Our study showcases a comprehensive profiling of chaperome shifts in human cancers and sets the stage for a systematic global analysis of PN alterations across the human diseasome towards novel hypotheses for therapeutic network re-adjustment in proteostasis disorders.Proteome balance is safeguarded by the proteostasis network (PN), an intricately regulated network of conserved processes that evolved to maintain native function of the diverse ensemble of protein species, ensuring cellular and organismal health. Proteostasis imbalances and collapse are implicated in a spectrum of human diseases, from neurodegeneration to cancer. The characteristics of PN disease alterations however have not been assessed in a systematic way. Since the chaperome is among the central components of the PN, we focused on the chaperome in our study by utilizing a curated functional ontology of the human chaperome that we connect in a high-confidence physical protein-protein interaction network. Challenged by the lack of a systems-level understanding of proteostasis alterations in the heterogeneous spectrum of human cancers, we assessed gene expression across more than 10,000 patient biopsies covering 22 solid cancers. We derived a novel customized Meta-PCA dimension reduction approach yielding M-scores as quantitative indicators of disease expression changes to condense the complexity of cancer transcriptomics datasets into quantitative functional network topographies. We confirm upregulation of the HSP90 family and also highlight HSP60s, Prefoldins, HSP100s, ER- and mitochondria-specific chaperones as pan-cancer enriched. Our analysis also reveals a surprisingly consistent strong downregulation of small heat shock proteins (sHSPs) and we stratify two cancer groups based on the preferential upregulation of ATP-dependent chaperones. Strikingly, our analyses highlight similarities between stem cell and cancer proteostasis, and diametrically opposed chaperome deregulation between cancers and neurodegenerative diseases. We developed a web-based Proteostasis Profiler tool (Pro 2), a new web-tool enabling intuitive visualisation of cancer chaperome deregulation maps. We stratify two cancer groups based on diverging chaperome deregulation and highlight similarities between cancer and stem cell proteostasis. Our study also exposes drastically opposed shifts between cancers and neurodegenerative diseases. Collectively, this study sets the stage for a systematic global analysis of PN alterations across the human diseasome.Protein homeostasis, or proteostasis, is maintained by the proteostasis network (PN), an intricately regulated modular network of interacting processes that evolved to balance the native proteome, supporting cellular and organismal health throughout lifespan. Imbalances and collapse of cellular proteostasis capacity, the capacity to buffer against cytotoxic damage and stress, is increasingly implicated in some of the most challenging diseases of our time, including neurodegeneration and cancers. The systems-level PN alterations in these diseases are not understood to date. Here, we address this challenge, focussing on the human chaperome, the ensemble of chaperones and co-chaperones, which represents a central conserved PN functional arm. We devised a novel data dimensionality reduction approach enabling quantitative contextual visualization of chaperome alterations in the heterogeneous spectrum of cancers based on gene expression data from thousands of patient biopsies. We developed Proteostasis Profiler (Pro Eukaryotic proteomes comprise a complex repertoire of diverse protein species that are organized in a modular interactome network in order to execute native function in support of proteostasis and a healthy cellular phenotype. Proteome balance is safeguarded by the proteostasis network (PN), an intricately regulated network of conserved processes that have evolved to safeguard the healthy folded proteome . CellulaImportant progress has been made in our understanding of proteostasis biology, building on fundamental insights on conserved proteostasis processes and their role in disease, such as chaperone-assisted protein folding and quality control –8, clearThe folding functional arm, the human chaperome, is highly conserved and of central importance in the PN, responsible for maintaining the native folded proteome. In cancers, mutations and genomic instability inevitably entail alterations of proteome composition and balance that are far less well explored than the consequences of nucleic acid sequence alterations. Post-translational alterations at the proteomic level are beyond the reach of DNA repair mechanisms and cancer cells are constantly challenged by the need to accommodate large amounts of proteotoxic stress in consequence of increased translational flux and proliferation as well as proteotoxic stressors. Proteome instability and pathological alterations in the abundance of key signalling or housekeeping molecules such as kinases, metabolic enzymes or molecular transporters have to be buffered by the PN to ensure cellular survival. The cancerous state poses characteristic requirements on the PN, such as high chaperone levels and elevated proteasome activity in order to ensure for sufficient correction or elimination of aberrant protein species in light of increased translational flux and metabolic stress . This chChallenged by the genetic complexity and heterogeneity, collective prevalence and unmet medical need of the wide spectrum of human cancers as well as the lack of a systems-level understanding of proteostasis alterations during carcinogenic transformation, we developed a novel integrated analytical pipeline and software toolkit for the quantitative profiling of chaperome changes across the human cancer landscape . We utilIn order to enable comprehensive, contextual, and quantitative representations of the complexity of chaperome alterations across a large number of patient biopsy disease datasets, we developed a new custom data dimensionality reduction and visualisation approach. Combining Meta-PCA, a novel principal component analysis (PCA) based two-step dimension reduction algorithm and its resulting quantitative M-scores of chaperome functional family disease alteration of gene expression with contextual polar plot visualisations, we provide intuitive quantitative maps of cancer chaperome gene expression changes .The mechanistic understanding of genotype-phenotype relationships in complex genetically heterogeneous diseases such as cancers requires the consideration of the cellular interactome network . To redu2), an integrated web-based suite of applications enabling intuitive quantitative analyses and comparative visualisation of differential expression of complex PN alterations across large disease dataset compendia such as the TCGA. Visualisation and analysis features include heat map clustering and polar plot display. Integrated meta-networks and interactome-guided 3D topographic maps ease comparative exploration of cancer chaperome deregulation in the context of interactome network wiring. Pro2 is designed to serve the scientific community as a user-friendly application for systems-level exploration of PN disease alterations, at reduced complexity.To make these resources easily available to the community, we developed Proteostasis Profiler , an intricately regulated network of conserved processes that have evolved to safeguard the native functional proteome and cellular health. The human chaperome, an ensemble of 332 chaperones and co-chaperones, represents a central functional arm within the PN in charge of maintaining the cellular folding landscape  4]. Mot. Mot4]. Notably, the human chaperome is predominantly upregulated across the majority of TCGA solid cancers with a ∆GSA group mean change of +0.50 as compared to 100 random sets of non-chaperome genes . This ovBesides marked differences in the pattern of cancer functional family changes, Euclidean clustering of cancer groups (rows) revealed two major clusters  are characterized by their capacity to replicate infinitely in culture, while maintaining a pluripotent state . This imt test), and as overall second most highly upregulated subunit besides CCT6A   in > 70% of cancers  39], ma, ma39],  t test) . Consist t test) , we foun t test) .Together these findings suggest that proteostasis shifts in cancer cells add to an altered, enhanced PN state that mimics the immortal and resilient stem cell phenotype, buffering genome instability and ensuing proteomic imbalances in support of sustained and increased cellular proliferation throughout cancerogenesis.Overall chaperome upregulation in cancers, with preferential enrichment for upregulation of ATP-dependent chaperones, alongside consistent downregulation of sHSPs, is diametrically opposed to chaperome deregulation trends previously observed in a study of chaperome alterations in human aging brains and in patient brains with age-onset neurodegenerative diseases . While sTo investigate this disease group difference further, we applied the analysis outlined for cancers above also on the gene expression datasets that had earlier revealed global chaperome repression in AD, HD, and PD . Our anaThese opposing chaperome deregulation signatures are in line with differing implications of proteostasis alterations in these diseases. While broad chaperome repression and proteostasis functional collapse is associated with aggregation and cytotoxicity of chronically expressed misfolding-prone proteins in neurodegenerative diseases , enhanceIn light of the diverse signatures of differential chaperome deregulation observed across cancers , and motWe devised Meta-PCA, a novel principal component analysis (PCA) based semi-supervised two-step dimension reduction approach that facilitates stratification of cancer patient and normal control samples within heterogeneous gene expression datasets . Based o2) tool associated to this study.Overall, these contextual quantitative representations enable an appreciation of the complex chaperome shifts in different cancer tissues derived from > 10,000 patient biopsy samples. The resulting compendium of differential cancer chaperome polar plots  is also The integration of disease-related differential transcriptomic changes with the cellular protein interactome network, or the edgotype, is instrumental to our understanding of genotype—phenotype relationships . TowardsWe started with 328,244 unique human PPIs (edges) between 16,995 proteins (nodes) downloaded from the BioGRID, IntAct, DIP, and MINT databases ,53],,. ZoomingIn order to enable focussed views on transcriptomic alterations of top-level chaperome functional families, we collapsed individual nodes onto functional family meta-nodes, and edges shared between families were collapsed as meta-edges such that meta-node sizes correspond to the number of family members and meta-edge thickness represents the number of shared interactions between families. We considered the meta-interactome derived from the curated high-confidence ME-CHAP interactome , where a2) suite of tools, to improve accessibility and applicability by the scientific community.This interactome-guided topographic display of differential chaperome alterations enables dimensionality and complexity reduction for the coherent display and comparative analysis of functional network shifts that can serve to compare differential changes i) in disease versus controls, ii) between diseases and disease classes, and iii) between perturbed or unperturbed states across large numbers of heterogeneous genomic datasets. Furthermore, this visualization lends itself for a systems-level assessment of PN deregulation topologies and their