The identification of small molecule inhibitors with anthelmintic activities that target conserved proteins among ruminant gastrointestinal nematodes.

Gastrointestinal nematode (GIN) infections are a major concern for the ruminant industry worldwide and result in significant production losses. Naturally occurring polyparasitism and increasing drug resistance that potentiate disease outcomes are observed among the most prevalent GINs of veterinary importance. Within the five major taxonomic clades, clade Va represents a group of GINs that predominantly affect the abomasum or small intestine of ruminants. However, the development of effective broad-spectrum anthelmintics against ruminant clade Va GINs has been challenged by a lack of comprehensive druggable genome resources. Here, we first assembled draft genomes for three clade Va species (Cooperia oncophora, Trichostrongylus colubriformis, and Ostertagia ostertagi) and compared them with closely related ruminant GINs. Genome-wide phylogenetic reconstruction showed a relationship among ruminant GINs structured by taxonomic classification. Orthogroup (OG) inference and functional enrichment analyses identified 220 clade Va-specific and Va-conserved OGs, enriched for functions related to cell cycle and cellular senescence. Further transcriptomic analysis identified 61 taxonomically and functionally conserved clade Va OGs that may function as drug targets for new broad-spectrum anthelmintics. Chemogenomic screening identified 11 compounds targeting homologs of these OGs, thus having potential anthelmintic activity. In in vitro phenotypic assays, three kinase inhibitors (digitoxigenin, K-252a, and staurosporine) exhibited broad-spectrum anthelmintic activities against clade Va GINs by obstructing the motility of exsheathed L3 (xL3) or molting of xL3 to L4. These results demonstrate valuable applications of the new ruminant GIN genomes in gaining better insights into their life cycles and offer a contemporary approach to discovering the next generation of anthelmintics.IMPORTANCEGastrointestinal nematode (GIN) infections in ruminants are caused by parasites that inhibit normal function in the digestive tract of cattle, sheep, and goats, thereby causing morbidity and mortality. Coinfection and increasing drug resistance to current therapeutic agents will continue to worsen disease outcomes and impose significant production losses on domestic livestock producers worldwide. In combination with ongoing therapeutic efforts, advancing the discovery of new drugs with novel modes of action is critical for better controlling GIN infections. The significance of this study is in assembling and characterizing new GIN genomes of Cooperia oncophora, Ostertagia ostertagi, and Trichostrongylus colubriformis for facilitating a multi-omics approach to identify novel, biologically conserved drug targets for five major GINs of veterinary importance. With this information, we were then able to demonstrate the potential of commercially available compounds as new anthelmintics.

Outcomes in Patients with Spinal Metastases Managed with Surgical Intervention.

BACKGROUND: Spinal metastases are a significant cause of morbidity in patients with advanced cancer, and management often requires surgical intervention. Although prior studies have identified factors that influence outcomes with surgery, the ability of these factors to predict outcomes remains unclear in the era of contemporary therapies, and there is a need to better identify patients who are likely to benefit from surgery. METHODS: We performed a single-center, retrospective analysis to evaluate risk factors for poor outcomes in patients with spinal metastases treated with surgery. The primary outcome was mortality at 180 days. RESULTS: A total of 128 patients were identified. Age ≥ 65 years at surgery (p = 0.0316), presence of extraspinal metastases (p = 0.0110), and ECOG performance scores >1 (p = 0.0397) were associated with mortality at 180 days on multivariate analysis. These factors and BMI ≤ 30 mg/kg2 (p = 0.0008) were also associated with worse overall survival. CONCLUSIONS: Age > 65, extraspinal metastases, and performance status scores >1 are factors associated with mortality at 180 days in patients with spinal metastases treated with surgery. Patients with these factors and BMI ≤ 30 mg/kg2 had worse overall survival. Our results support multidisciplinary discussions regarding the benefits and risks associated with surgery in patients with these risk factors.

Genomic Profiling of Lung Adenocarcinoma in Never-Smokers.

PURPOSE: Approximately 10%-40% of patients with lung cancer report no history of tobacco smoking (never-smokers). We analyzed whole-exome and RNA-sequencing data of 160 tumor and normal lung adenocarcinoma (LUAD) samples from never-smokers to identify clinically actionable alterations and gain insight into the environmental and hereditary risk factors for LUAD among never-smokers. METHODS: We performed whole-exome and RNA-sequencing of 88 and 69 never-smoker LUADs. We analyzed these data in conjunction with data from 76 never-smoker and 299 smoker LUAD samples sequenced by The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium. RESULTS: We observed a high prevalence of clinically actionable driver alterations in never-smoker LUADs compared with smoker LUADs (78%-92% v 49.5%; P < .0001). Although a subset of never-smoker samples demonstrated germline alterations in DNA repair genes, the frequency of samples showing germline variants in cancer predisposing genes was comparable between smokers and never-smokers (6.4% v 6.9%; P = .82). A subset of never-smoker samples (5.9%) showed mutation signatures that were suggestive of passive exposure to cigarette smoke. Finally, analysis of RNA-sequencing data showed distinct immune transcriptional subtypes of never-smoker LUADs that varied in their expression of clinically relevant immune checkpoint molecules and immune cell composition. CONCLUSION: In this comprehensive genomic and transcriptome analysis of never-smoker LUADs, we observed a potential role for germline variants in DNA repair genes and passive exposure to cigarette smoke in the pathogenesis of a subset of never-smoker LUADs. Our findings also show that clinically actionable driver alterations are highly prevalent in never-smoker LUADs, highlighting the need for obtaining biopsies with adequate cellularity for clinical genomic testing in these patients.

Improving Characterization of Understudied Human Microbiomes Using Targeted Phylogenetics.

Whole-genome bacterial sequences are required to better understand microbial functions, niche-specific bacterial metabolism, and disease states. Although genomic sequences are available for many of the human-associated bacteria from commonly tested body habitats (e.g., feces), as few as 13% of bacterium-derived reads from other sites such as the skin map to known bacterial genomes. To facilitate a better characterization of metagenomic shotgun reads from underrepresented body sites, we collected over 10,000 bacterial isolates originating from 14 human body habitats, identified novel taxonomic groups based on full-length 16S rRNA gene sequences, clustered the sequences to ensure that no individual taxonomic group was overselected for sequencing, prioritized bacteria from underrepresented body sites (such as skin and respiratory and urinary tracts), and sequenced and assembled genomes for 665 new bacterial strains. Here, we show that addition of these genomes improved read mapping rates of Human Microbiome Project (HMP) metagenomic samples by nearly 30% for the previously underrepresented phylum Fusobacteria, and 27.5% of the novel genomes generated here had high representation in at least one of the tested HMP samples, compared to 12.5% of the sequences in the public databases, indicating an enrichment of useful novel genomic sequences resulting from the prioritization procedure. As our understanding of the human microbiome continues to improve and to enter the realm of therapy developments, targeted approaches such as this to improve genomic databases will increase in importance from both an academic and a clinical perspective.IMPORTANCE The human microbiome plays a critically important role in health and disease, but current understanding of the mechanisms underlying the interactions between the varying microbiome and the different host environments is lacking. Having access to a database of fully sequenced bacterial genomes provides invaluable insights into microbial functions, but currently sequenced genomes for the human microbiome have largely come from a limited number of body sites (primarily feces), while other sites such as the skin, respiratory tract, and urinary tract are underrepresented, resulting in as little as 13% of bacterium-derived reads mapping to known bacterial genomes. Here, we sequenced and assembled 665 new bacterial genomes, prioritized from a larger database to select underrepresented body sites and bacterial taxa in the existing databases. As a result, we substantially improve mapping rates for samples from the Human Microbiome Project and provide an important contribution to human bacterial genomic databases for future studies.

Improving eukaryotic genome annotation using single molecule mRNA sequencing.

