Sarcoidosis is a granulomatous multisystem disorder of unclear etiology that involves any organ, most commonly the lung and the lymph nodes. It is hypothesized that the disease derives from the interaction between single or multiple environmental factors and genetically determined host factors. Multiple potential etiologic agents for sarcoidosis have been proposed without any definitive demonstration of causality.We report the case of two patients, husband (57 years old) and wife (55 years old), both suffering from sarcoidosis. They underwent a lymph node biopsy by mediastinoscopy which showed a “granulomatous epithelioid giant cell non-necrotising chronic lymphadenitis”. They had lived up to 3 years ago in the country in a farm, in contact with organic dusts, animals such as dogs, chickens, rabbits, pigeons; now they have lived since about 3 years in an urban area where there are numerous chemical industries and stone quarries. The aim of this case report was to focus on environmental factors that might be related to the pathogenesis of the sarcoidosis. Sarcoidosis is a granulomatous multisystem disorder of unclear etiology that involves any organ, most commonly the lung and the intrathoracic lymph nodes. Also heart, skin, and central nervous system are frequently affected. The diagnosis of sarcoidosis is further hindered by the lack of any reliable and specific diagnostic test, as there are no laboratory or imaging findings that enable the definitive diagnosis. Even the histological data are not conclusive because the non-necrotising granuloma, a hallmark of morphologic appearance of the disease, is not unique for sarcoidosis. Granulomas typically consist of a compact central area of macrophages that differentiate into epithelioid cells and then fuse to form multinucleated giant cells surrounded by lymphocytes.Mycobacterium spp. being the most commonly implicated organism. The role of various non-infectious organic and inorganic environmental agents has been studied in the pathogenesis of sarcoidosis.A genetic predisposition to sarcoidosis is epidemiologically evidenced by the demonstration of familial aggregation, differences in disease susceptibility and severity between racial groups, and the significantly increased incidence in monozygotic twins of affected individuals compared to other siblings ,2. It isWe report the case of two patients, husband and wife who had lived up to 3 years ago in the open country, in contact with organic dusts (hay and molds), and animals such as dogs, chickens, rabbits, pigeons; now they live in an urban area where there are numerous chemicals industries and stone quarries.The woman, 55 years old, is a housewife and a former smoker of 1 pack/years. She underwent a hysterectomy for uterine fibromatosis, and a surgical operation for right humerus osteochondroma. She has got a herpes simplex virus type I infection. At the time of our first observation she referred in the last 2 years dry cough with little sputum, dyspnea grade II according to the American Thoracic Society (ATS) scale and mild fever. At the beginning of the symptoms she performed a chest x-ray which showed “interstitial thickening” and a microbiological sputum examination which was negative for tuberculosis and fungal infection. The general practitioner gave her a non-specific antibiotic therapy with low benefit and then a steroid therapy with improvement in symptoms. At the time of our observation, she underwent a total-body CT scan which showed “a series of lung nodules distributed in all lobes; a solid slab in the mantle of the apical segment of the left lower lobe; several small adenomegalies in the anterior mediastinum, around the trachea and below aorta artery; no evidence of effusion in the pleural cavity” in the thorax   was normal.The 6-min walking test was normal and the pulmonary function tests showed a reduction in the diffusing capacity for carbon monoxide (DLO) Table . The artCO  in severe obesity. As his wife, he has got a herpes simplex virus type I infection. He takes regular therapy with aspirin, doxazosin, allopurinol, irbesartan, erythropoietin and insulin. He referred dyspnea ATS grade II-III in the absence of cough. He also reported to have taken steroids in the past at the starting dose of 50 mg/day and gradually tapered. This therapy was recommended in the absence of a definitive diagnosis and on the basis of symptoms and radiological abnormalities of “interstitial thickening” present in the chest x-ray. At the time of our first observation, the pulmonary function tests revealed a mild restrictive pattern with reduction of DLThe environmental exposition of the two patients concerns chemical agents because of the industries near where they lived in the last years and because of the presence of a sand quarry. The industries in the area where they live include those involved in the processing of tires. Thus, factors such as the inorganic powder used in the rubber industry can play an active role in the pathogenesis of their disease. Other factors potentially involved could be silica, silicone and the fibers of the glass. Three recent longitudinal studies regarding the World Trade Center disaster have emphasized the role of airborne inorganic particulate exposure in sarcoidosis -6. TheseGeographic clustering of disease in the southern United States and in other parts of the world has promoted further speculations concerning weather, soil, and foliage in the pathogenesis of sarcoidosis . Past stInterestingly, other studies suggest the hypothesis of either a person-to-person transmission or a shared exposure to an environmental agent. In a well documented clustering that has occurred in the relatively isolated population of the Isle of Man, individuals with sarcoidosis were more likely to have had prior contact with an individual who had sarcoidosis, compared to controls (39.6% versus 1.1%). These contacts included neighbors, unrelated cohabitants, and co-workers . SimilarRhodococcussp[Finally, several observations suggest that sarcoidosis may be caused by an infectious agent. There are many known infectious agents that can cause granulomas that resemble those of sarcoidosis, including mycobacteria, herpes viruses, histoplasmosis, treponematosis, sporotrichosis, coccidioidomycosis, schistosomiasis, listeria, the agent of Whipple’s disease and ococcussp. BecauseBoth our patients have got labial herpes simplex virus and this could suggest a potential role of the infection in the development of the disease. Moreover, before moving to the urban area, the two patients lived in the countryside, reporting exposure to molds and hay. Finally, they were exposed to chemical agents derived from the industry of the tires. Among those, silica is a mix of modern tires and it is known that silica could be potentially involved in the pathogenesis of the disease. However, all the environmental factors that could be considered potential triggers of the disease are not sufficient by themselves to cause sarcoidosis and must interact with a particular immune response of the host. Therefore, the genetic predisposition of the patients plays a key role in the development and progression of the disease.In conclusion, this case report wants to focus on environmental factors that may be potential causative agents for sarcoidosis. Important tool in investigating patients with sarcoidosis is a complete medical history that includes occupational and environmental factors, lifestyle habits and any previous infection. It could be also useful to study and monitor the areas with the highest incidence of this disease.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.IL and IS made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; LF and FV were involved in drafting the manuscript or revising it critically for important intellectual content; SV gave final approval of the version to be published.


After 3 months, she presented with raised nodules on her knees and elbows. After 8 months, she developed painful subcutaneous nodules on her forearms. We diagnosed sarcoidosis with both plaques and subcutaneous nodules associated with combination therapy with peginterferon α-2a and ribavirin. Sarcoidosis with both plaques and subcutaneous nodules is very rare. The patient had a sustained Th2 response in the liver. And a sustained Th1 response occurred only in the skin. It is likely that, for this reason, sarcoidosis was localized to the skin, and the patient developed sarcoidosis with both plaques and subcutaneous nodules.A 64-year-old woman with hepatitis C was treated using combination therapy with peginterferon  Combination therapy with interferon (IFN) and ribavirin is the standard treatment for hepatitis C. Both IFN and ribavirin induce a T helper cell (Th) response. Sarcoidosis is a systemic granulomatous disease that is triggered by a Th1 response. IFN-induced sarcoidosis is well documented, and some cases of cutaneous sarcoidosis have been reported . In the α-2a 180 mg/week and ribavirin 600 mg/day. After 2 months, the peginterferon α-2a dose was reduced by 50 percent due to a decrease in neutrophilic leukocytes. The HCV RNA level decreased, and liver enzymes remained normal. Three months after combination therapy was initiated, the patient presented with several asymptomatic, raised, brawny red nodules on the knees and elbows.A 64-year-old woman with hepatitis C was started on a treatment regimen of ursodeoxycholic acid. Liver enzymes could be normalized, but a negative hepatitis C virus (HCV) ribonucleic acid (RNA) value could not be achieved. Therefore, the patient began combination therapy with peginterferonγ-glutamyltransferase (γ-GTP) 16 IU/mL. The angiotensin-converting enzyme (ACE) level was slightly elevated at 22.7 U/L, while the lysozyme level was normal at 4.8 μL/mL. A chest X-ray showed no active disease, and the cardiac electrogram showed no abnormality. The patient's eye examination was unremarkable. Based on these findings, a diagnosis of sarcoidosis with both plaques and subcutaneous nodules was made. Since we suspected that sarcoidosis was caused by peginterferon α-2a and ribavirin therapy, these drugs were discontinued, and ursodeoxycholic acid therapy was restarted at the original dose. Observation of the superficial and subcutaneous nodules continued, with no additional therapy. Gradually, these nodules disappeared, and the patient was pain free. Four months after stopping the combination therapy, no subcutaneous nodules were found on palpation, and no superficial nodules could be detected with the unaided eye. An MRI scan of the lesions of the knees and elbows showed no abnormality. In addition, the ACE level normalized at 19.7 U/L, and the lysozyme level decreased to 4.2 μL/mL. After several years, the patient has had no reactivation of sarcoidosis, and liver enzymes are maintained within normal limits by the administration of ursodeoxycholic acid. At first, the nodules were characterized by disappearances and reappearances. However, 7 months after starting combination therapy, the patient complained of pain in her knees and elbows. Nodules in these regions were clearly visible Figures  and 2. Xα is known to stimulate the Th1 cells, and those produce leukotrienes that seem to be responsible for the development of the granulomas. Ribavirin also enhances the Th1 response. Therefore, combination therapy with IFN-α and ribavirin is a trigger for sarcoidosis. Many cases of cutaneous sarcoidosis have been reported as a result of combination therapy for hepatitis C [Sarcoidosis is an autoimmune systemic disease characterized by the Th1 immune response. IFN-atitis C . HoweverThe recognition of sarcoidosis with both plaques and subcutaneous nodules was based on several factors. In the current case, the Th1 response was not enhanced in the liver, but the Th2 response improved. It has been reported that patients with chronic hepatitis demonstrate an enhanced Th2 response . In the α-2a has a long duration of action, with maximum plasma concentration (Tmax) occurring from 72 to 96 hours. Therapeutic blood levels of the drug are sustained for 168 hours. In addition, peginterferon α-2a is administered by subcutaneous injection. Thus, the drug was confined to the skin. It is likely that sarcoidosis was localized to the skin for this reason.Peginterferon This paper describes a rare case of sarcoidosis with both plaques and subcutaneous nodules. Th2 cells reached other organs through the bloodstream because of the sustained Th2 response in the liver. However, the skin is far removed from the liver. Thus, only the patient's skin showed an enhanced Th1 response.


Caenorhabditis elegans by combining computational predictions and experimentally derived TF–DNA interaction data. We found that first introns of C. elegans genes, particularly those for families enriched in long first introns, are more conserved in length, have more conserved predicted TF interactions and are bound by more TFs than other introns. We detected a significant positive correlation between first intron size and the number of TF interactions obtained from chromatin immunoprecipitation assays or determined by yeast one-hybrid assays. TFs that bind first introns are largely different from those binding promoters, suggesting that the different interactions are complementary rather than redundant. By combining first intron and promoter interactions, we found that genes that share a large fraction of TF interactions are more likely to be co-expressed than when only TF interactions with promoters are considered. Altogether, our data suggest that C. elegans gene regulation may be additive through the combined effects of multiple regulatory regions.Gene expression is controlled through the binding of transcription factors (TFs) to regulatory genomic regions. First introns are longer than other introns in multiple eukaryotic species and are under selective constraint. Here we explore the importance of first introns in TF binding in the nematode  Saccharomyces cerevisiae, where TFs bind promoters immediately upstream of the transcription start site (TSS)  to regulatory genomic regions such as promoters and enhancers. The regulation of transcription is relatively simple in single cell eukaryotes such as the yeast te (TSS) . However introns ,3. Enhan introns ,5. HowevCaenorhabditis elegans is a primary model system for the study of development, as well as physiological processes such as fat storage, dietary response and aging  or gene-centered approaches (DNA-to-protein) ,11. TF-cal stage . These sal stage ,13, theral stage . Furthert intron . HoweverC. elegans TFs  assays can be used to identify the repertoire of TFs that can bind to a DNA sequence of interest ,19. Collgans TFs . Y1H assC. elegans. However, with the exception of 43 TFs that have been profiled genome-wide by ChIP , the propensity of first introns to interact with TFs has been largely unexplored. Here, we use a combined experimental and computational approach to investigate TF binding to C. elegans (first) introns. We find that the first intron is generally longer than other introns, which is in agreement with previous observations , alternative start sites (i.e. two or more distinct first coding exons) and presence downstream in an operon . Genes that were negative for all three criteria were defined as ‘conventional genes’ (15 621 genes). Promoter regions were defined from the start codon to up to 2-kb upstream unless there was a neighboring gene closer, in which case the region was extended until the start codon or the 3′-end of the neighboring gene.Genomic sequences and annotations were downloaded from C. elegans conventional gene were compared to all coding exons of the C. briggsae ortholog to determine the homologous segment using tBlastx. Matches were tabulated and genes where contiguous one-to-one alignments could be accurately detected were used for downstream analyses. The analysis of length conservation was performed for genes that have an identical exon–intron structure  in C. elegans and C. briggsae (3404 genes) to be able to determine and compare length conservation for all introns. For length conservation analysis, only genes that have a first intron longer than 500 bp in at least one of the species were considered (942 genes).Gene orthology was based on InParanoid data . Individhttp://intermine.modencode.org/ on 29 April 2012. The midpoint of the ChIPped region was defined as the binding site. Gene fragments were analyzed individually to determine whether the TF ChIP peak midpoints were located between the start and end of the fragment.ChIP interaction data from the modENCODE Project  were dowC. elegans TFs that belong to a broad range of TF families. We used a threshold of 0.09 as previously determined   to scoretermined . A prediCaenorhabditis elegans conventional genes with first introns longer than 250 bp were analyzed for gene family enrichment (gene families defined according to InterPro) using DAVID Bioinformatics Functional Annotation tool  and 40–50 genes were randomly selected from each bin. The first introns of the selected genes were cloned as previously described for promoter fragments (C. elegans (N2) genomic DNA by polymerase chain reaction, cloned into a gateway entry vector and subsequently transferred into the HIS3 and LacZ Y1H reporter Destination vectors. The reporter constructs were integrated into the genome of S. cerevisiae (Y1H-aS2) to create Y1H bait strains as described (Supplementary Table S1. Yeast bait strains corresponding to gene promoters were generated using the promoterome resource (Supplementary Table S2). Interactions that were detected in two of four replicates were considered positive. Auto-active and low-confidence DNA baits (i.e. baits with non-uniform background reporter expression) (10 for first intron baits) were removed from further analysis.We selected the first introns of 227 resource . eY1H asSupplementary Table S1). Promoter interactions for the same homeodomain genes were obtained from Reece-Hoyes  database . For eacf is the probability density distribution, x is the number of interactions and 1/λ is the average number of interactions.The promoter and first intron in-degree (number of TF interactions per regulatory element) distributions were modeled using the exponential distribution as previously shown for promoters in several species ,21,32,33The promoter + first intron in-degree distribution was modeled with a hypo-exponential distribution . This diP = 0.145) and the first intron (λI = 0.3) in-degree distributions and used to determine the expected hypo-exponential distribution for the promoter + first intron in-degree.The λ values were estimated for the promoter . We named the remaining set of genes ‘conventional genes’ (76% of C. elegans protein-coding genes). As shown in C. elegans conventional genes are longer than other introns , confirming previous observations  and whose first intron is ≥500-bp long in at least one species (942 genes). Genes with different exon–intron structure were not included in the analysis to be able to compare intron length conservation between introns at different positions. Additionally, introns that are shorter than 500 bp in both species were excluded from the analysis, as length conservation in this case may only reflect a general tendency for worm introns to be short. Briefly, we divided the length of the longer of the two introns by that of the shorter one. When intron length is conserved, the intron length ratio is close to 1, whereas larger discrepancies in intron length result in values >1 (P < 0.01). To confirm that this observation is not specific to the C. elegans and C. briggsae comparison, we compared 100 randomly selected C. elegans genes with orthologs in C. remanei and observed a 28% increase in conservation of first intron length relative to the second intron (data not shown). These results suggest that there is evolutionary pressure to maintain the length of first introns, which points to a functionally important role such as the regulation of gene expression.If first introns are important for gene regulation, one would expect that the length of long first introns is more conserved than the length of long non-first introns, as they could potentially contain more TFBSs and would thus be more sensitive to deletions. To test this prediction, we compared the intron length ratio between conventional genes of two related nematode species, alues >1 B. By usiTo test the potential importance of the first intron in gene regulation, we used TF binding as a proxy. We first evaluated the frequency of TF interactions with different gene regions using ChIP data for 43 TFs obtained from the modENCODE Project . We founC. elegans TFs , with first introns longer than 500 bp having on average 30-fold more TF interactions than first introns shorter than 100 bp.First introns bind on average only 3.4% of the number of TFs compared with promoters as determined by ChIP assays A. HoweveC. elegans TFs and, in most cases, only in a single developmental stage and laboratory condition  in a high-throughput manner . Although this number is lower than the average number of TFs that bind gene promoters   , our resGiven that some first introns, in particular longer ones, bind a large number of TFs, we hypothesized that these introns may compensate for low TF binding to the cognate promoter. To test this hypothesis, we compared the number of ChIP interactions between promoters and first introns on a gene-by-gene basis C. We didWe reasoned that TF interactions with first introns could contribute to gene regulation in two ways. First, TFs that bind the first intron could be overlapping with those that bind promoters, resulting in redundancy and, as a consequence, a robust regulation of gene expression ,41. SecoP = 0.286). The same was true when only first intron interactions were considered (P = 0.466). However, when promoter and first intron interactions were combined, gene-pairs with a high PDI similarity were enriched for gene-pairs with medium and high co-expression (P = 0.0016). This observation provides further support for the notion that promoter and first intron interactions are not redundant, but rather may independently contribute to gene expression.Gene regulation is achieved by the combinatorial action of TFs. We hypothesized that if two genes share a large fraction of TFs binding to their regulatory regions, they are more likely to be co-expressed than two genes that have different interacting TFs. To test this hypothesis, we focused on 24 homeodomain genes, a gene family enriched in long first introns . We firstab-1 and ceh-27 have a relatively high co-expression correlation (0.43), although they have a low PDI similarity (0.17) if only promoter interactions are considered (ceh-83 and ceh-89 have a relatively low co-expression correlation (0.26), although they have a high PDI similarity (0.5) when considering only promoter interactions, but have a low PDI similarity (0.17) if first intron interactions are included  per promoter best follows an exponential distribution, with most promoters binding very few TFs and a few promoters binding a disproportionately large number of TFs  often occur in specific gene families. For instance, GPCR, the endoglin/CD105 protein family, homeodomain TFs and neurotransmitter-gated ion channels on average have long first introns . ImportaC. elegans and C. briggsae than the rest of the conventional genes . Interestingly, in mice, neuronal and developmental genes have been associated with the greatest enrichment of putatively functional noncoding sequences in first introns as defined by sequence constraint, suggesting a more complex regulation for this set of genes  grants  and a postdoctoral fellowship from the Pew Latin American Fellows Program (to J.I.F.B. in part). Funding for open access charge: NIH.Conflict of interest statement. None declared.


