The cultivated plants were collected from nineteen local markets. The mean δ13C value for maize (grain) was −11.8±0.4 ‰ (n = 27). Leguminous cultigens  were characterized by significantly lower δ15N values and significantly higher %N than non-leguminous cultigens. Wild plants from thirteen sites were collected in the Moche River Valley area between sea level and ∼4,000 meters above sea level (masl). These sites were associated with mean annual precipitation ranging from 0 to 710 mm. Plants growing at low altitude sites receiving low amounts of precipitation were characterized by higher δ15N values than plants growing at higher altitudes and receiving higher amounts of precipitation, although this trend dissipated when altitude was >2,000 masl and MAP was >400 mm. For C3 plants, foliar δ13C was positively correlated with altitude and precipitation. This suggests that the influence of altitude may overshadow the influence of water availability on foliar δ13C values at this scale.The development of isotopic baselines for comparison with paleodietary data is crucial, but often overlooked. We review the factors affecting the carbon (δ This method is useful because the carbon and nitrogen isotopic compositions of consumer tissues are directly related to the carbon and nitrogen isotopic compositions of the foods consumed In all cases, interpretations of isotopic data depend on a thorough understanding of the range and variation in isotopic compositions of source materials Despite the fact that plants are known to be characterized by extremely variable carbon and nitrogen isotopic compositions 13C values for plants along a more limited altitudinal transect in Chile (Atacama Desert). A number of other studies have provided isotopic data on a much more limited scale from various sites in Argentina With respect to the Andean region of South America, the isotopic composition of plants is very poorly studied, both from ecological and paleodietary perspectives. The most comprehensive study of the latter type was conducted by Tieszen and Chapman The number of carbon and nitrogen isotopic studies in the Andean region has increased dramatically in the last ten years, facilitated by outstanding organic preservation in many areas. The majority of these studies have been conducted in Peru The purpose of this study is to systematically examine the carbon and nitrogen isotopic compositions of plants from the Moche River Valley in northern Peru collected at various altitudes from the coast to the highlands. These data provide a robust baseline for paleodietary, paleoecological, and related investigations in northern Peru that will utilize the carbon and nitrogen isotopic compositions of consumer tissues.The Andes are an area of marked environmental complexity and diversity. This diversity is driven largely by variation in altitude . As one lomas) lomas, riparian vegetation grows in the relatively lush river valleys that cut into the Andes, although the vast majority of this land is cultivated. Thickets of the leguminous algarroba tree regularly occur at low altitudes, and it is generally believed that much more extensive forests of these trees existed in the past The coastal region of Peru is dominated by the hyper-arid Peruvian desert. Cool sea-surface temperatures created by the northward flowing Peruvian Current, combined with a subtropical anticyclone, create remarkably stable and relatively mild temperatures along the roughly 2,000 km north-south extent of the Peruvian desert lomas may form within this zone Acacia, Polylepis) Immediately above the area of oceanic influence and up to an altitude of ∼1,800 m, the environment is cooler, although generally similar, in comparison to the coastal zone. Although mean annual precipitation increases, this zone can still be characterized as dry, with most locations receiving less than 400 mm of annual precipitation. In some circumstances, 13C) of terrestrial plants is the photosynthetic pathway utilized. Plants that fix carbon using the C3 pathway  are characterized by lower δ13C values (ca. −26 ‰) than plants utilizing the C4 (Hatch-Slack) pathway (ca. −12 ‰) 13C) is smaller in C4 plants than in C3 plants. In other words, C3 plants discriminate more strongly against the heavier isotope (13C) than C4 plants. The vast majority of C4 plants are tropical grasses, the most significant of which in New World archaeological contexts is maize (Zea mays), but also amaranth (Amaranthus caudatus). With respect to human diet, most wild C4 plants are not significant, and thus a large body of research has focused on assessing and quantifying the contribution of C4 cultigens  to the diet 3 and C4 plants. Referred to as CAM (Crassulacean acid metabolism) plants, these species fix carbon in a manner analogous to C4 plants overnight, but utilize the C3 photosynthetic pathway during the afternoon The most salient mechanism influencing the carbon isotopic composition  and photobiont or phycobiont . The carbon isotopic composition of lichens is determined largely by the type of photobiont involved. Lichens with green algae as the photobiont exhibit a wide range of carbon isotopic compositions (−35 to −17 ‰), while lichens with cyanobacteria as the photobiont tend to have higher, and a more restricted range of carbon isotopic compositions (−23 to −14 ‰) Additional plant groups that are not readily assigned into the aforementioned categories include mosses and lichens. Mosses, which are non-vascular plants, utilize the C13C values relative to canopy plants and plants growing in more open environments. Often referred to as the ‘canopy effect’, this is attributed to relatively 13C-depleted CO2 in the understory due to the utilization of recycled CO22] relative to the canopy Aside from the differences in carbon isotopic composition resulting from variable carbon fixation, a number of environmental factors have also been demonstrated to influence the carbon isotopic composition of plant tissues. For example, low-growing plants under dense forest cover tend to exhibit lower δ3 plants, with most studies finding little or no correlation between rainfall and/or water availability and plant δ13C for C4 plants 4 plant δ13C over a continental  rainfall gradient, although this relationship is atypical. It is believed that the relationship between aridity and plant δ13C is caused by increased stomatal closure when water availability is low, which is accompanied by decreased discrimination against 13C during photosynthesis and, in turn, comparatively less negative δ13C values Water availability has been observed to be negatively correlated with the carbon isotopic composition of plants 13C values. In a manner somewhat analogous to drought stress, salt stress induces increased stomatal closure, and therefore reduces discrimination against 13C by the plant Soil salinity has also been demonstrated to influence plant δ13C values have been found to increase with increasing altitude 13C and altitude is not entirely clear. Some have suggested exceptionally high carboxylation rates relative to stomatal conductance 13C. A very strong positive correlation has been observed between altitude and leaf mass per unit area A number of studies have found elevational gradients in plant carbon isotopic composition. Generally, foliar δ13C values, with higher irradiance being associated with less negative δ13C values in leaves. Such variation can occur within a single plant , and even along a single branch, with leaves growing in interior, shaded areas having lower δ13C values than leaves growing in exterior, exposed areas 13C associated with irradiance have been attributed to differences in intercellular CO2 concentration Irradiance has also been shown to influence foliar δ13C values of leaves, stems, roots, and other tissues 13C relative to non-photosynthetic tissues, typically by 2 to 4 ‰ 3 plants, with C4 plants often showing little variation between leaves and non-photosynthetic tissues, or leaves with relatively high δ13C values in some cases 13C. First, different tissues may contain variable proportions of molecules that are relatively enriched or depleted of 13C compared to total organic matter. Most notably, lipids 13C values, while the opposite is true for cellulose, sugars, and starches 13C of specific compounds  between different plant parts 13C variation. Damesin and Lelarge 13C are treated at length by Cernusak et al. Carbon isotopic composition is not necessarily equal among different plant parts. Numerous studies have observed variation in the δ13C within plant parts, over time. Specifically, emerging leaves, which are not yet photosynthetic and therefore more closely resemble other non-photosynthetic or heterotrophic plants parts, tend to have less negative δ13C values (by about 1 to 3 ‰) relative to fully emerged, photosynthetic leaves 13C values than those acquired heterotrophically, and this is likely partly responsible for the decrease in leaf δ13C over time In addition to variation among plant parts, a number of studies have found variation in δ13C values than terrestrial animals, except in cases where C4 plants dominate the diet of the latter. While this distinction holds in the vast majority of circumstances For the purpose of this paper, ‘marine plants’ refers specifically to macroalgae, or plants that are typically classified as kelps, seaweeds, and seagrasses. One of the most commonly reported distinctions in carbon isotopic composition is that marine animals tend to have higher δ3 and C4 plants, with two notable exceptions. Seagrasses (Zostera sp.), have extremely high δ13C values, typically higher than most terrestrial C4 plants  and HCO3−(aq) is a significant factor, as δ13C of HCO3−(aq) is ∼9 ‰ less negative than that of CO2(aq)2 further complicates matters 13C as it may differ due to variable water velocity 13C values, such as: salinity 2 concentration Marine plants are characterized by a high degree of variability in carbon isotopic composition. 4 plants . There i13C values (<−30 ‰). In general, the brown algae (kelps) have been noted to contribute significantly to nearshore ecosystems in terms of secondary production, with numerous studies examining the relative contributions of offshore phytoplankton and nearshore macroalgae Some red algae (Floridiophyceae) are characterized by consistently very low δ2, nitrogen is actively taken up from the soil in the vast majority of cases. The two most important nitrogenous species utilized by plants are nitrate (NO3−) and ammonium (NH4+). In general, nitrate is the most abundant form of mineralized nitrogen available to plants, but in some instances, such as waterlogged or acidic soils, ammonium may predominate 2), which is obtained by symbiotic bacteria residing in root nodules (rhizobia) Unlike carbon, which is obtained by plants as atmospheric CO2-fixation by plants (mostly members of Fabaceae), which are common in both wild and domestic contexts in many parts of the central Andes. Plants that utilize significant amounts of atmospheric N2 are characterized by comparatively low δ15N values, typically ∼0 ‰ 15N of atmospheric N2 is ∼0 ‰ 2-fixation is not associated with significant fractionation of 15N 3− and NH4+ tend to have δ15N values >0 ‰ 2-fixing plants have δ15N values that tend to be >0 ‰, although these are highly variable for a number of reasons as discussed in more detail below.There are two important aspects of variation in N source pertinent to the present study. The first relates to N15N variation is the uptake of fertilizer-derived N by plants. Animal fertilizers are characterized by extremely variable δ15N values depending on the relative proportions of N-bearing species in the fertilizer  15N values between 2 and 8 ‰ 15N values, often between 6 and 15 ‰ 15N values for animal fertilizers (>25 ‰) have been recorded for seabird guano 4+ volatilization. The addition of animal fertilizer N to the soil therefore adds an N source with an isotopic composition that is usually enriched in 15N relative to endogenous soil N. This results in higher δ15N values for plants growing in soils fertilized with animal waste than those plants growing in unfertilized soil or soils fertilized with chemical fertilizers The second potentially significant source-related cause of plant δ15N values 15N values due to the concentrated addition of animal waste, but studies have produced conflicting results, with some finding grazing to: increase plant δ15N values 15N values 15N values 15N in plant roots, but decrease δ15N in shoots Animal-derived N may be delivered to plants by means other than purposeful application of manures. Several studies have documented that the addition of N from animal carcasses  provide substantial quantities of N taken up by plants. These plants tend to be characterized by relatively high δ15N have been related to mycorrhizal  associations 15N values, Craine et al. 15N on the basis of mycorrhizal associations, with the following patterns : ericoid (−2 ‰), ectomycorrhizal (−3.2 ‰), arbuscular (−5.9 ‰). The comparatively low δ15N values of plants with mycorrhizal associations has been attributed to a fractionation of 8 to 10 ‰ against 15N during the transfer of N from fungi to plants Strong distinctions in plant δ15N values: (1) fractionations associated with NO3− assimilation in the root vs. shoot, (2) movement of nitrogenous compounds between nitrogen sources and sinks, (3) reliance on isotopically variable N sources as tissue forms over time.There are three main reasons that plants exhibit intraplant and temporal variation in their tissue δ3− and NH4+ are taken up by plant roots. NO3− can be immediately assimilated into organic N in the root, or it may be routed to the shoot and assimilated there. The assimilation of NO3− into organic N is associated with a fractionation of 15N of up to −20 ‰ 3− that is moved to the shoot has already been exposed to some fractionation associated with assimilation and is enriched in 15N compared to the NO3− that was assimilated in the root. On this basis, it is expected that shoots will have higher δ15N values than roots in plants fed with NO3−4+ is assimilated only in the root, plants with NH4+ as their primary N source are not expected to have significant root/shoot variation in δ15N Both NO15N in relation to sinks , which has been observed in several studies As plants grow they accumulate N in certain tissues (sources) and, over time, move this N to other tissues (sinks). In many species, annuals in particular, large portions of the plant’s resources are allocated to grain production or flowering. In these cases, significant portions of leaf and/or stem N is mobilized and allocated to the fruits, grains, or flowers 4+, NO3−) and the nitrogen isotopic composition of fertilizer-derived N changes over time as various soil processes  occur. The nature of this variation is complex and will depend on the type of fertilizer applied In agricultural settings, the variation within a plant over time may become particularly complex due to the application of nitrogenous fertilizers. The availability of different N-bearing species from the fertilizer  15N values and local precipitation and/or water availability. These effects have been demonstrated at regional or continental 15N values in herbivore tissues may be the product of physiological processes within the animal related to drought stress 15N values and plant δ15N values, while providing no support for the ‘physiological stress hypothesis’ Plant δ15N values appears to be extremely complex, with numerous variables contributing to the pattern. Several authors, including Handley et al. 15N, 15N enrichment will be favored for any process that increases the flux of organic matter to mineral N, or decreases the flux of mineral N into organic matter 3 volatilization would cause an overall enrichment in 15N of the soil-plant system.The nature of the relationship between rainfall and plant δ15N values are strongly influenced by the forms and isotopic composition of available N 3− relative to recycled NH4+ will strongly influence the δ15N of marine producers, including macroalgae. Systems that are nutrient poor (oligotrophic) tend to be more dependent on recycled NH4+, and systems that are nutrient rich (eutrophic) tend to be more dependent on upwelled NO3−. This results in nutrient-rich, upwelling systems being enriched in 15N relative to oligotrophic systems In comparison to terrestrial plants, the factors affecting the nitrogen isotopic composition of marine plants have not been investigated intensively other than the influence of anthropogenic nitrogen. As is the case with terrestrial plants, marine plant δWild plants were collected between 2011/07/18 and 2011/08/03. We used regional ecological classifications defined by Tosi Cultigens (edible portions) were collected from local markets between 2008/10/08 and 2008/11/09 . Plants For both wild plants and cultigens, geospatial data were recorded using a Garmin® Oregon® 450 portable GPS unit . After collection, plants were air-dried on site. Prior to shipping, plants were dried with a Salton® DH−1171 food dehydrator . Plants were separated according to tissue . For grasses, all aboveground tissues were considered to be leaf except where significant stem development was present, in which case, leaf and stem were differentiated. All geospatial data associated with these sampling sites are available as a Google Earth.kmz file in the Supporting Information .Plants were not sampled from privately-held land or from protected areas. Endangered or protected species were not sampled. Plant materials were imported under permit #2011−03853 from the Canadian Food Inspection Agency. No additional specific permissions were required for these activities.Samples were prepared according to Szpak et al. 13C and δ15N) and elemental compositions (%C and %N) were determined using a Delta V isotope ratio mass spectrometer  coupled to an elemental analyzer , located in the Laboratory for Stable Isotope Science (LSIS) at the University of Western Ontario . For samples with <2% N, nitrogen isotopic compositions were determined separately, with excess CO2 being removed with a Carbo-Sorb trap  prior to isotopic analysis.Isotopic  and USGS41 . In addition to USGS40 and USGS41, internal (keratin) and international  standard reference materials were analyzed to monitor analytical precision and accuracy. A δ13C value of −24.03±0.14 ‰ was obtained for 81 analyses of the internal keratin standard, which compared well with its average value of −24.04 ‰. A δ13C value of −10.46±0.09 ‰ was obtained for 46 analyses of IAEA-CH-6, which compared well with its accepted value of −10.45 ‰. Sample reproducibility was ±0.10 ‰ for δ13C and ±0.50% for %C (50 replicates). A δ15N value of 6.37±0.13 ‰ was obtained for 172 analyses of an internal keratin standard, which compared well with its average value of 6.36 ‰. A δ15N value of 20.3±0.4 ‰ was obtained for 76 analyses of IAEA-N-2, which compared well with its accepted value of 20.3 ‰. Sample reproducibility was ±0.14 ‰ for δ15N and ±0.10% for %N (84 replicates).Sample δ3 and C4 categories. For comparisons among plant functional types, and sampling sites, foliar tissue was used since other tissues were not as extensively sampled.Plants were grouped into the following major functional categories for analysis: herb/shrub, tree, grass/sedge, vine. Plants that are invasive and/or introduced species were included in the calculation of means for particular sites since their isotopic compositions should still be impacted by the same environmental factors as other plants. For all statistical analyses of carbon isotopic composition, grass/sedge and herb/shrub were further separated into Cρ). One-way analysis of variance (ANOVA) followed by either a Tukey’s HSD test (if variance was homoscedastic) or a Dunnett’s T3 test (if variance was not homoscedastic) was used to compare means. All statistical analyses and regressions were performed in SPSS 16 for Windows.Correlations between foliar isotopic compositions and environmental parameters  were assessed using Spearman’s rank correlation coefficient  to −25.6±1.9 ‰ (mashua). The mean δ13C value for maize, which was the only C4 plant examined, was −11.8±0.4 ‰. Mean δ15N values for cultigens were typically more variable than δ13C values, ranging from −0.2±0.4 ‰ (Phaseolus lunatus) to 7.9±1.3 ‰ (quinoa).The carbon and nitrogen isotopic compositions were analyzed for a total of 85 cultigen samples from eleven species. Carbon and nitrogen isotopic compositions for cultigens are presented in 13C among cultigens (Fp = 0.93), but there were for δ15N (maize included) (Fp<0.001). Results of post-hoc Dunnett’s T3 test for δ15N differences among individual cultigen species are presented in 15N values than non-leguminous species .When maize is excluded, there were no significant differences in δ species ; collectFp<0.001).Cultigen N content is presented in 4 plant taxa was generally higher at lower altitude sites receiving low amounts of rainfall. This fits with what is known about the global distribution of C4 plants A total of 139 species were sampled primarily from ten sites distributed along an altitudinal transect from 10 to 4,070 masl. The number of taxa sampled and environmental variables for each of the sampling locations are presented in 13C values for C3 plants ranged from −31.9 to −22.5 ‰, with a mean value of −27.6±1.9 ‰ (n = 122). Foliar δ13C values for C4 plants ranged from −15.6 to −11.6 ‰, with a mean value of −13.5±1.0 ‰ (n = 17). Foliar δ15N values for C3 plants ranged from −4.1 to 13.0‰, with a mean value of 3.7±4.0 ‰. Foliar δ15N values for C4 plants ranged from −3.2 to 15.0 ‰, with a mean value of 5.5±5.7 ‰. The single lichen analyzed (Usnea andina) was characterized by a δ13C value intermediate between C3 and C4 plants (−20.5 ‰) and a very low δ15N value (−6.5 ‰), consistent with previously reported results for lichens The carbon and nitrogen isotopic compositions were measured for all 139 species. Foliar tissue was analyzed from all species, and additional tissues analyzed included: 112 stems, 28 roots, 51 flowers, and 62 seeds. Carbon and nitrogen isotopic compositions for wild plants are presented in 15N among plant functional groups (Fp = 0.15). Foliar δ13C differed significantly among plant functional groups (Fp<0.001), although this was driven by differences between C3 and C4 groups; there were no significant differences in foliar δ13C between plant functional groups within C3 and C4 groups (There were no significant differences in foliar δ4 groups .15N (15N between tissues (Δ15N) being highly variable: Δ15Nstem−leaf = −0.3±2.3 ‰, Δ15Nroot−leaf = 0.4±3.1 ‰, Δ15Nflower−leaf = 0.5±1.4 ‰, Δ15Nseed−leaf = 0.5±1.7 ‰. Conversely, foliar tissue was typically characterized by lower δ13C values than all other tissues analyzed , there was no clear pattern of intraplant variation in δ13C: Δ13Cstem−leaf = 0.0±0.8 ‰, Δ13Croot−leaf = 0.5±0.7 ‰, Δ13Cflower−leaf = −0.3±0.6 ‰, Δ13Cseed−leaf = −0.2±1.3 ‰.There was no clear pattern of intraplant variation in δ15N  with difanalyzed , and intAcacia trees and shrubs alone, foliar δ15N values ranged from −1.0 to 9.6 ‰, suggesting that some species are not engaged in active N2-fixation. While wild legumes were characterized by lower foliar δ15N values relative to non-legumes , this difference was not statistically significant (Fp = 0.18).Foliar nitrogen isotopic compositions for wild legumes (Fabaceae) were highly variable, ranging from −1.4 to 9.6 ‰. Among 3 plant foliar δ13C and δ15N values for sampling locations with ≥5 species sampled are presented in 15N values at low altitude sites were 2 to 8 ‰ higher than mean foliar δ15N values at high altitude sites. Foliar δ15N was negatively correlated with mean annual precipitation  and altitude . Foliar δ13C was positively correlated with mean annual precipitation  and altitude . For comparative purposes, mean plant δ13C values for sites sampled along an altitudinal transect in northern Chile are presented in Mean wild C13C and δ15N values for marine plants are presented in 13C values for marine plants ranged from −18.7±0.7 ‰ (Gymnogongrus furcellatus) to −14.2±1.2 ‰ (Grateloupia doryphora). Mean δ15N values for marine plants ranged from 2.5±0.9 ‰ (Gymnogongrus furcellatus) to 7.8±0.1 ‰ (Cryptopleura cryptoneuron). Overall, marine plants were characterized by δ13C values that were intermediate between C3 and C4 plant isotopic compositions, although more similar to the latter. In comparison to wild plants growing at the three sites located closest to the coast, marine plants were not characterized by significantly higher δ15N values when the plants from the three terrestrial sites are treated separately (Fp = 0.71) or grouped together (Fp = 0.91).The carbon and nitrogen isotopic compositions were determined for a total of 25 marine plant samples from five species. Mean δ13C than wild C4 plants , similar to previously determined values for other parts of the world 13C value of −10.3 ‰  or by animal manures  that would not have been available in the region prior to the arrival of the Spanish. The same is true for nitrogen isotopic data obtained from modern agricultural plants globally, and as a general rule, the limitations of these data must be recognized. Nevertheless, some patterns are likely to be broadly applicable.For the most part, the δ15N values in legumes in that region is tenuous. Where the potential effects of nitrogenous fertilizers on legume δ15N values are unknown  rather than any biochemical or physiological process specific to any particular plant species. Ultimately, the best source of baseline isotopic data for paleodietary studies may be from archaeobotanical remains Aside from the differences in δThere were no clear distinctions between different plant functional groups  with respect to either carbon or nitrogen isotopic compositions. While some systematic variation may be expected due to variable nitrogen acquisition strategies (e.g. rooting depth) or differential distribution of biomolecules with distinct isotopic compositions, the diverse range of environmental conditions from which plants were sampled likely served to blur any isotopic distinctions between functional groups. Moreover, the sample sizes for different plant functional groups within any one site were too small for meaningful comparisons to be made.15N with respect to leguminous trees and shrubs, with some species having foliar δ15N values close to 0 ‰, and others having relatively high δ15N values. Previous studies have similarly found conflicting patterns of relatively high and low δ15N values in leguminous trees. Codron et al. 2-fixation among leguminous trees in an arid region of southern Africa, with trees growing at the most arid sites showing no evidence of N2-fixation. Fruit-bearing trees of the genus Prosopsis  are suggested to have been an important food source for various groups in the Andean region 15N values typical of N2-fixing trees (ca. 0 ‰) in Prosopis pallida from the Sechura Desert of northern Peru. Conversely, on the basis of the isotopic data recorded in this study for leguminous trees in the Moche River Valley, the assumption that Prosopis would be characterized by significantly lower δ15N values relative to other plants is tenuous. Given the potential importance of these foods in the diet, a more extensive study of the nitrogen isotopic composition of central Andean leguminous trees would be beneficial.There was no consistent pattern in plant δ3− as the N source 15N may vary considerably among tissues due to biochemical processes associated with growth and senescence over time 15N likely relates to a number of factors, including: variable reliance on different N sources  by different plant taxa and between sampling locations, differences in plant life cycles between different taxa, and spatial variation in the influence of environmental factors on the isotopic composition of source N.Plant nitrogen isotopic composition did not systematically vary between different tissues sampled. On the basis of hydroponic studies, significant intraplant variation (between roots and shoots) is only expected when plants are fed with NO13C than other tissues  located ∼6 km from the coast, had grain δ15N values of 6.3±0.3 ‰ 15N values would be expected to be more limited , but with little difference in elevation between sites. Conversely, we sampled along a more restricted rainfall gradient (∼700 mm), but a very large altitudinal gradient . Increased altitude and increased rainfall have opposing effects on foliar δ13C values, and the results of this study suggest the predominance of altitudinal effects on foliar carbon isotopic compositions in northern Peru. A similar pattern was observed along a comparable altitudinal gradient in northern Chile .Variation in plant isotopic compositions along environmental gradients is particularly important with respect to the reconstruction of the diet of humans and animals using isotopic data. While the majority of wild plants analyzed in this study would not have been consumed by humans, the results are very relevant to the reconstruction of animal management practices. There is considerable debate in the Andean region with respect to the herding practices of South American camelids , and whether or not animals recovered from coastal sites were raised locally, or imported from elsewhere 13C values are more difficult to evaluate. While the positive linear relationship between altitude and foliar δ13C is strong, the relative distribution of C3 and C4 plants would serve to counter these effects. Because there will be proportionately more C4 plants at dry, low altitude sites relative to moister, high altitude sites, the average δ13C value of available forage would still be higher at low altitude sites. Thus, markedly higher δ13C and δ15N values observed in some camelids from low altitude sites The potential consequences of altitudinal variation in plant δ13C values intermediate between C3 and C4 plants, with δ15N values comparable to terrestrial plants growing on the coast. DeNiro 13C and δ15N values in coastal Peruvian camelids. While the number of macroalgal species sampled in this study is not extensive, the isotopic data presented here are not consistent with this explanation. With the exception of instances in which marine plants grow in areas of exceptionally high influence of marine bird and/or mammalian excreta 15N values to be higher than the δ15N values of plants growing along the arid coast of Peru.Marine algae are known to have been an important dietary resource for many groups of people in the coastal regions of Peru and Chile 13C value of −11.8±0.4‰, which suggests that a δ13C value (adjusted for the Suess Effect) of ca. −10.3 ‰ would be appropriate for paleodietary models in the region. Leguminous cultigens were characterized by significantly lower δ15N values and higher N contents than non-leguminous cultigens; this distinction was not as clear for wild legumes. Marine plants were characterized by δ13C values intermediate between wild terrestrial C3 and C4 vegetation and δ15N values that were very similar to terrestrial plants growing at low altitudes. C4 plants were generally more abundant at lower altitude sites. Carbon and nitrogen isotopic compositions of wild plants were strongly influenced by local environmental factors. Foliar δ13C was positively correlated with altitude and negatively correlated with mean annual precipitation. Foliar δ15N was negatively correlated with altitude and mean annual precipitation.Maize from the study area has a mean δWhile the last twenty years have seen a proliferation of studies utilizing the isotopic analysis of archaeological materials for the purpose of reconstructing diet, the development of isotopic baselines for interpreting such data has lagged behind these investigations. This hampers our ability to realize the full potential of isotopic data. This study begins to fill part of that gap by providing an initial understanding of the baseline isotopic variation in plants from northern Peru. Further studies of this nature are required to better understand baseline isotopic variation in other regions.Dataset S1Sampling site locations for wild and market plants. This.kmz file can be executed in Google Earth (http://www.earth.google.com)(KML)Click here for additional data file.Table S1Isotopic and elemental data for all cultigens analyzed.(XLS)Click here for additional data file.


N–terminal domain of the human ether–a–go–go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N–terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N–terminal 135 residues of hERG contains a previously described Per–Arnt–Sim (PAS) domain (residues 26–135) as well as an amphipathic α–helix (residues 13–23) and an initial unstructured segment (residues 2–9). Deletion of residues 2–25, only the unstructured segment (residues 2–9) or replacement of the α–helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α–helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N–terminal α–helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.The cytoplasmic  The fifth and sixth transmembrane domains along with an intervening pore helix from each of the four subunits surrounds the ion conducting pore C–terminal to the pore domain is thought to contribute to the stabilization of the tetrameric structure N–terminus of each subunit contains a Per–Arnt–Sim (PAS) domain (residues 26–135) The human ether–a–go–go related gene  is able to restore deactivation gating in N–terminally truncated hERG N–terminal tail that precedes the PAS domain (residues 1–25) also result in significantly faster rates of deactivation N–terminal 16 residues can slow the deactivation kinetics of channels with most of the N–terminus deleted  or replacement of the amphipathic α–helical region with a flexible linker resulted in faster deactivation, suggesting that both regions are necessary but neither is sufficient to permit normal deactivation. Alanine scanning of the N–terminal tail indicated that residues R5 and G6 are involved in critical interactions that stabilize the open state of the channel. Although the majority of alanine mutations in the amphipathic α–helical region did not have a significant effect on the rate of deactivation, both I19A and R20A showed an enhanced rate of deactivation. This suggests that the α–helix may act as a spacer, rather than being involved in critical specific interactions with other domains of the channel.To clarify the role of the N–terminal cleavable glutathione–S–transferase (GST) fusion in E. coli C41 strain after overnight induction at 20°C. A freeze/thaw method was used to lyse the cells with 20 mM Tris buffer containing 5 mM 2–mercaptoethanol, 0.1% v/v Tween 20, and 150 mM NaCl. The lysate was incubated with glutathione beads  for 3 h and the protein eluted by TEV protease digestion overnight at 4°C. The protein was concentrated and passed through a Superdex 75 column , equilibrated in 10 mM HEPES pH 6.9, 150 mM NaCl, 5 mM N–octyl–D–glucoside (OG)  and 3 mM tris(2–carboxyethyl)phosphine (TCEP). The purified PAS domain eluted as a single peak at the expected molecular size for a monomer  was expressed as an 13C/15N PAS domain protein in solution containing 10 mM HEPES, 3 mM TCEP, 5 mM OG, and 7% D2O, at pH 6.9. All NMR experiments were performed on a Bruker Avance II 900 MHz NMR spectrometer at 298 K. 2D 1H–15N HSQC, 3D 1H–15N NOESY and 3D 1H–13C NOESY data were acquired using traditional methods while 3D HNCO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, C(CO)NH, H(CCO)NH and HCCH–TOCSY data were acquired using a non–uniform sampling method and maximum entropy reconstruction 2O before acquiring 3D HCCH–TOCSY and 1H–13C NOESY NMR spectra.The NMR sample consisted of 0.21 mM 1H–15N and 1H–13C NOESY data, 24 hydrogen bond constraints were derived from the 1H–13C NOESY data (based on amide protons that were still observable afte exchange of the sample into D2O buffer), and 178 dihedral angle constraints  were derived from TALOS All NMR spectra were analysed using XEASY3 Xenopus laevis β–globin gene . Mutagenesis was carried out using the Quickchange mutagenesis method  and confirmed by DNA sequencing. Wild–type (WT) and mutant channel cDNAs were linearized with BamHI and cRNA transcribed with T7 RNA polymerase using the mMessage mMachine kit .HERG cDNA  was subcloned into a pBluescript vector containing the 5′ untranslated region (UTR) and 3′ UTR of the Xenopus laevis oocytes were prepared as previously described 2 1.8, MgCl2 1.0 and HEPES 5.0) supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline and 10 µg mL−1 gentamicin, adjusted to pH 7.5 with NaOH and incubated at 18°C. All experiments were approved by the Garvan/St Vincent's Animal Ethics Committee .Xenopus laevis oocytes were injected with 5–10 ng cRNA and incubated at 18°C for 24–48 h prior to electrophysiological recordings. All experiments were undertaken at room temperature (21–22°C). Two–electrode, voltage–clamp experiments were performed using a Geneclamp 500B amplifier . Glass microelectrodes had tip resistances of 0.3–1.0 MΩ when filled with 3 M KCl. Oocytes were perfused with ND96 solution (see above). In all protocols a step depolarization of +20 mV from the holding potential of −90 mV was applied at the start of each sweep to enable off–line leak–current subtraction. We assumed that the current leakage was linear in the voltage range −160 to +40 mV. Data acquisition and analysis were performed using pCLAMP software  and Excel software . All parameter values were estimated as mean ± standard error of the mean (SEM) for n experiments, where n denotes the number of different oocytes studied for each construct.Imax) and fitted with a Boltzmann expression:maxg/g is the relative conductance, V0.5 is the half–activation voltage, Vt is the test potential and k is the ‘slope factor’. Alternatively, the data were fitted with the thermodynamic form of the Boltzmann expression:G0 is the work done at 0 mV, zg is the effective number of gating charges moving across the membrane electric field, E. F, is Faraday's constant, R is the universal gas constant and T is the absolute temperature. Equations (1) and (2) are equivalent, however from Equation (2) we can calculate the effect of mutations on changes in the chemical potential (ΔG0) and electrostatic potential (–zgEF) that drives activation and deactivation of the channel.Isochronal activation curves were measured using standard tail current analysis zgEF) as defined in Equation (2).To measure rates of deactivation, oocytes were depolarized from a holding potential of −90 mV to +40 mV for 1 s to fully activate the channels; they were then repolarized to potentials in the range −50 to −160 mV. A double exponential function was fitted to the decaying portion of tail currents. In order to compare rates of deactivation for different mutants at comparable driving forces, the voltages in the range −60 to −160 mV were converted to electrochemical driving forces  were 0.40 and 1.10 Å, respectively.The solution structure of hERG 1-135 domain was well folded with dispersed peaks in the 15N-HSQC . More th15N-HSQC  to be un channel . The RMSN–terminal tail structures are shown in Statistics highlighting the extremely high precision and stereochemical quality of the ensemble of hERG PAS domain and The NMR solution structure for the segment from residues S26 to K135 was almost identical to the crystal structure of the same region . The aveN–terminal tail, it was consistent with the secondary structure prediction using the NMR chemical shifts and inter–residue NOE constraints (In addition to the structure of the PAS domain (residues S26 to K135), the NMR solution structure revealed a randomly distributed α–helical tail  revealed that it was amphipathic with positively charged residues (R20 and K21), negatively charged residues (D16 and E23), and polar residues (T13 and T17), located on the same side of the helix  were located on the opposite face of the helix. Residues 1–9 are disordered with no clearly defined structure, while residues 10–12 adopted a turn conformation.Closer examination of the he helix , while nN–terminal tail, we compared the effects of deleting only the initial portion of the N–terminus (Δ2–9), with deletion of the entire N–terminal tail (Δ2–25), or replacement of the α–helix with a flexible linker , 8.7±0.3 ms  and 7.5±0.2 ms  compared to 33.7±1.0 ms .0.5) of Δ2–9 channels  was shifted in the depolarizing direction compared to WT hERG , without any change in slope. A small but statistically significant shift in activation V0.5 was also observed for GGSmut  channels, while Δ2–25 channels were similar to WT hERG (0) and electrostatic (–zgEF) potential that drives activation was calculated by fitting the activation data with a Boltzmann function in the form of Equation 2  than WT hERG . To compensate for changes in electrochemical driving force, the rates of deactivation calculated at voltages between −50 mV to −160 mV were plotted against the electrochemical potential for deactivation , the I–V relationship of peak tail currents at −70 mV plotted against the preceding voltage step . The res WT hERG . The chetivation . After cN–terminal tail, native residues from P2–E23 were individually replaced with alanine, or with valine in the case of A9. Measured rates for fast (τfast) and slow (τslow) components of deactivation, in addition to the relative contributions of these components are given in –1  for R5A and 12.7±1.3 ms (n = 7) for G6A compared with 28.5±2.8 ms (n = 11) for WT , alanine mutations I19A and R20A gave channels with faster rates of deactivation when compared with WT hERG; τfast at −30 kJmol–1 was 19.2±1.3 ms (n = 13) for I19A and 20.4±1.0 ms (n = 20) for R20A . Somewhat surprisingly, two mutations in the α –helical region, i.e., T13A  and D16A  exhibited slower deactivation rates than WT . Interestingly, the four residues with altered deactivation rates  lay on one face of the amphipathic helix  appeared to adopt a turn conformation but there were insufficient NOE constraints to be able to define it as an extension of the α–helix.The solution structure of hERG PAS domain determined in this study was found to be very similar to the crystal structure determined previously by Morais Cabral et al. N–terminal tail  were both required, but neither alone was sufficient, for the normal slow deactivation kinetics of WT hERG channels.Our functional, electrophysiological data indicated that removal of either the initial unstructured segment (Δ2–9) or replacement of the α–helix with a flexible linker (GGS mutant) both produced similar phenotypes, i.e. markedly faster rates of deactivation. This is essentially the same phenotype produced by deletion of the entire l Δ2–25, . These dN–terminal segment (P2 – N12) identified R5 and G6 as the most critical residues for deactivation. Both R5A and G6A mutants had enhanced rates of deactivation that could easily explain the faster deactivation observed with deletion of residues 2–9. Within the α–helical segment two alanine mutants resulted in enhanced rates of deactivation (I19A and R20A). These mutants resulted in a smaller perturbation to deactivation than R5A and G6A, nevertheless the combined effect of all four residues may explain why deletion of residues 2–25 caused a greater enhancement of the rate of deactivation than did deletion of residues 2–9.Alanine scanning mutagenesis of the initial unstructured i.e., T13A and D16A in the α–helix and P10A in the linker between the α–helix and the initial unstructured segment. It is possible that these three residues are important for ensuring that the tail does not bind too tightly to the open state of the channel. It is noted that T13 and D16 lie on the same side of the α–helix as I19 and R20, which are the only other residues in the α–helix that significantly perturbed deactivation. This suggests that this surface of the α–helix is involved in protein–protein interactions that affect the rate of deactivation. However, when we mapped the functional effect of alanine mutants onto the structure of the N–terminal α–helix, the residues with perturbed function did not lie along the entire length of the α–helix  is important for its function and/or regulation.It is important to recall that the structure we have solved is an isolated domain. It is possible that the N–terminal tail  of the channel where the PAS/N–terminal α–helix and the flexible N–terminal domains bind remain to be determined. Two obvious candidates are the S4–S5 linker N–terminal α–helix bind to the cyclic–nucleotide binding domain will require expression and purification of the cyclic–nucleotide binding domain.Deletion of the l Δ2–25, , the entl Δ2–25,  or majors Δ2–354  all resuFigure S115N-HSQC spectrum of hERG N–terminal domain (M1-K135). The spectrum was collected at 25°C on a Bruker 900 MHz Avance II spectrometer fitted with a cryoprobe. Backbone amide proton resonances are identified by their residue numbers. Sidechain N-H resonances are indicated by an sc suffix.(EPS)Click here for additional data file.Figure S2Amino acid sequence of hERG PAS domain and overview of NMR data. The secondary structure elements are labelled according to the hERG PAS domain PDB structure (2L0W). Hydrogen bonds constraints used in the structure calculation are indicated as black circles. Chemical shift index (CSI) prediction of the secondary structure is shown immediately above the amino acid sequence. Thick and thin bars indicate strong and weak NOE cross-peaks intensities for the sequential proton–proton NOE connectivities . The observed medium-range NOEs dNN, dαN, dαN, dαβ and dαN and are indicated by lines connecting the two residues that are related by the NOE.(EPS)Click here for additional data file.Figure S3Relative amplitudes of τfast and τslow that comprise deactivation rates. Tail currents recorded over a range of potentials (Vm) following a test pulse to +40 mV are fit with a double exponential function (see tion see . Relativ(EPS)Click here for additional data file.Table S1−1, for WT and all mutants investigated in this study.Time constants for fast component of deactivation, at −30 kJ mol(DOC)Click here for additional data file.Table S2Time constants for fast and slow components of deactivation and ratio of fast and slow components of deactivation, at -120 mV, for WT and all mutants investigated in this study.(DOC)Click here for additional data file.


