Accumulating evidence suggests that pathogenic TAR DNA-binding protein (TDP)-43 fragments contain a partial RNA-recognition motif domain 2 (RRM2) in amyotrophic lateral sclerosis (ALS)/frontotemporal lobar degeneration. However, the molecular basis for how this domain links to the conformation and function of TDP-43 is unclear. Previous crystal analyses have documented that the RRM2-DNA complex dimerizes under acidic and high salt conditions, mediated by the intermolecular hydrogen bonds of Glu246-Ile249 and Asp247-Asp247. The aims of this study were to investigate the roles of Glu246 and Asp247 in the molecular assembly of RRM2 under physiological conditions, and to evaluate their potential use as markers for TDP-43 misfolding due to the aberrantly exposed dimer interface. Unexpectedly, gel filtration analyses showed that, regardless of DNA interaction, the RRM2 domain remained as a stable monomer in phosphate-buffered saline. Studies using substitution mutants revealed that Glu246 and, especially, Asp247 played a crucial role in preserving the functional RRM2 monomers. Substitution to glycine at Glu246 or Asp247 induced the formation of fibrillar oligomers of RRM2 accompanied by the loss of DNA-binding affinity, which also affected the conformation and the RNA splicing function of full-length TDP-43. A novel monoclonal antibody against peptides containing Asp247 was found to react with TDP-43 inclusions of ALS patients and mislocalized cytosolic TDP-43 in cultured cells, but not with nuclear wild-type TDP-43. Our findings indicate that Glu246 and Asp247 play pivotal roles in the proper conformation and function of TDP-43. In particular, Asp247 should be studied as a molecular target with an aberrant conformation related to TDP-43 proteinopathy. Recent advances in proteomics have allowed the identification of a new marker protein, TAR DNA-binding protein 43 kDa (TDP-43), for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) C. elegans model of TDP-43 proteinopathy found a definite motor phenotype only when the transgene contained both RRM1 and RRM2 TDP-43 is composed of an N-terminal nuclear localizing signal (NLS), two RNA-interacting domains , and a C-terminus containing a glycine-rich domain. It is our consensus that the overexpression of carboxyl 25- and 35-kDa fragments containing the C-terminus is able to recapitulate many features of TDP-43 proteinopathy 4)2SO4, pH 4.2) through the hydrogen bonds of Glu246 (E246)-Ile249 (I249) and Asp247 (D247)-D247, which are located in the fourth β-strand These lines of evidence imply that an aberrant conformation of the RRM2 domain might link to TDP-43 proteinopathy. Although many works have characterized RRM1 as the predominant domain for RNA processing In light of the above studies and, in particular, the study by Kuo et al., we chose to investigate E246 and D247 of TDP-43 as a potential dimer interface and as markers for misfolded TDP-43, similar to the superoxide dismutase 1 (SOD1) epitope of dimer interface (SEDI) in SOD1 The protocols for genetic analysis and neuropathological procedures were approved by and performed under the guidelines of our institutional ethics committee. Informed consent was obtained from all individuals or their guardians before the analysis.E. coli expression plasmids for the RRM2 domain (residues 190–265) of human TDP-43 were prepared with a commercially available kit  and a conventional PCR protocol. In brief, PCR products obtained from pcDNA3-TDP-43-FLAG were subcloned into the BamHI site of pGEX6p-1 . Mutant RRM2 proteins at E246 and D247 were made with site-specific mutagenesis . Deletion mutants of TDP-43-EGFP for RRM1 (ΔRRM1) or RRM2 (ΔRRM2) were generated by PCR, with primer pairs used to delete the nucleotides encoding these domains. All constructs were verified by DNA sequencing in a Big Dye Terminator 3.1 . Primer sequences are provided in the Mammalian expression plasmids for human TDP-43 tagged with FLAG (pcDNA3-TDP-43-FLAG) or EGFP (pEGFP-N3-TDP-43) were constructed as described previously E. coli strain BL21(DE3)pLysS transformed with the expression plasmids was treated with 0.2 mM isopropyl thiogalactoside (IPTG) for 16 h at 22°C to induce protein expression. Proteins were cleaved from glutathione-S-transferase (GST) through digestion with a PreScission protease . The protein purity was validated by SDS-PAGE and Coomassie brilliant blue staining  assay was performed to estimate β-sheet formation of RRM2 domain. Recombinant RRM2 proteins of ,WT or substitution mutants at E246/D247 were incubated for 10 min at 22 or 70°C. After 24 hr post-incubation at 4°C, the protein solution (0.1 mM in PBS) was reacted with an equimolar amount of 2-(4′-Methylaminophenyl)benzothiazole , a derivative of ThT. Fluorescence was measured by the multi-plate reader  at 440 nm for excitation and at 490 nm for emission.Soluble RRM2 domains (2–3 mg/mL) with or without (TG)12 oligonucleotides, or extracts of cultured human embryonic kidney cells  expressing TDP-43-FLAG in PBS were applied to a Superdex 75 10/300 column or to a Superose 12 10/300 column equilibrated with PBS, respectively . Samples were subjected to high-performance liquid chromatography  at a flow rate of 0.5 mL/min in PBS. Before applying the column, protein samples were centrifuged for 20 min at 15000 g and 4°C to clear pellets. Recombinant proteins and (TG)12 oligonucleotides were detected by absorbance with 215 and 254 nm, respectively. TDP-43-FLAG was detected by Western blot with an anti-TDP-43 antibody. For the estimation of molecular weight, the columns were calibrated with thyroglobulin , ferritin , manganese superoxide dismutase , bovine serum albumin , ovalbumin , SOD1 , myoglobin , and aprotinin  as protein standards.2 and 100% humidity. Expression plasmids for TDP-43 tagged with FLAG or EGFP at the carboxyl terminus were transiently transfected into cells with a FuGene HD transfection kit according to the manufacturer's instructions . Cells were analyzed at 48 h after transfection.HEK293A cells were cultured in Dulbecco's minimum essential media (DMEM) with 4.5 g/L glucose containing 10% fetal bovine serum (FBS) and penicillin/streptomycin. Human neuroblastoma SHSY-5Y cells For immunofluorescence, cells were fixed with 4% paraformaldehyde (PFA) for 20 min at 22°C. After blocking in 5% normal goat serum in PBS with 0.2% TritonX100, cells were incubated with primary antibody. Cells were washed three times in PBS and reacted with a CF 568-conjugated secondary antibody at a dilution of 1∶1000 . For counterstaining of nuclei, 4′,6-diamidino-2-phenylindole  was used. Immunostained cells were observed with a confocal laser microscope .For the quantification of the number of cells with misfolded TDP-43, between 7 and 10 fluorescent photos were randomly taken and analyzed with Image J software . Cells with aggregates or nuclear exclusion were manually counted by an individual who was blinded to the identity of the transfected proteins.Proteins were subjected to heat (70°C for 10 min) to investigate thermostability and susceptibility of oligomerization. For SDS-PAGE, protein samples were denatured in an SDS sampling buffer with 100 mM dithiothreitol (DTT) at 70°C for 20 min. Proteins were separated with a Tris-Bis polyacrylamide gel. After SDS-PAGE, gels were analyzed by Coomassie brilliant blue  staining or Western blot. For Western blotting, peroxidase-conjugated secondary antibodies  and an enhanced chemiluminescence kit  were used. To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody  was used (1∶1000). Mouse monoclonal anti-actin antibody (1∶1000) was purchased from Millipore . Rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1∶1000) was purchased from Santa Cruz .To examine the effect of amino acid substitution on the detergent solubility of TDP-43 proteins, transfected cells were homogenized in lysis buffer  and centrifuged for 60 min at 20,400 g at 4°C. Supernatants were mixed with SDS sampling buffer (adjusted to 2% SDS and 100 mM DTT). After a single rinse with the lysis buffer, pellets were resuspended in 2% SDS sampling buffer with 100 mM DTT with five cycles of sonication for 20 seconds each. The supernatant and pellet were heat-denatured at 70°C for 20 min and analyzed by Western blotting as the detergent-soluble and -insoluble fractions, respectively.To quantify insolubility, the membranes were incubated with rat mAb anti-GFP  for TDP-43-EGFP and reblotted with an anti-actin antibody and analyzed with imaging software (Image J). TDP-43-EGFP proteins were standardized by actin, in which the ratio of TDP-43-EGFP to actin was obtained in either the detergent-soluble (ratio-sol) or detergent-insoluble (ratio-insol) fraction. Moreover, each ratio was standardized by the averaged ratio of the WT. The insolubility index was obtained by dividing ratio-insol by ratio-sol, and was expressed as fold change compared to WT.5′ TAGGATCCGG TCACCAGGAA GTTGGTTAAA TCA 3′ and 5′ CAACTTCAAG CTCCTAAGCC ACTGC 3′. The efficiency of exon 9 skipping was analyzed by densitometry with Image J.The RNA splicing of TDP-43 was estimated by using an exon 9 skipping assay with a TG13T5 minigene reporter plasmid, as reported previously but with minor modifications QSLCGEDLIIKGISVHISNA (241–260AA) and injected into C57Bl/6 mice after emulsification in Complete Freund's adjuvant (CFA). Hybridomas were generated by fusing mouse spleens and P3-X63-Ag8-U mouse myeloma cells through a conventional protocol with 50% polyethylene glycol. The first screening was positive/negative selection against recombinant wild type (WT) and then E246G/D247G RRM2 proteins by ELISA. The second selection involved immunohistochemistry with spinal cord sections of sporadic ALS patients who were previously confirmed to have TDP-43 pathology.Antigen peptides were designed within To clarify the affinity of the generated mAb against TDP-43 in cells, HEK293A cells transfected with TDP-43-FLAG were analyzed by immunoprecipitation and Western blotting. Protein G-coated magnetic beads (20 µL)  were coupled to 3B12A by incubation with 1 mL of the hybridoma supernatant at 4°C for 16 h. Total cell lysates from transfected HEK293A cells in RIPA buffer without SDS were incubated with 3B12A coupled to protein G magnetic beads in the same buffer at 4°C overnight. Beads were washed five times in RIPA containing 0.1% SDS and then eluted with 2× SDS sampling buffer containing 100 mM DTT at 70°C for 20 min. Immunoprecipitates