Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations.Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus  When attempting to prevent the spread of plant diseases, comprehensive inventories of viral diversity are fundamental both for effective quarantine and sanitation efforts, and to ensure that plant materials within biological resource centres (BRCs) can be safely distributed The major challenge of using classical nucleic acid sequence-informed detection tools such as polymerase chain reaction (PCR) or Southern hybridisation assays, is that despite being highly sensitive, these techniques are generally either species or, at best, genus-specific. In addition, such tools lack the capacity to detect, let alone identify, pathogens that are unknown, poorly characterized or highly variable. Although it might be argued that the most economically important pathogens tend to be well characterized and that it is therefore not a serious issue that many of the more obscure pathogens go undetected, it is becoming better appreciated that the “importance” of any particular pathogenic microbe is very difficult to define. Specifically, the environmental and economic impacts of a particular pathogen can vary widely with varying climatic and ecological conditions and there are large numbers of microbes that are presently not classified as pathogens , which will eventually emerge as important future pathogens Whereas “sequence-dependent” microbial detection methods, which are generally based on PCR or nucleic acid hybridisation, can only be used to target known pathogens, sequence-independent next generation sequencing (NGS) based approaches can potentially provide an ideal platform for identifying almost all known and unknown microbes present in any particular host organism In addition to numerous applications in the study of animal infecting viruses, NGS-based metagenomics approaches have also been used to detect plant infecting viruses A major shortcoming of these metagenomic approaches is, however, that they remain technically cumbersome and too expensive for routine diagnostic applications on collections of eukaryotic hosts - even if barcoded primers are used to bulk-sequence pooled samples from multiple sources Sugarcane streak Egypt Virus  and were maintained for use as positive controls during the application of diagnostic tools for SSEV detection in sugarcane plants passing through the quarantine station. Using the siRNA- and VANA-targeted NGS approaches, we discovered and characterized a novel highly divergent mastrevirus from these two plants. This novel virus was also identified in other sugarcane plants originating from Sudan that exhibited white spots on the base of their leaf blades that become fused laterally, so as to appear as chlorotic stripes. Accordingly, we have proposed naming this virus Sugarcane white streak Virus (SWSV). In addition, we present a detailed analysis of siRNAs derived from the SWSV and SSEV variants infecting the two analysed sugarcane plants.Here we describe the application of siRNA- and VANA-targeted NGS approaches to the analyses of two Egyptian sugarcane plants maintained for a number of years at the CIRAD Sugarcane Quarantine Station in Montpellier, France. These plants were both known to be infected with Sugarcane streak Egypt virus (SSEV) and had been kept in a quarantine greenhouse at the CIRAD Sugarcane Quarantine Station, in Montpellier, France. The two sugarcane plants, VARX and USDA , were both initially collected in Egypt during two independent sampling surveys in the late 1990s Leaves presenting typical symptoms of sugarcane streak disease were sampled from two sugarcane plants that had previously been found to be infected with et al. 5′-ATC GTA GCA GCC TTC GGA TCC TCC-3′ and LinkerMid52 primer for USDA: 5′-ATG TGT CTA GCC TTC GGA TCC TCC-3′), and 10 µl of HotStarTaq Plus Master Mix Kit (Qiagen). The following cycling conditions were used: one cycle of 95°C for 5 min, five cycles of 95°C for 1 min, 50°C for 1 min, 72°C for 1.5 min, 35 cycles of 95°C for 30 sec, 50°C for 30 sec, 72°C for 1.5 min +2 sec at each cycle. An additional final extension for 10 min at 72°C was then performed. DNA products were pooled , cleaned up using the Wizard SV Gel and PCR Clean-Up System (Promega) and sequenced on 1/8th of a 454 pyrosequencing plate using GS FLX Titanium reagents .One gram of leaf material from the VARX and USDA plants were ground in Hanks' buffered salt solution (HBSS) (1∶10) with four ceramic beads  using a tissue homogeniser . The homogenised plant extracts were centrifuged at 3,200×g for 5 min and 6 ml of the supernatants were further centrifuged at 8,228×g for 3 min. The resulting supernatants were then filtered through a 0.45 µm sterile syringe filter. The filtrate was then centrifuged at 148,000×g for 2.5 hrs at 4°C to concentrate viral particles. The resulting pellet was resuspended overnight at 4°C in 200 µl of HBSS. Non-encapsidated nucleic acids were eliminated by adding 15 U of bovine pancreas DNase I (Euromedex) and 1.9 U of bovine pancreas RNase A  followed by incubation at 37°C for 90 min. Total nucleic acids were finally extracted from virions using a NucleoSpin 96 Virus Core Kit  following the manufacturer's protocol. The amplification of extracted nucleic acids was performed as described by Victoria et al. The nucleic acid extraction and sequencing approach of Kreuze De novo assemblies of contigs were performed with a minimal contig size set at 100 bp and 200 bp for Illumina and 454 GS FLX Titanium reads, respectively. A posteriori mapping of reads against the complete genomes of SWSV (once the full genome had been cloned and sequenced) or SSEV or against parts of these genomes were also performed using CLC Genomics Workbench 5.15. Primary sequence outputs have been deposited in the sequence read archive of GenBank .Analyses of reads produced by either Illumina (siRNA sequencing) or 454 GS FLX Titanium (Amplified-VANA sequencing) were performed using CLC Genomics Workbench 5.15. Geminiviridae, including SSEV. These two primer pairs  were complementary to sequences within the rep gene of SWSV. Total DNAs from the two sugarcane plants described above (VARX and USDA) were extracted using the DNeasy Plant Mini Kit (Qiagen) and screened for SWSV using the two pairs of primers and GoTaq Hot Start Master Mix (Promega) following the manufacturer's protocol. Amplification conditions consisted of an initial denaturation at 95°C for 2 min, 35 cycles at 94°C for 10 sec, 55°C for 30 sec, 68°C for 3 min, and a final extension step at 68°C for 10 min. Amplification products of ∼2.8 Kbp were gel purified, ligated to pGEM-T (Promega) and sequenced by standard Sanger sequencing using a primer walking approach.Two partially overlapping SWSV specific PCR primer pairs were designed so as to avoid any potential cross-hybridization to 63 representative species of the family rep gene. Total RNAs from VARX were extracted using the RNeasy Plant Mini Kit (Qiagen). DNase treatment of extracted RNAs was carried out using RQ1 RNase-Free DNase (Promega) following the manufacturer's protocol. Viral cDNA synthesis was performed by incubation of 1 µl of DNase treated RNAs with 15 µl of RNase free water, 0.6 µM of each primers (SWSV_F2: 5′-ACC ATG TGC TGC CAG TAA TT-3′ and ECORI-2R: 5′-GAG TTG AAT TCC GGT ACC AAG GAC-3′), and 0.4 mM of mixed deoxynucloside triphosphate (dNTPs), 5 µl of 5X Qiagen OneStep RT-PCR Buffer and 1 µl of Qiagen OneStep RT-PCR Enzyme Mix. Tubes were first placed at 50°C for 30 min for cDNA synthesis. PCR amplification was then carried out using the following cycling conditions: One cycle of 95°C for 15 min, 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min. An additional final extension for 10 min at 72°C was then performed. Amplification products were gel purified, ligated to pGEM-T (Promega) and sequenced by standard Sanger sequencing.Reverse transcriptase priming and amplification of nucleic acids were carried out in order to detect the intron of the Sugarcane streak Egypt Virus, Sugarcane streak virus, Maize streak virus, Sugarcane streak Reunion virus, Eragrostis streak virus and Saccharum streak virus. PCR amplification was carried out using 1 µl of DNA template in a 25 µl reaction containing 0.2 µM of each broad spectrum primer (SSV_1732F: 5′-CAR TCV ACR TTR TTY TGC CAG TA-3′ and SSV_2176R: 5′-GAR TAC CTY TCH ATG MTH CAG A-3′) and GoTaq Hot Start Master Mix (Promega) following the manufacturer's protocol. The following cycling conditions were used: One cycle of 95°C for 2 min, 35 cycles of 94°C for 1 min, 53°C for 1 min, 72°C for 1 min. An additional final extension for 10 min at 72°C was then performed.DNAs extracted from 17 sugarcane plants originating from Sudan kept at −20°C or six freshly extracted from plants maintained at the CIRAD Sugarcane Quarantine Station were screened for SWSV. DNA extracted from one sugarcane seedling grown from true seeds (fuzz) was also screened for SWSV. PCR amplification was carried out using the two pairs of primers described above  using GoTaq Hot Start Master Mix (Promega) following the manufacturer's protocol. Amplification products of ∼2.8 Kbp were gel purified, ligated to pGEM-T (Promega) and sequenced as described above. Plants infected with SWSV were also screened for all known sugarcane-infecting mastreviruses:Six complete genomes of the novel mastrevirus were recovered from plants VARX, USDA, A0037, B0069, D0005 and E0144  and wereThe SWSV/SSEV full genome sequences and predicted unspliced complementary and virion strand transcripts were separately folded using Nucleic Acid Secondary Structure Predictor www.r-project.org) were used to determine whether the distribution of reads between paired and unpaired sites were different, Wilcoxon rank-sum tests  indicated whether there were significantly more reads at paired sites compared to unpaired sites and vice versa. Whereas the Kolmogorov-Smirnov tests were used to indicate whether any associations existed between siRNA locations and base pairing within nucleic acid secondary structures, the Wilcoxon rank-sum tests were used to determine whether detected associations were positive (siRNAs tended to occur at structured sites) or negative (siRNAs tended to occur outside of structured sites).Given the genomic coordinates of pairing nucleotides within the HCSS, we investigated whether there was any significant trend for more reads  occurring within secondary structures predicted to occur within (i) the full genomes, (ii) the virion-strand transcripts and (iii) the complementary-strand transcripts. The reads were mapped to the secondary structures and we counted how many nucleotides were located at paired and unpaired sites. While Kolmogorov-Smirnov tests  and two from the USDA plant (2706 nt and 649 nt), encoded proteins with between 