Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 µM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 µM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

The screening and prioritization of contaminants of emerging concern in the marine environment based on multiple biological response measures.

There is ample evidence that a range of anthropogenic chemicals occur in the aquatic environment, some of which have the potential to cause harm. Contaminants of Emerging Concern (CECs) are a subset of anthropogenic compounds that are poorly characterized in terms of effects and occurrences, and that are generally unregulated. Due to the sheer number of chemicals used, it is necessary to identify and prioritize those that may cause biological impacts. A key challenge of doing so is the lack of traditional ecotoxicological information. The utilization of in vitro exposure-response studies or benchmarks based on in vivo data can provide a basis for developing threshold values for evaluation of potential impacts. There are challenges, including understanding the accuracy and range of application for modeled measures and translating in vitro response information from receptor models to apical endpoints. Despite this, the use of multiple lines of evidence increases the range of available information, and supports a weight-of-evidence approach to inform the screening and prioritization of CECs in the environment. The objective of this work is to perform an evaluation of CECs detected in an urban estuary, and to identify those that are most likely to elicit a biological response. Monitoring data from marine water, wastewater, and fish and shellfish tissue samples from 17 different campaigns combined with multiple biological response measures were compared with appropriate threshold values. CECs were categorized based on their potential to elicit a biological response; the uncertainty, based on consistency of lines of evidence, was also evaluated. Two-hundred-fifteen CECs were detected. Fifty-seven were identified as High Priority (likely to cause a biological effect), and 84 as Watch List (potential to cause biological effects). Due to the extent of the monitoring and range of the lines of evidence, this approach and results are applicable to other urbanized estuarine systems.

Assessing hydroxylated polycyclic aromatic hydrocarbon (OHPAH) metabolites in bile of English sole (Parophrys vetulus) from Puget Sound, WA, USA by liquid chromatography/tandem mass spectrometry (LC-MS/MS).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that are widely monitored in marine biota from urbanized areas, due to their toxicity to aquatic organisms. Teleost fish can quickly metabolize PAHs into hydroxylated forms (OHPAHs) that, in some cases, are more toxic than the parent (unmetabolized) PAHs. But due to this fast metabolism, monitoring traditional parent PAHs in fish can cause underestimation on assessing PAH exposure. In addition, environmental levels of individual OHPAH metabolites are lacking in the literature worldwide. Therefore, we developed a rapid and accurate analytical method in which a number of individual OHPAHs metabolites are measured simultaneously in fish bile, via liquid chromatography coupled with tandem mass spectrometry, including low and high molecular weight mono- and diol-OHPAHs. We analyzed bile samples of 119 English sole (Parophrys vetulus) collected from 14 Puget Sound, WA, USA, sites, which has multiple sources of PAHs, including urban stormwater runoff, wastewater effluents, as well as an inactive creosote facility. The mean (± SD) biliary summed OHPAH (∑OHPAH) concentrations determined in English sole from urban, near-urban, and non-urban sites were 790 ± 1400 (n = 46), 310 ± 330 (n = 44) and 130 ± 200 (n = 29) ng/mL, respectively, with a maximum reaching 9400 ng/mL in a sample from an urban site. We compared these novel biliary OHPAH metabolite data with parent PAHs measured in stomach content of the same individual sole. Biliary ∑OHPAH concentrations were significantly correlated with the levels of ∑PAH in stomach content, however, with major differences in their distribution. We also demonstrated that biliary OHPAH metabolite data in English sole can potentially be used to distinguish different sampling sites due to a specific variety and intensity of PAH sources in the aquatic environment, which makes this a very important analytical approach for assessing PAH exposure in the environment.

Highly contaminated river otters (Lontra canadensis) are effective biomonitors of environmental pollutant exposure.

River otters (Lontra canadensis) are apex predators that bioaccumulate contaminants via their diet, potentially serving as biomonitors of watershed health. They reside throughout the Green-Duwamish River, WA (USA), a watershed encompassing an extreme urbanization gradient, including a US Superfund site slated for a 17-year remediation. The objectives of this study were to document baseline contaminant levels in river otters, assess otters' utility as top trophic-level biomonitors of contaminant exposure, and evaluate the potential for health impacts on this species. We measured a suite of contaminants of concern, lipid content, nitrogen stable isotopes (δ15N), and microsatellite DNA markers in 69 otter scat samples collected from twelve sites. Landcover characteristics were used to group sampling sites into industrial (Superfund site), suburban, and rural development zones. Concentrations of polychlorinated biphenyls (PCBs), polybrominated diphenyl ether flame-retardants (PBDEs), dichlorodiphenyl-trichloroethane and its metabolites (DDTs), and polycyclic aromatic hydrocarbons (PAHs) increased significantly with increasing urbanization, and were best predicted by models that included development zone, suggesting that river otters are effective biomonitors, as defined in this study. Diet also played an important role, with lipid content, δ15N or both included in all best models. We recommend river otter scat be included in evaluating restoration efforts in this Superfund site, and as a potentially useful monitoring tool wherever otters are found. We also report ΣPCB and ΣPAH exposures among the highest published for wild river otters, with almost 70% of samples in the Superfund site exceeding established levels of concern.

Helminth antigens modulate human PBMCs, attenuating disease progression in a humanised mouse model of graft versus host disease.

Fasciola hepatica is a trematode worm that causes fascioliasis, a neglected tropical disease in humans and livestock. To gain insight into the host-parasite interactions that facilitate infection, we have investigated the immunomodulatory properties of the parasite's tegumental coat (FhTeg), a major antigen source that is sloughed off and renewed every 2-3 h as the worm migrates through host tissue. Using mouse models of infection, we have previously shown that FhTeg induces a novel phenotype of dendritic cells that induce anergic CD4+ T-cells. We proposed that this induced state of hyporesponsiveness characterised by suppression of cell proliferation and cytokine secretion was one mechanism by which F. hepatica prevented host protective immunity to support the parasite survival. To determine if the same mechanisms are utilised during human infections, we have now examined the interaction of FhTeg with human PBMCs. FhTeg binds to and modulates cytokine production in human PBMCs, in particular targeting the CD4+ population resulting in reduced levels of TNF, IL-2 and IFNγ and increased markers of anergy. Furthermore, the adoptive transfer of FhTeg stimulated PBMCs to a humanised model of acute graft versus host disease (GvHD) attenuated disease progression by increasing survival and reducing pathological scores. These mice also displayed a significant decrease in the total number of human CD4+ cells expressing TNF, IL-2 and IFNγ in the spleen, liver and lung. This study therefore concurs with evidence from ruminant and murine models of infection suggesting that anergic CD4+ T cells are associated with successful Fasciola hepatica infection and highlights an important role for FhTeg in contributing to the overall immunosuppressive effects of this parasite.

Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

Fasciola hepatica Extracellular Vesicles isolated from excretory-secretory products using a gravity flow method modulate dendritic cell phenotype and activity.

Parasite-released extracellular vesicles (EVs) deliver signals to the host immune system that are critical to maintaining the long-term relationship between parasite and host. In the present study, total EVs (FhEVs) released in vitro by adults of the helminth parasite Fasciola hepatica were isolated using a recently described gravity flow method that protects their structural integrity. The FhEVs molecular cargo was defined using proteomic analysis and their surface topology characterised by glycan microarrays. The proteomic analysis identified 618 proteins, 121 of which contained putative N-linked glycosylation sites while 132 proteins contained putative O-linked glycosylation sites. Glycan arrays revealed surface-exposed glycans with a high affinity for mannose-binding lectins indicating the predominance of oligo mannose-rich glycoproteins, as well as other glycans with a high affinity for complex-type N-glycans. When added to bone-marrow derived dendritic cells isolated FhEV induced a novel phenotype that was categorised by the secretion of low levels of TNF, enhanced expression of cell surface markers (CD80, CD86, CD40, OX40L, and SIGNR1) and elevation of intracellular markers (SOCS1 and SOCS3). When FhEV-stimulated BMDCs were introduced into OT-II mice by adoptive transfer, IL-2 secretion from skin draining lymph nodes and spleen cells was inhibited in response to both specific and non-specific antigen stimulation. Immunisation of mice with a suspension of FhEV did not elicit significant immune responses; however, in the presence of alum, FhEVs induced a mixed Th1/Th2 immune response with high antigen specific antibody titres. Thus, we have demonstrated that FhEVs induce a unique phentotype in DC capable of suppressing IL-2 secretion from T-cells. Our studies add to the growing immuno-proteomic database that will be an important source for the discovery of future parasite vaccines and immunotherapeutic biologicals.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry.

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 µM) and arphamenine A (IC50 = 15.75 µM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

Concentrations and profiles of organochlorine contaminants in North Pacific resident and transient killer whale (Orcinus orca) populations.

Organochlorine (OC) profiles have been used as chemical "fingerprints" to infer an animal's foraging area. North Pacific killer whale (Orcinus orca) populations are exposed to different levels and patterns of OCs based on their prey, distribution, and amount of time spent in a particular area. To characterize concentrations and profiles of OCs found in various populations of North Pacific killer whales, polychlorinated biphenyls (PCBs), including dioxin-like congeners, DDTs, and hexachlorobenzene (HCB), were measured in biopsy blubber samples of photo-identified resident (fish-eating) and transient (mammal-eating) killer whales collected from 1994 through 2002 from Russian Far East waters to the waters of the west coast of the United States, representing 10 populations. We compared blubber OC concentrations based on ecotype (resident vs. transient), sex and reproductive maturity, and geographic area. We also examined OC mixtures to determine if we could detect segregated geographical areas (foraging areas) among the six populations with sufficient sample sizes. Transients had significantly higher OC concentrations than residents and adult male whales had consistently higher OC levels compared to adult females, regardless of ecotype. Our OC profile findings indicate segregated foraging areas for the North Pacific killer whales, consistent with observations of their geographic distributions. Several potential health risks have also been associated with exposure to high levels of contaminants in top-level predators including reproductive impairment, immune suppression, skeletal deformities, and carcinoma. The results of this baseline study provide information on the geographic distribution of OCs found in North Pacific killer whales, results which are crucial for assessing the potential health risks associated with OC exposure in this species.

Chemical tracers guide identification of the location and source of persistent organic pollutants in juvenile Chinook salmon (Oncorhynchus tshawytscha), migrating seaward through an estuary with multiple contaminant inputs.

Understanding the spatial extent, magnitude, and source of contaminant exposure in biota is necessary to formulate appropriate conservation measures to reduce or remediate contaminant exposure. However, obtaining such information for migratory animals is challenging. Juvenile Chinook salmon (Oncorhynchus tshawytscha), a threatened species throughout the US Pacific Northwest, are exposed to persistent organic pollutants (POPs), including polybrominated diphenyl ether (PBDE) flame retardants and polychlorinated biphenyls (PCBs), in many developed rivers and estuaries. This study used three types of complementary chemical tracer data (contaminant concentrations, POP fingerprints, and stable isotopes), to determine the location and source of contaminant exposure for natural- and hatchery-origin Chinook salmon migrating seaward through a developed watershed with multiple contaminant sources. Concentration data revealed that salmon were exposed to and accumulated predominantly PBDEs and PCBs in the lower mainstem region of the river, with higher PBDEs in natural- than hatchery-origin fish but similar PCBs in both groups, associated with differences in contaminant inputs and/or habitat use. The POP fingerprints of the natural-origin-fish captured from this region were also distinct from other region and origin sample groups, with much higher proportions of PBDEs in the total POP concentration, indicating a different contaminant source or habitat use than the hatchery-origin fish. Stable isotopes, independent tracers of food sources and habitat use, revealed that natural-origin fish from this region also had depleted δ15N signatures compared to other sample groups, associated with exposure to nutrient-rich wastewater. The PBDE-enhanced POP fingerprints in these salmon were correlated with the degree of depletion in nitrogen stable isotopes of the fish, suggesting a common wastewater source for both the PBDEs and the nitrogen. Identification of the location and source of contaminant exposure allows environmental managers to establish conservation measures to control contaminant inputs, necessary steps to improve the health of Chinook salmon and enhance their marine survival.

Toxic Burdens of Freshwater Biofilms and Use as a Source Tracking Tool in Rivers and Streams.

Biofilms, composed of periphyton, bacteria, and organic detritus, are the base of the food web in many streams and rivers. This media adsorbs and actively sequesters organic and inorganic contaminants from the water column. Here, we demonstrate the utility of using the contaminant concentrations in the biofilm matrix as an environmental media in source tracking and understanding biological impacts at higher trophic levels. Physical partitioning of polychlorinated biphenyl (PCB) and polybrominated diphenyl ether congeners is the dominant mode of uptake from water to biofilm and bioaccumulation factor: log Kow relationships suggest that PCB uptake is often near equilibrium between log Kow 5-7. We show that the concentrations of metals in biofilms are more effective at delineating and recording spatial and temporal differences in metal inputs than bed sediments and water samples. The burden of metals in the biofilm matrix explained adverse impacts and variability in periphyton metrics and ecological integrity in macroinvertebrates. This work provides new insights into the partitioning of organic chemicals onto biofilms and shows clear linkages between metals in the biofilm matrix and ecological health of invertebrates that depend on biofilms as a food source.

Bovine κ-Casein Fragment Induces Hypo-Responsive M2-Like Macrophage Phenotype.

Immunomodulatory nutraceuticals have garnered special attention due to their therapeutic potential for the amelioration of many chronic inflammatory conditions. Macrophages are key players in the induction, propagation and resolution of inflammation, actively contributing to the pathogenesis and resolution of inflammatory disorders. As such, this study aimed to investigate the possible therapeutic effects bovine casein derived nutraceuticals exert on macrophage immunological function. Initial studies demonstrated that sodium caseinate induced a M2-like macrophage phenotype that was attributed to the kappa-casein subunit. Kappa-casein primed macrophages acquired a M2-like phenotype that expressed CD206, CD54, OX40L, CD40 on the cell surface and gene expression of Arg-1, RELM-α and YM1, archetypical M2 markers. Macrophages stimulated with kappa-casein secreted significantly reduced TNF-α and IL-10 in response to TLR stimulation through a mechanism that targeted the nuclear factor-κB signal transduction pathway. Macrophage proteolytic processing of kappa-casein was required to elicit these suppressive effects, indicating that a fragment other than C-terminal fragment, glycomacropeptide, induced these modulatory effects. Kappa-casein treated macrophages also impaired T-cell responses. Given the powerful immuno-modulatory effects exhibited by kappa-casein and our understanding of immunopathology associated with inflammatory diseases, this fragment has the potential as an oral nutraceutical and therefore warrants further investigation.

Polycyclic aromatic hydrocarbons in Pacific herring (Clupea pallasii) embryos exposed to creosote-treated pilings during a piling-removal project in a nearshore marine habitat of Puget Sound.

We used manually spawned, field-deployed embryos of a common marine fish species, Pacific herring (Clupea pallasii), to evaluate accumulation of polycyclic aromatic hydrocarbons (PAHs) associated with an incomplete creosote-treated piling (CTP) removal project. Embryos near undisturbed 100-year-old CTPs (before removal) accumulated higher PAHs and exhibited higher cyp1a gene expression than embryos from reference areas. Embryos incubated close to CTP debris after CTP removal showed PAHs 90 times higher than reference areas up to a year after CTP removal. cyp1a fold-induction correlated with total embryo PAHs in all three years. Patterns of individual PAH chemicals differed slightly between embryos, wood sampled from CTPs, and passive samplers. This study illustrates the importance of using appropriate techniques and procedures to remove CTPs in aquatic environments to prevent release of toxic chemicals. Of particular concern is that incomplete CTP removal could expose sensitive life stages of fishes to chemicals that may reduce their survival.

The cathepsin-like cysteine peptidases of trematodes of the genus Fasciola.

Fasciolosis caused by trematode parasites of the genus Fasciola is a global disease of livestock, particularly cattle, sheep, water buffalo and goats. It is also a major human zoonosis with reports suggesting that 2.4-17 million people are infected worldwide, and 91.1 million people currently living at risk of infection. A unique feature of these worms is their reliance on a family of developmentally-regulated papain-like cysteine peptidases, termed cathepsins. These proteolytic enzymes play central roles in virulence, infection, tissue migration and modulation of host innate and adaptive immune responses. The availability of a Fasciola hepatica genome, and the exploitation of transcriptomic and proteomic technologies to probe parasite growth and development, has enlightened our understanding of the cathepsin-like cysteine peptidases. Here, we clarify the structure of the cathepsin-like cysteine peptidase families and, in this context, review the phylogenetics, structure, biochemistry and function of these enzymes in the host-parasite relationship.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells.

Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.

In silico analyses of protein glycosylating genes in the helminth Fasciola hepatica (liver fluke) predict protein-linked glycan simplicity and reveal temporally-dynamic expression profiles.

Glycoproteins secreted by helminth parasites are immunogenic and represent appealing components of vaccine preparations. Our poor knowledge of the pathways that mediate protein glycosylation in parasitic flatworms hinders our understanding of how proteins are synthesised and modified, and our ability to target these pathways for parasite control. Here we provide the first detailed description of genes associated with protein glycosylation in a parasitic flatworm, focusing on the genome of the liver fluke (Fasciola hepatica), which is a globally important trematode parasite of humans and their livestock. Using 190 human sequences as search queries against currently available F. hepatica genomes, we identified 149 orthologues with putative roles in sugar uptake or nucleotide sugar synthesis, and an array of glycosyltransferase and glycosidase activities required for protein N- and O-glycosylation. We found appreciable duplication within these orthologues, describing just 87 non-redundant genes when paralogues were excluded. F. hepatica lacks many of the enzymes required to produce complex N- and O-linked glycans, which explains the genomic basis for the structurally simple glycans described by F. hepatica glycomic datasets, and predicts pervasive structural simplicity in the wider glycome. These data provide a foundation for functional genomic interrogation of these pathways with the view towards novel parasite intervention strategies.

Infection by the Helminth Parasite Fasciola hepatica Requires Rapid Regulation of Metabolic, Virulence, and Invasive Factors to Adjust to Its Mammalian Host.

The parasite Fasciola hepatica infects a broad range of mammals with impunity. Following ingestion of parasites (metacercariae) by the host, newly excysted juveniles (NEJ) emerge from their cysts, rapidly penetrate the duodenal wall and migrate to the liver. Successful infection takes just a few hours and involves negotiating hurdles presented by host macromolecules, tissues and micro-environments, as well as the immune system. Here, transcriptome and proteome analysis of ex vivo F. hepatica metacercariae and NEJ reveal the rapidity and multitude of metabolic and developmental alterations that take place in order for the parasite to establish infection. We found that metacercariae despite being encased in a cyst are metabolically active, and primed for infection. Following excystment, NEJ expend vital energy stores and rapidly adjust their metabolic pathways to cope with their new and increasingly anaerobic environment. Temperature increases induce neoblast proliferation and the remarkable up-regulation of genes associated with growth and development. Cysteine proteases synthesized by gastrodermal cells are secreted to facilitate invasion and tissue degradation, and tegumental transporters, such as aquaporins, are varied to deal with osmotic/salinity changes. Major proteins of the total NEJ secretome include proteases, protease inhibitors and anti-oxidants, and an array of immunomodulators that likely disarm host innate immune effector cells. Thus, the challenges of infection by F. hepatica parasites are met by rapid metabolic and physiological adjustments that expedite tissue invasion and immune evasion; these changes facilitate parasite growth, development and maturation. Our molecular analysis of the critical processes involved in host invasion has identified key targets for future drug and vaccine strategies directed at preventing parasite infection.

Time Trends of Persistent Organic Pollutants in Benthic and Pelagic Indicator Fishes from Puget Sound, Washington, USA.

We modeled temporal trends in polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and dichlorodiphenyltrichloroethane and its metabolites (DDTs) in two indicator fish species representing benthic and pelagic habitats in Puget Sound, Washington, USA. English sole (Parophrys vetulus, benthic) index sites and larger-scale Pacific herring (Clupea pallasii, pelagic) foraging areas represented a wide range of possible contamination conditions, with sampling locations situated adjacent to watersheds exhibiting high, medium and low development. Consistency in analytical data throughout the study was maintained by either calculating method-bias-correction factors on paired samples as methods evolved or by analyzing older archived samples by current methods. PCBs declined moderately in two herring stocks from a low-development basin (2.3 and 4.0% annual rate of decline) and showed no change in the highly developed and moderately developed basins during a 16- to 21-year period. PCBs increased in English sole from four of ten sites (2.9-7.1%), and the remaining six exhibited no significant change. PBDEs and DDTs declined significantly in all herring stocks (4.2-8.1%), although analytical challenges warrant caution in interpreting DDT results. PBDEs declined in English sole from two high-development and one low-development site (3.7-7.2%) and remained unchanged in the remaining seven. DDTs increased in English sole from one high-development site (Tacoma City Waterway) and declined in two high-development and one low development site. As with herring, analytical challenges warrant caution in interpreting the English sole DDT results. It is likely that source controls and mitigation efforts have contributed to the declines in PBDEs and DDTs overall, whereas PCBs appear to have persisted, especially in the pelagic food web, despite bans in PCB production and use.