readjustment in human disease and therapeutic intervention. We implemented topographic map visualisations into the Proteostasis Profiler  as an integrated web-based resource and suite of tools, for interactive dimensionality-reduction, analysis and visualisation of disease-specific alterations of proteostasis functional arms, such as the chaperome, in the context of the interactome network. In this study we highlight Pro2 use-cases for the human chaperome across TCGA solid cancers in comparison to the three major neurodegenerative diseases using differential gene expression heat maps (∆GSA)  Figs , Meta-PCSA) Figs  and S3, SA) Figs  and S5. Cancer prevalence, genetic complexity and heterogeneity represent unmet medical need and a significant challenge to personalized medicine, calling for genome-informed therapeutic intervention strategies . While iHere, we focused on the human chaperome, a central PN component, and highly conserved facilitator and safeguard of the healthy folded proteome using an expert-curated human chaperome functional gene ontology comprising an ensemble of 332 chaperone and co-chaperone genes  to systehsf-1 knockdown in C. elegans [Evidence points towards functional associations between increased proteostasis buffering capacity and maintenance of “stemness”, immortality and proliferative potential in both cancer cells and pluripotent stem cells . For ins elegans , consist elegans . Connect elegans , 73. Whi elegans , and inc elegans , 76, we Contrary to stem cell proteostasis, which is set up to maintain pluripotency and proliferative capacity, neurodegenerative diseases such as Alzheimer’s (AD), Huntington’s (HD), and Parkinson’s disease (PD) display signs of proteostasis functional collapse. Misfolding diseases feature overexpression of aggregation—prone proteins such as Aβ in AD, α-synuclein in PD, or huntingtin in HD that entail a “toxic-gain-of-function” resulting in chaperome overload, gradually exceeding proteostasis capacity , while “C. elegans models of amyloid β (Aβ) and polyQ proteotoxicity, chaperones and co-chaperones far less well studied than HSP90 had equally strong protective effects [A recent study linked repression of ~30% of the human chaperome in aging brains and in neurodegenerative diseases to proteostasis functional collapse and pointed to the role of a chaperome sub-network as a conserved proteostasis safeguard . Intrigu effects . Similar effects  found on effects . Interes effects . Collect effects .2), an integrated web-based suite of tools enabling processing, analysis and visualisation of proteostasis alterations in human diseases at reduced dimensionality, towards hypotheses-building for mechanistic understanding and clinical translation.In summary, our study showcases a systematic profiling of the extent of chaperome deregulation, as a central PN functional arm, in a panel of human cancers and three major neurodegenerative disorders, accompanied by a resource of quantitative multi-dimensional maps with reduced complexity. Therapeutic PN regulation for increased or restored proteostasis capacity may be beneficial in both loss-of-function and gain-of-toxic-function diseases of protein misfolding . AttenuaFocussing on pan-cancer analysis of the human chaperome, we chose The Cancer Genome Atlas (TCGA) as the main source for our analyses, as an established dataset that is widely used and adopted by the scientific community. The Broad Institute TCGA GDAC Firehose was accessed to download TCGA RNAseqv2 raw counts data followed by application of the voom method for the transformation of count data to normalized log2-counts per million (logCPM) . Each ofWe applied Gene Set Analysis (GSA) , an advaWe subdivided the human chaperome into functional subsets of chaperones and co-chaperones, and further divided chaperones into two sets of ATP-dependent and ATP-independent chaperones according to the annotations provided by Brehme et al. 2014 [M denotes the M-score of chaperome (CHAP) family x, Gx is the vector of gene expression values corresponding to genes in CHAP family x, and Fx is the function we want to fit. For simplicity, we considered a linear first degree model as follows: Fx is a vector of weights Wx with identical length as the vector Gx, and we aim to find Wx for all x using PCA. Assuming equivalent biological function of each CHAPx among all tissues, we first calculate Fx for each tissue in order to separate disease from healthy samples for each tissue, and then combine all “relevant” PCs in order to obtain the main underlying PC, or ‘Meta-PC’, of the corresponding CHAP group. We outline the ‘Meta-PCA’ algorithm as follows:Gene Set Analysis (GSA) is a statistical hypothesis testing method that is by definition limited to confirmatory data analysis with respect to pre-existing hypotheses. In order to serve the goal of quantitative exploratory pan-cancer chaperome analysis, while retaining a maximum information content during model reduction, we devised Meta-PCA, a novel quantitative multi-step dimension reduction model fitting strategy based on principal component analysis (PCA). Principal component analysis (PCA) uses orthogonal transformation to convert a set of variables to linearly uncorrelated variables, such that they are ordered by their information content, which allows for removal of dimensions with lowest information content for dimensionality reduction in complex heterogeneous datasets. In order to stratify cancer from healthy biopsy gene expression samples based on chaperome functional family gene expression in highly convoluted datasets comprising multiple different cancer types, we designed Meta-PCA as a novel two-step method capable of handling this type of heterogeneous data. We hypothesized that each chaperome functional family or process can be described by a low number of variable dimensions, considering that genes within each group are either related or act together in molecular complexes. Therefore, we used a PCA-based approach for quantitative assessment and dimensionality reduction of functional chaperome alterations based on disease gene expression data. Challenged by highly varying sample counts in the different TCGA cancer group datasets, where datasets (tissues) with high sample numbers are at risk of dominating PCA results as compared to cancer groups with low sample numbers, we developed a custom approach that is not limited by a lack of underlying models for interpolation or undesirable loss of information, such as in up- or down-sampling, respectively, allowing us to consider all samples in the included TCGA cancer groups. Assuming distinct roles for each chaperome functional group we defineStep 0: For each CHAP group and tissue we assume a modelx in tissue t, x in tissue t to an M-score value, and x in tissue t.Step 1: Assuming Wtx, which is the vector of weights for CHAP group x and tissue t. Then we calculate PCA on the gene expression matrix (GEX) comprising all genes in CHAP group x, and all samples of tissue t, including ‘solid tissue normal’ and ‘primary solid tumors’. So in this step we have Step 2: The x does not change drastically between healthy and cancer, PC1 will represent an unwanted variable or even noise. So we have to filter out the t-test. For each tissue, we test the separation of −4.Step 3: We combine all Wx, which is the universal mapping of gene expressions in CHAP group x to its corresponding M-score, regardless of tissue type. Therefore, we calculate Wx asWx can be used as the universal function Fx , hereafter referred to as ‘edges’, were downloaded on 23 Dec 2016 from the BioGRID , IntAct Considering these curation levels, three physical chaperome (CHAP) interactomes of increasing confidence level are obtained :SE-CHAP: 666 unique physical edges between 220 nodes without curation for type or number of evidence (single evidence SE-CHAP)ME-CHAP: 222 unique physical edges between 128 nodes with multiple pieces of evidence detected by ≥ 2 different experimental methods OR reported by ≥ 2 independent studies (PMIDs) (multiple evidence ME-CHAP)MM-CHAP: 132 unique physical edges between 96 nodes detected by ≥ 2 different experimental methods only (multiple method MM-CHAP).Different PPI source databases may annotate an identical reported PPI to different PSI-MI terms situated at different depth of the same branch within the PSI-MI ontology tree. In these cases, PPIs that are actually only supported by one piece of evidence can unintentionally be mislabelled as multiple evidence PPIs. Our automated quality curation script resolves this problem through up-propagation within the PSI-MI—ontology tree. Assume one PPI is annotated with two different interaction detection methods, A and B, then 1) if PSI-MI ontology tree levels of method A and method B are identical but their PSI-MI terms (IDs) are different, then the methods are considered as different, otherwise A and B are considered the same and the interaction is eliminated from the MM-CHAP interactome, 2) if the level of method A is higher (deeper in the ontology tree) than the level of method B, then the code searches for its parent situated at the same level as method B and compares the parent method ID with B to determine if the methods are identical or different.We considered 2-dimensional physical interactome information to guide the spatial layout (x-y coordinates) of human chaperome functional ontology families in a 3-dimensional (x-y-z coordinates) topographic representation of chaperome M-score changes between disease and healthy tissue (z coordinate). Physical chaperome protein-protein interactome network data (PPIs) was obtained and curated as described above. We considered a network involving only high quality curated interactions supported by multiple pieces of evidence (ME-CHAP). We used the R package iGraph in order to collapse nodes corresponding to each level 1 functional ontology family into meta-nodes, and edges shared between all members of any two different level 1 functional families into meta-edges, such that meta-node size corresponds to the number of family members and meta-edge thickness represents the number of shared interactions between two families. Meta-node colour is set to reflect gene expression changes of each respective functional family in disease. We then applied a force-directed network graph layout algorithm to the meta-network according to Kamada and Kawai  and extr2) in order to enable visual exploration of the data and results described in this manuscript, obtained through our algorithms and visualisation tools. Pro2 is accessible directly at http://www.proteostasys.org. Pro2 is implemented using Django (https://www.djangoproject.com/))), a web framework written in Python language (https://www.python.org). All the charts in the tool are generated using the plotly platform (https://plot.ly). The Pro2 tool itself is hosted on the Heroku platform (https://www.heroku.com).We designed a web-based Proteostasis Profiler (Pro2) Github repository at https://github.com/brehmelab/Pro2.All R and Python scripts and code related to this manuscript are accessible through the Proteostasis Profiler Click here for additional data file.S2 FigA. Comparing upregulation and downregulation of gene expression of chaperones and co-chaperones using GSA reveals a general upregulation of chaperones and co-chaperones in cancer, with preferential upregulation of chaperones. Colour code indicates chaperone up-regulation of gene expression. Axes represent chaperone downregulation, co-chaperone upregulation and downregulation of gene expression. B. Box-and-whisker plots highlight fractions of differentially expressed genes in each chaperome subset for all TCGA cancers assessed, based on A. Differentially expressed genes in each set were obtained by linear modelling (Limma package in R) and considering genes with p value < 0.5 following Benjamini-Hochberg correction. Box boundaries, 25% and 75% quartiles; middle horizontal line, median; whiskers, quartile boundaries for values beyond 1.5 times the interquartile range; small circle, outlier. C. Assessing differential expression of ATP-dependent (n = 50) vs. ATP-independent (n = 38) chaperones highlights a preferential upregulation of ATP-dependent chaperones across TCGA cancers D. Box plots (drawn as in B.) show fractions of differentially expressed genes in the two sets of ATP-dependent and ATP-independent chaperones for all TCGA cancers assessed, based on C.Analysis of differential cancer gene expression of chaperome functional subsets, comparing chaperones and co-chaperones as well as ATP-dependent and ATP-independent chaperones . See als(PDF)Click here for additional data file.S3 FigChaperome gene expression shifts between healthy and cancer (pp. 1–22) or AD, PD and HD (pp. 23–25) tissue biopsy datasets are quantified by Meta-PCA and visualized in context by plotting resulting M-scores on polar maps as in (PDF)Click here for additional data file.S4 FigChaperome meta-interactome networks for cancers (pp. 1–22) or AD, PD and HD (pp. 23–25) are shown as in (PDF)Click here for additional data file.S5 Fig3D topographic maps of cancer (pp. 1–22) or AD, PD and HD (pp. 23–25) chaperome alterations are obtained by projecting gene expression changes between disease and healthy counterpart biopsy gene expression (z dimension) onto the high-confidence chaperome meta-interactome (ME-CHAP) graph layout (x-y dimensions).(PDF)Click here for additional data file.S1 TableTab S1A. List of 332 human chaperome genes, expert curated by functional ontology groups at six levels of increasing detail (Level 1 = broad >> level 6 = detailed). For each entry, HGNC gene symbol, EntrezID, and functional ontology annotation levels are indicated. Human chaperome as in [Tab S1B. List of 43 proteasome genes. For each entry, HGNC gene symbol, EntrezID, and functional ontology annotation levels are indicated. Proteasome complex annotation according to HGNC [me as in , Tab S1B to HGNC .(XLSX)Click here for additional data file.S2 TableTab S2A. 666 curated physical human protein—protein interactions between 220 chaperome nodes supported by any number of evidence, including interactions reported only by one single study (PMID). Tab S2B. 222 curated high-confidence physical human protein—protein interactions between 128 chaperome nodes supported by multiple pieces of evidence, including interactions reported by multiple methods or studies (PMIDs). Tab S2C. 132 curated high-confidence physical human protein—protein interactions between 96 chaperome nodes supported by multiple pieces of evidence, including only interactions reported by multiple methods.Human physical protein-protein interactions (PPIs) were obtained for 332 human chaperome members from curated public databases reporting human PPIs with PSI-MI method annotations . For each node, Entrez Gene ID and corresponding HGNC symbol, chaperome functional ontology annotation levels 1 and 2, interaction detection method MI code as annotated in the respective source database, and unique methods per PPI following curation, curation evidence level, PMIDs with corresponding literature evidence for each interaction, source databases, and respective MI method IDs are indicated. (XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Granulin-epithelin precursor (GEP) is a secretory growth factor, which has been demonstrated to control cancer growth, invasion, drug resistance and immune escape. Our previous studies and others also demonstrated its potential in targeted therapy. Comprehensive characterization of GEP partner on cancer cells are warranted. We have previously shown that GEP interacted with heparan sulfate on the surface of liver cancer cells and the interaction is crucial for GEP-mediated signaling transduction. This study aims to characterize GEP protein partner at the cell membrane with the co-immunoprecipitation and mass spectrometry approach.n = 77 pairs) were examined by real-time quantitative RT-PCR. GEP and GRP78 protein expressions were investigated by immunohistochemistry on paraffin sections.The membrane fraction from liver cancer model Hep3B was used for capturing binding partner with the specific monoclonal antibody against GEP. The precipitated proteins were analyzed by mass spectrometry. After identifying the GEP binding partner, this specific interaction was validated in additional liver cancer cell line HepG2 by co-immunoprecipitation using GRP78 and GEP antibodies, respectively, as the bait. GRP78 transcript levels in hepatocellular carcinoma (HCC) clinical samples . GRP78 and GEP transcript levels were significantly correlated , and the proteins were also detectable in the cytoplasm of liver cancer cells by immunohistochemical staining.We identified the GEP-binding protein as 78-kDa glucose-regulated protein . This interaction was validated in independent HCC cell lines. Increased GRP78 mRNA levels were demonstrated in liver cancer tissues compared with the paralleled liver tissues (GRP78 and GEP are interacting protein partners in liver cancer cells and may play a role in GEP-mediated cancer progression in HCC. The secretory growth factor granulin-epithelin precursor (GEP) is also known as progranulin or PC cell-derived growth factor. It constitutes of seven and half cysteine rich granulin subunits, which are known to regulate inflammation . The preHCC is the major form of primary liver cancers . With abWe have previously shown that heparan sulfate (HS) might act as the co-receptor, which is essential in the cell surface binding and the signaling transduction of GEP . Further2 incubator. GEP-overexpressed HepG2 (HepG2-FL) and Hep3B (Hep3B-FL) were generated by lipofection of GEP plasmid as reported previously [Two human liver cancer cell lines, Hep3B and HepG2 , were cultured in advanced minimum essential medium (AMEM) supplemented with 10% fetal bovine serum and L-glutamine at 37 °C in a 5% COHCC patients were recruited between October 2002 and September 2005 with written inform consent at Queen Mary Hospital in Hong Kong. Real-time RT-PCR expression assays were performed with the snap frozen HCC and the adjacent non-tumor (NT) tissue specimens. The tissues were formalin-fixed and paraffin-embedded for immunohistochemistry.Purification of recombinant GEP (rGEP) and the binding assay were described previously . Briefly2, 5 mM N-ethylmaleimide, 10 mM EDTA, complete protease inhibitor cocktail, pH 7.2). Cells debris was removed by centrifugation at 3000×g for 10 min. The supernatant containing the cellular protein complexes and the homogenized membrane-anchored protein complexes was collected. Supernatant was then centrifuged at 20000×g for an hour to collect the pellet, which contained the crude membrane fraction. The pellet was lysed in lysis buffer  under rotation at 4 °C for 1 h. Dissolved membrane proteins were collected from the supernatant after centrifugation at 20000×g for an hour. Membrane protein concentration was analyzed by DC Protein Assay reagents .The GEP-overexpressing-Hep3B cells, Hep3B-FL, were cultured for 3 days and were then washed with ice-cold PBS buffer with 0.1% glucose and Tris-buffered sucrose solution . The cells were harvested by scraping and collected by centrifugation for 5 min at 500×g at 4 °C. After washing with Tris-buffered sucrose solution, cells were resuspended and homogenized in solution A  was used for detection.The differential protein band at about 85 kDa was excised from the Coomassie blue-stained gel, and in-gel trypsin digestion was performed as previously described . Brieflyn = 77 pairs). The GRP78 relative levels had been normalized with control 18S and calibrator for RNA quantity and plate-to-plate variation. The mRNA expression of GEP has been determined previously in the same cohort [Real-time quantitative RT-PCR was performed as described previously . GRP78 pe cohort  and was GEP and GRP78 proteins were examined by immunohistochemistry . Monoclot-test or one-way analysis of variance as appropriate and described in the text. The Youden index (sensitivity + specificity - 1) was used to determine the optimal cutoff of GRP78 expression for prediction of survival outcome. Youden index was employed to maximize the sensitivity (true-positive fraction) and specificity  of the prediction simultaneously. Descriptive parameters were analyzed by chi-squared test with Bonferroni correction. P values less than 0.05 were considered significant. Statistical analyses were performed by SPSS .Continuous variables were assessed by Spearman’s correlation, comparison between groups by Student’s The rGEP bound on the surface of HCC cells through HS  could beP < 0.05). The details of the trypsinized peptides were listed in Table Co-immunoprecipitation (co-IP) of the lysate of the membrane fraction Hep3B-FLwith GEP antibody, compared with the controls lysate alone and antibody alone respectively, only one single extra band was observed in the co-IP experiment. The SDS-PAGE gel was stained by Coomassie blue and the protein band was excised for mass spectrometry analyzes. This unknown protein was identified as GRP78 according to the masses of the trypsinized peptides in two independent runs Table . Both inThen, membrane fraction of another HCC cells, HepG2-FL, was extracted for co-IP analysis to validate the interaction between GEP and GRP78. As show in the immunoblot of Fig. After identifying the GRP78 as a binding partner of GEP, the protein expression of GRP78 was investigated in the HCC clinical samples by immunohistochemistry (IHC). Clinical samples that have shown over-expression of GEP in our previous studies were further examined for GRP78 by IHC. Coincident to a previous finding in HCC , the proP = 0.002) , non-tumor , and the tumor-to-non-tumor fold change   Fig. . Moreove01) Fig. -d.Fig. 4P = 0.022)  curve analysis. Clinico-pathological features were analyzed against the expression level of GRP78 Table . Among t22) Fig. . Venous Re-localization of GRP78 to the cell surface has been reported previously. We tried to determine the re-localization in GEP expression and cell surface binding of GEP. Thapsigargin and tunicamycin, which induce ER stress by inhibiting the fusion of autophagosomes with lysosomes and inhibiting glycosylation, respectively, have been shown to induce re-localization of GRP78 . In bothIn this study, we used co-IP and mass spectrometry to identify GEP binding partner from the membrane-enriched protein fraction of HCC cells. We have identified GRP78 as a binding partner of GEP in Hep3B Table  and valiWith respect to the nature of GRP78 relocalization to cell surface, we attempted to determine if this re-localization increased endogenous GEP expression or on the cell surface of HCC cells; and if GRP78 contributed to cell surface binding of rGEP. Consistent with a previous study , higher Clinical significance of GRP78 expression has been analyzed. Coincident to a previous study of GRP78 at the protein level , we demoThe functional roles of GRP78 in HCC have been revealed in previous studies. Over-expression of GRP78 and the consequential activation of UPR have been indicated to confer drug resistance on HCC against sorafenib and doxorubicin , 37. GRPIn summary, this study identified GRP78 from the membrane fraction of HCC cells as a binding partner of GEP. Their interaction may shed light on the multifaceted roles of both GRP78 and GEP in HCC tumorigenesis, especially their mechanistic relationship in cancer progression and drug resistance.


