SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

BackgroundSerine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis.Methodology/principal findingsSmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting.Conclusions/significanceThe data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy

Trophic Dynamics of the Boreal Forests of the Kluane Region

The trophic dynamics of the Yukon boreal forest have been under investigation at the Kluane Lake Research Station since 1973. We monitored and conducted experiments on the major species in this ecosystem, except the large mammals (for logistic reasons). The central problem has been to determine the causes of the 9 – 10 year cycle of snowshoe hares, and to achieve this we carried out several large-scale experiments manipulating food supplies, predator pressure, and soil nutrient availability to test hypotheses that food, predation, or habitat quality regulate populations. The hare cycle is driven top-down by predators, and most hares die because they are killed by predators. Predators also cause stress in female hares, and the stress response seems to be responsible for the loss of reproductive potential in the decline and low phases of the hare cycle. Many of the specialist predators and some herbivores in this ecosystem fluctuate with the hare cycle. Arctic ground squirrels do, but red squirrels do not, being linked closely to white spruce seed masting years. Small rodents fluctuate in numbers in two patterns. Red-backed voles and four species of Microtus voles have a 3 – 4 year cycle that seems to be driven by food supplies and social behaviour. Deer mice, in contrast, have fluctuated dramatically in the 38 years we have monitored them, but not cyclically. White spruce seed production varies with temperature and rainfall, but was not affected by adding nutrients in fertilizer. Global warming and reduced hare browsing in the last 20 years have helped to increase the abundance of shrubs in these forests. It will be challenging to predict how this system will change as climatic warming proceeds, because even closely related species in the same trophic level respond differently to perturbations. We recommend continued monitoring of the major species in these boreal forests.La dynamique trophique de la forêt boréale du Yukon fait l’objet d’une étude à la station de recherche du lac Kluane depuis 1973. Nous avons fait des expériences et surveillé les espèces importantes de cet écosystème, sauf en ce qui a trait aux principaux mammifères (pour des raisons de logistique). Le problème central a consisté à déterminer les causes du cycle de 9 à 10 ans du lièvre d’Amérique. Pour ce faire, nous avons effectué plusieurs expériences à grande échelle dans le cadre desquelles nous avons manipulé les disponibilités alimentaires, la pression exercée par les prédateurs et la disponibilité en nutriments dans le sol afin de mettre à l’épreuve les hypothèses selon lesquelles la nourriture, la prédation ou la qualité de l’habitat régularisent les populations. Le cycle du lièvre est dicté par les prédateurs de haut en bas, et la plupart des lièvres meurent parce qu’ils sont tués par les prédateurs. Par ailleurs, les prédateurs sont une source de stress chez les lièvres femelles, et la réaction au stress semble responsable de la perte de capacité de reproduction dans la phase du déclin et la phase basse du cycle du lièvre. Grand nombre des prédateurs spécialistes et certains herbivores de cet écosystème fluctuent en fonction du cycle du lièvre. C’est le cas du spermophile arctique, mais ce n’est pas le cas de l’écureuil roux, car il est étroitement lié aux années de paisson de graines d’épinette blanche. Le nombre de petits rongeurs fluctue en fonction de deux modèles. Le campagnol à dos roux et quatre espèces de campagnols Microtus ont un cycle de trois à quatre ans qui semble dicté par les disponibilités alimentaires et le comportement social, tandis que la souris sylvestre a connu d’énormes fluctuations pendant les 38 années qui ont fait l’objet d’une surveillance, sans toutefois afficher de cycles. La production de graines d’épinette blanche varie en fonction des températures et des chutes de pluie, mais n’a pas été influencée par l’ajout de nutriments au fertilisant. Le réchauffement planétaire et le broutage réduit des lièvres ces 20 dernières années ont aidé à accroître l’abondance d’arbustes dans ces forêts. Il sera difficile de prévoir comment ce système changera au fur et à mesure du réchauffement climatique, car même les espèces étroitement liées du même niveau trophique réagissent aux perturbations de manière différente. Nous recommandons la surveillance continue des principales espèces de ces forêts boréales

Global protease activity profiling provides differential diagnosis of pancreatic cysts

Purpose: Pancreatic cysts are estimated to be present in 2%-3% of the adult population. Unfortunately, current diagnostics do not accurately distinguish benign cysts from those that can progress into invasive cancer. Misregulated pericellular proteolysis is a hallmark of malignancy, and therefore, we used a global approach to discover protease activities that differentiate benign nonmucinous cysts from premalignant mucinous cysts.Experimental Design: We employed an unbiased and global protease profiling approach to discover protease activities in 23 cyst fluid samples. The distinguishing activities of select proteases was confirmed in 110 samples using specific fluorogenic substrates and required less than 5 μL of cyst fluid.Results: We determined that the activities of the aspartyl proteases gastricsin and cathepsin E are highly increased in fluid from mucinous cysts. IHC analysis revealed that gastricsin expression was associated with regions of low-grade dysplasia, whereas cathepsin E expression was independent of dysplasia grade. Gastricsin activity differentiated mucinous from nonmucinous cysts with a specificity of 100% and a sensitivity of 93%, whereas cathepsin E activity was 92% specific and 70% sensitive. Gastricsin significantly outperformed the most widely used molecular biomarker, carcinoembryonic antigen (CEA), which demonstrated 94% specificity and 65% sensitivity. Combined analysis of gastricsin and CEA resulted in a near perfect classifier with 100% specificity and 98% sensitivity.Conclusions: Quantitation of gastricsin and cathepsin E activities accurately distinguished mucinous from nonmucinous pancreatic cysts and has the potential to replace current diagnostics for analysis of these highly prevalent lesions. Clin Cancer Res; 23(16); 4865-74. ©2017 AACR

Proteomic analysis of pollination-induced corolla senescence in petunia

Senescence represents the last phase of petal development during which macromolecules and organelles are degraded and nutrients are recycled to developing tissues. To understand better the post-transcriptional changes regulating petal senescence, a proteomic approach was used to profile protein changes during the senescence of Petunia×hybrida ‘Mitchell Diploid’ corollas. Total soluble proteins were extracted from unpollinated petunia corollas at 0, 24, 48, and 72 h after flower opening and at 24, 48, and 72 h after pollination. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in non-senescing (unpollinated) and senescing (pollinated) corollas, and image analysis was used to determine which proteins were up- or down-regulated by the experimentally determined cut-off of 2.1-fold for P <0.05. One hundred and thirty-three differentially expressed protein spots were selected for sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the identity of these proteins. Searching translated EST databases and the NCBI non-redundant protein database, it was possible to assign a putative identification to greater than 90% of these proteins. Many of the senescence up-regulated proteins were putatively involved in defence and stress responses or macromolecule catabolism. Some proteins, not previously characterized during flower senescence, were identified, including an orthologue of the tomato abscisic acid stress ripening protein 4 (ASR4). Gene expression patterns did not always correlate with protein expression, confirming that both proteomic and genomic approaches will be required to obtain a detailed understanding of the regulation of petal senescence

Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome

The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic β2 and β5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the β5 P1 binding pocket, compromising compound binding. Dual targeting of P. falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio