Functional characterization of a novel family of acetylcholine-gated chloride channels in Schistosoma mansoni.

Acetylcholine is the canonical excitatory neurotransmitter of the mammalian neuromuscular system. However, in the trematode parasite Schistosoma mansoni, cholinergic stimulation leads to muscle relaxation and a flaccid paralysis, suggesting an inhibitory mode of action. Information about the pharmacological mechanism of this inhibition is lacking. Here, we used a combination of techniques to assess the role of cholinergic receptors in schistosome motor function. The neuromuscular effects of acetylcholine are typically mediated by gated cation channels of the nicotinic receptor (nAChR) family. Bioinformatics analyses identified numerous nAChR subunits in the S. mansoni genome but, interestingly, nearly half of these subunits carried a motif normally associated with chloride-selectivity. These putative schistosome acetylcholine-gated chloride channels (SmACCs) are evolutionarily divergent from those of nematodes and form a unique clade within the larger family of nAChRs. Pharmacological and RNA interference (RNAi) behavioral screens were used to assess the role of the SmACCs in larval motor function. Treatment with antagonists produced the same effect as RNAi suppression of SmACCs; both led to a hypermotile phenotype consistent with abrogation of an inhibitory neuromuscular mediator. Antibodies were then generated against two of the SmACCs for use in immunolocalization studies. SmACC-1 and SmACC-2 localize to regions of the peripheral nervous system that innervate the body wall muscles, yet neither appears to be expressed directly on the musculature. One gene, SmACC-1, was expressed in HEK-293 cells and characterized using an iodide flux assay. The results indicate that SmACC-1 formed a functional homomeric chloride channel and was activated selectively by a panel of cholinergic agonists. The results described in this study identify a novel clade of nicotinic chloride channels that act as inhibitory modulators of schistosome neuromuscular function. Additionally, the iodide flux assay used to characterize SmACC-1 represents a new high-throughput tool for drug screening against these unique parasite ion channels.

flp-32 Ligand/receptor silencing phenocopy faster plant pathogenic nematodes.

Restrictions on nematicide usage underscore the need for novel control strategies for plant pathogenic nematodes such as Globodera pallida (potato cyst nematode) that impose a significant economic burden on plant cultivation activities. The nematode neuropeptide signalling system is an attractive resource for novel control targets as it plays a critical role in sensory and motor functions. The FMRFamide-like peptides (FLPs) form the largest and most diverse family of neuropeptides in invertebrates, and are structurally conserved across nematode species, highlighting the utility of the FLPergic system as a broad-spectrum control target. flp-32 is expressed widely across nematode species. This study investigates the role of flp-32 in G. pallida and shows that: (i) Gp-flp-32 encodes the peptide AMRNALVRFamide; (ii) Gp-flp-32 is expressed in the brain and ventral nerve cord of G. pallida; (iii) migration rate increases in Gp-flp-32-silenced worms; (iv) the ability of G. pallida to infect potato plant root systems is enhanced in Gp-flp-32-silenced worms; (v) a novel putative Gp-flp-32 receptor (Gp-flp-32R) is expressed in G. pallida; and, (vi) Gp-flp-32R-silenced worms also display an increase in migration rate. This work demonstrates that Gp-flp-32 plays an intrinsic role in the modulation of locomotory behaviour in G. pallida and putatively interacts with at least one novel G-protein coupled receptor (Gp-flp-32R). This is the first functional characterisation of a parasitic nematode FLP-GPCR.

An automated, high-resolution phenotypic assay for adult Brugia malayi and microfilaria.

Brugia malayi are thread-like parasitic worms and one of the etiological agents of Lymphatic filariasis (LF). Existing anthelmintic drugs to treat LF are effective in reducing the larval microfilaria (mf) counts in human bloodstream but are less effective on adult parasites. To test potential drug candidates, we report a multi-parameter phenotypic assay based on tracking the motility of adult B. malayi and mf in vitro. For adult B. malayi, motility is characterized by the centroid velocity, path curvature, angular velocity, eccentricity, extent, and Euler Number. These parameters are evaluated in experiments with three anthelmintic drugs. For B. malayi mf, motility is extracted from the evolving body skeleton to yield positional data and bending angles at 74 key point. We achieved high-fidelity tracking of complex worm postures (self-occlusions, omega turns, body bending, and reversals) while providing a visual representation of pose estimates and behavioral attributes in both space and time scales.

Extracellular vesicles secreted by Brugia malayi microfilariae modulate the melanization pathway in the mosquito host.

Vector-borne, filarial nematode diseases cause significant disease burdens in humans and domestic animals worldwide. Although there is strong direct evidence of parasite-driven immunomodulation of mammalian host responses, there is less evidence of parasite immunomodulation of the vector host. We have previously reported that all life stages of Brugia malayi, a filarial nematode and causative agent of Lymphatic filariasis, secrete extracellular vesicles (EVs). Here we investigate the immunomodulatory effects of microfilariae-derived EVs on the vector host Aedes aegypti. RNA-seq analysis of an Ae. aegypti cell line treated with B. malayi microfilariae EVs showed differential expression of both mRNAs and miRNAs. AAEL002590, an Ae. aegypti gene encoding a serine protease, was shown to be downregulated when cells were treated with biologically relevant EV concentrations in vitro. Injection of adult female mosquitoes with biologically relevant concentrations of EVs validated these results in vivo, recapitulating the downregulation of AAEL002590 transcript. This gene was predicted to be involved in the mosquito phenoloxidase (PO) cascade leading to the canonical melanization response and correspondingly, both suppression of this gene using RNAi and parasite EV treatment reduced PO activity in vivo. Our data indicate that parasite-derived EVs interfere with critical immune responses in the vector host, including melanization.

Development of an in vivo RNAi protocol to investigate gene function in the filarial nematode, Brugia malayi.

Our ability to control diseases caused by parasitic nematodes is constrained by a limited portfolio of effective drugs and a paucity of robust tools to investigate parasitic nematode biology. RNA interference (RNAi) is a reverse-genetics tool with great potential to identify novel drug targets and interrogate parasite gene function, but present RNAi protocols for parasitic nematodes, which remove the parasite from the host and execute RNAi in vitro, are unreliable and inconsistent. We have established an alternative in vivo RNAi protocol targeting the filarial nematode Brugia malayi as it develops in an intermediate host, the mosquito Aedes aegypti. Injection of worm-derived short interfering RNA (siRNA) and double stranded RNA (dsRNA) into parasitized mosquitoes elicits suppression of B. malayi target gene transcript abundance in a concentration-dependent fashion. The suppression of this gene, a cathepsin L-like cysteine protease (Bm-cpl-1) is specific and profound, both injection of siRNA and dsRNA reduce transcript abundance by 83%. In vivo Bm-cpl-1 suppression results in multiple aberrant phenotypes; worm motility is inhibited by up to 69% and parasites exhibit slow-moving, kinked and partial-paralysis postures. Bm-cpl-1 suppression also retards worm growth by 48%. Bm-cpl-1 suppression ultimately prevents parasite development within the mosquito and effectively abolishes transmission potential because parasites do not migrate to the head and proboscis. Finally, Bm-cpl-1 suppression decreases parasite burden and increases mosquito survival. This is the first demonstration of in vivo RNAi in animal parasitic nematodes and results indicate this protocol is more effective than existing in vitro RNAi methods. The potential of this new protocol to investigate parasitic nematode biology and to identify and validate novel anthelmintic drug targets is discussed.

Expression and Secretion of Circular RNAs in the Parasitic Nematode, Ascaris suum.

Circular RNAs (circRNAs) are a recently identified RNA species with emerging functional roles as microRNA (miRNA) and protein sponges, regulators of gene transcription and translation, and modulators of fundamental biological processes including immunoregulation. Relevant to this study, circRNAs have recently been described in the parasitic nematode, Haemonchus contortus, suggesting they may have functionally important roles in parasites. Given their involvement in regulating biological processes, a better understanding of their role in parasites could be leveraged for future control efforts. Here, we report the use of next-generation sequencing to identify 1,997 distinct circRNAs expressed in adult female stages of the gastrointestinal parasitic nematode, Ascaris suum. We describe spatial expression in the ovary-enriched and body wall muscle, and also report circRNA presence in extracellular vesicles (EVs) secreted by the parasite into the external environment. Further, we used an in-silico approach to predict that a subset of Ascaris circRNAs bind both endogenous parasite miRNAs as well as human host miRNAs, suggesting they could be functional as both endogenous and exogenous miRNA sponges to alter gene expression. There was not a strong correlation between Ascaris circRNA length and endogenous miRNA interactions, indicating Ascaris circRNAs are enriched for Ascaris miRNA binding sites, but that human miRNAs were predicted form a more thermodynamically stable bond with Ascaris circRNAs. These results suggest that secreted circRNAs could be interacting with host miRNAs at the host-parasite interface and influencing host gene transcription. Lastly, although we have previously found that therapeutically relevant concentrations of the anthelmintic drug ivermectin inhibited EV release from parasitic nematodes, we did not observe a direct effect of ivermectin treatment on Ascaris circRNAs expression or secretion.

Secreted filarial nematode galectins modulate host immune cells.

Lymphatic filariasis (LF) is a mosquito-borne disease caused by filarial nematodes including Brugia malayi. Over 860 million people worldwide are infected or at risk of infection in 72 endemic countries. The absence of a protective vaccine means that current control strategies rely on mass drug administration programs that utilize inadequate drugs that cannot effectively kill adult parasites, thus established infections are incurable. Progress to address deficiencies in the approach to LF control is hindered by a poor mechanistic understanding of host-parasite interactions, including mechanisms of host immunomodulation by the parasite, a critical adaptation for establishing and maintaining infections. The canonical type 2 host response to helminth infection characterized by anti-inflammatory and regulatory immune phenotypes is modified by filarial nematodes during chronic LF. Current efforts at identifying parasite-derived factors driving this modification focus on parasite excretory-secretory products (ESP), including extracellular vesicles (EVs). We have previously profiled the cargo of B. malayi EVs and identified B. malayi galectin-1 and galectin-2 as among the most abundant EV proteins. In this study we further investigated the function of these proteins. Sequence analysis of the parasite galectins revealed highest homology to mammalian galectin-9 and functional characterization identified similar substrate affinities consistent with this designation. Immunological assays showed that Bma-LEC-2 is a bioactive protein that can polarize macrophages to an alternatively activated phenotype and selectively induce apoptosis in Th1 cells. Our data shows that an abundantly secreted parasite galectin is immunomodulatory and induces phenotypes consistent with the modified type 2 response characteristic of chronic LF infection.

The repertoire of G protein-coupled receptors in the human parasite Schistosoma mansoni and the model organism Schmidtea mediterranea.

BACKGROUND: G protein-coupled receptors (GPCRs) constitute one of the largest groupings of eukaryotic proteins, and represent a particularly lucrative set of pharmaceutical targets. They play an important role in eukaryotic signal transduction and physiology, mediating cellular responses to a diverse range of extracellular stimuli. The phylum Platyhelminthes is of considerable medical and biological importance, housing major pathogens as well as established model organisms. The recent availability of genomic data for the human blood fluke Schistosoma mansoni and the model planarian Schmidtea mediterranea paves the way for the first comprehensive effort to identify and analyze GPCRs in this important phylum. RESULTS: Application of a novel transmembrane-oriented approach to receptor mining led to the discovery of 117 S. mansoni GPCRs, representing all of the major families; 105 Rhodopsin, 2 Glutamate, 3 Adhesion, 2 Secretin and 5 Frizzled. Similarly, 418 Rhodopsin, 9 Glutamate, 21 Adhesion, 1 Secretin and 11 Frizzled S. mediterranea receptors were identified. Among these, we report the identification of novel receptor groupings, including a large and highly-diverged Platyhelminth-specific Rhodopsin subfamily, a planarian-specific Adhesion-like family, and atypical Glutamate-like receptors. Phylogenetic analysis was carried out following extensive gene curation. Support vector machines (SVMs) were trained and used for ligand-based classification of full-length Rhodopsin GPCRs, complementing phylogenetic and homology-based classification. CONCLUSIONS: Genome-wide investigation of GPCRs in two platyhelminth genomes reveals an extensive and complex receptor signaling repertoire with many unique features. This work provides important sequence and functional leads for understanding basic flatworm receptor biology, and sheds light on a lucrative set of anthelmintic drug targets.

Towards nanovesicle-based disease diagnostics: a rapid single-step exosome assay within one hour through in situ immunomagnetic extraction and nanophotonic label-free detection.

Exosomes have been considered as high-quality biomarkers for disease diagnosis, as they are secreted by cells into extracellular environments as nanovesicles with rich and unique molecular information, and can be isolated and enriched from clinical samples. However, most existing exosome assays, to date, require time-consuming isolation and purification procedures; the detection specificity and sensitivity are also in need of improvement for the realization of exosome-based disease diagnostics. This paper reports a unique exosome assay technology that enables completing both magnetic nanoparticle (MNP)-based exosome extraction and high-sensitivity photonic crystal (PC)-based label-free exosome detection in a single miniature vessel within one hour, while providing an improved sensitivity and selectivity. High specificity of the assay to membrane antigens is realized by functionalizing both the MNPs and the PC with specific antibodies. A low limit of detection on the order of 107 exosome particles per milliliter (volume) is achieved because the conjugated MNP-exosome nanocomplexes offer a larger index change on the PC surface, compared to the exosomes alone without using MNPs. Briefly, the single-step exosome assay involves (i) forming specific MNP-exosome nanocomplexes to enrich exosomes from complex samples directly on the PC surface at the bottom of the vessel, with a >500 enrichment factor, and (ii) subsequently, performing in situ quantification of the nanocomplexes using the PC biosensor. The present exosome assay method is validated in analyzing multiple membrane proteins of exosomes derived from murine macrophage cells with high selectivity and sensitivity, while requiring only about one hour. This assay technology will provide great potential for exosome-based disease diagnostics.

Hyperspectral imaging-based exosome microarray for rapid molecular profiling of extracellular vesicles.

One of the challenges of exploiting extracellular vesicles (EVs) as a disease biomarker is to differentiate EVs released by similar cell types or phenotypes. This paper reports a high-throughput and label-free EV microarray technology to differentiate EVs by simultaneous characterization of a panel of EV membrane proteins. The EsupplV microarray platform, which consists of an array of antibodies printed on a photonic crystal biosensor and a microscopic hyperspectral imaging technique, can rapidly assess the binding of the EV membrane proteins with their corresponding antibodies. The EV microarray assay requires only a 2 µL sample volume and a detection time of less than 2 h. The EV microarray assay was validated by not only quantifying seven membrane proteins carried by macrophage-derived EVs but also distinguishing the EVs secreted by three macrophage phenotypes. In particular, the EV microarray technology can generate a molecular fingerprint of target EVs that can be used to identify the EVs' parental cells, and thus has utility for basic science research as well as for point-of-care disease diagnostics and therapeutics.

Emerging Roles of Urine-Derived Components for the Management of Bladder Cancer: One Man's Trash Is Another Man's Treasure.

Urinary bladder cancer (UBC) is the most common malignancy of the urinary tract in humans, with an estimated global prevalence of 1.1 million cases over 5 years. Because of its high rates of recurrence and resistance to chemotherapy, UBC is one of the most expensive cancers to treat, resulting in significant health care costs. The development of innovative molecular and cellular tools is necessary to refine patient stratification and help predict response to treatment. Urine is an underused resource of biological components shed from bladder tumors, such as exfoliated cells and extracellular vesicles, that could serve as molecular fingerprints and provide valuable biological insights into tumor phenotype and mechanisms of resistance to chemotherapy. Additionally, characterization of urine-derived extracellular vesicles and cells could be used as reliable biomarkers for prediction of response to neoadjuvant therapy.

RNA Sequencing Reveals Key Metabolic Pathways Are Modified by Short-Term Whole Egg Consumption.