Because etomidate induces prolonged adrenal suppression, even following a single bolus, its use as an infused anesthetic is limited. Our previous study indicated that a single administration of the novel etomidate analog methoxyethyletomidate hydrochloride (ET-26-HCl) shows little suppression of adrenocortical function. The aims of the present study were to (1) determine the minimum infusion rate of ET-26-HCl and compare it with those for etomidate and cyclopropyl-methoxycarbonylmetomidate (CPMM), a rapidly metabolized etomidate analog that is currently in clinical trials and (2) to evaluate adrenocortical function after a continuous infusion of ET-26-HCl as part of a broader study investigating whether this etomidate analog is suitable for long infusion in the maintenance of anesthesia.n = 32) were then randomly divided into four groups: etomidate, ET-26-HCl, CPMM, and vehicle control. Rats in each group were infused for 60 min with one of the drugs at its predetermined minimum infusion rate. Blood samples were drawn initially and then every 30 min after drug infusion to determine the adrenocorticotropic hormone-stimulated concentration of serum corticosterone as a measure of adrenocortical function.The up-and-down method was used to determine the minimum infusion rates for ET-26-HCl, etomidate and CPMM. Sprague-Dawley rats ; however, this reduction did not reach statistical significance. Thus, further studies are warranted examining the practicability of using ET-26-HCl as an infused anesthetic. Etomidate, one of the most frequently used intravenous anesthetics, has many favorable properties, such as its ability to maintain hemodynamic stability and generate low incidences of respiratory depression and anaphylaxis. However, the adrenocortical insufficiency caused by etomidate restricts its clinical applications. Several studies have shown that as little as a single administration of etomidate may induce adrenocortical insufficiency, and the increased propensity for this may last 48 h after the administration of etomidate .In the 1980s, etomidate was infused in a continuous manner in critical patients to maintain sedation; however, studies later showed that this dosage regimen increased mortality . PharmacGuide to the Care and Use of Experimental Animals . Five animals per cage were housed under standard conditions at a temperature of 22 °C and a humidity of 60% and with standard laboratory rat chow and water. The animals were allowed to acclimatize for 1 week.All animal protocols used in the present study were approved by the Ethics Committee of the West China Hospital, Sichuan University, China . Sprague Dawley rats weighing 225–350 grams were purchased from Chengdu Dassy Biological Technology Co. Ltd.  and cared for in accordance with the the Canadian Council on Animal Care’s Etomidate (2 mg/mL) formulated as an emulsion was purchased from B. Braun Melsungen AG, and propofol (10 mg/mL) formulated as an emulsion was purchased from AstraZeneca. The Laboratory of Anesthesia and Critical Care Medicine  synthesized ET-26-HCl using a previously published approach; ET-26-HCl was formulated as an aqueous solution (10 mg/mL) with 35% propylene glycol and then diluted with normal saline  to 6 mg/mL. Cyclopropyl-methoxycarbonylmetomidate (CPMM) was also synthesized by our laboratory according to the issued patent  and formulated as an aqueous solution (8 mg/mL) with 20% sulfobutylether-β-cyclodextrin .An infusion pump  was used to administer the sedatives and hypnotics, and a homoeothermic blanket was used to maintain the body temperature of the rats at 36–38 °C while they were anesthetized. A pulse oximeter placed on the upper right hind leg was used for monitoring oxygen saturation. Heart rate and rhythm and the respiratory rate were monitored with a BL-420S biological data acquisition and analysis system . Oxygen (100%) was delivered at a rate of 2 L/min to each anesthetized rat through a face mask connected to a coaxial circuit.n = 60) were randomly assigned to three groups that received either etomidate, ET-26-HCl, or CPMM.Rats  for each anesthetic was determined as previously described . The inin = 32) were then randomly divided into the following four groups, with eight rats in each group: etomidate, ET-26-HCl, CPMM, or propylene glycol vehicle control, and the infusion protocol started (see g) for 5 min. The supernatant was transferred to a clean Eppendorf centrifuge tube and subjected to a second centrifugation . After the second centrifugation, the supernatant was removed again, and a final high-speed centrifugation was performed to remove all red blood cells and particulate. The serum was transferred to a clean tube and immediately placed at −20 °C. The corticosterone concentration in each serum sample obtained at the various time points was quantified within 1–2 days using an enzyme-linked immunosorbent assay  and a 96-well plate reader.The infusion protocol for each rat began at 8:30–9:00 a.m. to minimize natural changes in corticosterone levels. After the rats were weighed and the caudal vein was catheterized, dexamethasone (0.2 mg/kg) was administered. Two hours later, the baseline blood sample was drawn. Rats . Figure preparation and curve fitting was performed with Prism version 5.0 .All data are presented as the mean ± standard deviation. For comparisons of serum corticosterone concentrations after infusion of vehicle and test compounds, a one-way analysis of variance was conducted followed by Tukey’s honest significant difference test or the Tamhane test. A value of The MIRs of etomidate, CPMM, and ET-26-HCl were 0.29, 0.95, and 0.62 mg/kg/min, respectively .The baseline concentrations of serum corticosterone (before the start of the drug infusion) were not significantly different among the four groups and averaged 185.86 ± 68.66 ng/mL. However, serum corticosterone concentrations increased over time in all groups after drug infusion and ACTH stimulation. Compared with the control group, rats administered etomidate demonstrated significantly lower corticosterone concentrations at 60 min, which is the end of the drug infusion, as well as 90, 120, 150, and 240 min after the beginning of drug infusion. Compared with the etomidate group, rats administered ET-26-HCl showed significant differences 90, 120, 180, 210, and 240 min after begin of drug infusion. Compared with rats administered CPMM, rats administered etomidate showed significant differences in serum corticosterone concentrations at the end of the drug infusion as well as 60, 90, 180, and 210 min after begin of drug infusion. ACTH-stimulated serum corticosterone concentrations in rats administered CPMM or ET-26-HCl were not significantly different from each other at any time .in vivo plasma concentration of drugs continuously infused at a constant rate reaches equilibrium at 4–5 half-lives. We determined that the MIR for etomidate was 0.285 mg/kg/min, for ET-26-HCl it was 0.62 mg/kg/min, and for CPMM it was 0.95 mg/kg/min. These results suggested that the anesthetic efficacy of ET-26-HCl was approximately one-half to one-third of that for etomidate, which is consistent with the results of our previous study (50 (effective dose for 50 percent of the group) of CPMM is 0.89 ± 0.18 mg/kg/min  or no (−) reaction to a painful stimulus, an up-and-down design method previously described . Each hyus study . In addig/kg/min . These aAfter determining the MIRs, we next examined the effects of etomidate, ET-26-HCl, and CPMM continuously infused for 1 h and inducing the same anesthesia depth on serum corticosterone concentrations. Compared with those in control rats, ACTH-stimulated serum corticosterone concentrations were significantly decreased by etomidate, while those following ET-26-HCl or CPMM administration were not associated with a significant difference. When ACTH-stimulated serum corticosterone concentrations in rats administered etomidate were compared with those in rats infused with vehicle, ET-26-HCl or CPMM, all time points examined after the drug infusions, except 180 min and 240 min, show significant differences with etomidate. The corticosterone concentrations tended to be reduced for the first hour following ET-26-HCl infusion (as compared to vehicle infusion); however, this reduction did not reach statistical significance. We concluded that ET-26-HCl does not induce obvious inhibition of adrenal function.The safe dosage range of etomidate has been diminishing owing to its inhibition of adrenocortical function, as the suppression following even a single bolus may last 72 h . This inThe method to evaluate minimum infusion rate of present study is feasible, and it could maintain a similar anesthesia depth of all drugs. The corticosterone concentrations tended to be reduced for the first hour following ET-26-HCl infusion (as compared to vehicle infusion) was mainly because of the molecular structure of ET-26-HCl still have some depression to the release of corticosterone; however, this reduction did not reach statistical significance. Thus, further studies are warranted examining the practicability of using ET-26-HCl as an infused anesthetic.10.7717/peerj.3693/supp-1Data S1Click here for additional data file.


Whole exome sequencing in four members of the largest family (R2010) identified a CLDN14 (DFNB29) variant , likely pathogenic, sensorineural hearing loss, autosomal recessive. Although not associated with deafness or disease, CLDN14 p. has been previously reported as a variant of uncertain significance (VUS). Targeted sequencing of 169 deafness probands identified one homozygote and one heterozygous carrier. Genealogical studies, cascade sequencing and haplotype analysis across four unrelated families showed all subjects with the unique audioprofile (n = 12) were also homozygous for p. and shared a 1.4 Mb DFNB29-associated haplotype on chromosome 21. Most significantly, sequencing 175 population controls revealed 1% of the population are heterozygous for CLDN14 p., consistent with a major founder effect in Newfoundland. The youngest CLDN14  homozygote passed newborn screening and had normal hearing thresholds up to 3 years of age, which then deteriorated to a precipitous loss >1 kHz during the first decade. Our study suggests that genetic testing may be necessary to identify at-risk children in time to prevent speech, language and developmental delay.Genetic isolates provide unprecedented opportunities to identify pathogenic mutations and explore the full natural history of clinically heterogeneous phenotypes such as hearing loss. We noticed a unique audioprofile, characterized by prelingual and rapid deterioration of hearing thresholds at frequencies >0.5 kHz in several adults from unrelated families from the island population of Newfoundland. Targeted serial Sanger sequencing of probands for deafness alleles (The online version of this article (doi:10.1007/s00439-016-1746-7) contains supplementary material, which is available to authorized users. GJB2 yields of less than 25%, suggesting a larger role for other recessive genes in prelingual severe cases  and configuration  using the standard behavioral audiogram. Although clinically heterogeneous, rare pathognomonic audiograms may present with surprising regularity in clinics within genetically isolated populations and where patients often share a common ancestor due to founder effects. For example, the Finnish and Pakistani populations have been invaluable for discovery of deafness genes as population bottlenecks (genetic drift) and/or inbreeding increase the likelihood of inheriting recessive alleles that are identical by descent. These populations are often characterized by large sibships, deep genealogies and higher consanguineous rates. The population of Newfoundland and Labrador (NL) was founded by ~20,000 Protestant English and Roman Catholic Irish settlers. Religious and geographic isolation within small coastal fishing (outport) communities Manion  has resuCLDN14 causing precipitous prelingual sensorineural hearing loss in children born with normal hearing thresholds. The essential role of CLDN14, a component of tight junctions, was first discovered through studies in consanguineous families from the genetically isolated population of Pakistan. Tight junctions have been shown to play a significant role in maintaining the structural integrity of cells within the inner ear. Other genes encoding tight junction proteins, such as MARVELD2 (DFNB49)  allele has previously been reported in multiple studies as a variant of uncertain significance  alleles are born with normal hearing thresholds and experience a rapid and progressive loss by 3–4 years of age. Extensive clinical recruitment and targeted screening suggest that CLDN14 p. represents a major founder variant in the Newfoundland population.A unique clinical audioprofile of steeply sloping sensorineural hearing loss was noted in several unrelated families. Herein, we report a founder missense variant in This project is part of a large study of hereditary hearing loss in the Canadian province of Newfoundland and Labrador. Informed consent, family history and permission to access medical records and audiograms were obtained from all participants as per approved institutional review board protocol #01.186 . Sensorineural hearing loss was determined when hearing thresholds were abnormal, and the air and bone conduction results within 10 decibels (dB) of each other . Both retrospective and prospective audiograms and health records were obtained.In the course of ongoing clinical recruitment, we noted a rare but consistent clinical audioprofile characterized as steeply sloping, sensorineural hearing loss above 0.5 kHz with mid- and high-frequency thresholds in the severe to profound range Fig. a–d. On tGenomic DNA was extracted from peripheral blood using a simple salting out protocol  and SNVs were filtered against publically available SNP databases . We assessed the impact of SNVs at the protein level with SIFT, PolyPhen-2, and MutationTaster. Filtered SNVs had a minimum of 20× coverage, a predicted moderate/high impact  and a minor allele frequency (MAF) of <1%. The apparent vertical transmission of hearing loss in several branches of the clan pedigree   and also in 175 ethnically matched controls. Microsatellites flanking candidate genes were genotyped according to standard procedures  noted that probands  all shared a unique hearing loss pattern. The proband of Family 2010  predicted to cause the substitution of arginine to a tryptophan residue at position 141 and to be deleterious by SIFT (and probably damaging by PolyPhen-2 and Panther). Although identified in both the probands (V-11) of Family 2010 and transmitted to her affected son (V-14), the c.421C>T variant did not co-segregate with mid–high-frequency loss in this family (data not shown).Targeted sequencing was carried out on probands for known deafness alleles  but none were found. Several gene candidates, as suggested by Audiogene . Under a recessive model, we filtered four homozygous variants . One of these, CLDN14 (DFNB29) is a known deafness gene expressed in the sensory epithelium of the organ of Corti of the inner ear  point variant  and has been reported in both European and African populations. The heterozygous CLDN14  allele is reported as a variant of uncertain significance in dbSNP (rs143797113), ExAC browser (MAF: 0.02564%), 1000 genomes (MAF: 0.04%), and the Grand Opportunity Exome Sequencing Project (MAF: 0.05%). This allele has also been reported in several control samples from other study cohorts within the USA  pathogenicity.Whole exome sequencing on Family 2010 using three affecteds V-9, VI-4 and V-17) and one unaffected parent (V-8)   is a likely pathogenic, recessive allele where homozygosity results in a distinct precipitous mid–high-frequency hearing loss and relatives inheriting a single copy (carriers) or wild type do not have this pattern. According to the American College of Medical Genetics standards and guideline  is a strong PS4 PM2 likely pathogenic variant. The cause of hearing loss in subjects with flat audioprofiles is not known, but is clearly not due to homozygosity for CLDN14 c.488C>T. Future studies will explore the genetic etiology of their hearing loss. Furthermore, screening our cohort of 169 deafness probands identified an additional homozygous subject (Family 2072) and two heterozygous carriers. In Family 2072, the proband’s mother (with the distinct audioprofile) was also found to be homozygous for CLDN14 p. and her father a carrier  resides on a 1.4 Mb ancestral haplotype shared across all four families  within the clan, which was incorporated into our DFNB29-associated deafness haplotype.Extensive genotyping in the vicinity of t p.Ala16Val residt p.Ala16Val residCLDN14 (DFNB29) variant , likely pathogenic, sensorineural hearing loss, autosomal recessive.Extension of the pedigrees and review of all clinical audiograms identified 16 subjects with hearing loss; 10/16 subjects showed the distinct precipitous mid–high-frequency hearing loss Fig. . SubjectCLDN14, c.488C>T, p. ) is likely pathogenic, and causes a precipitous, bilateral and rapid deterioration of hearing thresholds at frequencies >0.75 kHz in children, progressing gradually in adults. We have also determined that this likely pathogenic variant is amplified in the founder population of the island of Newfoundland and is present in ~1% of the population. The role of CLDN14 in nonsyndromic hearing loss was first described in two large consanguineous families from Pakistan with recessive profound congenital deafness  variant reported in this study has been identified in previous studies but not in association with disease. It was first reported as a VUS by Thorleifsson et al.  allele in 3 of 1230 study participants for another kidney function study. The heterozygous p. allele was also found in a Swedish GWAS study examining the polygenic nature of schizophrenia . In summary, these studies suggest that the likely pathogenic CLDN14 p. allele is both rare and widely distributed around the globe.The n et al.  in a larn et al. , who detDFNB1 locus (GJB2), which is often the only gene that is routinely screened in the clinical setting when there is a family history of hearing loss. This represents a massive ascertainment bias, as children who are DFNB1 negative are not followed up, due to the expenses associated with genetic testing. Recently, a large, ethnically diverse, cohort study demonstrated the importance of investigating DFNB1-negative deaf probands  pass newborn and early hearing tests. The proband’s son  was discharged after his test at 1 year of age indicated normal hearing. Preschool testing 4 years later showed significant deterioration of both mid and high frequencies  also have a consistent phenotype but challenges in management remain. Hearing aids benefit affected children and young adults (up to the third decade), but most adults do not find them beneficial. For example, PIDs V-9 (age: 50), V-10 (age: 51) and V-17 , a VUS previously identified in the USA, Iceland and Sweden. CLDN14 p. appears to be of Irish origin and causes a precipitous, prelingual recessive sensorineural hearing loss. This likely pathogenic variant is frequent in this island population of Northern European decent, and CLDN14 p. homozygotes have normal hearing thresholds at birth, then experience rapid, progressive nonsyndromic hearing loss in early childhood. Although missed by newborn hearing screening, genetic testing would ensure identification of at-risk children, allowing for appropriate monitoring and timely intervention, aural rehabilitation and counseling for families. Although GJB2 is routinely screened in the regional hospital diagnostic clinic, we recommend targeted screening of CLDN14, as well.A population-based study of hearing loss in the NL population has clarified the role of DFNB29 locus is in linkage disequilibrium with p.. Even though our study presents several lines of evidence to suggest pathogenicity, experimental functional analysis of p. is required.While exome sequencing is a powerful tool to elucidate disease causing, coding variant, it does not explore non-coding regions. Additionally, there is no experimental proof of the predicted amino acid substitution, and without functional data, we cannot be certain that the point mutation impacts protein location within tight junctions. For example, this variant could cause alternative splicing or alter gene expression. Although less likely, it is plausible that a causal, non-coding variant at the Supplementary material 1 (PDF 56 kb)Below is the link to the electronic supplementary material.


