Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells.Reservosomes are lysosome-related organelles found in Using Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones.T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation.Taken together, our results suggest that epimastigote forms of  Intracellular traffic of cholesterol has been widely studied in mammalian cells Trypanosoma cruzi, the etiologic agent of Chagas disease, does not synthesize cholesterol. Remarkably, the major sterol produced by this protozoan is ergosterol, whose biosynthetic pathway is an important target for chemotherapy T. cruzi endocytic pathway, the reservosome  placed at the posterior region of the parasite Preliminary biochemical analysis of isolated reservosomes revealed cholesterol (cholesteryl esters) and ergosterol as the major neutral lipids in these organelles In this work we established a protocol for lipid inclusion isolation, starting from a purified reservosome fraction, in order to determine their composition by GC-MS. We showed that rectangular and spherical lipid droplets are formed mostly by cholesterol and their formation is dependent on serum supply. Moreover, we demonstrated that epimastigotes are capable of consuming cholesterol stock in reservosomes, including rod like inclusions, during periods of serum starvation.T. cruzi epimastigotes (Y strain) were cultivated for 4–5 days at 28°C in LIT (liver-infusion tryptose) medium 6 epimastigotes per mL were cultivated in LIT supplemented with 1, 10 or 50% of FCS at 28°C for 24, 48, 72 and 96 h. Cellular growth was measured by direct counting in a Neubauer chamber.3. The plasma was added to a vertical angle centrifuge tube with 20.0 mL of saline solution . This material was ultracentrifuged at 150,000 g in a vertical angle Beckman RPVTi 50 rotor  at 4°C for 18 hours. The LDL fraction was localized and removed. KBr was added again to adjust the density to 1.2 g/cm3, and the material was ultracentrifuged at 150,000 g in the same rotor at 4°C for 18 hours. The purified lipoprotein were extensively dialyzed against PBS with 1 mM EDTA. The lipoprotein concentration was quantified using DC Protein Assay (Bio Rad). For the uptake assays and culture, the LDL was sterilized by filtration with a 0.22 µm membrane . The integrity of the sterile LDL was verified in a polyacrylamide (3–15%) slab gel under denaturing conditions.Low density lipoprotein (LDL) was purified from fresh human plasma as described Lipid extraction of fetal calf serum (FCS)  without protein precipitation was performed as described Trypanosoma cruzi epimastigotes (5×106 cells) from LIT medium supplemented with 1, 10 or 50% FCS were washed twice in PBS  and fixed in 4% freshly prepared formaldehyde in PBS for 20 min at room temperature. After washing twice in PBS, cells were incubated in 10 µg/mL Nile Red for 15 min at room temperature, protected from light. The parasites were washed in PBS, pH 7.2, and incubated in DAPI (5 µg/mL) for 5 min. The cellular suspensions were transferred to a black 96-well microplate and Nile Red fluorescence was determined in a Microplate Reader Spectra Max M2 (Molecular Devices): yellow-gold fluorescence of neutral lipid inclusions were acquired . An aliquot of each cell suspension was collected and adhered to 0.1% poly-L-lysine coated glass coverslips. Samples were mounted on 0.2 M n-propylgallate in glycerol∶PBS (9∶1) and the yellow-gold fluorescence images of neutral lipid inclusions were acquired using appropriated filters in a Zeiss Axioplan epifluorescence microscope coupled to an Olympus X30 CCD camera. Images were further processed using Adobe Photoshop CS2 . Nile Red had already been used to quantify and image lipid inclusion formation inside acidic organelles 2, 250 mM sucrose) supplemented with protease inhibitor cocktail (Sigma-Aldrich P2714). For lipid inclusion isolation, three milliliters of isolated reservosomes (1.5–2 mg/mL) were washed in TMS, pH 7.2, and centrifuged at 120,000×g for 15 min at 4°C in a Beckman SW28 centrifuge tube. The pellet was resuspended in 3 mL 200 mM Na2CO3, pH 11.5, at room temperature for 15 min. The sample was then sonicated at 10% of total amplitude  using a standard probe (13 mm radiating diameter), for three cycles of 10 s, with 5-s rest between cycles. Sample volume was completed to 7 mL with the alkaline solution and the suspension was transferred to a SW 40 centrifuge tube. Sample was overlaid with 3.5 mL 20 mM Tris-HCl, pH 7.5, containing 100 mM KCl and 2 mM MgCl2, and the tube was centrifuged at 274.000×g for 1 h a 4°C. A white band was collected at the top of the gradient, mixed with 3 mL TMS, pH 7.5, and deposited at the bottom of a Beckman SW50.1 centrifuge tube, overlaid with additional 2 mL 20 mM Tris-HCl, pH 7.5, 100 mM KCl and 2 mM MgCl2, and centrifuged at the same conditions described before. The white band was recovered and processed for electron microscopy or frozen at −20°C to subsequent biochemical analysis.A reservosome purified fraction from parasites cultivated in 10 and 50% FCS medium were obtained as described Alternatively, reservosome fraction was adhered to 0.1% poly-L-lysine coated glass coverslips for 20 min and fixed in 4% formaldehyde in PBS for 10 min at room temperature. The samples were washed in PBS, followed by Nile Red incubation (10 µg/mL in PBS) for 15 min, protected from light. Finally, reservosomes were washed in PBS and Nile Red labeled lipid inclusions observed as described before.To quantify the neutral lipid content of isolated reservosomes from 10 and 50% FCS by fluorimetric analysis, reservosomes isolated from both cultures were washed in PBS and prepared for Nile red fluorimetric analysis as described in the previous section.Epimastigotes  and isolated reservosomes were fixed in 2.5% glutaraldehyde and 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 60 min at room temperature, postfixed in 1% osmium tetroxide, 0.8% potassium ferrocyanide, 5 mM calcium chloride in 0.1 M cacodylate buffer, pH 7.2, for 60 min, dehydrated in an acetone series and embedded in Epoxi resin. Ultrathin sections were stained with 5% uranyl acetate and lead citrate and observed in a Zeiss 900 transmission electron microscope operating at 80 kV.4 in 0,1 M sodium phosphate buffer, pH 7.6, for 20 min, washed in water, incubated for 10 min in 4% uranyl acetate and observed in a Zeiss 900 transmission electron microscope operating at 80 kV.Isolated lipid inclusions were visualized under the electron microscope as described Morphometric measurements were performed with images acquired at the magnification of 30.000×(1 micrometer corresponds to 533 pixels) at 80 kV using MET ZEISS 900. All cells in each random section were registered until reaching 50 different cells. The areas occupied by reservosomes and by the lipid inclusions inside them were measured and processed using Image J.3)/methanol (CH3OH) solutions: CHCl3∶CH3OH∶H2O  and CHCl3∶CH3OH  g. Following centrifugation, the organic phase was transferred to a clean glass tube with Teflon-lined cap and stored at −20°C until further use. The sample was dried under highly pure nitrogen stream after the last extraction in each step as described by Isolated reservosomes and lipid inclusions (in 13×100 mm PYREX clear borosilicate glass tubes with Teflon-lined screw cap) were extracted three times in 10 volumes of chloroform (CHCl4) after heating at 200°C for 20 min. After that, the chromatography plates were digitized.Samples were analyzed by one-dimensional thin-layer chromatography (TLC) on Silica Gel 60 plates  for neutral lipids using n-hexane∶diethyl ether∶acetic acid (60∶40∶1 v/v). Cholesterol, cholesteryl-oleate, glycerol-tryoleate, diolein, oleoyl-glycerol and oleic acid  were used as standards. The lipids were visualized using a charring reagent . After washing with CHThe analysis of the sterol fraction by GC-MS was carried out as described m/z range, and a chromatogram was generated by plotting the spectra of diagnostic fragment-ion species for m/z 41, 43, and 55. Fatty-acid species were identified by comparison with the FAME 37 methylated FA mix standard .For GC-MS analysis, samples were redissolved in 100 µL DCM and 1 µL was used for analysis in a Trace-GC gas chromatographer  coupled to a mass spectrometer  (GC-MS). Samples were separated in a SP-2380 fused silica column . The injector was set at 200°C, and the following gradient was used: 70°C for 5 min, followed by 4°C/min up to 140°C, 2°C/min up to 185°C, and 185°C for 10 min. Helium was used as the carrier gas, with a flow rate of 1 mL/min. The molecules were ionized by electron impact at 70 eV and 200°C. The spectra were collected in the 30–400 6 cells were washed in PBS and incubated in Nile Red (10 µg/mL in PBS) for 15 min in the dark. Then, the parasites were washed in PBS, followed by incubation with 1 µM Sytox Blue (Invitrogen) for 5 min to determine cell viability. For positive control, 5×106 cells were permeabilized for 5 min with 0.1% Triton X-100 in PBS. The quantification of fluorescence was determined as describe before. In addition, parasites were processed for observation by transmission electron microscopy and morphometric analysis as described above.Aiming to determine the lipid reservoir consumption of epimastigotes from medium supplemented with 10 or 50% FCS, parasites were washed in PBS, transferred to serum-free LIT medium and incubated for 0, 8, 24, 48, or 72 h at 28°C. After each time, 5×10in vitro to verify cell proliferation in low and high concentrations of serum. Statistical analysis showed that cell proliferation is dependent on serum concentration, as observed in We decided to modulate FCS concentration in epimastigote studies Subsequently, we hypothesize that the epimastigote total lipid content would vary in concentration according to sterol uptake from the parasite diet. To test this hypothesis we collected the parasites from medium with 1, 10 and 50% FCS and incubated cells in Nile Red for neutral lipid fluorescence staining and performed a fluorimetric analysis . We obsein situ. Thus, our protocol appears to produce an enriched fraction of reservosome lipid inclusions, since no other structures, such as contaminant membranes and organelles, were evident.In order to determine the biochemical nature of reservosome lipid inclusions, we had first obtained a purified fraction of isolated reservosomes as previously performed before by our group Additionally, when the reservosome lipid fraction was subjected to GC-MS analysis, we found out that the major neutral lipids were cholesterol and cholesteryl esters . No otheThe more predominant fatty acid species covalently linked to cholesterol both in reservosome and lipid fractions was palmitate (C16:0), followed by oleate (C18:1), stearate (C18:0) and myristate (C14:0) .Epimastigotes originated from 10 and 50% FCS cultures were maintained in LIT medium without serum for 0, 8, 24, 48 and 72 hours. We could follow lipid consumption by fluorimetric analysis of Nile Red fluorescence  and obseT. cruzi depends on exogenous lipids from host to play essential roles in all developmental forms, as growth and host infection. Changes in phospholipid and neutral lipid profiles resulting from aging in culture or temperature shift of epimastigotes or trypomastigotes were already reported T. cruzi is available. In this way, reservosomes, the final step in the epimastigote endocytic pathway, exercise a pivotal role in storing and disposing lipids and proteins uptaken by endocytosis T. cruzi, the LDL receptor was not characterized, although LDL endocytosis and accumulation in reservosomes have already been demonstrated T. brucei require lipoproteins (LDL and HDL) and other serum components to growth and maintenance in axenic cultures, providing cholesterol, cholesterol esters and phospholipids to the parasites T. brucei and in other trypanosomatids such as T. b. rhodesiense, Leishmania donovani and Crithidia luciliaeT. cruzi epimastigote could operate by similar mechanisms.Since trypanosomatids do not synthesize cholesterol (or cholesteryl ester derivatives) In our current analysis, ergosterol represented only 1.5% of neutral lipids in reservosomes, being completely absent in the lipid inclusion fraction. This clarifies the real nature of reservosome lipid inclusions, and highlights the role of cholesterol from serum in the formation of lipid rods. The low concentration of ergosterol in reservosomes is in agreement with early work concerning ergosterol distribution in epimastigote subcellular fractions In this work we have obtained the first fatty acid profile of reservosomes. Like other lipids, fatty acid biosynthesis is constantly submitted to environmental conditions, which includes temperature, availability in the culture medium and aging Taken together, these results support the idea that the neutral lipid content of reservosomes is a consequence of intense acquisition of exogenous lipids. Reservosomes containing sword-shaped lipid inclusion profiles surrounded by a phospholipid monolayer have already been shown by our group T. cruzi epimastigotes to mobilize reservosome lipid inclusions remain largely unknown. The reservosome proteomic analysis failed to identify proteins usually related to the efflux and cellular distribution of cholesterol, like NPC1 and NPC2. However, other proteins were found that could be involved in lipid transport from reservosomes into cytoplasm, as an ABCA1 transporter and Rab18 T. cruzi, ABCA1 was localized at the plasma membrane, flagellar pocket and endocytic compartments. It could function in extruding cholesterol excess back to extracellular milieu, as it does in mammals. The presence of ABCA1 in endocytic compartments may be correlated to its own turnover or, alternatively, to the extrusion of cholesterol from reservosomes to the cytosol. Rab18 is commonly found in lipid bodies and endoplasmic reticulum of many cell types, where it is involved in releasing lipids from these internal reservoirs T. cruzi reservosomes The mechanisms used by Figure S1Thin layer chromatography from medium and parasites in different culture conditions. A – Liver Infusion Triptose (LIT) Medium; B – LIT with 10% Fetal Calf Serum (FCS); C – LIT with 10% delipidated Fetal Calf Serum; D – 10% Fetal Calf Serum; E – 10% delipidated Fetal Calf Serum (dFCS); F – T.cruzi Epimastigotes in LIT+10% FCS (1×108 cells); G - T.cruzi Epimastigotes in LIT+10% dFCS for 48 h (1×108 cells); H - T.cruzi Epimastigotes in LIT+10% dFCS+1 mg/mL human LDL (1×108 cells); I - T.cruzi Epimastigotes in LIT+10% dFCS+2 mg/mL human LDL (1×108 cells). Lipids: HC – Hydrocarbon; CHOE – Cholesteryl-ester; ERGE – Ergosteryl-ester; TG – Triacylglycerol; FA – Free Fatty Acids; CHO – Cholesterol; ERG – Ergosterol; MG – Monoacylglycerol; PL – Phospholipids; ND – Not Determined.(JPG)Click here for additional data file.Figure S2Fluorimetric analysis using Nile Red of epimastigotes in different culture conditions. Control epimastigotes, cultivated in 10% FCS, store twice as much neutral lipids than those grown in 10% delipidated FCS. After incubation with purified 1 mg/mL LDL the parasites presented 6 times more neutral lipids and after incubation with 2 mg/mL LDL, the store amount reaches 8 times the capacity of control epimastigotes. Fluorescence intensity was expressed in arbitrary units. The results are from two independent experiments.(PPT)Click here for additional data file.


The O2 concentration difference between the seawater and the test surface (ΔO2) was taken as a measure for the photosynthetic rate. Our results showed that O2 and pH levels were significantly higher on photosymbiotic foraminiferal surfaces in light than in dark conditions, and than on surfaces of symbiont-free foraminifera. Rates of photosynthesis at saturated light conditions did not change significantly between OA treatments . The pH at the cell surface decreased during incubations at elevated 2pCO, also during light incubations. Photosynthesis increased the surface pH but this increase was insufficient to compensate for ambient seawater pH decreases. We thus conclude that photosynthesis does only partly protect symbiont bearing foraminifera against OA.Ocean acidification (OA) can have adverse effects on marine calcifiers. Yet, phototrophic marine calcifiers elevate their external oxygen and pH microenvironment in daylight, through the uptake of dissolved inorganic carbon (DIC) by photosynthesis. We studied to which extent pH elevation within their microenvironments in daylight can counteract ambient seawater pH reductions, i.e. OA conditions. We measured the O Around phototrophic organisms  DBLs can maintain substantial gradients of O2 and pH microenvironment of 4 photosymbiotic and 2 symbiont-free benthic tropical foraminiferal species under different ocean acidification scenarios in light and dark conditions.We hypothesize that OA induced increases of seawater DIC might enhance photosynthesis of photosymbiotic calcifiers and consequently result in increased pH levels on their surfaces in daylight. Thus, the pH DBL might form a shield around the organism protecting it from OA. We studied whether this pH elevation within their microenvironment can protect photosymbiotic calcifiers (or at least partly compensate) from the effects of ocean acidification in daylight and therefore lend additional resistance compared to non photosymbiotic calcifiers. We tested this hypothesis by measuring the O2 and pH microenvironments under equal experimental conditions.Benthic foraminifera represent a good group of model organisms for this study, because compared to most other calcifiers, calcification is periodic rather than continous, and periods of calcification can be detected visually. Additionally, the process of chamber formation is very sensitive to mechanical disturbances and thus unlikely to occur in short term flume measurements see . ImpactsMarginopora vertebralis, Amphistegina radiata, Heterostegina depressa, and Peneroplis sp., and the symbiont-free species Quinquelloculina sp. and Miliola sp. were hand collected from coral rubble and other substrates containing foraminiferal assemblages by SCUBA diving during a cruise in the summer months of 2010 in the Whitsunday area, central section of the Great Barrier Marine Park. All necessary permits were obtained prior to field collection from the Great Barrier Marine Park Authority (Permit-No: G09/30237.1). Collection sites included, Bait Reef S 19°80.17′ E 149°07.55′, Daydream Island S 20°15.35′, E 148°48.73′, Shaw Island S 20°31.02′ E 149°04.48′ and Deloraine Island S 20°09.30′, E 149°04.50′  and salinity 35–36). A detailed description of the sampling sites can be found in Uthicke et al. Specimens of the photosymbiotic species −2 s−1), until they were transported to the Australian Institute of Marine Science (AIMS) in Townsville. Prior to experiments, specimens acclimatised in indoor climatic chambers>3 weeks in natural seawater  at 24° - 26°C, 10 µmol photons m−2 s−1, 12 h : 12 h diurnal cycling and fed with microalgae (Isocrysis sp.). Salinity of nearshore seawater available at the AIMS was diluted (32–34) due to high seasonal rainfall. During culturing and experimental treatments seawater salinity was therefore adjusted to 35 by the addition of sea salt . Salinities were measured using a refractometer .After collection, specimens were washed off substrates, cleaned by gentle washing and sieving and identified to species and genus level 2 enriched air into a semi-closed circulation system of filtered (1 µm) natural seawater. CO2 enriched air (0.2%) was humidified via a system of Erlenmeyer flasks and bubbled into an aerated reservoir tank (30 L), connected to incubation chambers, which contained the organisms (water flow rate 0.5–1.0 cm s−1). Gas flow rates and thereby 2pCO levels were regulated via mass flow controllers . The system was allowed to equilibrate for>48 h.Carbon perturbations experiments were performed by the addition of CO−1 (mean ± SD), to simulate average natural in situ flow conditions experienced by epifaunal and shallow infaunal foraminifera within the benthic boundary layer of reef environments All amperometric and potentiometric microsensor measurements were conducted in a Faraday cage to minimize electrical disturbance. Before the measurements specimens were carefully transferred with a fine brush from the incubation chambers into a flow-cell (1.2 ml volume), connected to the same circulation system. Net flow rates within the flow cell were adjusted volumetrically to 0.50±0.02 cm sIllumination was provided from above via a fiber-optic guide from a halogen light source . Light intensities were monitored with a quantum irradiance meter , combined with a light sensor for photosynthetic active radiation (PAR).2 microsensors with a guard cathode , precision 0.05 µM) were constructed and calibrated as previously described 90), precision 0.001, on the NBS scale), as previously described by de Beer 2+ concentrations were determined with LIX microelectrodes , precision 13 µM), which were prepared, calibrated and used as described et al. Clark-type OMarginopora vertebralis, which exhibits a flat surface structure (2 evolution within the DBL of phototrophic species was tested under varying light intensities (data not shown). A light intensity of 30 µmol photons m−2 s−1 was found saturating for all photosymbiotic species, without causing photo-inhibition in the tested low light species Amphistegina radiata and Heterostegina depressaUsing a fine brush, foraminifera were positioned horizontally in the middle of the flow cell resting on their central elevations, with the exception of tructure . Microse90 value of steady-state signals of the system O2, pH and Ca2+ probes were positioned on the test surface of photosymbiotic individuals and recorded for ∼30 min, while light levels were altered (light/dark changes). O2 (pA) reached>90% steady-state signals ∼2 min, pH values (mV) took<6 min, while Ca2+ (mV) values did not change significantly. To ensure steady-state, light levels were applied for 10–60 min prior to measurements. Steady-state profiles were measured in step sizes of 50 µm (up to 400 µm) and 100 µm about 1500 µm upward perpendicular to the foraminiferal test, through the diffusive boundary layer into the bulk seawater , 30 µmol photons m−2 s−1 under mean turbulent flow conditions (0.5 cm s−1) and consecutively after flow was turned off after 5, 10, 20, 30, 40, 50, 60, 90 and 100 min and again after flow was re-established within 5 min. Individuals of M. vertebralis were chosen for these measurements since they remained attached to the bottom of the flow cell in a fixed position for extended periods of time.To illustrate the effect of zero flow (i.e. static culture) conditions on pH microenvironments, individuals of 2 heterogeneity across the shell surfaces, specimens of every species were fine scale profiled at 432 µatm from front to back in flow direction , 1142 µatm (pH 7.85) and 2151 µatm (pH 7.60) with photosymbiotic species, and at two 2pCO levels (432 and 2151 µatm) with symbiont-free individuals (−2 s−1) and darkness were conducted for O2 on day 2, pH on day 3, and Ca2+ on day 4.To account to some extent for spatial heterogeneity across shell surfaces  during tividuals . After 22 and kept at 4°C until being shipped to the University of Sydney, where they were analysed by open cell potentiometric titration Seawater was renewed for each experimental treatment and kept at a constant salinity (35) and pH according to the treatment . TemperaFor microsensor measurements, healthy, intact foraminiferal specimens of similar size and pigment shading were selected and liveliness confirmed in all individuals by the observation of movement. Individuals were photographed  via the dissecting microscope, before and after the experimental treatments  concentrations for dilute aqueous solutions were calculated from pH levels. Differences in concentrations between the bulk seawater and the surface of the shells, denoted as ΔO2, ΔH+ and ΔCa2+, were attained from the measured profiles. Concentration differences were calculated as the lowest and highest spatial points of the profiles respectively. At very low metabolic rates and therefore increased resolution, profile noise was balanced by a line of best fit through the seawater baseline concentrations, and DBL gradients, to attain concentration differences. Since microsensor measurements of O2, pH and Ca2+ were performed consecutively on different days, they did not depict true spatial replicates of one location . Measurement position differences of ΔO2, ΔH+ and ΔCa2+ within individuals . All statistical analysis used the software R Means of ΔOorrected . Group cHeterostegina depressa at 2151 µatm and Amphistegina radiata individuals at 1141 and 2151 µatm showed visual signs of symbiont loss (i.e. bleaching) at the end of the 4 day incubations  at the surface of the shell  remained constant, indicating the absence of confounding factors , A. radiata (prior to the incubation) and H. depressa (prior to and during the incubation) at 2151 µatm. Under illumination mean ΔO2 was significantly elevated at all 2pCO treatments in photosymbiotic, compared to symbiont free species , U-test: W = 0, p = 1.90e−07). Beside individuals that exhibited symbiont loss, mean ΔO2 of photosymbiotic species did not change significantly between elevated and control 2pCO . In A. radiata, which displayed severe visual signs of bleaching, ΔO2 was strongly decreased at 2151 µatm  were measured in both Quinqueloculina (at 2151 µatm) and Miliola specimens . In darkness mean ΔO2 was negative in all photosymbiotic species indicating respiration (−11±3 µM), which was enhanced in M. vertebralis and H. depressa at 1141 µatm and reduced in A. radiata at increased 2pCO (−1.17±0.54 µM).Illumination significantly increased mean ΔOynthesis , 6. BesiO2 level , mean ΔOsed pCO2 . Symbion2, mean ΔH+ was significantly affected by 2pCO treatment, trophic level, species and symbiont-type  of all species was slightly increased indicating net respiration. Yet, all photosymbiotic species showed a negative mean ΔH+ at elevated 2pCO conditions in darkness , compared to photosymbiotic species and also slightly negative at 2151 µatm at both light levels (−0.49±0.17 nM).In contrast to ΔOont-type . Under ieatments . In dark2+ were generally very low and exhibited high variation in space and time (39±24 µM). Mean ΔCa2+ did not change significantly with any of the measured factors (2+ was still not significantly different from 0 (23±29 µM), indicating no net CaCO3 dissolution or Ca2+ uptake.Changes in mean ΔCa factors . At 21512 (i.e. netPS or respiration) and ΔH+ were both quite variable across profiles within and across individuals  per individual for all photosymbiotic species, but not in symbiont-free species , nor were intercepts and slopes of the regressions significantly different between the two 2pCO treatments. Interestingly, ΔH+ of all linear regressions at ΔO2 = 0 was negative (range: −3.193 to −0.063 nM), beside Quinqueloculina at 432 µatm, indicating that H+ concentrations on the foraminiferal surface are slightly decreased compared with the bulk seawater when the net O2 flux equals zero.Mean ΔOividuals . Yet, th species . The intdepressa , Table 32 and pH measurements of photosymbiotic and symbiont-free foraminifera. In light, net O2 evolution (photosynthesis) within the DBL of photosymbiotic species remained relatively unaffected by the 2pCO treatments and surface pH was significantly increased. Yet, H+ differences (ΔH+) were significantly enlarged within the DBL with increasing 2pCO. However, the H+ decreases only amounted to ∼27% (at 432 µatm) and ∼14% (at 2151 µatm) of the ambient seawater H+ concentration. Photosynthesis was thus was insufficient to compensate for the more than four-fold increased ambient H+ concentrations between the highest and lowest 2pCO treatment (2 (i.e. net photosynthesis) was not influenced by 2pCO in any species , thus H+ concentrations in response to constant DIC production−/consumption-rates  in light, in respect to the mean O2 difference (ΔO2), by profiling areas of high symbiont densities with pH sensor and areas of low symbiont density with O2 sensors  (>10 min) in the dark for CO2 fixation in the calvin cycle. This would however imply that CO2(aq) uptake and fixation of the holobiont outweighed respiratory CO2(aq) production in darkness. A third explanation could be that foraminifera actively up-regulate their microenvironmental pH in darkness, via active proton pumping or antiporter exchange 2pCO and to maintain pH homeostasis for vital cellular functions. A fourth explanation could be the excretion of nitrogen waste by the foraminifera in the dark in the form of NH3, which would elevate microenvironmental alkalinity, thus increase pH. The excretion of NH3 is widely distributed among marine protists 2pCO, due to increased energy demands and nutrient uptake.Interestingly, microenvironmental Hted pCO2 . One pos+ fluxes , and sinest pCO2 , indicat2+ over the shell surface was very low, but single profiles displayed strong gradients  and the 3D surface structure of the location 2 DBL clearly decreases along middle ridges of individuals, where laminar flow velocity was highest M. vertebralis specimens showed the steepest O2 and pH gradients, without enlarged DBL thickness (i.e. net O2 fluxes), indicating overall increased photosynthesis compared to all other species , as well as one-dimensional diffusional resistance.The extent to which surface O species , 3, 5. Y2 fluxes and pH dynamics across the surface of photosynthetic organisms , measured within and among the individuals, was therefore expected. Yet, spatial heterogeneity within individuals  represent ecological realistic values, mimicking pore-water flow and sediment surface friction velocities 2pCO, thus DIC manipulations  concentrations in the OA range between 2 and 3 mM  and back-light microscope (F). Images were taken after control (432 µatm) treatment incubations. A) Marginopora vertebralis, B) Amphistegina radiata, C) Heterostegina depressa, D) Peneroplis sp., E) Quinqueloculina sp. and F) Miliola sp. individuals. Sizes are stated as largest possible diameter of individuals.(TIF)Click here for additional data file.Figure S2Close up dissecting microscope images, taken before (432 µatm) and after the 1141 µatm treatment incubation. A) Marginopora vertebralis, B) Amphistegina radiata, C) Heterostegina depressa, D) Peneroplis sp., individuals. Sizes are given as largest possible diameter of individuals. In A. radiata, areas of bleaching are indicated by white dashed circles.(TIF)Click here for additional data file.Figure S3Close up dissecting microscope images, taken before (432 µatm) and after the 2151 µatm treatment incubation. A) Marginopora vertebralis, B) Amphistegina radiata, C) Heterostegina depressa, D) Peneroplis sp., individuals. Sizes are stated as largest possible diameter of individuals. Both A. radiata and H. depressa showed signs of bleaching. Symbiont clumping in A. radiata is indicated by white dashed circles.(TIF)Click here for additional data file.Figure S4th, 50th and 75th percentiles of ΔCa2+, calculated from profiles measured during the Box-plots representing the 252pCO−2 s−1) and dark conditions for individual species. treatment incubation, at light  photosymbiotic and B) symbiont-free species. Outliers (>1.5 interquartile range) and extreme values (>3 times interquartile range) are indicated by (O) and (*) respectively.(TIF)Click here for additional data file.


In conclusion, these data suggested that prolonged APTT + normal PT + PC < 100 × 109 cells/L is useful in evaluating the likelihood of DF and/or DHF.Alertness to dengue and making a timely diagnosis is extremely important in the treatment of dengue and containment of dengue epidemics. We evaluated the complementary role of clinical-practice-based laboratory data in facilitating suspicion/diagnosis of dengue. One hundred overall dengue (57 dengue fever [DF] and 43 dengue hemorrhagic fever [DHF]) cases and another 100 nondengue cases  were analyzed. We separately compared individual laboratory variables  and varied combined variables of DF and/or DHF cases with the corresponding ones of nondengue cases. The sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) in the diagnosis of DF and/or DHF were measured based on these laboratory variables. While trade-off between sensitivity and specificity, and/or suboptimal PPV/NPV was found at measurements using these variables, prolonged APTT + normal PT + PC < 100 × 10 It is estimated that more than 2.5 billion people are living in geographic locales where dengue is endemic, and 50–100 million people have been annually infected by dengue virus (DENV) . DHF is eability . Based oeability . Grades eability . The defeability , 7, and eability . It is nDengue epidemic was once absent in Taiwan after 1942 . It was In 2002 a dengue epidemic due to dengue virus serotype 2 (DENV-2) developed in southern Taiwan in which more than 5000 symptomatic cases were found and most of the affected patients were adults , 13, andPotentially eligible included patients who were adults aged ≧18 years with a tentative clinical diagnosis of DF/DHF treated at Emergency Services or during admission at KSCGMH between July and November 2002. After excluding those with missing data, a total of 200 patients were eventually included for analyses. Half of the included patients who were subsequently proven to be serologically dengue-positive were allocated as the overall dengue cases, while another half who were serologically dengue-negative were allocated as the nondengue cases. A definitive diagnosis of dengue was made when at least one of the following serologic test results was found: (i) a positive reverse-transcriptase polymerase detection of DENV, (ii) a positive enzyme-linked immunosorbent assay result for specific immunoglobulin M antibody for DENV in acute-phase serum, and (iii) a fourfold or higher increase in dengue-specific hemagglutination inhibition titers in convalescent serum as compared to that in acute-phase serum –16. The t-test or Mann-Whitney U test was used for comparison of continuous variables. A 2-tailed P was considered statistically significant.A retrospective chart review was carried out for collection of demographic, clinical, and clinical-practice-based laboratory information of the included patients. The retrieved WBC, PC, PT, APTT, ALT, and AST were assayed from the specimens sampled from the affected patients when the tentative diagnosis of DF/DHF was made. Each of these data was regarded as an individual variable. Each individual variable and varied combined variables of the overall dengue cases, DF cases, and DHF cases were separately compared to the corresponding ones of nondengue cases. Individual components in combined variables were results of assays of blood specimens sampled on the same day. Because a large number of variables were derived from combination of individual variables and because each of these combined variables were regarded as one individual variable in analysis, to make the complexity more legible, combined variable A and variable B were expressed as a variable A + B and so on. The Chi-squared test or Fisher exact test was used for comparison of dichotomous variables, while the Student's To assess the values of the aforementioned data in facilitating the suspicion/diagnoses of dengue in general and DF/DHF in particular, we calculated the sensitivity, specificity, accuracy, PPV, and NPV based on these individual laboratory variables and varied combinations of each of them . BecauseThe overall dengue case and nondengue case groups each included 100 patients, and the former was made up of 57 DF cases and 43 DHF cases. Similar demographics but a number of significant differences in clinical manifestations  and clinThe sensitivity, specificity, PPV, NPV and accuracy calculated from individual laboratory variables and varied combinations of them in the diagnosis of DF and/or DHF are summarized in 9 cells/L (100%), PC < 100 × 109 cells/L (97%), prolonged APTT (89.7%), prolonged APTT + PC < 150 × 109 cells/L (89.6%), prolonged APTT + normal PT (89.4%), prolonged APTT + normal PT + PC < 150 × 109 cells/L (89.4%), prolonged APTT + PC < 100 × 109 cells/L (88.6%), and prolonged APTT + normal PT + PC < 100 × 109 cells/L (87.9%). The specificity of PC < 150 × 109 cells/L and PC < 100 × 109 cells/L was 48.8% and 70%, respectively. Prolonged APTT + normal PT + PC < 100 × 109 cells/L had an accuracy of 84.6% , while PC < 100 × 109 cells/L had an accuracy of 83.5% .In the diagnosis of overall dengue, variables with a high sensitivity, in decreasing order, were PC < 150 × 109 cells/L (100%), PC < 100 × 109 cells/L (94.7%), prolonged APTT + normal PT + PC < 150 × 109 cells/L (91.7%), prolonged APTT (91.7%), prolonged APTT + normal PT (91.7%), prolonged APTT + PC < 100 × 109 cells/L (88.9%), and prolonged APTT + normal PT + PC < 100 × 109 cells/L (88.9%). Among them, prolonged APTT + normal PT + PC < 100 × 109 cells/L had an accuracy of 83.8% . The specificity of PC < 150 × 109 cells/L and PC < 100 × 109 cells/L was 29.0% and 70%, respectively.In the diagnosis of DF, variables with a high sensitivity, in decreasing order, were PC < 150 × 109 cells/L (100%), PC < 100 × 109 cells/L (100%), AST > 40 U/L (97.1%), PC < 150 × 109 cells/L + AST > 40 U/L (97.1%), PC < 100 × 109 cells/L + AST > 40 U/L (97.1%), ALT > 40 U/L + AST > 40 U/L (92.6%), PC < 150 × 109 cells/L + ALT > 40 U/L + AST > 40 U/L (92.6%), and PC < 100 × 109 cells/L + ALT > 40 U/L + AST > 40 U/L (92.6%). The specificity of PC < 150 × 109 cells/L and PC<100 × 109 cells/L was 29% and 70%, respectively. Variables with high accuracy (>80.0%) were PC < 100 × 109 cells/L + ALT > 40 U/L + AST > 40 U/L , prolonged APTT + normal PT + PC < 100 × 109 cells/L , prolonged APTT + normal PT + AST > 40 U/L , prolonged APTT + normal PT + ALT > 40 U/L , and PC < 100 × 109 cells/L + ALT > 40 U/L .In the diagnosis of DHF, variables with high sensitivity, in decreasing order, were PC < 150 × 109 cells/L, prolonged APTT + normal PT + PC < 100 × 109 cells/L, PC < 100 × 109 cells/L + ALT > 40 U/L, PC < 100 × 109 cells/L + ALT > 40 U/L + AST > 40 U/L, prolonged APTT + normal PT + AST > 40 U/L, and prolonged APTT + normal PT + ALT > 40 U/L were included for ROC analysis. AUC, along with the sensitivity, specificity, PPV, NPV, and accuracy in the diagnosis of DF and/or DHF is summarized in 9 cells/L and prolonged APTT + normal PT + PC < 100 × 109 cells/L each had a good predictive accuracy (AUC > 0.8) in the diagnoses of the overall dengue, DF, and DHF, while the remaining variables had a good predictive accuracy only in the diagnosis of DHF.PC < 100 × 10The immunopathogenesis of DF/DHF is characterized by an aberrant immune overactivation and cytokine overproduction that lead to the development of a great array of clinical and laboratory manifestations , 21–23. γ activates macrophages to phagocytosize the auto antibody-coated platelets, rendering thrombocytopenia [Myelosuppression in DF/DHF leads to leukopenia , and somytopenia . During ytopenia , 26.9 cells/L and/or prolonged APTT + normal PT with an elevated AST and/or an elevated ALT had a good accuracy in the diagnosis of DHF but not in the diagnosis of the overall dengue or DF.An APTT prolongation and normal PT often found in DHF suggest a defect in the intrinsic pathway of coagulation, which is caused by either downregulation of the synthesis or overconsumption of specific factor(s) that are presumably produced by hepatocytes . HepatitGiven significant differences in clinical manifestations between the overall dengue/DF/DHF cases and the nondengue cases in this series and in others , clinici9 cells/L had a high sensitivity but low specificity in the diagnosis of DF and/or DHF, it may be useful in screening dengue illness when this viral infection is rarely encountered. Prolonged APTT + normal PT + PC < 100 × 109 cells/L with high sensitivity  and a comparatively high specificity of 78.9% for DF and/or DHF in this series suggest that the combined variable is especially useful in screening dengue illness during a dengue epidemic or in countries where dengue is always endemic, as under these circumstances, it is likely that clinicians tend to make a tentative diagnosis of dengue in most febrile patients lacking obvious localizing signs to suggest an alternative diagnosis [While ROC plots provide a global comprehensive view of the test, sensitivity and specificity describe the test's ability to correctly distinguish between DF/DHF and nondengue patients . As PC <iagnosis , 30.9 cells/L, PC < 100 × 109 cells/L + ALT > 40 U/L, PC < 100 × 109 cells/L + ALT > 40 U/L + AST > 40 U/L, prolonged APTT + normal PT + AST > 40 U/L, and prolonged APTT + normal PT + ALT > 40 U/L were found to have a comparable sensitivity and specificity in the diagnosis of DHF. However, when facing a patient with an underlying liver dysfunction due to viral hepatitis and/or fatty liver, APTT + normal PT + PC < 100 × 109 cells/L is the variable of choice for screening DHF.In the diagnosis of DHF, APTT + normal PT + PC < 100 × 109 cells/L had a favorable sensitivity, specificity, PPV, and NPV in diagnosis of DF and/or DHF. To make the applicability of these clinical-practice-based laboratory data simplified and user-friendly, we propose prolonged APTT + normal PT + PC < 100 × 109 cells/L be used for screening and evaluating the likelihood of DF and/or DHF.Predictive value, a calculation of the percentage of correct negative or correct positive result, is applicable once the prevalence of a disease is taken into consideration . The potThe present study implies that daily-practice-based laboratory data play a complementary role in prompting the suspicion and/or facilitating the diagnosis of DF and/or DHF, which is especially important for clinicians who are inexperienced with these infectious entities. In addition to providing an appropriate therapeutic guidance, a timely suspicion and diagnosis of dengue infection may help the public health authorities launch necessary containment measures earlier, thus diminishing the amplitude of a dengue epidemic that would otherwise be a much larger one.As DF/DHF features dynamically changing clinical and laboratory manifestations within a few days , 31, 32,Our data were obtained from adult patients during a dengue epidemic due to DENV-2 in Taiwan. Of our serologically dengue-negative patients, 78% suffered viral infections other than dengue and 6% suffered bacterial sepsis, while the rest 22% experienced miscellaneous diseases (see footnote of


The aim of our study was to assess a possible association between plasma inflammatory biomarkers  and the extent of fibrosis or cirrhosis using a FibroScan® in HIV/HCV co-infected patients.vs. F3–F4).This cross-sectional study assessed 60 HIV/HCV co-infected patients who had paired plasma samples and FibroScan® values available. All included patients were controlled for HIV infection (HIV-1 RNA <50 copies/mL) and had detectable HCV RNA levels. Levels of three biomarkers were measured in all samples using commercial ELISA kits. Multivariate logistic regression models identified factors associated with the METAVIR stages of fibrosis (F0–F2 P = 0.01), aspartate aminotransferase (AST), alanine aminotransferase, platelet counts, and CD4 cell counts were associated with the stage of liver fibrosis and, thus, were introduced into the model. However, only AST  was independently associated with F3–F4 stage liver fibrosis.In univariate logistic regression analyses, in addition to sCD14 (odds ratio [OR] = 3.23, 95% confidence interval [95%CI] = 1.30–7.97, In our study of HIV/HCV co-infected patients, sCD14 plasma level, a biomarker of monocyte activation, was not independently associated with the F3–F4 stage of liver fibrosis. We hypothesize that the higher levels of inflammation markers observed in HIV/HCV co-infected patients, compared to HCV mono-infected patients, prevent this association being observed within this population. Microbial translocation has been described as associated with chronic inflammation in both HIV-infected and hepatitis (HBV or HCV)-infected patients In HIV-infected patients, several studies reported that high sCD14 plasma levels predict disease progression and are positively correlated with all-cause mortality The aim of our study was to assess the possible associations between plasma inflammatory biomarkers and the extent of liver fibrosis or cirrhosis in HIV/HCV co-infected patients.This cross-sectional study included co-infected HIV/HCV patients from the Infectious Diseases Unit of Bichat Hospital . All enrolled patients were receiving an antiretroviral-based therapy and were virologically suppressed for HIV replication (HIV-1 RNA <50 copies/mL), they had detectable HCV RNA levels, and paired plasma samples and FibroScan® values were available. Plasma samples were obtained between 2009 and 2011, and stored at −80°C in Bichat Hospital virology lab.This study's protocol was approved by an institutional ethics review board , and all patients provided their written informed consent.Levels of three biomarkers were measured in all samples. hsCRP , hsIL-6 , and sCD14  were measured using commercial ELISA assays. Lower levels of detection were 0.010 ng/mL, 0.039 pg/mL, and 0.125 µg/mL for hsCRP, IL-6, and sCD14, respectively.vs. F3–F4). Then we adjusted the model for those factors found, in univariate analyses, to be associated with METAVIR fibrosis stage (F0–F2 vs. F3–F4). We applied a forward-selection procedure for those variables with a univariate P-value of ≤0.20, and we retained a significance threshold of P = 0.05 to determine which factors were independently associated with the stage of liver fibrosis. Analyses were conducted with SAS software, version 9.2 .Sociodemographic characteristics and clinical, virological, and biological data from patients were assessed using descriptive statistical methods. We assessed the correlation between inflammatory biomarkers and FibroScan® values as continuous variables using Spearman’s rank test. We also used a logistic regression model, as a dichotomous variable, to determine if there was an association between the level of inflammatory biomarkers and METAVIR stage of liver fibrosis  were infected with HCV genotype 1, three (5%) with HCV genotype 2, six (10%) with HCV genotype 3, and fifteen (25%) with HCV genotype 4. Seventeen patients (28%) displayed METAVIR F3- or F4-stage liver fibrosis . The median time since diagnosis of HIV infection was 19 years (IQR = 11–23). The median CD4 cell count at the time of the study and nadir CD4 cell count were 562/mm3 (IQR = 291–771) and 177/mm3 (IQR = 70–255), respectively.The characteristics of the 60 included patients are shown in P = 0.002), but not with IL-6  or hsCRP-plasma levels  (P = 0.01).In the 60 patients, median sCD14, hsCRP, and IL-6 levels were 2.79 µg/mL (IQR = 2.39–3.46), 0.67 µg/L (IQR = 0.19–2.07), and 1.73 pg/mL (IQR = 0.47–4.91), respectively. The FibroScan® values correlated with sCD14 plasma levels  . In the P = 0.0009), platelet counts (P = 0.0055), and CD4 cell counts (P = 0.05) were associated with the stage of liver fibrosis and so were introduced into the model (P = 0.0009) was independently associated with the F3–F4 stage of liver fibrosis  .In this study, based on 60 HIV/HCV co-infected patients, we found high levels of inflammatory biomarkers. However, sCD14 plasma level, the biomarker of monocyte activation, was not found to be independently associated with F3–F4 stage liver fibrosis.A main limitation of our study was its limited sample size, which meant there was lack of statistical power; however, we assumed that the risk of selection bias was negligible. Furthermore, few studies have reported on the role of inflammatory biomarkers in HIV/HCV co-infection and, as yet, no large studies have been conducted.In our study, the median sCD14 plasma level was 2.79 µg/mL (IQR = 2.39–3.46), which is a similar level to that observed in the study of Marchetti et al.: they assessed inflammatory biomarkers in HIV/HCV co-infected patients and found that median sCD14 plasma levels ranged from 2.79 to 3.09 µg/mL and was linked to the severity of the liver disease We found similar results to those of Marchetti et al. regarding the ability of inflammatory markers to predict the degree of liver inflammation and its progression to cirrhosis in HIV/HCV co-infected patients: these authors reported that a greater extent of fibrosis was not associated with higher sCD14 plasma levels The difference between these studies is probably not related to differences in statistical power as (i) a similar number of patients were assessed in these studies; and (ii) sCD14 was associated with the stage of fibrosis, in univariate analyses, in both studies In our study, the level of the liver enzyme, AST, was the only factor independently associated with the extent of fibrosis. In HIV-infected patients, chronic elevation of AST and HCV infection have been previously described as independently associated with significant fibrosis In conclusion, in our series of HIV/HCV co-infected patients, sCD14 plasma level, the biomarker of monocyte activation, was not independently associated with the F3–F4 stage of liver fibrosis. Thus, we hypothesize that the higher levels of inflammation markers observed in HIV/HCV co-infected compared to HCV mono-infected patients may prevent such an association being observed in this population. Further studies that explore the possible roles of others biomarkers, for example the innate immune-activation marker, soluble CD163, may improve our understanding of the mechanisms of systemic immune activation.


Aspergillii that attack Avicel, cotton cellulose and other forms of crystalline cellulose.Cellulose consisting of arrays of linear beta-1,4 linked glucans, is the most abundant carbon-containing polymer present in biomass. Recalcitrance of crystalline cellulose towards enzymatic degradation is widely reported and is the result of intra- and inter-molecular hydrogen bonds within and among the linear glucans. Cellobiohydrolases are enzymes that attack crystalline cellulose. Here we report on two forms of glycosyl hydrolase family 7 cellobiohydrolases common to all Talaromyces emersonii Cel7A 3-dimensional structure, identifies the typical tunnel-like catalytic active site while Cbh1 shows an additional loop that partially obstructs the substrate-fitting channel. CelD does not have a CBD and shows a four amino acid residue deletion on the tunnel-obstructing loop providing a continuous opening in the absence of a CBD. Cbh1 and CelD are catalytically functional and while specific activity against Avicel is 7.7 and 0.5 U.mg prot-1, respectively specific activity on pNPC is virtually identical. Cbh1 is slightly more stable to thermal inactivation compared to CelD and is much less sensitive to glucose inhibition suggesting that an open tunnel configuration, or absence of a CBD, alters the way the catalytic domain interacts with the substrate. Cbh1 and CelD enzyme mixtures on crystalline cellulosic substrates show a strong combinatorial effort response for mixtures where Cbh1 is present in 2:1 or 4:1 molar excess. When CelD was overrepresented the combinatorial effort could only be partially overcome. CelD appears to bind and hydrolyze only loose cellulosic chains while Cbh1 is capable of opening new cellulosic substrate molecules away from the cellulosic fiber.Cellobiohydrolases Cbh1 and CelD have similar catalytic domains but only Cbh1 contains a carbohydrate-binding domain (CBD) that binds to cellulose. Structural superpositioning of Cbh1 and CelD on the Cellobiohydrolases both with and without a CBD occur in most fungal genomes where both enzymes are secreted, and likely participate in cellulose degradation. The fact that only Cbh1 binds to the substrate and in combination with CelD exhibits strong synergy only when Cbh1 is present in excess, suggests that Cbh1 unties enough chains from cellulose fibers, thus enabling processive access of CelD. Biofuel generation from sources such as cornstarch, sugarcane or sweet sorghum syrups, produces large amounts of biomass waste products. For example, over 90% of the plant is unused in the case of ethanol production from cornstarch -3. CurreBiomass polysaccharides consist of cellulose, hemicellulose and pectin. Cellulose is a linear, crystalline self-assembled nanofiber formed from the linear polymer containing exclusively glucose monomers linked through β-1,4-glycosydic bonds ,6 and isBecause cellulose is structurally complex and crystalline in nature, it is recalcitrant towards microbial or enzymatic attack. Unfortunately, current pretreatment methods – e.g., acid hydrolysis ,9 or pyrA complete enzymatic cellulose degrading system consists of at least three related partially redundant biochemical reactions. Endo-glucanases (EC 3.2.1.4) randomly hydrolyze internal glycosydic bonds to decrease the length of the cellulose chain and multiply polymer ends -14. Exo-Exo-glucanases hydrolyze cellulose chains by removing cellobiose either from the non-reducing end  or reducing end , which in both cases results in the release of reducing sugars (cellobiose) but little polymer length reduction . In fungThe catalytic domain (CD) of a typical cellobiohydrolase forms a channel-shaped cavity topped by several flexible loops resulting in a tunnel like structure -39, whicAspergillii completely sequenced genomes, we found between three and five genes encoding cellobiohydrolases, typically two in each of the CAZy families GH6 and GH7, respectively. Remarkably, for each GH family 6 or 7 fungal cellobiohydrolase examined, one enzyme was linked to a CBD and a second was not.From a comprehensive bioinformatics survey of seven Aspergillus niveus. The structural differences between these two enzymes were the lack of a CBD in CelD and a conserved loop that obstructed the catalytic tunnel in enzymes that were linked to a CBD, but missing in enzymes that did not link to a CBD, ensuring that the tunnel was always open. These structural differences reflected directly on kinetic properties, thermostability, inhibition by glucose and cellobiose and the way these proteins interacted with crystalline substrate molecules. We hypothesize that the cellobiohydrolase linked to a CBD is competent to release cellulosic chains from the crystalline nanofiber and the cellobiohydrolase without a CBD can only use loose ends, without the aid of a CBD.Here we describe two GH7-cellobiohydrolases, Cbh1 and CelD from Fungi and bacteria produce enzymes that at least partially degrade plant cell wall polysaccharides ,44. MoreAspergillus precisely encodes two GH7-cellobiohydrolases, one with, and another without a CBD. The one exception is Aspergillus flavus/A. oryzae , which A. niveus a filamentous fungus analogous to A. fumigatus  grew at high temperatures, up to 50°C, on medium containing complex carbon sources such as cellulose and hemicellulose. As expected, the genome of A. niveus encoded two GH7-cellobiohydrolases: Cbh1-like 1,4-beta-D-glucan cellobiohydrolase with a linker and CBD and a CelD-like 1,4-beta-D-glucan cellobiohydrolase with no linker and CBD. Genes, cbh1 and celD were expressed and proteins secreted into the medium when the fungus was grown on cellulose  as the carbon source (data not shown).cbh1 and celD genes have been expressed as client proteins in A. nidulans, under the control of the maltose promoter, translated and secreted via the glucoamylase signal peptide )ribed in . The celribed in , phosphoribed in , validatA. nidulans minimal medium (MM) and general cultivation techniques were used throughout this work and were based on [Standard based on ,62.Aspergillus “shuttle” expression plasmid for expression and secretion of client proteins was used [+ competent Escherichia coli TOP 10 F’ competent cells . Random ampicillin-resistant colonies were selected and grown in 5 ml LB-ampicillin broth, plasmids purified [A. nidulans strain FGSC A773 (pyrGpyroA) genome [pyrG+, zeocin resistant transformants were grown on 10 ml MM, pyridoxine and 5% maltose containing plates for 48 h at 37°C. Accumulation of Cbh1 and CelD in the medium was analyzed by SDS-PAGE and one transformant for each enzyme was used for further investigation.The pEXPYR was used . PCR-amppurified , restricpurified . Plasmid) genome  and reco7-108 spores/ml were inoculated in liquid minimal medium supplemented with 0.5 to 15% of maltose, distributed onto dishes (20 ml in 150 mm Petri-dishes and 500 ml onto cafeteria trays) and incubated (stationary) at 37°C for 2–3 days. The mycelial mat was lifted with a spatula and discarded and the medium collected by filtration, centrifuged at 10,000xg for 10 min prior to concentration by ultra-filtration , quantified by the Bradford method [10d method , validatd method  and usedxg and concentrated by ultra filtration on Amicon 10 kDa cut-off micro columns. The majority of the protein content recovered from culture filtrates was Cbh1 or CelD.For biochemical studies, the recombinant Cbh1 and CelD proteins were produced by heavy inoculation of fresh conidia onto Petri dishes containing MM supplemented with pyridoxine and 5% maltose and incubated at 37°C for 48 h. Cbh1 and CelD were routinely recovered with this stationary incubation method and proteolysis avoided due to the 2 day incubation period. The medium was harvested by filtration, centrifuged at 10,000Crude ultra filtrated protein extracts were resolved by SDS-PAGE, Comassie-blue stained (CB) and the SDS removed by successive washes with 25% isopropanol solution. After transferred to 50 mM ammonium acetate buffer pH 5.0 a 1% carboxymethylcellulose (CMC) solution was infused into the SDS-free polyacrylamide gel, incubated at 37°C for 120 min, and stained with Congo red -68.pNPC were collected and loaded onto a Superdex G-75® (10x30 mm) gel filtration column, equilibrated with 50 mM ammonium acetate buffer, pH 5.0 and eluted fractions showing enzymatic activity were analyzed by SDS-PAGE. Single band containing fractions were combined, concentrated and used for further biochemical analysis. The flow rate used for both chromatographic steps was 0.5 ml min-1. Purified Cbh1 and CelD fractions were validated by SDS-PAGE  were applied to ion exchange Resource Q® column equilibrated with 20 mM sodium phosphate buffer, pH 7.4 and proteins eluted with a linear 0 to 1 M sodium chloride gradient . Fractions active on G-75® 10x0 mm gel pNPC with the pH ranging from pH 3.0 to pH 8.0 using 50 mM phosphate/citrate buffer. Optimal enzyme operating temperatures for Cbh1 and CelD were measured at their optimal pH, 5.0, with temperatures ranging from 30°C to 80°C. Thermal stability of Cbh1 and CelD was tested at optimal pH and exposure for various times (up to one hour). Purified enzyme in 50 mM ammonium acetate (without substrate) was incubated at temperatures ranging from 40 to 70°C. Samples were drawn from a master mix and residual activity assayed with 18 mM pNPC.Optimal pH was measured at 50°C in the presence of 18 mM xg for 15 min, supernatant (free fraction) collected and concentrated in an Amicon, 10 kDa cutoff ultra filtration column mixed with 2X Laemmli buffer and subjected to SDS-PAGE. The bound fraction was released from the Avicel or cotton linter or Avicel slurry by addition of 40 μl of 2X Laemmli buffer, vigorous agitation and boiling for 10 min. The SDS protein-solubilized slurry was centrifuged and supernatant (bound fraction) subjected to SDS-PAGE.To reveal the functionality and specificity of the predicted CBM1 domain, the binding of Cbh1 or CelD was evaluated by a pull-down assay. 20 μg of Cbh1 or CelD was incubated at 4 C for 30 min in a rotary shaker, with 200 μl of 30% cotton cellulose or Avicel slurry in 50 mM, pH 5.0, ammonium acetate buffer. The reaction was centrifuged at 14,000pNPCellobiose, incubated for 120 min or as indicated at 50°C and the release of reducing sugars determined with the DNS method [Substrate specificity of cellobiohydrolases was determined by incubating 1 μg of Cbh1 or CelD with a, 1% slurry of cotton cellulose (Sigmacell 50), Avicel PH-101, PASC or 1% solution of CMC or 18 mM S method . SpecifiMichaelis-Menten kinetic constants were determined from Lineweaver–Burk plots. Reaction rates were measured using Avicel and cotton cellulose ranging from 0 to 100 mg/ml of insoluble substrate suspended in 50 mM ammonium acetate buffer pH 5.0. Reactions were carried out over a 120-min period at 50°C, boiled, and the released reducing sugars determined by DNS assay . The rawTalaromyces emersonii  from D: 1Q9H, ) was useD: 1Q9H, . To guarD: 1Q9H, , the objD: 1Q9H, . IncomplD: 1Q9H, .pNPC dissolved in 50 mM ammonium acetate buffer pH 5.0 with glucose or cellobiose added in a range from 0 to 100 mM at 50°C. After 15 min, enzyme activity was stopped by adding 100 μl of a 2% Na2CO3. The pNPC chromophore release was spectrophotometrically quantified at 410 nm with a Multimode Infinte M200 Reader .To analyze Cbh1 and CelD inhibition, 1 μg enzyme was incubated in 18 mM CaZy, Carbohydrate-Active enZYmes Database (http://www.cazy.org/); Cbh1, CaZy GH7 (Cel7A) cellobiohydrolase 1; CelD, CaZy GH7 (Cel7B) cellobiohydrolase D; CI, Crystalinity index; CD, Catalytic domain; CBD, Cellulose-binding domain; CMC, Carboxymethylcellulose; AV, Avicel; CC, Cotton cellulose; DNS, Dintrosalicilic acid; DS, Degree of synergism; FP, Whatman #3 filter paper; MM, Minimal Aspergillus medium; pNPC, Para-nitrophenylcellobiose; pNPG, Para-nitophenylglucose; PASC, Phosphoric acid swollen (Avicel) cellulose; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis.The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this article.Conceived and designed the experiments: RAP, MLTMP and AM. Made the 3D-structural modeling: MTM and FMS. Performed the experiments: ARLD, FS and TAG and contributed equally to this work. Analyzed the data: FS, ARLD, TAG, RAP and MTM. Wrote the paper: RAP, ARLD, FS, FMS, MLTMP and MTM. All authors read and approved the final manuscript.Table S1. Primers used in this study. Table S2. Cbh1 and CelD substrate binding competition. Table S3. Cbh1 and CelD pNPC and pNPG activity .Click here for fileFigure S1. Time course A. nidulans client expression and secretion of A. niveus Cbh1 (A) and CelD (B) and purified enzymes (C). Note that after the second day native Cbh1 and CelD are subjected to proteolytic degradation in the medium. Click here for fileFigure S2.Cellobiohydrolase substrate dependent kinetics with crystalline cellulosic fibers. Michaelis-Menten substrate dependent, avicel (open symbols) and cotton linters (closed) cellobiohydrolase activity of Cbh1 (A) and CelD (B). Nearly equal amounts of enzyme (9.3 nM Cbh1 and 10.7 nM CelD) were incubated with increasing amounts of substrate, avicel or cotton linters and specific velocity (μmol/min) determined after a 120 min reaction period at 40 C. Click here for fileFigure S3. Cbh1 and CelD differential thermal inactivation .Click here for file


Glucosylceramide synthase (GCS) is one enzyme that provides a major route for ceramide clearance. Recent evidence has indicated an important role for GCS in multidrug resistance (MDR) tumors. Doxorubicin (DOX)can modulate the expression of GCS in leukemia and ovary cell lines. However, few studies have investigated their relationship in breast cancer;We collected 84 excision biopsies from patients with invasive ductal breast cancer of whom 33 patients had undergone preoperative chemotherapy. Immunohistochemistry was used to analyze the expression of GCS protein and significantly showed that the expression of GCS was higher in the samples from patients treated with preoperative chemotherapy(p = 0.018). In order to investigate the underlying mechanism, breast cancer cell lines were cultured with different concentrations of DOX, and mRNA and protein levels of GCS were then detected;DOX significantly upregulated the expression of GCS at both the mRNA and protein level in ERα-positive MCF-7 cells.We then block down the Sp1 site of GCS promoter, which inhibited the DOX-mediated increase in GCS expression; and after Erα was inhibited in MCF-7 cells, the up-regulation of GCS by DOX also been inhibited.In conclusion, our data demonstrated the novel finding that DOX could modulate the expression of GCS through the Sp1 site of GCS promoter in ERα-positive breast cancer cells. Breast cancer is one of the most common causes of death in women due to cancer worldwide. Besides surgical methods, chemotherapy and endocrine therapies are also used in the treatment of breast cancer. The resistance of tumors to chemotherapy occurs not only to single cytotoxic drugs, but also as a cross-resistance to a range of drugs with different structures and cellular targets. This phenomenon is termed multidrug resistance (MDR), and is one of the contributing factors that prevents survival rates for breast cancer improving further The accumulation of recent evidence has pointed towards an important role for glucosylceramide synthase (GCS) in MDR. Sphingolipids, which include ceramide and sphingosine, are essential structural components of cell membranes. Furthermore, they also play an important role in regulating the proliferation, survival and apoptosis of cells. GCS is a pivotal enzyme that transfers UDP–glucose to ceramide to form glucosylceramide (GC) Anthracycline-based chemotherapy (treatment regimens that involve anthracyclines such as doxorubicin or epirubicin) has been used clinically for over two decades. Several studies have confirmed that doxorubicin (adriamycin) can modulate the expression of GCS in the leukemia cell line, HL-60, and an ovary cell line, NCI/ADR-RES Tissue samples from 84 patients with invasive ductal breast carcinoma who underwent complete dissection of the breast and axillary lymph nodes and 5 patients with accessory breast who underwent complete dissection of the tissue were collected at the Qilu Hospital Shandong University, China, between January and September 2007. Thirty-three of the patients had also undergone preoperative chemotherapy .Tumor samples were paraffin-embedded and histopathological variables, including tumor size, lymph node metastasis, histological subtype, and histological grade were determined by reviewing pathology reports and hematoxylin and eosin (H&E) stained sections. Patient and tumor characteristics are summarized in The use of these tissues was approved by the Research Ethics Committee of Shandong Medical University, and we obtained informed written consent for pathological evaluation from all participants involved in our study.2.Two drug-sensitive breast cancer cell lines, MCF-7 (ER-positive) and MDA-MB-231 (ER-negative), were obtained from the American National Cancer Institute. The multidrug-resistance breast cancer cell line, MCF-7/ADM, was selected from MCF-7 using doxorubicin treatment in stages 2O2-methanol solution. The slides were blocked with 10% normal goat serum for 10 min and were incubated with GCS antibody (1∶500) overnight at 4°C. The slides were then probed with HRP-labeled polymer conjugated to a secondary antibody for 30 minutes. The antibody against GCS was a kind gift from Dr. D. Marks . After the MCF-7/ADM cell line was confirmed to overexpress GCS protein, it was used as a positive control in this research. Otherwise, the normal breast tissues were also used as control.Immunohistochemical staining was carried out using the DAKO Envision Detection Kit . In brief, tissue blocks were cut into 4 µm-thick sections, dried, deparaffinized, and rehydrated. Antigen retrieval was performed in a microwave oven for 15 min in 10 mM citrate buffer at pH 6.0. For the cell culture experiments, after cells were embedded in 4% neutral formaldehyde for 2 h, PBS with 0.5% Tween-20 was added for 30 min at room temperature. For all samples, endogenous peroxidase activity was blocked with a 3% HStaining results were interpreted by a breast pathologist who was blinded to patient outcomes. Tumors with 1% or more positively stained nuclei were considered positive for estrogen receptor(ER) Cells were treated with various concentrations of DOX  for 24 or 48 hours. The concentrations of DOX were calculated according to the IC50 of the cells (IC50 =  drug concentration (µmol/L) that results in the 50% inhibition of cell growth).5′-CCT TTC CTC TCC CCA CCT TCC TCT-3′, antisense: 5′-GGT TTC AGA AGA GAG ACA CCT GGG-3′5′-CACACGGCACAGTAGCGAG-3′Total RNA was isolated using Trizol  and quantitative real-time PCR (qPCR) was used to detect GCS mRNA. qPCR was performed using SYBR Green Real-time PCR MasterMix . The primers for GCS were as follows: sense: All the cells were cultured for another 48 h after treatment, the confluent cells were then lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Triton-X100, 2 mM EDTA (PH 8.0), 5 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin for 20 min on ice. The complex was centrifuged at 12,000×g for 10 min at 4°C. As described previously 5′-ATTCCGGGGGCGGGGGCATG-3′) and mock ODNs (5′-CATGCCATCGCTACCGGGGC-3′) were transfected  into breast cancer cells suspended in RPMI 1640 using lipofectamine reagent  for 12 hours. After transfection, cells were incubated with 10% FCS-RPMI 1640 for 12 hours and then treated with DOX for 24 h Phosphorothioated double-stranded ODNs (decoy ODNs) containing the sequence of the GCS putative Sp1/GC-rich binding site  of lipofectamine  or 5 µl (1 mg/ml) of vector was diluted into 250 µl of 1640 culture medium without serum. After incubated for 10 min at room temperature, the diluted vector and lipofectamine were mixed together and incubated for 20 min. Then the mixture was adding to the cells washed three times by medium without serum. Six hours later, the medium was replaced with 1 ml of complete 1640 culture medium .As control, 10 µl of lipofectamine and 5 µl (1 mg/ml) of pSUPER.neo+GFP were also transfected.Before transfection, cells were seeded in 6-well plates at the density of 1×1050.Assays were performed as described previously 6 cells/ml. The cells were stained with Annexin V-FITC and gently vortexed. After 15 min incubation at 4–8°C in the dark, PI was added to the cells for another 5 min incubation at 4–8°C in the dark. The results were analyzed by flow cytometry .Apoptotic rates were assessed with flow cytometry using the Annexin V-fluorescein isothiocyanate/propidium iodide (PI) kit . Samples were washed with ice-cold PBS twice and resuspended in binding buffer at a density of 1×10The Chi-square test or the Fisher’s exact test was used to analyze the relationship between the expression of GCS and chemotherapy. Cellular data were presented as the mean ± standard deviation, and one-way ANOVA and Dunnett’s T3 was used to determine the statistical significance. P-values <0.05 were considered statistically significant. All calculations were performed using the SPSS16.0 for windows statistical software package .p  = 0.018). Further analysis showed that, in the ER-positive tumor samples, the CAF protocol increased the positive rate of GCS protein from 28.57% (10/35) to 60% (12/20) (p  = 0.022); however, in the ER-negative samples, CAF protocol did not induce the expression of GCS.The expression of GCS protein was detected in all samples by immunohistochemical staining . In the qPCR was adopted to detect the expression of GCS mRNA, which was approximately 10.2-fold higher in MCF-7 cells than in MDA-MB-231 cells. As shown in In MDA-MB-231 cells, the low concentration of DOX (0.1 µM) decreased the level of GCS mRNA by 35.47% (p<0.01) and 0.7 µM DOX increased GCS mRNA levels by only approximately 1.34-fold relative to the controls (p<0.01) . CompareGCS protein expression was analyzed by Western blotting, as shown in After cells were transfected with the Sp1 decoy ODNs, GCS mRNA levels were significantly decreased by approximately 99.97% in MCF-7 cells  and 99.9After cells were transfected with the ERα interference plasmid pSUPER-ERαi, GCS mRNA levels were significantly decreased by approximately 84.75% in MCF-7 cells  compared50 of MCF-7 cells increased from 0.3155±0.0179 µmol/L to 2.164±0.0899 µmol/L after they were treated with 0.2 µM DOX for 24 hours (p<0.01). After they were transfected with Sp1 decoy ODNs, the IC50 for DOX decreased to 0.01113±0.00007 µmol/L, but did not change when cells were treated with DOX for 24 h (p>0.05)  , Fig. 7C50 for DOX was 0.1241±0.0179 µmol/L, which increased to 0.1366±0.0116 µmol/L after they were treated with 0.2 µM DOX for 24 hours (p>0.05)  . After tsistance . These cThe apoptosis rate was detected by flow cytometry using the Annexin V-fluorescein isothiocyanate/propidium iodide (PI) kit. Annexin V-positive, PI-negative cells were scored as early apoptotic cells. The apoptosis rates estimated in the present study only included early apoptotic cells, which were marked as LR in In MDA-MB-231 cells, DOX increased the apoptosis rate from 7.03±2.61% to 12.4±1.67%. After transfection with Sp1 decoy ODNs, the apoptosis rate increased and the rise of apoptosis rate by DOX disappeared .Sphingolipids, which include ceramides and sphingosine, were first isolated and characterized in the late 1800s. However, they have been long regarded as structural and insert components of cell membranes. In recent years, many studies have shown that they are still associated with a myriad of cell processes, including proliferation, cell survival and death. Ceramide, an important member of sphingolipid metabolism, has been proven to be a second messenger in the process of apoptosis The regulation of ceramide levels involves many enzymes, such as ceramide synthase and sphingomyelinase, which are responsible for the generation of ceramide, sphingomyelin synthase and ceramidase Recently, accumulating evidence has pointed towards an important role for GCS in MDR. Many drug-resistant cell lines have been found to overexpress GCS, including breast cancer cell lines, while the inhibition of GCS by antisense oligonucleotides or by specific inhibitors could restore chemosensitivity in many cancer cell lines p<0.05)  .In order to confirm whether doxorubicin induces the expression of GCS, different concentrations of doxorubicin were added to sensitive breast cancer cell lines, MCF-7 and MDA-MB-231. The results demonstrated that doxorubicin upregulated the expression of GCS at both the mRNA and protein levels in the ERα-positive cell line, MCF-7 , while t In 2009, Ruckhäberle et al. analyzed microarray data regarding GCS mRNA expression in 1,681 breast tumors and found that GCS expression was associated with positive estrogen receptor (ER) status, lower histological grading, low Ki67 levels and ErbB2 negativity In conclusion, our data demonstrated the novel findings that DOX could modulate the expression of GCS through the Sp1 site of the GCS promoter in ERα-positive breast cancer cells.


Thuja sutchuenensis Franch. is a critically endangered plant endemic to the North-East Chongqing, China. Genetic variation was studied to assess the distribution of genetic diversity within and among seven populations from the single remnant locations, using inter-simple sequence repeat (ISSR) markers. A total of 15 primers generated 310 well defined bands, with an average of 20.7 bands per primer. The seven populations revealed a relatively high level of genetic diversity in the species. The percentage of polymorphic bands, Nei’s gene diversity and Shannon’s information index at the population and species level were 76.1%, 0.155, 0.252 and 100%, 0.165, 0.295, respectively. A low level of genetic differentiation among populations (GST = 0.102), in line with the results of Analyses of Molecular Variance (AMOVA), and a high level of gene flow (Nm = 4.407) were observed. Both the Unweighted Pair Group Method with Arithmatic Mean (UPGMA) cluster analysis and Principal Coordinates Analysis (PCoA) supported the grouping of all seven populations into two groups. In addition, Mantel test revealed no significant correlation between genetic and geographical distances . The low genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered species. Thuja sutchuenensis Franch., an evergreen coniferous species of genus Thuja in the cypress family Cupressaceae, is a critically endangered species endemic to mountain area in North-East Chongqing Municipality, China . The mixture was incubated at 65 °C for 30 min followed by two extractions with chloroform: isoamyl alcohol (24:1). The pellet obtained was washed with 75% ethanol, finally dissolved in TE (Tris-EDTA) buffer . Quality and concentration of total DNA were verified by spectrophotometry at 260 and 280 nm. DNA samples were stored at −20 °C for ISSR amplification.Total DNA was extracted from silica gel-dried needles of each plant (0.1 g) following the CTAB method , as grou2, 200 μM dNTPs, 1 μM primer and 0.6 U Taq DNA polymerase . Amplification reaction was performed in a Gene Amp PCR System 9600 . The cycle program included an initial 5 min denaturation at 94 °C, followed by 45 cycles of 30 s at 94 °C, 45 s at 50 °C (52 °C for the primer 895#) and 2 min at 72 °C, and 10 min final extension at 72 °C. PCR Products were electrophoresed in 1.6% agarose gel in 0.5× TBE at 100 V for 2 h, stained with ethidium bromide (0.5 μg/mL). The negative control was run by replacing template DNA with ddH2O. Gels with amplification fragments were visualized and photographed with DG-III . DL1000 bp ladder  was used as DNA molecular weight.A subset of 15 primers  chosen fP, %), Nei’s gene diversity (H), Shannon’s information index (I), the observed number of alleles (Na) and the effective number of alleles (Ne) [HT), genetic diversity within populations (HS), and the relative magnitude of genetic differentiation among populations (GST = (HT − HS)/HT) [Nm) was computed by the formula of Nm = (1 − GST)/2GST [Only distinct, reproducible, well-resolved fragments were scored as present (1) or absent (0) for each ISSR reaction. The data obtained were combined in a single matrix and genetic coefficients were firstly generated using the program POPGENE v1.32 to describe genetic variation at intra-and inter-population level: the percentage of polymorphic bands (les (Ne) ,38. The  HS)/HT) . An estiST)/2GST . The AMOST)/2GST .Genetic divergence between populations was investigated using Nei’s unbiased genetic distances and genetic identities . Nei’s uT. sutchuenensis was appreciable both at the population and species level. Low genetic differentiation was found among populations, which may be attributed to limited geographic distance and abundant gene flow. Both UPGMA cluster analysis and PCA supported the grouping of all seven populations into two groups. Based on these findings, strategies are proposed for the conservation of the species.In summary, our results indicated the genetic diversity of


Ebolaviruses cause hemorrhagic fever with up to 90% lethality and in fatal cases, are characterized by early suppression of the host innate immune system. One of the proteins likely responsible for this effect is VP24. VP24 is known to antagonize interferon signaling by binding host karyopherin α proteins, thereby preventing them from transporting the tyrosine-phosphorylated transcription factor STAT1 to the nucleus. Here, we report that VP24 binds STAT1 directly, suggesting that VP24 can suppress at least two distinct branches of the interferon pathway. Here, we also report the first crystal structures of VP24, derived from different species of ebolavirus that are pathogenic (Sudan) and nonpathogenic to humans (Reston). These structures reveal that VP24 has a novel, pyramidal fold. A site on a particular face of the pyramid exhibits reduced solvent exchange when in complex with STAT1. This site is above two highly conserved pockets in VP24 that contain key residues previously implicated in virulence. These crystal structures and accompanying biochemical analysis map differences between pathogenic and nonpathogenic viruses, offer templates for drug design, and provide the three-dimensional framework necessary for biological dissection of the many functions of VP24 in the virus life cycle. Ebolaviruses cause severe hemorrhagic fever that is exacerbated by immediate suppression of host immune function. VP24, one of only eight proteins encoded by ebolaviruses, functions in virus replication and assembly, and is thought to contribute to immune suppression by binding to a certain class of molecules called karyopherins to prevent them from transporting a transcription factor termed STAT1. Here we report that VP24 is also able to directly bind STAT1 by itself, and thereby likely contributes to immune suppression by an additional mechanism. Analysis of these multiple roles of VP24 and design of drugs against them have been hindered by the lack of structural information on VP24 and its lack of homology to any other known protein. Hence, here we also present X-ray structures of VP24 derived from two different ebolavirus species that are pathogenic and nonpathogenic to humans. These structures and accompanying deuterium exchange mass spectrometry identify the likely binding site of STAT1 onto VP24, map sites that are conserved or differ between pathogenic and nonpathogenic species, and provide the critical 3D templates by which we may dissect and interpret the many roles that VP24 plays in the virus life cycle. The ebolaviruses and marburgviruses are enveloped, non-segmented, negative-strand RNA viruses that belong to the family Filoviridae. There are five antigenically distinct ebolaviruses that are ∼40% different in amino acid sequence, and are each named after the location of the outbreak during which they were first identified: Zaire (now known simply as Ebola virus or EBOV), Sudan virus (SUDV), Taï Forest virus (TAFV), Reston virus (RESTV) and Bundibugyo virus (BDBV). Marburgviruses and most ebolaviruses cause severe hemorrhagic fever in both humans and nonhuman primates, with fatality up to 90%. The exception is RESTV, which appears to be non-pathogenic in humans, although it remains pathogenic to non-human primates For the pathogenic ebolaviruses, early suppression of host interferon (IFN) production and signaling plays a decisive factor in disease outcome STAT1 belongs to the STAT family of transcription factors, is a key mediator of the IFN response pathway The importance of STAT1 to the antiviral response is underlined by the fact that viruses (and other microbes) have evolved proteins that inhibit every step of STAT1 activation In addition to its role in interferon antagonism, ebolavirus VP24 has also been proposed to associate with membranes Here, we demonstrate that VP24 also binds directly to STAT1 itself, present the first X-ray crystal structures of VP24 from both Sudan and Reston viruses, and map a possible site of STAT1 interaction on the VP24 crystal structure by deuterium exchange mass spectrometry (DXMS). The biochemical and structural analysis presented here identifies a new function by which VP24 may contribute to and/or prolong innate immunosuppression, and provides the necessary three-dimensional templates for understanding the multiple roles of VP24 in the ebolavirus life cycle and design of antiviral compounds against them.1–683 (truncated prior to its phosphorylation site at Tyr701). BSA was used as a negative control. Indeed, VP24 is able directly associate with STAT11–683 . However, we wondered if VP24 could play a more direct role as well. To answer this question, we performed an ELISA to test binding of purified Ebola virus VP24 or Sudan virus VP24 to purified STAT1AT11–683 . Althoug1–233 and SUDV11–233) and Reston virus   using selenomethionine-incorporated protein expressed recombinantly in E. coli. We subsequently determined structures of SUDV1–233 and RESTV11–237 by molecular replacement, both at 2.0 Å resolution  . We detesolution .de novo predictions: as predicted, helices 5–8 are indeed observed, helix 8 is quite long, and a β sheet exists at the base of the structure. Differences between the prediction and the experimental structure are that a three-stranded sheet was predicted, but a five-stranded sheet is observed VP24 adopts a compact, single domain, α/β structure of novel fold , and is hydrophilic rather than hydrophobic . Five reSerial passage studies to confer lethality of EBOV to rodents resulted in five mutations in VP24 QLS(L/M) in the ebolaviruses but are GIYLTS in the marburgviruses , and other regions of VP24 that exchange with solvent more rapidly when in complex with STAT1, perhaps due to conformational change and increased mobility.Deuterium exchange mass spectrometry (DXMS) is able to rapidly map footprints of protein-protein binding sites and offers a broader picture than analysis of point mutants alone 1–683, VP24 peptidic regions 96–98 and 106–121 demonstrate slower H/D exchange kinetics, suggesting a site of protein-protein interaction  onto the RESTV30 sites . One of E. coli or 293T cells) and find that purified VP24 from all three viruses is monomeric in solution  and RESTV VP24 (P1211) structures, although it is currently unclear if it is biologically relevant  and VP24 from an ebolavirus that thus far, appears nonpathogenic to humans , although it is lethal to nonhuman primates. The overall folds of SUDV and RESTV VP24 are similar, as expected , VP24 could prevent or dampen antiviral responses through multiple routes. The combination of both ebolavirus VP24 and ebolavirus VP35  in the infected cell offers greater coverage of the different pathways by which antiviral responses occur. Interestingly, plasmocytoid dendritic cells (pDCs), which are major producers of type I interferon Hence, both P-STAT1 and U-STAT1 play multiple roles in antiviral defense, and may play somewhat different roles in different cell types. By affecting both P-STAT1 (by binding karyopherins and/or possibly by forming a karyopherin-STAT1-VP24 tertiary complex) and U-STAT1  was expressed in E. coli Rosetta-gami 2(DE3)pLysS cells. Truncation of the C terminus permitted protein solubility in the absence of detergents. The N-terminal truncation used in the first SUDV constructs was based on general construct screening. Cultures were grown in LB medium supplemented with ampicillin (100 µg ml−1), and expression was induced by the addition of 0.5 mM IPTG at 16°C. Harvested cells from overnight induction were resuspended in lysis buffer  for lysis at 25,000 psi using a Microfluidizer processor. The lysed mixture was then centrifuged for 50 minutes at 16,000 r.p.m. in a JA-17 rotor (Beckman Coulter). The supernatant was loaded on a HisTrap FF crude column  with a step gradient of 30 mM and 500 mM imidazole in lysis buffer. SUDV1–233 and SUDV11–233 VP24 were further purified by size exclusion on a HiLoad 16/60 Superdex 75 prep grade column (Amersham Pharmacia) in 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl.VP24 from Sudan virus . The culture was induced with IPTG after 15 min. Cells were harvested after 4 hr and purified as described above.Selenomethionine-incorporated SUDVFull-length SUDV (in pTriEx 5 vector (Novagen)) was also transiently expressed in mammalian HEK293T cells in a five-layer CellStack (corning). The cells were transfected at 60% confluency with 420 µg of DNA and 1.2 mg of PEI diluted in 42 ml of PBS. The PEI mixture was incubated at room temperature for 20 min. before adding to the cells. After 48 hours, cells were freeze-thawed three times and lysed in 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl. Protein was affinity purified with strep-tactin superflow beads (Qiagen), then further purified by size exclusion on a Superdex 200 10/300 GL . 10 µl of the peak fraction was run on SDS-PAGE, and probed by Western blot with an anti-strep antibody.All SUDV VP24 proteins contain a valine to alanine substitution at position 22 from the GenBank deposited sequence. Residue 22 is on helix α1 and is buried within the structure . The SudE. coli as previously described. Full-length EBOV VP24 was cloned into the pET46 Ek/LIC vector from cDNA that was a gift from Dr. Viktor Volchkov (Claude Bernard Université de Lyon 1). cDNA for RESTV VP24 was synthesized by GenScript . Both full length RESTV and RESTV11–237 were subcloned into pET46 Ek/LIC for expression.Full-length Ebola virus  VP24 and both full-length and N- and C-terminally truncated Reston (RESTV) virus VP24 were expressed and purified in 1–683 (human) in a pET46 Ek/LIC vector was expressed in E. coli Rosetta-gami 2(DE3)pLysS cells. Cultures were grown in LB medium supplemented with ampicillin (100 µg ml−1), and expression was induced by the addition of 0.5 mM IPTG at 25°C overnight. Harvested cells were then resuspended in lysis buffer  for lysis at 25,000 psi using a Microfluidizer processor. Next, the lysed mixture was centrifuged for 50 minutes at 16,000 r.p.m. in a JA-17 rotor (Beckman Coulter). The supernatant was loaded on a HisTrap FF crude column  with a gradient of 10 mM to 500 mM imidazole in lysis buffer. STAT11–683 was further purified by size exclusion on a Superdex 200 10/300 GL  in 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 5 mM BME. STAT1 cDNA was a gift from Dr. Christopher Basler (Mount Sinai School of Medicine).Truncated, unphosphorylated STAT11–233 crystallized in 0.1 M HEPES, pH 7.5, 0.1 M MgCl2, and 8% (v/v) PEG 550 mme. SUDV11–233 crystallized in 0.1 M HEPES, pH 7.0, 6% MPD, and 15% (w/v) D-(+)-sucrose. SeMet-derivatized SUDV11–233 crystallized in 0.1 M HEPES, pH 7.0, 10% MPD, and 1 mM DTT. RESTV11–237 crystallized in 0.1 M Bis-Tris, pH 5.5, 0.2 M NaCl, 8% (w/v) PEG 3350, and 14% (w/v) D-(+)-sucrose. All crystals were grown by the hanging-drop vapor diffusion method at 22°C. Crystals were flash frozen in liquid nitrogen and cryoprotected with their reservoir solutions supplemented with 40% PEG 550 mme for SUDV1–233, 40% sucrose for SUDV11–233 and RESTV11–237, and 40% MPD for SeMet-derivatized SUDV11–233.SUDV11–237 contains two molecules in the asymmetric unit. Diffraction data were collected at 100 K on SBC 19ID  and BCSB 5.0.2 , and were processed either with HKL2000 Both SUDV structures contain one molecule in the asymmetric unit while RESTV11–233 were generated by MAD  using Auto-Rickshaw 1–233 was determined by molecular replacement  and 93.1% (SUDV11–233) of residues are in the most favored region of Ramachandran plots, with no residues in the disallowed regions. The final model of SUDV1–233 contains residues 9–106 and 113–232 with residues 113 and 210–213 replaced with alanines. The final model of SUDV11–233 contains residues 13–61, 71–107, 114–209, and 212–228. Residue 209 was replaced with alanine.Experimental phases for SUDV11–237 was determined by molecular replacement in Auto-Rickshaw 11–233 as the initial search model. The structure was refined with Phenix.refine RESTV1–233, SUDV11–233, and RESTV11–237, respectively.Atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession codes 3VNE, 3VNF, and 4D9O for SUDV1–233 and EBOV1–233) was bound to ELISA plates (Corning Costar 3690) at 0.01 mg/ml in 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl, overnight at 4°C. Plates were then blocked for one hour at room temperature with 3% BSA. After washing with PBS containing 0.05% TWEEN 20, 50 µl of STAT11–683 with a C-terminal HA-tag was added at 0.03 mg/ml and allowed to bind for two hours at room temperature. Plates were again washed, 50 µl of anti-HA (Covance) was added at 1 µg/ml and incubated at room temperature for one hour. After a third washing step, 50 µl of HRP-conjugated secondary antibody (Thermo Scientific Pierce) was added and allowed to incubate for one hour at room temperature. Plates were developed with TMB Substrate Kit (Pierce) and read at 450 nm. BSA was used as a negative control.50 µl of each VP24  by rapid denaturation, optional disulfide-reduction and digestion by solid-phase pepsin into overlapping fragments of ∼3–15 amino acids. The perturbed masses of the resulting peptides, and therefore their deuterium content, are quantified by liquid chromatography- mass spectrometry.2O buffer  and then diluted into 6 µl of D2O buffer  at 0°C. At 10 s, 100 s and 1000 s, the deuterium exchange was quenched by adding 12 µl of optimized quench  and then samples were frozen at −80°C. In addition, nondeuterated samples (incubated in H2O buffer mentioned above) and equilibrium-deuterated samples (incubated in D2O buffer containing 0.5% formic acid overnight at 25°C) were prepared. The samples were later thawed at 5°C and passed over an AL-20-pepsin column (16 µl bed volume (Sigma)) at a flow rate of 20 µl/min Prior to the deuteration studies, quench conditions that produced an optimal pepsin fragmentation pattern were established as previously described SEQUEST software (Thermo Finnigan Inc.) was used to identify the sequence of the peptide ions. The centroids of the isotopic envelopes of nondeuterated, functionally deuterated and equilibrium-deuterated peptides were measured using DXMS Explorer  and then converted to corresponding deuteration levels Figure S1a) Crystallized constructs of VP24 from SUDV and RESTV. The first five residues of both the N- and C-termini are indicated respectively. The N-terminal 6xHis-tag was retained throughout purification and crystallization. (b) Stereo view of SUDV VP24 Faces 1, 2, and 3 in rainbow with blue indicating the N-terminus and red indicating the C-terminus.Additional details of VP24 structures. ((TIF)Click here for additional data file.Figure S2a) Sequence alignment of ebola- and marburgviruses. Secondary structures are assigned according to the crystal structures. Mostly conserved residues are in white boxes (red characters) while absolutely conserved residues are in red boxes (white characters). Grey stars indicate residues with alternate side-chain conformations observed in electron density maps. (b) Structural alignment of SUDV1–233 (pink), SUDV11–233 (green), and RESTV11–237 (blue) VP24. SUDV1–233 and SUDV11–233 align with an r.m.s.d. of 0.67 Å, and SUDV1–233 and RESTV11–237 align with an r.m.s.d. of 0.81 Å Click here for additional data file.Figure S3a) Zoomed-in view of the cavity on Face 1 in RESTV. In RESTV, Tyr 172 is rotated 180° away from Met 71. (b) Zoomed-in view of the Face 1 cavity in SUDV. Tyr 172 is rotated toward Met 71. (c) The pairwise VP24-VP24 interface observed between the two RESTV11–237 in the asymmetric unit (blue) superposed onto the essentially identical pair of SUDV1–233 molecules (magenta) formed by a crystallographic two-fold axis. Residues 51–90 and 167–222 are not illustrated in order to enhance clarity. Molecular surface representations of the pairwise interactions (light and dark grey) are inset. (d and e) Crystal lattice interactions in RESTV and SUDV. (d) Met10 (a result of construct design) and Val11  form hydrophobic interactions with the Face 1 cavity of a neighboring RESTV11–237 VP24 (molecule 1). The backbone of Met10 and Val11 hydrogen bonds to the surrounding residues. (e) Leu10 and Val11  bind into the same pocket of another SUDV1–233 VP24 (molecule 1). Additional hydrogen bonding with surrounding residues is also illustrated.Close-up of Face 1 cavity. ((TIF)Click here for additional data file.Figure S411–237. (a) Sequence differences (red) between RESTV and SUDV and (b) between RESTV and EBOV (Zaire) are mapped onto Faces 1, 2, and 3. RESTV and SUDV are 75% identical and RESTV and EBOV are 81% identical. Circles indicate region shown to interact with karyopherin α1 and regions highlighted by DXMS study. Interestingly, these regions primarily concentrate on Face 3. To a lesser degree, two regions, 71–79 and 181–198 are also accessible through Face 1. Residue 50, which has been shown to increase virulence in a mice serial passage study Sequence comparison of RESTV, SUDV, and EBOV mapped onto RESTV(TIF)Click here for additional data file.Figure S5a) SUDV (Sudan) VP24 (expressed in E. coli) was separated by size on a Superdex-200 10/30 prep grade column. Log of molecular mass standards  plotted with the elution chromatogram is shown inset within the graph. (b) RESTV (Reston) and (c) EBOV (Zaire) protein samples  were separated by size on a Superdex-200 column with elution buffer containing 2.5 mM CHAPS. Log of molecular mass standards  plotted with elution chromatogram is shown inset. A single peak of VP24 was eluted from each ebolavirus and corresponds to about 28 kD in mass (monomer). (d) Similarly, when SUDV VP24, expressed in mammalian HEK293T cells was separated by size exclusion on a Superdex-200 10/30 prep grade column, only a monomeric species was observed. The small peak at ∼8 ml corresponds to aggregated protein eluted in the void volume (much larger than tetramer). Log of molecular mass standards  plotted with elution chromatogram is shown inset. The elution buffer consists of 10 mM Tris-HCl, pH 8.0, and 0.3 M NaCl. (e) Peak fraction from the S200 column (d) probed by Western blot with an anti-strep tag antibody. Full-length purified SUDV and RESTV precipitated at pH 7.4, and 0.15 M NaCl. Therefore, gel filtration analysis was carried out at pH 8.0 and 0.3 M NaCl where the proteins remained soluble.Gel filtration analysis of full-length VP24. ((TIF)Click here for additional data file.Figure S6a) Residue 22 (magenta) is on helix α1 and forms hydrophobic interactions with neighboring residues E18, L26, L145, S146, M147, and L150. All SUDV structures contain a valine to alanine substitution at position 22. (b) Comparison of SUDV (grey) and RESTV  with residue 22 shown as stick. The loop region connecting helices α7 and α8 is slightly shifted in RESTV to accommodate the bulkier isoleucine side chain.Close-up view of VP24 residue V22. ((TIF)Click here for additional data file.