were analyzed by Western blot with a rabbit polyclonal anti-FLAG antibody (Abnova).Postmortem lumbar spinal cords from five patients with neuropathologically proven sporadic ALS (mean age: 64.6 years) and three age-matched neurologically normal subjects (mean age: 68.7 years) were analyzed. The mean postmortem delays of the ALS and control groups were 9.0 and 9.4 h, respectively. During autopsy, spinal cords were removed, and blocks of the lumbar levels of the spinal cords were immediately placed in 10% buffered formalin and embedded in paraffin. No pathological changes were noted in the spinal cords from control subjects.For immunohistochemistry, after heat retrieval by autoclaving (10 min at 121°C in 10 mM sodium citrate buffer), 6 µm thick sections were incubated overnight at 4°C with either 3B12A diluted 1∶250 with PBS containing 3% BSA (PBS-BSA) or rabbit polyclonal antibody against amino acids 1–260 of TDP-43 (ProteinTech) diluted 1∶2000 with PBS-BSA. Bound primary antibodies were detected with a Vectastain Elite ABC kit  with 3,3′-diaminobenzidine tetrahydrochloride used as the chromogen. The staining specificity was confirmed by replacing the primary antibody with the appropriate amount of PBS-BSA. No reaction product deposits were seen in these control-stained sections.For the double immunofluorescent study, the spinal cord sections were incubated simultaneously with rabbit polyclonal anti-ubiquitin antibody  and 3B12A mAb. Sections were washed three times in PBS, incubated with a mixture of anti-mouse IgG-Alexa fluor 488 and anti-rabbit IgG Alexa fluor 568 (Invitrogen), and observed by fluorescence microscopy .p-value<0.05 was judged as significant.Comparison of multiple experiments was performed by one-way analysis of variance (ANOVA) with Newman-Keuls test with the Prism statistical software package . A 4)2SO4) conditions, through the hydrogen bonds of E246-I249 and D247-D247. To investigate whether this dimerization links to pathological conditions in TDP-43 proteinopathy, we generated recombinant proteins for the RRM2 domain (aa 190–265) of human WT TDP-43 and several substitution mutants of E246 and/or D247, which are conserved acidic amino acids among various species  migrated in a single peak of ∼10 kDa , which iWe also examined whether RRM2 forms molecular complexes by perfluoro-octanoic acid (PFO)-PAGE, which preserves the protein-protein interactions of both cytosolic and membranous proteins We next compared the effects of amino acid replacement of either E246 or D247 in the oligomerization of RRM2 by PAGE and Superdex 75 size exclusion chromatography. To examine which of these two amino acid residues contributes to the proper conformation of RRM2, a single substitution mutant of each residue to glycine was generated. SDS-PAGE revealed that D247G displayed SDS-resistant and DTT-independent oligomers with higher molecular weight than the E246G mutant RRM2 . SuperdeWe further investigated the effect of the substitution of E246/D247 on the conformation and functions of full-length (FL) TDP-43. HEK293A cells were transiently transfected with WT TDP-43-EGFP or various mutants with substitutions at E246 and/or E247 , or E246Q/D247N (QN)). According to confocal fluorescence microscopy analysis, substitutions of E246 and/or D247 promoted the formation of multiple punctate aggregates or large inclusions, mostly in the nucleus but with occasional cytosolic distribution . AlthougWe next examined the effect of E246/D247 substitution of TDP-43 on the detergent (1% TritonX100) solubility of TDP-43. EGFP-tagged TDP-43 of WT, mutants with defective NLS (mNLS), and several substitution mutants of E246/D247 were overexpressed in HEK293A cells. Using an anti-GFP antibody, we performed Western blot analyses of the denatured cell lysates, which had been separated into detergent-soluble and -insoluble fractions. The results revealed that all types of TDP-43 were predominantly monomers. The expression levels of TDP-43-EGFP containing RRM2 (GG) were obviously lower than those of the others. Comparable amounts of the detergent-insoluble species of TDP-43-EGFP (GG) were obtained relative to the other types . This fiEffects of the substitution of the E246 and D247 residues on the molecular assembly of TDP-43 under physiological conditions were investigated by size fractionation. At 48 h after transfection of TDP-43-FLAG containing WT or GG RRM2, transfected HEK293A cells were resuspended in PBS, briefly sonicated, and centrifuged at 17,400× g for 5 min at 4°C. The supernatants were introduced into a Superose 12 column for size exclusion. As shown in The role of E246/D247 in the RNA splicing of TDP-43 was investigated with the established exon 9 splicing cassette of cystic fibrosis transmembrane conductance regulator (CFTR) WT TDP-43 efficiently promoted the splicing of exon 9 compared to the vector control (12), which indirectly reflects the splicing efficiency of RRM domains, was examined 12 for 1 h at 37°C before loading onto a Superdex 75 column. This pretreatment generated a single peak detected at 215 and 254 nm, the optimal wavelengths for proteins and nucleotides, respectively. The molecular mass was 20 kDa, corresponding to the combined size of monomeric RRM2 and (TG)12 . We further investigated the influence of the substitution of E246/D247 on the DNA-binding affinity of RRM2. Monomeric RRM2 (QN) maintained its binding affinity with (TG)12 even when exposed to 70°C for 10 min, as shown by the merged signals for absorbance at 215 and 254 nm  could not bind (TG)12. A small amount of residual free WT, QN and GG monomers still remained as a separate peak d 254 nm , *2, whiate peak , *1, whiCollectively, our results strongly indicated that the side chain structure of E246 and D247 played pivotal roles in preserving the conformation and RNA metabolism activity of TDP-43, although E246 and D247 are not essential for interacting with DNA.Our results revealed that substitution mutants at E246 and D247 led to an aberrant conformation and defective DNA/RNA processing of the RRM2 domain and TDP-43. A previous crystal study demonstrated that, under acidic and high salt conditions, the RRM2-DNA complex is dimerized through two hydrogen bonds of E246-I249 and D247-D247. Based on these findings, we hypothesized that the regional environment around these residues would be affected by protein misfolding of TDP-43 in ALS, as has been observed in the case of dimer interface residues of SOD1 in familial ALS E. coli affinity . Furtheraffinity . These raffinity , consistaffinity . It was affinity .Intriguingly, our novel mAb, 3B12A, which recognized D247 as an epitope, strongly reacted with recombinant RRM2 or full-length TDP-43 proteins by ELISA, but poorly reacted with nuclear WT TDP-43 in cells or unaffected human spinal cord sections. On the other hand, cytosolic TDP-43 in cultured cells or pathological hallmarks of ALS specimens, such as Lewy body-like hyaline inclusions or skein-like inclusions, were stained by 3B12A . These rThe RRM domains of TDP-43 have five β-strands, whereas those in many other RNA-binding proteins have four, which may underlie its propensity towards amyloid formation Despite the consensus that cytosolic TDP-43 inclusions are among the pathological hallmarks of ALS, knowledge on the role of the regional structures in the conformation of TDP-43 is limited. An increasing body of evidence indicates that TDP-43 proteinopathy underlies motor neuron degeneration caused by diverse genetic defects in various hereditary ALS, including ubiquilin 2 Our studies using substitution mutants of E246/D247 clearly showed that the side chain structure of these residues strictly governed the functional conformation of the RRM2 domain. The exact role of E246/D247 in the pathogenesis of ALS has yet to be determined. Clarification of the chemical modifications or binding proteins focusing on E246/D247 will advance our understanding of TDP-43 proteinopathy. Given the advancement of various promising therapeutic studies, the early diagnosis of ALS/FTLD is inevitable. Therefore, our finding that E246 and D247 may serve as molecular markers for early pathogenic cytosolic TDP-43 species could contribute to the future improvement of therapeutic interventions. In light of the recent notion that various pathogenic proteins found in neurodegenerative diseases are propagated similar to prion proteins, molecular targeting using an antibody such as 3B12A might be a promising approach to capture the intercellular species. Further investigation is necessary to clarify the initial events that induce the misfolding of TDP-43 and to expand our understanding of and cure TDP-43 proteinopathies, such as ALS and FTLD.Figure S1SDS-PAGE analysis of de novo recombinant RRM2 proteins of WT, various substitution mutants at E246 and/or D247 purified from E coli. 5 µg of recombinant RRM1 proteins of wild-type (WT), E246Q/D247N (QN), and E246G/D247G (GG) under denaturing conditions with 100 mM DTT. Gels were stained with Coomassie brilliant blue.(PDF)Click here for additional data file.Figure S2PFO-PAGE study to investigate the molecular size of the RRM2 domain in WT or various mutations of E246/D247. After heat treatment (at 37 or 70°C) or overnight agitation, recombinant RRM2 proteins of wild-type (WT), E246Q/D247N (QN), and E246G/D247G (GG) under denaturing conditions, were incubated in 1% perfluoro-octanoic acid (PFO) sampling buffer for 1 h and then separated by a 15% PAGE in buffer containing 0.5% PFO. Gels were stained with Coomassie brilliant blue (CBB). RRM2 with substitutions at E246 and D247 was readily oligomerized, especially by heat stress at 70°C for 10 min.(PDF)Click here for additional data file.Figure S3Effect of deletion of RRM2 domain or substitution at E246 and D247 of TDP-43 on the cellular distribution and morphology of SHSY-5Y cells. Confocal micrographs showing the expression patterns of various full-length TDP-43 constructs with or without mutations  or TDP-43 devoid of the RRM2 domain (ΔRRM2) in human neuronal SHSY-5Y cells. GG and D247G mutants show more nuclear and, occasionally, cytosolic aggregates. Scale bar indicates 10 µm.