Mycoplasma hyopneumoniae (M. hyopneumoniae) and prevalence and extension of CVPC in 1) experimentally infected pigs, 2) slaughtered pigs and 3) sequentially necropsied pigs in a longitudinal study. CVPC was scored by means of the European Pharmacopoeia recommended methodology. Specific IgG, IgG1 and IgG2 antibodies were assessed in serum. In addition, mucosal IgG and IgA antibodies were analyzed in broncho-alveolar lavage fluid (BALF) from experimentally challenged pigs. The systemic humoral immune response in experimentally infected pigs was delayed in onset whereas humoral respiratory mucosal immune response appeared more rapidly but declined earlier. Although low, BALF IgG antibodies showed the highest correlation with CVPC scores . In slaughter-aged pigs, both percentage of lungs with CVPC and mean lung lesion score were significantly higher in M. hyopneumoniae seropositive farms compared to the seronegative ones (p<0.001). Similarly, seropositive sequentially necropsied pigs showed more severe CVPC than seronegative ones. Overall, mean serological values might help to forecast prevalence and severity of EP-like lung lesions using a population based approach. Remarkably, the specific systemic humoral immune response was found to be predominated by the IgG2 subclass, suggesting a dominant Th1-mediated immune response to M. hyopneumoniae.Immunopathological events are key for the development of enzootic pneumonia (EP), which is macroscopically observed as cranioventral pulmonary consolidation (CVPC). This study aimed to investigate the putative association between the humoral immune response against  Mycoplasma hyopneumoniae (M. hyopneumoniae) is considered the most important primary bacterial respiratory pathogen involved in such lung lesions. When secondary or concomitant infections by other respiratory bacterial organisms are also implicated, enzootic pneumonia (EP) occurs [One of the main pulmonary lesions found in pigs on abattoir inspection is cranioventral pulmonary consolidation (CVPC) , 2. Myco) occurs .M. hyopneumoniae infections, at experimental, herd and abattoir levels [post-mortem lung evaluation is an end-point parameter that does not provide information on the ongoing respiratory problems in any of the cases [ante-mortem parameter that would provide reliable and real-time information in relation with CVPC prevalence and severity.CVPC quantification by means of lung lesion scoring is frequently used to estimate the incidence and severity of lung lesions associated to r levels –6. NeverM. hyopneumoniae infection pattern and development of the associated lung lesions [M. hyopneumoniae antibodies and CVPC development has been analyzed in several studies. However, while some studies found a significant positive correlation between both parameters [M. hyopneumoniae as a tool to assess lung lesions deserves further investigation. On the other hand, most of the commercially available enzyme-linked immunosorbent assay (ELISA) kits are designed to detect specific M. hyopneumoniae IgG antibodies in serum, but information on other humoral immune parameters and their relationship with CVPC is not available. Therefore, the present study sought to investigate the potential association between different humoral immune parameters, both at local and systemic levels, with the prevalence and severity of CVPC in pigs naturally and experimentally infected with M. hyopneumoniae.Immunopathological events are considered to play an important role in both  lesions . In connrameters , 7–10, orameters . NoteworM. hyopneumoniae experimentally inoculated animals [In order to accomplish the abovementioned objective, samples from three different published studies were used: 1) samples from  animals ; 2) samp animals . SamplesM. hyopneumoniae were experimentally inoculated as described previously [Ninety seven 6 week-old pigs free from eviously . One uniM. hyopneumoniae, 16 did not vaccinate and, from the remaining farms, no data was available [M. hyopneumoniae specific antibodies were determined from sera. Farms were then classified as seropositive or seronegative according to the mean S/P ratio obtained from those 20 sampled animals. In addition, mean farm IgG1 and IgG2 OD values were also obtained.A total of 54 batches of 54 different farms located in North-eastern, Central and South-eastern Spain, were included in the study . A batchvailable . The pigFifty-eight pigs from a farrow-to-finish farm located in North-eastern Spain with a history of EP associated respiratory problems were included in the analyses. These piglets belonged to the control group (non-vaccinated) of a previously published work . Blood sM. hyopneumoniae infection (CVPC) was assessed using different scoring systems depending on the study. Thus, in order to harmonize all lung lesion scores, those that were obtained through Madec and Kobisch (1982) [In each of these studies, extension of gross lung lesions compatible with h (1982)  (slaughth (1982)  (chronolh (1982) .M. hyopneumoniae IgG antibodies by means of a commercial indirect ELISA  and according to the manufacturer’s instructions. The relative amounts of specific IgG antibodies in samples were expressed as sample-to-positive (S/P) ratio based on optical densities (OD) as follows: S/P = OD sample-OD mean negative control/ OD mean positive control-OD mean negative control. Samples with an S/P of ≥0.5 were considered positive whereas samples with an S/P of <0.5 were considered negative. An S/P = 0 was given to those samples with OD values below the mean OD of the negative control.Sera derived from all the studies were tested for M. hyopneumoniae-specific IgG1 and IgG2 subtypes from sera were measured by using a modification of the above mentioned ELISA . Mouse anti-pig IgG1 and IgG2 monoclonal antibodies  were firstly tested in M. hyopneumoniae positive and negative sera diluted 1:30, 1:50, 1:70, 1:90, 1:120 and 1:150 with the sample diluent provided by the kit. As a result, 1:50 dilution  was appropriate to discriminate between M. hyopneumoniae seropositive and seronegative samples. After 30 min incubation, plates were washed and IgG1 and IgG2 antibodies diluted to 1:1000 were added. After incubating for 45 min, plates were washed again and alkaline phosphatase-labeled goat anti-mouse IgG polyclonal antibody  diluted to 1:1000 was added to each plate well. After incubating for 45 min, plates were washed and the reaction was visualized after 15 min incubation with the kit substrate reagent. Sample absorbance was obtained by reading at 405 nm and results expressed as OD values. For each plate, sample results were adjusted by subtracting the OD of the mean negative control provided by the kit. Furthermore, the cut-off was calculated as mean OD value of negative control plus three standard deviations (SD). The threshold was established at an OD value of 0.13 for both IgG1 and IgG2 antibodies.Values higher than this cut-off were considered positive whereas values below this cut-off were considered negative.Mycoplasma hyopneumoniae Antibody Test Kit; BioChek, UK) and conforming to the manufacturer’s instructions but for the initial dilution of the sample. For IgA antibody detection, an alkaline phosphatase-labelled goat anti-porcine IgA polyclonal antibody  was used as described previously [Undiluted and previously centrifuged BALF samples from the experimental study were tested for specific IgG antibodies by means of the above mentioned assay  or continuous . A non-parametric test (Mann-Whitney) was used to evaluate the effect of any categorical variable over the continuous ones. Besides, in order to study putative associations among continuous variables, a linear regression analysis was carried out. Contingency tables with the corresponding chi-square statistics were performed among categorical variables.Statistical analyses were performed using SPSS software, version 15.0 . The significance level (α) was set at M. hyopneumoniae humoral immune parameters, percentage of animals showing CVPC as well as the mean lung lesion score of the animals sacrificed at 21 or 28 dpi are represented in None of the control pigs showed CVPC. Among inoculated animals, local and systemic p>0.05) were found in terms of number of animals affected neither in mean lung lesion scores (CVPC was observed both in pigs sacrificed at 21 and 28 dpi and no significant differences (n scores .p = 0.09) (p>0.05) correlation was found between individual lung lesion scores and S/P ratios. Nevertheless, the mean S/P ratio of animals with CVPC (0.32±0.36) was significantly higher (p<0.05) than the one from animals without lung lesions (0.03±0.05) at 28 dpi.Number of seropositive animals was higher at 28 than at 21 dpi ( = 0.09) . AlthougM. hyopneumoniae were detected in sera at 21 dpi, even in those three animals that seroconverted for total IgG (p<0.0001) between OD values of both antibody subclasses was observed, but serum IgG2 amounts were found to be significantly higher (p<0.05) than IgG1 ones at 28 dpi. Furthermore, significantly higher (p<0.05) IgG1 and IgG2 levels were detected in those animals showing CVPC in comparison with those without lung lesions  at 21 dpi than at 28 dpi (p<0.05) of both specific IgG and IgA antibodies in BALF was observed in those animals showing CVPC in comparison with those without lung lesions (p = 0.07) (p<0.05) between IgG BALF OD values and lung lesion scores at individual level was observed. Nonetheless, this relationship was not observed with regard to IgA BALF OD values.Number of positive animals for BALF IgG and IgA antibodies was significantly higher (t 28 dpi . A signi = 0.07) . Althougp<0.001) mean lung lesion score as well as percentage of lungs affected by CVPC than seronegative ones (p>0.05) was found between the mean S/P value of the 20 randomly collected sera and the mean lung lesion score at individual farm level.All the 54 farms included in the present