EIP). Transport and catabolism (49% of CP) and cell growth and death (24.6% of CP) constituted the majority of CP category. The major constitutions of OS were immune system (23.2%), endocrine system (21.1%), and environmental adaptation (16%).To understand the classification of pathway mapping for A. thaliana  (Supplementary File S4), which are classified into 42 families. The most abundant TF family was the ERF group , followed by NAC , C2H2 , bHLH , bZIP , GRAS , G2-like , WRKY , HD-ZIP , MYB , and MYB related  (Table 5).Plant transcription factor database (TFDB 4.0) consists of 165 species covering the main lineages of the green plants . TFs ideTable 6.During the past few years, many genes related to salt tolerance or involving in response to salt stress have been discovered in a large number of plant species, and several excellent reviews and research articles are available . AccordiTable 5) to determine their expression levels by qRT-PCR. The results showed that they were up- or down-regulated in response to high-salt stress, including one ERF (ERF72) gene, two NAC genes (NAC019 and NAC026), one C2H2 gene (ZAT12), two bHLH genes (PIL1 and bHLH115), three WRKY genes , two HD-ZIP genes (HB-12 and HB-16), one MYB gene (MYB2). As shown in Figure 5, ERF72, NAC019, PIL1, NAC026, WRKY75, MYB2, and HB-12 were obviously up-regulated at 14 h of 500 mM NaCl shock.To validate our ESTs, we first selected 12 genes from the most abundant 11 groups of TFs , including four osmolyte biosynthesis genes , 6 transmembrane transporters and ion homeostasis genes , 2 reactive oxygen scavengers genes (MT2B and TRXH3), 5 stress proteins genes , and 2 signaling components genes (CPK13 and CML13). Of the 19 salt-induced genes, P5CS1, NHX1, AER, and TRXH3 were dramatically up-regulated at 14 h of salt stress.Furthermore, another 19 salt stress related genes from the seven groups in A. pumila, an ephemeral plant, has a good ecological adaptability with sensitive responsiveness to environmental changes during the growth and development stages. It is not a halophyte like salt cress , nor a glycophyte like A. thaliana. Although it does not possess salt glands, A. pumila has very good salt tolerance, thus providing a good insight into the adaptive mechanisms of salt stress in the Brassicaceae  and function annotations (Table 6). Many previously reported salt-related genes were also manifested in this library, for example VP1, NHX1, SOS2, SOS3, NAC, MYB, ERF, LEA, P5CS1, and others. The identification of putative salt stress-related genes in the dataset would be helpful in revealing the adaptation mechanisms for A. pumila in response to environmental stimuli.Further analyses showed that a large number of ESTs were related to biotic and abiotic stimulus responses according to the GO biological process category , but so were genes related to salt stress (Table 6), indicating that the salt shock treatment might suppress the process of photosynthesis and induce the salt stress defenses in A. pumila.The NaCl shock conditions applied to the plants used in the preparation of the cDNA library are effective in inducing the typical salt-tolerance process not only in halophytes but also in non-halophyte , and theP5CS1 and ALDH10A9, . Many ESTs related to proline synthesis were also found in the EST database. Under high salinity shock conditions, the expression of P5CS1 (Contig4970), encoding delta-1-pyrroline 5-carboxylase, a bifunctional enzyme for proline biosynthesis, was induced early at 0.5 h, continued to increase over the 24 h period .High levels of salinity (200–500 mM) can cause osmotic shock and plasmolysis in plant root cells, so osmotic and ionic stresses are generally regarded to be the two main components of salt stress or shock . After eALDH10A9 (Contig2945), which is functionally involved in the synthesis of the osmolyte glycine betaine, was also up-regulated during response to salt shock. Many ESTs related to sugar synthesis were present in the library. Previous study had found that trehalose functions as an osmoprotectant and prevents abiotic stress from damaging organisms (TPS10 (Contig2925), encoding a trehalose biosynthesis enzyme, was up-regulated at 0.5 h and 9 h of salinity shock. In A. thaliana, an alkenal reductase (AER) homolog was possibly involved in NAD/NADH homeostasis and played a role in antioxidant defense, but its molecular mechanism remains unclear .Expression of betaine aldehyde dehydrogenase gene rganisms . Trehalorganisms . The exp unclear . Express+ from the cytoplasm into the apoplast, and sequestering of Na+ from the cytosol to the vacuole by using specific plasma membrane and vacuole sodium/hydrogen antiporters . It is well documented that the plasma membrane and tonoplast NHX (+-adenosine triphosphatase (PM-H+-ATPase), the tonoplast H+-ATPase (V-ATPase) and the H+-inorganic pyrophosphatase    regulate, and VP .A. pumila, a vacuolar Na+/H+ antiporter gene NHX1 (JZ944758), CBL-interacting serine/threonine-protein kinase 9 , CBL-interacting serine/threonine-protein kinase 6 , multidrug resistance protein ABC transporter family (Contig4793) were present; +-ATPasePM-H (Contig4385), V-ATPase (Contig4804), VP1 (Contig4134) and several related ATPase (Contig2566 and Contig3622) were also present (Table 6). In A. pumila, NHX1 and VP1 expression were significantly up-regulated under high salt conditions, and the highest expression of NHX1 was observed at 9 h, whereas for VP1, it was 3 h and for V-ATPase it was 0.5 h, respectively . However, +-ATPasePM-H expression was only up-regulated after 0.5 and 24 h of NaCl treatment, and even down-regulated at 3 and 9 h. Thus, PM-H+-ATPase may be less important physiologically than VP in A. pumila under salt shock. Although, >16,000 cDNA clones were sequenced, some genes may be expressed at too low a level to be identified. The plasma membrane-localized Na+/H+ antiporter SOS1  might be involved in controlling the speed of water flux across cellular membranes under salt stress to help sustain osmotic homeostasis . qRT-PCRCAT3 (Contig5024) contained 53 ESTs, GSTF3 (Contig5012) contained 37 ESTs and GST1 (Contig4958) contained 15 ESTs (Table 2). These genes were also reported in the EST database of the halophytes Thellungiella salsuginea .Salinity stress leads to increases in amount of ROS such as singlet oxygen, peroxides, superoxide, hydroxyl radical, and hydrogen peroxide , resultilsuginea  and Salirachiata , as wellrachiata . qRT-PCRCitrus sinensis (Glycine soja (MTP-2B transcript (Contig4911) was up-regulated after 0.5 h of salt shock .Metallothioneins (MT) are a group of cysteine-rich, low-molecular-weight metal-binding proteins that are thought to be involved in metal ion metabolism and detoxification . MT-likesinensis , and theine soja . The MTPTRX3 (Contig4961) gene in A. pumila was rapidly up-regulated in response to salinity shock, and peaked at 3 h.In plants, thioredoxin plays a wide range of roles in many important biological processes, ranging from photosynthesis to growth, flowering and the development and germination of seeds, as well as cell-to-cell communication . It is aTable 6). Analyzing gene expression time-courses in response to high salinity shock revealed that RD21a transcription peaked at 14 h, and LEA peaked at 9 h, whereas PIP1B and HSP81-4 transcription peaked at 3 h .Under salt shock or stress conditions, many stress proteins were also produced to enhance salt tolerance . It was 2+, which is involved in stress signal transduction as reported by a number of authors  was slightly up-regulated as early as 0.5 h of salt treatment, peaked at 14 h and then returned normal levels at 24 h .Calmodulin (CaM) is an intracellular target of the secondary messenger Ca authors . Once bophatases . The expCPK gene has been shown to increase tolerance to low temperature, drought, and high salt in rice (CPK13 (Contig2514) gene was highly expressed after 9 h of salt treatment but returned to normal levels after 24 h. Altogether, time-course analyses of the expression of the genes studied confirm that salinity tolerance phenomena in A. pumila involve sequential events starting with the expression of salt-responsive genes, the primary induction of key genes, followed by expression of other genes.Calcium-dependent protein kinase (CPK) is involved in the strengthening of cell walls and in early stages of signal transduction, and it is a component of the immediate response to osmotic shock/plasmolysis . Overexp in rice . The CPKcis-acting DNA sequence common to the enhancer regions of specific functionally related genes (specific TFs) or by forming a large transcription preinitiation complex that interacts with RNA polymerase directly in all genes   . In plann factors, whereaselements .A. pumila (Supplementary File S4). Among these, high-frequency of TF families included ERF, NAC, C2H2, bHLH, bZIP, GRAS, G2-like, WRKY, HD-ZIP, and MYB (Table 5). Transcript levels of ERF72, PIL1, NAC019, WRKY53, HB-16, NAC026, WRKY75, MYB2, and HB-12 were significantly up-regulated in response to salinity shock .In the current study, 251 putative TFs (representing 2.8% of unigenes) were identified from NAC gene family is one of the largest plant-specific transcription factor families. Increasing evidence indicates that NAC domain-containing proteins are also involved in plant response to biotic and abiotic stresses. In A. thaliana, ANAC019, ANAC055, and ANAC072 were markedly up-regulated by drought, salt, and ABA treatment, and consequently improved plant drought resistance .sistance . SimilarA. thaliana, can regulate plant responses to multiple