Exploring the broad-spectrum pharmacological activity of two less studied Australian native fruits: chemical characterisation using LCMS-driven metabolomics.

Australian fruits such as native currant (Acrotriche depressa) and lemon aspen (Acronychia acidula) are under-examined in terms of their therapeutic potential. In this study, the in vitro antiproliferative activity of native currant and lemon aspen extracts (water and ethanol) against MCF7 breast adenocarcinoma cells was determined using the Alamar blue assay. The most potent extracts (native currant water, NC-W; native currant ethanol, NC-Et; lemon aspen ethanol, LA-Et) were further evaluated using flow cytometry to detect the potential induction of apoptosis in MCF7 cells whereas 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay was implemented to understand the impact of the extracts on the intracellular reactive oxygen species (ROS) levels in MCF7 cells. Furthermore, the antioxidant activity of the extracts was assessed using ABTS [2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate)], and CUPRAC (cupric reducing antioxidant capacity) assays. The antimicrobial susceptibility testing of NC-W, NC-Et, and LA-Et was carried out against Gram-positive (Staphylococcus aureus), Gram-negative (Escherichia coli), and yeast (Candida albicans) strains using a resazurin-based assay. Additionally, potential metabolites in the NC-W and NC-Et extracts were analysed with liquid chromatography-mass spectrometry (LC-MS) driven metabolomics and chemometrics to spot differential and major metabolites. A dose-dependent antiproliferative activity was conferred by the NC extracts against MCF7 cells. Of the two LA extracts, only LA-Et showed a dose-dependent antiproliferative activity at higher concentrations. Both NC extracts and LA-Et induced apoptosis in MCF7 cells. None of the extracts increased the production of ROS significantly in MCF7 cells compared to the untreated control. A dose-dependent antioxidant activity was observed in both antioxidant assays. Both NC and LA extracts showed a similar minimum inhibitory concentration (MIC) value against S. aureus. Only LA-Et showed activity against E. coli, while NC-W and NC-Et were less active. All extracts showed MIC values of >1500 µg mL-1 against C. albicans. The metabolomics analysis revealed an abundance of flavonoids, fatty acyl derivatives, carbohydrates, carboxylic acids and their derivatives, and alkaloid compounds as potential bioactive metabolites in the NC extracts. In conclusion, both NC and LA showed antiproliferative (against MCF7 breast adenocarcinoma cells through the induction of apoptosis), strong antioxidant and minimal antimicrobial properties.

A retrospective multistate analysis: Do regional football games impact emergency department patient volume?

OBJECTIVE: We sought to assess the effect of National Football League (NFL) games played by a regional sports team, the New England Patriots, on emergency department (ED) patient volume. METHODS: We conducted a multicenter, retrospective chart review at the following 3 tertiary centers in New England from 2012 to 2019: Beth Israel Deaconess Medical Center, Boston, MA; Dartmouth Hitchcock Medical Center, Lebanon, NH; and Maine Medical Center, Portland, ME. RESULTS: Within the NFL season, we observed a 2.6% overall decrease (-10.4 patients) in average total daily volume across the study sites on Sundays when Patriots games were played compared with Sundays when games were not played (P = 0.07; 95% confidence interval [CI], -22.37 to 1.62). We observed a 4.3% reduction (-19.0 patients) in average total daily volume across the study sites on Mondays during which Patriots games were played compared with Mondays without games (P = 0.15; 95% CI, -43.51 to 5.47). Subanalyses on the 5-hour period corresponding with each Patriots game showed reductions in mean patient volume per hour. Although our primary and subanalyses showed reductions in patient volume during Patriots games, these results were not statistically significant. CONCLUSIONS: Our data support prior studies that showed a minimal impact of major sporting events on ED patient volume at tertiary centers. These results add to the limited data on this topic and can inform administrators whether staffing adjustments are necessary during similar types of sporting events.

The Effect of a Rapid Assessment Zone on Emergency Department Operations and Throughput.

STUDY OBJECTIVE: We examine the effects of a front-end flow model designated the rapid assessment zone on multiple emergency department (ED) operational metrics. METHODS: This was a retrospective, before-after study of consecutive patient visits at an urban community ED. Six-month periods were compared before and after an intervention in 2017 that changed patient flow and the intake process. A lead nurse role splits patient flow immediately on patient arrival according to only age and chief complaint, allowing direct bedding without the bottlenecks of vital sign measurement, full triage assessment, or Emergency Severity Index assignment. A new patient care area (designated rapid assessment zone) preferentially expedites treatment of patients likely to remain ambulatory and serves as flexible acute care space when needed by individual cases and the ED. The outcomes measured were ED length of stay, arrival-to-provider time, the rate of leaving before treatment completion, and the rate of leaving before being seen. Data were analyzed with nonparametric testing, χ2 analysis, and multiple linear regression, controlling for patient visit characteristics, ED daily census volumes, and measurements of boarding patients. RESULTS: We analyzed 43,847 visits in the preintervention and 44,792 visits in the postintervention periods. The intervention was associated with the following changes: median ED length of stay from 203 to 171 minutes (-15.8%), median arrival-to-provider time from 28 to 13 minutes (-53.6%), leaving before treatment completion from 1.0% to 0.8% (-20%), and leaving before being seen from 3.1% to 0.5% (-84%). Regression analysis accounting for multiple confounders demonstrated that the reduced length of stay after rapid assessment zone implementation persisted across Emergency Severity Index levels 2 to 5 and all ED daily census levels. CONCLUSION: The rapid assessment zone model aims to decrease front-end bottlenecks and minimize serial intake assessments at a high-volume, urban ED. It was associated with improved patient throughput and decreased early patient departure. It may represent a useful model for similar centers.

The in vitro effects of interferon-gamma, alone or in combination with amphotericin B, tested against the pathogenic fungi Candida albicans and Aspergillus fumigatus.

OBJECTIVE: Recent studies into the antifungal activity of NK-cells against the Aspergillus fumigatus have presented differing accounts on their mode of antifungal activity. One of these mechanisms proposed that NK-cells may kill the fungus via the direct effects of exposure to Interferon gamma (IFN-γ). RESULTS: In this study we investigated the direct antifungal effects of recombinant human IFN-γ against a range of pathogenic fungi by measuring cellular damage using an XTT-based assay and cell viability through plate counts. It was found that 32 pg/ml of IFN-γ exhibited a significant but small antifungal effect on A. fumigatus (p = 0.02), Aspergillus flavus (p = 0.04) and Saccharomyces cerevisiae (p = 0.03), inhibiting growth by 6, 11 and 17% respectively. No significant inhibitory effects were observed in Candida species (p > 0.05 for all species tested) or Cryptococus neoformans (p = 0.98). Short term exposure (3 h) to a combination of amphotericin B (1 µg/ml) and IFN-γ (32 pg/ml) increased the effectiveness of amphotericin B against A. fumigatus and S. cerevisiae but not Candida albicans. These data suggest that IFN-γ does not possess strong antifungal activity but can enhance the effect of amphotericin B under some testing conditions against Aspergillus species.

Comparative proteomic analysis of two pathogenic Tritrichomonas foetus genotypes: there is more to the proteome than meets the eye.

Certain clinical isolates of Tritrichomonas foetus infect the urogenital tract of cattle while others infect the gastrointestinal tract of cats. Previous studies have identified subtle genetic differences between these isolates with the term "genotype" adopted to reflect host origin. The aim of this work was to seek evidence of host-specific adaptation and to clarify the relationship between T. foetus genotypes. To do this we characterised the proteomes of both genotypes using two-dimensional gel electrophoresis (2DE) coupled with LC-MS/MS. Our comparative analysis of the data revealed that both genotypes exhibited largely similar proteoform profiles; however differentiation was possible with 24 spots identified as having a four-fold or greater change. Deeper analysis using 2DE zymography and protease-specific fluorogenic substrates revealed marked differences in cysteine protease (CP) expression profiles between the two genotypes. These variances in CP activities could also account for the pathogenic and histopathological differences previously observed between T. foetus genotypes in cross-infection studies. Our findings highlight the importance of CPs as major determinants of parasite virulence and provide a foundation for future host-parasite interaction studies, with direct implications for the development of vaccines or drugs targeting T. foetus.