Fibrillar aggregates involved in neurodegenerative diseases have the ability to spread from one cell to another in a prion-like manner. The underlying molecular mechanisms, in particular the binding mode of the fibrils to cell membranes, are poorly understood. In this work we decipher the modality by which aggregates bind to the cellular membrane, one of the obligatory steps of the propagation cycle. By characterizing the binding properties of aggregates made of α-synuclein or huntingtin exon 1 protein displaying similar composition and structure but different lengths to mammalian cells we demonstrate that in both cases aggregates bind laterally to the cellular membrane, with aggregates extremities displaying little or no role in membrane binding. Lateral binding to artificial liposomes was also observed by transmission electron microscopy. In addition we show that although α-synuclein and huntingtin exon 1 fibrils bind both laterally to the cellular membrane, their mechanisms of interaction differ. Our findings have important implications for the development of future therapeutic tools that aim to block protein aggregates propagation in the brain. The aggregation of proteins into fibrillar high molecular-weight species is the hallmark of human degenerative diseases, including Alzheimer’s, Parkinson’s, or Huntington’s234567per se, of two types of β-2-microglobulin fibrils displaying different lengths, Xue and Radford proposed that the binding was mediated mainly through fibrils ends11112122242520The docking of extra-cellular aggregates to the plasma membrane of naïve neurons is a key step of the vicious propagation-amplification cycle. Next, the aggregates are internalized and reach the cytoplasm where they can recruit the otherwise soluble like endogenous protein thus elongating/amplifying. The circle completes with the release of the amplified aggregates into the extracellular milieu, where they can target naïve cells. Very little is known about the molecular basis by which protein fibrillar aggregates bind to the plasma membrane. Different mechanisms have been proposed. The binding could be passive1213101618Here we specifically assessed this issue by studying the binding of amyloid fibrils displaying similar composition and structure but different lengths to artificial liposomes and mammalian cells. We used two different and complementary models, namely α-synuclein (αSyn) involved in Parkinson’s disease, and huntingtin protein exon 1 (HTTExon1) which aggregation is responsible of Huntington’s disease. We observed using electron microscopy massive lateral binding of αSyn and HTTExon1 fibrils to unilamellar vesicles made of brain lipids. We could demonstrate that at identical monomer concentrations, long fibrils made of either αSyn or HTTExon1 bind to the same extent or even better both to artificial liposomes and cells than their short counterparts, thus demonstrating a preeminent role of fibrils surface in the binding. We also show that although αSyn and HTTExon1 fibrils bind both laterally to the cellular membrane, their mechanisms of interaction differ.To determine if amyloid fibrils bind to the cellular membrane laterally or through their extremities, we decided to characterize the binding of fibrils identical in everything but their length . ConsideWe used sonication to obtain fibrils of different lengths. We assembled αSyn and HTTExon1 into fibrils as described in the Methods section. Each sample was then divided in two: the first one was sonicated for only 10 min, to disperse the aggregates and generate individual long fibrils; the second sample was sonicated for 16 h to break the long fibrils into short ones. Representative images and the distributions of fibril lengths obtained by quantitative electron microscopy for long and short fibrils of each protein are shown in Fibrils with different conformations are susceptible to bind differently to the membraneWe conclude from these physico-chemical assessments that we generated αSyn and HTTExon1 fibrils of distinct lengths but identical structures.We used unilamellar vesicles composed of total brain lipids to image the binding of short and long αSyn and HTTExon1 fibrils to lipid membranes. αSyn and HTTExon1 long and short fibrils (50 μM) were incubated for 1 hour at room temperature in Dulbecco’s modified Eagle’s medium containing unilamellar vesicles made of brain lipids. Representative images obtained by electron microscopy are displayed in We conclude from these experiments that both αSyn and HTTExon1 fibrils bind laterally to artificial liposomes.Artificial liposomes cannot recapitulate the properties of cellular membranes. We therefore assessed the binding of short and long αSyn and HTTExon1 fibrils to cultured cells.We first controlled that neither the length nor the structure of short and long αSyn and HTTExon1 fibrils were modified upon incubation in a cellular environment. αSyn and HTTExon1 long and short fibrils (50 μM) were incubated in Dulbecco’s modified Eagle’s medium at room temperature with unilamellar vesicles made of brain lipids. Electron microscopy demonstrated that each type of fibrils retain their morphology  and lengNext we compared the affinity of long and short αSyn and HTTExon1 fibrils for the cellular membrane. αSyn and HTTExon1 fibrils were labelled with Atto 488 before sonication, to ensure that long and short fibrils have identical monomer concentration and fluorescence intensity. We controlled that the labelling altered neither the morphology nor the lengths of the fibrils.KD for the binding of long αSyn fibrils to Neuro 2A cells of 0.8 ± 0.2 μM   and purified as describedE. coli strain BL21(DE3)  and purified as describedRecombinant human wild-type αSyn was expressed in For fibril formation, αSyn was incubated in assembly buffer A  at 37 °C under continuous shaking in an Eppendorf Thermomixer set at 600 rpm; HTTExon1 was incubated in assembly buffer B  at 37 °C without agitation. Long and short fibrils were obtained by sonicating the fibrils with an ultrasond sonicator (Hielsher) set with an amplitude of 75 and a cycle of 0.5 s for 10 min or 16 h, respectively.For cell sorting experiments, αSyn and HTTExon1 fibrils were first washed in PBS and then labelled by the addition of 2 molar excess of the aminoreactive fluorescent dye Atto 488 N-hydroxysuccinimide ester (ATTO-Tec GmbH) before the sonication step. Labelling was performed following the manufacturer’s recommendation. Unreacted dye was removed by two cycles of sedimentation and suspension of the fibrils in PBS.Porcine brain lipid extract  was purchased from Avanti Polar Lipids . Unilamellar vesicles were prepared as previously describedProtein assemblies were examined by transmission electron microscopy (TEM) in a Jeol 1400 transmission electron microscope  following adsorption onto carbon-coated 200 mesh grids and negative staining with 1% uranyl acetate. The images were recorded with a Gatan Orius CCD camera . Length measurements were performed on individual fibrils with the software ImageJ . For each fibril type  lengths distribution were obtained by measuring the lengths of at least 1000 fibrils, using more than 20 negatively stained electron micrographs obtained from 3 to 10 independent experiments.αSyn (10 μM monomer concentration) and HTTExon1 (50 μM monomer concentration) long and short fibrils were incubated with Thioflavin T (10 μM). Thioflavin T fluorescence was recorded with a Cary Eclipse spectrofluorimeter  using excitation and emission wavelengths set at 440 and 480 nm, respectively.−1). Aliquots (10 μl) were removed at different time intervals following addition of the protease, immediately mixed with denaturing buffer  to arrest immediately the cleavage reaction, incubated for exactly 5 min at 90 °C and frozen at −80 °C until analysis on Tris-glycine SDS-polyacrylamide (15%) gel electrophoresis (PAGE). For HTTExon1 aliquots were taken for solubilization. 40 μl of the reaction mixture were withdrawn and immediately diluted in 200 μl of TFA:HFIP 1:1. Fibril solubilization was allowed for 2 hours. The solvent was then removed under a stream of nitrogen for 30 min. After removal from the gas flow the samples were immediately resuspended in 40 μl NaOH 0.1M and then diluted in 40 μl of denaturing buffer. For the analysis of αSyn fibrils cleavage, the gels were stained by coomassie. For the analysis of HTTExon1 fibrils cleavage, the gels were stained with SYPRO Orange  diluted 5000 fold in 10% acetic acid for 1h and visualized using LAS-3000 imager .αSyn (100 μM monomer concentration) and HTTExon1 (50 μM monomer concentration) long and short fibrils in their respective assembly buffers or incubated in Dulbecco’s modified Eagle’s medium with unilamellar vesicles from brain lipids were treated at 37 °C by Proteinase K .Murine neuroblastoma Neuro 2a cells (ATCC) were culture at 37 °C in humidified air with 5% CO−1 of propidium iodide. At time zero, Atto 488-labelled αSyn long or short fibrils at increasing monomer concentrations (0.1–5 μM) were added to the cells (106 cells per ml) and analysis were performed using a MoFlo Astrios Cell Sorter (Beckman Coulter). The cell population was gated by forward and side scatter and at least 15,000 cells were recorded. The fraction of fluorescent cells was calculated over a time frame where the signal was stable.To monitor the binding of αSyn long and short fibrils to the cell membranes, Neuro 2A cells lifted with EDTA were resuspended in serum-free, phenol red free-Dulbecco’s modified Eagle’s medium containing 1 μg.ml5 cells were platted in a 6 cm-diameter Petri dish 48 h before the experiment. They were then washed with PBS and incubated with Atto 488-labelled HTTExon1 long or short fibrils at increasing monomer concentrations (0.5–10 μM) for 2 h at 37 °C. After incubation cells were lifted with EDTA and analysed as described above.As the binding of HTTExon1 fibrils to cells is a slow processSubconfluent Neuro 2A cells cultured on ibidi-μ-Dishes  were loaded for 30 min at 25 °C with 2 μM of the fluorescent dye Fluo-4-acetoxymethyl ester (Fluo-4-AM) (Invitrogen) in the presence of 0.02% (w/v) Pluronic Acid F127 (Invitrogen) and 2.5 mM of the organic anion-transport inhibitor probenicid (Invitrogen). After washing the cells deesterification of the Fluo-4-AM was allowed for 10 min at 25 °C in serum-free Dulbecco’s modified Eagle’s Medium in the presence of 0.02% (w/v) Pluronic Acid F127 (Invitrogen). PBS, PBS + EGTA (5 mM), αSyn or HTTExon1 long or short fibrils (10 μM monomer concentration) with or without EGTA (5 mM) were then added to the cells. The cells were imaged over time on the Axio Observer ZI epifluorescence microscope (Zeiss) at a magnification of 20x. Images were acquired every 1 (αSyn) or 2 (HTTExon1) min for 40 (αSyn) or 60 (HTTExon1) min at 25 °C in humidified air with 5% CO2. The fluorescence was quantified for each cell of the field (≈50–100 cells/field) using the software ImageJ, after background subtraction for each image, and expressed as a fraction of the maximum fluorescence recorded upon addition of ionomycin (10 μM) to the cells.How to cite this article: Monsellier, E. et al. α-Synuclein and Huntingtin exon 1 amyloid fibrils bind laterally to the cellular membrane. Sci. Rep.6, 19180; doi: 10.1038/srep19180 (2016).


LS-coupling model are presented along with eigenvector compositions of the levels. On the basis of this analysis new designations are recommended for several levels.New measurements of Ar III wavelengths between 508 Å and 4183 Å are combined with measurements from the literature to find improved values for the energy of most of the known levels in Ar III. Parameters derived from fitting the new level energies to an  Atomic Energy Levels 2 when added to the level value of 4s5S2, gives the ionization energy as 328 550 cm−1. Although the values of δn for both Ca III and Sc III are in near agreement and would therefore lead to a relatively small value for the uncertainty of the calculated ionization energy, we feel that with such a small sampling of possible values of n it is necessary to place a standard uncertainty of approximately ± 100 cm−1 on the ionization energy. The value of 328 550 ± 100 cm−1 compares very well with the value of 328 600 cm−1 obtained by Edlén [In their paper on ionization energies of singly ionized rare earths, Sugar and Reader  showed tby Edlén  using a


Ultrafine Co3O4 nanoparticles were homogeneously embedded in ultrathin porous graphitic carbon in this material. The carbon sheets, which have large specific surface area, high electronic conductivity, and outstanding mechanical flexibility, are very effective to keep the stability of Co3O4 nanoparticales which has a large capacity. As a consequence, a very high reversible capacity of up to 1413 mA h g−1 at a current density of 0.1 A g−1 after 100 cycles, a high rate capability , and a superior cycling performance at an ultrahigh rate (760 mA h g−1 at 5 C after 1000 cycles) are achieved by this lithium-ion-battery anode material.A high-performance anode material for lithium storage was successfully synthesized by glucose as carbon source and cobalt nitrate as Co The electrochemical properties of such materials were tested as a LIBs anode. It presented very high reversible capacity and excellent rate performance and cycling stability. It might be possible to extend this technique to other kind of nanostructured matal oxide for important applications in high-performance LIBs, supercapacitors, adsorbents, catalysts and sensors in many scientific disciplines.Cobaltosic oxide CoO4 has re3O4@C@PGC nanosheets is schematically illustrated in 37386H12O6 and Co(NO3)2 were first dissolved in distilled water to get a homogeneous solution. After gradually heated to 80 °C, the solution color changed from pink to blue, the sample viscosity increased greatly, and then the polymerization of the glucose began, which resulted in the generation of a very thin frame homogeneously coated on the NaCl particle surface. After that, the composite powders were calcinated at 750 °C under N2. During this process, the Co(NO3)2 should be decomposed into cobalt oxide6H12O6 into Co NPs with the protection of inactive gas29in situ oxidized into Co3O4 NPs as the Co@C@PGC nanosheets were calcinated in air. Finally, the Co3O4@C@PGC nanosheets were obtained on the surface of the NaCl particles as shown by the images of The overall synthetic procedure of the Co3O4@C@PGC nanosheets were both shown in 6H12O6 takes place at step one, and the annealing in air at step two has little damage on the carbon. Except of graphite, all peaks in the XRD pattern of Co@C@PGC belong to Co (JCPDS 15-0806), which implies that the cobalt has been completely reduced from its oxide. Only Co3O4 phase is shown in the XRD pattern after calcinations in air except of graphite. It suggests that the calcination in step two is very effective to accomplish the conversion from the precursor (Co@C@PGC nanosheets) to the targeted sample (Co3O4@C@PGC nanosheets). The average sizes of NPs were both calculated based on the full width at half maximum intensity of (311) for Co3O4 and (111) for Co using Scherrer’s formula. The estimated average particle size of Co3O4 is very close to Co, which is about 20.4 nm. In addition, NaCl phase isn’t found in both XRD patterns. So NaCl particles were completely removed in the water-washing process.The XRD patterns of Co@C@PGC and Co3O4@C@PGC nanosheets was shown in the SEM image of 2 interconnected with each other to build up a porous architecture. This architecture can be reserved even after calcinations in air, which is evidenced by the SEM image shown in 3O4/C composites without adding NaCl was also observed by SEM, shown in 3O4@C@PGC nanosheets. Their thickness is more than 500 nm. They just pile up on each other, leaving very little space between them. The thin sheets observed in Co@C@PGC and Co3O4@C@PGC disappear thoroughly in Co3O4/C composites. It suggests that NaCl particles have played a crucial role in building up such porous architecture of nanosheets. A TEM image and HRTEM image of Co3O4@C@PGC nanosheets are shown in 3O4 NPs, with diameters ranging from 5 to 40 nm, are well-dispersed in the PGC nanosheet. The selected area electron diffraction pattern in the inset of 3O4 NPs. In fact, the Co3O4 NPs were firmly anchored by the porous graphite nanosheets. As shown in 3O4 crystal is perfectly encapsulated by thin and well-graphitized onion-like carbon shells. The HRTEM interference fringes of the Co3O4 crystal can be clearly decerned. They have a spacing of 0.24 nm, as denoted in 3O4. The spacing of the interference fringe of the shell is 0.34 nm, which is also equal to the spacing of (002) plane of graphite. And the thickness of graphite shell is about 9 nm. The morphology of Co@C@PGC nanosheets is also characterized by TEM and HRTEM  is usually used to evaluate the degree of crystallization of carbon materialsG/ID for the Co3O4@C@PGC nanosheets is ~1.3, indicating that the obtained nanosheets own well-crystallized graphitic structures3O4 NPs. The carbon content is determined by TGA and the result is shown in oC in air, the carbon will be completely burnt out. So, the weight loss in TGA result is equal to the carbon content. The carbon content of the Co3O4@C@PGC nanosheets is 29.1 wt% which is comparable to the Co3O4/C composites  film263O43O4 anode can be described by the following electrochemical conversion reactionThe initial three cycles of the representative cyclic voltammogram (CV) performed on a coin half-cell at room temperature between 0.01 and 3.0 V at a sweep rate of 0.1 mV sThe main reduction peak is shifted to ~1.15 V, and the peak intensity drops significantly from the second cycle, indicating the occurrence of some irreversible reactions associated with formation of the SEI film in the first cycle3O4@C@PGC nanosheets electrodes at a current density of 0.1 A g−1 between 0.01 and 3.00 V are shown in −1, respectively, resulting in an initial Coulombic efficiency of ~64%. The initial irreversible capacity loss of the Co3O4@C@PGC nanosheets could be associated with the inevitable formation of SEI and decomposition of electrolyte513O4@C@PGC nanosheets exhibit excellent cycle stability24Typical galvanostatic charge/discharge curves of the Co3O4@C@PGC nanosheets, Co3O4/C composite and Co3O4 NPs were investigated at a current density of 0.1 A g−1. The results were shown in 3O4@C@PGC nanosheets electrode has the best cyclic retention and the highest reversible capacity. After 100 cycles, the reversible capacity is as high as 1413 mA h g−1. The capacity is even higher than the theoretical capacity of Co3O4, which is common in many reported literatures192628295455563O4 NPs have large specific surface area where more active sites for lithium ions are located27−1 . It is obvious that the Co3O4@C@PGC electrode still needs ten more cycles to activate all active materials for its reversible capacity increases with cycle number until the 11th cycle503O4@C composite electrode shows a stable capacity as tested at 1 C. The reversible capacity of the Co3O4@C@PGC nanosheets electrode reaches a very high value of 1105 mA h g−1 at the eleventh cycle233O4@C composite electrode maintains a reversible capacity of 502 mA h g−1 during these cycles. At each tested specific current density including 2, 5, 10, and 15 C, the average reversible capacity of the Co3O4@C@PGC electrode  is much higher than Co3O4@C composite electrode . Even at 20 C, the average reversible capacity (345 mA h g−1) is still very close to the theoretical capacity of graphite. When the current rate finally returns to the initial value of 1 C after 61 cycles, the electrode can recover its initial capacity of 1030 mA h g−1 with a little bit loss. And it is still sustainable up to the 70th cycle. The high reversible capacity and excellent rate performance indicate that the electrode has a high energy density and power density. So the electrode occupies the up-right corner of Ragone plot, as shown in Fig. S63O4@C@PGC nanosheets electrode has been further investigated upon 1000 cycles at 5 C and the result is shown in −1, corresponding to a process of activating the active substance, which is common in the literature50−1 even after 1000 cycles.The electrode made by Co3O4@C@PGC nanosheets, Co3O4/C composite, and Co3O4 NPs after 10 cycles at a current density of 0.1 C, respectively. The corresponding Nyquist plots are shown in SEI) and contact resistance (Rf), the semicircle across the medium-frequency region represents the charge-transfer impedance (Rct) on the electrode/electrolyte interface, and the low-frequency linear tail corresponds to the Warburg impedance (Zw) associated with the diffusion of lithium ions in the bulk electrode (Re)73644623O4@C@PGC nanosheets electrode has the smallest semicircle diameter. So it possesses the lowest contact and charge-transfer impedances. That is to say, Li+ and electron are able to transport fastest during the electrochemical insertion/extraction reaction in the electrode, which makes it reasonable that the Co3O4@C@PGC nanosheets electrode shows the best rate performance.In order to uncover the mechanism which endow the electrode with superior electrochemical performance, electrochemical impedance spectroscopy (EIS), a promising tool for investigating diffusion issues, was conducted at frequencies from 100 kHz to 0.01 Hz on the electrode of Co3O4@C@PGC electrode possesses a much larger reversible capacity at any rate, as shown in 3O4@C@PGC nanosheets anode can be illustrated by the mechanism suggested in 3O4 NPs in the paper is small enough. It is very close to the minimal size below which pulverization can’t happen183O4 cores are firmly covered by well-graphitized onion-like carbon shells. Either the pulverization wouldn’t happen in this experiment, or, even if it happened, the particle would be split into finite particles which still connect with the carbon shell, just as illustrated by 3O4 NPs because of the reversible conversion reaction3O4 NPs during lithium ion insertion/extraction process. Finally, the volume change is accommodated by the abundant macropore and mesopore in the PGC nanosheets. So the structure of the material is so robust which guarantee the stability of the reversible capacity during cycle charge/discharge. The Co3O4 NPs were integrated into PGC nanosheets. Thus, a continuous network is constructed by the conductive carbon, which ensures the electron transport freely. The porous structure of the nanosheets provides a huge interfacial area with electrolyte. The electrode has abundant accesses for Li+ to interior active materials. The thin film structure of Co3O4@C@PGC nanosheets shortens the diffusion path of Li+. So the electrode can work very well at a very high specific current density. At last, the thin film structure is also very favorable for the adsorption of lithium ions on both sides, edges, and other defects of these nanosheets593O4 NPs3O4@C@PGC electrode is higher than the theoretical specific capacity of Co3O4.As compared with the results in literature32930313233576465666768693O4 NPs with an average grain diameter of about 20.4 nm were successfully fabricated. In this strategy, Co3O4@C@PGC nanosheets were compounded using glucose as the carbon source, cobalt nitrate as the Co3O4 precursor, and a surface of sodium chloride as the template. In this unique architecture, the PGC nanosheets with high elasticity can effectively preserve the structural and interfacial stabilization of Co3O4 NPs as well as accommodate the mechanical stress resulting from the severe volume change of Co3O4 NPs during lithium ion insertion/extraction, while the interconnected porous graphitic carbon networks with large surface area, superior electrical conductivity, high mechanical flexibility and stability lead to remarkably enhanced structural and electrical integrity as well as excellent kinetics for ion and electron transport of the overall electrode. As a result, such a nanostructured electrode exhibits an extremely durable high-rate capability: a capacity of 1030 mA h g−1 is achieved at 2 C, 560 mA h g−1 at 10 C, and 345 mA h g−1 at 20 C. Cycling at 5 C after 1000 cycles leads to a recovered capacity of 760 mA h g−1, still almost 2 times more than the capacity of graphite. The obtained good performance opens up new opportunities in the development of high performance next-generation LIB used for alternative energy and electric transportation.In summary, porous graphitic carbon nanosheets encapsulating small and uniform Co3)2.6H2O and 15 g of NaCl were dissolved in 50 ml of deionized water under magnetic stirring until a transparent blue solution was obtained. Then, the solution was transfered into a drying oven and dried at 80 oC for 24 h. The dry block of crystal was ground to fine powder in an agatemortar. The composite powder was heated in a tubular vacuum furnace at 750 oC for 2 h under flowing N2. Finally, it was annealed again at 300 oC for 4 h under air. Once cooled to room temperature, the as-synthesized black powder was washed by deionized water several times to remove NaCl, and then Co3O4@C@PGC nanosheets were obtained. For comparison, Co3O4/C composites were also synthesized by carbonizing the mixture of Co(NO3)2·6H2O and glucose without adding NaCl at the same conditions. Meanwhile, Co3O4 NPs were also synthesized by sintering Co3O4@C@PGC nanosheets at 600 oC for 2 h in air. And PGC nanosheets were obtained by immersing Co3O4@C@PGC nanosheets in plenty of nitric acid for 4 h at 70 oC with magnetic stirring to remove the Co3O4 NPs.All the chemicals were of analytical grade and were used without further purification. Firstly, 1.8 g of glucose , 0.582 g of Co surface areas and porosities of the products were determined by nitrogen adsorption and desorption using a Micromeritics ASAP 2020 analyzer.XRD measurements were taken on X-ray diffractometer  with Cu Kα radiation at a wavelength of 1.5406 Å to determine the phase composition and crystallinity. Field emission scanning electron microscope (FESEM) images were acquired on a JEOL JSM-6700F micro-scope operated at 5 kV. The transmission electron microscope (TEM), high-resolution TEM (HR-TEM) images were recorded on a JEOL JEM-2100F high-resolution transmission electron microscope at 200 kV. Thermogravimentry analysis (TGA) was performed with a Perkin-Elmer (TA Instruments) instrument up to 750 3O4@C@PGC nanosheets, Co3O4/C composites, Co3O4 NPs and PGC nanosheets), conductive material  and binder polyvinylidene fluoride  in a weight ratio of 8:1:1 with N-methyl-2-pyrrolidone  as solvent. The mixture was fully stirred for several hours in a weighing bottle into slurry. Then, it was uniformly pasted on Cu foil substrates. After vacuum drying at 120 oC for 12 h, the electrode disks (d = 12 mm) were punched and weighed. The working electrode has approximately 0.5–1.0 mg of active material. The diameter of counter electrode (16 mm) is larger than working electrode. The electrolyte is 1 M LiPF6 in a mixture of ethylene carbonate and diethyl carbonate (1:1 by weight) and a Celgard 2400 polypropylene membrane was used as the separator. The cells were assembled in an argon-filled glove box in which both the moisture and oxygen contents were controlled to be less than 0.1 ppm. Cyclic voltammetry (CV) was performed on an IVIUMSTAT electrochemical workstation at 0.1 mV s−1 in the range of 0.01−3.0 V. Galvanostatic charge-discharge cycling tests were performed using an LAND CT2001A battery testing system in the voltage range between 0.01 V and 3 V. Electrochemical impedance spectroscopy (EIS) experiments were carried out using an IVIUMSTAT electrochemical workstation in the frequency range of 0.01−100 kHz at room temperature. All of the specific capacities in this work were estimated by using the weight of the active materials.Electrochemical measurements were conducted on a coin-type test cell (CR2025) with metallic lithium sheet as both counter and reference electrode. The working electrode is fabricated by mixing active material .