abiotic stresses (A. pumila bHLH15 (Contig280) is homologous to AtHLH15, which responds to salt stress  was markedly up-regulated at 14 h of NaCl treatment .bHLHs, which represent the second-largest type of transcription factor in stresses . As fount stress . The WRKt stress . Over-exn plants . A. pumiA. pumila MYB2 (Contig4149) was clearly up-regulated at 14 h of salt shock. Taken together, these results indicated that TFs may contribute to the regulation of salt response in A. pumila, and lay the foundations for further investigations into the functions of TFs in A. pumila under salt stress.MYB TFs comprise one of the largest TF families and are involved in controlling various processes, including biotic and abiotic responses. AtMYB2 possesses an R2R3 MYB DNA-binding domain and is known to regulate the expression of salt- and dehydration-responsive genes . A. pumiA. pumila is a valuable resource, which will facilitate the comparative genomics study of salt stress response among green plants.The database  obtained from XH conceived and designed the experiments and performed the cDNA clones sequencing experiments. LY analyzed the ESTs data. YJ performed the gene expression experiments. JL and FL help to analyze the ESTs data. XH wrote the manuscript. All the authors discussed the results and contributed to the manuscripts.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Psoriasis is a chronic inflammatory skin disease, the immunologic model of which has been profoundly revised following recent advances in the understanding of its pathophysiology. In the current model, a crosstalk between keratinocytes, neutrophils, mast cells, T cells, and dendritic cells is thought to create inflammatory and pro-proliferative circuits mediated by chemokines and cytokines. Various triggers, including recently identified autoantigens, Toll-like receptor agonists, chemerin, and thymic stromal lymphopoietin may activate the pathogenic cascade resulting in enhanced production of pro-inflammatory and proliferation-inducing mediators such as interleukin (IL)-17, tumor necrosis factor (TNF)-α, IL-23, IL-22, interferon (IFN)-α, and IFN-γ by immune cells. Among these key cytokines lie therapeutic targets for currently approved antipsoriatic therapies. This review aims to provide a comprehensive overview on the immune-mediated mechanisms characterizing the current pathogenic model of psoriasis. Plaque-type psoriasis is a chronic inflammatory skin disease involving both the innate and the adaptive immune compartments, crosstalking with skin tissue cells. The interaction between hyperproliferative keratinocytes (KCs), inflammatory dendritic cells (DCs), neutrophils, mast cells, and T cells, induces to the development of psoriatic lesions, clinically characterized by sharply demarked, erythematous, and scaly plaques. In the last three decades, the pathogenic model for psoriasis has been profoundly revised according to a broader and deeper understanding of the immune mechanisms leading to plaque formation. Before the late 1990s, there was a debate on whether KC proliferation was due to intrinsic KC defects triggering an immune response or, viceversa, whether KC hyperproliferation was a secondary phenomenon induced by immune activation and inflammation. In 1995, a milestone study demonstrated psoriatic plaque resolution following selective apoptosis of activated T cells, without affecting KC survival or activation, thus demonstrating the crucial role of the immune system, particularly of T cells, in the disease ,3,4,5. IMore recently, the clinical interest in psoriasis-related comorbid conditions has fueled the investigation on immune-mediated circuits or mediators that could link psoriasis to its comorbidities. The view of psoriasis shifted from a mere skin disease to a skin disorder associated to systemic inflammation and a wide array of concomitant conditions, mirroring other tissue-specific diseases with systemic implications such as Crohn’s disease, and rheumatoid arthritis  . Hence, A large plethora of immune cells contribute, to different extents, to the pathogenesis of psoriasis. In this section, we will illustrate the role and the most relevant supporting evidence of each cell type.The role of T cells in the pathogenesis of psoriasis has been well described, and both CD4+ T cells  and CD8+ T cells  seem to be critical in the development of the skin lesions ,33,34,35Overall, in human lesional skin as well as in the bloodstream the number of both CD4+ and CD8+ T cells is increased ,32,34,35Based on their cytokine production, multiple subsets of CD4+ lymphocytes (Th) have been identified within the cellular infiltrates: Th1, Th17, Th9, follicular Th, and Th22 cells, as have their CD8+ counterparts (Tc). Specifically, Th1 and Tc1 peculiarly show (i) signal