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Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Prior studies examining the mutational landscape of GBM revealed recurrent alterations in genes that regulate the same growth control pathways. To this regard, ~ 40% of GBM harbor EGFR alterations, whereas BRAF variants are rare. Existing data suggests that gain-of-function mutations in these genes are mutually exclusive. This study was designed to explore the clinical, pathological, and molecular differences between EGFR- and BRAF-mutated GBM. We reviewed retrospective clinical data from 89 GBM patients referred for molecular testing between November 2012 and December 2015. Differences in tumor mutational profile, location, histology, and survival outcomes were compared in patients with EGFR- versus BRAF-mutated tumors, and microarray data from The Cancer Genome Atlas was used to assess differential gene expression between the groups. Individuals with BRAF-mutant tumors were typically younger and survived longer relative to those with EGFR-mutant tumors, even in the absence of targeted treatments. BRAF-mutant tumors lacked distinct histomorphology but exhibited unique localization in the brain, typically arising adjacent to the lateral ventricles. Compared to EGFR- and IDH1-mutant tumors, BRAF-mutant tumors showed increased expression of genes related to a trophoblast-like phenotype, specifically HLA-G and pregnancy specific glycoproteins, that have been implicated in invasion and immune evasion. Taken together, these observations suggest a distinct clinical presentation, brain location, and gene expression profile for BRAF-mutant tumors. Pending further study, this may prove useful in the stratification and management of GBM. [ABSTRACT FROM AUTHOR]

Meningiomas are common in adults (~35% of brain tumors) but rare in children, where they exhibit unique clinical, pathological and molecular features compared to adult counterparts. Thus, data generated from adult cohorts may be imperfectly suited to guiding diagnostic, prognostic and treatment decisions for children. We studied 50 meningioma patients ≤18 years with available clinical and pathological data to address the need for data obtained in the pediatric setting. As previously described, we noted a slight bias toward male patients and a higher proportion of spinal tumors compared to adults. Thirty‐eight of 50 specimens were further analyzed by next generation sequencing. Loss‐of‐function mutations in NF2 and chromosome 22 losses were common, but pathogenic variants in other genes (SMARCB1, FUBP1, BRAF, TERT promoter, CHEK2, SMAD and GATA3) were identified in a minority of cases. Copy number variants outside of chromosomes 22 and 1 were infrequent. H3K27 hypomethylation, a useful biomarker in adult tumors, was not found in our cohort. In exploring the correlation between mitotic count and recurrence‐free survival, we found a threshold of six mitoses per 10 high powered fields as the optimal cutoff in predicting recurrence‐free survival. If independently validated in larger studies, adjusted grading thresholds could enhance the clinical management of pediatric meningiomas. [ABSTRACT FROM AUTHOR]

Abstract Meningiomas are among the most common tumors of the adult central nervous system (CNS). They are classified by the World Health Organization into three pathologic grades with increasing severity: grade I are benign with favorable treatment outcomes and low recurrence rates while grade III display malignant behavior and poor progression-free survival. Previous studies have shown that inactivation of NF-2 is the most common genetic event in high-grade meningioma; however, there is dearth of molecular data to distinguish grade II (AM-II) from the even more aggressive grade III (AM-III). As part of a routine diagnostic workup, 19 AM-II and 5 AM-III were submitted for targeted sequencing on a panel of twenty-four genes relevant to CNS tumors. The data generated during the course of clinical care was collected and re-analyzed with the aim of identifying molecular features to distinguish AM-II and AM-III. Our cases contained several well-characterized, potentially actionable mutations, but we did not find any novel, recurrent sequence variants. Copy number variations were common in both AM-II and AM-III; chr22q loss was the most prevalent followed in decreasing frequency by losses of chr1p, chr14q, and chr10. In particular, chr10 loss was noted in 4 of 5 AM-III cases but none of the AM-II cases. This suggests that chr10 loss may serve as a diagnostic and perhaps a prognostic marker to differentiate AM-II from AM-III. If confirmed in larger studies, our finding could further aid the classification of meningioma. [ABSTRACT FROM AUTHOR]

Ascaris suum provides a powerful model for studying parasitic nematodes, including individual tissues such as the intestine, an established target for anthelmintic treatments. Here, we add a valuable experimental component to our existing functional, proteomic, transcriptomic and phylogenomic studies of the Ascaris suum intestine, by developing a method to manipulate intestinal cell functions via direct delivery of experimental treatments (in this case, double-stranded (ds)RNA) to the apical intestinal membrane. We developed an intestinal perfusion method for direct, controlled delivery of dsRNA/heterogeneous small interfering (hsi) RNA into the intestinal lumen for experimentation. RNA-Seq (22 samples) was used to assess influences of the method on global intestinal gene expression. Successful mRNA-specific knockdown in intestinal cells of adult A. suum was accomplished with this new experimental method. Global transcriptional profiling confirmed that targeted transcripts were knocked down more significantly than any others, with only 12 (0.07% of all genes) or 238 (1.3%) off-target gene transcripts consistently differentially regulated by dsRNA treatment or the perfusion experimental design, respectively (after 24 h). The system supports controlled, effective delivery of treatments (dsRNA/hsiRNA) to the apical intestinal membrane with relatively minor off-target effects, and builds on our experimental model to dissect A. suum intestinal cell functions with broad relevance to parasitic nematodes. [ABSTRACT FROM AUTHOR]

Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domestic ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer milieus offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis’ gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in cattle and humans. [ABSTRACT FROM AUTHOR]

Background: Paragonimiasis is an important and widespread neglected tropical disease. Fifteen Paragonimus species are human pathogens, but two of these, Paragonimus westermani and P. skrjabini, are responsible for the bulk of human disease. Despite their medical and economic significance, there is limited information on the gene content and expression of Paragonimus lung flukes. Results: The transcriptomes of adult P. westermani and P. skrjabini were studied with deep sequencing technology. Approximately 30 million reads per species were assembled into 21,586 and 25,825 unigenes for P. westermani and P. skrjabini, respectively. Many unigenes showed homology with sequences from other food-borne trematodes, but 1,217 high-confidence Paragonimus-specific unigenes were identified. Analyses indicated that both species have the potential for aerobic and anaerobic metabolism but not de novo fatty acid biosynthesis and that they may interact with host signaling pathways. Some 12,432 P. westermani and P. skrjabini unigenes showed a clear correspondence in bi-directional sequence similarity matches. The expression of shared unigenes was mostly well correlated, but differentially expressed unigenes were identified and shown to be enriched for functions related to proteolysis for P. westermani and microtubule based motility for P. skrjabini. Conclusions: The assembled transcriptomes of P. westermani and P. skrjabini, inferred proteins, and extensive functional annotations generated for this project (including identified primary sequence similarities to various species, protein domains, biological pathways, predicted proteases, molecular mimics and secreted proteins, etc.) represent a valuable resource for hypothesis driven research on these medically and economically important species. [ABSTRACT FROM AUTHOR]

Background: Paragonimiasis is a food-borne trematode infection acquired by eating raw or undercooked crustaceans. It is a major public health problem in the far East, but it also occurs in South Asia, Africa, and in the Americas. Paragonimus worms cause chronic lung disease with cough, fever and hemoptysis that can be confused with tuberculosis or other non-parasitic diseases. Treatment is straightforward, but diagnosis is often delayed due to a lack of reliable parasitological or serodiagnostic tests. Hence, the purpose of this study was to use a systems biology approach to identify key parasite proteins that may be useful for development of improved diagnostic tests. Methodology/Principal Findings: The transcriptome of adult Paragonimus kellicotti was sequenced with Illumina technology. Raw reads were pre-processed and assembled into 78,674 unique transcripts derived from 54,622 genetic loci, and 77,123 unique protein translations were predicted. A total of 2,555 predicted proteins (from 1,863 genetic loci) were verified by mass spectrometric analysis of total worm homogenate, including 63 proteins lacking homology to previously characterized sequences. Parasite proteins encoded by 321 transcripts (227 genetic loci) were reactive with antibodies from infected patients, as demonstrated by immunoaffinity purification and high-resolution liquid chromatography-mass spectrometry. Serodiagnostic candidates were prioritized based on several criteria, especially low conservation with proteins in other trematodes. Cysteine proteases, MFP6 proteins and myoglobins were abundant among the immunoreactive proteins, and these warrant further study as diagnostic candidates. Conclusions: The transcriptome, proteome and immunolome of adult P. kellicotti represent a major advance in the study of Paragonimus species. These data provide a powerful foundation for translational research to develop improved diagnostic tests. Similar integrated approaches may be useful for identifying novel targets for drugs and vaccines in the future. Author Summary: Paragonimiasis is a food-borne trematode infection that people acquire when they eat raw or undercooked crustaceans. Disease symptoms (including cough, fever, blood in sputum, etc.) can be similar to those observed in patients with tuberculosis or bacterial pneumonia, frequently resulting in misdiagnosis. Although the infection is relatively easy to treat, diagnosis is complicated. Available diagnostic assays rely on total parasite homogenate to facilitate the detection of Paragonimus-specific antibodies in patients. Though these blot-based assays have shown high sensitivity and specificity, they are inconvenient because total parasite homogenate is not readily available. This study used next generation genomic and proteomic methods to identify transcripts and proteins expressed in adult Paragonimus flukes. We then used sera from patients infected with P. kellicotti to isolate immunoreactive proteins, and these were analyzed by mass spectrometry. The annotated transcriptome and the associated proteome of the antibody immune response represent a significant advance in research on Paragonimus. This information will be a valuable resource for further research on Paragonimus and paragonimiasis. Thus this project illustrates the potential power of employing systems biology for translational research in parasitology. [ABSTRACT FROM AUTHOR]

