The central complex selects and coordinates the species- and situation-specific song production in acoustically communicating grasshoppers. Control of sound production is mediated by several neurotransmitters and modulators, their receptors and intracellular signaling pathways. It has previously been shown that muscarinic cholinergic excitation in the central complex promotes sound production whereas both GABA and nitric oxide/cyclic GMP signaling suppress its performance. The present immunocytochemical and pharmacological study investigates the question whether GABA and nitric oxide mediate inhibition of sound production independently. Muscarinic ACh receptors are expressed by columnar output neurons of the central complex that innervate the lower division of the central body and terminate in the lateral accessory lobes. GABAergic tangential neurons that innervate the lower division of the central body arborize in close proximity of columnar neurons and thus may directly inhibit these central complex output neurons. A subset of these GABAergic tangential neurons accumulates cyclic GMP following the release of nitric oxide from neurites in the upper division of the central body. While sound production stimulated by muscarine injection into the central complex is suppressed by co-application of sodium nitroprusside, picrotoxin-stimulated singing was not affected by co-application of this nitric oxide donor, indicating that nitric oxide mediated inhibition requires functional GABA signaling. Hence, grasshopper sound production is controlled by processing of information in the lower division of the central body which is subject to modulation by nitric oxide released from neurons in the upper division. Reproductive and agonistic behaviors of acoustically communicating grasshoppers include the production of species- and situation-specific sound patterns. Grasshoppers generate acoustic signals for mate attraction, courtship and rivalry by rhythmically rubbing their hindlegs against the forewings ( = stridulation)  is a midline spanning network of highly structured neuropils in the center of the insect midbrain. It includes four interconnected subunits : the proIn order to study the flow and processing of information within the central complex, we mapped the distribution of neurotransmitters (GABA), receptors (muscarinic ACh-receptors) and neural signaling-associated metabolites  that have been demonstrated to contribute to the cephalic control of stridulation in previous pharmacological studies. The results indicate, that suppression of sound production is mediated by GABA release from tangential neurons in the CBL, that seem to directly inhibit muscarinic receptor-expressing and stridulation activating columnar output neurons of the CX. In contrast, NO released in the CBU mediates its stridulation suppressing effect indirectly, via stimulation of cGMP accumulation in GABAergic terminals in the CBL. This hypothesis was supported by pharmacological experiments. The present study identifies the CBL as the neuropil where pathways that promote or suppress sound production converge on columnar output neurons of the central complex.Chorthippus biguttulus (L. 1758) were caught in public and non-protected areas in the vicinity of Göttingen, Germany, and kept separately in the laboratory for up to several weeks. Ch. biguttulus is a common species in middle Europe that is neither endangered nor protected. Additional Ch. biguttulus were reared from eggs that were collected in the previous summer and kept at 4°C for at least 4 months. The nymphs hatched after ∼1 week at 26°C and were raised to adulthood on wheat and supplemental food for crickets (Nekton Pforzheim) at a 16/8 h light dark cycle.Adult specimen of the grasshopper −2 M sodium nitroprusside  and 5×10−4 3-(5′hydroxymethyl-2′furyl)-1benzyl indazole , an activator of soluble guanylyl cyclase The following primary antisera were used: rabbit anti-mAChR  was generated against GABA. To test specifity of the polyclonal guinea-pig GABA antiserum on brain sections of onjugate .Schistocerca gregariaThe monoclonal anti-citrulline antiserum has been characterized by Martinelli et al. Ch. biguttulus, the species used in this study. Preadsorption with 1 µg/ml of a BSA-cGMP conjugate eliminated all immunostaining in the grasshopper S. gregariaCh. biguttulus.The specifity of the sheep anti-cGMP antiserum, which was raised against formaldehyde fixed cGMP, has been demonstrated by blotting different nucleosides on nitrocellulose membanes (for a detailed description of the specificity controls see http://rsb.info.nih.gov/ij/). Colocalisation was measured by a distance-based colocalistion analysis The terminology for brain structures follows Strausfeld −3 or 10−4 M. Drugs were applied through capillaries with two chambers pulled to a single tip. Coupling to a pressure pump (WPI model PV 820) enabled injections of small volumes (1–3 nl) from either chamber to the same site within the brain. Stimulating drugs were injected in regular intervals of 5 min to maintain a similar level of overall excitation. Inhibitory drugs were injected once (after the activity released by the stimulating drug was terminated) and their influence on subsequent injections of the stimulating drug evaluated.Adult male grasshoppers were attached with wax to a holder. The dorsal part of the head capsule was opened with razor blade and its frontal part with the brain was tipped forward to expose the dorsal  surface of the brain. Small pieces of reflecting foil  were glued to the femur of each hind leg to enable the recording of stridulatory movements with two optoelectronic devices. Voltage signals proportional to the elevation of each hind leg were digitized with a sampling rate of 3 kHz  and stored as data files on a regular PC. The software NEUROLAB One injection of a stimulating drug (muscarine or picrotoxin) usually elicited several song sequences separated by short pauses. The sum of the durations of all song sequences released by one stimulus was taken as the total duration of stridulation and the highest value obtained in one experiment at a particular stimulation site was set as 100%. Relative total durations of song production of three stimuli preceding the application of the inhibitory drug were averaged and used as a reference. Potential changes in the duration of muscarine- or picrotoxin-stimulated song production following one injection of sodium nitroprusside were evaluated with the non-parametric Friedman test for multiple dependent values followed by the Wilcoxon-Wilcox test to identify the significantly different data sets. Calculations were made with Excel  and figures assembled with Photoshop (Adobe Systems).Ch. biguttulus brain is shown in Muscarinic AChR immunoreactivity in the central complex  was obseCh. biguttulus is strongly innervated by bilateral pairs of GABA immunoreactive tangential neurons, with their somata in the inferior median protocerebrum  left the CX via the IT1-tract  to a fixed site in the central complex. After a few initial stimulations, each following picrotoxin stimulus elicited a similar duration of sound production. A single injection of the NO donor SNP was applied to the same site in the central complex and its influence on subsequent picrotoxin-stimulated sound production evaluated. In contrast to muscarine-stimulated sound production  are sufficient to release stridulation by disinhibition. The distribution of GABA in the CX of Ch. biguttulus  had their somata in the ventro-median protocerebrum and the pars intercerebralis (PI) where no GABA immunoreactive cell bodies were detected in Ch. biguttulus.GABA is the major fast inhibitory transmitter in the insect brain. Injections of GABA into the central complex of grasshoppers inhibit stridulation induced by cholinergic agonists after short latency and with short duration guttulus  is very c fibers  and not NO has been shown to inhibit muscarine-stimulated stridulation, when injected into the central body at the same site as muscarine. This inhibitory effect is mediated by activation of soluble guanylate-cyclase and subsequent increase of cytosolic cGMP levels Ch. biguttulus have previously been labeled by NADPHdiaphorase histochemistry and uNOS-immunocytochemistry Ch. biguttulus females. Systemic application of the NOS inhibitor aminoguanidine caused both, a substantial reduction of citrulline accumulation in the central complex and an increased responsiveness of female grasshoppers to males' calling songs Ch. biguttulus. In contrast, previous studies on S. gregariaLocusta migratoriaCh. biguttulusNO-producing neurites in the central complex of Ch. biguttulus. Cyclic GMP immunopositive neurites belonged to neurons that appeared similar to TL2 and TL3 neurons described in S. gregaria by Müller and coworkers Ch. biguttulus, additional NO-responsive neurons were detected in the CBU, the noduli and the LAL of S. gregaria and L. migratoriaCh. biguttulus, NO is exclusively produced in the CBU, which has been confirmed by three different methods to label NO-producing cells . In contrast, NO-responsive neurites are exclusively located in the CBL at an approximate distance of 40–60 µm from NO production sites. This distance lies well within the action range of NO that was estimated in other preparations  was exclusively detected in GABA-containing tangential neurons innervating layer 2 of the CBL. Consequently, these fibers represent the only candidate targets for NO released within the central body of arations : >200 µmA receptors. In both experiments, the injection capillary must have been placed in the CBL, since dendrites of muscarinic AChR-expressing neurons overlap with presynaptic terminals of GABAergic tangential neurons in this neuropil. A number of previous experiments in which substances with limited diffusion radius were injected to sites where muscarine stimulated sound production are also in line with this result. Muscarine-stimulated sound production was reversibly suppressed by 8-Br-cGMP and the inhibitor of cGMP-dependent phosphodiesterase Zaprinast Further support for the hypothesis that NO-responsive GABAergic tangential neurons could mediate the inhibitory effects of endogenous NO release in the central complex arises from pharmacological experiments in this and previous studies. In the present study, the NO donor SNP suppressed muscarine-stimulated sound production when injected to the same site in the central complex where muscarine elicited singing. In a series of similar experiments, SNP could not suppress stridulation that was elicited by picrotoxin-mediated inhibition of GABACh. biguttulus since various signaling pathways known to promote (ACh) or to suppress (GABA and NO-stimulated cGMP) sound production converge in this neuropil , the calyx of the mushroom bodies (A2) and the optic lobes (A3), three regions that showed high intense GABA-like-immunoreactivity in both, neurites and cell bodies (B). Scale bars: 50 µm in A2 and B2 and 100 µm in A1, A3, B1 and B3).Preadsorption study to demonstrate the specificity of the guinea-pig anti-GABA antiserum. The left panel (A) shows the results of staining experiments in which the primary antiserum has been preadsorbed with GABA while the right panel (B) shows the results of the positive control. Specific staining was entirely abolished through preincubation of the primary antisera with the 20 mg/ml GABA-BSA antigen in the central body (A(TIF)Click here for additional data file.Figure S2Ch. biguttulus outside the central complex. Intensely labeled neurites were detected in the medulla of the optic lobes (A and B) and emerging from in the inner core of the antennal lobe (C). The antibody used in our study labeled the same brain regions in D. melanogaster. Immunoreactivity was also detected in the calyx (D) and the pedunculus (E) of the mushroom bodies of Ch. biguttulus. Though this has so far not been described for D. melanogaster, physiological studies on honeybees implicated a functional role for muscarinergic signaling in this brain region.Muscarinic AChR immunoreactivity in the brain of (TIF)Click here for additional data file.