91 and 100% sequence identity with previously described SSEV proteins (Table1). BLASTx analysis revealed that an additional two contigs from the VARX plant (2122 and 127 nt) and three contigs from the USDA plant  were homologous with known mastreviruses but were nevertheless only distantly related to mastrevirus sequences currently deposited in GenBank (Table1).This approach was used in an attempt to detect both RNA and DNA viruses that may be present in the two sugarcane plants A posteriori mapping of VANA 454 reads obtained from the VARX and USDA plants against the complete SWSV genome (see below), revealed that 23.9% (625/2612) and 16.1% (264/1635) of the total reads were derived from this genome and that these yielded complete genome coverage at an average depth of 81X and 29X, respectively. Interestingly, a ∼120 nt long region of very low coverage (<4X) was identified, which mapped to the large intergenic region (LIR) of the SWSV genome  and 75% of reads  for the VARX and USDA plants, respectively .de novo assembly, six of which showed significant degrees of similarity to mastreviruses based on BLASTx A total of 15,275,640 raw reads were generated from the VARX sugarcane sample, which were then filtered down to 3,945,108 high quality reads in the 21 to 24 nt size range of siRNAs. From these reads, 226 contigs were obtained by Following the cloning and sequencing of the full genome of the new mastrevirus  it was determined that 0.59% of the Illumina reads obtained from the VARX plant could be mapped to this genome  to generIt is also noteworthy that besides differences in coverage depth, these various genomic regions of SWSV also showed differences in the siRNA size classes that they yielded. While there was an enrichment of the 21 and 22 nt siRNA size classes amongst the total siRNA reads mapping to the V1 and V2 ORFs, there was a depletion of the 21 nt siRNA size classes and an enrichment of the 24 nt size class amongst total siRNA reads mapping to the C1 ORF . The LIRSince the VARX plant was also infected with SSEV, a similar analysis of SSEV-derived siRNAs was performed. Mapping against the genome of SSEV (NC_001868) demonstrated that 0.17% of total reads (6572) were derived from it and that these reads covered 98.6% of the SSEV genome at an average depth of 55X, leaving only 4 gaps of between 5 and 15 nucleotides . AlthougBy collectively using the Illumina siRNA reads and the 454 VANA reads it was possible to assemble a single genome of the novel mastrevirus from both the VARX and USDA plants.SWSVIt has been previously determined that nucleic acid structures can have an appreciable impact on both the distribution of siRNA targets We detected a strong association between the absence of predicted secondary structures within the ssDNA SWSV genome and increased frequencies of corresponding 22, 23 and 24 nt long siRNAs , with which it shares 61% genome-wide identity. Whereas the Rep and MP amino acid sequences of SWSV are also most similar to those of WDIV , the CP is most similar to that of Panicum streak virus . Based on the 78% species demarcation threshold set by the Geminivirus study group of the ICTV Mastrevirus of the Family Geminiviridae (Digitaria streak virus (DSV) and WDIV, the Reps cluster with the African streak viruses and WDIV  out of the 23 screened .Complete SWSV genomes from four sugarcane plants  were cloned and sequenced. The genomes of these isolates have >91% genome-wide identity with those recovered from the VARX and USDA sugarcane plants. Phylogenetic analyses of the full genomes  and of tUsing primers that allow the amplification of all sugarcane-infecting mastreviruses, including Sugarcane streak Egypt Virus, Sugarcane streak virus, Maize streak virus, Sugarcane streak Reunion virus, Eragrostis streak virus and Saccharum streak virus, the five sugarcane plants originating from Sudan were shown to be free of co-infection with other known mastreviruses. Three of them are still maintained at the CIRAD sugarcane quarantine station  and exhibit white spots on the base of their leaf blades, around the blade joint where the two wedge shaped areas called “dewlaps” are located . These sThe SWSV genomes recovered from the various sugarcane plants were between 2828 and 2836 nt and are, in almost all respects, very similar to those of all other previously described mastreviruses. The one exceptional feature of the SWSV genomes is that in case of the VARX and USDA isolates alternative splicing of complementary sense transcripts likely results in the expression of both a standard Rep (which is predicted to be 396 amino acids long), and a rather unusual RepA of 418 amino acids long. This is the only known occurrence in any geminivirus of a RepA that is larger than Rep.Given the uniqueness of this apparent genome organisation in the USDA and VARX isolates the correct identification of the intron within the complementary sense transcript was verified. RT-PCR reactions targeting the complementary sense transcript clearly indicated the presence of a mixture of spliced and non-spliced complementary sense mRNA transcripts, and confirmed that the correct locations of the acceptor and donor sites of the 66 nt long SWSV intron had been identified .All the SWSV genome sequences determined here share evidence of the same ancestral recombination event in the short intergenic region - corresponding to genomic coordinates 1419–1468 in the USDA isolate . Corresponding coordinates are known to be very common sites of recombination in mastreviruses We have performed NGS-based analyses of both siRNA and VANA isolated from sugarcane plants originating from Egypt. Both sequence-independent NGS approaches revealed the presence of a novel mastrevirus, SWSV, which had so far escaped routine quarantine detection assays, possibly because it was present in mixed infection with SSEV. The procedures used for the discovery of SWSV pave the way towards the application of NGS-based quarantine detection procedures. Such procedures would likely be hierarchical with a first stage sequence-independent NGS step followed by sequence-dependent secondary assays. Whereas the first step would be to identify novel viruses within a single plant (perhaps one displaying apparent disease symptoms), the second step would be to use sequence dependent approaches to both confirm the presence of any novel virus(es) identified in the original host, and identify the presence of this(ese) virus(es) in larger plant collections. A major strength of such an approach is that it would also yield complete genome sequences.The present study also confirms that both VANA Begomovirus genus , very little comparable information has previously been available for mastreviruses.Virus-derived siRNAs naturally accumulate in virus-infected plants as a consequence of the action of Dicer enzymes as part of the RNA silencing-based plant antiviral defences The siRNA distributions observed here for SWSV and SSEV, perhaps unsurprisingly, seem to largely parallel those previously reported for begomoviruses. In particular, the differences in size classes observed between different genome regions suggest that mastreviruses are subject both to transcriptional gene silencing, based on 24 nt long siRNAs produced through the action of DCL3, and to post-transcriptional gene silencing (PTGS) mediated by the 21–22 nt long siRNAs produced through the action of the antiviral Dicers DCL4 and DCL2 . The action of the former mechanism is particularly evident in the siRNAs mapping to the SWSV intron but is also, to a lesser extent, evident in the siRNAs mapping to both the non-coding regions and the complementary sense ORFs of SWSV and SSEV. On the other hand, the 21–22 nt siRNA size classes associated with PTGS are particularly evident in the siRNAs mapping to the two virion sense ORFs which are known to be more actively transcribed in mastreviruses than their complementary sense counterparts For both SWSV and SSEV we detected a significant association between the frequencies of siRNAs and the presence/absence of predicted secondary structures within both the single stranded DNA (ssDNA) genomes of these viruses and their predicted single stranded RNA (ssRNA) complementary and virion strand transcripts. However, whereas significantly more siRNAs corresponded with unstructured regions of the ssDNA genome, for the transcripts significantly more siRNAs corresponded with structured regions of ssRNA. It is plausible that base-paired nucleotides within transcript RNA molecules are protected from siRNA binding and that the secondary structures evident both in transcripts produced by SWSV, SSEV and in mastrevirus genomes in general In both analysed Egyptian sugarcane accessions, VARX and USDA, SWSV was found to be present in co-infections with SSEV. Both sugarcane plants were independently collected in Egypt which suggests that SWSV infection of Egyptian sugarcane plants may not be a rare phenomenon. SWSV was also detected in SSEV-free plants that originated from Sudan. It is noteworthy that one of the Sudanese plants from which SWSV was isolated, E0144, was initially grown in Sudan in 1992 before being transferred to Barbados in 1998 and subsequently sent back to the CIRAD Sugarcane Quarantine Station in 2009 . Assuming that SWSV did not infect this plant in Barbados between 1998 and 2009, it is plausible that SWSV was present along the Nile basin at least from the late 1980s. Interestingly, as a consequence of indel polymorphisms in the 66 nt long SWSV intron, the Egyptian SWSV isolates VARX and USDA have a highly unusual genome organization and likely express a RepA protein that, while having the same N- and C-terminus sequences as Rep, is 22 amino acids longer than Rep.The recent discoveries of SWSV and other highly divergent mastreviruses Sugarcane yellow leaf virus (SCYLV), which remained unnoticed for at least 30 years during its spread throughout the world Given the relatively high degrees of sequence divergence observed between the different SWSV isolates described here (∼9%), it is plausible that the natural geographical range of SWSV is broader than just the Nile basin. Also, the global dissemination of sugarcane cuttings, the absence of SWSV diagnostic tools, and the fact that SWSV induces, at least in one case, extremely mild symptoms in sugarcane imply that SWSV may have already been unknowingly distributed throughout the sugarcane growing regions of the world. The failure of established sugarcane quarantine diagnostics in this regard provides a dramatic example of how potentially pathogenic viruses can evade the screening procedures of quarantine facilities and may spread worldwide through international plant material exchanges. In this regard the situation with SWSV might closely match that of Our study stresses both the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting, and the need to better assess viral diversity within plants that are destined for exotic habitats. It indicates that a combination of sequence-independent NGS-based partial viral genome sequencing coupled with sequence-dependent Sanger-based full genome cloning and sequencing is likely to reduce the number of non-intercepted virus pathogens passing through plant quarantine stations, while at the same time alerting authorities to the presence and potential spread of viruses with unknown pathogenic potentials.Figure S1a posteriori mapping against the complete genome of SSEV of reads produced by Illumina (siRNA sequencing). The genomic organization of SSEV is schematically shown at the top of the figure. (B) Size distribution of sequenced siRNAs obtained from the VARX plant mapping on the V1–V2 ORFs region of SSEV. Histograms represent the number of siRNA reads in each size class. and (C) Size distribution reads mapping on C1–C2 ORFs region of SSEV.