Homing in on getting out: characterisation of SERA6, a putative malarial protease with a role in egress

The human malaria parasite, P. falciparum, replicates within a membrane-bound intraerythrocytic parasitophorous vacuole (PV). The resulting daughter merozoites actively escape from the host cell in a process called egress. There is convincing evidence that proteases are key players in this step. These proteases could serve as excellent targets for the development of new antimalarial drugs. P. falciparum Serine Repeat Antigens (SERAs) form a family of 9 proteins all containing a central papain-like domain that identifies them as putative cysteine proteases. They are highly conserved throughout all Plasmodium species, and there is strong genetic evidence that they may play a role in egress. P. falciparum SERA6 is one of the most highly-expressed SERAs in asexual erythrocyte stages. In this study biochemical fractionation and indirect immunofluorescence analysis were used to confirm localisation of SERA6 to the PV. It was shown that SERA6 is a substrate for PfSUB1, a subtilisin-like protease which is crucial for egress and which is released into the PV just prior to egress. SERA6 is cleaved by PfSUB1 at 3 positions, releasing the papain-like domain. Processing of SERA6 by PfSUB1 is partially indispensable in in vitro P. falciparum parasites, as some mutations in SERA6 that block cleavage are not tolerated in the parasite. In addition, the putative catalytic Cys of SERA6 cannot be replaced with a non-catalytic Ala residue in the parasite, indicating SERA6 is an indispensable cysteine protease. Finally it was demonstrated that PfSUB1-mediated processing of the P. berghei orthologue of SERA6 converts it to an active cysteine protease. It can be proposed that SERA6 is a key player in a proteolytic cascade that leads to egress of the blood-stage malaria parasite

Subsecond pore-scale displacement processes and relaxation dynamics in multiphase flow

With recent advances at X‐ray microcomputed tomography (μCT) synchrotron beam lines, it is now possible to study pore‐scale flow in porous rock under dynamic flow conditions. The collection of four‐dimensional data allows for the direct 3‐D visualization of fluid‐fluid displacement in porous rock as a function of time. However, even state‐of‐the‐art fast‐μCT scans require between one and a few seconds to complete and the much faster fluid movement occurring during that time interval is manifested as imaging artifacts in the reconstructed 3‐D volume. We present an approach to analyze the 2‐D radiograph data collected during fast‐μCT to study the pore‐scale displacement dynamics on the time scale of 40 ms which is near the intrinsic time scale of individual Haines jumps. We present a methodology to identify the time intervals at which pore‐scale displacement events in the observed field of view occur and hence, how reconstruction intervals can be chosen to avoid fluid‐movement‐induced reconstruction artifacts. We further quantify the size, order, frequency, and location of fluid‐fluid displacement at the millisecond time scale. We observe that after a displacement event, the pore‐scale fluid distribution relaxes to (quasi‐) equilibrium in cascades of pore‐scale fluid rearrangements with an average relaxation time for the whole cascade between 0.5 and 2.0 s. These findings help to identify the flow regimes and intrinsic time and length scales relevant to fractional flow. While the focus of the work is in the context of multiphase flow, the approach could be applied to many different μCT applications where morphological changes occur at a time scale less than that required for collecting a μCT scan

Bladder cancer cells acquire competent mechanisms to escape Fas-mediated apoptosis and immune surveillance in the course of malignant transformation

Mechanisms of resistance against Fas-mediated cell killing have been reported in different malignancies. However, the biological response of immune escape mechanisms might depend on malignant transformation of cancer cells. In this study we investigated different mechanisms of immune escape in 2 well-differentiated low-grade (RT4 and RT112) and 2 poorly differentiated high-grade (T24 and TCCSUP) bladder cancer cell lines. Fas, the receptor of Fas-ligand, is expressed and shedded by human transitional bladder carcinoma cell lines RT4, RT112, T24 and TCCSUP. Cytotoxicity and apoptosis assays demonstrate that in spite of the Fas expression, poorly differentiated T24 and TCCSUP cells are insensitive towards either recombinant Fas-ligand or agonistic apoptosis-inducing monoclonal antibody against Fas. In poorly differentiated T24 and TCCSUP cell lines we were able to detect marked Fas-ligand protein by flow cytometry and Western blot analysis. In grade 1 RT4 and RT112 cells only minor expression of Fas-ligand possibly because of proteinase action. Fas-ligand mRNA translation or post-translational processing seems to be regulated differentially in the cancer cell lines depending on malignant transformation. In co-culture experiments we show that poorly differentiated cells can induce apoptosis and cell death in Jurkat cells and activated peripheral blood mononuclear cells. This in vitro study suggests that bladder cancer cells can take advantage of different mechanisms of immune evasion and become more competent in avoiding immune surveillance during transformation to higher-grade malignant disease. © 2001 Cancer Research Campaign www.bjcancer.co

A high throughput screen for next-generation leads targeting malaria parasite transmission

Spread of parasite resistance to artemisinin threatens current frontline antimalarial therapies, highlighting the need for new drugs with alternative modes of action. Since only 0.2–1% of asexual parasites differentiate into sexual, transmission-competent forms, targeting this natural bottleneck provides a tangible route to interrupt disease transmission and mitigate resistance selection. Here we present a high-throughput screen of gametogenesis against a ~70,000 compound diversity library, identifying seventeen drug-like molecules that target transmission. Hit molecules possess varied activity profiles including male-specific, dual acting male–female and dual-asexual-sexual, with one promising N-((4-hydroxychroman-4-yl)methyl)-sulphonamide scaffold found to have sub-micromolar activity in vitro and in vivo efficacy. Development of leads with modes of action focussed on the sexual stages of malaria parasite development provide a previously unexplored base from which future therapeutics can be developed, capable of preventing parasite transmission through the population

Debiasing the NEOWISE Cryogenic Mission Comet Populations

We use NEOWISE data from the four-band and three-band cryogenic phases of the Wide-field Infrared Survey Explorer mission to constrain size distributions of the comet populations and debias measurements of the short- and long-period comet (LPC) populations. We find that the fit to the debiased LPC population yields a cumulative size−frequency distribution (SFD) power-law slope (β) of −1.0 ± 0.1, while the debiased Jupiter-family comet (JFC) SFD has a steeper slope with β = −2.3 ± 0.2. The JFCs in our debiased sample yielded a mean nucleus size of 1.3 km in diameter, while the LPCs' mean size is roughly twice as large, 2.1 km, yielding mean size ratios (〈D_(LPC)〉/〈D_(JFC)〉) that differ by a factor of 1.6. Over the course of the 8 months of the survey, our results indicate that the number of LPCs passing within 1.5 au are a factor of several higher than previous estimates, while JFCs are within the previous range of estimates of a few thousand down to sizes near 1.3 km in diameter. Finally, we also observe evidence for structure in the orbital distribution of LPCs, with an overdensity of comets clustered near 110° inclination and perihelion near 2.9 au that is not attributable to observational bias

Cardiopulmonary Exercise Testing Provides Additional Prognostic Information in Cystic Fibrosis

RATIONALE: The prognostic value of cardiopulmonary exercise testing (CPET) for survival in cystic fibrosis (CF) in the context of current clinical management, when controlling for other known prognostic factors, is unclear. OBJECTIVES: To determine the prognostic value of CPET-derived measures beyond peak oxygen uptake (V.o2peak) following rigorous adjustment for other predictors. METHODS: Data from 10 CF centers in Australia, Europe, and North America were collected retrospectively. A total of 510 patients completed a cycle CPET between January 2000 and December 2007, of which 433 fulfilled the criteria for a maximal effort. Time to death/lung transplantation was analyzed using Cox proportional hazards regression. In addition, phenotyping using hierarchical Ward clustering was performed to characterize high-risk subgroups. MEASUREMENTS AND MAIN RESULTS: Cox regression showed, even after adjustment for sex, FEV1% predicted, body mass index (z-score), age at CPET, Pseudomonas aeruginosa status, and CF-related diabetes as covariates in the model, that V.o2peak in % predicted (hazard ratio [HR], 0.964; 95% confidence interval [CI], 0.944–0.986), peak work rate (% predicted; HR, 0.969; 95% CI, 0.951–0.988), ventilatory equivalent for oxygen (HR, 1.085; 95% CI, 1.041–1.132), and carbon dioxide (HR, 1.060; 95% CI, 1.007–1.115) (all P < 0.05) were significant predictors of death or lung transplantation at 10-year follow-up. Phenotyping revealed that CPET-derived measures were important for clustering. We identified a high-risk cluster characterized by poor lung function, nutritional status, and exercise capacity. CONCLUSIONS: CPET provides additional prognostic information to established predictors of death/lung transplantation in CF. High-risk patients may especially benefit from regular monitoring of exercise capacity and exercise counseling