Given the central role of HIV-1 diversity in the HIV pandemic and its impact on vaccine development, it is imperative that global molecular epidemiology surveillance continues. Constantly improving phylogenetics-based analytical techniques and rapidly expanding HIV sequence datasets promise to yield important insights into the origin, evolution and spread of HIV-1.In an effort to study the phylogeography and phylodynamics of the HIV-1 M epidemic in the early 20th century, we analysed 50 plasma samples from HIV-infected blood donors from Cameroon. Full length gag sequences were generated and aligned using MUSCLE along with a representative selection of HIV sequences from the rest of the world and all published gag sequences from Cameroon and other West African countries. A maximum likelihood phylogenetic tree was constructed from these sequences following removal of recombinant sequence fragments by a blinded fully exploratory screen for recombination using RDP3.All the Cameroonian sequences were derived from HIV-1 M viruses. The phylogenetic tree indicated that at least one of the newly-sequenced CRF02_AG viruses is an outlier of the CRF02_AG clade and may help resolve the controversy surrounding the origins of this clade. Furthermore, isolates from Cameroon were spread throughout the phylogenetic tree clustering with different subtypes and circulating recombinant forms, a finding consistent with West Africa being the geographic origin of the global HIV epidemic. Importunately, our blind recombination screen suggested that many divergent Cameroonian viruses previously identified as being unique recombinant forms, may be divergent, relatively non-recombinant, but under-sampled subtype-level lineages.Lineages diverging early after the origin of HIV-1 M are likely still circulating in Cameroon and could be suitable for retracing the movement and evolutionary dynamics of HIV-1 during the earliest stages of the pandemic. These lineages will be useful for reconstructing ancestral HIV-1M sequences for use as vaccine immunogens.


Further, the analysis of a biological specimen's associated microbiome can also be performed using RNA-seq data and this application is gaining interest in the scientific community. There are many existing bioinformatics tools designed for analysis and visualization of transcriptome data. Despite the availability of an array of next generation sequencing (NGS) analysis tools, the analysis of RNA-seq data sets poses a challenge for many biomedical researchers who are not familiar with command-line tools. Here we present RNA CoMPASS, a comprehensive RNA-seq analysis pipeline for the simultaneous analysis of transcriptomes and metatranscriptomes from diverse biological specimens. RNA CoMPASS leverages existing tools and parallel computing technology to facilitate the analysis of even very large datasets. RNA CoMPASS has a web-based graphical user interface with intrinsic queuing to control a distributed computational pipeline. RNA CoMPASS was evaluated by analyzing RNA-seq data sets from 45 B-cell samples. Twenty-two of these samples were derived from lymphoblastoid cell lines (LCLs) generated by the infection of naïve B-cells with the Epstein Barr virus (EBV), while another 23 samples were derived from Burkitt's lymphomas (BL), some of which arose in part through infection with EBV. Appropriately, RNA CoMPASS identified EBV in all LCLs and in a fraction of the BLs. Cluster analysis of the human transcriptome component of the RNA CoMPASS output clearly separated the BLs  from the LCLs (which have a blast-like phenotype) with evidence of activated MYC signaling and lower interferon and NF-kB signaling in the BLs. Together, this analysis illustrates the utility of RNA CoMPASS in the simultaneous analysis of transcriptome and metatranscriptome data. RNA CoMPASS is freely available at  Fusobacterium and colorectal carcinoma was made using two different NGS approaches Through its capacity to delve deeply into the genetic composition of a biological specimen, next generation sequencing (NGS) technology presents an unprecedented approach to pathogen discovery in the context of human disease. This unbiased approach to identify undiscovered human disease causing pathogens has already shown promise, resulting in the discovery of a novel Merkel cell polyomavirus in Merkel cell carcinoma While current sequence-based computational subtraction pipelines are used solely for pathogen discovery, RNA CoMPASS, takes advantage of the richness of RNA-seq data to provide host transcript expression data in addition to pathogen analysis. This concept, recently coined “dual RNA-seq” by Westermann and colleagues RNA-seq data sets from 22 Human B-Cell samples (lymphoblastoid cell lines [LCLs]) immortalized with Epstein-Barr Virus (EBV) were downloaded from the NCBI Sequence Read Archive (SRA010302). Samples were sequenced using an Illumina Genome Analyzer II machine running single end 50 base sequencing reactions. Similarly, 22 Human Burkitt's Lymphoma (BL) samples were obtained from the NCBI Sequence Read Archive (SRA048058). Samples were sequenced using an Illumina Genome Analyzer II machine running paired end 107 and 102 base sequencing reactions. The Akata RNA-seq data set was generated previously in our lab (SRA047981) www.novocraft.com)  with a reference genome ), splice junctions  is a graphical user interface (GUI) based parallel computation pipeline for the analysis of both exogenous and human sequences from RNA-seq data. Several open source programs and a single commercial program are utilized in this automated pipeline. For the deduplication steps, an in-house de-duplication algorithm is used. Alignments to the reference genome are carried out using Novoalign V2.07.18 (P<0.05 following the Benjamini-Hochberg correction for multiple testing. The fitted log(CPM) values for the subset of genes that were differentially expressed in the LCL samples relative to the BL samples were then clustered using the Euclidean distance and complete linkage algorithm to detect groups of co-expressed genes.Human transcript counts were imported into the R software environment and analyzed using the edgeR package http://www.novocraft.com/) . Any host genome can be uploaded to RNA CoMPASS. In our case, we used the human reference genome, hg19 (UCSC), plus splice junctions . To further reduce the computational burden incurred by BLASTing these unmapped host reads, RNA CoMPASS offers an optional stage prior to NT database BLASTing where the user can BLAST against a host transcript database. Because host transcript databases are much smaller than the NT database, host reads not aligned by Novoalign can be filtered out at lower computational cost than would otherwise be incurred by BLASTing these reads against the NT database.Exogenous sequence analysis proceeds concurrently with the endogenous analysis . UtiliziAfter BLASTing against the NT database, taxonomic analysis is performed by importing the BLAST results into MEGAN In RNA CoMPASS, we have implemented both the Java Parallel Processing Framework (JPPF) API and Portable Batch System (PBS) API in order to deploy it on either a small local cluster or a grid system managed by PBS submission. Our testing of RNA CoMPASS in both environments (data not shown for grid system) showed that our pipeline could efficiently analyze RNA-seq data sets achieving a significant speedup over analysis on a single machine. This will allow other investigators to use RNA CoMPASS on whichever type of computational environment they have access to. In our case, we employed RNA CoMPASS on a local 4-node cluster environment (Intel Xeon Mac Pros with 64–96 GB RAM).To evaluate the performance of RNA CoMPASS on a cluster environment versus a single node environment, we benchmarked 6 RNA-seq data sets with incrementally varied file sizes on both a single machine and on a local cluster with 4 nodes. The 6 files used for this analysis were extracted from a previously generated RNA-seq data set from a BL cell line (1 sequence pair from the Akata RNA-seq data set) To test the utility of RNA CoMPASS to identify pathogens within biological specimens we used RNA-seq data sets from two distinct B-cell types, lymphoblastoid cell lines (LCLs) and Burkitt's lymphoma (BL) samples. LCLs are not tumor cells but have instead been immortalized by infection with EBV. In contrast, the Burkitt's lymphoma cells lines are tumor cell lines, some of which underwent tumorigenesis in part through natural infection with EBV. Notably, however, although some Burkitt's lymphoma cell lines are infected with EBV, the EBV gene expression pattern and the cell phenotype of Burkitt's lymphomas and LCLs are distinct.Single-end RNA-seq data sets from 45 B-cell lines  were analyzed using RNA CoMPASS . Most saAs expected, EBV gene expression in LCLs is generally more robust and shows the expression profile expected in this cell type with all the latent proteins including EBNA 1,2, and 3 and LMP 1 and 2 being expressed . In contThe host transcriptome analysis component of RNA CoMPASS generates gene expression output files that can be used for cluster and pathway analysis. Gene expression output from RNA CoMPASS analysis of the 22 LCL and 23 BL samples was subjected to hierarchical clustering and differential gene expression analysis . FurtherTo investigate differences in LCLs compared to BLs, Ingenuity Pathway Analysis software (IPA: Ingenuity Systems) was used to assist in the analysis of signaling pathways and molecular functions associated with the differentially expressed cellular genes. Upstream regulator analysis within IPA predicted activation of MYC (z-score: 3.375), MYCN (z-score: 2.813), MAPK9 (z-score: 2.414), and MAPK1 (z-score: 2.138) pathways with an inhibition of Interferon alpha (z-score: −2.916), interferon gamma (z-score: −2.788), NF-kB (z-score: −2.746), interferon alpha-2 (z-score: −2.723), and interferon lambda (z-score: −2.000) pathways in BL relative to LCL samples  and S3. RNA CoMPASS is designed to take advantage of several open source programs in order to streamline and accelerate RNA-seq data analysis. RNA CoMPASS helps the researcher to manage the computational burden of processing large sets of RNA-Seq data by parallelizing the most compute intensive steps of the process and automatically managing files through each step of the pipeline. The simultaneous analysis of the host transcriptome along with the discovery of pathogens allows investigators to not only detect pathogens but also study the relationship between the pathogen and host transcription.In RNA CoMPASS, we have implemented both the Java Parallel Processing Framework (JPPF) API and Portable Batch System (PBS) API in order to deploy it on either a small local cluster or a grid system managed by PBS submission. Our testing of RNA CoMPASS in both environments showed that our pipeline could efficiently analyze RNA-seq data sets achieving a significant speedup over analysis on a single machine. This will allow other investigators to use RNA CoMPASS on whichever type of computational environment they have access to. In our case, we employed RNA CoMPASS on a local 4-node cluster environment (Intel Xeon Mac Pros with 64-96GB RAM) which achieves a speedup approaching the theoretical limit of 4 by splitting the computational tasks over 4 machines as the file size increases. This speedup of a 4 node cluster serves as a proof of principle that an even greater speedup could be obtained using a significant computational cluster involving hundreds of nodes. A recently published study Though the BLASTing step of RNA CoMPASS incurs moderate limits on the size of input file that can be processed , it allows for a more comprehensive analysis. In previous work, we would that sampling 10 million reads from an RNA sequencing experiment is likely to be well beyond the number needed to detect meaningful levels of exogenous agents We used LCLs and BL samples to evaluate the pathogen and host transcriptome analysis arms of RNA CoMPASS because of their differences in phenotypes. The LCLs were generated by infecting human B-cells with EBV, which typically display an activated B-cell like phenotype  and BART transcripts). The BL samples typically display a germinal center-like phenotype (type I latency – expressing EBNA1 and BART transcripts). The entire sequence file for all samples was used as input for RNA CoMPASS. Although the BL samples were sequenced using a paired-end approach, only one of the reads from each pair was analyzed in order to remain consistent among all samples and because a single-end read should provide sufficient evidence for pathogen discovery.RNA CoMPASS discovered a significantly larger proportion of EBV (Human Herpesvirus 4) reads within the LCLs as compared to the BLs. This is an anticipated result, which validates the usage of RNA CoMPASS to interrogate genetic material of exogenous origin.One of the most highly active pathways within BLs is the MYC pathway. In our study, the MYC and MYCN pathways were predicted to be the top two activated pathways in BLs relative to LCLs according to IPA's upstream regulator analysis. Several MYC targets have been reported in the literature Transcriptome analysis has shown that the MYC signature is the hallmark signaling difference between Burkitt's lymphomas and diffuse large B-cell lymphomas with: upregulation of MYC-target genes and downregulation of genes involved in the NF-kB and interferon responses On the other hand, the most highly inhibited pathways within BLs compared to LCLs were the interferon response pathway and the NF-kB pathway. The NF-kB pathway has been shown to play a vital role in EBV's ability to transform naïve B-cells as the EBV transforming latency protein, LMP1 continuously activates NF-kB A few other noteworthy genes that were observed as being differentially expressed include TCF3 and TOP2A, both of which have increased expression in BLs relative to LCLs. In a recent study using RNA-seq with RNA interference screening of BLs, Schmitz et al was able to identify mutations affecting the transcription factor TCF3 in vitro and in vivoThe other molecule, TOP2A has been shown to determine anthracycline-based drug (e.g. doxorubicin) response http://rnacompass.sourceforge.net. Installation guidelines, supporting documentation, along with example data are available from the sourceforge repository. The most up to date source code is available from the sourceforge site; the version of the source code at the time of publication is included in In summary, our results demonstrate the utility of RNA CoMPASS in analyzing large sequence datasets for the discovery of pathogens and host transcriptome analysis. The use of this pipeline is expected to enable more researchers to enter the filed of RNA Sequencing and to yield novel associations between pathogens and human diseases with important medical implications. This study shows the disparate expression profiles between Lymphoblastoid cell lines and the Burkitt's lymphoma samples thereby exhibiting the ability of RNA CoMPASS to analyze endogenous sequencing. RNA CoMPASS is publically available under the GPL: We are planning to implement the Circos plot capability for discovered pathogens as well as clustering analysis for host gene expression in later versions of RNA CoMPASS. These improvements will further streamline and complement the analysis of RNA-seq data in the discovery and analysis of pathogens associated with malignancies. In addition, we are investigating ways to further improve the speedup of the pipeline.Figure S1Performance of RNA CoMPASS based on individual tasks. The six Akata RNA-seq data set files used previously were benchmarked on completion of individual tasks and represented in the graphs. Runs on a single node are represented using blue columns while runs on a 4-node cluster are represented using red columns. The green line represents speedup time between the single node and 4-node environment. Note in particular that speedup of the BLAST portion of RNA CoMPASS and overall speedup approaches the theoretical limit of 4 as the data size is increased.(TIF)Click here for additional data file.Figure S2Predicted top activated upstream pathway of top 250 differentially expressed genes.(TIF)Click here for additional data file.Figure S3Predicted top inhibited upstream pathway of top 250 differentially expressed genes.(TIF)Click here for additional data file.Table S1Sample list.(XLS)Click here for additional data file.Table S2Top 250 differentially expressed genes between lymphoblastoid cell line and Burkitt's lymphoma samples.(XLS)Click here for additional data file.Table S3All differentially expressed genes between lymphoblastoid cell line and Burkitt's lymphoma samples.(XLS)Click here for additional data file.Software S1Source code for RNA CoMPASS.(TXT)Click here for additional data file.


Decades after the eradication of smallpox, its etiological agent, variola virus (VARV), remains a threat as a potential bioweapon. Outbreaks of smallpox around the time of the global eradication effort exhibited variable case fatality rates (CFRs), likely attributable in part to complex viral genetic determinants of smallpox virulence. We aimed to identify genome-wide single nucleotide polymorphisms associated with CFR. We evaluated unadjusted and outbreak geographic location-adjusted models of single SNPs and two- and three-way interactions between SNPs.Using the data mining approach multifactor dimensionality reduction (MDR), we identified five VARV SNPs in models significantly associated with CFR. The top performing unadjusted model and adjusted models both revealed the same two-way gene-gene interaction. We discuss the biological plausibility of the influence of the SNPs identified these and other significant models on the strain-specific virulence of VARV.We have identified genetic loci in the VARV genome that are statistically associated with VARV virulence as measured by CFR. While our ability to infer a causal relationship between the specific SNPs identified in our analysis and VARV virulence is limited, our results suggest that smallpox severity is in part associated with VARV strain variation and that VARV virulence may be determined by multiple genetic loci. This study represents the first application of MDR to the identification of pathogen gene-gene interactions for predicting infectious disease outbreak severity. Smallpox, the only human infectious disease to have been eradicated, remains a threat as a potential agent of bioterrorism. Mortality rates during natural outbreaks of smallpox varied widely, a feature partially attributed to strain-specific virulence of the etiological agent, the orthopoxvirus variola virus (VARV) . An undeWe analyzed the genome-wide single nucleotide polymorphisms (SNPs) of a collection of 35 temporally, geographically and epidemiologically diverse VARV isolates housed at the US Centers for Disease Control and Prevention’s secure repository (previously described in ) for assFrom the full-genome sequences of the 35 isolates we studied, we identified 1730 SNPs using kSNP with k=25 . We builp<0.001); therefore, in adjusted models we included a binary covariate where 0 represented strains isolated from non-African outbreaks and 1 represented strains isolated from African outbreaks. We fit one-way, two-way and three-way SNP-SNP interaction models using MDR. All models were fit using five-fold cross-validation and evaluated for significance using 1000 random permutations [Our outcome of interest was CFR, coded as a binary variable where 0 represented CFRs ≤ 10 percent and 1 represented CFRs > 10 percent. Of the 35 isolates, 18 were associated with CFRs > 10 percent. Preliminary analyses revealed that CFR is significantly associated with geographic location, namely, whether the outbreak occurred on the African continent  form. The IEV is the precursor to the extracellular cell-associated enveloped virion, which requires this protein for producing the actin tails that push it away from the cell surface and allow it to disseminate to adjacent cells. Loss of A36R results in a small plaque phenotype e in vivo. SNP 151Other models identified by our MDR analysis performed inferiorly to these; however, they allowed us to identify SNPs that were significantly associated with CFR. SNP 183083 was found in two relatively high-performing models, both the one-way and three-way unadjusted models. This SNP is in the gene for the CrmB secreted TNF-alpha-receptor-like protein. This is a TNF-alpha receptor expressed by poxviruses that blocks cytokine activity to interfere with the host immune function, thereby enhancing viral virulence . AdditioThis study represents a novel application of MDR to studying pathogen genome-wide associations with infectious disease outbreak severity. Of the models we evaluated using MDR, the best-performing model predicted smallpox outbreak CFR with a two-way VARV gene-gene interaction. While we are limited in our ability to infer a causal relationship between the interacting SNPs identified in our analysis and smallpox outbreak severity, the genes implicated have potential biological plausibility as determinants of VARV virulence and may represent targets for future laboratory-based investigations. The results of this study reinforce existing evidence that smallpox outbreak severity may be in part a consequence of the VARV strain associated with the outbreak and suggests that VARV virulence is a complex trait influenced by multiple genetic loci.CFR: Case fatality rate; EEV: Extracellular enveloped virus; IEV: Intracellular enveloped virion; MDR: Multifactor dimensionality reduction; SNP: Single nucleotide polymorphism; VARV: Variola virus.The authors declare that they have no competing interests.AGH participated in the study design, performed the statistical analysis and drafted the manuscript. SNG performed SNP analysis and contributed to the interpretation of the data and the drafting of the manuscript. JHM conceived of the study, participated in the study design and statistical analyses and contributed to interpretation of the data and the drafting of the manuscript. All authors read and approved the final manuscript.