study were found to be affected by CVPC. Globally, mean percentage of lungs affected by CVPC and mean lung lesion score (Ph. Eur.) were 68.1  and 5.15 , respectively. Based on the results obtained from the 20 randomly tested serum samples per farm, 40 (74.1%) farms were considered seropositive and the remaining 14 (25.9%) seronegative. Additionally, seropositive farms had a significantly higher (ive ones . Howeverp<0.0001) between both isotypes was detected and IgG2 response against M. hyopneumoniae was found to be significantly (p<0.0001) higher than the IgG1 one. Indeed, a significant positive correlation was found between S/P ratios and IgG2 OD values  whereas such relationship was not as good when considering IgG1 OD values . Though, no significant association (p>0.05) between the mean IgG2 OD value and the mean lung lesion score at individual farm level was found.All the 40 farms classified as seropositive showed positive IgG2 humoral immune responses and 24 of them (60%) had also IgG1 positive reactions. Similarly to what was observed at experimental level, a significant positive correlation , seropositive animals necropsied at 15, 18 and 22 weeks of age had higher lung lesion scores in comparison with the seronegative counterparts. Nonetheless, a statistical tendency between individual S/P ratios and lung lesion scores was observed at 22 weeks of age .M. hyopneumoniae IgG2 levels increased progressively from 15 weeks of age until the end of the study (22 weeks), when the mean maximum IgG2 levels in sera were reached (p<0.05) than IgG1 ones at both 18 and 22 weeks of age. Additionally, a positive significant correlation was found between S/P ratios and IgG2 OD values in 18  and 22  week-old pigs. Notwithstanding, individual IgG2 OD values were positively correlated with mean lung lesion scores in 12 , 15 , 18  and 22  week-old animals.Following the seroconversion pattern, mean  reached . On the ante-mortem parameter that would be used to reliably forecast the prevalence and severity of CVPC during the life of the pig. While serology  is the most common monitoring tool used to determine the M. hyopneumoniae status of a pig population [M. hyopneumoniae and CVPC [M. hyopneumoniae as a CVPC estimator by describing the association between different specific antibody isotype responses, both at local and systemic levels, and CVPC. Such putative relationship was evaluated in three different scenarios. First, experimental conditions allow modelling the acute phase of M. hyopneumoniae infection. Second, the relevance of CVPC is commonly assessed in slaughter-aged pigs [M. hyopneumoniae infectious status is known. Lastly, pigs chronologically necropsied enabled the assessment of such relationship at different time points along M. hyopneumoniae infection dynamics. Despite CVP is frequently attributed to M. hyopneumoniae infection [Pasteurella multocida and swine influenza virus) as well as influenced by farm management and housing conditions [M. hyopneumoniae circulation in herds from both the slaughterhouse and chronological studies [M. hyopneumoniae.This study was motivated by the interest to identify an easy pulation , severaland CVPC , 7–10. Oged pigs , 6, althnfection , 7, it mnditions , 16. Alt studies , 12, it M. hyopneumoniae experimentally infected pigs [M. hyopneumoniae attachment to epithelial cells, IgG antibodies are mainly found in the alveoli, being more active at lung parenchyma level through participation in opsonization and phagocytosis [M. hyopneumoniae develops faster than the systemic one, which is commonly delayed in onset but persists longer. Henceforth, in an early stage of M. hyopneumoniae infection, a mucosal-based antibody measurement  would be probably better as a CVPC predictor than a serological-based one.At the experimental level, the local humoral immune response was stronger than the systemic one after three weeks of inoculation. Inversely, once the systemic humoral immune response began to increase by 28 dpi, revealed by a higher number of seropositive animals and increased IgG1 and IgG2 responses, a decline of both IgG and IgA antibody levels in BALF was observed. In the present work, higher levels of specific IgG and IgA mucosal antibodies were detected in those animals with CVPC in comparison with those without lung lesions. In agreement, there is a remarkable increase in the number of cells producing immunoglobulins A and G in the lungs of ted pigs –19. Addiocytosis , 21. Theocytosis , the preM. hyopneumoniae acute infection, IgG at the systemic level may be more important in later phases. As supporting evidence of this notion, a relationship between pneumonic lesions and IgG serum levels against Mycoplasma pulmonis in mice has been indicated [M. hyopneumoniae specific IgG might be related with the severity of CVPC and that could help to forecast prevalence and severity of EP-like lung lesions using a population based approach. However, the S/P value was not a good measure to predict severity of CVPC at an individual animal or farm level. Unfortunately, the specific relationship between serological responses and lung lesion scores in the 20 sampled slaughtered animals per farm was not possible. Thus, the association between both parameters at an individual slaughter weight animal level could not be assessed.While the local humoral immune response could play a major role in the pathogenesis of ndicated . Noticeandicated . ResultsM. hyopneumoniae exposure [M. hyopneumoniae seroconversion to the slaughter [M. hyopneumoniae infections would probably escape detection, whereas animals infected close to the slaughter date might show CVPC but no seroconversion. For a better understanding of the humoral immunity value to forecast lifetime CVPC, samples from pigs that were followed up from birth to 22 weeks of age with sequential necropsies were evaluated. In such scenario, CVPC appeared earlier (at 12 weeks of age) than evident seroconversion (15 weeks of age) and both the percentage of seropositive pigs and animals with lung lesions increased progressively towards the end of the finishing period. Henceforth and similar to what was seen in slaughtered pigs, seropositivity was linked with more severe CVPC, but no significant correlation was observed between individual S/P ratios and lung lesion scores at any time point. While the chronological study was performed in non-vaccinated pigs, slaughtered animals belonged to batches from vaccinated and non-vaccinated herds. Although CVPC in slaughtered pigs was not importantly affected by M. hyopneumoniae vaccination [Lung lesions progress and regress throughout the lives of the pigs , 25, 26.exposure , 28, serexposure , 30. In laughter , 31. ThuM. hyopneumoniae infection and the ratio of these subclasses with respect to protection are not yet known. In the murine respiratory mycoplasma infection model, specific Th2 cell responses were found to be involved in the development of inflammatory lung lesions. In contrast, the ability to generate a Th1 cell-mediated lung immune response was related with more protection [A novel approach aimed to determine whether there was a measurable difference in IgG1 and/or IgG2 subclasses in pig sera with and without CVPC was raised. Predominance of one IgG subclass over another in an immune response suggests Th1/Th2 bias . Dominanotection . Remarkaotection .M. hyopneumoniae infections was predominate the IgG2 subclass. In fact, IgG2 OD values and S/P values  were found to be significantly and positively correlated. Moreover, the chronological study revealed that this antibody subclass level developed in parallel with the specific IgG response, indicating that IgG2 might probably be the main component of the systemic humoral immune response against M. hyopneumoniae. Contrarily, previous studies have reported a higher IgG1 than IgG2 antibody response in pigs challenged with M. hyopneumoniae at local [M. hyopneumoniae antigen. The latter might have had a certain influence on the observed IgG isotype bias, as it has been demonstrated that the antigen may determine the type of immune response that develops [M. hyopneumoniae. Interestingly, Th1 immune response is required for host defense against intracellular pathogens [M. hyopneumoniae is regarded as an extracellular bacteria [M. hyopneumoniae associated lung lesions may provide an incentive for further evaluation of the Th immune response in M. hyopneumoniae infection scenarios.In the present study, the systemic humoral immune response in both experimental and natural at local , 37 and at local  levels. at local  proteinsdevelops . Notwithdevelops , presentathogens , 33. Altbacteria , it has bacteria . AltogetM. hyopneumoniae infection in pigs. To our knowledge, this is the first study examining the involvement of different M. hyopneumoniae specific antibody isotype responses in lung lesion development. The results indicated an increased antibody-specific production that most likely is associated to lung lesion severity during infection. Since M. hyopneumoniae systemic humoral immune response is delayed in onset, a mucosal-based antibody measurement would be preferably chosen to predict lung lesion severity at early stages of infection. Being M. hyopneumoniae monitoring by ELISA one of the most common practices on swine farms, the obtained results may give an indication of the lung lesion severity in a certain batch. In addition, there was a clear trend towards the IgG2 response, which may reflect a dominant Th1-controlled antibody-mediated immune response. However, the measurement of other parameters related with the cellular immune response would be required to further study the role that the Th1/Th2 bias could have in M. hyopneumoniae infections.The present study provides insight into those potential mechanisms of immunity that might be implicated in the pathogenesis of S1 Table(XLSX)Click here for additional data file.