(A) Genome coverages obtained after (TIF)Click here for additional data file.Figure S2Two-dimensional genome-wide percentage pairwise nucleotide identity plot of monocot-infecting mastreviruses including the six novel SWSV isolates from this study.(TIF)Click here for additional data file.Figure S3(A) Maximum-likelihood phylogenetic tree of six SWSV isolates. The six isolates can be classified into 3 strains, SWSV-A , -B  and -C (A0037). (B) Genome-wide pairwise nucleotide similarity score matrix, the 94% strain demarcation threshold set by the Geminivirus study group of the ICTV  is indicated (green coloured below 94% and pink-red coloured above 94%).(TIF)Click here for additional data file.Figure S4Symptoms caused by SWSV on B0065, B0069 and D0005 plants.(TIF)Click here for additional data file.Figure S5Reverse transcriptase priming and amplification of nucleic acids were carried out in order to detect the rep gene C-sense intronic region. (A) Agarose gel detection of presence of a mixture of spliced and non-spliced complementary sense mRNA transcripts. 1: 1 Kb ladder; 2: Reverse transcriptase priming and amplification of nucleic acids without DNase treatment of extracted RNAs; 3: Reverse transcriptase priming and amplification of nucleic acids with DNase treatment of extracted RNAs. (B) 66 nt long SWSV intron nucleotidic sequence and splice donor and acceptor sites. The sequence of the intron (in lower case) and its flanking exons (upper case) are shown. The 5′ (donor) and 3′ (acceptor) splice sites are underlined (lower case).(TIF)Click here for additional data file.Table S1List of the sugarcane varieties from the CIRAD Sugarcane Quarantine Station (SQS) that were screened for the presence of all known sugarcane-infecting mastreviruses and SWSV.(DOC)Click here for additional data file.


Better understanding of the host-pathogen interaction and the detailed B. pertussis (Bp) target recognition pattern will help in guided vaccine design. We characterized the specific epitope antigen recognition profiles of serum antibodies (‘the reactome’) induced by whooping cough and B. pertussis (Bp) vaccines from a case–control study conducted in 1996 in infants enrolled in a Bp vaccine trial in Sweden .Pertussis  whole-cell (wc), acellular 5 (DPTa5), or with the 2 component (a2) vaccines and from infants receiving only DT (n = 10 for each group) were tested with high-content peptide microarrays containing 17 Bp proteins displayed as linear (n = 3175) peptide stretches. Slides were incubated with serum and peptide-IgG complexes detected with Cy5-labeled goat anti-human IgG and analyzed using a GenePix 4000B microarray scanner, followed by statistical analysis, using PAM  and the identification of uniquely recognized peptide epitopes.Sera from children with whooping cough, 239 (7.7%) in DTPwc, 259 (8.4%) in DTPa5, 105 (3.4%) DTPa2, 179 (5.8%) in the DT groups. Recognition of strongly recognized peptides was similar between whooping cough and DPTwc, but statistically different between whooping cough vs. DTPa5 (p < 0.05), DTPa2 and DT (p < 0.001 vs. both) vaccines. 6/3,085 and 2/3,085 peptides were exclusively recognized in (10/10) sera from children with whooping cough and DTPa2 vaccination, respectively. DTPwc resembles more closely the whooping cough reactome as compared to acellular vaccines.367/3,085 (11.9%) peptides were recognized in 10/10 sera from children with We could identify a unique recognition signature common for each vaccination group (10/10 children). Peptide microarray technology allows detection of subtle differences in epitope signature responses and may help to guide rational vaccine development by the objective description of a clinically relevant immune response that confers protection against infectious pathogens.The online version of this article (doi:10.1186/s12865-015-0090-3) contains supplementary material, which is available to authorized users. Pertussis (whooping cough) caused by B. pertussis (Bp), remains a major global public health problem [pertussis remains endemic in the Western countries [ problem ,2. Despiountries . In the ountries , Israel ountries  and USA ountries . In Caliountries   to identify differences in immune recognition induced by available Bp vaccines as compared to natural infection, and iii) to objectively define the qualitative differences in humoral target recognition induced by current vaccines [whooping cough and in 3 groups of infants randomized to different Bp vaccines from a trial conducted 1996 in Sweden [There is an unmet need i) to depict the immunological recognition matrix to understand the specific epitope recognition pattern induced by natural infection with vaccines . We assen Sweden  using a pertussis vaccine Stockholm trial I [whooping cough;;10 children who received a diphtheria (D) and tetanus (T), vaccine  as placebo, and developed whole cell (wc) (DTPwc) vaccine licensed in the United States ;10 children immunized with the diphtheria (D), tetanus (T), pertussis (P) DTPa5 vaccine ;10 children immunized with the 5 component acellular candidate DTPa2 vaccine ;10 ichildren immunized with the 2 component acellular candidate DT vaccine and did not develop whooping cough.10 children immunized with the Swedish-produced Samples were randomly selected among the serum samples from the  trial I , stored  trial I .10 childSera were collected 30 days after the last dose, except for the group which whooping cough .The Stockholm regional ethics committee North (Dnr 911258) has approved the study. All subjects provided informed consent. Both parents of the children provided informed consent on their behalf. The informed consent was provided in a written format, signed and is on file at the Swedish National Institute of Public Health, Stockholm, Sweden.Bp proteins . The slides contain three identical sub-arrays with 3,175 unique peptides on each subarray. Each sub-array contains 16 blocks arranged in a regular pattern, with spots arranged in a matrix 16 X 15. An image of a microarray is provided in the Additional file  earlier , were alExperiments were performed following a standardized protocol -16: 300 Data analysis consisted of 4 steps as described .All images and aligned files were visually inspected to check for artifacts and for spots erroneously flagged by the software. Images of background and foreground intensities were produced for every sub-array by using bioinformatics tools. All spots or areas which did not represent a high quality signal were removed from analysis. Further quality controls were also performed  and the All spots showing a response on the buffer slides - and for this reason identified as possible false positive - were removed from the analysis, as well as all spots that did not show any signal  in the data acquisition process. Low response spots, with a signal below a computed cut off  were also removed.the normalization process was performed using the simple linear model as described before ,14,16,17never in serum from any individual in a control reference group, e.g. in the current report the ‘reference’ individuals who received placebo . Strongly recognized peptides identified in each group were plotted according to index value and number of times they were recognized in the group of interest. Lastly, a 3D-graphical representation of the “reactome” [B. pertussis proteins was computed for every group, by plotting mean index value for every peptide, as well as the protein and position on the respective amino acid-sequence of the protein. A similar 3D-plot was computed to compare the signals in two study groups, plotting the Δ value between the mean index values in the two groups .Data analysis was performed using two different statistical methods: (i) PAM  , a predieactome”  of B. peDT (control) group vs. the remaining groups, a Chi-square test for the goodness of fit was used.All pre-processing and statistical analyses were performed customizing open-source packages of Bioconductor, R software ,22. In ahttp://www.immuneepitope.org/) and homologous sequences highlighted.In order to relate our results to the epitopes which have been identified previously in the literature, we searched the B-cell Immune Epitope Database  (IEDB) swhooping cough showed that 367 (11.9%) of 3,085 peptides were commonly recognized in in 10/10 serum samples. 