Courtesy stigma is the stigmatization a person perceives or experiences due to their association with a stigmatized individual or group. Most HIV-related stigma scales have been developed for people living with HIV/AIDS (PLWHAs), but not for their HIV-uninfected family members. To date, few measurement scales have been designed to measure the degree of stigma among both PLWHAs and their HIV-uninfected family members at the family level. We developed a set of courtesy stigma scales and estimated their reliability and validity from 256 PLWHAs and 256 of their HIV-uninfected family members. Exploratory and confirmatory factor analyses were performed in two independent samples: a development sample (N = 216) and a validation sample (N = 296), respectively. Two factors (“public stigma” and “self-perceived stigma”) had high internal consistency reliability  and good construct validity (standardized factor loading range: 0.37–0.95) in both samples. These findings document that the newly developed brief instrument is a psychometrically sound measure of HIV-related stigma among both PLWHAs and their HIV-uninfected family members. Stigma continues to be a major barrier to the treatment-as-prevention strategy for HIV interventions As posited by Goffman, perceived or experienced stigma could be passed on to family members of those with the stigmatizing attribute and has been coined “courtesy stigma” The Chinese culture may foster stigma Despite the presence of psychometrically sound measures of HIV-related stigma among a variety of populations, including PLWHAs The study protocol was approved by the Institutional Review Boards of Virginia Commonwealth University, the Guangxi Center for Disease Control and Prevention and the Anhui Medical University Institute of Biomedicine. In accordance with the approved protocol, written informed consent was obtained from all study participants prior to data collection.We conducted two cross-sectional studies among HIV affected families in Anhui and Guangxi, China in 2008 and 2010 The first cross-sectional study was conducted among PLWHAs and their family members in a rural area in Anhui province. The majority of farmers were infected with HIV through commercial plasma donations that occurred in the early-to-mid 1990s The second cross-sectional study was conducted in Nanning, Guangxi. The study methodology has been described elsewhere The development sample was taken from Anhui where 108 HIV-infected individuals and 108 of their HIV-uninfected family members participated in the interview. At this site, 118 HIV affected families were invited to participated, 10 families were excluded as one or two family members declined to participate or did not provide information regarding stigma. The major mode of HIV transmission at the study site was unsafe commercial blood donation practices. The validation sample was taken from Guangxi where 148 HIV-infected individuals and 148 of their HIV-uninfected family members participated in the interview. Of 170 HIV dyads invited to participate in this study, 20 dyads refused, and 2 dyads did not provide information about their perceived stigma and were excluded. The primary mode of HIV transmission in this region was injection drug use (IDU).strongly agree, agree, disagree, strongly disagree). Examples of the items included: “No one would be willing to take care of their children when HIV infected people die from AIDS.” and “People seldom buy food from HIV-infected individuals or their family”. To measure self-perceived stigma, we developed 9 items with a 4-point ordinal response format . Examples of items included: “Because of my family member's HIV status, I feel shame and self-blame” and “Because of my family member's HIV status, I feel that children are kept away from me by their parents”.Instruments were initially drafted in English and then translated into Chinese by research members who were fluent in both languages. The Chinese version of the items was then distributed to research team members who reviewed and modified the wording to make it appropriate for the Chinese context. The development of courtesy stigma scales was based on our previous studies CES-D  is a 10-item scale designed to measure depressive symptoms experienced in the past week In addition to the 22-item courtesy stigma scales, we administered depression and self-efficacy scales to provide concurrent validity evidence for the newly developed instrument. The general self-efficacy scale was used to measure a general sense of perceived self-efficacy Exploratory factor analysis (EFA) with oblique rotation was performed to assess the dimensionality of the CCSS items that underlie the sample data and to identify the measurement structure of the test. Two-factor solutions were inspected separately for PLWHAs and their HIV-uninfected relatives. Scree plots, factor patterns and factor structure coefficients, the number of items with high loadings on one factor and low on the remaining factors, and the theoretical meaningfulness of identified factors were considered in deciding the measurement model for the courtesy scales. The number of eigenvalues preceding the elbow in scree plots was retained for rotation; remaining eigenvalues were deemed unimportant and were subsequently dropped. The cumulative proportion of variance was obtained from successive factor solutions. The EFA procedures were carried out in MPlus version 7.1 using an asymptotically distribution-free estimator, i.e., weighted least squares estimator with robust standard errors and mean- and variance-adjusted chi-square values (WLSMV), given the ordered categorical nature of item distributions and an oblique rotation .Confirmatory factor analysis (CFA) was used in the validation sample to replicate the measurement model suggested by EFA in the development sample. Goodness of fit was assessed using chi-square test of exact fit , root mean square errors of approximation , Comparative Fit Index  and Tucker Lewis Index  Concurrent validity was assessed by evaluating associations between the courtesy stigma scales and depression and self-efficacy scores. Based on previous research The development sample consisted of 216 subjects (108 PLWHAs and 108 HIV-uninfected family members) who participated in the interview. The descriptive statistics are presented separately by HIV status in The EFA was conducted on the development sample. The scree plots obtained separately from HIV-infected and-uninfected samples suggested a two-factor solution see , as hypoTo assess their concurrent validity, we estimated the correlations of the stigma scales with depression and self-efficacy in two independent samples of the HIV-infected individuals and HIV-uninfected individuals. The validation sample consisted of 296 subjects (148 PLWHAs and 148 HIV-uninfected family members). The HIV-infected sample had a mean age of 40.7 years . One third (30.4%) of the HIV-infected sample was female, 25.7% had a primary school education or no education, 27% were farmers, and 73% were married at the time of interview. The HIV-uninfected sample had a mean age of 37.5 years . Sixty-one percent were female, 23% had a primary school education or no education, 24.3% were farmers, and 83.1% were married at the time of interview.To further validate the findings of the development sample from the EFA, we used confirmatory factor analysis (CFA) on the validation sample. Specifically, we fit the correlated two-factor model separately for the HIV-infected and HIV-uninfected samples. The CFA model specifications relied solely on the EFA results described above in order to cross-validate the measurement structure of stigma scales in two independent samples. Whereas the chi-square test of exact fit and RMSEA did not indicate good model fit, the CFI and TLI indices supported the model in both the HIV-infected sample  and HIV-uninfected sample . The standardized factor loading in public stigma was between 0.40–0.93 in HIV-infected sample and 0.62–0.93 in HIV-uninfected sample. The factor loading in self-perceived stigma was between 0.40–0.93 in HIV-infected sample and 0.37–0.95 in HIV-uninfected sample . The intThe results of the concurrent validity analyses indicated the expected correlations among HIV public stigma, self-perceived stigma, depression, and self-efficacy. Specifically, public stigma was significantly correlated with self-perceived stigma  and depression , but not with self-efficacy  among in the HIV positive sample. In the HIV-uninfected sample, public stigma was significantly correlated with self-perceived stigma  and depression , but not with self-efficacy . HIV self-perceived stigma was significantly correlated with depression , but not with self-efficacy  in the HIV-infected sample. In the HIV-uninfected sample, self-perceived stigma was significantly correlated with depression  and self-efficacy  .The findings of this study demonstrate that the CCSSs can reliably capture courtesy stigma among HIV-uninfected family members of PLWHAs. Because the same set of stigma scales can be used in both PLWHAs and their family members, comparisons of the two types of stigma can be directly performed at the dyadic or family level. Results from factor analyses showed that these scales shared the same factor structure in both HIV-infected and HIV-uninfected samples, indicating consistency across two different populations. High Cronbach's alpha coefficients supported the internal consistency and reliability of the HIV stigma scales. The alpha of 0.90 for public stigma and 0.88 for self-perceived stigma among the HIV-uninfected family members provides confidence that the CCSS reliably measures two dimensions of courtesy stigma.Concurrent validity of the CCSS scales was supported by significant correlations among the stigma scale with depression and self-efficacy, which previous studies have shown to be closely related to stigma While previous studies have assessed psychometric characteristics of HIV-related stigma scales in particular populations, few studies have empirically assessed an instrument that could be used with equal accuracy among PLWHA and their HIV-uninfected adult family members. Our findings document sound psychometric properties of the CCSS scales to measure HIV courtesy stigma among family members of HIV-infected individuals as well as among PLWHA themselves. The psychometric properties were very robust in the two different study populations with different HIV transmission modes  and across different investigation periods (2008 vs. 2010). The CCSSs have advantages over existing instruments in that it is brief, easy to administer, and applicable to diverse populations.Despite the many strengths of this study, some limitations should be noted. While EFA/CFA strongly supported the correlated two-factor structure of the CCSSs in two samples, fit indices from CFA in two independent samples provided mixed support for the measurement structure. Future studies with larger sample sizes are needed to further test the proposed measurement model for the CCSSs in independent samples. While our results show that there is a reliable scale for measuring courtesy stigma among HIV-uninfected people in the study population, there may be some limitations of the generalizability to different populations, especially to those in individualistic Western cultures. As the major HIV transmission routes in this study were blood donation and heroin injection, these measurement scales may not be appropriately used among HIV-infected people who contract HIV via other transmission modes .Our study provides a valuable tool for measuring HIV-related stigma for PLWHAs and courtesy stigma for their HIV negative family members in China and other countries with similar settings. The findings of this study may provide insight into new ways to improve current HIV-related stigma scales, particularly those targeting HIV-uninfected family members who perceive or experience courtesy stigma. The new scales can be used in the development and implementation of family-focused public health interventions to address stigma across HIV-affected families that extend beyond the PLWHAs.


In the NOD mouse, the incidence of type-1 diabetes is thought to be influenced by the degree of cleanliness of the mouse colony. Studies collectively demonstrate that exposure to bacterial antigen or infection in the neonatal period prevents diabetes  The development of the postnatal immune system is guided by the interactions of lymphocytes with self-MHC/peptide ligands derived from our body's own tissues and those from the environment, such as the commensal microbial flora of the gastrointestinal tract and the diet. The important role of the gastrointestinal microbiota has been emphasized by the evidence of reduced intestinal lymphatic tissue and underdeveloped lymphoid organs in germ-free mice The way in which antigenic stimulation guides the development and maintenance of a healthy immune system is of fundamental importance to our understanding of immunological tolerance. In the non-obese diabetic (NOD) mouse strain, the target pancreatic insulin producing beta cells are attacked and destroyed by activated immune cells, leading to type-1 diabetes. Several infectious, and non-infectious agents are known to prevent type-1 diabetes in NOD mice. They include; persistent viral infection MHV , mycobacThus, the incidence of type-1 diabetes in NOD mice is thought to reflect the degree of cleanliness of the colony. A widely accepted extrapolation from these data has been that NOD mice maintained under germ-free conditions have an increased incidence of diabetes. However, there is little evidence to support this view http://www.taconic.com/wmspage.cfm?parm1=292 until thirty weeks of age at Taconic Farms breeding facility, Germantown, NY, USA. All supplies were sterilized and entered into the isolator using strict aseptic techniques. Mice underwent monthly microbiological testing to ensure that germfree rodents were free of aerobic and anaerobic organisms (for a complete list of agents see http://www.taconic.com/wmspage.cfm?parm1=265). Urine glucose was measured every one or two weeks and mice with frank glucosuria -two consecutive reading of 4+ (estimated at 55 mmol/L urine glucose) were considered diabetic. Control female NOD mice were housed under specific pathogen-free (SPF) conditions at Taconic and the Scripps rodent colony, which were handled in accordance with the TSRI Animal Care and Use Committee, which approved this study (A3194-01). Blood glucose values (BGV) were determined using Glucofilm blood glucose strips . Mice were considered diabetic following two consecutive blood glucose readings above 18 mmol/L.A germfree caesarean derivation was performed on NOD mice using 10 females and 5 males. Ceasarean-derived mice were weaned onto sterile dams and housed in sterile isolators to maintain germfree status and fed an endotoxin free NIH31-M diet, designed to provide appropriate nutrition after being autoclaved Bacillus cereus) was detected at week 16. These mice exhibited a delayed onset, and reduced incidence of clinical disease (p<0.001) during the 30-week study period  was indistinguishable from that of NOD mice housed under SPF conditions in our colony (n = 20) , p<0.78.y period .MyD88-/- NOD mice The NOD genetic background is one important factor contributing to the normal development of T1D in female NOD mice in the absence of known intestinal microbiota. However, the critical influence of the environment was highlighted by the reduced incidence of diabetes following spontaneous monoculture with aerobic, spore-forming, bacteria. Diet has a well-established role in the development of T1D in NOD mice Taken together, these findings debunk the myth that germ-free NOD female mice have increased diabetes incidence and support the notion that modulation of intestinal microbiota can have beneficial effects on the development of autoimmune diabetes. Future examination of the influence of an array of commensal microbiota on diabetes incidence offers the potential for non-invasive approaches for individuals at risk of developing type-1 diabetes.


Comprehensive understanding of molecular mechanisms underlying viral infection is a major challenge towards the discovery of new antiviral drugs and susceptibility factors of human diseases. New advances in the field are expected from systems-level modelling and integration of the incessant torrent of high-throughput "-omics" data.in silico model was applied to comprehensively explore the molecular relationships between viruses and their associated diseases. This was done by merging virus-host and host-host physical protein-protein interactomes with the set of genes essential for viral replication and involved in human genetic diseases. This systems-level approach provides strong evidence that viral proteomes target a wide range of functional and inter-connected modules of proteins as well as highly central and bridging proteins within the human interactome. The high centrality of targeted proteins was correlated to their essentiality for viruses' lifecycle, using functional genomic RNAi data. A stealth-attack of viruses on proteins bridging cellular functions was demonstrated by simulation of cellular network perturbations, a property that could be essential in the molecular aetiology of some human diseases. Networking the Human Infectome and Diseasome unravels the connectivity of viruses to a wide range of diseases and profiled molecular basis of Hepatitis C Virus-induced diseases as well as 38 new candidate genetic predisposition factors involved in type 1 diabetes mellitus.Here, we describe the Human Infectome protein interaction Network, a novel systems virology model of a virtual virus-infected human cell concerning 110 viruses. This The Human Infectome and Diseasome Networks described here provide a unique gateway towards the comprehensive modelling and analysis of the systems level properties associated to viral infection as well as candidate genes potentially involved in the molecular aetiology of human diseases. Infectious diseases cause million of deaths as reported by the World Health Organization (WHO). In this context, emerging and re-emerging infectious diseases - such as flu, AIDS, tuberculosis, hepatitis - stress the need to accelerate and rationalize the development of better diagnostics, treatments and preventions. It is widely accepted that appearance and severity of infectious diseases depend on the ability of viruses to interfere with host cell functions and defence. Indeed, during virus-host co-evolution, hosts have selected an arsenal of complex defence mechanisms to eliminate viruses from the organism. Conversely, because of their high replication and propagation rate, viruses have evolved strategies, at least in part driven by molecular interactions, to circumvent host cellular defence and to maintain their control on the cellular machinery. Comprehensive understanding of how viruses and their host interact appears as a major milestone in the quest of deciphering viral infection processes and the molecular aetiology of some complex diseases.i.e. infectomics, it is now possible to investigate viral infection at the systems level. The virus-host interactome, i.e. the whole set of protein interactions occurring within virus-infected cells, has been investigated in the last decades by the mean of low scale experiments focusing on single viral proteins. Recently, interactomics relying on high-throughput technologies  have boosted virus-host systems biology [Owing to new advances in high-throughput profiling of an infection,  biology ,2. Impro biology -7. These biology .al. explored the diseasome network to investigate the molecular basis of genetic diseases throughout integration of the cellular protein interaction network and the comprehensive dataset of disease-gene associations [From the late nineteenth-century, Koch's postulates were routinely applied to identify microbial origins of diseases. More recently, molecular postulates were introduced to unravel mechanisms associated to virulence . Indeed,ciations ,12. Whetin silico topological perturbations associated to viral infection and profiled new candidate targets that could be suitable for drug design. Finally, a prototype of a Human Infectome Diseasome Network (HIDN) based on OMIM annotations of genes involved in human multi-factorial genetic diseases was designed. This framework was applied to explore in more details molecular basis of the Hepatitis C Virus-induced diseases and to question the dual role of viruses in the susceptibility to type 1 diabetes mellitus (T1D).Here, we aimed at providing a systems biology rational to Koch's postulates by deciphering the molecular basis and the complexity of viruses-induced diseases through the comprehensive analysis of virus-host connections to the human diseasome. A bottom-up approach was used to reconstruct the Human Infectome Network (HIN), as a systemic model of virus-infected human cell based on a protein-protein interactions scaffold. The topology of the protein interaction network was first explored and detailed insights on the functional essentiality of host proteins targeted by viruses were given. We next simulated TP = 1,148). The probability distribution of the viral targeting intensity, kv(TP) - i.e. the number of viral proteins interacting with a host protein - showed that 32%  of targeted cellular proteins interact with more than one viral protein. At the taxonomic level, we observed that 28% , 22,5%  and 19,4%  of the targeted cellular proteins interact with more than one virus, family and Baltimore's group respectively. These results are highly suggestive of convergent molecular mechanisms based on protein interactions independently selected during the evolution of viruses.In a systems virology perspective, a virus-infected cell was modelled as an integrated system driven by physical interactions between viral and host cellular proteins - the Human Infectome Network  are also directly and physically interconnected within the human cellular interactome. These results are in good agreement with previous observation made for the Hepatitis C Virus [h(VP)) - could directly correlate with the functional pleiotropy of viral proteins and with the diversity of molecular strategies they evolved to subvert the host cellular machinery and to evade from the cellular antiviral defence.Approximately 50% of the human proteins 10,378/20,219) have been shown to interact with at least one interacting partner. In our study, a total of 92% . The centrality (b) measures the number of shortest paths (spl)  that pass through a given protein. The centrality of a protein is used to estimate the theoretical flux of information that is potentially controlled by this protein. We also used the shortest path length observed between human proteins as an unbiased measure of functional relationships as observed within molecular pathways or complexes of proteins.To better characterize to what extent viral proteins affect the functioning and the robustness of the cellular network, we next analyzed topological properties of targeted proteins within HIN  = 38 versus mean kh(NTP) = 10; one-tailed Wilcoxon test; P-value < 2 × 10-16; see Additional file h(TP) = 5.34 × 10-04 versus mean bh(NTP) = 8.23 × 10-05; one-tailed Wilcoxon test; P-value < 2 × 10-16; see Additional file h(TP) = 2.91 versus mean splh(NTP) = 3.66; one-tailed Wilcoxon test; P-value < 2 × 10-16). These statistically significant trends validate, at a larger scale, previous analysis of experimental virus-host protein interaction networks [P-value < 2.2 × 0-16). A similar shift in the distribution was observed at the taxonomic level for viral species (mean degree 62 versus 28), family (mean degree 66 versus 29) and Baltimore's group (mean degree 68 versus 30), confirming previous observations [i.e. inspection bias due to Y2H screening technology or false positives) until the availability of comprehensive, totally unbiased and high-quality human and virus-host interactomes, it remains that this network-based analysis provides additional support to the hypothesis that viruses have evolved convergent strategies measurable at the proteome-wide level to control highly connected and central proteins in order to subvert essential functions of the cell.Figure networks . All thervations . AlthougP-Value < 0.05). The overlap of proteins observed between modules was finally used to define modules linkers . Interestingly, a statistically significant proportion of these linkers (~20% = 104/541) are targeted by at least one viral protein, suggesting that viruses have a tendency to perturb functional cross-talks between functional modules .The host cellular machinery is controlled by a highly complex, dynamic and regulated modular protein interaction network . To undeh(TP)) and the intensity of viral targeting (kv(TP)). Nevertheless, the strength of this correlation remains week . Although we cannot totally rule out that this partially results from virus-host and host-host interactome incompleteness, it is also highly suggestive of viral strategy based on targeting of poorly connected proteins within the human interactome.We observe a significant correlation between the level of connectivity of host targeted proteins  exhibit significant higher bridging centrality than non-overlapping proteins . Taken together, these results support the notion that viruses have evolved highly tuned mechanisms to control host cell functioning. This control can be achieved not only by directly targeting central modular functions, but also by controlling how these functions are co-regulated by targeting molecular bridges.Interestingly, when focused on the low-connectivity proteins, the analysis revealed that viral proteins significantly interact with bridging proteins, t Figure . BecauseIn silico simulation of molecular perturbations engendered by protein removal showed that the cellular network exhibits stronger robustness against deletion of bridging proteins as compared to hubs and central proteins removal . Based on the "guilty by association concept" [P-Value < 6.427 10e-12). Topological analysis also revealed that these essential host factors targeted either directly or indirectly by viruses exhibit a significant higher level of connectivity  and centrality  but similar values of bridging properties, as compared to not-essential host factors (NOT-EHFs), i.e. proteins that interact with at least one viral protein, but that was not identified as an EHFs by RNA genomic screens . This means that a human protein that directly interacts with a viral protein has roughly 2 times more chance to be involved in a human disease than human proteins not targeted by a virus . This proportion increased to 74,5%   when neighbourhood targeting was integrated in the analysis. This strongly suggests that targeted proteins and diseases are tightly connected at the molecular level either directly and/or indirectly. Conversely, 50%  of the diseases reported in OMIM were also found connected either directly or indirectly to at least one virus. Statistical enrichment analysis using OMIM ontology identified 34 diseases significantly linked to viruses, the majority of them being related to cancers  and neurodegenerative diseases . Interestingly, we also showed that the number of diseases associated to TP-Ns is partially correlated to the targeting intensity of these proteins by viral families .To decipher the molecular basis of viral infection pathophysiology, the OMIM annotations  of 1,729P-Value < 0.05). Interestingly, we have also shown that targeted DRPs exhibit significant higher bridging centrality as compared to not-targeted DRPs . This indicates that the viral attack on molecular bridges might participate at least in part to the molecular onset of human diseases.Network properties of proteins targeted directly or through their neighbourhood were also assessed in the context of the human diseasome. Surprisingly, DRPs directly and indirectly targeted exhibit significant higher bridging values than proteins not connected to any diseases  was designed Figure . In goods Figure . In a mos Figure , such ass Figure , left, cs Figure , right aP-Value < 1.0 10e-04) [The extended HIDN was next applied to decipher the molecular aetiology of autoimmune diseases for which viral infection is suspected to play an important role in interaction with genetic factors  Figure . A large 10e-04) . Moreove 10e-04) -27 (Addihttp://pbildb1.univ-lyon1.fr/virhostnet) and as a supplemental table  supported by at least two-independent experimental procedures. For the remaining 70% interactions, most of them come from large Y2H screens for which validation rates are closed to 80%, indicating the overall high-quality of our dataset and its robustness for meta-analysis study. This open resource can thus be used as a gold standard for the analysis of pathogen-host interactome but also for the investigation of viral aetiology of human diseases. This resource is available both throughout the VirHostNet website  and genome-wide association studies will allow deciphering of virus-host complex relationship as well as a better understanding of the role of viruses in the aetiology of some complex multi-factorial human diseases. The simplified model that is proposed here will have to be improved for instance by measuring how interactions between viral and host proteins are essentials for the virus or yet dispensable for the life of the host. It will be also of great interest to better formalise and quantify how do the viral proteins interact with the cellular proteins to completely abrogate the function of the host proteins and how often do they modulate their functions by hijacking the cellular proteins for their own needs.Comprehensive combination of virus-host interactome with other "-omics" data  concept, a new concept to pinpoint the functional importance of those proteins targeted by pathogens that will undoubtedly provide a rational for prioritizing new molecular susceptibility factors. Among the 38 "pris la main dans le sac" candidates indirectly interacting with disease related proteins, VISA is known to be involved in the production of IFN-β in an IFIH1-dependent pathway and could be crucial role in T1D susceptibility. Although too preliminary to draw general conclusions on the viral aetiology of autoimmunity, these observations nevertheless support the idea that a comprehensive analysis of virus-host protein-protein interactions and their immuno-modulatory consequences at the systems level is a powerful approach for the identification of susceptibility factors and new therapeutic strategies [The Human Infectome and Diseasome Network framework also helped us to revisit and complete Koch's postulates, initially developed to characterize microbial causation of infectious diseases, with the fresh eye of molecular systems virology . Indeed, for T1D -34. IFIHs of T1D . Interess of T1D . One nons of T1D ,38. Baserategies ,40.By merging the Human Infectome and Diseasome Networks, we provided the first bottom-up systems-level mapping of molecular associations between viral infection and human diseases based on virus-host physical protein-protein interactions. This association is consistent with the hypothesis that the onset and the pathogenesis of some chronic diseases, even mainly attributed to genetics, lifestyle and environment factors may also be modulated by infectious agents. The Human Infectome Diseasome Networks also provide an original platform and resource to rationally identify therapeutic targets.v; red) and host proteins  and two coloured edges types - virus-host  and host-host  protein-protein interactions ; with a finite series of graph vertices V defined in {Vh, Vv} and a set of graph edges E defined in {Evh, Ehh}. An integrative bottom-up strategy was used to reconstruct a high-quality prototype of HIN based on experimental protein-protein interactions data manually extracted from the literature and integrated from different public databases. Virus-host protein-protein interactions rely on a high-quality dataset manually curated and validated by a team of virology and molecular biology experts, as described in VirHostNet [http://pbildb1.univ-lyon1.fr/virhostnet).The Human Infectome Network (HIN) was modelled as a coloured graph. A coloured graph is a graph which has colours associated with each type of vertex or each type of edge. HIN is composed of two coloured vertex types - viral  percolation algorithm was performed on the cellular protein interaction network part of HIN to construct a network of interconnected cellular proteins' modules. More details on the methodology are available in Palla et al. [http://www.geneontology.org) was used to characterise the functional relevance of each module using functional enrichment approach based on exact Fisher testing and Benjamini and Hochberg multiple testing correction. The top biological process with significant enrichment  for more than half of the corresponding proteins was retained to attribute a function to a given module  for node v of interest, is defined by:The bridging coefficient is defined bywhere N(v) is the set of neighbours of the node v.al. to systematically quantify and compare the extent of network perturbations associated to nodes removal, in a simplified model of viral attack. Decrease of the largest component size was monitored as a function of the proportion of nodes removed and was compared for the different topological metrics  [We implemented the methodology developed by Jeong et trality) .The HIDN was mathematically formalized as a bipartite graph composed of two types of nodes corresponding to either diseases (black nodes) or viruses (coloured nodes). In HIDN, disease and viral nodes are connected by an edge if at least one protein related to a given disease is targeted by at least one protein encoded by this virus. Viral species and disease are connected by an edge if at least one disease related protein is directly targeted by at least one viral protein. The extended HIDN is a collapsed protein-protein representation of HIDN that includes the direct neighbours of the disease-related proteins that are targeted by viruses.P-value associated to multiple testing . A total of 20,219 non-redundant coding genes were used as the human proteome reference dataset for all the statistical enrichment tests.The Wilcoxon rank-sum test was used to statistically test for the differences between medians of variables not-normally distributed. The Wilcoxon rank-sum or Mann-Whitney U test is a non-parametric test which is used as an alternative to the two-sample Student's t-test, for assessing whether two independent samples of observations come from the same distribution. The Exact Fisher test was used to statistically test the differences observed between proportions and to test overrepresentation and functional enrichment. Benjamini and Hochberg correction was used to adjust DRPs: Disease Related Proteins; DRP-Ns: Disease Related Proteins Neighbourhoods; EHFs: Essential Host Factors; EHF-Ns: Essential Host Factors Neighbourhoods; HIN: Human Infectome Network; HIDN: Human Infectome-Diseasome Network; N-DRPs: Not Disease Related Proteins; N-EHFs: Not Essential Host Factors; N-TPs: Not Targeted Proteins; T1D: Type 1 Diabetes mellitus; TPs: Targeted Proteins; TP-N s: Targeted Proteins NeighbourhoodsVN designed, performed the study and drafted the manuscript. BDC was involved in data analysis. BDC and VL and CRC corrected the manuscript. All authors read and approved the final manuscript.Supplementary material. This file contains the supplementary material and data of the manuscript.Click here for fileVirus-host protein-protein interactions. The dataset of 2,099 virus-host protein-protein interactions.Click here for fileHuman protein-protein interactions (HPIN). The full and high-quality datasets of respectively 70,874 human and 36,144 human protein-protein interactions (HPIN).Click here for fileNetwork properties. Network properties with the full and the high-quality Human Protein Interaction Network.Click here for fileConnectivity and centrality probability distribution of targeted proteins (TP) within the human interactome part of HIN. P(kh) is the probability of a node to connect kh other nodes in the network. P(bh) is the probability of a node to have a centrality equal to bh in the network. Normalised log degree (top) and log centrality (bottom) distribution of not-targeted (blue) and targeted proteins (red). Solid lines represent linear regression fits. Vertical dashed lines give mean degree and centrality values. Each class is represented with conventional standard error.Click here for fileThe modular landscape of the Human Infectome Network. Virus-Host targeting profiles and Functional annotations. Each line represents a module of proteins (or a community) extracted from the HIN using CFinder algorithm. Each community is identified by a unique identification number (community_id) that is also used to describe the module in the SF1.tar.gz interactive network  of proteins forming the module, the number (n_inf) and proportion (prop_inf) of proteins targeted by viral proteins as well as the Exact Fisher test P-value  of the preferential targeting of the module are given. GO functional enrichment of each module has been computed and the gene ontology accession number (go_acc), its description (description), the proportion of protein in this module having the selected go term (see main text Methods) (go_prop_annot), the proportion of proteins of the modules being annotated by at least one term (go_n_annot) are given for both biological process and molecular function annotation.Click here for fileHuman Essential Host Factors (EHFs). The full dataset of 1,501 human Essential Host Factors (EHFs).Click here for fileDiseases Related Proteins (DRPs). The full dataset of Diseases Related Proteins and their associations with genetic diseases described in OMIM.Click here for fileT1DBase annotation of the 38 T1D candidates. Genomic Co-localization of the candidates with a susceptibility region to T1D (Genetic Confirmed Region) in either human, mouse or rat; expression in beta cells/Islets (Expression in Beta Cells/Islets) and manual curation by T1DBase (T1D Annotation) are annotated by Boolean ("1"). The number of publications with T1D candidate gene co-citation is also given (T1D Publications).Click here for file


Binge eating disorder is a prevalent adolescent disorder, associated with increased eating disorder and general psychopathology as well as an increased risk for overweight and obesity. As opposed to binge eating disorder in adults, there is a lack of validated psychological treatments for this condition in adolescents. The goal of this research project is therefore to determine the efficacy of age-adapted cognitive-behavioral therapy in adolescents with binge eating disorder – the gold standard treatment for adults with binge eating disorder.th or 5th Edition, will be centrally randomized to 4 months of cognitive-behavioral therapy (n = 30) or a waiting-list control condition (n = 30). Using an observer-blind design, patients are assessed at baseline, mid-treatment, post-treatment, and at 6- and 12-month follow-ups after the end of treatment. In 20 individual outpatient sessions, cognitive-behavioral therapy for adolescents focuses on eating behavior, body image, and stress; parents receive psychoeducation on these topics. Primary endpoint is the number of episodes with binge eating over the previous 28 days at post-treatment using a state-of-the art clinical interview. Secondary outcome measures address the specific eating disorder psychopathology, general psychopathology, mental comorbidity, self-esteem, quality of life, and body weight.In a single-center efficacy trial, 60 12- to 20-year-old adolescents meeting diagnostic criteria of binge eating disorder (full-syndrome or subthreshold) according to the Diagnostic and Statistical Manual of Mental Disorders 4This trial will allow us to determine the short- and long-term efficacy of cognitive-behavioral therapy in adolescent binge eating disorder, to determine cost-effectiveness, and to identify predictors of treatment outcome. Evidence will be gathered regarding whether this treatment will help to prevent excessive weight gain. If efficacy can be demonstrated, the results from this trial will enhance availability of evidence-based treatment of adolescent binge eating disorder.DRKS00000542German Clinical Trials Register:  Binge eating disorder (BED), a provisional category in the Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM-IV-TR) , was appSimilar to BED in adults, this disorder in adolescence is associated with increased eating disorder psychopathology  and general psychopathology , impaired quality of life, and overweight and obesity ,10,11. BOf great concern are findings that youth with binge eating or BED show increased weight gain and obesity onset over time ,28-31. GRelated to the recency of research about BED in adolescence, there is a lack of validated treatments for this age group. For adults with BED, cognitive-behavioral therapy (CBT) is the most well-established psychological treatment, as reflected in systematic reviews -39, metaAmong the few studies evaluating CBT or other psychological treatments for adolescents with recurrent binge eating, CBT was in a pilot randomized-controlled trial (RCT) compared with a delayed treatment control condition. This CBT trial was efficacious in yielding abstinence from recurrent binge eating over a three-month follow-up in 12- to 18-year-old adolescents (n = 26) . In addiIn this context, the aim of the Binge Eating Disorder in Adolescents (BEDA) study is to adapt an established CBT manual for adult BED to adolescent BED, and evaluate its short- and long-term efficacy, relative to a WL control condition in a single-center randomized trial. Additional objectives are to identify outcome predictors (moderators and mediators) and to evaluate cost-effectiveness.(1) Age-adapted individual CBT will be superior to WL in reducing the number of binge eating episodes in adolescents with BED 4 months following randomization.(2) Age-adapted individual CBT will be superior to WL in reducing the number of days with binge eating episodes; leading to greater abstinence from binge eating; reducing eating disorder psychopathology and the severity of depressive symptoms; increasing self-esteem and quality of life; and stabilizing body mass index. The therapeutic gains will occur at mid-treatment and will be maintained at 6 and 12 months following CBT.(3) Age adapted individual CBT will be cost-effective.BEDA is a single-center, prospective, randomized superiority trial, evaluating the efficacy and cost-effectiveness of CBT  compared to a WL control condition. The study design is depicted in Figure or BED according to DSM-5 based on objective and/or subjective episodes of binge eating or Subthreshold BED according to DSM-5. To ensure generalization of study results, exclusion criteria are kept to a minimum.A total of 60 12- to 20-year-old adolescents meeting age-adapted DSM criteria of BED will be randomized to either CBT (n = 30) or WL (n = 30). Inclusion criteria are summarized below. Based on the evidence on classification of BED in youth (see Introduction), age-adapted diagnostic criteria of BED are used in order to determine: BED according to DSM-IV-TR based on objective and/or subjective episodes of binge eating Inclusion:• Age 12–20 years• Diagnosis of BED according to age-adapted DSM criteria;• BED (DSM-IV-TR): At least 2 days with objective and/or subjective episodes of binge eating per week over the past 6 months; at least 3 out of 5 behavioral indicators; marked distress; absence of regular compensatory behaviors to avoid weight gain; absence of anorexia nervosa and BN;• BED (DSM-5): At least 1 episode of objective and/xor subjective episodes of binge eating per week over the past 3 months; at least 3 out of 5 behavioral indicators; marked distress; absence of regular compensatory behaviors to avoid weight gain; absence of anorexia nervosa and BN;• Subthreshold BED (DSM-5). All DSM-5 criteria for BED are met, except the binge eating occurs on average less than once a week and for less than 3 months.