A Plasmodium falciparum S33 proline aminopeptidase is associated with changes in erythrocyte deformability.

Infection with the apicomplexan parasite Plasmodium falciparum is a major cause of morbidity and mortality worldwide. One of the striking features of this parasite is its ability to remodel and decrease the deformability of host red blood cells, a process that contributes to disease. To further understand the virulence of Pf we investigated the biochemistry and function of a putative Pf S33 proline aminopeptidase (PfPAP). Unlike other P. falciparum aminopeptidases, PfPAP contains a predicted protein export element that is non-syntenic with other human infecting Plasmodium species. Characterization of PfPAP demonstrated that it is exported into the host red blood cell and that it is a prolyl aminopeptidase with a preference for N-terminal proline substrates. In addition genetic deletion of this exopeptidase was shown to lead to an increase in the deformability of parasite-infected red cells and in reduced adherence to the endothelial cell receptor CD36 under flow conditions. Our studies suggest that PfPAP plays a role in the rigidification and adhesion of infected red blood cells to endothelial surface receptors, a role that may make this protein a novel target for anti-disease interventions strategies.

Host origin determines pH tolerance of Tritrichomonas foetus isolates from the feline gastrointestinal and bovine urogenital tracts.

The ability for protozoan parasites to tolerate pH fluctuations within their niche is critical for the establishment of infection and require the parasite to be capable of adapting to a distinct pH range. We used two host adapted Tritrichomonas foetus isolates, capable of infecting either the digestive tract (pH 5.3-6.6) of feline hosts or the reproductive tract (pH 7.4-7.8) of bovine hosts to address their adaptability to changing pH. Using flow cytometry, we investigated the pH tolerance of the bovine and feline T. foetus isolates over a range of physiologically relevant pH in vitro. Following exposure to mild acid stress (pH 6), the bovine T. foetus isolates showed a significant decrease in cell viability and increased cytoplasmic granularity (p-value < 0.003, p-value < 0.0002) compared to pH 7 and 8 (p-value > 0.7). In contrast, the feline genotype displayed an enhanced capacity to maintain cell morphology and viability (p-value > 0.05). Microscopic assessment revealed that following exposure to a weak acidic stress (pH 6), the bovine T. foetus transformed into rounded parasites with extended cell volumes and displays a decrease in viability. The higher tolerance for acidic extracellular environment of the feline isolate compared to the bovine isolate suggests that pH could be a critical factor in regulating T. foetus infections and host-specificity.

Comparative transcriptomics reveals striking similarities between the bovine and feline isolates of Tritrichomonas foetus: consequences for in silico drug-target identification.

BACKGROUND: Few, if any, protozoan parasites are reported to exhibit extreme organ tropism like the flagellate Tritrichomonas foetus. In cattle, T. foetus infects the reproductive system causing abortion, whereas the infection in cats results in chronic large bowel diarrhoea. In the absence of a T. foetus genome, we utilized a de novo approach to assemble the transcriptome of the bovine and feline genotype to identify host-specific adaptations and virulence factors specific to each genotype. Furthermore, a subset of orthologs was used to characterize putative druggable targets and expose complications of in silico drug target mining in species with indefinite host-ranges. RESULTS: Illumina RNA-seq reads were assembled into two representative bovine and feline transcriptomes containing 42,363 and 36,559 contigs, respectively. Coding and non-coding regions of the genome libraries revealed striking similarities, with 24,620 shared homolog pairs reduced down to 7,547 coding orthologs between the two genotypes. The transcriptomes were near identical in functional category distribution; with no indication of selective pressure acting on orthologs despite differences in parasite origins/host. Orthologs formed a large proportion of highly expressed transcripts in both genotypes (bovine genotype: 76%, feline genotype: 56%). Mining the libraries for protease virulence factors revealed the cysteine proteases (CP) to be the most common. In total, 483 and 445 bovine and feline T. foetus transcripts were identified as putative proteases based on MEROPS database, with 9 hits to putative protease inhibitors. In bovine T. foetus, CP8 is the preferentially transcribed CP while in the feline genotype, transcription of CP7 showed higher abundance. In silico druggability analysis of the two genotypes revealed that when host sequences are taken into account, drug targets are genotype-specific. CONCLUSION: Gene discovery analysis based on RNA-seq data analysis revealed prominent similarities between the bovine and feline T. foetus, suggesting recent adaptation to their respective host/niche. T. foetus represents a unique case of a mammalian protozoan expanding its parasitic grasp across distantly related host lineages. Consequences of the host-range for in silico drug targeting are exposed here, demonstrating that targets of the parasite in one host are not necessarily ideal for the same parasite in another host.

In vitro combination therapy using low dose clotrimazole and photodynamic therapy leads to enhanced killing of the dermatophyte Trichophyton rubrum.

BACKGROUND: Superficial infections of the skin and mucous membranes caused by dermatophyte fungi are amongst the most common and challenging infections to treat. Previously we demonstrated the phototoxic effects of photodynamic therapy (PDT) towards Trichophyton rubrum, using a green laser to photoactivate Rose Bengal (RB). The aim of this study was to evaluate whether we could; (1) achieve a similar effect using an inexpensive light-emitting diode (LED) to photoactivate RB and (2) to evaluate whether our PDT regime could be combined with standard antifungal drug therapy and increase its effectiveness. METHODS: We designed and built our own inexpensive green (530 nm) LED source and tested its efficacy as part our RB-PDT regime in vitro against T. rubrum. We also examined the potential benefits of incorporating PDT as part of combination therapy and whether the order in which this was done had an impact. First we subjected spore suspensions to sub-inhibitory concentrations of a number of antifungal agents (CLT, MCZ and TRB) for 72 hours followed by RB-PDT. Secondly we subjected spore suspensions to sub-inhibitory PDT followed by drug treatment and evaluated if there were any changes to the minimum inhibitory concentrations (MICs) of the drugs tested. RESULTS: The optimal conditions for photoinactivation of T. rubrum using RB-PDT alone were 140 µM of RB and 24 J/cm2 of LED (equating to a 30-minute exposure). These parameters also caused a 100% reduction in the viability of the pathogenic yeast Candida albicans and the model fungus Saccharomyces cerevisiae. By combining our RB-PDT regime as an adjunct to antifungal drugs we were able to dramatically reduce the exposure times. Treatment of spore suspensions using a sub-inhibitory dose of clotrimazole (CLT) followed by RB-PDT, this order was critical, significantly reduced the exposure times required to achieve 100% inhibition of T. rubrum to 15 minutes as compared to RB-PDT alone. CONCLUSIONS: The combination of antifungal drug and RB-PDT represents an attractive alternative to the current antifungal therapies used to treat superficial fungal diseases. Our approach has the potential to reduce treatment times and drug dosages which can also reduce drug toxicity and improve patient compliance.

The polyene antifungals, amphotericin B and nystatin, cause cell death in Saccharomyces cerevisiae by a distinct mechanism to amphibian-derived antimicrobial peptides.

BACKGROUND: There is a pressing need to identify novel antifungal drug targets to aid in the therapy of life-threatening mycoses and overcome increasing drug resistance. Identifying specific mechanisms of action of membrane-interacting antimicrobial drugs on the model fungus Saccharomyces cerevisiae is one avenue towards addressing this issue. The S. cerevisiae deletion mutants Δizh2, Δizh3, Δaif1 and Δstm1 were demonstrated to be resistant to amphibian-derived antimicrobial peptides (AMPs). The purpose of this study was to examine whether AMPs and polyene antifungals have a similar mode of action; this was done by comparing the relative tolerance of the mutants listed above to both classes of antifungal. FINDINGS: In support of previous findings on solid media it was shown that Δizh2 and Δizh3 mutants had increased resistance to both amphotericin B (1-2 µg ml-1) and nystatin (2.5 - 5 µg ml-1) in liquid culture, after acute exposure. However, Δaif1 and Δstm1 had wild-type levels of susceptibility to these polyenes. The generation of reactive oxygen species (ROS) after exposure to amphotericin B was also reduced in Δizh2 and Δizh3. These data indicated that polyene antifungal and AMPs may act via distinct mechanisms of inducing cell death in S. cerevisiae. CONCLUSIONS: Further understanding of the mechanism(s) involved in causing cell death and the roles of IZH2 and IZH3 in drug susceptibility may help to inform improved drug design and treatment of fungal pathogens.

An in vitro study of the photodynamic effect of rose bengal on Trichophyton rubrum.

Onychomycosis, a fungal infection of the finger or toenails, is predominantly caused by Trichophyton rubrum. Treatment is difficult due to high recurrence rates and problems with treatment compliance. For these reasons, alternative therapies are needed. Here we describe the photoactivation of Rose Bengal (RB) using a green laser (λ = 532 nm) at fluences of 68, 133 and 228 J/cm(2) , and assess its fungicidal activity on T. rubrum spore suspensions. A 140 µM RB solution was able to induce a fungicidal effect on T. rubrum when photosensitized with the fluence of 228 J/cm(2) . RB photosensitization using a green laser provides a potential novel treatment for T. rubrum infections.

An investigation into the inhibitory effect of ultraviolet radiation on Trichophyton rubrum.

Fungal infection of nails, onychomycosis, is predominantly caused by Trichophyton rubrum. This infection is an important public health concern due to its persistent nature and high recurrence rates. Alternative treatments are urgently required. One such alternative is phototherapy involving the action of photothermal or photochemical processes. The aim of this novel study was to assess which wavelengths within the ultraviolet (UV) spectrum were inhibitory and equally important nail transmissible. Initial irradiations of T. rubrum spore suspensions were carried out using a tunable wavelength lamp system (fluence ≤3.1 J/cm(2)) at wavelengths between 280 and 400 nm (UVC to UVA) to evaluate which wavelengths prevented fungal growth. Light-emitting diodes (LEDs) of defined wavelengths were subsequently chosen with a view to evaluate and potentially implement this technology as a low-cost "in-home" treatment. Our experiments demonstrated that exposure at 280 nm using an LED with a fluence as low as 0.5 J/cm(2) was inhibitory, i.e., no growth following a 2-week incubation (p < 0.05; one-way ANOVA), while exposure to longer wavelengths was not. A key requirement for the use of phototherapy in the treatment of onychomycosis is that it must be nail transmissible. Our results indicate that the treatment with UVC is not feasible given that there is no overlap between the antifungal activity observed at 280 nm and transmission through the nail plate. However, a potential indirect application of this technology could be the decontamination of reservoirs of infection such as the shoes of infected individuals, thus preventing reinfection.

Comparative analysis of Tritrichomonas foetus (Riedmüller, 1928) cat genotype, T. foetus (Riedmüller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci.

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmüller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1, 2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine, 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensisCulberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2, 4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suisDavaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis.

Integrating the disaster cycle model into traditional disaster diplomacy concepts.

Disaster diplomacy is an evolving contemporary model that examines how disaster response strategies can facilitate cooperation between parties in conflict. The concept of disaster diplomacy has emerged during the past decade to address how disaster response can be leveraged to promote peace, facilitate communication, promote human rights, and strengthen intercommunity ties in the increasingly multipolar modern world. Historically, the concept has evolved through two camps, one that focuses on the interactions between national governments in conflict and another that emphasizes the grassroots movements that can promote change. The two divergent approaches can be reconciled and disaster diplomacy further matured by contextualizing the concept within the disaster cycle, a model well established within the disaster risk management community. In particular, access to available health care, especially for the most vulnerable populations, may need to be negotiated. As such, disaster response professionals, including emergency medicine specialists, can play an important role in the development and implementation of disaster diplomacy concepts.

The aminopeptidase inhibitor CHR-2863 is an orally bioavailable inhibitor of murine malaria.

Malaria remains a significant risk in many areas of the world, with resistance to the current antimalarial pharmacopeia an ever-increasing problem. The M1 alanine aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) are believed to play a role in the terminal stages of digestion of host hemoglobin and thereby generate a pool of free amino acids that are essential for parasite growth and development. Here, we show that an orally bioavailable aminopeptidase inhibitor, CHR-2863, is efficacious against murine malaria.

Sequence differences in the diagnostic region of the cysteine protease 8 gene of Tritrichomonas foetus parasites of cats and cattle.

In order to investigate the genetic variation between Tritrichomonas foetus from bovine and feline origins, cysteine protease 8 (CP8) coding sequence was selected as the polymorphic DNA marker. Direct sequencing of CP8 coding sequence of T. foetus from four feline isolates and two bovine isolates with polymerase chain reaction successfully revealed conserved nucleotide polymorphisms between feline and bovine isolates. These results provide useful information for CP8-based molecular differentiation of T. foetus genotypes.

The phylogeny, structure and function of trematode cysteine proteases, with particular emphasis on the Fasciola hepatica cathepsin L family.

Helminth parasites (nematodes, flatworms and cestodes) infect over 1 billion of the world's population causing high morbidity and mortality. The large tissue-dwelling worms express papain-like cysteine peptidases, termed cathepsins that play important roles in virulence including host entry, tissue migration and the suppression of host immune responses. Much of our knowledge of helminth cathepsins comes from studies using flatworms or trematode (fluke) parasites. The developmentally-regulated expression of these proteases correlates with the passage of parasites through host tissues and their encounters with different host macromolecules. Recent phylogenetic, biochemical and structural studies indicate that trematode cathepsins exhibit overlapping but distinct substrate specificities due to divergence within the protease active site. Here we provide an overview of the evolution, biochemistry and structure of these important enzymes and highlight how recent advances in proteomics and gene silencing techniques are allowing researchers to probe their biological functions. We focus mainly on members of the cathepsin L gene family of the animal and human pathogen, Fasciola hepatica, because of our deep understanding of their function, biochemistry and structure.

Aminopeptidases of malaria parasites: new targets for chemotherapy.

Novel targets for new drug development are urgently required to combat malaria, a disease that puts half of the world's population at risk. One group of enzymes identified within the genome of the most lethal of the causative agents of malaria, Plasmodium falciparum, that may have the potential to become new targets for antimalarial drug development are the aminopeptidases. These enzymes catalyse the cleavage of the N-terminal amino acids from proteins and peptides. P. falciparum appears to encode for at least nine aminopeptidases, two neutral aminopeptidases, one aspartyl aminopeptidase, one aminopeptidase P, one prolyl aminopeptidase and four methionine aminopeptidases. Recent advances in our understanding of these genes and their protein products are outlined in this review, including their potential for antimalarial drug development.

Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases.

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the X-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the X-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.

Helminth cysteine proteases inhibit TRIF-dependent activation of macrophages via degradation of TLR3.

Helminth pathogens prepare a Th2 type immunological environment in their hosts to ensure their longevity. They achieve this by secreting molecules that not only actively drive type 2 responses but also suppress type 1 responses. Here, we show that the major cysteine proteases secreted from the helminth pathogens Fasciola hepatica (FheCL1) and Schistosoma mansoni (SmCB1) protect mice from the lethal effects of lipopolysaccharide by preventing the release of inflammatory mediators, nitric oxide, interleukin-6, tumor necrosis factor alpha, and interleukin-12, from macrophages. The proteases specifically block the MyD88-independent TRIF-dependent signaling pathway of Toll-like receptor (TLR)4 and TLR3. Microscopical and flow cytometric studies, however, show that alteration of macrophage function by cysteine protease is not mediated by cleavage of components of the TLR4 complex on the cell surface but occurs by degradation of TLR3 within the endosome. This is the first study to describe a parasite molecule that degrades this receptor and pinpoints a novel mechanism by which helminth parasites modulate the innate immune responses of their hosts to suppress the development of Th1 responses.