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.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens, including fungal infections. Herein, we show that the simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from that of the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and neglected tropical diseases (NTDs) targeted for elimination by 2030.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens including fungal infections. Since these diseases target the most vulnerable communities who are disadvantaged by health and socio-economic factors, new agents should be, if possible, easy-to-prepare to allow for commercialization based on their low cost. In this study, we show that simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and NTDs targeted for elimination by 2030. Overall, the discovery of these new compounds with broad-spectrum activity opens new avenues for the development of treatments for several current human infections, either caused by fungi or by parasites, including other NTDs, as well as newly emerging diseases. ONE-SENTENCE SUMMARY: Simple derivatives of the well-known antifungal drug fluconazole were found to be highly effective in vivo against fungal infections, and also potent against the parasitic nematode Brugia, which causes lymphatic filariasis and against Trichuris, one of the soil-transmitted helminths that infects millions of people globally.

Aspartyl Protease Inhibitors as Anti-Filarial Drugs.

The current treatments for lymphatic filariasis and onchocerciasis do not effectively kill the adult parasitic nematodes, allowing these chronic and debilitating diseases to persist in millions of people. Thus, the discovery of new drugs with macrofilaricidal potential to treat these filarial diseases is critical. To facilitate this need, we first investigated the effects of three aspartyl protease inhibitors (APIs) that are FDA-approved as HIV antiretroviral drugs on the adult filarial nematode, Brugia malayi and the endosymbiotic bacteria, Wolbachia. From the three hits, nelfinavir had the best potency with an IC50 value of 7.78 µM, followed by ritonavir and lopinavir with IC50 values of 14.3 µM and 16.9 µM, respectively. The three APIs have a direct effect on killing adult B. malayi after 6 days of exposure in vitro and did not affect the Wolbachia titers. Sequence conservation and stage-specific gene expression analysis identified Bm8660 as the most likely primary aspartic protease target for these drug(s). Immunolocalization using antibodies raised against the Bm8660 ortholog of Onchocerca volvulus showed it is strongly expressed in female B. malayi, especially in metabolically active tissues such as lateral and dorsal/ventral chords, hypodermis, and uterus tissue. Global transcriptional response analysis using adult female B. pahangi treated with APIs identified four additional aspartic proteases differentially regulated by the three effective drugs, as well as significant enrichment of various pathways including ubiquitin mediated proteolysis, protein kinases, and MAPK/AMPK/FoxO signaling. In vitro testing against the adult gastro-intestinal nematode Trichuris muris suggested broad-spectrum potential for these APIs. This study suggests that APIs may serve as new leads to be further explored for drug discovery to treat parasitic nematode infections.

ADAS-viewer: web-based application for integrative analysis of multi-omics data in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder and is represented by complicated biological mechanisms and complexity of brain tissue. Our understanding of the complicated molecular architecture that contributes to AD progression benefits from performing comprehensive and systemic investigations with multi-layered molecular and biological data from different brain regions. Since recently different independent studies generated various omics data in different brain regions of AD patients, multi-omics data integration can be a useful resource for better comprehensive understanding of AD. Here we present a web platform, ADAS-viewer, that provides researchers with the ability to comprehensively investigate and visualize multi-omics data from multiple brain regions of AD patients. ADAS-viewer offers means to identify functional changes in transcript and exon expression (i.e., alternative splicing) along with associated genetic or epigenetic regulatory effects. Specifically, it integrates genomic, transcriptomic, methylation, and miRNA data collected from seven different brain regions (cerebellum, temporal cortex, dorsolateral prefrontal cortex, frontal pole, inferior frontal gyrus, parahippocampal gyrus, and superior temporal gyrus) across three independent cohort datasets. ADAS-viewer is particularly useful as a web-based application for analyzing and visualizing multi-omics data across multiple brain regions at both transcript and exon level, allowing the identification of candidate biomarkers of Alzheimer's disease.

Transcriptome analysis of alternative splicing in the pathogen life cycle in human foreskin fibroblasts infected with Trypanosoma cruzi.

Trypanosoma cruzi is an intracellular protozoan parasite that causes Chagas disease as a zoonotic pathogen. The parasite has been shown to remodel expression in the host transcriptome under different conditions. Although alternative splicing (AS) is involved in virtually every biological function in eukaryotes, including cellular differentiation and responses to immune reactions, host AS events that occur as a result of T. cruzi infection have yet to be explored. In this study, we bioinformatically investigated the transcriptome AS dynamics of T. cruzi (Y strain) infected human foreskin fibroblasts using RNA-Seq data captured over four timepoints (4, 24, 48, and 72 h post infection (hpi)). We identified 1768, 399, 250, and 299 differentially expressed exons (AS exons) at 4, 24, 48, and 72 hpi, respectively, showing that host AS mechanism may have a significant role in the intracellular life cycle of the parasite. We present an exon skipping event in HDAC7, which is a candidate gene that is important in the parasite's cell cycle. To sum up, this bioinformatics analysis of transcriptome may provide new potential insight into AS regulation in human foreskin fibroblast (HFF) cells infected by T. cruzi and into its implication to the parasite life cycle. Moreover, identified AS genes may provide new potential molecular candidates for improving treatment.