Increasing genome data show that introns, a hallmark of eukaryotes, already existed at a high density in the last common ancestor of extant eukaryotes. However, intron content is highly variable among species. The tempo of intron gains and losses has been irregular and several factors may explain why some genomes are intron-poor whereas other are intron-rich.We studied the dynamics of intron gains and losses in an α-amylase gene, whose product breaks down starch and other polysaccharides. It was transferred from an Actinobacterium to an ancestor of Agaricomycotina. This gene underwent further duplications in several species. The results indicate a high rate of intron insertions soon after the gene settled in the fungal genome. A number of these oldest introns, regularly scattered along the gene, remained conserved. Subsequent gains and losses were lineage dependent, with a majority of losses. Moreover, a few species exhibited a high number of both specific intron gains and losses in recent periods. There was little sequence conservation around insertion sites, then probably little information for splicing, whereas splicing sites, inside introns, showed typical and conserved patterns. There was little variation of intron size.Since most Basidiomycetes have intron-rich genomes and this richness was ancestral in Fungi, long before the transfer event, we suggest that the new gene was shaped to comply with requirements of the splicing machinery, such as short exon and intron sizes, in order to be correctly processed. Saccharomyces cerevisiae, Guillardia theta, Encephalitozoon cuniculi. Intron-rich species are often multicellular, for example vertebrates, the worm Caenorhabditis elegans, the fungus Phanerochaete chrysosporium, the sea squirt Ciona intestinalis. Intron-rich unicellular organisms also exist, like the green alga Chlamydomonas)us genes . In S. htronless  and in mtronless .The HGT α-amylase gene could be a suitable model for studying the evolution of information content around intron sites. However, we found a low level of information at positions −2 to +2 surrounding intron sites, contrasting with our results in animals , where tIn contrast, information at both 5' and 3' splicing sites was strong and typical of fungal introns , suggestAltogether, our data suggest that several features were important to confer to the transferred gene suitable characteristics regarding splicing efficiency: short introns; shortening the exons to a small size through multiple intron gains, although exon sizes were more variable than intron sizes; and rather regular intron spacing along the entire gene, perhaps for efficient nonsense mediated decay . It is nOur results need now to be generalized by investigating other genes recently transferred from bacteria, in both intron-rich and intron-poor genomes, in order to confirm whether introns colonized intronless genes rapidly, with a density related to the genome average.HGT: Horizontal gene transfer; CBM: Carbohydrate binding module; GH: Glycosyl hydrolase.The authors declare no competing interests.JLDL designed the study, acquired experimental and database data, analyzed data, and drafted the manuscript. MB brought expertise in Fungi, contributed material and phylogenetic data and analyzed and discussed data. SJ performed GH13 phylogeny, AHV contributed to discussion and manuscript writing, DC performed the datation study and contributed to discussion and manuscript writing. All authors read and approved the final manuscript.List of primers designed for detection of α-amylase genes orthologous to Phchr1|7087|. PCR conditions were: initial denaturation 94°C, 6 mn; denaturation 94°C, 25 s; annealing 58°C, 50s; elongation 72°C, 1 mn, 45 cycles using the Taq Gold polymerase (Applied Biosystems). Various combinations of forward and reverse primers were tried.Click here for fileGenBank or JGI accession numbers of sequences EF1α, RNA polymerase II LSU 1 and LSU2, used for datation estimates.Click here for fileAbbreviations used in Figure Click here for fileAlignment of the α-amylase protein sequences studied, built with MAFFT, showing the intron positions. Pink: phase 0 introns; green: phase 1 introns; blue: phase 2 introns. This alignment was used, without the N-terminal variable region , for gene tree reconstruction . The tree was rooted with two bacterial sequences. Abbreviations are given in Additional file Click here for fileReconciliation tree made from the gene tree (Additional file Click here for fileFungal species tree and divergence times estimated with BEAST using EF1α+LSU1+LSU2, with dates of divergence at nodes. Horizontal bars show the 95% highest posterior intervals of the divergence times.Click here for fileAnalysis of exon size distribution. For each gene, the effective number of exon was computed according to ref. 16. The statistical significance was estimated by 10,000 simulations. Ne: effective number of exons. ns: not significant; *: p<0.05. 5%: value of Ne below which are the smallest 5% simulated Ne values; 95%: value of Ne below which are the smallest 5% simulated Ne values.Click here for fileConsensus sequences at positions -2 and -1, and +1 and +2 around intron positions with different phases, drawn with Weblogo 3.2 . n is thClick here for fileConsensus splicing sites of introns drawn with Weblogo 3.2 . Left toClick here for file


The secretion of Interleukin-4 (IL4) is the characteristic of T-helper 2 responses. IL4 is a cytokine produced by CD4+ T cells in response to helminthes and other extracellular parasites. It has a critical role in guiding antibody class switching, hematopoiesis and inflammation, and the development of appropriate effector T-cell responses. In this study, it is the first time an attempt has been made to understand whether it is possible to predict IL4 inducing peptides. The data set used in this study comprises 904 experimentally validated IL4 inducing and 742 noninducing MHC class II binders. Our analysis revealed that certain types of residues are preferred at certain positions in IL4 inducing peptides. It was also observed that IL4 inducing and noninducing epitopes differ in compositional and motif pattern. Based on our analysis we developed classification models where the hybrid method of amino acid pairs and motif information performed the best with maximum accuracy of 75.76% and MCC of 0.51. These results indicate that it is possible to predict IL4 inducing peptides with reasonable precession. These models would be useful in designing the peptides that may induce desired Th2 response. Cellular immune response to a pathogen is mediated through the processing and presentation of antigen on the surface via major histocompatibility complex (MHC). The exogenous antigens are processed through lysosome and presented by MHC class II. The loaded peptide on MHC class II interacts with CD4+ T cells and a pattern of cytokine is synthesized and secreted. Depending upon the cytokines secreted, the T-helper cells polarize into diverse T-cell populations like Th1, Th2, Th17, or iTregs , cancer Although this identification demands experimentally validating the immune response generated by each antigen, this investigation is time-consuming, cumbersome, and expensive task since the possible antigens and corresponding fragments range in millions –11. ThusIn the past, several methods have been developed for identification of MHC class II binders that may activate T-helper cells. These T-helper cells induce different types of cytokines like IL-4 and IFN-gamma. Presently, available methods provide no information about the type of cytokine a MHC II binding peptide will induce. In order to address this issue, an attempt has been made to develop a method for predicting IL-4 inducing MHC II binding peptides.It is important to select the right dataset for developing a prediction method. The performance of the method is largely dependent on the datasets used for training a model. In this study datasets were generated from publicly available immune epitope database (IEDB) . We extrSince the main dataset includes only MHC class-II binders, the prediction algorithm (based on the above dataset) can only predict IL4 inducing peptides from MHC class-II binders. We are also interested in discriminating the IL4 inducing peptide from the random peptides. Thus, we created an alternate dataset for building prediction models that can be used for mapping IL4-inducing peptides in antigens. Our alternate dataset has random peptides as negative set instead of non-IL4-inducing peptides. We generated IL4 noninducing (negative examples), from SwissProt proteins.We first analyzed the IL4 inducing positive and noninducing negative sequences, to comprehend the preferred peptide length for both positive and negative peptides, by using R-package for creating boxplot . We alsoThe recognition of functional motifs in peptide or proteins constitutes an important element in functional annotation of sequences . In the We further explored 100 degenerate motifs using three kinds of classification: (i) none, (ii) Koolman-Rohm , and  using the following formula and a vector of dimension 20 is used to represent amino acid composition of a peptide:Likewise, the algorithm calculates dipeptide composition (DPC) and a vector of dimension 400, representing a peptide, using the following formula:Amino acids pairs (AAP) based method represents the input epitope by a vector of fixed vector length (400) by incorporating the information from each amino acid pair and their propensity in the given dataset. This approach had shown its potential in past for predicting B-cell epitope .Here, we converted positive and negative examples into binary codes, where each amino acid is represented by a vector of dimension 20 . Using these binary vectors, different peptides were represented, such as a 15-amino acid long peptide which is represented by a vector of dimension 300 (15 × 20).In this study, classification models have been developed using machine learning technique support vector machine (SVM). In order to implement or develop SVM models, we used software SVMlight . SVM modWe further employed a hybrid approach, where we combined the predictions from both motif and model based methods. In a hybrid approach, the weight of +1 was given to the peptide having IL4 motif (exclusively found in IL4 inducing peptides) and −1 was given to peptide having a non-IL4-motif (exclusively found in non-IL4-inducing peptides). We developed several hybrid models depending on the type of vector inputs used for SVM based prediction.In order to develop reliable prediction models, we trained and tested our models using fivefold cross-validation technique. In this analysis, the whole dataset is divided randomly into five equal parts, and each time four sets are used for training our models and remaining set is used for testing. This procedure is repeated five times so that each set is tested once and four times it is used for training. The final performance of the model is evaluated by averaging the performance of models on each set. The performance of models was measured using the following standard parameters, that is, sensitivity, specificity, accuracy, and Matthew's correlation coefficient (MCC).http://dx.doi.org/10.1155/2013/263952).In order to understand the properties of the IL4 inducers, we analyzed both IL4 inducer (IL4+) and noninducer (IL4−) MHC class II binding epitopes extracted from IEDB. There are several studies where authors have exploited physiochemical properties (PCPs) of peptides to discriminate one class of peptide from other classes . We examWe first compared the length of the two types of peptides and observed that average length of Il4 inducing and noninducing peptides is not significantly different. We could not find any significant relation between length of sequence and its potential to induce IL4 production .We also compared amino acid composition of IL4+ and IL4− peptides and found compositional biasness between two types of peptides. It was observed that residues E, F, K, and I are more abundant in IL4 inducing peptides, while IL4 noninducing sequences majorly include G, D, and L .We have analyzed the role of MHC alleles to skew the immune response to induce IL4 cytokine. The dataset that comprises 1759 epitopes was derived from 2845 IL4 assays and MHC alleles were undetermined in 1901 (66.8%) assays. The rest of assays (33.2%) were restricted to 91 MHC alleles. Out of these 91 alleles, 34 alleles were observed in IL4 positive as well as IL4 negative assays . We have also found 10 alleles for which IL4 assays were returned as exclusively negative and 47 alleles for which all the IL4 assays were resulted as exclusively positive . The mosThe amino acid compositional analysis described the overall dominant residues in IL4 inducing and noninducing peptides. However, this information does not specify the positional preference of specific amino acid residues at specific positions. In order to know the preference of a particular amino acid at different positions or at N- or C-terminals, we created the two-sample logo for our positive and negative IL4 peptides. Two-sample logo as depicted in In order to look at position specific proclivity of different PCPs as mentioned in the method section, we calculated the average of every PCP, at every position of N′ and C′ terminal as mentioned in the method section. For every PCP, we found various positions showing discriminating values in plot ; for example, 1, 2, 3, 5, and 12 positions of N′ terminal show high hydrophilicity in IL4+ peptides. Based on these observations we selected discriminating residues for PCP as mentioned in Table 1S. We selected some of the discriminating PCP and looked at the average of the sum of PCP of IL4+, IL4−, IL4 + Nt, IL4 − Nt, IL4 + Ct, and IL − Ct amino acid sequences  (see the Method section). We found hydrophilicity, pI, amphipathicity, steric, and charge properties, discriminating between IL4+ and IL4− data. On the other hand, the sum of PCP of selected residue positions (see the Method section) showed a significant difference in all the PCPs .We next tried to determine exclusive motifs or patterns in IL4 inducing peptides by using MERCI software. We used three types of classification, that is, none, Koolman-Rohm, and Betts-Russell, to determine 100 motifs in peptides. It was observed that Betts-Russell classification significantly discriminated 205 IL4 inducers from noninducers and Koolman-Rohm was significantly distinguished 150 non-IL4-inducers from IL4 inducers .Collectively, motifs generated from all types of classification discriminated 333 positive and 237 negative peptides. The best motifs generated from each classification are listed in We developed prediction models using SVM that is widely used in the past for classification models –30. In tWe further developed another SVM model based on binary profile of amino acids of the peptides, where a vector of dimension 20 represents each residue. The compositional variation plot for each residue in IL4 inducing and noninducing peptides and the performance of SVM model based on binary profile of N-/C-terminal residues were also analyzed (depicted in Table 3S).We adopted a hybrid approach for prediction of IL4 peptides by combining the prediction based on SVM model and motif search. The datasets were first sorted based on the exclusive motifs searched in positive and negative peptides using MERCI. The initial search identified 333 IL4 inducing and 237 IL4 noninducing MHC class II binding peptides, and these sequences were given weightage by adding +1 and −1, respectively, in SVM score in the hybrid method. Additionally, in this hybrid approach, we developed four different models using different input features each time . This teAll the models described above has been developed on main dataset that contain experimentally validated IL4 inducing and noninducing MHC class II binders. These models only can be used for predicting IL4 inducing peptides if users know that their query peptide is MHC class II binders. In order to provide service to the community we developed models on alternate dataset that can be used to discover IL4 peptides in proteins/antigens. As described in Materials and the Methods section our alternate dataset contains negative set/examples random peptide. We developed models on alternate dataset and achieved maximum accuracy of 70% .Performance on independent dataset is one of the best ways to validate a prediction model. As the IEDB database is continuously updated, we extracted the 71-peptide novel entries (deposited after extraction of our dataset) from IEDB for MHC class II binders for which IL4 assay was positive. Out of 71 peptides our best model correctly predicted 49 epitopes at default threshold.With the advent of the next generation sequencing techniques, the designing of rational vaccines based on the immunogenic features of peptides has become a need of the modern era. Identification of peptides or antigenic that can activate all arms of the immune system is important for designing effective immunotherapy or epitope/subunit vaccine. It means vaccine candidates (peptide/antigen) should have antigenic regions that can activate both B-cell and T-cell epitopes (MHC Class I or II peptides). The Th2 response is very important in vaccine or immunotherapy design against extracellular pathogen. IL4 is the principal cytokine that directs commitment of T cells to Th2 phenotype . TherefoWe extracted experimentally validated MHC class II binding peptides from IEDB with and without IL4 inducing potential. We initially analyzed both these datasets to select important features that could lay the basis for the IL4 inducing capability of the peptide. It is well documented that the binding of peptides to MHC complex is largely dependent on the length of the peptides ; thus weMHC alleles are well documented in literature for thier capability to skew the immune response –35. Our Distinction of different immune epitopes has already been reported with different PCPs in the past . In our Next, we tried to discriminate IL4 inducers and noninducers by use of machine learning technique. For analysis of positional feature of a sequence by SVM, binary patterns of the sequences were used as input. Since binary patterns of peptides could only be applied at a fixed length, we generated different binary inputs by varying the length of amino acids from 9 to 15 through both N and C terminals of a peptide. The performance of SVM model based on these inputs showed a MCC of 0.18. Further, we analyzed the residue and dipeptide compositional vector of IL4 inducer and noninducer sequences. It was observed that the models based on compositional profile performed better than models trained on binary patterns, possibly because this attribute of the sequence does not depend upon the length of the peptide as it has a fixed feature input of 20 and 400 for residue composition and dipeptide composition vector, respectively.We have also developed hybrid model, by employing the information from motif and machine learning. In the hybrid approach, the weightage has been given to sequences that could be predicted with exclusive motifs searched using MERCI. We observed that the performance was improved up to a value of MCC 0.51 using hybrid of MERCI and amino acid pair. This could be attributed to the role of propensity and exclusive motifs in prediction of IL4 inducing epitopes as it was also publicized for B-cell epitopes . The AAPin silico prediction method that can aid in understanding the IL4 inducing potential of the antigens in computer aided rational vaccine design for better control of diseases.Performance of our model on independent dataset was that 69% (49 out of 71) is satisfactory. This performance is comparable with 78.76% sensitivity at fivefold cross-validation on training dataset. In summary, we have developed the The tendency of an epitope to induce IL4 and skew the immune response towards Th2 makes it of great significance in immunotherapy and vaccine designing. Although the induction of IL4 response is a very complex issue that depends on a number of factors like cytokine milieu, MHC haplotype, the costimulatory molecules, and peptide itself, a peptide is an important factor that could be controlled easily while designing a vaccine or immunotherapy. However, Th2 response includes other cytokines like IL5 and IL10; we only focused on IL4 cytokine. Although the experimental evidence for Th2 peptides is limited, our computational analysis appears to support their existence.http://crdd.osdd.net/raghava/il4pred/).Keeping this limitation in mind, we have made an attempt to predict the peptides that may induce IL4 response. In this study we evaluate performance of our models using fivefold cross-validation as well as evaluating performance of our method on an independent dataset. It was observed that our model predicts IL4 inducing peptides with reasonable accuracy. In order to facilitate the scientific community working in the area of subunit vaccine, we have used the above models for developing a webserver IL4pred (We are providing three tables and four figures as supplementary document. In these three tables, we are providing the performance of SVM on our main dataset using features like physicochemical properties, compositional feature combined with length of peptide and binary profile of different window length. The figures in supplementary document represent the analysis of physiochemical properties and MHC alleles in our main dataset.Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are characterized by intraneuronal deposition of the nuclear TAR DNA-binding protein 43 (TDP-43) caused by unknown mechanisms. Here, we studied TDP-43 in primary neurons under different stress conditions and found that only proteasome inhibition by MG-132 or lactacystin could induce significant cytoplasmic accumulation of TDP-43, a histopathological hallmark in disease. This cytoplasmic accumulation was accompanied by phosphorylation, ubiquitination and aggregation of TDP-43, recapitulating major features of disease. Proteasome inhibition produced similar effects in both hippocampal and cortical neurons, as well as in immortalized motor neurons. To determine the contribution of TDP-43 to cell death, we reduced TDP-43 expression using small interfering RNA (siRNA), and found that reduced levels of TDP-43 dose-dependently rendered neurons more vulnerable to MG-132. Taken together, our data suggests a role for the proteasome in subcellular localization of TDP-43, and possibly in disease. Amyotrophic lateral sclerosis (ALS) [also referred to as Lou Gehrig's disease] is the most frequent form of motor neuron disease and is characterized by rapidly progressive degeneration of upper and lower motor neurons. ALS often leads to death within three years of diagnosis Histopathologically, FTLD can either present with inclusions consisting of fibrillary aggregates of the microtubule-associated protein tau, or inclusions which are tau-negative, but stain with antibodies to ubiquitin (FTLD-U) TARDBP on human chromosome 1. It was first identified as a binding partner of the human immunodeficiency virus TAR DNA element TDP-43 is an ubiquitous nuclear protein of 414 amino acids and 43 kD that is encoded by the gene TARDBP in familial and sporadic ALS TARDBP. In FTLD, where 40% of patients have a family history of neurological disease, other multiple chromosomal loci have been revealed MAPT gene were the first to be found in familial forms of FTLD-tau TARDBP have been reported in a number of patients with FTLD plus ALS PGRNVCPCHMP2BA pathogenic role for TDP-43 in disease is supported by the identification of mutations in Proteins that are targeted for degradation are enzymatically tagged with ubiquitin, which promotes disintegration by the proteasome machinery N-Methyl-D-aspartic acid (NMDA)), oxidative stress and mitochondrial damage (H2O2), proteasome inhibition  and ER stress (thapsigargin (TG), tunicamycin (TM)). Following treatment, the subcellular distribution of TDP-43 was determined immunocytochemically, complemented by Western blot analysis. Interestingly, only proteasome inhibition with MG-132 or lactacystin resulted in a pronounced change in TDP-43 localization with cytoplasmic accumulation, while TDP-43 remained primarily in nuclei of neurons exposed to staurosporine, NMDA, H2O2, TG or TM , while only MG-132 and lactacystin induced heat shock protein 70 (Hsp70) expression and widespread ubiquitination .Redistribution of TDP-43 from the nucleus to the cytoplasm is a characteristic feature found in neurons of brain and spinal cord sections from FTLD-TDP and ALS patients TG or TM Fig. 1B.in vitro (DIV) were treated with 5 µM MG-132 for 24 to 60 hours and analyzed by immunocytochemistry. While TDP-43 localized strictly to the nucleus in vehicle-treated controls, as observed by overlay with DAPI staining, proteasome inhibition resulted in staining of TDP-43 in the cytoplasm together with a reduction in nuclear TDP-43 staining . Similarly, TDP-43 was abundant in the nuclear fraction after 6 hours of incubation with MG-132. However, levels of nuclear TDP-43 progressively decreased after 12 hours of treatment with MG-132, and were undetectable after 60 hours . In parallel to the nuclear decrease, TDP-43 was increasingly recovered in cytoplasmic fractions until 12 hours of treatment with MG-132. Interestingly, TDP-43 then also progressively decreased in the cytoplasmic fraction after 24 and 36 hours, and was hardly detectable at 60 hours . Since there was no obvious difference in the intensity of cytoplasmic TDP-43 staining in MG-132 treated neurons between 16 and 36 hours or even later . Taken together, MG-132 treatment of cortical neurons resulted in cytoplasmic accumulation of TDP-43, which eventually became insoluble.Next, we addressed the subcellular distribution of TDP-43 in cortical neurons following MG-132 treatment. Nuclei of MG-132 and vehicle treated primary cortical neurons were separated from cytoplasmic fractions at different time points by sequential extraction en later Fig. 2D,, top). No changes in soluble