C9orf72. Here we report a study of the C9orf72 protein by examining the consequences of loss of C9orf72 functions. Deletion of one or both alleles of the C9orf72 gene in mice causes age-dependent lethality phenotypes. We demonstrate that C9orf72 regulates nutrient sensing as the loss of C9orf72 decreases phosphorylation of the mTOR substrate S6K1. The transcription factor EB (TFEB), a master regulator of lysosomal and autophagy genes, which is negatively regulated by mTOR, is substantially up-regulated in C9orf72 loss-of-function animal and cellular models. Consistent with reduced mTOR activity and increased TFEB levels, loss of C9orf72 enhances autophagic flux, suggesting that C9orf72 is a negative regulator of autophagy. We identified a protein complex consisting of C9orf72 and SMCR8, both of which are homologous to DENN-like proteins. The depletion of C9orf72 or SMCR8 leads to significant down-regulation of each other’s protein level. Loss of SMCR8 alters mTOR signaling and autophagy. These results demonstrate that the C9orf72-SMCR8 protein complex functions in the regulation of metabolism and provide evidence that loss of C9orf72 function may contribute to the pathogenesis of relevant diseases.The most common cause of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia is a hexanucleotide repeat expansion in  C9orf72 is one of many uncharacterized genes in the human genome. The presence of repeated nucleotides in the non-coding region of the C9orf72 gene (GGGGCC) has been linked to the neurodegenerative diseases Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD). However, how the presence of these repeats in the gene leads to neurodegeneration is unknown. One possible explanation is that the repeats lead to a reduced expression of the C9orf72 gene and loss of function of the C9orf72 protein. Although C9orf72 is well-conserved among multi-cellular organisms, its protein function remains to be determined. In this study, we demonstrated that loss of C9orf72 reduces mTOR signaling and enhances autophagy. mTOR signaling and autophagy are important for the cellular maintenance of metabolic balances, especially under stress conditions. C9orf72 protein exists in a complex with another DENN-like protein, SMCR8, which also regulates mTOR signaling and autophagy. We generated mice lacking C9orf72, which died prematurely and showed dramatic upregulation of TFEB, a crucial transcriptional regulator of autophagy and lysosomal genes, that integrates mTOR activity state and autophagic capacity. We propose that C9orf72 function is important for metabolic control and its deficiency can contribute to the development of neurodegenerative diseases. C9orf72), has been found to be the most common cause of both ALS and FTD [C9orf72 HRE leads to neurodegeneration remains to be determined, although both gain-of-toxicity and loss-of-function mechanisms have been proposed. The gain-of-toxicity mechanisms involve both RNA and protein products generated from the expanded hexanucleotide repeats. For example, RNAs containing the expanded repeats can interfere with the functions of specific RNA-binding proteins [C9orf72 gene is disrupted. Multiple studies have demonstrated that C9orf72 RNA and protein levels are reduced in patient cells and brains [C9orf72 orthologs in zebrafish and C. elegans has deleterious effects [Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of motor neurons. Frontotemporal dementia (FTD) is the second most common type of dementia in people younger than 65 and is characterized by degeneration of the frontal and temporal lobes of the brain. A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in the promoter or intron of the uncharacterized gene, chromosome 9 open reading frame 72  and enhanced autophagy flux. We further show that C9orf72 interacts with another DENN-like protein Smith-Magenis syndrome chromosome region candidate 8 (SMCR8), which also regulates mTOR signaling and autophagy. The results suggest that a deficiency in the function of C9orf72 may contribute to the pathogenesis of relevant neurodegenerative diseases.C9orf72 has one orthologous gene in the mouse, 3110043O21Rik, which is located on chromosome 4. For convenience, we refer to the mouse gene as C9orf72 hereafter. The mouse C9orf72 gene is predicted to produce seven transcripts, three of which are protein-coding, as compared to the human C9orf72 gene, which produces three transcripts and two protein isoforms. The mouse C9orf72 proteins share 98% identity with their human C9orf72 counterparts  mouse model lacking the protein. Human terparts . We genef72 gene . We furtf72 gene . Westernf72 gene , confirmf72 gene . This def72 gene . In postAlthough we observed no obvious neuronal defects in C9orf72 KO mice, it is possible that C9orf72 has functions in the nervous system in response to stresses. Thus, we asked if mTOR signaling is altered when C9orf72 is absent, since mTOR signaling is a central signaling pathway that senses the stresses related to nutrient availability, oxygen, and energy levels . Also, DSubsequently, we asked whether the observed reduction of mTOR activation in the absence of C9orf72 impacts the function of TFEB, a transcription factor that is a master regulator of lysosome biogenesis and autophagy-related genes, and a substrate of mTOR . In an ain vivo. Analysis of brain homogenates by western blotting from all examined C9orf72 KO mice showed a dramatic increase in endogenous TFEB levels compared with wild-type controls  mass spectrometry . Human Crichment . Notablyrichment . Conversrichment . The intrichment . Consistrichment .Since we identified SMCR8 as the most abundant protein interactor of C9orf72, we asked whether C9orf72 influences the level of SMCR8 protein. While examining the brain lysates from the C9orf72 KO mice, we observed a dramatic reduction in the level of SMCR8 protein. Although present in wild-type brains, SMCR8 was not detected in C9orf72-/- brain homogenates by western blotting . ExaminaTo further study the function of SMCR8, we obtained a CRISPR/Cas-9 generated SMCR8 KO cell line. This cell line contains a frameshift mutation in the first exon of SMCR8 resulting in the loss of the full-length protein product . Since wNext we studied how SMCR8 regulates C9ORF72 protein levels. We first asked whether the regulation occurs due to changes in protein stability or turnover. Since the level of C9ORF72 was too low to allow for chase experiments to probe their turnover in the SMCR8 KO cells, we overexpressed C-terminal-V5 tagged C9orf72 and N-terminal-mCherry tagged SMCR8, or an mCherry only control, into HEK293T cells and studied their protein levels. Compared with the mCherry control, the expression of mCherry-SMCR8 substantially increased the level of C9orf72-V5 . ImportaGiven the connections between C9orf72 and SMCR8, we asked whether loss of SMCR8 plays a role in mTOR signaling similar to that of C9orf72. In accordance with the results from C9orf72-/- MEF cells, knockout of SMCR8 led to a similar defect. In SMCR8 KO HAP1 cells, the phosphorylation of S6K1 after amino acid treatment was significantly decreased when compared with control cells .Next, we investigated if loss of SMCR8 also affected autophagy. First, we examined LC3 levels after shRNA-mediated knockdown of SMCR8 in HEK293T cells by immunoblotting. As observed in C9orf72-/- MEFs and C9orf72 shRNA treated HEK293T cells, knockdown of SMCR8 led to a decrease in the ratio of LC3II to LC3I, when compared with cells treated with scrambled shRNA . AdditioIn the present study, we have identified a function of C9orf72 in regulating mTOR signaling and autophagy. Loss of C9orf72 leads to deficiency in the phosphorylation of S6K1 and increase of TFEB protein levels and nuclear activity, demonstrating a regulatory role of C9orf72 in the mTOR signaling pathway upstream of autophagy.We identified the major interacting partner of C9orf72 protein as SMCR8. The most structurally homologous proteins to SMCR8 and C9orf72 in the human proteome are folliculin (FLCN) and folliculin-interacting proteins (FNIP1 or 2), respectively , 21. LikOur results demonstrate that loss of C9orf72 can alter the dynamics of autophagy. We observed a relative increase in LC3I levels upon loss of C9orf72 , in consThere have been recent reports describing C9orf72’s functions in autophagy , 41–43, Since multiple studies have reported that the hexanucleotide repeat expansion led to reduced expression of C9orf72 mRNAs and proteins in patient cells and brains –15, the The animal protocol (MO15M165) was approved by the Johns Hopkins Animal Care and Use Committee following the National Research Council’s guide to the Care and Use of Laboratory Animals.MluI and BglII followed by blunting and religation. The following shRNA sequences were used: 5’ctgtgttacctcctgaccagtcagattga 3’ (SMCR8); 5’cttccacagacagaacttagtttctacct 3’ (C9orf72). The autophagy luciferase assay plasmids were kindly provided by Brian Seed (Harvard) and the normalization plasmid pCMV-SEAP was from Addgene . GFP-TFEB was obtained from Addgene . GFP-TFEB used for MEF experiments was described before [C9orf72 cDNA (HsCD00398737) was obtained from Arizona State University and SMCR8 cDNA (HsCD00347993) from Harvard Plasmid Repositories. The C9orf72 constructs were generated using the Gateway cloning system  with a C-terminal 3xFlag or V5 tag. The SMCR8 constructs were generated with an N-terminal Flag or mCherry tag using Gateway or classical cloning methods, respectively. All shRNAs were cloned into the pRFP-C-RS vector (Origene), which was modified to remove the RFP coding sequence via digestion with d before . For GFPtm1.1(KOMP)Mbp were obtained from the KOMP repository. The ES cells with a strain background of C57BL/6N-Atm1Brd were microinjected into blastocysts, and the germline-transmitted allele was maintained on the C57BL/6 background. Male mice bearing the original targeting allele were crossed with SOX2-Cre recombinase transgenic female mice  to remove the LoxP-flanked neomycin selection cassette. The resulting allele was bred to heterozygotes and homozygotes that were used in this study. The genotyping primers were the following: gaatggagatcggagcacttatgg , gccttagtaactaagcttgctgccc , gcacaagctatgttcatttgg , gactaacagaagaacccgttgtg .Mouse ES cell lines containing a heterozygous allele of 3110043O21RikFor the low-protein diet assay, 16 week old, gender-matched littermates were fed a low-protein diet  or standard chow for 4 weeks prior to tissue collection. Mouse tissue was lysed in modified RIPA buffer  using a Dounce homogenizer, sonicated, and used for further analysis. For the survival analysis, Kaplan-Meyer curves were generated using GraphPad Prism software.2. Cell lines were transfected using Lipofectamine 2000 (ThermoFisher) according to the manufacturer’s instructions.All cells were maintained in DMEM supplemented with 10% FBS unless otherwise noted. The SMCR8 knockout HAP1 cells (HZGHC003606c011) were created at Horizon Genomics  by using CRISPR/Cas9 and maintained in IMDM supplemented with 10% FBS. All cell lines were cultured in 95% O2/5% COMouse embryonic fibroblasts were isolated from Day 13 embryos by trypsin digestion and their genotypes confirmed by PCR. The lines were immortalized by transfecting cells with the SV40-T antigen-expressing