Eggs are protein-rich, nutrient-dense, and contain bioactive ingredients that have been shown to modify gene expression and impact health. To understand the effects of egg consumption on tissue-specific mRNA and microRNA expression, we examined the role of whole egg consumption (20% protein, w/w) on differentially expressed genes (DEGs) between rat (n = 12) transcriptomes in the prefrontal cortex (PFC), liver, kidney, and visceral adipose tissue (VAT). Principal component analysis with hierarchical clustering was used to examine transcriptome profiles between dietary treatment groups. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis as well as genetic network and disease enrichment analysis to examine which metabolic pathways were the most predominantly altered in each tissue. Overall, our data demonstrates that whole egg consumption for 2 weeks modified the expression of 52 genes in the PFC, 22 genes in VAT, and two genes in the liver (adj p < 0.05). Additionally, 16 miRNAs were found to be differentially regulated in the PFC, VAT, and liver, but none survived multiple testing correction. The main pathways influenced by WE consumption were glutathione metabolism in VAT and cholesterol biosynthesis in the PFC. These data highlight key pathways that may be involved in diseases and are impacted by acute consumption of a diet containing whole eggs.

Neuropeptide physiology in helminths.

Parasitic worms come from two distinct, distant phyla, Nematoda (roundworms) and Platyhelminthes (flatworms). The nervous systems of worms from both phyla are replete with neuropeptides and there is ample physiological evidence that these neuropeptides control vital aspects of worm biology. In each phyla, the physiological evidence for critical roles for helminth neuropeptides is derived from both parasitic and free-living members. In the nematodes, the intestinal parasite Ascaris suum and the free-living Caenorhabditis elegans have yielded most of the data; in the platyhelminths, the most physiological data has come from the blood fluke Schistosoma mansoni. FMRFamide-like peptides (FLPs) have many varied effects (excitation, relaxation, or a combination) on somatic musculature, reproductive musculature, the pharynx and motor neurons in nematodes. Insulin-like peptides (INSs) play an essential role in nematode dauer formation and other developmental processes. There is also some evidence for a role in somatic muscle control for the somewhat heterogeneous grouping ofpeptides known as neuropeptide-like proteins (NLPs). In platyhelminths, as in nematodes, FLPs have a central role in somatic muscle function. Reports of FLP physiological action in platyhelminths are limited to a potent excitation of the somatic musculature. Platyhelminths are also abundantly endowed with neuropeptide Fs (NPFs), which appear absent from nematodes. There is not yet any data linking platyhelminth NPF to any particular physiological outcome, but this neuropeptide does potently and specifically inhibit cAMP accumulation in schistosomes. In nematodes and platyhelminths, there is an abundance of physiological evidence demonstrating that neuropeptides play critical roles in the biology of both free-living and parasitic helminths. While it is certainly true that there remains a great deal to learn about the biology of neuropeptides in both phyla, physiological evidence presently available points to neuropeptidergic signaling as a very promising field from which to harvest future drug targets.

Evaluation of the pathogenicity and virulence of three strains of Salmonella organisms in calves and pigs.

OBJECTIVE: To assess in pigs the pathogenicity and virulence of 3 strains of Salmonella spp capable of causing atypical salmonellosis in cattle. ANIMALS: 36 Holstein calves and 72 pigs experimentally infected with Salmonella spp. PROCEDURES: Representative Salmonella strains associated with 3 new disease phenotypes (protozoa-mediated hypervirulence, multisystemic cytopathicity, and encephalopathy) that have been characterized in cattle during the past 10 years were orally inoculated into pigs. Clinical manifestations were compared with those observed in cattle. Samples were collected from various tissues, and the presence of Salmonella organisms was assessed qualitatively and quantitatively by use of Salmonella-selective media. RESULTS: Of the 3 unique Salmonella disease phenotypes observed in cattle, only protozoa-mediated hypervirulence was observed in pigs. Hypervirulence was related to a more rapid onset of disease and higher pathogen burden in pigs than in cattle. This phenotype was observed in pigs inoculated with multiresistant Salmonella enterica serotypes Typhimurium or Choleraesuis bearing the Salmonella genomic island 1 (SGI1) integron. CONCLUSIONS AND CLINICAL RELEVANCE: Salmonella hypervirulence was identified in pigs noculated with SGI1-bearing strains exposed to free-living protozoa. Additionally, an SGI1-bearing strain of Salmonella Choleraesuis was detected that resulted in augmented virulence in pigs. Therefore, it appeared that protozoa-associated salmonellosis was analogous in pigs and cattle. Salmonella-mediated encephalopathy and multisystemic cytopathicity did not appear to be relevant diseases in pigs.