239 (7.7%) peptides were recognized in 10/10 serum samples from subjects who received the DTPwc vaccine, while 259 (8.4%), 105 (3.4%) and 179 (5.8%) peptides were recognized in sera obtained from 10/10 children receiving the DTPa5 vaccine, the DTPa2 vaccine or the DT vaccine, respectively (without a diagnosis of whooping cough). Figure Bp infection and DPTwc (p = NS), while it was statistically different between serum recognition patterns associated with whooping cough and the groups who received the acellular vaccines . Table Bp protein, stratified for the study groups.Three thousand eighty five peptides remained in the analysis after quality control, i.e. after false positive, empty and low intensity signal spots removal. Analysis of sera from children with that 367 1.9% of 3Bp epitopes. The IgG target recognition pattern induced by the DTPwc and the DTPa5 vaccines were similar to the serum pattern from children with whooping cough, while the DTPa2 group showed a shape similar to the recognition pattern detected in the DT group [see also Additional file Figure Bp serum recognition pattern from individuals with whooping cough and from the DTPa2 groups showed a characteristic shape of IgG recognition curves using the computed Δ value for mean indexes using the DT group as a reference  could be identified in 10/10 serum samples from patients with whooping cough; 2 peptides were derived from pertussis toxin components ; 3 peptides from adhesion proteins ; and 1 peptide from the transporter protein ompP. In serum samples from the DTPa2 group, 1 peptide was derived from the tracheal colonization factor (tcfA) and a different peptide epitope from the GTP-binding elongation factor (bipA). The two peptides specifically recognized in the DTPa2 group did not belong to the 2 proteins used as vaccine components; the sequence of these 8 peptides is reported in Table Bp epitope recognition in serum from children with whooping cough versus the control, i.e. the reference group who received DT . Alternatively, this method allows also comparing groups of individuals, i.e. individuals who received different kinds of Bp vaccines.The exclusive epitope recognition analysis yields peptide targets that are unique for each test cohort. A different kind of analysis, PAM, identifies epitopes that are both always strongly recognized in the reference, and always weakly recognized in the ‘test’ group (or vice versa) in serum from each individual in the group. This allows predicting whether a reactivity pattern is associated to a certain defined endpoint  placebo. The peptide epitope targets are only shown if they exhibited a constant reactivity pattern for each of the 10 individuals in the test groups. The DTPa2 group yielded 45, the DTPa5 100 and the whole cell vaccine 42 Bp targets that were differentially recognized as compared to the placebo group. Infection with Bordetella pertussis yielded 32 targets that were differentially recognized as compared to the group who received (DT) placebo. The identity of these targets and location within each target molecule is provided in the online Additional file Bp targets that would segregate infected versus non- Bp infected individuals. The detailed listing of the targets is provided in the Additional file Figure Bp epitopes were captured by the peptide microarray matrix: sixty-five B. pertussis B-cell epitopes were retrieved from the IEDB  appeared to vanish over time [Bp strains carrying mutations of virulence factors has been reported [Primary prevention remains the main intervention to limit pertussis occurrence and new transmission. The protective effect mediated by the ver time  and emerreported Bp induced by natural infection and 3 different vaccines in children enrolled in a clinical trial conducted in Sweden [DTPa5, ensured the best ratio between protection from whooping cough and acceptable rates of side effects.Our study explored the immune response against n Sweden : this trBp epitope database with the peptide microarray technology described in this report (supplementary Table S5), ii) show robust differences between different vaccines concerning epitope recognition patterns . These results suggest that peptide microarrays provide a platform to visualize quantitative and qualitative differences in humoral recognition patterns at the epitope level. This may be relevant since genetic changes in Bp have been reported [We have been able to i) identify a high number of B-cell epitopes that have been described in the literature and the reported -28.whooping cough displayed the broadest Bp epitope antibody recognition, with a certain number of peptides exclusively recognized in this group. Only serum analysis from individuals vaccinated with DTPwc showed a similar trend concerning the number of recognized Bp peptides, consistent with the fact that all the components of the bacterial wall are present in this vaccine preparation. The DTPa5 as well as the DTPa2 vaccine induced a significantly different humoral recognition pattern. The DTPa2 vaccine appeared to boost pre-existing Bp-reactive antibody responses as compared to the induction and expansion of new antibody reactivity pattern directed to new Bp target antigens.. This is reflected in stronger recognition of the proteins Bordetella resistance to killing (BrkA), Vag8 protein, Putative autotransporter (BapC). Conversely, humoral recognition of the filamentous hemagglutinin (FHA) appears to be induced by vaccination with the DTPwc vaccine as well as after natural infection with Bp.Serum from children with Bp in the course of whooping cough after resolution is long-lasting and more protracted as compared to the immune response induced by vaccines; it may offer new potential targets to improve vaccine design for pertussis once the nature of the antibody reactivity mediating immune protection will be deciphered. The DTPa2 vaccine has a very good safety profile, yet its effect has been questioned in the past particularly in terms of protection and its duration [DTPa2 vaccine acts by boosting a pre-existing ‘natural’ Bp recognition matrix, as compared to other vaccines which rather induced a shift in serum Bp epitope recognition patterns. Both DTPwc and DTPa5 showed a reactome similar to the Bp natural infection; future studies may address whether this would be related to increased protection induced by these vaccines compared to the DTPa2 [The immune responses induced by duration . This cohe DTPa2 .Bp target recognition induced by different vaccines: i) Vaccines could boost and modulate the recognition matrix for natural occurring and Bp specific antibodies directed against Bordetella spp, eliciting pre-existing humoral immune responses directed against Bp epitopes. This is consistent with the concept that ‘natural antibodies’ are a fundamental part of the immune system and play a crucial role in modulating the recognition (and response) to self and ‘non-self’ infectious antigens [Bp vaccination experience most likely the full-blown disease with the typical whooping cough presentation, yet some (non-vaccinated) individuals appear to experience limited disease, suggesting that ‘abortive’ cases or even immune protection may occur in the absence of vaccination [Bordetella species  [Bp.There are at least four different, not mutually exclusive explanations concerning the spectrum of antigens ,30 ii) Ccination ,32. Therolmesii) ,34 couldBp peptides recognized in sera from 10/10 infants with whooping cough), yet we identified also ‘private’ humoral responses unique for each individuals ; iii) Potentially cross-reactive antibodies which target closely related hemagglutinin or fimbriae from other bacterial species may be responsible for different efficacy of the vaccines iv) Vaccination leads not only to epitope-specific immune responses directed against targets contained in the vaccine, yet to other molecular targets as well (epitope spreading). This concept is appreciated in other areas of medicine and contributes to clinical efficacy of some vaccines. For instance, antigen-spreading mediates vaccination-induced regression in human melanoma [Most of the serum immune responses in the groups of our study were shared among the individuals in each group  whole cell vaccine exhibited decreased pertussis attack rates as compared to individuals primed with acellular vaccines [Peptide-microarray-guided analysis may also help to decipher the phenomenon of ‘epitope suppression’  which havaccines ,44: diffBp after natural infection in comparison to 3 vaccines. The report shows the potential of the high-content peptide microarray technique in infectious diseases, detecting epitopes by far more numerous and likely more immunogenic compared to the ones already reported in electronic databases. It also offers a new possibility to objectively decipher immune reactivity in clinically well-defined test groups undergoing vaccination strategies and allows to test for batch-to-batch consistency. Target recognition patterns in serum from individuals who experienced infection with Bp (and enjoyed protection for a longer period of time) could guide the development of Bp vaccines.Microarray analysis offered for the first time a comprehensive characterization of the immune response to


Autoimmune regulator (AIRE) gene, which promote thymic display of thousands of peripheral tissue antigens in a process critical for establishing central immune tolerance. We here used proteome arrays to perform a comprehensive study of autoimmune targets in APS1. Interrogation of established autoantigens revealed highly reliable detection of autoantibodies, and by exploring the full panel of more than 9000 proteins we further identified MAGEB2 and PDILT as novel major autoantigens in APS1. Our proteome-wide assessment revealed a marked enrichment for tissue-specific immune targets, mirroring AIRE’s selectiveness for this category of genes. Our findings also suggest that only a very limited portion of the proteome becomes targeted by the immune system in APS1, which contrasts the broad defect of thymic presentation associated with AIRE-deficiency and raises novel questions what other factors are needed for break of tolerance.Autoimmune polyendocrine syndrome type 1 (APS1) is a monogenic disorder that features multiple autoimmune disease manifestations. It is caused by mutations in the  Autoimmune responses can ultimately be defined at the molecular level by the specific interaction between T- or B-cell receptors and a distinct self-molecule. In tissue-specific autoimmune disorders the immune system typically target molecules that are exclusively expressed in the affected tissue and involve a combined cellular and humoral response with cognate specificities1256Candidiasis, hypoparathyroidism and Addison’s disease, of which two are required for the diagnosis8Autoimmune regulator (AIRE) gene10AIRE-driven antigen display ensures that naïve T-cells are exposed to tissue-specific molecules that otherwise would not be encountered in the thymus, and is therefore indispensible for the negative selection of autoreactive T-cells12AIRE’s control in the thymus1415AIRE. Although several autoantigens have been defined in APS1 through focused investigations1718192021Autoimmune polyendocrine syndrome type 1 (APS1) is an autosomal recessive disorder that features multiple tissue-specific autoimmune manifestations (Mendelian Inheritance in Man #240300). APS1 is clinically defined by three hallmark components; chronic mucocutaneous Patients with APS1 develop multiple autoimmune manifestations and harbor autoantibodies against a range of tissues. We used sera from 51 patients with APS1 and 21 healthy subjects to probe a human proteome arrays containing over 9000 full-length human proteins (ProtoArray®) for IgG autoantibodies. The great majority of array targets generated low signal intensities at around one thousandth of the saturation level for both patient and healthy control sera, and only a minimal fraction of the targets generated near saturated or saturated signal levels see . A numbePatients with APS1 develop complex combinations of autoimmune manifestations. We assessed correlations between the established autoantibodies to better understand how individual autoimmune responses were interlinked. Autoantibodies against the two closely related type 1 interferon specifies; IFNW1 and IFNA1, showed strong correlation (Spearman’s ρ = 0.79). Likewise, we observed correlations between autoantibodies against GAD2 and GAD1 (ρ = 0.70) that are both glutamic acid decarboxylases and co-expressed in pancreatic islet cells and gamma-aminobutyric acid (GABA) producing neurons. Interestingly, we also found correlation between TPH1 and DCC (ρ = 0.70), which belong to distinct enzyme families but are both involved in the monoamine synthesis and co-expressed in serotonin producing cells in the central nervous system and gut .AIRE-gene mutations have been described in patients with APS1AIRE gene. With the exception of two described AIRE gene mutations associated with distinct non-APS1 autoimmunity syndromes with dominant inheritance24AIRE gene2627AIRE genotype an autoimmune expression in APS1 we compared autoantibody responses against the known antigens between patients with different AIRE genotypes. We first performed a principal component analysis using autoantibody data for the known autoantigens and investigated whether patients grouped according to their AIRE genotypes. The 21 healthy controls formed a distinct cluster separated from the patients with APS1, but patients homozygous for R257X, patients homozygous for c967−979del13 and patients heterozygous for AIRE or with rare AIRE gene mutations were dispersed without tendency for internal clustering , were selected for further scrutiny. In this selection we found several targets that were located nearby and strongly correlated with known autoantigens on the array, suggesting these signals had appeared as result of printing contaminations , which left us with a set of 429 targets for further investigation. To select for patient specific signals we introduced cutoff values for all targets at three standard deviations above the average of the healthy control group and compared the frequency of positive individuals between the patients with APS1 and the healthy control group. The 35 targets most strongly associated with the APS1 patient group, with unadjusted p-values ranging from 8 × 10ions see . To idenions see . Our annions see . MAGEB2 ions see .Our proteome array data suggested MAGEB2 and PDILT were major immune targets in APS1. To confirm the novel candidates with an independent method we developed radio-ligand binding assays for MAGEB2 and PDILT and assessed an extended APS1 cohort and a broad clinical control material. Previous studies suggest MAGEB2 and PDILT are specifically expressed in male gonadal cells3334We validated the tissue-distribution of MAGEB2 and PDILT mRNA across a panel of eleven human tissues using digital droplet PCR. MAGEB2 and PDILT transcripts were detected at high levels in testicular tissue and were absent in the non-gonadal tissues . To defiAPS1 features multi-organ autoimmunity and autoantibody responses against a range of tissues. Although several immune targets have been defined in separate studies, the autoimmune expression of APS1 has not previously been captured in a comprehensive way. In this study we assessed the B-cell response against a panel of over 9000 human proteins to enable detailed profiling of known autoantigens and to identify novel immune targets in APS1. Review of multiple established autoantigens revealed highly robust autoantibody detection, and by exploring the full array panel we further identified two novel autoantigens, MAGEB2 and PDILT, that could be confirmed by independent methods and in a replication cohort.AIRE-deficiency is translated into autoimmune responses, as comprehensive studies of autoimmune targets have not been previously undertaken in patients with APS1 or Aire-deficient mice. Our broad characterization of B-cell targets provides a novel means to appreciate these events.Studies in APS1 and its animal model have shed light on the process whereby the immune system learns self toleranceAIRE-deficiency. Studies in Aire knock out mice have allowed detailed characterization of AIRE controlled gene expression - first using micro arrays and PCR and recently with increased precision by mRNA sequencing - and suggest around four thousand genes are positively controlled by Aire in medullary thymic epithelial cells121415AIRE and the number of molecules that become immune targets in APS1. It is important here to consider the possibility that some autoantigens in the panel could have failed to generate signals of technical reasons and therefore were missed in our analyses. Indeed autoantibody detection can depend on factors such as protein folding, posttranslational modification and immobilization of the antigen. However, our extensive review of previously established autoantigens, replicating all known targets in the panel, indicated a highly sensitive and reliable detection of autoantibodies. The great difference in number of AIRE controlled genes and number of detected autoantigens thereby did not appear to represent a technical bias but to reflect biology.One important observation that can be made is that only a very limited portion of the proteome appears to become targeted by autoantibodies in patients with APS1, as compared with the broad defect in thymic antigen presentation associated with AIRE-dependent antigen presentation. First it should be recognized that antigen recognition is MHC-dependent, and that only a subset of self-antigens are expected to possess the necessary requirements for being presented on MHC efficiently and to render sturdy immune responses. The potential impacts of B-cell tolerance and peripheral tolerance mechanisms must also be considered here, which may provide additional filters for the development of autoimmune responses. Recent studies in patients with APS1 have also shed light on the role of peripheral antigen presentation in autoimmune manifestationAIRE-dependent genes must also be expressed at sufficient level in the periphery in order to give rise to autoantibody responses.Several explanations must be considered why the autoimmune response is, as it appears, orders of magnitude more limited than could be expected from the defect in Human Protein AtlasAIRE-deficiency and the notion that AIRE is responsible for specifically compensating the absence of tissue-specific genes in the thymusAnother key observation that can be made from our survey of autoantibody specificities is that autoimmunity in APS1 preferentially targets molecules with restricted tissue expression profiles. This conforms to previous experience, and stands out with increased clarity here with the overview perspective from a proteome-wide screen. In our selection of major immune targets more than half were annotated as being tissue enriched and remaining as either being tissue enhanced, group enriched or not detected, according to mRNA sequencing data from the amino acid and derivate metabolism and cell-cell signaling. Also neurotransmitter biosynthesis and vitamin binding were enriched terms among the immune targets  concentration or deficient response to synthetic ACTH stimulation test (failure to reach 550 nmol/L in 30 or 60 min). Most patients had also been diagnosed with additional disease components, including; malabsorption, type 1 diabetes mellitus, hypogonadism, pernicious anemia, alopecia and vitiligo. Females with premature ovarian insufficiency not related to APS1Serum samples were collected from Finnish, Norwegian and Swedish patients with APS1. All patients met the clinical diagnostic criteria for APS1, requiring at least two of the hallmark components; chronic mucocutaneous All patients had given their informed consent for participation.Immune Response BioMarker Profiling, using the recommended detection reagent  and blocking buffer . The arrays were probed with sera at a dilution of 1:2000. Arrays were scanned using a GenePix 4000B microarray scanner, and the GenePix® Pro microarray (v6.1) software was used for alignment and data acquisition.Probing and scanning of the protein arrays  was conducted according to Invitrogen’s protocol for Statistical analyses of proteome array data were performed on log-transformed intensities. Fisher’s exact test was used for ranking protein array targets that differed between the patient with APS1 and healthy control group. A signal was considered positive if the intensity value was above the average of the healthy + 3SD.in vitro in presence of 35S Methionine (Promega TNT Systems). Immunoprecipitation was conducted in 96-well filtration plates (Millipore). A positive standard, an APS1 patient serum with known reactivity, and a negative standard, 4% BSA, were included to each plate. 30,000 CPM of radio-labeled protein and 2.5 μl of serum sample were added to each well. All sera were analyzed twice. Serum antibodies were immobilized to protein A Sepharose  during over-night incubation. Radioactivity was measured in a micro-beta counter . Autoantibody index values were calculated according to the following: / × 100.Autoantibodies against PDILT and MAGEB2 were measured using radio-ligand binding assays. Human cDNA-clones for PDILT  and MAGEB2  were cloned into a pTNT expression vector , and were transcribed and translated Total RNA from Origene was converted to cDNA using Life Technologies’ Double Stranded cDNA Synthesis Kit and the protocol with random hexamers  for the first strand synthesis. Bio-Rad’s ddPCR supermix for probes  was used and droplet generation was conducted according to BioRad’s standard protocol with TagMan probes to PDILT and MAGEB2 and β2M as a control . Samples were run on a Quantalife ddPCR machine and Quantasoft software was used to collect and analyze data.Staining for MAGEB2 and PDILT were performed on formalin fixed paraffin embedded human testicular tissue using the Autostainer 480 (Thermo Fisher Scientific). Tissue slides were first incubated with Ultra V block  for 5 minutes, and thereafter incubated with either anti-MAGEB2  at 1:1750 dilution or anti-PDILT  at 1:1000 dilution for 30 minutes. The slides were then incubated with labeled horseradish peroxidase-polymer for 30 minutes, followed by 3,3′-Diaminobenzidine (DAB) solution for 2 × 5 minutes. Slides were counterstained in Mayers hematoxylin  for 5 minutes using the Autostainer XL (Leica), and then rinsed in lithium carbonate water (diluted 1:5 from saturated solution) for 1 minute. The slides were dehydrated in graded ethanol and lastly coverlipped  using an automated glass coverslipper . The slides were scanned using the automated scanning system Aperio XT (Aperio Technologies).To find statistically overrepresented gene ontology terms (GOa-human database) assigned to the established autoantigens present on the array, we used the online GOstat softwareHow to cite this article: Landegren, N. et al. Proteome-wide survey of the autoimmune target repertoire in autoimmune polyendocrine syndrome type 1. Sci. Rep.6, 20104; doi: 10.1038/srep20104 (2016).


Diffuse astrocytoma  is a low-grade, primary brain neoplasm with high potential of recurrence as higher grade malignant form. We have analyzed differentially expressed membrane proteins from these tumors, using high-resolution mass spectrometry. A total of 2803 proteins were identified, 340 of them differentially expressed with minimum of 2 fold change and based on ≥2 unique peptides. Bioinformatics analysis of this dataset also revealed important molecular networks and pathways relevant to tumorigenesis, mTOR signaling pathway being a major pathway identified. Comparison of 340 differentially expressed proteins with the transcript data from Grade II diffuse astrocytomas reported earlier, revealed about 190 of the proteins correlate in their trends in expression. Considering progressive and recurrent nature of these tumors, we have mapped the differentially expressed proteins for their secretory potential, integrated the resulting list with similar list of proteins from anaplastic astrocytoma (WHO Grade III) tumors and provide a panel of proteins along with their proteotypic peptides, as a resource that would be useful for investigation as circulatory plasma markers for post-treatment surveillance of DA patients. It is characterized by slow growth with low probability of infiltration into neighboring brain tissue. Though relatively rare237910In the present study, we have analyzed protein expression changes in the microsomal fraction of clinical tissue samples with diffuse astrocytoma in comparison to control, using iTRAQ and high-resolution mass spectrometry, followed by extensive bioinformatics analysis to get further insights into molecular changes in these tumors and to generate a resource which could be useful for developing circulatory biomarkers for clinical applications such as post-treatment surveillance.All the samples were collected at the time of surgery with informed consent from patients and approval of the Institutional Ethics Committee, Nizam’s Institute of Medical Sciences (NIMS), Hyderabad, India and all the experiments were performed in accordance with recommended guidelines and regulations. Tumor tissue specimens were snap frozen in liquid nitrogen and stored at −80 °C until use. Multiple sections from the temporal neocortex were studied and the tumor grade was assigned on the basis of clinical evaluation and histopathology as per WHO guidelines. Out of forty-five astrocytoma specimens collected over the period of 2 years, nine of them were grouped as diffuse astrocytomas. Six age matched samples (20–40 years) of either sex were selected for present study. Brain tissue obtained from temporal lobe epilepsy surgeries were collected as experimental controls. A large amount of temporal cortical tissue needs to be removed in these surgeries to reach hippocampus which is usually the most likely seizure focus. The temporal cortex used as control did not show any histological abnormalities by light microscopy. Further, immunohistochemistry (IHC) with antibodies directed against phosphorylated neurofilament and synaptophysin proteins did not reveal any abnormal neurons in the cortex. These control subjects were in the 20–30 year old age group.et al.Tissues from tumor patients  or control subjects  were pooled separately and microsomal fraction was prepared according to the procedure of Cox Microsomal protein fraction from tumor or control tissues was subjected to trypsin digestion and the peptides were labelled with iTRAQ reagents according to the manufacturer’s instructions  and as described previouslyNanoflow electrospray ionization tandem mass spectrometric analysis was carried out using LTQ Orbitrap Velos  interfaced with Agilent’s 1200 Series nanoflow LC system. Peptides from each SCX fraction were enriched using a C18 trap column (75 μm × 2 cm) at a flow rate of 3 μl/min and fractionated on an analytical column (75 μm × 10 cm) at a flow rate of 350 nl/min using a linear gradient of 7–30% acetonitrile (ACN) over 65 min. Mass spectrometric analysis was performed in a data dependent manner using the Orbitrap mass analyzer at a mass resolution of 60,000 at m/z 400. For each MS cycle, twenty top most intense precursor ions were selected and subjected to MS/MS fragmentation and detected at a mass resolution of 15,000 at m/z 400. The fragmentation was carried out using higher-energy collision dissociation (HCD) mode. Collision energy (CE) between 39–42% was used for optimization and normalized CE of 40% was used to obtain release of reporter ions from all peptides detected in the full scan. The ions selected for fragmentation were excluded for next 30 sec. The automatic gain control for full FT MS and FT MS/MS was set to 1 million ions and 0.1 million ions respectively with a maximum time of accumulation of 500 ms. The lock mass option was enabled for accurate mass measurements.Protein identification, quantification and annotations of differentially expressed proteins were carried out as follows. The MS/MS data was analyzed using Proteome Discoverer  in Sequest mode using NCBI RefSeq database (release 52) containing 33,985 proteins. Search parameters included trypsin as the enzyme with 1 missed cleavage allowed; precursor and fragment mass tolerance were set to 20 ppm (around 97% of the peptides detected conformed to <10 ppm mass error) and 0.1Da, respectively; Methionine oxidation was set as a dynamic modification while methylthio modification at cysteine and iTRAQ modification at N-terminus of the peptide and lysines were set as static modifications. The peptide and protein information were extracted using high peptide confidence and top one peptide rank filters. The FDR was calculated by enabling the peptide sequence analysis using a decoy database. High confidence peptide identifications were obtained by setting a target FDR threshold of 1% at the peptide level. Mass spectrometric analysis resulted in identification of a total of 20,783 peptides. After removing peptides not labelled with all the four labels (n = 212) and those (n = 1968) shared between multiple proteins, 18,603 peptides were considered for identification of proteins. The labelling efficiency was thus 99%.Relative quantitation of proteins was carried out based on the intensities of reporter ions released during MS/MS fragmentation of peptides. The average relative intensities of the two reporter ions for each of the unique peptide identifiers for a protein were used to determine relative quantity of a protein and percentage variability. Appropriate filters at the level of peptides/peptide spectral matches (PSMs) and then at the protein level were applied to the quantification values as described in earlier publicationhttp://www.hprd.org)Gene Ontology annotations of the proteins identified were carried out based on Human Protein Reference Database , brevican core protein (BCAN), ectonucleotide pyrophosphatase/phosphodiesterase family member 6 (ENPP6) and heterogeneous nuclear ribonucleoprotein (HNRNP) K were studied by immunohistochemistry using commercially available Tissue microarray containing 13 Diffuse Astrocytomas cases and 4 control tissue cores (US BioMax). In brief, after deparaffinization and rehydration of formalin-fixed paraffin-embedded tumor tissue sections, antigen retrieval was performed by immersing the slide in antigen retrieval buffer  at 95 °C for 5 min. Endogenous peroxidases were blocked with 0.03% hydrogen peroxide, and nonspecific binding was blocked with 2% fetal calf serum in Tris-buffered saline with 0.1% Triton X-100 . Sections were then incubated for 1 h at RT with EGFR , BCAN , ENPP6  and HNRNP K  primary antibodies  followed by peroxidase-labelled polymer conjugate to anti-rabbit or anti-mouse immunoglobulins compatible with the primary antibody, for 1 h and developed with DAB system . Sections were counter stained with the Mayer’s hematoxylin, dehydrated and images were taken under microscope.DAs are low incidence tumors, yet important as they mostly occur in younger age group individuals with a high chance of recurrence and significantly long median survival time. Presently the general treatment modality is surgery followed by radiation, with mixed outcome. Better treatment strategies as well as post treatment surveillance are important unmet clinical needs. With this focus, we have studied differentially regulated proteins from the microsomal fraction from clinical tissues to understand molecular changes underlying DA and to identify proteins that may have strong secretory potential for application as post treatment surveillance markers. Considering low incidence of these tumors and sample paucity, our experimental approach has been to carry out quantitative LC-MS/MS analysis using iTRAQ, on microsomal fraction purified from pooled tissue biopsies from patients diagnosed with DA, followed by cross-comparison with transcript data from individual patient samples and/or verification of the functionally significant members by immunohistochemistry on tissue microarrays with individual samples. We also screened the proteins from the dataset applying bioinformatics for their secretory potential and identified a set of proteins that may serve as candidates for investigation towards application for post-treatment surveillance. Thus the study represents discovery-stage findings that could be used by us and others for clinical validations.A pool of biopsies from six male and female patients between 20–40 years of age group was used to prepare the microsomal fraction containing endoplasmic reticulum, golgi, intracellular vesicles, and plasma membrane proteins. This was analyzed to identify differentially expressed proteins using iTRAQ labeling of tryptic peptides followed by LC-MS/MS analysis using LTQ Orbitrap Velos mass spectrometer. Microsomal fraction from a pool of temporal lobe epilepsy surgery specimens was used as control. The workflow of the analysis is given in A total of 18,603 iTRAQ labelled peptides was identified which mapped to 2803 proteins, majority of them with multiple peptides. A total of 340 proteins were found to be differentially expressed with at least 2-fold change (218 upregulated and 122 down regulated proteins). The altered levels of each of the identified proteins were based on at least two peptides with two reporter ions for each peptide. We have identified and quantified 84 proteins with 2 peptides, 73 with 3 peptides and remaining 183 proteins with 4 or more peptides. For averaging the quantities of the proteins, we used only unique peptides identifying a protein with variability of less than 40% in the peptide ratio. Subcellular classification of the 340 differentially expressed proteins using Gene Ontology information from Human Protein Reference Database (HPRD) revealed majority (53%) of them as proteins known to be associated with the endoplasmic reticulum and plasma membrane . Supplemet al.www.oncomine.org) in DA tumors revealed a total of 195 proteins (57.4%) to be common  by Sun e common . Of thesChanges at the chromosome levels such as mutations, copy number variations are important factors that may affect downstream events relevant to tumor development. We also mapped differentially expressed proteins to the chromosome 12 which is implicated in glial tumors2426P-values associated with these molecular and cellular functions, networks and canonical pathways are shown in Ingenuity Pathway Knowledge Base classification of the protein differentials observed by us indicated following molecular and cellular functions, networks and canonical pathways. The top network identified includes molecules associated with cell-to-cell signaling and interactions, tissue development and cellular movement. Major molecular and cellular functions and canonical pathways enriched in the dataset are shown in PI3K/AKT/mTOR signaling is known to play important role in cell proliferation and cell growth and mTOR is a master regulator of cell growth through its ability to stimulate ribosome biogenesis and mRNA translation30RTK signalling is one of the most frequently observed pathway in human cancers and EGFR is one of the best known oncogenic RTK for several cancers including gliomas33Brevican core protein (BCAN) is a brain-specific chondroitin sulfate proteoglycan has been observed to be highly expressed during development, in response to injury and in primary brain tumors3738Ectonucleotide pyrophosphatase/phosphodiesterase family member 6 (ENPP6) was observed to be overexpressed in proteomic data. It is a glycosylphosphatidylinositol (GPI)-anchored alkaline lysophospholipase C predominantly expressed in brain myelin and kidney40DAs have a long median survival but invariably recur. After treatment, they may recur as Gr II or higher grade anaplastic astrocytomas (WHO grade III) or secondary glioblastomas (WHO Grade IV)54649On mapping of the differentially expressed protein dataset (n = 340) to SignalP 4.1 and TMHMM 2.0 software tools, or Exocarta database, we identified 84 signal sequence containing proteins, 106 with transmembrane domain containing proteins and 157 as exosomal proteins . Taking 5156We have identified 340 high confidence differentially expressed proteins with high resolution mass spectrometry, from the microsomal fraction of low-grade (Grade II) glioma - diffuse astrocytoma. mTOR activation, increase in ribosome biogenesis and protein translation were found to be major processes altered in these tumors; PI3Kc/mTOR pathway is also implicated in the large study carried out by TCGA. Differentially expressed proteins in this early stage, low-grade gliomas, include important regulatory proteins such as EGFR, HNRNP K and BCAN. Though not specific to DA they may be promising candidates for confirmatory diagnosis of these early stage tumors. We also provide a catalogue of differentially expressed proteins in Gr II and Gr III with secretory potential along with their proteotypic peptides which may be a useful resource for targeted investigation as surveillance markers for tumor recurrence - an important unmet clinical need.How to cite this article: Polisetty, R. V. et al. Microsomal membrane proteome of low grade diffuse astrocytomas: Differentially expressed proteins and candidate surveillance biomarkers. Sci. Rep.6, 26882; doi: 10.1038/srep26882 (2016).


Mycobacterium tuberculosis rpoB gene with rapid molecular tests GeneXpert MTB/RIF and Hain MTBDRplus. Such mutations are found in >95% of Mycobacterium tuberculosis strains resistant to RIF by conventional culture-based drug susceptibility testing (DST). However routine diagnostic screening with molecular tests uncovered specific “low level” rpoB mutations conferring resistance to RIF below the critical concentration of 1 μg/ml in some phenotypically susceptible strains. Cases with discrepant phenotypic (susceptible) and genotypic (resistant) results for resistance to RIF account for at least 10% of resistant diagnoses by molecular tests and urgently require new guidelines to inform therapeutic decision making. Eight strains with a “low level” rpoB mutation L511P were isolated by GHESKIO laboratory between 2008 and 2012 from 6 HIV-negative and 2 HIV-positive patients during routine molecular testing. Five isolates with a single L511P mutation and two isolates with double mutation L511P&M515T had MICs for RIF between 0.125 and 0.5 μg/ml and tested susceptible in culture-based DST. The eighth isolate carried a double mutation L511P&D516C and was phenotypically resistant to RIF. All eight strains shared the same spoligotype SIT 53 commonly found in Haiti but classic epidemiological investigation failed to uncover direct contacts between the patients. Whole Genome Sequencing (WGS) revealed that L511P cluster isolates resulted from a clonal expansion of an ancestral strain resistant to Isoniazid and to a very low level of RIF. Under the selective pressure of RIF-based therapy the strain acquired mutation in the M306 codon of embB followed by secondary mutations in rpoB and escalation of resistance level. This scenario highlights the importance of subcritical resistance to RIF for both clinical management of patients and public health and provides support for introducing rpoB mutations as proxy for MICs into laboratory diagnosis of RIF resistance. This study illustrates that WGS is a promising multi-purpose genotyping tool for high-burden settings as it provides both “gold standard” sequencing results for prediction of drug susceptibility and a high-resolution data for epidemiological investigation in a single assay.The World Health Organization recommends diagnosing Multidrug-Resistant Tuberculosis (MDR-TB) in high burden countries by detection of mutations in Rifampin (RIF) Resistance Determining Region of  Multidrug resistant tuberculosis (MDR-TB) is defined by resistance to at least two anti-tuberculosis drugs—Rifampin (RIF) and Isoniazid (INH). MDR-TB cases do not respond to the standard therapy and require prolonged and expensive treatment with toxic second-line antibiotics . ResistaMycobacterium tuberculosis (MTB) in medium supplemented with a critical concentration of RIF, currently set at 1 μg/ml  in Port-au-Prince, Haiti, detected several types of “low level” ar tests . StrainsThe study was approved by the Institutional Review Board of Weill Cornell Medical College  and the Institutional Review Board of GHESKIO Centres . Clinical and epidemiological data were extracted from patients’ charts. As this was a retrospective clinical chart review, the requirement for informed consent was waived by the institutional review boards.plus  or/and GeneXpert MTB/RIF  assays. Isolates found resistant with molecular tests were further analyzed by sequencing of genes linked to resistance to RIF (rpoB), INH , ethambutol (EMB) (embB), pyrazinamide (PZA) (pncA) and fluoroquinolones (gyrA) and by spoligotyping. Isolates were also tested with conventional phenotypic DST on solid and liquid media. Minimal Inhibitory Concentration (MIC) to RIF was determined with Alamar Blue Broth Microdilution assay in 96-well plates. Laboratory procedures were described in detail elsewhere –3533–35]rpoA or rpoC genes known to compensate for the fitness cost in RIF-resistant strains [WGS confirmed mutations in katG , embB  and rpoB  identified with Sanger sequencing. We found no mutations in  strains –36.Mutations in gyrA (E21Q) and gyrB (K526Q) were found in all sequenced strains. However they were not associated with resistance to Fluoroquinolones as strains tested susceptible to Ofloxacin in a concentration range of 1 to 0.031 μg/ml. Full list of annotated sequence variants is available upon request.6110 RFLP, Spoligotyping and MIRU-VNTR each capture changes in only small part of MTB genome and are often used in tandem combinations [Use of molecular tools improves the accuracy and resolution of classic epidemiological investigations , which iinations , 43. As inations , 44, 45,inations  and to dinations . ArguablOur results demonstrated superior resolution power of the WGS typing in high prevalence setting when compared to classic epidemiological investigation—it was able to confirm a possible indirect link between patients (A and B) and to uncover unknown links . At least four patients with history of treatment failure would likely have been misclassified as cases with acquired resistance, while WGS unequivocally demonstrated that they were cases of primary MDR-TB. Our unpublished data indicate that primary MDR-TB is an important factor driving MDR-TB epidemics in Haiti and so resources need to be allocated not only for MDR-TB diagnostics and treatment but also for the active tracing and screening of contacts of known TB patients. The eight isolates characterized in this study probably represent only the tip of the iceberg in the outbreak since mycobacterial culture is not routinely performed for diagnosis of TB in Haiti where GHESKIO laboratory is the only facility with culture capacity. For that reason the outbreak transmission chain could not be reconstructed and the index case was not identified.Recent WGS studies estimated the evolution rate in settings with low TB and HIV prevalence as 0.3–0.5 SNPs per genome per year resulting in accumulation of maximum 5 genetic changes in three years and 10 genetic changes in 10 years , 46. BasTo our knowledge this is the first report that delineates the fate of infections involving MTB strains with RIF MIC of 0.125 μg/ml, which is eight times lower than the currently accepted critical concentration of the drug. WGS confirmed that at least 7 out of the 8 cases in L511P cluster resulted from the clonal expansion of the same ancestral strain. Although the strain fitness was not determined in this study, it did not prevent transmission from mother to child, among distant family members, and between the casual social contacts. Immuno-compromised patient status was not a prerequisite for transmission since six of the eight patients who provided MTB isolates were HIV-negative.embB mutation at position M306. “Canonical” mutations in M306 codon of embB are necessary but not sufficient for development of a high-level resistance to EMB [embB mutation precluded the acquisition of a secondary rpoB mutation in two independent instances. Both times RIF resistance level escalated from the baseline 0.125 μg/ml—to intermediate level of 0.5 μg/ml in patient A (L511P & M515T) and to a high resistance level of >8 μg/ml in patient C (L511P & D516C).Under the selective pressure of treatment with first line TB drugs, the strain initially resistant only to INH and to the very low level of RIF, acquired e to EMB , 48. Howe to EMB . AccordiIn conclusion, this study reports the first results obtained from a project to genotype MDR-TB isolates in Haiti with WGS. While only a very limited number of strains were sequenced, we already gained novel insights into the public health significance of MTB strains with a sub-critical resistance to RIF by demonstrating their transmissibility and potential for escalation of resistance level under conditions of treatment with RIF-based regimens. We expect that introduction of routine WGS of MDR-TB strains in Haiti will provide necessary data to improve diagnosis, deepen understanding about genetic determinants of resistance and help shaping more effective public health policies to combat the disease.S1 TableList of drugs and genes adopted from . Drugs wNT—not tested* One of eight isolates with double mutations L511P&D516C was resistant to RIFE. coli genome shown in parentheses** Position in (DOCX)Click here for additional data file.