Biomass burning fuel consumption rates: a field measurement database

Landscape fires show large variability in the amount of biomass or fuel consumed per unit area burned. Fuel consumption (FC) depends on the biomass available to burn and the fraction of the biomass that is actually combusted, and can be combined with estimates of area burned to assess emissions. While burned area can be detected from space and estimates are becoming more reliable due to improved algorithms and sensors, FC is usually modeled or taken selectively from the literature. We compiled the peerreviewed literature on FC for various biomes and fuel categories to understand FC and its variability better, and to provide a database that can be used to constrain biogeochemical models with fire modules. We compiled in total 77 studies covering 11 biomes including savanna (15 studies, average FC of 4.6 t DM (dry matter) ha 1 with a standard deviation of 2.2),tropical forest (n = 19, FC = 126 +/- 77),temperate forest (n = 12, FC = 58 +/- 72),boreal forest (n = 16, FC = 35 +/- 24),pasture (n = 4, FC = 28 +/- 9.3),shifting cultivation (n = 2, FC = 23, with a range of 4.0-43),crop residue (n = 4, FC = 6.5 +/- 9.0),chaparral (n = 3, FC = 27 +/- 19),tropical peatland (n = 4, FC = 314 +/- 196),boreal peatland (n = 2, FC = 42 [42-43]),and tundra (n = 1, FC = 40). Within biomes the regional variability in the number of measurements was sometimes large, with e. g. only three measurement locations in boreal Russia and 35 sites in North America. Substantial regional differences in FC were found within the defined biomes: for example, FC of temperate pine forests in the USA was 37% lower than Australian forests dominated by eucalypt trees. Besides showing the differences between biomes, FC estimates were also grouped into different fuel classes. Our results highlight the large variability in FC, not only between biomes but also within biomes and fuel classes. This implies that substantial uncertainties are associated with using biome-averaged values to represent FC for whole biomes. Comparing the compiled FC values with co-located Global Fire Emissions Database version 3 (GFED3) FC indicates that modeling studies that aim to represent variability in FC also within biomes, still require improvements as they have difficulty in representing the dynamics governing FC

Pyrazoleamide compounds are potent antimalarials that target Na+ homeostasis in intraerythrocytic Plasmodium falciparum

The quest for new antimalarial drugs, especially those with novel modes of action, is essential in the face of emerging drug-resistant parasites. Here we describe a new chemical class of molecules, pyrazoleamides, with potent activity against human malaria parasites and showing remarkably rapid parasite clearance in an in vivo model. Investigations involving pyrazoleamide-resistant parasites, whole-genome sequencing and gene transfers reveal that mutations in two proteins, a calcium-dependent protein kinase (PfCDPK5) and a P-type cation-ATPase (PfATP4), are necessary to impart full resistance to these compounds. A pyrazoleamide compound causes a rapid disruption of Na+ regulation in blood-stage Plasmodium falciparum parasites. Similar effect on Na+ homeostasis was recently reported for spiroindolones, which are antimalarials of a chemical class quite distinct from pyrazoleamides. Our results reveal that disruption of Na+ homeostasis in malaria parasites is a promising mode of antimalarial action mediated by at least two distinct chemical classes