BACKGROUND: The advantages of Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology include long reads, low systematic bias, and high consensus read accuracy. Here we use these attributes to improve on the genome annotation of the parasitic hookworm Ancylostoma ceylanicum using PacBio RNA-Seq. RESULTS: We sequenced 192,888 circular consensus sequences (CCS) derived from cDNAs generated using the CloneTech SMARTer system. These SMARTer-SMRT libraries were normalized and size-selected providing a robust population of expressed structural genes for subsequent genome annotation. We demonstrate PacBio mRNA sequences based genome annotation improvement, compared to genome annotation using conventional sequencing-by-synthesis alone, by identifying 1609 (9.2%) new genes, extended the length of 3965 (26.7%) genes and increased the total genomic exon length by 1.9 Mb (12.4%). Non-coding sequence representation (primarily from UTRs based on dT reverse transcription priming) was particularly improved, increasing in total length by fifteen-fold, by increasing both the length and number of UTR exons. In addition, the UTR data provided by these CCS allowed for the identification of a novel SL2 splice leader sequence for A. ceylanicum and an increase in the number and proportion of functionally annotated genes. RNA-seq data also confirmed some of the newly annotated genes and gene features. CONCLUSION: Overall, PacBio data has supported a significant improvement in gene annotation in this genome, and is an appealing alternative or complementary technique for genome annotation to the other transcript sequencing technologies.

Genomic heterogeneity of ALK fusion breakpoints in non-small-cell lung cancer.

In lung adenocarcinoma, canonical EML4-ALK inversion results in a fusion protein with a constitutively active ALK kinase domain. Evidence of ALK rearrangement occurs in a minority (2-7%) of lung adenocarcinoma, and only ~60% of these patients will respond to targeted ALK inhibition by drugs such as crizotinib and ceritinib. Clinically, targeted anti-ALK therapy is often initiated based on evidence of an ALK genomic rearrangement detected by fluorescence in situ hybridization (FISH) of interphase cells in formalin-fixed, paraffin-embedded tissue sections. At the genomic level, however, ALK rearrangements are heterogeneous, with multiple potential breakpoints in EML4, and alternate fusion partners. Using next-generation sequencing of DNA and RNA together with ALK immunohistochemistry, we comprehensively characterized genomic breakpoints in 33 FISH-positive lung adenocarcinomas. Of these 33 cases, 29 (88%) had detectable DNA level ALK rearrangements involving EML4, KIF5B, or non-canonical partners including ASXL2, ATP6V1B1, PRKAR1A, and SPDYA. A subset of 12 cases had material available for RNA-Seq. Of these, eight of eight (100%) cases with DNA rearrangements showed ALK fusion transcripts from RNA-Seq; three of four cases (75%) without detectable DNA rearrangements were similarly negative by RNA-Seq, and one case was positive by RNA-Seq but negative by DNA next-generation sequencing. By immunohistochemistry, 17 of 19 (89%) tested cases were clearly positive for ALK protein expression; the remaining cases had no detectable DNA level rearrangement or had a non-canonical rearrangement not predicted to form a fusion protein. Survival analysis of patients treated with targeted ALK inhibitors demonstrates a significant difference in mean survival between patients with next-generation sequencing confirmed EML4-ALK rearrangements, and those without (20.6 months vs 5.4 months, P<0.01). Together, these data demonstrate abundant genomic heterogeneity among ALK-rearranged lung adenocarcinoma, which may account for differences in treatment response with targeted ALK inhibitors.

Genomic introgression mapping of field-derived multiple-anthelmintic resistance in Teladorsagia circumcincta.

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans.

Genome Sequence of Christensenella minuta DSM 22607T.

Obesity influences and is influenced by the human gut microbiome. Here, we present the genome of Christensenella minuta, a highly heritable bacterial species which has been found to be strongly associated with obesity through an unknown biological mechanism. This novel genome provides a valuable resource for future obesity therapeutic studies.

Genomes of Fasciola hepatica from the Americas Reveal Colonization with Neorickettsia Endobacteria Related to the Agents of Potomac Horse and Human Sennetsu Fevers.

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 domesticated 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 millieux 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 ruminants and humans.

Genomic diversity in Onchocerca volvulus and its Wolbachia endosymbiont.

Ongoing elimination efforts have altered the global distribution of Onchocerca volvulus, the agent of river blindness, and further population restructuring is expected as efforts continue. Therefore, a better understanding of population genetic processes and their effect on biogeography is needed to support elimination goals. We describe O. volvulus genome variation in 27 isolates from the early 1990s (before widespread mass treatment) from four distinct locales: Ecuador, Uganda, the West African forest and the West African savanna. We observed genetic substructuring between Ecuador and West Africa and between the West African forest and savanna bioclimes, with evidence of unidirectional gene flow from savanna to forest strains. We identified forest:savanna-discriminatory genomic regions and report a set of ancestry informative loci that can be used to differentiate between forest, savanna and admixed isolates, which has not previously been possible. We observed mito-nuclear discordance possibly stemming from incomplete lineage sorting. The catalogue of the nuclear, mitochondrial and endosymbiont DNA variants generated in this study will support future basic and translational onchocerciasis research, with particular relevance for ongoing control programmes, and boost efforts to characterize drug, vaccine and diagnostic targets.

Genome Sequences of 12 Bacterial Isolates Obtained from the Urine of Pregnant Women.

The presence of bacteria in urine can pose significant risks during pregnancy. However, there are few reference genome strains for many common urinary bacteria. We isolated 12 urinary strains of Streptococcus, Staphylococcus, Citrobacter, Gardnerella, and Lactobacillus These strains and their genomes are now available to the research community.

Genome Sequences of 11 Human Vaginal Actinobacteria Strains.

The composition of the vaginal microbiota is an important health determinant. Several members of the phylum Actinobacteria have been implicated in bacterial vaginosis, a condition associated with many negative health outcomes. Here, we present 11 strains of vaginal Actinobacteria (now available through BEI Resources) along with draft genome sequences.

Genome Sequences of 14 Firmicutes Strains Isolated from the Human Vagina.

Research on vaginal infections is currently limited by a lack of available fully sequenced bacterial reference strains. Here, we present strains (now available through BEI Resources) and genome sequences for a set of 14 vaginal isolates from the phylum Firmicutes These genome sequences provide a valuable resource for future research in understanding the role of Gram-positive bacteria in vaginal health and disease.

Genome Sequences of Nine Gram-Negative Vaginal Bacterial Isolates.

The vagina is home to a wide variety of bacteria that have great potential to impact human health. Here, we announce reference strains (now available through BEI Resources) and draft genome sequences for 9 Gram-negative vaginal isolates from the taxa Citrobacter, Klebsiella, Fusobacterium, Proteus, and Prevotella.

Dictyocaulus viviparus genome, variome and transcriptome elucidate lungworm biology and support future intervention.

The bovine lungworm, Dictyocaulus viviparus (order Strongylida), is an important parasite of livestock that causes substantial economic and production losses worldwide. Here we report the draft genome, variome, and developmental transcriptome of D. viviparus. The genome (161 Mb) is smaller than those of related bursate nematodes and encodes fewer proteins (14,171 total). In the first genome-wide assessment of genomic variation in any parasitic nematode, we found a high degree of sequence variability in proteins predicted to be involved host-parasite interactions. Next, we used extensive RNA sequence data to track gene transcription across the life cycle of D. viviparus, and identified genes that might be important in nematode development and parasitism. Finally, we predicted genes that could be vital in host-parasite interactions, genes that could serve as drug targets, and putative RNAi effectors with a view to developing functional genomic tools. This extensive, well-curated dataset should provide a basis for developing new anthelmintics, vaccines, and improved diagnostic tests and serve as a platform for future investigations of drug resistance and epidemiology of the bovine lungworm and related nematodes.

Cracking the nodule worm code advances knowledge of parasite biology and biotechnology to tackle major diseases of livestock.