(PDF)Click here for additional data file.Figure S4Generation of mouse monoclonal antibody against D247. A, Schematic representation of antigen design and screening procedure for mAb production. Supernatant of hybridoma obtained from mice, which had been immunized with peptides containing E246 and D247 (241–260 aa), was tested by ELISA for reactivity against recombinant WT RRM2 and E246G/D247G RRM2. B, ELISA showing the reactivity of 3B12A mAb to recombinant proteins for full-length TDP-43 and WT RRM2 domain. C–E, ELISA showing that 3B12A recognizes WT RRM2, with a specific reactivity for D247. 3B12A recognized recombinant RRM2 but did not recognize RRM1, mouse superoxide dismutase 1 (SOD1), or bovine serum albumin (BSA) (C). 3B12A recognized WT RRM2 and E246G mutant RRM2 but did not recognize D247G or E246G/D247G mutants (D). 3B12A did not recognize RRM2 containing the E246Q/D247N mutations, which substituted amino acids with similar side chains (E).(PDF)Click here for additional data file.Figure S53B12A mAb effectively stains cytosolic TDP-43. Confocal immunofluorescent micrographs of HEK293A cells expressing TDP-43-FLAG of wild-type  and defective NLS . In D–F, HEK293A cells were treated with 20 mM lactacystin to induce nuclear exclusion of WT TDP-43. Cells were doubly stained with antibodies against FLAG (red) and 3B12A (green). Scale bar indicates 10 µm.(PDF)Click here for additional data file.Figure S6Familial ALS-linked TDP-43 is marginally recognized by 3B12A. Confocal immunofluorescent micrographs of SHSY-5Y cells expressing mutant TDP-43-EGFP  which were stained by 3B12A mAb(red). Scale bar indicates 30 µm.(PDF)Click here for additional data file.Figure S73B12A mAb interacts with TDP-43 regardless of DNA interaction. A sandwich ELISA showing that 3B12A recognizes recombinant TDP-43 protein regardless of its interaction with biotin-labeled (TG)12 repeat oligonucleotides. Rabbit polyclonal anti-TDP-43 antibody was coated onto ELISA plate to capture recombinant TDP-43 protein. 3B12A was used for detection antibody after 1 hr incubation with biotin-labeled (TG)12 oligonucleotide (blue). The (TG)12, which interacted with captured TDP-43 was also quantified by obtaining peroxidase activity of streptavidin-conjugated horseradish peroxidase without 3B12A application (green).(PDF)Click here for additional data file.Figure S83B12A stains TDP-43–positive round cytosolic inclusions. Serial sections from a different ALS patient from those in (PDF)Click here for additional data file.Figure S9Non-aggregated forms of cytosolic TDP-43 are not ubiquitinated or phosphorylated. Confocal micrographs used for immunofluorescence analysis of SHSY-5Y cells that were transiently transfected with TDP-43-EGFP or the defective nuclear localizing signal form of TDP-43-EGFP (mNLS). After fixation with 4% paraformaldehyde, cells were stained with anti-ubiquitin (A–C) or anti-phosphorylated TDP-43 at S409/S410 (D–F) antibody. Note that the mNLS mutant form of TDP-43 is not labeled by these antibodies. Scale bar indicates 10 µm.(PDF)Click here for additional data file.Table S1Primer pairs used for plasmid construction.(PDF)Click here for additional data file.File S1Supplementary Materials and Methods.(PDF)Click here for additional data file.


Discovery of communities in complex networks is a fundamental data analysis problem with applications in various domains. While most of the existing approaches have focused on discovering communities of nodes, recent studies have shown the advantages and uses of link community discovery in networks. Generative models provide a promising class of techniques for the identification of modular structures in networks, but most generative models mainly focus on the detection of node communities rather than link communities. In this work, we propose a generative model, which is based on the importance of each node when forming links in each community, to describe the structure of link communities. We proceed to fit the model parameters by taking it as an optimization problem, and solve it using nonnegative matrix factorization. Thereafter, in order to automatically determine the number of communities, we extend the above method by introducing a strategy of iterative bipartition. This extended method not only finds the number of communities all by itself, but also obtains high efficiency, and thus it is more suitable to deal with large and unexplored real networks. We test this approach on both synthetic benchmarks and real-world networks including an application on a large biological network, and compare it with two highly related methods. Results demonstrate the superior performance of our approach over competing methods for the detection of link communities. Many complex systems in the real world exist in the form of networks, such as social networks, biological networks, Web networks, etc., which are collectively referred to as complex networks. One of the main problems in the study of complex networks is the detection of community structure Although previous research towards community detection mainly focused on the community of nodes, several recent works begin to switch the attention to community of links Recently, a number of approaches to the detection of link communities in graphs have been proposed. For instance, Ahn et al. izθ’s, where izθ denotes the propensity of node i to have links in the z-th community. Then, they take izθjzθ as the expected number of links in the z-th community connecting nodes i and j. Finally, they fit the parameters of this model using a method of maximum likelihood evaluation based on an expectation-maximization algorithm.Moreover, in face of the good performance and sound theoretical principles, generative models form a promising class of techniques for identifying communities from networks. Techniques that are being actively researched and developed zω’s and izφ’s, where zω denotes the size of a link community z, and izφ describes the degree of importance when node i forms links in this community. Then, the expected number of links in the z-th community connecting nodes i and j is denoted by zφizφjzω. Compared with Ball’s model, here we introduce an additional set of parameters zω’s to characterize the sizes of different communities, aiming to make it more flexible when describing link communities. Thereafter, in order to fit the parameters of this model, we define it as an optimization problem based on squared loss, and solve it by using a technique of nonnegative matrix factorization (NMF). At last, we extend the above method by introducing a strategy of iterative bipartition, namely NMFIB, which can not only determine the number of communities all by itself but also get these results with a high efficiency. Therefore, this combined approach is better suited for discovery in unexplored and large networks. Also of note is that, this iterative bipartition process can be used to improve other model-based methods, such as Ball’s method.In this work, based on Ball’s model, we propose a new generative model to describe the community structure of links. This model is parameterized by two sets of parameters In this section, we first introduce a model for the description of link communities in networks, and then present a method based on nonnegative matrix factorization to fit the model parameters. Thereafter, we offer an example to illustrate the method. At last, we extend the above method to a new one that automatically determines the number of communities.n of vertices and m undirected edges. Assume that the links can be partitioned into c communities using a soft community membership variable R, where ijzR denotes the probability that link  belongs to community z, subject to zω’s and izφ’s, where zω denotes the size of community z and is defined as twice the expected number of links (or weight) in this community, and izφ denotes the probability that community z selects node i when it generates edges. Thus, we have We define the generative model of link communities, which produces networks with a given number i, j) can be generated as follows. First, we choose a community z randomly with an expected size zω. Then by using probabilities izφ, jzφ, community z selects nodes i, j as a pair. Consequently, the expected number of links in community z, that lies between nodes i and j, can be evaluated asBased on the above model, an edge  belongs to community z. Then we haveFormally speaking, assume that the community assignments are represented by a set of variables i, j) to community r satisfying r = argmaxz {ijzR, z = 1,2,…,c}, and then get the hard partition of links.As the soft membership of communities cannot be used directly, we can simply assign each link nn× can be achieved by optimizing (4). In the rest of this section, a method based on nonnegative matrix factorization (NMF) is developed to solve the optimization problem in (4).X, where izX is defined asWe first introduce an auxiliary matrix The loss function in (4) can be rewritten as a constrained nonnegative matrix factorization problem,F denotes the Frobenius norm, and X, using (5) zω can be given asλ is a hyper-parameter that reflects the importance of the hard constraints. Violation to stronger hard constraints incurs a higher penalty to the objective function. In our experiments, we first get an initial value of X0 by setting λ  = 0. Then we restart the optimization with X = X0 and let λ to be a relatively large number, e.g. 1000, to minimize the chance of violating the parameter constraints. The purpose of the initialization is to restrict the model search so as to start from some good approximation. Similar to other forms of NMF, the objective function in (9) is not convex w.r.t. X, so that it is computationally intractable to find a global minimum. Therefore, a heuristic, gradient descent strategy is adopted to search for local minima. This gradient descent strategy can be implemented in a multiplicative updating algorithm similar to the method for SNMF X is introduced to (9), resulting in the following equivalent objective function,It is nontrivial to directly optimize (6) with the hard constraints. We relax this optimization problem by introducing a penalty term that represents the hard constraints into the objective function, arriving at minimizing the following objective function,For any stationary state, we haveiz(X)iz  = 0, we have the following equation,Using complementary slackness condition (Θ)X:This leads to the following update rule for When the above iteration rule converges, the converged solution satisfies the Karush-Kuhn-Tucker (KKT) conditions The convergence of the iterative updating rules follows the theorem below.Theorem 1:The objective function O