TDP-43 were observed with vehicle treatment. Levels of insoluble 43 kD TDP-43 increased progressively, with highest levels observed after 36 hours . Western blots of the urea buffer fractions also reveal increased amounts of insoluble high molecular TDP-43 species together with lower molecular weight fragments. Hardly any insoluble TDP-43 was present in the vehicle treated cells. Consistent with impaired proteasome activity, treatment with MG-132 induced expression of Hsp70, which was readily detectable after 12 hours and thereafter . In parallel, it caused a progressive increase in insoluble TDP-43, together with the appearance of high molecular weight aggregates. In comparison, the amount of insoluble TDP-43 in vehicle treated controls was very low. In contrast to the primary neuronal cultures, insoluble 25 and 35kD low-molecular weight fragments were much more abundant in the NSC-34 cells. Similar to the primary neurons, treatment with MG-132 resulted in a progressive increase in Hsp70 expression levels after 12 hours, together with a high degree of protein ubiquitination consistent with proteasome inhibition . Next, we counted the number of viable NSC-34 cells during MG-132 treatment . Cell numbers were comparable with vehicle until 36 hours of MG-132 treatment, and significantly lower numbers were observed only 48 hours after the commencement MG-132 treatment, suggesting that TDP-43 insolubility precedes cell death.Neuronal cell lines have been instrumental in studying both physiological and pathological processes in neurodegeneration Fig. S2A). Consistent with TDP-43 phosphorylation, dephosphorylation with alkaline phosphatase (AP) of the insoluble fractions from MG-132 treated NSC-34 cells resulted in a molecular weight shift to 43 kD . Furthermore, immunoprecipitation with a TDP-43 antibody and subsequent Western blotting with an antibody to ubiquitin showed that TDP-43 was highly ubiquitinated following MG-132 treatment of NSC-34 cells . Taken together, MG-132 treatment of NSC-34 cells results in a progressive increase in the insolubility of TDP-43 associated with phosphorylation and ubiquitination, resulting in an expression pattern very similar to primary neurons and, more importantly, FTLD-TDP and ALS.In addition to the characteristic 43kD band, the insoluble fractions contained increasing amounts of a 45kD TDP-43 reactive product . Interestingly, in our experiments, neither excitotoxicity, caspase activation, oxidative stress nor ER stress induced noticeable accumulation of TDP-43 in the cytoplasm. Therefore, pronounced TDP-43 accumulation in the cytoplasm is, among the tested cell stressors, a unique feature of proteasome inhibition. This possibly reflects that the subcellular distribution of TDP-43 is regulated, at least in part, by the proteasome system. Interestingly, proteasome inhibition is known to change the subcellular localization of a range of proteins in processes like centrosome recruitment and nuclear translocation Apart from proteasome inhibitors, additional cell stressors have been used to reproduce features of TDP-43 pathology in different cell culture systems. For example, fragmentation of TDP-43 in cell culture has been induced by staurosporine TDP-43 has been found to functionally interact with both hnRNP A1 and hnRNP A2/B1 TARDBP mutations Proteasome dysfunction has been implicated in several neurodegenerative disorders, including ALS A common effect of treatments that lead to TDP-43 pathology in cell culture models (e.g. TDP-43 fragmentation or subcellular re-distribution) is that they also induce cell death, which is sometimes not taken into account. Hence, the question remains as to whether the induction of the TDP-43 pathology is a result of cell death (or pathways that co-induce cell death and TDP-43 pathology), or alternatively, that the TDP-43 pathology contributes to the cell toxicity. Also in our study, we cannot fully exclude that cell death is accompanied by TDP-43 re-localization, however, these changes were observed prior to detection of cellular toxicity. Furthermore, moderately reducing TDP-43 levels in primary neurons via siRNA increased their vulnerability to MG-132 treatment significantly, while only strong reduction of TDP-43 levels by siRNA induced cell death on its own. Knockdown of TDP-43 induces cell death in Neuro-2a cells possibly through Rho family GTPases In summary, we showed that proteasome inhibition by MG-132 in primary neurons and the NSC-34 motor neuron cell line is sufficient to induce cytoplasmic accumulation, aggregation, fragmentation and insolubility of TDP-43, bearing similarities to the TDP-43 pathology in FTLD-TDP and ALS. Reducing TDP-43 levels exacerbated the toxic effects of MG-132-induced proteasome inhibition in cultured neurons. Therefore, our data support a second hit hypothesis in addition to TDP-43 dysfunction in these diseases. Furthermore, our data suggest that the subcellular distribution of TDP-43 under physiological conditions is, at least in part, regulated by the proteasome system.Human brain samples were obtained from the Australian Brain Bank Network and the Department of Pathology of the University of Zurich, Switzerland, with approval from the Human Ethics Review Committee of the University of Sydney and the Zurich University Hospital Research Ethics Committee. Written consent from donors or donors next of kin for brain donation was received by each brain bank. Immunohistochemical staining was carried out as previously described 6 cortical cells were plated on 10 cm culture dishes. 2 hours after cells have attached, the plating medium was exchanged to Neurobasal medium supplemented with B-27 supplement and Glutamax .All animal experiments have been approved by the Animal Ethics Committee of the University of Sydney . Primary hippocampal and cortical cultures were prepared as previously described 6 NSC-34 cells were plated on 10cm culture dishes and maintained in medium containing high glucose DME  supplemented with 10% FBS , 2 mM L-glutamine and penicillin/streptomycin (Gibco).The immortalized motor neuron cell line NSC-34 was cultured as previously described 2O2, 1 µM NMDA and 50 nM staurosporine , the compounds were dissolved in either sterile water (H2O2 and NMDA) or DMSO (staurosporine) to yield stock solutions, and for all subsequent treatments further diluted in Neuralbasal/B-27 medium. Treatments with these particular compounds were added directly to the culture medium for 1 hour, after which the medium was removed, the cultures washed once with culture medium, and then replenished with fresh Neuralbasal/B-27 medium and were then continued to be cultured for the indicated amount of time. For vehicle controls, the according final dilution of DMSO was used.Primary hippocampal and cortical cultures were treated with 5 µM MG-132 , 10 µM lactacystin , 20 µM thapsigargin and 100 µg/mL tunicamycin (both Sigma) after 20 DIV for up to 60 hours. These compounds were dissolved in DMSO to yield stock solutions. For all subsequent treatments, the compounds and DMSO were further diluted in Neurobasal/B-27 medium and added to cultures to the indicated final concentrations. For treatment with 5 µM HPrior to treatment with MG-132, the NSC-34 cells were treated with Mitomycin C (Sigma) at a final concentration of 10 ug/mL for 2.5 hours to arrest cell division. As for primary cultures, stock solutions of MG-132 and DMSO were dissolved in culture medium and cells treated at the indicated concentrations. For cell counting, cell were washed once with pre-warmed PBS to remove any unattached cells, and stained with 0.4% trypan blue solution (Sigma).Once the cultures had been treated for the required amount of time, the cells were washed once with pre-warmed PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Fixed cells were permeabilized with 0.2% Triton X in PBS for 5 minutes and blocked for 1 hour in 3%BSA/2% heat-inactivated goat serum (Sigma) in PBS. Cells were stained overnight at 4°C with primary antibodies to: TDP-43 , phosphorylated TDP-43 Ser403/4  hnRNP A1 and hnRNP A2/B1 , neurofilament , MAP2  FUS  and β3 tubulin . Nuclei were visualized with DAPI . Secondary antibodies  were coupled with the fluorochromes Alexa Fluor 555 (mouse) and 488 (rabbit). All antibodies were diluted in blocking buffer. Dead cells were visualized by uptake of propidium iodide (Sigma) that had been added to the cell culture medium 5 min prior to fixation g for 5 minutes to isolate nuclei. The cytoplasmic supernatant was re-centrifuged at 500 g for 5 minutes to remove any contaminating nuclei. The nuclei pellets were resuspended in high salt buffer  and incubated on ice for 20 minutes. This homogenate was centrifuged at 14000 rpm for 10 minutes, at 4°C, to obtain the nuclear supernatant. The pellet was resuspended in urea buffer , sonicated at 30% amplitude and centrifuged at 14000 rpm. The supernatant obtained is the insoluble fraction.Subcellular fractionation of primary cortical cells (20 DIV) treated with MG-132 or DMSO was carried out as previously described 3VO4, 5 µM Pepstatin A, 1 mM DTT). The samples were then sonicated at 30% amplitude and centrifuged at 50,000 g for 60 min at 4°C. The supernatant was collected as the soluble fraction. The resulting pellets were resuspended in RIPA buffer, re-sonicated and re-centrifuged as above twice more to remove any contaminating proteins. Only supernatants from the first centrifugation were analyzed. The remaining pellets were resuspended in urea buffer, sonicated at 30% amplitude and centrifuged at 50,000 g for 60 min at 4°C. The supernatant obtained is the insoluble fraction.Proteins were extracted from both primary cortical neurons and NSC-34 cells as previously described 2O for 5 min, followed by 0.2 M NaOH for 10 min and dH2O for 5 min before being reprobed for GAPDH, α-actin and α-tubulin. Quantification of bands was performed using BioRad Quantity One 1-D analysis software v4.6 (BioRad).Protein measurements of all obtained fractions were determined by the Bradford assay. Protein extracts were analyzed by immunoblotting, which was performed as previously described IPs were carried out as previously described Protein samples were incubated with calf intestine AP  in the corresponding AP buffer (50 mM Tris-HCl (pH 9.0), 1 mM MgCl, and Complete Protease Inhibitor (Roche)) at 37°C for 2 hours while shaking. Once complete, sample buffer was added and the samples analysed by Western blotting.For lentiviral production, 293T human embryonic kidney cells were used to generate lentiviral packaging, whilst SH-SY5Y cells were used for tittering of viral stocks, as previously described Statistical analysis was performed with the Prism 5.0 software . All values are given as mean ± SEM.Figure S1A) Western blot analysis reveals similar levels of H2AX upon all treatments: vehicle (DMSO), NMDA (1 µM), H2O2 (5 µM), staurosporine (50 nM), MG-132 (5 µM), lactacystin (10 µM), thapsigargin  and tunicamycin . While H2AX is not phosphorylated in vehicle-treated neurons, all other treatments caused a similar degree of phosphorylation of H2AX, a non-specific marker of cell death, at chosen doses. (B) Only MG-132 and lactacystin induce expression of Hsp 70 and high levels of ubiquitination, indicating proteasome dysfunction.Effects of different treatments on primary neurons. ((TIF)Click here for additional data file.Figure S2A) RIPA and subsequent urea extraction of proteins from MG-132 (5µM) and vehicle (DMSO) treated NSC-34 cells. TDP-43 (arrowhead) and its fragments (*) reduce in the RIPA-soluble fractions over 48 hours, whereas TDP-43 is unchanged in vehicle treated controls. Note the presence of soluble TDP-43 fragments in controls. In parallel, TDP-43 increased in the insoluble (Urea) fractions of MG-132 treated neurons together with fragments (*), a distinct 45kD species (§) and high molecular weight aggregates (#). (B) Consistent with proteasome inhibition by MG-132, levels of the heat-shock protein 70 (Hsp70) increases with time. (C) Similarly, the amount of insoluble highly ubiquitinated proteins increased progressively upon MG-132 treatment. Representative Western blots from three experiments are shown. (D) NSC-34 cell viability during proteasome inhibition. The number of attached viable NSC-34 cells was similar upon vehicle (DMSO) and MG-132 treatment up to 36 hours, while a significant decrease became only obvious after 48 hours . (E) Phosphorylation of insoluble TDP-43 from MG-132-treated NSC-34 cells as shown by molecular weight shift upon dephosphorylation with alkaline phosphatase (AP). Accordingly, TDP-43-reactive bands of approximately 45kD (§) and the high molecular weight smear (#) collapsed to 43kD. (F) Immunoprecipitation (IP) with an antibody to TDP-43 and subsequent detection with an antibody to ubiquitin shows the high degree of ubiquitination of the high molecular weight TDP-43 reactive species (#). Note the 50kD Fc-antibody band (α).Progressive insolubility of TDP-43 upon proteasome inhibition in the immortalized motor neuron cell line, NSC-34. ((TIF)Click here for additional data file.


Integrating knowledge from across the natural and social sciences is necessary to effectively address societal tradeoffs between human use of biological diversity and its preservation. Collaborative processes can change the ways decision makers think about scientific evidence, enhance levels of mutual trust and credibility, and advance the conservation policy discourse. Canada has responsibility for a large fraction of some major ecosystems, such as boreal forests, Arctic tundra, wetlands, and temperate and Arctic oceans. Stressors to biological diversity within these ecosystems arise from activities of the country's resource-based economy, as well as external drivers of environmental change. Effective management is complicated by incongruence between ecological and political boundaries and conflicting perspectives on social and economic goals. Many knowledge gaps about stressors and their management might be reduced through targeted, timely research. We identify 40 questions that, if addressed or answered, would advance research that has a high probability of supporting development of effective policies and management strategies for species, ecosystems, and ecological processes in Canada. A total of 396 candidate questions drawn from natural and social science disciplines were contributed by individuals with diverse organizational affiliations. These were collaboratively winnowed to 40 by our team of collaborators. The questions emphasize understanding ecosystems, the effects and mitigation of climate change, coordinating governance and management efforts across multiple jurisdictions, and examining relations between conservation policy and the social and economic well-being of Aboriginal peoples. The questions we identified provide potential links between evidence from the conservation sciences and formulation of policies for conservation and resource management. Our collaborative process of communication and engagement between scientists and decision makers for generating and prioritizing research questions at a national level could be a model for similar efforts beyond Canada.Generación de Preguntas de Investigación Prioritarias para Informar a las Políticas y Gestión de la Conservación a Nivel Nacional Although we recognize the benefits of ongoing curiosity-driven research, we believe there is clear value in aligning research in the natural and social sciences more closely with the information needs of decision makers. Scientific research can inform decisions regarding domestic environmental protection and enhance a country's ability to contribute to international solutions to global environmental challenges . In CanaTargeted research can have instrumental effects , which dRecent projects have been undertaken at the global  and natibiodiversity, defined here as “the variety of genes, species and ecosystems and the ecological processes that allow them to evolve and adapt to a changing world” , environmental nongovernmental organizations (n= 3), industry associations (n= 2), policy analysis or advisory organizations (n= 6), and academia (n= 7). The core team of eight organizers and four student assistants brought the total number of participants to 40. Twenty-eight of the participants attended a workshop in Ottawa, the national capitol, in April 2010. Although provincial and municipal governments also have substantive, often paramount, responsibilities for ecosystem management, involving participants from a full range of regional government organizations was not feasible logistically. Academic participants were drawn from across the country and many team members had extensive regional policy experience.A core team of eight organizers (the eight lead authors) designed and implemented the project. A larger group—who participated in solicitation and synthesis of questions—included policy advisors and decision makers in governments and nongovernmental organizations  and academics with expertise in conservation science. Of 57 invitees to that group, 43 responded and 28 accepted. The final group of participants was drawn from federal government agencies with national responsibilities . Questions were sought from policymakers and their advisors in government, policy specialists in the nongovernmental and private sectors, Aboriginal leaders and science advisors, researchers in the natural and social sciences, and the philanthropic community. We used a bilingual (English and French) website established for this exercise to collect questions over 5 weeks.yes, no, or it depends response, (5) contain a subject of the intervention, an intervention, and a measurable effect if related to interventions and effects, and (6) increase the effectiveness of policy about, and management of, resource use and biodiversity in the face of environmental stressors if answered.Instructions for individuals who wished to submit questions were similar to those for other priority-setting exercises , although policy applications were stressed. Individuals were asked, “What research question, if answered, would substantially advance the development of effective policies and management strategies for species, ecosystems, and ecological processes in Canada?” Aspirational criteria for questions were that they (1) be answerable through a realistic research design, (2) be answerable on the basis of facts rather than value judgments, (3) be of a spatial and temporal scale that could be addressed realistically by a research team or program, (4) not be answerable simply with a A total of 396 questions were received in 271 submissions. Questions were submitted by individuals with diverse organizational affiliations . Over 62% (159 of 255) of individuals who voluntarily provided background professional information had more than 10 years experience in natural resource management. Core team members culled candidate questions that failed to meet the aspirational criteria and combined and refined questions to avoid substantial redundancies. This process reduced the list to 242 questions for participants to consider in the workshop. All questions are included in the Supporting Information.At the workshop, participants selected the top 40 questions from those submitted through a process developed for similar priority-setting exercises in the United Kingdom and United States . The workshop opened with a plenary session that included participant introductions, an overview of how participants were selected, highlights from recent exercises, an explanation of the policy emphasis of this exercise, and a review of workshop processes and ground rules. The balance of the day was spent in three breakout sessions (75–90 minutes each) composed of three concurrent groups per session. Each breakout group discussed one loosely themed set of 25–30 candidate questions, narrowing the set to four candidates for the top 40 and two alternates. Participants in breakout groups sometimes combined candidate questions, developed alternate questions that captured key ideas from candidate questions, and referred to the original list of 396 questions. The 54 questions that resulted from the first day  were discussed during a plenary session on the second day, during which the top 40 were selected by consensus.After the workshop, the core team and participants, including those individuals unable to attend the workshop in person, refined the questions by consensus-based email dialogue. During the refinement phase, it became apparent that some questions were quite similar. Combining three pairs of questions allowed us to include other candidates that were not on the initial list of 40. Participation of scientists helped ensure that questions were sufficiently narrow to form the basis of a research program. Participation of policymakers helped ensure that the final questions were relevant.We grouped the top 40 questions into themes: ecosystem structure and function, land cover and habitats, populations and species, resource-based industries, parks and protected areas, environmental change, environmental values, economic benefits and costs, individual and community well-being, adaptive management, and policy and governance. The organization of these themes was drawn from the adaptive-management logic framework outlined in Ecosystem attributes that provide benefits to humans  are increasingly a focus of biodiversity policy . The alt1. To what extent can ecological function and the supply of ecosystem services be predicted on the basis of ecosystem composition and structure?2. How does managing ecosystems for particular ecological functions or services affect other elements of biodiversity?3. How can transition points or thresholds among ecosystem states best be identified and predicted?Current approaches to ecosystem management seek to link land-cover types, species’ habitats, and ecosystem stability and services in part to understand how natural and human-induced changes in land cover might affect ecological, social, and economic well-being e.g., .4. How are populations of animals and plants affected by different configurations of land cover?5. At what spatial and temporal scales are management or policy interventions most likely to achieve various objectives for conservation of land cover?6. What ecological principles and information standards employed in the identification of critical habitat under the Species at Risk Act are most likely to contribute to species recovery?7. To what extent can habitat restoration or rehabilitation compensate for loss of quantity or quality of existing species’ habitat?Many ecosystem functions and services depend on the maintenance of particular populations or species e.g., . Populat8. What investments and management interventions will be required to maintain or increase the abundance of harvested populations when harvesting is one of multiple stressors acting on those populations?9. What are the cumulative demographic and genetic effects of harvest on target and nontarget populations and species?10. How do transboundary migrations of terrestrial and aquatic animals affect efforts to manage populations of those species?Natural-resource industries account for a substantial portion of Canada's economic activity . Where past policies have failed to effectively manage biodiversity there have sometimes been enormous economic and social impacts. For example, collapse of the Newfoundland cod fishery led, in 1992, to the largest single layoff of workers in Canadian history . Policy 11. What are the potential effects of large-scale energy exploration, development, and extraction projects on ecosystems and species?12. How does management of terrestrial and aquatic food-production systems affect species, ecological functions and services, and economic benefits?13. What are the magnitude and extent of effects of terrestrial resource-development activities on aquatic ecosystems?14. How do extractive and nonextractive industrial activities affect ecological functions and biodiversity in aquatic ecosystems?Canada has an extensive system of terrestrial parks and other protected areas  and a growing system of freshwater and marine parks for which federal, provincial, territorial, and regional governments all have responsibilities. Parks are managed to meet different objectives and mandates to protect species and ecosystems, and provide recreational opportunities that sometimes conflict. There are gaps in knowledge of how networks of parks can and should be designed, connected, and managed to meet ecological, social, cultural, and economic objectives.15. To what