Ezrin Interacts with the SARS Coronavirus Spike Protein and Restrains Infection at the Entry Stage

Background

Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process.

Methodology/Principal Findings

We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion.

Conclusions/Significance

Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.     

Modeling changes in soil organic matter in Amazon forest to pasture conversion with the Century model

Land use and land cover changes in the Brazilian Amazon region have major implications for regional and even global carbon cycling. We analyzed the effects of the predominant land use change, conversion of tropical forest to pasture, on total soil C and N, using the Century ecosystem model and data collected from the Nova Vida ranch, Western Brazilian Amazon. We estimated equilibrium organic matter levels, plant productivity and residue carbon inputs under native forest conditions, then simulated deforestation following the slash and burn procedure. Soil organic matter dynamics were simulated for pastures established in 1989, 1987, 1983, 1979, 1972, 1951, and 1911. Using input data from the Nova Vida ranch, the Century model predicted that forest clearance and conversion to pasture would cause an initial decline in soil C and N stocks, followed by a slow rise to levels exceeding those under native forest. Simulated soil total C and N levels (2500 g C m^?2 and 245 g N m^?2 in the 0–20 cm layer) prior to conversion to pasture were close to those measured in the native forest. Simulated above‐ and below‐ground biomass for the forest and pasture were comparable with literature values from this region. The model predicted the long‐term changes in soil C and N under pasture inferred from the pasture chronosequence, but there was considerable variation in soil C stocks for pastures <20 years in age. Differences in soil texture between pastures were relatively small and could not account for much of the variability between different pastures of similar ages, in either the measured or simulated data. It is likely that much of the variability in C stocks between pastures of similar ages is related to initial C stocks immediately following deforestation and that this was the largest source of variability in the chronosequence. Internal C cycling processes in Century were evaluated using measurements of microbial biomass and soil δ¹³C. The relative magnitude and long‐term trend in microbial biomass simulated by the model were consistent with measurements. The close fit of simulated to measured values of δ¹³C over time suggests that the relative loss of forest‐derived C and its replacement by pasture‐derived C was accurately predicted by the model. After 80 years, almost 90% of the organic matter in the top 20 cm was pasture derived. While our analysis represents a single ‘case study’ of pasture conversion, our results suggest that modeling studies in these pasture systems can help to evaluate the magnitude of impacts on C and N cycling, and determine the effect of management strategies on pasture sustainability.     

Plant N-glycan profiling of minute amounts of material

Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring α(1,3)- or α(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.     

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography—mass spectrometry

Furosemide (FD; Lasix^®) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-μl sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6–14.0 mg), with a mean urine volume of 3024 ml (range 2620–3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analyses to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography—mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.     

CO2 emission from mineral soils following land‐cover change in Brazil

This paper presents a first approximation of net CO₂ fluxes from mineral soil due to land use, land‐use changes and forestry (LULUCF) activities in Brazil for the periods 1970–90 and 1975–95. The methodology employed is an adaptation of the approach proposed by the IPCC in ‘Revised 1996 guidelines for national greenhouse gas inventories’, which is based on the variations in soil C stocks as a function of changes in land‐use activities. The calculation was done separately for each Brazilian state and subsequently summarized for all of Brazil. The annual fluxes for Brazil indicate a net emission of CO₂ to the atmosphere, which decreased from 93.3 Tg CO₂ for the period 1970–90 to 46.4 Tg CO₂ for the period 1975–95. This corresponded to yearly net emission rates of 11.0 g CO₂ m^?2 year^?1 for the 1970–90 period and 5.5 g CO₂ m^?2 year^?1 for the 1975–95 period. Within each administrative region, considerable differences in the yearly emission rates between the states could be observed. Several sources of uncertainties could be identified. The most important uncertainties were linked to the impact factor values, which represent changes in native C stock associated with conversion of the native vegetation to agricultural use.     

Down-regulated expression of plant-specific glycoepitopes in alfalfa

Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N -glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N -glycosylation in alfalfa. The first was a knock-down of two plant-specific N -glycan maturation enzymes, β1,2-xylosyltransferase and α1,3-fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human β1,4-galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N -acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum . Both knock-down and knock-in strategies strongly, but not completely, inhibited the biosynthesis of α1,3-fucose- and β1,2-xylose-containing glycoepitopes in transgenic alfalfa. However, recombinant human β1,4-galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N -glycans.     

Assessment of the efficacy of antimalarial drugs recommended by the National Malaria Control Programme in Madagascar: Up-dated baseline data from randomized and multi-site clinical trials

Background

In order to improve the monitoring of the antimalarial drug resistance in Madagascar, a new national network based on eight sentinel sites was set up. In 2006/2007, a multi-site randomized clinical trial was designed to assess the therapeutic efficacy of chloroquine (CQ), sulphadoxine-pyrimethamine (SP), amodiaquine (AQ) and artesunate plus amodiaquine combination (ASAQ), the antimalarial therapies recommended by the National Malaria Control Programme (NMCP).

Methods

Children between six months and 15 years of age, with uncomplicated falciparum malaria, were enrolled. Primary endpoints were the day-14 and day-28 risks of parasitological failure, either unadjusted or adjusted by genotyping. Risks of clinical and parasitological treatment failure after adjustment by genotyping were estimated using Kaplan-Meier survival analysis. Secondary outcomes included fever clearance, parasite clearance, change in haemoglobin levels between Day 0 and the last day of follow-up, and the incidence of adverse events.

Results

A total of 1,347 of 1,434 patients (93.9%) completed treatment and follow-up to day 28. All treatment regimens, except for the chloroquine (CQ) treatment group, resulted in clinical cure rates above 97.6% by day-14 and 96.7% by day-28 (adjusted by genotyping). Parasite and fever clearance was more rapid with artesunate plus amodiaquine, but the extent of haematological recovery on day-28 did not differ significantly between the four groups. No severe side-effects were observed during the follow-up period.

Conclusion

These findings (i) constitute an up-dated baseline data on the efficacy of antimalarial drugs recommended by the NMCP, (ii) show that antimalarial drug resistance remains low in Madagascar, except for CQ, compared to the bordering countries in the Indian Ocean region such as the Comoros Archipelago and (iii) support the current policy of ASAQ as the first-line treatment in uncomplicated falciparum malaria.