plasmid pSG5 Large T using Lipofectamine 2000. The cells were passaged at least 5x to ensure the homogeneity of the cell population before use in experiments. To isolate embryonic stem cells, 14-week old C9orf72 heterozygous females were treated with Pregnant Mare Serum Gonadotropin via intraperitoneal injection followed by injection 24 hours later with human chorionic gonadotrophin to induce superovulation prior to mating with C9orf72 heterozygous males. Embryos were collected 48 hours after the second injection at the transgenic core facility at Johns Hopkins University and the genotypes confirmed by PCR. Wild type and C9ORF72-/- ES cells were cultured on 0.1% gelatin coated plates in 2i media consisting of half of DMEM/F12 and half of Neurobasal media containing N2-supplement (ThermoFisher Scientific 17502048), B-27 supplement (ThermoFisher Scientific 17504044), 0.05% BSA (ThermoFisher Scientific 15260037), 50 units Penicillin-Streptomycin, 1 μM PD03259010 (Stemgent 04–0006), 3 μM CHIR99021 (Stemgent 04–0004), 2 mM Glutamine, 150 μM Monothioglycerol (Sigma M6145) and 1,000 U/ml LIF.6 ES cells were harvested by dissociating with 0.05% trypsin-EDTA (ThermoFisher) and cultured in suspension condition in DFK5 media , 50 μM nonessential amino acids, 100 μM β-mercaptoehanol, 5 μM thymidine, 15 μM adenosine, 15 μM cytosine, 15 μM guanosine and 15 μM uridine) for 48 hours. After two days, the resulting embryonic bodies were treated with 2 μM retinoic acid and 600 nM of SAG in fresh DFK5 media and cultured another 4 days. Media was replaced every two days. For experiments, 1.5 x 106 cells were plated on each well of laminin-coated 6 well plates in DFK5 media containing 5 ng/mL glial-derived neurotrophic factor , 5 ng/mL brain-derived neurotrophic factor , 5 ng/mL neurotrophin-3  for 24 hours. After 24 hours, media were changed with DFKNB media consisting of half of DFK5 media and half of Neurobasal media with B27, 5 ng/mL GDNF, 5 ng/mL of BDNF and 5 ng/mL of NT-3.Motorneuron differentiation protocol was modified from a previously reported induction protocol using retinoic acid and Smoothened agonist  . BrieflyAll cells were starved using Earles’s balanced salt solution  for 50 min. Amino acid stimulation was applied by treating cells with essential amino acids (Gibco) and non-essential amino acids  in EBSS. Amino acids were diluted to match DMEM concentrations. Cells were treated in EBSS plus amino acids for 10–20 min prior to lysate collection. Lysates were processed as described above except that Phospho-stop inhibitor tablets (Roche) were added to the lysis buffer.Wild type or C9orf72-/- MEF cells were transfected with GFP-LC3 and RFP-Rab7, and cells were treated with DMEM containing 10% FBS  or EBSS , in the presence or absence of the lysosomal inhibitor Bafilomycin A1 (Baf) for 3 hours, before being fixed with 4% paraformaldehyde. High resolution images were acquired using a Z sweep function, permitting acquisition of total cellular fluorescence using a DeltaVision Elite microscope  with 60× PlanApo NA 1.4 Oil objective lens (Olympus) and images were deconvolvd using SoftWoRx software. Subsequently, individual cells were manually segmented; LC3-positve vesicles in the green channel and Rab7-positive vesicles in the red channel. Using a boolean function, the overlap between these segmented images was used to generate a third mask corresponding to co-localized LC3 and Rab7 vesicles (Yellow image) which are autolysosomes. Single vesicle areas were calculated from LC3, Rab7 and co-localization masks, and mean values for each experiment were normalized to ND or FM, as indicated.HEK293T cells were grown on class coverslips and transfected with the indicated constructs as described above. Images were captured using an SP8 confocal microscope (Leica) and processed using ImageJ software. For GFP-TFEB imaging in HEK293T cells, live cells were imaged while maintained in phenol red free DMEM containing 10% FBS. For LysoTrackerBlue staining, 50nM of LysoTrackerBlue was added into the media for an hour and the media was changed before imaging.Cells were collected in modified RIPA lysis buffer  and sonicated using a Diagenode Bioruptor for 15 min  and the resulting lysates were centrifuged at 16,000 x g for 10 min at 4°C. For mTOR assays, cells were collected in HEPES lysis buffer  and centrifuged at 16,000 x g for 10 min at 4°C. Protein concentrations were determined using the bicinchonic acid assay (ThermoFisher). For GFP-TFEB nuclear import analysis, cells were fractionated using Subcellular Fractionation Kit for Cultured Cells (ThermoFisher) following the manufacturer’s protocols. Then the cytoplasmic and membrane proteins were combined as the cytosolic fraction and the nuclear soluble and chromatin bound proteins were combined as the nuclear fraction. PARP was used as a nuclear marker and Caspase 3 as a cytoplasmic marker. For autophagic flux determination, wild type and C9orf72-/- MEFs were subjected to fresh fully supplemented medium or nutrient deprivation (EBSS) for 3 hours. Antibodies used were: mouse anti-Flag, rabbit anti-SMCR8 (Sigma), mouse anti-GFP, rabbit anti-GAPDH (ThermoFisher), rabbit anti-C9orf72, mouse anti-actin (Santa Cruz), rabbit anti-p62, rabbit anti p-70S6K, rabbit anti p-p70S6K, rabbit anti-PARP, rabbit anti-Caspase 3, rabbit anti-DYKDDDDK , rabbit anti-V5 (Novus), mouse anti-V5 (Invitrogen), mouse anti-TFEB (Mybiosources), mouse anti-Lamp1 , mouse anti-Lamp2 , and rabbit anti-LC3B (Abcam).13C6,15N4 L-Arginine, 13C6,15N2 L-Lysine) or light  DMEM and verified for near-completion of labeling by mass spectrometry. Heavy isotope-labeled cells were transfected with C9orf72-Flag and light isotope-labeled cells were mock transfected with Lipofectamine. After immunoprecipitation with Flag-tag beads, the resulting immunoprecipitates were pooled, concentrated, separated via SDS-PAGE, and subjected to trypsin in-gel digestion. The digested samples were subjected to LC-MS/MS analysis on an Orbitrap Elite mass spectrometer coupled with Easy nLC II liquid chromatography system. The mass spectrometry data were analyzed using the Proteome Discoverer 1.4 software suite against human Refseq 59 protein database. A 1% peptide-spectrum-match and peptide-level false discovery rate was applied for data analysis.HEK293T cells were incubated in heavy , incubated for 30 min on ice and centrifuged at 16,000 x g prior to immunoprecipitation. For Flag immunoprecipitations, the resulting supernatants were added to Flag-conjugated beads  and incubated for 2 h at 4°C with gentle rotation. The beads were washed 5x with IP buffer and the immunoprecipitates eluted by incubating the beads with SDS-PAGE loading dye for 5 min at 95° C. For SILAC analysis, immunoprecipitates were eluted using Flag peptide (Sigma) at 5μg/μl. For SMCR8 immunoprecipitation, anti-SMCR8 antibody (Abcam) was incubated with protein A Sepharose beads  and incubated at room temperature for 2 h and the beads treated as described above.Gaussia luciferase release assay [Gaussia luciferase assay kit (New Englabnd Biolabs) and the Phospha-light SEAP reporter system (ThermoFisher) using a microplate reader .The ATG4B-dependent processing of LC3 in autophagy was quantified with a se assay , 58. ATGGender matched four month old mice were intracardially perfused with ice-cold 4% paraformaldehyde. Brains were removed and post-fixed and equilibrated with 30% sucrose. Sections were prepared using Cryostar NX70 (ThermoScientific). Sections were washed with PBST three times to permeabilize cells, pre-incubated with 10% anti-goat serum for an hour at RT, incubated with an anti-p62 antibody  for overnight at 4°C, and then incubated with Alexa488-congugated secondary antibody after washing three times with PBS. Images were obtained using an SP8 confocal microscope (Leica) after samples were washed three times with PBS and mounted with Vectashield.Total RNA was isolated from cells with the RNeasy Plus Mini kit and cDNAs were synthesized with the QuantiTect reverse transcription kit (Qiagen). Primers for quantitative RT-qPCR were from PrimerBank unless otherwise noted . RT-qPCRt tests unless indicated otherwise.All quantitation and statistical tests were performed using ImageJ and GraphPad Prism software (Version 6.0). The p-values for all experiments were obtained using Student’s S1 FigAlignment depicting the predicted two isoforms of human C9orf72 and the three isoforms of mouse C9orf72. The accession numbers are as follows: NP_00124293 Isoform A Human; NP_659442 Isoform B Human; EDL05456 Isoform 1 Mouse; Q6DFW0.1 Isoform 2 Mouse; EDL05457 Isoform 3 Mouse.(TIF)Click here for additional data file.S2 FigA) Immunoblot analysis of whole protein lysates from representative MEF lines generated from C9orf72 KO and wild-type littermates using an antibody against LC3. C9orf72-/- MEFs show an increase in the ratio of LC3I to LC3II when compared with wild type cells. B) Quantification of the LC3II to LC3I ratio from representative C9orf72-/- and wild-type MEF lines from three independent experiments. C9orf72-/- MEFs show a significant increase in the ratio of LC3II to LC3I when compared with wild-type littermate controls . C) Immunoblot analysis of neurally differentiated cells derived from C9orf72 KO and wild-type embryonic stem cells. C9orf72-/- cells with enriched motor neurons show a dramatic increase in LC3I compared with wild-type controls. Student’s t test is used and data is presented as mean ± SEM.(TIF)Click here for additional data file.S3 FigA) Representative live cell images of RFP-Rab7/GFP-LC3 co-localization in C9orf72-/- MEFs. RFP-Rab7 and GFP-LC3 were transfected in wild-type or C9orf72-/- cells and treated with Bafilomycin in fully supplemented medium (FM) conditions. B) Representative image of western blot analysis of LC3 in fully supplemented medium conditions. C) Immunoblot analysis of LC3 levels before and after autophagy induction with nutrient starvation. HEK293T cells were transfected with C9orf72 shRNA or scrambled shRNA control. Approximately 72 hours after transfection, cells were treated with starvation medium (EBSS) with or without Bafilomycin for 2 hours and the resulting lysates were analyzed via immunoblotting. Scale bars: 10 μm.(TIF)Click here for additional data file.S4 FigA) Diagram depicting the luciferase release assay. This assay monitors the cleavage of LC3 by the autophagy-associated protease ATG4B. An actin-LC3-GLuc fusion protein, which is fused to the cytoskeleton, is expressed along with a constitutively secreted alkaline phosphatase (SEAP) normalization control. The cleavage of LC3 by ATG4B allows for the rapid secretion of the GLuc luciferase into the cell medium where it can be measured using standard luciferase assay methods. B) Quantification of ATG4B activity after shRNA-mediated knockdown of C9orf72. HEK293T cells were co-transfected with scrambled or C9orf72 shRNA, an ATG4B activity luciferase reporter, and a normalization control construct. Graph represents the cumulative luciferase signal from time points taken at 48 and 72 hours post-transfection from three independent experiments. Knockdown of C9orf72 significantly decreased secreted luciferase signal which corresponds to a decrease in ATG4B activity . C) Analysis of ATG4B protein levels after shRNA-mediated knockdown of C9orf72 in HEK293T cells. HEK293T cells were transfected with C9orf72 or control shRNA and lysates collected 72 hours after transfection and the indicated proteins detected by immunoblotting. D) Quantification of ATG4B protein levels after shRNA-mediated knockdown of C9orf72 in HEK293T from three independent experiments. Knockdown of C9orf72 does not significantly change ATG4B levels (n = 3). Student’s t test is used and data is presented as mean ± SEM.