Crossed beaks have been reported to occur in Appenzeller Barthuhn, a local Swiss chicken breed. The assumed causes for this beak deformity which are also seen in other bird species including domestic chickens, range from environmental influences to genetic factors. The aim of this project was to characterize the prevalence, the phenotype, and the underlying genetics of crossed beaks in Appenzeller Barthuhn chickens.The estimated prevalence of 7% crossed beaks in Appenzeller Barthuhn was significantly higher compared to two other local Swiss chicken breeds. A breeding trial showed significantly higher prevalence of offspring with deformed beaks from mating of affected parents compared to mating of non-affected parents.Examination of 77 Appenzeller Barthuhn chickens with crossed beaks showed a variable phenotype presentation. The deviation of the beak from the median plane through the head ranged from 1° to 61°. In more than 60% of the cases, the upper and lower beak were bent in the same direction, whereas the remaining cases showed different forms of crossed beaks. Computed tomographic scans and bone maceration of the head of two chickens with crossed beaks revealed that the maxilla and the mandibula were affected, while other parts of the skull appeared to be normal.LOC426217, a member of the keratin family, was postulated as a candidate gene for beak deformity in domestic chickens. Sequencing of the coding region revealed two significantly associated synonymous variants for crossed beaks in Appenzeller Barthuhn chickens. A genome-wide association study and a comparative analysis of runs of homozygosity based on high-density SNP array genotyping data of 53 cases and 102 controls showed no evidence of association.The gene The findings suggest a hereditary cause of crossed beaks in Appenzeller Barthuhn chickens. However, the observed variation in the phenotype, together with the inconclusive molecular genetic results indicates the need for additional research to unravel the genetic architecture of this beak deformity.The online version of this article (10.1186/s12917-018-1398-z) contains supplementary material, which is available to authorized users. Loxia curvirostra) showing distinctive mandibles, crossed at the tips, which enable them to extract seeds from conifer cones and other fruits [A bird’s beak is a defining feature of its head. It is important for the intake of food and water , 2, for r fruits . A bird’r fruits . The rhar fruits , 11. In r fruits ).In domestic chickens, beak deformities affect welfare and production. The occurrence of different types of crossed beaks was already reported almost a century ago in various breeds , 14. TheLOC426217 found five variants that differed significantly between chickens with crossed beaks and normal controls [Different reasons for the occurrence of beak deformities in birds have been proposed: they can result from the underlying bony structure, from a malocclusion of the mandibles or disturbances in the stratum germinativum of the epidermis , 17. Thecontrols .In recent years, breeders have occasionally reported the occurrence of crossed beaks in Appenzeller Barthuhn chickens Fig. . Along wA total of 165 breeding reports, provided by breeders, were analyzed to derive the prevalence of crossed beaks for the breeding seasons 2012–2017. Breeders reported their annual breeding success and the number of hatched animals showing crossed beaks. Estimated prevalence were compared by Pearson’s chi-squared test with Yates’ continuity correction using the R software .Animals from the 2016 breeding season were selected based on the shape of their beak at 12 weeks of age, and included in a targeted breeding experiment as the parental generation. This comprised in total of six breeding units of comparable size: three units consisting of affected animals and three units consisting of control animals  deviation from the Hardy-Weinberg equilibrium. Additionally, genotypes of individuals with a call rate below 90% were excluded. In total, 155 individual genotypes consisting of 341,115 SNPs remained for the genome-wide association study (GWAS). A mixed linear model was fitted with the GenABEL package [p-values were below the 5% Bonferroni-corrected threshold for 341,115 independent tests (pBONF 1.47 × 10− 7). Runs of homozygosity (ROH) were detected using PLINK v1.90 [Genomic DNA samples from 53 cases and 102 control Appenzeller Barthuhn chickens were genotyped with the Axiom™ Genome-Wide Chicken Array Kit (Affymetrix) for 580,961 SNP markers. PLINK v1.07  was appl package , includiNK v1.90  with a dp-value < 0.001) and Schweizerhuhn  .Across the three Swiss local chicken breeds, a total of 78 out of 2698 chicken (2.9%) were reported to show a crossed beak within the past six years Table . The anoIn the F1-generation of the breeding trial, 83 offspring of parents with crossed beaks and 102 offspring of parents with normal beaks were examined between 10 and 12 weeks of age  of crossed beak offspring was observed when comparing these two groups: 2.9% of the offspring from normal parents showed crossed beaks, whereas 15.7% affected animals were observed in the offspring of parents with crossed beaks  showed an upper and lower beak that deviated clearly either left or right , while no significant difference in the direction of the lateral bend of the lower beak  could be found. There was no significant difference between the angles of the upper and the lower beak . The angle between upper and lower beak varied between 0° (cases where both upper and lower beak deviated at exactly the same angle) and 61°.Upper and lower beaks which deviated from the median plane showed a wide range of angles from only 1° up to 50° in the upper beak and from 2° up to 61° in the lower beak. About 50% of all deviation angles of beaks were between 1° and 9° for the upper beak, and 2° and 12° for the lower beak, respectively. The upper beak was more often bent to the right side .In 51 out of 77 cases (66.2%), one or both tips of the beak were turned in the direction of the other tip, as if trying to find its counterpart Fig. . This waA total of 64 cases (83.1%) showed an excessively vertically downward bent upper beak. In 21 cases (27.3%), the lower beak was bent upwards and in 21 cases (27.3%) bent downwards Fig. . In 23 cThe rhamphotheca in animals with crossed beak showed no overall abnormality. Most commonly observed were brittle tips on overlong beaks, sometimes with pieces of tip missing Fig. . The norThe remainder of the head including the cranium, eyes, ears, feathers and wattles did not show any outward abnormality. The most rostral part of the rose comb was often bent with the upper beak. The nares, situated laterally and at the base of the upper beak were in some cases compressed on one side and stretched on the other side depending on the bending of the upper beak Fig. . The opeMaceration and computed tomographic images showed that the shape of several bones was affected by the crossed beak Fig. –h comparLOC426217 in 53 cases (of upper and lower beak deformity) and 114 controls revealed a total of 12 variants  difference in haplotype distribution between cases and controls was observed . The upper and lower beaks of the crossbill are aligned when they hatch and cross with growth [Within the last century, the sporadic occurrence of the crossed beak phenotype has been described by various authors in wildlife as well as in domestic birds including chickens , 39, 40.h growth . This deh growth . The samThis study reports for the first time a detailed phenotype characterization of crossed beak in chickens based on morphological measurements and anatomical examination. Nearly 80 years ago Landauer  developeAs hypothesized earlier a genetic cause is responsible for the development of crossed beaks in chickens. A significantly higher prevalence of affected animals was found in the Appenzeller Barthuhn breed compared with two other local chicken breeds. This information combined with the known differences in the breeds  suggestsLOC426217 located on chromosome 25 was recently proposed as a functional candidate for crossed beak [LOC426217 as a possible candidate gene for crossed beak, although the phenotype of that study included only lower beak deviation. As both associated variants do not cause a change in the amino acid sequence, their functional impact on the phenotype expression is not obvious. Furthermore, the subsequent haplotype association analysis also showed a significant difference between cases and controls in Appenzeller Barthuhn chickens. As the associated LOC426217 haplotype also occurs almost exclusively in the genotyped control cohorts of Appenzeller Spitzhaubenhuhn and Schweizerhuhn, it is unlikely that these variants explain the crossed beak phenotype. It could be speculated that other DNA variants, e.g. structural variants affecting larger segments of DNA, situated in the flanking region containing regulatory important elements of LOC426217 might be present on the associated haplotype in Appenzeller Barthuhn only which could lead to the development of crossed beaks. On the other hand, the identification of LOC426217 as a candidate gene for beak deformities is based on transcript analysis from lower mandibles [The gene sed beak , 16, theLOC426217 variants on chromosome 25 was not confirmed by the genome-wide study. The SNP array marker density was most likely sufficient so it could be assumed, that the previous result might be false positive. The current findings suggest that the crossed beak anomaly in Appenzeller Barthuhn chickens cannot be explained by simple Mendelian inheritance as was previously assumed [Finally, the GWAS and the analysis of runs of homozygosity did not support the association with any specific region of the chicken genome. However, the breeding analysis suggests a genetic background. The previously identified single locus association for the tested  assumed . The colPhenotypic variability in chickens with crossed beaks exists. Genetic predisposition in the Appenzeller Barthuhn chicken breed is evident. The targeted mating experiments confirmed a genetic influence on the occurrence of crossed beak chickens. Genetic analyses were inconclusive, indicating a complex mode of inheritance.Additional file 1:Scheme of the breeding trial. In total six breeding units of comparable size, made up the parental generation: three units consisting of affected animals  and three units consisting of control animals . For the F1-generation, eggs of the six parental groups were collected and incubated. After hatching, chicks were reared until the age of 12 weeks. (TIFF 1025 kb)Additional file 2:Phenotype details of Appenzeller Barthuhn chickens with crossed beak and all cases used in the study. (XLSX 19 kb)Additional file 3:Phenotyping tool for the measurement of beak angles. The head is fixed between two screws, which sit in the orbita of the skull, and one caudal screw, which supports the caudal part of the head. The photograph is taken from directly above. Based on the photograph, the 0° axis is defined as perpendicular to the two screws and through the median plane of the head . The axis of the beak is defined by drawing a line along the base and middle part of the upper or lower beak (yellow line). (TIFF 10326 kb)Additional file 4:Table with all cases for phenotype description and genetic analyses. (XLSX 12 kb)Additional file 5:LOC426217 of Appenzeller Barthuhn, Appenzeller Spitzhaubenhuhn and Schweizerhuhn. (XLSX 30 kb)Genotypes and diplotypes of Appenzeller Barthuhn and two other Swiss Chicken breeds. Genotypes of 12 variants and their diplotypes in Additional file 6:− 7). (B) MDS plot. (C) QQ plot. (TIFF 1480 kb)GWAS of 53 cases and 102 controls. (A) Manhattan plot. The red line marks the 5% Bonferroni-corrected threshold for 341,115 independent tests  position on the chromosome (CHR), and on the y-axis the proportions of cases (yellow) and controls (black) being homozygous. (TIFF 1394 kb)