Background: Paragonimiasis is a food-borne trematode infection acquired by eating raw or undercooked crustaceans. It is a major public health problem in the far East, but it also occurs in South Asia, Africa, and in the Americas. Paragonimus worms cause chronic lung disease with cough, fever and hemoptysis that can be confused with tuberculosis or other non-parasitic diseases. Treatment is straightforward, but diagnosis is often delayed due to a lack of reliable parasitological or serodiagnostic tests. Hence, the purpose of this study was to use a systems biology approach to identify key parasite proteins that may be useful for development of improved diagnostic tests. Methodology/Principal Findings: The transcriptome of adult Paragonimus kellicotti was sequenced with Illumina technology. Raw reads were pre-processed and assembled into 78,674 unique transcripts derived from 54,622 genetic loci, and 77,123 unique protein translations were predicted. A total of 2,555 predicted proteins (from 1,863 genetic loci) were verified by mass spectrometric analysis of total worm homogenate, including 63 proteins lacking homology to previously characterized sequences. Parasite proteins encoded by 321 transcripts (227 genetic loci) were reactive with antibodies from infected patients, as demonstrated by immunoaffinity purification and high-resolution liquid chromatography-mass spectrometry. Serodiagnostic candidates were prioritized based on several criteria, especially low conservation with proteins in other trematodes. Cysteine proteases, MFP6 proteins and myoglobins were abundant among the immunoreactive proteins, and these warrant further study as diagnostic candidates. Conclusions: The transcriptome, proteome and immunolome of adult P. kellicotti represent a major advance in the study of Paragonimus species. These data provide a powerful foundation for translational research to develop improved diagnostic tests. Similar integrated approaches may be useful for identifying novel targets for drugs and vaccines in the future. [ABSTRACT FROM AUTHOR]

Background: Alternative splicing (AS) of mRNA is a vital mechanism for enhancing genomic complexity in eukaryotes. Spliced isoforms of the same gene can have diverse molecular and biological functions and are often differentially expressed across various tissues, times, and conditions. Thus, AS has important implications in the study of parasitic nematodes with complex life cycles. Transcriptomic datasets are available from many species, but data must be revisited with splice-aware assembly protocols to facilitate the study of AS in helminthes. Methods: We sequenced cDNA from the model worm Caenorhabditis elegans using 454/Roche technology for use as an experimental dataset. Reads were assembled with Newbler software, invoking the cDNA option. Several combinations of parameters were tested and assembled transcripts were verified by comparison with previously reported C. elegans genes and transcript isoforms and with Illumina RNAseq data. Results: Thoughtful adjustment of program parameters increased the percentage of assembled transcripts that matched known C. elegans sequences, decreased mis-assembly rates (i.e., cis- and trans-chimeras), and improved the coverage of the geneset. The optimized protocol was used to update de novo transcriptome assemblies from nine parasitic nematode species, including important pathogens of humans and domestic animals. Our assemblies indicated AS rates in the range of 20-30%, typically with 2-3 transcripts per AS locus, depending on the species. Transcript isoforms from the nine species were translated and searched for similarity to known proteins and functional domains. Some 21 InterPro domains, including several involved in nucleotide and chromatin binding, were statistically correlated with AS genetic loci. In most cases, the Roche/454 data explored in this study are the only sequences available from the species in question; however, the recently published genome of the human hookworm Necator americanus provided an additional opportunity to validate our results. Conclusions: Our optimized assembly parameters facilitated the first survey of AS among parasitic nematodes. The nine transcriptome assemblies, their protein translations, and basic annotations are available from Nematode. net as a resource for the research community. These should be useful for studies of specific genes and gene families of interest as well as for curating draft genome assemblies as they become available. [ABSTRACT FROM AUTHOR]