(TIF)Click here for additional data file.S5 FigA) Representative immunoblot of p62 protein levels in C9orf72-/- MEFs and wild-type control cells. B) Quantification of p62 protein levels shows a decrease in the C9orf72-/- MEFs . C) Immunoblot analysis of liver homogenates from C9orf72-/- and wild-type animals fed a low-protein diet. C9orf72 KO mice show a decrease in LC3II compared with wild-type littermates. D) Quantification of LC3II levels in liver homogenates derived from C9orf72 KO and wild-type animals fed a low-protein diet. C9orf72 KO mice shows a significant decrease in LC3II when compared with wild-type littermates . Student’s t test is used and data is presented as mean ± SEM.(TIF)Click here for additional data file.S6 FigA) Representative MS spectrum of an identified peptide, ANELASVEK for SMRC8. The full MS spectrum of light and heavy forms of the peptide m/z 480.75 and m/z 484.76 and their relative intensity was shown in the spectrum. B) The MS/MS spectrum of the identified spectrum shown in panel (A). C) Validation of the C9orf72-SMCR8 interaction via co-expression in HEK293T cells. HEK293T cells were either co-transfected with Flag-tagged SMCR8 and V5-tagged C9orf72 or transfected with C9orf72-V5 alone and the resulting lysates were incubated with anti-Flag-conjugated beads. V5-tagged C9orf72 successfully co-immunoprecipitates with Flag-SMCR8 and does not bind to beads alone. D) Colocalization of C9orf72 and SMCR8 in HEK293T cells. Cells were transfected with GFP-tagged C9orf72 and mCherry-tagged SMCR8 and imaged using confocal microscopy. Arrow points to a cell expressing both constructs.(TIF)Click here for additional data file.S7 FigA) qPCR analysis of SMCR8 RNA levels in mouse brain. RNA was isolated from wild-type or C9orf72 KO littermates and SMCR8 transcript levels were assessed by qPCR. No significant change in transcript levels were observed between samples (n = 2) B) qPCR analysis of C9orf72 RNA levels in HAP1 SMCR8 knockout cells. RNA was isolated from control or SMCR8 KO cells and C9orf72 transcript levels assessed by qPCR. No decrease in transcript levels was observed in SMCR8 KO cells as compared to control . C) Immunoblot analysis of C9orf72-/- mouse and wild-type littermate brain homogenates shows no change in WDR41 protein levels in C9orf72 KO animals compared with wild-type control animals. D) Immunoblot analysis of control and SMCR8 KO HAP1 cells shows no change in WDR41 protein levels. E) Analysis of C9orf72 protein levels after MG132 or Bafilomycin treatment. HEK293 cells were transfected with C9orf72-V5 and treated with MG132 (5 μM) or Bafilomycin (100 nM) for 16 hours and the indicated proteins analyzed by immunoblotting. C9orf72 protein levels were increased after MG132 or Bafilomycin treatment. Student’s t test is used and data is presented as mean ± SEM.(TIF)Click here for additional data file.S8 FigA) Schematic representation of the CRISPR/Cas9 generated SMCR8 KO cell line. HAP1 cells were engineered to contain a 7-base pair deletion in exon 1 of the SMCR8 gene. This mutation is predicted to have an early stop codon and result in the loss of the full-length protein product. B) Validation of SMCR8 deletion. Sequencing results of genomic DNA extracted from control and SMCR8 knockout HAP1 cells confirming the presence of the 7-base pair deletion in exon 1. C) Immunoblot analysis of HAP1 cells. Immunoblot analysis of control and SMCR8 KO HAP1 cells using an antibody against SMCR8. We interpret the band detected in the knockout cells to be a cross-reactive protein product. D) Analysis of C9orf72 levels in SMCR8 knockdown cells. HEK293T cells were transfected with control shRNA or SMCR8-targeted shRNA and the resulting lysates analyzed via immunoblotting. Cells treated with SMCR8 shRNA show a decrease in C9orf72 levels when compared to cells treated with scrambled shRNA. E) Validation of SMCR8 shRNA. HEK293T cells were co-transfected with mCherry-tagged SMCR8 and either control shRNA or SMCR8 shRNA and the resulting lysates were analyzed by immunoblotting. SMCR8-targeted shRNA successfully knocked down overexpressed SMCR8, validating our shRNA.(TIF)Click here for additional data file.S1 TableThe top hits with the heavy/light SILAC ratio more than five are shown.(PDF)Click here for additional data file.S2 TableThe DNA primer sequences used for quantitative PCR are provided.(PDF)Click here for additional data file.S1 MovieA video of a representative C9orf72-/- mouse at end stage showing lethargic and weak behavior.(MOV)Click here for additional data file.


Approximately one in five breast cancers are driven by amplification and overexpression of the human epidermal growth factor receptor 2 (HER2) receptor kinase, and HER2-enriched (HER2E) is one of four major transcriptional subtypes of breast cancer. We set out to understand the genomics of HER2 amplification independent of subtype, and the underlying drivers and biology of HER2E tumors.We investigated published genomic data from 3155 breast tumors and 5391 non-breast tumors.HER2 amplification is a distinct driver event seen in all breast cancer subtypes, rather than a subtype marker, with major characteristics restricted to amplification and overexpression of HER2 and neighboring genes. The HER2E subtype has a distinctive transcriptional landscape independent of HER2A that reflects androgen receptor signaling as replacement for estrogen receptor (ER)-driven tumorigenesis. HER2 amplification is also an event in 1.8% of non-breast tumors.These discoveries reveal therapeutic opportunities for combining anti-HER2 therapy with anti-androgen agents in breast cancer, and highlight the potential for broader therapeutic use of HER2 inhibitors.The online version of this article (10.1186/s13058-018-0933-y) contains supplementary material, which is available to authorized users. Transcriptional profiling has enabled the classification of many cancers into distinct gene expression subtypes, allowing improved diagnosis and treatment selection. Breast cancer has four well-established, transcriptional subtypes in the prediction analysis of microarray 50 (PAM50) scheme: luminal A, luminal B, basal-like, and human epidermal growth factor receptor 2 (HER2)-enriched (HER2E) . These swww.prosigna.com). In retrospective analysis of breast cancer trials, HER2E tumors with HER2 amplification were observed to benefit most from anti-HER2 agents, luminal A and B tumors with HER2 amplification were likely to benefit from anti-HER2 targeted therapy, and basal-like tumors with HER2 amplification benefited least [Across cancers, classification of tumors by broad expression profiling is increasingly used for drug development and individual treatment decisions. PAM50, for example, is available in both Europe and the USA via the Prosigna™ assay (ed least –11. It ied least . AdditioWe explored the nature of HER2A breast cancer using genomic profiles of 3155 breast tumors from The Cancer Genome Atlas (TCGA) , the MetGenomic data are from TCGA , Metabrihttps://gdc.cancer.gov) and were analyzed with HTSeqGenie [th percentile nRPKM across all breast tumors < 1). This resulted in RNAseq data on 15464 genes for 994 breast tumors.RNA sequencing (RNAseq) data are from NCI's Genomic Data Commons (SeqGenie . Gene exhttps://gdc.cancer.gov). Data were processed with an internal pipeline based on PICNIC [Affymetrix SNP6 copy number data are from NCI's Genomic Data Commons , obtained from a balanced panel of 200 randomly selected ER+ (IHC) and 200 randomly selected ER- TCGA tumors, to mimic the ER proportion in the PAM50 training set. We also updated the symbols of three genes for which annotation has changed since the original PAM50 publication [CDCA1 to NUF2 (Entrez Gene ID 83540), KNTC2 to NDC80 (Entrez Gene ID 10403), and ORC6L to ORC6 (Entrez Gene ID 23594).Expression and copy number data were available for 895 tumors. We performed intrinsic subtype classification using the PAM50 predictor as described . Subtypelication : CDCA1 thttps://gdc.cancer.gov).Of the 895 tumors, 154 were basal-like, 73 HER2-enriched, 423 luminal A, 214 luminal B, and 31 normal-like by PAM50. We excluded the 31 normal-like tumors, based on the hypothesis that normal-like is an artifact of having too few tumor cells and an abundant presence of normal breast and/or stromal cells, supported by a tumor-normal mixing experiment and treatment-induced subtype switching . We alsoExome sequences of tumors and matched normals are from NCI's Genomic Data Commons and were analyzed with HTSeqGenie and the Genome Analysis Toolkit (GATK) for variant calling. Tumor-specific variants were obtained by comparing tumor and matched normal, excluding polymorphic variants from dbSNP version 132 that are not reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database.https://gdc.cancer.gov). Data were processed with an internal pipeline based on R/BioConductor packages methylumi and methyAnalysis, for quality and color balance assessment, color balance adjustment, background adjustment, normalization and methylation modeling. We used the lumi BioConductor package for the calculation of M values, as the log2 ratio of methylated to unmethylated probe intensity.Infinium HumanMethylation450 Beadchip methylation data are from NCI's Genomic Data Commons (https://gdc.cancer.gov). The HER2 antibody used for RPPA is the mouse monoclonal MS-325-P1 (Lab Vision) and recognizes the cytoplasmic domain of recombinant human erb-b2/HER2. This antibody has a predominant single band in western blot against cell lines and tumors, lacks nonspecific binding, has similar results to RPPA and western blot, and was therefore certified for use by RPPA [Reverse phase protein array (RPPA) level-3 data are from NCI's Genomic Data Commons /College of American Pathologists (CAP) guidelines for IHC, supplemented with fluorescent in-situ hybridization (FISH) results and/or copy number calls for tumors with equivocal or missing HER2 IHC .k-nearest neighbor approach with k = 10, using the R package impute. We excluded probes that were detected in less than 1% of tumors, and poor-quality probes  or without match to any genomic region or transcript [ERBB2, this resulted in the selection of probe ILMN_2352131 . Additional removal of genes of uncertain function (LOC symbols) resulted in expression data for 15682 genes in 1990 tumors.Metabric consists of a discovery cohort of 997 patients with breast cancer and a validation cohort of 995 patients with breast cancer . We acceanscript . RetainiAffymetrix SNP6 copy number data for 1991 tumors were processed with our internal pipeline, in the same way as for TCGA. We excluded 28 tumors with low-quality data and high background noise, and 710 tumors for which ploidy and normal contamination could not be estimated accurately. The importance of ploidy for our ploidy-based