extent can species be maintained over the long term within existing parks and protected areas in the absence of proactive, external management?16. What are the ecological consequences of existing human activities in parks and their implications for future management?17. What are the impacts of alternative configurations of, and management strategies for, aquatic reserves on ecological function, species, and social well-being?18. What are the effects of different configurations of, and management strategies for, protected areas on biological diversity across landscapes and beyond the boundaries of terrestrial protected areas?Canada is already affected by climate change, especially in northern regions . Underst19. How is climate change likely to affect the abundance, rates of growth, and distribution of and interactions among populations?20. How can undesirable ecological, social, and economic effects of future changes in freshwater availability be reduced or mitigated?21. How will northern coastal ecosystems respond to changes in climate and industrial activity if reduction in ice cover increases human access to those ecosystems?22. What policies and management strategies most effectively mitigate the undesirable effects of non-native invasive species, potential disease vectors, and emerging pathogens?23. What are the effects of different policies and management strategies on the capacities of humans and other species to adapt to environmental change?Environmental values are influenced by demographic, cultural, experiential, and educational variables . They ca24. How does the integration of conservation science into public education influence environmental values, ecological literacy, and behaviors that affect the environment?25. How does urbanization and immigration affect environmental values, behavior, and conservation outcomes?26. How do individual and societal values about collective responsibility for common- pool resources affect private conservation initiatives and stewardship efforts?International agencies, governments, and researchers have highlighted the economic value of biodiversity. Accounting for the full costs and benefits of ecological change is central for credible economic analyses . The way27. What are the true direct and indirect contributions of ecosystems to economic well-being and how are these benefits likely to change in the future?28. What are the comparative ecological, economic, and social impacts of economic instruments versus input or output controls for ecosystem management?29. What are the benefits and costs of horizontally and vertically integrating policies and regulations within and across different policy domains such as environment, health, and trade?Policymakers are concerned with aspects of individual, community, and societal well-being beyond economics. Environmental change affects human health e.g., , communi30. How do terrestrial and aquatic conservation policies directly or indirectly affect human health?31. What are the effects of conservation initiatives on the health, livelihoods, traditional cultures, and identities of Aboriginal peoples?32. What are the effects of different strategies for building community capacity on levels of citizen engagement in environmental stewardship, restoration, and conservation?Canadian governments are committed to working together with citizens using “an ecosystem and adaptive management approach to achieve shared outcomes” . Ecosyst33. How can local and traditional knowledge be most effectively communicated and synthesized with scientific knowledge to inform conservation science and management?34. What forms of scientific evidence, risk assessment, and knowledge transfer most effectively and increase the probability of achieving ecosystem-management objectives?35. What monitoring methods can effectively and efficiently assess long-term, incremental, and cumulative environmental changes?36. What are appropriate indicators to evaluate the effects of public, private, and nongovernmental-sector policies, programs, and initiatives on biodiversity?Canada is a parliamentary democracy with a complex federal structure and diverse citizens who expect to participate in collective decisions. This situation complicates the formation and implementation of national evidence-based and results-oriented policy. It is difficult to develop consistent policies across jurisdictions and to determine the most-appropriate level of governance to address a given environmental problem. It is also difficult to balance the legitimacy of governance (a function of structure and inclusive processes) with explicit action to conserve biodiversity as required by laws, international commitments, and moral values.37. What factors influence the likelihood of compliance with environmental legislation and regulations?38. How do policy, legal, or institutional arrangements shape the effectiveness of integrated management for terrestrial watersheds and adjacent coastal environments?39. How do the actions of Aboriginal governments and organizations influence conservation strategies and the assignment of responsibilities for environmental governance in Canada?40. How can environmental laws, policies, and regulations be integrated and harmonized across multiple jurisdictions and sectors to achieve conservation objectives?Answers to questions about conservation policy can be informed by scientific evidence about the ecological, social, and economic effects of governance interventions. The Some of the themes identified in this Canadian exercise coincide closely with those identified in the United States . Relative to the United States, however, there was less emphasis on environmental stressors (27% vs. 55% of the 40 questions) and more emphasis on governance and management issues (67% vs. 32% of the 40 questions). In Canada there was strong emphasis on the effect of aboriginal governance on federal and provincial conservation policy and, conversely, on the effect of governmental conservation policy on individual and community well-being for Canada's Aboriginal population.We suspect that the contrasting priorities within North America arise from cultural and political differences . In other fields of study, political context has been identified as an important factor influencing the effect of research , so it wThe questions we identified provide potential links between evidence from the conservation sciences and the formulation of environmental policies for conservation and resource management. Integrating knowledge from across the natural and social sciences is necessary to effectively address societal tradeoffs between use and conservation of biodiversity. The collaborative process of communication and engagement between scientists and decision makers can change the ways that decision makers think about evidence, enhance levels of mutual trust and credibility, and potentially help advance the conservation policy discourse in Canada and internationally.


In this work, we synthesized graphene oxide from silk cocoon embarking its new dimension as a magnetic fluorophore when compared with its present technical status, which at best is for extracting silk as a biomaterial for tissue engineering applications. We produced graphene oxide by pyrolysing the silk cocoon in an inert atmosphere. The collected raw carbon is oxidized by nitric acid that readily produces multilayer graphene oxide with nano carbon particulates. Structural properties of the graphene oxide were analyzed using scanning electron microscopy, transmission electron microscopy, Fourier transform infra-red spectroscopy, and Raman spectroscopy. The oxidized sample shows remarkable fluorescence, multi-photon imaging and magnetic properties. On increasing the excitation wavelength, the fluorescence emission intensity of the graphene oxide also increases and found maximum emission at 380 nm excitation wavelength. On studying the two photon absorption (TPA) property of aqueous graphene oxide using Z-scan technique, we found significant TPA activity at near infrared wavelength. In addition, the graphene oxide shows ferromagnetic behavior at room temperature. The observed fluorescence and magnetic property were attributed to the defects caused in the graphene oxide structure by introducing oxygen containing hydrophilic groups during the oxidation process. Previously silk cocoon has been used extensively in deriving silk-based tissue engineering materials and as gas filter. Here we show a novel application of silk cocoon by synthesizing graphene oxide based magnetic-fluorophore for bio-imaging applications.The online version of this article (doi:10.1007/s13205-013-0128-2) contains supplementary material, which is available to authorized users. Carbon, the sixth most abundant element, exhibits the unique property of forming a wide variety of structures and allotropes. Because pre-historic times, only two allotropes of carbon are known widely: graphite and diamond. However, provoked by the discovery of zero-dimensional allotrope (0D) fullerenes by Kroto et al. , there isp2 bonded carbon atoms. It is the “thinnest material” that is stable in free form  and potassium chlorate (KClO3) resulting in brilliantly transparent yellow pallets (Brodie 4) and potassium chlorate (KClO3) by dissolving them in concentrated sulfuric acid (H2SO4) , transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. The synthesized graphene oxide also exhibit excellent fluorescence property and two-photon activity at near-IR (NIR) excitations (800 nm), thus making it a very strong candidate for two-photon microscopy applications. Magnetic property measurements of graphene oxide showed ferromagnetic behavior at room temperature indicating its excellent potential in magneto-optic studies also.All chemical reagents were of analytical grade, and used without further purification and Tasar silk cocoon were collected from the state of Chhattisgarh in India as previously described  for 24 h. Then, the acid was decanted off and the brown mass was thoroughly washed with distilled water till it was free from acid and becomes neutral. The brown residue was finally dried in vacuum and subjected for further analysis.The collected cocoons were grounded in mortar and pestle. Three grams of grounded Tasar silk cocoon was pyrolysed in a muffle furnace at 400 °C for 2 h in argon (Ar) atmosphere. This resulted in the formation of black solid which was collected at room temperature. The powdered raw carbon was taken in a thimble made of Whatman filter paper. Then, this raw carbonized cocoon was subjected to soxhlet purification. Continuous washing of raw carbon powder were done by petroleum ether followed by acetone. The raw carbonized cocoon was oxidized by treating with concentrated nitric acid (HNOMorphology of both the raw carbonized cocoon (containing graphene) and oxidized carbonized cocoon (containing graphene oxide) were characterized using SUPRA 40VP field emission scanning electron microscope (FESEM) equipped with energy dispersive X-ray (EDX) facility (Carl Zeiss NTS GmbH). To analyze the soxhlet purified raw carbonized cocoon (containing graphene), 3 mg of sample was taken in 4 ml isopropanol solution and sonicated for 30 min. 50 μl of the sonicated solution was placed on brass stubs and vacuum dried. For analyzing graphene oxide, 3 mg sample was taken in 4 ml isopropanol and sonicated for 3 min. 50 μl of the sonicated solution was placed on brass stubs and vacuum dried and subjected to SEM analysis.TEM was performed using Tecnai 20 G2 300 kV, STWIN model. An acceleration voltage of 200 kV was used for TEM analysis. Samples for TEM analysis were prepared by dropping 5 μl of the re-dispersed raw and oxidized carbon on to a carbon-coated copper TEM grid with a mess size of 400.FT-IR spectra were recorded using Bruker Vertex 70 FT-IR spectrophotometer using KBr disc.After Raman spectra were recorded using a Raman spectrometer (WITEC model) with 514 nm excitation.Fluorescence microscopy was performed using Leica Microsystems DM 2500 microscope.Fluorescence spectra of graphene oxide were obtained using Perkin Elmer LS 55 fluorescence spectrometer with peltier temperature programmer (PTP-1). The fluorescence excitation spectrum was recorded at the highest emission wave length of 460 nm.