Efficiency of feed utilization is important for animal production because it can reduce greenhouse gas emissions and improve industry profitability. However, the genetic basis of feed utilization in livestock remains poorly understood. Recent developments in molecular genetics, such as platforms for genome-wide genotyping and sequencing, provide an opportunity to identify genes and pathways that influence production traits. It is known that transcriptional networks influence feed efficiency-related traits such as growth and energy balance. This study sought to identify differentially expressed genes in animals genetically divergent for Residual Feed Intake (RFI), using RNA sequencing methodology (RNA-seq) to obtain information from genome-wide expression profiles in the liver tissues of Nelore cattle.Differential gene expression analysis between high Residual Feed Intake  and low Residual Feed Intake  groups was performed to provide insights into the molecular mechanisms that underlie feed efficiency-related traits in beef cattle. A total of 112 annotated genes were identified as being differentially expressed between animals with divergent RFI phenotypes. These genes are involved in ion transport and metal ion binding; act as membrane or transmembrane proteins; and belong to gene clusters that are likely related to the transport and catalysis of molecules through the cell membrane and essential mechanisms of nutrient absorption. Genes with functions in cellular signaling, growth and proliferation, cell death and survival were also differentially expressed. Among the over-represented pathways were drug or xenobiotic metabolism, complement and coagulation cascades, NRF2-mediated oxidative stress, melatonin degradation and glutathione metabolism.Our data provide new insights and perspectives on the genetic basis of feed efficiency in cattle. Some previously identified mechanisms were supported and new pathways controlling feed efficiency in Nelore cattle were discovered. We potentially identified genes and pathways that play key roles in hepatic metabolic adaptations to oxidative stress such as those involved in antioxidant mechanisms. These results improve our understanding of the metabolic mechanisms underlying feed efficiency in beef cattle and will help develop strategies for selection towards the desired phenotype.The online version of this article (doi:10.1186/s12864-015-1464-x) contains supplementary material, which is available to authorized users. Feed efficiency-related traits are increasingly being studied because of their importance to the overall profitability of animal production. Moreover, the selection of more efficient animals reduces the land required for feed production, methane emissions and nitrogen excretion resulting from the digestion/metabolic process -3. HeritThere are several indices that are commonly used to estimate the feed efficiency of growing cattle; one of them being residual feed intake (RFI) which is independent of body weight and weight gain. RFI is used to identify individuals that deviate from their expected level of feed intake given their size and growth rate over at least a 70 day feeding period . BecauseDevelopments in molecular genetics, specifically high-throughput sequencing methods, offer a unique opportunity to identify genes and pathways that are associated with complex traits and diseases . CurrentRecent studies have identified putative quantitative trait loci (QTL) for feed efficiency on several chromosomes in Nelore populations ,11. HoweThe RFI phenotypes for this Nelore population were previously used to perform a genome-wide association study (GWAS) and the summary statistics for the population were described . Table 1After mapping reads with TopHat v2.0.6 ,14, CuffHPRT1) and Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein, Zeta (YWHAZ) and found expression patterns for these genes to be similar within each of the treatments. Finally, a principal component analysis (PCA) of FPKM values for all genes indicated that there were sufficient numbers of DE genes to differentiate the RFI groups  was used to partition the DE genes into up- and down-regulated groups with 43 DE genes being down-regulated and 69 up-regulated in the LRFI relative to the HRFI groups  and LRFI (efficient) groups identified 112 DE annotated genes. The sign of the logCOL1A1), glutathione S-transferase M1 (GSTM1), regulator of G-protein signaling 2 (RGS2), ring finger protein 150 (RNF150), solute carrier family 2 (facilitated glucose/fructose transporter), member 5 (SLC2A5) and vimentin (VIM).Six genes that were previously identified in a microarray study that profiled gene expression in the livers of Angus cattle selected for high and low RFI  were alsFABP1) also known as liver-type fatty acid-binding protein (L-FABP) was up-regulated in the LRFI group. Uncoupling protein 2  (UCP2) and fatty acid desaturase 2 (FADS2) with roles in carbohydrate and/or fatty acid metabolism and mitochondrial function were also found to be DE and up-regulated in the LRFI group.Other candidate genes previously described as functioning in the determination of traits related to feed efficiency were also found in this analysis -22. For vice versa. When analyzed using Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.7 using cattle as the background  = 0 and Var(a) = Where, Dry matter intake and average daily gain described elsewhere  were usePreparation of the mRNA samples for sequencing was performed by ESALQ Genomics Center , using the TruSeq RNA Sample Preparation Kit®  according to manufacturer’s instructions. Briefly, 100 mg of frozen liver was used to extract RNA using the TRIzol® reagent  and 2 μg of total RNA from each liver sample was used for library preparation. The concentration and purity of RNA was measured using NanoDrop™  and then sample integrity was assessed by Bioanalyzer . The mRNA was first enriched from the total RNA by using oligo dT magnetic beads, then the poly(A) RNA was fragmented and cDNA was synthesized. Next, the cDNA underwent end repair, the 3’ ends were adenylated and universal bar-coded adapters were ligated to the cDNA fragments to perform a solid phase PCR to produce the sequencing library. Following library construction, the sequencing library was evaluated and quantified using both an Agilent 2100 Bioanalyzer® and quantitative PCR with the KAPA Library Quantification kit® . Finally, libraries were pooled to perform multiplexing sequencing. Cluster generation and sequencing were performed on the Illumina HiSeq 2000®. Paired-end reads of 2 × 100 bp were produced.Bos taurus virtual transcriptome internally built by Tophat using the UMD3.1 reference genome. TopHat first extracted the transcript sequences and used Bowtie to align reads to the virtual transcriptome using a provided reference annotation file. The reads that could not be fully mapped to the virtual transcriptome were then mapped to the UMD3.1 reference genome. These reads were converted into genomic mappings and merged with the novel transcriptome mappings and splice junctions in the final output file. A total of 2 mismatches per read were allowed in alignment.Computations were performed on the HPC resources at the University of Missouri Bioinformatics Consortium (UMBC). Low-quality reads were filtered and adapter sequences trimmed using SeqClean software. TopHat v2.0.6 ,14 was tCufflinks v2.0.2  was initCuffdiff2 software was run to test for DE genes between the RFI groups with geometric normalization used to estimate transcript abundance. Correction for multiple testing  was performed using the Benjamini-Hochberg methodology. Cuffdiff2 calculated the FPKM for each transcript, primary transcript, and gene in each sample. A false discovery rate ≤ 0.05 was adopted to consider a gene as being DE.Data exploration and visualization was performed using the CummeRbund package  implemenDAVID v6.7  was usedwww.qiagen.com/ingenuity) was also used to discover and explore biological processes and the roles of DE genes. The Ingenuity Pathways Knowledge Base comprises relationships such as between genes, mRNAs and proteins to test for significantly overrepresented networks and pathways. We provided the fold changes and q-values of DE among genes from the Cuffdiff analysis to the IPA to perform the statistical analysis for the representation of each network and to visualize the results.The IPA (The RNA-seq data sets supporting the results of this study are available in the ENA repository (EMBL-EBI), under accession PRJEB7696.