Many infectious diseases caused by eukaryotic pathogens have a devastating, long-term impact on animal health and welfare. Hundreds of millions of animals are affected by parasitic nematodes of the order Strongylida. Unlocking the molecular biology of representatives of this order, and understanding nematode-host interactions, drug resistance and disease using advanced technologies could lead to entirely new ways of controlling the diseases that they cause. Oesophagostomum dentatum (nodule worm; superfamily Strongyloidea) is an economically important strongylid nematode parasite of swine worldwide. The present article reports recent advances made in biology and animal biotechnology through the draft genome and developmental transcriptome of O. dentatum, in order to support biological research of this and related parasitic nematodes as well as the search for new and improved interventions. This first genome of any member of the Strongyloidea is 443 Mb in size and predicted to encode 25,291 protein-coding genes. Here, we review the dynamics of transcription throughout the life cycle of O. dentatum, describe double-stranded RNA interference (RNAi) machinery and infer molecules involved in development and reproduction, and in inducing or modulating immune responses or disease. The secretome predicted for O. dentatum is particularly rich in peptidases linked to interactions with host tissues and/or feeding activity, and a diverse array of molecules likely involved in immune responses. This research progress provides an important resource for future comparative genomic and molecular biological investigations as well as for biotechnological research toward new anthelmintics, vaccines and diagnostic tests.

Extreme Sensory Complexity Encoded in the 10-Megabase Draft Genome Sequence of the Chromatically Acclimating Cyanobacterium Tolypothrix sp. PCC 7601.

Tolypothrix sp. PCC 7601 is a freshwater filamentous cyanobacterium with complex responses to environmental conditions. Here, we present its 9.96-Mbp draft genome sequence, containing 10,065 putative protein-coding sequences, including 305 predicted two-component system proteins and 27 putative phytochrome-class photoreceptors, the most such proteins in any sequenced genome.

Helminth.net: expansions to Nematode.net and an introduction to Trematode.net.

Helminth.net (http://www.helminth.net) is the new moniker for a collection of databases: Nematode.net and Trematode.net. Within this collection we provide services and resources for parasitic roundworms (nematodes) and flatworms (trematodes), collectively known as helminths. For over a decade we have provided resources for studying nematodes via our veteran site Nematode.net (http://nematode.net). In this article, (i) we provide an update on the expansions of Nematode.net that hosts omics data from 84 species and provides advanced search tools to the broad scientific community so that data can be mined in a useful and user-friendly manner and (ii) we introduce Trematode.net, a site dedicated to the dissemination of data from flukes, flatworm parasites of the class Trematoda, phylum Platyhelminthes. Trematode.net is an independent component of Helminth.net and currently hosts data from 16 species, with information ranging from genomic, functional genomic data, enzymatic pathway utilization to microbiome changes associated with helminth infections. The databases' interface, with a sophisticated query engine as a backbone, is intended to allow users to search for multi-factorial combinations of species' omics properties. This report describes updates to Nematode.net since its last description in NAR, 2012, and also introduces and presents its new sibling site, Trematode.net.

Genome of the human hookworm Necator americanus.

The hookworm Necator americanus is the predominant soil-transmitted human parasite. Adult worms feed on blood in the small intestine, causing iron-deficiency anemia, malnutrition, growth and development stunting in children, and severe morbidity and mortality during pregnancy in women. We report sequencing and assembly of the N. americanus genome (244 Mb, 19,151 genes). Characterization of this first hookworm genome sequence identified genes orchestrating the hookworm's invasion of the human host, genes involved in blood feeding and development, and genes encoding proteins that represent new potential drug targets against hookworms. N. americanus has undergone a considerable and unique expansion of immunomodulator proteins, some of which we highlight as potential treatments against inflammatory diseases. We also used a protein microarray to demonstrate a postgenomic application of the hookworm genome sequence. This genome provides an invaluable resource to boost ongoing efforts toward fundamental and applied postgenomic research, including the development of new methods to control hookworm and human immunological diseases.