Scoparia dulcis, is used as a herbal remedy by diabetics worldwide. Fresh Scoparia dulcis porridge elicited a low glycaemic index (GI) and anti-hyperglycaemic effects when fed to diabetic Wistar rats. Commercially produced Scoparia dulcis porridge (SDC) elicited medium GI. Present study was aimed at studying the anti-diabetic effects of consumption of commercially produced S. dulcis porridge.Leaf extracts of n = 35) on medication, with mild and moderate diabetes  was conducted. Within the first three months (study period 1) group 1 was the test and group 2 was the control. Following a wash-out period, the two groups were crossed over . Test group consumed commercially produced SDC for 3 days/week for three months and the control group any other food. At the onset and end of each study period glucose measurements , lipid measurements , toxicity parameters  were analyzed by enzyme assay kit methods using a KONELAB 20XT auto analyzer. Significances between groups were analyzed by one way ANOVA  and Mann Whitney test . Within group comparisons were carried out by Bonferroni post hoc test.A randomized crossover clinical trial with type 2 diabetic patients (p = 0.003) while HbA1c of group 2 decreased from 7.0 ± 0.3to 6.7 ± 0.3 while in the test group. Therefore, both test groups (1 and 2) elicited a decrease in HbA1c compared to respective control groups. Both test groups elicited a non significant decrease in FBG following the intervention . No significant differences (p >0.05) in insulin, cholesterol measurements  and atherogenic index between or within groups were observed. All other measurements  were normal and not significantly different between or within groups.During the crossover clinical trial HbA1c of group 1 decreased from 7.9 ± 0.5 to 6.5 ± 0.3 (p >0.05) of type 2 diabetic patients. The porridge had no effect on cholesterol measurements and no toxicity was observed at the dose tested. Therefore, the SDC porridge can be recommended as a suitable meal for diabetic patients.Porridge made with SDC leaf extract decreased FBG and HbA1c ( Scoparia dulcis is used as a herbal remedy by diabetics worldwide with proven hypoglycaemic effects  (15 kg) were mixed and extruded at 110 –120 °C using JR 600 Extruder (InstraPor). Extruded meal was dehydrated using a hot air dryer . Dried meal was grinded using a Hammer mill (particle size 100 % pass through 0.5 mm mesh). Extruded meal was mixed in 1:1 ratio with boiled and dried intact rice grains , macrovascular complications , type 1 diabetics, who refused consent, and/or speak only Tamil were excluded from the study.n = 37) were grouped randomly in to test (group 1) (Scoparia dulcis) and control (group 2) groups (17–20/group) (every other patient from a list was included in to one group by the researchers). Each patient was in the test group for 3 months and in the control group for another 3 months. Test group was advised to consume one packet of Scoparia dulcis porridge once a day for 3 days/ week for 3 months and control group was advised to consume a normal breakfast except a green leafy porridge. One patient from the test group discontinued participation due to a hypoglycaemic attack subsequent to consuming the porridge. Following commencement of the study, one patient from the control group left the country. At the end of three months (study period 1) the groups were crossed over following a washout period of two weeks and the study was repeated for another three months (study period 2) with no dropouts. Thus the analyses included 35 patients (Flow diagram 1) -0.203 x  x (1.210 if black)]. All analyses were carried out on the same day that the blood samples were collected. Considering the cost and practical difficulties, the week 12 values of insulin and HbA1c were considered as unchanged and taken as the basal values for the second study period (week 14).Blood (8 cc) were drawn from the median cubital vein of the patients (fasted for 8 – 10 h), by a trained phlebotomist, at the onset and end of each month during the study and serum was separated by centrifuging the samples at 3500 rpm. Liver enzymes , creatinine, urea, CRP, lipid measurements  and blood glucose measurements  were measured at the onset and at the end of the third month of the study. All parameters were measured by enzymatic kit assays  except for plasma insulin which was measured by an ELISA assay (DRG Diagnostics Insulin assay kit). eGFR was calculated using the standard equation , the initial (0 week and 14th week) FBG between groups 1 and 2 were not significantly different . Although not significant, FBG in both test groups declined following the intervention  HbA1c between group 1 and 2 were not significantly different . However, a significant decrease (p = 0.003) in HbA1c in group 1 was observed during the intervention. In group 2 a significant decline in HbA1c was not observed (p = 0.98). Mean serum insulin levels were not significantly different (p >0.05) between or within group 1 and group 2 during both the study periods.During the two study periods, initial (0 week and 14p >0.05) in any of the lipid parameters  within groups 1 and 2 during both study periods. However, the TG in group 2 (control) was significantly higher when compared to group 1 (test) (p = 0.036) at the end of first 3 months (study period 1). A significantly high (p = 0.022) HDL-C level was observed in the test group (group 2) compared to the control (group 1) at the end of the second 3 months (study period 2). TC and LDL-C were above the upper limit of normal and TG was near the upper limit of normal in groups 1 and 2 during both study periods.Table per se do not provide details on accurate risk levels, mainly in familial hypercholesterolaemia [Estimation of TC: HDL, also known as atherogenic or Castelli index and LDL: HDL are vital indicators of cardio vascular risk  as they rolaemia   was assessed. Atherogenic index of >0.21 indicates a high risk of atherosclerosis [p >0.05) between or within the groups 1 and 2 was observed for AI at any study period of the study.Although serum total cholesterol (TC) is normal, with low HDL level, the probability of development of atherosclerotic plaque is high . As TC pclerosis  (Table 3p >0.05) was not observed between or within the two groups for all tested liver enzymes. Mean measurement variations of liver enzymes were within the normal reference range during both study periods (1 and 2). Mean eGFR of all study participants was within the normal range and was between 74.2 – 87.5 mL/min/1.73 m2, irrespective of the duration of diabetes . No significant change was observed in urea, creatinine and CRP during both study periods.Table S. dulcis leaves as used in this study could elicit significant anti-hyperglycaemic effects. A 1 % reduction of HbA1c over three months is known to reduce the risk of micro-vascular complications by 37 %, DM related complications and death by 21 % [p = 0.003) in HbA1c in group 1 during the intervention and a non-significant  decline in HbA1c in group 2, consumption of SDC porridge may contribute towards a reduction of micro-vascular and other fatal complications in diabetics [S. dulcis porridge may help in reducing the morbidity and mortality due to diabetes.This clinical trial was designed to investigate the antidiabetic potential and toxic effects of consumption of a novel commercially produced herbal porridge in diabetics. The dried leaf solid dose in the porridge  was 13–33 times lower than the doses (250 – 500 mg/kgBW) used in previously reported studies [Scoparia dulcis leaf extract include decreased oxidative stress in RINm5F pancreatic cells [A significant change in mean plasma insulin was not observed between or within the groups 1 and 2 indicating that there was no change in insulin synthesis, secretion or binding to receptors during the study period when the patients were in the control group. However, a decrease in FBG and HbA1c in both test groups during both study periods (study periods 1 and 2) was observed. This could be due to an increment of both the insulin level (synthesis and secretion) and binding to receptors mediated by SDC  or mightic cells , increasic cells , decreasic cells  and incric cells . In thisic cells , which cThe study also revealed that 61.5 % and 59.0 % of the study population was hypercholesterolaemic and/or hypertriglyceridaemic respectively. More than 80 % of the T2DM patients in this study belonged to the high risk category according to the atherogenic index indicating a high susceptibility of these patients for cardio-vascular diseases.An elevated occurrence in lipid abnormalities in patients who have had diabetes for more than one year was observed during this study. The percentages of hypercholesterolaemic and hypertriglyeridaemic diabetic patients were 82.7 % and 90.5 % respectively. Insulin resistance elevates the VLDL and apolipoprotein (apo) B-containing lipoproteins concurrently diminishing the suppressive effect of insulin on apo B secretion either by reducing apoB degradation or by inhibition of microsomal TG transfer protein activity . This hiAbsence of any significant elevation of AST, ALT, ALP, creatinine or urea or a decline in eGFR in both test groups compared to control during both study periods (study period 1 and 2), indicate that there is no acute or short term liver or kidney toxicity by consumption of the porridge at the dose given. Further studies are recommended to prove the effects of consumption of SDC porridge continuously for a long period.CRP is an inflammatory marker and is generally elevated in diabetic patients . As CRP Precision of the results could have been increased with a higher number of patients, however due to the limited time period this could not be achieved. As the study was not a double blinded study and as the control group had not received a placebo, existence of bias to a certain extent in the present study is possible.Scoparia dulcis leaf extract reduce fasting blood glucose and HbA1c levels without elevating liver enzymes, creatinine, urea and CRP in type 2 diabetic patients. As a rice based herbal porridge is more palatable and fulfilling than a leaf extract or a tablet, the porridge used for this study would be a better breakfast meal for diabetics due to its medium GI [Porridge made with edium GI , anti-hy