Ivermectin inhibits extracellular vesicle secretion from parasitic nematodes.

Lymphatic filariasis (LF) is a disease caused by parasitic filarial nematodes that is endemic in 49 countries of the world and affects or threatens over 890 million people. Strategies to control LF rely heavily on mass administration of anthelmintic drugs including ivermectin (IVM), a macrocyclic lactone drug considered an Essential Medicine by the WHO. However, despite its widespread use the therapeutic mode of action of IVM against filarial nematodes is not clear. We have previously reported that filarial nematodes secrete extracellular vesicles (EVs) and that their cargo has immunomodulatory properties. Here we investigate the effects of IVM and other anti-filarial drugs on parasitic nematode EV secretion, motility, and protein secretion. We show that inhibition of EV secretion was a specific property of IVM, which had consistent and significant inhibitory effects across nematode life stages and species, with the exception of male parasites. IVM inhibited EV secretion, but not parasite motility, at therapeutically relevant concentrations. Protein secretion was inhibited by IVM in the microfilariae stage, but not in any other stage tested. Our data provides evidence that inhibiting the secretion of immunomodulatory EVs by parasitic nematodes could explain, at least in part, IVM mode of action and provides a phenotype for novel drug discovery.

The protein and microRNA cargo of extracellular vesicles from parasitic helminths - current status and research priorities.

Helminth parasites have a remarkable ability to persist within their mammalian hosts, which is largely due to their secretion of molecules with immunomodulatory properties. Although the soluble components of helminth secretions have been extensively studied, the discovery that helminths release extracellular vesicles (EVs) has added further complexity to the host-parasite interaction. Whilst several studies have begun to characterise the molecules carried by helminth EVs, work aimed at investigating their biological functions has been hindered by a lack of helminth-specific EV markers. To begin to address this, we summarised helminth EV literature to date. With a focus on the protein and microRNA (miRNA) cargo, we aimed to detect similarities and differences across those major groups of helminths for which data are available; namely nematodes, trematodes and cestodes. Pfam analysis revealed that although there is no universal EV marker for all helminth species, the EF-hand protein family was present in all EV datasets from cestodes and trematodes, and could serve as a platyhelminth EV biomarker. In contrast, M13 metallopeptidases and actin may have potential as markers for nematode EVs. As with proteins, many miRNA families appeared to be species-, stage-, or dataset-specific. Two miRNA families were common to nematode EVs (mir-10 and let-7); the miRNA cargo of EVs secreted by clade I species appeared somewhat different from species from other clades. Five miRNA families (mir-71, mir-10, mir-190, let-7 and mir-2) were shared by all trematode species examined. Our analysis has identified novel markers that may be used in studies aimed at characterising helminth EVs and interrogating their function at the host-parasite interface. In addition, we discuss the heterogeneity of methods used for helminth EV isolation and emphasise the need for a standardised approach in reporting on helminth EV data.

Whole egg consumption increases gene expression within the glutathione pathway in the liver of Zucker Diabetic Fatty rats.