−1 energy and 150 fs pulse width was focused on to the sample with a 20 cm focal lens. Across the focal plane of this lens, a 1-mm path length cuvette containing the clear aqueous sample solution was scanned in the laser propagation direction [so is the name Z scan  shows the SEM images of purified raw carbon and oxidized carbon. The raw carbon contains layers of sheets with carbon particulates and amorphous carbon. These amorphous carbons were removed during oxidation process and layered graphene oxide is separated out.TEM image of oxidized carbon shown in Fig. FT-IR spectroscopy was performed to study the presence of different functional groups. FT-IR spectra of graphene oxide in Fig. sp2 and sp3 hybridized forms of carbon in the graphene oxide . The D-band occurs due to the number of defects and broken symmetry of basal plane of graphitized carbon atom. The G-band corresponds to the Eg vibrational mode of sp2 hybridized carbon atoms in both rings and chain structures. The Raman spectrum of the oxidized soot is shown in Fig. −1. The first one is associated with the stretching vibrations of sp2 hybridized carbon atoms and the second one is attributed to the disorder-induced stretching modes of graphite. The D-line of oxidized soot has almost the same intensity as that of the G-line when compared with the insoluble raw soot residue  at the zigzag sites was one of the possible explanations for the fluorescent emission . Radiative recombination of this e–h pair in these localized sp2 clusters give rise to fluorescence property  of fluorescent excitation study of graphene oxide are ~0.78 eV (4.42–3.64 eV), ~1.24 eV (4.42–3.18 eV), and ~0.45 eV (3.64–3.19 eV). Hoffman demonstrated that for a triplet ground state carbene, the difference in energy (δE) between σ and π orbital must be less than 1.5 eV  of the system. The schematic of the experimental set-up for the measurement of the optical properties is shown in Fig. 2 of supplementary material. Figure T(z) is the normalized energy transmittance as a function of z position for a sample thickness L; I0 is the incident laser intensity (1.09 nJ pulse−1) at the focus of the lens; and λ (780 nm) with a minimum beam radius ω0 (30 μm) at the focus. Because all the parameters in Eq.  data at different z positions easily gives the value of β as 0.55 × 10−9 cm W−1.The efficiency of any nonlinear two-photon material is measured in terms of the TPA coefficient (Hc) of graphene oxide is 143.75 and saturation magnetization (Ms) is 0.005 emu g−1. Low coercivity values indicating the graphene oxide is a soft magnetic material. To understand the origin ferromagnetic behavior of graphene oxide several theoretical works has been done, but it is not yet fully understood. Theoretical study has been suggested that graphene oxide show ferromagnetism due to the presence of various defects and long-range ordered coupling among these defects. In graphene oxide, the presence of C–OH, –COOH, and C=O groups which creates various defects may be responsible for ferromagnetic behavior of graphene oxide. Introduction of this hydrophilic group on the topological structures of raw carbon obtained from pyrolysis of silk cocoon may leads to increase defects due to intra or inter molecular hydrogen bonding among them.Room temperature ferromagnetic behavior of graphene oxide is shown in Fig. In summary, we have demonstrated the synthesis of graphene oxide from Tasar silk cocoon by pyrolyzing at 400 °C in argon atmosphere followed by its oxidation with nitric acid. Different characterization techniques were utilized to characterize the raw carbon and oxidized carbon. Synthesized water soluble graphene oxide shows remarkable fluorescence property. On increasing the excitation wave length to 380 nm, emission peak was observed at 460 nm. Its tunable fluorescence property with different wavelength can be used in applications like bio-imaging. Soft ferromagnetic behavior of graphene oxide which also exhibits single and two-photon fluorescence characteristics can open the new door for wide potential application, such as, magneto-optics, carbon-based magneto resistance, spintronics, magnetic memory devices, and so on.Supplementary material 1 (DOC 1402 kb)Below is the link to the electronic supplementary material.


We investigate the effect of water adsorption on the electrical properties of graphene oxide (GO) films using the direct current (DC) measurement and alternating current (AC) complex impedance spectroscopy. GO suspension synthesized by a modified Hummer's method is deposited on Au interdigitated electrodes. The strong electrical interaction of water molecules with GO films was observed through electrical characterizations. The DC measurement results show that the electrical properties of GO films are humidity- and applied voltage amplitude-dependent. The AC complex impedance spectroscopy method is used to analyze the mechanism of electrical interaction between water molecules and GO films in detail. At low humidity, GO films exhibit poor conductivity and can be seen as an insulator. However, at high humidity, the conductivity of GO films increases due to the enhancement of ion conduction. Our systematic research on this effect provides the fundamental supports for the development of graphene devices originating from solution-processed graphene oxide. Many oxygen-containing groups, including hydroxyl, epoxy and carboxylic acid, were bonded to the two-dimension network. The presence of sp3-bonded hybridized carbon atoms weakens the conductivity and enhances the hydrophilic property of GO. Recently, exploring the feasibility of integrating GO into graphene-based electronic devices has motivated immense studies on the intrinsic electrical [Graphene oxide (GO), a single thin sheet of graphite oxide that conventionally serves as a precursor material for preparing graphene -3, has rectrical ,8 and meectrical ,10 propeectrical , electriectrical -14, tempectrical ,16, lighectrical , etc. Dusp3-bonded hybridized carbon atoms, it is worth noting that GO can capture water vapor from external environment easily, owing to its notable hydrophilicity [In earlier works ,8, severhilicity -20. Henchilicity ,19. The Graphite oxide was synthesized via the oxidative treatment of natural graphite using the modified Hummer's method . Then, g2 (300 nm) formed by thermal oxidization. Ti/Au layers with the thickness of 100:400 nm were deposited on SiO2 layer using magnetron sputtering. The Au electrodes with a 20-μm-wide gap were formed through photolithography followed by wet etching. Before being functionalized by GO films, the IDE was rinsed with distilled water and ethanol and dried in vacuum overnight. The device was fabricated by dispersing the GO suspension onto Au IDE. A few drops (4 μl) of the GO suspension were cast onto Au IDE by a micro-syringe. After drying at room temperature for 6 h, a discrete network of GO sheets was left on the Au IDE.Interdigitated electrodes (IDEs) were fabricated on an n-type silicon wafer with a top layer of SiOThe schematic diagram of the experimental setup is shown in Figure −1) is shown in Figure −1. The peaks between 1,800 and 1,050 cm−1 are due to C = O, C–H, C–OH, C–O–C and C–O stretching at 1,730, 1,629, 1,396, 1,240 and 1,060 cm−1, respectively.The photograph of the stable GO dispersion (1 mg mlI-V) characteristics of the GO-film-functionalized IDE, is measured with a voltage sweeping mode at various humidity points. In this configuration, one electrode of the IDE is loaded with the sweeping voltage bias, and the other electrode is grounded. Recent works have noted that the DC electrical property of GO films can be influenced by the amplitude of sweeping voltage [I-V characteristics of GO films with low (−1 to 1 V) and high (−4 to 4 V) sweeping voltages at various humidity levels, respectively.The DC electrical property, i.e., current–voltage  provides a powerful tool to analyze the electrical interaction of the investigated material with water molecules -24. To k+) arising from hydroxyl of GO is bonded to excess adsorbed water molecules to form hydronium (H3O+) ion. However, the hydronium ions formed by charged carriers were alone and not enough to yield the continuous conduction path due to insufficient adsorption of water molecules. Thus, this process does not bring out conductance obviously. However, the increasing formation of hydronium ions can be seen as an accumulate layer. The mechanism is illustrated by a cartoon image shown in Figure RGO and geometric capacitance CGO of the GO films, as shown in Figure At lower RH (<54% RH), only semicircle-type impedance is observed in Figure + afresh, leading to a formation of a liquid layer around the interlayer of GO sheets by two-dimensional capillary or swelling effect. This process increases the interlayer distance of GO sheets largely, which can be sufficient to accommodate a monolayer of water. The formation of a liquid layer provides a conduction path across, between GO films and electrodes as illustrated in Figure +). As a result, ion conduction, i.e., Warburg impedance Zw, appears. Therefore, Warburg impedance Zw is added in the equivalent circuit shown in Figure As the humidity increased stepwise above 54% RH, straight line-type impedance appears at the low-frequency region, and semicircle-type impedance appears at the high-frequency region shown in Figure Figure sp3-bonded hybridized carbon atoms. As RH increased stepwise above 54%, the conductivity of GO films increased sharply due to strong water-adsorption-induced ion conduction. The results are beneficial to the development of graphene-based electronics, especially graphene device arising from solution-processed GO. Finally, the exciting-frequency-dependent impedance of the GO films versus humidity was discussed, and this result suggested a potential application of GO films in impedance-type humidity sensor.We used the DC measurement and AC complex impedance spectroscopy methods to investigate the effect of ambient humidity on the electrical properties of GO films. The strong interaction of water molecules with GO films was observed through electrical characterizations. The DC measurement results show that the electrical properties of GO films were affected by ambient humidity and the amplitude of applied voltage. The electrical sensing mechanism of GO films was discussed by analyzing the characteristics of AC complex impedance spectroscopy. At low RH (<54%), GO films exhibited poor conduction property due to the presence of The authors declare that they have no competing interests.The experiment and initial manuscript were prepared by YY and XDC. XYL helped in the AFM scan. ZQW helped in the FT-IR analysis. BQZ and JFZ contributed valuable idea and useful discussion for this manuscript. All authors read and approved the final manuscript.