Our results suggest that during the first lithiation process the ZnFe2O4 nanoparticles undergo a conversion process and generate a composite structure of 1–3 nm Fe and Zn nanograins within Li2O matrix. During the delithiation process, volume contraction and the conversion of Zn and Fe take place with the disappearance of Li2O, followed by the complete conversion to Fe2O3 and ZnO not the original phase ZnFe2O4. The following cycles are dominated by the full reversible phase conversion between Zn, Fe and ZnO, Fe2O3. The Fe valence evolution during cycles evidenced by electron energy–loss spectroscopy (EELS) techniques also exhibit the reversible conversion between Fe and Fe2O3 after the first lithiation, agreeing well with the in situ TEM results. Such in situ TEM observations provide valuable phenomenological insights into electrochemical reaction of MTMOs, which may help to optimize the composition of anode materials for further improved electrochemical performance.A family of mixed transition–metal oxides (MTMOs) has great potential for applications as anodes for lithium ion batteries (LIBs). However, the reaction mechanism of MTMOs anodes during lithiation/delithiation is remain unclear. Here, the lithiation/delithiation processes of ZnFe Recently, Fe–based oxides  to meet the rapid development of portable electronics. Various transition metal oxides (TMOs), such as iron oxides124682O4 stands out from other Fe-based spinel series anodes because of its high abundance and high theoretical specific capacity of 1072 mAh g−1 et al. first reported the use of ZnFe2O4 as an anode synthesized by a pulsed laser deposition method−1 and after100 cycles 78% of the capacity (434 mAh g−1) was still retained. Then the electrochemical performances of diverse ZnFe2O4 nanostructure have been greatly enhanced, such as ZnFe2O4/C hollow spheres2O4 octahedrons2O4 nanofibers (ZFO–NF) and nanorods2O3, which is distinguishing from the previous in situ TEM study on Fe2O3 anodeet al.2O4 is reversible reactions of LiZn to ZnO and Fe to FeO after the first discharge process. Particularly, the dynamic electrochemical reaction of binary transition metal oxide for LIBs is still in a black box.ZnFeIn situ TEM technique has been recognized as an excellent option to monitor real–time observation of electrode materials with lithium and sodium on the nanometer scale26272823122O39S8/CNTin situ TEM technique. Up to now, the electrochemical reaction between ZnFe2O4 and Li has not been studied. Here, an all–solid nano–LIB was constructed inside a high–resolution TEM using ZnFe2O4 as working electrode to visualize the microstructure and phase evolution during electrochemical processes. It is found that upon lithiation the ZnFe2O4 nanoparticle was converted into numerous Fe and Zn nanograins within Li2O matrix with a severe volume expansion. During delithiation, the anode cannot be converted to its original phase ZnFe2O4 but transformed to Fe2O3 and ZnO. The Fe valence evolution of ZnFe2O4 nanoparticle is also studied by EELS measurements, which agrees well with the in situ TEM results. Our in situ TEM results for provided the direct experimental evidence of the reaction mechanism of ZnFe2O4 during lithium-ion insertion and extraction.2O4/graphene is shown in 2O4/graphene, it indicates there are many ZnFe2O4 particles with sizes of 120~180 nm anchored on graphene, and the transparent nature of the graphene implies that it is fully exfoliated into single or few-layer sheet. A high–magnification TEM image of an individual ZnFe2O4 nanoparticle is given in 2O4 nanoparticle is primarily composed of nanocrystals with a size ~10 nm. The smaller size of ZnFe2O4 nanocrystals can shorten Li+ diffusion pathways, increase the electron/ion conductance, and reduce the volume change induced by lithiation/delithiation, further enable the ZnFe2O4 nanocrystals to show an improved electrochemical performance. The high resolution transmission electron micrograph (HRTEM) was taken along the 2O4 (JCPDS card no. 89–1012). 2O4/graphene. All the diffraction rings can be perfectly indexed as a cubic structure of ZnFe2O4 (JCPDS card no. 89–1012); it further confirms that the resultant products are ZnFe2O4 phase.The microstructure characterization of the obtained ZnFe2O4 during lithiation–delithiation cycles, an in situ nanoscale electrochemical device of ZnFe2O4 was constructed, as schematically shown in 2O4/graphene anode, metal Li counter electrode, and the naturally grown solid electrolyte Li2O layer on metal Li. After contact between Li2O and ZnFe2O4/graphene anode was established, a constant potential of −1.0 V was applied to the ZnFe2O4/graphene against to the Li counter electrode to drive the first lithiation of ZnFe2O4. 2O4 nanoparticles with diameters of ~196 and 205 nm in the first electrochemical lithiation process. It suggests that the lithiation occurred on all surfaces within 37.0 s, indicating the fast lithium diffusion on the ZnFe2O4 nanoparticle surface. As shown in 2O4 nanoparticle anchored on graphene layer, and the first lithiation lasted for 21 s, giving a lithiation ratio of ~2.14 nm/s for this ZnFe2O4 nanoparticle. As for another ZnFe2O4 nanoparticle sited on the edge of the graphene , (220), (311) lattice planes of Li2O are marked by red lines, while the (110), (101) lattice planes of Fe and Zn have been labeled. Then the potential was reversed to + 3 V to initiate the delithiation process, and the TEM images of the electrode after full delithiation are shown in 2O3 respectively. Also the ED pattern . The pristine ZnFe2O4 nanoparticle was inflated and expanded its size to 223 nm after full lithiation  measured in 3d transition metal is used to determine the occupation number of 3d electrons. Here the IL3/IL2 of Fe has been obtained to correlate EELS features with the valence states of Fe in the first three lithiated and delithiated states. 2O4/graphene electrode to confirm the evolution of valence states of Fe elements in the electrochemical lithiation and delithiation cycles. The EELS spectrum of Fe in the original ZnFe2O4/graphene electrode is shown in 3/L2 intensity ratio of Fe is 5.3, confirming that the valence state of Fe is undoubtedly 3 + 3/L2 intensity ratio of Fe in the fully lithiated stage reduced to 2.3, which is smaller than that of Fe2+ (4.1 ± 0.2), agrees well with the valence state of 02O4/graphene anode that shown in 3/L2 intensity ratio of Fe increased to 5.1, this EELS can be regarded approximately complete oxidation, that is, Fe “3 +” fingerprint. This result confirms that after the first cycle the delithiated product was Fe2O3. It says the Fe element can renew its original state of Fe3+ after the first delithiation process, further demonstrating good reversibility of Fe metal. 3/L2 intensity ratio of Fe is 2.5 after the second full lithiation process, which is similar with that of Fe in the first lithiated stage, corresponding to the valance state of zero. Then the L3/L2 intensity ratio of Fe increased to 5.2 again calculated from the EELS spectrum given in 3/L2 intensity ratio of Fe in ZnFe2O4 was also noticed in the third cycle. The repeated changes in L3/L2 intensity ratio of Fe indicate the complete and reversible electrochemical transition between Fe0 and Fe3+ during the electrochemical processes, thus leading to high reversibility for iron oxide based anodes. The EELS results are agree well with the ED results shown in 2O4 during the electrochemical processes is a reversible phase transition between Fe and Fe2O3 after the first lithiation process.An EELS that assisted in TEM is a useful technique for analyzing the valences of some transition metal elements at the nanoscale. The transition of an electron from a 2p level to 3d orbitals leads to the formation of Lin situ TEM analysis, the possible reactions of ZnFe2O4 during the lithiation/delithiation process are proposed as follows. During the first lithiation process, Li+ ions can diffuse quickly on graphene sheets, leading to a uniform lithiation take place on the surface of ZnFe2O4 nanoparticles. As Li+ insertion continued, the lithiation front in the ZnFe2O4 gradually propagated and gave rise to a visible interface between the lithiated and unlithiated phases. As a result, lithiation is essentially the destruction of the crystal structure, lithium is intercalated into ZnFe2O4 and led to metallic Zn, Fe nanograins and Li2O appear in the product followed by the obvious volume expansion. The first lithiation can be expressed as: ZnFe2O4 + 8Li+ +  8e− → Zn + 2Fe + 4Li2O. During the delithiation process, Li-ion will firstly be extracted from the lithiated ZnFe2O4 leading to volume contraction, and lithiated product metallic Fe and Zn nanograins can be oxidized to metal oxide (Fe2O3 and ZnO) with the presence of Li2O through the conversion reaction, ZnFe2O4 is not the initial molecule that can be recovered. So the delithiation process can be described as: Zn + Li2O → ZnO + 2Li+ + 2e−, 2Fe + 3Li2O → Fe2O3 + 6Li+ + 6e−. In the following lithiation/delithiation cycles, the reversible conversion reaction of ZnO, Fe2O3 and metallic Zn, Fe nanoparticles take place and indicates good reversibility. Thus, after the first cycle, reversible reactions can be expressed as following equations: ZnO + 2Li+ + 2e−2O, Fe2O3 + 6Li+ + 6e−2O. 2O4 nanoparticles during lithiation/delithiation cycles.According to the above 2O4  Fe and Zn nanocrystallites within Li2O matrix followed by obvious volume expansion. In the first delithiation process, the HRTEM and ED results show that ZnFe2O4 is not the original molecule that can be recovered, but metallic Zn and Fe nanoparticles oxidized to their respective metal oxides ZnO and Fe2O3 with the disappearance of Li2O through the complete conversion reaction. The ED patterns and EELS spectra reveal that the electrochemical lithiation/delithiation processes of ZnFe2O4 nanoparticles as anode in LIBs are revealed to be reversible phase transition between Fe, Zn nanograins and Fe2O3, ZnO nanograins. The information obtained from our findings can help to further improve the electrochemical performance of this type material and also is insightful for exploring various types of electrode materials in LIB technology.In summary, the electrochemical reaction mechanism of ZnFe2O4/graphene was prepared by a hydrothermal route. Firstly, GO (100 mg) was dispersed in ethylene glycol (80 ml) with sonication for 30 min to form a homogeneous dispersion. Then, Zn(Ac)2 H2O (0.55 g), FeCl3 (0.81 g), and NaAc (3.6 g) were added into the above solution with stirring for 30 min, and the mixture was transferred into a Teflon–lined autoclave with a capacity of 100 mL, and maintained at 200 °C for 24 h. The precipitate was isolated by filtration and washed several times after cooling down to room temperature. Finally, the product was obtained by drying the precipitate at 60 °C for 12 h.Graphite oxide (GO) was synthesized by a modified Hummers’ methodin situ observation on the electrochemical behaviors of ZnFe2O4 anode with the assistance of HRTEM, electron diffraction (ED) and EELS measurements. The ZnFe2O4 nanoparticles anchored on graphene were used as the working electrode, and Li metal was coated onto a piezo–driven W probe and regarded as lithium source and counter electrode, an oxide layer of Li2O formed on Li metal when it is exposed to air act as the solid electrolyte. The detailed setup procedure for the nano-LIB can be found in literature2O4/graphene driven by the nanomanipulator of the TEM-STM holder is contact with the Li2O layer, a nano-LIBs cell is successfully constructed. After that, a potential of −1 V was applied to the ZnFe2O4 electrode against the Li source to drive Li+ transport to initiate lithiation process, and then the bias was reversed to + 3 V to facilitate delithiation. EELS measurements were performed on a TEM with the assistance of Gatan EELS attachments.The nano-LIBs experimental set-up was constructed inside a TEM (JEOL JEM–2100F) to enable the How to cite this article: Su, Q. et al. Study on the Electrochemical Reaction Mechanism of ZnFe2O4 by In Situ Transmission Electron Microscopy. Sci. Rep.6, 28197; doi: 10.1038/srep28197 (2016).