in (9) is non-increasing under the update rule in (10). O is invariant under these updates if and only if X becomes stationary.Proof: We adopt the auxiliary function approach used in Expectation-Maximization and NMF. The basic idea is to construct an auxiliary function X, then we are guaranteed to drive O(X) down.If we can minimize Note that,O(X). We can define the series of update rules as:X as in (10).So we get the update rule for AX in (10) is mc, where m is the number of edges and c is the number of communities. The time to calculate 1n(1nTX) is nc, where n is the number of nodes. The time of calculating X(TXX) is nc2, and the time of calculating 1n(1nTX)(TX1n(1nTX)) is also nc2. Consequently, the time to evaluate (10) once is O(mc+nc2), and hence the time complexity of our method is O(T(mc+nc2)), where T is the iteration number for convergence. Also, according to O(Tmc). Therefore, the time complexity of our method is competitive with that of Ball’s since nc is often competitive with m.Notice that, the time to calculate Here we depict the main idea of our method using a simple example, illustrated by G by optimizing (4), and get the best parameters of the model zω’s and izφ’s which are shown as zω’s and izφ’s, we can form the expected graphs of all the link communities in G according to (1), which are shown as G according to (2), which can be regarded as an ensemble of the expected graphs of all its communities, shown as G shown in First, we fit the expected graph to the given network c, i.e., the number of communities in a network. This is also a common drawback suffered by almost all methods based on generative models. The statistical model selection applied to generative models may, in principle, be able to find the number of communities in a consistent and satisfactory manner Nonetheless, the method mentioned above can still be improved. The main drawback is that, our model offers no criteria for determining the value of parameter c of communities is given, as large networks often have large values of c, the convergence rate of the core optimization algorithms  will necessarily become very slow. This is also an important limitation from existing model-based methods when dealing with large-scale networks in the real world.Furthermore, even if the number c  = 2, and then recursively subdivide the two parts. In dividing a subnetwork, we isolate it from the rest of the network and perform a ‘nested’ NMFIB on it, resulting in a link partition of the subnetwork with two smaller link communities. Subsequently, we decide whether to accept this bipartition by a special method based on the link partition quality. We summarize the algorithm NMFIB using the following recursive algorithm:To mitigate the above problems, we extend our original NMF method proposed above to a more practical one, namely NMFIB, meaning “NMF with iterative bipartition”. In NMFIB, we first divide a network into two link modules using NMF with the number of communities AlgorithmP = NMFIB(G)//G is a network, P is a link partition of G.P = {E(G)};//E(G) denotes the edge set of G.1. G into two link modules N1 and N2 by NMF;2. Divide E(N1)E(N2) = Φ, E(N1)E(N2) = E(N).//P;3. If the link partition quality cannot be improved by this bipartition, return //the quality function is to be introduced later.P1 =  NMFIB(N1);4. P2 =  NMFIB(N2);5. P = P1P2.6. Return G, so as to obtain a superior link community structure. There are several measures for community structures, but most of them are defined for node communities DSubsequently, our remaining task for NMFIB is focused on determining the termination condition for the repetitive process of subdividing the links of network m links and n nodes, P = {P1, P2, …, cP} is a partition of the links into c communities. The number of links in community z, zP, is zm = |zP|. The number of induced nodes, all nodes that those links touch, is zn = |∪eijPz,∈ {i, j}|. The link density zD of zP isFor a network with zm normalized by the minimum and maximum numbers of links among zn connected nodes. Thus, zD  = 1 when zP is a clique, or zD  = 0 when zP is a tree. In particular, we assume that zD  = 0 if zn  = 2 without loss of generality. In essence, zD measures how ‘clique-ish’ versus ‘tree-ish’ zP is. Then, the partition density, D, is the average of zD, weighted by the fraction of links that are present:This is D, the determination of acceptance of the bipartitions for link communities will be independent on the order in which the bipartitioning queue happens in the above iterative procedure. Let we divide an arbitrary community r into two sub-communities r1 and r2. The community structure will become P' = {P1, …, rP-1, rP1, rP2, rP+1, …, cP}, and the its D-value will beIt is worthy of note that, when using function D, will beD-value only depends on this community and its bipartition result, and thus the determination of acceptance of the bipartitions for link communities will be independent on the order of the bipartitioning queue.Then, considering a bipartition for an arbitrary community, the variation of its In order to evaluate the performance of our above method, we tested it both on benchmark synthetic networks and on some widely used real-world networks. The synthetic networks allow us to test the ability of different methods to detect known communities under controlled conditions, while the real networks allow us to observe its performance under practical conditions. Further, we applied our method on a large biological network derived from real world data.DFurthermore, we compared our method with two well-known and highly related methods. The first is a model-based method for link communities proposed by Ball et al All experiments are done on a single Dell Server (Intel(R) Xeon(R) CPU 5130 @ 2.00 GHz 2.00 GHz processor with 4 Gbytes of main memory). The source code of the algorithms used here can all be obtained from the authors. Especially, the code of our methods, which are written as two functions NMF  and NMFIB (our method with iterative bipartition) in Matlab, is available in Recently, several types of synthetic benchmarks have been proposed for node communities n  = 10000 nodes each, divided into two overlapping (link) communities. We placed x nodes in the first community only, i.e., these nodes have connections exclusively within the community, y nodes in the second community only, and the remaining z = n-x-y nodes in both communities, with equal numbers of connections to nodes in these two communities on average. We set the expected degree of all nodes to a fixed value <k>. We also varied the parameters x, y, z, and <k> to generate networks with stark community structures or no structure at all, so as to vary the difficulty of the network instances posed to the algorithms.As done in the experiment designed by Ball et al. in z  = 500, divided the remaining nodes evenly , and varied the value of <k> from 1 to 16 with an increment of 1. For the second set of tests, we again set the overlap at z  = 500 but fixed <k>  = 10 and varied the ratio between x and y. Finally, for the third set of tests, we set <k>  = 10, constrained x and y to be equal, and varied the amount of overlap z.We performed three sets of tests. In the first set of experiments, we fixed the size of the overlap between the communities at priori information for both our NMF method and Ball’s method in this comparison. In As the actual number of communities of the benchmark networks used here is known to be 2, for fairness we make it as a S be the set of truly overlapping nodes and V be the set of predicted overlapping nodes, the Jaccard index is J = |S∩V|/|S∪V|. This index is a standard measure of similarity between sets that rewards accurate identification of the overlap while penalizes both false positives and false negatives. Furthermore, we adopted the Jaccard index to compare the two algorithms’ ability for identifying overlapping (link) communities using the same sets of network instances. Let DD as the quality metric to compare their results. Fortunately, the extended map equation As real-world networks may have some unique topological properties not present in synthetic ones, we now consider some widely used real networks to further evaluate the performance of these algorithms. All the networks we used here are obtained from Newman’s website priori information needed by Ball’s method and our NMF method, and compare these three methods under the condition that the number of communities is the same. The compared results are shown in In the following, in order to evaluate these methods fairly and completely, we perform three sets of tests. First, we use the number of communities attained by Ahn’s method as a Further, we compared the performance of our NMF method with iterative bipartition (NMFIB), Ball’s method with iterative bipartition, as well as Ahn’s method. At this time, all these methods can determine the number of communities automatically. The comparison of these algorithms is shown in c got by the corresponding iterative bipartition method. The compared results are shown in Finally, we compare our NMF method with iterative bipartition  and our original NMF method  with the given number of communities To sum up, our method with iterative bipartition not only has a higher clustering quality compared with other methods, but also it can determine the number of communities automatically. Thus, it may be more suitable for use when detecting link communities on unexplored real networks.Saccharomyces cerevisiaeThe large real network we selected for a particular application is the protein-protein interaction (PPI) network of budding yeast p-value to quantify the significance of the term. To quantify the biological significance of a community structure, we used as quality metric the average number of significantly enriched GO terms with p-values less than a given threshold for all communities. The larger this average number of significant GO terms, the more biologically significant the community structure is.We used the Gene Ontology (GO) terms p-value thresholds tested. It stands as an example of the consistent superior performance of our method over all compared competing methods.Here we compared the results of our method NMFIB, Ball’s method with iterative bipartition and Ahn’s method, since all of them can automatically determine the number of communities. The results are shown in zω’s, to characterize the sizes of different