• Written informed consent of parent and adolescent for adolescents ages <18 years, written informed consent of adolescents at ages ≥18 years.Exclusion:• Current BN;• Current substance abuse;• Current suicidal ideation;• Psychotic disorder;• Bipolar disorder;• Serious unstable medical problems or conditions ;• Current intake of antipsychotic or weight-affecting drugs;• Current psychotherapy;• Inpatient psychiatric treatment within the past 3 months prior to screening;• Current conservative weight loss treatment;• Female adolescents in pregnancy or lactation;• Lack of compliance with major study procedures;• Current participation in another treatment trial.The study was approved by the Ethical Committee at the University of Leipzig Medical Center. Written informed consent of at least one parent and informed consent of the adolescent are obtained for adolescents <18 years after the study has been fully explained. For adolescents ≥18 years, written informed consent of the adolescent only is required. Patients and their parents can withdraw at any point without any penalty. The trial will be conducted in accordance to the guidelines for Good Clinical Practice . All perhttp://www.ess-stress.de), school-based recruitment , and recruitment in clinical settings .The ongoing study is conducted from September 2011 at the Outpatient Unit of the Integrated Research and Treatment Center (IFB) AdiposityDiseases at University of Leipzig Medical Center, Leipzig, Germany. Recruitment strategies include population-based recruitment  including familial factors ; and a greater focus on enhancing an autonomous motivation for treatment, since adolescents may not necessarily be motivated for treatment and/or treatment may be sought by third parties .All adolescent patients are guaranteed confidentiality regarding their parents unless they provide information that meets criteria for mandated reporting. Although family-based treatment is efficacious in adolescent BN , parentaThe age-adapted CBT manual includes the following phases: 1) initial treatment phase for motivational enhancement; (2) intensive treatment phase including modules on eating behavior, body image, and stress; and (3) self-management phase for relapse prevention. Therapeutic phases, goals, and techniques are depicted in Table  initial The age-adapted CBT with the adolescent lasts 4 months and comprises 20 individual face-to-face sessions (50 min) with the adolescent. Within the first month of treatment, two sessions per week are scheduled (sessions 1–8), and in months 2 to 4 one session per week is scheduled (sessions 9–20). CBT treatment is provided by at least three clinical psychologists with advanced training in behavior therapy and a specific training in CBT of BED. Treatment fidelity is ensured through a specific training in the CBT manual and regular supervision, which will also prevent drift in treatment delivery. Patients are allocated to therapists according to their availability, and each therapist will see eight or more patients. Treatment fidelity and therapist adherence will be established using continuous adherence controls based on audio documentation of the psychotherapeutic sessions by two independent, trained raters using five (25% of sessions) randomly-selected tapes per patient. The rating system is specific for CBT of eating disorders, and was developed and evaluated in an independent clinical study of CBT for adults with BED .Half of the patients will be randomly assigned to a WL control condition. All WL patients are guaranteed individual CBT after a waiting period of 4 months . The WL patients are instructed not to seek any other medical or psychological treatment for BED or obesity during the WL period without informing the project leader.The primary outcome measure is the number of binge eating episodes over the previous 28 days 4 months after randomization , which is a standard measure within treatment trials of adult BED . The num2). Body weight and height is objectively measured through calibrated instruments. Psychiatric comorbidity is determined per structured clinical interview K-DIPS [Secondary outcome measures include the number of binge eating episodes 2 months after randomization , and maintenance of treatment success regarding the number of binge eating episodes at 6 and 12 months following CBT . Additional secondary outcomes are also measured at 2 and 4 months after randomization and at 6 and 12 months following CBT: the number of days with binge eating; abstinence  from binge eating; eating disorder psychopathology ; additional aspects of non-normative eating behavior as determined through the Dutch Eating Behavior Questionnaire ,64; the w K-DIPS  6 and 12We chose these outcome measures because they exhibit good psychometric properties in the respective age group, are well-established in German, and are used in international research studies. The structured clinical interviews EDE and K-DIPS are conducted by trained raters. All raters are blind to study hypotheses and randomization, and have no therapeutic relationship with the patients. They underwent extensive training for conducting the interviews and receive ongoing supervision for standardized administration (drift prevention).Health resource utilization and loss of productivity is assessed using the Client Sociodemographic and Service Receipt Inventory , adaptedPotential predictor variables, assessed at baseline, consist of outcome variables, sociodemographic and anamnestic variables, and patient motivation and expectation ratings assessed through visual analogue scales. The predictor (moderator) variables will be selected based on the literature or on our theoretical understanding of the mode of action of the treatment.For exploratory identification of mediators, process measures are obtained by the patient every week of treatment. These consist of a selection of items of the self-report form of the EDE, the Eating Disorder Examination-Questionnaire -75.t-test (α = 0.05). With an assumed overall dropout rate of 10% of study patients (range: 0 to 11.5%) [RCTs analyzing the reduction of binge eating episodes by interpersonal psychotherapy , an Inteo 11.5%) ,48, 30 pAdolescents who meet the respective criteria and who give informed consent (and for adolescents aged <18 years whose parents give informed consent) are randomized. To ensure concealment of allocation, the randomization is centrally performed by the Coordination Center for Clinical Trials of the University of Leipzig. Randomization is computer-assisted, using Pocock’s minimization algorithm . The ranOutcome assessments are performed by independent raters who have no therapeutic relationship with the patients and are blind to study hypotheses and randomization. Blinding of patients and treatment is not possible in this trial, as both therapists and patients know the study arm from the particular modes of delivery.The primary outcome “reduction in the number of binge eating episodes over the previous 28 days 4 months after randomization” will be investigated with analysis of covariance using the ratio as the dependent variable (the number of binge eating episodes at 4 months following randomization divided by the number at baseline), treatment group as factor and the inverse number of binge eating episodes at baseline as a covariate based on the full analysis set . The fulSecondary outcome measures will be analyzed in an exploratory manner and will be evaluated by means of parametric or non-parametric tests, depending on the given scale level and type of distribution of the observed variables. Maintenance of treatment effects will be evaluated by random coefficient models. For all outcomes, effect sizes will be estimated and presented with 95% confidence intervals. Predictors of treatment outcome (moderators and mediators) will be identified in an exploratory manner using multivariable analyses.The trial is performed in cooperation with the Coordination Center for Clinical Trials of the University of Leipzig, which is responsible for monitoring and data management. Data are monitored for completeness, consistency, and plausibility. Errors in data entry are determined in a step-wise procedure, examining all data of five patients and, depending on error rates, examining all data in up to an additional 25% of the patients. Data quality is ensured through plausibility checks . Post-treatment data will be released only after study completion . No interim analyses are planned.The study data will be reported in accordance with the extended Consolidated Standards of Reporting Trials guidelines for non-pharmacological treatment studies .Adverse events are all unwanted medical events  occurring throughout the trial whether or not they have a causal association with the trial. Adverse events will be documented at every assessment and at every week of treatment throughout the trial. They are rated regarding their severity: serious adverse events are those having led to death, that are life-threatening, make inpatient treatment necessary, lead to sustained harm, or cause birth defects or deformities. Serious adverse events include mental or physical decompensations that indicate a need for hospitalization . Adverse events are recorded through a self-report assessment of somatic symptoms ,80 at evAn independent Data Monitoring and Safety Committee (DMSC) was established to meet once per year for independent study supervision. The DMSC is composed of three researchers with expertise in the area of the study. The type of information monitored includes patient recruitment, number of dropouts, and all adverse events including study withdrawals. Any serious adverse event will be immediately reported to the Ethical Committee at the University of Leipzig Medical Center and will be forwarded to the DMSC. The DMSC receives recruitment and retention updates on a regular basis.Recent evidence indicates that a substantial proportion of adolescents suffer from BED and associated psychopathology. Additionally, they are at risk of excessive weight gain, overweight, and obesity, with increased risk of associated medical sequelae. As the research interest in adolescent BED has been growing only recently, there is a lack of evidence-based psychological treatments for BED in this age group. One randomized pilot study demonstrated feasibility and suggested potential efficacy of a brief individual CBT for binge eating and associated psychopathology ; anotherIn this study, an evidence-supported CBT manual for adult BED was adapted for adolescents and is subjected to a confirmatory efficacy test comparing individual CBT to a WL control condition in a randomized psychotherapy trial with a follow-up period of one year. An extension of this long-term follow-up is planned, but will depend on organizational requirements after the end of the funding period. If efficacy can be demonstrated, the results from this trial will enhance availability and foster dissemination of evidence-based treatment of adolescent BED. Beyond the effects on BED and associated psychopathology, evidence will be gathered to determine if this treatment will also help to prevent excessive weight gain. The study will further allow for the identification of individuals for whom (moderators) CBT will work and allow for clarification of which mechanism (mediators) changes will be entailed. Such results would advance decisions for matching adolescents with BED into CBT, and may inform technical aspects of intervention conduct.This study is ongoing and will continue until April 2015.BED: Binge eating Disorder; BEDA: Binge eating disorder in adolescents; BN: Bulimia nervosa; CBT: Cognitive-behavioral therapy; DMSC: Data Safety and Monitoring Committee; DSM-IV-TR: Diagnostic and Statistical Manual of Mental Disorders Fourth Edition Text Revision; DSM-5: Diagnostic and Statistical Manual of Mental Disorders Fifth Edition; EDE: Eating Disorder Examination; IFB: Integrated Research and Treatment Center AdiposityDiseases; K-DIPS: Structured clinical interview Diagnostisches Interview bei Psychischen Störungen des Kindes- und Jugendalters; RCT: Randomized-controlled trial; WL: Waiting list.The author declares that she has no competing interests.AH, PhD, is a clinical psychologist, with a concentration on the psychological treatment of binge eating disorder and obesity in adults, adolescents, and children. She is Professor of Behavioral Medicine and Psychological Director of the Outpatient Unit of the Integrated Research and Treatment Center AdiposityDiseases at the Leipzig University Medical Center, Leipzig, Germany. She serves as the President of German Eating Disorders Society.