Abstract: Lymphatic filariasis is caused by three closely related nematode parasites: Wuchereria bancrofti, Brugia malayi and Brugia timori. These species have many ecological variants that differ in several aspects of their biology such as mosquito vector species, host range, periodicity, and morphology. Although the genome of B. malayi (the first genome sequenced from a parasitic nematode) has been available for more than five years, very little is known about genetic variability among the lymphatic dwelling filariae. The genetic diversity among these worms is not only interesting from a biological perspective, but it may have important practical implications for the Global Program to Eliminate Lymphatic Filariasis, as the parasites may respond differently to diagnostic tests and/or medical interventions. Therefore, better information on their genetic variability is urgently needed. With improved methods for nucleic acid extraction and recent advances in sequencing chemistry and instrumentation, this gap can be filled relatively inexpensively. Improved information on filarial genetic diversity may increase the chances of success for lymphatic filariasis elimination programs. [Copyright &y& Elsevier]

Background: Most filarial species in the genus Onchocerca depend on Wolbachia endobacteria to successfully carry out their life cycle. O. flexuosa is a Wolbachia-free species, but its genome contains Wolbachia-like sequences presumably obtained from Wolbachia via horizontal gene transfer. Proteogenomic studies have shown that many of these Wolbachia-like sequences are expressed in adult worms. Methods: Six Wolbachia-like sequences in O. flexuosa were chosen for further study based on their sequence conservation with Wolbachia genes, length of predicted open reading frames, and expression at the RNA and/or protein levels. In situ hybridization and immunohistochemical labeling were used to localize Wolbachia-like transcripts and peptides in adult worm tissues. Results: RNA probes representing three of the six target sequences produced hybridization signals in worm tissues. These probes bound to transcripts in the intestine and lateral chords of both sexes, in the hypodermis, median chords and uteri in females, and in sperm precursor cells in males. Antibodies raised to three peptides corresponding to these transcripts bound to specific bands in a soluble extract of adult O. flexuosa by Western blot that were not labeled by control antibodies in pre-immune serum. Two of the three antibodies produced labeling patterns in adult worm sections that were similar to those of the RNA probes, while the third produced a different pattern. Conclusions: A subset of the Wolbachia-like sequences present in the genome of the Wolbachia-free filarial species O. flexuosa are transcribed in tissues where Wolbachia reside in infected filarial species. Some of the peptides and/or proteins derived from these transcripts appear to be concentrated in the same tissues while others may be exported to other regions of the worm. These results suggest that horizontally transferred Wolbachia genes and gene products may replicate important Wolbachia functions in uninfected filarial worms. [ABSTRACT FROM AUTHOR]

Most filarial parasites in the subfamilies Onchocercinae and Dirofilariinae depend on Wolbachia endobacteria to successfully carry out their life cycle. Recently published data indicate that the few Wolbachia-free species in these subfamilies were infected in the distant past and have subsequently shed their endosymbionts. We used an integrated transcriptomic and proteomic analysis of Onchocerca flexuosa to explore the molecular mechanisms that allow worms of this species to survive without a bacterial partner. Roche/454 sequencing of the adult transcriptome produced 16,814 isogroup and 47,252 singleton sequences that are estimated to represent approximately 41% of the complete gene set. Sequences similar to 97 Wolbachia genes were identified from the transcriptome, some of which appear on the same transcripts as sequences similar to nematode genes. Computationally predicted peptides, including those with similarity to Wolbachia proteins, were classified at the domain and pathway levels in order to assess the metabolic capabilities of O. flexuosa and compare against the Wolbachia-dependent model filaria, Brugia malayi. Transcript data further facilitated a shotgun proteomic analysis of O. flexuosa adult worm lysate, resulting in the identification of 1,803 proteins. Three of the peptides detected by mass spectroscopy map to two ABC transport-related proteins from Wolbachia. Antibodies raised to one of the Wolbachia-like peptides labeled a single 38 kDa band on Western blots of O. flexuosa lysate and stained specific worm tissues by immunohistology. Future studies will be required to determine the exact functions of Wolbachia-like peptides and proteins in O. flexuosa and to assess their roles in worm biology. [ABSTRACT FROM AUTHOR]

Background: Wolbachia are among the most abundant symbiotic microbes on earth; they are present in about 66% of all insect species, some spiders, mites and crustaceans, and most filarial nematode species. Infected filarial nematodes, including many pathogens of medical and veterinary importance, depend on Wolbachia for proper development and survival. The mechanisms behind this interdependence are not understood. Interestingly, a minority of filarial species examined to date are naturally Wolbachia-free. Methodology/Principal Findings: We used 454 pyrosequencing to survey the genomes of two distantly related Wolbachiafree filarial species, Acanthocheilonema viteae and Onchocerca flexuosa. This screen identified 49 Wolbachia-like DNA sequences in A. viteae and 114 in O. flexuosa. qRT-PCR reactions detected expression of 30 Wolbachia-like sequences in A. viteae and 56 in O. flexuosa. Approximately half of these appear to be transcribed from pseudogenes. In situ hybridization showed that two of these pseudogene transcripts were specifically expressed in developing embryos and testes of both species. Conclusions/Significance: These results strongly suggest that the last common ancestor of extant filarial nematodes was infected with Wolbachia and that this former endosymbiont contributed to their genome evolution. Horizontally transferred Wolbachia DNA may explain the ability of some filarial species to live and reproduce without the endosymbiont while other species cannot. [ABSTRACT FROM AUTHOR]

Abstract: Genetic characterization of field isolates and clinical specimens of filarial nematodes is often limited by a shortage of DNA; therefore, we evaluated a multiple displacement amplification (MDA) based whole genome amplification method. The quality of amplified DNA was examined by conventional PCR, real-time PCR, and DNA hybridization. MDA of 5.0ng of adult Brugia malayi DNA and one-fifteenth of the DNA isolated from a single microfilaria resulted in 6.3 and 4.2μg of amplified DNA, respectively. Amplified DNA was equivalent to native genomic DNA for hybridization to B. malayi BAC library clones or to an oligonucleotide microarray with approximately 18,000 filarial DNA sequences. MDA is useful for whole genome amplification of filarial DNA from very small amounts of starting material. This technology will permit detailed studies of genetic diversity that were not previously feasible. [Copyright &y& Elsevier]

Background Paragonimus spp. (lung flukes) are among the most injurious foodborne helminths, infecting ∼23 million people and subjecting ∼292 million to infection risk. Paragonimiasis is acquired from infected undercooked crustaceans and primarily affects the lungs but often causes lesions elsewhere including the brain. The disease is easily mistaken for tuberculosis owing to similar pulmonary symptoms, and accordingly, diagnostics are in demand. Results We assembled, annotated, and compared draft genomes of 4 prevalent and distinct Paragonimus species: Paragonimus miyazakii, Paragonimus westermani, Paragonimus kellicotti , and Paragonimus heterotremus. Genomes ranged from 697 to 923 Mb, included 12,072–12,853 genes, and were 71.6–90.1% complete according to BUSCO. Orthologous group analysis spanning 21 species (lung, liver, and blood flukes, additional platyhelminths, and hosts) provided insights into lung fluke biology. We identified 256 lung fluke–specific and conserved orthologous groups with consistent transcriptional adult-stage Paragonimus expression profiles and enriched for iron acquisition, immune modulation, and other parasite functions. Previously identified Paragonimus diagnostic antigens were matched to genes, providing an opportunity to optimize and ensure pan- Paragonimus reactivity for diagnostic assays. Conclusions This report provides advances in molecular understanding of Paragonimus and underpins future studies into the biology, evolution, and pathogenesis of Paragonimus and related foodborne flukes. We anticipate that these novel genomic and transcriptomic resources will be invaluable for future lung fluke research. [ABSTRACT FROM AUTHOR]