communities, which may enable our model to be more flexible in describing link communities. Then, we fitted the model parameters by taking it as an optimization problem, and used an approach of nonnegative matrix factorization to solve it. Thereafter, we extended the above method by introducing a strategy of iterative bipartition. This leads to a new method, namely NMFIB, that we show to be more suitable for structure discovery in unexplored and large networks. Also, this iterative bipartition process is suitable to be used to improve other model-based methods, such as Ball’s method. We tested our method both on synthetic benchmarks and on real-world networks including an application on a large biological network, and compared it with two well-known and highly related methods. Experimental results demonstrated the superior performance of our method over the competing methods in the detection of link communities.In this work, we proposed a generative model for link communities based on Ball’s model izX (elements in the auxiliary nonnegative matrix X) that falls below a predetermined threshold, and improve the efficiency of our NMF method by using a technique of sparse matrix calculations. This is one of the directions for our future work. Moreover, in the current work, we only used partition density DOur model was mainly designed to describe link communities, but it can be also extended to find node communities. A simple and approximate method is assigning each node solely to the community to which it most strongly belongs in the overlapping (link) community structure. But in the future, we wish to improve our model from some other viewpoints, and make it able to describe link communities as well as node communities, naturally and in a similar way, rather than being a simple extension. Furthermore, as discussed, the efficiency of our method is competitive with that of Ball’s method, and it will become more efficient when introducing the strategy of iterative bipartition. Nevertheless, in order to deal with some very large networks such as the WWW, the Internet, etc, its efficiency needs still to be improved. Possibly, we can devise a pruning strategy that sets to zero any


Pulmonary hypertension (PH) represents an important complication of idiopathic pulmonary fibrosis (IPF) with a negative impact on patient survival. Herpes viruses are thought to play an etiological role in the development and/or progression of IPF. The influence of viruses on PH associated with IPF is unknown. We aimed to investigate the influence of viruses in IPF patients focusing on aspects related to PH. A laboratory mouse model of gamma-herpesvirus (MHV-68) induced pulmonary fibrosis was also assessed.Lung tissue samples from 55 IPF patients and 41 controls were studied by molecular analysis to detect various viral genomes. Viral molecular data obtained were correlated with mean pulmonary arterial pressure (mPAP) and arterial remodelling. Different clinical and morphological variables were studied by univariate and multivariate analyses at time of transplant and in the early post-transplant period. The same lung tissue analyses were performed in MHV-68 infected mice.p = 0.0003) and only herpes virus genomes were detected. Viral cases showed higher mPAP (p = 0.01), poorer performance in the six minute walking test  and higher frequency of primary graft (PGD) dysfunction after lung transplant (p = 0.02). Increased arterial thickening, particularly of the intimal layer (p = 0.002 and p = 0.004) and higher TGF-β expression (p = 0.002) were demonstrated in viral cases. The remodelled vessels showed increased vessel cell proliferation (Ki-67 positive cells) in the proximity to metaplastic epithelial cells and macrophages. Viral infection was associated with higher mPAP (p = 0.03), poorer performance in the 6MWT (p = 0.008) and PGD (p = 0.02) after adjusting for other covariates/intermediate factors. In MHV-68 infected mice, morphological features were similar to those of patients.A higher frequency of virus positive cases was found in IPF patients than in controls (Herpesviral infections may contribute to the development of PH in IPF patients. Idiopathic pulmonary fibrosis (IPF), morphologically usual interstitial pneumonia, is a chronic progressive disease of unknown aetiology with irreversible scarring in the lung Recent studies have shown that PH measured with right-heart catheterization or transthoracic echocardiography in IPF patients is associated with low DLCO, shorter walk distances, and desaturation during exercise However to date no clinical or biohumoral parameters are available as predictive markers of PH in IPF patients. Several authors have shown that PH in IPF relates poorly to the degree of pulmonary function test and is not always confined to advanced disease Pathogenic mechanisms of IPF and of its main complications are complex and still largely unknown although different hypotheses have been proposed, varying from previous chronic inflammation with subsequent widespread fibrosis to abnormal wound healing. Increasing evidence suggests that a key event in IPF is deregulated epithelial cell function with ongoing alveolar epithelial injury and an associated abnormal host repair response, leading to patchy and heterogeneous morphological changes Alveolar epithelial cells, one of the most important targets of the virus, are then involved in the activation of different signalling pathways responsible for fibrotic lung remodelling. In viral animal and cell models several profibrogenic cytokines/chemokines have been shown to be over-expressed and among these transforming growth factor beta 1 (TGF-β1) plays a key role The aim of the study was to investigate the relevance of respiratory viruses in IPF patients, with special emphasis on cases complicated by PH. For this purpose a large number of IPF patients and control subjects including other non-IPF diffuse parenchymal lung diseases (DPLDs) were studied. Viral molecular data were correlated with mean pulmonary arterial pressure (mPAP) and arterial remodelling. Different clinical and morphological variables were studied by univariate and multivariate analysis at time of transplant and in the early post-transplant period.A mouse model was used to examine whether murine gammaherpesvirus-68 (MHV-68), genetically and biologically similar to EBV, contributes to the development of vessel remodelling other than pulmonary fibrosis.Native lungs from 55 IPF patients who underwent lung transplantation (LT) between September 1998 and February 2010 in our Centre were studied . The control group consisted of 22 native lungs from other non-IPF -DPLDs-  and 19 normal lungs (13 non implanted donor lungs and 6 autopsy cases). Prior to LT after the diagnosis of IPF, the majority received oral prednisone while a minority were treated with prednisone and azathioprine.Each patient underwent pulmonary function testing, high resolution computed tomography and right heart catheterization. These tests were performed in all patients at the time of waiting list inclusion and before LT. For the present study clinical/functional parameters collected at the time of LT were considered.The diagnosis of interstitial lung disease was based on the diagnostic criteria of the American Thoracic Society/European Respiratory Society Consensus Classification System Moreover, short term post-transplant follow-up (12 months after LT) was also considered in all IPF cases, mainly focusing on primary graft dysfunction (PGD) and acute rejection, both considered causes of early and late graft dysfunction. PGD and acute rejection were graded as proposed by the International Society for Heart and Lung Transplantation (ISHLT) Working Group Written informed consent was obtained from all subjects. The work was approved by the Institutional Ethics Committee. Relevant clinical data of the IPF and non-IPF DPLD population are summarized in Nucleic acids were extracted from fresh lung tissues sampled from different areas (∼1 mg of tissue) of all 96 cases (55 IPF patients and 41 control subjects) using a modified RNAzol method Samples were considered as true positive when the reproducibility of PCR analysis was verified a second time. Amplicon specificity was verified by direct cycle sequencing as previously described Since EBV was identified with high frequency in IPF patients, RNA-in situ hybridization-ISH  was performed to identify the viral target cells. To quantify DNA viral genome copies real time PCR was also carried out in EBV positive cases whose additional DNA was available using artus® EBV TM PCR KIT The extent of fibrosis was evaluated in Masson’s trichrome stained sections analyzing 10 random fields using computer-assisted morphometric software (Image ProPlus® 5.1), as previously reported Arterial thickening was quantified using computer-assisted morphometric software on elastic-Van Gieson (EVG) stained lung sections focusing on muscular arteries of an average diameter of 300 µm (range: 100–500 µm). In particular, medial thickening (MT) was evaluated as previously described in at least 5 arterial sections. The same approach was used to measure intimal thickening (IT): MT% =  × 100; IT% =  × 100 For Ki-67 immunohistochemistry, after microwave antigen retrieval, the sections were treated with normal serum  and incubated for 60 min with the primary monoclonal antibody anti-Ki67  at a dilution of 1∶100. Sections were subsequently incubated with rabbit HRP polymer  for 30 min. Immunoreactivity was visualized with 3-3′-diaminobenzidine (Dako). Negative controls processed omitting the primary antibody did not show any reaction. Ki-67 expression and TUNEL positivity were evaluated only in blood vessel cell components. All evaluations were performed blindly.5 PFU MHV-68 and euthanized 7 days (n = 3), 14 days (n = 6) and 23 days (n = 3) post infection (post infection). Uninfected mice (n = 4) served as controls.Female, 5 to 8 week old CD-1 mice [Hsd:ICR (CD1)] were purchased from Harlan Laboratories, UK and housed at the University of Liverpool under specific pathogen-free conditions. Mice were intranasally infected with 4×10Immediately after death, lungs were collected and parts frozen at −80°C for RNA extraction: others were fixed in 10% buffered formalin and routinely embedded in paraffin wax. All experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of The National Institutes of Health. The protocol was approved by UK Home Office regulations and under Project Licence number 40/2483 and Personal Licence number 60/6501. All infections were performed under light isoflorane anaesthesia. All experiments were carried out in manner to minimize suffering.Consecutive paraffin-embedded sections were stained with haematoxylin and eosin for histology, with Masson’s trichrome stain for fibrosis extension and with EVG for vessel remodelling, both morphometrically measured as above. The evaluation of arterial thickening focused on pulmonary arteries with an average diameter of 82 µm ranging from 43 to 100 µm. The assessment of vascular remodelling also included an evaluation of apoptosis and proliferation of vessel cell components using the TUNEL technique and Ki-67 immunostaining, as described above. Ki-67 antibody used for this analysis was rabbit polyclonal . Immunohistochemistry was also employed for the detection of MHV-68 antigen, to highlight lung macrophages (expression of lysozyme) and to demonstrate TGF-β expression . Viral tRNA was demonstrated by RNA-ISH Data were analyzed using the SAS statistical software version 9.1.3 . For quantitative variables the results are expressed as mean values ± standard deviation if normally distributed, otherwise as median, Q1–Q3. The normality of distribution of quantitative variables was tested by means of Shapiro-Wilk statistics. For quantitative characteristics, differences between subjects with and without viral infection were evaluated by using the Mann-Whitney test. The prevalence of specific conditions was expressed as a percentage, and differences between groups were evaluated using the χ-squared test and exact Fisher’s test, as adequate.Unadjusted and adjusted relationships between virus and clinical/morphological features were tested with general linear models (GLM procedure). In all analyses, a two-tail level for significance was set at 0.05. Simple logistic regression models were applied to estimate odds ratios (ORs) for PGD using recipients’ and donors’ characteristics as predictors. A multivariate logistic model was used to estimate the adjusted OR of virus for PGD, controlling for PAP>25 mmHg. Considering that incidence was quite common in the study population, adjusted risk ratios (RRs) were approximated from adjusted ORs p = 0.0003). Normal lungs were all negative. Herpes viruses were the only detected genomes in IPF and EBV resulted as the most frequent, present in more than half of IPF patients. In 2 cases double infection was found. EBV was never detected in control cases . Gene sequencing of all amplicons showed a high homology (from 95% to 99%) with human viral genome sequences. RNA-ISH for EBV (EBER) identified viral RNA in 40% of EBV-PCR positive IPF cases. The positivity was detected in alveolar epithelial cells other than within monocytes/macrophages . Normal lungs did not show arterial remodelling  and metaplastic alveolar epithelial cells . Median TGF-β scores in the alveolar epithelium were significantly higher in IPF patients than in the DPLD group . In normal lungs, TGF-β expression was restricted to only scattered intraalveolar macrophages. Arterial remodelling in IPF cases showed a median total thickness score of 43.8% (Q1–Q3: 35.7–53.1%) with IT and MT scores of 17.7% (Q1–Q3: 13.5–24%) and 26.2% (Q1–Q3: 21.8–28.9%) respectively. These values were significantly lower in the DPLD group  and worse performance in the 6MWT  than virus negative cases  than virus negative cases. The intimal layer was most severely affected  (p = 0.002)   A,B,C,D.= 0.004)  while ce= 0.004)  A and B.= 0.002)  A,B,C,D.p = 0.03) after adjusting for other related covariates and intermediate factors . Moreover, viral presence was an independent marker of significantly poorer performance in the 6MWT (p = 0.008) using the GLM adjusting for covariates . When we considered the short-term post-transplant follow-up we found a higher frequency of PGD  and, although not statistically significant, an increased rate of acute rejection  in virus positive compared to negative IPF cases. A multivariate analysis identified an increased risk for PGD to be associated with viral infection independently of the major recipient/donor characteristics that are usually considered to influence PGD, including PH   .Analysis of MHV-68 DNA load showed, as previously demonstrated, that viral infection peaked on day 7 post infection The histological examination of uninfected control animals did not identify any pathological changes. On day 7 post infection, all infected animals exhibited increased, macrophage-dominated interstitial cellularity, mild type II pneumocyte hyperplasia and perivascular inflammatory infiltration. The latter infiltrates were associated with slight patchy collagen deposition. Scattered individual macrophages and metaplastic alveolar epithelial cells were found to express MHV-68 antigen. Viral latency, represented by the expression of viral tRNA, was detected in type II pneumocytes and alveolar macrophages. On day 14 post infection, mild multifocal, almost diffuse fibrosis of alveolar septa was observed with a median of 26% (range: 17–38%). In some animals, this was associated with random patches of collagen deposition. Numerous macrophages were seen within the interstitium and often around arteries. In addition, some type II pneumocytes exhibited TGF-β expression  A, B.Arteries were assessed for vessel remodelling and compared for the thickening scores. In uninfected control mice, the average score was 16.5. Seven and 14 days post infection, it was 18 and 24.5 respectively  A,B,C,D.In the present study different respiratory viruses were investigated in lungs from a large cohort of IPF patients and the control group  displaying the prevalent role of herpes viruses. The work highlights the broad impact of herpesviral infection, particularly EBV, in the disease emphasizing its association with a more severe disease phenotype as IPF with associated higher mPAP and worse 6MWT. A multiple logistic regression analysis demonstrated that herpesviral infection was independently associated with more marked vessel remodelling, higher value of PAP and worse performance in the 6MWT.Neither clinical data  nor morphological changes (fibrosis extension) had more significant impact than viral infection. Of particular note is that the presence of viral infection had also early-term negative influence on the graft function. IPF virus positive recipients showed a higher frequency of PGD, independent of other recipient and donor characteristics. Viral products and/or the activated immune system related to viral infection in native IPF lungs may potentiate the immune response to alloantigens after transplantation, thus resulting in early graft dysfunction. Only one previous study performed on 24 IPF patients reported more rapid disease progression in EBV positive cases. The majority of viral cases died from respiratory failure at a mean of 41 month follow up. In the study PH was not specifically considered The reported frequency of herpes viruses in IPF lungs ranges from 30 to 100%. These differences may be related to technical sensitivity, disease heterogeneity or selection of patients. The presence of herpes viruses detected in our cases may even be underestimated due the fact that the molecular investigation was performed on tissue samples from end-stage diseases. Viral clearance, after initial injury, could occur in some cases with persistence and progression of lung injury due to activation of an immunological response, as it occurs in other chronic virus-related diseases, e.g. dilated-cardiomyopathy-post-myocarditis To the best of our knowledge no attempt has been made to specifically investigate the influence of herpes viruses on arterial remodelling and PH in IPF patients.Several types of viruses, particularly those of the herpes family, have been found to be associated with vessel remodelling, development of atherosclerosis and clinical features of hypertension vivo has not been well defined, in vitro and experimental studies have demonstrated an important influence of this cytokine in muscle/fibroblast proliferation, endothelial-mesenchymal transition, and extracellular matrix production of intimal and medial layers Although the function of TGF in vascular cell growth in The demonstration of a direct causal relationship between herpesvirus infection and vessel remodelling/PH in IPF would require longitudinal studies of the same patients, an impossible task with lung tissue but attainable with bronchoalveolar lavage or peripheral blood samples. However this limitation has been partially overcome in the present study using a laboratory MHV-68 infected mouse model. Indeed, in these animals 2 weeks after infection significant arterial remodelling and increased TGF-β expression was seen, as those observed in clinical lung specimens from IPF patients with high mPAP.In summary, our results demonstrated for the first time a different phenotype of virus-positive IPF patients. In particular virus-positive IPF cases showed more pronounced vessel remodelling and a higher mPAP and significantly higher PGD after transplantation. While there is large mechanistic evidence of epithelial herpesvirus-associated alveolar injury, the effect of these viruses on the pulmonary vasculature in IPF merits investigation. A deeper knowledge of viral-induced pathways in endothelial cells could give new insights for a targeted therapeutic approach of this important complication in the subgroup of patients (virus positive cases). In this context, the high degree of similarity between MHV-68 infection of CD-1 mice and virus positive IPF indicates that this is an excellent model with which to study pathogenesis and interventions.Figure S1Cell apoptosis in IPF lung tissue. Endothelial cell apoptosis (TUNEL positive) well seen in a capillary surrounded by extensive fibrosis (arrows). Bar scale: 10 µm.(TIF)Click here for additional data file.Figure S2Cell apoptosis in MHV-68 infected CD1 mice lungs. Endothelial cell apoptosis  well seen in a capillary of high remodeled area. Note apoptotic body inside a macrophage (arrow head). Bar scale: 10 µm.(TIF)Click here for additional data file.Table S1Virus-positive vs virus-negative IPF .(DOC)Click here for additional data file.Table S2Unadjusted relative risks  for post-transplant PGD – recipients and donors characteristics used as predictors.(DOC)Click here for additional data file.


According to this process, a distance must be maintained between the plasma initial stage and the deposition stage to allow the plasma to diffuse to the substrate. Raman spectra revealed that a higher hydrogen concentration promoted the synthesis of a high-quality graphene film. The results demonstrate that plasma-assisted thermal CVD is a low-cost and effective way to synthesis high-quality graphene films at low temperature for graphene-based applications.Plasma-assisted thermal chemical vapor deposition (CVD) was carried out to synthesize high-quality graphene film at a low temperature of 600°C. Monolayer graphene films were thus synthesized on Cu foil using various ratios of hydrogen and methane in a gaseous mixture. The  The carriers in graphene are transported in the π-orbitals that are perpendicular to the surface so the optical transparency of a single layer of graphene can be as high as approximately 97%, and it can exhibit excellent electronic properties with reported mobilities of between 3,000 and 27,000 cm2/V·s [Graphene, a sp cm2/V·s -3. Vario cm2/V·s -7. Chemi cm2/V·s . Sulfuri cm2/V·s . This me cm2/V·s ,12. The  cm2/V·s -19. This cm2/V·s , and sur cm2/V·s ,22. Howe2) flow rates from 5 to 20 sccm at a temperature of as low as 600°C. Figure Throughout the experiments, plasma-assisted thermal CVD was used to synthesize graphene films on polycrystalline copper foils with various hydrogen  gas, flowing at 1 sccm, was the carbon source; it was mixed with various flows of H2 and fed into the tube for 5 min to form a monolayer of graphene. Subsequently, the sample was rapidly cooled by removing it from the hot zone of the thermal furnace. The synthesized graphene films were transferred onto the SiO2 (300 nm)/Si substrates by etching away the copper foil in an iron chloride (FeCl3) solution. Prior to wet etching, a 200-nm-thick thin film of PMMA (poly-methyl methacrylate) was spin-coated on the top of graphene/copper foil and then baking it at 130°C for 1 min. The PMMA/graphene thin films were washed with dilute hydrochloric acid solution to remove the metal ions and then rinsed in DI water. PMMA/graphene films were placed on the SiO2 (300 nm)/Si substrate, and the PMMA was then dissolved in an acetone bath over 24 h.Graphene films were grown on a 25-μm-thick copper foil  using the proposed plasma-assisted thermal CVD system by a method similar to one described elsewhere . Prior t4/H2 mixed plasma and CHx radicals. The gaseous CHx radicals recombined with each other after they had floated for a certain distance, and the metastable carbon atoms and molecules formed a sp2 structure on the copper surface. Most importantly, the most effective length for growing graphene between the plasma and the center of the hot zone was approximately 30 cm herein.Figure 4/H2 mixed gas with various proportions of H2[n = 1, 2, 3,…) [n > 1) in a strong electric field, ionizing the hydrogen atom as the electrons were excited to high energy levels. The transition from n = 3 to n = 2 is called H-alpha (Hα) and that from n = 4 to n = 2 is called H-beta (Hβ) with emitted wavelengths of approximately 656 and 486 nm, respectively. After ionization, the excited electron recombined with a proton to form a new hydrogen atom, yielding the Hx spectra. In this case, the ionized gas of CH4/H2 recombined as CHx radicals moved after a certain distance. Figure 2 flow rates and a gas pressure of 0.5 Torr. In this work, the recombination lines of the atomic  and molecular (H2 = 550 to 650 nm) hydrogen dominate the emission spectra. The emission spectra of CH radical (430 nm) and C2 dimers (541 nm) after the introduction of CH4 are obtained [α occurs when hydrogen is ionized where intensity increases with the H2 flow rate. The intensity of the CH spectral peak declined slightly as the H2 flow rate increased, revealing that increasing the H2 concentration improves the rate of decomposition of the mixture gas. The C2 dimers in plasma during the plasma-assisted thermal CVD are critical to the formation of various carbon materials [Figure ons of H2. Accordi 2, 3,…) ; electroobtained . Hα occuaterials . Further2 flow rates from 5 to 20 sccm at a low temperature of 600°C. Typical features of the monolayer graphene are observed. They include (1) a 0.5-G-to-2D intensity ratio and (2) a symmetric 2D band that is centered at approximately 2,680 cm−1 with a full width at half maximum (FWHM) of approximately 33 cm−1. The 2D band is related to the inter-valley double resonant Raman scattering, and the peak of the G band is produced by the Eg2 phonon at the center of the Brillouin zone around approximately 1,580 cm−1. The D band is associated with the breathing modes of the sp2 atoms and is activated by a defect. Sharp single Lorentzian 2D band was observed at approximately 2,700 cm−1 when the H2 flow rate exceeded 10 sccm. The intensity of the D band decreased with increasing H2 flow rate indicating not only increased crystallization of graphene but also in CVD graphene on copper, the formation of C-H bonds as out-of-plane defects. Overall, hydrogen plays an important role in the growth of graphene and in determining its quality. This result is consistent with Figure Figure 2 flow rate increases, Id/Ig declines from 0.33 to 0.13 and I2d/Ig increases from 0.98 to 2.29. The lower 2D band and higher D band reveal that the more disordered graphene growth, the lower is the H2 flow rate. Interestingly, the 2D-peak FWHMs (39 to 35 cm−1) of the series of samples varied slightly with the H2 flow rate because the low solubility of carbon in copper makes graphene growth self-limiting, and a higher H2 concentration improves the inter-valley double resonance in the Raman spectrum.Figure 2 flow rates. When the H2 flow rate increased, the Raman spectra of the samples have I2d/Ig ratios that increase from 0.98 to 2.29 and the FWHMs of the 2D band that decrease from 39 to 35, both indicate that the graphene film is high quality. Plasma-assisted thermal CVD is a more effective method for depositing high-quality graphene films on metal substrates.This study elucidates the effect of hydrogen on graphene grown on Cu by plasma-assisted thermal CVD at a low temperature of 600°C. The mechanism of growth of graphene by plasma-assisted thermal CVD was clarified by obtaining plasma emission spectra at various HThe authors declare that they have no competing interests.SHC (Chan) designed the study and wrote the paper. WTL and MCL analyzed the data. SHC (Chen), YCL, and CCK are advisors. All authors read and approved the final manuscript.


A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection. This substance is widely used not only as a stationery glue and as a laundry starch but also in biological applications: for instance, as a cryopreservation reagent for embryos  and 12%(w/w)], PVA3500 [13%(w/w)] and PVA2400 [15%(w/w)] were dissolved in water by brief heating (363 K) and stirring.As the water-soluble polymer, we used polyvinyl alcohol (PVA) with average polymerization degrees ranging from 2000 to 4500 . PVA2000, PVA2400 and PVA4500 were supplied by Japan VAM & POVAL Co. Ltd, Japan . PVA3500 was purchased from Wako Pure Chemical Industries Ltd (product No. 163-16355). Aqueous solutions of PVA4500 [8%(w/w) and 13%(w/w) PVA4500 gave a powder diffraction pattern of hexagonal ice after flash-cooling without any humidity control. Similarly, glue exposed to air of 92.3% RH (relative humidity) produced a powder diffraction pattern of cubic ice , but at 92.0% RH no ice ring was observed . Since the final resultant concentrations of PVA are affected by vapour pressure, i.e. atmospheric humidity  PVA4500 was used as the mounting glue in all of the experiments if not otherwise specified.For flash-cooling experiments, humidity conditions that vitrify the PVA solutions themselves were examined by X-ray scattering. Initial solutions of 8%. Dry air was supplied by an N2-gas generator  and an HOHMI YC-4R air compressor . Saturated-humidity air was produced by bubbling the dry air in a water bath kept at 301 K and cooling to room temperature (296 ± 1 K with a temperature stability of ±0.3 K h−1) during transportation. Next, it was mixed with the dry air and the total flow rate was brought to 3 l min−1 or greater using two SEC-N series digital mass-flow controllers  controlled by a PC running Windows 7. Although this handmade humidity generator was used in this study, we confirmed that a modified version of the commercial humidity generator HUM-1 , which can produce humid air at a high flow rate, also worked for this purpose. The humidity-control gas was transported to a humid air nozzle, which was produced from a flexible Teflon tube with an inner diameter of 10 mm. The outlet temperature and the relative humidity were measured inside the humid air nozzle using an SHT71 humidity sensor . This humidity-control device can control the humidity range between 0.1 and 94.5% RH. Since the humidity was regulated by the mixture of gases, humidity change took place at a rate of between 0.2 and 1% RH min−1.We developed a simple humidity-control apparatus equipped with a two-flow humidity generator Fig. 2a. Dry a2.3.2.3.1.−1). The humid air nozzle of the apparatus was placed under the sample  and the humidity was adjusted; typically, the initial humidity was 83% RH. The cryostream nozzle was removed from the sample position to avoid interference with the humid air . In the following steps performed at room temperature, the intensity of X-ray radiation should be minimized in order to reduce radiation damage. In the case of our beamline, SPring-8 BL38B1, an aluminium plate with a thickness of 600–1000 µm was used to attenuate the X-ray beam with the full photon flux of 1.17 × 1011 photons s−1 to 1/10 to 1/40 at a wavelength of 1 Å, respectively.The humidity-control apparatus was installed on the SPring-8 BL38B1 beamline equipped with a one-axis rotation goniometer , ADSC Quantum 210 or 315 CCD detectors and a GN2 nitrogen cryostream system (Rigaku Co.). All of the following operations were performed at room temperature  PVA4500 was used except for certain crystallization conditions. PVA interacts with ions, especially multivalent anions such as tartrate, phosphate and sulfate ions, and forms a physical hydrogel. Therefore, it is often difficult to thoroughly coat the crystals owing to a varying degree of hydrogel elasticity that depends on the concentration and the valences of the ions. It is known that plasticizing reagents such as glycerol, ethylene glycol and polyethylene glycol 400 prevent gel formation and that 5%(v/v) glycerol is sufficient to reduce gel formation  glycerol before transferring the crystal into the glue. (ii) Glycerol was added to the crystallization solutions to a final concentration of 5%(v/v). (iii) In the case of crystallization solutions containing a high concentration of multivalent ions (e.g. greater than 1 M), the glue was prepared with equal volumes of pure glycerol and the aqueous PVA solution.An aqueous glue solution consisting of 8% with a typical diameter larger than the crystal . A crystal was directly picked up from a crystallization droplet by the glue-coated loop without removal of the mother liquor surrounding the crystal . The scooped crystal was kept steady for a few seconds so that it could be thoroughly coated and covered by the glue on the loop . Next, the loop was mounted on the diffractometer and exposed to the humid air. This operation should be completed within 10–20 s to minimize transpiration of the crystalline moisture.A small amount of the glue solution was applied to and spread over a crystal-mounting loop (2.3.4.c). To avoid this, a suitable humidity level needs to be established immediately. The swelling of the glue and consequent formation of crystalline ice at cryogenic temperature is another concern. The protocol for coping with these problems is described in Fig. 3A destructive hydration shock could be induced if the glue were diluted by absorbing a large amount of the droplet solution in the crystal pickup Fig. 2c. To avHKL-2000 suite .Once the humidity had been optimized, it was kept constant without any further change. Before proceeding to the next step, the crystal was maintained at the optimum humidity for 15 min to complete water equilibration. Since lower humidity was preferred in many crystals, the glue was dehydrated and shrunk at optimum humidity . Lysozyme crystals were grown using hanging-drop vapour diffusion at 293 K. Hanging drops were prepared by mixing 5 µl of 35 mg ml2.4.2.Bacillus subtilis was expressed and purified as described previously , which was the lowest concentration to vitrify the mother liquor. However, the crystals were mechanically very fragile and sensitive to environmental changes. Indeed, the crystals gradually delaminated and crumbled once the cover slides of the hanging drops were opened.The bacterial hydrolase RsbQ from 2.4.3.et al.  glycerol.Bovine insulin was obtained from Sigma–Aldrich Co. (product No. I550) and crystallized based on the method reported by Gursky  al. 1992. The cry2.4.4.Blastochloris viridis photoreaction centre (BvRC) crystal was provided by Dr Takayuki Odahara. The crystals of BvRC were grown using sitting-drop vapour diffusion at 288 K with a small glass vessel for protein solution installed in a sealed plastic container. Sitting drops were prepared using 400 µl of 18 mg ml−1BvRC solution in 2.5%(w/v) triethylamine phosphate, 2.5%(w/v) 1,2,3-hexanetriol, 0.3% lauryl dimethylamine oxide (LDAO), 1.51 M ammonium sulfate, 60 mM sodium phosphate buffer pH 7.0. The volume of reservoir solution consisting of 5%(w/v) triethylamine phosphate, 1.89 M ammonium sulfate, 100 mM sodium phosphate buffer pH 7.0 was 12 ml in each setup. The glue for the HAG mount was a mixture prepared from equal volumes of pure glycerol and 8%(w/w) PVA4500 solution.The bacterial membrane protein 2.4.5.4 synthase (LTC4S) was provided by Dr Hideo Ago  n-dodecyl-β-d-maltoside (DDM) with an equal volume of reservoir solution consisting of 0.04%(w/v) DDM, 5 mM glutathione, 1.6–1.7 M ammonium sulfate, 0.5–0.8 M magnesium chloride, 20 mM MES–NaOH buffer pH 6.5. The crystals of LTC4S were transferred overnight into harvesting solution consisting of 2.4 M ammonium sulfate, 50 mM glutathione, 20 mM MES–NaOH buffer pH 6.5 . By employing an HAG mount using 13%(w/w) PVA3500 glue without any other cryoprotectants and an optimized humidity of 69.1% RH, cryoprotection of the RsbQ crystals was successfully achieved and a full diffraction data set was collected  or higher as a permeable cryoprotectant in the conventional cryocooling method, whereas they could be mounted and cryocooled with only 5%(v/v) glycerol in the HAG method. In this case the crystal was first soaked in reservoir solution supplemented with 5%(v/v) glycerol to replace the mother liquor and was then used in HAG mounting. The addition of only 5%(v/v) glycerol was sufficient to prevent gel formation by the 0.4 M phosphate ion contained in the mother liquor. By contrast, for both BvRC and LTC4S crystals the addition of 5%(v/v) glycerol was insufficient to prevent gel formation because both of the crystallization conditions contained high concentrations of sulfate ions (1.9–2.5 M). However, addition of glycerol to the polymer glue was effective for both crystals. The glue prepared by mixing equal volumes of glycerol and 8%(w/w) PVA4500, i.e. a final concentration of 50%(v/v) glycerol and 4%(w/w) PVA, was successfully subjected to cryocooling without ice formation  glycerol was damaged in only 2 min . During the dehydration process to 68.3% RH , a 0.73% shrinkage of the crystallographic a axis and a 1.45% stretching of the c axis were simultaneously observed. With further dehydration from 68.3 to 67.6% RH the crystal suddenly cracked and the mosaicity surged . Rehydration with increased humidity did not lead to recovery of the crystal from cracking.In addition to the cryoprotection effects, we also investigated the transformation of lysozyme crystal lattices in response to humidity changes. Firstly, we estimated the humidity ranges in which glue-coated crystals remain stable. Tetragonal crystals of hen egg-white lysozyme were mounted by the HAG method without any additional cryoprotectants and the humidity was reduced from 87.7% RH to the catastrophic point at which the crystal was damaged . The dehydration process gradually led to lattice transformation along the a and c axes to 78.51 and 38.35 Å, respectively. Moreover, during the following rehydration process the lattice parameters inversely traced the transitions that were observed in the dehydration process. This showed a linear relationship between the humidity and the lattice constants, although a small nonlinear response and increased mosaicity were observed in the first dehydration steps. Such a nonlinear response was also observed by exposing a crystal to constant humidity . In particular, the samples exposed to high humidity (87.3–88.6% RH) showed higher mosaicity (0.55–0.66°) than the others (<0.46°). In terms of the resolution, the data set with the highest resolution was obtained from the 82.7% RH condition, which is in the middle of the tested humidity range (Table 2i.e. a solution of only PVA treated with humidity above 92% RH gave ice-ring diffraction on cryocooling. It has been reported that the water vapour pressure of aqueous PVA (molecular mass 88 kDa) at concentrations of about 60% or below remains approximately at the saturated pressure of water vapour at 304.15 K  by exposure to unsaturated humid air, leading to successful vitrification.The effect of the HAG mount for cryocooling was examined using five lysozyme crystals, each with a different humidity condition, in which no ice-ring patterns were observed but for which the mosaicities increased after cryocooling Table 2b. In pae Table 2. Both ex4.−1) compared with that of the crystallization solution of lysozyme .(i) The nature of retaining moisture and buffering moisture exchange. The glue prevents direct exchange of water vapour between the crystal and the atmosphere. Instead, the glue releases its own moisture, rather than that of the crystal, to the dry atmosphere during crystal handling or humidity changes. This moderate moisture-exchange system protects fragile crystals from a hydration or dehydration shock. In addition, the hypotonic property of the polymer solution retains the moisture of a crystal. The osmotic pressure of the glue solution depends on the concentration of its solute and is also affected by the polymerization degree of the solute polymer according to the Flory–Huggins theory; a longer polymer gives a lower osmotic pressure at a given vapour pressure. Indeed, the osmolality of the glue is almost negligible  High viscosity and slow diffusion. The high viscosity of the glue guards the crystals from mechanical stress and slows the diffusion of solvents, solutes and PVA itself. The high viscosity may retard changes in the chemical environment surrounding a crystal. The effects might also mitigate osmotic shocks (Sugiyama (iii) Affinity for crystallization solutes. The glue might provide a similar chemical condition to the crystallization drop by absorbing the crystallization solution during crystal pickup. Even in dried conditions, the absorbed chemicals might be trapped by the glue through interaction with the hydroxyl groups of PVA, preventing the condensation of chemicals inside the crystal.(iv) Suppression of ice crystals. Similarly to permeable cryoprotectants, the hydrophilic polymer suppresses the formation of ice crystals in the solution around the crystals. Moreover, the hydrophilic glue can absorb the excess crystallization solvent that usually contributes to ice formation. In this sense, the use of the polymer glue is different from oil coating, in which case a small amount of the excess solvent may be left at the crystal–oil border.Our results demonstrate the high feasibility of the HAG method for crystal mounting and cryocooling. We speculate upon several possible reasons for the feasibility and efficacy of this method.c). However, glue-coated lysozyme crystals showed a different response to that of the uncoated crystals to humidity changes . In the hypertonic condition, the polymer concentration exponentially correlates with the osmotic pressure. In addition to the pressure, mechanical force induced by dehydration of the PVA hydrogel and salt displacement might cause significant stress to crystals. In some cases, the glue surface became lumpy and salt crystals appeared during the dehydration processes. The surface roughness of the glue might reflect the formation of PVA hydrogel, in which physical networks of polymer molecules are formed by hydrogen bonds and ionic interactions together with polar or ionic solutes. To avoid unrecoverable damage, it might be necessary to optimize the humidity and the amount of the plasticizing reagents.Meanwhile, the mechanism that causes the catastrophic damage upon dehydration remains unclear Fig. 5a. In thet al., 2012et al., 2012The controllability of the osmotic pressure will aid crystallographers to manage lattice constants and isomorphism when dealing with a series of crystals at room temperature. This feature might be useful for multi-crystal data collection using synchrotrons or X-ray free-electron lasers (Kern 5.The new crystal-mounting method, the HAG method, is an effective way of handling fragile protein crystals and is compatible with experiments at both room and cryogenic temperatures. Furthermore, treatment of samples with proper humidity could produce desired lattice constants, potentially resulting in improved isomorphism for multi-crystal data collection using a series of crystals. This feature is required for experiments with radiation-sensitive crystals or when conducting experiments at room temperature using a synchrotron or an X-ray free-electron laser X-ray source. We are in the process of further improvement of the method.