definition of HER2 amplification warranted stringent custom quality control. This resulted in high-quality DNA copy number alterations for 1253 breast tumors.Expression and copy number data were available for 1252 tumors, of which 167 were basal-like, 135 HER2-enriched, 518 luminal A, 287 luminal B, 142 normal-like, and 3 unclassified according to PAM50 calls provided by Metabric [IHC for ER was performed on 98% of tumors, and IHC for HER2 on 41% of tumors. We defined HER2 positivity as IHC 2+ or 3+. IntClust membership was available for 95% of tumors, of which 202 tumors are ER- by IHC.The United States Oncology trial 01062 is an adjuvant study assessing the addition of capecitabine to standard chemotherapy . DNA andExpression and copy number data were available for 1008 tumors . We excluded 21 normal-like tumors, resulting in 987 tumors. ER and PR status for these tumors were assessed with IHC and HER2 status was assessed with IHC and FISH.HER2 overexpression from RNAseq data (TCGA), Illumina microarray data (Metabric), and Nanostring data (USO1062), and for HER2 and phospho-HER2 protein abundance from RPPA data (TCGA). Voom + limma in R was used for differential gene expression analysis. PAM50 subtype and a measure of chromosomal instability and breakage (CIN) were included in the model when assessing differentially expressed genes between HER2A and non-HER2A breast tumors. CIN is a persistently high rate of loss and gain of chromosomes or chromosomal segments, and can confound differential gene expression when its prevalence is different between HER2A and non-HER2A tumors. We calculated CIN as the total number of segments on the autosomal chromosomes with distinct copy levels. ER expression, PR expression and proliferation score as calculated by the PAM50 algorithm were included in the model when assessing genes differentially expressed between HER2E and non-HER2E breast tumors. Gene set enrichment analysis was done using the camera function in the Bioconductor package limma. Camera is a gene set enrichment test that accounts for correlation between genes that belong to the same gene set [p value corrected for multiple testing ) [We assessed the performance of three HER2A classification schemes: four or more ploidy-corrected copies of HER2, five or more total copies of HER2, and four or more centromere-corrected copies of HER2  ) . The CoxDoane breast cancer classes up, Doane response to androgen up, and Farmer breast cancer cluster 7), and 31 genes suppressed by androgen . We did not include genes located on chromosome 17, to not confound the detection of AR-driven tumors with HER2 amplification. The expression of each signature gene was z-score-normalized across the ER- tumors per cohort . The AR-ness score for an ER- breast tumor was then defined as the average z-scored expression of 14 positive signature genes, minus the average z-scored expression of 31 negative signature genes. Out of 45 signature genes, 42 were available for Metabric. Only nine signature genes were present on the Nanostring platform used for USO1062. These clinical trial tumors were therefore not scored for AR-ness.We built a signature of 14 genes induced by androgen or reflective of active AR signaling  or a diverse panel of molecular data (gastric) –27. For We defined activating HER2 mutations as those with increased tyrosine kinase activity and cellular signaling, that increase cellular transformation and tumor formation, and/or sensitize tumor cells to HER2-targeted therapies in at least one of the referenced studies –31. ThisWe defined HER2-amplified (HER2A) tumors as having a ploidy-corrected copy number for HER2 ≥ 4 (i.e. ratio of copy number to ploidy ≥2). Ploidy corrects for the extensive background amplifications seen in breast tumors. This threshold maximized concordance with HER2 over-expression, clinical HER2 status, HER2 protein abundance, and phosphorylated HER2 protein abundance in TCGA and Metabric  vs. 10% (24)); GATA binding protein 3 (GATA3) mutations are more prevalent in HER2E tumors without HER2 amplification (1.8% (2) vs. 13.3% (11)); and BRCA1 deletions are more prevalent in HER2E tumors with HER2 amplification (21.8% (n = 26) vs. 4.5% (4))  and lipophilin B (SCGB1D2). These are chromosomal neighbors on 11q13, form a covalent complex [We assessed the genomic correlates of HER2 amplification by comparing amplified and non-amplified tumors across all subtypes. We focused on mutations and copy number alterations in 43 genes previously shown by TCGA to be frequently altered in breast cancer  Additio. The mut.8% n = 2 vs. 4.5% complex , and are complex , 34. HER complex , 15. TakDoane breast cancer classes up; Additional file While HER2E tumors are believed to be HER2-driven, only half of HER2E tumors are HER2A. This brought into question whether HER2E is a consistent subtype. We set out to better understand the molecular composition of the HER2E subtype, other than increased transcription of HER2 and GRB7, the two amplicon genes on the PAM50 panel. We performed gene set enrichment analysis between HER2E and non-HER2E TCGA tumors, accounting for ER expression, PR expression and PAM50 proliferation score, and omitting genes on chromosome 17 to reduce interference with HER2 amplification , another steroid hormone receptor that is highly expressed in some breast tumors, has overlapping target genes with ER, and is on average eight times more highly expressed in HER2E compared to non-HER2E tumors , and concordantly the 21 AR-driven basal-like tumors were enriched for HER2A  . Subdivision by subtype revealed that two thirds of HER2E tumors are HER2A, and AR-ness score is consistent across HER2A and non-HER2A tumors of this subtype, showcasing that HER2E is a consistent subtype independent of HER2A status Fig. . In MetaThe finding that ER- and/or HER2A tumors are frequently AR-driven suggests that those tumors may benefit from treatment regimens including AR antagonists. TN tumors with a luminal AR-driven (LAR) profile, defined as expressing AR and downstream AR targets and co-activators , have beTo support this hypothesis, we assessed prognosis in TCGA and Metabric. In TCGA, HER2E ER+ tumors were associated with the worst and luminal A tumors with the best overall survival, and this was the case in both HER2A and non-HER2A tumors  ER+ luminal A vs. ER+ HER2E, 0.301, 95% CI 0.109–0.833) Fig. . In MetaSpecifically for ER- tumors, there is a trend in TCGA towards worse survival in patients with ER- tumors with active AR signaling compared to ER- tumors with inactive AR signaling in both HER2A and non-HER2A tumors  Fig. . Though LRRC37A11P to CASC3. We confirmed the HER2 amplicon boundaries in the Metabric cohort  are not significantly co-amplified with HER2. We thus find no evidence for co-operating copy number drivers with HER2 outside chromosome 17.Outside of chromosome 17, we identified 10 significantly co-amplified regions containing 116 genes . Relative expression levels of broad amplicon genes outside of the core are only lower in lung adenocarcinoma and kidney HER2A tumors in comparison to breast (both FDR p = 0.021)  Fig. .HER2 amplification does not induce transcriptional changes outside of the amplicon in a coherent pan-cancer manner. Only 14 protein-coding genes, all located on 17q12-21, consistently distinguish pan-cancer HER2A from non-HER2A tumors   and enzaFocal amplification of the HER2 locus in non-breast cancers suggests that HER2 is a targetable driver in subsets of other cancers, and indeed, trastuzumab has been approved for gastric and gastro-esophageal junction cancers. Our analysis indicates that bladder, endometrial, and ovarian cancers have the most potential to benefit from HER2-targeted treatment, based on high HER2 levels, but other cancer types may also respond. For instance, the NSCLC cell line Calu-3 is HER2+ by IHC and western blot, and responds to T-DM1 and pertuzumab in vitro and in vivo . TrastuzBesides HER2 amplification, clinical trials are ongoing to evaluate the efficacy of anti-HER2 agents in HER2 mutant but not amplified tumors . We showed in a large pan-cancer cohort that non-HER2A tumors with either an activating or non-functional HER2 mutation do not have elevated levels of phosphorylated HER2. This supports recent in vitro and in vivo work by Zabransky and colleagues suggesting that HER2 missense mutations, when present alone, are insufficient drivers of growth and metastasis .We explored the genomics of HER2 amplification in 3155 breast tumors across three cohorts. While HER2 amplification is traditionally associated with the HER2E transcriptional subtype, we observed that the two are substantially distinct. We found HER2 amplification in other intrinsic subtypes, and used this to categorize the landscape of HER2 amplification, independent of subtype. We also found that HER2E has a distinctive transcriptional landscape independent of HER2A, and that this likely reflects AR signaling as a possible replacement for ER-driven signaling. We propose that the HER2E category be recognized as AR-related, and showed that HER2 amplification is an oncogenic driver, found in all subtypes, rather than a marker of any intrinsic subtype. Beyond breast cancer, HER2 amplification is consistently a pan-cancer event that builds on top of transcriptional subtypes, and we propose candidate cancers for HER2-targeted treatment.Additional file 1:ERBB2, ER, PR and AR for the tumors in these three cohorts. (XLSX 669 kb)Flow chart summarizing sample size and available data for the TCGA, Metabric and USO1062 cohort; Clinical metadata, subtype information, HER2A status, AR-ness score, HER2 copy number, and expression levels of Additional file 2:Robust development of the CN/ploidy definition, and comparison with two alternative HER2A measures. (PDF 115 kb)Additional file 3:p = 0.002), and to 86.1% for centromere-corrected copies (p = 2e-19). Concordance with HER2 protein levels drops from 94.1% to 92.8% (p = 0.18) and 86.6% (p = 7e-6), respectively. Concordance with pHER2 protein levels drops from 93.6% to 91.7% (p = 0.05) and 85% (p = 2e-7). Concordance with clinical HER2 status drops from 94.9% to 94.0% (p = 0.11) and 86.7% (p = 2e-10). Ploidy-corrected HER2A captures a larger fraction of the PAM50 HER2E subtype (90.4% concordance for HER2E vs. other subtypes) than either total  or centromere-corrected HER2A status . (E) HER2 overexpression in 1107 Metabric tumors is defined as log2 expression ratio ≥12.5. (F) Concordance of three HER2A classification schemes with HER2 expression, clinical HER2 status and PAM50 subtype in Metabric. The concordance to HER2 gene expression drops from 97.7% to 95.1% for total copies (McNemar test p = 1e-5), and to 90.9% for centromere-corrected copies (p = 5e-17). Concordance with clinical HER2 status drops from 94.1% to 91.5% (p = 3e-3) and 90.9% (p = 2e-6), respectively. Overlap with PAM50 HER2E drops from 88.6% to 86.8% (p = 2e-3) and 82.3% (p = 1e-14). (G) HER2 overexpression in 987 USO1062 tumors is defined as normalized log10 counts ≥1.02. (H) Concordance of HER2A status with HER2 overexpression, clinical HER2 status and PAM50 subtype in the USO1062 cohort. (PDF 147 kb)Definition of HER2-amplified breast cancer. (A) HER2 overexpression in 864 TCGA tumors is defined as log2 (nRPKM + 1) ≥8.2 . (B) Abundant HER2 protein in 367 TCGA tumors is defined as log2 (RPPA) ≥0.92. (C) Abundant phosphorylated HER2 (Tyr1248) is defined as log2 (RPPA) ≥0.605. (D) Concordance of three HER2A classification schemes with HER2 expression, HER2 protein, phosphorylated HER2, clinical HER2 status and PAM50 subtype in TCGA  Gene-gene expression correlation in TCGA breast tumors for 43 genes differentially expressed between HER2A and non-HER2A tumors when accounting for PAM50 subtype and chromosomal instability. Two sets of 3 or more highly correlated genes (gene-gene correlation >0.6) are highlighted on the right: 28 genes on 17q12-21 near HER2, and 3 SCGB genes at 11q13  and SCGB1D2 (FC 4.4). (PDF 390 kb)HER2 amplification is a discrete event on top of a luminal or basal state, with minor consistent correlation with gene expression. (A) Row-scaled expression of 50 PAM50 genes in 864 TCGA breast tumors, labeled on top by HER2A status and PAM50 subtype. (B) PAM50 scores for TCGA breast tumors categorized by PAM50 subtype and HER2A status. HER2A tumors, in red, are confidently classified as luminal A, luminal B, basal-like, or HER2E, with PAM50 scores within 3.3–4.3% of the PAM50 scores of non-HER2A tumors of the same subtype. (C) Shown are the odds that a genomic alteration in gene A will occur in an HER2A tumor of subtype X compared to the odds of gene A being altered in a non-HER2A tumor of subtype X. Each dot represents the enrichment of alterations in a gene in HER2A compared to non-HER2A tumors of a particular subtype, colored by PAM50, with mutations shown as diamond and copy number alterations as circle. Fisher’s exact Additional file 5:Mutation and copy number profiles of breast tumors. (A) Mutation profile for 23 genes previously shown to be significantly mutated in breast cancer  in 679 TAdditional file 6:p value <0.05, and fold change >2 (linear model). (B) List of 14 protein-coding genes with cancer-independent expression differences between HER2A and non-HER2A tumors in a panel of 2838 non-breast TCGA tumors, after accounting for cancer and chromosomal instability. These genes fulfilled the following criteria: adjusted p value <0.001, and fold change >2. (XLSX 24 kb)Protein-coding genes associated with HER2 amplification. (A) List of 43 protein-coding genes with subtype-independent expression differences between HER2A and non-HER2A breast tumors, after accounting for PAM50 subtype and differences in chromosomal instability, in the TCGA and Metabric cohort. These genes fulfilled the following criteria in at least one cohort: adjusted Additional file 7:Characterization of HER2E tumors and AR-ness signature. (A) Overview of genes differentially expressed between HER2E and non-HER2E TCGA tumors, accounting for ER expression, PR expression, and PAM50 proliferation score, and omitting genes on chromosome 17. (B) Gene set enrichment results for the C2 gene set collection from MSigDB   AR protein levels in 90 ER- TCGA tumors by AR-ness score. (E) Kaplan-Meier curve of overall survival (OS) in 977 Metabric tumors with a median follow up of 7.3 years, divided into 5 groups based on ER IHC status, PAM50 subtype, and AR activity (positive vs. negative AR-ness score). OS was truncated to 17 years of follow up. (F) Left, Kaplan-Meier curve of OS in 57 ER- HER2A tumors from Metabric, divided by AR-ness score. Right, Kaplan-Meier curve of OS in 121 ER- non-HER2A tumors from Metabric, divided by AR-ness score. OS was truncated to 17 years of follow up. (PDF 167 kb)Identification of ER- tumors that are AR-driven. (A) Top 10 C2 gene sets from MSigDB  enrichedAdditional file 9:t test p values per chromosome ranging from 0.37 (chr 5) to 5e-26 (chr 17), and <0.05 for 17/22 autosomes. (B) Copy number levels in 106 HER2A TCGA breast tumors, for genes on chromosome 17 from 35 Mb to 40 Mb (ordered by genomic location). The core HER2 amplicon on top is shown in green, the broad HER2 amplicon in yellow, and genes outside of the broad HER2 amplicon on 17q in magenta. Shown in green on top is correlation between copy number and expression (log2 nRPKM + 1) for each gene across the 864 tumors. (C) HER2 amplicon profile in 133 HER2A Metabric tumors. See Fig. p values for co-amplification of genes with HER2 in 1971 tumors from TCGA and Metabric cohorts. Amplification was defined as 4 or more ploidy-corrected copies. Chromosomes are colored alternatingly in black and gold.  We detected two regions with germline micro-deletions or micro-gains in normal breast tissue from TCGA: 34.4–34.6 Mb and 44.1–44.8 Mb. These regions are defined as loci with copy number levels either >2.4 or <1.6 in at least 5% of normal breast samples, and were removed from Fig. Characterization of the HER2 amplicon and HER2 co-amplification in breast cancer. (A) Chromosomal instability, shown as the number of breaks per kb per autosome, is higher in HER2A than non-HER2A TCGA tumors, with one-sided Additional file 10:p value <0.05). Percentage of amplification in the HER2A vs. non-HER2A tumors, Fisher’s exact FDR p value, and gene annotation are shown. Genes in the core or broad HER2 amplicon are listed, and known cancer genes as reported previously [HER2 co-amplification events: 14 regions with 931 genes are significantly co-amplified with HER2 in TCGA and Metabric combined cohorts , endometrium (p = 0.003), and lung squamous cell carcinoma (p = 0.009) compared to unaltered tumors. (B) Phospho-AKT (pan-AKT Ser473) levels do not differ by HER2 status . (C) Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) levels do not differ by HER2 status ). (D) Prevalence of HER2 mutations varies from 0.4 to 8.6%. (PDF 214 kb)HER2 amplification and mutation in other cancers. (A-C) For each cancer, tumors are grouped by HER2 amplification and mutation status. Tumors without HER2 amplification or mutation are shown in black, HER2A tumors (regardless of HER2 mutation) in red, unamplified tumors with an activating HER2 mutation (HER2MUT act) in green, and unamplified tumors with an untested or functionally inactive HER2 mutation (HER2MUT unk) in gold. (A) Phospho-ERBB3 (Tyr1289) levels are significantly higher in HER2A breast . (E) The three SCGB genes on 11q13 trend towards increased expression in cervix and ovarian cancer, but lack consistent pan-cancer association with HER2A (p = 0.72).  Fold change values were calculated as the difference between log2 average expression, with significance defined as t test p < 0.05. P values were obtained from multivariate linear models, predicting gene set expression by HER2A and cancer in 2838 non-breast tumors. (PDF 271 kb)HER2A tumors in other cancers share certain similarities with breast cancer. (A) Copy number of genes on chromosome 17 are shown in 98 HER2A non-breast tumors. Three distinct groups of HER2A tumors are labeled on the right: tumors with 17q arm-level amplification, defined as 5 or more copies for at least 80% of genes (cyan); tumors with 17q gain (copy number between 2.5 and 5 for 80% or more genes) (magenta); and tumors that are mainly 17q diploid with copy number <2.5 for the majority of 17q genes (green)", with "as per Fig. Additional file 13:PGAP3, ERBB2, MIEN1, GRB7), representative genes in the broad HER2 amplicon , and TOP2A (more telomeric on 17q), in HER2A tumors (red), non-HER2A tumors without HER2 overexpression (black), and non-HER2A tumors with HER2 overexpression . (A) Seven non-HER2A, o/e breast tumors. (B) Six non-HER2A, o/e gastric tumors. (C) Two non-HER2A, o/e endometrial tumors. (D) Two non-HER2A, o/e cervix tumors. (E) Two non-HER2A, o/e bladder tumors. (F) Two non-HER2A, o/e lung squamous cell carcinoma tumors. (G) One non-HER2A, o/e lung adenocarcinoma tumor. (H) One non-HER2A, o/e ovarian tumor. (I) Relative copy number levels for broad HER2 amplicon genes in 23 non-HER2A o/e tumors. Relative copy number levels exceed 2 (or are borderline at 1.9) in 6 out of 23 tumors. Tumors are colored by cancer, and genes in the broad HER2 amplicon are colored as per Fig. p value below the indicated value for a four-group comparison: HER2A, non-o/e; HER2A, o/e; non-HER2A, non-o/e; non-HER2A, o/e. (J) For breast cancer, included are 27 methylation probes with Kruskal-Wallis FDR p < 1e-15. (K) For gastric cancer, included are 37 methylation probes with p < 1e-5. (L) For cervix cancer, included are 28 methylation probes with p < 0.01. (M) For bladder cancer, included are 21 methylation probes with p < 0.01. (PDF 571 kb)Coordinated expression of HER2-neighboring genes in the absence of amplification. (A-H) Expression of genes in the core HER2 amplicon (


Objectives. To explore electroacupuncture's (EA's) effects on fasting blood glucose (FBG) and insulin resistance of type 2 diabetic mellitus (T2DM) model rats and give a possible explanation for the effects. Method. It takes high fat diet and intraperitoneal injection of streptozotocin  for model preparation. Model rats were randomly divided into T2DM Model group, EA weiwanxiashu (EX-B3) group, and sham EA group (n = 12/group). EA  was applied as intervention. FBG, area under curve (AUC) of oral glucose tolerance test (OGTT), insulin resistance index (HOMA-IR), pancreatic B cell function index (HOMA-B), skeletal muscle phosphorylated phosphatidylinositol-3-kinase (PI3K), glucose transporter 4 (GLUT4), and membrane GLUT4 protein expression were measured. Results. EA weiwanxiashu (EX-B3) can greatly upregulate model rat's significantly reduced skeletal muscle PI3K (Y607) and membrane GLUT4 protein expression (P < 0.01), effectively reducing model rats' FBG and AUC of OGTT (P < 0.01). The effects are far superior to sham EA group. Conclusion. EA weiwanxiashu (EX-B3) can upregulate skeletal muscle phosphorylated PI3K protein expression, to stimulate membrane translocation of GLUT4 and thereby increase skeletal muscle glucose intake to treat T2DM. Type 2 diabetes mellitus (T2DM) accounts for nearly 95% of total diabetic patients worldwide , 2. The Glucose transporter 4 (GLUT4) is highly expressed in major tissues responding to insulin including skeletal muscle, liver, and adipose tissue . It actiAcupuncture is proved to have certain effect on T2DM and was thereby recommended by World Health Organization (WHO) as possible therapeutic method for the disease . PreviouAccording to traditional Chinese medicine (TCM), weiwanxiashu (EX-B3) is specialized in treatment of diabetes and is believed to have outstanding effect . It is oThis study aims at observing EA weiwanxiashu's (EX-B3's) effects on hyperglycemia and insulin resistance of high fat diet combined with STZ-induced T2DM rats and exploring their relation with skeletal muscle GLUT4 protein expression.Experiment animals are 65 clean level male Sprague-Dawley rats (160 ± 5 g) purchased from S.P.F limited, Beijing (license: SCXK 2011-004). Animals were maintained in animal facility at Beijing University of Chinese Medicine with a controlled environment of 23°C, relative humidity 60%, and 12 h light/dark cycle. Rats were housed 6/cage, with free access to food and water. All the experiment procedures were performed in accordance with WHO's International Guiding Principles for Biomedical Research Involving Animals. And the study protocol was approved by the Joint Ethical Review Committee of Beijing University of Chinese Medicine (R-20131219-7).All the rats