transducer and activator of transcription 1 (STAT1) and T-bet expression as signature transcriptional factors ; (ii) reRecent studies have revealed that the majority of IL-17-producing T cells in both human and murine psoriasis express the γδ T cell receptor ,55. ThesVarious subtypes of DCs can be detected in normal and pathological skin . Howeverprimum movens in psoriasis pathogenesis: first, IFN-α regulates the development and maturation of T cells and myeloid DCs, that markedly express the IFN receptor . T. T89]. TThe early steps of the pathogenic cascade consists in the activation of IFN-α-producing pDCs triggered by TLR agonists, and/or the activation of autoreactive T cells producing IFN-γ, and other key-cytokines such as IL-17. While autoreactive T cells may potentially initiate the pathogenic process, leading to the psoriatic phenotype, pDCs, through their IFN-α-production, stimulate mDC to become highly inflammatory dermal DCs producing TNF-α, IL-23, IL-20, and NO. Besides IFN-α, they may be also stimulated by TSLP and TNF-α. Their IL-23 production stimulates IL-17 producing cells, which include Th17, Tc17, γδ T cells, ILC3, mast cells, and neutrophils. IL-17, in cooperation with other cytokines such as TNFα and IL-22, induces the development of the psoriasis phenotype through tissue cell activation. The most relevant tissue response is provided by keratinocytes that, releasing chemokines and other pro-inflammatory molecules (AMPs), sustain skin inflammation.Nowadays, psoriasis is the best-studied immune-mediated skin disease. Multiple cytokine-mediated signaling pathways can be traced within the psoriasis transcriptome, although just a few turned out to be crucial for the development of the psoriasis phenotype, with their blockade being therapeutically advantageous. The classic view of psoriasis pathogenesis was established on the IL-12/Th1 pathway but has now been profoundly revised, and “under the IL-17 light”, the pathogenic role of IFN-γ has been reconsidered, placing it in the early steps of the psoriatic cascade, and acting as an activating factor for antigen-presenting cells. The current pathogenic model is centered on the IL-23/IL-17 axis, and it is being dynamically integrated and remodeled according to new acquisitions, such as the recent identification of autoantigens and autoreactive T cells. However, many aspects still need to be elucidated, and their clarification will help to better understand the pathology of psoriasis and to improve the therapeutic strategy against this disease.


NF2, which is disrupted by mutation or loss in about 50% of tumors. Besides NF2, several recurrent driver mutations were recently uncovered through next-generation sequencing. Here, we performed whole-genome sequencing across 7 tumor-normal pairs to identify somatic genetic alterations in meningioma. As a result, Chromatin regulators, including multiple histone members, histone-modifying enzymes and several epigenetic regulators, are the major category among all of the identified copy number variants and single nucleotide variants. Notably, all samples contained copy number variants in histone members. Recurrent chromosomal rearrangements were detected on chromosome 22q, 6p21-p22 and 1q21, and most of the histone copy number variants occurred in these regions. These results will help to define the genetic landscape of meningioma and facilitate more effective genomics-guided personalized therapy.The major known genetic contributor to meningioma formation was  Meningiomas are the most common primary intracranial neoplasms in adults, accounting for 35.8% of all primary central nervous system (CNS) tumors diagnosed in the US , 2. In CNF2) on chromosome 22, encoding the protein Merlin. Loss of NF2 is found in approximately 50% of sporadic meningiomas [TRAF7, KLF4, AKT1, SMO, PIK3CA, NOTCH2, SMARCB1, CHEK2, SMARCE1 and POLR2A, particularly in the remaining half of meningiomas with wild-type NF2 [Previously, the only genetic driver of meningiomas to be identified was bi-allelic mutation or loss of the tumor suppressor gene neurofibromatosis 2  were also detected in this study. Especially, we identified many novel mutations and copy number variants in meningioma.To discover more candidate mutations, here we used whole-genome sequencing approaches on a set of 7 primary unradiated grade I meningiomas and paired normal blood samples. As a result, most of the previously reported meningioma mutations  were identified through whole-genome sequencing of the seven paired meningioma samples, including 103,289 inserted and deleted sequences (indels) and 290,389 single nucleotide polymorphisms (SNPs) . Among tNF2, CHEK2 and SMARCB1, and all the three tumor suppressors were deleted in the three cases  were observed. Notably, three non-coding RNA  were recurrently amplified. Of the 1,341 coding-changing mutations, several known meningioma-driver mutations  and a handful of novel candidate genes  were identified  and two known meningioma-driver mutations (AKT1E17K and SMARCB1R377H) were also detected. All the seven cases harbored mutations in MUC4 and six cases carried mutations in MUC16, however the mutation sites are almost different from each other . Of the three cases, case 5 also harbored a truncation mutation in NF2 and a mutation in SMARCB1. Case 7 co-occurred mutations in CHEK2, NOTCH2, TRAF7 and AKT1. Case 4 carried copy number alterations in two histone members, as well as candidate mutations in TRAF7, AKT1, KMT2C and ATF2.As shown in Table NF2 wild-type meningiomas which also lack other known meningioma-driver mutations: i) Case 2 contained deletions of BARD1, SMARCAL1 and two histone members, as well as mutations in NF1, DDR1, BCL11A and ATF6B; ii) Case 3 harbored deletions on Chromosome 2q containing CASP8, CASP10, BARD1 and SMARCAL1, and amplifications on Chromosome 6p containing DAXX and three histone members; iii) Case 6 harbored amplifications on Chromosome 6p containing NOTCH4 and four histone members.Of the remaining three In this study, numerous SNVs and CNVs have been identified by whole-genome sequencing. Of these SNVs, a high prevalence of C>T base transversions were observed. Different cancer types have different mutational signatures. For example, Lung adenocarcinoma and lung squamous cell carcinoma contain increased C>A transversions , while mMUC4, MUC12, MUC16 and MUC3A, were frequently observed. Mucins are a family (over 20 members) of high molecular weight, heavily glycosylated proteins produced by epithelial tissues. Increased mucin expression, especially MUC1 and MUC4, occurs in many adenocarcinomas [MUC16, also known as carcinoma antigen 125, is a prominent biomarker of ovarian cancer. Next-generation sequencing also found frequent mutations in several MUCIN family genes in a variety of cancer types [MUCINs mutated so frequently and their functions in the genesis and development of meningioma.Many candidates, not previously implicated in meningioma, have been uncovered in the current analysis. Of the identified somatic mutations, it is interesting that mutations in several mucin members, including rcinomas . MUC16, HDAC1, HDAC10, KMT2C, KDM4A and KDM1A), and other chromatin regulators . These chromatin regulators are the major category among all the identified alterations. AR42, a novel histone deacetylase inhibitor, has been reported to be a candidate drug for vestibular schwannoma and meningioma [Notably, we identified recurrent mutations or alterations in multiple chromatin regulators, including multiple histone members, histone-modifying enzymes  of the West China Hospital (File No. SKLB20140830-02), Sichuan, China, and informed consent were obtained from all the parents. All experiments were performed in accordance with relevant guidelines. All samples were fresh-frozen primary resections from individuals who were newly diagnosed as meningioma and hadn't treated with chemotherapy or radiation previously . GenomicLibrary construction (5 μg DNA), whole-genome sequencing and data analysis were carried out by WuXi AppTec, China. Briefly, DNA was sheared with Covaris S220 Sonicator (Covaris) to a target of 300–400 bp average size. Fragmented DNA was purified using Sample Purification Beads (Illumina). Adapter-ligated libraries were prepared with the TruSeq Nano DNA Sample Prep Kits (Illumina) according to Illumina-provided protocol. DNA concentrations of the resulting sequencing libraries were measured with the Qubit 2.0 fluorometer dsDNA HS Assay (Thermo Fisher Scientific). Quantities and sizes of the resulting sequencing libraries were analyzed using Agilent BioAnalyzer 2100 (Agilent). The libraries were used in cluster formation on an Illumina cBOT cluster generation system with HiSeq X HD PE Cluster Kits (illumina). Paired-end sequencing was performed by using an Illumina HiSeq X™ Ten following Illumina-provided protocols for 2×150 paired-end sequencing ). Data quality control, alignment with UCSC hg19, variant  calling, annotation, statistics and filter were performed by WuXi AppTec with public tools and databases including MuTect, VarScan2, GATK, CGATools, IGV, Circos, SAMtools, Polyphen2, SIFT, DBSNP, 1000genomes and COSMIC as previously reported [http://www.cbioportal.org/). Mutation profiles of a single gene across various tumor types were also generated via MutationMapper tools from cBioPortal. Gene interaction network and Gene Ontology (GO) enrichment were analyzed by STRING online tools (http://string-db.org/).Visualization of the distribution of different CNVs/SNVs subtypes were performed by using OncoPrinter tools from cBioPortal (