Comparative genomics and transcriptomics of 4 Paragonimus species provide insights into lung fluke parasitism and pathogenesis.

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.

Noda-Like RNA Viruses Infecting Caenorhabditis Nematodes: Sympatry, Diversity, and Reassortment.

Three RNA viruses related to nodaviruses were previously described to naturally infect the nematode Caenorhabditis elegans and its relative, Caenorhabditis briggsae Here, we report on a collection of more than 50 viral variants from wild-caught Caenorhabditis. We describe the discovery of a new related virus, the Melník virus, infecting C. briggsae, which similarly infects intestinal cells. In France, a frequent pattern of coinfection of C. briggsae by the Santeuil virus and Le Blanc virus was observed at the level of an individual nematode and even a single cell. We do not find evidence of reassortment between the RNA1 and RNA2 molecules of Santeuil and Le Blanc viruses. However, by studying patterns of evolution of each virus, reassortments of RNA1 and RNA2 among variants of each virus were identified. We develop assays to test the relative infectivity and competitive ability of the viral variants and detect an interaction between host genotype and Santeuil virus genotype, such that the result depends on the host strain.IMPORTANCE The roundworm Caenorhabditis elegans is a laboratory model organism in biology. We study natural populations of this small animal and its relative, C. briggsae, and the viruses that infect them. We previously discovered three RNA viruses related to nodaviruses and here describe a fourth one, called the Melník virus. These viruses have a genome composed of two RNA molecules. We find that two viruses may infect the same animal and the same cell. The two RNA molecules may be exchanged between variants of a given viral species. We study the diversity of each viral species and devise an assay of their infectivity and competitive ability. Using this assay, we show that the outcome of the competition also depends on the host.

Characterization of cell death in Escherichia coli mediated by XseA, a large subunit of exonuclease VII.

Exonuclease VII (ExoVII) of Escherichia coli is a single strandspecific DNA nuclease composed of two different subunits: the large subunit, XseA, and the small subunit, XseB. In this study, we found that multicopy single-stranded DNAs (msDNAs), Ec83 and Ec78, are the in vivo substrates of ExoVII; the enzyme cuts the phosphodiester bond between the fourth and fifth nucleotides from the 5'end. We used this msDNA cleavage to assess ExoVII activity in vivo. Both subunits were required for enzyme activity. Expression of XseA without XseB caused cell death, even though no ExoVII activity was detected. The lethality caused by XseA was rescued by surplus XseB. In XseA-induced death, cells were elongated and multinucleated, and their chromosomes were fragmented and condensed; these are the morphological hallmarks of apoptotic cell death in bacteria. A putative caspase recognition sequence (FVAD) was found in XseA, and its hypothetical caspase product with 257 amino acids was as active as the intact protein in inducing cell death. We propose that under ordinary conditions, XseA protects chromosome as a component of the ExoVII enzyme, but in some conditions, the protein causes cell death; the destruction of cell is probably carried out by the amino terminal fragment derived from the cleavage of XseA by caspase-like enzyme.

The Construction of Regulatory Network for Insulin-Mediated Genes by Integrating Methods Based on Transcription Factor Binding Motifs and Gene Expression Variations.

Type 2 diabetes mellitus is a complex metabolic disorder associated with multiple genetic, developmental and environmental factors. The recent advances in gene expression microarray technologies as well as network-based analysis methodologies provide groundbreaking opportunities to study type 2 diabetes mellitus. In the present study, we used previously published gene expression microarray datasets of human skeletal muscle samples collected from 20 insulin sensitive individuals before and after insulin treatment in order to construct insulin-mediated regulatory network. Based on a motif discovery method implemented by iRegulon, a Cytoscape app, we identified 25 candidate regulons, motifs of which were enriched among the promoters of 478 up-regulated genes and 82 down-regulated genes. We then looked for a hierarchical network of the candidate regulators, in such a way that the conditional combination of their expression changes may explain those of their target genes. Using Genomica, a software tool for regulatory network construction, we obtained a hierarchical network of eight regulons that were used to map insulin downstream signaling network. Taken together, the results illustrate the benefits of combining completely different methods such as motif-based regulatory factor discovery and expression level-based construction of regulatory network of their target genes in understanding insulin induced biological processes and signaling pathways.

Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli.