Poa secunda, both with and without competition from the invasive exotic grass, Bromus tectorum. We measured seedling survival and above-ground biomass at two experimental sites in western Montana, and found that the source of seeds selected for restoration can influence establishment at the restoration site. Cultivars had an overall advantage when compared with local genotypes, supporting evidence of greater vigor among cultivated varieties of native species. This advantage, however, declined rapidly in the presence of B. tectorum and most accessions were not significantly different for growth and survival in competition plots. Only one cultivar had a consistent advantage despite a strong decline in its performance when competing with invasive plants. As a result, cultivated varieties did not meet expectations for greater establishment and persistence relative to local genotypes in the presence of invasive, exotic species. We recommend the use of representative local or regional wild seed sources in restoration to minimize commercial selection, and a mix of individual accessions  in highly invaded settings to capture vigorous genotypes and increase the odds native plants will establish at restoration sites.Local, wild-collected seeds of native plants are recommended for use in ecological restoration to maintain patterns of adaptive variation. However, some environments are so drastically altered by exotic, invasive weeds that original environmental conditions may no longer exist. Under these circumstances, cultivated varieties selected for improved germination and vigor may have a competitive advantage at highly disturbed sites. This study investigated differences in early establishment and seedling performance between wild and cultivated seed sources of the native grass,  Ecological restoration of native plants depends on successful germination and establishment of seeded species, and the source of seeds for restoration may have consequences for both short- and long-term population viability , 2. IntrWhile local, wild-collected seed is recommended for ecological restoration, its availability is scarce and restoration practitioners frequently purchase commercially produced seed sources to meet native vegetation goals and reduce costs , 11. ComRecommendations to use locally adapted seed sources are based on the assumption that the environmental conditions to which plants are adapted remain relatively unchanged. However, some areas are so drastically altered by exotic, invasive weeds that original environmental conditions may no longer exist , 17. BecBromus tectorum L. (cheatgrass or downy brome) in North America is a well-documented example of land conversion as a result of biological invasion [Bromus tectorum currently dominates millions of acres of grassland in the Intermountain West and represents a significant challenge for the restoration of native plant species [Bromus tectorum is a winter annual and has a competitive advantage over native plants that germinate later during the growing season [B. tectorum are also responsible for increased fire frequency, which can significantly alter both abiotic and biotic conditions at restoration sites [The spread of invasion . Bromus  species . Bromus g season . Stands on sites . Thus, ton sites .Poa secunda J. Presl (Sandberg bluegrass) at sites impacted by B. tectorum. Poa secunda is a native, cool-season perennial grass commonly targeted for restoration because of its vast distribution [B. tectorum, the selection of suitable seed sources may change with the level of invasion at the restoration site.This study investigated the early seedling establishment and performance of wild and cultivated seed sources of ribution  and poteribution . We askePseudoroegneria spicata (Pursh) Á. Löve, Festuca idahoensis Elmer, Koeleria macrantha (Ledeb.) Schultes, and Poa secunda. Invasive species are widespread and include Centaurea stoebe L., Poa bulbosa L. and B. tectorum.We conducted this study at MPG Ranch, a conservation property consisting of 9,500 acres in western Montana . The ranP. secunda. The first (Lower) site was located at an elevation of 998 m and consisted of sandy loam soil having an average pH of 6.3. Prior to establishment of the experimental plots, the area had been used for soybean production. The second (Upper) site was located 1,023 m further up-slope, at an elevation of 1,076 m and consisted of loam soil with an average pH of 6.6. The Upper Site had previously been dominated by Agropyron cristatum (L.) Gaertn, (crested wheatgrass) with a P. bulbosa understory and used as pasture for cattle. Historical climate data for the area available from the PRISM Climate Group (2012) recorded an average annual precipitation of approximately 35 cm and a mean annual temperature of 7°C (range of -9–30°C). For the duration of this study, annual precipitation logged at the nearest MPG Ranch weather station was approximately 20 cm and the mean temperature was 9°C (-17–37°C).Our experiment was conducted at two sites at MPG Ranch to evaluate the interactions of local environmental conditions and competition by exotic invasive plants with wild and cultivated accessions of Poa secunda is an important component of sagebrush steppe and temperate grassland vegetation in the western United States [P. secunda serves as a primary source of forage for both wildlife and livestock at the beginning of the growing season [Poa secunda occurs from Alaska through the Intermountain West and Great Plains states. Individuals of Sandberg bluegrass germinate early in the spring, and reproduce by tillering and seed maturation in early summer. Seed production is commonly the result of apomixis, but outcrossed pollination and subsequent self-fertilization contribute to distinct genetic lines [d States . A nativg season . Poa secic lines .P. secunda complex [Three U.S. Department of Agriculture selected varieties  were included in the study because of their recommended use in the study region and similar, early-maturing attributes within the  complex . These cWe established and seeded our study sites in early fall 2012. The two sites were initially disked, chain harrowed, and raked to remove existing vegetation and litter to ensure a smooth and even seedbed. Fencing was installed around the perimeter of each experimental site to prevent herbivory by large ungulates.B. tectorum . Brome seeds were broadcast into experimental plots at a rate of 7,600 pure live seed m-2 [Poa secunda  accessions were replicated 50 times within each plot, and planted in random order at intervals of 30 cm to avoid intraspecific competition. To account for viability differences among seed sources, four seeds of the same accession were planted per replication to ensure high germination rates in the spring [The experiment was organized as a randomized complete block design with five paired-plot replicates at both study sites, resulting in ten plots per site .. One plseed m-2 , 32 and e spring . A colorBromus for competition plots to avoid edge effects. This experimental design resulted in a total of 300 Poa plants possible (50 replications for the 6 accessions) per plot.Each plot had dimensions of 1.8 x 15 m and each pair of weed-free and competition plots was separated by 1 m. All plots contained a 45 cm wide buffer around the edge; buffer zones were cleared for weed-free plots, and planted with Bromus canopy cover was taken at its maximum production period in July to determine if levels of competition were even between study sites. Bromus cover was measured using Daubenmire’s six cover classes [Poa plants was clipped and dried in an oven at 60°C for three days. Dry weights for aboveground biomass were measured to 0.0001 g using an analytical balance .Experimental plots were surveyed for germination during spring 2013 after seeds had overwintered. A survey of survival was subsequently conducted once every 3 weeks during the spring/summer growing season, resulting in four surveys for all experimental plots. A plot-wide survey of  classes . Sites wn ≤ 50). Prior to analyses, survival data were arcsine square-root transformed and biomass data were log transformed to satisfy assumptions of statistical tests [Data for analyses comprised experimental plot means for each accession with respect to the proportion of survivors and average biomass. We calculated the proportion of survivors as the number of surviving seedlings divided by the original number of replications per plot (50 per accession). Average biomass was calculated for the number of survivors per accession per plot  where block was nested within site. Block was considered a random factor and accession, site, and competition treatment were analyzed as fixed factors. The repeated measures model incorporated compound symmetry covariance structure which resulted in the smallest Akaike’s Information Criterion .A split-plot, mixed-model ANOVA (block nested within site) was used to calculate the significance of main and interaction effects for above-ground biomass of each cultivated and wild seed source between weed-free and competition plots. Block was treated as a random factor and accession, site, and competition treatment were analyzed as fixed factors. All calculations for survival and biomass data were made using the MIXED procedure in SAS version 9.3.1, using an alpha of 0.05 . The presence or absence of small mammal herbivory was added to the models as a covariate. Degrees of freedom were estimated using Satterwaite’s method  and pairBromus cover to ensure that the level of competition was consistent among all treatment plots in the study. The midpoint of the Daubenmire cover class range was averaged across each treatment plot. Average Bromus cover values were compared to determine if cover class was significantly different among competition plots.As a last step, we assessed P < 0.001). This outcome may reflect differences in the viability or germination and establishment of seeds among accessions. Herbivory was noted during the final two surveys, and was likely caused by small rodents. Approximately 21% of experimental Poa plants were impacted by herbivory at the time of the third survey, and this number increased to 28% by the fourth and final survey. Average Bromus canopy cover for competition plots ranged from 46–69%. Plot cover differences were not significant and we included competition effects in analyses measured as the presence or absence of brome.A total of 2,627 seeds germinated and established, and 1,773 were monitored after thinning to one plant per replication. One of the wild seed sources (MPG-5) had near-zero rates of germination and as a result, this seed source was removed from analyses. Despite efforts to account for differences in seed viability, cultivated seeds had higher rates of seedling emergence when compared with the two wild seed sources , and in all cases, survival declined over time  and survival of HP seedlings was greater than all other cultivated and wild accessions combined (P < 0.001).Results of the repeated measures analysis for surveys 1–3 revealed significant differences in seedling survival for the interactive effects of accession, site, survey, and competition treatment . Survivaver time . We did g season . OverallPoa accession, competition treatment and differences between experimental sites , although that effect was solely the result of greater survival of the HP cultivar . Among accessions, the HP cultivar had the greatest number of survivors regardless of site or competition treatment (P < 0.001). We also noted that survival of MPG-15 seedlings did not differ significantly from MH and RE seedlings, suggesting that some wild populations may perform as well as some cultivated accessions. Only MPG-10 and MH had a similar proportion of survivors in competition and weed-free plots. However, the number of survivors was low for these two accessions and we were unable to determine if this effect was due to the ability of these accessions to tolerate Bromus, or the result of a lack of statistical power.Cultivars were more likely to survive than wild plants by the time of the last survey , although that effect was no longer significant when the HP cultivar was removed from the contrast (P = 0.482). HP produced the greatest average biomass compared to all other accessions (P = 0.0004). On average, the five accessions produced more biomass at the Lower Site than the Upper Site, but this difference was only significant for MPG-15. At the Lower Site, wild accessions performed as well as a subset of cultivars , while MPG-15 was similar to cultivars MH and RE at the Upper Site. Regardless of accession, competition with Bromus negatively affected Poa biomass at the Lower Site (P = 0.003). However, there was no effect of competition at the Upper Site (P = 0.237), and this was likely the result of high levels of herbivory that reduced biomass of Poa in weed-free plots by the final survey. Biomass did not differ between sites in competition plots.reatment . CultivaBromus , but this effect was due to the performance of HP . Wild accessions did not differ from cultivars in weed-free plots (P = 0.372). Both wild accessions performed as well as at least two cultivars in weed-free plots , and they performed as well as one or more cultivars in competition plots . Three accessions  produced similar amounts of biomass between weed-free and competition plots, while two accessions (MPG-10 and RE) produced significantly lower biomass in competition plots relative to weed-free plots.Regardless of site, HP yielded the greatest biomass compared to all other accessions when in competition with Poa secunda at sites impacted by cheatgrass invasion. We noted differences in performance among seed sources for measured variables in nearly all cases, suggesting that the source of seeds can have a significant impact on plant performance and, as a result, restoration outcomes [In the present study, we investigated seedling establishment and growth among local and cultivated genotypes of outcomes . Mean bioutcomes , 38). Dioutcomes . Experimoutcomes .Bromus plots were only apparent late in the growing season and were significant at the final survey. This indicated a window of opportunity to minimize competition with weeds when restoration sites are cleared prior to seeding, and thereby improve establishment of desired species with continued management of exotics [Bromus for all measured variables, indicating its potential for tolerance of invasive species. One of Mountain Home’s reported uses is in post-fire restoration of Bromus dominated landscapes [Competition effects in  exotics , 42. Desndscapes , and theP. secunda would be to test the consequences of seed source over time once seedlings are established, and determine the replicability of these results when plants are grown in a matrix of the resident (native and non-native) plant community.Our study was a limited test of local adaptation because we were unable to reciprocally transplant seed sources, the resident plant community was removed prior to site establishment, and sites were weeded throughout the duration of the experiment . EvidencEnvironmental conditions such as soil, slope, precipitation, and elevation may have influenced differences in seedling performance between experimental sites. Low emergence rates across accessions could be explained by the drier than normal conditions that year. Early in the field season, survival of experimental plants was greater at the Upper Site where loam soils had more potential for moisture retention relative to sandy loam soil at the Lower Site . During Bromus and most accessions were not significantly different for growth and survival in competition treatments. Only the HP cultivar had an advantage, and the rapid decline in its performance in the presence of Bromus indicated this advantage may decline over time and requires further investigation. As a result, cultivated varieties as a group did not meet our expectations for greater establishment and persistence relative to local plants in the presence of invasive, exotic species. Other control measures would be needed to reduce competition with invasive plants, and promote the early establishment of desirable, native vegetation . Additirbicide; , 8, 50 . Non-curbicide; . MeasureIn cases where sites are heavily impacted by invasive species and other control measures are not feasible or are ineffective, our results suggest cultivated varieties may provide some advantage for establishment of natives. However, the ability to discern among cultivars is difficult unless data are available for competitive dominance and overall vigor (such as in the case of High Plains). If no data are available, the use of multiple cultivar accessions might increase the odds of including highly competitive genotypes in the restoration seed mix. This strategy, however, is not without risks and would be most useful in highly invaded settings. In contrast, if restoration sites are contiguous with undisturbed plant communities and/or exotic species are not predominant, we recommend the use of a composite seed mix representing appropriate local or regional variation either via wild collections or limited field production of region-specific varieties to avoid directional selection and altered patterns of genetic variation. This approach would maintain intraspecific patterns of genetic diversity, increase the odds of including vigorous wild genotypes, and may improve plant community diversity as well as resistance to biological invasion –8, 52.S1 FigPoa secunda accessions, placed randomly within each plot .Each site contained five blocks (Block 1–5), split (Plot A or B) between one weed-free plot (white border) and one paired cheatgrass plot (shaded border). Every plot contained 50 replicates of all six (TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


We monitored microbiological properties in two forest sites over a period of 17 years (1993–2010) within the International Cooperative Programme on Integrated Monitoring of Air Pollution Effects on Ecosystems (ICP IM). The two study sites were located in South Tyrol in the Italian Alps at altitudes of 1,737 m a.s.l.  and 570 m a.s.l. (submontane site IT02). Soil samples were collected in the late spring and autumn of 1993, 2000, and 2010, and were characterized by measuring respiration, key enzyme activities involved in the C, N, P, and S cycles and litter degradation, and the abundance of viable bacterial and fungal populations. Over the study period, an increase in mean annual air temperatures at both sites  was calculated from trendlines. Significantly lower mean annual air temperatures, higher temperature fluctuations, and higher annual precipitation rates were observed at site IT01 than at site IT02. Subalpine site IT01 was characterized by significantly lower microbial activity  and abundance than those at submontane site IT02. The year of sampling had a significant effect on all the parameters investigated, except for nitrification. Fungal abundance decreased consistently over the study period, while no consistent trend was noted among the other parameters investigated. Season only affected a few of the measured microbiological parameters: respiration and bacterial numbers were significantly higher in the spring than in the autumn, while the opposite was noted for xylanase and phosphatase activities. Soil fungi contributed essentially to xylanase and protease activities, while soil bacteria were mainly involved in degradation processes that required the activity of sulfatase. The objective of this study was to monitor soil microbiological properties in the late spring and autumn of 1993, 2000, and 2010. Soil samples were characterized with regard to respiration, the key enzyme activities involved in the C, N, P, and S cycles, and litter degradation. Culture-dependent methods were used to obtain information on the abundance of bacterial and fungal populations. We evaluated the effects of the sampling year, season, and altitude  on soil microbial activity at these sites. So far, the impact of global warming on microbial communities and activities in forest soils has been evaluated in short-term (2–3 years) soil warming experiments; however, no monitoring studies have been conducted for a period comparable to our 17-year study.The two investigated sampling sites IT01 (Ritten) and IT02 (Montiggl) are two long-term monitoring sites that were installed in the Italian Alps in 1992 within the framework of ICP IM. These sites represent two widely distributed and forestally significant forest types in South Tyrol; their characteristics are shown in Picea abies, Pinus cembra, and Larix decidua. The climate is subalpine-continental with a mean annual temperature of 4°C and mean annual rainfall of 1,000 mm. Before confining the study site, high stress factors arose from grazing and touring . The pedogenetic substratum consists of a mixed morainic and detritus cover of porphyritic pebbles and stones on quattziferous porphyry of the Permian period . The soil was classified (FAO) as haplic podsol. The site consists of coniferous forests close to the timber line and is dominated by Quercus pubenscens, Q. robur, Fraxinus ornus, and Ostrya carpinifolia. The climate is mild continental with submediterranean influences, with a mean annual temperature of 11°C and mean annual rainfall of 900 mm. Before confining the study site, high stress factors resulted from grazing, touring, clear-cutting, and reforestation with allochthonous species (Picea abies)  as dystric cambisol. The site consists of deciduous forests and is dominated by Air temperature at the two sites was constantly monitored according to WMO standards at open field stations using the sensor t026 TTEPRH . Bulk precipitation was monitored by open field pluviometers using the sensor t027 TP500 .ca. 5 kg) were collected from each of these sampling spots from the Ah horizon (top 10 cm). Sampling occurred in 1993, 2000, and 2010. To determine the effects of season, soil samples were collected in each sampling year from each site both in the late spring (May) and autumn (October). Immediately after sampling, soil samples were transported in cooled boxes to the laboratory, sieved (<2 mm), and immediately analyzed or stored at 2°C. Each soil sample was analyzed with three replicates.In each site, 10 sampling spots that were distributed uniformly over the site were chosen in 1992 when the sites were established. Soil samples , maximum water-holding capacity, and soil organic matter  as described , 46.2 produced during incubation. After 18 h of incubation at a temperature of 22°C, BaCl2 was added and excess NaOH was titrated with HCl to the endpoint of phenolphthalein. Substrate-induced respiration (SIR) was determined using the same method in soil samples that were amended with glucose and incubated at 22°C in bottles containing NaOH.Soil respiration  was determined according to the ICP IM Manual . Soil saSoil dehydrogenase activity was examined using trinitrophenyltetrazolium chloride  as a substrate and 0.1 M Tris-HCl, pH 7.8, as a buffer. After 16 h of incubation at 25°C, the produced triphenyl formazan (TPF) was extracted and measured spectrophotometrically .Soil xylanase activity was assessed using xylane  as a substrate and 2 M sodium acetate-acetic acid buffer, pH 5.5. After 24 h of incubation at 50°C, the reduced sugars released during the incubation period were determined colorimetrically .Protease activity was determined using casein  as a substrate and 0.05 M Tris-HCl, pH 8.1, as buffer. After 2 h of incubation at 50°C, the amino acids released during incubation were extracted and quantified colorimetrically , 45.p-nitrophenyl phosphate as a substrate and 0.5 M sodium acetate-acetic acid buffer, pH 5.0. After 1 h of incubation at 37°C, the p-nitrophenol released during the incubation was extracted and colorimetrically determined.Phosphomonoesterase activity (acidic phosphatase) was measured as described in the ICP IM Manual  by usingp-nitrophenyl sulfate as a substrate and 0.5 M sodium acetate-acetic acid buffer, pH 5.8. After 1 h of incubation at 37°C, the p-nitrophenol released during the incubation was extracted and colorimetrically determined .Arylsulfatase activity was determined using 0.02 M Nitrification was determined after the incubation of soil samples amended with ammonium chloride solution for 2 weeks (samples from site IT02) or 3 weeks (samples from site IT01 were incubated for longer due to a higher SOM content) at 25°C and the released amounts of ammonium and nitrate were determined colorimetrically. Oxidation of the substrate (ammonium chloride) was equivalent to the nitrification dynamics and was expressed as a percentage of the added nitrogen , 45.−1) to exclude fungal growth. Soil fungi were enumerated on malt extract agar  supplemented with 0.02% (w/v) dl-lactic acid and chloramphenicol (100 μg mL−1) to exclude bacterial growth. Plates were incubated at 25°C for 48–72 h.Culturable heterotrophic soil bacteria and soil fungi were quantified by the plate-count method for viable cells. Appropriate dilutions of soil suspensions were spread on the surfaces of agar plates. The numbers of heterotrophic bacteria were determined on Standard I agar, supplemented with cycloheximide  content, which constitutes the basis of nutrition for heterotrophic soil microorganisms , while sP<0.05) effect on the soil microbiological parameters investigated. Kruskal-Wallis ANOVA and Mood’s median test were additionally applied for non-parametric data; however, these tests confirmed the results obtained by ANOVA. Multivariate statistics  was applied to evaluate the relationship between the measured soil microbiological parameters and determining factors. Correlations between the determined properties in each site were tested by Spearman’s rank correlation coefficient since most of the data were non-parametric.Properties were determined for each soil sample (10 soil samples per site) in the spring and autumn of each sampling year, and the mean values of replicate determinations were used for statistical calculations . Normal distributions were evaluated by the Kolmogorov-Smirnov-test. ANOVA was applied to determine whether the sampling year, sampling season, and/or the sampling site had a significant . Site IT02 exhibited submediterranean influences with equinoctial pluvial regimes. This atmospheric condition, along with factors such as poor soil formation and low water retention capacity denoted occasional periods of summer aridity .P<0.05) colder climatic conditions and larger temperature fluctuations than those at site IT02. Mean annual air temperatures were 4.3±0.7°C at site IT01 and 10.9±0.7°C at site IT02. Minimum and maximum air temperatures were 1.9±0.7°C and 7.1±0.9°C at site IT01, and were markedly higher at IT02 (5.8±0.5°C and 15.9±1.0°C). Site IT01 was also characterized by significantly higher annual precipitation (977±142 mm) than that at IT02 (830±174 mm).Mean annual air temperatures and precipitation at the two sites during the study period are shown in Over the study period (1993–2010), an increase in the mean annual air temperature  at both sites was calculated from trendlines. A 34-year-climatic series (1977–2011) at site IT02 even showed an increase in mean annual air temperatures over time from 10°C to 11.4°C (data not shown). An almost identical increase in temperature was reported at a site in northern Italy (org (19%), and nitrogen (0.8%) than that from site IT02 ; however, the C/N ratios were similar . Soil from site IT01 was further characterized by its higher acidity (pH 3.3) than that from site IT02 (pH 4.1) (Marked differences were observed in the nutrient contents of soil at the two studied sites. Soil from site IT01 contained significantly higher amounts of SOM (33%), C similar . This is(pH 4.1) .The year of sampling had a significant effect on all investigated parameters, except nitrification  and 3. HOverall, an increase in air temperatures of 0.6°C (IT01) and 0.8°C (IT02) over the study period of 17 years was noted at the two sites. A 35-year monitoring period (1977–2011) also revealed an increase in temperature of 1.4°C at site IT02. As already described above, the impact of increased temperatures on microbial communities and activities in forest soils has been evaluated in soil warming experiments conducted over a period of up to 2 years , 49, 58;Season affected only a few of the measured microbiological parameters. Respiration and bacterial numbers were significantly higher in the spring than in the autumn, while the opposite was noted for the activities of xylanase and phosphatase. No significant differences were observed in any of the other parameters examined in this study between samples measured in the spring and autumn  and 3.i.e., winter communities had a higher capacity for the degradation of complex C substrates , while summer communities were better adapted to utilize labile C sources , can be explained by the microorganisms involved in litter degradation. The highest amount of litter accumulates in autumn. This was clearly shown by comparing SOM contents in the spring and autumn: For example, in 2000, SOM contents at site IT01 were 36.5% and 80.3% in the spring and autumn, respectively; values recorded at site IT02 were 44.1% and 81.9%, respectively. Although litter contains components that are not easy to degrade, the degradation of these compounds by fungal cellulases and xylanases starts in autumn, and, consequently these activities are higher in the autumn than in the spring. After the snow melts in spring, easily degradable compounds are available and mainly converted by bacteria. Consequently, bacterial numbers are higher in the spring than in the autumn, which is consistent with the results of our study. Seasonal variations in microbial communities in forest soil were attributed to an altered resource supply,  sources . Seasona sources , 28. For sources . This haAll measured parameters, except for nitrification and phosphatase activity, were significantly different between the two investigated sites; the subalpine site IT01 was generally characterized by significantly lower microbial activity and abundance than those observed at the submontane site IT02  and 3.This can be explained by the climatic conditions prevailing at the two sites. Site IT01, located at 1,737 m a.s.l., is exposed to significantly lower temperatures (lower by approximately 7°C on average over the study period), higher temperature fluctuations and higher precipitation (higher by 147 mm on average) than site IT02, which is located at 570 m a.s.l. High altitude ecosystems experience extreme temperature fluctuations with changing seasons and climates, higher precipitation, and a shorter vegetation period , 31. DueAn evaluation of the effects of the combinations of the three independent factors  clearly indicated that a number of activities were influenced by the combination of sampling year and season  as well as by the combination of sampling year and site . The combination of season and site only influenced bacterial numbers, while the combination of all three independent factors  only had a significant effect on SIR .2 and H2O, as demonstrated by the strong relationship between bacteria, respiration and SIR. Bacterial sulfatases degrade S-containing peptides and amino acids.Principal components (PC) were extracted for soil microbiological parameters measured over the whole study period at both sites in the spring and autumn. The first principal component (PC1) explained 41.0% of the variance in data while the second (PC2) accounted for 33.4%. Two clusters could be observed. PC1 was significantly (>0.7) determined by the activities of dehydrogenase, xylanase, and protease as well as fungal numbers , and thiNitrification and phosphatase activity did not contribute significantly to any of the clusters (to PC1 or PC2). Nitrification is mainly carried out by autotrophic bacteria (rarely and to a weak extent by heterotrophic bacteria) that were not detected by the culturable method for heterotrophic microorganisms used in the present study. Nitrification has mainly been attributed to acid-sensitive chemoautotrophic nitrifiers; however, this process has also been documented in acid forest soil , 29. SeaWhen plotting the PC1 and PC2 scores for all soil samples analyzed, we obtained a clear separation between the two studied sites . Within Data obtained by PCA were confirmed by correlation analysis  in whichin situ conditions  on the metabolic activity of soil  and +0.8°C (IT02) over the monitoring period (1993–2010) were calculated from trendlines. A significant effect of the sampling year was detected on all microbiological properties studied (except nitrification), but with no consistent trend, whereas the effect of seasonality resulted in a significant response in a few parameters only. Soil microbial activities may be weakly correlated with climatic factors because they are typically estimated under optimized rather than nditions . Enzyme nditions . However of soil , 45, 57. of soil . The rol of soil . The soi of soil , 23.i.e. microbial activity and abundance were lower in the subalpine site IT01 than in the submontane site IT02. These differences were mainly attributed to significant  differences in the climate conditions prevailing at these sites. However, other factors such as physical and chemical soil properties also play a role. Altitude, which strongly influences climate, was found to affect a number of soil biochemical properties  and a decrease in the microbial population size and diversity (Biomonitoring sites also have the objective to quantify the variations between sites . We founoperties . The reliversity , 30, 32 iversity , 44, 55 iversity .Therefore, further studies are warranted in order to obtain an insight into changing climate conditions on soil microorganisms. The combination of studies on soil microbial activity and diversity with special consideration of the soil microclimate should allow for a better understanding of the functional role of soil microorganisms in forest soils and will be the focus of future investigations.


Omalizumab is a widely used therapeutic anti-IgE antibody. Here we report the crystal structure of the omalizumab–Fab in complex with an IgE-Fc fragment. This structure reveals the mechanism of omalizumab-mediated inhibition of IgE interactions with both high- and low-affinity IgE receptors, and explains why omalizumab selectively binds free IgE. The structure of the complex also provides mechanistic insight into a class of disruptive IgE inhibitors that accelerate the dissociation of the high-affinity IgE receptor from IgE. We use this structural data to generate a mutant IgE-Fc fragment that is resistant to omalizumab binding. Treatment with this omalizumab-resistant IgE-Fc fragment, in combination with omalizumab, promotes the exchange of cell-bound full-length IgE with omalizumab-resistant IgE-Fc fragments on human basophils. This combination treatment also blocks basophil activation more efficiently than either agent alone, providing a novel approach to probe regulatory mechanisms underlying IgE hypersensitivity with implications for therapeutic interventions. Omalizumab is an antibody against IgE that is used to treat patients with asthma and chronic idiopathic urticaria. Here, the authors report the structure of omalizumab in complex with an IgE fragment and develop an approach to exchange rather than deplete IgE on human basophils to block their activation. Allergic diseases represent an over-reaction of the immune system to normally non-hazardous environmental substances, such as dust mites, pet dander, pollen or mold, and the incidence of allergies worldwide is rising1467891011The IgE-Fc region contains three domains (Cɛ2–4). All three domains affect IgE receptor binding, yet the Cɛ3–4 fragment independently binds FcɛRI and CD23 and contains the residues involved in IgE receptor interactions1314IgE has been a target for therapeutic development because of its central role in the allergic response. The anti-IgE monoclonal antibody omalizumab is currently indicated for the treatment of moderate to severe persistent asthma and chronic idiopathic urticaria. Omalizumab has demonstrated robust clinical efficacy1617203–4) to 2.5 Å  that is resistant to omalizumab neutralization but is able to bind CD23 and FcɛRI. Significant experimental evidence has accumulated suggesting that IgE-dependent homeostatic regulatory pathways respond to the loss of receptor-bound IgE induced by omalizumab treatment, and could offset or constrain the therapeutic benefit of the anti-IgE treatment8122627282930313–4 mutant, in combination with omalizumab, can effectively exchange cell-bound IgE with IgE-R419N-Fc3–4 and that this dual inhibitor treatment is more potent at blocking basophil activation than either inhibitor alone. This approach of simultaneously depleting antigen-specific IgE, while engaging FcɛRI and CD23 receptors with an IgE variant, can be used to further probe the role of IgE-dependent regulatory pathways during anti-IgE treatment and may provide a route to enhance current anti-IgE therapies.The omalizumab structure also facilitated the design of an IgE-Fc3–4 mutant (IgE-G335C-Fc3–4), which contains an engineered disulfide bond at position 335 that traps the IgE-Fc3–4 domain in a closed conformation with reduced conformational flexibility3–4 variant, we crystallized the IgE-G335C-Fc3–4:omalizumab–Fab complex . The purified complex . Side-chain electron density was well resolved throughout the IgE:omalizumab interface  between intact IgE and the IgE-Fc3–4 fragments.Given that the crystal structure lacks the Cɛ2 domain, it cannot account for possible contributions of the Cɛ2 to omalizumab:IgE interactions. Therefore, we also compared the binding kinetics of full-length IgE (clone Sus11) to that of the IgE-Fcepitopes , and, ass tested . Surpriss tested . This re3–4, the IgE-G335C-Fc3–4 mutant . IgE-G335C-Fc3–4 exhibited similar kinetics in omalizumab-binding studies as wild-type IgE-Fc3–4  for omalizumab with IgE-Fc3–4 bound to FcɛRIα complexes that was closer to full-length IgE alone and slower than unbound IgE-Fc3–4. The similarity in association rates between full-length IgE and IgE-Fc3–4:FcɛRIα complexes may be in part because of the fact that each binding partner contains a single exposed Cɛ3 domain (kd) for the omalizumab:IgE-Fc3–4:FcɛRIα complexes was also similar to that of the measured rates for IgE:FcɛRIα or omalizumab:IgE complexes  loops, with the exception of the light chain CDR3 loop, contact IgE. CDR residues previously shown to be required for omalizumab binding either contact IgE  and the omalizumab:IgE complex.We recently described a class of IgE inhibitors derived from Designed Ankyrin Repeat Protein (DARPin) libraries, capable of disrupting IgE:FcɛRI complexes23. D ref. , indicat3 versus E2_79 and FcɛRIα=401 Å3). The omalizumab steric conflicts extend along the length of the omalizumab light chain and FcɛRIα N-terminal domain, while the E2_79 steric conflicts are more localized near the IgE:FcɛRIα interface  has proven successful for controlling allergic diseases. During omalizumab therapy, omalizumab neutralizes free serum IgE and slowly decreases surface levels of IgE on allergic effector cells. Therefore, it is impossible to simultaneously replace depleted allergen-reactive species with benign IgE species because of the requirement for continued excess omalizumab to be present during treatment. Since IgE-dependent homeostatic regulatory pathways could potentially counteract anti-IgE therapies, we sought to explore the possibility of replacing rather than removing patient IgE. We hypothesized that omalizumab-resistant IgE variants or fragments could be used in combination with omalizumab to effectively exchange the native IgE on human cells bearing FcɛRI or CD23.in vivoex vivo experiments with human whole blood (24–48 h), we did not observe significant loss of cell surface IgE at doses corresponding to the mean omalizumab serum concentrations from clinical trialsOver the course of omalizumab treatment, free IgE levels and IgE surface levels on basophils decline relatively rapidly within days3–4 or omalizumab-resistant IgE-R419N-Fc3–4, and the JW8 human/mouse chimeric IgE (composed of a human IgE heavy chain and a mouse-λ-light chain). The JW8 mouse-λ-light chain allowed us to track IgE reloading on stripped basophils in parallel with the biotinylated IgE-Fc3–4 proteins. We ensured that all antibody reagents used in staining carried the mouse-κ-light chain, or were rat antibodies, to avoid nonspecific binding from the anti-mouse-λ-light-chain antibody used to track JW8-IgE.To track the addition, removal and exchange of IgE species, we employed biotinylated IgE-Fc3–4 or IgE-R419N-Fc3–4 species , only minimal reloading of wild-type IgE-Fc3–4 was observed, in contrast to the dramatic reloading of IgE-R419N-Fc3–4  as compared with omalizumab alone by day 3  should be minor if each agent acted only as a competitive inhibitor for JW8-IgE:FcɛRIα interactions, suggesting that these agents can act synergistically to block basophil activation.To determine whether IgE-R419N-Fcts for 3  or 6 dayts for 3  before Nts for 3 . Of noteby day 3 , suggestby day 3 . This retrations . Furthertivation . The addAnti-IgE therapy remains an important tool for the management of allergic disorders, and novel second-generation anti-IgE therapies will soon join omalizumab. Yet, the aetiology of allergic disorders is complex and optimal therapeutic responses will likely come from treatment regimens that target and modulate multiple immunological processes.Here we have clarified the structural basis for omalizumab-mediated inhibition of IgE binding to both FcɛRI and CD23. This analysis revealed similarities between the disruptive inhibitor E2_79 and omalizumab, and provides a mechanistic framework to develop antibody-based therapies that can accelerate the dissociation of IgE:FcɛRIα complexes. Such agents could rapidly disarm basophils and mast cells, allowing them to achieve a therapeutic effect faster.3–4 can act synergistically with omalizumab ex vivo at very low doses to inhibit basophil activation. This finding suggests that simultaneously targeting FcɛRI and IgE with competitive inhibitors could enhance therapeutic responses. Prior studies have demonstrated that non-activating ligands can antagonize FcɛRI responses to activating ligands by sequestering receptor-proximal signalling components3–4:FcɛRI complexes could suppress activation through such mechanisms, in addition to competing for receptor occupancy. Sustained FcɛRI receptor occupancy could also suppress homeostatic responses to the loss of IgE on omalizumab-treated basophils.We have also demonstrated that mutant IgE fragments, in conjunction with omalizumab treatment, can exchange native IgE on human cells for IgE fragments, maintaining the occupancy of both high- and low-affinity IgE receptors. We pursued this approach, given the experimental evidence that basophils increase in sensitivity on a per IgE basis during omalizumab treatment28298111231Finally, the dissociation of allergen-specific IgE on mast cells located in peripheral tissues is slow, and the effectiveness of anti-IgE within these compartments is poorly understoodex vivo results, the concept is far from clinical application. Unexpected antibody responses to the R419N IgE, although unlikely, could prevent the development of any universally benign IgE variant for exchange therapy, and could induce anaphylactic antibody responses to receptor-bound IgE fragments. Yet, our approach with IgE-R419N-Fc3–4 does have two distinct advantages in this regard. First, N-linked glycosylation events are effective at masking antibody epitopes, and could reduce the immunogenicity of IgE-R419N-Fc3–4 . All insect vectors have been previously published: IgE-Fc3–4 , Canada) with a vascular endothelial growth factor (VEGF) signal sequence from the pTTVH8G vector (NRC). Constructs were transfected using 25-kDa linear polyethylenimine (Polysciences), and transiently expressed in suspension HEK-6E cells (NRC) for 120 h according to the supplier's protocols. Cell supernatants were filtered through 0.45-μM Durapore filter (Millipore) and incubated with Ni-NTA resin (Qiagen) for 1 h at room temperature, washed with 10 resin bed volumes of wash buffer (25 mM imidazole in PBS at pH 7.4) and eluted with 2 resin bed volumes of elution buffer (300 mM imidazole in PBS at pH 7.4). The eluted protein was then concentrated using an Amicon Ultra-15 filter unit (Millipore) and further purified with gel filtration on a Superdex 200 10/300 GL column (GE). Protein used for experiments with human basophils was subsequently buffer-exchanged into sterile PBS pH 7.4 using an Amicon Ultra-15 filter unit (Millipore). The DARPins E2_79 and bi53_79 were cloned into the pQE-30 expression vector between BamHI and HindIII restriction sites, and expressed in XL-1 blue E. coli (NEB) at 37 degrees overnight following induction with isopropyl-β-D-thiogalactoside. Soluble DARPins were purified using Ni-NTA affinity chromatography, followed by gel filtration on a Superdex 200 10/300 GL column (GE).Fab fragments of Omalizumab (Novartis) were prepared by digestion over an immobilized ficin agarose resin (Pierce) in 10-mM citrate buffer with 25 mM cysteine and 5 mM EDTA at pH 6.0 for 5 h. The Fab fragments were purified in two steps with protein G (Pierce) and gel filtration to yield homogenous omalizumab–Fab. Protein G columns were washed with phosphate buffer over a pH gradient  and Fab was eluted with glycine buffer at pH 2.5. Insect-cell-derived IgE-Fc−1 was added to 0.1 μl of well solution. Crystals were harvested and frozen in the same crystallization buffer for data collection.Crystals were grown in hanging drops in 0.2 M Lithium Sulfate, 0.1 M Tris pH 8.5 and 41% PEG 400. A volume of 0.1 μl of complex at 9 mg ml1 space group with the unit cell dimensions a=100.10 Å, b=107.14 Å, c=151.04 Å and β=95.18°. A molecular replacement solution was obtained using Phaser-MR in the Phenix package (version 1.9)Diffraction data were collected in two 360° sweeps of one crystal on the microfocus beamline 12-2 at the Stanford Synchrotron Radiation Lightsource, and data were indexed, integrated and scaled using the HKL2000 suiteStructural alignments with omalizumab:IgE and E2_79:IgE complexes were made with the Cɛ3 domain at site 2 of the FcɛRIα complex. The alignment with the CD23:IgE complex was made with either Cɛ3 domain within the CD23 complex . The coordinates of each IgE-binding molecule  from these aligned complexes were then loaded into pdbset (CCP4) and centred within an arbitrarily defined unit cell to accommodate the full chain. The coordinates of the aligned and transformed structures were then input into sfall (CCP4) to generate an atom map from the polypeptide chain (solvent atoms and ligands were not included), and input into mapmsk (CCP4) to make a map mask. The resulting map masks were input into overlapmap (CCP4) using the MAP INCLUDE function, keeping only density of overlapping regions of each inhibitor with the receptor complex in question. The volume of the resulting overlap maps were then calculated with the volumes tool in Chimera3–4 or R419N-IgE-Fc3–4 was biotinylated using EZ-Link Sulfo-NHS-LC-biotin (Pierce), with a 30-fold molar excess of sulfo-NHS-LC-biotin for 30 min at room temperature. The reaction was stopped using 1.0 M Tris pH 7.4, and proteins were dialysed overnight into PBS pH 7.4, and sterile-filtered with 0.22-μm filter.IgE-Fc−1 as estimated from supplier's supplied concentration range (AbD Serotec) or were left untreated. These cells were then treated overnight at 37°, as specified in the figure legends with omalizumab, IgE-Fc variants or vehicle controls. The following day cells were washed three times with BF buffer at 4 °C and stained for analysis.Blood was drawn from two healthy volunteers and a third volunteer with a history of food allergy ranging in age from 21 to 37 . Blood was collected in heparin vacutainer tubes (BD), and washed in 10 blood volumes of BF buffer (RPMI-1640 (Life Technologies) supplemented with 10% fetal calf serum  and 1% penicillin/streptomycin (Gibco)). Washed blood cells were then suspended in their original blood volume in BF buffer and treated with or without E2_79 at 25 μM for 2 h at 37 °C to remove surface IgE. Stripped cells were then reloaded with IgE-JW8 at 120–300 ng mlAfter washing, treated cells and controls were incubated with human Fc-block (BD), and stained with the following antibodies as described in figure legends (at 1:100 dilution unless stated): IgE-FITC (eBiosciences clone: IgE21), FcɛRIα-APC (eBiosciences clone AER-37 at 1:50 dilution), anti-biotin AF-488 (eBiosciences clone: BK-1/39), anti-mouse-lambda-light-chain PE (BioLegend clone: RML-42), CD123 PE-Cy5 (BD clone: 9F5 at 1:20 dilution), HLA-DR PE-Cy7 (BioLegend clone L243 at 1:80 dilution), CD203c BV421 (BioLegend clone: NP4D6 at 1:50 dilution), CD19-PE (BD clone: HIB19 at 1:50), CD23-BV421 (BioLegend EBVCS-5 at 1:50 dilution) and Aqua Live Dead stain (Life Technologies). Stained cells were then lysed with RBS lysis buffer (BioLegend) for 5 min at room temperature, and washed with FACS buffer (PBS pH 7.4 supplemented with 10% FCS). Data were collected on a DxP FACSCAN from Cytek Development in Fremont, CA  using FlowJo CE and analysed using FlowJo (version 10).−1. Dissociation was assessed for 240 s under constant buffer flow. For each measurement, the chip surface was regenerated with 50 mM NaOH. Individual sensorgram curves were exported to Excel, and graphs were prepared with the GraphPad Prism 5.0 software . In all experiments, unspecific binding to flow cell 1 was subtracted from the signal on flow cell 2.SPR measurements were conducted on a BIAcore X100 device and evaluated with the BIAevaluation software . For kinetic analysis of different IgE fragments on omalizumab and FcɛRIα, 1,000 response units of omalizumab or rhFcɛRIα were immobilized in acetate buffer  on flow cell 2 of a CM5 chip . Flow cell 1 was activated and deactivated without immobilization, according to the manufacturer's protocol. The different IgE fragments (produced as described above) or full-length Sus11 IgE were diluted in HBS-EP+ running buffer  and injected for 120 s at a constant flow rate of 10 μl min−1 by using Percoll density centrifugation of dextran-sedimented supernatants with further purification with the Miltenyi basophil isolation kit II , as previously described5 cells per well in a 96-well plate in 50 μl of RPMI containing 10% heat-inactivated FCS, 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin (medium). Cells were kept in a cell incubator at 37 °C, 5% CO2. For desensitization, cells were treated with 50 μM disruptive anti-IgE inhibitor bi53_79 for 8 h in the presence of 10 ng ml−1 rhIL-3 and subsequently washed three times with 150 μl PBS to remove dissociated IgE and anti-IgE inhibitor from the supernatant. For resensitization, cells were incubated with 100 nM JW8-IgE  for 2 h in the presence of 10 ng ml−1 rhIL-3. Subsequently, cells were washed two times with 150 μl PBS and treated with omalizumab, IgE-R419N-Fc3–4 or a combination of these molecules for 3 or 6 days at the indicated concentrations. For determination of basophil activation, cells were stimulated with 1–1,000 ng ml−1 NIP(7)BSA  in the presence of 10 ng ml−1 rhIL-3 for 30 min at 37 °C, 5% CO2. Subsequently, cells were stained with 10 μl anti-CD63 FITC anti-CCR3-PE staining mix  for 20 min at room temperature. At least 3 × 103 basophils were acquired on a FACSCalibur device. Data were analysed with the FlowJo V10 software .Human primary basophils were isolated from whole blood of volunteers with approval from the local ethics committee (KEK Bern). Informed consent was obtained from all donors in accordance with the Helsinki Declaration. Human basophils were isolated from three different donors, with total IgE levels ranging from 35 to 78 kU lAccession codes: The reflection data and coordinates for the omalizumab:IgE complex have been deposited in the RCSB with PDB ID code 5HYS.How to cite this article: Pennington, L. F. et al. Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange. Nat. Commun. 7:11610 doi: 10.1038/ncomms11610 (2016).Supplementary Figures 1-11


Antibodies play major roles in immunity to malaria; however, a limited understanding of mechanisms mediating protection is a major barrier to vaccine development. We have demonstrated that acquired human anti-malarial antibodies promote complement deposition on the merozoite to mediate inhibition of erythrocyte invasion through C1q fixation and activation of the classical complement pathway. Antibody-mediated complement-dependent (Ab-C′) inhibition was the predominant invasion-inhibitory activity of human antibodies; most antibodies were non-inhibitory without complement. Inhibitory activity was mediated predominately via C1q fixation, and merozoite surface proteins 1 and 2 were identified as major targets. Complement fixation by antibodies was very strongly associated with protection from both clinical malaria and high-density parasitemia in a prospective longitudinal study of children. Ab-C′ inhibitory activity could be induced by human immunization with a candidate merozoite surface-protein vaccine. Our findings demonstrate that human anti-malarial antibodies have evolved to function by fixing complement for potent invasion-inhibitory activity and protective immunity.  •P. falciparum replicationAntibodies function with complement to inhibit •Antibodies fix C1q to block invasion and lyse merozoites•Complement-fixing antibodies are strongly associated with immunity in children•Antibody-complement inhibition can be induced by human vaccination Plasmodium falciparum.Antibodies are important in immunity to malaria, but their protective function has been unclear. Boyle and colleagues report that acquired and vaccine-induced human antibodies recruit complement to block infection of erythrocytes and blood-stage replication of  Plasmodium falciparum are an important component of acquired immunity against malaria, as demonstrated in pivotal studies in which immunoglobulin G (IgG) from immune adults was transferred to malaria-infected children and resulted in parasite clearance and recovery  or malaria-naive IgG  was investigated via immunoblot. C1q and C3b were detected at higher levels on merozoites incubated with NS and PNG IgG than on merozoites incubated with Australian IgG, reflecting activation of the classical complement cascade by anti-merozoite antibodies E and S1FTo test the importance of the activation of the classical complement cascade in relation to that of the alternative complement cascade, we compared the invasion-inhibitory activity of anti-malarial antibodies between NS and HIS heated at 50°C for 20 min; this treatment inhibits the alternative complement cascade by inactivating Factor B but leaves the classical complement cascade intact. The 50°C treatment of serum had no significant effect on the invasion-enhancing activity; invasion inhibition by PNG IgG was greater in the presence of NS and 50°C-treated serum than in the presence of standard HIS A. This iTo investigate whether MAC deposition and lysis could also contribute to inhibiting invasion, we assessed the ability of IgG and complement to lyse merozoites. Merozoite invasion occurs rapidly, such that 80% of invasion occurs within 10 min of mixing with RBCs ; therefoThe importance of Ab-C′ inhibition in naturally acquired immunity was assessed with purified IgG from Kenyan (n = 33) and PNG (n = 10) individuals. Overall, there was much greater Ab-C′ inhibition than direct inhibitory activity A. ComparIn Kenyan individuals, we assessed the relationship among Ab-C′ inhibition, direct inhibition, IgG subclass reactivity to merozoites, and antibody activity in standard complement-free growth-inhibition assays (GIAs). The prevalence of antibodies to merozoites was high, and merozoite-specific IgG was strongly and significantly correlated with Ab-C′ inhibition, but not with direct inhibitory activity and less strongly with activity in GIAs . Of noteHaving shown that Ab-C′ inhibition functions via fixation of C1q and the activation of the classical complement cascade, we evaluated the relationship between C1q deposition and Ab-C′ inhibition. We measured antibody-mediated C1q deposition on merozoites by immunoblot using IgG from nine Kenyan individuals with high, medium, or low Ab-C′ inhibition (n = 3 for each group). C1q deposition was notably higher with IgG from individuals who had high Ab-C′ inhibitory activity than with IgG from those with medium or low activity F. Next, To obtain epidemiologic evidence of the importance of antibody-mediated complement fixation in acquired immunity to malaria, we tested antibodies for C1q fixation from a longitudinal cohort of 206 5- to 14-year-old children who were resident in a malaria-endemic region of PNG ; all chiP. falciparum and preventing disease.To assess the role of antibody-mediated C1q fixation in protection from symptomatic malaria and high-density parasitemia, we grouped children into low, medium, and high categories of C1q-fixation activity; we then compared the relative risk of malaria between response groups. High C1q deposition was very strongly associated with protection from clinical malaria . The assTo identify merozoite antigens that are targets of Ab-C′ inhibition, we tested rabbit antibodies to several major merozoite surface antigens for inhibition of invasion in the presence of NS or HIS. Antibodies to MSP1-19, MSP1 block 2, and MSP2 substantially inhibited invasion in the presence of NS, but not HIS A. ActiviMSP2 and MSP3 are vaccine candidates that have progressed to clinical trials, but development has been hampered by the lack of immunologic correlates of protection, given that these antibodies are relatively non-inhibitory in standard GIAs . The funTo further confirm the role of complement fixation in mediating invasion inhibition and the significance of MSP2 as a target, we tested a human MSP2-specific monoclonal antibody (mAb) with and without a specific change (L234A or L235A [LALA]) in the amino acid sequence of the Fc region ; this chWe examined whether Ab-C′ inhibitory activity could be induced by immunization of malaria-naive individuals with recombinant merozoite surface proteins. We studied samples from the recent phase 1 clinical trial of the MSP2-C1 vaccine . IgG froAlthough the importance of antibody in immunity to malaria has been established , mechaniComparisons of Ab-C′ inhibitory activity in serum depleted and reconstituted with C1q or C5 highlight the importance of C1q in mediating inhibition of invasion. Further, C1q alone was able to significantly enhance the inhibitory activity of anti-malarial antibodies in the absence of other complement factors. This was further demonstrated by comparison of wild-type and altered human MSP2 mAbs; Ab-C′ inhibition was only seen with the wild-type and not the altered mAb. C1q-mediated antibody neutralization has been previously reported with influenza  and WestAlthough C1q-mediated enhancement appears central to invasion-inhibitory activity, complement deposition and lysis of merozoites are likely to have other implications in vivo, including enhancement of phagocytosis and induction of pro-inflammatory cytokines that might further mediate control of parasitemia. Complement activation, particularly as part of the innate immune response, has been implicated in pathogenesis as a result of induction of inflammatory responses  and PNG (Madang District). Unexposed serum pools were from Australian donors residing in Melbourne . IgG from serum pools was purified with Melon Gel according to the manufacturer’s (Thermo Scientific) instructions. Purified IgG was concentrated in 10-kDa MWCO (molecular weight cutoff) spin-purification tubes (Amicon) and buffer exchanged with PBS. For the longitudinal study of PNG children, plasma samples were obtained at enrollment from a prospective treatment-reinfection cohort of 206 children aged 5–14 years (median = 9.3) in Madang, PNG . Childre ISA 720   as described in Merozoites were incubated with 25% NS and test IgG or serum samples for 1, 5, 10, 15, or 30 min at 37°C. Merozoites were washed and solubilized for immunoblot analysis. C1q and C3 were detected with anti-C1q  and anti-C3 , respectively.6 merozoites/well. Plates were blocked and then incubated with sera samples (1/250), and then recombinant C1q (10 μg/ml) and C1q were detected with goat anti-C1q antibodies and anti-goat-HRP. C1q and C3 deposition was also detected with C5-depleted serum as a complement source. For ELISA analysis of MAC deposition, isolated merozoites were incubated with 25% NS serum and IgG from PNG or Australian (Melbourne) serum pools for 10 min at 37°C and then washed and coated into Nunc 96-well plates. Plates were blocked, and the presence of MAC was detected with anti-C5–C9 antibodies (rabbit) followed by anti-rabbit-HRP.For ELISA, plates were coated with purified merozoites at 5 × 10For immuno-electron microscopy, merozoites were incubated with NS or HIS with PNG IgG for 10 min. Merozoites were washed and fixed in 1% glutaraldehyde and then processed and imaged as described in Merozoites were incubated with 5% PNG or Melbourne IgG and 20% NS, HIS, or C1q-depleted serum for 10 min (37°C); for assays to assess the rate of merozoite lysis, an aliquot of sample was taken every minute for analysis by flow cytometry. Samples were diluted 1/100 in 200 μl cold PBS and 1% newborn calf serum, and the density of merozoites was counted with CountBright counting beads via flow cytometry.ELISAs were performed according to standard methods . PurifieDifferences in invasion-inhibitory IgG activity between NS and HIS and between C1q-depleted and -reconstituted serum were calculated with paired t tests in Stata/SE 11.2. Associations between antibody reactivity to intact merozoites by ELISA and functional activity in assays of Ab-C′ inhibition, direct inhibition, and growth inhibition were assessed with Spearman’s correlations calculated in Prism.Analysis of the cohort study was performed with Stata/SE 12.0. Differences in C1q deposition between groups were assessed by chi-square tests  or Wilcoxon rank-sum tests (for continuous variables). For assessment of associations between C1q deposition and protection, subjects were stratified into tertiles according to low (including those classified as “negative”), medium, or high deposition of C1q, as determined by optical-density values for each sample. Groups were compared for risk of clinical malaria  or high-density parasitemia  with the Cox proportional-hazards model . SurvivaM.J.B., L.R., G.F., and J.G.B. planned the study and interpreted results with input from all authors. M.J.B., L.R., Y.S.C., G.F., H.A.J., and C.L. performed experiments. F.H.O., K.K.A.T., D.J.C., I.M., J.S.M., K.M., J.S., and R.F.A. provided key reagents. M.J.B., L.R., G.F., and J.G.B. wrote the manuscript with input from all authors.


Striatal medium spiny neurons (MSNs) receive lateral inhibitory projections from other MSNs and feedforward inhibitory projections from fast-spiking, parvalbumin-containing striatal interneurons (FSIs). The functional roles of these connections are unknown, and difficult to study in an experimental preparation. We therefore investigated the functionality of both lateral (MSN-MSN) and feedforward (FSI-MSN) inhibition using a large-scale computational model of the striatal network. The model consists of 2744 MSNs comprised of 189 compartments each and 121 FSIs comprised of 148 compartments each, with dendrites explicitly represented and almost all known ionic currents included and strictly constrained by biological data as appropriate. Our analysis of the model indicates that both lateral inhibition and feedforward inhibition function at the population level to limit non-ensemble MSN spiking while preserving ensemble MSN spiking. Specifically, lateral inhibition enables large ensembles of MSNs firing synchronously to strongly suppress non-ensemble MSNs over a short time-scale (10–30 ms). Feedforward inhibition enables FSIs to strongly inhibit weakly activated, non-ensemble MSNs while moderately inhibiting activated ensemble MSNs. Importantly, FSIs appear to more effectively inhibit MSNs when FSIs fire asynchronously. Both types of inhibition would increase the signal-to-noise ratio of responding MSN ensembles and contribute to the formation and dissolution of MSN ensembles in the striatal network. The basal ganglia, a set of interconnected neural structures positioned deep in the brain, are believed to be critically involved in learning   inhibition enabling strongly activated MSNs or MSN ensembles to shut down competing MSNs/MSN ensembles Groves, . InteresLikewise, the anatomy of the feedforward inhibitory projection has fueled a number of hypotheses regarding its functionality, though with somewhat less experimental data available for evaluating each theory. The observation that FSI-to-MSN projections form clusters of synapses near the MSN soma and produce relatively large IPSPs in the postsynaptic cell  and feedforward (FSI-to-MSN) inhibition using a biophysically constrained, large scale computational model of the striatal network. In an effort to accurately capture the complex dynamics of inhibitory and excitatory inputs in the dendrites of MSNs, we explicitly included dendrites and almost all known ionic channels in both the FSI and MSN models. We found that lateral inhibition enabled large ensembles of synchronously firing MSNs to strongly suppress non-synchronous MSNs. We found that feedforward inhibition effectively suppressed MSN activity—especially non-synchronous MSN activity—but only when FSI cells fired asynchronously. These results suggest that the functional role of lateral inhibition may be to aid MSN ensemble synchronization and formation, while the functional role of feedforward inhibition may be to suppress less active MSN ensembles in favor of more active MSN ensembles. These findings will help to refine and inspire both new and existing conceptual models of the function of the striatum and of the basal ganglia.Determining the role of inhibition in the striatal network The model was developed in the NEURON 7 simulation environment , so we focus only on the most salient aspects of the single cell model in this description. Cell dimensions , and passive properties were set to match published values  contributed to a separate pool that did not regulate the BK and SK currents, in accord with published experimental results  of striatal tissue, we did not differentiate between projections among D1 and D2 expressing cells, though differences in connectivity between these populations have been shown to exist .Lateral projections (MSN to MSN) in the model randomly connect to compartments (uniform probability) in the secondary and tertiary dendrites of MSN to match published results  in the soma and primary dendrites of an MSN, in agreement with published findings  received an independent spike train. Each spike train was generated using the following algorithm  amplitudes and time courses for lateral and feedforward inhibition shown in Figures 3C–F were obtained by subtracting MSN membrane voltage traces from a 512 MSN network simulation with lateral (feedforward) inhibition active from the same network simulation without lateral (feedforward) inhibition active. To simplify the calculation, only one MSN (FSI) received enough depolarizing input to spike, while all other MSNs received unique synaptic input trains that placed them at various potentials below firing threshold. Only data from the first spike in the simulation was used for the plot. For each IPSP, we found the maximum and minimum value of the IPSP, and then used whichever value (max or min) had a greater absolute value. Therefore, there are no IPSPs of zero amplitude, or very small absolute value, on the plot.The traces shown in Figure 4 are calculated for glutamatergic inputs only. For Figure 4A, FSIs in the network received glutamatergic synaptic inputs at a frequency of 300 Hz. For Figure 4B, MSNs in the network received glutamatergic synaptic inputs at a frequency of 1000 Hz.Synaptic input frequencies in in vitro MSN from an adult (P-X) rat accumbens core neuron.The MSNmodel is a stylized representation of a nucleus accumbens core MSN, which has been previously described  model was adapted from a previously published model of a rat dorsal striatal parvalbumin-expressing FSI  indicates 95% confidence in equivalence. After discarding any fits with an R-squared value of less than 80%, this gave a mean T1 of 10.4 ms (SD of 3.9 ms) and a mean T2 of 10.2 ms (SD of 3.6 ms) for lateral inhibition in the model. For comparison, the in vitro data in Figure z = −0.18), 10.2 ms (z = 0.35), and 10.5 ms (z = −0.18) for T1, and 6.3 (z = 7.4), 7.3 (z = 5.5), and 8.1 ms (z = 4.0) for T2. For feedforward inhibition, fitting the model gave a mean T1 of 6.5 ms (SD of 3.7 ms), and a mean T2 of 4.4 ms (SD of 4.9 ms). The in vitro data in Figure z = −6.3) for T1 and 5.7 (z = −1.8) ms for T2. Therefore, the model's values for lateral T1 and feedforward T2 time constants match in vitro data, while the model's values for lateral T2 and feedforward T1 time constants do not.We fit a double exponential to each IPSP using the form V = exp(−t/T1) − exp(−t/T2), where V is voltage, t is time, and T1 and T2 are time constants, and used a one-sample z-test to compare the ms SD of .9 ms andms SD of .9 ms andWe examined the effects of lateral (MSN-to-MSN) and feedforward (FSI-to-MSN) inhibition on MSN firing rate in response to increasing frequencies of unstructured synaptic inputs Figure . LateralAccordingly, lateral inhibition appears to be capable of significantly limiting uncorrelated spiking in the striatal network model, despite the fact that MSNs form few connections per cell and contact mostly distal locations on the target MSN. In contrast, feedforward inhibition is relatively incapable of suppressing uncorrelated MSN firing, despite the fact that FSIs form multiple proximal connections with target MSNs.We next examined the effect of lateral inhibition on MSN neural ensembles responding to correlated and uncorrelated inputs. Specifically, we created a network simulation in which half of the MSNs (cells 1–1372) were responding as a partially synchronized ensemble and the other half (cells 1373–2744) were firing randomly Figures . EnsemblUpon comparing the activity in the network without lateral inhibition Figure  and the Therefore, lateral inhibition improves the timing precision of the ensemble response to coordinated input by suppressing the activity of MSNs (both intra-ensemble cells and non-ensemble cells) that are not precisely synchronized with the active ensemble.We next investigated whether synchronized feedforward inhibition could similarly suppress MSN activity. We created a network simulation in which all 2744 MSNs received noisy synaptic input, and all 121 FSIs received precisely the same synaptic input and therefore spiked in perfectly synchronized approximately 60 Hz bursts every 125 ms. Comparing the activity of the network without feedforward inhibition Figure , left toSince synchronized feedforward inhibition does not appear to significantly suppress MSN firing, we asked whether desynchronized feedforward inhibition might be more effective in suppressing MSN activity. To study this, we created a network simulation in which half the MSNs responded as a partially synchronized ensemble  while the other half of the MSNs were not synchronized at all—receiving only noisy synaptic inputs Figure . CompariAccordingly, feedforward inhibition appears to disproportionately limit non-synchronously firing MSNs in favor of synchronously firing MSNs, and appears to act more effectively when FSIs are desynchronized.To our knowledge, this model is the most accurate representation to date of the current state of knowledge of the connectivity and parameters of the striatal network. It is also the first striatal network model to incorporate multi-compartment MSN and FSI models .Our operating philosophy in building the single cell MSN model, and later the network model, was to include as much experimentally verified data as possible in order to remain objective. A reduced computational model may accurately reproduce some, perhaps most, of the behavior we present here. However, it is only possible to conclude this with reasonable certainty Importantly, we did not explicitly account for gap junctions between FSIs in the model, though these have been observed experimentally (Kita et al., Several other groups have created large computational models of the striatum using single compartment cells in order to study the functional effects of the lateral and feedforward inhibitory projections. Using large scale network models consisting of 100–4000 single compartment neurons, researchers have shown that lateral inhibitory interactions in the striatal network enhance the ability of MSNs to express a diverse array of spiking characteristics and form large cellular ensembles with other MSNs (Humphries et al., With respect to feedforward inhibition, a previous model found that feedforward inhibition actually increased MSN spiking (Humphries et al., The primary reason that lateral inhibition is able to more powerfully suppress MSN firing than feedforward inhibition is because of the significantly greater number of lateral inhibitory inputs per MSN. Additionally, we suggest that the dendritic localization of lateral inhibitory inputs may actually be advantageous for influencing MSN activity. In previous work, we demonstrated that the MSN model's dendrites integrate input independently and together “pull” the soma up toward the spike threshold (Wolf et al., in vivo (Berke, in vivo, which is supported by our model.A surprising finding of our study is that asynchronous FSI activity appears to have a more profound inhibitory effect on MSN spiking than synchronous FSI activity. Since each feedforward IPSP is already relatively large, we speculate that several FSIs firing in synchrony have only a small reinforcing effect. Given the brief feedforward IPSP time course of ~10 ms, asynchronous spiking would distribute the inhibitory effect of the FSI spiking over a longer time course, and induce more of a sustained suppression than synchronous FSI spiking. Importantly, experimental recordings have observed that FSIs are tonically active o Berke,  and tendo Berke, , 2011. TWe have demonstrated that lateral inhibition can significantly reduce uncorrelated MSN spiking as well as enable synchronously firing MSNs to strongly suppress non-synchronized MSNs. These findings are in line with the concept that the striatum functions as a competitive neural network, but suggest that it is not only the size of an MSN ensemble which determines the winner in the competition, but also the latency with which the ensemble responds to inputs. The ability of lateral inhibition to significantly constrain uncorrelated activity in the network Figure  also sugAdditionally, we suggest that the precise MSN spike timing conveyed by lateral inhibition is important for regulating synaptic plasticity—specifically, dopamine-dependent, spike-timing dependent plasticity. Corticostriatal plasticity has been shown to be driven by a type of spike-timing dependent plasticity in which the presence or absence of dopamine determines whether a synaptic connection strengthens or weakens (Wickens et al., in vitro experiments have shown that FSIs burst at the beginning and ending of MSN ensemble formation (Carrillo-Reid et al., In contrast, we showed that feedforward inhibition can disproportionately reduce spiking in non-synchronously firing MSNs while sparing synchronously firing MSNs. Recent studies have shown that even though FSIs fire non-synchronously Berke, , they doBoth forms of inhibition would increase the signal-to-noise ratio of MSN ensembles. Lateral inhibition, by enforcing precise synchronization of active MSN ensembles while suppressing non-synchronized cells, would increase the clarity of activated ensembles relative to background activity in the striatum and facilitate signal readout by the pallidum and other downstream structures. Feedforward inhibition would comprise a simple yet effective mechanism for turning off weak neural ensembles while sparing stronger ones, again increasing the clarity of signal presentation in the striatum.Taken together, our results suggest a conceptual framework within which models of striatal and basal ganglia function may be considered. For example, within the context of action selection, where an organism must choose from among several potentially conflicting choices, lateral inhibition would be expected to improve the ability of the network to learn new action representations, to efficiently select only one action at any given time, and to associate the outcome of an action with the correct choice representation. Feedforward inhibition would be expected to ensure the selection of the most appropriate action, to help the striatal network shift between actions, and to prevent multiple action representations from being active simultaneously. Importantly, subjects with impaired lateral inhibition would be expected to exhibit learning deficits as well as impairments in action initiation and control, as with chorea in Huntington's disease and akinesia and cognitive deficits in Parkinson's disease. Subjects with impaired feedforward inhibition would be expected to exhibit deficits in the selection of appropriate actions, as observed in Tourette's disease and obsessive-compulsive disorder. Continued research with animal models of these disorders, along with selective inactivation of either form of inhibition, will allow for the testing of the predictions generated from the comprehensive network model presented here.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oct-4, Nanog, Sox2 and by an augmentation of fgf5 gene expression – a marker of post-implantation epiblast. Together, these data indicate that LIF-depleted mouse ESCs undergo a transition from the LIF/STAT3-supported pluripotent state to the FGFR/ERK-committed primed-like state with expression of early differentiation markers mediated through activation of mTOR signaling.Leukemia inhibitory factor (LIF) is indispensable to maintain the pluripotent state of mouse embryonic stem cells (ESCs), but the mechanisms underlying the role of LIF/STAT3 pathway are yet poorly understood. Here we first showed that the LIF/STAT3-regulated signaling pathway contributes to the maintenance of self-renewal and pluripotency of mouse ESCs by suppressing mTOR , which is necessary for early differentiation. When LIF is withdrawn from culture medium, the mTOR activity rapidly increases as detected by phosphorylation of its targets – ribosomal protein S6 and translation factor 4EBP1. In turn, suppression of STAT3 phosphorylation on Tyr-705 by a specific small molecule WP1066 also activates phosphorylation of the mTOR target S6 ribosomal protein. LIF removal strongly activates ERK activity indicating that ERK can be involved in either direct phosphorylation of mTOR or phosphorylation of an upstream negative regulator of mTOR – TSC1/TSC2 proteins. According to western blotting data, LIF withdrawal leads to phosphorylation of TSC2 protein thereby relieving its negative effect on mTOR activity. mTOR activation is accompanied by a decrease of pluripotent gene expression  The mechanisms underlying pluripotency and self-renewal are of great interest, and a number of signaling pathways  have been shown to contribute to the circuitry of ESCs state.3 LIF/STAT3  signaling pathway is one of the first described pathways regulating pluripotency in mouse ESCs.4 LIF is a cytokine from the IL-6 family, which acts through the LIFR/gp130 receptor and activates STAT3 transcription factor at Tyr-705 that provides activation of pluripotency genes expression and downregulation of such differentiation markers as Gata3, Gata4, T/brachyury and Eomes.5 It has been also shown that LIF can activate Ras-MAPK and PI3K-Akt through the involvement of gp130 adaptor protein.7 Thus LIF/STAT3 signaling pathway promotes self-renewal and blocks the differentiation of mouse ESCs. Respectively, LIF removal from culture media is the first step to change mouse ESCs pluripotent state and to direct to differentiation. It is known that suppression of MEK/ERK by pharmacological inhibitors promotes their self-renewal, whereas ERK activation is essential for differentiation induction in embryonic stem cells suggesting a role of LIF pathway in maintaining the low levels of the ERK activity in undifferentiated cells.8Embryonic stem cells (ESCs) are pluripotent cells derived from the early blastocyst that are capable of self-renewing for a long time 9 mTOR is essential for growth and proliferation in early mouse embryo and proliferation of mouse ES cells. Thus, disruption of mTOR gene results in early post-implantation lethality; moreover, mES cells fail to be obtained from mTOR−/− blastocysts.11 Normal function of mTOR-signaling pathway is also essential for trophoblast development. There are contradictory data on the role of mTOR in human ESCs: according to Zhou et al. mTOR supports long-term self-renewal, while other reports suggest that mTOR-mediated activation of p70-S6K induces differentiation.13 DEPTOR, a negative regulator of mTOR signaling, has an important role in maintenance of pluripotent state of embryonic stem cells and its level dramatically decreases with differentiation of mouse ESCs.14mTOR  signaling pathway has an important role in a variety of cell processes including cell growth, translation regulation, cell metabolism, cell cycle regulation and autophagy. This signaling pathway integrates signals from growth factor receptors (insulin and IGF), energy status, amino acids abundance and several stress factors. The key protein of this pathway mTOR kinase was found to be a key component of two different molecular complexes: mTORC1 (rapamycin sensitive complex) and mTORC2 (rapamycin-insensitive complex).oct4, nanog, klf4 and sox2 and by an increase of fgf5 gene expression – the marker of post-implantation epiblast and primed pluripotent state. Treatment with MEK1,2 inhibitor PD0325901 canceled the mTOR activation thereby implying the involvement of MEK-ERK pathway in mTOR activation. Together, these data indicate that LIF depletion of mouse ESCs induces a transition from LIF/STAT3-supported pluripotency to FGFR/ERK-committed primed-like state mediated through activation of mTOR signaling.Here, we examined the activity of mTOR signaling pathway under conditions that permit self-renewal of mESCs (in the presence of LIF), and under conditions that promote differentiation (in the absence of LIF). mTOR activity in the mTORC1 and mTORC2 complexes increases after LIF withdrawal from the medium indicating that LIF/STAT3 signaling negatively effects the activity of mTOR. To understand the mechanism of mTOR activation, we checked the phosphorylation state of TSC2, an upstream negative regulator of mTOR. It turned out that in LIF-depleted cells the activated ERK is involved in phosphorylation of TSC2 protein thereby relieving its negative effect on mTOR activity. qRT-PCR analysis showed that the activation of mTOR upon LIF withdrawal occurs simultaneously in parallel with a decrease in transcription of genes 4 First, we compared the morphological and cell growth characteristics of mESCs growing in the absence of LIF. According to this data, within 24 h after LIF withdrawal the morphology of mESC E14TV2 cell line acquires some features characteristic for differentiating cells. Undifferentiated mESCs grow in compact and dense colonies, which are becoming less dense and more flattened after LIF withdrawal with the increasing number of individual separately growing cells . This confirms a conclusion that mTOR is under negative control of LIF/STAT3 pathway.An increase of mTOR kinase activity induced by LIF withdrawal accompanies an onset of mESCs differentiation. These events appear to be tightly linked and evidence for a functional significance of mTOR-pathway in triggering differentiation. To assess the role of mTOR-signaling pathway in mES cells, we used two inhibitors with different mechanisms and specificity: rapamycin  and PP242 . In accordance with the increasing role of mTOR after LIF withdrawal, the LIF-deprived mESCs become more sensitive to mTOR inhibition. Rapamycin and PP242 suppress the clonal growth and cell viability of LIF-depleted mESCs in more than twice as efficient as compared with LIF-supplemented cells (clonability and MTT assays) . Similarβ inhibitors is sufficient to maintain self-renewing properties of mouse ESCs (2i protocol).18 To elucidate a role of ERK1/2 in upregulation of mTOR signaling, we checked the phosphorylation state of the ERK1/2 on Thr202/Tyr204 residues in LIF-depleted mESCs. Data presented in TSC2 tumor suppressor gene. Tuberin is an important regulator of cell proliferation and tumor development as well as a negative regulator of mTOR.19 Tuberin can be directly phosphorylated at Thr1462 by PKB/Akt that relieves its inhibitory effect on mTOR mediated through Rheb. Tuberin also can be inhibitory phosphorylated at Ser664 by ERK1,2 that leads to abrogation of mTOR inhibition. Undifferentiated cells have low levels of TSC2 phosphorylation that correlates with a low level of mTOR activity . Firstly, we checked the phosphorylation state of Ulk-1 (Atg-1), which is a primary target for positive (pAMPK) or negative (mTOR) regulation of autophagy.22 Although Ulk-1 has many sites for its phosphorylation, the most important site for AMPK-dependent activation is Ser555, whereas phosphorylation on Ser757 by mTOR kinase leads to a suppression of autophagy. As expected, LIF withdrawal stimulates Ulk-1 phosphorylation on Ser757 that reflects upregulation of mTOR activity /AKT; and mitogen-activated protein kinase (MAPK).25 Overall, this approach offers the unique opportunity to study switching over the signaling pathways controlling self-renewal, pluripotency and differentiation. Thus, despite a weak effect on the cell cycle parameters the expression of genes determining pluripotent state drastically decreases, and the change of mESC morphology takes place as well  is sufficient to trigger Nanog gene downregulation through activation of the MEK/ERK pathway and differentiation of mESCs to primitive endoderm.26 However, this FGF2-receptor activation has minimal effect on other pluripotency genes, including Oct4 and Sox2.To induce differentiation, mouse ESCs are usually transferred to the LIF-free medium supplemented with a set of growth factors that promote differentiation to a certain lineage. LIF removal is one of the first triggers to commit differentiation, albeit it has a slight effect on the viability and proliferation characteristics . However as well . This ma27 As to the contribution of ERK in mTOR acivation, the ERK phosphorylation rapidly and greatly increases after LIF withdrawal, suggesting that the activation of FGFR/MEK/ERK pathway may contribute to mTOR phosphorylation. Indeed, very strong activation of ERK in LIF-depleted mESCs correlates with TSC2 phosphorylation and a MEK/ERK inhibitor abolishes it  state. In case of the exit from the pluripotent state induced by LIF withdrawal and described in the present work, the mTOR activation does not suppress a marker of autophagy LC3 or its conversion to membrane-bound LC3-II form , which blocks autophagolysosome formation.32Available data show that high mTOR activity decreases the autophagic flux, therefore, of interest to know how LIF withdrawal induced mTOR activation affects the level of autophagy in mESCs. There are recent data focusing on the role of autophagy in cell reprogramming. In particular, there is a robust induction of autophagy during the reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs) by four transcription factors . This process occurs independently of p53 activation, and is mediated by the synergistic downregulation of mTORC1 and the induction of autophagy-related genes.-II form . But our33 LIF-depleted cells, similar to primed pluripotent cells, still express, albeit on a low level, the canonical pluripotency genes Oct-4, Sox2 and Nanog, but not Klf4, and also can express specification markers Fgf5 and T/brachyury  origin of these stem cells.35 Interestingly, mTOR supports long-term self-renewal of human ESCs that are analogous to rodent EpiSCs and correspond to primed pluripotent state.38The interpretations of the observed phenomena can be as follows. Upon LIF withdrawal, mESCs are likely to undergo a transition from naive pluripotent state to so-called primed pluripotent state characteristic for rodent EpiSCs and primate ESC cells.rachyury . The EpiOct-4, Nanog, and Sox2. As Sox2 negatively regulates the mTOR gene promoter activity,39 the shutdown of Sox2 can upregulate mTOR transcription, although this mechanism cannot explain the immediate mTOR activation observed after LIF withdrawal. Rather, the interplay of LIF/STAT3 and FGFR/ERK pathways determines mTOR activity level, as well as the outcome of the pluripotent cell state: naive or primed pluripotency. In naive ESCs, LIF/STAT3 pathway is dominant over FGFR/ERK,40 but with LIF withdrawal the FGFR/ERK pathway is activated, leading to phophorylation of a negative mTOR kinase regulator, Tuberin (TSC2), and eventually to activation of mTOR.LIF withdrawal downregulates the expression of STAT3/Klf4 target pluripotent genes In summary, LIF withdrawal causes a reversible transition of mESCs from naive pluripotent state to FGFR/ERK-committed early differentiation mediated through activation of mTOR signaling. The resulting state has some features characteristic for the primed pluripotency.2. Recombinant mLIF was expressed using an expression vector pGEX-2Т-hLIF in Escherichia coli and was further purified on a Glutathione Sepharose column. For clonal viability assay cells were seeded in a clonal density (500 cell per well on a six-well plate) and were Giemsa-stained and calculated after 6 days of culture in different media. The mTOR inhibitors rapamycin and pp242 were purchased in Calbiochem .Mouse embryonic stem E14TV2 cell line was maintained on tissue culture dishes  coated with 0.2% porcine gelatin  in a Dulbecco's modified Eagle's medium DMEM/F12 (1 : 1)  supplemented with 0.1 mM 2-mercaptoethanol (Sigma), 10% fetal bovine serum , and 1000 units/ml of recombinant murine LIF at 37 °C in atmosphere of 5% COμg/ml RNase A and propidium iodide for 15 min at 37 °C. Samples were analyzed by using flow cytometer Coulter Epics XL FACscan . Cell cycle phase distribution analysis was performed with MODFIT LT 3.0 software .For cytometric analysis of cell cycle distribution, cells were harvested, washed with phosphate-buffered saline (PBS) and incubated for 30 min in PBS containing 0.01% of saponin (Sigma) at room temperature. Cells were washed twice with PBS and incubated with 100 2. The resulting formazan precipitate was dissolved in DMSO (Sigma), gently pipetted and dispensed into 96-well plate. The absorbance was measured at 570 nm wavelength using Multiskan EX . Each experiment was repeated six times followed by calculation of the standard error of the mean.Evaluation of cell viability was performed by using colorimetric MTT-assay -2,5-diphenyltetrazolium bromide . The amount of formazan product correlates with the quantity of live cells. Cells were seeded in 24-well plates and were treated with rapamycin or pp242. After 24 h treatment MTT (0.5 mg/ml dissolved in PBS) was added, and cells were incubated for 1.5 h at 37 °C in 5% COCells were seeded and grown on gelatine-coated coverslips rinsed with cold PBS, fixed in 4% formaldehyde for 15 min at RT, and permeabilized with 0.25% Triton X-100 for 20 min. After three washes cells were incubated in blocking solution (5% BSA from Sigma in PBS) and then incubated with primary antibodies against phospho-S6 , LC3  at 4 °C overnight. After washing samples were incubated for 1 h with secondary antibodies AlexaFlour-488/568-conjugated goat anti-rabbit/mouse F(ab′)2 fragment . Primary and secondary antibodies were dissolved in PBS with 5% BSA and 0.1% Tween-20 (Sigma). Nuclei were stained by 5 min incubation with To-Pro3 (Invitrogen) or DAPI. Images were analyzed with the confocal microscope .β-glycerophosphate). Equal amounts of protein extracts were run on polyacrylamide gel electrophoresis, transferred to PVDF-FL membranes (Millipore) and blotted with primary antibodies, according to manufacturer's recommendations. HRP-conjugated goat anti-rabbit and rabbit anti-mouse antibodies  were used as secondary antibodies. Proteins on membranes were visualized by means of ECL . The relative band intensity was quantified using the Gel-pro Analyzer software . Pre-stained molecular weight protein standard (Sea-blue Plus 2) was used as protein marker .For immunoblotting, cell lysates were obtained by incubating cells in RIPA buffer containing PBS solution, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS (Sigma), protease and phosphatase inhibitors , 5 mM EGTA, 10 mM α-tubulin (Sigma #T516 8); Sox2 (#4900), GAPDH (#2118) pULk1 Ser555 (#5869), pUlkSer757 (#6888), p-mTOR Ser2448 (#6536), pSTAT3 Tyr705 (#9145), pS6 Ser235/236 (#4858), p4EBP1 Thr37/46 (#2855), PKB/AktSer308 (#13038), PKB/AktSer473 (#4060), pERKThr202/Tyr204 (#4370) and pTSC2 Thr1462 (#3617) .Primary antibody: Oct3/4 (#sc-5279) and Nanog (#sc-376915) from Santa Cruz Biotechnology ; μg RNA, using random hexaprimers  and M-MuLV Revertase . qPCR was performed using the Real-Time PCR Reagent kit with SYBR Green dye and the reference dye ROX , on the 7500 Real-Time PCR System . The reaction parameters were according to the manufacturer's instructions .For qRT-PCR and RT-PCR total cellular RNA was isolated using TRIzol (Invitrogen) according to a manufacturer's protocol. Reverse transcription was performed with 2 oct-3/4: 5′-CAAGTTGGCGTGGAGACT-3′/5′-TTCATGTCCTGGGACTCCTC-3′ klf-4: 5′-GACTAACCGTTGGCGTGAG-3′/5′-CGGGTTGTTACTGCTGCAAG-3′ nanog: 5′-GATGCAAGAACTCTCCTCCA-3′/5′-CAATGGATGCTGGGATACTC-3′ gapdh: 5′-TGTGTCCGTCGTGGATCTGA-3′/5′-TTGCTGTTGAAGTCGCAGGAG-3′ beta-actin: 5′-CCGTAAAGACCTCTATGCCAAC-3′/5′-ATGGAGCCACCGATCCACA-3′ fgf5: 5′-GCTGTGTCTCAGGGGATTGT-3′/5′-CACTCTCGGCCTGTCTTTTC-3′ T/brachyury: 5′-TGACCAAGAACGGCAGGAGG -3′/5′-TGGGTCTCGGGAAAGCAGTG-3′ mixL: 5′-CGTCTTCCGACAGACCATGT-3′/5′-GTTCTGGAACCACACCTGGAT-3′ afp: 5′-TGGTTACACGAGGAAAGCCC-3′/5′-AATGTCGGCCATTCCCTCACG-3′ mtor: 5′-CAAGATGCTTGGGACGGGT-3′/5′-CATTCCGGCTCTTCAGTCCA-3′.in vitro. Cells were washed with PBS and lysed in ice-cold buffer containing 50 mM HEPES pH 7.4, 5 mM CHAPS, 5 mM DTT, 0.5% NP-40 for 30 min. After centrifugation protein concentration was measured and 100 μg of proteins from each sample were used for the assay. Samples were incubated for 1 h at 37 °C in the following buffer: 20 mM HEPES pH 7.4, 0.1% CHAPS, 5 mM DTT, 2 mM EDTA and 40 mM Ac-DEVD-AMC (Sigma). The emission fluorescence at 460 nm produced by cleaved Ac-DEVD-AMC was measured after excitation at 360 nm using fluorimeter GloMax-Multi Jr .Fluorescent caspase-3 substrate  was used to measure caspase-3 activity