Nutrigenomic evidence supports the idea that Type 2 Diabetes Mellitus (T2DM) arises due to the interactions between the transcriptome, individual genetic profiles, lifestyle, and diet. Since eggs are a nutrient dense food containing bioactive ingredients that modify gene expression, our goal was to examine the role of whole egg consumption on the transcriptome during T2DM. We analyzed whether whole egg consumption in Zucker Diabetic Fatty (ZDF) rats alters microRNA and mRNA expression across the adipose, liver, kidney, and prefrontal cortex tissue. Male ZDF (fa/fa) rats (n = 12) and their lean controls (fa/+) (n = 12) were obtained at 6 wk of age. Rats had ad libitum access to water and were randomly assigned to a modified semi-purified AIN93G casein-based diet or a whole egg-based diet, both providing 20% protein (w/w). TotalRNA libraries were prepared using QuantSeq 3' mRNA-Seq and Lexogen smallRNA library prep kits and were further sequenced on an Illumina HighSeq3000. Differential gene expression was conducted using DESeq2 in R and Benjamini-Hochberg adjusted P-values controlling for false discovery rate at 5%. We identified 9 microRNAs and 583 genes that were differentially expressed in response to 8 wk of consuming whole egg-based diets. Kyto Encyclopedia of Genes and Genomes/Gene ontology pathway analyses demonstrated that 12 genes in the glutathione metabolism pathway were upregulated in the liver and kidney of ZDF rats fed whole egg. Whole egg consumption primarily altered glutathione pathways such as conjugation, methylation, glucuronidation, and detoxification of reactive oxygen species. These pathways are often negatively affected during T2DM, therefore this data provides unique insight into the nutrigenomic response of dietary whole egg consumption during the progression of T2DM.

flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference.

The potato cyst nematode Globodera pallida is a serious pest of potato crops. Nematode FMRFamide-like peptides (FLPs) are one of the most diverse neuropeptide families known, and modulate sensory and motor functions. As neuromuscular function is a well-established target for parasite control, parasitic nematode FLP signaling has significant potential in novel control strategies. In the absence of transgenic parasitic nematodes and the reported ineffectiveness of neuronal gene RNAi in Caenorhabditis elegans, nothing is known about flp function in nematode parasites. In attempts to evaluate flp function in G. pallida, we have discovered that, unlike in C. elegans, these genes are readily susceptible to RNAi. Silencing any of the five characterized G. pallida flp genes (Gp-flp-1, -6, -12, -14, or -18) incurred distinct aberrant behavioral phenotypes consistent with key roles in motor function. Further delineation of these effects revealed that double-stranded RNA exposure time (> or = 18 h) and concentration (> or = 0.1 microg/ml) were critical to the observed effects, which were reversible. G. pallida flp genes are essential to coordinated locomotory activities, do not display redundancy, and are susceptible to RNAi, paving the way for the investigation of RNAi-mediated flp gene silencing as a novel plant parasite control strategy.

Praziquantel Interaction with Mammalian Targets in the Spotlight.

Chan et al. recently demonstrated that the antischistosomal drug praziquantel has a potent and specific interaction with human 5-HT2B receptors, and that the drug also elicits contraction of mouse mesenteric vasculature apparently mediated by the same receptor subtype We consider what this might mean about the drug's molecular therapeutic targets in both the worm and the host.

Profiling extracellular vesicle release by the filarial nematode Brugia malayi reveals sex-specific differences in cargo and a sensitivity to ivermectin.

The filarial nematode Brugia malayi is an etiological agent of Lymphatic Filariasis. The capability of B. malayi and other parasitic nematodes to modulate host biology is recognized but the mechanisms by which such manipulation occurs are obscure. An emerging paradigm is the release of parasite-derived extracellular vesicles (EV) containing bioactive proteins and small RNA species that allow secretion of parasite effector molecules and their potential trafficking to host tissues. We have previously described EV release from the infectious L3 stage B. malayi and here we profile vesicle release across all intra-mammalian life cycle stages (microfilariae, L3, L4, adult male and female worms). Nanoparticle Tracking Analysis was used to quantify and size EVs revealing discrete vesicle populations and indicating a secretory process that is conserved across the life cycle. Brugia EVs are internalized by murine macrophages with no preference for life stage suggesting a uniform mechanism for effector molecule trafficking. Further, the use of chemical uptake inhibitors suggests all life stage EVs are internalized by phagocytosis. Proteomic profiling of adult male and female EVs using nano-scale LC-MS/MS described quantitative and qualitative differences in the adult EV proteome, helping define the biogenesis of Brugia EVs and revealing sexual dimorphic characteristics in immunomodulatory cargo. Finally, ivermectin was found to rapidly inhibit EV release by all Brugia life stages. Further this drug effect was also observed in the related filarial nematode, the canine heartworm Dirofilaria immitis but not in an ivermectin-unresponsive field isolate of that parasite, highlighting a potential mechanism of action for this drug and suggesting new screening platforms for anti-filarial drug development.