were fed with ordinary rodent chow for 1 week for adaptation. After that, 12 rats were selected randomly according to body weight as normal control group (fed with ordinary rodent chow). Other animals (53 rats) were prepared as T2DM model rats by high fat diet combined with STZ injection according to previous researches –35: RatsRandom blood glucose (RBG) was measured 72 h after the injection, and oral glucose tolerance test (OGTT) was conducted 14 days after the injection. Animals with RBG > 16.7 mmol/L and OGTT's 2 h time-point blood glucose > 11.1 mmol/L were selected as successfully prepared T2DM models. Altogether 36 rats were included as model animals.During the 14 days between STZ injection and model evaluation, rats except those of the normal control group were fed with high fat diet.n = 12), EA weiwanxiashu (EX-B3) group (n = 12), and sham EA group (n = 12). The intervention began at the same day of grouping. And, since the day of grouping, all animals were fed with ordinary rodent chow.According to fasting blood glucose (FBG) measured 14 days after STZ injection, T2DM model rats were divided by random block design into 3 groups. They are T2DM model group  group, the study observes EA weiwanxiashu's (EX-B3's) intervention effects on FBG and glucose tolerance and explores the mechanism of the effects. Sham EA group was set to clarify the genuine effects of EA, when compared with EA weiwanxiashu (EX-B3) group.Details of Interventions. The intervention was applied during 18:00–20:30, once every day (from Monday to Saturday), for 4 weeks, with a one-day interval between every 2 weeks. FBG was tested 8:00 on Sunday morning , with rats fasted overnight since 22:00, Saturday night  Group. Rats were fixed the same way as the normal control or T2DM model group, wait 5 minutes for adaptation. After that, needles of 0.16 mm × 7 mm  were inserted obliquely  into weiwanxiashu (EX-B3) on both sides for 4 mm with help of a plastic tube to minimize the pain. When applying acupuncture, the needle was first put into a tube (which is 1.5 mm shorter than the needle) and pressed against skin of point area. By swiftly tapping on the needle handle outside the tube, the needle penetrated the skin for further adjustment of angle and depth. Soon after the insertion of needles, EA  was applied on weiwanxiashu (EX-B3) by connecting the positive charge of EA device  with needles on weiwanxiashu (EX-B3) and negative charge with a saline-infiltrated cotton ball pasted on rear foot of the same side. Weiwanxiashu (EX-B3) is located 7 mm lateral to the depression under the process of the 8th thoracic vertebrae.Sham EA Group. Rats were fixed the same way as EA weiwanxiashu (EX-B3) group. After that, needles were inserted horizontally (towards the tip of tail at an angle of 15 degrees) into nonpoint  for 2 mm. Soon after the insertion, EA  was applied by connecting nonpoint with a saline-infiltrated cotton ball pasted on the tip of the tail.Fixation of animals, point locating, needle insertion, and EA device connection on all rats during the 4-week intervention were done by the same persons, respectively.FBG was tested 1 day before the intervention and at the 7th, the 14th, the 21st, and the 28th day of the intervention using enzyme end-point method . Blood sOGTT was tested one day before the intervention and the day after the last intervention, to determine the effect of EA on rats' glucose tolerance. Protocol: All animals were fasted overnight from 22:00 to 8:00. After the test of FBG, rats were intragastrically given glucose solution at 2 g/kg body weight  and thenβ-actin.After sacrifice of rats, same part of the quadriceps femoris of all rats was collected for western blot test of GLUT4 protein expression. Results were shown as the ratio to Protocol of Western Blot Test. For each group, 3 samples were selected randomly according to FBG tested before scarification.μg/μL, and then denatured in 95°C water for 5 min.For each sample, 100 mg of the tissue was ground in a 1.5 mL grinder with 1 mL radioimmunoprecipitation (RIPA) lysis buffer and protein inhibitor mixture in ice bath. The homogenate was then collected in a 1.5 mL centrifuge tube, placed on ice for 30 min (vortexed every 5 min), and then centrifuged . The liquid supernatant was then collected for measurement of protein concentration with Bicinchoninic acid (BCA) kit, prepared at a balanced concentration of 4.67 μL–70 μg. Proteins of different groups were wet-transferred  to Polyvinylidene Fluoride (PVDF) microporous membrane (0.45 μm in diameter), blocked 1 h in 5% w/v skimmed milk, 1x TBS, and 0.1% Tween 20 at 25°C with gentle shaking, and then incubated with 1 : 2000 diluted antibody  in 0.5% w/v skimmed milk, 1x TBS, and 0.1% Tween 20 at 25°C with gentle shaking for 1 h and at 4°C for 16 h. After that, the primary antibody was discarded, and the membrane was washed 3 times (5 min each time) with 1x TBST, incubated with 1 : 2000 diluted secondary antibody in 0.5% w/v skimmed milk, 1x TBS, and 0.1% Tween 20 at 25°C with gentle shaking for 1 h, and washed 3 times (5 min each time) with 1x TBST. The photo was taken in darkroom. And integrated optical density (IOD) was calculated with ImageProPlus 6.0.For western blot test, polyacrylamide gel electrophoresis  was made using 10% separating gel. The sample volume is 15 β-actin.After sacrifice of rats, same part of the quadriceps femoris of all rats was collected. Membrane proteins were extracted with Eukaryotic Membrane Protein Extraction Reagent Kit . Other procedures are the same as protocol of western blot test listed above. Results were shown as the ratio to Comparative ratio of skeletal muscle membrane GLUT4 expression was shown as the ratio of skeletal muscle membrane GLUT4 expression to GLUT4 expression of the whole cell.β-actin.After sacrifice of rats, the same part of the quadriceps femoris of all rats was collected for western blot test of PI3K (Y607) protein expression. Protein were extracted with phosphatase inhibitor. Other procedures are the same as protocol of western blot test listed above . Results were shown as the ratio to P < 0.05 was defined as significant difference and P < 0.01 as extremely significant difference. One-way analysis of variance (ANOVA) was applied for the comparison of serum insulin content and skeletal muscle cytoplasmic and membrane GLUT4 expression. For other indices, multiple-group nonparametric test was applied due to variance nonhomogeneity.SPSS 19.0 was applied for the processing of data. Results were shown as P < 0.01), and no statistical difference can be observed between the 3 groups. After 4 weeks' intervention, FBG of T2DM model group, EA weiwanxiashu (EX-B3) group, and sham EA group is still remarkably higher than that of the normal control group (P < 0.01). EA weiwanxiashu (EX-B3) can greatly reduce T2DM model rats' FBG (P < 0.01). But no significant difference can be observed between EA weiwanxiashu (EX-B3) group and sham EA group  group, and sham EA group are significantly higher than that of normal control group (roup see .P < 0.01). And no statistical difference can be observed between the 3 groups. After intervention, AUC of OGTT of T2DM model group, EA weiwanxiashu (EX-B3) group, and sham EA group are still remarkably higher than that of the normal control group (P < 0.01). EA weiwanxiashu (EX-B3) group's result is greatly lower than T2DM model group (P < 0.01). No statistical difference can be observed between EA weiwanxiashu (EX-B3) group and sham EA group  group, and sham EA group are significantly higher than that of normal control group (roup see .P < 0.01). EA weiwanxiashu (EX-B3) can reduce rats' HOMA-IR to an extent significantly lower than T2DM model group (P < 0.01). HOMA-B of T2DM model group, EA weiwanxiashu (EX-B3) group, and sham EA group is greatly lower than that of normal control group (P < 0.01). EA weiwanxiashu (EX-B3) group can significantly increase the reading, compared with T2DM model group (P < 0.05) and sham EA group (P < 0.05)  group, and sham EA group is greatly higher than that of the normal control group (.05) see .P < 0.01). Neither EA weiwanxiashu (EX-B3) nor sham EA can effectively upregulate GLUT4 expression in skeletal muscle . EA weiwanxiashu (EX-B3) can greatly improve GLUT4 protein expression in the membrane, compared with T2DM model group (P < 0.01) and sham EA group (P < 0.05)  group, and sham EA group (.05) see .P < 0.01). EA weiwanxiashu (EX-B3) can effectively upregulate the ratio of GLUT4 in membrane of skeletal muscle, compared with T2DM model group and sham EA group , indicating stimulation of membrane translocation of GLUT4. However, the reading is still greatly lower than that of the normal control group (P < 0.01)  see .P < 0.05). EA weiwanxiashu (EX-B3) can greatly improve PI3K (Y607) expression in skeletal muscle (P < 0.01). Besides, its expression is remarkably higher than sham EA group (P < 0.01)  protein expression of T2DM model group is significantly lower than that of the normal control group (.01) see .shu point is where qi of its corresponding zang-fu organs gathers. Back-shu point is therefore especially useful for treatment of diseases of its related zang-fu organ [shu point of the pancreas and is therefore believed to be useful for treatment of T2DM. The point was frequently selected in clinical trial or animal experiment studies of T2DM in China, and many reports published in Chinese have proved its outstanding effect [According to TCM, back-fu organ . Weiwanxg effect –31. Howeg effect , 32 due Results of our study proved that EA weiwanxiashu (EX-B3) can significantly reduce model rats' highly rocketed FBG , AUC of The above changes can be performed by activation of GLUT4. In static state, GLUT4 is sequestered in intracellular vesicles in skeletal muscle cells and has no biological activity. When stimulated by insulin signal or muscle contraction , vesiclePrevious researches show that it is possible to regulate GLUT4 expression by EA –46. It iOur research results show that EA weiwanxiashu (EX-B3) can significantly improve hyperglycemia and insulin resistance of model rats and greatly improve skeletal muscle membrane GLUT4 expression but not cytoplasmic GLUT4 expression, indicating that EA can stimulate GLUT4 membrane translocation to reduce FBG and relieve insulin resistance. Test of skeletal muscle PI3K (Y607) protein expression demonstrates that, in our study, upregulation of GLUT4 membrane translocation is related to phosphorylation of PI3K.This is in accordance with previous research conclusion that insulin signal takes effects in the skeletal muscle by binding insulin with the alpha-subunits of insulin receptor on the surface of cell. After that, beta-subunit of insulin receptor will autophosphorylate and lead to activating tyrosine phosphorylation of insulin receptor substrate  . PhosphoSince in our study, EA weiwanxiashu (EX-B3) group exceeds sham EA group in regulation of skeletal phosphorylated PI3K expression , membranEA weiwanxiashu (EX-B3) is especially useful for intervention of T2DM.EA weiwanxiashu (EX-B3) can upregulate skeletal muscle phosphorylated PI3K protein expression, to stimulate membrane translocation of GLUT4 and thereby increase skeletal muscle glucose intake to reduce blood glucose and relieve insulin resistance.


P<0.001) and TNM stages (P<0.001). And higher PTP4A2 expression was an independent prognostic factor for adverse overall survival (P<0.05) and poor disease-free survival (P<0.05). Our results demonstrated that the overexpression of PTP4A2 was closely associated with poor survival outcome in patients with NPC and may represent a novel prognostic biomarker and therapeutic target for this disease.Protein tyrosine phosphatase 4A2 (PTP4A2) has been implicated as an oncogenic protein in several human cancers. However, the level of PTP4A2 expression and its prognostic significance in nasopharyngeal carcinoma (NPC) remains unknown. In this study, Western blotting (WB), quantitative real-time PCR (qT-PCR) and immunohischemistry (IHC) was applied to evaluated the expression levels of PTP4A2 in NPC cell lines and tumor tissues combining two independent cohorts. Receiver-operator curve (ROC) analysis was used to assessed the optimal cut-off score in training cohort (266 cases). This cut-off score was subjected to determine the association of PTP4A2 expression with patients’ clinical characteristics and survival outcome in the validation cohort (201 cases) and the overall population (467 cases). We found that PTP4A2 were significantly overexpressed in NPC cell lines compared with normal nasopharyngeal epithelial cell. Moreover, overexpression of PTP4A2 was positively correlated with advanced T classification ( Nasopharyngeal carcinoma (NPC), an Epstein-Bar virus (EBV)-related head and neck cancer, is a neoplastic disorder that arises from the epithelial lining of the nasopharynx . Though PTP4A1, PTP4A2, and PTP4P3 genes, respectively [Tyrosine phosphorylation and dephosphorylation, are ubiquitous regulatory events affecting the functional activities of protein that activate diverse cellular processes, such as cell cycle progression, apoptosis, transcriptional regulation, protein trafficking and protein degradation . Alteredectively , 7. Prevectively –10. Howeectively –13. Of nectively . HoweverIn this current study, we examined the expression status of PTP4A2 in two independent cohorts of NPC patients using immunohistochemistry (IHC) method. Receiver operating characteristic (ROC) curve analysis was performed to select the optimal cut-off value for PTP4A2 expression into high- and low-expression groups. We finally founded that up-regulated expression of PTP4A2 was significantly associated with aggressive clinical features and poor survival of NPC patients in both the training and validation cohorts.Western blotting and quantitative real-time PCR was applied to analysis the expression levels of PTP4A2 protein and mRNA in five NPC cell lines  and one immortalized primary nasopharyngeal epithelial cell line (NPEC2 Bmi-1). Our western blotting analysis demonstrated that NPC cell lines exhibited higher level of PTP4A2 protein expression as compared to that in NPEC2 Bmi-1 Figure : left. qTo better estimate the expression of PTP4A2 in NPC, we first subjected the IHC score of PTP4A2 in the training cohort to ROC curve analysis with respect to each clinical feature , N status(P=0.003), TNM stage(P<0.001), Viral capsid antigen immunoglobulin A (VCA IgA) (P<0.001) and Early antigen immunoglobulin A (EA IgA) (P<0.001). But in validation cohort, the difference was only observed in T classification (P=0.002) and TNM stage (P=0.012). As expected, we failed to discover a significant difference between PTP4A2 expression and other clinical characteristics in both cohorts, including patients’ age, gender and WHO type (P>0.05).Table P<0.05, Table P<0.001, Figure P<0.001, Table P<0.001, Figure P<0.001, Table P<0.001, Figure P<0.001, Table P<0.001, Figure P<0.001, Table P<0.05, Table P<0.05, Table The established prognostic factors of the NPC patients’ survival were first utilized to test the representativeness of all NPC cases in our study. Univariate analysis suggested a significant impact of well-known clinicopathologic prognostic features, such as such as T classification, N status and TNM stage on the patient survival rates  constitutes a large family of enzymes that can exert variable effects on signaling pathways , 19. Notal stage , 22. Oural stage  and coloal stage . In partal stage , 19, 22.et al demonstrated that PTP4A2 might activate small GTPases of the Rho family to promote invasion and metastasis [et al showed that PTP4A2 could regulated tumor cell migration and invasion in human lung cancer cells through an ERK-dependent signaling pathway [As we known, the majority of cancer deaths is a result of tumor metastasis rather than primary tumors, and the vascular invasion and metastasis are important steps in cancer progression. The potential function of PTP4A2 in cancer progression have been explored in recently investigations. A study by James tastasis . Other stastasis . Wang et pathway . We prev pathway . All theIn summary, this is the first study aimed at evaluating the possible use of PTP4A2 as a clinically relevant indicator for NPC aggressiveness and as a prognostic marker for patient survival in NPC. Nevertheless, further investigation into the cellular pathways by which PTP4A2 exerts its influence on the development and progression of NPC and prospective studies on the prognostic significance of PTP4A2 are required. We hope PTP4A2 might be helpful to render a more personalised treatment strategy in NPC.2.NPC cell lines , and one immortalized human nasopharyngeal epithelial cells (NPECs) induced by Bmi-1 (NPEC2 Bmi-1) were obtained from the group of Musheng Mu. The cells were cultured according to the instructions from the supplier. They were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% COParaffin-embedded pathological specimens of NPC patientsn=266) in training cohort were obtained form the archives of the Department of Pathology, between January 2002 and December 2004 at Sun Yat-sen University Cancer Center, Guangzhou, China. Another randomly collected, independent validation cohort of 201 NPC patients between January 1998 and December 2001 from the same department were obtained and assessed in parallel. The enrollment criteria were: 1) histopathologically and clinically proven primary carcinoma of nasopharynx; 2) no previous malignant disease or a second primary cancer; 3) no previous treatment or severe complications. The clinicopathologic information of two cohorts including age; gender; T classification; N status; TNM stage; WHO type; Viral capsid antigen immunoglobulin A (VCA IgA) and Early antigen immunoglobulin A (EA IgA) were described in Table 66 in traThe expression of PTP4A2 in NPC was examined by immunohistochemical techniques using a standard two-step technique as demonstrated previously . These sExpression levels of PTP4A2 protein were evaluated by microscopic examination of stained slides. The presence of cytoplasmic brown granules was considered to be positive for PTP4A2 expression. In Brief, the expression pattern was assessed as follows: the staining intensity was assigned as 0 (no staining), 1+ (weak), 2+ (moderate) or 3+ (strong) (I0-3). Then, the proportion of tumor cells with that intensity was divided by the total number of tumor cells and recorded in 5% increments from 0 to 100 . Each IHC score (range 0-300) was determined by adding the sum of the scores obtained for each intensity and the proportion of the area stained (F=I1×P1+I2×P2+I3×P3). The reproducibility of the scoring manner has been described previously . PTP4A2 Receiver-operator curve (ROC) analysis was applied to PTP4A2 to define cutoff scores for tumor “positivity” by a 0,1-criterion . BrieflyWhole cell extracts were prepared in radio immunoprecipitation assay (RIPA) buffer and centrifuged at 12000g for 15 minutes. Protein concentrations were measured using the bicinchoninic acid assay. Equal amounts of whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferrend on a ployvinylidene difluoride (PVDF) membrane . The membranes were then blocked with 5% skimmed milk and incubated with primary rabbit monoclonal antibodies against PTP4A2  and GAPDH , respectively. After washing the membrane, it was then incubated with secondary anti-mouse antibody from Santa Cruz Biotechnology . The immunoreactive signals were detected with enhanced chemiluminescence kit . The procedures followed were conducted in accordance with the manufacturer's instructions.Total RNA was extracted from the NPC cell lines using Trizol regent  and cDNA was synthesized by SuperScript Reverse Transcriptase kit  according to the manufacturer's instruction. The primer sequences used to amplify PTP4A2 were: 5′-AGCCAGGTTGCTGTGTTGCAG-3′ (forward) and 5′-CACAGCAATGCCCATTGGTA-3′ (reverse). GAPDH was used as an internal control for normalization.Statistical analysis was performed with the SPSS statistical software package . The relationship between PTP4A2 expression and the clinicopathologic features of the NPC patients in both cohorts were evaluated by a Pearson's chi-squared test. ROC analysis was employed to determine the cutoff value for PTP4A2 positivity. Univariate and multivariate survival analysis was performed with the Cox proportional hazards regression model. The corresponding Hazard ratio (HR) and 95% CI were taken from Cox regression models. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. Differences were considered significant if the P-value from a two-tailed test was<0.05.


Cystoisospora spp., Toxocara canis, Giardia sp.) and viral agents that are frequently encountered and known to cause gastrointestinal signs in dogs  as a comparative model.Previous data suggest that fecal S100A12 has clinical utility as a biomarker of chronic gastrointestinal inflammation (idiopathic inflammatory bowel disease) in both people and dogs, but the effect of gastrointestinal pathogens on fecal S100A12 concentrations is largely unknown. The role of S100A12 in parasite and viral infections is also difficult to study in traditional animal models due to the lack of S100A12 expression in rodents. Thus, the aim of this study was to evaluate fecal S100A12 concentrations in a cohort of puppies with intestinal parasites (Cystoisospora spp. (C. canis and C. ohioensis), Toxocara canis, Giardia sp., as well as canine coronavirus (CCV) and parvovirus (CPV). S100A12 concentrations were measured in all fecal samples using an in-house radioimmunoassay. Statistical analyses were performed using non-parametric 2-group or multiple-group comparisons, non-parametric correlation analysis, association testing between nominal variables, and construction of a multivariate mixed model.Spot fecal samples were collected from 307 puppies  in French breeding kennels, and fecal scores  were assigned. Fecal samples were tested for Cystoisospora spp. (P = 0.0384) and in dogs infected with parvovirus (P = 0.0277), whereas dogs infected with coronavirus had decreased fecal S100A12 concentrations (P = 0.0345). However, shedding of any single enteropathogen did not affect fecal S100A12 concentrations in multivariate analysis  in this study. Only fecal score and breed size had an effect on fecal S100A12 concentrations in multivariate analysis (P < 0.0001).Fecal S100A12 concentrations ranged from < 24−14,363 ng/g. Univariate analysis only showed increased fecal S100A12 concentrations in dogs shedding An infection with any single enteropathogen tested in this study is unlikely to alter fecal S100A12 concentrations, and these preliminary data are important for further studies evaluating fecal S100A12 concentrations in dogs or when using fecal S100A12 concentrations as a biomarker in patients with chronic idiopathic gastrointestinal inflammation. The presence of C. canis was associated with breed size and CPV2 infection .There was an association between an abnormal fecal score and the presence of Fecal S100A12 concentrations ranged from < 24−14,363 ng/g , with 20/307 dogs (6%) having a fecal S100A12 concentration above the upper limit of the reference interval (i.e. > 745 ng/g).Cystoisospora spp. . However, there was no significant difference between those dogs infected or not with either C. canis  or C. ohioensis-complex   nor Giardia spp.  was significantly associated with increased fecal S100A12 concentrations  = 0.08; P = 0.1956), C. ohioensis (ρ(301) = 0.08; P = 0.1721) and T. canis (ρ(301) = 0.06; P = 0.3451) did not correlate with fecal S100A12 concentrations. Fecal S100A12 concentrations were affected by CCV  or CPV2 infection  in univariate analysis  (Table (298) = -0.28, 95% CI: -0.35– -0.12; P < 0.001). Breed size was also significantly associated with fecal S100A12 concentrations   = 23.418, P < 0.0001) and breed size  = 11.655, P < 0.0019) remained significant  = 14.54, P = 0.0004) when evaluating the same parameters in multivariate models not including the parameter fecal score.On multivariate analyses, only the effect of fecal score . These preliminary data are important for further studies evaluating fecal S100A12 concentrations in dogs with chronic gastrointestinal diseases and are also of comparative relevance given the limitations of studying the effects of similar enteropathogens on fecal S100A12 concentrations in humans.Toxocara canis or Giardia sp. In contrast, the presence of the parasite genera Cystoisospora spp. - independent of their number or subspecies - as well as an infection with the viral pathogens CCV or CPV2 were shown to be associated with alterations in fecal S100A12 concentrations on univariate analysis but not in a multivariate model. Thus, infections with enteropathogens evaluated in this study are not primarily associated with alterations in fecal S100A12 concentrations, whereas the concentration of fecal S100A12 is affected by the fecal score which reflects the overall health of the digestive tract. This is important for further studies evaluating fecal S100A12 concentrations in patients with suspected chronic gastrointestinal inflammation [Giardia duodenalis and helminth infections [This study showed that fecal S100A12 concentrations in puppies are not affected by an infection with ammation . These ffections . Also, tfections  were comfections .Cystoisospora spp., CCV and CPV2 in univariate analysis) and those dogs testing negative in this study. This was still an unexpected finding given that S100A12 has been shown to have filariostatic or filaricidal properties (anti-parasite mechanism) through its binding to parasite paramyosin [Echinococcus granulosus was associated with a strong S100A12 response [E. granulosus results in a more granulomatous type of inflammation although other cells were also shown to express S100A12 at the site of infection-associated inflammation. These findings further suggest that limiting the availability of transition metal ions  is not the mechanism of action to combat parasite or viral infections as opposed to infections with bacterial pathogens [E. coli has been shown for canine calprotectin [No difference in fecal S100A12 concentrations were detected between puppies testing positive for enteropathogens  using samples from older puppies and adult dogs . Using tWe acknowledge that this study has some limitations. First, the small sample size in the analyses of subgroups of dogs carries the potential for type II error. Secondly, spot fecal samples were used for the detection of enteropathogens and also for measuring fecal S100A12 concentrations, but the biological variation and distribution of these enteropathogens and the S100A12 protein in fecal specimens in healthy dogs and dogs with gastrointestinal disease has not yet been reported. Lastly, histopathologic evaluation of gastrointestinal mucosal biopsies to determine whether enteropathogen shedding, a decreased fecal score, or both are associated with any clinically relevant mucosal lesions  was not performed in the dogs in this study.Cystoisospora spp., Toxocara canis, Giardia sp., CCV and CPV2 does not appear to primarily affect fecal S100A12 concentrations in dogs. Thus, the presence of any one of these endoparasites or viral agents appears to be an unlikely cause of altered fecal S100A12 concentrations, but the fecal score as an indicator of the overall digestive health is reflected by the fecal S100A12 concentration. These preliminary data are an important starting point for further studies evaluating fecal S100A12 concentrations in dogs or when using fecal S100A12 concentrations as a biomarker in patients with chronic idiopathic gastrointestinal inflammation. In addition, the dog could be considered as an alternative model for studying S100A12 biology in humans.We conclude that shedding of the enteropathogens


Quadriviridae, has a multipartite genome consisting of four monocistronic segments. Whereas most dsRNA virus capsids are based on dimers of a single protein, the ~450-Å-diameter, T = 1 RnQV1 capsid is built of P2 and P4 protein heterodimers, each with more than 1000 residues. Despite a lack of sequence similarity between the two proteins, they have a similar α-helical domain, the structural signature shared with the lineage of the dsRNA bluetongue virus-like viruses. Domain insertions in P2 and P4 preferential sites provide additional functions at the capsid outer surface, probably related to enzyme activity. The P2 insertion has a fold similar to that of gelsolin and profilin, two actin-binding proteins with a function in cytoskeleton metabolism, whereas the P4 insertion suggests protease activity involved in cleavage of the P2 383-residue C-terminal region, absent in the mature viral particle. Our results indicate that the intimate virus-fungus partnership has altered the capsid genome-protective and/or receptor-binding functions. Fungal virus evolution has tended to allocate enzyme activities to the virus capsid outer surface.Unlike their counterparts in bacterial and higher eukaryotic hosts, most fungal viruses are transmitted intracellularly and lack an extracellular phase. Here we determined the cryo-EM structure at 3.7 Å resolution of Rosellinia necatrix quadrivirus 1 (RnQV1), a fungal double-stranded (ds)RNA virus. RnQV1, the type species of the family  Rosellinia necatrix, a pathogenic ascomycete to a wide range of plants. At difference most dsRNA viruses, whose capsid is made of protein homodimers, RnQV1 is based on a single-shelled lattice built of 60 P2-P4 heterodimers. Despite a lack of sequence similarity, P2 and P4 have a similar α-helical domain, a structural signature shared with the dsRNA virus lineage. In addition to organizing the viral genome and replicative machinery, P2 and P4 have acquired new functions by inserting complex domains in preferential insertion sites. Whereas the P2 insertion domain has a fold like that of actin-binding proteins, the structure of the P4 insertion domain indicates proteolytic activity. Understanding the structure of a fungal virus capsid with enzyme activities could allow its development as nanoreactors for biotechnological application.Most fungal RNA viruses are transmitted by cytoplasmic interchange without leaving the host. We report the cryo-electron microscopy structure, at near-atomic resolution, of the double-stranded RNA Rosellinia necatrix quadrivirus 1 (RnQV1); this virus infects the fungus  The virus capsid is sufficiently robust to protect the genome and ensure its propagation during passage from one host cell or organism to another; it must simultaneously be labile enough to allow genome delivery within the host cell to initiate infection. The capsid is not only a metastable macromolecular assembly, but is also a dynamic structure whose protein components have transient conformations related to its distinct roles during the viral life cycle , 2. NumeReoviridae  and Relion [http://www2.mrc-lmb.cam.ac.uk/relion/index.php/Main_Page]. Graphics were produced by UCSF Chimera [http://www.cgl.ucsf.edu/chimera/]. A total of 53,683 particles were picked with the Xmipp automatic picking routine and 37,531 particles were selected manually. Alternatively, Relion reference-free 2D classification was used to discard bad particles and a similar data set of 42,267 particles was obtained. 3D classification with Relion resulted in four classes using the RnQV1-W1075 structure [To correct for beam-induced movement, the 18 central frames of each movie were aligned using whole-image motion correction , after wd Relion  [http:// Chimera  [http://tructure   was used for structural alignment between RnQV1 CP P2 and P4 proteins and their overlap with PcV CP A and B domains as well as L-A Gag CP. We used FATCAT [http://fatcat.burnham.org), CATH [http://www.cathdb.info) and Dali servers to search for structures related to the P2 Asp227-Tyr350 domain.The electrostatic potential for the RnQV1-W1118 capsid was calculated using DelPhi software  and surfd FATCAT  (http://g), CATH   with trypsin  as described . ResultiThe RnQV1-W1118 cryo-EM map is deposited in the Electron Microscopy Data Bank (accession no. emd-3619) and the atomic coordinates of the RnQV1-W1118 P2 and P4 proteins are deposited in the PDB (ID code 5ND1).S1 Fig(A) Surface-shaded RnQV1 capsid (left) and central slab of density (right) viewed along a twofold axis. The surface is colored based on local resolution as indicated in the color key . (B) Cryo-EM density map of the RnQV1 capsid asymmetric unit. Backbones of P2 (yellow) and P4 (blue) are superimposed on the density map.  Regions of the cryo-EM density map (grey mesh), with atomic models of (C) helices P4 α25 (yellow), P2 α6 (blue) and (D) two β-sheets of P4  and P2 . Atomic models are shown as ribbons and sticks with amino acid residues labeled.(TIF)Click here for additional data file.S2 FigPositive ion mode mass spectra of RnQV1 (A) P2 and (B) P4 bands analyzed by MALDI ionization. Mass spectra showed peaks in the mass/charge (m/z) 900–3500 range.(TIF)Click here for additional data file.S3 FigTop, ribbon diagrams of P2 (left) and P4 (right) SIID, rainbow-colored from blue (N terminus) to red (C terminus). Bottom, sequence alignment of P2 Cys738-Ser848 (blue) and P4 Gly817-Thr929 (yellow) segments. α-helices (rectangles) and β-strands (arrows) are rainbow-colored from blue (N terminus) to red (C terminus) for each SIID.(TIF)Click here for additional data file.S4 Fig(A) Dali sequence alignment of P2 (blue) and PcV domain A (orange). The α-helices (rectangles) and β-strands (arrows) are rainbow-colored from blue (N terminus) to red (C terminus). Triangles indicate nonaligned segments (sizes indicated). (B) Dali sequence alignment of P2 (blue) and L-A virus Gag (red). Triangles indicate nonaligned segments (sizes indicated).(TIF)Click here for additional data file.S5 Fig Dali sequence alignments of P4 (yellow) and PcV domain A (orange) (A), and P4 (yellow) and L-A virus Gag (red) (B). The α-helices (rectangles) and β-strands (arrows) are rainbow-colored from blue (N terminus) to red (C terminus). Triangles indicate nonaligned segments (sizes indicated).(TIF)Click here for additional data file.S6 FigT = 1 capsids of Penicillium chrysogenum virus (PcV), L-A virus (L-A), Penicillium stoloniferum virus F (PsV-F), picobirnavirus (PBV), Rosellinia necatrix quadrivirus 1 (RnQV1), rotavirus, bluetongue virus (BTV), cytoplasmic polyhedrosis virus (CPV), and orthoreovirus, viewed along a two-fold axis of icosahedral symmetry. The inner surface charge representations of this non-exhaustive group of dsRNA virus T = 1 capsids show the distribution of negative (red) and positive (blue) charges. dsRNA densities and other packaged proteins (such as RNA polymerases) were removed computationally.(TIF)Click here for additional data file.S7 Fig(A) Central section of the RnQV1 capsid 3D reconstruction viewed along a two-fold axis (protein is dark). Arrows indicate the five- (5), three- (3) and two-fold (2) icosahedral symmetry axes. (B) Magnified view of the box in A. Atomic model regions of P2 (blue) and P4 (yellow) are superimposed on the protein density (black) The last visible P4 residue (K1005) is indicated in two P4 molecules. (C) Five copies of P2 (blue) and five of P4 (yellow) are superimposed on the density map around a five-fold axis. The five P4 Lys1005 (red) are followed by five partially disorganized densities (yellow) that occlude the five-fold axis channel, in which it was not possible to include additional P4 residues.(TIF)Click here for additional data file.S8 Fig(A) Immature RnQV1 capsid. The 30 dimeric rod-shaped densities (orange) represent the processed P2 C-terminal α-helical domains. Capsid shell colored as in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Movie(MP4)Click here for additional data file.S2 Movie(MP4)Click here for additional data file.S3 Movie(MP4)Click here for additional data file.S4 MovieP2 is shown as blue sticks and P4 surfaces are represented with electrostatic potentials, showing the distribution of negative (red) and positive (blue) charges.(MP4)Click here for additional data file.S5 Movie(MP4)Click here for additional data file.S6 Movie(MP4)Click here for additional data file.